同半胱胺酸(homocysteine)抑制動脈內皮細胞生長之作用機轉

行政院國家科學委員會專題研究計畫 成果報告

同半胱胺酸(homocysteine)抑制動脈內皮細胞生長之作用

機轉

中 華 民 國 94 年 11 月 1 日

研究成果自評

這個一年期的研究計劃進行順利，我們的結果顯示

homocysteine 對血管內皮

細 胞 的 影 響 ， 無 論 是 在

DNA synthesis ，FGF2 suppression ，或 FGF promoter

activity 方 面 均 與 氧 化 態 低 密 度 脂 蛋 白 (OxLDL) 相 當 。 Homocysteine 對 FGF2

mRNA 的 downregulation 也與減低 FGF2 transcriptional activity 有關，這些發現與

我們已經發表的

oxLDL 的結果一致。

雖然短短一年時間的研究，我們已經可以相當程度的確定

homocysteine 對血

管內皮細胞的傷害是源自

FGF2 downregulation，這個機轉有其特異性，因為另一

個重要的

angiogenesis factor VEGF 的基因表現並不受 homocysteine 或 oxLDL 的影

響。這些實驗的結果是一個全新的發現，我們也已寫成

manuscript 正在 review

中，相信這個研究對

homocysteine 所造成之內皮細胞功能缺損進而引起冠狀動脈

硬化疾病的一些機轉有更深入的瞭解，進而藉由這些認識幫助我們發展預防及治

療冠狀動脈硬化疾病的新方針。

※主要研究成果摘要

Several promising results have been obtained for this one-year project, and are summarized below.

1) Homocysteine reduces FGF2 mRNA

and protein levels in cultured EC in a time- and concentration-dependent fashion, which is similar to oxLDL; 2) Homocysteine concomitantly

reduced cell cycle transition and cell viability;

3) Homocysteine suppressed FGF2 promoter activity;

4) The inhibition of DNA synthesis by homocysteine can be reversed by FGF2 but not VEGF;

5) Homocysteine-induced EC

apoptosis can be prevented by FGF2 but not VEGF.

Related publications and results are listed in the following (參照以下「參考文獻」及

「Results」).

※與本計劃相關之研究成果詳細說明 Endothelial dysfunction is an early manifestation of atherosclerosis but its

underlying mechanism is not fully understood.

In the past 5 years, under the financial support of three NSC grants: i) NSC89-2314-B-002-029 (1998-2000), ii) NSC89-2316-B-002-017-M52 (1999-2002) [擔任協同主持人], and iii) NSC91-2320-B- 002-185 (2002-2003) [擔任計劃主持 人], we have demonstrated that cytotoxic effects of oxidized low-density lipoproteins (oxLDL) on endothelial cells (EC) are attributable in part to suppression of fibroblast growth factor 2 (FGF2, also known as basic fibroblast growth factor, bFGF）expression. We have been working intensively on this “oxLDL-FGF2 suppression-EC dysfunction” hypothesis and have finally made the concept of this novel oxLDL-FGF2 signaling pathway recognized by publishing the results in the accredited Circulation.

The first grant (Principal Investigator: Dr. Yuan-Teh Lee) concluded by demonstrating in cultured human and bovine aortic EC that oxLDL reduced FGF2 mRNA levels in a time- and concentration-dependent manner. (published

in Circulation 2000) (參考文獻-1). This report

is the first proposal of a novel mechanism underlying oxLDL-induced EC dysfunction ⎯through downregulation of FGF2 gene expression. Second, data obtained above describe FGF2 gene regulation at the RNA level by oxLDL and help subsequent delineation of the signal transduction pathways mediating the

effects of oxLDL, which led to the next NSC grant 1999-2002.

The second grant of 1999-2002 NSC project (part of a program project directed by Dr. Yuan-Teh Lee) concluded by delineating the lipid components of oxLDL and determining their signal transduction pathways in inhibiting down regulation of FGF2 in cultured aortic EC. We found that oxLDL down-regulates endothelial FGF2 through a PTX-sensitive heterotrimeric G-protein pathway involving mediator

phospholipids similar, but not identical, to PAF. An abstract on oxLDL-FGF2 signaling was presented in the 1999 American Heart

Association Scientific Sessions (參考文獻-2),

and a comprehensive manuscript has been published in Circulation 2001 (參考文獻-3).

