Work from our laboratory documents pathological events, including myofiber oxidative damage and degeneration, myofibrosis, micro-vessel (diameter = 50–150 μm) remodeling, and collagenous investment of terminal micro-vessels (diameter ≤ 15 µm) in the calf muscle of patients with Peripheral Artery Disease (PAD). In this study, we evaluate the hypothesis that the vascular pathology associated with the legs of PAD patients encompasses pathologic changes to the smallest micro-vessels in calf muscle. Biopsies were collected from the calf muscle of control subjects and patients with Fontaine Stage II and Stage IV PAD. Slide specimens were evaluated by Quantitative Multi-Spectral and Fluorescence Microscopy. Inter-myofiber collagen, stained with Masson Trichrome (MT), was increased in Stage II patients, and more substantially in Stage IV patients in association with collagenous thickening of terminal micro-vessel walls. Evaluation of the Basement Membrane (BM) of these vessels reveals increased thickness in Stage II patients, and increased thickness, diameter, and Collagen I deposition in Stage IV patients. Coverage of these micro-vessels with pericytes, key contributors to fibrosis and BM remodeling, was increased in Stage II patients, and was greatest in Stage IV patients. Vascular pathology of the legs of PAD patients extends beyond atherosclerotic main inflow arteries and affects the entire vascular tree—including the smallest micro-vessels.

Peripheral artery disease (PAD), a severe atherosclerotic condition primarily of the elderly, afflicts 200 million individuals, worldwide, and is associated with lower extremity myopathy. Circulating markers of inflammation have been linked to risk and severity of PAD but the contribution of local inflammation to myopathy remains unknown. We evaluated, by ELISA, calf muscle of PAD patients (N = 23) and control subjects (N = 18) for local expression of inflammatory cytokines including Granulocyte/Monocyte Colony-Stimulating Factor (GM-CSF), Interleukin 17A (IL-17A), Interferon Y (IFN-Y), tumor necrosis factor a (TNF-a), and Interleukin 6 (IL-6). One or more of these cytokines were expressed in nineteen patients and 2 controls and coordinated expression of GM-CSF, IL-17A, IFN-Y, and TNF-a, a signature of activated, MHC Class II dependent autoreactive Th-cells, was unique to 11 patients. GM-CSF is the central driver of tissue-damaging myeloid macrophages. Patients with this cytokine signature had a shorter (P= 0.017) Claudication Onset Distance (17 m) compared with patients lacking the signature (102 m). Transforming Growth Factor β1 (TGFβ1) and Chemokine Ligand 5 (CCL5) were expressed coordinately in all PAD and control muscles, independently of GM-CSF, IL-17A, IFN-Y, TNF-a, or IL-6. TGFβ1 and CCL5 and their gene transcripts were increased in PAD muscle, consistent with increased age-associated inflammation in these patients. Serum cytokines were not informative of muscle cytokine expression. We have identified a cytokine profile of autoimmune inflammation in calf muscles of a significant proportion of claudicating PAD patients, in association with decreased limb function, and a second independent profile consistent with increased "inflammaging" in all PAD patients.

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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.