Compressive stress-relaxation of human atherosclerotic plaque.

Knowledge of the mechanical properties of human atherosclerotic plaque is fundamental to understanding atherosclerosis and its treatment. Data are scant, however, particularly with respect to the time-dependent nature of plaque behavior. Previous experiments in our lab showed that human plaques do not exhibit the traditional preconditioning behavior common to most soft tissues. In particular, the behaviors of three classes of plaques differed fundamentally in response to multiple, successive, cyclic compression protocols. In this report, we demonstrate that plaques exhibit different responses to successive relaxation tests in uniaxial compression. Not only is there significant relaxation, but there are composition-dependent differences in the general character of the relaxation responses. Such information on the time-dependent behavior is important for the design of clinical protocols such as stenting or angioplasty wherein the atherosclerotic vessel is subjected to persistent or multiple short duration loadings. This study presents a step toward a better understanding of the biomechanical behavior of atherosclerotic plaques; however, the need for much more data remains.

Cocaine-induced increase in the permeability function of human vascular endothelial cell monolayers.

The effects of cocaine on endothelial cell macromolecular transport, electrical resistance, and morphology were assessed. In confluent endothelial monolayers grown on microporus filters, cocaine (0.01 to 1 mmol/L) induced a rapid concentration-dependent increase in permeability to peroxidase and low density lipoprotein. Along with increased transport, the cocaine effect was paralleled by a decrease in transendothelial electrical resistance. Alterations in membrane resistance were fully reversible following washout of the drug, providing evidence that cocaine does not cause permanent injury to the integrity of the monolayer. Cocaines major metabolites, benzoylecgonine and ecgonine methyl ester, had minimal effect on electrical resistance properties, whereas monolayer impedance was markedly depressed by the novel cocaine/alcohol metabolite, cocaine ethyl ester (cocaethylene). Morphologic studies of cocaine-treated endothelial cells revealed a marked disruption of F-actin and the formation of intercellular gaps; no evidence of cell lysis and/or detachment was noted. Forskolin, a potent activator of adenylate cyclase known to promote the endothelial cell barrier function, impaired cocaine-induced changes in electrical resistance and morphology. Cocaine, however, had no effect on resting levels of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) in confluent endothelial monolayers. In summary, the results indicate that cocaine directly induces structural defects in the endothelial cell barrier which enhance the transport of macromolecular tracers, the mechanism does not appear to involve intracellular cAMP.

Increased insulin-like growth factor-1 protein in human left ventricular hypertrophy.

Animal models of cardiac hypertrophy demonstrated increased expression of insulin-like-growth factor-1 (IGF-1) in the heart. To study protein expression of insulin-like-growth factor 1 in left ventricular hypertrophy (LVH) in humans 11 hearts of autopsy cases with LVH were compared to 11 controls using immunohistochemical staining with anti-human IGF-1. LVH was defined as thickening of the left ventricular wall which ranged from 1.6 to 2.5 cm with hearts weights from 400 to 900 g. Immunohistochemical staining for IGF-1 was increased in the presence of LVH. In cases of LVH 37.9 +/- 3.5% of the cross-sectional myocardial area stained positively for IGF-1 compared to 6.8 +/- 2.9% in controls (P < 0.001). The findings support the hypothesis that IGF-1 has a role in the pathogenesis of LVH in humans. The increase of IGF-1 protein with LVH suggests reactivation of the cardiac IGF-1 genes in the hypertrophied adult cardiomyocyte.

Composition- and history-dependent radial compressive behavior of human atherosclerotic plaque.

