Nascent Vessels Research Articles

New targets of nascent lymphatic vessels in ocular diseases.

Recent advancements in the field of endothelial markers of lymphatic vessels and lymphangiogenic factors have shed light on the association between several ocular diseases and ocular nascent lymphatic vessels. The immune privilege of corneal tissue typically limits the formation of lymphatic vessels in a healthy eye. However, vessels in the eyes can potentially undergo lymphangiogenesis and be conditionally activated. It is evident that nascent lymphatic vessels in the eyes contribute to various ocular pathologies. Conversely, lymphatic vessels are present in the corneal limbus, ciliary body, lacrimal glands, optic nerve sheaths, and extraocular muscles, while a lymphatic vasculature-like system exists in the choroid, that can potentially cause several ocular pathologies. Moreover, numerous studies indicate that many ocular diseases can influence or activate nascent lymphatic vessels, ultimately affecting patient prognosis. By understanding the mechanisms underlying the onset, development, and regression of ocular nascent lymphatic vessels, as well as exploring related research on ocular diseases, this article aims to offer novel perspectives for the treatment of such conditions.

Ontogenesis of the Mouse Ocular Surface Lymphatic Vascular Network.

Ocular lymphatic vessels play major physiological role in eye homeostasis and their dysfunction can contribute to the progression of several eye diseases. In this study, we characterized their spatiotemporal development and the cellular mechanisms occurring during their ontogenesis in the mouse eye. Whole mount immunofluorescent staining and imaging by standard or lightsheet fluorescence microscopy were performed on late embryonic and early postnatal eye mouse samples. We observed that the ocular surface lymphatic vascular network develops at the early postnatal stages (between P0 and P5) from two nascent trunks arising at the nasal side on both sides of the nictitating membrane. These nascent vessels further branch and encircle the whole eye surface by sprouting lymphangiogenesis. In addition, we got evidence for the existence of a transient lymphvasculogenesis process generating lymphatic vessel fragments that will mostly formed the corneolimbal lymphatic vasculature which further connect to the conjunctival lymphatic network. Our results also support that CD206-positive macrophages can transdifferentiate and then integrate into the lymphatic neovessels. Several complementary cellular processes participate in the development of the lymphatic ocular surface vasculature. This knowledge paves the way for the design of new therapeutic strategies to interfere with ocular lymphatic vessel formation when needed.

Inhibition of transient receptor potential cation channel 6 promotes capillary arterialization during post-ischaemic blood flow recovery.

Capillary arterialization, characterized by the coverage of pre-existing or nascent capillary vessels with vascular smooth muscle cells (VSMCs), is critical for the development of collateral arterioles to improve post-ischaemic blood flow. We previously demonstrated that the inhibition of transient receptor potential 6 subfamily C, member 6 (TRPC6) channels facilitate contractile differentiation of VSMCs under ischaemic stress. We here investigated whether TRPC6 inhibition promotes post-ischaemic blood flow recovery through capillary arterialization in vivo. Mice were subjected to hindlimb ischaemia by ligating left femoral artery. The recovery rate of peripheral blood flow was calculated by the ratio of ischaemic left leg to non-ischaemic right one. The number and diameter of blood vessels were analysed by immunohistochemistry. Expression and phosphorylation levels of TRPC6 proteins were determined by western blotting and immunohistochemistry. Although the post-ischaemic blood flow recovery is reportedly dependent on endothelium-dependent relaxing factors, systemic TRPC6 deletion significantly promoted blood flow recovery under the condition that nitric oxide or prostacyclin production were inhibited, accompanying capillary arterialization. Cilostazol, a clinically approved drug for peripheral arterial disease, facilitates blood flow recovery by inactivating TRPC6 via phosphorylation at Thr69 in VSMCs. Furthermore, inhibition of TRPC6 channel activity by pyrazole-2 (Pyr2; BTP2; YM-58483) promoted post-ischaemic blood flow recovery in Apolipoprotein E-knockout mice. Suppression of TRPC6 channel activity in VSMCs could be a new strategy for the improvement of post-ischaemic peripheral blood circulation.

