Contraction is a primary function of vascular smooth

Contractile Properties of the Cultured Vascular Smooth Muscle Cells The Crucial Role Played by RhoA in the Regulation of Contractility Dan Bi, Junji Nishimura, Naohisa Niiro, Katsuya Hirano, Hideo Kanaide Abstract Vascular smooth muscle cells (VSMCs) have a remarkable degree of plasticity and in response to vascular injury, they can change to a dedifferentiated state that can be typically seen in cell cultures. Recently, Y27632, a Rho kinase inhibitor, has been reported to preferentially correct hypertension in a hypertensive rat model. We thus tested the hypothesis that the contraction of the cultured VSMCs might be more dependent on the function of RhoA than the VSMCs in fresh tissue. For this purpose, a tissue-like ring preparation was made using the cultured porcine coronary artery SMCs (CASMCs) and collagen gel (reconstituted ring: R-ring). The R-ring developed an isometric tension on stimulation by high external K or various receptor agonists. The phorbol ester (a protein kinase C (PKC) activator)-induced contraction of the intact R-ring was greatly inhibited, while the GTP S (an activator of RhoA)-induced and Ca 2 -independent contraction of permeabilized R-ring was greatly enhanced, in comparison to the fresh coronary artery ring. An immunoblot analysis showed the expression levels of RhoA and myosin phosphatase subunits (MYPT1 and PP1c ) to be up-regulated, while the levels of CPI-17 (PKC-potentiated protein phosphatase-1 inhibitory protein), h1-calponin and PKC isoforms were downregulated in cultured CASMCs. The knock down of RhoA by RNA interference decreased the contractility of the cultured CASMCs. It is concluded that the contractility of the cultured VSMCs thus appears to be much more dependent on the function of RhoA than VSMCs in fresh tissue. The expression level of RhoA thus plays a crucial role in regulating the contractility of cultured VSMCs. (Circ Res. 2005; 96:890-897.) Key Words: vascular smooth muscle contraction RhoA CPI-17 RNA interference vascular disease Contraction is a primary function of vascular smooth muscle cells (VSMCs) and those within adult blood vessels proliferate at an extremely low rate. However, in response to vascular injury, VSMCs dramatically increase their proliferation rate and become dedifferentiated due to their remarkable plasticity, which induces changes in their contractile properties. 1 It is thus of great interest to elucidate the changes in the contractile properties of the proliferating dedifferentiated VSMCs. We considered the most suitable model of cells to investigate this to be cultured VSMCs, because in vitro culturing of VSMCs mimics the dedifferentiation induced by vascular injury. 2 The contractility of cultured VSMCs had been indirectly evaluated by measuring the cell shape, cell shortening, or cell area. 3 5 However, new methods to directly measure an isometric tension development of the VSMCs, 6 SMCs, 7 and even of the nonmuscle cells such as fibroblasts 8 have recently been reported. As a result, it is now possible to measure the isometric tension development of cultured VSMCs. The contraction of VSMCs is primarily regulated by the transient changes in the intracellular Ca 2 concentration ([Ca 2 ] i ) and the subsequent phosphorylation of the myosin light chain (MLC) catalyzed by Ca 2 /calmodulin-dependent MLC kinase (MLCK). 9 However, the Ca 2 sensitivity of the contractile apparatus has been demonstrated to change during various types of stimulation. 10,11 Recent research on the mechanism regulating the increase in Ca 2 sensitivity showed two major pathways to be involved in this mechanism, namely, the RhoA-Rho kinase pathway and the protein kinase C (PKC)-CPI-17 (PKC-potentiated protein phosphatase-1 inhibitory protein) pathway. 12 Especially the RhoA-Rho kinase pathway has recently been implicated in the pathogenesis of the abnormal contraction of the VSMCs in vascular diseases, thus indicating that this pathway might play an important role in cultured VSMCs. 13 One of the major advantages of using cultured cells is that we can knock down the expression of a gene of interest in these cells by the use of RNA interference (RNAi). RNAi is a strategy of sequence-specific posttranscriptional gene silencing, and it has been successfully applied for the disruption of a gene transcript in plants and caenorhabditis elegans. Gene silencing by RNAi had been accomplished in mamma- Original received April 16, 2004; resubmission received November 29, 2004; revised resubmission received March 3, 2005; accepted March 9, 2005. From the Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. Correspondence to Hideo Kanaide, MD, PhD, Prof, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan. E-mail kanaide@molcar.med.kyushu-u.ac.jp 2005 American Heart Association, Inc. Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000163018.66460.85 890

