Shuhei Kawahara

Rho Kinase Inhibition by Fasudil Ameliorates Diabetes-Induced Microvascular Damage

OBJECTIVE—Leukocyte adhesion in retinal microvasuculature substantially contributes to diabetic retinopathy. Involvement of the Rho/Rho kinase (ROCK) pathway in diabetic microvasculopathy and therapeutic potential of fasudil, a selective ROCK inhibitor, are investigated. RESEARCH DESIGN AND METHODS—Localization of RhoA/ROCK and Rho activity were examined in retinal tissues of rats. Impact of intravitreal fasudil administration on retinal endothelial nitric oxide synthase (eNOS) and myosin phosphatase target protein (MYPT)-1 phosphorylation, intercellular adhesion molecule-1 (ICAM-1) expression, leukocyte adhesion, and endothelial damage in rat eyes were investigated. Adhesion of neutrophils from diabetic retinopathy patients or nondiabetic control subjects to cultured microvascular endothelial cells was quantified. The potential of fasudil for endothelial protection was investigated by measuring the number of adherent neutrophils and terminal transferase-mediated dUTP nick-end labeling–positive endothelial cells. RESULTS—RhoA and ROCK colocalized predominantly in retinal microvessels. Significant Rho activation was observed in retinas of diabetic rats. Intravitreal fasudil significantly increased eNOS phosphorylation, whereas it reduced MYPT-1 phosphorylation, ICAM-1 expression, leukocyte adhesion, and the number of damaged endothelium in retinas of diabetic rats. Neutrophils from diabetic retinopathy patients showed significantly higher adhesion to cultured endothelium and caused endothelial apoptosis, which was significantly reduced by fasudil. Blockade of the Fas-FasL interaction prevented endothelial apoptosis. The protective effect of fasudil on endothelial apoptosis was significantly reversed by Nω-nitro-l-arginine methyl ester, a NOS inhibitor, whereas neutrophil adhesion remained unaffected. CONCLUSIONS—The Rho/ROCK pathway plays a critical role in diabetic retinal microvasculopathy. Fasudil protects the vascular endothelium by inhibiting neutrophil adhesion and reducing neutrophil-induced endothelial injury. ROCK inhibition may become a new strategy in the management of diabetic retinopathy, especially in its early stages.

Role of TGF-β in proliferative vitreoretinal diseases and ROCK as a therapeutic target

Cicatricial contraction of preretinal fibrous membrane is a cause of severe vision loss in proliferative vitreoretinal diseases such as proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). TGF-β is overexpressed in the vitreous of patients with proliferative vitreoretinal diseases and is also detectable in the contractile membranes. Therefore, TGF-β is presumed to contribute to the cicatricial contraction of the membranes, however, the underlying mechanisms and TGF-βs importance among various other factors remain to be elucidated. Vitreous samples from PDR or PVR patients caused significantly larger contraction of hyalocyte-containing collagen gels, compared with nonproliferative controls. The contractile effect was strongly correlated with the vitreal concentration of activated TGF-β2 (r = 0.82, P < 0.0001). PDR or PVR vitreous promoted expression of α-smooth muscle actin (α-SMA) and phosphorylation of myosin light chain (MLC), a downstream mediator of Rho-kinase (ROCK), both of which were dramatically but incompletely suppressed by TGF-β blockade. In contrast, fasudil, a potent and selective ROCK inhibitor, almost completely blocked the vitreous-induced MLC phosphorylation and collagen gel contraction. Fasudil disrupted α-SMA organization, but it did not affect its vitreal expression. In vivo, fasudil significantly inhibited the progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells by electroretinographic and histological analyses. These results elucidate the critical role of TGF-β in mediating cicatricial contraction in proliferative vitreoretinal diseases. ROCK, a key downstream mediator of TGF-β and other factors might become a unique therapeutic target in the treatment of proliferative vitreoretinal diseases.

