Littérature scientifique sur le sujet « Platelet-Derived Factor PFs »

Purpose: The survival times of glioblastoma (GB) patients after the standard therapy including safe maximal resection followed by radiotherapy plus concomitant and adjuvant temozolomide are heterogeneous. In order to define a simple, reliable method for predicting whether patients with isocitrate dehydrogenase (IDH)-wildtype GB treated with the standard therapy will be short- or long-term survivors, we analyzed the correlation of preoperative blood counts and their combined forms with progression-free survival (PFS) and overall survival (OS) in these patients. Methods: Eighty-five patients with primary IDH-wildtype GB treated with the standard therapy between 2012 and 2019 were analyzed retrospectively. Cox proportional hazards models and Kaplan–Meier analysis were used to investigate the survival function of preoperative hematological parameters. Results: Preoperative high neutrophil-to-lymphocyte ratio (NLR, >2.42), high platelet count (>236 × 109/L), and low red blood cell (RBC) count (≤4.59 × 1012/L) were independent prognostic factors for poorer OS (p = 0.030, p = 0.030, and p = 0.004, respectively). Moreover, a high NLR was an independent prognostic factor for shorter PFS (p = 0.010). We also found that, like NLR, preoperative high derived NLR (dNLR, >1.89) was of poor prognostic value for both PFS (p = 0.002) and OS (p = 0.033). A significant correlation was observed between NLR and dNLR (r = 0.88, p < 0.001), which had a similar prognostic power for OS (NLR: AUC = 0.58; 95% CI: [0.48; 0.68]; dNLR: AUC = 0.62; 95% CI: [0.51; 0.72]). Two scores, one based on preoperative platelet and RBC counts plus NLR and the other on preoperative platelet and RBC counts plus dNLR, were found to be independent prognostic factors for PFS (p = 0.006 and p = 0.002, respectively) and OS (p < 0.001 for both scores). Conclusion: Cheap, routinely ordered, preoperative assessments of blood markers, such as NLR, dNLR, RBC, and platelet counts, can predict the survival outcomes of patients with IDH-wildtype GB treated with the standard therapy.

Pazopanib is an oral multitargeted tyrozine kinase inhibitor that is used in advanced renal cancer in most countries of the world. Pazopanib inhibits tyrosine kinase receptors involved in tumor angiogenesis and proliferation, including VEGF, platelet-derived growth factor (PDGF) and stem cell growth factor receptor c-Kit, which leads to inhibiting angiogenesis, growth and proliferation of tumor cells. In clinical trials, pazopanib demonstrated improvement of progression-free survival (PFS) versus placebo in previously untreated patients and patients treated with cytokines, as well as the absence of worsening PFS versus sunitinib in the first-line therapy of clear cell RCC in the good- and intermediate prognosis groups. In addition, pazopanib demonstrated a better safety profile than sunitinib. The results of use of pazopanib in broad clinical practice are consistent with data from randomized trials that confirms the high efficacy combined with good tolerability of this drug even in patients who do not meet the generally accepted criteria for the inclusion in clinical trials.

5041 Background: Prognostic factors (PF) for OS have yet to be fully defined for patients with metastatic RCC in the era of VEGF-targeted therapy. This study identifies PFs in this population and updated survival and validation results are presented. Methods: Baseline characteristics and outcomes on anti-VEGF-naïve metastatic RCC patients were collected from three US and four Canadian centers. Using a Cox proportional hazards model, 3 risk categories for predicting survival were identified on the basis of 6 pretreatment clinical features. Results: Six-hundred forty-five patients were included. The median (m) OS was 22 months (95% CI: 20.0–24.8) with a median follow-up of 25 months. Patients were treated with sunitinib (n = 396), sorafenib (n = 200) or bevacizumab (n = 49); 33% had prior immunotherapy. Four of the five PFs previously identified by MSKCC were independent predictors of short survival, including hemoglobin below the lower limit of normal (LLN) (p < 0.0001), corrected calcium above the upper limit of normal (ULN) (p = 0.0006), Karnofsky performance status <80% (p < 0.0001) and time from initial diagnosis to initiation of therapy ULN (pULN (p = 0.012) were independent adverse PFs. Patients were assigned one point for each poor PF and were segregated into three risk categories: favorable-risk (0 PFs, n = 133) median OS (mOS) 37.0 months; intermediate-risk (1 - 2 PFs, n = 292) mOS 28.5 months; and poor-risk (3–6 PFs, n = 139) mOS 9.4 months (log rank p < 0.0001). This model produced a c-index of 0.74 and the bootstrap procedure confirmed good internal validity. The discriminatory ability of the model and its parameter estimates were not affected after adjusting for prior use of immunotherapy or the type of anti-VEGF drug used. Conclusions: These data validate components of the MSKCC model with the addition of platelet and neutrophil counts. This model derived from a large population can be incorporated into patient care and clinical trials of VEGF-targeted agents. [Table: see text]

