Rho-associated Protein Kinase Inhibitor Y-27632 Research Articles

Peripheral Rho-associated protein kinase activation mediates acupuncture analgesia

BackgroundAcupuncture has proven effective for various types of pain and peripheral molecular signals around acupuncture-treated areas have been suggested to contribute to the analgesic effects of acupuncture; however, the underlying mechanism from these peripheral molecular signals to central ones remains unclear. We investigated whether peripheral Rho-associated protein kinase (ROCK) activation induced by acupuncture treatment mediates acupuncture analgesia. We also investigated the relationship between ROCK activation and extracellular signal-regulated kinase (ERK), which has previously been proven to mediate acupuncture analgesia and other related molecular changes during acupuncture. MethodsAcupuncture was treated at the bilateral GB34 acupoints of C57BL/6 mice, after which changes in ROCK activation and the location of its expression in the skin were analyzed. To verify the role of ROCK in acupuncture analgesia, we administrated ROCK inhibitor Y-27632 (0.3 μg/ul) into the skin before acupuncture treatment with formalin and complete Freund adjuvant (CFA) induced pain models, then the nociceptive responses were analyzed. ResultsAcupuncture treatment produced ROCK2 activation in the skin after 30 and 60 minutes, and the histological analyses revealed that ROCK2 was activated in the fibroblast of the dermis. The acupuncture-induced ROCK2 expression was significantly attenuated by the ERK inhibitor, whereas phospho-ERK expression was not inhibited by ROCK inhibitor. In both the formalin- and CFA-induced mouse pain models, acupuncture analgesia was blocked by ROCK inhibitor administration. ConclusionAcupuncture treatment-induced ROCK2 expression is a downstream effector of phospho-ERK in the skin and plays a crucial role in acupuncture analgesia.

Efficient establishment of an optimized culture condition for cashmere goat primary hair follicle stem cells.

Hair follicle stem cells (HFSCs) are an important basis for hair follicle morphogenesis and hair cycle growth. This cell type also represents an excellent model for studying the gene function and molecular regulation of the hair growth cycle, including proliferation, differentiation, and apoptosis. Basically, the functional investigation of hair growth-regulating genes demands a sufficient amount of HFSCs. However, efficient propagation of HFSCs in goats is a challenging process under the current culture conditions. Here, we investigated the effect of four components, including the Rho-associated protein kinase (ROCK) inhibitor Y-27632, leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and vitamin C, on cell growth and pluripotency in the basal culture medium (DMEM/F12 supplemented with 2% fetal bovine serum). We found that adding Y-27632, LIF, and bFGF independently increased the proliferation and pluripotency of goat HFSCs (gHFSCs), with Y-27632 having the most significant effect (P < 0.001). Fluorescence-activated cell sorting of the cell cycle revealed that Y-27632 promoted gHFSC proliferation by inducing the cell cycle from S to G2/M phase (P < 0.05). We further demonstrated that gHFSCs displayed superior proliferative capacity, clone-forming ability, and differentiation potential in the combined presence of Y-27632 (10 μM) and bFGF (10 ng/mL). We termed this novel culture condition as gHFEM, which stands for goat Hair Follicle Enhanced Medium. Taken together, these results indicate that gHFEM is an optimal condition for in vitro culture of gHFSCs, which will subsequently facilitate the study of HF growth and biology.

Multi-targeted anti-inflammatory drugs for the treatment of neurological disorders.

Multi-targeted anti-inflammatory drugs for the treatment of neurological disorders.

Transforming growth factor beta (TGFβ) pathway is essential for hypoblast and epiblast development in ovine post-hatching embryos

The developmental failures occurring between blastocyst hatching and implantation in farm ungulates are a major cause of pregnancy losses. At the expanded blastocyst stage, three cell lineages emerge in the embryo: trophoblast, hypoblast and epiblast, the latter being the most vulnerable during post-hatching development. Transforming growth factor beta (TGFβ) signaling pathway is involved in hypoblast and epiblast development; however, previous in vitro functional studies are limited to the expanded blastocyst stage. In this study, we have analyzed the effect of TGFβ inhibition with 10, 20 or 40 μM SB431542 during ovine post-hatching developmental period using a recently developed culture system able to recapitulate major developmental landmarks. We have found a negative effect of TGFβ inhibition on hypoblast and epiblast development that could be partially reverted by Rho-associated protein kinase (ROCK) inhibitor Y-27632. Our findings provide new insights into the molecular networks regulating embryo development beyond the expanded blastocyst and could help to elucidate the causes of early pregnancy losses in farm ungulates.

