Macrophage efferocytosis with VEGFC and lymphangiogenesis: rescuing the broken heart

Cardiac repair following ischemic injury is indispensable for survival and requires a coordinated cellular response involving the mobilization of immune cells from the secondary lymphoid organs to the site of damage. Efferocytosis, the engulfment of cell debris and dying cells by innate immune cells, along with lymphangiogenesis, the formation of new lymphatic vessels, are emerging as central to the cardiac healing response. In this issue of the JCI, Glinton et al. used state-of-the-art approaches to demonstrate that efferocytosis induced vascular endothelial growth factor C (VEGFC) in myeloid cells and stimulated lymphangiogenesis and cardiac repair. These findings provide impactful mechanistic information that can be leveraged to therapeutically target pathways in cardiac repair and ischemic heart failure.     

Adenine nucleotides stimulate migration in wounded cultures of kidney epithelial cells.

Adenine nucleotides speed structural and functional recovery when administered after experimental renal injury in the rat and stimulate proliferation of kidney epithelial cells. As cell migration is a component of renal regeneration after acute tubular necrosis, we have used an in vitro model of wound healing to study this process. High density, quiescent monkey kidney epithelial cultures were wounded by mechanically scraping away defined regions of the monolayer to simulate the effect of cell loss after tubular necrosis and the number of cells that migrated into the denuded area was counted. Migration was independent of cell proliferation. Provision of adenosine, adenine nucleotides, or cyclic AMP increased the number of migrating cells and accelerated repair of the wound. Other purine and pyrimidine nucleotides were not effective. Arginine-glycine-aspartic acid-serine peptide, which blocks the binding of extracellular fibronectin to its cell surface receptor, completely inhibited migration in the presence or absence of ADP. Very low concentrations of epidermal growth factor (K0.5 approximately 0.3 ng/ml) stimulated migration, whereas transforming growth factor-beta 2 was inhibitory (Ki approximately 0.2 ng/ml). Thus, adenosine and/or adenine nucleotides released from injured or dying renal cells, or administered exogenously, may stimulate surviving cells in the wounded nephron to migrate along the basement membrane, thereby rapidly restoring tubular structure and function.     

Inhibition of cyclooxygenase-2 aggravates doxorubicin-mediated cardiac injury in vivo

The clinical use of doxorubicin, an anthracycline chemotherapeutic agent, is limited by cardiotoxicity, particularly when combined with herceptin, an antibody that blocks the HER2 receptor. Doxorubicin induces cyclooxygenase-2 (COX-2) activity in rat neonatal cardiomyocytes. This expression of COX-2 limits doxorubicin-induced cardiac cell injury, raising the possibility that the administration of a prostaglandin may protect the heart during the in vivo administration of doxorubicin. Doxorubicin (15 mg/kg) administered to adult male Sprague Dawley rats induced COX-2 expression and activity in cardiac tissue. Prostacyclin generation measured as the excretion of 2,3-dinor-6-keto-PGF(1alpha) also increased, and this was blocked by a COX-2 inhibitor, SC236. In contrast, administration of a COX-1 inhibitor SC560 at a dose that reduced serum thromboxane B2 by more than 80% did not prevent the doxorubicin-induced increase in prostacyclin generation. Doxorubicin increased cardiac injury, detected as a rise in plasma cardiac troponin T, serum lactate dehydrogenase, and cardiomyocyte apoptosis; this was aggravated by coadministration of SC236 but not SC560. The degree of injury in animals treated with a combination of doxorubicin and SC236 was attenuated by prior administration of the prostacyclin analogue iloprost. These data raise the possibility of protecting the heart during the administration of doxorubicin by prior administration of prostacyclin.     

Combining stem cells in myocardial infarction: The road to superior repair?

