Cholesterol Primes Vascular Smooth Muscle to Induce Ca2 Sensitization Mediated by a Sphingosylphosphorylcholine–Rho-Kinase Pathway
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Noriyasu Morikage, Hiroko Kishi, Masafum
参见附件。
the Department of Molecular Physiology (N.M., H.K., M. Sato, F.G., S.S., S.K.) and First Department of Surgery (N.M., K.H., K.E.), Yamaguchi University School of Medicine
Mochida Pharmaceutical Co, Ltd (T.Y., M. Soma), Tokyo, Japan.
Abstract
Hypercholesterolemia is a major risk factor involved in abnormal cardiovascular events. Rho-kinase–mediated Ca2+ sensitization of vascular smooth muscle (VSM) plays a critical role in vasospasm and hypertension. We recently identified sphingosylphosphorylcholine (SPC) and Src family tyrosine kinase (Src-TK) as upstream mediators for the Rho-kinase–mediated Ca2+ sensitization. Here we report the strong linkage between cholesterol and the Ca2+ sensitization of VSM mediated by a novel SPC/Src-TK/Rho-kinase pathway in both humans and rabbits. The extent of the sensitization correlated well with the total cholesterol or low-density lipoprotein cholesterol levels in serum. However, an inverse correlation with the serum level of high-density lipoprotein cholesterol was observed, and a correlation with other cardiovascular risk factors was nil. When cholesterol-lowering therapy was given to patients and rabbits with hypercholesterolemia, the SPC-induced contractions diminished. Depletion of VSM cholesterol by -cyclodextrin resulted in a loss of membrane caveolin-1, a marker of cholesterol-enriched lipid raft, and inhibited the SPC-induced Ca2+ sensitization and translocation of Rho-kinase from cytosol to the cell membrane. Vasocontractions induced by membrane depolarization and by an adrenergic agonist were cholesterol-independent. Our data support the previously unreported concept that cholesterol potentiates the Ca2+ sensitization of VSM mediated by a SPC/Src-TK/Rho-kinase pathway, and are also compatible with a role for cholesterol-enriched membrane microdomain, a lipid raft. This process may play an important role in the development of abnormal vascular contractions in patients with hypercholesterolemia.
Key Words: Ca2+ sensitization contraction membrane lipid raft Rho-kinase sphingosylphosphorylcholine vascular smooth muscle
Introduction
Abnormal vascular contraction as a result of Ca2+ sensitization of vascular smooth muscles (VSMs) has attracted attention as a cause of hypertension and vasospasm.1 Kureishi et al showed that constitutively active Rho-kinase applied to the cytosol of permeabilized VSM induced Ca2+ sensitization and increased myosin light chain phosphorylation through a mechanism that is independent of a Ca2+-dependent myosin light chain kinase pathway.2 In addition, the Rho-kinase blocker Y27632 cures hypertension in rats without affecting normal blood pressure,3 and it also inhibits vasospasm of coronary arteries in a porcine model.4 However, the mechanism by which Rho-kinase–mediated Ca2+ sensitization of VSM is triggered in cardiovascular diseases has not been evident.
Hyperlipidemia is a major risk factor for abnormal cardiovascular events. Evidence for this is provided by the J-shaped curve showing a strong relationship between serum cholesterol levels and the relative risk for coronary disease5–7 (see also Figure I in the online data supplement, available at http://circres.ahajournals.org). Atheromatous plaques may be involved in cardiovascular events,8,9 but the extent of plaque formation and rupture does not necessarily correlate with cholesterol levels in patients. The clinical evidences that single treatment of hyperlipidemia induces marked reduction of the occurrence of the cardiovascular events strongly suggest more direct linkage between cholesterol and cardiovascular event. Because coronary artery spasm contributes significantly to acute coronary syndromes, abnormal vascular contraction may be another important cause of cardiovascular events.10 However, the relationship between serum cholesterol levels and the abnormal Ca2+ sensitization of VSM contraction has not been documented.
We have recently identified SPC and Src family tyrosine kinase (Src-TK) as upstream mediators for the Rho-kinase–mediated Ca2+ sensitization of the pig and bovine VSM contractions.11–13 The SPC has been regarded as a novel messenger for vasospasm.14 Therefore, in the present study, we tested the possible linkage between cholesterol and the Ca2+ sensitization of VSM contraction mediated by a novel SPC/Rho-kinase pathway. We investigated the possible cholesterol dependence of the SPC-induced Ca2+ sensitization in humans and rabbits with hypercholesterolemia. Our data show a direct enhancement of VSM Ca2+ sensitization by cholesterol via an SPC/Rho-kinase pathway.
Materials and Methods
Patient Profiles
The clinical histories of the 39 Japanese patients (26 men and 13 women) enrolled in the study were as follows: 16 with hypercholesterolemia, 12 with hypertension, 8 with diabetes, and 10 smoked cigarettes. All were about to undergo vascular or gastrointestinal surgery. Before surgery, the serum levels of total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides were determined. A total of 10 internal thoracic arteries were obtained from patients with angina pectoris who underwent coronary artery bypass surgery. Patients with gastrointestinal cancer who underwent gastrectomy or colectomy were the source of 17 gastroepiploic arteries and 12 colic arteries. Immediately after removal, the distal segments of the left internal thoracic artery (not used for surgical implantation), the gastroepiploic artery, and the colic artery were placed in ice-cold physiological salt solution (PSS).
Animals
We used Japanese white male rabbits (2.0 to 2.5 kg). We fed 24 rabbits standard chow or a cholesterol diet (0.2% to 0.5%) for 2 to 4 weeks to investigate the relationship between SPC-induced contractions and serum cholesterol levels. We fed 12 rabbits sequentially 0.5% (2 weeks) and 0.2% (8 weeks) cholesterol diet to investigate the effects of cholesterol-lowering therapy on SPC-induced contraction. We fed 16 rabbits a 0.5% cholesterol diet (2 weeks) to investigate effects of -cyclodextrin on VSM. We killed the animals with an overdose of pentobarbital (50 mg/kg IV) and rapidly removed the mesenteric arteries.
Preparation of Vascular Smooth Muscle Strips
The arteries were cut into helical strips lacking endothelium and adventitia, as described.15–18 The complete removal of the endothelium from the strips was confirmed by the lack of any relaxing response to 1 μmol/L bradykinin.
Simultaneous Measurement of [Ca2+]i and Force
The [Ca2+]i and force in the VSM strips (1.0x4.0 mm) loaded with fura-2 (1.25 μmol/L fura-2/acetoxymethyl ester [fura-2/AM]), were recorded simultaneously, using a fura-2 fluorometer (CAM 230; Japan Spectroscopic Co) equipped with an optical fiber system, as described.15–17 The developed tension and the fluorescence ratio were expressed as percentages of the response to 118 mmol/L K+ depolarization.
