Dysfunctional and Proinflammatory Regulatory T-Lymphocytes Are Essential for Adverse Cardiac Remodeling in Ischemic Cardiomyopathy.

BACKGROUND: Heart failure (HF) is a state of inappropriately sustained inflammation, suggesting the loss of normal immunosuppressive mechanisms. Regulatory T-lymphocytes (Tregs) are considered key suppressors of immune responses; however, their role in HF is unknown. We hypothesized that Tregs are dysfunctional in ischemic cardiomyopathy and HF, and they promote immune activation and left ventricular (LV) remodeling. METHODS: Adult male wild-type C57BL/6 mice, Foxp3-diphtheria toxin receptor transgenic mice, and tumor necrosis factor (TNF) α receptor-1 (TNFR1)-/- mice underwent nonreperfused myocardial infarction to induce HF or sham operation. LV remodeling was assessed by echocardiography as well as histological and molecular phenotyping. Alterations in Treg profile and function were examined by flow cytometry, immunostaining, and in vitro cell assays. RESULTS: Compared with wild-type sham mice, CD4+Foxp3+ Tregs in wild-type HF mice robustly expanded in the heart, circulation, spleen, and lymph nodes in a phasic manner after myocardial infarction, beyond the early phase of wound healing, and exhibited proinflammatory T helper 1-type features with interferon-γ, TNFα, and TNFR1 expression, loss of immunomodulatory capacity, heightened proliferation, and potentiated antiangiogenic and profibrotic properties. Selective Treg ablation in Foxp3-diphtheria toxin receptor mice with ischemic cardiomyopathy reversed LV remodeling and dysfunction, alleviating hypertrophy and fibrosis, while suppressing circulating CD4+ T cells and systemic inflammation and enhancing tissue neovascularization. Tregs reconstituted after ablation exhibited restoration of immunosuppressive capacity and normalized TNFR1 expression. Treg dysfunction was also tightly coupled to Treg-endothelial cell contact- and TNFR1-dependent inhibition of angiogenesis and the mobilization and tissue infiltration of CD34+Flk1+ circulating angiogenic cells in a C-C chemokine ligand 5/C-C chemokine receptor 5-dependent manner. Anti-CD25-mediated Treg depletion in wild-type mice imparted similar benefits on LV remodeling, circulating angiogenic cells, and tissue neovascularization. CONCLUSIONS: Proinflammatory and antiangiogenic Tregs play an essential pathogenetic role in chronic ischemic HF to promote immune activation and pathological LV remodeling. The restoration of normal Treg function may be a viable approach to therapeutic immunomodulation in this disease.

CCR2+ Monocyte-Derived Infiltrating Macrophages Are Required for Adverse Cardiac Remodeling During Pressure Overload.

Although chronic inflammation is a central feature of heart failure (HF), the immune cell profiles differ with different underlying causes. This suggests that for immunomodulatory therapy in HF to be successful, it needs to be tailored to the specific etiology. Here, the authors demonstrate that monocyte-derived C-C chemokine receptor 2 (CCR2)+ macrophages infiltrate the heart early during pressure overload in mice, and that blocking this response either pharmacologically or with antibody-mediated CCR2+ monocyte depletion alleviates late pathological left ventricular remodeling and dysfunction, T-cell expansion, and cardiac fibrosis. Hence, suppression of CCR2+ monocytes/macrophages may be an important immunomodulatory therapeutic target to ameliorate pressure-overload HF.

Leukocyte iNOS is required for inflammation and pathological remodeling in ischemic heart failure.

