Angiogenic Network Research Articles

Exploring the Extracellular Regulation of the Tumor Angiogenic Interaction Network Using a Systems Biology Model.

Tumor angiogenesis is regulated by pro- and anti-angiogenic factors. Anti-angiogenic agents target the interconnected network of angiogenic factors to inhibit neovascularization, which subsequently impedes tumor growth. Due to the complexity of this network, optimizing anti-angiogenic cancer treatments requires detailed knowledge at a systems level. In this study, we constructed a tumor tissue-based model to better understand how the angiogenic network is regulated by opposing mediators at the extracellular level. We consider the network comprised of two pro-angiogenic factors: vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2), and two anti-angiogenic factors: thrombospondin-1 (TSP1) and platelet factor 4 (PF4). The model’s prediction of angiogenic factors’ distribution in tumor tissue reveals the localization of different factors and indicates the angiogenic state of the tumor. We explored how the distributions are affected by the secretion of the pro- and anti-angiogenic factors, illustrating how the angiogenic network is regulated in the extracellular space. Interestingly, we identified a counterintuitive result that the secretion of the anti-angiogenic factor PF4 can enhance pro-angiogenic signaling by elevating the levels of the interstitial and surface-level pro-angiogenic species. This counterintuitive situation is pertinent to the clinical setting, such as the release of anti-angiogenic factors in platelet activation or the administration of exogenous PF4 for anti-angiogenic therapy. Our study provides mechanistic insights into this counterintuitive result and highlights the role of heparan sulfate proteoglycans in regulating the interactions between angiogenic factors. This work complements previous studies aimed at understanding the formation of angiogenic complexes in tumor tissue and helps in the development of anti-cancer strategies targeting angiogenesis.

Proteomic analysis of human mesenchymal stromal cell secretomes: a systematic comparison of the angiogenic potential

Human mesenchymal stromal cell (hMSC) secretomes have shown to influence the microenvironment upon injury, promoting cytoprotection, angiogenesis, and tissue repair. The angiogenic potential is of particular interest for the treatment of ischemic diseases. Interestingly, hMSC secretomes isolated from different tissue sources have shown dissimilarities with respect to their angiogenic profile. This study compares angiogenesis of hMSC secretomes from adipose tissue (hADSCs), bone marrow (hBMSCs), and umbilical cord Wharton’s jelly (hWJSCs). hMSC secretomes were obtained under xenofree conditions and analyzed by liquid chromatography tandem mass spectrometry (LC/MS-MS). Biological processes related to angiogenesis were found to be enriched in the proteomic profile of hMSC secretomes. hWJSC secretomes revealed a more complete angiogenic network with higher concentrations of angiogenesis related proteins, followed by hBMSC secretomes. hADSC secretomes lacked central angiogenic proteins and expressed most detected proteins to a significantly lower level. In vivo all secretomes induced vascularization of subcutaneously implanted Matrigel plugs in mice. Differences in secretome composition were functionally analyzed with monocyte and endothelial cell (EC) in vitro co-culture experiments using vi-SNE based multidimensional flow cytometry data analysis. Functional responses between hBMSC and hWJSC secretomes were comparable, with significantly higher migration of CD14++ CD16− monocytes and enhanced macrophage differentiation compared with hADSC secretomes. Both secretomes also induced a more profound pro-angiogenic phenotype of ECs. These results suggest hWJSCs secretome as the most potent hMSC source for inflammation-mediated angiogenesis induction, while the potency of hADSC secretomes was lowest. This systematic analysis may have implication on the selection of hMSCs for future clinical studies.

Stromal Vascular Fraction Vasculogenesis, Vessel Incorporation, and Integration with Intact Angiogenic Microvascular Networks in an Ex Vivo Cultured Tissue Model

