Vascular Inflammation and Smooth Muscle Contractility: The Role of Nox1-Derived Superoxide and LRRC8 Anion Channels.

Vascular inflammation underlies the development of hypertension, and the mechanisms by which it increases blood pressure remain the topic of intense investigation. Proinflammatory factors including glucose, salt, vasoconstrictors, cytokines, wall stress, and growth factors enhance contractility and impair relaxation of vascular smooth muscle cells. These pathways share a dependence upon redox signaling, and excessive activation promotes oxidative stress that promotes vascular aging. Vascular smooth muscle cell phenotypic switching and migration into the intima contribute to atherosclerosis, while hypercontractility increases systemic vascular resistance and vasospasm that can trigger ischemia. Here, we review factors that drive the initiation and progression of this vasculopathy in vascular smooth muscle cells. Emphasis is placed on the contribution of reactive oxygen species generated by the Nox1 NADPH oxidase which produces extracellular superoxide (O2•-). The mechanisms of O2•- signaling remain poorly defined, but recent evidence demonstrates physical association of Nox1 with leucine-rich repeat containing 8 family volume-sensitive anion channels. These may provide a pathway for influx of O2•- to the cytoplasm, creating an oxidized cytoplasmic nanodomain where redox-based signals can affect both cytoskeletal structure and vasomotor function. Understanding the mechanistic links between inflammation, O2•- and vascular smooth muscle cell contractility may facilitate targeting of anti-inflammatory therapy in hypertension.

Differential expression of the enzymes regulating myosin light chain phosphorylation are responsible for the slower relaxation of pulmonary artery than mesenteric artery in rats.

While arterial tone is generally determined by the phosphorylation of Ser19 in myosin light chain (p-MLC2), Thr18/Ser19 diphosphorylation of MLC2 (pp-MLC2) has been suggested to hinder the relaxation of smooth muscle. In a dual-wire myography of rodent pulmonary artery (PA) and mesenteric artery (MA), we noticed significantly slower relaxation in PA than in MA after 80 mM KCl-induced condition (80K-contraction). Thus, we investigated the MLC2 phosphorylation and the expression levels of its regulatory enzymes; soluble guanylate cyclase (sGC), Rho-A dependent kinase (ROCK) and myosin light chain phosphatase target regulatory subunit (MYPT1). Immunoblotting showed higher sGC-α and ROCK2 in PA than MA, while sGC-ß and MYPT1 levels were higher in MA than in PA. Interestingly, the level of pp-MLC2 was higher in PA than in MA without stimulation. In the 80K-contraction state, the levels of p-MLC2 and pp-MLC2 were commonly increased. Treatment with the ROCK inhibitor (Y27632, 10 µM) reversed the higher pp-MLC2 in PA. In the myography study, pharmacological inhibition of sGC (ODQ, 10 µM) slowed relaxation during washout, which was more pronounced in PA than in MA. The simultaneous treatment of Y27632 and ODQ reversed the impaired relaxation in PA and MA. Although treatment of PA with Y27632 alone could increase the rate of relaxation, it was still slower than that of MA without Y27632 treatment. Taken together, we suggest that the higher ROCK and lower MYPT in PA would have induced the higher level of MLC2 phosphorylation, which is responsible for the characteristic slow relaxation in PA.

CSE/H2S ameliorates colitis in mice via protection of enteric glial cells and inhibition of the RhoA/ROCK pathway.

The enteric glial cells (EGCs) participate in the homeostasis of the gastrointestinal tract, and RhoA/ROCK signaling pathway plays a vital role in colonic tight junctions. Hydrogen sulfide (H2S) has been reported to alleviate colitis. However, the effect and mechanism of endogenous H2S on colitis remain unclear. This study established a Cystathionine-γ-lyase (CSE) knockout mouse model, a significant source of H2S production in the gut. The role of CSE-produced H2S on EGCs and the RhoA/ROCK signaling pathway was investigated in experimental colitis using CSE knockout (KO) and wild-type (WT) mice. CSE gene knockout animals presented with disease progression, more deteriorated clinical scores, colon shortening, and histological damage. EGCs dysfunction, characterized by decreased expression of the glial fibrillary acidic protein (GFAP), C3, and S100A10, was observed in the colon of WT and KO mice, especially in KO mice. RhoA/ROCK pathway was significantly upregulated in colon of colitis mice, which was more evident in KO mice. Pretreatment with NaHS, an exogenous H2S donor, significantly ameliorated mucosal injury and inhibited the expression of proinflammatory factors. Furthermore, we found that NaHS promoted the transformation of EGCs from "A1" to "A2" type, with decreased expression of C3 and increased expression of S100A10. These findings suggest that CSE/H2S protects mice from colon inflammation, which may be associated with preserving EGCs function by promoting EGCs transformation and inhibiting the RhoA/ROCK pathway.

