Total Vessel Length Research Articles

Development of an ImageJ-based method for analysing the developing zebrafish vasculature

Zebrafish with fluorescently labelled blood vessels provide an excellent model for studying angiogenesis. Most commonly the growth of the intersegmental blood vessels is investigated in response to compounds or manipulation of gene expression and analysed using manual methods, typically scoring the connectivity of these blood vessels to the dorsal longitudinal anastomotic vessel. Such methods are laborious and best suited to time points after the connectivity of these vessels have been established. By contrast, reported image processing-based methods are difficult to implement and often depend on specialist software. This study aimed to develop and evaluate a computational method using the freely available ImageJ software to quantify the development of intersegmental blood vessel formation. This methodology developed allowed rapid analysis of vascular development. The outputs of total vessel length and number of junctions best documented defective vascular development at differing levels of severity and gave comparable results to the frequently used manual approach of calculating percentage connectivity of intersegmental vessels. This ImageJ-based method allowed objective quantitation of vascular network formation in zebrafish enabling a free, straightforward and rapid approach to determine the effect of novel compounds or genetic manipulation of the angiogenic process.

Sox2 regulates astrocytic and vascular development in the retina.

Sox2 is a transcriptional regulator that is highly expressed in retinal astrocytes, yet its function in these cells has not previously been examined. To understand its role, we conditionally deleted Sox2 from the population of astrocytes and examined the consequences on retinal development. We found that Sox2 deletion does not alter the migration of astrocytes, but it impairs their maturation, evidenced by the delayed upregulation of glial fibrillary acidic protein (GFAP) across the retina. The centro-peripheral gradient of angiogenesis is also delayed in Sox2-CKO retinas. In the mature retina, we observed lasting abnormalities in the astrocytic population evidenced by the sporadic loss of GFAP immunoreactivity in the peripheral retina as well as by the aberrant extension of processes into the inner retina. Blood vessels in the adult retina are also under-developed and show a decrease in the frequency of branch points and in total vessel length. The developmental relationship between maturing astrocytes and angiogenesis suggests a causal relationship between the astrocytic loss of Sox2 and the vascular architecture in maturity. We suggest that the delay in astrocytic maturation and vascular invasion may render the retina hypoxic, thereby causing the abnormalities we observe in adulthood. These studies uncover a novel role for Sox2 in the development of retinal astrocytes and indicate that its removal can lead to lasting changes to retinal homeostasis.

Abstract: Plug N Play: Auto-Assembly of a Hierarchical Vasculature in a Tissue Engineered Scaffold

INTRODUCTION: Tissue engineering applications in medicine and surgery remain severely limited by insufficient vascularization. Recently, sphingosine-1-phosphate (S1P), a bioactive sphingolipid stored in platelets and found in micromolar quantities in the plasma and nearly absent in the interstitium, has gained broad attention for its effects on endothelial motility and vascular maturation. It is thought that an S1P gradient between the lumen and the interstitium is integral to reparative angiogenesis. Herein we utilize S1P gradients within our tissue engineered hydrogels to promote self-assembly of endothelial cells into tubules that anastomose with a central millimeter sized channel with the aim of a fabricating a novel hierarchical pre-vascularized regenerative template. METHODS: Constructs were fabricated by suspending endothelial cells (EC) in type I collagen impregnated with S1P at a density of 2 million cells/ml. Within the collagen, a 1mm diameter channel was then lumenally seeded with ECs. Confocal, fluorescent, and light microscopy were performed to quantify and compare vascular morphology. Endpoints measured included endolumenal diameter, total vessel length, number of sprout endpoints, junction density, and extent of invasion. RESULTS: Axial sections demonstrated robust cellular invasion out of the central conduit. Sprout-like structures were found branching and anastomosing with the central conduit in a complex plexus. Constructs exhibited an average endothelial sprout density of 37 ± 8 sprouts/mm2with an average depth of invasion of 129 ± 58 um. The extent of plexus formation showed a statistically significant increase in total vessel length (p = 0.0003), total number of endothelial endpoints (p=0.0004), and junction density (p < 0.0001) in cultures treated with S1P. Lumen diameters were similar in caliber to deep dermal arteriolar vessels (p > 0.05). CONCLUSION: While current dermal replacement products are limited in clinical utility by the absence of an inherent vasculature, we have demonstrated the successful fabrication of a novel, rapidly prevascularized regenerative template containing an inherent hierarchical vasculature utilizing the powerful angiogenic regulator S1P. Such constructs hold tremendous promise for clinical translation and especially for use in challenging wounds.

