Endothelial Migration Research Articles

Neutrophil Protein Kinase R Mediates Endothelial Adhesion and Migration by the Promotion of Neutrophil Actin Polymerization.

Neutrophils protect against bacterial and fungal infections, but tight regulation of cell activation is essential for avoiding tissue damage in autoimmune disorders. Protein kinase R (PKR) is a serine/threonine kinase originally characterized by its role in the defense mechanisms against viral infection. Although PKR is involved in the signaling pathways of neurodegenerative diseases and metabolic disorders, its function in neutrophils is not well delineated. In this study, we demonstrate that human neutrophil PKR mediates adhesion to endothelial cells under physiological flow conditions but does not mediate rolling on those cells. Also, neutrophil PKR activation contributes to migration toward chemoattractants. Mechanistically, neutrophil PKR mediates the cell spreading and binding to ICAM-1 in static condition. Moreover, Ab microarray reveals that calcium/calmodulin-dependent protein kinase II is phosphorylated downstream of PKR and affects actin polymerization that is a cytoskeleton rearrangement indispensable for neutrophil migration induced by fMLF. In vivo, neutrophil recruitment into the dorsal air pouch of mice is reduced by PKR inhibitor treatment. Also, in mice with nephrotoxic serum nephritis, the compound treatment suppresses neutrophil accumulation in kidney glomerulus and subsequent development of albuminuria. Thus, in vascular inflammation, neutrophil PKR plays a critical role in the recruitment process, including endothelial adhesion and migration via leukocyte actin polymerization.

Hemin-Induced Endothelial Dysfunction and Endothelial to Mesenchymal Transition in the Pathogenesis of Pulmonary Hypertension Due to Chronic Hemolysis.

Pulmonary hypertension in sickle cell disease is an independent predictor of mortality, yet the pathogenesis of pulmonary vascular disease in chronic hemolytic disorders remains incompletely understood and treatment options are limited primarily to supportive care. The release of extracellular hemoglobin has been implicated in the development of pulmonary hypertension, and in this study we explored the direct effects of hemin, the oxidized moiety of heme, on the pulmonary artery endothelium. We found that low dose hemin exposure leads to significantly increased endothelial cell proliferation, migration, and cytokine release as markers of endothelial dysfunction. Protein expression changes in our pulmonary artery endothelial cells showed upregulation of mesenchymal markers after hemin treatment in conjunction with a decrease in endothelial markers. Endothelial to mesenchymal transition (EndoMT) resulting from hemin exposure was further confirmed by showing upregulation of the transcription factors SNAI1 and SLUG, known to regulate EndoMT. Lastly, given the endothelial dysfunction and phenotypic transition observed, the endothelial cytoskeleton was considered a potential novel target. Inhibiting myosin light chain kinase, to prevent phosphorylation of myosin light chain and cytoskeletal contraction, attenuated hemin-induced endothelial hyper-proliferation, migration, and cytokine release. The findings in this study implicate hemin as a key inducer of endothelial dysfunction through EndoMT, which may play an important role in pulmonary vascular remodeling during the development of pulmonary hypertension in chronic hemolytic states.

Tetrahedral Framework Nucleic Acids Connected with MicroRNA-126 Mimics for Applications in Vascular Inflammation, Remodeling, and Homeostasis.