Comparison of Netarsudil/Latanoprost Therapy with Latanoprost Monotherapy for Lowering Intraocular Pressure: A Systematic Review and Meta-analysis.

PURPOSE: Netarsudil is a Rho kinase inhibitor and the first new class of clinically useful ocular hypotensive agents. In this study, we conducted a systematic literature review and meta-analysis to summarize and synthesize the available evidence on the efficacy and safety of fixed-dose combination (FDC) therapy with netarsudil/latanoprost in patients with glaucoma. METHODS: We identified relevant studies in PubMed, Ovid Medline, Embase, and Cochrane Central until April 2021. The quality of the studies and the level of evidence were assessed using the Risk of Bias tool. Efficacy was measured as the mean difference in reducing intraocular pressure (IOP), and safety was assessed by the risk of conjunctival hyperemia (CH) due to FDC therapy, netarsudil monotherapy, or latanoprost monotherapy. RESULTS: Four studies met the predefined eligibility criteria and were included in the meta-analysis. The mean difference in the reduction in IOP after 2 weeks and 4 to 6 weeks of drug administration was -2.41 mmHg (95% confidence interval [CI], -2.95 to -1.87) and -1.77 mmHg (95% CI, -2.31 to -1.87), respectively, in patients receiving FDC therapy versus those receiving latanoprost monotherapy. On the other hand, latanoprost monotherapy had a greater effect in reducing IOP than netarsudil monotherapy after 4 to 6 weeks of administration (mean difference, 0.95 mmHg; 95% CI, 0.43 to 1.47). The risk of CH was significantly higher with both FDC therapy and netarsudil monotherapy compared to latanoprost monotherapy in week 12, where the relative ratio was 3.01 (95% CI, 1.95 to 4.66) and 2.33 (95% CI, 1.54 to 3.54), each. CONCLUSIONS: Netarsudil/latanoprost FDC therapy has a significantly greater effect on reducing IOP than latanoprost alone. The symptoms of CH were mostly mild, and only a few glaucoma patients discontinued the medication owing to CH in earlier clinical trials. Therefore, it would be beneficial to consider the administration of netarsudil/latanoprost FDC therapy in patients with glaucoma.

Advantages of Rho-associated kinases and their inhibitor fasudil for the treatment of neurodegenerative diseases.

Ras homolog (Rho)-associated kinases (ROCKs) belong to the serine-threonine kinase family, which plays a pivotal role in regulating the damage, survival, axon guidance, and regeneration of neurons. ROCKs are also involved in the biological effects of immune cells and glial cells, as well as the development of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Previous studies by us and others confirmed that ROCKs inhibitors attenuated the symptoms and progression of experimental models of the abovementioned neurodegenerative diseases by inhibiting neuroinflammation, regulating immune imbalance, repairing the blood-brain barrier, and promoting nerve repair and myelin regeneration. Fasudil, the first ROCKs inhibitor to be used clinically, has a good therapeutic effect on neurodegenerative diseases. Fasudil increases the activity of neural stem cells and mesenchymal stem cells, thus optimizing cell therapy. This review will systematically describe, for the first time, the effects of abnormal activation of ROCKs on T cells, B cells, microglia, astrocytes, oligodendrocytes, and pericytes in neurodegenerative diseases of the central nervous system, summarize the therapeutic potential of fasudil in several experimental models of neurodegenerative diseases, and clarify the possible cellular and molecular mechanisms of ROCKs inhibition. This review also proposes that fasudil is a novel potential treatment, especially in combination with cell-based therapy. Findings from this review add support for further investigation of ROCKs and its inhibitor fasudil for the treatment of neurodegenerative diseases.

Effect of manipulation on cartilage in rats with knee osteoarthritis based on the Rho-associated protein kinase/LIM kinase 1/Cofilin signaling pathways.

