John A. McClung

Up-Regulation of Heme Oxygenase Provides Vascular Protection in an Animal Model of Diabetes through Its Antioxidant and Antiapoptotic Effects

Heme oxygenase (HO) plays a critical role in the regulation of cellular oxidative stress. The effects of the reactive oxygen species scavenger ebselen and the HO inducers cobalt protoporphyrin and stannous chloride (SnCl2) on HO protein levels and activity, indices of oxidative stress, and the progression of diabetes were examined in the Zucker rat model of type 2 diabetes. The onset of diabetes coincided with an increase in HO-1 protein levels and a paradoxical decrease in HO activity, which was restored by administration of ebselen. Up-regulation of HO-1 expressed in the early development of diabetes produced a decrease in oxidative/nitrosative stress as manifested by decreased levels of 3-nitrotyrosine, superoxide, and cellular heme content. This was accompanied by a decrease in endothelial cell sloughing and reduced blood pressure. Increased HO activity was also associated with a significant increase in the antiapoptotic signaling molecules Bcl-xl and phosphorylation of p38-mitogen-activated protein kinase but no significant increases in Bcl-2 or BAD proteins. In conclusion, 3-nitrotyrosine, cellular heme, and superoxide, promoters of vascular damage, are reduced by HO-1 induction, thereby preserving vascular integrity and protecting cardiac function involving an increase in antiapoptotic proteins.

Heme Oxygenase-Mediated Increases in Adiponectin Decrease Fat Content and Inflammatory Cytokines Tumor Necrosis Factor-α and Interleukin-6 in Zucker Rats and Reduce Adipogenesis in Human Mesenchymal Stem Cells

Adiponectin, an abundant adipocyte-derived plasma protein that modulates vascular function in type 2 diabetes, has been shown to provide cytoprotection to both pancreatic and vascular systems in diabetes. Therefore, we examined whether up-regulation of heme oxygenase (HO)-1 ameliorates the levels of inflammatory cytokines and influences serum adiponectin in Zucker fat (ZF) rats. ZF rats displayed a decrease in both HO activity and HO-1 and HO-2 protein levels and an increase in tumor necrosis factor (TNF)-α and interleukin (IL)-6 compared with Zucker lean (ZL) rats. Treatment of ZF animals with 2 mg/kg cobalt protoporphyrin IX (CoPP) increased protein levels of HO-1 and HO activity, but HO-2 was unaffected. The increase in HO-1 was associated with a decrease in superoxide levels (p < 0.05) and an increase in plasma adiponectin (p < 0.005), compared with untreated ZF rats. CoPP treatment decreased visceral and s.c. fat content, and it reduced weight gain (p < 0.01). In addition, the inflammatory cytokines TNF-α and IL-6 were decreased (p < 0.04 and p < 0.008, respectively). Treatment of human bone marrow-derived adipocytes cultured with CoPP resulted in an increase in HO-1 and a decrease in superoxide levels. Up-regulation of HO-1 caused adipose remodeling, smaller adipocytes, and increased adiponectin secretion in the culture medium of human bone marrow-derived adipocytes. In summary, this study demonstrates that the antiobesity effect of HO-1 induction results in an increase in adiponectin secretion, in vivo and in vitro, a decrease in TNF-α and IL-6, and a reduction in weight gain. These findings highlight the pivotal role and symbiotic relationship of HO-1 and adiponectin in the modulation of the metabolic syndrome phenotype.

Circulating endothelial cells are elevated in patients with type 2 diabetes mellitus independently of HbA1c

Aims/hypothesisPatients with diabetes mellitus are well known to be at high risk for vascular disease. Circulating endothelial cells (CECs) have been reported to be an ex vivo indicator of vascular injury. We investigated the presence of CECs in the peripheral blood of 25 patients with diabetes mellitus and in nine non-diabetic control donors.MethodsEndothelial cells were isolated from peripheral blood with anti-CD-146–coated immunomagnetic Dynabeads, and were stained with acridine orange dye and counted by fluorescence microscopy. The cells were also stained for von Willebrand factor and Ulex europaeus lectin 1.ResultsPatients with diabetes mellitus had an elevated number of CECs (mean 69±30 cells/ml, range 35–126) compared with healthy controls (mean 10±5 cells/ml, range 3–18) (p<0.001). The increase in CECs did not correlate with the levels of HbA1c. Circulating endothelial cell numbers were elevated regardless of glucose levels, suggesting that, even with control of glucose levels, there is increased endothelial cell sloughing.ConclusionsOur study suggests that the higher number of CECs in patients with type 2 diabetes may reflect ongoing vascular injury that is not directly dependent on glucose control.

