cAMP/PKA-mediated in vitro angiogenesis: A novel interplay between PKA, RhoA/ROCK, and VEGFR2 signalling

Hafiza Idrees (Gießen)1, M. Aslam (Gießen)1

1Justus-Liebig-Universität Giessen Experimentelle Kardiologie Gießen, Deutschland


Background and Aims: We and others have previously demonstrated that cyclic AMP/protein kinase A (cAMP/PKA) signalling stabilise endothelial cell (EC) barrier function via modulating the activities of Rho GTPases, mainly RhoA and Rac1. In the present study we analysed whether cAMP/PKA also modulates EC angiogenesis capacity and whether Rho GTPases are involved in this function.

Methods: The study was carried out on cultured human umbilical vein ECs (HUVECs). RhoA and Rac1 activities were genetically manipulated by lentiviral-mediated overexpression of dominant negative (DN) and constitutively active (CA) forms of RhoA and Rac1. Pharmacologically, specific activation and inhibition of PKA was achieved by using the cAMP analogue N6-benzoyl-cAMP (5 mM) and protein kinase A inhibitor peptide (PKI) (1 µM), respectively. Rac1 was inhibited using NSC 23766 (10 µM), and RhoA was activated using Rho activator I. In vitro angiogenesis was analysed by 3-D spheroid assay and matrigel tube formation.

Results: Specific, pharmacological activation of PKA induced EC migration (wound healing assay), tube formation, and 3-D spheroid formation and potentiated VEGF-induced in vitro angiogenesis. These pro-angiogenic activities were abrogated by highly specific cell-permeable peptide inhibitor (PKI) of PKA. Activation of cAMP/PKA signalling resulted in increased VEGFR2 and Akt phosphorylation, increased Rac1 activity, and strong inhibition of the RhoA/Rock pathway. Activation of RhoA but not inhibition of Rac1 abrogated PKA-induced VEGFR2 phosphorylation and tube and 3-D spheroid formation. Lentivirus-mediated overexpression of the CA form of RhoA but not Rac1 abrogated PKA-induced VEGFR2 and Akt phosphorylation and angiogenesis. Moreover, pharmacological inhibition of Akt also abrogated PKA-mediated VEGFR2 phosphorylation and angiogenesis. Likewise, overexpression of DN RhoA or pharmacological inhibitors of Rho kinase (ROCK) promoted basal and potentiated PKA-mediated VEGFR2 and Akt phosphorylation and angiogenesis.

Conclusion: We describe for the first time a novel interplay between PKA, RhoA/ROCK, Akt, and VEGFR2 signalling and angiogenesis in ECs. Inhibition of RhoA/ROCK plays an important role in mediating PKA-induced EC angiogenesis, and activation of RhoA/ROCK signalling may offer an important target to promote therapeutic angiogenesis.
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