Raj Kishore

Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic signaling

Sonic hedgehog (Shh) is a crucial regulator of organ development during embryogenesis. We investigated whether intramyocardial gene transfer of naked DNA encoding human Shh (phShh) could promote a favorable effect on recovery from acute and chronic myocardial ischemia in adult animals, not only by promoting neovascularization, but by broader effects, consistent with the role of this morphogen in embryogenesis. After Shh gene transfer, the hedgehog pathway was upregulated in mammalian fibroblasts and cardiomyocytes. This resulted in preservation of left ventricular function in both acute and chronic myocardial ischemia by enhanced neovascularization, and reduced fibrosis and cardiac apoptosis. Shh gene transfer also enhanced the contribution of bone marrow–derived endothelial progenitor cells to myocardial neovascularization. These data suggest that Shh gene therapy may have considerable therapeutic potential in individuals with acute and chronic myocardial ischemia by triggering expression of multiple trophic factors and engendering tissue repair in the adult heart.

Estradiol Enhances Recovery After Myocardial Infarction by Augmenting Incorporation of Bone Marrow–Derived Endothelial Progenitor Cells Into Sites of Ischemia-Induced Neovascularization via Endothelial Nitric Oxide Synthase–Mediated Activation of Matrix Metalloproteinase-9

Background— Recent data have indicated that estradiol can modulate the kinetics of endothelial progenitor cells (EPCs) via endothelial nitric oxide synthase (eNOS)–dependent mechanisms. We hypothesized that estradiol could augment the incorporation of bone marrow (BM)–derived EPCs into sites of ischemia-induced neovascularization, resulting in protection from ischemic injury. Methods and Results— Myocardial infarction (MI) was induced by ligation of the left coronary artery in ovariectomized mice receiving either 17β-estradiol or placebo. Estradiol induced significant increases in circulating EPCs 2 and 3 weeks after MI in estradiol-treated animals, and capillary density was significantly greater in estradiol-treated animals. Greater numbers of BM-derived EPCs were observed at ischemic sites in estradiol-treated animals than in placebo-treated animals 1 and 4 weeks after MI. In eNOS-null mice, the effect of estradiol on mobilization of EPCs was lost, as was the functional improvement in recovery from acute myocardial ischemia. A decrease was found in matrix metalloproteinase-9 (MMP-9) expression in eNOS-null mice under basal and estradiol-stimulated conditions after MI, the mobilization of EPCs by estradiol was lost in MMP-9–null mice, and the functional benefit conferred by estradiol treatment after MI in wild-type mice was significantly attenuated. Conclusions— Estradiol preserves the integrity of ischemic tissue by augmenting the mobilization and incorporation of BM-derived EPCs into sites of neovascularization by eNOS-mediated augmentation of MMP-9 expression in the BM. Moreover, these data have broader implications with regard to our understanding of the role of EPCs in post-MI recovery and on the sex discrepancy in cardiac events.

Estrogen Receptors α and β Mediate Contribution of Bone Marrow–Derived Endothelial Progenitor Cells to Functional Recovery After Myocardial Infarction

Background— Estradiol (E2) modulates the kinetics of circulating endothelial progenitor cells (EPCs) and favorably affects neovascularization after ischemic injury. However, the roles of estrogen receptors &agr; (ER&agr;) and &bgr; (ER&bgr;) in EPC biology are largely unknown. Methods and Results— In response to E2, migration, tube formation, adhesion, and estrogen-responsive element–dependent gene transcription activities were severely impaired in EPCs obtained from ER&agr;-knockout mice (ER&agr;KO) and moderately impaired in ER&bgr;KO EPCs. The number of ER&agr;&Kgr;&Ogr; EPCs (42.4±1.5; P<0.001) and ER&bgr;KO EPCs (55.4±1.8; P= 0.03) incorporated into the ischemic border zone was reduced as compared with wild-type (WT) EPCs (72.5±1.3). In bone marrow transplantation (BMT) models, the number of mobilized endogenous EPCs in E2-treated mice was significantly reduced in ER&agr;KO BMT (WT mice transplanted with ER&agr;KO bone marrow) (2.03±0.18%; P= 0.004 versus WT BMT) and ER&bgr;KO BMT (2.62±0.07%; P= 0.02 versus WT) compared with WT BMT (2.87±0.13%) (WT to WT BMT as control) mice. Capillary density at the border zone of ischemic myocardium also was significantly reduced in ER&agr;KO BMT and ER&bgr;KO BMT compared with WT mice (WT BMT, 1718±75/mm2; ER&agr;KO BMT, 1107±48/mm2; ER&bgr;KO BMT, 1567±50/mm2). ER&agr; mRNA was expressed more abundantly on EPCs compared with ER&bgr;. Moreover, vascular endothelial growth factor was significantly downregulated on ER&agr;KO EPCs compared with WT EPCs both in vitro and in vivo. Conclusions— Both ER&agr; and ER&bgr; contribute to E2-mediated EPC activation and tissue incorporation and to preservation of cardiac function after myocardial infarction. ER&agr; plays a more prominent role in this process. Moreover, ER&agr; contributes to upregulation of vascular endothelial growth factor, revealing possible mechanisms of an effect of E2 on EPC biology. Finally, these data provide additional evidence of the importance of bone marrow–derived EPC phenotype in ischemic tissue repair.

