Toll-like receptors ligand immunomodulators for the treatment congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a rare disease that affects the development of the diaphragm, leading to abnormal lung development. Unfortunately, there is no established therapy for CDH. Retinoic acid pathways are implicated in the ethology of CDH and macrophages are known to play a role in repairing organ damage. METHODS: We have analyzed the effect of several Toll like receptor (TLR) ligands in the nitrofen-induced CDH model in pregnant rats widely used to study this disease and in the G2-GATA4Cre;Wt1fl/fl CDH genetic mice model. Morphometric and histological studies were carried out. Immune cell infiltration was assayed by immunochemistry and immunofluorescence and retinoic pathway gene expression analyzed in vivo and in vitro in macrophages. RESULTS: We found that administering a single dose of atypical TLR2/4 ligands (CS1 or CS2), 3 days after nitrofen, cured diaphragmatic hernia in 73% of the fetuses and repaired the lesion with complete diaphragm closure being on the other hand nontoxic for the mothers or pups. Moreover, these immunomodulators also improved pulmonary hypoplasia and alveolar maturation and vessel hypertrophy, enhancing pulmonary maturity of fetuses. We also found that CS1 treatment rescued the CDH phenotype in the G2-GATA4Cre;Wt1fl/fl CDH genetic mice model. Only 1 out of 11 mutant embryos showed CDH after CS1 administration, whereas CDH prevalence was 70% in untreated mutant embryos. Mechanistically, CS1 stimulated the infiltration of repairing M2 macrophages (CD206+ and Arg1+) into the damaged diaphragm and reduced T cell infiltration. Additionally, those TLR ligands induced retinol pathway genes, including RBP1, RALDH2, RARα, and RARß, in the affected lungs and the diaphragm and in macrophages in vitro. CONCLUSIONS: Our research has shown that TLR ligand immunomodulators that influence anti-inflammatory macrophage activation can be effective in treating CDH, being nontoxic for the mothers or pups suggesting that those TLR ligands are a promising solution for CDH leading to orphan drug designation for CS1. The immune system of the fetus would be responsible for repairing the damage and closure of the hernia in the diaphragm and enhanced proper lung development after CS1 treatment.

Correction: Díaz del Moral et al. Cardiomyocyte-Specific Wt1 Is Involved in Cardiac Metabolism and Response to Damage. J. Cardiovasc. Dev. Dis. 2023, 10, 211.

In the published publication [...].

Novel Insights into the Molecular Mechanisms Governing Embryonic Epicardium Formation.

The embryonic epicardium originates from the proepicardium, an extracardiac primordium constituted by a cluster of mesothelial cells. In early embryos, the embryonic epicardium is characterized by a squamous cell epithelium resting on the myocardium surface. Subsequently, it invades the subepicardial space and thereafter the embryonic myocardium by means of an epithelial-mesenchymal transition. Within the myocardium, epicardial-derived cells present multilineage potential, later differentiating into smooth muscle cells and contributing both to coronary vasculature and cardiac fibroblasts in the mature heart. Over the last decades, we have progressively increased our understanding of those cellular and molecular mechanisms driving proepicardial/embryonic epicardium formation. This study provides a state-of-the-art review of the transcriptional and emerging post-transcriptional mechanisms involved in the formation and differentiation of the embryonic epicardium.

Cardiomyocyte-Specific Wt1 Is Involved in Cardiac Metabolism and Response to Damage.

