RhoA/rho kinase pathway activation in age-associated endothelial cell dysfunction and thrombosis.

The small GTPase RhoA and the downstream Rho kinase (ROCK) regulate several cell functions and pathological processes in the vascular system that contribute to the age-dependent risk of cardiovascular disease, including endothelial dysfunction, excessive permeability, inflammation, impaired angiogenesis, abnormal vasoconstriction, decreased nitric oxide production and apoptosis. Frailty is a loss of physiological reserve and adaptive capacity with advanced age and is accompanied by a pro-inflammatory and pro-oxidative state that promotes vascular dysfunction and thrombosis. This review summarises the role of the RhoA/Rho kinase signalling pathway in endothelial dysfunction, the acquisition of the pro-thrombotic state and vascular ageing. We also discuss the possible role of RhoA/Rho kinase signalling as a promising therapeutic target for the prevention and treatment of age-related cardiovascular disease.

Alkannin Inhibited Hepatic Inflammation in Diabetic Db/Db Mice.

BACKGROUND/AIMS: The current study was designed to investigate the protective role of alkannin (ALK) on liver injury in diabetic C57BL/KsJ-db/db mice and explore its potential mechanisms. METHODS: An oral glucose tolerance test (OGTT) was performed. The levels of insulin, alanine aminotransferase (ALT), aspartate aminotransaminase (AST), total cholesterol (TC) and triglyceride (TG) were determined by commercial kits. The pro-inflammatory cytokines interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α were determined by ELISA. The levels of the ROCK/NF-κB pathway were determined by Western blotting. RESULTS: The contents of pro-inflammatory cytokines interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α were inhibited by ALK, metformin or fasudil in diabetic db/db mice. Further, Western blotting analysis showed that the expression of Rho, ROCK1, ROCK2, p-NF-κBp65, and p-IκBα was significantly reversed by ALK treatment. In human hepatic HepG2 cells, the hepatoprotective effects of ALK were further characterized. With response to palmitic acid-challenge, increased amounts of insulin, ALT, AST, TG, and TC were observed, whereas ALK pretreatment significantly inhibited their leakage in HepG2 cells without appreciable cytotoxic effects. The inflammation condition was recovered with ALK treatment as shown by changes of IL-1ß, IL-6 and TNF-α. Further, Western blotting analysis also suggested that ALK improves hepatic inflammation in a Rho-kinase pathway. CONCLUSION: The present study successfully investigated the role of Rho-kinase signalling in diabetic liver injury. ALK exhibited hepatoprotective effects in diabetic db/db mice, and it might act through improving hepatic inflammation through the Rho-kinase pathway.

Bortezomib induces thrombocytopenia by the inhibition of proplatelet formation of megakaryocytes.

Bortezomib is a potent proteasome inhibitor that has been extensively used to treat multiple myeloma. One of the most common grade 3 adverse events is cyclic thrombocytopenia. In this study, we studied the mechanism by which bortezomib induces thrombocytopenia in a mouse model. After the intravenous administration of bortezomib (2.5 mg/kg) via tail vein, platelet counts significantly decreased on days 2-4 and recovered to the normal range on day 6. Bortezomib (2.5 mg/kg) injected into mice in vivo did not affect colony-forming unit-megakaryocytes (CFU-Mk) or megakaryocytes in the bone marrow. However, proplatelet formation (PPF) significantly decreased on days 2 and 4, after bortezomib administration to mice. Meanwhile, CFU-Mk formation and the ploidy distribution of cultured megakaryocytes in vitro were not affected by bortezomib used at concentrations of ≤ 1 ng/mL. The PPF of megakaryocytes in vitro significantly decreased with 0.1, 1, 10, and 100 ng/mL bortezomib. Considering the bortezomib concentration in clinical studies, these data strongly suggest that decreased PPF activity induces thrombocytopenia. To elucidate the mechanism behind decreased PPF, Western blot was performed. Activated Rho expression increased after the incubation of murine platelets with bortezomib. Decreased PPF activity was eliminated by the addition of Y27632, a Rho kinase inhibitor, in vitro. Given that the Rho/Rho kinase pathway is a negative regulator of PPF, bortezomib increases activated Rho, inducing decreased PPF, which results in decreased platelet count.

