VEGFR1–mediated pericyte ablation links VEGF and PlGF to cancer-associated retinopathy

VEGF coordinates complex regulation of cellular regeneration and interactions between endothelial and perivascular cells; dysfunction of the VEGF signaling system leads to retinopathy. Here, we show that systemic delivery of VEGF and placental growth factor (PlGF) by protein implantation, tumors, and adenoviral vectors ablates pericytes from the mature retinal vasculature through the VEGF receptor 1 (VEGFR1)-mediated signaling pathway, leading to increased vascular leakage. In contrast, we demonstrate VEGF receptor 2 (VEGFR2) is primarily expressed in nonvascular photoreceptors and ganglion cells. Moreover, blockade of VEGFR1 but not VEGFR2 significantly restores pericyte saturation in mature retinal vessels. Our findings link VEGF and PlGF to cancer-associated retinopathy, reveal the molecular mechanisms of VEGFR1 ligand-mediated retinopathy, and define VEGFR1 as an important target of antiangiogenic therapy for treatment of retinopathy.     

Genetic and pharmacological inhibition of JNK ameliorates hypoxia-induced retinopathy through interference with VEGF expression

Many ocular pathologies, including retinopathy of prematurity (ROP), diabetic retinopathy, and age-related macular degeneration, result in vision loss because of aberrant neoangiogenesis. A common feature of these conditions is the presence of hypoxic areas and overexpression of the proangiogenic vascular endothelial growth factor (VEGF). The prevailing current treatment, laser ablation of the retina, is destructive and only partially effective. Preventive and less destructive therapies are much more desirable. Here, we show that mice lacking c-Jun N-terminal kinase 1 (JNK1) exhibit reduced pathological angiogenesis and lower levels of retinal VEGF production in a murine model of ROP. We found that hypoxia induces JNK activation and regulates VEGF expression by enhancing the binding of phospho-c-Jun to the VEGF promoter. Intravitreal injection of a specific JNK inhibitor decreases retinal VEGF expression and reduces pathological retinal neovascularization without obvious side effects. These results strongly suggest that JNK1 plays a key role in retinal neoangiogenesis and that it represents a new pharmacological target for treatment of diseases where excessive neoangiogenesis is the underlying pathology.     

Rat strain-dependent susceptibility to ischemia-induced retinopathy associated with retinal vascular endothelial growth factor regulation

Vascular endothelial growth factor (VEGF) is a potent inflammation, vascular permeability, and angiogenic factor. Variations of the VEGF gene are implicated in the pathogenesis of diabetic retinopathy. Previous studies have shown that Brown Norway (BN) rats have higher retinal VEGF levels and more severe retinal vascular leakage than Sprague-Dawley (SD) rats in response to ischemia and diabetes. To investigate the molecular mechanism of vascular leakage in this animal model, F2 progeny were generated by crossbreeding BN and SD rats. Neonatal rats were exposed to hyperoxia to induce oxygen-induced retinopathy (OIR) models. The F2 rats in response to ischemia have shown a linear distribution of retinal VEGF levels, which is significantly and positively correlated to retinal vascular leakage. We identified a single nucleotide polymorphism (SNP) at upstream stimulating factor-binding site in the VEGF promoter region between BN and SD rats. No differences were found in retinal vascular permeability or VEGF levels between F2 rats with BN, SD, and BN/SD alleles of VEGF SNP. The increased retinal VEGF levels are correlated to ischemia-induced retinal vascular leakage in the OIR rat model. The VEGF mRNA and promoter are not responsible for increased retinal VEGF level and vascular permeability. The up-regulation of VEGF expression activated by a yet to be identified upstream factor or mediator affecting VEGF stability may be associated with a high susceptibility to retinal vascular leakage in BN rats.     

