Selective Rho-associated Kinase Inhibitor Research Articles

Therapeutic potential of Rho-associated kinase inhibitor Y27632 in corneal endothelial dysfunction: an in vitro and in vivo study

To investigate the effects of a selective inhibitor of Rho-associated kinase (ROCK), Y-27632, on inbred Wuzhishan porcine corneal endothelial cells (PCECs) in vitro and in vivo studies. Primary PCECs were trypsinized from Wuzhishan miniature porcine corneal tissues. The optimal concentration of Y-27632 on PCECs was determined through MTT and 5-ethynyl-2'-deoxyuridine (EdU)-labeling assays. Seven New Zealand rabbits were used as a corneal endothelial dysfunction model, and a PCECs suspension supplemented with Y-27632 was injected into the anterior chamber of the rabbits. The progression of rabbit corneal opacity and edema were observed by slit lamp examination. The rabbits were sacrificed, and rabbit globes were enucleated for trypan blue-alizarin red staining, hematoxylin-eosin staining, and immunofluorescence analysis. Administration of 100 µmol/L Y-27632 facilitated PCECs' proliferation obviously. The rabbit corneas injected with PCECs suspension and 100 µmol/L Y-27632 were restored to transparency significantly after 14d. The 100 µmol/L Y-27632 treatment improves PCECs' proliferation significantly. And our results suggest that Y-27632 and PCECs can be used to treat corneal endothelial dysfunction.

AR12286 Alleviates TGF-β-Related Myofibroblast Transdifferentiation and Reduces Fibrosis after Glaucoma Filtration Surgery.

Scar formation can cause the failure of glaucoma filtration surgery. We investigated the effect of AR12286, a selective Rho-associated kinase inhibitor, on myofibroblast transdifferentiation and intraocular pressure assessment in rabbit glaucoma filtration surgery models. Cell migration and collagen contraction were used to demonstrate the functionality of AR12286-modulated human conjunctival fibroblasts (HConFs). Polymerase chain reaction quantitative analysis was used to determine the effect of AR12286 on the production of collagen Type 1A1 and fibronectin 1. Cell migration and collagen contraction in HConFs were activated by TGF-β1. However, compared with the control group, rabbit models treated with AR12286 exhibited higher reduction in intraocular pressure after filtration surgery, and decreased collagen levels at the wound site in vivo. Therefore, increased α-SMA expression in HConFs induced by TGF-β1 could be inhibited by AR12286, and the production of Type 1A1 collagen and fibronectin 1 in TGF-β1-treated HConFs was inhibited by AR12286. Overall, the stimulation of HConFs by TGF-β1 was alleviated by AR12286, and this effect was mediated by the downregulation of TGF-β receptor-related SMAD signaling pathways. In vivo results indicated that AR12286 thus improves the outcome of filtration surgery as a result of its antifibrotic action in the bleb tissue because AR12286 inhibited the TGF-β receptor-related signaling pathway, suppressing several downstream reactions in myofibroblast transdifferentiation.

Inhibition of Agonist-Induced Positive Inotropy by a Selective Rho-Associated Kinase Inhibitor, Y-27632

We examined the effect of Y-27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclo-hexanecarboxamide), a selective Rho-associated kinase (ROCK) inhibitor, on agonist-induced inotropy in isolated mouse left atria. Endothelin-1, angiotensin-II, and prostaglandin F2α (PGF2α) produced positive inotropy, which was significantly attenuated by Y-27632 (100 μM). On the other hand, isoproterenol-induced positive inotropy was not attenuated by the drug. These results provide the first evidence that the Rho/ROCK pathway is involved in endothelin-1-, angiotensin-II-, and PGF2α-induced positive inotropy, but not in β-adrenoceptor-mediated positive inotropy.

Azithromycin inhibits muscarinic 2 receptor-activated and voltage-activated Ca2+ permeant ion channels and Ca2+ sensitization, relaxing airway smooth muscle contraction.

