20-HETE inhibits the proliferation of vascular smooth muscle cells via transforming growth factor-β

Chan-Jung Liang,Harlan E Ives,Chuen-Mao Yang,Yunn-Hwa Ma

doi:10.1194/jlr.m700155-jlr200

Chan-Jung Liang, Harlan E Ives + Show 2 more

Open Access

https://doi.org/10.1194/jlr.m700155-jlr200

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Abstract

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, has been shown to modulate the growth of vascular smooth muscle cells (VSMCs). We asked whether 20-HETE modulates the proliferation of R22D cells, a clonal VSMC from neonatal rats, by releasing transforming growth factor-beta (TGF-beta). Incubation of R22D cells with 20-HETE for 24 h attenuated [(3)H]thymidine incorporation in a concentration-dependent manner without causing the release of lactate dehydrogenase. 20-HETE also inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation in R22D cells and human VSMCs. At 5 muM, 20-HETE reduced [(3)H]thymidine incorporation by 34 +/- 6%; anti-TGF-beta neutralizing antibody, but not nonspecific IgG, completely reversed the attenuated [(3)H]thymidine incorporation induced by 20-HETE. In addition, 20-HETE attenuated fetal bovine serum- and PDGF-induced expression of cyclin D1, a downstream effector of TGF-beta(1), which was reversed by anti-TGF-beta antibody. Further studies demonstrated that 20-HETE may increase TGF-beta release to a level high enough to inhibit [(3)H]thymidine incorporation without altering the steady-state mRNA level of TGF-beta. Nevertheless, pretreatment of indomethacin (a cyclooxygenase inhibitor) or paxilline (a potassium channel inhibitor) did not affect the inhibitory effect on DNA synthesis induced by 20-HETE. These results demonstrate for the first time a growth-inhibitory effect induced by 20-HETE, which may be mediated by TGF-beta.

Highlights

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, has been shown to modulate the growth of vascular smooth muscle cells (VSMCs)
We demonstrated that transforming growth factor-b (TGF-b) may mediate the inhibitory effect of 20-HETE on the growth of VSMCs from neonatal rat or human
Anti-TGF-b RII antibody against TGF-b receptor type II (20 mg/ml) significantly reversed the inhibitory effect of 20-HETE (i.e., a 23 6 1% inhibition) on [3H]thymidine incorporation to the control level (n 5 4; P, 0.05) (Fig. 3B). These results suggested that the inhibitory effect of 20-HETE may be mediated by TGF-b in R22D cells

Summary

MATERIALS AND METHODS

Materials 20-HETE (in ethanol) purchased from Cayman Chemical Co. (Ann Arbor, MI) was dried under N2 gas and resuspended in the serum-free medium before use; an equal volume of serumfree medium was used as vehicle. R22D cells (A, B, D), aortic VSMCs (C), or human VSMCs (E) were cultured in growth medium (B) or in serum-free medium for 24 h (A, C) before treatment with 20-HETE. Quiescent R22D cells (D) and human VSMCs (E) were incubated with platelet-derived growth factor (PDGF; 5 ng/ml) in the presence or absence of 20-HETE (5 mM). Confluent cells were cultured in serum-free medium for 24 h before incubation with 20-HETE at the indicated times. Cells were exposed to FBS (10%) or PDGF (5 ng/ml) at the indicated times with or without administration of 20-HETE (5 mM). Quiescent R22D cells were exposed to 20-HETE in the presence of TGF-b neutralizing antibody, anti-TGF-b RII antibody, or nonspecific IgG.

RESULTS

Findings

DISCUSSION