Cyclic stretch and hypertension induce retinal expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2: potential mechanisms for exacerbation of diabetic retinopathy by hypertension.

Systemic hypertension exacerbates diabetic retinopathy and other coexisting ocular disorders through mechanisms that remain largely unknown. Increased vascular permeability and intraocular neovascularization characterize these conditions and are complications primarily mediated by vascular endothelial growth factor (VEGF). Because systemic hypertension increases vascular stretch, we evaluated the expression of VEGF, VEGF-R2 (kinase insert domain-containing receptor [KDR]), and VEGF-R1 (fms-like tyrosine kinase [Flt]) in bovine retinal endothelial cells (BRECs) undergoing clinically relevant cyclic stretch and in spontaneously hypertensive rat (SHR) retina. A single exposure to 20% symmetric static stretch increased KDR mRNA expression 3.9 +/- 1.1-fold after 3 h (P = 0.002), with a gradual return to baseline within 9 h. In contrast, BRECs exposed to cardiac-profile cyclic stretch at 60 cpm continuously accumulated KDR mRNA in a transcriptionally mediated, time-dependent and stretch-magnitude-dependent manner. Exposure to 9% cyclic stretch increased KDR mRNA expression 8.7 +/- 2.9-fold (P = 0.011) after 9 h and KDR protein concentration 1.8 +/- 0.3-fold (P = 0.005) after 12 h. Stretched-induced VEGF responses were similar. Scatchard binding analysis demonstrated a 180 +/- 40% (P = 0.032) increase in high-affinity VEGF receptor number with no change in affinity. Cyclic stretch increased basal thymidine uptake 60 +/- 10% (P < 0.001) and VEGF-stimulated thymidine uptake by 2.6 +/- 0.2-fold (P = 0.005). VEGF-NAb reduced cyclic stretch-induced thymidine uptake by 65%. Stretched-induced KDR expression was not inhibited by AT1 receptor blockade using candesartan. Hypertension increased retinal KDR expression 67 +/- 42% (P < 0.05) in SHR rats compared with normotensive WKY control animals. When hypertension was reduced using captopril or candesartan, retinal KDR expression returned to baseline levels. VEGF reacted similarly, but Flt expression did not change. These data suggest a novel molecular mechanism that would account for the exacerbation of diabetic retinopathy by concomitant hypertension, and may partially explain the principal clinical manifestations of hypertensive retinopathy itself. Furthermore, these data imply that anti-VEGF therapies may prove therapeutically effective for hypertensive retinopathy and/or ameliorating the deleterious effects of coexistent hypertension on VEGF-associated disorders such as diabetic retinopathy.

Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase-akt-dependent pathways in retinal vascular cells.

Fibroblastic proliferation accompanies many angiogenesis-related retinal and systemic diseases. Since connective tissue growth factor (CTGF) is a potent mitogen for fibrosis, extracellular matrix production, and angiogenesis, we have studied the effects and mechanism by which vascular endothelial growth factor (VEGF) regulates CTGF gene expression in retinal capillary cells. In our study, VEGF increased CTGF mRNA levels in a time- and concentration-dependent manner in bovine retinal endothelial cells and pericytes, without the need of new protein synthesis and without altering mRNA stability. VEGF activated the tyrosine receptor phosphorylation of KDR and Flt1 and increased the binding of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit to KDR and Flt1, both of which could mediate CTGF gene induction. VEGF-induced CTGF expression was mediated primarily by PI3-kinase activation, whereas PKC and ERK pathways made only minimal contributions. Furthermore, overexpression of constitutive active Akt was sufficient to induce CTGF gene expression, and inhibition of Akt activation by overexpressing dominant negative mutant of Akt abolished the VEGF-induced CTGF expression. These data suggest that VEGF can increase CTGF gene expression in bovine retinal capillary cells via KDR or Flt receptors and the activation of PI3-kinase-Akt pathway independently of PKC or Ras-ERK pathway, possibly inducing the fibrosis observed in retinal neovascular diseases.

Retinal expression, regulation, and functional bioactivity of prostacyclin-stimulating factor.

