This study aimed to investigate the effects of hypoxia on RhoA/Rho-kinase (ROCK) signaling pathway and autophagy in pulmonary artery smooth muscle cells (PASMCs), and to explore the underlying mechanism of Umbelliferone (Umb) in ameliorating chronic hypoxic pulmonary hypertension. PASMCs were cultured from Sprague-Dawley (SD) rats and randomly divided into control group, hypoxia group, hypoxia + Umb intervention group and normoxia + Umb intervention group. Alpha smooth muscle actin (α-SMA) and LC3 were assessed by immunofluorescence staining. Protein expression of RhoA, ROCK2, p-MYPT1, LC3-II, Beclin-1, p62, C-Caspase 3, Bax and Bcl-2 was analyzed by Western blotting. In in vivo study, SD rats were divided into control group, hypoxia group and hypoxia + Umb intervention group. Weight ratio of the right ventricle (RV)/left ventricle plus septum (LV+S) was detected, and pulmonary arterial morphological features were examined by HE staining. The results indicated that compared with the control group, the LC3-II/LC3-I ratio and expression of Beclin-1 were significantly increased, while p62 expression was significantly decreased, and the expressions of RhoA, ROCK2 and p-MYPT1 were significantly increased in PASMCs of hypoxia group (P < 0.05). The changes of LC3-II/LC3-I ratio, the expressions of Beclin-1, p62, RhoA, ROCK2 and p-MYPT1 in PASMCs were reversed by Umb treatment (P < 0.05). Consistently, the pulmonary arterial wall was thickened and the RV/(LV+S) ratio was increased in hypoxic rats, which were significantly improved by Umb treatment (P < 0.05). These results suggest that Umb can improve hypoxia-induced pulmonary hypertension by inhibiting the RhoA/ROCK signaling pathway and autophagy in PASMCs.
Animals ; Autophagy ; Beclin-1/pharmacology* ; Hypertension, Pulmonary/etiology* ; Hypoxia/complications* ; Myocytes, Smooth Muscle/metabolism* ; Pulmonary Artery ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Umbelliferones/pharmacology* ; rho-Associated Kinases/pharmacology*

OBJECTIVETo study the mechanism of endothelial Rho/Rho kinase in extravascular migration of fibrosarcoma cell.

METHODSWe used an in vitro model of fibrosarcoma cell transmigration across a monolayer of human umbilical vein endothelial cell (HUVEC) cultured on collagen gel to observe extravascular migration of fibrosarcoma cells, and then calculated the electrical resistance of HUVEC monolayer and endothelial myosin light chain (MLC) phosphorylation in extravascular migration of fibrosarcoma cells.

RESULTSFibrosarcoma cells migrated through endothelial cells into collagen gel. The electrical resistance of a HUVEC monolayer reduced and endothelial MLC phosphorylation enhanced in the extravascular migration of fibrosarcoma cells. Endothelial Rho inhibitor (C3 transferase) and Rho kinase inhibitor (Y-27632) inhibited the extravascular migration of fibrosarcoma cells and inhibited the reduction of electrical resistance of a HUVEC monolayer and the enhancement of endothelial MLC phosphorylation in extravascular migration of fibrosarcoma cells.

CONCLUSIONEndothelial Rho/Rho kinase may regulate fibrosarcoma cell transendothelial migration through MLC phosphorylation.

Amides ; pharmacology ; Cell Movement ; physiology ; Cells, Cultured ; Enzyme Inhibitors ; pharmacology ; Fibrosarcoma ; pathology ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Phosphorylation ; Pyridines ; pharmacology ; rho GTP-Binding Proteins ; metabolism ; physiology ; rho-Associated Kinases ; metabolism ; physiology

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

OBJECTIVETo study the expression of Rho/Rho kinase of cardiac muscle in heart failure rats caused by pressure overload and the effects of fasudil on heart failure.

