Ting C. Zhao

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.

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.

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.

Effect of disruption of Akt-1 of lin(-)c-kit(+) stem cells on myocardial performance in infarcted heart.

AIMS We have demonstrated an important role of bone marrow-derived stem cells in preservation of myocardial function. We investigated whether Akt-1 of lin(-)c-kit(+) stem cells preserves ventricular function following myocardial infarction (MI). METHODS AND RESULTS Isolated lin(-)c-kit(+) cells were conjugated with anti-c-kit heteroconjugated to anti-vascular cell adhesion molecule to facilitate the attachment of stem cells into damaged tissues. Female severe combined immunodeficient mice were used as recipients. MI was created by ligation of the left descending artery. After 48 h, animals were divided into four groups: (i) sham (n = 5): animals underwent thoracotomy without MI; (ii) MI (n = 5): animals underwent MI and received medium; (iii) MI + wild-type (Wt) stem cells (n = 6): MI animals received 5 x 10(5) Wt lin(-)c-kit(+) stem cells; (iv) MI + Akt-1(-/-) stem cells (n = 6): MI animals received 5 x 10(5) Akt-1(-/-) lin(-)c-kit(+) stem cells. Two weeks later, left ventricular function was measured in the Langendorff mode. The peripheral administration of Wt armed stem cells into MI animals restored ventricular function, which was absent in animals receiving Akt-1(-/-) cells. Real-time PCR indicates a decrease in SRY3, a Y chromosome marker in hearts receiving Akt-1(-/-) cells. An increase in angiogenic response was demonstrated in hearts receiving Wt stem cells but not Akt-1(-/-) stem cells. CONCLUSION Our results demonstrate that the peripheral administration of Wt lin(-)c-kit(+) stem cells restores ventricular function and promotes angiogenic response following MI. These benefits were abrogated in MI mice receiving Akt-1(-/-) stem cells, suggesting the pivotal role of Akt-1 in mediating stem cells to protect MI hearts.

β-Adrenergic Receptor Mediated Protection against Doxorubicin-Induced Apoptosis in Cardiomyocytes: The Impact of High Ambient Glucose

Recent studies have demonstrated that the beta2-adrenergic receptor (beta2AR)-Galphai signaling pathway exerts a cardiac antiapoptotic effect. The goals of this study were to determine the intracellular signaling factors involved in beta2AR-mediated protection against doxorubicin-induced apoptosis in H9c2 cardiomyocyte and explore the impact of high ambient glucose on the antiapoptotic effect. Under physiological glucose environment (100 mg/dl), beta2AR stimulation prevented doxorubicin-induced apoptosis, which was attenuated by cotreatment with wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, or transfection of a dominant-negative Akt. Inhibition of Src kinase with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine or cSrc small interfering RNA 32 also attenuated the antiapoptotic effect. Inhibition of platelet-derived growth factor receptor (PDGFR) with AG1296 reversed the beta2AR-induced antiapoptotic effect. Transfection of an active Src cDNA (Y529F) alone was sufficient to render the cells resistant to apoptosis, and the resistance was blocked by wortmannin. Transfection of an active PI3K minigene (iSH2-p110) alone also induced resistance to apoptosis, and the resistance was reversed by an Akt-inhibitor but not by AG1296. High ambient glucose (450 mg/dl) caused two major effects: 1) it significantly reduced betaAR-induced PDGFR phosphorylation, Src kinase activity, and activation of PI3K signaling pathway; and 2) it partially attenuated beta2AR-induced antiapoptotic effect. These data provide in vitro evidence supporting a signaling cascade by which beta2AR exerts a protective effect against doxorubicin-induced apoptosis via sequential involvement of Galphai, Gbetagamma, Src, PDGFR, PI3K, and Akt. High ambient glucose significantly attenuates beta2AR-mediated cardioprotection by suppressing factors involved in this cascade including PDGFR, Src, and PI3K/Akt.