Dissertations / Theses on the topic 'Platelet factor 4'

Aim: Platelets contribute to chronic inflammation but their role in periodontitis is not well understood. The aim of this study was to compare platelet recruitment and activation in healthy and inflamed periodontium. Materials and Methods: Gingival crevicular fluid (GCF) samples were obtained from sites of healthy periodontium, gingivitis and periodontitis. Platelets were quantified in the GCF by staining and microscopy. GCF concentrations of platelet factor 4 (PF4) [PF4]GCF and glycoprotein IIbIIIa ([GPIIbIIIa]GCF) were determined by ELISA. Blood samples were obtained from the 3 patient groups. Platelets were isolated from whole blood and stimulated with lipopolysaccharide (LPS) from P. gingivalis to evaluate and compare the LPS-induced PF4 release. Results: Compared to controls, platelet recruitment was increased at gingivitis and periodontitis sites, based on platelet counts and [GPIIbIIIa]GCF. [PF4]GCF was elevated in periodontal pockets but not at gingivitis or healthy sites. Circulating plasma levels of PF4 were higher in patients with generalized severe periodontitis (SP), compared to patients with gingivitis or healthy periodontium. Platelets isolated from SP patients contained and released more PF4 in response to P. gingivalis LPS than platelets from gingivitis or periodontally healthy patients. Conclusions: Periodontitis is associated with increased platelet activation and PF4 release, both locally and systemically.
Dentistry, Faculty of
Graduate

Background and Objective: Periodontitis is a highly prevalent chronic inflammatory disease that causes tooth loss, morbidity and confers an increased risk for systemic disease. Tissue destruction during periodontitis is due in large part to collagen-degrading matrix metalloproteinases (MMPs) released by resident cells of the periodontium in response to pro-inflammatory cytokines. Platelets are immune-competent blood cells with a newly recognized role in chronic inflammation, however their role in the pathogenesis of periodontitis is undefined. Consequently, the objective of this study was to assess the effect of platelet factor 4 (PF4), a major platelet-derived cytokine, on MMP-1 (collagenase) expression in human gingival fibroblasts (HGFs). Methods: HGFs were cultured in the presence or absence of recombinant PF4. Pro-MMP-1 secretion was quantified by enzyme-linked immunosorbent assay (ELISA) analysis of the cell culture supernatants. MMP-1 transcription was quantified by real-time polymerase chain reaction (qPCR). Regulation of MMP-1 production by the p44/42 MAP kinase (MAPK) signaling pathway was examined in the presence or absence of PF4. Results: Exposure to PF4 caused a ~2-3-fold increase in MMP-1 transcription and secretion from cultured human gingival fibroblasts (HGFs). PF4 treatment also enhanced phosphorylation of p44/42 MAP kinase (MAPK), which has been previously shown to induce MMP-1 expression in fibroblasts. Blockade of p44/42 MAPK signaling with the cell-permeant inhibitors PD98059 and PD184352 abrogated PF4-induced pro-MMP-1 transcription upregulation and release from cultured HGFs. Conclusion: We conclude that platelet factor 4 upregulates MMP-1 expression in human gingival fibroblasts in a p44/42 MAPK-dependent manner. These findings point to a previously unidentified role for platelets in the pathogenesis of periodontal diseases.
Dentistry, Faculty of
Graduate

本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである
Kyoto University (京都大学)
0048
新制・課程博士
医学博士
甲第4772号
医博第1273号
新制||医||500(附属図書館)
UT51-91-E143
京都大学大学院医学研究科内科系専攻
(主査)教授 三河 春樹, 教授 泉 孝英, 教授 大島 駿作
学位規則第5条第1項該当

Immune heparin-induced thrombocytopenia (HIT) is a potentially serious complication of heparin therapy and is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. Early diagnosis of HIT is important to reduce morbidity and mortality. I developed an enzyme immunoassay that detects the binding of HIT IgG to PF4-heparin in the fluid phase. This required techniques to purify and biotinylate PF4. The fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. I was able to detect anti-PF4-heparin IgG in 93% of HIT patients. I also investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 88% of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration. While HIT patients possess antibodies to PF4-heparin, I observed that HIT antibodies will also bind to PF4 alone adsorbed on polystyrene ELISA wells but not to soluble PF4 in the absence of heparin. Having developed a technique to affinity-purify anti-PF4-heparin HIT IgG, I provide the first estimates of the avidity of HIT IgG. HIT IgG displayed relatively high functional affinity for both PF4-heparin (Kd=7-30nM) and polystyrene adsorbed PF4 alone (Kd=20-70nM). Furthermore, agarose beads coated with PF4 alone were almost as effective as beads coated with PF4 plus heparin in depleting HIT plasmas of anti-PF4-heparin antibodies. I conclude that the HIT antibodies which bind to polystyrene adsorbed PF4 without heparin are largely the same IgG molecules that bind PF4-heparin and thus most HIT antibodies bind epitope(s) on PF4 and not epitope(s) formed by part of a PF4 molecule and part of a heparin molecule. Binding of PF4 to heparin (optimal) or polystyrene/agarose (sub-optimal) promotes recognition of this epitope. Under conditions that are more physiological and sensitive than previous studies, I observed that affinity-purified HIT IgG will cause platelet aggregation upon the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. I quantitated the binding of affinity-purified HIT 125I-IgG to platelets as they activate in a plasma milieu. Binding of the HIT IgG was dependent upon heparin and some degree of platelet activation. Blocking the platelet Fc??? receptor-II with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. I conclude that anti-PF4-heparin IgG is the only component specific to HIT plasma that is required to induce platelet aggregation. The Fab region of HIT IgG binds to PF4-heparin that is on the surface of activated platelets. I propose that only then does the Fc portion of the bound IgG activate other platelets via the Fc receptor. My data support a dynamic model of platelet activation where released PF4 enhances further antibody binding and more release.

