Mesenchymal Stem Cell Transplantation Has a Regenerative Effect in Ischemic Myocardium: An Experimental Rat Model Evaluated by SPECT-CT Assessment.

Translational perspective: Ischemic heart disease remains a major medical problem with high mortality rates. Beside the great efforts devoted to research worldwide and the use of numerous experimental models, an absolute understanding of myocardial infarction and tissue loss has not yet been achieved. Furthermore, the regeneration of myocardial tissue and the improvement of myocardial activity after ischemia is one of the major areas of interest in the medical (and especially cardiovascular) community. In a novel experimental rat model, the beneficial effect of mesenchymal stem cell transplantation (MSCT) in a surgically induced ischemic myocardium was documented. From a clinical perspective, this work supports the surgical administration of MSCT in the infarcted area during coronary artery bypass surgery. AIMS: The regeneration of myocardial tissue and the improvement of myocardial activity after ischemia is one of the major areas of interest in cardiovascular research. We developed a novel experimental rat model and used it to examine the effect of mesenchymal stem cell transplantation (MSCT) on myocardial ischemia evaluated by SPECT-CT and immunohistochemistry. METHODS AND RESULTS: An open thoracotomy took place for forty adult female Wistar rats with (n = 30) or without (n = 10) surgical ligation of the left anterior descending coronary artery (LAD) in order to cause myocardial ischemia. Myocardial viability was evaluated via SPECT/CT 7 days before surgery, as well as at 7 and 14 days post-surgery. At day 0, 15 animals received homologous stem cells injected at the ischemic myocardium area. A SPECT/CT evaluation showed decreased activity of the myocardial cells in the left ventricle one week post-infarction. Regeneration of the ischemic myocardium fifteen days post-infarction was recorded only in animals subjected to stem cell transplantation. These findings were also confirmed by histology and immunohistochemical analysis, with the significantly higher expression of GATA4 and Nkx2.5. CONCLUSIONS: The positive effect of mesenchymal stem cell transplantation in the ischemic myocardium was recorded. The application of SPECT-CT allowed a clear evaluation of both the quality and quantity of the living myocardium post-infarction, leading to a new approach in the research of cardiovascular diseases. From a clinical perspective, MSCT may be beneficial when accompanied by myocardial revascularization procedures.

A Novel Experimental Rat Model for the In Vivo Assessment of Myocardial Ischemia Based on Single Photon Emission Computed Tomography.

BACKGROUND/AIM: Myocardial infarction, an acute medical situation with a high mortality rate worldwide, has been extensively studied in modern cardiovascular research, using different experimental models. However, a deep understanding of myocardial activity loss has not been fully investigated. We have developed a novel experimental rat model for noninvasive assessment of myocardial ischemia based on single photon emission computed tomography (SPECT/CT), in order to further understand and evaluate myocardial activity before and after surgical induction of myocardial ischemia. MATERIALS AND METHODS: Thirty adult female Wistar rats underwent open thoracotomy with (n=20) or without (n=10) surgical ligation of the left anterior descending coronary artery (LAD). The myocardial ischemia was confirmed with ECG and myocardial viability was evaluated via SPECT/CT at 7 days before as well as at 7 and 14 days post-surgery, after which animals were sacrificed and myocardial ischemic injury was further assessed histologically. RESULTS: All animals were evaluated with anatomical and functional criteria based on the SPECT/CT imaging results. A successful surgical technique causing ischemia and loss of myocardial function in all animals undergoing a LAD ligation was established. Furthermore, evaluation of the viable myocardium with SPECT/CT confirmed the reduction of functional myocardial cells of the left ventricle post-infarction, which was also documented histologically. CONCLUSION: Using our technique, the validity of this animal model to induce and evaluate myocardial ischemia was demonstrated. Our choice to apply SPECT-CT qualitative and quantitative evaluation of myocardial function leads to a new approach in experimentation with an anticipated significant impact in the ongoing cardiovascular laboratory research.

Low Prevalence of TERT Promoter, BRAF and RAS Mutations in Papillary Thyroid Cancer in the Greek Population.