e16207 Background: The aim of our study was to investigate the prognostic role of the neutrophil to lymphocyte ratio (NLR), derived neutrophil to lymphocyte ratio (dNLR), platelet to lymphocyte ratio (PLR), and lymphocyte to monocyte ratio (LMR) in PFS and OS of patients with neuroendocrine tumors (NETs) of the gastrointestinal tract. Methods: We analyzed medical records of 69 patients with stage I - IVB NETs of the gastrointestinal tract (25 men, 44 women; median age is 57 years [44.75-66.00]). All patients were treated according to the standard protocols from 2015 till 2020. We examined demographics, clinical stage, tumor morphology, and baseline levels of leukocytes, neutrophils, lymphocytes, monocytes, and PLT. We also analyzed the calculated value of NLR, dNLR, PLR and LMR. Results: The ROC analysis made it possible to determine that the factors, that have a significant impact on the PFS indicator are: absolute (cut-off: ≤2.26; p = 0.0274) and relative (cut-off: ≤30; p = 0.0158) lymphocyte count, NLR (cut-off: 1.85; p = 0.0230) and dNLR (cut-off: 1.40; p = 0.0209). The median PFS of patients whose baseline absolute lymphocyte count 2.26x109/l or less was 34.0 months and was significantly (61%) less than the median PFS in the group of patients with an absolute lymphocyte count greater than cut-off value (p = 0.0122; HR = 0.39: 95% CI 0.19-0.81). The median PFS of patients with relative lymphocyte count £30% was 25.0 months and was significantly (61%) less than the median PFS in the group of patients with lymphocyte count > 30% (p = 0.0082; HR = 0.39: 95% CI 0.20-0.79). The median PFS of patients with NLR > 1.85 was 26.0 months and was significantly (60%) less than the median PFS in the group of patients with NLR1≤.85 (p = 0.0095; HR = 0.40: 95% CI 0.21-0.80). The median PFS of patients with dNLR > 1.40 was 20.0 months and was significantly (60%) less than the median PFS in the group of patients with dNLR≤140 (p = 0.0120; HR = 0.40: 95% CI 0.20-0.82). In multivariant analyses, which contained all significance factors by univariate analysis, count of lymphocytes: lymphocytes≤30% significantly increased the risk of disease progression: p = 0.0344; HR = 0.97; 95% CI 0.94-0.99. Conclusions: Systemic inflammatory markers have demonstrated their diagnostic and predictive value in patients with neuroendocrine tumors of the gastrointestinal tract.