IL-10-induced TNF-alpha mRNA destabilization is mediated via IL-10 suppression of p38 MAP kinase activation and inhibition of HuR expression

Inflammation plays an essential role in vascular injury and repair. Mononuclear phagocytes are important contributors in these processes, in part, via adhesive interactions and secretion of proinflammatory cytokines. The antiinflammatory cytokine interleukin (IL)‐10 suppresses such responses via deactivation of monocytes/macrophages and repression of inflammatory cytokine expression. The mechanisms of IL‐10s suppressive action are, however, incompletely characterized. Here, we report that systemic IL‐10 treatment after carotid artery denudation in mice blunts inflammatory cell infiltration and arterial tumor necrosis factor (TNF) expression. At the molecular level, in a human monocytic cell line, U937 IL‐10 suppressed LPS‐induced mRNA expression of a number of inflammatory cytokines, mainly via posttranscriptional mRNA destabilization. Detailed studies on IL‐10 regulation of TNF‐ mRNA expression identified AU‐rich elements (ARE) in the 3 untranslated region as a necessary determinant of IL‐10mediated TNF‐α mRNA destabilization. IL‐10 sensitivity to TNF depends on the ability of IL‐10 to inhibit the expression and mRNA‐stabilizing protein HuR and via IL‐10 mediated repression of p38 mitogen‐activated protein (MAP) kinase activation. Because IL‐10 function and signaling are important components for control of inflammatory responses, these results may provide insights necessary to develop strategies for modulating vascular repair and other accelerated arteriopathies, including transplant vasculopathy and vein graft hyperplasia.—Johnson Rajasingh, Evelyn Bord, Corinne Luedemann, Jun Asai, Hiromichi Hamada, Tina Thorne, Gangjian Qin, David Goukassian, Yan Zhu, Douglas W. Losordo, and Raj Kishore. IL‐10‐induced TNF‐αalpha mRNA destabilization is mediated via IL‐10 suppression of p38 MAP kinase activation and inhibition of HuR expression. FASEB J. 20, E1393–E1403 (2006)

Functional disruption of α4 integrin mobilizes bone marrow-derived endothelial progenitors and augments ischemic neovascularization

The cell surface receptor α4 integrin plays a critical role in the homing, engraftment, and maintenance of hematopoietic progenitor cells (HPCs) in the bone marrow (BM). Down-regulation or functional blockade of α4 integrin or its ligand vascular cell adhesion molecule-1 mobilizes long-term HPCs. We investigated the role of α4 integrin in the mobilization and homing of BM endothelial progenitor cells (EPCs). EPCs with endothelial colony-forming activity in the BM are exclusively α4 integrin–expressing cells. In vivo, a single dose of anti–α4 integrin antibody resulted in increased circulating EPC counts for 3 d. In hindlimb ischemia and myocardial infarction, systemically administered anti–α4 integrin antibody increased recruitment and incorporation of BM EPCs in newly formed vasculature and improved functional blood flow recovery and tissue preservation. Interestingly, BM EPCs that had been preblocked with anti–α4 integrin ex vivo or collected from α4 integrin–deficient mice incorporated as well as control cells into the neovasculature in ischemic sites, suggesting that α4 integrin may be dispensable or play a redundant role in EPC homing to ischemic tissue. These data indicate that functional disruption of α4 integrin may represent a potential angiogenic therapy for ischemic disease by increasing the available circulating supply of EPCs.

Tumor necrosis factor-mediated E2F1 suppression in endothelial cells: differential requirement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signal transduction pathways.

Abstract— After balloon angioplasty, locally expressed tumor necrosis factor (TNF)-&agr; disrupts endothelial cell (EC) proliferation and reendothelialization of the injured vessel. We have previously reported that TNF inhibits the EC cycle and downregulates the transcription factor E2F1. Ectopic expression of E2F1 at the site of injury improves reendothelialization of the injured vessel. In this study, we report that c-Jun N-terminal kinase (JNK) 1 and p38 mitogen-activated protein kinases (MAPKs) are differentially required for E2F1 expression and activity in ECs. Overexpression of constitutively active JNK1 mimicked TNF-mediated inhibitory events, whereas dominant-negative JNK1 prevented these effects. E2F cis elements in the promoter of E2F1 gene mediate suppressive actions of TNF, because removal of these sites rendered E2F1 promoter activity insensitive to TNF. JNK1 physically interacted with E2F1 and inactivated it via direct phosphorylation. Additionally, TNF inhibited Rb phosphorylation and dissociation from E2F1. Overexpression of constitutively active p38 MAPK facilitated Rb-E2F1 dissociation, whereas that of dominant-negative p38 MAPK did not. Taken together, these data suggest a differential requirement of JNK1 and p38 MAPK in TNF regulation of E2F1. Targeted inactivation of JNK1 at arterial injury sites may represent a potential therapeutic intervention for ameliorating TNF-mediated EC dysfunction.