Permeabilization of Cell Membrane
Rabbit mesenteric artery smooth muscle strips (150 μmx2 mm) were membrane permeabilized using 0.12 mg/dL -toxin (20 minutes) or 30 μmol/L -escin (20 to 30 minutes), as described.2,11,15,18 Briefly, isometric tension was measured using a force transducer (UL-2g; Minebea, Tokyo, Japan) in a well on a bubble plate. The precise composition of the solutions has been described elsewhere.15,19 Ca2+ was buffered using 10 mmol/L EGTA. Normal relaxing solution for the experiment with membrane-permeabilized strips was of the following composition (mmol/L): potassium methanesulphonate, 74.1; magnesium methanesulphonate, 2; MgATP, 4.5; EGTA, 1; creatine phosphate, 10; PIPES, 30. In activating solution (pCa 6.3), 10 mmol/L EGTA was used, a specified amount of calcium methanesulfonate was added to give a desired concentration of free calcium ions, and the ionic strength was kept constant at 0.2 mol/L by adjusting the concentration of potassium methanesulfonate.
Construction and Purification of a Dominant Negative Form of Rho-Kinase
The cDNA encoding a dominant negative form of Rho-kinase (dn-ROK) was obtained and subcloned into the vector pGEX-6P-1, as described.12 The glutathione S-transferase (GST)-fusion protein was expressed in Escherichia coli and purified, as described.12
VSM Cell Culture
VSM cells from human coronary artery smooth muscle cells (BioWhittaker; Walkersville, Md) were grown on coverslips coated with 0.3% gelatin and cultured in SmGM-2 (Biowhittaker; Walkersville, Md) containing 0.5 ng/mL human epidermal growth factor, 5 μg/mL insulin, 2 ng/mL human fibroblast growth factor, 50 μg/mL gentamycin, 50 ng/mL amphotericin-B and 5% FBS. The serum was removed 24 hours before the experiment.
Immunofluorescence
Immunostaining was done using an anti–Rho-kinase primary antibody (Transduction Laboratories) and Alexa 488–labeled secondary antibody, as described.12,13 Western blotting confirmed the specificity of the antibody for Rho-kinase. No fluorescence was observed when the secondary antibody alone was used. Cells were observed by immunofluorescence using a confocal laser scan microscope (LSM 510, Carl Zeiss) and images were digitized using NIH Image version 1.62.
Cholesterol Depletion of VSM Strips
VSM strips were incubated for 4 hour at 37°C with 5 mmol/L -cyclodextrin in DMEM, as described20; then the lipids were extracted as described.21 The cholesterol or phospholipid contents were measured quantitatively using commercially available kits (cholesterol CII test kit, phospholipid C test kit; Wako Inc, Osaka, Japan).
Solutions
SPC was obtained from Sigma (St Louis, Mo) or Biomol (Plymouth Meeting, Pa). Y27632, eicosapentaenoic acid (EPA), and pravastatin were kindly provided by Welfide Corp (Osaka, Japan), Mochida Corp (Tokyo, Japan), and Sankyo Corp (Tokyo, Japan), respectively. The composition of the normal PSS was 123 mmol/L NaCl, 4.7 mmol/L KCl, 15.5 mmol/L NaHCO3, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgCl2, 1.25 mmol/L CaCl2, and 11.5 mmol/L D-glucose. All solutions were gassed with a mixture of 5% CO2 and 95% O2 (pH 7.4 at 37°C).
Results
SPC-Induced Ca2+ Sensitization of Human VSM Contraction
A large and sustained contraction of arteries obtained from patients with hypercholesterolemia was induced by SPC (30 μmol/L) (Figure 1b). This was not observed in arteries obtained from patients with normal serum cholesterol levels (Figure 1a). In contrast, the contractions induced by high K+ depolarization were similar in both sets of patients (0.80±0.12 g/mm2, n=9 and 0.97±0.18 g/mm2, n=11; P>0.05), indicating that the contractions induced by high K+ depolarization were independent of the serum cholesterol levels (Figure 1b). Only a slight or little elevation of [Ca2+]i accompanied the SPC-induced contraction (Figure 1b), indicating that SPC induces Ca2+ sensitization of human VSM. The SPC-induced contraction was significantly (P<0.01) enhanced in hypercholesterolemic patients; 76.2±13.8% of high K+-induced contractions for those with hypercholesterolemia, compared with 13.2±1.8% for those with normal cholesterol levels. The SPC-induced contractions were inhibited by Y27632, a specific inhibitor of Rho-kinase (Figure 1b). However, the contractions were not affected by removal of the endothelium or by the addition of NG-monomethyl-L-arginine (L-NMMA), an endothelial NO synthase blocker (data not shown). This suggested that molecular mechanisms leading to the contractions involved Rho-kinase and not the endothelium. No other cardiovascular risk factors, such as hypertension, diabetes, and cigarette smoking, nor the age or sex of the patients correlated with SPC-induced contraction (data not shown).
Involvement of Rho-Kinase, but Not G Protein, in SPC-Induced Contraction of Human and Mammalian VSMs
The specific Rho-kinase inhibitor Y27632 (2 μmol/L) blocked the SPC-induced contraction (ie, Ca2+ sensitization) of VSM obtained from hypercholesterolemic patients and rabbits (Figure 1c and 1d). However, the high K+ depolarization–induced contraction (ie, Ca2+-dependent contraction) was not affected by Y27632 (Figure 1c and 1d). Because nonspecific effects of Y27632 have been reported, these data confirm that Y27632 (at the concentration used) had no effect on the Ca2+-dependent contraction.
The involvement of Rho-kinase, rather than unknown Y27632-sensitive protein kinases, was confirmed by the application of a dominant negative Rho-kinase (dn-ROK) to the cytosol of the VSM permeabilized with -escin. The SPC-induced Ca2+ sensitization (at a constant concentration of Ca2+; pCa 6.3) was abolished by both Y27632 and the dn-ROK (Figure 1e and 1f). The dn-ROK had no effect on Ca2+-induced contractions or Ca2+ sensitization induced by phorbol ester, a protein kinase C activator, thus confirming specificity of the dominant negative Rho-kinase in Rho-kinase–mediated Ca2+ sensitization.
Translocation of Rho-kinase from the cytosol to the cell membrane plays an important role in activation of this enzyme.22 In the present study, SPC (30 μmol/L, for 30 minutes) induced translocation of cytosolic Rho-kinase to the cell membrane of VSM cells (Figure 1g). This was associated with the activation of Rho-kinase by SPC (data not shown), as described.13 These results strongly suggest that SPC induced Ca2+ sensitization of VSM through the activation and translocation of Rho-kinase.
