Most studies on the role of Rho-associated kinases on keratinocyte proliferation have used Y-27632 to inhibit ROCK [1,6,14]. Y-27632 is a potent inhibitor of ROCK 1 and 2, but it also has some inhibitory effects on PKC, cAMP-dependent protein kinase and myosin light-chain kinase . Therefore, we tested additional ROCK in- hibitors to determine whether they could also promote keratinocyte proliferation. Fasudil hydrochloride (HA- 1077) is a well-known vasodilator  that inhibits ROCK 1 and 2 as well as cAMP-dependent protein kin- ase. HA1000 hydrochloride is a hydroxylated metabolite of HA-1077 and is 100 times more selective for ROCK 1 and 2 than for other kinases. Finally, GSK 429286 is a potent ROCK1/2 inhibitor that is active at much lower concentrations (IC50 14 nM) . In parallel, cells were treated with 10 μM Y-27632, 20 μM fasudil hydroch- loride, 20 μM HA1000 hydrochloride, or 100 nM GSK 429286. Cells were passed for the times and population doublings shown in Figure 3, by which time it was clear that treatment with all four ROCK inhibitors had stimu- lated the keratinocytes to bypass senescence and in each case the resulting cells were still highly proliferative.
on cardiomyocyte apoptosis and myocar- dial infarct size following Rho-kinase acti- vation. The effects of inhibition of ERK- MAPK could be rescued by inhibition of Rho- kinase in vivo. Inhibition of Rho- kinase reversed cardiomyocyte apoptosis and the size of infarcts caused by inhibi- tion of ERK-MAPK in IPC. The combina- tion of PD98059 and fasudil restored protection, as reflected by decreased car- diomyocyte apoptosis (22%) and infarct size (12%) (P < 0.05 versus the IPC + PD98059 group). The infarct size did not decrease as significantly as cardiomyocyte apoptosis. These data show that cardio- protection of Rho-kinaseinhibition is me- diated by antiapoptosis more than de- creased infarct size after inhibition of ERK-MAPK. These results indicate that ERK-MAPK signaling is required in IPC to oppose the effect of Rho-kinase signal- ing on cell apoptosis.
The extent of functional recovery after stroke differs enormously amongst patients and is dictated by the concordant concerted activities of a variety of processes like neurogenesis and angiogenesis. Rho-kinaseinhibition has previously been implicated in neurogenesis under in vitro conditions and proven to be effective and safe in clinical settings even after an extended period of treatment. Indeed, a placebo-controlled clinical trial performed with patients who were able to receive the first injection of fasudil (60 mg, intravenous) within 48h of acute ischemic stroke onset and twice daily for 14 days thereafter exhibited radically improved neurological and clinical outcomes at 2 weeks after the initiation of treatment and at 1 month after the onset of symptoms, respectively (Shibuya et al., 2013; Ding et al., 2010). Intriguingly, a recent translational study has shown that inhibition of Rho-kinase by fasudil or Y-27632 can improve the functional recovery even after irreversible formation of brain lesions following photothrombotic stroke. Like the abovementioned trial, the animals here were also given an extended therapy, namely they received the first dose of Y-27632 or fasudil (both 30 mg) via oral gavage 3 days after the induction of stroke and then twice daily for 4 weeks (Lemmens et al., 2013). The present study unravels the remarkable therapeutic capacity of targeting Rho-kinase during acute phase of an ischemic stroke. Considering the eligibility of a selective subset of patients for thrombolysis and seminal role of Rho-kinase in evoking hemorrhagic transformation, these findings may be of considerable therapeutic importance (Ishiguro et al., 2012).