e15674 Background: Several factors including pro-angiogenic cytokines have been reported as predictive markers for the treatment effect of sorafenib in patients with HCC; however, many of them were determined based on the results of retrospective studies. Methods: In this prospective cohort study (UMIN000009771), we recruited advanced HCC patients who were treated with sorafenib, and periodically measured serum levels of eight pro-angiogenic cytokines, which were risk factors for the prognosis reported by us and others. The effect of the cytokine expressions before and at 2 (4) weeks after starting sorafenib and at PD, on progression free survival (PFS), overall survival (OS), and post progression survival (PPS) were evaluated. The effect of early appearance of adverse events within 30 days, which were reported as other risk factors, was also evaluated. Results: Eighty patients were enrolled. Median PFS and OS were 3.1 and 11.7 months, respectively. There was no statistically significant variable that could predict PFS, which included patients’ characteristics, cytokines before the therapy, and early onset of adverse events. However, PFS of the patients who showed the decrease of platelet-derived growth factor after 2 weeks (HR2.78, 95%CI 1.35-6.21, P=0.005) or vascular endothelial growth factor after 4 weeks (HR3.31, 95%CI 1.59-6.83, P=0.001) compared to the values before sorafenib treatment was short. OS was short in patients with poor performance status, large tumor size (>30mm), large tumor number (>5), high des-gamma-carboxyprothrombin (DCP, >100mAU/mL), high hepatocyte growth factor, and high angiopoietin-2 (Ang-2) before therapy, in addition to no hand foot syndrome within 30 days. Multivariate analysis with these factors revealed that high Ang-2 (HR2.25, 95%CI 1.08-4.86, p=0.029) and high DCP (HR3.55, 95%CI 1.25-12.7, p=0.014) were the independent risk factors for OS. High Ang-2 at PD could also be a marker of short PPS. Conclusions: The prediction of PFS, OS and PPS is possible by measuring serum pro-angiogenic cytokines at appropriate timing. Ang-2 is a key cytokine for predicting the prognosis of HCC patients treated with sorafenib.

PurposePazopanib is an oral angiogenesis inhibitor targeting vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and c-Kit. This randomized, double-blind, placebo-controlled phase III study evaluated efficacy and safety of pazopanib monotherapy in treatment-naive and cytokine-pretreated patients with advanced renal cell carcinoma (RCC).Patients and MethodsAdult patients with measurable, locally advanced, and/or metastatic RCC were randomly assigned 2:1 to receive oral pazopanib or placebo. The primary end point was progression-free survival (PFS). Secondary end points included overall survival, tumor response rate (Response Evaluation Criteria in Solid Tumors), and safety. Radiographic assessments of tumors were independently reviewed.ResultsOf 435 patients enrolled, 233 were treatment naive (54%) and 202 were cytokine pretreated (46%). PFS was significantly prolonged with pazopanib compared with placebo in the overall study population (median, PFS 9.2 v 4.2 months; hazard ratio [HR], 0.46; 95% CI, 0.34 to 0.62; P < .0001), the treatment-naive subpopulation (median PFS 11.1 v 2.8 months; HR, 0.40; 95% CI, 0.27 to 0.60; P < .0001), and the cytokine-pretreated subpopulation (median PFS, 7.4 v 4.2 months; HR, 0.54; 95% CI, 0.35 to 0.84; P < .001). The objective response rate was 30% with pazopanib compared with 3% with placebo (P < .001). The median duration of response was longer than 1 year. The most common adverse events were diarrhea, hypertension, hair color changes, nausea, anorexia, and vomiting. There was no evidence of clinically important differences in quality of life for pazopanib versus placebo.ConclusionPazopanib demonstrated significant improvement in PFS and tumor response compared with placebo in treatment-naive and cytokine-pretreated patients with advanced and/or metastatic RCC.

PURPOSE Antiangiogenic agents combined with chemotherapy have efficacy in the treatment of unresectable malignant pleural mesothelioma (MPM). Cediranib (AstraZeneca, Cheshire, United Kingdom), a vascular endothelial growth factor receptor and platelet-derived growth factor receptor inhibitor, demonstrated therapeutic potential in a prior phase I trial. We evaluated a phase II trial for efficacy. PATIENTS AND METHODS SWOG S0905 (ClinicalTrials.gov identifier: NCT01064648 ) randomly assigned cediranib or placebo with platinum-pemetrexed for six cycles followed by maintenance cediranib or placebo in unresectable chemotherapy-naïve patients with MPM of any histologic subtype. Primary end point was Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 progression-free survival (PFS). Secondary end points included overall survival, PFS by modified RECIST v1.1, response (modified RECIST and RECIST v1.1), disease control, and safety/toxicity. The trial was designed to detect a difference in RECIST v1.1 PFS at the one-sided 0.1 level using a stratified log-rank test. RESULTS Ninety-two eligible patients were enrolled (75% epithelioid and 25% biphasic or sarcomatoid). The cediranib arm had more grade 3 and 4 diarrhea, dehydration, hypertension, and weight loss. Cediranib improved PFS by RECIST v1.1 (hazard ratio, 0.71; 80% CI, 0.54 to 0.95; P = .062; 7.2 months v 5.6 months) and increased modified RECIST v1.1 response (50% v 20%; P = .006). By modified RECIST v1.1, cediranib numerically increased PFS (hazard ratio, 0.77; 80% CI, 0.59 to 1.02; P = .12; median, 6.9 months v 5.6 months). No significant difference in overall survival was observed. CONCLUSION The addition of cediranib to platinum-pemetrexed improved PFS by RECIST v1.1 and response rate by modified RECIST in patients with unresectable MPM. Whereas adding antiangiogenics to chemotherapy has been a successful strategy for some patients, the cediranib toxicity profile and small incremental survival benefit precludes additional development in MPM.

