Activation of the Pro-Oxidant PKCβII-p66Shc Signaling Pathway Contributes to Pericyte Dysfunction in Skeletal Muscles of Patients With Diabetes With Critical Limb Ischemia

Abstract

General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/pure/about/ebr-terms AbstractCritical limb ischemia (CLI), foot ulcers, former amputation and impaired regeneration are independent risk factors for limb amputation in diabetic subjects. The present work investigates whether and by which mechanism diabetes negatively impacts on functional properties of muscular pericytes (MPs), which are resident stem cells committed to reparative angiomyogenesis.We obtained muscle biopsies from diabetic patients undergoing major limb amputation and control subjects. Diabetic muscles collected at the rim of normal tissue surrounding the plane of dissection showed myofibres degeneration, fat deposition, and reduction of MPs vascular coverage. Diabetic MPs (D-MPs) display ultrastructural alterations, a differentiation bias towards adipogenesis at the detriment of myogenesis and an inhibitory activity on angiogenesis. Furthermore, they have an imbalanced redox state, with down-regulation of the anti-oxidant enzymes SOD-1 and catalase and activation of the pro- Myofibres Cross-Sectional AnalysisCryosections from diabetic and control muscle biopsies were stained with anti-laminin antibody and analyzed using an ImageJ macro. For each sample (n=3), the area of more than two thousand single fibres was measured. MP isolationHuman MPs were isolated following well-established procedures (12; 27). Briefly, muscle biopsies were finely minced and digested with collagenase II (100U/mL) for 45min at 37°C on shaking. The digestion mixture was centrifuged and re-suspended in growth medium (α-MEM supplemented with 20% FBS). The cell suspension was filtered through a 70μm cell strainer, dispensed in plastic dishes at clonal density (1000 cell/cm 2 ) and incubated at 37°Cand 5% CO2 in the growth medium. MPs were selected by plastic adherence in culture for at least 10 days when they form colonies positive for ALP, NG2 and CD146 (27). Transmission electron microscopy (TEM)For ultrastructural analysis, a pellet of MPs was fixed for 2h at 4°C in a mixture of 2% paraformaldehyde and 2% glutaraldehyde in 0.05mol/L pH7.3 cacodylate buffer, post-fixed in 1% osmium tetroxide, and embedded in Epon-Araldite. Thin sections were counterstained with uranyl acetate and lead citrate and examined with a Philips/FEI Morgagni electron microscope.7 Immuno-cytochemistryAfter fixation with 4% paraformaldehyde, cells were permeabilized with 0.3% Triton X-100 Microphotographs were acquired using the imaging software AxioVision Imaging System (Zeiss). When required for 3D image acquisition, an Olympus FV 1000 confocal laser scanning microscope with 60X oil immersion lens was used. MP flow cytometryMPs were stained for surface antigen expression using the following antibodies: CD44-APC, CD90-APC, and ALP-PerCP Cy5.5 (all from BD Biosciences). For Ser36-phospho-p66Shc quantification, cells were fixed and permeabilized...

Lack of Contribution of p66shc and Its Mitochondrial Translocation to Ischemia-Reperfusion Injury and Cardioprotection by Ischemic Preconditioning

“…However, alterations in p66shc expression/phosphorylation occur in pathological conditions in humans, such as in muscular pericytes of diabetic patients (Vono et al, 2016), in peripheral blood monocytes and renal tissue biopsies of patients with diabetic nephropathy (Xu et al, 2016), and also in peripheral blood monocytes of patients with acute coronary syndrome, but not with stable coronary artery disease (Franzeck et al, 2012). Since such risk factors and co-morbidities may abrogate the cardioprotection by preconditioning (Ferdinandy et al, 2014), it remains to be elucidated whether p66shc contributes toward cardioprotection under pathological conditions.…”

Effect of Stem Cells and Gene Transfected Stem Cells Therapy on the Pancreas of Experimentally Induced Type 1 Diabetes

“…It was proved that MSCs exhibit a surface marker profile ＋ve for CD44 (31). It was added that diabetes negatively impacts on functional properties of tissue resident SCs with consequent defective regeneration (32).…”

Skeletal muscle-derived Human Mesenchymal Stem Cells: influence of different culture conditions on proliferative and myogenic capabilities

“…hMSCs were isolated from skeletal muscle tissue using a protocol that includes mechanical mincing, enzymatic digestion with type II collagenase, filtration, and selection of the colonies on plastic surface at low confluence (Sacchetti et al, 2016;Vono et al, 2016). Briefly, human skeletal muscle biopsies are finely minced with a surgical knife and collected in a solution of collagenase type II (100 U/ml in PBS Ca2+/ Mg2+), subsequently left to incubate in a thermal shaker for 45 minutes at 37 °C.…”