Vascular endothelial growth factor A competitively inhibits platelet-derived growth factor (PDGF)-dependent activation of PDGF receptor and subsequent signaling events and cellular responses.
ABSTRACT: Certain platelet-derived growth factor (PDGF) isoforms are associated with proliferative vitreoretinopathy (PVR), a sight-threatening complication that develops in a subset of patients recovering from retinal reattachment surgery. Although these PDGF isoforms are abundant in the vitreous of patients and experimental animals with PVR, they make only a minor contribution to activating PDGF receptor ? (PDGFR?) and driving experimental PVR. Rather, growth factors outside of the PDGF family are the primary (and indirect) agonists of PDGFR?. These observations beg the question of why vitreal PDGFs fail to activate PDGFR?. We report here that vitreous contains an inhibitor of PDGF-dependent activation of PDGFR? and that a major portion of this inhibitory activity is due to vascular endothelial cell growth factor A (VEGF-A). Furthermore, recombinant VEGF-A competitively blocks PDGF-dependent binding and activation of PDGFR, signaling events, and cellular responses. These findings unveil a previously unappreciated relationship between distant members of the PDGF/VEGF family that may contribute to pathogenesis of a blinding eye disease.
Project description:Proliferative vitreoretinopathy (PVR) is a recurring and problematic disease for which there is no pharmacologic treatment. Platelet-derived growth factor (PDGF) in the vitreous is associated with experimental and clinical PVR. Furthermore, PDGF receptors (PDGFRs) are present and activated in epiretinal membranes of patient donors, and they are essential for experimental PVR. These observations suggest that PVR arises at least in part from PDGF/PDGFR-driven events. The goal of this study was to determine whether PDGFs were a potential therapeutic target for PVR.Experimental PVR was induced in rabbits by injecting fibroblasts. Vitreous specimens were collected from experimental rabbits or from patients undergoing vitrectomy to repair retinal detachment. A neutralizing PDGF antibody and a PDGF Trap were tested for their ability to prevent experimental PVR. Activation of PDGFR was monitored by antiphosphotyrosine Western blot analysis of immunoprecipitated PDGFRs. Contraction of collagen gels was monitored in vitro.Neutralizing vitreal PDGFs did not effectively attenuate PVR, even though the reagents used potently blocked PDGF-dependent activation of the PDGF alpha receptor (PDGFRalpha). Vitreal growth factors outside the PDGF family modestly activated PDGFRalpha and appeared to do so without engaging the ligand-binding domain of PDGFRalpha. This indirect route to activate PDGFRalpha had profound functional consequences. It promoted the contraction of collagen gels and appeared sufficient to drive experimental PVR.Although PDGF appears to be a poor therapeutic target, PDGFRalpha is particularly attractive because it can be activated by a much larger spectrum of vitreal growth factors than previously appreciated.
Project description:Proliferative vitreoretinopathy (PVR) exemplifies a disease that is difficult to predict, lacks effective treatment options, and substantially reduces the quality of life of an individual. Surgery to correct a rhegmatogenous retinal detachment fails primarily because of PVR. Likely mediators of PVR are growth factors in vitreous, which stimulate cells within and behind the retina as an inevitable consequence of a breached retina. Three classes of growth factors [vascular endothelial growth factor A (VEGF-A), platelet-derived growth factors (PDGFs), and non-PDGFs (growth factors outside of the PDGF family)] are relevant to PVR pathogenesis because they act on PDGF receptor ?, which is required for experimental PVR and is associated with this disease in humans. We discovered that ranibizumab (a clinically approved agent that neutralizes VEGF-A) reduced the bioactivity of vitreous from patients and experimental animals with PVR, and protected rabbits from developing disease. The apparent mechanism of ranibizumab action involved derepressing PDGFs, which, at the concentrations present in PVR vitreous, inhibited non-PDGF-mediated activation of PDGF receptor ?. These preclinical findings suggest that available approaches to neutralize VEGF-A are prophylactic for PVR, and that anti-VEGF-based therapies may be effective for managing more than angiogenesis- and edema-driven pathological conditions.
Project description:Proliferative vitreoretinopathy (PVR) thwarts the repair of rhegmatogenous retinal detachments. Currently, there is no effective prevention for PVR. Platelet-derived growth factor receptor ? (PDGFR?) is associated with PVR in humans and strongly promotes experimental PVR driven by multiple vitreal growth factors outside the PDGF family. We sought to identify vitreal factors required for experimental PVR and to establish a potential approach to prevent PVR. Vitreous was obtained from normal rabbits or those in which PVR was either developing or stabilized. Normal vitreous contained substantial levels of growth factors and cytokines, which changed quantitatively and/or qualitatively as PVR progressed and stabilized. Neutralizing a subset of these agents in rabbit vitreous eliminated their ability to induce PVR-relevant signaling and cellular responses. A single intravitreal injection of neutralizing reagents for this subset prevented experimental PVR. To identify growth factors and cytokines likely driving PVR in humans, we subjected vitreous from patients with or without PVR to a similar series of analyses. This analysis accurately identified those agents required for vitreous-induced contraction of cells from a patient PVR membrane. We conclude that combination therapy encompassing a subset of vitreal growth factors and cytokines is a potential approach to prevent PVR.