Skeletal Muscle Injury is Sufficient for Angiogenesis but Myofiber Regeneration is Required for Restoring Microvascular Integrity

Acute injury of skeletal muscle damages myofibers, microvessels, and supporting tissue components. To restore the structural and functional integrity of intact muscle, each tissue component must regenerate. In adult skeletal muscle, resident muscle stem cells (satellite cells) efficiently regenerate myofibers within 10 days post injury (dpi) and microvascular integrity recovers by 21 dpi. However, the requirement for crosstalk between regenerating myofibers and microvessels is poorly understood. We tested the hypothesis that myofiber regeneration is necessary for revascularization and restoration of microvascular integrity. To ablate satellite cells and impair myofiber regeneration, we studied male and female mice (age, ~4 mo) in which tamoxifen‐dependent Cre recombination induced the expression of diphtheria toxin in quiescent satellite cells, killing them prior to injury. For acute muscle injury, the gluteus maximus muscle (GM) was injected with BaCl2 (1.2%, 75 µL). Microvascular perfusion and permeability were evaluated by injecting fluorescent dextrans (70 & 155 kDa) into the circulation during intravital imaging of the GM at defined timepoints following injury. Despite the absence of myofiber regeneration, sprouting angiogenesis was robust and capillary perfusion was restored within 7 dpi, as observed in wild‐type mice. However, the permeability barrier of nascent microvessels was compromised as shown by profuse leakage of both fluorescent dextrans. At 21 dpi, the absence of regenerated myofibers was accompanied by sustained inflammation and myosteatosis. Areas of ectopic adipocyte accumulation were hypervascular and localized around venules. Adjacent areas contained empty myofiber “sheaths” which were underperfused; residual microvascular networks exhibited pruning. These findings suggest that, following acute injury of adult skeletal muscle, revascularization occurs in the absence of myogenesis but is defective and nascent vessels are unstable. We conclude that myofiber regeneration is essential to recovering a functionally intact microcirculation following muscle injury.

Longitudinal dynamics of microvascular recovery after acquired cortical injury

Acquired brain injuries due to trauma damage the cortical vasculature, which in turn impairs blood flow to injured tissues. There are reports of vascular morphological recovery following traumatic brain injury, but the remodeling process has not been examined longitudinally in detail after injury in vivo. Understanding the dynamic processes that influence recovery is thus critically important. We evaluated the longitudinal and dynamic microvascular recovery and remodeling up to 2 months post injury using live brain miniscope and 2-photon microscopic imaging. The new imaging approaches captured dynamic morphological and functional recovery processes at high spatial and temporal resolution in vivo. Vessel painting documented the initial loss and subsequent temporal morphological vascular recovery at the injury site. Miniscopes were used to longitudinally image the temporal dynamics of vascular repair in vivo after brain injury in individual mice across each cohort. We observe near-immediate nascent growth of new vessels in and adjacent to the injury site that peaks between 14 and 21 days post injury. 2-photon microscopy confirms new vascular growth and further demonstrates differences between cortical layers after cortical injury: large vessels persist in deeper cortical layers (> 200 μm), while superficial layers exhibit a dense plexus of fine (and often non-perfused) vessels displaying regrowth. Functionally, blood flow increases mirror increasing vascular density. Filopodia development and endothelial sprouting is measurable within 3 days post injury that rapidly transforms regions devoid of vessels to dense vascular plexus in which new vessels become increasingly perfused. Within 7 days post injury, blood flow is observed in these nascent vessels. Behavioral analysis reveals improved vascular modulation after 9 days post injury, consistent with vascular regrowth. We conclude that morphological recovery events are closely linked to functional recovery of blood flow to the compromised tissues, which subsequently leads to improved behavioral outcomes.

Longitudinal Assessment of Alkali Injury on Mouse Cornea Using Anterior Segment Optical Coherence Tomography.