Bi et al RhoA and Contractility in Cultured VSMCs 891 lian cells. 14,15 Recently, RNAi has been successfully applied to the VSMCs. 16,17 As a result, it is now possible to knock down a gene of interest in the cultured VSMCs. In the present study, we investigated the contractile properties of cultured porcine coronary artery SMCs (CASMCs) and compared them with those of fresh coronary artery rings (F-CA-rings). The obtained results indicated that the contractions of the cultured CASMCs are more dependent on the RhoA-mediated pathway than on the PKC-mediated pathway, in comparison to the F-CA-rings. The expression levels of RhoA, MYPT1 (a large subunit of MLC phosphatase, MLCP), and PP1c (a catalytic subunit of MLCP) in the cultured CASMCs were upregulated, whereas the levels of CPI-17, Rac1, two isoforms of Rho kinase, MLCK, h1- calponin, PKC, and PKC were downregulated. The knockdown of RhoA induced a decrease in the contractility of the cultured CASMCs. These results indicated that the expression level of RhoA thus plays a critical role in the regulation of contractility in the dedifferentiated VSMCs. Materials and Methods Cell Culture The cultured porcine CASMCs were prepared as previously described, 18 with minor modifications. Briefly, the media layer of the right coronary artery was treated with 1.0 mg/ml collagenase and 10 U/mL elastase for 60 minutes. The primary cultured cells were prepared by the collagen gel embedded method, using a collagen gel matrix (Cellmatrix Type I-P, Nitta gelatin) and a Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. The primary cultured cells were subcultured in a culture dish and maintained in a culture medium, as described, containing 50 g/ml sodium ascorbate, in order to maintain the contractility. 6 Production of a Reconstituted Ring The cultured CASMCs were collected by trypsinization and mixed with the culture medium (10 7 cells/ml) containing 0.6 mg/ml type I collagen (Cellmatrix Type I-P). The mixture (100 L, 10 6 cells) was placed around a silicone column ( 2 mm), which was attached on the center of the silicone disc ( 8 mm). After the collagen gel became solid, the culture medium was added and cultured for 24 to 48 hours. The ring preparation so obtained was used for measurements of isometric tension development. Measurement of Tension in the Intact or -Toxin Permeabilized Preparations Both the tension measurements and -toxin permeabilization were performed as described previously. 10 The F-CA-rings ( 2 mm, 1-mm long) were prepared from porcine right coronary artery after removing the adventitial tissue and endothelium. For the tension recordings, the ring preparations (F-CA or reconstituted ring, R-rings) were mounted onto two tungsten wires by passing the wires through the lumen of the rings. When needed, the rings were permeabilized with 5000 U/mL Staphylococcus -toxin ( hemolysin, Sigma) for 60 minutes in a solution composed of (in mmol/l) 10 EGTA, 100 potassium methanesulphonate, 3.38 MgCl 2, 2.2 Na 2 ATP, 10 creatine phosphate, and 20 Tris-maleate; ph 6.8. Measurement of isometric tension was performed at 24 to 25 C. MLC Phosphorylation The phosphorylation of MLC was determined as described previously. 19 The permeabilized R-rings were treated with pca 8 solution alone or with pca 8 plus 10 mol/l Y27632 or with pca 8 plus 10 mol/l GTP S. The sample was subjected to a urea-glycerol gel electrophoresis and a subsequent immunoblot analysis using anti- MLC (Sigma) or anti phospho-mlc at Ser19 (generously provided by Dr M. Ikebe, University of Massachusetts Medical School, Worcester, Mass) antibody. Immunoblot Analysis Proteins were extracted from cultured CASMCs or F-CA and subjected to immunoblot analysis as previously described. 20 Regarding the tissue samples, freshly dissected right coronary artery was rapidly frozen in liquid nitrogen and then was shattered by hammering. Thirty micrograms of total protein was separated with SDS- PAGE and transferred to polyvinylidene difluoride membrane (Bio- Rad). Membranes were incubated with the indicated antibodies. Antigen detection was performed with an ECL plus detection kit (Amersham). The following antibodies were used: Anti-RhoA, anti-mlck, anti h1-calponin (Santa Cruz Biotechnology), anti- Rac1, anti-rock I, anti-rock II, anti-pkc (BD Biosciences, San Jose, CA), anti CPI-17, anti-pkc, anti-pp1c (Upstate Biotechnology), and anti-mypt1 (Babco). RhoA Translocation RhoA translocation was determined using the methods described by Massey et al. 21 The -toxin permeabilized F-CA-rings or R-rings were lysed with ice-cold hypotonic buffer, after they were treated with pca 8 solution with or without 10 mol/l GTP S. After the membrane and cytosolic fractions were separated by differential centrifugation, equal amounts of proteins (15 g) were applied to SDS-PAGE. The RhoA protein was detected by an immunoblot