Chronic hibernating myocardium in sheep can occur without degenerating events and is reversed after revascularization.

INTRODUCTION: Our goal was to show that blunting of myocardial flow reserve is mainly involved in adaptive chronic myocardial hibernation without apparent cardiomyocyte degeneration. METHODS AND RESULTS: Sheep chronically instrumented with critical multivessel stenosis and/or percutaneous transluminal coronary angioplasty (PTCA)-induced revascularization were allowed to run and feed in the open for 2 and 5 months, respectively. Regional myocardial blood flow (MBF) with colored microspheres, regional and global left ventricular function and dimensions (2D echocardiography), and myocardial structure were studied. In sheep with a critical stenosis, a progressive increase in left ventricular end-diastolic and end-systolic cavity area and a decrease in fractional area change were found. Fraction of wall thickness decreased in all left ventricular wall segments. MBF was slightly but not significantly decreased at rest at 2 months. Morphological quantification revealed a rather small but significant increase in diffusely distributed connective tissue, cardiomyocyte hypertrophy, and presence of viable myocardium of which almost 30 % of the myocytes showed depletion of sarcomeres and accumulation of glycogen. The extent of myolysis in the transmural layer correlated with the degree of left ventricular dilation. Structural degeneration of cardiomyocytes was not observed. Balloon dilatation (PTCA) of one of the coronary artery stenoses at 10 weeks revealed recovery of fraction of wall thickness and near normalization of global subcellular structure at 20 weeks. CONCLUSION: These data indicate that chronic reduction of coronary reserve by itself can induce ischemic cardiomyopathy characterized by left ventricular dilatation, depressed regional and global function, adaptive chronic myocardial hibernation, reactive fibrosis and cardiomyocyte hypertrophy in the absence of obvious degenerative phenomena. SUMMARY: Reduction of myocardial flow reserve due to chronic coronary artery stenosis in sheep induces adaptive myocardial hibernation without involvement of degenerative phenomena.

Temporal and spatial variations in structural protein expression during the progression from stunned to hibernating myocardium.

BACKGROUND: Dysfunctional and normally perfused remote regions show equal myolysis and glycogen accumulation in pig hibernating myocardium. We tested the hypothesis that these arose secondary to elevations in preload rather than ischemia. METHODS AND RESULTS: Expression of structural protein (desmin, desmoplakin, titin, cardiotin, alpha-smooth muscle actin, lamin-A/C, and lamin-B2) in viable dysfunctional myocardium was analyzed by immunohistochemistry. We performed blinded analysis of paired dysfunctional left anterior descending coronary artery and normal remote subendocardial samples from stunned (24 hours; n=6), and hibernating (2 weeks; n=6) myocardium versus sham controls pigs (n=7). Within 24 hours, cardiac myocytes globally reexpressed alpha-smooth muscle actin. In stunned myocardium, cardiotin was globally reduced, whereas reductions in desmin were restricted to the dysfunctional region. Alterations progressed with the transition to hibernating myocardium, in which desmin, cardiotin, and titin were globally reduced. A qualitatively similar reorganization of cytoskeletal proteins occurred 3 hours after transient elevation of left ventricular end-diastolic pressure to 33+/-3 mm Hg. CONCLUSIONS: Qualitative cardiomyocyte remodeling similar to that in humans with chronic hibernation occurs rapidly after a critical coronary stenosis is applied, as well as after transient elevations in left ventricular end-diastolic pressure in the absence of ischemia. Thus, reorganization of cytoskeletal proteins in patients with viable dysfunctional myocardium appears to reflect chronic and/or cyclical elevations in preload associated with episodes of spontaneous regional ischemia.

Time course of atrial fibrillation-induced cellular structural remodeling in atria of the goat.

BACKGROUND: Previously we documented cellular structural changes of a non-degenerative nature in atrial myocytes after atrial fibrillation (AF) in the goat. The time course of these changes was not studied. METHODS AND RESULTS: Cellular structural changes were studied by light- and electron microscopy and immunohistochemistry in goat atria after 0-16 weeks AF. The first sign of cellular structural remodeling was a more homogeneous chromatin distribution, at 1 week of AF. Sub-structural changes in mitochondria and sarcoplasmic reticulum occurred gradually. Cellular degeneration was absent. The degree of myolysis and glycogen accumulation increased till 8 weeks of AF and did not increase further from thereon. After 16 weeks of AF, 42% of the myocytes in the right atrial free wall were affected by myolysis. The diameter of the atrial myocytes increased. Dedifferentiation of the atrial myocytes was suggested by altered expression patterns of structural proteins, such as the disappearance of cardiotin (1 week), the A-I junctional part of titin (4 weeks), desmin at the intercalated disk (ID) (8 weeks) and a gradual re-expression of alpha-smooth muscle actin. CONCLUSION: Remodeling of the cellular ultrastructure in atrial myocardium of the goat develops progressively during AF. Re-expression of fetal proteins indicate dedifferentiation of atrial myocytes, analogous to observations in hibernating myocardium of the ventricle.

