Protection of DDAH2 Overexpression Against Homocysteine-Induced Impairments of DDAH/ADMA/NOS/NO Pathway in Endothelial Cells

Background/Aims: Homocysteine-induced endothelial dysfunction favors the development of cardiovascular diseases through accumulation of endogenous nitric oxide (NO) synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). Dimethylarginine dimethylaminohydrolase 2 (DDAH2) is the major enzyme for the degradation of ADMA in endothelial cells. The purpose of this study was to determine whether suppressed DDAH2 expression contributed to impairments of DDAH/ADMA/NOS/NO pathway induced by homocysteine in endothelial cells and whether DDAH2 overexpression could prevent endothelial cell dysfunction caused by homocysteine. Methods: Liposome-mediated transfection of endothelial cells was performed to establish the cell line of DDAH2 overexpression. After treatment of cells with 1 mmol/L homocysteine for 24 h, the transcription and expression of DDAH1 and DDAH2, DDAH and NOS activities as well as ADMA and NO concentrations were measured. Results: Treatment of endothelial cells with homocysteine significantly suppressed the transcription and expression of DDAH2 but not DDAH1. This suppression was associated with the declined DDAH activity, increased ADMA accumulation, inhibited NOS activity and decreased NO production in endothelial cells. DDAH2 overexpression not only resisted homocysteine-induced decline of DDAH activity, but also decreased the accumulation of endogenous ADMA, subsequently attenuated the reductions of NOS activity and NO production induced by homocysteine. Conclusions: These results indicate that suppression of DDAH2 expression is a culprit for homocysteine-induced impairments of DDAH/ADMA/NOS/NO pathway in endothelial cells, and therapeutic manipulation of DDAH2 expression may be a promising strategy for preventing endothelial dysfunction and cardiovascular diseases associated with hyperhomocysteinemia.


Introduction
Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases. It has been showed that hyperhomocysteinemia is responsible for about 10% of total risk of cardiovascular diseases [1]. Homocysteine favors the development of cardiovascular diseases through its endothelial toxicity which leads to the decrease of nitric oxide (NO) bioavailability and impairment of endothelium-dependent vasodilatation, generally termed as endothelial dysfunction [2,3]. It is well recognized that endothelial dysfunction plays a pivotal role in the pathogenesis of cardiovascular diseases. However, there is no medical treatment that is effective to prevent the endothelial dysfunction and cardiovascular diseases associated with hyperhomocysteinemia. Although supplement of vitamin B could reduce plasma homocysteine concentrations, the endothelial dysfunction induced by homocysteine could not be improved by treatment with vitamin B [4][5][6][7][8]. Therefore, it is important to find an effective therapeutic approach for improving endothelial dysfunction and cardiovascular diseases induced by hyperhomocysteinaemia.
Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), has been identified as an independent risk factor of endothelial dysfunction [2,9,10]. Elevated ADMA concentration has been found in many cardiovascular diseases which are related to endothelial dysfunction [11][12][13]. In multivariable regression analysis, ADMA plasma concentrations were positively correlated with plasma total homocysteine [10,14], and elevated ADMA concentration is always associated with homocysteine-induced endothelial dysfunction [2,4,10,13,14]. These results indicate that ADMA is a mediator of endothelial dysfunction induced by homocysteine.
The major pathway of endogenous ADMA clearance is hydrolysis to citrulline and methylamines by dimethylarginine dimethylaminohydroase (DDAH), so DDAH is the key regulator of endogenous ADMA levels. Two isoforms of DDAH, DDAH1 and DDAH2 with tissue-specific distribution have been identified. It is intriguing that DDAH activity was significantly inhibited after homocysteine treatment in endothelial cells, but DDAH protein expression remained unchanged [15]. Although homocysteine was found to inhibit the activity of DDAH directly [16], overexpression of DDAH1 could not protect the mice with dietary hyperhomocysteinemia from endothelial dysfunction [17]. These results allowed authors to speculate that DDAH2 rather than DDAH1 might play a pivotal role in ADMA metabolism of endothelial cells and homocysteine-induced endothelial dysfunction. Accordingly, in the present study, the alterations of DDAH/ADMA/NOS/NO pathway induced by homocysteine were measured in endothelial cells, and then the effects of DDAH2 overexpression on homocysteine-induced impairments of DDAH/ADMA/NOS/NO pathway in endothelial cells were subsequently investigated. The results of this study would delineate a crucial role for suppression of DDAH2 expression as a culprit event in homocysteine-induced impairments of DDAH/ADMA/NOS/NO pathway and further elucidate the mechanism for endothelial dysfunction associated with hyperhomocysteinemia.

