EPAS1 expression contributes to maintenance of the primordial follicle pool in the mouse ovary.

Oxygen availability can have profound effects on cell fate decisions and survival, in part by regulating expression of hypoxia-inducible factors (HIFs). In the ovary, HIF expression has been characterised in granulosa cells, however, any requirement in oocytes remains relatively undefined. Here we developed a Hif2a/Epas1 germline-specific knockout mouse line in which females were fertile, however produced 40% fewer pups than controls. No defects in follicle development were detected, and quality of MII oocytes was normal, as per assessments of viability, intracellular reactive oxygen species, and spindle parameters. However, a significant diminishment of the primordial follicle pool was evident in cKO females that was attributed to accelerated follicle loss from postnatal day 6 onwards, potentially via disruption of the autophagy pathway. These data demonstrate the importance of HIF signalling in oocytes, particularly at the primordial follicle stage, and lend to the importance of controlling oxygen tension in the development of in vitro growth and maturation approaches for assisted reproduction.

The hypoxia-inducible factor EPAS1 is required for spermatogonial stem cell function in regenerative conditions.

In this study we explored the role of hypoxia and the hypoxia-inducible transcription factor EPAS1 in regulating spermatogonial stem cell (SSC) function in the mouse testis. We have demonstrated that SSCs reside in hypoxic microenvironments in the testis through utilization of the oxygen-sensing probe pimonidazole, and by confirming the stable presence of EPAS1, which is degraded at >5% O2. Through the generation of a germline-specific Epas1 knockout mouse line, and through modulation of EPAS1 levels in primary cultures of spermatogonia with the small drug molecule Daprodustat, we have demonstrated that EPAS1 is required for robust SSC function in regenerative conditions (post-transplantation and post-chemotherapy), via the regulation of key cellular processes such as metabolism. These findings shed light on the relationship between hypoxia and male fertility and will potentially facilitate optimization of in vitro culture conditions for infertility treatment pipelines using SSCs, such as those directed at pediatric cancer survivors.

Fibulin-3 is necessary to prevent cardiac rupture following myocardial infarction.

Despite the high prevalence of heart failure in the western world, there are few effective treatments. Fibulin-3 is a protein involved in extracellular matrix (ECM) structural integrity, however its role in the heart is unknown. We have demonstrated, using single cell RNA-seq, that fibulin-3 was highly expressed in quiescent murine cardiac fibroblasts, with expression highest prior to injury and late post-infarct (from ~ day-28 to week-8). In humans, fibulin-3 was upregulated in left ventricular tissue and plasma of heart failure patients. Fibulin-3 knockout (Efemp1-/-) and wildtype mice were subjected to experimental myocardial infarction. Fibulin-3 deletion resulted in significantly higher rate of cardiac rupture days 3-6 post-infarct, indicating a weak and poorly formed scar, with severe ventricular remodelling in surviving mice at day-28 post-infarct. Fibulin-3 knockout mice demonstrated less collagen deposition at day-3 post-infarct, with abnormal collagen fibre-alignment. RNA-seq on day-3 infarct tissue revealed upregulation of ECM degradation and inflammatory genes, but downregulation of ECM assembly/structure/organisation genes in fibulin-3 knockout mice. GSEA pathway analysis showed enrichment of inflammatory pathways and a depletion of ECM organisation pathways. Fibulin-3 originates from cardiac fibroblasts, is upregulated in human heart failure, and is necessary for correct ECM organisation/structural integrity of fibrotic tissue to prevent cardiac rupture post-infarct.

Inflammatory cell response following ST-elevation myocardial infarction treated with primary percutaneous coronary intervention and its impact on cardiovascular outcomes: A systematic review and meta-analysis.

