Increase In Endothelial Cell Migration Research Articles

Vascular endothelial effects of dibutyl phthalate: In vitro and in vivo evidence

Dibutyl phthalate (DBP) is widely used in many consumer and personal care products. Here, we report vascular endothelial response to DBP in three different exposure scenarios: after short-term exposure (24 h) of human endothelial cells (ECs) EA.hy926 to 10−6, 10−5, and 10−4 M DBP, long-term exposure (12 weeks) of EA.hy926 cells to 10−9, 10−8, and 10−7 M DBP, and exposure of rats (28 and 90 days) to 100, 500, and 5000 mg DBP/kg food. We examined different vascular functions such as migration of ECs, adhesion of ECs to the extracellular matrix, tube formation, the morphology of rat aorta, as well as several signaling pathways involved in controlling endothelial function. Short-term in vitro exposure to DBP increased migration of ECs through G protein-coupled estrogen receptor, extracellular signal-regulated kinase 1/2, and nitric oxide (NO) signaling and decreased adhesion to gelatin. Long-term in vitro exposure to DBP transiently increased EC migration and had a bidirectional effect on EC adhesion to gelatin and tube formation. These effects were accompanied by a sustained increase in NO production and endothelial NO synthase (eNOS) and Akt activity. In vivo, exposure to DBP for 90 days decreased the aortic wall-to-lumen ratio and increased eNOS and Akt phosphorylation in ECs of rat aorta. This comparative investigation has shown that exposure to DBP may affect vascular function by altering EC migration, adhesion to gelatin, and tube formation after short- and long-term in vitro exposure and by decreasing the aortic wall-to-lumen ratio in vivo. The eNOS-NO and Akt signaling could be important in mediating the effects of DBP in long-term exposure scenarios.

Soluble Endoglin Stimulates Inflammatory and Angiogenic Responses in Microglia That Are Associated with Endothelial Dysfunction.

Increased soluble endoglin (sENG) has been observed in human brain arteriovenous malformations (bAVMs). In addition, the overexpression of sENG in concurrence with vascular endothelial growth factor (VEGF)-A has been shown to induce dysplastic vessel formation in mouse brains. However, the underlying mechanism of sENG-induced vascular malformations is not clear. The evidence suggests the role of sENG as a pro-inflammatory modulator, and increased microglial accumulation and inflammation have been observed in bAVMs. Therefore, we hypothesized that microglia mediate sENG-induced inflammation and endothelial cell (EC) dysfunction in bAVMs. In this study, we confirmed that the presence of sENG along with VEGF-A overexpression induced dysplastic vessel formation. Remarkably, we observed increased microglial activation around dysplastic vessels with the expression of NLRP3, an inflammasome marker. We found that sENG increased the gene expression of VEGF-A, pro-inflammatory cytokines/inflammasome mediators (TNF-α, IL-6, NLRP3, ASC, Caspase-1, and IL-1β), and proteolytic enzyme (MMP-9) in BV2 microglia. The conditioned media from sENG-treated BV2 (BV2-sENG-CM) significantly increased levels of angiogenic factors (Notch-1 and TGFβ) and pERK1/2 in ECs but it decreased the level of IL-17RD, an anti-angiogenic mediator. Finally, the BV2-sENG-CM significantly increased EC migration and tube formation. Together, our study demonstrates that sENG provokes microglia to express angiogenic/inflammatory molecules which may be involved in EC dysfunction. Our study corroborates the contribution of microglia to the pathology of sENG-associated vascular malformations.

Hyaluronic Acid Oligomer Immobilization as an Angiogenic Trigger for the Neovascularization of TE Constructs.

