Pulmonary hypertension (PH) is a severe vascular complication of sickle cell disease (SCD); yet, not all patients with SCD develop PH, and PH also arises independently. This duality underscores the need to understand their intersecting biology. We integrated metabolomic, proteomic, and elemental analyses of human peripheral blood mononuclear cells (PBMCs) from individuals with SCD, PH, combined SCD-PH, and healthy controls to define shared and distinct mechanisms. PBMCs from SCD patients, regardless of PH status, displayed significantly elevated intracellular iron, consistent with chronic hemolysis and erythrophagocytosis. Multi-omic profiling revealed condition-specific immune-metabolic signatures: SCD PBMCs showed mitochondrial suppression and reduced oxidative phosphorylation; PH PBMCs showed dysregulated arginine and creatine metabolism, implicating nitric oxide and polyamine pathways; and SCD-PH PBMCs displayed amplified hemoglobin/iron handling, oxidative stress, and immune activation. Unsupervised clustering confirmed discrete phenotypes, with greatest overlap between SCD and SCD-PH, reflecting the additive impact of hemolysis-driven iron loading and PH-driven metabolic remodeling. Histological validation of SCD-PH lung tissue demonstrated iron accumulation in perivascular macrophages, supporting a mechanistic link between systemic PBMC remodeling and pulmonary vascular pathology. Together, these findings establish PBMCs as a readily accessible compartment that mirrors disease-specific metabolic and immune alterations. By capturing iron, arginine, and redox pathways across SCD, PH, and SCD-PH, our study positions PBMC profiling as a novel tool for mechanistic insight, patient stratification, and biomarker discovery and novel interventions.

Background: Pulmonary hypertension (PH) is a progressive disorder marked by persistently activated cell types within pulmonary vessels, such as adventitial fibroblasts. PH-associated fibroblasts show altered gene expression linked to proliferation, metabolism, and inflammation. The complement system is traditionally viewed as a serum-based immune effector. However, recent findings suggest a role of fibroblast-specific, local, and intracellular-produced complement (complosome) proteins in driving vascular inflammation and remodeling in PH. This study investigates fibroblast-specific complement (CFD) in regulating glycolysis, mitochondrial function, and inflammation and assesses the therapeutic potential of CFD inhibitors in PH. Methods and Results: In vivo observations in humans and animals demonstrated high C3, CFB, and especially CFD signals in fibroblasts of PH lesions. To understand the role of complements fibroblasts. Fibroblasts were isolated from distal pulmonary arteries of patients with idiopathic pulmonary arterial hypertension (IPAH-Fibs) and calves with severe PH (PH-Fibs), and control fibroblasts (CO-Fibs) from healthy donors and age-matched bovine controls. Transcriptomic and protein analyses using RNAseq/qRT-PCR and immunoblotting revealed that IPAH/PH-Fibs showed significantly higher C3, C5, CFB, and CFD expression than CO-Fibs. This increased expression was accompanied by elevated levels of C3 and C5 activation fragments (C3a) both intracellularly and in extracellular medium, suggesting intracellular and local complement activation. CFD, a key enzyme in the alternative complement pathway, is crucial for C3 activation, as downregulating CFD in PH-Fibs led to reduced C3a production, suggesting a link between CFD activity and C3a generation. C3a receptor (C3aR1) was detected on isolated mitochondria and cell membranes of adventitial fibroblasts. C3a signal was also detected in fibroblast conditioned media and mitochondrial fraction, supporting the hypothesis that C3a-induced metabolic and proinflammatory reprogramming occurs via C3aR1. CFD knockdown normalized expression of elevated metabolic genes (GLUT1, HK2, GPI, ENO1, and ACO1) and proinflammatory genes (MCP1, SDF1, IL-6, IL-13, and IL-33) in PH-Fibs. PH-fibs exhibited increased activation of glycolysis, TCA, and fatty acid metabolism pathways, which were significantly reduced by CFD knockdown. Further in this study, we repurposed commercially available CFD inhibitors, particularly Danicopan and Vimircopan. We used isolated primary adventitial fibroblasts and precision-cut lung slices (PCLS) to test therapeutic potential of CFD inhibitors. Treatment with CFD inhibitors effectively reduced intracellular and local complement activation, particularly of CFB and C3 in PH Fibs. Conclusion: This study highlights the crucial role of local and intracellular complement proteins, particularly CFD, in PH fibroblasts. Targeting CFD significantly reduced production of C3-activated fragments (C3a) and metabolites and genes associated with metabolic and pro-inflammatory reprogramming.

