George Drummond

Diabetes Impairs the Vascular Recruitment of Normal Stem Cells by Oxidant Damage, Reversed by Increases in pAMPK, Heme Oxygenase-1, and Adiponectin

Atherosclerosis progression is accelerated in diabetes mellitus (DM) by either direct endothelial damage or reduced availability and function of endothelial progenitor cells (EPCs). Both alterations are related to increased oxidant damage.

Physiological significance of heme oxygenase in hypertension

The last decade has witnessed an explosion in the elucidation of the role that the heme oxygenase system plays in human physiology. This system encompasses not only the heme degradative pathway, including heme oxygenase and biliverdin reductase, but also the products of heme degradation, carbon monoxide, iron, and biliverdin/bilirubin. Their role in diabetes, inflammation, heart disease, hypertension, transplantation, and pulmonary disease are areas of burgeoning research. The research has focused not only on heme itself but also on its metabolic products as well as endogenous compounds involved in a vast number of genetic and metabolic processes that are affected when heme metabolism is perturbed. It should be noted, however, that although the use of carbon monoxide and biliverdin/bilirubin as therapeutic agents has been successful, these agents can be toxic at high levels in tissue, e.g., kernicterus. Care must be used to ensure that when these compounds are used as therapeutic agents their deleterious effects are minimized or avoided. On balance, however, the strategies to target heme oxygenase-1 as described in this review offer promising therapeutic approaches to clinicians for the effective management of hypertension and renal function. The approaches detailed may prove to be seminal in the development of a new therapeutic strategy to treat hypertension.

Translational Significance of Heme Oxygenase in Obesity and Metabolic Syndrome

The global epidemic of obesity continues unabated with sequelae of diabetes and metabolic syndrome. This review reflects the dramatic increase in research on the role of increased expression of heme oxygenase (HO)-1/HO-2, biliverdin reductase, and HO activity on vascular disease. The HO system engages with other systems to mitigate the deleterious effects of oxidative stress in obesity and cardiovascular disease (CVD). Recent reports indicate that HO-1/HO-2 protein expression and HO activity have several important roles in hemostasis and reactive oxygen species (ROS)-dependent perturbations associated with metabolic syndrome. HO-1 protects tissue during inflammatory stress in obesity through the degradation of pro-oxidant heme and the production of carbon monoxide (CO) and bilirubin, both of which have anti-inflammatory and anti-apoptotic properties. By contrast, repression of HO-1 is associated with increases of cellular heme and inflammatory conditions including hypertension, stroke, and atherosclerosis. HO-1 is a major focus in the development of potential therapeutic strategies to reverse the clinical complications of obesity and metabolic syndrome.

Impact of silencing HO‐2 on EC‐SOD and the mitochondrial signaling pathway

The contribution of heme oxygenase HO‐2, the primary source of bilirubin and carbon monoxide (CO) under physiological conditions, to the regulation of vascular function has remained largely unexplored. Using siRNA HO‐2, we examined the effect of suppressed levels of HO‐2 on vascular antioxidant and survival proteins. In vivo HO‐2 siRNA treatment decreased the basal levels of EC‐SOD, pAKT proteins (serine‐473 and threonine‐308), without changing Akt protein expression. HO‐2 siRNA treatment increased 3‐nitrotyrosine (3‐NT) and apoptotic signaling kinase‐1 (ASK‐1) (P < 0.01). HO activity was decreased by the use of siRNA HO‐2. We extended these studies to the mitochondria, examining for the presence of HO‐1 and its role in the regulation of pro‐ and anti‐apoptotic proteins. HO activity was increased by the administration of CoPP resulting in the translocation of HO‐1 into the mitochondria, mainly to the inner face of the mitochondrial inner membrane. These findings suggest that HO‐2 is critical in the maintenance of heme homeostasis and also the regulation of apoptosis by controlling levels of EC‐SOD, Akt, 3‐NT, and ASK‐1. In addition, localization of HO‐1 in the mitochondrial compartment plays a critical role in mitochondria‐mediated apoptosis. J. Cell. Biochem. 100: 815–823, 2007.