OBJECTIVE: To investigate the effect of manipulation treatment on knee osteoarthritis rats and the effect on Rho-associated protein kinase (ROCK)/LIM-kinase1 (LIMK1)/Cofilin signaling pathway. METHOD: Fifty Specific pathogen Free Sprague-Dawley rats were randomly divided into five groups ( = 8 each): blank group, model group, manipulation group, celecoxib group, and manipulation combined with celecoxib group (MC group). The osteoarthritis model was established by injecting 0.2 mL 4% papain into the articular disc of the rats. After successfully establishing the model, we treated the manipulation group with pushing manipulation using one-finger-meditation to the Neixiyan (EX-LE4), Waixiyan (EX-LE5), Xuehai (SP10), Liangqiu (ST34), and Zusanli (ST36) acupoints for 10 min each time. Also, the celecoxib group was gavaged with 24 mg•kg•d celecoxib, while the MC group was treated using both of these two methods. After four weeks, the cartilage of the right femur was removed for hematoxylin-eosin staining of the cartilage tissue. The expressions of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in serum were observed using the enzyme-linked immunosorbent assay. Besides, we detected the expressions of ROCK, LIMK1, Phospho-LIM-kinase1 (Phospho-LIMK1), Cofilin, and Phospho-Cofilin by Western blot. RESULTS: Compared to the model group, the manipulation group, celecoxib group, and MC group all exhibited superior results concerning pathological morphologic changes of cartilage, as observed by hematoxylin-eosin staining and calculated using the Mankin score. Besides, in contrast to the blank group, the model group exhibited elevated serum levels of IL-1ß and TNF-α ( 0.01), while the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage were all higher ( 0.01). Also, the serum levels of IL-1ß and TNF-α in each treatment group were lower (0.01) than in the model group. Moreover, there were lower expressions of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the manipulation group and the MC group (< 0.01). Compared with the model group, the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the celecoxib group were not statistically different ( > 0.05). CONCLUSION: In this study, we established that manipulation has a better curative effect than celecoxib. Manipulation inhibits the development of cytoskeleton damage in cartilage and slows articular degeneration by regulating the expression of related proteins in the cytoskeletal signaling pathway.

RhoA/ROCK-1 Signaling Pathway and Oxidative Stress in Coronary Artery Disease Patients

Abstract Introduction: Coronary artery disease (CAD) is an ischemic condition that occurs as a result of partial or complete interruption of blood flow by narrowing or complete blockage of the vessels supplying the heart, which are called coronary arteries. Our objective in this study is to investigate the RhoA/Rho-associated kinase (ROCK)-1 signaling pathway and oxidative stress in CAD patients. Methods: A total of 81 individuals aged between 40-70 years - including 45 patients (15 females and 30 males) who were admitted to the Artvin State Hospital Cardiovascular Surgery Clinic and were diagnosed with CAD and 36 healthy volunteers (15 females and 21 males) - participated in this study. Serum samples were tested for total cholesterol, triglyceride, low-density lipoprotein, high-density lipoprotein, malondialdehyde (MDA), superoxide dismutase (SOD), RhoA, and ROCK-1 values. Results: Serum RhoA, MDA levels, and ROCK-1 activity in the CAD group were found to be statistically significantly higher than in the control group (P<0.001). Concordantly, serum SOD activity was found to be statistically significantly lower in the CAD group than in the control group (P<0.001). Conclusion: Inhibition of the activity of RhoA/ROCK-1 pathway would be beneficial in treating cardiovascular diseases since this pathway plays an important role in the development of these diseases.

Status of Rho kinase inhibitors in glaucoma therapeutics-an overview.

Medical management remains the cornerstone of glaucoma management despite advances in the surgical or laser procedures. After a leap of almost two decades of the advent of prostaglandin analogues, recently a new class of drug, Rho kinase (ROCK) inhibitors, has come to limelight because of their varied therapeutic potential in different clinical conditions of eye, especially glaucoma. Their efficacy of lowering intraocular pressure (IOP) by virtue of an entirely different mechanism of decreasing outflow resistance has ignited a series of clinical trials evaluating their potential as monotherapy or as adjunct to existing antiglaucoma medications, and three of them ripasudil, netarsudil and roclatan have even been approved for clinical use in the recent past. There are evidences suggesting their beneficial effects in glaucoma patients even via non-IOP-dependent mechanisms like neuroprotection by improving blood flow to the optic nerve and increasing ganglion cell survival. They can even act as antifibrotic agents and reduce bleb scarring after glaucoma surgery. Hence, their effective role in glaucomatous optic neuropathy is multifaceted primary being improved drainage through the conventional pathway. On the other hand, certain local adverse effects like conjunctival hyperaemia have been reported in substantial proportion of patients, while some others like blepharitis, subconjunctival haemorrhages and cornea verticillata constitute less common side effects. The purpose of this review is to summarize the discovery, evolution and recent update of clinical trials on Rho kinase inhibitors as antiglaucoma medicine and to delineate their role in existing management protocol.

