Presence Of Y-27632 Research Articles

Biomaterial systems for evaluating the influence of ECM mechanics on anti-fibrotic therapeutic efficacy

Cardiac fibrosis is characterized by excessive accumulation and deposition of ECM proteins. Cardiac fibrosis is commonly implicated in a variety of cardiovascular diseases, including post-myocardial infarction (MI). We have previously developed a dual-delivery nanogel therapeutic to deliver tissue plasminogen activator (tPA) and Y-27632 (a ROCK inhibitor) to address MI-associated coronary artery occlusion and downregulate cell-contractility mediated fibrotic responses. Initial in vitro studies were conducted on glass substrates. The study presented here employs the use of polyacrylamide (PA) gels and microgel thin films to mimic healthy and fibrotic cardiac tissue mechanics. Soft and stiff polyacrylamide substrates or high and low loss tangent microgel thin films were utilized to examine the influence of cell-substrate interactions on dual-loaded nanogel therapeutic efficacy. In the presence of Y-27632 containing nanogels, a reduction of fibrotic marker expression was noted on traditional PA gels mimicking healthy and fibrotic cardiac tissue mechanics. These findings differed on more physiologically relevant microgel thin films, where early treatment with the ROCK inhibitor intensified the fibrotic related responses.

Sustained intestinal epithelial monolayer wound closure after transient application of a FAK-activating small molecule.

M64HCl, which has drug-like properties, is a water-soluble Focal Adhesion Kinase (FAK) activator that promotes murine mucosal healing after ischemic or NSAID-induced injury. Since M64HCl has a short plasma half-life in vivo (less than two hours), it has been administered as a continuous infusion with osmotic minipumps in previous animal studies. However, the effects of more transient exposure to M64HCl on monolayer wound closure remained unclear. Herein, we compared the effects of shorter M64HCl treatment in vitro to continuous treatment for 24 hours on monolayer wound closure. We then investigated how long FAK activation and downstream ERK1/2 activation persist after two hours of M64HCl treatment in Caco-2 cells. M64HCl concentrations immediately after washing measured by mass spectrometry confirmed that M64HCl had been completely removed from the medium while intracellular concentrations had been reduced by 95%. Three-hour and four-hour M64HCl (100 nM) treatment promoted epithelial sheet migration over 24 hours similar to continuous 24-hour exposure. 100nM M64HCl did not increase cell number. Exposing cells twice with 2-hr exposures of M64HCl during a 24-hour period had a similar effect. Both FAK inhibitor PF-573228 (10 μM) and ERK kinase (MEK) inhibitor PD98059 (20 μM) reduced basal wound closure in the absence of M64HCl, and each completely prevented any stimulation of wound closure by M64HCl. Rho kinase inhibitor Y-27632 (20 μM) stimulated Caco-2 monolayer wound closure but no further increase was seen with M64HCl in the presence of Y-27632. M64HCl (100 nM) treatment for 3 hours stimulated Rho kinase activity. M64HCl decreased F-actin in Caco-2 cells. Furthermore, a two-hour treatment with M64HCl (100 nM) stimulated sustained FAK activation and ERK1/2 activation for up to 16 and hours 24 hours, respectively. These results suggest that transient M64HCl treatment promotes prolonged intestinal epithelial monolayer wound closure by stimulating sustained activation of the FAK/ERK1/2 pathway. Such molecules may be useful to promote gastrointestinal mucosal repair even with a relatively short half-life.

The role of rhoA/rho-kinase and PKC in the inhibitory effect of L-cysteine/H2S pathway on the carbachol-mediated contraction of mouse bladder smooth muscle.