Angiogenesis, the growth of new vessels from pre‐existing vessels, is vital to wound healing in the cases of myocardial infarction and peripheral arterial disease. Adipose‐derived stromal vascular fraction (SVF), consisting of endothelial cells, endothelial progenitor cells, pericytes, smooth muscle cells, fibroblasts and immune cells, can be transplanted to assist in wound healing as seen in various clinical trials. However, the spatiotemporal incorporation of the SVF within living tissues remains largely unknown. As such, understanding where and how SVF cells contribute to microvascular growth and remodeling will help guide their therapeutic use. The objective of this study was to evaluate the impact of SVF cells and their fate during microvascular growth utilizing a novel tissue culture model that enables time‐lapse observation of cell dynamics across intact networks. SVF was isolated from adipose of an adult male Wistar rat, labeled with DiI, and seeded onto mesentery tissues harvested from the same rat before culturing in MEM + 10% fetal bovine serum and 1% PenStrep for 3 days. Tissues were then labeled for lectin to identify vessels. Alternatively, SVF was isolated from adipose harvested from adult male or female SD‐EGFP rats, seeded onto lectin labeled mesentery from adult male Wistar rats, placed under the same culture conditions, and imaged every 24 hours. Vascularized area was increased in SVF treated versus control tissues (SVF = 84.5±1.3%; Control = 56.5%; p < 0.001; n = 4 tissues per group). The SVF related microvasculature was characterized by asterisk‐shaped clusters. GFP tracking of SVF revealed vessel segment formation via vasculogenesis containing endothelial cells and pericytes in previously avascular areas. In pre‐vascularized regions, SVF cells contributed to new vessel growth and integration with and along native vessels. SVF cells were observed both in apparent endothelial cell and wrapping pericyte locations. Interestingly, examples of new vessel segments derived from SVF cells were observed along LYVE‐1 positive lymphatic vessels. These results suggest that SVF transplantation increases vascular area through vasculogenesis and contributes to angiogenesis of pre‐existing vasculature. The results also support the use of the rat mesentery culture model as a novel tool for elucidating SVF cell transplant dynamics in an intact tissue environment.Support or Funding InformationFunded by NIH R01AG049821This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Potential Role of Dietary Platelet-Activating Factor Inhibitors in Cancer Prevention and Treatment.

Cancer is the second leading cause of mortality worldwide. The role of unresolved inflammation in cancer progression and metastasis is well established. Platelet-activating factor (PAF) is a key proinflammatory mediator in the initiation and progression of cancer. Evidence suggests that PAF is integral to suppression of the immune system and promotion of metastasis and tumor growth by altering local angiogenic and cytokine networks. Interactions between PAF and its receptor may have a role in various digestive, skin, and hormone-dependent cancers. Diet plays a critical role in the prevention of cancer and its treatment. Research indicates that the Mediterranean diet may reduce the incidence of several cancers in which dietary PAF inhibitors have a role. Dietary PAF inhibitors such as polar lipids have demonstrated inhibitory effects against the physiological actions of PAF in cancer and other chronic inflammatory conditions in vitro and in vivo. In addition, experimental models of radiotherapy and chemotherapy demonstrate that inhibition of PAF as adjuvant therapy may lead to more favorable outcomes. Although promising, there is limited evidence on the potential benefits of dietary PAF inhibitors on cancer prevention or treatment. Therefore, further extensive research is required to assess the effects of various dietary factors and PAF inhibitors and to elucidate the mechanisms in prevention of cancer progression and metastasis at a molecular level.

Quantitative assessment of angiogenic microvasculature using Ultrasound multiple scattering with microbubble contrast agents

In tumors, angiogenesis (formation of new blood vessels) is established as a biomarker of malignancy. A random, isotropic, high-density vessel network is linked to tumor invasiveness. Ultrasound quantification of angiogenic micro-architecture could help increase the specificity of ultrasound for cancer diagnosis. We exploit multiple scattering by microbubbles populating angiogenic networks to characterize an effective medium diffusivity via the diffusion constant (D). The inter-element response matrix is acquired using an L11-4v linear array transducer operating at 7.8 MHz. D is computed from the time evolution of the incoherent contribution to the backscattered intensity in the near field. D is measured in fibrosarcoma tumors subcutaneously implanted in rats (n = 16), and in control, healthy tissue (n = 18). D is measured for two different orientations (Coronal and Transverse) and the anisotropy of the microvasculature is evaluated via the ratio of the D values obtained in the two different orientations. D was found significantly different between control (1.38 ± 0.51 mm2/μs) and tumor (0.65 ± 0.27 mm2/μs) (p<0.01) and anisotropy of the angiogenic network was observed in control cases (1.62 ± 0.911). For further validation, these results were corroborated with vascular density measurements from acoustic angiography data, confirming increased vessel density in tumors compared to controls.

