For the enantioselective and simultaneous analysis of lactate and 3-hydroxybutyrate, a validated online two-dimensional high-performance liquid chromatography system using 4-nitro-7-piperazino-2,1,3-benzoxadiazole as a fluorescent derivatization reagent has been developed. For the reversed-phase separation in the first dimension, a Capcell Pak C18 ACR column (1.5 × 250 mm, particle size 3 μm) was used, and the target fractions were isolated by their hydrophobicity. In the second dimension, a polysaccharide-coated enantioselective column, Chiralpak AD-H (2.0 × 250 mm, 5 μm), was used. The system was validated by the calibration curve, intraday precision, interday precision, and accuracy using standards and real human samples, and satisfactory results were obtained. The present method was applied to human plasma and urine, and in the plasma, trace amounts of d-lactate (8.4 μM) and l-3-hydroxybutyrate (1.0 μM), besides high levels of l-lactate (860.9 μM) and d-3-hydroxybutyrate (59.4 μM), were successfully determined. In urine, trace levels of d-lactate (3.7 μM), d-3-hydroxybutyrate (2.3 μM), and l-3-hydroxybutyrate (3.3 μM) in addition to a relatively large amount of l-lactate (15.4 μM) were observed. The present online two-dimensional high-performance liquid chromatography system is useful for the simultaneous determination of all the lactate and 3-hydroxybutyrate enantiomers in human physiological fluids, and further clinical applications are ongoing.

Objective: Circulating progenitor cells possess vasculogenesis property and participate in repair of vascular injury. Cx (connexin) 43—a transmembrane protein constituting gap junctions—is involved in vascular pathology. However, the role of Cx43 in smooth muscle progenitor cells (SPCs) remained unclear. Approach and Results: Human SPCs cultured from CD34 + peripheral blood mononuclear cells expressed smooth muscle cell markers, such as smooth muscle MHC (myosin heavy chain), nonmuscle MHC, calponin, and CD140B, and Cx43 was the most abundant Cx isoform. To evaluate the role of Cx43 in SPCs, short interference RNA was used to knock down Cx43 expression. Cellular activities of SPCs were reduced by Cx43 downregulation. In addition, Cx43 downregulation attenuated angiogenic potential of SPCs in hind limb ischemia mice. Protein array and ELISA of the supernatant from SPCs showed that IL (interleukin)-6, IL-8, and HGF (hepatocyte growth factor) were reduced by Cx43 downregulation. Simultaneously, Cx43 downregulation reduced the phosphorylation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Akt (protein kinase B) pathway and reactivation of NF-κB and Akt using betulinic acid, and SC79 could restore the secretion of growth factors and cytokines. Moreover, FAK (focal adhesion kinase)-Src (proto-oncogene tyrosine-protein kinase Src) activation was increased by Cx43 downregulation, and inactivation of Akt–NF-κB could be restored by Src inhibitor (PP2), indicating that Akt–NF-κB inactivated by Cx43 downregulation arose from FAK-Src activation. Finally, the depressed cellular activities and secretion of SPCs after Cx43 downregulation were restored by FAK inhibitor PF-562271 or PP2. Conclusions: SPCs possess angiogenic potential to repair ischemic tissue mainly through paracrine effects. Gap junction protein Cx43 plays an important role in regulating cellular function and paracrine effects of SPCs through FAK-Src axis.

Calcium channel antagonists have been reported to have a favorable impact on cyclosporin A (CsA)-treated kidney transplant recipients. However, it is not clear whether this is because of their direct effect on antagonizing the toxicity of CsA to renal tubular cells. In this study, we have used Madin^Darby canine kidney tubular cells as a model to examine the effect of diltiazem, a calcium channel antagonist, on CsA-induced apoptosis. Moreover, to investigate the possible regulation of CsA cytotoxicity by intracellular calcium level, the effect of the calcium ionophore A23187 on CsA-induced apoptosis was also examined. We found that treatment of CsA (20 W WM) alone caused 20^30% cell death, which was apparently (30^40%) enhanced by diltiazem at 100 W Wg/ml, accompanied by more severe DNA fragmentation, activation of caspases, and a decreased level of Bcl-2. The caspase inhibitor ZVAD-fmk or Bcl-2 overexpression was capable of suppressing apoptosis induced by the synergistic effect of diltiazem and CsA. Moreover, the survival rate of cells treated with CsA (30 W WM) alone remained only 30%, however, it was markedly (V V40%) elevated by cotreatment with A23187 (75 ng/ml). The rescue of cells from CsAinduced apoptosis by A23187 was correlated with AKT activation, BAD phosphorylation, and caspase-3 inactivation. Taken together, our results suggest that the reported favorable impact of diltiazem on kidney grafts is likely not because of its direct protection on renal tubular cells. Instead, it enhances the toxicity of CsA to renal tubular cells. In addition, our findings raise a possibility that the intracellular calcium level and the AKT pathway may participate in the regulation of CsA cytotoxicity. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Objective-We examined the effect of thrombomodulin (TM) domains 2 and 3 (TMD23) on human early endothelial progenitor cells (EPCs). Methods and Results-TM was expressed and released by human EPCs cultured from peripheral blood mononuclear cells (PBMCs). Addition of TMD23 (100 ng/mL) to the cultured PBMCs increased the colony-forming units, chemotactic motility, matrix metalloproteinase activity, and interleukin-8 secretion but decreased tumor necrosis factor-␣ (TNF-␣) release. Analysis of the signal pathway showed that TMD23 activated Akt. Inhibition of phosphatidylinositol-3 kinase-Akt blocked the effects of TMD23 on chemotactic motility, matrix metalloproteinase-9, interleukin-8, and TNF-␣. In hindlimb ischemia mice, laser Doppler perfusion imaging of the ischemic limb during the 21 days after arterial ligation showed that the perfusion recovered best with intraperitoneal infusion of TMD23 plus local injection of early EPCs, followed by either infusion of TMD23 or injection of the cells. Animals without either treatment had the worst results. Animals treated with TMD23 also had lower circulating and tissue levels of TNF-␣. Conclusion-TM