analysis as described. The membrane to total RhoA ratio was considered to represent RhoA translocation. Knockdown of RhoA by RNAi The single-stranded sense and antisense RNAs, based on the pig RhoA mrna, 22 and their inverted sequences (control) were synthesized by Hokkaido System Sciences. The sequences were 5 -GUGGAUGGAAAGCAGGUAGAGTT-3 (RhoA sense), 5 - CUCUACCUGCUUUCCAUCCACTT-3 (RhoA antisense), 5 - GAGAUGGACGAAAGGUAGGUGTT-3 (RhoA inverted sense), 5 -CACCUACCUUUCGUCCAUCUCTT-3 (RhoA inverted antisense). The annealing of RNAs and transfection were performed using the method described by Elbashir et al. 14 Cationic lipid complexes containing 200 pmol of indicated small interfering RNA (sirna) and 6 L oligofectamine (Invitrogen) were added to a dish. The cells were exposed to Opti-MEM1 (Invitrogen) containing 25 nmol/l RNA for 40 to 45 hours and then the cells were used for the experiments. Measurement of [Ca 2 ] i in the CASMCs The changes in [Ca 2 ] i of the cells cultured on 35-mm dishes were monitored with front-surface fluorometry using fura-2 as previously described. 23 The [Ca 2 ] i levels were expressed as a percentage, assigning those obtained at rest and with 25 mol/l ionomycin to be 0% and 100%, respectively. Data Analysis The data were expressed as the mean SEM along with the number of observation ( n). Statistical differences between the sets of data were determined using either the unpaired or paired Student t tests. Results Contractility of the R-Ring Made of CASMCs In order to examine the contractility of the cultured VSMCs, we first established a method to measure the isometric tension development of the cultured CASMCs. We added sodium ascorbate to the culture medium to keep the cultured CASMCs contractile, according to the method reported by L Heureux et al. 6 Using these contractile CASMCs, we made a tissue-like R-ring preparation similar to the smooth muscle

892 Circulation Research April 29, 2005 Figure 1. Isometric tension development of the R-ring made of cultured CASMCs. R-ring responded to 118 mmol/l K (A), 1 mol/l bradykinin (BK) (B), 100 mol/l histamine (His) (C), and 1 mol/l endothelin 1 (ET-1) (D). Addition of 10 mol/l sodium nitroprusside (SNP) (E) or 1 mol/l Y27632 (F) during the contraction induced by 118 mmol/l K caused a relaxation. Traces shown are representative of 7 (A through D, and F) or 6 (E) independent experiments. cell populated collagen gel fiber described by Oishi et al. 24 As shown in Figure 1, we could determine the isometric tension development of the R-ring made of these CASMCs. The R-ring responded to a high external K solution (44.1 4.2 mg, n 7) as well as the various receptor agonists, including bradykinin (90.9 12.3 mg, n 7), histamine (91.9 14 mg, n 7), and endothelin-1 (8.6 1.9 mg, n 7; Figure 1A through 1D). Furthermore, the R-ring relaxed after the addition of sodium nitroprusside, as shown in Figure 1E. The application of 10 mol/l sodium nitroprusside during the contraction induced by 118 mmol/l K solution reduced the level of tension to 27 11% (n 6), assuming the levels of resting and 118 mmol/l K solution-induced contraction to be 0% and 100%, respectively. In addition, as shown in Figure 1F, the application of 1 mol/l Y27632, a Rho kinase inhibitor, 25 during the contraction induced by 118 mmol/l K solution reduced the level of tension to 3.8 1.8% (n 7). To examine the involvement of PKC in the contraction of the cultured contractile CASMCs, we applied phorbol-12,13- dibutyrate (PDBu), a PKC activator, as shown in Figure 2A. Although 0.3 mol/l PDBu induced a contraction of the R-ring, it was much smaller than the 118 mmol/l K solution induced contraction (23 5% of this contraction; n 5). We next examined the involvement of the G protein (presumably RhoA) mediated pathway in the contraction of the cultured CASMCs. For this purpose, we performed experiments using -toxin permeabilized preparation. In the presence of Ca 2 (pca 6), the enhancement of contraction induced by 10 mol/l GTP S, a nonhydrolyzable GTP analog that is known to activate G protein, in R-rings (26 3% of the 10 mol/l Ca 2 -induced contraction, n 3) was significantly (P 0.05) greater than that in F-CA-rings (17 2%, n 3, traces not shown). However, the most marked difference could be seen in the nominally Ca 2 free medium containing 10 mmol/l EGTA (pca 8). As shown in Figure 2A, the permeabilized preparation contracted at pca 8 with Figure 2. Effect of PDBu or GTP S on the R-rings (A and C) and F-CA-rings (B). A, Application of 0.3 mol/l PDBu to the intact R-ring induced only a small contraction. After permeabilization, 10 mol/l GTP S applied in the nominally Ca 2 free solution (pca 8) induced a significant contraction. B, PDBu induced a large contraction in the intact F-CA-rings. GTP S induced only a small contraction in the permeabilized F-CArings at pca 8. C, GTP S increased (P 0.01, n 5) and Y27632 (10 