We investigated the role of RhoA/Rho-kinase (ROCK) and PKC in the inhibitory effect of L-cysteine/hydrogen sulfide (H2S) pathway on the carbachol-mediated contraction of mouse bladder smooth muscle. Carbachol (10-8-10-4M) induced a concentration-dependent contraction in bladder tissues. L-cysteine (H2S precursor; 10-2M) and exogenous H2S (NaHS; 10-3M) reduced the contractions evoked by carbachol by ~ 49 and ~ 53%, respectively, relative to control. The inhibitory effect of L-cysteine on contractions to carbachol was reversed by 10-2M PAG (~ 40%) and 10-3M AOAA (~ 55%), cystathionine-gamma-lyase (CSE) and cystathionine-β-synthase (CBS) inhibitor, respectively. Y-27632 (10-6M) and GF 109203X (10-6M), a specific ROCK and PKC inhibitor, respectively, reduced contractions evoked by carbachol (~ 18 and ~ 24% respectively), and the inhibitory effect of Y-27632 and GF 109203X on contractions was reversed by PAG (~ 29 and ~ 19%, respectively) but not by AOAA. Also, Y-27632 and GF 109203X reduced the inhibitory responses of L-cysteine on the carbachol-induced contractions (~ 38 and ~ 52% respectively), and PAG abolished the inhibitory effect of L-cysteine on the contractions in the presence of Y-27632 (~ 38%). Also, the protein expressions of CSE, CBS, and 3-MST enzymes responsible for endogenous H2S synthesis were detected by Western blot method. H2S level was increased by L-cysteine, Y-27632, and GF 109203X (from 0.12 ± 0.02 to 0.47 ± 0.13, 0.26 ± 0.03, and 0.23 ± 0.06nmol/mg respectively), and this augmentation in H2S level decreased with PAG (0.17 ± 0.02, 0.15 ± 0.03, and 0.07 ± 0.04nmol/mg respectively). Furthermore, L-cysteine and NaHS reduced carbachol-induced ROCK-1, pMYPT1, and pMLC20 levels. Inhibitory effects of L-cysteine on ROCK-1, pMYPT1, and pMLC20 levels, but not of NaHS, were reversed by PAG. These results suggest that there is an interaction between L-cysteine/H2S and RhoA/ROCK pathway via inhibition of ROCK-1, pMYPT1, and pMLC20, and the inhibition of RhoA/ROCK and/or PKC signal pathway may be mediated by the CSE-generated H2S in mouse bladder.

Signaling Cascade Mediating the Effect of FTY720P on the Na+/K+ ATPase in LLC-PK1.

In renal ischemia, the Na+/K+ ATPase of the kidney epithelial cells translocates to intracellular compartments, resulting in altered kidney functions. Sphingosine-1-phosphate (S1P) was shown to play a protective role against this ischemic injury. Whether the sphingolipid targets the Na+/K+ ATPase is a possibility that has not been explored before. This work aims at investigating the effect of S1P on renal Na+/K+ ATPase using its analogue FTY720P and LLC-PK1 cells. The activity of the Na+/K+ ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the enzyme while its protein expression was studied by western blot analysis. FTY720P increased the activity of the ATPase in a dose and time dependent manner, with a highest effect observed at 15 minutes and a dose of 80 nM. The protein expression was also increased. The stimulation of the Na+/K+ ATPase disappeared completely in presence of JTE-013, a specific blocker of S1PR2, as well as in presence of Y-27632, a Rho kinase inhibitor, BAPTA-AM, a Ca2+ chelator, wortmannin, a PI3K inhibitor, carboxy-PTIO, a scavenger for nitric oxide (NO), and KT 5823, a PKG inhibitor. CYM 5520, a S1PR2 agonist mimicked the effect of FTY720P. FTY720P increased the expression of p-Akt, a direct effector of PI3K, however, this increase disappeared when Rho kinase was inhibited, revealing that Rho kinase acts upstream PI3K. Glyco-SNAP-1, a NO donor, activated the pump in both presence and absence of wortmannin, indicating that PI3K is upstream NO. Interestingly, glyco-SNAP-1 and 8-bromo-cGMP, a PKG activator, exerted no effect on the Na+/K+ ATPase in absence of free Ca2+ revealing that the NO mediated effect is calcium-dependent. The involvement of calcium was further confirmed by the translocation of NFAT to the nucleus. The presence of verapamil or extracellular EGTA abolished the stimulatory effect of FTY720P, indicating that the source of calcium is extracellular. The results suggest that FTY720P activates sequentially S1PR2, Rho kinase, PI3K, leading to NO release and PKG stimulation. The latter phosphorylates calcium channels in the cell membrane, leading to calcium influx, and translocation of the ATPase units to the membrane.

