Christina Magkou

Expression of the epidermal growth factor receptor (EGFR) and the phosphorylated EGFR in invasive breast carcinomas

IntroductionEpidermal growth factor receptor (EGFR) is involved in regulating cell growth in breast carcinomas. Its activated form, phosphorylated EGFR (pEGFR), is correlated with poor prognosis in lung cancer, but it has not yet been fully investigated in breast cancer. The aim of this study was to investigate the expressions of EGFR and pEGFR and their correlation with overall and disease-free survival, clinicopathological parameters and biological markers of invasion and angiogenesis (phosphorylated Akt [pAkt], urokinase plasminogen activator receptor [uPAR], matrix metalloproteinase [MMP]-14, vascular endothelial growth factor receptor [VEGFR]-1/Flt-1).MethodsA three-step immunohistochemical method was applied to paraffin-embedded sections from 154 patients with invasive breast carcinoma in order to detect expressions of the proteins EGFR, pEGFR, oestrogen receptor, progesterone receptor, c-erbB-2, pAkt, VEGFR-1/Flt-1, MMP-14 and uPAR. The results were evaluated statistically using the χ2 test. Overall and disease-free survival distribution curves were assessed using the Kaplan-Meier test and log-rank statistics, followed by Cox proportional hazards regression model.ResultsEGFR and pEGFR proteins were immunodetected in the membrane of the malignant cells (11.3% and 35.7%, respectively). EGFR expression was positively correlated with nuclear grade (P = 0.001) and negatively correlated with the hormonal receptor oestrogen receptor (P = 0.005). pEGFR was positively related to the Akt pathway (P = 0.008) and appeared to participate in invasion and metastasis (uPAR, P = 0.049; MMP-14, P = 0.025; VEGFR-1/Flt-1, P = 0.016). Univariate analysis showed that the EGFR/pEGFR phenotype was associated with poor overall survival (P = 0.019), a finding further supported by multivariate analysis (P = 0.013).ConclusionThese data provide evidence that pEGFR expression is related to angiogenesis (via VEGFR-1/Flt-1, MMP-14 and pAkt pathways) and invasiveness (via uPAR, MMP-14 and pAkt pathways) and that the EGFR/pEGFR phenotype is associated with poor patient survival in invasive breast cancer.

The clinicopathological and prognostic significance of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP-9 according to their localization in invasive breast carcinoma.

Aims:  To investigate the clinicopathological and prognostic significance of membrane type 1 matrix metalloproteinase (MT1‐MMP) and MMP‐9 proteins expression in invasive breast carcinoma and their relationship to tumour proliferation and expression of c‐erbB2 and peroxisome proliferator‐activated receptor (PPAR) gamma.

Expression of tissue inhibitor of matrix metalloproteinases (TIMP)-3 protein in invasive breast carcinoma: Relation to tumor phenotype and clinical outcome

IntroductionOur aim was to study the expression pattern of tissue inhibitor of metalloproteinases (TIMP)-3 protein in invasive breast carcinoma, and its clinicopathological and prognostic value as well as its relation to markers indicative of the tumor phenotype.MethodsImmunohistochemistry was performed on paraffin-embedded tissue specimens from 173 invasive breast carcinomas to detect the proteins TIMP-3, estrogen receptor (ER), progesterone receptor, p53, c-erbB-2, topoisomerase IIα and Bcl-2.ResultsTIMP-3 protein was immunodetected in the cytoplasm of the malignant cells and the peritumoral stroma, as well as in in situ carcinoma and normal epithelium. Reduced expression of TIMP-3 protein within cancer cells was correlated with carcinomas of high nuclear and histological grade (p = 0.032 and p = 0.015, respectively), and low ER expression (p = 0.053). Moreover, TIMP-3 immunopositivity was inversely correlated with the expression of p53 and topoIIα proteins (p = 0.002 and p = 0.008, respectively), whereas it was positively associated with Bcl-2 expression (p = 0.020). Reduced expression of TIMP-3 protein within cancer cells was found to have an unfavorable impact on disease-free survival (p = 0.052) in the entirety of the patient population, as well as in both subgroups of lymph-node-positive and mutant-p53-negative patients (p = 0.007 and p = 0.037, respectively). Stromal localization of TIMP-3 protein was found to have no clinicopathological or prognostic value.ConclusionThis is the first immunohistochemical study to show that TIMP-3 protein within cancer cells is associated with tumor phenotype. Reduced expression of TIMP-3 protein within cancer cells was found to correlate with an aggressive tumor phenotype, negatively affecting the disease-free survival of both subgroups of lymph node-positive and mutant-p53-negative patients.

Pretreatment with atorvastatin attenuates lung injury caused by high-stretch mechanical ventilation in an isolated rabbit lung model.