Invasive procedures to revascularize occluded blood vessels rely on the mechanical response of the diseased tissue. Failure rates associated with such procedures show the need for improvement. to understand the associated mechanics, the material properties of atherosclerotic plaque should be known; yet data are scant. The purpose of this study was to investigate the different mechanical responses exhibited by plaques with different compositions, focusing specifically on radial compressive behavior. A custom-built experimental system was developed that was fully computer controlled with a broad range of loading capabilities. A temperature-controlled, physiologic specimen bath allowed testing at 37 degrees C. Monotonically loaded specimens showed that plaque behavior was nonlinear under finite deformations. A multiple cycle protocol, executed in two phases, distinguished three types of mechanical response of different plaques. The differences in behavior were associated with histologic differences in plaque composition, and mechanically characterized by different "repeatability" (the stabilization of the cyclic response) and "recoverability" (the second loading phase retracing the first loading phase behavior). Type 1 behavior was categorized by repeatability and recoverability. Type 2 behavior displayed repeatability but only partial recovery during the second loading phase. Recovery was absent in type 3 behavior. The histologic observations demonstrated that calcified tissue was present only in specimens displaying type 1 behavior. Fibrous tissue and part of a modified media (due to disease) were present in specimens displaying type 2 behavior. An atheroma, along with a relatively thin modified media, was present in specimens displaying type 3 behavior. The differences in the maximum stretches attained at the end of phase I loading, the stretch offset from the first to the 15th cycle of phase I loading, and the hysteresis in the first and 15th cycles of phase I loading distinguished the specimen behaviors with statistical significance. These compression data showed that plaques exhibit composition- and history-dependent nonlinear and inelastic responses under finite deformations.

The use of bilateral adult renal allografts - a method to optimize function from donor kidneys with suboptimal nephron mass.

Alternatives to traditional organ donor usage has allowed expansion of the organ donor pool to help compensate for the increasing disparity between recipients and donors. The use of bilateral adult renal transplants is a novel idea to salvage older donor kidneys with suboptimal nephron mass that would otherwise be destined for discard. Ten renal transplants were performed utilizing both kidneys from adult cadaver donors with diminished nephron mass determined by calculated glomerular filtration rate or biopsy evidence of significant glomerulosclerosis (>10%). Nine of ten (90%) recipients have satisfactory renal function at a mean follow-up of 7 months. The single case of graft failure was due to documented medical non-compliance. Mean serum creatinine at 6 months was 1.5 mg/dl. Mean measured creatinine clearance was 43.2+/-3.4. These preliminary findings suggest that the use of bilateral renal transplants provide satisfactory early function and allows salvage of older donor kidneys with suboptimal nephron mass.

Role of atheroma liposomes and malondialdehyde-modified low-density lipoproteins in complement activation.

We investigated the ability of atheroma-associated liposomes and malondialdehyde (MDA)-modified low-density lipoproteins (MDA-LDL) to activate complement. Complement activation markers C3a, Bb, C4d and SC5b-9 were measured in both normal and complement-deficient sera. We found that MDA-LDL was able to generate C3a and SC5b-9, predominantly by the alternative pathway. High-density lipoproteins modified with MDA were also capable of C3a generation although to a lesser degree. The presence of atheroma-associated liposomes did not result in detectable levels of complement activation markers. We conclude that MDA-modified lipoproteins may represent a possible source for complement activation within atherosclerotic lesions.

Localization of thyrotropin-releasing hormone mRNA expression in the rat heart by in situ hybridization histochemistry.

Our laboratory has recently detected mRNA of thyrotropin-releasing hormone (TRH) in the rat heart. The density of mRNA for TRH is five-fold higher in the atria than in the left and right ventricle. We also found TRH receptor mRNA and 3H-TRH-binding sites in both ventricles. Cardiac contractility was stimulated after intracoronary administration of TRH. This study was performed to investigate the localization of TRH in the heart. We utilized in situ hybridization histochemistry (ISHH) to localize TRH mRNA expression in the rat heart. ISHH was performed on fresh frozen heart tissue sections which were hybridized with a specific 35S-TRH oligo probe and subsequently processed by autoradiography. The autoradiographic signals corresponding to TRH mRNA were analyzed with an image program. For positive controls TRH mRNA was identified in the hypothalamic paraventricular nucleus. This test confirms the specificity of the TRH oligo probe. Cardiac hybridization signals were observed predominantly in the atria and localized preferentially in atrial connective tissues, vascular adventitia and atrial cardiomyocytes. No hybridization signals were found in ventricular cardiomyocytes. These observations suggest that TRH is synthesized in atrial myocytes and atrial vascular structures. Based on studies which show synthesis of the TRH receptors in ventricular cardiomyocytes, we hypothesize that atrial TRH is an endocrine source for the stimulation of ventricular contractility and that endothelial and adventitial TRH may play a role(s) in the regulation of the growth and/or vasomotor tome of the cardiac vascular system.