RhoA/ROCK-1 Signaling Pathway and Oxidative Stress in Coronary Artery Disease Patients.

INTRODUCTION: Coronary artery disease (CAD) is an ischemic condition that occurs as a result of partial or complete interruption of blood flow by narrowing or complete blockage of the vessels supplying the heart, which are called coronary arteries. Our objective in this study is to investigate the RhoA/Rho-associated kinase (ROCK)-1 signaling pathway and oxidative stress in CAD patients. METHODS: A total of 81 individuals aged between 40-70 years - including 45 patients (15 females and 30 males) who were admitted to the Artvin State Hospital Cardiovascular Surgery Clinic and were diagnosed with CAD and 36 healthy volunteers (15 females and 21 males) - participated in this study. Serum samples were tested for total cholesterol, triglyceride, low-density lipoprotein, high-density lipoprotein, malondialdehyde (MDA), superoxide dismutase (SOD), RhoA, and ROCK-1 values. RESULTS: Serum RhoA, MDA levels, and ROCK-1 activity in the CAD group were found to be statistically significantly higher than in the control group (P<0.001). Concordantly, serum SOD activity was found to be statistically significantly lower in the CAD group than in the control group (P<0.001). CONCLUSION: Inhibition of the activity of RhoA/ROCK-1 pathway would be beneficial in treating cardiovascular diseases since this pathway plays an important role in the development of these diseases.

Role of RhoA/ROCK2 signaling pathway in trilobatin-induced reduction of cerebral ischemia-reperfusion injury in rats / 中华麻醉学杂志

Objective:To evaluate role of Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil protein kinase 2 (ROCK2) signaling pathway in trilobatin-induced reduction of cerebral ischemia-reperfusion (I/R) injury in rats.Methods:Eighty clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 230-280 g, were divided into 4 groups ( n=20 each) using a random number table method: sham operation group (group S), cerebral I/R group (group IR), cerebral I/R plus trilobatin group (group T) and cerebral I/R plus trilobatin plus RhoA/ROCK2 signaling pathway agonist AA group (group A). The model of focal cerebral I/R injury was developed by middle cerebral artery occlusion in anesthetized animals.Trilobatin 15 mg/kg was given by gavage twice a day for 3 consecutive days in T and A groups.RhoA/ROCK2 signaling pathway agonist AA 10 mg/kg was intraperitoneally injected before each administration by gavage in group A. Neurobehavioral score was assessed at 24 h of reperfusion, then the rats were sacrificed, and the hippocampal tissues were isolated for determination of the apoptosis rate of hippocampal neurons (by flow cytometry), cerebral infarction volume (by TTC staining), and expression of phosphorylated RhoA (p-RhoA), ROCK2 and cleaved caspase-3 (by Western blot) and for microscopic examination of ultrastructure of hippocampal neurons (with a transmission electron microscope). Results:Compared with group S, the neurobehavioral score, apoptosis rate of hippocampal neurons, and cerebral infarction volume were significantly increased, the expression of p-RhoA, ROCK2 and cleaved caspase-3 was up-regulated ( P<0.05), and the pathological damage to hippocampal neurons was aggravated in group IR.Compared with group IR, the neurobehavioral score, apoptosis rate of hippocampal neurons and cerebral infarction volume were significantly decreased, the expression of p-RhoA, ROCK2 and cleaved caspase-3 was down-regulated ( P<0.05), and the pathological damage to hippocampal neurons was alleviated in group T. Compared with group T, the neurobehavioral score, apoptosis rate of hippocampal neurons, and cerebral infarction volume were significantly increased, the expression of p-RhoA, ROCK2 and cleaved caspase-3 was up-regulated ( P<0.05), and the pathological damage to hippocampal neurons was aggravated in group A. Conclusions:RhoA/ROCK2 signaling pathway is involved in trilobatin-induced reduction of cerebral I/R injury, which may be related to inhibition of apoptosis in hippocampal neurons of rats.

Rho-Kinase Inhibitors for the Treatment of Refractory Diabetic Macular Oedema.

