Distinct Effects of Unfractionated Heparin versus Bivalirudin on Circulating Angiogenic Peptides

Background: Human studies of therapeutic angiogenesis, stem-cell, and progenitor-cell therapy have failed to demonstrate consistent clinical benefit. Recent studies have shown that heparin increases circulating levels of anti-angiogenic peptides. Given the widely prevalent use of heparin in percutaneous and surgical procedures including those performed as part of studies examining the benefit of therapeutic angiogenesis and cell-based therapy, we compared the effects of unfractionated heparin (UFH) on angiogenic peptides with those of bivalirudin, a relatively newer anticoagulant whose effects on angiogenic peptides have not been studied. Methodology/Principal Findings: We measured soluble fms-like tyrosine kinase-1 (sFLT1), placental growth factor (PlGF), vascular endothelial growth factor (VEGF), and soluble Endoglin (sEng) serum levels by enzyme linked immunosorbent assays (ELISA) in 16 patients undergoing elective percutaneous coronary intervention. Compared to baseline values, sFLT1 and PlGF levels increased by 26296313 % and 253654%, respectively, within 30 minutes of UFH therapy (p,0.01 for both; n = 8). VEGF levels decreased by 93.265 % in patients treated with UFH (p,0.01 versus baseline). No change in sEng levels were observed after UFH therapy. No changes in sFLT1, PlGF, VEGF, or sEng levels were observed in any patients receiving bivalirudin (n = 8). To further explore the direct effect of anticoagulation on circulating angiogenic peptides, adult, male wild-type mice received venous injections of clinically dosed UFH or bivalirudin. Compared to saline controls, sFLT1 an

S100A7-Downregulation Inhibits Epidermal Growth Factor-Induced Signaling in Breast Cancer Cells and Blocks Osteoclast Formation

S100A7 is a small calcium binding protein, which has been shown to be differentially expressed in psoriatic skin lesions, as well as in squamous cell tumors of the skin, lung and breast. Although its expression has been correlated to HER+ high-grade tumors and to a high risk of progression, the molecular mechanisms of these S100A7-mediated tumorigenic effects are not well known. Here, we showed for the first time that epidermal growth factor (EGF) induces S100A7 expression in both MCF-7 and MDA-MB-468 cell lines. We also observed a decrease in EGF-directed migration in shRNA-downregulated MDA-MB-468 cell lines. Furthermore, our signaling studies revealed that EGF induced simultaneous EGF receptor phosphorylation at Tyr1173 and HER2 phosphorylation at Tyr1248 in S100A7-downregulated cell lines as compared to the vector-transfected controls. In addition, reduced phosphorylation of Src at tyrosine 416 and p-SHP2 at tyrosine 542 was observed in these downregulated cell lines. Further studies revealed that S100A7-downregulated cells had reduced angiogenesis in vivo based on matrigel plug assays. Our results also showed decreased tumor-induced osteoclastic resorption in an intra-tibial bone injection model involving SCID mice. S100A7-downregulated cells had decreased osteoclast number and size as compared to the vector controls, and this decrease was associated with variations in IL-8 expression in in vitro cell cultures. This is a novel report on the role of S100A7 in EGF-induced signaling in breast cancer cells and in osteoclast formation

Introduction of a quality improvement curriculum in the Department of Internal Medicine, Lincoln Medical Center

Community hospitals with limited resources struggle to engage physicians in Quality improvement initiatives. We introduced Quality Improvement (QI) curriculum for residents in response to ACGME requirements and surveyed the residents understanding of QI and their involvement in QI projects before and after the introduction of the curriculum. The current article describes our experiences with the process, the challenges and possible solutions to have a successful resident led QI initiative in a community hospital. Methods: A formal QI curriculum was introduced in the Department of Internal Medicine from September to October 2015 using the Model for Improvement from Institute for Health care Improvement (IHI). Learners were expected to read the online modules, discuss in small group sessions and later encouraged to draft their QI projects using the Charter form and PDSA form available on the HI website. Online surveys were conducted a week prior and 3 months after completion of the curriculum Results: 80% (100/117) of residents completed the pre-curriculum survey and 52% (61/117) completed the survey post curriculum. 96.7% of residents report that physicians should lead QI projects and training rather than the hospital administrators. Residents had 20% increase in understanding and confidence in leading quality improvement projects post curriculum once initiated. Most Residents (72%) feel QI should be taught during residency. Active involvement of residents with interest was seen after the initiation of Open School Institute of health improvement (IHI) curriculum as compared to Institutional led QI’s. The resident interventions, pitfalls with change processes with an example of PDSA cycle are discussed. Conclusion: A Dedicated QI curriculum is necessary to prepare the physicians deliver quality care in an increasing complex health care delivery system. The strength of the curriculum is the ease of understanding the material, easily available to all, and can be easily replicated in a Community Hospital program with limited resources. Participation in QI by residents may promote constructive competitiveness among related hospitals in public system to improve delivery of safe care. Abbreviations: ACGME: Accreditation Council for Graduate Medical Education; IHI: Institute of Healthcare Improvement; PDSA: Plan-Do-Study-Act; PGY: QI: Quality improvemen