Possible contribution of hyalocytes to idiopathic epiretinal membrane formation and its contraction

Aim: To address the cellular components and the contractile mechanisms of the idiopathic epiretinal membrane (ERM). Methods: Ten surgically removed ERMs were fixed in 4% paraformaldehyde and analysed by whole-mount immunohistochemistry with anti-glial fibrillar acidic protein (GFAP) and alpha smooth-muscle actin (αSMA) antibodies. Type I collagen gel contraction assay, an established wound-healing assay in vitro, was performed using cultured bovine hyalocytes or normal human astrocytes (NHA) to evaluate the contractile property of the cells in the presence of tissue growth factor (TGF)-β2. The expression of αSMA was also analysed by western blot analysis to examine myofibroblastic transdifferentiation of the cells. Vitreous-induced collagen gel contraction was also evaluated. Results: All membranes were composed of αSMA immunopositive cells in contracted foci and GFAP immunopositive cells in the periphery. No apparent double positive cells were observed in any membranes examined. Cultured hyalocytes showed overexpression of αSMA and hypercontraction of collagen gels in response to TGF-β2, while glial cells showed marginal change. The vitreous from ERM patients also caused overexpression of αSMA and hypercontraction of the gels embedding hyalocytes, which were almost completely inhibited in the presence of anti-TGF-β2 neutralising antibody. Conclusions: Hyalocytes might be one of the critical components of ERM mediating its contractile property through the effect of TGF-β2 in the vitreous fluid.

Antiangiogenic properties of fasudil, a potent Rho-Kinase inhibitor

PurposeVascular endothelial growth factor (VEGF) plays a pivotal role in pathological angiogenesis. In this study, we addressed the therapeutic potential of fasudil, a potent Rho-kinase inhibitor, for VEGF-elicited angiogenesis and also for the intracellular signalings induced by VEGF.MethodsIn vitro, the inhibitory effects of fasudil on the VEGF-dependent VEGF receptor 2 (VEFGR2 or KDR), extracellular signal-related kinase (ERK) 1/2, Akt and myosin light chain (MLC) phosphorylation, as well as on the migration and proliferation of bovine retinal microvascular endothelial cells (BRECs) were analyzed with Western blotting, [3H]-thymidine uptake, and modified Boyden chamber assay. VEGF-elicited in vivo angiogenesis was analyzed with a mouse corneal micropocket assay coembedded with or without fasudil.ResultsVEGF caused enhanced MLC phosphorylation of BRECs, which was almost completely attenuated by 10μM fasudil. VEGF-dependent phosphorylation of ERK1/2 and Akt were also partially but significantly attenuated by treatment with fasudil without affecting VEGFR2 (KDR) phosphorylation. Moreover, both VEGF-induced [3H]-thymidine uptake and the migration of BRECs were significantly inhibited in the presence of fasudil. Finally, VEGF-elicited angiogenesis in the corneal micropocket assay was potently attenuated by coembedding with fasudil (P < 0.01).ConclusionsThese findings indicate that fasudil might have a therapeutic potential for ocular angiogenic diseases. The antiangiogenic effect of fasudil appears to be mediated through the blockade not only of Rho-kinase signaling but also of ERK and Akt signaling.

Functional characteristics of connective tissue growth factor on vitreoretinal cells.

Connective tissue growth factor (CTGF) level is elevated in eyes with proliferative vitreoretinal diseases, such as proliferative diabetic retinopathy and proliferative vitreoretinopathy (PVR), as we previously reported, but its functional characteristics on vitreoretinal cells are yet to be clarified. In this study, we demonstrated a growth-promoting effect of CTGF on cultured hyalocytes and bovine retinal pigment epithelial cells (BRPEs) with the induction of p44/p42 mitogen-activated protein kinase phosphorylation and [3H]thymidine incorporation. CTGF also stimulated the synthesis of fibronectin by hyalocytes and BRPEs without significant effect on collagen gel contraction by these cells. On the other hand, CTGF had no direct effects on the proliferation, migration, or in vitro tube formation by vascular endothelial cells. Nevertheless, CTGF promoted vascular endothelial growth factor (VEGF) gene expression by hyalocytes and BRPEs. Although the concentrations of both CTGF and VEGF in the human vitreous samples with proliferative vitreoretinal diseases were elevated, there was no significant correlation between these concentrations. These findings indicate that CTGF appears to be involved in the formation of proliferative membranes without direct regulation of their cicatricial contraction in the pathogenesis of proliferative vitreoretinal diseases. Whereas CTGF might have no direct effects or minimal effects, if any, on retinal neovascularization, it is possible that CTGF has indirect effects by modulating the expression of VEGF.

