Andreas Zakrzewicz

Angiotensin II–Induced Leukocyte Adhesion on Human Coronary Endothelial Cells Is Mediated by E-Selectin

Clinical data suggest a link between the activation of the renin-angiotensin system and cardiovascular ischemic events. Leukocyte accumulation in the vessel wall is a hallmark of early atherosclerosis and plaque progression. E-Selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) are adhesion molecules participating in mediating interactions between leukocytes and endothelial cells and have been found to be expressed in athero-sclerotic plaques. We investigated whether angiotensin II, the effector of the renin-angiotensin system, influences the endothelial expression of E-selectin, VCAM-1, and ICAM-1. In coronary endothelial cells derived from explanted human hearts, angiotensin II (10(-11) to 10(-5) mol/L) induced a concentration-dependent increase in E-selectin expression. The effect was measured by cell ELISA and duplex reverse-transcription polymerase chain reaction (RT-PCR) and reached its maximum at 10(-7) mol/L. Angiotensin II induced only a small increase in E-selectin expression in cardiac microvascular endothelial cells. VCAM-1 and ICAM-1 were not affected by angiotensin II stimulation. In addition, the effect of angiotensin II-induced E-selectin expression on leukocyte adhesion was quantified under flow conditions. Angiotensin II (10(-7) mol/L) increased leukocyte adhesion significantly to 67% of the maximal effect by tumor necrosis factor-alpha at a wall shear stress of 2 dyne/cm2. This adhesion was found to be E-selectin dependent, as demonstrated by blocking antibodies. The AT1-receptor antagonist DUP 753 significantly reduced E-selectin-dependent adhesion, whereas the AT2-receptor antagonist PD 123177 had no inhibitory effect. In addition, only AT1-receptor, but not AT2-receptor, mRNA could be detected by RT-PCR in coronary endothelial cells. Therefore, it is suggested that AT1 receptors mediate the effects of angiotensin II on E-selectin expression and leukocyte adhesion on coronary endothelial cells.

Beta2 integrins (CD11/CD18) promote apoptosis of human neutrophils.

Apoptosis of human polymorphonuclear neutrophils (PMN) is thought to be critical for the control of the inflammatory process, but the mechanisms underlying its regulation in physiological settings are still incompletely understood. This study was undertaken to test the hypothesis that the β2 integrin (CD11/CD18) family of leukocyte adhesion molecules contributes to the control of activated PMN by up‐regulating apoptosis. Apoptosis of isolated human PMN was investigated by 1) analysis of DNA content, 2) detection of DNA degradation, 3) morphological studies, and 4) measurement of CD16 expression on the cell surface. We found that β2 integrins potentiated the tumor necrosis factor α (TNF‐α) ‐induced apoptosis within 4 and 8 h after stimulation. The effect required aggregation of the β2 integrin Mac‐1 (CD11b/CD18), which was induced by antibody cross‐linking, and was independent of Fc receptors. An enhancement of apoptosis was also observed after migration of PMN through an endothelial cell monolayer. TNF‐α‐induced apoptosis as well as potentiation by β2 integrins was prevented by inhibition of tyrosine kinases with herbimycin A or genistein. The present study provides a new model for the regulation of PMN apoptosis by a functional cross‐talk between β2 integrins and TNF‐α with a promoting role for the β2 integrins. This mechanism, which allows enhanced elimination of previously emigrated PMN, may be critical to abate local inflammatory processes in vivo.—Walzog, B., Jeblonski, F., Zakrzewicz, A., Gaehtgens, P. β2 integrins (CD11/CD18) promote apoptosis of human neutrophils. FASEB J. 11, 1177–1186 (1997)

Evidence for modulation of genes involved in vascular adaptation by prolonged exposure of endothelial cells to shear stress

