Structural and Functional Dysregulation of the Brain Endothelium in HIV Infection and Substance Abuse

Blood–brain barrier (BBB) injury and dysfunction following infection with the human immunodeficiency virus (HIV) enables viral entry into the brain, infection of resident brain cells, neuronal injury and subsequent neurodegeneration leading to HIV-associated neurocognitive disorders (HAND). Although combination antiretroviral therapy has significantly reduced the incidence and prevalence of acquired immunodeficiency syndrome and increased the life expectancy of people living with HIV, the prevalence of HAND remains high. With aging of people living with HIV associated with increased comorbidities, the prevalence of HIV-related central nervous system (CNS) complications is expected to remain high. Considering the principal role of the brain endothelium in HIV infection of the CNS and HAND, the purpose of this manuscript is to review the current literature on the pathobiology of the brain endothelium structural and functional dysregulation in HIV infection, including in the presence of HIV-1 and viral proteins (gp120, Tat, Nef, and Vpr). We summarize evidence from human and animal studies, in vitro studies, and associated mechanisms. We further summarize evidence of synergy or lack thereof between commonly abused substances (cocaine, methamphetamine, alcohol, tobacco, opioids, and cannabinoids) and HIV- or viral protein-induced BBB injury and dysfunction.


Introduction
The blood-brain barrier (BBB) is a semi-permeable membrane that separates circulating blood from brain tissues, regulates the transport of oxygen and essential nutrients into the central nervous system (CNS), protects the brain from harmful substances, and maintains brain homeostasis [1][2][3][4].It is primarily composed of endothelial cells (ECs) lining the brain blood vessels, a basement membrane that encases the ECs on the abluminal side, pericytes, and astrocyte endfeet [2][3][4].The molecular components of the BBB responsible for its protective and barrier functions include endothelial tight junction (TJ) proteins (occludin, claudins, and zonula occludens (ZO)), adherent junction (AJ) proteins, and efflux transporters [2,3].Endothelial injury and subsequent BBB dysfunction are implicated in the pathogenesis of several CNS infections and diseases, including human immunodeficiency virus (HIV) CNS infection and neuroHIV.
In the early stages of HIV infection, endothelial injury following exposure to circulating infected leukocytes, virions, and viral proteins, as well as subsequent BBB dysfunction, enables HIV entry into the brain and infection of resident CNS cells, including productive microglial infection [5,6].Virions and viral proteins released by infected brain cells are associated with increased inflammation and oxidative stress, which leads to neuronal injury and subsequent neurodegeneration [7,8] (Figure 1).Increased neurodegeneration over time results in motor, cognitive, and behavioral abnormalities termed HIV-associated neurocognitive disorders (HAND) [9,10].HAND subclassification includes HIV-associated asymptomatic neurocognitive impairment, mild neurocognitive disorder, and HIV-associated dementia [10][11][12][13].Although the incidence and prevalence of the most severe form of HAND has decreased with the advent of combination antiretroviral therapy (ART), the overall prevalence of HAND and its associated morbidity remains high (up to 40%) [14,15].With increased life expectancy and aging of people living with HIV (PLWH) [16,17], the prevalence of HAND and HIV-associated CNS comorbidities is expected to remain high.Considering the principal role of the brain endothelium in HIV infection of the CNS and HAND, the purpose of this manuscript is to review the current literature on BBB structure and function in HIV infection and substance abuse.We summarize evidence from human and animal studies, in vitro studies, and associated mechanisms.We also summarize evidence of synergy, or lack thereof, between commonly abused substances (cocaine, methamphetamine, alcohol, tobacco, opioids, and cannabinoids) and HIV-or viral protein-induced BBB injury and dysfunction.
Cells 2024, 13, x FOR PEER REVIEW 2 of 20 neurodegeneration over time results in motor, cognitive, and behavioral abnormalities termed HIV-associated neurocognitive disorders (HAND) [9,10].HAND subclassification includes HIV-associated asymptomatic neurocognitive impairment, mild neurocognitive disorder, and HIV-associated dementia [10][11][12][13].Although the incidence and prevalence of the most severe form of HAND has decreased with the advent of combination antiretroviral therapy (ART), the overall prevalence of HAND and its associated morbidity remains high (up to 40%) [14,15].With increased life expectancy and aging of people living with HIV (PLWH) [16,17], the prevalence of HAND and HIV-associated CNS comorbidities is expected to remain high.Considering the principal role of the brain endothelium in HIV infection of the CNS and HAND, the purpose of this manuscript is to review the current literature on BBB structure and function in HIV infection and substance abuse.We summarize evidence from human and animal studies, in vitro studies, and associated mechanisms.We also summarize evidence of synergy, or lack thereof, between commonly abused substances (cocaine, methamphetamine, alcohol, tobacco, opioids, and cannabinoids) and HIV-or viral protein-induced BBB injury and dysfunction.