S100A7-downregulation decreases angiogenesis.

<p>(A) Effect of S100A7 on blood vessel formation in matrigel plugs containing MDA-MB-468 cell lines. 250 µl of Growth factor-reduced Matrigel Matrix High Concentration (BD Biosciences) was mixed with 6×10⁶ cells (250 µl of vector control or 250 µl of S100A7-downregulated cells) and implanted subcutaneously into SCID mice (two sites per mouse). After 4 weeks, one matrigel plug from each mouse injected with vector control or S100A7-shRNA cells was fixed in Tissue Tek and snap-frozen. The sections were stained for CD31 antibody by immunohistochemistry. (B) The other plug was grounded and the supernatant was analyzed for hemoglobin content using the Hemoglobin Assay Kit (Sigma Diagnostics). The results are interpreted as the blood volume per unit weight of matrigel (* p<0.05).</p

S100A7-downregulated cells decrease osteoclast formation and IL-8 expression levels.

<p>Osteoclast precursors were prepared (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001741#s4" target="_blank">Materials and Methods</a>), plated in 24-well plates and allowed to stand for 5–6 hours. (A) Inserts containing 5×10⁴ vector control cells, the S100A7 shRNA (downregulated) cells or RANKL without breast cancer cells (internal positive control) were then placed on the wells and the numbers of TRAP+ multinucleated cells generated under each condition were counted at the end of 48 hours. Each culture was done in triplicate. The experiments were repeated thrice with similar results. (B) The number of multinucleated fused osteoclast cells (A) in an average of triplicate wells is represented in the histogram. Results are expressed as the mean±SEM (*p<0.05). (C) Vector control and S100A7 shRNA-transfected stable MDA-MB-468 cell lines were grown to 80% confluence and supernatants were analyzed for IL-8 expression by ELISA. IL-8 expression was significantly less (*p<0.02) in the S100A7-downregulated cells as compared to the vector-transfected controls.</p

S100A7 downregulation decreases osteolytic lesions <i>in vivo.</i>

<p>Cells were directly introduced into the tibias of 4 week-old mice, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001741#s4" target="_blank">Materials and Methods</a>. Micro-CT scanning of live mice was done using a GE eXplore Micro-CT scanner (GE Healthcare Ltd, UK) at 93 µm resolution, 6 weeks after injection. The tibias were dissected out and scout view radiographs were taken with a micro-CT40 scanner at 12 µm resolution obtained from SCANCO Medical AG (Switzerland). There were osteolytic lesions, destruction of the cortex and extension of the tumor into the soft tissue in the mice injected with the control S100A7 cells (Panels A1 and A2). The cortex of the mice injected with the S100A7-downregulated cells was intact (Panels B1 and B2). Tibias were excised and processed for conventional histological examination. S100A7 control mice showed tumor-induced osteolysis (Panel C1), and the osteolysis was evident in all three injected mice as compared to mice injected with the S100A7-downregulated cells (Panel C2) (H & E staining, 20×). The letter ‘T’ in panel C1 represents tumor. The experiments were repeated twice with identical results.</p

EGF stimulation enhances S100A7 expression in breast cancer cells.

<p>70–80% confluent MCF-7 cells (A) and MDA-MB-468 cells (B) were washed twice and rinsed with PBS followed by 18 hrs of serum starvation. The cells were then stimulated with various concentrations of epidermal growth factor (EGF) for 30 minutes at 37°C. The cells were lysed and S100A7 expression was analyzed by Western blotting with anti-S100A7 antibody, as indicated. Equal protein loading in each lane was checked by stripping the blots and probing with β-Actin antibody (A and B, lower panels). The experiments were repeated thrice with identical results.</p