Restricted T-Cell Repertoire in the Epicardial Adipose Tissue of Non-ST Segment Elevation Myocardial Infarction Patients.

Aims: Human epicardial adipose tissue, a dynamic source of multiple bioactive factors, holds a close functional and anatomic relationship with the epicardial coronary arteries and communicates with the coronary artery wall through paracrine and vasocrine secretions. We explored the hypothesis that T-cell recruitment into epicardial adipose tissue (EAT) in patients with non-ST segment elevation myocardial infarction (NSTEMI) could be part of a specific antigen-driven response implicated in acute coronary syndrome onset and progression. Methods and Results: We enrolled 32 NSTEMI patients and 34 chronic coronary syndrome (CCS) patients undergoing coronary artery bypass grafting (CABG) and 12 mitral valve disease (MVD) patients undergoing surgery. We performed EAT proteome profiling on pooled specimens from three NSTEMI and three CCS patients. We performed T-cell receptor (TCR) spectratyping and CDR3 sequencing in EAT and peripheral blood mononuclear cells of 29 NSTEMI, 31 CCS, and 12 MVD patients. We then used computational modeling studies to predict interactions of the TCR beta chain variable region (TRBV) and explore sequence alignments. The EAT proteome profiling displayed a higher content of pro-inflammatory molecules (CD31, CHI3L1, CRP, EMPRINN, ENG, IL-17, IL-33, MMP-9, MPO, NGAL, RBP-4, RETN, VDB) in NSTEMI as compared to CCS (P < 0.0001). CDR3-beta spectratyping showed a TRBV21 enrichment in EAT of NSTEMI (12/29 patients; 41%) as compared with CCS (1/31 patients; 3%) and MVD (none) (ANOVA for trend P < 0.001). Of note, 11/12 (92%) NSTEMI patients with TRBV21 perturbation were at their first manifestation of ACS. Four patients with the first event shared a distinctive TRBV21-CDR3 sequence of 178 bp length and 2/4 were carriers of the human leukocyte antigen (HLA)-A*03:01 allele. A 3D analysis predicted the most likely epitope able to bind HLA-A3*01 and interact with the TRBV21-CDR3 sequence of 178 bp length, while the alignment results were consistent with microbial DNA sequences. Conclusions: Our study revealed a unique immune signature of the epicardial adipose tissue, which led to a 3D modeling of the TCRBV/peptide/HLA-A3 complex, in acute coronary syndrome patients at their first event, paving the way for epitope-driven therapeutic strategies.

Molecular Hallmarks of Ischemia with Non-Obstructive Coronary Arteries: The "INOCA versus Obstructive CCS" Challenge.

Up to 4 million patients with signs of myocardial ischemia have no obstructive coronary artery disease (CAD). The absence of precise guidelines for diagnosis and treatment in non-obstructive CAD encourages the scientific community to fill the gap knowledge, to provide non-invasive and less expensive diagnostic tools. The aim of our study was to explore the biological profile of Ischemia with Non-Obstructive Coronary Arteries (INOCA) patients with microvascular dysfunction compared to patients presenting with obstructive chronic coronary syndrome (ObCCS) in order to find specific hallmarks of each clinical condition. We performed a gene expression array from peripheral blood mononuclear cells (PBMCs) isolated from INOCA (n = 18) and ObCCS (n = 20) patients. Our results showed a significantly reduced gene expression of molecules involved in cell adhesion, signaling, vascular motion, and inflammation in INOCA as compared to the ObCCS group. In detail, we found lower expression of Platelet and Endothelial Cell Adhesion Molecule 1 (CD31, p < 0.0001), Intercellular Adhesion Molecule-1 (ICAM1, p = 0.0004), Tumor Necrosis Factor (TNF p = 0.0003), Transferrin Receptor (TFRC, p = 0.002), and Vascular Endothelial Growth Factor A (VEGFA, p = 0.0006) in the INOCA group compared with ObCCS. Meanwhile, we observed an increased expression of Hyaluronidase (HYAL2, p < 0.0001) in INOCA patients in comparison to ObCCS. The distinct expression of molecular biomarkers might allow an early and non-invasive differential diagnosis between ObCCS and INOCA, improving clinical management and treatment options, in the era of personalized medicine.

