KD025 Shifts Pulmonary Endothelial Cell Bioenergetics and Decreases Baseline Lung Permeability.

KD025 is a ROCK2 inhibitor currently being tested in clinical trials for the treatment of fibrotic lung diseases. The therapeutic effects of KD025 are partly due to its inhibition of profibrotic pathways and fat metabolism. However, whether KD025 affects pulmonary microvascular endothelial cell (PMVEC) function is unknown, despite evidence that alveolar-capillary membrane disruption constitutes major causes of death in fibrotic lung diseases. We hypothesized that KD025 regulates PMVEC metabolism, pH, migration, and survival, a series of interrelated functional characteristics that determine pulmonary barrier integrity. We used PMVECs isolated from Sprague Dawley rats. KD025 dose-dependently decreased lactate production and glucose consumption. The inhibitory effect of KD025 was more potent compared with other metabolic modifiers, including 2-deoxy-glucose, extracellular acidosis, dichloroacetate, and remogliflozin. Interestingly, KD025 increased oxidative phosphorylation, whereas 2-deoxy-glucose did not. KD025 also decreased intracellular pH and induced a compensatory increase in anion exchanger 2. KD025 inhibited PMVEC migration, but fasudil (nonspecific ROCK inhibitor) did not. We tested endothelial permeability in vivo using Evans Blue dye in the bleomycin pulmonary fibrosis model. Baseline permeability was decreased in KD025-treated animals independent of bleomycin treatment. Under hypoxia, KD025 increased PMVEC necrosis as indicated by increased lactate dehydrogenase release and propidium iodide uptake and decreased ATP; it did not affect Annexin V binding. ROCK2 knockdown had no effect on PMVEC metabolism, pH, and migration, but it increased nonapoptotic caspase-3 activity. Together, we report that KD025 promotes oxidative phosphorylation; decreases glycolysis, intracellular pH, and migration; and strengthens pulmonary barrier integrity in a ROCK2-independent manner.

Fasudil alleviates brain damage in rats after carbon monoxide poisoning through regulating neurite outgrowth inhibitor/oligodendrocytemyelin glycoprotein signalling pathway.

Carbon monoxide (CO) poisoning can lead to many serious neurological symptoms. Currently, there are no effective therapies for CO poisoning. In this study, rats exposed to CO received hyperbaric oxygen therapy, and those in the Fasudil group were given additional Fasudil injection once daily. We found that the escape latency in CO poisoning group (CO group) was significantly prolonged, the T1 /Ttotal was obviously decreased, and the mean escape time and the active escape latency were notably extended compared with those in normal control group (NC group, P < 0.05). After administration of Fasudil, the escape latency was significantly shortened, T1 /Ttotal was gradually increased as compared with CO group (>1 week, P < 0.05). Ultrastructural damage of neurons and blood-brain barrier of rats was serious in CO group, while the structural and functional integrity of neuron and mitochondria maintained relatively well in Fasudil group. Moreover, we also noted that the expressions of neurite outgrowth inhibitor (Nogo), oligodendrocyte-myelin glycoprotein (OMgp) and Rock in brain tissue were significantly increased in CO group, and the elevated levels of the three proteins were still observed at 2 months after CO poisoning. Fasudil markedly reduced their expressions compared with those of CO group (P < 0.05). In summary, the activation of Nogo-OMgp/Rho signalling pathway is associated with brain injury in rats with CO poisoning. Fasudil can efficiently down-regulate the expressions of Nogo, OMgp and Rock proteins, paving a way for the treatment of acute brain damage after CO poisoning.

Shen'ge powder decreases the cardiomyocyte hypertrophy in chronic heart failure by activating the Rho protein/Rho-associated coiledcoil forming protein kinase signaling pathway.

