Microparticle Delivery of Cyclic AMP to Pulmonary Microvascular Endothelial Cells

Abstract

Microparticles (MPs) are important for the exchange of information between cells. MPs are extracellular vesicles that range in size from 0.1 to 1 μm in diameter. They are constitutively released from many cell types and are thought to play a role in vascular homeostasis. Further, the number and content of MPs are altered in pathologies involving inflammation and vascular cell activation. MPs carry intracellular constituents from parent cells that affect target cell function. However, the mechanisms of delivery of the MP payload to target cells are unknown. Understanding the payload delivery system between MPs and target cells is the focus of this project.We recently discovered that cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger, is found in MPs released from pulmonary microvascular endothelial cells (PMVECs). Additionally, MPs isolated from PMVECs treated with β adrenergic agonists and phosphodiesterase (PDE) inhibitors have increased cAMP levels. This suggests that different levels of cAMP are packaged in MPs from the parent cell. MPs containing maximal cAMP were therefore used to study the delivery of MP payload to target cells.We developed mathematical models of MP payload delivery to subcellular compartments of target PMVECs to better understand how the cAMP signaling pathway may be altered by MP‐mediated cAMP delivery. Simulations indicate that if the cAMP contained in one MP were released into a near‐membrane compartment of a target cell, cAMP levels within that compartment would be sufficient to activate protein kinase A (PKA) and Exchange protein activated by cAMP (Epac) for several minutes. Release of cAMP from a single MP released into the larger volume of the bulk cytoplasm would have little effect on target cell signaling. However, several MPs are often internalized by a target cell and their combined cAMP content would sufficiently elevate bulk cytosolic cAMP levels to activate PKA.To experimentally test the predictions of this model, MPs were collected from PMVECs treated with rolipram (a PDE type 4 inhibitor; 10 μM, 5 min) and isoproterenol (a β adrenergic agonist; 1 μM, 10 min). These MPs were added to cells transfected with a cytosolic cAMP FRET sensor. Time lapse hyperspectral image stacks were acquired using a Nikon A1R confocal microscope. Custom hyperspectral analysis scripts in MatLab (MathWorks) were used to unmix fluorescent spectra and quantify cAMP concentration changes throughout the target cell. Preliminary results indicate that MPs significantly increased cAMP levels in target cells compared to control. Together, these data indicate that MPs can deliver cAMP as a functional payload. Future studies will determine whether the MP‐delivered cAMP is sufficient to trigger sustained responses in target cells and contribute to MP‐mediated signaling events.Support or Funding InformationThis work was supported by NIH T32HL076125, P01HL066299, S10RR027535, and AHA 11SDG7390037.