PurposeChemical burns due to alkalis cause extensive damage to the ocular surface leading to blindness. Assessment of ocular burn could be challenging due to severe opacity, inflammation, and angiogenesis. Anterior segment optical coherence tomography (AS-OCT) and OCT angiography (OCTA) may provide fast, non-invasive deep tissue visualization of pathology with high sensitivity in conjunction with slit-lamp analysis.MethodsC57-BL/6J mice were anesthetized with ketamine/dexmedetomidine, and corneal alkali burn was induced (n = 6) by placing filter paper soaked in 1-M sodium hydroxide for 30 seconds on the right eye while the left eye was kept as control. Longitudinal imaging was done with AS-OCT/OCTA and fluorescein angiography at various time intervals for 14 days.ResultsAS-OCT showed characteristic pathological changes in alkali-burned eyes with high sensitivity. Although OCT/OCTA showed three-dimensional and cross-sectional views of the anterior chamber and angiogenesis, fluorescein angiography showed nascent vessels with active leakage. Corneal swelling progressively increased by 125.26% on day 12 with a high prevalence of epithelial bullae, stromal cysts, stromal splitting, and Descemet's membrane detachment. Neovascularization was noted as early as day 4 in the burned eyes by both methods. Severe corneal opacity and anterior chamber inflammation were also detected by AS-OCT/OCTA.ConclusionsAS-OCT/OCTA is a promising, noninvasive, high-resolution imaging modality that can provide both qualitative and quantitative information regarding deep tissue pathology at a structural level.Translational RelevanceNoninvasive AS-OCT/OCTA and fluorescein methods show promise in clinical pathology evaluation for ocular injury management and prognostic indications, as the early presence of Descemet's membrane detachment and corneal swelling appears to be correlated with the severity and localization of corneal neovascularization.

Genetic Factors as the Basis of Sex Differences in Damage to Lung Endothelium and Regulation of Angiogenesis Cells in Modeling Pulmonary Emphysema in C57BL/6 Mice with Dyslipidemia and Hyperglycemia.

We studied the formation of injuries in lung endothelium and the response of angiogenesis cells during modeling of pulmonary emphysema in male and female C57BL/6 mice with metabolic disorders. Hemodynamic disturbances and reduction in the area of the microvasculature caused by combined pathology in male mice were more pronounced than in females. Mobilization and migration of angiogenic precursors were impaired in both male and female mice. In males, activity of recruiting endothelial progenitor cells, vascular smooth muscle cells, luminal cells of nascent vessels and pericytes into the lungs was additionally reduced. In females, accumulation of endothelial progenitor cells (CD45-CD31+CD34+), vascular smooth muscle cells, and pericytes in the lungs was observed, which indicated activation of endothelial regeneration. Sex differences in the reaction of the lung endothelium and angiogenesis cells can be explained by genetic factors of lipid and glucose metabolism.

Dual function of perivascular fibroblasts in vascular stabilization in zebrafish.

Blood vessels are vital to sustain life in all vertebrates. While it is known that mural cells (pericytes and smooth muscle cells) regulate vascular integrity, the contribution of other cell types to vascular stabilization has been largely unexplored. Using zebrafish, we identified sclerotome-derived perivascular fibroblasts as a novel population of blood vessel associated cells. In contrast to pericytes, perivascular fibroblasts emerge early during development, express the extracellular matrix (ECM) genes col1a2 and col5a1, and display distinct morphology and distribution. Time-lapse imaging reveals that perivascular fibroblasts serve as pericyte precursors. Genetic ablation of perivascular fibroblasts markedly reduces collagen deposition around endothelial cells, resulting in dysmorphic blood vessels with variable diameters. Strikingly, col5a1 mutants show spontaneous hemorrhage, and the penetrance of the phenotype is strongly enhanced by the additional loss of col1a2. Together, our work reveals dual roles of perivascular fibroblasts in vascular stabilization where they establish the ECM around nascent vessels and function as pericyte progenitors.

Intermedin promotes vessel fusion by inducing VE-cadherin accumulation at potential fusion sites and to achieve a dynamic balance between VE-cadherin-complex dissociation/reconstitution.