OBJECTIVE: The current study aimed to explore the role and the molecular mechanism of Shen'ge powder in cardiomyocyte hypertrophy in chronic heart failure (CHF). METHODS: Sprague-Dawley rats were selected for the study and divided randomly into four groups: control, model, Shen'ge powder, and fasudil group. An inverted microscope was used to determine the diameter of cardiomyocytes in each group. The survival and apoptotic rate of cardiomyocytes in each group was determined by the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. The messenger RNA levels and protein expression of RhoA, Rho-associated coiled-coil forming protein kinase (ROCK), myosin light-chain phosphatase (MLCP), and myosin light-chain kinase (MLCK) were determined by quantitative reverse transcription-polymerase chain reaction and Western blot analysis, respectively. RESULTS: CHF increased the diameter and apoptotic rate of cardiomyocytes and decreased the survival rate of cardiomyocytes. The levels of RhoA, ROCK, and MLCK were increased significantly in CHF model rats, and the level of MLCP was decreased. After treating with Shen'ge powder, the expression of RhoA, ROCK, and MLCK decreased dramatically and the expression of MLCP increased. CONCLUSION: Shen'ge powder could reduce cardiomyocyte hypertrophy in CHF by regulating the Rho/ROCK signaling pathway.

Fasudil alleviates pressure overload-induced heart failure by activating Nrf2-mediated antioxidant responses.

The RhoA/Rho-kinase cascade plays an important role in many aspects of cardiovascular function. This study aims to investigate the protective effects of fasudil, a Rho-kinase inhibitor, on pressure overload induced heart failure in rats. Pressure overload induced heart failure was induced in SD rats by banding the abdominal aorta for 8 weeks. The rats were divided into four groups: Sham, TAC, TAC plus low dose of fasudil, and TAC plus high dose of fasudil group. Low dose and high dose fasudil were 5 and 10 mg/kg/day, respectively. Rats in the Sham and TAC groups were treated with vehicle. Fasudil effectively inhibited TAC-induced heart failure, as evaluated by echocardiography and transmission electron microscopy. Fasudil could significantly promote superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activity and significantly decrease malondialdehyde (MDA) content in a dose-dependent maner in TAC rats. Consistently, fasudil evoked significant nuclear translocation of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) with increased DNA/promoter binding and transactivation of Nrf2 targets. In addition, fasudil increased the content of iron as well as transferrin receptor 1 (TfR1) in TAC rats. A mild oxidative stress induced by iron may activate the antioxidant enzymes by feedback response. Taken together, these results indicate that the protective effect of fasudil may be due to its strong antioxidative activities which related with the activated Nrf2 and its down-regulated genes. These findings provide a new treatment concept and support the benefit of fasudil treatment in heart failure.

Rho Kinase Inhibitor, Fasudil, Attenuates Contrast-induced Acute Kidney Injury.

In this study, we tested the hypothesis that fasudil, a Rho kinase inhibitor, would protect against contrast-induced acute kidney injury (CI-AKI) in a mouse model and attempted to elucidate the mechanism involved. Mice subjected to unilateral ligation of the left anterior renal pedicle were divided into four groups: (1) control group, (2) CI-AKI induced by contrast media (CM group), (3) CI-AKI plus low-dose fasudil (LD group) and (4) CI-AKI plus high-dose fasudil (HD group). Animals from groups 2-4 received iodixanol (4.0 g iodine/kg), and the control group received saline. At 12, 2 hr before iodixanol injection and 4 hr after iodixanol administration, the animals in groups 3-4 received 3 or 10 mg/kg fasudil, respectively. Renal blood flow, renal function parameters, kidney histology and the expression of proteins that regulates apoptosis and inflammation were determined 24 hr later. Fasudil treatment notably ameliorated contrast medium-induced medullary damage, restored renal function, suppressed renal tubular apoptosis, ameliorated redox imbalance and DNA damage. Fasudil had a nephroprotective effect that was partially attributed to its anti-inflammatory, anti-apoptotic and antioxidant effects of inhibiting the Rho/ROCK pathway.

The Rho GTPase signalling pathway in urothelial carcinoma.

