Effects of Short Term Adiponectin Receptor Agonism on Cardiac Function and Energetics in Diabetic db/db Mice.

Objective Impaired cardiac efficiency is a hallmark of diabetic cardiomyopathy in models of type 2 diabetes. Adiponectin receptor 1 (AdipoR1) deficiency impairs cardiac efficiency in non-diabetic mice, suggesting that hypoadiponectinemia in type 2 diabetes may contribute to impaired cardiac efficiency due to compromised AdipoR1 signaling. Thus, we investigated whether targeting cardiac adiponectin receptors may improve cardiac function and energetics, and attenuate diabetic cardiomyopathy in type 2 diabetic mice. Methods A non-selective adiponectin receptor agonist, AdipoRon, and vehicle were injected intraperitoneally into Eight-week-old db/db or C57BLKS/J mice for 10 days. Cardiac morphology and function were evaluated by echocardiography and working heart perfusions. Results Based on echocardiography, AdipoRon treatment did not alter ejection fraction, left ventricular diameters or left ventricular wall thickness in db/db mice compared to vehicle-treated mice. In isolated working hearts, an impairment in cardiac output and efficiency in db/db mice was not improved by AdipoRon. Mitochondrial respiratory capacity, respiration in the presence of oligomycin, and 4-hydroxynonenal levels were similar among all groups. However, AdipoRon induced a marked shift in the substrate oxidation pattern in db/db mice towards increased reliance on glucose utilization. In parallel, the diabetes-associated increase in serum triglyceride levels in vehicle-treated db/db mice was blunted by AdipoRon treatment, while an increase in myocardial triglycerides in vehicle-treated db/db mice was not altered by AdipoRon treatment. Conclusion AdipoRon treatment shifts myocardial substrate preference towards increased glucose utilization, likely by decreasing fatty acid delivery to the heart, but was not sufficient to improve cardiac output and efficiency in db/db mice

Atypical presentation of a locally advanced hepatocellular carcinoma: Extensive workup of an incidental finding on computed tomography performed for planning of transcatheter aortic valve implantation

This case report presents the diagnostic workup of liver malignancy incidentally detected in a 72-year-old male patient on routine body computed tomography angiography (CTA) performed for planning transcatheter aortic valve implantation (TAVI). The patient initially presented to an outside hospital with chest discomfort, where routine diagnostic procedures in the emergency room revealed grade III aortic valve stenosis. Routine CTA for TAVI planning in our department then revealed tumor thrombosis of the portal vein suspicious for hepatic malignancy. In contrast-enhanced ultrasound (CEUS) only the left hepatic lobe was inhomogeneously transformed with early arterial contrast enhancement. Magnetic resonance imaging (MRI) confirmed a primary hepatic malignancy involving the left liver. Transcutaneous biopsy with ultrasound guidance established the diagnosis of hepatocellular carcinoma (HCC). Incidental findings may be of prognostic relevance for the patient and in a number of cases, TAVI can be a prerequisite for the appropriate therapy

Growth factor requirements and growth curves.

(A) Expression (RT-PCR analysis) of mRNAs coding for EGF (epidermal growth factor), TGFα (transforming growth factor α), HB-EGF (Heparin-binding EGF-like growth factor), and bFGF (basic fibroblast growth factor). Bars depict the mean of a minimum of three replicates, whiskers the standard deviation. Significant differences relative to the expression in the T1338 cell line are indicated (**, pB) Effects of growth factor depletion on proliferation (BrdU ELISA). The combination of growth factors added into the medium is indicated by distinct levels of gray(white: EGF/bFGF; light grey: EGF; dark grey: bFGF; black: no growth factor added). The bars depict mean values and standard deviations. Significant reduction of BrdU incorporation is indicated by p-values (**, pC) Growth curves of the SLGC lines indicated were performed in the presence of EGF (red line), bFGF (black line) or both (blue line), or in the absence of growth factors (violet line). After six days (d6) cells were re-plated for studies with extended incubation times. Values for d9 were determined from both the original and the replated cultures. Significant differences between the growth curves at specific time points are indicated by p-values (*, pD) Growth factor ELISA. The amounts of EGF and bFGF present in T1371 and T1447 cultures were determined at days d2 and d9 after plating. The assays were performed in DMEM/Ham’s F12 containing fetal calf serum (10% FCS) or the serum supplements BIT (bovine albumin, insulin, and transferrin) and B27, respectively. The presence of exogenous growth factors is indicated by EGF/bFGF. Each assay encompassed a minimum of four replicates. Significant differences are indicated (**, p (TIF)</p