Intragenic mutations of CDKN2B and CDKN2A in primary human esophageal cancers.

The CDKN2A and CDKN2B genes, encoding p16 and p15 respectively, are located on chromosome 9p21, a locus at which frequent homozygous and heterozygous deletions occur in many primary human tumors, including esophageal carcinoma. CDKN2A and CDKN2B inhibit cyclin dependent kinase 4 (CDK4) and CDK6 and control cellular proliferation by preventing entry into the S phase of the cell cycle. Their inactivation may contribute to uncontrolled growth in human cancer. We previously described CDKN2A exon 2 mutations in a pilot study of 43 esophageal cancers. In order to determine whether CDKN2A and CDKN2B are frequent targets of 9p21 deletion in esophageal carcinogenesis, we have now analyzed 60 primary esophageal cancers for mutations in both exons 1 and 2 of CDKN2A and CDKN2B by direct sequencing of PCR amplified genomic DNAs. In conjunction with our previously published data, we have identified a total of eight nucleic acid substitutions among 60 esophageal carcinomas; here, we describe one new CDKN2B nonsense mutation and one new silent CDKN2B mutation that occurred somatically. Taken together, these results suggest that intragenic mutations in CDKN2A and CDKN2B occur in esophageal cancer, but that they are infrequent events. In view of the known high frequency of loss of heterozygosity at the chromosome 9p21 locus in esophageal cancers, the current data suggest that intragenic mutation is not the predominant mode of inactivation of CDKN2A and CDKN2B or that other genes are targets of deletion at this locus in these cancers.

Studies on TGF-beta 1 gene expression in the intima of the human aorta in regions with high and low probability of developing atherosclerotic lesions.

Certain regions of the human aorta are at greater risk for early and more severe atherosclerotic lesions development than others. Cornhill and coworkers (Cornhill FJ et al.: Arteriosclerosis 5:415, 1985) created maps for the probability of developing atherosclerosis defining the high-probability region (HPR) in the dorsal descending thoracic aorta and the low-probability region (LPR) in the ventral descending thoracic aorta. Our study examines the hypothesis that transforming growth factor beta -1 (TGF-beta 1), a well-known suppressor of growth and function in many human cell lines, is one of the inhibitors of human atherogenesis. The present experiment analyzes the expression of mRNA for TGF-beta 1 in both the HPR and the LPR of aortas from young (age 17 to 25 y) males of black (n = 8) and white (n = 7) race. The level of TGF-beta 1 gene expression was assessed in the aortic intima in both the HPR and the LPR, using National Institutes of Health Image 1.47, an Apple Macintosh application capable of digital image processing, analysis, and morphometric measurement. There was significantly lower (P = 0.002, alpha = 0.05) TGF-beta 1 gene expression in the HPR than in the LPR in the 22- to 25-y age group. There was no significant difference in the 17- to 21-y age group and between the HPR and the LPR in the entire study group.(ABSTRACT TRUNCATED AT 250 WORDS)

Rare incidence of human papillomavirus types 16 and 18 in primary and metastatic human prostate cancer.

