Myocardial Wound Healing Research Articles

Abstract We074: Macrophages Endocytose the Extracellular Matrix Component Hyaluronan

Background: Following myocardial infarction, macrophages clear cellular debris during remodeling of the extracellular matrix (ECM). The presence of macrophages coincides with a significant increase in the ECM component hyaluronan (HA). HA is produced in copious amounts following injury, such as myocardial infarction, and HA may interfere with myocardial wound healing. Because macrophages express hyaluronan receptors and are known to facilitate ECM remodeling, it is possible that macrophages remove HA early after MI. The extent to which macrophages clear HA is not known. Goal: To determine the extent to which macrophages endocytose hyaluronan (HA). Methods and Results: Bone marrow derived macrophages (BMDMs) were polarized into a pro-inflammatory (M1) phenotype using LPS and INF-g, or pro-reparative phenotype (M2) using IL-4 and IL-13. In addition to the polarization factors, cells were treated with 5,000 ng/ml of a fluorescein-labeled HA or a vehicle for 24 hours. At the time of collection, cells were stained with trypan blue to quench surface fluorescence and flow cytometry was used to measure the uptake of labeled HA. An increase in cellular fluorescence intensity after HA treatment indicates the extent of endocytosis of HA. Mean fluorescence intensity (MFI) values were recorded and used to calculate the change in fluorescence between the treated and control groups (DMFI). A significant increase in DMFI was observed when both M1 and M2 macrophages were compared to naïve (M0) controls. Conclusion: Macrophages endocytose HA, and polarized macrophages endocytose HA at a greater level than naïve macrophages. Hence, polarized macrophages may play a role in clearing HA in the heart following injury. We speculate that enhancement of macrophage-mediated clearance of HA may improve acute scar formation following myocardial infarction.

LPS differentially affects expression of CD14 and CCR2 in monocyte subsets of Post-STEMI patients with hyperglycemia

AimsFollowing ST-segment elevation myocardial infarction (STEMI), recruitment and activation of monocytes [classical (CD14++CD16-CCR2++), intermediate (CD14++CD16+CCR2+), non-classical (CD14LowCD16++CCR2Low)] are needed for myocardial wound healing. Monocyte surface receptor CC chemokine receptor type 2 (CCR2) is responsible for monocyte chemotaxis to sites of inflammation and the lipopolysaccharide (LPS)-binding protein co-receptor, CD14, is involved in pro-inflammatory monocyte activation. The purpose of this investigation was to determine the effects of ex-vivo LPS activation on monocyte subset CD14 and CCR2 expression in post-STEMI individuals with normal and elevated random blood glucose. MethodsPost-STEMI subjects were identified as normal random glucose (NG, <98 mg/dL, n = 13) or impaired random glucose (IG, ≥98 mg/dL, n = 26) and monocytes were analyzed for non-activated and LPS-activated (1 µg/mL for 4 h) CCR2 and CD14 expression. ResultsNon-activated intermediate monocytes from IG showed decreased CD14 expression when compared to NG, which was maintained following LPS-activation. The NG group showed a larger absolute reduction in classical CCR2 expression, leading to a significant difference between NG and IG following LPS-activation. ConclusionResults suggest a heightened response to pro-inflammatory activation in IG following STEMI, which may impair or delay post-STEMI myocardial healing, and thus increase the incidence of chronic heart failure. NIH 1R34HL121402.

Sirt7 Deficiency Attenuates Neointimal Formation Following Vascular Injury by Modulating Vascular Smooth Muscle Cell Proliferation.

