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.

Effects of Andrographolide on Intracellular pH Regulation, Cellular Migration, and Apoptosis in Human Cervical Cancer Cells †.

Cancer cells have been characterized with alkaline intracellular pH (pHi) values (≥7.2) to enable cancer proliferation, migration, and progression. The aim of the present study was to explore the concentration-dependent effects of Andrographolide, an active diterpenoid compound of herb Andrographis paniculata, on Na+/H+ exchanger isoform 1 (NHE1), cellular migration and apoptosis in human cervical cancer cells (HeLa). The pHi was detected by microspectrofluorometry method, and intracellular acidification was induced by NH4Cl prepulse technique. Viability and protein expression were determined by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Western blot, respectively. Human normal endocervical cells (End1), ectocervical cells (Ect1), and HeLa were bought commercially. The resting pHi value of HeLa (≈7.47) was significantly higher than that of End1 and Ect1 (≈7.30), and shifted from alkaline to acidic following acid/base impacts. In HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid | N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) -buffered superfusate, NHE1 and V-ATPase co-existed functionally for acid extrusion in HeLa, while only NHE1 existed functionally in End/Ect1. Andrographolide (3-1000 µM) concentration-dependently inhibited NHE1 activity. Cell-migration and expressions of NHE1, V-ATPase, PARP (poly-ADP-ribose-polymerase), pro-Caspase-3, and Bcl-2 were significantly reduced by pretreating with Andrographolide (≥100 µM) for 24-48 h in HeLa. Andrographolide inhibited cell viability of End1-cells/Ect1 and HeLa (≥100 and ≥30 µM, respectively). The present findings implicate the promising clinical applications of Andrographolide on cervical cancer treatment.

Isoorientin Decreases Cell Migration via Decreasing Functional Activity and Molecular Expression of Proton-Linked Monocarboxylate Transporters in Human Lung Cancer Cells.

Aggressive tumor cells mainly rely on glycolysis, and further release vast amounts of lactate and protons by monocarboxylate transporter (MCT), which causes a higher intracellular pH (pHi) and acidic extracellular pH. Isoorientin, a principle flavonoid compound extracted from several plant species, shows various pharmacological activities. However, effects of isoorientin on anticancer and MCT await to explore in human lung cancer cells. Human lung cancer tissues were obtained from cancer patients undergoing surgery, while the human lung adenocarcinoma cells (A549) were bought commercially. Change of pHi was detected by microspectrofluorometry method with a pH-sensitive fluorescent dye, BCECF. MTT and wound-healing assay were used to detect the cell viability and migration, respectively. Western blot techniques and immunocytochemistry staining were used to detect the protein expression. Our results indicated that the expression of MCTs1/4 and CD147 were upregulated significantly in human lung tissues. In experiments of A549 cells, under HEPES-buffer, the resting pHi was 7.47, and isoorientin (1-300µM) inhibited functional activity of MCT concentration-dependently (up to -42%). Pretreatment with isoorientin (3-100µM) for 24h, MCT activity and cell migration were significantly inhibited (-25% and -40%, respectively), while the cell viability was not affected. Moreover, the expression of MCTs1/4, CD147, and matrix metalloproteinase (MMP) 2/9 were significantly down regulated. In summary, MCTs1/4 and CD147 are significantly upregulated in human lung adenocarcinoma tissues, and isoorientin inhibits cells-migration by inhibiting activity/expression of MCTs1/4 and MMPs2/9 in human lung cancer cells. These novel findings suggest that isoorientin could be a promising pharmacological agent for lung cancer.

Functional effects of urotensin-II on intracellular pH regulators in human radial artery smooth muscle cells.

The regulation of intracellular pH (pHi) plays a vital role in various cellular functions. We previously demonstrated that three different acid extruders, the Na+-H+ exchanger (NHE), Na+-HCO3- co-transporter (NBC) and H+-linked monocarboxylate transporter (MCT), functioned together in cultured human radial artery smooth muscle cells (HRASMCs). However, the functions of acid-loading transporters in HRASMCs remain poorly understood. Urotensin II (U-II), one of the most potent vasoconstrictors, is highly expressed in many cardiovascular diseases. The aim of this present study was to determine the concentration effect of U-II (3 pM∼100 nM) on the functional activity of pHi regulators in HRASMCs. Cultured HRASMCs were derived from segments of human radial arteries obtained from patients undergoing bypass grafting. Changes in pHi recovery due to intracellular acidification and alkalization induced by NH4Cl prepulse and Na-acetate prepulse, respectively, were detected by microspectrofluorimetry with the pH-sensitive fluorescent dye BCECF. Our present study showed that (a) U-II increased the activity of NHE in a concentration-dependent manner but did not change that of NBC or MCT or resting pHi, (b) the Cl--OH- exchanger (CHE) facilitated base extrusion, and (c) U-II induced a concentration-dependent increase in the activity of CHE. In conclusion, for the first time, our results highlight a concentration-dependent increase in the activity of NHE and CHE, but not NBC and MCT, induced by U-II in HRASMCs.

