Effects of the Rho-Kinase Inhibitor Y-27632 on Extraocular Muscle Surgery in Rabbits.
ABSTRACT: To evaluate the effect of the Rho-kinase inhibitor Y-27632 on postoperative inflammation and adhesion following extraocular muscle surgery in rabbits.The superior rectus muscle reinsertion was performed on both eyes of 8 New Zealand white rabbits. After reinsertion, the rabbits received subconjunctival injections of the Rho-kinase inhibitor and saline on each eye. To assess acute and late inflammatory changes, Ki-67, CD11β+, and F4/80 were evaluated and the sites of muscle reattachment were evaluated for a postoperative adhesion score and histopathologically for collagen formation.F4/80 antibody expression was significantly different in the Rho-kinase inhibitor-injected group at both postoperative day 3 and week 4 (p = 0.038, 0.031). However, Ki-67 and CD11β+ were not different the between two groups. The difference in the SRM/conjunctiva adhesion score between the two groups was also significant (p = 0.034). Conclusion. Intraoperative subconjunctival injection of the Rho-kinase inhibitor may be effective for adjunctive management of inflammation and fibrosis in rabbit eyes following extraocular muscle surgery.
Project description:KEY CLINICAL MESSAGE: Not all orbital fractures are associated with clinical signs of swelling, ecchymosis, and subconjunctival hemorrhage. The "white-eyed" blowout fracture is more commonly seen in children and is associated with entrapment of the extraocular muscles. Early surgical intervention is indicated and it must have been in the differential diagnosis of the head injury patient with opthalmoplegia.
Project description:<h4>Purpose</h4>The trabecular meshwork (TM) cell-matrix interactions and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. The current study was designed to evaluate the role of a carbohydrate-binding protein, galectin-8 (Gal8), in TM cell adhesion and Rho signaling.<h4>Methods</h4>Normal human TM cells were assayed for Gal8 expression by immunohistochemistry and Western blot analysis. To assess the role of Gal8 in TM cell adhesion and Rho signaling, the cell adhesion and spreading assays were performed on Gal8-coated culture plates in the presence and the absence of anti-?? integrin antibody and Rho and Rho-kinase inhibitors. In addition, the effect of Gal8-mediated cell-matrix interactions on TM cell cytoskeleton arrangement and myosin light chain 2 (MLC2) phosphorylation was examined.<h4>Principal findings</h4>We demonstrate here that Gal8 is expressed in the TM and a function-blocking anti-?? integrin antibody inhibits the adhesion and spreading of TM cells to Gal8-coated wells. Cell spreading on Gal8 substratum was associated with the accumulation of phosphorylated myosin light chain and the formation of stress fibers that was inhibited by the Rho inhibitor, C3 transferase, as well as by the Rho-kinase inhibitor, Y27632.<h4>Conclusions/significance</h4>The above findings present a novel function for Gal8 in activating Rho signaling in TM cells. This function may allow Gal8 to participate in the regulation of aqueous outflow.
Project description:Thromboxane A2 receptor (TPr) has been reported to trigger vascular inflammation. Nuclear factor ? B (NF-?B) is a known transcription factor. The aims of the present study were to determine the contributions of NF-?B activation to TPr-triggered vascular inflammation and elucidate the mechanism(s) underlying TPr activation of NF-?B.The effects of TPr activators, [1S-[1 alpha,2 alpha(Z),3beta(1E,3S*), 4 alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) and U46619, on NF-?B activation, phosphorylation of rhoA/rho-associated kinases and liver kinase B1, cell adhesion and migration, proliferation, and endothelium-dependent vasorelaxation were assayed in cultured human umbilical vein endothelial cells, human monocytes, or isolated mouse aortas. Exposure of human umbilical vein endothelial cells to TPr agonists I-BOP and U46619 induced dose-dependent and time-dependent phosphorylation of inhibitor of ?B ? in parallel with aberrant expression of inflammatory markers cyclooxygenase-2, inducible nitric oxide synthase, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Inhibition of NF-?B by pharmacological or genetic means abolished TPr-triggered expression of inflammatory markers. Consistently, exposure of human umbilical vein endothelial cells to either I-BOP or U46619 significantly increased phosphorylation of inhibitor of ?B ?, I kappaB kinase, rhoA, rho-associated kinases, and liver kinase B1. Pretreatment of human umbilical vein endothelial cells with the TPr antagonist SQ29548 or rho-associated kinases inhibitor Y27632 or silencing of the LKB1 blocked TPr-enhanced phosphorylation of inhibitor of ?B ? and its upstream kinase, I kappaB kinase. Finally, exposure of isolated mouse aortas to either U46619 or I-BOP enhanced NF-?B activation and vascular inflammation in parallel with reduced endothelium-dependent relaxation in intact vessels.TPr stimulation instigates aberrant inflammation and endothelial dysfunction via rho-associated kinases/liver kinase B1/I kappaB kinase-dependent NF-?B activation in vascular endothelial cells.
