An absence of studies precludes understanding the effects of cART or other substances, including THC, used by individuals with HIV, on the abundance of exmiRNA and their associations with extracellular vesicles and extracellular components (ECs). Additionally, the evolution of exmiRNA levels throughout the course of SIV infection, THC treatment, cART treatment, or the combined THC and cART treatment remains uncertain. We sequentially assessed microRNAs (miRNAs) in blood plasma-derived extracellular vesicles (EVs) and endothelial cells (ECs). The EDTA blood plasma of male Indian rhesus macaques (RMs) was partitioned into five treatment groups, each encompassing paired EVs and ECs—VEH/SIV, VEH/SIV/cART, THC/SIV, THC/SIV/cART, or THC alone. The separation of EVs and ECs, a critical process, was accomplished by employing the PPLC nano-particle purification tool, a state-of-the-art technology featuring gradient agarose bead sizes and a fast fraction collector, ensuring the collection of preparative quantities of sub-populations of extracellular structures with high resolution. Small RNA sequencing (sRNA-seq), performed on custom sequencing platforms provided by RealSeq Biosciences (Santa Cruz, CA), was utilized to determine the global miRNA profiles of the paired extracellular vesicles (EVs) and endothelial cells (ECs). Bioinformatic tools were employed to analyze the sRNA-seq data. The validation of key exmiRNA was undertaken using specific TaqMan microRNA stem-loop RT-qPCR assays. BIIB129 concentration We studied the effect of cART, THC, or their combined administration on the presence and cellular arrangement of blood plasma exmiRNA in extracellular vesicles and endothelial cells from SIV-infected RMs. In our follow-up study (Manuscript 1 of this series, detailing that ~30% of exmiRNAs were within uninfected RMs), we verify the existence of exmiRNAs in both lipid-based carriers (EVs) and non-lipid-based carriers (ECs). The association levels for exmiRNAs in EVs are 295% to 356%, while the levels for ECs are 642% to 705%, respectively. rapid biomarker The distinct influence of cART and THC treatments on the exmiRNA enrichment and compartmentalization patterns is noteworthy. Within the VEH/SIV/cART cohort, a substantial decrease was seen in 12 EV-related and 15 EC-related miRNAs. Circulating levels of the muscle-specific miRNA, EV-associated miR-206, were significantly higher in the VEH/SIV/ART group in comparison to the VEH/SIV group. MiRNA-target enrichment analysis highlighted ExmiR-139-5p's role in endocrine resistance, focal adhesion, lipid and atherosclerosis processes, apoptosis, and breast cancer; its levels were considerably lower in the VEH/SIV/cART group compared to the VEH/SIV group, in all tissue compartments examined. In the case of THC treatment, 5 EV-correlated and 21 EC-correlated miRNAs were notably diminished in the VEH/THC/SIV group. The VEH/THC/SIV group showed a higher presence of EV-associated miR-99a-5p compared to the VEH/SIV group, exhibiting a distinct contrast to the significant reduction of miR-335-5p counts in both EVs and ECs of the THC/SIV group when juxtaposed with the VEH/SIV group. A noteworthy surge in the quantity of eight miRNAs (miR-186-5p, miR-382-5p, miR-139-5p, miR-652, miR-10a-5p, miR-657, miR-140-5p, and miR-29c-3p) was detected in EVs from the SIV/cART/THC treatment group, which was significantly greater than the levels in the VEH/SIV/cART group. MiRNA-target enrichment analysis indicated that a group of eight miRNAs play a role in endocrine resistance, focal adhesions, lipid metabolism and atherosclerosis, apoptosis, breast cancer, and addiction to cocaine and amphetamine. In electric cars and electric vehicles, concurrent THC and cART treatment resulted in a noticeably greater concentration of miR-139-5p relative to the control group of vehicle/SIV. Untreated and treated (cART, THC, or both) rheumatoid models (RMs) demonstrate persistent host responses to infection or treatments, evidenced by significant shifts in host microRNAs (miRNAs) within both extracellular vesicles (EVs) and endothelial cells (ECs), despite viral load suppression by cART and inflammation reduction by THC. To expand our understanding of miRNA alterations in extracellular vesicles and endothelial cells, and to investigate potential cause-and-effect relationships, we implemented a longitudinal miRNA profiling analysis, measuring miRNAs at both one and five months post-infection (MPI). MiRNA profiles tied to THC or cART treatment of SIV-infected macaques were observed in extracellular vesicles and endothelial cells. In all experimental cohorts (VEH/SIV, SIV/cART, THC/SIV, THC/SIV/cART, and THC), endothelial cells (ECs) displayed a substantially larger miRNA count compared to extracellular vesicles (EVs) during the longitudinal assessment from the first to fifth month post-initiation (MPI). Moreover, treatment with cART and THC demonstrated a longitudinal impact on the abundance and compartmentalization pattern of ex-miRNAs in the two types of carriers. A longitudinal study in Manuscript 1 showed that SIV infection decreased EV-associated miRNA-128-3p. Surprisingly, administering cART to SIV-infected RMs did not elevate miR-128-3p; rather, it caused a longitudinal increase in six other EV-associated miRNAs: miR-484, miR-107, miR-206, miR-184, miR-1260b, and miR-6132. Moreover, the administration of cART to THC-treated SIV-infected RMs exhibited a longitudinal decline in three EV-associated miRNAs (miR-342-3p, miR-100-5p, and miR-181b-5p), alongside a corresponding longitudinal increase in three EC-associated miRNAs (miR-676-3p, miR-574-3p, and miR-505-5p). Longitudinal miRNA alterations in SIV-infected RMs could signal disease progression, but similar alterations in the cART and THC groups could indicate a response to the treatment. Through paired analyses of EVs and ECs miRNAomes, this study provides a comprehensive cross-sectional and longitudinal report on host exmiRNA responses to SIV infection and how THC, cART, or a combination of both, affects the miRNAome during the course of SIV infection. In a general assessment, our collected data indicate novel changes in the exmiRNA profile of blood plasma subsequent to SIV infection. Our data further suggest that cART and THC treatments, both individually and in tandem, can modify the abundance and compartmentalization of multiple exmiRNAs associated with diverse diseases and biological processes.