Sirt7 is a recently identified sirtuin and has important roles in various pathological conditions, including cancer progression and metabolic disorders. It has previously been reported that Sirt7 is a key molecule in acute myocardial wound healing and pressure overload-induced cardiac hypertrophy. In this study, the role of Sirt7 in neointimal formation after vascular injury is investigated. Systemic (Sirt7-/-) and smooth muscle cell-specific Sirt7-deficient mice were subjected to femoral artery wire injury. Primary vascular smooth muscle cells (VSMCs) were isolated from the aorta of wild type (WT) and Sirt7-/-mice and their capacity for cell proliferation and migration was compared. Sirt7 expression was increased in vascular tissue at the sites of injury. Sirt7-/-mice demonstrated significant reduction in neointimal formation compared to WT mice. In vitro, Sirt7 deficiency attenuated the proliferation of serum-induced VSMCs. Serum stimulation-induced upregulation of cyclins and cyclin-dependent-kinase 2 (CDK2) was significantly attenuated in VSMCs of Sirt7-/-compared with WT mice. These changes were accompanied by enhanced expression of the microRNA 290-295 cluster, the translational negative regulator of CDK2, in VSMCs of Sirt7-/-mice. It was confirmed that smooth muscle cell-specific Sirt7-deficient mice showed significant reduction in neointima compared with control mice. Sirt7 deficiency attenuates neointimal formation after vascular injury. Given the predominant role in vascular neointimal formation, Sirt7 is a potentially suitable target for treatment of vascular diseases.

Abstract MP206: Cortical Bone Derived Stem Cells Modulate The Adaptive Immune Response Post-MI

Background: The inflammatory response mounted following myocardial infarction (MI) is closely coupled to myocardial wound healing processes such as angiogenesis, fibroblast maturation, and cardiomyocyte survival. A consortium of pro-reparative Tregs occupy the myocardium during ischemic injury and are essential in modulating cardiac homeostasis. However, Treg exposure to chronic, ischemic microenvironments compromises Tregs’ pro-reparative signatures and induces pathogenic remodeling of Tregs, compromising cardiac wound healing. Cortical Bone Derived Stem Cells (CBSCs) have superior engraftment capabilities compared to other stem cell types and can modulate the post-MI inflammatory response by establishing anti-inflammatory paracrine signaling dominance and the expansion of CD45+ leukocytes and CD4+ T cells residence in the MI heart. Therefore, we hypothesized CBSC cell therapy can modulate the recruitment and phenotype of Treg residence in the MI heart to permit their enhanced cardiogenic properties. Methods and Results: Animals that received intramyocardial injection of CBSCs along the infarct border zone of the permanently ligated LAD possessed smaller infarct sizes and improved cardiac function, that paralleled pro-reparative, TNFRII+ Treg expansion and repressed pathogenic TNFRI+, Treg residence 1- and 8- weeks post-MI compared to Mesenchymal Stem Cells (MSCs) or vehicle (PBS) treated animals. Similar TNFRI/II Treg dynamics were also observed in the spleen. Diphtheria toxin (DTR) mediated Treg ablation and S1P1 agonist administration, in the presence of CBSC therapy, reverted pro-reparative Treg expansion and solicited infarct size expansion and compromised cardiac function. The exposure of CBSC paracrine secretome to naïve CD4+ T cell cultures induces pro-reparative TNFRII+ Treg expansion. Paracrine profiling of CBSC secretome identifies Osteoprotegerin (OPG) enrichment and OPG depletion, via siRNA and lentivirus, compromises T cell survival signaling and pro-reparative, TNFRII+ Treg establishment both in vitro and in the MI heart. Conclusions: CBSCs can modulate the induction and preservation of pro-reparative TNFRII+ Tregs in the MI heart to solicit cardiac repair during acute and chronic ischemic injury.

Healing the Broken Heart; The Immunomodulatory Effects of Stem Cell Therapy.

