6093

ROCK1

P160ROCK|ROCK-I;Rho-associated, coiled-coil containing protein kinase 1;ROCK1;Rho-associated, coiled-coil containing protein kinase 1

Rho kinase|p160 ROCK-1|p160-ROCK|renal carcinoma antigen NY-REN-35|rho-associated protein kinase 1|rho-associated, coiled-coil-containing protein kinase 1|rho-associated, coiled-coil-containing protein kinase I

18q11.1

protein-coding

Count: Rock1; 81762

OBJECTIVE: To observe the effect of breviscapine on the pulmonary artery pressure and the Rho-kinase and Rho-kinase mRNA in pulmonary arterioles of rats treated with hypoxia, and therefore to explore the mechanisms of breviscapine on hypoxic pulmonary hypertension. METHODS: Eighteen adult male SD rats were randomly divided into 3 groups. One group was exposed to air (normal group), the second group was exposed to isobaric hypoxia for 3 weeks (hypoxic group), and the third group was exposed to hypoxia for 3 weeks and treated with breviscapine (preventive group). Cardiac catheterization was used to measure the mean pulmonary arterial pressure (mPAP). The heart was isolated, and the right ventricle (RV), left ventricle plus ventricular septum (LV + S) were weighed to calculate the ratio RV/(LV + S). The ratio of vascular wall thickness/vascular external diameter (WT%) and the ratio of vascular wall area/total vascular area (WA%) were measured by image analysis. The quantity of Rho-kinase and Rho-kinase mRNA in rat pulmonary arterioles were determined by immunohistochemistry and in situ hybridization respectively. RESULTS: The mPAP in the preventive group [(19.83 +/- 1.47) mm Hg, 1 mm Hg = 0.133 kPa] was significantly lower than that of the hypoxic group [(27.3 +/- 5.0) mm Hg], t = 4.28, P < 0.05. The RV/(LV + S) in the preventive group (0.29 +/- 0.03) was significantly lower than that in the hypoxic group (0.34 +/- 0.05, t = 2.39, P < 0.05). The WT% and WA% in the preventive group (25 +/- 5 and 45 +/- 5, respectively) were significantly lower than those in the hypoxic group (36 +/- 12 and 59 +/- 13, respectively, t = 4.89, 5.89, P < 0.05). The positive staining of ROCKI and ROCKII on pulmonary arterioles in the preventive group (1.18 +/- 0.10 and 1.30 +/- 0.12, respectively) were significantly lower than those in the hypoxic group (1.29 +/- 0.08 and 1.63 +/- 0.24, respectively, t = 3.90, 5.82, P < 0.05). The positive staining of ROCKI mRNA and ROCKII mRNA in the preventive group (1.23 +/- 0.13 and 1.22 +/- 0.06, respectively) were significantly lower than those in the hypoxic group (1.37 +/- 0.13 and 1.59 +/- 0.31, respectively, t = 3.94, 5.83, P < 0.05). CONCLUSION: Breviscapine was shown to prevent hypoxic pulmonary hypertension and decrease Rho-kinase and Rho-kinase mRNA.

BACKGROUND: Direct evidence for Rho-kinase activation in patients with pulmonary hypertension (PH) is still lacking. METHODS AND RESULTS: Rho-kinase activity in circulating neutrophils was examined by determining the ratio of phosphorylated/total forms of myosin-binding subunit, a substrate of Rho-kinase, in 40 consecutive PH patients and 40 healthy controls. Next, Rho-kinase expression and activity was examined in isolated human lung tissues (5 patients with idiopathic pulmonary arterial hypertension [IPAH], 5 controls) and vascular reactivity of isolated small human pulmonary arteries in vitro (4 IPAH, 4 controls). Rho-kinase activity in circulating neutrophils was significantly increased in the PH patients overall compared with controls (P<0.0001). Significant correlations were noted between Rho-kinase activity and the severity and duration of PAH (all P<0.05). Rho-kinase expression and activity in isolated lung tissues also were significantly increased in the IPAH patients compared with the controls (both P<0.0001). Endothelium-dependent relaxation was markedly impaired and serotonin-induced contraction (in the absence of the endothelium) markedly enhanced in the PAH patients compared with the controls, and the hypercontraction to serotonin was abolished by hydroxyfasudil, a specific Rho-kinase inhibitor. CONCLUSIONS: These results provide the first direct evidence for Rho-kinase activation in patients with PAH, suggesting the therapeutic importance of Rho-kinase in the disorder.

Calcium is the major intracellular messenger that triggers smooth muscle contraction. The study of calcium-binding proteins, such as calmodulin and its downstream effectors, reveals critical regulation of smooth muscle contraction by protein kinases and phosphatases. Moreover, the small GTP-binding protein RhoA and its downstream effector protein, Rho-kinase, have been shown to play a novel role in the regulation of smooth muscle contraction. Studies have shown that the activation of Rho-kinase is involved in the development of endothelial dysfunction, inflammation, restenosis, and increased vascular tone in a number of cardiovascular disorders. Because inhibitors of this pathway promote vasodilation independent of the mechanism that increases vasoconstrictor tone, it is our hypothesis that Rho-kinase is constitutively active in regulating vasoconstrictor tone in the pulmonary and systemic vascular beds. Studies in the literature suggest that the RhoA/Rho-kinase pathway has an important role in the pathogenesis of pulmonary hypertension.

