Isabel M. Mol

Brown fat activation reduces hypercholesterolaemia and protects from atherosclerosis development

Brown adipose tissue (BAT) combusts high amounts of fatty acids, thereby lowering plasma triglyceride levels and reducing obesity. However, the precise role of BAT in plasma cholesterol metabolism and atherosclerosis development remains unclear. Here we show that BAT activation by β3-adrenergic receptor stimulation protects from atherosclerosis in hyperlipidemic APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism that unlike hyperlipidemic Apoe−/− and Ldlr−/− mice expresses functional apoE and LDLR. BAT activation increases energy expenditure and decreases plasma triglyceride and cholesterol levels. Mechanistically, we demonstrate that BAT activation enhances the selective uptake of fatty acids from triglyceride-rich lipoproteins into BAT, subsequently accelerating the hepatic clearance of the cholesterol-enriched remnants. These effects depend on a functional hepatic apoE-LDLR clearance pathway as BAT activation in Apoe−/− and Ldlr−/− mice does not attenuate hypercholesterolaemia and atherosclerosis. We conclude that activation of BAT is a powerful therapeutic avenue to ameliorate hyperlipidaemia and protect from atherosclerosis.

Targeting integrins and enhanced permeability and retention (EPR) effect for optical imaging of oral cancer.

Near‐infrared (NIR) fluorescence optical imaging is a promising technique to assess the tumor margins during cancer surgery. This technique requires targeting by specific fluorescence agents to differentiate tumor from normal surrounding tissue. We assessed the feasibility of cancer detection using NIR fluorescence agents that target either αvβ3 integrins or the enhanced permeability and retention (EPR) effect in an orthotopic mouse model of oral cancer.

Optical imaging of oral squamous cell carcinoma and cervical lymph node metastasis

In oral cancer surgery, intraoperative optical imaging could help the surgeon to determine adequate tumor‐free margins.

Dual-wavelength imaging of tumor progression by activatable and targeting near-infrared fluorescent probes in a bioluminescent breast cancer model.

Bioluminescence imaging (BLI) has shown its appeal as a sensitive technique for in vivo whole body optical imaging. However, the development of injectable tumor-specific near-infrared fluorescent (NIRF) probes makes fluorescence imaging (FLI) a promising alternative to BLI in situations where BLI cannot be used or is unwanted (e.g., spontaneous transgenic tumor models, or syngeneic mice to study immune effects). In this study, we addressed the questions whether it is possible to detect tumor progression using FLI with appropriate sensitivity and how FLI correlates with BLI measurements. In addition, we explored the possibility to simultaneously detect multiple tumor characteristics by dual-wavelength FLI (∼700 and ∼800 nm) in combination with spectral unmixing. Using a luciferase-expressing 4T1-luc2 mouse breast cancer model and combinations of activatable and targeting NIRF probes, we showed that the activatable NIRF probes (ProSense680 and MMPSense680) and the targeting NIRF probes (IRDye 800CW 2-DG and IRDye 800CW EGF) were either activated by or bound to 4T1-luc2 cells. In vivo, we implanted 4T1-luc2 cells orthotopically in nude mice and were able to follow tumor progression longitudinally both by BLI and dual-wavelength FLI. We were able to reveal different probe signals within the tumor, which co-localized with immuno-staining. Moreover, we observed a linear correlation between the internal BLI signals and the FLI signals obtained from the NIRF probes. Finally, we could detect pulmonary metastases both by BLI and FLI and confirmed their presence histologically. Taken together, these data suggest that dual-wavelength FLI is a feasible approach to simultaneously detect different features of one tumor and to follow tumor progression with appropriate specificity and sensitivity. This study may open up new perspectives for the detection of tumors and metastases in various experimental models and could also have clinical applications, such as image-guided surgery.

PXR agonism decreases plasma HDL levels in ApoE*3-Leiden.CETP mice

Pregnane X receptor (PXR) agonism has been shown to affect multiple steps in both the synthesis and catabolism of HDL, but its integrated effect on HDL metabolism in vivo remains unclear. The aim of this study was to evaluate the net effect of PXR agonism on HDL metabolism in ApoE3-Leiden (E3L) and E3L.CETP mice, well-established models for human-like lipoprotein metabolism. Female mice were fed a diet with increasing amounts of the potent PXR agonist 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN). In E3L and E3L.CETP mice, PCN increased liver lipids as well as plasma cholesterol and triglycerides. However, whereas PCN increased cholesterol contained in large HDL-1 particles in E3L mice, it dose-dependently decreased HDL-cholesterol in E3L.CETP mice, indicating that CETP expression dominates the effect of PCN on HDL metabolism. Analysis of the hepatic expression of genes involved in HDL metabolism showed that PCN decreased expression of genes involved in HDL synthesis (Abca1, Apoa1), maturation (Lcat, Pltp) and clearance (Sr-b1). The HDL-increasing effect of PCN, observed in E3L mice, is likely caused by a marked decrease in hepatic SR-BI protein expression, and completely reversed by CETP expression. We conclude that chronic PXR agonism dose-dependently reduces plasma HDL-cholesterol in the presence of CETP.