In the failing heart, iNOS is expressed by both macrophages and cardiomyocytes. We hypothesized that inflammatory cell-localized iNOS exacerbates left ventricular (LV) remodeling. Wild-type (WT) C57BL/6 mice underwent total body irradiation and reconstitution with bone marrow from iNOS-/- mice (iNOS-/-c) or WT mice (WTc). Chimeric mice underwent coronary ligation to induce large infarction and ischemic heart failure (HF), or sham surgery. After 28 days, as compared with WTc sham mice, WTc HF mice exhibited significant (p < 0.05) mortality, LV dysfunction, hypertrophy, fibrosis, oxidative/nitrative stress, inflammatory activation, and iNOS upregulation. These mice also exhibited a ~twofold increase in circulating Ly6Chi pro-inflammatory monocytes, and ~sevenfold higher cardiac M1 macrophages, which were primarily CCR2- cells. In contrast, as compared with WTc HF mice, iNOS-/-c HF mice exhibited significantly improved survival, LV function, hypertrophy, fibrosis, oxidative/nitrative stress, and inflammatory activation, without differences in overall cardiac iNOS expression. Moreover, iNOS-/-c HF mice exhibited lower circulating Ly6Chi monocytes, and augmented cardiac M2 macrophages, but with greater infiltrating monocyte-derived CCR2+ macrophages vs. WTc HF mice. Lastly, upon cell-to-cell contact with naïve cardiomyocytes, peritoneal macrophages from WT HF mice depressed contraction, and augmented cardiomyocyte oxygen free radicals and peroxynitrite. These effects were not observed upon contact with macrophages from iNOS-/- HF mice. We conclude that leukocyte iNOS is obligatory for local and systemic inflammatory activation and cardiac remodeling in ischemic HF. Activated macrophages in HF may directly induce cardiomyocyte contractile dysfunction and oxidant stress upon cell-to-cell contact; this juxtacrine response requires macrophage-localized iNOS.

Elevating CXCR7 Improves Angiogenic Function of EPCs via Akt/GSK-3ß/Fyn-Mediated Nrf2 Activation in Diabetic Limb Ischemia.

RATIONALE: Endothelial progenitor cells (EPCs) respond to stromal cell-derived factor 1 (SDF-1) through chemokine receptors CXCR7 and CXCR4. Whether SDF-1 receptors involves in diabetes mellitus-induced EPCs dysfunction remains unknown. OBJECTIVE: To determine the role of SDF-1 receptors in diabetic EPCs dysfunction. METHODS AND RESULTS: CXCR7 expression, but not CXCR4 was reduced in EPCs from db/db mice, which coincided with impaired tube formation. Knockdown of CXCR7 impaired tube formation of EPCs from normal mice, whereas upregulation of CXCR7 rescued angiogenic function of EPCs from db/db mice. In normal EPCs treated with oxidized low-density lipoprotein or high glucose also reduced CXCR7 expression, impaired tube formation, and increased oxidative stress and apoptosis. The damaging effects of oxidized low-density lipoprotein or high glucose were markedly reduced by SDF-1 pretreatment in EPCs transduced with CXCR7 lentivirus but not in EPCs transduced with control lentivirus. Most importantly, EPCs transduced with CXCR7 lentivirus were superior to EPCs transduced with control lentivirus for therapy of ischemic limbs in db/db mice. Mechanistic studies demonstrated that oxidized low-density lipoprotein or high glucose inhibited protein kinase B and glycogen synthase kinase-3ß phosphorylation, nuclear export of Fyn and nuclear localization of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), blunting Nrf2 downstream target genes heme oxygenase-1, NAD(P)H dehydrogenase (quinone 1) and catalase, and inducing an increase in EPC oxidative stress. This destructive cascade was blocked by SDF-1 treatment in EPCs transduced with CXCR7 lentivirus. Furthermore, inhibition of phosphatidylinositol 3-kinase/protein kinase B prevented SDF-1/CXCR7-mediated Nrf2 activation and blocked angiogenic repair. Moreover, Nrf2 knockdown almost completely abolished the protective effects of SDF-1/CXCR7 on EPC function in vitro and in vivo. CONCLUSIONS: Elevated expression of CXCR7 enhances EPC resistance to diabetes mellitus-induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia. The benefits of CXCR7 are mediated predominantly by a protein kinase B/glycogen synthase kinase-3ß/Fyn pathway via increased activity of Nrf2.

Long-Term Outcome of Administration of c-kit(POS) Cardiac Progenitor Cells After Acute Myocardial Infarction: Transplanted Cells Do not Become Cardiomyocytes, but Structural and Functional Improvement and Proliferation of Endogenous Cells Persist for at Least One Year.

