Piper sarmentosum inhibits ICAM-1 and Nox4 gene expression in oxidative stress-induced human umbilical vein endothelial cells

Background Aqueous extract of Piper sarmentosum (AEPS) is known to possess antioxidant and anti-atherosclerotic activities but the mechanism responsible for it remains unclear. In early part of atherosclerosis, nuclear factor-kappa B (NF-κB) induces the expression of cellular adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1) and E-selectin. NADPH oxidase 4 (Nox4) is the predominant source of superoxide in the endothelial cells whereas superoxide dismutase 1 (SOD1), catalase (CAT) and glutathione peroxidase (GPx) are the antioxidant enzymes responsible for inactivating reactive oxygen species. The present study aimed to investigate the effects of AEPS on the gene expression of NF-κB, VCAM-1, ICAM-1, E-selectin, Nox4, SOD1, CAT and GPx in cultured human umbilical vein endothelial cells (HUVECs). Methods HUVECs were divided into four groups:- control; treatment with 180 μM hydrogen peroxide (H2O2); treatment with 150 μg/mL AEPS and concomitant treatment with AEPS and H2O2 for 24 hours. Total RNA was extracted from all the groups of HUVEC using TRI reagent. Subsequently, qPCR was carried out to determine the mRNA expression of NF-κB, VCAM-1, ICAM-1, E-selectin, Nox4, SOD1, CAT and GPx. The specificity of the reactions was verified using melting curve analysis and agarose gel electrophoresis. Results When stimulated with H2O2, HUVECs expressed higher level of ICAM-1 (1.3-fold) and Nox4 (1.2-fold) mRNA expression. However, AEPS treatment led to a reduction in the mRNA expression of ICAM-1 (p < 0.01) and Nox4 (p < 0.05) in the H2O2-induced HUVECs. AEPS also upregulated the mRNA expression of SOD1 (p < 0.05), CAT (p < 0.01) and GPx (p < 0.05) in oxidative stress-induced HUVECs. There was no significant change in the mRNA expression of VCAM-1 and E-selectin. Conclusion The expressional suppression of ICAM-1 and Nox4 and induction of antioxidant enzymes might be an important component of the vascular protective effect of AEPS.


Background
Atherosclerosis has been recognized as a chronic inflammatory disease and oxidative stress plays a pivotal role in its initiation and progression [1]. Endothelial dysfunction is considered to be an early marker for atherosclerosis [2]. Evidence suggests that increased production of reactive oxygen species (ROS) and vascular inflammation play important roles in endothelial dysfunction. Vascular disorders, through over expression of adhesion molecules and cytokines are involved in the development of atherosclerosis. Endothelial cells in human atherosclerotic lesions have increased cell adhesion molecules expression such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial selectin (E-selectin) [3,4]. The adhesion of monocytes to the arterial wall and their subsequent infiltration and differentiation into macrophages are the key events in the development of atherosclerosis. Nuclear factor-kappa B (NF-B) is known to play a critical role in the development of inflammatory response by upregulating the expression of VCAM-1, ICAM-1 and E-selectin [5]. It has been suggested that NF-B is an oxidative stress-responsive transcription factor. Antioxidants and free radical scavengers such as vitamin E derivatives, N-acetyl-cysteine and thiol reagents inhibit the activation of NF-B, strongly supporting the idea that reactive oxygen species (ROS) are involved in the activation process [6].
Under oxidative stress, macrophages generate ROS such as superoxides, leading to LDL oxidation [7]. In the vascular wall, ROS can be produced by several enzyme systems including NADPH oxidases, xanthine oxidase, uncoupled endothelial nitric oxide synthase, lipoxygenases and myeloperoxidase [8]. Although all these enzymes can contribute to oxidative stress, NADPH oxidases (Nox) are the predominant source of ROS in the vasculature. In diseased human coronary arteries, about 60% of total vascular superoxide is derived from Nox [9]. Vascular tissues express the Nox isoforms Nox1, Nox2, Nox4 and Nox5. In human umbilical vein endothelial cells (HUVECs), the expression level of Nox4 is 100-fold higher than that of Nox1, Nox2 or Nox5, suggesting that Nox4 is the major source of ROS in these cells [10]. Increased Nox4 expression is associated with early progression of atherosclerotic plaque [11].
To protect cells from the damage caused by ROS, organisms have evolved several defense mechanisms to rapidly and efficiently remove ROS. This includes the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). SOD catalyzes the dismutation of superoxide to hydrogen peroxide (H 2 O 2 ) while CAT and GPx convert H 2 O 2 to water [12].
Piper sarmentosum ( Figure 1) is a herbaceous plant that is commonly found in the tropical regions such as the Southeast Asia. The plant extracts have been reported to possess pharmacological properties like antituberculous [13], anti cancer [14], hypoglycaemic [15], anti-malarial [16], anti-nociceptive and anti-inflammatory [17]. As per recent research reports, the aqueous extracts of Piper sarmentosum (AEPS) leaves have been reported to improve endothelial function by promoting nitric oxide production in HUVECs [18]. Various extracts prepared from Piper sarmentosum leaves have exhibited potent effects as antioxidant in modulating oxidative stress in H 2 O 2 -induced HUVECs [19]. Moreover, administration of AEPS reduced atherosclerotic lesion in hypercholesterolemic rabbits [20]. Based on the cardiovascular protective effects of AEPS mentioned above, the present study was designed to investigate the effects of AEPS on the gene expression of NF-B, cellular adhesion molecules (VCAM-1, ICAM-1 and E-selectin), Nox4 and antioxidant enzymes (SOD1, CAT, GPx) in HUVECs.

