Thèses sur le sujet « Brain capillary endothelial cell »

The blood brain barrier (BBB) is a selective biological barrier located at the brain capillaries, that protects the central nervous system (CNS) by monitoring exchanges between blood and brain. The BBB controls and regulates the composition of the CNS environment and it still constitutes the main obstacle for drug delivery to the brain (Weiss N. et al., 2009). The significant scientific and industrial interest in the physiology and pathology of the BBB led to the development of vast number of in vitro BBB models. Even though no “ideal” model exists yet, some of the currently available ones are very useful to investigate permeability, transport mechanisms and cellular and molecular events which occur at the BBB level. New strategies for brain targeted drug delivery exploit endogenously expressed transporters to elicit drug passage across the BBB. Among them, nanoparticles represent a promising tool, since they are biocompatible and biodegradable, and they can be functionalized to target the BBB (De Boer A.G. and Gaillard P.J., 2007) (Beija M et al., 2012; Caruthers S.D. et al., 2007; Moghimi S.M. et al., 2005). In this study we settled in vitro BBB models to identify, with high-throughput screening, the most promising nanoliposomes (NL) for combined BBB crossing and binding of amyloid peptides, for joint therapy and diagnosis of Alzheimer’s disease (AD). Firstly, we characterized two in vitro models of BBB, based on immortalized cell lines of human and rat origin, the hCMEC/D3 and RBE4 cells, respectively. We tested the transendothelial electrical resistance (TEER) and the endothelial permeability (PE) of small hydrophilic compounds: our results, in agreement with data reported in literature, lead us to conclude that these cellular models are suitable for their employment as high-throughput screening tools. Subsequently, we tested NL mono-functionalized with three different peptides, the apolipoproteinE derived peptide (the ApoE monomer (mApoE), amino acids 141-150), its tandem dimer (dApoE) (141-150)2, and the Human Immunodeficency Virus type 1 (HIV-1) transactivator of transcription (TAT) peptide. We evaluated their uptake and PE; we selected the TAT functionalization as the best performing concerning cellular uptake, and the mApoE functionalization when considering both the internalization and PE. Once assessed the dynamics of mono-functionalized NL interactions with endothelial cells, we investigated mApoE- and dApoE-NL loading a curcumin-derivative (Re F. et al., 2011) to bind Aβ. We clearly demonstrated that the mApoE-functionalization allows a better drug cellular internalization, whereas dApoE-NL enhances drug PE at the highest extent. We then considered mApoE- and dApoE-NL exposing the Aβ targeting ligands phosphatidic acid (PA) or cardiolipin (CL), demonstrating that PA-mApoE-NL showed the highest cellular uptake and PE. We also studied TAT-NL exposing curcumin derivative3 (Airoldi C. et al., 2011) for Aβ binding, clearly indicating that TAT functionalization increased cellular uptake and PE of curcumin derivative3-NL. We also studied intracellular fate of NL double functionalized, exposing Aβ targeting ligands, and no co-localization was detected with acidic cellular compartments, suggesting that NL may escape from lysosomal degradative pathway. Taken together, these results indicate that the formulations herein analyzed are suitable tools for brain targeted drug and contrast agent delivery. We suggest further development of mApoE and dApoE-NL entrapped with a drug payload for their employment as BBB endothelial cell or brain targeted drug delivery tools, respectively. We also selected PAmApoE-NL and curcumin derivative3-TAT-NL as promising tools for their employment in combination for AD therapy and diagnosis. Further studies, based also on in vivo experiments, are needed to evaluate NL suitability for clinical exploitation. Finally, we inquired the endocytic mechanisms that mediates the entry of NL in the endothelial cells of BBB. We employed RNA interference technique to down-regulate caveolin1 expression. Our preliminary data suggest that caveolin1 and the related caveolaemediated endocytosis pathway may account for 40% of mApoE-NL cellular uptake. Future directions regard the down-regulation of other proteins specifically involved in different endocytic mechanisms, i.e. clathrin-mediated and adsoprptive endocytosis, in order to assess which endocytic mechanisms may account for ApoE and TAT-NL internalization.

The blood brain barrier (BBB), formed by endothelial cells lining the lumen of the brain capillaries, is a restrictively permeable interface that only allows transport of specific compounds into the brain. Cardiolipin (CL) is a mitochondrial- specific phospholipid known to be required for the activity and integrity of the respiratory chain. The current study examined the role of cardiolipin in maintaining an optimal mitochondrial function that may be necessary to support the barrier properties of the brain microvessel endothelial cells (BMECs). Endothelial cells have been suggested to obtain most of their energy through an-aerobic glycolysis based on studies of cells that were obtained from the peripheral vasculatures. However, here, we showed that the adult human brain capillary endothelial cell line (hCMEC/D3) appeared to produce ~60% of their basal ATP requirement through mitochondrial oxidative phosphorylation. In addition, RNAi mediated knockdown of the CL biosynthetic enzyme cardiolipin synthase (CLS), although did not grossly affect the mitochondrial coupling efficiency of the hCMEC/D3 cells, did seem to reduce their ability to increase their mitochondrial function under conditions of increased demand. Furthermore, the knockdown appeared to have acted as a metabolic switch causing the hCMEC/D3 cells to become more dependent on glycolysis. These cells also showed increase in [3H]-2-deoxyglucose uptake under a low glucose availability condition, which might have served as a mechanism to compensate for their reduced energy production efficiency. Interestingly, the increase in glucose uptake appeared correlated to an increase in [3H]-2-deoxyglucose glucose transport across the knockdown confluent hCMEC/D3 monolayers grown on Transwell® plates, which was used in our study as an in vitro model for the human BBB. This suggests that changes in the brain endothelial energy status may play a role in regulating glucose transport across the BBB. These observations, perhaps, also explain why the brain capillary endothelial cells were previously observed to possess higher mitochondrial content than those coming from non-BBB regions (Oldendorf et al. 1977).

In order to effectively treat disorders whose pathology is marked by neovascularization, a better understanding of the pathways that mediate the processes involved in angiogenesis is needed. To this end we have identified two important pathways that regulate endothelial cell capillary morphogenesis, a key process in angiogenesis. We have identified the small GTPase RhoB as being induced by vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs). Depletion of RhoB inhibited endothelial cell VEGF - mediated migration, sprouting, and cord formation. Cells depleted of RhoB showed a marked increase in RhoA activation in response to VEGF. Defects in cord formation in RhoB - depleted cells could be partially restored through treatment with the Rho inhibitor C3 transferase or ROCK I/II inhibitors, indicating increased RhoA activity and enhanced downstream signaling from RhoA contribute to the phenotype of decreased cord formation observed in cells depleted of RhoB. Interestingly, we did not observe a significant change in RhoC activity in RhoB - depleted cells suggesting differential regulation of RhoA and RhoC by RhoB in HUVECs. We have also identified microRNA - 30b (miR - 30b) as being negatively regulated by VEGF and as being a negative regulator of HUVEC capillary morphogenesis. Overexpression of miR - 30b significantly reduced HUVEC cord formation in vitro, while inhibition of miR - 30b enhanced cord formation. Neither overexpression nor inhibition of miR - 30b affected migration or viability of endothelial cells. Interestingly, miR - 30b regulated the expression of TGFβ2 but not TGFβ1, with overexpression of miR - 30b inducing expression of TGFβ2 mRNA and protein, and inducing phosphorylaton of Smad2 , suggesting TGFβ2 produced in response to miR - 30b overexpression functions in an iii autocrine manner to stimulate HUVECs . MiR - 30b effects on TGFβ2 expression were found to be regulated to an extent by ATF2, as miR - 30b overexpressing cells exhibited increased levels of phosphorylated ATF2 , with depletion of ATF2 via siRNA resulting in inhibition of miR - 30b - induced TGFβ2 expression. Treatment of HUVECs with TGFβ2 inhibited cord formation, while TGFβ1 had no effect, indicating a major difference in how endothelial cells respond to these two related growth factors. Inhibition of TGFβ2 with a neutralizing antibody restored cord formation in miR - 30b overexpressing cells to levels similar to control cells, thus identifying TGFβ2 expression as contributing to the inhibitory effects of miR - 30b overexpression on capillary morphogenesis. Thus, we have identified two signaling pathways regulated by VEGF in HUVECs that further our understanding of the process of angiogenesis and may provide novel targets for therapeutic intervention into diseases involving angiogenesis.

Oxygen free radicals have been implicated in several diseases including ischemic stroke, and myocardial infarction. They can trigger chain reactions like peroxidation of membrane phospholipids, leading to osmotic imbalance and cell death. Isoprostanes are stable products of lipid peroxidation that have a constrictor effect on the vasculature and bronchii. As isoprostanes are abundantly generated in tissues under oxidant stress, we have hypothesized that they could be related to endothelial dysfunction observed during ischemia/reperfasion by affecting endothelial cell survival. The effects of 8-iso-PGE2 and 8-iso-PGF2alpha, two abundantly produced isoprostanes, were studied on porcine endothelial cultures and isolated brain microvessels. Cell survival was evaluated by MTT reduction, double staining with DNA-binding fluorochromes and in situ DNA fragmentation labeling,
8-Iso-PGF2alpha (1--10 nM) induced 20--25% cell death in endothelial cultures after 24 h coincident with similar increase in the number of cells that become permeable to PI. On the contrary, 8-iso-PGE 2 did not affect endothelial cell survival. Approximately 9% of the cells suffered apoptosis. This percentage remained unchanged regardless the treatment. Several observations indicate a role for thromboxane A2 to mediate 8-iso-PGF2alpha-induced death: (1) the levels of thromboxane A2 increased dramatically in endothelial cultures after 8-iso-PGF2alpha-treatment; (2) inhibitors of thromboxane synthase, CGS12970 and U6355A and Ibuprofen, a non-selective inhibitor of cyclooxygenases, reverted the effect of the isoprostane; (3) analogs of thromboxane A2 U46619 and IBOP, reproduce the effect of 8-iso-PGF 2alpha after 24 h. 8-Iso-PGF2alpha also decreased endothelial viability on isolated brain microvessels. These results suggest, that 8-iso-PGF2alpha, might be a direct contributor to ischemia/reperfusion injury.

Through bona fide tight junctions and regulated transcytosis, brain endothelial cells (ECs) are able to establish a blood-brain barrier (BBB) that regulates access of leucocytes and solutes to the central nervous systenn (CNS). Occludin was the first transmembrane tight junction protein identified and it has been demonstrated that expression of mutant occludin proteins in epithelial cells dramatically affects neutrophil transmigration and monolayer permeability. To determine if similar strategies could influence transendothelial lymphocyte migration and other endothelial barrier properties, a brain EC line (GPNT) was transfected with a range of occludin proteins and characterised by functional assays. Expression of wild-type occludin reduced T cell migration and, as in epithelial cells, this was shown to be dependent on an unmodified N-terminal domain. The mechanism(s) by which lymphocytes physically cross the endothelial barrier remains a poorly described stage of lymphocyte extravasation. The possibility remains, however, that modulation of endothelial cell-cell junctions is a necessary pre-requisite to physically allow the passage of a leucocyte through the EC wall, i.e the paracellular pathway. The firm adhesion of circulating T cells to BBB ECs is predominantly via ICAM- 1, a cell adhesion molecule of the immunoglobulin superfamily expressed on the EC and previously demonstrated to be capable of signal transduction. A hypothesis was generated that I CAM-1 engagement facilitates diapedesis by activating signalling pathways that regulate cell-cell junctions. Using GPNT EC, the role of ICAM-1 in regulating junctional proteins and integrity was assessed. Following crosslinking of ICAM-1, both VE-cadherin and PECAM showed increased tyrosine phosphorylation with identical experiments showing that ICAM-1 crosslinking correlated with an increase in transmonolayer permeability to molecular tracers. Immunoprecipitated VE-cadherin showed no change in its association with p- or y-catenin following ICAM-1 crosslinking, however, there was increased association of both catenins with PECAM via a rho-dependent/ROCK- independent pathway. The existence of such pathways suggests that pharmacological targeting of ICAM-1-mediated signalling may be advantageous in the therapeutic management of neuroinflammatory diseases.

