Interaction of epibrassinolide and selenium ameliorates the excess copper in Brassica juncea through altered proline metabolism and antioxidants.

24-Epibrassinolide (EBL) and Selenium (Se) individually confer tolerance to various abiotic stresses, but their interactive effect in the regulation of copper (Cu) homeostasis in plants exposed to toxic levels of Cu is poorly investigated. This study provides an insight into the effects of EBL (foliar) and/or Se (through sand) on Brassica juncea plants exposed to toxic levels of Cu. The combined effect of EBL and Se on compartmentalization of Cu, oxidative stress markers, photosynthetic machinery and biochemical traits in B. juncea were analyzed. Application of EBL and Se through different mode modulated the compartmentalization of Cu in different parts of plants, enhanced the photosynthetic traits, and activities of various antioxidant enzymes and proline accumulation in B. juncea under excess copper levels. These enhanced levels of antioxidant enzymes, proline (osmolyte) accumulation triggered by combination of EBL and Se could have conferred tolerance to the B. juncea plants under toxic level of copper and also maintained Cu homeostasis in various parts of plants. This study indicates that combination of EBL and Se through different mode is an operative approach for Cu detoxification in plants and could be exploited for removal of excess copper from polluted soil.

A bis-quinoline ruthenium(II) arene complex with submicromolar cytotoxicity in castration-resistant prostate cancer cells.

A new Ru(II) arene chlorido organometallic complex [(η6-p-cymene)(L)RuCl]PF6 (named as pCYRuL) using 2-bis(quinolin-2-ylmethylene) hydrazine (L) was developed that exhibits potent anticancer activity against castration-resistant prostate cancer (CRPC) (IC50 = 0.71 µM), and it is 45 times more effective than the standard drug cisplatin (IC50 = 31.3 µM) in a castration-resistant human prostatic adenocarcinoma cell line (PC-3) but non-toxic in normal human kidney cells (HK2) as well as normal breast cells (MCF10A) and found that pCYRuL exerted anticancer activity via apoptosis induction and cell cycle arrest in the G2/M phase of PC-3 cells.

Design and formulation of mucoadhesive microspheres of sitagliptin.

Mucoadhesive microspheres of sitagliptin (SITCM), a new anti-diabetic drug was prepared with carbopol 934 P using Buchi B-90 nano spray drier and optimized to analyse the key effects and relations of three factors on formulation of SITCM were studied. The appearance of the microspheres was found to be shriveled to nearly spherical, with a narrow size of 2-8 µm. The drug loading and percentage yield was found to be 73 ± 0.2% and 92 ± 0.3%, respectively. In vitro release indicated Korsmeyer-Peppas pattern mucoadhesion of SITCM-8 was found to be 7.8 ± 0.3 h. In vivo studies in rats suggest that the sitagliptin was retained in the gastrointestinal tract for an extended period of time (∼12 h) and control group was reduced significantly (∼4 h). This study concludes that the mucoadhesive microsphere could be one of the most appropriate drug delivery approaches for the successful delivery of sitagliptin.

Update on diagnosis and management of refractory corneal infections.

Infectious keratitis is a medical emergency resulting in significant visual morbidity. Indiscriminate use of antimicrobials leading to the emergence of resistant or refractory microorganisms has further worsened the prognosis. Coexisting ocular surface diseases, delay in diagnosis due to inadequate microbiological sample, a slow-growing/virulent organism, or systemic immunosuppressive state all contribute to the refractory response of the ulcer. With improved understanding of these varied ocular and systemic factors contributing to the refractory nature of the microbes, role of biofilm formation and recent research on improving the bioavailability of drugs along with the development of alternative therapies have helped provide the required multidimensional approach to effectively diagnose and manage cases of refractory corneal ulcers and prevent corneal perforations or further dissemination of disease. In this review, we explore the current literature and future directions of the diagnosis and treatment of refractory keratitis.

Screening of biologically important Zn2+ by a chemosensor with fluorescent turn on-off mechanism.

