Endothelial Migration Research Articles

Mechano-Redox Control of Macrophage-1 Antigen De-Adhesion From ICAM-1 (Intercellular Adhesion Molecule 1) by Protein Disulfide Isomerase Promotes Directional Movement Under Flow.

Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMβ2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the βI domain of the β2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the βI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

Abstract 2654: Inhibition of lymphoma interactions withangiogenic and lymphangiogenic endotheliumusing αCD20-IgG1-huEndoP125A, an anti-CD20 endostatin fusion protein

Abstract Mantle Cell Lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma that frequently metastasizes. Lymphoma dissemination into distant sites is the primary cause of cancer mortality. Lymphoma metastasis requires development of supporting vasculature in which the interaction between malignant B cells and the vasculature or lymphatics is critical to tumor cell growth and dissemination. Human umbilical vein endothelial cells (HUVEC) or human lymphatic endothelial cells (HLEC) cultured on Matrigel, in vitro, assemble into networks of capillary like structures (CLS) or lymphatic like structures (LLS). When Mino or Jeko-1 MCL cells are co-cultured with vascular or lymphatic cells they migrate toward and anneal to nascent CLS or LLS. We constructed a novel antibody fusion protein, αCD20-IgG1-huEndoP125A, to inhibit such interactions. αCD20-IgG1-huEndoP125A links an αCD20 human IgG1 targeting domain and a mutant version of human endostatin, huEndoP125A, with enhanced anti-angiogenic properties. αCD20-IgG1-huEndoP125A completely inhibits CLS or LLS formation and prevents the alignment and annealing of MCL to CLS or LLS. αCD20-IgG1-huEndoP125A also significantly reduces both endothelial and MCL migration and invasion in vitro. Cell migration and trafficking is tightly regulated by chemokine interactions with corresponding chemokine receptors. We assayed for the presence of chemokines using a dot blot chemokine array. Jeko-1 and HUVEC cells were cultured on Matrigel, alone or together in co-culture, and either treated with αCD20-IgG1-huEndoP125A or left untreated. CXCL12 was expressed at high levels in HUVEC cultures undergoing CLS formation and expression was further increased in HUVEC and MCL co-cultures. HUVEC or HUVEC/MCL co-cultures treated with αCD20-IgG1-huEndoP125A showed markedly decreased CXCL12 expression. Flow cytometry showed that corresponding chemokine receptor CXCR4 was upregulated on MCL cells compared to normal B cells. CXCR4 was also upregulated on HUVEC undergoing CLS formation, and further increased on HUVEC in co-culture with MCL cells. αCD20-IgG1-huEndoP125A inhibited HUVEC CLS formation, reduced MCL migration to and alignment with nascent CLS or LLS, downregulated CXCL12 levels in HUVEC or coculture supernatants, and reduced CXCR4 expression on both HUVEC and MCL cells. Murine 38c13-huCD20 lymphoma cells were implanted s.c. into C3H mice and mice were treated i.p. with either PBS, Rituximab, or equimolar αCD20-IgG1-huEndoP125A. Mice treated with αCD20-IgG1-huEndoP125A showed significantly reduced tumor growth compared to mice treated with PBS or rituximab. αCD20-IgG1-huEndoP125A ability to inhibit angiogenesis, lymphangiogenesis, and lymphoma association with angiogenic or lymphatic vasculature may be a potent means of preventing lymphoma growth and metastasis. Citation Format: Christian Elledge, Seung-Uon Shin, Rathin Das, Yu Zhang, Hyun-Mi Cho, Sundaram Ramakrishnan, Ankita Sankar, Joseph Rosenblatt. Inhibition of lymphoma interactions withangiogenic and lymphangiogenic endotheliumusing αCD20-IgG1-huEndoP125A, an anti-CD20 endostatin fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2654.

Fucoidan and topography modification improved in situ endothelialization on acellular synthetic vascular grafts.

