Endothelial Monolayers Research Articles

The Anti-Vascular Endothelial Growth Factor Receptor 1 (VEGFR-1) D16F7 Monoclonal Antibody Inhibits Melanoma Adhesion to Soluble VEGFR-1 and Tissue Invasion in Response to Placenta Growth Factor.

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family involved in tumor-associated angiogenesis and melanoma invasion of the extra-cellular matrix (ECM) through activation of membrane VEGF receptor 1 (VEGFR-1). A soluble VEGFR-1 (sVEGFR-1) form is released in the ECM, where it sequesters proangiogenic factors and stimulates endothelial or tumor cell adhesion and chemotaxis through interaction with α5β1 integrin. The anti-VEGFR-1 monoclonal antibody (D16F7 mAb) inhibits VEGF-A or PlGF-mediated signal transduction without affecting ligand interaction, thus preserving sVEGFR-1 decoy function. The aim of this study was to investigate whether D16F7 mAb hampers melanoma spread by in vitro analysis of cell adhesion to sVEGFR-1, ECM invasion, transmigration through an endothelial cell monolayer and in vivo evaluation of tumor infiltrative potential in a syngeneic murine model. Results indicate that D16F7 mAb significantly inhibits melanoma adhesion to sVEGFR-1 and ECM invasion, as well as transmigration in response to PlGF. Moreover, treatment of melanoma-bearing mice with the anti-VEGFR-1 mAb not only inhibits tumor growth but also induces a significant reduction in bone infiltration associated with a decrease in PlGF-positive melanoma cells. Furthermore, D16F7 mAb reduces PlGF production by melanoma cells. Therefore, blockade of PLGF/VEGFR-1 signaling represents a suitable strategy to counteract the metastatic potential of melanoma.

Rictor maintains endothelial integrity under shear stress

Background: Endothelial injury induced by low shear stress (LSS) is an initiating factor in the pathogenesis of various cardiovascular diseases, including atherosclerosis, hypertension, and thrombotic diseases. Low shear stress activates the mammalian target of rapamycin complex 2 (mTORC2) signaling pathway. Rictor, the main constituent protein of mTORC2, is involved in vascular development. However, the impact of conditional Rictor ablation on endothelial homeostasis, especially on endothelial-specific markers, such as vascular endothelial-cadherin (VE-cadherin) and von Willebrand factor (VWF), under blood flow stimulation is unclear. Objective: We aimed to investigate whether endothelial Rictor is involved in maintaining vascular endothelial integrity and the potential role of Rictor in atheroprone blood flow-mediated endothelial injury. Methods and results: Immunofluorescence staining showed that endothelial Rictor was successfully knocked out in a mouse model. Scanning electron microscopy (EM) detection revealed disruption of the endothelial monolayer in the thoracic aorta of Rictor-deficient mice. Furthermore, scanning electron microscopy and transmission electron microscopy showed that Rictor deletion disrupted endothelial integrity and expanded cell junctions in the left common carotid artery region. In vitro, low shear stress disrupted actin filament polarity and the promoted the translocation of vascular endothelial-cadherin, the key component of adherens junctions (AJs) in human umbilical vein endothelial cells. After Rictor downregulation by small interfering RNA, the translocation of vascular endothelial-cadherin and stress fibers increased. Rictor knockdown inhibited low shear stress-induced von Willebrand factor upregulation, and downregulation of vascular endothelial-cadherin decreased low shear stress-induced von Willebrand factor expression. These results suggest that vascular endothelial-cadherin/von Willebrand factor is a possible mechanism mediated by Rictor in the pathological process of low shear stress-induced endothelial injury. Conclusion: Rictor is a key protein that regulates endothelial integrity under vascular physiological homeostasis, and Rictor mediates low shear stress-induced endothelial injury by regulating adherens junctions and von Willebrand factor.