Residual internal limiting membrane in epiretinal membrane surgery

Background/aim: To examine the degree of the residual internal limiting membrane (ILM) after epiretinal membrane (ERM) peeling. Methods: Sixty-one eyes of 59 patients with ERM were enrolled. After ERM peeling, residual ILM was visualised with Brilliant Blue G (BBG). The residual ILM pattern was divided into three groups: (1) residual type (ILM mostly remained), (2) half type (approximately half of ILM remaind), (3) no residual type (ILM mostly removed with ERM). If ILM remained, residual ILM was removed in all cases and histologically examined using the flat mount method in 10 cases. The correlation between the degree of ERM evaluated by preoperative best-corrected visual acuity (BCVA) and residual ILM pattern was also examined. Results: Twenty-eight eyes (45.9%) were of the residual type. Three eyes (4.9%) were of the half type, and 30 eyes (49.2%) were of no residual type. The mean preoperative BCVA showed no significant correlation with the residual ILM pattern. Flat mount immunohistochemistry revealed many remnant cells, both glial fibrillar acidic protein positive and negative, on residual ILMs in all specimens examined. No recurrence that needed surgical treatment was observed. Conclusion: Residual ILM with remnant cells seems to be frequent after ERM removal. Intraoperative staining with BBG may be helpful in determining the extent of ILM removal.

Blood vessel endothelial VEGFR-2 delays lymphangiogenesis: an endogenous trapping mechanism links lymph- and angiogenesis.

Angio- and lymphangiogenesis are inherently related processes. However, how blood and lymphatic vessels regulate each other is unknown. This work introduces a novel mechanism explaining the temporal and spatial relation of blood and lymphatic vessels. Vascular endothelial growth factor-A (VEGF-A) surprisingly reduced VEGF-C in the supernatant of blood vessel endothelial cells, suggesting growth factor (GF) clearance by the growing endothelium. The orientation of lymphatic sprouting toward angiogenic vessels and away from exogenous GFs was VEGF-C dependent. In vivo molecular imaging revealed higher VEGF receptor (R)-2 in angiogenic tips compared with normal vessels. Consistently, lymphatic growth was impeded in the angiogenic front. VEGF-C/R-2 complex in the cytoplasm of VEGF-A-treated endothelium indicated that receptor-mediated internalization causes GF clearance from the extracellular matrix. GF clearance by receptor-mediated internalization is a new paradigm explaining various characteristics of lymphatics.

Histopathology of Neovascular Tissue From Eyes With Proliferative Diabetic Retinopathy After Intravitreal Bevacizumab Injection

PURPOSE To examine the histopathologic effect of a single intravitreal injection of bevacizumab on newly formed vessels in eyes with proliferative diabetic retinopathy (PDR). DESIGN Interventional case series and laboratory investigation. METHODS Two days after intravitreal injection of bevacizumab (1.25 mg/eye), pars plana vitrectomy or trabeculectomy was performed for the treatment of PDR or neovascular glaucoma (NVG) associated with PDR. Ten surgically removed preretinal proliferative tissues and 6 deep scleral flaps containing trabecular meshwork were fixed in 2% glutaraldehyde or 4% paraformaldehyde and were subjected to transmission electron microscopic analysis, immunohistochemical analysis, and terminal deoxyuridiine triphosphate (dUTP) nick-end labeling staining. Two surgically removed preretinal proliferative tissues and 2 deep scleral flaps from patients with PDR and NVG, but without preoperative intravitreal injection of bevacizumab (IVB), served as controls. RESULTS In control tissues, vascular endothelial cells possessed many fenestrations and were accompanied by pericytes. Apoptotic vascular endothelial cells frequently were observed in tissue after intravitreal injection of bevacizumab, whereas they were not observed in control tissues. Additionally, no apparent fenestration was observed in newly formed vessels from either proliferative tissue or trabecular meshwork after intravitreal injection of bevacizumab. In both PDR and NVG tissues after intravitreal injection of bevacizumab, overexpression of smooth muscle actin was observed in newly formed vessels, suggesting that the treatment may have increased pericytes on the vasculature as compared with control tissue. CONCLUSIONS Intravitreal injection of bevacizumab may induce changes in immature, newly formed vessels of PDR or NVG tissue, leading to endothelial apoptosis with vascular regression, while inducing normalization of premature vessels by increasing pericyte coverage and reducing vessel fenestration.