Inhibition of U-II/UT signaling ameliorates cystitis-associated bladder hyperactivity by targeting the RhoA/Rho-kinase pathway.

Urotensin II (U-II) and its receptor (UT) are involved in the pathogenesis of various diseases; however, their association with the development of cystitis has not been elucidated. The present study was designed to investigate the functional role of U-II/UT signaling in cyclophosphamide (CYP)-induced cystitis. A total of 60 female rats were randomly divided into the control and CYP-treated groups. Intraperitoneal injection of CYP successfully induced cystitis in rats of the CYP-treated group. The protein and mRNA expression levels of U-II and UT were significantly enhanced in rat bladder tissues of the CYP-treated group. Furthermore, the results of the immunofluorescence staining analysis demonstrated that CYP treatment apparently increased the expression levels of UT in the urothelium layer, detrusor smooth muscle, and bladder interstitial Cajal-like cells. The selective antagonist of UT, SB657510 (10 µm), significantly suppressed the CYP-induced increase in the spontaneous contractions of muscle strips and ameliorated the bladder hyperactivity of CYP-treated rats. Moreover, CYP treatment significantly increased the protein expression levels of Ras homolog family member (Rho) A and Rho-associated protein kinase 2 in rat bladder tissues. Following pretreatment with the Rho-kinase inhibitor Y-27632 (10 µm), the inhibitory effects of SB657510 (10 µm) on the spontaneous contractions of muscle strips were eliminated. In conclusion, the results of the present study suggested that activation of U-II/UT signaling promoted the development of cystitis-associated-bladder hyperactivity by targeting the RhoA/Rho-kinase pathway, indicating that the U-II/UT signaling could serve as a novel target for the treatment of interstitial cystitis/bladder pain syndrome.

Comprehensive review on novel targets and emerging therapeutic modalities for pulmonary arterial Hypertension.

Pulmonary Arterial Hypertension (PAH) is the progressive increase in mean pulmonary arterial pressure (mPAP) (≥20 mmHg at rest). Current treatment strategies include the drugs targeting at nitric oxide pathway, endothelin receptors, prostaglandin receptors, thromboxane receptors and phosphodiesterase inhibitors, which provides the symptomatic relief. Despite of these treatments, the mortality amongst the PAH patients remains high due to non-reversal of the condition. This review primarily covers the introduction of PAH and the current treatments of the disease. This is followed by the newer disease targets expressed in the pathobiology of the disease like Rho Kinase Pathway, Vasoactive Intestinal Peptide Pathway, Receptor Tyrosine Kinases, Serotonin signalling pathway, Voltage-gated potassium (Kv) channel pathway. Newer formulation strategies for targeting at these specific receptors were covered and includes nano formulations like liposomes, Micelles, Polymeric Nanoparticles, Solid Lipid Nanoparticles (SLN), Bioresorbable stents, NONOates, Cell-Based Therapies, miRNA therapy for PAH. Novel targets were identified for their role in the pathogenesis of the PAH and needs to be targeted with new molecules or existing molecules effectively. Nanosystems have shown their potential as alternative carriers on the virtue of their better performance than traditional drug delivery systems.

Enhancement of the RhoA/Rho kinase pathway is associated with stress-related erectile dysfunction in a restraint water immersion stress model.

Stress is a risk factor for erectile dysfunction (ED); however, the pathology of stress-induced ED remains unclear. Accordingly, in this study, we investigated the mechanisms of stress-induced ED using a rat model. Ten-week-old male Wistar/ST rats were maintained in a cage filled with water to a height of 2 cm (stress group) or a normal cage (control group). We found that water immersion stress significantly enhanced the contractile response to noradrenaline in the corpus cavernosum (CC) (p < 0.05). Moreover, stress significantly decreased erectile function, as assessed by changes in intracavernous pressure (p < 0.01). In addition, Rho kinase-1 (ROCK-1) protein expression was significantly upregulated under stress conditions (p < 0.05), and phosphorylated myosin light chain (phospho-MLC) levels, contribute to smooth muscle contraction, were also upregulated (p < 0.01). Treatment with fasudil hydrochloride, a Rho kinase inhibitor, for 5 days significantly improved erectile function (p < 0.01) and normalized ROCK-1 and phospho-MLC levels (p < 0.01). Thus, the RhoA/Rho kinase pathway may be associated with stress-induced ED via contraction of CC. Stress also decreased the smooth muscle/collagen ratio of CC (p < 0.01), and fasudil treatment did not alleviate these effects (p = 0.50). These findings suggested that penile fibrosis gradually progressed under stress conditions and that fibrosis may be independent of the RhoA/Rho kinase pathway, implying that longer exposure to stress may promote ED. We conclude that stress-induced ED was caused by contraction of CC mediated by the RhoA/Rho kinase pathway.