Evidence from Human Studies
Studies have shown that HIV-infected patients have BBB injury that is associated with increased permeability, infiltration of circulating leukocytes into the CNS, and advanced disease.In these studies, patients with HAND had significantly more damage to the brain endothelium, and increased BBB permeability was observed in 50% of AIDS patients compared to 22% in asymptomatic PLWH [18][19][20].There is other evidence of increased BBB breakdown in HIV-infected humans.Contrast-enhanced magnetic resonance imaging of PLWH showed altered myoinositol/creatine (mI/Cr) in the basal ganglia associated with high plasma viral load [21].High cerebrospinal fluid (CSF) albumin and CSF/serum albumin quotient (QAlb) in PLWH correlates with increased neuronal injury (higher CSF neurofilament-L) and impaired brain bioenergetics (lower N-acetylaspartate-to-creatine ratio) in the parietal gray matter [22].
Evidence suggests that ART does not prevent or abrogate HIV-induced BBB injury and can increase BBB permeability.In PLWH who had an abnormal QAlb and N-acetylaspartateto-creatine ratio, one year of ART did not improve QA1b [22].Impaired BBB integrity has been reported in both HIV-infected subjects with HAND and neuroasymptomatic PLWH, despite a decreased QA1b post-ART [17].Although ART suppressed CSF and plasma viral loads, it did not significantly change albumin and neurofilament-L levels [19].A study of virally suppressed patients with HAND showed BBB disruptions in the basal ganglia and frontal white matter, despite a lack of peripheral inflammation [23].HIV-infected adults on suppressive ART also showed impaired BBB associated with increased levels of amyloid-β (1-42), and phosphorylated and total tau correlating with CSF albumin and QAlb [24].
Autopsy evidence of brain endothelium injury and impaired BBB in HIV-infected patients has included enlarged microvessels and loss of hippocampal capillaries associated with degeneration of perivascular tissues [25], fragmentation and loss of microvessels TJ proteins, including claudin-5, occludin, ZO-1 and ZO-2 in several brain regions (frontal cortex, basal ganglia, subcortical white matter, and cortical gray matter) [26][27][28].Brain tissues of HIV-infected humans has also showed significantly more capillary loss and decreased TJs in patients with HAND compared to HIV-infected subjects without HAND and seronegative controls subjects [20,23,[26][27][28][29].

Evidence from Animal Studies
Primate models: Simian immunodeficiency virus (SIV)-infected macaques, including animals with SIV encephalitis, showed endothelial activation and BBB injury, reduced expression of endothelial TJ proteins occludin and ZO-1, and fragmentation of TJs in the basal ganglia microvessels associated with increased pro-inflammatory cytokines and accumulation of perivascular macrophages in the CNS [30][31][32][33].SIV-infected macaques showed increased expression of vascular cell adhesion molecule (VCAM)-1 that correlated with endothelial activation, BBB injury, and increased production of cytokines such as tumor necrosis factor (TNF)-α and interferon-γ [32,34].SIV-infected animals also showed increased production of chemokines such as the chemokine ligand-5 (CCL5), macrophage inflammatory protein-1α and -1β, monocyte chemotactic protein-3, and interferon-inducible protein-10 [35].These chemokines likely contribute to increased diapedesis and recruitment of circulating leukocytes into the brain.
Induction of Tat expression in transgenic (Tg) mice resulted in BBB breakdown associated with increased leakage of circulating dyes into the brain, activation of perivascular macrophages, and microgliosis [42].HIV-1 Tg26 mice and gp120 Tg mice showed reduced ZO-1 expression and increased BBB permeability, including enhanced leakage of albumin into the brain associated with increased intercellular adhesion molecule (ICAM)-1, VCAM-1, and substance-P [37,38].Injection of HIV-1 gp120 proteins to rats induced ROS, caused the upregulation of MMP-2 and MMP-9, and reduced brain claudin-5 and laminin (major component of endothelial basement membrane) [39,40].Injection of HIV-1 Nef protein into rats also induced BBB disruption, reduced claudin-5, and increased the permeability of the BBB and other vascular tissues.This was associated with increased MMP-9 activity, inflammation, and leukocyte infiltration into tissues [43,44].MMP-9 inhibitors prevented Nef-mediated BBB injury [43].