SPC also induced Ca2+ sensitization of rabbit VSM permeabilized with -toxin (Figure 1h). Phenylephrine (0.1 μmol/L), a G-protein–coupled receptor agonist, induced VSM contraction only in the presence of GTP (10 μmol/L), which is required for G-protein activation. However, SPC (30 μmol/L) induced contraction of the same VSM strip permeabilized with -toxin even in the absence of GTP (Figure 1h). These results suggest that G-protein activation was not required for SPC-induced Ca2+ sensitization, thereby supporting a role for SPC as a sphingolipid mediator.
It has been reported that SPC-induced vasoconstriction was stereospecific and inhibited by pertussis toxin, suggesting the involvement of the G-protein–coupled receptor.23 Thus, we investigated the effect of pertussis toxin and the stereospecificity. The SPC-induced contraction was not inhibited by the treatment of pertussis toxin. However, D-erythro-SPC induced large contraction, whereas L-threo-SPC induced a little contraction, indicating the stereospecificity of the SPC-induced contraction. No GTP requirement of SPC-induced contraction in the permeabilized VSM and no inhibition by pertussis toxin suggest that there is no involvement of G proteins in the SPC-induced contraction. However, because there are stereospecificity of SPC-induced contraction, we suggest that SPC may affect the some receptor, which is not coupled with G proteins.
SPC-Induced Contractions of Human VSM Depend on Serum Cholesterol Levels
The extent of the SPC-induced contraction correlated well (P<0.01) with the serum total and LDL-cholesterol levels of the patients (Figure 2a and 2b). However, an inverse correlation was observed between the extent of contraction and the ratio of the HDL-cholesterol level to the total cholesterol level (Figure 2c). The responses to SPC also correlated well with serum triglyceride and non HDL-cholesterol levels (data not shown). In contrast, the contractions and [Ca2+]i elevations induced by high K+ depolarization were not cholesterol dependent. The relationship between the serum cholesterol level and the relative risk of coronary heart disease of the patients was originally derived from results of the Multiple Risk Factor Intervention Trial (MRFIT)5 that formed a typical J-shaped curve (supplemental Figure I). Interestingly, this curve closely resembled the curve obtained in the present study on the relationship between SPC-induced contraction and the serum total cholesterol level (Figure 2a). Identical J-shaped curves have also been observed in Japanese patients.7
There is no difference (P>0.05) in the extent of the SPC-induced contraction and cholesterol dependence between internal thoracic artery and gastroepiploic artery (Figure 2a through 2c).
SPC-Induced Contractions of Rabbit VSM Depend on Serum Cholesterol Levels
Similar results were obtained when vascular strips from rabbits were used. The rabbits had been fed either standard chow or a cholesterol-rich diet (Figure 2d through 2g). Again, the extent of the SPC-induced contraction correlated well (P<0.01) with the level of total cholesterol, LDL-cholesterol, and non HDL-cholesterol in serum, and an inverse correlation was observed with the HDL-cholesterol/total cholesterol ratio. In contrast, contractions induced by high K+ depolarization and the adrenergic agonist (phenylephrine) were cholesterol independent. The extent of high K+-induced contractions (force/cross-sectional area of the strip) was 1.25±0.17 g/mm2 for rabbits with normal cholesterol levels (n=17) versus 1.43±0.13 g/mm2 for those with hypercholesterolemia (n=7) (P>0.05). The extent of the phenylephrine-induced contractions (force/cross-sectional area of the strip) was 1.26±0.17 g/mm2 for normal cholesterol (n=17) versus 1.48±0.20 g/mm2 for rabbits with hypercholesterolemia (n=7) (P>0.05).
Effects of Cholesterol-Lowering Therapy on SPC-Induced VSM Contractions
Treatment of hypercholesterolemic patients with cholesterol-lowering drugs reduced the total cholesterol level in their serum (Figure 3a), with a concurrent reduction in the SPC-induced contractions (Figure 3b). In rabbits, the oral administration of EPA or pravastatin lowered significantly the total cholesterol level in serum and the SPC-induced VSM contractions, to the same degree (Figure 3c and 3d). Furthermore, SPC-induced contractions of rabbit VSM correlated well with the serum cholesterol level of rabbits, regardless of whether or not they had been given cholesterol-lowering therapy (Figure 3e).
Effects of Cholesterol Depletion by -Cyclodextrin on SPC-Induced VSM Contractions and Translocation of Rho-Kinase
Cholesterol depletion by treatment with -cyclodextrin20 diminished markedly the SPC-induced contractions of the artery from hypercholesterolemic rabbits, without affecting the contractions induced by high K+ depolarization or by phenylephrine (Figure 4). The specific depletion of cholesterol, rather than phospholipids, by -cyclodextrin treatment, was confirmed (Figure 4d). In addition, the acute and direct application of -cyclodextrin had no effect on the SPC-induced contractions (Figure 4c), indicating that the inhibitory effect is attributable to cholesterol depletion, rather than to a direct and nonspecific inhibitory effect on VSM contractions.
In addition, -cyclodextrin resulted in a loss of membrane caveolin-1, a marker of cholesterol-enriched lipid raft (supplemental Figure II) and abolished the translocation of Rho-kinase from the cytosol to the surface membrane (Figure 1g).
Discussion
Factors causing abnormal vascular contractions in patients with hypercholesterolemia were examined and the following novel findings were obtained. Rho-kinase–mediated Ca2+ sensitization of human and rabbit VSM contractions was induced by SPC and this effect correlated well with the serum level of total or LDL-cholesterol: the data closely resembled the J-shaped curve obtained in the MRFIT study5 for serum cholesterol levels and the relative risk of coronary heart disease (Figure 2a versus supplemental Figure I).
It has been reported that risk factors (such as hyperlipidemia, hypertension, smoking, and diabetes) may influence each other when they occur concurrently in patients. However, the SPC-induced contractions had no detectable relationship with other risk factors (smoking, diabetes, and hypertension). In addition, contractions induced by high K+ and phenylephrine were independent of serum cholesterol levels. These results clearly indicate that the SPC-induced contraction correlates with serum cholesterol levels alone and only cholesterol affects the abnormal contraction induced by SPC. Furthermore, cholesterol-lowering therapy led to a reduction in abnormal SPC-induced contractions, and the decrease also correlated with serum cholesterol levels (Figures 3 and 4). Selective depletion of cholesterol (but not phospholipids) in vascular tissue by -cyclodextrin also inhibited SPC-induced Ca2+ sensitization (Figure 4). However, this depletion did not affect "normal and physiological" vascular contractions induced by membrane depolarization and an adrenergic agonist (Figure 4). These findings strongly indicate that cholesterol accumulating in vascular tissue directly and specifically primes VSM to induce the Ca2+ sensitization mediated by a SPC via a Rho-kinase pathway, which has been reported to play a pivotal role in vasospasm.4 Taken together, these observations strongly suggest a causal relationship between the serum or vascular tissue cholesterol level and the SPC-induced Rho-kinase–mediated contractions.
