Evaluating Performance of Circular Staplers Using Comparative Test Methods for Evidence-Based Surgery.

BACKGROUND: Circular stapler anastomosis is a common surgical procedure. Despite technological advancements, anastomotic leak remains a postoperative concern. Assessment of new technologies is impeded by variations in test methods and analysis, precluding outcome reproducibility and direct comparisons of results across studies. The development of robust and reproducible preclinical test methods is critical to accelerating stapling technology advancements. METHODOLOGY: Leak pressure, staple line perfusion and security, and device removal force were quantified for triple-row (Tri-staple EEA, TriEEA) and double-row staplers (Echelon Circular Powered, ECP). Leak and perfusion testing were performed in vivo. Device removal force and staple line security testing were performed with synthetic medium using an Instron. Data were analyzed using unpaired student's t-test or Kruskal-Wallis test, with statistical significance defined as P < .05. RESULTS: Leak pressure was 73% higher in TriEEA vs ECP (P = .016). TriEEA staple line failure force was lower than ECP at 40 and 50 mmHg (P = .001 and P = .023, respectively). Perfusion to the staple line was higher (148%) for TriEEA than for ECP (P = .003) and the force required to remove the device from its stapled anastomosis was 78% lower for TriEEA than for ECP (P < .001). DISCUSSION/CONCLUSIONS: This report addresses a primary limitation in stapling research by presenting novel methodologies which enhance clinical relevance and provide sufficient detail for reproduction by independent investigators. These methods are applied to a comparison between triple-row and double-row staplers to demonstrate utility of new test methods in assessing key technology design features.

miR-467 regulates inflammation and blood insulin and glucose.

Obesity is associated with inflammation and insulin resistance (IR), but the regulation of insulin sensitivity (IS) and connections between IS and inflammation remain unclear. We investigated the role of miR-467a-5p, a miRNA induced by hyperglycaemia, in regulating inflammation and blood glucose handling. We previously demonstrated that miR-467a-5p is induced by hyperglycaemia and inhibits the production of thrombospondin-1 (TSP-1), a protein implicated in regulating inflammation. To investigate the role of miR-467 in blood glucose handling and tissue inflammation, WT C57BL/6 mice were fed chow or Western diet from 5 to 32 weeks of age and injected weekly with miR-467a-5p antagonist. Inhibiting miR-467a-5p resulted in 47% increase in macrophage infiltration and increased Il6 levels in adipose tissue, higher plasma insulin levels (98 ng/mL vs 63 ng/mL), and 17% decrease in glucose clearance without increase in weight or HDL/LDL. The antagonist effect was lost in mice on Western diet. Mice lacking TSP-1 lost some but not all of the miR-467 effects, suggesting Thbs1 (and other unknown transcripts) are targeted by miR-467 to regulate inflammation. miR-467a-5p provides a physiological feedback when blood glucose is elevated to avoid inflammation and increased blood glucose and insulin levels, which may prevent IR.

Three-dimensional structural analysis reveals a Cdk5-mediated kinase cascade regulating hepatic biliary network branching in zebrafish.

The intrahepatic biliary network is a highly branched three-dimensional network lined by biliary epithelial cells, but how its branching patterns are precisely established is not clear. We designed a new computer-based algorithm that quantitatively computes the structural differences of the three-dimensional networks. Utilizing the algorithm, we showed that inhibition of Cyclin-dependent kinase 5 (Cdk5) led to reduced branching in the intrahepatic biliary network in zebrafish. Further, we identified a previously unappreciated downstream kinase cascade regulated by Cdk5. Pharmacological manipulations of this downstream kinase cascade produced a crowded branching defect in the intrahepatic biliary network and influenced actin dynamics in biliary epithelial cells. We generated larvae carrying a mutation in cdk5 regulatory subunit 1a (cdk5r1a), an essential activator of Cdk5. cdk5r1a mutant larvae show similar branching defects as those observed in Cdk5 inhibitor-treated larvae. A small-molecule compound that interferes with the downstream kinase cascade rescued the mutant phenotype. These results provide new insights into branching morphogenesis of the intrahepatic biliary network.

Comparative systems pharmacology of HIF stabilization in the prevention of retinopathy of prematurity.

