Expression of vascular endothelial growth factor receptors 1, 2, and 3 in quiescent endothelia.

The vascular endothelial growth factor (VEGF) family is involved in angiogenesis, and therefore VEGFs are considered as targets for anti-angiogenic therapeutic strategies against cancer. However, the physiological functions of VEGFs in quiescent tissues are unclear and may interfere with such systemic therapies. In pathological conditions, increased levels of expression of the VEGF receptors VEGFR-1, VEGFR-2, and VEGFR-3 accompany VEGF activity. In this study we investigated normal human and monkey tissues for expression patterns of these receptors. Immunohistochemical staining methods at the light and electron microscopic level were applied to normal human and monkey tissue samples, using monoclonal antibodies (MAbs) against the three VEGFRs and anti-endothelial MAbs PAL-E and anti-CD31 to identify blood and lymph vessels. In human and monkey, similar distribution patterns of the three VEGFRs were found. Co-expression of VEGFR-1, -2, and -3 was observed in microvessels adjacent to epithelia in the eye, gastrointestinal mucosa, liver, kidney, and hair follicles, which is in line with the reported preferential expression of VEGF-A in some of these epithelia. VEGFR-1, -2, and -3 expression was also observed in blood vessels and sinusoids of lymphoid tissues. Furthermore, VEGFR-1, but not VEGFR-2 and -3, was present in microvessels in brain and retina. Electron microscopy showed that VEGFR-1 expression was restricted to pericytes and VEGFR-2 to endothelial cells in normal vasculature of tonsils. These findings indicate that VEGFRs have specific distribution patterns in normal tissues, suggesting physiological functions of VEGFs that may be disturbed by systemic anti-VEGF therapy. One of these functions may be involvement of VEGF in paracrine relations between epithelia and adjacent capillaries.     

Quantification and cell-to-cell variation of vascular endothelial growth factor receptors

The vascular endothelial growth factor receptors (VEGFR) play a significant role in angiogenesis, the formation of new blood vessels from existing vasculature. Systems biology offers promising approaches to better understand angiogenesis by computational modeling the key molecular interactions in this process. Such modeling requires quantitative knowledge of cell surface density of pro-angiogenic receptors versus anti-angiogenic receptors, their regulation, and their cell-to-cell variability. Using quantitative fluorescence, we systematically characterized the endothelial surface density of VEGFRs and neuropilin-1 (NRP1). We also determined the role of VEGF in regulating the surface density of these receptors. Applying cell-by-cell analysis revealed heterogeneity in receptor surface density and VEGF tuning of this heterogeneity. Altogether, we determine inherent differences in the surface expression levels of these receptors and the role of VEGF in regulating the balance of anti-angiogenic or modulatory (VEGFR1) and pro-angiogenic (VEGFR2) receptors.     

Vascular endothelial growth factor, vascular endothelial growth factor receptor-3 and cyclooxygenase-2 expression in psoriasis

OBJECTIVE: To investigate expression patterns and relationship of vascular endothelial growth factor (VEGF), vascular endothelial receptor-3 (VEGF-R3) (FLT-4) and cyclooxygenase-2 (COX-2) in psoriasis. STUDY DESIGN: Forty-three patients were included in this study. The clinical severity of psoriasis was assessed using the psoriasis area and severity index (PASI). Punch biopsy samples both from psoriatic and nonlesional skin were taken and VEGF, VEGF-R3 and COX-2 expressions determined. RESULTS: VEGF, VEGF-R3 and COX-2 expressions were detected in 90.9%, 78.0% and 86.4% of psoriatic and 84.1%, 71.8%, and 84.1% of nonlesional skin, respectively. Epidermal VEGF, VEGF-R3 and COX-2 expressions were detected in 56.8%, 77.8% and 34.1 of psoriatic and 75%, 78.1% and 65.9% of nonlesional skin, respectively. In dermis, VEGF, VEGF-R3 and COX-2 expression was observed in 88.6%, 77.5% and 84.1% of psoriatic and 81.8%, 64.1% and 77.3% of nonlesional skin, respectively. Among the PASI subgroups no statistically significant differences were detected for VEGF, VEGF-R3 and COX-2 expression. CONCLUSION: Our study demonstrated that VEGF, VEGF-R3 and COX-2 expression in psoriatic and nonlesional skin is significantly high in epidermis and dermis. Although there was significant concordance between VEGF and VEGF-R3 expressions in psoriatic lesions, there seems to be no concordance between the others.     