Objective:We hypothesized that pretreatment with atorvastatin improves alveolar capillary permeability and hemodynamics and, thus, confers protection against lung injury caused by high-stretch mechanical ventilation. Methods:Twenty-four isolated sets of normal rabbit lungs were utilized. Treated animals received atorvastatin (20 mg/kg body weight/day by mouth) for 3 days before surgery. Lungs were perfused constantly (300 mL/min) and ventilated for 1 hr with pressure-control ventilation at either 23 (high pressure; resulting in tidal volume approximately 22 mL/kg) or 11 (low pressure; tidal volume approximately 10 mL/kg) cm H2O peak inspiratory pressure and positive end-expiratory pressure of 3 cm H2O. Four groups were examined: high pressure–no statin, high pressure–statin pretreatment, low pressure–no statin, and low pressure–statin pretreatment. Results:The high-pressure–no statin group sustained more damage than the low-pressure groups. In high-pressure groups, lungs of statin-pretreated vs. no statin-pretreated animals sustained a significantly lower increase in ultrafiltration coefficient (an accurate marker of alveolar capillary permeability; high-pressure–statin pretreatment vs. high-pressure–no statin, −0.013 ± 0.017 g/min/mm Hg/100g vs. 1.723 ± 0.495 g/min/mm Hg/100g; p < .001), lower weight gain (i.e., less edema formation; 4.62 ± 1.50 grams vs. 17.75 ± 4.71 grams; p = .005), improved hemodynamics (i.e., lower increase in mean pulmonary artery pressure; 0.56 ± 0.51 mm Hg vs. 5.62 ± 1.52 mm Hg; p = .04), lower protein concentration in bronchoalveolar lavage fluid (p < .001), and fewer histologic lesions (p = .013). Apoptosis of lung parenchyma cells was not different (p = .97). There was no difference between low-pressure–statin pretreatment and low-pressure–no statin groups regarding these outcomes. Conclusion:In this model, atorvastatin improves alveolar capillary permeability and hemodynamics and, thus, attenuates lung injury caused by high-stretch mechanical ventilation.

Differential effect of the expression of TGF-β pathway inhibitors, Smad-7 and Ski, on invasive breast carcinomas: relation to biologic behavior

Theohari I, Giannopoulou I, Magkou C, Nomikos A, Melissaris S, Nakopoulou L. Differential effect of the expression of TGF‐β pathway inhibitors, Smad‐7 and Ski, on invasive breast carcinomas: relation to biologic behavior. APMIS 2011.

Prognostic significance of phosphorylated STAT‐1 expression in premenopausal and postmenopausal patients with invasive breast cancer

Magkou C, Giannopoulou I, Theohari I, Fytou A, Rafailidis P, Nomikos A, Papadimitriou C & Nakopoulou L 
(2012) Histopathology 60, 1125–1132

Metformin attenuates ventilator-induced lung injury

IntroductionDiabetic patients may develop acute lung injury less often than non-diabetics; a fact that could be partially ascribed to the usage of antidiabetic drugs, including metformin. Metformin exhibits pleiotropic properties which make it potentially beneficial against lung injury. We hypothesized that pretreatment with metformin preserves alveolar capillary permeability and, thus, prevents ventilator-induced lung injury.MethodsTwenty-four rabbits were randomly assigned to pretreatment with metformin (250 mg/Kg body weight/day per os) or no medication for two days. Explanted lungs were perfused at constant flow rate (300 mL/min) and ventilated with injurious (peak airway pressure 23 cmH2O, tidal volume ≈17 mL/Kg) or protective (peak airway pressure 11 cmH2O, tidal volume ≈7 mL/Kg) settings for 1 hour. Alveolar capillary permeability was assessed by ultrafiltration coefficient, total protein concentration in bronchoalveolar lavage fluid (BALF) and angiotensin-converting enzyme (ACE) activity in BALF.ResultsHigh-pressure ventilation of the ex-vivo lung preparation resulted in increased microvascular permeability, edema formation and microhemorrhage compared to protective ventilation. Compared to no medication, pretreatment with metformin was associated with a 2.9-fold reduction in ultrafiltration coefficient, a 2.5-fold reduction in pulmonary edema formation, lower protein concentration in BALF, lower ACE activity in BALF, and fewer histological lesions upon challenge of the lung preparation with injurious ventilation. In contrast, no differences regarding pulmonary artery pressure and BALF total cell number were noted. Administration of metformin did not impact on outcomes of lungs subjected to protective ventilation.ConclusionsPretreatment with metformin preserves alveolar capillary permeability and, thus, decreases the severity of ventilator-induced lung injury in this model.

