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(Circulation. 2003;107:1033.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Reduced Connexin43 Expression Inhibits Atherosclerotic Lesion Formation in Low-Density Lipoprotein Receptor–Deficient Mice

Brenda R. Kwak, PhD; Niels Veillard, MS; Graziano Pelli; Flore Mulhaupt; Richard W. James, PhD; Marc Chanson, PhD; François Mach, MD

From the Division of Cardiology (B.R.K., N.V., G.P., F. Mulhaupt, F. Mach), Division of Endocrinology and Diabetes, Department of Medicine (R.W.J.), and Department of Pediatrics (M.C.), University Hospital, Geneva, Switzerland.

Correspondence to François Mach, MD, Foundation for Medical Research, Division of Cardiology, 64 Avenue Roseraie, 1211 Geneva, Switzerland. E-mail francois.mach{at}medecine.unige.ch

	Abstract

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Background— Gap junctions allow the direct exchange of ions and small molecules between cells in contact, thus coordinating physiological processes such as cell growth and differentiation. We have recently demonstrated increased expression of the gap junction protein connexin43 (Cx43) in specific subsets of cells in atherosclerotic lesions. Because the development of atherosclerosis depends critically on paracrine cell-to-cell interactions, we hypothesized that direct intercellular communication via gap junctions may be another factor controlling atherogenesis.

Methods and Results— The role of Cx43 in atherogenesis was examined by use of both a genetic and a pharmacological approach. First, atherosclerosis-susceptible LDL receptor–deficient (LDLR^-/-) mice with normal (Cx43^+/+) or reduced (Cx43^+/-) levels of Cx43 were fed a cholesterol-rich diet for 14 weeks. The progression of atherosclerosis was reduced by 50% (P<0.01) in the thoracoabdominal aorta and in the aortic roots of Cx43^+/-LDLR^-/- mice compared with Cx43^+/+LDLR^-/- controls. Atheroma in Cx43^+/-LDLR^-/- mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells. Next, we observed that HMG-CoA reductase inhibitors, or "statins," lipid-lowering drugs well known for their pleiotropic antiatherogenic effects, reduced Cx43 expression in primary human vascular cells in vitro. Atheroma of LDLR^-/- mice treated orally with pravastatin contained fewer inflammatory cells and exhibited thicker fibrous caps than controls. This was associated with reduced Cx43 expression in lesions of statin-treated mice.

Conclusions— These data indicate a critical role for Cx43-mediated gap junctional communication in atherosclerotic plaque formation.

Key Words: atherosclerosis • ion channels • drugs

	Introduction

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Atherosclerosis, the principal cause of death in adult populations of Western societies, is a chronic immunoinflammatory disease that generally begins in young adolescents and has clinical manifestations in middle age or later.^1,2 This multifactorial process is characterized initially by the subendothelial intimal accumulation of lipid-rich macrophages and T lymphocytes ("fatty streaks"), followed by lesions composed of layers of foam cells and proliferating smooth muscle cells (SMCs) with deposition of extracellular matrix ("atheroma"). Crucial events during atherosclerotic lesion formation are the coordinated interactions between circulating blood cells and cells that reside within the arterial wall.^3,4

Intercellular channels present in gap junctions provide a simple method of synchronizing responses in multicellular organisms through the direct exchange of ions and small molecules between adjacent cells.^5,6 This type of intercellular signaling allows rapidly coordinated activities such as contraction of the heart and transmission of neuronal signals at electrical synapses. In addition, gap junctional communication (GJC) plays a role in slower physiological processes such as cell growth and development. Molecular cloning studies have demonstrated that gap junctions are formed by members of a family of related proteins called connexins (Cxs).⁷ There are 20 different Cxs in the human and mouse genome.⁸ Cx43 is the most widespread and abundant member of this family.

Cxs are dynamic proteins with half-lives ranging from 1 to 5 hours, indicating that gap junction channels are fully exchanged several times a day.^9,10 This may provide a mechanism to regulate direct cytoplasmic cross-talk between cells under normal or pathological conditions. Interestingly, the expression of Cx43 is upregulated in specific subsets of cells during atherogenesis. Indeed, relatively high expression levels of Cx43 are observed in migrating and proliferating SMCs in the neointima,^11,12 in atheroma-associated macrophage foam cells,^13,14 and in endothelial cells (ECs) covering the shoulder region of atherosclerotic lesions.¹²

To the best of our knowledge, no study to date has evaluated whether GJC participates in the process of atherosclerotic plaque formation. Here, we have used both a genetic and a pharmacological approach to reduce Cx43 expression. In mice with reduced levels of Cx43, we show beneficial effects on both the progression and the composition of atherosclerotic lesions. These results provide novel in vivo evidence for a key role of gap junctions in atherogenesis.

