Glen E. Cooke

HIV-1 infection and AIDS dementia are influenced by a mutant MCP-1 allele linked to increased monocyte infiltration of tissues and MCP-1 levels

Studies in humans and in experimental models of HIV-1 infection indicate an important role for monocyte chemoattractant protein-1 (MCP-1; also known as CC chemokine ligand 2), a potent chemoattractant and activator of mononuclear phagocytes (MP) in the pathogenesis of HIV-associated dementia (HAD). We determined the influence of genetic variation in MCP-1 on HIV-1 pathogenesis in large cohorts of HIV-1-infected adults and children. In adults, homozygosity for the MCP-1 –2578G allele was associated with a 50% reduction in the risk of acquiring HIV-1. However, once HIV-1 infection was established, this same MCP-1 genotype was associated with accelerated disease progression and a 4.5-fold increased risk of HAD. We examined the molecular and cellular basis for these genotype–phenotype associations and found that the mutant MCP-1 –2578G allele conferred greater transcriptional activity via differential DNA–protein interactions, enhanced protein production in vitro, increased serum MCP-1 levels, as well as MP infiltration into tissues. Thus, MCP-1 expression had a two-edged role in HIV-1 infection: it afforded partial protection from viral infection, but during infection, its proinflammatory properties and ability to up-regulate HIV-1 replication collectively may contribute to accelerated disease progression and increased risk of dementia. Our findings suggest that MCP-1 antagonists may be useful in HIV-1 infection, especially for HAD, and that HIV+ individuals possessing the MCP-1 –2578G allele may benefit from early initiation of antiretroviral drugs that effectively cross the blood–brain barrier. In a broader context, the MCP-1 –2578G allele may serve as a genetic determinant of outcome of other disease states in which MP-mediated tissue injury is central to disease pathogenesis.

Plasma atherogenic markers in congestive heart failure and posttransplant (heart) patients.

OBJECTIVES We hypothesized that plasma factors important for the development of atherosclerosis play a major role in the occurrence of cardiac allograft vasculopathy (CAV). BACKGROUND Cardiac allograft vasculopathy is a major cause of death among heart transplant recipients, has a poorly understood pathogenesis and has similarities to atherosclerotic coronary disease. METHODS The study population consisted of 93 postcardiac transplant recipients. Thirty-one patients with congestive heart failure (CHF) and 18 healthy individuals served as control subjects. Posttransplant coronary anatomy was evaluated by angiography and intravascular ultrasound. Laboratory analyses of lipids, homocysteine, vitamin B12 and folate, fibrinogen, von Willebrand factor antigen (vWFAg) and renin were obtained on all participants. RESULTS Posttransplant patients were found to have elevated serum triglycerides, total cholesterol/ high-density lipoprotein cholesterol ratio, lipoprotein (a), homocysteine, vWFAg, fibrinogen and renin and lower high-density lipoprotein cholesterol. Most of these laboratory atherogenic factors were also elevated to a similar degree in the CHF control population. Although most atherogenic markers were elevated, there was little correlation with CAV severity. Cardiac allograft vasculopathy severity varied with time after transplantation, 3-hydroxy-methyl-glutaryl-coenzyme A reductase inhibitor use and prior cytomegalovirus infection. Even within the normal range, lower RBC folate levels were associated with increased severity of CAV. CONCLUSIONS The posttransplant course is associated with increased clinical and laboratory atherogenic factors, some of which likely contribute to the severity of coronary vasculopathy. Compared with normal control subjects, many of these markers are already increased in pretransplant CHF patients with or without occlusive coronary artery disease.

The PlA Polymorphism of Glycoprotein IIIa Functions as a Modifier for the Effect of Estrogen on Platelet Aggregation