The repair of damaged endothelium is crucial for vascular homeostasis maintenance, which comprises the recovery of early stage impaired endothelial cells and migration of surrounding unimpaired endothelial cells. MicroRNAs (miRNAs) play an indispensable role in balancing gene expression in organisms. For vascular tissues, miR-126 is one of the most important regulators and might have substantial application potential in maintaining vascular homeostasis. In this study, a type of sticky-end-modified tetrahedral framework nucleic acids (tFNAs-SE) was employed to successfully link the miR-126 5p mimic duplex, which was termed tFNAs-miR-126 5p mimics (tFNAs-MMs). Existing vascular endothelial growth factors (VEGF), tFNAs-MMs can improve cell viability, resist apoptosis, and recover the state and functions of LPS-induced impaired human umbilical vein endothelial cells (HUVECs). The angiogenesis ability of impaired HUVECs was recovered by tFNAs-MMs in vitro and in vivo. The mechanisms underlying these phenomena were demonstrated to be related to the downregulation of caspase3 and negative regulators of VEGF (SPRED1 and PIK3R2). Moreover, tFNAs-MMs promoted the migration and proliferation of HUVECs. Briefly, the strategy of sticky-end-modified tFNAs connecting miRNA mimics is available for miRNA gain of function, while tFNAs-MMs might be a promising agent for repairing early stage vascular damage and maintaining vascular homeostasis.

Jiedu Huoxue Decoction for Cytokine Storm and Thrombosis in Severe COVID-19: A Combined Bioinformatics and Computational Chemistry Approach

Jiedu Huoxue Decoction (JHD), a recommended traditional prescription for patients with severe COVID-19, has appeared in the treatment protocols in China. Based on bioinformatics and computational chemistry methods, including molecular docking, molecular dynamics (MD) simulation, and Molecular Mechanics Generalized Born Surface Area (MM/GBSA) calculation, we aimed to reveal the mechanism of JHD in treating severe COVID-19. The compounds in JHD were obtained and screened on TCMSP, SwissADME, and ADMETLab platforms. The compound targets were obtained from TCMSP and STITCH, while COVID-19 targets were obtained from Genecards and NCBI. The protein-protein interaction network was constructed by using STRING. Gene Ontology (GO) and KEGG enrichment were performed with ClueGO and R language. AutoDock vina was employed for molecular docking. 100 ns MD simulation of the optimal docking complex was carried out with AmberTools 20. A total of 84 compounds and 29 potential targets of JHD for COVID-19 were collected. The key phytochemicals included quercetin, luteolin, β-sitosterol, puerarin, stigmasterol, kaempferol, and wogonin, which could regulate the immune system. The hub genes included IL6, IL10, VEGFA, IL1B, CCL2, HMOX1, DPP4, and ACE2. ACE2 and DPP4 were related to SARS-CoV-2 entering cells. GO and KEGG analysis showed that JHD could intervene in cytokine storm and endothelial proliferation and migration related to thrombosis. The molecular docking, 100 ns MD simulation, and MM/GBSA calculation confirmed that targets enriched in the COVID-19 pathway had high affinities with related compounds, and the conformations of the puerarin-ACE2, quercetin-EGFR, luteolin-EGFR, and quercetin-IL1B complexes were stable. In a word, JHD could treat COVID-19 by intervening in cytokine storm, thrombosis, and the entry of SARS-CoV-2, while regulating the immune system. These mechanisms were consistent with JHD's therapeutic concept of “detoxification” and “promoting blood circulation and removing blood stasis” in treating COVID-19. The research provides a theoretical basis for the development and application of JHD.

Cell-Crossing Functional Network Driven by microRNA-125a Regulates Endothelial Permeability and Monocyte Trafficking in Acute Inflammation