Tissue engineered (TE) substitutes of clinically relevant sizes need an adequate vascular system to ensure function and proper tissue integration after implantation. However, the predictable vascularization of TE substitutes is yet to be achieved. Molecular weight variations in hyaluronic acid (HA) have been pointed to trigger angiogenesis. Thus, this study investigates HA oligomer immobilization as a promoter for TE construct vascularization. As a proof-of-concept, the surface of methacrylated gelatin (GelMA) hydrogels were functionalized with high molecular weight (HMW; 1.5 to 1.8 MDa) and low molecular weight (LMW; < 10 kDa) HA, previously modified with aldehyde groups to enable the immobilization through Schiff's base formation. The ability of A-HA to bind amine-presenting surfaces was confirmed by Surface Plasmon Resonance (SPR). Human Umbilical Vein Endothelial Cells (HUVECs) seeded over hydrogels functionalized with LMW HA showed higher proliferation and expression of angiogenic markers (KDR and CD31), than those grown in HMW HA conjugated- or plain surfaces, in line with the activation of HA ERK1/2 mediated downstream signaling. Moreover, when cocultured with human dental pulp cells (hDPCs) encapsulated into the GelMA, an increase in endothelial cell migration was observed for the LMW HA functionalized formulations. Overall LMW HA functionalization enhanced endothelial cell response showing potential as an angiogenesis inducer for TE applications.

Effect of a coronary-heart-disease-associated variant of ADAMTS7 on endothelial cell angiogenesis

Background and aimsRecent studies have unveiled an association between ADAMTS7 gene variation and coronary artery disease (CAD) caused by atherosclerosis. We investigated if the ADAMTS7 Serine214-to-Proline substitution arising from a CAD-associated variant affected angiogenesis, since neovascularization plays an important role in atherosclerosis. Methods and resultsADAMTS7 knockdown in vascular endothelial cells (ECs) attenuated their angiogenesis potential, whereas augmented ADAMTS7-Ser214 expression had the opposite effect, leading to increased ECs migratory and tube formation ability. Proteomics analysis showed an increase in thrombospondin-1, a reported angiogenesis inhibitor, in culture media conditioned by ECs with ADAMTS7 knockdown and a decrease of thrombospondin-1 in media conditioned by ECs with ADAMTS7-Ser214 overexpression. Cleavage assay indicated that ADAMTS7 possessed thrombospondin-1 degrading activity, which was reduced by the Ser214-to-Pro substitution. The pro-angiogenic effect of ADAMTS7-Ser214 diminished in the presence of a thrombospondin-1 blocking antibody. ConclusionsThe ADAMTS7 Ser217-to-Pro substitution as a result of ADAMTS7 polymorphism affects thrombospondin-1 degradation, thereby promoting atherogenesis through increased EC migration and tube formation.

Transcriptional Upregulation of CaMKIIδ through the JAK/STAT3 Pathway is Necessary for the IL‐6‐Dependent Increase in Endothelial Cell Migration

Multifunctional Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is a well‐characterized family of proteins comprised of four separate isoforms: α, β, δ, and γ. These isoforms are differentially expressed in a tissue‐specific manner and contribute to diverse physiological and pathophysiological conditions. However, little is known regarding CaMKII isoform expression and function in the endothelium. Previous data from our lab has shown that CaMKIIδ6 is the primary isoform expressed in human umbilical vein endothelial cells (HUVECs) and promote thrombin‐induced loss of barrier function. Interleukin 6 (IL‐6) is a proinflammatory cytokine that induces loss of barrier function in HUVECs through a JAK/STAT3 signaling cascade. The purpose of this study was to test if CaMKIIδ mediates IL‐6‐induced increase in endothelial barrier permeability. Using qPCR, we determined that CaMKIIδ is the most abundant isoform of the enzyme present in HUVECs. Upon treatment with IL‐6, significant increases in both CaMKIIδ transcription and protein expression were observed. Treatment of HUVECs with JAK inhibitor ruxolitinib and siRNA for STAT3 decreased CaMKIIδ expression. However, adenoviral overexpression of various mutant CaMKII isoforms or siRNA knock down of CaMKIIδ did not affect IL‐6‐dependent loss of barrier function as measured by electric cell‐substrate impedence sensing (ECIS). It has been reported that IL‐6 signaling not only induces a loss of endothelial barrier function, but also increases endothelial cell migration. Indeed, siRNA knock down of CaMKIIδ attenuated IL‐6‐induced endothelial cell migration in a scratch‐wound assay. For the first time, our findings thus far have identified transcriptional induction of CaMKIIδ by IL‐6 that is necessary for the subsequent increase in endothelial cell migration.Support or Funding InformationRO1HL49426‐21 to Harold A. SingerThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modification of Cardiac Progenitor Cell-Derived Exosomes by miR-322 Provides Protection against Myocardial Infarction through Nox2-Dependent Angiogenesis.