Genome-wide coexpression of steroid receptors in the mouse brain: Identifying signaling pathways and functionally coordinated regions

Significance Steroid hormones coordinate the activity of many brain regions by binding to nuclear receptors that act as transcription factors. This study uses genome-wide correlation of gene expression in the mouse brain to discover (i) brain regions that respond in a similar manner to particular steroids, (ii) signaling pathways that are used in a steroid receptor and brain region-specific manner, and (iii) potential target genes and relationships between groups of target genes. The data constitute a rich repository for the research community to support further new insights in neuroendocrine relationships and to develop novel ways to manipulate brain activity in research or clinical settings. Steroid receptors are pleiotropic transcription factors that coordinate adaptation to different physiological states. An important target organ is the brain, but even though their effects are well studied in specific regions, brain-wide steroid receptor targets and mediators remain largely unknown due to the complexity of the brain. Here, we tested the idea that novel aspects of steroid action can be identified through spatial correlation of steroid receptors with genome-wide mRNA expression across different regions in the mouse brain. First, we observed significant coexpression of six nuclear receptors (NRs) [androgen receptor (Ar), estrogen receptor alpha (Esr1), estrogen receptor beta (Esr2), glucocorticoid receptor (Gr), mineralocorticoid receptor (Mr), and progesterone receptor (Pgr)] with sets of steroid target genes that were identified in single brain regions. These coexpression relationships were also present in distinct other brain regions, suggestive of as yet unidentified coordinate regulation of brain regions by, for example, glucocorticoids and estrogens. Second, coexpression of a set of 62 known NR coregulators and the six steroid receptors in 12 nonoverlapping mouse brain regions revealed selective downstream pathways, such as Pak6 as a mediator for the effects of Ar and Gr on dopaminergic transmission. Third, Magel2 and Irs4 were identified and validated as strongly responsive targets to the estrogen diethylstilbestrol in the mouse hypothalamus. The brain- and genome-wide correlations of mRNA expression levels of six steroid receptors that we provide constitute a rich resource for further predictions and understanding of brain modulation by steroid hormones.

Dual wavelength tumor targeting for detection of hypopharyngeal cancer using near-infrared optical imaging in an animal model

Optical imaging is a promising technique to visualize cancer tissue during surgery. In this study, we explored the use of combinations of near‐infrared (NIR) fluorescence agents that emit fluorescence signal at different wavelengths and each target specific tumor characteristics. Two combinations of agents (ProSense680 combined with 2DG CW800 and MMPSense680 combined with EGF CW800) were used to detect hypopharyngeal cancer in an animal model. ProSense680 and MMPSense680 detect increased activity of cathepsins and matrix metalloproteinases, respectively. These enzymes are mainly found in the invasive tumor border due to degradation of the extracellular matrix. 2DG CW800 detects tumor cells with high glucose metabolism and EGF CW800 is internalized by the epidermal growth factor receptor of tumor cells. Whole‐body imaging revealed clear demarcation of tumor tissue using all four agents. The tumor‐to‐background ratio (standard deviation, p‐value) was 3.69 (0.72, p < 0.001) for ProSense680; 4.26 (1.33, p < 0.001) for MMPSense680; 5.81 (3.59, p = 0.02) for 2DG CW800 and 4.84 (1.56, p < 0.001) for EGF CW800. Fluorescence signal corresponded with histopathology and immunohistochemistry, demonstrating signal of ProSense680 and MMPSense680 in the invasive tumor border, and signal of 2DG CW800 and EGF CW800 in the tumor tissue. In conclusion, we demonstrated the feasibility of dual wavelength tumor detection using different targeting strategies simultaneously in an animal model. Combined targeting at different wavelengths allowed simultaneous imaging of different tumor characteristics. NIR fluorescence optical imaging has the potential to be translated into the clinic in order to improve the complete removal of tumors by real‐time image‐guided surgery.

