Yinke Yang

Significant improvement of heart function by cotransplantation of human mesenchymal stem cells and fetal cardiomyocytes in postinfarcted pigs

BACKGROUND Viable cardiomyocytes after myocardial infarction (MI) are unable to repair the necrotic myocardium due to their limited capability of regeneration. The present study investigated whether intramyocardial transplantation of human mesenchymal stem cells (hMSCs) or cotransplantation of hMSCs plus human fetal cardiomyocytes (hFCs; 1:1) reconstituted impaired myocardium and improved cardiac function in MI pigs. METHODS AND RESULTS Cultured hMSCs were transfected with green fluorescent protein (GFP). Six weeks after MI induction and cell transplantation, cardiac function was significantly improved in MI pigs transplanted with hMSCs alone. However, the improvement was even markedly greater in MI pigs cotransplanted with hMSCs plus hFCs. Histological examination demonstrated that transplantation of hMSCs alone or hMSCs plus hFCs formed GFP-positive engrafts in infarcted myocardium. In addition, immunostaining for cardiac alpha-myosin heavy chain and troponin I showed positive stains in infarcted regions transplanted with hMSCs alone or hMSCs plus hFCs. CONCLUSIONS Our data demonstrate that transplantation of hMSCs alone improved cardiac function in MI pigs with a markedly greater improvement from cotransplantation of hMSCs plus hFCs. This improvement might result from myocardial regeneration and angiogenesis in injured hearts by engrafted cells.

Cardiomyocytes overexpressing TNF-α attract migration of embryonic stem cells via activation of p38 and c-Jun amino-terminal kinase

Tumor necrosis factor‐α (TNF‐α) plays an important role in the pathogenesis of myocardial infarction. Stem cells are able to regenerate infarcted myocardium. This study investigated whether TNF‐α was able to induce migration of embryonic stem cells (ESCs) in vitro. We used a Transwell assay in which neonatal rat cardiomyocytes, with or without transfection of TNF‐α cDNA, were plated in the lower compart‐ ments and mouse ESCs tagged with green fuorescent protein were added to the upper compartments. TNF‐α level was significantly increased in the medium of the lower compartments seeded with TNF‐α‐transfected cardiomyocytes. Compared with the controls, overex‐ pression of TNF‐α significantly enhanced migration of ESCs to the lower compartments. This enhancement was attenuated by preincubation of ESCs with the antibody against the type II TNF‐α receptor (TNF‐RII), but not by the antibody against the type I TNF‐α receptor (TNF‐RI). Western blot analysis showed that the phosphorylated protein levels of p38 and c‐Jun amino‐terminal kinase (JNK) were significantly in‐ creased in TNF‐α‐treated ESCs. Inhibition of the activ‐ ity of p38 or JNK significantly attenuated TNF‐α‐in‐ duced ESC migration. Our data demonstrate that excessive TNF‐α stimulates TNF‐RII and enhances mi‐ gration of ESCs in vitro. Activation of p38 and JNK is required for TNF‐α‐enhanced ESC migration.—Chen, Y., Ke, Q., Yang, Y., Rana, J. S., Tang, J., Morgan, J. P., Xiao, Y.‐F. Cardiomyocytes overexpressing TNF‐α at‐ tract migration of embryonic stem cells via activation of p38 and c‐Jun amino‐terminal kinase. FASEB J. 17, 2231‐2239 (2003)