METHODSThe heart failure models were successfully induced by coarctation of ascending aorta after 20 weeks in this study. Thirty female Wistar operated rats were divided randomly into three groups (n = 10) for 4 week treatment. (1) Sham operation group: normal saline, 0.1 ml, i.p,Bid. (2) Heart failure group: normal saline, 0.1 ml,i.p,Bid. (3) Fasudil group: fasudil 5 mg/kg, i.p, Bid. The hemodynamic parameters, the ratio of LV weight to body weight, the expressions of RhoA and Rho kinase mRNA, and the concentration of calcium ion same as [Ca(2+)](i) were investigated in the three groups.

RESULTSHemodynamic parameters were significantly changed in heart failure group than those in sham operation group, such as left ventricular diastolic end pressure increased [(13.00 +/- 0.30) mm Hg vs (3.78 +/- 0.31) mm Hg, P < 0.01], left ventricular systolic pressure decreased [(97.20 +/- 7.21) mm Hg vs (129.45 +/- 7.52) mm Hg, P < 0.01]. Those results could be significantly changed by use of fasudil, P < 0.01. The ratio of LV weight to body weight was significantly increased in heart failure group than that in sham operation group [(4.77 +/- 0.08) mg/g vs (2.51 +/- 0.12) mg/g, P < 0.01]. Fasudil could significantly decrease the ratio of LV weight to body weight compared with that in heart failure group [(4.05 +/- 0.08) mg/g vs (4.77 +/- 0.08) mg/g, P < 0.01]. Cardiac muscle RhoA, Rho kinase mRNA level and [Ca(2+)](i) were higher in heart failure group than those in sham operation group [Ca(2+)](i) (475.93 +/- 28.22) nmol/L vs (79.25 +/- 3.33) nmol/L, P < 0.01. Compared with those in heart failure group, the expressions of RhoA, Rho kinase mRNA level decreased significantly, P < 0.01, and the levels of cardiomyocyte [Ca(2+)](i) had no change in fasudil group [(462.78 +/- 16.72) nmol/L vs (475.93 +/- 28.22) nmol/L, P > 0.05].

CONCLUSIONSThese results indicated that heart failure was probably related to activating of RhoA, Rho kinase. Fasudil may contribute to the observed beneficial effects on heart failure such as the decrease of RhoA, Rho kinase mRNA expression and not increase of [Ca(2+)](i) level. Rho/Rho kinase may be a novel, potent signaling of heart failure.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Female ; Heart Failure ; metabolism ; physiopathology ; Myocardium ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; Rats ; Rats, Wistar ; rho-Associated Kinases ; metabolism

OBJECTIVETo investigate the mechanisms of Aldosterone stimulating hepatic stellate cells(HSCs) contraction via Ca2+-independent pathways.

METHODSHSC-T6 cell line was pre-disposed with Aldo 10mumol/L. The cell contraction was detected by silicone-rubber-membrane cultivation directly. The concentration variation of intracellular free calcium in rat HSC was observed by laser confocal microscopy. Besides, HSC-T6 cell line was under pre-disposal treatment with the blocking agents of Aldo receptor -antisterone, protein kinase C (PKC) special blocking agent-Stauro, Rho kinase blocking agent-Y27632 and MLCK special blocking agent-ML-7 respectively prior to stimulation with aldosterone. RT-PCR was used to detect the expression of Rock2, RhoAGTP and RhoGEF in Ca2+- independent pathways mediated by Rho-kinase.

RESULTSAldo could induce HSCs contraction. The concentration of intracellular free calcium in rat HSCs had no change after pre-disposal treatment with Aldo. The mRNA expressions of Rock2, RhoAGTP and RhoGEF increased significantly after treatment with Aldo (0.770+/-0.049, 0.960+/-0.096, 0.180+/-0.006, P is less than 0.01).When inhibited with antisterone, the mRNA expressions of the three elements were (0.440+/-0.166, 0.370+/-0.180 and 0.050+/-0.001, P is less than 0.01), lower than that of Aldo group, but higher in ML-7+Stauro + Aldo groups (0.940+/-0.066, 1.330+/-0.192 and 0.160+/-0.007, P is less than 0.05) as compared to the control group (0.140+/-0.023, 0.540+/-0.111 and 0.110+/-0.012). In the Y27632 + ML-7 + Stauro+Aldo group, the mRNA expression of RhoGEF (0.290+/-0.004, P is less than 0.01)was higher than that of the ML-7 + Stauro + Aldo group (0.160+/-0.007).