BACKGROUND: Inflammatory responses post STEMI may mediate major adverse cardiovascular events (MACE). This is the first systematic review to map leukocyte response following a STEMI and its association with outcomes. METHODS: We systematically searched EMBASE and Medline for studies of STEMIs undergoing primary PCI. Eligible studies reported leukocytes or its subtype plus either 30-day and/or 1-year MACE. Random effects model for pooled proportions was used to estimate 30-day and 1-year mortality and MACE. Meta-regression was used to estimate the effect of leukocyte counts on cardiovascular outcomes. Publication bias was assessed using Egger's regression-based test. The review was registered with PROSPERO (CRD42019124991). RESULTS: Of the 3,813 studies meeting the preliminary search criteria, 24 cohort studies were eligible for inclusion, representing 19,074 persons [76.4% male (n = 14,539); mean age 61.6 years]. Leukocytes had a mean of 10.5x109 (SD 4.7) on admission and 11.1x109 (SD 3.3) at day one post STEMI. Neutrophils increased day one post STEMI, while lymphocytes decreased. There was limited data on other leukocyte subtypes and beyond day one. Estimated 30-day and 1-year all-cause mortality were 6.5% (95% CI 4.8-8.2, p <0.001) and 9.7% (95% CI 5.6-13.8, p <0.001), while the estimated 30-day and 1-year MACE were 14.9% (95% CI 5.3-24.4, p < 0.001) and 15.2% (95% CI 7.2-23.2, p < 0.001). The meta-analysis was limited by a high degree of heterogeneity between studies. CONCLUSIONS: This review highlights the urgent need to better characterise inflammation post STEMI to identify mediators for the persistently high mortality and morbidity associated with STEMI.

Extracellular Matrix Protein-1 as a Mediator of Inflammation-Induced Fibrosis After Myocardial Infarction.

Irreversible fibrosis is a hallmark of myocardial infarction (MI) and heart failure. Extracellular matrix protein-1 (ECM-1) is up-regulated in these hearts, localized to fibrotic, inflammatory, and perivascular areas. ECM-1 originates predominantly from fibroblasts, macrophages, and pericytes/vascular cells in uninjured human and mouse hearts, and from M1 and M2 macrophages and myofibroblasts after MI. ECM-1 stimulates fibroblast-to-myofibroblast transition, up-regulates key fibrotic and inflammatory pathways, and inhibits cardiac fibroblast migration. ECM-1 binds HuCFb cell surface receptor LRP1, and LRP1 inhibition blocks ECM-1 from stimulating fibroblast-to-myofibroblast transition, confirming a novel ECM-1-LRP1 fibrotic signaling axis. ECM-1 may represent a novel mechanism facilitating inflammation-fibrosis crosstalk.

ß-Adrenergic Receptor Stimulation Maintains NCX-CaMKII Axis and Prevents Overactivation of IL6R-Signaling in Cardiomyocytes upon Increased Workload.

Excessive ß-adrenergic stimulation and tachycardia are potent triggers of cardiac remodeling; however, their exact cellular effects remain elusive. Here, we sought to determine the potency of ß-adrenergic stimulation and tachycardia to modulate gene expression profiles of cardiomyocytes. Using neonatal rat ventricular cardiomyocytes, we showed that tachycardia caused a significant upregulation of sodium-calcium exchanger (NCX) and the activation of calcium/calmodulin-dependent kinase II (CaMKII) in the nuclear region. Acute isoprenaline treatment ameliorated NCX-upregulation and potentiated CaMKII activity, specifically on the sarcoplasmic reticulum and the nuclear envelope, while preincubation with the ß-blocker propranolol abolished both isoprenaline-mediated effects. On a transcriptional level, screening for hypertrophy-related genes revealed tachycardia-induced upregulation of interleukin-6 receptor (IL6R). While isoprenaline prevented this effect, pharmacological intervention with propranolol or NCX inhibitor ORM-10962 demonstrated that simultaneous CaMKII activation on the subcellular Ca2+ stores and prevention of NCX upregulation are needed for keeping IL6R activation low. Finally, using hypertensive Dahl salt-sensitive rats, we showed that blunted ß-adrenergic signaling is associated with NCX upregulation and enhanced IL6R signaling. We therefore propose a previously unrecognized protective role of ß-adrenergic signaling, which is compromised in cardiac pathologies, in preventing IL6R overactivation under increased workload. A better understanding of these processes may contribute to refinement of therapeutic options for patients receiving ß-blockers.

Paracrine Factors Released by Stem Cells of Mesenchymal Origin and their Effects in Cardiovascular Disease: A Systematic Review of Pre-clinical Studies.

