The role of Fumarate hydratase 1 (FH1) in atherosclerosis

J. Wrobel (Köln)¹, D. Kagan (Köln)², S. Steinhofer (Köln)², D. Schatton (Köln)³, M. Babaki (Köln)⁴, M. Depuydt (Leiden)⁵, E. Bartok (Bonn)⁶, H. Nemade (Köln)², S. B. L. Jung (Köln)⁷, K. Tinaz (Köln)¹, P. Schelemei (Köln)², S. Wei (Utrecht)⁸, M. Mokry (Utrecht)⁹, M. De Winther (Amsterdam,)¹⁰, M. Pasparakis (Köln)¹¹, M. Mollenhauer (Köln)¹, S. Baldus (Köln)⁷, C. Frezza (Köln)³, H. Winkels (Köln)^1
1Herzzentrum der Universität zu Köln Klinik III für Innere Medizin Köln, Deutschland; ²Universitätsklinikum Köln Klinik III für Innere Medizin - Experimentelle Kardiologie Köln, Deutschland; ³Institute for Metabolomics in Ageing Cluster of Excellence Cellular Stress Responses in Aging-associated Diseases (CECAD) Köln, Deutschland; ⁴Center for Molecular Medicine Cologne (CMMC) Köln, Deutschland; ⁵Leiden Academic Centre for Drug Research LACDR/Biopharmaceutics Faculty of Science Leiden, Niederlande; ⁶Universitätsklinikum Bonn Institut für Experimentelle Hämatologie und Transfusionsmedizin Bonn, Deutschland; ⁷Herzzentrum der Universität zu Köln Klinik für Kardiologie, Angiologie, Pneumologie und Internistische Intensivmedizin Köln, Deutschland; ⁸University Medical Center Utrecht Experimental Cardiology Laboratory Utrecht, Niederlande; ⁹Central Diagnostics Laboratory, University Medical Center Utrecht Utrecht, Niederlande; ¹⁰University Medical Center Amsterdam Amsterdam,, Niederlande; ¹¹Universität zu Köln CECAD Research Center, Institute of Genetics Köln, Deutschland

Background: Atherosclerosis is a lipid-driven vascular inflammation in which macrophages play a central role in plaque initiation and progression. Excessive cholesterol uptake drives metabolic dysregulation in macrophages, promoting foam cell formation and disease progression. The mitochondrial TCA cycle enzyme fumarate hydratase (FH1) has been implicated in macrophage metabolic regulation and pro-atherosclerotic cytokine release, but its role in foam cell formation and atherosclerosis remains entirely unknown.

Aim: To investigate the role of FH1 in foam cell formation and atherosclerotic plaque progression, and to explore its potential clinical relevance in humans.

Methods/Results: In silico analysis of single-cell RNA sequencing of leukocytes from murine atherosclerotic aortas revealed decreased Fh1 expression in Trem2⁺ macrophages. Immunofluorescent imaging of atherosclerotic plaques in low-density lipoprotein receptor knock-out (LDLr^-/-) mice fed western-type diet for 12 weeks confirmed reduced Fh1 abundance in CD68^pos BODIPY^pos foamy macrophages compared to CD68^pos BODIPY^neg macrophages (n=3; P<0.001). FACS confirmed decreased FH1 abundance in aortic foamy macrophages in LDLr-/- mice (n=4/group; P=0.029). In silico CRISPR screen-analysis of BV2 myeloid cells undergoing foam cell formation ranked Fh1 as the TCA enzyme most associated with oxLDL uptake (n=5/group; P<0.001). Interestingly, in vitro, Fh1 expression was unchanged in oxLDL-treated bone marrow-derived macrophages (BMDMs), but co-stimulation with LPS, mimicking plaque inflammation, significantly reduced FH1 (n=3/group; P=0.001). Compared to vehicle, pre-stimulation with the FH1 inhibitor (FHIN1) reduced DIL-labeled oxLDL uptake in BMDMs (n=3/group; P=0.015). Genetic Fh1-deficiency in BMDMs reduced expression of foam cell associated genes including Trem2 (n=3/group; P=0.001), Cd36 (P=0.034) and Plin2 (P=0.044). In vivo, macrophage-specific Fh1 deletion (Cx3cr1-CreERT2 x Fh1-flox-flox (Cx3cr1^Fh1) mice) showed reduced Fh1 expression in peritoneal Cx3cr1⁺ leukocytes after 1 week of tamoxifen-containing HFD ((TAM-HFD) n =3/group; P=0.034). In comparison to controls, transplantation of Cx3cr1^Fh1 bone marrow into lethally-irradiated LDLr^-/- mice increased atherosclerotic burden after 12 weeks of HFD followed by 4 weeks TAM-HFD (n=7/group; P=0.021). Ongoing work is investigating phenotypic and inflammatory changes in the plaque microenvironment. In 1071 patients undergoing carotid endarterectomy, higher FH1 expression in plaques was associated with lower occurrence of stroke/TIA at the time of surgery (OR 0.84; 95% CI 0.73–0.96; P=0.009), independent of risk factors and lipid biomarkers.

Conclusion: Fh1 is downregulated in foam cells both in vitro and in vivo, and higher Fh1 expression in human plaques is associated with reduced cerebrovascular events, suggesting potential predictive relevance. FH1 deficiency decreased oxLDL uptake, altered foam cell gene expression, and increased atherosclerotic burden in mice. These findings highlight FH1 as a potential target for atherosclerosis prevention and therapy.