Micronized Acellular Matrix Biomaterial Leverages Eosinophils for Postinfarct Cardiac Repair.

After ischemic injury, immune cells mediate maladaptive cardiac remodeling. Extracellular matrix biomaterials may redirect inflammation toward repair. Pericardial fluid contains pro-reparative immune cells, potentially leverageable by biomaterials. Herein, we explore how pericardial delivery of a micronized extracellular matrix biomaterial affects cardiac healing. In noninfarcted mice, pericardial delivery increases pericardial and myocardial eosinophil counts. This response is sustained after myocardial infarction, stimulating an interleukin 4 rich milieu. Ultimately, the biomaterial improves postinfarct vascularization and cardiac function; and eosinophil-knockout negates these benefits. For the first time, to our knowledge, we demonstrate the therapeutic potential of pericardial biomaterial delivery and the eosinophil's critical role in biomaterial-mediated postinfarct repair.

Pericardial Immune Cells and Their Evolving Role in Cardiovascular Pathophysiology.

The pericardium plays several homeostatic roles to support and maintain everyday cardiac function. Recent advances in techniques and experimental models have allowed for further exploration into the cellular contents of the pericardium itself. Of particular interest are the various immune cell populations present in the space within the pericardial fluid and fat. In contrast to immune cells of the comparable pleura, peritoneum and heart, pericardial immune cells appear to be distinct in their function and phenotype. Specifically, recent work has suggested these cells play critical roles in an array of pathophysiological conditions including myocardial infarction, pericarditis, and post-cardiac surgery complications. In this review, we spotlight the pericardial immune cells currently identified in mice and humans, the pathophysiological role of these cells, and the clinical significance of the immunocardiology axis in cardiovascular health.

Pericardial Inflammatory Mediators That Can Drive Postoperative Atrial Fibrillation in Cardiac Surgery Patients.

Postoperative atrial fibrillation (POAF) is a common dysrhythmia that affects a significant number of patients undergoing cardiac surgery. Many studies aim to better understand this complex postsurgical complication by analysing circulating biomarkers in patients who develop POAF. More recently, the pericardial space was shown to contain inflammatory mediators that could trigger POAF. In this review we summarise recent studies that examine the immune mediators present in the pericardial space and their potential implications for the pathophysiology of POAF in cardiac surgery patients. Ongoing research in this area should better delineate the multifactorial etiology of POAF, where specific markers may be targeted to reduce the incidence of POAF and improve outcomes for this patient population.

The role of macrophage subsets in and around the heart in modulating cardiac homeostasis and pathophysiology.

Cardiac and pericardial macrophages contribute to both homeostatic and pathophysiological processes. Recent advances have identified a vast repertoire of these macrophage populations in and around the heart - broadly categorized into a CCR2+/CCR2- dichotomy. While these unique populations can be further distinguished by origin, localization, and other cell surface markers, further exploration into the role of cardiac and pericardial macrophage subpopulations in disease contributes an additional layer of complexity. As such, novel transgenic models and exogenous targeting techniques have been employed to evaluate these macrophages. In this review, we highlight known cardiac and pericardial macrophage populations, their functions, and the experimental tools used to bolster our knowledge of these cells in the cardiac context.

Acellular biomaterial modulates myocardial inflammation and promotes endogenous mechanisms of postinfarct cardiac repair.

OBJECTIVE: After myocardial infarction, we previously showed that epicardial implantation of porcine small intestinal submucosal extracellular matrix (SIS-ECM) improves postinfarct cardiac function through fibroblast-mediated angiogenic and antifibrotic pathways. Herein, we characterize how SIS-ECM also coordinates a reparative cardiac inflammatory response. METHODS: RNA sequencing and multiplex characterized modulation of fibroblast transcriptional and paracrine activity by SIS-ECM. Inhibitors of fibroblast growth factor 2 and toll-like receptor 9 elucidated mechanism. Mice received coronary ligation (infarction) and either SIS-ECM implantation (treatment) or sham surgery (control). Flow cytometry of SIS-ECM and the murine myocardium quantified monocytes, neutrophils, and proangiogenic subtypes. Microscopy tracked fibroblasts and immune cells, and characterized myocardial angiogenesis. RESULTS: SIS-ECM increased fibroblast transcription of inflammatory pathways and production of angiogenic vascular endothelial growth factor and inflammatory cytokines via fibroblast growth factor 2 and toll-like receptor 9-dependent pathways. Two-photon microscopy showed that SIS-ECM became engrafted by native fibroblasts and leukocytes, subsequently increasing release of inflammatory cytokines and angiogenic vascular endothelial growth factor. On flow cytometry, SIS-ECM implantation increased day-7 myocardial counts of neutrophils, inflammatory monocytes, and proangiogenic vascular endothelial growth factor recptor 1 subtypes. SIS-ECM has a higher proportion of proangiogenic leukocytes compared with the myocardium. Resonant confocal microscopy showed neovascularization near SIS-ECM. CONCLUSIONS: SIS-ECM promotes engraftment by native fibroblasts and leukocytes, and modulates fibroblast activity via fibroblast growth factor 2 and toll-like receptor 9 to potentiate a proangiogenic inflammatory response. Subsequently, the material increases myocardial counts of reparative proangiogenic leukocytes that can induce neovascularization. This reparative inflammatory response may explain previously reported functional improvements. Fibroblast growth factor 2 and toll-like receptor 9 mechanisms can be leveraged to design next-generation materials for postinfarct cardiac repair.

