Piero Tanganelli

Bronchopulmonary dysplasia of the premature baby

Prematurely born infants who required assisted ventilation may develop chronic lung disease or bronchopulmonary dysplasia (BPD). The cells involved in the reparative process of the premature lung are not well defined. The repair of injured tissues is a highly standardized process and the most important cells are activated (modulated) fibroblasts (myofibroblasts). A key cytokine in controlling repair is transforming growth factor‐β (TGF‐β). To characterize the cells involved in the repair process of the premature lung, we employed immunocytochemical techniques and examined the lungs of 39 autopsied premature babies who had neonatal respiratory distress syndrome (RDS). All were treated in neonatal intensive care units and required mechanical ventilation and supplemental oxygen; all survived for at least 12 hours. Antibodies were employed against vimentin, α‐smooth muscle (α‐SM) actin, total muscle actin, desmin, MAC387, and TGF‐β.

Mass spectrometry‐based proteomics: The road to lung cancer biomarker discovery

Lung cancer is the leading cause of cancer death in men and women in Western nations, and is among the deadliest cancers with a 5-year survival rate of 15%. The high mortality caused by lung cancer is attributable to a late-stage diagnosis and the lack of effective treatments. So, it is crucial to identify new biomarkers that could function not only to detect lung cancer at an early stage but also to shed light on the molecular mechanisms that underlie cancer development and serve as the basis for the development of novel therapeutic strategies. Considering that DNA-based biomarkers for lung cancer showed inadequate sensitivity, specificity, and reproducibility, proteomics could represent a better tool for the identification of useful biomarkers and therapeutic targets for this cancer type. Among the proteomics technologies, the most powerful tool is mass spectrometry. In this review, we describe studies that use mass spectrometry-based proteomics technologies to analyze tumor proteins and peptides, which might represent new diagnostic, prognostic, and predictive markers for lung cancer. We focus in particular on those findings that hold promise to impact significantly on the clinical management of this disease.

Biological features (inflammation and neoangiogenesis) and atherosclerotic risk factors in carotid plaques and calcified aortic valve stenosis: two different sites of the same disease?

Neoangiogenesis and inflammation have a pivotal role in atherosclerosis. Observations support the hypothesis that calcified aortic valve stenosis is an inflammatory process, similar to atherosclerosis in tissue features and risk factors. We studied 2 groups of cases: 47 were affected by hemodynamic atherosclerotic carotid plaque (group 1) and 35 by severe calcified aortic valve stenosis (group 2). We compared the groups for atherosclerosis risk factors, morphologic features, and immunohistochemical phenotypes. In both groups, men, smokers, and hypertensive subjects prevailed, and histologic analysis showed an elevated score for T-lymphocyte infiltrates, neoangiogenesis, calcium, and sclerosis. Adhesion molecule expression was present in both lesions. Expression of intercellular adhesion molecule 1 correlated with inflammatory infiltrates (group 1, P = .0007; group 2, P = .06). Neoangiogenesis also correlated with inflammatory infiltrates (group 1, P = .035; group 2, P = .045). In valves, neoangiogenesis correlated with calcium (P = .048). Carotid plaque and calcified valve stenosis showed common risk factors and biologic hallmarks of a chronic inflammatory process. Inflammation and neoangiogenesis have a crucial role in plaque evolution and in the progression of aortic valve stenosis.

Effect of the administration of n-3 polyunsaturated fatty acids on circulating levels of microparticles in patients with a previous myocardial infarction

Background Increased levels of microparticles exposing tissue factor circulate in the blood of patients with coronary heart disease, possibly disseminating their pro-thrombotic and pro-inflammatory potential. Because diets rich in n-3 (polyunsaturated) fatty acids have been associated with reduced incidence of coronary heart disease-related events, we investigated the in vivo effects of treatments with n-3 fatty acids on levels of circulating microparticles and their tissue factor- dependent procoagulant activity in patients with a previous myocardial infarction. Design and Methods Forty-six post-myocardial infarction patients were assigned to receive either 5.2 g of n-3 fatty acids daily (n=23) or an olive oil placebo (n=23) for 12 weeks. Circulating microparticles were isolated from peripheral blood. The number of microparticles, their cellular source and tissue factor antigen were determined by flow cytometry, and their procoagulant potential assayed by a fibrin generation test. Results The total number of microparticles, endothelium-derived microparticles and microparticle tissue factor antigen were not significantly different between the two groups. However, the number of platelet-derived microparticles [from a median of 431 (126–1796, range) ×106/L to a median of 226 (87–677, range)] ×106/L and monocyte-derived microparticles [from a median of 388 (9–1681, range) ×106/L to a median of 265 (7–984, range) ×106/L] in plasma were significantly (p<0.05) decreased by n-3 fatty acids, while they were unchanged in the placebo group. Total microparticle tissue factor-procoagulant activity was also reduced in the n-3 fatty acid group compared to that in the placebo group. Conclusions Treatment with n-3 fatty acids after myocardial infarction exerts favorable effects on levels of platelet- and monocyte-derived microparticles, thus possibly explaining some of the anti-inflammatory and anti-thrombotic properties of these natural compounds.