Pharmacological inhibition of Notch activation with GSI decreased expression of BiP and Rhokinase, confirming that Notch3 is an upstream regulator. The functional significance of these systems was further explored using 4-PBA and fasudil, which corrected many of the abnormal molecular changes in CADASIL VSMCs, including Notch3 downstream target genes, Rhokinase activity, and ER stress markers. In addi- tion, in CADASIL VSMCs, 4-PBA and fasudil normalized increased phosphorylation of cytoskeleton-as- sociated proteins, vinculin and cofilin, which are structural proteins that influence cytoskeletal organiza- tion. These inhibitors had no effect on VSMC proliferation or apoptosis but ameliorated the cytoskeletal remodeling defects in CADASIL, indicating that ER stress– and Rhokinase–mediated functional respons- es are not generalized phenomena, but are highly specific. Of significance, 4-PBA reduced Rhokinase activity, while fasudil decreased BiP expression, indicating crosstalk between ER stress and Rhokinase in the context of increased Notch3 activity in CADASIL. Interactions between these pathways have been reported in other systems, as evidenced by (i) reduction in renal Notch signaling in response to Rhokinaseinhibition (52), (ii) Notch3-induced activation of Rhokinase in pluripotent stem cells (51), (iii) decreased vascular Rho A signaling in Notch3-deficient mice (33), (iv) amelioration of ER stress by Rhokinase inhib- itors (30), and (v) Notch3 association with ER stress (32).
As main results, sympathetic tone (with or without vagal blockade) was found to be increased, whereas baroreflex sensitivity of heart rate was depressed in models of re- novascular hypertension, irrespective of residual renal mass. Differential results relate to parasympathetic tone (with or without beta 1-adrenergic blockade) that was depressed in the 2K1C model only. In addition, left ven- tricular hypertrophy was present in experimental reno- vascular hypertension with reduced renal mass (1K1C) only. Renal norepinephrine excretion was reduced in the 1K1C model exclusively. Hypothetically, renal reduced catecholamine excretion and/or impaired renal catechol- amine degradation may be considered as mechanisms of sympathoactivation in the 1K1C model. Finally, Rho- kinaseinhibition improved baroreflex function solely in experimental renovascular hypertension with reduced renal mass (1K1C), whereas AT1 blockade improved
To confirm that cRO can fully phenocopy the expected morphogenetic effects of Rhokinaseinhibition, unilateral gut defects were analyzed at the tissue and cellular level. We found previously (Reed et al., 2009) that global exposure to RO leads to aberrant cell rearrangements and abnormal epithelial morphogenesis in the shortened gut tube (compare Fig. 3A-D with 3E-H). As expected, the irradiated, decaged side of cRO- exposed guts exhibited similar defects in cellular architecture, including failure to form a single-layer epithelium and to apically localize -catenin (Fig. 3I-L, right side epithelia), comparable to the defects induced globally by RO (Reed et al., 2009) (Fig. 3E-H). By contrast, the non-irradiated side of cRO- exposed guts had completely normal epithelial architecture (e.g. compare left side epithelium in Fig. 3J-L with 3B-D). The irradiated gut cells also failed to undergo the normal tissue- elongating rearrangements observed in control embryos (supplementary material Fig. S5). Thus, cRO phenocopies RO at the organ, tissue and cellular level in a light-dependent and spatially localized manner.
