Olive leaf extract counteracts epithelial to mesenchymal transition process induced by peritoneal dialysis, through the inhibition of TGFß1 signaling.

The mesothelial cells (MCs) play an important role in the morpho-functional alterations of the peritoneal membrane (PM) undergoing peritoneal dialysis (PD). MCs, through the epithelial-mesenchymal transition process (EMT), progressively acquire a myofibroblast-like phenotype, promoting peritoneal fibrosis (PF) and failure of peritoneal membrane function. Transforming growth factor ß1 (TGFß1), through canonical and non-canonical pathways, promotes the epithelial-mesenchymal transition (EMT) process leading to PF. To investigate the therapeutic potential of an olive leaf extract (OLE) on preserving peritoneal membrane function, we evaluated the effect of OLE on the TGFß1-induced EMT in mesothelial cells, Met5A, and elucidated the underlying molecular mechanisms. As assessed by changes in the expression of epithelial, mesenchymal, and fibrotic cell markers (such as E-cadherin, N-cadherin, α-SMA, fibronectin, vimentin), levels of matrix metalloproteinases (MMP2 and MMP9), and cell migration, OLE inhibited the TGFß1-induced EMT. Importantly, the beneficial effect of OLE was mediated by reduction of the TGFß1-induced activation of Smad2/3 signaling and the mitigation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Smad/non-Smad signaling pathways, activated by TGFß1, both reduce expression of epithelial marker E-cadherin which has a crucial role in EMT initiation. Interestingly, we observed that in presence of OLE activity of the E-cadherin, promoter was increased and concomitantly OLE reduced the nuclear content of its co-repressor SNAIL. Our results suggest the potential therapeutic of OLE to counteract fibrotic process in peritoneal dialysis patients.

Olive leaf extract counteracts cell proliferation and cyst growth in an in vitro model of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive enlargement of kidney cysts, leading to chronic kidney disease. Since the available treatment for ADPKD is limited, there is emerging interest for natural compounds as potential therapeutic candidates. The aim of our study was to investigate whether an olive leaf extract may be able to counteract the cyst growth in an in vitro model of ADPKD. We treated WT9-12 cells with an olive leaf extract (OLE). In monolayer culture we evaluated cell viability by the MTT assay, protein expression by western-blot analysis and apoptosis by DNA laddering and TUNEL assays. For functional studies we used transient transfection and ChIP assays. Intracellular calcium measurement was performed with a spectrofluorimeter using a fluorescent probe. 3D-cell-culture was used for cyst growth studies. OLE reduced the WT9-12 cell growth rate and affected intracellular signaling due to high c-AMP levels, as OLE reduced PKA levels, enhanced p-AKT, restored B-Raf-inactivation and down-regulated p-ERK. We elucidated the molecular mechanism by which OLE, via Sp1, transactivates the p21WAF1/Cip1 promoter, whose levels are down-regulated by mutated PKD1. We demonstrated that p-AKT up-regulation also played a crucial role in the OLE-induced anti-apoptotic effect and that OLE ameliorated intracellular calcium levels, the primary cause of ADPKD. Finally, using a 3D-cell-culture model we observed that OLE reduced the cyst size. Therefore, multifaceted OLE may be considered a new therapeutic approach for ADPKD treatment.

Rapamycin-induced autophagy protects proximal tubular renal cells against proteinuric damage through the transcriptional activation of the nerve growth factor receptor NGFR.

Experimental evidence demonstrated that macroautophagy/autophagy exerts a crucial role in maintain renal cellular homeostasis and represents a protective mechanism against renal injuries. Interestingly, it has been demonstrated that in the human proximal tubular renal cell line, HK-2, the MTOR inhibitor rapamycin enhanced autophagy and mitigated the apoptosis damage induced by urinary protein overload. However, the underlying molecular mechanism has not yet been elucidated. In our study we demonstrated, for the first time, that in HK-2 cells, the exposure to low doses of rapamycin transactivated the NGFR promoter, leading to autophagic activation. Indeed, we observed that in HK-2 cells silenced for the NGFR gene, the rapamycin-induced autophagic process was prevented, as the upregulation of the proautophagic markers, BECN1, as well as LC3-II, and the autophagic vacuoles evaluated by transmission electron microscopy, were not found. Concomitantly, using a series of deletion constructs of the NGFR promoter we found that the EGR1 transcription factor was responsible for the rapamycin-mediated transactivation of the NGFR promoter. Finally, our results provided evidence that the cotreatment with rapamycin plus albumin further enhanced autophagy via NGFR activation, reducing the proapoptotic events promoted by albumin alone. This effect was prevented in HK-2 cells silenced for the NGFR gene or pretreated with the MTOR activator, MHY1485. Taken together, our results describe a novel molecular mechanism by which rapamycin-induced autophagy, mitigates the tubular renal damage caused by proteinuria, suggesting that the use of low doses of rapamycin could represent a new therapeutic strategy to counteract the tubule-interstitial injury observed in patients affected by proteinuric nephropathies, avoiding the side effects of high doses of rapamycin.

