Thomas Moore-Morris

Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis

Myofibroblasts produce the fibrous scar in hepatic fibrosis. In the carbon tetrachloride (CCl4) model of liver fibrosis, quiescent hepatic stellate cells (HSC) are activated to become myofibroblasts. When the underlying etiological agent is removed, clinical and experimental fibrosis undergoes a remarkable regression with complete disappearance of these myofibroblasts. Although some myofibroblasts apoptose, it is unknown whether other myofibroblasts may revert to an inactive phenotype during regression of fibrosis. We elucidated the fate of HSCs/myofibroblasts during recovery from CCl4- and alcohol-induced liver fibrosis using Cre-LoxP–based genetic labeling of myofibroblasts. Here we demonstrate that half of the myofibroblasts escape apoptosis during regression of liver fibrosis, down-regulate fibrogenic genes, and acquire a phenotype similar to, but distinct from, quiescent HSCs in their ability to more rapidly reactivate into myofibroblasts in response to fibrogenic stimuli and strongly contribute to liver fibrosis. Inactivation of HSCs was associated with up-regulation of the anti-apoptotic genes Hspa1a/b, which participate in the survival of HSCs in culture and in vivo.

Resident fibroblast lineages mediate pressure overload–induced cardiac fibrosis

Activation and accumulation of cardiac fibroblasts, which result in excessive extracellular matrix deposition and consequent mechanical stiffness, myocyte uncoupling, and ischemia, are key contributors to heart failure progression. Recently, endothelial-to-mesenchymal transition (EndoMT) and the recruitment of circulating hematopoietic progenitors to the heart have been reported to generate substantial numbers of cardiac fibroblasts in response to pressure overload-induced injury; therefore, these processes are widely considered to be promising therapeutic targets. Here, using multiple independent murine Cre lines and a collagen1a1-GFP fusion reporter, which specifically labels fibroblasts, we found that following pressure overload, fibroblasts were not derived from hematopoietic cells, EndoMT, or epicardial epithelial-to-mesenchymal transition. Instead, pressure overload promoted comparable proliferation and activation of two resident fibroblast lineages, including a previously described epicardial population and a population of endothelial origin. Together, these data present a paradigm for the origins of cardiac fibroblasts during development and in fibrosis. Furthermore, these data indicate that therapeutic strategies for reducing pathogenic cardiac fibroblasts should shift from targeting presumptive EndoMT or infiltrating hematopoietically derived fibroblasts, toward common pathways upregulated in two endogenous fibroblast populations.

Origin of myofibroblasts in the fibrotic liver in mice

Significance Liver resident activated hepatic stellate cells (aHSCs), and activated portal fibroblasts (aPFs) are the major source of the fibrous scar in the liver. aPFs have been implicated in liver fibrosis caused by cholestatic liver injury, whereas fibrosis in hepatotoxic liver injury is attributed to aHSCs. However, the contribution of aPFs to cholestatic fibrosis is not well characterized because of difficulties in cell purification and the lack of identified aPF-specific markers. We have developed a novel flow cytometry-based method of aPFs purification from the nonparenchymal cell fraction of collagen-α1(I)-GFP mice and have identified potential aPF-specific markers. The goal of this study is to determine whether aPFs contribute to cholestatic liver fibrosis and identify the mechanism(s) of their activation. Hepatic myofibroblasts are activated in response to chronic liver injury of any etiology to produce a fibrous scar. Despite extensive studies, the origin of myofibroblasts in different types of fibrotic liver diseases is unresolved. To identify distinct populations of myofibroblasts and quantify their contribution to hepatic fibrosis of two different etiologies, collagen-α1(I)-GFP mice were subjected to hepatotoxic (carbon tetrachloride; CCl4) or cholestatic (bile duct ligation; BDL) liver injury. All myofibroblasts were purified by flow cytometry of GFP+ cells and then different subsets identified by phenotyping. Liver resident activated hepatic stellate cells (aHSCs) and activated portal fibroblasts (aPFs) are the major source (>95%) of fibrogenic myofibroblasts in these models of liver fibrosis in mice. As previously reported using other methodologies, hepatic stellate cells (HSCs) are the major source of myofibroblasts (>87%) in CCl4 liver injury. However, aPFs are a major source of myofibroblasts in cholestatic liver injury, contributing >70% of myofibroblasts at the onset of injury (5 d BDL). The relative contribution of aPFs decreases with progressive injury, as HSCs become activated and contribute to the myofibroblast population (14 and 20 d BDL). Unlike aHSCs, aPFs respond to stimulation with taurocholic acid and IL-25 by induction of collagen-α1(I) and IL-13, respectively. Furthermore, BDL-activated PFs express high levels of collagen type I and provide stimulatory signals to HSCs. Gene expression analysis identified several novel markers of aPFs, including a mesothelial-specific marker mesothelin. PFs may play a critical role in the pathogenesis of cholestatic liver fibrosis and, therefore, serve as an attractive target for antifibrotic therapy.

