Yi-Tang Tseng

Targeting human CD34+ hematopoietic stem cells with anti-CD45 × anti-myosin light-chain bispecific antibody preserves cardiac function in myocardial infarction

We have previously shown that targeting human CD34(+) hematopoietic stem cells (HSC) with a bispecific antibody (BiAb) directed against myosin light chain (MLC) increases delivery of cells to the injured hearts and improves cardiac performance in the nude rat. In this study, we have sought to validate our previous observations and to perform more detailed determination of ventricular function in immunocompetent mice with myocardial infarction (MI) that were treated with armed CD34(+) HSC. We examined whether armed CD34(+) HSC would target the injured heart following MI and restore ventricular function in vitro. MI was created by ligation of the left anterior descending artery. After 48 h, adult ICR mice received either 0.5 x 10(6) human CD34(+) HSC armed with anti-CD45 x anti-MLC BiAb or an equal volume of medium through a single tail vein injection. Two weeks after stem cell administration, ventricular function of hearts from mice receiving armed CD34(+) HSC was significantly greater compared with the same parameters from control mice. Immunohistochemistry confirmed the accumulation of CD34(+) HSC in MI hearts infused with stem cells. Angiogenesis was significantly enhanced in CD34(+) HSC-treated heart as determined by vascular density per area. Furthermore, histopathological examination revealed that the retained cardiac function observed in CD34(+) HSC-treated mice was associated with decreased ventricular fibrosis. These results suggest that peripheral administration of armed CD34(+) HSC results in localization of CD34(+) HSC to injured myocardium and restores myocardial function.

Placental biogenic amine transporters : cloning and expression

During intrauterine development, catecholamine turnover (production and clearance rates) is higher than under any other circumstances. This is mediated in large part by placental clearance of circulating catecholamines via a cocaine-sensitive, neuronal transporter-dependent mechanism. In order to confirm the molecular mechanisms for placental transport, we screened an ovine placental cDNA library for biogenic amine transporters. We report here the identification of two biogenic amine transporters with sequences very similar to their neuronal counterparts. One is an ovine serotonin transporter (oSERT) with > 90% homology to the human neuronal SERT. Expression studies confirm transport and competitive binding affinities consistent with a SERT transporter. We have also isolated a partial sequence for the ovine norepinephrine transporter (oNET). These results confirm the placental expression of plasma membrane biogenic amine transporters. We suggest the exaggerated fetal vulnerability to uptake inhibitors, like cocaine, may be due to blockade of placental biogenic amine transport.

A Novel Phosphoinositide 3-Kinase-dependent Pathway for Angiotensin II/AT-1 Receptor-mediated Induction of Collagen Synthesis in MES-13 Mesangial Cells

Chronic activation of the angiotensin II (ANG II) type 1 receptor (AT-1R) is critical in the development of chronic kidney disease. ANG II activates mesangial cells (MCs) and stimulates the synthesis of extracellular matrix components. To determine the molecular mechanisms underlying the induction of MC collagen, a mouse mesangial cell line MES-13 was employed. ANG II treatment induced an increase in collagen synthesis, which was abrogated by co-treatment with losartan (an AT-1R antagonist), wortmannin (a phosphoinositide 3-kinase (PI3K) inhibitor), an Akt inhibitor, and stable transfection of dominant negative-Akt1. ANG II induced a significant increase in PI3K activity, which was abolished by co-treatment with losartan or 2′,5′-dideoxyadenosine (2′,5′-DOA, an adenylyl cyclase inhibitor) but not by PD123319 (an AT-2R antagonist) or H89 (a protein kinase A (PKA) inhibitor). The Epac (exchange protein directly activated by cAMP)-specific cAMP analog, 8-pHPT-2′-O-Me-cAMP, significantly increased PI3K activity, whereas a PKA-specific analog, 6-benzoyladenosine-cAMP, showed no effect. The ANG II-induced increase in PI3K activity was also blocked by co-treatment with PP2, an Src inhibitor, or AG1478, an epidermal growth factor receptor (EGFR) antagonist. ANG II induced phosphorylation of Akt and p70S6K and EGFR, which was abrogated by knockdown of c-Src by small interference RNA. Knockdown of Src also effectively abolished ANG II-induced collagen synthesis. Conversely, stable transfection of a constitutively active Src mutant enhanced basal PI3K activity and collagen production, which was abrogated by AG1478 but not by 2′,5′-DOA. Moreover, acute treatment with ANG II significantly increased Src activity, which was abrogated with co-treatment of 2′,5′-DOA. Taken together, these results suggest that ANG II induces collagen synthesis in MCs by activating the ANG II/AT-1R-EGFR-PI3K pathway. This transactivation is dependent on cAMP/Epac but not on PKA. Src kinase plays a pivotal role in this signaling pathway between cAMP and EGFR. This is the first demonstration that an AT1R-PI3K/Akt crosstalk, along with transactivation of EGFR, mediates ANG II-induced collagen synthesis in MCs.

