Reparative Macrophages Research Articles

Compression Decreases Anatomical and Functional Recovery and Alters Inflammation after Contusive Spinal Cord Injury.

Experimental models of spinal cord injury (SCI) typically utilize contusion or compression injuries. Clinically, however, SCI is heterogeneous and the primary injury mode may affect secondary injury progression and neuroprotective therapeutic efficacy. Specifically, immunomodulatory agents are of therapeutic interest because the activation state of SCI macrophages may facilitate pathology but also improve repair. It is unknown currently how the primary injury biomechanics affect macrophage activation. Therefore, to determine the effects of compression subsequent to spinal contusion, we examined recovery, secondary injury, and macrophage activation in C57/BL6 mice after SCI with or without a 20 sec compression at two contusion impact forces (50 and 75 kdyn). We observed that regardless of the initial impact force, compression increased tissue damage and worsened functional recovery. Interestingly, compression-dependent damage is not evident until one week after SCI. Further, compression limits functional recovery to the first two weeks post-SCI; in the absence of compression, mice receiving contusion SCI recover for four weeks. To determine whether the recovery plateau is indicative of compression-specific inflammatory responses, we examined macrophage activation with immunohistochemical markers of purportedly pathological (CD86 and macrophage receptor with collagenous structure [MARCO]) and reparative macrophages (arginase [Arg1] and CD206). We detected significant increases in macrophages expression of MARCO and decreases in macrophage Arg1 expression with compression, suggesting a biomechanical-dependent shift in SCI macrophage activation. Collectively, compression-induced alterations in tissue and functional recovery and inflammation highlight the need to consider the primary SCI biomechanics in the design and clinical implementation of immunomodulatory therapies.

Interaction of 12/15-lipoxygenase with fatty acids alters the leukocyte kinetics leading to improved postmyocardial infarction healing.

The metabolic transformation of fatty acids to form oxylipids using 12/15-lipoxygenase (LOX) can promote either resolving or nonresolving inflammation. However, the mechanism of how 12/15-LOX interacts with polyunsaturated fatty acids (PUFA) in postmyocardial infarction (post-MI) healing is unclear. Here, we reported the role of 12/15-LOX in post-MI cardiac remodeling in a PUFA [10% (wt/wt), 22 kcal]-enriched environment. Wild-type (WT; C57BL/6J) and 12/15-LOX-null (12/15-LOX-/-) male mice of 8-12 wk of age were fed a PUFA-enriched diet for 1 mo and subjected to permanent coronary artery ligation. Post-MI mice were monitored for day 1 or until day 5 along with standard diet-fed MI controls. No-MI surgery mice served as naïve controls. PUFA-fed WT and 12/15-LOX-/- mice improved ejection fraction and reduced lung edema greater than WT mice at day 5 post-MI (P < 0.05). Post-MI, neutrophil density was decreased in PUFA-fed WT and 12/15-LOX-/- mice at day 1 (P < 0.05). Deletion of 12/15-LOX in mice led to increased cytochrome P-450-derived bioactive lipid mediator epoxyeicosatrienoic acids (EETs), i.e., 11,12-EpETrE and 14,15-EpETrE, which were further enhanced by acute PUFA intake post-MI. Macrophage density was decreased in WT + PUFA and 12/15-LOX-/- mice compared with their respective standard diet-fed WT controls at day 5 post-MI. 12/15-LOX-/- + PUFA mice displayed an increased expression of chemokine (C-C motif) ligand 2 and reparative macrophages markers (Ym-1, Mrc-1, and Arg-1, all P < 0.05) in the infarcted area. Furthermore, 12/15-LOX-/- mice, with or without PUFA, showed reduced collagen deposition at day 5 post-MI compared with WT mice. In conclusion, deletion of 12/15-LOX and short-term exposure of PUFA promoted leukocyte clearance, thereby limiting cardiac remodeling and promoting an effective resolution of inflammation.NEW & NOTEWORTHY This study determined that 1) deletion of 12/15-lipoxygenase (LOX) promotes the generation of epoxyeicosatrienoic acids, the cytochrome P-450-derived metabolites in postmyocardial infarction (post-MI) healing; 2) acute exposure of fatty acids to 12/15-LOX-/- mice drives leukocyte (neutrophils and macrophages) clearance post-MI; and 3) metabolic transformation of fats is the significant contributor in leukocyte clearance to drive either resolving or nonresolving inflammation post-MI.