Multicell migration tracking within angiogenic networks by deep learning-based segmentation and augmented Bayesian filtering.

.Cell migration is a key feature for living organisms. Image analysis tools are useful in studying cell migration in three-dimensional (3-D) in vitro environments. We consider angiogenic vessels formed in 3-D microfluidic devices (MFDs) and develop an image analysis system to extract cell behaviors from experimental phase-contrast microscopy image sequences. The proposed system initializes tracks with the end-point confocal nuclei coordinates. We apply convolutional neural networks to detect cell candidates and combine backward Kalman filtering with multiple hypothesis tracking to link the cell candidates at each time step. These hypotheses incorporate prior knowledge on vessel formation and cell proliferation rates. The association accuracy reaches 86.4% for the proposed algorithm, indicating that the proposed system is able to associate cells more accurately than existing approaches. Cell culture experiments in 3-D MFDs have shown considerable promise for improving biology research. The proposed system is expected to be a useful quantitative tool for potential microscopy problems of MFDs.

Quantitative analysis of the angiogenic microvasculature in tumor using multiple scattering and dual-frequency transducers

The objective is to characterize angiogenic networks using contrast-enhanced multiple scattering. The methods relies on the measurement of the diffusion constant from the time evolution of the incoherent backscattered intensity. Dual-frequency arrays allow to emit a pulse at a frequency close to resonance frequency of microbubbles, and to receive the backscattered echoes at higher harmonics, resulting in better contrast-to-tissue ratio compared to single frequency transducers. At 8 MHz, we demonstrated that the diffusion constant enables the quantification of the vascular density and anisotropy in tumor-related vasculature in rat models in vivo. We show here that the results could be further improved using a dual-frequency approach, using two linear arrays with different central frequencies (7 MHz/18 MHz) and using a custom made dual frequency transducer (3 MHz/15 MHz). The minimum detectable distance between two vessel-mimicking cellulose tubes filled with contrast agents was measured via the diffusion constant. The dual-frequency approach drastically reduced the smallest detectable distance between tubes, which was found to be 25µm using a single frequency, 20µm using two linear arrays and 10µm using the custom dual-frequency array, respectively. These results show that measuring the diffusion constant with a dual frequency array can enable the quantification of vessel density with high accuracy

Dynamic, heterogeneous endothelial Tie2 expression and capillary blood flow during microvascular remodeling

Microvascular endothelial cell heterogeneity and its relationship to hemodynamics remains poorly understood due to a lack of sufficient methods to examine these parameters in vivo at high resolution throughout an angiogenic network. The availability of surrogate markers for functional vascular proteins, such as green fluorescent protein, enables expression in individual cells to be followed over time using confocal microscopy, while photoacoustic microscopy enables dynamic measurement of blood flow across the network with capillary-level resolution. We combined these two non-invasive imaging modalities in order to spatially and temporally analyze biochemical and biomechanical drivers of angiogenesis in murine corneal neovessels. By stimulating corneal angiogenesis with an alkali burn in Tie2-GFP fluorescent-reporter mice, we evaluated how onset of blood flow and surgically-altered blood flow affects Tie2-GFP expression. Our study establishes a novel platform for analyzing heterogeneous blood flow and fluorescent reporter protein expression across a dynamic microvascular network in an adult mammal.

