Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing.

Transition between activation and quiescence programs in hematopoietic stem and progenitor cells (HSC/HSPCs) is perceived to be governed intrinsically and by microenvironmental co-adaptation. However, HSC programs dictating both transition and adaptability, remain poorly defined. Single cell multiome analysis divulging differential transcriptional activity between distinct HSPC states, indicated for the exclusive absence of Fli-1 motif from quiescent HSCs. We reveal that Fli-1 activity is essential for HSCs during regenerative hematopoiesis. Fli-1 directs activation programs while manipulating cellular sensory and output machineries, enabling HSPCs co-adoptability with a stimulated vascular niche. During regenerative conditions, Fli-1 presets and enables propagation of niche-derived Notch1 signaling. Constitutively induced Notch1 signaling is sufficient to recuperate functional HSC impairments in the absence of Fli-1. Applying FLI-1 modified-mRNA transduction into lethargic adult human mobilized HSPCs, enables their vigorous niche-mediated expansion along with superior engraftment capacities. Thus, decryption of stem cell activation programs offers valuable insights for immune regenerative medicine.

Developmental angiocrine diversification of endothelial cells for organotypic regeneration.

Adult organs are vascularized by specialized blood vessels. In addition to inter-organ vascular heterogeneity, each organ is arborized by structurally and functionally diversified populations of endothelial cells (ECs). The molecular pathways that are induced to orchestrate inter- and intra- organ vascular heterogeneity and zonation are shaped during development and fully specified postnatally. Notably, intra-organ specialization of ECs is associated with induction of angiocrine factors that guide cross-talk between ECs and parenchymal cells, establishing co-zonated vascular regions within each organ. In this review, we describe how microenvironmental tissue-specific biophysical, biochemical, immune, and inflammatory cues dictate the specialization of ECs with zonated functions. We delineate how physiological and biophysical stressors in the developing liver, lung, and kidney vasculature induce specialization of capillary beds. Deciphering mechanisms by which vascular microvasculature diversity is attained could set the stage for treating regenerative disorders and promote healing of organs without provoking fibrosis.

Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis.

Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2)3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) 'resets' these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens4,5. In three-dimensional matrices-which do not have the constraints of bioprinted scaffolds-the 'reset' vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call 'Organ-On-VascularNet'. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting.

Molecular determinants of nephron vascular specialization in the kidney.

Although kidney parenchymal tissue can be generated in vitro, reconstructing the complex vasculature of the kidney remains a daunting task. The molecular pathways that specify and sustain functional, phenotypic and structural heterogeneity of the kidney vasculature are unknown. Here, we employ high-throughput bulk and single-cell RNA sequencing of the non-lymphatic endothelial cells (ECs) of the kidney to identify the molecular pathways that dictate vascular zonation from embryos to adulthood. We show that the kidney manifests vascular-specific signatures expressing defined transcription factors, ion channels, solute transporters, and angiocrine factors choreographing kidney functions. Notably, the ontology of the glomerulus coincides with induction of unique transcription factors, including Tbx3, Gata5, Prdm1, and Pbx1. Deletion of Tbx3 in ECs results in glomerular hypoplasia, microaneurysms and regressed fenestrations leading to fibrosis in subsets of glomeruli. Deciphering the molecular determinants of kidney vascular signatures lays the foundation for rebuilding nephrons and uncovering the pathogenesis of kidney disorders.

Severe Cardiac Dysfunction and Death Caused by Arrhythmogenic Right Ventricular Cardiomyopathy Type 5 Are Improved by Inhibition of Glycogen Synthase Kinase-3ß.

