ROCK Inhibitor Y-27632 Research Articles

Crucial Role of ROCK2-Mediated Phosphorylation and Upregulation of FHOD3 in the Pathogenesis of Angiotensin II-Induced Cardiac Hypertrophy.

Cardiac hypertrophy is characterized by increased myofibrillogenesis. Angiotensin II (Ang-II) is an essential mediator of the pressure overload-induced cardiac hypertrophy in part through RhoA/ROCK (small GTPase/Rho-associated coiled-coil containing protein kinase) pathway. FHOD3 (formin homology 2 domain containing 3), a cardiac-restricted member of diaphanous-related formins, is crucial in regulating myofibrillogenesis in cardiomyocytes. FHOD3 maintains inactive through autoinhibition by an intramolecular interaction between its C- and N-terminal domains. Phosphorylation of the 3 highly conserved residues (1406S, 1412S, and 1416T) within the C terminus (CT) of FHOD3 by ROCK1 is sufficient for its activation. However, it is unclear whether ROCK-mediated FHOD3 activation plays a role in the pathogenesis of Ang-II-induced cardiac hypertrophy. In this study, we detected increases in FHOD3 expression and phosphorylation in cardiomyocytes from Ang-II-induced rat cardiac hypertrophy models. Valsartan attenuated such increases. In cultured neonate rat cardiomyocytes, overexpression of phosphor-mimetic mutant FHOD3-DDD, but not wild-type FHOD3, resulted in myofibrillogenesis and cardiomyocyte hypertrophy. Expression of a phosphor-resistant mutant FHOD3-AAA completely abolished myofibrillogenesis and attenuated Ang-II-induced cardiomyocyte hypertrophy. Pretreatment of neonate rat cardiomyocytes with ROCK inhibitor Y27632 reduced Ang-II-induced FHOD3 activation and upregulation, suggesting the involvement of ROCK activities. Silencing of ROCK2, but not ROCK1, in neonate rat cardiomyocytes, significantly lessened Ang-II-induced cardiomyocyte hypertrophy. ROCK2 can directly phosphorylate FHOD3 at both 1412S and 1416T in vitro and is more potent than ROCK1. Both kinases failed to phosphorylate 1406S. Coexpression of FHOD3 with constitutively active ROCK2 induced more stress fiber formation than that with constitutively active ROCK1. Collectively, our results demonstrated the importance of ROCK2 regulated FHOD3 expression and activation in Ang-II-induced myofibrillogenesis, thus provided a novel mechanism for the pathogenesis of Ang-II-induced cardiac hypertrophy.

A dual-layer macro/mesoporous structured TiO2 surface improves the initial adhesion of osteoblast-like cells

A dual-layer TiO2 surface with hierarchical macro and mesoporous structure was prepared by a combinational approach of micro-arc oxidation followed by evaporation-induced self-assembly of nano-crystallites. The mesoporous layer contains pores with an average size of <10nm and consists of anatase TiO2 nanocrystallites. The dual-layer hierarchical macro/mesoporous structured TiO2 surface improves the hydrophilicity and fibronectin adsorption ability in comparison with the sole macroporous or smooth TiO2 surface. With the formation of an additional mesoporous layer on macroporous TiO2 surface, the attached number of human osteogenic sarcoma cells (SaOS-2) increases in the initial incubation of 4h but it does not show significant difference after 24h compared to that attached on the macroporous or smooth surfaces. Whereas, it was noticed that SaOS-2 cells have larger spread area and more stress fibers on the macro/mesoporous structured surface than those on the other surfaces. To understand the intracellular mechanism of the initial cell adhesion on the macro/mesoporous surface, the Rho/ROCK pathway was investigated to reveal the topography-induced biological functions by introducing the ROCK inhibitor Y-27632 during cell culture. In the presence of Y-27632, cells on the macroporous surface and macro/mesoporous surface both show stellate appearance, with poor assembly stress fibers and long cell membrane protrusions. Cells on the smooth surface have larger spread areas compared to the former two surfaces. And the attached cells significantly reduced but there are no differences among the three surfaces. It reveals that the ROCK inhibitor invalidates the promotion of initial cell adhesion on the macro/mesoporous structure. This study may shed light on the mechanism behind the enhancement effect of macro/mesoporous structure for initial cell adhesion.