Expression of proteases and Pearson correlation.

(A) Comparison of the relative amounts of ADAM (a disintegrin and metalloprotease domain) proteases as well as BACE (beta-secretase) and MMP2 (matrix metallopeptidase 2) in the SLGC lines indicated (data from proteome array). Based on the assumption that the antibodies spotted on the filters of the proteome array possessed similar KDs, the relative expression of the proteases was calculated. The sum of the signals of all proteases was arbitrary set at 100%. The bars indicate to what percentage the individual proteases contribute to this expression. (B) Similar assay as in (A). The relative Cathepsin D levels were compared to the relative expression of all other proteases, depicted in panel (A). (C) Similar analysis as in (A) comparing the relative expression of the protease inhibitors TIMP (tissue inhibitor of metalloproteinases) 1, 2, 3 and 4.–Parts D and E graphically summarize some of the correlation data shown in Figs 8 and 9. In (B) the coefficients are indicated on the y-axis, highlighting expression levels with a positive correlation to the Sox2 (blue) or CD133 (violet) dots. In (C) the Pearson correlation coefficients were calculated relative to the levels of the neural proteins Tau and GFAP, as well as the hyaluronan receptor CD44, the integrin αv, and the N- and E-cadherin, respectively. In all cases, the expression levels that entered the calculations were determined with the same whole cell extracts. The calculations were confirmed with biological replicates. (TIF)</p

Expression of cadherins and CAMs (cell adhesion molecules).

(A) Comparison of the relative amounts of Cadherins expressed in the SLGC lines indicated (data from proteome arrays). Based on the assumption that the antibodies spotted on the filters of the proteome arrays possessed similar KDs, the relative expression of the cadherins was calculated. The sum of the signals of all cadherins was arbitrary set at 100%. The bars indicate to what percentage the individual cadherins contribute to this expression. C4, C11, and C13, cadherins 4, 11 and 13; E-C, E-cadherin; N-C, N-cadherin; P-C, placental cadherin; VE-C, VE-(vascular endothelial) cadherin/CD144. (B) Quantification of Western blot analyzes investigating the expression of N-cadherin in the SLGC lines indicated. Bars represent the mean expression normalized against Actin, whiskers represent the variation between technical replicates. (C) Similar experiment as in Fig 5 using biological replicates of the various SLGC lines (flow cytometry analysis). The established cell lines U87 (glioblastoma) and/or CaCo2 (colon carcinoma), which lack stemness were used as references. The antibodies bind to the VE-cadherin/CD144 and PECAM-1 (platelet endothelial cell adhesion molecule -1)/CD31, respectively. Bars depict the percentage of positive cells determined in one representative experiment. (D) Comparison of the relative amounts of CAMs expressed in the SLGC lines indicated (data from proteome arrays). The sum of the signals of all CAMs was arbitrary set at 100%. The bars indicate to what percentage the individual CAMs contribute to the total CAM expression. Cea-CAM-5 or -1, Carcinoembryonic antigen-related cell adhesion molecule -5 or -1; CHL-1, cell adhesion molecule L1-like; N-CAM, nerve cell adhesion molecule; VCAM, Vascular cell adhesion protein 1; PECAM, platelet endothelial cell adhesion molecule; ICAM-2, intercellular adhesion molecule 2; Neurotrimin, GPI-anchored cell adhesion molecule that might promote neurite outgrowth; NCAM-1, neural adhesion molecule-1; NCAM-L1, neural cell adhesion molecule L1; EpCAM, epithelial cell adhesion molecule. (TIF)</p

Expression analyses HER family.