Thrombogenesis remains the primary failure of synthetic vascular grafts. Endothelial coverage is crucial to provide an antithrombogenic surface. However, most synthetic materials do not support cell adhesion, and transanastomotic endothelial migration is limited. Here, a surface modification strategy using fucoidan and topography was developed to enable fast in situ endothelialization of polyvinyl alcohol, which is not endothelial cell-adhesive. Among three different immobilization approaches compared, conjugation of aminated-fucoidan promoted endothelial monolayer formation while minimizing thrombogenicity in both in vitro platelet rich plasma testing and ex vivo non-human primate shunt assay. Screening of six topographical patterns showed that 2μm gratings increased endothelial cell migration without inducing inflammation responses of endothelial cells. Mechanistic studies demonstrated that fucoidan could attract fibronectin, enabling integrin binding and focal adhesion formation and activating focal adhesion kinase (FAK) signaling, and 2μm gratings further enhanced FAK-mediated cell migration. In a clinically relevant rabbit carotid artery end-to-side anastomosis model, 60% in situ endothelialization was observed throughout the entire lumen of 1.7mm inner diameter modified grafts, compared to 0% of unmodified graft, and the four-week graft patency also increased. This work presents a promising strategy to stimulate in situ endothelialization on synthetic materials for improving long-term performance.

MiR-126 promotes M1 to M2 macrophage phenotype switching via VEGFA and KLF4.

Macrophage polarization and microRNA play crucial roles in the development of atherosclerosis (AS). The M1 macrophage phenotype contributes to the formation of plaques, while the M2 macrophage phenotype resolves inflammation and promotes tissue repair. MiR-126 has been found to play a role in regulating macrophage polarization in the context of AS. However, the exact mechanism of miR-126 requires further research. The foam cell model was established by stimulating THP-1 with oxidized low-density lipoprotein (ox-LDL). We transfected foam cells with miR-126 mimic and its negative control. The transfection of miR-126 was implemented by riboFECT CP transfection kit. The levels of miR-126 and M1/M2 associated genes in foam cells were quantified using reverse transcription-quantitative PCR (RT-qPCR). Additionally, the expressions of CD86+ and CD206+ cells in foam cells were determined by flow cytometry. Western blotting and RT-qPCR were used to determine the protein and mRNA levels of the vascular endothelial growth factor A (VEGFA) and the transcriptional regulator Krüppel-like factor 4 (KLF4), respectively. Additionally, we detected endothelial cell migration after co-culturing endothelial cells and macrophages. MG-132 was used to indirectly activate the expression of VEGFA, and the expression of KLF4 was also evaluated. The activation of apoptosis and production of foam cells were boosted by the addition of ox-LDL. We transfected foam cells with miR-126 mimic and its negative control and observed that miR-126 greatly suppressed foam cell development and inhibited phagocytosis. Moreover, it caused pro-inflammatory M1 macrophages to switch to the anti-inflammatory M2 phenotype. This was reflected by the increase in anti-inflammatory gene expression and the decrease in pro-inflammatory gene expression. Additionally, miR-126 dramatically decreased the expressions of VEGFA and KLF4. The protein-protein interaction network analysis showed a significantly high correlation between miR-126, VEGFA, and KLF4. MiR-126 may also promote EC migration by activating macrophage PPAR γ expression and effectively suppressing macrophage inflammation. MG-132 indirectly activated the expression of VEGFA, and the expression of KLF4 also significantly increased, which indicates a direct or indirect relationship between VEGFA and KLF4. Our study shows that miR-126 can reverse ox-LDL-mediated phagocytosis and apoptosis in macrophages. Consequently, the potential role of miR-126 was manifested in regulating macrophage function and promoting vascular endothelial migration.

MicroRNA detection in carotid atherosclerosis: prospects for clinical use

Carotid atherosclerosis is a significant cause of cerebrovascular disease. However, with many candidate markers, precise assessment of its development and progression risks is still limited. This paper reviews state-of-the-art concepts of microRNA as an atherogenesis biomarker throughout various stages including endothelial dysfunction, cholesterol/lipid metabolism, inflammation, oxidative stress, angiogenesis regulation, and proliferation and migration of vascular smooth muscle cells. Based on the available literature, we have described most significant microRNAs for each stage characterized in brief. We have visualized interactions between microRNAs and validated target genes with MIENTURNET and suggest and justify a set of microRNAs for further pilot studies of carotid atherosclerosis.

