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京都大学0048新制・課程博士博士(医学)甲第10718号医博第2702号新制||医||857(附属図書館)UT51-2004-G565京都大学大学院医学研究科外科系専攻(主査)教授 鈴木 茂彦, 教授 月田 承一郎, 教授 中村 孝志学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

The Role of Matrix Proteins in Cardiac Pathology

The extracellular matrix (ECM) and ECM-regulatory proteins mediate structural and cell-cell interactions that are crucial for embryonic cardiac development and postnatal homeostasis, as well as organ remodeling and repair in response to injury. These proteins possess a broad functionality that is regulated by multiple structural domains and dependent on their ability to interact with extracellular substrates and/or cell surface receptors. Several different cell types (cardiomyocytes, fibroblasts, endothelial and inflammatory cells) within the myocardium elaborate ECM proteins, and their role in cardiovascular (patho)physiology has been increasingly recognized. This has stimulated robust research dissecting the ECM protein function in human health and disease and replicating the genetic proof-of-principle. This review summarizes recent developments regarding the contribution of ECM to cardiovascular disease. The clear importance of this heterogeneous group of proteins in attenuating maladaptive repair responses provides an impetus for further investigation into these proteins as potential pharmacological targets in cardiac diseases and beyond

Nuclear Insulin-Like Growth Factor Binding Protein-3 As a Biomarker in Triple-Negative Breast Cancer Xenograft Tumors: Effect of Targeted Therapy and Comparison With Chemotherapy

Triple-negative breast cancer (TNBC) typically has a worse outcome than other breast cancer subtypes, in part owing to a lack of approved therapeutic targets or prognostic markers. We have previously described an oncogenic pathway in basal-like TNBC cells, initiated by insulin-like growth factor binding protein-3 (IGFBP-3), in which the epidermal growth factor receptor (EGFR) is transactivated by sphingosine-1-phosphate (S1P) resulting from sphingosine kinase (SphK)-1 activation. Oncogenic IGFBP-3 signaling can be targeted by combination treatment with the S1P receptor modulator and SphK inhibitor, fingolimod, and the EGFR kinase inhibitor, gefitinib (F + G). However, the interaction of this treatment with chemotherapy has not been documented. Since we observed nuclear localization of IGFBP-3 in some TNBC tumors, this study aimed to evaluate the prognostic significance of nuclear IGFBP-3 in pre-clinical models of basal-like TNBC treated with F + G and doxorubicin. Orthotopic xenograft tumors were grown in nude mice from the human basal-like TNBC cell lines MDA-MB-468 and HCC1806, and were treated with gefitinib, 25 mg/Kg, plus fingolimod, 5 mg/Kg, 3-times weekly. In some studies, doxorubicin was also administered once weekly for 6 weeks. Tumor tissue proteins were quantitated by immunohistochemistry (IHC). Interaction between doxorubicin and F + G was also studied in proliferation assays in vitro. In both tumor models, tissue staining for IGFBP-3 was predominantly nuclear. Combination of F + G significantly enhanced mouse survival, decreased nuclear IGFBP-3 and Ki67 staining, and increased apoptosis (cleaved caspase-3) staining. Kaplan–Meier survival analysis showed that a high tumor IGFBP-3 IHC score (>median), like a high Ki67 score, was significantly associated with shorter survival time, whereas a high apoptosis score was associated with prolonged survival. Studied in vitro in both cell lines, low-dose doxorubicin that had little effect alone, strongly enhanced the cytostatic effect of low-dose F + G combination. However, in both in vivo models, doxorubicin at maximum-tolerated dose neither inhibited tumor growth when administered alone, nor enhanced the significant inhibitory effect of F + G. We conclude that doxorubicin may not add benefit to the inhibitory effect of F + G unless its dose-limiting toxicity can be overcome. Nuclear IGFBP-3 appears to have potential as a prognostic marker in TNBC and could be evaluated for clinical utility

Novel Perspectives in Chronic Kidney Disease-Specific Cardiovascular Disease

Chronic kidney disease (CKD) affects > 10% of the global adult population and significantly increases the risk of cardiovascular disease (CVD), which remains the leading cause of death in this population. The development and progression of CVD—compared to the general population—is premature and accelerated, manifesting as coronary artery disease, heart failure, arrhythmias, and sudden cardiac death. CKD and CV disease combine to cause multimorbid cardiorenal syndrome (CRS) due to contributions from shared risk factors, including systolic hypertension, diabetes mellitus, obesity, and dyslipidemia. Additional neurohormonal activation, innate immunity, and inflammation contribute to progressive cardiac and renal deterioration, reflecting the strong bidirectional interaction between these organ systems. A shared molecular pathophysiology—including inflammation, oxidative stress, senescence, and hemodynamic fluctuations characterise all types of CRS. This review highlights the evolving paradigm and recent advances in our understanding of the molecular biology of CRS, outlining the potential for disease-specific therapies and biomarker disease detection

