Beta-Secretase 1 Recruits Amyloid-Beta Precursor Protein to ROCK2 Kinase, Resulting in Erroneous Phosphorylation and Beta-Amyloid Plaque Formation.

The amyloidogenic processing of APP depends on two events: its phosphorylation by ROCK2 (at Thr654) and the phosphorylation of the APP-cleaving enzyme BACE1 (at Ser498). However, the mechanisms and structural details of APP-ROCK2 and BACE1-ROCK2 binding are unknown. Using direct physical methods in combination with an in silico approach, we found that BACE1 binds into the substrate-binding groove of ROCK2 with a low affinity (Kd = 18 µM), while no binding of APP to ROCK2 alone could be detected. On the other hand, a strong association (Kd = 3.5 nM) of APP to the weak ROCK2-BACE1 complex was observed, although no stable ternary complex was detected, i.e., BACE1 was displaced by APP. We constructed a sequential functional model: (1) BACE1 weakly binds to ROCK2 and induces an allosteric conformational change in ROCK2; (2) APP strongly binds to the ROCK2-BACE1 complex, and BACE1 is released; and (3) ROCK2 phosphorylates APP at Thr654 (leading to a longer stay in the early endosome during APP processing). Direct fluorescence titration experiments showed that the APP646-664 or APP665-695 fragments did not bind separately to the ROCK2-BACE1 complex. Based on these observations, we conclude that two binding sites are involved in the ROCK2-APP interaction: (1) the substrate-binding groove, where the APP646-664 sequence containing Thr654 sits and (2) the allosteric binding site, where the APP665-695 sequence binds. These results open the way to attack the allosteric site to prevent APP phosphorylation at Thr654 by ROCK2 without inhibiting the activity of ROCK2 towards its other substrates.

Nephroprotective effects of honokiol in a high-fat diet-streptozotocin rat model of diabetic nephropathy.

AIMS: Diabetic nephropathy (DN) is the foremost basis of end-stage kidney failure implicating endoplasmic reticulum (ER) stress and dysregulation of Rho kinase/Rock pathway. Magnolia plants are used in traditional medicine systems in Southeast Asia owing to bioactive phytoconstituents. Earlier, honokiol (Hon) exhibited therapeutic potential in experimental models of metabolic, renal, and brain disorders. In the present study, we evaluated potential of Hon against DN and possible molecular mechanisms. MAIN METHODS: In the existing experiments, high-fat diet (HFD) (17 weeks) and streptozotocin (STZ) (40 mg/kg once) induced DN rats were orally treated with Hon (25, 50, 100 mg/kg) or metformin (150 mg/kg) for 8 weeks. KEY FINDINGS: Hon attenuated albuminuria, blood biomarkers (e.g., urea nitrogen, glucose, C-reactive protein, and creatinine) and ameliorated lipid profile, electrolytes levels (Na+/K+), and creatinine clearance against DN. Hon significantly decreased renal oxidative stress and inflammatory biomarkers against DN. Histomorphometry and microscopic analysis revealed nephroprotective effects of Hon marked by a decrease in leukocyte infiltration, renal tissue damage, and urine sediments. RT-qPCR showed that Hon treatment attenuated mRNA expression of transforming growth factor-ß1 (TGF-ß1), endothelin-1 (ET-1), ER stress markers (GRP78, CHOP, ATF4, and TRB3), and Rock 1/2 in DN rats. Data from ELISA supported a decrease in levels of TGF-ß1, ET-1, ER stress markers, and Rock1/2 by Hon. SIGNIFICANCE: Hon attenuated hyperglycemia, redox imbalance, and inflammation and improved renal functions in rats. Hon alleviates DN pathogenesis possibly by attenuating ER stress and Rock pathway.

A systematic review on the effects of ROCK inhibitors on proliferation and/or differentiation in human somatic stem cells: A hypothesis that ROCK inhibitors support corneal endothelial healing via acting on the limbal stem cell niche.

