Platelet-specific TGFβ Deficiency Aggravates Atherosclerosis, Vascular Inflammation, and Hypercholesterolemia in Mice

Abstract

Background

Atherosclerosis involves inflammatory and thrombotic mechanisms, to which platelets, CD4⁺ T effector cells, and transforming growth factor β (TGFβ) all contribute importantly. Platelets are the principal source of circulating TGFβ, which profoundly regulates CD4⁺ T effector cell responses. The impact of platelet-derived TGFβ on atherosclerosis is, however, unknown.

Objectives

The present work investigated how platelet-specific TGFβ-deficiency impacts CD4⁺ T effector cell responses and atherogenesis.

Methods

Murine platelet-selective TGFβ deficiency (plt-TGFβ^−/−) was created by a Pf4-Cre approach, and an atherosclerotic mouse model was established by functional abrogation of Ldlr and 10 to 15 weeks of a high-fat diet in plt-TGFβ^−/− mice and their non-plt-TGFβ^−/− littermates.

Results

En face Oil Red O staining of the aorta showed more atherosclerotic lesion formation in plt-TGFβ^−/− mice, with significant increases in both lesion size and lesion coverage of the total aortic area. Cryosections of the aortic root confirmed the aggravation of atherogenesis. Platelet-derived TGFβ deficiency increased circulating platelets and plasma levels of total cholesterol, LDL cholesterol, and triglycerides after a 10- or 15-week high-fat diet period. RNA sequencing and proteomic analyses of the aorta showed signs of CD4⁺ T effector cell and macrophage activation in plt-TGFβ^−/− mice.

Conclusion

Platelet-specific TGFβ deficiency aggravates atherosclerosis, via increasing arterial inflammation and plasma levels of cholesterol. Our findings demonstrate that platelet-derived TGFβ is prominently athero-protective.

Keywords

platelets - transforming growth factor β - CD4⁺ T cells - atherosclerosis - hyperlipidemia

Contributors' Statement

S.T., Y.S., and Z.S. performed experiments, interpreted the data, and wrote the manuscript; Y.M. performed research, interpreted the data, and revised the manuscript; A.G. and J.Z. performed experiments, interpreted the data, and revised the manuscript; D.F.J.K., W.L., J.A., H.H., M.W., M.H., M.Z., and J.P. designed the study, interpreted the data, and revised the manuscript; C.M. and N.L. designed the study, interpreted the data, organized the research, and wrote the manuscript.

^‡ These authors contributed equally to this article.