Antiangiogenic activity of zinc and zinc-sorafenib combination using the chick chorioallantoic membrane assay: a descriptive study

Background: Zinc, a trace element, is known for downregulating several proangiogenic growth factors and cytokines. However, its antiangiogenic activity is not adequately studied. The present study was aimed to evaluate the possible antiangiogenic activity of zinc via the chick chorioallantoic membrane (CAM) assay. Also, the antiangiogenic activity of the combination therapy of zinc with various doses of sorafenib, a tyrosine kinase inhibitor, was evaluated.Methods: A pilot study was initially conducted so as to select suitable doses of zinc and sorafenib. The antiangiogenic activity after combining zinc 2.5 μg/embryo with sorafenib 1, and 2 μg/embryo was also evaluated. The antiangiogenic activity was quantified in terms of total length of blood vessels, number of junctions, number of branching points, and mean length of the blood vessels.Results: Zinc 2.5 μg/embryo showed significant (p &lt;0.05) antiangiogenic activity, as compared to the control group. However, its effect was not comparable to that of sorafenib 2 μg/embryo. The combination of zinc 2.5 μg/embryo with sorafenib 2 μg/embryo did not show an additive/synergistic effect. The combination of zinc 2.5 μg/embryo with sorafenib 1 μg/embryo produced an antiangiogenic activity which was comparable (p &gt;0.05) to that of sorafenib 2 μg/embryo.Conclusions: Zinc caused significant antiangiogenic activity in the CAM assay. The lack of addition/synergism in the zinc-sorafenib combination could have been due to the variability in the dose/ratio selection. Addition of zinc to sorafenib therapy could improve treatment tolerability, reduce cost of therapy, and reduce the emergence of drug resistance. Future mechanistic studies could identify the exact pharmacodynamics of zinc as an angiogenesis inhibitor.

Diffuse coronary artery disease among other atherosclerotic plaque characteristics by coronary computed tomography angiography for predicting coronary vessel-specific ischemia by fractional flow reserve

Background and aimsCoronary computed tomography angiography (CCTA) permits effective identification of diffuse CAD and atherosclerotic plaque characteristics (APCs). We sought to examine the usefulness of diffuse CAD beyond luminal narrowing and APCs by CCTA to detect vessel-specific ischemia. Methods407 vessels (n = 252 patients) from the DeFACTO diagnostic accuracy study were retrospectively analyzed for percent plaque diffuseness (PD). Percent plaque diffuseness (PD) was obtained on per-vessel level by summation of all contiguous lesion lengths and divided by total vessel length, and was logarithmically transformed (log percent PD). Additional CCTA measures of stenosis severity including minimal lumen diameter (MLD), and APCs, such as positive remodeling (PR) and low attenuation plaque (LAP), were also included. Vessel-specific ischemia was defined as fractional flow reserve (FFR) ≤0.80. Multivariable regression, discrimination by area under the receiver operating characteristic curve (AUC), and category-free net reclassification improvement (cNRI) were assessed. ResultsBackward stepwise logistic regression revealed that for every unit increase in log percent PD, there was a 58% (95% CI: 1.01–2.48, p = 0.048) rise in the odds of having an abnormal FFR, independent of stenosis severity and APCs. The AUC indicated no further improvement in discriminatory ability after adding log percent PD to the final parsimonious model of MLD, PR, and LAP (AUC difference: 0.003, 95% CI: −0.003–0.010, p = 0.33). Conversely, adding log percent PD to the base model of MLD, PR, and LAP improved cNRI by 0.21 (95% CI: 0.01–0.41, p < 0.001). ConclusionsAccounting for diffuse CAD may help improve the accuracy of CCTA for detecting vessel-specific ischemia.

OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY OF A29 BIRDSHOT CHORIORETINOPATHY COMPLICATED BY RETINAL NEOVASCULARIZATION

To describe the quantitative optical coherence tomography angiography analysis of the superficial and deep retinal capillary plexus in a case of A29 birdshot chorioretinopathy (BCR) complicated by retinal neovascularization. Multimodal retinal imaging, including optical coherence tomography angiography with quantitated vessel density analysis of the superficial and deep retinal capillary plexus, was performed in a case of A29 BCR. Capillary density (mm) was defined as the total vessel length (mm) per area (mm). A 39-year-old healthy white man presented with vitreous hemorrhage and retinal neovascularization at baseline. Clinical presentation was consistent with BCR and human leukocyte antigen testing confirmed A29 positivity. Optical coherence tomography angiography was performed 6 years after the diagnosis and revealed capillary density reduction in the superficial and especially in the deep retinal capillary plexus. Quantitative vessel density analysis was compared with the mean capillary density of 11 age-matched normal eyes and demonstrated 6% and 18% reduction of the superficial plexus in the right and left eyes of our patient, respectively. For the deep plexus, a reduction of 25% and 32% was found in the right and left eyes, respectively. In this patient with BCR, capillary density reduction was most marked at the deep retinal capillary plexus level and may explain the development of retinal neovascularization in this case. Quantitative optical coherence tomography angiography may be a useful tool to study and to monitor retinal microvascular changes in patients with BCR and should be further explored as a biomarker of therapeutic response.

