Frauke V. Härtel

cAMP/PKA antagonizes thrombin-induced inactivation of endothelial myosin light chain phosphatase: role of CPI-17

AIMS Activation of cAMP signalling abrogates thrombin-induced hyperpermeability. One of the mechanisms underlying this protective effect is the inactivation of endothelial contractile machinery, one of the major determinants of endothelial barrier function, mainly via the activation of myosin light chain phosphatase (MLCP). To date, the mechanisms of cAMP-mediated MLCP activation are only partially understood. Here the contribution of two cAMP effectors, PKA and Epac, in the regulation of endothelial contractile machinery and barrier function was studied. METHODS AND RESULTS Endothelial contractile machinery and barrier function were analysed in cultured human umbilical vein endothelial cells (HUVEC). The cAMP analogues 8-CPT-cAMP and 6-Bnz-cAMP were used to activate Epac and PKA, respectively, and forskolin (FSK) was used to activate adenylyl cyclase. The cells were challenged by thrombin to inhibit MLCP via the RhoA/Rock pathway. Activation of either PKA or Epac partially blocked thrombin-induced hyperpermeability. Simultaneous activation of PKA and Epac had additive effects that were comparable to that of FSK. Activation of PKA but not Epac inhibited thrombin-induced phosphorylation of MLC and the MLCP regulatory subunit MYPT1, partly via inhibition of the RhoA/Rock pathway. FSK activated the MLCP catalytic subunit PP1 via dephosphorylation and dissociation of the PP1 inhibitory protein CPI-17. FSK blunted thrombin-induced CPI-17 phosphorylation, CPI-17/PP1 complex formation, and PP1 inactivation. Down-regulation of CPI-17 attenuated thrombin-induced hyperpermeability and abolished the antagonistic effect of the PKA activator, whereas the Epac activator retained its antagonistic effect. CONCLUSION cAMP/PKA regulates the endothelial barrier via inhibition of the contractile machinery, mainly by the activation of MLCP via inhibition of CPI-17 and RhoA/Rock. The permeability-lowering effect of the cAMP/Epac pathway is independent of CPI-17.

ATP antagonism of thrombin-induced endothelial barrier permeability

OBJECTIVES Thrombin induces endothelial barrier failure by activating the contractile machinery of endothelial cells. Contractile activation is due to an increase in myosin light chain (MLC) phosphorylation. Here, it was investigated whether stimulation of endothelial cells with ATP can interrupt this thrombin-induced pathomechanism. METHODS In cultured human umbilical vein endothelial cells, cytosolic calcium [Ca(2+)](i) (Fura 2 method), phosphorylation of MLC, isometric tension and permeability for albumin were studied. RESULTS Thrombin (0.2 U/ml) increased [Ca(2+)](i) from a basal level of 78+/-8 to 570+/-63 nM (mean+/-S.D., n=5, P<0.05), MLC phosphorylation from 71+/-7 to 163+/-18%, isometric tension from 157+/-17 to 232+/-26 microN, and permeability from 2.8+/-0.4 to 11.6+/-1 x 10(-6) cm/s. Co-presence of ATP (10 microM) and thrombin did not alter the [Ca(2+)](i) rise, but reduced MLC phosphorylation to 59.8+/-10%, isometric tension to 174+/-14 microN, and permeability to 5.4+/-0.6 x 10(-6) cm/s. The thrombin-induced rise in MLC phosphorylation was sensitive to reduction of [Ca(2+)](i) It was accompanied by an increase in Rho activation, and was inhibited by Y-27632 (10 microM), a Rho-kinase blocker. The ATP-induced decrease in MLC phosphorylation was not sensitive to [Ca(2+)](i). It was not accompanied by changes in RhoA activation, and could not by suppressed by Y-27632. CONCLUSIONS ATP antagonizes the Ca(2+)- and Rho-dependent effects of thrombin on MLC phosphorylation most likely by a Ca(2+)- and Rho-independent activation of MLC phosphatase. It thereby functionally antagonizes the thrombin-induced increase in monolayer tension and permeability.

Insulin Stabilizes Microvascular Endothelial Barrier Function via Phosphatidylinositol 3-Kinase/Akt-Mediated Rac1 Activation

Objective—Insulin is a key regulator of metabolism, but it also confers protective effects on the cardiovascular system. Here, we analyze the mechanism by which insulin stabilizes endothelial barrier function. Methods and Results—Insulin reduced basal and antagonized tumor necrosis factor-&agr;-induced macromolecule permeability of rat coronary microvascular endothelial monolayers. It also abolished reperfusion-induced vascular leakage in isolated-perfused rat hearts. Insulin induced dephosphorylation of the regulatory myosin light chains, as well as translocation of actin and vascular endothelial (VE)-cadherin to cell borders, indicating a reduction in contractile activation and stabilization of cell adhesion structures. These protective effects were blocked by genistein or Hydroxy-2-naphthalenylmethylphosphonic acid tris acetoxymethyl ester (HNMPA-[AM]3), a pan-tyrosine-kinase or specific insulin-receptor-kinase inhibitor, respectively. Insulin stimulated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and NO production, and it activated Rac1. Inhibition of PI3K/Akt abrogated Rac1 activation and insulin-induced barrier protection, whereas inhibition of the endothelial nitric oxide synthase/soluble guanylyl cyclase pathway partially inhibited them. Inhibition of Rac1 abrogated the assembly of actin at cell borders. Accordingly, it abolished the protective effect of insulin on barrier function of the cultured endothelial monolayer, as well as the intact coronary system of ischemic-reperfused hearts. Conclusion—Insulin stabilizes endothelial barrier via inactivation of the endothelial contractile machinery and enhancement of cell-cell adhesions. These effects are mediated via PI3K/Akt- and NO/cGMP-induced Rac1 activation.

