Function of Krüppel‑like factor 2 in the shear stress‑induced cell differentiation of endothelial progenitor cells to endothelial cells

Chu,Hai‑Rong; Sun,Yu‑Cong; Gao,Yu; Guan,Xiu‑Mei; Yan,Hong; Cui,Xiao‑Dong; Zhang,Xiao‑Yun; Li,Xin; Li,Hong; Cheng,Min

doi:10.3892/mmr.2019.9819

Function of Krüppel‑like factor 2 in the shear stress‑induced cell differentiation of endothelial progenitor cells to endothelial cells

Authors:
- Hai‑Rong Chu
- Yu‑Cong Sun
- Yu Gao
- Xiu‑Mei Guan
- Hong Yan
- Xiao‑Dong Cui
- Xiao‑Yun Zhang
- Xin Li
- Hong Li
- Min Cheng
View Affiliations

Affiliations: Medicine Research Center, Clinical Medical College, Weifang Medical University, Weifang, Shandong 261053, P.R. China
Published online on: January 3, 2019 https://doi.org/10.3892/mmr.2019.9819
Pages: 1739-1746

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The present study aimed to evaluate the effects of Krüppel‑like factor 2 (KLF2) on the differentiation of endothelial progenitor cells (EPCs) to endothelial cells (ECs) induced by shear stress, and to investigate the corresponding mechanisms. Cultured rat late EPCs were exposed to shear stress (12 dyn/cm2) for different lengths of time. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was used to measure the initial KLF2 mRNA levels in each group. Subsequently, the EPCs were treated with anti‑integrin β1 or β3 antibodies to block integrin β1 and β3, respectively, or cytochalasin D to destroy F‑actin, and the subsequent expression levels of KLF2 in EPCs were measured. Then, KLF2 small interfering RNAs (siRNAs) were transfected into EPCs, and RT‑qPCR was used to measure the mRNA expression level of KLF2. Additionally, flow cytometry was applied to evaluate the protein levels of cluster of differentiation 31 (CD31) and the von Willebrand factor (vWF), and the regulatory effects of KLF2 in the promoter region of vWF were determined via a luciferase assay. High shear stress upregulated KLF2 expression, while blocking integrin β1/β3 or destroying F‑actin resulted in a corresponding decrease in KLF2 expression. Downregulation of KLF2 expression by siKLF2 inhibited the differentiation of EPCs to ECs under shear stress conditions, while the expression of EC‑specific markers decreased, including CD31 and vWF. Various lengths of the vWF promoter region induced vWF expression, and EPCs co‑transfected with KLF2 significantly increased the vWF expression levels compared with the group treated with vWF alone (P<0.01). In conclusion, shear stress may upregulate KLF2 expression, which may be associated with the integrin‑actin cytoskeleton system. Most importantly, the shear stress‑induced differentiation of EPCs may be mediated by KLF2.

