1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Hypertension, Pulmonary ; drug therapy ; Hypoxia ; pathology ; Protein Kinase Inhibitors ; pharmacology ; Rats ; rho-Associated Kinases ; antagonists & inhibitors

BACKGROUNDAfter injury, axonal regeneration of the adult central nervous system (CNS) is inhibited by myelin-derived growth-suppressing proteins. These axonal growth inhibitory proteins are mediated via activation of Rho, a small GTP-binding protein. The activated form of Rho, which is bound to GTP, is the direct activator of Rho kinase (ROCK) through serial downstream effector proteins to inhibit axonal regeneration. The objective of this study was to observe the therapeutic effect of inactivation of the Rho-ROCK signaling pathway to promote neurologic recovery after spinal cord injuries in rats.

METHODSOne hundred and twenty adult female Sprague-Dawley rats were randomly divided into three groups. Laminectomies alone were conducted in 40 rats in the sham group. Laminectomies and spinal cord transections were performed in 40 rats in the control group (treated with normal saline administered intraperitoneally). Laminectomies and spinal cord transections were performed in 40 rats in the fasudil-treated group (treated with fasudil administered intraperitoneally). Neurologic recovery was evaluated before surgery and 3 days, and 1, 2, 3, and 4 weeks after surgery using the Basso-Beattie-Bresnahan (BBB) scale of hind limb movement. At the same time, the expression of RhoA mRNA was determined with RT-PCR. Histopathologic examinations and immunofluorescence staining of NF were performed 1 month after surgery.

RESULTSCompared with the control group, the BBB scores of the fasudil-treated group were significantly increased and the expression of RhoA mRNA was significantly decreased. In the fasudil-treated group, a large number of NF-positive regenerating fibers was observed; some fibers crossed the slit of the lesion.

CONCLUSIONInactivation of the Rho-ROCK signaling pathway promotes CNS axonal regeneration and neurologic recovery after spinal cord injuries in rats.

Animals ; Female ; Fluorescent Antibody Technique ; Nerve Regeneration ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; physiology ; Spinal Cord Injuries ; pathology ; physiopathology ; psychology ; therapy ; rho-Associated Kinases ; antagonists & inhibitors ; physiology ; rhoA GTP-Binding Protein ; antagonists & inhibitors ; physiology

OBJECTIVETo reveal the roles of Rho kinase (ROCK) in the mechanisms of complications in diabetes by reviewing the correlations between ROCK and related complications in diabetes.

DATA SOURCESThe data used in the present article were mainly from PubMed with relevant English articles published from 1998 to 2010. The search terms were "ROCK" and "diabetes".

STUDY SELECTIONOriginal articles including the roles of ROCK or its inhibitors in diabetic complications and review articles about the biological character of ROCK were selected.

RESULTSThe activity and expression of ROCK were up-regulated in the models of type 1 or type 2 diabetes animals and the cultured cells with concentrations of high glucose, ROCK activation was associated with the development or progression of complications in diabetes. Inhibition of RhoA/ROCK pathway prevented or ameliorated the pathologic changes of diabetic complications, and ROCK has been regarded as a key target for treatment of these complications.

CONCLUSIONRhoA/ROCK signaling plays important roles in the pathogenesis of long-term complications in diabetes and ROCK inhibitors are becoming a promising solution to treatments of complications in diabetes.

Animals ; Cardiomyopathies ; etiology ; Diabetes Complications ; etiology ; therapy ; Humans ; Sexual Dysfunction, Physiological ; etiology ; Signal Transduction ; Urinary Bladder Diseases ; etiology ; rho-Associated Kinases ; antagonists & inhibitors ; chemistry ; physiology ; rhoA GTP-Binding Protein ; antagonists & inhibitors ; chemistry ; physiology

OBJECTIVETo investigate the changes in endothelial cytoskeletal reorganization and the role of Rho in the signal transduction pathway.

METHODSECV304 cells were cultured and randomly divided into following groups: i.e. sham (with normal rat serum treatment), burn (with burn rat serum treatment), Y (with 30 micromol/L Rho kinase inhibitor Y-27632 treatment), burn plus Y (pretreatment of cells with burn serum before treated with 30 micromol/L Y-27632), Y plus burn (pretreatment of cells with Y-27632 for 1 hour before treated with burn serum), LPA (with normal rat serum and 13 micromol/L LPA), and LPA plus Y (pretreatment of cells with LPA before treated with Y-27632) groups. The indices were examined at 6, 7 and 8 posttreatment hours (PTH) in all groups except in Y group. The endothelial morphology was observed with HE staining. Endothelial cytoskeleton was observed by dual-fluorescence labeling of filamenta (F) with Rhodamine-phalloidin and monomer (G) with oregon green labeled DNAase. The actin content in the cells in all groups was measured with flow cytometry.

