However, no studies have examined the impact of cART or other substances, including THC, used by PLWH, on the presence of exmiRNA, or its link with extracellular vesicles (EVs) and extracellular components (ECs). Besides, the temporal evolution of exmiRNA levels after SIV infection, after THC treatment, cART treatment, or the treatment using both THC and cART therapy remain uncertain. We serially analyzed microRNAs (miRNAs) linked to extracellular vesicles (EVs) derived from blood plasma 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. A superior separation of EVs and ECs was achieved with the PPLC nano-particle purification tool, a cutting-edge technology featuring gradient agarose bead sizes and a rapid fraction collector, which yielded preparative quantities of sub-populations of extracellular structures with high resolution. RealSeq Biosciences' (Santa Cruz, CA) custom sequencing platform, specializing in small RNA sequencing (sRNA-seq), was employed to identify the global miRNA profiles of the paired endothelial cells (ECs) and extracellular vesicles (EVs). The sRNA-seq data's analysis leveraged the application of several bioinformatics tools. Key exmiRNA validation employed specific TaqMan microRNA stem-loop RT-qPCR assays. read more The effect of cART, THC, or their combined administration on the concentration and localization of blood plasma exmiRNA within extracellular vesicles and endothelial cells was investigated in 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. germline epigenetic defects The disparate effects of cART and THC therapies are clearly reflected in the exmiRNA enrichment and compartmentalization patterns. A reduction in the levels of 12 EV-associated and 15 EC-associated miRNAs was statistically significant in the VEH/SIV/cART study group. Within the VEH/SIV/ART group, blood concentrations of EV-associated miR-206, a muscle-specific miRNA, were superior to those in the VEH/SIV group. Significant downregulation of ExmiR-139-5p, a microRNA implicated in endocrine resistance, focal adhesion, lipid metabolism, atherosclerosis, apoptosis, and breast cancer, was observed in the VEH/SIV/cART group relative to the VEH/SIV group, regardless of the tissue compartment as assessed by miRNA-target enrichment analysis. Concerning the effects of THC treatment, 5 EV-connected and 21 EC-connected miRNAs showed a considerable reduction in the VEH/THC/SIV group. A comparative analysis of EV-associated miR-99a-5p levels revealed a higher concentration in the VEH/THC/SIV group relative to the VEH/SIV group. Conversely, a statistically significant decrease in miR-335-5p was seen in both EVs and ECs of the THC/SIV group in contrast to the VEH/SIV group. EVs from the SIV/cART/THC group exhibited a significant increase in the number 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), each being far greater in number in comparison to the EV samples from the VEH/SIV/cART group. Eight miRNAs identified through miRNA-target enrichment analyses are implicated in endocrine resistance, focal adhesions, lipid metabolism and atherosclerosis, apoptosis, breast cancer, and addiction to cocaine and amphetamines. In electric vehicles and electric cars, combined THC and cART treatments showed a substantial increase in the observed number of miR-139-5p molecules when compared to the VEH/SIV control group. Persistent host responses to infection or treatments, as evidenced by significant alterations in host microRNAs (miRNAs) within both extracellular vesicles (EVs) and endothelial cells (ECs) from untreated and treated (with cART, THC, or both) rheumatoid models (RMs), persist despite cART's viral load reduction and THC's anti-inflammatory effects. To further investigate the pattern of microRNA alterations within extracellular vesicles and endothelial cells, and to explore potential causal relationships, we performed a longitudinal analysis of miRNA profiles, measured at one and five months post-infection (MPI). In SIV-infected macaques, we identified miRNA signatures associated with THC or cART treatment, present in both extracellular vesicles and endothelial cells. Across all experimental groups (VEH/SIV, SIV/cART, THC/SIV, THC/SIV/cART, and THC), endothelial cells (ECs) demonstrated a greater number of microRNAs (miRNAs) than extracellular vesicles (EVs), as measured longitudinally from 1 MPI to 5 MPI. The application of cART and THC treatments demonstrated a longitudinal impact on both the amount and compartmentalization of ex-miRNAs in both carriers. Manuscript 1 reveals that SIV infection caused a longitudinal decrease in EV-associated miRNA-128-3p. However, administering cART to SIV-infected RMs did not increase miR-128-3p, but instead caused a longitudinal elevation in six other EV-associated miRNAs, including miR-484, miR-107, miR-206, miR-184, miR-1260b, and miR-6132. The combination therapy of THC and cART in SIV-infected RMs resulted in a longitudinal reduction in three EV-associated miRNAs (miR-342-3p, miR-100-5p, miR-181b-5p) and a longitudinal elevation of three EC-associated miRNAs (miR-676-3p, miR-574-3p, miR-505-5p). The dynamic nature of miRNAs in SIV-infected RMs may potentially indicate disease progression, whereas similar dynamic variations in miRNAs in the cART and THC Groups may be suggestive of treatment effectiveness. The paired analyses of EVs and ECs miRNAomes presented a comprehensive, cross-sectional and longitudinal view of how the host exmiRNA system reacts to SIV infection, considering the effects of THC, cART, and the combined treatments on the miRNAome. Our findings, viewed collectively, highlight previously unidentified alterations in the exmiRNA composition of blood plasma following exposure to SIV. Analysis of our data reveals that cART and THC treatment, used alone or together, might impact the quantity and localization of various exmiRNAs implicated in a range of diseases and biological functions.

