Extracellular Vesicles: How a Circulating Biomarker Can Double As a Regulator of Blood Pressure.

Abstract

HomeHypertensionVol. 75, No. 1Extracellular Vesicles Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBExtracellular VesiclesHow a Circulating Biomarker Can Double As a Regulator of Blood Pressure Pooneh Bagher Pooneh BagherPooneh Bagher Correspondence to Pooneh Bagher, 8447 Riverside Parkway, Bryan, TX 77807. Email E-mail Address: [email protected] From the Department of Medical Physiology, Texas A&M University Health Science Center, Bryan, Texas. Search for more papers by this author Originally published25 Nov 2019https://doi.org/10.1161/HYPERTENSIONAHA.119.13549Hypertension. 2020;75:40–43This article is a commentary on the followingCirculating Extracellular Vesicles in Normotension Restrain Vasodilation in Resistance ArteriesOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: November 25, 2019: Ahead of Print See related article, pp 218–228An extracellular vesicle (EV) is a general term for a group of heterogeneous membrane-bound structures that are secreted from a wide range of cell types, including those comprising the cardiovascular and immune systems.1 From cardiovascular disease to cancer, EVs are quickly being recognized as potentially useful biomarkers for screening disease progression in part due to their presence in easily accessible bodily fluids such as urine, blood, and saliva.2 Additionally, EVs are emerging as vehicles of intercellular communication between parent cells (source of EVs) and recipient cells (target of EV action) throughout the body. Thus, it is not surprising that since the first observation of EVs released from maturing reticulocytes during erythrocyte development,3 the burgeoning field has focused on how and why cells synthesize and release these sub-micron structures, in particular, during pathophysiological states. Considering that EVs are circulating factors, the effect on vascular structures, the primary conduit, and distributor of blood has been relatively understudied.4–6 In this issue of Hypertension, Good et al7 demonstrate a novel role for EVs in regulating arterial reactivity and ultimately blood pressure.8Extracellular vesicles can be divided into multiple subcategories—the most relevant to the work of Good et al being exosomes and microvesicles (see van Niel et al1 for additional classifications). Exosomes, typically 50 to 150 nm in diameter, are intraluminal vesicles released from multivesicular endosomes as they fuse with the plasma membrane of the parent cell (Figure, left). This vesicular release occurs both via endosomal sorting complexes required for transport-dependent and endosomal sorting complexes required for transport-independent mechanisms. Much like neurotransmitter release from synaptic boutons, exosome secretion can be a calcium-dependent process which utilizes SNARE proteins to initiate the fusion of the multivesicular endosome membrane with the plasma membrane to cause release of the exosomes and other vesicular cargo in a paracrine fashion. Microvesicles, which range from 50 nm up to 1 µm in diameter, can also act as paracrine mediators; however, the primary pathway of release is through outward budding and fission of the plasma membrane (Figure, left). Despite the recognition of these distinct pathways for EV release, a number of technical limitations hinder the ability to clearly differentiate EV sources experimentally.9 Indeed, there is currently no gold-standard method of EV isolation. Despite these limitations, cell-type specific markers have been used to identify the potential parent cell of origin. Although Good et al demonstrated that the size, morphology, and concentration of the EVs were unchanged in hypertension, they did observe differential expression of cell surface markers (CD31+ and CD45+) in hypertensive rats compared with both prehypertensive rats and age-matched normotensive controls. These data support the idea that EV composition changes following the development of hypertension.Download figureDownload PowerPointFigure. Schematic of two methods of Extracellular Vesicle (EV) release. EVs can be released from a wide range of cells, such as those of cardiovascular and immune origin (left). A generalized cell is used to demonstrate two subtypes of EV: exosomes and microvesicles. Exosomes (white circles) are released into the bloodstream due to the fusion of multivesicular endosomes (blue) with the plasma membrane. Microvesicles (yellow circles containing green circles) are released into the bloodstream via outward budding and fission. Due to the release of EVs into the bloodstream, these systemically circulating biomarkers have the ability to influence target cells at both local and remote sites, such as endothelial cells (light blue) and smooth muscle cells (dark blue) that comprise the vasculature as demonstrated by Good et al (right). EVs restrains vasodilation which helps maintain normotensive blood pressure (top right). A number of changes occur during and following the development of hypertension (dark gray arrow and font). Increased vasoconstriction to adrenergic stimuli ultimately results in an increase in blood pressure. It can be hypothesized that the inability for hypertensive EVs to restrain vasodilation is a compensatory mechanism to