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

Abstract

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.

Genetic ablation of serotonin receptor 2B improves aortic valve hemodynamics in a high-cholesterol diet mouse model

“…6). Immune cell recruitment plays a key role in other 5-HT 2B -mediated fibrotic disease and has been shown to contribute to the Notch1 +/model of CAVD, suggesting it may be involved here (23,33,34). This may also indicate a phenotype arising from cells other than myofibroblasts.…”

Machine learning-based microarray analyses indicate low-expression genes might collectively influence PAH disease

“…Second, bone-marrow derived cells are found to be more essential to many disease processes. Particularly, transplantation of patient-derived bone marrow has been enough to induce PAH in mice [18], and transplantation of mutant mice with wild-type bone marrow ameliorates it [19] in more than one model [20]. Therefore, these cultured blood cells actually have important etiological roles in identifying PAH.…”

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

“…SB204741 is a well-validated and specific 5-HT 2B receptor antagonist commonly used in pharmacological studies targeting this ligand [28,29]. SB204741 dosing varies widely across the literature, but we use 1 mg kg -1 d -1 to allow for longer time course administration with good results [30].…”

“…Our data show Htr2b ablation nor receptor inhibition alter Runx2 expression or osteogenic calcification, suggesting the hemodynamic phenotype observed in mutant mice is not based on canonical osteogenic signaling pathways (Fig 6). Immune cell recruitment plays a key role in other 5-HT 2B -mediated fibrotic disease and has been shown to contribute to the Notch1 +/model of CAVD, suggesting it may be involved here [25,30,39]. This may also indicate a phenotype arising from cells other than myofibroblasts.…”

“…SB204741 is known to be a selective antagonist of the 2B receptor subtype but has relatively low potency [28]. While we have had success using a 1 mg kg -1 d -1 in previous studies in the cardiovascular system, studies using SB204741 in other diseases like chronic liver disease or pancreatic cancer require higher dosages of up to 10 mg kg -1 d -1 [30,42,43]. Additionally, our window of intervention may have been too early as six months of age is still relatively early for the disease to present.…”