Opening of the endothelial barrier and targeted infiltration of leukocytes into the affected tissue are hallmarks of the inflammatory response. The molecular mechanisms regulating these processes are still widely elusive. In this study, we elucidate a novel regulatory network, in which miR-125a acts as a central hub that regulates and synchronizes both endothelial barrier permeability and monocyte migration. We found that inflammatory stimulation of endothelial cells induces miR-125a expression, which consecutively inhibits a regulatory network consisting of the two adhesion molecules VE-Cadherin (CDH5) and Claudin-5 (CLDN5), two regulatory tyrosine phosphatases (PTPN1, PPP1CA) and the transcription factor ETS1 eventually leading to the opening of the endothelial barrier. Moreover, under the influence of miR-125a, endothelial expression of the chemokine CCL2, the most predominant ligand for the monocytic chemokine receptor CCR2, was strongly enhanced. In monocytes, on the other hand, we detected markedly repressed expression levels of miR-125a upon inflammatory stimulation. This induced a forced expression of its direct target gene CCR2, entailing a strongly enhanced monocyte chemotaxis. Collectively, cell-type-specific differential expression of miR-125a forms a synergistic functional network controlling monocyte trafficking across the endothelial barrier towards the site of inflammation. In addition to the known mechanism of miRNAs being shuttled between cells via extracellular vesicles, our study uncovers a novel dimension of miRNA function: One miRNA, although disparately regulated in the cells involved, directs a biologic process in a synergistic and mutually reinforcing manner. These findings provide important new insights into the regulation of the inflammatory cascade and may be of great use for future clinical applications.

Relationship Between Endothelial and Angiogenesis Biomarkers Envisage Mortality in a Prospective Cohort of COVID-19 Patients Requiring Respiratory Support.

PurposeEndothelial damage and angiogenesis are fundamental elements of neovascularisation and fibrosis observed in patients with coronavirus disease 2019 (COVID-19). Here, we aimed to evaluate whether early endothelial and angiogenic biomarkers detection predicts mortality and major cardiovascular events in patients with COVID-19 requiring respiratory support.MethodsChanges in serum syndecan-1, thrombomodulin, and angiogenic factor concentrations were analysed during the first 24 h and 10 days after COVID-19 hospitalisation in patients with high-flow nasal oxygen or mechanical ventilation. Also, we performed an exploratory evaluation of the endothelial migration process induced by COVID-19 in the patients' serum using an endothelial cell culture model.ResultsIn 43 patients, mean syndecan-1 concentration was 40.96 ± 106.9 ng/mL with a 33.9% increase (49.96 ± 58.1 ng/mL) at day 10. Both increases were significant compared to healthy controls (Kruskal–Wallis p < 0.0001). We observed an increase in thrombomodulin, Angiopoietin-2, human vascular endothelial growth factor (VEGF), and human hepatocyte growth factor (HGF) concentrations during the first 24 h, with a decrease in human tissue inhibitor of metalloproteinases-2 (TIMP-2) that remained after 10 days. An increase in human Interleukin-8 (IL-8) on the 10th day accompanied by high HGF was also noted. The incidence of myocardial injury and pulmonary thromboembolism was 55.8 and 20%, respectively. The incidence of in-hospital deaths was 16.3%. Biomarkers showed differences in severity of COVID-19. Syndecan-1, human platelet-derived growth factor (PDGF), VEGF, and Ang-2 predicted mortality. A multiple logistic regression model with TIMP-2 and PDGF had positive and negative predictive powers of 80.9 and 70%, respectively, for mortality. None of the biomarkers predicted myocardial injury or pulmonary thromboembolism. A proteome profiler array found changes in concentration in a large number of biomarkers of angiogenesis and chemoattractants. Finally, the serum samples from COVID-19 patients increased cell migration compared to that from healthy individuals.ConclusionWe observed that early endothelial and angiogenic biomarkers predicted mortality in patients with COVID-19. Chemoattractants from patients with COVID-19 increase the migration of endothelial cells. Trials are needed for confirmation, as this poses a therapeutic target for SARS-CoV-2.

Electron Transfer Flavoprotein (ETF) α Controls Blood Vessel Development by Regulating Endothelial Mitochondrial Bioenergetics and Oxygen Consumption.