Rho-Associated Protein Kinase Inhibitor and Hypoxia Synergistically Enhance the Self-Renewal, Survival Rate, and Proliferation of Human Stem Cells.

IntroductionHigh-efficacy single-cell cloning of human-induced pluripotent cells (IPSCs) remains a major challenge. The development of a culture method that supports single-cell passaging while maintaining reproducibility, homogeneity, scalability, and cell expansion to clinically relevant numbers is necessary for clinical application.MethodsTo address this issue, we combined the use of the rho-associated protein kinase (ROCK) inhibitor Y-27632 and hypoxic conditions in culture to produce a novel, efficient single-cell culture method for human IPSCs and embryonic stem cells.ResultsThrough immunocytochemistry, alkaline phosphatase assays, and flow cytometry, we demonstrated that our method enabled high single-cell proliferation while maintaining self-renewal and pluripotency abilities.DiscussionWe showed the beneficial effect of the interaction between hypoxia and ROCK inhibition in regulating cell proliferation, pluripotency, and single-cell survival of pluripotent cells.

ProBDNF and Brain-Derived Neurotrophic Factor Prodomain Differently Modulate Acetylcholine Release in Regenerating and Mature Mouse Motor Synapses.

The effects of brain-derived neurotrophic factor (BDNF) processing by-products (proBDNF and BDNF prodomain) on the activity of mouse neuromuscular junctions (NMJs) were studied in synapses formed during the reinnervation of extensor digitorum longus muscle (m. EDL) and mature synapses of the diaphragm. The parameters of spontaneous miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were analyzed in presence of each of the BDNF maturation products (both – 1 nM). In newly formed NMJs, proBDNF caused an increase in the resting membrane potential of muscle fibers and a decrease in the frequency of MEPPs, which was prevented by tertiapin-Q, a G-protein-coupled inwardly rectifying potassium channels (GIRK) blocker but not by p75 receptor signaling inhibitor TAT-Pep5. proBDNF had no effect on the parameters of EPPs. BDNF prodomain in newly formed synapses had effects different from those of proBDNF: it increased the amplitude of MEPPs, which was prevented by vesamicol, an inhibitor of vesicular acetylcholine (ACh) transporter; and reduced the quantal content of EPPs. In mature NMJs, proBDNF did not influence MEPPs parameters, but BDNF prodomain suppressed both spontaneous and evoked ACh release: decreased the frequency and amplitude of MEPPs, and the amplitude and quantal content of EPPs. This effect of the BDNF prodomain was prevented by blocking GIRK channels, by TAT-Pep5 or by Rho-associated protein kinase (ROCK) inhibitor Y-27632. At the same time, the BDNF prodomain did not show any inhibitory effects in diaphragm motor synapses of pannexin 1 knockout mice, which have impaired purinergic regulation of neuromuscular transmission. The data obtained suggest that there is a previously unknown mechanism for the acute suppression of spontaneous and evoked ACh release in mature motor synapses, which involves the activation of p75 receptors, ROCK and GIRK channels by BDNF prodomain and requires interaction with metabotropic purinoreceptors. In general, our results show that both the precursor of BDNF and the product of its maturation have predominantly inhibitory effects on spontaneous and evoked ACh release in newly formed or functionally mature neuromuscular junctions, which are mainly opposite to the effects of BDNF. The inhibitory influences of both proteins related to brain neurotrophin are mediated via GIRK channels of mouse NMJs.

Combined Inhibition of Fyn and c-Src Protects Hippocampal Neurons and Improves Spatial Memory via ROCK after Traumatic Brain Injury.