mol/l) decreased (P 0.01, n 3) the MLC phosphorylation level of the permeabilized R-ring at pca 8 (P 0.01, n 8). MLC was monophosphorylated at Ser 19, and no diphosphorylated MLC could be detected. the addition of 10 mol/l GTP S. The level of this contraction reached 31 7% (n 7) of the maximal 10 mol/l Ca 2 -induced contraction. Figure 2B shows the effects of 0.3 mol/l PDBu or 10 mol/l GTP S on the contractions of the F-CA-rings. The PDBu-induced contractions relative to the 118 mmol/l K solution induced contraction (129 7.2%; n 4) in the F-CA-rings were significantly (P 0.01) greater than those in the R-rings, whereas inactive phorbol (4 -phorbol-12,13-didecanoate; Sigma) had no effect (trace not shown). On the other hand, the GTP Sinduced contractions at pca 8 relative to the 10 mol/l Ca 2 -induced maximum contractions (5.7 1.5%; n 5) in the permeabilized F-CA-rings were significantly (P 0.01) smaller than those in the R-rings. In order to further characterize the GTP S-induced contraction, we measured the MLC phosphorylation. As shown

Bi et al RhoA and Contractility in Cultured VSMCs 893 Figure 3. Immunoblot analysis of the selected proteins in the F-CA and cultured CASMCs. A, Representative photographs of immunoblot of the indicated protein in F-CA and the cultured CASMCs (Culture). B, Summary of results obtained from 3 to 5 independent experiments performed in a similar manner as that shown in A. Expressions of RhoA (P 0.01), MYPT1 (P 0.05), and PP1c (P 0.01) were upregulated, whereas those of Rac1, CPI-17, ROCK I, ROCK II, h1-calponin, MLCK, PKC, and PKC were downregulated (all P 0.01). in Figure 2C, GTP S-induced Ca 2 -independent contraction of R-ring accompanied the increased level of MLC phosphorylation that was revealed to be at Ser 19 determined by the anti phospho-mlc at Ser19 antibody. No diphosphorylated MLC could be detected. In addition, the relatively high level of MLC phosphorylation (20 2%, n 8) at rest (pca 8) decreased to 5 1% (n 3, P 0.01) in the presence of 10 mol/l Y27632. Change in the Expression of the Selected Proteins in Cultured CASMCs These results indicated that the contraction of the cultured VSMCs might be more dependent on the function of RhoA, but less dependent on the function of PKC, than that of the normal VSMCs. We thus next examined the expression levels of the selected proteins in cultured CASMCs and compared them with those in F-CA. As shown in Figure 3, the expression levels of RhoA, MYPT1, and PP1c increased in the cultured CASMCs in comparison to those in F-CA. On the other hand, the expression levels of Rac1, CPI-17, Rho kinases (ROCK I and ROCK II), h1-calponin, MLCK, PKC, and PKC decreased in the cultured CASMCs. To further confirm the role of RhoA in the GTP S-induced Ca 2 - independent contractions, we measured the translocation of RhoA. The membrane-bound active form of RhoA was not significantly changed in the permeabilized F-CA-rings treated with GTP S at pca 8, whereas it significantly increased in the R-rings under the same conditions (Figure 4). Knockdown of RhoA by RNAi To reduce the expression level of RhoA, we used RNAi. As shown in Figure 5A, the expression of RhoA of the CASMCs transfected by sirna targeting RhoA was much lower than Figure 4. RhoA translocation induced by GTP S in F-CA-rings and R-rings at pca 8. -Toxin permeabilized F-CA-rings or R-rings were treated with pca 8 solution with or without 10 mol/l GTP S. After the membrane (M) and cytosolic (C) fractions were separated, equal amounts of proteins (15 g) were applied to SDS-PAGE. Representative photograph and a summary (n 4 in F-CA-rings and n 3 in R-rings) are shown. Ratio of membrane-bound to total RhoA (M/T) significantly (P 0.05) increased in the GTP S-treated R-rings, but not in the F-CA-rings. that of the control cells (Figure 5B; P 0.01) that were transfected with sirna of the reversed sequence. On the other hand, there was no difference in the expression levels of Rac1, ROCK I, ROCK II, and CPI-17 between RhoA knocked-down and control cells. Figure 5B shows a summary of the data obtained from three experiments done in a similar manner as in Figure 5A. These results indicated that RhoA protein could be selectively knocked down by RNAi technology. In addition, there was no significant difference in [Ca 2 ] i Figure 5. Immunoblot of the RhoA knocked-down CASMCs by RNAi. A, At 48 hours after transfection, equal amounts of lysate proteins (30 g) of control and RhoA knocked-down (KD) cells were analyzed on an immunoblot with an antibody against RhoA, Rac1, ROCK I, ROCK II, or CPI-17. In case of CPI-17, the sample from F-CA was also run as a positive control. B, Summary of the densitometric analysis of the three independent experiments performed in a similar manner as in A. Expression levels of the indicated proteins in the RhoA knocked-down cells are shown, with those in the control cells set at 100%.