Rho-associated protein kinase-dependent moesin phosphorylation is required for PD-L1 stabilization in breast cancer.

Expression of programmed cell death ligand (PD-L1) is associated with poor prognosis in breast cancer. Understanding the regulation of PD-L1 expression in breast cancer could provide a new strategy for breast cancer treatment. Here, we demonstrate that moesin (MSN) phosphorylation by Rho-associated protein kinase (ROCK) stabilizes PD-L1 protein levels. Our results indicate that phosphorylated MSN may compete with the E3 ubiquitin ligase SPOP for binding PD-L1. ROCK inhibition via the Y-27632 inhibitor or MSN silencing decreased PD-L1 expression, resulting in T-cell activation both invitro and invivo. Administration of Y-27632 into immunocompetent Balb/c mice bearing breast tumors suppressed tumor progression and enhanced CD4+ and CD8+ T-cell infiltration. RNA-seq analysis of Y-27632-treated mouse tumors revealed that ROCK inhibition upregulated several immune response genes. However, the combination of Y-27632 and an anti-PD-1 antibody did not show additive or synergistic effects due to reduced PD-L1 in the presence of Y-27632. Our study unravels a previously unappreciated mechanism of PD-L1 regulation through the ROCK-MSN pathway. Moreover, we found that ROCK inhibitors could be combined with breast cancer immunotherapy.

Conditional cell reprogramming for modeling host-virus interactions and human viral diseases.

Conventional cancer and transformed cell lines are widely used in cancer biology and other fields within biology. These cells usually have abnormalities from the original tumor itself, but may also develop abnormalities due to genetic manipulation, or genetic and epigenetic changes during long‐term passages. Primary cultures may maintain lineage functions as the original tissue types, yet they have a very limited life span or population doubling time because of the nature of cellular senescence. Primary cultures usually have very low yields, and the high variability from any original tissue specimens, largely limiting their applications in research. Animal models are often used for studies of virus infections, disease modeling, development of antiviral drugs, and vaccines. Human viruses often need a series of passages in vivo to adapt to the host environment because of variable receptors on the cell surface and may have intracellular restrictions from the cell types or host species. Here, we describe a long‐term cell culture system, conditionally reprogrammed cells (CRCs), and its applications in modeling human viral diseases and drug discovery. Using feeder layer coculture in presence of Y‐27632 (conditional reprogramming, CR), CRCs can be obtained and rapidly propagated from surgical specimens, core or needle biopsies, and other minimally invasive or noninvasive specimens, for example, nasal cavity brushing. CRCs preserve their lineage functions and provide biologically relevant and physiological conditions, which are suitable for studies of viral entry and replication, innate immune responses of host cells, and discovery of antiviral drugs. In this review, we summarize the applications of CR technology in modeling host‐virus interactions and human viral diseases including severe acute respiratory syndrome coronavirus‐2 and coronavirus disease‐2019, and antiviral discovery.

A Rho-Associated Coiled-Coil Containing Kinase Inhibitor, Y-27632, Improves Viability of Dissociated Single Cells, Efficiency of Colony Formation, and Cryopreservation in Porcine Pluripotent Stem Cells.