Project description:Implantation of the embryo into the uterine compartment is a multistep event involving attachment of the embryo to the endometrial epithelia, followed by invasion of the embryo through the endometrial stroma. RHOA, RAC1, and CDC42 are members of the Rho GTPase family of proteins, which control cell functions such as cell migration and cytoskeletal reorganization. Herein, using a heterologous in vitro coculture model, we show that implantation of mouse blastocysts into human endometrial stromal cells (hESCs) is regulated by Rho GTPase activity in hESCs. Whereas iRNA-mediated silencing of RAC1 expression in hESCs led to inhibition of embryo implantation, silencing of either RHOA or CDC42 in hESCs promoted embryo implantation in coculture assays. Analysis of downstream signaling pathways demonstrated that RAC1 silencing was associated with decreased focal adhesion disassembly and resulted in large focal adhesion complexes in hESCs. In contrast, RHOA or CDC42 silencing resulted in perturbed focal adhesion assembly, leading to a decrease in the number of focal adhesions observed. Furthermore, inhibition of Rho signaling using a Rho kinase inhibitor, Y27632, led to decreased activation of protein tyrosine kinase 2 (PTK2, also called focal adhesion kinase) and decreased focal adhesion assembly. Importantly, perturbation of focal adhesion turnover in hESCs, mediated by PTK2 silencing, resulted in inhibition of embryo implantation into hESC monolayers. These findings suggest that Rho GTPase-PTK2-dependent remodeling of the endometrial stromal cell compartment may be critical for successful embryo implantation.
Project description:Transforming growth factor-? (TGF-?) activity has been implicated in subconjunctival scarring in eyes following glaucoma filtration surgery (GFS). The purpose of this study is to determine whether an inhibitor for activin receptor-like kinase (ALK) 5 (also known as TGF-? receptor type I) could suppress TGF-? activity and thereby promote filtering bleb survival after GFS in a rabbit model.An ALK-5 inhibitor, SB-505124, was used. A docking study was performed to investigate the interaction between the inhibitor and the receptor. Immunofluorescence for connective tissue growth factor (CTGF) and ?-smooth muscle actin (?-SMA) was performed in cultured rabbit subconjunctival fibroblasts. Immunoblotting for phosphorylated Smad2 (pSmad2), CTGF, and ?-SMA was also performed. In an in vivo rabbit GFS model, SB-505124 was delivered in a lactose tablet during surgery. Eyes were examined by slit-lamp and intraocular pressure (IOP) was measured until the time of bleb failure or up to 28 days after surgery. Tissue sections on day 5 after surgery were histologically evaluated after staining with hematoxylin and eosin. The sections were also immunostained for CTGF and ?-SMA. In addition, cell outgrowth from dissected subconjunctival tissues placed in a cell culture flask with media was investigated.The docking study indicated hydrogen bond interactions between SB-505124 and amino acids His-283 and Ser-280 of ALK-5. Suppression of pSmad2, CTGF, and ?-SMA by SB-505124 was observed in cultured fibroblasts. Filtering blebs in the GFS with SB-505124 group were maintained for more than 10 days, and the period of bleb survival was significantly longer than that in controls. IOP levels after surgery seemed to be related to bleb survival. Histologically, subconjunctival cell infiltration and scarring at the surgical site in the GFS with SB-505124 and mitomycin C (MMC) groups were much subsided compared to controls. Suppression of CTGF and ?-SMA by SB-505124 was also observed by immunofluorescence. Cell outgrowth from explants dissected from eyes to which SB-505124 was applied during GFS was robust while outgrowth was poor from those treated with MMC.The ALK-5 inhibitor SB-505124 was efficacious both in vitro and in vivo in suppressing the TGF-? action. The inhibitor may provide a novel therapy for preventing ocular inflammation and scarring.
