ADAM17‐enriched Exosomes Contribute to Neuronal Activation in Hypertension

Abstract

ADAM17 is the prime mediator of Angiotensin Converting Enzyme 2 (ACE2) shedding, a process by which the ACE2 ectodomain is cleaved from the cell membrane and released in the surrounding milieu. ACE2 is a compensatory enzyme converting Angiotensin (Ang)-II to Ang-(1-7). Recent studies have reported the presence of activated ADAM17 on exosomes, suggesting the possibility of shedding propagation from one cell to another. Here, we hypothesized that in hypertensive conditions, exosomes are enriched in ADAM17 and contribute to neuronal activation, possibly leading to enhanced sympathetic activity. Human plasma and cerebrospinal fluid were collected from both African Americans (AA) and Caucasian Americans (CA), normotensive and hypertensive patients (n=3/group). Exosomes were extracted using total exosome isolation kit (Invitrogen) and quantified by estimation of proteins (BCA kit). The ADAM17 expression in exosomes was measured by capillary western (protein Simple) and the exosomes’ morphology was studied using electron microscopy (Cryo-TEM). To investigate the role of these exosomes, Neuro2A cells were treated with phorbol myristate, an ADAM17 activator, or Ang-II (100 nM) for 4 hours, followed by extraction of exosomes (Invitrogen) and estimation of their ADAM17 levels (capillary western). After 24 h, Neuro2A cells were transfected with GCaMP6s, a fluorescent calcium indicator, and exposed for 4 h with PBS (control), Ang-II (100 nM) or exosomes isolated from hypertensive patients before Ca++-mediated fluorescence was quantified (Cytation 5). Exosomes extracted from patients’ plasma showed a significant 3-fold increase in ADAM17 expression in hypertensive individuals when compared to normotensive counterparts (Normotensive AA: 3.1 ±0.6, CA: 4.5 ±0.3; Hypertensive AA: 7.1 ±0.2, CA: 7.0 ±0.5, au: arbitrary units; n=3: one-way ANOVA, P< 0.01 and P< 0.001 vs. normotensive). In normotensive patients ADAM17 content was higher in CA but was similar in AA and CA under hypertensive conditions. Exosomes extracted from Neuro2A cells showed a 3-fold increase in ADAM17 expression in both Ang-II- and PMA-treated groups when compared to control, thereby confirming the expression of ADAM17 in exosomes from stimulated neuronal cells. The Ca++-mediated fluorescence intensity in Neuro2A cells treated with exosomes from hypertensive patients or Ang-II was significantly increased when compared to control cells (control: 4.0 ±0.6, Ang-II: 20 ±0.2, exosomes: 15.1 ±0.2, au, n=3, one-way ANOVA, P<0.001 vs. control, n=3). Our data show that upon activation the mature form of ADAM17 is released in the form of exosomes in the brain or plasma from hypertensive patients, and in cells following Ang-II stimulation, possibly contributing to the enhanced ACE2 shedding in those individuals. In addition, neurons exposed to Ang-II and ADAM17-enriched exosomes (i.e. isolated from hypertensive patients) show increased Ca++-mediated fluorescence confirming an activated neuron phenotype that could contribute to elevated excitatory activity on neuronal cells and ultimately to an enhanced sympathetic activity.