Production of Homogeneous Cultured Human Corneal Endothelial Cells Indispensable for Innovative Cell Therapy.

Cultured human corneal endothelial cells (cHCECs) are anticipated to become an alternative to donor corneas for the treatment of corneal endothelial dysfunction. The aim of this study was to establish proper culture protocols to successfully obtain a reproducibly homogeneous subpopulation (SP) with matured cHCEC functions and devoid of cell-state transition suitable for cell-injection therapy. The presence of SPs in cHCECs was investigated in terms of surface cluster-of-differentiation (CD) marker expression level by flow cytometry, as combined analysis of CD markers can definitively specify the SP (effector cells) conceivably the most suitable for cell therapy among diverse SPs. The culture conditions were evaluated by flow cytometry in terms of the proportion (E-ratio) of effector cells designated by CD markers. Flow cytometry analysis identifying CD44-CD166+CD133-CD105-CD24-CD26- effector cells proved convenient and reliable for standardizing the culture procedures. To ascertain the reproducible production of cHCECs with E-ratios of more than 90% and with no karyotype abnormality, the preferred donor age was younger than 29 years. The continuous presence of Rho-associated protein kinase (ROCK)-inhibitor Y-27632 greatly increased the E-ratios, whereas the presence of transforming growth factor-beta/Smad-inhibitor SB431542 greatly reduced the number of recovered cHCECs. The seeding cell density during culture passages proved vital for maintaining a high E-ratio for extended passages. The continuous presence of ROCK-inhibitor Y-27632 throughout the cultures greatly improved the E-ratio. Our findings elucidated the culture conditions needed to obtain reproducible cHCECs with high E-ratios, thus ensuring homogeneous cHCECs with matured functions for the treatment of corneal endothelial dysfunction.

Dasatinib inhibits actin fiber reorganization and promotes endothelial cell permeability through RhoA-ROCK pathway.

Treatment with dasatinib, a tyrosine kinase inhibitor, is associated with edema, pleural effusion, and pulmonary edema. We investigated the effect of dasatinib on the barrier function of human microvascular endothelial cells‐1 (HMEC‐1) in vitro and in vivo. The permeability of HMEC‐1 to fluorescein isothiocyante (FITC)‐dextran increased in Transwell chambers within 5 min following the addition of therapeutic concentrations of dasatinib. The change in permeability was associated with increased activation of RhoA GTPase and its effector Rho‐associated coiled‐coil kinase 1(ROCK1). RhoA inhibitor C3 transferase almost completely inhibited dasatinib‐induced increase in permeability. Under similar conditions, imatinib had no effect on permeability or activation of RhoA. Since integrin‐induced cell spreading suppresses RhoA activation, we examined the effect of dasatinib on cell spreading on fibronectin substrate. Dasatinib impaired endothelial cell spreading in a concentration‐dependent manner and induced disorganization of actin fibers. Tyrosine kinases play an essential role in transmitting signals from integrins to RhoA and we examined tyrosine phosphorylation of several cytoskeletal proteins. Dasatinib markedly inhibited tyrosine phosphorylation of p130 Crk‐associated substrate (p130cas), paxillin and vinculin. These results suggest that the inhibition of tyrosine phosphorylation of the focal adhesion plaque components by dasatinib may alter the assembly of actin fibers resulting in the activation of RhoA/ROCK pathway. Consistent with these findings, dasatinib‐induced increase in the permeability was blocked by ROCK inhibitor y27632. In vivo administration of y27632, significantly inhibited the dasatinib‐induced extravasation of Evans blue in mice and dasatinib‐induced increase in microvascular permeability was attenuated in ROCK1‐deficient mice. These findings suggest that ROCK inhibitors could serve as therapeutic modalities to ameliorate the dasatinib‐induced pulmonary changes.