RESULTSIn sham and control group, the cells were in fusiform or polygonal shape, with satisfactory growth; filamentous actin (F-actin) was mainly distributed in the peripheral site of the cytoplasm and formed peripheral filamental band. The cells became confluent to form a single layer with reticular structure. Globular actin (G-actin) was concentrated in the nucleus and per nucleus. In burn group, after 6 hours of burn serum treatment, the ability of cells to adhere to vessel wall was weakened, and a striking reorganization of the actin cytoskeleton and the formation of the stress fibers were found. Furthermore, the fluorescent intensity of the peripheral filament bands was weakened, and dispersed actin monomers were seen in the cytoplasm. This reaction was enhanced along with elapse of stimulation time. In burn plus Y or Y plus burn group, the cells grew and adhered well to the wall of culture vessel. The distribution of the filamentous actin was the same as the sham group, while the stress fiber decreased in amount obviously. The structure of globular actin was condensed with little G-actin in the cytoplasm. The changes in actin cytoskeleton in LPA group was similar to that in burn group. The effects of LPA on actin reorganization could also be reversed by Y-27632. The content of F-actin in burn group at 6 PTH (0.63 +/- 0.07) was lower than that in sham group (0.75 +/- 0.08), while the content of G-actin in burn group (1.28 +/- 0.27) was higher than that in sham group (1.16 +/- 0.16, P > 0.05).

CONCLUSIONBurn serum induces vascular endothelial actin cytoskeleton reorganization in endothelial cells via the Rho-dependent signal pathway. Similar to the effect of LPA, this effect could be reversed by Y-27632.

Actins ; metabolism ; Amides ; pharmacology ; Animals ; Burns ; blood ; metabolism ; Cells, Cultured ; Cytoskeleton ; metabolism ; Endothelial Cells ; metabolism ; Endothelium, Vascular ; Humans ; Male ; Pyridines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Serum ; metabolism ; Signal Transduction ; rho-Associated Kinases ; antagonists & inhibitors ; metabolism

AIM: To investigate the effects of pinocembrin on angiotensin II (Ang II)-induced vascular contraction, and to explore its molecular mechanism of actions. METHODS: The isometric vascular tone was measured in rat thoracic aortic rings with denuded endothelium. Phosphorylation level of myosin phosphatase target unit 1 (MYPT1), and protein levels of Rho kinase 1 (ROCK1, ROKβ or p160ROCK) and angiotensin II type-1 receptor (AT1R) were determined by Western blot analysis. RESULTS: Pinocembrin produced a relaxant effect on endothelium-denuded aortic rings contracted by Ang II (100 nmol·L(-1)) in a dose-dependent manner. In endothelium-denuded aortic rings stimulated by Ang II, pretreatment with pinocembrin (25 and 100 μmol·L(-1)) for 20 min significantly attenuated MYPT1 phosphorylation and ROCK1 protein levels. Meanwhile, the protein level of AT1R in response to Ang II was not affected by pinocembrin in rat aortic rings. CONCLUSION These findings indicate that pinocembrin inhibits vasoconstriction induced by Ang II in rat endothelium-denuded aortic rings, and the mechanism at least in part, is due to the blockade of the RhoA/ROCK pathway.
Angiotensin II ; metabolism ; Animals ; Aorta ; drug effects ; enzymology ; metabolism ; physiopathology ; Flavanones ; pharmacology ; In Vitro Techniques ; Male ; Myocardial Contraction ; drug effects ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Vasoconstriction ; drug effects ; rho-Associated Kinases ; antagonists & inhibitors ; genetics ; metabolism

OBJECTIVETo study changes in paired-immunoglobulin-like receptor B (PirB) expression after hypoxic-ischemic brain damage (HIBD) as well as the role for targeted inhibition of PirB activity in nerve regeneration in rats.

METHODSNewborn Sprague-Dawleyrats rats were divided into: a sham operation group (n=30), a HIBD group (n=30), and an anti PirB antibody treatment group (n=6). In the HIBD group, HIBD was induced by right carotid artery ligature and subsequent exposure to hypoxia (8% O2) for 3 hours. In the sham operation group, right carotid artery was dissected as in the HIBD group but no ligature and hypoxic exposure was not applied. In the two groups, 6 animals were sacrificed at 0, 6, 12, 24 and 72 hours after the operation and hypoxic exposure. In the antibody treatment group, after carotid artery ligation and hypoxia exposure as in the HIBD group, an anti PirB antibody was injected intracerebrally and animals were sacrificed 72 hours after the injection. Immediately after sacrifice of the animals at designated time points, brain tissue specimens were collected. The presence and content of PirB protein were assessed by immunohistochemistry and Western blot analysis respectively, the abundance of PirB mRNA was determined by RT-PCR, and the Rho kinase (Rock) activity was determined by immunoprecipitation.

