Microenvironmental Regulation of Macrophage Transcriptomic and Metabolomic Profiles in Pulmonary Hypertension

Abstract

The recruitment and subsequent polarization of inflammatory monocytes/macrophages in the perivascular regions of pulmonary arteries is a key feature of pulmonary hypertension (PH). However, the mechanisms driving macrophage polarization within the adventitial microenvironment during PH progression remain unclear. We previously established that reciprocal interactions between fibroblasts and macrophages are essential in driving the activated phenotype of both cell types although the signals involved in these interactions remain undefined. We sought to test the hypothesis that adventitial fibroblasts produce a complex array of metabolites and proteins that coordinately direct metabolomic and transcriptomic re-programming of naïve macrophages to recapitulate the pathophysiologic phenotype observed in PH. Media conditioned by pulmonary artery adventitial fibroblasts isolated from pulmonary hypertensive (PH-CM) or age-matched control (CO-CM) calves were used to activate bone marrow derived macrophages. RNA-Seq and mass spectrometry-based metabolomics analyses were performed. Fibroblast conditioned medium from patients with idiopathic pulmonary arterial hypertension or controls were used to validate transcriptional findings. The microenvironment was targeted in vitro using a fibroblast-macrophage co-culture system and in vivo in a mouse model of hypoxia-induced PH. Both CO-CM and PH-CM actively, yet distinctly regulated macrophage transcriptomic and metabolomic profiles. Network integration revealed coordinated rewiring of pro-inflammatory and pro-remodeling gene regulation in concert with altered mitochondrial and intermediary metabolism in response to PH-CM. Pro-inflammation and metabolism are key regulators of macrophage phenotype in vitro, and are closely related to in vivo flow sorted lung interstitial/perivascular macrophages from hypoxic mice. Metabolic changes are accompanied by increased free NADH levels and increased expression of a metabolic sensor and transcriptional co-repressor, C-terminal binding protein 1 (CtBP1), a mechanism shared with adventitial PH-fibroblasts. Targeting the microenvironment created by both cell types with the CtBP1 inhibitor MTOB, inhibited macrophage pro-inflammatory and metabolic re-programming both in vitro and in vivo. In conclusion, coordinated transcriptional and metabolic reprogramming is a critical mechanism regulating macrophage polarization in response to the complex adventitial microenvironment in PH. Targeting the adventitial microenvironment can return activated macrophages toward quiescence and attenuate pathological remodeling that drives PH progression.

Murine models of sickle cell disease and beta‐thalassemia demonstrate pulmonary hypertension with distinctive features

“…To better understand the differential underpinnings of PH in Hbb th3/+ and Berk-ss mice the metabolism in lung and RV tissue were evaluated. Lung metabolic reprogramming is a hallmark of PH 36 and to date studies have focused on the specific metabolic adaptations of different cell types (e.g., fibroblasts, 37 , 38 macrophages, 39 and smooth muscle cells 40 ) in the pulmonary vasculature that participate in the proliferation of the pulmonary adventitia and fibrosis. While results from evaluation of the RV are reported on in rodent PH models of SU5416-hypoxia-normoxia exposure, 33 limited information is available as to whether such metabolic adaptations are observed in the lungs and RV of murine models of hemoglobinopathies linked to PH.…”

Complement-containing small extracellular vesicles from adventitial fibroblasts induce proinflammatory and metabolic reprogramming in macrophages

“…Importantly, other studies have demonstrated that similar to macrophages in other injured tissues, proinflammatory activation of macrophages within the microenvironment of the injured artery wall in PH is associated with metabolic reprogramming characterized by aerobic glycolysis ( 16 – 18 ). The relevance of these studies is supported by our recent report, in which we showed that the proinflammatory and proremodeling phenotype of mouse bone marrow–derived macrophages (mBMDMs) treated with conditioned medium from cultured Fibs (control [CO] and PH) approximated the Inflammatory and metabolic phenotype of perivascular macrophages flow-sorted from the lungs of mice exposed to hypoxia in vivo ( 14 ).…”

“…Given that the fibroblast is the principal resident cell in the perivascular compartment, our past studies have focused on fibroblast-macrophage interactions, believing this would provide insight into their specific interactions relevant to PH, especially since the majority of monocytes/macrophages present and contributing to the disease process are recruited, non-resident cells (11). Ex-vivo studies have shown that vascular adventitial fibroblasts, obtained from humans with pulmonary arterial hypertension (PAH) and calves with severe hypoxia-induced PH, can recruit, retain, and activate monocytes/macrophages toward pro-inflammatory and proremodeling phenotype, at least in part, through soluble cytokine release (2,(12)(13)(14)(15).…”

Dysregulated Immunity in Pulmonary Hypertension: From Companion to Composer

“…While macrophage cell populations reside (and reproduce locally) in lungs under physiological conditions, their population increases rapidly in response to injury and pathological stimuli (e.g., PH) by mobilization of monocytes from distal sites (spleen and bone marrow) and their recruitment into specific organs ( Florentin et al, 2018 ). The active recruitment of monocytes to the site of injury and their polarization is orchestrated by the vascular microenvironment and is mainly facilitated by stimuli released from the local tissue milieu, e.g., including damage-associated molecular patterns, chemokines, apoptotic bodies, or fibroblast-derived factors ( Amsellem et al, 2017 ; Lin et al, 2019 ; Fan et al, 2021 ; Li et al, 2021 ).…”