Background-Bone marrow implantation (BMI) was shown to enhance angiogenesis in a rat ischemic heart model. This preclinical study using a swine model was designed to test the safety and therapeutic effectiveness of BMI. Methods and Results-BM-derived mononuclear cells (BM-MNCs) were injected into a zone made ischemic by coronary artery ligation. Three weeks after BMI, regional blood flow and capillary densities were significantly higher (4.6-and 2.8-fold, respectively), and cardiac function was improved. Angiography revealed that there was a marked increase (5.7-fold) in number of visible collateral vessels. Implantation of porcine coronary microvascular endothelial cells (CMECs) did not cause any significant increase in capillary densities. Labeled BM-MNCs were incorporated into Ϸ31% of neocapillaries and corresponded to Ϸ8.7% of macrophages but did not actively survive as myoblasts or fibroblasts.There was no bone formation by osteoblasts or malignant ventricular arrhythmia. Time-dependent changes in plasma levels for cardiac enzymes (troponin I and creatine kinase-MB) did not differ between the BMI, CMEC, and medium-alone implantation groups. BM-MNCs contained 16% of endothelial-lineage cells and expressed basic fibroblast growth factorӷvascular endothelial growth factorϾangiopoietin 1 mRNAs, and their cardiac levels were significantly upregulated by BMI. Cardiac interleukin-1␤ and tumor necrosis factor-␣ mRNA expression were also induced by BMI but not by CMEC implantation. BM-MNCs were actively differentiated to endothelial cells in vitro and formed network structure with human umbilical vein endothelial cells. Conclusions-BMI may constitute a novel safety strategy for achieving optimal therapeutic angiogenesis by the natural ability of the BM cells to secrete potent angiogenic ligands and cytokines as well as to be incorporated into foci of neovascularization.

A point mutation in the human insulin receptor gene in a patient with type A insulin resistance alters the amino acid sequence within the tetrabasic processing site of the proreceptor molecule from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser. Epstein-Barr virus-transformed lymphocytes from this patient synthesize an insulin receptor precursor that is normally glycosylated and inserted into the plasma membrane but is not cleaved to mature alpha and beta subunits. Insulin binding to these cells is severely reduced but can be increased about fivefold by gentle treatment with trypsin, accompanied by the appearance of normal alpha subunits. These results indicate that proteolysis of the proreceptor is necessary for its normal full insulin-binding sensitivity and signal-transducing activity and that a cellular protease that is more stringent in its specificity than trypsin is required to process the receptor precursor.

S100A12, also called EN-RAGE (extracellular newly identified receptor for advanced glycation end products binding protein) or calcium-binding protein in amniotic fluid-1, is a ligand for RAGE. It has been shown that S100A12 induces adhesion molecules such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in the vascular endothelial cell and mediates migration and activation of monocytes/macrophages through RAGE binding and that infusion of lipopolysaccharide into mice causes time-dependent increase of S100A12 in the plasma. Therefore, circulating S100A12 protein may be involved in chronic inflammation in the atherosclerotic lesion. In this study, we developed an ELISA system that uses specific monoclonal antibodies against recombinant human S100A12 to measure plasma S100A12 levels in patients with diabetes. On using our S100A12 ELISA system, the coefficients of variation of intra- and interassay were less than 4 and 9%, respectively. The analytical lower detection limit was 0.2 ng/ml. When plasma S100A12 levels were measured by this system, the concentrations were more than twice as high in the patients with diabetes, compared with those without. Using univariate analysis in all subjects, plasma S100A12 concentrations correlated with hemoglobin A1c, fasting glucose, high-sensitivity C-reactive protein and white blood cell count. Stepwise multiple regression analyses, however, revealed that only white blood cell count and hemoglobin A1c remained significant independent determinants of plasma S100A12 concentration. These results suggest that plasma S100A12 protein levels are regulated by factors related to subclinical inflammation and glucose control in patients with type 2 diabetes.