Project description:The vitreous contains a plethora of growth factors that are strongly implicated in the formation of fibroproliferative diseases such as proliferative vitreoretinopathy. Although platelet-derived growth factors (PDGFs) are present in the vitreous, vitreal growth factors outside of the PDGF family activated the PDGF alpha receptor (PDGFRalpha) and promoted disease progression in a rabbit model of proliferative vitreoretinopathy (H. Lei, G. Velez, P. Hovland, T. Hirose, D. Gilbertson, and A. Kazlauskas (2008) submitted for publication.) In this report we investigated the mechanism by which non-PDGFs activated PDGFRalpha. We found that non-PDGFs increased the cellular level of reactive oxygen species (ROS) and that this event was necessary and sufficient for phosphorylation of PDGFRalpha. We speculated that the underlying mechanism was ROS-mediated inhibition of phosphotyrosine phosphatases, which antagonize receptor auto-phosphorylation. However, this did not appear to be the case. Non-PDGFs promoted tyrosine phosphorylation of catalytically inactive PDGFRalpha, and thereby indicated that at least one additional tyrosine kinase was involved. Indeed, preventing expression or blocking the kinase activity of Src family kinases suppressed non-PDGF-dependent tyrosine phosphorylation of PDGFRalpha. Thus non-PDGFs increased the level of ROS, which activated Src family kinases and resulted in phosphorylation of PDGFRalpha. Finally, although non-PDGFs induced only modest phosphorylation of PDGFRalpha, proliferation and survival of cells in response to non-PDGFs was significantly enhanced by expression of PDGFRalpha. These studies reveal a novel mechanism for activation of PDGFRalpha that appears capable of enhancing the responsiveness of cells to growth factors outside of the PDGF family.
Project description:Platelet-derived growth factors (PDGFs) play important roles in skeletal development and bone fracture healing, yet how PDGFs execute their functions remains incompletely understood. Here we show that PDGF-AA, but not -AB or -BB, could activate the BMP-Smad1/5/8 pathway in mesenchymal stem cells (MSCs), which requires BMPRIA as well as PDGFR?. PDGF-AA promotes MSC osteogenic differentiation through the BMP-Smad1/5/8-Runx2/Osx axis and MSC migration via the BMP-Smad1/5/8-Twist1/Atf4 axis. Mechanistic studies show that PDGF-AA activates BMP-Smad1/5/8 signaling by feedback down-regulating PDGFR?, which frees BMPRI and allows for BMPRI-BMPRII complex formation to activate smad1/5/8, using BMP molecules in the microenvironment. This study unravels a physical and functional interaction between PDGFR? and BMPRI, which plays an important role in MSC differentiation and migration, and establishes a link between PDGF-AA and BMPs pathways, two essential regulators of embryonic development and tissue homeostasis.
Project description:Proliferative vitreoretinopathy (PVR) is a nonneovascular blinding disease and the leading cause for failure in surgical repair of rhegmatogenous retinal detachments. Once formed, PVR is difficult to treat. Hence, there is an acute interest in developing approaches to prevent PVR. Of the many growth factors and cytokines that accumulate in vitreous as PVR develops, neutralizing vascular endothelial growth factor (VEGF) A has recently been found to prevent PVR in at least one animal model. The goal of this study was to test if Food and Drug Administration-approved agents could protect the eye from PVR in multiple animal models and to further investigate the underlying mechanisms. Neutralizing VEGF with aflibercept (VEGF Trap-Eye) safely and effectively protected rabbits from PVR in multiple models of disease. Furthermore, aflibercept reduced the bioactivity of both experimental and clinical PVR vitreous. Finally, although VEGF could promote some PVR-associated cellular responses via VEGF receptors expressed on the retinal pigment epithelial cells that drive this disease, VEGF's major contribution to vitreal bioactivity occurred via platelet-derived growth factor receptor ?. Thus, VEGF promotes PVR by a noncanonical ability to engage platelet-derived growth factor receptor ?. These findings indicate that VEGF contributes to nonangiogenic diseases and that anti-VEGF-based therapies may be effective on a wider spectrum of diseases than previously appreciated.