Le facteur 4 plaquettaire (PF4/CXCL4) est un tétramère constitué de quatre sous-unités identiques de 7,8 kDa qui est libéré en grande quantité par les plaquettes lors de l’hémostase primaire (ensemble des phénomènes permettant un colmatage initial d’une lésion vasculaire). L’étude de la formation d’un caillot de fibrine en présence de PF4 montre une augmentation de la turbidité finale du caillot : le PF4 modifie le réseau formé. Etant donné que la plupart des acteurs de la fibrinolyse se lie au caillot de fibrine et que le PF4 modifie sa structure, nous avons pensé qu’il serait intéressant de rechercher si le PF4 influençait aussi la fibrinolyse. La lyse d'un caillot est effectuée par la plasmine issue de l'activation du plasminogène par son activateur d’origine tissulaire (t-PA) en présence d’un cofacteur qui n'est autre que la fibrine. Nous avons étudié la lyse de caillots de plasma, obtenus par activation de la cascade de la coagulation, en condition statique et à l'aide d'un modèle de thrombose artérielle (système Chandler loop). Dans les deux cas, une diminution du temps de demi-lyse a été observée en présence de PF4. Cependant, la lyse de caillots préparés par simple ajout de thrombine sur du fibrinogène ne permet pas de retrouver cet effet du PF4. Ceci suggère que l’influence du PF4 sur la structure du caillot n’est pas à l’origine de l’effet sur sa lyse et que le PF4 n’influence pas (ou très peu) l'activation du plasminogène, ainsi que l'activité de la plasmine résultante. Cette hypothèse a été confirmée par l’étude de l’activité amydolytique du t-PA et de la plasmine (quelle soit ajoutée ou générée). En système purifié, les inhibiteurs plasmatiques de la fibrinolyse sont absents. Les deux principaux sont l'inhibiteur de l'activateur du plasminogène de type 1 (PAI-1) et l’α2-antiplasmine (α2-AP). La lyse de caillots préparés à partir de plasma déficient en α2-AP montre une diminution du temps de demi-lyse en présence de PF4 (comme pour le plasma normal), alors qu’avec le plasma dépourvu de PAI-1 le temps de demi-lyse n'est plus influencé. De plus, l’ajout de PAI-1 dans le système purifié entraine une diminution du temps de demi-lyse en présence de PF4. Ceci suggère que le PF4 prévient directement ou indirectement l'inhibition du t-PA par PAI-1. L’étude de la cinétique d'inhibition de l’activité amidolytique du t-PA par le PAI-1, la détermination de la stœchiométrie de cette inhibition, et l’analyse de ces cinétiques par immuno-empreinte montrent que le PF4 est un modulateur de la fibrinolyse qui agit en retardant la formation d'un complexe initial entre le t-PA et le PAI-1. Cette nouvelle fonction du PF4 est cohérente, et vient en complément de celle décrite récemment d’inhibiteur de l'activation du TAFI
Platelet factor 4 (PF4/CXCL4) is a tetramer constituted of four identical 7,8 kDa subunits released in large quantities by platelets during primary heamostasis (allowing initial clogging of a vascular injury). Study of fibrin clot formation in the presence of PF4 shows an increase of the final clot turbidity: PF4 modifies the formed network. Given that most fibrinolysis actors are bound to the fibrin clot and that PF4 modifies its structure we thought it would be interesting to investigate if PF4 also influences fibrinolysis. Clot lysis is performed by plasmin originating from activation of its precursor by tissue plasminogen activator (t-PA) with fibrin itself as cofactor of the reaction. We have studied lysis of plasma clots formed by activation of the coagulation cascade in static condition and in a Chandler loop model mimicking arterial thrombosis. Half-times of lysis decreased in the presence of PF4 in both systems. However, PF4 had no longer detectable influence on the half-time of lysis with clots formed by direct addition of thrombin on purified fibrinogen. Observation suggested that the observed decrease of the half-time of lysis induced by PF4 did not originate from its influence on fibrin clot formation and that PF4 had little effect if any on plasminogen activation or plasmin activity. We confirmed this hypothesis by comparing amydolytic activities of t-PA and plasmin (added or generated