OBJECTIVES: The reported data regarding the incidence and significance of human papillomavirus (HPV) in prostate cancer have been inconsistent. In the present study the incidence of HPV 16 and 18 was evaluated in an expanded series of primary as well as metastatic prostate cancer specimens, in order to evaluate a potential role of HPV infection in development and progression of prostate cancer. This is the first study attempting to establish the significance of HPV in metastatic prostate cancer. METHODS: The presence of high risk human papillomaviruses HPV 16 and 18 was analyzed using the polymerase chain reaction (PCR) amplification method and Southern blot hybridization analysis in a total of 61 prostatic tissue specimens: 43 primary prostate adenocarcinoma formalin-fixed, paraffin-embedded specimens, with varying degrees of differentiation (mean Gleason score 5.8, range 3 to 9); 17 pelvic lymph nodes positive for metastatic deposits; and 1 normal prostate specimen. RESULTS: This human papillomavirus typing indicates that only 1 out of the 43 prostatic specimens analyzed was positive for HPV 16 and 1 metastatic lymph node was positive for HPV 18, as revealed by Southern analysis. These results demonstrate the infrequent detection of HPV 16 and 18 DNA in all the primary prostatic adenocarcinoma specimens and metastatic lymph nodes analyzed in this study population. CONCLUSIONS: The negative HPV status for primary and metastatic prostate cancer demonstrated in this study provides a strong argument against an etiological role of HPV infection in the development and progression of the disease.

Dolichoectatic aneurysm of common carotid artery: an animal model with histological correlation.

There is a limited understanding of the pathogenesis of dolichoectatic (dolicho = long; ectatic = dilated) aneurysms of elastic arteries. Therefore, we developed a model of dolichoectatic changes in elastic arteries by injecting porcine elastase into the media of the carotid artery of male New Zealand white rabbits. After 3 months, gross examination of the carotid arteries in vivo revealed dilated and tortuous vessels. The carotid arteries were then harvested, and cross-sections of the vessels were stained by the Verhoff-van Giesson stain. Histologically, the internal elastic lamina was dissolved in the most dilated areas. The elastic lamellae of the media were also digested and there was reorientation of the innermost medial smooth muscle layer. These gross and histologic changes were present in 80% of the treated carotid arteries and in none of the contralateral control vessels. Our study suggests the importance of the elastic lamellae for the maintenance of tubular shape and length of the carotid artery and describes a new chronic animal model of dolichoectatic aneurysm of the common carotid artery.

Increased prevalence of aortic fatty streaks in cholesterol-fed rabbits administered intravenous cocaine: the role of vascular endothelium.

Several recent postmortem studies suggest an increased prevalence of atherosclerosis in young habitual cocaine abusers. However, little is known about the effects of cocaine abuse on the vascular endothelium and its relationship to atherosclerosis. Therefore, the consequence of chronic administration of intravenous cocaine on the induction of aortic sudanophilia was examined. Male New Zealand White rabbits were fed a 0.5% cholesterol diet for 10 wk. During this period, animals were randomized to receive either cocaine-hydrochloride (0.25 mg/kg) intravenously (n = 17) twice daily; or an equivalent volume of 0.9% physiologic saline, control group (n = 16). Mean values for total circulating leukocytes and platelets and total plasma cholesterol and triglycerides were similar in both groups throughout the protocol. At the completion of the study, aortic sudanophilia was measured and expressed as a percentage of regional involvement (R1 = proximal 4 cm, R2 = middle 6 cm, and R3 = distal 10 cm). Statistical significance among groups was achieved in the proximal thoracic aorta (p = 0.057). No significant differences in sudanophilia were noted in the middle and distal segments. When animals were placed in subgroups according to percent total plaque involvement, there was a significant increased distribution of rabbits with a greater extent of sudanophilia in the cocaine-treated group as compared with control (p = 0.01, chi-square analysis). Immunocytochemical studies using the macrophage-specific and muscle actin-specific monoclonal antibodies demonstrated that sudanophilic areas in both groups were predominantly composed of macrophage-derived foam cells. Evaluation of plaque morphology showed an increase in intimal plaque thickness and in the number of macrophages and smooth muscle cells in cocaine-treated animals; however, group differences were not statistically significant. Because no significant differences were found in the cellular composition of atherosclerotic plaques between groups, further studies were performed to assess the effects of cocaine on the permeability function of cultured endothelial cell monolayers as a possible mechanism of increased sudanophilia. Cocaine (100 microM)-treated endothelial cell monolayers demonstrated an increased permeability to horseradish peroxidase during all time intervals studied (0-6 hr). Permeability differences were statistically significant at 30 min and 1 hr (p = 0.003 and 0.02, respectively). Collectively, these observations suggest that administration of cocaine to cholesterol-fed rabbits increases the prevalence of aortic sudanophilia via at least one possible mechanism involving enhanced vascular permeability.

