BACKGROUND:To date, we have lacked an understanding of how coronary artery disease (CAD) affects the extracellular vesicle (EV) profile of human pericardial fluid (PF) and there is a paucity of data querying whether PF-derived EVs have functional benefits. This study characterizes PF-derived EVs and assesses their impact on angiogenesis in vitro and in vivo. METHODS:PF was collected from patients with and without CAD. PF-derived EVs of different sizes were isolated and characterized using microfluidic resistive pulse sensing. Human coronary artery endothelial cells (HCAECs) were exposed to EVs. Uptake of EVs by HCAECs and their impact on cell proliferation was evaluated. HCAECs were analyzed for their migratory and angiogenic properties. The in vivo effects of PF-derived EVs were assessed using murine ischemia models. To elucidate putative mechanisms, proteomic analysis was performed followed by in silico pathway analysis and functional validation. RESULTS:Small and medium-sized EVs (sEV and mEV, respectively) were isolated from PF. HCAECs exposed to sEVs isolated from CAD patients exhibited a significant increase in proliferations, wound closure, and tube formation. Injection of sEVs isolated from the PF of patients with CAD into pericardial cavity of MI mice reduced cardiac fibrosis and improved cardiac function. Differential protein expression implicated key pathways in angiogenesis, apoptosis mitigation, and fibrosis as protective effects of PF-sEVs. CONCLUSION:We identify that PF-derived EVs exert cardioprotective effects by promoting angiogenesis and reducing fibrosis. Future studies should evaluate whether our findings can be recapitulated in a large animal model and a pilot clinical trial.

The pericardial space is a biologically active inflammatory and cellular microenvironment with growing relevance to cardiac disease and surgical practice. Pericardial fluid contains bioactive mediators that frequently exceed systemic concentrations, and the anatomical proximity of this compartment to the myocardium highlights its potential role in cardiac pathology and post-surgical complications. Despite increasing recognition of its importance, the immune biology of the pericardial space remains incompletely characterized, and significant gaps persist in our understanding of its molecular and cellular dynamics. Available evidence indicates that pericardial cytokines, chemokines, and immune cell populations exhibit disease- and injury-specific patterns with relevance in myocardial ischemia, heart failure, cardiac transplantation, and post-operative atrial fibrillation. Following cardiac surgery, pericardial fluid sampled at pericardial opening and from post-operative mediastinal drainage demonstrates a compartmentalized inflammatory response with elevated pro-inflammatory mediators that exceed corresponding systemic levels. In the setting of ischemic injury, pericardial fluid is enriched in markers of tissue remodelling and fibrosis. After myocardial infarction, neutrophils become the dominant inflammatory cell, while resident pericardial macrophages are depleted but subsequently recover during the reparative phase. Collectively, these findings support a framework in which the pericardial space functions as a dynamic signalling reservoir that both reflects and modulates cardiac inflammatory and reparative processes. This review summarizes the current evidence on pericardial immune cells, cytokines, chemokines, and emerging molecular mediators across cardiac surgical populations and the spectrum of ischemic injury, heart failure, and post-operative atrial fibrillation.

The pericardium plays an important homeostatic role for the neighboring heart, providing both lubrication and structural support. In vivo models have further identified a protective role for the pericardium in modulating cardiac remodeling following myocardial infarction, possibly through the actions of tissue-resident pericardial macrophages. Using patient-derived pericardial samples, we establish that human pericardial immune cells directly inhibit cardiac fibroblast fibrotic activity, and this action is dampened following myocardial infarction. Using single-cell RNA sequencing of patient pericardial fluid cells, we identify two pericardial macrophage subsets that are uniquely altered in response to myocardial infarction, which contributes to a shift in their effector molecule expression profiles. We confirm that fibronectin-expressing human pericardial macrophages are the primary driver of the pericardial antifibrotic actions through the release of cystatin C. Finally, we establish cystatin C as a myeloid cell-derived cardioprotective effector molecule in an in vivo model of myocardial infarction. Collectively, we uncover a molecular mechanism of the local immune environment that regulates cardiac remodeling after myocardial infarction.

After injury, the adult human heart fails to regenerate and forms a persistent fibrotic scar. By contrast, fibrosis is transient in the injured zebrafish heart, facilitating cell recruitment and providing regenerative cues. The mechanisms that restrain excessive fibrosis while enabling regeneration remain poorly understood. Here we show that fibulin-2 (Fbln2) regulates specific populations of activated epicardial cells to balance the response to cardiac injury. Using genetic tools for Fbln2 dosage, we find that attenuation of epicardial activation stimulates regenerative programs. Mechanistically, we identify epicardial nuclear protein 1b (Nupr1b) as an Fbln2 effector. Using gain- and loss-of-function approaches, we show that Nupr1b controls epicardial myofibroblast abundance. Notably, epicardial-specific overexpression of nupr1b rescued fbln2 mutant phenotypes. These findings shed light on how modulation of epicardial cell state transitions through Fbln2-Nupr1b signaling regulates regenerative responses after cardiac injury.

