Marwan Emara

N-acetylcysteine improves the hemodynamics and oxidative stress in hypoxic newborn pigs reoxygenated with 100% oxygen.

Neonatal asphyxia may lead to cardiac and renal complications perhaps mediated by oxygen free radicals. Using a model of neonatal hypoxia-reoxygenation, we tested the hypothesis that N-acetylcysteine (NAC) would improve cardiac function and renal blood flow. Eighteen piglets (aged 1-4 days old, weighing 1.4-2.2 kg) were anesthetized and acutely instrumented for continuous monitoring of pulmonary and renal artery flow (cardiac index [CI] and renal artery flow index [RAFI], respectively) and mean blood pressure. Alveolar hypoxia was induced for 2 h, followed by resuscitation with 100% oxygen for 1 h and 21% oxygen for 3 h. Animals were randomized to sham-operated, hypoxic control, and NAC treatment (i.v. bolus of 150 mg/kg given at 10 min of reoxygenation followed by 100 mg/kg per h infusion) groups. Myocardial and renal tissue glutathione content and lipid hydroperoxide levels were assayed, and histology was examined. After 2 h of hypoxia, all animals were acidotic (pH 6.96 ± 0.04) and in cardiogenic shock with depressed renal blood flow. Upon reoxygenation, CI and RAFI increased but gradually deteriorated later. The NAC treatment prevented the decreased CI, stroke volume, mean blood pressure, systemic oxygen delivery, RAFI, and renal oxygen delivery at 2 to 4 h of reoxygenation observed in hypoxic controls (versus shams, all P < 0.05). The myocardial and renal tissue glutathione content was significantly higher in the NAC treatment group (versus controls). The CI and RAFI at 4 h of reoxygenation correlated with the tissue glutathione redox ratio (r = 0.5 and 0.6, respectively, P < 0.05). There were no significant differences in heart rate, pulmonary artery pressure, systemic oxygen uptake, and tissue lipid hydroperoxide levels between groups. No histologic injury was found in the heart or kidney. In this porcine model of neonatal hypoxia and 100% reoxygenation, NAC improved cardiac function and renal perfusion, with improved tissue glutathione content.

Serum levels of matrix metalloproteinase-2 and -9 and conventional tumor markers (CEA and CA 19-9) in patients with colorectal and gastric cancers

Abstract Background: Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, play an important role in tumor invasion and metastasis. This study aimed to determine the serum levels of MMP-2, MMP-9, 130- and 225-kDa gelatinolytic bands and conventional tumor markers, carcinoembryonic antigen (CEA) and cancer antigen (CA) 19-9, in patients with gastrointestinal cancers. The relationship between these parameters and clinicopathological factors was also studied. Methods: Sera from controls (n=19), and patients with colorectal (n=47) and gastric (n=34) cancer were collected prospectively. The gelatinolytic activities of MMP-2, MMP-9, 130- and 225-kDa bands were determined using gelatin zymography. CEA and CA 19-9 were determined using immunoradiometric assay (IRMA). Results: Serum levels of MMP-9, 130- and 225-kDa gelatinolytic bands, CEA, and CA 19-9, but not MMP-2, in colorectal and gastric cancer were significantly higher than that of controls. No significant correlation was found between histological grade or clinical stage and levels of MMP-9, 130- and 225-kDa gelatinolytic bands, which were correlated (r=0.61–0.89, p<0.005). Conclusions: Our findings suggest that zymographic determination of MMP-9, 130- and 225-kDa gelatinolytic bands in colorectal and gastric cancer may be useful in studying these types of cancer in parallel with conventional tumor markers. Clin Chem Lab Med 2009;47:993–1000.

The effect of dobutamine on platelet aggregatory function in newborn piglets with hypoxia and reoxygenation.

