CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-κB/RelA in maintaining the homeostasis of human vascular cells

Abstract

Vascular cell functionality is critical to blood vessel homeostasis. Constitutive NF-κB activation in vascular cells results in chronic vascular inflammation, leading to various cardiovascular diseases. However, how NF-κB regulates human blood vessel homeostasis remains largely elusive. Here, using CRISPR/Cas9-mediated gene editing, we generated RelA knockout human embryonic stem cells (hESCs) and differentiated them into various vascular cell derivatives to study how NF-κB modulates human vascular cells under basal and inflammatory conditions. Multi-dimensional phenotypic assessments and transcriptomic analyses revealed that RelA deficiency affected vascular cells via modulating inflammation, survival, vasculogenesis, cell differentiation and extracellular matrix organization in a cell typespecific manner under basal condition, and that RelA protected vascular cells against apoptosis and modulated vascular inflammatory response upon tumor necrosis factor α (TNFα) stimulation. Lastly, further evaluation of gene expression patterns in IκBα knockout vascular cells demonstrated that IκBα acted largely independent of RelA signaling. Taken together, our data reveal a protective role of NF-κB/RelA in modulating human blood vessel homeostasis and map the human vascular transcriptomic landscapes for the discovery of novel therapeutic targets.

Key words： NF-κB; RelA; Stem cell; Inflammation; Apoptosis

References

1 Anders S, Pyl PT, Huber W (2015) HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics 31:166–169

2 Baker RG, Hayden MS, Ghosh S (2011) NF-kappaB, inflammation, and metabolic disease. Cell Metab 13:11–22

3 Barnes PJ, Karin M (1997) Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336:1066–1071

4 Brand K, Page S, Rogler G, Bartsch A, Brandl R, Knuechel R, Page M, Kaltschmidt C, Baeuerle PA, Neumeier D (1996) Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. J Clin Invest 97:1715–1722

5 Breitbach M, Kimura K, Luis TC, Fuegemann CJ, Woll PS, Hesse M, Facchini R, Rieck S, Jobin K, Reinhardt J (2018) In Vivo Labeling by CD73 Marks Multipotent Stromal Cells and Highlights Endothelial Heterogeneity in the Bone Marrow Niche. Cell Stem Cell 22(262–276):e267

6 Caplan AI, Correa D (2011) The MSC: an injury drugstore. Cell Stem Cell 9:11–15

7 Chen G, Chen Y, Chen H, Li L, Yao J, Jiang Q, Lin X, Wen J, Lin L (2011a) The effect of NF-kappaB pathway on proliferation and apoptosis of human umbilical vein endothelial cells induced by intermittent high glucose. Mol Cell Biochem 347:127–133

8 Chen G, Qiao Y, Yao J, Jiang Q, Lin X, Chen F, Lin F, Lin M, Lin L, Zhu P (2011b) Construction of NF-kappaB-targeting RNAi adenovirus vector and the effect of NF-kappaB pathway on proliferation and apoptosis of vascular endothelial cells. Mol Biol Rep 38:3089–3094

9 Chiba T, Kondo Y, Shinozaki S, Kaneko E, Ishigami A, Maruyama N, Umezawa K, Shimokado K (2006) A selective NFkappaB inhibitor, DHMEQ, reduced atherosclerosis in ApoE-deficient mice. J Atheroscler Thromb 13:308–313

10 Courtois G, Smahi A, Reichenbach J, Doffinger R, Cancrini C, Bonnet M, Puel A, Chable-Bessia C, Yamaoka S, Feinberg J (2003) A hypermorphic IkappaBalpha mutation is associated with autosomal dominant anhidrotic ectodermal dysplasia and T cell immunodeficiency. J Clin Invest 112:1108–1115

11 da Silva Meirelles L, Caplan AI, Nardi NB (2008) In search of the in vivo identity of mesenchymal stem cells. Stem Cells 26:2287–2299

12 Ding Q, Regan SN, Xia Y, Oostrom LA, Cowan CA, Musunuru K (2013) Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell 12:393–394

13 Duan S, Yuan G, Liu X, Ren R, Li J, Zhang W, Wu J, Xu X, Fu L, Li Y (2015) PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype. Nat Commun 6:10068

14 Fagerlund R, Behar M, Fortmann KT, Lin YE, Vargas JD, Hoffmann A (2015) Anatomy of a negative feedback loop: the case of IkappaBalpha. J R Soc Interface 12:0262