The establishment of porcine epiblast stem cells (pEpiSCs) and induced pluripotent stem cells (piPSCs) derived from diametrical derivations is of great importance in developing biomedical models. However, pEpiSCs and piPSCs have been technically much harder to culture than mouse embryonic stem cells, showing problematic properties such as spontaneous differentiation and apoptosis after cryopreservation. Therefore, we demonstrated that Y-27632 as a Rho-associated coiled-coil containing kinase inhibitor could prevent dissociated pEpiSCs and piPSCs from undesirable differentiation and apoptosis in cryopreservation protocols. pEpiSC 2, 8 lines, Sendai virus-induced pluripotent stem cells (Sev-iPSCs), and lentivirus-induced pluripotent stem cells were cultured with 10 μM Y-27632 before collecting dissociated cells retrieved from colonies using various enzymes. Dissociated single cells were transferred into freezing mediums (open pulled straw vitrification, STEM-CELLBANKER® (SCB), 10% dimethylsulfoxide in serum) for cryopreservation. The rates of viability and colony formation obtained from dissociated porcine stem cells after freezing/thawing were examined in the presence of Y-27632. The characteristics of pluripotency and in vitro differentiation were also examined in these stem cells treated with Y-27632 after cryopreservation. As a result, the viability and efficiency of colony formation of dissociated pEpiSCs (2, 8 lines) and Sev-iPSCs treated with 10 μM Y-27632 using the SCB cryopreservation protocol were significantly increased when compared with those of nontreated Y-27632 (p < 0.05). Pluripotency genes (OCT-3/4, NANOG, and SOX2) were positively expressed in Y-27632-treated porcine pluripotent stem cells. Also, in vitro differentiation of these stem cells was successfully induced in the presence of 10 μM Y-27632. These results indicated that treatment of Y-27632 for single-cell dissociation and the SCB cryopreservation protocol could facilitate handling porcine pluripotent stem cells and provide the widespread use of these stem cells.

A novel technique for in situ uniaxial tests of self-assembled soft biomaterials.

We introduce a novel method to form 3D biomimetic tissues from a droplet of a cell-extracellular matrix (ECM) mixture on a sensor stage and to quantify tissue force and stiffness as a function of time under optical microscopes. This method exploits advances in micro-nano fabrication and capillarity for self-assembly and self-alignment of tissues on the stage. It allows simultaneous investigation of the microstructure of the tissue in situ while its mechanical response is quantified, thus linking tissue biophysics with physiology and revealing structural-functional properties of 3D tissues. We demonstrate the functionality of the stage by studying the mechanical behavior of different cell-collagen mixtures under mechanical, chemical and electrical stimulation. This includes force evolution in cell-free collagen during curing, myotubes differentiated from muscle cell-collagen/Matrigel ECM subjected to electrical stimulation, and fibroblast-collagen tissue subjected to cancer cell conditioned media (CM) and a Rho-kinase inhibitor, Y27632. Muscle contraction decreases with increasing frequency of electrical stimulation, and fibroblasts respond to CM by increasing contractility for a short time and completely relax in the presence of Y27632 but restore force with Y27632 washout.

Ex vivo induction of regulatory T cells from conventional CD4+ T cells is sensitive to substrate rigidity.

The immune system maintains a balance between protection and tolerance. Regulatory T cells (Tregs) function as a vital tolerance mechanism in the immune system to suppress effector immune cells. Additionally, Tregs can be utilized as a form of immunotherapy for autoimmune disorders. As T cells have previously been shown to exhibit sensitivity to the rigidity of an activating substrate upon activation via IL-2 secretion, we herein explore the previously unknown effect of substrate rigidity on the induction of Tregs from conventional naïve mouse CD4+ T cells. Substrates with modulatable rigidities ranging from a hundred kilopascals to a few megapascals were fabricated via poly(dimethylsiloxane). We found that there was a significant increase in Treg induction at lower substrate rigidities (i.e., E ~ 100 kPa) compared to higher rigidity levels (i.e., E ~ 3 MPa). To confirm that this significant difference in induction rate was truly related to T-cell mechanosensing, we administered compound Y-27632 to inhibit myosin contractility. In the presence of Y-27632, the myosin-based contractility was disrupted and, as a result, the difference in Treg induction caused by the substrate rigidity was abrogated. This study demonstrates that mechanosensing is involved in Treg induction and raises questions about the underlying molecular mechanisms involved in this process. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3001-3008, 2018.