Retinal neovascularisation without ischaemia in the spontaneously diabetic Torii rat

Aims/hypothesis The spontaneously diabetic Torii (SDT) rat has recently been established as a model of type 2 human diabetes mellitus. Male SDT rats develop severe diabetic ocular complications. This study investigated the nature of the ocular complications in this model and addressed the question of whether the SDT rat is a good model of human proliferative diabetic retinopathy.Methods Male SDT rats aged 50 weeks were studied for a period of 8 months. Under deep anaesthesia, one eye of each animal was enucleated following perfusion with fluorescein dextran and a retinal flat mount was prepared to study vascular structure. The other eye was enucleated and investigated histologically by haematoxylin–eosin and azan staining and by immunohistochemistry using antibodies against vascular endothelium (Griffonia simplicifolia isolectin B4 antibody) and vascular endothelial growth factor (VEGF).Results From the vascular structure study, 17 of 32 rats (53%) showed proliferative retinopathy without vascular non-perfusion. The histological study revealed traction retinal folds in rats with proliferative retinopathy. Azan staining showed some proliferative matrix in rats with normal retinal structure and those with proliferative retinopathy compared with normoglycaemic controls. Staining with Griffonia simplicifolia isolectin B4 antibody showed no specific vascular changes in any of the rats, while VEGF staining revealed higher immunoreactivity in the retina of rats with normal retinal structure and those with proliferative retinopathy, but only low immunoreactivity in the control animals.Conclusions/interpretation There appear to be differences between the SDT rat model of diabetic retinopathy and human proliferative diabetic retinopathy, as the SDT rat develops retinal neovascularisation without retinal ischaemia. This very unique display of ocular neovascularisation may be caused by increased expression of VEGF.     

Melatonin attenuated retinal neovascularization and neuroglial dysfunction by inhibition of HIF‐1α‐VEGF pathway in oxygen‐induced retinopathy mice

Retinopathy of prematurity (ROP) is a retinopathy characterized by retinal neovascularization (RNV) occurring in preterm infants treated with high concentrations of oxygen and may lead to blindness in severe cases. Currently, anti‐VEGF therapy is a major treatment for ROP, but it is costly and may cause serious complications. The previous study has demonstrated that melatonin exerted neuroprotective effect against retinal ganglion cell death induced by hypoxia in neonatal rats. However, whether melatonin is anti‐angiogenic and neuroglial protective in the progression of ROP remains unknown. Thus, this study was to investigate the effect of melatonin on RNV and neuroglia in the retina of oxygen‐induced retinopathy (OIR) mice. The results showed a reduction in retinal vascular leakage in OIR mice after melatonin treatment. Besides, the size of retinal neovascular and avascular areas, the number of preretinal neovascular cell nuclei, and the number of proliferative vascular endothelial cells within the neovascular area were significantly decreased in mice treated with melatonin. After oxygen‐induced injury, the density of astrocytes was decreased, accompanied by morphologic and functional changes of astrocytes. Besides, retinal microglia were also activated. Meanwhile, the levels of inflammatory factors were elevated. However, these pathologic processes were all hindered by melatonin treatment. Furthermore, HIF‐1α‐VEGF pathway was activated in the retina of OIR mice, yet was suppressed in melatonin‐treated OIR mice retinas. In conclusion, melatonin prevented pathologic neovascularization, protected neuroglial cells, and exerts anti‐inflammation effect via inhibition of HIF‐1α‐VEGF pathway in OIR retinas, suggesting that melatonin could be a promising therapeutic agent for ROP.     

IQGAP1, a novel vascular endothelial growth factor receptor binding protein, is involved in reactive oxygen species--dependent endothelial migration and proliferation