Prostacyclin-stimulating factor (PSF) acts on vascular endothelial cells to stimulate the synthesis of the vasodilatory molecule prostacyclin (PGI2). We have examined the expression, regulation, and hemodynamic bioactivity of PSF both in whole retina and in cultured cells derived from this tissue. PSF was expressed in all retinal cell types examined in vitro, but immunohistochemical analysis revealed PSF mainly associated with retinal vessels. PSF expression was constitutive in retinal pericytes (RPCs) but could be modulated in bovine retinal capillary endothelial cells (RECs) by cell confluency, hypoxia, serum starvation, high glucose concentrations, or inversely by soluble factors present in early vs. late retinopathy, such as TGF-beta, VEGF, or bFGF. In addition, RPC-conditioned media dramatically increased REC PGI2 production, a response inhibited by blocking PSF with a specific antisense oligodeoxynucleotide (ODN). In vivo, PGI2 increased retinal blood flow (RBF) in control and diabetic animals. Furthermore, the early drop in RBF during the initial weeks after inducing diabetes in rats, as well as the later increase in RBF, both correlated with levels of retinal PSF. RBF also responded to treatment with RPC-conditioned media, and this effect could be partially blocked using the antisense PSF ODN. We conclude that PSF expressed by ocular cells can induce PGI2, retinal vascular dilation, and increased retinal blood flow, and that alterations in retinal PSF expression may explain the biphasic changes in RBF observed in diabetes.

17 Beta-estradiol increases VEGF receptor-2 and promotes DNA synthesis in retinal microvascular endothelial cells.

PURPOSE: Estrogen is known to promote angiogenesis in gonads. The presence of estrogen receptors in the vascular endothelium of organs other than gonads has been reported. The goal of this study was to determine whether estrogen promotes the proliferation of retinal microvascular endothelial cells and to explore the mechanism of it. METHODS: DNA was quantitated using primary cultures of bovine retinal endothelial cells that were incubated with different doses of 17 beta-estradiol (E2), VEGF, or both. The changes in expression level of VEGF and VEGF receptor-2 (VEGFR2) were measured using northern blot analysis after treatment with E2. The presence of estrogen receptors in the endothelial cells was studied by immunohistochemistry and western blot analysis. RESULTS: 17 Beta-estradiol (E2) increased the DNA level in bovine retinal capillary endothelial cells (BRECs) by 177% at 1 nM (P < 0.05) and 150% at 10 nM (P < 0.05) by comparison with unstimulated BREC. One hundred nanomole tamoxifen completely blocked the E2-induced DNA synthesis in BRECs. Ten nanomole E2 augmented vascular endothelial growth factor (VEGF)-induced DNA synthesis in BRECs significantly (160%, P < 0.01). Ten nanomole E2 also increased VEGF mRNA expression, which peaked after 24 hours (6.7 times, P < 0.05), and VEGF receptor-2 (VEGFR2) mRNA expression, which peaked after 9 hours (2.4 times, P < 0.05). The mRNA expression level of VEGFR2 peaked with 10 nM E2 (P < 0.05) and that of VEGF reached maximum with 1 nM E2 (15 times, P < 0.001). VEGFR2 and VEGF proteins increased in parallel with their mRNA levels. Immunocytochemistry showed estrogen receptor expression in BRECs, and western blot analysis indicated the presence of a 67-kDa protein that was compatible with the estrogen receptor. CONCLUSIONS: These findings suggest that E2 may stimulate BREC growth by the receptor-mediated pathway and that E2 may augment the VEGF-dependent angiogenesis partly through the upregulation of VEGFR2.

Estrogen protects against cellular infiltration by reducing the expressions of E-selectin and IL-6 in endotoxin-induced uveitis.