The RhoA/ROCK Pathway Ameliorates Adhesion and Inflammatory Infiltration Induced by AGEs in Glomerular Endothelial Cells

A recent study demonstrated that advanced glycation end products (AGEs) play a role in monocyte infiltration in mesangial areas in diabetic nephropathy. The Ras homolog gene family, member A Rho kinase (RhoA/ROCK) pathway plays a role in regulating cell migration. We hypothesized that the RhoA/ROCK pathway affects adhesion and inflammation in endothelial cells induced by AGEs. Rat glomerular endothelial cells (rGECs) were cultured with AGEs (80 μg/ml) in vitro. The ROCK inhibitor Y27632 (10 nmol/l) and ROCK1-siRNA were used to inhibit ROCK. We investigated levels of the intercellular adhesion molecule 1 (ICAM-1) and monocyte chemoattractant protein1 (MCP-1) in rGECs. Db/db mice were used as a diabetes model and received Fasudil (10 mg/kg/d, n = 6) via intraperitoneal injection for 12 weeks. We found that AGEs increased the expression of ICAM-1 and MCP-1 in rGECs, and the RhoA/ROCK pathway inhibitor Y27632 depressed the release of adhesion molecules. Moreover, blocking the RhoA/ROCK pathway ameliorated macrophage transfer to the endothelium. Reduced expression of adhesion molecules and amelioration of inflammatory cell infiltration in the glomerulus were observed in db/db mice treated with Fasudil. The RhoA/ROCK pathway plays a role in adhesion molecule expression and inflammatory cell infiltration in glomerular endothelial cells induced by AGEs.

Mechanical mismatch between Ras transformed and untransformed epithelial cells.

The organization of the actin cytoskeleton plays a key role in regulating cell mechanics. It is fundamentally altered during transformation, affecting how cells interact with their environment. We investigated mechanical properties of cells expressing constitutively active, oncogenic Ras (RasV12) in adherent and suspended states. To do this, we utilized atomic force microscopy and a microfluidic optical stretcher. We found that adherent cells stiffen and suspended cells soften with the expression of constitutively active Ras. The effect on adherent cells was reversed when contractility was inhibited with the ROCK inhibitor Y-27632, resulting in softer RasV12 cells. Our findings suggest that increased ROCK activity as a result of Ras has opposite effects on suspended and adhered cells. Our results also establish the importance of the activation of ROCK by Ras and its effect on cell mechanics.

Development of Poly Lactic/Glycolic Acid (PLGA) Microspheres for Controlled Release of Rho-Associated Kinase Inhibitor.

Purpose The purpose of this study was to investigate the feasibility of poly lactic/glycolic acid (PLGA) as a drug delivery carrier of Rho kinase (ROCK) inhibitor for the treatment of corneal endothelial disease. Method ROCK inhibitor Y-27632 and PLGA were dissolved in water with or without gelatin (W1), and a double emulsion [(W1/O)/W2] was formed with dichloromethane (O) and polyvinyl alcohol (W2). Drug release curve was obtained by evaluating the released Y-27632 by using high performance liquid chromatography. PLGA was injected into the anterior chamber or subconjunctiva in rabbit eyes, and ocular complication was evaluated by slitlamp microscope and histological analysis. Results Y-27632 incorporated PLGA microspheres with different molecular weights, and different composition ratios of lactic acid and glycolic acid were fabricated. A high molecular weight and low content of glycolic acid produced a slower and longer release. The Y-27632 released from PLGA microspheres significantly promoted the cell proliferation of cultured corneal endothelial cells. The injection of PLGA did not induce any evident eye complication. Conclusions ROCK inhibitor-incorporated PLGA microspheres were fabricated, and the microspheres achieved the sustained release of ROCK inhibitor over 7–10 days in vitro. Our data should encourage researchers to use PLGA microspheres for treating corneal endothelial diseases.

ROCK inhibitor Y-27632 attenuated early endothelial dysfunction caused by occupational environmental concentrations of carbon black nanoparticles

The unique properties of carbon black nanoparticles (CBNs) make them feasible for new applications and raise concerns about cardiovascular disorders.