View Figures

View References

1	Rafieian-Kopaei M, Setorki M, Doudi M, Baradaran A and Nasri H: Atherosclerosis: Process, indicators, risk factors and new hopes. Int J Prev Med. 5:927–946. 2014.PubMed/NCBI
2	Skrzypkowska M, Myśliwska J, Słomiński B, Siebert J, Gutknecht P and Rybastanisławowska M: Quantitative and functional characteristics of endothelial progenitor cells in newly diagnosed hypertensive patients. J Hum Hypertens. 29:324–330. 2015. View Article : Google Scholar : PubMed/NCBI
3	Stancu CS, Toma L and Sima AV: Dual role of lipoproteins in endothelial cell dysfunction in atherosclerosis. Cell Tissue Res. 349:433–446. 2012. View Article : Google Scholar : PubMed/NCBI
4	Zhang X, Cui X, Cheng L, Guan X, Li H, Li X and Cheng M: Actin stabilization by jasplakinolide affects the function of bone marrow-derived late endothelial progenitor cells. PLoS One. 7:e508992012. View Article : Google Scholar : PubMed/NCBI
5	Cui X, Zhang X, Guan X, Li H, Li X, Lu H and Cheng M: Shear stress augments the endothelial cell differentiation marker expression in late EPCs by upregulating integrins. Biochem Biophys Res Commun. 425:419–425. 2012. View Article : Google Scholar : PubMed/NCBI
6	Cheng M, Guan X, Li H, Cui X, Zhang X, Li X, Jing X, Wu H and Avsar E: Shear stress regulates late EPC differentiation via mechanosensitive molecule-mediated cytoskeletal rearrangement. Plos One. 8:e676752013. View Article : Google Scholar : PubMed/NCBI
7	Bieker JJ: Kruppel-like factors: Three fingers in many pies. J Biol Chem. 276:34355–34358. 2001. View Article : Google Scholar : PubMed/NCBI
8	Dekker RJ, van Thienen JV, Rohlena J, de Jager SC, Elderkamp YW, Seppen J, de Vries CJ, Biessen EA, van Berkel TJ, Pannekoek H and Horrevoets AJ: Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes. Am J Pathol. 167:609–618. 2005. View Article : Google Scholar : PubMed/NCBI
9	Dekker RJ, Boon RA, Rondaij MG, Kragt A, Volger OL, Elderkamp YW, Meijers JC, Voorberg J, Pannekoek H and Horrevoets AJ: KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium. Blood. 107:4354–5363. 2006. View Article : Google Scholar : PubMed/NCBI
10	He S, Yang L, Li D and Li M: Kruppel-like factor 2-mediated suppression of MicroRNA-155 reduces the proinflammatory activation of macrophages. Plos One. 10:e1390602015.
11	Das M, Lu J, Joseph M, Aggarwal R, Kanji S, McMichael BK, Lee BS, Agarwal S, Ray-Chaudhury A, Iwenofu OH, et al: Kruppel-like factor 2 (KLF2) regulates monocyte differentiation and functions in mBSA and il-1β-induced arthritis. Curr Mol Med. 12:113–125. 2012. View Article : Google Scholar : PubMed/NCBI
12	Anderson KP, Kern CB, Crable SC and Lingrel JB: Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Krüppel-like factor: Identification of a new multigene family. Mol Cell Biol. 15:5957–5965. 1995. View Article : Google Scholar : PubMed/NCBI
13	Kuo CT, Veselits ML, Barton KP, Lu MM, Clendenin C and Leiden JM: The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis. Genes Dev. 11:2996–3006. 1997. View Article : Google Scholar : PubMed/NCBI
14	Taniguchi H, Jacinto FV, Villanueva A, Fernandez AF, Yamamoto H, Carmona FJ, Puertas S, Marquez VE, Shinomura Y, Imai K and Esteller M: Silencing of Kruppel-like factor 2 by the histone methyltransferase EZH2 in human cancer. Oncogene. 31:1988–1994. 2012. View Article : Google Scholar : PubMed/NCBI
15	Stroncek JD, Grant BS, Brown MA, Povsic TJ, Truskey GA and Reichert WM: Comparison of endothelial cell phenotypic markers of late-outgrowth endothelial progenitor cells isolated from patients with coronary artery disease and healthy volunteers. Tissue Eng Part A. 15:3473–3486. 2009. View Article : Google Scholar : PubMed/NCBI
16	Doddaballapur A, Michalik KM, Manavski Y, Lucas T, Houtkooper RH, You X, Chen W, Zeiher AM, Potente M, Dimmeler S and Boon RA: Laminar shear stress inhibits endothelial cell metabolism via KLF2-mediated repression of PFKFB3. Arterioscler Thromb Vasc Biol. 35:137–145. 2015. View Article : Google Scholar : PubMed/NCBI
17	National Research Council: Guide for the Care and Use of Laboratory Animals. The National Academies Press. (Washington, DC). 1996.
