Demethylbellidifolin Inhibits Adhesion of Monocytes to Endothelial Cells via Reduction of Tumor Necrosis Factor alpha and Endogenous Nitric Oxide Synthase Inhibitor Level

• Abstract
• Materials and Methods
• References

Abstract

The effect of demethylbellidifolin (DMB), a major compound of Swertia davidi Franch, on the adhesion of monocytes to endothelial cells induced by oxidized low-density lipoprotein (ox-LDL) was studied. Adhesion of monocytes to endothelial cells was induced by treatment with ox-LDL (100 μg/mL) for 48 h. Levels of tumor necrosis factor-α (TNF-α) and asymmetric dimethylarginine (ADMA, an endogenous inhibitor of NOS) in conditioned medium and the activity of dimethylarginine dimethylaminohydrolase (DDAH) in endothelial cells were measured. DMB (3 or 10 μmol/L) significantly inhibited the adhesion of monocytes to endothelial cells, attenuated an increase in levels of TNF-α and ADMA, and a decrease in the activity of DDAH by ox-LDL. The present results suggest that DMB inhibits the increased adhesion of monocytes to endothelial cells induced by ox-LDL, and that the effect of DMB is related to reduction of the ADMA concentration via reduction of TNF-α  production in cultured endothelial cells treated with ox-LDL.

It has been found that endogenous inhibitors of nitric oxide synthase such as ADMA, are significantly increased in animals and patients with atherosclerosis [1], [2]. Inflammatory cytokines such as TNF-α are significantly increased in animals and patients with atherosclerosis [3], [4], and exhibit numerous biological effects, including up-regulation of adhesion molecules expression, activation of the adhesion of monocyte and endothelial cell [5]. Recently, it has been shown that TNF-α  treatment significantly elevated the level of ADMA in cultured endothelial cells [6]. Xanthones, phenolic compounds that commonly occur in plants, have extensive pharmacological actions [7]. Recently, it was reported that some xanthones inhibited the increased expression of intercellular adhesion molecule-1 (ICAM-1) induced by TNF-α in cultured endothelial cells [8]. Our recent work has shown that daviditin A, a xanthone compound, protects the endothelium against damage induced by lysophosphatidylcholine (LPC) concomitantly with a decrease of the ADMA level [9]. Demethylbellidifolin (DMB, 1,3,5,8-tetrahydroxyxanthone) was extracted from Swertia davidi Franch (Gentianaceae) (Fig. [1]). In the present study, we examined the relationship of the effects between inhibition of adhesion of monocytes and ADMA level by DMB in cultured endothelial cells treated with ox-LDL.

Adhesion of monocytes to endothelial cells and concentration of TNF-α: Treatment with ox-LDL (100 μg/mL) for 48 h caused a significant increase in the adhesion of monocytes to endothelial cells and concentration of TNF-α. DMB (1, 3 or 10 μmol/L) significantly attenuated the increase in adhesion of monocytes and concentration of TNF-α by ox-LDL. Similarly, probucol also significantly attenuated the increased adhesion of monocytes and level of TNF-α (Fig. [2]).

Concentrations of ADMA and activity of DDAH: Treatment with ox-LDL (100 μg/ml) for 48 h significantly increased concentrations of ADMA and decreased the activity of DDAH. DMB (3 or 10 μmol/L) significantly inhibited an increase in the concentration of ADMA and a decrease in the activity of DDAH by ox-LDL. Probucol (5 μmol/L) also markedly inhibited the elevated concentration of ADMA (Fig. [3]).

NO, an important local regulatory factor in cardiovascular tissues, is synthesized from L-arginine by NOS in endothelial cells. L-Arginine analogues such as ADMA, which is present in blood of both humans and animals, can inhibit NOS in vivo and in vitro. There is growing evidence that endothelial dysfunction in some cardiovascular diseases is associated with an elevation of ADMA [10]. It has been shown that an elevation of serum ADMA is associated with increased adhesiveness of monocytes in hypercholesterolemic humans [11]. In the present study, treatment with ox-LDL increased the adhesion of monocytes to endothelial cells concomitantly with an increased level of ADMA. These findings support the hypothesis that the increased adhesion of monocytes to endothelial cells induced by ox-LDL is related to an elevation of the ADMA level.

