Background: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by profound vascular remodeling in which pulmonary arteries narrow because of medial thickening and occlusion by neointimal lesions, resulting in elevated pulmonary vascular resistance and right heart failure. Therapies targeting the neointima would represent a significant advance in PAH treatment; however, our understanding of the cellular events driving neointima formation, and the molecular pathways that control them, remains limited. Methods: We comprehensively map the stepwise remodeling of pulmonary arteries in a robust, chronic inflammatory mouse model of pulmonary hypertension. This model demonstrates pathological features of the human disease, including increased right ventricular pressures, medial thickening, neointimal lesion formation, elastin breakdown, increased anastomosis within the bronchial circulation, and perivascular inflammation. Using genetic lineage tracing, clonal analysis, multiplexed in situ hybridization, immunostaining, deep confocal imaging, and staged pharmacological inhibition, we define the cell behaviors underlying each stage of vascular remodeling and identify a pathway required for neointima formation. Results: Neointima arises from smooth muscle cells (SMCs) and not endothelium. Medial SMCs proliferate broadly to thicken the media, after which a small number of SMCs are selected to establish the neointima. These neointimal founder cells subsequently undergoing massive clonal expansion to form occlusive neointimal lesions. The normal pulmonary artery SMC population is heterogeneous, and we identify a Notch3-marked minority subset of SMCs as the major neointimal cell of origin. Notch signaling is specifically required for the selection of neointimal founder cells, and Notch inhibition significantly improves pulmonary artery pressure in animals with pulmonary hypertension. Conclusions: This work describes the first nongenetically driven murine model of pulmonary hypertension (PH) that generates robust and diffuse occlusive neointimal lesions across the pulmonary vascular bed and does so in a stereotyped timeframe. We uncover distinct cellular and molecular mechanisms underlying medial thickening and neointima formation and highlight novel transcriptional, behavioral, and pathogenic heterogeneity within pulmonary artery SMCs. In this model, inflammation is sufficient to generate characteristic vascular pathologies and physiological measures of human PAH. We hope that identifying the molecular cues regulating each stage of vascular remodeling will open new avenues for therapeutic advancements in the treatment of PAH.

Rationale:: Revascularization of injured, ischemic, and regenerating organs is essential to restore organ function. In the postinfarct heart, however, the mechanisms underlying the formation of new coronary arteries are poorly understood. Objective:: To study vascular remodeling of coronary arteries after infarction. Methods and Results:: We performed permanent left coronary ligation on Connexin40-GFP mice expressing green fluorescent protein ( GFP ) in endothelial cells of coronary arteries but not veins, capillaries, or endocardium. GFP ⁺ endothelial foci were identified within the endocardium in the infarct zone. These previously undescribed structures, termed endocardial flowers, have a distinct endothelial phenotype (Cx40 ⁺ , VEGFR2 ⁺ , and endoglin ⁻ ) to the surrounding endocardium (Cx40 ⁻ , VEGFR2 ⁻ , and endoglin ⁺ ). Endocardial flowers are contiguous with coronary vessels and associated with subendocardial smooth muscle cell accumulation. Genetic lineage tracing reveals extensive endothelial plasticity in the postinfarct heart, showing that endocardial flowers develop by arteriogenesis of Cx40 ⁻ cells and by outgrowth of pre-existing coronary arteries. Finally, endocardial flowers exhibit angiogenic features, including early VEGFR2 expression and active proliferation of adjacent endocardial and smooth muscle cells. Conclusions:: Arterial endothelial foci within the endocardium reveal extensive endothelial cell plasticity in the infarct zone and identify the endocardium as a site of endogenous arteriogenesis and source of endothelial cells to promote vascularization in regenerative strategies.

Left ventricular non-compaction (LVNC) is a rare cardiomyopathy associated with a hypertrabeculated phenotype and a large spectrum of symptoms. It is still unclear whether LVNC results from a defect of ventricular trabeculae development and the mechanistic basis that underlies the varying severity of this pathology is unknown. To investigate these issues, we inactivated the cardiac transcription factor Nkx2-5 in trabecular myocardium at different stages of trabecular morphogenesis using an inducible Cx40-creERT2 allele. Conditional deletion of Nkx2-5 at embryonic stages, during trabecular formation, provokes a severe hypertrabeculated phenotype associated with subendocardial fibrosis and Purkinje fiber hypoplasia. A milder phenotype was observed after Nkx2-5 deletion at fetal stages, during trabecular compaction. A longitudinal study of cardiac function in adult Nkx2-5 conditional mutant mice demonstrates that excessive trabeculation is associated with complex ventricular conduction defects, progressively leading to strain defects, and, in 50% of mutant mice, to heart failure. Progressive impaired cardiac function correlates with conduction and strain defects independently of the degree of hypertrabeculation. Transcriptomic analysis of molecular pathways reflects myocardial remodeling with a larger number of differentially expressed genes in the severe versus mild phenotype and identifies Six1 as being upregulated in hypertrabeculated hearts. Our results provide insights into the etiology of LVNC and link its pathogenicity with compromised trabecular development including compaction defects and ventricular conduction system hypoplasia. During fetal heart morphogenesis, formation of the mature ventricular wall requires coordinated compaction of the inner trabecular layer and growth of the outer layer of myocardium. Arrested trabecular development has been implicated in the pathogenesis of hypertrabeculation associated with ventricular non-compaction cardiomyopathy. However much uncertainty still exists among clinicians concerning the physiopathology of ventricular non-compaction cardiomyopathy, including its clinical characteristics, prognosis, classification and even the definition of hypertrabeculation. In particular, distinguishing between pathological and non-pathological subtypes of non-compaction is currently a major issue. Here we show that deletion of the gene encoding the transcription factor Nkx2-5 at critical steps during trabecular development recapitulates pathological features of hypertrabeculation, providing the first model of ventricular non-compaction cardiomyopathy in adult mice. We demonstrate that excessive trabeculation due to failure of trabecular compaction during fetal development is associated with Purkinje fiber hypoplasia and subendocardial fibrosis. Longitudinal functional studies reveal that these mice present all the clinical signs of symptomatic left ventricular non-compaction cardiomyopathy, including conduction defects, strain defects and progressive heart failure. Our results, including transcriptomic analysis, suggest that pathological features of non-compaction are primarily developmental defects. This study clarifies the origin of the pathological outcomes associated with LVNC and may provide helpful information for clinicians concerning the etiology of this rare cardiomyopathy.

The ventricular conduction system coordinates heartbeats by rapid propagation of electrical activity through the Purkinje fiber (PF) network. PFs share common progenitors with contractile cardiomyocytes, yet the mechanisms of segregation and network morphogenesis are poorly understood. Here, we apply genetic fate mapping and temporal clonal analysis to identify murine cardiomyocytes committed to the PF lineage as early as E7.5. We find that a polyclonal PF network emerges by progressive recruitment of conductive precursors to this scaffold from a pool of bipotent progenitors. At late fetal stages, the segregation of conductive cells increases during a phase of rapid recruitment to build the definitive PF network through a non-cell autonomous mechanism. We also show that PF differentiation is impaired in Nkx2-5 haploinsufficient embryos leading to failure to extend the scaffold. In particular, late fetal recruitment fails, resulting in PF hypoplasia and persistence of bipotent progenitors. Our results identify how transcription factor dosage regulates cell fate divergence during distinct phases of PF network morphogenesis.