Combination antiretroviral therapy (cART) has dramatically changed the outlook of infection with Human Immunodeficiency Virus, type 1 (HIV), from one with no treatment to that of a manageable chronic disease, and people with HIV (PWH) now have a lifespan getting closer to those uninfected albeit with a larger burden of comorbidities, including vascular disease (1). Cerebrovascular disease (CBVD) is associated with aging and is more frequent among PWH (2). It poses a significant disease burden in this vulnerable population, especially as nearly half of PWH in USA are age 50 and older. CBVD is a leading cause of vascular cognitive impairment and dementia that may further contribute to HIV-associated cognitive impairment (3–7).

HIV is a neurotropic virus and is known to enter the central nervous system (CNS) as early as 8 days post infection (8). Of interest, HIV can also infect pericytes, a key cellular component of the blood–brain barrier (BBB) (9). Systemic chronic inflammation associated with HIV infection may also contribute to BBB permeability (10). Similarly, cART effect on endothelial cell may facilitate BBB disruption (11). The aging HIV population also faces traditional risk factors, including diabetes, dyslipidemia, and cardiovascular disease, that can contribute to CBVD. Large epidemiological studies suggest that CBVD is more common in PWH compared to HIV uninfected individuals and is considered a major driver of CNS injury (3–7).

Advanced neuroimaging techniques such as arterial spin labeling (ASL) and resting-state functional magnetic resonance imaging (rs-fMRI) allow non-invasive assessment of cerebral blood flow (CBF) and cerebrovascular reactivity (CVR), respectively (12–17). CBF is a measure of brain perfusion and can be used to assess both local changes in perfusion pressure and redistribution of blood flow within that region’s vascular network. CBF is tightly coupled to neuronal function, though several components of the functional neurovascular unit (neurons, astrocytes, endothelial, and pericytes) contribute to overall CBF regulation (18). CVR reflects a compensatory change in cerebral arteries in response to a vasoactive stimulus such as CO₂ to meet the changing blood supply demands of the CNS (13). Since decreased CVR is associated with CBVD, CVR mapping can be used as an imaging marker to assess cerebral vascular territories with or at risk of CBVD (19). There are a few reports that have investigated CBF in PWH. Ances et al. (20) have shown reduced resting CBF in the basal ganglia and visual cortex of PWH compared to HIV negative healthy controls (HC). CBF abnormalities have also been documented in PWH with cognitive impairment (20–22). However, rs-fMRI based CVR without CO₂ challenge (15, 16) is a relatively newer measure of CNS neurovascular function, especially in the context of HIV infection.

In addition to these neuroimaging vascular markers, plasma levels of microparticles (MPs) of endothelial and glial origin can be informative of BBB alteration. Endothelial cells (ECs) lining the inner walls of cerebral blood vessels are critical for maintenance of neurovascular homeostasis, and injury to these cells can affect many critical functions, including CBF, CVR, and BBB function. In addition, damage to endothelial cells as well as their activation can result in release of MPs by the ECs and also leakage of glial MPs into the periphery.

Microparticles are small membrane-bound micro-vesicles of 0.1–1 μM in diameter (23, 24). They act as signaling shuttles that can deliver the intracellular cargo from the cell of their origin to the recipient cell and can contribute to inflammation in various chronic conditions, including HIV infection and vascular disease, as shown by our group (25) and others (26–35). Circulating microparticles of glial origin (expressing GFAP-Glial Fibrillary acidic protein) have been considered a marker of neurovascular injury during HIV infection and other CNS disorders (36–40). Further, our group (41, 42) and others (43, 44) have shown that sonic hedgehog (Shh+) signaling during astrocyte-endothelial communication is neuroprotective and is found to be disrupted during HIV infection and can lead to increased BBB permeability. These studies indicate that MPs of glia and endothelial origins could be used as biomarkers of BBB injury. However, it is not known how these peripheral markers correlate with neurovascular neuroimaging markers.