The Wilms tumor suppressor gene (Wt1) encodes a C2H2-type zinc-finger transcription factor that participates in transcriptional regulation, RNA metabolism, and protein-protein interactions. WT1 is involved in the development of several organs, including the kidneys and gonads, heart, spleen, adrenal glands, liver, diaphragm, and neuronal system. We previously provided evidence of transient WT1 expression in about 25% of cardiomyocytes of mouse embryos. Conditional deletion of Wt1 in the cardiac troponin T lineage caused abnormal cardiac development. A low expression of WT1 has also been reported in adult cardiomyocytes. Therefore, we aimed to explore its function in cardiac homeostasis and in the response to pharmacologically induced damage. Silencing of Wt1 in cultured neonatal murine cardiomyocytes provoked alterations in mitochondrial membrane potential and changes in the expression of genes related to calcium homeostasis. Ablation of WT1 in adult cardiomyocytes by crossing αMHCMerCreMer mice with homozygous WT1-floxed mice induced hypertrophy, interstitial fibrosis, altered metabolism, and mitochondrial dysfunction. In addition, conditional deletion of WT1 in adult cardiomyocytes increased doxorubicin-induced damage. These findings suggest a novel role of WT1 in myocardial physiology and protection against damage.

GATA4 induces liver fibrosis regression by deactivating hepatic stellate cells.

In response to liver injury, hepatic stellate cells activate and acquire proliferative and contractile features. The regression of liver fibrosis appears to involve the clearance of activated hepatic stellate cells, either by apoptosis or by reversion toward a quiescent-like state, a process called deactivation. Thus, deactivation of active hepatic stellate cells has emerged as a novel and promising therapeutic approach for liver fibrosis. However, our knowledge of the master regulators involved in the deactivation and/or activation of fibrotic hepatic stellate cells is still limited. The transcription factor GATA4 has been previously shown to play an important role in embryonic hepatic stellate cell quiescence. In this work, we show that lack of GATA4 in adult mice caused hepatic stellate cell activation and, consequently, liver fibrosis. During regression of liver fibrosis, Gata4 was reexpressed in deactivated hepatic stellate cells. Overexpression of Gata4 in hepatic stellate cells promoted liver fibrosis regression in CCl4-treated mice. GATA4 induced changes in the expression of fibrogenic and antifibrogenic genes, promoting hepatic stellate cell deactivation. Finally, we show that GATA4 directly repressed EPAS1 transcription in hepatic stellate cells and that stabilization of the HIF2α protein in hepatic stellate cells leads to liver fibrosis.

Deletion of the Wilms' Tumor Suppressor Gene in the Cardiac Troponin-T Lineage Reveals Novel Functions of WT1 in Heart Development.

Expression of Wilms' tumor suppressor transcription factor (WT1) in the embryonic epicardium is essential for cardiac development, but its myocardial expression is little known. We have found that WT1 is expressed at low levels in 20-25% of the embryonic cardiomyocytes. Conditional ablation of WT1 using a cardiac troponin T driver (Tnnt2 Cre ) caused abnormal sinus venosus and atrium development, lack of pectinate muscles, thin ventricular myocardium and, in some cases, interventricular septum and cardiac wall defects, ventricular diverticula and aneurisms. Coronary development was normal and there was not embryonic lethality, although survival of adult mutant mice was reduced probably due to perinatal mortality. Adult mutant mice showed electrocardiographic anomalies, including increased RR and QRS intervals, and decreased PR intervals. RNASeq analysis identified differential expression of 137 genes in the E13.5 mutant heart as compared to controls. GO functional enrichment analysis suggested that both calcium ion regulation and modulation of potassium channels are deeply altered in the mutant myocardium. In summary, together with its essential function in the embryonic epicardium, myocardial WT1 expression is also required for normal cardiac development.

The Insulin-like Growth Factor Signalling Pathway in Cardiac Development and Regeneration.

Insulin and Insulin-like growth factors (IGFs) perform key roles during embryonic development, regulating processes of cell proliferation and survival. The IGF signalling pathway comprises two IGFs (IGF1, IGF2), two IGF receptors (IGFR1, IGFR2), and six IGF binding proteins (IGFBPs) that regulate IGF transport and availability. The IGF signalling pathway is essential for cardiac development. IGF2 is the primary mitogen inducing ventricular cardiomyocyte proliferation and morphogenesis of the compact myocardial wall. Conditional deletion of the Igf1r and the insulin receptor (Insr) genes in the myocardium results in decreased cardiomyocyte proliferation and ventricular wall hypoplasia. The significance of the IGF signalling pathway during embryonic development has led to consider it as a candidate for adult cardiac repair and regeneration. In fact, paracrine IGF2 plays a key role in the transient regenerative ability of the newborn mouse heart. We aimed to review the current knowledge about the role played by the IGF signalling pathway during cardiac development and also the clinical potential of recapitulating this developmental axis in regeneration of the adult heart.