through plasminogen activation). In purified system, fibrinolysis inhibitors are absent. The two main inhibitors are plasminogen activator inhibitor-1 (PAI-1) and α2-antiplasmin (α2-AP). Lysis of clots obtained from α2-AP deficient plasma showed a decrease of the half-time of lysis in the presence of PF4 (as in normal plasma), whereas in PAI-1 deficient plasma half-time of lysis was unchanged. Moreover if PAI-1 was added to the purified system, half-time of lysis decreased in the presence of PF4. Observations therefore suggested that PF4 prevented directly or indirectly t-PA inhibition by PAI-1. Kinetics of the amidolytic activity of t-PA inhibition by PAI-1 in the presence or not of PF4, determination of its stoichiometry and Western blot analysis of these inhibition kinetics revealed that PF4 is a fibrinolysis modulator which delays formation of the initial (Michaelis) complex between t-PA and PAI-1. This new feature of PF4 is consistent and complementary with its recently described role as a modulator of TAFI activation

Que o envenenamento pela serpente Bothrops jararaca causa distúrbios hemorrágicos sistêmicos, com alteração da coagulação e fibrinólise sangüíneas, é notório. Contudo, pouco se sabe sobre a ação in vivo desse veneno sobre as plaquetas. Em estudos recentes, demonstrou-se que esse veneno causa trombocitopenia, distúrbios da agregação e diminuição do número de corpos densos plaquetários, que, dessarte, sugeriam a ativação das plaquetas circulantes. Com o escopo de comprovar esta hipótese e melhor caracterizar as ações in vivo desse veneno sobre as plaquetas, serviu-se de um modelo experimental que empregava coelhos para o envenenamento pela B. jararaca. No grupo experimental, os animais foram injetados i.v. com o veneno da B. jararaca (60 µg/kg) e no grupo controle com salina. Previamente à administração de salina ou veneno, os coelhos tiveram suas plaquetas marcadas ex vivo com NHS-biotina. Para a avaliação das alterações plaquetárias, amostras de sangue foram coletadas seqüencialmente, em intervalos de tempo que variaram de 1 a 144 horas após a administração do veneno ou salina. Durante o envenenamento, houve trombocitopenia, hipofibrinogenemia, elevação dos níveis plasmáticos do fator de von Willebrand, diminuição da função plaquetária no sangue total induzida pela botrocetina e pelo colágeno e diminuição da secreção de ATP. Não obstante, os níveis plasmáticos de fator plaquetário 4, um marcador específico da ativação plaquetária in vivo, e os níveis intraplaquetários de serotonina se mantiveram constantes. Pela citometria de fluxo, observou-se um decréscimo significativo da expressão do epítopo da GPIIb-IIIa reconhecido pelo anticorpo monoclonal P2, porém isso não foi observado ao utilizar-se anticorpos policlonais. A expressão de fibrinogênio ou dos produtos de degradação do fibrinogênio/fibrina (PDF) na membrana plaquetária também não sofreu alteração significativa ao longo do tempo. Houve, todavia, elevações significativas da P-selectina plaquetária, um receptor cuja expressão é indicativa de ativação plaquetária, e do epítopo induzido por ligantes (LIBS1) da GPIIIa. A porcentagem de plaquetas reticuladas na circulação, assim como os tempos de sobrevivência plaquetária, não foram estatisticamente diferentes entre os dois grupos. As análises histológicas e imuno-histoquímicas dos órgãos dos coelhos mostraram que as plaquetas circulantes são retidas entre redes de fibrina nos capilares pulmonares. Os resultados obtidos sugerem que a trombina engendrada pelos componentes pró-coagulantes deste veneno desempenha uma função essencial na patogenia dos distúrbios da coagulação e plaquetários observados neste modelo de envenenamento. O aumento da expressão de P-selectina no grupo experimental comprovou a hipótese inicial de que as plaquetas dos coelhos envenenados são verdadeiramente ativadas na circulação. Os dados ora apresentados demonstram definitivamente que a diminuição do fibrinogênio ou o aumento dos PDF não são a causa fundamental da disfunção plaquetária observada no envenenamento botrópico e que outro(s) composto(s) parece(m) estar envolvido(s) com estes distúrbios plaquetários.