Regulation of human endothelial cell migration by oral contraceptive estrogen receptor ligands

Ethinylestradiol (EE) and estetrol (E4) are the two main estrogenic agents used in combined oral contraceptives. These compounds have different binding affinity to and efficacy on estrogen receptors (ER) subtypes. We previously reported that treatment with estrogenic agents enhances angiogenesis via nongenomic, G protein-coupled estrogen receptor (GPER)-dependent mechanisms. However, the impact of EE and E4 on human endothelial function has been little investigated. EE and E4 (10−9– 10−7 M) significantly enhanced migration of human umbilical vein endothelial cells (HUVECs) using scratch and Boyden chamber assays. Mechanistically, both agents increased accumulation of phosphorylated protein tyrosine kinase 2 on tyrosine 397 (FAK Y397), a key player in endothelial cell motility, after 30-min treatment. Treatment with increasing concentrations of EE, but not E4, enhanced accumulation of the glycolysis activator PFKFB3. Of note, effects of EE and E4 on endothelial migration and signalling proteins were abolished by addition of the GPER antagonist G36 (10−6 M). Thus, EE and E4 induced comparable endothelial responses in vitro, suggesting no apparent alterations of vascular remodelling and regeneration capacity by oral contraceptives containing these agents.

Andrographolide suppresses hypoxia-induced embryonic hyaloid vascular system development through HIF-1a/VEGFR2 signaling pathway.

Ocular abnormalities and the development of retinal vasculature may cause postnatal retinopathy. In the past decade, tremendous progress has been made in identifying the mechanisms that regulate retina vasculature. However, the means of regulating embryonic hyaloid vasculature development is largely unknown. This study aims to determine whether and how andrographolide regulates embryonic hyaloid vasculature development. Murine embryonic retinas were used in this study. Whole mount isolectin B4 (IB4) staining, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and immunofluorescence staining (IF) were performed to determine whether andrographolide is critical for embryonic hyaloid vasculature development. BrdU incorporation assay, Boyden chamber migration assay, spheroid sprouting assay, and Matrigel-based tube formation assay were performed to evaluate whether andrographolide regulates the proliferation and migration of vascular endothelial cells. Molecular docking simulation and Co-immunoprecipitation assay were used to observe protein interaction. Hypoxia conditions exist in murine embryonic retinas. Hypoxia induces HIF-1a expression; high-expressed HIF-1a interacts with VEGFR2, resulting in the activation of the VEGF signaling pathway. Andrographolide suppresses hypoxia-induced HIF-1a expression and, at least in part, interrupts the interaction between HIF-1a and VEGFR2, causing inhibiting endothelial proliferation and migration, eventually inhibiting embryonic hyaloid vasculature development. Our data demonstrated that andrographolide plays a critical role in regulating embryonic hyaloid vasculature development.

Argonaute 2 Restores Erectile Function by Enhancing Angiogenesis and Reducing Reactive Oxygen Species Production in Streptozotocin (STZ)-Induced Type-1 Diabetic Mice.

Severe vascular and nerve damage from diabetes is a leading cause of erectile dysfunction (ED) and poor response to oral phosphodiesterase 5 inhibitors. Argonaute 2 (Ago2), a catalytic engine in mammalian RNA interference, is involved in neurovascular regeneration under inflammatory conditions. In the present study, we report that Ago2 administration can effectively improve penile erection by enhancing cavernous endothelial cell angiogenesis and survival under diabetic conditions. We found that although Ago2 is highly expressed around blood vessels and nerves, it is significantly reduced in the penis tissue of diabetic mice. Exogenous administration of the Ago2 protein restored erectile function in diabetic mice by reducing reactive oxygen species production-signaling pathways (inducing eNOS Ser1177/NF-κB Ser536 signaling) and improving cavernous endothelial angiogenesis, migration, and cell survival. Our study provides new evidence that Ago2 mediation may be a promising therapeutic strategy and a new approach for diabetic ED treatment.

A Phytoprostane from Gracilaria longissima Increases Platelet Activation, Platelet Adhesion to Leukocytes and Endothelial Cell Migration by Potential Binding to EP3 Prostaglandin Receptor.