Reported herein the synthesis, characterization and biologically important zinc ion binding propensity of a weakly fluorescent chemosensor, 4-methyl-2,6-bis((E)-(2-(4-phenylthiazol-2-yl)hydrazono)methyl)phenol (1). 1H NMR spectroscopic titration experiment reveals the binding knack of 1 to the essential Zn2+. The photo-physical studies of 1 exhibit an enhancement in the fluorescence by several folds upon binding with the zinc ions attributed to PET-off process, with a binding constant value of 5.22×103M-1. 1 exhibits an excellent detection range for Zn2+ with lower detection limit value of 2.31×10-8M. The selectivity of 1 was studied with various mono and divalent metal cations and it was observed that most cations either quenches the fluorescence or remains unchanged except for Cd2+, which shows a slight enhancement in fluorescence intensity of 1. The ratiometric displacement of Cd2+ ions by Zn2+ ions shows an excellent selectivity towards in-situ detection of Zn2+ ions. Photo-physical studies also support the reversible binding of 1 to Zn2+ ions having on and off mechanism in presence of EDTA. Such recognition of the biologically important zinc ions finds potential application in live cell imaging.

Suspended animation: the past, present and future of major cardiothoracic trauma.

About 50% of the trauma victims die at the scene mostly because of exsanguinating haemorrhage. Most trials of resuscitation fail in face of the ongoing bleeding. Ongoing research/studies to save these victims by inducing rapid hypothermia using cardiopulmonary bypass as an emergency initial measure along with delayed resuscitation show improved outcomes. A comprehensive review of this research and analysis of studies showed that rapid induction of hypothermia within 5 min of cardiac arrest is associated with better survival and improved neurological outcome. This led us to conclude that suspended animation is a lifesaving modality for the treatment of trauma victims, otherwise hurtling towards certain death. This should be integrated into regular clinical practice. The US Food and Drug Administration has given its approval for clinical trials on such an intervention.

Bio-affinity of copper(II) complexes with nitrogen and oxygen donor ligands: Synthesis, structural studies and in vitro DNA and HSA interaction of copper(II) complexes.

Reported herein the binding affinity between Human Serum Albumin and the DNA binding and cleavage activity of three copper(II) complexes, [Cu(phen)(o-van)ClO4] (1), [Cu(phen)(gly)]ClO4 (2) and [Cu(L1)2(H2O)2] (3) wherein 1 and 2 are synthesized with 1,10-phenanthroline (phen) and co-ligands (o-van: o-vanillin; gly: glycine) and 3 with a ligand 2-hydroxy-3-methoxybenzylidene-4H-1,2,4-triazol-4-amine (H1L1). Complex 2 crystallizes in monoclinic (P21/n) space group shows square pyramidal geometry. The complex 3 crystallizes in monoclinic (P21/a) space group. All the three complexes exhibit binding affinity towards the transport protein Human Serum albumin (HSA). Quantitative evaluation of the thermodynamics of interaction and the results obtained from fluorescence spectroscopy suggest that metal coordinated glycynate, o-vanillin and perchlorate groups have a major role to play in the binding process, the latter two being stronger in the binding of complex 1. The coordinated water in complex 3 also plays an important role in the binding, which makes binding of complex 3 with HSA stronger than that of complex 2. Experimental results indicate that the binding affinity of the complexes towards CT-DNA is in the order 1>3>2 implying that complex 1 binds stronger than complex 3 and 2.The DNA cleaving activity of all the three complexes was explored in the presence of reactive oxygen compound, H2O2. All the three complexes have primarily shown the DNA cleaving activity.

Aprotinin preserves cellular junctions and reduces myocardial edema after regional ischemia and cardioplegic arrest.