BACKGROUND: Arrhythmogenic cardiomyopathy/arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disease characterized by fibrofatty replacement of the myocardium, resulting in heart failure and sudden cardiac death. The most aggressive arrhythmogenic cardiomyopathy/ARVC subtype is ARVC type 5 (ARVC5), caused by a p.S358L mutation in TMEM43 (transmembrane protein 43). The function and localization of TMEM43 are unknown, as is the mechanism by which the p.S358L mutation causes the disease. Here, we report the characterization of the first transgenic mouse model of ARVC5. METHODS: We generated transgenic mice overexpressing TMEM43 in either its wild-type or p.S358L mutant (TMEM43-S358L) form in postnatal cardiomyocytes under the control of the α-myosin heavy chain promoter. RESULTS: We found that mice expressing TMEM43-S358L recapitulate the human disease and die at a young age. Mutant TMEM43 causes cardiomyocyte death and severe fibrofatty replacement. We also demonstrate that TMEM43 localizes at the nuclear membrane and interacts with emerin and ß-actin. TMEM43-S358L shows partial delocalization to the cytoplasm, reduced interaction with emerin and ß-actin, and activation of glycogen synthase kinase-3ß (GSK3ß). Furthermore, we show that targeting cardiac fibrosis has no beneficial effect, whereas overexpression of the calcineurin splice variant calcineurin Aß1 results in GSK3ß inhibition and improved cardiac function and survival. Similarly, treatment of TMEM43 mutant mice with a GSK3ß inhibitor improves cardiac function. Finally, human induced pluripotent stem cells bearing the p.S358L mutation also showed contractile dysfunction that was partially restored after GSK3ß inhibition. CONCLUSIONS: Our data provide evidence that TMEM43-S358L leads to sustained cardiomyocyte death and fibrofatty replacement. Overexpression of calcineurin Aß1 in TMEM43 mutant mice or chemical GSK3ß inhibition improves cardiac function and increases mice life span. Our results pave the way toward new therapeutic approaches for ARVC5.

ZBTB17 (MIZ1) Is Important for the Cardiac Stress Response and a Novel Candidate Gene for Cardiomyopathy and Heart Failure.

BACKGROUND: Mutations in sarcomeric and cytoskeletal proteins are a major cause of hereditary cardiomyopathies, but our knowledge remains incomplete as to how the genetic defects execute their effects. METHODS AND RESULTS: We used cysteine and glycine-rich protein 3, a known cardiomyopathy gene, in a yeast 2-hybrid screen and identified zinc-finger and BTB domain-containing protein 17 (ZBTB17) as a novel interacting partner. ZBTB17 is a transcription factor that contains the peak association signal (rs10927875) at the replicated 1p36 cardiomyopathy locus. ZBTB17 expression protected cardiac myocytes from apoptosis in vitro and in a mouse model with cardiac myocyte-specific deletion of Zbtb17, which develops cardiomyopathy and fibrosis after biomechanical stress. ZBTB17 also regulated cardiac myocyte hypertrophy in vitro and in vivo in a calcineurin-dependent manner. CONCLUSIONS: We revealed new functions for ZBTB17 in the heart, a transcription factor that may play a role as a novel cardiomyopathy gene.

Induction of the calcineurin variant CnAß1 after myocardial infarction reduces post-infarction ventricular remodelling by promoting infarct vascularization.

AIMS: Ventricular remodelling following myocardial infarction progressively leads to loss of contractile capacity and heart failure. Although calcineurin promotes maladaptive cardiac hypertrophy, we recently showed that the calcineurin splicing variant, CnAß1, has beneficial effects on the infarcted heart. However, whether this variant limits necrosis or improves remodelling is still unknown, precluding translation to the clinical arena. Here, we explored the effects and therapeutic potential of CnAß1 overexpression post-infarction. METHODS AND RESULTS: Double transgenic mice with inducible cardiomyocyte-specific overexpression of CnAß1 underwent left coronary artery ligation followed by reperfusion. Echocardiographic analysis showed depressed cardiac function in all infarcted mice 3 days post-infarction. Induction of CnAß1 overexpression 1 week after infarction improved function and reduced ventricular dilatation. CnAß1-overexpressing mice showed shorter, thicker scars, and reduced infarct expansion, accompanied by reduced myocardial remodelling. CnAß1 induced vascular endothelial growth factor (VEGF) expression in cardiomyocytes, which resulted in increased infarct vascularization. This paracrine angiogenic effect of CnAß1 was mediated by activation of the Akt/mammalian target of rapamycin pathway and VEGF. CONCLUSIONS: Our results indicate that CnAß1 exerts beneficial effects on the infarcted heart by promoting infarct vascularization and preventing infarct expansion. These findings emphasize the translational potential of CnAß1 for gene-based therapies.