Insulin Resistance in Vascular Endothelial Cells Promotes Intestinal Tumor Formation

The risk of several cancers, including colorectal cancer, is increased in patients with obesity and type 2 diabetes, conditions characterized by hyperinsulinemia and insulin resistance. Because hyperinsulinemia itself is an independent risk factor for cancer development, we examined tissue-specific insulin action in intestinal tumor formation. In vitro, insulin increased proliferation of primary cultures of intestinal tumor epithelial cells from ApcMin/+ mice by over 2-fold. Surprisingly, targeted deletion of insulin receptors in intestinal epithelial cells in ApcMin/+ mice did not change intestinal tumor number or size distribution on either a low or high-fat diet. We therefore asked whether cells in the tumor stroma might explain the association between tumor formation and insulin resistance. To this end, we generated ApcMin/+ mice with loss of insulin receptors in vascular endothelial cells. Strikingly, these mice had 42% more intestinal tumors than controls, no change in tumor angiogenesis, but increased expression of vascular cell adhesion molecule-1 (VCAM-1) in primary culture of tumor endothelial cells. Insulin decreased VCAM-1 expression and leukocyte adhesion in quiescent tumor endothelial cells with intact insulin receptors and partly prevented increases in VCAM-1 and leukocyte adhesion after treatment with tumor necrosis factor-α. Knockout of insulin receptors in endothelial cells also increased leukocyte adhesion in mesenteric venules and increased the frequency of neutrophils in tumors. We conclude that although insulin is mitogenic for intestinal tumor cells in vitro, its action on tumor cells in vivo is via signals from the tumor microenvironment. Insulin resistance in tumor endothelial cells produces an activated, proinflammatory state that promotes tumorigenesis. Improvement of endothelial dysfunction may reduce colorectal cancer risk in patients with obesity and type 2 diabetes

Determining Plasma Protein Variation Parameters as a Prerequisite for Biomarker Studies—A TMT-Based LC-MSMS Proteome Investigation

Specific plasma proteins serve as valuable markers for various diseases and are in many cases routinely measured in clinical laboratories by fully automated systems. For safe diagnostics and monitoring using these markers, it is important to ensure an analytical quality in line with clinical needs. For this purpose, information on the analytical and the biological variation of the measured plasma protein, also in the context of the discovery and validation of novel, disease protein biomarkers, is important, particularly in relation to for sample size calculations in clinical studies. Nevertheless, information on the biological variation of the majority of medium-to-high abundant plasma proteins is largely absent. In this study, we hypothesized that it is possible to generate data on inter-individual biological variation in combination with analytical variation of several hundred abundant plasma proteins, by applying LC-MS/MS in combination with relative quantification using isobaric tagging (10-plex TMT-labeling) to plasma samples. Using this analytical proteomic approach, we analyzed 42 plasma samples prepared in doublets, and estimated the technical, inter-individual biological, and total variation of 265 of the most abundant proteins present in human plasma thereby creating the prerequisites for power analysis and sample size determination in future clinical proteomics studies. Our results demonstrated that only five samples per group may provide sufficient statistical power for most of the analyzed proteins if relative changes in abundances >1.5-fold are expected. Seventeen of the measured proteins are present in the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Biological Variation Database, and demonstrated remarkably similar biological CV’s to the corresponding CV’s listed in the EFLM database suggesting that the generated proteomic determined variation knowledge is useful for large-scale determination of plasma protein variations

Localization of microfibrillar-associated protein 4 (MFAP4) in human tissues: clinical evaluation of serum MFAP4 and its association with various cardiovascular conditions.

Microfibrillar-associated protein 4 (MFAP4) is located in the extracellular matrix (ECM). We sought to identify tissues with high levels of MFAP4 mRNA and MFAP4 protein expression. Moreover, we aimed to evaluate the significance of MFAP4 as a marker of cardiovascular disease (CVD) and to correlate MFAP4 with other known ECM markers, such as fibulin-1, osteoprotegerin (OPG), and osteopontin (OPN). Quantitative real-time PCR demonstrated that MFAP4 mRNA was more highly expressed in the heart, lung, and intestine than in other elastic tissues. Immunohistochemical studies demonstrated high levels of MFAP4 protein mainly at sites rich in elastic fibers and within blood vessels in all tissues investigated. The AlphaLISA technique was used to determine serum MFAP4 levels in a clinical cohort of 172 patients consisting of 5 matched groups with varying degrees of CVD: 1: patients with ST elevation myocardial infarction (STEMI), 2: patients with non-STEMI, 3: patients destined for vascular surgery because of various atherosclerotic diseases (stable atherosclerotic disease), 4: apparently healthy individuals with documented coronary artery calcification (CAC-positive), and 5: apparently healthy individuals without signs of coronary artery calcification (CAC-negative). Serum MFAP4 levels were significantly lower in patients with stable atherosclerotic disease than CAC-negative individuals (p<0.05). Furthermore, lower serum MFAP4 levels were present in patients with stable atherosclerotic disease compared with STEMI and non-STEMI patients (p<0.05). In patients with stable atherosclerotic disease, positive correlations between MFAP4 and both fibulin-1 (ρ = 0.50; p = 0.0244) and OPG (ρ = 0.62; p = 0.0014) were found. Together, these results indicate that MFAP4 is mainly located in elastic fibers and is highly expressed in blood vessels. The present study suggests that serum MFAP4 varies in groups of patients with different cardiovascular conditions. Further studies are warranted to describe the role of serum MFAP4 as a biomarker of stable atherosclerotic disease

Immunohistochemical staining of MFAP4 in the basal membrane of the testis (A) and prostate (F) and in the spiral arteries of the uterus (C, E).

<p>Tissues were stained using the monoclonal anti-MFAP4 (HG-HYB 7–14) antibody and counterstained with Mayeŕs hematoxylin. Negative control sections with omission of the monoclonal anti-MFAP4 (HG-HYB 7–14) antibody in the prostate (B) and in the uterus (D). <i>Scale bars:</i> (A, B) 200 µm, (C, D, F) 100 µm and (E) 20 µm.</p