Cardiovascular Disease (CVD) is a leading cause of mortality within the United States. Current treatments being administered to patients who suffered a myocardial infarction (MI) have increased patient survival, but do not facilitate the replacement of damaged myocardium. Recent studies demonstrate that stem cell-based therapies promote myocardial repair; however, the poor engraftment of the transferred stem cell populations within the infarcted myocardium is a major limitation, regardless of the cell type. One explanation for poor cell retention is attributed to the harsh inflammatory response mounted following MI. The inflammatory response coupled to cardiac repair processes is divided into two distinct phases. The first phase is initiated during ischemic injury when necrosed myocardium releases Danger Associated Molecular Patterns (DAMPs) and chemokines/cytokines to induce the activation and recruitment of neutrophils and pro-inflammatory M1 macrophages (MΦs); in turn, facilitating necrotic tissue clearance. During the second phase, a shift from the M1 inflammatory functional phenotype to the M2 anti-inflammatory and pro-reparative functional phenotype, permits the resolution of inflammation and the establishment of tissue repair. T-regulatory cells (Tregs) are also influential in mediating the establishment of the pro-reparative phase by directly regulating M1 to M2 MΦ differentiation. Current studies suggest CD4+ T-lymphocyte populations become activated when presented with autoantigens released from the injured myocardium. The identity of the cardiac autoantigens or paracrine signaling molecules released from the ischemic tissue that directly mediate the phenotypic plasticity of T-lymphocyte populations in the post-MI heart are just beginning to be elucidated. Stem cells are enriched centers that contain a diverse paracrine secretome that can directly regulate responses within neighboring cell populations. Previous studies identify that stem cell mediated paracrine signaling can influence the phenotype and function of immune cell populations in vitro, but how stem cells directly mediate the inflammatory microenvironment of the ischemic heart is poorly characterized and is a topic of extensive investigation. In this review, we summarize the complex literature that details the inflammatory microenvironment of the ischemic heart and provide novel insights regarding how paracrine mediated signaling produced by stem cell-based therapies can regulate immune cell subsets to facilitate pro-reparative myocardial wound healing.

Abstract 723: Cortical Bone Derived Stem Cells Modulates T Cell Response After Myocardial Injury

Rationale: The ischemic environment following myocardial infarction induces a harsh, pro-inflammatory state that facilitates the removal of necrotic tissue. Manipulation of T cell responses after cardiac injury directly impacts the outcome of the reparative response. Previous studies have demonstrated Cortical Bone Derived Stem Cells (CBSCs) improve cardiac function after myocardial infarction. CBSCs produce a diverse paracrine profile; whether this can lead to the modulation of T cell response by manipulating T cell subsets and allowing for increased cardiac wound healing post-MI remains unknown. Objective: To determine whether CBSCs secrete a paracrine profile that promotes wound healing by modulating T cell response following myocardial injury. Methods and Results: CD3+ T-lymphocytes were isolated from the spleen of C57BL/6J male mice ages 8 to 12 weeks via Fluorescent Activated Cell Sorting (FACs). CBSC secretome was introduced to T cell populations via a transwell culture system in the presence of CD3/28 coated beads with IL2. T-regulatory (Treg) populations were quantified via FACs 24 hours after in vitro CBSC exposure. Treg populations exposed to CBSC secretome in hypoxic (1% O2) conditions expanded to 26.9% of the CD4+ compartment, a 4.5 fold increase compared to control conditions. This result was further confirmed via qRT-PCR analysis. T cell culture secretome was quantified via proteome profiling of 111 soluble cytokine proteins 24 hours post CBSC exposure. CBSCs express a robust chemotaxis signature that promotes immune cell recruitment. T cells cultured in the presence of CBSC (pre-conditioned or co-culture conditions) exhibit increased CCL5, CCL22, CD40, CXCL1, and Lipocalin-2. C-reactive protein, a pro-inflammatory marker, was decreased by ~5.5 fold in the presence of CBSCs, promoting wound healing. Conclusions: CBSCs secrete a diverse paracrine profile that promotes Treg expansion during hypoxic conditions. Expansion of Treg populations post-CBSC exposure in the post-IR heart leads to increased inflammation resolution and improved myocardial wound healing, allowing for increased cardiac function and repressed cardiac remodeling.