Heme oxygenase-mediated increases in adiponectin decrease fat content and inflammatory cytokines, TNF and IL-6, in Zucker rats and reduce adipogenesis in human mesenchymal stem cells

Adiponectin, an abundant adipocyte-derived plasma protein that modulates vascular function in type 2 diabetes, has been shown to provide cytoprotection to both pancreatic and vascular systems in diabetes. Therefore, we examined whether up-regulation of heme oxygenase (HO)-1 ameliorates the levels of inflammatory cytokines and influences serum adiponectin in Zucker fat (ZF) rats. ZF rats displayed a decrease in both HO activity and HO-1 and HO-2 protein levels and an increase in tumor necrosis factor (TNF)-α and interleukin (IL)-6 compared with Zucker lean (ZL) rats. Treatment of ZF animals with 2 mg/kg cobalt protoporphyrin IX (CoPP) increased protein levels of HO-1 and HO activity, but HO-2 was unaffected. The increase in HO-1 was associated with a decrease in superoxide levels (p < 0.05) and an increase in plasma adiponectin (p < 0.005), compared with untreated ZF rats. CoPP treatment decreased visceral and s.c. fat content, and it reduced weight gain (p < 0.01). In addition, the inflammatory cytokines TNF-α and IL-6 were decreased (p < 0.04 and p < 0.008, respectively). Treatment of human bone marrow-derived adipocytes cultured with CoPP resulted in an increase in HO-1 and a decrease in superoxide levels. Up-regulation of HO-1 caused adipose remodeling, smaller adipocytes, and increased adiponectin secretion in the culture medium of human bone marrow-derived adipocytes. In summary, this study demonstrates that the antiobesity effect of HO-1 induction results in an increase in adiponectin secretion, in vivo and in vitro, a decrease in TNF-α and IL-6, and a reduction in weight gain. These findings highlight the pivotal role and symbiotic relationship of HO-1 and adiponectin in the modulation of the metabolic syndrome phenotype.

Long-Term Treatment with the Apolipoprotein A1 Mimetic Peptide Increases Antioxidants and Vascular Repair in Type I Diabetic Rats

Apolipoprotein A1 mimetic peptide (D-4F), synthesized from D-amino acid, enhances the ability of high-density lipoprotein to protect low-density lipoprotein (LDL) against oxidation in atherosclerotic disease. Using a rat model of type I diabetes, we investigated whether chronic use of D-4F would lead to up-regulation of heme oxygenase (HO)-1, endothelial cell marker (CD31+), and thrombomodulin (TM) expression and increase the number of endothelial progenitor cells (EPCs). Sprague-Dawley rats were rendered diabetic with streptozotocin (STZ) and either D-4F or vehicle was administered, by i.p. injection, daily for 6 weeks (100 μg/100 g b.wt.). HO activity was measured in liver, kidney, heart, and aorta. After 6 weeks of D-4F treatment, HO activity significantly increased in the heart and aorta by 29 and 31% (p < 0.05 and p < 0.49), respectively. Long-term D-4F treatment also caused a significant increase in TM and CD31+ expression. D-4F administration increased antioxidant capacity, as reflected by the decrease in oxidized protein and oxidized LDL, and enhanced EPC function and/or repair, as evidenced by the increase in EPC endothelial nitric-oxide synthase (eNOS) and prevention of vascular TM and CD31+ loss. In conclusion, HO-1 and eNOS are relevant targets for D-4F and may contribute to the D-4F-mediated increase in TM and CD31+, the antioxidant and anti-inflammatory properties, and confers robust vascular protection in this animal model of type 1 diabetes.

Type 5 phosphodiesterase inhibitors in the treatment of erectile dysfunction and cardiovascular disease.

Since the discovery of sildenafil in 1989 as a highly selective inhibitor of the phosphodiesterase type-5 (PDE-5) receptor, 2 additional PDE-5 inhibitors, tadalafil and vardenafil, have emerged as safe and effective treatments of erectile dysfunction (ED). Enzymes in the PDE family catalyze the hydrolysis of the intracellular signaling molecules cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which is the second messenger of nitric oxide (NO) and a principal mediator of smooth muscle relaxation and vasodilation. Sildenafil was initially introduced for clinical use as the result of extensive research on chemical agents targeting PDE-5 that might potentially be useful in the treatment of coronary heart disease. Erection is largely a hemodynamic event, which is regulated by vascular tone and blood flow balance in the penis. Endothelial dysfunction, an early component of atherosclerosis, may inhibit a vascular event such as erection and is rarely confined to the arteries supplying blood to the penis, but more likely occurs throughout the vascular bed. In addition to the effects of the NO-cGMP signaling pathway on cavernosal smooth muscle, clinical findings have suggested that vascular tone in the pulmonary, coronary, and other vascular tissues expressed by PDE-5 is also influenced by this signal transduction mechanism. This has led to the emergence of novel therapeutic indications for sildenafil over a range of cardiovascular conditions that are either well-established risk factors or comorbidities with ED. Recently, the U.S. Food and Drug Administration approved sildenafil as an orally active therapy for the treatment of primary pulmonary hypertension. The drug will be marketed under the trade name of Revatio, not Viagra, the name used for the ED indication. The approved dose for primary pulmonary hypertension is 20 mg 3 times daily.