Acidic Stress Triggers Sodium-Coupled Bicarbonate Transport and Promotes Survival in A375 Human Melanoma Cells.

Melanoma cells preserve intracellular pH (pHi) within a viable range despite an acidic ambient pH that typically falls below pH 7.0. The molecular mechanisms underlying this form of acidic preservation in melanoma remain poorly understood. Previous studies had demonstrated that proton transporters including the monocarboxylate transporter (MCT), the sodium hydrogen exchanger (NHE), and V-Type ATPase mediate acid extrusion to counter intracellular acidification in melanoma cells. In this report, the expression and function of the Sodium-Coupled Bicarbonate Transporter (NCBT) family of base loaders were further characterized in melanoma cell lines. NCBT family members were found to be expressed in three different melanoma cell lines - A375, MeWo, and HS695T - and included the electrogenic sodium-bicarbonate cotransporter isoforms 1 and 2 (NBCe1 and NBCe2), the electroneutral sodium-bicarbonate cotransporter (NBCn1), and the sodium-dependent chloride-bicarbonate exchanger (NDCBE). These transporters facilitated 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-dependent pHi recovery in melanoma cells, in response to intracellular acidification induced by ammonium chloride prepulse. Furthermore, the expression of NCBTs were upregulated via chronic exposure to extracellular acidification. Given the current research interest in the NCBTs as a molecular driver of tumourigenesis, characterising NCBT in melanoma provides impetus for developing novel therapeutic targets for melanoma treatment.

B-type natriuretic peptide enhances fibrotic effects via matrix metalloproteinase-2 expression in the mouse atrium in vivo and in human atrial myofibroblasts in vitro.

B-type natriuretic peptide (BNP) was approved by the US Food and Drug Administration in 2001 for the treatment of heart failure. However, the effects of BNP in clinical applications are controversial and uncertain. Recently, study indicated that high BNP levels are associated with an increased risk of developing atrial fibrillation. In this study, we investigated the direct effects of BNP on TNF-α-induced atrial fibrosis mice, as well as its effects on human atrial myofibroblasts. We found that injecting TNF-α-induced mice with recombinant human BNP enhanced atrial fibrosis via matrix metalloproteinase-2 (MMP-2) expression and collagen accumulation. Furthermore, we found that BNP stimulated MMP-2 expression in human atrial myofibroblasts. Treatment of human atrial myofibroblasts with cycloheximide had no effect on this outcome; however, treatment of cells with MG132 enhanced BNP-induced MMP-2 expression, indicating that protein stability and inhibition of proteasome-mediated protein degradation pathways are potentially involved. Inhibition of SIRT1 significantly decreased BNP-induced MMP-2 expression. Additionally, confocal and coimmunoprecipitation data indicated that BNP-regulated MMP-2 expression are likely to be mediated through direct interaction with SIRT1, which is thought to deacetylate MMP-2 and to increase its protein stability in human atrial myofibroblasts. Finally, we confirmed that SIRT1 is expressed and cytoplasmically redistributed as well as colocalized with MMP-2 in mouse fibrotic atrial tissue. We suggest a possible fibrosis-promoting role of BNP in the atrium, although the antifibrotic properties of BNP in the ventricle have been reported in previous studies, and that the coordination between MMP-2 and SIRT1 in BNP-induced atrial myofibroblasts participates in atrial fibrosis.

Dysfunctional high density lipoprotein failed to rescue the function of oxidized low density lipoprotein-treated endothelial progenitor cells: a novel index for the prediction of HDL functionality.