BACKGROUND Although estrogen has been shown to contribute to retardation of the development of coronary heart disease in premenopausal women, the efficacy of hormone replacement therapy for coronary heart disease prevention in women with established coronary heart disease remains controversial. Hence, additional research is needed to clarify the effects of hormone replacement therapy on the cardiovascular and clotting systems. We investigated the effect of estrogen on platelet aggregation induced by standard agonists (epinephrine and adenosine diphosphate), with and without the platelet antagonist aspirin. Furthermore, we analyzed our data according to the presence or absence of a prevalent polymorphism of the glycoprotein (GP) IIIa subunit of the platelet fibrinogen receptor GPIIb-IIIa, PlA2. METHODS AND RESULTS The effect of estrogen on aggregation of platelets was studied in healthy men (n = 20, 10 PlA1/A1 and 10 PlA1/A2) and premenopausal healthy women (n = 10, 5 PlA1/A1 and 5 PlA1/A2). The PlA1/A1 and PlA1/A2 individuals were matched for age and race. Platelet response to agonists was investigated in the presence of (1) estrogen (10(-11) to 10(-8) mol/L), (2) aspirin (0.056 to 56 micromol/L), (3) estrogen plus aspirin, and (4) estrogen plus ICI 182 780 (ICI, 10(-9) mol/L, an inhibitor of the estrogen receptor). We found that physiologic concentrations of estrogen strongly and significantly inhibited the aggregation of PlA1/A2 platelets (P<.005 for epinephrine and P<.05 for adenosine diphosphate, induced aggregation, respectively) in both men and women. Concentrations of estrogen that were 1000-fold greater were required to observe the same level of inhibition with PlA1/A1 platelets. In the presence of aspirin, estrogen failed to provide additional inhibitory effect on aggregation of both PlA1/A1 and PlA1/A2 platelets. The estrogen-specific inhibitor ICI blocked the effect of estrogen on aggregation, suggesting that this effect is mediated by the estrogen receptor. CONCLUSIONS Estrogen inhibits the aggregation of platelets, but such inhibition is highly dependent on the presence or absence of the PlA2 polymorphism of GPIIIa. However, in the presence of aspirin, the inhibitory effect of estrogen on aggregation was no longer detectable.

Endothelial nitric oxide synthase gene: prospects for treatment of heart disease.

Nitric oxide functions as a signaling molecule with a well-established role in vascular homeostasis. It is synthesized from the oxidation of L-arginine by the enzyme, endothelial nitric oxide synthase (eNOS). The eNOS gene has a number of polymorphic sites, including SNPs, dinucleotide repeats and variable number tandem repeat sequences, and the opportunity exists to investigate polymorphic functional correlates as well as disease-specific associations, especially in cardiovascular disease, including coronary artery disease, and its most severe consequence, myocardial infarction. A number of clinical and functional correlative studies involving eNOS polymorphisms have been reported and are presented. The promise and complexity of pharmacogenetics is illustrated using eNOS as an example because of its relationship with cardiovascular biology and pathology. In this review, we will discuss the impact of nitric oxide, eNOS, genetic regulation, clinical investigation and, ultimately, prospects for treatment of heart disease.

Higher Prevalence of GPIIIa PlA2 Polymorphism in Siblings of Patients With Premature Coronary Heart Disease

BACKGROUND The Pl(A2) polymorphism of GPIIIa has been associated with unstable coronary syndromes in some studies, but the association has remained debated. None of the previous studies have focused on families at high risk. Risk factors tend to cluster within kindreds with high prevalence of premature coronary heart disease (CHD). Therefore, a heightened prevalence of the Pl(A2) polymorphism among siblings of patients with CHD would support the hypothesis that Pl(A2) is linked, directly or indirectly, to CHD. OBJECTIVES To measure the prevalence of the Pl(A2) polymorphism among siblings of patients with CHD before the age of 60 years and to seek an association between the Pl(A2) polymorphism and established atherosclerotic and thrombogenic risk factors. METHODS From January 1994 to April 1996, we genotyped 116 asymptomatic siblings (60 Caucasians, 56 Afro-Caribbeans) of patients with CHD manifestations before the age of 60 years for the Pl(A) polymorphism (also called HPA-1). A control cohort was used for comparison, consisting of individuals that were matched for race and geographic area but were free of CHD (n = 268, 168 Caucasians and 100 Afro-Caribbeans). In addition, we have characterized the sibling cohort for other atherogenic and thrombogenic risk factors. RESULTS The prevalence of Pl(A2)-positive individuals (Pl(A2)[+], Pl(A1/A2) heterozygotes plus Pl(A2/A2) homozygotes) in the sibling cohort was high: 41.4%. When analyzed separately, the prevalence of Pl(A2)(+) siblings was 53.3% among Caucasians and 28.6% among Afro-Caribbeans. There was no association between Pl(A2) and other established atherogenic or thrombogenic risk factors. Interestingly, the clustering of other risk factors was lesser among Pl(A2)(+) siblings than their Pl(A1) counterparts. CONCLUSIONS This study supports the hypothesis that the prevalence of Pl(A2)(+) individuals is high in kindreds with premature CHD. Hence, like the established risk factors that tend to cluster in families with premature CHD and contribute strongly to the accelerated atherosclerotic process affecting these individuals, the Pl(A2) polymorphism of GPIIIa may represent an inherited risk that promotes the thromboembolic complications of CHD. That these asymptomatic Pl(A2)(+) siblings had overall less established risk factors than their Pl(A1) counterparts might represent an explanation for why they remained asymptomatic despite their Pl(A2) positivity.

