Characterization of intracellular buffering power in human induced pluripotent stem cells and the loss of pluripotency is delayed by acidic stimulation and increase of NHE1 activity.

The homeostasis of intracellular pH (pHi ) affects many cellular functions. Our previous study has established a functional and molecular model of the active pHi regulators in human induced pluripotent stem cells (hiPSCs). The aims of the present study were to further quantify passive pHi buffering power (ß) and to investigate the effects of extracellular pH and Na+ -H+ exchanger 1 (NHE1) activity on pluripotency in hiPSCs. pHi was detected by microspectrofluorimetry with pH-sensitive dye-BCECF. Western blot, immunofluorescence staining, and flow cytometry were used to detect protein expression and pluripotency. Our study in hiPSCs showed that (a) the value of total (ßtot ), intrinsic (ßi ), and CO2 -dependent ( ßCO2 ) buffering power all increased while pHi increased; (b) during the spontaneous differentiation for 4 days, the ß values of ßtot and ßCO2 changed in a tendency of decrease, despite the absence of statistical significance; (c) an acidic cultured environment retained pluripotency and further upregulated expression and activity of NHE1 during spontaneous differentiation; (d) inhibition on NHE1 activity promoted the loss of pluripotency. In conclusion, we, for the first time, established a quantitative model of passive ß during differentiation and demonstrated that maintenance of NHE1 at a higher level was of critical importance for pluripotency retention in hiPSCs.

Dipeptidyl Peptidase-4 Inhibitor Decreases Allograft Vasculopathy Via Regulating the Functions of Endothelial Progenitor Cells in Normoglycemic Rats.

PURPOSE: Chronic rejection induces the occurrence of orthotopic allograft transplantation (OAT) vasculopathy, which results in failure of the donor organ. Numerous studies have demonstrated that in addition to regulating blood sugar homeostasis, dipeptidyl peptidase-4 (DPP-4) inhibitors can also provide efficacious therapeutic and protective effects against cardiovascular diseases. However, their effects on OAT-induced vasculopathy remain unknown. Thus, the aim of this study was to investigate the direct effects of sitagliptin on OAT vasculopathy in vivo and in vitro. METHODS: The PVG/Seac rat thoracic aorta graft to ACI/NKyo rat abdominal aorta model was used to explore the effects of sitagliptin on vasculopathy. Human endothelial progenitor cells (EPCs) were used to investigate the possible underlying mechanisms. RESULTS: We demonstrated that sitagliptin decreases vasculopathy in OAT ACI/NKyo rats. Treatment with sitagliptin decreased BNP and HMGB1 levels, increased GLP-1 activity and stromal cell-derived factor 1α (SDF-1α) expression, elevated the number of circulating EPCs, and improved the differentiation possibility of mononuclear cells to EPCs ex vivo. However, in vitro studies showed that recombinant B-type natriuretic peptide (BNP) and high mobility group box 1 (HMGB1) impaired EPC function, whereas these phenomena were reversed by glucagon-like peptide 1 (GLP-1) receptor agonist treatment. CONCLUSIONS: We suggest that the mechanisms underlying sitagliptin-mediated inhibition of OAT vasculopathy probably occur through a direct increase in GLP-1 activity. In addition to the GLP-1-dependent pathway, sitagliptin may regulate SDF-1α levels and EPC function to reduce OAT-induced vascular injury. This study may provide new prevention and treatment strategies for DPP-4 inhibitors in chronic rejection-induced vasculopathy.

Melanocyte Differentiation From Induced Pluripotent Stem Cells Derived From Human Adipose-Derived Stem Cells.

The pigment melanin is produced by melanocytes, is primarily responsible for skin color, and protects it against ultraviolet rays that can cause the destruction of genetic material within the keratinocytes. To elucidate the mechanisms of many diseases associated with melanocytes, such as melanoma and albinism, or burns with uneven pigment distribution, the disease model needs to be established first. In this study, we aimed to construct the melanocyte model from patients in a short period.Sandai virus vector containing 4 stemness genes (Oct4, Sox2, Klf4, c-Myc) was transfected into human adipose-derived stem cells to produce induced pluripotent stem cells (iPSCs). Immunofluorescence staining was used to confirm the expression of specific proteins for iPSCs, including Tra-1-60, Tra-1-81, Oct-4, Sox-2, and Nango. polymerase chain reaction results also showed that specific genes of iPSCs with the ability to cause the differentiation of cells into the 3 germ layers were expressed. In our in vivo experiments, iPSCs were subcutaneously injected into nude mice to induce teratoma formation for 2 months. The morphology of the 3 germ layers was confirmed by hematoxylin and eosin staining. Furthermore, melanocytes were purified by serial induction medium, and their presence was confirmed by flow cytometry and the expression of different markers for melanocytes.