Azithromycin (AZM) has been used for the treatment of asthma and chronic obstructive pulmonary disease (COPD); however, the effects and underlying mechanisms of AZM remain largely unknown. The effects of AZM on airway smooth muscles (ASMs) and the underlying mechanisms were studied using isometric muscle force measurements, the examination of lung slices, imaging, and patch-clamp techniques. AZM completely inhibited acetylcholine (ACH)-induced precontraction of ASMs in animals (mice, guinea pigs, and rabbits) and humans. Two other macrolide antibiotics, roxithromycin and Klaricid, displayed a decreased inhibitory activity, and the aminoglycoside antibiotics penicillin and streptomycin did not have an inhibitory effect. Precontractions were partially inhibited by nifedipine (selective inhibitor of L-type voltage-dependent Ca2+ channels (LVDCCs)), Pyr3 (selective inhibitor of TRPC3 and/or STIM/Orai channels, which are nonselective cation channels (NSCCs)), and Y-27632 (selective inhibitor of Rho-associated kinase (ROCK)). Moreover, LVDCC- and NSCC-mediated currents were inhibited by AZM, and the latter were suppressed by the muscarinic (M) 2 receptor inhibitor methoctramine. AZM inhibited LVDCC Ca2+ permeant ion channels, M2 receptors, and TRPC3 and/or STIM/Orai, which decreased cytosolic Ca2+ concentrations and led to muscle relaxation. This relaxation was also enhanced by the inhibition of Ca2+ sensitization. Therefore, AZM has potential as a novel and potent bronchodilator. The findings of this study improve the understanding of the effects of AZM on asthma and COPD.

Effect of the Rho Kinase Inhibitor Y-27632 on Corneal Endothelial Wound Healing.

The purpose of this study was to investigate the feasibility of using Rho-associated kinase (ROCK) inhibitor eye drops for treating severe corneal endothelial damage due to surgical invasion. A rabbit corneal endothelial damage model was created by mechanically scraping half the area of the corneal endothelium of eighteen eyes of Japanese white rabbits. A selective ROCK inhibitor, Y-27632 (10 mM), was applied topically for 2 weeks, and then the anterior segment was evaluated by slitlamp microscopy. The corneal endothelium was evaluated by phalloidin staining and immunohistochemical analysis. We then conducted pilot clinical research and applied Y-27632 eye drops topically to three patients who exhibited severe corneal edema due to corneal endothelial damage. In the corneal endothelial damage rabbit model, more Ki67-positive cells were detected in Y-27632-treated eyes than in control eyes. Five of six corneas became transparent in Y-27632-treated eyes, whereas zero of six corneas became transparent in the control eyes (P < 0.01). Actin fibers were distributed at the cell cortex in the eyes treated with Y-27632, whereas actin distribution was partially disrupted, and stress fibers were observed in control eyes. N-cadherin and Na+/K+-ATPase were expressed in almost all cells in Y-27632-treated eyes, but expression decreased in control eyes. Preliminary human cases confirmed that ROCK inhibitor eye drops were considerably effective for treatment of corneal edema associated with cataract surgery. ROCK inhibitor may be developed as an eye drop for treating acute corneal endothelial damage to prevent progression of bullous keratopathy. (University Hospital Medical Information Network Clinical Trial Registry no. UMIN000003625; www.umin.ac.jp/ctr).

ROCK has a crucial role in regulating prostate tumor growth through interaction with c-Myc

Rho-associated kinase (ROCK) has an essential role in governing cell morphology and motility, and increased ROCK activity contributes to cancer cell invasion and metastasis. Burgeoning data suggest that ROCK is also involved in the growth regulation of tumor cells. However, thus far, the molecular mechanisms responsible for ROCK-governed tumor cell growth have not been clearly elucidated. Here we showed that inhibition of ROCK kinase activity, either by a selective ROCK inhibitor Y27632 or by specific ROCK small interfering RNA (siRNA) molecules, attenuated not only motility but also the proliferation of PC3 prostate cancer cells in vitro and in vivo. Importantly, mechanistic investigation revealed that ROCK endowed cancer cells with tumorigenic capability, mainly by targeting c-Myc. ROCK could increase the transcriptional activity of c-Myc by promoting c-Myc protein stability, and ROCK inhibition reduced c-Myc-mediated expression of mRNA targets (such as HSPC111) and microRNA targets (such as miR-17-92 cluster). We provided evidence demonstrating that ROCK1 directly interacted with and phosphorylated c-Myc, resulting in stabilization of the protein and activation of its transcriptional activity. Suppression of ROCK-c-Myc downstream molecules, such as c-Myc-regulated miR-17, also impaired tumor cell growth in vitro and in vivo. In addition, c-Myc was shown to exert a positive feedback regulation on ROCK by increasing RhoA mRNA expression. Therefore, inhibition of ROCK and its stimulated signaling might prove to be a promising strategy for restraining tumor progression in prostate cancer.

