Increased Cardiovascular Disease Risk In The HIV-Positive Population on ART: Potential Role of HIV-Nef and Tat

Abstract

With effective antiretroviral therapy (ART), many HIV-infected people die of diseases other than acquired immune deficiency syndrome (AIDS). In particular, coronary artery disease has emerged as one of most critical complications of HIV infection and a major cause of morbidity and mortality. Although reportedly antiretroviral combination therapy itself may accelerate atherosclerosis by enhancing dyslipidemia, most recent epidemiological studies support the notion that HIV infection itself contributes to cardiovascular disease. However, it is still a mystery how the virus can contribute to cardiovascular disease development even while suppressed by ARTs. This review discusses the current understanding of interactions between HIV infection and cardiovascular diseases in both clinical and experimental studies with special focus on those viral proteins which are still produced by HIV. This will help infectious disease/vascular biology experts to gain insights into the pathophysiological mechanisms of HIV associated cardiovascular disease and new trends to treat and prevent cardiovascular disease in the HIV infected population.

Cardiovascular disease (CVD) as a severe complication in HIV patients

Due to the successful control of HIV viremia and HIV-induced AIDS through antiretroviral therapy (ART), CVD has emerged as a leading cause of death in those infected with human immunodeficiency virus (HIV) [1–3]. Interestingly, HIV-infected individuals have an increased risk of CVD both in the absence and presence of virologically suppressive ART [4–8]. Triant et al have shown that acute myocardial infarction (AMI) rates and cardiovascular risk factors were increased in HIV patients compared with non-HIV patients as indicated by a relative risk of 1.75 (95% CI 1.51–2.02, p<0.0001) for AMI [6]. This increased risk is magnified exponentially in the older HIV-infected population. Freiberg et al analyzed over 81,000 HIV-positive and HIV-negative individuals from the Virtual Cohort of the Veterans Aging Cohort Study from 2003 to 2008, and showed that even after accounting for other traditional risk factors, HIV infection resulted in a two-fold higher risk of heart attack [9].

Atherosclerosis in the HIV infected population

Several studies have addressed the nature of atherosclerotic lesions in the HIV infected population [10–13]. Fitch et al. reported [10, 14] that the presence of plaque and numbers of noncalcified plaque segments were increased in the HIV-infected group compared with HIV negative controls. They concluded that HIV patients without significant metabolic abnormalities may still develop non-calcified plaque and therefore are at increased risk for CAD [10]. Another study assessed the increased risk of atherosclerosis in HIV-infected patients by measuring carotid intima-medial thickness in 145 HIV patients on ART for at least 6 months. They revealed that 34 (23.4%) of these patients had carotid plaques that were associated with three independent risk factors: old age [odds ratio (OR) 6.16, 95% confidence interval (CI) (1.09–34.88; p=0.040), hypertension (OR 12.62, 95% CI 1.72–92.49; p=0.013) and higher low-density lipoprotein cholesterol (LDL-C) (OR 1.08, 95% CI 1.01–1.16; p=0.039) [11].

Is ART a culprit for HIV-related CVD?

Although the increased risk of CVD in HIV-infected patients is still not fully understood yet, one explanation could be HIV virus- or ART-induced hyperlipidemia and hypercholesterolemia, conditions known to promote progression of coronary atherosclerosis [15]. Indeed, HIV itself [16, 17] and a few antiretroviral drugs [18], in particular HIV protease inhibitors, reportedly can cause dyslipidemia in the HIV-positive population, thus contributing to the increased risk for CVD. However, the heightened risk of CVD persists even in the current treatment era, in which new generation ART have significantly reduced dysmetabolic side effects (e.g. insulin resistance, dyslipidemia, and hypertension) [19, 20].

