Extracellular Vesicles as Delivery Systems in Disease Therapy.

Extracellular vesicles (EVs)/exosomes are nanosized membrane-bound structures that are released by virtually all cells. EVs have attracted great attention in the scientific community since the discovery of their roles in cell-to-cell communication. EVs' enclosed structure protects bioactive molecules from degradation in the extracellular space and targets specific tissues according to the topography of membrane proteins. Upon absorption by recipient cells, EV cargo can modify the transcription machinery and alter the cellular functions of these cells, playing a role in disease pathogenesis. EVs have been tested as the delivery system for the mRNA COVID-19 vaccine. Recently, different therapeutic strategies have been designed to use EVs as a delivery system for microRNAs and mRNA. In this review, we will focus on the exciting and various platforms related to using EVs as delivery vehicles, mainly in gene editing using CRISPR/Cas9, cancer therapy, drug delivery, and vaccines. We will also touch upon their roles in disease pathogenesis.

HIV Promotes Atherosclerosis via Circulating Extracellular Vesicle MicroRNAs.

People living with HIV (PLHIV) are at a higher risk of having cerebrocardiovascular diseases (CVD) compared to HIV negative (HIVneg) individuals. The mechanisms underlying this elevated risk remains elusive. We hypothesize that HIV infection results in modified microRNA (miR) content in plasma extracellular vesicles (EVs), which modulates the functionality of vascular repairing cells, i.e., endothelial colony-forming cells (ECFCs) in humans or lineage negative bone marrow cells (lin- BMCs) in mice, and vascular wall cells. PLHIV (N = 74) have increased atherosclerosis and fewer ECFCs than HIVneg individuals (N = 23). Plasma from PLHIV was fractionated into EVs (HIVposEVs) and plasma depleted of EVs (HIV PLdepEVs). HIVposEVs, but not HIV PLdepEVs or HIVnegEVs (EVs from HIVneg individuals), increased atherosclerosis in apoE-/- mice, which was accompanied by elevated senescence and impaired functionality of arterial cells and lin- BMCs. Small RNA-seq identified EV-miRs overrepresented in HIVposEVs, including let-7b-5p. MSC (mesenchymal stromal cell)-derived tailored EVs (TEVs) loaded with the antagomir for let-7b-5p (miRZip-let-7b) counteracted, while TEVs loaded with let-7b-5p recapitulated the effects of HIVposEVs in vivo. Lin- BMCs overexpressing Hmga2 (a let-7b-5p target gene) lacking the 3'UTR and as such is resistant to miR-mediated regulation showed protection against HIVposEVs-induced changes in lin- BMCs in vitro. Our data provide a mechanism to explain, at least in part, the increased CVD risk seen in PLHIV.

MicroRNA-423-5p Mediates Cocaine-Induced Smooth Muscle Cell Contraction by Targeting Cacna2d2.

Cocaine abuse increases the risk of atherosclerotic cardiovascular disease (CVD) and causes acute coronary syndromes (ACS) and hypertension (HTN). Significant research has explored the role of the sympathetic nervous system mediating the cocaine effects on the cardiovascular (CV) system. However, the response of the sympathetic nervous system alone is insufficient to completely account for the CV consequences seen in cocaine users. In this study, we examined the role of microRNAs (miRNAs) in mediating the effect of cocaine on the CV system. MiRNAs regulate many important biological processes and have been associated with both response to cocaine and CV disease development. Multiple miRNAs have altered expression in the CV system (CVS) upon cocaine exposure. To understand the molecular mechanisms underlying the cocaine response in the CV system, we studied the role of miRNA-423-5p and its target Cacna2d2 in the regulation of intracellular calcium concentration and SMC contractility, a critical factor in the modulation of blood pressure (BP). We used in vivo models to evaluate BP and aortic stiffness. In vitro, cocaine treatment decreased miR-423-5p expression and increased Cacna2d2 expression, which led to elevated intracellular calcium concentrations and increased SMC contractility. Overexpression of miR-423-5p, silencing of its target Cacna2d2, and treatment with a calcium channel blocker reversed the elevated SMC contractility caused by cocaine. In contrast, suppression of miR-423-5p increased the intracellular calcium concentration and SMC contractibility. In vivo, smooth muscle-specific overexpression of miR-423-5p ameliorated the increase in BP and aortic stiffness associated with cocaine use. Thus, miR-423-5p regulates SMC contraction by modulating Cacna2d2 expression increasing intracellular calcium concentrations. Modulation of the miR-423-5p-Cacna2d2-Calcium transport pathway may represent a novel therapeutic strategy to improve cocaine-induced HTN and aortic stiffness.