Within this two-part series, this is the introductory manuscript, Manuscript 1. Our initial investigations into the concentration and spatial distribution of blood plasma extracellular microRNAs (exmiRNAs) within extracellular entities, such as blood plasma extracellular vesicles (EVs) and extracellular condensates (ECs), are presented in this report, specifically focusing on the context of untreated HIV/SIV infection. This study (Manuscript 1) proposes to (i) evaluate the abundance and cellular compartmentalization of exmiRNAs within extracellular vesicles and endothelial cells in a healthy, uninfected context, and (ii) assess how SIV infection influences the concentration and compartmentalization of exmiRNAs within these cellular components. A considerable amount of work has been undertaken in investigating the epigenetic control of viral infections, especially with regard to the crucial role played by exmiRNAs in the development of viral diseases. Regulating cellular processes is the function of microRNAs (miRNAs), small non-coding RNA molecules, approximately 20-22 nucleotides long, which exert their influence by either degrading targeted messenger RNA or repressing protein translation. Despite their initial association with the cellular microenvironment, circulating microRNAs are now recognized in a variety of extracellular locales, including blood serum and plasma. MicroRNAs (miRNAs), during their time in the circulatory system, are protected from ribonuclease-mediated degradation by virtue of their association with lipid and protein carriers, including lipoproteins and various extracellular entities like exosomes and extracellular components. MiRNAs demonstrably participate in numerous biological processes and diseases such as cell proliferation, differentiation, apoptosis, stress responses, inflammation, cardiovascular diseases, cancer, aging, neurological diseases, and the pathology of HIV/SIV infections. Research on the roles of lipoproteins and EV-associated exmiRNAs in various disease processes has progressed significantly; nevertheless, the connection between exmiRNAs and endothelial cells is still an area of investigation. Correspondingly, the effect of SIV infection on the presence and spatial arrangement of exmiRNAs in extracellular vesicles is unknown. Electric vehicle (EV) research suggests that a large proportion of circulating miRNAs might not be associated with EVs. The carriers of exmiRNAs have not been systematically analyzed, due to the lack of a robust method for distinguishing exosomes from other extracellular particles, including endothelial cells. Cytokine Detection The EDTA blood plasma of 15 SIV-uninfected male Indian rhesus macaques (RMs) was processed to isolate paired EVs and ECs. Extracellular vesicles (EVs) and exosomes (ECs) were isolated from EDTA blood plasma from SIV-infected (SIV+, n = 3) RMs not receiving cART at two time points post-infection, one month (1 MPI) and five months (5 MPI). Gradient agarose bead sizes and a high-speed fraction collector, integral components of the innovative PPLC technology, were critical for separating EVs and ECs. This resulted in high-resolution separation and recovery of significant quantities of sub-populations of extracellular particles. Employing small RNA sequencing (sRNA-seq) on a custom sequencing platform from RealSeq Biosciences (Santa Cruz, CA), the global miRNA profiles of the matched extracellular vesicles (EVs) and endothelial cells (ECs) were determined. Bioinformatic tools were employed to analyze the sRNA-seq data. Using specific TaqMan microRNA stem-loop RT-qPCR assays, the validation of key exmiRNAs was carried out. Results from our investigation show that exmiRNAs in blood plasma are not confined to a particular type of extracellular particle but instead co-occur with both lipid-based carriers (EVs) and non-lipid-based carriers (ECs), with a statistically significant proportion (~30%) observed in association with ECs.