Fhl1, a new spatially specific protein, regulates vein graft neointimal hyperplasia

Abstract

	•This study firstly used spatial transcriptomics technique to analyse the neointima and generated a specific neointimal transcriptomic atlas.
	•Fhl1 exhibits specific and stable expression in the spatial region of the neointima. It has thus far the highest enrichment of expression in the neointima in NIH phases, suggesting that it is a prominent molecular biomarker of neointima.
	•We generated rats with a Fhl1 deletion and found that insufficient Fhl1 expression caused an increase in the severity of vascular inflammation and proliferation during neointimal hyperplasia. Adenovirus-mediated FHL1 overexpression in human saphenous vein have beneficial effects in preventing neointimal hyperplasia. These highlight its potential as a therapeutic target for mitigating vein graft failure associated with cardiovascular procedures.
	•Spatial transcriptomics profiles and morphological observations demonstrated that a newly generated cell population outside the grafted vein with hybrid phenotype between SMCs and fibroblasts contributes to neointimal formation.

Keywords

Fhl1 / neointimal hyperplasia / spatial transcriptomics / vein graft remodelling

References

[1] MozaffarianD, Benjamin EJ, GoAS, et al. Heart disease and stroke statistics–2015 update: a report from the American Heart Association. Circulation. 2015; 131(4): e29-s322.

[2] RuelM, VermaS, BhattDL. What is the optimal revascularization strategy for left main coronary stenosis? JAMA Cardiol. 2017; 2(10): 1061-1062.

[3] AhmedI, Yandrapalli S. Internal Mammary Artery Bypass. StatPearls; 2022.

[4] MahmoudSA, Widrich J. Endoscopic Vein Harvesting. StatPearls [Internet]; 2023.

[5] MutoA, ModelL, ZieglerK, Eghbalieh SD, DardikA. Mechanisms of vein graft adaptation to the arterial circulation: insights into the neointimal algorithm and management strategies. Circ J. 2010; 74(8): 1501-1512.

[6] SalinasHM, KhanSI, McCormackMC, et al. Prevention of vein graft intimal hyperplasia with photochemical tissue passivation. J Vasc Surg. 2017; 65(1): 190-196.

[7] GaudinoM, Antoniades C, BenedettoU, et al. Mechanisms, consequences, and prevention of coronary graft failure. Circulation. 2017; 136(18): 1749-1764.

[8] OwensCD, GasperWJ, RahmanAS, Conte MS. Vein graft failure. J Vasc Surg. 2015; 61(1): 203-216.

[9] MenzoianJO, KosharAL, RodriguesN. Alexis Carrel, Rene Leriche, Jean Kunlin, and the history of bypass surgery. J Vasc Surg. 2011; 54(2): 571-574.

[10] ArnettDK, Blumenthal RS, AlbertMA, et al. 2019 ACC/AHA guideline on the primary prevention of cardiovascular disease: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. J Am Coll Cardiol. 2019; 74(10): e177-e232.

[11] BreslauRC, Deweese JA. Successful endophlebectomy of autogenous venous bypass graft. Ann Surg. 1965; 162(2): 251-254.

[12] OwensCD, WakeN, ConteMS, Gerhard-Herman M, BeckmanJA. In vivo human lower extremity saphenous vein bypass grafts manifest flow mediated vasodilation. J Vasc Surg. 2009; 50(5): 1063-1070.

[13] UchidaK, Sasahara M, MorigamiN, HazamaF, Kinoshita M. Expression of platelet-derived growth factor B-chain in neointimal smooth muscle cells of balloon injured rabbit femoral arteries. Atherosclerosis. 1996; 124(1): 9-23.

[14] WadeyK, LopesJ, BendeckM, George S. Role of smooth muscle cells in coronary artery bypass grafting failure. Cardiovasc Res. 2018; 114(4): 601-610.

[15] XuK, Al-AniMK, PanX, ChiQ, DongN, Qiu X. Plant-derived products for treatment of vascular intima hyperplasia selectively inhibit vascular smooth muscle cell functions. Evid Based Complement Alternat Med. 2018; 2018: 3549312.