TNF-α is widely synthesized in various cells including macrophages and endothelial cells and has been found to up-regulate the expression of adhesion molecules and increase the adhesion of monocytes to endothelium [5]. Recently, it has been reported that treatment with TNF-α significantly elevated the level of ADMA in cultured human endothelial cells [6]. The present results shown that treatment with ox-LDL significantly increased the levels of TNF-α and ADMA in cultured endothelial cells, which further proves that the increased level of ADMA induced by ox-LDL may be related to an elevation of TNF-α production.

Xanthones, main components extracted from Swertia davidi, have extensive pharmacological actions including antioxidation and anti-inflammation [7]. The results of the present study revealed that DMB significantly attenuated the increased adhesion of monocytes to endothelial cells in cultured endothelial cells treated with ox-LDL. The effect of DMB may be related to a reduction of the ADMA level by inhibition of TNF-α  production, because DMB also attenuated significantly the elevation of TNF-α and ADMA level in cultured endothelial cells treated with ox-LDL.

ADMA is synthesized by protein arginine methyltransferases (PRMTs) and degraded by DDAH [10]. There is evidence that lipid-induced dysregulation of DDAH plays an important role in the elevation of ADMA in hypercholesterolemia [1]. As mentioned above, the elevated level of ADMA with TNF-α also is related to the decreased activity of DDAH in cultured human endothelial cells [6]. The present results showed that DMB significantly decreased the levels of ADMA concomitantly with an improvement of DDAH activity in cultured endothelial cells treated with ox-LDL. These findings suggest that the decreased level of ADMA with DMB is related to an increase of DDAH activity by reduction of the TNF-α level.

In summary, the present results suggest that DMB inhibits the adhesion of monocytes to endothelial cells induced by ox-LDL, and that the protective effect of DMB on the endothelium is related to a decrease of ADMA concentration via a reduction of TNF-α production.

Materials and Methods

Reagents: DMB (purity: 99.0 %), which was extracted from Swertia davidi Franch (Gentianaceae) (School of Pharmaceutical Sciences, Central South University, China). Asymmetric dimethylarginine (ADMA) was obtained from Sigma. DMEM was obtained from Gibco. Tumor necrosis factor-α (TNF-α) assay kit was obtained from Sen-xiong science and technology industrial Co. Ltd (Shanghai, China). Probucol (Sigma) was used as a positive control.

Native LDL (nLDL) was isolated and oxidized as previously described [12], [13]; TNF-α concentration was assayed by enzyme-linked immunosorbent assay (ELISA). Cell culture and monocyte-endothelial cell adhesion were measured as previously described. [14]. The content of ADMA was measured by high-performance liquid chromatography (HPLC) as described previously with some modifications [15]. The activity of DDAH in endothelial cells was estimated by directly measuring the amount of ADMA metabolized by the enzyme [16]. Results are expressed as means ± SEM. Data were analyzed by ANOVA followed by the unpaired Student’s t-test for multiple comparisons. The significance level was chosen as P < 0.05.

References

• 1 Böger R H, Bode-Böger S M, Szuba A, Tsao P S, Chan J R, Tangphao O, Blaschke T F, Cooke J P. Asymmetric dimethylarginine: a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia.  Circulation. 1998;  98 1842-7
  PubMedSearch in Google Scholar
• 2 Yu X J, Li Y J, Xiong Y. Increase of an endogenous inhibitor of nitric oxide synthesis in serum of high cholesterol fed rabbits.  Life Sci. 1994;  54 753-8
  CrossrefPubMedSearch in Google Scholar
• 3 Rus H, Niculescu F, Vlaicu R. Tumor necrosis factor-α in human arterial wall with atherosclerosis.  Atherosclerosis. 1991;  89 247-54
  PubMedSearch in Google Scholar
• 4 Tanaka H, Sukhova G, Schwartz D, Libby P. Proliferating arterial smooth muscle cells after balloon injury express TNF-α but not interleukin-1 or basic fibroblast growth factor.  Arterioscler Thromb Vasc Biol. 1996;  16 12-18
  PubMedSearch in Google Scholar
• 5 Chen Y H, Lin S J, Chen J W, Ku H H, Chen Y L. Magnolol attenuates VCAM-1 expression in vitro in TNF-alpha-treated human aortic endothelial cells and in vivo in the aorta of cholesterol-fed rabbits.  Br J Pharmacol. 2002;  135 37-47
  PubMedSearch in Google Scholar
• 6 Ito A, Tsao P S, Adimoolam S, Kimoto M, Ogawa T, Cooke J P. Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase.  Circulation. 1999;  99 3092-5
  CrossrefPubMedSearch in Google Scholar
• 7 Peres V, Nagem T J, De oliveira F F. Tetraoxygenated naturally occurring xanthones.  Phytochemistry. 2000;  55 683-710
  CrossrefPubMedSearch in Google Scholar
• 8 Madan B, Singh I, Kumar A, Prasad A K, Raj H G, Parmar V S. Xanthones as inhibitors of microsomal lipid peroxidation and TNF-alpha induced ICAM-1 expression on human umbilical vein endothelial cells (HUVECs).  Bioorg Med Chem. 2002;  10 3431-6
  PubMedSearch in Google Scholar
• 9 Jiang D J, Jiang J L, Tan G S, Du Y H, Xu K P, Li Y J. Protective effects of daviditin A against endothelial damage induced by lysophosphatidylcholine.  N-S Arch Pharmacol;. 2003;  367 600-6
  PubMedSearch in Google Scholar
• 10 Cooke J P. Does ADMA cause endothelial dysfunction?.  Arterioscler Thromb Vasc Biol. 2000;  20 2032-7
  CrossrefPubMedSearch in Google Scholar

