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Involvement of Rho-kinase in hypertensive vascular disease: a novel therapeutic target in hypertension ¹

Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan, and the
^* Department of Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, Japan

²Correspondence: The Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3–1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: shimo{at}cardiol.med.kyushu-u.ac.jp

SPECIFIC AIM

The Rho/Rho-kinasepathway plays an important role in regulation of vascular smooth muscle cell (VSMC) contraction and other cellular functions such as proliferation and migration. However, direct evidence for the involvement of Rho-kinase in hypertensive vascular disease is lacking. The present study examined the role of Rho-kinase in functional and structural alterations of hypertensive blood vessels in spontaneously hypertensive rats (SHR).

PRINCIPAL FINDINGS

1. Functional up-regulation of Rho-kinase is involved in the hyper-reactivity of hypertensive blood vessels of SHR
To examine whether or not Rho-kinase is involved in hypertensive blood vessels, we examined SHR and Wister-Kyoto rats as a control. Isolated arterial segments were mounted on organ chamber myographs and force measurements were performed. Contractions of isolated coronary and carotid arteries in response to phenylephrine and serotonin were significantly augmented in SHR compared with WKY. Hydroxyfasudil, a specific Rho-kinase inhibitor, preferentially and significantly inhibited the contractile responses in SHR but not those in WKY. In small mesenteric arteries, contractile responses to those agonists were comparable between the 2 strains, however, hydroxyfasudil again significantly inhibited the contractile responses in SHR but not those in WKY. We then directly examined whether Rho-kinase is involved in the augmented Ca² sensitization of coronary arteries in SHR using cell permeabilization method. The augmented GTP S-induced Ca²⁺ sensitization in SHR was mediated by Rho-kinase since hydroxyfasudil normalized the augmented Ca²⁺ sensitization in SHR to the levels seen in WKY.

2. The expression and activity of Rho-kinase are augmented in hypertensive blood vessels
Expression of Rho-kinase mRNA measured by RT-PCR analysis was significantly greater in blood vessels from SHR than those from WKY (Fig. 1A ). To quantify the activity of Rho-kinase in blood vessels, we performed Western blot analysis for phophorylated myosin binding subunit (MBS) of myosin phosphatase in isolated carotid arteries using a specific antibody for MBS phosphorylated at Ser-854 by Rho-kinase. The extent of MBS phosphorylations upon stimulation by serotonin (1 µM) was significantly greater in SHR than in WKY. The increased MBS phosphorylations in SHR were significantly inhibited to the basal levels by hydroxyfasudil (Fig. 1B ).

View larger version (21K): [in this window] [in a new window]   Figure 1. Expression and function of Rho-kinase in blood vessels from WKY and SHR. A) RT-PCR analysis for Rho-kinase mRNA expression in isolated carotid arteries. Expression of Rho-kinase mRNA was significantly greater in blood vessels from SHR than in those from WKY. ß-actin was used as an internal control. Results are expressed as mean ± SE. B) Western blot analysis for MBS phosphorylations (MBS-P) in isolated carotid arteries. Carotid artery strips were incubated in physiological salt solution for 30 min with or without hydroxyfasudil (3 µM). After 5 min exposure to serotonin (1 µM), they were quickly frozen to terminate the reaction. The extracted protein was loaded for SDS-PAGE/immunoblot analysis. The extent of MBS phosphorylation in carotid arteries upon stimulation by serotonin (5-HT) (1 µM) was significantly greater in SHR than in WKY, while the basal levels of MBS phosphorylations were comparable between the 2 strains. The serotonin-induced increase in MBS phosphorylations in SHR were significantly inhibited to the basal levels by a Rho-kinase inhibitor, hydroxyfasudil (3 µM). Results are expressed as mean ± SE.

3. Functional up-regulation of Rho-kinase precedes the development of hypertension in SHR
We examined the contractile responses of isolated mesenteric arteries from 4-wk-old rats (SHR are not hypertensive yet). Hydroxyfasudil preferentially and significantly inhibited the agonists-induced contractions in young SHR but not those in WKY. Moreover, the extent of inhibitory effect of hydroxyfasudil was greater in young SHR than in adult SHR.

4. Rho-kinase plays a key role in the vascular lesion formation in hypertension
To elucidate the involvement of Rho-kinase in the vascular lesion formation in hypertensive blood vessels, we examined the inhibitory effect of long-term (5 months) oral treatment with fasudil on the vascular lesion formation in SHR. After oral absorption, fasudil is metabolized to hydroxyfasudil. Long-term treatment with fasudil effectively inhibited the vascular lesion formation such as medial thickening and perivascular fibrosis even at a nonhypotensive dose (Fig. 2A , B ). We confirmed that the long-term treatment with fasudil also effectively inhibited the Rho-kinase activity in both the heart and blood vessels of SHR (Fig. 2C ).

View larger version (53K): [in this window] [in a new window]   Figure 2. Inhibitory effect of the long-term treatment with a Rho-kinase inhibitor, fasudil, on the vascular lesion formation in SHR. A) Representative micrographs of small coronary arteries (Masson-trichrome staining) from WKY, control SHR (SHR, C), SHR treated with a low-dose (SHR, LD) and a high-dose of fasudil (SHR, HD). The bar indicates 100 µm. B) Quantitative analysis of medial thickening (as shown by wall-to-lumen ratio, upper panel) and perivascular fibrosis (lower panel) in small coronary arteries. Results are expressed as mean ± SE. P < 0.01 vs. WKY, *P < 0.05, **P < 0.01 vs. SHR, C. C) Western blot showing Rho-kinase inhibition demonstrated by the decrease in phosphorylated MBS (MBS-P) caused by the long-term (5 months) treatment with fasudil in the heart and the carotid artery (CA) of SHR (representative of 3 independent experi-ments).

CONCLUSIONS

In summary, the present study provides the novel findings that 1) up-regulation of Rho-kinase in blood vessels is involved in the vascular hyper-reactivity of SHR through inhibition of MLCPh, 2) up-regulation of Rho-kinase precedes the development of hypertension in SHR, and 3) Rho-kinase is substantially involved in the vascular lesion formation in SHR. To the best of our knowledge, this is the first report that directly demonstrates the involvement of Rho-kinase in hypertensive vascular disease. Although essential hypertension is one of the major predisposing risk factors of cardiovascular disease, its pathogenesis still remains to be fully elucidated. Intracellular signaling pathway mediated by the small G protein Rho and its target molecule Rho-kinase plays an important role in regulation of VSMC contraction and other cellular functions such as proliferation, hypertrophy, adhesion, and migration of vascular cells including VSMC, inflammatory cells, and fibroblasts. This study provides the first evidence that up-regulation of Rho-kinase plays a key role in the pathogenesis of hypertensive vascular disease. Thus, Rho-kinase could be regarded as a novel therapeutic target for hypertensive vascular disease.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic diagram showing a hypothesis how Rho-kinase is involved in the pathogenesis of hypertensive vascular disease. It is highly possible that Rho-kinase-mediated pathway is involved in a) hypercontraction of VSMC, b) proliferation and migration of VSMC, c) recruitment of inflammatory cells, and d) perivascular fibrosis. These Rho-kinase-related functional and structual alterations may be involved in the pathogenesis of hypertensive vascular disease.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0735fje ; to cite this article, use FASEB J. (February 20, 2001) 10.1096/fj.00-0735fje

This Article Full Text (PDF) All Versions of this Article: 15/6/1062 00-0735fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by MUKAI, Y. Articles by TAKESHITA, A. Search for Related Content PubMed PubMed Citation Articles by MUKAI, Y. Articles by TAKESHITA, A.