CC chemokine receptor 2 functions in osteoblastic transformation of valvular interstitial cells

Calcific aortic valve disease (CAVD) emerges as a challenging clinical issue, which is associated with high cardiovascular mortality. It has been demonstrated that osteoblastic transformation of AVICs is a key mechanism of CAVD and C-C motif chemokine receptors (CCRs) may favor this process. Thus, we aimed to investigate whether CCRs were involved in osteoblastic transformation of AVICs during the development CAVD.

We first analyzed microarray data (GSE51472 and GSE12644) to identify differentially expressed genes between CAVD aortic valve tissue and normal samples, followed by verification of immunohistochemistry, qPCR and western blotting. Primary aortic valvular interstitial cells (AVICs) were incubated with specific inhibitors and/or siRNA of CCR2 and CCL2 under pro-calcifying medium. The levels of CCL2 in the medium were measured by ELISA. In addition, we used recombinant CCL2 to activate CCR2 in calcifying AVICs. Alizarin red S staining and calcium deposition were used to evaluate the degree of calcification. Western blotting was used to determine osteoblastic transformation of AVIC and total Akt and phosphorylated Akt expression.

CCR2 levels were remarkably up-regulated in calcified aortic valve and calcifying AVICs. Silencing CCR2 inhibited the osteoblastic transformation and calcification of AVICs. In addition, recombinant CCL2 activated CCR2 and accelerated AVICs calcification through PI3K/Akt pathway.

We characterize abnormal activation of CCL2/CCR2 axis as a promoter of AVICs osteoblastic transformation and calcification. Our findings implicate the CCL2/CCR2-PI3K/Akt pathway as a potential target for treatment of CAVD.