In spite of being well established that Bothrops jararaca snake venom causes blood coagulation and fibrinolysis disturbances in patients, scant information about blood platelet disorders during envenomation is available. In recent investigations, thrombocytopenia, platelet aggregation disturbances and decreased numbers of platelet dense bodies were observed following venom administration, suggesting that circulating platelets had been activated. In order to prove this hypothesis and to gain a better characterization of the in vivo role of this venom on platelets, an experimental model of B. jararaca envenomation was utilized. Rabbits were injected i.v. either with B. jararaca venom (60 µg/kg) (experimental group) or saline (control group). Previously to saline or venom administration, rabbit platelets were labeled ex vivo with NHS-biotin. To evaluate platelet disturbances, blood samples were collected consecutively, at time intervals that varied from 1 to 144 hours after venom or saline administration. During envenomation, there were thrombocytopenia, hypofibrinogenemia, elevation of von Willebrand factor plasma levels, reduced botrocetin- and collagen-induced platelet aggregation in whole blood, and decreased ATP secretion. However, plasma levels of platelet factor 4, a specific marker of in vivo platelet activation, and intraplatelet serotonin levels remained constant. By flow cytometry, a significant decrease on the expression of GPIIb-IIIa epitope recognized by P2 monoclonal antibody was observed; however, this was not observed when polyclonal antibodies were employed. Fibrinogen or fibrin(ogen) degradation product (FDP) expression on platelet surface showed no significant alteration. Nonetheless, significant elevations of platelet P-selectin, a receptor whose expression is indicative of platelet activation, and of ligand-induced binding sites (LIBS1) of GPIIIa were noted. The percentage of circulating reticulated platelets, as well as platelet survival times, were not statistically different between the two groups. Histopathological and immunohistochemical analyses of rabbit organs demonstrated that circulating platelets were sequestered among fibrin deposits in pulmonary capillaries. These results suggest that thrombin generated by procoagulating components of B. jararaca venom has an essential role in the pathogenesis of platelet and coagulation disorders in this experimental model. Increased expression of P-selectin in the experimental group proves the initial hypothesis that platelets of envenomed rabbits are indeed activated in the circulation. The data presented herein demonstrate definitively that decreased fibrinogen or increased FDP levels are not the primary cause of the platelet dysfunction observed in bothropic envenomation, but other substances seem to be responsible for it.

Malaria is an infectious disease caused by Plasmodium parasites, transmitted by the female Anopheles mosquito. Malaria can cause mild symptoms as well as more severe complications which may lead to death. Annually, there are half a million deaths worldwide with millions more newly infected with malaria. Although antimalarials exist, due to the prevalence of drug resistance, novel, more effective treatments are needed to combat malaria by aiding the host immune response. One of the earliest signs of a malarial infection is a decrease in the concentration of platelets in the blood of infected individuals -clinically referred to as thrombocytopenia. Platelets have been shown to play a protective role during a malaria infection by targeting the parasite vacuole via platelet factor 4 (PF4). PF4 is only released from activated platelets upon contact with infected red blood cells (iRBCs), and not uninfected red blood cells (uRBCs). The molecules responsible for platelet activation and subsequent release of PF4 from iRBCs were to be determined in this thesis. Platelet activation via infected lysate from trophozoite stage parasites was originally observed but not consistent. Purified trophozoites were unable to activate platelets hence the molecule of interest was unable to be determined. The second part of this thesis was to measure the contribution of platelets binding to RBCs in thrombocytopenia. It has been previously shown that platelets bind preferentially to iRBCs compared with uRBCs. No definite mechanism of thrombocytopenia has been elucidated to date, although a number, such as splenic pooling and endothelial activation, have been thought to play a role. In this thesis, it was hypothesised that platelet binding and subsequent clearance may be responsible for malaria-induced thrombocytopenia. Before the onset of thrombocytopenia, preferential binding of platelets to iRBCs was observed and although platelets were cleared more quickly in infected mice, the contribution of platelet bound RBCs was unable to be definitely identified.