Multiple sclerosis (MS) is considered an immune-mediated disorder of the central nervous system (CNS) characterized by multifocal areas of inflammation and demyelination. Lesion formation depends on migration of lymphocytes from the systemic compartment into the CNS through the blood-brain barrier. We have defined molecular and functional properties of human brain derived microvascular endothelial cells and of T lymphocytes that regulate the migration process and analysed how changes in these properties, in response to inflammatory conditions, contribute to the pathogenesis of multiple sclerosis. Rates of migration of T lymphocytes derived from patients with MS across either a fibronectin and/or endothelial cell barrier in vitro were increased compared to cells obtained from healthy controls. In the MS patients, migration rate correlated with disease activity, defined by clinical and MRI criteria and with IFN gamma production by T cells. Migration was reduced in patients receiving current therapies for MS. Adhesion molecules, chemokines and matrix metalloproteinases were all found to regulate the migration of MS derived lymphocytes through proteins of the basement membrane and human brain endothelial cells. We further demonstrated that the migratory properties of lymphocytes and both permeability and chemokine production by human brain endothelial cells, can be regulated via signalling through the specific kinin B1 receptor that is up-regulated on lymphocytes and human brain endothelial cells during inflammation, providing a potential novel therapeutic approach to modulate lymphocyte-endothelial cell interactions.

The clearance of neurotoxic amyloid beta (Aβ) from the brain represents a novel therapeutic target for Alzheimer's disease (AD). The ability of two blood-brain barrier (BBB) drug transporters, P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP), to transport Aβ was investigated using a human brain endothelial cell (BEC) line, hCMEC/D3. P-gp expression by hCMEC/D3 cells was stable over a high passage number, polarised on the apical membrane, consistent with the blood side in vivo, and comparable, albeit slightly reduced, to primary isolated human BECs. The P-gp inhibitors tariquidar and vinblastine prevented the efflux of rhodamine 123 from hCMEC/D3 cells, indicative of functional P-gp expression. hCMEC/D3 cells therefore constituted a suitable model to investigate P-gp substrate interactions in vitro. P-gp, and to a lesser extent BCRP, inhibition, increased the net influx and decreased the efflux of 0.1 nM¹²⁵ I Aβ 1-40 in hCMEC/D3 cells. Both P-gp and BCRP inhibition increased the apical-to-basolateral but not the basolateral-to-apical permeability of hCMEC/D3 cells to nM¹²⁵ I Aβ 1-40. This data is consistent with P-gp and BCRP, acting in vivo to prevent blood-borne Aβ peptides entering the brain but not to clear Aβ load from the brain.

Inflammation or injury of the central nervous system generally results in the activation of brain endothelial cells and a change in the composition and expression of the extracellular matrix (ECM) network of the basal lamina of the brain vasculature. The main contributors to brain damage are interleukin (IL)-1-mediated inflammatory processes. The main aim of this project is to investigate whether the ECM associated with brain endothelial cells is modified in response to ischaemic injury in vitro, and to test the hypothesis that alteration of ECM composition following injury is a critical regulator of IL-1-induced endothelial cell activation. The in vitro blood-brain barrier model used in this thesis was composed of brain endothelial cells and astrocytes and this model displayed classic BBB characteristics. Oxygen-glucose deprivation (OGD) for 2.5 hours followed by 4 hours of reperfusion ± 10 ng/ml IL-1β induced changes in rat brain endothelial cell (rBEC) morphology, occludin and ZO-1 distribution and cytokine-induced neutrophil chemoattractant (CINC)-1 release. Immunohistochemistry on brain sections from animals subjected to middle cerebral artery occlusion (MCAO) demonstrated upregulation of laminin α4 protein following 48 hours and 6 days of reperfusion. Fibronectin expression was also increased in the brain vessels of animals subjected to MCAO following 48 hours of reperfusion. Similarly, 2.5 hours OGD and 2 hours reperfusion ±IL-1β induced changes in laminin α4, β1 and γ1, type IV collagen α1 chain and fibronectin mRNA expression in vitro. ECM molecules also influenced rBEC morphology, adhesion, proliferation, organisation and TEER. Integrin β1 mediated rBEC adhesion to type I collagen, type IV collagen and cellular fibronectin but not laminin-511 and the combination of laminin-411 and -511. Laminin-511 and the combination of laminin-411 and -511 significantly increased CINC-1 release compared to type I collagen. Laminin-411 and -511 also upregulated occludin protein expression and maintained occludin distribution following IL-1β treatment in rBECs. To conclude, ECM associated with brain endothelial cells was modified in response to ischaemic injury ±IL-1β in vitro, and vascular ECM proteins altered rBEC activation in response to IL-1β. Therefore, a change in ECM composition following injury is a critical regulator of IL-1-induced endothelial cell activation.

Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true â barrier characteristicsâ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.

Doctor of Philosophy (Medicine)
Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true ‘barrier characteristics’ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.

Neisseria meningitidis is the major cause of meningitis and sepsis, two kind of diseases that can affect children and young adults within a few hours, unless a rapid antibiotic therapy is provided. The meningococcal disease dates back to the 16th century. The first description of the disease caused by this pathogen was stated by Viesseux in 1805 as 33 deaths occurred in Geneva, Switzerland [1]. It took about seventy years before two Italians (Marchiafava and Celli) in 1884 identified micrococcal infiltrates within the cerebrospinal fluid [2]. The worldwide presence of meningococcal serogroups may vary within regions and countries. With the coming of antimicrobial agents, like sulphonamides, and with the development of an appropriate health care and prevention programme, the fatality rate cases has dropped from 14% to 9%, although 11% to 19% of patients continued to have post-infection issues such as neurological disorders, hearing or limb loss [3]. The bacteria can be divided into 13 different serogroups and, among these, up to 99% of infection is ascribed to the serogroups named A, B, C, 29E, W-135 and Y (Fig. 2). All the serogroups have been listed in 20 serotypes on the presence of PorB antigen, 10 serotypes on the presence of PorA antigen, and in other immunotypes on the presence of other bacterial proteins and on the presence of a characteristic lipopolysaccharide called LOS (lipooligosaccharide) [4]. The transmission from a carrier to an other person occurs by liquid droplet and the natural reservoir of Neisseria meningitidis is the human throat, in particular it usually invades the human nasopharynx where it can survive asymptomatically. The reported annual incidence goes from 1 to 5 cases per 100000 inhabitants in industrialized countries, while in non developed-countries the incidence goes up to 50 cases per 100000 inhabitants. More then 50% of cases occur within children below 5 years of age, and the peak regards those under the first year of age. This fact is due to the loss of maternal antibodies by the newborn. In non-epidemic period, the percentage of healthy carriers range from 10 to 20%, and notably the condition of chronic carrier is not so uncommon [5, 6]. Only in a small percentage of cases the colonization progresses until the insurgence of the pathogenesis. This happens because in the majority of cases specific antibodies or the human complement system are able to destroy the pathogens in the blood flow allowing a powerful impairment of the dissemination. In a small group of population the colonization of the upper respiratory tract is followed by a rapid invasion of the epithelial cells, and from there bacteria can reach the blood flow and invade the central nervous system (CNS), inducing the establishment of an acute inflammatory response. How the balance between being an healthy carrier or a infected patient can change so rapidly it is still unknown. Some factors that can play a role in this switch could be the virulence of the bacterial strain, the responsiveness of the host immune system, the mucosal integrity, and some environmental factors [7]. Neisserial heparin binding antigen (NHBA) is a surface- exposed lipoprotein from Neisseria meningitidis that was originally identified by reverse vaccinology [8]. NHBA in Nm has a predicted molecular weight of 51 kDa. The protein contains an Arg-rich region (-RFRRSARSRRS-) located at position 296–305 that is highly conserved among different Nm strains. The protein is specific for Neisseria species, as no homologous proteins were found in non redundant prokaryotic databases. Full length NHBA can be cleaved by two different proteases in two different manners: NalP, a neisserial protein with serine protease activity cleaves the entire protein at its C-terminal producing a 22 kDa protein fragment (commonly named C2) which starts with Ser293 and hence comprises the highly conserved Arg-rich region. The human proteases lactoferrin (hLf) cleaves NHBA immediately downstream of the Arg-rich region releasing a shorter fragment of approximately 21 kDa (commonly named C1) [9] . Although it is known that a crucial step in the pathogenesis of bacterial meningitidis is the disturbance of cerebral microvascular endothelial function, resulting in blood-brain barrier breakdown, the bacterial factor(s) produced by Nm responsible for this alteration remains to be established. The integrity of the endothelia is controlled by the protein VE-cadherin, mainly localized at cell-to-cell adherens junctions where it promotes cell adhesion and controls endothelial permeability [10]. It has been reported that alteration in the endothelial permeability can be ascribed to phosphorylation events induced by soluble factors such as VEGF or TGF-beta[11] [12]. Our work demonstrates that the NHBA- derived fragment C2 (but not C1) increases the endothelial permeability of HBMEC (human brain microvasculature endothelial cells) grown as monolayer onto the membrane of a transwell system. Indeed, the exposure of the apical domain of the endothelium to C2 allows the passage of the fluorescent tracer BSA-FITC, from the apical side to the basal one, early after the treatment. Interestingly, the effect of C2 on the endothelium integrity is such to allow the passage of bacteria, E. coli but, notably, also N. meningitidis MC58, from the apical to the basolateral side of the transwell and it depends on the production of mitochondrial ROS. Remarkably, we have found that the administration of C2 to endothelia results in a ROS-dependent reduction of the total VE-cadherin content. This event requires after VE-cadherin phosphorylation, the endocytosis and the subsequent degradation of the protein. Collectively our data suggest the possibility that C2 might be involved in the mechanisms of invasion owned by the bacterium to cross host tissues.
Neisseria meningitidis è uno dei patogeni in grado di causare meningite oltre che sepsi in soggetti infettati, due patologie che colpiscono maggiormente bambini e adolescenti entro poche ore dal contagio a meno di una tempestiva terapia antibiotica. La malattia meningococcica risale al sedicesimo secolo. La prima descrizione della malattia causata da questo agente patogeno avvenne ad opera di Viesseux nel 1805 come conseguenza di 33 decessi occorsi a Ginevra, Svizzera [1]. Circa 70 anni dopo, due italiani (Marchiafava e Celli) nel 1884 identificarono per la prima volta degli infiltrati meningococcichi nel fluido cerebrospinale [2]. La presenza di Neisseria meningitidis nel mondo varia in base a paesi e regioni e risulta essere ciclica. Grazie alla scoperta di agenti antimicrobicidi come i sulfonamidici e grazie alla diffusione di un adeguato protocollo di prevenzione sanitaria i casi di mortalita` dovuti a questo agente patogeno sono rapidamente diminuiti dal 14 al 9%. Ciò nonostante una percentuale compresa tra l’11 e il 19% dei soggetti ha continuato ad avere problemi post-infezione come disordini neurologici, o perdità dell’udito [3]. Esistono attualmente 13 sierogruppi e, di questi, il 99% delle infezioni è causato dai tipi A, B, C, 29E, W-135 e Y. I sierogruppi sono stati a loro volta classificati in 20 sierotipi sulla base della presenza dell’antigene proteico PorB, in 10 sierotipi sulla base dell’antigene PorA e in altri immunotipi a seconda della loro capacita` di indurre una risposta immunitaria nell’ospite grazie alla presenza di altre proteine batteriche del patogeno, e per la presenza di un particolare lipopolisaccaride chiamato LOS (lipooligosaccaride) [4]. Neisseria meningitidis è in grado di colonizzare l’epitelio della mucosa orofaringea, dove vi può sopravvivere in maniera asintomatica per l’ospite. La trasmissione inter-individuale avviene attraverso secrezioni dell’apparato respiratorio. L’ incidenza annuale risulta essere di 1- 5 casi ogni 100000 abitanti nei paesi industrializzati, mentre nei paesi ancora in via di sviluppo questa sale a 50 casi per 100000 abitanti. Più del 50% dei casi riguarda bambini sotto i 5 anni d’età, con un’elevata incidenza per coloro che hanno meno di un anno di vita. Questo fatto dipende dall’emivita degli anticorpi materni solitamente in grado di proteggere il neonato per circa 3-4 mesi dopo la nascita. In periodi definiti non-epidemici la percentuale dei portatori sani varia tra il 10 e il 20% della popolazione, e per l’appunto la condizione di portatore asintomatico non è poi così infrequente [5, 6]. Soltanto in un numero ristretto di casi la colonizzazione del batterio progredisce manifestando la patogenesi meningococcica: ciò è per la maggior parte dovuto alla presenza di specifici anticorpi, o per l’attività del sistema del complemento dell’ospite che è in grado di controllare ed eliminare il patogeno impedendone così la sua disseminazione attraverso il flusso sanguigno. Tuttavia, in un piccolo gruppo della popolazione, la colonizzazione del tratto respiratorio superiore è seguita da una rapida invasione delle cellule epiteliali della mucosa, da dove il batterio è in grado di entrare nel torrente ematico, e raggiungere il sistema nervoso centrale inducendo una forte risposta infiammatoria. Quale sia l’evento che perturbi l’equilibrio tra essere portatore asintomatico e paziente infetto ancora non è noto. Alcuni fattori sembrano giocare un ruolo chiave in questo cambiamento come la virulenza del ceppo batterico, la capacità della risposta immunitaria dell’ospite, l’integrità della mucosa e alcuni fattori ambientali [7]. La proteina NHBA, Neisserial Heparin Binding Antigen, è una lipoproteina esposta sulla superficie del batterio, originariamente identificata attraverso la tecnica della “reverse vaccinology” [8]. NHBA in Nm ha un peso molecolare predetto di 51 kDa. La proteina altresì contiene una regione ricca in Arginine (-RFRRSARSRRS-) localizzata in posizione 296 -305 ed altamente conservata in vari ceppi di Neisseria [9]. Tale proteina è altamente conservata in Neisseria e non ha omologie di sequenza con nessun’altra proteina registrata nei database procariotici. Due diverse proteasi possono tagliare la proteina intera NHBA producendo due frammenti differenti: nel primo caso la proteasi batterica NalP taglia la proteina intera in posizione C-terminale producendo un frammento di 22 kDa (comunemente chiamato C2) che inzia con la Ser293 e quindi comprendendo lo stretch di Arginine. Invece, nel secondo caso, la lattoferrina umana (hLf) taglia NHBA immediatamente a monte della sequenza di Arginine, producendo un frammento più corto di circa 21 kDa (comunemente chiamato C1). Sebbene sia risaputo che un passaggio cruciale nella patogenesi mediata da Neisseria meningitidis sia l’alterazione della funzione di barriera della microvascolatura encefalica, che può dunque risultare in una rottura della barriera emato- encefalica stessa, non è ancora chiaro quali siano i fattori rilasciati o prodotti dal batterio in grado di indurre un simile effetto. L’integrità dell’endotelio è controllata dalla proteina VE-caderina, localizzata sulle giunzioni aderenti che regolano il contatto cellula- cellula. Tale proteina promuove e regola dunque la permeabilità endoteliale [10]. E’ stato ben documentato che l’alterazione della permeabilità endoteliale può essere dovuta a processi di fosforilazione indotti da fattori solubili come VEGF o TGF-beta[11] [12]. Il nostro lavoro documenta come, a differenza del frammento C1, il frammento C2 prodotto dal taglio della proteina intera NHBA, sia in grado di aumentare la permeabilità delle cellule endoteliali HBMEC (human brain microvasculature endothelial cells) fatte crescere a monostrato sulla membrana di un sistema di transwell. L’esposizione della porzione apicale dell’endotelio polarizzato al frammento C2 consente il passaggio di un tracciante fluorescente, BSA-FITC, dal lato superiore a quello inferiore del transwell, in tempi rapidi a seguito del trattamento. E’ interessante notare che l’effetto di C2 sull’endotelio è tale da permettere il passaggio dal lato superiore a quello inferiore del transwell non solo di E. coli, usato come modello batterico preliminare, ma anche dello stesso Neisseria meningitidis MC58, in maniera ROS dipendente. Degno di nota è il fatto che abbiamo osservato che la somministrazione di C2 alle cellule endoteliali provoca una riduzione ROS dipendente del contenuto totale di VE-caderina. A seguito della sua fosforilazione, infatti, VE-caderina viene endocitata all’interno della cellula per poi essere degradata probabilmente attraverso il trasporto di essa verso il proteasoma. I nostri dati suggeriscono pertanto che C2 sia uno dei meccanismi di invasione possieduti da Neisseria per invadere i tessuti dell'ospite.

