Identification of Fasudil as a collaborator to promote the anti-tumor effect of lenvatinib in hepatocellular carcinoma by inhibiting GLI2-mediated hedgehog signaling pathway.

Lenvatinib is a frontline tyrosine kinase inhibitor for patients with advanced hepatocellular carcinoma (HCC). However, just 25% of patients benefit from the treatment, and acquired resistance always develops. To date, there are neither effective medications to combat lenvatinib resistance nor accurate markers that might predict how well a patient would respond to the lenvatinib treatment. Thus, novel strategies to recognize and deal with lenvatinib resistance are desperately needed. In the current study, a robust Lenvatinib Resistance index (LRi) model to predict lenvatinib response status in HCC was first established. Subsequently, five candidate drugs (Mercaptopurine, AACOCF3, NU1025, Fasudil, and Exisulind) that were capable of reversing lenvatinib resistance signature were initially selected by performing the connectivity map (CMap) analysis, and fasudil finally stood out by conducting a series of cellular functional assays in vitro and xenograft mouse model. Transcriptomics revealed that the co-administration of lenvatinib and fasudil overcame lenvatinib resistance by remodeling the hedgehog signaling pathway. Mechanistically, the feedback activation of EGFR by lenvatinib led to the activation of the GLI2-ABCC1 pathway, which supported the HCC cell's survival and proliferation. Notably, co-administration of lenvatinib and fasudil significantly inhibited IHH, the upstream switch of the hedgehog pathway, to counteract GLI2 activation and finally enhance the effectiveness of lenvatinib. These findings elucidated a novel EGFR-mediated mechanism of lenvatinib resistance and provided a practical approach to overcoming drug resistance in HCC through meaningful drug repurposing strategies.

Fasudil alleviates the vascular endothelial dysfunction and several phenotypes of Fabry disease.

Fabry disease (FD), a lysosomal storage disorder, is caused by defective α-galactosidase (GLA) activity, which results in the accumulation of globotriaosylceramide (Gb3) in endothelial cells and leads to life-threatening complications such as left ventricular hypertrophy (LVH), renal failure, and stroke. Enzyme replacement therapy (ERT) results in Gb3 clearance; however, because of a short half-life in the body and the high immunogenicity of FD patients, ERT has a limited therapeutic effect, particularly in patients with late-onset disease or progressive complications. Because vascular endothelial cells (VECs) derived from FD-induced pluripotent stem cells display increased thrombospondin-1 (TSP1) expression and enhanced SMAD2 signaling, we screened for chemical compounds that could downregulate TSP1 and SMAD2 signaling. Fasudil reduced the levels of p-SMAD2 and TSP1 in FD-VECs and increased the expression of angiogenic factors. Furthermore, fasudil downregulated the endothelial-to-mesenchymal transition (EndMT) and mitochondrial function of FD-VECs. Oral administration of fasudil to FD mice alleviated several FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat insensitivity. Our findings demonstrate that fasudil is a novel candidate for FD therapy.

Rho-kinase inhibitor restores glomerular fatty acid metabolism in diabetic kidney disease.

The small GTPase Rho and its effector Rho-kinase (ROCK) are activated in the diabetic kidney, and recent studies decade have demonstrated that ROCK signaling is an integral pathway in the progression of diabetic kidney disease. We previously identified the distinct role of ROCK1, an isoform of ROCK, in fatty acid metabolism in diabetic glomeruli. However, the effect of pharmacological intervention for ROCK1 is not clear. In the present study, we show that the inhibition of ROCK1 by Y-27632 and fasudil restores fatty acid oxidation in the glomeruli. Mechanistically, these compounds optimize fatty acid utilization and redox balance in mesangial cells via AMPK phosphorylation and the subsequent induction of PGC-1α. A further in vivo study showed that the inhibition of ROCK1 suppressed the downregulation of the fatty acid oxidation-related gene expression in glomeruli and mitochondrial fragmentation in the mesangial cells of db/db mice. These observations indicate that ROCK1 could be a promising therapeutic target for diabetic kidney disease through a mechanism that improves glomerular fatty acid metabolism.

Fasudil inhibits the expression of C/EBP homologous protein to protect against liver injury in acetaminophen-overdosed mice.