Yuan-Jian Li, MD

Department of Pharmacology

School of Pharmaceutical Sciences

Central South University

Xiang-Ya Road #88

Changsha 410078

P. R. China

Phone: +86-731-4805441

Fax: +86-731-2650442

Email: yuan_jianli@yahoo.com

References

• 1 Böger R H, Bode-Böger S M, Szuba A, Tsao P S, Chan J R, Tangphao O, Blaschke T F, Cooke J P. Asymmetric dimethylarginine: a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia.  Circulation. 1998;  98 1842-7
  PubMedSearch in Google Scholar
• 2 Yu X J, Li Y J, Xiong Y. Increase of an endogenous inhibitor of nitric oxide synthesis in serum of high cholesterol fed rabbits.  Life Sci. 1994;  54 753-8
  CrossrefPubMedSearch in Google Scholar
• 3 Rus H, Niculescu F, Vlaicu R. Tumor necrosis factor-α in human arterial wall with atherosclerosis.  Atherosclerosis. 1991;  89 247-54
  PubMedSearch in Google Scholar
• 4 Tanaka H, Sukhova G, Schwartz D, Libby P. Proliferating arterial smooth muscle cells after balloon injury express TNF-α but not interleukin-1 or basic fibroblast growth factor.  Arterioscler Thromb Vasc Biol. 1996;  16 12-18
  PubMedSearch in Google Scholar
• 5 Chen Y H, Lin S J, Chen J W, Ku H H, Chen Y L. Magnolol attenuates VCAM-1 expression in vitro in TNF-alpha-treated human aortic endothelial cells and in vivo in the aorta of cholesterol-fed rabbits.  Br J Pharmacol. 2002;  135 37-47
  PubMedSearch in Google Scholar
• 6 Ito A, Tsao P S, Adimoolam S, Kimoto M, Ogawa T, Cooke J P. Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase.  Circulation. 1999;  99 3092-5
  CrossrefPubMedSearch in Google Scholar
• 7 Peres V, Nagem T J, De oliveira F F. Tetraoxygenated naturally occurring xanthones.  Phytochemistry. 2000;  55 683-710
  CrossrefPubMedSearch in Google Scholar
• 8 Madan B, Singh I, Kumar A, Prasad A K, Raj H G, Parmar V S. Xanthones as inhibitors of microsomal lipid peroxidation and TNF-alpha induced ICAM-1 expression on human umbilical vein endothelial cells (HUVECs).  Bioorg Med Chem. 2002;  10 3431-6
  PubMedSearch in Google Scholar
• 9 Jiang D J, Jiang J L, Tan G S, Du Y H, Xu K P, Li Y J. Protective effects of daviditin A against endothelial damage induced by lysophosphatidylcholine.  N-S Arch Pharmacol;. 2003;  367 600-6
  PubMedSearch in Google Scholar
• 10 Cooke J P. Does ADMA cause endothelial dysfunction?.  Arterioscler Thromb Vasc Biol. 2000;  20 2032-7
  CrossrefPubMedSearch in Google Scholar

Yuan-Jian Li, MD

Department of Pharmacology

School of Pharmaceutical Sciences

Central South University

Xiang-Ya Road #88

Changsha 410078

P. R. China

Phone: +86-731-4805441

Fax: +86-731-2650442

Email: yuan_jianli@yahoo.com