There is increased recycling of soluble fibronectin from the cell surface for fibrillogenesis. This recycling is regulated by TGF-β in a transcription- and SMAD-independent manner via specific TβRII and integrin α5β1 interactions. The recycling of fibronectin is Rab11 dependent and is required for TGF-β–induced cell migration.

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Background: Myocardial infarction (MI) induces an intense injury response which ultimately generates a collagen-dominated scar. While required to prevent ventricular rupture, the fibrotic process is often sustained in a manner detrimental to optimal recovery. Cardiac myofibroblasts are the cells tasked with depositing and remodeling collagen and are a prime target to limit the fibrotic process post-MI. Serotonin 2B receptor (5-HT 2B ) signaling has been shown to be harmful in a variety of cardiopulmonary pathologies and could play an important role in mediating scar formation after MI. Methods: We employed two pharmacologic antagonists to explore the effect of 5-HT 2B inhibition on outcomes post-MI and characterized the histological and microstructural changes involved in tissue remodeling. Inducible, 5-HT 2B ablation driven by Tcf21 MCM and Postn MCM were used to evaluate resident cardiac fibroblast- and myofibroblast-specific contributions of 5-HT 2B , respectively. RNA sequencing was used to motivate subsequent in vitro analyses to explore cardiac fibroblast phenotype. Results: 5-HT 2B antagonism preserved cardiac structure and function by facilitating a less fibrotic scar, indicated by decreased scar thickness and decreased border zone area. 5-HT 2B antagonism resulted in collagen fiber redistribution to thinner collagen fibers which were more anisotropic, enhancing left ventricular contractility, while fibrotic tissue stiffness was decreased, limiting the hypertrophic response of uninjured cardiomyocytes. Using a tamoxifen-inducible Cre, we ablated 5-HT 2B from Tcf21 -lineage resident cardiac fibroblasts and saw similar improvements to the pharmacologic approach. Tamoxifen-inducible Cre-mediated ablation of 5-HT 2B after onset of injury in Postn -lineage myofibroblasts also improved cardiac outcomes. RNA sequencing and subsequent in vitro analyses corroborate a decrease in fibroblast proliferation, migration, and remodeling capabilities through alterations in Dnajb4 expression and Src phosphorylation. Conclusions: Together, our findings illustrate that 5-HT 2B expression in either cardiac fibroblasts or activated myofibroblasts directly contributes to excessive scar formation, resulting in adverse remodeling and impaired cardiac function after MI.

Rationale: Pulmonary arterial hypertension (PAH) is a deadly disease of the pulmonary vasculature for which no disease modifying therapies exist. Small vessel stiffening and remodeling are fundamental pathologic features of PAH 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 if BM-derived PACs directly contributed to experimental pulmonary hypertension (PH) by promoting small vessel stiffening through serotonin 2B receptor (5-HT2B) 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 pharmacologic 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 PAH 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.

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