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Mechanisms of Adenosine A1 Receptor‐Mediated Enhancement of Vasa Vasorum Endothelial Cell Barrier Function

The vasa vasorum (VV) is a microcirculatory network that provides oxygen and nutrients to the adventitia and media of large blood vessels. Previously, we have demonstrated that angiogenic expansion of pulmonary artery (PA) VV in the chronically hypoxic calves exhibit increased permeability for circulating inflammatory cells and macromolecules. Extracellular adenosine (Ado) increased transendothelial electrical resistance (TER) of VVEC isolated from control (Denver altitude) and, in lesser extent, hypoxic (2 weeks, PB=430 mmHg) neonatal calves. Ado‐induced VVEC barrier enhancement involved activation of Ado A1 receptors (A1R), PI3K/AKT pathway, and actin remodeling. In the current study we demonstrated that inhibition of Rac1 by transduction of VVEC with dominant‐negative adenoviral Rac N17 construct almost completely abolished adenosine‐induced increase in TER supporting a critical role of Rac1 activation in Ado‐induced VVEC barrier enhancement. Inhibition of Rac1 activity also leads to the decrease of AKT phosphorylation, suggesting that Rac1 is an upstream regulator of AKT signaling in VVEC. In addition, Ado increased autophoshorylation of p21‐activated kinase 1 (PAK1), a well‐known downstream target of Rac1, reflecting its activation. Pharmacologic inhibition of PAK1 decreased basal TER and abolished Ado‐induced TER increase indicating involvement of PAK1 in VVEC barrier maintenance. Importantly, A1R expression was demonstrated in the VV of pulmonary arteries of Sprague Dawley (SD) rats, indicating that VV localization of A1R is not restricted to bovine VV. Intravenous co‐injection of Rhodamine‐conjugated Griffonia Simplicifolia lectin and FITC‐conjugated dextran in SD rats followed by two photon confocal microscopy of dissected pulmonary arteries revealed increased permeability of PA VV in chronically hypoxic SD rats. Thus, this model shows a great promise for the in vivo validation of the protective role A1R agonists in VV barrier integrity. In summary, we demonstrate for the first time that A1Rs enhance VVEC barrier function through the activation of Rac1/PAK pathway and actin cytoskeleton remodeling. We propose that A1Rs can be recognized as a vascular bed‐specific and novel therapeutic target to regulate VV barrier function and pathologic vascular remodeling in chronic hypoxia.