	Methods

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Mice
All animal studies were approved by local veterinary authorities. Heterozygous Cx43 mice¹⁵ and LDL receptor (LDLR)–deficient mice,¹⁶ both on a C57BL/6J background (Jackson Laboratory, Bar Harbor, Me) were crossed, and offspring were subsequently intercrossed to obtain Cx43^+/-LDLR^-/- mice and Cx43^+/+LDLR^-/- littermate controls. Genotypes were determined by polymerase chain reaction (PCR), following the suppliers’ instructions.

At 10 weeks, 7 males of each group consumed a high-cholesterol diet (1.25% cholesterol, 0% cholate; Research Diets). After 14 weeks, mice were killed, blood was taken, and aortas were subsequently perfused with 0.9% NaCl. Aortas were separated into 3 parts, of which the roots and arches were snap-frozen in OCT compound and the abdominal parts were fixed in 2% paraformaldehyde. The extent of atherosclerosis was assessed in aortic roots and thoracoabdominal aortas according to standardized methods.¹⁷ Quantification was performed by computer image analysis using Zeiss KS400 Software. Thus, we calculated, for each aortic root, an average of lesion area from 6 sections 50 µm distant from each other by dividing the area of lipid deposition (stained with Sudan IV) by the total valve surface. The thoracoabdominal aorta was opened longitudinally along the ventral midline, and the lesion areas in en face preparation were stained with Sudan IV. The percentage of lipid deposition was calculated by dividing the Sudan IV–stained area by the total thoracoabdominal surface.

In a separate study, 10 male LDLR^-/- mice 10 weeks old consumed the high-cholesterol diet, and 5 of them also received pravastatin at 10 mg · kg^-1 · d^-1 in their drinking water. After 14 weeks, mice were killed and processed as described above.

Lipid Analysis
Mouse plasma cholesterol and triglyceride concentrations were determined before and after the cholesterol-rich diet as described elsewhere.¹⁸

Immunohistochemistry
Five-micrometer serial sections were cut from aortic arches and immunolabeled with antibodies recognizing Cx43 (Zymed), SM-MHC (kindly provided by Prof Gabbiani, Pathology Department, Geneva, Switzerland), or MOMA-2 (Biosource). Protocols and tests for specificity are described elsewhere in detail.¹² Collagens type I and III were stained with picrosirius red.¹⁹

Cell Isolation and Culture
As approved by the local Ethical Committee, human vascular ECs and SMCs were isolated from saphenous veins obtained after varicose vein surgery and cultured as described previously.²⁰ They were used at passages 2 to 5. Subconfluent cultures were stimulated for 48 hours with atorvastatin (Parke-Davis), pravastatin (Bristol-Myers Squibb), or simvastatin (Merck Sharp Dohme) and L-mevalonate (Sigma).

Western Blotting
Cell harvesting, protein quantification, and immunoblotting were performed as described before.²⁰ Blots were incubated with anti-Cx43 (Zymed, 1:250) or anti-human ß-actin (PharMingen; 1:5000).

Dye Coupling
Dye transfer assays on subconfluent cultures of primary human SMCs, cultured for 48 hours with or without 10 µmol/L simvastatin, were performed as described before.²¹

Statistical Analysis
Data are presented as mean±SEM. Data sets containing multiple groups were analyzed by ANOVA. Mean values between 2 groups were compared by a 2-tailed Student’s t test, after an F test for homogeneity of variances had been performed. If data failed to meet the requirements for use of the parametric t test, a Mann-Whitney U test was used. Differences were considered statistically significant at a value of P<0.05.

