ROCK Inhibition by Fasudil Ameliorates Diabetes-Induced Microvascular Damage Short running title: Fasudil ameliorates diabetic microvasculopathy

OBJECTIVE: Leukocyte adhesion in retinal microvasuculature substantially contributes to diabetic retinopathy (DR). Involvement of the Rho/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 rats’ retinal tissues. Impact of intravitreal fasudil administration on retinal eNOS and MYPT-1 phosphorylation, ICAM-1 expression, leukocyte adhesion, and endothelial damage in rat eyes were investigated. Adhesion of neutrophils from DR patients or non-diabetic control subjects to cultured microvascular endothelial cells was quantified. Fasudil’s potential for endothelial protection was investigated by measuring the number of adherent neutrophils and TUNEL-positive endothelial cells. RESULTS: RhoA and ROCK co-localized predominantly in retinal micro-vessels. Significant Rho activation was observed in retinas of diabetic rats. Intravitreal fasudil significantly increased eNOS phosphorylation, while it reduced MYPT-1 phosphorylation, ICAM-1 expression, leukocyte adhesion, and the number of damaged endothelium in retinas of diabetic rats. Neutrophils from DR 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 L-NAME, a NOS inhibitor, while neutrophil adhesion remained unaffected. CONCLUSION: 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 DR, especially in its early stages. Diabetic retinopathy (DR), a prevalent complication of diabetes, is a leading cause of vision loss (1). Early non-proliferative stages of DR are characterized by retinal microvascular damage that leads to vascular hyperpermeability. While visual acuity is rarely affected in this early stage, progression of the diseased states leads to proliferative retinopathy that can cause severe vision loss. Vitreous hemorrhage caused by a rupture of neovascular vessels and tractional retinal detachment associated with a cicatrical contraction of proliferative membranes are hallmark of the later DR stages. Despite considerable recent advances in vitreoretinal surgery, generally performed in later DR stages, a satisfying visual acuity cannot be always achieved. Even in the early DR stages that might be detected by routine eye exams, management of general factors, such as blood glucose level (2) and blood pressure (3), currently constitute the only preventive measures. New approaches for amelioration and treatment of DR, especially in the earlier stages, are thus urgently needed. The various clinical findings in the retinal vasculature throughout DR, such as capillary occlusion and leakage, are related to endothelial damage secondary to increased leukocyte adhesion at least in part (4,5). Diabetic leukocyte adhesion in retinal microvessels is mediated by adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1) and leukocyte β2-integrins (CD18/CD11a and CD18/CD11b) (6). In experimental diabetes, the expressions of these molecules are increased, and their blockade prevents leukocyte adhesion and endothelial damage (7). The interaction of endothelial Fas with Fas ligand (FasL), expressed on adherent neutrophils, causes endothelial damage and apoptosis in experimental diabetes (8). However, the role of the Fas/FasL pathway in human diabetic microvasculopathy remains to be investigated. Rho/Rho-kinase (ROCK) pathway plays an important role in pathologic vascular conditions, such as cerebral and coronary spasm (9), hypertension (10), and arteriosclerosis (11). Fasudil, a potent and selective ROCK inhibitor, is relatively safe and effective in the treatment of cardiovascular disease (12). Recent studies have shown that the Rho/ROCK pathway is also involved in the pathogenesis of diabetic complications in rat renal cortex (13) and aorta (14). In vitro, Rho activity is increased in bovine aortic endothelial cells treated with high glucose (15). Rho/ROCK pathway promotes leukocyte adhesion to the microvasculature by affecting the expression and function of adhesion molecules. For instance, Rho/ROCK activation increases ICAM-1 expression (16). In addition, Rho/ROCK signaling is associated with ICAM-1 clustering (17) and activation of ezrin, radixin, and moesin (ERM) in endothelial cells, which jointly form the anchoring structures for leukocyte integrins (18,19). Activation of the Rho/ROCK pathway stimulates phosphorylation of myosin regulatory light chain (MLC) (20), causing firm adhesion of leukocytes to the microvasculature by promoting the higher affinity state of integrins (21) and their assembly (22). Endothelial nitric oxide synthase (eNOS) generates nitric oxide (NO), a potent vasodilator (23) and anti-apoptotic factor (24,25). eNOS expression and function through phosphorylation is decreased in diabetic rat aorta (26) and myocardium (27). In vitro, eNOS phosphorylation is also reduced by high glucose in coronary artery endothelial cells (28) or bovine aortic