Guanylyl Cyclase Activation Reverses Resistive Breathing–Induced Lung Injury and Inflammation

Inspiratory resistive breathing (RB), encountered in obstructive lung diseases, induces lung injury. The soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway is down-regulated in chronic and acute animal models of RB, such as asthma, chronic obstructive pulmonary disease, and in endotoxin-induced acute lung injury. Our objectives were to: (1) characterize the effects of increased concurrent inspiratory and expiratory resistance in mice via tracheal banding; and (2) investigate the contribution of the sGC/cGMP pathway in RB-induced lung injury. Anesthetized C57BL/6 mice underwent RB achieved by restricting tracheal surface area to 50% (tracheal banding). RB for 24 hours resulted in increased bronchoalveolar lavage fluid cellularity and protein content, marked leukocyte infiltration in the lungs, and perturbed respiratory mechanics (increased tissue resistance and elasticity, shifted static pressure-volume curve right and downwards, decreased static compliance), consistent with the presence of acute lung injury. RB down-regulated sGC expression in the lung. All manifestations of lung injury caused by RB were exacerbated by the administration of the sGC inhibitor, 1H-[1,2,4]oxodiazolo[4,3-]quinoxalin-l-one, or when RB was performed using sGCα1 knockout mice. Conversely, restoration of sGC signaling by prior administration of the sGC activator BAY 58-2667 (Bayer, Leverkusen, Germany) prevented RB-induced lung injury. Strikingly, direct pharmacological activation of sGC with BAY 58-2667 24 hours after RB reversed, within 6 hours, the established lung injury. These findings raise the possibility that pharmacological targeting of the sGC-cGMP axis could be used to ameliorate lung dysfunction in obstructive lung diseases.

Acid-induced acute lung injury in mice is associated with P44/42 and c-Jun N-terminal kinase activation and requires the function of tumor necrosis factor α receptor I.

ABSTRACT Aspiration of hydrochloric acid (HCl)–containing gastric juice leads to acute lung injury (ALI) and hypoxemic respiratory failure due to an exuberant inflammatory response associated with pulmonary edema from increased vascular and epithelial permeability. The aim of this study was to determine the role and signaling mechanisms of tumor necrosis factor &agr; (TNF-&agr;) in experimental ALI from HCl aspiration using a combination of genetic animal models and pharmacologic inhibition strategies. To this end, HCl was instilled intratracheally to mice, followed by respiratory system elastance measurement, bronchoalveolar lavage, and lung tissue harvesting 24 h after injection. Hydrochloric acid instillation induced an inflammatory response in the lungs of wild-type mice, evidenced as increased bronchoalveolar lavage total cells, neutrophils, and total protein; histologic lung injury score; and respiratory system elastance, whereas TNF-&agr; receptor I mRNA levels were maintained. These alterations could be prevented by pretreatment with etanercept or genetic deletion of the 55-kd TNF-&agr; receptor I, but not by deletion of the TNF-&agr; gene. Hydrochloric acid induced a 6-fold increase in apoptotic, caspase 3–positive cells in lung sections from wild-type mice, which was abrogated in mice lacking TNF-&agr; receptor I. In immunoblotting and immunohistochemistry studies, HCl stimulated signaling via p44/42 and c-Jun N-terminal kinase, which was blocked in TNF-&agr; receptor I knockout mice. In conclusion, ALI induced by HCl requires TNF-&agr; receptor I function and associates with activation of downstream proinflammatory signaling pathways p44/42 and c-Jun N-terminal kinase.

Inspiratory resistive breathing induces MMP-9 and MMP-12 expression in the lung

Inspiratory resistive breathing (IRB) is characterized by large negative intrathoracic pressures and was shown to induce pulmonary inflammation in previously healthy rats. Matrix metalloproteinases (MMP)-9 and -12 are induced by inflammation and mechanical stress in the lung. We hypothesized that IRB induces MMP-9 and -12 in the lung. Anesthetized, tracheostomized rats breathed spontaneously through a two-way valve, connected to an inspiratory resistance, with the tidal inspiratory tracheal pressure set at 50% of the maximum. Quietly breathing animals served as controls. After 3 and 6 h of IRB, respiratory mechanics were measured, bronchoalveolar lavage (BAL) was performed, lung injury score was estimated, and lung MMP-9 was estimated by zymography and ELISA. MMP-9 and MMP-12 immunohistochemistry was performed. Isolated normal alveolar macrophages were incubated with BAL from rats that underwent IRB. After 18 h, MMP-9 and -12 levels were measured in supernatants, and immunocytochemistry was performed. Macrophages were treated with IL-1β, IL-6, or TNF-α, and MMP-9 in supernatants was measured. After 6 h of IRB, leukocytes in BAL increased, and IL-1β and IL-6 levels were elevated. Elasticity and injury score were increased after 3 and 6 h of IRB. Lung MMP-9 levels increased after 6 h of IRB. MMP-9 and MMP-12 were detected in alveolar macrophages and epithelial (bronchial/alveolar) cells after 3 and 6 h of IRB. MMP-9 and MMP-12 were found in supernatants after treatment with 6 h of IRB BAL. Cytosolic immunostaining was detected after treatment with 3 and 6 h of IRB BAL. All cytokines induced MMP-9 in culture supernatants. In conclusion, IRB induces MMP-9 and -12 in the lung of previously healthy rats.