Resolvin D1 Programs Myocardial Infarction‐induced Cardiorenal Syndrome in Heart Failure Pathology

Resolvins are innate immune responsive lipid mediators produced after myocardial infarction (MI) to resolve inflammation. MI‐induced bidirectional interaction between progressive left ventricle remodeling and kidney dysfunction is known to advance cardiorenal syndrome (CRS). Whether resolvins limits MI‐induced cardiorenal dysfunction is unclear. Thus, to define the role of resolvin D1 (RvD1) in post‐MI CRS, we tested the hypothesis using 8 to 12 weeks old male C57BL/6 mice that were subjected to coronary artery ligation surgery. RvD1 (3ug/kg/day) was injected 3 hour post‐MI for day (d)1 or continued till d5. No‐MI naïve and saline‐injected MI mice were used as controls. RvD1‐injected group promoted neutrophils (CD11b+/Ly6G+; 2.0±0.3%) egress from infarcted LV compared with MI‐control group (4.2±2.0%) at d5 post‐MI. Further, RvD1 injected mice showed higher reparative macrophages (F4/80+/Ly6Clow/CD206+; 20.0±1.0%) phenotype in the infarcted LV compared with MI‐control group (14.4±2.1%) at d5 post‐MI measured by flow cytometry. MI‐induced LV remote hypertrophy was limited in RvD1‐injected mice compared with MI‐control mice confirmed using wheat germ agglutinin staining. Post‐MI nephrin expression was diffused to glomerular membrane at d5 in MI‐control mice indicating renal injury impacting kidney glomerulus structure but to a lesser extent in RvD1‐injected mice validated using immunofluorescence staining. Administration of RvD1 attenuated MI‐induced renal inflammation decreasing neutrophil gelatinase‐associated lipocalin (2.0±0.1 fold p&lt;0.05) gene and protein expression in the kidney at d1 post‐MI (2.2±0.1 fold; p&lt;0.5) and d5 compared to MI‐control. In conclusion, RvD1 not only clears MI‐induced inflammation by increasing resolving leukocytes but also is renoprotective in nature in heart failure pathology.Support or Funding InformationHL132989 and AT006704 to GVH

Shock wave treatment after hindlimb ischaemia results in increased perfusion and M2 macrophage presence.

Shock wave therapy (SWT) has been shown to induce angiogenesis in ischaemic muscle. However, the mechanism of action remains unknown. Macrophages are crucial for angiogenic responses after ischaemic injury. The M2 macrophage subset enables tissue repair and induces angiogenesis. It was hypothesized that the angiogenic effects of SWT are at least partly caused by enhanced macrophage recruitment. C57BL/6 mice were subjected to hind limb ischaemia with subsequent SWT or sham treatment. Muscles were analysed via immunofluorescence staining, reverse-transcription polymerase chain reaction and western blot. Gene expression and proteins involved in macrophage recruitment were analysed and tissue sections were stained for macrophages, including subsets, capillaries and arterioles. Laser Doppler perfusion imaging was performed to assess functional outcome. Treated muscles showed increased expression of the pivotal macrophage recruiting factor monocyte chemotactic protein 1 (MCP-1). Higher levels of macrophage marker CD14 were found. Increased numbers of macrophages after SWT could be confirmed by immunofluorescence staining. The expression of the M2 polarization promoting chemokine interleukin 13 was significantly elevated in the treatment group. Elevated mRNA expression of the M2 scavenger receptor CD163 was found after SWT. Immunofluorescence staining confirmed increased numbers of M2 macrophages after treatment. It was found that SWT resulted in higher number of capillaries and arterioles. Assessment of functional outcome revealed significantly improved limb perfusion in treated animals. Shock wave therapy causes increased macrophage recruitment and enhanced polarization towards reparative M2 macrophages in ischaemic muscle resulting in angiogenesis and improved limb perfusion and therefore represents a promising new treatment option for the treatment of ischaemic heart disease. Copyright © 2016 John Wiley & Sons, Ltd.

Glucose regulates the intrinsic inflammatory response of the heart to surgically induced hypothermic ischemic arrest and reperfusion.