Our results clearly demonstrate that deletion of TRPV4 (TRPV4KO EC) results in increased Rho activation and angiogenesis in vitro, ex vivo, and in vivo. Further, our in vitro results showed that inhibition of the Rho pathway with Y-27632 normalized abnormal EC function and angiogenesis. Based on these findings, we hypothesized that Y-27632 treatment may reduce tumor growth in TRPV4KO mice. To accomplish this, we injected Lewis Lung Carcinoma (LLC) cells into TRPV4KO mice. Once the tumors became palpable (after ~7 days), we injected Y-27632 (i.p., 10 mg/kg) every day for 14 days. Saline injected mice served as controls. Additionally, to examine if Rhokinaseinhibition normalizes tumor vasculature and improves cancer therapy, we treated mice with anti-cancer drug, Cisplatin (i.p., 3 mg/kg), once per week, alone and in combination with Y-27632. When used in combination with Y-27632, Cisplatin was administered 2–4 days after Y-27632 treatment. Overall, the TRPV4KO mice were divided into four groups: 1) Control 2) Y-27632 3) Cisplatin and 4) Y-27632 + Cisplatin. Tumor growth and angiogenesis was monitored as previously described . We found that time dependent tumor growth in the control animals reached around 2000 mm 3 at 21 days,
pharmacological target for clinical application. 15 Studies have demonstrated that inhibition of tonic contraction of corporal smooth muscle by intracavernosal injection or topi- cal application of Rho-kinase inhibitors to the penis results in increased blood ﬂ ow into erectile tissue and causing an erection. 31 Rho-kinaseinhibition restored erectile responses and dynamic infusion cavernosometry parameters by alle- viating increased apoptosis, decreased immune histochem- ical staining of α -SMA and increased caspase-3 activity in a NO-independent manner. Moreover, histological and mole- cular dysregulation were alleviated by Rho-kinaseinhibition, 14,19,41,42 which might improve its ef ﬁ cacy as com- pared with the currently available anti-ED drugs. However, Table 5 Traditional Plants Used as Rho Kinases (ROCK) Inhibitors as a Therapeutic Agent for the Treatment of Erectile Dysfunction
fibers (data not shown). In addition, SB431542 rescued total des- mosomal protein levels in cKO cells by immunoblotting (Figure 7E) and membranous desmosomal protein levels in cKO cells by immu- nofluorescence (Figure 7F) and surface biotinylation assays (Figure 8A). Inhibition of ALK5 was also sufficient to rescue expression of biochemical differentiation markers (Figure 7E) in keratinocytes with mTORC1 loss of function. Dispase assays on cKO keratino- cyte monolayers demonstrated functional rescue of cell adhesion strength upon agitation with ALK5 inhibition (Figure 8B). Finally, to verify the specificity of the effects of ALK5 pharmacologic inhi- bition, we treated cells with ALK5 si RNA. Surprisingly, even rela- tively modest ALK5 knockdown by siRNA reduced p-MLC2 levels in keratinocytes with mTORC1 loss of function (Figure 8C and Supplemental Figure 5, A and B), as well as actin stress fiber forma- tion (Figure 8D and Supplemental Figure 5D). ALK5 knockdown in cKO cells was sufficient to rescue cell-cell adhesion as evidenced by total (Supplemental Figure 5, A and B) and membranous (Fig- ure 8D and Supplemental Figure 5D) desmosomal protein levels (DSP1/2, DSG1, DSG3, PKP3), and some measures of biochemical differentiation (loricrin, TGM1) were also rescued (Supplemental Figure 5A). Interestingly, similar effects were seen in WT cells with ALK5 inhibition (Supplemental Figure 4I) and ALK5 knockdown (Supplemental Figure 5C), suggesting a role for basal ALK5 signal- ing in the regulation of adhesion and differentiation. Cumulatively, these data support a model in which mTORC1 silencing leads to increased TGF-β receptor levels and signaling, which in turn medi- ates ROCK hyperactivity in keratinocytes. Increased cytoskeletal tension downstream of ROCK results in decreased desmosomal gene expression and decreased cell-cell adhesion, which con- tributes to impaired biochemical differentiation in the epidermis (Supplemental Figure 6).