Embryonic circulating endothelial progenitor cells.

The development of vascular system in vertebrates has been traditionally explained by early vasculogenic assembly of angioblasts followed by angiogenic outgrowth of pre-existing vessels. The discovery of adult endothelial progenitor cells (Asahara et al. in Science 275(5302):964-967, 1997) challenged this view, since postnatal vascular growth could be accomplished by recruitment of circulating cells with the ability to differentiate into endothelial cells. However, the existence of embryonic circulating endothelial progenitor cells and their actual contribution to vascular development is far less known. We review in this paper the literature concerning the features, origin and physiological functions of embryonic and foetal circulating endothelial progenitors. Our review includes the early (E7.5) progenitors isolated from yolk sac, the hematovascular progenitors identified in the foetal liver, the yolk sac-derived erythro-myeloid progenitors, circulating hematopoietic cells from the G2-GATA4 lineage and the endothelial colony-forming cells isolated from the placenta and umbilical cord blood. We highlight the need of further characterization of these populations and the relationships between them.

Contribution of a GATA4-Expressing Hematopoietic Progenitor Lineage to the Adult Mouse Endothelium.

Different sources have been claimed for the embryonic origin of the coronary endothelium. Recently, the potential of circulating cells as progenitors of the cardiac endothelium has also been suggested. In a previous study we have shown that circulating progenitors are recruited by the embryonic endocardium and incorporated into the coronary vessels. These progenitors derive from a mesodermal lineage characterized by the expression of Gata4 under control of the enhancer G2. Herein, we aim to trace this specific lineage throughout postnatal stages. We have found that more than 50% of the adult cardiac endothelium derives from the G2-GATA4 lineage. This percentage increases from embryos to adults probably due to differential proliferation and postnatal recruitment of circulating endothelial progenitors. In fact, injection of fetal liver or placental cells in the blood stream of neonates leads to incorporation of G2-GATA4 lineage cells to the coronary endothelium. On the other hand, labeling of the hematopoietic lineage by the stage E7.5 also resulted in positive coronary endothelial cells from both, embryos and adults. Our results suggest that early hematopoietic progenitors recruited by the embryonic ventricular endocardium can become the predominant source of definitive endothelium during the vascularization of the heart.

Epicardial cell lineages and the origin of the coronary endothelium.

The embryonic epicardium generates a population of epicardial-derived mesenchymal cells (EPDC) whose contribution to the coronary endothelium is minor or, according to some reports, negligible. We have compared four murine cell-tracing models related to the EPDC in order to elucidate this contribution. Cre recombinase was expressed under control of the promoters of the Wilms' tumor suppressor (Wt1), the cardiac troponin (cTnT), and the GATA5 genes, activating expression of the R26REYFP reporter. We have also used the G2 enhancer of the GATA4 gene as a driver due to its activation in the proepicardium. Recombination was found in most of the epicardium/EPDC in all cases. The contribution of these lineages to the cardiac endothelium was analyzed using confocal microscopy and flow cytometry. G2-GATA4 lineage cells are the most frequent in the endothelium, probably due to the recruitment of circulating endothelial progenitors. The contribution of the WT1 cell lineage increases along gestation due to further endothelial expression of WT1. GATA5 and cTnT lineages represent 4% of the cardiac endothelial cells throughout the gestation, probably standing for the actual EPDC contribution to the coronary endothelium. These results suggest caution when using a sole cell-tracing model to study the fate of the EPDC.

Retinoids in Stellate Cells: Development, Repair, and Regeneration.