Background: Pulmonary vascular remodeling is a progressive pathological process characterized by functional alterations within pulmonary artery smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs). Mechanisms driving the transition to a diseased phenotype remain elusive. Methods: We combined transcriptomic and proteomic profiling with phenotypic characterization of source-matched cells from healthy controls and individuals with idiopathic pulmonary arterial hypertension (IPAH). Bidirectional cellular crosstalk was examined using direct and indirect co-culture models, and phenotypic responses were assessed via transcriptome analysis. Results: PASMC and PAAF undergo distinct phenotypic shifts during pulmonary vascular remodeling, with limited shared features, such as reduced mitochondrial content and hyperpolarization. IPAH-PASMC exhibit increased glycosaminoglycan production and downregulation of contractile machinery, while IPAH-PAAF display a hyperproliferative phenotype. We identified alterations in extracellular matrix components, including laminin and collagen, alongside pentraxin-3 and hepatocyte growth factor, as potential regulators of PASMC phenotypic transitions mediated by PAAF. Conclusions: While PASMCs and PAAFs retain their core cellular identities, they acquire distinct disease-associated states. These findings provide new insights into the dynamic interplay of pulmonary vascular mesenchymal cells in disease pathogenesis. Funding: This work was supported by Cardio-Pulmonary Institute EXC 2026 390649896 (GK) and Austrian Science Fund (FWF) grant I 4651-B (SC).

Hypoxia is a major cause of pulmonary hypertension (PH) worldwide, and it is likely that interstitial pulmonary macrophages contribute to this vascular pathology. We observed in hypoxia-exposed mice an increase in resident interstitial macrophages, which expanded through proliferation and expressed the monocyte recruitment ligand CCL2. We also observed an increase in CCR2+ macrophages through recruitment, which express the protein thrombospondin-1, which functionally activates TGF-β to cause vascular disease. Blockade of monocyte recruitment with either CCL2-neutralizing antibody treatment or CCR2 deficiency in the bone marrow compartment suppressed hypoxic PH. These data were supported by analysis of plasma samples from humans who traveled from low (225 m) to high (3500 m) elevation, revealing an increase in thrombospondin-1 and TGF-β expression following ascent, which was blocked by dexamethasone prophylaxis. In the hypoxic mouse model, dexamethasone prophylaxis recapitulated these findings by mechanistically suppressing CCL2 expression and CCR2+ monocyte recruitment. These data suggest a pathologic cross talk between 2 discrete interstitial macrophage populations, which can be therapeutically targeted.

Dysregulated redox signaling contributes to pulmonary hypertension (PH) and vascular depletion of the redox enzyme extracellular superoxide dismutase (EC-SOD) from smooth muscle cells [EC-SOD SMC knockout (KO)] worsens chronic hypoxic PH. Given the important role of macrophages in PH, this study aimed to determine if interstitial macrophages (IMs) and their interactions with hyaluronan (HA), a component of extracellular matrix (ECM), are modulated by vascular EC-SOD. Floxed wild-type, EC-SOD SMC KO, and SOD mimetic- or vehicle-treated mice were exposed to hypobaric hypoxia [∼10% fraction of inspired oxygen ([Formula: see text])], for 4, 14, or 21 days. Using flow cytometry, we demonstrated that the transient increase in IMs at day 4 was exacerbated in EC-SOD SMC KO mice and prevented with SOD mimetic pretreatment. Highlighting the importance of targeting vascular oxidative stress in the early response to hypoxia, pretreatment with a single dose of EC-SOD mimetic decreased right ventricular systolic pressure, right ventricular hypertrophy, and small vessel muscularization at day 21. To assess IM phenotypic reprogramming in hypoxia, RNA-seq was performed on flow-sorted IMs revealing baseline proinflammatory activation and enhanced activation of vascular and ECM remodeling pathways in response to hypoxia in EC-SOD SMC KO IMs compared with controls. To further investigate the ECM remodeling response, we quantified IMs expressing the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), and IM-hyaluronan binding. Lyve1+ IMs and Lyve1+ HA+ IMs were increased in response to hypoxia in EC-SOD SMC KO mice and accumulated in the perivascular space of the lung. In conclusion, vascular EC-SOD limits IM accumulation and proinflammatory profibrotic IM signaling, including perivascular accumulation of Lyve1+ IMs and their binding to hyaluronan.NEW & NOTEWORTHY Expression of the redox enzyme EC-SOD limits PH severity. Using vascular-selective EC-SOD depletion and SOD mimetic treatment in chronic hypoxic PH, we demonstrated that EC-SOD limits the hypoxia-induced accumulation of IMs. IMs from mice with low vascular EC-SOD were proinflammatory at baseline and enhanced ECM remodeling pathway activation in response to hypoxia. We identified Lyve1+ IMs as a perivascular, ECM-interacting subset that accumulate in hypoxia and could contribute to vascular remodeling in PH.

