Ibolya E. András

Mechanisms of the blood-brain barrier disruption in HIV-1 infection

Summary1. Alterations of brain microvasculature and the disruption of the blood–brain barrier (BBB) integrity are commonly associated with human immunodeficiency virus type 1 (HIV-1) infection. These changes are most frequently found in human immunodeficiency virus-related encephalitis (HIVE) and in human immunodeficiency virus-associated dementia (HAD).2. It has been hypothesized that the disruption of the BBB occurs early in the course of HIV-1 infection and can be responsible for HIV-1 entry into the CNS.3. The current review discusses the mechanisms of injury to brain endothelial cells and alterations of the BBB integrity in HIV-infection with focus on the vascular effects of HIV Tat protein. In addition, this review describes the mechanisms of the BBB disruption due to HIV-1 or Tat protein interaction with selected risk factors for HIV infection, such as substance abuse and aging.

HIV-1 Tat protein alters tight junction protein expression and distribution in cultured brain endothelial cells

Disruption of the blood‐brain barrier (BBB) is widely believed to be the main route of human immunodeficiency virus (HIV) entry into the central nervous system (CNS). Although mechanisms of this process are not fully understood, alterations of tight junction protein expression can contribute, at least in part, to this phenomenon. Tight junctions are critical structural and functional elements of cerebral microvascular endothelial cells and the BBB. The aim of the present study was to examine the effects of HIV‐1 Tat protein on expression of tight junction proteins. Primary cultures of brain microvascular endothelial cells (BMEC) were employed in these experiments. A 24‐hr exposure of BMEC to Tat1–72 resulted in a decrease of claudin‐1, claudin‐5, and zonula occludens (ZO)‐2 expression, whereas total levels of occludin and ZO‐1 remained unchanged. In addition, a short (3‐hr) exposure of BMEC to Tat1–72 induced cellular redistribution of claudin‐5 immunoreactivity. Tat1–72‐induced alterations of claudin‐5 expression also were confirmed in vivo where Tat1–72 was injected into the right hippocampus of mice. These findings indicate that HIV‐1 Tat protein can markedly affect expression and distribution of specific tight junction proteins in brain endothelium. Alterations of only distinct tight junction proteins suggest a finely tuned effect of Tat1–72 on the BBB. Because tight junction proteins are critical for the barrier function of the BBB, such alterations can lead to disturbances of the BBB integrity and contribute to HIV trafficking into the brain.

Expression of glutamate receptors on cultured cerebral endothelial cells

Activation of glutamate receptors has been shown to mediate a large number of neuronal processes such as long‐term potentiation and ischemic damage. In addition to neurons and glia, glutamate receptors may occur on cerebral endothelial cells (CECs). The aim of the present study was to determine which glutamate receptors are expressed in CECs and to demonstrate the functional presence of such channels. By using reverse transcriptase‐polymerase chain reaction, we showed that primary cultures of rat CECs express N‐methyl‐D‐aspartate (NMDA) receptors (NR1 subunit, which is necessary for the formation of functional NMDA receptors, and NR2A–C subunits), 2‐amino‐3‐(3‐hydroxy‐5‐methyl‐4‐isoxazolyl‐propionate (AMPA) receptors (GLUR1–4 subunits), and metabotropic receptors (mGLUR). Exposure of the cultures to 2 mM glutamate, a well‐established mediator of ischemic damage, for 30 min increased significantly the phosphorylation of calcium/calmodulin‐dependent protein kinase II even after 10‐ and 60‐min recovery times. This effect could be prevented by the NMDA blocker MK‐801. The presence of multiple glutamate receptor types may confer a finely tuned responsiveness of the cerebral endothelium to glutamate in physiological and pathological conditions. J. Neurosci. Res. 54:814–819, 1998.

Signaling mechanisms of HIV-1 Tat-induced alterations of claudin-5 expression in brain endothelial cells

Exposure of brain microvascular endothelial cells (BMEC) to human immunodeficiency virus-1 (HIV-1) Tat protein can decrease expression and change distribution of tight junction proteins, including claudin-5. Owing to the importance of claudin-5 in maintaining the blood–brain barrier (BBB) integrity, the present study focused on the regulatory mechanisms of Tat-induced alterations of claudin-5 mRNA and protein levels. Real-time reverse-transcription-polymerase chain reaction revealed that claudin-5 mRNA was markedly diminished in BMEC exposed to Tat. However, U0126 (an inhibitor of mitogen-activated protein kinase kinase1/2, MEK1/2) protected against this effect. In addition, inhibition of the vascular endothelial growth factor receptor type 2 (VEGFR-2) by SU1498, phosphatidylinositol-3 kinase (PI-3 K) by LY294002, nuclear factor-κB (NF-κB) by peptide SN50, and intracellular calcium by BAPTA/AM partially prevented Tat-mediated alterations in claudin-5 protein levels and immunoreactivity patterns. In contrast, inhibition of protein kinase C did not affect claudin-5 expression in Tat-treated cells. The present findings indicate that activation of VEGFR-2 and multiple redox-regulated signal transduction pathways are involved in Tat-induced alterations of claudin-5 expression. Because claudins constitute the major backbone of tight junctions, the present data are relevant to the disturbances of the BBB in the course of HIV-1 infection.