Active compounds extracted from extra virgin olive oil counteract mesothelial-to-mesenchymal transition of peritoneal mesothelium cells exposed to conventional peritoneal dialysate: in vitro and in vivo evidences.

During peritoneal dialysis (PD), peritoneal mesothelial cells undergo a transition from an epithelial phenotype to a mesenchymal phenotype that, together with the inflammatory process, promotes tissue fibrosis and a failure of peritoneal membrane function. To date, there is no definitive treatment for the progressive thickening and angiogenesis of the peritoneal membrane associated with PD. In this study we tested, in vitro and in vivo, the ability of active compounds extracted from extra virgin olive oil (AC-EVOO) to counteract the mesothelial-to-mesenchymal transition process (MMT) observed in mesothelial cells chronically exposed to the conventional peritoneal dialysate (DL). In particular, we used a cultivar from southern Italy known to have a high polyphenol content. Our results showed that, in mesothelial cells exposed to DL, the combined treatment with AC-EVOO prevented the genic and protein upregulation of key mesenchymal and inflammatory markers, as well as the MCs' migratory capacity. Concomitantly, we tested the antifibrotic efficacy of AC-EVOO in mesothelial cells obtained from effluents of patients undergoing PD, whose "fibroblast-like" phenotype was defined by flow-cytometry assay. We observed that in these cells AC-EVOO significantly mitigated, but did not reverse, the MMT process. In conclusion, our preliminary results suggest that AC-EVOO can interfere with critical factors in the process of differentiation, preventing myofibroblast formation, but once fibrosis has already progressed it is unable to promote the redifferentiation to the epithelial phenotype. Further studies are needed to establish whether AC-EVOO could represent a new therapeutic target to prevent peritoneal fibrosis.

High doses of hydroxytyrosol induce apoptosis in papillary and follicular thyroid cancer cells.

PURPOSE: Recent evidences indicates that hydroxytyrosol, one of the main olive oil phenols, possess antitumor effects because of its pro-oxidant properties and the capacity to inhibit proliferation and to promote apoptosis in several tumor cell lines, although most of the results were obtained for breast and digestive systems cancers. METHODS: In this study, we evaluated the activities of hydroxytyrosol against papillary (TPC-1, FB-2) and follicular (WRO) thyroid cancer cell lines. RESULTS: Cellular viability revealed that high doses of hydroxytyrosol reduced cancer cells viability concomitantly with a reduction of cyclin D1 expression and an up-regulation of cell cycle key modulator p21 levels. In the same experimental conditions, Annexin V-PI staining and DNA laddering revealed that hydroxytyrosol exerts proapoptotic effects on papillary and follicular cancer cells. Furthermore, by Western blot analysis, we observed that hydroxytyrosol treatment reduced thyroid cancer cells viability by promoting apoptotic cell death via intrinsic pathway. CONCLUSIONS: Collectively, our results demonstrated for the first time that in thyroid cancer cells hydroxytyrosol promoted apoptosis at higher doses with respect to other cancer cells lines. Therefore, further studies will reveal the mechanisms by which thyroid cancer cells are more resistant to the proapoptotic effect exerted by hydroxytyrosol as well as the potential application as novel target therapeutic in thyroid cancer.

Early cytomegalovirus-specific T-cell response and estimated glomerular filtration rate identify patients at high risk of infection after renal transplantation.