Rho-Associated Kinase Inhibitor Eye Drop Treatment as a Possible Medical Treatment for Fuchs Corneal Dystrophy

To report a case of Fuchs corneal dystrophy that was successfully treated by Rho-associated kinase (ROCK) inhibitor eye drops, subsequent to transcorneal freezing of damaged corneal endothelial cells. A 52-year-old Japanese man with a diagnosis of late-onset Fuchs corneal dystrophy was referred to our hospital as a candidate for keratoplasty. Best-corrected vision was 20/20 in the right eye and 20/63 in the left eye. Multiple guttae were observed in both eyes. The right cornea was clear, but the left showed severe central edema, with a central corneal thickness of 703 μm. We were unable to perform specular microscopy in the central cornea, but endothelial cells were observed in the midperiphery at a density of 757 cells per square millimeter. The patient was treated by a corneal endothelial denudation in the prepupillary region followed by the topical administration of a selective ROCK inhibitor, Y-27632, as eye drops for 1 week. Follow-up of 24 months is reported. Corneal clarity recovered and vision improved to 20/20 two weeks after the treatment. At 6 months, vision had improved to 20/16 and central corneal thickness measured was 568 μm, significantly lower than its pretreatment value. Endothelial function and vision have been well maintained up to the most recent observation, 24 months after the treatment. The average corneal endothelial density in the central and peripheral cornea was 1549.3 ± 89.7 and 705.0 ± 61.1 cells per square millimeter, respectively. The case highlights the possibility of medical treatments involving the use of ROCK inhibitor eye drops as an alternative to graft surgery for certain forms of corneal endothelial disease.

Rho-associated kinase inhibitors: A novel glaucoma therapy

The rho-associated kinase (ROCK) signaling pathway is activated via secreted bioactive molecules or via integrin activation after extracellular matrix binding. These lead to polymerization of actin stress fibers and formation of focal adhesions. Accumulating evidence suggests that actin cytoskeleton-modulating signals are involved in aqueous outflow regulation. Aqueous humor contains various biologically active factors, some of which are elevated in glaucomatous eyes. These factors affect aqueous outflow, in part, through ROCK signaling modulation. Various drugs acting on the cytoskeleton have also been shown to increase aqueous outflow by acting directly on outflow tissue. In vivo animal studies have shown that the trabecular meshwork (TM) actin cytoskeleton in glaucomatous eyes is more disorganized and more randomly oriented than in non-glaucomatous control eyes. In a previous study, we introduced ROCK inhibitors as a potential glaucoma therapy by showing that a selective ROCK inhibitor significantly lowered rabbit IOP. Rho-associated kinase inhibitors directly affect the TM and Schlemm's canal (SC), differing from the target sight of other glaucoma drugs. The TM is affected earlier and more strongly than ciliary muscle cells by ROCK inhibitors, largely because of pharmacological affinity differences stemming from regulatory mechanisms. Additionally, ROCK inhibitors disrupt tight junctions, result in F-actin depolymerization, and modulate intracellular calcium level, effectively increasing SC-cell monolayer permeability. Perfusion of an enucleated eye with a ROCK inhibitor resulted in wider empty spaces in the juxtacanalicular (JCT) area and more giant vacuoles in the endothelial cells of SC, while the endothelial lining of SC was intact. Interestingly, ROCK inhibitors also increase retinal blood flow by relaxing vascular smooth muscle cells, directly protecting neurons against various stresses, while promoting wound healing. These additional effects may help slow progressing visual field loss in glaucoma patients, making ROCK inhibitors an even more desirable anti-glaucoma agent. All evidence indicates that aqueous humor outflow is affected by cytoskeleton physiology and this information may provide valuable insight into understanding glaucoma pathology and treatment.