Publisher Correction: New insight into the mechanisms of ectopic fat deposition improvement after bariatric surgery.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

New insight into the mechanisms of ectopic fat deposition improvement after bariatric surgery.

Non-alcoholic fatty-liver disease (NAFLD) is frequent in obese patients and represents a major risk factor for the development of diabetes and its complications. Bariatric surgery reverses the hepatic features of NAFLD. However, its mechanism of action remains elusive. We performed a comprehensive analysis of the mechanism leading to the improvement of NAFLD and insulin resistance in both obese rodents and humans following sleeve-gastrectomy (SG). SG improved insulin sensitivity and reduced hepatic and monocyte fat accumulation. Importantly, fat accumulation in monocytes was well comparable to that in hepatocytes, suggesting that Plin2 levels in monocytes might be a non-invasive marker for the diagnosis of NAFLD. Both in vitro and in vivo studies demonstrated an effective metabolic regeneration of liver function and insulin sensitivity. Specifically, SG improved NAFLD significantly by enhancing AMP-activated protein kinase (AMPK) phosphorylation and chaperone-mediated autophagy (CMA) that translate into the removal of Plin2 coating lipid droplets. This led to an increase in lipolysis and specific amelioration of hepatic insulin resistance. Elucidating the mechanism of impaired liver metabolism in obese subjects will help to design new strategies for the prevention and treatment of NAFLD.

Methylation analysis of HOXA10 regulatory elements in patients with endometriosis.

OBJECTIVE: The pathogenesis of endometriosis is still mysterious, being retrograde menstruation and coelomic metaplasia the most accepted hypotheses. Recently, it has been proposed that endometriosis is caused by fine-tuning alterations of the female genital system development during the foetal life and that in utero exposition to endocrine disruptors can be one of the factors causing the disease, possibly acting on the methylation status of the genome. In this study, we have evaluated the methylation status of HOXA10 gene regulation regions in a cohort of 22 endometriosis patients respect to a control group of 6 healthy women. RESULTS: The methylation study was carried out on three CpG islands, previously described hypermethylated in the endometrium of endometriosis patients and include 22 CpG sites, 21 CpG sites and 10 CpG sites, respectively identified through the online platform MethPrimer. The analysis did not find significant differences between patients with endometriosis and healthy control individuals. These results confirm previous studies on genome wide methylation analysis in endometriosis patients. Therefore, other epigenetically altered genes should be considered more related to the pathogenesis of endometriosis.

Epicardial adipose tissue microbial colonization and inflammasome activation in acute coronary syndrome.

BACKGROUND: Epicardial adipose tissue (EAT) has a close functional and anatomic relationship with epicardial coronary arteries. Accumulating evidence suggests that host microbiome alterations may play a role in several inflammatory/immune disorders, triggering a robust proinflammatory response also involving interleukin-1ß (IL-1ß) and the NALP3 inflammasome. In the current study, we explore the hypothesis that in patients with non-ST elevation acute coronary syndrome (ACS), EAT contains potentially pro-atherosclerotic bacteria that might elicit inflammasome activation. METHODS: EAT samples were obtained during coronary artery bypass grafting from ACS (n=18) and effort stable angina (SA; n=16) patients, and as controls, from patients with angiographically normal coronary arteries undergoing surgery for mitral insufficiency (MVD; n=13). In all patients, NALP3 and proIL-1ß mRNA expressions were evaluated with qRT-PCR. In 3 patients from each group, EAT microbiota composition was determined using next-generation sequencing technologies. RESULTS: In EAT, mRNA expression of both NALP3 and pro-IL1ß was significantly higher in ACS than in SA and MVD (P=0.028 and P=0.005, respectively). A broad range of bacterial species (n=76) was identified in both ACS and SA, with different predominant species. In contrast, microbial DNA was barely observed in MVD. CONCLUSIONS: Our study demonstrated the presence of bacterial DNA directly into EAT, surrounding diseased coronary arteries, of patients with ACS. Furthermore, ACS is associated with NALP3/inflammasome pathway activation in EAT. Our data suggest that the EAT environment is susceptible to microbial colonization that might stimulate a proinflammatory response. These findings add new elements to the pathogenesis of ACS and suggest novel therapeutic targets.

Insulin Resistance, Microbiota, and Fat Distribution Changes by a New Model of Vertical Sleeve Gastrectomy in Obese Rats.