Anterior uveitis associated with Behcet's disease and ankylosing spondylitis preferentially occurs in adult men, which may suggest the effects of sex hormones on acute anterior uveitis. Recently, estrogen receptors in the vascular endothelium have been reported to be involved in several pathological conditions. In the present study, we examined the gender differences in susceptibility to endotoxin-induced uveitis (EIU) and the effects of estrogen on anterior inflammation. EIU was induced in adult male, female, and ovariectomized female Lewis rats (200 g) by hind footpad injection of 200 microg of LPS. In EIU, cellular infiltration was more marked in male than in female rats, and ovariectomy increased cellular infiltration. Treatment with 10 microg of 17beta-estradiol significantly reduced the cell number in male and ovariectomized female rats with EIU. Estrogen receptor immunoreactivity was found in the nucleus of vascular endothelium and in some stromal cells of the iris-ciliary body. Semiquantitative PCR revealed that E-selectin and IL-6 gene expressions were increased in rats following LPS injection, and an overdose of tamoxifen, an estrogen receptor antagonist, reversed the effect of 17beta-estradiol on E-selectin, but not its effect on IL-6. These observations suggested that the down-modulation of these inflammatory genes by estrogen may contribute to the reduction in cellular infiltration in acute anterior uveitis.

Quantitative analysis of diabetic macular edema after scatter laser photocoagulation with the scanning retinal thickness analyzer.

PURPOSE: To define the effect of scatter laser photocoagulation on foveal retinal thickness. METHODS: A commercial scanning retinal thickness analyzer was used to measure retinal thickness. The foveal retinal thickness was measured at the central area of the fundus (0.4 x 0.4 mm). The method was applied to 20 consecutive patients (mean age, 52.4 +/-16.9 years) with diabetic retinopathy. Measurements were performed before and 6 weeks after scatter photocoagulation. Patients were examined by fluorescein angiography and slit-lamp biomicroscopy to detect macular edema. RESULTS: Mean foveal thickness before scatter photocoagulation was 187+/-45 microm, increasing to 221+/-46 microm after the treatment (P = 0.0001). The foveal thickness increased in 12 eyes (60%). Laser treatment increased macular permeability in two eyes (10%). Biomicroscopic examination revealed central macular thickening in one eye (5%). Visual acuity was reduced in four eyes (20%). CONCLUSIONS: Our results suggest that subclinical macular edema occurs after scatter laser photocoagulation. The retinal thickness analyzer is a sensitive tool for early detection of macular edema after laser treatment, because increases in retinal thickness as small as 34 microm cannot be assessed by slit-lamp biomicroscopy.

Increased expression of KDR/Flk-1 (VEGFR-2) in murine model of ischemia-induced retinal neovascularization.

Although the vascular endothelial growth factor (VEGF)/VEGF receptor system plays a critical role in the pathogenesis of ischemic retinal neovascular diseases such as diabetic retinopathy, regulation of VEGF receptor expression in ischemic retina has not been fully investigated in vivo. Accordingly, we studied the regulation of Flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2) expression in a mouse model of ischemia-induced retinal neovascularization. Immunohistochemistry for Flt-1 and KDR/Flk-1 revealed that, in hypoxic retina, the immunoreactivity of KDR/Flk-1 was increased in both intensity and extent of involvement in the vessels near the avascular area, particularly at the neovascular tufts, but that the pattern of Flt-1 expression in hypoxic retina was almost the same as that of control animals. The number of KDR/Flk-1-positive vessels was significantly increased in hypoxic retina (P < 0.01). In addition, expression of both Flt-1 and KDR/Flk-1 was observed in nonvascular cells of the neural retina. Northern blot analysis demonstrated that the mRNA levels of KDR/Flk-1 were greater in the neovascular retina of hypoxic animals than in control animals. We suggest that the increased expression of KDR/Flk-1 in vascular cells might potentiate the VEGF-mediated angiogenesis that accompanies many ischemic retinal diseases.

Contribution of E-selectin to cellular infiltration during endotoxin-induced uveitis.