Thesis (M.S.)--University of California, San Diego, 2008.
Title from first page of PDF file (viewed April 7, 2008). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 64-70).

Unphysiological chronic signalling by the antiviral cytokine interferon-a (IFN-a) in the brain induces cerebral type I interferonopathies which can have debilitating neurological consequences. Here we investigated the role of protein phosphorylation and the contribution of key cell types of the brain to the pathogenesis of these diseases. Using a mouse model for IFN-a-induced neurodegeneration, termed GFAP-IFNa (GIFN) mice, we identified widespread protein phosphorylation as a new mechanism by which IFN-a mediates its effects. Analyses revealed the changes in protein phosphorylation aligned with the clinical hallmarks and pathological outcome of disease. Further, these changes were mediated by a few kinase families, which was reflected in IFN-a-treated microglia and astrocytes, key immune-responsive cells in the brain. Together, this revealed an unappreciated role of protein phosphorylation mediating the cellular response to IFN-a. The findings also showed that microglial and astrocyte responses to IFN-a contribute to disease in GIFN mice. However, hallmarks of cerebral type I interferonopathies involve vasculopathy. To specifically delineate the role of vascular cells, we generated GIFN mice whose vascular cells are unresponsive to IFN-a (GIFN x IFN-a receptor floxed x Tek-Cre), termed GIT mice. GIT mice appeared resoundingly like wild-type (WT) mice and unlike GIFN mice with improved weights, motor function and behaviour, reduced mortality, rescued cerebral pathology, WT-like microvasculature, intact blood brain barrier integrity and reduced peripheral cell infiltration; all in the presence of chronic IFN-a expression and markers of inflammation. These results demonstrate for the first time IFN-a signalling in vascular cells is a key pathogenic mechanism in type I interferonopathies. Hence, directly targeting the vasculature presents a promising therapeutic avenue to treat type I interferonopathic brain disease.

Alzheimer's disease (AD) is characterized by accumulation and deposition of beta-amyloid (Abeta) peptides in the brain. Deposition of Abeta peptides in cerebrovascular system results in chronic vascular inflammation, neurovascular uncoupling and insufficiency, contributing to neurodegeneration. The purpose of this study was to investigate the inflammatory response in human brain endothelial cells (HBEC) induced by aggregated Abeta 1-40 and the molecular mechanism of the Abeta-stimulated inflammatory gene expression. Primary or immortalized HBEC (iHBEC) were treated with aggregated Abeta1-40, control peptides (scrambled Abeta1-40 or poly-asparagine) or 2mM NaOH (vehicle), in the presence or absence of a specific JNK inhibitor (SP600125, 30 muM) at different time points, and inflammatory gene expression was analyzed by reverse-transcriptase polymerase-chain reaction (RT-PCR). TranSignal Protein/DNA Array was used to profile the activities of transcription factors (TF) in Abeta-stimulated HBEC. Nuclear extracts were isolated to determine interaction of AP-1 with DNA by electrophoretic mobility shift assay (EMSA) and supershift assay. The levels of total c-Jun and phosphorylated c-Jun at Ser73/Ser63 were analyzed through Western blotting, and AP-1 transcriptional activity was evaluated by reporter gene assays. In addition, to test indirect effects of Abeta on iHBEC, conditioned media from Abeta1-42-stimulated primary microglia was used to treat iHBEC at different time points in the presence of SP600125. The results obtained in this study have shown that aggregated Abeta1-40 peptides strongly stimulated the expression of monocyte chemoattractant protein 1 (MCP-1), interleukin 8 (IL-8), interleukin 6 (IL-6) and growth-related oncogene (GRO). TF profiling has revealed that Abeta treatment strongly activated AP-1 in the cells. Electrophoretic migration shift assay (EMSA) and supershift assay have confirmed that AP-1 was strongly activated in HBEC at 2 and 4 h post-Abeta-treatment and physically interacted with AP-1-binding DNA sequence and that c-Jun is a component of the activated AP-1 dimeric complex. Abeta-stimulated AP-1 activity was further confirmed by reporter gene assay. In summary, these experiments have demonstrated that binding and internalization of Abeta peptides at HBEC cellular membrane, possibly through RAGE, stimulated JNK signaling cascade pathway in HBEC which resulted in increased phosphorylation of c-Jun at Ser-73 and that activated AP-1 was responsible for increased expression of inflammatory genes in the cells. Aside from direct action on HBEC, Abeta peptides also stimulated microglia to release pro-inflammatory factors, which in turn, stimulated the expression of inflammatory genes in HBEC. The JNK-AP1 signaling pathway and inflammatory factors characterized in this study may potentially serve as therapeutic targets to relieve Abeta-induced inflammation in Alzheimer's disease. (Abstract shortened by UMI.)

Traumatic brain injury (TBI) is a growing health concern worldwide that affects a broad range of the population. As TBI is the leading cause of disability and mortality in children, several pre-clinical models have been developed using rodents at a variety of different ages; however, key brain maturation events are overlooked that leave some age groups more or less vulnerable to injury. Thus, there has been a large emphasis on producing relevant animal models to elucidate molecular pathways that could be of therapeutic potential to help limit neuronal injury and improve behavioral outcome. TBI involves a host of different biochemical events, including disruption of the cerebral vasculature and breakdown of the blood brain barrier (BBB) that exacerbate secondary injuries. A better of understanding of the mechanism(s) underlying cerebral vascular regulation will aid in establishing more effective treatment strategies aimed at improving cerebral blood flow restoration and preventing further neuronal loss. Our studies reveal an age-at- injury dependence on the Angiopoetin-Tie2 axis, which mediates neuroprotection in a model of juvenile TBI following cortical controlled impact (CCI) that is not seen in adult mice. The protection observed was mediated, in part, by the microvascular response to CCI injury and prompted further detailed analysis of the larger arteriole network across several mouse strains and models of TBI. Our second study revealed both a model and species dependent effect on a specialized network of arteriole vessels, called collaterals after trauma. We demonstrated that a repetitive mild TBI (rmTBI) can induce collateral remodeling in C57BL/6 but not CD1 mice; however, CCI injury had no effect on collateral changes in either strain. Together, these findings demonstrate an age-dependent and species/model dependent effect on vascular remodeling that highlights the importance of individualized therapeutics to TBI.
Ph. D.

Pathological angiogenesis, or new blood vessel formation, is involved in many pathologies, including cancer and serious eye diseases. While traditional anti-angiogenic therapies target vascular endothelial growth factor receptors to reduce or inhibit new vessel formation, this approach has several downsides, including unpleasant side effects and low efficacy over time. Therefore, identifying new targets to treat pathological angiogenesis is still needed. CMG2, one of the two identified anthrax toxin receptors, has been proposed as an alternative target to treat pathological angiogenesis. CMG2’s role as a cell surface receptor that mediates anthrax toxin internalization is very well documented. One physiological function for CMG2, not related to anthrax intoxication, is suggested by the observation that loss-of-function mutations in CMG2 cause hyaline fibromatosis syndrome (HFS), a genetic disease that results in accumulations of extra-cellular matrix (ECM) protein in different parts of the body. While the complete molecular mechanism for CMG2’s role in regulating angiogenesis has not been determined, this dissertation addresses multiple ways CMG2 regulates pathological angiogenesis. We have discovered that CMG2 plays a role in mediating ECM homeostasis via endocytosis of ECM proteins and protein fragments as a way to generate angiogenic signals from the cell. We have also demonstrated that a fragment from Col IV, S16, is endocytosed into the cells by interacting with CMG2, and S16 treatment to endothelial cells leads to a significant reduction in cell migration. Also, an endothelial cell migration assay with CMG2 knockout cells results in abolished directional migration, indicating that CMG2 is required for endothelial cell chemotaxis. Notably, we have identified that bFGF, VEGF, and PDGF are involved in CMG2 mediated chemotaxis but not insulin and sphingosine-1-phosphate (S1P). While recent literature reports show that CMG2 works closely with RhoA GTPase, which is commonly known to regulate cell migration, we have also observed that inhibition of RhoA also reduced cell chemotaxis towards VEGF but not S1P. These results could be leveraged to develop new classes of therapeutic molecules to treat pathological angiogenesis induced by multiple various growth factors via targeting CMG2.