CONCLUSIONAldo could induce HSCs contraction via Ca2+-independent pathways and Rho-Rock pathway involved in the process.

Aldosterone ; pharmacology ; Animals ; Cell Line ; Hepatic Stellate Cells ; drug effects ; metabolism ; physiology ; Rats ; Signal Transduction ; drug effects ; rho-Associated Kinases ; metabolism

OBJECTIVETo investigate the expressions of sphingosine-1-phosphate receptors 1-3 (S1P1- 3) in the corpus cavernosum of castrated male rats and its relationship with the NOS/NO/cGMP and RhoA/Rho kinase signaling pathways.

METHODSWe equally randomized 18 eight-week-old healthy male SD rats into a sham-operation control, a castration, and a testosterone replacement (TR) group and harvested the bilateral testes and epididymides from the rats in the latter two groups, followed by 4 weeks of subcutaneous injection of testosterone propionate at 3 mg per kilogram of the body weight per day for those in the TR group and that of plant oil for those in the control and castration groups. At the age of 12 weeks, we measured the serum testosterone (T) level and maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP) of the animals and determined the expressions of SlP1-3, eNOS, P-eNOS, ROCK1, and ROCK2 in the corpus cavernosum by Western blot and immunohistochemistry.

RESULTSThe serum T level was significantly decreased in the rats of the castration group as compared with those of the control and TR groups ([0.41 ± 0.04] vs [16.01 ± 1.02] and [15.84 ± 1.32] nmol/L, P < 0.01), with no statistically significant difference between the latter two groups. The ICPmax/MAP at 0 V, 3 V, and 5 V electric stimulation was remarkably lower in the rats of the castration group (0.088 ± 0.014, 0.323 ± 0.014, and 0.432 ± 0.012) than in those of the control group (0.155 ± 0.011, 0.711 ± 0. 010, and 0.819 ± 0.024) and TR group (0.153 ± 0.012, 0.696 ± 0.017, and 0.763 ± 0.027) (P < 0.01), with no significant difference between the latter two groups. With GAPDH as internal control, the animals of the castration group showed markedly reduced expressions of S1P1 ([49.99 ± 3.39]%), eNOS ([46.82 ± 3.81]%) , and P-eNOS ([45.42 ± 4.35]%) in comparison with those in the control group ([72.57 ± 3.06], [89.76 ± 3.98], and [82.53 ± 8.92] and TR group ([71.77 ± 4.43], [87.19 ± 4.23], and [79.82 ± 7.38]%) (P < 0.01) , while the expressions of S1P2, S1P3, ROCK1, and ROCK2 were significantly upregulated in the castration group ([82.35 ± 4.13], [61.03 ± 5.14], [74.50 ± 4.02], and [69.83 ± 5.75]%) as compared with those in the control group ([41.67 ± 1.68], [31.66 ± 2.67], [35.69 ± 5.56], and [39.85 ± 7.17]%) and TR group ([42.80 ± 3.87], [32.25 ± 4.22], 38.06 ± 5.21], and [42.36 ± 4.44]%) (P < 0.01).

CONCLUSIONAndrogen deficiency induces significant reduction of ICPmax/ MAP in male rats, which is possibly associated with the decline of S1P1 in the corpus cavernosum, inhibition of the eNOS/NO/cGMP signaling pathway, increased expressions of S1P2 and S1P3, and activation of the RhoA/Rho kinase signaling pathway.