	Results

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Comparison of LDLR-Deficient Mice With Normal or Reduced Cx43 Levels
To examine the contribution of Cx43 in atherogenesis, Cx43^+/- mice were bred with LDLR^-/- mice. Cx43^+/-LDLR^-/- mice and Cx43^+/+LDLR^-/- littermates were identified by PCR (Figure 1a). At 10 weeks of age on normal chow diet, male Cx43^+/- LDLR^-/- and Cx43^+/+LDLR^-/- mice had similar body weights and comparable plasma levels of cholesterol and triglycerides (Table). Atherosclerosis was induced experimentally by feeding the mice a cholesterol-rich diet for 14 weeks, after which they were killed. Western blotting of aorta protein extracts confirmed reduced Cx43 expression in Cx43^+/-LDLR^-/- mice compared with Cx43^+/+LDLR^-/- littermates (Figure 1a). No significant differences in body weight, plasma lipid profile, or leukocyte count were observed between Cx43^+/-LDLR^-/- and Cx43^+/+ LDLR^-/- mice after 14 weeks of cholesterol-rich diet (Table).

View larger version (40K): [in this window] [in a new window]   Figure 1. Reduced atherosclerotic lesion formation in mice with reduced Cx43 by a genetic approach. a, PCR analysis of offspring from crosses of Cx43+/-LDLR-/- and LDLR-/- resulted in Cx43+/-LDLR-/- (lanes 1 to 3) and Cx43+/+LDLR-/- (lanes 4 to 6) littermates. Bands at 1000 bp represent Cx43 mutant allele, at 500 bp Cx43 wild-type allele, and at 200 bp LDLR mutant allele. Western blot for Cx43 and ß-actin (loading control) of aorta homogenates from these mice confirmed reduced Cx43 expression in heterozygous mice. b, Staining with Sudan IV (red) demonstrates reduced lesion formation in thoracoabdominal aorta (top) and aortic root (bottom) of Cx43+/-LDLR-/- mice (left) vs Cx43+/+ LDLR-/- littermates (right). c, Quantification of atherosclerotic lesion formation in 7 Cx43+/-LDLR-/- (red) and 7 Cx43+/+LDLR-/- (blue) mice. Values are expressed as mean±SEM, *P<0.01.

View this table: [in this window] [in a new window]   Characteristics of LDLR-Deficient Mice With Normal or Reduced Cx43 Levels

Reduction in Lesion Size in LDLR-Deficient Mice With Reduced Cx43 Levels
Atherosclerotic lesion development was first examined in whole-mount en face preparations of the descending aorta stained for lipids with Sudan IV (Figure 1b, top). In control Cx43^+/+LDLR^-/- mice on the cholesterol-rich diet for 14 weeks, >15% of the aorta was covered with lesions (red staining in Figure 1b, top right). In comparison, fewer and smaller lesions were observed in the aortas of Cx43^+/-LDLR^-/- mice (Figure 1b, top left). Examination of aortic roots (Figure 1b, bottom) stained with Sudan IV for lipids showed decreased atherosclerotic plaque formation in Cx43^+/-LDLR^-/- mice compared with Cx43^+/+ DLR^-/- mice. By computer image analysis, we quantified the lipid/valve ratio and the percentage of lipid deposition within abdominal aortas. The results showed that lesion development was reduced by 50% (P<0.01) both at the level of the aortic roots and in the descending aortas of mice featuring genetically reduced amounts of Cx43 (Figure 1c).

Lesion Composition in LDLR-Deficient Mice With Normal or Reduced Cx43 Levels
We then analyzed the composition of the atherosclerotic plaques in aortic arch sections of Cx43^+/-LDLR^-/- and Cx43^+/+ LDLR^-/- mice. Atherosclerotic lesions typically developed at lesion-prone sites, such as the lesser curvature of the aortic arch and the outflow tracts of the brachiocephalic, common carotid, and subclavian arteries. Lesion development at the lesser curvature was less extensive in Cx43^+/-LDLR^-/- mice than in their Cx43^+/+LDLR^-/- littermates, and the lesions of the 2 groups displayed different phenotypes (Figure 2, a and b). We evaluated important determinants of plaque stability such as the cellular composition of the atherosclerotic plaque, ie, inflammatory cells versus SMCs, interstitial collagen content, and lipid deposition. Lesions in Cx43^+/-LDLR^-/- mice were characterized by smaller lipid cores and thicker fibrous caps compared with lesions of Cx43^+/+LDLR^-/- littermates (Figure 2, a and b). The fibrous caps in mice with genetically reduced Cx43 expression contained more interstitial collagen and larger numbers of SMCs (Figure 2, c through f). In addition, lesions in Cx43^+/- LDLR^-/- mice contained fewer inflammatory cells than lesions in Cx43^+/+LDLR^-/- mice: 133±19 and 207±21 mononuclear cells/mm², respectively (P<0.05).