Tie2 as a novel key factor of microangiopathy in systemic sclerosis

BackgroundThe angiopoietin(Ang)/Tie2 system is a key regulator of vascular biology. The expression of membrane bound (mb) Tie2 and Ang-1 ensures vessel stability, whereas Ang-2, inducible by vascular endothelial growth factor (VEGF), hypoxia, and inflammation, acts as an antagonist. Tie2 signalling is also attenuated by soluble Tie2 (sTie2), the extracellular domain of the receptor, which is shed upon stimulation with VEGF. Herein, we investigate the role of Ang/Tie2 in the peripheral vasculopathy in systemic sclerosis (SSc) including animal models.MethodsThe expression of Ang-1/-2 and Tie2 in skin/serum of SSc patients was compared with healthy controls by immunohistochemistry (IHC)/ELISA. Expression of Ang/Tie2 was analysed in different animal models: VEGF transgenic (tg) mice, hypoxia model, bleomycin-induced skin fibrosis, and tight skin 1 (TSK1) mice.ResultsIn SSc, dermal microvessels abundantly expressed Ang-2, but not Ang-1 compared with healthy controls. The percentage of mbTie2+ microvessels was profoundly decreased whereas the levels of sTie2 were increased already in early disease. Both in skin and sera of SSc patients, the Ang1/2 ratio was reduced, being lowest in patients with digital ulcers indicating vessel destabilizing conditions. We next studied potential influencing factors in animal models. The VEGF tg mouse model, the hypoxia, and the inflammation-dependent bleomycin model all showed a similar dysregulation of Ang/Tie2 as in SSc, which did not apply for the non-inflammatory TSK1 model.ConclusionPeripheral microvasculopathy in SSc results from a complex dysregulation of angiogenic signalling networks including the VEGF and the Ang/Tie2 system. The profoundly disturbed Ang-/Tie-2 balance might represent an important target for vascular therapeutic approaches in SSc.

Assessment of Heparanase-Mediated Angiogenesis Using Microvascular Endothelial Cells: Identification of λ-Carrageenan Derivative as a Potent Anti Angiogenic Agent.

Heparanase is overexpressed by tumor cells and degrades the extracellular matrix proteoglycans through cleavage of heparan sulfates (HS), allowing pro-angiogenic factor release and thus playing a key role in tumor angiogenesis and metastasis. Here we propose new HS analogs as potent heparanase inhibitors: Heparin as a positive control, Dextran Sulfate, λ-Carrageenan, and modified forms of them obtained by depolymerization associated to glycol splitting (RD-GS). After heparanase activity assessment, 11 kDa RD-GS-λ-Carrageenan emerged as the most effective heparanase inhibitor with an IC50 of 7.32 ng/mL compared to 10.7 ng/mL for the 16 kDa unfractionated heparin. The fractionated polysaccharides were then tested in a heparanase-rich medium-based in vitro model, mimicking tumor microenvironment, to determine their effect on microvascular endothelial cells (HSkMEC) angiogenesis. As a preliminary study, we identified that under hypoxic and nutrient poor conditions, MCF-7 cancer cells released much more mature heparanase in their supernatant than in normal conditions. Then a MatrigelTM assay using HSkMEC cultured under hypoxic conditions in the presence (or not) of this heparanase-rich supernatant was realized. Adding heparanase-rich media strongly enhanced angiogenic network formation with a production of twice more pseudo-vessels than with the control. When sulfated polysaccharides were tested in this angiogenesis assay, RD-GS-λ-Carrageenan was identified as a promising anti-angiogenic agent.

Ensemble Averages, Soliton Dynamics and Influence of Haptotaxis in a Model of Tumor-Induced Angiogenesis

In this work, we present a numerical study of the influence of matrix degrading enzyme (MDE) dynamics and haptotaxis on the development of vessel networks in tumor-induced angiogenesis. Avascular tumors produce growth factors that induce nearby blood vessels to emit sprouts formed by endothelial cells. These capillary sprouts advance toward the tumor by chemotaxis (gradients of growth factor) and haptotaxis (adhesion to the tissue matrix outside blood vessels). The motion of the capillaries in this constrained space is modelled by stochastic processes (Langevin equations, branching and merging of sprouts) coupled to continuum equations for concentrations of involved substances. There is a complementary deterministic description in terms of the density of actively moving tips of vessel sprouts. The latter forms a stable soliton-like wave whose motion is influenced by the different taxis mechanisms. We show the delaying effect of haptotaxis on the advance of the angiogenic vessel network by direct numerical simulations of the stochastic process and by a study of the soliton motion.

How tumour-induced vascular changes alter angiogenesis: Insights from a computational model

A computational model was developed to describe experimentally observed vascular changes induced by the introduction of a tumour on a mouse equipped with a dorsal skinfold chamber. The vascular structure of the host tissue was segmented from in vivo images and transposed into the computational framework. Simulations of tumour-induced vascular changes were performed and include the destabilizing effects of the growth factor VEGF on the integrity of the vessels walls. The integration of those effects, that include alteration of the vessel wall elasticity and wall breaching, were required to realistically reproduce the experimental observations. The model was then used to investigate the importance of the vascular changes for oxygen delivery and tumour development. To that end, we compared simulations obtained with a dynamic vasculature with those obtained with a static one. The results showed that the tumour growth was strongly impeded by the constant vascular changes. More precisely, it is the angiogenic process itself that was affected by vascular changes occurring in bigger upstream vessels and resulting in a less efficient angiogenic network for oxygen delivery. As a consequence, tumour cells are mostly kept in a non-proliferative hypoxic state. Tumour dormancy thus appears as one potential consequence of the intense vascular changes in the host tissue.