In Figure 3C, cholesterol levels in EPA and pravastatin groups appear to be higher than in the control group. However, SPC-induced VSM contraction was not different in those groups compared with control. These findings suggest the possibility of a cholesterol-independent effect of EPA and pravastatin on Ca2+ sensitization. Indeed, it is well known that statin has cholesterol-independent effects via inhibition of isoprenylation of small G proteins including Rho, which is the upstream regulator of Rho-kinase. Therefore, the effects of EPA and/or pravastatin appear to be mediated not only by cholesterol reduction but also by the possible involvement of cholesterol-independent mechanism (eg, cholesterol-independent inhibition of the Rho-kinase pathway). Although we did not examine precisely these possibilities in the present study, the inhibitory effects of pravastatin on the Ca2+ sensitization of hyperlipidemic animals may also support the beneficial clinical effects of statins on the vascular diseases associated with hyperlipidemia.
It has to be considered that lipoproteins contain a considerable amount of SPC, especially HDL. Thus, it is also likely that high HDL levels may cause a desensitization of the possible SPC receptors and thus decrease SPC-induced contraction. We did not evaluate directly this possibility in this study, although -cyclodextrin effects support the involvement of membrane cholesterol and its enriched membrane domain, membrane lipid raft.
According to Figures 1a and 2, there was nearly no effect of SPC in vessels from humans with normal serum cholesterol. In contrast, SPC to a significant extent contracted vessels from normal cholesterol rabbits (Figure 2d through 2g). In the present study, we did not have strong evidence to explain such difference between human and rabbit VSM. However, the normal cholesterol levels are much lower in rabbit (<30 mg/dL) than in human (150 to 200 mg/dL), and rabbit is a vegetarian eating very low cholesterol food. Therefore, rabbit may be more sensitive to cholesterol loading than humans.
A large number of epidemiological studies conducted in Europe, the United States, and Japan indicate that hypercholesterolemia is a major risk factor for abnormal cardiovascular events and even the treatment of subjects with hypercholesterolemia alone can decrease the occurrence of cardiovascular events.5–7,24–29 Several associated factors, including plaque disruption and thrombosis, have been implicated in the pathogenesis of hypercholesterolemia-associated increase in the risk of cardiovascular events.8,9 However, we found no documentation regarding a factor responsible for the J-shaped curve obtained in the MRFIT clinical megastudy5 and the Japanese Atherosclerosis Society (JAS) study.7 For example, when plotted against the cholesterol level, neither the extent of plaque disruption nor contraction induced by vasoactive substances followed the J-shaped curve. In the present study, we demonstrate for the first time that the Ca2+ sensitization mediated by a novel SPC/Rho-kinase pathway follows the J-shaped curve.
In the present study, Ca2+ sensitization was evaluated directly using 2 techniques: firstly, the simultaneous measurement of [Ca2+]i and force; and secondly, the permeabilization of cell membrane using -escin and -toxin. A Rho-kinase inhibitor (Y27632) (Figure 1) and a dn-ROK (Figure 1e) were used to demonstrate that SPC induces Ca2+ sensitization through Rho-kinase activation. This observation is also supported by the SPC-induced translocation of cytosolic Rho-kinase to the cell membrane (Figure 1g). Using a porcine model, other researchers found that Rho-kinase plays a key role in the induction of VSM hypercontractions such as vasospasm.4 Our results, taken together with these findings, strongly suggest that Ca2+ sensitization of VSM mediated by a SPC/Rho-kinase pathway is activated by hypercholesterolemia and triggers abnormal vascular contractions. Vasospasms, for example, could increase the relative risk of coronary disease, which was seen to be associated with a rise in serum cholesterol levels in the MRFIT study.5
The molecular mechanism(s) by which cholesterol potentiates VSM Ca2+ sensitization are not completely clarified. Cholesterol depletion by -cyclodextrin blocked not only SPC-induced contraction (Figure 4) but also translocation of Rho-kinase to cell membrane (Figure 1). Because -cyclodextrin induces a selective destruction of a cholesterol-enriched membrane microdomain, lipid raft,20 it is likely that Rho-kinase translocated to lipid raft. Although disruption of lipid rafts is known to affect many signal transduction components and disruption of lipid rafts by -cyclodextrin may be trivial, these effects of -cyclodextrin seem to be specific for cholesterol depletion and VSM contraction (see also a detailed description in the online data supplement). It was clearly demonstrated that membrane lipid raft contains more cholesterol than other nonraft membrane in smooth muscle.30 Indeed, depletion of cholesterol by -cyclodextrin resulted in marked reduction of staining of caveolin-1, a well-known marker of membrane lipid raft, in cultured VSM, as shown in supplemental Figure II. In addition, we previously reported that Src-TK mediates SPC-induced contraction and activation of Rho-kinase and that SPC induces translocation of Fyn, a member of Src-TK localizing to lipid raft.13 Taken together, these findings are compatible with a crucial role of cholesterol-enriched membrane lipid raft in the VSM Ca2+ sensitization. In addition, future studies must address the question of whether plasma cholesterol levels (and not just -cyclodextrin treatment) indeed have an influence on VSM lipid raft composition.
In conclusion, our data indicate that SPC may be a novel trigger for cholesterol-dependent Ca2+ sensitization of VSM contractions mediated by Rho-kinase in humans and rabbits. Furthermore, the SPC/Rho-kinase pathway may initiate the abnormal vascular contractions associated with hypercholesterolemia. We predict that therapy to lower the levels of cholesterol in the serum of patients would reduce the occurrence of cardiovascular events associated with abnormal vascular contractions. Finally, our results also suggest that cholesterol and its enriched lipid rafts may contribute to the SPC/Src-TK/Rho-kinase–mediated Ca2+ sensitization of VSM, which may provide molecular mechanism for the beneficial, even acute, effects of cholesterol-lowering therapy on cardiovascular events.28,29
Acknowledgments
We thank S. Miwa for technical assistance and M. Ohara (Fukuoka) for critical comments on the manuscript.
Sources of Funding
This work was supported in part by a research grant from Ube Industries and grants-in-aid for scientific research from the Ministry of Education, Science, Technology, Sports and Culture, Japan.
Disclosures
None.