PURPOSE: The selectin family is a group of early-reactive adhesion molecules that plays a role in the rolling phase of leukocytes in cellular infiltration. It has been reported that P-selectin is expressed on vascular endothelium in the iris-ciliary body 15 minutes after lipopolysaccharide (LPS) treatment in endotoxin-induced uveitis (EIU) and may contribute to the initial phase of ocular inflammation. The objective of the present study was to identify the expression pattern of E-selectin, another member of the selectin family, and to investigate the role of E-selectin during the course of EIU. METHODS: Endotoxin-induced uveitis was induced in male Lewis rats by a footpad injection of 200 microg LPS. The time-dependent expressions of E-selectin in EIU in the iris- ciliary body and the retina were studied by immunohistochemistry using wholemounts and paraffin-embedded sections and by monitoring the level of E-selectin mRNA expression. A monoclonal antibody to E-selectin and a control antibody were each injected intravenously to evaluate the effects of E-selectin inhibition on ocular inflammation at the time of maximum uveitis. In the anterior uvea, the effect was evaluated by the number of infiltrated cells and by the protein concentration in the aqueous humor 24 hours after LPS treatment; in the retina, the myeloperoxidase (MPO) activity was measured 48 hours after LPS treatment. The effect of the combined injection of anti-P-selectin and anti-E-selectin antibodies was also studied. It was then determined whether the delayed inhibition of E-selectin (6, 12, or 24 hours after LPS injection) could contribute to the early resolution of the uveitis. RESULTS: E-selectin immunoreactivity was observed on the vessel walls of the iris and retina 7 hours after LPS treatment in wholemounts and paraffin-embedded sections and remained positive for 24 hours after LPS treatment. The expression of E-selectin messenger RNA gene peaked at 6 hours and again at 18 hours after LPS treatment in the iris- ciliary body and retina. The expression returned to the basal level 24 hours after LPS treatment in the iris- ciliary body and 48 hours after LPS treatment in the retina. The selective inhibition of E-selectin significantly blocked the cellular infiltration into the aqueous humor (P < 0.005) but had a milder effect on the protein concentration in the aqueous humor (P=0.0536). The inhibition of E-selectin and P-selectin almost abrogated cellular infiltration (P < 0.001). Myeloperoxidase activity in the retina 48 hours after LPS treatment was again significantly decreased by the inhibition of E-selectin alone and by the inhibition of E-selectin and P-selectin (P < 0.0001). A single injection of anti-E-selectin antibody 6, 12, or 24 hours after LPS injection effectively blocked cellular infiltration in the aqueous humor 24 and 48 hours after LPS treatment. CONCLUSIONS: In EIU, E-selectin may be expressed on the vascular endothelium and remain after the period of expression of P-selectin and until approximately the time of maximum uveitis. The present results suggest that, in contrast to the role of P-selectin as an initiator of cellular infiltration, E-selectin may contribute to continuing cellular infiltration into the inflammatory site during inflammation, and its inhibition may contribute to the early resolution of the uveitis.

Quantitative analysis of foveal retinal thickness in diabetic retinopathy with the scanning retinal thickness analyzer.

PURPOSE: This study sought to measure foveal retinal thickness in patients with diabetic retinopathy and to investigate the relationship between foveal thickness and visual acuity, biomicroscopic findings, and angiographic features. METHODS: A commercial scanning retinal thickness analyzer was used to measure retinal thickness. A laser slit was projected onto the retina and scanned in 400 milliseconds across the central area of the fundus. The image where the laser slit intersects with the retina was digitally recorded and analyzed. Retinal thickness was measured in 35 patients (35 eyes; patient age, 57 +/- 13 years) with diabetic retinopathy. Patients also were examined by fluorescein angiography and slit-lamp biomicroscopy to detect foveal thickening. RESULTS: Linear regression analysis indicated a significant correlation between foveal thickness and visual acuity (adjusted R2 = 0.72, P < 0.001). Foveal thickness was abnormal in 6 (100%) of 6 eyes in which foveal thickening was detected with slit-lamp biomicroscopy. Foveal thickness also was abnormal in 9 (31%) of 29 eyes that appeared normal by biomicroscopic examination. Foveal thickness was 136 +/- 65 microns in 7 eyes without leakage, 175 +/- 35 microns in 13 eyes with questionable leakage, and 291 +/- 120 microns in 7 eyes with definite leakage (P = 0.0075). CONCLUSIONS: Retinal thickness analysis is shown to be more sensitive than slit-lamp biomicroscopy for detecting small changes in retinal thickness. Retinal thickness analysis may prove to be a useful, noninvasive modality for the development or regression of macular edema.