Dobutamine, a &bgr;-adrenoceptor agonist that is often used to treat myocardial dysfunction in asphyxiated neonates, may act on the adrenoceptors of platelets resulting in activation. Little information is available on the effect and mechanistic pathway of dobutamine on the platelet aggregatory function in neonatal asphyxia. Newborn piglets were acutely instrumented and exposed to hypoxia for 2 h and reoxygenation for 4 h. Piglets were randomized to receive dobutamine infusion (5, 10, or 20 µg/kg per min) or saline (hypoxic-control) at 2 to 4 h of reoxygenation (n = 8 each), and sham-operated animals were not exposed to hypoxia and reoxygenation (n = 6). Platelet number, collagen-stimulated whole blood aggregation, and plasma concentrations of thromboxane B2 were studied. The effects of &agr;- and &bgr;-adrenoceptor antagonists (phentolamine and propranolol, respectively) on platelet aggregation to in vitro administration of dobutamine (3µM) were also examined. Shock and metabolic acidosis developed similarly in all hypoxia-reoxygenated groups. At 4 h of reoxygenation, platelet numbers in all groups decreased, with no differences among groups. Platelet aggregation deteriorated significantly with a rightward shift of concentration-response curve in piglets receiving 10 and 20 µg/kg per min of dobutamine. The group that received 20 µg/kg per min of dobutamine had increased plasma thromboxane B2 concentrations from baseline (P < 0.05). The platelet aggregatory response induced by 3 µM of dobutamine was improved by the coadministration of the &bgr;-but not the &agr;-adrenoceptor antagonist. We observed platelet aggregatory dysfunction in hypoxic-reoxygenated newborn piglets treated with high-dose dobutamine. Further investigation is needed to examine the differential effects of dobutamine and hypoxia-reoxygenation in platelet aggregation in newborns.

Temporal platelet aggregatory function in hypoxic newborn piglets reoxygenated with 18%, 21%, and 100% oxygen.

Thromboembolic and bleeding complications are common after asphyxia. We studied the temporal effects of different oxygen concentrations used in resuscitating hypoxic newborn piglets on platelet aggregatory function. Alveolar normocapnic hypoxia (fractional inspired oxygen concentration = 0.15) was induced in piglets (1-4 d, 1.7-2.5 kg) for 2 h, followed by reoxygenation with 18%, 21%, or 100% oxygen for 1 h and then 21% for 2 h (n = 8-9 per group). Control piglets underwent surgery with no hypoxia-reoxygenation (n = 5). Platelet counts and collagen-stimulated (2-10 &mgr;g/mL) whole blood aggregation were studied at normoxic baseline and at 3 h, 2 d, and 4 d of recovery. Platelet activation markers including plasma thromboxane B2 and matrix metalloproteinase 2 and 9 levels were measured. At 2 h hypoxia (mean PaO2 30-35 mmHg), all piglets were hypotensive and acidotic (mean pH 7.19-7.24). In 100% reoxygenation piglets, the concentration-response curves of collagen-stimulated platelet aggregation were significantly shifted upward at 3 h and 2 d of recovery with no differences in the collagen concentration required to induce 50% of maximum aggregation, and this normalized to baseline on 4 d. In the 18% and 21% reoxygenated groups, there were no changes in platelet aggregation during the experiment. Platelet counts were not different between groups and over time. Hypoxic-reoxygenated piglets had increased plasma thromboxane B2 (100% group) and matrix metalloproteinase-2 levels (21% and 100% groups) (versus respective baseline, P < 0.05), with no difference between experimental groups. These findings suggest transient platelet activation in hypoxic newborn piglets resuscitated with 100% but not with 18% and 21% oxygen, of which the clinical significance requires further investigation.

Effects of N-Acetylcysteine on Intestinal Reoxygenation Injury in Hypoxic Newborn Piglets Resuscitated with 100% Oxygen