BACKGROUND: Assessing the risk of cytomegalovirus (CMV) viremia in kidney transplant recipients (KTR) may be helpful to indicate in which patient it is worth starting antiviral treatment during preemptive strategy. METHODS: In 40 CMV-seropositive KTR preemptively treated with ganciclovir, we used interferon (IFN)-γ ELISpot test to evaluate whether monitoring T cells directed against phosphoprotein (pp) 65 and immediate early (IE)-1 antigens could predict the onset of viremia. RESULTS: CMV viremia occurred in 24 patients (60%) within 120 days after transplantation. Non-viremic patients had higher anti-pp65, anti-IE-1 T cells, and estimated glomerular filtration rate (eGFR) in the first 90 days after transplantation. At logistic regression, anti-pp65, anti-IE-1 T cells, and eGFR measured at day 30 were significantly associated with CMV infection. Cutoff values of 15 spot-forming cells (SFCs)/200,000 peripheral blood mononuclear cells (PBMCs) for anti-IE, 40 SFCs/200,000 PBMCs for anti-pp65, and 46.6 mL/min/1.73 m(2) for eGFR, respectively, predicted the risk of CMV infection with high sensitivity and specificity (area under the receiver operating characteristic curve >0.75). Using a classification tree model, we identified as high-risk patients those showing anti-pp65 <42 SFCs/200,000 PBMCs and eGFR <62 mL/min/1.73 m(2) , as well as anti-pp65 ≥42 and anti-IE-1 <6.5 SFCs/200,000 PBMCs. CONCLUSION: Monitoring CMV-specific T-cell responses and eGFR in the first month post transplant can identify patients at high risk of CMV infection, for whom preemptive antiviral therapy is recommended.

High nerve growth factor blood concentration in renal transplantation: a new prognostic marker?

BACKGROUND: Nerve growth factor (NGF) belongs to the family of neurotropic proteins NGF is markedly expressed in proteinuric renal diseases and in end-stage renal disease; it might be involved in kidney physiopathology. To date, little is known about NGF concentrations in kidney transplant recipients (KTRs). Because NGF exerts its action on cell survival and differentiation, tissue repair, and inflammatory responses, it may also be implicated in the pathogenesis of chronic allograft nephropathy. The aim of this study was to determine circulating NGF concentrations in KTRs and to ascertain their use as a prognostic marker for kidney transplant outcomes. METHODS: Using enzyme-linked immunosorbent assay, we performed quantification of NGF in the serum of 40 prevalent KTRs at baseline and at 6 months. RESULTS: NGF concentrations in KTRs averaged 1.16 ± 0.67 ng/mL. They negative-linearly correlated with recipient age. Logistic multivariate regression analysis showed NGF to be independently associated with increased proteinuria over the 6-month follow-up. CONCLUSIONS: Our data demonstrated that serum concentrations of NGF in KTRs were elevated and that they could be considered to be a prognostic marker in kidney transplantation.

Sperm metabolism in pigs: a role for peroxisome proliferator-activated receptor gamma (PPARγ).

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor expressed predominantly in adipose tissue, also implicated in energy homeostasis. In this study, we used western blotting and immunofluorescence techniques to demonstrate for the first time that pig spermatozoa express PPARγ. To investigate the functional role of PPARγ in pig sperm, we evaluated its action on different events that characterize the biology of sperm cells, i.e. motility, capacitation, viability and acrosome reaction, using the PPARγ-agonist 15-deoxy-12,14-prostaglandin J2 (PGJ2). In responses to PGJ2 treatment, motility, cholesterol efflux and tyrosine phosphorylation were increased, which broadens the role of PPARγ from that previously described in the literature, as it also acts to improve sperm functionality. To further our understanding of the significance of PPARγ in pig sperm, we focused its effects on lipid and glucose metabolism. Evaluation of triglyceride content and lipase, acyl-CoA dehydrogenase and G6PDH activities suggests that PPARγ induces energy expenditure in pig spermatozoa. These data represent a meaningful advance in the field of sperm energy metabolism. Taken together, our results demonstrate for the first time that PPARγ is expressed by pig sperm, thus improving its functionalities in terms of motility, capacitation, acrosome reaction, survival and metabolism.

Farnesoid X receptor inhibits tamoxifen-resistant MCF-7 breast cancer cell growth through downregulation of HER2 expression.