While impairment of vascular homeostasis induced by hypercholesterolemia is the first step of cardiovascular diseases, the molecular mechanism behind such impairment is not well known. Here, we reported that high-cholesterol diet (HCD) induced defective vessel sprouting in zebrafish larvae. Electron transfer flavoprotein subunit α (ETFα) (encoded by the ETFA gene), a protein that mediates transfer of electrons from a series of mitochondrial flavoenzymes to the respiratory chain, was downregulated in HCD-fed zebrafish and in endothelial cells treated with oxidized low-density lipoprotein. Knockdown of ETFα with morpholino antisense oligonucleotides reproduced vascular sprouting defects in zebrafish larvae, while replenishing with exogeneous ETFA mRNA could successfully rescue these defects. ETFA knockdown in endothelial cells reduces cell migration, proliferation, and tube formation in vitro. Finally, knockdown of ETFA in endothelial cells also reduced fatty acid oxidation, oxygen consumption rate, and hypoxia-inducible factor-1α (HIF1α) protein levels. Taken together, we demonstrate that downregulation of ETFα is involved in hypercholesterolemia-induced defective vessel sprouting in zebrafish larvae via inhibition of endothelial proliferation and migration. The molecular mechanism behind this phenomenon is the decrease of HIF1α induced by downregulation of ETFα in endothelial cells. This work suggests that disturbance of ETFα-mediated oxygen homeostasis is one of the mechanisms behind hypercholesterolemia-induced vascular dysfunction.

Establishment of a Combined Diagnostic Model of Abdominal Aortic Aneurysm with Random Forest and Artificial Neural Network.

Objectives. Abdominal aortic aneurysm (AAA), a disease with high mortality, is limited by the current diagnostic methods in the early screening. This study aimed to screen novel and significant biomarkers and construct a diagnostic model for AAA by using a novel machine learning method, i.e., an ensemble of the random forest (RF) algorithm and artificial neural network (ANN). Methods and Results. Through a search of the Gene Expression Omnibus (GEO) database, two large-sample gene expression datasets (GSE57691 and GSE47472) were downloaded and preprocessed. Differentially expressed genes (DEGs) in GSE57691 were identified by R software, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). Essential metabolic pathways related to positive regulation of cell death and NAD binding were found. Then, RF was used to identify key genes from the DEGs, and an AAA diagnostic model was established by ANN. A transcription factor (TF) regulatory network of key genes related to angiogenesis and endothelial migration was constructed. Finally, a validation dataset was used to validate the model and the area under the receiver operating characteristic curve (AUC) value was high. Conclusion. Potential AAA-associated gene biomarkers were identified by RF, and a novel early diagnostic model of AAA was established by ANN. The AUC indicated that the diagnostic model had a highly satisfactory diagnostic performance. In conclusion, this study will provide a promising theoretical basis for further clinical and experimental studies.

The effects of sulfated hyaluronan in breast, lung and colorectal carcinoma and monocytes/macrophages cells: Its role in angiogenesis and tumor progression.

Hyaluronan (HA) is a component of the extracellular matrix (ECM) it is the main non-sulfated glycosaminoglycan able to modulate cell behavior in the healthy and tumor context. Sulfated hyaluronan (sHA) is a biomaterial derived from chemical modifications of HA, since this molecule is not naturally sulfated. The HA sulfation modifies several properties of the native molecule, acquiring antitumor properties in different cancers. In this study, we evaluated the action of sHA of ~30-60 kDa with different degrees of sulfation (0.7 sHA1 and 2.5 sHA3) on tumor cells of a breast, lung, and colorectal cancer model and its action on other cells of the tumor microenvironment, such as endothelial and monocytes/macrophage cells. Our data showed that in breast and lung tumor cells, sHA3 is able to modulate cell viability, cytotoxicity, and proliferation, but no effects were observed on colorectal cancer cells. In 3D cultures of breast and lung cancer cells, sHA3 diminished the size of the tumorsphere and modulated total HA levels. In these tumor models, treatment of monocytes/macrophages with sHA3 showed a downregulation of the expression of angiogenic factors. We also observed a decrease in endothelial cell migration and modulation of the hyaluronan-binding protein TSG-6. In the breast in vivo xenograft model, monocytes/macrophages preincubated with sHA1 or sHA3 decreased tumor vasculature, TSG-6 and HA levels. Besides, in silico analysis showed an association of TSG-6, HAS2, and IL-8 with biological processes implicated in the progression of the tumor. Taken together, our data indicate that sHA in a breast and lung tumor context is able to induce an antiangiogenic action on tumor cells as well as in monocytes/macrophages (Mo/MØ) by modulation of endothelial migration, angiogenic factors, and vessel formation.

