Michael T. Johnstone

Impaired nitric oxide-mediated vasodilation in patients with non-insulin-dependent diabetes mellitus☆

OBJECTIVES This study sought to determine whether nitric oxide-mediated vasodilation is abnormal in patients with non-insulin-dependent diabetes mellitus. BACKGROUND Multiple investigations, both in experimental models and in patients with insulin-dependent diabetes mellitus, demonstrate impaired endothelium-dependent vasodilation. Decreased availability of endothelium-derived nitric oxide may contribute to the high prevalence of vascular disease in diabetes. METHODS Vascular reactivity was measured in the forearm resistance vessels of 21 patients with non-insulin-dependent diabetes mellitus and 23 matched healthy control subjects. No patient had hypertension or hypercholesterolemia. Each subject was pretreated with aspirin to inhibit endogenous production of vasoactive prostanoids. Methacholine chloride (0.3 to 10 microg/min) was administered through a brachial artery cannula to assess vasodilation to endothelium-derived nitric oxide. Sodium nitroprusside (0.3 to 10 microg/min) was infused to evaluate vasodilation to an exogenous nitric oxide donor. Verapamil (10 to 300 microg/min) was administered to distinguish impaired nitric-oxide-mediated vasodilation from general dysfunction of vascular smooth muscle. Forearm blood flow was determined by venous occlusion plethysmography, and dose-response curves were generated for each agent. To assess the role of vasoconstrictor prostanoids, a subset of eight diabetic subjects were reexamined in the absence of aspirin treatment. RESULTS Basal forearm blood flow in diabetic and nondiabetic subjects was comparable. The forearm blood flow responses to both methacholine chloride and nitroprusside were significantly attenuated in diabetic compared with nondiabetic subjects (p < 0.005 by analysis of variance for both agents). In contrast, the response to verapamil was not significantly different between the groups (p > 0.50). The forearm blood flow responses to these agents were not significantly affected by cyclooxygenase inhibition. CONCLUSIONS Nitric oxide-mediated vasodilation is impaired in non-insulin-dependent diabetes mellitus. Vasoconstrictor prostanoids do not contribute significantly to vascular dysfunction. The attenuated response to exogenous as well as endogenous nitric oxide donors suggests that the abnormality is due to increased inactivation of nitric oxide or to decreased reactivity of the vascular smooth muscle to nitric oxide.

In Vivo Molecular Imaging of Acute and Subacute Thrombosis Using a Fibrin-Binding Magnetic Resonance Imaging Contrast Agent

Background—Plaque rupture with subsequent thrombosis is recognized as the underlying pathophysiology of most acute coronary syndromes and stroke. Thus, direct thrombus visualization may be beneficial for both diagnosis and guidance of therapy. We sought to test the feasibility of direct imaging of acute and subacute thrombosis using MRI together with a novel fibrin-binding gadolinium-labeled peptide, EP-1873, in an experimental animal model of plaque rupture and thrombosis. Methods and Results—Fifteen male New Zealand White rabbits (weight, ≈3.5 kg) were made atherosclerotic by feeding a high-cholesterol diet after endothelial aortic injury. Plaque rupture was then induced with the use of Russell’s viper venom (RVV) and histamine. Subsequently, MRI of the subrenal aorta was performed before RVV, after RVV, and after EP-1873. Histology was performed on regions suggested by MRI to contain thrombus. Nine rabbits (60%) developed plaque rupture and thrombus, including 25 thrombi visually apparent on MRI as “hot spots” after injection of EP-1873. Histological correlation confirmed all 25 thrombi (100%), with no thrombi seen in the other regions of the aorta. In the remaining 6 rabbits (control) without plaque rupture, no thrombus was observed on the MR images or on histology. Conclusions—We demonstrate the feasibility of in vivo “molecular” MRI for the detection of acute and subacute thrombosis using a novel fibrin-binding MRI contrast agent in an animal model of atherosclerosis and acute/subacute thrombosis. Potential clinical applications include thrombus detection in acute coronary syndromes and stroke.