Using lung immunohistochemistry and stereology, platelets were found to accumulate and co-localize with leukocytes, particularly monocytes, within the mural and adventitial space of remodeled vessels of patients with pulmonary arterial hypertension. The presumed signaling between these cell types invites further studies into the role of platelet-monocyte aggregates in pulmonary hypertension.

The complement system is central to the innate immune response, playing a critical role in proinflammatory and autoimmune diseases such as pulmonary hypertension (PH). Recent discoveries highlight the emerging role of intracellular complement, or the “complosome,” in regulating cellular processes such as glycolysis, mitochondrial dynamics, and inflammatory gene expression. This study investigated the hypothesis that intracellular complement proteins C3, CFB, and CFD are upregulated in PH fibroblasts (PH-Fibs) and drive their metabolic and inflammatory states, contributing to PH progression. Our results revealed a pronounced upregulation of CFD, CFB, and C3 in PH-Fibs from human samples and bovine models, both in vivo and in vitro. The finding of elevated levels of C3 activation fragments, including C3b, C3d, and C3a, emphasized enhanced C3 activity. PH-Fibs exhibited notable metabolic reprogramming and increased levels of proinflammatory mediators such as MCP1, SDF1, IL-6, IL-13, and IL-33. Silencing CFD via shRNA reduced CFB activation and C3a production, while normalizing glycolysis, tricarboxylic acid (TCA) cycle activity, and fatty acid metabolism. Metabolomic and gene expression analyses of CFD-knockdown PH-Fibs revealed restored metabolic and inflammatory profiles, underscoring CFD’s crucial role in these changes. This study emphasizes the crucial role of intracellular complement in PH pathogenesis, highlighting the potential for complement-targeted therapies in PH.

Here, we present a protocol for automated quantification and viability analysis of pulmonary arterial cells (PACs) using a cellular image cytometer. We describe steps for obtaining PACs from the Pulmonary Hypertension Breakthrough Initiative, culturing and seeding them on microfluidic chips, and integrating chips into 6-well plates. Cells are stained with Hoechst 33342 and propidium iodide for live/dead analysis. Automated imaging and software-assisted quantification standardize workflows and broaden applications in pulmonary vascular research. For complete details on the use and execution of this protocol, please refer to Al Hilal et al.1.