Abstract 15395: Caveolin-1 is a Primary Determinant of Endothelial Stiffening Associated With Dyslipidemia, Disturbed Flow and Ageing

Objective: Endothelial stiffness is emerging as a major determinant in endothelial function. Here, we analyzed the role of caveolin-1 (Cav-1) in determining the stiffness of endothelial cells exposed to oxidized low density lipoprotein (oxLDL) under static and hemodynamic conditions in vitro and of aortic endothelium in vivo in mouse models of dyslipidemia and ageing. Approach and Results: Elastic moduli of cultured endothelial cells and of endothelial monolayer of freshly isolated mouse aortas were measured using atomic force microscopy. We found that a loss of Cav-1 abrogates the uptake of oxLDL (n=5-8) and oxLDL-induced endothelial stiffening (n=4-5) but has no effect on endothelial stiffness in the absence of oxLDL (n=4). A loss of Cav-1 also abrogated endothelial stiffening induced by disturbed flow, which in turn is oxLDL dependent (n=6). Mechanistically, Cav-1 is required for the expression of CD36 (cluster of differentiation 36) scavenger receptor (n=4) but not for endothelial stiffening induced by a direct application of 7ketocholesterol (n=8). Genetic deletion of Cav-1 also led to profound changes in endothelial stiffness in vivo : it abrogated endothelial stiffening observed in the disturbed flow region of the aortic arch (n=5-6), abrogated endothelial stiffening induced by feeding the mice high fat diet (4-6 weeks, n=5) and significantly blunted endothelial stiffening that develops with advanced age (n=5-6). This effect was independent of stiffening of the sub-endothelium layer (n=5-8). Conclusions: Taken together, this study demonstrates that Cav-1 plays a critical role in endothelial stiffening induced by oxLDL in vitro and by dyslipidemia, disturbed flow and ageing in vivo . Mechanistically, Cav-1 regulates endothelial stiffening via CD36-mediated uptake of oxLDL, whereas exposure to 7ketocholesterol circumvents this step.

Rho-associated coiled-coil kinase contributes an important role in Xingnaojing injection-induced adverse drug reactions

Xingnaojing injection (XNJI) is widely used for acute cerebral hemorrhage. However, the adverse drug reactions (ADRs) caused by XNJI have attracted broad attention. In this study, mice were intravenously administrated with a single dose of XNJI (10 or 20 mL/kg) to evaluate the vascular permeability. Then, pathological examinations on ears and lungs were performed to further determine the characteristics of ADRs induced by XNJI. In addition, human umbilical vein endothelial cells (HUVECs) were stimulated with XNJI (1/128, 1/64, and 1/32 XNJI pure product dilution) and stained with rhodamine-phalloidin to analyze the alterations on cytoskeleton. In addition, endothelial monolayer was incubated with XNJI and the effect on permeability was tested. Western blot testing was conducted to measure the involvement of Rho-associated coiled-coil kinase (ROCK) in XNJI-induced ADRs and fasudil hydrochloride was applied to additionally confirm the contribution of ROCK. The results indicated that XNJI may lead to dose-associated vascular leakage. None visibly abnormal symptom was noticed in the mice treated with XNJI at 10 mL/kg, while inflammation in ears and lungs was observed in the mice administrated with 20 mL/kg XNJI. In in vitro experiment, stimulation with high concentration of XNJI (1/32 XNJI pure product dilution) could enhance the permeability of confluent endothelial monolayer and induce rearrangement of actin cytoskeleton. Activation of ROCK, which was shown as augmented expressions of p-MLC2 and p-MYPT1, was observed in cells and mice treated with XNJI. ROCK inhibitor could attenuate XNJI-induced dysfunction of endothelial barrier in in vitro experiment and alleviate the vascular leakage and inflammation noticed in mice treated with XNJI. This study indicates that overdose of XNJI can destroy vascular endothelial function and induce increased vascular permeability, which may cause ADRs in clinical practice. Meanwhile, ROCK plays an important role in XNJI-induced ADRs.

