Microvascular Stenosis in Critical Limb Ischemia: Role of Partial Endothelial to Mesenchymal Transition

Critical limb ischemia (CLI) is a widespread and debilitating manifestation of atherosclerosis. Unfortunately, revascularization strategies are often precluded or unsuccessful, resulting in amputation. A major reason for treatment failure is likely co-existing abnormalities in ­­the microvasculature. However, the specific microvascular defects present in end-stage PAD in humans remain unknown. The purpose of this study was to delineate abnormalities in the microvascular wall in the critically ischemic skeletal muscle of patients with CLI. To elucidate the microvascular landscape in CLI, we studied human tibialis anterior and gastrocnemius muscles harvested from below-knee amputations of 10 individuals with CLI. Control muscles are from individuals without PAD. Capillary and arteriole density were both increased in CLI samples. Surprisingly, the arterioles in CLI patients, were found to be stenotic. Moreover, the endothelial cells themselves underwent a reorientation and were rounded, obstructing the lumen. Notably, 9% of arterioles in CLI patients were completely occluded, while an additional 33% were stenotic. These aberrant endothelial cells showed a striking shift in N-cadherin localization, from diffuse staining to strong junctional and apical enrichment. Furthermore, the obstructive endothelial cells expressed mesenchymal cell markers S100A4 and Snail, indicating partial endothelial-to-mesenchymal transition (EndMT). To determine the mechanism of activation for the partial EndMT, immunostaining for pSMAD2/3 revealed higher signal in endothelial cells of CLI arterioles. As well, the mural cells of CLI arterioles had increased expression of TGFß1, together implicating TGFß singling in driving EndMT in CLI arterioles. These studies reveal EC-based microvascular stenosis as a previously unidentified feature of CLI