endothelial cells (29). Since Rho/ROCK activation dephosphorylates eNOS in human umbilical venous cells (30), we hypothesize that ROCK inhibition may be anti-apoptotic in retinal endothelium during diabetes. In the present study, we investigate the mechanisms of endothelial damage in diabetic microvasculopathy using neutrophils from diabetic patients. We reveal the critical role of Rho/ROCK pathway in retinal microvascular damage associated with diabetes. Moreover, we examine the potential benefit of fasudil as a therapeutic agent in amelioration and treatment of diabetes-induced microvascular damage especially in the early DR stages. RESEARCH DESIGN AND METHODS Materials. Anti-RhoA mouse monoclonal antibody (mAb), anti-ROCK1 (sc-5561), ROCK2 (sc-5561) rabbit polyclonal antibody (pAb) and anti-CD-34 mAb were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor 488-conjugated anti-rabbit pAb and anti-mouse pAb, Alexa Fluor 555-conjugated anti-mouse pAb, and DAPI were obtained from Molecular Probes (Eugene, OR). For flow cytometry, anti-rat mAbs (CD11a, CD11b, CD18, FasL) were purchased from BD PharMingen (San Diego, CA). Anti-human mAbs (CD11a, CD11b, CD18) were from BD PharMingen except for anti-FasL ( eBioscience, San Diego, CA). Recombinant human tumor necrosis factor-α (rhTNF-α) was purchased from Sigma (Tokyo, Japan). Fasudil, a potent and selective ROCK inhibitor, was a generous gift of Asahi Kasei Pharma (Tokyo, Japan). Immunofluorescence microscopy. Paraffin embedded sections were dehydrated and subsequently incubated with antibodies against RhoA, ROCK1 and ROCK2. After washing, sections were incubated with Alexa Fluor 488-conjugated secondary antibody. For double staining, anti-CD34 mAb and Alexa Fluor 555-conjugated secondary antibody were used for staining endothelial cells. Counterstaining of nuclei was performed with DAPI. Staining was observed under a fluorscence microscope (BZ-9000, KEYENCE Corp, Osaka, Japan). Animal procedure. Wistar rats (male, 4 weeks) were obtained from Kyudo (Fukuoka, Japan). The procedures adhered to the guidelines from the Association for Research in Vision and Ophthalmology for animal use in research. Each rat received single 65 mg/kg intraperitoneal injections of streptozotocin (STZ, WAKO, Tokyo, Japan). Rats with blood glucose levels > 400 mg/dl, 24 hours after STZ injection, were considered diabetic. Intravitreal injections were carried out as previously described (31). Fasudil was dissolved in intraocular irrigating solution (OpeguardMA, Senju Pharmaceutical, Osaka). Intravitreal injections of fasudil (5μl, 360μM) were performed over a period of 1 minute with a 33-gauge needle on a Hamilton syringe (Reno, NV) every 3 days after diabetes onset. Assuming a vitreous humor volume of 60μl, the calculated final intraocular concentration of fasudil was 30μM. Sham injections (Opeguard) were performed in normal control and diabetic rats. All animals were euthanized 2 weeks after diabetes induction. Rho activity in the retina. GTP bound Rho (Rho-GTP) was examined to evaluate Rho activity in the rat retinal tissues. Rho activity in control and diabetic retinas was measured using a Rho activation assay kit (Upstate Biotechnology, Lake Placid, NY). Whole retinal lysates were extracted from one eye (about 60μg protein) per each condition. Total protein concentrations were measured and the same amounts of protein were applied in each sample. Rho-GTP bound to rhotekin-agarose was detected by Western blotting with anti-Rho mAb, according to the manufacturer’s protocol. MYPT-1 and eNOS phosphorylation. Retinal lysates for Western blotting were prepared as previously described (32). The blots were blocked with skim milk and incubated overnight at 4°C with rabbit phospho-MYPT-1 (1:1000; Thr 853, CycLex Co, Nagano, Japan) or rabbit phospho-eNOS (1:1000; Ser1177, Cell Signaling, Beverly, MA), horseradish peroxidase-labeled goat anti-rabbit IgG (1:4000; Bio-Rad, Richmond, CA) for 1hour at room temperature. Visualization was performed with an enhanced chemiluminesence detection system (Amersham, Arlington Heights, IL). The membranes were also reblotted with rabbit anti-MYPT-1(1:2000; Cell Signaling) and rabbit anti-eNOS (1:1000; sc-654; Santa Cruz Biotechnology). Lane loading differences were normalized by reblotting the membranes with an antibody against GAPDH. Signal intensities were expressed as percentage ratios of phospho-MYPT-1/GAPDH or phospho-eNOS/GAPDH. Blood processing and neutrophil isolation. This study was approved by the Institutional Review Board and performed in accordance with the ethical standards of the 1989 Declaration of Helsinki. Written informed consents were obtained from participants. Patients were type 2 diabetics with DR, who had undergone vitrectomy due to macular edema or proliferative changes. Neutrophils were isolated from whole blood as previously described (33). The purity of neutrophils was > 96%, as confirmed by Giemsa staining. Cells were resuspended in RPMI1640 medium (Sigma) with 5% fetal bovine serum (FBS) and used immediately for experiments. ICAM-1 enzyme-linked immunosorbent assay (ELISA). ICAM-1 concentration in the retinal lysates was measured by ELISA (R&D Systems, Minneapolis, MN). To normalize the ICAM-1 protein levels, total protein concentrations were measured using the bicinchoninic acid kit (Bio-Rad, Hercules, CA). Flow cytometry. To quantify CD11a, CD11b, CD18, and FasL expressions on the surface of neutrophils, the cells were incubated with CD11a mAb (1:50), CD11b mAb (1:50), CD18 mAb (1:50) and FasL mAb (1:25), which were labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), or biotin. To detect biotinylated mAbs, allophycocyanin-coupled streptavidin was used as the secondary reagent (BD PharMingen, 1:50). Neutrophils were then washed with staining buffer and surface fluorescence of 10 cells was analyzed with a FACSscan (Becton Dickinson, San Jose, CA). Results are expressed as fluorescence intensity on a logarithmic scale. Leukocyte adhesion to retinal vasculature. Retinal vessels and adhering leukocytes in control and diabetic animals were labeled with FITC-conjugated concanavalin A lectin (ConA, Vector Laboratories, Burlingame, CA), as previously described (4,34). Briefly, rats were perfused with 50 ml PBS 5-10 minutes to remove intravascular content, including erythrocytes and non-adherent leukocytes. To allow drainage, a 16-gauge needle was placed into the right atrium. The perfusion was continued with FITC-coupled ConA (40μg/ml in PBS). Retinal flat-mounts were prepared for evaluation of leukocyte accumulation. The total number of adherent leukocytes per retina was counted using a fluorescence microscope. Endothelial damage in rat retinas. Dead or injured endothelial cells in rat retinas were visualized by in vivo staining with Propidium Iodide (PI, Molecular Probes) (4; 8). PI (1mg/ml) and DAPI (10mg/ml) were injected intravenously via femoral vein. After 12 hours, the vasculature and adherent leukocytes in rat retinas were labeled with ConA. The retinas were then studied under a fluorescence microscope. The total number of PI positive cells per retina was counted. Neutrophil endothelial adhesion assay. The adhesive property of peripheral blood neutrophils, isolated from DR patients or normal subjects, to confluent monolayers of human dermal microvascular endothelial cells (HMVECs; Cambrex, East Rutherford, NJ) was evaluated using an established co-culture system (7). After pretreatment with fasudil (0, 5, or 20μM) for 1 hour at 37°C, HMVECs were treated with rhTNF-α (10ng/ml) for 12 hours at 37°C in humidified 5% CO2 atmosphere to enhance leukocyte adhesion. Neutrophils were resuspended at 10 cells/ml and labeled with 1mM Calcein-AM (BD PharMingen) for 30 minutes at 37°C in RPMI 1640. Labeled neutrophils were washed and co-incubated (20×10 cells/ml, 500μl per well) with pretreated HMVECs for 1hour at 37°C. Non-adherent cells were washed out, and the number of adherent neutrophils in 4 different areas of each well was counted and averaged using Image J software (NIH). Endothelial apoptosis induced by adherent neutrophils. HMVECs were incubated for 10 minutes with Hoechst 33342 (red fluorescence). After pretreatment with fasudil for 1 hour, HMVECs were stimulated for 12 hours with rhTNF-α (10 ng/ml) at 37°C. Unlabeled neutrophils (50×10cells/ml) were co-cultured with HMVEC for 12 hours at 37°C as described above. Apoptotic and potentially necrotic cells (green fluorescence) were detected by Terminal transferase dUTP nick-end labeling (TUNEL) staining with the TdT Fluorescein in situ apoptosis detection kit (R&D Systems), according to the manufacturer’s protocol. Apoptotic HMVECs demonstrated double labeling and appeared yellow. The number of apoptotic cells in 4 different areas per well was counted using fluorescence microscopy. To determine the involvement of NO and Fas/FasL signaling in endothelial apoptosis, HMVECs were preincubated with L-NAME (1mM, Sigma), an inhibitor of nitric oxide synthase, before fasudil treatment, and neutrophils were incubated with soluble Fas receptor (sFasR, Kamiya Biomedical, Seattle, WA), which inhibits FasL-induced apoptosis by acting as a decoy receptor, before co-culture for 1hour at 37°C. To investigate the involvement of Rho/ROCK pathway in Fas/FasL signaling, HMVECs were preincubated with fasudil in the presence or absence of L-NAME before the addition of soluble FasL (Sigma) for 1hour at 37°C. Statistical analysis. All results were expressed as mean±SEM. Statistical differences were assessed using the nonparametric Kruskal-Wallis variance analysis. To adjust for inflated α errors due to multiple comparisons, the corrected significant P value was defined using the Bonferroni correction. Statistical differences between two groups were analyzed by Mann-Whitney U test.