Fibroblasts under pressure: cardiac fibroblast responses to hypertension and antihypertensive therapies.

Approximately 50% of Americans have hypertension, which significantly increases the risk of heart failure. In response to increased peripheral resistance in hypertension, intensified mechanical stretch in the myocardium induces cardiomyocyte hypertrophy and fibroblast activation to withstand increased pressure overload. This changes the structure and function of the heart, leading to pathological cardiac remodeling and eventual progression to heart failure. In the presence of hypertensive stimuli, cardiac fibroblasts activate and differentiate to myofibroblast phenotype capable of enhanced extracellular matrix secretion in coordination with other cell types, mainly cardiomyocytes. Both systemic and local renin-angiotensin-aldosterone system activation lead to increased angiotensin II stimulation of fibroblasts. Angiotensin II directly activates fibrotic signaling such as transforming growth factor ß/SMAD and mitogen-activated protein kinase (MAPK) signaling to produce extracellular matrix comprised of collagens and matricellular proteins. With the advent of single-cell RNA sequencing techniques, heterogeneity in fibroblast populations has been identified in the left ventricle in models of hypertension and pressure overload. The various clusters of fibroblasts reveal a range of phenotypes and activation states. Select antihypertensive therapies have been shown to be effective in limiting fibrosis, with some having direct actions on cardiac fibroblasts. The present review focuses on the fibroblast-specific changes that occur in response to hypertension and pressure overload, the knowledge gained from single-cell analyses, and the effect of antihypertensive therapies. Understanding the dynamics of hypertensive fibroblast populations and their similarities and differences by sex is crucial for the advent of new targets and personalized medicine.

Realizing the price of academic freedom.

Since 2010, the number of life science doctoral graduates opting into academic postdoctoral employment has steadily declined. In recent years, this decline has made routine headlines in academic news cycles, and faculty members, universities, and funding bodies alike have begun to take notice. In November 2022, the National Institutes of Health (NIH) convened a special interest group to address the problems in postdoctoral recruitment and retention. In response, the American Physiological Society Science Policy Committee highlighted several key issues in postdoctoral training and working conditions and offered the NIH solutions to consider. There are known issues that affect postdoctoral recruitment and retention efforts: low wages relative to other employment sectors, a heavy workload, and poor job prospects to name a few. Unfortunately, these concerns are frequently dismissed as "the price of doing business in academia," and postdoctoral scholars are promised that if they overcome the trials and tribulations of this training period, the reward at the end, a career with academic freedom to pursue your own interests, justifies the means. However, academic freedom cannot and should not be used as the band-aid in a system where most of us will never actually experience academic freedom. Instead, we should systematically embrace solutions that improve the personal and professional health of early career researchers in all levels of training and independence if the goal is to truly shore up the academic workforce.

Surfaceome mapping of primary human heart cells with CellSurfer uncovers cardiomyocyte surface protein LSMEM2 and proteome dynamics in failing hearts.

Cardiac cell surface proteins are drug targets and useful biomarkers for discriminating among cellular phenotypes and disease states. Here we developed an analytical platform, CellSurfer, that enables quantitative cell surface proteome (surfaceome) profiling of cells present in limited quantities, and we apply it to isolated primary human heart cells. We report experimental evidence of surface localization and extracellular domains for 1,144 N-glycoproteins, including cell-type-restricted and region-restricted glycoproteins. We identified a surface protein specific for healthy cardiomyocytes, LSMEM2, and validated an anti-LSMEM2 monoclonal antibody for flow cytometry and imaging. Surfaceome comparisons among pluripotent stem cell derivatives and their primary counterparts highlighted important differences with direct implications for drug screening and disease modeling. Finally, 20% of cell surface proteins, including LSMEM2, were differentially abundant between failing and non-failing cardiomyocytes. These results represent a rich resource to advance development of cell type and organ-specific targets for drug delivery, disease modeling, immunophenotyping and in vivo imaging.

The cardiac wound healing response to myocardial infarction.

