Absence of transforming growth factor beta 1 in murine platelets reduces neointima formation without affecting arterial thrombosis

Eva Schütz; Magdalena L. Bochenek; Dennis R. Riehl; Markus Bosmann; Thomas Münzel; Stavros Konstantinides; Katrin Schäfer

doi:10.1160/TH17-02-0112

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2017; 117(09): 1782-1797
DOI: 10.1160/TH17-02-0112

Cellular Haemostasis and Platelets

Schattauer GmbH

Absence of transforming growth factor beta 1 in murine platelets reduces neointima formation without affecting arterial thrombosis

Authors

Eva Schütz

¹Center for Cardiology, Cardiology I, University Medical Center Mainz, Mainz, Germany

³Center for Translational Vascular Biology (CTVB), University Medical Center Mainz, Mainz, Germany
Magdalena L. Bochenek

¹Center for Cardiology, Cardiology I, University Medical Center Mainz, Mainz, Germany

²Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany

³Center for Translational Vascular Biology (CTVB), University Medical Center Mainz, Mainz, Germany
Dennis R. Riehl

²Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany
Markus Bosmann

²Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany

³Center for Translational Vascular Biology (CTVB), University Medical Center Mainz, Mainz, Germany
Thomas Münzel

¹Center for Cardiology, Cardiology I, University Medical Center Mainz, Mainz, Germany

³Center for Translational Vascular Biology (CTVB), University Medical Center Mainz, Mainz, Germany
Stavros Konstantinides

²Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany

³Center for Translational Vascular Biology (CTVB), University Medical Center Mainz, Mainz, Germany
Katrin Schäfer

¹Center for Cardiology, Cardiology I, University Medical Center Mainz, Mainz, Germany

³Center for Translational Vascular Biology (CTVB), University Medical Center Mainz, Mainz, Germany

Abstract

Summary

Platelet degranulation at the site of vascular injury prevents bleeding and may affect the chronic vascular wound healing response. Transforming Growth Factor (TGF)-β1 is a major component of platelet α-granules known to accumulating in thrombi. It was our aim to determine the role of TGFβ1 released from activated platelets for neointima formation following arterial injury and thrombosis. Mice with platelet-specific deletion of TGFβ1 (Plt.TGFβ-KO) underwent carotid artery injury. Immunoassays confirmed the absence of active TGFβ1 in platelet releasates and plasma of Plt.TGFβ-KO mice. Whole blood analyses revealed similar haematological parameters, and tail cut assays excluded major bleeding defects. Platelet aggregation and the acute thrombotic response to injury in vivo did not differ between Plt.TGFβ-KO and Plt.TGFβ-WT mice. Morphometric analysis revealed that absence of TGFβ1 in platelets resulted in a significant reduction of neointima formation with lower neointima area, intima-to-media ratio, and lumen stenosis. On the other hand, the media area was enlarged in mice lacking TGFβ1 in platelets and contained increased amounts of proteases involved in latent TGFβ activation, including MMP2, MMP9 and thrombin. Significantly increased numbers of proliferating cells and cells expressing the mesenchymal markers platelet-derived growth factor receptor-β or fibroblast-specific protein-1, and the macrophage antigen F4/80, were observed in the media of Plt.TGFβ-KO mice, whereas the medial smooth muscle-actin-immuno-positive area and collagen content did not differ between genotypes. Our findings support an essential role for platelet-derived TGFβ1 for the vascular remodelling response to arterial injury, apparently independent from the role of platelets in thrombosis or haemostasis.

Supplementary Material to this article is available online at www.thrombosis-online.com.