offset this prolonged increase in blood pressure. Although the bioactive component of EVs has not been identified, loss of its activity is not sufficient to overcome the increased vasoconstriction in hypertension (bottom right). EVs and vasculature are not to scale.EVs As Regulators of Vascular ReactivityMesenteric resistance arteries, as were used by Good et al, are comprised of a layer of endothelial cells lining the lumen of the vessel in a longitudinal direction, oriented in the direction of blood flow. These endothelial cells are wrapped circumferentially by 1 to 3 layers of vascular smooth muscle cells, oriented perpendicular to the direction of blood flow. Although other cells, such as perivascular nerves, are also present in the vascular wall, this was not a primary focus of their manuscript and thus will not be discussed further.10 As small changes in diameter in these resistance arteries can lead to significant alterations in blood flow, the authors utilized pressure myography on freshly isolated cannulated mesenteric arteries from normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats to examine vascular responses to both vasoconstricting and vasodilating agents. The authors preconstricted these arteries with an alpha-adrenergic receptor agonist, phenylephrine. Activation of these receptors has been well documented (and confirmed by Good et al) to cause a greater contractile response in SHR arteries compared with WKY.11 The authors utilized the muscarinic receptor agonist, acetylcholine (ACh) to examine EC-dependent vasodilation in the absence and presence of EVs from hypertensive or normotensive donors. Further experiments were performed in the presence of the eNOS (endothelial-dependent nitric oxide synthase) inhibitor, L-nitro-arginine methyl ester to determine the NO-dependence of these vasodilatory responses. Indeed, one of the key aspects of the experimental design for the study (summarized in Table) was the preexposure of arteries isolated from normotensive and hypertensive rats to either EVs from normotensive or hypertensive rats. In addition, arteries from wild-type C57BL/6J mice were preexposed to EVs from normotensive and hypertensive humans followed by examination of vasodilatory responses to ACh. This approach allowed the authors to differentiate between changes in arterial function due to the underlying disease state from those occurring due to the EV exposure itself.Table. Overview of the Effect of EVs on Vascular Responses.Source of Arterial Tissue/Animal AgeEV source±treatmentC57BL/6J mice (normotensive) 10–20 wks oldWKY rats (normotensive) 12-wk oldSHR rats (hypertensive) 12-wk oldWKY EVs–↓ Vasodilation compared with control (Figure 3A)Vasodilation unchanged compared with control (Figure 3B)WKY EVs+L-NAME–Vasodilation similar to WKY EVs alone and L-NAME alone (Figure 4C and 4F)–SHR EVs–Vasodilation unchanged compared with control (Figure 3C)Vasodilation unchanged compared with control (Figure 3D)SHR EVs+L-NAME–↓ Vasodilation compared with SHR EVs alone but vasodilation similar to L-NAME alone (Figure 4D and 4F)–Delipidated WKY EVs–↓ Vasodilation compared with control but vasodilation unchanged compared with WKY EVs (Figure 5A)–Delipidated SHR EVs–↓ Vasodilation compared with control and SHR EV-treated conditions (Figure 5B)–WKY EVs from 6-wk-old rats*–↓ Vasodilation compared with control (Figure 5C and 5E)–SHR EVs from 6-wk-old rats*–↓ Vasodilation compared with control (Figure 5D and 5E)–EVs from normotensive humans↓ Vasodilation compared with control (Figure 5F)––EVs from hypertensive humansVasodilation unchanged compared with control (Figure 5F)––Dose-response curves to acetylcholine were used to examine vasodilation throughout. Note, trends are stated as not all comparisons were statistically significant. LNAME indicates L-nitro-arginine methyl ester; SHR, spontaneously hypertensive; and WKY, Wistar Kyoto.*SHRs do not develop hypertension by 6 wks of age, thus this cohort was prehypertensive. Six-wk-old WKY are age-matched controls.Figure numbers/letters correspond to figure panels from the article of Good et al.7The authors observed a reduction in vasodilation in normotensive arteries following exposure to EVs isolated from blood samples from normotensive (human and rodent), delipidated hypertensive, and prehypertensive donors. This reduction in vasodilation was not observed in normotensive arteries preexposed to EVs isolated from the blood of either rodent or human hypertensive donors. Although there was no additive effect in WKY arteries exposed simultaneously to L-nitro-arginine methyl ester and either WKY or SHR EVs, the differential dilatory response of these arteries exposed to WKY EVs or SHR EVs alone suggested a role for nitric oxide (NO)-mediated signals in normotension. Interestingly, preexposure of arteries from normotensive rats to EVs from hypertensive rats only caused a reduced vasodilatory response after delipidation. As delipidation disrupts the vesicular membrane allowing the vesicular contents to be available for cell signaling, these data suggest hypertensive EVs still contained the vasodilation dampening signal, but that during the development of hypertension the bioavailability and presumably the localization changes. EVs isolated from either prehypertensive or age-matched control