Effect of angiopoietin-like protein 4 on rat pulmonary microvascular endothelial cells exposed to LPS

Wang,Yuxi; Chen,Hailong; Li,Hailong; Zhang,Jingwen; Gao,Yanyan

doi:10.3892/ijmm.2013.1420

Effect of angiopoietin-like protein 4 on rat pulmonary microvascular endothelial cells exposed to LPS

Authors:
- Yuxi Wang
- Hailong Chen
- Hailong Li
- Jingwen Zhang
- Yanyan Gao
View Affiliations

Affiliations: Dalian Medical University, Dalian, Liaoning, P.R. China, Department of General Surgery, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, P.R. China, Department of Central Laboratory, Dalian Municipal Central Hospital, Dalian, Liaoning, P.R. China
Published online on: June 20, 2013 https://doi.org/10.3892/ijmm.2013.1420
Pages: 568-576
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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

Pulmonary microvascular endothelial cells (PMVECs) possess highly proliferative and angiogenic capacities and are localized at the critical interface between the blood and microvessel wall; they are the primary targets of inflammatory cytokines during lung inflammation. Angiopoietin-like protein 4 (Angptl4) is a circulating protein that has recently been implicated in the regulation of angiogenesis and metastasis. This study aimed to investigate the effect of Angptl4 on rat PMVECs (RPMVECs) exposed to lipopolysaccharide (LPS). The cell culture was stimulated with LPS. Total Angptl4 cDNA was obtained from Source BioScience. The PCR product was cloned into the pcDNA3.1-eGFP or the pcDNA3.1‑eGFP‑Angptl4 vector, which were then transfected into the RPMVECs using SuperFect transfection reagent. The Angptl4 mRNA levels, protein levels and cell morphology of the RPMVECs in the experimental groups were detected using real time‑PCR, western blot analysis, MTT assay, ELISA and confocal microscopy methods, respectively. The Angptl4 expression vector, pcDNA3.1‑eGFP-Angptl4, was successfully constructed. The Angptl4 mRNA level in the LPS-pcDNA3.1-eGFP-transfected group (blank control) was slightly increased and was significantly higher in the experimental group compared with the empty vector and blank control group with significant differences. Pro-apoptotic caspase-8, -9 and Bax protein were inhibited, while p-AKT/AKT and p-Mek1/2 protein expression was also decreased. The rosiglitazone group had significantly decreased levels of the inflammatory cytokine, tumor necrosis factor (TNF)-α (P<0.01). The overexpression of Angptl4 inhibited the LPS-induced increase in the permeability of the RPMVECs, which was associated with the depolymerization of central F-actin in the RPMVECs. In conclusion, our study demonstrates that the overexpression of Angptl4 exerts protective, anti‑inflammatory and anti‑angiogenic effects. It represents a novel therapeutic target gene for the treatment of acute lung injury induced by LPS.

