An intracellular complement system drives metabolic and proinflammatory reprogramming of vascular fibroblasts in pulmonary hypertension
Ram Raj Prasad, Sushil Kumar, Hui Zhang, Min Li, Cheng-Jun Hu, Suzette Riddle, Brittany A. McKeon, M.G. Frid, Konrad Hoetzenecker, Slaven Crnkovic, Grazyna Kwapiszewska, Rubin M. Tuder, Kurt R. Stenmark
Ram Raj Prasad, Sushil Kumar, Hui Zhang, Min Li, Cheng-Jun Hu, Suzette Riddle, Brittany A. McKeon, M.G. Frid, Konrad Hoetzenecker, Slaven Crnkovic, Grazyna Kwapiszewska, Rubin M. Tuder, Kurt R. Stenmark
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Research Article Pulmonology Vascular biology

An intracellular complement system drives metabolic and proinflammatory reprogramming of vascular fibroblasts in pulmonary hypertension

Abstract

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.

Authors

Ram Raj Prasad, Sushil Kumar, Hui Zhang, Min Li, Cheng-Jun Hu, Suzette Riddle, Brittany A. McKeon, M.G. Frid, Konrad Hoetzenecker, Slaven Crnkovic, Grazyna Kwapiszewska, Rubin M. Tuder, Kurt R. Stenmark

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Figure 4

CFD regulates the activation of C3 in PA adventitial fibroblasts.

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CFD regulates the activation of C3 in PA adventitial fibroblasts. (A) qR...
(A) qRT-PCR data show the mRNA level of CFD in scrambled-shRNA (Scr-sh) and CFD-knockdown (CFD-sh) PH-Fibs. (B) Western blot and (C) corresponding quantitative data of immunoblots from scrambled-shRNA and CFD-knockdown PH-Fibs. C3a denotes the activated component of C3, and CFBb represents the active fragment of CFB. The suppression of CFD through shRNA led to decreased levels of active components C3a and CFBb. (D) qRT-PCR data shows the overexpression of CFD in CO-Fibs utilizing the pLV-CFD expression vector. (E) Western blot and (F) corresponding quantitative data of immunoblots from control vector– and pLV-CFD expression vector–transfected CO-Fibs. C3a and CFBb indicate activation of C3 and CFB on overexpression of CFD in CO-Fibs. Paired 2-tailed t test was used to compare 2 groups of samples. Data are presented as mean ± SEM from bovine control fibroblasts (CO-Fibs Vector and CO-Fibs CFD Exp), n = 3; and PH fibroblast (PH-Fibs Scr-sh and PH-Fibs CFD-sh), n = 4. *P ≤ 0.05, **P < 0.01, ***P < 0.001.