OBJECTIVE Shear stress is known to modulate gene expression. However, the molecular link between blood flow and long-time vessel adaptation is still unclear. In this study, the variations of gene expression by prolonged shear stress exposure was investigated in order to identify genes possibly involved in flow dependent vascular adaptation. METHODS Human umbilical vein endothelial cells (HUVECs) were exposed to laminar shear stress (6 dyn/cm(2); 24 h) and analyzed by differential display (DDRT-PCR). Flow-modulation of differentially expressed genes by different exposure times (4, 24, 48 h) and in human cardiac microvascular endothelial cells (HCMECs) (24 h exposure) was analyzed by RT-PCR and northern blotting. RESULTS DDRT-PCR analysis displayed 13 down- and 20 up-regulated products in response to flow. Four known genes were identified: Angiopoietin-2, a protein reported to reduce vessel stability, was progressively (4-48 h) down-regulated by shear stress. The induction of the anti-angiogenic metalloproteinase METH-1 was maximal after 4 h exposure and sustained over the time (24-48 h). Growth arrest-specific mRNA 3 (gas3) and calpactin 1 light chain (p11) were up-regulated only by prolonged exposure (24-48 h). Analysis of the expression of angiopoietin-2, METH-1, gas3, and p11 in shear stress exposed (24 h) HCMECs showed modulation patterns comparable to those observed in HUVECs. CONCLUSION Since angiopoietin-2 and METH-1 are known to be involved in vessel regression/stabilization, the reported modulation of these genes by prolonged shear stress exposure strongly suggests their participation in flow-dependent vascular adaptation.

Cdc2-Like Kinases and DNA Topoisomerase I Regulate Alternative Splicing of Tissue Factor in Human Endothelial Cells

Tumor necrosis factor (TNF)-α–stimulated human umbilical vein endothelial cells express 2 naturally occurring forms of tissue factor (TF), the primary initiator of blood coagulation: the soluble alternatively spliced isoform and the full-length TF isoform. The regulatory pathways enabling this phenomenon are completely unknown. Cdc2-like kinases and DNA topoisomerase I regulate alternative splicing via phosphorylation of serine/arginine-rich proteins. In this study, we examined effects of serine/arginine-rich protein kinases on TF splicing following stimulation with TNF-α. Human endothelial cells were pretreated with specific inhibitors or small interfering RNAs against Cdc2-like kinases and DNA topoisomerase I before stimulation with TNF-α. TF levels were determined by semiquantitative RT-PCR, real-time PCR, and Western blotting. Cellular procoagulant activity was analyzed in a chromogenic TF activity assay. All 4 known Cdc2-like kinases forms were expressed in human endothelial cells. Selective inhibition of Cdc2-like kinases and DNA topoisomerase I elicited distinct changes in TF biosynthesis in TNF-α–stimulated endothelial cells, which impacted endothelial procoagulant activity. This study is the first to demonstrate that serine/arginine-rich protein kinases modulate splicing of TF pre-mRNA in human endothelial cells and, consequently, endothelial procoagulant activity under inflammatory conditions.

Vascular endothelial growth factor is expressed in endothelial cells isolated from skeletal muscles of nitric oxide synthase knockout mice during prazosin-induced angiogenesis.

In skeletal muscles, angiogenesis can be induced by increases in wall shear stress. To identify molecules involved in the angiogenic process, a method based on the use of BS-1 lectin-coated magnetic beads was developed to isolate a cellular fraction enriched in microvascular endothelial cells which are directly exposed to wall shear stress. Using such cellular fractions from skeletal muscles of C57 mice in which angiogenesis was induced by administration with the alpha(1)-adrenergic antagonist prazosin, we found the concentration of vascular endothelial growth factor (VEGF) increased in correlation to the duration of the prazosin stimulus. In contrast, the angiopoietin-2/tie-2 system was not changed even after 4days of prazosin treatment. In neuronal nitric oxide synthase (nNOS) knockout mice, the VEGF concentration was also elevated after prazosin treatment but remained almost unchanged in endothelial nitric oxide synthase (eNOS) knockout mice. However, eNOS (and not nNOS) knockout mice expressed higher levels of VEGF under non-stimulated conditions as compared to C57 mice. These results suggest that VEGF produced in endothelial cells is involved in angiogenesis in skeletal muscles of mice responding to the administration of systemic vasodilators. NO derived from eNOS and nNOS may be an important regulator of the angiogenic response in skeletal muscles in vivo.