Accelerated Wound Healing and Keratinocyte Proliferation through PI3K/Akt/pS6 and VEGFR2 Signaling by Topical Use of Pleural Fluid.

Impaired wound healing is an ongoing issue that cancer patients undergoing chemotherapy or radiotherapy face. Our previous study regarding lung-cancer-associated pleural fluid (LCPF) demonstrated its propensity to promote endothelial proliferation, migration, and angiogenesis, which are crucial features during cutaneous wound healing. Therefore, the current study aimed to investigate the effect of pleural fluid on cutaneous wound closure in vitro and in vivo using HaCaT keratinocytes and a full-thickness skin wound model, respectively. Both heart-failure-associated pleural fluid (HFPF) and LCPF were sequentially centrifuged and filtered to obtain a cell-free status. Treatment with HFPF and LCPF homogeneously induced HaCaT proliferation with cell cycle progression, migration, and MMP2 upregulation. Western blotting revealed increased PI3K/Akt phosphorylation and VEGFR2/VEGFA expression in HaCaT cells. When treated with the PI3K inhibitor, LCPF-induced keratinocyte proliferation was attenuated with decreased pS6 levels. By applying the VEGFR2 inhibitor, LCPF-induced keratinocyte proliferation was ameliorated by pS6 and MMP2 downregulation. The effect of LCPF-induced cell junction rearrangement was disrupted by co-treatment with a VEGFR2 inhibitor. Compared with a 0.9% saline dressing, LCPF significantly accelerated wound closure and re-epithelization when used as a dressing material in a full-thickness wound model. Histological analysis revealed increased neo-epidermis thickness and dermis collagen synthesis in the LCPF-treated group. Furthermore, LCPF treatment activated basal keratinocytes at the wound edge with the upregulation of Ki-67, VEGFA, and MMP2. Our preliminaries provided the benefit of wet dressing with pleural fluid to improve cutaneous wound closure through enhanced re-epithelization and disclosed future autologous application in cancer wound treatment.

Raised SPINK1 levels play a role in angiogenesis and the transendothelial migration of ALL cells

The present study was designed to assess whether raised Serine protease inhibitor Kazal type 1 (SPINK1) expressions modulates angiogenesis. Human umbilical vein endothelial cells (HUVECs) exposed to SPINK1 were noted to exhibit raised expressions of interleukin-8 (IL-8) as well as VCAM-1 and ICAM-1 cell adhesion molecules in a dose-dependent manner. In co-culture system of HUVECs and Acute lymphoblastic leukemia (ALL) cells, SPINK1 exposure also resulted in enhanced endothelial cell motility and ALL cells trans-endothelial migration. High concentrations of SPINK1 caused in vitro cellular reorganization into tubes in Matrigel-cultured HUVECs and induced in vivo vascularization and brain infiltration of NOD/SCID ALL model mice. The further transcriptomic analysis indicated that SPINK1 treatment altered several biological processes of endothelial cells and led to activation of the MAPK pathway. This study is the first to determine the neovascularization effects of raised SPINK1.