Plant phytoprostanes (PhytoPs) are lipid oxidative stress mediators that share structural similarities with mammal prostaglandins (PGs). They have been demonstrated to modulate inflammatory processes mediated by prostaglandins. The present study aims to test the effects of the most abundant oxylipin from Gracilaria longissima, ent-9-D1t-Phytoprostane (9-D1t-PhytoP), on platelet activation and vascular cells as well as clarify possible interactions with platelets and the endothelial EP3 receptor Platelet and monocyte activation was assessed by flow cytometry in the presence of purified 9-D1t-PhytoP. Cell migration was studied using the human Ea.hy926 cell line by performing a scratch wound healing assay. The RNA expression of inflammatory markers was evaluated by RT-PCR under inflammatory conditions. Blind docking consensus was applied to the study of the interactions of selected ligands against the EP3 receptor protein. The 9D1t-PhytoP exerts several pharmacological effects; these include prothrombotic and wound-healing properties. In endothelial cells, 9D1t-PhytP mimics the migration stimulus of PGE2. Computational analysis revealed that 9D1t-PhytP forms a stable complex with the hydrophobic pocket of the EP3 receptor by interaction with the same residues as misoprostol and prostaglandin E2 (PGE2), thus supporting its potential as an EP3 agonist. The potential to form procoagulant platelets and the higher endothelial migration rate of the 9-D1t-PhytoP, together with its capability to interact with PGE2 main target receptor in platelets suggest herein that this oxylipin could be a strong candidate for pharmaceutical research from a multitarget perspective.

Endostatin level in coronavirus patients: a prospective study

Background Endostatin is a protein linked to the C-terminal of collagen in the basement membrane of endothelial cells. It functions as an antiangiogenic protein. It inhibits endothelial proliferation, migration, and tube formation. Studies have observed that it downregulates endothelial-signaling pathways of proangiogenic activity as well as upregulates the angiogenic gene. Aim This work was designated to explore the levels of endostatin in coronavirus disease 2019, trying to investigate its role as a prognostic marker. Patients and methods Patients with positive PCR for SARS-CoV-2 were divided into two groups (survivors and nonsurvivors). Computed tomography chest was for each patient. The nonsurvivor group comprised 19 patients and the survivor group had 31 patients. In addition, 50 normal volunteers participated in this study and served as a control group. Serum endostatin was measured by ELISA and was compared with other measured laboratory parameters. Results Endostatin was statically increased in nonsurvivors than both survivors and normal volunteers (P 1=0.007, P 2=<0.001). However, this significant increase was still observed in survivors on comparing with normal individuals. There was a correlation between endostatin and different parameters in coronavirus disease 2019 patients. Conclusion The high level of endostatin in this prospective study and its correlation with laboratory makers and clinical manifestations leads us to consider it as a valuable prognostic factor. Moreover, it can predict mortality and severity of patients. It was important to conduct a study on large scale to confirm this value.

The matricellular protein CCN3 supports lung endothelial homeostasis and function.

Aberrant vascular remodeling contributes to the progression of many aging-associated diseases, including idiopathic pulmonary fibrosis (IPF), where heterogeneous capillary density, endothelial transcriptional alterations, and increased vascular permeability correlate with poor disease outcomes. Thus, identifying disease-driving mechanisms in the pulmonary vasculature may be a promising strategy to limit IPF progression. Here, we identified Ccn3 as an endothelial-derived factor that is upregulated in resolving but not in persistent lung fibrosis in mice, and whose function is critical for vascular homeostasis and repair. Loss and gain of function experiments were carried out to test the role of CCN3 in lung microvascular endothelial function in vitro through RNAi and the addition of recombinant human CCN3 protein, respectively. Endothelial migration, permeability, proliferation, and in vitro angiogenesis were tested in cultured human lung microvascular endothelial cells (ECs). Loss of CCN3 in lung ECs resulted in transcriptional alterations along with impaired wound-healing responses, in vitro angiogenesis, barrier integrity as well as an increased profibrotic activity through paracrine signals, whereas the addition of recombinant CCN3 augmented endothelial function. Altogether, our results demonstrate that the matricellular protein CCN3 plays an important role in lung endothelial function and could serve as a promising therapeutic target to facilitate vascular repair and promote lung fibrosis resolution.

Interferon-γ drives macrophage reprogramming, cerebrovascular remodelling, and cognitive dysfunction in a zebrafish and a mouse model of ion imbalance and pressure overload.