In order to examine the effect of cART on the neurovascular function, we examined longitudinal changes in CVR, CBF, and MPs of astrocytic and endothelial origin in PWH before and after initiation of cART.

This study was reviewed and approved by the Research Subjects Review Board at the University of Rochester, and all study participants signed a written informed consent prior to undergoing study procedures. Details of the study cohort have been previously reported (45). For the analyses reported here, data on 88 unique study participants, 35 (32 male) treatment-naïve PWH and 53 (27 male) age-matched HIV− healthy controls were available at baseline. However, the total number of study participants in the HIV+ cohort decreased from 35 to 32 at 12 weeks and further declined to 15 at year 2. Similarly, the total number of study participants in HIV− cohort went from 53 at baseline visit to 25 at year 2 visit. After initiation of cART the mean CD4 counts were 580 cells/μl (SD: 261) at week 12 and 848 cells/μl (SD: 271) at year 2. Similarly mean viral load was 513 copies/mL (n = 8, SD: 2629) at week 12 and 20 copies/mL (n = 10, SD: 42) at year 2. In rest of the study participants, viral loads were below detectable limit. Demographic and clinical characteristics of study participants are presented in Table 1.

All participants underwent a comprehensive clinical, laboratory, and neuroimaging evaluation. PWH were assessed at baseline (BSL, prior to initiation of cART) and 12 weeks (W12), and 2 years (Y2) post-cART initiation, while healthy controls were assessed at BSL, and YR 2 only. All PWH were antiretroviral treatment-naïve prior to enrollment and met the following laboratory parameters within 30 days of baseline evaluation: hemoglobin ≥ 9.0 g/dL, serum creatinine ≤2 × ULN, AST (SGOT), ALT (SGPT), and alkaline phosphatase ≤ 2 × upper limit of normal. Participants with severe premorbid or comorbid psychiatric disorders, such as diagnoses of schizophrenia, bipolar disorder, and active depression, were excluded. Additional exclusionary criteria were stroke, head trauma resulting in the loss of consciousness for more than 30 min, multiple sclerosis, brain infections (except for HIV-1), space-occupying brain lesions requiring acute or chronic therapy, dementia as established by the HAND definition, active alcohol and drug abuse within 6 months of study entry, and conditions preventing MRI scanning (claustrophobia and metallic implants). Study participants meeting criteria for HIV-associated mild neurocognitive disorder (MND) or HIV-associated asymptomatic neurocognitive impairment (ANI) were eligible to participate. Exclusion criteria for healthy controls differ only for HIV status.

The results from this study suggest that CVR, endothelial, and glia MPs are early markers of neurovascular dysfunction in cART naïve PWH. Most of these markers improved shortly after cART treatment; however, the beneficial effect of cART treatment diminished over time. Abnormalities in CVR were found in both subcortical gray matter regions (CN and TH) and white global white matter (GWM), indicating a diffuse involvement of the brain microcirculation. The trend of decreased CVR during chronic cART-treated PWH was associated with a decrease in CBF, especially in the GWM.

Two recent studies have also assessed CVR in PWH using alternative approaches to ours. Callen et al. (55) used changes in CBF induced by intravenous acetazolamide and found decreased CVR in the frontal lobe and basal ganglia. Chow et al. (56) used transcranial Doppler ultrasound (TCD) and breath-holding to assess mean flow velocity changes to infer CVR. Higher CVR was associated with a higher score on the Montreal Cognitive Assessment.

Since the cohort of PWH we enrolled was relatively young (average age 34 years) with preserved immune function before starting cART (average nadir and baseline CD4+ cell count > 400 cell/uL), it could be envisioned that PWH as they age would be further exposed to CBVD and in particular to cerebral small vessel disease (CSVD). CVR is closely linked to the regulation of the microcirculation, thus, is reduced in CSVD (57). Consistent with these implications are recent studies showing a higher prevalence of CSVD in PWH compared to HC (2, 58).

CBF in our study did not differ between PWH cART naïve prior to starting treatment and controls, suggesting that more significant alteration in tissue perfusion is necessary before changes in CBF can be captured. In contrast, CVR is more closely related to CSVD (59).