Rho kinase inhibitors (ROCKi) have attracted growing multidisciplinary interest, particularly in Ophthalmology where the question as to how they promote corneal endothelial healing remains unresolved. Concurrently, stem cell biology has rapidly progressed in unravelling drivers of stem cell (SC) proliferation and differentiation, where mechanical niche factors and the actin cytoskeleton are increasingly recognized as key players. There is mounting evidence from the study of the peripheral corneal endothelium that supports the likelihood of an internal limbal stem cell niche. The possibility that ROCKi stimulate the endothelial SC niche has not been addressed. Furthermore, there is currently a paucity of data that directly evaluates whether ROCKi promotes corneal endothelial healing by acting on this limbal SC niche located near the transition zone. Therefore, we performed a systematic review examining the effects ROCKi on the proliferation and differentiation of human somatic SC, to provide insight into its effects on various human SC populations. An appraisal of electronic searches of four databases identified 1 in vivo and 58 in vitro studies (36 evaluated proliferation while 53 examined differentiation). Types of SC studied included mesenchymal (n = 32), epithelial (n = 11), epidermal (n = 8), hematopoietic and other (n = 8). The ROCK 1/2 selective inhibitor Y-27632 was used in almost all studies (n = 58), while several studies evaluated ≥2 ROCKi (n = 4) including fasudil, H-1152, and KD025. ROCKi significantly influenced human somatic SC proliferation in 81% of studies (29/36) and SC differentiation in 94% of studies (50/53). The present systemic review highlights that ROCKi are influential in regulating human SC proliferation and differentiation, and provides evidence to support the hypothesis that ROCKi promotes corneal endothelial division and maintenance via acting on the inner limbal SC niche.

Paeoniflorin alleviates liver injury in hypercholesterolemic rats through the ROCK/AMPK pathway.

Paeoniflorin (PF) is the main active component in Paeonia lactiflora Pall, and it has multiple effects. However, the precise mechanism of PF in hypercholesterolemia is unclear. In this study, rats were either fed a high-cholesterol diet (HCD) for 4 weeks to establish the hypercholesterolemic model or administered normal saline or PF (20 mg/kg/day). PF significantly reduced liver weight and the liver index. PF reduced hepatic lipid deposition and inflammation, improved serum lipid metabolism, and significantly inhibited serum and hepatic oxidative stress and the inflammatory response. PF treatment caused a marked decrease in the phosphorylated myosin phosphatase target subunit (p-MYPT)-1, nuclear sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase (FAS) levels, and an increase in the low-density lipoprotein receptor (LDLR) and phosphorylated-AMP-activated protein kinase (p-AMPK). Thus, PF could alleviate liver injury in hypercholesterolemic rats, and the specific mechanism may be related to the antioxidant, anti-inflammatory properties, and ROCK/AMPK/SREBP-1c signaling pathway.

Rho-Kinase as a Target for Cancer Therapy and Its Immunotherapeutic Potential.

Cancer immunotherapy is fast rising as a prominent new pillar of cancer treatment, harnessing the immune system to fight against numerous types of cancer. Rho-kinase (ROCK) pathway is involved in diverse cellular activities, and is therefore the target of interest in various diseases at the cellular level including cancer. Indeed, ROCK is well-known for its involvement in the tumor cell and tumor microenvironment, especially in its ability to enhance tumor cell progression, migration, metastasis, and extracellular matrix remodeling. Importantly, ROCK is also considered to be a novel and effective modulator of immune cells, although further studies are needed. In this review article, we describe the various activities of ROCK and its potential to be utilized in cancer treatment, particularly in cancer immunotherapy, by shining a light on its activities in the immune system.

Renal ROCK Activation and Its Pharmacological Inhibition in Patients With Diabetes.

Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine/threonine kinase with essential roles in cytoskeletal functions. Substantial evidence implicates ROCK as a critical regulator in the inception and progression of diabetic nephropathy through a mechanism involving mesangial fibrosis, podocyte apoptosis, and endothelial inflammation. Despite these experimental observations, human data is lacking. Here we show that the phosphorylated form of myosin phosphatase targeting subunit 1 (MYPT1), a ROCK substrate, was increased in both the glomerular and tubulointerstitial areas in patients with histologically confirmed diabetic nephropathy. We also conducted a retrospective pilot analysis of data from patients with diabetes to assess the renoprotective effects of fasudil, an ATP-competitive ROCK inhibitor licensed in Japan for the prevention of vasospasm following subarachnoid hemorrhage. Fifteen subjects (male, n = 8; female, n = 7; age 65.7 ± 14.7 years; body height, 161.1 ± 12.6 cm; body weight, 57.6 ± 13.7 kg; body mass index, 22.4 ± 3.7 kg/m2) were enrolled to evaluate blood pressure and the renal outcome after fasudil treatment. Of note, proteinuria was significantly reduced at the end of the fasudil treatment without affecting the blood pressure or estimated glomerular filtration rate. Taken together, these findings suggest that the administration of fasudil could be associated with a better renal outcome by inhibiting the ROCK activity in patients with diabetes.

