title: Adenosine A1 receptors promote vasa vasorum endothelial cell barrier integrity via Gi and Akt-dependent actin cytoskeleton remodeling.
abstract: BACKGROUND
  In a neonatal model of hypoxic pulmonary hypertension, a dramatic pulmonary artery adventitial thickening, accumulation of inflammatory cells in the adventitial compartment, and angiogenic expansion of the vasa vasorum microcirculatory network are observed. These pathophysiological responses suggest that rapidly proliferating vasa vasorum endothelial cells (VVEC) may exhibit increased permeability for circulating blood cells and macromolecules. However, the molecular mechanisms underlying these observations remain unexplored. Some reports implicated extracellular adenosine in the regulation of vascular permeability under hypoxic and inflammatory conditions. Thus, we aimed to determine the role of adenosine in barrier regulation of VVEC isolated from the pulmonary arteries of normoxic (VVEC-Co) or chronically hypoxic (VVEC-Hyp) neonatal calves.
  
  
  RESULTS
  We demonstrate via a transendothelial electrical resistance measurement that exogenous adenosine significantly enhanced the barrier function in VVEC-Co and, to a lesser extent, in VVEC-Hyp. Our data from a quantitative reverse transcription polymerase chain reaction show that both VVEC-Co and VVEC-Hyp express all four adenosine receptors (A1, A2A, A2B, and A3), with the highest expression level of A1 receptors (A1Rs). However, A1R expression was significantly lower in VVEC-Hyp compared to VVEC-Co. By using an A1R-specific agonist/antagonist and siRNA, we demonstrate that A1Rs are mostly responsible for adenosine-induced enhancement in barrier function. Adenosine-induced barrier integrity enhancement was attenuated by pretreatment of VVEC with pertussis toxin and GSK690693 or LY294002, suggesting the involvement of Gi proteins and the PI3K-Akt pathway. Moreover, we reveal a critical role of actin cytoskeleton in VVEC barrier regulation by using specific inhibitors of actin and microtubule polymerization. Further, we show that adenosine pretreatment blocked the tumor necrosis factor alpha (TNF-α)-induced permeability in VVEC-Co, validating its anti-inflammatory effects.
  
  
  CONCLUSIONS
  We demonstrate for the first time that stimulation of A1Rs enhances the barrier function in VVEC by activation of the Gi/PI3K/Akt pathway and remodeling of actin microfilament.
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, Georgia, United States of America. usiddaramappa@georgiahealth.edu
authors.forename: Siddaramappa Nagavedi
authors.initials: SN
authors.lastname: Umapathy
authors.forename: Nagavedi
authors.initials: N
authors.lastname: Siddaramappa Umapathy
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Kaczmarek
authors.forename: Nooreen
authors.initials: N
authors.lastname: Fatteh
authors.forename: Nana
authors.initials: N
authors.lastname: Burns
authors.forename: Rudolf
authors.initials: R
authors.lastname: Lucas
authors.forename: Kurt R
authors.initials: KR
authors.lastname: Stenmark
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Verin
authors.forename: Evgenia V
authors.initials: EV
authors.lastname: Gerasimovskaya
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002867:Chromones / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D010069:Oxadiazoles / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D043682:Receptor, Adenosine A1 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014651:Vasa Vasorum / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
chemical_list: D002867:Chromones; C528328:GSK690693; D009025:Morpholines; D010069:Oxadiazoles; D043682:Receptor, Adenosine A1; D014409:Tumor Necrosis Factor-alpha; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D051057:Proto-Oncogene Proteins c-akt; D019206:GTP-Binding Protein alpha Subunits, Gi-Go