title: Transforming growth factor-beta 1 produced by vascular smooth muscle cells predicts fibrosis in the gastrocnemius of patients with peripheral artery disease.
abstract: BACKGROUND
  Lower leg ischemia, myopathy, and limb dysfunction are distinguishing features of peripheral artery disease (PAD). The myopathy of PAD is characterized by myofiber degeneration in association with extracellular matrix expansion, and increased expression of transforming growth factor-beta 1 (TGF-β1; a pro-fibrotic cytokine). In this study, we evaluated cellular expression of TGF-β1 in gastrocnemius of control (CTRL) and PAD patients and its relationship to deposited collagen, fibroblast accumulation and limb hemodynamics.
  
  
  METHODS
  Gastrocnemius biopsies were collected from PAD patients with claudication (PAD-II; N = 25) and tissue loss (PAD-IV; N = 20) and from CTRL patients (N = 20). TGF-β1 in slide-mounted specimens was labeled with fluorescent antibodies and analyzed by quantitative wide-field, fluorescence microscopy. We evaluated co-localization of TGF-β1 with vascular smooth muscle cells (SMC) (high molecular weight caldesmon), fibroblasts (TE-7 antigen), macrophages (CD163), T cells (CD3) and endothelial cells (CD31). Collagen was stained with Masson Trichrome and collagen density was determined by quantitative bright-field microscopy with multi-spectral imaging.
  
  
  RESULTS
  Collagen density increased from CTRL to PAD-II to PAD-IV specimens (all differences p < 0.05) and was prominent around microvessels. TGF-β1 expression increased with advancing disease (all differences p < 0.05), correlated with collagen density across all specimens (r = 0.864; p < 0.001), associated with fibroblast accumulation, and was observed exclusively in SMC. TGF-β1 expression inversely correlated with ankle-brachial index across PAD patients (r = -0.698; p < 0.001).
  
  
  CONCLUSIONS
  Our findings support a progressive fibrosis in the gastrocnemius of PAD patients that is caused by elevated TGF-β1 production in the SMC of microvessels in response to tissue hypoxia.
authors: Department of Surgery, University of Nebraska Medical Center, Omaha, NE, USA. dha@unmc.edu.|Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, USA. dha@unmc.edu.
authors: Duy M
authors: DM
authors: Ha
authors: Department of Surgery, University of Nebraska Medical Center, Omaha, NE, USA. lauren.carpentermd@gmail.com.
authors: Lauren C
authors: LC
authors: Carpenter
authors: Department of Surgery, University of Nebraska Medical Center, Omaha, NE, USA. koutakisp@unmc.edu.
authors: Panagiotis
authors: P
authors: Koutakis
authors: Department of Surgery, University of Nebraska Medical Center, Omaha, NE, USA. stanley1swanson@gmail.com.
authors: Stanley A
authors: SA
authors: Swanson
authors: Department of Surgery, University of Nebraska Medical Center, Omaha, NE, USA. zhenzhu@unmc.edu.
authors: Zhen
authors: Z
authors: Zhu
authors: Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, USA. MinaHanna@creighton.edu.
authors: Mina
authors: M
authors: Hanna
authors: Department of Surgery and VA Research Service, VA Nebraska-Western Iowa Health Care System, Omaha, NE, USA. Holly.DeSpiegelaere@va.gov.
authors: Holly K
authors: HK
authors: DeSpiegelaere
authors: Department of Surgery, University of Nebraska Medical Center, Omaha, NE, USA. ipipinos@unmc.edu.|Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, USA. ipipinos@unmc.edu.|Department of Surgery and VA Research Service, VA Nebraska-Western Iowa Health Care System, Omaha, NE, USA. ipipinos@unmc.edu.|983280 Nebraska Medical Center, Omaha, NE, 68198-7690, USA. ipipinos@unmc.edu.
authors: Iraklis I
authors: II
authors: Pipinos
authors: Department of Surgery, University of Nebraska Medical Center, Omaha, NE, USA. gpcasale@unmc.edu.|987690 Nebraska Medical Center, Omaha, NE, 68198-7690, USA. gpcasale@unmc.edu.
authors: George P
authors: GP
authors: Casale
mesh_terms: D016022:Case-Control Studies; D003094:Collagen / Q000378:metabolism; D003710:Demography; D005260:Female; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D005355:Fibrosis; D006801:Humans; D008297:Male; D055806:Microvessels / Q000378:metabolism / Q000473:pathology; D008875:Middle Aged; D018482:Muscle, Skeletal / Q000473:pathology*; D009131:Muscle, Smooth, Vascular / Q000473:pathology*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D058729:Peripheral Arterial Disease / Q000473:pathology*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
chemical_list: D053773:Transforming Growth Factor beta1; D003094:Collagen