Importance of Junctional Adhesion Molecule-C for Neointimal Hyperplasia and Monocyte Recruitment in Atherosclerosis-Prone Mice–Brief Report
Objective— Although junctional adhesion molecule (JAM)-C has been implicated in the control of inflammatory leukocyte recruitment, its role in neointima formation after arterial injury has not been elucidated.
Methods and Results— In apolipoprotein E–deficient (Apoe−/−) mice fed an atherogenic diet, antibody blockade of JAM-C significantly reduced neointimal hyperplasia after wire injury of carotid arteries without altering medial area and decreased neointimal macrophage but not smooth muscle cell (SMC) content. An increased expression of JAM-C was detected in colocalization with luminal SMCs 1 day after injury and neointimal SMCs after 3 weeks. Blocking JAM-C inhibited monocytic cell arrest and leukocyte adhesion to carotid arteries perfused ex vivo and in vivo. Furthermore, monocyte adhesion to activated coronary artery SMCs under flow conditions in vitro was diminished by blocking JAM-C.
Conclusions— Our data provide the first evidence for a crucial role of JAM-C in accelerated lesion formation and leukocyte recruitment in atherosclerosis-prone mice.
The recruitment of inflammatory cells to the vessel wall determines neointimal hyperplasia after injury.1,2 A key role herein can be ascribed to junctional adhesion molecules (JAMs) of the immunoglobulin superfamily, which mediate firm arrest but also subsequent transmigration of leukocytes across endothelial cells (ECs).2–4 JAM-C has been identified to be localized in tight junctions of ECs,4,5 in smooth muscle cells (SMCs),6 but also human platelets7 and blocking antibodies to JAM-C were shown to inhibit leukocyte accumulation in different mouse models of inflammation.5–7 In Apoe−/− mice, JAM-C was described to be highly expressed in ECs and intimal SMCs.6 Recently, genetic deficiency in JAM-C was demonstrated to result in respiratory, digestive, and immune disorders, unveiling its importance in diverse biological processes.8
We here addressed the role of JAM-C in neointima formation after wire-induced arterial injury in Apoe−/− mice using a blocking antibody.
Materials and Methods
For detailed Materials and Methods, please see the supplemental materials (available online at http://atvb.ahajournals.org).
Mouse Model of Carotid Artery Injury
Apoe−/− mice on high-fat diet received monoclonal H33 or isotype control9,10 3/wk and underwent wire-induced injury of carotid arteries. Movats’ pentachrome and immunofluorescence staining was performed on paraffin-embedded artery sections.
Adhesion Assay, Ex Vivo Perfusion, and Intravital Microscopy
MonoMac6 cell adhesion to human coronary SMCs was analyzed in a parallel-wall flow-chamber. Leukocyte arrest to carotid arteries was analyzed ex vivo or in vivo.
Results and Discussion
Carotid arteries of Apoe−/− mice fed an atherogenic diet and treated with a specific neutralizing anti–JAM-C (H33)9,10 or isotype control antibody were analyzed 3 weeks after wire-induced denudation-injury. Compared with control-treated mice, a significant reduction in neointimal plaque formation but not medial areas was observed in H33-treated mice (Figure 1A and 1B). Quantitative immunostaining revealed a significant decrease in the relative content of neointimal macrophages (Figure 1C and 1D), whereas SMC content was not altered (Figure 1E). The inhibition of plaque formation may thus be attributable to an attenuation of monocyte infiltration.
In contrast to healthy vessels, atherosclerotic arteries display increased expression of JAM-C in EC layers and intimal SMCs, and oxLDL induces the redistribution of JAM-C from inter endothelial contacts supporting leukocyte recruitment.6 Compared to marginal staining in uninjured arteries, an enhanced expression of JAM-C could be observed in injured segments of Apoe−/− carotid arteries (not shown). One day after injury, double-immunofluorescence staining revealed the expression of JAM-C in colocalization with luminal SMCs (Figure 2A), whereas remaining ECs could only rarely be detected (not shown). This is in line with previous findings that SMCs can replace ECs in denuded vessels and display a proadhesive phenotype with a constitutive upregulation of different chemokines11 and implies that luminal SMCs can express and present JAM-C to circulating leukocytes. Three weeks after injury the expression of JAM-C was detected in colocalization with luminal ECs but also SMCs and neointimal plaque SMCs (Figure 2A).