Dans les systèmes biologiques, les barrières tissulaires permettent le transport sélectif de molécules du sang au tissu approprié. Un exemple de barrière tissulaire est la barrière hémato-encéphalique (BHE). La BHE protège le cerveau du sang et maintient l'homéostasie du microenvironnement du cerveau, ce qui est essentiel à l'activité et à la fonction neuronale. La caractérisation de cette BHE est importante, car un dysfonctionnement de cette barrière est souvent révélateur de toxicité ou de maladie. Bien que le nombre d'articles publiés dans le domaine du développement et de la caractérisation de la BHE ait été multiplié ces dernières années, la validité des modèles utilisés est encore un sujet de débat. L'avènement de l'électronique organique a créé une occasion unique pour coupler les mondes de l'électronique et de la biologie, à l'aide de dispositifs tels que le transistor électrochimique organique (OECT). OECT constitue un outil très sensible et économique pour diagnostiquer l’intégrité d’une barrière tissulaire. Dans cette étude, nous avons tout d’abord développé trois différents modèles de BHE. Nous avons optimisé l’adhésion des cellules endothéliales cérébrales sur la matière active du transistor. Nous avons ainsi pu établir l'intégration des OECTs avec des cellules immortalisées humaines micro vasculaires cérébrales endothéliales (h CMEC/D3) en tant que modèle in vitro de BHE. Nous avons démontré que la fonction de tissu de la BHE peut être détectée en utilisant cette nouvelle technique. En outre, par cette technique, une perturbation de la barrière (par exemple, provoquée par un composé toxique) pourra être détectée électriquement au moyen d'une mesure de courant
In biological systems many tissue types have evolved a barrier function to selectively allow the transport of matter from the lumen to the tissue beneath; one example is the Blood Brain Barrier (BBB). The BBB protects the brain from the blood and maintains homeostasis of the brain microenvironment, which is crucial for neuronal activity and function. Characterization of the BBB is very important as its disruption or malfunction is often indicative of toxicity/disease. Though the number of published papers in the field of in vitro BBB has multiplied in recent years, the validity of the models used is still a subject of debate.The advent of organic electronics has created a unique opportunity to interface the worlds of electronics and biology, using devices such as the Organic ElectroChemical Transistor (OECT), which provide a very sensitive way to detect minute ionic currents in an electrolyte as the transistor amplifies the gate current.In this study, we test three different type of BBB in order to develop a stable BBB model. We optimize the adhesion of brain endothelial cell on OECT conducting polymer. We show the integration of OECTs with immortalized human cerebral microvascular endothelial cells as a model of human blood brain barrier, and demonstrate that the barrier tissue function can be detected. Moreover, by this technique, a disruption in the barrier (e.g. caused by a toxic compound) is assessed electrically through a measurement of the drain current. Results show the successful development and validation of an in vitro BBB model. Dynamic monitoring of the barrier properties of the BBB barrier tissue was possible using the OECT

Background: Strategies to maintain BBB integrity in diseases with a hypoxia/reoxygenation (H/R) component involve preventing glutathione (GSH) loss from endothelial cells. GSH efflux transporters include multidrug resistance proteins (Mrps). Therefore, characterization of Mrp regulation at the BBB during H/R is required to advance these transporters as therapeutic targets. Our goal was to investigate, in vivo, regulation of Abcc1, Abcc2, and Abcc4 mRNA expression (i.e., genes encoding Mrp isoforms that transport GSH) by nuclear factor E2-related factor (Nrf2) using a well-established H/R model. Methods: Female Sprague-Dawley rats (200-250 g) were subjected to normoxia (Nx, 21% O-2, 60 min), hypoxia (Hx, 6% O-2, 60 min) or H/R (6% O-2, 60 min followed by 21% O-2, 10 min, 30 min, or 1 h) or were treated with the Nrf2 activator sulforaphane (25 mg/kg, i.p.) for 3 h. Abcc mRNA expression in brain microvessels was determined using quantitative real-time PCR. Nrf2 signaling activation was examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) respectively. Data were expressed as mean +/- SD and analyzed via ANOVA followed by the post hoc Bonferroni t test. Results: We observed increased microvascular expression of Abcc1, Abcc2, and Abcc4 mRNA following H/R treatment with reoxygenation times of 10 min, 30 min, and 1 h and in animals treated with sulforaphane. Using a biotinylated Nrf2 probe, we observed an upward band shift in brain microvessels isolated from H/R animals or animals administered sulforaphane. ChIP studies showed increased Nrf2 binding to antioxidant response elements on Abcc1, Abcc2, and Abcc4 promoters following H/R or sulforaphane treatment, suggesting a role for Nrf2 signaling in Abcc gene regulation. Conclusions: Our data show increased Abcc1, Abcc2, and Abcc4 mRNA expression at the BBB in response to H/R stress and that Abcc gene expression is regulated by Nrf2 signaling. Since these Mrp isoforms transport GSH, these results may point to endogenous transporters that can be targeted for BBB protection during H/R stress. Experiments are ongoing to examine functional implications of Nrf2-mediated increases in Abcc transcript expression. Such studies will determine utility of targeting Mrp isoforms for BBB protection in diseases with an H/R component.

Pharmacology
Ph.D.
Background: Epidemiology, clinical trials and meta-analysis studies have established that Hyperhomocysteinemia (HHcy) is an independent risk factor for stroke. However, the exact molecular mechanism underlying the HHcy-induced risk of stroke is unclear. Our study aims to investigate the role of HHcy in stroke. Methods and results: We established a mice mode of focal ischemic stroke, termed transient Middle Cerebral Artery Occlusion (tMCAO) and conducted surgery on a mice model of HHcy (plasma homocysteine level ~150μM), in which a Zn2+ inducible human cystathionine β-synthase (CBS) transgene was introduced to circumvent the neonatal lethality of the CBS gene deficiency (Tg-hCBS Cbs-/- mice). Fourteen-week-old male mice were used in the experiment. A student’s t-test was used for the evaluation of the statistical significance between the two groups. For the comparison across multiple groups, one-way ANOVA was used. We found that HHcy 1) increased the infarction volume from 42.3 ± 4.9 mm
Temple University--Theses

Blood-brain barrier (BBB) is a unique membranous barrier, which segregates brain from the circulating blood. It works as a physical and metabolic barrier between the central nervous system (CNS) and periphery. In mammals, endothelial cells were shown to be of BBB and are characterized by the tight junctions along with efflux system which are responsible for the restriction of movement of molecules within the cells. Efflux system consists of multidrug resistance proteins such as P-glycoprotein (P-gp). P-gp removes substances out back from the brain to the blood before they reach to the brain. So the barrier is impermeable to many compounds such as amino acids, ions, small peptides and proteins, making it the most challenging factor for the development of new drugs for targeting CNS. Curcumin is a bioactive compound that has a number of health promoting benefits such as anti-inflammatory, anticancer, anti-oxidant agent; as well as a role in neurodegenerative diseases, but low oral bioavailability is the major limiting factor. Low water solubility and rapid metabolism are the two important factors responsible for poor bioavailability of curcumin. Galaxolide is a musk compound and previously known for the bioaccumulation of toxic components in the aquatic animals by interference with the activity of multidrug/multixenobiotic resistance efflux transporters (MDR/MXR). The bioavailability of curcumin can be enhanced when administered with galaxolide. This study was carried out to investigate the effect of galaxolide on the permeation of curcumin through the epithelial cell monolayers. MDCKII-MDR1 cell monolayer is used an in vitro blood-brain barrier model while Caco-2 monolayer is used as an in vitro intestinal model, which also expresses the P-glycoprotein. The curcumin and galaxolide were separately solubilised in the DMSO and used in combination to perform permeation study, to determine the effect of galaxolide on curcumin permeation through epithelial cell monolayers. The galaxolide shows an efflux protein inhibition activity and this activity was used to enhance permeation of curcumin through the Caco-2 monolayer. In summary, galaxolide is a novel permeation enhancer molecule, which can be used for the improvement of drug delivery of other bioactive compounds in future.

Lipid oxidation is one of the leading causes of food quality degradation. Manufacturers typically add antioxidants or purge a productâ s package of oxygen to inhibit oxidation and the resulting off-flavors. Synthetic antioxidants (e.g. BHT, BHA) and some natural antioxidants (e.g. α-tocopherol) have found widespread use in this application. Unfortunately, the public views synthetic additives in a negative light and the current natural antioxidants have been unable to match the protection afforded by the synthetic antioxidants. The search for underutilized and natural antioxidants has led scientists to investigate many different plant-based extracts for use in food and in the treatment and prevention of disease. The objectives of this research were (1) to use ORAChromatography to identify peanut root extract fractions with high antioxidant capacity, (2) identification of compounds in peanut root extracts using HPLC and mass spectrometry, (3) test for the presence of aflatoxins in the extracts, (4) test peanut root extract in food model system for oxidation reduction capabilities, and (5) Testing peanut root extractâ s ability to decrease protein oxidation in cell culture. Crude peanut root extracts have high antioxidant activities that do not vary by cultivar. The ORAC activities of the peanut root fractions separated by HPLC with a C18 column varied (600.3 â 6564.4 μM TE/g dry extract), as did the total phenolic contents (23.1 â 79.6 mg GAE/g dry extract). Peanut root fractions had aflatoxins contamination well above the 20 ppb limit. Peanut root extracts and the known antioxidants tested were found to have no significant effect in inhibiting oxidation of peanut paste or HBMEC. Peanut root extracts were not shown to have any positive effects, but further research is necessary to eliminate peanut root extracts as a possible food ingredient and health supplement.
Ph. D.