BACKGROUND: Cardiac surgery with cardiopulmonary bypass (CPB) and cardioplegic arrest has been associated with myocardial edema attributable to vascular permeability, which is regulated in part by thrombin-induced alterations in cellular junctions. Aprotinin has been demonstrated to prevent activation of the thrombin protease-activated receptor, and we hypothesized that aprotinin preserves myocardial cellular junctions and prevents myocardial edema in a porcine model of regional ischemia and cardioplegic arrest. METHODS AND RESULTS: Fourteen pigs were subjected to 30 minutes of regional ischemia, followed by 60 minutes of CPB, with 45 minutes of crystalloid cardioplegia, then 90 minutes of post-CPB reperfusion. The treatment group (n=7) was administered aprotinin (40,000 kallikrein inhibitor units [KIU]/kg loading dose, 40,000 KIU/kg pump prime, and 10,000 KIU/kg per hour continuous infusion). Control animals (n=7) received normal saline. Myocardial vascular endothelial (VE)-cadherin, beta-catenin and gamma-catenin, and associated mitogen-activated protein kinase (MAPK) pathways were assessed by immunoblot and immunoprecipitation. Histologic analysis of the cellular junctions was done by immunofluorescence. Myocardial tissue water content was measured. VE-cadherin, beta-catenin, and gamma-catenin levels were significantly greater in the aprotinin group (all P<0.05). Immunfluorescence confirmed that aprotinin prevented loss of coronary endothelial adherens junction continuity. Aprotinin reduced tyrosine phosphorylation in myocardial tissue sections. Phospho-p38 activity was approximately 30% lower in the aprotinin group (P=0.007). The aprotinin group demonstrated decreased myocardial tissue water content (81.2+/-0.5% versus 83.5+/-0.3%; P=0.01) and reduced intravenous fluid requirements (2.9+/-0.2 L versus 4.0+/-0.4 L; P=0.03). CONCLUSIONS: Aprotinin preserves adherens junctions after regional ischemia and cardioplegic arrest through a mechanism potentially involving the p38 MAPK pathway, resulting in preservation of the VE barrier and reduced myocardial tissue edema.

Effects of L-arginine on fibroblast growth factor 2-induced angiogenesis in a model of endothelial dysfunction.

BACKGROUND: Nitric oxide availability, which is decreased in advanced coronary artery disease associated with endothelial dysfunction, is an important mediator of fibroblast growth factor-2 (FGF-2)-induced angiogenesis. This could explain the disappointing results of FGF-2 therapy in clinical trials despite promising preclinical studies. We examined the influence of L-arginine supplementation to FGF-2 therapy on myocardial microvascular reactivity and perfusion in a porcine model of endothelial dysfunction. METHODS AND RESULTS: Eighteen pigs were fed either a normal (NORM, n=6) or high cholesterol diet, with (HICHOL-ARG, n=6) or without (HICHOL, n=6) L-arginine. All pigs underwent ameroid placement on the circumflex artery and 3 weeks later received surgical FGF-2 treatment. Four weeks after treatment, endothelial-dependent coronary microvascular responses and lateral myocardial perfusion were assessed. Endothelial cell density was determined by immunohistochemistry. FGF-2, fibroblast growth receptor-1, endothelial-derived nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and syndecan-4 levels were determined by immunoblotting. Pigs from the HICHOL group showed endothelial dysfunction in the circumflex territory, which was normalized by L-arginine supplementation. FGF-2 treatment was ineffective in the HICHOL group (circumflex/left anterior descending blood flow ratios: 1.01 (rest) and 1.01 (pace), after and before treatment). Addition of L-arginine improved myocardial perfusion in response to FGF-2 at rest (ratio 1.13, P=0.02 versus HICHOL) but not during pacing (ratio 0.94, P=NS), and was associated with increased protein levels of iNOS and eNOS. CONCLUSIONS: L-arginine supplementation can partially restore the normal response to endothelium-dependent vasorelaxants and myocardial perfusion in response to FGF-2 treatment in a swine model of hypercholesterolemia-induced endothelial dysfunction. These findings suggest a role for L-arginine in combination with FGF-2 therapy for end-stage coronary artery disease.

Mitogen-activated protein kinase inhibition and cardioplegia-cardiopulmonary bypass reduce coronary myogenic tone.