Endothelial cell (EC) proliferation and migration are important for reendothelialization and angiogenesis. We have demonstrated that reactive oxygen species (ROS) derived from the small GTPase Rac1-dependent NAD(P)H oxidase are involved in vascular endothelial growth factor (VEGF)-mediated endothelial responses mainly through the VEGF type2 receptor (VEGFR2). Little is known about the underlying molecular mechanisms. IQGAP1 is a scaffolding protein that controls cellular motility and morphogenesis by interacting directly with cytoskeletal, cell adhesion, and small G proteins, including Rac1. In this study, we show that IQGAP1 is robustly expressed in ECs and binds to the VEGFR2. A pulldown assay using purified proteins demonstrates that IQGAP1 directly interacts with active VEGFR2. In cultured ECs, VEGF stimulation rapidly promotes recruitment of Rac1 to IQGAP1, which inducibly binds to VEGFR2 and which, in turn, is associated with tyrosine phosphorylation of IQGAP1. Endogenous IQGAP1 knockdown by siRNA shows that IQGAP1 is involved in VEGF-stimulated ROS production, Akt phosphorylation, endothelial migration, and proliferation. Wound assays reveal that IQGAP1 and phosphorylated VEGFR2 accumulate and colocalize at the leading edge in actively migrating ECs. Moreover, we found that IQGAP1 expression is dramatically increased in the VEGFR2-positive regenerating EC layer in balloon-injured rat carotid artery. These results suggest that IQGAP1 functions as a VEGFR2-associated scaffold protein to organize ROS-dependent VEGF signaling, thereby promoting EC migration and proliferation, which may contribute to repair and maintenance of the functional integrity of established blood vessels.     

IGFBP3 suppresses retinopathy through suppression of oxygen-induced vessel loss and promotion of vascular regrowth

Vessel loss precipitates many diseases. In particular, vessel loss resulting in hypoxia induces retinal neovascularization in diabetic retinopathy and in retinopathy of prematurity (ROP), major causes of blindness. Here we define insulin-like growth factor binding protein-3 (IGFBP3) as a new modulator of vascular survival and regrowth in oxygen-induced retinopathy. In IGFBP3-deficient mice, there was a dose-dependent increase in oxygen-induced retinal vessel loss. Subsequent to oxygen-induced retinal vessel loss, Igfbp3(-/-) mice had a 31% decrease in retinal vessel regrowth versus controls after returning to room air. No difference in serum insulin-like growth factor 1 (IGF1) levels was observed among groups. Wild-type mice treated with exogenous IGFBP3 had a significant increase in vessel regrowth. This correlated with a 30% increase in endothelial progenitor cells in the retina at postnatal day 15, indicating that IGFBP3 could be serving as a progenitor cell chemoattractant. In a prospective clinical study, we measured IGFBP3 (and IGF1) plasma levels weekly and examined retinas in all premature infants born at gestational ages <32 weeks at high risk for ROP. The mean level of IGFBP3 at 30-35 weeks postmenstrual age (PMA) for infants with proliferative ROP (ROP stages 3>, n = 13) was 802 microg/liter, and for infants with no ROP (ROP stage 0, n = 38) the mean level was 974 microg/liter (P < 0.03). These results suggest that IGFBP3, acting independently of IGF1, helps to prevent oxygen-induced vessel loss and to promote vascular regrowth after vascular destruction in vivo in a dose-dependent manner, resulting in less retinal neovascularization.     

Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo

Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Because VEGF promotes the proliferation of vascular endothelial cells, we examined the possibility that it also stimulates the proliferation of neuronal precursors in murine cerebral cortical cultures and in adult rat brain in vivo. VEGF (>10 ng/ml) stimulated 5-bromo-2'-deoxyuridine (BrdUrd) incorporation into cells that expressed immature neuronal marker proteins and increased cell number in cultures by 20-30%. Cultured cells labeled by BrdUrd expressed VEGFR2/Flk-1, but not VEGFR1/Flt-1 receptors, and the effect of VEGF was blocked by the VEGFR2/Flk-1 receptor tyrosine kinase inhibitor SU1498. Intracerebroventricular administration of VEGF into rat brain increased BrdUrd labeling of cells in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), where VEGFR2/Flk-1 was colocalized with the immature neuronal marker, doublecortin (Dcx). The increase in BrdUrd labeling after the administration of VEGF was caused by an increase in cell proliferation, rather than a decrease in cell death, because VEGF did not reduce caspase-3 cleavage in SVZ or SGZ. Cells labeled with BrdUrd after VEGF treatment in vivo include immature and mature neurons, astroglia, and endothelial cells. These findings implicate the angiogenesis factor VEGF in neurogenesis as well.     