Expression of vascular endothelial growth factor and basic fibroblast growth factor receptors in lung cancer

OBJECTIVE: To determine the expression of two angiogenic factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor receptors (FGFR), in non-small cell lung carcinoma (NSCLC) in relation to tumor stage (TN0, TN1, TN2) and in association with the expression of p53 protein, a potential suppressor of tumor angiogenesis. STUDY DESIGN: The immunohistochemical (IHC) expression of VEGF and FGFR was examined in paraffin sections of 56 NSCLC in relation to the presence of lymph node metastases and p53 expression. Nodal status of NSCLC determined: 27 tumors, N0; 16, N1; and 13, N2 stage. Semiquantitative analysis with a score corresponding to IHC staining intensity and percentage of positive cells was used. Statistical analysis was performed with the chi 2 test. RESULTS: A significant association was noted between VEGF and FGFR expression in NSCLC. No relation was found between VEGF, FGFR expression and lymph node metastasis or p53 expression. CONCLUSION: We assume that VEGF and FGFR act in a synergistic manner in NSCLC and that their expression is not related to lymph node metastases. Angiogenesis is a very complex phenomenon and heterogeneous within tumors. Also, it is affected by microenviromental factors.     

Increased mitochondrial H2O2 production promotes endothelial NF-kappaB activation in aged rat arteries

Previous studies have shown that the aging vascular system undergoes pro-atherogenic phenotypic changes, including increased oxidative stress and a pro-inflammatory shift in endothelial gene expression profile. To elucidate the link between increased oxidative stress and vascular inflammation in aging, we compared the carotid arteries and aortas of young and aged (24 mo old) Fisher 344 rats. In aged vessels there was an increased NF-kappaB activity (assessed by luciferase reporter gene assay and NF-kappaB binding assay), which was attenuated by scavenging H(2)O(2). Aging did not alter the vascular mRNA and protein expression of p65 and p50 subunits of NF-kappaB. In endothelial cells of aged vessels there was an increased production of H(2)O(2) (assessed by 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester fluorescence), which was attenuated by the mitochondrial uncoupler FCCP. In young arteries and cultured endothelial cells, antimycin A plus succinate significantly increased FCCP-sensitive mitochondrial H(2)O(2) generation, which was associated with activation of NF-kappaB. In aged vessels inhibition of NF-kappaB (by pyrrolidenedithiocarbamate, resveratrol) significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness. Thus increased mitochondrial oxidative stress contributes to endothelial NF-kappaB activation, which contributes to the pro-inflammatory phenotypic alterations in the aged vaculature. Our model predicts that by reducing mitochondrial H(2)O(2) production and/or directly inhibiting NF-kappaB novel anti-aging pharmacological treatments (e.g., calorie restriction mimetics) will exert significant anti-inflammatory and vasoprotective effects.     

Vascular endothelial growth factor restores erectile function through modulation of the insulin-like growth factor system and sex hormone receptors in diabetic rat