We investigated the isolated working rat heart as a model to study early transcriptional remodeling induced in the setting of open heart surgery and stress hyperglycemia. Hearts of male Sprague Dawley rats were cold-arrested in Krebs-Henseleit buffer and subjected to 60 min normothermic reperfusion in the working mode with buffer supplemented with noncarbohydrate substrates plus glucose (25 mM) or mannitol (25 mM; osmotic control). Gene expression profiles were determined by microarray analysis and compared with those of nonperfused hearts. Perfused hearts displayed a transcriptional signature independent from the presence of glucose showing a more than twofold increase in expression of 71 genes connected to inflammation, cell proliferation, and apoptosis. These transcriptional alterations were very similar to the ones taking place in the hearts of open heart surgery patients. Prominent among those alterations was the upregulation of the three master regulators of metabolic reprogramming, MYC, NR4A1, and NR4A2. Targeted pathway analysis revealed an upregulation of metabolic processes associated with the proliferation and activation of macrophages and fibroblasts. Glucose potentiated the upregulation of a subset of genes associated with polarization of tissue reparative M2-like macrophages, an effect that was lost in perfused hearts from rats rendered insulin resistant by high-sucrose feeding. The results expose the heart as a significant source of proinflammatory mediators released in response to stress associated with cardiac surgery with cardiopulmonary bypass, and suggest a major role for glucose as a signal in the determination of resident cardiac macrophage polarization.

Macrophage-Associated Osteoactivin/GPNMB Mediates Mesenchymal Stem Cell Survival, Proliferation, and Migration Via a CD44-Dependent Mechanism.

Although MSCs have been widely recognized to have therapeutic potential in the repair of injured or diseased tissues, it remains unclear how functional activities of mesenchymal stem cells (MSCs) are influenced by the surrounding inflammatory milieu at the site of tissue injury. Macrophages constitute an essential component of innate immunity and have been shown to exhibit a phenotypic plasticity in response to various stimuli, which play a central role in both acute inflammation and wound repair. Osteoactivin (OA)/Glycoprotein non-metastatic melanoma protein B (GPNMB), a transmembrane glycoprotein that plays a role in cell differentiation, survival, and angiogenesis. The objective of this study was to investigate the potential role of OA/GPNMB in macrophage-induced MSC function. We found that reparative M2 macrophages express significantly greater levels of OA/GPNMB than pro-inflammatory M1 macrophages. Furthermore, using loss of function and rescue studies, we demonstrated that M2 macrophages-secreted OA/GPNMB positively regulates the viability, proliferation, and migration of MSCs. More importantly, we demonstrated that OA/GPNMB acts through ERK and AKT signaling pathways in MSCs via CD44, to induce these effects. Taken together, our results provide pivotal insight into the mechanism by which OA/GPNMB contributes to the tissue reparative phenotype of M2 macrophages and positively regulates functional activities of MSCs. J. Cell. Biochem. 117: 1511-1521, 2016. © 2015 Wiley Periodicals, Inc.

Extracardiac-Lodged Mesenchymal Stromal Cells Propel an Inflammatory Response Against Myocardial Infarction via Paracrine Effects.

Transplantation of stem cells, including mesenchymal stromal cells (MSCs), improves the recovery of cardiac function after myocardial infarction (MI) in experimental studies using animal models and in patients. However, the improvement of cardiac function following MSC transplantation remains suboptimal in both preclinical and clinical studies. Understanding the mechanism of cell therapy may improve its therapeutic outcomes, but the mode of action mediating stem cell promotion of cardiac repair is complex and not fully understood. Recent studies suggest that the immunomodulatory effects of MSCs on the macrophage M1/M2 subtype transition allow the transplanted stem cells to inhibit inflammation-induced injury and promote cardiac repair in acute MI. However, equally compelling evidence shows that there is poor survival and minimal graft persistence of transplanted MSCs within the infarcted heart tissues, negating the view that graft survival per se is required for the observed high rate and long duration of the transition from proinflammatory M1 to reparative M2 macrophages in the infarcted myocardium. Therefore, we raised a novel hypothesis that the therapeutic effects of MSC transplantation for acute MI depends not primarily on the grafted cells in infarct myocardium, but that MSCs migrating to and being lodged in the extracardiac organs, demonstrating good graft survival and persistence, may render the therapeutic effects in MI. More specifically, MSC transplantation promotes the transition from M1 to M2 in extracardiac organs, such as spleen and bone marrow, and therapeutic effects are conferred to the infarcted myocardium via paracrine effects. In MSC transplantation, the conversion from proinflammatory M1 to anti-inflammatory M2 monocytes may occur remotely from the heart and may serve as one of the major pathways in regulating the dual effects of inflammation. This hypothesis, if proven valid, may represent an important new mechanism of action to be considered for the future of MSC transplantation in the treatment of MI.