(A) Left: Comparison of the relative amounts of members of the EGF-receptor/HER family (data from proteome arrays). Based on the assumption that the antibodies spotted on the filters of the proteome arrays possessed similar KDs, the relative expression was calculated. The sum of the signals from the four receptors was arbitrary set at 100%. The bars indicate to what percentage the individual HERs contribute to this expression. Right: similar comparison considering Epiregulin, HB-EGF, Amphiregulin, all of which may activate EGFR/HER1. (B) Western blot analysis of the expression of the proteins indicated in the blots. Upper panel of left: The full-length(fl) EGF (epidermal growth factor) receptor and truncated EGFR proteins are shown. Right: Similar blots using antibodies against the PDGF (platelet-derived growth factor) receptors α and β. Lower panel on left: comparison of GFAP and CD133 expression. (C) Relative expression of CDK4 and CDK6: expression was normalized against the corresponding Actin signals. The mean values of three independent biological replicates are shown; whiskers indicate variations. (D) RT-PCR analysis of p53 transcripts. p53* indicates that the PCR product is longer than usual due to a mutation in a splice site. The triangle indicates increasing passages (p2, p5, p9, p26, p35); gapdh served as a reference gene. (E) Western blot analysis of the expression of IDH1 (IDH1: cytoplasmic, 46.7 kDa), IDH2 (IDH2: mitochondrial isoform 1: 50.9 kDa, isoform 2: 45.2 kDa) and p53 in the SLGC lines indicated. The loading control GAPDH is shown below the respective blots. Brackets indicate that the analyses were performed using the same nitrocellulose filter.—GAPDH, glycerol aldehyde-3-phosphate dehydrogenase. (TIF)</p

Characteristics of SLGC lines and correlations.

Glioblastoma multiforme (GBM) and the GBM variant gliosarcoma (GS) are among the tumors with the highest morbidity and mortality, providing only palliation. Stem-like glioma cells (SLGCs) are involved in tumor initiation, progression, therapy resistance, and relapse. The identification of general features of SLGCs could contribute to the development of more efficient therapies. Commercially available protein arrays were used to determine the cell surface signature of eight SLGC lines from GBMs, one SLGC line obtained from a xenotransplanted GBM-derived SLGC line, and three SLGC lines from GSs. By means of non-negative matrix factorization expression metaprofiles were calculated. Using the cophenetic correlation coefficient (CCC) five metaprofiles (MPs) were identified, which are characterized by specific combinations of 7–12 factors. Furthermore, the expression of several factors, that are associated with GBM prognosis, GBM subtypes, SLGC differentiation stages, or neural identity was evaluated. The investigation encompassed 24 distinct SLGC lines, four of which were derived from xenotransplanted SLGCs, and included the SLGC lines characterized by the metaprofiles. It turned out that all SLGC lines expressed the epidermal growth factor EGFR and EGFR ligands, often in the presence of additional receptor tyrosine kinases. Moreover, all SLGC lines displayed a neural signature and the IDH1 wildtype, but differed in their p53 and PTEN status. Pearson Correlation analysis identified a positive association between the pluripotency factor Sox2 and the expression of FABP7, Musashi, CD133, GFAP, but not with MGMT or Hif1α. Spherical growth, however, was positively correlated with high levels of Hif1α, CDK4, PTEN, and PDGFRβ, whereas correlations with stemness factors or MGMT (MGMT expression and promoter methylation) were low or missing. Factors highly expressed by all SLGC lines, irrespective of their degree of stemness and growth behavior, are Cathepsin-D, CD99, EMMPRIN/CD147, Intβ1, the Galectins 3 and 3b, and N-Cadherin.</div

Proteins characterizing the metaprofiles.