Project description:BACKGROUND: Brain radiation necrosis (RN) occurring after radiotherapy is a serious complication. We and others have performed several treatments for RN, using anticoagulants, corticosteroids, surgical resection and bevacizumab. However, the mechanisms underlying RN have not yet been completely elucidated. For more than a decade, platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) have been extensively studied in many biological processes. These proteins influence a wide range of biological responses and participate in many normal and pathological conditions. In this study, we demonstrated that PDGF isoforms (PDGF-A, B, C, and D) and PDGFRs (PDGFR-? and ?) are involved in the pathogenesis of human brain RN. We speculated on their roles, with a focus on their potential involvement in angiogenesis and inflammation in RN. METHODS: Seven surgical specimens of RN, obtained from 2006 to 2013 at our department, were subjected to histopathological analyses and stained with hematoxylin and eosin. We qualitatively analyzed the protein expression of each isoform of PDGF by immunohistochemistry. We also examined their expression with double immunofluorescence. RESULTS: All PDGFs were expressed in macrophages, microglia, and endothelial cells in the boundary of the core of RN, namely, the perinecrotic area (PN), as well as in undamaged brain tissue (UB). PDGF-C, D and PDGFR-? were also expressed in reactive astrocytes in PN. PDGFs and PDGFR-? were scarcely detected in UB, but PDGFR-? was specifically expressed in endothelial cells not only in PN but also in UB. CONCLUSIONS: PDGFs/PDGFRs play critical roles in angiogenesis and possibly in inflammation, and they contribute to the pathogenesis of RN, irrespective of the original tumor pathology and applied radiation modality. Treatments for the inhibition of PDGF-C, PDGF-D, and PDGFR-? may provide new approaches for the treatment of RN induced by common radiation therapies.
Project description:INTRODUCTION:Proliferative vitreoretinopathy (PVR), which is regulated by growth factors and cytokines, is the leading cause of failure in vitreoretinal surgery. In this study, we aimed to investigate the role of the human serum and vitreous inflammation-related factors in the development of proliferative vitreoretinopathy (PVR). METHODS:Blood and vitreous samples were obtained from patients undergoing pars plana vitrectomy. Inflammation-related factors were detected using an immunology multiplex assay on a Luminex® xMAP® platform. Patients with PVR and rhegmatogenous retinal detachment (RRD) were compared with macular hole (MH) or epiretinal membrane (ERM) patients without any other ocular or systemic disease. RESULTS:Thirty-six serum samples and 34 vitreous samples were obtained. Thirty-one different growth factors and cytokines were detected in serum samples. However, none of the circulating growth factors and cytokines were found to be different from the controls. Ten different growth factors and cytokines were measured in the vitreous samples. The concentration levels of PDGF-AA, TGF-?, VEGF, IL-6, IL-8, and TNF? were found to have significantly increased in the vitreous of PVR patients. CONCLUSION:Our study found that none of the circulating inflammation-related factors were changed in PVR or RRD patients, indicating the absence of a system inflammatory biomarkers to predict the development of proliferative vitreoretinopathy. As a supplement to previous research, the concentrations of PDGF-AA, TGF-?, VEGF, IL-6, IL-8, and TNF? were significantly upregulated in the vitreous of PVR patients. These factors should be considered for preventing PVR.
Project description:Nearly all studies of angiogenesis have focused on uni-family ligand-receptor binding, e.g., VEGFs bind to VEGF receptors, PDGFs bind to PDGF receptors, etc. The discovery of VEGF-PDGFRs binding challenges this paradigm and calls for investigation of other ligand-receptor binding possibilities. We utilized surface plasmon resonance to identify and measure PDGF-to-VEGFR binding rates, establishing cut-offs for binding and non-binding interactions. We quantified the kinetics of the recent VEGF-A:PDGFRβ interaction for the first time with KD = 340 pM. We discovered new PDGF:VEGFR2 interactions with PDGF-AA:R2 KD = 530 nM, PDGF-AB:R2 KD = 110 pM, PDGF-BB:R2 KD = 40 nM, and PDGF-CC:R2 KD = 70 pM. We computationally predict that cross-family PDGF binding could contribute up to 96% of VEGFR2 ligation in healthy conditions and in cancer. Together the identification, quantification, and simulation of these novel cross-family interactions posits new mechanisms for understanding anti-angiogenic drug resistance and presents an expanded role of growth factor signaling with significance in health and disease.
Project description:Malignant cells are capable of influencing the microenvironment in a manner that facilitates tumor cell survival. Bidirectional crosstalk between chronic lymphocytic leukemic (CLL) cells and marrow-derived mesenchymal stromal cells (MSCs) activates both cell types. In this study, we observed that the conditioned medium (CM) obtained from CLL cells was able to induce Akt activation in MSC. Subsequent studies investigated the mechanism of MSC activation mediated by CLL-CM. Platelet-derived growth factor receptors (PDGFRs) were selectively activated in MSCs by CLL-CM and found to be critical receptors for CLL-CM-driven MSC proliferation and MSC Akt activation. The known ligands of PDGFR, platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF), were detected in CLL-CM, but PDGF was the predominant ligand involved in the CM-mediated PDGFR activation. Both PDGF and VEGF were found to be elevated in the plasma of CLL patients with a positive association for high-risk factors and more advanced stage. Finally, we demonstrated that PDGF induced MSC VEGF production through a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. These results show that PDGF-PDGFR signaling influences at least the MSC in the microenvironment of CLL and may play a role in the induction of an angiogenic switch known to be permissive for disease progression.