Cardiac surgery elicits pericardial inflammatory responses that are distinct compared with postcardiopulmonary bypass systemic inflammation.

Objectives: Cardiac surgery using cardiopulmonary bypass contributes to a robust systemic inflammatory process. Local intrapericardial postsurgical inflammation is believed to trigger important clinical implications, such as postoperative atrial fibrillation and postsurgical intrathoracic adhesions. Immune mediators in the pericardial space may underlie such complications. Methods: In this prospective pilot clinical study, 12 patients undergoing isolated coronary artery bypass graft surgery were enrolled. Native pericardial fluid and venous blood samples (baseline) were collected immediately after pericardiotomy. Postoperative pericardial fluid and venous blood samples were collected 48-hours after cardiopulmonary bypass and compared with baseline. Flow cytometry determined proportions of specific immune cells, whereas multiplex analysis probed for inflammatory mediators. Results: Neutrophils are the predominant cells in both the pericardial space and peripheral blood postoperatively. There are significantly more CD163lo macrophages in blood compared with pericardial effluent after surgery. Although there are significantly more CD163hi macrophages in native pericardial fluid compared with baseline blood, after surgery there are significantly fewer of these cells present in the pericardial space compared with blood. Postoperatively, concentration of interleukin receptor antagonist 6, and interleukin 8 were significantly higher in the pericardial space compared with blood. After surgery, compared with blood, the pericardial space has a significantly higher concentration of matrix metalloproteinase 3, matrix metalloproteinase 8, and matrix metalloproteinase 9. The same trend was observed with transformational growth factor ß. Conclusions: Cardiac surgery elicits an inflammatory response in the pericardial space, which differs from systemic inflammatory responses. Future work should determine whether or not this distinct local inflammatory response contributes to postsurgical complications and could be modified to influence clinical outcomes.

A monocyte-leptin-angiogenesis pathway critical for repair post-infection.

During infection, inflammatory monocytes are thought to be key for bacterial eradication, but this is hard to reconcile with the large numbers of neutrophils that are recruited for each monocyte that migrates to the afflicted tissue, and the much more robust microbicidal functions of the neutrophils. However, unlike neutrophils, monocytes have the capacity to convert to situationally specific macrophages that may have critical functions beyond infection control1,2. Here, using a foreign body coated with Staphylococcus aureus and imaging over time from cutaneous infection to wound resolution, we show that monocytes and neutrophils are recruited in similar numbers with low-dose infection but not with high-dose infection, and form a localization pattern in which monocytes surround the infection site, whereas neutrophils infiltrate it. Monocytes did not contribute to bacterial clearance but converted to macrophages that persisted for weeks after infection, regulating hypodermal adipocyte expansion and production of the adipokine hormone leptin. In infected monocyte-deficient mice there was increased persistent hypodermis thickening and an elevated leptin level, which drove overgrowth of dysfunctional blood vasculature and delayed healing, with a thickened scar. Ghrelin, which opposes leptin function3, was produced locally by monocytes, and reduced vascular overgrowth and improved healing post-infection. In sum, we find that monocytes function as a cellular rheostat by regulating leptin levels and revascularization during wound repair.

Neutrophils in homeostasis and tissue repair.

Neutrophils are the most abundant innate immune cell and are equipped with highly destructive molecular cargo. As such, these cells were long thought to be short-lived killer cells that unleash their full cytotoxic programs on pathogens following infection and on host bystander cells after sterile injury. However, this view of neutrophils is overly simplistic and as a result is outdated. Numerous studies now collectively highlight neutrophils as far more complex and having a host of homeostatic and tissue-reparative functions. In this review, we summarize these underappreciated roles across organs and injury models.

Comprehensive characterization of the postoperative pericardial inflammatory response: Potential implications for clinical outcomes.

