Inflammatory response of tracheobronchial epithelial cells to endotoxin.

Respiratory epithelial cells play a crucial role in the inflammatory response in endotoxin-induced lung injury, an experimental model for acute lung injury. To determine the role of epithelial cells in the upper respiratory compartment in the inflammatory response to endotoxin, we exposed tracheobronchial epithelial cells (TBEC) to lipopolysaccharide (LPS). Expression of inflammatory mediators was analyzed, and the biological implications were assessed using chemotaxis and adherence assays. Epithelial cell necrosis and apoptosis were determined to identify LPS-induced cell damage. Treatment of TBEC with LPS induced enhanced protein expression of cytokines and chemokines (increases of 235-654%, P < 0.05), with increased chemotactic activity regarding neutrophil recruitment. Expression of the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was enhanced by 52-101% (P < 0.0001). This upregulation led to increased adhesion of neutrophils, with >95% adherence to TBEC after LPS stimulation, which could be blocked by either ICAM-1 (69%) or VCAM-1 antibodies (55%) (P < 0.05). Enhanced neutrophil-induced necrosis of TBEC was observed when TBEC were exposed to LPS. Reduced neutrophil adherence by ICAM-1 or VCAM-1 antibodies resulted in significantly lower TBEC death (52 and 34%, respectively, P < 0.05). Therefore, tight adherence of neutrophils to TBEC appears to promote epithelial cell killing. In addition to indirect effector cell-induced TBEC death, direct LPS-induced cell damage was seen with increased apoptosis rate in LPS-stimulated TBEC (36% increase of caspase-3, P < 0.01). These data provide evidence that LPS induces TBEC killing in a necrosis- and apoptosis-dependent manner.

Effects of tacrolimus or sirolimus on proliferation of vascular smooth muscle and endothelial cells.

Local strategies directed against vascular smooth muscle cell (VSMC) proliferation such as drug-eluting stents reduce the occurrence of restenosis. However, these approaches may also inhibit endothelial cell (EC) proliferation and, thus, impair reendothelialization. We compared the effects of tacrolimus on human VSMC and EC proliferation and migration to sirolimus, a compound with similar molecular structure. Thymidine incorporation was determined in growth factor-stimulated VSMC and EC. The drug concentration at which maximal VSMC proliferation was inhibited by 50% (IC50) was about 10-fold higher for tacrolimus (3.8 x 10 M) than for sirolimus (4.1 x 10 M; P = 0.055). It is interesting that the molar IC50 value in EC was around 10-fold higher for tacrolimus (2.3 x 10 M) than for sirolimus (7.1 x 10 M; P < 0.01). The profile of these drugs on VSMC and EC migration was similar to the one found in the proliferation assays. Inhibition of VSMC proliferation by both tacrolimus and sirolimus was associated with upregulation of the cell-cycle inhibitor p27. Thus, tacrolimus is less potent than sirolimus for inhibiting VSMC proliferation or migration. However, tacrolimus exerts markedly less antiproliferative effects on EC compared with sirolimus. In combination with its potent antiinflammatory effects, tacrolimus may represent a promising compound for the use in drug-eluting stents.

Selective inhibition of protein kinase Cbeta2 prevents acute effects of high glucose on vascular cell adhesion molecule-1 expression in human endothelial cells.

BACKGROUND: Enhanced expression of adhesion molecules by the endothelium may account for vascular damage in diabetics and nondiabetic patients who develop stress hyperglycemia during acute myocardial infarction. We analyzed the phosphorylation of protein kinase Cbeta2 (PKCbeta2) at serine/threonine residues, which may contribute to the endothelial dysfunction during acute hyperglycemia. Furthermore, this study was designed to investigate whether selective blockade of this regulatory mechanism may prevent the development of endothelial hyperadhesiveness. METHODS AND RESULTS: Incubation of the human aortic endothelial cells with high glucose (22.2 mmol/L) resulted in significant increase of vascular cell adhesion molecule (VCAM)-1 protein expression (172+/-15% versus control; P<0.01). Phorbol 12-myristate 13-acetate, a potent activator of PKC, mimicked the effect of high glucose on VCAM-1 expression. High glucose led to a rapid increase (181+/-22% versus control; P<0.01) of membrane-bound PKCbeta, reflecting activation of this enzyme. The nonselective inhibitor of PKCbeta1 and PKCbeta2 isoforms LY379196, as well as CGP53353, a highly selective inhibitor of PKCbeta2, prevented in a dose-dependent manner upregulation of VCAM-1. Incubation with high glucose was associated with increased PKCbeta2 phosphorylation at the Ser-660 residue, and both LY379196 and CGP53353 prevented this event. Exposure of the cells to high glucose also reduced the protein level of the inhibitory subunit of nuclear factor-kappaB, IkappaBalpha, leading to its enhanced binding activity. Selective inhibition of PKCbeta abolished IkappaBalpha degradation. CONCLUSIONS: Our findings demonstrate for the first time that phosphorylation of Ser-660 represents a selective regulatory mechanism for glucose-induced upregulation of VCAM-1. Therefore, PKCbeta2-selective inhibitors may be promising drugs for treatment of endothelial dysfunction during acute hyperglycemia and possibly in diabetes.