Commencing the two-part series is Manuscript 1, the first manuscript in this study. This report details the results of our initial studies on the presence and distribution of extracellular microRNAs (exmiRNAs), particularly within blood plasma extracellular vesicles (EVs) and extracellular condensates (ECs), in individuals with untreated HIV/SIV infection. The goals of this manuscript (Manuscript 1) include (i) determining the concentration and cellular location of exmiRNAs in extracellular vesicles (EVs) and endothelial cells (ECs) in a healthy uninfected state and (ii) assessing the consequences of SIV infection on the abundance and compartmentalization of exmiRNAs in these cellular structures. Significant attention has been given to the epigenetic regulation of viral infections, especially the role of exmiRNAs in controlling viral disease progression. Cellular processes are modulated by microRNAs (miRNAs), which are small non-coding RNA molecules, around 20-22 nucleotides long, achieving this regulation by targeting mRNA for degradation or suppressing the initiation of protein synthesis. Despite their initial association with the cellular microenvironment, circulating microRNAs are now recognized in a variety of extracellular locales, including blood serum and plasma. Ribonucleases are prevented from degrading circulating microRNAs (miRNAs) due to their complex with lipid and protein carriers, such as lipoproteins and other extracellular compartments, including extracellular vesicles (EVs) and extracellular components (ECs). Various biological processes and diseases, including cell proliferation, differentiation, apoptosis, stress responses, inflammation, cardiovascular diseases, cancer, aging, neurological diseases, and the pathogenesis of HIV/SIV, are impacted by the functional roles of miRNAs. While the function of lipoproteins and exmiRNAs, which are frequently associated with extracellular vesicles, has been explored in relation to various disease states, a connection between exmiRNAs and endothelial cells has not been established. The effect of SIV infection on the quantity and arrangement of exmiRNAs inside extracellular particles is presently unknown. Academic publications on electric vehicles (EVs) have shown that a considerable number of circulating microRNAs (miRNAs) are probably not connected with EVs. A methodical investigation into the means of exmiRNA transport has not been performed due to the difficulty in separating exosomes from other extracellular particles, including endothelial cells. Brain biomimicry Paired EVs and ECs were subsequently separated from the EDTA blood plasma of SIV-uninfected male Indian rhesus macaques (RMs, n = 15). Subsequently, paired EVs and ECs were also isolated from the EDTA blood plasma of cART-naive SIV-infected (SIV+, n = 3) RMs at two time points: one month and five months post-infection (1 MPI and 5 MPI). With the aid of PPLC, a groundbreaking, innovative technology incorporating gradient agarose bead sizes and a high-throughput fraction collector, the separation of EVs and ECs was achieved. This method efficiently provides high-resolution separation and retrieval of preparative 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. Analysis of the sRNA-seq data was conducted using a variety of bioinformatic tools. Employing specific TaqMan microRNA stem-loop RT-qPCR assays, key exmiRNAs were validated. Our findings demonstrate that exmiRNAs within blood plasma are not confined to a single extracellular particle type, but rather are linked to both lipid-based carriers (EVs) and non-lipid-based carriers (ECs), with a notable (approximately 30%) portion of the exmiRNAs found in association with ECs.