Expressional and Functional Characterization of Intracellular pH Regulators and Effects of Ethanol in Human Oral Epidermoid Carcinoma Cells.

BACKGROUND/AIMS: To functionally characterize intracellular pH (pHi) regulating mechanisms, such as Na+-H+ exchanger (NHE) and Na+-HCO3- co-transporter (NBC), and further examine effects of ethanol on the pHi regulating mechanism in human oral epidermoid carcinoma (OEC-M1) cells. METHODS: OEC-M1 cells were a gift from Tri-Service General Hospital. Changes of pHi were detected by microspectrofluroimetry with a pH-sensitive fluorescent dye, BCECF. Isoforms of transporters were examined by Western blot technique. RESULTS: i) the steady-state pHi value shifted from alkaline (7.35∼7.49) to acidic (7.0∼7.03) following acid/base impacts; ii) in HEPES-buffer system, pHi recovery following induced-acidification was totally blocked by either removing [Na]o+ or adding HOE 694 (a NHE1 specific inhibitor), which demonstrates existence of NHE1; iii) in HCO3-/CO2-buffer system, the pHi recovery following induced-acidification was entirely blocked by either removing [Na]o+ or adding HOE 694 plus DIDS (a NBC specific inhibitor), which suggests existence of Na+- and HCO3-dependent acid-extruder, i.e. NBC; iv) the isoforms of the two acid extruders were NHE1, NBCn1, NBCe1 and NDCBE; v) ethanol (10-1000 mM) showed a biphasic and concentration-dependent effect on resting pHi (i.e. increase then decrease) by changing the activity of NHE1 and NBC accordingly; vi) treatment with ethanol for 24 hr (> 300 mM) significantly inhibited the expression of NHE1, NBCn1 and NDCBE, while up-regulated NBCe1. CONCLUSIONS: Ethanol affects pHi in a concentration-dependent manner by changing function and expression of NHE1 and NBC isoforms in OEC-M1 cells.

Tanshinone IIA Inhibits High Glucose-Induced Collagen Synthesis via Nuclear Factor Erythroid 2-Related Factor 2 in Cardiac Fibroblasts.

BACKGROUND/AIMS: Diabetes is associated with increased incidence of myocardial dysfunction, which is partly characterized by interstitial and perivascular fibrosis. Cardiac fibroblasts have been identified as an important participant in the development of cardiac fibrosis. Exposure of cultured cardiac fibroblasts to high glucose resulted in increased collagen synthesis. Tanshinone IIA can alleviate the ventricular fibrosis that develops in a number of different experimental conditions. However, whether tanshinone IIA can prevent high glucose-induced collagen synthesis in cardiac fibroblasts remains unknown. The aim of this study was to evaluate the effects of tanshinone IIA on high glucose-induced collagen synthesis in cardiac fibroblasts. METHODS: Rat cardiac fibroblasts were cultured in high glucose (25 mM) media in the absence or presence of tanshinone IIA and the changes in collagen synthesis, transforming growth factor-ß1 (TGF-ß1) production and related signaling molecules were assessed by 3H-proline incorporation, quantitative polymerase chain reaction, enzyme linked immunosorbent assay, and Western blotting. RESULTS: The results indicate cardiac fibroblasts exposed to high glucose condition show increased cell proliferation and collagen synthesis and these effects were abolished by tanshinone IIA treatment. Furthermore, the inhibitory effect of tanshinone IIA on high glucose induced cell proliferation and collagen synthesis may be associated with its activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and the inhibition of TGF-ß1 production and Smad2/3 phosphorylation. CONCLUSION: In summary, our results highlights the critical role tanshinone IIA plays as an antioxidant in attenuating high glucose-mediated collagen synthesis through inhibiting TGF-ß1/Smad signaling in cardiac fibroblasts which provide a mechanistic basis for the clinical application of tanshinone IIA in the treating diabetic-related cardiac fibrosis.

Lipopolysaccharide pretreatment increases protease-activated receptor-2 expression and monocyte chemoattractant protein-1 secretion in vascular endothelial cells.

