Role of the ATP-binding cassette transporter Abcg2 in the phenotype and function of cardiac side population cells

Recently, the side population (SP) phenotype has been introduced as a reliable marker to identify subpopulations of cells with stem/progenitor cell properties in various tissues. We and others have identified SP cells from postmitotic tissues, including adult myocardium, in which they have been suggested to contribute to cellular regeneration following injury. SP cells are identified and characterized by a unique efflux of Hoechst 33342 dye. Abcg2 belongs to the ATP-binding cassette (ABC) transporter superfamily and constitutes the molecular basis for the dye efflux, hence the SP phenotype, in hematopoietic stem cells. Although Abcg2 is also expressed in cardiac SP (cSP) cells, its role in regulating the SP phenotype and function of cSP cells is unknown. Herein, we demonstrate that regulation of the SP phenotype in cSP cells occurs in a dynamic, age-dependent fashion, with Abcg2 as the molecular determinant of the cSP phenotype in the neonatal heart and another ABC transporter, Mdr1, as the main contributor to the SP phenotype in the adult heart. Using loss- and gain-of-function experiments, we find that Abcg2 tightly regulates cell fate and function. Adult cSP cells isolated from mice with genetic ablation of Abcg2 exhibit blunted proliferation capacity and augmented cell death. Conversely, overexpression of Abcg2 is sufficient to enhance cell proliferation, although with a limitation of cardiomyogenic differentiation. In summary, for the first time, we reveal a functional role for Abcg2 in modulating the proliferation, differentiation, and survival of adult cSP cells that goes beyond its distinct role in Hoechst dye efflux.     

Telomere length changes in patients with aplastic anaemia

To investigate telomere changes in patients with aplastic anaemia (AA) and clinical factors influencing the telomere dynamics, telomere length (TL) was measured in peripheral blood mononuclear cells using Southern blot analysis of 42 patients with AA and 39 healthy normal controls. Nineteen patients received supportive treatment only, while the remaining 23 patients received immunosuppressive therapy with anti-thymocyte globulin or anti-lymphocyte globulin +/- cyclosporin A. In AA patients, TL was on average 1.41 kb shorter than that of age-matched normal controls (P < 0.001). In patients treated with immunosuppression, the mean TL of non-responders was significantly shorter than that of age-matched normal controls (P < 0.001), while no difference in TL was detected in responders compared with controls. Positive correlation was observed between the extent of telomere shortening, the severity of neutropenia (P = 0.05) and the degree of mean corpuscular volume elevation (P = 0.005) at the time of the study. However, there was no correlation with time elapsed since diagnosis (P = 0.214). These findings suggest that haematopoietic stem cells in patients with AA rapidly lose TL at the onset of the disease. The TL shortening may reflect the severity of impairment of haematopoiesis.     

NAD(P)H oxidase-dependent platelet superoxide anion release increases platelet recruitment

Platelets, although not phagocytotic, have been suggested to release O. Since O-producing reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases can be specifically activated by certain agonists and are found in several nonphagocytotic tissues, we investigated whether such an enzyme is the source of platelet-derived O. We further studied which agonists cause platelet O release and whether platelet-derived O influences thrombus formation in vitro. Collagen, but not adenosine 5'-diphosphate (ADP) or thrombin, increased O formation in washed human platelets. This was a reduced nicotinamide adenine dinucleotide (NADH)-dependent process, as shown in platelet lysates. Consistent with a role of a platelet, NAD(P)H oxidase expression of its subunits p47(phox) and p67(phox) and inhibition of platelet O formation by diphenylene-iodoniumchloride (DPI) and by the specific peptide-antagonist gp91ds-tat were observed. Whereas platelet-derived O did not influence initial aggregation, platelet recruitment to a preformed thrombus following collagen stimulation was significantly attenuated by superoxide dismutase (SOD) or DPI. It was also inhibited when ADP released during aggregation was cleaved by the ectonucleotidase apyrase. ADP in supernatants of collagen-activated platelets was decreased in the presence of SOD, resulting in lower ADP concentrations available for recruitment of further platelets. Exogenous O increased ADP- concentrations in supernatants of collagen-stimulated platelets and induced irreversible aggregation when platelets were stimulated with otherwise subthreshold concentrations of ADP. These results strongly suggest that collagen activation induces NAD(P)H oxidase-dependent O release in platelets, which in turn enhances availability of released ADP, resulting in increased platelet recruitment.     