Thyroid cancer is a common endocrine malignancy and displays a variety of histological patterns ranging from adenoma to malignant tumors. Molecular diagnostics have given significant insights into the genetic basis of thyroid tumorigenesis, known to be linked with signaling pathways affected by oxidative stress. We report for the first time a genotype study of TERT promoter combined with BRAF and RAS mutations in Papillary Thyroid Cancer (PTC) cases in the Greek population. Polymerase Chain Reaction and sequencing were used to identify TERT promoter (C228T, C250T, CC243-243TT) mutations, the BRAF (T1799A) mutation and mutations in codons 12, 13, 61 of the HRAS, KRAS and NRAS genes. The most common C228T TERT promoter mutation was identified in 2 PTC cases co-existing with the BRAF mutation. The BRAF T1799A mutation was detected in 10 PTC cases, while two different NRAS mutations in codon 61 (C181A and A182G) were found in 2 PTC cases. These mutations occur in a mutually exclusive manner. Our results indicate that despite the low frequencies, the study of the specific mutations should be encouraged because they are indicative of aggressive forms of thyroid cancer of the papillary histotype in this patient cohort, thus providing insights towards their therapeutic management.

Deregulation of p53/survivin apoptotic markers correlated to PTEN expression in pterygium neoplastic cells.

PURPOSE: Pterygium is a distinct clinicopathological entity characterized by degenerated and neoplastic-like features. Concerning its rise on normal conjunctiva epithelia, the role of specific gene deregulations including apoptotic/anti-apoptotic factors and significant suppressor genes in signaling transduction pathways is under investigation. In the current study, we co-analyzed p53, survivin and PTEN proteins in pterygia and normal conjunctiva. METHODS: Using a liquid-based cytology assay, 50 cell specimens were obtained by a smooth scraping on conjunctiva epithelia and fixed accordingly. Among them, 38 were pterygia and the remaining (n=12) normal epithelia (control group). Immunocytochemistry assays were implemented on the corresponding slides by applying ani-p53, survivin, and PTEN antibodies. Digital image analysis was performed for evaluating objectively the corresponding immunostaining intensity levels. RESULTS: The majority of the examined pterygia cases overexpressed the markers p53:22/38-57.9%, survivin:30/38-78.9%, and PTEN:25/38-65.7%. Interestingly, overall p53/PTEN co-expression was found to be statistically significant (p=0.022). CONCLUSIONS: Survivin overexpression leads to an increased anti-apoptotic activity playing a central molecular role in the pathogenesis and progression of pterygia. Furthermore, although p53 expression is observed in these lesions, its impact seems to be low compared to survivin's influence on them. Additionally, the role of PTEN in the process is potentially significant providing a suppressor balance to the p53/ survivin complex.

HER2/CHR 17 tissue microarray chromogenic in situ hybridization analysis in colon adenocarcinoma: multiple gene signals vs protein expression.

PURPOSE: HER2-dependent signalling pathway is deregulated in a subset of colon adenocarcinomas. Although HER2 protein expression patterns demonstrate a broad diversity in these tumors, the critical parameter for targeting the gene is the detection of gene amplification. Our aim was to investigate the correlation between HER2 protein levels and chromosome 17 (chr 17) copies. METHODS: Sixty paraffin-embedded samples of primary colon adenocarcinomas were cored at 1 mm diameter and transferred to the microarray block. Immunohistochemistry (IHC) was performed using anti-HER2 monoclonal antibody. Chromogenic in situ hybridization (CISH) was performed using HER2 gene/chromosome 17 centromeric probes. RESULTS: HER2 protein overexpression (score: 2+/3+) was observed in 20/60 (33.3%) samples. CISH analysis detected 11/60 (18.33%) amplified cases, whereas chromosome 17 aneuploidy (polysomy) was identified in 13/60 (21.66%) cases. Significant associations were detected correlating HER2 expression with grade of the tumors (p=0.03), Chr 17 with stage (p=0.01), gene copies with protein expression (p=0.008), and also Chr 17 centromere signals with overall gene signals (p=0.001). CONCLUSION: Multiple HER2 gene copies lead to different protein expression patterns (score 1+ to 3+) but pure gene amplification is only a subset of them. Identification of chromosome polysomy is a critical parameter in detecting original gene amplification in conventional one-color CISH methods.

Increased expression of epidermal growth factor receptor (EGF-R) in patients with different forms of lung fibrosis.