Keywords

Neointima - platelets - thrombosis - transforming growth factor beta 1 - vascular injury

Full Text

References

References
1 Amsterdam EA, Wenger NK, Brindis RG. et al. 2014 AHA/ACC guideline for the management of patients with non-ST-elevation acute coronary syndromes: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. Circulation 2014; 130: e344-e426.
2 Ross R, Glomset J, Harker L. Response to injury and atherogenesis. Am J Pathol 1977; 86: 675-684.
3 Chandrasekar B, Tanguay JF. Platelets and restenosis. J Am Coll Cardiol 2000; 35: 555-562.
4 Teeuwen K, van der Schaaf RJ, Adriaenssens T. et al. Randomized Multicenter Trial Investigating Angiographic Outcomes of Hybrid Sirolimus-Eluting Stents With Biodegradable Polymer Compared With Everolimus-Eluting Stents With Durable Polymer in Chronic Total Occlusions: The PRISON IV Trial. JACC Cardiovasc Interv 2017; 10: 133-143.
5 Assoian RK, Komoriya A, Meyers CA. et al. Transforming growth factor-beta in human platelets. Identification of a major storage site, purification, and characterization. J Biol Chem 1983; 258: 7155-7160.
6 Grainger DJ, Wakefield L, Bethell HW. et al. Release and activation of platelet latent TGF-beta in blood clots during dissolution with plasmin. Nat Med 1995; 01: 932-937.
7 Fafeur V, Terman BI, Blum J. et al. Basic FGF treatment of endothelial cells down-regulates the 85-KDa TGF beta receptor subtype and decreases the growth inhibitory response to TGF-beta 1. Growth Factors 1990; 03: 237-245.
8 Ward MR, Agrotis A, Jennings G. et al. Vascular types I and II transforming growth factor-beta receptor expression: differential dependency on tyrosine ki-nases during induction by TGF-beta. FEBS Lett 1998; 422: 197-200.
9 Battegay EJ, Raines EW, Seifert RA. et al. TGF-beta induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. Cell 1990; 63: 515-524.
10 Petrov VV, Fagard RH, Lijnen PJ. Stimulation of collagen production by transforming growth factor-beta1 during differentiation of cardiac fibroblasts to myofibroblasts. Hypertension 2002; 39: 258-263.
11 Sigel AV, Centrella M, Eghbali-Webb M. Regulation of proliferative response of cardiac fibroblasts by transforming growth factor-beta 1. J Mol Cell Cardiol 1996; 28: 1921-1929.
12 Nabel EG, Shum L, Pompili VJ. et al. Direct transfer of transforming growth factor beta 1 gene into arteries stimulates fibrocellular hyperplasia. Proc Natl Acad Sci USA 1993; 90: 10759-10763.
13 Kanzaki T, Tamura K, Takahashi K. et al. In vivo effect of TGF- beta 1. Enhanced intimal thickening by administration of TGF- beta 1 in rabbit arteries injured with a balloon catheter. Arterioscler Thromb Vasc Biol 1995; 15: 1951-1957.
14 Wolf YG, Rasmussen LM, Ruoslahti E. Antibodies against transforming growth factor-beta 1 suppress intimal hyperplasia in a rat model. J Clin Invest 1994; 93: 1172-1178.
15 Merrilees M, Beaumont B, Scott L. et al. Effect of TGF-beta(1) antisense S-oligo-nucleotide on synthesis and accumulation of matrix proteoglycans in balloon catheter-injured neointima of rabbit carotid arteries. J Vasc Res 2000; 37: 50-60.
16 Yamamoto K, Morishita R, Tomita N. et al. Ribozyme oligonucleotides against transforming growth factor-beta inhibited neointimal formation after vascular injury in rat model: potential application of ribozyme strategy to treat cardiovascular disease. Circulation 2000; 102: 1308-1314.
17 Majesky MW, Lindner V, Twardzik DR. et al. Production of transforming growth factor beta 1 during repair of arterial injury. J Clin Invest 1991; 88: 904-910.