Cellular Physiology and Biochemistry
Cell culture Human umbilical vein endothelial cell line (HUVEC, ATCC, Manassas, VA, USA) were cultured in RPMI1640 medium supplemented with 10% (v/v) fetal bovine serum and maintained at 37°C in a humidified atmosphere with 5% CO 2 . Cells were routinely trypsinized when they reached confluence using 0.02% (w/v) EDTA and 0.25% (w/v) trypsin. This cell line retains many characteristics of primary endothelial cells, including the expression of DDAH and NOS as well as the synthesis of NO.

Experimental protocol
The pcDNA3.1-DDAH2 expression plasmid was constructed, and the stable DDAH2-transfected endothelial cells were selected by incubation with G418 (800 µg/ml) for approximate 14 days as previously described [18]. DDAH2 overexpression was screened by RT-PCR and Western blotting. Subconfluent untransfected, pcDNA3.1-DDAH2 transfected and empty vector transfected cells were cultured in the absence or presence of homocysteine (1 mmol/L) for 24 h. After treatment, the cells were harvested for measurements of mRNA transcription and protein expression of DDAH1 and DDAH2, as well as the enzyme activities of DDAH and NOS, media were collected to determine the concentrations of ADMA, nitrite/nitrate and LDH.

Western blots
Cells were lysed with RIPA buffer, and equivalent amounts of protein (assessed by the Bradford protein assay) were loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins were transferred onto a nitrocellulose membrane. The membranes were incubated with 5% skimmed milk at room temperature for 1 h, and then incubated with polyantibodies against DDAH1, DDAH2 and β-actin over night at 4°C, followed by incubation with second goat anti-rabbit polyclonal antibody at room temperature for 1h, respectively. After washing again, protein bands were detected with the method of chemiluminescence and exposed to X-ray films. Densitometric quantifications of DDAH1 and DDAH2 protein bands were performed using a Bio-Rad Image Analyzer densitometry system and normalized to their corresponding β-actin protein band.

Assays of DDAH, NOS and LDH activities
Cells were resuspended in 1 ml sodium phosphate buffer (0.1 mM, pH 6.5) and then sonicated. The supernatant was assayed for DDAH activity by the conversion of L-citrulline from ADMA as previously described [19]. One unit of the enzyme was defined as the amount that catalyzed formation of 1 µmol/L L-citrulline from ADMA per min at 37°C and normalized to the protein content of the supernatant.
The NOS activity was assayed as previously described [18]. One unit of enzyme activity was defined as amount catalyzing formation of 1 nmol/L NO from L-arginine per minute at 37°C and normalized to the protein content of the supernatant.
Leakage of LDH was measured as an index of cell damage by the conversion of lactic acid to pyruvic acid with a commercial assay kit. The amount of the resultant pyruvic acid was determined spectrophotometrically at 440 nm. One unit of the enzyme activity was defined as the amount that catalyzed formation of 1 mmol/L pyruvic acid per min at 37°C and normalized to the cell number. Accordingly, LDH activity was expressed as units per milliliters of medium per 10 6 cells.