INTRODUCTION: Emerging evidence supports the role of epidermal growth factor-receptor (EGFR) in fibrogenesis. The aim of our study was to investigate the expression profiles of EGFR in three forms of IIPs, including idiopathic pulmonary fibrosis (IPF), cryptogenic organizing pneumonia (COP), and nonspecific interstitial pneumonia (NSIP). PATIENTS AND METHODS: Twenty newly diagnosed patients with IPF, 15 with COP, and 15 with NSIP (cellular, n = 4 and fibrotic, n = 11) were investigated. Fifteen paraffin blocks obtained from the normal part of lungs removed for benign lesions were used as controls. Immunohistochemistry was carried out using specific monoclonal antibody. Results were verified by qRT-PCR. RESULTS: A significant EGFR upregulation, both in protein and mRNA level, was observed in IPF, COP, and fibrotic NSIP samples compared to controls. EGFR was primarily localized in the hyperplastic alveolar epithelium surrounding areas of fibrosis in IPF, COP, and fibrotic NSIP samples, as assessed by double immunohistochemistry analysis with surfactant protein-A. EGFR mRNA levels were positively associated with indicators of lung fibrosis (type 1 collagen mRNA levels) and negatively correlated with functional prognostic parameters. CONCLUSIONS: We conclude that EGFR is upregulated in the hyperplastic alveolar epithelium in all three fibrotic forms of IIPs indicating a potential role during abnormal reepithelization.

Assessment of angiogenic markers and female sex hormone receptors in pregnancy tumor of the gingiva.

PURPOSE: Oral pregnancy tumors (OPTs) arise on the inflamed gingiva of women after the first trimester of pregnancy. The expression of angiogenic markers and female hormone receptors was assessed. MATERIALS AND METHODS: Immunohistochemistry was used to analyze the expression of estrogen and progesterone receptors and the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF) and its receptor, fibroblast growth factor (FGF), and hypoxia inducible factors 1α and 3α (HIF1α and HIF3α). Experimental groups included 9 OPTs, 10 oral pyogenic granulomas from nonpregnant women of the same age, and 9 oral pyogenic granulomas from postmenopausal women. RESULTS: VEGF expression in stromal histiocytes and endothelial cells of small vessels was positively correlated in the OPT group (P < .05 by χ(2) test). VEGF receptor also was overexpressed in stromal histiocytes and endothelial cells of OPTs compared with oral pyogenic granulomas from nonpregnant and postmenopausal women (P < .005 by χ(2) test). No correlation was detected among estrogen and progesterone receptors, FGF and HIF1α and HIF3α (ER and PgR respectively) in the 3 experimental groups. CONCLUSIONS: VEGF-associated angiogenesis is most likely involved in the pathogenesis of the lesion. These results imply that local inhibition of VEGF activity could be an adjuvant therapeutic approach for OPTs to control hemorrhage, which can be massive at the surgical excision of such lesions during pregnancy.

Pulmonary autotaxin expression contributes to the pathogenesis of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic form of diffuse lung disease occurring mainly in older adults. Increased lysophosphatidic acid (LPA) concentrations have been reported in the alveolar space of both idiopathic pulmonary fibrosis patients and a corresponding animal model, whereas the genetic deletion or pharmacological inhibition of LPA receptor 1 attenuated the development of the modeled disease, suggesting a direct involvement of LPA in disease pathogenesis. In this report, increased concentrations of autotaxin (ATX; ENPP2), the enzyme largely responsible for extracellular LPA production, were detected in both murine and human fibrotic lungs. The genetic deletion of ATX from bronchial epithelial cells or macrophages attenuated disease severity, establishing ATX as a novel player in IPF pathogenesis. Furthermore, the pharmacological inhibition of ATX attenuated the development of the modeled disease, suggesting that ATX is a possible therapeutic target in IPF.

Comparative study of the immunohistochemical expression of tissue inhibitors of metalloproteinases 1 and 2 between clearly invasive carcinomas and "in situ" trophoblast invasion.

Tissue inhibitors of metalloproteinases (TIMPs) play an important role in extracellular matrix homeostasis by regulating MMP activity. Although they were initially considered inhibitors of tumor growth and metastasis, recently their role in cancer progression has been controversial. The aim of our study was to compare the immunohistochemical expression of TIMP1 and TIMP2 between an uncontrollably invasive phenomenon (cancer) and an "in situ" process (trophoblast invasion) in an effort to assess any differential role of these molecules between these two distinct phenomena and therefore to understand better their contribution in cancer invasion and migration. We performed an immunohistochemical analysis of 50 carcinomas (colorectal, gastric, breast, pulmonary, and renal) and 40 first trimester gestations. The marker expression was evaluated semiquantitatively, separately in cancer parenchymal and trophoblastic cells as well as in malignant stromal and decidual cells, according to a percentage scale (0, <10, 10-50, and >50%) and according to staining intensity (0, +, ++, and +++). Our results showed that there was no statistically significant difference in TIMP1 expression between cancer parenchymal cells and trophoblastic cells. On the other hand, TIMP1 was expressed more often in decidual cells than in cancer stromal cells. Immunostaining for TIMP2 was more extensive and intense both in trophoblastic and decidual cells than in cancer parenchymal and stromal cells, respectively. The reduced expression of TIMP2 in metastatic carcinomas by comparison with non-metastatic gestation specimens underlines its importance in cancer invasion and migration. On the other hand, TIMP1 was more expressed in decidua than cancer stroma, but at the same time showed no statistically significant difference between cancer parenchyma and trophoblasts, highlighting its multifunctional activity in cancer progression.

