Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Master of Science

Program

Medical Biophysics

Supervisor

Pickering, J. G.

Abstract

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.

Summary for Lay Audience

The muscles that exist in our legs require a tremendous amount of blood to keep healthy. In some individuals, the blood supply to the legs is impaired because of plaque build-up in the arteries that supply the legs with blood. When severe, this can result in severe leg pain, skin ulcers that don’t heal, and it ultimately it may require the leg to be amputated. Diabetics are especially prone to this serious problem.

The main treatment for severe blood vessel disease in the legs is surgical bypass or stenting. However, these procedures are not effective for many individuals and an amputation is still necessary. This is a major and world-wide problem.

One reason surgery is ineffective for so many individuals is that not only is there plaque build-up in the leg arteries, but there is also narrowing of the tiny blood vessels – called arterioles – that dive into the muscles themselves. However, because these vessels are so small, they are exceedingly difficult to study.

In this thesis, I have established that, in legs that have blood flow that is sufficiently poor than an amputation is necessary, there is a serious problem with the small arterioles. I examined tissue from these legs using highly sophisticated microscopy techniques. I discovered that the inner lining of arterioles is no longer thin and smooth. Instead, the cells that make up the inner lining, called endothelial cells, become so bulky that they clog the small channels, preventing the blood from flowing. I also discovered that these endothelial cells actually convert into a different type of cell that has a tendency to stick to its nearby neighbours, a bad situation for blood vessels.

These discoveries could explain why bypass procedures fail in many people, because small clogged arterioles cannot be bypassed. The findings could also open possibilities for new strategies to improve the blood flow in individuals with narrowing of the leg arteries.

Normal Arteriole.mov (80161 kB)
Online Video 4.4.1

Occluded Arteriole.mov (64009 kB)
Online Video 4.4.2

Available for download on Saturday, July 24, 2021

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