Anna Severino

Anakinra, a Recombinant Human Interleukin-1 Receptor Antagonist, Inhibits Apoptosis in Experimental Acute Myocardial Infarction

Background— Experimental interleukin-1 receptor antagonist gene overexpression has shown that interleukin-1 receptor antagonist is cardioprotective during global cardiac ischemia. The aim of the present study was to test the impact of an exogenous recombinant human interleukin-1 receptor antagonist (anakinra) in experimental acute myocardial infarction. Methods and Results— Two animal studies were conducted: one of immediate anakinra administration during ischemia in the mouse and one of delayed anakinra administration 24 hours after ischemia in the rat. Seventy-eight Institute of Cancer Research mice and 20 Wistar rats underwent surgical coronary artery ligation (or sham operation) and were treated with either anakinra 1 mg/kg or NaCl 0.9% (saline). Treatment was administered during surgery and then daily for 6 doses in the mice and starting on day 2 daily for 5 doses in the rats. Twenty-eight mice underwent infarct size assessment 24 hours after surgery, 6 saline-treated mice and 22 mice treated with increasing doses of anakinra (1 mg/kg [n=6], 10 mg/kg [n=6], and 100 mg/kg [n=10]); 6 mice were euthanized at 7 days for protein expression analysis. The remaining animals underwent transthoracic echocardiography before surgery and 7 days later just before death. Cardiomyocyte apoptosis was measured in the peri-infarct regions. The antiapoptotic effect of anakinra was tested in a primary rat cardiomyocyte culture during simulated ischemia and in vitro on caspase-1 and -9 activities. At 7 days, 15 of the 16 mice (94%) treated with anakinra were alive versus 11 of the 20 mice (55%) treated with saline (P=0.013). No differences in infarct size at 24 hours compared with saline were observed with the 1- and 10-mg/kg doses, whereas a 13% reduction in infarct size was found with the 100-mg/kg dose (P=0.015). Treatment with anakinra was associated with a significant reduction in cardiomyocyte apoptosis in both the immediate and delayed treatment groups (3.1±0.2% versus 0.5±0.3% [P<0.001] and 4.2±0.4% versus 1.1±0.2% [P<0.001], respectively). Compared with saline-treated animals, anakinra-treated mice and rats showed signs of more favorable ventricular remodeling. In vitro, anakinra significantly prevented apoptosis induced by simulated ischemia and inhibited caspase-1 and -9 activities. Conclusions— Administration of anakinra within 24 hours of acute myocardial infarction significantly ameliorates the remodeling process by inhibiting cardiomyocyte apoptosis in 2 different experimental animal models of AMI. This may open the door for using anakinra to prevent postischemic cardiac remodeling and heart failure.

Distribution of the serine protease HtrA1 in normal human tissues.

The human HtrA family of proteases consists of three members: HtrA1, HtrA2, and HtrA3. In bacteria, the chief role of HtrA is recognition and degradation of misfolded proteins in the periplasm, combining a dual activity of chaperone and protease. In humans, the three HtrA homologues appear to be involved in diverse functions such as cell growth, apoptosis, allergic reactions, fertilization, control of blood pressure, and blood clotting. Previous studies using RNA blot hybridization have shown that the expression of HtrA1 is ubiquitous in normal human tissues. Here we show by immunohistochemistry (IHC) that HtrA1 is widely expressed, although different tissue distributions and/or levels of expression were detected in the different tissues examined. In particular, high to medium HtrA1 expression was detected in mature layers of epidermis, in secretory breast epithelium, in liver, and in kidney tubules of cortex, in concordance with its secretory properties. Furthermore, we show a higher protein expression level in the epithelium of proliferative endometrium, in contrast to epithelium of secretory endometrium, which is almost completely negative for this protein. This suggests a possible role for HtrA1 in the modulation of tissue activity in this organ. The various expression levels in human tissues indicate several possible roles for HtrA1 in different cell types.

The serine protease HtrA1 specifically interacts and degrades the tuberous sclerosis complex 2 protein.