Myocardial infarction (MI) is defined as evidence of myocardial necrosis consistent with prolonged ischemia. In response to MI, the myocardium undergoes a series of wound healing events that initiate inflammation and shift to anti-inflammation before transitioning to tissue repair that culminates in scar formation to replace the region of the necrotic myocardium. The overall response to MI is determined by two major steps, the first of which is the secretion of proteases by infiltrating leukocytes to breakdown extracellular matrix (ECM) components, a necessary step to remove necrotic cardiomyocytes. The second step is the generation of new ECM that comprises the scar; and this step is governed by the cardiac fibroblasts as the major source of new ECM synthesis. The leukocyte component resides in the middle of the two-step process, contributing to both sides as the leukocytes transition from pro-inflammatory to anti-inflammatory and reparative cell phenotypes. The balance between the two steps determines the final quantity and quality of scar formed, which in turn contributes to chronic outcomes following MI, including the progression to heart failure. This review will summarize our current knowledge regarding the cardiac wound healing response to MI, primarily focused on experimental models of MI in mice. This article is categorized under: Cardiovascular Diseases > Molecular and Cellular Physiology Immune System Diseases > Molecular and Cellular Physiology.

Harnessing the Plasma Proteome to Mirror Current and Predict Future Cardiac Remodeling After Myocardial Infarction.

To identify plasma proteins that mirror current and predict future remodeling after myocardial infarction (MI), we retrospectively interrogated plasma proteomes of day (D)0 control (n = 16) and D3 MI (n = 15) from C57BL/6 J mice (20 ± 1 months). A total of 165 unique proteins were correlated with cardiac physiology variables. We prospectively tested the hypothesis that candidates identified retrospectively would predict cardiac physiology at an extended timepoint (D7 MI) in a second cohort of mice (n = 4 ± 1 months). We also examined human plasma from healthy controls (n = 18) and patients 48 h after presentation for MI (n = 41). Retrospectively, we identified 5 strong reflectors of remodeling (all r ≥ 0.60 and p < 0.05). Prospectively, ApoA1, IgA, IL-17E, and TIMP-1 mirrored current and predicted future remodeling. In humans, cytokine-cytokine receptor signaling was the top enriched KEGG pathway for all candidates. In summary, we identified plasma proteins that serve as useful prognostic indicators of adverse remodeling and progression to heart failure.

MMP-12 polarizes neutrophil signalome towards an apoptotic signature.

While macrophages are well-known to polarize across the inflammatory spectrum, neutrophils have only recently been found to activate in a similar fashion in response to pro- or anti-inflammatory stimuli. Matrix metalloproteinase (MMP)-12 mediates neutrophil physiology with direct signaling mechanisms yet to be investigated. We hypothesized MMP-12 may modify neutrophil signaling. Bone marrow neutrophils were stimulated with interleukin (IL-1ß; pro-inflammatory), IL-4 (anti-inflammatory), or MMP-12. The secretome was mapped by multi-analyte profiling and intracellular signaling evaluated by array. IL-1ß induced a cytokine-mediated inflammatory LPS-like signalome, with upregulation of pro-inflammatory cytokines such as interferon gamma (IFNγ,15.2-fold,p = 0.001), chemokine (C-X-C motif) ligand 1 (CXCL1,8.4-fold,p = 0.005), and tumor necrosis factor alpha (TNFα,11.2-fold,p = 0.004). IL-4 induced strong intracellular signaling with upregulation of mitogen-activated protein kinase kinase (MEK1;1.9-fold,p = 0.0005) and downregulation of signal transducer and activator of transcription 4 (STAT4;0.77-fold,0.001). MMP-12 increased IL-4 secretion 20-fold and induced a robust apoptotic neutrophil signalome with upregulation of forkhead box O1 (FOXO1;1.4-fold,p < 0.0001) and downregulation of WNT signaling with MMP-12 cleavage of the adherens junction components ß-catenin, cahderin-3, and catenin-α2. In conclusion, neutrophils shifted phenotype by stimuli, with MMP-12 inducing a unique apoptotic signalome with higher resemblance to the anti-inflammatory signalome. SIGNIFICANCE: This study revealed that neutrophils demonstrate unique polarization signaling responses to specific stimuli, with the matrix metalloproteinase (MMP)-12 signalome showing similarity to the IL-4 signalome. MMP-12 polarized neutrophils towards a strong apoptotic signature by upregulating FOXO1 and downregulating WNT signaling. Our results highlight that neutrophils display more plasticity than previously appreciated.

