Potential Role of Axonal Chemorepellent Slit2 in Modulating Adventitial Inflammation in a Rat Carotid Artery Balloon Injury Model.

Leukocyte infiltration of adventitial and perivascular tissues is an early event in the development of vascular remodeling after injury. We investigated whether Slit/Robo-an axonal chemorepellent system in vertebrate and invertebrate development-is activated during the inflammatory phase that follows endothelial denudation. Using the rat carotid artery model of angioplasty, we conducted a time course analysis of mRNAs encoding Slit ligands (Slit2 and Slit3) and Robo receptors (Robo1, Robo2, and Robo4), as well as proinflammatory cell adhesion molecule (CAM) genes. Adventitial inflammatory cells were counted in immunostained arterial sections. E-selectin, vascular CAM-1, and intercellular CAM-1 were upregulated 2-3 hours after injury, followed by infiltration of neutrophils and monocytes as evidenced by real-time polymerase chain reaction, in situ hybridization, and immunohistochemistry. Slit2, Slit3, and Robo genes exhibited no expression changes at 3 hours; however, they were markedly upregulated 1 day after angioplasty. Intercellular CAM-1 expression was reduced by 50%, and the number of adventitial neutrophils decreased by >75% 1 day after angioplasty. Slit2 has been shown to be a potent chemorepelent of leukocytes, endothelial cells, and smooth muscle cells. Thus, we decided to further investigate the localization of Slit2 in injured vessels. Immunohistochemical stainings revealed the presence of Slit2 within the vessel wall and in the perivascular vasa vasorum of naive and injured arteries. Double immunohistochemical analyses showed that infiltrating monocytes expressed Slit2 in the perivascular and adventitial tissues of injured arteries 1 and 3 days postangioplasty. In addition, recombinant full-length Slit2 and Slit2-N/1118, an N-terminal fragment of Slit2, inhibited stromal cell-derived factor 1-mediated migration of circulating rat peripheral blood mononuclear cells. In summary, adventitial activation of CAM genes and neutrophil infiltration preceded upregulation of Slit/Robo genes. Sli2 expression colocalized with infiltrating inflammatory cells in the adventitial layer. This temporospatial association suggests that leukocyte chemorepellent Slit2 may be involved in halting the adventitial accumulation of inflammatory cells in injured vessels.

Sequential patterns of chemokine- and chemokine receptor-synthesis following vessel wall injury in porcine coronary arteries.

Inflammation plays a central role in vascular repair, and spreads into perivascular tissue (PVT) following angioplasty. Chemokines (CK) and chemokine receptors (CKR) are key determinants of inflammatory chemotaxis. We sought to assess the arterial and perivascular expression of the CK CCL2 and CXCL2, and the CKR CCR2, CCR5, and CXCR4 in balloon-injured porcine coronary arteries. Vascular cells that express specific CK and CKR mRNA during post-angioplasty time course were detected by in situ hybridization (ISH), and expression was quantified by real time RT-PCR in PVT. CCL2 was maximal in PVT from 2 to 24h post injury, coincident with local macrophage-activation. Expression was upregulated in media and adventitia from 24h to 3 days, and in neointima at 7 days. CXCL2 was detected in media at 2 and 4h, and also in some neointimal cells. CCR2 and CCR5 were maximal in PVT at 24h and 3 days, respectively. Expression shifted to media and adventitia at 2 and 3 days, and to neointima and adventitia at 7 days, and was low at 14 days. CXCR4 was low in PVT, but was upregulated in media and adventitia at 2 and 3 days, as well as in neointima and adventitia at 7 days. In conclusion, PVT is the primary source of inflammatory CK and CKR early post-angioplasty. Specific sequential patterns of CK- and CKR-synthesis are identified that may regulate phase-specific chemotaxis by spatio-temporally differential expression during coronary response to injury.

HERP1 inhibits myocardin-induced vascular smooth muscle cell differentiation by interfering with SRF binding to CArG box.

OBJECTIVE: Myocardin is a coactivator of serum response factor (SRF) required for vascular smooth muscle cell (VSMC) differentiation. HERP1 is a transcriptional repressor, which is abundantly expressed in vascular system and is known to function as a target gene of Notch. However, the role of HERP1 in the pathogenesis of vascular lesions remains unknown. The present study characterizes the expression of HERP1 in normal and diseased vessels, and tests the hypothesis that HERP1 inhibits SRF/myocardin-dependent SMC gene expression. METHODS AND RESULTS: Immunohistochemistry revealed that HERP1 and myocardin expression was localized to SMC in the neointima of balloon-injured rat aorta and in human coronary atherosclerotic lesions. Expression of both HERP1 and myocardin was elevated in cultured VSMCs compared with medial SMC. Overexpressed HERP1 inhibited the myocardin-induced SMC marker gene expression in 10T1/2 cells. HERP1 protein interfered with the SRF/CArG-box interaction in vivo and in vitro. Immunoprecipitation assays showed that HERP1 physically interacts with SRF. CONCLUSIONS: HERP1 expression was associated with the SMC proliferation and dedifferentiation in vitro and in vivo. HERP1 may play a role in promoting the phenotypic modulation of VSMCs during vascular injury and atherosclerotic process by interfering with SRF binding to CArG-box through physical association between HERP1 and SRF.