Myocardial infarction irreversibly destroys millions of cardiomyocytes in the ventricle, making it the leading cause of heart failure worldwide. Over the past two decades, many progenitor and stem cell types were proposed as the ideal candidate to regenerate the heart after injury. The potential of stem cell therapy has been investigated thoroughly in animal and human studies, aiming at cardiac repair by true tissue replacement, by immune modulation, or by the secretion of paracrine factors that stimulate endogenous repair processes. Despite some successful results in animal models, the outcome from clinical trials remains overall disappointing, largely due to the limited stem cell survival and retention after transplantation. Extensive interest was developed regarding the combinational use of stem cells and various priming strategies to improve the efficacy of regenerative cell therapy. In this review, we provide a critical discussion of the different stem cell types investigated in preclinical and clinical studies in the field of cardiac repair. Moreover, we give an update on the potential of stem cell combinations as well as preconditioning and explore the future promises of these novel regenerative strategies.     

Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice

Cardiac progenitor cells are a potential source of cell therapy for heart failure. Although recent studies have shown that transplantation of cardiac stem/progenitor cells improves function of infarcted hearts, the precise mechanisms of the improvement in function remain poorly understood. The present study demonstrates that transplantation of sheets of clonally expanded stem cell antigen 1–positive (Sca-1–positive) cells (CPCs) ameliorates cardiac dysfunction after myocardial infarction in mice. CPC efficiently differentiated into cardiomyocytes and secreted various cytokines, including soluble VCAM-1 (sVCAM-1). Secreted sVCAM-1 induced migration of endothelial cells and CPCs and prevented cardiomyocyte death from oxidative stress through activation of Akt, ERK, and p38 MAPK. Treatment with antibodies specific for very late antigen-4 (VLA-4), a receptor of sVCAM-1, abolished the effects of CPC-derived conditioned medium on cardiomyocytes and CPCs in vitro and inhibited angiogenesis, CPC migration, and survival in vivo, which led to attenuation of improved cardiac function following transplantation of CPC sheets. These results suggest that CPC transplantation improves cardiac function after myocardial infarction through cardiomyocyte differentiation and paracrine mechanisms mediated via the sVCAM-1/VLA-4 signaling pathway.     

Control of the adaptive response of the heart to stress via the Notch1 receptor pathway

In the damaged heart, cardiac adaptation relies primarily on cardiomyocyte hypertrophy. The recent discovery of cardiac stem cells in the postnatal heart, however, suggests that these cells could participate in the response to stress via their capacity to regenerate cardiac tissues. Using models of cardiac hypertrophy and failure, we demonstrate that components of the Notch pathway are up-regulated in the hypertrophic heart. The Notch pathway is an evolutionarily conserved cell-to-cell communication system, which is crucial in many developmental processes. Notch also plays key roles in the regenerative capacity of self-renewing organs. In the heart, Notch1 signaling takes place in cardiomyocytes and in mesenchymal cardiac precursors and is activated secondary to stimulated Jagged1 expression on the surface of cardiomyocytes. Using mice lacking Notch1 expression specifically in the heart, we show that the Notch1 pathway controls pathophysiological cardiac remodeling. In the absence of Notch1, cardiac hypertrophy is exacerbated, fibrosis develops, function is altered, and the mortality rate increases. Therefore, in cardiomyocytes, Notch controls maturation, limits the extent of the hypertrophic response, and may thereby contribute to cell survival. In cardiac precursors, Notch prevents cardiogenic differentiation, favors proliferation, and may facilitate the expansion of a transient amplifying cell compartment.     

Gene transfer of connexin43 into skeletal muscle

Cellular cardiomyoplasty using skeletal myoblasts may be beneficial for infarct repair. One drawback to skeletal muscle cells is their lack of gap junction expression after differentiation, thus preventing electrical coupling to host cardiomyocytes. We sought to overexpress the gap junction protein connexin43 (Cx43) in differentiated skeletal myotubes, using retroviral, adenoviral, and plasmid-mediated gene transfer. All strategies resulted in overexpression of Cx43 in cultured myotubes, but expression of Cx43 from constitutive viral promoters caused significant death upon differentiation. Dye transfer studies showed that surviving myotubes contained functional gap junctions, however. Retrovirally transfected myoblasts did not express Cx43 after grafting into the heart, possibly due to promoter silencing. Adenovirally transfected myoblasts expressed abundant Cx43 after forming myotubes in cardiac grafts, but grafts showed signs of injury at 1 week and had died by 2 weeks. Interestingly, transfection of already differentiated myotubes with adenoviral Cx43 was nontoxic, implying a window of vulnerability during differentiation. To test this hypothesis, Cx43 was expressed from the muscle creatine kinase (MCK) promoter, which is active only after myocyte differentiation. The MCK promoter resulted in high levels of Cx43 expression in differentiated myotubes but did not cause cell death during differentiation. MCK-Cx43-transfected myoblasts formed viable cardiac grafts and, in some cases, Cx43-expressing myotubes were in close apposition to host cardiomyocytes, possibly allowing electrical coupling. Thus, high levels of Cx43 during skeletal muscle differentiation cause cell death. When, however, expression of Cx43 is delayed until after differentiation, using the MCK promoter, myotubes are viable and express gap junction proteins after grafting in the heart. This strategy may permit electrical coupling of skeletal and cardiac muscle for cardiac repair.     