BACKGROUND: Cardioplegia-cardiopulmonary bypass (C/CPB) is associated with coronary microcirculatory dysfunction. Regulation of the microcirculation includes myogenic tone. Mitogen-activated protein kinases (MAPK) have been implicated in coronary vasomotor function. We hypothesized that vasomotor dysfunction of the coronary microcirculation is mediated in part by alterations in extracellular signal regulated kinase 1/2 (ERK1/2) activity following C/CPB in humans. METHODS AND RESULTS: Atrial myocardium was harvested from patients (n=15) before and after blood cardioplegia and short-term reperfusion under conditions of CPB. Myogenic tone of coronary arterioles was measured by videomicroscopy. Microvessel tone was determined post-C/CPB and after PD98059, a MAPK/ERK kinase 1/2 (MEK1/2) inhibitor. MAPK phosphatase-1 (MKP-1) and activated ERK1/2 were measured by Western blot. MKP-1 gene expression was determined by Northern blot. In situ hybridization and immunohistochemistry were used to localize myocardial MKP-1 and activated ERK1/2, respectively. Myogenic tone was reduced in coronary arterioles post-C/CPB (-10.5+/-0.9%, P<0.01 versus control/pre-C/CPB, n=5). Myogenic tone was decreased in coronary microvessels after 30 micromol/L (n=5) and 50 micromol/L (n=5) PD98059 treatment (-11.0+/-0.8% and -14.6+/-2.0%, respectively, both P<0.01 versus control/pre-C/CPB). Myocardial levels of activated ERK1/2 were reduced post-C/CPB (0.6+/-0.1, post/pre-C/CPB ratio, P<0.05, n=5) while MKP-1 levels increased (4.2+/-0.6, post/pre-C/CPB ratio, P<0.05, n=5). Myocardial MKP-1 gene expression increased post-C/CPB (3.0+/-0.8, post/pre-C/CPB ratio, P<0.05, n=5). MKP-1 and activated ERK1/2 localized to coronary arterioles in myocardial sections. CONCLUSIONS: Coronary myogenic tone is dependent on ERK1/2 and decreased after C/CPB. C/CPB reduces levels of activated ERK1/2, potentially by increased levels of MKP-1. The ERK1/2 signal transduction pathway in part mediates coronary microvascular dysfunction after C/CPB in humans.

Differences in gene expression profiles of diabetic and nondiabetic patients undergoing cardiopulmonary bypass and cardioplegic arrest.

BACKGROUND: Diabetes mellitus is an independent risk factor for early postoperative mortality and complications after coronary artery bypass grafting (CABG). We sought to compare the cardiac gene expression responses to cardiopulmonary bypass (CPB) and cardioplegic arrest (C) in patients with and without diabetes. METHODS AND RESULTS: Twenty atrial myocardium samples were harvested from 5 type II insulin-dependent diabetic and 5 matched nondiabetic patients undergoing CABG, before and after CPB/C. Oligonucleotide microarray analyses of 12625 genes were performed on the 10 sample pairs using matched pre-CPB tissues as controls. Array results were validated with Northern blotting and immunoblotting. Compared with pre-CPB/C, post-CPB/C myocardial tissues revealed 851 upregulated and 480 downregulated genes with a threshold P< or =0.025 (signal-to-noise ratio, 4.04) in the diabetic group, compared with 480 upregulated and 626 downregulated genes (signal-to-noise ratio, 3.04) in the nondiabetic group (P<0.001). There were 18 genes that were upregulated >4-fold in diabetic and nondiabetic patients (including inflammatory/transcription activators FOS, CYR 61, and IL-6, apoptotic gene NR4A1, stress gene DUSP1, and glucose-transporter gene SLC2A3). However, 28 genes showed such marked upregulation in the diabetic group exclusively (including inflammatory/transcription activators MYC, IL8, IL-1beta, growth factor vascular endothelial growth factor, amphiregulin, and glucose metabolism-involved gene insulin receptor substrate 1), and 27 genes in the nondiabetic group only, including glycogen-binding subunit PPP1R3C. CONCLUSIONS: Gene expression profile after CPB/C is quantitatively and qualitatively different in patients with diabetes. These results have important implications for the design of tailored myocardial protection and operative strategies for diabetic patients undergoing CPB/C.