PurposeThis phase II trial assessed the activity and tolerability of an oral dose of imatinib mesylate 400 mg twice daily in patients with recurrent or persistent epithelial ovarian or primary peritoneal carcinoma. The association between the expression of certain markers and clinical outcome was investigated.Patients and MethodsPrimary measure of clinical efficacy was progression-free survival (PFS) at 6 months. Mutational analysis of KIT, immunohistochemistry (IHC) and enzyme-linked immunosorbent assay for markers (KIT, platelet-derived growth factor [PDGF] receptor [-R], AKT2, phosphorylated AKT [p-AKT], stem cell factor [SCF], and PDGF) were performed.ResultsFifty-six eligible patients were evaluated. Nine patients were progression free for at least 6 months including one complete responder. The median PFS and survival were 2 and 16 months, respectively. The most common grade 3 and 4 toxicities were neutropenia, GI, dermatologic effects, pain, and electrolyte disturbances. At least one target of imatinib (KIT, PDGFR-α, or PDGFR-β) was expressed in all tumors, and most tumors expressed all three receptors. Higher expression of p-AKT and PDGFR-β were associated with shorter PFS, and higher IHC scores (% immunopositive cells × staining intensity) of SCF and p-AKT were associated with decreased overall survival. No sequence mutations were detected in the KIT gene. Higher pretreatment plasma concentrations of PDGF-AB, PDGF-BB, and vascular endothelial growth factor (VEGF) were individually associated with shorter PFS and survival.ConclusionImatinib mesylate was well tolerated but had minimal single-agent activity in patients with recurrent ovarian or primary peritoneal carcinoma. No marker was identified that would predict activity of imatinib; however, tumor p-AKT and plasma VEGF levels were associated with poor outcome.

PurposeGiven the molecular pathophysiology of thyroid cancer and the spectrum of kinases inhibited by sorafenib, including Raf kinase, vascular endothelial growth factor receptors, platelet-derived growth factor receptor, and RET tyrosine kinases, we conducted an open-label phase II trial to determine the efficacy of sorafenib in patients with advanced thyroid carcinoma.Patients and MethodsEligible patients with metastatic, iodine-refractory thyroid carcinoma received sorafenib 400 mg orally twice daily. Responses were measured radiographically every 2 to 3 months. The study end points included response rate, progression-free survival (PFS), and best response by Response Evaluation Criteria in Solid Tumors.ResultsThirty patients were entered onto the study and treated for a minimum of 16 weeks. Seven patients (23%; 95% CI, 0.10 to 0.42) had a partial response lasting 18+ to 84 weeks. Sixteen patients (53%; 95% CI, 0.34 to 0.72) had stable disease lasting 14 to 89+ weeks. Seventeen (95%) of 19 patients for whom serial thyroglobulin levels were available showed a marked and rapid response in thyroglobulin levels with a mean decrease of 70%. The median PFS was 79 weeks. Toxicity was consistent with other sorafenib trials, although a single patient died of liver failure that was likely treatment related.ConclusionSorafenib has clinically relevant antitumor activity in patients with metastatic, iodine-refractory thyroid carcinoma, with an overall clinical benefit rate (partial response + stable disease) of 77%, median PFS of 79 weeks, and an overall acceptable safety profile. These results represent a significant advance over chemotherapy in both response rate and PFS and support further investigation of this agent in these patients.