Effects of endothelin-1 on calcium-independent contraction of bovine trabecular meshwork

Endothelin-1 (ET-1) is known to induce contraction of trabecular meshwork (TM) and is probably involved in the pathogenesis of glaucoma. Calcium (Ca(2+))-independent contraction has been shown in TM, and its inhibition may represent an interesting way of influencing outflow facility, and thus intraocular pressure (IOP). This study investigates the role of ET-1 and its receptors ET-A and ET-B (ET-AR and ET-BR) in TM Ca(2+)-independent contractility. Isometric tension measurements of bovine TM (BTM) strips were performed using a force-length transducer system. Intra- and extracellular Ca(2+) buffering was achieved by means of EGTA and BAPTA-AM. Under Ca(2+)-free conditions, ET-1-induced contractility of TM was assessed also in the presence of the specific inhibitors for ET-AR and ET-BR, BQ123 and BQ788 respectively. In order to clarify the intracellular mediators of Ca(2+)-independent contractility induced by ET-1, TM contraction was further measured in the presence of Y-27632, a selective inhibitor of Rho-associated kinases (ROCKs). The expression of ROCK1 and of its activating protein RhoA in BTM cells was investigated using western blot analysis. ET-1 induced a significant contraction of native BTM after intra- and extracellular Ca(2+)-depletion (45% +/- 8% of the maximally inducible contraction). Both endothelin receptor inhibitors BQ123 and BQ788 significantly reduced TM Ca(2+)-independent contraction in response to ET-1 (8.4 +/- 3.3% and 20.3 +/- 4.8% respectively). In the presence of the ROCK inhibitor Y-27632, ET-1-induced contraction of TM under Ca(2+)-free conditions was almost completely abolished (4.3% +/- 1.7%, p < 0.001). A clear signal for RhoA at 24 kDa and ROCK1 at 160 kDa could be detected in lysates of native tissue and cultured BTM cells with western blot. This study shows evidence that a significant portion of ET-1-induced contraction of TM is Ca(2+)-independent. In this contraction pathway, both ET-AR and ET-BR are involved with RhoA and its kinases as intracellular mediators. Ca(2+)-independent contraction of TM in response to ET-1 may represent a specific target to modulate IOP.

Lovastatin induces the expression of bradykinin type 2 receptors in cultured human coronary artery endothelial cells

Cardioprotective bradykinin type-2 receptors (BK-2Rs) are downregulated in the myocardial endothelium of both human and rat failing hearts. Statins are cardioprotective drugs that reduce the level of plasma cholesterol but also exert cholesterol-independent pleiotropic effects. Here we examined the effect of lovastatin on BK-2R expression in cultured human coronary artery endothelial cells. The effect of lovastatin on the expression of BK receptors in human coronary artery endothelial cells (HCAECs) was examined by real-time PCR, Western blot analysis and immunocytochemistry. Lovastatin induced a time- and concentration-dependent increase in both BK-2R and BK-1R mRNA expression in the cultured HCAECs. Also, the number of functional BK-2Rs capable of inducing BK-mediated NO production and cGMP signaling was increased in the lovastatin-treated HCAECs. Mevalonate, the direct metabolite of HMG-CoA reductase, reversed the effect of lovastatin. Furthermore, lovastatin inhibited Rho activation and a selective inhibitor of Rho-associated kinases, Y-27632, induced a similar increase in BK-2R expression as lovastatin. In contrast, a specific inhibitor of COX-2, NS398, significantly inhibited the lovastatin-induced expression of BK-2Rs. Here we show for the first time that lovastatin induces the expression of BK-2Rs in cultured human coronary artery endothelial cells through a novel cholesterol-independent pleiotropic mechanism that involves RhoA kinase inhibition and COX-2 activation. Thus, reported beneficial effects of statins in cardiovascular diseases may be partly mediated by an increased expression of cardioprotective BK-2Rs in the endothelial cells of the coronary tree. Moreover, the use of COX-2 inhibitors may affect the level of endothelial BK-2Rs in a negative fashion.