Footnotes
Original received August 20, 2003; first resubmission received August 8, 2005; second resubmission received February 9, 2006; revised second resubmission received May 31, 2006; accepted June 27, 2006.
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the Department of Molecular Physiology (N.M., H.K., M. Sato, F.G., S.S., S.K.) and First Department of Surgery (N.M., K.H., K.E.), Yamaguchi University School of Medicine
Mochida Pharmaceutical Co, Ltd (T.Y., M. Soma), Tokyo, Japan.
Abstract
Hypercholesterolemia is a major risk factor involved in abnormal cardiovascular events. Rho-kinase–mediated Ca2+ sensitization of vascular smooth muscle (VSM) plays a critical role in vasospasm and hypertension. We recently identified sphingosylphosphorylcholine (SPC) and Src family tyrosine kinase (Src-TK) as upstream mediators for the Rho-kinase–mediated Ca2+ sensitization. Here we report the strong linkage between cholesterol and the Ca2+ sensitization of VSM mediated by a novel SPC/Src-TK/Rho-kinase pathway in both humans and rabbits. The extent of the sensitization correlated well with the total cholesterol or low-density lipoprotein cholesterol levels in serum. However, an inverse correlation with the serum level of high-density lipoprotein cholesterol was observed, and a correlation with other cardiovascular risk factors was nil. When cholesterol-lowering therapy was given to patients and rabbits with hypercholesterolemia, the SPC-induced contractions diminished. Depletion of VSM cholesterol by -cyclodextrin resulted in a loss of membrane caveolin-1, a marker of cholesterol-enriched lipid raft, and inhibited the SPC-induced Ca2+ sensitization and translocation of Rho-kinase from cytosol to the cell membrane. Vasocontractions induced by membrane depolarization and by an adrenergic agonist were cholesterol-independent. Our data support the previously unreported concept that cholesterol potentiates the Ca2+ sensitization of VSM mediated by a SPC/Src-TK/Rho-kinase pathway, and are also compatible with a role for cholesterol-enriched membrane microdomain, a lipid raft. This process may play an important role in the development of abnormal vascular contractions in patients with hypercholesterolemia.
Key Words: Ca2+ sensitization contraction membrane lipid raft Rho-kinase sphingosylphosphorylcholine vascular smooth muscle
Introduction
Abnormal vascular contraction as a result of Ca2+ sensitization of vascular smooth muscles (VSMs) has attracted attention as a cause of hypertension and vasospasm.1 Kureishi et al showed that constitutively active Rho-kinase applied to the cytosol of permeabilized VSM induced Ca2+ sensitization and increased myosin light chain phosphorylation through a mechanism that is independent of a Ca2+-dependent myosin light chain kinase pathway.2 In addition, the Rho-kinase blocker Y27632 cures hypertension in rats without affecting normal blood pressure,3 and it also inhibits vasospasm of coronary arteries in a porcine model.4 However, the mechanism by which Rho-kinase–mediated Ca2+ sensitization of VSM is triggered in cardiovascular diseases has not been evident.
Hyperlipidemia is a major risk factor for abnormal cardiovascular events. Evidence for this is provided by the J-shaped curve showing a strong relationship between serum cholesterol levels and the relative risk for coronary disease5–7 (see also Figure I in the online data supplement, available at http://circres.ahajournals.org). Atheromatous plaques may be involved in cardiovascular events,8,9 but the extent of plaque formation and rupture does not necessarily correlate with cholesterol levels in patients. The clinical evidences that single treatment of hyperlipidemia induces marked reduction of the occurrence of the cardiovascular events strongly suggest more direct linkage between cholesterol and cardiovascular event. Because coronary artery spasm contributes significantly to acute coronary syndromes, abnormal vascular contraction may be another important cause of cardiovascular events.10 However, the relationship between serum cholesterol levels and the abnormal Ca2+ sensitization of VSM contraction has not been documented.
We have recently identified SPC and Src family tyrosine kinase (Src-TK) as upstream mediators for the Rho-kinase–mediated Ca2+ sensitization of the pig and bovine VSM contractions.11–13 The SPC has been regarded as a novel messenger for vasospasm.14 Therefore, in the present study, we tested the possible linkage between cholesterol and the Ca2+ sensitization of VSM contraction mediated by a novel SPC/Rho-kinase pathway. We investigated the possible cholesterol dependence of the SPC-induced Ca2+ sensitization in humans and rabbits with hypercholesterolemia. Our data show a direct enhancement of VSM Ca2+ sensitization by cholesterol via an SPC/Rho-kinase pathway.
Materials and Methods
Patient Profiles
The clinical histories of the 39 Japanese patients (26 men and 13 women) enrolled in the study were as follows: 16 with hypercholesterolemia, 12 with hypertension, 8 with diabetes, and 10 smoked cigarettes. All were about to undergo vascular or gastrointestinal surgery. Before surgery, the serum levels of total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides were determined. A total of 10 internal thoracic arteries were obtained from patients with angina pectoris who underwent coronary artery bypass surgery. Patients with gastrointestinal cancer who underwent gastrectomy or colectomy were the source of 17 gastroepiploic arteries and 12 colic arteries. Immediately after removal, the distal segments of the left internal thoracic artery (not used for surgical implantation), the gastroepiploic artery, and the colic artery were placed in ice-cold physiological salt solution (PSS).
Animals
We used Japanese white male rabbits (2.0 to 2.5 kg). We fed 24 rabbits standard chow or a cholesterol diet (0.2% to 0.5%) for 2 to 4 weeks to investigate the relationship between SPC-induced contractions and serum cholesterol levels. We fed 12 rabbits sequentially 0.5% (2 weeks) and 0.2% (8 weeks) cholesterol diet to investigate the effects of cholesterol-lowering therapy on SPC-induced contraction. We fed 16 rabbits a 0.5% cholesterol diet (2 weeks) to investigate effects of -cyclodextrin on VSM. We killed the animals with an overdose of pentobarbital (50 mg/kg IV) and rapidly removed the mesenteric arteries.
Preparation of Vascular Smooth Muscle Strips
The arteries were cut into helical strips lacking endothelium and adventitia, as described.15–18 The complete removal of the endothelium from the strips was confirmed by the lack of any relaxing response to 1 μmol/L bradykinin.
Simultaneous Measurement of [Ca2+]i and Force
The [Ca2+]i and force in the VSM strips (1.0x4.0 mm) loaded with fura-2 (1.25 μmol/L fura-2/acetoxymethyl ester [fura-2/AM]), were recorded simultaneously, using a fura-2 fluorometer (CAM 230; Japan Spectroscopic Co) equipped with an optical fiber system, as described.15–17 The developed tension and the fluorescence ratio were expressed as percentages of the response to 118 mmol/L K+ depolarization.