Identification of a novel proliferation-inducing determinant using lentiviral expression cloning.

One of the major challenges in the post-genome era is the correlation between genes and function or phenotype. We have pioneered a strategy for screening of cDNA libraries, which is based on sequential combination of lentiviral and oncoretroviral expression systems and can be used to identify proliferation-modulating genes. Screening of a lentiviral expression library derived from adult human brain cDNA resulted in cloning of the potent proliferation-inducing determinant termed pi1 (proliferation inducer 1). Transduction experiments using GFP-expressing oncoretroviruses to target proliferation-competent cells suggested that overexpression of pi1 initiates proliferation of human umbilical vein endothelial cells (HUVECs). Growth induction of HUVECs as well as Swiss3T3 fibroblasts was confirmed by Brd-uridine incorporation assays, which correlated increased DNA synthesis with expression of pi1. The identified pi1 cDNA is 297 bp long and encodes a 10 kDa polypeptide. Since deregulation of proliferation control accounts for a number of today's untreatable human diseases such as neurodegenerative disorders and cancer, discovery of novel proliferation-modulating genes is essential for developing new strategies for gene therapy and tissue engineering.

High glucose causes upregulation of cyclooxygenase-2 and alters prostanoid profile in human endothelial cells: role of protein kinase C and reactive oxygen species.

BACKGROUND: Prostaglandins generated by cyclooxygenase (COX) have been implicated in hyperglycemia-induced endothelial dysfunction. However, the role of individual COX isoenzymes as well as the molecular mechanisms linking oxidative stress and endothelial dysfunction in diabetes remains to be clarified. METHODS AND RESULTS: Human aortic endothelial cells were exposed to normal (5.5 mmol/L) and high (22.2 mmol/L) glucose. Glucose selectively increased mRNA and protein expression of COX-2. Its upregulation was associated with an increase of thromboxane A2 and a reduction of prostacyclin (PGI2) release. Glucose-induced activation of PKC resulted in the formation of peroxynitrite and tyrosine nitration of PGI2 synthase. NO release was reduced despite 2-fold increase of endothelial NO synthase expression. Phorbol ester caused an increase of COX-2 and endothelial NO synthase expression similar to that elicited by glucose. These effects were prevented by the PKC inhibitor calphostin C. N-acetylcysteine, vitamin C, and calphostin C prevented ROS formation, restored NO release, and reduced colocalization of nitrotyrosine and PGI2 synthase. Expression of p22(phox), a subunit of NAD(P)H oxidase, was increased, and diphenyleneiodonium inhibited ROS formation. By contrast, indomethacin did not affect glucose-induced ROS generation. CONCLUSIONS: Thus, high glucose, via PKC signaling, induces oxidative stress and upregulation of COX-2, resulting in reduced NO availability and altered prostanoid profile.

Cell-type-specific characteristics modulate the transduction efficiency of adeno-associated virus type 2 and restrain infection of endothelial cells.