Inhibition of the cardiac angiogenic response to surgical FGF-2 therapy in a Swine endothelial dysfunction model.

BACKGROUND: Discrepancy exists between the potent effects of therapeutic angiogenesis in laboratory animals and the marginal results observed in patients with advanced coronary artery disease. In vitro and small animal data suggest that angiogenesis may depend on locally available nitric oxide (NO), but the impact of endothelial dysfunction on therapeutic angiogenesis in the myocardium has been unclear. We compared the effects of clinically applicable angiogenesis methods in swine in which endothelial dysfunction was experimentally induced to that observed in normal swine. METHODS AND RESULTS: Miniswine were fed either a regular (N=13) or hypercholesterolemic diet (N=13) for 20 weeks. Hypercholesterolemic swine showed coronary endothelial dysfunction on videomicroscopy. Animals from both groups received 100 microg of perivascular sustained-release fibroblast growth factor (FGF)-2 in the lateral myocardial territory, previously made ischemic by placement of an ameroid constrictor around the circumflex artery. After 4 weeks of FGF-2 therapy, lateral myocardial perfusion was significantly lower in hypercholesterolemic than in normocholesterolemic swine, both at rest and during pacing (0.44+/-0.04 versus 0.81+/-0.15 mL/min/g at rest, respectively; P=0.006; and 0.50+/-0.06 versus 0.71+/-0.10 mL/min/g during pacing; P=0.02). Hypercholesterolemic swine showed no net increase in perfusion from FGF-2 treatment. Endothelial cell density and FGF receptor-1 expression were significantly lower in the lateral territory of hypercholesterolemic versus normocholesterolemic animals. CONCLUSIONS: The cardiac angiogenic response to FGF-2 treatment using clinically applicable methods was markedly inhibited in hypercholesterolemic swine with coronary endothelial dysfunction. These findings suggest that coronary endothelial dysfunction is major obstacle to the efficacy of clinical angiogenesis protocols and constitutes a target toward making angiogenesis more effective in patients with advanced coronary disease.

Normalization of coronary microvascular reactivity and improvement in myocardial perfusion by surgical vascular endothelial growth factor therapy combined with oral supplementation of l-arginine in a porcine model of endothelial dysfunction.

OBJECTIVE: Vascular endothelial growth factor acts in part through nitric oxide release, the availability of which is decreased in endothelial dysfunction associated with advanced coronary artery disease. This could explain the relatively disappointing results of vascular endothelial growth factor therapy in clinical studies compared with animal studies. We examined the influence of L-arginine supplementation to vascular endothelial growth factor therapy on myocardial microvascular reactivity and perfusion in a porcine model of endothelial dysfunction. METHODS: Twenty-four pigs were fed either a normal (NORM, n = 8) or high-cholesterol diet with (CHOL-ARG, n = 8) or without (CHOL, n = 8) L-arginine. All pigs underwent ameroid placement on the circumflex artery and then 3 weeks later received surgical vascular endothelial growth factor treatment. Four weeks after treatment, endothelial-dependent coronary microvascular responses and lateral myocardial perfusion were assessed. Endothelial cell density was determined by means of immunohistochemistry. Vascular endothelial growth factor, endothelial nitric oxide synthase, and Akt levels were determined by means of immunoblotting. RESULTS: Pigs from the CHOL group showed endothelial dysfunction in the circumflex territory, which was normalized by L-arginine supplementation. Vascular endothelial growth factor treatment was ineffective in the CHOL group (circumflex/left anterior descending coronary artery blood flow ratios: 0.95 [rest] and 0.74 [pace] before-after treatment; P < .05 compared with the NORM group). Addition of L-arginine restored the angiogenic effect of vascular endothelial growth factor (ratios: 1.13 [rest] and 1.20 [pace]; P < .05) and was associated with increased endothelial cell density, as well as vascular endothelial growth factor, endothelial nitric oxide synthase, and Akt protein levels in the ischemic territory. CONCLUSIONS: L-Arginine supplementation can restore normal endothelium-dependent vasorelaxation and angiogenic response to vascular endothelial growth factor in a swine model of chronic myocardial ischemia with hypercholesterolemia-induced endothelial dysfunction. These findings suggest a putative role for L-arginine in combination with vascular endothelial growth factor therapy for end-stage coronary artery disease.