Prognostic value of cytokinetic parameters in lung cancer after in vivo bromodeoxyuridine labelling.

BACKGROUND: To improve the overall survival rate in lung cancer by adjustment of treatment protocols on basis of tumour characteristics of individual patients, independent predictive parameters are required. Therefore, we evaluated the prognostic value of cytokinetic parameters such as bromodeoxyuridine (BrdU) labelling index (LI), S-phase fraction (SPF), unlabelled S-phase fraction (USPF), S-phase duration (Ts) and potential tumour doubling time (Tpot), next to more established parameters. MATERIALS AND METHODS: To this end a series of 92 bronchoscopic specimens of in vivo BrdU labelled lung cancer patients, 72 presenting with non-small cell lung carcinoma (NSCLC) and 20 patients with small cell lung carcinoma (SCLC), were analysed flow cytometrically. Clinical as well as cytokinetic data were collected and related to survival times over a follow-up period of 2 to 7 years. RESULTS: We found that Tpot was a significant independent discriminator of good and poor prognosis in NSCLC. In particular, in non-squamous NSCLC a short Tpot, short Ts and high LI predicted for shorter survival time. In squamous cell carcinoma, a high USPF may predict a shorter survival period, although the correlation was only borderline-significant. CONCLUSIONS: We conclude that these parameters may in future be of use in drawing up more adequate treatment schedules for individual lung cancer patients.

Role of differential cell proliferation in the tail bud in aberrant mouse neurulation.

In the mouse mutant curly tail, the phenotypes spina bifida and curled tail result from a delay in closure of the posterior neuropore (PNP). At the developmental stage when this delay can first be recognized, the caudal region of the embryo demonstrates a transiently enhanced curvature of the body axis which likely inhibits elevation, convergence, and fusion of the neural folds. The enhanced curvature is thought to be the result of a decreased proliferation in the ventrally located gut endoderm and notochord, together with a normal proliferation of the overlying neuroepithelium of the PNP. However, the proliferation defect and the enhanced curvature were originally demonstrated at the same developmental stage, while it is expected that reduced proliferation should precede enhanced curvature and delayed PNP closure. The caudal region originates from the tail bud and we therefore propose that the enhanced curvature is induced by a disturbed dorso-ventral proliferation pattern in the tail bud. Using flow cytometry, proliferation patterns were determined separately for the dorsal and ventral halves of the tail bud of curly tail and of control embryos as well as of recombinant embryos having the curly tail phenotype with a genetic background which is matched to the BALB/c control strain. In general, it appeared that about half of the cell cycle duration in tail bud cells was occupied by S phase, about 40% by G0/G1 and the rest by G2/M. For the control embryos, no dorso-ventral differences in relative phase duration were demonstrated. However, curly tail and recombinant embryos at the 21-25 somite stage, prior to the onset of enhanced curvature, exhibited ventrally a higher proportion of G0/G1 phase cells than dorsally, and a complementary relationship for S phase cells. We interpret these observations as indicating a prolonged G1 phase at the ventral side of the tail bud, resulting in a prolongation of the cell cycle and thus a decreased proliferation. In 26-30 somite stage embryos, prior to the normalization of curvature in curly tail embryos, the dorso-ventral proliferation balance was re-established. We conclude that a reduced proliferation in the ventral part of the tail bud of the curly tail embryo precedes both the onset of enhanced curvature and the previously observed reduction in proliferation of the hindgut and notochord, and is a likely candidate for an early event in the pathogenetic sequence leading to the curly tail phenotype.

Detection of malignant cells in cerebrospinal fluid using fluorescence in situ hybridization.

Cytologic examination of cerebrospinal fluid (CSF) is the diagnostic gold standard for leptomeningeal metastasis (LMM). However, this technique is only moderately sensitive when routine staining procedures are applied. The use of fluorescence in situ hybridization (FISH) to identify malignant cells may have an additional value in diagnosing LMM, since numerical chromosomal aberrations (NCA) can be detected at the single cell level. We tested the feasibility of FISH to detect tumor cells in CSF by analyzing 22 samples with proven LMM with a probe for chromosome 1 (1q12) to detect NCA in the cells. A control group consisted of samples from 10 patients with inflammatory neurologic disease. In 7 LMM samples no cells or only lysed cells were found, due to time delay before fixation. Of the other 15 LMM samples analyzed, 13 showed NCA (87%), while no NCA were found in the control group. Our results indicate that FISH may be a useful additional diagnostic tool to the cytodiagnosis of CSF in cases of LMM. We expect that FISH can become an additional marker for malignancy at the single cell level in patients with LMM, which may also be of use to determine the effect of therapy for LMM.