Urothelial carcinoma remains a clinical challenge: non-muscle-invasive disease has a high rate of recurrence and risk of progression, and outcomes for patients with advanced disease are poor, owing to a lack of effective systemic therapies. The Rho GTPase family of enzymes was first identified >30 years ago and contains >20 members, which are divided into eight subfamilies: Cdc42, Rac, Rho, RhoUV, RhoBTB, RhoDF, RhoH, and Rnd. Rho GTPases are molecular on-off switches, which are increasingly being understood to have a critical role in a number of cellular processes, including cell migration, cell polarity, cell adhesion, cell cycle progression, and regulation of the cytoskeleton. This switch is an evolutionarily conserved system in which GTPases alternate between GDP-bound (inactive) and GTP-bound (active) forms. The activities of these Rho GTPases are many, context-dependent, and regulated by a number of proteins that are being progressively elucidated. Aberrations of the Rho GTPase signalling pathways have been implicated in various malignancies, including urothelial carcinoma, and understanding of the role of Rho GTPases in these diseases is increasing. This signalling pathway has the potential for therapeutic targeting in urothelial carcinoma. Research in this area is nascent, and much work is necessary before current laboratory-based research can be translated into the clinic.

Multitarget Therapeutic Effect of Fasudil in APP/PS1transgenic Mice.

INTRODUCTION: Therapeutic strategies targeting Alzheimer's disease-related molecule ß- amyloid (Aß), Tau protein and ß-site amyloid precursor protein cleaving enzyme (BACE) have been recently explored. However, the treatment effect for single target is not ideal. Based on multiaspect roles of Rho kinase inhibitor Fasudil on neuroprotection, neurorepair and immunomodulation, we observed therapeutic potential of Fasudil and explored possible mechanisms in amyloid precursor protein/ presenilin-1 transgenic (APP/PS1 Tg) mice, an animal model of Alzheimer's disease. METHODS: APP/PS1 Tg mice were treated with Fasudil (25 mg/kg/day) for 2 months by intraperitoneal injection. Mouse behavior tests were recorded every day. The expression of Aß deposition, Tau protein phosphorylation, BACE and postsynaptic density 95 (PSD-95) in hippocampus was assayed. The levels in the brain of Toll-like receptors (TLRs)-nuclear factor kappa B/p65（NF-κB/p65)- myeloid differentiation primary response gene 88 (MyD88) inflammatory cytokine axis were measured. RESULTS: Fasudil treatment ameliorated learning and memory deficits, accompanied by reduced Aß deposition, Tau protein phosphorylation, and BACE expression, as well as increased PSD-95 expression in hippocampus. Fasudil intervention also inhibited TLR-2/4, p-NF-κB/p65, MyD88, interleukin-1beta, interleukin-6 and tumor necrosis factor-α for TLRs-NF-κB-MyD88 inflammatory cytokine axis and the induction of interleukin-10. CONCLUSION: Fasudil exhibited multitarget therapeutic effect in APP/PS1 Tg mice. The study provides preclinical evidence that Fasudil treatment ameliorated memory deficits in APP/PS1 Tg mice, accompanied by the reduction of Aß deposition and Tau protein phosphorylation, the decrease of BACE and the increase of PSD-95, as well as inhibition of TLRs-NF-κB-MyD88 inflammatory cytokine axis. However, these results still need to be repeated and confirmed before clinical application.

Hypoxia-induced pulmonary arterial hypertension augments lung injury and airway reactivity caused by ozone exposure.

Ozone (O3)-related cardiorespiratory effects are a growing public health concern. Ground level O3 can exacerbate pre-existing respiratory conditions; however, research regarding therapeutic interventions to reduce O3-induced lung injury is limited. In patients with chronic obstructive pulmonary disease, hypoxia-associated pulmonary hypertension (HPH) is a frequent comorbidity that is difficult to treat clinically, yet associated with increased mortality and frequency of exacerbations. In this study, we hypothesized that established HPH would confer vulnerability to acute O3 pulmonary toxicity. Additionally, we tested whether improvement of pulmonary endothelial barrier integrity via rho-kinase inhibition could mitigate pulmonary inflammation and injury. To determine if O3 exacerbated HPH, male C57BL/6 mice were subject to either 3 weeks continuous normoxia (20.9% O2) or hypoxia (10.0% O2), followed by a 4-h exposure to either 1ppm O3 or filtered air (FA). As an additional experimental intervention fasudil (20mg/kg) was administered intraperitoneally prior to and after O3 exposures. As expected, hypoxia significantly increased right ventricular pressure and hypertrophy. O3 exposure in normoxic mice caused lung inflammation but not injury, as indicated by increased cellularity and edema in the lung. However, in hypoxic mice, O3 exposure led to increased inflammation and edema, along with a profound increase in airway hyperresponsiveness to methacholine. Fasudil administration resulted in reduced O3-induced lung injury via the enhancement of pulmonary endothelial barrier integrity. These results indicate that increased pulmonary vascular pressure may enhance lung injury, inflammation and edema when exposed to pollutants, and that enhancement of pulmonary endothelial barrier integrity may alleviate such vulnerability.