The kidney regulates regeneration, but don't upset the balance.

Better understanding of the cellular pathophysiological process undergoing kidney injury and repair will be hopefully result in the design of more targeted therapies to prevent injury, hasten repair, and minimize chronic progressive kidney diseases. The relevance of CSF-1 signalling for kidney organ development and inflammatory disease has been highlighted by numerous studies. Interestingly, there are different functions of CSF-1 in acute kidney injury versus chronic kidney disease (CKD). Within CKD, an enhanced expression of CSF-1 results in more damage, and thus disruption of the CSF-1/CSF-1R interaction/activation is protective. A reverse scenario is seen during acute kidney injury, where inhibition of CSF-1 leads to delayed recovery of kidney function and less regenerative (M2) macrophages. However, the major factor to stimulate epithelial cell repair and the cell type(s) generating the factor in response to acute kidney injury remained unclear. In their recent report Wang et al. used a specific CSF-1 knockout in the proximal tubular cells, induced acute kidney injury, and analyzed the recovery of kidney function. They nicely demonstrated a strong positive effect of renal and proximal tubular secreted CSF-1. It mediates the differentiation of infiltrated monocytes into M2 macrophages, also denoted as reparative macrophages. Mice with a deletion of CSF-1 within the proximal tubular cells exhibited a delayed recovery from acute kidney injury. These findings may pave the path to therapeutic intervention in acute kidney injury.

Neutrophils orchestrate post-myocardial infarction healing by polarizing macrophages towards a reparative phenotype.

Acute myocardial infarction (MI) is the leading cause of mortality worldwide. Anti-inflammatory strategies to reduce neutrophil-driven acute post-MI injury have been shown to limit acute cardiac tissue damage. On the other hand, whether neutrophils are required for resolving post-MI inflammation and repair is unknown. We show that neutrophil-depleted mice subjected to MI had worsened cardiac function, increased fibrosis, and progressively developed heart failure. Flow cytometry of blood, lymphoid organs and digested hearts revealed reduced numbers of Ly6Chigh monocytes in infarcts of neutrophil-depleted mice, whereas the number of macrophages increased, which was paralleled by reduced splenic Ly6Chigh monocyte mobilization but enhanced proliferation of cardiac macrophages. Macrophage subtype analysis revealed reduced cardiac expression of M1 markers, whereas M2 markers were increased in neutrophil-depleted mice. Surprisingly, we found reduced expression of phagocytosis receptor myeloid-epithelial-reproductive tyrosine kinase, a marker of reparative M2c macrophages which mediate clearance of apoptotic cells. In agreement with this finding, neutrophil-depleted mice had increased numbers of TUNEL-positive cells within infarcts. We identified neutrophil gelatinase-associated lipocalin (NGAL) in the neutrophil secretome as a key inducer of macrophages with high capacity to engulf apoptotic cells. The cardiac macrophage phenotype in neutrophil-depleted mice was restored by administration of neutrophil secretome or NGAL. Neutrophils are crucially involved in cardiac repair after MI by polarizing macrophages towards a reparative phenotype. Therapeutic strategies to reduce acute neutrophil-driven inflammation after MI should be carefully balanced as they might interfere with the healing response and cardiac remodelling.

Lp-PLA2 Antagonizes Left Ventricular Healing After Myocardial Infarction by Impairing the Appearance of Reparative Macrophages.