Exogenous adenine nucleotides replete endothelial cell adenosine triphosphate after oxidant injury by adenosine uptake

We studied the ability of human umbilical vein endothelial cells to recover from oxidant-induced ATP depletion. When endothelial cell ATP levels were depressed to 0.93 +/- 0.14 pmol/micrograms protein (compared with 4.96 +/- 0.6 pmol/micrograms protein in control cells) by hydrogen peroxide generated with 25 mU/ml glucose-glucose oxidase over 45 minutes, ATP levels returned to 1.73 +/- 0.21 pmol/micrograms protein during a 3-hour recovery period after oxidant injury ceased. When 25 microM ATP, ADP, AMP, or adenosine was added to the recovery media, intracellular ATP was significantly (p less than 0.001) increased to greater than 4.4 pmol/micrograms cell protein for each metabolite. HPLC of supernatants from oxidant-injured endothelial cells incubated with ATP, ADP, and AMP demonstrated extracellular metabolism of the adenine nucleotides to adenosine. When adenosine transport was inhibited with dipyridamole and nitrobenzylthioinosine, recovery of intracellular ATP by exogenous ATP, ADP, AMP, and adenosine was significantly (p less than 0.001) inhibited. Such cells were intact, as demonstrated by lack of LDH release. When oxidant stress was prolonged to 90 minutes, ATP depletion was irreversible, regardless of exogenously supplied adenosine; such cells demonstrated loss of cell integrity as demonstrated by release of intracellular LDH. Our results demonstrated that exogenous adenine nucleotides enhance recovery of oxidant-induced ATP depletion through metabolism to adenosine and subsequent adenosine uptake. Prolonged oxidant injury resulted in irreversible ATP depletion and loss of cell integrity that was not altered by exogenously supplied adenosine.     

Isolation and expansion of resident cardiac progenitor cells

After myocardial infarction, loss of viable cardiomyocytes severely impairs cardiac function. Recently, stem cell transplantation has been put forward as a promising approach to repair the damaged heart. Although several clinical trials have already been performed, the dominant beneficial effects are probably due to neoangiogenesis and arteriogenesis. However, replacement of cardiomyocytes is vital to improve cardiac function in the long term. Stem cells and progenitor cells, with the capacity to differentiate into cardiomyocytes, have been described in both embryonic and adult tissues. Upon stimulation, cardiac progenitor cells proliferate and differentiate into cardiomyocytes, vascular smooth muscle cells, and endothelial cells. Currently however, high proliferation rates and differentiation of cardiac progenitor cells beyond the fetal stage have not yet been achieved. Full differentiation into adult cardiomyocytes in vitro and in vivo might be important for efficient integration with the host environment and therefore more research is needed to study factors that influence proliferation and differentiation. Here we will discuss the isolation of cardiac progenitor cells, their potential to differentiate into various cell types needed for cardiac repair, the possible mechanisms behind these events, and how these cells may be implemented in future clinical settings.     