Enhancing Functional Platelet Release In Vivo from in Vitro-Grown Megakaryocytes Using the Protein Kinase Inhibitor SU6656

The clinical demand for platelet transfusions is increasing, threatening the ability to obtain sufficient healthy donors to provide these platelets. Advances in regenerative medicine research have opened the possibility of generating sufficient in vitro-grown megakaryocytes and consequent platelets to supply a portion of the clinical platelet transfusion demand. We have shown that infusing megakaryocytes for obtaining released, functional platelets is a viable alternative strategy than trying to release platelets in vitro. However, for both approaches, in vitro-cultured megakaryocytes have lower ploidy and release fewer platelets than likely occurs in vivo by primary cells. SU6656 inhibitor, a Src kinase inhibitor, has been shown to influence ploidization in several megakaryocyte-like line with purported increase in proplatelets release. However, in our hands, other agents - such as the ROCK inhibitor Y27632 - while increasing polyploidization markedly, inhibited platelet release per infused megakaryocyte in vivo. We grew megakaryocytes from CD34+ cells for 12 days with or without SU6656 (2.5 µM) supplementation during the last 4 days. We found that the SU6656 inhibitor only increased the number of CD34+-derived megakaryocytes by ~15% at the end of the 12 day growth, but more markedly increase the percent of large megakaryocytes measured by FSC parameter in flow cytometry evaluation from 28 up to 41% and percent of high granular megakaryocytes from 27 to 45%. These changes were accompanied with a shift in average ploidy from 4.9 to 6.9 (p<0.0003, N=6). Notably, SU6656-treated megakaryocytes released ~4-fold more platelets per infused megakaryocytes in immunocompromized NSG mice than untreated similarly in vitro-grown megakaryocytes. By 24 hrs, there were 6.5-fold platelets from the infused SU6656-treated megakaryocytes than control untreated (p<0.037, N=6). Released platelets from the drug-treated and untreated megakaryocytes had similar levels of percent thiazole orange positivity as an indication that they were young platelets. Importantly, baseline annexin V, CD62p and PAC1 binding prior to agonist exposure were also similarly and increased to the same extent after thrombin (1U/ml) stimulation. Additionally, incorporation into a growing cremaster laser injury-induced thrombus in vivo was similar further indicating retained function by the platelets released from the drug-treated megakaryocytes. A number of strategies such as modifying the level of transcription factors have been proposed to increase the size, ploidy or proplatelets release from in vitro-grown megakaryocytes. In none of these cases have these released platelets in vivo biology been examined and demonstrated to replicate high release number per megakaryocyte and retained functionality. We show that terminal exposure of in vitro-grown megakaryocytes to the non-specific inhibitor SU6656 significantly increases in vivo yield while leaving in vivo half-life and functionality intact. The exact pathway affected by SU6656 that leads to these results is now being pursued. DisclosuresNo relevant conflicts of interest to declare.

Vincristine promotes migration and invasion of colorectal cancer HCT116 cells through RhoA/ROCK/ Myosin light chain pathway.

Vincristine is an antitumor vinca alkaloid isolated from vinca rosea, and is a medication used to treat a number of types of cancer. In this study, we investigated the impact of vincristine on oncogenic phenotypes of human colorectal cancer HCT116 cells. MTT assay demonstrated that vincristine showed a obviously inhibitory effect on cell growth compared to non-treated cells. However, Transwell assay showed that vincristine promoted migration and invasion of HCT116 cells in vitro in a concentration-dependent manner between 0.5 and 15 μM vincristine treatment, whereas cell growth showed no remarkable difference within the same concentration range. Additionally, Western blot analysis showed that vincristine significantly elevated RhoA activity and Myosin light chain (MLC) phosphorylation, suggesting the involvement of RhoA/ROCK pathway in the vincristine-induced enhancement of cellular motility. Furthermore, we found that both the siRNA for RhoA and ROCK inhibitor Y27632 attenuated the phosphorylation of MLC, as well as vincristine-induced migration and invasion. These data indicate that vincristine enhanced migration and invasion of HCT116 cells possibly through stimulating RhoA/ROCK/MLC signaling pathway.