18 Nikol S, Isner JM, Pickering JG. et al. Expression of transforming growth factor-beta 1 is increased in human vascular restenosis lesions. J Clin Invest 1992; 90: 1582-1592.
19 Lev PR, Salim JP, Marta RF. et al. Platelets possess functional TGF-beta receptors and Smad2 protein. Platelets 2007; 18: 35-42.
20 Hu JH, Wei H, Jaffe M. et al. Postnatal Deletion of the Type II Transforming Growth Factor-beta Receptor in Smooth Muscle Cells Causes Severe Aorto-pathy in Mice. Arterioscler Thromb Vasc Biol 2015; 35: 2647-2656.
21 Yang P, Schmit BM, Fu C. et al. Smooth muscle cell-specific Tgfbr1 deficiency promotes aortic aneurysm formation by stimulating multiple signaling events. Sci Rep 2016; 06: 35444
22 Li W, Li Q, Jiao Y. et al. Tgfbr2 disruption in postnatal smooth muscle impairs aortic wall homeostasis. J Clin Invest 2014; 124: 755-767.
23 Azhar M, Yin M, Bommireddy R. et al. Generation of mice with a conditional allele for transforming growth factor beta 1 gene. Genesis 2009; 47: 423-431.
24 Tiedt R, Schomber T, Hao-Shen H. et al. Pf4-Cre transgenic mice allow the generation of lineage-restricted gene knockouts for studying megakaryocyte and platelet function in vivo. Blood 2007; 109: 1503-1506.
25 Konstantinides S, Schäfer K, Koschnick S. et al. Leptin-dependent platelet aggregation and arterial thrombosis suggests a mechanism for atherothrombotic disease in obesity. J Clin Invest 2001; 108: 1533-1540.
26 Dellas C, Schäfer K, Rohm IK. et al. Leptin signalling and leptin-mediated activation of human platelets: importance of JAK2 and the phospholipases Cgam-ma2 and A2. Thromb Haemost 2007; 98: 1063-1071.
27 Schäfer K, Konstantinides S, Riedel C. et al. Different mechanisms of increased luminal stenosis after arterial injury in mice deficient for urokinase- or tissue-type plasminogen activator. Circulation 2002; 106: 1847-1852.
28 Soslau G, Morgan DA, Jaffe JS. et al. Cytokine mRNA expression in human platelets and a megakaryocytic cell line and cytokine modulation of platelet function. Cytokine 1997; 09: 405-411.
29 Hoying JB, Yin M, Diebold R. et al. Transforming growth factor beta1 enhances platelet aggregation through a non-transcriptional effect on the fibrinogen receptor. J Biol Chem 1999; 274: 31008-31013.
30 Meyer A, Wang W, Qu J. et al. Platelet TGF-beta1 contributions to plasma TGF-beta1, cardiac fibrosis, and systolic dysfunction in a mouse model of pressure overload. Blood 2012; 119: 1064-1074.
31 Ishibashi T, Miller SL, Burstein SA. Type beta transforming growth factor is a potent inhibitor of murine megakaryocytopoiesis in vitro. Blood 1987; 69: 1737-1741.
32 Kawasaki T, Dewerchin M, Lijnen HR. et al. Mouse carotid artery ligation induces platelet-leukocyte-dependent luminal fibrin, required for neointima development. Circ Res 2001; 88: 159-166.
33 Golino P, Ambrosio G, Ragni M. et al. Inhibition of leucocyte and platelet adhesion reduces neointimal hyperplasia after arterial injury. Thromb Haemost 1997; 77: 783-788.
34 Evans DJ, Jackman LE, Chamberlain J. et al. Platelet P2Y(12) receptor influences the vessel wall response to arterial injury and thrombosis. Circulation 2009; 119: 116-122.
35 Patil SB, Jackman LE, Francis SE. et al. Ticagrelor effectively and reversibly blocks murine platelet P2Y12-mediated thrombosis and demonstrates a requirement for sustained P2Y12 inhibition to prevent subsequent neointima. Arterioscler Thromb Vasc Biol 2010; 30: 2385-2391.
36 Rath D, Chatterjee M, Holtkamp A, Tekath N, Borst O, Vogel S, Müller K, Gawaz M, Geisler T. Evidence of an interaction between TGF-β1 and the SDF-1/CXCR4/CXCR7 axis in human platelets. Thromb Res 2016; 144: 79-84.