[16] KallenbachK, Salcher R, HeimA, KarckM, Mignatti P, HaverichA. Inhibition of smooth muscle cell migration and neointima formation in vein grafts by overexpression of matrix metalloproteinase-3. J Vasc Surg. 2009; 49(3): 750-758.

[17] CooleyBC, NevadoJ, MelladJ, et al. TGF-beta signaling mediates endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling. Sci Transl Med. 2014; 6(227): 227ra34.

[18] LiL, TerryCM, BlumenthalDK, et al. Cellular and morphological changes during neointimal hyperplasia development in a porcine arteriovenous graft model. Nephrol Dial Transplant. 2007; 22(11): 3139-3146.

[19] CollinsMJ, LiX, LvW, et al. Therapeutic strategies to combat neointimal hyperplasia in vascular grafts. Expert Rev Cardiovasc Ther. 2012; 10(5): 635-647.

[20] HanXJ, ChenM, HongT, et al. Lentivirus-mediated RNAi knockdown of the gap junction protein, Cx43, attenuates the development of vascular restenosis following balloon injury. Int J Mol Med. 2015; 35(4): 885-892.

[21] BarresiMJF, Gilbert SF. Developmental Biology. Sinauer Associates; 2019.

[22] JunkerJP, NoelES, GuryevV, et al. Genome-wide RNA tomography in the zebrafish embryo. Cell. 2014; 159(3): 662-675.

[23] AspM, Bergenstrahle J, LundebergJ. Spatially resolved transcriptomes-next generation tools for tissue exploration. Bioessays. 2020; 42(10): e1900221.

[24] WaylenLN, NimHT, MartelottoLG, RamialisonM. From whole-mount to single-cell spatial assessment of gene expression in 3D. Commun Biol. 2020; 3(1): 602.

[25] TevesJM, WonKJ. Mapping cellular coordinates through advances in spatial transcriptomics technology. Mol Cells. 2020; 43(7): 591-599. doi:10.14348/molcells.2020.0020

[26] StahlPL, SalmenF, VickovicS, et al. Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science. 2016; 353(6294): 78-82.

[27] BaronM, TagoreM, HunterMV, et al. The stress-like cancer cell state is a consistent component of tumorigenesis. Cell Syst. 2020; 11(5): 536-546. e7.

[28] MoncadaR, Barkley D, WagnerF, et al. Integrating microarray-based spatial transcriptomics and single-cell RNA-seq reveals tissue architecture in pancreatic ductal adenocarcinomas. Nat Biotechnol. 2020; 38(3): 333-342.

[29] JiAL, RubinAJ, ThraneK, et al. Multimodal analysis of composition and spatial architecture in human squamous cell carcinoma. Cell. 2020; 182(2): 497-514. e22.

[30] ChaoqunW, Haoliang L, SuiqingH, et al. LIM and cysteine-rich domains 1 mediated flow-dependent vein graft remodeling. J Biol Regul Homeostat Agents. 2024; 38(2): 1127-1137.

[31] TangY, JiaY, FanL, et al. MFN2 prevents neointimal hyperplasia in vein grafts via destabilizing PFK1. Circ Res. 2022; 130(11): e26-e43.

[32] SalmenF, StahlPL, MollbrinkA, et al. Barcoded solid-phase RNA capture for spatial transcriptomics profiling in mammalian tissue sections. Nat Protoc. 2018; 13(11): 2501-2534.

[33] JemtA, SalmenF, LundmarkA, et al. An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries. Sci Rep. 2016; 6: 37137.

[34] JiaY, MaoC, MaZ, et al. PHB2 maintains the contractile phenotype of VSMCs by counteracting PKM2 splicing. Circ Res. 2022; 131(10): 807-824.

[35] LeeSM, TsuiSK, ChanKK, et al. Chromosomal mapping, tissue distribution and cDNA sequence of four-and-a-half LIM domain protein 1 (FHL1). Gene. 1998; 216(1): 163-170.