Ethanol Modulation of TNF‐alpha Biosynthesis and Signaling in Endothelial Cells: Synergistic Augmentation of TNF‐alpha Mediated Endothelial Cell Dysfunctions by Chronic Ethanol

Despite reported cardio-protective effects of low alcohol intake, chronic alcoholism remains a risk factor in the pathogenesis of coronary artery disease. Dose related bimodal effects of alcohol on cardiovascular system might reflect contrasting influences of light versus heavy alcohol consumption on the vascular endothelium. Chronic ethanol induced damage to various organs has been linked to the increased release of TNF-alpha (TNF). We have previously shown that TNF, expressed at the sites of arterial injury, suppresses re-endothelialization of denuded arteries and inhibits endothelial cell (EC) proliferation in vitro. Here we report that in vitro chronic ethanol exposure enhances agonist-induced TNF mRNA and protein expression in EC. Ethanol-mediated increment in TNF expression involves increased de novo transcription without affecting mRNA stability. DNA binding assays revealed that ethanol-induced TNF up regulation was AP1 dependent. Functionally, TNF induced EC dysfunction, including reduced proliferation, migration and cyclin A expression, were all markedly enhanced in the presence of ethanol. Additionally, expression of cyclin D1 was significantly attenuated in cells co-treated with TNF and ethanol while each treatment alone had little effect on cyclin D1 expression. Furthermore, exposure to ethanol potentiated and prolonged agonist-induced activation of JNK. Inhibition of JNK by over-expression of dominant negative JNK1 substantially reversed ethanol/TNF-mediated inhibition of cyclin A expression and EC proliferation, suggesting modulation of JNK1 signaling as the mechanism for ethanol/TNF-induced EC dysfunctions. Taken together, these data indicate that chronic ethanol consumption may negatively influence post angioplasty re-endothelialization thereby contributing to the development of restenosis.

Functionally Novel Tumor Necrosis Factor-α–Modulated CHR-Binding Protein Mediates Cyclin A Transcriptional Repression in Vascular Endothelial Cells

Abstract— Local expression of tumor necrosis factor-&agr; (TNF-&agr;) at the sites of arterial injury after balloon angioplasty, suppresses endothelial cell (EC) proliferation and negatively affects reendothelialization of the injured vessel. We have previously reported that in vitro exposure of ECs to TNF-&agr; induced EC growth arrest and apoptosis. These effects were mediated, at least in part, by downregulation of cell cycle regulatory proteins. In the present study, we report potential mechanism(s) for TNF-&agr;–mediated suppression of cyclin A in ECs. TNF-&agr; exposure to ECs completely abrogated cyclin A mRNA expression via mechanisms involving both transcriptional and posttranscriptional modifications. TNF-&agr; inhibited de novo cyclin A mRNA synthesis and suppressed cyclin A promoter activity. Utilizing deletion mutants of human cyclin A promoter, we have identified CDE-CHR (C ell cycle–D ependent E lements–C ell cycle genes H omology R egion) region of cyclin A promoter as a target for TNF-&agr; suppressive action. Experiments to investigate CDE-CHR binding proteins/factors revealed a TNF-&agr;–mediated increase in specific DNA binding activity to the CHR elements. This increase in binding activity by TNF-&agr; was mediated via the induction of a functionally novel 84-kDa protein that binds specifically to CHR in Southwestern assays. UV cross-linking and SDS-PAGE analysis of proteins eluted from specific complex confirmed the presence of this 84-kDa protein. Moreover, induction of this protein by TNF-&agr; was protein synthesis dependent. Additionally, exposure of ECs to TNF-&agr; markedly reduced cyclin A mRNA stability. Targeted disruption of this protein could potentially be a therapeutic strategy to rescue EC proliferation in vivo.

Inhibition of Melanoma Angiogenesis by Telomere Homolog Oligonucleotides

Telomere homolog oligonucleotides (T-oligos) activate an innate telomere-based program that leads to multiple anticancer effects. T-oligos act at telomeres to initiate signaling through the Werner protein and ATM kinase. We wanted to determine if T-oligos have antiangiogenic effects. We found that T-oligo-treated human melanoma (MM-AN) cells had decreased expression of vascular endothelial growth factor (VEGF), VEGF receptor 2, angiopoeitin-1 and -2 and decreased VEGF secretion. T-oligos activated the transcription factor E2F1 and inhibited the activity of the angiogenic transcription factor, HIF-1α. T-oligos inhibited EC tubulogenesis and total tumor microvascular density matrix invasion by MM-AN cells and ECs in vitro. In melanoma SCID xenografts, two systemic T-oligo injections decreased by 60% (P < .004) total tumor microvascular density and the functional vessels density by 80% (P < .002). These findings suggest that restriction of tumor angiogenesis is among the hosts innate telomere-based anticancer responses and provide further evidence that T-oligos may offer a powerful new approach for melanoma treatment.