PlA2, a variant of GPIIIa implicated in coronary thromboembolic complications

Ischemic heart disease (IHD) and adverse events after coronary stent placement represent polygenic disorders. They correspond to complex traits induced not only by multiple genes but also by the interaction of these genes with the environment. Multiple loci likely to be implicated in IHD are

Platelet PlA2 Polymorphism and Thromboembolic Events: From Inherited Risk to Pharmacogenetics

Nearly three million individuals develop a myocardial infarction (MI) in the U.S. each year, and more than 10% of these individuals die as a consequence of such an event. De~nite progress has been made in the management of patients admitted to hospitals for acute MI. However, as shown with three successive GUSTO trials on thrombolysis for MI patients carried over a decade, mortality at 30 days has reached a plateau of around 7% for those individuals who are eligible for ~brinolytic therapy [1–3]. Nearly one third of the patients who die of MI do so prior to reaching a hospital and therefore cannot bene~t from the progress of modern medicine [4]. Many MIs are triggered by the rupture or ulceration of an atherosclerotic plaque [5–7]. While many plaque ruptures are believed to remain asymptomatic, some lead to the formation of an occlusive thrombus [7,8]. The HMG Co-A reductase inhibitors, pravastatin, lovastatin, and simvastatin have been shown in large primary and secondary prevention studies (WESCOP, CARE, LIPID, AFCAPS/TexCAPS, and 4S) to prevent 25–35% of all MI deaths [9–13]. Hence it was suggested that plaque stabilization with a statin corresponds to an ef~cient way to prevent death in patients with coronary heart disease (CHD). Although such a strategy represents a substantial step forward in our ability to manage patients with CHD, it remains that 65–75% of the patients are not protected from MI death by taking a statin, indicating the limitations of lipid-lowering therapy. Indeed, multiple risk factors seem to conspire and lead to such dreadful events [14]. It is time to develop novel strategies to upscale our ability to recognize individuals at particular risk for MI and to target our powerful therapeutic means in the most ef~cient way. A new promising strategy to improve the treatment of CHD will be based on the premise of genomic therapeutics. The concept is simple: therapeutic decisions will be based not only on traditional assessment of patients but also on genomic information obtained for these patients. Over the past 30 years, scientists have accumulated a large amount of information about environmental risk factors that contribute to atherogenesis and thrombogenesis. Much less is known about the genetic risk factors that contribute to these processes, although there is clear evidence of a genetic contribution to CHD [15–19]. As part of an effort to discover novel inherited thrombogenic risk factors, we measured the density of the glycoprotein (GP)IIb-IIIa receptors (integrin a2bb3) on the surface of platelets obtained from the siblings of patients with premature CHD (de~ned as manifestation of CHD prior to age 60) [20]. We discovered that for many siblings, the GPIIIa subunit of the ~brinogen receptor was lacking an antigenic epitope required for recognition by a monoclonal antibody (SZ21) commonly used to detect GPIIIa on western blots [21]. We were able to establish, in collaboration with Dr. Paul Bray, that the loss of GPIIIa epitope recognition was not due to a new mutation, but instead was secondary to the PlA2 polymorphism [22–24]. In our experimental conditions, the GPIIIa subunit of PlA2/A2 homozygous platelets reacted very weekly with SZ21 compared with GPIIIa from PlA1/A1 homozygous platelets, whereas PlA1/A2 heterozygous platelets displayed an intermediate af~nity for this antibody [22]. The ~nding that many siblings from patients with CHD displayed the PlA2 polymorphism on the surface of their platelets lead us to generate the hypothesis that PlA2 represents a risk factor for CHD [21]. In a pair-matched case-control study, we showed that the PlA2 prevalence in patients admitted to the coronary care unit of a tertiary academic hospital was twice that of a control group admitted to the same hospital but with no history of vascular diseases [25]. The difference between the prevalence of control and case pa-

The safety and efficacy of aspirin and clopidogrel as a combination treatment in patients with coronary heart disease.