Metabolic surgery improves insulin resistance and type 2 diabetes possibly because of weight loss. We performed a novel sleeve gastrectomy in rats that resects ∼80% of the glandular portion, leaving the forestomach almost intact (glandular gastrectomy [GG]) and compared subsequent metabolic remodeling with a sham operation. GG did not affect body weight, at least after 10 weeks; improved hepatic and peripheral insulin sensitivity likely through increased Akt, glycogen synthase kinase 3, and AMPK phosphorylation; and reduced ectopic fat deposition and hepatic glycogen overaccumulation. Body adipose tissue was redistributed, with reduction of intraabdominal fat. We found a reduction of circulating ghrelin levels, increased GLP-1 plasma concentration, and remodeling of gut microbiome diversity characterized by a lower relative abundance of Ruminococcus and a higher relative abundance of Lactobacillus and Collinsella These data suggest that at least in rat, the glandular stomach plays a central role in the improvement of insulin resistance, even if obesity persists. GG provides a new model of the metabolically healthy obese phenotype.

Early effect of Roux-en-Y gastric bypass on insulin sensitivity and signaling.

BACKGROUND: A large body of literature indicates the rapidity with which Roux-en-Y gastric bypass (RYGB) improves glycemic control. However, the underlying physiologic mechanisms are still a matter of debate. SETTING: Catholic University, School of Medicine, Rome, Italy. METHODS: Ten morbidly obese patients, before and 4 weeks after RYGB, and 10 healthy controls were studied. We measured insulin sensitivity as the homeostasis model assessment-estimated insulin resistance (HOMA-IR) and by the euglycemic hyperinsulinemic clamp, and phosphorylation of protein kinase B (Akt) on Ser473 and Thr308 and of GSK3 α-ß on Ser 9 and Ser21 in skeletal muscle biopsy specimens by Western blot analysis. RESULTS: Obese patients before RYGB displayed reduced insulin sensitivity (M value) and clearance and increased fasting Akt phosphorylation on Ser473 compared with controls. M significantly increased after surgery (from 2.6 ± 0.6 to 2.8 ± 0.7 mg/kg fat free mass/min, P = .026) but remained far below the values in controls (10.0 ± 3.8 mg/kg fat free mass/min, P<.001). Insulin clearance increased from 453.5 ± 117.5 to 555.2 ± 61.6 (P = .00076), becoming similar to that of controls 582.2 ± 59.0 mU/m(2)/min. HOMA-IR decreased from 4.1 ± 0.07 to 2.3 ± 0.5 (P = .004), becoming comparable with controls (2.2 ± 0.9). The hyperphosphorylation of Akt on Ser473 observed at fasting before RYGB was significantly reduced thereafter, becoming similar to that of healthy controls; the other phosphorylation states remained unchanged. CONCLUSIONS: Following RYGB, we found a prompt improvement of hepatic insulin resistance with normalization of hepatic insulin clearance and a small amelioration of whole-body insulin sensitivity. The supranormal levels of Akt Ser473 observed at fast in the skeletal muscle tissue at baseline were normalized after RYGB, and their changes correlated with those of both hepatic and peripheral insulin resistance. Although other mechanisms of action, such as the effect of weight loss and reduced food intake, cannot be excluded, the reduction of muscle Akt hyperphosphorylation on the serine residue can play a role in the early improvement of insulin sensitivity.

Increased PTPN22 expression and defective CREB activation impair regulatory T-cell differentiation in non-ST-segment elevation acute coronary syndromes.