Healing after myocardial infarction (MI) involves the biphasic accumulation of inflammatory Ly-6C(high) and reparative Ly-6C(low) monocytes/macrophages. Excessive inflammation disrupts the balance between the 2 phases, impairs infarct healing, and contributes to left ventricle remodeling and heart failure. Lipoprotein-associated phospholipase A2 (Lp-PLA2), a member of the phospholipase A2 family of enzymes, produced predominantly by leukocytes, participates in host defenses and disease. Elevated Lp-PLA2 levels associate with increased risk of cardiovascular events across diverse patient populations, but the mechanisms by which the enzyme elicits its effects remain unclear. This study tested the role of Lp-PLA2 in healing after MI. In response to MI, Lp-PLA2 levels markedly increased in the circulation. To test the functional importance of Lp-PLA2, we generated chimeric mice whose bone marrow-derived leukocytes were Lp-PLA2-deficient (bmLp-PLA2 (-/-)). Compared with wild-type controls, bmLp-PLA2 (-/-) mice subjected to MI had lower serum levels of inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, and decreased number of circulating inflammatory myeloid cells. Accordingly, bmLp-PLA2 (-/-) mice developed smaller and less inflamed infarcts with reduced numbers of infiltrating neutrophils and inflammatory Ly-6C(high) monocytes. During the later, reparative phase, infarcts of bmLp-PLA2 (-/-) mice contained Ly-6C(low) macrophages with a skewed M2-prone gene expression signature, increased collagen deposition, fewer inflammatory cells, and improved indices of angiogenesis. Consequently, the hearts of bmLp-PLA2 (-/-) mice healed more efficiently, as determined by improved left ventricle remodeling and ejection fraction. Lp-PLA2 augments the inflammatory response after MI and antagonizes healing by disrupting the balance between inflammation and repair, providing a rationale for focused study of ventricular function and heart failure after targeting this enzyme acutely in MI.

The Reparative Function of Cardiomyocytes in the Infarcted Myocardium

Effective repair of the infarcted heart requires spatial containment of the inflammatory reaction within the infarcted area. A recently published study demonstrates that in the pro-inflammatory environment of the infarct border zone, surviving cardiomyocytes restrain inflammation by activating a Reg3β-dependent cascade that recruits reparative macrophages and promotes neutrophil clearance.

Inflammation and repair in the ischaemic myocardium.

Shortly after myocardial infarction, various circulating leukocyte subsets accumulate in the heart. Leukocyte recruitment is highly coordinated and relies on cell production in the bone marrow, mobilization to the blood, and chemokine-mediated infiltration to the destination tissue. Neutrophils, which are phagocytic and inflammatory, are among the first leukocytes to accumulate in large numbers. Within a day, neutrophils disappear and are replaced by a subset of monocytes that further contribute to inflammation and phagocytosis. After a few days, monocyte-derived reparative macrophages accrue, quell inflammation, and foster angiogenesis and tissue remodelling. Studies suggest a well-balanced response comprising these three waves is essential to optimal infarct healing.

P8 Hydrogen sulfide and SPRC mitigate infarct of myocardial infarction via governing migration and infilitration of macrophages

Background Myocardial infarction (MI), associated with the inflammatory response, has long been postulated to be key determinant of rapid necrosis of cardiac myocytes. As a part of the robust inflammatory response, activated macrophages infiltrating into the necrotic myocardium is prerequisite for healing process and scar formation during the post-infarction remodeling. Hydrogen sulfide (H 2 S) and S-propargyl-l-cysteine (SPRC) have been shown to have potent cyto-protective effects. Thus, the present study was to evaluate the roles and mechanisms of NaHS and SPRC on macrophage during infarct repair. Results Echocardiographic analysis showed that ejection fraction (EF) and fractional shortening (FS) were improved in mice pretreated with NaHS and SPRC (67 ± 6%, 77 ± 8%) compared to saline (47 ± 6%) at day 5 post-MI. Concomitantly, 3, 5 and 8 days after MI, the cardiac tissues of NaHS and SPRC groups exerted an increase in immunohistochemical staining for macrophage markers (Galectin-3). And the mRNA levels of Galectin-3 and F4/80 were also increased significantly. In addition, both SPRC and NaHS trigged decreased levels of IL-1beta, IL-6, and TNF- α , which were mainly produced by M1 macrophages, but increased levels of IL-10 and CD163, which were partially due to the activation of reparative M2 Macrophages. In addition, transwell assay revealed that NaHS and SPRC accelerated the migration of peritoneal macrophages, and the immunofluorescence analysis indicated a promoted reorganization of actin cytoskeleton. Conclusion NaHS and SPRC may have potential as an anti-infarct of MI, through governing migration and infilitration of macrophages.