多發性骨髓瘤(Multiple myeloma) 為骨髓內漿細胞異常增生的惡性腫瘤，到目前為止仍然難以治癒。其發生發展是一個複雜的多步驟事件，涉及腫瘤細胞中遺傳和表觀遺傳的改變，以及骨髓微環境的支持。現已確定骨髓瘤細胞和骨髓微環境之間的相互作用對於骨髓瘤的病理發生，以及骨髓瘤細胞的生長，遷移和抗藥性起著關鍵作用。血小根因子四(Platelet factor 4, or PF4) 是一種抗血管生成的趨化因子。它不僅在體外抑制血管內皮細胞增殖和遷移，而且在體內抑制腫瘤的生長。此前，我們發現PF4 基因在多發性骨髓瘤中等位缺失以及DNA 高度甲基化，因而導致其在骨髓瘤病人及細胞系中的表達缺失或降低。在本研究中，我們利用體內和體外實驗鑒定了PF4 對骨髓瘤細胞以及血管生成的作用，並闡明了其作用機制。
首先，我們在體外鑒定了PF4 在骨髓瘤細胞中的功能。我們發現PF4 抑制骨髓瘤細胞系以及從病人骨髓中分離出來的骨髓瘤細胞的生長，以及促進其凋亡。其促凋亡活性與caspase-3 和PARP 的激活有關。我們也檢測了PF4 在骨髓瘤中對血管生成的作用。我們首先分離了病人骨髓中的內皮細胞。結果顯示PF4抑制骨髓瘤內皮細胞的生長和管狀物的形成。這些結果證明PF4 在骨髓瘤中可能是一個抑癌因子。
接下來我們進一步檢測了PF4 在體內的抑癌功能。在第一種模型中，骨髓瘤細胞被皮下移植到重症聯合兔疫缺陷型(NOD-SCID) 小鼠中。尾靜脈注射200ngPF4 明顯的抑制了腫瘤的生長，並延長了小鼠的成活率。第二種小鼠模型稱為兔鼠融合模型(SCID-rab model) 。在這一模型中，大白兔的腿骨先被皮下移植到(NOD-SCID) 小鼠中，再將骨髓瘤細胞注射入已植入的大白兔腿骨的骨腔中。兩周後，小鼠被尾靜脈注射入20 或200ng PF4 。結果顯示200ng PF4 顯著抑制了腫瘤的生長。通過兔疫組化分析大白兔腿骨切片，我們進一步證明了PF4 在腫瘤細胞中的增瘟，凋亡以及血管生成的作用。我們的發現因此證實了PF4 是骨髓瘤中的一個抑制因子。
為了鑒定PF4 在骨髓瘤中的作用機制，我們用Protein/DNA 微陣列(Protein/DNA array) 分析了PF4 參與的信號通路。結果顯示PF4 調節了若干個轉錄因子，其中包括STAT3 。凝膠遷移(EMSA) 和螢光素酪報告基因(luciferase reporter assay )檢測進一步證實PF4 抑制了STAT3 的DNA 結合能力以及轉錄活性。因此PF4 可能通過抑制STAT3 信號通路而抑制骨髓瘤的生長。我們進一步發現PF4 能抑制組成性的以及自介素6 (IL-6) 誘導的STAT3的激活。我們發現PF4 下調了STAT3 下游的靶基因，包括Mc1-1， Survivin 以及血管內皮細胞生長因子(VEGF)。而過表達組成性激活的STAT3 能逆轉PF4 所誘導的細胞凋亡。在兔鼠敵合模型中，通過兔疫組化分析大白兔腿骨切片，我們發現PF4 能抑制STAT3 的入核。SOCS3 是STAT3 其中的一個抑制因子，我們發現PF4 能誘導SOCS3 的表達。而干擾掉SOCS3 能使PF4 喪失其抑制STAT3 激活的能力。這些結果表明PF4 可能通過誘導SOCS3 的表達，從而抑制STAT3 信號通路，引起骨髓瘤的生長抑制以及抗血管生成。
總而言之，本研究表明PF4 是骨髓髓中一個重要調節因子。在體外和體內，PF4 通過抑制STAT3 信號通路，從而抑制腫瘤細胞的生長，促進凋亡以及抑制血管生成。本文為PF4 的臨床研究，作為一種新的治療骨髓瘤藥物，提高骨髓瘤病人的治療效果提供基礎。
Multiple myeloma (MM) is an incurable hematological malignancy characterized by accumulation of clonal plasma cells in bone marrow (BM). The development and progression of MM is a complex multistep tumorigenic event involving both genetic and epigenetic changes in the tumor cell as well as the support by the BM microenvironment. It has been well established that the physical interaction of MM cells with the BM milieu are crucial for MM pathogenesis, MM cell growth, survival, migration and drug resistance. Platelet factor 4 (PF4), a potent antiangiogenic chemokine, not only inhibits endothelial cell proliferation and migration in vitro but also solid tumor growth in vivo. Our group previously demonstrated loss of PF4 expression in patient MM samples and MM cell lines due to concurrent allelic loss and DNA hypermethylation. In this study, we characterized the effects of PF4 on MM cells and angiogenesis in the BM milieu both in vitro and in vivo and elucidated the mechanism of PF4 effects on MM.