BACKGROUND: Critical impairment of adaptive immune response has been observed in patients with acute coronary syndromes (ACS) with reduced expansion of regulatory T cells (Treg) and enhanced effector T-cell responsiveness, both associated with poorer outcomes. OBJECTIVES: This study investigated the mechanisms underlying T-cell dysregulation in ACS. METHODS: We evaluated both early and downstream T-cell receptor activation pathways after ex vivo stimulation with anti-CD3 and anti-CD28 crosslink in CD4(+) T cells from 20 patients with non-ST-segment elevation myocardial infarction (NSTEMI), 20 with stable angina (SA), and 20 controls. We reassessed 10 NSTEMI and 10 SA patients after 1 year. RESULTS: Phospho-flow analysis revealed reduced phosphorylation of the zeta-chain-associated protein kinase of 70 kDa at the inhibitory residue tyrosine 292, enhancing T-cell activation, in NSTEMI helper T cells versus SA and controls (each, p < 0.001), resulting from increased expression of the protein tyrosine phosphatase, nonreceptor type, 22 (PTPN22) (p < 0.001 for both comparisons), persisting at follow-up. We also observed reduced phosphorylation (p < 0.001 versus controls) and lower levels of binding to interleukins 2 and 10 core promoter regions of the transcription factor cyclic adenosine monophosphate response element-binding protein (CREB) in NSTEMI (p < 0.05 vs. controls), which recovered at 1 year. Finally, in NSTEMI patients, helper T cells had a reduced ability in T-cell receptor-induced Treg generation (p = 0.002 vs. SA; p = 0.001 vs. controls), partially recovered at 1 year. Restoring CREB activity and silencing PTPN22 enhanced NSTEMI patients' ability to generate Treg. CONCLUSIONS: The persistent overexpression of PTPN22 and the transient reduction of CREB activity, associated with impaired Treg differentiation, might play a role in ACS.

Growth properties of cardiac stem cells are a novel biomarker of patients' outcome after coronary bypass surgery.

BACKGROUND: The efficacy of bypass surgery in patients with ischemic cardiomyopathy is not easily predictable; preoperative clinical conditions may be similar, but the outcome may differ significantly. We hypothesized that the growth reserve of cardiac stem cells (CSCs) and circulating cytokines promoting CSC activation are critical determinants of ventricular remodeling in this patient population. METHODS AND RESULTS: To document the growth kinetics of CSCs, population-doubling time, telomere length, telomerase activity, and insulin-like growth factor-1 receptor expression were measured in CSCs isolated from 38 patients undergoing bypass surgery. Additionally, the blood levels of insulin-like growth factor-1, hepatocyte growth factor, and vascular endothelial growth factor were evaluated. The variables of CSC growth were expressed as a function of the changes in wall thickness, chamber diameter and volume, ventricular mass-to-chamber volume ratio, and ejection fraction, before and 12 months after surgery. A high correlation was found between indices of CSC function and cardiac anatomy. Negative ventricular remodeling was not observed if CSCs retained a significant growth reserve. The high concentration of insulin-like growth factor-1 systemically pointed to the insulin-like growth factor-1-insulin-like growth factor-1 receptor system as a major player in the adaptive response of the myocardium. hepatocyte growth factor, a mediator of CSC migration, was also high in these patients preoperatively, as was vascular endothelial growth factor, possibly reflecting the vascular growth needed before bypass surgery. Conversely, a decline in CSC growth was coupled with wall thinning, chamber dilation, and depressed ejection fraction. CONCLUSIONS: The telomere-telomerase axis, population-doubling time, and insulin-like growth factor-1 receptor expression in CSCs, together with a high circulating level of insulin-like growth factor-1, represent a novel biomarker able to predict the evolution of ischemic cardiomyopathy following revascularization.

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 family's 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.

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.

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.

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-1beta, 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.

Identification of protein disulfide isomerase as a cardiomyocyte survival factor in ischemic cardiomyopathy.

OBJECTIVES: The aim of the study was to analyze the molecular mechanisms activated during postinfarction remodeling in human hearts. BACKGROUND: The molecular mechanisms of initial response to ischemic insult in the heart and the pathways involved in compensation and remodeling are still largely unknown. METHODS: Up-regulation or down-regulation of gene expression in the human viable peri-infarct (vs. remote) myocardial region was investigated by complementary deoxyribonucleic acid array technology and confirmed at a single-gene/protein level with reverse transcriptase polymerase chain reaction and immunohistochemistry. An in vitro model of cardiomyocyte hypoxia in HL1 cells was used to validate anti-apoptotic effects of the candidate gene/protein and to assess the associated downstream cascade. Finally, a mouse model of myocardial infarction was used to test the in vivo effects of exogenous transfection with the candidate gene/protein. RESULTS: Protein disulfide isomerase (PDI), a member of the unfolded protein response, is 3-fold up-regulated in the viable peri-infarct myocardial region, and in a postmortem model, its expression is significantly inversely correlated with apoptotic rate and with presence of heart failure (HF) and biventricular dilatation. Induced PDI expression in HL1 cells conferred protection from hypoxia-induced apoptosis. Adenoviral-mediated PDI gene transfer to the mouse heart resulted in 2.5-fold smaller infarct size, significantly reduced cardiomyocyte apoptosis in the peri-infarct region, and smaller left ventricular end-diastolic diameter versus mice treated with a transgene-null adenoviral vector. CONCLUSIONS: These results suggest that PDI promotes survival after ischemic damage and that zinc-superoxide dismutase is one of the PDI molecular targets. Pharmacological modulation of this pathway might prove useful for future prevention and treatment of HF.