L'ictus isquèmic és una de les principals causes de mortalitat, discapacitat de llarg termini i de morbiditat al món. Actualment, els únics tractaments eficaços són el tractament trombolític intravenós amb activador del plasminogen tissular (tPA) i les novedoses trombectomies intra-arterials, que requereixen ser administrades en la fase hiperaguda de la malaltia (<4,5 hores per al tractament amb tPA i <6 hores per les emergents la teràpies endovasculars). Per tant, és necessari investigar noves teràpies que podrien ser utilitzades per tractar un gran nombre de pacients en una fase posterior de la malaltia per reparar el teixit danyat. Alguns enfocaments terapèutics apunten que per assolir aquest objectiu és necessari potenciar la remodelació vascular i la neurogènesi en el cervell isquèmic. L'objectiu d'aquesta tesi és l'estudi de la modulació de la metaloproteinasa de matriu-13 (MMP-13) en la neurorreparación i el d’investigar diverses MMPs com a biomarcadors que serveixin per monitoritzar la millora de la funció motora durant la teràpia neuro-rehabilitadora. Utilitzant un model d’isquèmia cerebral per oclusió de l’arteria cerebral mitja en ratolins, es va trobar que expressió de la MMP-13 està implicada en el dany i en mecanismes de reparació post-isquèmia. Després de la isquèmia-reperfusió, la deficiència en MMP-13 redueix la mida de l'infart, millora l’estatus funcional en la fase més aguda i protegeix de l’aparició de transformacions hemorràgiques, però interfereix negativament en la recuperació del teixit cortical mitjançant la reducció en la migració neuroblast en proliferació, en el remodelat vascular i en un microambient amb factors tròfics reduïts. Al mateix temps les cèl·lules endotelials progenitores amb un silenciament transitori de l’expressió de MMP-13 presenten una funció aberrant en assajos de tubulogènesis, in vitro. Com que la deficiència de MMP-13 protegia del dany cerebral en el model animal d’isquèmia al temps que participava en la recuperació del teixit en fases de reparació, aquesta tesi també estudia el perfil temporal dels nivells plasmàtics de MMP-3, MMP-12 i MMP-13 durant la teràpia neuro-rehabilitadora per investigar el seu paper com a potencials biomarcadors del teixit en recuperació. Els nostres resultats mostren que alts nivells de plasma MMP-13, així com MMP-12, estan fortament associats amb la severitat i extensió de la isquèmia, però no els de MMP-3. Curiosament, els pacients amb un millor status en la funció motora durant la rehabilitació presenten els nivells més elevats de MMP-3 i reduïts de MMP-12 / -13. També, els valors de MMP-3 estan estretament associats amb la millor recuperació motora després de l'accident cerebrovascular, el que indica el seu potencial com a biomarcador de la millora en la funció motora. Aquests resultats són importants per imaginar el futur ús d'un biomarcador per tal de poder ajustar, personalitzar i modular teràpies de neuro-rehabilitació d'acord a les necessitats dels pacients. En conclusió aquesta tesi mostra el paper de les MMPs en el dany i reparació de teixits després de l'ictus isquèmic i ressalta la importància de monitoritzar i modular aquestes proteases en favor de la recuperació dels pacients.
Ischemic stroke is a leading cause of mortality, long-time disability and morbidity in the world. Currently, the only effective treatments are the intravenous thrombolytic therapy with tissue plasminogen activator (tPA) and the new emerging intra-arterial thrombectomies, which require to be administered in the hyperacute phase of stroke (<4.5 hours for tPA treatment and <6 hours for endovascular therapy). Therefore, it is necessary to investigate new therapies that could be used to treat a large number of patients in a later phase to repair and rewire the damaged tissue. One approach to achieve these objectives is to enhance vascular remodeling and neurogenesis in the ischemic brain. The aim of this thesis is to study the modulation of extracellular matrix metalloproteinase-13 (MMP-13) in brain injury and neurorepair and to investigate several MMPs as serving biomarkers to monitor motor and functional improvement during neurorehabilitation therapy. Using a distal middle cerebral artery occlusion model of ischemia in mice, we found that the modulation of MMP-13 is involved in ischemic damage and repair mechanisms. After ischemia-reperfusion, MMP-13 deficiency reduces infarct size, improves functional outcome and protects from hemorrhagic transformation, but impairs cortical tissue recovery by reducing proliferating neuroblast migration and vessel remodelling in a microenvironment with reduced trophic factors. At the same time MMP-13-silenced endothelial progenitor cells present an aberrant in vitro tubulogenesis function. As MMP-13 deficiency protects brain injury acutely and participates in post-stroke tissue recovery, this thesis also studies the temporal profile of plasma MMP-3, MMP-12 and MMP-13 during rehabilitation therapy to investigate their potential role as biomarkers of tissue recovery. Our results show that high levels of plasma MMP-13, as well as MMP-12, are strongly associated with stroke severity acutely, but not MMP-3. Interestingly, those patients with a better motor outcome during rehabilitation present elevated MMP-3 and decreased MMP-12/-13 levels. MMP-3 is closely associated with the better post-stroke motor recovery, indicating the potential as a biomarker of final functional improvement. These are important results that envision the use of biomarkers that could help to adjust, personalize and modulate neurorehabilitation therapies according to patients’ needs. In conclusion this thesis shows the role of MMPs in tissue injury and repair after stroke and the importance to monitor and modulate these proteases in favor of patients’ recovery.

The ß-carbolines norharman and harman structurally resemble the synthetic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that is known for its ability to damage neuromelanin-containing dopaminergic neurons of the substantia nigra and thereby induce parkinsonism. MPTP is, however, not normally present in the environment whereas the ß-carbolines are present in cooked food and tobacco smoke. In this thesis it was demonstrated that norharman and harman had affinity to melanin and were retained in neuromelanin-containing neurons of frogs up to 30 days post-injection (the longest survival time examined). It was also demonstrated that norharman induced neurodegeneration, activation of glia cells and motor impairment in mice. Furthermore, this compound induced ER stress and cell death in PC12 cells. An in vitro model of dopamine melanin-loaded PC12 cells was developed in order to study the effect of melanin on norharman-induced toxicity. In this model, melanin seemed to attenuate toxicity induced by low concentrations of norharman. After exposure to the highest concentration of norharman, melanin clusters were disaggregated and there was an increased expression of stress proteins and caspases-3, known to be involved in apoptosis. The polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene was demonstrated to have a CYP1A1-dependent localization in endothelial cells in the choroid plexus, in the veins in the leptomeninges and in the cerebral veins of mice pre-treated with CYP1-inducers. These results demonstrate that the distribution of environmental compounds could be influenced by the presence of neuromelanin and expression of CYP enzymes in the brain and that norharman may induce neurotoxic effects in vivo and in vitro.

Title: Role and potential of endothelial progenitor cells in cerebral vasospasm Abstract: Background and aim: Despite many treatment approaches, cerebral vasospasm and delayed ischemic neuronal damage (DIND) still represent a serious threat to patients with subarachnoid haemorrhage (SAH). Endothelial progenitor cells (EPC) have been involved as prognostic indicators in several vascular diseases and mesenchymal stem cells already have shown some benefits in ischemic injury. Aim of this study was to investigate the therapeutic potential of endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) in attenuating or preventing vasospasm and DIND in patients with SAH. Methods: Given the emergent role of DIND as a result of multifactorial hypoperfusion stress in the outcome of SAH patients, the efficacy of EPC and MSC in reducing neuronal damage has been evaluated in an in vitro model of ischemia, namely the oxygen glucose deprivation (OGD), on primary rat cortical neuronal cultures. Further, we tested in a clinical observational study SAH patients with and without vasospasm for different recruitment patterns of circulating EPC. To this purpose arterial blood samples were collected at various timepoints from admission to discharge of the patients. On these samples real-time quantitative PCR (RT-qPCR) was performed to detect gene expression relative to EPCs, since cytofluorimetric analysis appeared not sensitive enough to detect this rare cell population. Results: Though present results need further confirmation, in vitro it was observed that both MSC and EPC treatment through conditioned medium or co-culture in transwell- although with some differences - mediate a survival advantage for OGD stressed neurons. Furthermore stem cell mediated treatment showed efficacy even when applied 24 hours after OGD stress induction. RT-qPCR results from a small sample of SAH patients might indicate an early mobilization of EPC related gene expression in subjects that do not develop vasospasm with a peak around day 4, whereas the expression of these genes remain invariably low in patients that develop vasospasm as in controls not affected by SAH. Conclusions: MSCs and EPCs seem to have an important potential role in preventing DIND in vitro as well as EPC recruitment might associate with lack of vasospasm in SAH patients. Further studies are needed to confirm these results and to prove a causal relationship between EPCs and vasospasm protection.

Die bakterielle Meningitis (BM) ist trotz antibiotischer Therapie eine Erkrankung mit einer hohen Mortalität und Morbidität. Kopfschmerzen und Meningismus sind Hauptsymtome und ein klinischer Hinweis für die Aktivierung trigeminaler Fasern. Ziel dieser Arbeit war es zu prüfen ob die freigesetzten Neuropeptide einen proinflammatorischen Effekt auf zerebrale Endothelzellen, einen wesentlichem Bestandteil der Blut-Hirn-Schranke haben. Wir verwendeten primär kultivierte zerebrale Kapillarendothelzellen (BMEC) der Ratte und als Stimulus Neuropeptide und/oder Pneumokokkenzellwände (PCW). Beide Neuropeptide, CGRP mehr als SP, verstärken den Effekt von PCW auf die mRNA Expression und Freisetzung von TNF-alpha, IL-1beta, IL-6, IL-10 und MIP-2 aus den BMEC. CGRP und SP haben nur eine geringe Wirkung. PCW regulieren die Dichte der CRLR (CGRP1-R) bzw. NK-1 Rezeptoren und erklären damit die kostimulatorische Wirkung. Zudem untersuchten wir den Effekt von PCW und/oder CGRP auf die Adrenomedullin (AM)- Synthese. AM ist ein vasodilatorisch wirkendes Peptid, dass vorwiegend in Endothelzellen konstitutiv gebildet wird und am CRLR Rezeptor wirkt. PCW und CGRP verstärken die Synthese von AM. Mit dieser Arbeit konnte gezeigt werden, dass PCW zur Hochregulation von Neuropeptidrezeptoren führt und CGRP und SP über diese Rezeptoren einen modulatorischen Effekt auf die Zytokinproduktion in BMEC haben. Ein genaues Verständnis dieser Interaktionen könnte die Entwicklung immunmodulatorischer Interventionen und damit eine Verbesserung der Prognose der bakteriellen Meningitis bewirken.
Despite antibiotic treatment bacterial meningitis is still associated with a high mortality and morbidity. Headache and meningismus as key symptoms, provide clear evidence for the activation of trigeminal nerve fibers. Aim of the study was to test whether the released neuropeptides have a proinflammatory effect in cerebral endothelial cells the major compartment of the blood brain barrier. We used primary brain microvascular endothelial cells of the rat (BMEC) which were stimulated with CGRP, SP and/or pneumococcal cell walls (PCW). Both neuropeptides CGRP more than SP enhanced PCW-induced mRNA expression and the release of TNF-alpha, IL-1-beta, IL-6, IL-10 and MIP-2. Neuropeptides alone were not able to induce these cytokines. PCW upregulate the density of CRLR receptor and regulate the NK-1 receptor and therefore may explain the costimulatory effect. Furthermore the effect of PCW and/or CGRP on adrenomedullin synthesis in BMEC was investigated. Adrenomedullin is a vasodilatatory peptide, which is constitutivly produced by endothelial cells and act on the CRLR receptor. PCW as well as CGRP enhance the synthesis of AM. Our data suggest that PCW upregulate neuropeptide receptors and modulate via these specific receptors the cytokine production. A detailed understanding of these interactions may open new immunmodulatory interventions and therefore may contribute to a better prognosis of bacterial meningitis.

Endothelial cells (EC) lining the cerebral blood vessels are responsible for the formation and maintenance of a unique structural and functional barrier, the blood-brain barrier (BBB), that normally prevents the movement of white blood cells from blood into brain. Inflammatory and infectious diseases of the central nervous system (CNS), including multiple sclerosis, encephalitis, meningitis and infarction, are characterized by increased BBB permeability and infiltration of the brain by leukocytes. The mechanisms that regulate the traffic of leukocytes across the BBB have not yet been elucidated. A group of EC surface molecules called adhesion molecules, have been implicated in interactions between inflammatory cells and extracerebral EC. They include E-selectin, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and platelet/endothelial cell adhesion molecule-1 (PECAM-1). Blocking these molecules with antibodies (Abs) reduces leukocyte traffic across EC. One of these molecules, ICAM-1, has been demonstrated in inflammatory CNS lesions. The objective of this study was to investigate the expression and function of four EC adhesion molecules in an in vitro model of the human BBB. The results indicate that primary cultures of unstimulated human brain microvessel EC (HBMEC) form high resistance monolayers and express low levels of E-selectin, VCAM-1 and ICAM-1 that can be significantly upregulated following stimulation with cytokines and LPS. PECAM-1 is strongly expressed by all cells constitutively. Treatment of HBMEC with TNF increases monolayer permeability, and augments adhesion and migration of polymorphonuclear leukocytes (PMN) and T-lymphocytes across the monolayers. Adhesion of resting T-lymphocytes to activated endothelium is mediated by VCAM-1 and ICAM-1. ICAM-1, E-selectin and PECAM-1 participate in T-lymphocyte migration across HBMEC monolayers. Adhesion of PMN to activated HBMEC is mediated by ICAM-1 and E-selectin, while ICAM-1 is involved in the migration process. Anti-adhesion molecule Abs reduce adhesion and/or migration but have no effect on the increased endothelial permeability secondary to leukocyte migration. These studies indicate that EC adhesion molecules, induced by cytokines, mediate cerebral EC-leukocyte interactions which, in association with a concurrent increase in endothelial permeability, may facilitate leukocyte traffic across the BBB in the early stages of CNS inflammation.