Stellate cells, either hepatic (HSCs) or pancreatic (PSCs), are a type of interstitial cells characterized by their ability to store retinoids in lipid vesicles. In pathological conditions both HSCs and PSCs lose their retinoid content and transform into fibroblast-like cells, contributing to the fibrogenic response. HSCs also participate in other functions including vasoregulation, drug detoxification, immunotolerance, and maintenance of the hepatocyte population. PSCs maintain pancreatic tissue architecture and regulate pancreatic exocrine function. Recently, PSCs have attracted the attention of researchers due to their interactions with pancreatic ductal adenocarcinoma cells. PSCs promote tumour growth and angiogenesis, and their fibrotic activity increases the resistance of pancreatic cancer to chemotherapy and radiation. We are reviewing the current literature concerning the role played by retinoids in the physiology and pathophysiology of the stellate cells, paying attention to their developmental aspects as well as the function of stellate cells in tissue repair and organ regeneration.

The Wilms' tumor suppressor gene regulates pancreas homeostasis and repair.

The Wilms' tumor suppressor gene (Wt1) encodes a zinc finger transcription factor that plays an essential role in the development of kidneys, gonads, spleen, adrenals and heart. Recent findings suggest that WT1 could also be playing physiological roles in adults. Systemic deletion of WT1 in mice provokes a severe deterioration of the exocrine pancreas, with mesothelial disruption, E-cadherin downregulation, disorganization of acinar architecture and accumulation of ascitic transudate. Despite this extensive damage, pancreatic stellate cells do not become activated and lose their canonical markers. We observed that pharmacological induction of pancreatitis in normal mice provokes de novo expression of WT1 in pancreatic stellate cells, concomitant with their activation. When pancreatitis was induced in mice after WT1 ablation, pancreatic stellate cells expressed WT1 and became activated, leading to a partial rescue of the acinar structure and the quiescent pancreatic stellate cell population after recovery from pancreatitis. We propose that WT1 modulates through the RALDH2/retinoic acid axis the restabilization of a part of the pancreatic stellate cell population and, indirectly, the repair of the pancreatic architecture, since quiescent pancreatic stellate cells are required for pancreas stability and repair. Thus, we suggest that WT1 plays novel and essential roles for the homeostasis of the adult pancreas and, through its upregulation in pancreatic stellate cells after a damage, for pancreatic regeneration. Due to the growing importance of the pancreatic stellate cells in physiological and pathophysiological conditions, these novel roles can be of translational relevance.

Mesothelial-mesenchymal transitions in embryogenesis.

Most animals develop coelomic cavities lined by an epithelial cell layer called the mesothelium. Embryonic mesothelial cells have the ability to transform into mesenchymal cells which populate many developing organs contributing to their connective and vascular tissues, and also to organ-specific cell types. Furthermore, embryonic mesothelium and mesothelial-derived cells produce essential signals for visceral morphogenesis. We review the most relevant literature about the mechanisms regulating the embryonic mesothelial-mesenchymal transition, the developmental fate of the mesothelial-derived cells and other functions of the embryonic mesothelium, such as its contribution to the establishment of left-right visceral asymmetries or its role in limb morphogenesis.

Role of the Wilms' tumor suppressor gene Wt1 in pancreatic development.