Three parameter flow cytometric analysis for simultaneous detection of cytokeratin, proliferation associated antigens and DNA content.

The analysis of the cell cycle distributions by univariate flow cytometric DNA measurement has been widely applied in the clinic to determine kinetic parameters of human malignancies. A common problem with measurements of cell cycle phase distributions in tumor biopsy material is the presence of non-malignant diploid cells. Furthermore, such a static measurement might not be accurate enough to describe the dynamic process of cell proliferation. For this purpose alternative methods have been developed to include BrdUrd incorporation or the presence of intrinsic proliferation associated markers such as PCNA or Ki67-Ag into the analysis. However, the presence of nonmalignant diploid cells will influence also these bivariate analyses, especially in case of DNA-diploidy of the tumor cells. Here we present a three parameter flow cytometric assay based on the simultaneous detection of cytokeratin, DNA and a proliferation associated marker, such as BrdUrd, PCNA or Ki67-Ag. Based on the presence of cytokeratin, epithelial cells can be selected for a detailed cell cycle analysis. This method can be applied to frozen tissue, which makes this assay useful for multicentre clinical studies.

Alterations in cytoskeletal and nuclear matrix-associated proteins during apoptosis.

Evidence exists that apoptosis or programmed cell death plays an important role in tumor growth. Morphologically apoptosis is characterized by membrane blebbing and nuclear fragmentation in individual cells. In this study, we investigated changes in the nuclear organization in spontaneously occurring apoptotic cells in several cancer cell lines using several antibodies to nuclear matrix constituents. It appeared that nuclear matrix components remained detectable in cells undergoing spontaneous apoptosis. The same results were found when apoptosis was induced by cycloheximide in the non-small cell lung cancer cell line MR65. Using this induction method, the percentage of apoptotic cells in MR65 cells increased, allowing a more detailed and extensive examination of nuclear matrix alterations together with cytoskeletal changes. To study the expression of cytokeratins, type A- and B lamins, a nuclear matrix-associated 13 kDa U1RNP particle and the Ki67-antigen, immunocytochemistry in combination with confocal scanning laser microscopy was used. Apoptotic cells were identified based on nuclear morphology and the in situ nick translation assay. Whereas immunoreactivity against lamins and Ki67-Ag was rapidly lost during apoptosis, expression of the 13 kDa protein and, in early apoptotic stages, also cytokeratin expression, was observed to remain present. Dead cells lacked reactivity with all the antibodies tested. The persistence of nuclear matrix components is therefore a useful marker for the detection of apoptosis.

S-phase arrest of nutrient deprived lung cancer cells.

The human small cell lung cancer cell line NCI-H82 was used to study the effect of nutritional status on cell proliferative parameters. Incorporation of bromodeoxyuridine (BrdUrd) was used to characterize actively proliferating cells and to obtain information on cell cycle dynamics. During several days, in which the culture medium was not changed, a gradual decrease in overall cell growth, labeling index, and vitality was observed. Simultaneously, an increase in the number of S-phase cells that did not incorporate BrdUrd was noticed. From a more detailed kinetic study on d 6 of nutrient depletion, it appeared that, although the cells incorporated BrdUrd, they stopped cycling. When the same cells were regrown in fresh culture medium, a delay of 10 h in G1-phase entry and exit was measured. After this delay the cells resumed the cell cycle at normal phase transit rates. In addition, BrdUrd unlabeled S-phase cells were gradually lost from the culture. Bivariate flow cytometric DNA/proliferating cell nuclear antigen (PCNA) and DNA/Ki67-antigen analyses confirmed a delay in G1 phase entry and exit. In this paper we show that nutrient depletion can cause cell cycle arrest as indicated by the occurrence of BrdUrd unlabeled S-phase cells. This arrest could lead to overestimation of kinetic parameters such as S-phase transit time (Ts) and potential time (Tpot) as determined after in vivo labeling of tumors.

Evaluation of proliferation parameters in in vivo bromodeoxyuridine labelled lung cancers.