EFFECTS OF Tempol ON SURVIVAL OF RANDOM PATTERN SKIN FLAPS IN RATS

To study the effects of the new small molecular oxygen free radical scavenger Tempol on the survival and vasculogenesis of the long random pattern skin flap (LRPSF) and its mechanism. Eighty-four male Sprague Dawley rats were randomly divided into control and Tempol groups (42 rats in each group). LRPSF of 9 cm×3 cm in size were prepared on the backs of rats in two groups based on the Mcfarlane flap. Rats were administered with Tempol (100 mg/kg) in the Tempol group and with normal saline in the control group by intraperitoneal injection at 15 minutes before operation and at 1-7 day after operation. The rat and the skin flap survival conditions were observed after operation; the survival rate of skin flap was measured, and the vascular structure, vascular volume, and total length of blood vessels were analyzed with Micro-CT three-dimensional imaging after 7 days; HE staining was used to observe the structure of the skin flaps and inflammation, immumohistochemical staining to observe vascular endothelial growth factor (VEGF) expression; water-soluble tetrazolium-1 method was used to measure the content of superoxide dismutase (SOD) and malondialdehyde (MDA), and ELISA to detect the expressions of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) after 1, 3, and 7 days. All of rats survived after operation, without hemorrhage, edema, and infection. With the extension of time, necrosis occurred in the distal part of the skin flaps in 2 groups, but the necrosis degree of the Tempol group was lower than that of control group; meanwhile, the blood vessel distribution and continuity were better than those of control group. The skin flaps survival rate, vascular volume, and total length of blood vessels of Tempol group were significantly higher than those of control group after 7 days (P<0.05). The clearer skin flaps structure, lighter inflammation reaction and inflammation cell infiltration, and higher VEGF staining intensity were observed in the Tempol group than the control group after 7 days. There was no significant difference in SOD, MDA, and TNF-α, and IL-6 contents between the 2 groups at immediate after operation. SOD significantly increased, but MDA, TNF-α, and IL-6 contents significantly decreased in the Tempol group when compared with control group after 1, 3, and 7 days (P<0.05). Tempol can significantly promote the LRPSF survival rates, its mechanism is closely related to the promotion of vasculogenesis and reduction of oxidative stress and inflammation.

Angiogenic Effects of Dimeric Dipeptide Mimetic of Loop 4 of Nerve Growth Factor.

Angiogenic action of compound GK-2, a dimeric dipeptide mimetic of loop 4 of nerve growth factor (NGF), was studied in in vitro and in vivo experiments. Experiments on human endothelial cell culture HUVEC showed that compound GK-2 significantly (p<0.05) stimulated the initial stage of angiogenesis, and its angiogenic activity was not inferior to the reference neurotrophin NGF. In experiments with hindlimb ischemia modeled in rats, GK-2 (1 mg/kg intraperitoneally for 14 days) significantly increased the total length of capillary vessels (p<0.003) and the number of vessels per 1 mm2 ischemic tissue (p<0.001) in comparison with the control. Our findings indicate that under experimental conditions compound GK-2 exhibits not only angiogenic, but also anti-ischemic activity.

Assessment of fibrotic tissue and microvascular architecture by in-line phase-contrast imaging in a mouse model of liver fibrosis.