RATIONALE: Cardiac progenitor cells (CPCs) improve left ventricular remodeling and function after acute or chronic myocardial infarction. However, the long-term (>5 weeks) effects, potential tumorigenicity, and fate of transplanted CPCs are unknown. OBJECTIVE: To assess the outcome of CPC therapy at 1 year. METHODS AND RESULTS: Female rats underwent a 90-minute coronary occlusion; 4 hours after reperfusion, they received intracoronarily vehicle or 1 million male, syngeneic CPCs. One year later, CPC-treated rats exhibited smaller scars and more viable myocardium in the risk region, along with improved left ventricular remodeling and regional and global left ventricular function. No tumors were observed. Some transplanted (Y-chromosome(POS)) CPCs (or their progeny) persisted and continued to proliferate, but they failed to acquire a mature cardiomyocyte phenotype and were too few (4-8% of nuclei) to account for the benefits of CPC therapy. Surprisingly, CPC transplantation triggered a prolonged proliferative response of endogenous cells, resulting in increased formation of endothelial cells and Y-chromosome(NEG) CPCs for 12 months and increased formation, for at least 7 months, of small cells that expressed cardiomyocytic proteins (α-sarcomeric actin) but did not have a mature cardiomyocyte phenotype. CONCLUSIONS: The beneficial effects of CPCs on left ventricular remodeling and dysfunction are sustained for at least 1 year and thus are likely to be permanent. Because transplanted CPCs do not differentiate into mature myocytes, their major mechanism of action must involve paracrine actions. These paracrine mechanisms could be very prolonged because some CPCs engraft, proliferate, and persist at 1 year. This is the first report that transplantation of any cell type in the heart induces a proliferative response that lasts at least 1 year. The results strongly support the safety and clinical utility of CPC therapy.

Evidence for the involvement of sphingosine-1-phosphate in the homing and engraftment of hematopoietic stem cells to bone marrow.

The α-chemokine stromal-derived factor 1 (SDF-1), which binds to the CXCR4 receptor, directs migration and homing of CXCR4+ hematopoietic stem/progenitor cells (HSPCs) to bone marrow (BM) stem cell niches. Nevertheless, it is also known that CXCR4-/- fetal liver-derived hematopoietic stem cells engraft into BM and that blockade of CXCR4 by its antagonist AMD3100 does not prevent engraftment of HSPCs. Because of this finding of SDF-1-CXCR4-independent BM homing, the unique role of SDF-1 in HSPC homing has recently been challenged. While SDF-1 is the only chemokine that chemoattracts HSPCs, other chemoattractants for these cells have recently been described, including the bioactive phosphosphingolipid sphingosine-1-phosphate (S1P). To address the potential role of S1P in homing of HSPCs to BM, we performed hematopoietic transplants into mice deficient in BM-expressed sphingosine kinase 1 (Sphk1-/-) using hematopoietic cells from normal control mice as well as cells from mice in which floxed CXCR4 (CXCR4fl/fl) was conditionally deleted. We observed the presence of a homing and engraftment defect in HSPCs of Sphk1-/- mice that was particularly profound after transplantation of CXCR4-/- BM cells. Thus, our results indicate that BM-microenvironment-expressed S1P plays a role in homing of HSPCs. They also support the concept that, in addition to the SDF-1-CXCR4 axis, other chemotactic axes are also involved in homing and engraftment of HSPCs.

Effects of Intracoronary Infusion of Escalating Doses of Cardiac Stem Cells in Rats With Acute Myocardial Infarction.

BACKGROUND: Although c-kit(pos) cardiac stem cells (CSCs) preserve left ventricular (LV) function and structure after myocardial infarction, CSC doses have been chosen arbitrarily, and the dose-effect relationship is unknown. METHODS AND RESULTS: Rats underwent a 90-minute coronary occlusion followed by 35 days of reperfusion. Vehicle or CSCs at 5 escalating doses (0.3×10(6), 0.75×10(6), 1.5×10(6), 3.0×10(6), and 6.0×10(6) cells/heart) were given intracoronarily 4 h after reperfusion. The lowest dose (0.3×10(6)) had no effect on LV function and morphology, whereas 0.75, 1.5, and 3.0×10(6) significantly improved regional and global LV function (echocardiography and hemodynamic studies). These 3 doses had similar effects on echocardiographic parameters (infarct wall thickening fraction, LV end-systolic and end-diastolic volumes, LV ejection fraction) and hemodynamic variables (LV end-diastolic pressure, LV dP/dtmax, preload adjusted maximal power, end-systolic elastance, preload recruitable stroke work) and produced similar reductions in apoptosis, scar size, infarct wall thinning, and LV expansion index and similar increases in viable myocardium in the risk region (morphometry). Infusion of 6.0×10(6) CSCs markedly increased postprocedural mortality. Green fluorescent protein and 5-bromo-2'-deoxyuridine staining indicated that persistence of donor cells and formation of new myocytes were negligible with all doses. CONCLUSIONS: Surprisingly, in this rat model of acute myocardial infarction, the dose-response relationship for intracoronary CSCs is flat. A minimal dose between 0.3 and 0.75×10(6) is necessary for efficacy; above this threshold, a 4-fold increase in cell number does not produce greater improvement in LV function or structure. Further increases in cell dose are harmful.