Previous studies have shown that intracavernous injection of vascular endothelial growth factor (VEGF) restored erectile function in diabetic rats. However, the mechanism of VEGF in diabetes-related erectile dysfunction (ED) has not been fully investigated. We hypothesize that intracavernous injection of VEGF may reverse diabetes-related ED through modulation of the insulin-like growth factor system and sex hormone receptors. To test this hypothesis the erectile function of treated and control rats was analyzed by measurement of intracavernous pressure (ICP) following electrostimulation of the cavernous nerves. Mean ICP was significantly lower in non-treated diabetic rats compared to controls. After VEGF injection, ICP was significantly higher than in non-treated diabetic rats. IGFBP-3 mRNA and protein expression was significantly higher in non-treated diabetic rat crura than controls, while VEGF-treated animals had control levels. ER-beta and PR mRNA and protein expression was significantly lower in non-treated diabetic rat crura. After VEGF injection, ER-beta and PR mRNA and protein expression was similar to control levels. Expression of AR and ER-alpha was the same in all groups. These findings suggest that orthotopic injection of VEGF may improve the functional recovery of diabetes-related ED through modulation of the insulin-like growth factor system and sex hormone receptors. To our knowledge, this is the first study demonstrating that VEGF treatment restores erectile function through restoration of the insulin-like growth factor system and sex hormone receptor genes at the mRNA and protein levels in diabetic rat crura. These results may be important in understanding the pathogenesis of diabetes-related ED and also in providing better strategies for management of this disease.     

Hyperoxia causes decreased expression of vascular endothelial growth factor and endothelial cell apoptosis in adult retina

Mice or humans with photoreceptor degenerations experience permeability and dropout of retinal capillaries. Loss of photoreceptors results in decreased oxygen usage and thinning of the retina with increased oxygen delivery to the inner retina. To investigate the possibility that increased tissue oxygen plays a role in the vascular damage, we exposed adult mice to hyperoxia, which also increases oxygen in the retina. After 1, 2, or 3 weeks of hyperoxia, there was a statistically significant decrease in retinal vascular density that was not reversible, and endothelial cell apoptosis was demonstrated by TUNEL staining. Mice exposed to hyperoxia and mice with photoreceptor degeneration both showed decreased expression of VEGF in the retina. After complete or near-complete degeneration of photoreceptors, there was increased expression of VEGF in RPE cells, which may explain the association of photoreceptor degeneration and neovascularization in or around the RPE. Increased expression of VEGF in photoreceptors of transgenic mice failed to prevent hyperoxia-induced retinal capillary dropout. These data suggest that increased oxygen in the retina, either by increased inspired oxygen or by photoreceptor degeneration, results in endothelial cell death and dropout of capillaries. Decreased expression of VEGF may be a contributing factor, but the situation may be more complicated for mature retinal vessels than it is for immature vessels, because VEGF replacement does not rescue mature retinal vessels, suggesting that other factors may also be involved.     

Differential roles of vascular endothelial growth factor receptors 1 and 2 in dendritic cell differentiation

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal differentiation is an important mechanism of tumor escape from immune control. A major role for vascular endothelial growth factor (VEGF) and its receptors, VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, has been documented in hemopoietic development. To study the roles of each of these receptors in DC differentiation, we used an in vitro system of myeloid DC differentiation from murine embryonic stem cells. Exposure of wild-type, VEGFR1(-/-), or VEGFR2(-/-) embryonic stem cells to exogenous VEGF or the VEGFR1-specific ligand, placental growth factor, revealed distinct roles of VEGF receptors. VEGFR1 is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic differentiation, but only marginally affects final DC maturation. SU5416, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR1 signaling was sufficient for blocking NF-kappaB activation in bone marrow hemopoietic progenitor cells. VEGF and placental growth factor affect the early stages of myeloid/DC differentiation. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR1.     

Inhibition of vascular permeability factor/vascular endothelial growth factor-mediated angiogenesis by the Kruppel-like factor KLF2

The Kruppel-like factor KLF2 was recently identified as a novel regulator of endothelial pro-inflammatory and pro-thrombotic function. Here it is shown that overexpression of KLF2 potently inhibits vascular permeability factor/vascular endothelial growth factor (VEGF-A)-mediated angiogenesis and tissue edema in the nude ear mouse model of angiogenesis. In vitro, KLF2 expression retards VEGF-mediated calcium flux, proliferation and induction of pro-inflammatory factors in endothelial cells. This effect is due to a potent inhibition of VEGFR2/KDR expression and promoter activity. These observations identify KLF2 as a regulator of VEGFR2/KDR and provide a foundation for novel approaches to regulate angiogenesis.     

