Durable engraftment of genetically modified FVIII-secreting autologous bone marrow stromal cells in the intramedullary microenvironment.

Genetically modified FVIII-expressing autologous bone marrow-derived mesenchymal stromal cells (BMSCs) could cure haemophilia A. However, culture-expanded BMSCs engraft poorly in extramedullary sites. Here, we compared the intramedullary cavity, skeletal muscle, subcutaneous tissue and systemic circulation as tissue microenvironments that could support durable engraftment of FVIII-secreting BMSC in vivo. A zinc finger nuclease integrated human FVIII transgene into PPP1R12C (intron 1) of culture-expanded primary canine BMSCs. FVIII-secretory capacity of implanted BMSCs in each dog was expressed as an individualized therapy index (number of viable BMSCs implanted × FVIII activity secreted/million BMSCs/24 hours). Plasma samples before and after implantation were assayed for transgenic FVIII protein using an anti-human FVIII antibody having negligible cross-reactivity with canine FVIII. Plasma transgenic FVIII persisted for at least 48 weeks after implantation in the intramedullary cavity. Transgenic FVIII protein levels were low after intramuscular implantation and undetectable after both intravenous infusion and subcutaneous implantation. All plasma samples were negative for anti-human FVIII antibodies. Plasma concentrations and durability of transgenic FVIII secretion showed no correlation with the therapy index. Thus, the implantation site microenvironment is crucial. The intramedullary microenvironment, but not extramedullary tissues, supported durable engraftment of genetically modified autologous FVIII-secreting BMSCs.

Microarray profiling shows distinct differences between primary tumors and commonly used preclinical models in hepatocellular carcinoma.

BACKGROUND: Despite advances in therapeutics, outcomes for hepatocellular carcinoma (HCC) remain poor and there is an urgent need for efficacious systemic therapy. Unfortunately, drugs that are successful in preclinical studies often fail in the clinical setting, and we hypothesize that this is due to functional differences between primary tumors and commonly used preclinical models. In this study, we attempt to answer this question by comparing tumor morphology and gene expression profiles between primary tumors, xenografts and HCC cell lines. METHODS: Hep G2 cell lines and tumor cells from patient tumor explants were subcutaneously (ectopically) injected into the flank and orthotopically into liver parenchyma of Mus Musculus SCID mice. The mice were euthanized after two weeks. RNA was extracted from the tumors, and gene expression profiling was performed using the Gene Chip Human Genome U133 Plus 2.0. Principal component analyses (PCA) and construction of dendrograms were conducted using Partek genomics suite. RESULTS: PCA showed that the commonly used HepG2 cell line model and its xenograft counterparts were vastly different from all fresh primary tumors. Expression profiles of primary tumors were also significantly divergent from their counterpart patient-derived xenograft (PDX) models, regardless of the site of implantation. Xenografts from the same primary tumors were more likely to cluster together regardless of site of implantation, although heat maps showed distinct differences in gene expression profiles between orthotopic and ectopic models. CONCLUSIONS: The data presented here challenges the utility of routinely used preclinical models. Models using HepG2 were vastly different from primary tumors and PDXs, suggesting that this is not clinically representative. Surprisingly, site of implantation (orthotopic versus ectopic) resulted in limited impact on gene expression profiles, and in both scenarios xenografts differed significantly from the original primary tumors, challenging the long-held notion that orthotopic PDX model is the gold standard preclinical model for HCC.

Structural stability of neoangiogenic intramyocardial microvessels supports functional recovery in chronic ischemic myocardium.