The Wilms tumor suppressor gene (Wt1) encodes a transcription factor involved in the development of a number of organs, but the role played by Wt1 in pancreatic development is unknown. The pancreas contains a population of pancreatic stellate cells (PSC) very important for pancreatic physiology. We described elsewhere that hepatic stellate cells originate from the WT1-expressing liver mesothelium. Thus, we checked if the origin of PSCs was similar. WT1 expression is restricted to the pancreatic mesothelium. Between embryonic day (E) 10.5 and E15.5, this mesothelium gives rise to mesenchymal cells that contribute to a major part of the PSC and other cell types including endothelial cells. Most WT1 systemic mutants show abnormal localization of the dorsal pancreas within the mesentery and intestinal malrotation by E14.0. Embryos with conditional deletion of WT1 between E9.5 and E12.5 showed normal dorsal pancreatic bud and intestine, but the number of acini in the ventral bud was reduced approximately 30% by E16.5. Proliferation of acinar cells was reduced in WT1 systemic mutants, but pancreatic differentiation was not impaired. Thus, mesothelial-derived cells constitute an important subpopulation of pancreatic mesodermal cells. WT1 expression is not essential for pancreas development, although it influences intestinal rotation and correct localization of the dorsal pancreas within the mesogastrium. Developmental Dynamics 247:924-933, 2018. © 2018 Wiley Periodicals, Inc.

Comparative developmental biology of the cardiac inflow tract.

The vertebrate heart receives the blood through the cardiac inflow tract. This area has experienced profound changes along the evolution of vertebrates; changes that have a reflection in the cardiac ontogeny. The development of the inflow tract involves dynamic changes due to the progressive addition of tissue derived from the secondary heart field. The inflow tract is the site where oxygenated blood coming from lungs is received separately from the systemic return, where the cardiac pacemaker is established and where the proepicardium develops. Differential cell migration towards the inflow tract breaks the symmetry of the primary heart tube and determines the direction of the cardiac looping. In air-breathing vertebrates, an inflow tract reorganization is essential to keep separate blood flows from systemic and pulmonary returns. Finally, the sinus venosus endocardium has recently been recognized as playing a role in the constitution of the coronary vasculature. Due to this developmental complexity, congenital anomalies of the inflow tract can cause severe cardiac diseases. We aimed to review the recent literature on the cellular and molecular mechanisms that regulate the morphogenesis of the cardiac inflow tract, together with comparative and evolutionary details, thus providing a basis for a better understanding of these mechanisms.

A right-handed signalling pathway drives heart looping in vertebrates.

Most animals show external bilateral symmetry, which hinders the observation of multiple internal left-right (L/R) asymmetries that are fundamental to organ packaging and function. In vertebrates, left identity is mediated by the left-specific Nodal-Pitx2 axis that is repressed on the right-hand side by the epithelial-mesenchymal transition (EMT) inducer Snail1 (refs 3, 4). Despite some existing evidence, it remains unclear whether an equivalent instructive pathway provides right-hand-specific information to the embryo. Here we show that, in zebrafish, BMP mediates the L/R asymmetric activation of another EMT inducer, Prrx1a, in the lateral plate mesoderm with higher levels on the right. Prrx1a drives L/R differential cell movements towards the midline, leading to a leftward displacement of the cardiac posterior pole through an actomyosin-dependent mechanism. Downregulation of Prrx1a prevents heart looping and leads to mesocardia. Two parallel and mutually repressed pathways, respectively driven by Nodal and BMP on the left and right lateral plate mesoderm, converge on the asymmetric activation of the transcription factors Pitx2 and Prrx1, which integrate left and right information to govern heart morphogenesis. This mechanism is conserved in the chicken embryo, and in the mouse SNAIL1 acts in a similar manner to Prrx1a in zebrafish and PRRX1 in the chick. Thus, a differential L/R EMT produces asymmetric cell movements and forces, more prominent from the right, that drive heart laterality in vertebrates.

Role of Vitamin A/Retinoic Acid in Regulation of Embryonic and Adult Hematopoiesis.