Expression of vascular endothelial growth factor receptors and their ligands in rat uterus during the postpartum involution period

Abstract

Vascular endothelial growth factor (VEGF) and its specific receptors, FLt1/fms, Flk1/KDR and FLt4, play important roles in vasculogenesis, and physiological and pathological angiogenesis. Whether angiogenic growth factors are involved in regulating angiogenic processes during the postpartum involution period (PP) of the rat uterus is unknown. We used immunohistochemistry to analyze the expression levels of VEGF, the fms-like tyrosine kinase 1 (FLt1/fms), the kinase insert domain-containing region 1 (Flk1/KDR), Fms-related tyrosine kinase 4 (FLt4) and vascular endothelial growth inhibitor (VEGI) in the rat uterus during the days 1, 3, 5, 10 and 15 of the PP to determine the temporal and spatial expressions of VEGF and its receptors during the PP. Throughout the PP, cytoplasmic and membrane staining of VEGI, VEGF and their receptors were observed in the lumens, crypts and glandular epithelial cells as well as in connective tissue and vascular endothelial and smooth muscle cells in the endometrium. We found that the intensity of the immunoreactions in the endometrium varied with the morphological changes that occurred during involution. Immunoreactions for VEGI, VEGF and their receptor, Flk1/KDR, in the luminal epithelial cells were stronger than those in the glandular epithelial and stromal cells, particularly during PP 1, 3 and 5, which suggests that these peptides may contribute to re-epithelialization of the endometrium. On the other hand, Flt1/fms immunoreactivity was strong mainly in the stromal cells during the PP. The presence of VEGF and its receptors (FLt1/fms, Flk1/KDR, FLt4) in the stromal cells and blood vessels during the PP suggests that they may contribute to regulating stromal repair and angiogenesis in the involuting uterus of the rat.     

Short-term hyperhomocysteinemia-induced oxidative stress activates retinal glial cells and increases vascular endothelial growth factor expression in rat retina

Hyperhomocysteinemia is associated with an increase in the incidence of vascular diseases, including retinal vascular diseases. We examined the effects of high plasma levels of homocysteine on retinal glial cells and vascular endothelial growth factor (VEGF) expression. Male Sprague-Dawley rats were fed either a 3.0 g/kg homocystine diet or a control diet for 2 week. The homocystine-diet group had higher plasma levels of homocysteine and thiobarbituric acid reactive substances (TBARSs) and lower plasma levels of folate, retinol, alpha-tocopherol, and retinal expression of CuZn superoxide dismutase (SOD) than the controls. The rats fed the homocystine-diet showed an increase in vimentin, glial fibrillary acidic protein (GFAP), and VEGF immunoreactivity in the retina as compared to the controls. The increase in vimentin immunoreactivity in the hyperhomocysteinemic rats was correlated with changes in GFAP immunoreactivity in astrocytes within the ganglion cell layer. We found for the first time that short-term hyperhomocysteinemia-induced oxidative stress activates retinal glial cells and increases VEGF expression in the retina.     

Model of competitive binding of vascular endothelial growth factor and placental growth factor to VEGF receptors on endothelial cells

Placental growth factor (PlGF) competes with vascular endothelial growth factor (VEGF) for binding to VEGF receptor (VEGFR)-1 but does not bind VEGFR2. Experiments show that PlGF can augment the response to VEGF in pathological angiogenesis and in models of endothelial cell survival, migration, and proliferation. This synergy has been hypothesized to be due to a combination of the following: signaling by PlGF through VEGFR1 and displacement of VEGF from VEGFR1 to VEGFR2 by PlGF, causing increased signaling through VEGFR2. In this study, the relative contribution of PlGF-induced VEGF displacement to the synergy is quantified using a mathematical model of ligand-receptor binding to examine the effect on ligand-receptor complex formation of VEGF and PlGF acting together. Parameters specific to the VEGF-PlGF system are used based on existing data. The model is used to simulate in silico a specific in vitro experiment in which VEGF-PlGF synergy is observed. We show that, whereas a significant change in the formation of endothelial surface growth factor-VEGFR1 complexes is predicted in the presence of PlGF, the increase in the number of VEGFR2-containing signaling complexes is less significant; these results were shown to be robust to significant variation in the kinetic parameters of the model. Synergistic effects observed in that experiment thus appear unlikely to be due to VEGF displacement but to a shift from VEGF-VEGFR1 to PlGF-VEGFR1 complexes and an increase in total VEGFR1 complexes. These results suggest that VEGFR1 signaling can be functional in adult-derived endothelial cells.     