We hypothesize that combining angiopoietin-1 (ANG-1) or ANG-2 with vascular endothelial growth factor (VEGF) improves myocardial perfusion and contractile function by modulating vascular adaptation of neoangiogenic microvessels in a chronic ischemic swine model. Four weeks after occlusion of the left circumflex coronary artery (LCx), animals were injected with AdVEGF(165) (n=6), AdVEGF(165)+AdANG-1 (n=6), AdVEGF(165)+AdANG-2 (n=6) or control vector (n=5) into the left ventricular posterolateral wall. Regional perfusion by fluorescent microspheres and segmental myocardial tissue velocity by tissue Doppler imaging (TDI) were assessed at baseline, 4 weeks post occlusion and 4 weeks post therapy. Despite similar vascular growth following VEGF+ANG-1 and VEGF+ANG-2 treatments, transmural myocardial contractility improved only when VEGF was paired with ANG-1. In contrast, regional systolic function deteriorated uniformly across subepicardial, mid-myocardial and subendocardial segments in VEGF and VEGF+ANG-2 treated groups. Contractile improvement was associated with enhanced vascular stability through augmented arteriole formation, tight structural integration between VE-cadherin and beta-catenin at endothelial junctions and improved cross-talk between endothelium and myocardium. Structural stability of developing intramyocardial microvessels contributes to systolic function during ischemic neovascularization. Coordinated regulation of angiogenic revascularization that supports vascular stability is a key aspect in improving therapeutic outcomes in ischemic myocardium.

Which is the optimal risk stratification system for surgically treated localized primary GIST? Comparison of three contemporary prognostic criteria in 171 tumors and a proposal for a modified Armed Forces Institute of Pathology risk criteria.

BACKGROUND: This study aims to validate and compare the performance of the National Institute of Health (NIH) criteria, Huang modified NIH criteria, and Armed Forces Institute of Pathology (AFIP) risk criteria for gastrointestinal stromal tumors (GISTs) in a large series of localized primary GISTs surgically treated at a single institution to determine the ideal risk stratification system for GIST. METHODS: The clinicopathological features of 171 consecutive patients who underwent surgical resection for GISTs were retrospectively reviewed. Statistical analyses were performed to compare the prognostic value of the three risk criteria by analyzing the discriminatory ability linear trend, homogeneity, monotonicity of gradients, and Akaike information criteria. RESULTS: The median actuarial recurrence-free survival (RFS) for all 171 patients was 70%. On multivariate analyses, size >10 cm, mitotic count >5/50 high-power field, tumor necrosis, and serosal involvement were independent prognostic factors of RFS. All three risk criteria demonstrated a statistically significant difference in the recurrence rate, median actuarial RFS, actuarial 5-year RFS, and tumor-specific death across the different stages. Comparison of the various risk-stratification systems demonstrated that our proposed modified AFIP criteria had the best independent predictive value of RFS when compared with the other systems. CONCLUSION: The NIH, modified NIH, and AFIP criteria are useful in the prognostication of GIST, and the AFIP risk criteria provided the best prognostication among the three systems for primary localized GIST. However, remarkable prognostic heterogeneity exists in the AFIP high-risk category, and with our proposed modification, this system provides the most accurate prognostic information.

Intraabdominal schwannomas: a single institution experience.

INTRODUCTION: Intraabdominal schwannomas are rare, benign tumors. This study presents a single institution experience with 12 such tumors. METHODS: Between 1991 to 2006, 12 patients with a pathologically proven intraabdominal schwannoma were identified from a series of 216 mesenchymal tumors and were reviewed retrospectively. RESULTS: There were nine females and three male patients with a median age of 58 years (range 35-88 years). Eleven patients were symptomatic, and the tumors were located in the stomach (n = 8), jejunum, colon, rectum, and lesser sac. Multiple preoperative investigations including endoscopies with biopsies and computed tomography (CT) scans were performed, but none yielded a correct definitive preoperative diagnosis. The median tumor size was 52 mm (range 18-95 mm). Pathological examination demonstrated the 11 gastrointestinal tract (GIT) schwannomas to be solid homogenous tumors, which were highly cellular and were composed of spindle cells with positive staining for S100 protein. The pathological appearance of the lesser sac schwannoma was distinct as it demonstrated cystic degeneration with hemorrhage and Antoni A and B areas on microscopy typical of soft tissue schwannomas. All 12 patients were disease-free at a median follow-up of 22 months (range 1-120 months). CONCLUSION: Intraabdominal schwannomas are rare tumors, which are most frequently located within the GIT. GIT schwannomas are difficult if not impossible to diagnose preoperatively as endoscopic and radiologic findings are nonspecific. The treatment of choice is complete surgical excision because of diagnostic uncertainty, and the long-term outcome is excellent as these lesions are uniformly benign.