Endothelial Notch signaling directly regulates the small GTPase RND1 to facilitate Notch suppression of endothelial migration

To control sprouting angiogenesis, endothelial Notch signaling suppresses tip cell formation, migration, and proliferation while promoting barrier formation. Each of these responses may be regulated by distinct Notch-regulated effectors. Notch activity is highly dynamic in sprouting endothelial cells, while constitutive Notch signaling drives homeostatic endothelial polarization, indicating the need for both rapid and constitutive Notch targets. In contrast to previous screens that focus on genes regulated by constitutively active Notch, we characterized the dynamic response to Notch. We examined transcriptional changes from 1.5 to 6 h after Notch signal activation via ligand-specific or EGTA induction in cultured primary human endothelial cells and neonatal mouse brain. In each combination of endothelial type and Notch manipulation, transcriptomic analysis identified distinct but overlapping sets of rapidly regulated genes and revealed many novel Notch target genes. Among the novel Notch-regulated signaling pathways identified were effectors in GPCR signaling, notably, the constitutively active GTPase RND1. In endothelial cells, RND1 was shown to be a novel direct Notch transcriptional target and required for Notch control of sprouting angiogenesis, endothelial migration, and Ras activity. We conclude that RND1 is directly regulated by endothelial Notch signaling in a rapid fashion in order to suppress endothelial migration.

Hypoxia pretreatment improves the therapeutic potential of bone marrow mesenchymal stem cells in hindlimb ischemia via upregulation of NRG-1.

Mesenchymal stem cells (MSCs) are considered a promising treatment for ischemic diseases, but their use is limited due to poor survival after injection. Hypoxia can significantly enhance the survival of MSCs. This study aimed to investigate hypoxia pretreatment of bone marrow mesenchymal stem cells (BM-MSCs) in hindlimb ischemia (HI) and the underlying mechanism. The HI mouse model was established and human BM-MSCs were injected into ischemic skeletal muscles. The blood flow reperfusion and capillary density were measured. In vitro, human BM-MSC cells were treated with hypoxia. The expression of NRG-1 and associated angiogenic factors were measured after knockdown or overexpression of NRG-1. The conditioned medium (CdM) of BM-MSCs was prepared and co-cultured with human umbilical vein endothelial cells (HUVECs), and then, the proliferation, migration, and angiogenesis of HUVECs were detected. After hypoxia pretreatment, NRG-1 expression, clone formation, proliferation, and angiogenic factor secretion from BM-MSCs were increased, while knockdown of NRG-1 reversed these results. In normoxia condition, overexpression of NRG-1 enhanced above factors. Additionally, hypoxia pretreatment of BM-MSCs induced the proliferation and migration of HUVECs and angiogenesis. Moreover, the injection of hypoxia pretreatment of BM-MSCs improved blood reperfusion and capillary density in HI mice, while knockdown of NRG-1 reversed the effect. Furthermore, the PI3K inhibitor and activator reversed the effect of NRG-1 overexpression and knockdown on angiogenesis. We concludes thathypoxia pretreatment of BM-MSCs facilitates angiogenesis and alleviates HI injury via NRG-1/PI3K/AKT pathway.

Histone 2B Facilitates Plasminogen-Enhanced Endothelial Migration through Protease-Activated Receptor 1 (PAR1) and Protease-Activated Receptor 2 (PAR2).

Plasminogen and its multiple receptors have been implicated in the responses of many different cell types. Among these receptors, histone 2B (H2B) has been shown to play a prominent role in macrophage responses. The contribution of H2B to plasminogen-induced endothelial migration, an event relevant to wound healing and angiogenesis, is unknown. Plasminogen enhanced the migration of endothelial cells, which was inhibited by both Protease-Activated Receptor-1 (PAR1) and 2 (PAR2) antagonists. H2B was detected on viable endothelial cells of venous and arterial origin, and an antibody to H2B that blocks plasminogen binding also inhibited the plasminogen-dependent migration by these cells. The antibody blockade was as effective as PAR1 or PAR2 antagonists in inhibiting endothelial cell migration. In pull-down experiments, H2B formed a complex with both PAR1 and PAR2 but not β3 integrin, another receptor implicated in endothelial migration in the presence of plasminogen. H2B was found to be associated with clathrin adapator protein, AP2µ (clathrin AP2µ) and β-arrestin2, which are central to the internationalization/signaling machinery of the PARs. These associations with PAR1-clathrin adaptor AP2µ- and PAR2-β-arrestin2-dependent internalization/signaling pathways provide a mechanism to link plasminogen to responses such as wound healing and angiogenesis.

