Carlo Alberto Beltrami

Apoptosis in the Failing Human Heart

BACKGROUND Loss of myocytes is an important mechanism in the development of cardiac failure of either ischemic or nonischemic origin. However, whether programmed cell death (apoptosis) is implicated in the terminal stages of heart failure is not known. We therefore studied the magnitude of myocyte apoptosis in patients with intractable congestive heart failure. METHODS Myocardial samples were obtained from the hearts of 36 patients who underwent cardiac transplantation and from the hearts of 3 patients who died soon after myocardial infarction. Samples from 11 normal hearts were used as controls. Apoptosis was evaluated histochemically, biochemically, and by a combination of histochemical analysis and confocal microscopy. The expression of two proto-oncogenes that influence apoptosis, BCL2 and BAX, was also determined. RESULTS Heart failure was characterized morphologically by a 232-fold increase in myocyte apoptosis and biochemically by DNA laddering (an indicator of apoptosis). The histochemical demonstration of DNA-strand breaks in myocyte nuclei was coupled with the documentation of chromatin condensation and fragmentation by confocal microscopy. All these findings reflect apoptosis of myocytes. The percentage of myocytes labeled with BCL2 (which protects cells against apoptosis) was 1.8 times as high in the hearts of patients with cardiac failure as in the normal hearts, whereas labeling with BAX (which promotes apoptosis) remained constant. The near doubling of the expression of BCL2 in the cardiac tissue of patients with heart failure was confirmed by Western blotting. CONCLUSIONS Programmed death of myocytes occurs in the decompensated human heart in spite of the enhanced expression of BCL2; this phenomenon may contribute to the progression of cardiac dysfunction.

Structural basis of end-stage failure in ischemic cardiomyopathy in humans.

BACKGROUND Ischemic cardiomyopathy is characterized by myocyte loss, reactive cellular hypertrophy, and ventricular scarring. However, the relative contribution of these tissue and cellular processes to late failure remains to be determined. METHODS AND RESULTS Ten hearts were obtained from individuals undergoing cardiac transplantation as a result of chronic coronary artery disease in its terminal stage. An identical number of control hearts were collected at autopsy from patients who died from causes other than cardiovascular disease, and morphometric methodologies were applied to the analysis of the left and right ventricular myocardium. Left ventricular hypertrophy evaluated as a change in organ weight, aggregate myocyte mass, and myocyte cell volume per nucleus showed increases of 85%, 47%, and 103%, respectively. Corresponding increases in the right ventricle were 75%, 74%, and 112%. Myocyte loss, which accounted for 28% and 30% in the left and right ventricles, was responsible for the difference in the assessment of myocyte hypertrophy at the ventricular, tissue, and cellular levels. Left ventricular muscle cell hypertrophy was accomplished through a 16% and 51% increase in myocyte diameter and length, whereas right ventricular myocyte hypertrophy was the consequence of a 13% and 67% increase in these linear dimensions, respectively. Moreover, a 36% reduction in the number of myocytes included in the thickness of the left ventricular wall was found. Collagen accumulation in the form of segmental, replacement, and interstitial fibrosis comprised an average 28% and 13% of the left and right ventricular myocardia, respectively. The combination of cell loss and myocardial fibrosis, myocyte lengthening, and mural slippage of cells resulted in 4.6-fold expansion of left ventricular cavitary volume and a 56% reduction in the ventricular mass-to-chamber volume ratio. CONCLUSIONS These results are consistent with the contention that both myocyte and collagen compartments participate in the development of decompensated eccentric ventricular hypertrophy in the cardiomyopathic heart of ischemic origin.

Myocyte Turnover in the Aging Human Heart

Rationale: The turnover of cardiomyocytes in the aging female and male heart is currently unknown, emphasizing the need to define human myocardial biology. Objective: The effects of age and gender on the magnitude of myocyte regeneration and the origin of newly formed cardiomyocytes were determined. Methods and Results: The interaction of myocyte replacement, cellular senescence, growth inhibition, and apoptosis was measured in normal female (n=32) and male (n=42) human hearts collected from patients 19 to 104 years of age who died from causes other than cardiovascular diseases. A progressive loss of telomeric DNA in human cardiac stem cells (hCSCs) occurs with aging and the newly formed cardiomyocytes inherit short telomeres and rapidly reach the senescent phenotype. Our data provide novel information on the superior ability of the female heart to sustain the multiple variables associated with the development of the senescent myopathy. At all ages, the female heart is equipped with a larger pool of functionally competent hCSCs and younger myocytes than the male myocardium. The replicative potential is higher and telomeres are longer in female hCSCs than in male hCSCs. In the female heart, myocyte turnover occurs at a rate of 10%, 14%, and 40% per year at 20, 60, and 100 years of age, respectively. Corresponding values in the male heart are 7%, 12%, and 32% per year, documenting that cardiomyogenesis involves a large and progressively increasing number of parenchymal cells with aging. From 20 to 100 years of age, the myocyte compartment is replaced 15 times in women and 11 times in men. Conclusions: The human heart is a highly dynamic organ regulated by a pool of resident hCSCs that modulate cardiac homeostasis and condition organ aging.