Background: Neonatal asphyxia may lead to the development of ischemia-reperfusion induced intestinal injury, which is related to oxygen-derived free radical production. N-Acetylcysteine (NAC) is a thiol-containing antioxidant which increases intracellular stores of glutathione. Objectives: Using a swine model of neonatal hypoxia-reoxygenation, we examined whether administration of NAC after resuscitation improved intestinal perfusion and reduced intestinal damage. Methods: Twenty-four piglets (1–4 days old, 1.4–2.2 kg) were anesthetized and acutely instrumented for continuous monitoring of superior mesenteric arterial flow and oxygen delivery. Alveolar hypoxia was induced for 2 h, followed by resuscitation with 100% oxygen for 1 h and 21% oxygen for 3 h. Animals were randomized to sham-operated, hypoxic control and NAC treatment (150 mg/kg i.v. at 0 or 10 min of reoxygenation followed by infusion 100 mg/kg/h) groups. During hypoxia-reoxygenation, intestinal tissue glutathione content, caspase-3 activity and reoxygenation injury were examined. Results: After 2 h of hypoxia, piglets were acidotic and hypotensive, with significantly depressed blood flow and oxygen delivery to the small intestine. Upon reoxygenation, hemodynamics recovered as did oxygen supply to the small intestine. After 4 h of reoxygenation, the NAC treatment improved mesenteric flow and oxygen delivery. Despite reducing the increase in caspase-3 activities after hypoxia-reoxygenation by NAC treatment, no significant differences in the glutathione content and histological grading of ileal injury were found among the experimental groups. Conclusions: In newborn piglets with hypoxia-reoxygenation, NAC may improve mesenteric blood flow and oxygen delivery without significant effect on tissue glutathione content. The protective role of NAC in the reoxygenated intestine after severe hypoxia warrants further investigation.

In vitro inhibition of human MMP-2 collagenolytic and gelatinolytic activity by neutralizing monoclonal antibodies.

Matrix metalloproteinases (MMPs) have been implicated in the degradation of the extracellular matrix in normal and pathological tissue remodelling. Among the MMPs, MMP-2 is the most commonly studied protease that has been involved in cancer, inflammation, infective diseases, degenerative diseases of the brain and vascular diseases. In this study, monoclonal antibodies (MAbs) were generated against human MMP-2, purified, characterized and tested for their ability to inhibit the enzymatic activity of MMP-2. Out of 12 positive clones generated against MMP-2, 2 clones (F2-1-11 and G8-25-5) were selected for further characterization. The selected clones react specifically with human pro and active form of MMP-2 in enzyme linked immunosorbant assay (ELISA), dot immunobinding assay (DIA) and Western blot and do not cross react with other human metalloproteinases or MMP-2 from other species. Additionally, these MAbs (F2-1-11 and G8-25-5) selectively inhibit collagenolytic and gelatinolytic activity of APMA ((p-aminophenylmercuric acetate)-activated-pro-MMP-2 and MMP-2, respectively.

Angiostatins decrease in the kidney of newborn piglets after hypoxia-reoxygenation

Little is known about the expression of kidney angiostatin in the hypoxia and reoxygenation of neonates. In this study, we compared the effect of 21% and 100% reoxygenation on kidney levels of angiostatin and its related factors in newborn piglets subjected to hypoxia-reoxygenation. Newborn piglets were subjected to 2h hypoxia followed by 1h of reoxygenation with either 21% or 100% oxygen and observed for 4days. There were 3 isoforms (38, 43 and 50kDa) of angiostatins identified in the kidney tissue of newborn piglets with the 38kDa being the major isoform (~60%). The 38kDa, but not 43 and 50kDa, angiostatin isoform correlated significantly with the levels of total angiostatin and plasminogen (r=0.95 and r=0.58, respectively). On day 4 of recovery in 100% hypoxic-reoxygenated group, there were decreases in kidney tissue levels of plasminogen, total angiostatin, angiostatin (38 and 43kDa, but not 50kDa), whereas no significant changes were found in the 21% hypoxic-reoxygenated group when compared to the sham-operated piglets with no hypoxia-reoxygenation. Both 21% and 100% hypoxic-reoxygenated groups did not show significant changes in kidney tissue levels of 50kDa angiostatin, MMP-2, MMP-9 and HIF-1alpha. In comparison to 21% oxygen, neonatal resuscitation with 100% oxygen decreased the kidney tissue levels of plasminogen and angiostatin that may play a role in neonatal kidney injury and altered renal development in adulthood.

