Background: The incidence of cardiovascular disease (CVD) is higher in HIV + patients than it is in the average population, and combination antiretroviral therapy (cART) is a recognized risk factor for CVD. However, the molecular mechanisms that link cART and CVD are currently unknown. Our study explores the role of the activation of p90RSK, a reactive oxygen species (ROS)-sensitive kinase, in engendering senescent phenotype in macrophages and accelerating atherogenesis in patients undergoing cART. Methods: Peripheral whole blood from cART-treated HIV + individuals and non-treated HIV − individuals was treated with H 2 O 2 (200 μM) for 4 minutes, and p90RSK activity in CD14 + monocytes was measured. Plaque formation in the carotids were also analyzed in these individuals. Macrophage senescence was determined by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. The involvement of p90RSK-NRF2 signaling in cART-induced senescence was assessed by p90RSK specific inhibitor (FMK-MEA) or dominant negative p90RSK (DN-p90RSK), and NRF2 activator (NRF2A). Further, the severity of atherosclerosis was determined in myeloid cell-specific wild type and DN-p90RSK transgenic mice. Results: Monocytes from HIV + patients exhibited higher levels of p90RSK activity and were also more sensitive to ROS than monocytes from HIV − individuals. A multiple linear regression analysis involving cART, Reynolds CV risk score, and basal p90RSK activity revealed that cART and basal p90RSK activity were the two significant determinants of plaque formation. Many of the antiretroviral drugs individually activated p90RSK, which simultaneously triggered all components of the macrophage senescent phenotype. cART inhibited antioxidant response element reporter activity via ERK5 S496 phosphorylation. NRF2A reversed the H 2 O 2-induced over-activation of p90RSK in cART-treated macrophages by countering the induction of senescent phenotype. Lastly, the data obtained from our gain-or loss-of-function mice conclusively showed the crucial role of p90RSK in inducing senescent phenotype in macrophages and atherogenesis. Conclusions: cART increased monocyte/macrophage sensitivity to ROS in HIV + individuals by suppressing NRF2-ARE activity via p90RSK-mediated ERK5 S496 phosphorylation, which coordinately elicited senescent phenotypes and pro-inflammatory responses. As such, our report underscores the importance of p90RSK regulation in monocytes/macrophages as a viable biomarker and therapeutic target for preventing CVD, especially in HIV + patients treated with cART.

People living with HIV are at higher risk of atherosclerosis (AS). The pathogenesis of this risk is not fully understood. To assess the regulatory networks involved in AS we sequenced mRNA of the peripheral blood mononuclear cells (PBMCs) and measured cytokine and chemokine levels in the plasma of 13 persons living with HIV and 12 matched HIV-negative persons with and without AS. microRNAs (miRNAs) are known to play a role in HIV infection and may modulate gene regulation to drive AS. Hence, we further assessed miRNA expression in PBMCs of a subset of 12 HIV+ people with and without atherosclerosis. We identified 12 miRNAs differentially expressed between HIV+ AS+ and HIV+ , and validated 5 of those by RT-qPCR. While a few of these miRNAs have been implicated in HIV and atherosclerosis, others are novel. Integrating miRNA measurements with mRNA, we identified 27 target genes including SLC4A7, a critical sodium and bicarbonate transporter, that are potentially dysregulated during atherosclerosis. Additionally, we uncovered that levels of plasma cytokines were associated with transcription factor activity and miRNA expression in PBMCs. For example, BACH2 activity was associated with IL-1β, IL-15, and MIP-1α. IP10 and TNFα levels were associated with miR-124-3p. Finally, integration of all data types into a single network revealed increased importance of miRNAs in network regulation of the HIV+ group in contrast with increased importance of cytokines in the HIV+ AS+ group.