Restoration of uridine 5′-triphosphate-suppressed delayed rectifying K+ currents by an NO activator KMUP-1 involves RhoA/Rho kinase signaling in pulmonary artery smooth muscle cells

We have demonstrated that KMUP-1 (7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) blunts monocrotaline-induced pulmonary arterial hypertension by altering Ca2+ sensitivity, K+-channel function, endothelial nitric oxide synthase activity, and RhoA/Rho kinase (ROCK) expression. This study further investigated whether KMUP-1 impedes uridine 5′-triphosphate (UTP)-inhibited delayed rectifying K+ (KDR) current in rat pulmonary arteries involved the RhoA/ROCK signaling. Pulmonary artery smooth muscle cells (PASMCs) were enzymatically dissociated from rat pulmonary arteries. KMUP-1 (30μM) attenuated UTP (30μM)-mediated membrane depolarization and abolished UTP-enhanced cytosolic Ca2+ concentration. Whole-cell patch-clamp electrophysiology was used to monitor KDR currents. A voltage-dependent KDR current was isolated and shown to consist of a 4-aminopyridine (5mM)-sensitive component and an insensitive component. The 4-aminopyridine sensitive KDR current was suppressed by UTP (30μM). The ROCK inhibitor Y27632 (30μM) abolished the ability of UTP to inhibit the KDR current. Like Y27632, KMUP-1 (30μM) similarly abolished UTP-inhibited KDR currents. Superfused protein kinase A and protein kinase G inhibitors (KT5720, 300nM and KT5823, 300nM) did not affect UTP-inhibited KDR currents, but the currents were restored by adding KMUP-1 (30μM) to the superfusate. KMUP-1 reversal of KDR current inhibition by UTP predominantly involves the ROCK inhibition. The results indicate that the RhoA/ROCK signaling pathway plays a key role in eliciting PASMCs depolarization caused by UTP, which would result in pulmonary artery constriction. KMUP-1 blocks UTP-mediated PASMCs depolarization, suggesting that it would prevent abnormal pulmonary vasoconstriction.

Quantifying the kinetics and morphological changes of the fusion of spheroid building blocks

Tissue fusion, whereby two or more spheroids coalesce, is a process that is fundamental to biofabrication. We have designed a quantitative, high-throughput platform to investigate the fusion of multicellular spheroids using agarose micro-molds. Spheroids of primary human chondrocytes (HCH) or human breast cancer cells (MCF-7) were self-assembled for 24 h and then brought together to form an array comprised of two spheroids (one doublet) per well. To quantify spheroid fusogenicity, we developed two assays: (1) an initial tack assay, defined as the minimum amount of time for two spheroids to form a mechanically stable tissue complex or doublet, and (2) a fusion assay, in which we defined and tracked key morphological parameters of the doublets as a function of time using wide-field fluorescence microscopy over a 24 h time-lapse. The initial tack of spheroid fusion was measured by inverting the micro-molds and centrifuging doublets at various time points to assess their connectedness. We found that the initial tack between two spheroids forms rapidly, with the majority of doublets remaining intact after centrifugation following just 30 min of fusion. Over the course of 24 h of fusion, several morphological changes occurred, which were quantified using a custom image analysis pipeline. End-to-end doublet lengths decreased over time, doublet widths decreased for chondrocytes and increased for MCF-7, contact lengths increased over time, and chondrocyte doublets exhibited higher intersphere angles at the end of fusion. We also assessed fusion by measuring the fluorescence intensity at the plane of fusion, which increased over time for both cell types. Interestingly, we observed that doublets moved and rotated in the micro-wells during fusion and this rotation was inhibited by ROCK inhibitor Y-27632 and myosin II inhibitor blebbistatin. Understanding and optimizing tissue fusion is essential for creating larger tissues, organs, or other structures using individual microtissues as building parts.

Calcineurin/NFAT Activation-Dependence of Leptin Synthesis and Vascular Growth in Response to Mechanical Stretch.