To explore the value of in-line phase-contrast imaging with computed tomography (ILPCI-CT) by synchrotron radiation (SR) for liver fibrosis. Liver fibrosis models were set up in 13 BALB/c mice by peritoneal injections of thioacetamide and evaluated by ILPCI-CT. Histological staging was used to categorize liver fibrosis into normal, mild fibrosis and advanced fibrosis groups. Microvessel density (MVD), the ratio of total vessel length to volume (L/V), the ratio of total number of branching points to liver volume (P/V) and the distribution of vessel diameter were assessed. The CT images showed slightly high-density shadows around the portal tracts in the fibrosis group. Three-dimensional reconstruction can detect vascular and nodular changes on the surface of fibrotic livers. The MVDs between the three groups were significantly different (P = 0.024). L/V was significantly different between the three groups (P = 0.014). There was a positive correlation between MVD and P/V. Fibrous material can be detected by ILPCI-CT even in the early stage of fibrosis. MVD, L/V, P/V and the distribution of vessel diameter were consistent with fibrosis-related angiogenesis progress. Three-dimensional reconstruction is a promising method to visualize morphological changes of the fibrotic liver. • ILPCI-CT can detect fibrous material even in the early stage of liver fibrosis. • MVD, L/V, P/V, and the distribution of vascular diameter reflect pathological angiogenesis. • 3D reconstruction could be a promising approach for detecting liver fibrosis.

The Dorsal Skin Fold Chamber as new model of delayed wound healing to monitor the impact of pro-angiogenic starPEG-heparin hydrogels on angiogenesis and wound healing outcome during skin regeneration

Event Abstract Back to Event The Dorsal Skin Fold Chamber as new model of delayed wound healing to monitor the impact of pro-angiogenic starPEG-heparin hydrogels on angiogenesis and wound healing outcome during skin regeneration Christian Beescho1, Teresa Schöpfer2, Lucas Schirmer3, Passant Atallah3, Uwe Freudenberg3, Carsten Werner3, Jan Christoph Simon2, Stefan Langer1 and Sandra Franz2 1 University Hospital Leipzig, Plastic and Reconstructive Surgery, Germany 2 University Hospital Leipzig, Dermatology and Venerology, Germany 3 Max Bergmann Center of Biomaterials, Leibniz Institute of Biofunctinoal Polymer Materials, Germany The Dorsal Skin Fold Chamber (DSFC) model is a useful tool that allows in vivo monitoring of vascularization of biomaterials. Angiogenesis is indispensable in wound healing. Delayed wound healing is associated with impaired new blood vessel formation, which calls for the development of pro-angiogenic wound dressings. We have recently shown that poly(ethylene glycol) (starPEG)-heparin hydrogels can deliver the angiogenic factors FGF-2 and VEGF and encourage angiogenesis in a CAM assay[1]. However, whether these hydrogels promote angiogenesis during wound healing and improve the wound healing outcome needs to be shown. We have therefore established the DSFC as a wound healing model that allows real-time in vivo imaging of angiogenesis and a dynamic characterization of inflammation and re-epithelialization. Dorsal skin of C57BL6 mice was shaved and depilated prior to DSFC implantation. Within the microscopy window, a 5 mm full-thickness wound was created. The chamber was filled with NaCl and closed with a transparent coverslip. At day 1, exudate was gently removed to receive a clear observation window for intravital microscopy (IVM). A starPEG-heparin hydrogel was applied onto the wound and the chamber was closed again. IVM was performed on day 1, 3, 6, 8, 10, 13, and 19 after wounding using FITC/dextran (i.v. injection) to visualize vessels in the wound area. Angiogenesis was analyzed with ZEN software (Zeiss), measuring the functional vessel density (defined as total blood vessel length per area) in the wound and wound edges. To correlate angiogenic events with the wound healing outcome inflammation, granulation tissue formation and re-epithelialization were analyzed in wound biopsies post mortem in the same kinetic as IVM using qRT-PCR and immunohistochemistry. Wound healing in the DSFC is delayed as seen by a late wound closure at day 13. The DSFC impairs the contraction of the M. panniculus carnosus that mediates murine wound closure. Instead, wounds close with new tissue formed by proliferating myofibroblasts and keratinocytes. Delayed wound healing in the DSFC is characterized by prolonged inflammation with a slow increase on infiltrating immune cells, reaching a peak at day 13 and resolving till day 19. Using IVM, angiogenesis could be observed during the whole study. New capillaries resulted in formation of tubular-structured blood vessels. The application of hydrogels onto wounds does not impair the wound healing process. We have adapted the DSFC as a useful model for standardized testing of angiogenic biomaterials during wound healing. Advantages of this model imply in vivo imaging of angiogenesis up to 3 weeks in a human-like wound healing model without muscle induced wound contraction that allows direct correlation of angiogenic effects with wound healing events. We are currently analyzing the impact of star-PEG-heparin hydrogels loaded with pro-angiogenic factors on angiogenesis and wound healing outcome using our DSFC wound healing model.

Hypoxia Inhibits De Novo Vascular Assembly of Adipose-Derived Stromal/Stem Cell Populations, but Promotes Growth of Preformed Vessels.