Characterizing nuclear morphology and expression of eNOS in vascular endothelial cells subjected to a continuous range of wall shear stress magnitudes and directionality

Complex patterns of hemodynamic wall shear stress occur in regions of arterial branching and curvature. Areas within these regions can be highly susceptible to atherosclerosis. Although many studies have characterized the response of vascular endothelial cells to shear stress in a categorical manner, our study herein addresses the need of characterizing endothelial behaviors over a continuous range of shear stress conditions that reflect the extensive variations seen in the vasculature. We evaluated the response of human umbilical vein endothelial cell monolayers to orbital flow at 120, 250, and 350 revolutions per minute (RPM) for 24 and 72 h. The orbital shaker model uniquely provides a continuous range of shear stress conditions from low and multidirectional at the center of each well of a culture plate to high and unidirectional at the periphery. We found distinct patterns of endothelial nuclear area, nuclear major and minor diameters, nuclear aspect ratio, and expression of endothelial nitric oxide synthase over this range of shear conditions and relationships were fit with linear and, where appropriate, power functions. Nuclear area was particularly sensitive with increases in the low and multidirectional WSS region that incrementally decreased as WSS became higher in magnitude and more unidirectional over the radius of the cell layers. The patterns of all endothelial behaviors exhibited high correlations (positive and negative) with metrics of shear stress magnitude and directionality that have been shown to strongly associate with atherosclerosis. Our findings demonstrate the exquisite sensitivity of these endothelial behaviors to incremental changes in shear stress magnitude and directionality, and provide critical quantitation of these relationships for predicting the susceptibility of an arterial segment to diseases such as atherosclerosis, particularly within complex flow environments in the vasculature such as around bifurcations.

Endothelial cell activation by interleukin-1 and extracellular matrix laminin-10 occurs via the YAP signalling pathway

Laminin-10 (LM-10) is a key regulator of blood-brain barrier (BBB) repair after hypoxia and inflammation. Here we investigated the signalling mechanisms regulated by LM-10 in human brain endothelial cell line hCMEC/D3 in response to interleukin(IL)-1beta(β) in vitro. LM-10 promoted endothelial proliferation and repair of an endothelial monolayer after scratch injury, and upregulated IL-1β-induced ICAM-1 and VCAM-1 expression. IL-1β and LM-10 regulated YAP signalling pathway in endothelial cells leading to differential expression of YAP target genes, ctgf and serpine-1, providing evidence that the YAP signalling pathway could be a new therapeutic target for the treatment of BBB dysfunction in CNS diseases.

Caveolin-1 is a primary determinant of endothelial stiffening associated with dyslipidemia, disturbed flow, and ageing

Endothelial stiffness is emerging as a major determinant in endothelial function. Here, we analyzed the role of caveolin-1 (Cav-1) in determining the stiffness of endothelial cells (EC) exposed to oxidized low density lipoprotein (oxLDL) under static and hemodynamic conditions in vitro and of aortic endothelium in vivo in mouse models of dyslipidemia and ageing. Elastic moduli of cultured ECs and of the endothelial monolayer of freshly isolated mouse aortas were measured using atomic force microscopy (AFM). We found that a loss of Cav-1 abrogates the uptake of oxLDL and oxLDL-induced endothelial stiffening, as well as endothelial stiffening induced by disturbed flow (DF), which was also oxLDL dependent. Mechanistically, Cav-1 is required for the expression of CD36 (cluster of differentiation 36) scavenger receptor. Genetic deletion of Cav-1 abrogated endothelial stiffening observed in the DF region of the aortic arch, and induced by a high fat diet (4–6 weeks) and significantly blunted endothelial stiffening that develops with advanced age. This effect was independent of stiffening of the sub-endothelium layer. Additionally, Cav-1 expression significantly increased with age. No differences in elastic modulus were observed between the sexes in advanced aged wild type and Cav-1 knockout mice. Taken together, this study demonstrates that Cav-1 plays a critical role in endothelial stiffening induced by oxLDL in vitro and by dyslipidemia, disturbed flow and ageing in vivo.