Interaction Between Poor Glycemic Control and 9p21 Locus on Risk of Coronary Artery Disease in Type 2 Diabetes

CONTEXT A common allele on chromosome 9p21 has been repeatedly associated with increased risk of coronary artery disease (CAD) in the general population. However, the magnitude of this effect in the population with diabetes has not been well characterized. OBJECTIVE To examine the association of the 9p21 variant with CAD in individuals with type 2 diabetes and evaluate its interaction with poor glycemic control. DESIGN, SETTING, AND PARTICIPANTS (1) Case-control study of 734 type 2 diabetes patients (322 with angiographically diagnosed CAD and 412 with no evidence of CAD) who were recruited between 2001 and 2006 at the Joslin Clinic, Beth Israel Deaconess Medical Center; and (2) independent cohort study of 475 type 2 diabetes patients from the Joslin Clinic whose survival status was monitored from their recruitment between 1993 and 1996 until December 31, 2004. Participants for both studies were genotyped for a representative single-nucleotide polymorphism at 9p21 (rs2383206) and characterized for their long-term glycemic control by averaging multiple hemoglobin A(1c) (HbA(1c)) measurements taken in the years before study entry. MAIN OUTCOME MEASURES For the case-control study, association between single-nucleotide polymorphism rs2383206 and CAD defined as angiographically documented stenosis greater than 50% in a major coronary artery or a main branch thereof was assessed and for the cohort study, cumulative 10-year mortality was documented. RESULTS Individuals who were homozygous for the risk allele were significantly more frequent among case than control participants (42.3% vs 28.9P = .0002). This association was unaffected by adjustment for cardiovascular risk factors, but the effect of the risk genotype was significantly magnified (adjusted P for interaction = .048) in the presence of poor glycemic control (worst tertile of the distribution of HbA(1c) at examination). Relative to the CAD risk for patients with neither a 9p21 risk allele nor poor glycemic control, the CAD odds for participants having 2 risk alleles but not poor glycemic control were increased 2-fold (odds ratio [OR], 1.99; 95% confidence interval [CI], 1.17-3.41), whereas the odds for study participants with the same genotype and with poor glycemic control were increased 4-fold (OR, 4.27; 95% CI, 2.26-8.01). The interaction was stronger (adjusted P = .005) when a measure of long-term glycemic control (7-year average rather than most recent HbA(1c)) was used with ORs of 7.83 (95% CI, 3.49-17.6) for participants having 2 risk alleles and a history of poor glycemia and 1.54 (95% CI, 0.72-3.30) for participants with the same genotype but without this exposure. A similar interaction between 9p21 variant and poor glycemic control was observed with respect to cumulative 10-year mortality in the cohort study (43.6% in patients with 2 risk alleles and poor glycemic control, 23.1% in individuals with only the 2 risk alleles, 30.0% in individuals with only poor glycemic control, and 31.6% in individuals with neither factor, P for interaction, = .036). CONCLUSION In this study population, the CAD risk associated with the 9p21 variant was increased in the presence of poor glycemic control in type 2 diabetes.

Comparison of effect of intensive lipid lowering with atorvastatin to less intensive lowering with lovastatin on C-reactive protein in patients with stable angina pectoris and inducible myocardial ischemia

In patients with stable angina pectoris and evidence of myocardial ischemia, intensive therapy with atorvastatin was associated with a more rapid decline in CRP than low-dose lovastatin. In the atorvastatin group, half of the long-term decrease occurred within 4 weeks of intensive therapy. Intensive therapy with statins to reach low LDL targets (<80 mg/dl) may offer greater plaque stabilization in higher-risk patients.

Tag polymorphisms at the A20 (TNFAIP3) locus are associated with lower gene expression and increased risk of coronary artery disease in type 2 diabetes

A20 or tumor necrosis factor (TNF)-induced protein 3 (TNFAIP3) is a negative regulator of nuclear factor-κB (NF-κB). We have investigated whether polymorphisms in this gene are associated with increased atherosclerosis in diabetic patients. Five tag single nucleotide polymorphisms (SNPs) were typed in 479 type 2 diabetic patients from Boston, including 239 coronary artery disease (CAD)-positive case subjects and 240 CAD-negative control subjects. Two tag SNPs (rs5029930 and rs610604) were independently associated with CAD; adjusted odds ratios (ORs) for minor allele carriers were 2.3 (95% CI 1.4–3.8, P = 0.001) and 2.0 (1.3–2.9, P = 0.0008), respectively. The association with rs610604 was dependent on glycemic control, with ORs of 3.9 among subjects with A1C ≤7.0% and 1.2 for those with A1C >7.0% (P for interaction = 0.015). A similar interaction pattern was found among 231 CAD-positive and 332 CAD-negative type 2 diabetic patients from Italy (OR 2.2, P = 0.05 vs. OR 0.9, P = 0.63 in the low vs. high A1C strata, P for interaction = 0.05). Quantitative RT-PCR in blood mononuclear cells from 83 nondiabetic subjects showed that rs610604 and rs5029930 minor allele homozygotes have 30–45% lower levels of A20 mRNA than major allele homozygotes, and heterozygotes have intermediate levels (P = 0.04 and 0.028, respectively). These findings point to variability in the A20/TNFAIP3 gene as a modulator of CAD risk in type 2 diabetes. This effect is mediated by allelic differences in A20 expression.