The NMDA and AMPA/KA Receptors are Involved in Glutamate-Induced Alterations of Occludin Expression and Phosphorylation in Brain Endothelial Cells

Glutamate levels increase dramatically in cerebral ischemia and stroke. This may lead to opening of the blood–brain barrier (BBB) and induce further brain damage. Because endothelial tight junctions are critical elements of the BBB integrity, the aim of this study was to investigate the mechanisms of glutamate-induced alterations of the tight-junction protein occludin in cultured brain microvascular endothelial cells (BMECs). Transient exposure to glutamate resulted in cellular redistribution of occludin, followed by a decrease in the total level of this protein and diminished barrier function of BMECs. Inhibition of the N-methyl-d-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate (AMPA/KA) receptors attenuated glutamate-induced changes in occludin redistribution but not in the total protein levels. Treatment with glutamate also increased tyrosine phosphorylation and decreased threonine phosphorylation of occludin. Inhibition of the NMDA receptors by MK-801 partially protected against glutamate-induced elevation of occludin tyrosine phosphorylation. In addition, pretreatment with MK-801-attenuated glutamate-mediated disruption of endothelial barrier function. Blocking of the AMPA/KA receptors by 6,7-dinitroquinoxaline-2. 3-dione (DNQX) protected against hypophosphorylation of threonine residues of occludin; however, it did not affect disruption of endothelial integrity. These findings indicate the opposite effects of the NMDA and AMPA/KA receptors on occludin phosphorylation and disruption of the BBB functions.

PPARα and PPARγ attenuate HIV-induced dysregulation of tight junction proteins by modulations of matrix metalloproteinase and proteasome activities

The blood‐brain barrier (BBB) plays an important role in HIV trafficking into the brain and the development of the central nervous system complications in HIV infection. Tight junctions are the main structural and functional elements that regulate the BBB integrity. Exposure of human brain microvascular endothelial cells (hCMEC/D3 cell line) to HIV‐infected monocytes resulted in decreased expression of tight junction proteins, such as junctional adhesion molecule‐A (JAM)‐A, occludin, and zonula occludens (ZO)‐1. Control experiments involved exposure to uninfected monocytes. Alterations of tight junction protein expression were associated with increased endothelial permeability and elevated transendothelial migration of HIV‐ infected monocytes across an in vitro model of the BBB. Notably, overexpression of the peroxisome proliferator‐activated receptor (PPAR)α or PPARγ attenuated HIV‐mediated dysregulation of tight junction proteins. With the use of exogenous PPARy agonists and silencing of PPARα or PPARγ, these protective effects were connected to down‐regulation of matrix metalloproteinase (MMP) and proteasome activities. Indeed, the HIV‐ induced decrease in the expression of JAM‐A and occludin was restored by inhibition of MMP activity. Moreover, both MMP and proteasome inhibitors attenuated HIV‐ mediated altered expression of ZO‐1. The present data indicate that down‐regulation of MMP and proteasome activities constitutes a novel mechanism of PPAR‐induced protections against HIV‐induced disruption of brain endothelial cells.— Huang, W., Eum, S. Y., Andras, I. E., Hennig, B., Toborek, M. PPARa and PPARy attenuate HIV‐induced dysregulation of tight junction proteins by modulations of matrix metalloproteinase and proteasome activities. FASEBJ. 23, 1596–1606 (2009)

HIV-TAT protein upregulates expression of multidrug resistance protein 1 in the blood-brain barrier

Central nervous system (CNS) complications of human immunodeficiency virus (HIV) infection remain a serious health risk in HIV/acquired immunodeficiency syndrome despite significant advances in highly active antiretroviral therapy (HAART). Specific drugs used for HAART are substrates for the efflux transport systems, such as the multidrug resistance-associated proteins (MRPs), which are present on brain microvascular endothelial cells (BMEC) and astrocytes, that is, the main cell types that form the blood-brain barrier (BBB). Thus, drugs employed in HAART are actively removed from the CNS and do not efficiently inhibit HIV replication in the brain. To study the potential mechanisms of this process, the aim of the present research was to address the hypothesis that HIV Tat protein can contribute to upregulation of MRP expression at the BBB level. Tat is a protein produced and released by HIV-infected cells, which may play an important role in brain vascular pathology in the course of HIV infection. Among the family of MRPs, exposure to Tat specifically induced MRP1 messenger ribonucleic acid and protein expression both in BMEC and astrocytes. These alterations were accompanied by enhanced MRP1-mediated efflux functions. Furthermore, activation of the mitogen-activated protein kinase signaling cascade was identified as the mechanism involved in Tat-mediated overexpression of MRP1. These results indicate that Tat exposure can lead to alterations of the BBB functions and decrease HAART efficacy in the CNS through overexpression of drug efflux transporters.