Direct Tie2 Agonists Stabilize Vasculature for the Treatment of Diabetic Macular Edema.

PurposeDiabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients.MethodsWe explored direct agonists of Tie2, a receptor known to stabilize vasculature and prevent leakage. We identified a multivalent PEG–Fab conjugate, Tie2.1-hexamer, that oligomerizes Tie2 and drives receptor activation and characterized its activities in vitro and in vivo.ResultsTie2.1-hexamer normalized and stabilized intercellular junctions of stressed endothelial cell monolayers in vitro, suppressed vascular leak in mice under conditions where anti-VEGF alone was ineffective, and demonstrated extended ocular exposure and robust pharmacodynamic responses in non-human primates.ConclusionsTie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor.Translational RelevanceOur study presents a promising potential therapeutic for the treatment of DME.

Construction of functional magnetic scaffold with temperature control switch for long-distance vascular injury

Fusion of endothelial monolayer for rapid reendothelialization is the key to vascular transplantation, which is crucial for remodeling long-distance (≥10 mm) vascular injury (LDVJ) and reducing the long-term repair period. Although silk fibroin (SF) has been widely prepared as a scaffold to repair vascular injuries, it is still challenging to remodel LDVJs. The design and manufacture of functional scaffolds with a rapid reendothelialization enhancement effect are helpful to solve this problem. Here, we developed a well-designed functional magnetic scaffold (MDP) with a temperature-controlled switch, intelligently regulating the release of magnetic nanoparticles (MNPs) to induce macrophages migration and M2 polarization. The results show that the radial support force of MDP is appropriately five times higher than that of pure silk fibroin scaffold (SFC), and the degradation of MDP is delayed, which is beneficial to maintaining the integrity of the scaffold. MDP induces the migration of macrophages, upregulates the expression of repair cytokines such as TGF-β, IL-10 and VEGF, and promotes the proliferation of mouse aortic vascular smooth muscle (MOVAS) cells. More importantly, the enhanced migration of macrophages at the phase transition temperature is more conducive to the proliferation and accumulation of vascular repair-related cells, which is conducive to rapid reendothelialization. In addition, MDP also significantly regulates the polarization of macrophages to M2 type, which is conducive to the secretion of repair cytokines to further promote vascular recovery. The evaluation of endothelial cells' adhesion and function also proved that MDP could increase the expression of vascular endothelial cadherin (VE-Cad) and collagen IV (COL IV) and has the potential for rapid reendothelialization. The replacement model in vivo animal experiments further proved that MDP exhibited a better repair effect than SFC, providing a new option for the repair of LDVJ in the future.

Septin and actin contributions to endothelial cell-cell junctions and monolayer integrity.

Septins in endothelial cells (ECs) have important roles supporting the integrity of the endothelial monolayer. Cell-cell junctions in EC monolayers are highly dynamic, with continuous retractions and protrusions. Depletion of septins in ECs leads to disruption of cell-cell junctions, which are composed of VE-cadherin and other junctional proteins. In EC monolayers, septins are concentrated at the plasma membrane at sites of cell-cell contact, in curved- and scallop-shaped patterns. These membrane-associated septin accumulations are located in regions of positive membrane curvature, and those regions are often associated with and immediately adjacent to actin-rich protrusions with negative membrane curvature. EC septins associate directly with plasma membrane lipids, based on findings with site-specific mutations of septins in ECs, which is consistent with biochemical and cell biological studies in other systems. Loss of septins leads to disruption of the EC monolayer, and gaps form between cells. The number and breadth of cell-cell contacts and junctions decreases, and the number and frequency of retractions, ruffles, and protrusions at cell edges also decreases. In addition, loss of septins leads to decreased amounts of F-actin at the cortical membrane, along with increased amounts of F-actin in stress fibers of the cytoplasm. Endothelial monolayer disruption from loss of septins is also associated with decreased transendothelial electric resistance (TEER) and increased levels of transendothelial migration (TEM) by immune and cancer cells, owing to the gaps in the monolayer. A current working model is that assembly of septin filaments at regions of positive membrane curvature contributes to a mechanical footing or base for actin-based protrusive forces generated at adjoining regions of the membrane. Specific molecular interactions between the septin and actin components of the cytoskeleton may also be important contributors. Regulators of actin assembly may promote and support the assembly of septin filaments at the membrane, as part of a molecular feedback loop between the assembly of septin and actin filaments.