Prospects for Creation of Cardioprotective and Antiarrhythmic Drugs Based on Opioid Receptor Agonists

It has now been demonstrated that the μ, δ₁, δ₂, and κ₁ opioid receptor (OR) agonists represent the most promising group of opioids for the creation of drugs enhancing cardiac tolerance to the detrimental effects of ischemia/reperfusion (I/R). Opioids are able to prevent necrosis and apoptosis of cardiomyocytes during I/R and improve cardiac contractility in the reperfusion period. The OR agonists exert an infarct‐reducing effect with prophylactic administration and prevent reperfusion‐induced cardiomyocyte death when ischemic injury of heart has already occurred; that is, opioids can mimic preconditioning and postconditioning phenomena. Furthermore, opioids are also effective in preventing ischemia‐induced arrhythmias.     

X-linked inhibitor of apoptosis protein-mediated attenuation of apoptosis, using a novel cardiac-enhanced adeno-associated viral vector

Successful amelioration of cardiac dysfunction and heart failure through gene therapy approaches will require a transgene effective at attenuating myocardial injury, and subsequent remodeling, using an efficient and safe delivery vehicle. Our laboratory has established a well-curated, high-quality repository of human myocardial tissues that we use as a discovery engine to identify putative therapeutic transgene targets, as well as to better understand the molecular basis of human heart failure. By using this rare resource we were able to examine age- and sex-matched left ventricular samples from (1) end-stage failing human hearts and (2) nonfailing human hearts and were able to identify the X-linked inhibitor of apoptosis protein (XIAP) as a novel target for treating cardiac dysfunction. We demonstrate that XIAP is diminished in failing human hearts, indicating that this potent inhibitor of apoptosis may be central in protecting the human heart from cellular injury culminating in heart failure. Efforts to ameliorate heart failure through delivery of XIAP compelled the design of a novel adeno-associated viral (AAV) vector, termed SASTG, that achieves highly efficient transduction in mouse heart and in cultured neonatal rat cardiomyocytes. Increased XIAP expression achieved with the SASTG vector inhibits caspase-3/7 activity in neonatal cardiomyocytes after induction of apoptosis through three common cardiac stresses: protein kinase C-γ inhibition, hypoxia, or β-adrenergic receptor agonist. These studies demonstrate the potential benefit of XIAP to correct heart failure after highly efficient delivery to the heart with the rationally designed SASTG AAV vector.     

Current perspectives on protective roles of erythropoietin in cardiovascular system: erythropoietin receptor as a novel therapeutic target

Erythropoietin (EPO) is a principal regulator that promotes proliferation and terminal differentiation of erythroid progenitor cells. EPO receptors are expressed not only in hematopoietic lineage cells but also in the cardiovascular system. We performed animal experiments using transgene-rescued EPO receptor null mutant mice (EpoR-/- rescued) that express the EPO receptor exclusively in the hematopoietic cells. The results of these experiments suggest that endogenous EPO/EPO receptor system in the heart exerts cardioprotective effects against myocardial injury induced by ischemia followed by reperfusion and pressure-overload induced left ventricular dysfunction. Many animal experiments have shown that the administration of recombinant human EPO also elicits cardioprotective effects against myocardial injury induced by ischemia and reperfusion. In contrast to the promising results of these animal experiments, recent clinical trials failed to demonstrate the reduction in infarct size or improvement of cardiac function by the administration of recombinant human EPO in patients with acute myocardial infarction who underwent primary percutaneous coronary intervention. It should be tested in future clinical studies whether a relatively low dose of recombinant human EPO or its derivatives that have no erythropoietic action reduces infarct size and ameliorates cardiac dysfunction in patients with acute myocardial infarction. In this article, we review implications of anemia associated with chronic heart failure, roles of the endogenous EPO/EPO receptor system, and the effects of the administration of erythropoiesis-stimulating agents in pathologic conditions of the heart by focusing on the EPO receptor as a potential candidate of novel therapeutic targets in cardiovascular diseases.     

If these myocytes could talk,they would speak the language of metabolites

Cardiac wound healing following ischemic injury requires a well-described spatiotemporal progression of events involving multiple cell types and cell-cell interactions. While cellular crosstalk among immune cell, endothelial cell, and fibroblast populations is known to regulate these progressive phases, the role of cardiac myocytes in controlling the wound-healing program is unclear. In this issue of the JCI, Li et al. describe a mechanism of cellular crosstalk between cardiac myocytes and fibroblasts that disrupts nonmyocyte cell function and worsens wound healing outcomes following myocardial infarction (MI). This tour de force study used an arsenal of multidisciplinary approaches to identify a central role for the ectonucleotidase ENPP1 in this process. These findings have clear therapeutic implications, as the authors identified a small molecular inhibitor of ENPP1 that improved post-MI outcomes in mice. These exciting data provide impactful mechanistic information that advance the field’s understanding of cardiac repair and remodeling.     