Factor VII Gene Polymorphism, Factor VII Levels, and Prevalent Cardiovascular Disease: The Framingham Heart Study

Elevated factor VII levels have been associated with increased cardiovascular risk in some studies. The arginine/glutamine (Arg/Gln) polymorphism of the factor VII gene has been previously shown to modify factor VII levels. However, the presence of a gene/environment interaction on factor VII levels or a link with cardiovascular disease (CVD) remains uncertain. We studied subjects from the Framingham Heart Study to determine (1) the extent to which this genetic polymorphism affects factor VII levels; (2) whether interactions exist between this polymorphism and environmental factors on factor VII levels; and (3) the association between the polymorphism and CVD. Genotype data and factor VII antigen levels were available in 1816 subjects. Factor VII levels differed significantly among genotypes in an additive fashion: Gln homozygous, 82.7+/-2.5%; heterozygous, 92.2+/-0.7%; and Arg homozygous, 100. 5+/-0.4% (P<0.0001). The polymorphism was the strongest, single predictor of factor VII levels, explaining 7.7% of the total variance of factor VII levels, whereas other traditional risk factors combined explained an additional 11.5% of the variance. There was an interaction (P=0.02) between the genotype and total cholesterol on factor VII levels, such that the correlation coefficient and slope (factor VII level/total cholesterol) were greatest in Gln/Gln subjects. Among 3204 subjects characterized for genotype and CVD, there was no significant relationship between the genotype and CVD (P=0.12). In the Framingham Heart Study, the Arg/Gln polymorphism was significantly associated with factor VII antigen levels. The strength of the association suggests that genetic variation plays an important role in determining factor VII levels. However, despite being associated with factor VII levels, the Arg/Gln polymorphism was not associated with prevalent CVD.

Angiotensin Receptor Blockade With Candesartan Attenuates Atherosclerosis, Plaque Disruption, and Macrophage Accumulation Within the Plaque in a Rabbit Model

Background—Little is known about whether direct angiotensin receptor blockade can reduce atherosclerosis and plaque disruption. This study evaluated the effect of angiotensin receptor blockade on both the development of atherosclerosis and the disruption of plaque in a modified Constantinides animal model. Methods and Results—Twenty-eight New Zealand White rabbits underwent aortic balloon injury followed by a 1% cholesterol diet for 8 weeks. Thirteen rabbits received candesartan at 0.5 mg · kg−1 · d−1 beginning 2 days before aortic balloon injury and continued for the total 8 weeks of the cholesterol diet. The rabbits were then pharmacologically triggered and humanely killed, and their aortas were analyzed. The degree of atherosclerosis was determined by intima-media ratio of the infrarenal portion of the aorta. The frequency of intra-aortic thrombosis, a measure of plaque disruption, and the percentages of macrophage area and collagen-staining area of the plaque were determined. Candesartan-treated rabbits had less atherosclerosis (intima-media infrarenal aorta ratio of 1.18±0.08 versus 1.57±0.08 [mean±SEM] for the placebo group, P<0.001); fewer thrombi (3 of 13 versus 11 of 15; P<0.05); lower percentage area of macrophages to total plaque (18.8±2.7% versus 27±2.5%, P<0.05); and higher collagen to total plaque area (45±3% versus 35±2%, P<0.01). Conclusions—These results demonstrate that angiotensin receptor blockade attenuates the degree of atherosclerosis and reduces both plaque disruption and macrophage accumulation while increasing collagen deposition in the aortas of this animal model.

