Abstract 40: Increased Expression of Transforming Growth Factor-Beta 1 in the Gastrocnemius of Patients with Peripheral Artery Disease is a Localized Response in the Ischemic Muscle

Abstract

Introduction: Patients with peripheral artery disease (PAD) develop a myopathy in their ischemic lower extremities, which is characterized by myofiber degeneration and myofibrosis. We previously showed that increased expression of transforming growth factor-beta 1 (TGF-β1), which stimulates collagen deposition, is associated with increased fibrosis in PAD gastrocnemius. Expression was localized to the microvasculature, suggesting that elevated TGF-β1 may be a systemic condition rather than a local response in PAD patients. Hypothesis: We hypothesize that increased expression of TGF-β1 in PAD gastrocnemius is a localized response in the ischemic muscle, which contributes to the myofibrosis of PAD. To test this hypothesis, we evaluated TGF-β1 expression and collagen deposition in the gastrocnemius of the diseased and contralateral legs of patients with unilateral PAD. Methods: Gastrocnemius biopsies were collected from both limbs of PAD patients with unilateral disease (n=10). Muscle homogenates were analyzed by qPCR for TGF-β1 transcripts expressed as fold change in relation to myosin heavy chain. TGF-β1 in paraffin-embedded gastrocnemius sections was labeled for fluorescence analysis and expression measured as grey scale units (gsu) by quantitative fluorescence microscopy. Sections were stained by Masson Trichrome and collagen density was measured by spectral analysis. Results: TGF-β1 transcripts were increased in the diseased compared to contralateral muscle (mean +/- s.e.; 4.65 +/- 0.95 vs. 0.61 +/- 0.15; p = 0.007) as was TGF-β1 protein expression (919 +/- 169 gsu vs. 593 gsu +/- 115; p = 0.013). Increased TGF-β1 expression was accompanied by increased collagen deposition in the diseased versus contralateral muscle (2162 gsu +/- 99 vs. 1739 gsu +/- 73; p = 0.007). Conclusions: TGF-β1 expression in PAD gastrocnemius does not represent a systemic condition that may interact with local ischemic events and thereby contribute to PAD myopathy. Instead, it is an integral and localized response in ischemic muscle, which stimulates fibrosis in the diseased muscle. Consequently, successful intervention in the fibrotic response of PAD muscle may be dependent on addressing tissue hypoxia or metabolic shifts associated with ischemia.