Dysregulated immune response contributes to inefficiency of treatment strategies to control hypertension and reduce the risk of end-organ damage. Uncovering the immune pathways driving the transition from the onset of hypertensive stimulus to the manifestation of multi-organ dysfunction are much-needed insights for immune targeted therapy. To aid visualization of cellular events orchestrating multi-organ pathogenesis, we modelled hypertensive cardiovascular remodelling in zebrafish. Zebrafish larvae exposed to ion-poor environment exhibited rapid angiotensinogen up-regulation, followed by manifestation of arterial hypertension and cardiac remodelling that recapitulates key characteristics of incipient heart failure with preserved ejection fraction. In the brain, time-lapse imaging revealed the occurrence of cerebrovascular regression through endothelial retraction and migration in response to the ion-poor treatment. This phenomenon is associated with macrophage/microglia-endothelial contacts and endothelial junctional retraction. Cytokine and transcriptomic profiling identified systemic up-regulation of interferon-γ and interleukin 1β and revealed altered macrophage/microglia transcriptional programme characterized by suppression of innate immunity and vasculo/neuroprotective gene expression. Both zebrafish and a murine model of pressure overload-induced brain damage demonstrated that the brain pathology and macrophage/microglia phenotypic alteration are dependent on interferon-γ signalling. In zebrafish, interferon-γ receptor 1 mutation prevents cerebrovascular remodelling and dysregulation of macrophage/microglia transcriptomic profile. Supplementation of bone morphogenetic protein 5 identified from the transcriptomic approach as a down-regulated gene in ion-poor-treated macrophages/microglia that is rescued by interferon-γ blockage, mitigated cerebral microvessel loss. In mice subjected to transverse aortic constriction-induced pressure overload, typically developing cerebrovascular injury, neuroinflammation, and cognitive dysfunction, interferon-γ neutralization protected them from blood-brain barrier disruption, cerebrovascular rarefaction, and cognitive decline. These findings uncover cellular and molecular players of an immune pathway communicating hypertensive stimulus to structural and functional remodelling of the brain and identify anti-interferon-γ treatment as a promising intervention strategy capable of preventing pressure overload-induced damage of the cerebrovascular and nervous systems.

STAT3 signaling induced by the IL-6 family of cytokines modulates angiogenesis.

Aberrant angiogenesis is a hallmark of cardiovascular and retinal neovascular disease. The STAT3 signaling pathway represents a potential pharmacological target for these diseases due to its impact on angiogenesis. Surprisingly, some STAT3 activators, such as the IL-6 cytokine family member oncostatin M (OSM), enhance angiogenesis, whereas others, such as ciliary neurotropic factor (CNTF), reduce it. This study aimed to clarify these conflicting effects. In contrast to the anti-angiogenic cytokine CNTF, the pro-angiogenic cytokine OSM was able to activate intracellular signaling pathways beyond the STAT3 pathway, including the ERK and AKT pathways. These differences translated into transcriptomic and metabolic shifts. siRNA-mediated STAT3 knockdown experiments showed a decrease in VEGF-induced endothelial migration and sprouting, enhancing the pro-angiogenic drive of OSM and switching the CNTF response from anti-angiogenic to pro-angiogenic. These effects correlated with a transcriptomic shift representing enhanced STAT1 and ERK activity following STAT3 knockdown, including a compensatory prolonged phosphorylated STAT1 activity. In conclusion, the angiogenic effect of STAT3 appears to be determined by cytokine-induced STAT3 specificity and simultaneous activity of other intracellular signaling pathways, whereas the STAT3 pathway, predominantly recognized for its pro-angiogenic phenotypes, reveals novel anti-angiogenic potential.

Transcriptomic Signature of Horseshoe Crab Carcinoscorpius rotundicauda Hemocytes' Response to Lipopolysaccharides.