Preparation of aqueous extract of Piper sarmentosum
Leaves of Piper sarmentosum were collected in Sungai Buloh, Malaysia and identified by a plant taxonomist from Forest Research Institute of Malaysia (voucher specimen: FRI 45870). The leaves were washed with tap water, cut into small pieces, sun-dried and grounded to powder form. Ten percent of AEPS was prepared by soaking 100 g of the powdered leaves in 900 ml of purified water and incubated in a high speed mixer at 80°C for 3 hours. After being left to cool, the extract was filtered using a mesh and further concentrated. The aqueous extract was then freeze-dried to powdered form of the extract and kept at 4°C until the experiments.

Cell culture and treatment protocols
Human umbilical cords were obtained under sterile condition. Informed consent was obtained from each subject and the present study was approved by the Ethical Research Committee of Universiti Kebangsaan Malaysia Medical Center (Project Code: FF-148-2009). HUVECs were obtained from umbilical cord veins by 0.1% Collagenase Type I (Gibco-Invitrogen Corp., Grand Island, N.Y.) digestion. Cells were grown in medium 200 (Cascade Biologics, USA) supplemented with LSGS (low serum growth supplement; Cascade Biologics, USA) at 37°C in a humidified atmosphere of 5% CO 2 and 95% air. HUVECs were confirmed by the typical endothelial cell cobblestone morphology and the positive expressions of vonWillebrand factor and CD31 in immunocytochemistry. The culture medium was changed every other day until the cells reached confluence. HUVECs from passage 3 at 80% confluency were used for this experiment. The cells were divided into four groups:-control; treatment with 180 μM H 2 O 2 ; treatment with 150 μg/mL AEPS and concomitant treatment with 150 μg/mL AEPS and 180 μM H 2 O 2 . All treatments were given for 24 hours. The dose of H 2 O 2 used was based on the IC 50 of H 2 O 2 while the dose of AEPS was based on the EC 50 of AEPS adopted from a previous study [19]. The rationale to use H 2 O 2 in this study was to induce oxidative stress. Oxidative stress has been associated with endothelial dysfunction which later progresses into the development of atherosclerosis.

Quantitative reverse transcription polymerase chain reaction (qPCR)
After treatment for 24 hours, total RNA from HUVECs was extracted using TRI Reagent (Molecular Research Center, Cincinnati, USA) as previous research protocol [21]. Polyacryl carrier (Molecular Research Center, Cincinnati, USA) was added to precipitate the total RNA. Extracted RNA pellet was then washed with 75% ethanol and dried prior to dissolving it in RNase and DNase free water (Invitrogen, Carlsbad, USA). Extracted total RNA was assessed for its purity and quantity using Nanodrop ND-100 spectrophotometer (Wilmington DE, USA) and stored at -80°C before use. Complimentary DNA (cDNA) was synthesized using SuperScript III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad, USA). A total of 20 μl of volume reaction which consisted of 10 μl of 2X RT Reaction Mix, 2 μl of RT Enzyme, 5 μl of total RNA and 3 μl of DEPC-treated water was incubated at 25°C for 10 minutes for primer annealing then at 50°C for 30 minutes for reverse transcription. Following this, the reaction was terminated at 85°C for 5 minutes, chilled on ice for 1 minute and 1 μl of E. coli RNase H was added to the mixture. The cDNA was further incubated at 37°C for 20 minutes and stored at -20°C until use. Subsequently qPCR was carried out to determine the mRNA expression of NF-B, VCAM-1, ICAM-1, E-selectin, Nox4, SOD1, CAT and GPx. Glycerylaldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene. Primer 3 software [22] was used to design the primers from NIH GenBank database. The primer sequences for NF-B, VCAM-1, ICAM-1, E-selectin, Nox4, SOD1, CAT and GPx are as listed in Table 1. The qPCR reaction was performed with 1 μl of cDNA, 5 μM of each forward and reverse primer and 12.5 μl of IQ SYBR Green Supermix (Bio-Rad, USA) in BioRad iCycler (Bio-Rad, USA) with reaction profile of: 40 cycles of 95°C (10 seconds) and 61°C (30 seconds). The reaction kinetic of each primer set and protocol was verified with melting profile and product size was further confirmed with 2% agarose gel electrophoresis stained with ethidium bromide (Sigma, St Louis, USA). The threshold cycle (C T ) value was determined and the relative mRNA expression of the genes was calculated as follows: 2ΔΔ CT with ΔΔC T = C T GAPDH -C T gene of interest.