Currently, there is a controversial discussion whether living with HIV or being on ART produces greater cardiovascular risk than being treatment-naïve. Untreated HIV infection has been found to associate with increased levels of IL-6, a pro-inflammatory cytokine and a stimulus for hepatic C-reactive protein production, a surrogate marker for systemic inflammation. Higher levels of IL-6 strongly predict cardiovascular events and overall mortality in antiretroviral-untreated and treated HIV infection [21]. This and several other studies suggest that ongoing HIV replication and immune depletion significantly contribute to increased prevalence of elevated inflammation biomarkers, altered coagulation, and monocyte activation, and this contribution is independent of the substantial contribution from comorbid conditions [22–24]. Hsue and colleagues [25] concluded that HIV infection correlates with premature atherosclerosis even in the absence of detectable viremia, overt immunodeficiency and exposure to ART, and appears to be independent of traditional cardiac risk factors. They found that Intima-media thickness (IMT) and C-reactive protein was strongly associated with the presence of HIV disease rather than viral load or CD4+ T cell count. However, antiretroviral drug exposure was also associated with higher IMT [25]. Nevertheless, the Women’s Interagency HIV Study measured Carotid Artery Stiffness via ultrasound 6.5 years after measurement of T Cell activation, and showed that even after the initiation of ART, persistently activated T Cells strongly correlated with increased carotid artery stiffness only in HIV patients, but not in HIV-negative patients [26]. This conclusion that HIV itself increases the risk of CVD is further supported by the National Institutes of Health’s Strategies for Management of Antiretroviral Therapy (SMART) study, which utilizes CD4+ cell counts to determine the starting and stopping point of intermittent therapy. It reported that the interruption of ART was surprisingly associated with increased risk of CVD [27]. This study suggests that ART is not the prevailing reason for increased CVD risk in those infected with HIV, and that HIV viral activities might also contribute. Hansen et. al showed impaired aortic endothelial function, increased c-IMT, and increased arterial stiffness in a Transgenic (Tg) mouse model expressing HIV viral proteins env, tat, nef, vpu, vpr, gp120 and rev. They also found markers for vascular remodeling such as decreased elastin content, increased cathepsin K and cathepsin S activity, and increased mechanical residual stress in the arteries of these Tg mice [28]. This is in line with previous conclusions that HIV infection itself has emerged as an independent contributor to cardiovascular disease in this population [5–7, 29].

Effects of smoking, alcohol and drug abuse for CVD in HIV patients

Smoking has been found to be an independent factor that is associated with CVD [30, 31]. Reportedly, 40–70% of HIV-infected population is current smokers. A cohort study of 33,308 HIV patients showed that the risk of myocardial infarction and CVD decreased with each passing year after smoking cessation, and that after 3 years the risk was almost half that of the first year. Smoking tobacco inhibits effective T cells function, which may result in increased risk of infection such as pulmonary infections. In addition, HIV infection is associated with a chronic state of persistent inflammation which increases the risk for CVD, chronic obstructive pulmonary disease and non-AIDS defining cancers.

The Veterans Aging Cohort Study showed that among HIV-infected men, alcohol abuse was associated with a higher prevalence of CVD compared with infrequent and moderate drinking, even after adjusting for traditional CVD risk factors, antiretroviral therapy, and CD4 count [32]. In addition, recreational drug use including cocaine and methamphetamine contributes to cardiac toxicity [33].

Endothelial dysfunction in HIV patients

Endothelial dysfunction is associated with an impaired ability of the vascular lining to maintain normal homeostasis. In vivo, it predominantly but not exclusively describes the reduced ability of arteries to dilate in response to flow-induced nitric oxide (NO) production. In vitro, correlates for vascular dysfunction include the NO-quenching reactive oxygen species (ROS), endothelial chemokine/adhesion protein expression and endothelial cell death (apoptosis or necrosis) because of the contribution of local inflammation and microthromboses to endothelial dysfunction in vivo. Endothelial dysfunction can progress to atherosclerosis and has been shown to predict future cardiovascular events in most population studies [34, 35]. The untreated HIV state has been associated with impaired endothelial function [36, 37]. This condition can be best experimentally assessed using whole virus or viral envelope protein, HIV gp120, which is essential for virus entry by binding CXCR4 or CCR5 receptors, and by activating these receptors to affect T lymphocytes, macrophages, cardiomyocytes, endothelial cells and central nervous system cells [38–41]. HIV gp120 was found in inflammatory cells, endothelial cells and cardiomyocytes in heart tissues from HIV patients with or without HIV cardiomyopathy, while HIV DNA or RNA was only in inflammatory cells [42]. Studies with HIV envelope as recombinant gp120 protein from CXCR4 binding strains have shown it induces endothelial apoptosis by CXCR4-dependent caspase activation [42–43]. Endothelium-derived nitric oxide, synthesized by the endothelial NO synthase (eNOS), is a major mediator of endothelium-dependent vasorelaxation and was reduced by HIV gp120 in TNF-α-activated endothelial cells [44]. Interestingly, exposure to cigarette smoke and HIV gp120 causes a synergistic increase in endothelial cell death[45].

However, as discussed in the paragraphs above, ART and its associated reduction in viral replication does not fully normalize endothelial activation and dysfunction. Thus, a better understanding of mechanisms for continued endothelial dysfunction in ART-treated HIV patients is needed to devise new therapeutic strategies to decrease cardiovascular risk in HIV-infected patients.