Macrophages secrete murinoglobulin-1 and galectin-3 to regulate neutrophil degranulation after myocardial infarction.

Inflammation presides early after myocardial infarction (MI) as a key event in cardiac wound healing. Ischemic cardiomyocytes secrete inflammatory cues to stimulate infiltration of leukocytes, predominantly macrophages and neutrophils. Infiltrating neutrophils degranulate to release a series of proteases including matrix metalloproteinase (MMP)-9 to break down extracellular matrix and remove necrotic myocytes to create space for the infarct scar to form. While neutrophil to macrophage communication has been explored, the reverse has been understudied. We used a proteomics approach to catalogue the macrophage secretome at MI day 1. Murinoglobulin-1 (MUG1) was the highest-ranked secreted protein (4.1-fold upregulated at MI day 1 vs. day 0 pre-MI cardiac macrophages, p = 0.004). By transcriptomics evaluation, galectin-3 (Lgals3) was 2.2-fold upregulated (p = 0.008) in MI day 1 macrophages. We explored the direct roles of MUG1 and Lgals3 on neutrophil degranulation. MUG1 blunted while Lgals3 amplified neutrophil degranulation in response to phorbol 12-myristate 13-acetate or interleukin-1ß, as measured by MMP-9 secretion. Lgals3 itself also stimulated MMP-9 secretion. To determine if MUG1 regulated Lgals3, we co-stimulated neutrophils with MUG1 and Lgals3. MUG1 limited degranulation stimulated by Lgals3 by 64% (p < 0.001). In vivo, MUG1 was elevated in the infarct region at MI days 1 and 3, while Lgals3 increased at MI day 7. The ratio of MUG1 to Lgals3 positively correlated with infarct wall thickness, revealing that MUG1 attenuated infarct wall thinning. In conclusion, macrophages at MI day 1 secrete MUG1 to limit and Lgals3 to accentuate neutrophil degranulation to regulate infarct wall thinning.

Faster skin wound healing predicts survival after myocardial infarction.

Both skin wound healing and the cardiac response to myocardial infarction (MI) progress through similar pathways involving inflammation, resolution, tissue repair, and scar formation. Due to the similarities, we hypothesized that the healing response to skin wounding would predict future response to MI. Mice were given a 3-mm skin wound using a disposable biopsy punch and the skin wound was imaged daily until closure. The same set of animals was given MI by permanent coronary artery ligation 28 days later and followed for 7 days. Cardiac physiology was measured by echocardiography at baseline and MI days 3 and 7. Animals that survived until day 7 were grouped as survivors, and animals that died from MI were grouped as nonsurvivors. Survivors had faster skin wound healing than nonsurvivors. Faster skin wound healing predicted MI survival better than commonly used cardiac functional variables (e.g., infarct size, fractional shortening, and end diastolic dimension). N-glycoproteome profiling of MI day 3 plasma revealed α2-macroglobulin and ELL-associated factor 1 as strong predictors of future MI death and progression to heart failure. A second cohort of MI mice validated these findings. To investigate the clinical relevance of α2-macroglobulin, we mapped the plasma glycoproteome in patients with MI 48 h after admission and in healthy controls. In patients, α2-macroglobulin was increased 48 h after MI. Apolipoprotein D, another plasma glycoprotein, detrimentally regulated both skin and cardiac wound healing in male but not female mice by promoting inflammation. Our results reveal that the skin is a mirror to the heart and common pathways link wound healing across organs.NEW & NOTEWORTHY Faster skin wound healers had more efficient cardiac healing after myocardial infarction (MI). Two plasma proteins at D3 MI, EAF1 and A2M, predicted MI death in 66% of cases. ApoD regulated both skin and cardiac wound healing in male mice by promoting inflammation. The skin was a mirror to the heart and common pathways linked wound healing across organs.

