The recombinant bifunctional protein αCD133-GPVI promotes repair of the infarcted myocardium in mice.

BACKGROUND: Bone-marrow-derived progenitor cells are important in myocardial repair mechanisms following prolonged ischemia. Cell-based therapy of diseased myocardium is limited by a low level of tissue engraftment. OBJECTIVES: The aim of this study was the development of the bifunctional protein αCD133-glycoprotein (GP)VI as an effective treatment for supporting vascular and myocardial repair mechanisms. RESULTS: We have generated and characterized a bifunctional molecule (αCD133-GPVI) that binds both to the subendothelium of the injured microvasculature and to CD133(+) progenitor cells with high affinity. αCD133-GPVI enhances progenitor cell adhesion to extracellular matrix proteins and differentiation into mature endothelial cells. In vivo studies showed that αCD133-GPVI favors adhesion of circulating progenitor cells to the injured vessel wall (intravital microscopy). Also, treatment of mice undergoing experimental myocardial infarction with αCD133-GPVI-labeled progenitor cells reduces infarction size and preserves myocardial function. CONCLUSIONS: The bifunctional trapping protein αCD133-GPVI represents a novel and promising therapeutic option for limiting heart failure of the ischemic myocardium.

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

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.

cAMP induced Rac 1-mediated cytoskeletal reorganization in microvascular endothelium.

It is well established that cAMP stabilizes endothelial barrier functions, in part by regulation of VE-cadherin via EPAC/Rap 1. The aim of the present study was to investigate whether cAMP activates Rac 1 in microvascular endothelium. In human dermal microvascular endothelial cells (HDMEC), treatment with forskolin/rolipram (F/R) to increase cAMP by as well as the Epac/Rap 1-stimulating cAMP analogue 8-pCPT-2'-O-methyl-cAMP (O-Me-cAMP) stabilized endothelial barrier properties as revealed by raised transendothelial electrical resistance (TER). Under these conditions, immunostaining of VE-cadherin and claudin 5 were increased and linearized. This was paralleled by activation of Rac 1 by 153 +/- 16% (F/R) or 281 +/- 65% (O-Me-cAMP) whereas activity of Rho A was unchanged. F/R and O-Me-cAMP increased the peripheral actin belt and recruited the Rac 1 effector cortactin to cell junctions, similar to direct activation of Rac 1 by CNF-1. Thrombin was used to further test the physiologic relevance of cAMP-mediated Rac 1 activation. Thrombin-induced drop of TER was paralleled by intercellular gap formation, inactivation of Rac 1 and activation of Rho A at 5 and 15 min whereas baseline conditions where re-established following 60 min. Both, F/R and O-Me-cAMP completely blocked the thrombin-induced barrier breakdown. F/R completely abolished thrombin-induced Rac 1 inactivation and Rho A activation whereas O-Me-cAMP only partially blocked Rac 1 inactivation. Taken together, these results indicate that Rac 1 activation likely contributes to the barrier-stabilizing effects of cAMP in microvascular endothelium and that these effects may in part be mediated by Epac/Rap 1.

Differential role of Rho GTPases in endothelial barrier regulation dependent on endothelial cell origin.

From studies using macrovascular endothelium, it was concluded that Rho A activation generally leads to endothelial barrier breakdown. Here, we characterized the role of Rho GTPases in endothelial barrier regulation in four different cell lines, both microvascular and macrovascular. Rho A activation by cytotoxic necrotizing factor y (CNFy) induced stress fiber formation in all cell lines. This was paralleled by gap formation and barrier breakdown in microvascular mesenteric endothelial cells (MesEnd), human dermal microvascular endothelial cells (HDMEC) as well as in macrovascular pulmonary artery endothelial cells (PAEC) but not in microvascular myocardial endothelial cells (MyEnd). In MyEnd cells, activation of Rac 1 and Cdc42 by CNF-1 strengthened barrier properties whereas in MesEnd, HDMEC and PAEC all three GTPases were activated which increased permeability in PAEC but not in MesEnd and HDMEC. In PAEC, CNF-1-induced decrease of barrier properties was blocked by the Rho kinase inhibitor Y27632 indicating that co-activation of Rho A dominated the barrier response. Inactivation of Rac 1 by toxin B or by lethal toxin (LT) compromised barrier properties in all cell lines. Taken together, Rac 1 requirement for endothelial barrier maintenance but not the destabilizing role of Rho A seems to be ubiquitous.