Feasibility study of particulate extracellular matrix (P-ECM) and left ventricular assist device (HVAD) therapy in chronic ischemic heart failure bovine model.

Myocardial recovery with left ventricular assist device (LVAD) support is uncommon and unpredictable. We tested the hypothesis that injectable particulate extracellular matrix (P-ECM) with LVAD support promotes cell proliferation and improves cardiac function. LVAD, P-ECM, and P-ECM + LVAD therapies were investigated in chronic ischemic heart failure (IHF) calves induced using coronary embolization. Particulate extracellular matrix emulsion (CorMatrix, Roswell, GA) was injected intramyocardially using a 7 needle pneumatic delivery tool. Left ventricular assist devices (HVAD, HeartWare) were implanted in a left ventricle (LV) apex to proximal descending aorta configuration. Cell proliferation was identified using BrdU (5 mg/kg) injections over the last 45 treatment days. Echocardiography was performed weekly. End-organ regional blood flow (RBF) was quantified at study endpoints using fluorescently labeled microspheres. Before treatment, IHF calves had an ejection fraction (EF) of 33 ± 2% and left ventricular end-diastolic volume of 214 ± 18 ml with cardiac cachexia (0.69 ± 0.06 kg/day). Healthy weight gain was restored in all groups (0.89 ± 0.03 kg/day). EF increased with P-ECM + HVAD from 36 ± 5% to 75 ± 2%, HVAD 38 ± 4% to 58 ± 5%, and P-ECM 27 ± 1% to 66 ± 6%. P-ECM + HVAD demonstrated the largest increase in cell proliferation and end-organ RBF. This study demonstrates the feasibility of combined LVAD support with P-ECM injection to stimulate new cell proliferation and improve cardiac function, which warrants further investigation.

Identification of heme oxygenase 1 (HO-1) as a novel negative regulator of mobilization of hematopoietic stem/progenitor cells.

Activation of complement cascade (ComC) play and important role in mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB). While there are vast experimental data on the mechanisms and factors that induce or promote mobilization of HSPCs, there is relatively less data on negative regulators of this process. We demonstrate for the first time that heme oxygenase-1 (HO-1) that has a well-documented anti-inflammatory potential plays an important and heretofore unrecognized role in retention of HSPCs in BM niches by i) modulating negatively activation of mobilization promoting ComC, ii) maintaining stromal derived factor-1 (SDF-1) level in the BM microenvironment and iii) attenuating chemotactic responsiveness of HSPCs to SDF-1 and sphingosine-1 phosphate (S1P) gradients in PB. Furthermore, our data showing a positive mobilizing effect by a non-toxic small-molecule inhibitor of HO-1 (SnPP) suggest that blockade of HO-1 would be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate better the molecular mechanisms responsible for the potential effect of HO-1 in homing of HSPCs after transplantation.

Co-activation of nuclear factor-κB and myocardin/serum response factor conveys the hypertrophy signal of high insulin levels in cardiac myoblasts.