Disruption of the blood-brain barrier (BBB) is hypothesized to play an important role in the disease biology of schizophrenia (SZ). Brain microvascular endothelial cells (BMECs) have paracellular and transcellular proteins, transporters, as well as important extracellular matrix proteins, which collectively contribute to maintaining proper BBB function. While previous studies have provided some insights into the role of the BBB in SZ pathophysiology, there is a significant gap in our understanding of the cellular-molecular underpinnings of its major component, BMECs. Human induced pluripotent stem cells (hiPSCs) provide an exciting new avenue for exploring the role of BMECs in SZ. We hypothesize that BMECs have intrinsic deficits that lead to BBB dysfunction in SZ. In this study, we first aimed to test whether the existing hiPSC-derived BMEC protocols work with our patient-specific hiPSC samples. Secondly, we sought to investigate any potential deficits between BMECs derived from healthy control (HC) and SZ subjects. We successfully adapted the established protocol and confirmed the identity of these hiPSC-derived BMECs with relevant cell markers such as CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. We also evaluate barrier function by measuring trans-endothelial electrical resistance (TEER) and efflux transporters activity of ABCB1 and ABCC1. We observed evidence of poor cellular adhesion and disrupted tight junctions in a subset of SZ hiPSC-derived BMECs, where approximately 70% of them demonstrated extensive BBB disruption (reduced TEER). These findings suggest that there may be cell-autonomous disease-specific deficits in BMECs in SZ that result in BBB dysfunction.
2022-06-07T00:00:00Z

碩士
長庚科技大學
健康產業科技研究所
107
As the population of the world ages, the elderly population and aging-related disorders are also increasing. Therefore, in recent years, most studies have focused on the aging-related disorders, the central nervous system (CNS) degenerative diseases particularly. These CNS disorders include traumatic brain injury, stroke, neuroinflammation, and neurodegeneration. It is known that endothelial cells of Blood-Brain-Barrier (BBB) in patients with neurodegenerative diseases usually have an inflammatory state, but the mechanisms are still unclear. Here, we use the brain microvascular endothelial cells (bEnd.3) as a cell model to investigate the inflammatory responses induction by proinflammatory factors IL-1 and TNF- and then the effects of some natural products on the responses. First, we found that both IL-1 and TNF- can induce several inflammatory proteins’ expression, including MMP-9 by regulating their gene and protein. Moreover, the pharmacological inhibitors were used to investigate the mechanisms. By pretreatment of cells with reactive oxygen species (ROS) inhibitors, we demonstrated that IL-1 and TNF- induced inflammatory proteins’ expression via a ROS-dependent signaling pathway, including NADPH oxidase (NOX) or mitochondria. We further found that IL-1 and TNF- stimulated activation of many signaling molecules, such as c-Src, EGFR, AKT, and MAPKs (ERK1/2, p38, and JNK1/2), which may be involved in the regulatory pathway by the specific pharmacological inhibitors in bEnd.3 cells. In transcriptional factors, IL-1 and TNF- stimulated activation of NF-B and AP-1. Finally, we found that IL-1 and TNF- induced the configurational change of ZO-1, as a cell tight junction protein, in bEnd.3 cells by immunofluorescence stain. The induction of MMP-9 by IL-1 and TNF- may be involved in the event. Furthermore, many previous studies have indicated that some natural products have anti-inflammatory, anti-oxidative, or immune-enhancing effects, which may have fewer side effects. Therefore, these natural products could be used for the prevention or treatment of chronic inflammation-related diseases. However, the effects of most natural products still lack scientific evidence. Here, we found some natural products can attenuate IL-1- and TNF--induced MMP-9 expression, suggesting that these natural products may contain anti-inflammatory components to protect the brain BBB from damage. We will further explore the active ingredients of these natural products and their mechanisms in the future.

Le glioblastome multiforme (GBM) représente la tumeur cérébrale primaire la plus agressive et la plus vascularisée chez l’adulte. La survie médiane après le diagnostic est de moins d’un an en l’absence de traitement. Malheureusement, 90% des patients traités avec de la radiothérapie après la résection chirurgicale d’un GBM développent une récidive tumorale. Récemment, le traitement des GBM avec radiothérapie et témozolomide, un agent reconnu pour ses propriétés antiangiogéniques, a permis de prolonger la survie médiane à 14,6 mois. Des efforts sont déployés pour identifier des substances naturelles capables d’inhiber, de retarder ou de renverser le processus de carcinogenèse. Epigallocatechin-3-gallate (EGCG), un polyphénol retrouvé dans le thé vert, est reconnu pour ses propriétés anticancéreuses et antiangiogéniques. L’EGCG pourrait sensibiliser les cellules tumorales cérébrales et les cellules endothéliales dérivées des tumeurs aux traitements conventionnels. Le chapitre II décrit la première partie de ce projet de doctorat. Nous avons tenté de déterminer si l’EGCG pourrait sensibiliser la réponse des GBM à l’irradiation (IR) et si des marqueurs moléculaires spécifiques sont impliqués. Nous avons documenté que les cellules U-87 étaient relativement radiorésistantes et que Survivin, une protéine inhibitrice de l’apoptose, pourrait être impliquée dans la radiorésistance des GBM. Aussi, nous avons démontré que le pré-traitement des cellules U-87 avec de l’EGCG pourrait annuler l’effet cytoprotecteur d’une surexpression de Survivin et potentialiser l’effet cytoréducteur de l’IR. Au chapitre III, nous avons caractérisé l’impact de l’IR sur la survie de cellules endothéliales microvasculaires cérébrales humaines (HBMEC) et nous avons déterminé si l’EGCG pouvait optimiser cet effet. Bien que les traitements individuels avec l’EGCG et l’IR diminuaient la survie des HBMEC, le traitement combiné diminuait de façon synergique la survie cellulaire. Nous avons documenté que le traitement combiné augmentait la mort cellulaire, plus spécifiquement la nécrose. Au chapitre IV, nous avons investigué l’impact de l’IR sur les fonctions angiogéniques des HBMEC résistantes à l’IR, notamment la prolifération cellulaire, la migration cellulaire en présence de facteurs de croissance dérivés des tumeurs cérébrales, et la capacité de tubulogenèse. La voie de signalisation des Rho a aussi été étudiée en relation avec les propriétés angiogéniques des HBMEC radiorésistantes. Nos données suggèrent que l’IR altère significativement les propriétés angiogéniques des HBMEC. La réponse aux facteurs importants pour la croissance tumorale et l’angiogenèse ainsi que la tubulogenèse sont atténuées dans ces cellules. En conclusion, ce projet de doctorat confirme les propriétés cytoréductrices de l’IR sur les gliomes malins et propose un nouveau mécanisme pour expliquer la radiorésistance des GBM. Ce projet documente pour la première fois l’effet cytotoxique de l’IR sur les HBMEC. Aussi, ce projet reconnaît l’existence de HBMEC radiorésistantes et caractérise leurs fonctions angiogéniques altérées. La combinaison de molécules naturelles anticancéreuses et antiangiogéniques telles que l’EGCG avec de la radiothérapie pourrait améliorer l’effet de l’IR sur les cellules tumorales et sur les cellules endothéliales associées, possiblement en augmentant la mort cellulaire. Cette thèse supporte l’intégration de nutriments avec propriétés anticancéreuses et antiangiogéniques dans le traitement des gliomes malins pour sensibiliser les cellules tumorales et endothéliales aux traitements conventionnels.
Glioblastoma multiform (GBM) represents the most aggressive and vascularised primary cerebral neoplasm in adults. Median length of survival without further therapy is usually less than one year from the time of diagnosis. Unfortunately, 90% of patients receiving radiotherapy following GBM resection develop a tumor recurrence. More recently, treatment of GBM with combined radiotherapy and temozolomide, an agent recognized for its antiangiogenic activity, increased the median survival to 14,6 months. Efforts have been oriented towards identifying naturally occurring substances capable of inhibiting, delaying or reversing the multi-stage carcinogenesis process. Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, has been recognized for its anticancerous and antiangiogenic property. EGCG may represent a potential agent capable of sensitizing brain tumor cells and their derived endothelial cells (ECs) to conventional treatments. In chapter II, the first part of this doctorate project aimed at determining if EGCG, in synergy with radiotherapy, can sensitize GBM’s response to radiation and whether specific molecular markers are involved. We documented that U-87 cells were relatively radioresistant and that Survivin, an inhibitor of apoptosis protein, may be involved in GBM’s radioresistance. We also found that pre-treatment of U-87 cells with EGCG could overcome the cytoprotective effect of Survivin overexpression and potentiate the cytoreductive effect of irradiation (IR). In chapter III, we characterized the impact of IR on human brain microvascular endothelial cell (HBMEC) survival and determined whether EGCG, could optimize this effect. We found that although EGCG treatment and IR individually decreased HBMEC survival, the combined treatment synergistically reduced survival. We documented that the combined treatment increased cell death, more specifically necrosis. In chapter IV, we investigated the impact of IR exposure on the angiogenic functions i.e. cell proliferation, cell migration in response to brain tumor-derived growth factors, and capacity for tubulogenesis of surviving human brain tumor-derived ECs. The Rho signalling pathway was also investigated in relation to the functional properties of radioresistant HBMEC. Our data suggests that IR significantly alters radioresistant HBMEC migration response to tumor-secreted growth factors and tubulogenesis. Response to growth factors important for tumor expansion and angiogenesis is significantly attenuated in these cells. In conclusion, this doctorate project confirmed IR’s cytoreductive properties on malignant gliomas. We proposed a novel mechanism to explain GBMs’ radioresistance. This project documented for the first time IR’s cytotoxic effect in HBMEC. It also described the existence of radioresistant HBMEC and characterized their altered angiogenic functions. The combination of natural anticancerous and antiangiogenic molecules such as EGCG with radiotherapy could improve IR’s effect on human malignant glioma cells and microvascular ECs, especially through increased necrosis of HBMEC. The thesis supports integrating nutrients bearing anticancerous and antiangiogenic properties, such as EGCG, in the management of gliomas to sensitize tumor and tumor-associated ECs to conventional therapies.

Nowadays, the biggest challenge in tissue engineering consists in developing structures and in the application of strategies to emulate the anatomical and cellular complexity and vascularization of native tissues to maintain cell viability and functionality. The presence of functional blood vessel networks is essential to ensure adequate nutrient flow and oxygen diffusion throughout the support structure, two key requirements for maintaining cell viability. This work aimed to develop a complex in vitro model that mimics the native vascular network. To this end, a multilayered membrane made of six bilayers of chitosan (CHI)/alginate (ALG) or CHI/ALG-RGD (tripeptide of Arginine (R)-Glycine (G)- Aspartic acid (D) responsible for the cellular adhesion to the extracellular matrix (ECM)) were produced via Layer-by-Layer (LbL) assembly technology on the ALG printed structures. The ALG structures coated with the multilayered membranes were embedded in xanthan gum, chemically modified with methacrylated groups in order to obtain a mechanically robust hydrogel structure after photocrosslinking by UV light exposure. The liquification of the ALG printed structures, coated with the CHI/ALG, CHI/ALG-RGD or without the multilayers membranes, with ethylenediaminetetraacetic acid (EDTA), led to the formation of microchannels in which human umbilical vein endothelial cells (HUVECs) were cultured for 24 hours. The obtained results demonstrate that the microchannels encompassing CHI/ALG-RGD multilayered membranes contributed to a larger cellular adhesion, demonstrating their potential to be applied in tissue engineering and regenerative medicine strategies.
Atualmente, o maior desafio em engenharia de tecidos consiste no desenvolvimento de estruturas e aplicação de estratégias que visem mimetizar a complexidade anatómica e celular, assim como a vascularização de tecidos nativos, de forma a manter a viabilidade e funcionalidade das células. A presença de estruturas funcionais à base de vasos sanguíneos é essencial para garantir o fluxo adequado de nutrientes, assim como a difusão de oxigénio em toda a estrutura de suporte, dois requisitos essenciais para manter a viabilidade celular. Este trabalho teve como objetivo desenvolver um modelo complexo in vitro que mimetize a rede vascular nativa. Com esse intuito, membranas multicamadas compreendendo seis bicamadas de quitosana (CHI)/alginato (ALG) e CHI/ALG-RGD (tripéptido de Arginina (R)-Glicina (G)-Ácido aspártico (D) responsável pela adesão de células à matriz extracelular) foram produzidas, via tecnologia de deposição camada-a-camada (do inglês Layer-by-Layer assembly technology), em estruturas impressas de ALG. As fibras de ALG revestidas com os filmes multicamadas foram embebidas em goma xantana, quimicamente modificada com grupos metacrilatos, de modo a obter uma estrutura de hidrogel mecanicamente robusta após foto-reticulação por ação da luz UV. A liquefação das estruturas impressas de ALG, contendo as multicamadas de CHI/ALG ou CHi/ALG-RGD, com ácido etilenodiamino tetra-acético (EDTA), levou à formação de microcanais nos quais se cultivaram células endoteliais humanas, extraídas da veia umbilical durante 24 horas. Os resultados obtidos demonstraram que os microcanais compreendendo as membranas multicamadas à base de CHI/ALG-RGD contribuíram para uma maior adesão celular, demonstrando o seu potencial para estratégias de engenharia de tecidos e medicina regenerativa.
Mestrado em Biotecnologia

Previous work in our laboratory indicates that the diameter of brain arteries and arterioles can be increased by N-methyl-D-aspartate (NMDA) receptor activation. We looked for expression of NMDA receptors and endothelial cell responses to NMDA receptor agonists and antagonists in the mouse brain endothelial cell line bEnd.3. Using RT-PCR and Western blotting we found evidence supporting the presence of NMDA receptor subunits NR1 and NR2C. Treatment of bEnd.3 cells with combinations of 100 μM glutamate and D-serine significantly increased intracellular calcium. However, we saw no direct evidence that NO was produced in response to NMDA receptor activation using the Griess method. We did observe an NMDA receptor-dependent increase in protein nitrosylation. This increase is unlikely related to enhanced NO levels since it was not correlated with NO production and was not inhibited by the endothelial NO synthase inhibitor L-NIO.