S100A9 is a functional effector of infarct wall thinning after myocardial infarction.

Neutrophils infiltrate into the left ventricle (LV) early after myocardial infarction (MI) and launch a proinflammatory response. Along with neutrophil infiltration, LV wall thinning due to cardiomyocyte necrosis also peaks at day 1 in the mouse model of MI. To understand the correlation, we examined a previously published data set that included day 0 (n = 10) and MI day (D) 1 (n = 10) neutrophil proteome and echocardiography assessments. Out of 123 proteins, 4 proteins positively correlated with the infarct wall thinning index (1/wall thickness): histone 1.2 (r = 0.62, P = 0.004), S100A9 (r = 0.60, P = 0.005), histone 3.1 (r = 0.55, P = 0.01), and fibrinogen (r = 0.47, P = 0.04). As S100A9 was the highest ranked secreted protein, we hypothesized that S100A9 is a functional effector of infarct wall thinning. We exogenously administered S100A8/A9 at the time of MI to mice [C57BL/6J, male, 3-6 mo of age, n = 7 M (D1), and n = 5 M (D3)] and compared with saline vehicle control-treated mice [n = 6 M (D1) and n = 6 M (D3)] at MI days 1 and 3. At MI day 3, the S100A8/A9 group showed a 22% increase in the wall thinning index compared with saline (P = 0.02), along with higher dilation and lower ejection fraction. The decline in cardiac physiology occurred subsequent to increased neutrophil and macrophage infiltration at MI day 1 and increased macrophage infiltration at D3. Our results reveal that S100A9 is a functional effector of infarct wall thinning.NEW & NOTEWORTHY S100A9 is a functional marker of infarct wall thinning.

Infarct in the Heart: What's MMP-9 Got to Do with It?

Over the past three decades, numerous studies have shown a strong connection between matrix metalloproteinase 9 (MMP-9) levels and myocardial infarction (MI) mortality and left ventricle remodeling and dysfunction. Despite this fact, clinical trials using MMP-9 inhibitors have been disappointing. This review focuses on the roles of MMP-9 in MI wound healing. Infiltrating leukocytes, cardiomyocytes, fibroblasts, and endothelial cells secrete MMP-9 during all phases of cardiac repair. MMP-9 both exacerbates the inflammatory response and aids in inflammation resolution by stimulating the pro-inflammatory to reparative cell transition. In addition, MMP-9 has a dual effect on neovascularization and prevents an overly stiff scar. Here, we review the complex role of MMP-9 in cardiac wound healing, and highlight the importance of targeting MMP-9 only for its detrimental actions. Therefore, delineating signaling pathways downstream of MMP-9 is critical.

Neutrophil crosstalk during cardiac wound healing after myocardial infarction.

Myocardial infarction (MI) initiates an intense inflammatory response that induces neutrophil infiltration into the infarct region. Neutrophils commence the pro-inflammatory response that includes upregulation of cytokines and chemokines (e.g., interleukin-1 beta) and degranulation of pre-formed proteases (e.g., matrix metalloproteinases -8 and -9) that degrade existing extracellular matrix to clear necrotic tissue. An increase or complete depletion of neutrophils both paradoxically impair MI resolution, indicating a complex role of neutrophils in cardiac wound healing. Following pro-inflammation, the neutrophil shifts to a reparative phenotype that promotes inflammation resolution and aids in scar formation. Across the shifts in phenotype, the neutrophil communicates with other cells to coordinate repair and scar formation. This review summarizes our current understanding of neutrophil crosstalk with cardiomyocytes and macrophages during MI wound healing.

Neutrophil signaling during myocardial infarction wound repair.