Rationale: In pulmonary hypertension (PH), females generally exhibit better right ventricular (RV) function than males, despite having higher PH prevalence. While PH alters RV metabolism, the mechanisms of sex-specific RV protection and the metabolic effects on individual cell types remain unknown. Previous studies showed high intensity interval training (HIIT) exercise lowered RV systolic pressures (RVSP) and improved cardiac index (CI) in rat monocrotaline-induced mild PH. We hypothesized that HIIT would improve RV hemodynamics and restore RV metabolism in rat Sugen Hypoxia (SuHx) severe PH in a sexually dimorphic manner.Methods: 8-week-old Sprague Dawley rats (n=14 male, n=8 female) received Sugen (SU5416) 20mg/kg and were housed for 3 weeks in hypobaric hypoxia followed by 3 weeks normoxia. SuHx rats were randomized 1:1 to remain sedentary (SuHx-Sed) or undergo HIIT exercise (SuHx-HIIT) on a treadmill 5x weekly for 6 weeks. Control rats (n=6 male, n=4 female) were housed in normoxia for 6 weeks. Rats underwent critical speed testing at baseline and 6 weeks. Hemodynamics were obtained followed by collection of peripheral blood and heart tissues. Blood was fractionated into plasma, red blood cells (RBCs), and peripheral blood mononuclear cells (PBMCs). The RV was dissected and digested to isolate myocytes, macrophages, and endothelial cells. Metabolomic profiling was performed on all samples using Ultra-High Performance Liquid Chromatography-Mass Spectrometry.Results: At baseline, female rats demonstrated better exercise endurance than males, but this sex difference was abrogated in SuHx-Sed and SuHx-HIIT conditions. SuHx-Sed males developed more severe PH and had higher RV contractility than SuHx-Sed females. Female rats developed higher mPAP and RVESP in response to HIIT exercise. One male and one female rat in the SuHx-HIIT group died during exercise before the end of the study period. HIIT had no significant effects on RV contractility, RV/PA coupling, RV cardiac output or cardiac index on surviving rats. Statistically significant sexual dimorphisms in the metabolome were seen in both peripheral and RV cellular compartments, and changes in metabolites in response to SuHx and SuHX with HIIT were observed in a preliminary analysis of samples from 12 rats. Conclusions: HIIT exercise may be detrimental to RV hemodynamics in severe rat SuHx PH, in contrast to previous studies showing benefit in mild PH. Metabolomic analyses are ongoing to determine whether changes in the peripheral compartment correlate with changes in the RV cellular compartment, and whether metabolomic shifts in response to SuHx and SuHX with HIIT exhibit sexual dimorphisms.

Introduction: Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease with no cure, and limited access to diseased lung tissue has directed research toward rodent models. Recent studies suggest that proteomics, metabolomics, or lipidomics alone can provide valuable insights into PAH pathobiology; however, an integrated multi-omics approach combining these domains has not yet been explored. Such an approach could offer a more comprehensive view of disease mechanisms. This study hypothesizes that interactions among the proteome, metabolome, and lipidome may reveal novel candidate biomarkers and enhance our understanding of PAH pathobiology. Methods and Results: We analyzed peripheral blood samples from 77 control (CTL, 55.5±16.2yrs, 61% female) subjects and 402 PAH (52.3±15.7yrs, 75% female) patients (defined by a mean pulmonary arterial pressure above 25 mmHg) collected and biobanked at Stanford University between 2008 and 2014. Mass spectrometry was used to perform untargeted proteomics, metabolomics, and lipidomics. To correct for systematic biases, we applied global data adjustment and variance stabilizing normalization, while imputing 0.25% missing values. This resulted in a final dataset of 3,300 peptides, 121 metabolites, and 540 lipids. We identified 832 peptides, 45 metabolites, and 222 lipids that were up- or down-regulated in PAH patients compared to CTL subjects (Fig. 1a-c). Circulating levels of 2-Hydroxyglutarate (2HG), identified by recursive feature elimination wrapped within a random forest algorithm, revealed excellent performance as a differentiator of PAH in a testing cohort withheld from model training (AUC = 0.95, Sensitivity = 89.1%, Specificity = 87.5%, see Fig. 1d). This was further validated in an external cohort of 18 CTL and 8 PAH patients (AUC = 0.9, Sensitivity = 89.5%, Specificity = 72.2%). Latent network interaction discovery (SLIDE, Rahimikollu 2024) identified seven significant latent networks that could differentiate PAH from CTL subjects. Fig. 1e shows an example of one network showing 2HG interacting with 2-Oxoglutarate and a glycosphingolipid (GM3(d34:1)). The network also consists of acylcarnitine molecules interacting with fatty acids and complement. Other latent networks revealed evidence of hypoxia, glycolytic reprogramming, and inflammation. Conclusion: Untargeted multi-omics analysis of PAH patient plasma uncovered a prominent hypoxic signature linked to metabolic dysfunction. Key intermediaries, such as 2HG and 2-Oxoglutarate, highlight intricate crosstalk between hypoxia-inducible factor signaling, acylcarnitines, gangliosides, and inflammatory markers in PAH pathogenesis. This study provides the first comprehensive view of how the proteome, metabolome, and lipidome collectively interact within established pathobiological pathways of PAH, aligning with and expanding upon findings in the broader PAH literature.