Exercise Counterbalances Rho/ROCK2 Signaling Impairment in the Skeletal Muscle and Ameliorates Insulin Sensitivity in Obese Mice.

Physical exercise is considered a fundamental strategy in improving insulin sensitivity and glucose uptake in skeletal muscle. However, the molecular mechanisms underlying this regulation, primarily on skeletal muscle glucose uptake, are not fully understood. Recent evidence has shown that Rho-kinase (ROCK) isoforms play a pivotal role in regulating skeletal muscle glucose uptake and systemic glucose homeostasis. The current study evaluated the effect of physical exercise on ROCK2 signaling in skeletal muscle of insulin-resistant obese animals. Physiological (ITT) and molecular analysis (immunoblotting, and RT-qPCR) were performed. The contents of RhoA and ROCK2 protein were decreased in skeletal muscle of obese mice compared to control mice but were restored to normal levels in response to physical exercise. The exercised animals also showed higher phosphorylation of insulin receptor substrate 1 (IRS1 Serine 632/635) and protein kinase B (Akt) in the skeletal muscle. However, phosphatase and tensin homolog (PTEN) and protein-tyrosine phosphatase-1B (PTP-1B), both inhibitory regulators for insulin action, were increased in obesity but decreased after exercise. The impact of ROCK2 action on muscle insulin signaling is further underscored by the fact that impaired IRS1 and Akt phosphorylation caused by palmitate in C2C12 myotubes was entirely restored by ROCK2 overexpression. These results suggest that the exercise-induced upregulation of RhoA-ROCK2 signaling in skeletal muscle is associated with increased systemic insulin sensitivity in obese mice and further implicate that muscle ROCK2 could be a potential target for treating obesity-linked metabolic disorders.

Safety and efficacy of topically administered netarsudil-latanoprost fixed dose combination (FDC; Rocklatan™) in normal and glaucomatous dogs with ADAMTS10-open-angle glaucoma (ADAMTS10-OAG).

OBJECTIVE: The aim of the study was to evaluate safety and efficacy of topically administered 0.02% netarsudil-0.005% latanoprost fixed-dose combination (FDC) (Rocklatan™; Aerie Pharmaceutical) in normal and glaucomatous dogs with ADAMTS10-open-angle glaucoma (ADAMTS10-OAG). ANIMALS STUDIED: Five normal and five glaucomatous beagle dogs with ADAMTS10-OAG were the study animals. PROCEDURES: In each dog, left (OS) or right eye (OD) was randomly selected for netarsudil-latanoprost FDC treatment. Contralateral eyes served as latanoprost-treated controls. The study was divided into four consecutive study periods: following a 4-day baseline period, two sequential 8-day study periods followed with once daily (q24h) and twice daily (q12h) treatments and ending with a washout period. Efficacy was measured by diurnal intraocular pressure (IOP) and pupil diameter. Safety was assessed by routine ophthalmic examination, gonioscopy, and pachymetry. Differences in least square means of quantitative outcome measures were compared between FDC and latanoprost treatments by using the linear Gaussian model. RESULTS: Baseline IOPs were 13.6 ± 0.7 mmHg (mean ± SEM) in normal and 28.3 ± 1.4 mmHg in OAG-affected dogs. There was a significant decrease in mean diurnal IOP following FDC administration in both normal (q24h: -2.1 mmHg; q12h: -4.1 mmHg) and glaucomatous dogs (q24h: -14.2 mmHg; q12h: -17.7 mmHg; p < .0001). There was no significant difference in the treatment effect when comparing FDC to latanoprost. Both FDC and latanoprost administration resulted in similarly significant pupil constriction (p < .0001). The FDC administration was well-tolerated but resulted in conjunctival hyperemia. CONCLUSIONS: Once or twice daily administration of netarsudil-latanoprost FDC (Rocklatan™) and latanoprost was equally effective in lowering IOP in normal and OAG-affected dogs. There was no netarsudil-related added treatment effect.

