NADPH Oxidase-Derived Overproduction of Reactive Oxygen Species Impairs Postischemic Neovascularization in Mice with Type 1 Diabetes

Abstract

We hypothesized that diabetes-induced oxidative stress may affect postischemic neovascularization. The response to unilateral femoral artery ligation was studied in wild-type or gp91 phox -deficient control or type 1 diabetic mice or in animals treated with the anti-oxidant N-acetyl-L-cysteine (NAC) or with in vivo electrotransfer of a plasmid encoding dominant-negative Rac1 (50 g) for 21 days. Postischemic neovascularization was reduced in diabetic mice in association with down-regulated vascular endothelial growth factor-A protein levels. In diabetic animals vascular endothelial growth factor levels and postischemic neovascularization were restored to nondiabetic levels by the scavenging of reactive oxygen species (ROS) by NAC administration or the inhibition of ROS generation by gp91 phox deficiency or by administration of dominant-negative Rac1. Finally, diabetes reduced the ability of adherent bone marrow-derived mononuclear cells (BM-MNCs) to differentiate into endothelial progenitor cells. Treatment with NAC (3 mmol/L), apocynin (200 mol/L), or the p38MAPK inhibitor LY333351 (10 mol/L) up-regulated the number of endothelial progenitor cell colonies derived from diabetic BM-MNCs by 1.5-, 1.6-, and 1.5-fold, respectively (P < 0.05). In the ischemic hindlimb model, injection of diabetic BM-MNCs isolated from NACtreated or gp91 phox -deficient diabetic mice increased neovascularization by ϳ1.5-fold greater than untreated diabetic BM-MNCs (P < 0.05). Thus, inhibition of NADPH oxidase-derived ROS overproduction improves the angiogenic and vasculogenic processes and restores postischemic neovascularization in type 1 diabetic mice.

Exercise training improves endothelial function via adiponectin-dependent and independent pathways in type 2 diabetic mice

“…Prior work showed activation of the vascular isoform of NAD(P)H oxidase contributes to superoxide formation in diabetes (13,38,54). Additionally, inhibition of ROS generation both by genetic deficiency and by pharmacological blockage of gp91 phox decreased production of superoxide in diabetes (13,38,54). We showed that gp91 phox protein expression in db/db is increased, suggesting that upregulation of gp91 phox is associated with apocynin-inhibitable NAD(P)H oxidase-derived superoxide production (54).…”

“…There are multiple cellular sources of superoxide such as NAD(P)H oxidase, xanthine oxidase, the mitochondrial respiratory chain, arachidonic acid metabolism, and uncoupled eNOS when substrate or cofactors are not available (5, 7). Prior work showed activation of the vascular isoform of NAD(P)H oxidase contributes to superoxide formation in diabetes (13,38,54). Additionally, inhibition of ROS generation both by genetic deficiency and by pharmacological blockage of gp91 phox decreased production of superoxide in diabetes (13,38,54).…”

Plasminogen Activator Inhibitor-1 Controls Bone Marrow-Derived Cells Therapeutic Effect Through MMP9 Signaling: Role in Physiological and Pathological Wound Healing

“…Immediately after isolation, 5 Â 10 6 BMMNC were plated on 35-mm cell culture dishes coated with rat plasma vitronectin (Sigma, St. Quentin Fallavier, France) and gelatin (0.1%) and maintained in endothelial basal medium (EBM2; BioWhittaker, Paris, France). After 6 days in culture, nonadherent cells were removed and adherent cells underwent immunochemical analysis, as previously described [18]. To detect the uptake of 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindocarbocyaninelabeled acetylated low-density lipoprotein (AcLDL-Dil), cells were incubated in medium containing AcLDL-Dil (Tebu, Le Perray en Yvelines, France) at 37 C for 1 hour.…”

“…BMMNC have been shown to differentiate into cells with an endothelial phenotype in vitro [18], and PAI-1 deficiency might trigger BMMNC differentiation leading to activation of vessel growth. We found that the percentage of cells with Dil LDL/BS-1 lectin-positive staining was increased by 1.3-fold in cultured PAI-1À/À compared to WT BMMNC-derived cells (Fig.…”

Hypertension Impairs Postnatal Vasculogenesis

“…3,4 Alternatively, 5 mL of blood was collected in EDTA-containing tubes. After centrifugation on a Ficoll gradient and red cell lysis, circulating blood mononuclear cells were then analyzed in flow analysis cytometry system (FACS).…”