Models to study atherosclerosis: a mechanistic insight

The recent failure of candidate drugs like cholesterol ester transfer protein (CETP) and acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors calls for a revised approach for screening anti-atherosclerotic drugs and development of new models of atherosclerosis. For this it is important to understand the mechanism of the disease in a particular model. Models simultaneously showing hyperlipidemia, inflammation and associated complications of diabetes and hypertension will serve the purpose better as they mimic the actual clinical condition. Besides this, analyzing candidate molecules in vivo, in vitro and at various levels of atherosclerosis progression is important. Models based on various cells and process involved in atherosclerosis should be used for screening candidate molecules. The challenge lies in bridging the gap between genetically friendly small animal and human-like bigger animal models. Sequencing of the mouse and human genome, development of a single nucleotide polymorphism (SNP) database and in silico quantitative trait loci (QTL) linkage analysis may enhance the understanding of atherosclerosis and help develop new therapeutic targets

Ramipril-like activity of Spondias mombin linn against no-flow ischemia and isoproterenol-induced cardiotoxicity in rat heart

The cardioprotective property of Spondias mombin (SM) was investigated and compared with that of the ACE inhibitor, ramipril. Alterations to markers of myocardial injury and indices of antioxidant capacity by isoproterenol (ISP) intoxication were significantly corrected in groups treated with SM. The inflammatory index was increased by 24% in ISP-intoxicated group compared with control (P < 0.001) but reduced in the groups administered ISP and treated with 100 or 250 mg/kg SM by 17% (P < 0.001) and 11% (P < 0.05) respectively. Serum lactate dehydrogenase activity and cholesterol level which were significantly increased in ISP-intoxicated group compared with control were reduced in groups administered ISP and treated with SM. Serum phosphate levels in groups administered ISP and treated with SM were significantly lower than values obtained for the ISP-intoxicated group (P < 0.001). Tissue catalase and superoxide dismutase activities as well as glutathione level were significantly increased in groups administered ISP and treated with SM compared to ISP-intoxicated group while MDA and nitrite levels were decreased. Disruption in the structure of cardiac myofibrils by ISP intoxication was reduced by treatment with SM. Comparable results were obtained for ramipril. These results are indicative of the potent cardioprotective property of SM

Nitrite content and antioxidant enzyme levels in the blood of schizophrenia patients

Rationale: Recent studies have suggested augmentation in the inflammatory response as well as involvement of nitric oxide (NO) in mood disorders. Polymorphonuclear leukocytes (PMN), NO and free radicals have been associated with inflammatory response; however, the status of NO in the PMN has not been investigated so far in schizophrenia patients. Objectives: The present study was undertaken to investigate levels of nitrite (a metabolite of NO), malonaldehyde (MDA, lipid peroxidation product) and antioxidant enzymes such as superoxide dismutase (SOD), catalase and glutathione peroxidase (Gpx) in the PMN of schizophrenia patients. Methods: Patients with schizophrenia (n=62) were diagnosed according to DSM-IV and were free of anti-psychotic medications/ECT for at least 3 months. Mean age of the patients was 29.06± 1.17 years, with a male to female ratio of 4:1, and mean duration of illness was 3.7± 0.6 years. The control group consisted of 82 healthy subjects with a mean age of 37.0± 1.26 and a male to female ratio of 5:1. PMN were isolated from the blood. Nitrite, MDA and antioxidant enzymes were estimated by standard biochemical techniques in the PMN of normal healthy controls and schizophrenia patients. Platelet and plasma nitrite levels were also estimated in controls and schizophrenia patients. Results: Nitrite content in the PMN was reduced to 68%, while plasma and platelet nitrite content in schizophrenia patients was not significantly changed in comparison to controls. Malonaldehyde (MDA) content in PMN was significantly augmented in schizophrenia patients but activity of SOD, catalase and Gpx remain unaltered. Conclusion: Results obtained indicate a significant decrease in NO synthesis and an increase in MDA in the PMN of schizophrenia patients, while antioxidant enzyme activities were not altered in the PMN of schizophrenia patients. This suggests that the decrease in PMN NO synthesis by PMN might lead to oxidative stress in schizophrenia patients

Neutrophil extracellular trap formation by nitric oxide donors: involvement of nitric oxide synthase and myeloperoxidase derived free radicals