Retinopathy of prematurity (ROP) causes 100,000 new cases of childhood blindness each year. ROP is initiated by oxygen supplementation necessary to prevent neonatal death. We used organ systems pharmacology to define the transcriptomes of mice that were cured of oxygen-induced retinopathy (OIR, ROP model) by hypoxia-inducible factor (HIF) stabilization via HIF prolyl hydroxylase inhibition using the isoquinolone Roxadustat or the 2-oxoglutarate analog dimethyloxalylglycine (DMOG). Although both molecules conferred a protective phenotype, gene expression analysis by RNA sequencing found that Roxadustat can prevent OIR by two pathways: direct retinal HIF stabilization and induction of aerobic glycolysis or indirect hepatic HIF-1 stabilization and increased serum angiokines. As predicted by pathway analysis, Roxadustat rescued the hepatic HIF-1 knockout mouse from retinal oxygen toxicity, whereas DMOG could not. The simplicity of systemic treatment that targets both the liver and the eye provides a rationale for protecting the severely premature infant from oxygen toxicity.

Eotaxin-Rich Proangiogenic Hematopoietic Progenitor Cells and CCR3+ Endothelium in the Atopic Asthmatic Response.

Angiogenesis is closely linked to and precedes eosinophilic infiltration in asthma. Eosinophils are recruited into the airway by chemoattractant eotaxins, which are expressed by endothelial cells, smooth muscles cells, epithelial cells, and hematopoietic cells. We hypothesized that bone marrow-derived proangiogenic progenitor cells that contain eotaxins contribute to the initiation of angiogenesis and inflammation in asthma. Whole-lung allergen challenge of atopic asthma patients revealed vascular activation occurs within hours of challenge and before airway inflammation. The eotaxin receptor CCR3 was expressed at high levels on submucosal endothelial cells in patients and a murine model of asthma. Ex vivo exposure of murine endothelial cells to eotaxins induced migration and angiogenesis. In mechanistic studies, wild-type mice transplanted with eotaxin-1/2-deficient bone marrow had markedly less angiogenesis and inflammation in an atopic asthma model, whereas adoptive transfer of proangiogenic progenitor cells from wild-type mice in an atopic asthma model into the eotaxin-1/2-deficient mice led to angiogenesis and airway inflammation. The findings indicate that Th2-promoting hematopoietic progenitor cells are rapidly recruited to the lung upon allergen exposure and release eotaxins that coordinately activate endothelial cells, angiogenesis, and airway inflammation.

Liver Surface Nodularity Score Allows Prediction of Cirrhosis Decompensation and Death.

Purpose To determine whether use of the liver surface nodularity (LSN) score, a quantitative biomarker derived from routine computed tomographic (CT) images, allows prediction of cirrhosis decompensation and death. Materials and Methods For this institutional review board-approved HIPAA-compliant retrospective study, adult patients with cirrhosis and Model for End-Stage Liver Disease (MELD) score within 3 months of initial liver CT imaging between January 3, 2006, and May 30, 2012, were identified from electronic medical records (n = 830). The LSN score was measured by using CT images and quantitative software. Competing risk regression was used to determine the association of the LSN score with hepatic decompensation and overall survival. A risk model combining LSN scores (<3 or ≥3) and MELD scores (<10 or ≥10) was created for predicting liver-related events. Results In patients with compensated cirrhosis, 40% (129 of 326) experienced decompensation during a median follow-up period of 4.22 years. After adjustment for competing risks including MELD score, LSN score (hazard ratio, 1.38; 95% confidence interval: 1.06, 1.79) was found to be independently predictive of hepatic decompensation. Median times to decompensation of patients at high (1.76 years, n = 48), intermediate (3.79 years, n = 126), and low (6.14 years, n = 152) risk of hepatic decompensation were significantly different (P < .001). Among the full cohort with compensated or decompensated cirrhosis, 61% (504 of 830) died during the median follow-up period of 2.26 years. After adjustment for competing risks, LSN score (hazard ratio, 1.22; 95% confidence interval: 1.11, 1.33) and MELD score (hazard ratio, 1.08; 95% confidence interval: 1.06, 1.11) were found to be independent predictors of death. Median times to death of patients at high (0.94 years, n = 315), intermediate (2.79 years, n = 312), and low (4.69 years, n = 203) risk were significantly different (P < .001). Conclusion The LSN score derived from routine CT images allows prediction of cirrhosis decompensation and death. ©RSNA, 2016 Online supplemental material is available for this article.