Strain-dependent modulation of macrophage polarization within scaffolds

Implanted synthetic substrates for the regeneration of cardiovascular tissues are exposed to mechanical forces that induce local deformation. Circulating inflammatory cells, actively participating in the healing process, will be subjected to strain once recruited. We investigated the effect of deformation on human peripheral blood mononuclear cells (hPBMCs) adherent onto a scaffold, with respect to macrophage polarization towards an inflammatory (M1) and reparative (M2) phenotype and to early tissue formation. HPBMCs were seeded onto poly-ε-caprolactone bisurea strips and subjected to 0%, 7% and 12% cyclic strain for up to one week. After 1 day, cells subjected to 7% deformation showed upregulated expression of pro and anti-inflammatory chemokines, such as MCP-1 and IL10. Immunostaining revealed presence of inflammatory macrophages in all groups, while immunoregulatory macrophages were detected mainly in the 0 and 7% groups and increased significantly over time. Biochemical assays indicated deposition of sulphated glycosaminoglycans and collagen after 7 days in both strained and unstrained samples. These results suggest that 7% cyclic strain applied to hPBMCs adherent on a scaffold modulates their polarization towards reparative macrophages and allows for early synthesis of extracellular matrix components, required to promote further cell adhesion and proliferation and to bind immunoregulatory cytokines.

Abstract 48: Cd73 Regulation of Netrin-1 Expression in Brain Ischemia

Focal cerebral ischemia is accompanied by inflammation, which further exacerbates injury. CD73 is an ectoenzyme with 5’-nucleotidase activity, which generates anti-inflammatory adenosine, from precursor AMP. In the CNS, CD73 is located on the choroid plexus cuboidal epithelium and on leptomeninges, two critical entry ports for anti- (M2) and pro-inflammatory (M1) macrophages, respectively. We previously showed that acute brain ischemia (48h) in CD73 -/- mice resulted in larger infarct volumes related to greater influx of M1 macrophages. Netrin-1 (Ntn-1) is secreted protein with leukorepulsive and neurite outgrowth- promoting properties. We hypothesized that macrophage influx is regulated by CD73 through its modulation of Ntn-1 expression after stroke. We examined the Ntn-1 expression in acute (24h) and delayed phases (7days) after induction of brain ischemia in wild type (WT) and CD73 -/- mice after photothrombotic middle cerebral artery occlusion. We demonstrated that Ntn-1 mRNA and protein expression was induced during the acute phase of brain ischemia, and suppressed in the delayed phase in WT compared to CD73 -/- mice. Netrin-1 mRNA expression at the 24 hr time point was higher in WT vs CD73 -/- mice (2.7 ± 0.2 vs 1.4 ± 0.4, respectively, p&lt;0.05). However, 7 days following brain ischemia, Ntn-1 protein expression was suppressed in WT compared to CD73 -/- mice (0.4 ± 0.1 to 0.9 ± 0.2; p&lt;0.03 respectively). In CD73 -/- mice, mRNA for CCR2 and IRF5 , were upregulated at day 7, whereas IRF4 the major polarization transcription factor for anti-inflammatory macrophages, was downregulated. These findings are consistent with the hypothesis that in acute stroke, CD73 acts to suppres influx of pro-inflammatory macrophages through induction of chemorepulsive Ntn-1 protein, but promotes influx of reparative macrophages by suppressing Ntn-1 expression at delayed phase of stroke. Our data are the first to identify a potential link between CD73 and Ntn-1 expressions in the ischemic brain, underscoring CD73 as an important modulator of the immune inflammatory response in stroke.

