Atherosclerosis is the leading cause of death worldwide. Intraplaque angiogenesis strongly contributes to the maturation of a stable plaque to an unstable phenotype by allowing intraplaque hemorrage, the leakage of immune and red blood cells inside the plaque, causing the plaque to enlarge and become rupture-prone.

Cell migration and proliferation, processes involved in angiogenesis, are controlled by different chemokines with their receptors. Among them, CXCR4 and its ligand CXCL12, have been shown to participate in atherosclerosis. However, their role in plaque instability has not yet been evaluated. Therefore, we aim to unravel the role of CXCL12-CXCR4 axis in intraplaque angiogenesis mediated plaque instability and test if therapeutic targeting of this axis can prevent plaque destabilization and rupture.

We analyzed a large dataset of microarray transcriptional profiling of human carotid atherosclerotic plaques taken from symptomatic patients undergoing carotid endarterectomy surgery (GSE163154). Among the top 7 differentially expressed chemokine receptors, CXCR4 had the highest expression and was increased in unstable plaques with intraplaque hemorrhage compared to stable plaques. Using immunofluorescent labeling, we confirmed the presence of CXCR4 in advanced human coronary artery plaques. In particular, CXCR4 was found throughout the lesion expressed by ECs forming intraplaque neovessels. Moreover, the same expression pattern was observed in murine atherosclerotic vein graft lesions, a mouse model that closely resembles human atherosclerosis, including intraplaque angiogenesis and hemorrhage.

To evaluate whether CXCR4 expression in intraplaque ECs can directly affect ECs migration, a key process in angiogenesis, an in vitro scratch-wound healing assay was performed. ECs were treated with a potent CXCR4 antagonist, AMD3100, and either CXCL12 or VEGF (a CXCR4-independent control). AMD3100 did not affect VEGF-induced EC migration, but reduced CXCL12-induced EC migration by 40%, suggesting that endothelial CXCR4 is likely to play a key role in CXCL12-mediated angiogenesis. To test this, we examined the effects of CXCR4 inhibition on complex neovessel formation using an ex-vivo murine aortic ring assay. Rings treated with AMD3100 and CXCL12 produced 60% less neovessels when compared to control rings treated with CXCL12 alone, confirming that the CXCR4-CXCL12 axis directly influences angiogenesis.

Together, our data indicate that CXCR4 is abundantly expressed in advanced atherosclerotic lesions and likely contributes to plaque instability by promoting intraplaque neovascularization. In future studies we will use conditional CXCR4 knockout mice combined with accelerated atherosclerosis vein graft surgery to specifically assess the role of endothelial CXCR4 in the process of intraplaque angiogenesis and hemorrage in in vivo atherosclerosis.