Background: Peripheral Artery Disease (PAD) is caused by atherosclerotic narrowing of arteries supplying the legs. PAD-induced myopathy is characterized by myofiber degeneration and progressive fibrosis. Qualitative histological review suggests pathological changes in the microvasculature of PAD muscle, in association with advancing fibrosis. We tested the hypothesis that microvessel architecture, pericyte coverage, and collagen profiles systematically change with advancing disease and are consistent with advancing microvascular pathology. Methods: Biopsies of PAD patients at Fontaine Stage II (n=15) and IV (n=16), and controls (n=15) were labeled with antibodies specific for Col I, Col IV, αSMA, or CD31 and analyzed by quantitative wide-field, fluorescence microscopy. Thickness of the basement membrane (BM), peri-microvascular Col I density, and BM lumen diameter were measured. Pericytes were identified by abluminal location within the microvascular BM and αSMA + labeling. Group differences were tested by ANOVA and a post hoc pairwise T Test with Bonferroni correction. Correlations were determined by linear regression analysis. Results: Thickness of the BM was greater in Stage II patients (1.58 μm) compared to controls (1.42 μm) (p<0.043) and in Stage IV (1.75 μm) compared to Stage II patients (p<0.021). Microvascular BM lumen diameter was increased (p<0.001) in Stage IV patients (3.97 μm) compared to control (3.25 μm) and Stage II patients (3.29 μm). Thickened PAD microvessels had greater pericyte coverage than control microvessels. BM thickness correlated positively with microvascular BM lumen diameter (R 2 = 0.513, p<0.001). Peri-microvascular Col I deposition correlated positively with microvascular lumen diameter (R 2 = 0.162, p=0.006), and was greater in Stage IV compared to Stage II patients (p=0.040). Conclusions: Increased perivascular Col I deposition, BM thickening, and BM lumen diameter represent advancing microvascular disease in PAD patients. Pericytes, which deposit BM collagen, are more abundant in thickened microvessels. Pericyte replication and secretion of Col IV may be determining factors in the microvascular pathology of PAD muscle.

Background: The myopathy of Peripheral Artery Disease (PAD), a consequence of ischemia caused by atherosclerotic plaques in arteries supplying the legs, is characterized by myofiber degeneration and fibrosis of the vascular walls and extramyofiber matrix. In association with fibrosis we have found a robust expression of the profibrotic cytokine TGFβ1 in vascular smooth muscle cells. Additionally, we have observed microvascular endothelial “swelling” in PAD muscle. Published data indicate that inhibitors of the angiotensin system reduce fibrosis in multiple organ systems and improve walking performance, in diverse patient populations. We hypothesize that “swelling” of microvasculature endothelial cells represents abnormal accumulation of basement membrane Collagen Type IV (Col-IV) and that treatment with Ramipril will improve the microvasculature. Methods and Results: Gastrocnemius biopsies of PAD patients at Fontaine Stage II (N=5) before and after six months of Ramipril intervention and control patients (N=4) were labeled with an antibody specific for Col-IV. Images were acquired with an automated wide-field microscope and then processed with Image Pro Plus® and AutoQuant® deconvolution software. We used Col IV label to measure wall thickness and lumen diameter of 230 to 360 microvessels per patient, with a custom MatLab program based on the Expectation Maximization algorithm coupled with a Gaussian Mixture Model. Microvessel wall thickness was significantly greater (p < 0.04) in PAD patients before (1.54 ± 0.04 μ) and after (1.61 ± 0.06 μ) Ramipril treatment compared to control (1.42 ± 0.02). Lumen diameter was significantly greater (p < 0.02) in Post-Ramipril (3.49 ± 0.04 μ) compared to Pre-Ramipril (3.18 ± 0.08 μ) and control (3.00 ± 0.11 μ) patients. Conclusions: The increase of microvessel lumen diameter with Ramipril treatment is expected to increase microvascular perfusion and thereby improve walking distance. Our study will be expanded to increase cohort size and evaluate the association of microvascular measurements with microperfusion determined by Contrast Enhanced Ultrasonography and with walking performance.