Rapamycin Inhibits Proliferation of Hemangioma Endothelial Cells by Reducing HIF-1-Dependent Expression of VEGF

Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1). VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas

Effectiveness of a national quality improvement programme to improve survival after emergency abdominal surgery (EPOCH): a stepped-wedge cluster-randomised trial

Background: Emergency abdominal surgery is associated with poor patient outcomes. We studied the effectiveness of a national quality improvement (QI) programme to implement a care pathway to improve survival for these patients. Methods: We did a stepped-wedge cluster-randomised trial of patients aged 40 years or older undergoing emergency open major abdominal surgery. Eligible UK National Health Service (NHS) hospitals (those that had an emergency general surgical service, a substantial volume of emergency abdominal surgery cases, and contributed data to the National Emergency Laparotomy Audit) were organised into 15 geographical clusters and commenced the QI programme in a random order, based on a computer-generated random sequence, over an 85-week period with one geographical cluster commencing the intervention every 5 weeks from the second to the 16th time period. Patients were masked to the study group, but it was not possible to mask hospital staff or investigators. The primary outcome measure was mortality within 90 days of surgery. Analyses were done on an intention-to-treat basis. This study is registered with the ISRCTN registry, number ISRCTN80682973. Findings: Treatment took place between March 3, 2014, and Oct 19, 2015. 22 754 patients were assessed for elegibility. Of 15 873 eligible patients from 93 NHS hospitals, primary outcome data were analysed for 8482 patients in the usual care group and 7374 in the QI group. Eight patients in the usual care group and nine patients in the QI group were not included in the analysis because of missing primary outcome data. The primary outcome of 90-day mortality occurred in 1210 (16%) patients in the QI group compared with 1393 (16%) patients in the usual care group (HR 1·11, 0·96–1·28). Interpretation: No survival benefit was observed from this QI programme to implement a care pathway for patients undergoing emergency abdominal surgery. Future QI programmes should ensure that teams have both the time and resources needed to improve patient care. Funding: National Institute for Health Research Health Services and Delivery Research Programme

Effectiveness of a national quality improvement programme to improve survival after emergency abdominal surgery (EPOCH): a stepped-wedge cluster-randomised trial

BACKGROUND: Emergency abdominal surgery is associated with poor patient outcomes. We studied the effectiveness of a national quality improvement (QI) programme to implement a care pathway to improve survival for these patients. METHODS: We did a stepped-wedge cluster-randomised trial of patients aged 40 years or older undergoing emergency open major abdominal surgery. Eligible UK National Health Service (NHS) hospitals (those that had an emergency general surgical service, a substantial volume of emergency abdominal surgery cases, and contributed data to the National Emergency Laparotomy Audit) were organised into 15 geographical clusters and commenced the QI programme in a random order, based on a computer-generated random sequence, over an 85-week period with one geographical cluster commencing the intervention every 5 weeks from the second to the 16th time period. Patients were masked to the study group, but it was not possible to mask hospital staff or investigators. The primary outcome measure was mortality within 90 days of surgery. Analyses were done on an intention-to-treat basis. This study is registered with the ISRCTN registry, number ISRCTN80682973. FINDINGS: Treatment took place between March 3, 2014, and Oct 19, 2015. 22 754 patients were assessed for elegibility. Of 15 873 eligible patients from 93 NHS hospitals, primary outcome data were analysed for 8482 patients in the usual care group and 7374 in the QI group. Eight patients in the usual care group and nine patients in the QI group were not included in the analysis because of missing primary outcome data. The primary outcome of 90-day mortality occurred in 1210 (16%) patients in the QI group compared with 1393 (16%) patients in the usual care group (HR 1·11, 0·96-1·28). INTERPRETATION: No survival benefit was observed from this QI programme to implement a care pathway for patients undergoing emergency abdominal surgery. Future QI programmes should ensure that teams have both the time and resources needed to improve patient care. FUNDING: National Institute for Health Research Health Services and Delivery Research Programme