Myocardial infarction (MI) is the primary cause of cardiovascular mortality, and therapeutic strategies to prevent or mitigate the consequences of MI are a high priority. Cardiac progenitor cells (CPCs) have been used to treat cardiac injury post-MI, and despite poor engraftment, they have been shown to inhibit apoptosis and to promote angiogenesis through poorly understood paracrine effects. We previously reported that the direct injection of exosomes derived from CPCs (CPCexo) into mouse hearts provides protection against apoptosis in a model of acute ischemia/reperfusion injury. Moreover, we and others have reported that reactive oxygen species (ROS) derived from NADPH oxidase (NOX) can enhance angiogenesis in endothelial cells (ECs). Here we examined whether bioengineered CPCexo transfected with a pro-angiogenic miR-322 (CPCexo-322) can improve therapeutic efficacy in a mouse model of MI as compared to CPCexo. Systemic administration of CPCexo-322 in mice after ischemic injury provided greater protection post-MI than control CPCexo, in part, through enhanced angiogenesis in the border zones of infarcted hearts. Mechanistically, the treatment of cultured human ECs with CPCexo-322 resulted in a greater angiogenic response, as determined by increased EC migration and capillary tube formation via increased Nox2-derived ROS. Our study reveals that the engineering of CPCexo via microRNA (miR) programing can enhance angiogenesis, and this may be an effective therapeutic strategy for the treatment of ischemic cardiovascular diseases.

Participation of membrane calcium channels in erythropoietin-induced endothelial cell migration

Calcium (Ca2+) plays an important role in angiogenesis, as it activates the cell migration machinery. Different proangiogenic factors have been demonstrated to induce transient Ca2+ increases in endothelial cells. This has raised interest in the contribution of Ca2+ channels to cell migration, and in a possible use of channel-blocking compounds in angiogenesis-related pathologies. We have investigated the ability of erythropoietin (Epo), a cytokine recently involved in angiogenesis, to induce Ca2+ influx through different types of membrane channels in EA.hy926 endothelial cells. The voltage-dependent Ca2+ channel antagonists amlodipine and diltiazem inhibited an Epo-triggered transient rise in intracellular Ca2+, similarly to a specific inhibitor (Pyr3) and a blocking antibody against the transient potential calcium channel 3 (TRPC3). Unlike diltiazem, amlodipine and the TRPC3 inhibitors prevented the stimulating action of Epo in cell migration and in vitro angiogenesis assays. Amlodipine was also able to inhibit an increase in endothelial cell migration induced by Epo in an inflammatory environment generated with TNF-α. These results support the participation of Ca2+ entry through voltage-dependent and transient potential channels in Epo-driven endothelial cell migration, highlighting the antiangiogenic activity of amlodipine.

MicroRNA-148b Targets the TGF-β Pathway to Regulate Angiogenesis and Endothelial-to-Mesenchymal Transition during Skin Wound Healing.

