OBJECTIVE-We previously showed epidermal growth factor receptor (EGFR) transactivation to be key mechanism in the regulation of resistance artery myogenic tone. Type 2 diabetes is associated with microvascular complications. We hypothesized that elevated EGFR phosphorylation contributes to resistance artery dysfunction in type 2 diabetes. RESEARCH DESIGN AND METHODS AND RESULTS-Diabetic db/db and nondiabetic (control) mice were treated with EGFR inhibitor (AG1478; 10 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) for 2 weeks. Isolated coronary artery and mesenteric resistance artery (MRA) were mounted in an arteriograph. Pressure-induced myogenic tone was increased in MRA and coronary artery from diabetic mice and normalized by AG1478. Phenylephrine-induced contraction and nitric oxide donor-induced relaxation were similar in all groups. Endothelium-dependent relaxation in response to shear stress and acetylcholine of MRA and coronary artery from diabetic mice was altered and associated with reduced endothelial nitric oxide synthase (eNOS) expression and phosphorylation. Treated diabetic mice with AG1478 improved coronary artery and MRA endothelial function and restored eNOS expression. Immunostaining and Western blot analysis showed increased endothelial and smooth muscle cell EGFR phosphorylation of MRA and coronary artery from diabetic mouse, which was reduced by AG1478. Primary cultured endothelial cells from resistance arteries treated with high glucose for 48 h showed an increase of EGFR phosphorylation associated with eNOS expression and phosphorylation decrease in response to calcium ionophore. Pretreatment of endothelial cells with AG1478 prevented the effect of high glucose.CONCLUSIONS-This study provides evidence of the role of elevated EGFR phosphorylation in coronary artery and MRA dysfunction in diabetic db/db mice. Therefore, EGFR should be a potential target for overcoming diabetic small artery complications.

Apoptosis is characterized by various cell morphological and biochemical features, one of which is the internucleosomal degradation of genomic DNA. The role of the human chromatin-bound Ca 2؉ -and Mg 2؉ -dependent endonuclease (CME) DNAS1L3 and its inhibition by poly(ADP-ribosyl)ation in the DNA degradation that accompanies apoptosis was investigated. The nuclear localization of this endonuclease is the unique feature that distinguishes it from other suggested apoptotic nucleases. Purified recombinant DNAS1L3 was shown to cleave nuclear DNA into both high molecular weight and oligonucleosomal fragments in vitro. Furthermore, exposure of mouse skin fibroblasts expressing DNAS1L3 to inducers of apoptosis resulted in oligonucleosomal DNA fragmentation, an effect not observed in cells not expressing this CME, as well as in a decrease in cell viability greater than that apparent in the control cells. Recombinant DNAS1L3 was modified by recombinant human poly(ADP-ribose) polymerase (PARP) in vitro, resulting in a loss of nuclease activity. The DNAS1L3 protein also underwent poly(ADP-ribosyl)ation in transfected mouse skin fibroblasts in response to inducers of apoptosis. The cleavage and inactivation of PARP by a caspase-3-like enzyme late in apoptosis were associated with a decrease in the extent of DNAS1L3 poly(ADPribosyl)ation, which likely releases DNAS1L3 from inhibition and allows it to catalyze the degradation of genomic DNA.Apoptosis, or programmed cell death, is an evolutionarily conserved process that is important in normal development, physiological homeostasis, and certain pathological conditions. It is mediated by a variety of intracellular enzymes, among which are endonucleases that catalyze the internucleosomal fragmentation of DNA, which is one of the hallmarks of apoptotic death (1, 2). Candidates for such endonucleases include the caspase-activated enzymes DFF40 (or CAD) (3-8) and NUC70 (9), divalent cation-dependent neutral (1, 2) or acidic (10, 11) endonucleases, leukemia-associated endo-exonucleases (12), and Ca 2ϩ -and Mg 2ϩ -dependent endonucleases (CMEs) 1 (13-18). CMEs introduce double strand breaks and single strand nicks into DNA, generating fragments with 5Ј-phosphate and 3Ј-hydroxyl termini, a mode of DNA fragmentation consistent with the products of chromatin degradation in apoptotic cells (19 -21). CME activity is increased by a variety of stimuli that induce apoptosis (17, 21), and treatments that prevent apoptosis also prevent the induction of CME activity. A role for CMEs in apoptosis has also been supported by studies demonstrating the inhibition of DNA fragmentation by Ca 2ϩ chelators or Zn 2ϩ (13)(14)(15)22). One of earliest nuclear events in apoptosis is the poly(ADPribosyl)ation of various proteins by poly(ADP-ribose) polymerase (PARP), an enzyme that is activated by the presence of DNA strand breaks (23,24). PARP catalyzes the modification of histones, topoisomerases I and II, SV40 large T antigen, DNA polymerase ␣, proliferating cell nuclear antigen, and various other DNA-bindi...