TPS3636 Background: Colorectal cancer (CRC) is the second leading cause of cancer-related death in western countries. With advances in the treatment of metastatic CRC (mCRC) in the last decade using combination chemotherapy and bevacizumab, a monoclonal antibody against the vascular endothelial growth factor (VEGF), progression free survival (PFS) in first and second line setting was substantially improved. Tumour angiogenesis is also driven by other factors but VEGF including Platelet Derived Growth Factor (PDGF) and Fibroblast Growth Factor (FGF). BIBF1120 is a potent, orally available triple angiokinase inhibitor that blocks the receptor tyrosine kinase activity of human VEGFR1-3, FGFR1 and -3 and PDGFRα and -β. Thus, BIBF1120 could be an exciting addition to the treatment of patients with chemorefractory CRC. Methods: Randomized, double-blind, placebo-controlled, multicentre, phase II trial of BIBF1120 (orally, 200 mg, bid, d1-d14, Arm A) vs. placebo (Arm B) in patients receiving mFOLFOX6 (oxaliplatin, 85 mg/m2, fluorouracil, 400 mg/m2 bolus and 2400 mg/m2 over 46 h, leucovorin, 200 mg/m2) q14 d for patients (ECOG performance status 0-1) with chemorefractory mCRC who received one prior line of chemotherapy. Scheduled are 90 patients per treatment arm. Primary endpoint: PFS, Secondary endpoints: objective response, overall survival, duration of overall response, safety. Additionally there will be an explorative analysis of predictive biomarkers for BIBF1120. 4 of planned 180 patients have been enrolled. We expect the last patient out in June 2013. (Trial identifier: NCT01362361.)

BACKGROUND AND AIMS: Congenital Hepatic Fibrosis (CHF) is caused by mutations in PKHD1, a gene encoding for fibrocystin, a protein of unknown function, expressed in cholangiocyte cilia and centromers. In CHF, biliary dysgenesis is accompanied by severe progressive portal fibrosis and portal hypertension. The mechanisms responsible for portal fibrosis in CHF are unclear. The αvβ6 integrin mediates local activation of TGFβ1 and is expressed by reactive cholangiocytes during cholestasis. To understand the mechanisms of fibrosis in CHF we studied the expression of αvβ6 integrin and its regulation in Pkhd1del4/del4 mice. METHODS: In Pkhd1del4/del4 mice we studied, at different ages (1-12 months): a) portal fibrosis (Sirius Red) and portal hypertension (spleen weight/body weight); b) αvβ6 mRNA and protein expression (RT-PCR, IHC); c) α-SMA and TGFβ1 mRNA expression (RT-PCR); d) portal inflammatory infiltrate (IHC for CD45 and FACS analysis of whole liver infiltrate); f) cytokines secretion from cultured monolayers of primary cholangiocytes (Luminex assay); g) cytokine effects on monocyte/macrophage proliferation (MTS assay) and migration (Boyden chamber); h) TGFβ1 and TNFα effects on β6 integrin mRNA expression by cultured cholangiocytes before and after inhibition of the TGFβ receptor type II (TGFβRII); i) TGFβ1 effects on collagen type I (COLL1) mRNA expression by cultured cholangiocytes. RESULTS: Pkhd1del4/del4 mice showed a progressive increase in αvβ6 integrin expression on biliary cyst epithelia. Expression of αvβ6 correlated with portal fibrosis (r=0.94, p<0.02) and with enrichment of a CD45+ve cell infiltrate in the portal space (r=0.97, p<0.01). Gene expression of TGFβ1 showed a similar age-dependent increase. FACS analysis showed that 50-75% of the CD45+ve cells were macrophages (CD45/CD11b/F4/80+ve). Cultured polarized Pkhd1del4/del4 cholangiocytes secreted from the basolateral side significantly increased amounts of CXCL1 and CXCL10 (p<0.05). Both cytokines were able to stimulate macrophage migration (p<0.05). Basal expression of β6 mRNA by cultured Pkhd1del4/del4 cholangiocytes (0.015±0.002 2^-dCt) was potently stimulated by the macrophage-derived cytokines TGFβ1 (0.017±0.002 2^-dCt, p<0.05) and TNFα (0.018±0.003 2^-dCt, p<0.05). Inhibition of TGFβRII completely blunted TGFβ1 (0.014±0.003 2^-dCt, p<0.05) but not TNFα effects (0.017±0.001 2^-dCt, p=ns) on β6 mRNA. COLL1 mRNA expression by cultured Pkhd1del4/del4 cholangiocytes (0.0009±0.0003 2^-dCt) was further and significantly increased after TGFβ1 stimulation (0.002±0.0005 2^-dCt, p<0.05). CONCLUSIONS: Pkhd1del4/del4 cholangiocytes possess increased basolateral secretory functions of chemokines (CXCL1, CXCL10) able to orchestrate macrophage homing to the peribiliary microenvironment. In turn, by releasing TGFβ1 and TNFα, macrophages up-regulate αvβ6 integrin in Pkhd1del4/del4 cholangiocytes. αvβ6 integrin activates latent TGFβ1, further increasing the fibrogenic properties of cholangiocytes.