Background and Aims: Hypertension and obesity are important risk factors of cardiovascular disease. They are both associated with high leptin levels and have been shown to promote vascular hypertrophy, through the RhoA/ROCK and ERK1/2 phosphorylation. Calcineurin/NFAT activation also induces vascular hypertrophy by upregulating various genes. This study aimed to decipher whether a crosstalk exists between the RhoA/ROCK pathway, Ca2+/calcineurin/NFAT pathway, and ERK1/2 phosphorylation in the process of mechanical stretch-induced vascular smooth muscle cell (VSMC) hypertrophy and leptin synthesis.Methods and Results: Rat portal vein (RPV) organ culture was used to investigate the effect of mechanical stretch and exogenous leptin (3.1 nM) on VSMC hypertrophy and leptin synthesis. Results showed that stretching the RPV significantly upregulated leptin secretion, mRNA, and protein expression, which were inhibited by the calcium channel blocker nifedipine (10 μM), the selective calcineurin inhibitor FK506 (1 nM), and the ERK1/2 inhibitor PD98059 (1 μM). The transcription inhibitor actinomycin D (0.1 μM) and the translation inhibitor cycloheximide (1 mM) significantly decreased stretch-induced leptin protein expression. Mechanical stretch or leptin caused an increase in wet weight changes and protein synthesis, considered as hypertrophic markers, while they were inhibited by FK506 (0.1 nM; 1 nM). In addition, stretch or exogenous leptin significantly increased calcineurin activity and MCIP1 expression whereas leptin induced NFAT nuclear translocation in VSMCs. Moreover, in response to stretch or exogenous leptin, the Rho inhibitor C3 exoenzyme (30 ng/mL), the ROCK inhibitor Y-27632 (10 μM), and the actin depolymerization agents Latrunculin B (50 nM) and cytochalasin D (1 μM) reduced calcineurin activation and NFAT nuclear translocation. ERK1/2 phosphorylation was inhibited by FK506 and C3.Conclusions: Mechanical stretch-induced VSMC hypertrophy and leptin synthesis and secretion are mediated by Ca2+/calcineurin/NFAT activation. RhoA/ROCK and ERK1/2 activation are critical for mechanical stretch-induced calcineurin activation.

Correlation between ECM guidance and actin polymerization on osteogenic differentiation of human adipose-derived stem cells

The correlation between extracellular matrix (ECM) components, cell shape, and stem cell guidance can shed light in understanding and mimicking the functionality of stem cell niches for various applications. This interplay on osteogenic guidance of human adipose-derived stem cells (hASCs) was focus of this study. Proliferation and osteogenic markers like alkaline phosphatase activity and calcium mineralization were slightly increased by the ECM components laminin (LA), collagen I (COL), and fibronectin (FIB); with control medium no differentiation occurred. ECM guided differentiation was rather dependent on osterix than on Runx2 pathway. FIB significantly enhanced cell elongation even in presence of actin polymerization blockers cytochalasin D (CytoD) and ROCK inhibitor Y-27632, which generally caused more rounded cells. Except for the COL surface, both inhibitors increased the extent of osterix, while the Runx2 pathway was more sensitive to the culture condition. Both inhibitors did not affect hASC proliferation. CytoD enabled osteogenic differentiation independently from the ECM, while it was rather blocked via Y-27632 treatment; on FIB the general highest extent of differentiation occurred. Taken together, the ECM effect on hASCs occurs indirectly and selectively via a dominant role of FIB: it sustains osteogenic differentiation in case of a tension-dependent control of actin polymerization.

Pretreatment with Rho-kinase inhibitor ameliorates lethal endotoxemia-induced liver injury by improving mitochondrial function