Hyperinsulinemia contributes to cardiac hypertrophy and heart failure in patients with the metabolic syndrome and type 2 diabetes. Here, high circulating levels of tumor necrosis factor (TNF)-α may synergize with insulin in signaling inflammation and cardiac hypertrophy. We tested whether high insulin affects activation of TNF-α-induced NF-κB and myocardin/serum response factor (SRF) to convey hypertrophy signaling in cardiac myoblasts. In canine cardiac myoblasts, treatment with high insulin (10(-8) to 10(-7) m) for 0-24 h increased insulin receptor substrate (IRS)-1 phosphorylation at Ser-307, decreased protein levels of chaperone-associated ubiquitin (Ub) E3 ligase C terminus of heat shock protein 70-interacting protein (CHIP), increased SRF activity, as well as ß-myosin heavy chain (MHC) and myocardin expressions. Here siRNAs to myocardin or NF-κB, as well as CHIP overexpression prevented (while siRNA-mediated CHIP disruption potentiated) high insulin-induced SR element (SRE) activation and ß-MHC expression. Insulin markedly potentiated TNF-α-induced NF-κB activation. Compared with insulin alone, insulin+TNF-α increased SRF/SRE binding and ß-MHC expression, which was reversed by the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and by NF-κB silencing. In the hearts of db/db diabetic mice, in which Akt phosphorylation was decreased, p38MAPK, Akt1, and IRS-1 phosphorylation at Ser-307 were increased, together with myocardin expression as well as SRE and NF-κB activities. In response to high insulin, cardiac myoblasts increase the expression or the promyogenic transcription factors myocardin/SRF in a CHIP-dependent manner. Insulin potentiates TNF-α in inducing NF-κB and SRF/SRE activities. In hyperinsulinemic states, myocardin may act as a nuclear effector of insulin, promoting cardiac hypertrophy.

Complement component 3 is necessary to preserve myocardium and myocardial function in chronic myocardial infarction.

Activation of the complement cascade (CC) with myocardial infarction (MI) acutely initiates immune cell infiltration, membrane attack complex formation on injured myocytes, and exacerbates myocardial injury. Recent studies implicate the CC in mobilization of stem/progenitor cells and tissue regeneration. Its role in chronic MI is unknown. Here, we consider complement component C3, in the chronic response to MI. C3 knockout (KO) mice were studied after permanent coronary artery ligation. C3 deficiency exacerbated myocardial dysfunction 28 days after MI compared to WT with further impaired systolic function and LV dilation despite similar infarct size 24 hours post-MI. Morphometric analysis 28 days post-MI showed C3 KO mice had more scar tissue with less viable myocardium within the infarct zone which correlated with decreased c-kit(pos) cardiac stem/progenitor cells (CPSC), decreased proliferating Ki67(pos) CSPCs and decreased formation of new BrdU(pos) /α-sarcomeric actin(pos) myocytes, and increased apoptosis compared to WT. Decreased CSPCs and increased apoptosis were evident 7 days post-MI in C3 KO hearts. The inflammatory response with MI was attenuated in the C3 KO and was accompanied by attenuated hematopoietic, pluripotent, and cardiac stem/progenitor cell mobilization into the peripheral blood 72 hours post-MI. These results are the first to demonstrate that CC, through C3, contributes to myocardial preservation and regeneration in response to chronic MI. Responses in the C3 KO infer that C3 activation in response to MI expands the resident CSPC population, increases new myocyte formation, increases and preserves myocardium, inflammatory response, and bone marrow stem/progenitor cell mobilization to preserve myocardial function.

Cigarette smoke adversely affects functions and cell membrane integrity in c-kit+ cardiac stem cells.

Cigarette smoking is a major risk factor for numerous diseases including cardiovascular diseases. Exposure to cigarette smoke (CS) leads to increased cardiovascular risk, myocardial injury, and mortality. Stem cell therapy is one of the promising therapeutic options available to treat myocardial injuries. Understanding the impact of cigarette smoke extract (CSE) on stem cell function would be valuable in determining the risk passed on during transplant. In this study, the impact of CSE on cardiac stem cell (CSC) functions was investigated using c-kit+ rat cardiac stem cells as the experimental model. Here, we hypothesized that CSE attenuates CSC membrane integrity, causes cytotoxicity, and affects many CSC functions via multiple mechanisms including modulation of extracellular stress-regulated kinase (ERK) (44/42) signaling and oxidative stress. The effects of CSE on CSCs were examined in vitro. Based on a published method, CSE was prepared. CSE-induced ERK signaling was detected by western blotting. CSE-induced modulation of catalase activity was also measured. Functional modulations due to CSE were examined via several methods including Apostain, BrdU, and LDH assays. In agreement with the CSE-induced activation of ERK, CSE-induced reduction in viability, migration, and increase in both cytotoxicity and para-cellular permeability were observed in CSCs. These results suggest that CSE impaired CSC responses that contribute to decreased ability of CSC to respond to stress or injury leading to exacerbation of the damage. Our findings will contribute to the understanding of the discipline and might contribute to the development of stem cell therapy approaches in the future.