Orientador: Marta Helena Krieger
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O óxido nítrico (NO) é um multifuncional agente biológico que nas últimas décadas tem sido alvo de uma infinidade de estudos e constitui hoje um dos mais importantes mediadores de processos intra e extracelulares. Diversos estudos demonstram sua capacidade de prevenção da ativação e adesão plaquetária ou leucocitária e inibição da proliferação e migração de células musculares lisas vasculares (VSMCs), entretanto, em condições de baixa disponibilidade do NO esses processos são prejudicados. Atualmente há o grande interesse no desenvolvimento de compostos capazes de liberar NO de forma modulada e estável, e, nesse sentido, os complexos nitrosilos de rutênio têm se destacado por suas características excepcionais. É amplamente reconhecido que a modulação fenotípica de VSMCs tem papel crítico na progressão de diversas doenças vasculares proeminentes. Sabe-se que o fator de crescimento derivado de plaquetas (PDGF-BB) é um dos principais estimulantes desse processo. Este estudo se propôs a caracterizar os efeitos inibitórios do complexo de rutênio trans-[Ru(NO)Cl(cyclam)](PF6)2, nomeado Ru(cyclam)NO, na modulação fenotípica, resposta proliferativa e migratória de VSMCs induzidas por PDGF-BB. VSMCs foram obtidas por técnica de cultura primária. A citotoxicidade do complexo, na faixa de concentração de 100 ?M a 1500 ?M, foi determinada em ensaios de redução do MTT e incorporação neutral red (NR), e comparadas a do nitroprussiato de sódio (SNP). A concentração 100 ?M foi definida para os demais protocolos experimentais. Western blotting, ensaios transwell e wound healing, e incorporação de timidina triciada foram utilizados para determinação da modulação fenotípica, migração e proliferação celular, respectivamente, e níveis de nitrato no meio foram determinados por quimiluminescência para avaliação do perfil de liberação de NO. O complexo demonstrou baixa citotoxicidade, mesmo na maior concentração e após 48 horas de exposição, reduzindo ao máximo em 30% a porcentagem de células viáveis em ambos os ensaios, demonstrando ser menos tóxico que o SNP. Níveis de nitrato no meio atigiram a concentração máxima após 30 minutos (11 ?M ± 4,8), de maneira mais lenta em relação ao SNP, cuja concentração máxima foi após 5 minutos (13 ?M ± 3,7). A proliferação das VSMCs induzida por PDGF-BB foi inibida, reduzindo à metade a radioatividade incorporada, bem como a expressão do marcador de proliferação PCNA. Observou-se redução de 45% na migração induzida por PDGF-BB nos ensaios transwell, e no wound-healing, embora qualitativo, a redução é notável. A modulação fenotípica da VSMC foi observada pela redução em 60% da expressão da proteína alfa-actina, característica do fenótipo maduro, e foi quase totalmente prevenida pelo tratamento com o complexo. Tal prevenção pode ser mediada pelo fator de transcrição ELK-1, que favorece a expressão de genes de diferenciação, e cuja fosforilação foi estimulada pelo PDGF-BB, porém inibida em quase 50% pelo pré-tratamento com Ru(cyclam)NO. As respostas observadas nos tratamentos com Ru(cyclam)NO foram promissoras, e, embora seu mecanismo de ação x ainda não esteja completamente esclarecido, este complexo demonstrou atividade biológica singular, e sua aplicação em condições clínicas onde há descontrole de processos de proliferação e migração de VSMCs, como a reestenose, apresenta-se como uma proposta interessante
Abstract: Nitric oxide (NO) is a multifuctional biological agent that in the recent decades has been the subject of a plethora of studies and today is one of the most important intracellular and extracellular processes mediators. Several studies have demonstrated its ability to prevent leukocyte or platelet adhesion and activation, and inhibition of vascular smooth muscle cells (VSMCs) proliferation and migration. However, low availability of NO conditions determines impairement of these processes. There is a keen interest in the development of compounds capable of releasing NO modulated so stable, and the nitrosyl ruthenium complexes have gained prominence for its exceptional features. It is widely recognized that the phenotypic modulation of VSMCs, and its uncontrolled proliferation and migration, plays a critical role in the progression of several prominent vascular diseases. It is known that the platelet-derived growth factor (PDGF) is a primary stimulant of the process. This study aimed to determine the inhibitory effects of the ruthenium complex NO donor trans-[Ru(NO)Cl(cyclam)](PF6)2, named Ru(cyclam)NO, in the phenotypic switching, on migratory and proliferative responses of VSMCs induced by PDGF-BB, and its biological response. VSMCs were obtained from primary culture methodology. The complex cytotoxicity were determined by MTT reduction and incorporation of neutral red (NR) assays, in the range of concentration from 100 ?M to 1500 ?M, and compared to sodium nitroprusside (SNP). For the following experimental protocols the concentration of the 100 ?M was set. Western blotting, transwell and wound healing assays, and incorporation of tritiated thymidine were used for determination of phenotypic switching, cell migration and proliferation, respectively. Evaluation of the NO profile release was determined as nitrate levels in the culture medium by chemiluminescence. The complex Ru(cyclam)NO showed low cytotoxicity even at the highest concentration evaluated and after 48 hours of exposition, reducing only 30% the percentage of viable cells in both trials, showing be less toxic than the SNP. Medium nitrate levels exibhited the highest concentration after 30 min (11 ?M ± 4.8), slower when compared to SNP, which reached the maximum concentration after 5 minutes (13 ?M ± 3.7). The proliferation of VSMCs induced by PDGF-BB was inhibited by half of the radioactivity incorporated counting, as well as the reduction on the expression of the proliferation marker PCNA. Observed a reduction by 45% in the migration induced by PDGF-BB determined in transwell assays, and on the wound-healing, although a qualitative result, the reduction of migration induced by PDGF-BB. The 60% reduction by PDGF-BB treatment of the contractile protein expression ?-SMA, characteristic of mature phenotype, revealed the modulation of VSMCs phenotype, and it was almost completely prevented by treatment with the complex. Such prevention was associated with inhibition by almost 50% of phosphorylation of the transcription factor ELK-1 stimulated by PDGF-BB. The responses determined with complex treatments revealed promising for future development of cardiovascular devices. Although its xii mechanism of action is not completely understood, this complex showed singular biological activity, and its application in some clinical conditions where there is uncontrolled proliferation and migration of VSMCs presents as a substancial proposal
Mestrado
Fisiologia
Mestra em Biologia Funcional e Molecular

