Paper Title
HIV PROMOTES ATHEROSCLEROSIS VIA CIRCULATING EXTRACELLULAR VESICLE MICRORNAS TARGETING MRNA EXPRESSION IN ENDOTHELIAL PROGENITOR CELLS (SHORT TITLE: EXTRACELLULAR MICRORNAS IN HIV-ATHEROSCLEROSIS)

Abstract
Rationale: People living with HIV (PLHIV), even withundetectable viral load, have a significantly higher risk of cerebrocardiovascular diseases (CVD) than HIV negative(HIVneg) subjects. The mechanisms underlying thiselevated CVD risk remain elusive. Extracellular vesicles (EVs) mediate cell-to-cell communication through the transfer of bioactive molecules, including microRNAs (miRs).We hypothesized thatHIV infection result in the release of plasma EVs with modified cargo, which confer the elevated CVD risk. Objective: To determine if plasma EVs, specifically their miR cargo,were responsible for the increased CVD risk in PLHIV. Methods and Results: 73 PLHIV and 25 HIVneg subjects were studied. PLHIV had increased atherosclerosis burden as assessed using carotid intima-media thickness (cIMT, 1.02 vs 0.71, P<0.05) by Doppler ultrasound, and decreased circulatingendothelial colony forming cell (ECFC)/ endothelial progenitor cell (EPC) levels (2.39 vs 5.76 CFU-F, P<0.001). Plasma from PLHIV was fractionated into EVs (HIVposEVs) and plasma-depleted of EVs (HIVplasmadepEVs) and injected weekly into atherogenic apoE-/- mice for 3 months. HIVposEVs, but not HIVplasmadepEVsor EVs derived from HIV negative subjects (HIVnegEVs) increased atherosclerosis plaque burden compared to saline control. Increased atherogenesis was accompanied byelevated senescence and apoptosis of endothelial and smooth muscle cells in the arterial wall, as well as senescence and impaired functionality of bone marrow EPCs. Small RNA-seq analysis identified candidate EV-miRs that are overrepresented in HIVposEVsvs.HIVnegEVs. These include let-7b-5p and miR-103a. Tailored EVs (TEVs) loaded withthe antagomirs for let-7b-5p (miRZip-let-7b) or miR-103a (miRZip-103a) were generated by overexpressing the respective antagomirs in bone marrow mesenchymal stromal cells (MSCs). These TEVspartially counteracted the effects of HIVposEVson the accelerated senescence ofbone marrow EPCs and aortic ECs, whereas EVs overexpressinglet-7b recapitulated in part the effects of HIVposEVs.Remarkably, EPCs and ECs overexpressing HMGA2(the let-7b-5p target gene) with 3’UTR deletion and resistant to let-7b regulationshowed protection against HIVposEVs-induced senescent and functional changes—an effect thatwas similar to miRZip-let-7bTEVs. Conclusion: Circulating EVs mediate the effects of HIV infection on accelerated atherosclerosis via altered EV-miR cargo, including upregulation of let-7b-5p, whichdownregulates HMGA2 expression in EPCs and ECs, resulting in their senescence and consequent functional impairment, contributing to vascular senescence and atherosclerosis development. NON-STANDARD ABBREVIATIONS AND ACRONYMS ART: Antiretroviral therapy. BM: Bone marrow. CFU: Colony forming units. cIMT: Carotid intima-media thickness. CVD: Cerebrocardiovascular disease. ECs: Endothelial cells. EPCs: Endothelial progenitor cells. EV-miRs: extracellular vesicles microRNAs. EVs: Extracellular vesicles. FDR: False discovery rate. HIVnegEVs: Extracellular vesicles from HIV negative plasma. HIVposEVs: Extracellular vesicles from PLHIV plasma. HIVplasmadepEVs: HIV plasmadepleted of EVs IACUC: Institutional Animal Care and Use Committee. IPA: Ingenuity Pathway Analysis. miRs: microRNAs. MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide. NTA: Nanoparticle Tracking Analysis. ORO: Oil Red O. PLHIV: People Living with HIV. SMCs: Smooth muscle cells. sRNA-Seq: small RNA Sequencing. TEVs: Tailored extracellular vesicles. TUNEL: Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling. Β-gal: β-galactosidase.