3D Imaging Reveals Complex Microvascular Remodeling in the Right Ventricle in Pulmonary Hypertension.

BACKGROUND: Pathogenic concepts of right ventricular (RV) failure in pulmonary arterial hypertension focus on a critical loss of microvasculature. However, the methods underpinning prior studies did not take into account the 3-dimensional (3D) aspects of cardiac tissue, making accurate quantification difficult. We applied deep-tissue imaging to the pressure-overloaded RV to uncover the 3D properties of the microvascular network and determine whether deficient microvascular adaptation contributes to RV failure. METHODS: Heart sections measuring 250-µm-thick were obtained from mice after pulmonary artery banding (PAB) or debanding PAB surgery and properties of the RV microvascular network were assessed using 3D imaging and quantification. Human heart tissues harvested at the time of transplantation from pulmonary arterial hypertension cases were compared with tissues from control cases with normal RV function. RESULTS: Longitudinal 3D assessment of PAB mouse hearts uncovered complex microvascular remodeling characterized by tortuous, shorter, thicker, highly branched vessels, and overall preserved microvascular density. This remodeling process was reversible in debanding PAB mice in which the RV function recovers over time. The remodeled microvasculature tightly wrapped around the hypertrophied cardiomyocytes to maintain a stable contact surface to cardiomyocytes as an adaptation to RV pressure overload, even in end-stage RV failure. However, microvasculature-cardiomyocyte contact was impaired in areas with interstitial fibrosis where cardiomyocytes displayed signs of hypoxia. Similar to PAB animals, microvascular density in the RV was preserved in patients with end-stage pulmonary arterial hypertension, and microvascular architectural changes appeared to vary by etiology, with patients with pulmonary veno-occlusive disease displaying a lack of microvascular complexity with uniformly short segments. CONCLUSIONS: 3D deep tissue imaging of the failing RV in PAB mice, pulmonary hypertension rats, and patients with pulmonary arterial hypertension reveals complex microvascular changes to preserve the microvascular density and maintain a stable microvascular-cardiomyocyte contact. Our studies provide a novel framework to understand microvascular adaptation in the pressure-overloaded RV that focuses on cell-cell interaction and goes beyond the concept of capillary rarefaction.

Ultrasound Neuromodulation of an Anti-Inflammatory Pathway at the Spleen Improves Experimental Pulmonary Hypertension.

BACKGROUND: Inflammation is pathogenically implicated in pulmonary arterial hypertension; however, it has not been adequately targeted therapeutically. We investigated whether neuromodulation of an anti-inflammatory neuroimmune pathway involving the splenic nerve using noninvasive, focused ultrasound stimulation of the spleen (sFUS) can improve experimental pulmonary hypertension. METHODS: Pulmonary hypertension was induced in rats either by Sugen 5416 (20 mg/kg SQ) injection, followed by 21 (or 35) days of hypoxia (sugen/hypoxia model), or by monocrotaline (60 mg/kg IP) injection (monocrotaline model). Animals were randomized to receive either 12-minute-long sessions of sFUS daily or sham stimulation for 14 days. Catheterizations, echocardiography, indices of autonomic function, lung and heart histology and immunohistochemistry, spleen flow cytometry, and lung single-cell RNA sequencing were performed after treatment to assess the effects of sFUS. RESULTS: Splenic denervation right before induction of pulmonary hypertension results in a more severe disease phenotype. In both sugen/hypoxia and monocrotaline models, sFUS treatment reduces right ventricular systolic pressure by 25% to 30% compared with sham treatment, without affecting systemic pressure, and improves right ventricular function and autonomic indices. sFUS reduces wall thickness, apoptosis, and proliferation in small pulmonary arterioles, suppresses CD3+ and CD68+ cell infiltration in lungs and right ventricular fibrosis and hypertrophy and lowers BNP (brain natriuretic peptide). Beneficial effects persist for weeks after sFUS discontinuation and are more robust with early and longer treatment. Splenic denervation abolishes sFUS therapeutic benefits. sFUS partially normalizes CD68+ and CD8+ T-cell counts in the spleen and downregulates several inflammatory genes and pathways in nonclassical and classical monocytes and macrophages in the lung. Differentially expressed genes in those cell types are significantly enriched for human pulmonary arterial hypertension-associated genes. CONCLUSIONS: sFUS causes dose-dependent, sustained improvement of hemodynamic, autonomic, laboratory, and pathological manifestations in 2 models of experimental pulmonary hypertension. Mechanistically, sFUS normalizes immune cell populations in the spleen and downregulates inflammatory genes and pathways in the lung, many of which are relevant in human disease.

