Dynamics of angiogenesis during wound healing: a coupled in vivo and in silico study.

OBJECTIVE: The most critical determinant of restoration of tissue structure during wound healing is the re-establishment of a functional vasculature, which largely occurs via angiogenesis, specifically endothelial sprouting from the pre-existing vasculature. MATERIALS AND METHODS: We used confocal microscopy to capture sequential images of perfused vascular segments within the injured panniculus carnosus muscle in the mouse dorsal skin-fold window chamber to quantify a range of microcirculatory parameters during the first nine days of healing. This data was used to inform a mathematical model of sequential growth of the vascular plexus. The modeling framework mirrored the experimental circular wound domain and incorporated capillary sprouting and endothelial cell (EC) sensing of vascular endothelial growth factor gradients. RESULTS: Wound areas, vessel densities and vessel junction densities obtained from the corresponding virtual wound were in excellent agreement both temporally and spatially with data measured during the in vivo healing process. Moreover, by perturbing the proliferative ability of ECs in the mathematical model, this leads to a severe reduction in vascular growth and poor healing. Quantitative measures from this second set of simulations were found to correlate extremely well with experimental data obtained from animals treated with an agent that targets endothelial proliferation (TNP-470). CONCLUSION: Our direct combination and comparison of in vivo longitudinal analysis (over time in the same animal) and mathematical modeling employed in this study establishes a useful new paradigm. The virtual wound created in this study can be used to investigate a wide range of experimental hypotheses associated with wound healing, including disorders characterized by aberrant angiogenesis (e.g., diabetic models) and the effects of vascular enhancing/disrupting agents or therapeutic interventions such as hyperbaric oxygen.

Cytochrome P450 1B1 mRNA untranslated regions interact to inhibit protein translation.

CYP1B1 mRNA is expressed constitutively in all normal extrahepatic human tissues, though the protein is usually undetectable. In contrast, CYP1B1 protein is expressed at high levels in tumors. In this study CYP1B1 mRNA and protein expression was measured in a panel of cell lines indicating that CYP1B1 regulation is altered in tumor cell lines in vitro. Interrogation of ONCOMINE revealed that CYP1B1 mRNA is not significantly overexpressed in tumors compared to normal tissues, suggesting CYP1B1 is subject to posttranscriptional control. Analysis of the CYP1B1 mRNA revealed a complex 5' untranslated region (UTR) containing a small upstream open-reading frame (uORF). These features are present in mRNAs subject to translational control so the effect of the 5'UTR was tested using in vitro translation in CHO-K1 cells. The 5'UTR significantly inhibited luciferase reporter gene translation, and mutation of the uORF start codon abolished the inhibitory effect. The 5'UTR also interacted with the microRNA-27b recognition element in the CYP1B1 mRNA 3'UTR to almost completely inhibit translation. CYP1B1 is subject to a high degree of translational control, which may explain the absence of protein expression in normal cells. Alterations in translational control during malignant transformation may help to explain the tumor-specific expression of CYP1B1 protein.

Synchrotron X-ray microtomography for assessment of bone tissue scaffolds.

X-ray microtomography (microCT) is a popular tool for imaging scaffolds designed for tissue engineering applications. The ability of synchrotron microCT to monitor tissue response and changes in a bioactive glass scaffold ex vivo were assessed. It was possible to observe the morphology of the bone; soft tissue ingrowth and the calcium distribution within the scaffold. A second aim was to use two newly developed compression rigs, one designed for use inside a laboratory based microCT machine for continual monitoring of the pore structure and crack formation and another designed for use in the synchrotron facility. Both rigs allowed imaging of the failure mechanism while obtaining stress-strain data. Failure mechanisms of the bioactive glass scaffolds were found not to follow classical predictions for the failure of brittle foams. Compression strengths were found to be 4.5-6 MPa while maintaining an interconnected pore network suitable for tissue engineering applications.

Cytochrome P450 1B1 expression in rat esophageal tumorigenesis promoted by gastric and duodenal reflux.

Cytochrome P450 1B1 (CYP1B1) mRNA is constitutively expressed in most normal extra-hepatic tissues; however the protein is not detectable in these tissues but is expressed in a wide variety of tumors. CYP1B1 is responsible for the activation of a number of carcinogens present in tobacco smoke and food. A surgical model of rat esophageal tumorigenesis, promoted by gastric or duodenal reflux was used to determine CYP1B1 expression in premalignant esophageal tissue. Immunohistochemistry was performed using a modified amplified fluorescein tyramide protocol. CYP1B1 was not observed in normal esophageal mucosa, submucosa, or muscularis mucosa. Animals exposed to gastric reflux developed mild hyperplasia. Varying degrees of hyperplasia were observed in the duodenal reflux group. All regions of hyperplasia showed moderate or strong CYP1B1 immunoreactivity. Duodenal reflux induced a small number of premalignant changes: immunoreactivity was absent from the epithelium of squamous dysplasia (0/10), Barrett's esophagus (0/7), and majority of dysplastic Barrett's esophagus (1/4). Moderate or strong immunoreactivity was observed in the majority (7/8) of squamous cell carcinomas (SCCs) in situ. Immunoreactivity was also observed in the lamina propria and submucosa in association with inflammation, regardless of the severity of inflammation. The expression of CYP1B1 in hyperplasia, SCCs in situ, or in association with inflammation may increase the production of carcinogenic metabolites, which may promote esophageal tumorigenesis.

Investigation of pericytes, hypoxia, and vascularity in bladder tumors: association with clinical outcomes.

The contribution of endothelial cell growth to angiogenesis has been widely studied; however, the involvement of pericytes is less well documented, especially in human tumors. In this study we aimed to quantify and assess the prognostic significance of pericyte coverage, the extent of hypoxia, and microvessel density (MVD) in normal bladder mucosa and urothelial carcinoma. Antibody to alpha-smooth muscle actin was used to assess the distribution of pericytes (mural/smooth muscle cells) in the microvessels of normal human bladder (n = 4) mucosa and in urothelial carcinoma (n = 47) samples; this was quantitated using microvessel pericyte index (MPI). The MVD was measured using two different methods (n = 47) and hypoxia was assessed using glucose transporter-1 (Glut-1) staining (n = 30). There was a 70% reduction in MPI in urothelial carcinomas compared to normal bladder mucosa (p < 0.0012); MPI did not correlate with tumor stage or grade. Ta and T1 superficial tumors were divided into two groups with a MPI of <15% or >15%. Progression-free survival was significantly shorter for tumors with MPI >15% (p = 0.0036). MVD had no prognostic value using either evaluation method. Glut-1 immunoreactivity was not prognostic in superficial urothelial carcinoma samples. Tumors with a higher MPI showed a greater Glut-1 immunoreactivity (p = 0.0051). Microvessels in urothelial carcinoma have a considerable loss of pericyte coverage compared to normal bladder mucosa. The data from this preliminary study indicate that progression-free survival was shorter in patients whose superficial tumors had higher pericyte coverage of the microvessels. This may be due to increased levels of hypoxia, as demonstrated by a significant increase in Glut-1 staining.