Acetaminophen (APAP) overdoses can cause severe liver injury. In this study, the protective effect of fasudil against APAP-induced liver injury was investigated. APAP (400 mg/kg) was administered to male C57BL/6J mice to induce liver injury, and fasudil (20 or 40 mg/kg) was injected 30 min before APAP administration. Fasudil markedly suppressed APAP-induced elevation in serum transaminase activity and hepatic necrosis and significantly reduced an increase in nitrotyrosine and DNA fragmentation. However, fasudil did not affect cytochrome P450 2E1 expression, N-acetyl-p-benzoquinone imine production or c-jun N-terminal kinase activation. In contrast, fasudil significantly inhibited an APAP-induced increase in expression of the transcription factor C/EBP homologous protein (CHOP) in the liver, accompanied by transcriptional suppression of ER stress-related molecules such as Ero1α, Atf4 and Grp78. These findings indicate that suppression of CHOP expression by fasudil exhibits a remarkable protective effect against APAP liver injury by regulating ER stress. We suggest that fasudil is a promising therapeutic candidate for treating APAP-induced liver injury.

RhoA/ROCK Signaling Is Involved in Pathological Retinal Neovascularization.

OBJECTIVE: The aim of the study was to evaluate the effect of the RhoA/ROCK inhibitor Fasudil on retinal neovascularization (NV) in vivo and angiogenesis in vitro. METHODS: C57BL/6 was used to establish an OIR model. First, RhoA/ROCK expression was first examined and compared between OIR and healthy controls. Then, we evaluated the effect of Fasudil on pathological retinal NV. Whole-mount retinal staining was performed. The percentage of NV area, the number of neovascular tufts (NVT), and branch points (BP) were quantified. Finally, human umbilical vein endothelial cells (HUVECs) were used to investigate the effect of Fasudil on angiogenesis. RESULTS: Real-time PCR and Western blotting showed that ROCK expression in retinal tissue was statistically upregulated in OIR. Furthermore, we found that Fasudil attenuated the percentage of NV area, the number of NVT, and BP significantly. In addition, Fasudil could suppress the proliferation and migration of HUVECs induced by VEGF. CONCLUSIONS: RhoA/ROCK might be involved in the pathogenesis of OIR. And its inhibitor Fasudil could suppress retinal NV in vivo and angiogenesis in vitro. Fasudil may be a potential treatment strategy for retinal vascular diseases.

Inhibition of Rho-kinase ameliorates decreased spine density in the medial prefrontal cortex and methamphetamine-induced cognitive dysfunction in mice carrying schizophrenia-associated mutations of the Arhgap10 gene.

Copy-number variations in the ARHGAP10 gene encoding Rho GTPase-activating protein 10 are associated with schizophrenia. Model mice (Arhgap10 S490P/NHEJ mice) that carry "double-hit" mutations in the Arhgap10 gene mimic the schizophrenia in a Japanese patient, exhibiting altered spine density, methamphetamine-induced cognitive dysfunction, and activation of RhoA/Rho-kinase signaling. However, it remains unclear whether the activation of RhoA/Rho-kinase signaling due to schizophrenia-associated Arhgap10 mutations causes the phenotypes of these model mice. Here, we investigated the effects of fasudil, a brain permeable Rho-kinase inhibitor, on altered spine density in the medial prefrontal cortex (mPFC) and on methamphetamine-induced cognitive impairment in a touchscreen­based visual discrimination task in Arhgap10 S490P/NHEJ mice. Fasudil (20 mg/kg, intraperitoneal) suppressed the increased phosphorylation of myosin phosphatase-targeting subunit 1, a substrate of Rho-kinase, in the striatum and mPFC of Arhgap10 S490P/NHEJ mice. In addition, daily oral administration of fasudil (20 mg/kg/day) for 7 days ameliorated the reduced spine density of layer 2/3 pyramidal neurons in the mPFC. Moreover, fasudil (3-20 mg/kg, intraperitoneal) rescued the methamphetamine (0.3 mg/kg)-induced cognitive impairment of visual discrimination in Arhgap10 S490P/NHEJ mice. Our results suggest that Rho-kinase plays significant roles in the neuropathological changes in spine morphology and in the vulnerability of cognition to methamphetamine in mice with schizophrenia-associated Arhgap10 mutations.