Inhibition of miR-223 reduces inflammation but not adverse cardiac remodelling after myocardial ischemia-reperfusion in vivo

Background: Coronary artery occlusion results in ischemic heart tissue and subsequent death of cardiomyocytes, followed by an inflammatory response to clear the infarcted area from dead cells. Invading inflammatory cells are suggested to contribute to myocardial ischemia-reperfusion (I/R) injury and adverse remodelling. Given the importance of the inflammatory phase during cardiac wound healing, better understanding is needed to develop novel interventions. In the present study, we investigated the role of the inflammatory-related miR-223 in the ischemic heart. Furthermore, we determined the effect of miR-223 modulation on inflammation and cardiac remodelling in a mouse model of myocardial I/R. Methods: Mice underwent 30 minutes of ischemia and received, 5 minutes before reperfusion, 8 mg/kg antagomiR-223 or mismatch-miR treatment, and consecutive injections at day 1 and 2 post-I/R. MiR-223 expression was quantified by in situ hybridization and PCR. Inflammatory cell influx was quantified by immunohistochemistry. By using magnetic resonance imaging (MRI), cardiac dimensions and function were assessed before and 28 days after surgery. Results: MiR-223 expression significantly increased 1 and 3 days after I/R, corresponding with the inflammatory phase upon cardiac injury. MiR-223 expression mainly increased in myocytes. Inhibition of miR-223 by antagomir treatment significantly reduced total leukocyte (CD45+ cells) and macrophages (Mac-3+ cells) influx at 3 days of reperfusion. End-diastolic volume (EDV) and end-systolic volume (ESV) showed a similar increase in both treatment groups, as well as a comparable decline in ejection fraction (EF) post-I/R. Conclusions: Although inhibition of miR-223 resulted in less inflammatory influx after reperfusion, this did not lead to less adverse cardiac remodelling. More research on the complex temporal and spatial role of miR-223 during the process of myocardial wound healing is necessary in order to understand the role of miR-223 upon I/R injury and whether it can be used as a novel therapeutic strategy.

Abstract 211: Regulatory T cells improve healing after myocardial infarction

Background: The proinflammatory activation of innate immunity by myocardial ischemic injury has been recognized for long time. Our recent data have indicated that activation of CD4+ T cells, presumably by auto-antigen recognition, is a prerequisite for formation of a stable scar and prevention from left ventricular dilation after experimental myocardial infarction in mice. We here hypothesized that regulatory CD4+CD25+Foxp3+CD4+ T cells might improve left ventricular wound healing and prevent from adverse remodeling after myocardial infarction. Results: Experimental myocardial infarction in mice induced the proliferation and activation of CD4+CD25+Foxp3+ regulatory T cells, as demonstrated by intracellular expression of the Ikaros family transcription factor Helios, in heart draining lymph nodes. Pretreatment of mice with an anti-CD25 antibody before myocardial infarction efficiently depleted CD4+CD25+Foxp3+ regulatory T cells and increased mortality after myocardial infarction as compared to mice treated with an isotype-matched control antibody of irrelevant specificity, i.e., 25% survival in anti-CD25 treated mice vs. 55,9% survival in control antibody treated animals. Therapeutic activation of regulatory CD4+CD25+Foxp3+ T cells by a superagonistic anti-CD28 antibody (CD28-SA) applied at day 2 after myocardial infarction prevented, compared to mice treated with an isotype-matched control antibody, from left ventricular rupture and resulted in improved survival (47.1% survival in the control group vs. 76.6% survival in the CD28-SA treated group). CD28-SA treatment lead to expansion of CD4+CD25+Foxp3+ T-cells in the peripheral blood and increased their frequency in the infarcted myocardium. This was associated with increased expression of several molecules known to facilitate wound healing by promoting the formation of a stable scar such as osteopontin and coagulation factor XIII. Conclusion: CD4+CD25+Foxp3+ regulatory T-cells are a prerequisite for proper myocardial wound healing and can be therapeutically activated to improve outcome after experimental myocardial infarction.