Overexpression of TGFbeta1 by adeno-associated virus type-2 vector protects myocardium from ischemia-reperfusion injury

Transforming growth factor beta(1) (TGFbeta(1)) has been purported to protect tissues from ischemia-reperfusion (I-R) injury. This study was designed to examine if overexpression of TGFbeta(1) using adeno-associated virus type 2 (AAV) protects cardiomyocytes from reoxygenation injury. TGFbeta(1) was overexpressed in cultured HL-1 mouse cardiomyocytes by transfection with AAV/TGFbeta(1)(Latent) or with AAV/TGFbeta(1)(ACT) (active TGFbeta(1)). TGFbeta(1) upregulation reduced cardiomyocyte apoptosis and necrosis induced by 24 h of hypoxia followed by 3 h of reoxygenation concomitant with reduction in reactive oxygen species release, activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and NF-kappaB expression. Transfection with AAV/TGFbeta(1)(ACT) was superior to that with AAV/TGFbeta(1)(Latent). To determine if AAV/TGFbeta(1)(ACT) upregulation in vivo would induce cardioprotection from I-R injury, rat hearts were injected with AAV/TGFbeta(1)(ACT) or phosphate-buffered saline (PBS). Six weeks later, TGFbeta(1)(ACT) was upregulated throughout the myocardium. Following I-R, AAV/TGFbeta(1)(ACT)-overexpressing rats had much smaller infarct size (P<0.01 vs PBS group), which was also related to reduced activation of NADPH oxidase and NF-kappaB, and lower levels of malondialdehyde in I-R tissues. These data demonstrate that overexpression of TGFbeta(1) by AAV can protect cardiac tissues from reperfusion injury, possibly via antioxidant mechanism. These findings suggest potential of TGFbeta(1)(ACT) gene therapy for cardioprotection from I-R injury.     

Parthenogenetic stem cells for tissue-engineered heart repair

Uniparental parthenotes are considered an unwanted byproduct of in vitro fertilization. In utero parthenote development is severely compromised by defective organogenesis and in particular by defective cardiogenesis. Although developmentally compromised, apparently pluripotent stem cells can be derived from parthenogenetic blastocysts. Here we hypothesized that nonembryonic parthenogenetic stem cells (PSCs) can be directed toward the cardiac lineage and applied to tissue-engineered heart repair. We first confirmed similar fundamental properties in murine PSCs and embryonic stem cells (ESCs), despite notable differences in genetic (allelic variability) and epigenetic (differential imprinting) characteristics. Haploidentity of major histocompatibility complexes (MHCs) in PSCs is particularly attractive for allogeneic cell-based therapies. Accordingly, we confirmed acceptance of PSCs in MHC-matched allotransplantation. Cardiomyocyte derivation from PSCs and ESCs was equally effective. The use of cardiomyocyte-restricted GFP enabled cell sorting and documentation of advanced structural and functional maturation in vitro and in vivo. This included seamless electrical integration of PSC-derived cardiomyocytes into recipient myocardium. Finally, we enriched cardiomyocytes to facilitate engineering of force-generating myocardium and demonstrated the utility of this technique in enhancing regional myocardial function after myocardial infarction. Collectively, our data demonstrate pluripotency, with unrestricted cardiogenicity in PSCs, and introduce this unique cell type as an attractive source for tissue-engineered heart repair.     

Endothelin-1 is an autocrine/paracrine factor in the mechanism of angiotensin II-induced hypertrophy in cultured rat cardiomyocytes.

