Abstract 19258: Altered Redox State Impairs the Functional Competence of Skeletal Muscle Pericytes in Patients With Complicated Type 2 Diabetes
Introduction: Type 2 diabetes mellitus (T2D) in association with critical limb ischemia (CLI) is detrimental to skeletal muscle function and metabolism and compromises muscle intrinsic regenerative ability.
Hypothesis: Assess whether T2D+CLI negatively impact on muscular regenerative cells such as muscle pericytes (MPs) functional properties.
Methods: Skeletal muscle biopsies were obtained under ethical approval at occasion of osteosarcoma diagnosis (H-MPs, n=8 ) or major amputation (T2D+CLI-MPs, n=11). MPs were characterized by flow cytometry (CD90, CD44, and Alkaline Phosphatase-AP), immunofluorescence (PDGFR-β, α-SMA, CD146, and NG2) and immunicytochemistry (AP). MP proliferation (BrdU assay), migration (scratch assay), apoptosis (caspase assay), myogenic differentiation (myosine heavy chain-MyHC staining), HUVECs tubulogenesis (Matrigel) and ROS production (MitoSox-Red) were assessed in vitro in at least n=3 donors per group. Molecular analysis were performed by RT-PCR.
Results: Both H-MPs and T2D+CLI-MPs expressed typical pericyte markers, but T2D+CLI-MPs proliferated slowly (0.16±0.06 vs. 1.98±0.46 vs. H-MPs) and differentiated less into MyHC positive fibres (4.78±0.34 vs. 16.15±5.032 vs. H-MPs) while migration and apoptosis were not modified. The addition of T2D+CLI-MPs or their conditioned media to HUVECs impaired endothelial tubulization on Matrigel (respectively 10.40±1.39 vs. 13.73±0.64, and 11.88±1.04 vs. 16±1.4, vs. HUVECs alone). Furthermore ROS content increased in T2D+CLI-MPs (3.70±0.45 vs. 0.57±0.24, vs. H-MPs) and was associated with a down-regulation of SOD-1 (0.53±0.11 vs. 1.03±0.11) and catalase (0.41±0.1 vs. 0.97±0.2 vs. H-MPs) and an up-regulation of p66Shc (2.84±0.6 vs. 0.81±0.02 vs. H-MPs). Antioxidant treatment with N-acetylcysteine reverted altered proliferation (1.55±0.06 vs. 1.21±0.02) and antiangiogenic effect (13.63±1.00 vs. 7.367±0.75) compared to untreated counterparts. For all experiments significance was p<0.05.
Conclusions: T2D+CLI hampers MPs biological functions relevant to muscular repair. Reversion of T2D+CLI-MPs alterations by antioxidant treatment suggests possible therapeutic targets for attenuation of peripheral complications.
Author Disclosures: R. Vono: None. C. Gargioli: None. E. Sangalli: None. D. Maselli: None. A. Gotti: None. S. Losa: None. G. Spinetti: None. P. Madeddu: None.
- © 2015 by American Heart Association, Inc.