A Validated Multiscale In-Silico Model for Mechano-sensitive Tumour Angiogenesis and Growth

Vascularisation is a key feature of cancer growth, invasion and metastasis. To better understand the governing biophysical processes and their relative importance, it is instructive to develop physiologically representative mathematical models with which to compare to experimental data. Previous studies have successfully applied this approach to test the effect of various biochemical factors on tumour growth and angiogenesis. However, these models do not account for the experimentally observed dependency of angiogenic network evolution on growth-induced solid stresses. This work introduces two novel features: the effects of hapto- and mechanotaxis on vessel sprouting, and mechano-sensitive dynamic vascular remodelling. The proposed three-dimensional, multiscale, in-silico model of dynamically coupled angiogenic tumour growth is specified to in-vivo and in-vitro data, chosen, where possible, to provide a physiologically consistent description. The model is then validated against in-vivo data from murine mammary carcinomas, with particular focus placed on identifying the influence of mechanical factors. Crucially, we find that it is necessary to include hapto- and mechanotaxis to recapitulate observed time-varying spatial distributions of angiogenic vasculature.

Talin Modulation by a Synthetic N-Acylurea Derivative Reduces Angiogenesis in Human Endothelial Cells.

Talin is a focal adhesion protein that activates integrins and recruits other focal adhesion proteins. Talin regulates the interactions between integrins and the extracellular matrix, which are critical for endothelial cells during angiogenesis. In this study, we successfully synthesized a novel talin modulator, N-((2-(1H-indol-3-yl)ethyl)carbamoyl)-2-(benzo[d][1,3]dioxol-5-yloxy)acetamide, referred to as KCH-1521. KCH-1521 was determined to bind talin and modulate downstream signaling molecules of talin. After 24 h of treatment, KCH-1521 changed the cell morphology of human umbilical vein endothelial cells (HUVECs) and reduced focal adhesion protein expression including vinculin and paxillin. Talin downstream signaling is regulated via focal adhesion kinase (FAK), kinase B (AKT), and extracellular signal-regulated kinase (ERK) pathways, however, treatment with KCH-1521 decreased phosphorylation of FAK, AKT, and ERK, leading to reduction of cell proliferation, survival, and angiogenesis. Interestingly, the expression of various angiogenic genes was significantly decreased after treatment with KCH-1521. Also, in vitro tube forming assay revealed that KCH-1521 reduced angiogenic networks in a time-dependent manner. To investigate the reversibility of its effects, KCH-1521 was removed after treatment. HUVECs recovered their morphology through rearrangement of the cytoskeleton and the expression of angiogenic genes was also recovered. By further optimization and in vivo studies of KCH-1521, a novel drug of talin modulation could be used to achieve therapeutic anti-angiogenesis for vascular diseases and cancers.

Quantification of antiangiogenic treatment effects on tissue heterogeneity in glioma tumour xenograft model using a combination of DCE-MRI and 3D-ultramicroscopy.

This study aimed at assessing the effects of an anti-angiogenic treatment, which neutralises vascular endothelial growth factor (VEGF), on tumour heterogeneity. Murine glioma cells have been inoculated into the right brain frontal lobe of 16 mice. Anti-VEGF antibody was administered to a first group (n = 8), while a second group (n = 8) received a placebo. Magnetic resonance acquisitions, performed at days 10, 12, 15 and 23 following the implantation, allowed the derivation of a three-dimensional features dataset characterising tumour heterogeneity. Three-dimensional ultramicroscopy and standard histochemistry analysis have been performed to verify in vivo results. Placebo-treated mice displayed a highly-vascularised area at the tumour periphery, a monolithic necrotic core and a chaotic dense vasculature across the entire tumour. In contrast, the B20-treated group did not show any highly vascularised regions and presents a fragmented necrotic core. A significant reduction of the number of vessel segments smaller than 17μm has been observed. There was no difference in overall tumour volume and growth rate between the two groups. Region-specific analysis revealed that VEGF inhibition affects only: (1) highly angiogenic compartments expressing high levels of VEGF and characterised by small capillaries, and also (2) the formation and structure of necrotic regions. These effects appear to be transient and limited in time. • VEGF inhibition affects only the highly angiogenic region and small capillaries network • VEGF inhibition is transient in time • Tumour volume is not affected by anti-angiogenic treatment • VEGF inhibition also influences the architecture of necrotic regions.