Inhibition of RhoA/Rho kinase signaling pathway by fasudil protects against kainic acid-induced neurite injury.

AIM: RhoA/Rho kinase pathway is essential for regulating cytoskeletal structure. Although its effect on normal neurite outgrowth has been demonstrated, the role of this pathway in seizure-induced neurite injury has not been revealed. The research examined the phosphorylation level of RhoA/Rho kinase signaling pathway and to clarify the effect of fasudil on RhoA/Rho kinase signaling pathway and neurite outgrowth in kainic acid (KA)-treated Neuro-2A cells and hippocampal neurons. METHOD: Western blotting analysis was used to investigate the expression of key proteins of RhoA/Rho kinase signaling pathway and the depolymerization of actin. After incubated without serum to induce neurite outgrowth, Neuro-2A cells were fixed, and immunofluorescent assay of rhodamine-phalloidin was applied to detect the cellular morphology and neurite length. The influence of KA on neurons was detected in primary hippocampal neurons. Whole-cell patch clamp was conducted in cultured neurons or hippocampal slices to record action potentials. RESULT: KA at the dose of 100-200 µmol/L induced the increase in phosphorylation of Rho-associated coiled-coil-containing protein kinase and decrease in phosphorylation of Lin11, Isl-1 and Mec-3 kinase and cofilin. The effect of 200 µmol/L KA was peaked at 1-2 hours, and then gradually returned to baseline after 8 hours. Pretreatment with Rho kinase inhibitor fasudil reversed KA-induced activation of RhoA/Rho kinase pathway and increase in phosphorylation of slingshot and 14-3-3, which consequently reduced the ratio of G/F-actin. KA treatment induced inhibition of neurite outgrowth and decrease in spines both in Neuro-2a cells and in cultured hippocampal neurons, and pretreatment with fasudil alleviated KA-induced neurite outgrowth inhibition and spine loss. CONCLUSION: These data indicate that inhibiting RhoA/Rho kinase pathway might be a potential treatment for seizure-induced injury.

The effect of metformin on indomethacin-induced gastric ulcer: Involvement of nitric oxide/Rho kinase pathway.

Gastric ulcer is a very common disease that represent an economic burden. Non-steroidal anti-inflammatory drugs induce ulcer in old patients and in patients with comorbidities. Indomethacin is widely used to induce gastric ulcer in animal models. Diabetic patients are highly susceptible to develop gastric ulcer. Metformin, the first line medication for the treatment of type II diabetes melilites that have many off label uses in non-diabetic patients, has been recently reported to have anti-inflammatory activities. Therefore, this research was conducted to assess the possible healing effects of metformin on gastric ulcers induced by indomethacin in rats. Indomethacin (48 mg/kg) single dose increased stomach acidity, ulcer index and induced histopathological changes. Indomethacin also decreased mucin levels and increased the activity of tumor necrosis factor-α (TNF-α), nuclear factor kappa-B (NF-κB), Rho-associated protein kinas-1 (ROCK-1) and decreased the levels of the protective nitric oxide (NO). After the induction of ulcer, rats were treated by omeprazole (30 mg/kg) or metformin (50, 100 or 200 mg/kg). Omeprazole and metformin were found to decrease stomach acidity and ulcer index, restored the histological features and increased mucin levels. Both also decreased the levels of NF-κB, TNF-α, ROCK-1 and increased NO. Metformin exerted ulcer healing effects comparable to that of omeprazole. This can be attributed, at least partly, to its anti-inflammatory activity and increasing NO levels.