Objective: There is a paucity of data on the inflammatory response that takes place in the pericardial space after cardiac surgery. This study provides a comprehensive assessment of the local postoperative inflammatory response. Methods: Forty-three patients underwent cardiotomy, where native pericardial fluid was aspirated and compared with postoperative pericardial effluent collected at 4, 24, and 48 hours' postcardiopulmonary bypass. Flow cytometry was used to define the levels and proportions of specific immune cells. Samples were also probed for concentrations of inflammatory cytokines, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Results: Preoperatively, the pericardial space mainly contains macrophages and T cells. However, the postsurgical pericardial space was populated predominately by neutrophils, which constituted almost 80% of immune cells present, and peaked at 24 hours. When surgical approaches were compared, minimally invasive surgery was associated with fewer neutrophils in the pericardial space at 4 hours' postsurgery. Analysis of the intrapericardial concentrations of inflammatory mediators showed interleukin-6, MMP-9, and TIMP-1 to be highest postsurgery. Over time, MMP-9 concentrations decreased significantly, whereas TIMP-1 levels increased, resulting in a significant reduction of the ratio of MMP:TIMP after surgery, suggesting that active inflammatory processes may influence extracellular matrix remodeling. Conclusions: These results show that cardiac surgery elicits profound alterations in the immune cell profile in the pericardial space. Defining the cellular and molecular mediators that drive pericardial-specific postoperative inflammatory processes may allow for targeted therapies to reduce immune-mediated complications.

An Intact Pericardium Ischemic Rodent Model.

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart's resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

Ischemic heart disease: Cellular and molecular immune contributions of the pericardium.

Ischemic heart disease promotes complex inflammatory and remodeling pathways which contribute to the development of chronic heart failure. Although blood-derived and local cardiac mediators have traditionally been linked with these processes, the pericardial space has more recently been noted as alternative contributor to the injury response in the heart. The pericardial space contains fluid rich in physiologically active mediators, and immunologically active adipose tissue, which are altered during myocardial infarction. Key immune cells in the pericardial fluid and adipose tissue have been identified which act as mediators for cell recruitment and function after myocardial infarction have been identified in experimental models. Here, we provide an overview of the current understanding of the inflammatory mechanisms of the pericardial space and their role in post-myocardial infarction remodeling and the potential for the use of the pericardial space as a delivery vehicle for treatments to modulate heart healing.

Prevention of Post-Operative Adhesions: A Comprehensive Review of Present and Emerging Strategies.

Post-operative adhesions affect patients undergoing all types of surgeries. They are associated with serious complications, including higher risk of morbidity and mortality. Given increased hospitalization, longer operative times, and longer length of hospital stay, post-surgical adhesions also pose a great financial burden. Although our knowledge of some of the underlying mechanisms driving adhesion formation has significantly improved over the past two decades, literature has yet to fully explain the pathogenesis and etiology of post-surgical adhesions. As a result, finding an ideal preventative strategy and leveraging appropriate tissue engineering strategies has proven to be difficult. Different products have been developed and enjoyed various levels of success along the translational tissue engineering research spectrum, but their clinical translation has been limited. Herein, we comprehensively review the agents and products that have been developed to mitigate post-operative adhesion formation. We also assess emerging strategies that aid in facilitating precision and personalized medicine to improve outcomes for patients and our healthcare system.

Post-Operative Adhesions: A Comprehensive Review of Mechanisms.

Post-surgical adhesions are common in almost all surgical areas and are associated with significant rates of morbidity, mortality, and increased healthcare costs, especially when a patient requires repeat operative interventions. Many groups have studied the mechanisms driving post-surgical adhesion formation. Despite continued advancements, we are yet to identify a prevailing mechanism. It is highly likely that post-operative adhesions have a multifactorial etiology. This complex pathophysiology, coupled with our incomplete understanding of the underlying pathways, has resulted in therapeutic options that have failed to demonstrate safety and efficacy on a consistent basis. The translation of findings from basic and preclinical research into robust clinical trials has also remained elusive. Herein, we present and contextualize the latest findings surrounding mechanisms that have been implicated in post-surgical adhesion formation.

An overview of human pericardial space and pericardial fluid.

The pericardium is a double-layered fibro-serous sac that envelops the majority of the surface of the heart as well as the great vessels. Pericardial fluid is also contained within the pericardial space. Together, the pericardium and pericardial fluid contribute to a homeostatic environment that facilitates normal cardiac function. Different diseases and procedural interventions may disrupt this homeostatic space causing an imbalance in the composition of immune mediators or by mechanical stress. Inflammatory cells, cytokines, and chemokines are present in the pericardial space. How these specific mediators contribute to different diseases is the subject of debate and research. With the advent of highly specialized assays that can identify and quantify various mediators we can potentially establish specific and sensitive biomarkers that can be used to differentiate pathologies, and aid clinicians in improving clinical outcomes for patients.

Gata6+ Pericardial Cavity Macrophages Relocate to the Injured Heart and Prevent Cardiac Fibrosis.