The cellular basis of dilated cardiomyopathy in humans

The present investigation was designed to evaluate whether end-stage cardiac failure in patients affected by dilated cardiomyopathy (DC) was dependent upon extensive myocyte cell death with reduction in muscle mass or was the consequence of collagen accumulation in the myocardium independently from myocyte cell loss. In addition, the mechanisms of ventricular dilation were analysed in order to determine whether the changes in cardiac anatomy were important variables in the development of intractable congestive heart failure. DC is characterized by chamber dilation, myocardial scarring and myocyte hypertrophy in the absence of significant coronary atherosclerosis. However, the relative contribution of each of these factors to the remodeling of the ventricle is currently unknown. Moreover, no information is available concerning the potential etiology of collagen deposition in the myocardium and the changes in number and size of ventricular myocytes with this disease. Morphometric methodologies were applied to the analysis of 10 DC hearts obtained from patients undergoing cardiac transplantation. An identical number of control hearts was collected from individuals who died from causes other than cardiovascular diseases. DC produced a 2.2-fold and 4.2-fold increase in left ventricular weight and chamber volume resulting in a 48% reduction in mass-to-volume ratio. In the right ventricle, tissue weight and chamber size were both nearly doubled. Left ventricular dilation was the result of a 59% lengthening of myocytes and a 20% increase in the transverse circumference due to slippage of myocytes within the wall. Myocardial scarring represented by segmental, replacement and interstitial fibrosis occupied approximately 20% of each ventricle, and was indicative of extensive myocyte cell loss. However, myocyte number was not reduced and average cell volume increased 2-fold in both ventricles. In conclusion, reactive growth processes in myocytes and architectural rearrangement of the muscle compartment of the myocardium appear to be the major determinants of ventricular remodeling and the occurrence of cardiac failure in DC.

Effects of Age and Heart Failure on Human Cardiac Stem Cell Function

Currently, it is unknown whether defects in stem cell growth and differentiation contribute to myocardial aging and chronic heart failure (CHF), and whether a compartment of functional human cardiac stem cells (hCSCs) persists in the decompensated heart. To determine whether aging and CHF are critical determinants of the loss in growth reserve of the heart, the properties of hCSCs were evaluated in 18 control and 23 explanted hearts. Age and CHF showed a progressive decrease in functionally competent hCSCs. Chronological age was a major predictor of five biomarkers of hCSC senescence: telomeric shortening, attenuated telomerase activity, telomere dysfunction-induced foci, and p21(Cip1) and p16(INK4a) expression. CHF had similar consequences for hCSCs, suggesting that defects in the balance between cardiomyocyte mass and the pool of nonsenescent hCSCs may condition the evolution of the decompensated myopathy. A correlation was found previously between telomere length in circulating bone marrow cells and cardiovascular diseases, but that analysis was restricted to average telomere length in a cell population, neglecting the fact that telomere attrition does not occur uniformly in all cells. The present study provides the first demonstration that dysfunctional telomeres in hCSCs are biomarkers of aging and heart failure. The biomarkers of cellular senescence identified here can be used to define the birth date of hCSCs and to sort young cells with potential therapeutic efficacy.

Unexpected expression of α- and β-globin in mesencephalic dopaminergic neurons and glial cells

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the substantia nigra (SN) (A9 neurons) and the ventral tegmental area (VTA) (A10 cells). A9 neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinsons disease. Here, we report that gene expression analysis of A9 dopaminergic neurons (DA) identifies transcripts for α- and β-chains of hemoglobin (Hb). Globin immunoreactivity decorates the majority of A9 DA, a subpopulation of cortical and hippocampal astrocytes and mature oligodendrocytes. This pattern of expression was confirmed in different mouse strains and in rat and human. We show that Hb is expressed in the SN of human postmortem brain. By microarray analysis of dopaminergic cell lines overexpressing α- and β-globin chains, changes in genes involved in O2 homeostasis and oxidative phopshorylation were observed, linking Hb expression to mitochondrial function. Our data suggest that the most famed oxygen-carrying globin is not exclusively restricted to the blood, but it may play a role in the normal physiology of the brain and neurodegenerative diseases.

Immunocytochemical expression of tissue specific transcription factor-1 in lung carcinoma.

