Examining the role of extacellular vesicles in blood pressure regulation

Abstract

Extracellular vesicles (EVs) are membrane-enclosed structures that aid in intercellular communication and other biological processes. Recent studies implicate EVs in various acute and chronic disease states, including hypertension. We have previously demonstrated that the gut microbiota plays a causal role in the pathogenesis of hypertension. However, the mechanisms by which the microbiota influences host blood pressure are largely unknown. We hypothesized that EVs act as mediators between the gut microbiota and host, exerting bidirectional effects to regulate blood pressure. For our experiments, we used the spontaneously hypertensive stroke prone rat (SHRSP), a well-known animal model of essential hypertension, and its corresponding normotensive control, the Wistar-Kyoto rat (WKY). To demonstrate the impact of EVs on host blood pressure, we transplanted plasma EVs from SHRSP and WKY donors into SHRSP and WKY recipients via intravenous injection. Treatment duration was 4 weeks, during which systolic blood pressure (SBP) measurements were taken. In SHRSP, WKY-EVs had a statistically significant SBP lowering effect over time compared with SHRSP-EVs (p=0.03), with a reduction of SBP of 23 mmHg at 4 weeks (166 ± 1 vs. 189 ± 3 mmHg; p=0.05). In WKYs, EV type had a significant main effect on SBP (p=0.05), with those receiving SHRSP-EVs exhibiting elevated SBP. We then performed flow cytometry analysis on various tissues from the treatment groups to investigate if immune and inflammatory processes could underlie EV action. We found that SHRSP that received SHRSP-EVs (SHRSP control) had increased Th17 cells (p<0.0001) and decreased regulatory T (Treg) cells (p<0.05) in ileum compared to WKY that received WKY-EVs (WKY control). These results are consistent with previous reports citing Th17/Treg imbalance toward Th17 dominance as a critical factor in hypertension. Interestingly, WKY-EV treatment in SHR restored Th17/Treg balance, decreasing Th17 numbers in brain (p<0.0001), ileum (p<0.0001) and cecum (p<0.01) and increasing Treg numbers in ileum (p<0.001) compared to SHRSP control. Conversely, WKY that received SHR-EVs had fewer Tregs in ileum (p<0.05) compared to WKY control, demonstrating that SHRSP-EV treatment in WKY resulted in deleterious inflammatory effects on gut. Finally, to examine if the effects of EVs on SBP may be mediated through alteration of the gut microbiota, we administered SHRSP-EVs or WKY-EVs to germ free rats lacking a gut microbiota. Germ free rats receiving SHRSP-EVs exhibited significantly elevated SBP as compared to those receiving WKY-EVs (150 ± 5 vs. 129 ± 4 mmHg; p=0.01). This finding further demonstrates the SBP elevating effect of SHRSP-EVs, and suggests EV alteration of the gut microbiota is not required to influence host blood pressure. Future studies will seek to establish how microbiota may alter circulating EV profile and function to promote the pathogenesis of hypertension. Overall, our data suggest that EVs play an important role in regulation of host blood pressure and gut inflammation, in part by alteration of the Th17/Treg balance.