The Effects of Modulating eNOS Activity and Coupling on Leukocyte-endothelial Interactions in Rat Mesenteric Postcapillary Venules

Background: Leukocyte-endothelial interactions associated with vascular injury are attenuated by endothelial-derived nitric oxide (NO). Endothelial NO synthase (eNOS) in the presence of tetrahydrobiopterin (BH4) produces NO from L-arginine and is termed eNOS coupling. However, when the ratio of dihydrobiopterin (BH2) to BH4 is increased, eNOS becomes uncoupled and produces superoxide instead of NO. Protein kinase C epsilon (PKC ε) positively regulates eNOS activity. This study examined modulating eNOS activity and coupling by superfusing BH2 (100 μM) by itself, combined with PKC ε activator (10μM) or PKC ε inhibitor, or combined with BH4 (100μM) and PKC ε activator in rat mesenteric venules

The Effects of Modulating eNOS Activity and Coupling on Leukocyte-Endothelial Interactions in Rat Mesenteric Postcapilary Venules

Leukocyte-endothelial interactions associated with vascular injury are attenuated by endothelial-derived nitric oxide (NO). Endothelial NO synthase (eNOS) in the presence of the essential cofactor, tetrahydrobiopterin (BH4) (oxidized form of BH4) produces NO from L-arginine and is termed eNOS coupling, under normal conditions. However, when the ratio of dihydrobiopterin (BH2) to BH4 is increased, eNOS becomes uncoupled and produces superoxide (SO) instead of NO and is subsequently converted to hydrogen peroxide as a result. Uncoupled eNOS contributes to oxidative stress and decreases NO bioavailability. When NO is decreased, endothelial dysfunction is the result and promotes an inflammatory event characterized by increased leukocyteendothelial interactions. We have found that superfusion of BH2 induces inflammatory responses. Protein kinase C epsilon (PKC Ɛ) positively regulates eNOS activity. To understand the interaction relationship between eNOS regulation and eNOS coupling/uncoupling cofactor status (i.e. BH4 and BH2) in vascular injury associated inflammatory responses, the PKC Ɛ activator/inhibitor peptide will be combined with BH4/BH2• Both PKC Ɛ activator/inhibitor peptide are cell myristoylated peptides that either promote or inhibit PKC Ɛ translocation to the cell membrane to increase or decrease eNOS activity, respectively. The role of the regulation of eNOS activity under increased BH4 with PKC Ɛ activator and inhibitor in a BH2-induced inflammation in vivo model has not be characterized. This study examined modulating eNOS activity and coupling/uncoupling on inflammatory responses by superfusing BH2 (100 )µM) by itself, combined with PKC Ɛ activator (10µM) or PKC Ɛ inhibitor (10µM), or combined with BH4 (100µM) and PKC Ɛ activator, or PKC Ɛ inhibitor in rat mesenteric venules. Intravital microscopy was used to evaluate leukocyte endothelial interactions within a 2 hr period.We found that BH2 (n=5, P2 (100 µM)combined with PKC Ɛ + (10 µM, n=5, P4 (n=6) significantly attenuated BH2 induced leukocyte rolling, adherence, and transmigration. The BH2 induced response on all three leukocyte endothelial interactions was significantly attenuated by PKC Ɛ- by itself ( 10 µM, n=6, P4 (n=5, P\u3c0.01). These results were further confirmed by hematoxylin/eosin staining which showed significantly decreased vascular leukocyte adherence and transmigration in PKC Ɛ+ with BH4 (n=6, P4 (n=5, P2(n=6, P0.01) treated rats compared to BH2. The data suggest that eNOS uncoupling may be an important mechanism mediating inflammation-induced vascular injury and that promoting eNOS coupling or inhibiting uncopuled eNOS can attenuate BH2-induced inflammation

DNA methylation age in paired tumor and adjacent normal breast tissue in Chinese women with breast cancer

Background Few studies have examined epigenetic age acceleration (AA), the diference between DNA methylation (DNAm) predicted age and chronological age, in relation to somatic genomic features in paired cancer and normal tissue, with less work done in non-European populations. In this study, we aimed to examine DNAm age and its associations with breast cancer risk factors, subtypes, somatic genomic profles including mutation and copy number alterations and other aging markers in breast tissue of Chinese breast cancer (BC) patients from Hong Kong. Methods We performed genome-wide DNA methylation profling of 196 tumor and 188 paired adjacent normal tissue collected from Chinese BC patients in Hong Kong (HKBC) using Illumina MethylationEPIC array. The DNAm age was calculated using Horvath’s pan-tissue clock model. Somatic genomic features were based on data from RNA sequencing (RNASeq), whole-exome sequencing (WES), and whole-genome sequencing (WGS). Pearson’s correlation (r), Kruskal–Wallis test, and regression models were used to estimate associations of DNAm AA with somatic features and breast cancer risk factors. Results DNAm age showed a stronger correlation with chronological age in normal (Pearson r=0.78, P<2.2e−16) than in tumor tissue (Pearson r=0.31, P=7.8e−06). Although overall DNAm age or AA did not vary signifcantly by tissue within the same individual, luminal A tumors exhibited increased DNAm AA (P=0.004) while HER2-enriched/basal-like tumors exhibited markedly lower DNAm AA (P=<.0001) compared with paired normal tissue. Consistent with the subtype association, tumor DNAm AA was positively correlated with ESR1 (Pearson r=0.39, P=6.3e−06) and PGR (Pearson r=0.36, P=2.4e−05) gene expression. In line with this, we found that increasing DNAm AA was associated with higher body mass index (P=0.039) and earlier age at menarche (P=0.035), factors that are related to cumulative exposure to estrogen. In contrast, variables indicating extensive genomic instability, such as TP53 somatic mutations, high tumor mutation/copy number alteration burden, and homologous repair defciency were associated with lower DNAm AA.</p