To characterize the effects of PF4 on MM cells in vitro, assays on cell growth, cell cycle arrest and apoptosis were performed and we found that PF4 inhibited growth and induced apoptosis in both MM cell lines and MM cells from patients. The proapoptotic activity of PF4 is associated with activation of caspase-3 and poly (ADP) ribose polymerase (PARP). We also investigated the effects of PF4 on angiogenesis in MM using endothelial cells isolated from patient's BM aspirates (MMECs). Our results showed that PF4 suppressed MMECs growth and tube formation on matrigel in a dose-dependent manner.
Given the ability of PF4 to suppress MM cell growth and angiogenesis in vitro, we evaluated its tumor suppressive function in vivo. In human subcutaneously matrigel xenograft mouse model, tail vein injection of 200ng PF4 significantly reduced MM tumor growth and prolonged survival. We next used the SCID-rab mouse model which recapitulates the human BM milieu in vivo. In this model, MM cells were directly injected into the rabbit bone which was subcutaneously implanted into the NOD-SCID mice. Two weeks after injection, SCID mice were treated with various dose of PF4 (20 or 200ng per injection, three times per week) or PBS by tail vein injection. ELISA assay for hIg (lambda) showed that tumor growth in 200ng PF4-treated mice was markedly reduced by 58% compared with the control group, which was further confirmed by immunohistochemistry analysis of CD 138 staining on rabbit bone section. Consistent with the in vitro results, induction of apoptosis in MM cells and inhibition of angiogenesis by PF4 could also be demonstrated in vivo, as evidenced by the findings on ki67, Cleaved caspase-3, CD31 and VEGF staining on rabbit bone sections from treated versus control mice. Our findings thus confirmed that PF4 is a novel tumor suppressor in MM.
However, the molecular mechanism of how PF4 inhibits MM tumorigenesis is still unclear. To identify the signal pathway PF4 involved in MM, Protein/DNA array was performed. We found that PF4 regulated several transcription factors including STAT3 in U266 cells. EMSA and luciferase reporter assay further confirmed that PF4 suppressed STAT3 DNA binding and transcriptional activity. So it is possible that PF4 mediates its tumor suppressive function, through suppressing STAT3 pathway in MM cells. We further found that pre-treatment of PF4 blocked both constitutive and interleukin-6-induced STAT3 activation in a time-dependent manner in human MM cells. PF4 could also down-regulate the STAT3-regulated gene products including Mcl-I, Survivin and vascular endothelial growth factor (VEGF). Moreover, enforced expression of constitutively active STAT3 rescued cells from PF4-induced apoptosis. In SCID-rab mouse model, we also found that PF4 inhibited STAT3 nuclear translocation by immunostaining of rabbit bone sections. When examined further, we found that PF4 induced the expression of one of the STAT3 inhibitor SOCS3, and gene silencing of SOCS3 by small interfering RNA abolished the ability of PF4 to inhibit STAT3 activation, suggesting a critical role of SOCS3 in the action of PF4. Our findings therefore suggest that by inducing SOCS3 expression, PF4 abrogates STAT3 activity, thus induces tumor growth inhibition and anti-angiogenesis.
Together, these novel studies have shown that PF4 is an important regulator of MM tumorigenesis. By abrogating STAT3 signaling it targets cell growth, induces apoptosis, suppresses angiogenesis both in vitro and in vivo in MM. These scientific observations provide the framework for clinical studies of this chemokine, as a novel drug for treatment of MM to improve patient outcome in MM.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Liang, Pei.