Apoptosis of Cardiomyocytes in Explanted and Transplanted Hearts Comparison of Results From In Situ TUNEL, ISEL, and ISOL Reactions

We assessed the efficiency of detecting myocyte apoptosis within human hearts using in situ enzymatic reactions in paraffin-embedded tissue samples: in situ end labeling (ISEL), terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), and in situ oligoligation (ISOL). The reactions were carried out in explanted hearts (idiopathic dilatative cardiomyopathy, n = 6; ischemic heart disease, n = 3) and in endomyocardial biopsy specimens (EMBs; n = 32) obtained from transplanted human hearts. The results were verified by DNA laddering. The ISOL reaction led to a significantly (P = .027) smaller number of false-positive results (2/41 [5%]) compared with assessment by ISEL (9/41 [22%]) or TUNEL (9/41 [22%]). Only 1 ISEL+ apoptotic cardiomyocyte was found in specimens from explanted hearts. Among the EMBs, 1 specimens had TUNEL+ apoptotic cardiomyocytes and 1 specimen had ISEL+ apoptotic cardiomyocytes. This implies that verifying results by independent methods must be used for TUNEL and ISEL techniques. A smaller number of false-positive results makes interpretation of ISOL results easier, although the sensitivity of this reaction remains to be established.

Parallel decrease of tissue factor surface exposure and increase of tissue factor microparticle release by the n-3 fatty acid docosahexaenoate in endothelial cells.

Tissue factor (TF) is expressed on the endothelium in response to inflammatory mediators, giving endothelial cells a pro-thrombotic phenotype. Since fish-derived n-3 fatty acids (FA) have been associated with reduced incidence of myocardial infarction, we investigated the endothelial effects of the most abundant n-3 FA, docosahexaenoate (DHA), on TF expression. Human umbilical vein endothelial cells were pre-incubated with DHA (or stearate and arachidonate as controls) for 48-72 hours, and then stimulated with bacterial lipopolysaccharide (LPS) or tumor necrosis factor-alpha. Pre-incubation of endothelial cells with DHA (but not stearate or arachidonate) concentration-dependently reduced surface protein exposure, independent of TF mRNA or total protein expression regulation. Conversely, DHA treatment in conjunction with activating stimuli, induced the release of endothelial TF-exposing microparticles from endothelial cells, quantitatively accounting for the decreased TF cell surface exposure. In conclusion, DHA treatment, with a time-course consistent with its incorporation in membrane phospholipids, increases the release of TF-exposing microparticles from endothelial cells, accounting for decreased endothelial cell TF surface exposure, thus potentially modifying the overall endothelial control of microparticle-related effects.

Stress Echocardiography as a Gatekeeper to Donation in Aged Marginal Donor Hearts: Anatomic and Pathologic Correlations of Abnormal Stress Echocardiography Results

BACKGROUND Owing to the shortage of donor hearts, the criteria for acceptance have been considerably expanded. Pharmacologic stress echocardiography is highly accurate in identifying prognostically significant coronary artery disease, but brain death and catecholamine storm in potential heart donors may substantially alter the cardiovascular response to stress. This study assessed correlates of an abnormal resting/stress echocardiography results in potential donors. METHODS From April 2005 to December 2007, 18 marginal candidate donors (9 men) aged 58 +/- 5 years were initially enrolled. After legal declaration of brain death, all marginal donors underwent bedside echocardiography, with baseline and (when resting echocardiography was normal) dipyridamole (0.84 mg/kg in 6 min) or dobutamine (up to 40 microg/kg/min) stress echo. Non-eligible hearts (with abnormal rest or stress echo findings) were excluded and underwent cardioautoptic verification. RESULTS Resting echocardiography showed wall motion abnormalities in 5 patients (excluded from donation). Stress echocardiography was performed in the remaining 13 (dipyridamole in 11; dobutamine in 2). Results were normal in 7, of which 6 were uneventfully transplanted in marginal recipients. Results were abnormal in 6, and autoptic verification performed showed coronary artery disease in 5, and initial cardiomyopathy in 1. CONCLUSIONS Bedside pharmacologic stress echocardiography can safely be performed in candidate heart donors, is able to unmask occult coronary artery disease or cardiomyopathy, and shows potential to extend donor criteria in heart transplantation. Further experience with using marginal donors is needed before exact guidelines can be established.