In a series of 44 bronchial biopsies from patients suspected of having endobronchial lung carcinoma, the validity of proliferating cell nuclear antigen (PCNA) and Ki67 antigen as proliferative indicators was evaluated in ethanol fixed, paraffin embedded tissue. The percentages of cells positive for these markers were compared to the in vivo bromodeoxyuridine (BrdU) labelling index. A good correlation was found between PCNA immunoreactivity and BrdU labelling index, while Ki67-antigen expression showed a significant relation with BrdU labelling index and with PCNA expression. All three parameters showed a trend towards similar values for the individual cases. Based on the fact that Ki67 antigen is expressed in all cycling cells, whereas replicon-associated PCNA and BrdU only reflect the S-phase fraction, the differences between Ki67-antigen scores on the one hand and BrdU and PCNA scores on the other were smaller than expected. In order to determine the degree of concordance between immunohistochemically and flow cytometrically detected proliferation variables, BrdU incorporation was measured using both methods in duplicate bronchial specimens. Discrepancies in labelling indices were observed predominantly in DNA diploid samples, with consistently lower values in the flow cytometrically analysed specimens. In tumour specimens with an aneuploid DNA content, flow cytometric determination of proliferative activity yielded results similar to those obtained by tissue section examination. We conclude that the scores for PCNA and Ki67 antigen, immunohistochemically detected in ethanol fixed, paraffin embedded tissue reflect functional proliferative activity.

Cytokinetic analysis of lung cancer by in vivo bromodeoxyuridine labelling.

Cytokinetic parameters of various types of lung cancer were determined in bronchoscopy specimens after in vivo labelling with the thymidine analogue bromodeoxyuridine (BrdU). The S-phase fraction and BrdU labelling index were measured flow cytometrically, allowing calculation of the S-phase transit time and potential tumour doubling time. The methodology used was found to be feasible for obtaining cytokinetic data from 76% of the bronchial biopsy samples. Despite the difference in clinical behaviour and growth pattern between small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), no significant differences were observed between the mean values of the cytokinetic parameters of SCLC and NSCLC. The estimated cell loss factor was higher in NSCLC than in SCLC. It appears that the growth of a tumour, as clinically observed, is to a considerable extent influenced by cell loss. In accord with this assumption is the fact that we have observed non-BrdU labelled S-phase cells, both in tumour biopsies and in apparently normal tissue. The presence of these so-called unlabelled S-phase cells in relation to cell loss is discussed.

Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.

A new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoy's, Bouin's or Zamboni's fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37 degrees C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.

Renewal of enterochromaffin cells in the rat caecum.

The localization, morphology, and neurohormonal peptide content of neuroendocrine cells have been extensively investigated. Relatively little is known about the kinetics of growth and differentiation of these cells. We studied the kinetics of enterochromaffin (EC) cells in the caecum of the rat, by applying the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU), to identify cells in S-phase, administered in pulse-chase and synchronous continuous labeling experiments. By double indirect immunofluorescence staining of tissue sections, using antibodies against serotonin and BrdU, percentages of BrdU positive EC cells could be enumerated, from which cell-kinetic parameters were derived. The following conclusions were drawn: 1) EC cells are renewed by proliferation of EC cells and by recruitment from proliferating precursor cells. 2) Caecal EC cells appear to consist of a relatively rapidly renewing and migrating fraction (60-65%) with a turnover time of approximately 16 days and a relatively slowly renewing and possibly stationary fraction (35-40%) with an estimated turnover time of approximately 150 days. 3) Seventy percent of the EC cells are localized in the lower half of mucosal crypts, 30% in the upper half. After prolonged labeling the percentage of labeled EC cells in the lower crypt half always exceeds that in the upper crypt half. This decrease in labeled EC cells during migration towards the mucosal surface indicates loss of endocrine cells, possibly owing to loss of endocrine characteristics.

Distribution of adenosine deaminase-complexing protein in murine tissues.

It has been suggested that mouse and rat lack adenosine deaminase-complexing protein because in these species exclusively the small molecular weight form of adenosine deaminase (ADA-S) is found. This suggestion is based on the assumption that the adenosine deaminase binding capacity is an inherent functional characteristic of adenosine deaminase-complexing protein. We report on the presence of adenosine deaminase-complexing protein immunoreactivity in mouse and rat determined with a species cross-reactive polyclonal anti-adenosine deaminase-complexing protein serum. In the mouse the tissue and subcellular distribution and the electrophoretic mobility in starch and polyacrylamide gels of the protein correspond with those of adenosine deaminase-complexing protein, but it does not bind the small molecular weight form of adenosine deaminase. Furthermore, in human, mouse, and rat kidney cortex adenosine deaminase and adenosine deaminase-complexing protein did not colocalize by immunohistochemistry. It is suggested that the function of adenosine deaminase-complexing protein is not adenosine deaminase-related.