To create a closed vascular system, angiogenic sprouts must meet and connect in a process called vessel fusion, which is a prerequisite for establishment of proper blood flow in nascent vessels. However, the molecular machinery underlying this process remains largely unknown. Herein, we report that intermedin (IMD), a calcitonin family member, promotes vessel fusion by inducing endothelial cells (ECs) to enter a “ready‐to‐anchor” state. IMD promotes vascular endothelial cadherin (VEC) accumulation at the potential fusion site to facilitate anchoring of approaching vessels to each other. Simultaneously, IMD fine‐tunes VEC activity to achieve a dynamic balance between VEC complex dissociation and reconstitution in order to widen the anastomotic point. IMD induces persistent VEC phosphorylation. Internalized phospho‐VEC preferentially binds to Rab4 and Rab11, which facilitate VEC vesicle recycling back to the cell‐cell contact for reconstruction of the VEC complex. This novel mechanism may explain how neovessels contact and fuse to adjacent vessels to create a closed vascular system.

Tetraspanin18 regulates angiogenesis through VEGFR2 and Notch pathways.

ABSTRACTThe VEGF pathway is critically required for vasculogenesis, the formation of the primary vascular network. It is also required for angiogenesis resulting in sprouting and pruning of vessels to generate mature arborizing structures. The Notch pathway is essential for arterial–venous specification and the maturation of nascent vessels. We have determined that Tspan18, a member of the Tetraspanin family, is expressed in developing vessels but not in mature vasculature in zebrafish and mouse wound healing. Moreover, reduction at Tspan18 level resulted in aberrant vascular patterning, impaired vessel stability and defective arterial–venous specification. Tspan18 deficiency reduced VEGF, VEGFR2, Notch3 and EphrinB2, and increased EphB4, VEGFR3, Semaphorin3, Neuropilin and PlexinD1 expression. Furthermore, vascular defects of Tspan18 deficiency could be rescued by ectopic expression of VEGFR2 and Notch, but not by knockdown of Semaphorin or Plexin. Functional studies showed that knockdown of Tspan18 led to reduced endothelial cell migration, invasion and tube formation. Tspan18 has dynamic expression, regulates vascular development and maturation in the embryo with re-expression in adult life in wound healing.

Tuberous Sclerosis (tsc2+/-) Model Eker Rats Reveals Extensive Neuronal Loss with Microglial Invasion and Vascular Remodeling Related to Brain Neoplasia.

Tuberous sclerosis complex (TSC) is a genetic disorder characterized by frequent noncancerous neoplasia in the brain, which can induce a range of severe neuropsychiatric symptoms in humans, resulting from out of control tissue growth. The causative spontaneous loss-of-function mutations have been also identified in rats. Herein, we studied histopathological and molecular changes in brain lesions of the Eker rat model carrying germline mutation of the tsc2 gene, predisposed to multiple neoplasias. Predominant subcortical tumors were analyzed, along with a rare form occurring within the pyriform lobe. The uniform composition of lesions supports the histochemical parity of malformations, with immunofluorescence data supporting their neuro-glial origin. Massive depletion of mature neurons and axonal loss were evident within lesions, with occasional necrotic foci implying advanced stage of pathology. Enrichment of mesenchymal-derived cell markers with hallmarks of neurogenesis and active microglia imply enhanced cell proliferation, with local immune response. The depletion of capillaries within the core was complemented by the formation of dense mesh of nascent vessels at the interface of neoplasia with healthy tissue, implying large-scale vascular remodeling. Taken as a whole, these findings present several novel features of brain tumors in Eker rat model, rendering it suitable for studies of the pathobiology and progression of primary brain tumors, with therapeutic interventions.

Intermedin Promotes Vessel Fusion by Inducing Endothelial Cells into a 'Ready to Anchor' State

To create a closed vascular system, angiogenic sprouts need to meet and connect in a process called vessel fusion, which is a prerequisite for establishing proper blood flow in nascent vessels. However, the molecular machinery underlying this process remains largely unknown. Herein, we report that intermedin (IMD), a calcitonin family member, promotes vessel fusion by inducing endothelial cells (ECs) into a “ready to anchor” state. IMD promotes VE-cadherin (VEC) accumulation at the potential fusion point to facilitate approaching vessels anchoring to each other. Simultaneously, IMD fine-tunes VEC activity to achieve a dynamic balance between VEC complex dissociation/reconstitution to widen the anastomotic point. IMD induces VEC endocytosis via Src-triggered VEC autophosphorylation, and concurrently promotes VEC endosome transport back to the cell-cell contact via Rab4 and Rab11 to rebuild the VEC complex. This novel mechanism may explain how the neovessels contact and fuse to adjacent vessels to create a closed vascular system