Macrophage Subpopulations Are Essential for Infarct Repair With and Without Stem Cell Therapy

This study sought to investigate the hypothesis that the favorable effects of mesenchymal stromal cells (MSCs) on infarct repair are mediated by macrophages. The favorable effects of MSC therapy in myocardial infarction (MI) are complex and not fully understood. We induced MI in mice and allocated them to bone marrow MSCs, mononuclear cells, or saline injection into the infarct, with and without early (4 h before MI) and late (3 days after MI) macrophage depletion. We then analyzed macrophage phenotype in the infarcted heart by flow cytometry and macrophage secretome in vitro. Left ventricular remodeling and global and regional function were assessed by echocardiography and speckle-tracking based strain imaging. The MSC therapy significantly increased the percentage of reparative M2 macrophages (F4/80(+)CD206(+)) in the infarcted myocardium, compared with mononuclear- and saline-treated hearts, 3 and 4 days after MI. Macrophage cytokine secretion, relevant to infarct healing and repair, was significantly increased after MSC therapy, or incubation with MSCs or MSC supernatant. Significantly, with and without MSC therapy, transient macrophage depletion increased mortality 30 days after MI. Furthermore, early macrophage depletion produced the greatest negative effect on infarct size and left ventricular remodeling and function, as well as a significant incidence of left ventricular thrombus formation. These deleterious effects were attenuated with macrophage restoration and MSC therapy. Some of the protective effects of MSCs on infarct repair are mediated by macrophages, which are essential for early healing and repair. Thus, targeting macrophages could be a novel strategy to improve infarct healing and repair.

Abstract 255: Modulation Of Cardiac Macrophages As A Strategy For Infarct Healing

Heart failure is the leading cause of death in the developed world and myocardial infarction (MI) is the most common cause. Macrophages are key cells that orchestrate the initial inflammatory as well as later stage wound healing responses following MI. These functions are carried out by pro-inflammatory (M1) and reparative (M2) macrophages respectively. Optimal healing response after MI requires a balancing act of the biphasic macrophage response, so as to not prolong inflammatory signals detrimental to wound healing. Taking advantage of the fact that interleukin-4 (IL-4) activates macrophages towards M2, we hypothesize that delivering IL-4 to the post-MI heart can alter the ratio of M2 to M1 macrophages in the infarct area and induce a better healing response. In this study, we validate our approach in vitro and perform in vitro optimization of a suitable delivery system. RAW 264.7 macrophages were stimulated with IL-4 (10ng/uL) or LPS/IFN-γ (100ng/mL and 10ng/mL) for 24h and gene expression markers (qPCR) and Nitric Oxide (NO) levels (Griess assay) analyzed as indication of M1or M2 activation. Mouse aortic endothelial cells were treated with conditioned media from these cells for 24h and tube formation assessed on matrigel. A bioactive, protease-cleavable polyethylene glycol (PEG) hydrogel delivery system was evaluated for release of functional IL-4 to LPS-activated macrophages. Empty or IL-4 encapsulating hydrogel was placed on a trans-well above LPS-stimulated macrophages. Collagenase I at 0.1mg/mL was applied over 48h to degrade the gels and release IL-4 (n≥3 and p&lt;0.05 considered significant by one-way ANOVA). We demonstrate that IL-4 significantly upregulates M2 markers (MRC-1 and Arg-1) while IFN-γ and LPS upregulate M1 markers (NO and TNF-alpha). We observe enhanced tube density in endothelial cells treated with M2 media while M1 inhibited tube formation. Hydrogel release study shows a significant reduction in NO levels of LPS-stimulated macrophages when IL-4 is released, demonstrating that IL-4 is released from the gel in its bioactive form. In conclusion, we show that macrophages can indeed respond to changing stimuli and adopt distinct activation types and our PEG based hydrogel could be a potential delivery system for in vivo IL-4 delivery.

The Ron receptor tyrosine kinase, macrophage heterogeneity and obesity associated disease. (P5070)

Abstract Macrophages play a central role in the progression of obesity associated disease. Macrophages are generally classified into two distinct programs, termed M1 and M2 macrophages, polar ends of a spectrum of potential macrophage activation states. The ability to tip the balance of macrophage activation away from inflammatory M1 cells and toward a reparative M2 phenotype could be beneficial in the treatment of chronic inflammation associated with obesity. The Ron receptor is expressed on tissue resident macrophages and plays a critical role in suppressing inflammation while promoting hallmarks of M2 macrophage activation. Here we show that Ron plays a protective role in the progression of atherosclerosis and hepatic steatosis in obese animals, associated with decreased inflammatory cytokine expression and decreased lipid deposition in these tissues. In lean animals, Ron is highly expressed on resident macrophages in adipose tissue. Upon weight gain, the expression of Ron is decreased and there is shift in the relative percentages of M1/M2 cells in Ron deficient mice, favoring an inflammatory environment. The percentage of Ron+ cells was also reduced in the liver and atherosclerotic plaques, while pro-inflammatory cytokine expression in isolated macrophages was enhanced in the absence of Ron. We predict that Ron is a potential marker for reparative macrophages and that the gene expression profile of macrophages from obese animals will correlate with Ron expression in these cells.

