Objective-Plasma apolipoprotein CIII (apoCIII) independently predicts risk for coronary heart disease (CHD). We recently reported that apoCIII directly enhances adhesion of human monocytes to endothelial cells (ECs), and identified the activation of PKC␣ as a necessary upstream event of enhanced monocyte adhesion. This study tested the hypothesis that apoCIII activates PKC␣ in human monocytic THP-1 cells, leading to NF-B activation. Methods and Results-Among inhibitors specific to PKC activators, phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609 limited apoCIII-induced PKC␣ activation and THP-1 cell adhesion. ApoCIII increased PC-PLC activity in THP-1 cells, resulting in PKC␣ activation. Pertussis toxin (PTX) inhibited apoCIII-induced PC-PLC activation and subsequent PKC␣ activation, implicating PTX-sensitive G protein pathway. ApoCIII further activated nuclear factor-B (NF-B) through PKC␣ in THP-1 cells and augmented ␤1-integrin expression. The NF-B inhibitor peptide SN50 partially inhibited apoCIII-induced ␤1-integrin expression and THP-1 cell adhesion. ApoCIII-rich VLDL had similar effects to apoCIII alone. Conclusions-PTX-sensitive G protein pathway participates critically in PKC␣ stimulation in THP-1 cells exposed to apoCIII, activating NF-B, and increasing ␤1-integrin. This action causes monocytic cells to adhere to endothelial cells. Key Words: apolipoproteins Ⅲ atherosclerosis Ⅲ adhesion molecules Ⅲ leukocytes Ⅲ signal transduction P revious clinical studies recognized apoCIII as an independent risk factor for coronary heart disease (CHD), 1-3 especially as a component of apoB lipoproteins. 1,4 Excessive apoCIII delays lipolysis of apoB lipoproteins 5 and inhibits their uptake by normal high-affinity receptors on hepatocytes, 6 causing hypertriglyceridemia. However, the direct effect of apoCIII on vascular cells or leukocytes remained untested. We recently reported that apoCIII alone or apoCIII-rich VLDL or LDL augment THP-1 cell adhesion to vascular ECs under static or flow conditions. 7 Protein kinase C (PKC) ␣ participated critically in this process. Ca 2ϩ , phospholipids, and diacylglycerol (DAG) activate PKC␣, one of the conventional PKC isoforms. 8,9 The literature currently provides little information regarding possible direct effects of apoCIII on these molecules, and the mechanism for PKC␣ activation by apoCIII remains undetermined. PKC participates importantly in several mechanisms in atherogenesis including monocyte-endothelial interaction. 8,10 However, the downstream pathway of PKC in apoCIII-treated THP-1 cells remains incompletely understood, although activation of RhoA partially contributes to the effect of apoCIII. 7 The present study demonstrates that pertussis toxin (PTX)-sensitive G protein and phosphatidylcholine-specific phospholipase C (PC-PLC) mediate apoCIII-induced PKC␣ activation, and that apoCIII-induced PKC␣ activation induces activation of nuclear factor-B (NF-B), a key regulator for inflammation in atherogenesis. Indeed, such activation leads to increased...

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The reaction of cytidine with hydrazine to give N4-aminocytidine was greatly promoted by addition of a less-than-stoichiometric amount of bisulfite, and the product was isolated in a good yield. N4-Aminocytidine was strongly mutagenic to bacteria (Salmonella typhimurium TA100 and TA1535, and E. coli WP2 uvrA) and to phage (phi X174 am3). The activity did not require the presence of mammalian microsomal fraction in the system. The mutagenic potency of N4-aminocytidine in these systems was two orders of magnitude greater than that of N4-amino-2'-deoxycytidine, and more than two orders of magnitude greater than that of N4-hydroxycytidine. The greater activity of the riboside than the deoxyriboside was ascribed to the lack of deoxycytidine kinase in these cells. This compound may be useful as a powerful mutagen to induce a transition mutation in microorganisms.

