The Effect of Lethal Radiation and PDGFBR Signaling on the Contribution of SMC in Atherosclerotic Lesions

Thrombosis after rupture or erosion of unstable atherosclerotic lesions is the leading cause of death worldwide. However, despite decades of research, the factors and mechanisms that control lesion stability are poorly understood. Human pathological studies have shown that lesions containing a thin fibrous cap and an abundance of CD68+ relative to ACTA2+ cells are at risk for rupture. CD68 is presumed to be a marker of macrophages (MΦ) and ACTA2 is the most common marker of smooth muscle cells (SMC). However, use of marker proteins alone is insufficient to identify cell lineage and thus, there are major ambiguities as to the origins of these cells as well as the pathways that control their functions within lesions. Herein, we showed that ACTA2+ cells in the fibrous cap derived from SMC seem to be the most effective regulators of lesion stability by three independent methods.
The first method elucidated the effects of radiotherapy on SMC in atherosclerosis. Radiotherapy has well-documented long-term adverse effects on cardiovascular disease (CVD), however, the reasons for this increased risk are not well characterized. Our studies tested whether radiation impairs protective adaptive SMC responses during vascular disease. To do so, SMC-lineage tracing mice were exposed to lethal radiation (1,200 cGy) with bone marrow transplantation (BMT) prior to atherogenesis or vessel injury. In the irradiated animals, there was a complete loss of SMC investment in 100% of carotid artery, aortic arch, and brachiocephalic artery (BCA) lesions, with associated decreases in multiple indices of atherosclerotic lesion stability within the BCA. This was compared to non-irradiated control animals that showed SMC investment in lesions. Importantly, there was no decrease in ACTA2 expression in the cap overall, suggesting after radiation these non-SMC derived ACTA2+ cells are not able to fully maintain lesion stability. Interestingly, we observed anatomic heterogeneity, as SMC ac-cumulated normally into lesions of the aortic root and abdominal aorta. These results revealed an undefined and unintended variable in previous studies using lethal irradiation and may help explain why patients exposed to radiation have increased risk for CVD.
The second method utilized a SMC-specific conditional knockout of the platelet derived growth factor beta receptor (PDGFBR) in Apoe-/- mice that was associated with >94% loss of SMC investment within lesions and the fibrous cap. Unexpectedly, this was not associated with detectable changes in lesion size or indices of plaque stability following 18 weeks of WD feeding. This was due at least in part to compensatory increases in the fraction of ACTA2+ fibrous cap cells derived from endothelial cells and MΦ transitioning to a mesenchymal state (EndoMT or MMT). However, this compensation was transient as mice fed WD for 26 weeks showed evidence of plaque instability including reduced collagen content and increased intraplaque hemorrhage, despite persistent increases in EndoMT and MMT. In these studies, we challenged the dogma that ACTA2+ cells within the fibrous cap of advanced lesions are nearly if not entirely de-rived from SMC by showing that up to 40% are derived from other sources.
Finally, we provided evidence based on Imatinib intervention studies and delayed knockout of PDGFBR in SMC that maintenance of a SMC- and ACTA2-rich fibrous cap is dependent on sustained PDGFBR signaling. Using SMC-lineage tracing mice, PDGFBR was knocked out in Myh11+ cells between 16-18 weeks of WD. BCA lesions were analyzed eight weeks later and showed significantly reduced ACTA2+ SMC and collagen content in the fibrous cap. Global antagonism of PDGFBR in SMC-lineage tracing mice by daily Imatinib administration also resulted in significant loss of total SMC and ACTA2+ cells in the fibrous cap and caused morbidity and mortality in 100% of the mice within seven days of injections, compared to 0% in the saline control group. Taken together, these intervention studies demonstrate that sustained PDGFBR signaling is required for maintenance of a protective fibrous cap, and antagonism is associated with deleterious effects on the lesion.
These studies demonstrate that radiation inhibits, and PDGFBR signaling in SMC induces their investment and retention within the fibrous cap, where SMC normally augment lesion stability, but that other cell types also play an under-appreciated role in con-tributing to lesion stability. Furthermore, these studies make a case that fortifying the fibrous cap may be a novel therapeutic target to stabilize existing atherosclerotic lesions.