PDGF which is released during platelet activation like the other ∝ granule components (fibrinogen, F VIII/vWF, PF4) could bind to platelet membrane Following this hypothesis, we have studied the binding of 125I pure human PDGF to washed human platelets activated by collagen. This binding was specific and time dependent and reached a plateau with 20 μg/ml of collagen. With 200 fold excess of unlabeled PDGF, the binding of 125I-PDGF decreased progressively to 10 .whereas unlabeled Epidermal Growth Factor did not compete with 125I-PDGF. Saturation curve and scatchard analysis have shown one class of sites 3,000 sites/cell with an apparent Kd = 10-8 M. The demonstration of PDGF binding to platelets led us to investigate the effects of PDGF on platelet function. PDGF inhibited the aggregation and 14C serotonin release induced by thrombin or collagen. This inhibition was dose dependent and more effective with human PDGF. A total inhibition of collagen-induced platelet aggregation was obtained with 50 ng/ml of human PDGF and 200 ng/ml of porcine PDGF. The aggregation and 14C serotonin release induced by arachidonic acid were not inhibited by PDGF. The metabolism of phosphoinositide was also investigated on washed human platelets prelabeled with 32P orthophosphate. We found that PDGF (200 ng/ml) induced a decrease of 32P associated with phosphatidylinositol 4 biphosphate (72 %) after 3 min, with a parallel increase of 32P-phosphatidylinositol 4 Phosphate (120 %) and 32P-phosphatidylinositol (120 %).In conclusion i) PDGF binds to activated platelets, ii) PDGF inhibits platelet aggregation and secretion, iii) PDGF modifies phosphoinositide metabolism. These results are in favour of a role of PDGF in a negative feed back control of platelet activation.