Publikationsverlauf

Eingereicht: 29. April 2025

Angenommen: 02. September 2025

Accepted Manuscript online:
03. September 2025

Artikel online veröffentlicht:
22. September 2025

• References

• 1 Libby P. The changing landscape of atherosclerosis. Nature 2021; 592 (7855): 524-533
• 2 Weber C, Habenicht AJR, von Hundelshausen P. Novel mechanisms and therapeutic targets in atherosclerosis: inflammation and beyond. Eur Heart J 2023; 44 (29) 2672-2681
• 3 Li N. CD4+ T cells in atherosclerosis: regulation by platelets. Thromb Haemost 2013; 109 (06) 980-990
• 4 Gawaz M, Geisler T, Borst O. Current concepts and novel targets for antiplatelet therapy. Nat Rev Cardiol 2023; 20 (09) 583-599
• 5 Gisterå A, Hansson GK. The immunology of atherosclerosis. Nat Rev Nephrol 2017; 13 (06) 368-380
• 6 Gisterå A, Ketelhuth DFJ, Malin SG, Hansson GK. Animal models of atherosclerosis—supportive notes and tricks of the trade. Circ Res 2022; 130 (12) 1869-1887
• 7 Hu H, Zhu L, Huang Z. et al. Platelets enhance lymphocyte adhesion and infiltration into arterial thrombus. Thromb Haemost 2010; 104 (06) 1184-1192
• 8 Gerdes N, Zhu L, Ersoy M. et al. Platelets regulate CD4⁺ T-cell differentiation via multiple chemokines in humans. Thromb Haemost 2011; 106 (02) 353-362
• 9 Zhou L, Lopes JE, Chong MM. et al. TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature 2008; 453 (7192): 236-240
• 10 Meyer A, Wang W, Qu J. et al. Platelet TGF-β1 contributions to plasma TGF-β1, cardiac fibrosis, and systolic dysfunction in a mouse model of pressure overload. Blood 2012; 119 (04) 1064-1074
• 11 Bjørklund MM, Hollensen AK, Hagensen MK. et al. Induction of atherosclerosis in mice and hamsters without germline genetic engineering. Circ Res 2014; 114 (11) 1684-1689
• 12 Gisterå A, Ketelhuth DF. Immunostaining of lymphocytes in mouse atherosclerotic plaque. Methods Mol Biol 2015; 1339: 149-159
• 13 van Duijn J, Kritikou E, Benne N. et al. CD8+ T-cells contribute to lesion stabilization in advanced atherosclerosis by limiting macrophage content and CD4+ T-cell responses. Cardiovasc Res 2019; 115 (04) 729-738
• 14 Gostynska S, Venkatesan T, Subramani K. et al. Megakaryocyte/platelet-derived TGF-β1 inhibits megakaryopoiesis in bone marrow by regulating thrombopoietin production in liver. Blood Adv 2022; 6 (11) 3321-3328
• 15 Bellucci S, Ignatova E, Jaillet N, Boffa MC. Platelet hyperactivation in patients with essential thrombocythemia is not associated with vascular endothelial cell damage as judged by the level of plasma thrombomodulin, protein S, PAI-1, t-PA and vWF. Thromb Haemost 1993; 70 (05) 736-742
• 16 Panova-Noeva M, Marchetti M, Russo L. et al. ADP-induced platelet aggregation and thrombin generation are increased in Essential Thrombocythemia and Polycythemia Vera. Thromb Res 2013; 132 (01) 88-93
• 17 Petersen-Uribe Á, Kremser M, Rohlfing AK. et al. Platelet-derived PCSK9 is associated with LDL metabolism and modulates atherothrombotic mechanisms in coronary artery disease. Int J Mol Sci 2021; 22 (20) 11179
• 18 Gisterå A, Ketelhuth DFJ. Lipid-driven immunometabolic responses in atherosclerosis. Curr Opin Lipidol 2018; 29 (05) 375-380
• 19 Andersson PO, Stockelberg D, Jacobsson S, Wadenvik H. A transforming growth factor-beta1-mediated bystander immune suppression could be associated with remission of chronic idiopathic thrombocytopenic purpura. Ann Hematol 2000; 79 (09) 507-513
• 20 Gisterå A, Robertson AK, Andersson J. et al. Transforming growth factor-β signaling in T cells promotes stabilization of atherosclerotic plaques through an interleukin-17-dependent pathway. Sci Transl Med 2013; 5 (196) 196ra100
• 21 Lievens D, Habets KL, Robertson AK. et al. Abrogated transforming growth factor beta receptor II (TGFβRII) signalling in dendritic cells promotes immune reactivity of T cells resulting in enhanced atherosclerosis. Eur Heart J 2013; 34 (48) 3717-3727
• 22 Chen PY, Qin L, Li G, Tellides G, Simons M. Smooth muscle FGF/TGFβ cross talk regulates atherosclerosis progression. EMBO Mol Med 2016; 8 (07) 712-728
• 23 Chen PY, Qin L, Li G. et al. Endothelial TGF-β signalling drives vascular inflammation and atherosclerosis. Nat Metab 2019; 1 (09) 912-926
• 24 Davì G, Patrono C. Platelet activation and atherothrombosis. N Engl J Med 2007; 357 (24) 2482-2494