Induction of Cardiac Cytochrome P450 in Cocaine-Treated Mice

Cytochrome P450 (P450) is a ubiquitous family of enzymes responsible for the metabolism of a wide variety of drugs and their metabolites, including cocaine. To investigate the effects of cocaine on myocardial injuries and cardiac P450 expression, BALB/c mice were injected daily intraperitoneally with cocaine (30 mg/kg) or cocaine plus pretreatment of P450 inhibitors for 14 days. Tumor necrosis factor-α (TNF-α) content and creatine phosphokinase (CPK) activity in mice hearts and serums were significantly increased after long-term treatment with cocaine. Pretreatment with the P450 inhibitor, cimetidine (Cime, 50 mg/kg) or metyrapone (Mety, 40 mg/kg) abolished or significantly attenuated the effects of cocaine on TNF-α and CPK activity. Western blot analysis shows that mouse cardiac tissues express the P450 isoforms CYP1A1, CYP1A2, and CYP2J2. The protein levels normalized with cyclophilin A were 1.20 ± 0.07, 0.67 ± 0.03, and 1.48 ± 0.01 for CYP1A1, CYP1A2, and CYP 2J2, respectively. After cocaine administration, CYP2J2 increased by 43.6% and CYP1A1 increased by 108.5%, but CYP1A2 was not significantly altered. However, the cytochrome P450 inhibitors Cime and Mety suppressed the cocaine-induced increase in CYP1A1 and CYP2J2 expression. Moreover, application of Cime or Mety alone did not alter the level of cardiac TNF-α or the expression of P450. Our results demonstrate that long-term exposure to cocaine causes an increase in cardiac CYP1A1 and CYP2J2 concentration. We speculate that induction of P450 isoforms may cause cardiac injury due to cocaine metabolites locally catalyzed by P450 or the increase in P450 expression itself.

Embryonic stem cells attenuate viral myocarditis in murine model.

We used mice to test our hypothesis that in response to viral invasion, stem cells may migrate into the heart and attenuate the effect of viral myocarditis. Male BALB/c mice were divided into three groups: mouse embryonic stem (ES) cell control, encephalomyocarditis virus (EMCV), and EMCV + ES cells. After administration of ES cells via tail vein, mice were immediately inoculated with EMCV. Mice were sacrificed at different days after EMCV inoculation. Mortality was recorded. Inflammatory cell infiltration and necrosis (major pathological changes of viral myocarditis) were evaluated by hematoxylin-eosin staining. ES cell migration and differentiation were identified by immunofluorescence. The survival rate in the EMCV + ES cell group (80%) was significantly increased (p < 0.05) over the EMCV-alone group (64%). Also, the incidence of inflammatory cell infiltration and myocardial lesions was lower in the EMCV + ES cell mice. Furthermore, the result of green fluorescent protein (GFP) and a-actinin analysis indicated that ES cells migrated into the heart and differentiated into myocytes after virus inoculation. In conclusion, ES cells significantly increased the survival of viral myocarditis mice and also decreased the necrosis and infiltration of inflammatory cells. These results demonstrated the ability of stem cells to mitigate the effects of viral infection on the heart and illustrated their potential therapeutic application to other mammalian species, including humans.

Intracellular accumulation of mercury enhances P450 CYP1A1 expression and Cl- currents in cultured shark rectal gland cells.

The effects of acute and subchronic exposure to mercury on the Cl- current (ICl) were investigated in cultured shark rectal gland (SRG) cells. The effects of intracellular accumulation of mercury on cytochrome P450 (P450) were also assessed. Bath perfusion of a cocktail solution containing forskolin, 1-isobutyl-3-methylxanthine, and 8-bromoadenosine monophosphate enhanced ICl. Addition of 10 microM HgCl2 significantly inhibited the cAMP-activated ICl (p < 0.05, n = 11). Intracellular dialysis with ATP gamma S did not prevent the inhibitory effect of mercury on ICl. In contrast, incubation of SRG cells with 10 microM HgCl2 for 48 hrs markedly increased ICl (p < 0.01, n = 12). Dephosphorylation of the channel by intracellular dialysis with phosphatase I and II abolished the mercury-incubated increase in ICl. The P450-mediated metabolite of arachidonic acid, 11,12-epoxyeicosatrienoic acid (11,12-EET), significantly increased ICl. However, application of 11,12-dihydroxyeicosatrienoic acid (11,12-DHT) did not alter ICl. Mercury incubation for 48 hrs did not alter the protein expression of Cl- channels, but caused an induction of CYP1A1 in cultured SRG cells. In addition, co-incubation of SRG cells with mercury and the P450 inhibitor clotrimazole prevented the mercury-incubated increase in ICl. Our results demonstrate that acute and subchronic application of mercury has opposing effects on ICl in cultured SRG cells. The acute effect of mercury on ICl may result from mercury blockade of Cl- channels. The subchronic effect of mercury on ICl may be due to an induction of P450 CYP1A1 and its mediated metabolites, but not due to an over-expression of Cl- channels.