Targeting circular RNA-MET for anti-angiogenesis treatment via inhibiting endothelial tip cell specialization

Endothelial tip cell specialization plays an essential role in angiogenesis, which is tightly regulated by the complicated gene regulatory network. Circular RNA (circRNA) is a type of covalently closed non-coding RNA that regulates gene expression in eukaryotes. Here, we report that the levels of circMET expression are significantly upregulated in the retinas of mice with oxygen-induced retinopathy, choroidal neovascularization, and diabetic retinopathy. circMET silencing significantly reduces pathological angiogenesis and inhibits tip cell specialization invivo. circMET silencing also decreases endothelial migration and sprouting invitro. Mechanistically, circMET regulates endothelial sprouting and pathological angiogenesis by acting as a scaffold to enhance the interaction between IGF2BP2 and NRARP/ESM1. Clinically, circMET is significantly upregulated in the clinical samples of the patients of diabetic retinopathy. circMET silencing could reduce diabetic vitreous-induced endothelial sprouting and retinal angiogenesis invivo. Collectively, these data identify a circRNA-mediated mechanism that coordinates tip cell specialization and pathological angiogenesis. circMET silencing is an exploitable therapeutic approach for the treatment of neovascular diseases.

Pericyte-derived extracellular vesicle-mimetic nanovesicles ameliorate erectile dysfunction via lipocalin 2 in diabetic mice.

Diabetes mellitus is one of the main causes of erectile dysfunction (ED). Men with diabetic ED do not respond well to oral phosphodiesterase-5 inhibitors owing to neurovascular dysfunction. Pericyte-derived extracellular vesicle-mimetic nanovesicles (PC-NVs) are known to promote nerve regeneration in a mouse model of cavernous nerve injury. Here, we report that administration of PC-NVs effectively promoted penile angiogenesis and neural regeneration under diabetic conditions, thereby improving erectile function. Specifically, PC-NVs induced endothelial proliferation and migration and reduced cell apoptosis under diabetic conditions. In addition, PC-NVs induced neural regeneration in STZ-induced diabetic mice in dorsal root ganglion and major pelvic ganglion explants in vivo and ex vivo under high-glucose conditions. We found that lipocalin 2 (Lcn2) is a new target of PC-NVs in this process, demonstrating that PC-NVs exert their angiogenic and nerve-regeneration effects by activating MAP kinase and PI3K/Akt and suppressing P53 signaling pathway in an Lcn2-dependent manner. Our findings provide new conclusive evidence that PC-NVs can promote neurovascular regeneration and recovery of erectile function under diabetic conditions via an Lcn2-dependent mechanism. Thus, local administration of PC-NVs may be a promising treatment strategy for the treatment of diabetic ED.

Scarless vocal fold regeneration by urine-derived stem cells and small intestinal submucosa hydrogel composites through enhancement of M2 macrophage Polarization, neovascularization and Re-epithelialization

Vocal fold (VF) scarring is a major etiology of voice disorders and a leading cause for permanent intractable dysphonia. To date, several approaches have been used to address VF injury and scarring, which included scar excision and biomaterial injection and/or implantation. Despite the considerable research progress made in this field, these have not been established as routine clinical procedures due to various restrictions. Tissue engineered scaffolds for delivering growth factors in vivo and implementation of simultaneous delivery of multi-potent cells may achieve better outcome. In this study, we have developed Urine-derived stem cells - Small intestinal submucosa hydrogel composites (USCs@SIS), which are injectable and capable of sustaining the survival and metabolism of the USCs in the scarred VF. The three-dimensional complex showed a robust ability of anti-inflammatory polarization, neovascularization, re-epithelialization and anti-fibrosis in a rabbit model for heat-injured VFs. In vitro experiment proved that the hydrogel composites could promote polarization of M2 macrophages, tube formation by human umbilical vein endothelial cells, proliferation and migration by human bronchial epithelial cells, and suppression of differentiation of human fetal lung fibroblasts and human primary fibroblast cells of VF. Application of the USCs@SIS thereby may overcome the substantial limitations of the current strategies for scarless VF regeneration.