Impact of HER2 and PTEN simultaneous deregulation in non-small cell lung carcinoma: correlation with biological behavior.

BACKGROUND: HER2/neu overexpression due to gene amplification is an important factor in breast cancer, modifying the sensitivity to anti-HER2 monoclonal antibody therapy. The clinical significance of HER2 expression in non small cell lung carcinoma (NSCLC) is currently under evaluation. The tumor suppressor gene PTEN negatively regulates the HER2/PI3K/Akt signalling pathway. The purpose of this study was to evaluate the role of simultaneous alteration in HER2 and PTEN protein expression in relation to biological behaviour of NSCLCs. MATERIALS AND METHOD: Protein expression was determined by immunohistochemistry in sixty-one (n=61) NSCLC cases along with CISH for HER2 gene analysis and detection of chromosome 17 aneuploidy. Patients were followed-up for a period of 34 to 41 months after surgery. RESULTS: HER2 overexpression (2+/3+ score) was detected in 17 (27.9%) patients while loss of PTEN expression was observed in 24 (39.3%) cases, low expression in 29 (47.6%) and overexpression in 8 (13.1%). Simultaneous HER2 overexpression and PTEN low/loss of expression were correlated with metastasis (71.4% vs 36.2% p=0.03) . Analysis in the subgroup of 22 patients of pTNM stage III with lymph node status N1 or N2 revealed that there was a relationship between the number of positive regional lymph node groups and simultaneous deregulation of the two genes (p=0.04). Multivariate analysis determined that HER2 overexpression was associated with an increasing risk of developing metastases (OR: 4.3; 95%CI: 1.2-15.9; p: 0.03) while PTEN overexpression was associated with lower risk (OR: 0.1; 95%CI: 0.1, 1.0; p: 0.05). CONCLUSIONS: Simultaneous HER2/PTEN deregulation is a significant genetic event that leads to a more aggressive phenotype of NSCLC.

Comparative expression profiling in pulmonary fibrosis suggests a role of hypoxia-inducible factor-1alpha in disease pathogenesis.

RATIONALE: Despite intense research efforts, the etiology and pathogenesis of idiopathic pulmonary fibrosis remain poorly understood. OBJECTIVES: To discover novel genes and/or cellular pathways involved in the pathogenesis of the disease. METHODS: We performed expression profiling of disease progression in a well-characterized animal model of the disease. Differentially expressed genes that were identified were compared with all publicly available expression profiles both from human patients and animal models. The role of hypoxia-inducible factor (HIF)-1alpha in disease pathogenesis was examined with a series of immunostainings, both in the animal model as well as in tissue microarrays containing tissue samples of human patients, followed by computerized image analysis. MEASUREMENTS AND MAIN RESULTS: Comparative expression profiling produced a prioritized gene list of high statistical significance, which consisted of the most likely disease modifiers identified so far in pulmonary fibrosis. Extending beyond target identification, a series of meta-analyses produced a number of biological hypotheses on disease pathogenesis. Among them, the role of HIF-1 signaling was further explored to reveal HIF-1alpha overexpression in the hyperplastic epithelium of fibrotic lungs, colocalized with its target genes p53 and Vegf. CONCLUSIONS: Comparative expression profiling was shown to be a highly efficient method in identifying deregulated genes and pathways. Moreover, tissue microarrays and computerized image analysis allowed for the high-throughput and unbiased assessment of histopathologic sections, adding substantial confidence in pathologic evaluations. More importantly, our results suggest an early primary role of HIF-1 in alveolar epithelial cell homeostasis and disease pathogenesis, provide insights on the pathophysiologic differences of different interstitial pneumonias, and indicate the importance of assessing the efficacy of pharmacologic inhibitors of HIF-1 activity in the treatment of pulmonary fibrosis.