Computational Screening of Tip and Stalk Cell Behavior Proposes a Role for Apelin Signaling in Sprout Progression.

Angiogenesis involves the formation of new blood vessels by sprouting or splitting of existing blood vessels. During sprouting, a highly motile type of endothelial cell, called the tip cell, migrates from the blood vessels followed by stalk cells, an endothelial cell type that forms the body of the sprout. To get more insight into how tip cells contribute to angiogenesis, we extended an existing computational model of vascular network formation based on the cellular Potts model with tip and stalk differentiation, without making a priori assumptions about the differences between tip cells and stalk cells. To predict potential differences, we looked for parameter values that make tip cells (a) move to the sprout tip, and (b) change the morphology of the angiogenic networks. The screening predicted that if tip cells respond less effectively to an endothelial chemoattractant than stalk cells, they move to the tips of the sprouts, which impacts the morphology of the networks. A comparison of this model prediction with genes expressed differentially in tip and stalk cells revealed that the endothelial chemoattractant Apelin and its receptor APJ may match the model prediction. To test the model prediction we inhibited Apelin signaling in our model and in an in vitro model of angiogenic sprouting, and found that in both cases inhibition of Apelin or of its receptor APJ reduces sprouting. Based on the prediction of the computational model, we propose that the differential expression of Apelin and APJ yields a “self-generated” gradient mechanisms that accelerates the extension of the sprout.

Interfacial self-assembly of endothelial cells toward angiogenic network formation in the composite hydrogel culture systems

Fabrication of a pre-vascularized tissue in vitro is an extensive research activity. The idea behind this approach is that a network of newly formed micro-vessels may be engineered in vitro by the seeding of a scaffold with endothelial cells. To this aim, understanding the effect of physicochemical properties of the scaffolding material and the method of cell seeding, in regulating endothelial cells’ behavior in the in vitro constructs, is an emerging requirement. In this study, the effect of interfacial self-assembly and contact guidance for the endothelial cell behavior and angiogenic network formation have been studied. This has been done by the fabrication of in vitro three-dimensional tissue constructs, using multilayer surface-modified polymer fibers, and two different methods of cell seeding by human umbilical vein endothelial cells. In the first method, human umbilical vein endothelial cells, fibers, and fibrin gel matrix were combined simultaneously. In the second method, the human umbilical vein endothelial cells and fibers, having various surface coatings, were sandwiched between two layers of fibrin gel matrix with or without fibroblast cell monolayer over the fibrin gel. In the optimal conditions, the effect of fibers in conjunction with the interfacial self-assembly enhanced a tube-like and interconnected network structure formation. This design could therefore have a major impact in the generation of the pre-vascularized tissue-engineered constructs.