Small molecule protein kinase inhibitors approved by regulatory agencies outside of the United States.

Owing to genetic alterations and overexpression, the dysregulation of protein kinases plays a significant role in the pathogenesis of many autoimmune and neoplastic disorders and protein kinase antagonists have become an important drug target. Although the efficacy of imatinib in the treatment of chronic myelogenous leukemia in the United States in 2001 was the main driver of protein kinase inhibitor drug discovery, this was preceded by the approval of fasudil (a ROCK antagonist) in Japan in 1995 for the treatment of cerebral vasospasm. There are 21 small molecule protein kinase inhibitors that are approved in China, Japan, Europe, and South Korea that are not approved in the United Sates and 75 FDA-approved inhibitors in the United States. Of the 21 agents, eleven target receptor protein-tyrosine kinases, eight inhibit nonreceptor protein-tyrosine kinases, and two block protein-serine/threonine kinases. All 21 drugs are orally bioavailable or topically effective. Of the non-FDA approved drugs, sixteen are prescribed for the treatment of neoplastic diseases, three are directed toward inflammatory disorders, one is used for glaucoma, and fasudil is used in the management of vasospasm. The leading targets of kinase inhibitors approved by both international regulatory agencies and by the FDA are members of the EGFR family, the VEGFR family, and the JAK family. One-third of the 21 internationally approved drugs are not compliant with Lipinski's rule of five for orally bioavailable drugs. The rule of five relies on four parameters including molecular weight, number of hydrogen bond donors and acceptors, and the Log of the partition coefficient.

Real-world data of clazosentan in combination therapy for aneurysmal subarachnoid hemorrhage: a multicenter retrospective cohort study.

Aneurysmal subarachnoid hemorrhage (aSAH) may lead to cerebral vasospasm, significantly associated with morbidity and mortality. In double-blind, placebo-controlled phase 3 studies, clazosentan reduces cerebral vasospasm-related morbidity and all-cause mortality in patients with aSAH. There are no reports about the clinical efficacy of clazosentan combination therapy with some other drugs. Initially, we explored the efficacy of clazosentan combination therapy with cilostazol, statin, and antiepileptic drugs. Subsequently, we assessed the add-on effect of fasudil to clazosentan combination therapy for aSAH patients. This multicenter, retrospective, observational cohort study included Japanese patients with aSAH between June 2022 and March 2023. The primary outcome was the ordinal score on the modified Rankin Scale (mRS; range, 0-6, with elevated scores indicating greater disability) at discharge. Among the 47 cases (women 74.5%; age 64.4 ± 15.0 years) undergoing clazosentan combination therapy, 29 (61.7%) resulted in favorable outcomes. Overall, vasospasm occurred in 16 cases (34.0%), with four cases (8.5%) developing vasospasm-related delayed cerebral ischemia (DCI). Both hypotension and vasospasm-related DCI were related to unfavorable outcome at discharge. Fasudil were added in 18 (38.3%) cases. Despite adding fasudil to clazosentan combination therapy, the incidence of aSAH-related vasospasm did not decrease. Added-on fasudil to combination therapy related to pulmonary edema, vasospasm, and vasospasm-related DCI, and unfavorable outcomes. Clazosentan combination therapy could potentially result in favorable outcomes for aSAH patients to prevent post-aSAH vasospasm-related DCI. The add-on effect of fasudil to combination therapy did not demonstrate a significant impact in reducing aSAH-related vasospasm or improving outcomes at discharge.

Inhibition of RhoA/ROCK signalling pathway activity improves neural damage and cognitive deficits in the fluorosis model.