894 Circulation Research April 29, 2005 addition, the maximal contractions induced by 10 mol/l Ca 2 solution in the RhoA knocked-down cells were also much smaller than those in the control cells (75 17 versus 42 16 mg; P 0.05), thus indicating a possible involvement of RhoA in the Ca 2 -induced contraction of R-rings. As shown in Figure 8C, the addition of 1 mol/l Y27632 inhibited the 10 mol/l Ca 2 -induced contraction of the R-rings to 11.1 1.4% (n 3) of this contraction. However, 1 mol/l Y27632 had no effect on the 10 mol/l Ca 2 - induced contraction of the F-CA-rings (Figure 8D; 97 2%, n 3; P 0.01 compared with the R-rings). Discussion In the present study, we investigated the contractility of the cultured CASMCs by measuring the isometric tension development of the R-rings and then compared it with that of the F-CA-rings. The contraction induced by PDBu was much smaller than that induced by high K in R-rings, although it was much greater than that by high K in F-CA-rings. In contrast, GTP S induced a significant contraction of the permeabilized R-rings at pca 8 in comparison to 10 mol/l Ca 2 -induced contraction, whereas it induced only slight contractions in F-CA-rings under the same condition. Figure 6. Effects of the RhoA knockdown on [Ca 2 ] i of the cultured CASMCs. A, Changes in [Ca 2 ] i during stimulation by 100 mmol/l K depolarization, 1 mol/l bradykinin (BK), and 100 mol/l histamine (His) in the RhoA knocked-down CASMCs (RhoA KD) and the control CASMCs (Control). Level of [Ca 2 ] i in the presence of 25 mol/l ionomycin was designated as 100%. B, Summary of the results obtained from 4 independent experiments performed in a similar manner as in A. There was no significant difference in the [Ca 2 ] i elevation induced by 100 mmol/l K depolarization, bradykinin, or histamine between RhoA knocked-down CASMCs and the control CASMCs. transients induced by 100 mmol/l K -depolarization, bradykinin, or histamine between RhoA knocked-down and control cells, as shown in Figure 6. On the other hand, RhoA knocked-down cells showed a very low contractility in comparison to the control cells. The contractions induced by 118 mmol/l K -depolarization as well as the various receptor agonists, including bradykinin, histamine, and endothelin-1 became significantly smaller than those of the control (Figure 7), whereas the contractions of the RhoA knocked-down cells induced by 1 mol/l calyculin A, a membrane permeant Type 1 and 2A phosphatase inhibitor, remained unchanged (104 44% of the control, n 4, P 0.05). Figure 8 shows the GTP S-induced contraction of the -toxin permeabilized preparation made of control or RhoA knocked-down cells at pca 8. The GTP S-induced contraction of the RhoA knocked-down cells (42 3%; n 7; Figure 8B) was significantly (P 0.05 by paired Student t test) smaller than that of the control cells (46 4%; n 7; Figure 8A), although the GTP S-induced Ca 2 -independent contraction of the RhoA knocked-down cells could not be completely inhibited. In Figure 7. Effect of RhoA knockdown on the contractility. A, Contractions induced by 118 mmol/l K,1 mol/l bradykinin (BK), 100 mol/l histamine (His), and 1 mol/l endothelin-1 (ET-1) of the RhoA knocked-down CASMCs (RhoA KD) were smaller than those of the control CASMCs (Control). B, Summary of the effects of RhoA knockdown on the contractility. Data are the mean SEM (n 4). Contractions of the RhoA knocked-down CASMCs were significantly (P 0.05) smaller than the control not only with agonist stimulation but also with 118 mmol/l K depolarization. Level of contraction induced by 118 mmol/l K depolarization in the control was designated as 100%. Statistical differences between the sets of data were determined using paired Student t tests.