HIV-Tat protein induces P-glycoprotein expression in brain microvascular endothelial cells

Among the different factors which can contribute to CNS alterations associated with HIV infection, Tat protein is considered to play a critical role. Evidence indicates that Tat can contribute to brain vascular pathology through induction of endothelial cell activation. In the present study, we hypothesized that Tat can affect expression of P‐glycoprotein (P‐gp) in brain microvascular endothelial cells (BMEC). P‐gp is an ATP‐dependent cellular efflux transporter which is involved in the removal of specific non‐polar molecules, including drugs used for highly active antiretroviral therapy (HAART). Treatment of BMEC with Tat1−72 resulted in P‐gp overexpression both at mRNA and protein levels. These alterations were confirmed in vivo in brain vessels of mice injected with Tat1−72 into the hippocampus. Furthermore, pre‐treatment of BMEC with SN50, a specific NF‐κB inhibitor, protected against Tat1−72‐stimulated expression of mdr1a gene, i.e. the gene which encodes for P‐gp in rodents. Tat1−72‐mediated changes in P‐gp expression were correlated with increased rhodamine 123 efflux, indicating the up‐regulation of transporter functions of P‐gp. These results suggest that Tat‐induced overexpression of P‐gp in brain microvessels may have significant implications for the development of resistance to HAART and may be a contributing factor for low efficacy of HAART in the CNS.

HIV-1 Tat Protein-Induced Alterations of ZO-1 Expression are Mediated by Redox-Regulated ERK1/2 Activation

HIV-1 Tat protein plays an important role in inducing monocyte infiltration into the brain and may alter the structure and functions of the blood—brain barrier (BBB). The BBB serves as a frontline defense system, protecting the central nervous system from infected monocytes entering the brain. Therefore, the aim of the present study was to examine the mechanisms of Tat effect on the integrity of the BBB in the mouse brain. Tat was injected into the right hippocampi of C57BL/6 mice and expression of tight junction protein zonula occludens-1 (ZO-1) was determined in control and treated mice. Tat administration resulted in decreased mRNA levels of ZO-1 and marked disruption of ZO-1 continuity. These changes were associated with accumulation of inflammatory cells in brain tissue of Tat-treated mice. Further experiments indicated that Tat-mediated alterations of redox-related signaling may be responsible for decreased ZO-1 expression. Specifically, injections with Tat resulted in activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and pretreatment with U0126, a specific inhibitor of ERK kinase, effectively ameliorated the Tat-induced diminished ZO-1 levels. In addition, administration of N-acetylcysteine (NAC), a precursor of glutathione and a potent antioxidant, attenuated both Tat-induced ERK1/2 activation and alterations in ZO-1 expression. These results indicate that Tat-induced oxidative stress can play an important role in affecting the integrity of the BBB through the ERK1/2 pathway.

NADPH oxidase and lipid raft-associated redox signaling are required for PCB153-induced upregulation of cell adhesion molecules in human brain endothelial cells

Exposure to persistent organic pollutants, such as polychlorinated biphenyls (PCBs), can lead to chronic inflammation and the development of vascular diseases. Because cell adhesion molecules (CAMs) of the cerebrovascular endothelium regulate infiltration of inflammatory cells into the brain, we have explored the molecular mechanisms by which ortho-substituted polychlorinated biphenyls (PCBs), such as PCB153, can upregulate CAMs in brain endothelial cells. Exposure to PCB153 increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as elevated adhesion of leukocytes to brain endothelial cells. These effects were impeded by inhibitors of EGFR, JAKs, or Src activity. In addition, pharmacological inhibition of NADPH oxidase or disruption of lipid rafts by cholesterol depleting agents blocked PCB153-induced phosphorylation of JAK and Src kinases and upregulation of CAMs. In contrast, silencing of caveolin-1 by siRNA interference did not affect upregulation of ICAM-1 and VCAM-1 in brain endothelial cells stimulated by PCB153. Results of the present study indicate that lipid raft-dependent NADPH oxidase/JAK/EGFR signaling mechanisms regulate the expression of CAMs in brain endothelial cells and adhesion of leukocytes to endothelial monolayers. Due to its role in leukocyte infiltration, induction of CAMs may contribute to PCB-induced cerebrovascular disorders and neurotoxic effects in the CNS.