After injury, the exposed subendothelial matrix can trigger the adhesion of platelets, which support leukocyte recruitment.1,12 On human platelets, JAM-C serves as a ligand for leukocyte-expressed Mac-1 facilitating platelet–leukocyte interactions.7 Whereas double-immunofluorescence staining revealed the accumulation of P-selectin+ platelets in arteries 1 day after injury, no colocalization with JAM-C was seen. Likewise, JAM-C was not detectable by FACS analysis on resting or thrombin-activated mouse platelets (supplemental Figure III), contrasting findings in human platelets.7
To further assess a contribution of JAM-C in early leukocyte recruitment, monocytic cell arrest was assessed in carotid arteries perfused ex vivo 1 day after injury. A marked accumulation of Mono Mac 6 cells was observed in untreated or isotype control preperfused arteries, which was significantly reduced after preperfusion with H33 (Figure 2B). Similarly, intravital microscopy revealed a significant reduction in the number of leukocytes adherent to carotid arteries 1 day after injury in vivo in H33-treated mice, as compared to untreated or isotype control-treated mice (Figure 2C). In vitro, Mono Mac 6 cell arrest supported by oxLDL-activated coronary human SMCs was markedly inhibited by pretreatment with H33 (Figure 2D). Furthermore, Mono Mac 6 cell adhesion to oxLDL-activated SMCs was markedly reduced in the presence of a blocking Mac-1 antibody, and the combination of both H33 and anti–Mac-1 antibodies resulted in an inhibition that was more pronounced than either treatment alone, whereas respective isotype controls had no effect (Figure 2D). These data imply that in conjunction with other ligands known to be expressed in oxLDL-activated SMCs, eg, ICAM-1,6 leukocyte-expressed Mac-1 might directly interact with JAM-C on SMCs. These data identify a prominent role of JAM-C in leukocyte adhesion and suggests that JAM-C on SMCs supports monocyte recruitment in vivo, thus contributing to neointima formation after injury.
The expression and interaction of endothelial JAM-C with JAM-B at intercellular junctions has been demonstrated.5,10 Although JAM-C can function as a counter-receptor for Mac-1 independently of JAM-B,7 antibodies to JAM-C can abolish the formation of JAM-B/JAM-C heterodimers10 and diminish monocyte extravasation by promoting reverse migration rather than by reducing orthograde transmigration.8 Whereas these mechanisms might contribute to the decrease in neointimal macrophages in H33-treated mice at later time points during reendothelialization, early adhesive interactions supported by JAM-C on exposed SMCs might mechanistically differ. In keeping with our data, JAM-C on SMCs may also engage in direct interactions with leukocytic Mac-1 to mediate adhesion to sites of inflammation or injury.
In conclusion, our data for the first time demonstrate that blocking JAM-C inhibits neointima formation and monocyte accumulation after arterial injury in Apoe−/− mice. Although potential side effects may preclude a systemic inhibition, JAM-C may constitute a therapeutic target for a local modulation of arterial remodeling in atherosclerosis.
We thank P. Hammel for antibody generation.
Sources of Funding
This work was supported by the Deutsche Forschungsgemeinschaft (FOR809-TP3/TP6), Avenir Program Inserm, Ligue Nationale contre le Cancer, Swiss National Science Foundation (310000-120184/1), and Swiss Cancer League (OCS-01653-02-2005).
E.S. and Y.D.-T. contributed equally to this study.
Received March 11, 2009; revision accepted May 24, 2009.
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