Effects of intravitreal injection of KH902, a vascular endothelial growth factor receptor decoy, on the retinas of streptozotocin-induced diabetic rats

Aims: KH902 is a fusion protein that can bind vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) through its binding ligand taken from the domains of VEGF receptor 1 and VEGF receptor 2 (VEGFR2). This study was to investigate the effects of intravitreal injection of KH902 on the retinas of streptozotocin‐induced diabetic rats. Methods: Two weeks after induction of diabetes, the left eyes of diabetic rats in each group received an intravitreal injection of phosphate‐buffered saline (PBS), Avastin or KH902 solution, respectively. Four weeks after intravitreal injection, retinal electrophysiological function and the integrity of inner blood retinal barrier (iBRB) were measured by electroretinogram and Evans blue perfusion. The protein levels of VEGF signal pathway were assayed by western blot. The expression and distribution of claudin‐5 and occludin were analysed by double immunofluorescent staining under confocal microscope. The expression of VEGFR2 and PlGF was measured by immunohistochemistry. Results: Four weeks after intravitreal injection, KH902‐treated rats had better retinal electrophysiological function, less retinal vessel leakage and lower levels of VEGFR2, PI3K, AKT, p‐AKT, p‐ERK and p‐SRC than PBS or Avastin‐treated rats. The distribution of claudin‐5 and occludin in the retinal vessels of diabetic rats treated by KH902 was smoother and more uniform than those of diabetic rats treated by PBS or Avastin. The expression of PlGF and VEGFR2 in KH902‐treated rats was decreased compared with those in PBS or Avastin‐treated rats. Conclusions: KH902 could improve retinal electrophysiological function and inhibit the breakdown of iBRB by inhibiting the expression of VEGFR2, PlGF and PI3K, and the activation of SRC, AKT and ERK.     

Plasminogen kringle 5 reduces vascular leakage in the retina in rat models of oxygen-induced retinopathy and diabetes

Aims/hypothesis Retinal vascular leakage is an early pathological feature in diabetic retinopathy and can lead to macular oedema and loss of vision. Previously we have shown that plasminogen kringle 5 (K5), an angiogenic inhibitor, inhibits retinal neovascularisation in the rat model of oxygen-induced retinopathy (OIR). The purpose of this study was to examine the effect of K5 on vascular leakage in the retina.Methods Neonatal rats were exposed to hyperoxia to induce OIR. Diabetes was induced in adult rats by injecting streptozotocin. Vascular permeability was measured by Evans blue method. Expression of vascular endothelial growth factor (VEGF) was evaluated using immunohistochemistry and western blot analysis.Results Rats with OIR and diabetes showed abnormal vascular hyperpermeability in the retina and iris. Intravitreal injection of K5, reduced vascular permeability in both animal models, but did not affect permeability in normal rats. K5 reduced vascular permeability at doses substantially lower than that required for inhibition of retinal neovascularisation. The K5-induced reduction in vascular permeability correlated with its down-regulation of VEGF expression in the retina. Moreover, K5 inhibited IGF-1-induced hyperpermeability, which is known to arise through up-regulation of endogenous VEGF expression. However, K5 had no effect on the hyperpermeability induced by injection of exogenous VEGF.Conclusions/interpretation Very low doses of K5 reduce pathological vascular leakage in the retina. K5 thus has therapeutic potential in the treatment of diabetic macular oedema. This effect can be ascribed, at least in part, to the down-regulation of endogenous VEGF expression.Abbreviations K5 plasminogen kringle 5 - OIR oxygen-induced retinopathy - STZ streptozotocin - VEGF vascular endothelial growth factor - P postnatal day     