37 Smith JD, Bryant SR, Couper LL. et al. Soluble transforming growth factor-beta type II receptor inhibits negative remodeling, fibroblast transdifferentiation, and intimal lesion formation but not endothelial growth. Circ Res 1999; 84: 1212-1222.
38 Lijnen HR, Lupu F, Moons L, Carmeliet P, Goulding D, Collen D. Temporal and topographic matrix metalloproteinase expression after vascular injury in mice. Thromb Haemost 1999; 81: 799-807.
39 Rath D, Chatterjee M, Muller I. et al. Platelet expression of transforming growth factor beta 1 is enhanced and associated with cardiovascular prognosis in patients with acute coronary syndrome. Atherosclerosis 2014; 237: 754-759.
40 Osadnik T, Strzelczyk JK, Regula R. et al. The Relationships between Polymorphisms in Genes Encoding the Growth Factors TGF-beta1, PDGFB, EGF, bFGF and VEGF-A and the Restenosis Process in Patients with Stable Coronary Artery Disease Treated with Bare Metal Stent. PLoS One 2016; 11: e0150500.
41 Feinberg MW, Watanabe M, Lebedeva MA. et al. Transforming growth factor-beta1 inhibition of vascular smooth muscle cell activation is mediated via Smad3. J Biol Chem 2004; 279: 16388-16393.
42 Grainger DJ, Metcalfe JC, Grace AA. et al. Transforming growth factor-beta dynamically regulates vascular smooth muscle differentiation in vivo. J Cell Sci 1998; 111: 2977-2988.
43 Inamoto S, Kwartler CS, Lafont AL. et al. TGFBR2 mutations alter smooth muscle cell phenotype and predispose to thoracic aortic aneurysms and dissections. Cardiovasc Res 2010; 88: 520-529.
44 Ryan ST, Koteliansky VE, Gotwals PJ. et al. Transforming growth factor-beta-dependent events in vascular remodeling following arterial injury. J Vasc Res 2003; 40: 37-46.
45 Kulkarni AB, Huh CG, Becker D. et al. Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci USA 1993; 90: 770-774.
46 Shull MM, Ormsby I, Kier AB. et al. Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature 1992; 359: 693-699.
47 Tsunawaki S, Sporn M, Ding A. et al. Deactivation of macrophages by transforming growth factor-beta. Nature 1988; 334: 260-262.
48 Haudenschild CC, Schwartz SM. Endothelial regeneration II. Restitution of endothelial continuity. Lab Invest 1979; 41: 407-418.
49 van den Brand MJ, Rensing BJ, Morel MA. et al. The effect of completeness of revascularization on event-free survival at one year in the ARTS trial. J Am Coll Cardiol 2002; 39: 559-564.
50 Heimark RL, Twardzik DR, Schwartz SM. Inhibition of endothelial regeneration by type-beta transforming growth factor from platelets. Science 1986; 233: 1078-1080.
51 Takehara K, LeRoy EC, Grotendorst GR. TGF-beta inhibition of endothelial cell proliferation: alteration of EGF binding and EGF-induced growth-regulatory (competence) gene expression. Cell 1987; 49: 415-422.
52 Goumans MJ, Valdimarsdottir G, Itoh S. et al. Balancing the activation state of the endothelium via two distinct TGF-beta type I receptors. EMBO J 2002; 21: 1743-1753.
53 Castañares C, Redondo-Horcajo M, Magán-Marchal N. et al. Signaling by ALK5 mediates TGF-beta-induced ET-1 expression in endothelial cells: a role for migration and proliferation. J Cell Sci 2007; 120: 1256-1266.
54 Oh SP, Seki T, Goss KA. et al. Activin receptor-like kinase 1 modulates transforming growth factor-β1 signaling in the regulation of angiogenesis. Proc Natl Acad Sci USA 2000; 97: 2626-2631.

Supplementary Material

Online Supplementary Material (PDF) (PDF) (opens in new window)