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

Pulmonary hypertension (PH) is a severe cardiopulmonary disease characterized by complement-dependent, fibroblast-induced perivascular accumulation and pro-inflammatory 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 pro-inflammatory 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 to CO-Fibs, and sEV biological activity was tested on mouse and bovine bone marrow-derived macrophages (BMDMs) and showed similar responses. PH-Fib-sEVs induced augmented expression of pro-inflammatory cytokines/chemokines and metabolic genes in BMDMs, compared to CO-Fib-sEVs. Pharmacological blockade of exosome release from PH-Fibs resulted in significant attenuation of pro-inflammatory activation of BMDMs. "Bottom-up" proteomic analyses revealed significant enrichment of complement and Coagulation cascades in PH-Fib-sEVs, including augmented expression of complement component C3. We therefore examined whether PH-Fib-sEVs-mediated pro-inflammatory activation of BMDMs was complement C3-dependent. Treatment of PH-Fibs with siC3-RNA significantly attenuated the capacity of PH-Fib-sEVs for pro-inflammatory activation of BMDMs. PH-Fib-sEVs mediated pro-glycolytic alterations and complement-dependent activation of macrophages toward a pro-inflammatory phenotype, as confirmed by metabolomic studies. Thus, fibroblast-released sEVs can serve as critical mediators of complement-induced perivascular/microenvironmental inflammation in PH.