Glutathione-S-transferase protects against oxidative injury of endothelial cell tight junctions.

Oxidative damage of endothelial tight junction permeability is involved in the pathophysiology of a variety of vascular diseases. The authors studied the role of the antioxidant enzyme, human glutathione-S-transferase A4-4 (hGSTA4-4), in regulating expression of major molecules of tight junction in vascular endothelial cells under oxidative stress induced by H(2)O(2). A vascular endothelial cell line, mouse pancreatic endothelial cells (MS1), was transduced with recombinant adenoviral vector containing hGSTA4-4 gene. hGSTA4-4 induced expression of tight junction proteins occludin and zonula occludens (ZO)-1 under oxidative stress. Increased hGSTA4-4 expression correlated with increased transepithelial electrical resistance and decreased tyrosine phosphorylation of occludin and ZO-1 following exposure to H(2)O(2). In addition, morphologic dissociation of occludin, ZO-1, and F-actin during oxidative stress was reduced in hGSTA4-4-expressing cells. To explore a genetic approach for vascular diseases associated with disruption of tight junction proteins, we introduced the same viral vector to blood vessels of mice, rats, and rabbits ex vivo and found strong expression of hGSTA4-4 in endothelial cells. These results demonstrate that oxidative stress mediated disruption of tight junctions in endothelial cells may be attenuated by hGSTA4-4 expression.

Synergistic vascular toxicity and fatty acid anilides in the toxic oil syndrome.

The underlying etiology of the toxic oil syndrome may be related to any of several toxic contaminants. The hypothesis is made that two or more toxic compounds may act synergistically to cause vascular damage in the toxic oil syndrome. To support this hypothesis, previous studies are reviewed concerning the remarkable synergistic toxic action of allylamine and beta-aminopropionitrile on the media of blood vessels. Although these toxins are not directly related to the toxic oil syndrome, this previous experimental work emphasizes the possibility that unexplored synergistic actions may be important. Furthermore, the hypothesis that contaminating fatty acid anilides in toxic oil undergo alterations during cooking is supported by high pressure liquid chromatographic analysis. The theoretic metabolism of fatty acid anilides is discussed. Recent data concerning the toxic actions of the anilides of oleic and linoleic acid are given. These data suggest that these anilides induce immunologic alterations that may be similar to those seen in the toxic oil syndrome. In addition, the heated anilides appear to have increased toxicity, supporting the concept that the use of toxic oil in cooking may increase its toxicity.

Identification of biochemical pathways for the metabolism of oxidized low-density lipoprotein derived aldehyde-4-hydroxy trans-2-nonenal in vascular smooth muscle cells.

Oxidation of low-density lipoproteins (LDL) generates high concentrations of unsaturated aldehydes, such as 4-hydroxy trans-2-nonenal (HNE). These aldehydes are mitogenic to vascular smooth muscle cells and sustain a vascular inflammation. Nevertheless, the processes that mediate and regulate the vascular metabolism of these aldehydes have not been examined. In this communication, we report the identification of the major metabolic pathways and products of [(3)H]-HNE in rat aortic smooth muscle cells in culture. High-performance liquid chromatography separation of the radioactivity recovered from these cells revealed that a large (60-65%) proportion of the metabolism was linked to glutathione (GSH). Electrospray mass spectrometry showed that glutathionyl-1,4 dihydroxynonene (GS-DHN) was the major metabolite of HNE in these cells. The formation of GS-DHN appears to be due aldose reductase (AR)-catalyzed reduction of glutathionyl 4-hydroxynonanal (GS-HNE), since inhibitors of AR (tolrestat or sorbinil) prevented GS-DHN formation, and increased the fraction of the glutathione conjugate remaining as GS-HNE. Gas chromatography-chemical ionization mass spectroscopy of the metabolites identified a subsidiary route of HNE metabolism leading to the formation of 4-hydroxynonanoic acid (HNA). Oxidation to HNA accounted for 25-30% of HNE metabolism. The formation of HNA was inhibited by cyanamide, indicating that the acid is derived from an aldehyde dehydrogenase (ALDH)-catalyzed pathway. The overall rate of HNE metabolism was insensitive to inhibition of AR or ALDH, although inhibition of HNA formation by cyanamide led to a corresponding increase in the fraction of HNE metabolized by the GSH-linked pathway, indicating that ALDH-catalyzed oxidation competes with glutathione conjugation. These metabolic pathways may be the key regulators of the vascular effects of HNE and oxidized LDL.

Papillary-cystic neoplasm of the pancreas.