Human adipose-derived stromal/stem cells are distinct from dermal fibroblasts as evaluated by biological characterization and RNA sequencing.

Human adipose-derived stromal/stem cells (ASC) have immunomodulatory properties and the potential to differentiate into several cell lines, important for application in regenerative medicine. However, the contamination with dermal fibroblasts (FIB) can impair the beneficial effects of ASC in cell therapy. It is then essential to develop new strategies that contribute to the distinction between these two cell types. In this study, we performed functional assays, high-throughput RNA sequencing (RNA-Seq) and quantitative PCR (qPCR) to find new markers that can distinguish ASC and FIB. We showed that ASC have adipogenic and osteogenic differentiation capacity and alkaline phosphatase activity, not observed in FIB. Gene expression variation analysis identified more than 2000 differentially expressed genes (DEG) between these two cell types. We validated 16 genes present in the list of DEG, including the alkaline phosphatase gene (ALPL). In conclusion, we showed that ASC and FIB have distinct biological properties as demonstrated by alkaline phosphatase activity and differentiation capacity, besides having different gene expression profiles. SIGNIFICANCE OF THE STUDY: Although many differences between stromal stem cells derived from human adipose tissue (ASC) and human dermal fibroblasts (FIB) are described, it is still difficult to find specific markers to differentiate them. This problem can interfere with the therapeutic use of ASC. This work aimed to find new markers to differentiate these two cell populations. Our findings suggest that these cells can be distinguished by biological and molecular characteristics, such as adipogenic and osteogenic differentiation, alkaline phosphatase activity and differential gene expression profiles. The DEG were related to the regulation of the cell cycle, development process, structural organization of the cell and synthesis of the extracellular matrix. This study helps to find new cellular markers to distinguish the two populations and to better understand the properties of these cells, which can improve cell therapy.

Rejuvenation of Senescent Endothelial Progenitor Cells by Extracellular Vesicles Derived From Mesenchymal Stromal Cells.

Mesenchymal stromal cell (MSC) transplantation is a form of the stem-cell therapy that has shown beneficial effects for many diseases. The use of stem-cell therapy, including MSC transplantation, however, has limitations such as the tumorigenic potential of stem cells and the lack of efficacy of aged autologous cells. An ideal therapeutic approach would keep the beneficial effects of MSC transplantation while circumventing the limitations associated with the use of intact stem cells. This study provides proof-of-concept evidence that MSC-derived extracellular vesicles represent a promising platform to develop an acellular therapeutic approach that would just do that. Extracellular vesicles are membranous vesicles secreted by MSCs and contain bioactive molecules to mediate communication between different cells. Extracellular vesicles can be taken up by recipient cells, and once inside the recipient cells, the bioactive molecules are released to exert the beneficial effects on the recipient cells. This study, for the first time to our knowledge, shows that extracellular vesicles secreted by MSCs recapitulate the beneficial effects of MSCs on vascular repair and promote blood vessel regeneration after ischemic events. Furthermore, MSCs from aged donors can be engineered to produce extracellular vesicles with improved regenerative potential, comparable to MSCs from young donors, thus eliminating the need for allogenic young donors for elderly patients.