Characterization of pulsed-dye laser-mediated vasodilatation in a rabbit femoral artery model of vasoconstriction.

Vasoconstriction is a clinical problem associated with invasive vascular procedures, microvascular reconstruction and subarachnoid hemorrhage. We sought to characterize the ability of pulsed-dye laser irradiation to reverse and prevent vasoconstriction in an anesthetized rabbit model of surgically and pharmacologically induced vasoconstriction. Five groups of experiments were performed to study the effect of pulsed-dye laser irradiation delivered through a 320 microns core ball-tip fiber into the femoral artery. The studies demonstrated that pulsed-dye irradiation can reproducibly cause vascular dilatation. The zone of vasodilatation propagated equally proximal and distal to the site of irradiation within the vessel. When saline was infused into the vessel to replace flowing blood during delivery of laser irradiation, no significant vasodilatation occurred. After laser irradiation reversed surgical and pharmacologic vasoconstriction, the vessel was resistant to further pharmacologic vasoconstriction. This resistance to pharmacologic vasoconstriction did not occur if the vessel was pharmacologically predilated before delivery of laser irradiation. Pathologic analysis of the vessels revealed endothelial damage and mild to moderate medial necrosis, most significant at the site of energy delivery. These studies provide characterization of pulsed-dye laser-mediated vasodilatation in an in vivo model. Delivery of pulsed-dye laser energy has potential clinical application and warrants further investigation.

Cocaine: an independent risk factor for aortic sudanophilia. A preliminary report.

Several recent autopsy reports indicate an increased prevalence of coronary atherosclerosis in ischemic heart disease temporally associated with cocaine abuse. The objective of this study was to conduct a retrospective analysis of sudanophilic lesions in young asymptomatic individuals who abused cocaine. Twenty-six cases (15-34-year-old black males) were examined from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. Sixteen subjects (mean age 25 +/- 1 years) had a positive toxicologic screen for cocaine and/or its major metabolites at autopsy and were confirmed habitual cocaine abusers. The remaining 10 cases (mean age 24 +/- 2 years) were subjects with a negative toxicologic screen at autopsy and no history of illicit drug abuse. Post-mortem blood was collected for lipoprotein analysis and determination of smoking status. The aorta and right coronary arteries were stained with Sudan IV and the degree and extent of sudanophilia was quantitated by image analysis. Multiple linear regression analysis of cocaine, age, smoking status, VLDL+LDL-C/HDL-C ratio and HDL-C as predictor variables of percentage intimal surface involvement, revealed an association between cocaine abuse and the extent of sudanophilia in both the thoracic and abdominal aorta (P = 0.002 and 0.049, respectively). Analysis of risk factors or of cocaine abuse as predictors of sudanophilia did not achieve statistical significance in the right coronary artery. These preliminary results suggest that habitual use of cocaine, through unknown mechanism(s), increases aortic sudanophilia independent of traditional risk factors.

Effect of aging on aortic morphology in populations with high and low prevalence of hypertension and atherosclerosis. Comparison between occidental and Chinese communities.