Rho kinase (ROCK) inhibition has been reported to improve various inflammatory diseases including endotoxemia. Mitochondrial dysfunction might be the key to the pathophysiology of sepsis-induced organ failure. Therefore, this study aimed to explore whether ROCK inhibition protects against the liver injury by regulating mitochondrial function in endotoxemia model mice. The mice were randomly divided into four groups (N=6–8 per group): control, LPS, LPS+Y-27632 (LPS+Y), and LPS+Mito-TEMPO (LPS+M). For induction of endotoxin-induced acute liver injury, the mice were intraperitoneally administered lipopolysaccharide (LPS, 20mg/kg). The ROCK inhibitor Y-27632 (or mitochondrial antioxidant Mito-TEMPO) was intraperitoneally administered at 18 and 1h before injection of LPS. The mice were euthanized 8h after LPS administration. The liver and blood samples were taken and preserved for analysis. Results of this study showed that pretreatment with Y-27632 or Mito-TEMPO significantly attenuated the liver injury as compared to the LPS group. This was confirmed by decreased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and by reduced hepatocellular apoptosis and histologic damage. Pretreatment with Y-27632 or Mito-TEMPO markedly reduced the LPS-induced inflammatory response and oxidative stress the in liver. Furthermore, it showed similar protective effects on the hepatic mitochondrial function, including an increased activity of complexes I and IV and mitochondrial superoxide dismutase (MnSOD), and an upregulated expression of mtDNA-encoded genes. Taken together, these data demonstrate that Mito-TEMPO can potently inhibit the endotoxin-induced mitochondrial and hepatic abnormalities and indicate that mitochondrial dysfunction might play a key role in the endotoxemia-induced acute liver injury. Moreover, our study shows that ROCK inhibition protects against the endotoxemia-induced liver injury by improving the mitochondrial function.

Early Alterations of Bile Canaliculi Dynamics and the Rho Kinase/Myosin Light Chain Kinase Pathway Are Characteristics of Drug-Induced Intrahepatic Cholestasis.

Intrahepatic cholestasis represents 20%-40% of drug-induced injuries from which a large proportion remains unpredictable. We aimed to investigate mechanisms underlying drug-induced cholestasis and improve its early detection using human HepaRG cells and a set of 12 cholestatic drugs and six noncholestatic drugs. In this study, we analyzed bile canaliculi dynamics, Rho kinase (ROCK)/myosin light chain kinase (MLCK) pathway implication, efflux inhibition of taurocholate [a predominant bile salt export pump (BSEP) substrate], and expression of the major canalicular and basolateral bile acid transporters. We demonstrated that 12 cholestatic drugs classified on the basis of reported clinical findings caused disturbances of both bile canaliculi dynamics, characterized by either dilatation or constriction, and alteration of the ROCK/MLCK signaling pathway, whereas noncholestatic compounds, by contrast, had no effect. Cotreatment with ROCK inhibitor Y-27632 [4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] and MLCK activator calmodulin reduced bile canaliculi constriction and dilatation, respectively, confirming the role of these pathways in drug-induced intrahepatic cholestasis. By contrast, inhibition of taurocholate efflux and/or human BSEP overexpressed in membrane vesicles was not observed with all cholestatic drugs; moreover, examples of noncholestatic compounds were reportedly found to inhibit BSEP. Transcripts levels of major bile acid transporters were determined after 24-hour treatment. BSEP, Na+-taurocholate cotransporting polypeptide, and organic anion transporting polypeptide B were downregulated with most cholestatic and some noncholestatic drugs, whereas deregulation of multidrug resistance-associated proteins was more variable, probably mainly reflecting secondary effects. Together, our results show that cholestatic drugs consistently cause an early alteration of bile canaliculi dynamics associated with modulation of ROCK/MLCK and these changes are more specific than efflux inhibition measurements alone as predictive nonclinical markers of drug-induced cholestasis.

Y-27632 reduces the MMP2 and MMP9 expression in endothelial cell via inhibition of ROCK signal pathway

To explore the effect of ROCK inhibitor Y-27632 on the matrix metalloproteinase 2 and 9 (MMP2 and MMP9) gene expression and activity in tumor necrosis factor α (TNF-α)-treated human umbilical vein endothelial cell (HUVEC). HHUVEC was divided into 3 groups, a control group, a TNF-α group, and a TNF-α plus Y-27632 group. The expressions of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), MMP2 and MMP9 were examined by real-time PCR. The MMP2/9 activity was measured by gelatin zymography. Compared to the control group, the mRNA expressions of ICAM-1, VCAM-1, MMP2 and MMP9 were increased TNF-α-treated cells, which were suppressed by ROCK inhibitor (P<0.01). The MMP2/9 activity was elevated in TNF-α-treated cells, which was reversed by ROCK inhibitor (P<0.05). ROCK inhibitor can suppress TNF-α-induced inflammation in endothelial cells through down-regulation of MMP2/9.