Abstract 163: Interleukin-13 Expression Attenuates Left Ventricular Dilation and Mortality After Myocardial Infarction

Background: Monocyte infiltration and extracellular matrix breakdown/ de novo formation critically influence wound healing and remodeling after myocardial infarction (MI). Interleukin-13 (IL-13) has potent pro-fibrotic properties and is a differentiation factor for monocytes/ macrophages. Therefore, we studied whether IL-13 affects myocardial wound healing and remodelling after MI. Methods and results: MI was induced by permanent ligation of the left coronary artery in WT and IL-13-/- mice. Expression of IL-13 was upregulated in the myocardium after MI. IL-13 expression was in part dependent on angiotensin II signalling as in vivo treatment with the Angiotensin-receptor blocker losartan attenuated myocardial IL-13 expression in response to MI. Intracellular FACS analysis of myocardial leukocytes further demonstrated IL-13 expression in a subset of myeloid CD11b+ cells within the myocardium. IL-13-/- mice showed higher mortality than WT mice after MI without difference in infarct size. Echocardiography further demonstrated increased left ventricular dilation in IL-13-/- when compared to WT mice. Histological analysis of the scar collagen did not reveal a difference between WT and IL-13-/- mice. FACS analysis of leukocytes from the infarct zone showed less MHC-II+ CD11b+ cells within the myocardium of IL-13-/- mice on day 7 indicating that IL-13 is required for the differentiation of heart infiltrating monocytes. Conclusions: Expression of IL-13 in response to MI beneficially modulates wound healing after MI.

Macrophage Depletion Impairs Wound Healing and Increases Left Ventricular Remodeling after Myocardial Injury in Mice

Macrophages have been suggested to be beneficial for myocardial wound healing. We investigated the role of macrophages in myocardial wound healing by inhibition of macrophage infiltration after myocardial injury. We used a murine cryoinjury model to induce left ventricular damage. Infiltrating macrophages were depleted during the 1st week after cryoinjury by serial intravenous injections of clodronate-containing liposomes. After injury, the presence of macrophages, which secreted high levels of transforming growth factor-beta and vascular endothelial growth factor-A, led to rapid removal of cell debris and replacement by granulation tissue containing inflammatory cells and blood vessels, followed by myofibroblast infiltration and collagen deposition. In macrophage-depleted hearts, nonresorbed cell debris was still observed 4 weeks after injury. Secretion of transforming growth factor-beta and vascular endothelial growth factor-A as well as neovascularization, myofibroblast infiltration, and collagen deposition decreased. Moreover, macrophage depletion resulted in a high mortality rate accompanied by increased left ventricular dilatation and wall thinning. In conclusion, infiltrating macrophage depletion markedly impairs wound healing and increases remodeling and mortality after myocardial injury, identifying the macrophage as a key player in myocardial wound healing. Based on these findings, we propose that increasing macrophage numbers early after myocardial infarction could be a clinically relevant option to promote myocardial wound healing and subsequently to reduce remodeling and heart failure.

Time-dependent changes in myocardial structure following discrete injury in mice deficient of matrix metalloproteinase-3

Myocardial scars from radiofrequency (RF) ablation can increase in size in the post-injury period, resulting in remodeling of the extracellular matrix (ECM). The matrix metalloproteinases (MMPs) contribute to adverse myocardial remodeling following injury. However, the role of specific MMP types in RF scar enlargement remains unclear. One MMP type, MMP-3, degrades a wide range of ECM substrates and can activate other MMPs. This project examined LV remodeling in wild type (WT) and MMP-3 deficient ( mmp-3 –/–) mice following RF injury. RF lesions (0.5 mm probe, 80 °C, 30 s) were created on the LV epicardium of WT (C57/BL6) and mmp-3 –/– mice and were terminally studied at 1 h, 3, 7, and 28 days post-RF ( n = 10 each). Heart mass indexed to tibial length (mg/mm) was similar in the WT and mmp-3 –/– mice at 1 h (8.1 ± 0.3 vs. 7.6 ± 0.3), but lower in the mmp-3 –/– mice at 28 days post-RF (11.9 ± 0.4 vs. 10.5 ± 0.4, P < 0.05). Scar volumes were greater in the mmp-3 –/– mice at 3 days, but similar in the two groups at 28 days. Immunohistochemical localization showed fewer macrophages and lymphocytes at the scar border at 3 days in the mmp-3 –/– hearts, but similar staining for these cells in WT and mmp-3 –/– hearts at 7 and 28 days post-RF. Post-RF, the early increase in scar volume was accelerated in mmp-3 –/– mice and associated with abnormal inflammatory cell infiltration/migration to the area of injury. These findings define a mechanistic role for MMP-3 in RF scar expansion and provide a temporal window during which interruption of MMP-3 activation may impair post-RF myocardial wound healing.