Evidence for cocaine and methylecgonidine stimulation of M2 muscarinic receptors in cultured human embryonic lung cells

Muscarinic cholinoceptor stimulation leads to an increase in guanylyl cyclase activity and to a decrease in adenylyl cyclase activity. This study examined the effects of cocaine and methylecgonidine (MEG) on muscarinic receptors by measurement of cyclic GMP and cyclic AMP content in cultured human embryonic lung (HEL299) cells which specifically express M2 muscarinic receptors. A concentration‐dependent increase in cyclic GMP production was observed in HEL299 cells incubated with carbachol, cocaine, or MEG for 24 h. The increase in cyclic GMP content was 3.6 fold for 1 μM carbachol (P<0.01), 3.1 fold for 1 μM cocaine (P<0.01), and 7.8 fold for 1 μM MEG (P<0.001), respectively. This increase in cyclic GMP content was significantly attenuated or abolished by the muscarinic receptor antagonist atropine or the M2 blocker methoctramine. In contrast, cocaine, MEG, and carbachol produced a significant inhibition of cyclic AMP production in HEL299 cells. Compared to the control, HEL299 cells treated with 1 μM cocaine decreased cyclic AMP production by 30%. MEG and carbachol at 1 μM decreased cyclic AMP production by 37 and 38%, respectively. Atropine or methoctramine at 1 or 10 μM significantly attenuated or abolished the cocaine‐induced decrease in cyclic AMP production. However, the antagonists alone had neither an effect on cyclic GMP nor cyclic AMP production. Pretreatment of HEL299 cells with pertussis toxin prevented the cocaine‐induced reduction of cyclic AMP production. Western blot analysis showed that HEL299 cells specifically express M2 muscarinic receptors without detectable M1 and M3. Incubation of HEL299 cells with cocaine, carbachol, and atropine did not alter the expression of M2 protein levels. However, the inducible isoform of nitric oxide synthase (iNOS) was induced in the presence of cocaine or carbachol and this induction was significantly attenuated after addition of atropine or methoctramine. The present data show that cocaine and MEG significantly affect cyclic GMP and cyclic AMP production in cultured HEL299 cells. Our results also show that these effects result from the drug‐induced stimulation of M2 muscarinic receptors accompanied with no alterations of receptor expression. However, the induction of iNOS by cocaine may result in the increase in cyclic GMP production.

Enhancement of nitric oxide production by methylecgonidine in cultured neonatal rat cardiomyocytes

In the present experiments, we investigated the effects of methylecgonidine (MEG) on nitric oxide (NO) production in cultured neonatal rat cardiomyocytes. Incubation of cultured cardiomyocytes with carbachol or MEG for 48 h significantly enhanced NO production. No release was increased from 1.48±0.13 μM (mg protein)−1 for control to 5.73±0.19 μM (mg protein)−1 for 1 μM carbachol treated cells (P<0.001). In addition, incubation with 1 μM MEG enhanced NO production to 5.55±0.28 μM (mg protein)−1. The effects of MEG on NO production were concentration‐dependent. The muscarinic antagonist atropine prevented the enhancement of NO production induced by carbachol or MEG. Compared to MEG‐induced NO production, cocaine was much less potent. The enhancement of NO production by carbachol or MEG was even greater in cultured cardiomyocytes transfected with the M2 cDNA. After 48‐h incubation with 1 μM carbachol or 1 μM MEG, NO production was increased by 6.5 and 6.7 fold, respectively, in cardiomyocytes overexpressing M2 receptors. Coincubation with atropine or NG‐nitro‐L‐arginine methyl ester abolished the enhancement of NO production. In contrast, NO production enhanced by carbachol or MEG in M1‐ or M3‐transfected cardiomyocytes was similar to the level in non‐transfected cells. Western blot analysis showed that the protein levels of M1, M2, and M3 were significantly increased in cardiomyocytes transfected with the receptor cDNAs, but MEG had no effect on the expressions. It is interesting that both carbachol and MEG caused a significant increase in constitutive endothelial NO synthase (eNOS) only in M2‐transfected cardiomyocytes, not in non‐transfected, M1‐ or M3‐transfected cells. Again, atropine blocked the MEG‐produced induction of eNOS. Our data demonstrate that MEG significantly enhanced NO production in cultured cardiomyocytes and that the enhancement of NO production may result from MEG stimulation of muscarinic M2 receptors.