Transforming growth factor beta (TGF-β) is crucial for regulation of the endothelial cell (EC) homeostasis. Perturbation of TGF-β signaling leads to pathological conditions in the vasculature, causing cardiovascular disease and fibrotic disorders. The TGF-β pathway is critical in endothelial-to-mesenchymal transition (EndMT), but a gap remains in our understanding of the regulation of TGF-β and related signaling in the endothelium. This study applied a gain- and loss-of function approach and an in vivo model of skin wound healing to demonstrate that miR-148b regulates TGF-β signaling and has a key role in EndMT, targeting TGFB2 and SMAD2. Overexpression of miR-148b increased EC migration, proliferation, and angiogenesis, whereas its inhibition promoted EndMT. Cytokine challenge decreased miR-148b levels in ECs while promoting EndMT through the regulation of SMAD2. Finally, in a mouse model of skin wound healing, delivery of miR-148b mimics promoted wound vascularization and accelerated closure. In contrast, inhibition of miR-148b enhanced EndMT in wounds, resulting in impaired wound closure that was reversed by SMAD2 silencing. Together, these results demonstrate for the first time that miR-148b is a key factor controlling EndMT and vascularization. This opens new avenues for therapeutic application of miR-148b in vascular and tissue repair.

Pro-angiogenic impact of SDF-1α gene-activated collagen-based scaffolds in stem cell driven angiogenesis

Ensuring an adequate angiogenic response during wound healing is a prevailing clinical challenge in biomaterials science. To address this, we aimed to develop a pro-angiogenic gene-activated scaffold (GAS) that could activate MSCs to produce paracrine factors and influence angiogenesis and wound repair. A non-viral polyethyleneimine (PEI) nanoparticles carrying a gene encoding for stromal derived factor-1 alpha (SDF-1α) was combined with a collagen-chondroitin sulfate scaffold to produce the GAS. The ability of this platform to enhance the angiogenic potential of mesenchymal stem cells (MSCs) was then assessed. We found that the MSCs on GAS exhibited early over-expression of SDF-1α mRNA with the activation of angiogenic markers VEGF and CXCR4. Exposing endothelial cells to conditioned media collected from GAS supported MSCs promoted a 20% increase in viability and 33% increase in tubule formation (p < 0.05). Furthermore, the conditioned media promoted a 50% increase in endothelial cell migration and wound closure (p < 0.005). Gene expression analysis of the endothelial cells revealed that the functional response was associated with up-regulation of angiogenic genes; VEGF, CXCR4, eNOS and SDF-1α. Overall, this study shows collagen-based scaffolds combined with SDF-1α gene therapy can provide enhanced pro-angiogenic response, suggesting a promising approach to overcome poor vasculature during wound healing.

Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells - Liquid biopsies for monitoring complications of pregnancy.

Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot®) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte™) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (<18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications.

P36 Pericyte seeded biofunctionalised vascular graft for improved endothelial coverage

Coronary bypass is the most common intervention for coronary obstruction. When autologous vessels are unavailable, synthetic grafts are employed. The challenge of rapid endothelialisation to reduce complications is still far from being achieved. This study describes a novel approach to synthetic graft design, based on the spatially defined presentation of functionalities within the scaffold, contributing to the biological activity of the pre-seeded pro-angiogenic human saphenous vein-derived pericytes (SVP). Indeed, the electrospun polycaprolactone (PCL) presented two distinct functionalities on either side: the osteopontin-derived adhesion peptide (Adh) to facilitate endothelial adhesion on the luminal side and the heparin binding peptide (Hep) to create a reservoir of SVP-produced growth factors on the adventitial side. Conjugated peptides retained functionality within the scaffold, with Adh increasing endothelial cells (EC) adhesion and Hep binding the SVP-produced VEGF and releasing it to improve EC growth over time. Additionally, seeding of the scaffold with SVP increased EC migration and proliferation, suggesting their supporting role in re-endothelialisation of the graft. When seeded on the dual peptide scaffold, SVP acted synergistically with the peptides determining a greater EC coverage, as compared to each component alone. SVP-seeded scaffolds were cryopreserved for up to 2 weeks in GMP-grade medium preserving cell viability and functionality. In conclusion, we engineered a progenitor cell-seeded vascular graft, which incorporates spatially organised biomolecules that act synergistically with the cells to support EC coverage.