Vascularization is critical for cell survival within tissue-engineered grafts. Adipose-derived stromal/stem cells (ASCs) are widely used in tissue engineering applications as they are a clinically relevant source of stem cells and endothelial progenitor cells. ASCs have previously been shown to self-assemble into pericyte-stabilized vascular networks in normoxic (20% O2) cultures. This capacity for de novo vascular assembly may accelerate graft vascularization in vivo rather than relying solely on angiogenic ingrowth. However, oxygen depletion within large cell-seeded grafts will be rapid, and it is unclear how this worsening hypoxic environment will impact the vascular assembly of the transplanted cells. The objectives of this study were to determine whether ASC-derived vessels could grow in hypoxia and to assess whether the vessel maturity (i.e., individual cells vs. preformed vessels) influenced this hypoxic response. Utilizing an in vitro vascularization model, ASCs were encapsulated within fibrin gels and cultured in vitro for up to 6 days in either normoxia (20% O2) or hypoxia (0.2% or 2% O2). In a subsequent experiment, vessels were allowed to preform in normoxia for 6 days before an additional 6 days of either normoxia or hypoxia. Viability, vessel growth, pericyte coverage, proliferation, metabolism, and angiogenic factor expression were assessed for each experimental approach. Vessel growth was dramatically inhibited in both moderate and severe hypoxia (47% and 11% total vessel length vs. normoxia, respectively), despite maintaining high cell viability and upregulating endogenous expression of vascular endothelial growth factor in hypoxia. Bromodeoxyuridine labeling indicated significantly reduced proliferation of endothelial cells in hypoxia. In contrast, when vascular networks were allowed to preform for 6 days in normoxia, vessels not only survived but also continued to grow more in hypoxia than those maintained in normoxia. These findings demonstrate that vascular assembly and growth are tightly regulated by oxygen tension and may be differentially affected by hypoxic conditions based on the maturity of the vessels. Understanding this relationship is critical to developing effective approaches to engineer viable tissue-engineered grafts in vivo.

RNAi-mediated knockdown of vascular endothelial growth factor inhibits vascularization and tumor growth in renal cell carcinoma.

In the present study, we aimed to examine the effects of the knockdown of vascular endothelial growth factor(VEGF) by RNA interference (RNAi) on vascularization and tumor growth in renal cell carcinoma(RCC). For this purpose, a lentiviral vector expressing VEGF-shRNA was constructed and transfected into 293Tcells. The efficiency of RNAi was determined by infecting human 786-O RCC cells with viral particles and measuring the VEGF mRNA levels by reverse transcription-quantitative polymerase chain reaction(RT-qPCR). The effect of transfection with VEGF‑shRNA on the secreted VEGF levels was also examined and the inhibitory effects on vascularization were also examined using a chick chorioallantoic membrane(CAM) assay. An RCC xenograft model was established in nude mice by implanting 786-Ocells to form subcutaneous tumors. VEGF expression was observed by immunohistochemical(IHC) staining of the xenograft tumors. The tumor volume and tumor inhibition rate were also recorded. The apoptosis of the cancer cells was measured by TUNEL assay and the efficiency of tumor inhibition was estimated. The interference rate of VEGF‑shRNA was 72.2%in the 786-O cells. Our results revealed that VEGF mRNA expression, the secreted VEGF level in the 786-O cells and the total vessel length were markedly reduced in the VEGF‑shRNA-transfected cells compared with the controls(all P<0.05). Compared with the controls, injections of lentivirus expressing VEGF-shRNA significantly inhibited tumor growth, and reduced tumor mass and VEGF expression in the tumor tissue(all P<0.05). The apoptotic index in the treatment group was significantly higher than that in the controls(bothP<0.05). Thus, our data indicate that the inhibition of VEGF expression by RNAi reduces VEGF mRNA levels, and inhibits angiogenesis and tumor growth in RCC, providing a future treatment option for RCC.