View Figures

View References

1	Ge H, Yang G, Yu X, Pourbahrami T and Li C: Oligomerization state-dependent hyperlipidemic effect of angiopoietin-like protein 4. J Lipid Res. 45:2071–2079. 2004. View Article : Google Scholar : PubMed/NCBI
2	Xu A, Lam MC, Chan KW, et al: Angiopoietin-like protein 4 decreases blood glucose and improves glucose tolerance but induces hyperlipidemia and hepatic steatosis in mice. Proc Natl Acad Sci USA. 102:6086–6091. 2005. View Article : Google Scholar : PubMed/NCBI
3	Sukonina V, Lookene A, Olivecrona T and Olivecrona G: Angiopoietin-like protein 4 converts lipoprotein lipase to inactive monomers and modulates lipase activity in adipose tissue. Proc Natl Acad Sci USA. 103:17450–17455. 2006. View Article : Google Scholar : PubMed/NCBI
4	Mandard S, Zandbergen F, van Straten E, Wahli W, Kuipers F, Muller M and Kersten S: The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity. J Biol Chem. 281:934–944. 2006. View Article : Google Scholar : PubMed/NCBI
5	Zhu P, Tan MJ, Huang RL, et al: Angiopoietin-like 4 protein elevates the prosurvival intracellular O₂(−):H₂O₂ratio and confers anoikis resistance to tumors. Cancer Cell. 19:401–415. 2011. View Article : Google Scholar : PubMed/NCBI
6	Kersten S: Regulation of lipid metabolism via angiopoietin-like proteins. Biochem Soc Trans. 33:1059–1062. 2005. View Article : Google Scholar : PubMed/NCBI
7	Mandard S, Zandbergen F, Tan NS, et al: The direct peroxisome proliferator-activated receptor target fasting-induced adipose factor (FIAF/PGAR/ANGPTL4) is present in blood plasma as a truncated protein that is increased by fenofibrate treatment. J Biol Chem. 279:34411–34420. 2004. View Article : Google Scholar
8	Koliwad SK, Kuo T, Shipp LE, et al: Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor) is a direct glucocorticoid receptor target and participates in glucocorticoid-regulated triglyceride metabolism. J Biol Chem. 284:25593–25601. 2009. View Article : Google Scholar
9	Padua D, Zhang XH, Wang Q, et al: TGF beta primes breast tumors for lung metastasis seeding through angiopoietin-like 4. Cell. 133:66–77. 2008. View Article : Google Scholar : PubMed/NCBI
10	Belanger AJ, Lu H, Date T, et al: Hypoxia up-regulates expression of peroxisome proliferator-activated receptor gamma angiopoietin-related gene (PGAR) in cardiomyocytes: role of hypoxia inducible factor 1alpha. J Mol Cell Cardiol. 34:765–774. 2002. View Article : Google Scholar
11	Liu F, Li W, Pauluhn J, Trubel H and Wang C: Lipopolysaccharide-induced acute lung injury in rats: comparative assessment of intratracheal instillation and aerosol inhalation. Toxicology. 304:158–166. 2013. View Article : Google Scholar : PubMed/NCBI
12	Feingold KR, Shigenaga JK, Cross AS, Moser A and Grunfeld C: Angiopoietin like protein 4 expression is decreased in activated macrophages. Biochem Biophys Res Commun. 421:612–615. 2012. View Article : Google Scholar : PubMed/NCBI
13	Ichikawa H, Naito Y, Takagi T, et al: A specific peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand, pioglitazone, ameliorates gastric mucosal damage induced by ischemia and reperfusion in rats. Redox Rep. 7:343–346. 2002. View Article : Google Scholar
14	Konturek PC, Brzozowski T, Kania J, et al: Pioglitazone, a specific ligand of the peroxisome proliferator-activated receptor gamma reduces gastric mucosal injury induced by ischaemia/reperfusion in rat. Scand J Gastroenterol. 38:468–476. 2003. View Article : Google Scholar
15	Villegas I, Martin AR, Toma W and de la Lastra CA: Rosiglitazone, an agonist of peroxisome proliferator-activated receptor gamma, protects against gastric ischemia-reperfusion damage in rats: role of oxygen free radicals generation. Eur J Pharmacol. 505:195–203. 2004. View Article : Google Scholar
16	Wada K, Nakajima A, Takahashi H, et al: Protective effect of endogenous PPARgamma against acute gastric mucosal lesions associated with ischemia-reperfusion. Am J Physiol Gastrointest Liver Physiol. 287:G452–G458. 2004. View Article : Google Scholar : PubMed/NCBI
17	Takagi T, Naito Y, Ichikawa H, et al: A PPAR-gamma ligand, 15-deoxy-Delta12,14-prostaglandin J(2), inhibited gastric mucosal injury induced by ischemia-reperfusion in rats. Redox Rep. 9:376–381. 2004. View Article : Google Scholar : PubMed/NCBI
18	Naito Y, Takagi T, Matsuyama K, Yoshida N and Yoshikawa T: Pioglitazone, a specific PPAR-gamma ligand, inhibits aspirin-induced gastric mucosal injury in rats. Aliment Pharmacol Ther. 15:865–873. 2001. View Article : Google Scholar : PubMed/NCBI
19	Slomiany A, Nishikawa H and Slomiany BL: Screening and modulation of extracellular signals by mucous barrier. Serum glycosylphosphatidylinositol phospholipase D (GPI-PLD) releases protective mucous barrier from oral mucosa. J Physiol Pharmacol. 53:21–38. 2002.