Vascular Tumor Burden as a New Quantitative CT Biomarker for Predicting Metastatic RCC Response to Antiangiogenic Therapy.

Purpose To quantify initial changes in the vascular tumor burden (VTB), a measure of the area of vascularized tumor on computed tomographic (CT) images, and predict tumor response to antiangiogenic therapy in patients with metastatic renal cell carcinoma (RCC). Materials and Methods For this institutional review board-approved HIPAA-compliant secondary analysis of a prospective phase III trial, adult patients with digital CT images and metastatic clear-cell RCC treated with sunitinib were included (n = 275). Target lesions were selected by using Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 guidelines, and the CT images obtained after one cycle of sunitinib therapy were evaluated in comparison with baseline images. Tumor-response software was created to quantify tumor metrics (length, area, VTB, and mean attenuation) and automate response assessment. Progression-free survival (PFS) in responders and nonresponders according to VTB criteria was compared by using the Cox proportional hazard ratio (HR). The intraclass correlation coefficient (ICC) was used to assess interobserver agreement among three readers evaluating 28 randomly selected patients. Results VTB criteria nonresponders (n = 120) according to the initial posttherapy CT study were 5.7 times more likely to experience progression of disease (HR = 5.70; 95% confidence interval [CI]: 4.07, 7.97; P < .001) than responders (n = 155). There was not a statistically significant difference in PFS between RECIST nonresponders (n = 255) and responders (n = 20; HR = 1.54; 95% CI: 0.85, 2.77; P = .148). In a patient-level analysis, interobserver agreement was very good for assessing percentage change in length, area, and VTB (ICC = 0.82 [95% CI: 0.67, 0.91], 0.89 [95% CI: 0.79, 0.94], and 0.88 [95% CI: 0.79, 0.94], respectively) but was very poor for assessing percentage change in mean attenuation (ICC = 0.17 [95% CI: -0.05, 0.45]). Conclusion A quantitative CT imaging biomarker designed to measure initial changes in the VTB separated patients into responders and nonresponders, each with significantly different PFS, and showed very good interobserver agreement in patients with metastatic RCC treated with sunitinib. © RSNA, 2016 Online supplemental material is available for this article.

Liver Surface Nodularity Quantification from Routine CT Images as a Biomarker for Detection and Evaluation of Cirrhosis.

Purpose To determine the accuracy, reproducibility, and intra- and interobserver agreement of a computer-based quantitative method to measure liver surface nodularity (LSN) from routine computed tomographic (CT) images as a biomarker for detection and evaluation of cirrhosis. Materials and Methods For this institutional review board-approved HIPAA-compliant retrospective study, adult patients with healthy livers (n = 24) or various stages of hepatitis C virus-induced chronic liver disease (n = 70) with routine nonenhanced and portal venous phase contrast agent-enhanced liver CT imaging with thick-section (5.0 mm) and thin-section (1.25-1.50 mm) axial images obtained between January 1, 2006, and March 31, 2011, were identified from the electronic medical records. A computer algorithm was developed to measure LSN and derive a score. LSN scores, splenic volume, and the ratio of left lateral segment (LLS) to total liver volume (TLV) were measured from the same multiphasic liver CT examinations. Accuracy for differentiating cirrhotic from noncirrhotic livers was assessed by area under the receiver operating characteristic curve. Intra- and interobserver agreement was assessed by intraclass correlation coefficient. Results Median LSN scores from nonenhanced thick-section CT images in cirrhotic livers (3.16; 56 livers) were significantly higher than in noncirrhotic livers (2.11; 38 livers; P < .001). LSN scores from the four CT imaging types (94 patients for each type) were very strongly correlated (range of Spearman r, 0.929-0.960). LSN scores from portal venous phase contrast-enhanced thick-section CT images had significantly higher accuracy (area under the receiver operating characteristic curve, 0.929) than splenic volume (area under the receiver operating characteristic curve, 0.835) or LLS-to-TLV ratio measurements (area under the receiver operating characteristic curve, 0.753) for differentiating cirrhotic from noncirrhotic livers (P = .038 and .003, respectively; n = 94). Intra- and interobserver agreements that used nonenhanced thick CT images were very good (intraclass correlation coefficient, 0.963 and 0.899, respectively). Conclusion Quantitative measurement of LSN on routine CT images accurately differentiated cirrhotic from noncirrhotic livers and was highly reproducible. (©) RSNA, 2016 Online supplemental material is available for this article.

