HIV-1 viral proteins gp120 and Tat induce oxidative stress in brain endothelial cells

Abstract

The blood–brain barrier (BBB) has an important role in the development of AIDS dementia. The HIV-1 envelope glycoprotein (gp120) and transregulatory protein (Tat) of HIV-1 are neurotoxic and cytotoxic and have been implicated in the development of HIV dementia. They are known to cause oxidative stress and are associated with disruption of the BBB. Here, we used an immortalized endothelial cell line from rat brain capillaries, RBE4, to determine whether gp120 and Tat can induce oxidative stress in an in vitro model of the BBB. RBE4 cells were exposed to gp120 or Tat and the levels of reduced glutathione (GSH), oxidized glutathione (GSSG), catalase (CAT) activity, glutathione peroxidase (GPx) activity, and glutathione reductase (GR) activity, and malondialdehyde (MDA) used as measures of oxidative stress. Both gp120 and Tat significantly decreased the levels of intracellular GSH, GPx, and GR and increased the levels of MDA in RBE4 cells, showing that the cells were oxidatively challenged. The ratio of GSH/GSSG, a widely accepted indicator of oxidative stress, was also significantly decreased. These studies show that both of these viral proteins can induce oxidative stress in immortalized BBB endothelial cells.

Introduction

AIDS is often accompanied by neurological disorders and neuropathological abnormalities. A third of the adults and half of the children with AIDS develop HIV-1-associated dementia (HAD) [23]. Cognitive impairment, postural disorders, and tremors are among the most common symptoms encountered in patients suffering from AIDS dementia complex [12]. The role of the blood–brain barrier (BBB) is important in the development of HIV dementia because it serves as the conduit by which free virus and infected immune cells enter the brain from the circulatory system [8], [25], [27]. It also prevents the effective accumulation of many anti-retrovirals in the brain [9], [35] and releases substances in response to immunological (including viral) challenges [7], [14], [20], [22], [26], [31]. A mild disruption of the BBB is more frequent in AIDS patients with dementia, as compared with AIDS patients without dementia or sero-negative controls [36], and such problems may be related to the trafficking of immune cells across the BBB [5]. The BBB is composed of a continuous layer of cerebrovascular endothelial cells connected by intercellular tight junctions. This barrier exists between blood and brain interstitial fluid and tightly regulates the entry of compounds into the brain. The BBB helps to maintain the homeostatic environment of the brain, supplies the brain's nutritive needs, and plays a role in communication between the brain and peripheral tissues [6], [13], [18], [19], [30], [33]. Under physiological conditions, the integrity of the BBB is protected from oxidative stress because the BBB has high levels of antioxidant enzymes. These peroxide detoxifying enzymes play an important role in protecting the functional integrity of the BBB, which is essential for proper functioning of the brain [28].

In the last few years, there has been a body of evidence that HIV-1 envelope protein gp120 or Tat (trans-activator of viral replication) may cause BBB changes. There is a possibility that the binding of these proteins to brain endothelial cells may cause many of the changes seen in the BBB in HIV-dementia patients [4]. For example, one study found a significant increase in permeability (up to 47%) in cultured endothelial cells after exposure to gp120 [11]. Another study confirmed that gp120 is capable of changing and activating the vascular component of the BBB in vivo [37]. Yet another study on HIV-1 Tat protein demonstrated that exposure of endothelial cells to HIV-1 Tat protein resulted in a dose-dependent increase in oxidative stress and a decrease in intracellular glutathione (GSH) [36].

GSH, which is an important intracellular non-protein thiol compound in mammalian cells, has a pivotal role in the maintenance of membrane integrity. It may also play an important role in the proper functioning of the BBB [2]. Although brain endothelial cells posses high intracellular levels of antioxidative defense mechanisms, such as GSH, GSH peroxidase (GPx), and catalase (CAT), exposure to toxins and drugs may render the BBB susceptible to toxic damage and change its specific functions [21].

This present study concentrates on the oxidative stress induced in vitro in an immortalized cell line of rat brain endothelial cells, RBE4 cells, when exposed to the HIV-1 proteins gp120 and Tat. Previous studies only investigated limited oxidative stress parameters. Therefore, in this study, we have included the most widely used parameters.