Resveratrol protects against atherosclerosis, but does not add to the antiatherogenic effect of atorvastatin, in APOE*3-Leiden.CETP mice

Resveratrol is a major constituent of traditional Asian medicinal herbs and red wine and is suggested to be a potential antiatherosclerotic drug due to its proposed hypolipidemic, anti-inflammatory and antioxidative properties. The aim of this study was to evaluate whether resveratrol protects against atherosclerosis development in APOE*3-Leiden.CETP (E3L.CETP) mice and adds to the antiatherogenic effect of mild statin treatment, currently the most widely used antiatherogenic therapy. E3L.CETP mice were fed a cholesterol-rich diet without (control) or with resveratrol (0.01% w/w), atorvastatin (0.0027% w/w) or both for 14 weeks. During the study plasma lipid, inflammatory and oxidative stress parameters were determined. Resveratrol reduced atherosclerotic lesion area (-52%) in the aortic root, comparable to atorvastatin (-40%) and the combination of both drugs (-47%). The collagen/macrophage ratio in the atherosclerotic lesion, a marker of plaque stability, was increased by resveratrol (+108%), atorvastatin (+124%) and the combination (+154%). Resveratrol decreased plasma cholesterol levels (-19%) comparable to atorvastatin (-19%) and the combination (-22%), which was completely confined to (very)low-density lipoprotein cholesterol levels in all groups. Post hoc analyses showed that the antiatherogenic effect of atorvastatin could be explained by cholesterol lowering, while the antiatherosclerotic effect of resveratrol could be attributed to factors additional to cholesterol lowering. Markers of inflammation and oxidative stress were not different, but resveratrol improved macrophage function. We conclude that resveratrol potently reduces atherosclerosis development and induces a more stable lesion phenotype in E3L.CETP mice. However, under the experimental conditions tested, resveratrol does not add to the antiatherogenic effect of atorvastatin.

Detection of Oral Squamous Cell Carcinoma and Cervical Lymph Node Metastasis Using Activatable Near-Infrared Fluorescence Agents

OBJECTIVE To assess the feasibility of optical imaging using activatable near-infrared fluorescence (NIRF) agents to detect oral cancer and cervical lymph node metastasis in vivo. DESIGN In vivo study. SETTING University medical center. SUBJECTS Female nude mice aged 4 to 6 weeks. INTERVENTION Luciferase-expressing OSC-19-luc cells were injected into the tongues of nude mice. A control group of nude mice was injected in the tongue with a physiologic saline solution. Tumor growth was followed by bioluminescence imaging. After 3 weeks, animals were randomly allocated to intravenous administration of 1 of 2 activatable NIRF agents: ProSense680 or MMPSense680. Fluorescence imaging of the mice was performed, and the tumor to background ratio (TBR) was determined on histologic sections of the tongue and cervical lymph nodes after resection at necropsy. MAIN OUTCOME MEASURE Fluorescence signals. RESULTS The fluorescence signals in tongue tumor and cervical lymph node metastases were significantly higher than those in control animals. The mean (SD) TBR of ProSense680 in the tongue was 15.8 (8.1) and in the lymph nodes was 11.8 (3.6). For MMPSense680, the mean (SD) TBR in the tongue was 18.6 (9.4) and in the lymph nodes was 10.5 (4.0). CONCLUSIONS Oral cancer and cervical lymph node metastases can be detected by targeting increased proteolytic activity at the tumor borders using NIRF optical imaging. These NIRF agents could be used for real-time image-guided surgery, which has the potential to improve the complete surgical resection of oral cancer.

Spatial and temporal expression of immunoglobulin superfamily member 1 in the rat

Loss-of-function mutations in the immunoglobulin superfamily member 1 (IGSF1) gene cause an X-linked syndrome of central hypothyroidism, macroorchidism, variable prolactin and GH deficiency, delayed pubertal testosterone rise, and obesity. To understand the pathophysiology of this syndrome, knowledge on IGSF1s place in normal development is imperative. Therefore, we investigated spatial and temporal protein and mRNA expression of IGSF1 in rats using immunohistochemistry, real-time quantitative PCR (qPCR), and in situ hybridization. We observed high levels of IGSF1 expression in the brain, specifically the embryonic and adult choroid plexus and hypothalamus (principally in glial cells), and in the pituitary gland (PIT1-lineage of GH, TSH, and PRL-producing cells). IGSF1 is also expressed in the embryonic and adult zona glomerulosa of the adrenal gland, islets of Langerhans of the pancreas, and costameres of the heart and skeletal muscle. IGSF1 is highly expressed in fetal liver, but is absent shortly after birth. In the adult testis, IGSF1 is present in Sertoli cells (epithelial stages XIII-VI), and elongating spermatids (stages X-XII). Specificity of protein expression was corroborated with Igsf1 mRNA expression in all tissues. The expression patterns of IGSF1 in the pituitary gland and testis are consistent with the pituitary hormone deficiencies and macroorchidism observed in patients with IGSF1 deficiency. The expression in the brain, adrenal gland, pancreas, liver, and muscle suggest IGSF1s function in endocrine physiology might be more extensive than previously considered.