Pericyte Recruitment in a Diabetic Mouse Model of Corneal Neovascularization

Angiogenesis, the generation of new blood vessels from a previously existing vasculature, is reinforced by the dynamic migration and signaling of wrapping support cells, termed pericytes, covering the capillary endothelium. Understanding pericyte‐endothelial cell interactions involved in angiogenesis is essential for developing therapies targeted at improving blood vessel growth and stabilization during disease, such as in diabetic wound healing. Previous work has established a connection between diminished pericyte coverage and delayed angiogenesis in diabetic wound healing models. To date, the location and identity of pericytes in healthy versus diabetic angiogenic networks has yet to be explored. Therefore, the objective of this study is to compare the extent of pericyte coverage in angiogenic networks of diabetic versus healthy mice.Adult female heterozygous Ins2(Akita) diabetic mice and wild type mice (n=6 per group) underwent a silver nitrate chemical burn on the surface of the eye to induce corneal angiogenesis. Angiogenic sprouting into the previously avascular tissue was imaged daily using bright field microscopy. Three and seven‐days after injury, corneas were harvested and stained with CD31 (endothelial cell marker), NG2 (pericyte marker), and myosin heavy‐chain 11 (Myh11, pericyte and smooth muscle cell marker).Using whole‐mount confocal imaging, Myh11+, NG2+ perivascular coverage over CD31+ endothelial cells was quantified in the vascular areas of the mouse models. There was a negative correlation between overlapping coverage of the two pericyte markers and glucose concentration (mg/dL) at days three and seven post‐stimulation (r‐square= 0.888 and 0.893, respectively). Subconjuctival hemorrhages per tissue in diabetic models were increased by 3.64‐fold and 4.00‐fold after three and seven days between glucose level and number of hemorrhages (r‐square= 0.787 and 0.831, respectively).Diabetic neovessels in this injury model have diminished pericyte coverage relative to healthy mice, which is consistent with the presence of abundant subconjuctival hemorrhages in diabetic corneas. The comparison between data collected and glucose concentration in the blood is essential to understanding the relationship between these readings with the severity of diabetic effects, with an increase in glucose concentration equating to a decrease in pericyte‐supported vasculature. Together, these data suggest that reduced pericyte coverage on neovessels may contribute to the impaired angiogenesis that occurs during diabetic wound healing.Support or Funding InformationUniversity of Virginia Vice President for Research, Harrison Foundation, and the Arnold and Mabel Beckman Foundation

In vivo characterization of the integration and vascularization of a silk-derived surgical scaffold

The application of acellular matrices, biomaterials, and polymeric scaffolds in reconstructive surgery facilitates postsurgical tissue remodeling and is increasingly used clinically in order to improve tissue healing and implant coverage. This study presents an invivo investigation of the integration of the knitted, silk-derived surgical scaffold, SERI(®) with regard to angiogenesis and wound healing. SERI(®) Surgical Scaffold was implanted into a full-thickness skin defect in male C57BL/6J mice (n=45) via the dorsal skinfold chamber (DSC). Skin tissue samples were collected for histology on days 2, 5, 7, 10, 14, and 21 (n=5 per time point) post implantation. Immunohistochemistry was performed for various angiogenic and inflammatory markers, as well as collagen deposition (CD31, VEGF, CD3, CD45, Desmin, and Sirius red). Vascular corrosion casting was used to assess the neovasculature within the silk and was visualized with scanning electron microscopy. We observed both early and late stages of inflammation during the healing process characterized by the infiltration of regenerating tissue by different subsets of leukocytes. Histological analysis displayed capillary-containing granulation tissue with full scaffold integration. In addition, collagen deposition within the scaffold and full skin defect was significantly increased over time. Qualitative analysis of the regenerated vasculature through corrosion casting and scanning electron microscopy revealed a complex, angiogenic network of capillaries originating from the wound bed. Based on these findings, SERI(®) displays the potential to be a promising resorbable bioengineered material for use in reconstructive surgery.

Differential Expression of Angiogenic Gene Networks during Post-natal Lung Alveolarization

Prematurity of birth is the leading cause of death in children under 5 years of age. Preterm birth rates are steadily increasing across the globe due to advancements in medical knowledge and procedures that allow progressively earlier fetuses to survive outside of the mother’s womb. Bronchopulmonary dysplasia is a chronic lung disorder that can result from mechanical ventilation and long-term oxygen use in preterm neonates. This condition is characterized by disrupted pulmonary alveolar septation, alveolar hypoplasia, and fewer, larger alveoli that lead to decreased surface area available for gas exchange. Despite notable advances in preterm care, bronchopulmonary dysplasia results in a large number of short- and long-term morbidities, thus more advanced treatments that facilitate lung development in this subset of pediatric patients are sorely needed. In this study, we examined alterations in the gene expression profiles of 81 genes involved in angiogenesis during the process of normal alveolarization in mouse lungs. Our data revealed that alveolarization can be largely divided into early (1 week postnatal) and late (4 to 8 weeks postnatal) stages based on angiogenic gene expression profiles. In summary, our findings provide molecularlevel data illustrating the dynamic alterations in pulmonary angiogenesis during postnatal alveolarization. This knowledge can be used to provide a better understanding of normal lung development, and set the stage for discovering targeted therapies that can assist in pulmonary functioning for preterm infants.