ω-3 and ω-6 Fatty Acids Modulate Conventional and Atypical Protein Kinase C Activities in a Brain Fatty Acid Binding Protein Dependent Manner in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a highly infiltrative brain cancer with a dismal prognosis. High levels of brain fatty acid binding protein (B-FABP) are associated with increased migration/infiltration in GBM cells, with a high ratio of arachidonic acid (AA) to docosahexaenoic acid (DHA) driving B-FABP-mediated migration. Since several protein kinase Cs (PKCs) are overexpressed in GBM and linked to migration, we explored a possible relationship between B-FABP and levels/activity of different PKCs, as a function of AA and DHA supplementation. We report that ectopic expression of B-FABP in U87 cells alters the levels of several PKCs, particularly PKCζ. Upon analysis of PKCζ RNA levels in a panel of GBM cell lines and patient-derived GBM neurospheres, we observed a trend towards moderate positive correlation (r = 0.624, p = 0.054) between B-FABP and PKCζ RNA levels. Analysis of PKC activity in U87 GBM cells revealed decreased typical PKC activity (23.4%) in B-FABP-expressing cells compared with nonexpressing cells, with no difference in novel and atypical PKC activities. AA and DHA modulated both conventional and atypical PKC activities in a B-FABP-dependent manner, but had no effect on novel PKC activity. These results suggest that conventional and atypical PKCs are potential downstream effectors of B-FABP/fatty acid-mediated alterations in GBM growth properties.

EPINEPHRINE VERSUS DOPAMINE TO TREAT SHOCK IN HYPOXIC NEWBORN PIGS RESUSCITATED WITH 100% OXYGEN: EPINEPHRINE VERSUS DOPAMINE IN ASPHYXIA

Shock and tissue hypoperfusion are common after asphyxia. We compared systemic and regional hemodynamic effects of epinephrine and dopamine in the treatment of shock and hypotension in asphyxiated newborn piglets resuscitated with 100% oxygen. Twenty-four piglets (1-3 days old; weight, 1.4-2.6 kg) were acutely instrumented to measure cardiac index (CI), carotid, mesenteric and renal arterial blood flows, and mean systemic (SAPs) and pulmonary arterial pressures (PAPs). Piglets had normocapnic alveolar hypoxia (FIO2 = 0.08-0.10) for 50 min and reoxygenated with FIO2= 1.0 for 1 h then FIO2 = 0.21 for 3.5 h. After 2 h reoxygenation, either dopamine (2 &mgr;g kg−1 min−1) or epinephrine (0.2&mgr;g kg−1 min−1) was given for 30 min in a blinded randomized manner, which was then increased to maintain SAP (within 10% of baseline, pressure-driven dose) for 2 h. Hypoxia caused hypotension (SAP, 44% ± 3% of baseline), cardiogenic shock (CI, 41% ± 4%), and metabolic acidosis (mean pH, 7.04-7.09). Upon reoxygenation, hemodynamic parameters immediately recovered but gradually deteriorated during 2 h with SAP at 45 ± 1 mmHg, CI at 74 ± 9% of baseline, and pH 7.32 ± 0.03. Low doses of either drug had no significant systemic and renal hemodynamic response. Epinephrine (0.3-1.5 &mgr;g kg−1 min−1) for 2 h increased SAP and CI (with higher stroke volume) and decreased pulmonary vascular resistance (with reduced PAP-SAP ratio), whereas the responses with dopamine (10-25 &mgr;g kg−1 min−1) were modest. Low-dose epinephrine improved mesenteric and carotid arterial flows, whereas the pressure-driven doses of epinephrine and dopamine increased carotid and mesenteric arterial flows, respectively. To treat shock in asphyxiated newborn piglets resuscitated with 100% oxygen, epinephrine exhibits an inotropic action compared with dopamine, whereas both catecholamines can increase carotid and mesenteric perfusion.ABBREVIATIONS - CI-cardiac index; FIO2-fractional inspired oxygen concentration; GSH-glutathione; GSSG-oxidized glutathione; LCCAFI-left common carotid arterial flow index; LRAFI-left renal arterial flow index; PAP-pulmonary arterial pressure; SAP-systemic arterial pressure; SMAFI-superior mesenteric arterial flow index