Low level of selenium increases the efficacy of 24-epibrassinolide through altered physiological and biochemical traits of Brassica juncea plants.

This study was conducted to provide an insight into the effect of Se (through soil) induced changes in Brassica juncea plants in the presence and absence of 24-epibrassinolide (EBL; foliar). The Se treatments showed dual response, 10 µM of Se significantly increased growth, water relations, photosynthetic attributes along with carbonic anhydrase activity whereas its higher concentrations proved inhibitory in concentration dependent manner. The follow-up application of EBL to the Se stressed plants improved growth, water relations, photosynthesis and simultaneously enhanced the various antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase with the excess accumulation of proline. In addition to this, 10 µM Se increases the efficacy of 10(-8) M of EBL and both in combination showed maximum increase for the growth and photosynthetic traits of plants. On the other hand, the elevated level of antioxidant enzymes as well as proline could have conferred tolerance to the Se-stressed plants resulting in improved growth, water relations and photosynthesis.

24-epibrassinolide mitigates the adverse effects of manganese induced toxicity through improved antioxidant system and photosynthetic attributes in Brassica juncea.

The objective of this study was to establish relationship between manganese-induced toxicity and antioxidant system response in Brassica juncea plants and also to investigate whether brassinosteroids activate antioxidant system to confer tolerance to the plants affected with manganese induced oxidative stress. Brassica juncea plants were administered with 3, 6, or 9 mM manganese at 10-day stage for 3 days. At 31-day stage, the seedlings were sprayed with deionized water (control) or 10(-8) M of 24-epibrassinolide, and plants were harvested at 45-day stage to assess growth, leaf gas-exchange traits, and biochemical parameters. The manganese treatments diminished growth along with photosynthetic attributes and carbonic anhydrase activity in the concentration-dependent manner, whereas it enhanced lipid peroxidation, electrolyte leakage, accumulation of H2O2 as well as proline, and various antioxidant enzymes in the leaves of Brassica juncea which were more pronounced at higher concentrations of manganese. However, the follow-up application of 24-epibrassinolide to the manganese stressed plants improved growth, water relations, and photosynthesis and further enhanced the various antioxidant enzymes viz. catalase, peroxidase, and superoxide dismutase and content of proline. The elevated level of antioxidant enzymes as well as proline could have conferred tolerance to the manganese-stressed plants resulting in improved growth and photosynthetic attributes.

Mitogen-activated protein kinase pathways and cardiac surgery.

Mitogen-activated protein kinases are serine-threonine protein kinases that are involved in several processes important to cardiac surgery such as vascular permeability, cytokine production, vasomotor function, and reperfusion injury. Mitogen-activated protein kinases are expressed in multiple cell types including cardiomyocytes, vascular endothelial cells, and vascular smooth muscle cells. Mitogen-activated protein kinases function in cellular signal transduction cascades and are activated by a diverse range of stimuli including ischemia, shear stress, and vasoactive agents. Three major mitogen-activated protein kinase families were identified as the extracellular signal-regulated kinases, c-Jun NH(2)-terminal protein kinases, and p38 kinases. Extensive investigation has established roles for extracellular signal-regulated kinases, c-Jun NH(2)-terminal protein kinases, and p38 kinases in cardiovascular signal transduction pathways. Activity of these signal cascades may contribute to the increased pulmonary vascular permeability and myocardial reperfusion injury observed after cardiac surgery with cardioplegia and cardiopulmonary bypass. Recent findings from our laboratory suggest that alterations in the activity of myocardial extracellular signal-regulated kinase pathways occur as a result of cardioplegia-cardiopulmonary bypass in humans. In addition, these differences in extracellular signal-regulated kinase activity were shown to mediate coronary microcirculatory dysfunction associated with cardioplegia-cardiopulmonary bypass. The resulting deficit in coronary microcirculatory regulation may potentially lead to detrimental effects on organ perfusion and function. As mitogen-activated protein kinase pathways are further characterized, our potential to develop methods to prevent morbidity associated with cardiac surgery and cardiopulmonary bypass may be greatly improved.