View larger version (138K): [in this window] [in a new window]   Figure 2. Atheroma with thicker fibrous cap in mice with reduced Cx43 by a genetic approach. Photographs of cryosections of Cx43+/-LDLR-/- (a, c, and d) and Cx43+/+ LDLR-/- (b, e, and f) mice. a, b, d, and f, Stained with Sirius red for collagen; c and e, incubated with antibodies against SM-MHC and enzymatically detected (alkaline phosphatase, red signal). Tissue is counterstained with hemalin. a and b, Bar=1200 µm; c through f, bar=75 µm. LC indicates lesser curvature; BC, brachiocephalic artery; CCA, common carotid artery; and SA, subclavian artery. Similar results were obtained in independent experiments with aortic arches from 5 mice in each group.

Statins Reduce Cx43 Expression in Primary Human Vascular Cells In Vitro
Next, we examined whether HMG-CoA reductase inhibitors (statins), lipid-lowering drugs well known for their pleiotropic antiatherogenic effects, influenced Cx43 expression in primary vascular cells of human origin. Three different statins, the lipophilic simvastatin and atorvastatin and the hydrophilic pravastatin, effectively reduced the expression of Cx43 in ECs (Figure 3a) and in SMCs (not shown). This effect was dose-dependent for all 3 statins and in both types of human vascular cells. The effect of simvastatin on Cx43 expression in SMCs, for example, was observed between 0.08 to 10 µmol/L (Figure 3b). The statin-induced reduction in Cx43 expression was abolished in the presence of L-mevalonate (Figure 3a). Cx43 was typically immunolocalized along the cell membranes of contacting SMCs. Simvastatin treatment strongly reduced the amount of Cx43 immunolabeling; however, the subcellular localization of Cx43 was not affected by the statin (Figure 3c). To investigate the functional consequences of statin-induced reduction of Cx43 expression, we carried out dye transfer assays in SMCs. Microinjection of the gap junction–permeable fluorescent tracer Lucifer yellow into 1 cell resulted in its diffusion to multiple neighboring cells. Pretreatment with simvastatin, however, significantly (P<0.01) reduced the Lucifer yellow diffusion (Figure 3d).

View larger version (34K): [in this window] [in a new window]   Figure 3. Statins reduce Cx43 expression and GJC in human vascular cells. a, Western blot for Cx43 and ß-actin of human vascular ECs unstimulated or treated with (1) 10 µmol/L simvastatin, (2) 10 µmol/L atorvastatin, (3) 20 µmol/L pravastatin, (4) 10 µmol/L simvastatin, and (5) 500 µmol/L L-mevalonate for 48 hours. b, Western blot for Cx43 and ß-actin of human vascular SMCs unstimulated or treated with simvastatin at (1) 10 µmol/L, (2) 2 µmol/L, (3) 0.4 µmol/L, (4) 0.08 µmol/L, or (5) 10 µmol/L and (6) 500 µmol/L L-mevalonate for 48 hours. Quantification of Western blots is expressed as ratio of Cx43/ß-actin signal for each sample. Similar results were obtained in independent experiments with cells from 4 different donors. *P<0.01 vs unstimulated cells, **P<0.05 vs cells treated with 10 µmol/L simvastatin. c, Immunostaining for Cx43 (FITC, green signal) on human vascular SMCs unstimulated (left) or treated with simvastatin 2 µmol/L (middle) or 10 µmol/L (right). Cells are counterstained with Evans blue. d, Diffusion of Lucifer yellow (LY) in unstimulated SMCs (top, 1) or treated with 10 µmol/L simvastatin (bottom, 2) for 48 hours. Quantification of dye coupling: number of Lucifer yellow–labeled cells (mean±SEM) in 28 injections for each condition on cells from 3 different donors. *P<0.01 vs unstimulated cells. c, Bar=5 µm; d, bar=30 µm.