Statistical analysis
Data was tested for normality using Kolmogorov-Smirnov test and all variables were normally distributed. Data was expressed as means of fold change ± SEM. Statistical analysis between two groups was performed

Effects of AEPS on NF-B mRNA expression in HUVECs
Both AEPS and H 2 O 2 did not show any significant changes in the mRNA expression of NF-B (Figure 2).

Effects of AEPS on Nox4 mRNA expression in HUVECs
The aqueous extract of PS significantly reduced Nox4 mRNA expression in HUVECs compared to the control group ( Figure 4). When stimulated with H 2 O 2 , HUVECs expressed higher (1.2-fold) level of Nox4 mRNA expression. However, the H 2 O 2 -induced Nox4 mRNA expression was significantly down regulated by AEPS.

Effects of AEPS on SOD1, CAT and GPx mRNA expression in HUVECs
HUVECs treated with AEPS had significantly higher level of SOD1, CAT and GPx mRNA expressions compared to the control group ( Figure 5A, 5B, 5C). The highest increase was observed in the CAT expression (2.2-fold) followed by GPx (1.3 fold) and SOD1 (1.2fold). In the oxidative stress-induced group, HUVECs treated with H 2 O 2 also showed significantly higher level of SOD1, CAT and GPx mRNA expressions compared with the control group. HUVECs treated with both AEPS and H 2 O 2 had significantly higher level of SOD1, CAT and GPx mRNA expressions than the control group but no significant difference from the single H 2 O 2 -treated and AEPS-treated groups.