Mesenchymal stem cell (MSC) therapy has gained significant traction in the context of cardiovascular repair, and have been proposed to exert their regenerative effects via the secretion of paracrine factors. In this systematic review, we examined the literature and consolidated available evidence for the "paracrine hypothesis". Two Ovid SP databases were searched using a strategy encompassing paracrine mediated MSC therapy in the context of ischemic heart disease. This yielded 86 articles which met the selection criteria for inclusion in this study. We found that the MSCs utilized in these articles were primarily derived from bone marrow, cardiac tissue, and adipose tissue. We identified 234 individual protective factors across these studies, including VEGF, HGF, and FGF2; which are proposed to exert their effects in a paracrine manner. The data collated in this systematic review identifies secreted paracrine factors that could decrease apoptosis, and increase angiogenesis, cell proliferation, and cell viability. These included studies have also demonstrated that the administration of MSCs and indirectly, their secreted factors can reduce infarct size, and improve left ventricular ejection fraction, contractility, compliance, and vessel density. Furthering our understanding of the way these factors mediate repair could lead to the identification of therapeutic targets for cardiac regeneration.

Small molecule STING inhibition improves myocardial infarction remodeling.

AIMS: Myocardial infarction (MI) is a major global cause of death. Massive cell death leads to inflammation, which is necessary for ensuing wound healing. Extensive inflammation, however, promotes infarct expansion and adverse remodeling. The DNA sensing receptor cyclic GMP-AMP synthase and its downstream signaling effector stimulator of interferon genes (cGAS-STING) is central in innate immune reactions in infections or autoimmunity. Cytosolic double-strand DNA activates the pathway and down-stream inflammatory responses. Recent papers demonstrated that this pathway is also active following MI and that its genetic targeting improves outcome. Thus, we investigated if pharmacologic pathway inhibition is protective after MI in order to test its translational potential. MAIN METHODS: We investigated novel and selective small-molecule STING inhibitors that inhibit STING palmitoylation and multimerization and thereby downstream pathway activation in a preclinical murine MI model. We assessed structural and functional cardiac remodeling, infarct expansion and fibrosis, as well as cardiomyocyte hypertrophy and the expression of inflammatory genes. KEY FINDINGS: Pharmacologic STING inhibition did not reduce mortality due to myocardial rupture in non-reperfused MI. Infarct size at day one was comparable. However, three weeks of pharmacologic STING inhibition after reperfused MI decreased infarct expansion and scarring, increased left ventricular systolic function to levels approaching normal values, and reduced myocardial hypertrophy. SIGNIFICANCE: Selective small-molecule STING inhibition after myocardial infarction has the potential to improve wound healing responses and pathological remodeling and thereby attenuate the development of ischemic heart failure.

The Role of Pathological Aging in Cardiac and Pulmonary Fibrosis.

Aging promotes a range of degenerative pathologies characterized by progressive losses of tissue and/or cellular function. Fibrosis is the hardening, overgrowth and scarring of various tissues characterized by the accumulation of extracellular matrix components. Aging is an important predisposing factor common for fibrotic heart and respiratory disease. Age-related processes such as senescence, inflammaging, autophagy and mitochondrial dysfunction are interconnected biological processes that diminish the regenerative capacity of the aged heart and lung and have been shown to play a crucial role in cardiac fibrosis and idiopathic pulmonary fibrosis. This review focuses on these four processes of aging in relation to their role in fibrosis. It has long been established that the heart and lung are linked both functionally and anatomically when it comes to health and disease, with an ever-expanding aging population, the incidence of fibrotic disease and therefore the number of fibrosis-related deaths will continue to rise. There are currently no feasible therapies to treat the effects of chronic fibrosis therefore highlighting the importance of exploring the processes of aging and its role in inducing and exacerbating fibrosis of each organ. The focus of this review may help to highlight potential avenues of therapeutic exploration.

Novel role of extracellular matrix protein 1 (ECM1) in cardiac aging and myocardial infarction.