Stromal cell derived factor-1α promotes C-Kit+ cardiac stem/progenitor cell quiescence through casein kinase 1α and GSK3ß.

A population of c-kit(+) cardiac stem/progenitor cells (CSPC) has been identified in the heart and shown to contribute to myocardial regeneration after infarction. Previously, we have shown the chemokine, stromal cell derived factor 1α (SDF1) is necessary for the myocardial response to infarction where chronic infusion of the CXCR4 antagonist, AMD3100, exacerbated MI. Notably, AMD3100 increased CSPC proliferation. The effect of SDF1 on CSPC proliferation was further investigated in primary cultures of magnetically sorted c-kit(+) CSPCs. SDF1 facilitated CSPC quiescence by blocking cell cycle progression at the G0 to G1 transition. SDF1 decreased casein kinase 1α (CK1α) consequently attenuating ß-catenin phosphorylation, destabilization, and degradation. Increased levels of ß-catenin with SDF1 were effective, increasing TCF/LEF reporter activity. SDF downregulation of CK1α was dependent on proteasomal degradation and decreased mRNA expression. CK1α siRNA knockdown verified SDF1-dependent CSPC quiescence requires CK1α downregulation and stablilization of ß-catenin. Conversely, ß-catenin knockdown increased CSPC proliferation. SDF1 also increased GSK3ß Y216 phosphorylation responsible for increased activity. SDF1 mediated CK1α downregulation and increase in GSK3ß activity affected cell cycle through Bmi-1 downregulation, increased cyclin D1 phosphorylation, and decreased cyclin D1 levels. In conclusion, SDF1 exerts a quiescent effect on resident c-kit(+) CSPCs by decreasing CK1α levels, increasing GSK3ß activity, stabilizing ß-catenin, and affecting regulation of the cell cycle through Bmi-1 and cyclin D1. SDF1-dependent quiescence is an important factor in stem and progenitor cell preservation under basal conditions, however, with stress or injury in which SDF1 is elevated, quiescence may limit expansion and contribution to myocardial regeneration.

Targeting phosphatidylinositol 3-kinase-Akt through hepatocyte growth factor for cardioprotection.

Several growth factors have been shown to protect the cardiomyocyte from the detrimental effects of acute ischemia-reperfusion injury, through the activation of a variety of cell-surface receptors and the subsequent recruitment of a number of intracellular signal transduction pathways. Among these growth factors, hepatocyte growth factor (HGF), also named as scatter factor, acts by recruiting the phosphatidylinositol 3-kinase (PI3K)-Akt signal transduction pathway, linked to cardioprotection, at the time of myocardial infarction and myocardial reperfusion. HGF has been reported to increase in the early phase of myocardial infarction, and has been shown to have mitogenic, angiogenic, antiapoptotic and antifibrotic activities in cardiac myocytes and endothelial cells. Also, endogenous HGF may play an important role in the regeneration of endothelial cells and cardiomyocytes by promoting angiogenesis and inhibiting apoptosis during remodeling of the ischemic myocardium. Thus, HGF has the potential to emerge as a cardioprotective agent for the treatment of several pathological cardiac conditions. Here we review the role of HGF with respect to its ability to confer direct myocardial protection in the setting of ischemia-reperfusion injury, focusing on the main underlying signaling pathway involved.

Long-term engraftment and angiogenic properties of lentivirally transduced adipose tissue-derived stromal cells.