MicroRNA‐26b‐5p Regulates Bone Marrow Mesenchymal Stem Cell Differentiation to Pericytes via Targeting Platelet‐Derived Growth Factor Receptor‐β and Interacting with Long non‐Coding RNA Maternally Expressed Gene 3 in Liver Fibrosis and Angiogenesis

Background/AimsPericytes are important regulators of vascular morphogenesis and function in multiple biological processes, including liver fibrosis, which is closely associated with angiogenesis. However, the origin and functional role of pericytes during liver fibrosis have not been deciphered yet.MethodsMice received irradiation and transplantation of EGFP‐labeled bone marrow (BM) mesenchymal stem cells (BMSCs), which obtained from EGFP transgenic mice and had been sorted and purified by immunomagnetic cell sorting with anti‐CD146 microbeads. Four weeks later, the BM was reconstructed and the chimera mice with EGFP‐labeled BMSCs were fed with methionine‐choline‐deficient and high fat (MCDHF) diet to build liver injury model. Expression of pericyte markers or fibrosis markers was characterized by qRT‐PCR, Western blot, immunofluerescent staining and microarray analysis. Biotin‐avidin pull‐down and lucifer reporter assay were performed to validate the binding of microRNA (miR)‐26b‐5p and its target platelet‐derived growth factor receptor beta (PDGFR‐β). GO, KEGG and Reactome enrichment analysis was conducted to clarify the function of differentially expressed genes.ResultsIncreased expression of pericyte markers (e.g. PDGFR‐β, neural glial antigen 2, regulator of G protein signaling 5) was detected in MCDHF mice with a positive correlation with fibrosis markers (e.g. TGF‐β1, α‐smooth muscle actin, procollagen α1(I) and (III)). Numerous PDGFR‐β+ pericytes was found in the fibrotic liver not only around mature vessels but also around nascent vessels, and a significant proportion (72.5%) of pericytes in mouse fibrotic liver derived from BMSCs. BMSCs differentiated into pericytes under TGF‐β1 treatment in vitro. Long non‐coding RNA maternally expressed gene 3 (LncMEG3), which had been focused on its effects on angiogenesis recently, positively regulated PDGFR‐β expression during BMSC differentiation to pericytes. Bioinformatics analysis from three databases (miRanda, TargetScan, and RNA22v2) suggested that miR‐26b‐5p was predicted to target PDGFR‐β. MiR‐26b‐5p negatively regulated BMSC differentiation to pericytes via directly targeting PDGFR‐β in cultured BMSCs. GO, KEGG and Reactome enrichment analysis revealed that miR‐26b‐5p overexpression affected a list of genes associated with cell differentiation, angiogenesis and extracellular matrix. LncMEG3 acted as a molecular sponge of miR‐26b‐5p thus contributed to the de‐repression of miRNA target PDGFR‐β. MiR‐26b‐5p agomir blocked BMSC differentiation to pericytes and attenuates liver fibrosis and angiogenesis in vivo.ConclusionmiR‐26b‐5p regulates BMSC differentiation to pericytes via targeting PDGFR‐β and interacting with lncMEG3, which may represent an effective therapeutic strategy for liver fibrosis.Support or Funding InformationThis work was supported by grants from the National Natural and Science Foundation of China (81430013, 81500465) and the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality(IDHT20150502)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Human Pluripotent Stem Cell-Derived Multipotent Vascular Progenitors of the Mesothelium Lineage Have Utility in Tissue Engineering and Repair

In this report we describe a human pluripotent stem cell-derived vascular progenitor (MesoT) cell of the mesothelium lineage. MesoT cells are multipotent and generate smooth muscle cells, endothelial cells, and pericytes and self-assemble into vessel-like networks invitro. MesoT cells transplanted into mechanically damaged neonatal mouse heart migrate into the injured tissue and contribute to nascent coronary vessels in the repair zone. When seeded onto decellularized vascular scaffolds, MesoT cells differentiate into the major vascular lineages and self-assemble into vasculature capable of supporting peripheral blood flow following transplantation. These findings demonstrate invivo functionality and the potential utility of MesoT cells in vascular engineering applications.