Carcinoscorpius rotundicauda (C. rotundicauda) is one of the four species of horseshoe crabs (HSCs). The HSC hemocytes store defense molecules that are released upon encountering invading pathogens. The HSCs rely on this innate immunity to continue its existence as a living fossil for more than 480 million years. To gain insight into the innate mechanisms involved, transcriptomic analysis was performed on isolated C. rotundicauda hemocytes challenged with lipopolysaccharides (LPS), the main components of the outer cell membrane of gram-negative bacteria. RNA-sequencing with Illumina HiSeq platform resulted in 232,628,086 and 245,448,176 raw reads corresponding to 190,326,253 and 201,180,020 high-quality mappable reads from control and LPS-stimulated hemocytes, respectively. Following LPS-stimulation, 79 genes were significantly upregulated and 265 genes were downregulated. The differentially expressed genes (DEGs) were related to multiple immune functional categories and pathways such as those of the cytoskeleton, Toll and Imd, apoptosis, MAP kinase (MAPK), inositol phosphate metabolism, phagosome, leucocyte endothelial migration, and gram-negative bacterial infection, among others. This study provides important information about the mechanisms of response to LPS, which is relevant for the understanding the HSCs' immune response.

Vascularized polypeptide hydrogel modulates macrophage polarization for wound healing

Wound repair involves a sophisticated process that includes angiogenesis, immunoregulation and collagen deposition. However, weak revascularization performance and the lack of biochemical cues to trigger immunomodulatory function currently limit biomaterial applications for skin regeneration and tissue engineering. Herein, we fabricate a new bioactive polypeptide hydrogel (QK-SF) constituted by silk fibroin (SF) and a vascular endothelial growth factor mimetic peptide KLTWQELYQLKYKGI (QK) for tissue regeneration by simultaneously promoting vascularization and macrophage polarization. Our results showed that this QK-SF hydrogel can be prepared via an easy manufacturing process, and exhibited good gel stability and low cytotoxicity to cultured human umbilical vein endothelial cells (HUVECs) via both live/dead and cell counting kit-8 assays. Importantly, this QK-SF hydrogel triggered macrophage polarization from M1 into M2, as exemplified by the enhanced expression of the M2 marker and decreased expression of the M1 marker in RAW264.7 cells. Furthermore, the QK-SF hydrogel showed high capacity for inducing endothelial growth, migration and angiogenesis, which were proved by increased expression of angiogenesis-related genes in HUVECs. Consistent with in vitro findings, in vivo data show that the QK-SF hydrogel promoted M2 polarization, keratinocyte differentiation, and collagen deposition in the mouse skin wound model in immunohistochemistry assay. Furthermore, this QK-SF hydrogel can reduce inflammation, induce angiogenesis and promote wound healing as exemplified by the increased vessel formation and decreased wound area in the mouse skin wound model. Altogether, these results indicate that the bioactive QK-SF hydrogel plays dual functional roles in promoting angiogenesis and immunoregulation for tissue regeneration. Statement of significanceThe QK-SF hydrogel plays dual functional roles in promoting angiogenesis and immunoregulation for tissue repair and wound healing. The QK-SF hydrogel can be prepared via an easy manufacturing process, and exhibited good gel stability and low cytotoxicity to cultured HUVECs. The QK-SF hydrogel triggered macrophage polarization from M1 into M2. The QK-SF hydrogel showed high capacity for inducing endothelial growth, migration and angiogenesis. The QK-SF hydrogel promoted M2 polarization, keratinocyte differentiation, and collagen deposition.

ROBO4 Inhibits Irradiation-Induced Microvascular Permeability, Endothelial to Mesenchymal Transition, and Improves Hematopoietic Reconstitution