Background-Although potential participation of bone marrow-derived circulating endothelial progenitor cells (EPCs) to neoangiogenesis has been proposed, the precise molecular mechanisms of EPC recruitment to vascular endothelium has not been fully elucidated. Methods and Results-Peripheral blood mononuclear cells were isolated from healthy volunteers and cultured for 7 days to obtain EPCs. Tumor necrosis factor-␣-activated human umbilical vein endothelial cells (HUVECs) supported significantly more rolling and adhesion of EPCs compared with inactivated HUVEC monolayer. Pretreatment of activated HUVEC with an adhesion-blocking mAb to E-selectin significantly reduced EPCs adhesion to HUVECs. When HUVECs were transduced with a recombinant adenovirus of E-selectin (AdRSVE-sel) or that of ␤-galactosidase (AdRSVLacZ), E-selectin-transduced but not LacZ-transduced HUVECs exhibited significantly more EPC rolling as well as adhesion. Further, effect of AdRSVE-sel or AdRSVLacZ was examined in mouse hind limb ischemic model. AdRSVE-sel-transduced mice showed significantly less limb necrosis and higher laser Doppler ratio when compared with AdRSVLacZ-transduced mice. Interestingly, blood flow recovery of ischemic limb observed in AdRSVE-seltransduced mice was more prominent when combined with EPC administration compared with that of AdRSVLacZtransduced mice. Conclusions-Endothelial E-selectin plays a crucial role in EPC-endothelial interaction in vitro. The importance of E-selectin was also confirmed in vivo even in the absence of exogenous EPC. These data provide molecular background for novel cell-based therapy for ischemic atherosclerosis.

Objective-Ultrasound (US)-mediated destruction of contrast microbubbles causes capillary rupturing that stimulates arteriogenesis, whereas intramuscular implantation (im) of bone marrow mononuclear cells (BM-MNCs) induces angiogenesis. We therefore studied whether US-targeted microbubble destruction combined with transplantation of BM-MNCs can enhance blood flow restoration by stimulating both angiogenesis and arteriogenesis. Methods and Results-US-mediated destruction of phospholipid-coated microbubbles was applied onto ischemic hindlimb muscle and subsequently BM-MNCs were transfused. A significant enhancement in blood flow recovery after BubbleϩUSϩBM-MNC infusion (34% increase, PϽ0.05) was observed compared with BubbleϩUS (25%). The ratio of capillary/muscle fiber increased by BubbleϩUSϩBM-MNC-i.v (260%, PϽ0.01) than that in the BubbleϩUS group (172%), into which BM-MNCs were incorporated (angiogenesis). Smooth muscle ␣-actin-positive arterioles were also increased, and angiography showed augmented collateral vessel formation (arteriogenesis). Platelet-derived proinflammatory factors activated by BubbleϩUS induces the expression of adhesion molecules (P-selectin and ICAM-1), leading to the attachment of transplanted BM-MNCs on the endothelium. Flow assay confirmed that the platelet-derived factors cause the adhesion of BM-MNCs onto endothelium under laminar flow. Conclusions-This

Previously, we discovered single-nucleotide polymorphisms (SNPs) associated with Immunoglobulin A (IgA) nephropathy in selectin genes, which were 712C>T(P238S) in L selectin, -642A>G in the promoter region of L selectin, and 1402C>T(H468Y) in E selectin. Interestingly, these SNPs were in nearly complete linkage disequilibrium, thus two haplotypes, disease-associated TGT and wild-type (Wt) CAC, were constructed. To investigate the functional significance of TGT haplotype, a stable CHO transfectant expressing a P238S-L-selectin variant (CHO-varL) and a recombinant adenovirus vector containing an H468Y-E-selectin variant (Ad-varE) were established and compared to their Wt counterparts. Under flow, CHO-varL exhibited significantly less adhesion over IL-1beta-activated HUVEC monolayers compared to CHO-wtL. Furthermore, a luciferase reporter construct, containing a promoter region of the L-selectin variant (luc-varL), exhibited significantly less transcription activity compared to Wt (luc-wtL). These results suggest that the adhesive interactions and expression level of L selectin in disease-associated haplotypes are significantly compromised, indicating a potential role of these SNPs in the pathogenesis of inflammatory diseases, including IgA nephropathy.

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