Retinopathy of prematurity

Retinopathy of prematurity (ROP) is a common blinding disease in children in the developed world despite current treatment, and is becoming increasingly prevalent in the developing world. ROP progresses in two phases. The first phase begins with delayed retinal vascular growth after birth and partial regression of existing vessels, followed by a second phase of hypoxia-induced pathological vessel growth. Two major risk factors of ROP are the use of oxygen and a decreased gestation period. Excessive oxygen contributes to ROP through regulation of vascular endothelial growth factor (VEGF). Suppression of VEGF by oxygen in phase I of ROP inhibits normal vessel growth, whereas elevated levels of VEGF induced by hypoxia in phase II of ROP precipitate pathological vessel proliferation. Insulin-like growth factor 1 (IGF-1) is a critical non-oxygen-regulated factor in ROP. We have found that serum levels of IGF-1 in premature babies directly correlate with the severity of clinical ROP. IGF-1 acts indirectly as a permissive factor by allowing maximal VEGF stimulation of vessel growth. Lack of IGF-1 in preterm infants prevents normal retinal vascular growth in phase I of ROP, despite the presence of VEGF. As infants mature, rising levels of IGF-1 in phase II of ROP allows VEGF stimulated pathological neovascularization. These findings suggest that restoration of IGF-1 to normal levels might be useful in preventing ROP in preterm infants.     

Kallikrein-binding protein inhibits retinal neovascularization and decreases vascular leakage

AIMS/HYPOTHESIS: Kallikrein-binding protein (KBP) is a serine proteinase inhibitor (serpin). It specifically binds to tissue kallikrein and inhibits kallikrein activity. Our study was designed to test its effects on retinal neovascularization and vascular permeability. METHODS: Endothelial cell proliferation was determined by [(3)H] thymidine incorporation assay and apoptosis quantified by Annexin V staining and flow cytometry. Effect on retinal neovascularization was determined by fluorescein angiography and count of pre-retinal vascular cells in an oxygen-induced retinopathy (OIR) model. Vascular permeability was assayed by the Evans blue method. Vascular endothelial growth factor (VEGF) was measured by Western blot analysis and ELISA. RESULTS: Kallikrein-binding protein specifically inhibited proliferation and induced apoptosis in retinal capillary endothelial cells. Intravitreal injection of KBP inhibited retinal neovascularization in an OIR model. Moreover, KBP decreased vascular leakage in the retina, iris and choroid in rats with OIR. Blockade of kinin receptors by specific antagonists showed significantly weaker inhibition of endothelial cells, when compared to that of KBP, suggesting that the anti-angiogenic activity of KBP is not through inhibiting kallikrein activity or kinin production. KBP competed with (125)I-VEGF for binding to endothelial cells and down-regulated VEGF production in endothelial cells and in the retina of the OIR rat model. CONCLUSION/INTERPRETATION: Kallikrein-binding protein is a multi-functional serpin, and its vascular activities are independent of its interactions with the kallikrein-kinin system. Inhibition of VEGF binding to its receptors and down-regulation of VEGF expression could represent a mechanism for the vascular activities of KBP.     

VEGF-C differentially regulates VEGF-A expression in ocular and cancer cells; promotes angiogenesis via RhoA mediated pathway

Vascular angiogenesis is regulated by a number of cytokines of which vascular endothelial growth factor (VEGF)-A/and its receptor vascular endothelial growth factor receptor 2 (VEGFR2) play an indisputable role. Similarly lymphangiogenesis is regulated by VEGF-C and its receptor VEGFR3. Currently for treating vasculogenesis diseases such as proliferative retinopathies and cancer, a number of anti-VEGF-A therapies are approved for clinical use. Although clinical efficacies achieved are remarkable, they are found to be transitory in nature, followed by restoration of anti-VEGF therapy resistant angiogenesis. Recently the regulatory role of VEGF-C in initiating and potentiating neo-angiogenesis has been uncovered. Although the interactive nature of VEGF-A and C is known, the dynamics of their expression under knockdown conditions is yet to be established. Here in this study we have utilized siRNA to knockdown both VEGF-A and C either independently or in combination. Analysis of VEGF-A and C expression (only in cancer cell lines MCF7, A549 and H460 but not in the ocular cell line RPE19) has shown enhanced expression levels of VEGF-C with increase in knockdown of VEGF-A. However, VEGF-C knockdown has resulted in decreased expression levels of VEGF-A both in RPE19 and MCF7 cells in a dose dependent manner. In addition, VEGF-C knockdown also resulted in decreased expression of RhoA. Further, knockdown studies of RhoA even with supplementation of VEGF-C or A has resulted in decreased endothelial cell proliferation and stress fiber formation, indicating that VEGF-C does promote angiogenesis via RhoA mediated pathway.     