Pleiotropic effects of the rho-kinase inhibitor fasudil after subarachnoid hemorrhage: a review of preclinical and clinical studies.

There is growing evidence that Rho-kinase contributes to cardiovascular disease, which has made Rho-kinase a target for the treatment of human diseases. To date, the only Rho-kinase inhibitor employed clinically in humans is fasudil, which has been used for the prevention of cerebral vasospasm and subsequent ischemic injury after surgery for subarachnoid hemorrhage (SAH). A number of pathological processes, in particular hemodynamic dysfunctions and inflammatory reactions, are thought to be related in the pathogenesis of delayed cerebral vasospasm and subsequent ischemic injury after SAH. This review focuses on fasudil's pleiotropic therapeutic effects: amelioration of hemodynamic dysfunction and inflammation, and discusses in detail the clinical studies on fasudil administered after the occurrence of SAH.

Effects of the Rho kinase inhibitor, hydroxyfasudil, on bladder dysfunction and inflammation in rats with HCl-induced cystitis.

OBJECTIVE: To evaluate the effect of the Rho kinase inhibitor, hydroxyfasudil, on bladder function in a rat model of HCl-induced chemical cystitis, and to elucidate the possible mechanisms associated with its therapeutic effect. METHODS: Female Sprague-Dawley rats with HCl-induced cystitis were given hydroxyfasudil (10 mg/kg, i.p.) for 7 days. Treatment efficacy was determined by comparing bladder function and histopathology to sham and untreated control rats. Bladder function was determined by cystometric analysis. Rho kinase activity was determined by quantitative reverse transcription polymerase chain reaction and signal inhibition of downstream Ras homolog member A/Rho kinase signaling molecules by western blot and immunohistochemistry. RESULTS: Treatment with hydroxyfasudil significantly improved bladder intercontraction intervals. Rats treated with hydroxyfasudil also showed a significant reduction of histopathological features associated with cystitis. Western blot and immunohistochemistry findings showed that hydroxyfasudil inhibited downstream molecules of Rho kinase that ameliorated changes associated with HCl-induced chemical cystitis, such as inflammatory cell recruitment and smooth muscle cell proliferation. CONCLUSION: The findings from the present study suggest a promising therapeutic role for hydroxyfasudil in bladder inflammation associated with cystitis.

Small molecule screening with laser cytometry can be used to identify pro-survival molecules in human embryonic stem cells.

Differentiated cells from human embryonic stem cells (hESCs) provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs) provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.

Fasudil, a Rho-kinase inhibitor, attenuates bleomycin-induced pulmonary fibrosis in mice.

The mechanisms underlying the pathogenesis of idiopathic pulmonary fibrosis (IPF) involve multiple pathways, such as inflammation, epithelial mesenchymal transition, coagulation, oxidative stress, and developmental processes. The small GTPase, RhoA, and its target protein, Rho-kinase (ROCK), may interact with other signaling pathways known to contribute to pulmonary fibrosis. This study aimed to determine the beneficial effects and mechanisms of fasudil, a selective ROCK inhibitor, on bleomycin-induced pulmonary fibrosis in mice. Our results showed that the Aschcroft score and hydroxyproline content of the bleomycin-treated mouse lung decreased in response to fasudil treatment. The number of infiltrated inflammatory cells in the bronchoalveolar lavage fluid (BALF) was attenuated by fasudil. In addition, fasudil reduced the production of transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA), and plasminogen activator inhibitor-1 (PAI-1) mRNA and protein expression in bleomycin-induced pulmonary fibrosis. These findings suggest that fasudil may be a potential therapeutic candidate for the treatment of pulmonary fibrosis.