Proangiogenic Properties of Thrombospondin-4.

OBJECTIVE: Thrombospondin-4 (TSP-4) is 1 of the 5 members of the thrombospondin protein family. TSP-1 and TSP-2 are potent antiangiogenic proteins. However, angiogenic properties of the 3 other TSPs, which do not contain the domains associated with the antiangiogeneic activity of TSP-1 and TSP-2, have not been explored. In our previous studies, we found that TSP-4 is expressed in the vascular matrix of blood vessels of various sizes and is especially abundant in capillaries. We sought to identify the function of TSP-4 in the regulation of angiogenesis. APPROACH AND RESULTS: The effect of TSP-4 in in vivo angiogenesis models and its effect on angiogenesis-related properties in cultured cells were assessed using Thbs4(-/-) mice, endothelial cells (EC) derived from these mice, and recombinant TSP-4. Angiogenesis was decreased in Thbs4(-/-) mice compared with wild-type mice. TSP-4 was detected in the lumen of the growing blood vessels. Mice expressing the P387 TSP-4 variant, which was previously associated with coronary artery disease and found to be more active in its cellular interactions, displayed greater angiogenesis compared with A387 form. Lung EC from Thbs4(-/-) mice exhibited decreased adhesion, migration, and proliferation capacities compared with EC from wild-type mice. Recombinant TSP-4 promoted proliferation and the migration of EC. Integrin α2 and gabapentin receptor α2δ-1 were identified as receptors involved in regulation of EC adhesion, migration, and proliferation by TSP-4. CONCLUSION: TSP-4, an extracellular matrix protein previously associated with tissue remodeling, is now demonstrated to possess proangiogenic activity.

Hyperglycemia diverts dividing osteoblastic precursor cells to an adipogenic pathway and induces synthesis of a hyaluronan matrix that is adhesive for monocytes.

Isolated rat bone marrow stromal cells cultured in osteogenic medium in which the normal 5.6 mm glucose is changed to hyperglycemic 25.6 mm glucose greatly increase lipid formation between 21-31 days of culture that is associated with decreased biomineralization, up-regulate expression of cyclin D3 and two adipogenic markers (CCAAT/enhancer binding protein α and peroxisome proliferator-activated receptor γ) within 5 days of culture, increase neutral and polar lipid synthesis within 5 days of culture, and form a monocyte-adhesive hyaluronan matrix through an endoplasmic reticulum stress-induced autophagic mechanism. Evidence is also provided that, by 4 weeks after diabetes onset in the streptozotocin-induced diabetic rat model, there is a large loss of trabecular bone mineral density without apparent proportional changes in underlying collagen matrices, a large accumulation of a hyaluronan matrix within the trabecular bone marrow, and adipocytes and macrophages embedded in this hyaluronan matrix. These results support the hypothesis that hyperglycemia in bone marrow diverts dividing osteoblastic precursor cells (bone marrow stromal cells) to a metabolically stressed adipogenic pathway that induces synthesis of a hyaluronan matrix that recruits inflammatory cells and establishes a chronic inflammatory process that demineralizes trabecular cancellous bone.

Automated Analysis of Anterior Chamber Inflammation by Spectral-Domain Optical Coherence Tomography.

PURPOSE: This study was designed to determine the feasibility of anterior segment optical coherence tomography (AS-OCT) to objectively image and quantify the degree of AC inflammation. DESIGN: Prospective evaluation of a diagnostic test. PARTICIPANTS: Patients with anterior segment involving uveitis. METHODS: Observational case series of patients with uveitis. Single-line and 3-dimensional (3D) volume AS-OCT scans were manually graded to evaluate for the presence or absence of cells in the AC. Clinical grading scores were correlated to the number of cells seen in each line scan. An automated algorithm was developed to measure the number of cells seen in the 3D volume scan and compared with manual measurements and clinical grading scores. MAIN OUTCOME MEASURES: Degree of anterior segment inflammation. RESULTS: A total of 114 eyes from 76 patients were imaged, 83 eyes with line scans and 31 eyes with volume scans. The average number of cells on line scans was 0.13 for grade 0, 1.2 for grade 1/2+, 2.6 for grade 1+, 5.7 for grade 2+, 15.5 for grade 3+, and 41.2 for grade 4+. Spearman correlation coefficient comparing clinical grade with the individual AS-OCT line scans was 0.967 (P < 0.0001). The range of cells in the automated cell count of 3D volume scans was 13.60 to 1222; the range for manual cell counts was from 9.2 to 2245. The Spearman correlation coefficients were r = 0.7765 (P < 0.0001) and r = 0.7484 (P < 0.0001) comparing the manual and automated cell counts with the clinical grade, respectively. Spearman correlation coefficient comparing the automatic cell counts with manual cell count in the 3D volume scan was 0.997 (P < 0.0001). CONCLUSIONS: Anterior segment OCT can be used to image and grade the degree of AC inflammation. Clinical grading strongly correlates with the number of cells on AS-OCT line scans and volume scans. The automated algorithm to measure cell count had a high correlation to manual measurement of cells in the 3D volume scans. This modality could be used to objectively grade response to treatment.