Lipid metabolic disorders play critical roles in atherogenesis. Traditionally, it has been suggested that reduced high density lipoprotein (HDL) levels might be an important morbidity indicator for cardiovascular diseases. Therefore, it has been argued that therapeutically raising HDL levels may reduce atherogenesis in patients with dyslipidemia. However, recent clinical trials to elevate serum HDL levels by pharmacologic approaches failed to demonstrate clinical efficacy. Thus, to investigate the functionality of HDL and to explore the possible clinical relevance as well as to define an effective indicator that can represent HDL function may provide another key and reference to disclose the clinical treatment of dyslipidemia. We analyzed the association between the data of dichlorofluorescein assay (assay the functionality of HDL), the effect of HDL on oxidized low density lipoprotein (oxLDL)-stimulated endothelial progenitor cells (EPCs) in vitro, levels of circulating EPCs, and ex vitro EPC colony forming units of each case, we defined the indicator (relative HDL index (RHDL index) = dichlorofluorescein assay result of each subject/dichlorofluorescein assay reading of our young healthy controls) that may represent functionality of HDL. HDL from healthy adults protected oxLDL-treated EPCs by modulating p38 mitogen-activated protein kinase and Rho activation and by promoting nitric oxide production. HDL from subject with RHDL index ≧2 also failed to restore the functionality of oxLDL-treated EPCs via cell-signaling pathways in vitro. The RHDL index significantly correlated with patients' circulating EPC number or EPC colony forming units ex vivo. In conclusions, we explored the RHDL index as a score to predict a patient's EPC functions in vivo and ex vitro.

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.

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.

Eotaxin-2 induces monocytic apoptosis in patients who have undergone coronary artery bypass surgery and in THP-1 cells in vitro regulated by thrombomodulin.

Cardiopulmonary bypass (CPB) induces cytokine production and causes postoperative monocytic inflammatory responses, which are associated with patient outcomes. In fact, monocytes regulate immunity through dynamic networks of survival and cellular apoptosis as well as thrombomodulin (TM)-associated differenciiation. Whether CPB affects the plasma level of eotaxin-2, a potent chemoattractant, or stimulates monocyte apoptosis among patients undergoing elective coronary artery bypass graft (CABG) surgery is also unknown. Thus, we aimed to investigate this subject and explored the feasible roles of TM in the phenomena. Firstly, clinical data showed that after CABG surgery, patients with lower plasma eotaxin-2 levels and higher TM expression levels exhibited reduced monocytic apoptosis, compared with that in patients with lower TM expression levels. Subsequently, to explore the hypothesis that eotaxin-2 induces monocytic apoptosis mediation by TM expression, we used in vitro monocytic THP-1 cells. The results indicated that treatment of THP-1 cells with eotaxin-2 markedly increased apoptosis. Knockdown of TM significantly increased, and overexpression of TM significantly reversed eotaxin-2-induced monocyte apoptosis, which was compared with that of only eotaxin-2-treated THP-1 cells. TM may regulate mitochondria-mediated apoptosis by its PI3K/Akt axis signaling pathway, which acts as an extinguisher for p53 and BAX activation, as well as limit further downstream release of cytochrome c and cleavage of caspases 8 and 3; we suggest that TM interacts with the cofilin cytoskeleton, which further supports a role for TM in eotaxin-induced THP-1 cell apoptosis. Based on clinical observation and in vitro study, we conclude that TM expression on monocytes is associated with their apoptosis. The above mechanisms may be relevant to clinical phenomena in which patients exhibiting more monocytic apoptosis are complicated by higher plasma levels of eotaxin-2 and lower TM expression on monocytes after CABG surgery.

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.

Functional and molecular characterization of transmembrane intracellular pH regulators in human dental pulp stem cells.

OBJECTIVE: Homeostasis of intracellular pH (pHi) plays vital roles in many cell functions, such as proliferation, apoptosis, differentiation and metastasis. Thus far, Na+-H+ exchanger (NHE), Na+-HCO3- co-transporter (NBC), Cl-/HCO3- exchanger (AE) and Cl-/OH- exchanger (CHE) have been identified to co-regulate pHi homeostasis. However, functional and biological pHi-regulators in human dental pulp stem cells (hDPSCs) have yet to be identified. DESIGN: Microspectrofluorimetry technique with pH-sensitive fluorescent dye, BCECF, was used to detect pHi changes. NH4Cl and Na+-acetate pre-pulse were used to induce intracellular acidosis and alkalosis, respectively. Isoforms of pHi-regulators were detected by Western blot technique. RESULTS: The resting pHi was no significant difference between that in HEPES-buffered (nominal HCO3--free) solution or CO2/HCO3-buffered system (7.42 and 7.46, respectively). The pHi recovery following the induced-intracellular acidosis was blocked completely by removing [Na+]o, while only slowed (-63%) by adding HOE694 (a NHE1 specific inhibitor) in HEPES-buffered solution. The pHi recovery was inhibited entirely by removing [Na+]o, while adding HOE 694 pulse DIDS (an anion-transporter inhibitor) only slowed (-55%) the acid extrusion. Both in HEPES-buffered and CO2/HCO3-buffered system solution, the pHi recovery after induced-intracellular alkalosis was entirely blocked by removing [Cl-]o. Western blot analysis showed the isoforms of pHi regulators, including NHE1/2, NBCe1/n1, AE1/2/3/4 and CHE in the hDPSCs. CONCLUSIONS: We demonstrate for the first time that resting pHi is significantly higher than 7.2 and meditates functionally by two Na+-dependent acid extruders (NHE and NBC), two Cl--dependent acid loaders (CHE and AE) and one Na+-independent acid extruder(s) in hDPSCs. These findings provide novel insight for basic and clinical treatment of dentistry.