A novel biodegradable biliary stent in the normal duct hepaticojejunal anastomosis: an 18-month follow-up in a large animal model.

Creating a well-functioning hepaticojejunostomy (HJ) anastomosis with nondilated bile ducts remains a challenge. Our aim was to study the use in a large animal model of a novel, braided polylactide barium sulfate biodegradable biliary stent (BDBS) without external connection and with no need for later removal. Fifty swine were randomly operated on for Roux-Y HJ with or without BDBS in the anastomosis, and followed up (dynamic biligraphy, x-ray, serum determinations, anastomosis inner diameter, and histology) for 1.5, 3, 6, 12, and 18 months. During the follow-up, one nonstented animal died because of anastomotic leakage. In x-ray BDBS was seen in place until 1.5 months in all of the stented animals. In the nonstented animals HJ anastomosis inner diameter was decreased at 18 months [6.3 (5.0-7.0) mm vs 7.4 (7.0-9.0) mm, p = 0.05] and liver clearance reduced at 12 and 18 months compared to stented animals. Serum liver values and liver and bile duct histology did not differ between the groups. We conclude that this novel BDBS is easy to insert into the HJ anastomosis with nondilated ducts. It is nontoxic, dissolves safely, and may be associated with a larger and better draining anastomosis at 18-month follow-up. These results encourage us to proceed to clinical studies.

Intravoxel incoherent imaging of renal fibrosis induced in a murine model of unilateral ureteral obstruction.

PURPOSE: To evaluate non-invasive imaging biomarkers for assessing renal fibrosis. DWI is used to assess renal function; intravoxel incoherent motion (IVIM) provides additional measures of perfusion-related diffusion (D*, blood flow; f, perfusion fraction). We aim to determine if reduced ADC seen in renal fibrosis is attributable to perfusion-related diffusion changes or to known reduction in tissue diffusivity (D). MATERIALS AND METHODS: Unilateral ureteral obstruction (UUO) was created in six mice to induce renal fibrosis. DWI was performed the day before and 7 days post-UUO. A range of b-values from 0 to 1200 s/mm(2) were used. IVIM parameters were obtained using region of interests drawn over the renal parenchyma. Histopathological analysis of both kidneys was performed in all mice. Results were analyzed using the paired t-test with P<0.05 considered statistically significant. RESULTS: D and f were significantly lower in the ligated kidneys at Day 7 compared to before ligation and no significant difference was found for D*. Comparing non-ligated and ligated kidneys within the same mouse at Day 7, significantly lower D values were observed in the ligated kidneys, while no significant difference was found for f and D*, although the values of f were generally lower. Histopathological analysis confirmed development of fibrosis and reduction in glomeruli in all the ligated kidneys at Day 7. CONCLUSION: Our study shows that the reduction in ADC seen in renal fibrosis is attributable not only to reduced D as previously encountered but also a decrease in vascularity as assessed by f. Reduction in f is possibly related to a reduction in glomeruli.

Stepwise training for reconstructive microsurgery: the journey to becoming a confident microsurgeon in singapore.

Microsurgery training in Singapore began in 1980 with the opening of the Experimental Surgical Unit. Since then, the unit has continued to grow and have held microsurgical training courses biannually. The road to becoming a full-fledged reconstructive surgeon requires the mastering of both microvascular as well as flap raising techniques and requires time, patience and good training facilities. In Singapore, over the past 2 decades, we have had the opportunity to develop good training facilities and to refine our surgical education programmes in reconstructive microsurgery. In this article, we share our experience with training in reconstructive microsurgery.