Our previous studies demonstrated that traumatic brain injury (TBI) and ventricular administration of thrombin caused hippocampal neuron loss and cognitive dysfunction via activation of Src family kinases (SFKs). Based on SFK localization in brain, we hypothesized SFK subtypes Fyn and c-Src, as well as SFK downstream molecule Rho-associated protein kinase (ROCK), contribute to cell death and cognitive dysfunction after TBI. We administered nanoparticle wrapped small interfering RNA (siRNA)-Fyn and siRNA-c-Src, or ROCK inhibitor Y-27632 to adult rats subjected to moderate lateral fluid percussion (LFP)-induced TBI. Spatial memory function was assessed from 12 to 16 days, and NeuN stained hippocampal neurons were assessed 16 days after TBI. The combination of siRNA-Fyn and siRNA-c-Src, but neither alone, prevented hippocampal neuron loss and spatial memory deficits after TBI. The ROCK inhibitor Y-27632 also prevented hippocampal neuronal loss and spatial memory deficits after TBI. The data suggest that the combined actions of three kinases (Fyn, c-Src, ROCK) mediate hippocampal neuronal cell death and spatial memory deficits produced by LFP-TBI, and that inhibiting this pathway prevents the TBI-induced cell death and memory deficits.

Deleted in liver cancer 1 suppresses the growth of prostate cancer cells through inhibiting Rho-associated protein kinase pathway

ObjectiveDeleted in liver cancer 1 (DLC1) is a GTPase-activating protein that is reported as a suppressor in certain human cancers. However, the detailed biological function of DLC1 is still unclear in human prostate cancer (PCa). In the present study, we aimed to explore the function of DLC1 in PCa cells. MethodsSilencing and overexpression of DLC1 were induced in an androgen-sensitive PCa cell line (LNCaP) using RNA interference and lentiviral vector transduction. The Cell Counting Kit-8 assay was performed to determine cell proliferation. The cell cycle was examined by performing a propidium iodide staining assay. ResultsOur results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of LNCaP cells. Moreover, DLC1 expression was negatively correlated with Rho-associated protein kinase (ROCK) expression in LNCaP cells. Importantly, this study showed that the ROCK inhibitor Y27632 restored the function of DLC1 in LNCaP cells and reduced the tumorigenicity of LNCaP cells in vivo. ConclusionOur results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of PCa cells and negatively correlated with ROCK expression in PCa cells and tissue.

The ROCK inhibitor Y-27632 ameliorates blood-spinal cord barrier disruption by reducing tight junction protein degradation via the MYPT1-MLC2 pathway after spinal cord injury in rats

The blood-spinal cord barrier (BSCB) is a physiological barrier between the blood and spinal cord parenchyma. This study aims to determine whether Y-27632, a Rho-associated protein kinase (ROCK) inhibitor, can protect the BSCB using in vivo models. The Evans blue fluorescence assay was used to detect leakage of the BSCB. Western blotting was used to define alterations in ROCK-related and tight junction (TJ) protein expression. Immunofluorescence triple-staining was used to evaluate histologic alterations in TJs. Locomotor function was evaluated using the open-field test, the Basso-Beattie-Bresnahan score, and footprint analysis.Two peaks of BSCB leakage after spinal cord injury (SCI) occurred at 24 h and 5 days. The ROCK inhibitor reduced the BSCB leakage at the second peak after SCI. Moreover, the ROCK inhibitor ameliorated the integrity of the BSCB and improved motor function recovery after SCI by regulating the phosphorylation of myosin phosphatase subunit-1 (MYPT1) and cofilin. ROCK inhibitors might protect the BSCB, which provides a new strategy for transitioning SCI treatment from the bench to bedside.

RAS induced senescence of skin keratinocytes is mediated through Rho-associated protein kinase (ROCK).

Cellular senescence is a well-documented response to oncogene activation in many tissues. Multiple pathways are invoked to achieve senescence indicating its importance to counteract the transforming activities of oncogenic stimulation. We now report that the Rho-associated protein kinase (ROCK) signaling pathway is a critical regulator of oncogene-induced senescence in skin carcinogenesis. Transformation of mouse keratinocytes with oncogenic RAS upregulates ROCK activity and initiates a senescence response characterized by cell enlargement, growth inhibition, upregulation of senescence associated β-galactosidase (SAβgal) expression, and release of multiple pro-inflammatory factors comprising the senescence-associated secretory phenotype (SASP). The addition of the ROCK inhibitor Y-27632 and others prevents these senescence responses and maintains proliferating confluent RAS transformed keratinocyte cultures indefinitely. Mechanistically, oncogenic RAS transformation is associated with upregulation of cell cycle inhibitors p15Ink4b , p16Ink4a , and p19Arf and downregulation of p-AKT, all of which are reversed by Y-27632. RNA-seq analysis of Y-27632 treated RAS-transformed keratinocytes indicated that the inhibitor reduced growth-inhibitory gene expression profiles and maintained expression of proliferative pathways. Y-27632 also reduced the expression of NF-κB effector genes and the expression of IκBζ downstream mediators. The senescence inhibition from Y-27632 was reversible, and upon its removal, senescence reoccurred in vitro with rapid upregulation of cell cycle inhibitors, SASP expression, and cell detachment. Y-27632 treated cultured RAS-keratinocytes formed tumors in the absence of the inhibitor when placed in skin orthografts suggesting that factors in the tumor microenvironment can overcome the drive to senescence imparted by overactive ROCK activity.