Improved Myocardial Perfusion in Chronic Diabetic Mice by the Up-Regulation of pLKB1 and AMPK Signaling

Previous studies related impaired myocardial microcirculation in diabetes to oxidative stress and endothelial dysfunction. Thus, this study was aimed to determine the effect of up‐regulating pAMPK‐pAKT signaling on coronary microvascular reactivity in the isolated heart of diabetic mice. We measured coronary resistance in wild‐type and streptozotocin (STZ)‐treated mice, during perfusion pressure changes. Glucose, insulin, and adiponectin levels in plasma and superoxide formation, NOx levels and heme oxygenase (HO) activity in myocardial tissue were determined. In addition, the expression of HO‐1, 3‐nitrotyrosine, pLKB1, pAMPK, pAKT, and peNOS proteins in control and diabetic hearts were measured. Coronary response to changes in perfusion pressure diverged from control in a time‐dependent manner following STZ administration. The responses observed at 28 weeks of diabetes (the maximum time examined) were mimicked by L‐NAME administration to control animals and were associated with a decrease in serum adiponectin and myocardial pLKB1, pAMPK, pAKT, and pGSK‐3 expression. Cobalt protoporphyrin treatment to induce HO‐1 expression reversed the microvascular reactivity seen in diabetes towards that of controls. Up‐regulation of HO‐1 was associated with an increase in adiponectin, pLKB1, pAKT, pAMPK, pGSK‐3, and peNOS levels and a decrease in myocardial superoxide and 3‐nitrotyrosine levels. In the present study we describe the time course of microvascular functional changes during the development of diabetes and the existence of a unique relationship between the levels of serum adiponectin, pLKB1, pAKT, and pAMPK activation in diabetic hearts. The restoration of microvascular function suggests a new therapeutic approach to even advanced cardiac microvascular derangement in diabetes. J. Cell. Biochem. 109: 1033–1044, 2010.

Heme Oxygenase-Derived Carbon Monoxide Restores Vascular Function in Type 1 Diabetes

Increased heme oxygenase-1 (HO-1) expression improves vascular function by decreasing superoxide and increasing antioxidant levels. We therefore examined if HO-1 induction increased serum adiponectin levels and ameliorated vascular dysfunction in Type 1 diabetes. Administration of either carbon monoxide (CORM-3) or the HO-1 inducers, Resveratrol, and cobalt protoporphyrin (CoPP), increased serum levels of adiponectin (high molecular weight) in diabetic (streptozotocin; STZ-induced) Sprague Dawley rats. Resveratrol and CoPP administration increased HO-1 protein expression and HO activity in the aorta and significantly (p<0.05) increased serum adiponectin levels, compared to untreated diabetic rats. The results obtained with the CO releasing molecule, CORM-3, indicate a direct involvement of CO leading to increased levels of adiponectin. The increase in adiponectin was associated with a significant decrease in circulating endothelial cells (CEC) (p<0.002), decreased EC fragmentations and a significant increase in thrombomodulin (TM) and CD31(+) cells (p<0.05). Increased adiponectin levels were associated with a decrease in TNF-alpha-induced ICAM-1 and VCAM-1 and caspase 3 activity in endothelial cells while phosphorylation of eNOS at Ser-1179 increased. The adiponectin mediated increase in peNOS and pAKT was prevented by the phosphatidylinositol-3 kinase inhibitor, LY294002. In conclusion, there appears to be a temporal HO-1-adiponectin relationship that has a key role in vascular protection in Type 1 diabetes via a mechanism that involves increased levels of carbon monoxide.

Upregulation of Heme Oxygenase-1 Combined with Increased Adiponectin Lowers Blood Pressure in Diabetic Spontaneously Hypertensive Rats through a Reduction in Endothelial Cell Dysfunction, Apoptosis and Oxidative Stress

