Pulmonary hypertension (PH) is a severe cardiopulmonary disease characterized by complement-dependent, fibroblast-induced perivascular accumulation and proinflammatory activation of macrophages. We hypothesized that, in PH, nanoscale-sized small extracellular vesicles (sEVs), released by perivascular/adventitial fibroblasts, are critical mediators of complement-dependent proinflammatory activation of macrophages. Pulmonary adventitial fibroblasts were isolated from calves with severe PH (PH-Fibs) and age-matched controls (CO-Fibs). PH-Fibs exhibited increased secretion of sEVs, compared with CO-Fibs, and sEV biological activity was tested on mouse and bovine bone marrow–derived macrophages (BMDMs) and showed similar responses. Compared with sEVs derived from CO-Fibs, sEVs derived from PH-Fibs (PH-Fib-sEVs) induced augmented expression of proinflammatory cytokines/chemokines and metabolic genes in BMDMs. Pharmacological blockade of exosome release from PH-Fibs resulted in significant attenuation of proinflammatory activation of BMDMs. “Bottom-up” proteomic analyses revealed significant enrichment of complement and coagulation cascades in PH-Fib-sEVs, including augmented expression of the complement component C3. We therefore examined whether the PH-Fib-sEV–mediated proinflammatory activation of BMDMs was complement C3 dependent. Treatment of PH-Fibs with siC3-RNA significantly attenuated the capacity of PH-Fib-sEVs for proinflammatory activation of BMDMs. PH-Fib-sEVs mediated proglycolytic alterations and complement-dependent activation of macrophages toward a proinflammatory phenotype, as confirmed by metabolomic studies. Thus, fibroblast-released sEVs served as critical mediators of complement-induced perivascular/microenvironmental inflammation in PH.

Fibroblasts, among the most prevalent and widely distributed cell types in the human body, play a crucial role in defining tissue structure. They do this by depositing and remodeling extracellular matrixes and organizing functional tissue networks, which are essential for tissue homeostasis and various human diseases. Pulmonary hypertension (PH) is a devastating syndrome with high mortality, characterized by remodeling of the pulmonary vasculature and significant cellular and structural changes within the intima, media, and adventitia layers. Most research on PH has focused on alterations in the intima (endothelial cells) and media (smooth muscle cells). However, research over the past decade has provided strong evidence of the critical role played by pulmonary artery adventitial fibroblasts in PH. These fibroblasts exhibit the earliest, most dramatic, and most sustained proliferative, apoptosis-resistant, and inflammatory responses to vascular stress. This review examines the aberrant phenotypes of PH fibroblasts and their role in the pathogenesis of PH, discusses potential molecular signaling pathways underlying these activated phenotypes, and highlights areas of research that merit further study to identify promising targets for the prevention and treatment of PH.

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.

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.