Excessive fluoride intake poses health risks to humans and animals. Many studies have indicated that fluoride exposure can damage the cytoskeleton and synapses, which has negative effects on the intellectual development of humans and animals. Our previous study suggested that the RhoA/ROCK signalling pathway is activated by NaF exposure in HT-22 cells and plays a vital role in cytoskeletal assembly and synaptogenesis. However, the mechanism underlying RhoA/ROCK-mediated cytoskeletal injury induced by fluoride remains unclear. In this study, Neuro-2A cells and ICR mice were used to investigate the effects of RhoA/ROCK activation inhibition on NaF-induced synaptic dysfunction and cognitive impairment. We detected the expression of GAP, RhoA, ROCK1/2, and (p)-MLC in vivo and in vitro model. The results showed that NaF exposure activated the RhoA/ROCK/MLC signalling pathway. We measured the effects of RhoA/ROCK inhibition on synaptic injury and intellectual impairment induced by NaF exposure. In vitro, Y-27632 suppressed activated RhoA/ROCK, attenuated morphological and ultrastructural damage, and decreased the survival rate and synapse-functional protein expression caused by NaF. In vivo, the results showed that the RhoA/ROCK/MLC pathway was inhibited by fasudil and improved pathological damage in the hippocampus, cognitive impairment, and decreased expression of neurofunctional proteins induced by NaF. Overall, these results suggest that fasudil and Y-27632 can reverse neurotoxicity caused by fluoride exposure. Furthermore, inhibition of RhoA/ROCK may be a future treatment for CNS injury, and more detailed studies on other neurodegenerative disease models are required to confirm its effectiveness.

Neurodegenerative Disease Associated Pathways in the Brains of Triple Transgenic Alzheimer's Model Mice Are Reversed Following Two Weeks of Peripheral Administration of Fasudil.

The pan Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor fasudil acts as a vasodilator and has been used as a medication for post-cerebral stroke for the past 29 years in Japan and China. More recently, based on the involvement of ROCK inhibition in synaptic function, neuronal survival, and processes associated with neuroinflammation, it has been suggested that the drug may be repurposed for neurodegenerative diseases. Indeed, fasudil has demonstrated preclinical efficacy in many neurodegenerative disease models. To facilitate an understanding of the wider biological processes at play due to ROCK inhibition in the context of neurodegeneration, we performed a global gene expression analysis on the brains of Alzheimer's disease model mice treated with fasudil via peripheral IP injection. We then performed a comparative analysis of the fasudil-driven transcriptional profile with profiles generated from a meta-analysis of multiple neurodegenerative diseases. Our results show that fasudil tends to drive gene expression in a reverse sense to that seen in brains with post-mortem neurodegenerative disease. The results are most striking in terms of pathway enrichment analysis, where pathways perturbed in Alzheimer's and Parkinson's diseases are overwhelmingly driven in the opposite direction by fasudil treatment. Thus, our results bolster the repurposing potential of fasudil by demonstrating an anti-neurodegenerative phenotype in a disease context and highlight the potential of in vivo transcriptional profiling of drug activity.

Genomic and Reverse Translational Analysis Discloses a Role for Small GTPase RhoA Signaling in the Pathogenesis of Schizophrenia: Rho-Kinase as a Novel Drug Target.

Schizophrenia is one of the most serious psychiatric disorders and is characterized by reductions in both brain volume and spine density in the frontal cortex. RhoA belongs to the RAS homolog (Rho) family and plays critical roles in neuronal development and structural plasticity via Rho-kinase. RhoA activity is regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Several variants in GAPs and GEFs associated with RhoA have been reported to be significantly associated with schizophrenia. Moreover, several mouse models carrying schizophrenia-associated gene variants involved in RhoA/Rho-kinase signaling have been developed. In this review, we summarize clinical evidence showing that variants in genes regulating RhoA activity are associated with schizophrenia. In the last half of the review, we discuss preclinical evidence indicating that RhoA/Rho-kinase is a potential therapeutic target of schizophrenia. In particular, Rho-kinase inhibitors exhibit anti-psychotic-like effects not only in Arhgap10 S490P/NHEJ mice, but also in pharmacologic models of schizophrenia (methamphetamine- and MK-801-treated mice). Accordingly, we propose that Rho-kinase inhibitors may have antipsychotic effects and reduce cognitive deficits in schizophrenia despite the presence or absence of genetic variants in small GTPase signaling pathways.

Effects of Butylphthalide Combined with Fasudil on Inflammatory Factors, Cognitive Function and Vascular Endothelial Function in Patients with Subarachnoid Hemorrhage Complicated with Cerebral Vasospasm.