Calcium Channels, Rho-Kinase, Protein Kinase-C, and Phospholipase-C Pathways Mediate Mercury Chloride-Induced Myometrial Contractions in Rats.

Adverse effects of mercury on female reproduction are reported; however, its effect on myogenic activity of uterus and mechanism thereof is obscure. Present study was undertaken to unravel the mechanistic pathways of mercuric chloride (HgCl2)-induced myometrial contraction in rats. Isometric tension in myometrial strips of rats following in vitro exposure to HgCl2 was recorded using data acquisition system-based physiograph. HgCl2 produced concentration-dependent (10nM-100μM) uterotonic effect which was significantly (p < 0.05) reduced in Ca2+-free solution and inhibited in the presence of nifedipine (1μM), a L-type Ca2+ channel blocker, thus suggesting the importance of extracellular Ca2+ and its entry through L-type calcium channels in HgCl2-induced myometrial contractions in rats. Cumulative concentration-response curve of HgCl2 was significantly (p < 0.05) shifted towards right in the presence of Y-27632 (10μM), a Rho-kinase inhibitor, suggesting the involvement of Ca2+-sensitization pathway in mediating HgCl2-induced myometrial contraction. HgCl2-induced myometrial contraction was also significantly (p < 0.05) inhibited in the presence of methoctramine or para-fluoro-hexahydro-siladifenidol, a selective M2 and M3 receptor antagonists, respectively, which evidently suggest that mercury also interacts with M2 and M3 muscarinic receptors to produce myometrial contractions. U-73122 and GF-109203X, the respective inhibitors of PLC and PKC-dependent pathways, downstream to the receptor activation, also significantly (p < 0.05) attenuated the uterotonic effect of HgCl2 on rat uterus. Taken together, present study evidently reveals that HgCl2 interacts with muscarinic receptors and activates calcium signaling cascades involving calcium channels, Rho-kinase, protein kinase-C, and phospholipase-C pathways to exert uterotonic effect in rats. Graphical Abstract Graphical abstract depicting the mechanism of mercury-induced myometrial contraction in rats. M receptor: Muscarinic receptor; PIP2: phospho-inositol bisphosphate; PLC: phospholipase-C; DAG: diacyl glycerol; IP3: inositol triphosphate; IP3R: inositol triphosphate receptor; PKC; protein kinase-C; MLCP: myosin light chain phosphatise; MYPT: myosin phosphatase; SR: sarco-endoplasmic reticulum.

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.

Functional involvement of protein kinase C, Rho-kinase and TRPC3 decreases while PLC increases with advancement of pregnancy in mediating oxytocin-induced myometrial contractions in water buffaloes (Bubalus bubalis)

Present study unravels the involvement of different calcium signaling pathways in oxytocin-induced contractions in myometrium of non-pregnant and pregnant buffaloes during early and mid-pregnancy stages. Uteri of pregnant animals were more sensitive than of non-pregnant buffaloes. Phasic contractions and frequency of contraction significantly increased with advancement of pregnancy, while tonic contractions non-significantly and amplitude significantly decreased from six months pregnancy onward. Oxytocin produced concentration-dependent-contraction on isolated myometrial strips of pregnant and non-pregnant buffaloes and the dose response curves (DRCs) of oxytocin were significantly (P < 0.05) shifted to right in the presence of nifedipine (1 μM), in Ca2+-free Ringer Locke solution (RLS), ruthenium red (30 μM), ruthenium red + nifedipine, cyclopiazonic acid (CPA; Ca2+ free RLS as well as RLS), CPA (10 μM)+nifedipine, U-73122 (1 μM) + nifedipine and SKF96365 (25 μM) on uteri of non-pregnant and pregnant (early and mid) animals. The DRCs were also significantly shifted towards right in the presence of Y-27632 (10 μM), GF109203X (5 μM) and Pyr3 (10 μM) on uteri of non-pregnant and early pregnancy stage buffaloes while only in the presence of U-73122 (1 μM) on uteri of mid-pregnancy stage buffaloes. Our finding suggest that and L-type Ca2+ channels, IP3-RyR-gated, and store-operated calcium channels including transient receptor potential channel (TRPC) pathways play significant role in mediating oxytocin-induced contractions in myometrium of pregnant and non-pregnant buffaloes. SERCA plays major role only during early-pregnancy while functional role of protein kinase C (PKC), Rho-kinase and TRPC3 pathways decreased and role of G-protein coupled receptor-phospholipase C (GPCR-PLC) pathway increased with advancement of pregnancy.