Tamoxifen (Tam) treatment is a first-line endocrine therapy for estrogen receptor-α-positive breast cancer patients. Unfortunately, resistance frequently occurs and is often related with overexpression of the membrane tyrosine kinase receptor HER2. This is the rationale behind combined treatments with endocrine therapy and novel inhibitors that reduce HER2 expression and signaling and thus inhibit Tam-resistant breast cancer cell growth. In this study, we show that activation of farnesoid X receptor (FXR), by the primary bile acid chenodeoxycholic acid (CDCA) or the synthetic agonist GW4064, inhibited growth of Tam-resistant breast cancer cells (termed MCF-7 TR1), which was used as an in vitro model of acquired Tam resistance. Our results demonstrate that CDCA treatment significantly reduced both anchorage-dependent and anchorage-independent epidermal growth factor (EGF)-induced growth in MCF-7 TR1 cells. Furthermore, results from western blot analysis and real-time reverse transcription-PCR revealed that CDCA treatment reduced HER2 expression and inhibited EGF-mediated HER2 and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation in these Tam-resistant breast cancer cells. Transient transfection experiments, using a vector containing the human HER2 promoter region, showed that CDCA treatment downregulated basal HER2 promoter activity. This occurred through an inhibition of nuclear factor-κB transcription factor binding to its specific responsive element located in the HER2 promoter region as revealed by mutagenesis studies, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Collectively, these data suggest that FXR ligand-dependent activity, blocking HER2/MAPK signaling, may overcome anti-estrogen resistance in human breast cancer cells and could represent a new therapeutic tool to treat breast cancer patients that develop resistance.

Timing and priorities for cystic fibrosis patients candidates to lung transplantation.

Bilateral lung transplantation is actually considered a valuable option for patients with endstage lung disease related to cystic fibrosis. Timing is crucial to transplant successfully as many patients as possible and it is mainly based on the progressive worsening of pulmonary function tests and quality of life. We reviewed the charts of all patients accepted for lung transplantation at our institution, in order to assess the role of several functional and demographic parameters; we compared the group of patients able to successfully wait for transplantation (Group A) with patients dying on the waiting list (Group B). Twenty-eight patients were accepted: 15 were successfully transplanted (2 at other institutions) (mean waiting time: 117 days), 7 died waiting (mean waiting time: 108 days) and 6 are still on the list. We recorded FEV-1, FVC, PaO2, PaCO2, supplemental O2 requirement, 6-minute walking test, right ventricular ejection fraction (RVEF) and cardio-pulmonary hemodynamics measured at right heart catheterization; we recorded also age at time of diagnosis and at time of evaluation, sex, weight and Schwachman score. These parameters were compared between Group A and B. Age at time of evaluation, sex, weight and Schwachman score did not present any difference between the two groups, as well as pulmonary function tests, PaO2, 6-minute walk test and RVEF. A statistically significant difference was found in terms of PaCO2 (43.9 +/- 9.3 in Group A vs 69.1 +/- 32.4 in Group B, heart rate at rest (102 +/- 21 vs 131 +/- 12) mean pulmonary artery pressure (20.6 +/- 2.9 vs 36 +/- 15.7), pulmonary vascular resistances (350 +/- 96 vs 460 +/- 119.4), cardiac index (3.2 +/- 0.6 vs 5.4 +/- 0.9). On the base of our initial experience we conclude that a careful evaluation of CF candidates for lung transplantation is recommended. A deterioration of pulmonary function tests and quality of life are useful parameters to accept patients in the waiting list; however priority should be attributed also on the base of cardio-pulmonary hemodynamics. A larger series of patients is required to draw definitive conclusions.

Tissue-type plasminogen activator therapy versus primary coronary angioplasty: impact on myocardial tissue perfusion and regional function 1 month after uncomplicated myocardial infarction.