Objective: To investigate the effect of butylphthalide combined with fasudil in the treatment of subarachnoid hemorrhage (SAH) with cerebral vasospasm (CVS) on inflammatory factors, cognitive function and vascular endothelial function. Methods: It is a retrospective study in which a total of 104 patients with SAH with CVS admitted to Baoding First Central Hospital from July 2020 to February 2022 were selected and randomly divided into two groups by drawing lots. Patients in the control group were treated with basic symptomatic treatment, while those in the observation group were treated with butylphthalide soft capsule combined with fasudil hydrochloride injection on the basis of the control group. Before and after treatment, serum neuron specific enolase (NSE), tumor necrosis factor-α(TNF-α), interleukin-8(IL-8), hypersensitive C-reactive protein (CRP), Birmingham Cognitive Screen test (BCoS) score, serum soluble intercellular adhesion molecule-1(ICAM-1), serum endothelin-1(ET-1), vascular endothelial growth factor (VEGF) levels, and endothelium-dependent vasodilation function (FMD) in the two groups were compared. Results: After treatment, the expression levels of NSE, TNF-α, IL-8 and CRP in the two groups were significantly decreased, and the expression levels of all indicators in the observation group were lower than that in the control group (p<0.05). After treatment, the scores of orientations, attention, memory, language, practice and action in the two groups were significantly increased, and the scores of all dimensions in the observation group were higher than those in the control group (p<0.05). After treatment, S-ICAM-1, ET-1, VEGF, FMD decreased in both groups, and all indicators of the observation group were lower than those of the control group, with statistically significant differences (p<0.05). Conclusion: Butylphthalide combined with fasudil therapy was found as effective in reducing inflammatory factors, ameliorating cognitive function and vascular endothelial function in patients with subarachnoid hemorrhage complicated with cerebral vasospasm.

Involvement of α1B-adrenoceptors and Rho kinase in contractions of rat aorta and mouse spleen.

α1-adrenoceptors link via the G-protein Gq/G11 to both Ca2+ entry and release from stores, but may also activate Rho kinase, which causes calcium sensitization. This study aimed to identify the subtype(s) of α1-adrenoceptor involved in Rho kinase-mediated responses in both rat aorta and mouse spleen, tissues in which contractions involve multiple subtypes of α1-adrenoceptor. Tissues were contracted with cumulative concentrations of noradrenaline (NA) in 0.5 log unit increments, before and in the presence of an antagonist or vehicle. Contractions produced by NA in rat aorta are entirely α1-adrenoceptor mediated as they are competitively blocked by prazosin. The α1A-adrenoceptor antagonist RS100329 had low potency in rat aorta. The α1D-adrenoceptor antagonist BMY7378 antagonized contractions in rat aorta in a biphasic manner: low concentrations blocking α1D-adrenoceptors and high concentrations blocking α1B-adrenoceptors. The Rho kinase inhibitor fasudil (10 µM) significantly reduced aortic contractions in terms of maximum response, suggesting inhibition of α1B-adrenoceptor mediated responses. In the mouse spleen, a tissue in which all 3 subtypes of α1-adrenoceptor are involved in contractions to NA, fasudil (3 µM) significantly reduced both early and late components to the NA contraction, the early component involving α1B- and α1D-adrenoceptors, and the late component involving α1B- and α1A-adrenoceptors. This suggests that fasudil inhibits α1B-adrenoceptor mediated responses. It is concluded that α1D- and α1B-adrenoceptors interact in rat aorta and α1D-, α1A- and α1B-adrenoceptors interact in the mouse spleen to produce contractions and these interactions suggest that one of the receptors preferentially activates Rho kinase, most likely the α1B-adrenoceptor.

Rho-kinase inhibition improves haemodynamic responses and circulating ATP during hypoxia and moderate intensity handgrip exercise in healthy older adults.