A Workflow to Track and Analyze Endothelial Migration During Vascular Development in Zebrafish Embryos Using Lightsheet Microscopy.

Zebrafish allow unrivalled in vivo imaging of vascular development due to their optical translucency and the availability of transgenic lines which fluorescently label cells and tissues of interest. Advances in light sheet fluorescence microscopy allow longer and faster imaging of live embryos at higher resolutions than previously possible, which facilitates study of dynamic cellular and molecular mechanisms underlying vessel formation and function. Here we describe a workflow using lightsheet microscopy to quantify endothelial cell (EC) migration dynamics during vascular development. Tracking movement of EC nuclei and analyzing the properties of EC migration trajectories permit detailed studies of angiogenesis and vascular remodeling in different contexts.

In Vitro Co-culture of Fibroblast and Endothelial Cells to Assess Angiogenesis.

Angiogenesis relies on the spatial and temporal coordination of endothelial migration and proliferation to form new blood vessels. This occurs through synchronous activation of multiple downstream pathways which facilitate vascular development. Proangiogenic growth factors and supporting extracellular matrix allow the formation of capillary-like tubules, reminiscent of microvascular beds, in vitro. In this chapter, we describe a methodology for the establishment of vascular networks by co-culture of endothelial cells and fibroblasts to facilitate the study of tubulogenic and angiogenic potential. We detail the use of siRNA mediated knockdown to deplete target genes of interest, in either the endothelial or fibroblast cells, to allow the assessment of their role in angiogenesis. Finally, we detail how these vascular networks may be stained using immunofluorescence to allow quantification of angiogenic potential in vitro.

Topical application of TAK1 inhibitor encapsulated by gelatin particle alleviates corneal neovascularization.

Rationale: Corneal neovascularization (CoNV) is a severe complication of various types of corneal diseases, that leads to permanent visual impairment. Current treatments for CoNV, such as steroids or anti-vascular endothelial growth factor agents, are argued over their therapeutic efficacy and adverse effects. Here, we demonstrate that transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) plays an important role in the pathogenesis of CoNV.Methods: Angiogenic activities were assessed in ex vivo and in vitro models subjected to TAK1 inhibition by 5Z-7-oxozeaenol, a selective inhibitor of TAK1. RNA-Seq was used to examine pathways that could be potentially affected by TAK1 inhibition. A gelatin-nanoparticles-encapsulated 5Z-7-oxozeaenol was developed as the eyedrop to treat CoNV in a rodent model.Results: We showed that 5Z-7-oxozeaenol reduced angiogenic processes through impeding cell proliferation. Transcriptome analysis suggested 5Z-7-oxozeaenol principally suppresses cell cycle and DNA replication, thereby restraining cell proliferation. In addition, inhibition of TAK1 by 5Z-7-oxozeaenol blocked TNFα-mediated NFκB signalling, and its downstream genes related to angiogenesis and inflammation. 5Z-7-oxozeaenol also ameliorated pro-angiogenic activity, including endothelial migration and tube formation. Furthermore, topical administration of the gelatin-nanoparticles-encapsulated 5Z-7-oxozeaenol led to significantly greater suppression of CoNV in a mouse model compared to the free form of 5Z-7-oxozeaenol, likely due to extended retention of 5Z-7-oxozeaenol in the cornea.Conclusion: Our study shows the potential of TAK1 as a therapeutic target for pathological angiogenesis, and the gelatin nanoparticle coupled with 5Z-7-oxozeaenol as a promising new eyedrop administration model in treatment of CoNV.