Endothelial Injury and BBB Dysfunction in HIV Infection and Substance Abuse
Substance abuse is associated with increased risk of HIV transmission and infection, and PLWH with substance use disorders are more likely to be diagnosed late, have poor adherence to ART, and experience accelerated disease progression, increased morbidity, neuropathological complications, and cognitive impairment [98,99].Substances of abuse that have been investigated for effects on the brain endothelium in HIV/AIDS include cocaine, methamphetamine (meth), alcohol, nicotine/tobacco, opioids, and cannabinoids.

Alcohol
Alcohol abuse negatively impacts brain function and can exacerbate HIV-associated CNS injury.Binge exposure of HIV Tg rats to ethanol, as well as in vitro exposure of rats and human brain ECs to ethanol and gp120, altered endothelial barrier properties (increased BBB permeability and decreased claudin-3, occludin, and JAM-2) via mechanisms involving the transient receptor potential melastatin-7 ion channel [116].In vitro studies of HBMECs exposed to ethanol and HIV-1 Nef, Vpr, Tat, or gp120 proteins showed that ethanol potentiated viral protein-induced ECs apoptosis and TNF-α production [96].Tat and alcohol individually and synergistically altered the BBB, increased macrophage transendothelial migration, and increased neurotoxicity [117].However, another study showed that alcohol and gp120 individually increased HBMEC permeability, ROS production, and cytoskeletal remodeling, but found no synergistic effect [118].

Tobacco
In vivo and in vitro studies showed that nicotine, cotinine, and tobacco smoke extracts have limited effect on the BBB but their combination with protease inhibitors (saquinavir [SQV] and ritonavir [RTV/r]) or gp120 proteins significantly increased BBB injury and dysfunction.Exposure of rats' brain ECs to nicotine, SQV, or RTV increased ROS, and combination of nicotine and SQV or SQV/r synergistically increased ROS and decreased ZO-1 [119].In rats treated with nicotine, cotinine, SQV or SQV/r, individual treatment had no major effect on ZO-1 or BBB permeability, but a combination of nicotine or tobacco smoke extracts with SQV or SQV/r significantly decreased ZO-1 and increased BBB permeability (by 2 to 4-fold).These effects were associated with Notch-4 downregulation [119].Nicotine, cotinine, and their combination target efflux transporters and dose-dependently increased BBB permeability to 14 C-sucrose and SQV, and increased SQV accumulation in several brain regions (frontal, parietal, and occipital cortex, hippocampus, thalamus, hypo-thalamus, and caudate/putamen) [119,120].HBMECs exposed to tobacco smoke extracts or gp120 showed decreased viability, mitochondrial membrane potential, ZO-1, occludin, and TEER, and increased BBB permeability.Combined treatment of HBMECs with tobacco smoke extracts and gp120 resulted in a synergistic decrease in TEER and an increase in paracellular permeability, associated with a 2-to 4-fold decrease in endothelial oxygen consumption, mitochondrial membrane potential, ZO-1, and occludin [121].These data suggest that tobacco use in HIV infection may be associated with increased risk of BBB oxidative injury and dysfunction.