Neuropilin-1 regulates vascular endothelial growth factor-mediated endothelial permeability

Neuropilin-1 (Npn-1) is a cell surface receptor that binds vascular endothelial growth factor (VEGF), a potent mediator of endothelial permeability, chemotaxis, and proliferation. In vitro, Npn-1 can complex with VEGF receptor-2 (VEGFR2) to enhance VEGFR2-mediated endothelial cell chemotaxis and proliferation. To determine the role of Npn-1/VEGFR2 complexes in VEGF-induced endothelial barrier dysfunction, endothelial cells were stably transfected with Npn1 or VEGFR2 alone (PAE/Npn and PAE/KDR, respectively), or VEGFR2 and Npn-1 (PAE/KDR/Npn-1). Permeability, estimated by measurement of transendothelial electrical resistance (TER), of PAE/Npn and PAE/KDR cell lines was not altered by VEGF165. In contrast, TER of PAE/KDR/Npn-1 cells decreased in dose-dependent fashion following VEGF165 (10 to 200 ng/mL). Activation of VEGFR2, and 2 downstream signaling intermediates (p38 and ERK1/2 MAPK) involved in VEGF-mediated permeability, also increased in PAE/KDR/Npn-1. Consistent with these data, inhibition of Npn-1, but not VEGFR2, attenuated VEGF165-mediated permeability of human pulmonary artery endothelial cells (HPAE), and VEGF121 (which cannot ligate Npn-1) did not alter TER of HPAE. Npn-1 inhibition also attenuated both VEGF165-mediated pulmonary vascular leak and activation of VEGFR2, p38, and ERK1/2 MAPK, in inducible lung-specific VEGF transgenic mice. These data support a critical role for Npn-1 in regulating endothelial barrier dysfunction in response to VEGF and suggest that activation of distinct receptor complexes may determine specificity of cellular response to VEGF.     

Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization.

Neovascular diseases of the retina are a major cause of blindness worldwide. Hypoxia is thought to be a common precursor to neovascularization in many retinal diseases, but the factors involved in the hypoxic neovascular response have not been fully identified. To investigate the role of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in retinal neovascularization, the expression of VEGF/VPF mRNA and protein were studied in a mouse model of proliferative retinopathy. RNA (Northern) blot analysis revealed that retinal VEGF/VPF mRNA expression increased 3-fold between 6 and 12 hr of relative retinal hypoxia and remained elevated during the development of neovascularization. In situ hybridization localized VEGF/VPF mRNA to cells bodies in the inner nuclear layer of the retina. Immunohistochemical confocal microscopy demonstrated that VEGF/VPF protein levels increase with a time course similar to that of the mRNA. The cells in the inner nuclear layer of the retina that produce VEGF/VPF were identified morphologically as Müller cells. These data suggest that VEGF/VPF expression in the retina plays a central role in the development of retinal ischemia-induced ocular neovascularization.     

Role of protein tyrosine phosphatase 1B in vascular endothelial growth factor signaling and cell-cell adhesions in endothelial cells

Vascular endothelial growth factor (VEGF) binding induces phosphorylation of VEGF receptor (VEGFR)2 in tyrosine, which is followed by disruption of VE-cadherin-mediated cell-cell contacts of endothelial cells (ECs), thereby stimulating EC proliferation and migration to promote angiogenesis. Tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Little is known about the role of endogenous PTPs in VEGF signaling in ECs. In this study, we found that PTP1B expression and activity are markedly increased in mice hindlimb ischemia model of angiogenesis. In ECs, overexpression of PTP1B, but not catalytically inactive mutant PTP1B-C/S, inhibits VEGF-induced phosphorylation of VEGFR2 and extracellular signal-regulated kinase 1/2, as well as EC proliferation, whereas knockdown of PTP1B by small interfering RNA enhances these responses, suggesting that PTP1B negatively regulates VEGFR2 signaling in ECs. VEGF-induced p38 mitogen-activated protein kinase phosphorylation and EC migration are not affected by PTP1B overexpression or knockdown. In vivo dephosphorylation and cotransfection assays reveal that PTP1B binds to VEGFR2 cytoplasmic domain in vivo and directly dephosphorylates activated VEGFR2 immunoprecipitates from human umbilical vein endothelial cells. Overexpression of PTP1B stabilizes VE-cadherin-mediated cell-cell adhesions by reducing VE-cadherin tyrosine phosphorylation, whereas PTP1B small interfering RNA causes opposite effects with increasing endothelial permeability, as measured by transendothelial electric resistance. In summary, PTP1B negatively regulates VEGFR2 receptor activation via binding to the VEGFR2, as well as stabilizes cell-cell adhesions through reducing tyrosine phosphorylation of VE-cadherin. Induction of PTP1B by hindlimb ischemia may represent an important counterregulatory mechanism that blunts overactivation of VEGFR2 during angiogenesis in vivo.     