Reduction of myocardial reperfusion injury by aprotinin after regional ischemia and cardioplegic arrest.

BACKGROUND: Surgical coronary revascularization with cardiopulmonary bypass and cardioplegia has been associated with reperfusion injury. The serine protease inhibitor aprotinin has been suggested to reduce reperfusion injury, yet a clinically relevant study examining regional ischemia under conditions of cardiopulmonary bypass and cardioplegia has not been performed. METHODS: Pigs were subjected to 30 minutes of regional myocardial ischemia by distal left anterior descending coronary artery occlusion, followed by 60 minutes of cardiopulmonary bypass with 45 minutes of cardioplegic arrest and 90 minutes of post-cardiopulmonary bypass reperfusion. The treatment group (n = 6) was administered aprotinin systemically (40,000 kallikrein-inhibiting units [KIU]/kg intravenous loading dose, 40,000 KIU/kg pump prime, and 10,000 KIU x kg(-1) x h(-1) intravenous continuous infusion). Control animals (n = 6) received crystalloid solution. Global and regional myocardial functions were analyzed by the left ventricular+dP/dt and the percentage segment shortening, respectively. Left ventricular infarct size was measured by tetrazolium staining. Tissue myeloperoxidase activity was measured. Myocardial sections were immunohistochemically stained for nitrotyrosine. Coronary microvessel function was studied by videomicroscopy. RESULTS: Myocardial infarct size was decreased with aprotinin treatment (27.0% +/- 3.5% vs 45.3% +/- 3.0%, aprotinin vs control; P <.05). Myocardium from the ischemic territory showed diminished nitrotyrosine staining in aprotinin-treated animals versus controls, and this was significant by grade (1.3 +/- 0.2 vs 3.2 +/- 0.2, aprotinin vs control; P <.01). In the aprotinin group, coronary microvessel relaxation improved most in response to the endothelium-dependent agonist adenosine diphosphate (44.7% +/- 3.2% vs 19.7% +/- 1.7%, aprotinin vs control; P <.01). No significant improvements in myocardial function were observed with aprotinin treatment. CONCLUSIONS: Aprotinin reduces reperfusion injury after regional ischemia and cardioplegic arrest. Protease inhibition may represent a molecular strategy to prevent postoperative myocardial injury after surgical revascularization with cardiopulmonary bypass.

Gene expression profile after cardiopulmonary bypass and cardioplegic arrest.

OBJECTIVE: This study examines the cardiac and peripheral gene expression responses to cardiopulmonary bypass and cardioplegic arrest. METHODS: Atrial myocardium and skeletal muscle were harvested from 16 patients who underwent coronary artery bypass grafting before and after cardiopulmonary bypass and cardioplegic arrest. Ten sample pairs were selected for patient similarity, and oligonucleotide microarray analyses of 12,625 genes were performed using matched precardiopulmonary bypass tissues as controls. Array results were validated with Northern blotting, real-time polymerase chain reaction, in situ hybridization, and immunoblotting. Statistical analyses were nonparametric. RESULTS: Median durations of cardiopulmonary bypass and cardioplegic arrest were 74 and 60 minutes, respectively. Compared with precardiopulmonary bypass, postcardiopulmonary bypass myocardial tissues revealed 480 up-regulated and 626 down-regulated genes with a threshold P value of.025 or less (signal-to-noise ratio: 3.46); skeletal muscle tissues showed 560 and 348 such genes, respectively (signal-to-noise ratio: 3.04). Up-regulated genes in cardiac tissues included inflammatory and transcription activators FOS; jun B proto-oncogene; nuclear receptor subfamily 4, group A, member 3; MYC; transcription factor-8; endothelial leukocyte adhesion molecule-1; and cysteine-rich 61; apoptotic genes nuclear receptor subfamily 4, group A, member 1 and cyclin-dependent kinase inhibitor 1A; and stress genes dual-specificity phosphatase-1, dual-specificity phosphatase-5, and B-cell translocation gene 2. Up-regulated skeletal muscle genes included interleukin 6; interleukin 8; tumor necrosis factor receptor superfamily, member 11B; nuclear receptor subfamily 4, group A, member 3; transcription factor-8; interleukin 13; jun B proto-oncogene; interleukin 1B; glycoprotein Ib, platelet, alpha polypeptide; and Ras-associated protein RAB27A. Down-regulated genes included haptoglobin and numerous immunoglobulins in the heart, and factor H-related gene 2, protein phosphatase 1, regulatory subunit 3A, and growth differentiation factor-8 in skeletal muscle. CONCLUSIONS: By establishing a profile of the gene-expression responses to cardiopulmonary bypass and cardioplegia, this study allows a better understanding of their effects and provides a framework for the evaluation of new cardiac surgical modalities directly at the genome level.