Skeletal muscle haemodynamics and circulating adenosine triphosphate (ATP) responses during hypoxia and exercise are blunted in older (OA) vs. young (YA) adults, which may be associated with impaired red blood cell (RBC) ATP release. Rho-kinase inhibition improves deoxygenation-induced ATP release from OA isolated RBCs. We tested the hypothesis that Rho-kinase inhibition (via fasudil) in vivo would improve local haemodynamic and ATP responses during hypoxia and exercise in OA. Healthy YA (25 ± 3 years; n = 12) and OA (65 ± 5 years; n = 13) participated in a randomized, double-blind, placebo-controlled, crossover study on two days (≥5 days between visits). A forearm deep venous catheter was used to administer saline/fasudil and sample venous plasma ATP ([ATP]V ). Forearm vascular conductance (FVC) and [ATP]V were measured at rest, during isocapnic hypoxia (80% SpO2${S_{{\rm{p}}{{\rm{O}}_{\rm{2}}}}}$ ), and during graded rhythmic handgrip exercise that was similar between groups (5, 15 and 25% maximum voluntary contraction (MVC)). Isolated RBC ATP release was measured during normoxia/hypoxia. With saline, ΔFVC was lower (P < 0.05) in OA vs. YA during hypoxia (∼60%) and during 15 and 25% MVC (∼25-30%), and these impairments were abolished with fasudil. Similarly, [ATP]V and ATP effluent responses from normoxia to hypoxia and rest to 25% MVC were lower in OA vs. YA and improved with fasudil (P < 0.05). Isolated RBC ATP release during hypoxia was impaired in OA vs. YA (∼75%; P < 0.05), which tended to improve with fasudil in OA (P = 0.082). These data suggest Rho-kinase inhibition improves haemodynamic responses to hypoxia and moderate intensity exercise in OA, which may be due in part to improved circulating ATP. KEY POINTS: Skeletal muscle blood flow responses to hypoxia and exercise are impaired with age. Blunted increases in circulating ATP, a vasodilator, in older adults may contribute to age-related impairments in haemodynamics. Red blood cells (RBCs) are a primary source of circulating ATP, and treating isolated RBCs with a Rho-kinase inhibitor improves age-related impairments in deoxygenation-induced RBC ATP release. In this study, treating healthy older adults systemically with the Rho-kinase inhibitor fasudil improved blood flow and circulating ATP responses during hypoxia and moderate intensity handgrip exercise compared to young adults, and also tended to improve isolated RBC ATP release. Improved blood flow regulation with fasudil was also associated with increased skeletal muscle oxygen delivery during hypoxia and exercise in older adults. This is the first study to demonstrate that Rho-kinase inhibition can significantly improve age-related impairments in haemodynamic and circulating ATP responses to physiological stimuli, which may have therapeutic implications.

Rho-Kinase Inhibition Improves the Outcome of Focal Subcortical White Matter Lesions.

BACKGROUND: Subcortical white matter lesions are exceedingly common in cerebral small vessel disease and lead to significant cumulative disability without an available treatment. Here, we tested a rho-kinase inhibitor on functional recovery after focal white matter injury. METHODS: A focal corpus callosum lesion was induced by stereotactic injection of N5-(1-iminoethyl)-L-ornithine in mice. Fasudil (10 mg/kg) or vehicle was administered daily for 2 weeks, starting one day after lesion induction. Resting-state functional connectivity and grid walk performance were studied longitudinally, and lesion volumes were determined at one month. RESULTS: Resting-state interhemispheric functional connectivity significantly recovered between days 1 and 14 in the fasudil group (P<0.001), despite worse initial connectivity loss than vehicle before treatment onset. Grid walk test revealed an increased number of foot faults in the vehicle group compared with baseline, which persisted for at least 4 weeks. In contrast, the fasudil arm did not show an increase in foot faults and had smaller lesions at 4 weeks. Immunohistochemical examination of reactive astrocytosis, synaptic density, and mature oligodendrocytes did not reveal a significant difference between treatment arms. CONCLUSIONS: These data show that delayed fasudil posttreatment improves functional outcomes after a focal subcortical white matter lesion in mice. Future work will aim to elucidate the mechanisms.

Combination of constraint-induced movement therapy with fasudil amplifies neurogenesis after focal cerebral ischemia/reperfusion in rats.