Angiotensin converting enzyme inhibition reduces retinal overexpression of vascular endothelial growth factor and hyperpermeability in experimental diabetes

Aims/hypothesis. Angiotensin converting enzyme (ACE) inhibition has been recently suggested to have retinoprotective actions in diabetic patients but the mechanism of this effect is not known. In vitro, angiotensin II stimulates expression of vascular endothelial growth factor (VEGF), a permeability-inducing and endothelial cell specific angiogenic factor which has been implicated in the pathogenesis of diabetic retinopathy in humans and in experimental animals. We sought to determine the effects of ACE inhibition on retinal VEGF expression and permeability in experimental diabetic retinopathy.¶Methods. Streptozotocin-induced diabetic rats and control animals were assigned at random to receive ACE inhibitor treatment or vehicle. At 24 weeks the retinal VEGF protein gene expression was assessed by northern blot analysis and in situ hybridisation. Retinal permeability to albumin was measured using a double isotope technique.¶Results. Experimental diabetes was associated with cell specific two to fourfold increase in retinal VEGF protein gene expression (p < 0.01) and a 2-fold increase in retinal vascular permeability to albumin (p < 0.01). The localization of VEGF expression in the retina was not altered in animals with experimental diabetes. Angiotensin converting enzyme inhibitor treatment of diabetic rats reduced diabetes-associated changes in VEGF gene expression and vascular permeability.¶Conclusion/interpretation. These findings implicate the renin-angiotensin system in the VEGF overexpression and hyperpermeability which accompany diabetic retinopathy and provide a potential mechanism for the beneficial effects of ACE inhibition in diabetic retinal disease. [Diabetologia (2000) 43: 1360–1367]     

Effects of vascular endothelial growth factors and their receptors on megakaryocytes and platelets and related diseases

It is well known that vascular endothelial growth factors (VEGFs) and their receptors (vascular endothelial growth factor receptors, VEGFRs) are expressed in different tissues, and VEGF‐VEGFR loops regulate a wide range of responses, including metabolic homeostasis, cell proliferation, migration and tubuleogenesis. As ligands, VEGFs act on three structurally related VEGFRs (VEGFR1, VEGFR2 and VEGFR3 [also termed FLT1, KDR and FLT4, respectively]) that deliver downstream signals. Haematopoietic stem cells (HSCs), megakaryocytic cell lines, cultured megakaryocytes (MKs), primary MKs and abnormal MKs express and secrete VEGFs. During the development from HSCs to MKs, VEGFR1, VEGFR2 and VEGFR3 are expressed at different developmental stages, respectively, and re‐expressed, e.g., VEGFR2, and play different roles in commitment, differentiation, proliferation, survival and polyplodization of HSCs/MKs via autocrine, paracrine and/or even intracrine loops. Moreover, VEGFs and their receptors are abnormally expressed in MK‐related diseases, including myeloproliferative neoplasms, myelodysplastic syndromes and acute megakaryocytic leukaemia (a rare subtype of acute myeloid leukaemia), and they lead to the disordered proliferation/differentiation of bone marrow cells and angiogenesis, indicating that they are closely related to these diseases. Thus, targeting VEGF‐VEGFR loops may be of potential therapeutic value.