Tumor necrosis factor-alpha is expressed by monocytes/macrophages following cardiac microembolization and is antagonized by cyclosporine.

The time course of expression of TNF-alpha in myocardial wound healing following ischemic injury was investigated in the porcine heart. Microembolization was used to induce focal ischemia and necrosis in hearts of 39 adult pigs. The animals were sacrificed after 3, 6, 12, 24 h, 3 and 7 days, and after 4 weeks, and the myocardial tissue was studied by immunofluorescence using specific antibodies. TNF-alpha containing cells were identified as monocytes/macrophages by double staining with a muramidase antibody. Monocytes/macrophages were the only source of TNF-alpha. Microembolization caused multiple necrotic foci with loss of myocytes in the left ventricular myocardium. These foci contained numerous monocytes/macrophages and showed an inflammatory reaction typical of wound healing followed by replacement with scar tissue. The number of TNF-alpha positive cells increased after 24 h, peaked between 3-7 days and slowly decreased thereafter. Expression of TNF-alpha in monocytes/macrophages was significantly reduced after pretreatment of pigs with cyclosporine or dexamethasone. It is concluded that 1.) in myocardial tissue monocytes/macrophages are the only cell type expressing TNF-alpha, 2.) TNF-alpha is involved in wound healing after ischemia, and 3.) synthesis of TNF-alpha and inflammatory angiogenesis can be inhibited be treatment with either cyclosporine or dexamethasone.

Cardiac cryolesions as an experimental model of myocardial wound healing.

The standard coronary ligation model for experimental myocardial infarction results in variable areas and patterns of necrosis; therefore, the healing of such infarctions is also variable. The authors developed an experimental myocardial injury model using simple cryoinjury, which allows standardization of the size, depth, and location of the wound. Thirty-eight left ventricular cryolesions were created in 19 dogs, which were then killed from 3 to 35 days after injury. A consistent decrease in the depth of scar (p less than 0.005) and accumulation of collagen (p less than 0.0001) over time characterized this healing myocardial wound. Histologic examination revealed that the cellular pattern of healing myocardial cryolesions is similar to that of a healing myocardial infarction but with less variability. The authors advocate the use of cardiac cryolesions as a model of experimental myocardial wound healing.

Autologous pericardium versus a xenograft substitute in myocardial wound healing

This study compared repair of myocardial wounds covered with autologous pericardium to healing of wounds covered with gluteraldehyde-preserved bovine pericardium in an experimental canine model. Right (RV) and left (LV) full thickness ventriculotomies were made and closed. In the control group ( n = 12), the pericardium was closed over the wound; in the experimental group ( n = 12), wounds were covered with bovine pericardium. Animals were sacrificed at 14, 21, 28, and 42 days. After excising the pericardium, 6 mm punch biopies of normal RV, RV wound, normal LV, and LV wound were assayed for hydroxyproline (HPro). Both autologous and bovine pericardium became densely adherent to the wounds. Bovine pericardium was mildly adherent over unwounded areas, while autologous pericardium was usually free. Normal RV contained more than twice as much HPro as normal LV (5.4 ± 0.57 μg/mg vs 1.7 ± 0.35 μg/mg, P < 0.0002). A gradual rise in HPro over time was seen in both groups, but this increase was statistically significant only at 42 days ( P < 0.05). There was no significant difference in HPro between wounds covered with autologous pericardium and those covered with bovine grafts ( P = 0.13) at any of the sample times in this study. In this experimental canine model, the pericardium does not appear to play a prominent role in myocardial wound healing by contributing collagen-producing fibroblasts. Furthermore, the bovine pericardial xenograft becomes densely adherent to LV and RV incisions. In the clinical setting, such may make reoperation more hazardous when the heart has been previously incised or coronary bypass grafts have been constructed.