Permeabilization of Cell Membrane
Rabbit mesenteric artery smooth muscle strips (150 μmx2 mm) were membrane permeabilized using 0.12 mg/dL -toxin (20 minutes) or 30 μmol/L -escin (20 to 30 minutes), as described.2,11,15,18 Briefly, isometric tension was measured using a force transducer (UL-2g; Minebea, Tokyo, Japan) in a well on a bubble plate. The precise composition of the solutions has been described elsewhere.15,19 Ca2+ was buffered using 10 mmol/L EGTA. Normal relaxing solution for the experiment with membrane-permeabilized strips was of the following composition (mmol/L): potassium methanesulphonate, 74.1; magnesium methanesulphonate, 2; MgATP, 4.5; EGTA, 1; creatine phosphate, 10; PIPES, 30. In activating solution (pCa 6.3), 10 mmol/L EGTA was used, a specified amount of calcium methanesulfonate was added to give a desired concentration of free calcium ions, and the ionic strength was kept constant at 0.2 mol/L by adjusting the concentration of potassium methanesulfonate.
Construction and Purification of a Dominant Negative Form of Rho-Kinase
The cDNA encoding a dominant negative form of Rho-kinase (dn-ROK) was obtained and subcloned into the vector pGEX-6P-1, as described.12 The glutathione S-transferase (GST)-fusion protein was expressed in Escherichia coli and purified, as described.12
VSM Cell Culture
VSM cells from human coronary artery smooth muscle cells (BioWhittaker; Walkersville, Md) were grown on coverslips coated with 0.3% gelatin and cultured in SmGM-2 (Biowhittaker; Walkersville, Md) containing 0.5 ng/mL human epidermal growth factor, 5 μg/mL insulin, 2 ng/mL human fibroblast growth factor, 50 μg/mL gentamycin, 50 ng/mL amphotericin-B and 5% FBS. The serum was removed 24 hours before the experiment.
Immunofluorescence
Immunostaining was done using an anti–Rho-kinase primary antibody (Transduction Laboratories) and Alexa 488–labeled secondary antibody, as described.12,13 Western blotting confirmed the specificity of the antibody for Rho-kinase. No fluorescence was observed when the secondary antibody alone was used. Cells were observed by immunofluorescence using a confocal laser scan microscope (LSM 510, Carl Zeiss) and images were digitized using NIH Image version 1.62.
Cholesterol Depletion of VSM Strips
VSM strips were incubated for 4 hour at 37°C with 5 mmol/L -cyclodextrin in DMEM, as described20; then the lipids were extracted as described.21 The cholesterol or phospholipid contents were measured quantitatively using commercially available kits (cholesterol CII test kit, phospholipid C test kit; Wako Inc, Osaka, Japan).
Solutions
SPC was obtained from Sigma (St Louis, Mo) or Biomol (Plymouth Meeting, Pa). Y27632, eicosapentaenoic acid (EPA), and pravastatin were kindly provided by Welfide Corp (Osaka, Japan), Mochida Corp (Tokyo, Japan), and Sankyo Corp (Tokyo, Japan), respectively. The composition of the normal PSS was 123 mmol/L NaCl, 4.7 mmol/L KCl, 15.5 mmol/L NaHCO3, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgCl2, 1.25 mmol/L CaCl2, and 11.5 mmol/L D-glucose. All solutions were gassed with a mixture of 5% CO2 and 95% O2 (pH 7.4 at 37°C).
Results
SPC-Induced Ca2+ Sensitization of Human VSM Contraction
A large and sustained contraction of arteries obtained from patients with hypercholesterolemia was induced by SPC (30 μmol/L) (Figure 1b). This was not observed in arteries obtained from patients with normal serum cholesterol levels (Figure 1a). In contrast, the contractions induced by high K+ depolarization were similar in both sets of patients (0.80±0.12 g/mm2, n=9 and 0.97±0.18 g/mm2, n=11; P>0.05), indicating that the contractions induced by high K+ depolarization were independent of the serum cholesterol levels (Figure 1b). Only a slight or little elevation of [Ca2+]i accompanied the SPC-induced contraction (Figure 1b), indicating that SPC induces Ca2+ sensitization of human VSM. The SPC-induced contraction was significantly (P<0.01) enhanced in hypercholesterolemic patients; 76.2±13.8% of high K+-induced contractions for those with hypercholesterolemia, compared with 13.2±1.8% for those with normal cholesterol levels. The SPC-induced contractions were inhibited by Y27632, a specific inhibitor of Rho-kinase (Figure 1b). However, the contractions were not affected by removal of the endothelium or by the addition of NG-monomethyl-L-arginine (L-NMMA), an endothelial NO synthase blocker (data not shown). This suggested that molecular mechanisms leading to the contractions involved Rho-kinase and not the endothelium. No other cardiovascular risk factors, such as hypertension, diabetes, and cigarette smoking, nor the age or sex of the patients correlated with SPC-induced contraction (data not shown).
Involvement of Rho-Kinase, but Not G Protein, in SPC-Induced Contraction of Human and Mammalian VSMs
The specific Rho-kinase inhibitor Y27632 (2 μmol/L) blocked the SPC-induced contraction (ie, Ca2+ sensitization) of VSM obtained from hypercholesterolemic patients and rabbits (Figure 1c and 1d). However, the high K+ depolarization–induced contraction (ie, Ca2+-dependent contraction) was not affected by Y27632 (Figure 1c and 1d). Because nonspecific effects of Y27632 have been reported, these data confirm that Y27632 (at the concentration used) had no effect on the Ca2+-dependent contraction.
The involvement of Rho-kinase, rather than unknown Y27632-sensitive protein kinases, was confirmed by the application of a dominant negative Rho-kinase (dn-ROK) to the cytosol of the VSM permeabilized with -escin. The SPC-induced Ca2+ sensitization (at a constant concentration of Ca2+; pCa 6.3) was abolished by both Y27632 and the dn-ROK (Figure 1e and 1f). The dn-ROK had no effect on Ca2+-induced contractions or Ca2+ sensitization induced by phorbol ester, a protein kinase C activator, thus confirming specificity of the dominant negative Rho-kinase in Rho-kinase–mediated Ca2+ sensitization.
Translocation of Rho-kinase from the cytosol to the cell membrane plays an important role in activation of this enzyme.22 In the present study, SPC (30 μmol/L, for 30 minutes) induced translocation of cytosolic Rho-kinase to the cell membrane of VSM cells (Figure 1g). This was associated with the activation of Rho-kinase by SPC (data not shown), as described.13 These results strongly suggest that SPC induced Ca2+ sensitization of VSM through the activation and translocation of Rho-kinase.