Ca2+]i-independent contractile force generation by rat hepatic stellate cells in response to endothelin-1

The contractile force generated by hepatic stellate cells in response to endothelin-1 contributes to sinusoidal blood flow regulation and hepatic fibrosis. This study's aim was to directly test the widely held view that changes in cytosolic Ca2+ concentration ([Ca2+]i) mediate stellate cell force generation. Contractile force generation by primary cultures of rat hepatic stellate cells grown in three-dimensional collagen gels was directly and quantitatively measured using a force transducer. Stellate cell [Ca2+]i, myosin activation, and migration were quantified using standard techniques. [Ca2+]i was modulated using ionomycin, BAPTA, KCl, and removal of extracellular Ca2+. Removal of extracellular Ca2+ did not alter endothelin-1-stimulated force development or [Ca2+]i. Ionomycin, a Ca2+ ionophore, triggered an increase in [Ca2+]i that was three times greater than that stimulated by endothelin-1, but only induced 16% of the force and 38% of the myosin regulatory light chain (MLC) phosphorylation induced by endothelin-1. Physiological increases in [Ca2+]i induced by hyperkalemia had no effect on contractile force. Loading BAPTA, a Ca2+ chelator, in stellate cells completely blocked endothelin-1-induced increases in [Ca2+]i but had no effect on endothelin-1-stimulated force generation or MLC phosphorylation. In contrast, Y-27632, a selective rho-associated kinase inhibitor, inhibited endothelin-1-stimulated force generation by at least 70% and MLC phosphorylation by at least 80%. Taken together, these observations indicate that changes in [Ca2+]i are neither necessary nor sufficient for contractile force generation by rat stellate cells. Our results challenge the current model of contractile regulation in hepatic stellate cells and have important implications for our understanding of hepatic pathophysiology.

Effects of Endothelin-1 on Human Trabecular Meshwork Cell Contraction

Trabecular meshwork (TM) cells are now considered to play an active role in the aqueous outflow mechanism, since they exhibit smooth muscle-like contractile properties. Endothelin-1 (ET-1), a potent vasoconstrictor peptide, has been proposed to play a role in the local regulation of aqueous outflow and intra-ocular pressure control. We propose an in vitro culture model as a method in the study of ET-1-induced human trabecular meshwork (HTM) cell contractility. Experiments were performed on semi-confluent HTM cells (primary cultures established from normotensive human donor eyes) at the 2nd passage with PBS as control, ET-1, sarafotoxin 6c (a selective endothelin B receptor agonist) and Y-27632 (a selective inhibitor of rho-associated kinase). The contractile status of the cells was evaluated by a morphometric analysis of the cell area, assuming that HTM cells in culture are able to reduce their area as a consequence of cytoskeletal contraction rather than regulatory volume decrease. Our data indicate that image analysis of the HTM cell area can be a reliable method in the study of TM cell contractility, since it utilizes human cells and offers versatile opportunities for the pharmacological evaluation of drugs controlling HTM cell status.

Rho A negatively regulates cytokine-mediated inducible nitric oxide synthase expression in brain-derived transformed cell lines: negative regulation of IKKalpha.

The present study describes the role of RhoA as a negative regulator of iNOS expression via the inactivation of NF-kappaB in transformed brain cell lines [C(6) glioma, human astrocytoma (T98G, A172), neuroblastoma (NEB), and immortal rat astrocytes]. Treatment with lovastatin resulted in the induction of LPS/IFN-gamma-mediated iNOS mRNA and increased nitric oxide (NO) production. The addition of mevalonate and geranylgeranylpyrophosphate (GGPP) reversed the lovastatin-mediated effect, whereas FPP had no effect. An inhibitor of geranylgeranyltransferase inhibitor (GGTI 298) further induced the cytokine and lovastatin-mediated iNOS expression, suggesting the involvement of geranylgeranylated proteins in the regulation of iNOS. Bacterial toxin B (inactivates RhoA, B, and C; CDC42; Rac proteins), C3 ADP-ribosyltransferase (C3) toxin from C. botulinum (inactivates RhoA, B, and C proteins), and Y-27632 (selective inhibitor of Rho-associated kinases) increased the LPS/IFN-gamma-mediated iNOS expression. Lovastatin treatment induced NO by increasing NF-kappaB translocation and its association with the CREB-binding protein (CBP/p300) via the downregulation of RhoA. Inhibition of RhoA resulted in increased activation of IKKalpha. Cotransfection studies with dominant-negative form of RhoA and iNOS-luciferase or NF-kappaB-luciferase reporter constructs further support these observations. Taken together, these studies show that downregulation of RhoA by lovastatin resulted in increased iNOS expression via the activation of NF-kappaB-CBP/p300 pathway in transformed brain cells.

