Azizah Ugusman

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

BackgroundAqueous 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).MethodsHUVECs 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.ResultsWhen 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.ConclusionThe expressional suppression of ICAM-1 and Nox4 and induction of antioxidant enzymes might be an important component of the vascular protective effect of AEPS.

Piper sarmentosum increases nitric oxide production in oxidative stress: a study on human umbilical vein endothelial cells

OBJECTIVE: Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway 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 aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H2O2 for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction. RESULTS: Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs. CONCLUSION: Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.

Flavonoids of Piper sarmentosum and its cytoprotective effects against oxidative stress

Abnormalities in endothelial cell structure and function may lead to diseases such as thrombosis and atherosclerosis. Oxidative stress plays an important role in the pathogenesis of various cardiovascular diseases including atherosclerosis. Previous studies have shown a relationship between a diet rich in flavonoid and a reduced incidence of cardiovascular diseases. Piper sarmentosum (PS) is a plant with high flavonoid content and it possesses antioxidant and anti-atherosclerotic activities. Therefore this study aimed to investigate the flavonoids present in aqueous extract of PS (AEPS) and its cytoprotective effects in oxidative stress-induced human umbilical vein endothelial cells (HUVEC). AEPS contained high total phenolic content (91.02 ± 0.02 mg QE/g DM) and total flavonoid content (48.57 ± 0.03 mg GAE/g DM). Screening using high performance liquid chromatography (HPLC) technique showed the presence of rutin and vitexin as the main flavonoids in AEPS. HUVEC were exposed to 180 µM H2O2 and treated with various concentrations of rutin or vitexin (10 to 400 µM) for 24 hours. Both rutin and vitexin at the concentration of 150-400 µM significantly increased the viability of H2O2-induced HUVEC as denoted by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Therefore rutin and vitexin as the main flavonoids present in PS may be involved in the protective effects of PS against oxidative stress.

Deletion of AMPKα1 attenuates the anticontractile effect of perivascular adipose tissue (PVAT) and reduces adiponectin release

Perivascular adipose tissue (PVAT) surrounds most blood vessels and secretes numerous active substances, including adiponectin, which produce a net anticontractile effect in healthy individuals. AMPK is a key mediator of cellular energy balance and may mediate the vascular effects of adiponectin. In this study, we investigated the role of AMPK within PVAT in mediating the anticontractile effect of PVAT.

High fat diet attenuates the anticontractile activity of aortic PVAT via a mechanism involving AMPK and reduced adiponectin secretion

Background and aim: Perivascular adipose tissue (PVAT) positively regulates vascular function through production of factors such as adiponectin but this effect is attenuated in obesity. The enzyme AMP-activated protein kinase (AMPK) is present in PVAT and is implicated in mediating the vascular effects of adiponectin. In this study, we investigated the effect of an obesogenic high fat diet (HFD) on aortic PVAT and whether any changes involved AMPK. Methods: Wild type Sv129 (WT) and AMPKα1 knockout (KO) mice aged 8 weeks were fed normal diet (ND) or HFD (42% kcal fat) for 12 weeks. Adiponectin production by PVAT was assessed by ELISA and AMPK expression studied using immunoblotting. Macrophages in PVAT were identified using immunohistochemistry and markers of M1 and M2 macrophage subtypes evaluated using real time-qPCR. Vascular responses were measured in endothelium-denuded aortic rings with or without attached PVAT. Carotid wire injury was performed and PVAT inflammation studied 7 days later. Key results: Aortic PVAT from KO and WT mice was morphologically indistinct but KO PVAT had more infiltrating macrophages. HFD caused an increased infiltration of macrophages in WT mice with increased expression of the M1 macrophage markers Nos2 and Il1b and the M2 marker Chil3. In WT mice, HFD reduced the anticontractile effect of PVAT as well as reducing adiponectin secretion and AMPK phosphorylation. PVAT from KO mice on ND had significantly reduced adiponectin secretion and no anticontractile effect and feeding HFD did not alter this. Wire injury induced macrophage infiltration of PVAT but did not cause further infiltration in KO mice. Conclusions: High-fat diet causes an inflammatory infiltrate, reduced AMPK phosphorylation and attenuates the anticontractile effect of murine aortic PVAT. Mice lacking AMPKα1 phenocopy many of the changes in wild-type aortic PVAT after HFD, suggesting that AMPK may protect the vessel against deleterious changes in response to HFD.

An investigation of the antiplatelet effects of succinobucol (AGI-1067)

Abstract Succinobucol is a phenolic antioxidant with anti-inflammatory and antiplatelet effects. Given the importance of oxidant stress in modulating platelet–platelet and platelet–vessel wall interactions, the aim of this study was to establish if antioxidant activity was responsible for the antiplatelet activity of succinobucol. Platelet aggregation in response to collagen and adenosine diphosphate (ADP) was studied in rabbit whole blood and platelet-rich plasma using impedance aggregometry. The effect of oxidant stress on aggregation, platelet lipid peroxides, and vascular tone was studied by incubating platelets, washed platelets or preconstricted rabbit iliac artery rings respectively with a combination of xanthine and xanthine oxidase (X/XO). To study the effect of succinobucol in vivo, anaesthetized rats were injected with up to 150 mg/kg succinobucol and aggregation measured in blood removed 15 mins later. Succinobucol (10−5–10−4 M) significantly attenuated platelet aggregation to collagen and ADP in whole blood and platelet-rich plasma. X/XO significantly increased aggregation to collagen and platelet lipid peroxides and this was reversed by succinobucol. Addition of X/XO to denuded rabbit iliac arteries caused a dose-dependent relaxation which was significantly inhibited by succinobucol. In vivo administration up to 150 mg/kg had no effect on heart rate or mean arterial blood pressure but significantly inhibited platelet aggregation to collagen ex vivo. In conclusion, succinobucol displays anti-platelet activity in rabbit and rat blood and reverses the increase in platelet aggregation in response to oxidant stress.