The Vascular Endothelial Growth Factor-A phosphorylates Murine Double Minute-2 on its Serine 166 via the Extracellular Signal-Regulated Kinase 1/2 and p90 Ribosomal S6 Kinase in primary human endothelial cells

Murine Double Minute-2 (Mdm2) has been identified as an essential regulator of skeletal muscle angiogenesis and the pro-angiogenic activity of endothelial cells. We have recently demonstrated that the pro-angiogenic Vascular Endothelial Growth Factor-A (VEGF-A) is a potent upstream regulator of Mdm2 phosphorylation on its Serine 166 (p-Ser166-Mdm2), a protein modification leading to an increase in endothelial cell migration. Here, we investigated the kinase signaling pathways that could be responsible for mediating VEGF-A-dependent Mdm2 phosphorylation. Incubation of primary human dermal microvascular endothelial cells with recombinant VEGF-A for 15 min led to increased phosphorylation levels of VEGF-receptor-2, Mdm2, Akt, Extracellular Signal-Regulated Kinase 1/2 (ERK1/2), and p90 Ribosomal S6 Kinase (p90RSK) proteins. In addition to being linked to VEGF-A signaling, Akt, ERK1/2 and p90RSK have been shown to potentially lead to Mdm2 phosphorylation. We therefore next analyzed which of these kinases could be responsible for VEGF-A-dependent Mdm2 phosphorylation on Serine 166 by using kinase-specific pharmacological inhibitors. Inhibition of ERK1/2 phosphorylation by UO126 entirely abrogated the response of p-Ser166-Mdm2 to VEGF-A treatment, while Akt phosphorylation inhibition by wortmannin led to further elevations in p-Ser166-Mdm2. p90RSK has been identified as a potential candidate downstream of ERK1/2 that could induce Mdm2 Ser166 phosphorylation. Two independent p90RSK inhibitors, FMK and BI-D1870, each led to an entire loss of p-Ser166-Mdm2 responsiveness to VEGF-A. Taken together, our results demonstrate that VEGF-A driven Mdm2 phosphorylation on Ser166 is dependent on the ERK1/2/p90RSK signaling pathway in primary human endothelial cells, furthering our understanding of the complex relationship between Mdm2 and VEGF-A in a physiological context.

Organ culture storage of pre-prepared corneal donor material for Descemet's membrane endothelial keratoplasty

PurposeTo evaluate the effect of media composition and storage method on pre-prepared Descemet's membrane endothelial keratoplasty (DMEK) grafts.Methods50 corneas were used. Endothelial wound healing and proliferation in different media were...

Phosphorylation of murine double minute-2 on Ser166 is downstream of VEGF-A in exercised skeletal muscle and regulates primary endothelial cell migration and FoxO gene expression.

We demonstrated in a previous study that murine double minute (Mdm)-2 is essential for exercise-induced skeletal muscle angiogenesis. In the current study, we investigated the mechanisms that regulate Mdm2 activity in response to acute exercise and identified VEGF-A as a key stimulator of Mdm2 phosphorylation on Ser(166) (p-Ser166-Mdm2). VEGF-A and p-Ser166-Mdm2 protein levels were measured in human and rodent muscle biopsy specimens after 1 bout of exercise. VEGF-A-dependent Mdm2 phosphorylation was demonstrated in vivo in mice harboring myofiber-specific deletion of VEGF-A (mVEGF(-/-)) and in vitro in primary human and rodent endothelial cells (ECs). Exercise increased VEGF-A and p-Ser166-Mdm2 protein levels respectively by 157 and 68% in human muscle vs. pre-exercise levels. Similar results were observed in exercised rodent muscles compared to sedentary controls; however, exercise-induced Mdm2 phosphorylation was significantly attenuated in mVEGF(-/-) mice. Recombinant VEGF-A elevated p-Ser166-Mdm2 by 50-125% and stimulated migration by 33% in ECs when compared to untreated cells, whereas the Mdm2 antagonist Nutlin-3a abrogated VEGF-driven EC migration. Finally, overexpression of a Ser166-Mdm2 phosphorylation mimetic increased EC migration, increased Mdm2 to FoxO1 binding (+55%), and decreased FoxO1-dependent gene expression compared with ECs overexpressing WT-Mdm2. Our results suggest that VEGF-mediated Mdm2 phosphorylation on Ser(166) is a novel proangiogenic pathway within the skeletal muscle.