PURPOSE: Spontaneous axonal plasticity and functional restoration after stroke may be limited by Nogo-A, a myelin-associated inhibitor, via activation of the Rho/Rho-associated protein kinase (ROCK) pathway. Constraint-induced movement therapy (CIMT) is a rehabilitation technique based on neuroplasticity and neural recombination. We recently reported that CIMT promoted neurogenesis after cerebral ischemia/reperfusion in part by inhibiting the Nogo-A-RhoA-ROCK pathway. Here, we examine the hypothesis that CIMT combined with the ROCK inhibitor fasudil further amplifies neurogenesis during stroke recovery. METHODS: Four groups of rats were randomized as follows: Cerebral ischemia-reperfusion (IR), Fasudil, CIMT and CIMT + Fasudil. Seven days after stroke, CIMT and/or intraperitoneal infusion of fasudil were initiated and continued for 3 weeks. The behavioral outcomes and immunohistochemical markers of neurogenesis were quantified. RESULTS: Compared with other groups, the combination of CIMT with fasudil after IR significantly improved motor and memory function recovery. In addition, BrdU, BrdU/doublecortin and BrdU/GFAP all increased significantly in the brain tissue of the combined treatment group compared to the CIMT or Fasudil group. CONCLUSION: These results suggest that the effects of CIMT on neurogenesis are amplified by fasudil during the recovery phase after stroke.

TNF-α Plus IL-1ß Induces Opposite Regulation of Cx43 Hemichannels and Gap Junctions in Mesangial Cells through a RhoA/ROCK-Dependent Pathway.

Connexin 43 (Cx43) is expressed in kidney tissue where it forms hemichannels and gap junction channels. However, the possible functional relationship between these membrane channels and their role in damaged renal cells remains unknown. Here, analysis of ethidium uptake and thiobarbituric acid reactive species revealed that treatment with TNF-α plus IL-1ß increases Cx43 hemichannel activity and oxidative stress in MES-13 cells (a cell line derived from mesangial cells), and in primary mesangial cells. The latter was also accompanied by a reduction in gap junctional communication, whereas Western blotting assays showed a progressive increase in phosphorylated MYPT (a target of RhoA/ROCK) and Cx43 upon TNF-α/IL-1ß treatment. Additionally, inhibition of RhoA/ROCK strongly antagonized the TNF-α/IL-1ß-induced activation of Cx43 hemichannels and reduction in gap junctional coupling. We propose that activation of Cx43 hemichannels and inhibition of cell-cell coupling during pro-inflammatory conditions could contribute to oxidative stress and damage of mesangial cells via the RhoA/ROCK pathway.

Dual efficacy of Fasudil at improvement of survival and reinnervation of flap through RhoA/ROCK/PI3K/Akt pathway.

Fasudil is reported to be effective at protecting against ischaemic diseases, and at augmenting axon growth. In this study, we aim to evaluate its efficacy in promoting flap survival and reinnervation. Ninety-two Institute of Cancer Research (ICR) mice were used and divided into the control, Fasudil, LY294002, Fasudil+LY294002 groups, receiving a daily intraperitoneal injection of normal saline, Fasudil (10 mg/kg), LY294002 (5 mg/kg), and Fasudil (10 mg/kg) + LY294002 (5 mg/kg), respectively. On days 0 and 5, the blood perfusion and diameter of the iliolumbar artery in the pedicle of the flaps in the four groups were evaluated using laser speckling contrast imaging (LSCI). On day 5, the flaps were photographed and the necrosis rate of the flaps was calculated using Photoshop CS6. In addition, tissues were harvested from the flaps and divided into two parts. One part underwent routine cryosection and immunofluorescent staining using the antibody against CD31 for evaluation of the microvascular density in the four groups. In the other part, the expression of RhoA, ROCK1+2, p-CPI-17, p-MYPT, p-PTEN, p-PI3K, p-Akt, and vascular endothelial growth factor (VEGF) within the flaps were determined using western blotting. Moreover, at days 0, 7, 15, and 30 after flap surgery, the axons within the flaps were evaluated using immunofluorescent staining with the antibody against Neurofilament-200. It turned out that the necrosis rate was (24.4 ± 7.7)%, (5.2 ± 1.6)%, (29.8 ± 4.2)%, and (30.9 ± 7.1)%, respectively, in the control, Fasudil, LY294002, LY294002+Fasudil groups. There was a significant reduction in the necrosis rate of the flaps in the Fasudil group (P < .001). The LSCI and immunofluorescent staining demonstrated that Fasudil could significantly expand the diameter of the iliolumbar artery in the pedicle, boost the overall blood perfusion, and increase the microvascular density of the flaps in the Fasudil group (P < .05), which could all be abolished by PI3K inhibitor LY294002. On day 5, the expression of p-CPI-17, p-MYPT, and p-PTEN were downregulated, whereas pPI3K, p-Akt, and VEGF were upregulated in the Fasudil group (P < .001). As for reinnervation, Neurofilament-200 fluorescent staining revealed that at days 15 and 30 after flap harvest, only in the Fasudil group could new axons be observed. It can be concluded that Fasudil could simultaneously improve the survival and axon growth after flap harvest, a dual efficacy achieved by inhibition of the RhoA/ROCK pathway, which in turn activates /PI3K/AKT pathway.