Opioids
Opioids abuse and overdoses have dramatically increased in the USA and worldwide, and there is evidence that opioids impact HIV immuno-and neuro-pathogenesis [122,123].In vitro studies showed that chronic low doses of morphine (MOR) had no major effect on HBMECs [124].However, evidence from other in vivo and in vitro studies suggests that in the presence of HIV proteins, opioids alter the BBB properties and function.Administration of MOR and Tat to mice increased leukocytes trafficking into the CNS and the combination of Tat and MOR potentiated CCL5 upregulation in the CNS [125].Tat Tg mice treated with fentanyl [126] or MOR [127,128] showed increased BBB disruption and brain inflammatory markers [126][127][128] and increased BBB leakage and recruitment of circulating macrophages into the striatum, hippocampus parenchyma, and perivascular space [127,128].However, buprenorphine, a partial opioid agonist, decreased CCL2-mediated adhesion of monocytes to HBMECs and transendothelial migration [129].These results led to suggestions that buprenorphine can reduce HIV-mediated neuroinflammation in addition to its use for the treatment of opioid addiction [129].Exposure of brain microvascular ECs to MOR and/or Tat altered the expression of ZO-1, occludin and JAM-2, and decreased TEER, which was associated with intracellular calcium release, activation of myosin light-chain kinase, and increased production of pro-inflammatory cytokines [130].MOR also increased the accumulation of tenofovir, emtricitabine, and dolutegravir in HBMECs and hCMEC/D3 cells [131].However, these in vitro findings may not necessarily translate to increased CNS entry of these antiretrovirals in vivo.Although MOR increased BBB leakage in Tat Tg mice, it significantly reduced CNS entry of abacavir and dolutegravir, and increased brain Pglycoprotein levels [127], which suggests that MOR may block CNS entry of antiretrovirals by upregulating BBB efflux transporters.

Cannabinoids
Cannabinoids have been shown to have anti-inflammatory and analgesic properties, and to have a protective effect on the BBB following brain injury.Cannabinoids increased TEER, decreased endothelial VCAM-1, reduced BBB permeability and ischemia-induced BBB damage [132,133], reduced ischemia-induced IL-6 and lactate dehydrogenase in astrocytes [133], increased endogenous antioxidant via NF-κB pathways, reduced cytokines (including TNF-α, IL-1β, and IL-6) expression, and decreased BBB permeability in mice following traumatic brain injury [134].Cannabinoid-type 2 receptor (CB2R) is expressed in ECs, glial cells, neurons, and brain macrophages [135], and has been shown to mediate cannabinoid effects in vitro and in vivo.Activation of CB2R decreased inflammatory responses in human macrophages and HBMECs, preserved endothelial AJ and TJ proteins, and decreased BBB permeability [136].CB2R activation decreased inflammation-induced ICAM-1 and VCAM-1, decreased monocytes' adhesion to the brain endothelium, and decreased BBB permeability in vivo [137,138].CB2R agonists increased endothelial AJs and TJs occludin, ZO-1, and claudin-5, restored BBB properties, reduced neuroinflammation and neurodegeneration, and attenuated brain injury in murine models of brain hemorrhage [139,140] and traumatic brain injury [141,142].
Cannabis is widely used among PLWH.Considering the evidence above, that cannabinoids attenuated inflammation and BBB damage following brain injury, it has been suggested that cannabinoids' anti-inflammatory properties could counter HIV-induced neuroinflammation, BBB damage, and CNS injury [135,143].CB1R and CB2R agonists decreased gp120-induced synapse loss and neuronal death in vitro [144], decreased gp120-induced downregulation of ZO-1, claudin-5, and JAM-1 in HBMECs, and decreased gp120-induced calcium influx, BBB permeability, and monocyte transmigration in vitro and in vivo [79].In cross-sectional studies of an observational cohort, analyses of the blood and CSF of HIVinfected adults and seronegative controls, cannabis users (moderate and daily users), and non-users, showed that recent use of cannabis was associated with reduced inflammatory markers in both the CSF and blood [143].Compared to HIV+ non-cannabis users, frequent and daily cannabis use was associated with lower BBB permeability (lower CSF/serum albumin ratio and CSF levels of soluble urokinase plasminogen activator receptor), lower CSF neurofilament-L [145], and lower CSF CCL2 and interferon gamma-induced protein-10 levels associated with better neurocognitive (learning) performance [146].Longitudinal studies of older HIV-infected adults who were frequent cannabis users, occasional users, and non-users, showed better global cognitive performance among occasional users compared to HIV+ non-cannabis users.However, recent cannabis use was linked to worse cognition, especially regarding memory function [147].In autopsy studies of brain tissues from HIV-infected humans on long-term ART, subjects with HAND [148], HIV encephalitis, and CNS comorbidities [149] had increased CB1R and CB2R in several brain regions, in neurons, glial cells, and meningeal and perivascular macrophages [148,149].Increased CB1R was also associated with worse cognition, including poorer memory function and speed of information processing [148].Furthermore, although CB2R agonists decreased gp120-induced neuronal injury, they had no effect on Tat-induced synapse loss or neuronal death [144].Thus, it is not yet clear whether cannabinoids abrogate or exacerbate HIVinduced CNS injury and HAND, or the potential effect of the dosage, potency (e.g., delta-9tetrahydrocannabinol content), and frequency of cannabis use, and polysubstance use.