rats were able to blunt the vasodilatory response. Vasodilation responses after exposure of hypertensive arteries to hypertensive and normotensive EVs were unchanged. Collectively, these data suggest that (1) something within the EVs dampen the typical acetylcholine mediated endothelial-dependent dilation, and that this occurs before observable changes in hypertension, (2) hypertension causes changes to EV-dependent NO-mediated signaling, and (3) delipidation of EVs from measurably hypertensive rats can recover the dampening of the vasodilatory response, presumably through the release of an otherwise masked vasoactive agent. Once again, these data support the idea that EV composition changes after hypertension development but further demonstrate that these changes have functional consequences on the resistance vasculature, where blood pressure regulation occurs.10The primary observations of Good et al from the offset may seem counterintuitive. Under physiological conditions, EVs function as a brake on the vasodilatory response presumably raising blood pressure. Under the pathophysiological state of hypertension, this brake was no longer present. It could be hypothesized that EVs change after disease progression as a compensatory mechanism, presumably to allow vasodilation to again occur unopposed. Although this type of compensation seems plausible, SHR rats still developed elevated blood pressure, suggesting that this adaptation was not sufficient to fully overcome the EC-dysfunction and hypercontractility of the smooth muscle cells.From a therapeutic perspective, it seems unlikely that the application of the findings of Good et al would be to make EVs isolated from a hypertensive animal more like those from a normotensive one, as this would ultimately dampen vasodilation further leading to even higher blood pressure. Thus, the question is not how can a hypertensive EV be more like a normotensive EV but what changes are occurring during the progression from normo- to hypertensive and how might that contribute to the pathological condition. What types of vasoreactive cargo are being released into the circulation and why does the localization of the bioreactive component change as the disease progresses? Some of these answers may lie in a better understanding of what is available for signaling after delipidation of hypertensive EVs, that is not present otherwise. An alternative perspective would be to harness the bioreactive component of normotensive EVs for pathologies where vasodilation may need to be dampened.Study LimitationsAlthough the therapeutic potential of the findings of Good et al may not be immediately obvious, addressing some of the limitations of the study may provide the link between their fundamental observation and its clinical significance. As the authors utilized mesenteric arteries from normotensive mice to examine the bioreactivity of human EVs, it is unclear if human arteries would exhibit a similar functional outcome. It is currently assumed that human arteries would follow the trends observed in the mouse (Table) because the vasoreactivity of mouse arteries exposed to human EVs were similar to rat arteries exposed to rat EVs. Future studies using human arteries not only from patients with normotension and hypertension but also patients with hypotension may provide insight into potential clinical applications of the current findings. EVs can be released from a range of cells in response to a number of stimuli with their contents and bioactivity dependent on the nature of the stimulus.1 The authors observed an increase in the cell surface markers CD31+ and CD45+ in EVs isolated from hypertensive rats; however, similar studies were not performed in human EVs. So, although it is known that CD31+ and CD45+ expression can indicate a range of parent cells including leukocytes, platelets, and endothelial cells, future studies should be performed to definitively delineate the EV sources and confirm if rodent studies are representative of the human condition.Another limitation of the current study was the inability to identify the exact bioactive component responsible for restraining vasodilation after arterial exposure to normotensive EVs and delipidated hypertensive EVs. Although the authors imply that eNOS may be a potential candidate, they did not provide experimental evidence to support a change in eNOS localization and bioavailability after the development of hypertension. Identification of the bioactive component and a better understanding of how its availability changes with disease progression, if harnessed, could be the most significant finding for future therapeutic applications arising from the current work.ConclusionsOverall, these data highlight that circulating biomarkers have the capability of regulating vasoreactivity and that this can have important functional consequences as diseases progress. It also brings to light the need to better understand how disease progression itself can change the bioreactivity of EVs, in particular, as it relates to their role as an intercellular communication pathway and, in this instance, a master regulator of systemic blood pressure.Sources of FundingNone.DisclosuresNone.FootnotesCorrespondence to Pooneh Bagher, 8447 Riverside Parkway, Bryan, TX 77807. Email [email protected]edu