SPC also induced Ca2+ sensitization of rabbit VSM permeabilized with -toxin (Figure 1h). Phenylephrine (0.1 μmol/L), a G-protein–coupled receptor agonist, induced VSM contraction only in the presence of GTP (10 μmol/L), which is required for G-protein activation. However, SPC (30 μmol/L) induced contraction of the same VSM strip permeabilized with -toxin even in the absence of GTP (Figure 1h). These results suggest that G-protein activation was not required for SPC-induced Ca2+ sensitization, thereby supporting a role for SPC as a sphingolipid mediator.
It has been reported that SPC-induced vasoconstriction was stereospecific and inhibited by pertussis toxin, suggesting the involvement of the G-protein–coupled receptor.23 Thus, we investigated the effect of pertussis toxin and the stereospecificity. The SPC-induced contraction was not inhibited by the treatment of pertussis toxin. However, D-erythro-SPC induced large contraction, whereas L-threo-SPC induced a little contraction, indicating the stereospecificity of the SPC-induced contraction. No GTP requirement of SPC-induced contraction in the permeabilized VSM and no inhibition by pertussis toxin suggest that there is no involvement of G proteins in the SPC-induced contraction. However, because there are stereospecificity of SPC-induced contraction, we suggest that SPC may affect the some receptor, which is not coupled with G proteins.
SPC-Induced Contractions of Human VSM Depend on Serum Cholesterol Levels
The extent of the SPC-induced contraction correlated well (P<0.01) with the serum total and LDL-cholesterol levels of the patients (Figure 2a and 2b). However, an inverse correlation was observed between the extent of contraction and the ratio of the HDL-cholesterol level to the total cholesterol level (Figure 2c). The responses to SPC also correlated well with serum triglyceride and non HDL-cholesterol levels (data not shown). In contrast, the contractions and [Ca2+]i elevations induced by high K+ depolarization were not cholesterol dependent. The relationship between the serum cholesterol level and the relative risk of coronary heart disease of the patients was originally derived from results of the Multiple Risk Factor Intervention Trial (MRFIT)5 that formed a typical J-shaped curve (supplemental Figure I). Interestingly, this curve closely resembled the curve obtained in the present study on the relationship between SPC-induced contraction and the serum total cholesterol level (Figure 2a). Identical J-shaped curves have also been observed in Japanese patients.7
There is no difference (P>0.05) in the extent of the SPC-induced contraction and cholesterol dependence between internal thoracic artery and gastroepiploic artery (Figure 2a through 2c).
SPC-Induced Contractions of Rabbit VSM Depend on Serum Cholesterol Levels
Similar results were obtained when vascular strips from rabbits were used. The rabbits had been fed either standard chow or a cholesterol-rich diet (Figure 2d through 2g). Again, the extent of the SPC-induced contraction correlated well (P<0.01) with the level of total cholesterol, LDL-cholesterol, and non HDL-cholesterol in serum, and an inverse correlation was observed with the HDL-cholesterol/total cholesterol ratio. In contrast, contractions induced by high K+ depolarization and the adrenergic agonist (phenylephrine) were cholesterol independent. The extent of high K+-induced contractions (force/cross-sectional area of the strip) was 1.25±0.17 g/mm2 for rabbits with normal cholesterol levels (n=17) versus 1.43±0.13 g/mm2 for those with hypercholesterolemia (n=7) (P>0.05). The extent of the phenylephrine-induced contractions (force/cross-sectional area of the strip) was 1.26±0.17 g/mm2 for normal cholesterol (n=17) versus 1.48±0.20 g/mm2 for rabbits with hypercholesterolemia (n=7) (P>0.05).
Effects of Cholesterol-Lowering Therapy on SPC-Induced VSM Contractions
Treatment of hypercholesterolemic patients with cholesterol-lowering drugs reduced the total cholesterol level in their serum (Figure 3a), with a concurrent reduction in the SPC-induced contractions (Figure 3b). In rabbits, the oral administration of EPA or pravastatin lowered significantly the total cholesterol level in serum and the SPC-induced VSM contractions, to the same degree (Figure 3c and 3d). Furthermore, SPC-induced contractions of rabbit VSM correlated well with the serum cholesterol level of rabbits, regardless of whether or not they had been given cholesterol-lowering therapy (Figure 3e).
Effects of Cholesterol Depletion by -Cyclodextrin on SPC-Induced VSM Contractions and Translocation of Rho-Kinase
Cholesterol depletion by treatment with -cyclodextrin20 diminished markedly the SPC-induced contractions of the artery from hypercholesterolemic rabbits, without affecting the contractions induced by high K+ depolarization or by phenylephrine (Figure 4). The specific depletion of cholesterol, rather than phospholipids, by -cyclodextrin treatment, was confirmed (Figure 4d). In addition, the acute and direct application of -cyclodextrin had no effect on the SPC-induced contractions (Figure 4c), indicating that the inhibitory effect is attributable to cholesterol depletion, rather than to a direct and nonspecific inhibitory effect on VSM contractions.
In addition, -cyclodextrin resulted in a loss of membrane caveolin-1, a marker of cholesterol-enriched lipid raft (supplemental Figure II) and abolished the translocation of Rho-kinase from the cytosol to the surface membrane (Figure 1g).
Discussion
Factors causing abnormal vascular contractions in patients with hypercholesterolemia were examined and the following novel findings were obtained. Rho-kinase–mediated Ca2+ sensitization of human and rabbit VSM contractions was induced by SPC and this effect correlated well with the serum level of total or LDL-cholesterol: the data closely resembled the J-shaped curve obtained in the MRFIT study5 for serum cholesterol levels and the relative risk of coronary heart disease (Figure 2a versus supplemental Figure I).
It has been reported that risk factors (such as hyperlipidemia, hypertension, smoking, and diabetes) may influence each other when they occur concurrently in patients. However, the SPC-induced contractions had no detectable relationship with other risk factors (smoking, diabetes, and hypertension). In addition, contractions induced by high K+ and phenylephrine were independent of serum cholesterol levels. These results clearly indicate that the SPC-induced contraction correlates with serum cholesterol levels alone and only cholesterol affects the abnormal contraction induced by SPC. Furthermore, cholesterol-lowering therapy led to a reduction in abnormal SPC-induced contractions, and the decrease also correlated with serum cholesterol levels (Figures 3 and 4). Selective depletion of cholesterol (but not phospholipids) in vascular tissue by -cyclodextrin also inhibited SPC-induced Ca2+ sensitization (Figure 4). However, this depletion did not affect "normal and physiological" vascular contractions induced by membrane depolarization and an adrenergic agonist (Figure 4). These findings strongly indicate that cholesterol accumulating in vascular tissue directly and specifically primes VSM to induce the Ca2+ sensitization mediated by a SPC via a Rho-kinase pathway, which has been reported to play a pivotal role in vasospasm.4 Taken together, these observations strongly suggest a causal relationship between the serum or vascular tissue cholesterol level and the SPC-induced Rho-kinase–mediated contractions.