High-dose fasudil preserves postconditioning against myocardial infarction under hyperglycemia in rats: role of mitochondrial KATP channels.

BACKGROUND: The current study was carried out to determine whether fasudil hydrochloride (fasudil), a Rho-kinase inhibitor, has myocardial postconditioning (PostC) activity under hyperglycemia as well as normoglycemia, and if so, whether the effects could be mediated by mitochondrial ATP-sensitive potassium (m-KATP) channels. METHODS: Male Sprague-Dawley rats were anesthetized with sodium pentobarbital. After opening the chest, all rats underwent 30-min coronary artery occlusion followed by 2-h reperfusion. The rats received low-dose (0.15 mg/kg) or high-dose (0.5 mg/kg) fasudil or diazoxide, an m-KATP channel opener, at 10 mg/kg, just before reperfusion under normoglycemic or hyperglycemic conditions. In another group, rats received 5-hydroxydecanoic acid (5HD), an m-KATP channel blocker, at 10 mg/kg, before high-dose fasudil. Myocardial infarct size was expressed as a percentage of area at risk (AAR). RESULTS: Under normoglycemia, low-dose and high-dose fasudil and diazoxide reduced myocardial infarct size (23 ± 8%, 21 ± 9% and 21 ± 10% of AAR, respectively) compared with that in the control (42 ± 7%). Under hyperglycemia, low-dose fasudil (40 ± 11%) and diazoxide (44 ± 14%) could not exert this beneficial effect, but high-dose fasudil reduced myocardial infarct size in the same manner as under normoglycemia (21 ± 13%). 5HD prevented fasudil-induced reduction of myocardial infarct size (42 ± 13%). CONCLUSION: Fasudil induces PostC against myocardial infarction via activation of m-KATP channels in the rat. Although hyperglycemia attenuates the PostC, high-dose fasudil can restore cardioprotection.

Coptisine exert cardioprotective effect through anti-oxidative and inhibition of RhoA/Rho kinase pathway on isoproterenol-induced myocardial infarction in rats.

OBJECTIVE: Because myocardial infarction is a major cause of morbidity and mortality worldwide, protecting the heart from the ischemia is the focus of intense research. Coptisine is an isoquinoline alkaloid extracted form Coptidis Rhizoma. This study aims to elucidate if coptisine is responsible for cardioprotection using myocardial infarction (MI) rat models and investigate its potential mechanism of action. METHODS: Myocardial infarction was produced in rats with 85 mgkg(-1) isoproterenol administered subcutaneously twice at an interval of 24 h. The rats were randomized into 7 groups: (I) Normal; (II) ISO; (III) ISO+fasudil; (IV) ISO+isosorbide dinitrate (ISDN) and (V-VII) ISO+coptisine (25, 50 and 100 mgkg(-1)). Cardiac function and markers of cardiac ischemic were assessed after MI. RESULTS: Rats pretreated with coptisine (25, 50 and 100 mgkg(-1)) for 21 days and received subcutaneously injected with ISO (85 mgkg(-1)) on the 20th and 21st day at an interval of 24 h. The results suggested that coptisine has strong antioxidant activity, and it can maintain cell membrane integrity, ameliorate mitochondrial respiratory dysfunction, reduce myocardial cells apoptosis, inhibit RhoA/ROCK expression induced by high-dose isoproterenol administration. CONCLUSIONS: Coptisine provided cardioprotection in a model of myocardial infarction, and therefore should be considered as a novel adjunctive therapy for attenuating myocardial damage.

Rho kinase inhibition by fasudil exerts antioxidant effects in hypercholesterolemic rats.