Protective effects of curcumin against rat intestinal inflammation­related motility disorders.

Intestinal inflammation frequently occurs alongside dysmotility, which is characterized by altered myosin light chain phosphorylation levels. Curcumin, an active component from the ginger family, is reported to confer anti­inflammatory effects. However, the effects of curcumin on both diarrhea and constipation associated inflammation remains to be elucidated. The present study was designed to investigate the effects of curcumin on diarrhea and constipation and to determine the related mechanisms. Sprague­Dawley rats were used to establish diarrhea and constipation models via intracolonic acetic acid (4%) instillation or cold water gavage for 2 weeks, respectively. Blood samples were collected to measure the serum levels of the cytokines TNF­α and IL­1ß using ELISA kits. Western blotting was performed to measure NF­κB, RhoA, Rho­related kinase 2, phosphorylated MLC20, phosphorylated myosin phosphorylated target subunit 1, 130k Da­MLC kinase (MLCK), c­kit tyrosine kinase protein expression, and reverse transcription­quantitative PCR was conducted to measure MLCK expression levels. The results indicated that curcumin reversed the elevations in the pro­inflammatory cytokines IL­1ß and TNF­α by inhibiting the NF­κB pathway in rats with diarrhea and constipation. The results also indicated that myosin light chain (MLC) phosphorylation in intestinal smooth muscle was positively and negatively associated with the motility of inflammation­related diarrhea and constipation in rats, respectively. Curcumin significantly reversed the increased MLC phosphorylation in the jejunum of the rats with diarrhea, significantly enhanced the reductions in inflammatory mediators, including TNF­α and IL­1ß, of rats with constipation and significantly ameliorated the related hyper­motility and hypo­motility in rats with both diarrhea and constipation. In conclusion, the potential roles of the MLC kinase, c­kit tyrosine and Rho A/Rho­associated kinase 2 pathways, which are involved in curcumin­induced amelioration of inflammation­related diarrhea and constipation, were explored in the present study. Results from the present study suggested that curcumin has potential therapeutic value for treating intestinal inflammation and inflammation­related motility disorders.

Exaggerated blood pressure response to fasudil or nifedipine in hypertensive Ren-2 transgenic rats: role of altered baroreflex.

Basal calcium sensitization is decreased in spontaneously hypertensive rats, although their blood pressure (BP) response to acute Rho-kinase inhibition is enhanced. Using fasudil (Rho-kinase inhibitor) or nifedipine (L-VDCC blocker), we evaluated the contribution of calcium sensitization and calcium entry to BP maintenance in hypertensive transgenic Ren-2 rats (TGR) focusing on the influence of major vasoactive systems and/or baroreflex efficiency on BP responses to these two drugs. Homozygous TGR and normotensive Hannover Sprague-Dawley (HanSD) control rats aged 5, 11, or 22 weeks were used. The acute BP-lowering effects of fasudil or nifedipine were studied in intact rats, nitric oxide-deficient L-NAME-pretreated rats and rats subjected to combined blockade of the renin-angiotensin system (RAS), sympathetic nervous system (SNS) and nitric oxide synthase (NOS). Fasudil- or nifedipine-induced BP reduction increased during hypertension development in TGR. By contrast, the nifedipine-induced BP response decreased, whereas the fasudil-induced BP response increased with age in HanSD controls. Our data indicated a major contribution of nifedipine-sensitive calcium entry and relative attenuation of calcium sensitization in hypertensive rats compared with normotensive controls. The BP responses to fasudil or nifedipine were enhanced by NOS inhibition and combined blockade in normotensive HanSD rats but not in hypertensive TGR. In conclusion, calcium  sensitization is attenuated by endogenous nitric oxide in normotensive HanSD rats but not in hypertensive TGR. Moreover, BP reduction elicited by acute Rho-kinase inhibition is partially compensated by enhanced sympathetic vasoconstriction. The decreased compensation in hypertensive rats with impaired baroreflex efficiency explains their greater BP response to fasudil than in normotensive animals.

Geniposide improves hepatic inflammation in diabetic db/db mice.