To elucidate the cellular mechanism by which angiotensin II (ANG II) induces cardiac hypertrophy, we investigated the possible autocrine/paracrine role of endogenous endothelin-1 (ET-1) in ANG II-induced hypertrophy of neonatal rat cardiomyocytes by use of synthetic ET-1 receptor antagonist and antisense oligonucleotides to preproET-1 (ppET-1) mRNA. Northern blot analysis and in situ hybridization revealed that ppET-1 mRNA was expressed in cardiomyocytes, but, to a lesser extent, in nonmyocytes as well. ANG II upregulated ppET-1 mRNA level by threefold over control level as early as 30 min, and it stimulated release of immunoreactive ET-1 from cardiomyocytes in a dose- and time-dependent manner. ET-1 stimulated ppET-1 mRNA levels after 30 min in a similar fashion as ANG II. Tetradecanoylphorbol-acetate (10(-7) M) mimicked the effects of ANG II and ET-1 on induction of ppET-1 mRNA. ANG II-induced ppET-1 gene expression was completely blocked by protein kinase C inhibitor H-7 or by down-regulation of endogenous protein kinase C by pretreatment with phorbol ester. ET-1 and ANG II stimulated twofold increase [3H]leucine incorporation into cardiomyocytes, whose effects were similarly and dose dependently inhibited by endothelin A receptor antagonist (BQ123). Introduction of antisense sequence against coding region of ppET-1 mRNA into cardiomyocytes resulted in complete blockade with ppET-1 mRNA levels and [3H]leucine incorporation stimulated by ANG II. These results suggest that endogenous ET-1 locally generated and secreted by cardiomyocytes may contribute to ANG II-induced cardiac hypertrophy via an autocrine/paracrine fashion.     

Liquid-chromatographic measurements of inosine, hypoxanthine, and xanthine in studies of fructose-induced degradation of adenine nucleotides in humans and rats

We applied a sensitive, precise liquid-chromatographic method of analysis for inosine, hypoxanthine, and xanthine to the study of fructose metabolism in humans and in rats. In the rat, intravenous loading with fructose induced, within minutes, substantial increases in the concentrations of inosine, hypoxanthine, and xanthine in plasma and urine. In plasma, these concentrations peaked after 5 min, then practically disappeared within 10 min. As expected, the fructose-induced increase in hypoxanthine was greatly amplified by pretreating the rats with allopurinol, an inhibitor of xanthine oxidase. In a healthy human subject, intravenous administration of fructose also induced prompt, substantial, and rapidly reversing increases in the concentrations of these metabolites of adenine nucleotides in plasma. The finding that fructose induced almost-immediate increases in the plasma concentrations of inosine, hypoxanthine, and xanthine is consistent with previous studies in rats, in which parenteral administration of fructose induced almost-immediate decreases of total adenine nucleotides (ATP + ADP + AMP) in the liver, and increased concentrations of uric acid and allantoin in the plasma.     

Secondary Sphere Formation Enhances the Functionality of Cardiac Progenitor Cells

Loss of cardiomyocytes impairs cardiac function after myocardial infarction (MI). Recent studies suggest that cardiac stem/progenitor cells could repair the damaged heart. However, cardiac progenitor cells are difficult to maintain in terms of purity and multipotency when propagated in two-dimensional culture systems. Here, we investigated a new strategy that enhances potency and enriches progenitor cells. We applied the repeated sphere formation strategy (cardiac explant → primary cardiosphere (CS) formation → sphere-derived cells (SDCs) in adherent culture condition → secondary CS formation by three-dimensional culture). Cells in secondary CS showed higher differentiation potentials than SDCs. When transplanted into the infarcted myocardium, secondary CSs engrafted robustly, improved left ventricular (LV) dysfunction, and reduced infarct sizes more than SDCs did. In addition to the cardiovascular differentiation of transplanted secondary CSs, robust vascular endothelial growth factor (VEGF) synthesis and secretion enhanced neovascularization in the infarcted myocardium. Microarray pathway analysis and blocking experiments using E-selectin knock-out hearts, specific chemicals, and small interfering RNAs (siRNAs) for each pathway revealed that E-selectin was indispensable to sphere initiation and ERK/Sp1/VEGF autoparacrine loop was responsible for sphere maturation. These results provide a simple strategy for enhancing cellular potency for cardiac repair. Furthermore, this strategy may be implemented to other types of stem/progenitor cell-based therapy.