Endothelial Outgrowth Cells Shift Macrophage Phenotype and Improve Kidney Viability in Swine Renal Artery Stenosis

Endothelial outgrowth cells (EOC) decrease inflammation and improve endothelial repair. Inflammation aggravates kidney injury in renal artery stenosis (RAS), and may account for its persistence upon revascularization. We hypothesized that EOC would decrease inflammatory (M1) macrophages and improve renal recovery in RAS. Pigs with 10 weeks of RAS were studied 4 weeks after percutaneous transluminal renal angioplasty (PTRA+stenting) or sham, with or without adjunct intrarenal delivery of autologous EOC (10×10(6)), and compared with similarly treated normal controls (n=7 each). Single-kidney function, microvascular and tissue remodeling, inflammation, oxidative stress, and fibrosis were evaluated. Four weeks after PTRA, EOC were engrafted in injected RAS-kidneys. Stenotic-kidney glomerular filtration rate was restored in RAS+EOC, RAS+PTRA, and RAS+PTRA+EOC pigs, whereas stenotic-kidney blood flow and angiogenesis were improved and fibrosis attenuated only in EOC-treated pigs. Furthermore, EOC increased cell proliferation and decreased the ratio of M1 (inflammatory)/M2 (reparative) macrophages, as well as circulating levels and stenotic-kidney release of inflammatory cytokines. Cultured-EOC released microvesicles in vitro and induced phenotypic switch (M1-to-M2) in cultured monocytes, which was inhibited by vascular endothelial growth factor blockade. Finally, a single intrarenal injection of rh-vascular endothelial growth factor (0.05 μg/kg) in 7 additional RAS pigs also restored M1/M2 ratio 4 weeks later. Intrarenal infusion of EOC after PTRA induced a vascular endothelial growth factor-mediated attenuation in macrophages inflammatory phenotype, preserved microvascular architecture and function, and decreased inflammation and fibrosis in the stenotic kidney, suggesting a novel mechanism and therapeutic potential for adjunctive EOC delivery in experimental RAS to improve PTRA outcomes.

Neuroimmune Guidance Cue Semaphorin 3E Is Expressed in Atherosclerotic Plaques and Regulates Macrophage Retention

The persistence of myeloid-derived cells in the artery wall is a characteristic of advanced atherosclerotic plaques. However, the mechanisms by which these cells are retained are poorly understood. Semaphorins, a class of neuronal guidance molecules, play a critical role in vascular patterning and development, and recent studies suggest that they may also have immunomodulatory functions. The present study evaluates the expression of Semaphorin 3E (Sema3E) in settings relevant to atherosclerosis and its contribution to macrophage accumulation in plaques. Immunofluorescence staining of Sema3E, and its receptor PlexinD1, demonstrated their expression in macrophages of advanced atherosclerotic lesions of Apoe(-/-) mice. Notably, in 2 different mouse models of atherosclerosis regression, Sema3E mRNA was highly downregulated in plaque macrophages, coincident with a reduction in plaque macrophage content and an enrichment in markers of reparative M2 macrophages. In vitro, Sema3E mRNA was highly expressed in inflammatory M1 macrophages and in macrophages treated with physiological drivers of plaque progression and inflammation, such as oxidized low-density lipoprotein and hypoxia. To explore mechanistically how Sema3E affects macrophage behavior, we treated macrophages with recombinant protein in the presence/absence of chemokines, including CCL19, a chemokine implicated in the egress of macrophages from atherosclerotic plaques. Sema3E blocked actin polymerization and macrophage migration stimulated by the chemokines, suggesting that it may immobilize these cells in the plaque. Sema3E is upregulated in macrophages of advanced plaques, is dynamically regulated by multiple atherosclerosis-relevant factors, and acts as a negative regulator of macrophage migration, which may promote macrophage retention and chronic inflammation in vivo.