Background: Hematopoietic Stem Cells Transplantation involves irradiation preconditioning which causes bone marrow endothelial cells dysfunction. While much emphasis is on the reconstitution of an adequate number of HSCs to repopulate the hematopoietic system after the death of malignant cells, EC preservation and normal functioning are indispensable to overcome the preconditioning damages. ROBO4 is an endothelial-specific receptor that is important in maintaining the vasculature. The purpose of this study is to ascertain ROBO4's role in regulating irradiation-induced damage of the endothelium and its subsequent effect on hematopoietic reconstruction. Method: Microvascular endothelial cells were treated with γ-radiation to establish an endothelial cell injury model. The expression of ROBO4 in the microvascular endothelial cells was manipulated employing lentiviral mediated RNAi (shRNA) and gene overexpression technology before irradiation treatment. The permeability of endothelial cells and its mesenchymal transition (Endo-MT) was measured using qPCR, immunocytochemistry, and immunoblotting to analyze the effect on the expression and distribution of junctional molecules (CD144, Connexin 43, and Occludin, etc.), endothelial cell-specific molecules (PECAM-1, VEGFR2 and TIE2, etc.), and specific mesenchymal cell markers (Fibronectin, Vimentin, S100A4, and ACTA2, etc.). Using Transwell and wound healing migration, Matrigel tube formation, and Transwell and FITC-Dextran permeability assays, we determined the changes in endothelial functions after ROBO4 gene manipulation and irradiation. In addition, a co-culture model of endothelial cells and hematopoietic stem cells isolated from C57BL/6 mice was constructed, and the effects of irradiated endothelial cells on the proliferation, differentiation and cell cycle of hematopoietic stem cells were analyzed using flow cytometry. Results: Ionizing radiation upregulates ROBO4 expression but disrupts endothelial junctional molecules. ROBO4 deletion enhances the irradiation-induced degradation of endothelial junctions hence increasing the permeability of the endothelium and also facilitating endothelial to mesenchymal transition (Endo-MT) by activating both the canonical (Smads) and non-canonical (PI3K/AKT/mTOR) pathways. Endothelial ROBO4 overexpression substantially reduces microvascular permeability and mitigates Endo-MT following irradiation. Even though ROBO4 overexpression does not dramatically affect endothelial functions, tube formation and migration potentials of endothelial cells significantly increase upon the knockdown of ROBO4 and irradiation. We also found that EC ROBO4 silencing inhibits the proliferation of HSPC and promotes HSC quiescence. On the other hand, ROBO4 overexpression lessened the inhibitory effects of the injured endothelium on hematopoietic stem cell expansion. Lastly, we saw that ROBO4 overexpression inhibits erythrocytosis and granulocytosis after irradiation compared to the control groups suggesting that EC ROBO4 may play a regulatory role in HSC differentiation. Conclusion: ROBO4 maintains microvascular integrity after radiation preconditioning treatment by regulating permeability and Endo-MT, protects endothelial function, and reduces the inhibitory effect of injured endothelium on hematopoietic reconstitution.

Abstract 13094: Extracellular Vesicles Associate With Cardio-Metabolic Status and Endothelial Dysfunction in a Cohort of African-American Women

Extracellular vesicles (EVs) are emerging as promising biomarkers for cardiovascular disease (CVD) and contributors to CVD pathogenesis. Plasma EVs access the endothelium from various vascular beds allowing them to impact endothelial function and inform on the global particulate secretome produced by parenchymal, vascular and immune cells. In this study we examined associations between plasma EVs and biochemical and physiological parameters in a cohort of African American (AA) women at risk for CVD. We also examined EV effect on coronary endothelial function. This study will serve as baseline data for a longitudinal study aimed to determine the effects of exercise on EVs and endothelial function in a similar cohort.A cohort of 24 AA women between 22-71 yrs, with BMI between 26-47 kg/m 2 and ASCVD risk scores between 2.3-22.1 were included in the study. EVs were isolated from fasted heparinized plasma using size exclusion chromatography. EV size and numbers were determined using nanoparticle tracking analysis. Lipids, inflammatory cytokines, and metabolic panels were measured at the time of blood draw. Human coronary endothelial cells were treated with EVs from participants and endothelial barrier function and migration were measured using ECIS technology. Associations were determined using multivariable linear regression analysis, adjusted for BMI and ASCVD risk.We found a negative association (beta=-0.712, p<0.01) between total triglycerides (TG), TG rich lipoprotein (Lp), and EV size; EV concentration was positively associated (beta= 0.51-0.61, p<0.01) with TG rich Lp, LDL cholesterol and TNFa. Reduced endothelial barrier function due to EVs was positively associated with monocyte counts (beta=0.42, p<0.05). EVs effect on increased endothelial migration was strongly and negatively associated with monocyte counts (beta=-0.85, p<0.001), IL8 and CXCl10 (beta=-0.56-0.46, p<0.05).Collectively these data show that EVs number and size strongly associate with circulating lipids, while EV impact on endothelial dysfunction is associated with inflammatory markers. EVs are therefore promising indicators of endothelial dysfunction that associate with inflammatory and metabolic markers even after adjusting for BMI and ASCVD risk.