A case of an unusual pancreatic tumor with a characteristic papillary-cystic microscopic morphology is presented. Review of four previously reported similar cases suggests a distinct clinical picture of a large abdominal mass occurring in a young person which apparently, after resection, does not rapidly recur. The histopathology of this tumor consists of papillary and cystic patterns, regular homogeneous cells with a few mitoses, glassy eosinophilic cytoplasm, and mucin and PAS positivity. Ultrastructural detail, including eccentric nucleoli, numerous mitochondria, sparse endoplasmic reticulum, and little evidence of secretory activity, suggests a duct cell origin for this rare tumor.

Use of low temperatures for glutathione histochemical stain.

We performed glutathione (GSH) staining at a low temperature to prevent GSH release from the section and, hence, improve morphology. Fresh frozen sections of liver, lung, kidney, heart, and stomach were incubated for GSH activity in an ice bath (2-5 degrees C) for 5-10 min. Low temperature incubation prevented GSH diffusion out of cells and minimized migration of granules into vessels and outside of tissue. Incubation at low temperature generally reduced the intensity of the stain compared to the standard method. We conclude that low temperature incubation improves GSH localization in cells, probably by regulating the rate, formation, and the size of GSH-mercury orange complexes.

Allylamine cardiotoxicity--IV. Metabolism to acrolein by cardiovascular tissues.

Acrolein was detected in homogenates of rat aorta, lung, skeletal muscle, and heart incubated with allylamine. Semicarbazide and hydralazine, which protect against allylamine-induced myocardial injury in vivo in the rat, inhibited acrolein formation. Hydrogen peroxide, a product of oxidative deamination, was generated during allylamine oxidation. Acrolein was also produced from allylamine by bovine plasma amine oxidase and porcine kidney diamine oxidase but not by rat liver or brain homogenates. Allylamine competitively inhibited benzylamine oxidation in rat aorta, but pargyline-sensitive monoamine oxidase was not involved in acrolein production. The high activity in aorta, the competition with benzylamine, and the sensitivity to benzylamine oxidase inhibitors indicate that benzylamine oxidase is the active enzyme in oxidizing allylamine. The formation of acrolein may be the basis of the cardiotoxic action of allylamine.

In vivo metabolism of the cardiovascular toxin, allylamine.

Previous evidence from this laboratory demonstrated that allylamine, a known cardiovascular toxin, is metabolized in vitro to acrolein, which has been hypothesized to act as a distal toxin. In this study, 3-hydroxypropylmercapturic acid was isolated and identified by MS, NMR, and 2D-NMR spectroscopy as the sole urinary metabolite of allylamine metabolism in vivo. Parallel experiments showed reduced glutathione (GSH) depletion in several organs (most marked in aorta, blood, and lung), which is consistent with GSH conjugation of the proposed acrolein intermediate. These findings indicate that allylamine was metabolized in vivo to a highly reactive aldehyde which was converted to a mercapturic acid through a GSH conjugation pathway; the exact mechanisms of cellular damage remain unclear.

Juxtaposition of the atrial appendages.

Juxtaposition of the atrial appendages is a rare congenital cardiac anomaly almost universally associated with severe conotruncal abnormalities, especially transposition of the aorta. This presentation describes a case of right sided juxtaposition of the atrial appendages without concomitant conotruncal malformations. This finding contradicts previous hypotheses concerning the genesis of juxtaposition.

Myocardial infarct size: clinicopathologic agreement and discordance.

An accurate postmortem method of planimetrically estimating the extent of myocardial infarction was employed in 16 cases. Delineation of necrotic myocardium was enhanced by a macroscopic staining technique, which utilizes a tetrazolium dye. Comparison of infarct size with peak serum creatine phosphokinase levels showed a general correlation between the two that was not statistically significant. Two markedly disparate cases serve to emphasize the need for clinical awareness of the temporal relationship between myocardial infarction and creatinine phosphokinase analysis as well as the possibility of other anatomic sources of elevation of serum enzyme levels. Comparison of infarct sizes in cardiogenic shock and nonshock patients confirms the existence of a significant relationship between a larger myocardial infarct and shock. However, the data from several patients in the group again emphasize the possibility of maintaining a reasonable blood pressure in the face of a massive myocardial infarction or, more importantly, of manifesting "cardiogenic" shock when only a small amount of left ventricular damage has been sustained. The latter possibility may be related to other anatomic events, e.g., bowel infarction, hemorrhage, or possibly right ventricular ischemia, infarction, or dysfunction.

Myofibroblastic proliferation on mitral valve chordae tendineae: a distinctive lesion associated with alcoholic liver disease.