Transient hypoxia induces ERK-dependent anti-apoptotic cell survival in endothelial cells

Ischemia-induced apoptosis of endothelial cells may contribute to tissue injury, organ failure, and transplantation rejection. However, little is known about survival mechanisms capable to counteract endothelial apoptosis. This study investigated the potential role of an endogenous anti-apoptotic response elicited by transient hypoxia, capable to avert ongoing apoptosis in endothelial cells. Experiments were carried out in three different types of cultured endothelial cells (human umbilical vein, pig aorta, and from rat coronary microvasculature). As a pro-apoptotic challenge endothelial cells were cultured in serum-free medium and subjected to hypoxia for 2 h. We found that transient hypoxia reduced caspase 3 activation within 1 h of hypoxia. Accordingly, the number of apoptotic cells was reduced after 24 h of reoxygenation. This was true for all three cell types analyzed. Analysis of Akt and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways revealed that hypoxia induced a transient activation of ERK 2 but not of Akt. ERK 2 phosphorylation preceded the phosphorylation of pro-apoptotic molecule Bad at Ser112, an inhibitory phosphorylation site specific for ERK. The protective effects of hypoxia regarding Bad phosphorylation, caspase 3 activation, and apoptosis were abolished by MEK 1/2 inhibitors, PD98059 or UO126, as well as by antisense oligonucleotides directed against ERK 1/2. Furthermore, inhibition of this pathway inhibited hypoxia-induced increase in mitochondrial membrane potential. The present study demonstrates that transient hypoxia induces a novel survival mechanism that protects endothelial cells against apoptosis. This endogenous process involves MEK/ERK-mediated inhibition of the pro-apoptotic molecule Bad and caspase 3.

Lithium prevents early cytosolic calcium increase and secondary injurious calcium overload in glycolytically inhibited endothelial cells

Cytosolic free calcium concentration ([Ca(2+)]i) is a central signalling element for the maintenance of endothelial barrier function. Under physiological conditions, it is controlled within narrow limits. Metabolic inhibition during ischemia/reperfusion, however, induces [Ca(2+)]i overload, which results in barrier failure. In a model of cultured porcine aortic endothelial monolayers (EC), we addressed the question of whether [Ca(2+)]i overload can be prevented by lithium treatment. [Ca(2+)]i and ATP were analysed using Fura-2 and HPLC, respectively. The combined inhibition of glycolytic and mitochondrial ATP synthesis by 2-desoxy-d-glucose (5mM; 2-DG) plus sodium cyanide (5mM; NaCN) caused a significant decrease in cellular ATP content (14±1 nmol/mg protein vs. 18±1 nmol/mg protein in the control, n=6 culture dishes, P<0.05), an increase in [Ca(2+)]i (278±24 nM vs. 71±2 nM in the control, n=60 cells, P<0.05), and the formation of gaps between adjacent EC. These observations indicate that there is impaired barrier function at an early state of metabolic inhibition. Glycolytic inhibition alone by 10mM 2-DG led to a similar decrease in ATP content (14±2 nmol/mg vs. 18±1 nmol/mg in the control, P<0.05) with a delay of 5 min. The [Ca(2+)]i response of EC was biphasic with a peak after 1 min (183±6 nM vs. 71±1 nM, n=60 cells, P<0.05) followed by a sustained increase in [Ca(2+)]i. A 24-h pre-treatment with 10mM of lithium chloride before the inhibition of ATP synthesis abolished both phases of the 2-DG-induced [Ca(2+)]i increase. This effect was not observed when lithium chloride was added simultaneously with 2-DG. We conclude that lithium chloride abolishes the injurious [Ca(2+)]i overload in EC and that this most likely occurs by preventing inositol 3-phosphate-sensitive Ca(2+)-release from the endoplasmic reticulum. Though further research is needed, these findings provide a novel option for therapeutic strategies to protect the endothelium against imminent barrier failure.

BDNF and its TrkB receptor in human fracture healing.

Fracture healing is a physiological process of repair which proceeds in stages, each characterized by a different predominant tissue in the fracture gap. Matrix reorganization is regulated by cytokines and growth factors. Neurotrophins and their receptors might be of importance to osteoblasts and endothelial cells during fracture healing. The aim of this study was to examine the presence of brain-derived neurotrophic factor (BDNF) and its tropomyosin-related kinase B receptor (TrkB) during human fracture healing. BDNF and TrkB were investigated in samples from human fracture gaps and cultured cells using RT-PCR, Western blot, and immunohistochemistry. Endothelial cells and osteoblastic cell lines demonstrated a cytoplasmic staining pattern of BDNF and TrkB in vitro. At the mRNA level, BDNF and TrkB were expressed in the initial and osteoid formation phase of human fracture healing. In the granulation tissue of fracture gap, both proteins--BDNF and TrkB--are concentrated in endothelial and osteoblastic cells at the margins of woven bone suggesting their involvement in the formation of new vessels. There was no evidence of BDNF or TrkB during fracture healing in chondrocytes of human enchondral tissue. Furthermore, BDNF is absent in mature bone. Taken together, BDNF and TrkB are involved in vessel formation and osteogenic processes during human fracture healing. The detection of BDNF and its TrkB receptor during various stages of the bone formation process in human fracture gap tissue were shown for the first time. The current study reveals that both proteins are up-regulated in human osteoblasts and endothelial cells in fracture healing.