The role of extracellular vesicle-associated MicroRNAs in HIV-associated atherosclerosis

艾滋病毒携带者(PLWH)患心血管疾病(CVD)的风险显著高于 给那些艾滋病毒阴性的同龄人。然而，驱动这一现象的潜在机制增加了心血管疾病的风险。 仍然难以捉摸。胞外囊泡(EV)/外切体是由所有细胞释放的，广泛分布于 血浆、尿液和其他生物液体。电动汽车含有多种生物活性分子，包括mRNAs， MicroRNAs(MiRs)、蛋白质和脂类，它们可以转移到目标细胞，改变它们的功能。 EVS的货物反映了宿主细胞的病理生理状态。可以想象，潜伏的艾滋病毒 在早期感染、长期接受抗逆转录病毒治疗(ART)和慢性 免疫激活/炎症(出于赠款撰写目的称为HIV阳性)可能会影响宿主 细胞，特别是外周血中的细胞，改变血浆EV的释放和运输。这些电动汽车 进而可以作为细胞间通讯的信息载体，影响动脉壁的细胞， 包括内皮细胞(ECs)和平滑肌细胞(SMC)以及内皮祖细胞 (EPC)在骨髓中，促进动脉粥样硬化的形成。为了验证这一假设，我们将血浆从 抗逆转录病毒治疗中病毒载量无法检测到的血浆EVS(HIVposEV)和血浆耗尽EVS 并将这些组分注射到致动脉粥样硬化的apoE-/-小鼠体内。值得注意的是，艾滋病毒，但不是 与从HIV阴性的EV分离的EV相比，HIV血浆DEPEV增加了动脉粥样硬化斑块负担 受试者(HIV-Ev)和PBS。这些变化伴随着衰老和细胞凋亡的增加。 动脉壁内皮细胞和平滑肌细胞的数量减少，骨髓内皮祖细胞的活性和功能受损。这些 令人兴奋的数据支持这一假设，即来自PLWH的循环EV重建了HIV阳性的影响 (无活动性艾滋病毒感染)动脉粥样硬化。HIVposEV和HIVNegEV中的RNA-seq显示差异miR 配置文件。EV-miRs在PLWH中过度表达的靶基因在心血管病理中的丰富 小路。我们设计了定制的电动汽车(TEV)，装载了LET-7B-5P和LET-7B-5P的对角 MiR103a-2，两个候选EV-miR通过转导间充质细胞在HIVposEV中高表达。 这些TEV抵消了HIVposEV对EPC加速衰老的影响，而let-7b- 5P过表达的EV在一定程度上概括了HIVposEV的作用。此外，EPC过度表达 HMGA2(顶级let-7b-5p靶基因)缺乏3‘UTR(抵抗miR调节)显示出保护作用 类似于miRZip-let-7b TEV对抗HIVposEvs诱导的衰老。在这份提案中，我们将确定是否 设计用来调节关键的HIVposEV相关MIR的TEV可以抵消慢性 炎症和免疫激活、抗逆转录病毒治疗和与艾滋病毒阳性相关的潜伏的艾滋病毒蓄积物 动脉粥样硬化的发展。此外，我们还将进一步剖析这些不利因素背后的机制。 通过检测HIV阳性对心血管系统的影响来研究HIVposEV和/或TEV对 内皮细胞、平滑肌细胞和内皮祖细胞的功能和转录组。我们将确定可能的miR-mRNA通路。 重要的是，我们将使用TEV、DIRECT MIR在PLWH的EPC样本中验证我们的鼠标发现 拮抗作用，以及靶基因过度表达的方法。

People living with HIV (PLWH) have a significantly higher risk of cardiovascular diseases (CVD) compared to their HIV negative counterparts. However, the underlying mechanisms that drive this increased CVD risk remain elusive. Extracellular vesicles (EVs)/exosomes are released by all cells and widely distributed in plasma, urine, and other biological fluids. EVs contain diverse bioactive molecules, including mRNAs, microRNAs (miRs), proteins, and lipids, which can be transferred to target cells altering their functionality. The cargo of EVs reflects the pathophysiological states of the host cells. It is conceivable that the latent HIV reservoir established during early infection, long-term exposure to antiretroviral therapy (ART), and chronic immune activation/inflammation (referred to as HIV positivity for grant writing purpose) may impact host cells, particularly those in the peripheral blood, altering the release and the cargo of plasma EVs. These EVs in turn may serve as information vehicles for cell-to-cell communication, affecting cells of the arterial wall, including endothelial cells (ECs) and smooth muscle cells (SMCs), as well as endothelial progenitor cells (EPCs) in the bone marrow, promoting atherogenesis. To test this hypothesis, we fractionated plasma from PLWH on ART with undetectable viral load into plasma EVs (HIVposEVs) and plasma-depleted of EVs (HIVplasmadepEVs) and injected these fractions into atherogenic apoE-/- mice. Remarkably, HIVposEVs, but not HIVplasmadepEVs, increased atherosclerosis plaque burden compared to EVs isolated from HIV negative subjects (HIVnegEVs) and PBS. These changes were accompanied by increased senescence and apoptosis of ECs and SMCs in the arterial wall and impaired viability and functionality of bone marrow EPCs. These exciting data support the hypothesis that circulating EVs from PLWH reconstitute the effects of HIV positivity (without active HIV infection) on atherosclerosis. RNA-seq in HIVposEVs and HIVnegEVs revealed differential miR profiles. The target genes of EV-miRs overrepresented in PLWH are enriched in cardiovascular pathology pathways. We have engineered tailored EVs (TEVs) that are loaded with the antagomirs for let-7b-5p and miR103a-2, two candidate EV-miRs overrepresented in HIVposEVs by transducing mesenchymal stromal cells. These TEVs counteracted the effects of HIVposEVs on the accelerated senescence of EPCs, whereas let-7b- 5p overexpressing EVs recapitulated, in part, the effects of HIVposEVs. Furthermore, EPCs overexpressing HMGA2 (a top let-7b-5p target gene) lacking the 3'UTR (resistant to miR regulation) showed protection similar to miRZip-let-7b TEVs against HIVposEVs-induced senescence. In this proposal, we will determine if TEVs engineered to modulate key HIVposEVs-associated miRs counteract the effects of the chronic inflammation and immune activation, ART, and latent HIV reservoir associated with HIV positivity on atherosclerosis development. In addition, we will further dissect the mechanisms underlying the adverse effect of HIV positivity on the cardiovascular system by examining the effects of HIVposEVs and/or TEVs on the functionality and transcriptome of ECs, SMCs and EPCs. We will identify putative miR-mRNA pathways. Importantly, we will validate our mouse findings in EPC samples from PLWH using TEVs, direct miR antagonism, and target gene overexpression approaches.