Adenovirus Vector E4 Gene Promotes Angiogenesis through Modulation of Junctional Connexin 40 and 43 Expression.

Gap junction channels formed of connexin (Cx) proteins provide a means for intercellular communication via direct linkage of the cytoplasm of adjacent cells and play important roles in the pathophysiology of vascular and cardiac homeostasis and disease. We have previously reported that introduction of E1/E3-deleted adenovirus (AdE4⁺) to endothelial cells (EC) selectively activates angiogenesis and increases cell survival via activation of the junctional adhesion molecule vascular endothelial-cadherin which targets the PI3K/Akt pathway. We now show that AdE4⁺ also potentiates Cx expression in EC in vitro and mouse heart tissue in vivo. Infection of EC with AdE4⁺, but not AdE4⁻, vectors resulted in time- and dose-dependent induction of Cx40 and suppression of Cx43 protein and mRNA as determined by immunoblot and Northern blot respectively. Maximal induction of Cx40 and suppression of Cx43 protein and mRNA were observed by 48 hours after infection, and between multiplicity-of-infection of 100 and 200. Immunohistochemical analysis of infected EC revealed that AdE4⁺ induced Cx40 expression localized to the plasma membrane at intercellular junctions, and attenuated total Cx43 expression. Cx40 expression was also markedly increased and Cx43 significantly decreased in the heart tissue of mice treated with AdE4⁺ via intra-tracheal administration compared to control mice. Pretreatment of EC with either PKA inhibitor (H89) or PI3K inhibitor (LY294002) abrogated the effects of AdE4⁺ in regulating Cx40 and Cx43, and was associated with diminished AdE4⁺-enhanced EC survival. Furthermore, neither PKG inhibitor (KT5823) nor guanylyl cyclase inhibitor (NS2028) affected AdE4⁺-modulation of Cx. It is of interest to note that cAMP has been shown to trigger Cx trafficking by way of PKA activation, as we have also observed. However, neither forskolin- nor cholera toxin-induced cAMP affected Cx expression in AdE4⁺-infected EC, indicating that PKA activation by AdE4⁺ may be independent of cAMP. Taken together, alteration of the Cx expression profile, as mediated through PKA and PI3K signaling pathways, may play a role in AdE4⁺-associated EC survival. These findings of Cx modulation by AdE4⁺ may not only explain why E4⁺ adenoviral vectors may induce increased survival of endothelial cells in vivo, but also may help us to understand the mechanisms of the pathophysiology of vascular and heart disorders by regulation of Cx.