Neutrophils are key effector cells of the innate immune system, serving as a first line of defense in the response to injury and playing essential roles in the wound healing process. Following myocardial infarction (MI), neutrophils infiltrate into the infarct region to propagate inflammation and begin the initial phase of cardiac wound repair. Pro-inflammatory neutrophils release proteases to degrade extracellular matrix (ECM), a necessary step for the removal of necrotic myocytes as a prelude for scar formation. Neutrophils transition their phenotype over time to regulate MI inflammation resolution and stabilize scar formation. Neutrophils contribute to the evolution from inflammation to resolution and scar formation by serving anti-inflammatory and repair functions. As anti-inflammatory cells, neutrophils contribute ECM proteins during scar formation, in particular fibronectin, galectin-3, and vimentin. The diverse and polarizing functions that contribute to MI wound repair make this innate immune cell a viable target to improve MI outcomes. Thus, understanding the signaling involved in neutrophil physiology in the context of MI may help to identify novel therapeutic targets.

Exogenous IL-4 shuts off pro-inflammation in neutrophils while stimulating anti-inflammation in macrophages to induce neutrophil phagocytosis following myocardial infarction.

INTRODUCTION: Macrophages and neutrophils are primary leukocytes involved in the inflammatory response to myocardial infarction (MI). While interleukin (IL)-4 is an in vitro anti-inflammatory stimulus, the MI myocardium does not express a considerable amount of IL-4 but does express IL4 receptors. We hypothesized that continuous exogenous IL-4 infusion starting 24 h after MI would promote a polarization switch in inflammatory cells towards a reparative phenotype. METHODS: C57BL/6J male mice (3-6 months of age) were subcutaneously infused with either saline (n = 17) or IL-4 (20 ng/g/day; n = 17) beginning 24 h after MI and evaluated at MI day 3. RESULTS: Macrophages and neutrophils were isolated ex vivo from the infarct region and examined. Exogenous IL-4 decreased pro-inflammatory Ccl3, Il12a, Tnfa, and Tgfb1 in neutrophils and increased anti-inflammatory Arg1 and Ym1 in macrophages (all p < .05). Tissue clearance by IL-4 treated neutrophils was not different, while selective phagocytosis of neutrophils doubled in IL-4 treated macrophages (p < .05). Of 24,339 genes examined by RNA-sequencing, 2042 genes were differentially expressed in macrophages from IL-4 stimulated infarct (all FDR p < .05). Pdgfc gene expression was ranked first, increasing 3-fold in macrophages stimulated with IL-4 (p = 1 × 10-9). Importantly, changes in macrophage physiology and transcriptome occurred in the absence of global LV effects. Bone marrow derived monocytes stimulated with mouse recombinant PDGF-CC protein (10 µg/ml) or PDGF-CC blocking antibody (200 ng/ml) did not change Arg1 or Ym1 expression, indicating the in vivo effect of IL-4 to stimulate macrophage anti-inflammatory gene expression was independent of PDGF-CC. CONCLUSIONS: Our results indicate that exogenous IL-4 promotes inflammation resolution by turning off pro-inflammation in neutrophils while stimulating anti-inflammation in macrophages to mediate removal of apoptotic neutrophils.

Cardiac fibroblast activation during myocardial infarction wound healing: Fibroblast polarization after MI.

Cardiac wound healing after myocardial infarction (MI) evolves from pro-inflammatory to anti-inflammatory to reparative responses, and the cardiac fibroblast is a central player during the entire transition. The fibroblast mirrors changes seen in the left ventricle infarct by undergoing a continuum of polarization phenotypes that follow pro-inflammatory, anti-inflammatory, and pro-scar producing profiles. The development of each phenotype transition is contingent upon the MI environment into which the fibroblast enters. In this mini-review, we summarize our current knowledge regarding cardiac fibroblast activation during MI and highlight key areas where gaps remain.

Understanding the mechanisms that determine extracellular matrix remodeling in the infarcted myocardium.

Myocardial Infarction (MI) initiates a series of wound healing events that begins with up-regulation of an inflammatory response and culminates in scar formation. The extracellular matrix (ECM) is intricately involved in all stages from initial break down of existing ECM to synthesis of new ECM to form the scar. This review will summarize our current knowledge on the processes involved in ECM remodeling after MI and identify the gaps that still need to be filled.