Latrunculin B modulates electrophysiological characteristics and arrhythmogenesis in pulmonary vein cardiomyocytes.

AF (atrial fibrillation) is the most common sustained arrhythmia, and the PVs (pulmonary veins) play a critical role in triggering AF. Stretch causes structural remodelling, including cytoskeleton rearrangement, which may play a role in the genesis of AF. Lat-B (latrunculin B), an inhibitor of actin polymerization, is involved in Ca(2+) regulation. However, it is unclear whether Lat-B directly modulates the electrophysiological characteristics and Ca(2+) homoeostasis of the PVs. Conventional microelectrodes, whole-cell patch-clamp, and the fluo-3 fluorimetric ratio technique were used to record ionic currents and intracellular Ca(2+) within isolated rabbit PV preparations, or within isolated single PV cardiomyocytes, before and after administration of Lat-B (100 nM). Langendorff-perfused rabbit hearts were exposed to acute and continuous atrial stretch, and we studied PV electrical activity. Lat-B (100 nM) decreased the spontaneous electrical activity by 16±4% in PV preparations. Lat-B (100 nM) decreased the late Na(+) current, L-type Ca(2+) current, Na(+)/Ca(2+) exchanger current, and stretch-activated BKCa current, but did not affect the Na(+) current in PV cardiomyocytes. Lat-B reduced the transient outward K(+) current and ultra-rapid delayed rectifier K(+) current, but increased the delayed rectifier K(+) current in isolated PV cardiomyocytes. In addition, Lat-B (100 nM) decreased intracellular Ca(2+) transient and sarcoplasmic reticulum Ca(2+) content in PV cardiomyocytes. Moreover, Lat-B attenuated stretch-induced increased spontaneous electrical activity and trigger activity. The effects of Lat-B on the PV spontaneous electrical activity were attenuated in the presence of Y-27632 [10 μM, a ROCK (Rho-associated kinase) inhibitor] and cytochalasin D (10 μM, an actin polymerization inhibitor). In conclusion, Lat-B regulates PV electrophysiological characteristics and attenuates stretch-induced arrhythmogenesis.

Short-duration increases in intraluminal pressure improve vasoconstrictor responses in aged skeletal muscle feed arteries.

We tested the hypothesis that exposure to a short-duration (1h) increase in intraluminal pressure, to mimic pressure associated with a bout of exercise, would attenuate age-induced impairments of vascular smooth muscle (VSM) constrictor responses in soleus muscle feed arteries (SFA) via the Rho pathway. SFA from young (4months) and old (24months) Fischer 344 rats were cannulated and pressurized to 90 or 130cmH2O for 1h. Following the 1-h treatment, pressure in P130 arteries was lowered to 90cmH2O for examination of vasoconstrictor responses to norepinephrine (NE), angiotensin II (Ang II), and phenylephrine (PE). To assess the role of the Rho pathway, vasoconstrictor responses were assessed in the absence or presence of a RhoA-kinase inhibitor (Y27632) or RhoA-kinase activator (LPA). Vasoconstrictor responses to NE, Ang II, and PE were impaired in old P90 SFA. Pretreatment of old SFA with increased pressure improved vasoconstrictor responses to NE, PE and Ang II. The beneficial effect of the pressure pretreatment in old SFA was eliminated in the presence of Y27632. In the presence of LPA, vasoconstrictor responses to Ang II were improved in old SFA such that responses were not different than young P90 SFA. These results indicate that a short-duration exposure to increased intraluminal pressure, to mimic pressure associated with a bout of exercise, attenuates or reverses the age-related decrement in VSM constrictor responses in SFA and that the beneficial response is mediated through Rho kinase.

Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.

Chronic ouabain treatment induces Rho kinase activation.

Ouabain is an endogenous Na(+)/K(+)-ATPase inhibitor whose chronic administration induces hypertension. Endogenous ouabain levels increase in human essential hypertension. On the other hand, Rho/Rho kinase (ROCK) pathway has been implicated in various animal models of hypertension. In the current work, we evaluated the possible involvement of Rho kinase in ouabain-induced hypertension. Ouabain was administered daily (20 µg/kg, i.p.) to Wistar rats for 6 weeks. After the ouabain treatment, we evaluated the possible changes in vascular responses to KCl and phenylephrine alone and in the presence of Rho kinase inhibitor Y27632. We also determined the expressions of ROCKs, Rho A and phosphorylation of myosin binding subunit of myosin light chain phosphatase (pMYPT) and activation of Rho A. Agonist-induced contractions in the presence of Y27632 are significantly decreased and Y27632-induced relaxations in aortas precontracted with phenylephrine are significantly enhanced with the chronic treatment of ouabain. Although the expressions of ROCK I and ROCK II remained unchanged, pMYPT expression was significantly increased in ouabain-treated group. Moreover, Rho A expression and activation were decreased after treatment with ouabain. Although Rho kinase expression did not change in aortas, increased basal Rho kinase activation may contribute to the development of ouabain-induced hypertension. Our current data present the first evidence that Rho kinase is involved in the development of ouabain-induced hypertension in rats.

The Rho kinase inhibitor Y-27632 facilitates the differentiation of bone marrow mesenchymal stem cells.

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell-based regenerative therapies, but technical challenges have limited cell yield and thus the success of these potential treatments. The Rho GTPases and downstream Rho kinases (Rho coiled-coil kinases or ROCKs) are central regulators of cytoskeletal dynamics during the cell cycle and thus help determine the balance between stem cells self-renewal, lineage commitment, and apoptosis. Here, we examined if suppression of ROCK signaling enhances the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) differentiation into neurons and neuroglial cells. BMSCs were cultured in epidermal growth factor (EGF, 10µg/l) and basic fibroblastic growth factor (bFGF, 10µg/l) in the presence or absence of the Rho kinase inhibitor Y-27632 (10µM). The expression levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence and Western blotting. The average number of NSE-positive cells increased from 83.20±8.677 (positive ratio 0.2140±0.0119) to 109.20±8.430 (positive ratio 0.3193±0.0161) per visual field in the presence of Y-27632, while GFAP-positive cell number increased from 96.30±8.486 (positive ratio 0.18±0.0152) to 107.50±8.683 (positive ratio 0.27±0.0115) (P<0.05 for both). Both NSE and GFAP protein expression levels were enhanced significantly by Y-27632 treatment (NSE: 0.74±0.05 vs. 1.03±0.06; GFAP: 0.64±0.08 vs. 0.97±0.05, both P<0.01) as indicated by Western blots. The Rho kinase inhibitor Y-27632 concomitant with EGF and bFGF stimulation promotes BMSC differentiation into neural cells. Control of Rho kinase activity may enhance the efficiency of stem cell-based treatments for neurodegenerative diseases.

