Abstract 13514: Apoptogenic Protein is a Novel Phenotypic Marker of the Contractile Vascular Smooth Muscle Cells

Abstract

Introduction: Apoptogenic protein (Apop) is a mitochondrial protein originally identified in the atherosclerotic cells cultured from thoracic aortas of ApoE-deficient mice. Functional analysis revealed that the expression of Apop using an expression vector induces apoptotic death of smooth muscle cells (SMC) in culture through the release of cytochrome c from mitochondria, followed by the activation of the caspase cascade. Here, we show that Apop is expressed in the contractile SMC, and can be a novel phenotypic marker of the SMC. Methods and Results: Human aortic smooth muscle cells (HASMC) and human coronary atery smooth muscle cells (HCASMC) were cultured in HuMedia-SB2 supplanted with hEGF, hFGF, insulin and 5% FBS. Differentiated HASMC were cultured on laminin-coated dishes in HuMedia-SD2 supplemented with heparin and 1% FBS. Gene expression level was analyzed by real time PCR. SM2 and SMemb were used as differentiation marker and synthetic SMC marker, respectively. The differentiated SMC showed a higher level of expression of SM2, and the ratio of SM2/SMemb was higher compared to the synthetic SMC. Apop gene expression was higher in differentiated HASMC compared to the synthetic phase cells. Similarly, HCASMC showed a higher level of Apop and SM2 expression and SM2/SMemb expression ratio in differentiated cells compared to the cells in synthetic phase. These results indicate that Apop is expressed in differentiated SMC at a higher level than synthetic SMC. CArG box, which is often present in phenotypic marker genes expressed in differentiated SMC, exists in the intron1 region of Apop gene. Serum response factor (SRF) binds to CArG box, and regulates the expression of the genes expressed in differentiated SMC. The silencing of SRF using siRNA reduced the expression of Apop in differentiaed HASMC, indicating that SRF regulates the expression of Apop in differentiated cells. Moreover, immunostaining using human atherosclerotic coronary artery specimens revealed that Apop gene is co-expressed with the differentiation marker, SM2 in the coronary artery SMC. Conclusion: These results indicate that Apop is a novel phenotypic marker of the differentiated SMC, and may be a novel therapeutic target for the prevention of the development of atherosclerosis.