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-X(L) 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.

Ischemia and apoptosis in an animal model of permanent infarct-related artery occlusion.

Apoptosis is a pathologic feature of cardiomyocytes in acute myocardial infarction (AMI) and heart failure. The temporal course of apoptosis in the peri-infarct area in the weeks following an AMI is still uncompletely defined. In order to study the time course of apoptosis after AMI, 16 rabbits underwent left coronary artery ligation and were sacrificed at 16, 26, 35, and 56 days after surgery. Increased apoptotic rate (AR) was observed at in the peri-infarct region than in remote myocardium (5.4% [2.5-9.6] vs 0.4% [0.1-0.9], respectively, P<0.001) and than in sham-operated cases (0.01% [0-0.02], P<0.001). A gradual decrease of AR in the peri-infarct region was observed over time with a 90% reduction at 8 weeks after coronary ligation.

Multiple interference of the human papillomavirus-16 E7 oncoprotein with the functional role of the metastasis suppressor Nm23-H1 protein.

High-risk human papillomaviruses (HPV) are linked to human cervical and other ano-genital cancers. Integration of the viral genome in the transformed epithelial cells is restricted to the coding regions for the E6 and E7 oncoproteins. Nevertheless, E7 plays the major role in cell transformation. We report a novel interaction between HPV-16 E7 and the Nm23-H1 and Nm23-H2 proteins identified in yeast by the two-hybrid system and confirmed by co-immunoprecipitation in the human keratinocyte HaCaT cell line. Expression of the E7 oncoprotein in HaCaT cells induces modified keratinocyte proliferation and differentiation patterns, and leads to down-modulation and functional inactivation of the metastasis suppressor Nm23-H1 protein. Both transcriptional down-regulation and protein degradation contribute to reduce Nm23-H1 intracellular content. Besides metastasis suppression, Nm23-H1 displays multiple functions in cell cycle regulation and differentiation, development, DNA regulation and caspase-independent apoptosis. As a consequence of Nm23-H1 inhibition, HPV-16 E7 expressing HaCaT cells, acquire invasiveness capabilities and resistance to granzyme A-induced apoptosis. We propose that impairment of the multifunctional role of Nm23-H1 is an important feature consistent with the complex strategy carried out by HPV-16 E7 to promote cell transformation and tumor progression.

In vitro and in vivo tumor growth inhibition by a p16-mimicking peptide in p16INK4A-defective, pRb-positive human melanoma cells.

The cell cycle regulatory pathway responsible for the control of the late-G1 checkpoint is found recurrently altered in human malignant melanoma, often due to lack of functional p16 or pRb (pRb-1) proteins. Here we examined the ability of p16-derived peptides to mimic p16 function in two exemplary human melanoma cell lines: the p16-defective, pRb-positive A375M cells and p16-positive, pRb-defective A2058 cells. The synthetic p16-mimicking peptides strongly induced apoptosis in p16-, pRb+ A375M cells in vitro, while they had significantly less activity on p16+, pRb- A2058 cells. The most active p16-mimicking peptide, p16-AP9, also potently inhibited in vivo growth of the A375M melanoma. Treated tumors showed a threefold smaller volume (P < 0.025) and a significant reduction of the mitotic index and of PCNA expression. Growth of A2058 cells in vivo was not affected by treatment with the p16-mimicking peptide. Our results demonstrate that p16-mimicking peptides can induce apoptosis in vitro and that can inhibit tumor growth in vivo in p16-defective, pRb-expressing human melanoma cells, suggesting that p16-mimicking peptides can represent a promising tool for targeted therapy in selected cancer phenotypes.