β-Adrenergic Receptor-PI3K Signaling Crosstalk in Mouse Heart: Elucidation of Immediate Downstream Signaling Cascades

Sustained β-adrenergic receptors (βAR) activation leads to cardiac hypertrophy and prevents left ventricular (LV) atrophy during LV unloading. The immediate signaling pathways downstream from βAR stimulation, however, have not been well investigated. The current study was to examine the early cardiac signaling mechanism(s) following βAR stimulation. In adult C57BL/6 mice, acute βAR stimulation induced significant increases in PI3K activity and activation of Akt and ERK1/2 in the heart, but not in lungs or livers. In contrast, the same treatment did not elicit these changes in β1/β2AR double knockout mice. We further showed the specificity of β2AR in this crosstalk as treatment with formoterol, a β2AR-selective agonist, but not dobutamine, a predominantly β1AR agonist, activated cardiac Akt and ERK1/2. Acute βAR stimulation also significantly increased the phosphorylation of mTOR (the mammalian target of rapamycin), P70S6K, ribosomal protein S6, GSK-3α/β (glycogen synthase kinase-3α/β), and FOXO1/3a (the forkhead box family of transcription factors 1 and 3a). Moreover, acute βAR stimulation time-dependently decreased the mRNA levels of the muscle-specific E3 ligases atrogin-1 and muscle ring finger protein-1 (MuRF1) in mouse heart. Our results indicate that acute βAR stimulation in vivo affects multiple cardiac signaling cascades, including the PI3K signaling pathway, ERK1/2, atrogin-1 and MuRF1. These data 1) provide convincing evidence for the crosstalk between βAR and PI3K signaling pathways; 2) confirm the β2AR specificity in this crosstalk in vivo; and 3) identify novel signaling factors involved in cardiac hypertrophy and LV unloading. Understanding of the intricate interplay between β2AR activation and these signaling cascades should provide critical clues to the pathogenesis of cardiac hypertrophy and enable identification of targets for early clinical interaction of cardiac lesions.

Human placental norepinephrine transporter mRNA: expression and correlation with fetal condition at birth.

The purpose of this study was to determine the primary form of human placental norepinephrine transporter (hNET) mRNA expressed in the human placenta and to compare the level of expression in normal pregnancies and in pregnancies complicated by drug exposure or other forms of physiological derangement. We used the hNET cDNA to measure RNA extracted from placenta and examined placental RNA following complicated and uncomplicated pregnancies. To compare transporter expression and its relation to fetal condition at birth, umbilical arterial plasma catecholamine levels, umbilical arterial blood gases and placental transporter mRNA level were compared by linear regression analysis. Uncomplicated pregnancies had a higher level of placental norepinephrine transporter mRNA than complicated pregnancies. An inverse relationship between umbilical cord norepinephrine level and transporter expression was demonstrated. We conclude that placental transporter expression represents an important and newly described metabolic function of the placenta. Placental catecholamine clearance mediated via the placental NET may be important in the pathophysiology of disorders associated with placental dysfunction, impaired placental blood flow or intrauterine growth retardation. This may also explain the adverse effects of drugs, such as cocaine, which block catecholamine transport.

Temporally controlled overexpression of cardiac-specific PI3Kα induces enhanced myocardial contractility—a new transgenic model

The phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates multiple cellular processes including cell survival/apoptosis and growth. In the cardiac context, PI3Kalpha plays important roles in cardiac growth. We have shown that cardiac PI3K activity is highly regulated during development, with the highest levels found during the fetal-neonatal transition period and the lowest levels in the adult. There is a close relationship between cardiomyocyte proliferation and cardiac PI3K activity. In adult transgenic mice, however, the prolonged constitutive activation of PI3Kalpha in the heart results in hypertrophy. To develop a strategy to allow temporally controlled overexpression of cardiac PI3Kalpha, we engineered a tetracycline (tet) transactivator tet-off controlled transgenic mouse line with a conditional overexpression of a cardiac-specific fusion protein of the SH2 domain of p85 and p110alpha. Cardiac PI3K activity and Akt phosphorylation were significantly increased in adult mice after transgene induction following the removal of doxycycline for 2 wk. The heart weight-to-body weight ratio was not changed, and there were no signs of cardiomyopathy. The overexpression of PI3Kalpha resulted in increased left ventricular (LV) developed pressure and the maximal and minimal positive values of the first derivative of LV pressure, but not heart rate, as assessed in Langendorff hearts. Mice overexpressing PI3Kalpha also had increases in the levels of Ca(2+)-regulating proteins, including the L-type Ca(2+) channels, ryanodine receptors, and sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a. Thus the temporally controlled overexpression of cardiac PI3Kalpha does not induce hypertrophy or cardiomyopathy but results in increased contractility, probably via the increased expression of multiple Ca(2+)-regulating proteins. These distinct phenotypes suggest a fundamental difference between transgenic mice with temporal or prolonged activation of cardiac PI3Kalpha.