AIMS: To investigate the immunocytochemical expression of the tissue specific transcription factor-1 (TTF-1) on cytological specimens of small cell lung carcinoma (SCLC) and to establish its value in the cytological diagnosis of lung cancer. METHODS: For each case, the diagnosis was made on cytological specimens and confirmed on subsequent bronchial biopsy specimens. TTF-1 was detected immunocytochemically using the avidinbiotin complex technique with a rabbit antiserum. Expression of TTF-1 was evaluated in 41 cases of SCLC and 17 cases of non-small cell carcinoma (NSCC). The latter were subdivided into eight cases of adenocarcinomas and nine cases of squamous cell carcinomas (SCC). RESULTS: Positive nuclear immunoreactivity to TTF-1 was identified in 38 (92.7%) of the 41 cases of SCLC, in five (62.5%) of eight cases of adenocarcinoma, and one (11%) of nine cases of SCC. A significant difference was observed between the two main groups, SCLC and NSCC. A comparison between SCLC and adenocarcinoma and SCC showed that TTF-1 expression was significantly different. TTF-1 immunoreactivity was not detected in the inflammatory cells of the same cases. CONCLUSIONS: TTF-1 is strictly associated with SCLC; it was weakly expressed in the various subtypes of NSCC. Although TTF-1 is not specific for SCLC, it can be used to highlight neoplastic cells to good effect when a large inflammatory component is present, and to differentiate SCLC from lymphoid infiltrates.

Portal vein thrombosis in hepatocellular carcinoma: age and sex distribution in an autopsy study

Abstract The purpose of this work was to investigate the prevalence, associated features and effect on survival of portal vein thrombosis (PVT) complicating hepatocellular carcinoma (HCC). The autopsy data of a series of 72 consecutive patients (57 male, 15 female) with HCC were reviewed. PVT was found in 32/72 patients (44%), and tended to be more common in female patients (10/15 versus 22/57, P = 0.052). Stratifying the data according to gender, it appeared that the mean age of patients with PVT compared to those without was greater in woman (71.9 ± 5.9 versus 63.2 ± 6.9 years, P = 0.024) and younger in men (58.8 ± 8.9 versus 66.0 ± 9.9 years, P = 0.007). When PVT was present, it was more likely that a definite diagnosis of HCC had been obtained before autopsy (P = 0.0001) and that death had been caused by bleeding complications (P = 0.007). Median survival times were similar, irrespective of the presence of PVT. During the natural history of HCC, PVT occurs in a substantial proportion of patients. Hormonal factors may have a permissive role in thrombus formation or neoplastic vascular invasion. Although in the presence of PVT a diagnosis of HCC is rarely missed and bleeding complications are likely to occur, patient survival does not seem to be significantly affected.

Multipotent Progenitor Cells Are Present in Human Peripheral Blood

To determine whether the peripheral blood in humans contains a population of multipotent progenitor cells (MPCs), products of leukapheresis were obtained from healthy donor volunteers following the administration of granulocyte colony-stimulating factor. Small clusters of adherent proliferating cells were collected, and these cells continued to divide up to 40 population doublings without reaching replicative senescence and growth arrest. MPCs were positive for the transcription factors Nanog, Oct3/4, Sox2, c-Myc, and Klf4 and expressed several antigens characteristic of mesenchymal stem cells. However, they were negative for markers of hematopoietic stem/progenitor cells and bone marrow cell lineages. MPCs had a cloning efficiency of ≈3%, and following their expansion, retained a highly immature phenotype. Under permissive culture conditions, MPCs differentiated into neurons, glial cells, hepatocytes, cardiomyocytes, endothelial cells, and osteoblasts. Moreover, the gene expression profile of MPCs partially overlapped with that of neural and embryonic stem cells, further demonstrating their primitive, uncommitted phenotype. Following subcutaneous transplantation in nonimmunosuppressed mice, MPCs migrated to distant organs and integrated structurally and functionally within the new tissue, acquiring the identity of resident parenchymal cells. In conclusion, undifferentiated cells with properties of embryonic stem cells can be isolated and expanded from human peripheral blood after granulocyte colony-stimulating factor administration. This cell pool may constitute a unique source of autologous cells with critical clinical import.

TTF-1 protein expression in pleural malignant mesotheliomas and adenocarcinomas of the lung

TTF-1 is a tissue-specific transcription factor expressed in the epithelial cells of thyroid and lung. This study investigates the immunohistochemical expression of TTF-1 in pleural malignant mesotheliomas (MM) and adenocarcinomas (AC) of the lung, respectively. For this purpose, 33 biopsy specimens of pulmonary AC and 24 specimens of MM were studied. TTF-1 immunoreactivity was identified in 19 of 33 cases of AC (57.5%) and in none of the 24 cases of MM. Positivity for TTF-1 was 100% specific and 57.5% sensitive for lung AC. Alternatively, negativity for TTF-1 was 57.5% specific and 100% sensitive for MM. These results suggest that TTF-1 can be favourably added to the immunohistochemical diagnostic panel for distinction between AC of the lung involving the pleura and pleural MM.