Human adipose tissue-derived stromal cells (ADSCs) are being evaluated for cardiovascular repair. We developed an ex vivo method for producing angiogenic ADSCs transduced with a self-inactivating lentiviral vector (LV) expressing the enhanced green fluorescence protein (EGFP) from an internal cytomegalovirus (CMV) promoter to track these cells after in vivo engraftment. ADSCs from visceral adipose tissue were transduced using a LV incorporating the Rous Sarcoma Virus (RSV) long terminal repeat (LTR) sequences and the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to enhance EGFP gene expression. We compared infection protocols with non-concentrated lentiviral supernatant or pellet fractions after ultracentrifugation, testing transduction efficiency, and reporter gene expression by quantitative flow cytometry at 5 and 28 days. Transduction of ADSCs with pellet after ultracentrifugation provided the highest transduction rate [flow cytometry titers: 6.5 ± 0.3 × 10(5) transduction units (TU)/mL and 20 ± 1.2 × 10(6) TU/mL at day 5 with non-concentrated lentiviral supernatant and pellet, respectively, with titer in the supernatant after ultracentrifugation remaining undetectable]. Reporter gene expression did not affect cell viability, morphology, proliferation, differentiation, self-renewal, or angiogenic activity. Furthermore, reporter gene expression did not significantly affect Fas/CD95-induced apoptosis. The in vivo implantation of transduced ADSCs into a mouse ischemic leg model resulted in efficient engraftment and angiogenesis. ADSC gene labeling using LVs is feasible and efficient, without impairment of stem cell characteristics, cell engraftment, and angiogenic activity. Such transduced ADSCs can be efficiently tracked in vitro and in vivo and may serve as vehicle for therapeutic genes.

The heme oxygenase 1 inducer (CoPP) protects human cardiac stem cells against apoptosis through activation of the extracellular signal-regulated kinase (ERK)/NRF2 signaling pathway and cytokine release.

Intracoronary delivery of c-kit-positive human cardiac stem cells (hCSCs) is a promising approach to repair the infarcted heart, but it is severely limited by the poor survival of donor cells. Cobalt protoporphyrin (CoPP), a well known heme oxygenase 1 inducer, has been used to promote endogenous CO generation and protect against ischemia/reperfusion injury. Therefore, we determined whether preconditioning hCSCs with CoPP promotes CSC survival. c-kit-positive, lineage-negative hCSCs were isolated from human heart biopsies. Lactate dehydrogenase release assays demonstrated that preconditioning CSCs with CoPP markedly enhanced cell survival after oxidative stress induced by H(2)O(2), concomitant with up-regulation of heme oxygenase 1, COX-2, and anti-apoptotic proteins (BCL2, BCL2-A1, and MCL-1) and increased phosphorylation of NRF2. Apoptotic cytometric assays showed that pretreatment of CSCs with CoPP enhanced the cells' resistance to apoptosis induced by oxidative stress. Conversely, knocking down HO-1, COX-2, or NRF2 by shRNA gene silencing abrogated the cytoprotective effects of CoPP. Further, preconditioning CSCs with CoPP led to a global increase in release of cytokines, such as EGF, FGFs, colony-stimulating factors, and chemokine ligand. Conditioned medium from cells pretreated with CoPP conferred naive CSCs remarkable resistance to apoptosis, demonstrating that cytokines released by preconditioned cells play a key role in the anti-apoptotic effects of CoPP. Preconditioning CSCs with CoPP also induced an increase in the phosphorylation of Erk1/2, which are known to modulate multiple pro-survival genes. These results potentially provide a simple and effective strategy to enhance survival of CSCs after transplantation and, therefore, their efficacy in repairing infarcted myocardium.

The COX-2/PGI2 receptor axis plays an obligatory role in mediating the cardioprotection conferred by the late phase of ischemic preconditioning.