β-Adrenergic receptors (βAR) regulate cardiomyocyte proliferation during early postnatal life

Cardiomyocyte development switches from hyperplasmic to hypertrophic growth between postnatal days 3 and 4 in rats. The mechanisms responsible for this transition have been controversial. β‐Adrenergic receptor (βAR) activation of mitogenic responses in vitro has been reported. We hypothesized that tonic activation of the βAR signaling regulates cell division in neonatal cardiomyocytes via effects on signaling ki‐nases known to be important in cell cycle regulation. The purpose of the current study was to elucidate the roles of βAR in rat cardiomyocyte growth in vivo. We demonstrated that βAR blockade induced a significant reduction in cardiomyocyte proliferation as measured by the BrdU labeling index. Blockade of βAR did not affect p38 or p44/42 MAPK activities. We further demonstrated that βAR blockade induced a prompt deactivation of the p70 ribosomal protein S6 kinase (p70 S6K). To confirm these results, we measured p70 S6K activity directly. Basal activity of p70 S6K in neonatal cardiomyocytes was fourfold higher than that of insulin‐treated adult rat liver. The activity of p70 S6K was reduced by 60% within 1 min after βAR blockade. We conclude that the βAR are involved in regulation of neonatal cardiomyocyte proliferation and that this mi‐togenic control may be mediated via the p70 S6K pathway.

High ambient glucose induces angiotensin-independent AT-1 receptor activation, leading to increases in proliferation and extracellular matrix accumulation in MES-13 mesangial cells

Diabetic nephropathy is associated with mesangial ECM (extracellular matrix) accumulation. We have shown that AT-1R [Ang II (angiotensin II) type I receptor] signalling induces ECM proteins via transactivation of PI3K (phosphoinositide 3-kinase) in mesangial cells. In the present study, we examined the mechanisms underlying the effect of high ambient glucose on cell proliferation and ECM expansion in a mesangial context. High glucose induced increases in PI3K activity, proliferation and ECM accumulation in mesangial cells. These effects were abrogated by losartan, an AT-1R antagonist, but not by [Sar1,Thr8]-Ang II (Sar is sarcosine), an inactive analogue of Ang II, or by a neutralizing antibody against Ang I/II. Overexpression of a constitutively active PI3Kalpha or AT-1R alone was sufficient to induce similar changes by high glucose. In contrast, overexpression of an inactive AT-1R lowered the basal levels and rendered the cells non-responsive to high glucose. Moreover, cells overexpressing wild-type AT-1R had enhanced sensitivity to acute Ang II stimulation. These cells, however, did not respond to conditioned medium obtained from mesangial cells cultured in high glucose. We further demonstrated that iAng (intracellular Ang II) can be induced by high glucose but only under certain conditions. Efficient suppression of iAng by short hairpin RNA against angiotensinogen, however, did not affect high glucose-induced effects on MES-13 cells. These results suggest that high ambient glucose induces activation of AT-1R in an Ang II-independent manner to transactivate PI3K, resulting in proliferation and ECM accumulation in mesangial cells.

Metformin Protects Cardiomyocyte from Doxorubicin Induced Cytotoxicity through an AMP-Activated Protein Kinase Dependent Signaling Pathway: An In Vitro Study

Doxorubicin (Dox) is one of the most widely used antitumor drugs, but its cumulative cardiotoxicity have been major concerns in cancer therapeutic practice for decades. Recent studies established that metformin (Met), an oral anti-diabetic drug, provides protective effects in Dox-induced cardiotoxicity. Met has been shown to increase fatty acid oxidation, an effect mediated by AMP activated protein kinase (AMPK). Here we delineate the intracellular signaling factors involved in Met mediated protection against Dox-induced cardiotoxicity in the H9c2 cardiomyoblast cell line. Treatment with low dose Met (0.1 mM) increased cell viabilities and Ki-67 expressions while decreasing LDH leakages, ROS generations and [Ca2+]i. The protective effect was reversed by a co-treatment with compound-C, an AMPK specific inhibitor, or by an over expression of a dominant-negative AMPKα cDNA. Inhibition of PKA with H89 or a suppression of Src kinase by a small hairpin siRNA also abrogated the protective effect of the low dose Met. Whereas, with a higher dose of Met (1.0 mM), the protective effects were abolished regardless of the enhanced AMPK, PKA/CREB1 and Src kinase activity. In high dose Met treated cells, expression of platelet-derived growth factor receptor (PDGFR) was significantly suppressed. Furthermore, the protective effect of low dose Met was totally reversed by co-treatment with AG1296, a PDGFR specific antagonist. These data provide in vitro evidence supporting a signaling cascade by which low dose Met exerts protective effects against Dox via sequential involvement of AMPK, PKA/CREB1, Src and PDGFR. Whereas high dose Met reverses the effect by suppressing PDGFR expression.

Expression of a pulmonary endothelial norepinephrine transporter

Summary. To determine whether pulmonary endothelial cells express the plasma membrane norepinephrine transporter (NET), an ovine-specific riboprobe was prepared from a partial NET cDNA clone isolated by PCR from a placental cDNA library. Northern hybridization detected a predominant 5.8 kb and a weaker transcript at 3.6 kb in RNAs isolated from near-term ovine fetal pulmonary endothelial cells. These data should be useful for future studies examining the molecular mechanisms underlying pulmonary endothelial NET expression and regulation.