In Figure 3C, cholesterol levels in EPA and pravastatin groups appear to be higher than in the control group. However, SPC-induced VSM contraction was not different in those groups compared with control. These findings suggest the possibility of a cholesterol-independent effect of EPA and pravastatin on Ca2+ sensitization. Indeed, it is well known that statin has cholesterol-independent effects via inhibition of isoprenylation of small G proteins including Rho, which is the upstream regulator of Rho-kinase. Therefore, the effects of EPA and/or pravastatin appear to be mediated not only by cholesterol reduction but also by the possible involvement of cholesterol-independent mechanism (eg, cholesterol-independent inhibition of the Rho-kinase pathway). Although we did not examine precisely these possibilities in the present study, the inhibitory effects of pravastatin on the Ca2+ sensitization of hyperlipidemic animals may also support the beneficial clinical effects of statins on the vascular diseases associated with hyperlipidemia.
It has to be considered that lipoproteins contain a considerable amount of SPC, especially HDL. Thus, it is also likely that high HDL levels may cause a desensitization of the possible SPC receptors and thus decrease SPC-induced contraction. We did not evaluate directly this possibility in this study, although -cyclodextrin effects support the involvement of membrane cholesterol and its enriched membrane domain, membrane lipid raft.
According to Figures 1a and 2, there was nearly no effect of SPC in vessels from humans with normal serum cholesterol. In contrast, SPC to a significant extent contracted vessels from normal cholesterol rabbits (Figure 2d through 2g). In the present study, we did not have strong evidence to explain such difference between human and rabbit VSM. However, the normal cholesterol levels are much lower in rabbit (<30 mg/dL) than in human (150 to 200 mg/dL), and rabbit is a vegetarian eating very low cholesterol food. Therefore, rabbit may be more sensitive to cholesterol loading than humans.
A large number of epidemiological studies conducted in Europe, the United States, and Japan indicate that hypercholesterolemia is a major risk factor for abnormal cardiovascular events and even the treatment of subjects with hypercholesterolemia alone can decrease the occurrence of cardiovascular events.5–7,24–29 Several associated factors, including plaque disruption and thrombosis, have been implicated in the pathogenesis of hypercholesterolemia-associated increase in the risk of cardiovascular events.8,9 However, we found no documentation regarding a factor responsible for the J-shaped curve obtained in the MRFIT clinical megastudy5 and the Japanese Atherosclerosis Society (JAS) study.7 For example, when plotted against the cholesterol level, neither the extent of plaque disruption nor contraction induced by vasoactive substances followed the J-shaped curve. In the present study, we demonstrate for the first time that the Ca2+ sensitization mediated by a novel SPC/Rho-kinase pathway follows the J-shaped curve.
In the present study, Ca2+ sensitization was evaluated directly using 2 techniques: firstly, the simultaneous measurement of [Ca2+]i and force; and secondly, the permeabilization of cell membrane using -escin and -toxin. A Rho-kinase inhibitor (Y27632) (Figure 1) and a dn-ROK (Figure 1e) were used to demonstrate that SPC induces Ca2+ sensitization through Rho-kinase activation. This observation is also supported by the SPC-induced translocation of cytosolic Rho-kinase to the cell membrane (Figure 1g). Using a porcine model, other researchers found that Rho-kinase plays a key role in the induction of VSM hypercontractions such as vasospasm.4 Our results, taken together with these findings, strongly suggest that Ca2+ sensitization of VSM mediated by a SPC/Rho-kinase pathway is activated by hypercholesterolemia and triggers abnormal vascular contractions. Vasospasms, for example, could increase the relative risk of coronary disease, which was seen to be associated with a rise in serum cholesterol levels in the MRFIT study.5
The molecular mechanism(s) by which cholesterol potentiates VSM Ca2+ sensitization are not completely clarified. Cholesterol depletion by -cyclodextrin blocked not only SPC-induced contraction (Figure 4) but also translocation of Rho-kinase to cell membrane (Figure 1). Because -cyclodextrin induces a selective destruction of a cholesterol-enriched membrane microdomain, lipid raft,20 it is likely that Rho-kinase translocated to lipid raft. Although disruption of lipid rafts is known to affect many signal transduction components and disruption of lipid rafts by -cyclodextrin may be trivial, these effects of -cyclodextrin seem to be specific for cholesterol depletion and VSM contraction (see also a detailed description in the online data supplement). It was clearly demonstrated that membrane lipid raft contains more cholesterol than other nonraft membrane in smooth muscle.30 Indeed, depletion of cholesterol by -cyclodextrin resulted in marked reduction of staining of caveolin-1, a well-known marker of membrane lipid raft, in cultured VSM, as shown in supplemental Figure II. In addition, we previously reported that Src-TK mediates SPC-induced contraction and activation of Rho-kinase and that SPC induces translocation of Fyn, a member of Src-TK localizing to lipid raft.13 Taken together, these findings are compatible with a crucial role of cholesterol-enriched membrane lipid raft in the VSM Ca2+ sensitization. In addition, future studies must address the question of whether plasma cholesterol levels (and not just -cyclodextrin treatment) indeed have an influence on VSM lipid raft composition.
In conclusion, our data indicate that SPC may be a novel trigger for cholesterol-dependent Ca2+ sensitization of VSM contractions mediated by Rho-kinase in humans and rabbits. Furthermore, the SPC/Rho-kinase pathway may initiate the abnormal vascular contractions associated with hypercholesterolemia. We predict that therapy to lower the levels of cholesterol in the serum of patients would reduce the occurrence of cardiovascular events associated with abnormal vascular contractions. Finally, our results also suggest that cholesterol and its enriched lipid rafts may contribute to the SPC/Src-TK/Rho-kinase–mediated Ca2+ sensitization of VSM, which may provide molecular mechanism for the beneficial, even acute, effects of cholesterol-lowering therapy on cardiovascular events.28,29
Acknowledgments
We thank S. Miwa for technical assistance and M. Ohara (Fukuoka) for critical comments on the manuscript.
Sources of Funding
This work was supported in part by a research grant from Ube Industries and grants-in-aid for scientific research from the Ministry of Education, Science, Technology, Sports and Culture, Japan.
Disclosures
None.
Footnotes
Original received August 20, 2003; first resubmission received August 8, 2005; second resubmission received February 9, 2006; revised second resubmission received May 31, 2006; accepted June 27, 2006.
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