The current study was designed to investigate the protective role of geniposide (GE) in liver injury in diabetic C57BL/KsJ-db/db mice and to explore the underlying mechanisms. The oral glucose tolerance test was performed, and the levels of insulin, alanine aminotransferase (ALT), aspartate aminotransaminase (AST), total cholesterol (TC) and triglyceride (TG) were determined. The levels of the pro-inflammatory cytokines interleukin (IL)-1ß, IL-6 and tumour necrosis factor-α were decreased by GE, metformin and fasudil in diabetic db/db mice. Western blotting analysis showed that the expression levels of Rho, ROCK1, ROCK2, p-NF-κBp65 and p-IκBα were significantly reversed by GE treatment. These findings demonstrated that GE exhibits a protective effect on diabetic hepatic inflammation.

Rho Kinase Pathway Alterations in the Brain and Leukocytes in Huntington's Disease.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the huntingtin gene. Therapeutic approaches targeting mutant huntingtin (mtHtt) or its downstream toxic consequences are under development, including Rho kinase pathway inhibition. We investigated the messenger RNA (mRNA) expression of Rho kinase pathway genes, including RhoA (Ras homolog family member A), ROCK1 (Rho-associated kinase1), PRK2 (protein kinase C-related protein kinase 2), Profilin1, cofilin1, MYPT1 (myosin phosphatase target subunit 1), and LIMK1 (LIM domain kinase 1) in HD human blood leukocytes, postmortem brain, and in R6/2 HD mouse brain tissue using qPCR. RhoA, ROCK1, PRK2, Profilin1, cofilin1, and MYPT1 were significantly increased in HD blood compared to controls. In frontal cortex of HD postmortem brain tissue, the expression of RhoA, ROCK1, PRK2, Profilin1, and MYPT1 were also significantly increased. In the brain from 4-week-old R6/2 mice, the expression of Rock1, Prk2, Cofilin1, and MYPT1 was significantly increased while RhoA, Rock1, Profilin1, Cofilin1, and Mypt1 were increased and Limk1 mRNA decreased in 13-week-old R6/2 mice. Western blot analysis using human postmortem tissues for ROCK1 and Profilin1 demonstrated significantly increased protein levels, which correlated with the mRNA increases. Collectively, we have shown the panel of Rho kinase pathway genes to be highly altered in human HD blood, postmortem brain tissue, and in R6/2 mice. These studies confirm that HD upregulates the Rho kinase pathway and identifies mRNAs that could serve as peripheral markers in HD patients and translational markers in HD mouse models.

Rosuvastatin intensifies the beneficial effects of rho-kinase inhibitor in reversal of monocrotaline-induced pulmonary hypertension.

INTRODUCTION: It remains controversial whether statins have a beneficial effect on pulmonary arterial hypertension (PAH). This study is intended to evaluate whether statin, co-administered with Rho-kinase inhibitor, could enhance its efficacy. Although Rho-kinase inhibitors, including fasudil, have been reported to improve pulmonary hypertension in experimental and clinical studies, the combination of these agents has not been tested in the treatment of pulmonary hypertension (PH). MATERIAL AND METHODS: The effects of such a regimen on hemodynamics, right ventricle hypertrophy, and Rho-associated protein kinase (ROCK) activity in experimental monocrotaline (MCT)-induced pulmonary hypertension were examined. Fourteen days after monocrotaline injection (60 mg/kg), male rats were treated orally for another 14 days with fasudil (15 mg/kg per day), or with a combination of fasudil + rosuvastatin (10 mg/kg per day). RESULTS: The drug combination reversed the MCT-induced increase in right ventricle pressure (RVP) and reduced right ventricular hypertrophy (RV/LV + S ratio) more than Rho kinase inhibitor alone. The simultaneous administration of fasudil and rosuvastatin caused a further decrease of RhoA kinase activity in isolated lung tissues as compared to fasudil alone. CONCLUSIONS: The results indicate that rosuvastatin intensifies the beneficial effects of Rho-kinase inhibitor on the Rho/Rho-kinase pathway and such a combination may represent an option for the treatment of pulmonary arterial hypertension.

Erectile dysfunction in heart failure rats is associated with increased neurogenic contractions in cavernous tissue and internal pudendal artery.