The use of aspirin and clopidogrel in combination has become part of the standard clinical care of patients with coronary artery disease. The use of this combination provides significant benefits compared with the use of aspirin alone in patients with acute coronary syndromes, and in patients treated with percutaneous coronary intervention with stent placement (both bare metal and drug-eluting stents). Clinical trials have demonstrated significant efficacy of this dual therapy; however, there is the potential for significant bleeding complications from the synergistic antiplatelet effects. In total, it appears that when there is vessel injury (mechanical from perctutaneous coronary intervention or a ruptured plaque), dual antiplatelet therapy with aspirin and clopidogrel results in improved outcomes, albeit with a small but significant inherent risk of increased bleeding.

Evidence for the Role of Epstein Barr Virus Infections in the Pathogenesis of Acute Coronary Events

Background The role of viral infections in the pathogenesis of atherosclerosis remains controversial largely due to inconsistent detection of the virus in atherosclerotic lesions. However, viral infections elicit a pro-inflammatory cascade known to be atherogenic and to precipitate acute ischemic events. We have published in vitro data that provide the foundation for a mechanism that reconciles these conflicting observations. To determine the relation between an early viral protein, deoxyuridine triphosphate nucleotidohydrolase (dUTPase), produced following reactivation of Epstein Barr Virus (EBV) to circulating pro-inflammatory cytokines, intercellular adhesion molecule-1 (ICAM-1) and acute coronary events. Methodology/Principal Findings Blood samples were obtained from 299 patients undergoing percutaneous coronary intervention for stable angina (SA), unstable angina (UA), or acute myocardial infarction (AMI). Plasma concentrations of pro-inflammatory cytokines and neutralizing antibody against EBV-encoded dUTPase were compared in the three patient groups. AMI was associated with the highest measures of interleukin-6 (ANOVA p<0.05; 4.6±2.6 pg/mL in patients with AMI vs. 3.2±2.3 pg/mL in SA). ICAM-1 was significantly higher in patients with AMI (ANOVA p<0.05; 304±116 pg/mL in AMI vs. 265±86 pg/mL SA). The highest values of ICAM-1 were found in patients having an AMI and who were antibody positive for dUTPase (ANOVA p = 0.008; 369±183 pg/mL in AMI and positive for dUTPase vs. 249±70 pg/mL in SA negative for dUTPase antibody). Conclusions/Significance These clinical data support a model, based on in vitro studies, by which EBV may precipitate AMI even under conditions of low viral load through the pro-inflammatory action of the early protein dUTPase that is produced even during incomplete viral replication. They further support the putative role of viral infections in the pathogenesis of atherosclerosis and coronary artery events.

Endothelial Nitric Oxide Synthase Polymorphisms Associated with Abnormal Nitric Oxide Production Are Not Over-represented in Children with Down Syndrome

OBJECTIVE Down syndrome patients are at increased risk for developing pulmonary hypertension (PHTN). Nitric oxide (NO) is an important factor for pulmonary vasoreactivity. Various endothelial nitric oxide synthase (eNOS) polymorphisms have been shown to affect NO. The goal of this study was to determine whether there was a difference in prevalence of eNOS polymorphisms between Down syndrome patients vs. non-Down syndrome patients. METHODS Down syndrome patients were recruited as well as non-Down syndrome patients. Gene polymorphisms for eNOS-3 (GG, GT, TT), eNOS-4 (bb, ba, aa), and eNOS-P (TT, TC, CC) were determined. Three forms of the 3 genes were compared in cross-tabulation tables with Down syndrome patients vs. non-Down syndrome patients and Down syndrome patients with heart defects vs. those without defects. Association was tested with chi-square and significance was set at P < or = .05. RESULTS Fifty-one Down syndrome patients and 411 controls were studied. Twenty-one Down syndrome patients had heart defects and 6 of these patients had documented PHTN. There was no difference in gender between Down syndrome patients (males 56.9%) and controls (males 50.4%), P = .38. Prevalence of eNOS polymorphisms between Down syndrome patients and controls was not different for the genes (eNOS-3, P = .94; eNOS-4, P = .40; eNOS-P, P = .18). There was no difference in gene polymorphisms between Down syndrome patients with heart defects vs. those without defects (eNOS-3, P = .19; eNOS-4, P = .29; eNOS-P, P = .99). CONCLUSION Prevalence of various eNOS polymorphisms between Down syndrome patients and controls was not different. Other polymorphisms that are associated with PHTN should be studied to determine whether they may be the cause of the increased risk of PHTN in Down syndrome patients.