Hamartin and tuberin are products of the tumor suppressor genes TSC1 and TSC2, respectively. Mutations affecting either gene result in the tuberous sclerosis syndrome, a neurologic genetic disorder characterized by the formation of multiple benign tumors or hamartomas. In this study, we report the identification of TSC2, but not TSC1, as a substrate of HtrA1, a member of the human HtrA family proteins of serine proteases. We show the direct interaction and colocalization in the cytoplasm of HtrA1 and TSC2 and that HtrA1 cleaves TSC2 both in vitro and in vivo. Finally, we show that alterations in HtrA1 expression cause modifications in phosphorylation status of two downstream targets of TSC2: 4E-BP1 and S6K. Our data suggest that, under particular physiologic or pathologic conditions, HtrA1 degrades TSC2 and activates the downstream targets. Considering that HtrA1 levels are significantly increased during embryogenesis, we speculate that one of the targets of HtrA1 activity during fetal development is the TSC2-TSC1 pathway. Mol Cancer Res; 8(9); 1248–60. ©2010 AACR.

The Role of PDI as a Survival Factor in Cardiomyocyte Ischemia

Acute myocardial infarction (AMI) leads to activation of unfolded protein response (UPR) following endoplasmic reticulum (ER) stress. Failing in the restoration of the proper folding activity in the ER can lead to apoptosis and cell death. While it can be easy to detect transcripts and proteins expression alterations during a pathological state, it can be difficult to address the importance of changes in protein expression in the physiopathological context. We found protein disulfide isomerase (PDI) increased expression in human autoptic heart samples correlating with cell survival following AMI. PDI enzymatic activity resulted to be important to achieve cardiomyocyte protection from hypoxic stress, dependent on its ability to relieve ER stress preventing accumulation of nonfolded proteins in the ER, and to enhance superoxide dismutase 1 (SOD-1) activity. Furthermore, adenoviral-mediated PDI overexpression in an in vivo mouse model of AMI prevented adverse cardiac remodeling reducing cardiomyocyte apoptosis. Finally, we suggest a method to detect alterations in normal redox state in PDI (and eventually in the PDI familys proteins) during pathologies in which ER stress is induced. Diabetes pathology correlates with increased risk of AMI and worse cardiac remodeling. We found an alteration in PDI redox state in the diabetic heart and suggest using this system for the detection of the redox state alteration to screen for therapies able to restore the proper redox state.

Sudden coronary death, fatal acute myocardial infarction and widespread coronary and myocardial inflammation

Background: T-lymphocyte activation within atherosclerotic plaque, and widespread to the myocardium, has been shown in patients with acute coronary syndromes. Objective: To investigate the presence of T-lymphocyte infiltrate at different stages of acute coronary syndromes by studying patients with sudden coronary death, acute myocardial infarction (AMI) and healed infarction, in comparison with patients with myocarditis and patients with non-ischaemic heart failure. Methods: 72 cases were studied at autopsy: 12 dying of sudden coronary death (group 1), 12 dying <4 weeks (group 2) and 12 dying >4 months after AMI (group 3), 12 with active lymphocytic myocarditis (group 4), 12 with hypertensive heart disease (group 5), and 12 control subjects (group 6). Light microscopy was performed to measure the number of activated T-lymphocytes (CD3+/DR+) in the myocardium and coronary artery wall, and intercellular adhesion molecule-1 (ICAM-1) expression in the myocardium. Results: Activated T-lymphocyte infiltrates and ICAM-1 myocardial expression in both remote and peri-infarction regions and activated T-lymphocytes within the epicardial coronary artery wall of both the infarct- and non-infarct-related arteries were found in groups 1, 2 and 3, whereas myocardial, but not coronary, infiltrates were found in groups 4 (p<0.001 vs groups 1, 2 and 3 for coronary infiltrates). Groups 5 and 6 had no evidence of myocardial or coronary inflammation (p<0.001 vs groups 1, 2 and 3). Conclusions: The study shows the presence of a lymphocytic infiltrate in both coronary arteries and myocardium and a proinflammatory phenotype shift in the myocardium associated with acute coronary thrombosis in patients dying suddenly, shortly, or even late after coronary thrombosis.