Early HIV-encoded proteins and endothelial dysfunction

The ongoing viral contribution to vascular dysfunction and CVD may be partially explained by the presence of HIV-infected reservoir cells, as reservoir cells and associated cytokine signaling are suggested to be important in the development and progression of cardiomyopathy and encephalopathy [46]. According to a prevailing view, infected cells hide in tissues such as the lymphatic system [47]. But how can these reservoir T cells possibly influence atherosclerosis development when systemic levels of released HIV proteins and proinflammatory cytokines are largely reduced when virus production is halted? Certainly, low-level transcription of HIV genes continues even after years of ART [48–50], but their relevance for disease is unclear. Interestingly, further analysis has shown that the majority of these transcripts represent the “early” HIV genes Tat, Rev, and Nef. As Rev is a nuclear protein [51] with no significant effects on vascular biology, we will focus on HIV-Tat and HIV-Nef proteins in the following paragraphs.

1. Effects of HIV-Tat on the vasculature

HIV Trans-Activator of Transcription (Tat) protein, a regulatory protein, is essential for efficiency of viral transcription [52] and has been implicated in several disease conditions ranging from pulmonary hypertension to sleep disorder [42] [53] [54]. Several in vivo studies suggest that Tat causes aberrant cell signaling and leads to altered endothelial cell morphology, gene expression, and survival. HIV-Tat has been suggested to play a role in HIV-related Kaposi sarcoma by promoting endothelial cell proliferation and tumor angiogenesis [55]; and Tat protein was shown to promote inflammation by activating human endothelial cells [56]. In contrast to its roles in angiogenesis, Tat can also cause apoptosis of primary microvascular endothelial cells of lung origin via either tumor necrosis factor secretion or the Fas pathway [57].

2. Role of HIV-Nef in endothelial dysfunction

Negative factor (Nef) is decreased to a much lesser extent than other HIV gene products after initiation of antiretroviral treatment [58, 59] suggesting that Nef could play a role in mechanisms of cardiovascular dysfunction in HIV patients on ART. In general, Nef is known as an important HIV pathogenic factor [60]. Nef is also responsible for T cell activation in infected cells [61, 62] and enhances virus production in vivo [63]. In fact, transgenic mice expressing CD4-promoter-driven Nef develop a spectrum of pathologies including AIDS-like disease [64] and vasospasm in the heart [65], and certain Nef gene variants were linked to pulmonary hypertension [66, 67]. We have recently reported that HIV-infected T cells are more potent than free virus in activating coronary arterial endothelial cells [68]. There is evidence that this effect is Nef-dependent. Nef-deleted virus shows only residual activity, suggesting that Nef, when compared to all other proteins including envelope gp120 and Tat, is the main contributor of HIV-induced endothelial activation.

Our and other groups [66, 68] have demonstrated Nef protein in endothelium of coronary and pulmonary arteries in SIV-HIV-Nef-infected macaques, which showed a relatively high percentage of blood cells constantly probing vascular endothelial cells [69], Thus, endothelial cells, especially those in developing atherosclerotic plaques, are constantly in direct contact with circulating monocytes and T cells and in prime physical position to receive Nef transfer. In the proinflammatory conditions associated with viremic and even aviremic HIV infection, endothelial cell activation leads to increased vascular adhesion protein and chemokine expression. This activation promotes T cell and monocyte adherence to endothelial cells, often followed by diapedesis. Importantly, Nef expressing T cells have been shown to exhibit increased adherence to endothelial cells based on their impaired diapedesis and migration into the subendothelium space [70].

In summary, the present review highlights that HIV early gene-encoded proteins, in particular Tat and Nef, could play a role in the development of CVD in the HIV-infected population independent of traditional risk factors (e.g. ART treatment and smoking/alcohol/drug use) (Table 1). By understanding the mechanisms of these risk factors, we may ultimately discover new ways to treat and prevent CVD in the HIV infected population.

Table 1.

Concluded the risk factors and their main contributions to CVD in HIV infected population.

Category of risk factor	Risk factor	Main contribution to CAD
ART	Protease inhibitors	Induce ROS activation and may induce endothelial cell apoptosis Induces dyslipidemia
	NRTI	Increase platelet reactivity
	NNRTI	Induce monocytes to adhere to vascular endothelium Induces dyslipidemia
HIV	Virus	Induces MCP-1 production
	Nef protein	Induces MCP-1 production, ROS activity and endothelial apoptosis eNOS downregulation
	Tat Protein	Induces expression of MCP-1 and adhesion molecules, including VCAM-1.ICAM-1 and E-selectin
Behaviors	Smoking, alcohol and drug abuse	Inflammation, Immune dysfunction, Synergy with gp120 to induce vascular endothelial cell death
Co-infection	Virus such as Hepatitis C,human herpesvirus 4 and Cytomegalovirus Bacteria	inflammation

Schematic 1.

Discussed mechanism of HIV-induced endothelial activation and dysfunction in untreated viremic (A) HIV patients and those on anti-retroviral treatment (B).