Statins Reduce Cx43 Expression in Mouse Atherosclerotic Lesions In Vivo
We evaluated the effects of statins on Cx43 expression in atheroma of LDLR^-/- mice after 14 weeks of cholesterol-rich diet. The hydrophilic pravastatin was administered daily in the drinking water during the period of the diet. No significant differences in body weight, plasma lipid profile, or leukocyte counts were observed between statin-treated and control LDLR^-/- mice (not shown). Immunohistological examination on sections of aortic arches revealed that atherosclerotic plaques of pravastatin-treated mice had thicker fibrous caps (Figure 4; compare a and b with f and g) containing more interstitial collagen (Figure 4, red staining, a and f) and concentric layers of SMCs (Figure 4, blue area below arrowhead in b and g). In addition, lesions in pravastatin-treated mice contained significantly fewer inflammatory cells than lesions in control mice: 126±28 and 214±25 cells/mm², respectively (see also Figure 4, c and h). Interestingly, immunostainings on consecutive cryosections revealed reduced Cx43 expression throughout the whole atherosclerotic lesion in pravastatin-treated mice (Figure 4, compare red staining in b and g). Thus, reduced Cx43 expression in SMCs (Figure 4, compare d and e with i and j) was associated with more interstitial collagen in the fibrous cap but also in the media (Figure 4, compare a and b with f and g). Furthermore, reduced Cx43 expression in the lipid core was associated with fewer inflammatory cells (Figure 4, compare c and d with h and i). Western blotting of aorta protein extracts confirmed reduced Cx43 expression in statin-treated mice compared with controls (Figure 4k).

View larger version (106K): [in this window] [in a new window]   Figure 4. Effects of pravastatin on mouse atheroma. Photographs of cryosections of LDLR-/- mice treated with pravastatin and cholesterol-rich diet (a through e) or with diet alone (f through j). Panels a and f are stained with Sirius Red for collagen. Panels b, d, g and i are incubated with primary antibodies against Cx43, panels c and h with antibodies against MOMA-2, and panels e and j with antibodies against SM-MHC. Antibody binding is with detected alkaline phosphatase (red signal). Tissue is counterstained with hemalin. Bar represent 300 µm in panels a, b, f, and g, and 75 µm in panels c through e and h through j. M indicates media; FC, fibrous cap; LC, lipid core; IC, inflammatory cells; and SMC, smooth muscle cells. Similar results were obtained in independent experiments with aortic arches from 5 mice in each group. k, Western blot for Cx43 and ß-actin (loading control) of pooled aorta homogenates from 3 control (1) and 3 statin-treated (2) mice confirmed reduced Cx43 expression in latter group.

	Discussion

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Homozygous Cx43-deficient mice die at birth because of cardiac malformations.¹⁵ However, breeding Cx43^+/- and LDLR^-/- mice allowed us to assess the effect of diminished Cx43 expression on atherosclerotic plaque formation. Our results provide strong evidence that reduced GJC is associated with decreased development of atherosclerotic lesions, reduced number of atheroma-associated inflammatory cells, and increased thickness of the fibrous cap covering the lesions.

On a normal chow diet, LDLR^-/- mice exhibit total plasma cholesterol levels of 200 mg/dL, which is only 2-fold higher than those of wild-type mice, and they do not develop gross aortic atherosclerotic lesions up to 1 year of age.¹⁶ To induce lesion development in LDLR^-/- mice, a high-cholesterol diet leading to increased plasma cholesterol, which serves as an atherogenic stimulus, is required.¹⁶ Accordingly, we found increased levels of plasma cholesterol and triglyceride levels in our LDLR^-/- mice after 14 weeks of high-cholesterol diet. Importantly, we observed no differences in serum lipid profiles between Cx43^+/-LDLR^-/- and Cx43^+/+LDLR^-/- mice. The 2 groups of mice also displayed similar increases in body weight and leukocyte count after the diet. In contrast, we observed that Cx43 protein levels were reduced by half in Cx43^+/-LDLR^-/- mice compared with Cx43^+/+LDLR^-/- littermates, an observation reported previously for Cx43^+/- mice.²² Thus, Cx43^+/-LDLR^-/- mice may serve as a well-defined model for studies aimed at elucidating the role of Cx43-mediated GJC in atherogenesis.

