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Tumor hypoxia has been shown to have prognostic value in clinical trials involving radiation, chemotherapy, and surgery. Tumor oxygenation studies at microvascular levels can provide understanding of oxygen transport on scales at which oxygen transfer to tissue occurs. To fully grasp the significance of blood oxygen delivery and hypoxia at microvascular levels during tumor growth and angiogenesis, the spatial and temporal relationship of the data must be preserved and mapped. Using tumors grown in window chamber models, hyperspectral imaging can provide serial spatial maps of blood oxygenation in terms of hemoglobin saturation at the microvascular level. We describe our application of hyperspectral imaging for in vivo microvascular tumor oxygen transport studies using red fluorescent protein (RFP) to identify all tumor cells, and hypoxia-driven green fluorescent protein (GFP) to identify the hypoxic fraction. 4T1 mouse mammary carcinoma cells, stably transfected with both reporter genes, are grown in dorsal skin-fold window chambers. Hyperspectral imaging is used to create image maps of hemoglobin saturation, and classify image pixels where RFP alone is present (tumor cells), or both RFP and GFP are present (hypoxic tumor cells). In this work, in vivo calibration of the imaging system is described and in vivo results are shown.

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Hyperactivity of the axis ACE/AngII/AT1R of the renin‐angiotensin system is associated with occurrence of acute thrombotic event. Recently a novel concept of a counterrugulatory axis, ACE2/Ang‐(1‐7)/Mas, has emerged. We hypothesized that ACE2 would be protective against thrombosis. Thrombus was induced in the vena cava of SHR and WKY rats by FeCl3 solution. ACE2 and ACE protein expression and activities in the thrombus were determined by Western blot and fluorogenic kinetic assays, respectively. Real time thrombus formation was visualized by intravital microscopy of the vessels of nude mice. Ferric chloride‐induced thrombus weight was 40% higher in the SHR compared to WKY rats. This was associated with a 20% decreased in ACE2 activity in the thrombus of the SHR. In contrast, ACE2 protein expression and ACE activity did not differ between the thrombus of WKY rats and SHR. Inhibition of ACE2 by DX600 increased the thrombus weight by 30%, preferentially in the SHR. Furthermore, treatment with XNT resulted in a 30% attenuation of thrombus formation in both the SHR and WKY. In addition, XNT treatment prolonged the time for complete vessel occlusion and reduced thrombus size when observed under real‐time intravital microscopy. Our data demonstrated that a decrease in ACE2 activity is associated with increased thrombus formation in the SHR. Furthermore, activation of ACE2 attenuates thrombus formation.

In this paper we report the design, testing and use of a scannerless probe specifically for minimally invasive imaging of deep tissue in vivo with an epi-fluorescence modality. The probe images a 500 µm diameter field of view through a 710 µm outer diameter probe with a maximum tissue penetration depth of 15 mm specifically configured for eGFP imaging. Example results are given from imaging the pituitary gland of rats and zebrafish hearts with lateral resolution of 2.5 µm., "Real-time visualization of human prolactin alternate promoter usage in vivo using a double-transgenic rat model," Mol. Endocrinol.Fast handheld two-photon fluorescence microendoscope with a 475 μm × 475 μm field of view for in vivo imaging," Opt.