Tongxinluo enhances the effect of atorvastatin on the treatment of atherosclerosis with chronic obstructive pulmonary disease by maintaining the pulmonary microvascular barrier.

Atherosclerosis (AS) is a common comorbidity of chronic obstructive pulmonary disease (COPD), and systemic inflammation is an important mechanism of COPD with AS. Tongxinluo (TXL) improves the function of vascular endothelial cells. We aimed to prove that impairment of pulmonary microvascular barrier function is involved in COPD-mediated aggravation of AS and investigate whether TXL enhances the effect of Ato (atorvastatin) on COPD with AS by protecting pulmonary microvascular endothelial barrier function. In vivo, a COPD with atherosclerotic apolipoprotein E knockout (AS ApoE-/-) mouse model was established by cigarette smoke combined with a high-fat diet. The animals were administered TXL, Ato, and TXL + Ato once a day for 20 weeks. Lung function, lung microvascular permeability, lung inflammation, systemic inflammation, serum lipid levels, atheromatous plaque formation, and endothelial damage biomarkers were measured. In vitro, human pulmonary microvascular endothelial cells (HPMECs) were pretreated with TXL and incubated with cigarette smoke extract to establish the model. The permeability of the endothelial monolayer, inflammatory cytokines, endothelial damage biomarkers, and tight junction (Tj) proteins were determined. Cigarette smoking significantly exacerbated the high-fat diet-induced pulmonary function decline, pulmonary microvascular endothelial barrier dysfunction, inflammation, and atherosclerotic plaques. These changes were reversed by TXL-Ato; the combination was more effective than Ato alone. Furthermore, TXL protected the HPMEC barrier and inhibited inflammation in HPMECs. COPD aggravates AS, possibly through the destruction of pulmonary microvascular barrier function; thus, lung inflammation triggers systemic inflammation. In treating COPD with AS, TXL enhances the antiatherosclerotic effect of Ato, protecting the pulmonary microvascular barrier.

C-Jun-mediated miR-19b expression induces endothelial barrier dysfunction in an in vitro model of hemorrhagic shock