Blockade of vascular endothelial growth factor receptor 2 inhibits intraplaque haemorrhage by normalization of plaque neovessels.

BackgroundPlaque angiogenesis is associated with atherosclerotic lesion growth, plaque instability and negative clinical outcome. Plaque angiogenesis is a natural occurring process to fulfil the increasing demand of oxygen and nourishment of the vessel wall. However, inadequate formed, immature plaque neovessels are leaky and cause intraplaque haemorrhage.ObjectiveBlockade of VEGFR2 normalizes the unbridled process of plaque neovessel formation and induces maturation of nascent vessels resulting in prevention of intraplaque haemorrhage and influx of inflammatory cells into the plaque and subsequently increases plaque stability.Methods and ResultsIn human carotid and vein graft atherosclerotic lesions, leaky plaque neovessels and intraplaque haemorrhage co‐localize with VEGF/VEGFR2 and angiopoietins. Using hypercholesterolaemic ApoE3*Leiden mice that received a donor caval vein interposition in the carotid artery, we demonstrate that atherosclerotic vein graft lesions at t28 are associated with hypoxia, Hif1α and Sdf1 up‐regulation. Local VEGF administration results in increased plaque angiogenesis. VEGFR2 blockade in this model results in a significant 44% decrease in intraplaque haemorrhage and 80% less extravasated erythrocytes compared to controls. VEGFR2 blockade in vivo results in a 32% of reduction in vein graft size and more stable lesions with significantly reduced macrophage content (30%), and increased collagen (54%) and smooth muscle cell content (123%). Significant decreased VEGF, angiopoietin‐2 and increased Connexin 40 expression levels demonstrate increased plaque neovessel maturation in the vein grafts. VEGFR2 blockade in an aortic ring assay showed increased pericyte coverage of the capillary sprouts.ConclusionInhibition of intraplaque haemorrhage by controlling neovessels maturation holds promise to improve plaque stability.

AJAP1 and Epithelial Cell Adhesion for Epicardial and Coronary Vessel Formation

The coronary vasculature forms to supply oxygen and nutrients to the contractile myocardium when it can no longer be supported by simple diffusion of blood from the cardiac lumen. This is an important step in cardiovascular development that is preceded by formation of the epicardium that contributes epicardial‐derived cells (EPDCs). These EPDCs proceed to form some cells within the nascent coronary vessels. Mice with proepicardium (PE)‐specific deletion of Tbx5 (Tbx5epi−/−) have delayed attachment of the epithelial‐like epicardial cells to the myocardium. This delay precipitates a cascade of deleterious events that impair coronary vessel formation and lead to punctate cardiac hemorrhaging and myocardial hypoxia in Tbx5epi−/− mice. In addition, Tbx5epi−/− mice exhibit reduced cardiac Ajap1 mRNA expression. We previously demonstrated that Ajap1 is expressed within the PE, epicardium and coronary vessels of the developing mouse heart, but little is known about its contribution to cardiovascular morphogenesis other than it is speculated to function as a cell adhesion molecule.Study ObjectiveThe goal of this study was to elucidate contributions of Ajap1 to epithelial cell behaviors that are integral to formation of the epicardium and the subsequent development of the coronary vessels. Based upon our preliminary in vivo data, we hypothesized that the reduction in cardiac Ajap1 expression of our Tbx5epi−/− mice acts as a catalyst to impair epicardium and, subsequently, coronary vessel formation.MethodsTo test this hypothesis, we first analyzed the contribution of AJAP1 to cell behavior in an in vitro system that models mammalian epicardial cells. We utilized the stably transformed MCF7 human breast adenocarcinoma cell line to assess contributions of AJAP1 to epithelial cell function as these cells retain epithelial characteristics in vitro and have endogenous AJAP1 expression. We achieved AJAP1 gene silencing in MCF7s through a small interfering RNAmediated approach that led to a significant reduction in both AJAP1 mRNA and protein expression in these cells. Next, control and AJAP1‐silenced epithelial cells were subjected to cell adhesion assays to assess the ability of these cells to adhere to various basement membrane matrices. Cells were allowed to adhere to these matrices for 1 – 24 hours and adherent cells were quantified. Finally, we analyzed Ajap1 protein expression in embryonic and adult mouse hearts to gauge how its levels change throughout important stages of cardiovascular development.ResultsData from our gene‐silenced epithelial cells revealed a significant difference in cell adhesion abilities between the control and AJAP1‐transfected cells. Cardiac Ajap1 expression changed notably throughout mouse development and after gestation.ConclusionsWe concluded that AJAP1 contributes to the in vitro cell adhesion properties in an epithelial model system of the mammalian epicardium. These data shed light on a mechanism by which AJAP1 contributes to cell adhesion in a mammalian model of epicardial cell behavior. This may be instrumental to understanding coronary vessel formation.Support or Funding InformationSupport: PCOM Center for Chronic Disorders of AgingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CD146 coordinates brain endothelial cell–pericyte communication for blood–brain barrier development