Bacterial meningitis occurs when blood-borne bacteria are able to penetrate highly specialized brain endothelial cells (BECs) and gain access to the meninges. Neisseria meningitidis (Nm) is a human-exclusive pathogen for which suitable in vitro models are severely lacking. Until recently, modeling BEC-Nm interactions has been almost exclusively limited to immortalized human cells that lack proper BEC phenotypes. Specifically, these in vitro models lack barrier properties, and continuous tight junctions. Alternatively, humanized mice have been used, but these must rely on known interactions and have limited translatability. This motivates the need to establish novel human-based in vitro BEC models that have barrier phenotypes to research Nm-BEC interactions. Recently, a human induced pluripotent stem cell (iPSC) model of BECs has been developed that possesses superior BEC phenotypes and closely mimics the in vivo blood vessels present at the blood-meningeal barrier. Here, iPSC-BECs were tested as a novel cellular model to study Nm-host pathogen interactions, with focus on host responses to Nm infection. Two wild type strains and three mutant strains of Nm were used to confirm that these followed similar phenotypes to previously described models. Importantly, the recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, at distinct time points of infection, and the secretion of IFN γ and RANTES by iPSC-BECs. Nm was directly observed to disrupt tight junction proteins ZO-1, Occludin, and Claudin-5 at late time points of infection, which became frayed and/or discontinuous upon infection. This destruction is preceded by, and might be dependent on, SNAI1 activation (a transcriptional repressor of tight junction proteins). In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability was observed at late infection time points. Notably, bacterial transmigration correlated with junctional disruption, indicating that the paracellular route contributes for bacterial crossing of BECs. Finally, RNA-Sequencing (RNA-Seq) of sorted, infected iPSC-BECs was established through the use of fluorescence-activated cell sorting (FACS) techniques following infection. This allowed the detection of expression data of Nm-responsive host genes not previously described thus far to play a role during meningitidis. In conclusion, here the utility of iPSC-BECs in vitro to study Nm infection could be demonstrated. This is the first BEC in vitro model to express all major BEC tight junctions and to display high barrier potential. Altogether, here this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes and suggests that the paracellular route contributes to Nm traversal of BECs
Eine bakterielle Meningitis tritt auf, wenn durch Blut übertragene Bakterien hochspezialisierte Hirnendothelzellen (BEC) durchdringen und Zugang zu den Meningen erhalten. Neisseria meningitidis (Nm) ist ein human-exklusiver Erreger, für dessen Untersuchung es an geeigneten In-vitro-Modellen mangelt. Bis vor kurzem war die Modellierung von BEC-Nm-Wechselwirkungen fast ausschließlich auf immortalisierte humane Zellen beschränkt, denen wichtige BEC-Phänotypen fehlen. Besonders hervorzuheben sind das Fehlen physiologischer Barriereeigenschaften durch unkontinuierliche dichte Zell-Zell-Verbindungen. Als alternative Modellorganismen können humanisierte Mäuse verwendet werden, die sich jedoch auf bekannte Wirt-Erreger-Wechselwirkungen stützen und durch Speziesunterschiede eine eingeschränkte Übersetzbarkeit aufweisen. Dies begründet die Notwendigkeit, neuartige humane In-vitro-BEC-Modelle zu etablieren, die physiologische Barrierephänotypen aufweisen, um Nm-BEC-Wechselwirkungen zu untersuchen. Kürzlich wurde ein humanes Modell entwickelt, welches auf aus induziert pluripotenten Stammzellen (iPSCs) abgeleiteten humanen BECs basiert und sich durch einen physiologischen Blut-Hirn-Schranken-Phänotyp auszeichnet. Die iPSC-BECs wurden in dieser Arbeit als neuartiges zelluläres Modell getestet, um Nm-Wirt-Pathogen-Wechselwirkungen zu untersuchen, wobei der Schwerpunkt auf Wirtsreaktionen auf Nm-Infektionen lag. Zwei Wildtypstämme und drei Mutantenstämme von Nm wurden verwendet, um zu bestätigen, dass diese ähnlichen Phänotypen wie in zuvor beschriebenen Modellen folgten. Hervorzuheben ist, dass die Rekrutierung des kürzlich veröffentlichten Pilus-Adhäsin-Rezeptors CD147 unter Meningokokken-Mikrokolonien in iPSC-BECs verifiziert werden konnte. Es wurde auch beobachtet, dass Nm die Expression der entzündungsfördernden und neutrophilen spezifischen Chemokine IL6, CXCL1, CXCL2, CXCL8 und CCL20 zu bestimmten Zeitpunkten der Infektion sowie die Sekretion von IFN-γ und RANTES durch iPSC-BECs signifikant erhöht. Es wurde zudem beobachtet, dass Nm die Tight Junction-Proteine ZO-1, Occludin und Claudin-5 zu späten Zeitpunkten der Infektion zerstört, verursacht durch die Infektion wurde ein ausgefranster und/oder diskontinuierlicher Tight Junction-Phänotyp beobachtet. Dieser Zerstörung geht die SNAI1-Aktivierung (ein Transkriptionsrepressor für Tight Junction-Proteine) voraus und könnte von ihr abhängig sein. In Übereinstimmung mit dem Verlust der Tight Junctions wurde zu späten Infektionszeitpunkten ein starker Verlust des transendothelialen elektrischen Widerstands und eine Zunahme der Natriumfluoreszein-Permeabilität beobachtet. Bemerkenswerterweise korrelierte die bakterielle Transmigration mit dem Verlust der Tight Junctions, was darauf hinweist, dass der parazelluläre Weg zur bakteriellen Überwindung von BECs eine entscheidende Rolle spielt. Schließlich wurde die RNA-Sequencing (RNA-Seq) von sortierten, infizierten iPSC-BECs durch die Verwendung von fluoreszenzaktivierten Zellsortiertechniken (FACS) nach der Infektion durchgeführt. Dies ermöglichte erstmalig den Nachweis von Expressionsdaten von Nm-responsiven Wirtsgenen, welche bei der Meningitidis eine Rolle zu spielen scheinen. Zusammenfassend konnte im Rahmen der vorliegenden Arbeit der Nutzen von iPSC-BECs In-Vitro-Modellen zur Untersuchung von Nm-Infektionen gezeigt werden. Dies ist das erste BEC-In-vitro-Modell, das alle wichtigen BEC-Tight Junctions exprimiert und ein hohes Barrierepotential aufweist. Insgesamt liefert das eingesetzte Modell neue Einblicke in die Nm-Pathogenese, einschließlich der Beeinflussung der Barriereeigenschaften und der Tight Junction-Komplexe durch Nm, und gibt erste Hinweise darauf, dass die parazelluläre Route zum Nm-Übertritt von BEC-Barrieren eine entscheidende Rolle spielt

The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB.

碩士
國立中正大學
化學工程所
94
The purpose of this study was to investigate the electrophoretic mobility and zeta potential of human brain microvessel endothelial cell (HBMEC) and solid lipid nanoparticle (SLN) with different cacao butter content, stirring rate, stored time and HBMEC coculture with SLN under the influence of pH value. The surface potential of SLN and HBMEC was estimated from the notion of ion condensation of Levin’s theory and Perturbation method. The result revealed that, the absolute value of the zeta potential, electrophoretic mobility, surface charge density and surface potential increased with an increace in pH value. For the same pH value, the more surface charge of biocolloidal particle the more absolute value of the zeta potential, electrophoretic mobility, surface charge density and surface potential. For the discrepancy between surface potential and zeta pontial (Dsz) of HBMEC and SLN, we can find the maximum of Dsz at pH=7 and decreased with increased and decreased pH value.

碩士
國立中正大學
化學工程所
95
The purpose of this study was to investigate the electrophoretic mobility and zeta potential of human brain microvessel endothelial cells (HBMEC), cationic solid lipid nanoparticles (SLN) with different ratio of cationic lipid (stearylamine/dimethyldioctadecylammonium bromide) which were formulated with different surfactant (Tween 80 or Span 20) and HBMEC coculture with SLN under the influence of the various concentration of glutamate. The surface charge density of SLN and HBMEC were estimated form the electrophoretic mobility of capillary electrophoresis converted through Ohshima’s electrokinetic theory of charged particle. The result revealed that, the molar fraction of stearylamine of the cationic SLN increased, the size increased, but the electrophoretic mobility and zeta potential decreased. The electrophoretic mobility, zeta potential, surface charge density of SLN decreased with an increase of the concentration of glutamate. For fixed ratio of cationic lipid, the zeta potential of SLN formulated with mixtured surfactant increased with an increased of weight fraction of Tween 80. For HBMEC cocultured with SLN, the higher the value of zeta potential of SLN, the more the reduction of the electrophoretic mobility and zeta potential of HBMEC. After SLN uptake of HBMEC, the electrophoretic mobility and zeta potential decreased.

Tese de doutoramento em Química (Química Biológica), apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra
A difusão passiva através da membrana celular é a principal via para a permeação de xenobióticos através de endotélios tight, tais como a barreira hemato-encefálica. A velocidade da permeação passiva através de bicamadas lipídicas de um dado fármaco é, portanto, um passo crítico na previsão da sua farmacocinética. Neste trabalho, foi realizado um estudo detalhado sobre a cinética e a termodinâmica da interacção da clorpromazina, um antipsicótico utilizado no tratamento da esquizofrenia, com membranas lipídicas compostas por diferentes misturas de lípidos. Utilizámos a calorimetria de titulação isotérmica, uma técnica muito eficiente para o estudo de interacções moleculares, que permite a medição do coeficiente de partição de moléculas entre o meio aquoso e a membrana, bem como dos parâmetros termodinâmicos associados a esta partição. A complexidade das membranas utilizadas neste estudo foi aumentada progressivamente, desde a bicamada simples de POPC puro até composições de lípidos características das monocamadas exterior e interior das células endoteliais. Os efeitos da presença de carga, de colesterol (Chol), de fosfolípidos com etanolamina (POPE) e de esfingomielina (SPM), bem como da coexistência de fases, foram avaliados na interacção de CPZ com as bicamadas lipídicas. A introdução de 10% molar de POPS carregado negativamente em membranas de POPC aumenta a partição da CPZ, devido à atracção electrostática entre as cargas opostas, sendo que a variação de entalpia torna-se mais favorável. Por outro lado, a presença de Chol aumenta a ordem das bicamadas e diminui fortemente a afinidade da CPZ para com a bicamada, tanto em termos da quantidade de CPZ que se associa com a membrana bem como da entalpia de interacção, que se torna desfavorável. Para membranas na fase líquida ordenada (POPC:SPM:Chol 1:5:4) observa-se um coeficiente de partição muito mais pequeno do que para as membranas na fase líquida desordenada, com uma contribuição entalpica muito desfavorável. A partir dos parâmetros obtidos para todas as bicamadas lipídicas estudadas, observa-se que a partição de CPZ em membranas na fase líquida desordenada tem uma contribuição entalpica significativamente favorável, enquanto que a associação de CPZ com membranas na fase líquida ordenada é somente impulsionada pelo aumento de entropia no sistema. Os coeficientes de partição obtidos permitiram o cálculo das afinidades de CPZ para com as diferentes membranas da célula e a previsão da concentração relativa de CPZ em cada pool de lípidos em equilíbrio. x A permeação passiva através de bicamadas lipídicas depende da afinidade do fármaco para com a membrana e também das constantes de velocidade para a interacção (inserção, desorpção e translocação). Neste trabalho, desenvolvemos uma metodologia que permite a caracterização quantitativa da constante de translocação através das membranas na fase líquida desordenada, usando ITC. Apresentamos também resultados preliminares sobre o uso desta técnica para obter as constantes de velocidade para inserção em e desorção de bicamadas lipídicas na fase líquida ordenada. Além disso, este trabalho oferece uma visão importante sobre a heterogeneidade de fases das misturas lipídicas caracterizadas. Os resultados obtidos com as misturas estudadas de POPC:SPM:Chol permitem a avaliação crítica dos diagramas de fases disponíveis na literatura. Também foram encontradas fortes evidências para a coexistência de fase líquida desordenada e líquida ordenada na mistura quaternária representante da monocamada citoplasmática da membrana, POPC:Chol:POPE:POPS 4:3:2:1. Adicionalmente, foram encontradas evidências de rearranjos estruturais nas membranas com POPS e/ou POPE, induzidos pela partição de compostos anfifílicos catiónicos, como a CPZ. Estes resultados podem ser relevantes para elucidar a comunicação estrutural entre as monocamadas exterior e interior da membrana plasmática, em função da hipótese da existência de rafts. Além disso, podem também contribuir para o entendimento dos efeitos da CPZ sobre a actividade dos transportadores de membrana, tais como a P-glicoproteína.
Bolsa de doutoramento concedida pela FCT (SFRH/BD/38951/2007)

