Veena Dhawan

Effect of garlic supplementation on oxidized low density lipoproteins and lipid peroxidation in patients of essential hypertension

Reactive oxygen species (ROS) has been implicated in the pathogenesis of many diseases including hypertension. Therefore, certain compounds with antioxidative capacity are believed to be protective against such diseases. Some components of garlic are known to possess antioxidative properties. Therefore, in the present study we investigated the effect of short-term garlic supplementation in essential hypertensive patients (EH) on indices of oxidative stress. Twenty patients of EH as diagnosed by JNC VI criteria (Group I) and 20 age and sex-matched normotensive controls were enrolled for the study. Both groups were given garlic pearls (GP) in a dose of 250 mg per day for 2 months. Baseline samples were obtained at start of the study, i.e. 0 day, and thereafter, 2 months (follow-up). Lipids and lipoprotein subfractions, plasma-oxidized low-density lipoproteins (ox-LDL), plasma and urinary concentration of 8-iso-Prostaglandin F2α (8-iso-PGF2α) as a biomarker of oxidative stress in vivo, and the total antioxidant status (TOS) of these individuals were determined. We observed a moderate hypercholesterolemia and a significantly raised blood pressure in hypertensive patients as compared to the controls. The indices of oxidative stress, i.e. plasma ox-LDL and plasma and urinary concentration of 8-iso-PGF2α were significantly increased in EH group. Further, hypertensive patients had a significantly low TOS as compared to the control group. With in 2 months of GP supplementation, there was a significant decline in both systolic (SBP) and diastolic blood pressures (DBP) and a significant reduction in ox-LDL and 8-iso-PGF2α levels in Group I patients. Further, a moderate increase in the TOS was also observed in this group as compared to their control counterparts. These findings suggest that dietary supplementation of garlic may be beneficial in reducing blood pressure and oxidative stress in hypertensive individuals (Mol Cell Biochem 266: 109–115, 2004)

Garlic supplementation prevents oxidative DNA damage in essential hypertension

Oxygen-free radicals and other oxygen/nitrogen species are constantly generated in the human body. Most are intercepted by antioxidant defences and perform useful metabolic roles, whereas others escape to damage biomolecules like DNA, lipids and proteins. Garlic has been shown to contain antioxidant phytochemicals that prevent oxidative damage. These include unique water-soluble organosulphur compounds, lipid-soluble organosulphur compounds and flavonoids. Therefore, in the present study, we have tried to explore the antioxidant effect of garlic supplementation on oxidative stress-induced DNA damage, nitric oxide (NO) and superoxide generation and on the total antioxidant status (TAS) in patients of essential hypertension (EH). Twenty patients of EH as diagnosed by JNC VI criteria (Group I) and 20 age and sex-matched normotensive controls (Group II) were enrolled in the study. Both groups were given garlic pearls (GP) in a dose of 250 mg per day for 2 months. Baseline samples were taken at the start of the study, i.e. 0 day, and thereafter 2 months follow-up. 8-Hydroxy-2′-deoxyguanosine (8-OHdG), lipids, lipid peroxidation (MDA), NO and antioxidant vitamins A, E and C were determined. A moderate decline in blood pressure (BP) and a significant reduction in 8-OHdG, NO levels and lipid peroxidation were observed in Group I subjects with GP supplementation. Further, a significant increase in vitamin levels and TAS was also observed in this group as compared to the control subjects. These findings point out the beneficial effects of garlic supplementation in reducing blood pressure and counteracting oxidative stress, and thereby, offering cardioprotection in essential hypertensives.

Receptor for advanced glycation end products (RAGE) and its inflammatory ligand EN-RAGE in non-diabetic subjects with pre-mature coronary artery disease.

