Complement Factor D Plays a Crucial Role in Metabolic and Pro-inflammatory Reprogramming of Vascular Fibroblasts in Pulmonary Hypertension

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Abstract 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.