Bi et al RhoA and Contractility in Cultured VSMCs 895 Figure 8. GTP S-induced contraction of the control and RhoA knocked-down CASMCs after permeabilization. Application of GTP S (10 mol/l) at pca 8 induced a smaller contraction in the RhoA knocked-down CASMCs (B) than in the control CASMCs (A). Traces shown are representative of 7 independent experiments. C, 1 mol/l Y27632 can relax the 10 mol/l Ca 2 -induced contraction in the permeabilized R-ring. D, 1 mol/l Y27632 had no effect on the 10 mol/l Ca 2 -induced contraction in the permeabilized F-CA-ring. An immunoblot analysis revealed that the expressions of PKC isoforms and CPI-17 decreased, whereas those of RhoA, MYPT1, and PP1c increased in the cultured CASMCs, compared with those of the F-CA. The knockdown of RhoA by RNAi markedly decreased the contractility. These results indicated that the contraction of the cultured CASMCs might be more dependent on the function of RhoA, but less dependent on the function of PKC, than fresh tissue. The expression level of RhoA appeared to be crucial in the regulation of contractility of the cultured VSMCs. Although the cultured VSMCs have been reported to lose their contractility, 26 L Heureux et al 6 recently reported a method to obtain the contractile cultured VSMCs that displayed a differentiated phenotype as demonstrated by the reexpression of VSMC-specific markers and actual tissue contraction in response to physiological stimuli. Using this method, we could also obtain the contractile cultured CASMCs that expressed VSMC-specific marker, h1- calponin, and functionally expressed cgmp-dependent protein kinase (PKG). Because it has been reported that the synthetic and proliferative phenotype cells do not express PKG, 27 these observations (contractility, h1-calponin expression, and functional expression of PKG) supported the notion that the CASMCs used in the present study are more differentiated, compared with the conventional synthetic cultured VSMCs. The contraction of the VSMCs is primarily regulated by the level of [Ca 2 ] i. The increase in [Ca 2 ] i causes the calmodulin-mediated activation of MLCK, which phosphorylates the MLC and thereby induces contraction. A decrease in [Ca 2 ] i inactivates MLCK and permits the dephosphorylation of MLC by MLCP, thus causing relaxation. 9 However, the extent of [Ca 2 ] i elevation has been reported to not always be parallel to the extent of contraction, 11,28 thus suggesting that a secondary regulatory pathway might play a role in the control of smooth muscle contractility. 10,11 Such secondary pathways may include the PKC-CPI-17 mediated or RhoA- Rho kinase mediated inhibition of MLCP. 12 However, the contraction induced by PDBu was much smaller than that induced by high K in R-rings, although it was much greater than that by high K in F-CA-rings. In support of this physiological observation, an immunoblot analysis revealed the expression levels of CPI-17 and PKC isoforms (PKC and PKC ) to be downregulated. From these results, the PKC-mediated pathway was considered to possibly not be a major pathway for the contraction of cultured CASMCs. GTP S is known to increase Ca 2 sensitivity of myofilament of VSMCs, but the presence of intracellular Ca 2 is thought to be a prerequisite condition for GTP S-induced contraction of the permeabilized preparation. 29 Consistent with this, the Ca 2 sensitizing effects by GTP S could be observed in both R-rings and F-CA-rings in the presence of Ca 2 (pca 6). However, the Ca 2 sensitizing effects by GTP S were greater in R-rings than in F-CA-rings. This difference could be clearly seen at pca 8. The permeabilized preparation of the R-rings significantly contracted even at pca 8 with the addition of 10 mol/l GTP S, in comparison to the 10 mol/l Ca 2 -induced contraction, whereas only little contraction could be obtained in F-CArings under the same condition. In good agreement with this physiological observation, the immunoblot analysis revealed that the expression of RhoA in cultured CASMCs increased and the membrane-bound active form of RhoA of the R-rings was also increased during activation with GTP S. In order to further confirm the role of RhoA, we next examined the effect of the RhoA knockdown on the contractility of the cultured CASMCs. We first checked the validity of the method of RNAi. The transfection of the sirna targeting RhoA selectively knocked down the expression of RhoA. The R-ring preparation made of RhoA knocked-down cells showed markedly decreased contractility, compared with the control. This decrease in contractility of the RhoA knocked-down cells was not due to the impaired function of the Ca 2 mobilization, because there was no difference in [Ca 2 ] i

896 Circulation Research April 29, 2005 transient between RhoA knocked-down and control cells. To further exclude this possibility, we also performed the experiments using the permeabilized preparation. The RhoA knockdown inhibited not only the GTP S-induced Ca 2 - independent contractions, but also the 10 mol/l Ca 2 - induced contraction. These results supported the idea that the contractility of the cultured CASMCs may be mainly dependent on the function of RhoA. Because the GTP S-induced Ca 2 -independent contractions of R-rings accompanied the increased level of MLC phosphorylation, we examined the kinases that were responsible for MLC phosphorylation. This increased MLC phosphorylation was found out to be monophosphorylation at Ser19 (Figure 2C). We thus considered it unlikely that ZIP-like kinase and/or integrin-linked kinase are involved in this contraction, because these kinases were reported to be nonselective to Ser19 and Thr18. 