Genetic ablation of serotonin receptor 2B improves aortic valve hemodynamics of Notch1 heterozygous mice in a high-cholesterol diet model.

Calcific aortic valve disease (CAVD) is a deadly disease that is rising in prevalence due to population aging. While the disease is complex and poorly understood, one well-documented driver of valvulopathy is serotonin agonism. Both serotonin overexpression, as seen with carcinoid tumors and drug-related agonism, such as with Fenfluramine use, are linked with various diseases of the valves. Thus, the objective of this study was to determine if genetic ablation or pharmacological antagonism of the 5-HT2B serotonin receptor (gene: Htr2b) could improve the hemodynamic and histological progression of calcific aortic valve disease. Htr2b mutant mice were crossed with Notch1+/- mice, an established small animal model of CAVD, to determine if genetic ablation affects CAVD progression. To assess the effect of pharmacological inhibition on CAVD progression, Notch1+/- mice were treated with the 5-HT2B receptor antagonist SB204741. Mice were analyzed using echocardiography, histology, immunofluorescence, and real-time quantitative polymerase chain reaction. Htr2b mutant mice showed lower aortic valve peak velocity and mean pressure gradient-classical hemodynamic indicators of aortic valve stenosis-without concurrent left ventricle change. 5-HT2B receptor antagonism, however, did not affect hemodynamic progression. Leaflet thickness, collagen density, and CAVD-associated transcriptional markers were not significantly different in any group. This study reveals that genetic ablation of Htr2b attenuates hemodynamic development of CAVD in the Notch1+/- mice, but pharmacological antagonism may require high doses or long-term treatment to slow progression.

Cadherin-11 blockade reduces inflammation-driven fibrotic remodeling and improves outcomes after myocardial infarction.

Over one million Americans experience myocardial infarction (MI) annually, and the resulting scar and subsequent cardiac fibrosis gives rise to heart failure. A specialized cell-cell adhesion protein, cadherin-11 (CDH11), contributes to inflammation and fibrosis in rheumatoid arthritis, pulmonary fibrosis, and aortic valve calcification but has not been studied in myocardium after MI. MI was induced by ligation of the left anterior descending artery in mice with either heterozygous or homozygous knockout of CDH11, wild-type mice receiving bone marrow transplants from Cdh11-deficient animals, and wild-type mice treated with a functional blocking antibody against CDH11 (SYN0012). Flow cytometry revealed significant CDH11 expression in noncardiomyocyte cells after MI. Animals given SYN0012 had improved cardiac function, as measured by echocardiogram, reduced tissue remodeling, and altered transcription of inflammatory and proangiogenic genes. Targeting CDH11 reduced bone marrow-derived myeloid cells and increased proangiogenic cells in the heart 3 days after MI. Cardiac fibroblast and macrophage interactions increased IL-6 secretion in vitro. Our findings suggest that CDH11-expressing cells contribute to inflammation-driven fibrotic remodeling after MI and that targeting CDH11 with a blocking antibody improves outcomes by altering recruitment of bone marrow-derived cells, limiting the macrophage-induced expression of IL-6 by fibroblasts and promoting vascularization.

Bone Marrow-Derived Proangiogenic Cells Mediate Pulmonary Arteriole Stiffening via Serotonin 2B Receptor Dependent Mechanism.

RATIONALE: Pulmonary arterial hypertension is a deadly disease of the pulmonary vasculature for which no disease-modifying therapies exist. Small-vessel stiffening and remodeling are fundamental pathological features of pulmonary arterial hypertension that occur early and drive further endovascular cell dysfunction. Bone marrow (BM)-derived proangiogenic cells (PACs), a specialized heterogeneous subpopulation of myeloid lineage cells, are thought to play an important role in pathogenesis. OBJECTIVE: To determine whether BM-derived PACs directly contributed to experimental pulmonary hypertension (PH) by promoting small-vessel stiffening through 5-HT2B (serotonin 2B receptor)-mediated signaling. METHODS AND RESULTS: We performed BM transplants using transgenic donor animals expressing diphtheria toxin secondary to activation of an endothelial-specific tamoxifen-inducible Cre and induced experimental PH using hypoxia with SU5416 to enhance endovascular injury and ablated BM-derived PACs, after which we measured right ventricular systolic pressures in a closed-chest procedure. BM-derived PAC lineage tracing was accomplished by transplanting BM from transgenic donor animals with fluorescently labeled hematopoietic cells and treating mice with a 5-HT2B antagonist. BM-derived PAC ablation both prevented and reversed experimental PH with SU5416-enhanced endovascular injury, reducing the number of muscularized pulmonary arterioles and normalizing arteriole stiffness as measured by atomic force microscopy. Similarly, treatment with a pharmacological antagonist of 5-HT2B also prevented experimental PH, reducing the number and stiffness of muscularized pulmonary arterioles. PACs accelerated pulmonary microvascular endothelial cell injury response in vitro, and the presence of BM-derived PACs significantly correlated with stiffer pulmonary arterioles in pulmonary arterial hypertension patients and mice with experimental PH. RNA sequencing of BM-derived PACs showed that 5-HT2B antagonism significantly altered biologic pathways regulating cell proliferation, locomotion and migration, and cytokine production and response to cytokine stimulus. CONCLUSIONS: Together, our findings illustrate that BM-derived PACs directly contribute to experimental PH with SU5416-enhanced endovascular injury by mediating small-vessel stiffening and remodeling in a 5-HT2B signaling-dependent manner.