OBJECTIVE Inflammation participates in atherosclerosis from its inception onwards. RAGE (receptor for advanced glycation end products) and its natural pro-inflammatory ligand, EN-RAGE (extracellular newly identified RAGE-binding protein) have been implicated in various inflammatory diseases. In present study, we determined the expression of RAGE and EN-RAGE in peripheral blood mononuclear cells (PBMCs) of subjects with pre-mature coronary artery disease (CAD) for the first time. METHODS AND RESULTS The study patients were angiographically proven non-diabetic patients with pre-mature CAD (Group I; N=100) and control group comprised of subjects with coronary risk factors and without coronary artery lesions (Group II; N=40). Semi-quantitative RT-PCR was performed to determine transcriptional expression of RAGE and EN-RAGE in PBMCs. Soluble RAGE (sRAGE) and C-reactive protein (hsCRP) levels were determined in serum of all study subjects using immunoassays. A significantly increased transcriptional expression of RAGE and EN-RAGE in PBMCs (p<0.01) of Group I patients was observed. Increased circulating hsCRP (p<0.01) levels and decreased sRAGE (p<0.01) levels were observed in Group I as compared with the Group II subjects. Severity of disease determined by Gensini score was found to be positively correlated with transcriptional expression of RAGE (r=0.530) and EN-RAGE (r=0.323). EN-RAGE expression revealed a strong association with RAGE (r=0.326), hsCRP (r=0.251) and a negative association with sRAGE (r=-0.222). CONCLUSIONS Increased expression of RAGE and EN-RAGE in non-diabetic pre-mature CAD and various associations discussed may amplify several cellular perturbations and thus significantly contribute to the pathophysiology of CAD.

Receptor for advanced glycation end products (RAGE) in vascular and inflammatory diseases.

Historically, the receptor for advanced glycation end products (RAGE) was thought to exclusively play an important role under hyperglycemic conditions. However, more and more evidence suggests that RAGE in fact is an inflammation perpetuating multi-ligand receptor and participates actively in various vascular and inflammatory diseases even in normoglycaemic conditions. Various ligands include advanced glycation end products (AGEs), S100 proteins and amphoterins etc. Besides full-length RAGE, numerous truncated forms of the receptor have also been described including the well-characterized soluble RAGE (sRAGE). sRAGE has an ability to act as a decoy to avoid interaction of RAGE with its pro-inflammatory ligands. Ligand engagement of RAGE activates multiple signaling pathways and also forms a positive feedback loop for its own enhanced expression. This review will discuss the role of multi-ligand receptor i.e. RAGE in context to various vascular diseases, which have a pathophysiologically important inflammatory component in normoglycaemic conditions.

C-reactive protein (CRP) up-regulates expression of receptor for advanced glycation end products (RAGE) and its inflammatory ligand EN-RAGE in THP-1 cells: Inhibitory effects of atorvastatin

BACKGROUND Receptor for advanced glycation end products (RAGE) may play an important role in inflammatory processes and endothelial activation. Extracellular newly identified RAGE binding protein (EN-RAGE), natural pro-inflammatory ligand for RAGE. The role of C-reactive protein (CRP) as a mediator in inflammation and atherosclerosis is the subject of recent investigations worldwide. In the present study, we investigated the effect of CRP on RAGE and EN-RAGE gene expression in THP-1 monocytic cell line. MAP kinases (ERK, p38 and JNK) were exploited as possible signaling pathways involved in the signal transduction by CRP. Further, atorvastatin was used as a therapeutic modality for modulation of these genes in the presence of CRP. MATERIALS AND METHODS Time and dose-dependent experiments were carried out in the presence of CRP. Specific MAPK pathways inhibitors were used to elucidate the signaling pathways involved. Effect of atorvastatin was also determined in the presence of CRP on the expression of these genes. RESULTS Time and dose-dependent experiments revealed that, treatment of THP-1 cells with 100 microg of CRP/ml/10(6) cells for 24 h, augmented the expression of RAGE and EN-RAGE genes by 2.5-3.5 folds and 3.5-4.5 folds respectively. CRP acted via FcgammaRII and utilized ERK, p38 and JNK pathways to transduce signals. Atorvastatin in a dose of 20 muM, was able to attenuate up-regulation of CRP-induced genes (p<0.01) and effects were both dose and time-dependent. CONCLUSION Our data strongly suggests that blockade of RAGE-EN-RAGE by statins at an early stage may prevent inflammation in atherosclerosis and counteract the harmful effects mediated by CRP.