AIMS: The rates of erectile dysfunction (ED) in heart failure (HF) are extremely high. This study tested the hypothesis that rats with HF display ED and that HF leads to increased sympathetic-mediated contractile tone of the cavernous tissue and/or internal pudendal arteries (IPA) as potential mechanisms contributing to ED. MAIN METHODS: HF was induced in Wistar rats by ligation of the left anterior descending coronary artery. Changes in the ratio of intracavernosal pressure/mean arterial pressure (ICP/MAP) after electrical stimulation of major pelvic ganglion were determined in vivo. Cavernosal and IPA contractions were induced by electric field stimulation (EFS) and phenylephrine. RhoA, Rho kinase 2 (ROCK 2) and myosin phosphatase target protein 1 (MYPT-1) protein expression and phosphorylation levels were also determined. KEY FINDINGS: HF rats display impaired erectile function represented by decreased ICP/MAP responses. EFS-mediated contractions were increased by HF in cavernous tissue and IPA. Contractions induced by phenylephrine were increased in cavernous tissue of HF rats, but decreased in IPA rings. Moreover, HF decreased RhoA protein expression, but increased ROCK 2 and MYPT-1 phosphorylation levels in cavernous tissue. In conclusion, rats with HF induced by myocardial infarction display ED in vivo and increased sympathetic-mediated contractile responses in cavernous tissue and IPA. Increased sympathetic-mediated contractile responses were associated with increased ROCK 2 and MYPT-1 phosphorylation in cavernosal tissue, suggesting the involvement of ROCK signaling pathway in ED genesis. SIGNIFICANCE: Our findings suggest new mechanisms linking HF to ED, providing potential therapeutic targets for treating ED associated to HF.

PVP formulated fullerene (C60) increases Rho-kinase dependent vascular tissue contractility in pregnant Sprague Dawley rats.

Pregnancy is a unique physiological state, in which C60 fullerene is reported to be distributed in both maternal and fetal tissues. Tissue distribution of C60 differs between pregnant and non-pregnant states, presumably due to functional changes in vasculature during pregnancy. We hypothesized that polyvinylpyrrolidone (PVP) formulated C60 (C60/PVP) increases vascular tissue contractility during pregnancy by increasing Rho-kinase activity. C60/PVP was administered intravenously to pregnant and non-pregnant female Sprague Dawley rats. Vascular responses were assessed using wire myography 24h post-exposure. Increased stress generation was observed in uterine artery, thoracic aorta and umbilical vein. Rho-Rho-kinase mediated force maintenance was increased in arterial segments from C60/PVP exposed pregnant rats when compared to PVP exposed rats. Our findings suggest that intravenous exposure to C60/PVP during pregnancy increases vascular tissue contractility of the uterine artery through elements of Rho-Rho-kinase signaling during late stages of pregnancy.

Suppression of the Rho/Rho-kinase pathway and prevention of cerebral vasospasm by combination treatment with statin and fasudil after subarachnoid hemorrhage in rabbit.

The Rho/Rho-kinase pathway is considered important in the pathogenesis of sustained smooth muscle cell contraction during cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH). The aims of this study were to investigate whether combination treatment, with pitavastatin as an inhibitor of RhoA and fasudil as an inhibitor of Rho-kinase, prevents the cerebral vasospasm. SAH was simulated using the double-hemorrhage rabbit model, and pitavastatin, or fasudil, or both (combination treatment) were administrated. The basilar artery (BA) cross-sectional area only in the combination treatment group was statistically larger than in the SAH group (p<0.05). BA Rho-kinase, as measured by ELISA, was statistically reduced only in the combination treatment group compared with the SAH group (p<0.05). In the other two treatment groups, pitavastatin or fasudil treatment group showed larger BA cross-sectional areas and lower value for BA Rho-kinase, but there were no statistically significant differences compared with the SAH group. The expression of endothelial nitric oxide synthase (eNOS), evaluated by immunohistochemistry in the pitavastatin group and the combination group, was higher than in the SAH group. Results indicate that combination treatment could extensively prevent cerebral vasospasm due to the synergic effect of combining pitavastatin and fasudil on the Rho/Rho-kinase pathway and on eNOS.