Human papillomavirus-16 E7 interacts with siva-1 and modulates apoptosis in HaCaT human immortalized keratinocytes†

The viral factor E7 plays a key role in the well‐established association between “high‐risk” Human Papillomavirus (HPV) infection and the development of epithelial malignant tumors, as uterine cervix and ano‐genital cancer. To delve into the molecular mechanisms of HPV‐mediated cell transformation, we searched for novel potential cellular targets of the HPV‐16 E7 oncoprotein, by means of the yeast two‐hybrid technique, identifying a protein–protein interaction between HPV‐16 E7 and the pro‐apoptotic cellular factor Siva‐1. Using co‐precipitation assays and the “PepSets” technique, we confirmed this physical interaction and mapped accurately, for both proteins, the amino acid residues involved. Additionally, we found that HPV‐16 E7 competed in vitro with the binding of the Bcl‐XL anti‐apoptotic factor to Siva‐1, an interaction that has a major inference in UV radiation‐induced apoptosis. In HaCaT immortalized human keratinocytes, forced HPV‐16 E7 expression by retroviral infection caused Siva‐1 transcript up‐regulation, detected by cDNA macroarray hybridization and real‐time quantitative PCR, paralleled by an increased amount of protein. Confirming the anti‐apoptotic role of HPV‐16 E7 in the HaCaT cellular model, evaluated by nuclear morphology, we also found that Siva‐1 expression produced a significant increase of the apoptotic rate in UV radiation‐exposed HaCaT cells, and that this effect resulted explicitly counteracted by HPV‐16 E7. Being apoptosis a key physiological process for the elimination of irreversibly injured cells, the anti‐apoptotic role of HPV‐16 E7, performed at least by its interference with Siva‐1, can be considered an additional mechanism for the survival of damaged, potentially transforming, cell clones. J. Cell. Physiol. 212: 118–125, 2007.

Delayed neutrophil apoptosis in patients with unstable angina: relation to C-reactive protein and recurrence of instability

AIMS To investigate spontaneous polymorphonuclear neutrophils (PMNs) apoptosis in unstable angina (UA) and its association with recurrence of instability. METHODS AND RESULTS We compared PMNs apoptotic rate at 4 and 24 h in patients with UA, stable angina (SA), and controls (H) with two different protocols by flow cytometry. We measured apoptotic rate of isolated PMNs (Protocol 1) in 30 UA patients, 13 SA patients, and 34 H; and apoptosis of PMNs in whole blood culture (Protocol 2) in further 10 UA patients, 7 SA patients, and 6 H. Serum high-sensitivity C-reactive protein was also measured. Polymorphonuclear neutrophils of UA patients showed a decreased apoptotic rate compared with SA patients and H at 4 h in Protocol 1 (both P < 0.01), and at 24 h in Protocol 2 (P < 0.05 and <0.01, respectively). In overall population, a negative correlation was found between apoptotic rate at 4 h and high-sensitivity C-reactive protein levels (P < 0.01). Six among 40 patients with UA had early recurrence of symptoms and their apoptotic rate was significantly reduced compared with UA patients without recurrence of symptoms (P = 0.024). CONCLUSIONS Our study demonstrates delayed PMN apoptosis in UA. This alteration might be involved in the persistence of inflammatory activation and affects recurrence of instability.

Pattern of Expression of Cyclin T1 in Human Tissues

Cyclin T1 was recently identified, together with cdk9 (previously named PI-TALRE), as part of the TAK multiprotein complex, a co-factor targeted by the human immunodeficiency virus Type 1 (HIV-1) protein named Tat, suggesting a role for this complex in transcription elongation. Although studies on mRNA and protein expression have shown that cyclin T1 is ubiquitous in adult human tissues, no data have yet been reported regarding the expression of this protein in different cell lineages. Using a polyclonal antiserum raised against cyclin T1, we investigated the pattern of expression of this protein in adult human tissues by immunohistochemistry. Cyclin T1 was expressed ubiquitously, although different levels of expression were found in various organs. Some specialized tissues, such as blood, lymphoid tissues, and cells of connective tissue origin, showed high cyclin T1 expression. These specific expression patterns are only partially justified by some well-known specialized functions of cyclin T1 in certain cell types, such as its involvement in peripheral blood lymphocytes and monocyte differentiation. The high expression level found in other tissues suggests new possible roles for cyclin T1 in cell types other than those of lymphoid tissue.