30,31 On the other hand, Rho kinase has been shown to directly phosphorylate MLC at Ser19 to induce contraction in a Ca 2 -independent manner. 32,33 It is thus possible that this mechanism might play a role in cultured VSMCs because of the increased expression of RhoA in the cultured CASMCs. Consistent with this speculation, the relatively high level of MLC phosphorylation at rest (pca 8) could be inhibited in the presence of 10 mol/l Y27632. In R-rings, all the contractions induced by depolarization, 10 mol/l Ca 2, and agonists, except for those induced by calyculin A, were inhibited by RhoA knockdown, indicating that RhoA-mediated pathway is thus likely involved in all three types of contractions. Consistent with this, it has recently been reported that membrane depolarization by 60 mmol/l KCl as well as noradrenalin (3 mol/l) stimulation induced similar time-dependent, sustained increases in the amount of an active GTP-bound form of RhoA. 34 This notion was also supported by the observations that the 118 mmol/l K -induced contraction (Figure 1F), as well as the 10 mol/l Ca 2 -induced contraction (Figure 8C), could be effectively inhibited by 1 mol/l Y27632 in R-rings. In contrast, 10 mol/l Ca 2 -induced contraction of F-CA-rings could not be inhibited by 1 mol/l Y27632 (Figure 8D). It was thus indicated that the contractions are more dependent on RhoA-Rho kinase pathway in R-rings than in F-CA-rings. This was thought to be the major reason why the GTP Sinduced contraction relative to that induced by 10 mol/l Ca 2 could not be completely inhibited after RhoA knockdown, because both contractions were equally inhibited by RhoA knockdown that cannot completely inhibit the expression of RhoA. Concerning the mechanism for the greater dependency of contraction on RhoA-Rho kinase pathway in R-rings than in F-CA-rings, the increased MYPT1 and PP1 expressions and the decreased MLCK and h1-calponin expressions were thought to be involved, in addition to the increased RhoA expression. In R-rings, the Ca 2 -MLCK pathway may not effectively induce contraction, because the increased MYPT1 and PP1c expressions should be coupled with the increase in the MLCP activity. In contrast, RhoA-Rho kinase pathway may be more effective in inducing contraction, because this pathway also inhibits MLCP. 12 The h1-calponin could be another possible candidate for the increase in GTP S-induced contraction of the cultured CASMCs, because calponin has been shown to inhibit the myosin ATPase. 35 In conclusion, we measured the isometric tension development of the cultured CASMCs. The excitation-contraction coupling of the cultured CASMCs was much different from that of the fresh tissue, in terms of the dependency on Ca 2, G protein, or PKC, possibly due to the upregulation of RhoA, MYPT1, and PP1c and/or downregulation of PKC isoforms, CPI-17 and h1-calponin. We thus were tempted to speculate that similar changes in the contractile properties might be involved in the abnormal contractility seen in various vascular diseases. In good agreement with this speculation, Uehata et al 25 reported that Y27632 dramatically corrected hypertension in several hypertensive rat models, although it did not change the blood pressure of the nonhypertensive rats. If there was no difference in the contractile mechanism of VSMCs between hypertensive and normal rats, Y27632 should have similar effects on the blood pressure of the normal rats. Acknowledgments This study was supported in part by the grant from the 21st Century COE Program and Grants-in-Aid for Scientific Research (nos. 15590758, 16590695) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank Dr M. Ikebe (University of Massachusetts Medical School, Worcester, Mass) for supplying the anti phospho-mlc at Ser19 antibody. We also thank Brian Quinn for linguistic comments. References 1. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004;84:767 801. 2. Martin KA, Rzucidlo EM, Merenick BL, Fingar DC, Brown DJ, Wagner RJ, Powell RJ. The mtor/p70 S6K1 pathway regulates vascular smooth muscle cell differentiation. Am J Physiol Cell Physiol. 2004;286: C507 C517. 3. Kobayashi S, Kanaide H, Hasegawa M, Yamamoto H, Nakamura M. Rat vascular smooth muscle cells in culture contract upon Ca 2 repletion after depletion. Br J Exp Pathol. 1985;66:719 728. 4. Li S, Sims S, Jiao Y, Chow LH, Pickering JG. Evidence from a novel human cell clone that adult vascular smooth muscle cells can convert reversibly between noncontractile and contractile phenotypes. Circ Res. 1999;85:338 348. 5. Gohla A, Schultz G, Offermanns S. Role for G(12)/G(13) in agonistinduced vascular smooth muscle cell contraction. Circ Res. 2000;87: 221 227. 6. L Heureux N, Stoclet JC, Auger FA, Lagaud GJ, Germain L, Andriantsitohaina R. A human tissue-engineered vascular media: a new model for pharmacological studies of contractile responses. FASEB J. 2001;15:515 524. 7. Oishi K, Itoh Y, Isshiki Y, Kai C, Takeda Y, Yamaura K, Takano- Ohmuro H, Uchida MK. Agonist-induced isometric contraction of smooth muscle cell-populated collagen gel fiber. Am J Physiol Cell Physiol. 2000;279:C1432 C1442. 8. Kolodney MS, Wysolmerski RB. Isometric contraction by fibroblasts and endothelial cells in tissue culture: a quantitative study. J Cell Biol. 1992;117:73 82. 9. Kamm KE, Stull JT. The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacol Toxicol. 1985;25:593 620. 10. Nishimura J, Kolber M, van Breemen C. Norepinephrine and GTPgamma-S increase myofilament Ca 2 sensitivity in alpha-toxin permeabilized arterial smooth muscle. Biochem Biophys Res Commun. 1988;157: 677 683. 11. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature. 1994;372:231 236.