A key role for ROCK in TNF-α-mediated diabetic microvascular damage.

PURPOSE Leukocyte adhesion releases tumor necrosis factor (TNF)-α that contributes to endothelial damage in early diabetic retinopathy (DR). Rho/Rho-kinase (ROCK) signaling mediates retinal endothelial damage in early DR. However, whether ROCK regulates TNF-α-mediated diabetic vascular damage is unknown. Here, the contribution of ROCK to TNF-α-mediated microvascular damage is investigated. METHODS In DR patients and nondiabetic control subjects, the levels of membranous (m) TNF-α on neutrophils, soluble (s) TNF-α and its receptors in sera, were measured. In cultured microvascular endothelial cells, phosphorylation of myosin phosphatase target protein (MYPT)-1, a downstream target of ROCK, was investigated with TNF-α or DR sera pretreatment. TNF-α-induced intercellular adhesion molecule-1 (ICAM-1) and endothelial nitric oxide synthase (eNOS) phosphorylation were measured with and without ROCK inhibition by fasudil or ROCK-specific small-interfering RNA (siRNA). In isolated neutrophils from control subjects, MYPT-1 phosphorylation was investigated in the presence of TNF-α. The impact of ROCK inhibition by fasudil on TNF-α-induced integrin (CD18, CD11a, CD11b) and intracellular cytoskeletal changes were investigated. RESULTS The serum levels of mTNF-α, sTNF-α, and its receptors were significantly elevated in DR patients. TNF-α as well as DR sera promoted MYPT-1 phosphorylation in endothelial cells, which was significantly reduced by anti-TNF-α neutralizing antibody. TNF-α-induced ICAM-1 expression, eNOS dephosphorylation, cytoskeletal changes, and CD11b/18 expression in neutrophils were significantly suppressed by fasudil as well as ROCK-specific siRNA. CONCLUSIONS ROCK is a key mediator of TNF-α signaling in diabetic microvessels. The important role of TNF-α in early DR provides a new rationale for ROCK inhibition beyond the previously shown mechanisms.

Vascular endothelial growth factor expression by hyalocytes and its regulation by glucocorticoid.

Aim: Tumour necrosis factor-α (TNF-α) is one of the major inflammatory cytokines involved in the pathogenesis of various vitreoretinal diseases. The authors investigated the effect of hypoxia, TNF-α and dexamethasone on vascular endothelial growth factor (VEGF) expression by cultured hyalocytes. Methods: Hyalocytes were isolated from bovine vitreous. Hypoxic and TNF-α-dependent effects on cultured hyalocytes were investigated using several assays to determine VEGF protein expression, hypoxia-inducible factor (HIF)-1α protein levels, HIF-1α-DNA-binding ability and VEGF mRNA stability. The effects of dexamethasone on VEGF expression and its intracellular signalling under hypoxic or TNF-α stimulated conditions were also examined. Results: Hypoxic conditions and TNF-α stimulation induce VEGF expression in hyalocytes. These stimuli also stabilise HIF-1α protein and increase its DNA-binding ability. Dexamethasone significantly inhibits both HIF-1α protein levels and HIF-1α-DNA-binding activity, and also decreases the hypoxic- and TNF-α -dependent induction of VEGF expression in hyalocyte. However, dexamethasone has no significant effect on the stability of VEGF mRNA. Conclusions: Hyalocytes may be involved in various vitreoretinal diseases by increasing HIF-1α protein stability and HIF-1α-DNA binding, and thus increasing VEGF production under pathological conditions. Dexamethasone seems to be capable of inhibiting hypoxic and TNF-α dependent VEGF production, presumably via its inhibitory effects on HIF-1α protein levels and its DNA-binding activity.