Changes in IP3 receptor expression and function in aortic smooth muscle of atherosclerotic mice

Peroxynitrite is an endothelium-independent vasodilator that induces relaxation via membrane hyperpolarization. The activation of IP3 receptors triggers the opening of potassium channels and hyperpolarization. Previously we found that relaxation to peroxynitrite was maintained during the development of atherosclerosis due to changes in the expression of calcium-regulatory proteins. In this study we investigated: (1) the mechanism of peroxynitrite-induced relaxation in the mouse aorta, (2) the effect of atherosclerosis on relaxation to peroxynitrite and other vasodilators, and (3) the effect of atherosclerosis on the expression and function of the IP3 receptor. Aortic function was studied using wire myography, and atherosclerosis was induced by fat-feeding ApoE-/- mice. The expression of IP3 receptors was studied using Western blotting and immunohistochemistry. Relaxation to peroxynitrite was attenuated by the IP3 antagonists 2-APB and xestospongin C and also the Kv channel blocker 4-aminopyridine (4-AP). Atherosclerosis attenuated vasodilation to cromakalim and the AMPK activator A769662 but not peroxynitrite. Relaxation was attenuated to a greater extent by 2-APB in atherosclerotic aortae despite the reduced expression of IP3 receptors. 4-AP was less effective in ApoE-/- mice fat-fed for 4 months. Peroxynitrite relaxation involves an IP3-induced calcium release and KV channel activation. This mechanism becomes less important as atherosclerosis develops, and relaxation to peroxynitrite may be maintained by increased calcium extrusion.

Piper sarmentosum attenuates TNF-α-induced VCAM-1 and ICAM-1 expression in human umbilical vein endothelial cells

Objectives Inflammation plays a key role in the pathogenesis of atherosclerosis. Piper sarmentosum is an herb with antioxidant and anti-atherosclerotic activities. The aim of this study was to evaluate the anti-inflammatory properties of an aqueous extract of P. sarmentosum (AEPS) in human umbilical vein endothelial cells (HUVECs). Methods HUVECs were divided into six groups: control, treatment with 10 ng/ml TNF-α, and co-treatment of 10 ng/ml TNF-α with four different concentrations of AEPS (100, 150, 250, and 300 μg/ml) for 24 h. Subsequently, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) protein expression, U937 monocyte cells adhesion, and nuclear factor-kappaB (NF-κB) p65 expression in HUVECs were measured. Results Treatment of TNF-α-stimulated HUVECs with AEPS at different concentrations resulted in decreased VCAM-1 and ICAM-1 protein expression in a dose-dependent manner. Furthermore, AEPS also inhibited TNF-α-stimulated U937 monocyte cells adhesion to HUVECs. In addition, AEPS reduced TNF-α-induced NF-κB p65 expression in a dose-dependent manner. Conclusions The results indicated that AEPS suppressed TNF-α-induced VCAM-1 and ICAM-1 expression NF-κB signaling.

The Assessment of Finger Photoplethysmography Fitness Index (PPGF) among Young Men with Cardiovascular Disease Risk Factors: A Cross Sectional Study

Two new vascular health markers which are derived from finger photoplethysmography (PPG) waveform have been introduced based on Malaysian population, namely PPG fitness index (PPGF) and vascular risk prediction index (VRPI). The objectives of this study were to investigate the associations between PPGF and other cardiovascular disease (CVD) markers such as carotid femoral pulse wave velocity (PWVCF), to compare PPGF between those with and without CVD risk factors and to determine the sensitivity of VRPI in identifying young subjects with CVD risk factors. A total of 114 men age 20 to 40 yrs with and without CVD risk factors were recruited. Risk factors included hypertension, smoking, dyslipidemia, abdominal obesity and family history of premature CVD. Subjects were divided into healthy, those with one risk factor and those with at least two risk factors. Their weight, height, peripheral and central blood pressure (BP), PWVCF and PPGF were measured and the sensitivity of VRPI in predicting subjects with CVD risk factor was calculated. Data was analyzed via SPSS version 15 and p 0.05). The independent variables for PPGF were forward pressure (Beta = 0.35, p < 0.01), PWVCF (Beta = -0.26, p < 0.01), systolic BP (Beta = -0.26, p = 0.04) and height (Beta = 0.24, p < 0.01). The sensitivity of VRPI was 82.02%. In conclusion, PPGF was correlated to PWVCF and may be a potential marker of arterial stiffness. In addition, VRPI is sensitive to be used as an early screening of CVD risk factors.