Bi et al RhoA and Contractility in Cultured VSMCs 897 12. Somlyo AP, Somlyo AV. Signal transduction by G-proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol (London). 2000;2:177 185. 13. Shimokawa H. Rho-kinase as a novel therapeutic target in treatment of cardiovascular diseases. J Cardiovasc Pharmacol. 2002;39:319 327. 14. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001;411:494 498. 15. Hannon GJ. RNA interference. Nature. 2002;418:244 251. 16. Sussmann M, Sarbia M, Meyer-Kirchrath J, Nusing RM, Schror K, Fischer JW. Induction of hyaluronic acid synthase 2 (HAS2) in human vascular smooth muscle cells by vasodilatory prostaglandins. Circ Res. 2004;94:592 600. 17. Deroanne C, Vouret-Craviari V, Wang B, Pouyssegur J. EphrinA1 inactivates integrin-mediated vascular smooth muscle cell spreading via the Rac/PAK pathway. J Cell Sci. 2003;116:1367 1376. 18. Shogakiuchi Y, Kanaide H, Nakamura M. Cytosolic calcium transients differ between porcine coronary arterial and aortic smooth muscle cells in primary culture. Circ Res. 1991;68:818 826. 19. Maeda Y, Hirano K, Nishimura J, Sasaki T, Kanaide H. Rho-kinase inhibitor inhibits both myosin phosphorylation-dependent and -independent enhancement of myofilament Ca 2 sensitivity in the bovine middle cerebral artery. Br J Pharmacol. 2003;140:871 880. 20. Hirano K, Derkach DN, Hirano M, Nishimura J, Takahashi S, Kanaide H. Transduction of the N-terminal fragments of MYPT1 enhances myofilament Ca 2 sensitivity in an intact coronary artery. Arterioscler Thromb Vasc Biol. 2004;24:464 469. 21. Massey AR, Miao L, Smith BN, Liu J, Kusaka I, Zhang JH, Tang J. Increased RhoA translocation in renal cortex of diabetic rats. Life Sci. 2003;72:2943 2952. 22. Nishimura J, Sakihara C, Zhou Y, Kanaide H. Expression of rho A and rho kinase mrnas in porcine vascular smooth muscle. Biochem Biophys Res Commun. 1996;227:750 754. 23. Hirano K, Hirano M, Kanaide H. Enhancement by captopril of bradykinin-induced calcium transients in cultured endothelial cells of the bovine aorta. Eur J Pharmacol. 1993;244:133 137. 24. Oishi K, Ishibashi T, Nakamura S, Mita M, Uchida MK. Protein kinase C promotes spontaneous relaxation of streptolysin-o-permeabilized smooth muscle cells from the guinea-pig stomach. Life Sci. 1999;64: 1975 1987. 25. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 1997;389:990 994. 26. Chamley JH, Campbell GR, McConnell JD, Groschel-Stewart U. Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and in subculture. Cell Tissue Res. 1977;177: 503 522. 27. Lincoln TM, Dey N, Sellak H. cgmp-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol. 2001;91:1421 1430. 28. Kanaide H. Measurement of [Ca 2 ] i in smooth muscle strips using frontsurface fluorimetry. Methods Mol Biol. 1999;114:269 277. 29. Nishimura J, van Breemen C. Direct regulation of smooth muscle contractile elements by second messengers. Biochem Biophys Res Commun. 1989;163:929 935. 30. Niiro N, Ikebe M. Zipper-interacting protein kinase induces Ca 2 -free smooth muscle contraction via myosin light chain phosphorylation. J Biol Chem. 2001;276:29567 29574. 31. Deng JT, Van Lierop JE, Sutherland C, Walsh MP. Ca 2 -independent smooth muscle contraction. a novel function for integrin-linked kinase. J Biol Chem. 2001;276:16365 16373. 32. Amano M, Ito M, Kimura K, Fukata Y, Chihara K, Nakano T, Matsuura Y, Kaibuchi K. Phosphorylation and activation of myosin by Rhoassociated kinase (Rho-kinase). J Biol Chem. 1996;271:20246 20249. 33. Kureishi Y, Kobayashi S, Amano M, Kimura K, Kanaide H, Nakano T, Kaibuchi K, Ito M. Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem. 1997;272:12257 12260. 34. Sakurada S, Takuwa N, Sugimoto N, Wang Y, Seto M, Sasaki Y, Takuwa Y. Ca 2 -dependent activation of Rho and Rho kinase in membrane depolarization-induced and receptor stimulation-induced vascular smooth muscle contraction. Circ Res. 2003;93:548 556. 35. Winder SJ, Walsh MP. Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation. J Biol Chem. 1990;265: 10148 10155.