The insulin receptor exists as two isoforms, A and B, that result from alternative splicing of exon 11 in the primary transcript. We have shown previously that the alternative splicing is developmentally and hormonally regulated. Consequently, these studies were instigated to identify sequences within the primary RNA transcript that regulate the alternative splicing. Minigenes containing exons 10, 11, and 12 and the intervening introns were constructed and transfected into HepG2 cells, which contain both isoforms of the insulin receptor. The cells were able to splice the minigene transcript to give both A (؊ exon 11) and B-like (؉ exon 11) RNAs. A series of internal deletions within intron 10 were tested for their ability to give A and B RNAs. Intron 10 contained two sequences that modulated exon 11 inclusion; a 48-nucleotide purine-rich sequence at the 5 end of intron 10 that functions as a splicing enhancer and causes an increase in exon 11 inclusion, and a 43-nucleotide sequence at the 3 end of intron 10 upstream of the branch point sequence that favors skipping of exon 11. Increasing the length of the polypyrimidine tract at the 3 end of intron 10 caused exon 11 to be spliced constitutively, indicating that a weak splice site is required for alternative splicing. Finally, point mutations, insertions, and deletions within exon 11 itself were able to regulate inclusion of the exon both positively and negatively. The human insulin receptor (IR)1 is encoded by a single gene that is located on chromosome 19 and composed of 22 exons. The mature IR exists as two isoforms, designated A and B, which result from alternative splicing of the primary transcript (1-3). The A isoform lacks exon 11, is expressed ubiquitously, and is the only isoform in lymphocytes, brain, and spleen; the B isoform contains exon 11 and is expressed predominantly in liver, muscle, adipocytes, and kidney (4 -6). Exon 11 is composed of 36 nucleotides that encode a 12-amino acid segment (residues 717-728) of the carboxyl terminus of the ␣-subunit of IR. A number of investigators have suggested that the isoform ratio could be altered in non-insulin-dependent diabetes mellitus (7-10), but other studies have produced conflicting results (11-14). We have found that alterations in isoform ratio in skeletal muscle were associated with hyperinsulinemia rather than diabetes (15). Similar results have been found in the rhesus monkey (16). Along these lines, Sell and co-workers (17) have shown that alternative splicing of the IR gene is regulated by insulin in the Fao hepatoma cell line. Furthermore, we have shown that the alternative splicing is hormonally and developmentally regulated in both the HepG2 hepatoma and 3T3-L1 adipocyte cell lines (18). The changes in splicing were accompanied by increases in insulin sensitivity, as measured by a number of parameters (19). These data indicate that regulation of the alternative splicing of the IR is important for insulin sensitivity and responsiveness.Splicing of pre-mRNA depends on the presence of relatively...

Background: S100A12, also known as EN-RAGE (extracellular newly identified receptor for advanced glycation end products binding protein) is a ligand for RAGE, and has been proposed to contribute to the development of atherosclerosis. In this study, we examined the plasma S100A12 concentration in patients with ESRD and undergoing hemodialysis (HD) and evaluated the relation between S100A12 level and carotid intimal media thickness (IMT) by ultrasound. Methods: We measured plasma S100A12 concentration in 72 HD patients and 42 control subjects. IMT of the carotid artery was measured by high-resolution B-mode ultrasonography in 46 HD patients. Results: The mean plasma S100A12 level was 2.3-fold higher in HD patients than in control subjects (25.0 ± 2.32 vs. 10.7 ± 0.97 ng/ml, p < 0.001). Stepwise multiple regression analysis identified circulating white blood cell count as a positive independent determinant and total cholesterol and serum albumin levels as negative independent determinants of plasma S100A12 concentration. The maximum IMT was positively correlated with plasma S100A12 level. Stepwise multiple regression analysis also identified plasma S100A12 as a significant independent determinant of the maximum IMT. Conclusion: These findings suggest that S100A12 protein is involved in the acceleration of atherosclerosis in HD patients.

The present study demonstrated that HB-EGF processed and released via PC-PLC-PKC signaling is an intermediate molecule for TGF-beta-mediated EGFR transactivation, and subsequent activation of ERK and p38MAPK is involved in FN expression via transcriptional regulation without requiring new protein synthesis.

Ang II-mediated FN expression was regulated by autocrine effects of HB-EGF and TGF-beta, suggesting a novel paradigm for cross-talk between Ang II and growth factor receptor signaling pathways.

SummaryBackground and objectives S100A12 is an endogenous receptor ligand for advanced glycation end products. Cardiovascular disease remains a major cause of morbidity and mortality in patients with chronic kidney disease. In this study, we report cross-sectional data on 550 hemodialysis patients and assess the relationship between plasma S100A12 level and cardiovascular disease.Design, setting, participants, & measurements A cross-sectional study of 550 maintenance hemodialysis patients was conducted. We investigated the past history of cardiovascular disease and quantified the plasma level of S100A12 protein in all participants.Results Plasma S100A12 level was higher in hemodialysis patients with cardiovascular disease (n ϭ 197; 33.8 Ϯ 28.1 ng/ml) than in those without it (n ϭ 353; 20.2 Ϯ 16.1 ng/ml; P Ͻ 0.001). In multivariate logistic regression analysis, the plasma S100A12 level (odds ratio [OR], 1.28; 95% confidence interval [CI], 1.13 to 1.44; P Ͻ 0.001) was identified as an independent factor associated with the prevalence of cardiovascular disease. The other factors associated with the prevalence of cardiovascular diseases were the presence of diabetes mellitus (OR, 2.81; 95% CI, 1.79 to 4.41; P Ͻ 0.001) and high-sensitivity CRP level (OR, 1.02; 95% CI, 1.00 to 1.05; P ϭ 0.046). Furthermore, the plasma S100A12 level (OR, 1.30; 95% CI, 1.09 to 1.54; P ϭ 0.004) was significantly associated with cardiovascular disease even in hemodialysis patients without diabetes mellitus (n ϭ 348). ConclusionsThese results suggest that the plasma S100A12 protein level is strongly associated with the prevalence of cardiovascular disease in hemodialysis patients.