Rhokinase is one of the best-characterized effectors of small GTPase RhoA and plays a crucial role in various cellular func- tions, such as smooth muscle contraction, cell migration, and cell proliferation. Rhokinase in cardiovascular diseases has re- ceived extensive attention and been studied in animal experi- mental models and human. Rhokinase plays an important role in inflammatory responses and arteriosclerotic lesions in hu- mans and animals; 26-28 multiple studies have demonstrated that
The presence of maternal citron transcripts and the late embryonic PNS phenotype suggested that maternally-derived Citron permits early proliferation, but is lost later in embryogenesis. After embryogenesis there is little proliferation in the nervous system until the CNS begins to divide rapidly at the end of the second larval instar (Truman et al., 1993), by which stage the zygotic mutant phenotype should be evident. citron mutant larval brains contained massively enlarged nuclei (Fig. 5A,B), DNA stains showing large diffuse aggregates of apparently hyperploid cells. In addition, dissociated mutant larval brain cells from any allelic combination were binucleate at a far higher rate (26/103) than normal (1/100; Fig. 5C). Chromosome preparations from mutant brains (Fig. 5D-F) showed tetraploid metaphase figures (Fig. 5D), confirming that citron mutant brain cells can complete S phase and enter mitosis following a previously failed mitosis or cytokinesis. We also observed hyperploid cells undergoing anaphase (Fig. 5E), suggesting that a failure of chromosome disjunction was not the primary defect. Furthermore, the frequency of diploid anaphases and telophases was similar in all citron allelic combinations (approximately 37% of mitotic cells) and did not significantly differ from wild-type control (39%). Consistent with this, immunohistochemical analysis showed that centrosomes separate normally in the polyploid cells, since we observed an assembly of microtubule spindles apparently emanating from multiple centrosomes (Fig. 5G-G′′,H-H′′). Some mutant brain cells were massively hyperploid (Fig. 5F,H ′ ) showing that in the absence of Citron, brain cells can undergo multiple mitotic cycles without dividing. However, with increasing cell ploidy, microtubules form abnormal spindles (Fig. 5G,H) and chromosomes fail to assemble at the metaphase equator (Fig. Fig. 3. Subcellular localisation of Citron-GFP to the contractile ring during cytokinesis depends on the normal activation of Rho signalling. (A-H) Citron-GFP was expressed in the 3-6 hour embryonic epidermis using paired-Gal4. DNA is stained with Hoechst 33258 (B,F), Citron-GFP is stained with anti-GFP (C,G), and microtubules are stained with anti-α-tubulin antibodies (D,H). (A,E) Merged images with Citron-GFP stained green, DNA stained blue and microtubules stained red. (A-D) A wild-type embryo showing Citron-GFP in the contractile ring (arrow) as it constricts around the central spindle microtubules. Citron-GFP is not localised in the adjacent anaphase cell that is yet to constrict (arrowhead). (E-G) An embryo mutant for the Rho activator pebble showing typical tetranucleate and binucleate cells and bipolar and tripolar spindles of cells failing cytokinesis. Citron-GFP shows no localisation to the contractile ring in pebble mutant telophase cells. It is found diffusely through the cytoplasm of telophase cells and not at the positions where contractile rings would normally form
Interestingly and importantly, our current study also preliminarily suggested that in the setting of hyperlipid- emia, 50 mg/Kg body weight per day of metformin ad- ministration was beneficial for NO production increment and CRP level reduction which was consistent to previous studies [14,22,23]. Nevertheless, the mechanisms associ- ated with these benefits are still incompletely clear. In light of our current study, we speculated that the benefits derived from metformin therapy of rats with hyperlipid- emia might be partially associated with its effects on inhi- biting Rhokinase activity. Nevertheless, since metformin had no effects on cholesterol biosynthesis as revealed in our study, inhibiting Rhokinase activity of metformin might not be related to its inhibition of isoprenylation of small GTP-binding proteins. Rather, on the basis of previ- ous studies [14,24,25], we considered that some alternative mechanisms might be associated with diminishment of Rhokinase activity with metformin therapy. First of all, basic researches suggest that cardio-protective effects of metformin are partially mediated by AMP-activated protein kinase (AMPK) activation which is responsible for eNOS up-regulation and NO production