BackgroundOur previous data demonstrated that miR-19b expression was increased in human lung microvascular endothelial cells in-vitro-, in-vivo and in patients with hemorrhagic shock, leading to a decrease in syndecan-1 mRNA and protein and resulting in loss of endothelial barrier function. However, the mechanism underlying increased miR-19b expression remains unclear. The objective of the current study was to determine if c-Jun mediates the early responsive microRNA, miR-19b, to cause endothelial barrier dysfunction.MethodHuman lung microvascular endothelial cells (HLMEC) or HEK293T cells were transfected with c-Jun overexpressing vector, c-Jun siRNA, miR-19b promoter vector, miR-19b mutated promoter vector, miR-19b oligo inhibitor, then subjected to hypoxia/reoxygenation as in-vitro model of hemorrhagic shock. Levels of protein, miRNA, and luciferase activity were measured. Transwell permeability of endothelial monolayers were also determined. Plasma levels of c-Jun were measured in injured patients with hemorrhagic shock.ResultHypoxia/reoxygenation induced primary (pri-)miR-19b, mature miR-19b, and c-Jun expression over time in a comparable timeframe. c-Jun silencing by transfection with its specific siRNA reduced both pri-miR-19b and mature miR-19b levels. Conversely, c-Jun overexpression enhanced H/R-induced pri-miR-19b. Studies using a luciferase reporter assay revealed that in cells transfected with vectors containing the wild-type miR-19b promoter and luciferase reporter, c-Jun overexpression or hypoxia/ reoxygenation significantly increased luciferase activity. c-Jun knockdown reduced the luciferase activity in these cells, suggesting that the miR-19b promoter is directly activated by c-Jun. Further, chromatin immunoprecipitation assay confirmed that c-Jun directly bound to the promoter DNA of miR-19b and hypoxia/reoxygenation significantly increased this interaction. Additionally, c-Jun silencing prevented cell surface syndecan-1 loss and endothelial barrier dysfunction in HLMECs after hypoxia/reoxygenation. Lastly, c-Jun was significantly elevated in patients with hemorrhagic shock compared to healthy controls.ConclusionTranscription factor c-Jun is inducible by hypoxia/reoxygenation, binds to and activates the miR-19b promoter. Using an in-vitro model of hemorrhagic shock, our findings identified a novel cellular mechanism whereby hypoxia/ reoxygenation increases miR-19b transcription by inducing c-Jun and leads to syndecan-1 decrease and endothelial cell barrier dysfunction. This finding supports that miR-19b could be a potential therapeutic target for hemorrhage shock.

Role of Endothelial STAT3 in Cerebrovascular Function and Protection from Ischemic Brain Injury.

STAT3 plays a protective role against ischemic brain injury; however, it is not clear which brain cell type mediates this effect, and by which mechanism. We tested the hypothesis that endothelial STAT3 contributes to protection from cerebral ischemia, by preserving cerebrovascular endothelial function and blood–brain barrier (BBB) integrity. The objective of this study was to determine the role of STAT3 in cerebrovascular endothelial cell (EC) survival and function, and its role in tissue outcome after cerebral ischemia. We found that in primary mouse brain microvascular ECs, STAT3 was constitutively active, and its phosphorylation was reduced by oxygen-glucose deprivation (OGD), recovering after re-oxygenation. STAT3 inhibition, using two mechanistically different pharmacological inhibitors, increased EC injury after OGD. The sub-lethal inhibition of STAT3 caused endothelial dysfunction, demonstrated by reduced nitric oxide release in response to acetylcholine and reduced barrier function of the endothelial monolayer. Finally, mice with reduced endothelial STAT3 (Tie2-Cre; STAT3flox/wt) sustained larger brain infarcts after middle cerebral artery occlusion (MCAO) compared to wild-type (WT) littermates. We conclude that STAT3 is vital to maintaining cerebrovascular integrity, playing a role in EC survival and function, and protection against cerebral ischemia. Endothelial STAT3 may serve as a potential target in preventing endothelial dysfunction after stroke.

Anisotropic topographies restore endothelial monolayer integrity and promote the proliferation of senescent endothelial cells.

Thrombogenicity remains a major issue in cardiovascular implants (CVIs). Complete surficial coverage of CVIs by a monolayer of endothelial cells (ECs) prior to implantation represents a promising strategy but is hampered by the overall logistical complexity and the high number of cells required. Consequently, extensive cell expansion is necessary, which may eventually lead to replicative senescence. Considering that micro-structured surfaces with anisotropic topography may promote endothelialization, we investigated the impact of gratings on the biomechanical properties and the replicative capacity of senescent ECs. After cultivation on gridded surfaces, the cells showed significant improvements in terms of adherens junction integrity, cell elongation, and orientation of the actin filaments, as well as enhanced yes-associated protein nuclear translocation and cell proliferation. Our data therefore suggest that micro-structured surfaces with anisotropic topographies may improve long-term endothelialization of CVIs.