The response of heat shock proteins (HSPs) to stress-induced stimuli is now well documented and understood. Specific HSPs like HSP70 play an important role in vascular diseases like atherosclerosis and hypertension. However, the involvement of other HSPs in these vascular pathologies has been largely ignored. HSP60 plays a particularly critical role in vascular cell growth, an important component of many vascular pathologies. HSP60 directly induces vascular smooth muscle cell proliferation. The mechanism may involve an HSP60-induced stimulation of the rate of nuclear protein import in the smooth muscle cell. HSP60 expression levels also correlate with the severity of the disease. In conclusion, HSP60 may have an important role to play in vascular diseases like atherosclerosis. HSP60 may be a promising future pharmaceutical target to focus upon in order to deter the pathological effects of disease conditions like hypertension and atherosclerosis.

Background Pericardial fluid (PF) contains cells, proteins, and inflammatory mediators, such as cytokines, chemokines, growth factors, and matrix metalloproteinases. To date, we lack an adequate understanding of the inflammatory response that acute injury elicits in the pericardial space. Objective To characterize the inflammatory profile in the pericardial space acutely after ischemia/reperfusion. Methods Pigs were used to establish a percutaneous ischemia/reperfusion injury model. PF was removed from pigs at different time points post-anesthesia or post-ischemia/reperfusion. Flow cytometry was used to characterize the immune cell composition of PF, while multiplex analysis was performed on the acellular portion of PF to determine the concentration of inflammatory mediators. There was a minimum of 3 pigs per group. Results While native PF mainly comprises macrophages, we show that neutrophils are the predominant inflammatory cell type in the pericardial space after injury. The combination of acute ischemia/reperfusion (IR) and repeatedly accessing the pericardial space significantly increases the concentration of interleukin-1 beta (IL-1β) and interleukin-1 receptor antagonist (IL-1ra). IR significantly increases the pericardial concentration of TGFβ1 but not TGFβ2. We observed that repeated manipulation of the pericardial space can also drive a robust pro-inflammatory response, resulting in a significant increase in immune cells and the accumulation of potent inflammatory mediators in the pericardial space. Conclusion In the present study, we show that both IR and surgical manipulation can drive robust inflammatory processes in the pericardial space, consisting of an increase in inflammatory cytokines and alteration in the number and composition of immune cells.

Human pericardial fluid (PF) is a rich reservoir of biologically active markers. The acellular compartment of PF can drive cardiac fibroblast activity in vitro. This process is mediated through the transforming growth factor-β pathway. Of clinical importance, the PF of patients with coronary artery disease has an increased profibrotic capacity compared with the PF of patients without coronary artery disease.

In a recent study published in Nature,Malamud et al.identified how neutrophil MICL recognizes neutrophil extra-cellular traps(NETs).This recognition suppresses further neutrophil activation and NET production,thereby preventing a vicious cycle of inflammation. Neutrophils are circulating immune cells that rapidly migrate into infected or injured tissues.Upon recruitment,these cells amplify inflammation by releasing cytokines,proteases,reactive oxygen species(ROS),and neutrophil extracellular traps(NETs)—a sticky web of DNA containing histones and other effector molecules.Of these programs,NETs may be the most potent because they can non-specifically kill cells by disrupting their cell membranes.1,2 During a bacterial or fungal infection,NETs are helpful as they efficiently immobilize and clear the pathogens.

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.

Kupffer cells (KCs) are localized in liver sinusoids but extend pseudopods to parenchymal cells to maintain their identity and serve as the body’s central bacterial filter. Liver cirrhosis drastically alters vascular architecture, but how KCs adapt is unclear. We used a mouse model of liver fibrosis and human tissue to examine immune adaptation. Fibrosis forced KCs to lose contact with parenchymal cells, down-regulating “KC identity,” which rendered them incapable of clearing bacteria. Commensals stimulated the recruitment of monocytes through CD44 to a spatially distinct vascular compartment. There, recruited monocytes formed large aggregates of multinucleated cells (syncytia) that expressed phenotypical KC markers and displayed enhanced bacterial capture ability. Syncytia formed via CD36 and were observed in human cirrhosis as a possible antimicrobial defense that evolved with fibrosis.

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.

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.

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.

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.