Effective Rho-associated protein kinase inhibitor treatment to dissociate human iPS cells for suspension culture to form embryoid body-like cell aggregates

Treatment conditions using Y-27632 in the preparation of cell suspension of dissociated human pluripotent stem cells (hiPSCs) were investigated in the context of embryoid body (EB)-like cell aggregates. The effectiveness of a pretreatment with Y-27632 before cell dissociation and that of a Y-27632 treatment during cell dissociation were investigated from the viewpoint of simplicity and robustness. The duration of Y-27632 treatment in the preparation process affected the circularity and agglomeration of dissociated hiPSCs. A single application of pretreatment failed to prevent the onset of blebbing. However, a pretreatment promoted the agglomeration of dissociated hiPSCs when combined with the addition of Y-27632 to cell suspension. Our results indicate that pretreatment enhances the agglomeration potential of dissociated hiPSCs. When cell dissociation was performed in the presence of Y-27632, dissociated hiPSCs possessed the highest circularity and significant agglomerating property. It was shown that treatment with Y-27632 during cell dissociation is a simple and robust method to prepare dissociated hiPSCs for suspension culture to form EB-like cell aggregates.

Effect of the ROCK inhibitor Y-27632 on Proliferation and Differentiation of Equine Bronchial Epithelial Cells

The airway epithelium plays an important role in physiological and pathological processes including inflammation, innate immunity and regenerative responses in the respiratory tract with regard to airway diseases such as human asthma, COPD and Equine Recurrent Airway Obstruction (RAO). Airway epithelial cell lines which would serve as an in vitro cell model are, indeed, missing for the horse. The objective of the study is to establish long-term equine bronchial epithelial cell cultures using the ROCK inhibitor Y-27632 and characterize them. Fresh isolated EBEC were cultured in the presence and absence of 10µM Y-27632 under conventional and air-liquid-interface culture conditions. Cell viability, morphology, proliferation and differentiation states were examined. Under conventional culture, Y-27632 induced higher growth rate of primary EBEC over a short period of time when compared to non-treated cells and increased the passage number. Epithelial cells grew faster and retain their epithelial cell morphology for a number of passages, with less contamination with fibroblasts. EBEC cultured on membrane inserts revealed high TEER values in the presence of Y-27632 and switching to Air-Liquid-Interface (ALI) did not cause decrement of TEER values. Inmunostaing of EBEC showed a different pattern of CK, TJP-1 and Vim expression indicating rapid differentiation of Y-27632-treated cells. Also, light and scanning electron microscopic imaging showed high amount of cilia, microvilli and ridges as well as more defined polygonal-shaped epithelial cells in the presence of Y-27632. Taken together, these results suggest that the ROCK inhibitor Y-27632 permits establishing conditional long-term in vitro airway epithelial cell models to investigate their role in equine airway diseases and as pharmacological targets.

Establishment and serial passage of cell cultures derived from LuCaP xenografts

LuCaP serially transplantable xenografts derived from primary and metastatic human prostate cancer encompass the molecular and cellular heterogeneity of the disease and are an invaluable resource for in vivo preclinical studies. A limitation of this model, however, has been the inability to establish and passage cell cultures derived from the xenografts. Here, we describe a novel spheroid culture system that supports long-term growth of LuCaP cells in vitro. Xenografts were minced and digested with collagenase. Tissue dissociation was terminated while the majority of cells remained as clusters rather than single cells. The cell clusters were suspended in StemPro medium supplemented with R1881 and Y-27632, a Rho kinase inhibitor, and placed in ultralow attachment dishes for spheroid culture. Serial passage was achieved by partial digestion to small clusters with trypsin/EDTA in the presence of Y-27632. Cell viability, growth and phenotype were monitored with LIVE/DEAD®, MTS, qRT-PCR, and immunocytochemical assays. Cells from six LuCaP xenografts formed proliferating spheroids that were serially passaged a minimum of three times and cryopreserved. Two of the cell lines, LuCaP 136 and LuCaP 147, were further passaged and characterized. Both expressed biomarkers characteristic of the xenografts of origin, were determined to be of independent origin by STR fingerprinting, and were free of mycoplasma. LuCaP 147 formed tumors similar to the original xenograft when injected into mice. The ability to culture LuCaP cells affords new opportunities for fast, cheap, and efficient preclinical studies and extends the value of the LuCaP xenograft models.