A comparative morphologic study of aortic changes with aging was conducted in different populations in an attempt to separate the effects of hypertension and atherosclerosis. Chinese and the occidental populations were chosen, as they are known to have a high prevalence of hypertension and atherosclerosis, respectively. Aortic tissue was collected from occidental (American and Australian) and Chinese populations from three geographic locations. Postmortem specimens were obtained from four fixed locations: ascending aorta (A), descending thoracic aorta (B), and abdominal aorta (suprarenal [C] and above the aortic bifurcation [D]). Histologic sections were used to measure aortic circumference, medial thickness, intimal thickness, and grade of atherosclerosis. Kidney sections were used to confirm the presence or absence of hypertension. A total of 302 cases (age range, 19 to 104 years; Male-to-female ration, 2:1) were studied: 112 Americans, 80 Australians, and 110 Chinese. Cases were divided into three age groups: 19 to 44; 45 to 64; and 65 years and older. The aortic circumference progressively decreased from sites A to D in all populations and age groups. The aortic circumference increased with age, and the increase was independent of the aortic location. When the populations were separated, however, the greater increase was at location A in the Chinese (P = .008) and locations D in the occidental (P = .13), a population contrast that was significant only in location A. Intimal thickness increased with advancing age and was maximal in the abdominal aorta. The population differences also were significant for intimal thickness and were significantly greater in the occidental population in B, C, and D locations, whereas for atherosclerosis significance was only seen in location D. Hypertension (as defined by the morphologic changes in the kidney) after adjusting for age, height, and weight resulted in no statistical significant effect on aortic circumference or on intimal thickness, but did show a significant increase in atherosclerosis score at locations B, C, and D. Also after adjusting for age, height, and weight, the Chinese had a significantly larger aortic circumference in location A compared with the occidental population, whereas in location D the occidentals with hypertension had a significantly larger circumference compared with Chinese, probably due to an interaction of atherosclerosis and hypertension. After similar adjustments, the medial thickness in locations A and C, the intimal thickness in B, C, and D, and atherosclerosis score in D were significantly greater in occidental than Chinese populations.(ABSTRACT TRUNCATED AT 400 WORDS)

Recovery of heart tissue following focal injury induced by dietary restriction of potassium.

Sixty-four Sprague-Dawley rats (initially weighing 200-225 grams) were divided into three groups. Group 1, the experimental group, was fed a potassium depleted diet for 42 days, followed by a potassium repleted diet for up to an additional 14 days. Group 2, the dietary control group, received a potassium deficient diet, but was continuously supplemented by drinking water containing potassium chloride 150 meq/L. Group 3, the control group remained on normal rat chow and tap water during the entire investigation. Quantitative morphometric analysis was used to assess the percent of myocardium occupied by lesion. These data were analyzed by an analysis of variance (ANOVA) for repeated measures, comparing the three groups with one another; a second analysis compared the myocardial lesions of the dietary experimental group during the potassium depletion and repletion periods. At the end of the dietary depletion period (day 42) focal areas of cardiac myocyte necrosis and mononuclear infiltrate were found in the experimental group. Morphometric assessment on day 42 revealed a volume fraction (Vv) of 8.61 (+/- 4.41)%, which was significantly greater (p = 0.0018), as compared with both control groups. Lesion area significantly regressed in two and one half days after potassium was supplemented in the dietary experimental group to 0.58 (+/- 0.34)% Vv (p = 0.0005). Six days after potassium was replaced in the diet, there was no significant difference between the experimental and control groups, and only a limited connective tissue scar was noted in the experimental group. The mechanism of the rapid regression of lesions and the production of only limited connective tissue scar is suggested but requires further elucidation.

Vascular reactivity during the progression of atherosclerotic plaque. A study in Watanabe heritable hyperlipidemic rabbits.