ROCK inhibitors 4: Structure-activity relationship studies of 7-azaindole-based rho kinase (ROCK) inhibitors.

Rho kinase (ROCK) inhibitors are of therapeutic value for the treatment of disorders such as hypertension and glaucoma, and potentially of wider use against diseases such as cancer and multiple sclerosis. We previously reported a series of potent and selective ROCK inhibitors based on a substituted 7-azaindole scaffold. Here we extend the SAR exploration of the 7-azaindole series to identify leads for further evaluation. New compounds such as 16, 17, 19, 21 and 22 showed excellent ROCK potency and protein kinase A (PKA) selectivity, combined with microsome and hepatocyte stability.

The Vasopressin Receptor 2 Mutant R137L Linked to the Nephrogenic Syndrome of Inappropriate Antidiuresis (NSIAD) Signals through an Alternative Pathway that Increases AQP2 Membrane Targeting Independently of S256 Phosphorylation.

NSIAD is a rare X-linked condition, caused by activating mutations in the AVPR2 gene coding for the vasopressin V2 receptor (V2R) associated with hyponatremia, despite undetectable plasma vasopressin levels. We have recently provided in vitro evidence that, compared to V2R-wt, expression of activating V2R mutations R137L, R137C and F229V cause a constitutive redistribution of the AQP2 water channel to the plasma membrane, higher basal water permeability and significantly higher basal levels of p256-AQP2 in the F229V mutant but not in R137L or R137C. In this study, V2R mutations were expressed in collecting duct principal cells and the associated signalling was dissected. V2R-R137L and R137C mutants had significantly higher basal pT269-AQP2 levels -independently of S256 and PKA-which were reduced to control by treatment with Rho kinase (ROCK) inhibitor. Interestingly, ROCK activity was found significantly higher in V2R-R137L along with activation of the Gα12/13-Rho-ROCK pathway. Of note, inhibition of ROCK reduced the basal elevated osmotic water permeability to control. To conclude, our data demonstrate for the first time that the gain-of-function mutation of the V2R, R137L causing NSIAD, signals through an alternative PKA-independent pathway that increases AQP2 membrane targeting through ROCK-induced phosphorylation at S/T269 independently of S256 of AQP2.

Physical exercise increases ROCK activity in the skeletal muscle of middle-aged rats.

The physical exercise is a potential strategy to control age-related metabolic disorders, such as insulin resistance, impaired glucose homeostasis, and type 2 diabetes. Rho-kinase (ROCK) increases skeletal muscle glucose uptake through Insulin Receptor Substrate 1 (IRS1) phosphorylation. Here, we investigated the role of physical exercise in ROCK pathway in the skeletal muscle of Fischer middle-aged rats. Firstly, we observed the ROCK distribution in different skeletal muscle fiber types. ROCK signaling pathway (ROCK1 and ROCK2) and activity (pMYPT1) were higher in the soleus, which was associated with increased insulin signaling pathway (pIR, pIRS1, pPDK, pGSK3ß). Middle-aged rats submitted to physical exercise, showed the upregulation of ROCK2 content and normalized RhoA (ROCK activator enzyme) levels in soleus muscle compared with middle-aged sedentary rats. These molecular changes in middle-aged exercised rats were accompanied by higher insulin signaling (pIRS1, pGSK3ß, pAS160, GLUT4) in the soleus muscle. Reinforcing these findings, when pharmacological inhibition of ROCK activity was performed (using Y-27632), the insulin signaling pathway and glucose metabolism-related genes (Tpi, Pgk1, Pgam2, Eno3) were decreased in the soleus muscle of exercised rats. In summary, ROCK signaling seems to contribute with whole-body glucose homeostasis (∼50 %) through its higher upregulation in the soleus muscle in middle-aged exercised rats.