Endothelial-Mesenchymal Transition in Regenerative Medicine

Endothelial-mesenchymal transition (EndMT) is a fundamental cellular mechanism that regulates embryonic development and diseases such as cancer and fibrosis. Recent developments in biomedical research have shown remarkable potential to harness the EndMT process for tissue engineering and regeneration. As an alternative to traditional or artificial stem cell therapies, EndMT may represent a safe method for engineering new tissues to treat degenerative diseases by mimicking a process that occurs in nature. This review discusses the signaling mechanisms and therapeutic inhibitors of EndMT, as well as the role of EndMT in development, disease, acquiring stem cell properties and generating connective tissues, and its potential as a novel mechanism for tissue regeneration

TGFβ3 Inhibits E-Cadherin Gene Expression in Palate Medial-Edge Epithelial Cells Through a Smad2-Smad4- LEF1 Transcription Complex

Dissociation of medial-edge epithelium (MEE) during palate development is essential for mediating correct craniofacial morphogenesis. This phenomenon is initiated by TGFβ3 upon adherence of opposing palatal shelves, because loss of E-cadherin causes the MEE seam to break into small epithelial islands. To investigate the molecular mechanisms that cause this E-cadherin loss, we isolated and cultured murine embryonic primary MEE cells from adhered or non-adhered palates. Here, we provide the first evidence that lymphoid enhancer factor 1 (LEF1), when functionally activated by phosphorylated Smad2 (Smad2- P) and Smad4 (rather than β-catenin), binds with the promoter of the E-cadherin gene to repress its transcription in response to TGFβ3 signaling. Furthermore, we found that TGFβ3 signaling stimulates epithelial-mesenchymal transformation (EMT) and cell migration in these cells. LEF1 and Smad4 were found to be necessary for up-regulation of the mesenchymal markers vimentin and fibronectin, independently of β- catenin. We proved that TGFβ3 signaling induces EMT in MEE cells by forming activated transcription complexes of Smad2-P, Smad4 and LEF1 that directly inhibit Ecadherin gene expression

Rapamycin reduces elevated VEGF and HIF-1 levels in hemangioma endothelial cells.

<p><b>A:</b> Immunoblotting showing the effects of rapamycin on p70S6K phosphorylation, HIF-1α expression and VEGF-A₁₆₅ expression in normal (HDMEC) or hemangioma (EC2, EC17B, EC21A) endothelial cell lysates. <b>B and C:</b> Luminex analysis showing reduced expression of HIF-1α (B) and VEGF-A₁₆₅ (C) in hemangioma endothelial cells treated with rapamycin. Data represent mean (n = 3)±SD; *<i>P</i><0.05 compared to vehicle.</p

Elevated HIF-1 expression in hemangioma endothelial cells caused by VEGF/PI3K signaling.

<p><b>A:</b> Immunoblotting showing high expression of HIF-1α in hemangioma endothelial cells (EC2, EC17B, EC21A) compared to normal endothelial cells (HDMEC). <b>B:</b> Immunocytochemistry showing constitutive nuclear localization of HIF-1α in hemangioma endothelial cells. <b>C and D:</b> Luminex analyses demonstrating that elevated HIF-1α levels in hemangioma endothelial cells are reduced in the presence of VEGF-A₁₆₅ neutralizing antibodies (VEGF Ab) or a chemical inhibitor of PI3K (LY294002). Data represent mean (n = 3)±SD; *<i>P</i><0.01 compared to IgG or vehicle.</p

Rapamycin inhibits proliferation of hemangioma endothelial cells.

<p><b>A:</b> Flow cytometry analysis of normal (HDMEC) or hemangioma (EC2, EC17B, EC21A) endothelial cell proliferation by BrdU incorporation in the presence of rapamycin. <b>B:</b> Quantification of flow cytometry assessing the effects of rapamycin on BrdU incorporation. Data represent mean (n = 3)±SD; *<i>P</i><0.01 compared to vehicle.</p

Elevated VEGF levels in hemangioma endothelial cells are HIF-1-dependent.

<p><b>A:</b> Immunoblotting showing the expression knockdown effects of HIF-1α siRNA on HIF-1α and VEGF-A₁₆₅ in normal (HDMEC) and hemangioma (EC2, EC17B, EC21A) endothelial cells. <b>B and C:</b> Luminex analysis showing suppression of HIF-1α and VEGF-A₁₆₅ protein levels in hemangioma endothelial cells by HIF-1α siRNA. Data represent mean (n = 3)±SD; *<i>P</i><0.05 compared to control siRNA.</p