BACKGROUND: This study investigated whether lipopolysaccharide (LPS) increase protease-activated receptor-2 (PAR-2) expression and enhance the association between PAR-2 expression and chemokine production in human vascular endothelial cells (ECs). METHODS: The morphology of ECs was observed through microphotography in cultured human umbilical vein ECs (EA. hy926 cells) treated with various LPS concentrations (0, 0.25, 0.5, 1, and 2 µg/mL) for 24 h, and cell viability was assessed using the MTT assay. Intracellular calcium imaging was performed to assess agonist (trypsin)-induced PAR-2 activity. Western blotting was used to explore the LPS-mediated signal transduction pathway and the expression of PAR-2 and adhesion molecule monocyte chemoattractant protein-1 (MCP-1) in ECs. RESULTS: Trypsin stimulation increased intracellular calcium release in ECs. The calcium influx was augmented in cells pretreated with a high LPS concentration (1 µg/mL). After 24 h treatment of LPS, no changes in ECs viability or morphology were observed. Western blotting revealed that LPS increased PAR-2 expression and enhanced trypsin-induced extracellular signal-regulated kinase (ERK)/p38 phosphorylation and MCP-1 secretion. However, pretreatment with selective ERK (PD98059), p38 mitogen-activated protein kinase (MAPK) (SB203580) inhibitors, and the selective PAR-2 antagonist (FSLLRY-NH2) blocked the effects of LPS-activated PAR-2 on MCP-1 secretion. CONCLUSIONS: Our findings provide the first evidence that the bacterial endotoxin LPS potentiates calcium mobilization and ERK/p38 MAPK pathway activation and leads to the secretion of the pro-inflammatory chemokine MCP-1 by inducing PAR-2 expression and its associated activity in vascular ECs. Therefore, PAR-2 exerts vascular inflammatory effects and plays an important role in bacterial infection-induced pathological responses.

Nicorandil Inhibits Cyclic Strain-Induced Interleukin-8 Expression in Human Umbilical Vein Endothelial Cells.

BACKGROUND: Nicorandil, a mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP) channel opener, exerts protective effects on the cardiovascular system. This study examined the effect of nicorandil on cyclic strain-induced interleukin-8 (IL-8) expression in human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs were exposed to cyclic strain in the presence or absence of nicorandil (1-10 µmol/l); we then analyzed IL-8 expression. We also assessed the effects of nicorandil on heme oxygenase-1 (HO-1) expression and cyclic strain-modulated IL-8 expression after HO-1 silencing in HUVECs. SUMMARY: HUVECs exposed to cyclic strain showed increased IL-8 messenger RNA expression and protein secretion. Nicorandil (1-10 µmol/l) inhibited cyclic strain-induced IL-8 expression, whereas 5-hydroxydecanoate (100 µmol/l), a selective inhibitor of the mitoKATP channel, completely reversed the inhibitory effects of nicorandil on cyclic strain-induced IL-8 expression. We demonstrated that nicorandil increased HO-1 expression in HUVECs. In addition, cobalt protoporphyrin (10 µmol/l), an inducer of HO-1 expression, mimicked the effects of nicorandil and inhibited IL-8 expression under cyclic strain, whereas zinc protoporphyrin IX (10 µmol/l), an inhibitor of HO-1 expression, antagonized the effect of nicorandil. HO-1 silencing significantly abrogated the inhibitory effects of nicorandil on cyclic strain-induced IL-8 expression, suggesting that HO-1 plays a role in the mechanism of action of nicorandil. KEY MESSAGES: This study is the first to report that nicorandil inhibits cyclic strain-induced IL-8 expression through the induction of HO-1 expression in HUVECs. This finding provides valuable new insight into the molecular pathways contributing to the vasoprotective effects of nicorandil.

The Effect and Underlying Mechanism of Ethanol on Intracellular H(+) -Equivalent Membrane Transporters in Human Aorta Smooth Muscle Cells.

BACKGROUND: The presence of intracellular pH (pHi ) regulators, including Na(+) -H(+) exchanger (NHE), Na(+) -HCO3- co-transporter (NBC), Cl(-) /OH(-) exchanger (CHE), and Cl(-) /HCO3- exchanger (AE), have been confirmed in many mammalian cells. Alcohol consumption is associated with increased risk of cardiovascular disorder. The aims of the study were to identify the possible transmembrane pHi regulators and to explore the effects of ethanol (EtOH) (10 to 300 mM) on the resting pHi and pHi regulators in human aorta smooth muscle cells (HASMCs). METHODS: HASMCs were obtained from patients undergoing heart transplant. The pHi was measured by microspectrofluorimetry with the pH-sensitive dye, BCECF-AM. RESULTS: The following results are obtained. (i) In cultured HASMCs, the resting pHi was 7.19 ± 0.04 and 7.13 ± 0.02 for HEPES- and CO2 /HCO3--buffered solution, respectively. (ii) Two different Na(+) -dependent acid-equivalent extruders, including NHE and Na(+) -coupled HCO3- transporter, functionally coexisted. (iii) Two different Cl(-) -dependent acid loaders (CHE and AE) were functionally identified. (iv) EtOH induced a biphasic, concentration-dependent change in resting pHi (+0.25 pH unit at 100 mM but only +0.05 pH unit at 300 mM) in bicarbonate-buffered solution, while caused a concentration-dependent decrease in resting pHi (-0.06 pH unit at 300 mM) in HEPES-buffered solution. (v) The effect of EtOH on NHE activity was also biphasic: increase of 40% at lower concentration of 10 mM, followed by decrease of 30% at higher concentration of 300 mM. (vi) The increase in Na(+) -coupled HCO3- transporter activity by EtOH was concentration dependent. (vii) The effect of EtOH on CHE and AE activities was both biphasic: increase of ~25% at 30 mM, followed by decrease of 10 to 25% at 100 mM, and finally increase of 15 to 20% at 300 mM. CONCLUSIONS: This study demonstrated that 2 acid extruders and 2 acid loaders coexisted functionally in HASMCs and that EtOH induced a biphasic, concentration-dependent change in resting pHi by altering the activity of the 2 acid extruders, NHE and Na(+) -coupled HCO3- transporter, and the 2 acid loaders, CHE and AE.