Establishment of an Efficient Primary Culture System for Human Hair Follicle Stem Cells Using the Rho-Associated Protein Kinase Inhibitor Y-27632.

BackgroundHair follicle tissue engineering is a promising strategy for treating hair loss. Human hair follicle stem cells (hHFSCs), which play a key role in the hair cycle, have potential applications in regenerative medicine. However, previous studies did not achieve efficient hHFSC expansion in vitro using feeder cells. Therefore, there is a need to develop an efficient primary culture system for the expansion and maintenance of hHFSCs.MethodsThe hHFSCs were obtained by two-step proteolytic digestion combined with microscopy. The cell culture dishes were coated with human fibronectin and inoculated with hHFSCs. The hHFSCs were harvested using a differential enrichment procedure. The effect of Rho-associated protein kinase (ROCK) inhibitor Y-27632, supplemented in keratinocyte serum-free medium (K-SFM), on adhesion, proliferation, and stemness of hHFSCs and the underlying molecular mechanisms were evaluated.ResultsThe hHFSCs cultured in K-SFM, supplemented with Y-27632, exhibited enhanced adhesion and proliferation. Additionally, Y-27632 treatment maintained the stemness of hHFSCs and promoted the ability of hHFSCs to regenerate hair follicles in vivo. However, Y-27632-induced proliferation and stemness in hHFSCs were conditional and reversible. Furthermore, Y-27632 maintained propagation and stemness of hHFSCs through the ERK/MAPK pathway.ConclusionAn efficient short-term culture system for primary hHFSCs was successfully established using human fibronectin and the ROCK inhibitor Y-27632, which promoted the proliferation, maintained the stemness of hHFSCs and promoted the ability to regenerate hair follicles in vivo. The xenofree culturing method used in this study provided a large number of high-quality seed cells, which have applications in hair follicle tissue engineering and stem cell therapy.

Bartonella type IV secretion effector BepC induces stress fiber formation through activation of GEF-H1

Bartonella T4SS effector BepC was reported to mediate internalization of big Bartonella aggregates into host cells by modulating F-actin polymerization. After that, BepC was indicated to induce host cell fragmentation, an interesting cell phenotype that is characterized by failure of rear-end retraction during cell migration, and subsequent dragging and fragmentation of cells. Here, we found that expression of BepC resulted in significant stress fiber formation and contractile cell morphology, which depended on combination of the N-terminus FIC (filamentation induced by c-AMP) domain and C-terminus BID (Bartonella intracellular delivery) domain of BepC. The FIC domain played a key role in BepC-induced stress fiber formation and cell fragmentation because deletion of FIC signature motif or mutation of two conserved amino acid residues abolished BepC-induced cell fragmentation. Immunoprecipitation confirmed the interaction of BepC with GEF-H1 (a microtubule-associated RhoA guanosine exchange factor), and siRNA-mediated depletion of GEF-H1 prevented BepC-induced stress fiber formation. Interaction with BepC caused the dissociation of GEF-H1 from microtubules and activation of RhoA to induce formation of stress fibers. The ROCK (Rho-associated protein kinase) inhibitor Y27632 completely blocked BepC effects on stress fiber formation and cell contractility. Moreover, stress fiber formation by BepC increased the stability of focal adhesions, which consequently impeded rear-edge detachment. Overall, our study revealed that BepC-induced stress fiber formation was achieved through the GEF-H1/RhoA/ROCK pathway.

Activation of yes-associated protein mediates sphingosine-1-phosphate-induced proliferation and migration of pulmonary artery smooth muscle cells and its potential mechanisms.