18	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
19	Obi S, Masuda H, Shizuno T, Sato A, Yamamoto K, Ando J, Abe Y and Asahara T: Fluid shear stress induces differentiation of circulating phenotype endothelial progenitor cells. Am J Physiol Cell Physiol. 303:C595–C606. 2012. View Article : Google Scholar : PubMed/NCBI
20	Ahsan T and Nerem RM: Fluid shear stress promotes an endothelial-like phenotype during the early differentiation of embryonic stem cells. Tissue Eng Part A. 16:3547–3553. 2010. View Article : Google Scholar : PubMed/NCBI
21	Chien S: Mechanotransduction and endothelial cell homeostasis: The wisdom of the cell. Am J Physiol Heart Circ Physiol. 292:H1209–H1224. 2007. View Article : Google Scholar : PubMed/NCBI
22	Ehsani Ardakani MJ, Safaei A, Arefi Oskouie A, Haghparast H, Haghazali M, Mohaghegh Shalmani H, Peyvandi H, Naderi N and Zali MR: Evaluation of liver cirrhosis and hepatocellular carcinoma using protein-protein interaction networks. Gastroenterol Hepatol Bed Bench. 9 Suppl 1:S14–S22. 2016.PubMed/NCBI
23	Cecchi E, Giglioli C, Valente S, Lazzeri C, Gensini GF, Abbate R and Mannini L: Role of hemodynamic shear stress in cardiovascular disease. Atherosclerosis. 214:249–256. 2011. View Article : Google Scholar : PubMed/NCBI
24	Doddaballapur A, Michalik KM, Manavski Y, Lucas T, Houtkooper RH, You X, Chen W, Zeiher AM, Potente M, Dimmeler S and Boon RA: Laminar shear stress inhibits endothelial cell metabolism via KLF2-mediated repression of PFKFB3. Arterioscler Thromb Vasc Biol. 35:137–145. 2015. View Article : Google Scholar : PubMed/NCBI
25	Lehoux S and Jones EA: Shear stress, arterial identity and atherosclerosis. Thromb Haemost. 115:467–473. 2016. View Article : Google Scholar : PubMed/NCBI
26	Olivon VC, Fraga-Silva RA, Segers D, Demougeot C, de Oliveira AM, Savergnini SS, Berthelot A, de Crom R, Krams R, Stergiopulos N and da Silva RF: Arginase inhibition prevents the low shear stress-induced development of vulnerable atherosclerotic plaques in ApoE-/- mice. Atherosclerosis. 227:236–243. 2013. View Article : Google Scholar : PubMed/NCBI
27	Boon RA, Leyen TA, Fontijn RD, Fledderus JO, Baggen JM, Volger OL, van Nieuw Amerongen GP and Horrevoets AJ: KLF2-induced actin shear fibers control both alignment to flow and JNK signaling in vascular endothelium. Blood. 115:2533–2542. 2010. View Article : Google Scholar : PubMed/NCBI
28	Vartanian KB, Berny MA, Mccarty OJ, Hanson SR and Hinds MT: Cytoskeletal structure regulates endothelial cell immunogenicity independent of fluid shear stress. Am J Physiol Cell Physiol. 298:C333–C341. 2010. View Article : Google Scholar : PubMed/NCBI
29	Lu C, Zhang J, Zhang D, Uzan G and Li M: EPCs in vascular repair: How can we clear the hurdles between bench and bedside? Front Biosci (Landmark Ed). 19:34–48. 2014. View Article : Google Scholar : PubMed/NCBI
30	Li X, Song Y, Wang D, Fu C, Zhu Z, Han Y, Li C, Wang N and Zhu Y: LIF maintains progenitor phenotype of endothelial progenitor cells via Krüppel-like factor 4. Microvasc Res. 84:270–277. 2012. View Article : Google Scholar : PubMed/NCBI
31	Cheng BB, Qu MJ, Wu LL, Shen Y, Yan ZQ, Zhang P, Qi YX and Jiang ZL: MicroRNA-34a targets Forkhead box j2 to modulate differentiation of endothelial progenitor cells in response to shear stress. J Mol Cell Cardiol. 74:4–12. 2014. View Article : Google Scholar : PubMed/NCBI
32	Wei S, Huang J, Li Y, Zhao J, Luo Y, Meng X, Sun H, Zhou X, Zhang M and Zhang W: Novel zinc finger transcription factor ZFP580 promotes differentiation of bone marrow-derived endothelial progenitor cells into endothelial cells via eNOS/NO pathway. J Mol Cell Cardiol. 87:17–26. 2015. View Article : Google Scholar : PubMed/NCBI
33	Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, Magner M, Isner JM and Asahara T: Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med. 5:434–438. 1999. View Article : Google Scholar : PubMed/NCBI
34	Hough C, Cameron CL, Notley CR, Brown C, O'Brien L, Keightley AM, Berber E and Lillicrap D: Influence of a GT repeat element on shear stress responsiveness of the VWF gene promoter. J Thromb Haemost. 6:1183–1190. 2008. View Article : Google Scholar : PubMed/NCBI