Eicosanoid regulation of vascular endothelial growth factor expression and angiogenesis in microvessel endothelial cells

12(R)-Hydroxy-5,8,14-eicosatrienoic acid (HETrE) is a potent inflammatory and angiogenic eicosanoid in ocular and dermal tissues. Previous studies suggested that 12(R)-HETrE activates microvessel endothelial cells via a high affinity binding site; however, the cellular mechanisms underlying 12(R)-HETrE angiogenic activity are unexplored. Because the synthesis of 12(R)-HETrE is induced in response to hypoxic injury, we examined its interactions with vascular endothelial growth factor (VEGF) in rabbit limbal microvessel endothelial cells. Addition of 12(R)-HETrE (0.1 nm) to the cells increased VEGF mRNA levels with maximum 5-fold increase at 45 min. The increase in VEGF mRNA was followed by an increase in immunoreactive VEGF protein. 12(R)-HETrE (0.1 nm) rapidly activated the extracellular signal-regulated kinases (ERKs) ERK1 and ERK2. Moreover, preincubation of cells with PD98059, a selective inhibitor of MEK-1, inhibited 12(R)-HETrE-induced VEGF mRNA. Addition of VEGF antibody to cells grown in Matrigel-coated culture plates inhibited 12(R)-HETrE-induced capillary tube-like formation, suggesting that VEGF mediates, at least in part, the angiogenic response to 12(R)-HETrE. The results indicate that in microvessel endothelial cells, 12(R)-HETrE induces VEGF expression via activation of ERK1/2 and that VEGF mediates, at least in part, the angiogenic activity of 12(R)-HETrE. Given the fact that both VEGF and 12(R)-HETrE are produced in the cornea after hypoxic injury, their interaction may be an important determinant in the development of neovascularized tissues.     

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.     

Role of vascular endothelial growth factor in regulation of physiological angiogenesis

Evidence accumulating over the last decade has established the fundamental role of vascular endothelial growth factor (VEGF) as a key regulator of normal and abnormal angiogenesis. The biological effects of VEGF are mediated by two tyrosine kinase receptors, Flt-1 (VEGFR-1) and KDR (VEGFR-2). The signaling and biological properties of these two receptors are strikingly different. VEGF is essential for early development of the vasculature to the extent that inactivation of even a single allele of the VEGF gene results in embryonic lethality. VEGF is also required for female reproductive functions and endochondral bone formation. Substantial evidence also implicates VEGF as an angiogenic mediator in tumors and intraocular neovascular syndromes, and numerous clinical trials are presently testing the hypothesis that inhibition of VEGF may have therapeutic value.     

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 expression and regulation of murine collagen-induced arthritis

We have examined the expression and function of the angiogenic factor, vascular endothelial growth factor (VEGF) during the evolution of type II collagen-induced arthritis (CIA). Biologically active VEGF was expressed along a time course that paralleled the expression of two specific VEGF receptors, Flk-1 and Flt-1, and the progression of joint disease. Moreover, levels of VEGF expression correlated with the degree of neovascularization, as defined by vWF levels, and arthritis severity. Macrophage- and fibroblast-like cells, which infiltrated inflamed sites and were then activated by other inflammatory mediators, are probably important sources of VEGF and may thus regulate angiogenesis during the development of CIA. Administration of anti-VEGF antiserum to CIA mice before the onset of arthritis delayed the onset, reduced the severity, and diminished the vWF content of arthritic joints. By contrast, administration of anti-VEGF antiserum after the onset of the disease had no effect on the progression or ultimate severity of the arthritis. These data suggest that VEGF plays a crucial role during an early stage of arthritis development, affecting both neovascularization and the progression of experimentally induced synovitis.     

RT-PCR method to quantify vascular endothelial growth factor expression.

The vascular endothelial growth factor (VEGF) is implicated in the progression of cancers. Its expression is well correlated with tumor growth and metastases. The availability of a rapid and sensitive method to detect the amounts of VEGF mRNA in biological samples of limited size, very small biopsies, or samples containing relatively few cells could provide an interesting prognostic tool for clinicians. We have developed an RT-PCR method that allows us to detect the VEGF mRNA from as little as 3 micrograms total mRNA. We have also shown that this protocol can be generalized to all cell lines tested. This method constitutes a very potent tool for the analysis of VEGF mRNA expression in different contexts.     