Construction of DDAH2 overexpressing cells
In the present study, the endothelial cell line of DDAH2 overexpression was constructed, shown by the higher levels of DDAH2 mRNA by RT-PCR (Fig.1 A) and DDAH2 protein by Western blotting (Fig.1 B) in DDAH2-transfected cells than that in untransfected cells or empty vector transfected cells.

Impairments of DDAH/ADMA/NOS/NO pathway by homocystein
Exposure of endothelial cells to 1 mmol/L homocysteine for 24 h suppressed DDAH2 mRNA transcription (Fig. 2 A & B, P<0.01) and protein expression (Fig. 2 C & D, P<0.01) but did not affect DDAH1 transcription (Fig. 3 A & B, P=NS) or expression (Fig. 3 C & D, P=NS). In parallel with the suppression of DDAH2 expression, DDAH activity was inhibited after homocysteine incubation in endothelial cells compared with corresponding control cells without homocysteine treatment (P<0.01, Fig. 4 A).
Incubation of endothelial cells with homocysteine significantly enhanced the accumulation of ADMA (P<0.01, Fig. 4B) and subsequently inhibited NOS activity (P<0.01, Fig. 4 C) versus their corresponding control cells. In accordance with the alteration in NOS activity, the concentration of nitrite/nitrate, the stable end product of NO, was finally reduced in homocysteine-treated endothelial cells compared with their corresponding control without homocysteine treatment (P<0.01, Fig. 4 D).