ZIP kinase phosphorylated and activated by Rho kinase/ROCK contributes to cytokinesis in mammalian cultured cells.

Cytokinesis of animal cells requires contraction of a contractile ring, composed of actin filaments and myosin II filaments. Phosphorylation of myosin II regulatory light chain (MRLC) promotes contraction of the actomyosin ring by activating myosin II motor activity. Both Rho-associated coiled-coil kinase (Rho kinase/ROCK) and Zipper-interacting protein kinase (ZIP kinase/ZIPK) have been reported to phosphorylate MRLC at the contractile ring. However, it remains unclear whether these kinases function independently of each other. Here, we clarified that ROCK colocalizes and forms a complex with ZIPK at telophase. As ROCK is reported to phosphorylate and activate ZIPK in vitro, we hypothesized that ZIPK phosphorylated by ROCK contributes to control cytokinesis. To address this, we expressed EGFP-ZIPK wild type (WT), a non-phosphorylatable mutant (T265A) or a phosphorylation-mimicking mutant (T265D) in HeLa cells and treated these cells with a ROCK inhibitor. Decrease in phosphorylated MRLC and a delay of furrow ingression by the ROCK inhibitor were rescued by the expression of EGFP-ZIPK-T265D, but not EGFP-ZIPK-WT or -T265A. This suggests that ROCK regulates MRLC phosphorylation followed by furrow ingression, through ZIPK phosphorylation.

Tsantan Sumtang Restored Right Ventricular Function in Chronic Hypoxia-Induced Pulmonary Hypertension Rats.

Background: Tsantan Sumtang originated from Four Tantras, which consisted of Choerospondias axillaris (Roxb.) B. L. Burtt and A. W. Hill, Santalum album L., and Myristica fragrans Houtt. The three herbs are in ratio 1:1:1. This medication is widely used for cardiovascular diseases. Aims: The purpose of this study was to explore the effect of Tsantan Sumtang on right ventricular (RV) function in hypoxia-induced pulmonary hypertension (HPH) rats and investigate the underlying mechanism. Methods: Sixty male Sprague-Dawley (SD) rats were divided into control, hypoxia, and hypoxia + Tsantan Sumtang (1.0, 1.25, and 1.5 g•kg-1•d-1) groups. Chronic hypoxia was induced by putting the rats inside a hypobaric chamber for four weeks and adjusting the inner pressure and oxygen content to match an altitude of 4500 m. Echocardiography was used to assess RV function and right ventricular-pulmonary arterial (RV-PA) coupling. The physiological parameters of the animals were also evaluated. Morphological characteristics of RV were assessed by hematoxylin and eosin (H&E) staining and TEM. Masson's trichrome staining, immunohistochemical staining, western blotting, and TUNEL assay were used to assess fibrosis and apoptosis levels. The antioxidant and anti-apoptosis properties of Tsantan Sumtang were also evaluated. The effect of Tsantan Sumtang on ROCK signaling pathway was evaluated using real-time quantitative PCR and western blotting. Results: We established an HPH rat model as indicated by the significant increases in the physiological parameters of the rats. Tsantan Sumtang showed a significant cardiac-protective function and an improved effect on RV-PA coupling. Moreover, Tsantan Sumtang treatment inhibited fibrosis and alleviated apoptosis and oxidative stress in RV. In terms of mechanism, Tsantan Sumtang reduced the expression of ROCK (ROCK1, ROCK2) in RV, inhibited cardiac remodeling-related transcription factors (NFATc3, P-STAT3), and regulated apoptosis-related proteins. Conclusion: Tsantan Sumtang was able to restore RV function, improve RV-PA coupling, recover hemodynamic and hematological indexes, and protect RV against structural maladaptive remodeling in the HPH rats. These findings demonstrated that Tsantan Sumtang protects the function of RV in HPH rats. The antioxidant and anti-apoptosis properties of Tsantan Sumtang may be responsible for inhibiting the ROCK signaling pathway.

p90 ribosomal S6 kinase (RSK) phosphorylates myosin phosphatase and thereby controls edge dynamics during cell migration.