The aims of the present study were to examine the molecular mechanisms underlying sphingosine-1-phosphate (S1P)-induced rat pulmonary artery smooth muscle cells (PASMCs) proliferation/migration and to determine the effect of yes-associated protein (YAP) activation on S1P-induced PASMCs proliferation/migration and its potential mechanisms. S1P induced YAP dephosphorylation and nuclear translocation, upregulated microRNA-130a/b (miR-130a/b) expression, reduced bone morphogenetic protein receptor 2 (BMPR2), and inhibitor of DNA binding 1(Id1) expression, and promoted PASMCs proliferation and migration. Pretreatment of cells with Rho-associated protein kinase (ROCK) inhibitor Y27632 suppressed S1P-induced YAP activation, miR-130a/b upregulation, BMPR2/Id1 downregulation, and PASMCs proliferation/migration. Knockdown of YAP using small interfering RNA also suppressed S1P-induced alterations of miR-130a/b, BMPR2, Id1, and PASMCs behavior. In addition, luciferase reporter assay indicated that miR-130a/b directly regulated BMPR2 expression in PASMCs. Inhibition of miR-130a/b functions by anti-miRNA oligonucleotides attenuated S1P-induced BMPR2/Id1 downregulation and the proliferation and migration of PASMCs. Taken together, our study indicates that S1P induces activation of YAP through ROCK signaling and subsequently increases miR-130a/b expression, which, in turn, downregulates BMPR2 and Id1 leading to PASMCs proliferation and migration.

Mitochondria as a Platform for Dictating the Cell Fate of Cultured Human Corneal Endothelial Cells.

PurposeAiming to clarify the role of mitochondria in cell fate decision of cultured human corneal endothelial cell (cHCEC) subpopulations.MethodsThe mitochondrial respiratory ability were examined with Mito stress and Mito fuel flex test assays using an extracellular flux analyzer (XFe24; Agilent Technologies; Santa Clara, CA) for human corneal endothelium tissues, mature cHCECs and a variety of cell state transitioned cHCECs. Tricarboxylic acid cycle and acetyl-coenzyme A–related enzymes was analyzed by proteomics for cell lysates using liquid chromatography–tandem mass spectrometry for cHCEC subpopulations.ResultsThe maximum oxygen consumption rate was found to become stable depending on the maturation of cHCECs. In the Mito stress tests, culture supplements, epidermal growth factor, SB203580, and SB431543 significantly repressed oxygen consumption rate, whereas a Rho-associated protein kinase inhibitor Y-27632 increased. Tricarboxylic acid cycle and mitochondria acetyl-coenzyme A–related enzymes were selectively upregulated in mature cHCECs, but not in cell state transitioned cHCECs. The maximum oxygen consumption rate was found to be higher in healthy human corneal endothelium tissues than those with deeply reduced cell density. An upregulated tricarboxylic acid cycle was linked with metabolic rewiring converting cHCECs to acquire the mitochondria-dependent oxidative phenotype.ConclusionsMitochondrial metabolic intermediates and energy metabolism are tightly linked to the endothelial cell fate and function. These findings will help us to standardize a protocol for endothelial cell injection.

Y-27632 Promotes the Repair Effect of Umbilical Cord Blood-Derived Endothelial Progenitor Cells on Corneal Endothelial Wound Healing.