In epithelial cells, alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts leads to the expression of the FGFR2(IIIb) isoform, whereas in mesenchymal cells, the same process results in the synthesis of FGFR2(IIIc). Expression of the FGFR2(IIIc) isoform during prostate tumor progression suggests a disruption of the epithelial character of these tumors. To visualize the use of FGFR2 exon IIIc in prostate AT3 tumors in syngeneic rats, we constructed minigene constructs that report on alternative splicing. Imaging these alternative splicing decisions revealed unexpected mesenchymal-epithelial transitions in these primary tumors. These transitions were observed more frequently where tumor cells were in contact with stroma. Indeed, these transitions were frequently observed among lung micrometastases in the organ parenchyma and immediately adjacent to blood vessels. Our data suggest an unforeseen relationship between epithelial mesenchymal plasticity and malignant fitness.alternative splicing ͉ mesenchymal-epithelial transitions ͉ tumor plasticity R egulation of alternative splicing is essential for normal gene expression (1), and alterations of this regulation are linked to disease (2), as illustrated by the association between cancer and splicing defects (3-7). An elegant example of this is provided by the splicing of transcripts encoding fibroblast growth factor receptor 2 (FGFR2). The status of FGFR2 alternative splicing depends on the interplay between several cis-acting elements in the FGFR2 premRNA and transacting factors, some of which are cell-type-specific (8). In mesenchymal cells, exon IIIb is silenced by the action of an exonic splicing silencer (9) and two flanking intronic splicing silencers (10-12). This silencing is mediated by Polypyrimidine tract-binding protein (PTB), hnRNP A1, and heretofore unknown factors. In epithelial cells, exon IIIb silencing is countered by several intronic elements. The best characterized are the intronic activating sequence 2, the intronic splicing activator and repressor (ISAR, also known as IAS3), and several GCAUG repeats (13-18). These elements have a dual function in epithelial cells, because they are also involved in silencing exon IIIc (14-18).FGFR2 splicing has been studied in tumors derived from the R-3327 Dunning rat prostate tumor, which arose spontaneously from the dorsal lobe of the prostate in a Copenhagen rat (19). Some R-3327-derived tumors (DT or DT3) express FGFR2(IIIb), which is consistent with their epithelial phenotype (20), whereas AT tumors (e.g., AT3), which have lost epithelial markers and display many mesenchymal indicators (21), express FGFR2(IIIc) (20). The significance of these alternative decisions for tumor behavior is underscored by the fact that forced expression of FGFR2(IIIb) suppresses tumor progression of AT3 tumors (22). Most importantly, however, the differential splicing of FGFR2 transcripts in these two cell types highlights broad differences in gene expression programs. Arguably, monitoring alternative sp...

Arteriovenous malformation (AVM) refers to a vascular anomaly where arteries and veins are directly connected through a complex, tangled web of abnormal AV fistulae without a normal capillary network. Hereditary hemorrhagic telangiectasia (HHT) types 1 and 2 arise from heterozygous mutations in endoglin (ENG) and activin receptor-like kinase 1 (ALK1), respectively. HHT patients possess AVMs in various organs, and telangiectases (small AVMs) along the mucocutaneous surface. Understanding why and how AVMs develop is crucial for developing therapies to inhibit the formation, growth, or maintenance of AVMs in HHT patients. Previously, we have shown that secondary factors such as wounding are required for Alk1-deficient vessels to develop skin AVMs. Here we present evidences that AVMs establish from nascent arteries and veins rather than from remodeling of a preexistent capillary network in the wound-induced skin AVM model. We also show that VEGF can mimic the wound effect on skin AVM formation, and VEGF neutralizing antibody can prevent skin AVM formation and ameliorate internal bleeding in Alk1-deficient adult mice. With topical applications at different stages of AVM development, we demonstrate that the VEGF blockade can prevent the formation of AVM and cease the progression of AVM development. Taken together, the presented experimental model is an invaluable system for precise molecular mechanism of action of VEGF blockades as well as for preclinical screening of drug candidates for epistaxis and gastrointestinal bleedings.

In response to sustained increase in contractile activity, mammalian skeletal muscle undergoes adaptation with enhanced mitochondrial biogenesis and fiber type switching. The peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was recently identified as a key regulator for these adaptive processes. To investigate the sequence elements in the PGC-1alpha gene that are responsible for activity-dependent transcriptional activation, we have established a unique system to analyze promoter activity in skeletal muscle of living mice. Expression of PGC-1alpha-firefly luciferase reporter gene in mouse tibialis anterior muscle transfected by electric pulse-mediated gene transfer was assessed repeatedly in the same muscle by using optical bioluminescence imaging analysis before and after low-frequency (10 Hz) motor nerve stimulation. Nerve stimulation (2 h) resulted in a transient 3-fold increase (P < 0.05) in PGC-1alpha promoter activity along with a 1.6-fold increase (P < 0.05) in endogenous PGC-1alpha mRNA. Mutation of two consensus myocyte enhancer factor 2 (MEF2) binding sites (-2901 and -1539) or a cAMP response element (CRE) (-222) completely abolished nerve stimulation-induced increase in PGC-1alpha promoter activity. These findings provide direct evidence that contractile activity-induced PGC-1alpha promoter activity in skeletal muscle is dependent on the MEF2 and the CRE sequence elements. The experimental methods used in the present study have general applicability to studies of gene regulation in muscle.