Protective Effects of Parecoxib, a Cyclo-Oxygenase-2 Inhibitor, in Postinfarction Remodeling in the Rat

Objective: Selective cyclo-oxygenase-2 (COX-2) inhibitors have been shown to preserve hemodynamic performance in experimental models of acute myocardial infarction (AMI) in rodents. The impact of COX-2 inhibition on apoptosis, vascular density, and postinfarction remodeling has not yet been fully characterized. The aim of the present study was to evaluate the effects of parecoxib, a selective COX-2 inhibitor, in an experimental AMI model in the rat. Methods: Twenty-four male Wistar rats (10 weeks of age, weighing 350-500 g) underwent surgical left coronary artery ligation. Four animals died within 24 hours. Starting on day 2, 10 rats received parecoxib (0.75 mg/kg intraperitoneal) daily for 5 days and the remaining 10 received NaCl-0.9%. Animals underwent transthoracic echocardiography before surgery and 7 days later for the measurement of end-diastolic and end-systolic diameter and wall thickness; thereafter, animals were sacrificed and histological analysis was performed to evaluate cardiomyocyte apoptosis and small arteriolar density. Data are expressed as mean and standard error. Results: Three saline-treated (30%) and zero parecoxib-treated animals died before day 7. Compared with saline-treated animals, rats treated with parecoxib had a smaller end-diastolic diameter (6.3 ± 0.1 vs. 7.0 ± 0.1 mm, P = 0.018) and end-systolic diameter (2.7 ± 0.1 vs. 3.9 ± 0.1 mm, P = 0.027), and had a greater fractional shortening (57 ± 1 vs. 45 ± 2%, P = 0.050). Systolic thickness in the anterior (infarct) wall was also significantly greater in the parecoxib-treated animals (3.2 ± 0.1 vs. 2.7 ± 0.1 mm, P = 0.008), while the posterior wall was not significantly affected (P = 0.08). Aneurysmal dilatation of the left ventricle was more frequent in saline-treated versus parecoxib-treated animals (43 vs. 0%, P = 0.025). Parecoxib treatment was associated with lower apoptotic rates (1.0 ± 0.2 vs. 4.0 ± 0.4%, P < 0.001) and preservation of arteriolar density (20 ± 5 vs. 8 ± 2 mm/mm3, P = 0.018) in the peri-infarct area, without differences in circulating interleukin-1β, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma levels. Conclusion: Administration of parecoxib significantly ameliorates the remodeling process after AMI, possibly through prevention of apoptosis and preservation of myocardial vascularity. These findings aid in the understanding of the role of COX-2 in ischemic damage and remodeling.

Cyclo-oxygenase-2 (COX-2) inhibition reduces apoptosis in acute myocardial infarction

Department of Medicine, Virginia Commonwealth University, Richmond, VA USA A. Abbate (A. Abbate, G. W. Vetrovec); Institute of Cardiology, Catholic University, Rome, Italy (G. G. L. Biondi-Zoccai, A. Severino, M. Campioni, G. Liuzzo, F. Crea, L. M. Biasucci); Department of Vascular Pathology, Istituto Dermopatico Immacolata, Rome, Italy (F. Limana, M. C. Capogrossi, S. Straino); Section of Oncology, Campus Bio-Medico University, Rome, Italy (D. Santini); Department of Experimental Medicine and of Pathology, Universita degli Studi “La Sapienza”, Rome, Italy (S. Scarpa, F. Vasaturo); Centro Cardiologico Fondazione Monzino, Milano, Italy (A. Germani); Department of Biochemistry and Biophysics “F. Cedrangolo”, Section of Pathologic Anatomy, Second University of Naples, Naples, Italy (A. Baldi)