CVR is a marker of endothelial function; therefore, we assessed the relationship between CVR and plasma levels of endothelial-derived MPs. We measured MPs that expressed endothelial marker CD144 (CD144+ MPs) and also MPs double positive for CD144 and CD62L (aka L-selectin), which is a marker of endothelial activation (CD144+CD62L+ MPs). Endothelial markers, including ICAM-1 and VCAM-1 were also measured. The increased plasma levels of CD144+ MPs in PWH compared to controls are consistent with the expected effect of inflammation before cART initiation and the subsequent decline with reduced inflammation during cART. In contrast, VCAM-1 level before cART were surprisingly lower in PWH and normalized with treatment. This finding needs to be confirmed in further investigations. On the other hand, ICAM-1 did not differ between groups.

Systemic inflammation associated with HIV infection could also lead to an altered BBB. In this study, we observed that GFAP+ MPs were elevated in cART naïve PWH, suggesting an increased BBB permeability. The beneficial response to cART treatment continued during the two-year follow-up. We next examined the temporal correlation between CVR and blood biomarkers. CVR in CN correlated negatively with endothelial and glial MP levels at baseline study visit, only in PWH. At year-2, despite a smaller number of study participants, glial MPs continue to show an even stronger negative correlation with CVR in caudate. Endothelial MPs also correlated negatively with CBF in GWM and Thalamus at baseline visit in PWH. However, after cART both MPs and CBF decreased, making now their relationship positively correlated but likely not causally related. From these results, CVR and endothelial and glia MPs provide a consistent mechanistic relationship when assessing cerebral microcirculation. When assessing CVR directly is not possible, for example in resource-limited settings where MRI facilities are not readily available, these blood biomarkers could be more easily implemented.

Our study has a few limitations. First, the sample size of our study is small due to both enrollment restrictions/difficulties with a cART-naïve population and a high attrition rate. Second, as the study recruited study participants that were treatment-naïve to cART, a young study population limited the evaluation of the effects of cART in older PWH. Also, PWH in this geographical location are heavily skewed toward male sex-at-birth. Owing to that, we were able to enroll only three cART naïve female study participants and thus were not able to evaluate the effect of sex. Furthermore, the calculation of CVR from resting-state fMRI has limitations as well. While methods have been developed to qualitatively assess a relative CVR index based on resting-state data (15, 16), the gold standard to quantify and assess CVR is using a vasoactive stimulus task-based breathing challenge (60). An additional limitation is the post label delay (PLD) of 1.5 s used in ASL, shorter than the 1.8 s currently recommended (61). It should be noted that our protocol was implemented prior to this recommendation. However, the relative young age of the cohort enrolled likely minimizes the impact of the shorter PLD. Despite these caveats, our study design allows for a unique approach to study and separate the effects of acute and chronic HIV infection and cART treatment on brain cerebrovascular function.

To the best of our knowledge, this is the only study that specifically investigates longitudinal changes in CVR, CBF, endothelial, and glia MPs before and after cART initiation. Our findings indicate that untreated HIV infection, even with relatively preserved immune function, affects brain microcirculation. While cART normalizes endothelial and glia MPs levels, effects on CVR and CBF seem less effective over time, possibly predisposing PWH to CSVD.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The studies involving humans were approved by University of Rochester Research Subject Review Board. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

MS and MU: study design, conducting experiments, acquiring data, analyzing data, and writing the manuscript. VS, AP, and YZ: conducting experiments, acquiring data, and analyzing data. KM: conducting experiments, acquiring data, analyzing data, and writing the manuscript. AT: data entry and database management. LW: statistical support. MT and JZ: manuscript review for intellectual content. XQ: statistical support and manuscript review for intellectual content. GS: study design, funding acquisition, and manuscript writing and review for intellectual content. All authors contributed to the article and approved the submitted version.

This work was supported by the National Institutes of Health, grant numbers R01MH099921 and R01AG054328.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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