BACKGROUND: Pharmacologic studies with cyclooxygenase-2 (COX-2) inhibitors suggest that the late phase of ischemic preconditioning (PC) is mediated by COX-2. However, nonspecific effects of COX-2 inhibitors cannot be ruled out, and the selectivity of these inhibitors for COX-2 vs. COX-1 is only relative. Furthermore, the specific prostaglandin (PG) receptors responsible for the salubrious actions of COX-2-derived prostanoids remain unclear. OBJECTIVE: To determine the role of COX-2 and prostacyclin receptor (IP) in late PC by gene deletion. METHODS: COX-2 knockout (KO) mice (COX-2(-/-)), prostacyclin receptor KO (IP(-/-)) mice, and respective wildtype (WT, COX-2(+/+) and IP(+/+)) mice underwent sham surgery or PC with six 4-min coronary occlusion (O)/4-min R cycles 24 h before a 30-min O/24 h R. RESULTS: There were no significant differences in infarct size (IS) between non-preconditioned (non-PC) COX-2(+/+), COX-2(-/-), IP(+/+), and IP(-/-) mice, indicating that neither COX-2 nor IP modulates IS in the absence of PC. When COX-2(-/-) or IP(-/-) mice were preconditioned, IS was not reduced, indicating that the protection of late PC was completely abrogated by deletion of either the COX-2 or the IP gene. Administration of the IP selective antagonist, RO3244794 to C57BL6/J (B6) mice 30 min prior to the 30-min O had no effect on IS. When B6 mice were preconditioned 24 h prior to the 30-min O, IS was markedly reduced; however, the protection of late PC was completely abrogated by pretreatment of RO3244794. CONCLUSIONS: This is the first study to demonstrate that targeted disruption of the COX-2 gene completely abrogates the infarct-sparing effect of late PC, and that the IP, downstream of the COX-2/prostanoid pathway, is a key mediator of the late PC. These results provide unequivocal molecular genetic evidence for an essential role of the COX-2/PGI2 receptor axis in the cardioprotection afforded by the late PC.

Insights into gene therapy for critical limb ischemia: the devil is in the details.

Patients with critical limb ischemia (CLI) without potential for revascularization are currently without alternate therapies. Several gene therapy trials have tested angiogenesis factors, hepatic growth factor, vascular endothelial growth factor, and basic fibroblast growth factor, in rescuing CLI patients from amputation and mortality, and for improved quality of life including decreased pain, improved healing, and blood flow. Trial results have been variable, with HGF gene therapy being the most successful. New studies examining each of these angiogenic factors provide insights that will be useful for the design of effective therapeutic strategies.

Chronic AMD3100 antagonism of SDF-1alpha-CXCR4 exacerbates cardiac dysfunction and remodeling after myocardial infarction.

The role of the SDF-1alpha-CXCR4 axis in response to myocardial infarction is unknown. We addressed it using the CXCR4 antagonist, AMD3100, to block SDF-1alpha interaction with CXCR4 after chronic coronary artery ligation. Chronic AMD3100 treatment decreased ejection fraction and fractional shortening in mice 20days after myocardial infarction compared with vehicle-treated mice (echocardiography). Morphometric analysis showed hearts of AMD3100-treated infarcted mice to have expanded scar, to be hypertrophic (confirmed by myocyte cross-section area) and dilated, with increased LV end systolic and end diastolic dimensions, and to have decreased scar collagen content; p-AKT levels were attenuated and this was accompanied by increased apoptosis. Despite increased injury, c-kit(pos) cardiac progenitor cells (CPCs) were increased in the risk region of AMD3100-treated infarcted mice; CPCs were CD34(neg)/CD45(neg) with the majority undergoing symmetric cell division. c-kit(pos)/MHC(pos) CPCs also increased in the risk region of the AMD3100-treated infarcted group. In this group, GSK-3beta signaling was attenuated compared to vehicle-treated, possibly accounting for increased proliferation and increased cardiac committed MHC(pos) CPCs. Increased proliferation following AMD3100 treatment was supported by increased levels of cyclin D1, a consequence of increased prolyl isomerase, Pin1, and decreased cyclin D1 phosphorylation. In summary, pharmacologic antagonism of CXCR4 demonstrates that SDF-1alpha-CXCR4 signaling plays an important role during and after myocardial infarction and that it exerts pleiotropic salubrious effects, protecting the myocardium from apoptotic cell death, facilitating scar formation, restricting CPC proliferation, and directing CPCs toward a cardiac fate.

Hepatocyte growth factor/Met gene transfer in cardiac stem cells--potential for cardiac repair.

The adult heart has been recently recognized as a self-renewing organ that contains a pool of committed resident cardiac stem cells (CSCs) and cardiac progenitor cells (CPCs). These adult CSCs and CPCs can be induced by cytokines and growth factors to migrate, differentiate, and proliferate in situ and potentially replace lost cardiomyocytes. Ligand-receptor systems, such as the tyrosine kinase receptor mesenchymal-epithelial transition factor (Met) and its ligand hepatocyte growth factor (HGF), are potential candidates for boosting migration, engraftment and commitment of CSCs. Here, we discuss the possible application of HGF/Met gene therapy to enhance the ability of CSCs to promote myocardial regeneration.