This study was designed to investigate the effect of increased levels of HO-1 on hypertension exacerbated by diabetes. Diabetic spontaneously hypertensive rat (SHR) and WKY (control) animals were treated with streptozotocin (STZ) to induce diabetes and stannous chloride (SnCl2) to upregulate HO-1. Treatment with SnCl2 not only attenuated the increase of blood pressure (p<0.01), but also increased HO-1 protein content, HO activity and plasma adiponectin levels, decreased the levels of superoxide and 3-nitrotyrosine (NT), respectively. Reduction in oxidative stress resulted in the increased expression of Bcl-2 and AKT with a concomitant reduction in circulating endothelial cells (CEC) in the peripheral blood (p<0.005) and an improvement of femoral reactivity (response to acetylcholine). Thus induction of HO-1 accompanied with increased plasma adiponectin levels in diabetic hypertensive rats alters the phenotype through a reduction in oxidative stress, thereby permitting endothelial cells to maintain an anti-apoptotic environment and the restoration of endothelial responses thus preventing hypertension.

ORIGINAL RESEARCH—BASIC SCIENCE: Effect of Hemin and Carbon Monoxide Releasing Molecule (CORM-3) on cGMP in Rat Penile Tissue

INTRODUCTION Cyclic guanosine monophosphate (cGMP) levels can be regulated by heme oxygenase-1 and 2 (HO-1 and HO-2)-derived carbon monoxide (CO). AIMS Assessment of the effect of upregulating CO in rat corpora cavernosa (CC) on cavernous cGMP. METHODS Three experimental groups were studied: first group (N = 40), short-term HO induction over 2 weeks by injection of intraperitoneal increasing doses of hemin; the second group (N = 40) was subjected to intracavernosal injection of CO donor, CORM-3, or its inactive form (iCORM-3) over 2 weeks; the third group (N = 60) was subdivided into three subgroups: the first one received a combined hemin and CORM-3, the second one received hemin and its inhibitor stannus mesoporphyrin (SnMP), and third one received a combined hemin, CORM-3, and SnMP. MAIN OUTCOME MEASURES In CC, HO-1 and HO-2 gene expression, Northern blot and Western blot, cGMP levels, and HO enzyme activity. RESULTS In the first group, maximum induction of HO-1 gene expression, HO enzyme activity, and cGMP occurred with 4-mg hemin dose with a successive increase over 2 weeks. In the second group, CORM-3 increased cGMP by twofold compared with iCORM-3, and also increased HO-1 protein. In the third group, SnMP inhibited the enhancing effect of CORM-3 and HO on erectile signaling molecules; i.e., HO-1 gene, enzyme activity, and cGMP. CONCLUSIONS CORM-3- or hemin-mediated CO release could increase cavernous tissue cGMP.

ORIGINAL RESEARCHORIGINAL RESEARCH—BASIC SCIENCE: Effect of Hemin and Carbon Monoxide Releasing Molecule (CORM-3) on cGMP in Rat Penile Tissue

INTRODUCTION Cyclic guanosine monophosphate (cGMP) levels can be regulated by heme oxygenase-1 and 2 (HO-1 and HO-2)-derived carbon monoxide (CO). AIMS Assessment of the effect of upregulating CO in rat corpora cavernosa (CC) on cavernous cGMP. METHODS Three experimental groups were studied: first group (N = 40), short-term HO induction over 2 weeks by injection of intraperitoneal increasing doses of hemin; the second group (N = 40) was subjected to intracavernosal injection of CO donor, CORM-3, or its inactive form (iCORM-3) over 2 weeks; the third group (N = 60) was subdivided into three subgroups: the first one received a combined hemin and CORM-3, the second one received hemin and its inhibitor stannus mesoporphyrin (SnMP), and third one received a combined hemin, CORM-3, and SnMP. MAIN OUTCOME MEASURES In CC, HO-1 and HO-2 gene expression, Northern blot and Western blot, cGMP levels, and HO enzyme activity. RESULTS In the first group, maximum induction of HO-1 gene expression, HO enzyme activity, and cGMP occurred with 4-mg hemin dose with a successive increase over 2 weeks. In the second group, CORM-3 increased cGMP by twofold compared with iCORM-3, and also increased HO-1 protein. In the third group, SnMP inhibited the enhancing effect of CORM-3 and HO on erectile signaling molecules; i.e., HO-1 gene, enzyme activity, and cGMP. CONCLUSIONS CORM-3- or hemin-mediated CO release could increase cavernous tissue cGMP.