Critical Roles of Xirp Proteins in Cardiac Conduction and Their Rare Variants Identified in Sudden Unexplained Nocturnal Death Syndrome and Brugada Syndrome in Chinese Han Population.

BACKGROUND: Sudden unexplained nocturnal death syndrome (SUNDS) remains an autopsy negative entity with unclear etiology. Arrhythmia has been implicated in SUNDS. Mutations/deficiencies in intercalated disc components have been shown to cause arrhythmias. Human cardiomyopathy-associated 1 (XIRP1) and 3 (XIRP2) are intercalated disc-associated, Xin repeats-containing proteins. Mouse Xirp1 is necessary for the integrity of intercalated disc and for the surface expression of transient outward and delayed rectifier K+ channels, whereas mouse Xirp2 is required for Xirp1 intercalated disc localization. Thus, XIRP1 and XIRP2 may be potentially causal genes for SUNDS. METHODS AND RESULTS: We genetically screened XIRP genes in 134 sporadic SUNDS victims and 22 Brugada syndrome (BrS) cases in a Chinese Han population. We identified 16 rare variants (6 were in silico predicted as deleterious) in SUNDS victims, including a novel variant, XIRP2-E215K. There were also four rare variants (2 were in silico predicted as deleterious) detected in BrS cases, including a novel variant, XIRP2-L2718P. Interestingly, among these 20 variants, we detected 2 likely pathogenic variants: a nonsense variant (XIRP2-Q2875*) and a frameshift variant (XIRP2-T2238QfsX7). Analyzing available Xirp2 knockout mice, we further found that mouse hearts without Xirp2 exhibited prolonged PR and QT intervals, slow conduction velocity, atrioventricular conduction block, and an abnormal infranodal ventricular conduction system. Whole-cell patch-clamp detected altered ionic currents in Xirp2-/- cardiomyocytes, consistent with the observed association between Xirp2 and Nav1.5/Kv1.5 in co-immunoprecipitation. CONCLUSIONS: This is the first report identifying likely pathogenic XIRP rare variants in arrhythmogenic disorders such as SUNDS and Brugada syndrome, and showing critical roles of Xirp2 in cardiac conduction.

Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells.

AIM: To establish a functional and molecular model of the intracellular pH (pHi) regulatory mechanism in human induced pluripotent stem cells (hiPSCs). METHODS: hiPSCs (HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital (IRB No. B-106-09). Changes in the pHi were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K+/nigericin method. NH4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power (ß) was calculated from the ΔpHi induced by perfusing different concentrations of (NH4)2SO4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pHi regulators and pluripotency markers. RESULTS: In this study, our results indicated that (1) the steady-state pHi value was found to be 7.5 ± 0.01 (n = 20) and 7.68 ± 0.01 (n =20) in HEPES and 5% CO2/HCO3 --buffered systems, respectively, which were much greater than that in normal adult cells (7.2); (2) in a CO2/HCO3 --buffered system, the values of total intracellular buffering power (ß) can be described by the following equation: ßtot = 107.79 (pHi)2 - 1522.2 (pHi) + 5396.9 (correlation coefficient R 2 = 0.85), in the estimated pHi range of 7.1-8.0; (3) the Na+/H+ exchanger (NHE) and the Na+/HCO3 - cotransporter (NBC) were found to be functionally activated for acid extrusion for pHi values less than 7.5 and 7.68, respectively; (4) V-ATPase and some other unknown Na+-independent acid extruder(s) could only be functionally detected for pHi values less than 7.1; (5) the Cl-/ OH- exchanger (CHE) and the Cl-/HCO3 - anion exchanger (AE) were found to be responsible for the weakening of intracellular proton loading; (6) besides the CHE and the AE, a Cl--independent acid loading mechanism was functionally identified; and (7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pHi value and diminished the functional activity and protein expression of the NHE and the NBC. CONCLUSION: For the first time, we established a functional and molecular model of a pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.

Cafestol, a coffee diterpene, inhibits urotensin II-induced interleukin-8 expression in human umbilical vein endothelial cells.