The main goal of the 2002-2003 NSC project (myself as Principal Investigator) is to further characterize the role of phospholipids in oxLDL-mediated suppression of FGF2 gene expression. In this one-year project, we have constructed the FGF2 promoter constructs for reporter gene assay, and identified phospholipid lysophosphatidylcholine (LPC) as an important lipid mediator for oxLDL-induced FGF2 downregulation. In these experiments, we have found that: i) LPC dose- and time-dependently decreased FGF2 protein in cultured bovine endothelial cells, ii) FGF2 mRNA levels were decreased in parallel, iii) the FGF2 promoter

activity was not affected by LPC, and iv) the FGF2 mRNA stability was decreased by LPC, indicating a post-transcriptional regulation of FGF2. A comprehensive manuscript is in preparation (參考文獻-4).

Meanwhile, several collaborative projects related to FGF2 regulation were carried out with fruitful results, including the investigation of apoptotic and signal transduction mechanisms involved in oxLDL-FGF2 pathways. The data have been either published or presented in several AHA scientific meeting (參考文獻-5, 6), and a comprehensive manuscript is under review (參考 文獻-7). We also executed a side project under this grant support, demonstrating that

homocysteine, another important cardiovascular risk factor, inhibited endothelial survival by reducing FGF2 expression, which is similar to oxLDL-FGF2 regulatory pathways. These results were presented in the 2002 AHA and 2005 ACC meeting (參考文獻-8, 9) and a comprehensive manuscript has been submitted to Circulation

Research for review (參考文獻-10).

This proposal represents continuous efforts of our studies on signaling mechanisms related to FGF2, an important EC mitogen and angiogen, in endothelial damage. The research focus of this project is put on homocysteine, an important atherosclerosis risk factor as well as an

study will be the first to demonstrate cytokine gene regulation by homocysteine. These results can also further our understanding of

homocysteine-induced endothelial damage, and may enable new approaches to the prevention and treatment of patients with elevated plasma homocysteine.

參考文獻

1. Chen CH, Jiang W, Via DP, Luo S, Li TR,

Lee YT, Henry PD. Oxidized Low-Density Lipoprotein Inhibits Endothelial Cell Growth by Suppressing Basic Fibroblast Growth Factor Expression. Circulation 2000;101:171-177.

2. Chang PY, Luo S, Lee YT, Henry PD, Chen

CH. Platelet-activating factor-like

phospholipids mediate oxidized LDL- induced downregulation of bFGF in endothelial cells.

Circulation 1999;100 (suppl. 1): I-694.

3. Chang PY, Luo S, Jiang T, Lee YT, Lu SC, Henry PD, Chen CH. Oxidized low-density lipoprotein down-regulates endothelial bFGF through a pertussis toxin–sensitive G-protein pathway: mediator role of platelet-activating factor–like phospholipids. Circulation 2001;104:588-593.

4. Chang PY, Chen CH, Lu SC, Huang WH, Chen YJ, Lee YT. Lysophosphatidylcholine mediates oxidized LDL-induced bFGF suppression in aortic endothelail cells.

Circulation 2005 (in preparation). 5. Chen CH, Jiang T, Yang JH, et al.

Low-density lipoprotein in hypercholesterolemic human plasma induces vascular endothelial cell apoptosis by inhibiting fibroblast growth

factor 2 transcription. Circulation. 2003; 107:2102–2108.

6. Jiang W, Lu J, Chang PY, Yang JH, Henry PD,

Marcelli M, Chen CH (2002). Oxidized LDL induces endothelial cell apoptosis by impairing the FGF2-PI3K-Akt pathway. Circulation. 2002;105:e86-e119 [Abstract].

7. Jiang W, Lu J, Yang JH, Chang PY, Lee YT.

Marcelli M, Henry PD, Liao WSL, Chen CH. Essential role of Akt-dependent FGF2

autoregulation in endothelial cell survival in the presence of oxidized LDL. Circ Res. 2005 Under review.

8. Chang PY, Huang WH, Lu SC, Lee YT (2002).

Homocysteine down-regulates basic fibroblast growth factor expression in endothelial cells.

Circulation. 2002;105:e86-e119 [Abstract].

9. Chang PY, Lu SC, Lee CM, Huang WH, Chen YJ, Chen CH, Lee YT. Homocysteine inhibits arterial endothelial cell survival by selectively reducing endogenous fibroblast growth factor 2. Circ Res. 2005 Under review.