Cell migration is essential to embryonic development, wound healing, and cancer cell dissemination. Cells move via leading-edge protrusion, substrate adhesion, and retraction of the cell's rear. The molecular mechanisms by which extracellular cues signal to the actomyosin cytoskeleton to control these motility mechanics are poorly understood. The growth factor-responsive and oncogenically activated protein extracellular signal-regulated kinase (ERK) promotes motility by signaling in actin polymerization-mediated edge protrusion. Using a combination of immunoblotting, co-immunoprecipitation, and myosin-binding experiments and cell migration assays, we show here that ERK also signals to the contractile machinery through its substrate, p90 ribosomal S6 kinase (RSK). We probed the signaling and migration dynamics of multiple mammalian cell lines and found that RSK phosphorylates myosin phosphatase-targeting subunit 1 (MYPT1) at Ser-507, which promotes an interaction of Rho kinase (ROCK) with MYPT1 and inhibits myosin targeting. We find that by inhibiting the myosin phosphatase, ERK and RSK promote myosin II-mediated tension for lamella expansion and optimal edge dynamics for cell migration. These findings suggest that ERK activity can coordinately amplify both protrusive and contractile forces for optimal cell motility.

In-Silico QSAR Modelling of Predicted Rho Kinase Inhibitors Against Cardio Vascular Diseases.

BACKGROUND: Rho-kinase is an essential downstream target of GTP-binding protein RhoA, and plays a crucial role in the calcium-sensitization pathway. Rho-kinase pathway is critically involved in phosphorylation state of myosin light chain, leading to increased contraction of smooth muscles. Inhibition of this pathway has turned out to be a promising target for several indications such as cardiovascular diseases, glaucoma and inflammatory diseases. METHODS: The present work focuses on a division-based 2D quantitative structure-activity relationship (QSAR) analysis along with a docking study to predict structural features that may be essential for the enhancement of selectivity and potency of the target compounds. Furthermore, a set of indoles and azaindoles were also projected based on the regression equation as novel developments. Molecular docking was applied for exploring the binding sites of the newly predicted set of compounds with the receptor. RESULTS: Results of the docked conformations suggested that introduction of non-bulky and substituted groups in the hinge region of ROCK-II ATP binding pocket would improve the activity by decreasing the bulkiness or length of the compounds. CONCLUSION: ADME studies were performed to ascertain the novelty and drug-like properties of the designed molecules, respectively.

Intravitreal injection of a Rho-kinase inhibitor (fasudil) combined with bevacizumab versus bevacizumab monotherapy for diabetic macular oedema: a pilot randomised clinical trial.

Click here to listen to the Podcast BACKGROUND/AIMS: To compare the efficacy of combined intravitreal injection of bevacizumab and a Rho-kinase inhibitor, fasudil (intravitreal bevacizumab (IVB)/intravitreal fasudil (IVF)), with IVB alone for centre-involving diabetic macular oedema (DME). METHODS: In this prospective randomised clinical trial, 44 eyes with centre-involving DME were randomised into two groups. The combined group received three consecutive injections of IVB (1.25 mg) and IVF (50 µM/L) monthly, while the monotherapy group received only one IVB (1.25 mg) injection per month for 3 months. Changes in best-corrected visual acuity (BCVA) and central macular thickness (CMT) were compared between the two groups at months 3 and 6. The primary outcome measure was the mean change in BCVA at month 6. RESULTS: Mean BCVA was significantly improved in both groups at month 3 (P<0.001), but it persisted up to month 6 only in the IVB/IVF group. Improvement of BCVA was greater in the IVB/IVF group at both time points (P=0.008, P<0.001). In the IVB/IVF and IVB groups, 54.5% versus 10% of the eyes gained≥15 ETDRS letters at month 6 (P=0.026). Between months 3 and 6, mean BCVA significantly decreased by 5±7 ETDRS letters in the IVB group (P=0.002), while no significant deterioration was observed in the IVB/IVF group. Corresponding with the BCVA changes, CMT was significantly reduced in both groups at month 3 (p=0.006, p<0.001) but this reduction sustained only in the IVB/IVF group up to month 6 (p<0.001). CONCLUSION: Adjunctive intravitreal injection of a Rho-kinase inhibitor may enhance and prolong the therapeutic effects of anti-vascular endothelial growth factor drugs for centre- involving DME.