Macrophages play an important role in structural cardiac remodeling and the transition to heart failure following myocardial infarction (MI). Previous research has focused on the impact of blood-derived monocytes on cardiac repair. Here we examined the contribution of resident cavity macrophages located in the pericardial space adjacent to the site of injury. We found that disruption of the pericardial cavity accelerated maladaptive post-MI cardiac remodeling. Gata6+ macrophages in mouse pericardial fluid contributed to the reparative immune response. Following experimental MI, these macrophages invaded the epicardium and lost Gata6 expression but continued to perform anti-fibrotic functions. Loss of this specialized macrophage population enhanced interstitial fibrosis after ischemic injury. Gata6+ macrophages were present in human pericardial fluid, supporting the notion that this reparative function is relevant in human disease. Our findings uncover an immune cardioprotective role for the pericardial tissue compartment and argue for the reevaluation of surgical procedures that remove the pericardium.

Heat shock protein 60 involvement in vascular smooth muscle cell proliferation.

AIM: Heat shock protein 60 (Hsp60) is a mediator of stress-induced vascular smooth muscle cell (VSMC) proliferation. This study will determine, first, if the mitochondrial or cytoplasmic localization of Hsp60 is critical to VSMC proliferation and, second, the mechanism of Hsp60 induction of VSMC proliferation with a focus on modification of nucleocytoplasmic trafficking. METHODS AND RESULTS: Hsp60 was overexpressed in primary rabbit VSMCs with or without a mitochondrial targeting sequence (AdHsp60mito-). Both interventions induced an increase in VSMC PCNA expression and proliferation. The increase in VSMC PCNA expression and growth was not observed after siRNA-mediated knockdown of Hsp60 expression. Nuclear protein import in VSMC was measured by fluorescent microscopy using a microinjected fluorescent import substrate. Nuclear protein import was stimulated by both AdHsp60 and AdHsp60mito- treatments. AdHsp60 treatment also induced increases in nucleoporin (Nup) 62, Nup153, importin-α, importin-ß and Ran expression as well as cellular ATP levels compared to control. AdHsp60mito- treatment induced an up-regulation in importin-α, importin-ß and Ran expression compared to control. Hsp60 knockdown did not change nuclear protein import nor the expression of any nuclear transport receptors or nucleoporins. Both heat shock treatment and Hsp60 overexpression promoted the interaction of Ran with Hsp60. CONCLUSIONS: VSMC proliferation can be modulated via an Hsp60 dependent, cytosol localized mechanism that in part involves a stimulation of nuclear protein import through an interaction with Ran. This novel cellular signaling role for Hsp60 may be important in growth-based vascular pathologies like atherosclerosis and hypertension.

Neutrophil heterogeneity: Bona fide subsets or polarization states?

Neutrophils are key components of the innate immune system that play important roles during infection, injury, and chronic disease. In recent years, neutrophil heterogeneity has become an emerging focus with accumulating evidence of neutrophil populations with distinct functions under both steady-state and pathologic conditions. Despite these advances, it remains unclear whether these different populations represent bona fide subsets or simply activation/polarization states in response to local cues. In this review, we summarize the varied neutrophils populations that have been described under both basal and during inflammation. We discuss the evidence that supports the existence of neutrophils subsets. Finally, we identify potential gaps in our knowledge that may further advance our current understanding of neutrophil heterogeneity.

Splenic Ly6Ghigh mature and Ly6Gint immature neutrophils contribute to eradication of S. pneumoniae.

The spleen plays an integral protective role against encapsulated bacterial infections. Our understanding of the associated mechanisms is limited to thymus-independent (TI) antibody production by the marginal zone (MZ) B cells, leaving the contribution of other splenic compartments such as the red pulp (RP) largely unexplored despite asplenic patients succumbing to the infection in the first 24 h, suggesting important antibody-independent mechanisms. In this study, using time-lapse intravital imaging of the spleen, we identify a tropism for Streptococcus pneumoniae in this organ mediated by tissue-resident MZ and RP macrophages and a protective role for two distinct splenic neutrophil populations (Ly6Ghi and Ly6Gintermediate) residing in the splenic RP. Splenic mature neutrophils mediated pneumococcal clearance in the spleen by plucking bacteria off the surface of RP macrophages that caught the majority of bacteria in a complement-dependent manner. This neutrophil phagocytic capacity was further enhanced after TI antibody production. Resident immature neutrophils (Ly6Gintermediate) in the spleen undergo emergency proliferation and mobilization from their splenic niche after pneumococcal stimulation to increase the effector mature neutrophil pool. We demonstrate that splenic neutrophils together with two macrophage populations and MZ B cells regulate systemic S. pneumoniae clearance through complementary mechanisms.