For all proteins that are significant (>95% percentile) for at least one metaprofile (MP1-5), their decoding along all MPs is shown. Solid dots indicate those above 95%; the shallow dots are those above 90% (see also Fig 4A). Furthermore, the proteins are sorted to match their principal MPs. All proteins that can be assigned to exactly one MP are clustered, and these clusters are color coded (MP1: blue, MP2: red, MP3: orange, MP4: green, and MP5: violet). As no unique protein could be linked to MP1, we used for clustering those that have expression above 90% but below 95% for the other MPs. All other proteins are assigned to a minimum of two metaprofiles (e.g., Galectin 3, NCAM L1) or all metaprofiles (CD147).—For abbreviations, see the legend to Fig 3.</p

Expression of neuronal proteins, cell cycle regulators and ABCG2.

(A-C) Western blot analyzes: The loading control Actin is shown below the corresponding blots. Brackets indicate that the same nitrocellulose filter was used for the respective detections.—AADC/DDC, aromatic L-amino acid decarboxylase/DOPA decarboxylase [isoforms due to alternative transcript splicing: 53.9 kDa; 40–48 kDa; 37.1 kDa]; ABCG2, ATP-binding cassette subfamily G2 member 2; CDK4 and 6, cyclin-dependent kinases 4 and 6; DARPP32, 32 kDa dopamine and cyclic adenosine 3’,5’-monophosphate regulated phosphoprotein; NF, neurofilaments H, M and L [heavy polypeptide, 220 kDa; medium polypeptide, 160 kDa; light polypeptide, 68 kDa]; Pax 6, paired box protein 6 (isoform 1: 46.7 kDa; isoform 5a: 48.2 kDa; additional isoforms: molecular weight below 40 kDa); Tau, microtubule-associated protein Tau. SC, derived from orthotopic tumor.</p

Characterization of SLGC lines.

(A) Immunocytochemistry analyzes: primary antibodies indicated in the figure were revealed with goat anti-mouse DyLight®488 (green) and goat anti-rabbit Cy3 (red), respectively. Except for T1389, all microphotographs are z-stacks. DAPI (4′,6-diamidino-2-phenylindole) nuclear counterstain (blue) is shown. Bars, 50 μm. CD15, Stage-Specific Embryonic Antigen1 (SSEA1); CD133, Prominin-1; GFAP, glial fibrillary acidic protein; Nestin, type IV intermediate filament; Sox2, SRY (sex determining region Y)-box transcription factor 2; Vimentin, type III intermediate filament.—(B) Capacity of SLGCs to generate orthotopic tumors in the SCID mouse model. HE (hematoxylin and Eosin) staining of 4.5 μm slices. The SLGCs were inoculated into the right hemisphere. Arrows point to the inoculation site, and arrowheads mark tumor borders. Bars, 50 μm; SC, cell line derived from an orthotopic tumor.—(C) Coronal T2-weighted MRI scan of SCID mice 6 weeks after inoculation with T1447 and 6 months after inoculation with T1338 cells. The left panel of MRI images depicts distinct sections of the same orthotopic tumor. The MRI of the T1338-inoculated mouse did not show any signs of tumor formation in the entire scan.—(D) Immunohistochemistry analysis of two SCID mouse brains (a, b) inoculated with T1338 cells. Triple stain with the antibody pairs rabbit-Sox2/goat-anti-rabbit-Cy3 and mouse-Stem-121/goat-anti-mouse-DyLight® 488, followed by nuclear counterstain with DAPI. Staining of 4.5 μm slices was done 12 months after xenotransplantation. White arrows point to Sox2-positive T1338 cells, red arrow heads to erythrocytes. Bars, 50 μm. (TIF)</p