15 Fang J, Yang J, Wu X, Zhang G, Li T, Wang X, Zhang H, Wang CC, Liu GH, Wang L (2018) Metformin alleviates human cellular aging by upregulating the endoplasmic reticulum glutathione peroxidase. Aging Cell 7:e12765

16 Galley HF, Webster NR (2004) Physiology of the endothelium. Br J Anaesth 93:105–113

17 Gareus R, Kotsaki E, Xanthoulea S, van der Made I, Gijbels MJ, Kardakaris R, Polykratis A, Kollias G, de Winther MP, Pasparakis M (2008) Endothelial cell-specific NF-kappaB inhibition protects mice from atherosclerosis. Cell Metab 8:372–383

18 Hajra L, Evans AI, Chen M, Hyduk SJ, Collins T, Cybulsky MI (2000) The NF-kappa B signal transduction pathway in aortic endothelial cells is primed for activation in regions predisposed to atherosclerotic lesion formation. Proc Natl Acad Sci USA 97:9052–9057

19 Hiraoka A, Yano Ki K, Kagami N, Takeshige K, Mio H, Anazawa H, Sugimoto S (2001) Stem cell growth factor: in situ hybridization analysis on the gene expression, molecular characterization and in vitro proliferative activity of a recombinant preparation on primitive hematopoietic progenitor cells. Hematol J 2:307–315

20 Ijaz T, Wakamiya M, Sun H, Recinos A III, Tilton RG, Brasier AR (2016) Generation and characterization of a novel transgenic mouse harboring conditional nuclear factor-kappa B/RelA knockout alleles. BMC Dev Biol 16:32

21 Jakkampudi A, Jangala R, Reddy BR, Mitnala S, Nageshwar Reddy D, Talukdar R (2016) NF-kappaB in acute pancreatitis: Mechanisms and therapeutic potential. Pancreatology 16:477–488

22 Janssen-Heininger YM, Poynter ME, Baeuerle PA (2000) Recent advances towards understanding redox mechanisms in the activation of nuclear factor kappaB. Free Radic Biol Med 28:1317–1327

23 Khan SY, Awad EM, Oszwald A, Mayr M, Yin X, Waltenberger B, Stuppner H, Lipovac M, Uhrin P, Breuss JM (2017) Premature senescence of endothelial cells upon chronic exposure to TNFα can be prevented by N-acetyl cysteine and plumericin. Sci Rep 7:39501

24 Kida Y, Kobayashi M, Suzuki T, Takeshita A, Okamatsu Y, Hanazawa S, Yasui T, Hasegawa K (2005) Interleukin-1 stimulates cytokines, prostaglandin E2 and matrix metalloproteinase-1 production via activation of MAPK/AP-1 and NF-kappaB in human gingival fibroblasts. Cytokine 29:159–168

25 Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12:357–360

26 Kirton JP, Xu Q (2010) Endothelial precursors in vascular repair. Microvasc Res 79:193–199

27 Kubben N, Zhang W, Wang L, Voss TC, Yang J, Qu J, Liu GH, Misteli T (2016) Repression of the antioxidant NRF2 pathway in premature aging. Cell 165:1361–1374

28 Kucharczak J, Simmons MJ, Fan Y, Gelinas C (2003) To be, or not to be: NF-kappaB is the answer–role of Rel/NF-kappaB in the regulation of apoptosis. Oncogene 22:8961–8982

29 Lee TH, Sottile J, Chiang HY (2015) Collagen inhibitory peptide R1R2 mediates vascular remodeling by decreasing inflammation and smooth muscle cell activation. PLoS ONE 10:e0117356

30 Li Y, Zhang W, Chang L, Han Y, Sun L, Gong X, Tang H, Liu Z, Deng H, Ye Y (2016) Vitamin C alleviates aging defects in a stem cell model for Werner syndrome. Protein Cell 7:478–488

31 Liu GH, Qu J, Shen X (2008) NF-kappaB/p65 antagonizes Nrf2-ARE pathway by depriving CBP from Nrf2 and facilitating recruitment of HDAC3 to MafK. Biochim Biophys Acta 1783:713–727

32 Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD, Suzuki K, Kurian L, Walsh C (2011) Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome. Nature 472:221–225

33 Liu GH, Qu J, Suzuki K, Nivet E, Li M, Montserrat N, Yi F, Xu X, Ruiz S, Zhang W (2012) Progressive degeneration of human neural stem cells caused by pathogenic LRRK2. Nature 491:603–607

34 Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550

35 Mallavia B, Recio C, Oguiza A, Ortiz-Munoz G, Lazaro I, Lopez-Parra V, Lopez-Franco O, Schindler S, Depping R, Egido J (2013) Peptide inhibitor of NF-kappaB translocation ameliorates experimental atherosclerosis. Am J Pathol 182:1910–1921