Improvement of in vivo anticancer and antiangiogenic potential of thalidomide derivatives

The strategy of antiangiogenic drugs is based on inhibiting formation of new blood vessels as alternative to limit cancer progression. In this work, we investigated the antitumor and antiangiogenic potential of eight thalidomide derivatives. Most of the molecules was not cytotoxic but 2a, 2d and 3d revealed weak antiproliferative activity on HL-60, Sarcoma 180 (S180) and normal peripheral blood mononuclear cells. Thalidomide, 2a and 2b were able to inhibit tumor growth (53.5%, 67.9% and 67.4%, respectively) in S180-bearing mice and presented moderate and reversible toxicity on liver, kidneys and spleens. Both analogs (2a and 2b) inhibited cell migration of endothelial (HUVEC) and melanoma cells (MDA/MB-435) at 50μg/mL. Immunohistochemistry labeling assays with CD-31 (PECAM-1) antibody showed microvascular density (MVD) was significantly reduced in thalidomide, 2a and 2b groups (30±4.9, 64.6±1.8 and 46.5±19.5%, respectively) (p<0.05). Neovascularization evaluated by Chorioallantoic Membrane Assay (CAM) with compounds 2a and 2b showed reduction of vessels’ number (12. 9±2.3 and 14.8±3.3%), neovascularization area (13.1±1.7 and 14.3±1.7%) and total length of vessels (9.2±1.5 and 9.9±1.9%). On the other hand, thalidomide did not alter vascularization parameters. Consequently, addition of thiosemicarbazone pharmacophore group into the phthalimidic ring improved the in vivo antitumor and antiangiogenic potential of the analogs 2a and 2b.

Design of Polymer Scaffolds for Tissue Engineering Applications

Agent-based models (ABMs) provide a flexible multilayer platform for incorporating various modeling techniques into a single hybrid model for designing optimal biomaterial scaffolds for angiogenesis in tissue engineering applications. Scaffold geometrical variables are considered as design variables. The growth-factor concentration profile is the only process variable considered in this study. The product variables used to illustrate the combined effects of scaffold design variables and process variables on the outcome of angiogenesis include the density and depth of capillary invasion within the scaffold. The scaffold design process and the ABM developed to simulate angiogenesis are described in this article. The performance of the ABM and the vascularization of the polymer scaffolds are evaluated by simulation studies. The effects of pore size, pore size distribution, and interconnectivity on the total blood vessel length, invasion depth, and total number of sprouts formed during the vascularization process are reported. The integration of the simulation of angiogenesis with ABMs and scaffold design techniques provides an iterative process for designing optimal scaffold structures. Such an approach facilitates faster design of optimized scaffolds with significantly less cost and enables better understanding of the mechanisms of angiogenesis of polymer scaffolds for tissue engineering applications.

Regulation of valve endothelial cell vasculogenic network architectures with ROCK and Rac inhibitors

ObjectiveThe age- and disease-dependent presence of microvessels within heart valves is an understudied characteristic of these tissues. Neovascularization involves endothelial cell (EC) migration and cytoskeletal reorientation, which are heavily regulated by the Rho family of GTPases. Given that valve ECs demonstrate unique mesenchymal transdifferentiation and cytoskeletal mechanoresponsiveness, compared to vascular ECs, this study quantified the effect of inhibiting two members of the Rho family on vasculogenic network formation by valve ECs. Approach and resultsA tubule-like structure vasculogenesis assay (assessing lacunarity, junction density, and vessel density) was performed with porcine aortic valve ECs treated with small molecule inhibitors of Rho-associated serine-threonine protein kinase (ROCK), Y-27632, or the Rac1 inhibitor, NSC-23766. Actin coordination, cell number, and cell migration were assessed through immunocytochemistry, MTT assay, and scratch wound healing assay. ROCK inhibition reduced network lacunarity and interrupted proper cell–cell adhesion and actin coordination. Rac1 inhibition increased lacunarity and delayed actin-mediated network formation. ROCK inhibition alone significantly inhibited migration, whereas both ROCK and Rac1 inhibition significantly reduced cell number over time compared to controls. Compared to a vascular EC line, the valve ECs generated a network with larger total vessel length, but a less smooth appearance. ConclusionsBoth ROCK and Rac1 inhibition interfered with key processes in vascular network formation by valve ECs. This is the first report of manipulation of valve EC vasculogenic organization in response to small molecule inhibitors. Further study is warranted to comprehend this facet of valvular cell biology and pathology and how it differs from vascular biology.

Topical fentanyl stimulates healing of ischemic wounds in diabetic rats.