Role of 8-iso-prostaglandin f2α and 25-hydroxycholesterol in the pathophysiology of endometriosis

OBJECTIVE To investigate the involvement of 8-iso-PGF(2alpha) and 25-hydroxycholesterol (25-OH-Chol) in the pathophysiology of endometriosis. DESIGN Observational case-control study using enzyme immunoassay and high-performance liquid chromatography (HPLC). SETTING Postgraduate Institute of Medical Education and Research. PATIENT(S) Forty-five women undergoing laparoscopy (n = 25), laparotomy (n = 19), or tubal ligation (n =1). INTERVENTION(S) Venipuncture and laparoscopic peritoneal fluid (PF) collection. MAIN OUTCOME MEASURE(S) The levels of 8-iso-PGF(2alpha) were determined both in urine and PF of all the patients using enzyme immunoassay. The levels of 25-OH-Chol were determined by using reversed phase HPLC both in the plasma and PF samples. Oxidative damage to DNA was assessed by agarose gel electrophoresis. RESULT(S) Significantly increased levels of 8-iso-PGF(2alpha) were observed both in urine and PF of women with endometriosis compared with control women. Similarly, higher levels of 25-OH-Chol were observed both in plasma and PF of patients compared with controls and the difference was statistically significant. A clear-cut tailing pattern was observed in DNA of patients with endometriosis, indicating significant DNA damage. CONCLUSION(S) Our observations implicate oxidative stress in the pathophysiology of endometriosis. For the first time, we demonstrate that 8-iso-PGF(2alpha) and oxysterols (the known promoters of steroidogenesis) might be the culprits in this disease.

In vitro effects of atorvastatin on lipopolysaccharide-induced gene expression in endometriotic stromal cells

OBJECTIVE To investigate the in vitro effects of atorvastatin on lipopolysaccharide (LPS)-induced gene expression in endometrial-endometriotic stromal cells. DESIGN In vitro experimental study using flow cytometry, ELISA, semiquantitative reverse transcriptase polymerase chain reaction, and Western blot. SETTING Postgraduate Institute of Medical Education and Research. PATIENT(S) Twenty-five women undergoing laparoscopy (n = 10) and laparotomy (n = 15). INTERVENTION(S) Endometriotic cyst wall (group I) and endometrial biopsy (group II) collection. MAIN OUTCOME MEASURE(S) The endometrial-endometriotic stromal cells were isolated from ectopic (group I) and eutopic (group II) endometrium by established methods, cultured, and stimulated with LPS (1 μg/mL), followed by atorvastatin treatment in a time- and dose-dependent manner to investigate the effects of LPS on proliferation (Ki-67) and expression of cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), receptor for advanced glycation end products (RAGE), extracellular newly identified RAGE binding protein (EN-RAGE), peroxisome proliferator activated receptor-γ (PPAR-γ), and liver X receptor-α (LXR-α) genes in endometrial-endometriotic stromal cells and on levels of insulin-like growth factor binding protein-1 (IGFBP-1) and 17β-E(2) in endometrial-endometriotic stromal cell culture supernatant. RESULT(S) Significant inhibition of Ki-67 and LPS-induced expression of inflammatory and angiogenic genes (COX-2, VEGF, RAGE, and EN-RAGE) was observed in atorvastatin-treated endometrial-endometriotic stromal cells. In contrast, a significant dose- and time-dependent increase in expression of anti-inflammatory genes (PPAR-γ and LXR-α) and levels of IGFBP-1 was observed after atorvastatin treatment in both the groups. However, atorvastatin treatment had no effect on 17β-E(2) levels in endometrial/endometriotic stromal cell culture supernatant. CONCLUSION(S) The data of the present study provide new insights for the implication of atorvastatin treatment for endometriosis in humans.