Target induction of heme oxygenase-1 attenuates anti-HLA I antibody induced transplant vasculopathy in an allogeneic aortic transplantation model in mice

Abstract Introduction After solid organ transplantation every second patient develops transplant vasculopathy (TV), which still remains the major obstacle for long-term graft survival. The main risk factor for developing TV are de novo synthesized donor-specific antibodies binding molecules of the MHC complex (HLA complex in humans, respectively) on the surface of endothelial cells (EC). EC become activated, vascular smooth muscle cells proliferate, and monocytes are recruited, contributing the development of the neointima a hallmark of TV. We have demonstrated that the anti-HLA I antibody (w6/32) significantly increases transmigration of monocytes across an endothelial monolayer in vitro. These interactions were dependent on modulating the anti-inflammatory enzyme heme oxygenase (HO)-1. Purpose The aim of the project is to investigate which adhesion molecule might mediate the antibody-induced transmigration of monocytes sensitive to HO-1 modulation and whether this modulation has beneficial effects in an allogenic aortic transplantation model in mice. Methods Before w6/32 stimulation, HO-1 was modulated by pretreating Human Coronary Artery Endothelial Cells (HCAEC) either with CoPPIX as a HO-1 activator or with statins to test for a therapeutic approach. Expression of a variety of established adhesion receptors in HCAEC was screened after HO-1 modulation and w6/32 stimulation. Antibody-induced transmigration and adhesion of THP-1 were quantified in the presence of CD62E-blocking antibodies. Thoracic aorta was transplanted into the abdominal aorta between fully MHC mismatched mice and applying the corresponding anti-MHC I antibody (SF1-1.1) induced TV. Mice were treated with CoPPIX, statins or CORM to evaluate the effects of HO-1 modulation or its metabolite CO on TV development. Statistical evaluation was performed with t-test or ANOVA. Results HO-1 modulation in HCAEC diminished w6/32-induced THP-1 transmigration. CD62E-expression in HCAECs was enhanced after w6/32 stimulation on mRNA and protein level, but preincubation with CoPPIX or statins reduced these effects. Incubation of HCAEC with a CD62E-blocking antibody after w6/32 stimulation significantly diminished the rate of transmigration normalized to isotype-stimulated HCAEC (isotype 100%, w6/32 + IgG 226% vs. w6/32 + CD62E-block 138%, p<0.05.). Treatment of mice with CoPPIX resulted in a significant reduction of the SF1-1.1-induced number of transmigrated monocytes (SF1-1.1: 10 vs. CoPPIX: 5.4; p<0.05). Application of statins (SF1-1.1: 10 vs. statin 6.6; p<0.05) or CORM (CORM: 6.1 vs. iCORM: 11.3, p<0.05) significantly reduces monocyte number as well. In all treatment groups, the SF1-1.1-induced neointima formation was ameliorated. Conclusion w6/32-induced monocyte transmigration is CD62E-dependent. SF1-1.1-induced monocytic infiltrate and intimal hyperplasia in a murine aortic transplantation model is also CD62E dependent and amenable to HO-1 modulation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This sudy was funded by the German Research Foundation to Jan Larmann