Animals ; Male ; Nitric Oxide Synthase Type III ; metabolism ; Orchiectomy ; Penis ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Lysosphingolipid ; metabolism ; Testosterone ; blood ; pharmacology ; rho-Associated Kinases ; metabolism

Animals ; Asthma ; drug therapy ; metabolism ; Inflammation ; Lung ; drug effects ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Phytotherapy ; Pyrazines ; pharmacology ; therapeutic use ; RNA, Messenger ; metabolism ; rho GTP-Binding Proteins ; metabolism ; rho-Associated Kinases ; metabolism

OBJECTIVETo investigate of the regulatory effect of Rho-kinase pathway activation on angiotensin II (Ang II)-induced contraction of human airway smooth muscle cells (HASMCs) in vitro.

METHODSCultured primary HASMCs were divided into control group, AngII group, AngII + irbesartan group and AngII + Y-27632 group with corresponding treatment. AngII-induced contraction of HASMCs was evaluated using collagen gel lattices and observed morphologically using immunofluorescence assay. Western Blotting was significantly performed to examine the protein expression of Rho-kinase signal pathway.

RESULTSAngII-induced HASMC contraction was inhibited by treatments with irbesartan and Y-27632 as shown by gel contraction assay (P<0.001). Y-27632 treatment produced a stronger inhibitory effect than irbesartan on the expression of phosphorylated moesin, a substrate of Rho kinase (P<0.05).

CONCLUSIONAngII induces the contraction of HASMCs partially as a result of activation of Rho-kinase pathway.

Amides ; pharmacology ; Angiotensin II ; pharmacology ; Asthma ; physiopathology ; Biphenyl Compounds ; pharmacology ; Bronchi ; cytology ; Humans ; Muscle Contraction ; drug effects ; Muscle, Smooth ; cytology ; Primary Cell Culture ; Pyridines ; pharmacology ; Signal Transduction ; drug effects ; Tetrazoles ; pharmacology ; 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

To explore the angiotensin peptide [Ang (1-7)]-mediated inhibition of Ang II in human hepatic stellate cells (HSCs) and determine the involvement of the ACE2-Ang (1-7)-Mas axis. The human HSC line, LX2, was used in all experiments, and divided into control (unstimulated) and Ang II-stimulated (10-6 mol/L) groups. The Ang II-stimulated cells were further divided among several pre-treatment (prior to Ang II) groups: ROCK-inhibited (Y27632 blocking agent, 10-6 mol/L); irbesartan-inhibited (AT-1 receptor antagonist, 10-6 mol/L); and Mas receptor-inhibited (A779 Mas receptor antagonist, 10-6 mol/L). To explore the potential inhibitory effects of various Ang family members, the Ang II-stimulated and pre-treated LX2 cells were exposed to Ang (1-7) (10-6 mol/L) for 24 h. Western blot, reverse transcription-polymerase chain reaction (RT-PCR), and QuantiGene assay were used to assess changes in protein and mRNA expression levels of RhoA, ROCK, and connective tissue growth factor (CTGF). Compared with the control group, Ang II-stimulated cells showed significantly increased levels of RhoA protein (0.337+/-0.074 vs. 0.870+/-0.093), ROCK2 mRNA (0.747+/-0.061 vs. 0.368+/-0.023), and CTGF mRNA (0.262+/-0.007 vs. 0.578+/-0.028) (all, P less than 0.01). Pre-treatment with irbesartan or Y27632 eliminated these responses. Ang (1-7) inhibited the Ang II-stimulated up-regulation of RhoA, ROCK, and CTGF. Ang (1-7) can inhibit the Ang II-stimulated up-regulation of RhoA, ROCK and CTGF in hepatic stellate cells, indicating that the ACE2-Ang (1-7)-Mas axis, an important branch of the renin-Ang-aldosterone system is involved in the occurrence and development of liver fibrosis.
Angiotensin I ; pharmacology ; Angiotensin II ; pharmacology ; Cells, Cultured ; Connective Tissue Growth Factor ; metabolism ; Hepatic Stellate Cells ; drug effects ; metabolism ; Humans ; Peptide Fragments ; pharmacology ; RNA, Messenger ; genetics ; Signal Transduction ; rho-Associated Kinases ; metabolism ; rhoA GTP-Binding Protein ; metabolism