Diabetic macular oedema (DMO) is one of the leading causes of vision loss associated with diabetic retinopathy (DR). New insights in managing this condition have changed the paradigm in its treatment, with intravitreal injections of antivascular endothelial growth factor (anti-VEGF) having become the standard therapy for DMO worldwide. However, there is no single standard therapy for all patients DMO refractory to anti-VEGF treatment; thus, further investigation is still needed. The key obstacles in developing suitable therapeutics for refractory DMO lie in its complex pathophysiology; therefore, there is an opportunity for further improvements in the progress and applications of new drugs. Previous studies have indicated that Rho-associated kinase (Rho-kinase/ROCK) is an essential molecule in the pathogenesis of DMO. This is why the Rho/ROCK signalling pathway has been proposed as a possible target for new treatments. The present review focuses on the recent progress on the possible role of ROCK and its therapeutic potential in DMO. A systematic literature search was performed, covering the years 1991 to 2021, using the following keywords: "rho-Associated Kinas-es", "Diabetic Retinopathy", "Macular Edema", "Ripasudil", "Fasudil" and "Netarsudil". Better insight into the pathological role of Rho-kinase/ROCK may lead to the development of new strategies for refractory DMO treatment and prevention.

Efficacy and safety of Fasudil on vasospasm caused by subarachnoid hemorrhage in elderly patients / 中华老年医学杂志

Objective:To evaluate the efficacy and safety of fasudil on vasospasm caused by subarachnoid hemorrhage in elderly patients.Methods:A total of 100 elderly patients with subarachnoid hemorrhage admitted to our hospital from January 2015 to May 2018 were enrolled as research objects.They were randomly divided into the Fasudil group(n=50, receiving the Rho kinase inhibitor Fasudil therapy)and the Nimodipine group(n=50, receiving Nimodipine therapy). The cerebral vasospasm and cerebral infarction lesions, the ability of daily life, clinical prognostic score, the incidence of symptomatic cerebral vasospasm and adverse reactions during treatment were evaluated and compared between the two groups.Results:After treatment, the incidences of cerebral vasospasm and cerebral infarction in Fasudil group were 2.04%(1/49)and 6.12%(3/49), respectively, which were lower than those in the Nimodipine group[12.50%(6/48)and 20.83%(10/48), respectively]( χ2=6.134 and 6.794, P=0.047 and 0.033). The scores of daily living ability was better in the Fasudil group than in the Nimodipine group(16.09±1.06 vs.22.91±1.66, t=7.721, P=0.026). The incidence of adverse reactions was lower in the Fasudil group than in the Nimodipine group(4.08% or 2/49 vs.16.7% or 8/48, χ2=6.362, P=0.040). There was no statistically significant difference in the proportion of patients with good prognosis between Fasudil group and Nimodipine group. Conclusions:Rho kinase inhibitor Fasudil can effectively prevent and improve cerebral vasospasm caused by subarachnoid hemorrhage, which is beneficial for improving the clinical prognosis and quality of life of the elderly patients with subarachnoid hemorrhage.

Lysophosphatidic acid receptor 4 regulates osteogenic and adipogenic differentiation of progenitor cells via inactivation of RhoA/ROCK1/ß-catenin signaling.

Recent evidence revealed that lysophosphatidic acid receptor 4 (LPAR4) plays a role in osteogenesis and bone remodeling in mice. However, the molecular mechanism by which LPAR4 controls osteogenic and adipogenic differentiation of mesenchymal progenitor cells remains pending. In the current study, our data showed that Lpar4 was expressed in bone and adipose tissue and the expression increased during osteoblast and adipocyte differentiation. Lpar4 overexpression in stromal ST2 and preosteoblastic MC3T3-E1 cells inhibited osteogenic differentiation. By contrast, Lpar4 overexpression in ST2 and mesenchymal C3H10T1/2 cells enhanced adipogenic differentiation. Conversely, depletion of endogenous Lpar4 in the progenitor cells induced osteogenic differentiation and inhibited adipogenic differentiation. Furthermore, enhanced osteoblast differentiation and alleviated fat accumulation were observed in marrow of mice after in vivo transfection of Lpar4 siRNA. Mechanism investigations revealed that LPAR4 inhibited the activation of ras homolog family member A (RhoA)/Rho-associated kinases 1 (ROCK1) and canonical Wnt signal pathways. ROCK1 was shown to be able to activate Wnt/ß-catenin pathway. We further demonstrated that the overexpression of ROCK1 stimulated osteogenic differentiation and restrained adipogenic differentiation from stromal progenitor cells. Moreover, overexpression of ROCK1 attenuated the inhibition of osteogenic differentiation by LPAR4. The current study has provided evidences demonstrating that RhoA/ROCK1 activates ß-catenin signaling to promote osteogenic differentiation and conversely restrain adipogenic differentiation. The inactivation of RhoA/ROCK1/ß-catenin signaling is involved in LPAR4 regulation of the directional differentiation of marrow stromal progenitor cells.