Ras homolog gene family member A (RhoA) through Rho kinase kinase (ROCK), one of its downstream effectors, regulates a wide range of cell physiological functions, including vascular smooth muscle cell (SMC) proliferation, by degrading cyclin-dependent kinase inhibitor, p27. Our previous studies found that heparin inhibition of pulmonary artery SMC (PASMC) proliferation and pulmonary hypertension was dependent on p27 up-regulation. To investigate whether ROCK, a regulator of p27, is involved in regulation of heparin inhibition of PASMC proliferation, we analyzed ROCK expression in the lungs from mice and from human PASMCs exposed to hypoxia, and investigated the effect of ROCK expression in vitro by RhoA cDNA transfection. We also investigated the effect of guanine nucleotide exchange factor (GEF)-H1, an upstream regulator of RhoA, on heparin inhibition of PASMC proliferation by GEF-H1 cDNA transfection. We found that: (1) hypoxia increased ROCK expression in mice and PASMCs; (2) overexpression of RhoA diminished the inhibitory effect of heparin on PASMC proliferation and down-regulated p27 expression; and (3) overexpression of GEF-H1 negated heparin inhibition of PASMC proliferation, which was accompanied by increased GTP-RhoA and decreased p27. This study demonstrates that the RhoA/ROCK pathway plays an important role in heparin inhibition on PASMC proliferation, and reveals that heparin inhibits PASMC proliferation through GEF-H1/RhoA/ROCK/p27 signaling pathway, by down-regulating GEF-H1, RhoA, and ROCK, and then up-regulating p27.

BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) continues to be one of the most serious intractable diseases that might start with activation of several triggers representing the genetic susceptibility of a patient. To elucidate what essentially contributes to the onset and progression of IPAH, we investigated factors playing an important role in IPAH by searching discrepant or controversial expression patterns between our murine model and those previously published for human IPAH. We employed the mouse model, which induced muscularization of pulmonary artery leading to hypertension by repeated intratracheal injection of Stachybotrys chartarum, a member of nonpathogenic and ubiquitous fungus in our envelopment. METHODS: Microarray assays with ontology and pathway analyses were performed with the lungs of mice. A comparison was made of the expression patterns of biological pathways between our model and those published for IPAH. RESULTS: Some pathways in our model showed the same expression patterns in IPAH, which included bone morphogenetic protein (BMP) signaling with down-regulation of BMP receptor type 2, activin-like kinase type 1, and endoglin. On the other hand, both Wnt/planar cell polarity (PCP) signaling and its downstream Rho/ROCK signaling were found alone to be activated in IPAH and not in our model. CONCLUSIONS: Activation of Wnt/PCP signaling, in upstream positions of the pathway, found alone in lungs from end stage IPAH may play essential roles in the pathogenesis of the disease.

The fawn-hooded rat (FHR) develops severe pulmonary hypertension (PH) when raised for the first 3-4 wk of life in the mild hypoxia of Denver's altitude (5,280 ft.). The PH is associated with sustained pulmonary vasoconstriction and pulmonary artery remodeling. Furthermore, lung alveolarization and vascularization are reduced in the Denver FHR. We have recently shown that RhoA/Rho kinase signaling is involved in both vasoconstriction and vascular remodeling in animal models of hypoxic PH. In this study, we investigated the role of RhoA/Rho kinase signaling in the PH of Denver FHR. In alpha-toxin permeabilized pulmonary arteries from Denver FHR, the contractile sensitivity to Ca2+ was increased compared with those from sea-level FHR. RhoA activity and Rho kinase I protein expression in pulmonary arteries of Denver FHR (10-wk-old) were higher than in those of sea-level FHR. Acute inhalation of the Rho kinase inhibitor fasudil selectively reduced the elevated pulmonary arterial pressure in Denver FHR in vivo. Chronic fasudil treatment (30 mg.kg-1.day-1, from birth to 10 wk old) markedly reduced the development of PH and improved lung alveolarization and vascularization in Denver FHR. These results suggest that Rho kinase-mediated sustained vasoconstriction, through increased Ca2+ sensitivity, plays an important role in the established PH and that RhoA/Rho kinase signaling contributes significantly to the development of PH and lung dysplasia in mild hypoxia-exposed FHR.

Ras homolog gene family member A (RhoA) through Rho kinase kinase (ROCK), one of its downstream effectors, regulates a wide range of cell physiological functions, including vascular smooth muscle cell (SMC) proliferation, by degrading cyclin-dependent kinase inhibitor, p27. Our previous studies found that heparin inhibition of pulmonary artery SMC (PASMC) proliferation and pulmonary hypertension was dependent on p27 up-regulation. To investigate whether ROCK, a regulator of p27, is involved in regulation of heparin inhibition of PASMC proliferation, we analyzed ROCK expression in the lungs from mice and from human PASMCs exposed to hypoxia, and investigated the effect of ROCK expression in vitro by RhoA cDNA transfection. We also investigated the effect of guanine nucleotide exchange factor (GEF)-H1, an upstream regulator of RhoA, on heparin inhibition of PASMC proliferation by GEF-H1 cDNA transfection. We found that: (1) hypoxia increased ROCK expression in mice and PASMCs; (2) overexpression of RhoA diminished the inhibitory effect of heparin on PASMC proliferation and down-regulated p27 expression; and (3) overexpression of GEF-H1 negated heparin inhibition of PASMC proliferation, which was accompanied by increased GTP-RhoA and decreased p27. This study demonstrates that the RhoA/ROCK pathway plays an important role in heparin inhibition on PASMC proliferation, and reveals that heparin inhibits PASMC proliferation through GEF-H1/RhoA/ROCK/p27 signaling pathway, by down-regulating GEF-H1, RhoA, and ROCK, and then up-regulating p27.