Effects of Indomethacin on Intracellular pH and Na⁺/H⁺ Exchanger in the Human Monocytes.

The ability to maintain optimal intracellular pH (pH(i)) is an essential requirement for all cells. Na⁺-H⁺ exchanger (NHE), a ubiquitously expressed transmembrane protein, has been found widely as a major acid extruder in many different cell types, including human monocytes. We therefore investigated the mechanism of the active pH(i) recovery from intracellular acidosis (induced by NH4Cl prepulse) using intracellular 2',7'-bis (2-carboxethyl)-5(6)-carboxyl-fluorescein (BCECF) fluorescence in cultured human monocytes. Indomethacin is a potent, nonselective inhibitor of cyclooxygenases. Due to its toxicity, the clinical use of indomethacin as an analgesic-antipyretic agent is limited. However, it has recently been found that indomethacin can effectively treat many inflammatory/immune disorders. In this study, we further investigated the effect of indomethacin on the pHi and explored the underlying mechanism. In HEPES (nominally HCO3⁻-free) Tyrode solution, a pH(i) recovery from induced intracellular acidosis could be blocked completely by 30 µM HOE 694, a specific NHE1 inhibitor, or by removing [Na⁺]0. Therefore, in the present study, we provided functional evidence, physiologically and pharmacologically, that the HCO3⁻-independent acid extruder was mostly likely the NHE1 which was involved in acid extrusion in the human monocytes. Moreover, indomethacin (1 µM-1 mM) decreased pH(i) levels in a concentration-dependent manner and significantly suppressed the activity of the NHE1, suggesting that indomethacin-induced intracellular acidosis is caused both by the inhibition of NHE1 activity and the non-specified NHE1-independent acidifying mechanism. In conclusion, our present study demonstrates that NHE1 exists functionally in human monocytes, and the indomethacin-induced pHi decreasing is summation effects on NHE1-dependent and -independent mechanism.

Assessment of the Risk Factors and Outcomes for Postoperative Atrial Fibrillation Patients Undergoing Isolated Coronary Artery Bypass Grafting.

BACKGROUND: Atrial fibrillation is the most common complication of cardiac surgery and is associated with significant morbidity and mortality. Recognizing patients at high risk for developing postoperative atrial fibrillation (POAF) may help identify those who could benefit from strategies to prevent POAF. This study was conducted to delineate outcomes and to assess risk factors for POAF among Taiwanese patients undergoing coronary artery bypass grafting (CABG). METHODS: From January 2009 until February 2012, this prospective study included 266 consecutive patients admitted to our hospital with coronary artery disease. All patients underwent isolated CABG. Patients with preoperative permanent atrial fibrillation and concomitant surgery were excluded. Multiple risk factors associated with the incidence of POAF were collected and evaluated. RESULTS: POAF occurred in 126 of 226 patients (47.37%). Univariate analysis revealed that significant risk factors for the condition were age, gender, diabetes, dyslipidemia, smoking, impaired renal function, impaired cardiac function, and increased serum electrolytes. Multivariate analysis showed dyslipidemia [hazard ratio (HR): 0.418; 95% confidence interval (Cl): 0.190-0.915, p = 0.029], impaired renal function as indicated by an estimated glomerular filtration rate < 60 mL/min/1.73 m(2) (HR: 3.174; 95% CI: 1.432-7.037, p = 0.004), and serum sodium (HR: 1.112; 95% Cl: 1.047-1.182, p = 0.001) prior to cardiopulmonary bypass as significant. Moreover, POAF was associated with lower 30-day, 1- and 3-year cumulative survival rates and higher early postoperative complications. CONCLUSIONS: Patients with isolated CABG who were administered ß-blockers, angiotensin converting enzyme inhibitor/angiotensin receptor blockers treatment, and lipid therapy before CABG were associated with reduced POAF, while those with impaired renal function and higher serum sodium before CABG predisposed POAF in a Taiwanese population. KEY WORDS: Atrial fibrillation (AF); Coronary artery bypass grafting (CABG); Coronary artery disease (CAD); Postoperative atrial fibrillation (POAF).