Protein Kinase N Promotes Stress-Induced Cardiac Dysfunction Through Phosphorylation of Myocardin-Related Transcription Factor A and Disruption of Its Interaction With Actin.

BACKGROUND: Heart failure is a complex syndrome that results from structural or functional impairment of ventricular filling or blood ejection. Protein phosphorylation is a major and essential intracellular mechanism that mediates various cellular processes in cardiomyocytes in response to extracellular and intracellular signals. The RHOA-associated protein kinase (ROCK/Rho-kinase), an effector regulated by the small GTPase RHOA, causes pathological phosphorylation of proteins, resulting in cardiovascular diseases. RHOA also activates protein kinase N (PKN); however, the role of PKN in cardiovascular diseases remains unclear. METHODS: To explore the role of PKNs in heart failure, we generated tamoxifen-inducible, cardiomyocyte-specific PKN1- and PKN2-knockout mice by intercrossing the αMHC-CreERT2 line with Pkn1flox/flox and Pkn2flox/flox mice and applied a mouse model of transverse aortic constriction- and angiotensin II-induced heart failure. To identify a novel substrate of PKNs, we incubated GST-tagged myocardin-related transcription factor A (MRTFA) with recombinant GST-PKN-catalytic domain or GST-ROCK-catalytic domain in the presence of radiolabeled ATP and detected radioactive GST-MRTFA as phosphorylated MRTFA. RESULTS: We demonstrated that RHOA activates 2 members of the PKN family of proteins, PKN1 and PKN2, in cardiomyocytes of mice with cardiac dysfunction. Cardiomyocyte-specific deletion of the genes encoding Pkn1 and Pkn2 (cmc-PKN1/2 DKO) did not affect basal heart function but protected mice from pressure overload- and angiotensin II-induced cardiac dysfunction. Furthermore, we identified MRTFA as a novel substrate of PKN1 and PKN2 and found that MRTFA phosphorylation by PKN was considerably more effective than that by ROCK in vitro. We confirmed that endogenous MRTFA phosphorylation in the heart was induced by pressure overload- and angiotensin II-induced cardiac dysfunction in wild-type mice, whereas cmc-PKN1/2 DKO mice suppressed transverse aortic constriction- and angiotensin II-induced phosphorylation of MRTFA. Although RHOA-mediated actin polymerization accelerated MRTFA-induced gene transcription, PKN1 and PKN2 inhibited the interaction of MRTFA with globular actin by phosphorylating MRTFA, causing increased serum response factor-mediated expression of cardiac hypertrophy- and fibrosis-associated genes. CONCLUSIONS: Our results indicate that PKN1 and PKN2 activation causes cardiac dysfunction and is involved in the transition to heart failure, thus providing unique targets for therapeutic intervention for heart failure.

Metformin Ameliorates Lipotoxic ß-Cell Dysfunction through a Concentration-Dependent Dual Mechanism of Action.

BACKGROUND: Chronic exposure to elevated levels of free fatty acids contributes to pancreatic ß-cell dysfunction. Although it is well known that metformin induces cellular energy depletion and a concomitant activation of AMP-activated protein kinase (AMPK) through inhibition of the respiratory chain, previous studies have shown inconsistent results with regard to the action of metformin on pancreatic ß-cells. We therefore examined the effects of metformin on pancreatic ß-cells under lipotoxic stress. METHODS: NIT-1 cells and mouse islets were exposed to palmitate and treated with 0.05 and 0.5 mM metformin. Cell viability, glucose-stimulated insulin secretion, cellular adenosine triphosphate, reactive oxygen species (ROS) levels and Rho kinase (ROCK) activities were measured. The phosphorylation of AMPK was evaluated by Western blot analysis and mRNA levels of endoplasmic reticulum (ER) stress markers and NADPH oxidase (NOX) were measured by real-time quantitative polymerase chain reaction analysis. RESULTS: We found that metformin has protective effects on palmitate-induced ß-cell dysfunction. Metformin at a concentration of 0.05 mM inhibits NOX and suppresses the palmitate-induced elevation of ER stress markers and ROS levels in a AMPK-independent manner, whereas 0.5 mM metformin inhibits ROCK activity and activates AMPK. CONCLUSION: This study suggests that the action of metformin on ß-cell lipotoxicity was implemented by different molecular pathways depending on its concentration. Metformin at a usual therapeutic dose is supposed to alleviate lipotoxic ß-cell dysfunction through inhibition of oxidative stress and ER stress.

Rho-kinase inhibitor attenuates airway mucus hypersecretion and inflammation partly by downregulation of IL-13 and the JNK1/2-AP1 signaling pathway.