Topically applied opioids promote angiogenesis and healing of ischemic wounds in rats. We examined if topical fentanyl stimulates wound healing in diabetic rats by stimulating growth-promoting signaling, angiogenesis, lymphangiogenesis and nerve regeneration. We used Zucker diabetic fatty rats that develop obesity and diabetes on a high fat diet due to a mutation in the Leptin receptor. Fentanyl blended with hydrocream was applied topically on ischemic wounds twice daily, and wound closure was analyzed regularly. Wound histology was analyzed by hematoxylin and eosin staining. Angiogenesis, lymphangiogenesis, nerve fibers and phospho-platelet derived growth factor receptor-β (PDGFR-β) were visualized by CD31-, lymphatic vessel endothelium-1, protein gene product 9.5- and anti-phospho PDGFR-β-immunoreactivity, respectively. Nitric oxide synthase (NOS) and PDGFR-β signaling were analyzed using Western immunoblotting. Fentanyl significantly promoted wound closure as compared to phosphate-buffered saline (PBS). Histology scores were significantly higher in fentanyl-treated wounds, indicative of increased granulation tissue formation, reduced edema and inflammation, and increased matrix deposition. Fentanyl treatment resulted in increased wound angiogenesis, lymphatic vasculature, nerve fibers, nitric oxide, NOS and PDGFR-β signaling as compared to PBS. Phospho-PDGFR-β co-localized with CD31 co-staining for vasculature. Topically applied fentanyl promotes closure of ischemic wounds in diabetic rats. Increased angiogenesis, lymphangiogenesis, peripheral nerve regeneration, NO and PDGFR-β signaling are associated with fentanyl-induced tissue remodeling and wound healing.

Trawling fishery of the western Mediterranean Sea: Métiers identification, effort characteristics, landings and income profiles

Due to the diversity of fleet characteristics and catch composition in Mediterranean fisheries, the General Fisheries Commission for the Mediterranean has placed emphasis on the direct control of fishing capacity and effort rather than catch limitation. This paper aims to analyse the evolution of fishing effort, landings and métiers in three trawling ports of the western Mediterranean between 2002 and 2011. Four métiers were identified, using multivariate techniques, in the fishery: Red mullet, European hake, Norway lobster and Red shrimp. The mean catch composition within the same métier was similar between ports, although the relative importance of species differed slightly from one port to another. Variation in fishing effort exerted was observed between métiers reflecting differences in the importance and usage of each métier by port. Temporal variations were found among different métiers at seasonal and annual scales. These temporal variations showed that métiers Red mullet and European hake were alternated. Positive significant correlations were observed between landings and different fishing effort variables with an exception of the relation between landings and engine power in the Norway lobster métier with negative significant correlations. Significant differences in total vessel length, gross tonnage, total CPUE and total income were detected among the four métiers depending on each port with few exceptions. These findings are considered a keystone for more practical implications and to assist fisheries' managers in the decision-making process. As demonstrated in the current paper, the management of multi-specific fisheries should start off considering the characteristics of each métier separately. In the sound of métier-based management, the patterns observed in the effort temporal distribution between métiers can indicate the most suitable time to reduce effort on specific target species.

Abstract 1439: Combination of metformin plus orlistat prevents tumor progression: novel role of the metabolic hormone fibroblast growth factor 21 (FGF21)

Abstract Caloric Restriction (CR) is known to be the only model to consistently increase the lifespan and postpone age-related diseases in all species. CR increases glycogenolysis, lipolysis with fatty acid and ketone body utilization and accelerates protein catabolism. We previously reported that CR induces metabolic and signaling changes that affect the tumor microenvironment preventing the growth of 4T1 mammary tumors and their metastases. 4T1 is a highly metastatic mouse breast cancer model which resembles breast cancer in patients. Obesity causes subclinical inflammation in adipose tissue that contributes to insulin resistance and cancer progression. Chronic inflammation predisposes to different forms of cancer and diabetes, and is correlated with increased risk of breast cancer. CR reduces insulin resistance, adiposity and inflammation and the anti-diabetic drug metformin (MET) is a CR mimetic agent. In this study, we evaluated whether the combination of MET with the anti-obesity drug orlistat (OR) enhances the CR mimetic and anti-cancer properties of MET on 4T1 mammary tumor cells. Female 8 week-old BALB/c mice received: vehicle, MET (in drinking water, 3mg/ml), OR i.p. injection (240mg/kg/day) or MET + OR. After 3 weeks on drugs, 4T1 cells (105) were injected into mice and drugs continued till the end of the experiment. Treated-mice had a reduced adiposity (p&amp;lt;0.05) without any signs of toxicity. At 30 days, MET + OR co-treatment reduced tumor volume (p&amp;lt;0.01) and displayed a range of cellular alterations such as decreased proliferative index (p&amp;lt;0.05); increased apoptotic rate (p&amp;lt;0.01) and altered intra-tumor collagen deposit. MET + OR treatment reduced total vessel length (p&amp;lt;0.01) and the number of spontaneous lung metastases (p&amp;lt;0.01). In addition, combinations of MET (1-2.5mM) + OR (2.5-5µM) decreased in vitro 4T1 mammary cell proliferation (p&amp;lt;0.01), adhesion (p&amp;lt;0.05) and migration (p&amp;lt;0.01) compared to vehicle and drugs alone. Interestingly, we found that levels of fibroblast growth factor 21 (FGF21), an endocrine factor that regulates glucose and lipid metabolism, were significantly increased with MET + OR treatment (p&amp;lt;0.05). It has been shown that increased circulating levels of FGF21 correlated with decreased obesity and increased sensitivity to insulin; but up to date the role of FGF21 in tumor progression was not elucidated. We found that treatment of 4T1 cells with MET + OR increased the secretion of FGF21 (p&amp;lt;0.05) measured by ELISA assays. To elucidate the role of FGF21; we initially demonstrated that addition of recombinant protein FGF21 (0-10nM) did not change 4T1 cell proliferation. In contrast, the reduced cell proliferation and adhesion induced by MET + OR, were abolished by the pre- and co-treatment with anti-FGF21 neutralizing antibody; indicating that FGF21 may play an important role. Our results provide a new rationale basis for the use of MET plus OR as cancer therapy. Citation Format: Shobika Sivaram, Erdene Baljinnyam, Kousaku Iwatsubo, Lydia I. Puricelli, Mariana S. De Lorenzo. Combination of metformin plus orlistat prevents tumor progression: novel role of the metabolic hormone fibroblast growth factor 21 (FGF21). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1439. doi:10.1158/1538-7445.AM2014-1439