Hearts from 1,676 consecutive autopsies were examined over a 4 1/2 year period between 1980 and 1984. Forty-seven (4.3%) of 1,083 adult hearts were found to have from one to nine distinctive bulbous thickenings (BTs) involving the mitral valve chordae tendineae. By light- and electron-microscopy, the BTs were found to consist of numerous myofibroblasts, collagen, and elastin layered over otherwise normal chordae and occasionally involving adjacent valve leaflets. No evidence of inflammation, rheumatic or otherwise, was found in histologic sections of the mitral, aortic, and tricuspid valves, or in samples of myocardium from all chambers. No BT was present in 593 hearts from infants and children, indicating that the lesions were acquired. Review of autopsy diagnoses showed that 14 (29.8%) of the 47 patients with BT had alcoholic hepatitis or micronodular cirrhosis, as opposed to 80 (7.7%) of the 1,036 patients without BT. This difference was highly statistically significant (P less than 0.01). The prevalence of viral liver disease was similar in the two groups. Of all patients with alcoholic liver disease, those with BT tended to be male and older. BT appears to be a distinctive process that is strongly correlated with alcoholic liver disease.

Heart donation after lightning strike.

We present the first reported case of heart donation after lightning strike. Approximately 150 to 300 generally young and healthy people die from lightning strike in the United States each year. These unfortunate victims may make good heart donors. Pertinent pathophysiology of lightning strike is briefly reviewed.

Extensive myocytolysis as a marker of sudden cardiac death.

Colliquative myocytolysis, an intracellular vacuolization of myocardial cells, is considered an indicator of acute myocardial ischemia. Although often present in ischemic cardiac death, this lesion is often either overlooked, or not utilized as a criterion for diagnosing ischemia. This study reports light microscopic and ultrastructural studies of six autopsied patients who died suddenly and showed extensive myocytolysis as the only pathologic finding. In two of these patients, the clinical history and electrocardiogram findings documented an acute ischemic episode 1 to 3 hours antemortem. Six control patients with matched age and postmortem intervals did not show the intracellular vacuolization characteristic of myocytolysis. The postmortem intervals ranged from 2 to 12 hours. Ultrastructurally, myocytolysis was seen as a well-defined large intracellular vacuole without any lining membrane and associated with disrupted myofibrils, prominent Z band degeneration, and a few fat droplets. The mitochondria showed swelling and disruption, as well as electron dense amorphous inclusions. The swelling and disruption of mitochondria are well-known postmortem artefacts, and the mitochondrial amorphous inclusions, large intracellular vacuoles, prominent Z band degeneration, and fat droplets are not seen with autolysis. We conclude that myocytolysis, especially if extensive and restricted to an area supplied by a major coronary artery, can be a helpful pathologic marker of myocardial ischemia, particularly in sudden deaths without any other histologic findings.

Lactobacillus endocarditis: a chronic active infection.

Lactobacilli are gram positive rod-shaped or filamentous bacteria that are a rare cause of endocarditis. We review the literature of less than 50 reported cases, and present a case of a 43-year-old male with a protracted, one-year history consistent with endocarditus. The pathologic findings in the heart at autopsy indicate a long, chronic course of smoldering infection with extensive secondary fibroelastosis of valvular structure and adjacent endocardium, although superimposed more acute vegetation with viable organisms consistent with the premortem blood culture of Lactobacillus acidophilus were abuntly present. Thus, the pathologic and clinical features of this case, and our review of the available literature, suggest that infective endocarditis caused by Lactobacillus species has an indolent nature and results in severe, chronic alterations of valvular structure.

Attenuation of isoproterenol-mediated myocardial injury in rat by an inhibitor of polyamine synthesis.

OBJECTIVE: Ornithine decarboxylase (ODC) is an initial rate-limiting enzyme in the synthesis of polyamines (putrescine, spermidine, and spermine) that play a role in cell growth and differentiation. Recent studies have shown that spermidine and spermine cause injury to a variety of cells including myocytes in vitro. In this investigation, we used alpha-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ODC activity and polyamine synthesis to test the hypothesis that polyamines contribute to myocardial injury in rat. METHODS: Male Sprague Dawley rats were treated with (i) saline (0.2 ml/day, s.c.), (ii) isoproterenol (ISO) (5 mg/kg/day for 8 days, s.c.) to produce necrotizing myocardial injury, or with (iii) DFMO + ISO. DFMO was started 2 days before the initiation of ISO and both ISO and DFMO were continued until the end of the experimental period. Myocardial injury was assessed by determining the increased release of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) into the plasma, and by morphometric analysis of the lesion area in heart sections stained with Gomori trichrome. RESULTS: ISO induced the release of CPK and LDH by 6 hr and 24 hr, respectively, and produced subendocardial necrosis, which was both acute and resolving following 8 days of ISO. DFMO treatment inhibited ISO-induced increases in (i) ODC activity and putrescine and spermidine levels in heart, (ii) CPK and LDH activity in plasma, and (iii) the area of subendocardial lesions. CONCLUSIONS: These observations suggest that polyamines are one of the intracellular factors that contribute to ISO-mediated cardiac injury in the rat.