36 Morris O, Liu X, Domingues C, Runchel C, Chai A, Basith S, Tenev T, Chen H, Choi S, Pennetta G (2016) Signal integration by the IkappaB protein pickle shapes drosophila innate host defense. Cell Host Microbe 20:283–295

37 Nedeljkovic ZS, Gokce N, Loscalzo J (2003) Mechanisms of oxidative stress and vascular dysfunction. Postgrad Med J 79:195–199

38 Osborn L, Kunkel S, Nabel GJ (1989) Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. Proc Natl Acad Sci USA 86:2336–2340

39 Pan H, Guan D, Liu X, Li J, Wang L, Wu J, Zhou J, Zhang W, Ren R, Zhang W (2016) SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2. Cell Res 26:190–205

40 Patsch C, Challet-Meylan L, Thoma EC, Urich E, Heckel T, O’Sullivan JF, Grainger SJ, Kapp FG, Sun L, Christensen K (2015) Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nat Cell Biol 17:994–1003

41 Perkins ND (2007) Integrating cell-signalling pathways with NFkappaB and IKK function. Nat Rev Mol Cell Biol 8:49–62

42 Perkins ND, Gilmore TD (2006) Good cop, bad cop: the different faces of NF-kappaB. Cell Death Diff 13:759–772

43 Rudijanto A (2007) The role of vascular smooth muscle cells on the pathogenesis of atherosclerosis. Acta Medica Indones 39:86–93

44 Salminen A, Huuskonen J, Ojala J, Kauppinen A, Kaarniranta K, Suuronen T (2008) Activation of innate immunity system during aging: NF-kB signaling is the molecular culprit of inflamm-aging. Ageing Res Rev 7:83–105

45 Schober A, Weber C (2005) Mechanisms of monocyte recruitment in vascular repair after injury. Antioxid Redox Signal 7:1249–1257

46 Simeonidis S, Stauber D, Chen G, Hendrickson WA, Thanos D (1999) Mechanisms by which IkappaB proteins control NFkappaB activity. Proc Natl Acad Sci USA 96:49–54

47 Tas SW, Vervoordeldonk MJ, Tak PP (2009) Gene therapy targeting nuclear factor-kappaB: towards clinical application in inflammatory diseases and cancer. Curr Gene Therapy 9:160–170

48 Tilstra JS, Clauson CL, Niedernhofer LJ, Robbins PD (2011) NFkappaB in Aging and Disease. Aging Dis 2:449–465

49 Uccelli A, Moretta L, Pistoia V (2008) Mesenchymal stem cells in health and disease. Nat Rev Immunol 8:726–736

50 Wang L, Yi F, Fu L, Yang J, Wang S, Wang Z, Suzuki K, Sun L, Xu X, Yu Y (2017) CRISPR/Cas9-mediated targeted gene correction in amyotrophic lateral sclerosis patient iPSCs. Protein Cell 8:365–378

51 Wang S, Hu B, Ding Z, Dang Y, Wu J, Li D, Liu X, Xiao B, Zhang W, Ren R (2018) ATF6 safeguards organelle homeostasis and cellular aging in human mesenchymal stem cells. Cell Discov 4:2

52 Wu Z, Zhang W, Song M, Wang W, Wei G, Li W, Lei J, Huang Y, Sang Y, Chan P (2018) Differential stem cell aging kinetics in Hutchinson-Gilford progeria syndrome and Werner syndrome. Protein Cell 9:333–350

53 Yang J, Li J, Suzuki K, Liu X, Wu J, Zhang W, Ren R, Zhang W, Chan P, Izpisua Belmonte JC (2017) Genetic enhancement in cultured human adult stem cells conferred by a single nucleotide recoding. Cell Res 27:1178–1181

54 Yu G, Wang LG, Han Y, He QY (2012) clusterProfiler: an R package for comparing biological themes among gene clusters. Omics 16:284–287

55 Yue R, Shen B, Morrison SJ (2016) Clec11a/osteolectin is an osteogenic growth factor that promotes the maintenance of the adult skeleton. eLife 5

56 Zhang W, Li J, Suzuki K, Qu J, Wang P, Zhou J, Liu X, Ren R, Xu X, Ocampo A (2015) Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging. Science 348:1160–1163

57 Zhang W, Song M, Qu J, Liu G (2018) Epigeneic modifications in cardiovascular aging and diseases. Circ Res (in press)