Neuroprotective effect of progesterone on acute phase changes induced by partial global cerebral ischaemia in mice

The possible neuroprotective effect of progesterone, a steroid hormone, on acute phase changes in a mouse model of cerebral ischaemia induced by bilateral common carotid artery occlusion (BCAO) was studied. A total of 72 male mice were included in the study. The BCAO model was used to induce partial global cerebral ischaemia. Morphological assessment included measurement of infarct size and brain oedema. Post‐ischaemic seizure susceptibility was assessed using a subconvulsive dose of pentylenetetrazole (30 mgkg−1 i.p.). Biochemical estimations included tumour necrosis factor α (TNF‐α) levels and enzyme parameters such as lipid peroxidation, superoxide dismutase, catalase and glutathione peroxidase, and protein estimation. BCAO induced a significant infarct size and oedema in the saline‐treated control group, along with an increase in oxidative stress, indicated by increased lipid peroxidation and decreased levels of antioxidants such as superoxide dismutase, catalase and glutathione peroxidase. Progesterone (15 mgkg−1 i.p.) administration showed a neuroprotective effect by significantly reducing the cerebral infarct size as compared with the control group. Post‐ischaemic seizure susceptibility was also reduced as the number of positive responders decreased. Brain oedema subsided, but not significantly. Progesterone significantly reduced TNF‐α levels compared with the ischaemia group. Progesterone improved levels of all the anti‐oxidants, indicating activity against oxidative stress induced by BCAO. The results demonstrate the neuroprotective effect of progesterone against ischaemic insult, suggesting a role for the steroid as a neuroprotective agent.

Chronic-arginine supplementation improves endothelial cell vasoactive functions in hypercholesterolemic and atherosclerotic monkeys

Chronic exposure to l-arginine results in regression of atherosclerotic lesions and reversal of endothelial dysfunction. We investigated whether chronic l-arginine supplementation induces regression of atherosclerotic lesions and reversal of endothelial dysfunction in atherogenic rhesus monkeys and the mechanism which leads to these effects. About 12 male rhesus monkeys were fed 1% cholesterol and 18 g butter for 6 months to create an experimental model of hypercholesterolaemia and atherosclerosis (Group I) and 12 monkeys were fed standard stock diet for 6 months (Group II). After, 6 months these two groups were further divided into 2 sub-groups which in addition to their respective diets were fed 2.5% l-arginine in drinking water for additional 6 months (Group III and Group IV). Systemic nitric oxide (NO) formation was assessed as plasma nitrite and cGMP formation every 3 months. Oxygen free radical (OFR) generation and malondialdehyde production as an index of lipid peroxidation were determined. Changes in isometric tension were compared in isolated ring segments of thoracic aorta from normal and hypercholesterolemic animals.Cholesterol feeding progressively reduced plasma nitrite and cGMP generation (p<0.05). Dietary l-arginine partly restored the levels of plasma nitrite and cGMP (p<0.05) but did not change plasma cholesterol levels. l-arginine significantly reduced aortic intimal thickening, blocked the production of carotid and coronary intimal plaques and completely preserved endothelium-dependent vasodilator function. Further, l-arginine significantly inhibited generation of the reactive oxygen species (ROS) generation and lipid peroxidation.Chronic oral supplementation with l-arginine blocks the progression of plaques via restoration of nitric oxide synthase substrate availability and reduction of vascular oxidative stress. (Mol Cell Biochem 269: 1–11, 2005)

Inhibitory effects of Terminalia arjuna on platelet activation in vitro in healthy subjects and patients with coronary artery disease.

Terminalia arjuna (TA) is a medicinal plant used as a cardiotonic in ayurveda. Besides others, scientific evidence dictates its strong hypolipidemic and antioxidant properties. However, anti-inflammatory and antiplatelet aggregatory properties of TA are not known. The present study demonstrates in vitro effects of its ethanolic bark extract (TAE) on platelet function indices. Twenty patients of angiographically proven coronary artery disease (CAD) were included in Group I and 20 age and sex-matched controls were included in Group II. Platelet activation was monitored by determining P-selectin (CD62P) expression, intracellular free calcium (Ca2+) release and platelet aggregation. In vitro effect of TA on platelets function indices was determined by incubating the platelets with TAE in a time and dose-dependent manner in presence/absence of ADP. TAE was able to significantly inhibit platelet aggregation both in patient and control groups. Significant attenuation in Ca2+ release and expression of CD62P was also observed with TAE. Our data clearly demonstrates that the bark extract of TA decreases platelet activation and may possess antithrombotic properties. The possible mechanism of action could be by desensitizing platelets to the agonist by competing with platelet receptor or by interfering with signal transduction. Thus, TA can be exploited for its therapeutic potential in CAD and related cardiovascular disorders.