Eph-B4 Mediates Vein Graft Adaptation By Regulation of eNOS

Vein graft adaptation is characterized by loss of expression of the tyrosine kinase receptor ephrin type-B receptor 4 (Eph-B4), the embryonic determinant of venous identity, without increased expression of its ligand ephrin-B2, the embryonic determinant of arterial identity. Endothelial nitric oxide synthase (eNOS) is an important mediator of vessel remodeling. We hypothesized that the mechanism of action of Eph-B4 during vein graft adaptation might be via regulation of downstream eNOS activity. Human and mouse vein graft specimens were examined for eNOS activity by Western blot, and vessel remodeling was assessed in vein grafts in wild-type (WT) or eNOS-knockout (KO) mice. Mouse endothelial cells (EC) were stimulated with ephrin-B2/Fc without and with preclustering, without and with L-NAME (1 mM), and assessed by Western blot and immunofluorescence for eNOS and Eph-B4 phosphorylation. NO production was assessed using a NO-specific chemiluminescence analyzer. Cell migration was assessed using a transwell assay. Human and mouse vein grafts both showed increased eNOS phosphorylation compared with normal veins (n = 3; P < .05). Vein grafts from eNOS-KO mice showed less dilation and less wall thickening compared with WT vein grafts (n = 7; P < .05). Ephrin-B2/Fc stimulated both Eph-B4 and eNOS phosphorylation in a bimodal temporal distribution (n = 4; P < .05), with preclustered ephrin-B2/Fc causing prolonged peak Eph-B4 and eNOS phosphorylation as well as altered subcellular localization (n = 4; P < .05). Ephrin-B2/Fc increased NO release from ECs (n = 3; P < .01) as well as a trend towards increased EC migration (n = 6; P = .11) in an eNOS-dependent fashion. eNOS is a mediator of vein graft adaptation to the arterial environment. Eph-B4 stimulates eNOS phosphorylation and mediates vein graft adaptation by regulation of eNOS activity.

Abstract 662: X-box Binding Protein 1 Splicing Promotes Wound Healing via Modulating Endothelial Nitric Oxide Synthase

Instruction: X-box binding protein 1 (XBP1) is a pivotal transcription factor in the pathogenesis of the immune system and in the endoplasmic reticulum stress response. Our previous studies have proved that XBP1 is involved in VEGF-mediated endothelial cell (EC) proliferation and angiogenesis. However, whether XBP1 can influence EC migration is unknown. Hypothesis: We assessed the hypothesis that XBP1 splicing is involved in endothelial migration contributing to wound healing and angiogenesis. Method and results: In this study, we used EC monolayer wound healing, tube formation and transwell migration models to study the involvement of XBP1 splicing in EC migration. We found that scratching on EC monolayer triggered XBP1 splicing, which was attenuated by the presence of LY294002, FAK-14 and L-NAME, suggesting that PI3K/Akt, FAK and eNOS may be involved in scratching-mediated upregulation of XBP1 splicing. Over-expression of spliced XBP1 (XBP1s) via Ad-XBP1s gene transfer increased EC migration, while knockdown of IRE1α or XBP1 by ShRNA lentiviruses suppressed EC migration. Over-expression of XBP1s upregulated eNOS at mRNA and protein levels leading to the increase of nitric oxide production. The eNOS promoter reporter analysis revealed that XBP1s down-regulated eNOS transcription. The pSI-Check2-eNOS-3’UTR reporter analysis indicated that XBP1s might upregulate eNOS mRNA via 3’UTR-mediated mRNA stabilization. The microRNA array assays found that XBP1s down-regulated microRNAs mir-24, mir-125 and mir-214. Transfection of EC with the miRNA mimics of those three miRNAs attenuated XBP1s-induced eNOS mRNA upregulation and EC migration. Conclusion: These results suggest that XBP1 splicing may increase eNOS mRNA stabilization and nitric oxide production via down-regulating miRNAs that target eNOS mRNA 3’-UTR, leading to EC migration and contributing to wound healing and angiogenesis. Significance: XBP1 splicing may provide a new therapeutic strategy to tackle endothelium injury-related cardiovascular disease.