To investigate the proliferation of umbilical cord blood-derived endothelial progenitor cells (UCB EPCs) and the differentiation efficiency toward corneal endothelial cell (CEC)-like cells induced by rho-associated protein kinase (ROCK) inhibitor Y-27632 and to determine the most effective strategy for repairing corneal endothelium injuries in rabbits. UCB EPCs were cultured in Endothelial Cell Growth Medium-2 (EGM-2) media or conditioned media (CM) from human CECs, with and without the addition of Y-27632. Bromo-deoxyuridine (BrdU) immunocytochemistry and cell counting kit-8 assays were used to examine the proliferation of the cells. Real-time polymerase chain reaction, western blot, and immunocytochemistry were used to detect the CEC markers. Nd:YAG laser was used to establish an appropriate endothelium injury model based on rabbit corneas. The following intracameral injections were then performed to repair the model: 100 μL Opti-MEM I reduced serum medium (model group), 2 × 105 UCB EPCs diluted in 100 μL Opti-MEM I reduced serum medium (EPC group), 100 μM Y-27632 diluted in 100 μL Opti-MEM I reduced serum medium (Y-27632 group), and 2 × 105 UCB EPCs supplemented with 100 μM Y-27632 (final volume 100 μL, EPC/Y-27632 group). The follow-up tests focused on corneal transparency, central corneal thickness, intraocular pressure, and in vivo confocal microscopy, which were performed to evaluate the healing of the wounds. Culturing UCB EPCs in CM supplemented with 10 μM Y-27632 resulted in higher proliferation rates compared with EGM-2 media and CM. There were significantly improved protein levels of Zona Occludens 1, N-cadherin, Na+-K+-ATPase α1, Na+-K+-ATPase β1, and Pax6 and improved mRNA levels of collagen type IV and VIII and AQP1. The combined intracameral injection of Y-27632 and UCB EPCs accelerated the recovery of corneal transparency, regression of corneal edema, and healing of the corneal endothelium compared with the injections of Y-27632 and UCB EPCs on their own. Y-27632 not only promotes the proliferation of UCB EPCs but also contributes to differentiation of UCB EPCs toward CECs in the presence of CM. The intracameral injection of Y-27632 itself promotes the healing of corneal endothelium wounds. On this basis, supplementing UCB EPCs with Y-27632 accelerates the healing of corneal endothelium wounds.

Mechanism of contractile dysfunction induced by serotonin in coronary artery in spontaneously hypertensive rats.

Hypertension is one of the risk factors for coronary heart disease. The present study investigated the mechanism of contractile dysfunction induced by serotonin (5-HT) in coronary artery in spontaneously hypertensive rats (SHRs). Coronary arteries were isolated form SHRs and Wistar rats. Arterial ring contraction was measured using a multi myograph system. Intracellular calcium concentration was measured with a Ca2+ probe fluo-4/AM in vascular smooth muscle cells (VSMCs) isolated from coronary arteries. Signaling pathway-related proteins were assayed by western blotting. A 5-HT2A receptor blocker, sarpogrelate, completely eliminated coronary artery contraction induced by 5-HT. PLCβ inhibitor U73122 also significantly inhibited the response to 5-HT. Compared with the Wistar rats, serotonin (5-HT)- and CaCl2-induced coronary vasoconstriction in the SHRs was significantly reduced. Rho-associated protein kinase inhibitor Y27632, PKC inhibitor rottlerin, and L-type calcium channel blocker nifedipine inhibited the 5-HT-induced coronary artery contraction in a dose-dependent manner in SHRs and Wistar rats. However, the inhibitory effects were reduced in SHRs. In addition, store-operated Ca2+ (SOC) induced an obvious Ca2+ influx in coronary arterial smooth muscle cells, whereas SOC-mediated contraction was very slight in coronary arteries. At the same time, it was found that 5-HT2AR, IP3R, and Cav1.2 protein expression and PKCδ activity were decreased, and STIM1 and Orai1 were increased in VSMCs from coronary arteries of SHRs compared with Wistar rats. These results implicate calcium-handling dysfunction mediated by the 5-HT2A receptor and downstream signaling pathway might lead to a reduction in 5-HT-induced contraction in SHR coronary arteries.

Rho-associated protein kinase and cyclophilin a are involved in inorganic phosphate-induced calcification signaling in vascular smooth muscle cells

Arterial calcification, a risk factor of cardiovascular events, develops with differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. Cyclophilin A (CypA) is a peptidyl-prolyl isomerase involved in cardiovascular diseases such as atherosclerosis and aortic aneurysms, and rho-associated protein kinase (ROCK) is involved in the pathogenesis of vascular calcification. CypA is secreted in a ROCK activity-dependent manner and works as a mitogen via autocrine or paracrine mechanisms in VSMCs. We examined the involvement of the ROCK-CypA axis in VSMC calcification induced by inorganic phosphate (Pi), a potent cell mineralization initiator. We found that Pi stimulated ROCK activity, CypA secretion, extracellular signal-regulated protein kinase (ERK) 1/2 phosphorylation, and runt-related transcription factor 2 expression, resulting in calcium accumulation in rat aortic smooth muscle cells (RASMCs). The ROCK inhibitor Y-27632 significantly suppressed Pi-induced CypA secretion, ERK1/2 phosphorylation, and calcium accumulation. Recombinant CypA was found to be associated with increased calcium accumulation in RASMCs. Based on these results, we suggest that autocrine CypA is mediated by ROCK activity and is involved in Pi-induced ERK1/2 phosphorylation following calcification signaling in RASMCs.

