Role of Rac 1 and cAMP in endothelial barrier stabilization and thrombin‐induced barrier breakdown

Abstract

Barrier stabilizing effects of cAMP as well as of the small GTPase Rac 1 are well established. Moreover, it is generally believed that permeability-increasing mediators such as thrombin disrupt endothelial barrier functions primarily via activation of Rho A. In this study, we provide evidence that decrease of both cAMP levels and of Rac 1 activity contribute to thrombin-mediated barrier breakdown. Treatment of human dermal microvascular endothelial cells (HDMEC) with Rac 1-inhibitor NSC-23766 decreased transendothelial electrical resistance (TER) and caused intercellular gap formation. These effects were reversed by addition of forskolin/rolipram (F/R) to increase intracellular cAMP but not by the cAMP analogue 8-pCPT-2'-O-Methyl-cAMP (O-Me-cAMP) which primarily stimulates protein kinase A (PKA)-independent signaling via Epac/Rap 1. However, both F/R and O-Me-cAMP did not increase TER above control levels in the presence of NSC-23766 in contrast to experiments without Rac 1 inhibition. Because Rac 1 was required for maintenance of barrier functions as well as for cAMP-mediated barrier stabilization, we tested the role of Rac 1 and cAMP in thrombin-induced barrier breakdown. Thrombin-induced drop of TER and intercellular gap formation were paralleled by a rapid decrease of cAMP as revealed by fluorescence resonance energy transfer (FRET). The efficacy of F/R or O-Me-cAMP to block barrier-destabilizing effects of thrombin was comparable to Y27632-induced inhibition of Rho kinase but was blunted when Rac 1 was inactivated by NSC-23766. Taken together, these data indicate that decrease of cAMP and Rac 1 activity may be an important step in inflammatory barrier disruption.

Lymphatic Endothelial Cells Adapt Their Barrier Function in Response to Changes in Shear Stress

“…It is worth noting that we used a slightly lower dose of NSC23766 (50 mM) than other studies (200 mM) of endothelial barrier function to achieve this inhibition. 22,35 In addition, our finding that the enhancement of R b was largely responsible for the overall elevation in TER (Fig. 8) supports the concept that Rac1-mediated membrane protrusions and tightening of intercellular junctions are key features of the HMLEC-d response to elevated t. Combined, these data suggest that Rac1 mediates the shear stress-induced increase in endothelial barrier function, including in lymphatic endothelial cells.…”

“…12,17,[22][23][24][25][26] We tested the role of this pathway by inhibiting PKA activity with the specific inhibitor H-89 (Fig. 7).…”

Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin

“…The second arm of the thrombin signaling cascade causes down-regulation of cAMP (28,34). Protease-activated receptor activation by thrombin both inhibits adenylate cyclase via G i (35) and activates PDE3, a cGMP-inhibited cAMP phosphodiesterase (36,37).…”

Small Rho GTPase-mediated actin dynamics at endothelial adherens junctions

“…[58][59][60] Barrier-protective cAMP signaling activates Rac1 indirectly via the RasGTPase Rap1. 61,62 In addition, Rac1 activation downstream of thrombin promotes reassembly of cell-cell junctions during the endothelial barrier recovery phase. 22,63 To promote (re)assembly of cell-cell junctions, Rac1 induces formation of lamellipodia protrusions.…”