OBJECTIVES: This study sought to compare the impact of primary coronary angioplasty and thrombolytic therapy for acute myocardial infarction (AMI) on 1-month infarct size and microvascular perfusion. BACKGROUND: The effect of the reperfusion strategies of primary coronary angioplasty and thrombolytic therapy on microvascular integrity still remains to be determined. METHODS: Sixty-two consecutive patients with a first AMI, undergoing intravenous tissue-type plasminogen activator (t-PA) therapy (32 patients, Group I) or primary angioplasty (30 patients, Group II), were studied. Only patients with 1-month Thrombolysis in Myocardial Infarction (TIMI) flow grade 2 or 3 were selected for the study. Patients in whom primary angioplasty was unsuccessful or those with clinical evidence of failed reperfusion were excluded. Microvascular perfusion was assessed at 1 month by intracoronary injection of sonicated microbubbles. Contrast score index (CSI) and wall motion score index (WMSI) were derived using qualitative methods. RESULTS: At baseline there were no significant differences between groups for age, risk factors, time to hospital presentation, Killip class on admission, prevalence of multivessel disease or anterior infarct site, infarct area extension before reperfusion, peak creatine kinase levels and postinfarction treatment. Conversely, significant differences between groups were found at follow-up for percent residual infarct related-artery (IRA) stenosis (70 +/- 12 vs 36 +/- 14 [mean +/- SD], p = 0.0001), CSI (1.02 +/- 0.4 vs. 1.49 +/- 0.5, p = 0.0003) and WMSI (1.67 +/- 0.3 vs. 1.45 +/- 0.3, p = 0.015). In particular, in the subset of patients with TIMI grade 3 flow, a perfusion defect occurred in one or more segments subtended by the IRA in 72% of Group I versus 31% of Group II patients (p < 0.00001) and in 27% of Group I versus 8% of Group II segments (p < 0.00001). CONCLUSIONS: The present study shows, in a highly selected cohort with successful IRA recanalization, that primary angioplasty is more effective than thrombolysis in preserving microvascular flow and preventing extension of myocardial damage at 1-month after AMI.

Occult lung cancer in patients with bullous emphysema.

BACKGROUND: The incidence of lung cancer is increased in patients with bullous emphysema. METHODS: A series of 95 patients undergoing excision of bullous lung tissue was reviewed to determine the incidence and long term outcome of occult carcinoma present in the resected material. RESULTS: Four patients (4.2%) had peripheral foci of large cell carcinoma in the resection specimen (three bullectomies and one lobectomy). CONCLUSIONS: Resected bullous lung tissue should be carefully examined for areas of bronchogenic carcinoma. The results of incidental complete excision are favourable.

Transesophageal dipyridamole echocardiography for diagnosis of coronary artery disease.

The value of transthoracic dipyridamole echocardiography has been extensively documented. However, in some patients, because of a poor acoustic window, the rest transthoracic examination is not always feasible and the transesophageal approach is more convenient. Therefore, transesophageal echocardiography with high dose dipyridamole (up to 0.84 mg/kg body weight over 10 min) was performed in 32 patients in whom the transthoracic dipyridamole test either was not feasible (n = 29) or yielded ambiguous results (n = 3). The transesophageal echocardiographic test results were considered abnormal when new dipyridamole-induced regional wall motion abnormalities were observed. All 32 patients underwent coronary angiography; significant coronary artery disease was defined as greater than or equal to 70% lumen diameter narrowing in at least one major vessel. All patients also performed a bicycle exercise test 1 day before transesophageal dipyridamole echocardiography. Transesophageal stress studies were completed in all patients, with a maximal imaging time (in tests with a negative result) of 20 min. No side effects or intolerance to drug or transducer was observed. The left ventricle was always visualized in the four-chamber and transgastric short-axis views. High quality two-dimensional echocardiographic images were obtained in all patients both at rest and at peak dipyridamole infusion and were digitally analyzed in a quad-screen format. Coronary angiography showed coronary artery obstruction in 24 patients: 6 had single-, 9 double- and 9 triple-vessel disease. The transesophageal dipyridamole test showed a specificity of 100% and an overall sensitivity of 92%. The sensitivity of this test for single-, double- and triple-vessel disease was 67%, 100% and 100%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of coronary artery by-pass grafts: reliability of MRI in clinical practice.

In order to test MRI ability to detect the number and the sites of coronary artery by-pass grafts (CABGs), 22 patients with CABGs were studied. The detection of a neo-vessel in even one of the examination slices was considered as positive for the study, disregarding the difference between its origin and course. With such a criterion, MRI total percentage of vascular bridges identification resulted in 76.1% (51/67) with very low values for CABGs implanted on diagonal, obtuse marginal and posterior descending vessels (11/24 = 45.8%). These results lead to the conclusion that, although MRI has some advantages in the identification of CABGs implanted on the main coronary vessels in the early post-operative period, its extensive use cannot be proposed at the present state of the art.