RESULTSAt 72 hours after operation, PirB mRNA abundance and protein content in the brain were significantly increased as compared with the measurements at 0 hour after operation in the HIBD group (P<0.05); ROCK activity was significantly increased in the HIBD group as compared with the sham operation and anti PirB antibody groups (P<0.05).

CONCLUSIONSPirB might be involved in HIBD through a Rho-ROCK-dependent mechanism and antibody-mediated neutralization of PirB in the brain may offer a novel therapeutic strategy for HIBD.

Animals ; Animals, Newborn ; Hypoxia-Ischemia, Brain ; physiopathology ; therapy ; Nerve Regeneration ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptors, Immunologic ; antagonists & inhibitors ; genetics ; physiology ; Signal Transduction ; rho-Associated Kinases ; metabolism

The present paper serves as a review of the associations between lower urinary tract symptoms (LUTS) and erectile dysfunction (ED), with a focus on common and combined pathways for treatment. LUTS and ED are common conditions seen in general urologic practice. Research has started to establish epidemiologic and pathophysiologic links between the two conditions and a strong association confirmed across multiple studies. Men seeking care for one condition should always be interviewed for complaints of the other condition. Proposed common pathways include alpha-1 adrenergic receptor imbalance, Rho-kinase overactivity, endothelial cell dysfunction and atherosclerosis-induced ischemia. Medical therapy has replaced surgery as the first-line treatment for LUTS in most patients, with the incorporation of alpha-adrenergic receptor antagonists (alpha-ARAs) and 5-alpha-reductase inhibitors (5-ARIs) into everyday practice. Treatment with alpha-ARAs contributes to some improvement in ED, whereas use of 5-ARIs results in worsened sexual function in some patients. Phosphodiesterase-5 (PDE-5) inhibitors have revolutionized the treatment of ED with a simple oral regimen, and new insights demonstrate a benefit of combined use of PDE-5 inhibitors and alpha-ARAs. The mechanisms of action of these medications support these observed benefits, and they are being studied in the basic science and clinical settings. In addition, novel mechanisms for therapy have been proposed based on clinical and research observations. The minimally invasive and surgical treatments for LUTS are known to have adverse effects on ejaculatory function, while their effects on erectile function are still debated. Much remains to be investigated, but it is clear that the associations between LUTS and ED lay the foundation for future therapies and possible preventative strategies.
5-alpha Reductase Inhibitors ; Adrenergic alpha-Antagonists ; therapeutic use ; Atherosclerosis ; complications ; Endothelium, Vascular ; Erectile Dysfunction ; etiology ; therapy ; Humans ; Male ; Phosphodiesterase Inhibitors ; therapeutic use ; Prostatic Hyperplasia ; complications ; surgery ; Receptors, Adrenergic, alpha ; physiology ; Urologic Diseases ; etiology ; therapy ; rho-Associated Kinases ; metabolism

OBJECTIVETo investigate the role of Rho-kinase signaling pathway in human airway smooth muscle cell (ASMCs) migration and cytoskeletal reorganization induced by endothelin-1 (ET-1).

METHODSPrimary cultured human ASMCs obtained by tracheal explant culture method were examined for cell migration in response to ET-1 treatment using modified Boyden chambers. The changes in actin cytoskeletal reorganization were observed under confocal laser scanning microscope, and the phosphorylation of myosin-phosphatase target 1 (p-MYPT1) was examined using Western blot analysis.

RESULTSAt the concentration of 0.1, 1, 10, and 100 nmol/L, ET-1 induced migration of the ASMCs, and 10 nmol/L ET-1 produced the most obvious effect (P<0.01). Rho-kinase inhibitor Y-27632 showed a dose-dependent inhibitory effect on ET-1-induced ASMC migration, and in cells exposed to 10 nmol/L ET-1, Y-27632 at 10 micromol/L significantly blocked ASMC migration (P<0.01). ET-1 (10 nmol/L) exposure resulted in reorganization of actin cytoskeleton and formation of stress fibers in the ASMCs, which were obviously inhibited by Y-27632. Compared with the control group, the AMSCs showed significant enhancement of p-MYPT1 protein expression after ET-1 exposure for 15 and 30 min (P<0.01), but prolonged exposure failed to result in the expression enhancement (P>0.05).