The effects of varying degrees of atherosclerotic plaque on vascular responsiveness in aortas of Watanabe heritable hyperlipidemic (WHHL) rabbits and New Zealand White (normal cholesterolemic) rabbits were studied. Ring segments from the aortic arch and thoracic aorta were mounted in organ chambers for isometric tension recording and measurement of endothelium-derived relaxing factor. WHHL rabbits were divided into three groups according to age: group 1, 3-5 months; group 2, 6-9 months; and group 3, 12-14 months. Atherosclerotic changes (expressed as a percent of total surface area) in the aortic arches in groups 1, 2, and 3 were 11 +/- 3% (mild), 28 +/- 6% (moderate), and 54 +/- 8% (severe) respectively; only occasional plaques were present in the thoracic aorta in all groups. Maximal contractions elicited with phenylephrine progressively decreased with increasing degrees of atherosclerotic plaque. Contractions evoked by histamine were augmented in all groups of WHHL rabbits when compared with controls, whereas those to serotonin were augmented only in vessels with mild atherosclerosis. As the severity of the intimal lesions increased, endothelium-dependent relaxations to acetylcholine, ATP, and calcium ionophore A23187 progressively decreased. Endothelium-independent relaxation to nitroglycerin was virtually complete in all segments. However, vessels with severe atherosclerosis were less sensitive to this agent as illustrated by a significant increase in the ED50 value. Scanning electron microscopy revealed a predominant loss of endothelial cells in the central regions of fibrous plaques. Thus, in WHHL rabbits, hypercholesterolemia and atherosclerosis result in an increased responsiveness of vascular smooth muscle to histamine and serotonin. Endothelium-mediated relaxation of vascular smooth muscle is reduced with the progression of atherosclerosis primarily due to a loss of endothelial cells.

Postmortem viability and early changes in organ culture of human and rabbit aortic endothelial cells.

In order to understand the role of the postmortem interval (PMI) on endothelial cell changes both in human and rabbit aortas, we have examined the ultrastructural cytomorphologic alterations of these cells. Human aorta (HA) and rabbit aorta (RA) were maintained in calcium-free, glucose-supplemented Hank's balanced salt solution (HBSS). Rabbit endothelial cells (REC) on the aorta (organ culture) assayed morphologically survive for at least 12 h in culture solution. The predominant morphological change in the RA was the formation of multiple subendothelial vacuoles (SEV). These vacuoles may form as the results of increased permeability of endothelial cells to ions and fluid or cell contraction. Cell to cell connection remained intact. Individual and dispersed endothelial cells were observed 8 h after removal from the animal when incubated in calcium-free HBSS. These necrotic endothelial cells were scattered among viable endothelial cells. Human aortic endothelial cells were also well preserved in the same media for periods of 6-8 h postmortem. Increased extracellular calcium (1.3 mM) in the incubation media caused accelerated cell death. These findings suggest that aortic endothelial cells can be preserved for longer periods of postmortem time than would be expected and that the use of calcium-free HBSS media supplemented with glucose improves endothelial cell viability in vitro.

Measurement of ATP synthesis and flocculent matrix densities in mitochondria as a function of 'in vitro' ischemia in the heart and liver of rats.

We intended to determine the levels of adenosine triphosphate (ATP) synthesis at the time when mitochondria ultrastructurally show flocculent densities in the matrix space. For this purpose, mitochondria were isolated from rat heart and rat liver after the tissues have been maintained under controlled ischemic conditions in vitro at 37 degrees C for intervals of 15, 30, 45, 60, 120, 180, and 240 (heart) min. The isolated mitochondria were tested for new ATP synthesis by luciferin/luciferase luminescence in the presence of substrate and adenosine 5'-diphosphate (ADP). The luminescence peaks were standardized and related to an external measure by measuring absorbance of ATP at 259 nm where the extinction coefficient is 15,400. Mitochondrial yield was monitored by measuring succinate dehydrogenase activity in the first homogenate and in the final mitochondrial pellet. Alternatively, cytochrome oxidase activity was used and the protein in the mitochondrial pellet was also determined. We found that the yield of mitochondria was above 53-54% in both liver and heart at 2 h of ischemia. Longer intervals were accompanied by lower yields. The ability to synthesize new ATP declined at different time intervals in ischemia of the heart compared to the liver. After 30 min ischemia, the synthesis in heart mitochondria is 18% of control, while the synthesis of liver mitochondria reaches 16% of control after 45 min of in vitro ischemia. Flocculent densities in heart mitochondria appeared at 45 min ischemia in vitro and in vivo, and at 60 min in liver mitochondria. We conclude that the decline of ATP synthesis is a significant early change in mitochondria and antedates the appearance of flocculent densities.