Neointimal formation in the porcine aortic organ culture. I. Cellular dynamics over 1 month

Abstract

Since intimal smooth muscle cells (SMC) are an important feature of atherosclerotic fibrofatty plaques, we tested the hypothesis that endothelial cells (EC) regulate SMC proliferation in the process of neointimal formation. Using a porcine thoracic aortic organ culture (OC) system, we previously showed in a preliminary study that short-term porcine aortic explants incubated in 5% fetal bovine serum for 7 days promote neointimal formation only in the presence of endothelium or in conditioned media collected from proliferating OC with endothelium present. We now report that these organ cultures can be maintained in culture for up to 4 weeks. The surface cells stained positively for dil-acetylated-low-density lipoprotein throughout the 4-week period indicating the continued presence of EC while the neointimal cells stained positively for the smooth muscle actin-specific monoclonal antibodies alpha-SM1 and HHF-35 indicating their smooth muscle nature. The number of EC did not change during the 4-week incubation period, however, EC turnover peaked at 5 days and remained elevated but constant throughout. The number of intimal SMC doubled between day 0 (18.4 +/- 0.2 cells/field) and day 7 (41.5 +/- 0.9) and between day 7 and day 14 (75.8 +/- 10.1). The intimal SMC number then stabilized thereafter (day 21: 79.5 +/- 7.8, day 28: 73.8 +/- 12.1). Cell proliferation played a large part since autoradiography studies using nondenuded OC showed that intimal SMC thymidine index on day 0 was 1.4% +/- 0.4, peaked at the end of day 5 (25.7% +/- 4.1) and gradually decreased thereafter. The peak in the intimal SMC thymidine index occurred during a period of high EC turnover (maximum EC thymidine index on day 5: 21.8% +/- 2.1), and the stabilization of intimal SMC number observed beyond 2 weeks of incubation occurred when EC replication was reduced (day 14: 6.7% +/- 0.21). Incubation of organ cultures denuded of their endothelium in 5% fetal bovine serum showed a marked decrease in neointimal formation. However, denuded OC when incubated in conditioned medium collected from 4-day-old nondenuded OC (4DCM) enhanced neointimal formation of denuded OC. In contrast, the neointima of denuded OC incubated in 24-day-old nondenuded OC conditioned medium was similar to that of controls. The thymidine index of intimal SMC of denuded OC treated with 4DCM was markedly higher than that found with the control treatment at all time points.(ABSTRACT TRUNCATED AT 400 WORDS)