1. The inhibition of Rho kinase (ROCK) ameliorates many cardiovascular dysfunctions, but the role of ROCK in oxidative stress in hypercholesterolemic rats has not been explored. The aim of the current study was to investigate the antioxidant effects and the potential related mechanisms of fasudil, a selective ROCK inhibitor, in high-cholesterol diet (HCD)-induced hypercholesterolemic rats. 2. Hypercholesterolemia was induced in rats by feeding with a HCD for 4 weeks. Starting from day 15, physiological saline (1 mL/100 g) or ROCK inhibitor, fasudil (10 or 30 mg/kg), was injected intraperitoneally for another 14 days. 3. The results showed that fasudil significantly suppressed ROCK activity, potently elevated the activities of antioxidant enzymes and the expression of endothelial nitric oxide synthase, as well as the concentration of nitric oxide in the serum and cardiac tissue. In addition, fasudil notably suppressed the extent of lipid peroxidation and attenuated the histopathological changes in the heart and liver of hypercholesterolemic rats. 4. These antioxidant effects of fasudil suggest that ROCK activation is involved in oxidative stress in hypercholesterolemic rats.

Simvastatin modulates the adhesion and growth of hepatocellular carcinoma cells via decrease of integrin expression and ROCK.

Hepatocellular carcinoma (HCC) has become a global health concern and is one of the leading causes of cancer death after lung and gastric cancers. It has been suggested that the 3-hydroxy-3-methyl-glutarylcoenzyme-CoA (HMG-CoA) reductase inhibitor simvastatin exhibits anticancer properties. To this end, we analyzed the influence of simvastatin on the cell growth and adhesion of HCC and evaluated the yet poorly characterized mechanism of action of simvastatin in HCC. HepG2 and Huh7 cells were treated with simvastatin (16-64 µM) for different time periods. Cell proliferation using the MTT assay and tumor cell adhesion to endothelial cell monolayers were evaluated. ß1, ß3 and α2 integrin adhesion receptors and the downstream target of simvastatin Rho-dependent kinase (ROCK) were analyzed by Western blot. Further blocking studies with the ROCK-inhibitor H1152 and anti-integrin ß1 and ß3 antibodies were carried out. Simvastatin treatment inhibited dose-dependently tumor cell growth and attachment to endothelium. The inhibitory effect of simvastatin on cell adhesion was associated with decreased expression of ß1, ß3 and α2 integrins. Furthermore, simvastatin strongly reduced the expression of ROCK-I and activated MYPT, an indicator of ROCK activity. Also, the ROCK-inhibitor H1152 reduced the adhesive capacity of the tumor cells. Anti-adhesive effects of simvastatin were prevented by exogenous mevalonate, a downstream product of HMG-CoA. Tumor cell adhesion to endothelium was significantly impaired following incubation with functional anti-ß1 antibody. Simvastatin modifies the expression of cell adhesion molecules leading to reduced tumor cell growth and invasion. These beneficial effects of simvastatin may be mediated by ROCK. The data presented may point to novel treatment options for HCC.

Effect of tongxinluo on vasoconstriction induced by the chronic injury of the adventitia in the rat carotid artery.

OBJECTIVE: Tongxinluo (TXL) is a traditional Chinese medicine that is developed on the meridian theory of traditional Chinese medicine, with the function of alleviating the angina. The present study was undertaken to explore the molecular mechanism of TXL in treating the pectoris angina through observing the effectiveness of TXL superfine powder on the vasoconstriction and the activation of RhoA/Rho-kinase pathway induced by the injury of the adventitia. METHODS: 36 male Wistar Kyoto rats were assigned to 3 treatments (n=12): vehicle, TXL (400 mg kg(-1) day(-1)) and fasudil (15 mg kg(-1) day(-1)). After 1 week of treatment, adventitia injury was induced by positioning a silicone collar around the right carotid artery for 1 week. Blood flow and vascular reactivity to serotonin were determined 1 week after injurying, the both sides of carotids were harvested for morphometry, Western blotting analysis and RT-PCR analysis. RESULTS: Adventitia injury leaded to histological changes of vasoconstriction with the lumen cross-sectional area of 44.7% (p<0.001) decreasing and the media diameter of 62.31% (p<0.001) increasing, accompanying by the reduction of the blood flow and the increase of vascular reactivity sensitivity to serotonin. Treatment with both TXL superfine powder and fasudil can prevent the development of vasoconstriction, improve the carotid blood flow and normalize the vascular hypersensitivity to serotonin. Adventitia injuring of the rat carotid increased the expression of Rho-kinase mRNA and p-MYPT1(Thr696) protein by 1.78-fold (p<0.05) and >2-fold respectively (p<0.05). TXL reduced the expression of Rho-kinase mRNA and p-MYPT1(Thr696) protein by 54.2% (p<0.05) and 57.1% (p<0.05) respectively in collared arteries. Fasudil restrained the p-MYPT1(Thr696) protein expression by 63.8% (p<0.05) in collared arteries, did not affect the collar-induced the increase of Rho-kinase mRNA expression (p>0.05). CONCLUSIONS: Treatment with TXL, similar to that with fasudil, can effectively prevent collar-induced vasoconstriction and vascular hyperreactivity to serotonin through inhibiting the RhoA/Rho-kinase pathway.