[36] ShathasivamT, Kislinger T, GramoliniAO. Genes, proteins and complexes: the multifaceted nature of FHL family proteins in diverse tissues. J Cell Mol Med. 2010; 14(12): 2702-2720.

[37] SatoK, KimuraM, SugiyamaK, et al. Four-and-a-half LIM domains 1 (FHL1) protein interacts with the rho guanine nucleotide exchange factor PLEKHG2/FLJ00018 and regulates cell morphogenesis. J Biol Chem. 2016; 291(48): 25227-25238.

[38] McGrathMJ, CottleDL, NguyenMA, et al. Four and a half LIM protein 1 binds myosin-binding protein C and regulates myosin filament formation and sarcomere assembly. J Biol Chem. 2006; 281(11): 7666-7683.

[39] D’ArcyCE, FeeneySJ, McLeanCA, et al. Identification of FHL1 as a therapeutic target for Duchenne muscular dystrophy. Hum Mol Genet. 2014; 23(3): 618-636.

[40] DingJ, CongY, LiF, et al. Muscle death participates in myofibrillar abnormalities in FHL1 knockout mice. Biochem Biophys Res Commun. 2020; 523(1): 105-111.

[41] Fisker HagAM, Pedersen SF, KjaerA. Gene expression of LOX-1, VCAM-1, and ICAM-1 in pre-atherosclerotic mice. Biochem Biophys Res Commun. 2008; 377(2): 689-693.

[42] IiyamaK, HajraL, IiyamaM, et al. Patterns of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression in rabbit and mouse atherosclerotic lesions and at sites predisposed to lesion formation. Circ Res. 1999; 85(2): 199-207.

[43] KwapiszewskaG, Wygrecka M, MarshLM, et al. Fhl-1, a new key protein in pulmonary hypertension. Circulation. 2008; 118(11): 1183-1194.

[44] TierneyJW, EvansBC, Cheung-FlynnJ, et al. Therapeutic MK2 inhibition blocks pathological vascular smooth muscle cell phenotype switch. JCI Insight. 2021; 6(19): e142339.

[45] MacabreyD, Deslarzes-Dubuis C, LongchampA, et al. Hydrogen sulphide release via the angiotensin converting enzyme inhibitor zofenopril prevents intimal hyperplasia in human vein segments and in a mouse model of carotid artery stenosis. Eur J Vasc Endovasc Surg. 2022; 63(2): 336-346.

[46] SharonyR, Pintucci G, SaundersPC, et al. Matrix metalloproteinase expression in vein grafts: role of inflammatory mediators and extracellular signal-regulated kinases-1 and -2. Am J Physiol Heart Circ Physiol. 2006; 290(4): H1651-H1659.

[47] TomasJJ, StarkVE, KimJL, et al. Beta-galactosidase-tagged adventitial myofibroblasts tracked to the neointima in healing rat vein grafts. J Vasc Res. 2003; 40(3): 266-275.

[48] BorinTF, Miyakawa AA, CardosoL, et al. Apoptosis, cell proliferation and modulation of cyclin-dependent kinase inhibitor p21(cip1) in vascular remodelling during vein arterialization in the rat. Int J Exp Pathol. 2009; 90(3): 328-337.

[49] WuW, WangC, ZangH, et al. Mature vascular smooth muscle cells, but not endothelial cells, serve as the major cellular source of intimal hyperplasia in vein grafts. Arterioscler Thromb Vasc Biol. 2020; 40(8): 1870-1890.

[50] WeiY, JiangH, ChaiC, et al. Endothelium-mimetic surface modification improves antithrombogenicity and enhances patency of vascular grafts in rats and pigs. JACC Basic Transl Sci. 2023; 8(7): 843-861.

[51] MannMJ, Whittemore AD, DonaldsonMC, et al. Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial. Lancet. 1999; 354(9189): 1493-1498.

[52] AlexanderJH, HafleyG, HarringtonRA, et al. Efficacy and safety of edifoligide, an E2F transcription factor decoy, for prevention of vein graft failure following coronary artery bypass graft surgery: PREVENT IV: a randomized controlled trial. JAMA. 2005; 294(19): 2446-2454.

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2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.