Non-Muscle MLCK Contributes to Endothelial Cell Hyper-Proliferation through the ERK Pathway as a Mechanism for Vascular Remodeling in Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by endothelial dysfunction, uncontrolled proliferation and migration of pulmonary arterial endothelial cells leading to increased pulmonary vascular resistance resulting in great morbidity and poor survival. Bone morphogenetic protein receptor II (BMPR2) plays an important role in the pathogenesis of PAH as the most common genetic mutation. Non-muscle myosin light chain kinase (nmMLCK) is an essential component of the cellular cytoskeleton and recent studies have shown that increased nmMLCK activity regulates biological processes in various pulmonary diseases such as asthma and acute lung injury. In this study, we aimed to discover the role of nmMLCK in the proliferation and migration of pulmonary arterial endothelial cells (HPAECs) in the pathogenesis of PAH. We used two cellular models relevant to the pathobiology of PAH including BMPR2 silenced and vascular endothelial growth factor (VEGF) stimulated HPAECs. Both models demonstrated an increase in nmMLCK activity along with a robust increase in cellular proliferation, inflammation, and cellular migration. The upregulated nmMLCK activity was also associated with increased ERK expression pointing towards a potential integral cytoplasmic interaction. Mechanistically, we confirmed that when nmMLCK is inhibited by MLCK selective inhibitor (ML-7), proliferation and migration are attenuated. In conclusion, our results demonstrate that nmMLCK upregulation in association with increased ERK expression may contribute to the pathogenesis of PAHby stimulating cellular proliferation and migration.

6'-Sialylactose abolished lipopolysaccharide-induced inflammation and hyper-permeability in endothelial cells.

Disruption of the endothelial barrier function and reduction in cell migration leads to endothelial dysfunction. One of the most abundant human milk oligosaccharides, 6'-sialylactose (6'-SL), is reported to exert various biological functions related to inflammatory responses. In this study, we evaluated the effects of 6'-SL on lipopolysaccharide (LPS)-induced inflammation caused by endothelial barrier damage. Our results showed that LPS at 500ng/mL strongly not only abolished cell migration but also hyperactivated MAPK and NF-κB pathways. 6'-SL suppressed LPS-induced endothelial inflammation via ERK1/2, p38, and JNK MAPK pathways. 6'-SL supported endothelial junctions by upregulating PECAM-1 expression and mRNA levels of tight junctions, such as ZO-1 and occludin, which were downregulated by LPS stimulation. It significantly inhibited the nuclear translocation of NF-κB, along with the downregulation of inflammatory cytokines, including TNF-α, IL-1β, MCP-1, VCAM-1, and ICAM-1. Furthermore, 6'-SL abolished NF-κB-mediated STAT3 in controlling endothelial migration and hyperpermeability via downregulating STAT3 activation and nuclear translocation. Finally, LPS induced over-expression of VCAM-1 and ZO-1 disassembly in both atheroprone and atheroprotective areas of mouse aorta, which were reversed by 6'-SL treatment. Altogether, our findings suggest that 6'-SL is a potent therapeutic agent for modulating inflammatory responses and endothelial hyperpermeability.

TRNA-derived fragment tRF-1001: A novel anti-angiogenic factor in pathological ocular angiogenesis

Transfer RNA-derived fragments (tRFs) are a novel class of non-coding RNA transcripts and play important roles in several physiological/pathological processes. However, the role of tRFs in ocular angiogenesis remains elusive. Herein, we investigate whether the intervention of tRF-1001 expression could suppress pathological ocular angiogenesis. The results show that the levels of tRF-1001 expression were reduced in the retinas of an oxygen-induced retinopathy (OIR) model, choroidal neovascularization model, and endothelial sprouting model invitro. Increased tRF-1001 expression could suppress ocular angiogenesis and endothelial sprouting invivo and reduce endothelial migration, specification, and sprouting invitro. Mechanistically, tRF-1001 regulated endothelial angiogenic effects via tRF-1001/METTL3/RBPJ-MAML1 signaling. The levels of tRF-1001 expression were downregulated in the aqueous humor of age-related macular degeneration (AMD) patients. tRF-1001 upregulation could suppress AMD aqueous humor-induced endothelial sprouting and pathological angiogenesis. Collectively, tRF-1001 acts as an anti-angiogenic factor during ocular angiogenesis. Targeting tRF-1001-mediated signaling is a therapeutic option for ocular neovascular diseases.