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Terry B Rogers,
Shibani Pati,
Shirley Gaa,
Dushon Riley,
Aarif Y Khakoo,
Shalin Patel,
Robert D Wardlow,
Cecilia A Frederick, Gentzon Hall,
Li-Ping He,
W Jonathan Lederer
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ABSTRACT: Since massive irreversible loss of cardiac myocytes occurs following myocardial injury, injection of human mesenchymal stem cells (hMSCs) has emerged as a promising therapeutic intervention. Despite the growing enthusiasm for this approach, the understanding of how hMSCs evoke cardiac improvement is ever more controversial. The present study critically tests hypothesis that hMSCs provide specific benefit directly to damaged ventricular myocytes. Cultures of neonatal mouse ventricular cardiac myocytes (nMCM) were subjected to two distinct acute stress protocols; incubations with either endotoxin, lipopolysaccharide (LPS) or toxic cytokine, IL-1β. Myocyte injury was assessed in intracellular Ca(2+) signaling assays in fluo-3-loaded nMCMs that were imaged with high temporal resolution by fluorescent microscopy. Following LPS or IL-1β treatment there was profound myocyte injury, manifest by chaotic [Ca(2+)](i) handling, quantified as a 3- to 5-fold increase in spontaneous [Ca(2+)](i) transients. Antibody neutralization experiments reveal such damage is mediated in part by interleukin-18 and not by tumor necrosis factor-α (TNF-α). Importantly, normal [Ca(2+)](i) signaling was preserved when cardiomyocytes were co-cultured with hMSCs. Since normal [Ca(2+)](i) handling was maintained in transwell cultures, where nMCMs and hMSCs were separated by a permeable membrane, a protective paracrine signaling cascade is operable. hMSCs provoke a genetic reprogramming of cardiomyocytes. LPS provokes release of TNFα from nMCMs which is blocked by hMSCs grown in co- or transwell cultures. Consistent with cytokine release, flow cytometry analyses reveal that hMSCs also block the LPS- and IL-1β-dependent activation of cardiac transcription factor, NF-κB. Importantly, hMSC-conditioned medium restores normal Ca(2+) signaling in LPS- and IL-1β-damaged nMCMs. These results reveal new evidence that hMSCs elicit protective and reparative effects on cardiac tissue through molecular reprogramming of the cardiac myocytes themselves. Thus these studies provide novel new insight into the cellular and molecular mechanisms that underlie the therapeutic benefit of hMSCs in the setting of heart failure. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".
Journal of Molecular and Cellular Cardiology 02/2011; 50(2):346-56. · 5.17 Impact Factor
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ABSTRACT: The Nuclear Factor-kappaB (NF-kappaB) signaling pathway has been linked to several pathologic processes in the myocardium including cardiomyocyte proinflammatory cytokine release, ischemia/reperfusion injury, hypertrophy and apoptosis. However, very little is known about the intracellular mechanisms that govern NF-kappaB activity in the myocardial cells. Recent advances in our understanding of the regulation of NF-kappaB signaling in non-myocyte systems suggest that the activity of the NF-kappaB pathway is tightly regulated by a diversity of stress-activated signaling intermediates through direct post-translational modification of various components of the NF-kappaB pathway. In this review, we will focus on these recent revelations and their implications not only in cardiac pathologies, but in the development of new therapeutic strategies to manage heart disease.
Journal of Molecular and Cellular Cardiology 11/2006; 41(4):580-91. · 5.17 Impact Factor
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ABSTRACT: Coupled to hundreds of receptors, G-proteins modulate signal transduction pathways and are important hormonal targets. The first objective was to determine the effect of pregnancy and estradiol on myometrial guanosine triphosphatase activity. The second objective was to begin dissecting the molecular mechanism(s) underlying alterations in guanosine triphosphatase activity.
Myometrial tissue was obtained from pregnant, nonpregnant, and ovariectomized untreated and estradiol-treated guinea pigs. Myometrial membranes were prepared by homogenization and differential centrifugation. Basal high-affinity specific guanosine triphosphatase activity was quantitated by enzymatic assay and expressed in rhomol 32Pi per milligram protein per minute. Guanosine triphosphatase activity was stimulated using oxytocin, isoproterenol, and prostaglandin F2alpha. Specific G-protein subunits were quantitated using Western blots. G-protein associated gene expression was semiquantitated using HGU133A gene array chips from Affymetrix.
Basal myometrial guanosine triphosphatase activity was increased in pregnant compared with nonpregnant animals. Estradiol increased basal myometrial guanosine triphosphatase activity, compared with untreated controls. The effect of estradiol on stimulated activity was agonist dependent. Both Galphas and Galphai isoform 1 protein levels were increased in myometrium from late pregnant compared with nonpregnant animals. By late gestation, the messenger ribonucleic acid levels of those genes were unaltered, compared with the nonpregnant animal. In general, the impact of pregnancy on G-protein family member gene messenger ribonucleic acid expression was modest. Only the small guanosine triphosphatase Rap1b demonstrated altered expression more than 2-fold during either myometrial quiescence (midpregnancy) or activation (term pregnancy) (up 3-fold during quiescence). Genomic network analyses revealed that the expression of another small guanosine triphosphatase, Rab7, was exclusively up-regulated (80%) during quiescence. During late pregnancy, network analysis showed that only G-protein beta was exclusively altered (up-regulated). Estradiol mimicked the pregnancy effect on both transcription and translation of G-protein family members for some but not all potentially relevant genes.
The increase in functional myometrial guanosine triphosphatase activity during pregnancy may reflect increased synthesis of 1 or more small guanosine triphosphatase.