Results

Figure 1. Homocysteine inhibited DNA synthesis, cell viability and induced cell death in cultured

bovine aortic endothelial cells (BAECs). A, Dose-dependent effects. Cells were treated with various concentrations of homocysteine for 24 h, and DNA synthesis(•), cell viability(□), and cell death(Δ) were assessed. Values are mean±SEM (n=3). *P<0.05, **P<0.01 vs corresponding untreated controls. B, Time-dependent effects. BAEC were incubated with 50(▼) or 100(∇) μM homocysteine or 100 μM cysteine (○) for 0–96 hours followed by cell viability assay. Values are mean±SEM (n=3). *P<0.05 vs PBS control.

Figure 2. Opposing effects of homocysteine and FGF2 on DNA synthesis and cell cycle transitions. A,

BAEC were treated with 100 μM homocysteine (Hcy), 50 ng/mL fibroblast growth factor 2 (FGF2), and 50 ng/mL vascular endothelial growth factor 165 (VEGF-165), alone or in combination. DNA synthesis was assessed by 3H-thymidine incorporation in cells after 24 hours of treatment. Values are mean±SEM (n=3). *P<0.05, **P<0.01 vs PBS control. B, BAEC were incubated with 100 μM homocysteine in the presence or absence of 50 ng/mL FGF2 or 50 ng/mL VEGF for 24 hours before being subjected to fluorescence-activated cell sorter analysis. The figure is representative of 3 separate experiments with similar results. Distributions of the cells in the G1, S, and G2/M phases are tabulated. Values are mean±SEM (n=3). *P<0.05 vs PBS control.

Figure 3. Effects of homocysteine on intracellular and extracellular FGF2 (A and B) and intracellular

VEGF (C) concentrations. A, Dose-dependent effects on FGF2. FGF2 concentrations were assayed by ELISA in BAEC incubated with homocysteine at 25 to 500 μM for 24 hours. Intracellular FGF2 (left panel) and extracellular FGF2 (right panel) are shown. Values are mean±SEM (n=3). *P<0.05, **P<0.01 vs PBS control. B, Time-dependent effects on FGF2. BAEC were incubated with homocysteine or cysteine (100 μM each) for indicated time periods. Cells treated with PBS for 96 hours were used as control. The intracellular FGF2 levels were assessed by Western blot analysis (left panel), which is representative of 3 separate experiments with similar results. The extracellular FGF2 were determined by ELISA (right panel). *P<0.05, **P<0.01 vs PBS control. C, VEGF expression. Intracellular VEGF concentrations were assayed by ELISA in BAEC incubated with homocysteine at 25 to 500 μM for 24 hours (left panel), or with homocysteine 100 μM for 24-96 hours (right panel). Values are mean±SEM (n=3). *P<0.05, **P<0.01 vs PBS control.

Figure 4. Homocysteine suppressed FGF2 transcription and mRNA expression in cultured human

coronary artery EC (HCAEC). A, FGF2 reporter gene constructs and reporter gene assay. Different lengths of human FGF2 5’-flanking sequences were fused to luciferase gene in pGL3 vector. +1 indicates the transcription start site. All of the constructs were cotransfected with phRL-TK (internal control) into HCAEC, and incubated in the presence or absence of 100 μM homocysteine (Hcy), 100 μM cysteine, or 100 μM methionine (Met) for 24 h. Luciferase activities were expressed as fold increases compared to pGL3-basic. Values are mean±SEM (n=3-5). *P<0.05 vs PBS control. B, Measurement of mRNA by reverse transcription polymerase chain reaction (RT-PCR). HCAEC were incubated with PBS (C) or 100 μM homocysteine (Hcy) for 24 hours, and cellular total RNA were

subjected to RT-PCR analysis with specific primers for FGF2, β-actin, FGFR1, and VEGFR2. Densitometry is shown. The data are representative of 3 separate experiments with similar results.

Figure 5. Apoptotic effects of excessively high concentrations of homocysteine on BAEC. Cells were

incubated with 500 μM homocysteine for 24 hours, in the presence or absence of 50 ng/mL FGF2 or 50 ng/mL VEGF, and apoptosis was assessed by TUNEL assay as described in Methods. Control cells were treated with PBS. The percentages of TUNEL–positive cells are expressed as mean±SEM (n=3). *P<0.05, **P<0.01 vs PBS control.

Figure 5