Thymosin Beta 4 Is Dispensable for Murine Cardiac Development and Function

Rationale: Thymosin beta 4 (T&bgr;4) is a 43–amino acid factor encoded by an X-linked gene. Recent studies have suggested that T&bgr;4 is a key factor in cardiac development, growth, disease, epicardial integrity, and blood vessel formation. Cardiac-specific short hairpin (sh)RNA knockdown of t&bgr;4 has been reported to result in embryonic lethality at E14.5–16.5, with severe cardiac and angiogenic defects. However, this shRNA t&bgr;4-knockdown model did not completely abrogate T&bgr;4 expression. To completely ablate T&bgr;4 and to rule out the possibility of off-target effects associated with shRNA gene silencing, further studies of global or cardiac-specific knockouts are critical. Objective: We examined the role of T&bgr;4 in developing and adult heart through global and cardiac specific t&bgr;4-knockout mouse models. Methods and Results: Global t&bgr;4-knockout mice were born at mendelian ratios and exhibited normal heart and blood vessel formation. Furthermore, in adult global t&bgr;4-knockout mice, cardiac function, capillary density, expression of key cardiac fetal and angiogenic genes, epicardial marker expression, and extracellular matrix deposition were indistinguishable from that of controls. Tissue-specific t&bgr;4-deficient mice, generated by crossing t&bgr;4-floxed mice to Nkx2.5-Cre and &agr;MHC-Cre, were also found to have no phenotype. Conclusions: We conclude that T&bgr;4 is dispensable for embryonic viability, heart development, coronary vessel development, and adult myocardial function.

Targeted ablation of nesprin 1 and nesprin 2 from murine myocardium results in cardiomyopathy, altered nuclear morphology and inhibition of the biomechanical gene response.

Recent interest has focused on the importance of the nucleus and associated nucleoskeleton in regulating changes in cardiac gene expression in response to biomechanical load. Mutations in genes encoding proteins of the inner nuclear membrane and nucleoskeleton, which cause cardiomyopathy, also disrupt expression of a biomechanically responsive gene program. Furthermore, mutations in the outer nuclear membrane protein Nesprin 1 and 2 have been implicated in cardiomyopathy. Here, we identify for the first time a role for the outer nuclear membrane proteins, Nesprin 1 and Nesprin 2, in regulating gene expression in response to biomechanical load. Ablation of both Nesprin 1 and 2 in cardiomyocytes, but neither alone, resulted in early onset cardiomyopathy. Mutant cardiomyocytes exhibited altered nuclear positioning, shape, and chromatin positioning. Loss of Nesprin 1 or 2, or both, led to impairment of gene expression changes in response to biomechanical stimuli. These data suggest a model whereby biomechanical signals are communicated from proteins of the outer nuclear membrane, to the inner nuclear membrane and nucleoskeleton, to result in changes in gene expression required for adaptation of the cardiomyocyte to changes in biomechanical load, and give insights into etiologies underlying cardiomyopathy consequent to mutations in Nesprin 1 and 2.

Origins of cardiac fibroblasts

Cardiac fibroblasts produce the extracellular matrix (ECM) scaffold within which the various cellular components of the heart are organized. As well as providing structural support, it is becoming evident that the quality and quantity of ECM is a key factor for determining cardiac cell behavior during development and in pathological contexts such as heart failure involving fibrosis. Cardiac fibroblasts have long remained a poorly characterized cardiac lineage. Well characterized markers are now paving the way for a better understanding of the roles of these cells in various developmental and disease contexts. Notably, the relevance of processes including endothelial-tomesenchymal transition and the recruitment of circulating fibroblast progenitors in heart failure has been challenged. This review describes the latest findings on cardiac fibroblast markers and developmental origins, and discusses their importance in myocardial remodeling. Effective modulation of cardiac fibroblast activity would likely contribute to successful treatment of various cardiac disorders.