Collagen cross-links mediated by the lysyl oxidase and lysyl hydroxylase families of enzymes significantly contribute to the biomechanical strength and rigidity of tissues, influencing cell signaling and the downstream cell phenotype. In the clinic, the proteolytically liberated N-terminal cross-linked peptide of collagen I (NTX) is used as a biomarker of bone and connective tissue turnover, which is altered in several disease processes. Despite the clinical utility of these collagen breakdown products, the majority of the cross-linked peptide species have not been identified in proteomic datasets. Here, we evaluate several parameters for the preparation and identification of these peptides from the collagen I-rich Achilles tendon. Our refined approach, which involves chemical digestion for protein solubilization coupled with mass spectrometry, enables the identification of NTX cross-links in a range of modification states. We then applied a spectral library approach to identify differences in collagen cross-links in bovine pulmonary hypertension. The presented method offers unique opportunities to understand extracellular matrix remodeling events in development, aging, wound healing, and fibrotic disease that modulate collagen architecture through lysyl hydroxylase and lysyl oxidase enzymes.

Inflammation drives the initiation and progression of pulmonary hypertension (PH). Platelets, increasingly recognized as immune cells, are activated and increased in the lungs of patients with PH. Platelet activation leads to the release of α-granule chemokines, many of which are implicated in PH. We hypothesized that hypoxia-induced secretion of platelet α-granule stored proteins and PH would be prevented in Nbeal2 -/-, α-granule deficient mice. WT and Nbeal2 -/- mice were maintained in normoxia or exposed to 10% hypobaric hypoxia for 3, 14, 21, or 35 days. We observed macrothrombocytopenia, increased circulating neutrophils and monocytes, and increased lung interstitial macrophages in Nbeal2 -/- mice at baseline. Hypoxia-induced platelet activation was attenuated, and hypoxia-induced increase in lung PF4 and platelets was delayed in Nbeal2 -/- mice compared to WT mice. Finally, although pulmonary vascular remodeling (PVR) and PH were attenuated at day 21, Nbeal2 -/- mice were not protected against hypoxia-induced PVR and PH at day 35. While this mutation also impacted circulating monocytes, neutrophils, and lung IMs, all of which are critical in the development of experimental PH, we gained further support for the role of platelets and α-granule proteins, such as PF4, in PH progression and pathogenesis and made several observations that expand our understanding of α-granule deficient mice in chronic hypoxia.

Background: Schistosoma-induced pulmonary arterial hypertension (Sch-PAH) is one of the most common forms of PAH, but it has been understudied. About 5% of patients chronically infected with S. mansoni that have a chronic severe liver disease termed Schistosoma hepatosplenic disease (Sch-HSD) will develop Sch-PAH, likely due to the development of portal hypertension and chronic inflammation with the development of vascular shunts that allow the transfer of Schistosoma eggs to the pulmonary circulation where they cause inflammation. Sch-PAH is characterized by remodeled vessels with an accumulation of inflammatory cells in the adventitia, likely critical to disease pathogenesis. In the present study, we propose to characterize the circulatory immune profile of patients with Sch-HSD and Sch-PAH with the goal of identifying peripheral inflammatory cell differences. Methods: SchHSD, Sch-PAH, and health control peripheral blood mononuclear samples were collected, banked, and analyzed using multi-parameter mass cytometry time of flight (CyTOF). A panel of 35 antibodies was used to identify and phenotype the cells. Results: N=4 SchPAH, 5 SchHSD, and 7 healthy controls (HCs) were analyzed. We observed that CD4+ T cells, and particularly Tregs, decreased in both SchHSD and SchPAH compared to HCs. There were fewer naïve and Th1 CD4+ and fewer CD8+ T cells in both SchHSD and SchPAH compared to HCs. CD1c+ dendritic cells and intermediate monocytes were increased in both SchHSD and SchPAH compared to HCs. We observed more double-positive CD4+ CD8+ T cells, particularly in the Sch-HSD group, and also an increased number of activated T cells in this group. Two CD8+ subsets, an effector CD8+ T cell, and a Th1-like CD8+ T cell, were more increased in Sch-HSD.In contrast, plasmacytoid DCs, which produce type I interferon, were increased only in the Sch-PAH group. Conclusions: Although Sch-HSD and Sch-PH are both chronic forms of Schistosomiasis, this preliminary study indicates both have a distinct immune profile that may be related to the pathogenesis of the disease. Key distinguishing factors between SchPAH and SchHSD may be more plasmacytoid DCs in SchPAH and more double-positive CD4-CD8 T cells and effector CD8 T cells in SchHSD.