Vascular endothelial growth factor and vascular endothelial growth factor receptor-2 expression in the chick embryo area vasculosa

Vascular endothelial growth factor is an angiogenic factor in vivo and in vitro that plays a crucial role in the control of blood vessel development and in pathological angiogenesis. The vascularized extraembryonic membranes of the chick embryo include the area vasculosa and the chorioallantoic membrane. In this study, we investigated the expression of vascular endothelial growth factor and of its receptor-2, specifically expressed by the endothelial cells, in the chick area vasculosa at days 6, 10 and 14 of incubation. Our results indicate that, in all the three developmental stages examined, vascular endothelial growth factor is clearly expressed in the endodermal cells immediately adjacent to the mesodermal endothelial cells which, in turn, expressed vascular endothelial growth factor receptor-2. These observations suggest that during the development of the vascular system, endodermal cells, expressing vascular endothelial growth factor, initiate angiogenesis by stimulating directly mesodermal cells, which express vascular endothelial growth factor receptor-2. Moreover, our data demonstrate that vascular endothelial growth factor receptor-2 expression is also maintained by endothelial cells in the later stages of development, until day 14 of incubation. In accord with other literature data, this suggests that vascular endothelial growth factor is required not only for proliferation, but also for the survival of endothelial cells.     

Vascular endothelial growth factor stimulates osteoblastic differentiation of cultured human periosteal-derived cells expressing vascular endothelial growth factor receptors

Angiogenesis plays an important role in bone development and postnatal bone fracture repair. Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) are primarily involved in angiogenesis. This study investigated the expression of VEGF isoforms, VEGFR-1, and VEGFR-2 during the osteoblastic differentiation of cultured human periosteal-derived cells. In addition, the effect of exogenous VEGF on the osteoblastic differentiation of cultured human periosteal-derived cells was also examined. The expression of the VEGF isoforms (VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆), VEGFR-1, and VEGFR-2 was observed in the periosteal-derived cells. Administration of KRN633, a VEGFR-1 and VEGFR-2 inhibitor, decreased the alkaline phosphatase (ALP) activity during the osteoblastic differentiation of cultured human periosteal-derived cells. However, the administration of VEGFR2 Kinase Inhibitor IV, a VEGFR-2 inhibitor, did not affect the ALP activity. The addition of recombinant human VEGF₁₆₅ elevated the ALP activity and increased the calcium content in the periosteal-derived cells. Treating the periosteal-derived cells with recombinant human VEGF₁₆₅ resulted in an increase in Runx2 transactivation in the periosteal-derived cells. These results suggest that exogenous VEGF stimulates the osteoblastic differentiation of cultured human periosteal-derived cells and VEGF might act as an autocrine growth factor for the osteoblastic differentiation of cultured human periosteal-derived cells.     

Evidence of the presence of a specific vascular endothelial growth factor in fetal bovine retina

The presence of a vascular endothelial cell growth factor (VEGF) in the retina was reported in a previous study. The present experiments show that VEGF exhibits a pronounced synergism with the serum-derived factor and the vascular endothelium (VE) effectors in stimulating the proliferation of vascular VE cells. VEGF shows a chromatographic multiplicity with the 25,000-D component as the smallest subunit. Mg2+ is the specific divalent cation that retains the VEGF molecule in the aggregated form and enhances the activity, both total and specific. In addition, VEGF is highly specific for endothelial cells and is distinctly different from FGF, EGF, and insulin in terms of molecular weight (MW) and cell specificity. Under our assay conditions, VEGF has no stimulatory effect on other cell lines examined, including lens epithelial cells, corneal epithelial cells, corneal keratocytes, Walker 256 carcinoma, and fibroblasts. These findings indicate that VEGF possesses characteristic properties not reported for other growth factors, and that VEGF is distinctly different from the growth factors isolated from the retina in other laboratories. The present study suggests that VEGF in the retina represents a new type of growth factor. The need to employ a highly defined assay condition could have eluded the detection of this factor in other laboratories.     