La barrière hémato-encéphalique (BHE) protège le système nerveux central (SNC) en contrôlant le passage des substances sanguines et des cellules immunitaires. La BHE est formée de cellules endothéliales liées ensemble par des jonctions serrées et ses fonctions sont maintenues par des astrocytes, celles ci sécrétant un nombre de facteurs essentiels. Une analyse protéomique de radeaux lipidiques de cellules endothéliales de la BHE humaine a identifié la présence de la voie de signalisation Hedgehog (Hh), une voie souvent liées à des processus de développement embryologique ainsi qu’au niveau des tissus adultes. Suite à nos expériences, j’ai déterminé que les astrocytes produisent et secrètent le ligand Sonic Hh (Shh) et que les cellules endothéliales humaines en cultures primaires expriment le récepteur Patched (Ptch)-1, le co-récepteur Smoothened (Smo) et le facteur de transcription Gli-1. De plus, l’activation de la voie Hh augmente l’étanchéité des cellules endothéliales de la BHE in vitro. Le blocage de l’activation de la voie Hh en utilisant l’antagoniste cyclopamine ainsi qu’en utilisant des souris Shh déficientes (-/-) diminue l’expression des protéines de jonctions serrées, claudin-5, occcludin, et ZO-1. La voie de signalisation s’est aussi montrée comme étant immunomodulatoire, puisque l’activation de la voie dans les cellules endothéliales de la BHE diminue l’expression de surface des molécules d’adhésion ICAM-1 et VCAM-1, ainsi que la sécrétion des chimiokines pro-inflammatoires IL-8/CXCL8 et MCP-1/CCL2, créant une diminution de la migration des lymphocytes CD4+ à travers une monocouche de cellules endothéliales de la BHE. Des traitements avec des cytokines pro-inflammatoires TNF-α and IFN-γ in vitro, augmente la production de Shh par les astrocytes ainsi que l’expression de surface de Ptch-1 et de Smo. Dans des lésions actives de la sclérose en plaques (SEP), où la BHE est plus perméable, les astrocytes hypertrophiques augmentent leur expression de Shh. Par contre, les cellules endothéliales de la BHE n’augmentent pas leur expression de Ptch-1 ou Smo, suggérant une dysfonction dans la voie de signalisation Hh. Ces résultats montrent que la voie de signalisation Hh promeut les propriétés de la BHE, et qu’un environnement d’inflammation pourrait potentiellement dérégler la BHE en affectant la voie de signalisation Hh des cellules endothéliales.
The blood-brain barrier (BBB), composed of tightly bound endothelial cells (ECs), regulates the entry of blood-borne molecules and immune cells into the CNS. Recent studies indicate that the Hedgehog (Hh) signaling pathway in adult tissues plays an important role in vascular proliferation, differentiation and tissue repair. Using a lipid membrane raft-based proteomic approach, I have identified the Hedgehog (Hh) pathway as a signaling cascade involved in preserving and upkeeping BBB functions. My study shows that human astrocytes express and secrete Sonic Hh (Shh) and conversely, that human BBB-ECs bear the Hh receptor Patched-1 (Ptch-1), the signal transducer Smoothened (Smo) as well as transcription factors of the Gli family. Furthermore, activation of the Hh pathway in BBB-ECs restricts the passage of soluble tracers in vitro. By blocking the Hh signaling in vitro and by using Shh knock-out (-/-) embryonic mice, I demonstrate a reduced expression of TJ molecules claudin-5, occludin and ZO-1. Hh activation also decreases the surface expression of cell adhesion molecules ICAM-1 and VCAM-1, and decreases BBB-ECs secretion of pro-inflammatory chemokines IL-8/CXCL8 and monocytes chemoattractant protein 1 MCP-1/CCL2, resulting in a reduction of migrating CD4+ lymphocytes across human BBB-EC monolayers. In vitro treatment with inflammatory cytokines TNF-α and IFN-γ, upregulates the production of astrocytic Shh and the BBB-EC surface expression of Ptch-1 and Smo. In active Multiple Sclerosis (MS) lesions, in which the BBB is disrupted, Shh expression is drastically upregulated in hypertrophic astrocytes, while Ptch-1 and Smo expression is down-regulated or left unchanged, suggesting that a deregulation in the Hh signaling pathway may prevent the barrier stabilizing properties of Hh. Our data demonstrate an anti-inflammatory and BBB-promoting effect of astrocyte-secreted Hh and suggest that a pro-inflammatory environment disrupt the BBB by impacting, at least in part, on Hh signaling in brain ECs.

L'ingénierie tissulaire est reconnue comme une méthode potentielle pour réparer ou régénérer les tissus endommagés. Malgré de grandes avancées dans l'ingénierie tissulaire, la réussite de la construction de tissus complexes avec des réseaux vascularisés reste un défi. Dans les modèles d'angiogenèse actuels, les cellules endothéliales sont ensemencées au hasard, n'offrant pas de structure organisée. La technologie de bioimpression par laser offre une résolution d'impression précise. Par cette technique, les structures microvasculaires peuvent être construites pour la fabrication d'organes complexes, ou pour modéliser la progression de la maladie ou les modèles de réponse aux médicaments. Dans cette étude, des techniques de bio-impression au laser ont été utilisées pour étudier le guidage de l'angiogenèse in vitro. Deux techniques basées sur le laser, le transfert direct induit par laser (LIFT) et le transfert latéral induit par laser (LIST) sont utilisées. Comparée à LIFT, la technologie LIST offrait des conditions idéales pour l'impression cellulaire telles que la concentration cellulaire requise pour la formation du tubes endothéliaux et l'uniformité du motif désiré. Nous avons réalisé le modelage de la formation de structures de type capillaire dans des motifs organisés via l'impression LIST. Les constructions de type capillaire formées présentent des motifs uniformes. Les structures formées ont été analysées par microscopie confocale et reconstruction d'images 3D. Bien que le développement de la lumière endothéliale soit incomplet, la technique développée possède le potentiel d'atteindre une stabilisation et un développement de la lumière si l'on recrute un deuxième type de cellule tel que les fibroblastes ou les péricytes.
Tissue engineering has been well acknowledged as a potential method to repair or regenerate damaged tissues in the human body, fulfilling the limitations and shortage in autologous and organ transplantations. Despite great advances in engineering tissues with simple geometry and low requirement for oxygen and blood supply such as cartilage, skin and cornea, success in constructing 3D complex tissues with vascularized networks remains a major challenge. Angiogenesis plays an important role in vascular development in vivo. In current angiogenesis models, endothelial cells are seeded randomly not offering precise and desired patterning. Laser-based bioprinting technology offers precise and high cell printing resolution. By using laser-based bioprinting technology, microvascular structures can be constructed as a platform for complex organ fabrication, disease progression and drug response models. In this study, laser-based bioprinting techniques are employed to study angiogenesis guidance in vitro by patterning endothelial cells. Two laser-based techniques, Laser-Induced Forward Transfer (LIFT) and Laser-Induced Side Transfer (LIST) are used as patterning tools. Compared to LIFT, LIST technology provided ideal conditions for cell printing such as required cell concentration for endothelial tube formation and pattern uniformity. In this study, we achieved the guidance of capillary-like structure formation in desired patterns via LIST printing. The formed capillary-like constructs featured precise patterns and uniformity. The structures were analyzed by confocal microscopy, 3D image reconstruction and frozen section procedure. Though lumen development was incomplete, it possesses the potential to attain further stabilization and lumen development if recruiting a second cell type such as fibroblast or pericyte.

La barrière hémato-encéphalique (BHE) est une structure retrouvée au niveau des capillaires cérébraux. Elle représente un véritable obstacle pour les actifs qui doivent se rendre au cerveau pour y exercer un effet pharmacologique. Durant les étapes du développement du médicament, des modèles cellulaires in vitro sont utilisés pour l’évaluation de la perméabilité au cerveau des nouveaux médicaments. Le modèle assemblé avec des cellules endothéliales (CEs) isolées des capillaires des cerveaux de souris présente un intérêt particulier pour la recherche en raison de sa facilité d’obtention et sa pertinence pour le criblage des médicaments. Le but de ce projet a été de construire et de caractériser un modèle monocouche de CEs primaires de souris. En parallèle, un modèle monocouche de la lignée murine b.End3 a été investigué. L’évaluation de ces modèles a été basée sur les valeurs de TEER et de perméabilité aux marqueurs fluorescents, ainsi que sur la présence des protéines spécifiques de la BHE. La validation du modèle a été établie par la corrélation des résultats de perméabilité obtenus avec le modèle développé (in vitro) avec ceux obtenus chez la souris (in vivo). L’intégrité et l’expression des protéines spécifiques de la BHE du modèle primaire se sont montrées supérieures au modèle bEnd.3. La corrélation in vitro/in vivo du modèle primaire a abouti à un r2 = 0,765 comparé au r2 = 0,019 pour le modèle bEnd.3. Ce travail de recherche montre que le modèle primaire monocouche issu de cellules endothéliales cérébrales de souris est un modèle simple et fiable pour la prédiction de la perméabilité des actifs à travers la BHE.
The blood-brain barrier (BBB), a central nervous system structure, is found in the cerebral capillaries. It represents a major obstacle for the drugs that have to reach the brain in order to exercise their pharmacological effect. In the early stages of the drug development, in vitro cell models are used to evaluate the brain permeability of new drugs. Models assembled using primary endothelial cells (ECs) isolated from mouse brain capillaries are of particular interest for research, as for their ease of obtaining and relevance for the drug screening. Thus, the goal of this project was to build and characterize a primary mouse monolayer model. At the same time, a murine b.End3 cell line monolayer model was investigated. The evaluation of these models was based on the TEER and fluorescent marker permeability values, as well as on the presence of the BBB hallmark proteins. The model validation was established by the correlation of the permeability data obtained with the in vitro model and the data obtained in mice (in vivo). As a result, the primary mouse model showed superior monolayer integrity and higher expression of the tight junction and membrane transporter proteins when compared with the bEnd.3 cell line model. The in vitro/in vivo correlation of the primary model resulted in r2 = 0.765 compared to the bEnd.3 model with r2 = 0.019. This research work shows that the primary monolayer mouse model is a simple and reliable model for predicting the drug permeability across the BBB.