Prevention of vasa vasorum neovascularization attenuates early neointima formation in experimental hypercholesterolemia

Abstract

Vasa vasorum (VV) neovascularization is a key feature of early atherosclerosis and adds substantial endothelial exchange-surface to the coronary vessel wall. Thus, it is conceivable that VV neovascularization favors the entry of pro-inflammatory and pro-atherosclerotic blood components into the coronary vessel wall. We sought to investigate the effects of Thalidomide (Th), a potent anti-angiogenic drug on vasa vasorum (VV) neovascularization, vessel wall inflammation, and neointima formation in early experimental atherosclerosis. Female domestic swine, 3 months old, were fed normal (N, n = 12) or high-cholesterol diet (HC, n = 12) for 3 months. In each group six pigs were randomized to 200 mg Thalidomide daily for the diet period (N + Th, HC + Th). LADs were scanned with micro-CT (20 microm cubic voxel size) to determine VV spatial density (#/mm2). Fresh-frozen coronary tissue was used for western blotting (VEGF, TNF-alpha, LOX-1, Ikappabetaalpha and Gro-alpha) and electrophoretic mobility shift assay (EMSA, NFkappabeta). Treatment with Thalidomide preserved VV spatial density [2.7 +/- 0.3 (N), 6.4 +/- 0.7 (HC), 3.5 +/- 0.8 (HC + Th); p = ns HC + Th vs. N] and inhibited the expression of VEGF, TNF-alpha and LOX-1, but not NFkappabeta activity in the coronary vessel wall. Immunofluorescence analyses revealed co-localization of vWF but not SMA and NFkappabeta, TNF-alpha as well as VEGF in HC and HC + Th coronaries. Intima-media thickness was significantly inhibited in HC + Th compared to HC. Serum levels of hs-CRP and TNF-alpha did not differ among the groups. Our study supports a role of VV neovascularization in the development of and a therapeutic potential for anti-angiogenic intervention in early atherosclerosis.

Figure 1

Upper panel: Volume rendered micro-CT images of coronary arteries and their vasa vasorum. Coronaries from hypercholesterolemic (HC) animals exhibit significant vasa vasorum neovascularization in comparison to coronaries from normal (N) and Thalidomide treated animals (HC+Th and N+Th). The lower panel shows a bar graph of the histologically quantified intima-media thickening in all four groups. Chronic administration of Thalidomide prevented intima-media thickening in the hypercholesterolemic animals.

Figure 2

Histology (H&E stains, representative examples from each study group). A. Normal, B. Normal + Thalidomide, C. Hypercholesterolemia, D. Hypercholesterolemia plus Thalidomide. Treatment with Thalidomide significantly inhibited intima-media thickening in hypercholesterolemic coronary arteries (D) compared to animals on hypercholesterolemic diet alone (C). Scale: 100μm.

Figure 3

Western blot analysis of the local expression of VEGF, TNF- α , and the receptor of oxidized LDL (LOX-1) in coronary artery tissue. Thalidomide treatment significantly decreased the expression of VEGF (top panel) and TNF-α (mid panel) in the coronary vessel wall. In addition, the expression of the LOX-1 receptor was significantly blunted (bottom panel). * p<0.05 vs. N, # p< 0.02 vs. HC+Th/N+Th (in upper panel), † p<0.001 vs. N and HC+Th/N+Th (in mid panel), § p<0.01 vs. N and HC+Th/N+Th (in lower panel).

Figure 4

Upper panel. Western Blot analysis of phosphorylated inhibitory kappB kinase-alpha (IkB-alpha, n=4 in all groups) showed increased expression in HC, HC+Th and N+Th animals (* p<0.05). Lower panel. Western Blot analysis of growth related oncogene protein-alpha (Gro-alpha, n=4 in N, HC and N+Th and n=5 in HC+Th) demonstrated no significant differences between all groups (N vs. N+Th, p=0.06).

Figure 5

Upper panel shows a representative electrophoretic mobility shift assay (EMSA) for NFkB on HeLa cells (first 3 lanes, lane 1 without tissue) and coronary artery samples (lanes 5–12, SC = specific competitor, NS = non-specific competitor). Lower panel highlights the quantitative results as the difference between non-specific and specific competitor. Compared with normal controls (N), nuclear translocation of NFkB is increased in coronary arteries from animals on a high-cholesterol diet without (HC) or with thalidomide treatment (HC+T) for 12 weeks as well as in animals on a normal diet with thalidomide treatment (N+T) for 12 weeks (*p<0.05 vs. N).

Figure 6

Co-localization of NFkB p65, VEGF and TNF-α with vWF. A, D, G are NFkB p65, VEGF, and TNF-α, respectively. B, E, and H are vWF. C, F, I are overlays of A and B, D and E, and G and H. Scale: 50μm.

Figure 7

Co-localization of each NFkB, VEGF and TNF-α with vWF. Except for VEGF-vWF in the N+Th group there was no further co-localization found in the N and N+Th groups. Both HC and HC+Th groups showed co-localization of all four markers.