Intrahepatic bile duct adenoma in a patient with chronic hepatitis B accompanied by elevation of alpha-fetoprotein

A 51-year-old male patient with chronic hepatitis B was referred to our hospital due to a 1-cm liver nodule on ultrasonography. Alpha-fetoprotein (AFP) was slightly elevated. The nodule showed prolonged enhancement on dynamic liver magnetic resonance imaging and appeared as a hyperintensity on both diffusion-weighted and T2-weighted imaging. The nodule was followed up because it was small and typical findings of hepatocellular carcinoma (HCC) were not observed in the dynamic imaging investigations. However, liver contrast-enhanced ultrasonography performed 1 month later showed enhancement during the arterial phase and definite washout during the delayed phase. Also, AFP had increased to over 200 ng/mL even though AST and ALT were decreased after administering an antiviral agent. He was presumptively diagnosed as HCC and underwent liver segmentectomy. Microscopy findings of the specimen indicated bile duct adenoma. After resection, the follow-up AFP had decreased to within the normal range. This patient represents a case of bile duct adenoma with AFP elevation mimicking HCC on contrast-enhanced ultrasonography.     

Dexmedetomidine-Induced Contraction in the Isolated Endothelium-Denuded Rat Aorta Involves PKC-δ-mediated JNK Phosphorylation

Vasoconstriction mediated by the highly selective alpha-2 adrenoceptor agonist dexmedetomidine leads to transiently increased blood pressure and severe hypertension. The dexmedetomidine-induced contraction involves the protein kinase C (PKC)-mediated pathway. However, the main PKC isoform involved in the dexmedetomidine-induced contraction remains unknown. The goal of this in vitro study was to examine the specific PKC isoform that contributes to the dexmedetomidine-induced contraction in the isolated rat aorta. The endothelium-denuded rat aorta was suspended for isometric tension recording. Dexmedetomidine dose-response curves were generated in the presence or absence of the following inhibitors: the pan-PKC inhibitor, chelerythrine; the PKC-α and -β inhibitor, Go6976; the PKC-α inhibitor, safingol; the PKC-β inhibitor, ruboxistaurin; the PKC-δ inhibitor, rottlerin; the c-Jun NH₂-terminal kinase (JNK) inhibitor, SP600125; and the myosin light chain kinase inhibitor, ML-7 hydrochloride. Western blot analysis was used to examine the effect of rottlerin on dexmedetomidine-induced PKC-δ expression and JNK phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) and to investigate the effect of dexmedetomidine on PKC-δ expression in VSMCs transfected with PKC-δ small interfering RNA (siRNA) or control siRNA. Chelerythrine as well as SP600125 and ML-7 hydrochloride attenuated the dexmedetomidine-induced contraction. Go6976, safingol, and ruboxistaurin had no effect on the dexmedetomidine-induced contraction, whereas rottlerin inhibited the dexmedetomidine-induced contraction. Dexmedetomidine induced PKC-δ expression, whereas rottlerin and PKC-δ siRNA transfection inhibited dexmedetomidine-induced PKC-δ expression. Dexmedetomidine also induced JNK phosphorylation, which was inhibited by rottlerin. Taken together, these results suggest that the dexmedetomidine-induced contraction involves PKC-δ-dependent JNK phosphorylation in the isolated rat aorta.     

Long‐term hepatitis B surface antigen (HBsAg) kinetics during entecavir treatment in Korean patients—Functional cure unlikely

The aim of this study was to investigate the on‐treatment kinetics of quantitative HBsAg during entecavir therapy to predict the treatment period needed to achieve functional cure. From a cohort of 1009 CHB treatment‐naïve patients who were started on entecavir, the kinetics of quantitative HBsAg decline was assessed in 410 patients by a linear mixed model. The difference in the kinetics of quantitative HBsAg was determined based on the HBeAg positivity, HBeAg seroclearance and presence of baseline liver cirrhosis. Among the 410 patients, 213 patients (52.0%) were HBeAg‐positive and 217 patients (66.1%) were male with a median age of 48 years. During a median follow‐up of 53.5 months, the quantitative HBsAg level showed a slow but consistent decrease. The expected log qHBsAg levels as a function of time during entecavir treatment in HBeAg(+) and HBeAg(?) patients were 3.4773‐0.0039 × Months and 3.1853‐0.0036 × Months, respectively. The estimated time to clearance of quantitative HBsAg in our study was greater than 74.1 years in HBeAg‐positive patients and 73.5 years in HBeAg‐negative patients. The calculated time to achieve functional cure is lifelong without regard to HBeAg seroclearance or presence of liver cirrhosis. The mathematical modelling from a long‐term follow‐up of chronic hepatitis B patients on entecavir shows that HBsAg clearance requires decades of treatment. Thus, lifelong therapy is inevitable in entecavir‐treated patients to achieve functional cure.