INTRODUCTION: The prevalence of heart failure increases in the aging population and following myocardial infarction (MI), yet the extracellular matrix (ECM) remodeling underpinning the development of aging- and MI-associated cardiac fibrosis remains poorly understood. A link between inflammation and fibrosis in the heart has long been appreciated, but has mechanistically remained undefined. We investigated the expression of a novel protein, extracellular matrix protein 1 (ECM1) in the aging and infarcted heart. METHODS: Young adult (3-month old) and aging (18-month old) C57BL/6 mice were assessed. Young mice were subjected to left anterior descending artery-ligation to induce MI, or transverse aortic constriction (TAC) surgery to induce pressure-overload cardiomyopathy. Left ventricle (LV) tissue was collected early and late post-MI/TAC. Bone marrow cells (BMCs) were isolated from young healthy mice, and subject to flow cytometry. Human cardiac fibroblast (CFb), myocyte, and coronary artery endothelial & smooth muscle cell lines were cultured; human CFbs were treated with recombinant ECM1. Primary mouse CFbs were cultured and treated with recombinant angiotensin-II or TGF-ß1. Immunoblotting, qPCR and mRNA fluorescent in-situ hybridization (mRNA-FISH) were conducted on LV tissue and cells. RESULTS: ECM1 expression was upregulated in the aging LV, and in the infarct zone of the LV early post-MI. No significant differences in ECM1 expression were found late post-MI or at any time-point post-TAC. ECM1 was not expressed in any resident cardiac cells, but ECM1 was highly expressed in BMCs, with high ECM1 expression in granulocytes. Flow cytometry of bone marrow revealed ECM1 expression in large granular leucocytes. mRNA-FISH revealed that ECM1 was indeed expressed by inflammatory cells in the infarct zone at day-3 post-MI. ECM1 stimulation of CFbs induced ERK1/2 and AKT activation and collagen-I expression, suggesting a pro-fibrotic role. CONCLUSIONS: ECM1 expression is increased in ageing and infarcted hearts but is not expressed by resident cardiac cells. Instead it is expressed by bone marrow-derived granulocytes. ECM1 is sufficient to induce cardiac fibroblast stimulation in vitro. Our findings suggest ECM1 is released from infiltrating inflammatory cells, which leads to cardiac fibroblast stimulation and fibrosis in aging and MI. ECM1 may be a novel intermediary between inflammation and fibrosis.

Chronic stress induced disturbances in Laminin: A significant contributor to modulating microglial pro-inflammatory tone?

Over the last decade, evidence supporting a link between microglia enhanced neuro-inflammatory signalling and mood disturbance has continued to build. One issue that has not been well addressed yet are the factors that drive microglia to enter into a higher pro-inflammatory state. The current study addressed the potential role of the extracellular matrix protein Laminin. C57BL6 adult mice were either exposed to chronic stress or handled for 6 consecutive weeks. Changes in Laminin, microglial morphology and pro-inflammatory cytokine expression were examined in tissue obtained from mice exposed to a chronic restraint stress procedure. These in vivo investigations were complemented by an extensive set of in vitro experiments utilising both a primary microglia and BV2 cell line to examine how Laminin influenced microglial pro-inflammatory tone. Chronic stress enhanced the expression of Laminin, microglial de-ramification and pro-inflammatory cytokine signalling. We further identified that microglia when cultured in the presence of Laminin produced and released significantly greater levels of pro-inflammatory cytokines; took longer to return to baseline following stimulation and exhibited enhanced phagocytic activity. These results suggest that chronic restraint stress is capable of modulating Laminin within the CNS, an effect that has implications for understanding environmental mediated disturbances of microglial function.

The Processes and Mechanisms of Cardiac and Pulmonary Fibrosis.

Fibrosis is the formation of fibrous connective tissue in response to injury. It is characterized by the accumulation of extracellular matrix components, particularly collagen, at the site of injury. Fibrosis is an adaptive response that is a vital component of wound healing and tissue repair. However, its continued activation is highly detrimental and a common final pathway of numerous disease states including cardiovascular and respiratory disease. Worldwide, fibrotic diseases cause over 800,000 deaths per year, accounting for ~45% of total deaths. With an aging population, the incidence of fibrotic disease and subsequently the number of fibrosis-related deaths will rise further. Although, fibrosis is a well-recognized cause of morbidity and mortality in a range of disease states, there are currently no viable therapies to reverse the effects of chronic fibrosis. Numerous predisposing factors contribute to the development of fibrosis. Biological aging in particular, interferes with repair of damaged tissue, accelerating the transition to pathological remodeling, rather than a process of resolution and regeneration. When fibrosis progresses in an uncontrolled manner, it results in the irreversible stiffening of the affected tissue, which can lead to organ malfunction and death. Further investigation into the mechanisms of fibrosis is necessary to elucidate novel, much needed, therapeutic targets. Fibrosis of the heart and lung make up a significant proportion of fibrosis-related deaths. It has long been established that the heart and lung are functionally and geographically linked when it comes to health and disease, and thus exploring the processes and mechanisms that contribute to fibrosis of each organ, the focus of this review, may help to highlight potential avenues of therapeutic investigation.