Early atherosclerosis in a chronic proteasome inhibition model lacks association with vasa vasorum neovascularisation

Background: Neovascularisation of the vasa vasorum (VV) is noted in the early stage of atherosclerosis; however, its pathophysiological role remains unclear. Proteasome inhibition has been shown to yield anti-angiogenic effects and to induce and aggravate atherosclerosis in normal (N) and hypercholesterolemic (HC) pigs. Purpose: To test the hypothesis that early atherogenesis develops without concomitant VV neovascularization in the setting of proteasome inhibitor treatment. Methods: Female pigs (n=4 per group) were randomized to either N or HC group without or with additional treatment of the proteasome inhibitor MLN-273 (0.08 mg/kg SQ twice weekly, N+PSI or HC+PSI). The coronary arteries were harvested after twelve weeks and processed for micro-CT imaging and tissue analyses. Results: As presented in the table, compared with N, VV density was increased in HC but not N+PSI or HC+PSI (2.8±0.2 vs. 5.0±0.9, 2.5±0.8, and 2.1±0.1 n/mm2, p<0.05; figure) as was vascular-area-fraction (0.64±0.13 vs. 1.80±0.34, 0.68±0.21, and 0.60±0.07%, p<0.01). Compared with N, the percentage of Ki- 67-positive VV tended to be higher in HC but not in N+PSI or HC+PSI (5.0±1.4 vs. 16.4±4.2, 13.9±9.2, and 1.0±0.4%, p<0.01). HIF-1α expression in the outer media, compared with N, was higher in HC, N+PSI, and HC+PSI (45.0±3.1 vs. 65.9±3.3, 74.8±4.7, and 77.8±3.0%, p<0.01 for HC vs. HC+PSI). VEGF staining per field in the adventitia, compared to N, was increased in HC, but not in N+PSI and HC+PSI (7.08±2.14 vs. 24.76±4.34, 2.52±1.70, and 1.03±0.54%, p<0.01). Conclusions: The current study shows that chronic proteasome inhibition prevents the increase in VV density in HC pigs but does not cause a significant decrease in VV density in N pigs. The underlying pharmaceutical mechanism seems to include the HIF-1α-VEGF-pathway. As chronic proteasome inhibition was shown before to induce and aggravate atherosclerosis in N and HC pigs, the current findings would suggest that the early stage of atherosclerosis did not necessarily require VV neovascularization for its development. Acknowledgement/Funding: National Institutes of Health grants; Mayo Foundation; Mayo Stiftung