Project description:Non-infectious anterior uveitis (AU) is a potentially sight threatening inflammatory condition. The current gold standard for treatment is topical steroids, but low ocular bioavailability and compliance issues with the intensive dosing regimen limit the efficacy of this treatment. Liposomes as a drug delivery system may help to overcome these problems. We studied the efficacy of a PEG-liposomal formulation of liposomal steroids, administered as a single subconjunctival dose, in the treatment of experimental uveitis in rabbit eyes. Rabbits that received subconjunctival liposomal triamcinolone acetonide phosphate (LTAP) or liposomal prednisolone phosphate (LPP) had significantly lower mean inflammatory scores than untreated controls on Day 4 after induction of uveitis (LPP vs controls, p?=?0.049) and 8 (LPP vs controls, p?=?0.007; LTAP vs controls, p?=?0.019), and lower scores than rabbits given topical PredForte1% 4 times a day on Day 8 (p?=?0.03). After antigen rechallenge, the subconjunctival liposomal steroid groups continued to have greater suppression of inflammation than untreated controls on Day 11 (p?=?0.02). Localization of liposomes in inflamed ocular tissue was confirmed by histology and immunostaining, and persisted in the eye for at least one month. Our study demonstrates that a single subconjunctival injection of liposomal steroids induces effective and sustained anti-inflammatory action.
Project description:KEY POINTS:Transforming growth-factor-? (TGF-?) and RhoA/Rho-kinase are independently implicated in the airway hyper-responsiveness associated with asthma, but how these proteins interact is not fully understood. We examined the effects of pre-treatment with TGF-? on expression and activity of RhoA, Rho-kinase and ARHGEF1, an activator of RhoA, as well as on bradykinin-induced contraction, in airway smooth muscle. TGF-? enhanced bradykinin-induced RhoA translocation, Rho-kinase-dependent phosphorylation and contraction, but partially suppressed bradykinin-induced RhoA activity (RhoA-GTP content). TGF-? enhanced the expression of ARHGEF1, while a small interfering RNA against ARHGEF1 and a RhoGEF inhibitor prevented the effects of TGF-? on RhoA and Rho-kinase activity and contraction, respectively. ARHGEF1 expression was also enhanced in airway smooth muscle from asthmatic patients and ovalbumin-sensitized mice. ARHGEF1 is a key TGF-? target gene, an important regulator of Rho-kinase activity and therefore a potential therapeutic target for the treatment of asthmatic airway hyper-responsiveness. ABSTRACT:Transforming growth factor-? (TGF-?), RhoA/Rho-kinase and Src-family kinases (SrcFK) have independently been implicated in airway hyper-responsiveness, but how they interact to regulate airway smooth muscle contractility is not fully understood. We found that TGF-? pre-treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho-kinase (Y27632), but not the SrcFK inhibitor PP2, prevented this enhancement. In cultured human airway smooth muscle cells (hASMCs), TGF-? pre-treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC20 , MYPT-1 and the actin-severing protein cofilin, but not of RhoA, ROCK2 or c-Src. In hASMCs, acute treatment with BK triggered subcellular translocation of ARHGEF1 and RhoA and enhanced auto-phosphorylation of SrcFK and phosphorylation of MYPT1 and MLC20 , but induced de-phosphorylation of cofilin. TGF-? pre-treatment amplified the effects of BK on RhoA translocation and MYPT1/MLC20 phosphorylation, but suppressed the effects of BK on RhoA-GTP content, SrcFK auto-phosphorylation and cofilin de-phosphorylation. In hASMCs, an ARHGEF1 small interfering RNA suppressed the effects of BK and TGF-? on RhoA-GTP content, RhoA translocation and MYPT1 and MLC20 phosphorylation, but minimally influenced the effects of TGF-? on cofilin expression and phosphorylation. ARHGEF1 expression was also enhanced in ASMCs of asthmatic patients and in lungs of ovalbumin-sensitized mice. Our data indicate that TGF-? enhances BK-induced contraction, RhoA translocation and Rho-kinase activity in airway smooth muscle largely via ARHGEF1, but independently of SrcFK and total RhoA-GTP content. A role for smooth muscle ARHGEF1 in asthmatic airway hyper-responsiveness is worthy of further investigation.