increase [14,26]. Accordingly [27,28], Rho kinas activated is partially dependent on reactive oxidative species, and up-regulation of eNOS expression and NO production ameliorate oxidative stress therefore inhibiting Rhokinase activation. Secondly, since high blood glucose is a potential stimulus for Rhokinase activation, and Rho isoprenylation and Rhokinase over-activation contrib- ute to insulin resistance which reciprocally induces high blood glucose [25,29], therefore we considered that it was possible that metformin declined Rho kin- ase activity by means of improving glucose metabol- ism. In our current study, we observed that rats with high-fat and high-cholesterol diet administration for 6 weeks, fasting blood glucose was somewhat increased in the hyperlipidemia groups than that of the sham group, indicating that hyperlipidemia per se might po- tentially compromise glucose metabolism. Neverthe- less, with 4 weeks of metformin therapy, fasting blood glucose levels in the metformin and combined groups were reduced in comparison to the control and atorva- statin groups, suggesting that metformin therapy was favorable for improving glucose metabolism despite in non-diabetes condition. Although all the hyperlipid- emia groups were not qualified to the criteria of dia- betes mellitus, we considered that increased fasting blood glucose might be associated with Rhokinase activ- ity enhancement, and in the metformin and combined
that regulates chondrocyte differentiation. Our data show that PP2 does not affect RhoA activity in chondrocytes, suggesting that Src kinases do not act upstream of RhoA. ROCK inhibi- tion caused a slight increase in the expression of Lyn and Frk transcripts, suggesting that RhoA/ROCK does not act prima- rily by suppressing the expression of these Src kinase genes, at least at the RNA level. However, these data do not exclude the possibility that the Rho pathway controls activity of Src kinases at the post-transcriptional level. In addition, it is feasi- ble that both signalling systems act in parallel pathways. Experiments are under way to examine these possibilities. Another protein that probably interacts with Src kinases in the control of chondrocyte physiology is FAK. Our data show that reduced FAK phosphorylation is associated with the loss of stress fibres and cell rounding in response to PP2. FAK is a direct substrate of Src kinases, but the residue examined (Y397) is auto-phosphorylated by FAK in response to integrin Figure 9
Figure 4 illustrates the use of a rho-diagram to evaluate the results of changing these search parameters. The closed rectangles plot the results of setting the parent ion tolerance in the range 4 > ∆m > 0 Da (∆m ) m - M, where m was the measured parent ion mass, and M was the mass calculated from the peptide sequence). The open diamonds plot the results of searching the same data set, with 0 > ∆m > -4 Da. Clearly, the plots were quite different: the first parameter setting produces a plot that showed no evidence of significant false-positives in any interval, while the second setting pro- duces a plot that only slowly separates itself from diagonal. This behavior indicated that the second setting produced far fewer true-positives than the first. This effect was predictable: the low resolution of the quadrupole mass spectrometer used to make the measurements has the practical effect of interpreting the contribution of 13 C isotopes as an effective mass shift to higher measured mass. The additional effects of space-charging in the ion trap also tended to shift the measurement toward
vitro. As α 5 β 1 antagonists block angiogenesis in vivo, these antagonists may induce caspase-3 and -8 activation in vivo. CAMs stimulated with bFGF were treated with saline, vehicle control (DMSO), caspase-3 or -8 inhibitors, and anti-integrin Ab’s in the presence or absence of caspase inhibitors. Angiogenesis was inhibit- ed by anti- α 5 β 1 Ab’s (Figure 5, a–d); this inhibition was partially reversed by cell-permeable caspase-3 inhibitors (P = 0.04, Figure 5a) and fully reversed by caspase-8 inhibitors (P = 0.05, Figure 5c). Caspase-3 inhibitors par- tially blocked caspase-3 activity in vivo (Figure 5b), while caspase-8 inhibitors completely blocked its activity in vivo (Figure 5d). Furthermore, caspase-3 and -8 inhibitors prevented in vivo endothelial cell DNA frag- mentation induced by α 5 β 1 inhibition (Figure 5e). Cas- pase-9 inhibitors had little effect on angiogenesis (not shown). Caspase inhibitors alone had no effect on angio- genesis or on unstimulated CAMs. These results indicate that α 5 β 1 antagonists activate caspases-8 and -3 in vivo, thereby inhibiting angiogenesis.