Activated Protein C Inhibits Proliferation and Tumor Necrosis Factor α-Stimulated Activation of p38, c-Jun NH2-Terminal Kinase (JNK) and Akt in Rheumatoid Synovial Fibroblasts

Photograph of a scene at Remington Park Race Track

NVP-BYL719, a novel PI3K-alpha selective inhibitor, overcomes acquired erlotinib resistance via upregulation of the PI3K/Akt/ mTOR pathway in pancreatic ductal adenocarcinoma

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with few treatment options. Epidermal growth factor receptor (EGFR) inhibitor erlotinib combined with gemcitabine only marginally improves survival, possibly due to development of acquired erlotinib resistance. In this study, we explore mechanisms of erlotinib resistance and propose PI3K and EGFR co-inhibition strategy to overcome this. Methods: We developed in-vitro erlotinib resistant pancreatic cancer cell models by subculturing two cell lines (BxPC-3, PANC-1) in dose-esclating erlotinib for 9 months, and tested erlotinib, IGF1R inhibitor NVP-AEW541 and PI3K-alpha inhibitor NVP-BYL719, alone or in combination. Cell proliferation, RTK-phosphorarray, western blotting, confocal microscopy, cell cycle, apoptotic, clonogenic and migration assays were performed. We investigated the anti-tumour effect of erlotinib plus NVP-BYL719 in a primary patient-derived tumour xenograft mice model. Results: Both acquired resistant cell lines showed activation of PI3K/Akt pathwy. They were cross-resistant to NVP-AEW541 and MEK inhibitor, but remained sensitive to NVP-BYL719. Importantly, erlotinib was synergistic with NVP-BYL719 in BxPC-ER (synergy index or S.I.=1.71) and PANC-ER (S.I.=1.44), more so than the respective parent cell lines. Treatment of resistant cell lines with erlotinib plus NVP-BYL719 caused substantial G1 cell cycle arrest (71% and 58%, respectively), inhibition of colony formation (69% and 72%), and considerable necrosis and apoptosis (65% and 53%), of greater magnitude than those observed in BxPC-3 and PANC-1. In-vivo, erlotinib plus NVP-BYL719 significantly reduced tumor volume by 11.20 mm3 (95% CI: 4.30-13.15mm3, P=0.012) compared to control. Conclusion: Erlotinib-resistant PDAC cells appeared to become dependent on the PI3K/Akt pathway through oncogenic addiction, making them highly susceptible to PI3K/ EGFR co-inhibition. The pre-clinical efficacy of erlotinib plus NVP-BYL719 combination was established. Erlotinib plus NVP-BYL719 may have therapeutic relevance in pancreatic cancer, especially in erlotinib-refractory disease or those with activated PI3K pathway

CD47 promotes age-associated deterioration in angiogenesis, blood flow and glucose homeostasis

The aged population is currently at its highest level in human history and is expected to increase further in the coming years. In humans, aging is accompanied by impaired angiogenesis, diminished blood flow and altered metabolism, among others. A cellular mechanism that impinges upon these manifestations of aging can be a suitable target for therapeutic intervention. Here we identify cell surface receptor CD47 as a novel age-sensitive driver of vascular and metabolic dysfunction. With the natural aging process, CD47 and its ligand thrombospondin-1 were increased, concurrent with a reduction of self-renewal transcription factors OCT4, SOX2, KLF4 and cMYC (OSKM) in arteries from aged wild-type mice and older human subjects compared to younger controls. These perturbations were prevented in arteries from aged CD47-null mice. Arterial endothelial cells isolated from aged wild-type mice displayed cellular exhaustion with decreased proliferation, migration and tube formation compared to cells from aged CD47-null mice. CD47 suppressed ex vivo sprouting, in vivo angiogenesis and skeletal muscle blood flow in aged wild-type mice. Treatment of arteries from older humans with a CD47 blocking antibody mitigated the age-related deterioration in angiogenesis. Finally, aged CD47-null mice were resistant to age- and diet-associated weight gain, glucose intolerance and insulin desensitization. These results indicate that the CD47-mediated signaling maladapts during aging to broadly impair endothelial self-renewal, angiogenesis, perfusion and glucose homeostasis. Our findings provide a strong rationale for therapeutically targeting CD47 to minimize these dysfunctions during aging

Confocal micrographs of CFPAC-1 cells demonstrating the influence of ApoA-II on lipid uptake.