Atherosclerosis (AS)-associated cardiovascular disease is an important cause of mortality in an aging population of people living with HIV (PLWH). This elevated risk has been attributed to viral infection, anti-retroviral therapy, chronic inflammation, and lifestyle factors. However, the rates at which PLWH develop AS vary even after controlling for length of infection, treatment duration, and for lifestyle factors. To investigate the molecular signaling underlying this variation, we sequenced 9368 peripheral blood mononuclear cells (PBMCs) from eight PLWH, four of whom have atherosclerosis (AS+). Additionally, a publicly available dataset of PBMCs from persons before and after HIV infection was used to investigate the effect of acute HIV infection. To characterize dysregulation of pathways rather than just measuring enrichment, we developed the single-cell Boolean Omics Network Invariant Time Analysis (scBONITA) algorithm. scBONITA infers executable dynamic pathway models and performs a perturbation analysis to identify high impact genes. These dynamic models are used for pathway analysis and to map sequenced cells to characteristic signaling states (attractor analysis). scBONITA revealed that lipid signaling regulates cell migration into the vascular endothelium in AS+ PLWH. Pathways implicated included AGE-RAGE and PI3K-AKT signaling in CD8+ T cells, and glucagon and cAMP signaling pathways in monocytes. Attractor analysis with scBONITA facilitated the pathway-based characterization of cellular states in CD8+ T cells and monocytes. In this manner, we identify critical cell-type specific molecular mechanisms underlying HIV-associated atherosclerosis using a novel computational method.

Mesenchymal stem cells (MSCs) are rare progenitor cells present in adult bone marrow that have the capacity to differentiate into a variety of tissue types, including bone, cartilage and fat. The biological activities of MSCs suggest a number of potential clinical applications, where each particular application is related to a specific MSC activity mediated by a different mechanism. Osiris Therapeutics has developed a technology for isolation and expansion of hMSCs from adult bone marrow for clinical use. Data from pre-clinical and clinical studies suggest that the ability of MSCs to migrate to inflammatory sites, modulate immune response, down-regulate inflammation, and accelerate tissue repair in the local environment may have therapeutic effects. Therefore in developing therapeutic applications, the MSCs should be verified to display one or more of above-mentioned functions, calling for the need to develop predictive functional assays. Modulation of the immune response is an apparent in vivo therapeutic property of the MSC necessary for successful Graft versus Host Disease (GVHD) treatment. Based on previous knowledge regarding mechanisms underlying MSC-mediated immunosuppressive effects, several markers for developing an MSC potency assay have been proposed. In the present study a relationship between selected markers and hMSC-mediated immunosuppression was investigated in vitro. Results show that co-culture of hMSCs with anti-CD3/CD28-activated peripheral blood mononuclear cells (hPBMCs) caused inhibition of lymphocyte proliferation. The hMSC effect on lymphocyte proliferation is dose-dependent, causing > 50% inhibition at approximately 1:10–1:25 MSC: T-lymphocyte ratio. Supernatants of parallel co-cultures taken on days 1, 3, and 5 were analyzed for prostaglandin 2 (PGE2), tumor necrosis factor-α (TNF-α), and tryptophan. The results showed increased levels of PGE2, decreased levels of TNF-α and increased depletion of tryptophan related to indoleamine 2,3-dioxygenase (IDO) enzyme activity, associated with increasing number of MSCs in each well. The quantity of PGE2 on day 1 and the level of tryptophan on day 5 in the MSC-PBMC co-culture supernatants correlated to the level of inhibition of proliferation, with the PGE2 range from approximately 11,000 to 22,000 pg/mL and 50% tryptophan depletion resulting in a 50% inhibition of the lymphocyte proliferation point. Further studies demonstrated that the addition of TNF-α to MSCs induced PGE2 secretion at a level which was similar to that detected in the co-culture studies of MSCs-PBMC. Thus, a strong correlation between inducible PGE2 secretion/IDO enzyme activity and the inhibition of lymphocyte proliferation by hMSCs in vitro indicates key molecules responsible for hMSC functional activity related to the immunological responses involved with diseases such as GVHD, solid organ transplantation and autoimmune diseases.