Adeno-associated viruses (AAVs) are promising vectors for various gene therapy applications due to their long-lasting transgene expression and wide spectrum of target cells. Recently, however, it has become apparent that there are considerable differences in the efficiencies of transduction of different cell types by AAVs. Here, we analyzed the efficiencies of transduction and the transport mechanisms of AAV type 2 (AAV-2) in different cell types, emphasizing endothelial cells. Expression analyses in both cultured cells and the rabbit carotid artery assay showed a remarkably low level of endothelial cell transduction in comparison to the highly permissive cell types. The study of the endosomal pathways of AAV-2 with fluorescently labeled virus showed clear targeting of the Golgi area in permissive cell lines, but this phenomenon was absent in the endothelial cell line EAhy-926. On the other hand, the response to the block of endosomal acidification by bafilomycin A1 also showed differences among the permissive cell types. We also analyzed the effect of proteasome inhibitors on endothelial cells, but their impact on the primary cells and in vivo was not significant. On the contrary, analysis of the expression pattern of heparan sulfate proteoglycans (HSPGs), the primary receptors of AAV-2, revealed massive deposits of HSPG in the extracellular matrix of endothelial cells. The matrix-associated receptors may therefore compete for virus binding and reduce transduction in endothelial cells. Accordingly, in endothelial cells detached from their matrix, AAV-2 transduction was significantly increased. Altogether, these results point to a more complex cell-type-specific mode of transduction of AAV-2 than previously appreciated.

Statin prevents tissue factor expression in human endothelial cells: role of Rho/Rho-kinase and Akt pathways.

BACKGROUND: Tissue factor plays a pivotal role in thrombus formation in acute coronary syndromes. However, the regulatory mechanisms underlying tissue factor expression are poorly understood. Statins are effective in patients with acute coronary syndromes. Hence, the aim of this study was to clarify in human endothelial cells the signaling pathways of thrombin-induced tissue factor expression and potential inhibitory effects of statins. METHODS AND RESULTS: In human aortic endothelial cells, simvastatin prevented tissue factor induction by thrombin (4 U/mL) in a concentration-dependent manner. The increase in tissue factor activity on the cell surface was also blocked by simvastatin. Simvastatin also prevented the upregulation of tissue factor expression and activity in human aortic smooth muscle cells. Mevalonate (100 micromol/L) reversed the inhibitory effect of simvastatin on tissue factor expression. Thrombin induced rapid activation of Rho A and p38 MAP kinase. The Rho-kinase inhibitor Y-27632 and the p38 MAP kinase inhibitor SB203580 prevented tissue factor induction. Akt was dephosphorylated by thrombin; the phosphoinositol 3-kinase inhibitor wortmannin enhanced its dephosphorylation as well as thrombin-induced tissue factor expression. Simvastatin prevented thrombin-induced Rho A activation but not p38 MAP kinase activation. Akt dephosphorylation by thrombin was blocked by both simvastatin and Y-27632. CONCLUSIONS: Endothelial tissue factor induction by thrombin is regulated by Rho/Rho-kinase, Akt, and p38 MAP kinase. Simvastatin prevents its induction through inhibition of Rho/Rho-kinase and activation of Akt. These findings provide new insights into the action of statins in acute coronary syndromes.

Felodipine inhibits nuclear translocation of p42/44 mitogen-activated protein kinase and human smooth muscle cell growth.

OBJECTIVE: Smooth muscle cell (SMC) proliferation contributes to vascular structural changes in cardiovascular disease. Ca(2+) antagonists exert antiproliferative effects and may also be clinically beneficial in the patients. However, the underlying mechanisms of action remain elusive. Activation of mitogen-activated protein kinases (MAPK), in particular p42/44mapk plays a central role in cell proliferation. We hypothesise that Ca(2+) antagonists inhibit cell proliferation by interfering with the p42/44mapk pathway in human SMC. METHODS: SMC were cultured from human aorta. Cell proliferation was analysed by [3H]thymidine incorporation. Activation of p42/44mapk and the nuclear target protein Elk-1 was analysed by phosphorylation and p42/44mapk nuclear translocation by confocal microscope. RESULTS: PDGF-BB (10 ng/ml) stimulated [3H]thymidine incorporation, phosphorylated p42/44mapk, caused nuclear translocation of the enzymes and phosphorylated the nuclear target protein Elk-1. Felodipine (10(-7) to 10(-5) mol/l) inhibited [3H]thymidine incorporation to PDGF-BB, had no effect on p42/44mapk phosphorylation. However, p42/44mapk nuclear translocation and Elk-1 activation stimulated by PDGF-BB were prevented by the Ca(2+) antagonist. CONCLUSION: Activation of p42/44mapk, subsequent nuclear translocation and activation of Elk-1 are essentially associated with human SMC proliferation. The Ca(2+) antagonist felodipine prevents p42/44mapk nuclear translocation (but not its activation) associated with inhibition of human SMC growth.