Reducing Cx43 levels in mice by this genetic approach markedly decreased the development of atherosclerotic lesions, as assessed at 2 localizations. One mechanism that may be responsible for decreased lesion progression in Cx43^+/- LDLR^-/- mice is inhibition of leukocyte infiltration into the lesions. Indeed, despite similar leukocyte counts in peripheral blood, we observed almost 2-fold fewer inflammatory cells in the lesions of Cx43^+/-LDLR^-/- mice compared with Cx43^+/+ LDLR^-/- controls. This suggests that GJC between ECs and leukocytes may promote leukocyte extravasation. Interestingly, in rat aorta, high levels of endothelial Cx43 are associated with areas facing turbulent blood flow that are vulnerable to atherosclerosis.²³ Furthermore, endothelial Cx43 expression has been detected in the shoulder region of advanced mouse atherosclerotic lesions, a localization known to experience disturbed flow that serves as a site of preference for leukocyte adhesion/migration.¹²

The composition of atherosclerotic plaques in Cx43^+/- LDLR^-/- mice was strikingly different from control littermates. Lesions of Cx43^+/-LDLR^-/- mice had smaller lipid cores and fewer macrophages, which serve as a major depot for lipids within atherosclerotic plaques. In addition, the lesions contained more SMCs, a finding that correlated with the enhanced content of interstitial collagen, the product of SMCs. The content of SMCs versus macrophages and the extent of collagen within the lesion and the size of the lipid core are features that in human lesions are related to the vulnerability of atherosclerotic lesions to rupture, with all its devastating consequences, among them acute myocardial infarction.²⁴ Thus, reducing Cx43 levels in mice by a genetic approach may favor potential plaque-stabilizing processes rather than affecting plaque size alone.

Inhibitors of HMG-CoA reductase (statins) lower plasma cholesterol in humans and reduce atherosclerosis-related morbidity and mortality.²⁵ Because several in vitro studies have identified numerous pleiotropic effects of statins on vascular cells that could modulate atherogenesis and plaque rupture via mechanisms other than lowering cholesterol,²⁶ we evaluated the effects of statins on gap junctions in primary human vascular cells. As demonstrated by Western blotting, statins dose-dependently inhibited Cx43 expression. The lower concentrations of simvastatin (0.4 to 0.08 µmol/L) used in this study are within the range of expected tissue levels derived from prescribed pharmacological dosages.²⁷ The effect of the statins on Cx43 expression is abolished in the presence of L-mevalonate, indicating that inhibition of HMG-CoA reductase is responsible for the reduction in Cx43.²⁸ The statin-induced reduction in Cx43 expression was associated with a marked reduction in the cell-to-cell diffusion of a fluorescent tracer. Thus, in human atheroma-associated cells, treatment with statins reduced Cx43 expression, leading to reduced GJC.

The clinical benefits of lipid lowering with statins are often attributed to changes in atherosclerotic plaque composition leading to plaque stability.²⁴ Interestingly, statins do not reduce plasma lipid levels in mice because of compensatory upregulation of HMG-CoA reductase.^28,29 Consequently, possible beneficial effects of statin treatment on the composition of mouse atherosclerotic plaques can be interpreted without this confounding variable. Similar to Cx43^+/-LDLR^-/- mice, lesions of statin-treated mice contained more SMCs and fewer macrophages, exhibited enhanced content of interstitial collagen, and had smaller lipid-rich areas. Importantly, these beneficial changes in plaque morphology were associated with reduced Cx43 expression in statin-treated mice.

In conclusion, using 2 independent approaches to reduce Cx43 expression in mice, we provide here novel evidence for a key role of GJC in atherosclerotic plaque formation. One of the most difficult challenges remaining will be to elucidate the mechanisms by which reduced Cx43-mediated intercellular communication leads to altered atherogenesis. For gap junction channels, this question will ultimately lead to the identification of molecules important for cell growth and differentiation, the intercellular signaling of which would be altered between atheroma-associated cells. The recent development of mice in which Cx43 can be tissue-specifically deleted^30,31 may be of great help to ascertain in which cell type Cx43 is essential for the atherogenic response. Whatever the mechanism, these findings identify Cx43-mediated intercellular communication as a new potential therapeutic target in atherogenesis. In this respect, the recent development of Cx-specific blocking peptides³² is of particular interest.

	Acknowledgments

 
This work was supported by grants from the Swiss National Science Foundation (3234-066311.01 and 3100-067777.02 to Dr Kwak, 3200-065121.01 to Dr Mach, 3100-067120.01 to Dr Chanson, and 3100-64788.01 to Dr James) and from the Fondation Leenaards.

Received August 28, 2002; revision received November 7, 2002; accepted November 11, 2002.

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