Endothelial cAMP deactivates ischemia-reperfusion-induced microvascular hyperpermeability via Rap1-mediated mechanisms

Abstract

Approaches to reduce excessive edema due to the microvascular hyperpermeability that occurs during ischemia-reperfusion (I/R) are needed to prevent muscle compartment syndrome. We tested the hypothesis that cAMP-activated mechanisms actively restore barrier integrity in postischemic striated muscle. We found, using I/R in intact muscles and hypoxia-reoxygenation (H/R, an I/R mimic) in human microvascular endothelial cells (HMVECs), that hyperpermeability can be deactivated by increasing cAMP levels through application of forskolin. This effect was seen whether or not the hyperpermeability was accompanied by increased mRNA expression of VEGF, which occurred only after 4 h of ischemia. We found that cAMP increases in HMVECs after H/R, suggesting that cAMP-mediated restoration of barrier function is a physiological mechanism. We explored the mechanisms underlying this effect of cAMP. We found that exchange protein activated by cAMP 1 (Epac1), a downstream effector of cAMP that stimulates Rap1 to enhance cell adhesion, was activated only at or after reoxygenation. Thus, when Rap1 was depleted by small interfering RNA, H/R-induced hyperpermeability persisted even when forskolin was applied. We demonstrate that) VEGF mRNA expression is not involved in hyperpermeability after brief ischemia, ) elevation of cAMP concentration at reperfusion deactivates hyperpermeability, and) cAMP activates the Epac1-Rap1 pathway to restore normal microvascular permeability. Our data support the novel concepts that ) different hyperpermeability mechanisms operate after brief and prolonged ischemia and) cAMP concentration elevation during reperfusion contributes to deactivation of I/R-induced hyperpermeability through the Epac-Rap1 pathway. Endothelial cAMP management at reperfusion may be therapeutic in I/R injury. Here, we demonstrate that ) stimulation of cAMP production deactivates ischemia-reperfusion-induced hyperpermeability in muscle and endothelial cells;) VEGF mRNA expression is not enhanced by brief ischemia, suggesting that VEGF mechanisms do not activate immediate postischemic hyperpermeability; and ) deactivation mechanisms operate via cAMP-exchange protein activated by cAMP 1-Rap1 to restore integrity of the endothelial barrier.

Epac1 protects the retina against ischemia/reperfusion-induced neuronal and vascular damage

“…Epac1 has been shown to reduce myocardial damage in response to I/R [ 20 ]. Additionally, work in muscle and endothelial cells showed that Epac1 reduced hyper-permeability following exposure to I/R [ 21 ]. We previously reported that Epac1 regulated ZO-1 and occludin in the mouse retina and in retinal endothelial cells [ 22 ].…”

IL-1β suppression of VE-cadherin transcription underlies sepsis-induced inflammatory lung injury

“…Mouse IL-1β/IL-1F2 Quantikine ELISA Kit (catalog SMLB00C), human IL-1β/IL-1F2 Quantikine tection elicited acutely by raising endothelial cell concentration of cAMP (40). In these studies, cAMP restored endothelial permeability in response to multiple permeability-increasing stimuli such as thrombin (40) and ischemia/reperfusion (41). The barrier protection was ascribed to cAMP-dependent activation of guanine nucleotide exchange factor (EPAC), which promoted exchange of GTP in the small GTPase Rap1 (40,42).…”

Rap1 Small GTPase Regulates Vascular Endothelial-Cadherin-Mediated Endothelial Cell–Cell Junctions and Vascular Permeability

“…We previously reported administration of 007 to suppress vascular endothelial growth factor (VEGF)-induced vascular hyperpermeability in murine skin. 34) Similarly, 007-induced activation of the Epac/Rap1 pathway was shown to inhibit microvascular hyperpermeability caused by PAF in the rat mesentery, 68) disruption of the endothelial barrier caused by toxins derived from Pseudomonas aeruginosa, a major nosocomial infection agent, 69) ischemia-reperfusion-induced microvascular hyperpermeability in skeletal muscle, 70) and cytokine-induced retinal vascular permeability. 71) Furthermore, cAMP-elevating GPCR agonists such as prostaglandin I2 and its stable analogs like iloprost and beraprost, prostaglandin A2, and adrenomedullin are known to exert Rap1-dependent protective effects against pulmonary endothelial barrier dysfunction during acute pulmonary injury induced by LPS administration and mechanical ventilation.…”