Immune Netw.  2009 Jun;9(3):106-113. 10.4110/in.2009.9.3.106.

IL-8/CXCL8 Upregulates 12-Lipoxygenase Expression in Vascular Smooth Muscle Cells from Spontaneously Hypertensive Rats

Affiliations
  • 1Department of Microbiology, College of Medicine, Yeungnam University, Daegu, Korea. heesun@med.yu.ac.kr
  • 2Department of Parmacology and Aging-associated Vascular Disease Research Center, College of Medicine, Yeungnam University, Daegu, Korea.

Abstract

BACKGROUND: We previously demonstrated remarkable differences in the expression of IL-8/CXCL8 in aortic tissues and vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) compared to VSMC from normotensive Wistar-Kyoto rats (WKY). In the present study, we investigated the direct effect of IL-8/CXCL8 on expression of 12-lipoxygenase (LO), a hypertensive modulator, in SHR VSMC. METHODS: Cultured aortic VSMC from SHR and WKY were used. Expression of 12-LO mRNA was determined by real-time polymerase chain reaction. Phosphorlyation of ERK1/2 and production of 12-LO and angiotensin II subtype 1 (AT1) receptor were assessed by Western blots. IL-8/CXCL8-stimulated DNA synthesis was determined by measuring incorporation of [3H]-thymidine. And effect of IL-8/CXCL8 on vascular tone was determined by phenylephrine-induced contraction of thoracic aortic rings. RESULTS: Treatment with IL-8/CXCL8 greatly increased 12-LO mRNA expression and protein production compared to treatment with angiotensin II. IL-8/CXCL8 also increased the expression of the AT1 receptor. The increase in 12-LO induced by IL-8/CXCL8 was inhibited by treatment with an AT1 receptor antagonist. The induction of 12-LO mRNA production and the proliferation of SHR VSMC by IL-8/CXCL8 was mediated by the ERK pathway. The proliferation of SHR VSMC and the vascular contraction in the thoracic aortic ring, both of which were induced by IL-8/CXCL8, were inhibited by baicalein, a 12-LO inhibitor. CONCLUSION: These results suggest that the potential role of IL-8/CXCL8 in hypertensive processes is likely mediated through the 12-LO pathway.

Keyword

IL-8/CXCL8; 12-lipoxygenase; rat vascular smooth muscle cell

MeSH Terms

Angiotensin II
Animals
Arachidonate 12-Lipoxygenase
Blotting, Western
Contracts
DNA
Flavanones
MAP Kinase Signaling System
Muscle, Smooth, Vascular
Rats
Rats, Inbred SHR
Real-Time Polymerase Chain Reaction
RNA, Messenger
Angiotensin II
Arachidonate 12-Lipoxygenase
DNA
Flavanones
RNA, Messenger

Figure

  • Figure 1 IL-8/CXCL8 increases 12-LO mRNA expression and protein production in SHR VSMC. (A) SHR and WKY VSMC were isolated from the thoracic aorta and cultured on plastic dishes at early passages (3 to 7). Total RNA was analyzed by real-time PCR. Bars represent means±SD from three independent experiments. *p<0.05 vs. WKY VSMC. (B) WKY VSMC were untreated or treated with IL-8/CXCL8 (100 ng/ml) for 2 h. Total RNA was then analyzed by real-time PCR. Bars represent means±SD from three independent experiments. (C) SHR VSMC were untreated or treated with IL-8/CXCL8 (100 ng/ml) or Ang II (100 nmol/L) for 2 h. Total RNA was then analyzed by real-time PCR. Bars represent means±SD from three independent experiments. a: p<0.05 vs. untreated VSMC. b: p<0.05 vs. treated with IL-8/CXCL8. (D) SHR VSMC were untreated or treated with IL-8/CXCL8 (100 ng/ml) or Ang II (100 nmol/L) for 2 and 4 h. Cell lysates were separated on 10% SDS-polyacrylamide gels and then immunoblotted with the 12-LO antibody. Data shown are representative of three independent experiments.

  • Figure 2 IL-8/CXCL8 increases AT1 receptor expression in SHR VSMC, and IL-8/CXCL8-induced expression of 12-LO mRNA is mediated through the AT1 receptor. (A) WKY and SHR VSMC were untreated or treated with IL-8/CXCL8 (100 ng/ml) for 2 h, and total RNA was analyzed by real-time PCR. Bars represent means±SD from four independent experiments. *p<0.05 vs. untreated VSMC. (B) VSMC were untreated or treated with IL-8/CXCL8 (100 ng/ml) for 2 and 4 h. Cell lysates were separated on 10% SDS-polyacrylamide gels and then immunoblotted with the AT1 receptor antibody. Data shown are representative of three independent experiments. (C) VSMC were untreated or treated with IL-8/CXCL8 (100 ng/ml) or IL-8/CXCL8 plus losartan (AT1 receptor antagonist, 10µmol/L) for 2 h or 4 h, and the total RNA and cell lysates were isolated. Total RNA was analyzed by real-time PCR. Cell lysates were separated on 10% SDS-polyacrylamide gels and then immunoblotted with the 12-LO antibody. Bars represent means±SD from three independent experiments. *p<0.05 vs. VSMC treated with IL-8/CXCL8. Data shown are representative of three independent experiments.

  • Figure 3 Expression of IL-8/CXCL8-induced 12-LO is mediated through the ERK pathway, and proliferation of SHR VSMC by IL-8/CXCL8 is inhibited by baicalein and PD98059. (A, B) VSMC were untreated (NT) or pretreated with PD98059 (ERK inhibitor, 10 µM) for 30 min. Cells were left untreated or treated with IL-8/CXCL8 (100 ng/ml) for 2 h, and the total RNA and cell lysates were isolated. The total RNA was analyzed by real-time PCR (A), and cell lysates were separated on 10% SDS-polyacrylamide gels and then immunoblotted with the phospho-ERK1/2 antibody (B). Bars represent means±SD from three independent experiments. *p<0.05 vs. VSMC treated with IL-8/CXCL8. Data shown are representative of three independent experiments. (C, D) SHR VSMC were treated with 12-HETE (500 nmol/L), with IL-8/CXCL8 (100 ng/ml), with baicalein (12-LO inhibitor, 10 µmol/L, B), or with PD98059 (10 µmol/L, C) for 48 h in medium containing [3H]-thymidine (1 µCi/ml). [3H]-thymidine incorporation is shown on the Y-axis. Bars represent means±SD from three independent experiments run in triplicate. *p<0.05 vs. untreated VSMC. **p<0.01 vs. VSMC treated with IL-8/CXCL8 alone. a: p<0.05 vs. VSMC treated with IL-8/CXCL8 alone.

  • Figure 4 Effect of IL-8/CXCL8 on the phenylephrine-induced contraction of thoracic aortic rings. Phenylephrine (1~100 µmol/L) was added to isolated thoracic aortic rings pretreated with IL-8/CXCL8 (200 ng/mL) and/or the 12-LO inhibitor baicalein (10 µmol/L) for 1 h. All contractions are expressed as grams (g) of contractile tension in control rings not exposed to IL-8/CXCL8 or baicalein. Data are means±SD of three independent experiments. a: p<0.05 vs. control rings. b: p<0.05 vs. rings treated with IL-8/CXCL8 alone.


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Hye Young Kim, Hye Ju Cha, Jin Hee Choi, Young Jin Kang, So Young Park, Hee Sun Kim
J Bacteriol Virol. 2015;45(2):138-150.    doi: 10.4167/jbv.2015.45.2.138.


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