Discussion
The present study confirmed that homocysteine inhibited DDAH activity leading to the accumulation of endogenous ADMA and reduction of NO synthesis in cultured endothelial cells. More importantly, this study revealed that these inhibitory effects of homocysteine were mediated by suppression of DDAH2 transcription and expression but not DDAH1. DDAH2 overexpression could preserve endothelial cells from the inhibition of homocysteine. These results provide new insight into the elucidation of the mechanism and potential therapeutic approach for endothelial dysfunction induced by homocysteine.
Homocysteine is a sulfhydryl-containing amino acid derived from methionine metabolism that is dependent on folic acid (vitamin B9), vitamin B12 and B6 as cofactors. Accordingly, deficiencies of these vitamins have also been shown to elevate plasma total homocysteine leading to endothelial dysfunction [20], and supplement of these vitamins is capable of lowering or normalizing plasma homocysteine levels [21]. However, increasing evidence demonstrates that supplementations of B vitamins and/or folic acid are not sufficient to improve vascular endothelial dysfunction and even produce adverse cardiovascular effects in animals and humans with hyperhomocysteinemia [4][5][6][7][8]22]. Therefore such treatments are no longer recommended. Endothelial dysfunction, characterized by impairment of endothelium-dependent vasodilatation or endothelium-derived nitric oxide (NO) synthesis, plays a crucial role in pathophysiological process of hyperhomocysteinemia-induced cardiovascular diseases [23,24]. ADMA has emerged as a key factor for homocysteine-associated endothelial dysfunction [2,4,10,[13][14][15]. The present study confirmed that homocysteine treatment significantly enhanced ADMA accumulation and subsequently suppressed NOS activity and NO generation in cultured endothelial cells. Similar increase of ADMA accumulation induced by homocysteine was found not only in cultured endothelial cells [15] and neurons [25], but also in animals with endothelial dysfunction [2] and patients with atherosclerosis [10,13]. These results indicate that ADMA is a mediator of diminishing NO bioavailability induced by homocysteine.
The mechanisms underlying homocysteine-induced endogenous ADMA accumulation are not fully understood. Although previous study reported that the inhibition of DDAH activity contributed to homocysteine-induced ADMA accumulation in endothelial cells 15 , it is still unknown whether suppressed DDAH2 expression contributes to the inhibition of DDAH activity and accumulation of endogenous ADMA induced by homocysteine in endothelial cells, because the DDAH2 and DDAH1 expression or transcription were not simultaneously examined in previous studies [2,4,10,[13][14][15]24]. The present study provides the evidence that the suppression of DDAH2 expression is a primary cause for homocysteineinduced impairments of DDAH/ADMA/NOS/NO pathway in endothelial cells. This study demonstrated that homocysteine markedly suppressed DDAH2 transcription and expression without affecting DDAH1, resulting in the decrease of DDAH activity, increase of endogenous ADMA accumulation, inhibition of NOS activity and NO generation in endothelial cells. Similar findings have been made by other investigators in hearts of dogs with congestive heart failure [26] and in lungs of rats with pulmonary hypertension [27]. Homocysteineinduced oxidation stress might be implicated in the suppression of DDAH transcription or expression, because DDAH is a redox-sensitive enzyme resulting from a critical sulfhydryl group within its catalytic site [16]. Taken together, these results indicate that suppressed expression of DDAH2 rather than DDAH1 contributes to inhibition of DDAH activity and NO synthesis induced by homocysteine in endothelial cells.
Given that suppressed DDAH2 expression plays the determinant role in the endothelial dysfunction associated with homocysteine and there is no effective pharmacological agent to protect against homocysteine-induced impairments of DDAH/ADMA/NOS/NO pathway, the endothelial cell line of DDAH2 overexpressing was constructed and used to investigate whether DDAH2 overexpression could resist the impairment induced by homocysteine. The DDAH2-transfected endothelial cells displayed apparent enhancement of DDAH2 transcription and expression compared with untransfected cells, indicating that the endothelial cell line of DDAH2 overexpression was successfully constructed. This study further demonstrated that DDAH2 overexpression could prevent the suppression of DDAH activity, accumulation of endogenous ADMA, inhibition of NOS activity and NO synthesis in endothelial cells treated with homocysteine. These results were corroborated by previous reports that silencing DDAH2 but not DDAH1 expression with siRNA given intravenously to rats significantly inhibited vascular endothelium-dependent relaxation response to acetylcholine [28] and that DDAH2 overexpression remarkably attenuated angiotensin IIinduced increase of plasma ADMA, vascular injury and cardiac oxidative stress in DDAH2 transgenic mice [29]. On the contrary, overexpression of DDAH1 did not protect mice with dietary hyperhomocysteinemia from endothelial dysfunction [17]. The potential explanations for this discrepancy could be that DDAH2 is the predominantly isoform in endothelial cells on the one hand and that DDAH2 possesses some ADMA-independent protective effects on vascular injury of homocysteine on the other hand [29]. Collectively, these results indicate that the acceleration of ADMA degradation by DDAH2 overexpression is an adequate strategy to preserve NOS activity and NO synthesis or endothelial function in hyperhomocysteinemia.

Cellular Physiology and Biochemistry
In order to evaluate the harm to endothelial cells by homocysteine, the activity of LDH in culture medium was measured to reflect the cell viability or degree of cell injury in the present study. Results showed that DDAH2 overexpression markedly attenuated homocysteineinduced enhance of LDH activity in the culture medium of endothelial cells, suggesting DDAH2 overexpression could preserve endothelial cells from the injury of homocysteine. Previous study has also demonstrated that DDAH2 overexpression could decline angiotensin II-induced endothelial cells injury [29]. These results implied that the protection of DDAH2 overexpression against homocysteine-induced cell injury might contribute to preservation of NO synthesis in endothelial cells.
In summary, the present study demonstrated for the first time that suppressed DDAH2 expression contributed to homocysteine-induced changes in DDAH activity, endogenous ADMA accumulation, NOS activity and NO synthesis in cultured endothelial cells. Overexpression of DDAH2 could effectively attenuate these impairments of DDAH/ ADMA/NOS/NO pathway induced by homocysteine. Therefore, DDAH2 may be a promising target for the treatment of endothelial dysfunction under hyperhomocysteinemia, and therapeutic manipulation of DDAH2 expression may also represent an effectively strategy for preservation endothelial NO synthesis from impairment of homocysteine.