<p>SMOFlipid was labelled with DiD (red), ApoA-II was labelled green with a secondary antibody and DAPI stained the nuclei blue. (A1) cells treated with ApoA-II alone, (A2) cells treated with lipid alone, (A3) cells treated with reconstituted lipoproteins after adding ApoA-II to DiD labeled SMOFlipid (SMOF/A-II) demonstrating increased uptake and (A4) cells treated with (SMOF/A-II) after pretreating with anti-SR-B1antibodies which reduced the lipid uptake (scale bar, 20 μm). (B) Cells were pretreated with unlabeled lipid at 1:10 dilution and (B2) after 2 h SMOF/A-II was added, it appeared to be endocytosed in cytoplasmic vesicles as shown in (B3) and (B4). ApoA-II (green) is attached with the cell membrane (yellow arrow head) and as well in the cytoplasm in part associated with the lipid (red arrow head) in Differential Interference Contrast (DIC) overlay image (scale bar 25 μm). (C) Live cell imaging experiment demonstrated greater uptake of lipid in cells when reconstituted lipoproteins after adding ApoA-II to DiD labeled SMOFlipid was added to the media for lipid concentrations of 1:20 and 1:10 (scale bar 100 μm). (D) The intensity of the DiD staining was greater when 4 samples were examined with eight to twelve areas of interest for each study where ApoA2 increased the uptake of lipid 25.6±2.2, <i>P</i> = 0.02 and 54.8±2.2, <i>P</i> = 0.004 for dilutions of 1:20 and 1:10 respectively.</p

Apolipoprotein A-II Plus Lipid Emulsion Enhance Cell Growth via SR-B1 and Target Pancreatic Cancer <i>In Vitro</i> and <i>In Vivo</i>

<div><p>Background</p><p>Apolipoprotein A-II (ApoA-II) is down regulated in the sera of pancreatic ductal adenocarcinoma (PDAC) patients, which may be due to increase utilization of high density lipoprotein (HDL) lipid by pancreatic cancer tissue. This study examined the influence of exogenous ApoA-II on lipid uptake and cell growth in pancreatic cancer (PC) both <i>in vitro</i> and <i>in vivo</i>.</p><p>Methods</p><p>Cryo transmission electron microscopy (TEM) examined ApoA-II’s influence on morphology of SMOFLipid emulsion. The influence of ApoA-II on proliferation of cancer cell lines was determined by incubating them with lipid+/-ApoA-II and anti-SR-B1 antibody. Lipid was labeled with the fluorophore, DiD, to trace lipid uptake by cancer cells <i>in vitro</i> by confocal microscopy and <i>in vivo</i> in PDAC patient derived xenograft tumours (PDXT) by fluorescence imaging. Scavenger receptor class B type-1(SR-B1) expression in PDAC cell lines and in PDAC PDXT was measured by western blotting and immunohistochemistry, respectively.</p><p>Results</p><p>ApoA-II spontaneously converted lipid emulsion into very small unilamellar rHDL like vesicles (rHDL/A-II) and enhanced lipid uptake in PANC-1, CFPAC-1 and primary tumour cells as shown by confocal microscopy. SR-B1 expression was 13.2, 10.6, 3.1 and 2.3 fold higher in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cell lines than the normal pancreatic cell line (HPDE6) and 3.7 fold greater in PDAC tissue than in normal pancreas. ApoA-II plus lipid significantly increased the uptake of labeled lipid and promoted cell growth in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cells which was inhibited by anti SR-B1 antibody. Further, ApoA-II increased the uptake of lipid in xenografts by 3.4 fold.</p><p>Conclusion</p><p>Our data suggest that ApoA-II enhance targeting potential of lipid in pancreatic cancer which may have imaging and drug delivery potentialities.</p></div

SMOFlipid ApoA-II targets pancreatic ductal adenocarcinoma (PDAC).

<p>(A) Spectral reflectance fluorescence images of 8 week old xenografts taken 48h after tail vein injection of PBS, DiD labeled SMOFlipid without or with ApoA-II using Carestream molecular imaging. Note the widespread distribution of DiD is better localized to the tumours when ApoA-II is included with lipid. Arrows indicate the site of tumours. (B) Spectral fluorescence images of explanted tumours and organs taken 48h after injection from mice after a tail vein injection of either PBS, lipid/DiD (n = 2) or lipid/DiD plus ApoA-II (n = 2) (right panel) along with the H&E photo micrograph of the tumour from two mice (left panel), retained the morphological characteristics with the original patient’s PC tumours in lipid and lipid plus ApoA-II treated mice. Note the uptake of fluorescence by the tumours, liver and spleen. (C) Graph represents the uptake by the tumour relative to the uptake of the liver as a fraction of area. These results were then normalised to the value for mice receiving lipid only (defined as 1) when the tumour uptake for the mice receiving lipid and ApoA-II had 3.4 fold increased uptake. Values are mean ± SD; <i>n</i> = 3.</p