Host response to implanted dacron grafts. A comparison between mesh and velour.

Segments of open Dacron mesh grafts were subcutaneously implanted in rats and harvested for a period of up to 12 weeks after operation at serial intervals. The gross and histologic events of the host response to the external surface were compared to that of segments of low-porosity Dacron velour implanted in a similar fashion. Mature collagen, generously vascularized with new capillaries, was noted throughout the mesh within three to four weeks, while a tightly bonded inner fibrous layer had formed from the surrounding tissues. Major segments of velour floated in amorphous caseous material for up to five weeks. These pools of debris with their concomitant inflammatory response slowly resolved during a ten-week period. This prolonged healing may contribute to eventual graft closure by progressive fibrosis and extrinsic contracture.

Inhibition of polyamine synthesis impairs the secretion of atrial natriuretic peptide.

The aim of the present study was to evaluate in vivo the role of polyamines in the secretion of atrial natriuretic peptide (ANP). alpha-Difluoromethylornithine (DFMO) which inhibits ornithine decarboxylase activity and polyamine synthesis was given in drinking water and through intraperitoneal administration to Sprague-Dawley rats. Carotid artery was cannulated for collection of blood samples and measurement of blood pressure following the administration of arginine-vasopressin (AVP). Analysis of polyamines in cardiac tissue indicated that DFMO treatment decreased contents of putrescine and spermidine in cardiac tissue by 80% and 48%, respectively. Quantitation of ANP in plasma by radioimmunoassay indicated that both basal and stimulated levels of ANP in DFMO-treated animals were 21.5% and 50% of those in control rats. The administration of putrescine restored the levels of basal and AVP-stimulated levels of ANP in plasma which confirmed that DFMO effect on ANP secretion occurred specifically through the polyamine pathway.

Polyamine regulation of atrial natriuretic peptide in cultured cardiocytes.

In the following investigation, we have studied the role of polyamines in the regulation of atrial natriuretic peptide (ANP) using ventricular cardiocytes which in culture synthesize and secrete ANP. Polyamines are cellular cations ubiquitous in eukaryotes, and ANP is a hormone synthesized and secreted by the cardiac atria in adult animal. The cardiocytes were isolated from neonatal rat hearts by enzymatic dissociation using trypsin and collagenase. The functional role of polyamines in regulation of ANP was assessed by exposing the cardiocytes to difluoromethylornithine (DFMO) which is an inhibitor of ornithine decarboxylase, an initial rate-limiting enzyme in the biosynthesis of polyamines. The results showed that DFMO reduced the levels of putrescine (diamine) and spermidine (triamine) in cultured cardiocytes, and it decreased the levels of ANP in media and cellular extracts of cardiocytes as a function of its dose. An addition of putrescine (100 microM) restored within 5-15 min the levels of ANP in media of both control and polyamine-depleted cardiocytes. These results suggest that polyamines are one of the cellular factors involved in regulation of ANP secretion in cultured cardiocytes.

Oxidative modification of lipids and proteins in aniline-induced splenic toxicity.

Our earlier studies with aniline suggested the involvement of oxidative stress as an early toxic event in the spleen. In order to understand the status and consequences of the damaging oxidative reactions, especially during the progression of characteristic splenic lesions, time-dependent subchronic studies were conducted in rats. Male Sprague-Dawley rats were treated with 65 mg/kg/day aniline in the drinking water, while control rats received drinking water only. The animals were euthanized after 1, 2, or 3 months of aniline exposure. Total iron content was remarkably greater in the aniline-treated rats than in age-matched controls. There were time-dependent increases in splenic lipid peroxidation of aniline-treated rats. Malondialdehyde-protein adducts were quantitated by a competitive ELISA and showed greater concentrations in the spleens of aniline-treated rats, further substantiating our lipid peroxidation results. Protein oxidation in the spleens of aniline-treated rats was also greater, with a maximum increase of approximately 76% at 3 months. Western blot analysis for oxidized proteins showed two distinct protein bands at approximately 114 kD and approximately 69 kD in both post-nuclear and mitochondrial fractions of the spleens. Furthermore, densitometric analysis of the blot showed increased band intensities of the oxidized proteins in both these spleen fractions from aniline-treated rats, suggesting the susceptibility of these proteins to aniline-induced oxidative stress. The most prominent morphological changes in the spleens of aniline-treated rats included thickening of the capsule, and capsular cells with nuclear prominence and hyperchromia indicative of capsular hyperplasia. These capsular changes and fibrosis of capsule, splenic trabeculae, and red pulp were noted at all three time points after aniline exposure. Our studies thus suggest that aniline-induced oxidative stress in the spleen is an ongoing event that leads to oxidative modifications of biomolecules. Such oxidative modifications, directly or indirectly, could contribute to the splenic toxicity leading to deleterious consequences, including capsular hyperplasia and fibrosis, as observed in this study, and possibly tumorigenesis in chronic aniline exposure conditions.