Intrinsic subtypes and tumor grades in breast cancer are associated with distinct 3-D power Doppler sonographic vascular features

PurposeThis study aimed to investigate the three-dimensional (3-D) power Doppler ultrasonographic (PDUS) vascular features of breast carcinoma according to intrinsic subtypes, nodal stage, and tumor grade. Materials and methodsTotal 115 receiving mastectomy breast carcinomas (mean size, 2.5cm; range, 0.7–6.5cm), including 102 invasive ductal carcinomas (IDC), 10 ductal carcinomas in situ (DCIS), and 3 invasive lobular carcinomas (ILC) diagnosed after mastectomy, were used in this retrospective study. Sixty IDC had nodal status and histopathologic tumor grades available for analysis. Vascular features, including number of vascular trees (NV), longest path length (LPL), total vessel length (TVL), number of bifurcations (NB), distance metric (DM), inflection count metric (ICM), vessel diameter (VD), and vessel-to-volume ratio (VVR) were extracted using 3-D thinning method. The Mann–Whitney U test, Student's t-test, one-way ANOVA, and Kruskal–Wallis test were performed as appropriate. ResultsThere was no significant difference of vascular features among IDC, DCIS and ILC. Except VD, vascular features in luminal type were significantly lower compared to HER2-enriched or triple negative types (p<0.05). Compared to ER+ (estrogen receptor positive) tumors, all features in ER− (estrogen receptor negative) tumors were significantly higher (p<0.01). Despite some significantly higher vascular features in high grade IDC compared to low and intermediate grade, there was no significant correlation between vascular features and nodal stages. ConclusionDifferences in 3-D PDUS vascular features among intrinsic types of IDC are attributed to their ER status. Vascular features extracted by 3-D PDUS correlate with tumor grades but not nodal stage in IDC.

Parameter sensitivity analysis of a lumped-parameter model of a chain of lymphangions in series

Any disruption of the lymphatic system due to trauma or injury can lead to edema. There is no effective cure for lymphedema, partly because predictive knowledge of lymphatic system reactions to interventions is lacking. A well-developed model of the system could greatly improve our understanding of its function. Lymphangions, defined as the vessel segment between two valves, are the individual pumping units. Based on our previous lumped-parameter model of a chain of lymphangions, this study aimed to identify the parameters that affect the system output the most using a sensitivity analysis. The system was highly sensitive to minimum valve resistance, such that variations in this parameter caused an order-of-magnitude change in time-average flow rate for certain values of imposed pressure difference. Average flow rate doubled when contraction frequency was increased within its physiological range. Optimum lymphangion length was found to be some 13-14.5 diameters. A peak of time-average flow rate occurred when transmural pressure was such that the pressure-diameter loop for active contractions was centered near maximum passive vessel compliance. Increasing the number of lymphangions in the chain improved the pumping in the presence of larger adverse pressure differences. For a given pressure difference, the optimal number of lymphangions increased with the total vessel length. These results indicate that further experiments to estimate valve resistance more accurately are necessary. The existence of an optimal value of transmural pressure may provide additional guidelines for increasing pumping in areas affected by edema.