Platelet (plt) factor 4 (PF4) is an alpha granule protein which can modulate T lymphocyte function. T cells may help regulate megakaryocytopoiesis. Therefore, we hypothesized that T cell-PF4 interactions might play a role in autoregulating marrow megakaryocyte (MEG) production. To test this idea, we studied MEG colony formation in plasma clot cultures containing human serum derived solely from pit poor normal AB plasma, enriched hematopoietic progenitor cells (HPC), autologous T cells, and exogenous PF4. Highly purified PF4 (single band on SDS gel) was prepared from outdated human pits by a combination of heparin-agarose, Sephacryl G-200, and Sephadex G-50 column chromatography. HPC were prepared by depleting normal light density marrow mononuclear cells of adherent monocytes, and T cells. T cells were further fractionated into helper (Leu 3+) and suppressor (Leu 2+) subtypes by solid phase immunoabsorption ("panning"). MEG colonies were enumerated by indirect immunofluorescence with an anti-human platelet glycoprotein antiserum. HPC(5×105/ml) were co-cultured with Leu 3+, or Leu 2+ T cells at target;T cell ratios of 2:1 (n=3; n=4 respectively) and l:l(n=4; n=4 respectively) in the presence of 2.5 μg/ml PF4. Under these growth conditions, MEG colony formation was unchanged (p>0.5) when compared to colonies formed by HPC in the absence of PF4. When the above experiments were repeated (n=2-3/condition) at a higher PF4 concentration [25 μg/ml], MEG colony formation was markedly (>60%) inhibited. To determine if PF4 directly inhibited MEG or erythroid progenitor cell growth (CFU-Meg; CFU-E) in vitro, HPC were cloned in PF4 (25μg/ml) without added T cells. Mean ± SEM of MEG and CFU-E derived colonies formed without vs. with PF4 was as follows:These results suggest that: 1) PF4 may be a non-T cell dependent, lineage specific inhibitor of CFU-MEG, and 2) PF4 may play a role in autoregulating human megakaryocytopoiesis.

We studied on the effect of PAI derived from human platelets on two different kinds of t-PAs; one was purchased from BioPool, Sweden, (human uterus or melanoma cell derived one-chain t-PA: one-chain u-mt-PA) and another was kindly supplied from Sumitomo Pharm. Co., Osaka, (recombinant two-chain t-PA: two-chain rt-PA). As the PAI-rich solution derived from platelets. We used the platelets extract, which was prepared as follows: The concentrated platelet-rich plasma (Red Cross Japan) was washed with phosphate buffer containing 0.4 % Triton X-100. The inhibitory activities of the PAI were measured by the method of parabolic rate assay and were calcurated from the activities remained in the mixtures of PAI and the t-PAs. The reactions of the PAI and the t-PAs were also analysed by the method of fibrin autography. The PAI suppressed the activity of two-chain rt-PA completely within 5 minutes, but could not suppressed that of one-chain u-mt-PA completely within such a short incubation time. These facts also recognized on fibrin autography. The molecular weights of both free t-PAs were recognized at 60 kDa and that of the complex of the PAI and two-chain rt-PA was recognized at 110 kDa, respectively. The fibrinolytic zone of the complex of PAI and one-chain u-mt-PA, however, could not be recognized. This indicates two possibilities; one is too small amounts of complex forming to detect and another is that SDS can not reveal the t-PA activity from the complex of PAI and one-chain u-mt-PA on fibrin autography. It remained unclear that the difference of the reactions of these t-PAs to PAI had been induced from whether the difference between one- and two-chain, or that between rt-PA and u-mt-PA. If the former is reasonable, it can be said that the molecular form of t-PA has an important significance in physiological fibrinolytic mechanism.