• 25 Mackman N, Bergmeier W, Stouffer GA, Weitz JI. Therapeutic strategies for thrombosis: new targets and approaches. Nat Rev Drug Discov 2020; 19 (05) 333-352
• 26 Grainger DJ. Transforming growth factor beta and atherosclerosis: so far, so good for the protective cytokine hypothesis. Arterioscler Thromb Vasc Biol 2004; 24 (03) 399-404
• 27 Huo Y, Schober A, Forlow SB. et al. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat Med 2003; 9 (01) 61-67
• 28 McCaffrey TA, Consigli S, Du B. et al. Decreased type II/type I TGF-beta receptor ratio in cells derived from human atherosclerotic lesions. Conversion from an antiproliferative to profibrotic response to TGF-beta1. J Clin Invest 1995; 96 (06) 2667-2675
• 29 Karolczak K, Watala C. Blood platelets as an important but underrated circulating source of TGFβ. Int J Mol Sci 2021; 22 (09) 4492
• 30 Lev PR, Salim JP, Marta RF, Osorio MJ, Goette NP, Molinas FC. Platelets possess functional TGF-beta receptors and Smad2 protein. Platelets 2007; 18 (01) 35-42
• 31 Li X, Mara AB, Musial SC. et al. Coordinated chemokine expression defines macrophage subsets across tissues. Nat Immunol 2024; 25 (06) 1110-1122
• 32 Cochain C, Vafadarnejad E, Arampatzi P. et al. Single-cell RNA-Seq reveals the transcriptional landscape and heterogeneity of aortic macrophages in murine atherosclerosis. Circ Res 2018; 122 (12) 1661-1674
• 33 Deng H, Xu Y, Hu X. et al. MEKK3-TGFβ crosstalk regulates inward arterial remodeling. Proc Natl Acad Sci U S A 2021; 118 (51) e2112625118
• 34 Zhu L, Huang Z, Stålesen R, Hansson GK, Li N. Platelets provoke distinct dynamics of immune responses by differentially regulating CD4+ T-cell proliferation. J Thromb Haemost 2014; 12 (07) 1156-1165
• 35 Klingenberg R, Gerdes N, Badeau RM. et al. Depletion of FOXP3+ regulatory T cells promotes hypercholesterolemia and atherosclerosis. J Clin Invest 2013; 123 (03) 1323-1334
• 36 Wang S, Link F, Han M. et al. The interplay of TGF-β1 and cholesterol orchestrating hepatocyte cell fate, EMT, and signals for HSC activation. Cell Mol Gastroenterol Hepatol 2024; 17 (04) 567-587
• 37 Nicholson AC, Hajjar DP. Transforming growth factor-beta up-regulates low density lipoprotein receptor-mediated cholesterol metabolism in vascular smooth muscle cells. J Biol Chem 1992; 267 (36) 25982-25987
• 38 Goldstein JL, Brown MS. The LDL receptor. Arterioscler Thromb Vasc Biol 2009; 29 (04) 431-438
• 39 Reilly NA, Lutgens E, Kuiper J, Heijmans BT, Jukema JW. Effects of fatty acids on T cell function: role in atherosclerosis. Nat Rev Cardiol 2021; 18 (12) 824-837
• 40 Lambert MP. Platelets in liver and renal disease. Hematology (Am Soc Hematol Educ Program) 2016; 2016 (01) 251-255
• 41 Dooley S, ten Dijke P. TGF-β in progression of liver disease. Cell Tissue Res 2012; 347 (01) 245-256
• 42 Ghafoory S, Varshney R, Robison T. et al. Platelet TGF-β1 deficiency decreases liver fibrosis in a mouse model of liver injury. Blood Adv 2018; 2 (05) 470-480
• 43 Fabregat I, Moreno-Càceres J, Sánchez A. et al; IT-LIVER Consortium. TGF-β signalling and liver disease. FEBS J 2016; 283 (12) 2219-2232
• 44 Braczkowski MJ, Kufel KM, Kulińska J. et al. Pleiotropic action of TGF-beta in physiological and pathological liver conditions. Biomedicines 2024; 12 (04) 925
• 45 Chen H, Chen X, Wang G. Platelets: a review of their function and effects in liver diseases. Liver Res 2020; 4: 129-135
• 46 Balaphas A, Meyer J, Perozzo R. et al. Platelet transforming growth factor-β1 induces liver sinusoidal endothelial cells to secrete interleukin-6. Cells 2020; 9 (05) 1311
• 47 Ragusa R, Basta G, Neglia D. et al. PCSK9 and atherosclerosis: looking beyond LDL regulation. Eur J Clin Invest 2021; 51 (04) e13459
• 48 Preiss D, Tobert JA, Hovingh GK, Reith C. Lipid-modifying agents, from statins to PCSK9 inhibitors: JACC Focus Seminar. J Am Coll Cardiol 2020; 75 (16) 1945-1955
• 49 Seidah NG, Awan Z, Chrétien M, Mbikay M. PCSK9: a key modulator of cardiovascular health. Circ Res 2014; 114 (06) 1022-1036
• 50 Zhou X, Johnston TP, Johansson D. et al. Hypercholesterolemia leads to elevated TGF-beta1 activity and T helper 3-dependent autoimmune responses in atherosclerotic mice. Atherosclerosis 2009; 204 (02) 381-387