"November 2011."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 139-161).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
Abstract in English --- p.I
Abstract in Chinese --- p.IV
List of Publications --- p.VI
Acknowledgements --- p.VII
List of abbreviations --- p.IX
List of Tables --- p.XII
List of Figures --- p.xm
Table of Contents --- p.XV
Chapter Chapter1 --- Introduction and Literature Review --- p.1
Chapter 1.1 --- Multiple myeloma-General description --- p.1
Chapter 1.1.1 --- Epidemiology of MM --- p.1
Chapter 1.1.2 --- Stages of MM --- p.1
Chapter 1.2 --- The bone marrow (BM) microenvironment in MM --- p.3
Chapter 1.3 --- Signal pathways in MM cells --- p.5
Chapter 1.3.1 --- JAK/STAT3 in cancers and MM --- p.5
Chapter 1.3.1.1 --- IL-6 and its receptor --- p.7
Chapter 1.3.1.2 --- Activation of downstream signals-The "on" signals --- p.9
Chapter 1.3.1.3 --- Inactivation of downstream signaling --- p.11
Chapter 1.3.1.3.1 --- Phosphatases --- p.12
Chapter 1.3.1.3.2 --- SOCS family --- p.13
Chapter 1.3.1.3.3 --- The PIAS family --- p.14
Chapter 1.3.2. --- NF-κB pathway --- p.15
Chapter 1.3.3 --- RAS-MAPK pathway --- p.17
Chapter 1.3.4 --- Phosphatidyl inositol-3 kinase (PI3K)/AKT --- p.18
Chapter 1.4 --- Angiogenesis in MM --- p.18
Chapter 1.4.1 --- The process of angiogenesis --- p.18
Chapter 1.4.2 --- Angiogenesis in caner --- p.20
Chapter 1.4.3 --- Angiogenesis in MM --- p.22
Chapter 1.5 --- Animal models in MM --- p.24
Chapter 1.6 --- Treatment of MM --- p.27
Chapter 1.6.1 --- Chemotherapy --- p.27
Chapter 1.6.2 --- Autologous stem cell transplantation --- p.28
Chapter 1.6.3 --- Biologically based therapies --- p.28
Chapter 1.7 --- Platelet factor 4 (PF4) --- p.30
Chapter 1.8 --- Structure of PF 4 --- p.30
Chapter 1.9 --- Role of PF4 in physiological process --- p.32
Chapter 1.9.1 --- Inhibition of megakaryocytopoiesis --- p.32
Chapter 1.9.2 --- PF4 and coagulation --- p.33
Chapter 1.10 --- Role of PF4 in pathological process --- p.34
Chapter 1.10.1 --- PF4 and cancer --- p.34
Chapter 1.10.2 --- PF4 is an angiogenic inhibitor --- p.35
Chapter 1.11 --- Clinical applications of PF4 --- p.37
Chapter 1.12 --- Summary and project aims --- p.37
Chapter Chapter 2 --- Materials and Methods --- p.40
Chapter 2.1 --- Reagents and antibodies --- p.40
Chapter 2.2 --- MM Cell lines --- p.40
Chapter 2.3 --- CD138⁺ primary MM cells --- p.41
Chapter 2.4 --- CD31⁺ MM endothelial cells (MMECs) --- p.42
Chapter 2.5 --- WST-1 assay --- p.43
Chapter 2.6 --- Trypan blue exclusion --- p.43
Chapter 2.7 --- Cell cycle analysis --- p.44
Chapter 2.8 --- Apoptosis analysis --- p.44
Chapter 2.9 --- In vitro tube formation assay --- p.45
Chapter 2.10 --- SCID-rab mice model --- p.45
Chapter 2.10.1 --- Construction of SCID-rab mice --- p.45
Chapter 2.10.2 --- Establishment and monitoring of myeloma in SCID-rab mice --- p.46
Chapter 2.10.3 --- Enzyme-linked immunosorbent assay (ELISA) --- p.46
Chapter 2.10.4 --- PF4 treatment --- p.47
Chapter 2.10.5 --- Immunohistochemistry --- p.48
Chapter 2.11 --- Protein/DNA arrays --- p.49
Chapter 2.12 --- Electrophoretic mobility shift assay (EMSA) --- p.50
Chapter 2.13 --- Luciferase reporter assay --- p.52
Chapter 2.14 --- Western blotting --- p.53
Chapter 2.15 --- RNA extraction --- p.54
Chapter 2.16 --- Real-time Polymerase Chain Reaction (Real-time PCR) --- p.54
Chapter 2.17 --- Nuclear transfection --- p.55
Chapter 2.18 --- Statistical analysis --- p.55
Chapter Chapter3 --- The role of PF4 in MM: in vitro studies --- p.58
Chapter 3.1 --- Results --- p.58
Chapter 3.1.1 --- PF4 inhibited growth of human MM cell lines --- p.58
Chapter 3.1.2 --- PF4 did not cause cell cycle arrest --- p.59
Chapter 3.1.3 --- PF4 induced apoptosis of myeloma cell lines --- p.63
Chapter 3.1.4 --- PF4 caused cell apoptosis in primary MM cells cultured in vitro --- p.64
Chapter 3.1.5 --- PF4 suppressed MMECs growth --- p.69
Chapter 3.1.6 --- PF4 suppressed MMECs tube formation --- p.69
Chapter 3.2 --- Discussion --- p.73
Chapter 3.2.1 --- Negative regulation of PF4 in MM cells growth in vitro --- p.73
Chapter 3.2.2 --- PF4 induces apoptosis in MM cell lines and primary MM cells --- p.74
Chapter 3.2.3 --- PF4 inhibits angiogenesis in MM in vitro --- p.76
Chapter 3.3 --- Summary --- p.79
Chapter Chapter4 --- The role ofPF4 in MM tumorigenesis: in vivo studies --- p.82
Chapter 4.1 --- Results --- p.82
Chapter 4.1.1 --- PF4 inhibited MM tumor growth and prolonged survival in subcutaneous matrigel xenograft model --- p.82
Chapter 4.1.2 --- PF4 inhibited MM tumor growth and prolonged survival in SCID-rab mouse model --- p.85