Presence of neutrophil extracellular traps (NET) at the site of inflammation and infection is demonstrated in various pathological conditions. Neutrophils migration and augmentation in NO availability are mostly associated with inflammation and pathogen infections. So far role of NO has not been explored in NET formation. Present study reports for the first time that incubation of human neutrophils with NO donors, sodium nitroprusside (SNP) or S-nitroso-N-acetyl-penicillamine (SNAP) led to the NET release and free radical generation, which was blocked in the presence of free radical scavengers (ebselen, NAC) or pretreatment of PMNs with inhibitors of NOS reductase domain (7-NI, DPI, imidazole) and myeloperoxidase (ABAH), while NOS oxygenase domain inhibitors had no effect. We thus propose that NO mediated NET release was dependent on the NO synthase (NOS) uncoupling and MPO derived free radicals, but independent of NADPH-oxidase. Moreover, NO dependent free radical generation, NET release and phosphorylation of MEK/ERK, p38MAPK was blocked by inhibitors of MEK/ERK (U0126), p38MAPK (SB202190 ), PKC (Rottlerin) suggesting for the first time role of ERK and p38MAPK in NET formation. The results of the present study seem to have important bearing on the role of NO in inflammatory pathological conditions

Increased myeloperoxidase enzyme activity in plasma is an indicator of inflammation and onset of sepsis

Introduction: Circulating lipopolysaccharides released from bacteria may activate both neutrophils and monocytes. The activated neutrophils release myeloperoxidase (MPO), a specific enzyme with strong oxidative activity. The aim of this study was to evaluate MPO enzyme activity in plasma of critically ill patients and to check the hypothesis that these concentrations in plasma would be higher in sepsis and systemic inflammatory conditions, as neutrophils release their contents before proliferating in response to stress. Material and Methods: Blood samples were collected from 105 critically ill patients admitted to the in:tensive care unit, consisting of those with systemic inflammatory response syndrome (n = 42), sepsis (n = 37), and septic shock (n = 26). Plasma MPO enzyme activity was determined by o-dianisidine-H<SUB>2</SUB>O<SUB>2</SUB> method, modified for 96-well plates. Results: The plasma MPO enzyme activity in sepsis patients was significantly higher than that in the control group (mean, 2.4 ± 1.8 in sepsis and 1.86 ± 1.2 nmol per milligram protein per 10 minutes in systemic inflammatory response syndrome vs 0.32 ± 0.11 nmol per milligram protein per 10 minutes in healthy controls). Mean plasma lactate levels in sepsis (7.8 ± 1.2 mmol/L) and shock patients (9.5 ± 1.2 mmol/L) and cytokines like tumor necrosis factor-α, interleukin-8, and interleukin-1β were simultaneously evaluated to establish onset of inflammation and sepsis. These results show that neutrophil activation occurring during inflammation and sepsis could be detected by plasma MPO concentration. Conclusion: The plasma MPO concentrations may be a marker of the neutrophil proliferation and severity of inflammation

Molecular and biochemical characterization of nitric oxide synthase isoforms and their intracellular distribution in human peripheral blood mononuclear cells

Nitric oxide synthase (NOS) expression and catalytic status in human peripheral blood mononuclear cells (PBMCs) is debatable, while its sub-cellular distribution remains unascertained. The present study characterizes NOS transcripts by real time PCR, NOS protein by immunoprecipitation (IP)/Western blot (WB), nitric oxide (NO) generation by DAF-2DA and NOS sub-cellular distribution by immunogold electron microscopy in resting PBMCs, monocytes and lymphocytes obtained from healthy donors. We observed constitutive expression of full length NOS isoforms (nNOS, iNOS and eNOS) in PBMCs: with the highest expression of iNOS in comparison to nNOS and eNOS. Isolated monocytes expressed more eNOS transcript and protein as compared to nNOS and iNOS. Lymphocytes however had more iNOS transcripts and protein than nNOS and eNOS. NOS was catalytically active in PBMCs, monocytes as well as in lymphocytes as evident by NO generation in the presence of substrate and cofactors, which was significantly reduced in the presence of NOS inhibitor. Immunogold electron microscopy and morphometric analysis revealed the distinct pattern of NOS distribution in monocytes and lymphocytes and also exhibited differences in the nuclear–cytoplasmic ratio. nNOS localization was much more in the cytosol than in the nucleus among both monocytes and lymphocytes. Interestingly, iNOS distribution was comparable in both cytosol and nucleus among monocytes, but in lymphocytes iNOS was predominantly localized to the cytosol. The present study exhibits constitutive presence of all the NOS isoforms in PBMCs and reports the distinct pattern of NOS distribution among monocytes and lymphocytes

Anti-platelet effects of Curcumaoil in experimental models of myocardialischemia-reperfusion and thrombosis