A porcine model of selective retinal capillary closure induced by embolization with fluorescent microspheres.

PURPOSE: To investigate the feasibility of creating an animal model of selective retinal capillary closure to mimic the capillary closure that occurs in diabetic retinopathy. METHODS: Fluorescent microspheres of 10- or 15-µm diameter were delivered to one eye of anesthetized pigs via a customized cannula advanced through the carotid arterial system to the origin of the external ophthalmic artery that supplies blood to the eye in this species. After preliminary trials in 10 pigs, embolization was performed in one eye of 34 animals that were allowed to survive for 7, 14, or 28 days. Embolized eyes were assessed by fluorescein angiography, electroretinography (ERG), and, after enucleation, light (LM) and electron (EM) microscopy. RESULTS: The microspheres were detectable in the retina immediately after embolization, were restricted to the nerve fiber layer of the retina, and remained thereafter within the retina for periods up to 28 days. They effectively occluded embolized capillaries and some precapillary arterioles. No systemic or cerebral adverse effects were noted, thus allowing survival and subsequent follow-up. Embolization caused a reduction in the b-wave amplitude and the oscillatory potentials of the rod-cone bright-flash ERG but did not affect the amplitude of the a-wave. Embolization induced extracellular and intracellular edema confined to the inner and mid retina, and as a result the retinas of embolized eyes were thicker than those of fellow, nonembolized eyes. The outer retina and RPE were unaffected. CONCLUSIONS: This survival model of retinal embolization with microspheres should be useful in the study of the retinal effects of the capillary closure that may occur in diabetic eyes.

Platelet-rich plasma has no effect on increasing free fat graft survival in the nude mouse.

BACKGROUND: Free fat grafts have an unpredictable survival rate, which may be dependent on host bed vascularity. Therefore, the authors hypothesized that the presence of growth factors in platelet-rich plasma (PRP), may enhance free fat graft survival. METHODS: Free fat grafts and autologous PRP were harvested from a healthy female and processed using the Coleman technique and the Medtronic Magellan system respectively. The experiment comprised two groups of 12 nude mice each with injection of free fat grafts into the scalp. The experimental group comprised the combination of 0.8 ml of free fat graft and 0.2 ml of PRP. The control group comprised the combination of 0.8 ml of free fat graft and 0.2 ml of normal saline. The mice were euthanized after 16 weeks and the fat grafts explanted and measured for weight and volume. Histology was performed with Oil Red O stain. Statistical analysis of the weight and volume in between groups was performed using the independent samples T-test (SPSS v11). The Mann-Whitney test was used to compare the ranking of six histological parameters between the two groups. RESULTS: The mean weight and volume for the experimental arm were 0.503 g and 0.545 ml respectively. The mean weight and volume for the control arm were 0.500 g and 0.541 ml respectively. The weight, volume and histological parameters between the two groups were not statistically significant. A mouse from each group died of unknown causes. CONCLUSION: PRP did not enhance free fat graft survival in the nude mouse.

Effective inhibition of xenografts of hepatocellular carcinoma (HepG2) by rapamycin and bevacizumab in an intrahepatic model.

PURPOSE: Hepatocellular carcinoma (HCC) displays a characteristic hypervascularity and depends on angiogenesis for tumor growth, which thus provides a potential target for therapeutic approaches to HCC. In this study, through the use of combined micro-positron emission tomography (PET)/computed tomography (CT), we investigate if such a combined targeting of vascular endothelial growth factor (VEGF) activity and expression might retard HCC growth in an orthotopic intrahepatic xenograft model. PROCEDURES: Xenograft models were created by intraportal vein injection of HepG2 cell suspensions in severe combined immunodeficient mice. The mice were then treated with (1) rapamycin (RAPA), a mammalian target of rapamycin pathway inhibitor; (2) bevazicumab (BEV), a VEGF monoclonal antibody; and (3) a RAPA/BEV combination. RESULTS: Assessment of HCC progression using CT with Omnipaque and PET with 2-deoxy-2-(F-18)-fluoro-D: -glucose showed that mice treated with RAPA/BEV had the lowest standardized uptake values (SUVs). At week 2, mice treated with RAPA/BEV, RAPA, and BEV all showed a marked decrease in the SUV(max) readings with the greatest drop being observed in the RAPA/BEV group (1.33 + 0.26, 1.81 + 0.2, 2.05 + 0.4 vs. vehicle control 2.11 + 0.53). CONCLUSIONS: Our results, supported by micro-PET/CT, suggest that RAPA/BEV represents a potential novel antiangiogenic therapy for the treatment of HCC.

