Introduction: Connexins (Cx) are transmembrane proteins, which can oligomerize to intercellular channels, so called gap junctions. Apart from mediating communication and transport between adjacent cells, Cx have been shown to regulate endothelial permeability and vascular inflammation. While an atheroprotective role is assumed for Cx37 and Cx40, the involvement of Cx43 in atherogenesis is controversially discussed.

Materials and methods: Carotid plaque specimens were collected from 40 patients undergoing endarterectomy due to severe stenosis (>70%) of the internal carotid artery. Two age- and sex-matched patient cohorts were created, based on classification of trichrome stained plaque specimens into stable (n=20) and vulnerable (n=20) plaques. Immunohistochemical stainings were performed to detect Cx43, CD31, CD68 and CD3 in plaque cross-sections. Degree of neovascularization was evaluated by determining the amount of CD31⁺ neovessels. Numbers of Cx43-expressing cells, CD68⁺ macrophages and CD3⁺ T cells was assessed in the plaque shoulder regions and the fibrous cap. Quanti-gene RNA-assays were performed to determine intraplaque gene expression.

Results: Patients with vulnerable plaque had a higher incidence of acute cerebral ischemic events (stroke, TIA) than those with stable plaque (p=0.034). Immunohistochemical analyses of plaque cross-sections revealed a high content of Cx43⁺cells in plaque shoulders and to a lesser extent in the fibrous cap. Cx43 was mainly expressed by CD68⁺ macrophages and CD31⁺ endothelial cells. Both plaque shoulders and fibrous caps showed higher numbers of Cx43-expressing cells in vulnerable compared to stable plaque specimens (p<0.0001). Cx43⁺cell content in the plaque shoulder correlated with numbers of CD31-expressing neovessels (r=0.3777, p=0.0231), the local T cell (r=0.4485, p=0.0061) and macrophage burden (r=0.6864, p<0.0001), the mean area of the lipid core (r=0.591, p=0.0002) as well as intraplaque expression of TNF-α (r=0.5148, p=0.0016) and chemokine receptor 5 (r=0.5526, p=0.0006). Interestingly, in plaques without neovascular processes, Cx43 expression was almost absent in the endothelium bordering the lumen. No correlation was found with the degree of luminal stenosis, while an inverse association with mean fibrous cap thickness (r=-0.6435, p<0.0001) was observed. Almost identical correlations were found for Cx43⁺cells in the fibrous cap.

Conclusion: The correlation of Cx43+ cell counts with plaque neovascularization and further markers of plaque vulnerability such as fibrous cap thickness and lipid core size strongly suggests the involvement of Cx43 in atherosclerosis progression.