Increased levels of vascular endothelial growth factor and advanced glycation end products in aqueous humor of patients with diabetic retinopathy.

Clinical studies have shown a relationship between diabetic retinopathy and vascular endothelial growth factor (VEGF) levels in ocular fluid. Advanced glycation end products (AGEs) have been implicated in diabetes complications, including diabetic retinopathy. Nepsilon-(carboxymethyl) lysine (CML) is a glycoxidation product that may be a marker of oxidative stress. In this study, we used enzyme-linked immunosorbent assays to determine the levels of VEGF, non-CML AGE and CML in the aqueous humor and serum of 82 Japanese patients with type 2 diabetes and 60 non-diabetic subjects. VEGF, non-CML AGE, and CML concentrations in aqueous humor and serum were then compared with the severity of diabetic retinopathy. Immunohistochemical detection analysis of non-CML AGE and CML was also performed using retinal tissues from patients with progressive diabetic retinopathy. Aqueous levels of VEGF, non-CML AGE and CML increased along with the progression of diabetic retinopathy compared to age-matched controls. After coagulation therapy, the VEGF, non-CML AGE, and CML levels were significantly reduced. Immunostaining showed diffuse co-localization of non-CML AGE and CML around microvessels and in the glial cells of proliferative membranes from patients with progressive diabetic retinopathy. These findings suggest that glycation and glycoxidation reactions (or oxidation, as revealed by CML) may contribute to both the onset and progression of diabetic retinopathy.     

Complexity in the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-receptors signaling

The adult vasculature results from a network of vessels that is originally derived in the embryo by vasculogenesis, a process whereby vessels are formed de novo from endothelial cell (EC) precursors, known as angioblasts. During vasculogenesis, angioblasts proliferate and come together to form an initial network of vessels, also known as the primary capillary plexus. Sprouting and branching of new vessels from the preexisting vessels in the process of angiogenesis remodel the capillary plexus. Normal angiogenesis, a well-balanced process, is important in the embryo to promote primary vascular tree as well as an adequate vasculature from developing organs. On the other hand, pathological angiogenesis which frequently occurs in tumors, rheumatoid arthritis, diabetic retinopathy and other circumstances can induce their own blood supply from the preexisting vasculature in a route that is close to normal angiogenesis. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is perhaps the most important of pro-angiogenic cytokine because of its ability to regulate most of the steps in the angiogenic cascade. The main goal of this review article is to discuss the complex nature of the mode of action of VPF/VEGF on vascular endothelium. To this end, we conclude that more research needs to be done for completely understanding the VPF/VEGF biology with relation to angiogenesis.     

Complexity in the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-receptors signaling

The adult vasculature results from a network of vessels that is originally derived in the embryo by vasculogenesis, a process whereby vessels are formed de novo from endothelial cell (EC) precursors, known as angioblasts. During vasculogenesis, angioblasts proliferate and come together to form an initial network of vessels, also known as the primary capillary plexus. Sprouting and branching of new vessels from the preexisting vessels in the process of angiogenesis remodel the capillary plexus. Normal angiogenesis, a well-balanced process, is important in the embryo to promote primary vascular tree as well as an adequate vasculature from developing organs. On the other hand, pathological angiogenesis which frequently occurrs in tumors, rheumatoid arthritis, diabetic retinopathy and other circumstances can induce their own blood supply from the preexisting vasculature in a route that is close to normal angiogenesis. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is perhaps the most important of pro-angiogenic cytokine because of its ability to regulate most of the steps in the angiogenic cascade. The main goal of this review article is to discuss the complex nature of the mode of action of VPF/VEGF on vascular endothelium. To this end, we conclude that more research needs to be done for completely understanding the VPF/VEGF biology with relation to angiogenesis. (Mol Cell Biochem 264: 51–61, 2004)