Objective-Type 2 diabetes is associated with increased advanced glycation end product (AGE) formation and vasculopathy.We hypothesized that AGEs contribute to resistance artery dysfunction. Methods and Results-Type 2 diabetic dbϪ /db Ϫ (diabetic) and nondiabetic db Ϫ /db ϩ (control) mice were treated with the AGE inhibitor (aminoguanidine: 50 mg/Kg/d) for 3 months. Isolated mesenteric resistance arteries (MRAs) were mounted in an arteriograph. Pressure-induced myogenic tone (MT) was increased in diabetic mice but was unaffected by aminoguanidine treatment. Phenylephrine-induced contraction and nitric oxide donor-induced endotheliumindependent relaxation were similar in all groups. In diabetic mice, endothelium-dependent relaxation in response to shear-stress or acetylcholine was altered and was associated with reduced eNOS protein and mRNA expression. Aminoguanidine treatment improved endothelial function and restored eNOS expression. AGE formation and hypoxia markers (plasminogen activator inhibitor 1 and Bnip3) were increased in MRA from diabetic mice and normalized with Aminoguanidine. Primary cultured endothelial cells (ECs) isolated from resistance arteries subjected to high glucose for 48 hours showed decreased eNOS expression and phosphorylation in response to calcium ionophore. High glucose decreased antioxidant protein (MnSOD) and increased prooxidant proteins (gp91phox) expression leading to increased oxidative stress generation, as assessed by DHE staining and endothelial NADH/NADPH oxidase activity. The preincubation of ECs with aminoguanidine restored eNOS-phosphorylation and expression as well as the balance between pro-and antioxidant factors induced by high glucose. Conclusions-We provide evidence of a link between AGEs, oxidative stress, and resistance artery EC dysfunction in type 2 diabetic mice. Thus, AGEs and oxidative stress may be a potential target for overcoming diabetic microvessels complications. (Arterioscler Thromb Vasc Biol. 2008;28:1432-1438) Key Words: resistance artery Ⅲ oxidative stress Ⅲ AGEs Ⅲ type 2 diabetic mice R esistance arteries are exposed to hemodynamic forces, including pressure and shear stress. Endothelial cells (ECs) have been proposed to be the primary sensors of wall shear stress for the transduction of mechanical stimuli into biological responses. 1 Resistance arteries play a crucial role in blood pressure control, tissue perfusion, and metabolism because they are prime determinants of local blood flow to subsequent tissue perfusion. Resistance artery tone is mainly regulated by mechanical factors (pressure and flow; mechanotransduction) and vasoactive agents. 2 The control of resistance artery tone is dependent on a complex interplay between ECs and vascular smooth muscle cells (VSMCs). In general, flow induces endothelium-dependent vasodilation via release of nitric oxide (NO), prostacylin I2, and endothelium-derived hyperpolarizing factor from ECs. 3 On the other hand, pressure-induced contraction (myogenic tone [MT]) is endothelium-independent and is media...

The nonsteroidal antiinflammatory drugs (NSAIDs) indomethacin and salicylic acid and the short chain fatty acid butyrate are effective colon cancer chemopreventive agents that increase reactive oxygen species (ROS) generation in colon cancer cells. Here we demonstrate that these agents sensitize the normally resistant human HT-29 colon cancer cell line to apoptosis induced by TNF-alpha or a Fas ligating antibody. The role of ROS in this sensitization is supported by the finding that direct exposure of the cells to H2O2 is sufficient for sensitization. Neither TNF-alpha nor Fas ligation alter basal or chemopreventive agent-activated ROS generation, suggesting that the death ligands and chemopreventive agents act in a complementary fashion. The dual chemopreventive agent/death ligand treatments do not increase Fas, TNF receptor 1, Bak or c-myc expression (although salicylic acid moderately induces of Fas expression). Cell death does correlate with alterations in NF-kappa B activity: the NSAIDs, butyrate and H2O2 enhance c-Rel complex formation by TNF-alpha and provide an overall enhancement of NF-kappa B activation by Fas. The antioxidant N-acetylcysteine (NAC) blocks cell death and NF-kappa B activation induced by Fas ligation, suggesting a potential role for NF-kappa B in Fas-induced apoptosis in these cells. The effects of NAC on TNF-alpha-induced cell death are more complex, with NAC being marginally protective and itself enhancing the formation of c-Rel containing complexes at higher concentrations (25 mM). The influence of NSAIDs and butyrate on ROS generation and death ligand sensitivity may be relevant to their ability to suppress colon carcinogenesis.