Abstract 654: XBP1 Splicing is Involved in Endothelial and Smooth Muscle Cells Interaction

Background: The X-box binding protein 1 (XBP1) is a stress inducible transcription factors, essential for cell survival under stress conditions. Our previous studies have revealed that XBP1 participates in endothelial cell (EC) proliferation, autophagic response and survival and in smooth muscle cell (SMC) proliferation. Whether XBP1 is involved in EC/SMC interaction remains unclear. Methods and Results: In this study, we first performed hindlimb ischemia model in XBP1loxP/loxP and SM22-Cre+/XBP1loxP/loxP mice. The XBP1 deficiency in SMCs significantly attenuated the recovery of foot blood perfusion in ischemic mice. Immunofluorescence staining revealed that ECs lost contact to each other in XBP1 deficient mice, suggesting that XBP1 in SMCs may contribute to EC/SMC interaction. The conditioned medium from Ad-XBP1s-infected SMCs increased EC migration and proliferation. The conditioned medium from Ad-XBP1s-infected ECs also increased SMC migration but suppressed SMC proliferation. The migration effect could be attenuated by depletion with anti-col4A1 antibody. Proteomics analysis and Western blot assays demonstrated that over-expression of the spliced XBP1 in SMCs increased the secretion of a 50KD band of collagen IVα1. RT-PCR analysis indicated that this 50KD band might be derived from an unconventional splicing, in which exon 4 and exon 42 directly joins together, resulting in a 533 amino acid peptide with the internal part shortened. Further experiments with immunohistological staining revealed that a functional XBP1 was essential to collagen IVα1 expression during embryonic development. Very little amount of collagen IV could be observed in XBP1-/- embryos. Conclusion: XBP1 in SMCs may modulate EC-SMC interaction via regulation of collagen IV expression and splicing, which may contribute to vasculature remodelling.

P18 Regulates Tubulogenesis in the Pulmonary Endothelium Through p38/mTOR‐dependent Pathway

Vasculogenesis and angiogenesis, the formation of new blood vessels, are processes requiring multiple endothelial cell (EC) functions, including proliferation, migration and adhesion. Blood vessel lumen formation is necessary for the creation of a functional vessel to transport blood, fluids, and nutrients and occurs via tubulogenesis. This process of lumen formation is not completely understood, however there is evidence for the role of the endosome. The endosomal adaptor protein p18 anchors the late endosome/lysosome to facilitate mTOR/MAPK activation. We have demonstrated that p18 binds to the early endosome to enhance VE‐cadherin recycling to EC junctions and improve EC barrier function via an mTOR/MAPK independent pathway. As EC permeability is a key component of tubulogenesis, we hypothesize that p18 localized to the endosome regulates tubulogenesis in the pulmonary EC, potentially through regulation of MAPK and mTOR activity. Lung EC were transfected with cDNA encoding p18wt display significantly increased EC migration, adhesion to gelatin and in vitro and in vivo tube formation, but had no effect of proliferation. Inhibition of mTOR (rapamycin) or p38MAPK (SB203580) significantly attenuated the pro‐tubulogenic role of p18wt. Conversely, overexpression of the non‐endosomal binding p18 (p18N39) and p18 siRNA knockdown attenuated VEGF‐induced adhesion and proliferation. Thus our data demonstrates a key role for endocytic protein p18 in promoting tubulogenesis within the pulmonary vasculature through activation of mTOR and p38. Thus we implicate p18 as a novel therapeutic target to regulate vessel formation within the pulmonary vasculature.