Aortic valve disease and gamma-glutamyltransferase: Accumulation in tissue and relationships with calcific degeneration

OBJECTIVE Degenerative aortic valve disease is characterized by some of the histological features of atherosclerotic lesions. Gamma-glutamyltransferase (GGT) has been recently implicated in pathogenesis of atherosclerosis, as well as in modulation of cells involved in calcium metabolism. We aimed to evaluate the possible implication of this enzyme activity in aortic valve disease. METHODS GGT immunohistochemistry was performed on valve leaflets of 64 patients with aortic valve stenosis undergoing valve replacement. Fractional GGT activity in plasma and tissue was analysed in a subgroup of cases by molecular exclusion chromatography. RESULTS A close association was found between tissue extracellular GGT staining and lipid deposits (p<0.0001). GGT was expressed by CD68-positive cells around neovessels, as well as by MMP-9- and TRAP-positive multinucleated cells in the vicinity of bone metaplasia areas. Total plasma GGT levels were associated with low HDL-c (p=0.028) and high triglycerides (p=0.017). Total GGT activity in tissue was negatively correlated with the extent of valves calcification (p=0.03). Both serum and tissue GGT levels were negatively associated with severity of valve stenosis, as judged by peak transvalvular pressure gradients (p<0.0003 and p<0.002, respectively). CONCLUSIONS Accumulation of GGT activity inside the lipid component of valves leaflets suggests a common mechanism of lesion shaping underlying both atherosclerosis and degenerative aortic valve disease. Moreover, the finding of GGT expression in cells with an osteoclast-like phenotype, and its negative correlation with both valves calcification and degree of valvular stenosis lend additional support to the recently envisaged involvement of GGT in the homeostasis of calcified tissues.

High plasma levels of the soluble receptor for advanced glycation endproducts in patients with symptomatic carotid atherosclerosis

Background  Advanced glycation endproducts (AGEs), particularly carboxymethyl(lysine)‐adducts (CML), exert part of their cellular effects by binding to a receptor, named receptor for AGEs (RAGE). The soluble form of this receptor (sRAGE) has been shown to have an athero‐protective role. We hypothesized the existence of a relationship between the AGE–RAGE axis and the occurrence of symptoms related to carotid atherosclerosis in nondiabetic conditions.

Different Factors Affecting Human ANP Amyloid Aggregation and Their Implications in Congestive Heart Failure

Aims Atrial Natriuretic Peptide (ANP)-containing amyloid is frequently found in the elderly heart. No data exist regarding ANP aggregation process and its link to pathologies. Our aims were: i) to experimentally prove the presumptive association of Congestive Heart Failure (CHF) and Isolated Atrial Amyloidosis (IAA); ii) to characterize ANP aggregation, thereby elucidating IAA implication in the CHF pathogenesis. Methods and Results A significant prevalence (85%) of IAA was immunohistochemically proven ex vivo in biopsies from CHF patients. We investigated in vitro (using Congo Red, Thioflavin T, SDS-PAGE, transmission electron microscopy, infrared spectroscopy) ANP fibrillogenesis, starting from α-ANP as well as the ability of dimeric β-ANP to promote amyloid formation. Different conditions were adopted, including those reproducing β-ANP prevalence in CHF. Our results defined the uncommon rapidity of α-ANP self-assembly at acidic pH supporting the hypothesis that such aggregates constitute the onset of a fibrillization process subsequently proceeding at physiological pH. Interestingly, CHF-like conditions induced the production of the most stable and time-resistant ANP fibrils suggesting that CHF affected people may be prone to develop IAA. Conclusions We established a link between IAA and CHF by ex vivo examination and assessed that β-ANP is, in vitro, the seed of ANP fibrils. Our results indicate that β-ANP plays a crucial role in ANP amyloid deposition under physiopathological CHF conditions. Overall, our findings indicate that early IAA-related ANP deposition may occur in CHF and suggest that these latter patients should be monitored for the development of cardiac amyloidosis.