Thrombospondin-4 regulates fibrosis and remodeling of the myocardium in response to pressure overload.

Thrombospondin-4 (TSP-4) expression increases dramatically in hypertrophic and failing hearts in rodent models and in humans. The aim of this study was to address the function of TSP-4 in the heart. TSP-4-knockout (Thbs4(-/-)) and wild-type (WT) mice were subjected to transverse aortic constriction (TAC) to increase left ventricle load. After 2 wk, Thbs4(-/-) mice had a significantly higher heart weight/body weight ratio than WT mice. The additional increase in the heart weight in TAC Thbs4(-/-) mice was due to increased deposition of extracellular matrix (ECM). The levels of interstitial collagens were higher in the knockout mice, but the size of cardiomyocytes and apoptosis in the myocardium was unaffected by TSP-4 deficiency, suggesting that increased reactive fibrosis was the primary cause of the higher heart weight. The increased ECM deposition in Thbs4(-/-) mice was accompanied by changes in functional parameters of the heart and decreased vessel density. The expression of inflammatory and fibrotic genes known to be influential in myocardial remodeling changed as a result of TSP-4 deficiency in vivo and as a result of incubation of cells with recombinant TSP-4 in vitro. Thus, TSP-4 is involved in regulating the adaptive responses of the heart to pressure overload, suggesting its important role in myocardial remodeling. Our study showed a direct influence of TSP-4 on heart function and to identify the mechanism of its effects on heart remodeling.

Randomized controlled trial of osteoconductive fixation screws for anterior cruciate ligament reconstruction: a comparison of the Calaxo and Milagro screws.

PURPOSE: To compare the outcome of 2 bioabsorbable screws for tibial interference fixation in anterior cruciate ligament reconstruction with reference to rate of absorption, osteoconductive properties, and clinical outcome. METHODS: Patients undergoing primary anterior cruciate ligament reconstruction with hamstring autograft in a single unit were invited to participate in this study. Patients were randomized to receive either the Calaxo screw (Smith & Nephew, Andover, MA) or Milagro screw (DePuy Mitek, Raynham, MA) for tibial fixation. Patients were reviewed with subjective and objective evaluation by use of the International Knee Documentation Committee form, Lysholm score, KT-1000 arthrometry (MEDmetric, San Diego, CA), and clinical examination. Magnetic resonance imaging was performed at 1 year and computed tomography scanning at 1 week and at 6, 12, and 24 months. RESULTS: Sixty patients agreed to participate in the study, with 32 patients randomized to the Calaxo screw and 28 to the Milagro screw for tibial fixation. There was no significant difference in subjective or objective clinical outcome between the 2 groups. At 24 months, 88% of Calaxo screws showed complete screw resorption compared with 0% of Milagro screws (P < .001). Tibial cysts were present in 88% of the Calaxo group and 7% of the Milagro group (P = .001). At 24 months, the mean volume of new bone formation for the Calaxo group was 21% of original screw volume. Ossification of the Milagro screw was unable to be accurately assessed as a result of incomplete screw resorption. CONCLUSIONS: Both screws showed similar favorable objective and subjective outcomes at 2 years. The Calaxo screw resorbed completely over a period of 6 months and was associated with a high incidence of intra-tunnel cyst formation. The Milagro screw increased in volume over a period of 6 months, followed by a gradual resorption, which was still ongoing at 2 years. Both screws were associated with tunnel widening, and neither showed evidence of significant tunnel ossification. We conclude that, despite satisfactory clinical outcomes, the addition of "osteoconductive" materials to bioabsorbable screws is not associated with bone formation at the screw site at 2 years. LEVEL OF EVIDENCE: Level I, randomized controlled trial.