Inhibition of agonist-induced positive inotropy by a selective Rho-associated kinase inhibitor, Y-27632.

We examined the effect of Y-27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclo-hexanecarboxamide), a selective Rho-associated kinase (ROCK) inhibitor, on agonist-induced inotropy in isolated mouse left atria. Endothelin-1, angiotensin-II, and prostaglandin F(2)(alpha) (PGF(2)(alpha)) produced positive inotropy, which was significantly attenuated by Y-27632 (100 microM). On the other hand, isoproterenol-induced positive inotropy was not attenuated by the drug. These results provide the first evidence that the Rho/ROCK pathway is involved in endothelin-1-, angiotensin-II-, and PGF(2)(alpha)-induced positive inotropy, but not in beta-adrenoceptor-mediated positive inotropy.

Cardiovascular effects of Y-27632, a selective Rho-associated kinase inhibitor, assessed in the halothane-anesthetized canine model

Y-27632, (+)-( R)- trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate, is a selective Rho-associated kinase inhibitor, which has been suggested to possess multiple clinical applications based on the in vitro observations. Since information regarding in vivo cardiovascular effects of Y-27632 is still limited, we assessed them using the halothane-anesthetized, closed-chest canine model. Administration of Y-27632 in a dose of 0.01 mg/kg, i.v. significantly decreased total peripheral vascular resistance together with an increase of cardiac output without affecting other cardiovascular parameters. Moreover, additional administration of Y-27632 in a dose of 0.1 mg/kg, i.v. significantly decreased blood pressure and left ventricular end-diastolic pressure, increased the heart rate and cardiac contractility, enhanced atrioventricular conduction and shortened the repolarization process as well as the effective refractory period. These results indicate that Y-27632 exerts a potent arterio-venodilator action with cardiostimulatory effects possibly through the sympathetic reflex in the in vivo canine model.

Cardiac Effects of a Selective Rho-Associated Kinase Inhibitor, Y-27632, Assessed in Canine Isolated, Blood-Perfused Heart Preparations

Chronotropic, inotropic and coronary effects of Y-27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride, monohydrate), a specific inhibitor of Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK), were assessed using canine isolated, blood-perfused heart preparations. Y-27632 slightly enhanced sinoatrial automaticity and significantly increased coronary blood flow, while it decreased ventricular contraction. The concentrations of Y-27632 needed to cause the currently observed changes were similar to those inhibiting ROCK in a previous in vitro study. These results suggest that the constitutional ROCK in the heart mainly regulates the ventricular contractility and coronary vascular tone rather than the sinoatrial automaticity.

Activation of Rho/Rho-Associated Kinase Pathway Is Involved in the Induction of Vascular Fibrogenesis.