The blood-brain barrier (BBB) establishes a protective interface between the central neuronal system and peripheral blood circulation and is crucial for homeostasis of the CNS. BBB formation starts when the endothelial cells (ECs) invade the CNS and pericytes are recruited to the nascent vessels during embryogenesis. Despite the essential function of pericyte-EC interaction during BBB development, the molecular mechanisms coordinating the pericyte-EC behavior and communication remain incompletely understood. Here, we report a single cell receptor, CD146, that presents dynamic expression patterns in the cerebrovasculature at the stages of BBB induction and maturation, coordinates the interplay of ECs and pericytes, and orchestrates BBB development spatiotemporally. In mouse brain, CD146 is first expressed in the cerebrovascular ECs of immature capillaries without pericyte coverage; with increased coverage of pericytes, CD146 could only be detected in pericytes, but not in cerebrovascular ECs. Specific deletion of Cd146 in mice ECs resulted in reduced brain endothelial claudin-5 expression and BBB breakdown. By analyzing mice with specific deletion of Cd146 in pericytes, which have defects in pericyte coverage and BBB integrity, we demonstrate that CD146 functions as a coreceptor of PDGF receptor-β to mediate pericyte recruitment to cerebrovascular ECs. Moreover, we found that the attached pericytes in turn down-regulate endothelial CD146 by secreting TGF-β1 to promote further BBB maturation. These results reveal that the dynamic expression of CD146 controls the behavior of ECs and pericytes, thereby coordinating the formation of a mature and stable BBB.

Transplantation of bioengineered rat lungs recellularized with endothelial and adipose-derived stromal cells

Bioengineered lungs consisting of a decellularized lung scaffold that is repopulated with a patient’s own cells could provide desperately needed donor organs in the future. This approach has been tested in rats, and has been partially explored in porcine and human lungs. However, existing bioengineered lungs are fragile, in part because of their immature vascular structure. Herein, we report the application of adipose-derived stem/stromal cells (ASCs) for engineering the pulmonary vasculature in a decellularized rat lung scaffold. We found that pre-seeded ASCs differentiated into pericytes and stabilized the endothelial cell (EC) monolayer in nascent pulmonary vessels, thereby contributing to EC survival in the regenerated lungs. The ASC-mediated stabilization of the ECs clearly reduced vascular permeability and suppressed alveolar hemorrhage in an orthotopic transplant model for up to 3 h after extubation. Fibroblast growth factor 9, a mesenchyme-targeting growth factor, enhanced ASC differentiation into pericytes but overstimulated their proliferation, causing a partial obstruction of the vasculature in the regenerated lung. ASCs may therefore provide a promising cell source for vascular regeneration in bioengineered lungs, though additional work is needed to optimize the growth factor or hormone milieu for organ culture.