We measured the effect of Rho-kinase on inflammation and mucus hypersecretion in the airways of mouse models of asthma. Additionally, we aimed to determine if these effects were the result of JNK 1/2-AP1 pathway inhibition.We sensitized and challenged female C57BL/6 mice using house dust mites (HDM) followed by treatment with an inhibitor of Rho-kinase. Lung tissue was harvested to evaluate inflammation and mucus secretion in the airways of asthma mice. Cytokine expression in broncho-alveolar lavage fluid (BALF) was established by ELISA and airway responsiveness, and was determined by the invasive lung function test. JNK1/2, p-JNK1/2, AP-1, and p-AP-1 protein expression was determined by Western blot analysis. Asthma model mice that were treated with Rho-kinase inhibitor showed a significantly decrease in inflammation score, inflammatory cells, and airway responsiveness. Additionally, we found that IL-13 expressions in BALF and mucus secretion were decreased in HDM-challenged mice treated with Rho-kinase inhibitor. Furthermore, Rho-kinase inhibitor treatment decreased the expression of JNK1/2 and AP-1 phosphorylation. Our findings indicated that the Rho-kinase inhibitor decreased HDM-induced mucus secretion as well as airway inflammation in asthma mice through regulation of the JNK1/2-AP-1 pathway.

Comparative Study of ROCK1 and ROCK2 in Hippocampal Spine Formation and Synaptic Function.

Rho-associated kinases (ROCKs) are serine-threonine protein kinases that act downstream of small Rho GTPases to regulate the dynamics of the actin cytoskeleton. Two ROCK isoforms (ROCK1 and ROCK2) are expressed in the mammalian central nervous system. Although ROCK activity has been implicated in synapse formation, whether the distinct ROCK isoforms have different roles in synapse formation and function in vivo is not clear. Here, we used a genetic approach to address this long-standing question. Both Rock1+/- and Rock2+/- mice had impaired glutamatergic transmission, reduced spine density, and fewer excitatory synapses in hippocampal CA1 pyramidal neurons. In addition, both Rock1+/- and Rock2+/- mice showed deficits in long-term potentiation at hippocampal CA1 synapses and were impaired in spatial learning and memory based on the water maze and contextual fear conditioning tests. However, the spine morphology of CA1 pyramidal neurons was altered only in Rock2+/- but not Rock1+/- mice. In this study we compared the roles of ROCK1 and ROCK2 in synapse formation and function in vivo for the first time. Our results provide a better understanding of the functions of distinct ROCK isoforms in synapse formation and function.

Metformin Ameliorates Lipotoxic β-Cell Dysfunction through a Concentration-Dependent Dual Mechanism of Action

BACKGROUND: Chronic exposure to elevated levels of free fatty acids contributes to pancreatic β-cell dysfunction. Although it is well known that metformin induces cellular energy depletion and a concomitant activation of AMP-activated protein kinase (AMPK) through inhibition of the respiratory chain, previous studies have shown inconsistent results with regard to the action of metformin on pancreatic β-cells. We therefore examined the effects of metformin on pancreatic β-cells under lipotoxic stress.METHODS: NIT-1 cells and mouse islets were exposed to palmitate and treated with 0.05 and 0.5 mM metformin. Cell viability, glucose-stimulated insulin secretion, cellular adenosine triphosphate, reactive oxygen species (ROS) levels and Rho kinase (ROCK) activities were measured. The phosphorylation of AMPK was evaluated by Western blot analysis and mRNA levels of endoplasmic reticulum (ER) stress markers and NADPH oxidase (NOX) were measured by real-time quantitative polymerase chain reaction analysis.RESULTS: We found that metformin has protective effects on palmitate-induced β-cell dysfunction. Metformin at a concentration of 0.05 mM inhibits NOX and suppresses the palmitate-induced elevation of ER stress markers and ROS levels in a AMPK-independent manner, whereas 0.5 mM metformin inhibits ROCK activity and activates AMPK.CONCLUSION: This study suggests that the action of metformin on β-cell lipotoxicity was implemented by different molecular pathways depending on its concentration. Metformin at a usual therapeutic dose is supposed to alleviate lipotoxic β-cell dysfunction through inhibition of oxidative stress and ER stress.