Long-term changes in hepatobiliary physiology after Roux-en-Y hepaticojejunostomy.

Hepaticojejunostomy (HJ) is a common operation used to by-pass extrahepatic biliary obstructions and to establish biliary-enteric continuity after resections for benign and malignant diseases. Little is known about the effect of this procedure on hepatobiliary physiology. The aim of the present study was to investigate in a swine model the changes in biliary dynamics, bile composition, and hepatic histology induced by Roux-Y HJ. Twenty-four swine (57 (47 to 76) kg) underwent cholecystectomy, with HJ (Group I; n = 12) or without any biliodigestive anastomosis (Group II, n = 12), and were followed up for 6 or 12 mo by repeated weight scaling, blood, serum, and bile analysis, (99m)Technetium (Tc), diethyliminodiacetic acid (HIDA) dynamic biligraphy, and histological analysis. During follow-up, HJ was associated with less weight gain, colonization of the bile duct with aerobic bacteria Escherichia coli dominating (in 75% of the animals), a shortened hilum-intestine transit time but reduced liver clearance in dynamic biligraphy, and fibrous periportal changes in liver histology (in 50% of the animals). We conclude that during 1 y follow-up HJ with no anastomotic stricture formation is associated with improved extrahepatic bile drainage, but with ascending contamination of bile ducts with bacteria, which might be involved with the fibrous periportal changes in the liver resulting in diminished excretion of Tc-HIDA from the hepatocytes into the bile. The clinical significance of these changes, and the reduced weight gain observed is a topic of further investigations.

Angiopoietin-1 promotes functional neovascularization that relieves ischemia by improving regional reperfusion in a swine chronic myocardial ischemia model.

This study investigates the long-term angiogenic effects of ANG-1 and VEGF in a swine chronic myocardial ischemia model. Four-weeks after gradual occlusion of the left circumflex coronary artery by ameroid constrictor, animals were injected with recombinant adenoviral vectors carrying either human ANG-1 (n=9), human VEGF(165) (n=10) or empty vector (n=7) into the left ventricle free wall supplied by the constricted artery. Left ventricular perfusion in animals that received AdANG-1 (3.25+/-0.16 ml/min/g, p<0.05) recovered robustly 4 weeks after gene transfer while ischemia persisted in the AdVEGF (1.09+/-0.13 ml/min/g) and empty vector (1.20+/-0.03 ml/min/g) groups. Microvascular densities in the left ventricles of animals that received AdANG-1 (19.61+/-1.76/0.572 mm(2) myocardial tissue, p<0.05) and AdVEGF (18.17+/-1.43/0.572 mm(2) myocardial tissue, p<0.05) were significantly higher than animals that received empty vector (13.53+/-0.92/0.572 mm(2) myocardial tissue) 12 weeks after gene transfer. ANG-1, but not VEGF, contributed to enhanced regional perfusion by increasing arteriolar density (1.9+/-0.4/0.572 mm(2) myocardial tissue vs. 0.7+/-0.2/0.572 mm(2) myocardial tissue, p<0.05) of large-sized (50-100 microm) arterioles. These data demonstrate that gene transfer of ANG-1 and VEGF enhances angiogenesis, but ANG-1 promotes sustained improvement of ventricular perfusion that expedites recovery of ischemic myocardium via arteriogenesis.