Abstract 2862: Tumor-targeting salmonella typhimurium a1-r inhibited angiogenesis of osteosarcoma cells visualized by color-coded imaging in the in vivo absorbable gelatin sponge assay

Abstract INTRODUCTION We already have reported a color-coded imaging model that showed the sponge agngiogenesis assay using absorbable gelatin sponge implanted in nestin-driven green fluorescent protein (ND-GFP) nude mice and that osteosarcoma cells promote angiogenesis in this sponge assay. We report here that Salmonella typhimurium A1-R (A1-R) inhibits angiogenesis of osteosarcoma cells in the sponge angiogenesis assay in ND-GFP mice. METHODS Sponge was initially transplanted subcutaneously in the flank of transgenic ND-GFP nude mice. Seven days after transplantation of sponge, skin flaps were made and 143B-RFP human osteosarcoma cells expressing red fluorescent protein (RFP) were injected into the transplanted sponge. After establishment of tumors in the sponge, the control-group mice were treated with PBS via tail-vein injection and A1-R treated group with A1-R likewise. Skin flaps were made at days 14, 21, and 28 after transplantation of the sponge to allow imaging of vascularization in the sponge using a variable-magnification small animal imaging system and confocal fluorescence microscopy. RESULTS: Vessel length in the sponge was measured after treatment with FV10-ASW Fluoview software. Nascent blood vessels grew in the sponge in a time-dependent manner. A random 3 fields were quantified in each group). The mean length of ND-GFP expressing blood vessels in the sponge in mice with A1-R treated group were 9.40, 12.40, and 10.12 mm/mm2, at days 14, 21, and 28, respectively. The mean length of ND-GFP expressing blood vessels in the sponge in mice of the control group were 8.86, 14.50, and 15.81 mm/mm2 on day 14, 21, and 28, respectively. ND-GFP expressing blood vessels of the mice in the osteosarcoma cells treated with A1-R had shorter vessels than the control group (on days 28, P < 0.05), suggested the extent of nascent blood vessel growth was significantly inhibited by A1-R treatment. Besides, we evaluated the RFP colored area of the sponge in both groups, which almost represent the viable tumor size. The mean RFP colored area of A1-R treated group in the sponge were 4.49, 7.53 and 8.15 mm2, at days 14, 21, and 28, respectively and that of control group were 3.78, 6.40, and 7.49 mm2 at days 14, 21, and 28, respectively. The size of possibly viable RFP colored tumor area between A1-R treated group and control group at days 28 did not show significant area difference, indicated A1-R treated osteosarcoma cells showed shorter nascent vessel length than control group, not due to the tumor size difference but due to the difference of angiogenesis inhibitory effect. As a result of both outcomes above, the extent of nascent blood vessel growth was significantly inhibited by A1-R treatment on days 28. CONCLUSION: In the present study, we reported the effect of angiogenesis inhibition of A1-R in vivo sponge assay, and suggest A1-R has potential for anti-angiogenic target therapy for osteosarcoma. Citation Format: Tasuku Kiyuna. Tumor-targeting salmonella typhimurium a1-r inhibited angiogenesis of osteosarcoma cells visualized by color-coded imaging in the in vivo absorbable gelatin sponge assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2862. doi:10.1158/1538-7445.AM2017-2862

Regulation of endothelial Fas expression as a mechanism of promotion of vascular integrity by mural cells in tumors.

Angiogenesis is a multi‐step process that culminates in vascular maturation whereby nascent vessels stabilize to become functional, and mural cells play an essential role in this process. Recent studies have shown that mural cells in tumors also promote and maintain vascular integrity, with wide‐reaching clinical implications including the regulation of tumor growth, metastases, and drug delivery. Various regulatory signaling pathways have been hitherto implicated, but whether regulation of Fas‐dependent apoptotic mechanisms is involved has not yet been fully investigated. We first compared endothelial FAS staining in human pancreatic ductal adenocarcinomas and colon carcinomas and show that the latter, characterized by lower mural cell coverage of tumor vasculature, demonstrated higher expression of FAS than the former. Next, in an in vitro coculture system of MS‐1 and 10T1/2 cells as endothelial and mural cells respectively, we show that mural cells decreased endothelial Fas expression. Then, in an in vivo model in which C26 colon carcinoma cells were inoculated together with MS‐1 cells alone or with the further addition of 10T1/2 cells, we demonstrate that mural cells prevented hemorrhage. Finally, knockdown of endothelial Fas sufficiently recapitulated the protection against hemorrhage seen with the addition of mural cells. These results together suggest that regulation of endothelial Fas signaling is involved in the promotion of vascular integrity by mural cells in tumors.