Effect of lidocaine on Rho/ROCK signaling pathway during endotoxin-induced acute lung injury in rats / 中华麻醉学杂志

Objective To evaluate the effect of lidocaine on Ras homologue (Rho)/Rho kinase (ROCK) signaling pathway during endotoxin-induced lung injury (LI) in rats.Methods Forty SPF male Wistar rats,aged 5-8 weeks,weighing 200-250 g,were divided into 5 groups (n=8 each) using a random number table method:control group (group C),lipopolysaccharide (LPS) group (group LPS)and lidocaine at 3 different doses groups (L1-3 groups).LI was induced by intraperitoneal LPS 5 mg/kg (0.1 ml).The equal volume of normal saline was given intraperitoneally in group C.Lidocaine 2,4 and 8 mg/kg was intraperitoneally injected at 1 h before LPS administration in L1-3 groups,respectively.The animals were sacrificed at 6 h after LPS or normal saline administration.Broncho-alveolar lavage fluid (BALF) was collected for determination of concentrations of interleukin-1 beta (IL-1β),IL-6 and tumor necrosis factor-alpha (TNF-α) (by enzyme-linked immunosorbent assay).The lung tissues were obtained for examination of the pathological changes which were scored and for measurement of the wet/dry weight ratio (W/D ratio) and activities of myeloperoxidase (MPO) and for determination of the expression of Rho,ROCK1,ROCK2,myosin phosphatase target protein 1 (MYPT1),phosphorylated MYPT1 (p-MYPT1)and ZO-1 (by Western blot).The phosphorylation of MYPT1 was calculated.Results Compared with group C,the activity of MPO,lung injury score,W/D ratio and concentrations of IL-1β,IL-6 and TNF-αin BALF were significantly increased,the expression of Rho,ROCK1 and ROCK2 was up-regulated,the phosphorylation of MYPT-1 was increased,and the expression of ZO-1 was down-regulated in the other four groups (P＜0.05).Compared with group LPS,the activity of MPO,lung injury score,W/D ratio and concentrations of IL-1β,IL-6 and TNF-α in BALF were significantly decreased,the expression of Rho,ROCK1 and ROCK2 was down-regulated,the phosphorylation of MYPT-1 was decreased,and the expression of ZO-1 was up-regulated in L1-L3 groups (P＜0.05).Conclusion Lidocaine can inhibit activation of Rho/ROCK signaling pathway during endotoxin-induced LI in rats,and the effect may be related to the antiinflammatory mechanism of lidocaine.

Mechanism underlying hydrogen-induced improvement of intestinal barrier function in septic mice: RhoA-mDia1 signaling pathway / 中华麻醉学杂志

Objective To investigate the relationship between the mechanism underlying hydrogeninduced improvement of intestinal barrier function and RhoA-mDia1 signaling pathway in septic mice.Methods Sixty male C57BL/6 mice,weighing 20-25 g,aged 6-8 weeks,were divided into 3 groups (n =20 each) using a random number table method:control group (group C),sepsis group (group S),and sepsis plus H2 group (group SH).Sepsis was produced by cecal ligation and puncture (CLP) in mice anesthetized with chloral hydrate.Mice in group SH inhaled air containing 2％ hydrogen for 1 h starting from 1 and 6 h after CLP.At 24 h after CLP,blood samples were obtained by cardiac puncture to measure the activity of serum diamine oxidase (DAO) and to count colony-forming unit (CFU) after bacterial culture,the small intestinal samples were obtained for microscopic examination of the ultrastructure of intestinal epithelial cells (with a transmission electron mnicroscope) and for measurement of the expression and distribution of tight junction protein ZO-1 in intestinal epithelial cells (by immunofluorescence) and expression of RhoA,expression and phosphorylation of myosin phosphatase target subunit 1 (MYPT1) and expression of mDia1 (by Western blot).The p-MYPT1/MYPT1 ratio was calculated.Results Compared with group C,the serum DAO activity and whole blood CFU counts were significantly increased,the expression of RhoA was up-regulated,and the expression of ZO-1 was down-regulated in S and SH groups,the p-MYPT1/MYPT1 ratio was significantly increased,and the expression of mDia1 was down-regulated in group S,and the p-MYPT1/MYPT1 ratio was significantly decreased,and the expression of mDia1 was up-regulated in group SH (P＜0.05).Compared with group S,the serum DAO activity,whole blood CFU counts and p-MYPT1/MYPT1 ratio were significantly decreased,the expression of RhoA was down-regulated,the expression of ZO-1 and mDia1 was up-regulated (P＜0.05),and the ultrastructure changes of intestinal tissues were significantly improved in group SH.Conclusion The mechanism by which hydrogen improves intestinal barrier function is related to inhibiting RhoA activity and increasing mDia1 activity in intestinal epithelial cells of septic mice.