Isolation and culture of neonatal mouse cardiomyocytes.

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes. The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Cardiac fibroblasts: from development to heart failure

Cardiac fibroblasts are a major cell population of the heart and are characterized by their capacity to produce extracellular matrix (ECM). In hearts subjected to pressure overload, excessive fibroblast accumulation is responsible for fibrosis of the myocardium, a major clinical issue. Hence, understanding mechanisms generating fibroblasts in this context has become a key question in the cardiovascular field. Recent studies now point to the activation of resident fibroblasts as the underlying cause of fibrosis. However, de novo generation of fibroblasts from endothelium and circulating hematopoietic cells has also been proposed to significantly contribute to fibrosis. Here, we discuss the latest findings on fibroblast origins, with a particular emphasis on the pressure overload model, and the implication of these findings for the development of anti-fibrotic therapies that are currently lacking.

Atrial natriuretic peptide regulates adipose tissue accumulation in adult atria

Significance Atrial fibrillation is the most frequent cardiac arrhythmia and is a major cause of stroke. Recently, it has been shown that the adipose tissue that accumulates at the surface of the heart contributes to the pathogenesis of atrial fibrillation by favoring fibrosis of the neighboring myocardium. However, the cellular origin of adult cardiac fat tissue is unknown. Here, we show that resident progenitor cells of the external layer of the heart, referred to as the “epicardium,” are a source of adipocytes through an epithelial-to-mesenchymal transition process. The atrial natriuretic peptide, which is secreted by atrial myocytes, is a potent factor in the differentiation of epicardial progenitors in adipocytes. Our data uncover cross-talk between myocardial mechanical properties and adipose tissue expansion. The abundance of epicardial adipose tissue (EAT) is associated with atrial fibrillation (AF), the most frequent cardiac arrhythmia. However, both the origin and the factors involved in EAT expansion are unknown. Here, we found that adult human atrial epicardial cells were highly adipogenic through an epithelial–mesenchymal transition both in vitro and in vivo. In a genetic lineage tracing the WT1CreERT2+/−RosatdT+/− mouse model subjected to a high-fat diet, adipocytes of atrial EAT derived from a subset of epicardial progenitors. Atrial myocardium secretome induces the adipogenic differentiation of adult mesenchymal epicardium-derived cells by modulating the balance between mesenchymal Wingless-type Mouse Mammary Tumor Virus integration site family, member 10B (Wnt10b)/β-catenin and adipogenic ERK/MAPK signaling pathways. The adipogenic property of the atrial secretome was enhanced in AF patients. The atrial natriuretic peptide secreted by atrial myocytes is a major adipogenic factor operating at a low concentration by binding to its natriuretic peptide receptor A (NPRA) receptor and, in turn, by activating a cGMP-dependent pathway. Hence, our data indicate cross-talk between EAT expansion and mechanical function of the atrial myocardium.

Concise Review: Pluripotent Stem Cell-Derived Cardiac Cells, A Promising Cell Source for Therapy of Heart Failure: Where Do We Stand?

Heart failure is still a major cause of hospitalization and mortality in developed countries. Many clinical trials have tested the use of multipotent stem cells as a cardiac regenerative medicine. The benefit for the patients of this therapeutic intervention has remained limited. Herein, we review the pluripotent stem cells as a cell source for cardiac regeneration. We more specifically address the various challenges of this cell therapy approach. We question the cell delivery systems, the immune tolerance of allogenic cells, the potential proarrhythmic effects, various drug mediated interventions to facilitate cell grafting and, finally, we describe the pathological conditions that may benefit from such an innovative approach. As members of a transatlantic consortium of excellence of basic science researchers and clinicians, we propose some guidelines to be applied to cell types and modes of delivery in order to translate pluripotent stem cell cardiac derivatives into safe and effective clinical trials. Stem Cells 2016;34:34–43