American journal of obstetrics and gynecology 08/2006; 195(1):275-87. · 3.28 Impact Factor
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Aarif Y Khakoo,
Shibani Pati,
Stasia A Anderson,
William Reid,
Mohamed F Elshal,
Ilsa I Rovira,
Ahn T Nguyen,
Daniela Malide,
Christian A Combs, Gentzon Hall,
Jianhu Zhang,
Mark Raffeld,
Terry B Rogers,
William Stetler-Stevenson,
Joseph A Frank,
Marvin Reitz,
Toren Finkel
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ABSTRACT: Emerging evidence suggests that both human stem cells and mature stromal cells can play an important role in the development and growth of human malignancies. In contrast to these tumor-promoting properties, we observed that in an in vivo model of Kaposi's sarcoma (KS), intravenously (i.v.) injected human mesenchymal stem cells (MSCs) home to sites of tumorigenesis and potently inhibit tumor growth. We further show that human MSCs can inhibit the in vitro activation of the Akt protein kinase within some but not all tumor and primary cell lines. The inhibition of Akt activity requires the MSCs to make direct cell-cell contact and can be inhibited by a neutralizing antibody against E-cadherin. We further demonstrate that in vivo, Akt activation within KS cells is potently down-regulated in areas adjacent to MSC infiltration. Finally, the in vivo tumor-suppressive effects of MSCs correlates with their ability to inhibit target cell Akt activity, and KS tumors engineered to express a constitutively activated Akt construct are no longer sensitive to i.v. MSC administration. These results suggest that in contrast to other stem cells or normal stromal cells, MSCs possess intrinsic antineoplastic properties and that this stem cell population might be of particular utility for treating those human malignancies characterized by dysregulated Akt.
Journal of Experimental Medicine 06/2006; 203(5):1235-47. · 13.85 Impact Factor
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ABSTRACT: TNF-alpha is recognized as a significant contributor to myocardial dysfunction. Although several studies suggest that members of the NF-kappaB family of transcription factors are essential regulators of myocardial TNF-alpha gene expression, recent developments in our understanding of the modulation of NF-kappaB activity through posttranslational modification of NF-kappaB subunits suggest that the present view of NF-kappaB-dependent cytokine expression in heart is incomplete. Therefore, the goal of the present study was to examine the role of p65 subunit phosphorylation in the regulation of TNF-alpha production in cultured neonatal ventricular myocytes. Bacterial LPS-induced TNF-alpha production is accompanied by a 12-fold increase in phosphorylation of p65 at Ser536, a modification associated with enhancement of p65 transactivation potential. Pharmacological inhibition of IKK-beta reduced LPS-induced TNF-alpha production 38-fold, TNF-alpha mRNA levels 6-fold, and IkappaB-alpha phosphorylation 5-fold and degraded IkappaB-alpha 2-fold and p65 phosphorylation 6-fold. Overexpression of dominant-negative p65 reduced TNF-alpha production 3.5-fold, whereas overexpression of dominant-negative IKK-beta reduced LPS-induced TNF-alpha production 2-fold and p65 phosphorylation 2-fold. Overexpression of dominant-negative IKK-alpha had no effect on p65 phosphorylation or TNF-alpha production, revealing that IKK-beta, not IKK-alpha, plays a central role in regulation of p65 phosphorylation at Ser536 and TNF-alpha production in heart. Finally, we demonstrated, using a chromatin immunoprecipitation assay, that LPS stimulates recruitment of Ser536-phosphorylated p65 to the TNF-alpha gene promoter in cardiac myocytes. Taken together, these data provide compelling evidence for the role of NF-kappaB signaling in TNF-alpha gene expression in heart and highlight the importance of this proinflammatory gene-regulatory pathway as a potential therapeutic target in the management of cytokine-induced myocardial dysfunction.
AJP Heart and Circulatory Physiology 12/2005; 289(5):H2103-11. · 3.71 Impact Factor
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ABSTRACT: Although tumor necrosis factor (TNF)-alpha is implicated in numerous cardiac pathologies, the intracellular events leading to its production by heart cells are largely unknown. The goal of the present study was to identify the role of the transcription factor nuclear factor (NF)-kappaB in this process. Among the many inducers of TNF-alpha expression in myeloid cells, only lipopolysaccharide (LPS) led to its induction in cultured neonatal myocytes. LPS also activated the NF-kappaB pathway, as evidenced by the degradation of the inhibitory protein IkappaB and the appearance of NF-kappaB-binding complexes in nuclear extracts. Furthermore, inhibitors of NF-kappaB activation, such as lactacystin, MG132, and pyrrolidine dithiocarbamate, were found to completely block the production of TNF-alpha in response to LPS stimulation, indicating a requirement of NF-kappaB for TNF-alpha expression. However, interleukin-1beta and phorbol 12-myristate 13-acetate also activated NF-kappaB but did not evoke TNF-alpha expression, revealing that this factor is not sufficient for cytokine production. Detailed examination of the NF-kappaB cascade revealed that cardiac cells displayed a unique pattern of IkappaB degradation in response to LPS, with IkappaBbeta but not IkappaBalpha being degraded upon stimulation. Additionally, two specific p65-containing DNA-binding complexes were observed in the nuclear extracts of neonatal cardiomyocytes: an inducible complex that is necessary for TNF-alpha expression and a constitutive species. Taken together, these results reveal that NF-kappaB is not only involved in cytokine production but also may be linked to other pathways that subserve a constitutive, protective mechanism for the heart cell.
AJP Heart and Circulatory Physiology 04/2002; 282(3):H872-9. · 3.71 Impact Factor