Cafestol, a diterpene molecule found in the berries of Coffea arabica L. (Rubiaceae), has been shown to exercise anti-angiogenic and anti-tumorigenic effects. However, cafestol's cellular mechanism has yet to be fully investigated. We previously demonstrated that urotensin II enhanced interleukin-8 secretion by endothelial cells, thereby increasing endothelial cell proliferation. Urotensin II may also participate in angiogenesis and tumor infiltration by macrophages. However, the effects of cafestol on urotensin II-induced interleukin-8 expression and cellular proliferation have not been determined. Here, we showed that pretreatment with cafestol inhibited urotensin II-stimulated endothelial cell proliferation. Further experiments demonstrated that cafestol increased translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and expression of enhanced heme oxygenase-1. Moreover, cafestol inhibited expression of urotensin II-induced interleukin-8. Cafestol's inhibitory effects on interleukin-8 expression and cellular proliferation induced by urotensin II were significantly abrogated by heme oxygenase-1 silencing, suggesting it may be involved in mediating the effects of cafestol. This study reports that cafestol inhibits urotensin II-induced interleukin-8 expression and cell proliferation via Nrf2/heme oxygenase-1-dependent mechanism in endothelial cells. These findings provide novel insight into the signaling pathways that may be important in mediating the effects of cafestol.

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.

A Thrombomodulin Gene Polymorphism (C1418T) Is Associated with Early Outcomes in Patients Undergoing Coronary Artery Bypass Graft Surgery with a Conventional Cardiopulmonary Bypass during Hospitalization.

Background: Thrombomodulin (TM) is a type of cell membrane-bound anticoagulant protein cofactor in the thrombin-mediated activation of protein C. Previous evidence has shown an association between TM polymorphisms and systemic inflammation. Conventional cardiopulmonary bypass (CPB), beating-heart CPB, and off-pump techniques have been widely used in cardiac surgery. However, these techniques may also cause systemic inflammatory responses in the patients. Whether TM polymorphisms are associated with systemic inflammation after cardiac surgery is still unclear. Methods: We analyzed the TM gene C1418T polymorphisms in 347 patients who underwent coronary artery bridge graft (CABG) surgery using allele-specific primers in a PCR assay. The clinical data during the hospital stay were collected and tested for correlations with the TM gene C1418T polymorphisms. Results: We separated the patients into two groups based on their TM C1418T genotype (CC genotype group and CT/TT genotype group). The days spent in an intensive care unit (ICU) and the incidence of fever in the ICU were significantly lower in the beating-heart CPB and off-pump groups than in the conventional CPB group. Additionally, the TM gene C1418T polymorphisms did not affect the early outcomes in patients in the beating-heart CPB and off-pump groups. Interestingly, in the conventional CPB group, patients with the CC genotype had a lower rate of fever, shorter duration of fever, and delay of ICU when compared with the CT/TT genotype. Conclusion: Surgeons may use a patient's TM gene C1418T polymorphism to predict the strength of systemic inflammation and speculate on early outcomes during hospitalization before conventional CPB is performed.

Assessment of veterinary drugs in plants using pharmacokinetic approaches: The absorption, distribution and elimination of tetracycline and sulfamethoxazole in ephemeral vegetables.

The present study was carried out to demonstrate novel use of pharmacokinetic approaches to characterize drug behaviors/movements in the vegetables with implications to food safety. The absorption, distribution, metabolism and most importantly, the elimination of tetracycline (TC) and sulfamethoxazole (SMX) in edible plants Brassica rapa chinensis and Ipomoea aquatica grown hydroponically were demonstrated and studied using non-compartmental pharmacokinetic analysis. The results revealed drug-dependent and vegetable-dependent pharmacokinetic differences and indicated that ephemeral vegetables could have high capacity accumulating antibiotics (up to 160 µg g-1 for TC and 38 µg g-1 for SMX) within hours. TC concentration in the root (Cmax) could reach 11 times higher than that in the cultivation fluid and 3-28 times higher than the petioles/stems. Based on the volume of distribution (Vss), SMX was 3-6 times more extensively distributed than TC. Both antibiotics showed evident, albeit slow elimination phase with elimination half-lives ranging from 22 to 88 hours. For the first time drug elimination through the roots of a plant was demonstrated, and by viewing the root as a central compartment and continuous infusion without a loading dose as drug administration mode, it is possible to pharmacokinetically monitor the movement of antibiotics and their fate in the vegetables with more detailed information not previously available. Phyto-pharmacokinetic could be a new area worth developing new models for the assessment of veterinary drugs in edible plants.

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.