Monocrotaline-induced pulmonary arterial hypertension: Time-course of injury and comparative evaluation of macitentan and Y-27632, a Rho kinase inhibitor

Novel pharmacological approaches are needed to improve outcomes of patients with idiopathic pulmonary hypertension. Rho-associated protein kinase (ROCK) inhibitors have shown beneficial effects in preclinical models of pulmonary arterial hypertension (PAH), because of their role in the regulation of pulmonary artery vasoconstrictor tone and remodeling. We compared a ROCK inhibitor, Y-27632, for the first time with the dual endothelin receptor antagonist, macitentan, in a monocrotaline-induced rat pulmonary hypertension model. Different methods (echocardiography, hemodynamics, histology of right ventricle and pulmonary vessels, and circulating biomarkers) showed consistently that 100 mg/kg daily of Y-27632 and 10 mg/kg daily of macitentan slowed the progression of PAH both at the functional and structural levels. Treatments started on day 14 after monocrotaline injection and lasted 14 days.The findings of all experimental methods show that the selective ROCK inhibitor Y-27632 has more pronounced effects than macitentan, but a major limitation to its use is its marked peripheral vasodilating action.

Fatty acid desaturase 2 is up-regulated by the treatment with statin through geranylgeranyl pyrophosphate-dependent Rho kinase pathway in HepG2 cells

Statins have been reported to increase the plasma concentration of arachidonic acid (AA), an omega-6 long chain polyunsaturated fatty acid (LCPUFA) in several clinical studies indicating that statins affect the endogenous synthesis of LCUFAs. In the present study, we investigated the roles of the intrinsic mevalonate cascade and Rho-dependent pathway in LCPUFA synthesis, especially focusing on fatty acid desaturases (Fads) 2, using the human hepatocellular carcinoma cell line HepG2. Cell number and the activity of caspase-3 and 7 (caspase-3/7) was measured using a commercial kit. Gene expression was analyzed by quantitative real-time PCR. Protein expression was detected by Western blot analysis. Atorvastatin decreased cell viability and increased caspase-3/7 activity in a dose-dependent manner. At lower concentrations, atorvastatin stimulated both mRNA and protein expression of Fads2, and increased mRNA expression of FADS1 and ELVOL5. Both mevalonate and geranylgeranyl-pyrophosphate (GGPP), but not cholesterol, fully reversed atorvastatin-induced upregulation of Fads2, and mevalonate-effected reversal was inhibited by treatment with the Rho-associated protein kinase inhibitor Y-27632. These data clearly demonstrated that in human HepG2 cells, statins affect the endogenous synthesis of LCPUFAs by regulation of not only Fads2, but also Fads1 and Elovl5, through the GGPP-dependent Rho kinase pathway.

Lentiviral Vector-Mediated Expression of Exoenzyme C3 Transferase Lowers Intraocular Pressure in Monkeys.

Primary open-angle glaucoma (POAG) is considered a lifelong disease characterized by optic nerve deterioration and visual field damage. Although the disease progression can usually be controlled by lowering the intraocular pressure (IOP), therapeutic effects of current approaches do not last long. Gene therapy could be a promising method for persistent treatment of the disease. Our previous study demonstrated that gene transfer of exoenzyme C3 transferase (C3) to the trabecular meshwork (TM) to inhibit Rho GTPase (Rho), the upstream signal molecule of Rho-associated kinase (ROCK), resulted in lowered IOP in normal rodent eyes. In the present study, we show that the lentiviral vector (LV)-mediated C3 expression inactivates RhoA in human TM cells by ADP ribosylation, resulting indisruption of the actin cytoskeleton and altered cell morphology. In addition, intracameral delivery of the C3 vector to monkey eyes leads to persistently lowered IOP without obvious signs of inflammation. This is the first report of using a vector to transduce the TM of an alive non-human primate with a gene that alters cellular machinery and physiology. Our results in non-human primates support that LV-mediated C3 expression in the TM may have therapeutic potential forglaucoma, the leading cause of irreversible blindness in humans.