A comparison of strategies for immortalizing mouse embryonic fibroblasts.

The genetically amenable mouse model has led to a large collection of genetically defined lines from which mouse embryonic fibroblasts (MEFs) have been derived. Despite their widespread use, MEFs are time consuming to generate and have a limited lifespan. Immortalizing primary MEFs with the desired genetic manipulations greatly reduces culture maintenance time, enables the generation of near limitless amounts of protein lysate, and facilitates biological replicates during experimentation. In this work, we have evaluated several approaches for MEF immortalization. When cultivated at 3% O2, some primary MEF lines could be proliferated for > 40 passages with a median doubling rate of 45 ± 55 h (n = 8). However, serial passaging at 3% O2 achieved spontaneous immortalization with varying success. If cultures seemed to be reaching their Hayflick limit when cultivated at 3% O2, supplementing the culture media with 5 µM ROCK inhibitor Y-27632 helped to extend proliferation and achieve spontaneous immortalization. MEFs immortalized via SV40 Ta infection reliably produced cell lines with a median doubling rate of 25 ± 9 h (n = 9) and viability greater than 90%. In addition to a discussion of the characteristics of cell lines generated with various immortalization strategies, pros and cons of each strategy are included as are recommendations for generating immortalized MEFs.

Topical delivery of a Rho-kinase inhibitor to the cornea via mucoadhesive film

The application of inhibitors of the Rho kinase pathway (ROCK inhibitors) to the surface of the eye in the form of eyedrops has beneficial effects which aid the recovery of diseased or injured endothelial cells that line the inner surface of the cornea. The aim of this study was to test the plausibility of delivering a selective ROCK inhibitor, Y-27632, to the cornea using a thin polymeric film. Mucoadhesive polymeric thin films were prepared incorporating Y-27632 and diffusional release into PBS was determined. Topical ocular delivery from the applied film was investigated using freshly excised porcine eyes and eyedrops of equivalent concentration acted as comparators; after 24h the formulations were removed and the corneas extracted. Drug-loaded thin polymeric films, with high clarity and pliability were produced. ROCK inhibitor Y-27632 was weakly retained within the film, with release attaining equilibrium after 1h. This in turn facilitated its rapid ocular delivery, and an approximately three-fold greater penetration of Y-27632 into cryoprobe-treated corneas was observed from the thin film (p<0.01) compared to eyedrops. These findings support the further development of ROCK inhibitor delivery to the cornea via release from thin mucoadhesive films to treat vision loss cause by corneal endothelial dysfunction.

Smooth muscle filamin A is a major determinant of conduit artery structure and function at the adult stage.

Human mutations in the X-linked FLNA gene are associated with a remarkably diverse phenotype, including severe arterial morphological anomalies. However, the role for filamin A (FlnA) in vascular cells remains partially understood. We used a smooth muscle (sm)-specific conditional mouse model to delete FlnA at the adult stage, thus avoiding the developmental effects of the knock-out. Inactivation of smFlnA in adult mice significantly lowered blood pressure, together with a decrease in pulse pressure. However, both the aorta and carotid arteries showed a major outward hypertrophic remodeling, resistant to losartan, and normally occurring in hypertensive conditions. Notably, arterial compliance was significantly enhanced in the absence of smFlnA. Moreover, reactivity of thoracic aorta rings to a variety of vasoconstrictors was elevated, while basal contractility in response to KCl depolarization was reduced. Enhanced reactivity to the thromboxane A2 receptor agonist U46619 was fully reversed by the ROCK inhibitor Y27632. We discuss the possibility that a reduction in arterial stiffness upon smFlnA inactivation might cause a compensatory increase in conduit artery diameter for normalization of parietal tension, independently of the ROCK pathway. In conclusion, deletion of smFlnA in adult mice recapitulates the vascular phenotype of human bilateral periventricular nodular heterotopia, culminating in aortic dilatation.