Background: Pulmonary hypertension (PH) is a severe cardiopulmonary disorder characterized by high blood pressure in the pulmonary arteries (PAs), resulting in significant morbidity and reduced life expectancy. Disrupted cell-cell communication triggers inflammation and vascular remodeling, eventually leading to PH. Recently we have shown complement-containing sEVs from adventitial fibroblasts induce proinflammatory and metabolic reprogramming in macrophages. Most studies to date have focused on sEVs derived from cultured cells or body fluids, such as plasma, serum, urine, semen, and milk. More recent studies have revealed two distinct types of nanovesicles present in tissues: matrix-bound nanovesicles (MBVs) and liquid-phase nanovesicles, also known as sEVs. The roles of these nanovesicles in ECM remodeling and cell-cell communication, particularly within intact tissue in the context of PH, remain unexplored. Methods and Results: Lung tissue from young control bovines and two-week hypoxic bovine models, which develop severe PH in 2week of hypoxia, was inflated with agarose and sectioned using a vibratome to prepare precision-cut lung slices (PCLS). Distal PAs of similar size, along with adjacent airways, were excised, and the regions of interest (1-2 mm x 1-2 mm) were processed for transmission electron microscopy (TEM). Thin sections (∼70 nm) of resin-embedded tissue were then imaged by TEM. Our observations revealed two distinct types of sEVs in the tissue: one group, MBV, located in close proximity to extracellular matrices (ECM), such as collagen and elastin fibers, and another group as liquid phase nanovesicles / sEVs located in extracellular space. To quantify these vesicles, we isolated sEVs from dPA and found a significant increase in sEVs quantity in the PH dPA compared to control dPA. Additionally, pathways analysis proteomic data of the decellularized ECM from dPA showed enrichment of complement and coagulation cascades, suggesting that MBVs are closely associated with ECM proteins and may play a role in complement-mediated ECM remodeling. To confirm the proteomic findings and complement abundance in individual nanovesicles, immuno-TEM with C3 immunogold labeling of purified sEVs from dPA revealed a higher expression of complement C3 protein in PH samples than controls. Conclusions: This study identifies two distinct types of sEVs in pulmonary tissue, with a higher quantity of sEVs and complement C3 protein expression in PH. These findings also suggest that MBVs may contribute to complement-mediated ECM remodeling and macrophage activation toward a proinflammatory phenotype, potentially driving disease progression in pulmonary hypertension (Fig.1). Further exploration of these pathways could lead to new therapeutic strategies.

Collagen cross-links created by the lysyl oxidase and lysyl hydroxylase families of enzymes are a significant contributing factor to the biomechanical strength and rigidity of tissues, which in turn influence cell signaling and ultimately cell phenotype. In the clinic, the proteolytically liberated N-terminal cross-linked peptide of collagen I (NTX) is used as a biomarker of bone and connective tissue turnover, which is altered in several disease processes. Despite the clinical utility of these collagen breakdown products, the majority of the cross-linked peptide species have not been identified in proteomic datasets. Here we evaluate several parameters for the preparation and identification of these peptides from the collagen I-rich Achilles tendon. Our refined approach involving chemical digestion for protein solubilization coupled with mass spectrometry allows for the identification of the NTX cross-links in a range of modification states. Based on the specificity of the enzymatic cross-linking reaction we utilized follow-up variable modification searches to facilitate identification with a wider range of analytical workflows. We then applied a spectral library approach to identify differences in collagen cross-links in bovine pulmonary hypertension. The presented method offers unique opportunities to understand extracellular matrix remodeling events in development, aging, wound healing, and fibrotic disease that modulate collagen architecture through lysyl-hydroxylase and lysyl-oxidase enzymes.