Vitamin A is an essential micronutrient throughout life. Its physiologically active metabolite retinoic acid (RA), acting through nuclear retinoic acid receptors (RARs), is a potent regulator of patterning during embryonic development, as well as being necessary for adult tissue homeostasis. Vitamin A deficiency during pregnancy increases risk of maternal night blindness and anemia and may be a cause of congenital malformations. Childhood Vitamin A deficiency can cause xerophthalmia, lower resistance to infection and increased risk of mortality. RA signaling appears to be essential for expression of genes involved in developmental hematopoiesis, regulating the endothelial/blood cells balance in the yolk sac, promoting the hemogenic program in the aorta-gonad-mesonephros area and stimulating eryrthropoiesis in fetal liver by activating the expression of erythropoietin. In adults, RA signaling regulates differentiation of granulocytes and enhances erythropoiesis. Vitamin A may facilitate iron absorption and metabolism to prevent anemia and plays a key role in mucosal immune responses, modulating the function of regulatory T cells. Furthermore, defective RA/RARα signaling is involved in the pathogenesis of acute promyelocytic leukemia due to a failure in differentiation of promyelocytes. This review focuses on the different roles played by vitamin A/RA signaling in physiological and pathological mouse hematopoiesis duddurring both, embryonic and adult life, and the consequences of vitamin A deficiency for the blood system.

A population of hematopoietic stem cells derives from GATA4-expressing progenitors located in the placenta and lateral mesoderm of mice.

GATA transcription factors are expressed in the mesoderm and endoderm during development. GATA1-3, but not GATA4, are critically involved in hematopoiesis. An enhancer (G2) of the mouse Gata4 gene directs its expression throughout the lateral mesoderm and the allantois, beginning at embryonic day 7.5, becoming restricted to the septum transversum by embryonic day 10.5, and disappearing by midgestation. We have studied the developmental fate of the G2-Gata4 cell lineage using a G2-Gata4Cre;R26REYFP mouse line. We found a substantial number of YFP+ hematopoietic cells of lymphoid, myeloid and erythroid lineages in embryos. Fetal CD41+/cKit+/CD34+ and Lin-/cKit+/CD31+ YFP+ hematopoietic progenitors were much more abundant in the placenta than in the aorta-gonad-mesonephros area. They were clonogenic in the MethoCult assay and fully reconstituted hematopoiesis in myeloablated mice. YFP+ cells represented about 20% of the hematopoietic system of adult mice. Adult YFP+ hematopoietic stem cells constituted a long-term repopulating, transplantable population. Thus, a lineage of adult hematopoietic stem cells is characterized by the expression of GATA4 in their embryonic progenitors and probably by its extraembryonic (placental) origin, although GATA4 appeared not to be required for hematopoietic stem cell differentiation. Both lineages basically showed similar physiological behavior in normal mice, but clinically relevant properties of this particular hematopoietic stem cell population should be checked in physiopathological conditions.

C3G promotes a selective release of angiogenic factors from activated mouse platelets to regulate angiogenesis and tumor metastasis.

Previous observations indicated that C3G (RAPGEF1) promotes α-granule release, evidenced by the increase in P-selectin exposure on the platelet surface following its activation. The goal of the present study is to further characterize the potential function of C3G as a modulator of the platelet releasate and its implication in the regulation of angiogenesis. Proteomic analysis revealed a decreased secretion of anti-angiogenic factors from activated transgenic C3G and C3G∆Cat platelets. Accordingly, the secretome from both transgenic platelets had an overall pro-angiogenic effect as evidenced by an in vitro capillary-tube formation assay with HUVECs (human umbilical vein endothelial cells) and by two in vivo models of heterotopic tumor growth. In addition, transgenic C3G expression in platelets greatly increased mouse melanoma cells metastasis. Moreover, immunofluorescence microscopy showed that the pro-angiogenic factors VEGF and bFGF were partially retained into α-granules in thrombin- and ADP-activated mouse platelets from both, C3G and C3GΔCat transgenic mice. The observed interaction between C3G and Vesicle-associated membrane protein (Vamp)-7 could explain these results. Concomitantly, increased platelet spreading in both transgenic platelets upon thrombin activation supports this novel function of C3G in α-granule exocytosis. Collectively, our data point out to the co-existence of Rap1GEF-dependent and independent mechanisms mediating C3G effects on platelet secretion, which regulates pathological angiogenesis in tumors and other contexts. The results herein support an important role for platelet C3G in angiogenesis and metastasis.