Cardiopulmonary bypass reduces peripheral microvascular contractile function by inhibition of mitogen-activated protein kinase activity.

BACKGROUND: Mitogen-activated protein kinases (MAPK) have been implicated in pathophysiologic responses to cardiopulmonary bypass (CPB). MAPK are deactivated by phosphatases, such as MAPK phosphatase-1 (MKP-1). We hypothesized that MAPK mediate peripheral microvascular contractile dysfunction caused by CPB in humans. METHODS: Skeletal muscle was harvested before and after CPB. Protein levels of MKP-1 and activated extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 were measured. MKP-1 gene expression was measured. Peripheral microvessel responses to vasopressors were studied by videomicroscopy. Contractile function also was measured after MAPK inhibition with PD98059 (ERK1/2) and SB203580 (p38). ERK1/2, p38, and MKP-1 were localized by immunohistochemistry and in situ hybridization. RESULTS: ERK1/2 and p38 activity was decreased in peripheral tissue after CPB. MKP-1 was increased after CPB. Contractile responses of peripheral arterioles to phenylephrine and vasopressin were decreased after CPB. Microvessel reactivity also was reduced after treatment with PD98059 and SB203580. ERK1/2, p38, and MKP-1 localized to peripheral arterioles in tissue sections. CONCLUSIONS: CPB reduces ERK1/2 and p38 activity in peripheral tissue, potentially by MKP-1. Contractile responses of peripheral arterioles to phenylephrine and vasopressin are dependent on ERK1/2 and p38 and are decreased after CPB. These results suggest that alterations in MAPK pathways in part regulate peripheral microvascular dysfunction after CPB in humans.

Inhibition of the cardiac angiogenic response to exogenous vascular endothelial growth factor.

BACKGROUND: The angiogenic effects of vascular endothelial growth factor (VEGF) are mediated by the stimulation of endothelial nitric oxide synthase (eNOS) and nitric oxide release. Nitric oxide availability is decreased in patients with coronary disease, a possible explanation for the humble results of VEGF in clinical trials. We sought to examine the effects of exogenous VEGF in a model of endothelial dysfunction. METHODS: Miniswine fed either a regular (N = 6, group NORM) or hypercholesterolemic diet (N = 6, HICHOL) underwent ameroid placement on the circumflex artery. Three weeks later, baseline myocardial perfusion was assessed by microsphere injections, and all pigs were treated with VEGF. Four weeks later, microsphere injections were repeated and the hearts harvested. Endothelial-dependent coronary microvascular reactivity, and VEGF and eNOS expression were assessed. RESULTS: HICHOL pigs showed significant endothelial dysfunction in the ischemic territory. Post-treatment myocardial blood flow in the circumflex territory was significantly higher in the NORM compared to the HICHOL group. VEGF and eNOS levels were increased in the ischemic territory in the NORM group but decreased in the HICHOL group. CONCLUSIONS: The cardiac angiogenic response to VEGF was markedly inhibited in a hypercholesterolemia-induced porcine model of endothelial dysfunction. Coronary endothelial dysfunction could be an obstacle to the efficacy of clinical angiogenesis protocols and a putative therapeutic target.