Evaluation of a medical ethics consultation service: Opinions of patients and health care providers

PURPOSE To study the perceived effectiveness of bioethics consultation as evaluated by both professional staff and patients and their families. PATIENTS AND METHODS An evaluation questionnaire was forwarded to physicians, nurses and patients or family members who were associated with 20 sequential cases referred for ethics consultation during a 2-year period. Respondents were asked to rate the consult as very helpful, somewhat helpful, or not helpful concerning a variety of issues related to the care of the patients. Responses were reviewed for agreement or disagreement between the three sample groups. RESULTS Ninety-six percent of physicians and 95% of nurses sampled felt that the consult was helpful, compared with only 65% of the patient/family responses. Lack of communication between patients and staff was cited as a problem in all cases in which the consult was deemed unhelpful by patients and family. CONCLUSIONS Patients or family members and professional staff have different perceptions regarding the value of bioethics consultation. When asked for comments, patients/family members cite lack of communication with professional staff as their primary reason for responding negatively. Earlier mobilization of consultative staff, rigorous training of consultants in techniques of conflict resolution, and education of primary caregivers regarding recognition of communication problems may serve to narrow this perceptual divergence.

Increased prevalence of coronary artery disease, silent myocardial ischemia, complex ventricular arrhythmias, atrial fibrillation, left ventricular hypertrophy, mitral annular calcium, and aortic valve calcium in patients with chronic renal insufficiency.

Cardiovascular morbidity and mortality is high in patients with chronic renal insufficiency. Patients with chronic renal insufficiency have an increased prevalence of coronary artery disease, silent myocardial ischemia, complex ventricular arrhythmias, atrial fibrillation, left ventricular hypertrophy, mitral annular calcium, and aortic valve calcium than patients with normal renal function. These risk factors for cardiovascular morbidity and mortality contribute to the increased incidence of cardiovascular morbidity and mortality seen in patients with chronic renal insufficiency.

Warfarin use and the risk of valvular calcification.

Summary.  Background: Warfarin affects the synthesis and function of the matrix Gla‐protein, a vitamin K‐dependent protein, which is a potent inhibitor of tissue calcification. Objectives: To investigate the incidence of mitral valve calcium (MVC), mitral annular calcium (MAC) and aortic valve calcium (AVC) in patients with non‐valvular atrial fibrillation (AF) treated with warfarin vs. no warfarin. Patients and methods: Of 1155 patients, mean age 74 years, with AF, 725 (63%) were treated with warfarin and 430 (37%) without warfarin. The incidence of MVC, MAC and AVC was investigated in these 1155 patients with two‐dimensional echocardiograms. Unadjusted logistic regression analysis was conducted to examine the association between the use of warfarin and the incidence of MVC, MAC or AVC. Logistic regression analyses were also conducted to investigate whether the relationship stands after adjustment for confounding risk factors such as age, sex, race, ejection fraction, smoking, hypertension, diabetes, dyslipidemia, coronary artery disease (CAD), glomerular filtration rate, calcium, phosphorus, calcium‐phosphorus product, alkaline phosphatase, use of aspirin, beta blockers, angiotensin‐converting enzyme inhibitors or angiotensin receptor blockers, and statins. Results: There was a significant association between the use of warfarin and the risk of calcification [unadjusted odds ratio = 1.71, 95% CI = (1.34–2.18)]. The association still stands after adjustment for confounding risk factors. MVC, MAC or AVC was present in 473 of 725 patients (65%) on warfarin vs. 225 of 430 patients (52%) not on warfarin (P < 0.0001). Whether this is a causal relationship remains unknown. Conclusions: Use of warfarin in patients with AF is associated with an increased prevalence of MVC, MAC or AVC.

Chronic renal dysfunction as an independent risk factor for the development of cardiovascular disease

Cardiac disease is the leading cause of death in patients having end-stage renal disease (ESRD). Patients with ESRD have a higher risk for developing coronary artery disease (CAD) than one would estimate from the presence of traditional risk factors such as hypertension, diabetes, hyperlipidemia, and cigarette smoking. Patients with milder forms of renal dysfunction who do not require dialysis also appear to have an increased risk for CAD. ESRD is associated with anemia, hyperhomocystinemia, increased calcium–phosphate product, hypoalbuminemia, increased troponin, increased markers of inflammation, increased oxidant stress, and decreased nitric oxide activity, factors that could contribute to increased CAD risk. Patients with ESRD require aggressive management of traditional risk factors for CAD, which include hypertension, hyperlipidemia, hyperhomocystinemia, and hypercoagulability. Milder forms of renal dysfunction could also be predictors of occult CAD and should be screened for in assessing cardiac risk in asymptomatic individuals.