Title: CD98 Regulates Vascular Smooth Muscle Cell Proliferation in Atherosclerosis
Abstract: CD98 is a transmembrane protein made of two subunits – CD98 heavy chain (CD98hc) and one of six light chains – and is involved in cell proliferation and survival. However, its influence on atherosclerotic development is unknown. The aim of this study was to determine if CD98hc expression in VSMC plays a role in the morphologic make-up of atherosclerotic plaques by regulating VSMC function. The animal models were double knockout mice (SMC-specific deletion of CD98hc in an atherosclerotic prone mouse) compared to controls which were parent mice only prone to atherosclerosis. Aortic samples from these two groups were used to obtain VSMC for the culture experiments studying in vitro VSMC proliferation and apoptosis. Eight- to 10-week-old mice were placed on a high fat diet (HFD) for up to 16 weeks to aid in developing target atherosclerotic plaques and sacrificed at 0, 4, 8, 12, and 16 weeks after starting the HFD. The aortic sinus, root, and aorta were cut into 5 um sections for Oil Red-O staining (esterified lipids with microscopy), picrosirius red staining (collagen with polarized light microscopy) and immunohistochemistry. Immunohistochemistry of the aortic root sections were stained for SM22a (smooth muscle cells), MOMA-2 (macrophages) and CD98hc. Proper preparation and staining allowed quantification of the extent of atherosclerosis, presence of collagen, macrophages and smooth muscle cell infiltration into the plaque. Serum samples at start of HFD and at harvest were analyzed for serum cholesterol, triglyceride, and circulating leukocyte populations (T cells, B cells, monocytes, neutrophils). The in vivo knockout mouse study verses controls determined the effect of CD98hc deficiency on VSMC function on the atherosclerotic plaque. Eight- to 10-week-old mice from each group were placed on a HFD for 8 weeks and 3 days before sacrifice were injected with an agent taken up during proliferation. Upon sacrifice, the mouse aortas were collected, thinly sectioned, digested and divided into two for detect of proliferation or apoptosis. The culture results demonstrated dramatically reduced cell counts, cell proliferation and migration of the CD98 deficient samples when compared to controls. Analysis of aortic VSCM after 8 weeks of HFD showed a reduction in knockout mouse VSMC proliferation as well as increased apoptosis compared to controls. The long-term in vivo atherosclerosis study demonstrated that total plaque area was similar in both groups. However, the knockout mice showed reduced presence of VSMC within the plaque (2.1 ± 0.4% vs 4.3 ± 0.4% SM22a-positive area per plaque area; P < .05), decreased collagen content, as well as increased necrotic core area (25.8 ± 1.9% vs 10.9 ± 1.6%; P < .05) compared to controls. These results are consistent with the conclusions expressed.