Extensive research on the mechanism of action and medicinal importance of curcumin obtained from turmeric (Curcuma longa) has unfolded its potential therapeutic value against many chronic ailments. Curcuma oil (C.oil), the highly lipophilic component from Curcuma longa has been documented for its neuroprotective efficacy against rat cerebral ischemia-reperfusion injury; however its effect on myocardial reperfusion injury remains unexplored. In the present study, effect of C.oil (500 mg/kg, po) was evaluated against myocardial ischemia-reperfusion induced injury in the rat model. C.oil failed to confer protection against cardiac injury, however significant reversal of ADP induced platelet aggregation (p &lt; 0.05) was evident in the same animals. Moreover, collagen and thrombin induced platelet aggregation (p &lt; 0.001) as well as tyrosine phosphorylation of various proteins in activated platelets was also suppressed. C.oil also offered significant protection against collagen-epinephrine induced thromboembolism in mice as well as augmented total time to occlusion against FeCl3 induced arterial thrombosis in rats. C.oil however had no effect on coagulation parameters (TT, PT and aPTT) and exerted a mild effect on the bleeding time. Bioavailability of C.oil, as assessed by monitoring ar-turmerone, α,β-turmerone and curlone, was 13%, 11% and 7% respectively, indicating high systemic exposure. Moreover, longer mean residence time (MRT) of ar-turmerone (13.2 h), α,β-turmerone (11.6 h) and Curlone (14.0 h) and plasma elimination half lives in the range of 5.5 to 7.2 h correlated with single 500 mg/kg dose regimen of C.oil. In the present study, C.oil thus seems to be an efficacious and safe anti-platelet agent which was protective against intravascular thrombosis

Macropinosome formation is M-CSF dependent.

<p>A and B. Wild-type macrophages were differentiated with M-CSF for 7 days and visualized by phase-contrast microscopy following the treatments described below. Macrophages differentiated with M-CSF were pretreated 30 min with DMSO drug vehicle (A), or 5 µM of cFMS (i.e., M-CSF receptor) tyrosine kinase inhibitor, GW2580 (B). Pretreatment was carried out without either serum or M-CSF. Withdrawal of M-CSF caused disappearance of the macrophage vacuoles. Subsequently, these macrophage cultures were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) without (A) or with GW2580 (B). Macrophages treated with M-CSF without GW2580 showed numerous vacuoles shown to be macropinosomes in Video S1. In contrast, there was complete inhibition of macropinosome formation when macrophage cultures were treated with GW2580 (also see Video S2). Scale bar in B = 75 µm and also applies to A. (C) Wild-type macrophages were incubated 24 h with 1 mg/ml LDL without or with 5 µM GW2580, and then cholesterol accumulation was assessed. Macrophages incubated without LDL had 111±3 nmol cholesterol/mg protein. ** = <i>p</i><0.01.</p

LDL-derived cholesterol accumulation occurs independently of class I PI3K isoforms.

<p>Cholesterol accumulation after 24 h-incubation without or with 1 mg/ml LDL was assessed in M-CSF differentiated macrophages cultured from wild-type, PI3Kγ-KI, PI3Kβ-KI, and PI3Kδ-KI mice.</p

The effect of possible inhibitors of fluid-phase pinocytosis on LDL uptake and net cholesterol accumulation in wild-type M-CSF-differentiated macrophages.

<p>Wild-type bone marrow-derived macrophages were pretreated 1 h with drug vehicle or the indicated drug. For cholesterol accumulation, macrophages then were incubated with 1 mg/ml LDL and inhibitor for 6 h. For ¹²⁵I-LDL uptake, macrophages then were incubated with 200 µg/ml ¹²⁵I-LDL and inhibitor for 6 h. All incubations were performed in serum-free medium containing 50 ng/ml M-CSF. The percent inhibition of net cholesterol accumulation compares macrophages treated with LDL alone and LDL with inhibitor (except the experiment that compares macrophages treated with LDL and dynamin peptide inhibitor with LDL and control peptide) after basal cholesterol values were subtracted from each. The percent inhibition of ¹²⁵I-LDL uptake compares macrophages treated with ¹²⁵I-LDL alone and ¹²⁵I-LDL with inhibitor. Control values for macrophages incubated with LDL alone or ¹²⁵I-LDL alone are indicated in parentheses. For cholesterol accumulation, control values are expressed as nmol net cholesterol accumulation/mg cell protein. For ¹²⁵I-LDL uptake, control values are expressed as µg ¹²⁵I-LDL uptake/mg cell protein. The range of cell-associated and degraded ¹²⁵I-LDL for all treatments except bafilomycin A1-treated macrophages was 15–19% and 82–86%, respectively. Cell-associated and degraded ¹²⁵I-LDL for bafilomycin A1-treated macrophages was 91% and 9%, respectively. * = <i>p</i><0.05. ** = <i>p</i><0.01. *** = <i>p</i><0.001. ND = not determined. “+” indicates a decrease in the number of macropinosomes and “−” indicates no effect on macropinosomes. “−” in percent inhibition columns indicates stimulation rather than inhibition.</p