Antinociceptive effects of AS1892802, a novel Rho kinase inhibitor, in rat models of inflammatory and noninflammatory arthritis.

Rho kinase (ROCK) is involved in various physiological functions, including cell motility, vasoconstriction, and neurite extension. Although a functional role of ROCK in nociception in the central nervous tissue has been reported in neuropathy, the peripheral function of this protein in hyperalgesia is not known. In this study, antinociceptive effects of AS1892802 [1-[(1S)-2-hydroxy-1-phenylethyl]-3-[4-(pyridin-4-yl)phenyl]urea], a novel and highly selective ROCK inhibitor, were investigated in two rat models of arthritis. Orally administered AS1892802 exhibited potent antinociceptive effect in both an adjuvant-induced arthritis (AIA) model (inflammatory arthritis model) and a monoiodoacetate-induced arthritis (MIA) model (noninflammatory arthritis model), with an ED(50) of 0.15 mg/kg (MIA model). Fasudil, a ROCK inhibitor, and tramadol were also effective in both models; however, diclofenac was effective only in the AIA model. The onset of antinociceptive effect of AS1892802 was as fast as those of tramadol and diclofenac. AS1892802 did not induce gastric irritation or abnormal behavior. Because AS1892802 rarely penetrates the central nervous tissue and is also effective by intra-articular administration, it seemed to function peripherally. These results suggest that AS1892802 has an attractive analgesic profile for the treatment of severe osteoarthritis pain.

Fasudil hydrochloride induces osteoblastic differentiation of stromal cell lines, C3H10T1/2 and ST2, via bone morphogenetic protein-2 expression.

Rho-kinase (ROK), downstream of the mevalonate pathway, is detrimental to vessels, and suppressing its activity is a target for the treatment of human disease such as coronary artery disease and pulmonary hypertension. Recent studies have shown that ROK has a crucial role in bone metabolism. However, the role of ROK in stromal cells is still unclear. The present study was undertaken to investigate the effect of a ROK inhibitor, fasudil hydrochloride, on stromal cell lines, C3H10T1/2 and ST2. In both cells, Fasudil significantly stimulated alkaline phosphatase activity and enhanced cell mineralization. Moreover, fasudil significantly increased the mRNA expression of collagen-I, osteocalcin, and bone morphogenetic protein-2 (BMP-2). Supplementation of noggin, a BMP-2 antagonist, significantly reversed the fasudil-induced collagen-I and osteocalcin mRNA expression in both cells. These findings suggest that fasudil induces the osteoblastic differentiation of stromal cells via enhancing BMP-2 expression, and that this drug might be beneficial for not only atherosclerosis but also osteoporosis by promoting bone formation.

Oily fraction of Semecarpus anacardium Linn nuts involves protein kinase C activation for its pro-inflammatory response.

The oily fraction (non polar fraction-NPF) of S. anacardium (SA) significantly increased the expression of protein kinase C-delta (PKC-delta) in macrophages in concentration dependent manner, which was similar to phorbol myristate acetate (PMA) response. Further, H-7 (1-(5-isoquinolinesulphonyl)-2-methylpiperazine), an inhibitor of PKC significantly inhibited this NPF mediated response in a concentration dependent manner. In the post treatment kinetics, H-7 showed this inhibition only up to 6 min post NPF/PMA addition, but in similar condition, quercetin, a flavone with reported antioxidant property, showed this inhibition only up to 2 min. The results clearly suggest that oily fraction of SA nuts enhances the expression of PKC protein, which may be responsible for its reported pro-inflammatory property.