Chapter 4.1.3 --- PF4 reduced human MM cell proliferation, angiogenesis and induced apoptosis in SCID-rab mice --- p.88
Chapter 4.2 --- Discussion --- p.91
Chapter 4.2.1 --- PF4 inhibited human tumor growth in subcutaneous matrigel xenograft mouse model --- p.91
Chapter 4.2.2 --- SCID-rab mouse model was successfully established and PF4 inhibited human MM turnor growth in this model --- p.92
Chapter 4.2.3 --- PF4 inhibited human MM cell proliferation, angiogenesis and induced apoptosis in SCID-rab mice --- p.95
Chapter 4.3 --- Summary --- p.96
Chapter Chapter 5 --- The molecular mechanisms of PF4 in MM tumorigenesis --- p.98
Chapter 5.1 --- Results --- p.98
Chapter 5.1.1 --- ProteinlDNA array hybridization and Quantification of protein/DNA array spots --- p.98
Chapter 5.1.2 --- PF4 suppressed DNA binding and transcriptional activity of STAT3 --- p.102
Chapter 5.1.3 --- PF4 inhibited constitutive STAT3 phosphorylation in MM cells --- p.104
Chapter 5.1.4 --- PF4 inhibited IL-6-induced STAT3 activation --- p.105
Chapter 5.1.5 --- PF4 suppressed STAT3 regulated gene expression --- p.107
Chapter 5.1.6 --- Enforced expression of constitutively active STAT3 rescued cells from PF4-induced apoptosis --- p.109
Chapter 5.1.7 --- PF4 induced the expression of SOCS3 --- p.111
Chapter 5.1.8 --- PF4-induced inhibition of STAT3 activation was reversed by gene silencing of SOCS3 --- p.111
Chapter 5.1.9 --- PF4 inhibited nuclear accumulation of STAT3 and induced expression of SOCS3 in vivo --- p.114
Chapter 5.2 --- Discussion --- p.115
Chapter 5.2.1 --- PF4 regulated several TFs --- p.115
Chapter 5.2.2 --- PF4 inhibited constitutive activation of STAT3 --- p.118
Chapter 5.2.3 --- PF4 inhibited IL-6 induced activation of STAT3 --- p.120
Chapter 5.2.4 --- PF4 suppressed STAT3 regulated gene expression --- p.121
Chapter 5.2.5 --- PF4 induced the expression of SOCS3 --- p.124
Chapter 5.3 --- Summary --- p.125
Chapter Chapter 6 --- Conclusion and future studies --- p.128
Chapter 6.1 --- Conclusion --- p.128
Chapter 6.2 --- Future studies --- p.135
Appendices --- p.137
References list --- p.139

Basic fibroblast growth factor (bFGF) plays an instrumental role in the cascade of events leading to restenosis, vascular re-occlusion due to excessive smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) deposition following arterial intervention procedures such as balloon angioplasty. The mechanism of bFGF activation following vascular injury has remained elusive. bFGF is stored bound to heparan sulfate proteoglycans in the ECM of the arterial media; release from extracellular sequestration may activate bFGF and initiate SMC proliferation and migration. bFGF mobilization at injured sites may be induced by platelet degranulation products. We have carried out in vitro studies demonstrating that platelet-derived heparanase and platelet factor-4 (PF4) liberate bFGF from the ECM of vascular SMCs, resulting in the induction of SMC proliferation and migration. Increases in proliferation and migration were inhibited by treatment with a bFGF-neutralizing antibody, suggesting that proliferation and migration in response to heparanase or PF4 are mediated by bFGF activation. When platelets were seeded on top of SMCs, degranulation products were found to release bFGF from the ECM, increasing cell proliferation and cell migration. These increases in SMC proliferation and migration were completely inhibited by the addition of a bFGF-neutralizing antibody. In order to investigate the role of heparanase and PF4 in vivo, each was delivered to the uninjured rat carotid artery. Heparanase and PF4 were both found to release bFGF, induce substantial SMC proliferation and increase the expression of several growth factor receptors thought to promote restenosis. An antibody that neutralizes platelet-derived heparanase was developed and evaluated in a rat carotid balloon injury model. Perivascular delivery of anti-heparanase IgG was found to inhibit bFGF depletion from the arterial wall by approximately 60% (p < 0.001) at 4 days. This correlated with the reduction in intimal thickening observed at 14 days. Platelet degranulation products, such as heparanase and PF4, may liberate bFGF from extracellular sequestration, activating the growth factor and inducing the SMC proliferation and migration that contribute to luminal narrowing following vascular injury. In addition, platelet-derived heparanase is likely to play a key role in initiating events leading to restenosis via bFGF mobilization.