The endothelium as physiological source of properdin: role of wall shear stress.

Properdin is a positive regulator of the alternative pathway of complement activation. It can be released by peripheral blood cells but is not synthesized in the liver and the physiological source of properdin in plasma is unknown. The endothelium is an extra-hepatic source for several complement components and shear stress can modulate their expression. The aim of this study was to analyze shear stress-exposed endothelial cells (EC) as physiological source for plasma properdin. Human umbilical vein EC (HUVEC) and human cardiac microvascular EC (HCMEC) were exposed to shear stress using a cone-and-plate apparatus and properdin expression was analyzed by RT-PCR, Northern, and Western blot. mRNA for properdin is barely detectable in untreated EC but strongly induced by laminar shear stress exposure (6 dyn/cm(2); 24 h). Properdin is induced also at the protein level and is released in the extracellular compartment. Properdin up-regulation requires a shear stress of 2-3 dyn/cm(2), is not transient, and is reversible by restoration of static conditions. Turbulent flow exposure results in two times higher induction of properdin than laminar flow exposure. The ability of endothelial cells exposed to shear stress to synthesize properdin proposes the endothelium as physiological source for plasma properdin and suggests a link between flow conditions and the modulation of the alternative pathway. Furthermore, the stronger properdin induction by turbulent flow may suggest an involvement in the pathology of atherosclerosis.

Regulation of endothelial connexin40 expression by shear stress

Endothelial connexin (Cx)40 plays an important role in signal propagation along blood vessel walls, modulating vessel diameter and thereby blood flow. Blood flow, in turn, has been shown to alter endothelial Cx40 expression. However, the timing and shear stress dependence of this relationship have remained unclear, as have the signal transduction pathways involved and the functional implications. Therefore, the aim of this study was to quantify the effects of shear stress on endothelial Cx40 expression, to analyze the role of phosphoinositide 3-kinase (PI3K)/Akt signaling involved, and to assess the possible functional consequences for the adaptation of microvascular networks. First-passage human umbilical vein endothelial cells were exposed to defined shear stress conditions and analyzed for Cx40 using real-time RT-PCR and immunoblot analysis. Shear stress caused long-term induction of Cx40 protein expression, with two short-term mRNA peaks at 4 and 16 h, indicating the dynamic nature of the adaptation process. Maximum shear stress-dependent induction was observed at shear levels between 6 and 10 dyn/cm(2). Simulation of this pattern of shear-dependent Cx expression in a vascular adaptation model of a microvascular network led to an improved fit for the simulated results to experimental measurements. Cx40 expression was greatly reduced by inhibiting PI3K or Akt, with PI3K activity being required for basal Cx40 expression and Akt activity taking part in its shear stress-dependent induction.

Clinical significance of prostacyclin and thromboxane in cancer of the female breast and genital tract

Studies investigating the role of thromboxane A2 and prostacyclin in cancer of the female breast and genital tract are reviewed. Whereas thromboxane A2 was found to promote the tumour growth and metastasis, prostacyclin exerted a protective effect in maintaining vascular and platelet homeostasis. Thus, monitoring of prostacyclin and thromboxane levels in plasma and urine of cancer patients may be essential for the evaluation of tumour growth and metastasis. Of all modulators of thromboxane and prostacyclin biosynthesis, nafazatrom was found to exhibit promising results for the treatment of advanced breast cancer, although its use in the routine therapy is questionable at this stage.