Conclusions
The BBB is central to HAND neuropathogenesis.BBB dysfunction during the early stages of HIV infection enables viral entry into the brain, infection of brain cells, and subsequent inflammation, oxidative injury of CNS cells, neurodegeneration, and HAND.This comprehensive review of current evidence from studies in humans, human autopsy brain samples, animals, and in vitro BBB models shows that there is increased injury of the cerebral endothelium, BBB breakdown, and permeability in both symptomatic and asymptomatic HIV infections.HIV-1 proteins such as gp120, Tat, and Nef also induce BBB injury and dysfunction in vivo (Table 1) and in vitro (Table 2), and commonly abused substances such as cocaine, meth, opioids, and tobacco potentiate HIV-and viral proteininduced BBB injury and dysfunction (Tables 1-3).

Table 1.
In vivo effects of HIV-1, viral proteins, and substances of abuse on the BBB integrity and function, and associated mechanisms.

Table 2.
In vitro effects of HIV-1, viral proteins, and substances of abuse on the BBB integrity and function, and associated mechanisms.
Meth or Meth and Tat increased ROS and MDA, reduced CAT, GPx, and SOD activity [111,112]; decreased viability and induced apoptosis in ECs [111,112,115].
Table 3. Interactions between substances of abuse and HIV, viral proteins, and antiretroviral drugs on the BBB integrity and function.
Abbreviations (Tables 1-3 This review also discussed the molecular mechanisms involved in BBB injury and dysfunction induced by HIV, viral proteins, and substances of abuse (overall findings are summarized in Tables 1-3).Synergistic increases in BBB damage with substance abuse and HIV or viral proteins would result in increased risk of CNS infection and HAND in HIV-infected substance abusers.As current evidence suggests that ART does not prevent or abrogate HIV-induced BBB injury, it would be important to determine whether ART alters HIV-induced neuropathology, and if any such CNS ART effects are influenced by antiretrovirals drug classes and CNS penetration.
Current evidence suggests that cannabinoids reduce inflammation and HIV-induced BBB injury.However, human studies show conflicting findings, including recent and frequent/daily cannabis use being associated with improved BBB function and better learning in cross-sectional studies and associated with worse cognition/poor memory function in longitudinal studies.Furthermore, human autopsy studies showed that HAND is associated with increased CB1R and CB2R, and demonstrate that increased CB1R is associated with worse memory function and speed of information processing.Thus, whether cannabinoids are neuroprotective or harmful in HIV CNS pathologies and HAND remains to be established.

Figure 1 .Figure 1 .
Figure 1.Schematic illustration of HAND neuropathogenesis.Following contact with the brain endothelium, circulating HIV-infected leukocytes, virions, and viral proteins (e.g., gp120, Tat, Nef) induce blood-brain barrier (BBB) injury, associated with increased inflammation and production ofFigure 1.Schematic illustration of HAND neuropathogenesis.Following contact with the brain endothelium, circulating HIV-infected leukocytes, virions, and viral proteins (e.g., gp120, Tat, Nef) induce blood-brain barrier (BBB) injury, associated with increased inflammation and production of reactive oxygen species (ROS), damage to endothelial tight junction (TJ) proteins (occludin, claudins, zonula occludens), and damage to adherent junctions (junctional adhesion molecules (JAMs)) proteins.This results in BBB dysfunction, including increased permeability and infiltration of HIV and infected cells into the central nervous system (CNS).Once in the brain, HIV infects resident brain cells, including microglia (productive infection) and astrocytes.Productively infected brain cells further release HIV and viral proteins, inflammatory cytokines, chemokines, and ROS.These factors further increase BBB injury and dysfunction, CNS infection, and induce neuronal injury.Repeated and prolonged neuronal injury causes neurodegeneration, which can result in HIV-associated neurocognitive disorders (HAND).Created with BioRender.com(accessed on 14 June 2024).