Pericellular versican regulates the fibroblast-myofibroblast transition: a role for ADAMTS5 protease-mediated proteolysis.

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5(-/-) mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFß signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5(-/-) cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcan(hdf/+)) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5(-/-) cells resulted from increased PCM versican content, we generated Adamts5(-/-);Vcan(hdf/+) mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5(-/-) mice. In Adamts5(-/-) fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates.

Rab13-dependent trafficking of RhoA is required for directional migration and angiogenesis.

Angiogenesis requires concomitant remodeling of cell junctions and migration, as exemplified by recent observations of extensive endothelial cell movement along growing blood vessels. We report that a protein complex that regulates cell junctions is required for VEGF-driven directional migration and for angiogenesis in vivo. The complex consists of RhoA and Syx, a RhoA guanine exchange factor cross-linked by the Crumbs polarity protein Mupp1 to angiomotin, a phosphatidylinositol-binding protein. The Syx-associated complex translocates to the leading edge of migrating cells by membrane trafficking that requires the tight junction recycling GTPase Rab13. In turn, Rab13 associates with Grb2, targeting Syx and RhoA to Tyr(1175)-phosphorylated VEGFR2 at the leading edge. Rab13 knockdown in zebrafish impeded sprouting of intersegmental vessels and diminished the directionality of their tip cells. These results indicate that endothelial cell mobility in sprouting vessels is facilitated by shuttling the same protein complex from disassembling junctions to the leading edges of cells.

Deep learning-enabled ultra-widefield retinal vessel segmentation with an automated quality-optimized angiographic phase selection tool.

OBJECTIVES: To demonstrate the feasibility of a deep learning-based vascular segmentation tool for UWFA and evaluate its ability to automatically identify quality-optimized phase-specific images. METHODS: Cumulative retinal vessel areas (RVA) were extracted from all available UWFA frames. Cubic splines were fitted for serial vascular assessment throughout the angiographic phases of eyes with diabetic retinopathy (DR), sickle cell retinopathy (SCR), or normal retinal vasculature. The image with maximum RVA was selected as the optimum early phase. A late phase frame was selected at a minimum of 4 min that most closely mirrored the RVA from the selected early image. Trained image analysts evaluated the selected pairs. RESULTS: A total of 13,980 UWFA sequences from 462 sessions were used to evaluate the performance and 1578 UWFA sequences from 66 sessions were used to create cubic splines. Maximum RVA was detected at a mean of 41 ± 15, 47 ± 27, 38 ± 8 s for DR, SCR, and normals respectively. In 85.2% of the sessions, appropriate images for both phases were successfully identified. The individual success rate was 90.7% for early and 94.6% for late frames. CONCLUSIONS: Retinal vascular characteristics are highly phased and field-of-view sensitive. Vascular parameters extracted by deep learning algorithms can be used for quality assessment of angiographic images and quality optimized phase selection. Clinical applications of a deep learning-based vascular segmentation and phase selection system might significantly improve the speed, consistency, and objectivity of UWFA evaluation.

Cross-talk between vascular endothelial growth factor and matrix metalloproteinases in the induction of neovascularization in vivo.

Matrix metalloproteinases (MMPs), a specialized group of enzymes capable of proteolytically degrading extracellular matrix proteins, have been postulated to play an important role in angiogenesis. It has been suggested that MMPs can regulate neovascularization using mechanisms other than simple remodeling of the capillary basement membrane. To determine the interplay between vascular endothelial growth factor (VEGF) and MMPs, we investigated the induction of angiogenesis by recombinant active MMPs and VEGF in vivo. Using a rat corneal micropocket in vivo angiogenesis assay, we observed that the active form of MMP-9 could induce neovascularization in vivo when compared with the pro- form of the enzyme as a control. This angiogenic response could be inhibited by neutralizing VEGF antibody, which suggests that MMPs acts upstream of VEGF. Additional in vitro studies using extracellular matrix loaded with radiolabeled VEGF determined that active MMPs can enzymatically release sequestered VEGF. Interestingly, in vivo angiogenesis induced by VEGF could be inhibited by MMP inhibitors, indicating that MMPs also act downstream of VEGF. In addition, inflammation plays an important role in the induction of angiogenesis mediated by both VEGF and MMPs. Our results suggest that MMPs act both upstream and downstream of VEGF and imply that potential combination therapies of VEGF and MMP inhibitors may be a useful therapeutic approach in diseases of pathological neovascularization.