CONCLUSIONRho-kinase signaling pathway may play an important role in ET-1-induced ASMC migration and reorganization of actin cytoskeleton.

Amides ; pharmacology ; Bronchi ; cytology ; Cell Movement ; drug effects ; Cells, Cultured ; Cytoskeleton ; drug effects ; metabolism ; Endothelin-1 ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Humans ; Microscopy, Confocal ; Muscle, Smooth ; cytology ; Pyridines ; pharmacology ; Signal Transduction ; drug effects ; rho-Associated Kinases ; antagonists & inhibitors ; metabolism

The present study was designed to test whether Rho-kinase (ROCK) specific inhibitor fasudil (HA-1077) could contribute to migration and vasculogenesis of endothelial cells differentiated from rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. rBMSCs were separated by gradient centrifugation on lymphocytes separation medium from bone marrow of Sprague-Dawley rats, and were cultured, purified and expanded in vitro. Cells of passage 2 to 3 were induced to differentiate into endothelial lineage cells by HG-DMEM plus EGM-2. These cells were identified as endothelial cells with positive factor VIII and Ulex europaeus agglutinin-1 expressions and DiI-Ac-LDL uptake. HA-1077 and VEGF synergistically promoted cell migration, especially in response to transwell chamber assay. When the cells were cultured on ECMatrix™, they showed cellular protrusions and/or cords of aligned cells resembling primitive capillary-like structures at 8 to 12 h of incubation. HA-1077 promoted cell migration and formation of capillary-like tubes. The length of the total capillary tubes was longer than that in the control group (P<0.05). When the cells were exposed to a combination of VEGF and HA-1077, the number of the capillary-like networks and the stability of tube increased. The results obtained suggest that HA-1077 can promote migration and vasculogenesis of endothelial cells differentiated from rBMSCs in vitro.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cell Movement ; drug effects ; Endothelial Cells ; cytology ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; rho-Associated Kinases ; antagonists & inhibitors

ObjectiveTo investigate the potential role of the RhoA/Rock signaling pathway in the formation of prostate cancer and the effects of the Rock inhibitor fasudil on the invasion, migration and apoptosis of human prostate cancer cells.

METHODSHuman prostate cancer cell lines PC3 and DU145 were treated with fasudil at the concentrations of 5, 10, 20, 40, 80, and 160 μmol/L, respectively, and those as negative controls cultured in the Ham's-F12 medium, all for 24 hours. Then, MTT assay was used to measure the cell inhibition rate and half maximal inhibitory concentration (IC50) value of fasudil, with 1/4 of IC50 as the medication dose for further investigation. The expressions of RhoA, RockⅠ, and RockⅡ proteins in the PC3 and DU145 cells were detected by Western blot and immunohistochemistry, and the invasion, migration and apoptosis of the cells were determined using the Transwell chamber, scratch wound healing assay and flow cytometry.

RESULTSFasudil inhibited the proliferation of the PC3 cells from (9.29±1.23)% at 5 μmol/L to (81.37±3.97)% at 160 μmol/L and that of DU145 from (7.59±1.54)% to (76.53±2.67)%, both in a dose-dependent manner (P<0.05 ). Significantly fewer PC3 and DU145 cells migrated into the lower compartment in the experimental group (39.2±8.4 and 34.2±6.7) than in the negative control (116.8±9.3 and 112.5±10.8) (P<0.05 ). The wound healing rates of the PC3 and DU145 cells were remarkably lower in the former (［37.26±1.17］% and ［32.38±2.73］%) than in the latter (［78.12±4.16］% and ［69.47±6.71］%) (P<0.05 ). Annexin V-FITC/PI double staining showed markedly increased apoptosis rates of PC3 and DU145 cells treated with fasudil (［31.88±2.49］% and ［28.65±2.99］%) as compared with the negative controls (［7.51±2.28］% and ［7.13±1.61］%) (P<0.05 ). The expressions of RockⅠ and RockⅡ were significantly reduced in the fasudil-treated cells in comparison with those of the control group (P<0.05 ) while that of RhoA showed no significant difference between the two groups (P>0.05 ).

CONCLUSIONSThe RhoA/Rock signaling pathway may play an important role in the formation of prostate cancer. Fasudil can significantly inhibit the proliferation, migration, and invasion and promote the apoptosis of human prostate cancer PC3 and DU145 cells by reducing RhoA/Rho kinase activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Humans ; Male ; Prostatic Neoplasms ; drug therapy ; pathology ; Signal Transduction ; rho-Associated Kinases ; antagonists & inhibitors