Transcriptional Profiling of Cultured, Embryonic Epicardial Cells Identifies Novel Genes and Signaling Pathways Regulated by TGFßR3 In Vitro.

The epicardium plays an important role in coronary vessel formation and Tgfbr3-/- mice exhibit failed coronary vessel development associated with decreased epicardial cell invasion. Immortalized Tgfbr3-/- epicardial cells display the same defects. Tgfbr3+/+ and Tgfbr3-/- cells incubated for 72 hours with VEH or ligands known to promote invasion via TGFßR3 (TGFß1, TGFß2, BMP2), for 72 hours were harvested for RNA-seq analysis. We selected for genes >2-fold differentially expressed between Tgfbr3+/+ and Tgfbr3-/- cells when incubated with VEH (604), TGFß1 (515), TGFß2 (553), or BMP2 (632). Gene Ontology (GO) analysis of these genes identified dysregulated biological processes consistent with the defects observed in Tgfbr3-/- cells, including those associated with extracellular matrix interaction. GO and Gene Regulatory Network (GRN) analysis identified distinct expression profiles between TGFß1-TGFß2 and VEH-BMP2 incubated cells, consistent with the differential response of epicardial cells to these ligands in vitro. Despite the differences observed between Tgfbr3+/+ and Tgfbr3-/- cells after TGFß and BMP ligand addition, GRNs constructed from these gene lists identified NF-ĸB as a key nodal point for all ligands examined. Tgfbr3-/- cells exhibited decreased expression of genes known to be activated by NF-ĸB signaling. NF-ĸB activity was stimulated in Tgfbr3+/+ epicardial cells after TGFß2 or BMP2 incubation, while Tgfbr3-/- cells failed to activate NF-ĸB in response to these ligands. Tgfbr3+/+ epicardial cells incubated with an inhibitor of NF-ĸB signaling no longer invaded into a collagen gel in response to TGFß2 or BMP2. These data suggest that NF-ĸB signaling is dysregulated in Tgfbr3-/- epicardial cells and that NF-ĸB signaling is required for epicardial cell invasion in vitro. Our approach successfully identified a signaling pathway important in epicardial cell behavior downstream of TGFßR3. Overall, the genes and signaling pathways identified through our analysis yield the first comprehensive list of candidate genes whose expression is dependent on TGFßR3 signaling.

Common pathways regulate Type III TGFß receptor-dependent cell invasion in epicardial and endocardial cells.

Epithelial-Mesenchymal Transformation (EMT) and the subsequent invasion of epicardial and endocardial cells during cardiac development is critical to the development of the coronary vessels and heart valves. The transformed cells give rise to cardiac fibroblasts and vascular smooth muscle cells or valvular interstitial cells, respectively. The Type III Transforming Growth Factor ß (TGFßR3) receptor regulates EMT and cell invasion in both cell types, but the signaling mechanisms downstream of TGFßR3 are not well understood. Here we use epicardial and endocardial cells in in vitro cell invasion assays to identify common mechanisms downstream of TGFßR3 that regulate cell invasion. Inhibition of NF-κB activity blocked cell invasion in epicardial and endocardial cells. NF-κB signaling was found to be dysregulated in Tgfbr3(-/-) epicardial cells which also show impaired cell invasion in response to ligand. TGFßR3-dependent cell invasion is also dependent upon Activin Receptor-Like Kinase (ALK) 2, ALK3, and ALK5 activity. A TGFßR3 mutant that contains a threonine to alanine substitution at residue 841 (TGFßR3-T841A) induces ligand-independent cell invasion in both epicardial and endocardial cells in vitro. These findings reveal a role for NF-κB signaling in the regulation of epicardial and endocardial cell invasion and identify a mutation in TGFßR3 which stimulates ligand-independent signaling.