B-cells in pulmonary arterial hypertension: friend, foe or bystander?

There is an unmet need for new therapeutic strategies that target alternative pathways to improve the prognosis of patients with pulmonary arterial hypertension (PAH). As immunity has been involved in the development and progression of vascular lesions in PAH, we review the potential contribution of B-cells in its pathogenesis and evaluate the relevance of B-cell-targeted therapies. Circulating B-cell homeostasis is altered in PAH patients, with total B-cell lymphopenia, abnormal subset distribution (expansion of naïve and antibody-secreting cells, reduction of memory B-cells) and chronic activation. B-cells are recruited to the lungs through local chemokine secretion, and activated by several mechanisms: 1) interaction with lung vascular autoantigens through cognate B-cell receptors; 2) costimulatory signals provided by T follicular helper cells (interleukin (IL)-21), type 2 T helper cells and mast cells (IL-4, IL-6 and IL-13); and 3) increased survival signals provided by B-cell activating factor pathways. This activity results in the formation of germinal centres within perivascular tertiary lymphoid organs and in the local production of pathogenic autoantibodies that target the pulmonary vasculature and vascular stabilisation factors (including angiotensin-II/endothelin-1 receptors and bone morphogenetic protein receptors). B-cells also mediate their effects through enhanced production of pro-inflammatory cytokines, reduced anti-inflammatory properties by regulatory B-cells, immunoglobulin (Ig)G-induced complement activation, and IgE-induced mast cell activation. Precision-medicine approaches targeting B-cell immunity are a promising direction for select PAH conditions, as suggested by the efficacy of anti-CD20 therapy in experimental models and a trial of rituximab in systemic sclerosis-associated PAH.

Rat microbial biogeography and age-dependent lactic acid bacteria in healthy lungs.

The laboratory rat emerges as a useful tool for studying the interaction between the host and its microbiome. To advance principles relevant to the human microbiome, we systematically investigated and defined the multitissue microbial biogeography of healthy Fischer 344 rats across their lifespan. Microbial community profiling data were extracted and integrated with host transcriptomic data from the Sequencing Quality Control consortium. Unsupervised machine learning, correlation, taxonomic diversity and abundance analyses were performed to determine and characterize the rat microbial biogeography and identify four intertissue microbial heterogeneity patterns (P1-P4). We found that the 11 body habitats harbored a greater diversity of microbes than previously suspected. Lactic acid bacteria (LAB) abundance progressively declined in lungs from breastfed newborn to adolescence/adult, and was below detectable levels in elderly rats. Bioinformatics analyses indicate that the abundance of LAB may be modulated by the lung-immune axis. The presence and levels of LAB in lungs were further evaluated by PCR in two validation datasets. The lung, testes, thymus, kidney, adrenal and muscle niches were found to have age-dependent alterations in microbial abundance. The 357 microbial signatures were positively correlated with host genes in cell proliferation (P1), DNA damage repair (P2) and DNA transcription (P3). Our study established a link between the metabolic properties of LAB with lung microbiota maturation and development. Breastfeeding and environmental exposure influence microbiome composition and host health and longevity. The inferred rat microbial biogeography and pattern-specific microbial signatures could be useful for microbiome therapeutic approaches to human health and life quality enhancement.

Pericytes contribute to pulmonary vascular remodeling via HIF2α signaling.

Vascular remodeling is the process of structural alteration and cell rearrangement of blood vessels in response to injury and is the cause of many of the world's most afflicted cardiovascular conditions, including pulmonary arterial hypertension (PAH). Many studies have focused on the effects of vascular endothelial cells and smooth muscle cells (SMCs) during vascular remodeling, but pericytes, an indispensable cell population residing largely in capillaries, are ignored in this maladaptive process. Here, we report that hypoxia-inducible factor 2α (HIF2α) expression is increased in the lung tissues of PAH patients, and HIF2α overexpressed pericytes result in greater contractility and an impaired endothelial-pericyte interaction. Using single-cell RNAseq and hypoxia-induced pulmonary hypertension (PH) models, we show that HIF2α is a major molecular regulator for the transformation of pericytes into SMC-like cells. Pericyte-selective HIF2α overexpression in mice exacerbates PH and right ventricular hypertrophy. Temporal cellular lineage tracing shows that HIF2α overexpressing reporter NG2+ cells (pericyte-selective) relocate from capillaries to arterioles and co-express SMA. This novel insight into the crucial role of NG2+ pericytes in pulmonary vascular remodeling via HIF2α signaling suggests a potential drug target for PH.