Abstract-Postnatal

(22% ؎ 2% versus 13% ؎ 3%, P < .05 and KDR (49% ؎ 12% versus 19% ؎ 7%, P < .05). Fluorescence and confocal laser microscopy demonstrated the uptake of apoptotic bodies by the EPCs. Apoptotic bodies-depleted medium had no effect, whereas the incubation with suspension of apoptotic bodies induced effects similar to those of ABRM. Our results suggest that apoptotic bodies from ECs are taken up by EPCs, increasing their number and differentiation state. Such a mechanism may facilitate the repair of injured endothelium and may represent a new signaling pathway between progenitor and damaged somatic cells.

Cell adhesion to endothelial cells stimulated by tumor necrosis factor-a (TNF) is due to induction of surface receptors, such as vascular cell adhesion molecule-1 (VCAM-1). The antiaxidant pyrrolidine dithiocarbamate (PDTC) specifically inhibits activation of nuclear factor-KB (NF-KB). Since *B motifs are present in VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) promoters, we used PDTC to study the regulatory mechanisms of VCAM-1 and ICAM-1 induction and subsequent monocyte adhesion in TNF-treated human umbilical vein endothelial cells (HUVECs). PDTC or JV-acetylcysteine dose dependently reduced TNF-induced VCAM-1 but not ICAM-1 surface protein (also in human umbilical arterial endothelial cells) and mRNA expression (by 70% at 100 junol/L PDTC) in HUVECs as assessed by flow cytometry and porymerase chain reaction. Gel-shift analysis in HUVECs demonstrated that PDTC prevented NF-KB mobilization by TNF, suggesting that only VCAM-1 induction was controlled by NF-KB. Since HUVECs released superoxide anions in response to TNF, and H 2 O 2

The reduction of CD11b expression and inhibition of CD11b-dependent monocyte adhesion to endothelium may crucially contribute to the clinical benefit of HMG-CoA reductase inhibitors in CHD, independent of cholesterol-lowering effects.

Our data suggest that aspirin inhibits NF-kappa B mobilization, induction of VCAM-1 and E-selectin, and subsequent monocyte adhesion in endothelial cells stimulated by TNF, thereby providing an additional mechanism for therapeutic effects of aspirin.

Incorporation of the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) but not eicosapentaenoic acid or n-6 arachidonic acid into human umbilical vein endothelial cell (HUVEC) phospholipids dose-dependently reduced tumor necrosis factor-alpha (TNF-alpha)-induced surface expression of vascular cell adhesion molecule-1 (VCAM-1). In parallel, DHA inhibited TNF-alpha-stimulated monocytic U937 cell adhesion to HUVECs but did not affect TNF-alpha- or interferon gamma-induced expression of intercellular adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 or VCAM-1 induction by interleukin-1 beta. DHA appeared to attenuate VCAM-1 transcription, as it reduced induction of VCAM-1 mRNA by TNF-alpha. VCAM-1 induction is regulated by activation of nuclear factor-kappa B, which can be mediated by a TNF-alpha-responsive phosphatidylcholine-specific phospholipase C (PC-PLC). Gel-shift analysis showed inhibition of TNF-alpha-induced nuclear factor-kappa B mobilization by DHA. While the PC-PLC inhibitor D609 dose-dependently prevented VCAM-1 induction by TNF-alpha, 1,2-diacyl-glycerol (DAG) stimulated VCAM-1 expression, suggesting that VCAM-1 induction by TNF-alpha may be mediated by activation of PC-PLC. Treatment with DHA resulted in a fourfold enrichment in PC. In addition, DHA or D609 but not eicosapentaenoic acid or arachidonic acid suppressed activation of PC-PLC by TNF-alpha, estimated as [14C]DAG synthesis in prelabeled HUVECs. Incorporation of DHA into phospholipids selectively attenuates VCAM-1 induction by TNF-alpha and subsequent monocytic cell adhesion by inhibition of TNF-alpha-stimulated PC-PLC activation in HUVECs.

—Apoptosis is important in normal development as well as in diseases such as atherosclerosis. However, the regulation of apoptosis is still not completely understood. We now show that the transcription factor nuclear factor-κB (NF-κB) controls the induction of apoptosis in human and rat vascular smooth muscle cells (SMCs). SMCs in high-density culture exhibited a high NF-κB activity and were insensitive to induction of apoptosis. Inhibition of NF-κB by adenovirus-mediated overexpression of its inhibitor IκBα caused a marked increase in cell death at low but not high cell density. Elevating endogenous IκBα levels by inhibiting its degradation with proteasomal inhibitors resulted in induction of apoptosis in low-density SMCs, as detected by increased binding of annexin V, reduced mitochondrial membrane potential, and increased hypodiploid DNA. In high-density cultures, protection against apoptosis was associated with the expression of inhibitor of apoptosis protein-1 (IAP-1). Transfer of IκBα reduced human IAP-1 mRNA levels, which suggested that IAP-1 is transcriptionally regulated by NF-κB. This was confirmed through identification of a motif with NF-κB–like binding activity in the human IAP-1 promoter region. Moreover, antisense inhibition of IAP-1 sensitized high-density SMCs to the induction of cell death. Together, our data imply that SMCs at high density are protected by an antiapoptotic mechanism that involves increased expression of NF-κB and IAP-1. Interference with pathways that control the susceptibility to programmed cell death may be helpful in the treatment of diseases where dysregulation of apoptosis is involved, eg, atherosclerosis and restenosis.

Despite the fact that lysophosphatidic acid (LPA) has been identified as a main platelet-activating lipid of mildly oxidized low-density lipoprotein (LDL) and human atherosclerotic lesions, it remains unknown whether it is capable of activating platelets in blood. We found that LPA at concentrations slightly above plasma levels induces platelet shape change, aggregation, and platelet-monocyte aggregate formation in blood. 1-alkyl-LPA (16:0 fatty acid) was almost 20-fold more potent than 1-acyl-LPA (16:0). LPA directly induced platelet shape change in blood and platelet-rich plasma obtained from all blood donors. However, LPA-stimulated platelet aggregation in blood was donor dependent. It could be completely blocked by apyrase and antagonists of the platelet adenosine diphosphate (ADP) receptors P2Y 1 and P2Y 12 . These substances also inhibited LPA-induced aggregation of platelet-rich plasma and aggregation and serotonin secretion of washed platelets. These results indicate a central role for ADP-mediated P2Y 1 and P2Y 12 receptor activation in supporting LPA-induced platelet aggregation. Platelet aggregation and platelet-monocyte aggregate formation stimulated by LPA was insensitive to inhibition by aspirin. We conclude that LPA at concentrations approaching those found in vivo can induce platelet shape change, aggregation, and platelet-monocyte aggregate formation in whole blood and suggest that antagonists of platelet P2Y 1 and P2Y 12 receptors might be useful preventing LPA-elicited thrombus formation in patients with cardiovascular