56 Abnormal extracellular accumulation of collagens and reorganization of the contractile cytoskeleton network in the vascular smooth muscle cells are characteristic features of angiosclerosis and end-organ damage during cardiovascular diseases. Rho-associated kinase (ROCK) activation is a key factor controling myosin phosphoryation and thus, its availability to form stress fibers. We have found previously that vasoconstrictor agents, such as angiotensin II (Ang II) or endothelin-1 (ET-1) play a major role in the activation of collagen I gene expression and synthesis within blood vessels . In the present study we investigated whether ROCK activation is involved in the profibrotic action of these peptides. Experiments were performed in transgenic mice harboring the luciferase reporter gene under the control of the collagen I- chain α2 promoter. Bolus iv. administration of pressive doses of Ang II or ET-1 induced an early (1 h) two-fold increase of collagen I gene activity in aortas. Co-administration of Y-27632, a selective inhibitor of ROCK, blocked the vasoconstrictor peptide-induced collagen I gene activation. Similar effects were obtained in freshly isolated aortas in vitro: Y-27632 blunted the Ang II- and ET-1-induced collagen I gene activation and the clustering of contractile cytoskeleton as evidenced by immunicytochemistry and confocal microscopy. Cytochalasin D, a potent inhibitor of actin polymerization also prevented the effect of Ang II and ET-1 on collagen I gene. In addition, aortic smooth muscle cells transfected with a constitutively active ROCK exhibited a two-fold increase in collagen I gene activity vs. control. These data suggest that ROCK is a major intracellular signaling pathway required for the early in vivo activation of collagen I gene induced by vasoactive peptides such as Ang II and ET-1 within the vasculature. It would be interesting to pursue in chronic studies whether ROCK inhibitors can be effeciently used as drugs against the development of vascular fibrogenesis.

Activation of Rho/Rho-Associated Kinase Pathway Is Involved in the Induction of Vascular Fibrogenesis.

56 Abnormal extracellular accumulation of collagens and reorganization of the contractile cytoskeleton network in the vascular smooth muscle cells are characteristic features of angiosclerosis and end-organ damage during cardiovascular diseases. Rho-associated kinase (ROCK) activation is a key factor controling myosin phosphoryation and thus, its availability to form stress fibers. We have found previously that vasoconstrictor agents, such as angiotensin II (Ang II) or endothelin-1 (ET-1) play a major role in the activation of collagen I gene expression and synthesis within blood vessels . In the present study we investigated whether ROCK activation is involved in the profibrotic action of these peptides. Experiments were performed in transgenic mice harboring the luciferase reporter gene under the control of the collagen I- chain α2 promoter. Bolus iv. administration of pressive doses of Ang II or ET-1 induced an early (1 h) two-fold increase of collagen I gene activity in aortas. Co-administration of Y-27632, a selective inhibitor of ROCK, blocked the vasoconstrictor peptide-induced collagen I gene activation. Similar effects were obtained in freshly isolated aortas in vitro: Y-27632 blunted the Ang II- and ET-1-induced collagen I gene activation and the clustering of contractile cytoskeleton as evidenced by immunicytochemistry and confocal microscopy. Cytochalasin D, a potent inhibitor of actin polymerization also prevented the effect of Ang II and ET-1 on collagen I gene. In addition, aortic smooth muscle cells transfected with a constitutively active ROCK exhibited a two-fold increase in collagen I gene activity vs. control. These data suggest that ROCK is a major intracellular signaling pathway required for the early in vivo activation of collagen I gene induced by vasoactive peptides such as Ang II and ET-1 within the vasculature. It would be interesting to pursue in chronic studies whether ROCK inhibitors can be effeciently used as drugs against the development of vascular fibrogenesis.

Small guanosine triphosphatase Rho/Rho-associated kinase as a novel regulator of intracellular redistribution of lysosomes in invasive tumor cells

To investigate the role of RhoA on the intracellular membrane dynamics of lysosomes in rat hepatoma cells (MM1), we analyzed the localization of lysosomal aspartic proteinase cathepsin D by confocal immunofluorescence microscopy in the dominant active RhoA-transfected cells. Here we show that the transfection of the dominant active form of human small guanosine triphosphatase (GTPase) RhoA in MMI cells, a highly invasive cell line, causes the redistribution and spreading of small punctate structures stained for cathepsin D throughout the cytoplasm. We found that the microtubule organization was markedly different in the two cell lines: uniformly developed and polymerized microtubule filaments were seen in the mock transfectants; however, the dynamic organization of microtubules was less pronounced in the active RhoA transfectants. Furthermore, we found for the first time that a selective inhibitor of Rho-associated kinase (p160ROCK), Y-27632, impeded the subcellular spreading of cathepsin D staining and promoted reclustering of cathepsin D toward the perinuclear region in the active RhoA-transfected cells. To our knowledge, this is the first indication that the RhoA/ROCK-mediated signaling pathway is involved in the intracellular membrane dynamics of lysosomes by regulating the cytoskeletal microtubule organization as well as the actin cytoskeletons.