Pro-urokinase and prekallikrein are both associated with platelets Implications for the intrinsic pathway of fibrinolysis and for therapeutic thrombolysis

The contact‐dependent intrinsic pathway of fibrinolysis involving factor XII, prekallikrein (PK) and pro‐urokinase (pro‐UK) remains poorly understood. Casein autography of washed, intact platelets revealed both PK and pro‐UK. Accordingly, platelets may mediate physiological thrombolysis by this pathway since factor XIIa activates PK and kallikrein activates pro‐UK. Acid washing dissociated PK but not pro‐UK from platelets. Exogenous pro‐UK was specifically incorporated by platelets from the ambient fluid and similarly could not be dissociated from intact platelets. Therefore, platelets may also mediate an effect from therapeutically administered pro‐UK by prolonging its half‐life.

Effect of Intensive Lipid Lowering, With or Without Antioxidant Vitamins, Compared With Moderate Lipid Lowering on Myocardial Ischemia in Patients With Stable Coronary Artery Disease The Vascular Basis for the Treatment of Myocardial Ischemia Study

Background—Lipid lowering with statins prevents adverse cardiac events. Both lipid-lowering and antioxidant therapies may favorably affect vasomotor function and thereby improve ischemia. Methods and Results—In a randomized, double-blind, placebo-controlled trial, 300 patients with stable coronary disease, a positive exercise treadmill test, 48-hour ambulatory ECG with ≥1 episode of ischemia, and fasting total cholesterol of 180 to 250 mg/dL were assigned to 1-year treatment with intensive atorvastatin to reduce LDL to <80 mg/dL (n=96), intensive atorvastatin to reduce LDL to <80 mg/dL plus antioxidant vitamins C (1000 mg/d) and E (800 mg/d) (n=101), or diet and low-dose lovastatin, if needed, to reduce LDL to <130 mg/dL (n=103; control group). Ischemia end points, including ambulatory ECG monitoring and exercise treadmill testing, and endothelial assessment using brachial artery flow-mediated dilation were obtained at baseline and at 6 and 12 months. Baseline characteristics were similar in all groups. LDL decreased from ≈153 mg/dL at baseline in the 2 atorvastatin groups to ≈83 mg/dL at 12 months (each P<0.0001) and from 147 to 120 mg/dL in the control group (P<0.0001). During ambulatory ECG monitoring, mean number of ischemic episodes per 48 hours decreased 31% to 61% in each group (each P<0.001; P=0.15 across groups), without a change in daily heart rate activity. Mean duration of ischemia for 48 hours decreased 26% to 62% in each group (each P<0.001; P=0.06 across groups). Mean exercise duration to 1-mm ST-segment depression significantly increased in each group, but total exercise duration and mean sum of maximum ST depression were unchanged. Angina frequency decreased in each group. There was no incremental effect of supplemental vitamins C and E on any ischemia outcome. Flow-mediated dilation studies indicated no meaningful changes. Conclusions—Intensive lipid lowering with atorvastatin to an LDL level of 80 mg/dL, with or without antioxidant vitamins, does not provide any further benefits in ambulatory ischemia, exercise time to onset of ischemia, and angina frequency than moderate lipid lowering with diet and low-dose lovastatin to an LDL level of <120 mg/dL.

The pathophysiology of the onset of morning cardiovascular events

Evidence obtained over the past decade indicates that myocardial infarction (MI) and sudden death are not random events but rather, in many cases, may be triggered by the daily activities of the subject. The importance of physical or mental stresses as triggers is suggested by the parallel morning increased onsets of MI, sudden cardiac death, and stroke. Unstable angina and MI are usually precipitated by thrombus formation over a disrupted plaque that causes partial or complete obstruction of coronary artery blood flow. This process may be caused by physiologic factors that lead to rupture of a vulnerable plaque and subsequent thrombosis. Beta-blockers and aspirin, which can diminish these physiologic processes, have been shown to blunt or abolish the morning peak of onset of acute MI. It is hypothesized that occlusive coronary thrombosis occurs when an atherosclerotic plaque becomes vulnerable to rupture, and mental or physical stress causes the plaque to rupture. Increases in coagulability or vasoconstriction triggered by daily activities may also contribute to complete occlusion of the coronary artery lumen. Recognition of the circadian variation of the onset of acute cardiovascular disease suggests the need for pharmacologic protection of patients during the vulnerable periods and provides clues to the mechanism of disease onset, the investigation of which may lead to improved methods of prevention.