20	Lappas M, Permezel M and Rice GE: 15-Deoxy-Delta(12,14)-prostaglandin J(2) and troglitazone regulation of the release of phospholipid metabolites, inflammatory cytokines and proteases from human gestational tissues. Placenta. 27:1060–1072. 2006. View Article : Google Scholar
21	Chen SF, Fei X and Li SH: A new simple method for isolation of microvascular endothelial cells avoiding both chemical and mechanical injuries. Microvasc Res. 50:119–128. 1995. View Article : Google Scholar : PubMed/NCBI
22	Cheng C, Liu H, Ge H, et al: Lipopolysaccharide induces expression of SSeCKS in rat lung microvascular endothelial cell. Mol Cell Biochem. 305:1–8. 2007. View Article : Google Scholar : PubMed/NCBI
23	Zhang H and Sun GY: LPS induces permeability injury in lung microvascular endothelium via AT(1) receptor. Arch Biochem Biophys. 441:75–83. 2005. View Article : Google Scholar : PubMed/NCBI
24	Adkison JB, Miller GT, Weber DS, et al: Differential responses of pulmonary endothelial phenotypes to cyclical stretch. Microvasc Res. 71:175–184. 2006. View Article : Google Scholar : PubMed/NCBI
25	Gao G, Kapushoc ST, Simpson AM, Thiemann OH and Simpson L: Guide RNAs of the recently isolated LEM125 strain of Leishmania tarentolae: an unexpected complexity. RNA. 7:1335–1347. 2001. View Article : Google Scholar : PubMed/NCBI
26	Essler M, Staddon JM, Weber PC and Aepfelbacher M: Cyclic AMP blocks bacterial lipopolysaccharide-induced myosin light chain phosphorylation in endothelial cells through inhibition of Rho/Rho kinase signaling. J Immunol. 164:6543–6549. 2000. View Article : Google Scholar
27	Harhaj NS and Antonetti DA: Regulation of tight junctions and loss of barrier function in pathophysiology. Int J Biochem Cell Biol. 36:1206–1237. 2004. View Article : Google Scholar : PubMed/NCBI
28	Koishi R, Ando Y, Ono M, et al: Angptl3 regulates lipid metabolism in mice. Nat Genet. 30:151–157. 2002. View Article : Google Scholar : PubMed/NCBI
29	Oike Y, Yasunaga K, Ito Y, et al: Angiopoietin-related growth factor (AGF) promotes epidermal proliferation, remodeling, and regeneration. Proc Natl Acad Sci USA. 100:9494–9499. 2003. View Article : Google Scholar : PubMed/NCBI
30	Kim I, Kim HG, Kim H, et al: Hepatic expression, synthesis and secretion of a novel fibrinogen/angiopoietin-related protein that prevents endothelial-cell apoptosis. Biochem J. 346:603–610. 2000. View Article : Google Scholar : PubMed/NCBI
31	Brunckhorst MK, Wang H, Lu R, et al: Angiopoietin-4 promotes glioblastoma progression by enhancing tumor cell viability and angiogenesis. Cancer Res. 70:7283–7293. 2010. View Article : Google Scholar : PubMed/NCBI
32	Lu B, Moser A, Shigenaga JK, Grunfeld C and Feingold KR: The acute phase response stimulates the expression of angiopoietin like protein 4. Biochem Biophys Res Commun. 391:1737–1741. 2010. View Article : Google Scholar : PubMed/NCBI
33	Hermann LM, Pinkerton M, Jennings K, et al: Angiopoietin-like-4 is a potential angiogenic mediator in arthritis. Clin Immunol. 115:93–101. 2005. View Article : Google Scholar : PubMed/NCBI
34	Cazes A, Galaup A, Chomel C, et al: Extracellular matrix-bound angiopoietin-like 4 inhibits endothelial cell adhesion, migration, and sprouting and alters actin cytoskeleton. Circ Res. 99:1207–1215. 2006. View Article : Google Scholar : PubMed/NCBI
35	Yang YH, Wang Y, Lam KS, et al: Suppression of the Raf/MEK/ERK signaling cascade and inhibition of angiogenesis by the carboxyl terminus of angiopoietin-like protein 4. Arterioscler Thromb Vasc Biol. 28:835–840. 2008. View Article : Google Scholar : PubMed/NCBI
36	Galaup A, Cazes A, Le Jan S, et al: Angiopoietin-like 4 prevents metastasis through inhibition of vascular permeability and tumor cell motility and invasiveness. Proc Natl Acad Sci USA. 103:18721–18726. 2006. View Article : Google Scholar : PubMed/NCBI
37	Li KQ, Li WL, Peng SY, et al: Anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection. Chin Med J (Engl). 117:1364–1369. 2004.PubMed/NCBI
38	Dhillon AS and Kolch W: Untying the regulation of the Raf-1 kinase. Arch Biochem Biophys. 404:3–9. 2002. View Article : Google Scholar : PubMed/NCBI
39	Wu F, Wang H, Li J, Liang J and Ma S: Homoplantaginin modulates insulin sensitivity in endothelial cells by inhibiting inflammation. Biol Pharm Bull. 35:1171–1177. 2012. View Article : Google Scholar : PubMed/NCBI
40	Masamune A, Watanabe T, Kikuta K and Shimosegawa T: Roles of pancreatic stellate cells in pancreatic inflammation and fibrosis. Clin Gastroenterol Hepatol. 7:S48–S54. 2009. View Article : Google Scholar : PubMed/NCBI
41	Faulkner A, Rosen S and Norman C: The right information may matter more than frequency-place alignment: simulations of frequency-aligned and upward shifting cochlear implant processors for a shallow electrode array insertion. Ear Hear. 27:139–152. 2006. View Article : Google Scholar