Schistosomiasis-induced pulmonary hypertension (PH) presents a significant global health burden, yet the underlying mechanisms remain poorly understood. Here, we investigate the involvement of platelets and the complement system in the initiation events leading to Schistosoma-induced PH. We demonstrate that Schistosoma exposure leads to thrombocytopenia, platelet accumulation in the lung, and platelet activation. In addition, we observed increased plasma complement anaphylatoxins C3a and C5a, indicative of complement system activation, and elevated platelet expression of C1q, C3, decay activating factor (DAF), and complement C3a and C5a receptors. Our findings suggest the active involvement of platelets in responding to complement system signals induced by Schistosoma exposure and form the basis for future mechanistic studies on how complement may regulate platelet activation and promote the development of Schistosoma-induced PH.NEW & NOTEWORTHY Schistosomiasis-induced pulmonary hypertension (PH) is a significant global health burden, yet the underlying mechanisms remain poorly understood. We demonstrate that Schistosoma exposure leads to platelet accumulation in the lung and platelet activation. We observed increased plasma levels of C3a and C5a, indicative of complement system activation, and elevated expression of platelet complement proteins and receptors. These findings underscore the role of platelets and complement in the inflammatory responses associated with Schistosoma-induced PH.

Pulmonary hypertension (PH) significantly impairs exercise capacity and the quality of life in patients, which is influenced by dysfunctions in multiple organ systems, including the right ventricle, lungs, and skeletal muscles. Recent research has identified metabolic reprogramming and mitochondrial dysfunction as contributing factors to reduced exercise tolerance in PH patients. In this study, we investigated the therapeutic potential of enhancing mitochondrial function through the activation of the mitochondrial deacetylase SIRT3, using SIRT3 activator Honokiol combined with the SIRT3 co-factor nicotinamide adenine dinucleotide (NAD), in a Sugen/Hypoxia-induced PH rat model. Our results show that Sugen/Hypoxia-induced PH significantly impairs RV, lung, and skeletal muscle function, leading to reduced exercise capacity. Treatment with Honokiol and NAD notably improved exercise endurance, primarily by restoring SIRT3 levels in skeletal muscles, reducing proteolysis and atrophy in the gastrocnemius, and enhancing mitochondrial complex I levels in the soleus. These effects were independent of changes in cardiopulmonary hemodynamics. We concluded that targeting skeletal muscle dysfunction may be a promising approach to improving exercise capacity and overall quality of life in PH patients.

BackgroundSchistosomiasis is a common cause of pulmonary hypertension (PH) worldwide. Type 2 inflammation contributes to the development of Schistosoma-induced PH. Specifically, interstitial macrophages (IMs) derived from monocytes play a pivotal role by producing thrombospondin-1 (TSP-1), which in turn activates TGF-β, thereby driving the pathology of PH. Resident and recruited IM subpopulations have recently been identified. We hypothesized that in Schistosoma-PH, one IM subpopulation expresses monocyte recruitment factors, whereas recruited monocytes become a separate IM subpopulation that expresses TSP-1.MethodsMice were intraperitoneally sensitized and then intravenously challenged with S. mansoni eggs. Flow cytometry on lungs and blood was performed on wildtype and reporter mice to identify IM subpopulations and protein expression. Single-cell RNA sequencing (scRNAseq) was performed on flow-sorted IMs from unexposed and at day 1, 3 and 7 following Schistosoma exposure to complement flow cytometry based IM characterization and identify gene expression.ResultsFlow cytometry and scRNAseq both identified 3 IM subpopulations, characterized by CCR2, MHCII, and FOLR2 expression. Following Schistosoma exposure, the CCR2+ IM subpopulation expanded, suggestive of circulating monocyte recruitment. Schistosoma exposure caused increased monocyte-recruitment ligand CCL2 expression in the resident FOLR2+ IM subpopulation. In contrast, the vascular pathology-driving protein TSP-1 was greatest in the CCR2+ IM subpopulation.ConclusionSchistosoma-induced PH involves crosstalk between IM subpopulations, with increased expression of monocyte recruitment ligands by resident FOLR2+ IMs, and the recruitment of CCR2+ IMs which express TSP-1 that activates TGF-β and causes PH.