Extracellular vesicle-induced cyclic AMP signaling

Abstract

Second messenger signaling is required for cellular processes. We previously reported that extracellular vesicles (EVs) from stimulated cultured endothelial cells contain the biochemical second messenger, cAMP. In the current study, we sought to determine whether cAMP-enriched EVs induce second messenger signaling pathways in naïve recipient cells. Our results indicate that cAMP-enriched EVs increase cAMP content sufficient to stimulate PKA activity. The implications of our work are that EVs represent a novel intercellular mechanism for second messenger, specifically cAMP, signaling.

Keywords: Cyclic adenosine monophosphate; Extracellular vesicles; Microvesicle; Second messenger.

Fig. 1.

Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) of extracellular vesicle (EVs). (A) EVs were isolated from cell culture medium of control or isoproterenol and rolipram-treated (I/R) cells then fixed and stained for TEM. EVs were intact, dense and heterogenous vesicles. (B) The particle size distribution for extracellular vesicles isolated from control and IR-treated cells. NTA results indicate a range of heterogenous, submicron EVs with a similar median size distribution, n = 3.

Fig. 2.

IR EVs trigger increased cAMP levels in PMVECs. IR EVs or vehicle control were added to PMVECs transfected with the H188 cAMP probe. cAMP levels were estimated as described in the Materials and Methods. (A) cAMP levels increased in the cells treated with IR EVs but not in cells treated with vehicle control (left panels). cAMP levels are as indicated by the color bar − hot colors indicate high cAMP and cool colors indicate the low cAMP. Outlines of the cell border (indicated by the white line at the cellular periphery) were obtained using the donor + acceptor signal. (B) A representative time-course of cAMP accumulation triggered by the addition of EVs (blue circles) of vehicle control (red squares) at 90 s (indicated by arrow). Data are representative of 6 experiments. (C) Addition of IR EVs triggered significant increases in intracellular cAMP levels in target PMVECs. Data were measured at the timepoint 5 min after addition of IR EVs. (* p < 0.01; n = 6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

Simulations of EV-mediated cAMP release in the near-membrane compartment of an endothelial cell. (A) cAMP levels in the near-membrane compartment (C1) triggered by release of cAMP from an individual EV into C1 at 10 s. [cAMP] EV cAMP concentrations are as indicated in the legend. (B) cAMP levels in the cytosolic compartment (C2) triggered by release of cAMP from an individual EV into C1. EV cAMP concentrations are as indicated in the legend of panel (A). It is apparent that EVs containing relatively high cAMP concentrations can activate PKA in near-membrane compartments (PKA EC₅₀ ≈ 0.1 μM cAMP). It is likely that PKA activity in C1 would remain high enough to induce sustained activation of PKA in C1. However, PKA activation would be lower in amplitude and transient in the remainder of the cell (C2).

Fig. 4.

Simulations of EV-mediated cAMP release in the cytosolic compartment of an endothelial cell. (A) cAMP levels in the near-membrane compartment (C1) triggered by release of cAMP from an individual EV into C2 at 10 s. EV cAMP concentrations are as indicated in the legend. (B) cAMP levels in C2 triggered by release of cAMP from an individual EV into C2. EV cAMP concentrations are as indicated in the legend of panel (A). Release of cAMP from an EV with high cAMP concentration into a large cellular volume (e.g. the bulk cytosol depicted by C2) would lead to sustained activation of PKA in C2 for >5 min and a transient increase in cAMP in C1.

Fig. 5.

EV treatment stimulates VASP phosphorylation. (A) Densitometry quantification of protein levels of phosphorylated-VASP (S157) normalized to total VASP of endothelial cells treated for 10 min with vehicle (control), I/R-EVs, or directly with rolipram and isoproterenol to stimulate PKA (I/R-treated). VASP phosphorylation increases with I/R-EV treatment and I/R treatment compared to vehicle *p < 0.05, n = 4 (B) Representative western blot. Protein levels in cells treated with vehicle (C), IR (I/R), or I/R-EVs (tEVs); n = 4. (C) PKA activity is increased in cell lysates treated with Isoproterenol (Iso) or I/R EVs; n = 3, *p < 0.05.

Fig. 6.

EVs do not deliver I/R. Mass spec analysis of cell lysates indicate both rolipram and isoproterenol are present in cell lysates(A and C), however, isoproterenol is not in abundance in EVs and rolipram is undetectable (B and D). Figs. A and B represent mass spec runs for isoproterenol detection and Figs. C and D for rolipram. Relative abundance is the amount of substance (Iso or Rol)/total cell count used for the experiment.

Fig. 7.

EV-mediated PKA signaling is not β-receptor-dependent. (A) Propranolol, a β-receptor inhibitor was used to pretreat PMVECs (Prop; 25 μM; 1 h) followed by isoproterenol or I/R EVs. Densitometry quantification of protein levels of phosphorylated-VASP (S157) normalized to total VASP and representative Western blot. n = 3 (B) PKA ELISA in the presence of propranolol (Prop; 25 μM; 1 h) reveals that inhibition of the β-receptor does not prevent PKA activity. n = 3, *p < 0.05.