Project description:Elevated sympathetic tone and activation of the renin-angiotensin system are pathophysiologic hallmarks of hypertension, and the interactions between these systems are particularly deleterious. The importance of Rho kinase as a mediator of the effects of angiotensin-II (AngII) in the periphery is clear, but the role of Rho kinase in sympathoexcitation caused by central AngII is not well established. We hypothesized that AngII mediates its effects in the brain by the activation of the RhoA/Rho kinase pathway. Chronically instrumented, conscious rabbits received the following intracerebroventricular infusion treatments for 2 weeks via osmotic minipump: AngII, Rho kinase inhibitor Fasudil, AngII plus Fasudil, or a vehicle control. AngII increased mean arterial pressure over the course of the infusion, and this effect was prevented by the coadministration of Fasudil. AngII increased cardiac and vascular sympathetic outflow as quantified by the heart rate response to metoprolol and the depressor effect of hexamethonium; coadministration of Fasudil abolished both of these effects. AngII increased baseline renal sympathetic nerve activity in conscious animals and impaired baroreflex control of sympathetic nerve activity; again Fasudil coinfusion prevented these effects. Each of these end points showed a statistically significant interaction between AngII and Fasudil. Quantitative immunofluorescence of brain slices confirmed that Rho kinase activity was increased by AngII and decreased by Fasudil. Taken together, these data indicate that hypertension, elevated sympathetic outflow, and baroreflex dysfunction caused by central AngII are mediated by Rho kinase activation and suggest that Rho kinase inhibition may be an important therapeutic target in sympathoexcitatory cardiovascular diseases.
Project description:BACKGROUND: Metabolic disorders, caused by excessive calorie intake and low physical activity, are important cardiovascular risk factors. Rho-kinase, an effector protein of the small GTP-binding protein RhoA, is an important cardiovascular therapeutic target and its activity is increased in patients with metabolic syndrome. We aimed to examine whether Rho-kinase inhibition improves high-fat diet (HFD)-induced metabolic disorders, and if so, to elucidate the involvement of AMP-activated kinase (AMPK), a key molecule of metabolic conditions. METHODS AND RESULTS: Mice were fed a high-fat diet, which induced metabolic phenotypes, such as obesity, hypercholesterolemia and glucose intolerance. These phenotypes are suppressed by treatment with selective Rho-kinase inhibitor, associated with increased whole body O2 consumption and AMPK activation in the skeletal muscle and liver. Moreover, Rho-kinase inhibition increased mRNA expression of the molecules linked to fatty acid oxidation, mitochondrial energy production and glucose metabolism, all of which are known as targets of AMPK in those tissues. In systemic overexpression of dominant-negative Rho-kinase mice, body weight, serum lipid levels and glucose metabolism were improved compared with littermate control mice. Furthermore, in AMPK?2-deficient mice, the beneficial effects of fasudil, a Rho-kinase inhibitor, on body weight, hypercholesterolemia, mRNA expression of the AMPK targets and increase of whole body O2 consumption were absent, whereas glucose metabolism was restored by fasudil to the level in wild-type mice. In cultured mouse myocytes, pharmacological and genetic inhibition of Rho-kinase increased AMPK activity through liver kinase b1 (LKB1), with up-regulation of its targets, which effects were abolished by an AMPK inhibitor, compound C. CONCLUSIONS: These results indicate that Rho-kinase inhibition ameliorates metabolic disorders through activation of the LKB1/AMPK pathway, suggesting that Rho-kinase is also a novel therapeutic target of metabolic disorders.
Project description:Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation. At low doses, the thromboxane A2 analogue U46619 does not activate integrin alphaIIbbeta3 or trigger platelet aggregation, but it induces shape changes. In the present study, we addressed whether low doses of U46619 trigger tyrosine phosphorylation independently of integrin alphaIIbbeta3 activation and ADP secretion, and synergize with adrenaline (epinephrine) to induce aggregation in acetylsalicylic acid (aspirin)-treated platelets. Low doses of U46619 triggered tyrosine phosphorylation of different proteins, including FAK (focal adhesion kinase), Src and Syk, independently of signals ensuing from integrin alphaIIbbeta3 or ADP receptors engaged by secreted ADP. The G(12/13)-mediated Rho/Rho-kinase pathway was also increased by low doses of U46619; however, this pathway was not upstream of tyrosine phosphorylation, because this occurred in the presence of the Rho-kinase inhibitor Y-27632. Although low doses of U46619 or adrenaline alone were unable to trigger platelet aggregation and integrin alphaIIbbeta3 activation, the combination of the two stimuli effectively induced these responses. PP2, a tyrosine kinase inhibitor, and Y-27632 inhibited platelet activation induced by low doses of U46619 plus adrenaline and, when used in combination, totally suppressed this platelet response. In addition, the two inhibitors selectively blocked tyrosine kinases and the Rho/Rho-kinase pathway respectively. These findings suggest that both tyrosine phosphorylation and the Rho/Rho-kinase pathway are required to activate platelet aggregation via G(12/13) plus G(z) signalling.