Membrane microviscosity regulates endothelial cell motility.

Endothelial cell (EC) movement is an initiating and rate-limiting event in the neogenesis and repair of blood vessels. Here, we explore the hypothesis that microviscosity of the plasma membrane (PM) is a key physiological regulator of cell movement. Aortic ECs treated with membrane-active agents, such as alpha-tocopherol, cholesterol and lysophospholipids, exhibited a biphasic dependency on membrane microviscosity, in which moderate increases enhanced EC migration, but increases beyond a threshold markedly inhibited migration. Surprisingly, angiogenic growth factors, that is, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), also increased membrane microviscosity, as measured in live cells by fluorescence recovery after photobleaching (FRAP). The localization of Rac to the PM was modified in cells treated with membrane-active agents or growth factors, suggesting a molecular mechanism for how membrane microviscosity influences cell movement. Our data show that angiogenic growth factors, as well as certain lipophilic molecules, regulate cell motility through alterations in membrane properties and the consequent relocalization of critical signalling molecules to membranes.

Temporal diabetes and diuresis-induced alteration of nerves and vasculature of the urinary bladder in the rat.

OBJECTIVE: • To characterize the temporal changes of the nerves and vasculature of the bladder in diabetic rats. MATERIALS AND METHODS: • A total of 36 Sprague-Dawley rats were divided into three groups: streptozotocin-induced diabetics, 5% sucrose-induced diuretics and age-matched controls. • The characteristics of the nerves and vasculature in the equatorial cross-sectional areas of the bladder were examined by immunofluorescence staining of their specific markers, neurofilament 200 (NF200) and CD31, at 1, 9 or 20 weeks after induction. • The distributions of the nerves and blood vessels were observed and the densities were quantified. RESULTS: • Diabetes caused a significant reduction in body weight. Bladder weight increased in diabetic and diuretic rats, but not in controls. • The total cross-sectional wall area and detrusor muscle area at the equatorial midline were greater in bladders of diabetic and diuretic rats than in controls. • Neurofilament 200-immunoreactive (NF200-IR) nerves were mainly distributed in the detrusor muscle. CD31-immunoreactive blood vessels were mainly distributed in the mucosa/submucosa. • There were no significant differences in the NF200-IR nerve terminal area among control, diabetic and diuretic groups. However nerve density was decreased at 9 and 20 weeks in the muscle, and at 20 weeks in the mucosa/submucosa in diabetic and diuretic animals. • Blood vessel density decreased in the diabetic and diuretic groups at 20 weeks in the muscle. CONCLUSIONS: • Diabetes induced time-dependent changes in the density of the nerves and vasculature in the bladder tissues. • Diabetes-related polyuria plays an important role in these changes.

Mandible Biomechanics and Continuously Erupting Teeth: A New Defect Model for Studying Load-Bearing Biomaterials.

Animals with elodont dentition and unfused mandible symphyses are hypothesized to have symmetric incisor morphology. Since these animals maintain their teeth by gnawing, they may provide physiologic feedback on mechanical function when unilateral mandible defects are created that manifest as ipsilateral changes in tooth structure. This defect model would potentially generate important information on the functional/mechanical properties of implants. Rats' and rabbits' mandibles and teeth are analyzed with µCT at baseline and post-intervention (n = 8 for each). Baseline incisors were compared. In a unilateral mandible pilot study, defects-ranging from critical size defect to complete ramus osteotomies-were created to assess effect on dentition (rats, n = 7; rabbits, n = 6). Within 90% confidence intervals, animals showed no baseline left/right differences in their incisors. There are apparent dental changes associated with unilateral defect type and location. Thus, at baseline, animals exhibit statistically significant incisor symmetry and there is an apparent relationship between mandible defect and incisor growth. The baseline symmetry proven here sets the stage to study the degree to which hemi-mandible destabilizing procedures result in measurable & reproducible disruption of dental asymmetry. In a validated model, an implant designed to function under load that prevents incisor asymmetry would provide supporting evidence that the implant has clinically useful load-bearing function.