A mechanobiological model of endothelial cell migration and proliferation

How angiogenesis is regulated by local environmental cues is still not fully understood despite its importance to many regenerative events. Although mechanics is known to influence angiogenesis, the specific cellular mechanisms influenced by mechanical loading are poorly understood. This study adopts a lattice-based modelling approach to simulate endothelial cell (EC) migration and proliferation in order to explore how mechanical stretch regulates their behaviour. The approach enables the explicit modelling of ECs and, in particular, their migration/proliferation (specifically, rate and directionality) in response to such mechanical cues. The model was first used to simulate previously reported experiments of EC migration and proliferation in an unloaded environment. Next, three potential effects (increased cell migration, increased cell proliferation and biased cellular migration) of mechanical stretch on EC behaviour were simulated using the model and the observed changes in cell population characteristics were compared to experimental findings. Combinations of these three potential drivers were also investigated. The model demonstrates that only by incorporating all three changes in cellular physiology (increased EC migration, increased EC proliferation and biased EC migration in the direction perpendicular to the applied strain) in response to dynamic loading, it is possible to successfully predict experimental findings. This provides support for the underlying model hypotheses for how mechanics regulates EC behaviour.

OR23: THE INTEGRIN β4 CONNECTING SEGMENT DOMAIN IS REQUIRED FOR HLA CLASS I-MEDIATED ENDOTHELIAL CELL ACTIVATION

Endothelial cell (EC) activation is a major consequence of HLA class I (HLA-I) antibody-mediated rejection. Previous studies have shown that the molecular association between HLA-I molecule and integrin β 4 (ITGB4) is required for EC migration and proliferation by regulating ERK1/2 signaling pathway. However, the proximal signaling pathway that regulates HLA-I and ITGB4 association remains unclear. We determined the domains of the ITGB4 cytoplasmic tail required for HLA-I mediated phosphorylation pathways and correlated these findings with functional studies. Primary human aortic endothelial cells (HAEC) were used for this study. To identify the ITGB4 intracellular domain that associates with HLA-I, we generated constructs encoding ITGB4 full length (WT) or deletion mutants that lack the Calx- β , fibronectin type III repeat (FNIII 1–4) or connecting segment (CS). These constructs were sub-cloned into adenovirus-based vector and adenovirus encoding WT or mutants were generated. Endogenous ITGB4 expression was suppressed using siRNA transfection. HAEC lacking endogenous ITGB4 were infected with adenovirus and protein phosphorylation and cell migration were further analyzed. Flow cytometry and Western blot analysis showed that both the WT and deletion mutant-infected HAEC had similar levels of cell surface or total expression of ITGB4. Coimmunoprecipitation studies showed that stimulation with the F(ab′)2 fragment of HLA-I antibody increased HLA-I association with ITGB4 in a time dependent manner in WT adenovirus infected HAEC. In contrast, HAEC infected with the CS deletion mutant significantly inhibited HLA-I association with ITGB4 compared to WT, Calx- β or FNIII mutant infected cells. In addition, the CS deletion mutant decreased HLA-I induced ERK1/2 phosphorylation as measured by Western blot. Using in vitro wound healing assay, we found that over expression of ITGB4 increased EC migration compared to LacZ infected cells, whereas CS deletion decreased HLA-I induced EC migration. Furthermore, HLA-I antibody increased the association of ITGB4 with the adaptor protein Shc. This association was decreased by CS deletion. Our results demonstrate that HLA-I binds to ITGB4 via the connecting segment domain that is required for ERK1/2 signaling and EC activation.