Contribution of serum and cellular semicarbazide-sensitive amine oxidase to amine metabolism and cardiovascular toxicity.

Semicarbazide-sensitive amine oxidase (SSAO) plays a role in the in vivo and in vitro toxicity of several environmental and endogenous amines. We investigated the role of SSAO as a component of cell culture medium (through addition of fetal calf serum (FCS)) compared to intracellular SSAO in the in vitro cytotoxicity of three amines and metabolites. Smooth muscle cells and beating cardiac myocytes were grown in 96-well plates and exposed to various concentrations and combinations of FCS in medium, amines (allylamine, AA; benzylamine, BZA; and methylamine, MA), and amine metabolites (aldehydes: acrolein, benzaldehyde, and formaldehyde; hydrogen peroxide, H2O2; ammonia, NH3). Amine and amine metabolite cytotoxicity was quantified by monitoring cell viability. SSAO activity was measured in FCS, cardiovascular cells, or rat plasma by a radioenzymatic assay using [14C]BZA. Our data show that AA and its aldehyde metabolite, acrolein, were the most toxic compounds to both cell types. However, AA toxicity was FCS-dependent in both cell types, while BZA, MA, and amine metabolite (i.e., aldehydes, H2O2, and NH3) cytotoxicity showed little FCS dependence. In these experiments, medium containing 10% FCS had a calculated amine metabolic capacity that was 30- to 50-fold that of the cultured smooth muscle cellular content in a single well of a 96-well plate. Our study demonstrates that SSAO in FCS contributes to amine metabolism and cytotoxicity to rat cardiovascular cells in vitro and how critical it is to evaluate serum for its role in mechanisms of amine toxicity in vitro and in vivo.

Phenylhydroxylamine: role in aniline-associated splenic oxidative stress and induction of subendocardial necrosis.

To elucidate the role of N-phenylhydroxylamine (PHA, N-hydroxylated metabolite of aniline) in the selective toxicity of aniline to the spleen, dose-dependent studies were conducted with PHA in rats. Male Sprague-Dawley rats were given four doses each (1 dose/day) of 0.025, 0.05, 0.1, or 0.2 mmol/kg PHA in 0.5 ml of aqueous agar (0.25%) by gavage. The control animals received an equal volume of vehicle only. The animals were euthanized 24 h following the last dose. PHA toxicity in the blood was evident from a dose-dependent increase of methemoglobin. The most affected organ was spleen, which appeared dark and enlarged (splenomegaly) and showed increased spleen-to-body weight ratios, which were 28, 40, 66, and 87% at PHA doses of 0.025, 0.05, 0.1, and 0.2 mmol/kg, respectively. Splenic lipid peroxidation (malondialdehyde content) was higher in all PHA-treated groups, whereas splenic protein oxidation (carbonyl content) increased in only the 0.05, 0.1, and 0.2 mmol/kg groups. The total iron content in the spleen also showed increases of 88, 135, 168, and 209% at PHA doses of 0.025, 0.05, 0.1, and 0.2 mmol/kg, respectively. These biochemical changes were accompanied by a dose-dependent vascular congestion in the spleen, a characteristic feature of aniline toxicity. Although the ratio of organ to body weight increased for both testes and heart at the highest dose, striking morphological changes were observed only in heart. The cardiac lesions consisted of a both acute and resolving multifocal subendocardial necrosis involving predominently the left ventricle. Our results suggest that PHA is a splenotoxin and thus contributes to the toxicity of aniline, while at a high dose, it is also cardiotoxic, perhaps due to anoxia associated with the marked methemoglobinemia. These results further support the involvement of oxidative stress in the splenotoxicity of aniline which may be caused by its reactive metabolite(s) such as PHA.