Druggable targets in the Rho pathway and their promise for therapeutic control of blood pressure.

The prevalence of high blood pressure (also known as hypertension) has steadily increased over the last few decades. Known as a silent killer, hypertension increases the risk for cardiovascular disease and can lead to stroke, heart attack, kidney failure and associated sequela. While numerous hypertensive therapies are currently available, it is estimated that only half of medicated patients exhibit blood pressure control. This signifies the need for a better understanding of the underlying cause of disease and for more effective therapies. While blood pressure homeostasis is very complex and involves the integrated control of multiple body systems, smooth muscle contractility and arterial resistance are important contributors. Strong evidence from pre-clinical animal models and genome-wide association studies indicate that smooth muscle contraction and BP homeostasis are governed by the small GTPase RhoA and its downstream target, Rho kinase. In this review, we summarize the signaling pathways and regulators that impart tight spatial-temporal control of RhoA activity in smooth muscle cells and discuss current therapeutic strategies to target these RhoA pathway components. We also discuss known allelic variations in the RhoA pathway and consider how these polymorphisms may affect genetic risk for hypertension and its clinical manifestations.

Netarsudil ophthalmic solution 0.02% for the treatment of patients with open-angle glaucoma or ocular hypertension.

Once-daily (p.m.) netarsudil ophthalmic solution 0.02% (Rhopressa) is approved in the United States for lowering elevated intraocular pressure (IOP) in patients with open-angle glaucoma or ocular hypertension. Netarsudil, a Rho kinase (ROCK) inhibitor that lowers IOP primarily by increasing trabecular outflow, produces statistically and clinically significant reductions in mean IOP from baseline, with comparable effects on nocturnal and diurnal IOP. In three phase III trials of patients with elevated IOP, the ocular hypotensive efficacy of once-daily netarsudil 0.02% met the criteria for noninferiority to twice-daily timolol 0.5% at all time points over 3 months in patients with baseline IOP less than 25 mmHg. The most frequent adverse event (AE) was generally mild conjunctival hyperemia, the severity of which did not increase with continued dosing. Netarsudil was associated with minimal treatment-related serious or systemic AEs, likely due to the lack of systemic exposure. This report summarizes the available preclinical and clinical data on netarsudil.

Fasudil ameliorates the ischemia/reperfusion oxidative injury in rat hearts through suppression of myosin regulatory light chain/NADPH oxidase 2 pathway.

Fasudil is a potent Rho-kinase (ROCK) inhibitor and can relax smooth muscle or cardiac muscle contraction through decreasing the phosphorylation level of myosin regulatory light chain (p-MLC20 or p-MLC2v), while p-MLC2v can function as a transcription factor to promote the NADPH oxidase 2 (NOX2) expression in rat hearts subjected to ischemia/reperfusion (I/R). This study aims to explore whether fasudil can protect the rat hearts against I/R oxidative injury through suppressing NOX2 expression via reduction of p-MLC2v level. The SD rat hearts were subjected to 1h-ischemia plus 3h-reperfusion, which showed myocardial injuries (myocardial fiber loss and disarray, increase of creatine kinase release and myocardial infarction/apoptosis), increase in ROCK activity and nuclear p-MLC2v level concomitant with up-regulation of NOX2 and H2O2 production; these phenomena were attenuated by fasudil in a dose-dependent manner. Next, we verified the cardioprotective effect of fasudil and the underlying mechanisms in hypoxia-reoxygenation (H/R) -treated H9c2 cells. Consistent with the results in vivo, the H/R-treated H9c2 cells showed cellular injury (increase in apoptotic ratio), elevation in ROCK activity and nuclear p-MLC2v level, accompanied by up-regulation of NOX2 and H2O2 production; these effects were blocked in the presence of fasudil in a dose-dependent way. Based on these observations, we conclude that beneficial effect of fasudil against myocardial I/R or H/R oxidative injury is related to the suppression of NOX2 expression through decrease of the p-MLC2v level. Our findings also highlight that intervention of MLC2v phosphorylation by drugs may provide a novel strategy to protect heart from I/R oxidative injury.