ABSTRACT The assembly of the musculoskeletal system in Drosophila relies on the integration of chemical and mechanical signaling between the developing muscles with ectodermal cells specialized as “tendon cells.” Mechanical tension generated at the junction of ﬂight muscles and tendon cells of the notum epithelium is required for muscle morphogenesis, and is balanced by the epithelium in order to not deform. We report that Drosophila Rhokinase (DRok) is necessary in tendon cells to assemble stable myotendinous junctions (MTJ), which are required for muscle morphogenesis and survival. In addition, DRok is required in tendon cells to maintain epithelial shape and cell orientation in the notum, independently of chascon ( chas ). Loss of DRok function in tendon cells results in mis- orientation of tendon cell extensions and abnormal accumulation of Thrombospondin and bPS-integrin, which may cause abnormal myotendinous junction formation and muscle morphogenesis. This role does not depend exclusively on nonmuscular Myosin-II activation (Myo-II), indicating that other DRok targets are key in this process. We propose that DRok function in tendon cells is key to promote the establishment of MTJ attachment and to balance mechanical tension generated at the MTJ by muscle compaction.
IFA analysis of serum revealed a novel autoantibody against brain tissue. An intracellular enzyme, Rho-associated protein kinase 2 (ROCK2), was identified as target-antigen. ROCK2 was expressed in affected brain tissue and archival bladder tumor samples of this patient. Brain histopathology revealed appositions of cytotoxic CD8 + T cells on ROCK2-positive neurons. ROCK2 antibodies were not found in the sera of 20 patients with bladder cancer and 17 with renal cancer, both without neurological symptoms, 49 healthy controls, and 39 patients with other antineuronal autoantibodies. In conclusion, novel onconeural antibodies targeting ROCK2 are associated with paraneoplastic encephalitis and should be screened for when paraneoplastic neurological syndromes, especially in patients with urogenital cancers, occur.
the addition of 0.1 µM isoprenaline and tension was re- adjusted to 0.5 g, immediately followed by two changes of fresh KH-buffer. After another equilibration period of 30 min EGF (0.1, 1, 3, 10 or 30 ng/ml) or PDGF (0.1, 1, 3, 10 or 30 ng/ml) was applied or cumulative concentration response curves (CRCs) were constructed to stepwise increasing concentrations of histamine (1 nM – 100 µM), PGE 2 (1 nM – 3µM) or PGF 2α (1 nM – 10 µM). When max- imal agonist-induced contraction was obtained, the tra- cheal rings were washed several times and maximal relaxation was established using isoprenaline. When used, the inhibitors of Rho-kinase (Y-27632, 1 µM), MAPK- ERK-kinase (MEK) (U-0126, 3 µM) or COX (indometh- acin, 3 µM) were applied to the organ bath 30 min before agonist addition. This was also the case for the FP-recep- tor-and EP 1 -receptor-antagonists AL-8810 and AH-6809 (10 µM both, applied individually to separate prepara- tions), respectively.
Our another finding was that the expression of RhoA/ ROK was down-regulated after isoflurane preconditioning. RhoA/ROK-kinase signaling pathways which is a major cellular target for regulating Ca2+ sensitivity of agonist-in- duced contraction  is a most important contraction mechanisms of artery smooth muscle cell, the activation of RhoA leads to stimulation of ROK that can favor myosin light chain (MLC) phosphorylation, actin-myosin inter- action and cell contraction . However, whether miRs regulate expression of RhoA/ROK in arterial grafts induced by isoflurane exposure remain unknown. Yang et al.  demonstrated that Iso inhibit KCl-induced PI3K-C2α-par- ticipation, Rhokinase-mediated MLC phosphorylation, and vasoconstriction in rat aortic smooth muscle. Here, our data indicated that isoflurane preconditioning supressed the expression of RhoA/ROK and activated endothelial eNOS, resulting in vasodilation of LIMA, so we demon- strated that isoflurane preconditioning may be very useful for reperfusion of ischemic myocardium after revasculariza- tion for left anterior descending coronary artery. However, cellular mechanistic experiments may be needed in the futuer.
This study showed that the highly selective Rho-kinase inhibitor SAR407899 is a relative potent relaxing agent of corpora cavernosa from different animal species and man. These results, in stimulation of penile erection, may be useful in the prevention and therapy of a num- ber of erectile dysfunctions, particularly those depending on hyper-functioning of the RhoA/Rho-kinase system, such as diabetes and hypertension. Future studies are required to confirm the potential of this compound and other more powerful molecules for ED.