ROCK/PKA Inhibition Rescues Hippocampal Hyperexcitability and GABAergic Neuron Alterations in a Oligophrenin-1 Knock-Out Mouse Model of X-Linked Intellectual Disability.

Oligophrenin-1 (Ophn1) encodes a Rho GTPase activating protein whose mutations cause X-linked intellectual disability (XLID) in humans. Loss of function of Ophn1 leads to impairments in the maturation and function of excitatory and inhibitory synapses, causing deficits in synaptic structure, function and plasticity. Epilepsy is a frequent comorbidity in patients with Ophn1-dependent XLID, but the cellular bases of hyperexcitability are poorly understood. Here we report that male mice knock-out (KO) for Ophn1 display hippocampal epileptiform alterations, which are associated with changes in parvalbumin-, somatostatin- and neuropeptide Y-positive interneurons. Because loss of function of Ophn1 is related to enhanced activity of Rho-associated protein kinase (ROCK) and protein kinase A (PKA), we attempted to rescue Ophn1-dependent pathological phenotypes by treatment with the ROCK/PKA inhibitor fasudil. While acute administration of fasudil had no impact on seizure activity, seven weeks of treatment in adulthood were able to correct electrographic, neuroanatomical and synaptic alterations of Ophn1 deficient mice. These data demonstrate that hyperexcitability and the associated changes in GABAergic markers can be rescued at the adult stage in Ophn1-dependent XLID through ROCK/PKA inhibition.SIGNIFICANCE STATEMENT In this study we demonstrate enhanced seizure propensity and impairments in hippocampal GABAergic circuitry in Ophn1 mouse model of X-linked intellectual disability (XLID). Importantly, the enhanced susceptibility to seizures, accompanied by an alteration of GABAergic markers were rescued by Rho-associated protein kinase (ROCK)/protein kinase A (PKA) inhibitor fasudil, a drug already tested on humans. Because seizures can significantly impact the quality of life of XLID patients, the present data suggest a potential therapeutic pathway to correct alterations in GABAergic networks and dampen pathological hyperexcitability in adults with XLID.

Rho-kinase inhibition by fasudil modulates pre-synaptic vesicle dynamics.

The Rho kinase (ROCK) signaling pathway is an attractive therapeutic target in neurodegeneration since it has been linked to the prevention of neuronal death and neurite regeneration. The isoquinoline derivative fasudil is a potent ROCK inhibitor, which is already approved for chronic clinical treatment in humans. However, the effects of chronic fasudil treatments on neuronal function are still unknown. We analyzed here chronic fasudil treatment in primary rat hippocampal cultures. Neurons were stimulated with 20 Hz field stimulation and we investigated pre-synaptic mechanisms and parameters regulating synaptic transmission after fasudil treatment by super resolution stimulated emission depletion (STED) microscopy, live-cell fluorescence imaging, and western blotting. Fasudil did not affect basic synaptic function or the amount of several synaptic proteins, but it altered the chronic dynamics of the synaptic vesicles. Fasudil reduced the proportion of the actively recycling vesicles, and shortened the vesicle lifetime, resulting overall in a reduction of the synaptic response upon stimulation. We conclude that fasudil does not alter synaptic structure, accelerates vesicle turnover, and decreases the number of released vesicles. This broadens the known spectrum of effects of this drug, and suggests new potential clinical uses.