Discussion
It is well known that the transcription factor NF-B is essential in regulation of the gene expression of cell adhesion molecules such as VCAM-1, ICAM-1 and Eselectin [5]. Therefore, we hypothesized that AEPS has the ability to modulate the expression of NF-B and cell adhesion molecules in H 2 O 2 -induced HUVECs. In the present study, both AEPS and H 2 O 2 did not have any significant effect on the gene expression of NF-B in HUVECs ( Figure 2). In another study, H 2 O 2 induces NF-B activation in porcine aortic endothelial cells but not in human aortic endothelial cells, suggesting that porcine endothelial cells might be more sensitive to H 2 O 2 compared to human endothelial cells [23]. However, treatment with H 2 O 2 caused an upregulation of ICAM-1 mRNA expression ( Figure 3B). The H 2 O 2 -induced ICAM-1 mRNA expression was significantly down regulated by AEPS. Both AEPS and H 2 O 2 did not have any significant effect on the gene expression of VCAM-1 and E-selectin ( Figure 3A, 3C).
In this study, the effects of H 2 O 2 on the cellular adhesion molecules expression are in accordance with earlier research [24]. In the study, treatment of HUVECs with 50 μmol/L H 2 O 2 for 24 hours increased the level of ICAM-1 mRNA expression but it did not induce the expression of VCAM-1 and E-selectin. Hydrogen peroxide also did not seem to activate NF-B. This could be caused by a difference in sensitivity of endothelial cells among different species. Human endothelial cells are relatively resistant to oxidative damage compared to endothelial cells cultured from other species [24]. Treatment with 400 μM H 2 O 2 upregulates VCAM-1 expression in porcine aortic endothelial cells but not on HUVECs and human aortic endothelial cells, suggesting that porcine endothelial cells might be more sensitive to H 2 O 2 compared with human endothelial cells [23]. However, at a higher dose (> 1000 μM) of H 2 O 2 , HUVECs can significantly upregulate VCAM-1 expression [23]. The dose used in this study was 180 μM which was much lower than that. The lower dose of H 2 O 2 and the relative resistance of HUVECs to oxidative damage may contribute to the non-significant changes in VCAM-1 and E-selectin.  The present study demonstrated that AEPS down regulated the mRNA expression of the ROS-producing enzyme Nox4 (Figure 4), and at the same time, upregulated the expression of ROS-inactivating enzymes; SOD1, CAT and GPx ( Figure 5A, 5B, 5C) in HUVECs. Although several ROS-generating systems have been described in endothelial and other vascular cells, NADPH oxidases (Nox) have now been recognized to be the major source of ROS in the vasculature [9]. The Nox enzyme complex consists of two essential membrane-bound subunits, gp91phox and p22phox, which composed of cytochrome b558, and several cytosolic regulatory components. The enzyme is dormant in resting cells, but on stimulation, the cytosolic subunits translocate to the cytochrome b558 at the membrane leading to activation of the enzyme and the release of large amounts of superoxides [25].
NADPH oxidase-mediated ROS production is regulated at two levels: gene expression of the Nox subunits and the enzyme activity [26]. To the best of our  knowledge, the present study is the first of its kind to report that AEPS decreased the gene expression of Nox4, which is the predominant Nox isoform found in endothelial cells. The present study also showed that treatment of HUVECs with H 2 O 2 upregulated Nox4 mRNA expression. In another study, H 2 O 2 was capable of upregulating the Nox subunit p22phox mRNA and protein expression in endothelial cells [25]. The H 2 O 2induced Nox4 mRNA expression was significantly down regulated by AEPS (Figure 4). This could be one of the mechanisms by which AEPS reduced endothelial oxidative stress.
The antioxidant enzymes represent a first line of defense against ROS by metabolizing them to innocuous byproducts. The first enzymatic reaction in the reduction pathway of oxygen occurs during the dismutation of two molecules of superoxides when they are converted to H 2 O 2 and oxygen. The enzyme involved at this step is one of two isoforms of superoxide dismutase (SOD); CuZnSOD or SOD1 which is present in the cytosol while MnSOD or SOD2 is located in the mitochondrial matrix. Although H 2 O 2 is not a free radical itself, it is reactive and it is rapidly converted into the highly reactive hydroxyl anion in the presence of ferrous ion via the Fenton reaction unless it is efficiently removed. Two enzymes participate in the removal of H 2 O 2 from the cellular environment; glutathione peroxidase (GPx) and catalase (CAT). Glutathione peroxidase is present in both the cytosol and mitochondria while CAT is present mainly in the peroxisomes. Catalase and GPx detoxify H 2 O 2 into water and oxygen [12].
Antioxidant enzymes; SOD, CAT and GPx are thought to be effective for augmentation of antioxidant defenses in endothelial cells [27]. As shown in Figure 5, treatment with AEPS upregulated the expression of SOD1, CAT and GPx in HUVECs. These results  suggested that the protective effects of AEPS against oxidative stress may be related to the increased ability to upregulate the antioxidant enzymes expression. The greatest inductions were in the levels of CAT mRNA; indicating that CAT plays an important role in scavenging H 2 O 2 . In other reports, upregulation of antioxidant enzymes by resveratrol protects aortic smooth muscle cells [28] and HUVECs [29] against oxidative stress. When HUVECs were exposed to H 2 O 2 , there was an increase in the mRNA expression of SOD1, CAT and GPx. This could be part of the defense mechanism of the cells to protect themselves better from the damage that was induced by H 2 O 2 [30].
This study showed that AEPS significantly reduced Nox4 expression and increased SOD1, CAT and GPx expression. This indicates an increase in cellular defense mechanism against oxidative stress and implies the vasculature-protective effect of AEPS. Previous study showed that AEPS has direct ROS scavenging ability [31]. Therefore, this study further improves the previous knowledge on the vasculature-protective effects of AEPS as it shows that AEPS can effectively increase cell defense mechanism against oxidative stress.
The leaves of Piper sarmentosum contained biologically active flavonoid compounds such as myricetin, apigenin, quercetin and rutin which are potent antioxidants [13,32]. Since the aqueous extract of Piper sarmentosum leaves used in the present study was not a purified component, the limitation of this study was the inability to determine the specific components of the plant that mediated the observed effects. However, it is suggested that the effects were due to the flavonoid compounds mentioned above.

Conclusions
The present study describes some novel effects of AEPS. By decreasing the expression of ICAM-1 and Nox4 and enhancing the expression of SOD1, CAT and GPx, AEPS represents a unique approach in reducing endothelial oxidative stress. The findings indicate a new insight into the mechanism involved in the vascular protective effect of AEPS.