Characterization of multiple signaling pathways of insulin in the regulation of vascular endothelial growth factor expression in vascular cells and angiogenesis

The effects of insulin on vascular endothelial growth factor (VEGF) expression in cultured vascular cells and in angiogenesis were characterized. Insulin increased VEGF mRNA levels in mouse aortic smooth muscle cells from 10(-9) to 10(-7) m with an initial peak of 3.7-fold increases at 1 h and a second peak of 2.8-fold after 12 h. The first peak of VEGF expression was inhibited by LY294002, an inhibitor of phosphatidylinositol (PI) 3-kinase, and by the overexpression of dominant negative forms of p85 subunit of PI 3-kinase or Akt. Inhibitors of MEK kinase, PD98059, or overexpression of dominant negative forms of Ras was ineffective. In contrast, the chronic effect of insulin on VEGF expression was partially inhibited by both LY294002 or PD98059 as well as by the overexpression of dominant negatives of PI 3-kinase or Ras. The importance of PI 3-kinase-Akt pathway on VEGF expression was confirmed in mouse aortic smooth muscle cells isolated from insulin receptor substrate -1 knockout (IRS-1-/-) mice that showed parallel reductions of 46-49% in insulin-stimulated VEGF expression and PI 3-kinase-Akt activation. Insulin-induced activation of PI 3-kinase-Akt on hypoxia-induced VEGF expression and neovascularization was reduced by 40% in the retina of neonatal hypoxia model using IRS-1-/- mice. Thus, unlike other cells, insulin can regulate VEGF expression by both IRS-1/PI 3-kinase-Akt cascade and Ras-MAPK pathways in aortic smooth muscle cells. The in vivo results provide direct evidence that insulin can modulate hypoxia-induced angiogenesis via reduction in VEGF expression in vivo.     

Expression and regulation of vascular endothelial growth factor in human pulmonary epithelial cells

Vascular endothelial growth factor (VEGF) is a potent endothelial cell growth and permeability factor highly expressed in rodent alveolar epithelium after injury and repair. To investigate VEGF synthesis in human lung epithelial cells, we examined VEGF expression by cultured cells under basal conditions and after cytokine treatment or oxidative stress. Basal VEGF expression was detected in transformed human epithelial cell lines (A549 and 1HAEo-) and in primary human bronchial epithelial cells with RT-PCR, Western blot, and immunocytochemistry. Among the cytokines tested, only transforming growth factor-beta1 increased the levels of excreted VEGF(165) as measured by ELISA. Under hypoxia (0% O(2) for 24 h), the VEGF(165) level increased fivefold, and this effect was O(2) concentration dependent. VEGF concentrations in the medium of all the cell types studied reached values similar to those found in bronchoalveolar lavage fluids from normal patients. Endothelial cells (human umbilical vein endothelial cells) exposed to conditioned medium from primary bronchial epithelial cell cultures showed an increased growth rate, which was inhibited in the presence of a specific neutralizing antibody to VEGF. These results suggest that lung epithelial cells participate in the endothelial repair and angiogenesis that follow lung injury through the synthesis of VEGF.     

Inhibition of vascular endothelial growth factor expression by TRUE gene silencing

Pathogenic angiogenesis in various diseases including cancer, autoimmune diseases, and age-related macular degeneration is thought to be regressed with anti-angiogenic drugs. TRUE gene silencing is a new technology to eliminate a specific mRNA using synthetic sgRNA and cellular tRNase Z^L. To discover anti-angiogenic sgRNAs, we applied TRUE silencing to the VEGF gene. We examined eight sgRNAs for efficacy in targeting exogenous human VEGF mRNA. Many of them worked efficiently in 293 and HeLa cells. Two of them downregulated the endogenous VEGF gene expression in HeLa cells very efficiently, and the efficacy of these two sgRNAs surpassed that of siRNA extremely.