Intravascular papillary endothelial hyperplasia in the cecum: a case report

Background Intravascular papillary endothelial hyperplasia (IPEH), also known as Masson's tumor, is a benign, non-neoplastic vascular lesion that is characterized by reactive proliferation of papillary endothelial cells associated with a thrombus. These lesions typically develop in the vascular regions of the head and neck, oral cavity, or extremities; however, other organ systems have been affected. IPEH in the gastrointestinal tract is rare, with only a few cases reported to date. Thus, the pathogenesis and clinical features of IPEH in the gastrointestinal tract are not entirely understood. Moreover, the local excision of certain subtypes of IPEH can be curative; this makes timely diagnosis essential. We present the case of a patient with IPEH in the cecum that was discovered while investigating the cause of severe anemia.Case presentationA 29-year-old woman visited a general practitioner (GP) with the complaint of abdominal pain. She was diagnosed with acute appendicitis and was prescribed antibiotics. After treatment, her abdominal pain disappeared. However, she was found to be severely anemic (hemoglobin level, 6.5 g/dl). To determine the cause of her anemia, the GP referred her to our hospital for further examination and treatment. Computed tomography scan revealed cecal wall thickening. Further, a lower gastrointestinal endoscopy revealed a 2-cm raised mass-like lesion in the cecum. This lesion was pathologically identified as an inflammatory granuloma. The cause of her anemia was determined to be bleeding from the lesion in the cecum. She underwent laparoscopic ileocecal resection.Histopathological examination of the surgical specimen revealed a spongy structure comprising many small papillary fibrous tissues lined by a typical monolayer endothelium. Further, immunohistochemical analysis showed that the cells of the endothelium monolayer expressed CD31, CD34. The Ki-67 labeling index was < 1%. Based on these findings, the lesion was identified as an IPEH in the cecum. The patient’s postoperative course was uneventful, and there was no evidence of recurrence during the 1.3 years of follow-up.Conclusions IPEH rarely arises within the abdominal cavity. Surgery remains the only treatment for IPEH and is associated with an excellent prognosis and a low recurrence rate. More aggressive lesions such as angiosarcoma should be excluded when considering the histologic diagnoses of IPEH, and expert pathologic review is vital. This is the first report of IPEH occurring in the cecum and represents a novel cause of gastrointestinal bleeding which the clinician should consider when evaluating a patient with atypical or difficult gastrointestinal bleeding sources.

Potential role of extracellular granzyme B in wet age-related macular degeneration and fuchs endothelial corneal dystrophy.

Age-related ocular diseases are the leading cause of blindness in developed countries and constitute a sizable socioeconomic burden worldwide. Age-related macular degeneration (AMD) and Fuchs endothelial corneal dystrophy (FECD) are some of the most common age-related diseases of the retina and cornea, respectively. AMD is characterized by a breakdown of the retinal pigment epithelial monolayer, which maintains retinal homeostasis, leading to retinal degeneration, while FECD is characterized by degeneration of the corneal endothelial monolayer, which maintains corneal hydration status, leading to corneal edema. Both AMD and FECD pathogenesis are characterized by disorganized local extracellular matrix (ECM) and toxic protein deposits, with both processes linked to aberrant protease activity. Granzyme B (GrB) is a serine protease traditionally known for immune-mediated initiation of apoptosis; however, it is now recognized that GrB is expressed by a variety of immune and non-immune cells and aberrant extracellular localization of GrB substantially contributes to various age-related pathologies through dysregulated cleavage of ECM, tight junction, and adherens junction proteins. Despite growing recognition of GrB involvement in multiple age-related pathologies, its role in AMD and FECD remains poorly understood. This review summarizes the pathophysiology of, and similarities between AMD and FECD, outlines the current knowledge of the role of GrB in AMD and FECD, as well as hypothesizes putative contributions of GrB to AMD and FECD pathogenesis and highlights the therapeutic potential of pharmacologically inhibiting GrB as an adjunctive treatment for AMD and FECD.

Role of moesin and its phosphorylation in VE-cadherin expression and distribution in endothelial adherens junctions

Background and aimVascular endothelial cadherin (VE-cadherin) is an important element of adherens junctions (AJs) between endothelial cells. Its expression and proper distribution are critical for AJ formation and vascular integrity. Our previous studies have demonstrated that moesin phosphorylation mediated the hyper-permeability in endothelial monolayer and microvessels. However, the role of moesin and its phosphorylation in VE-cadherin expression and distribution is not clear. Methods and resultsIn vivo, expression of VE-cadherin was significantly reduced in retina and other various tissues in moesin knock out mice (Msn-/Y). In vitro, by regulating moesin expression with siRNA and adenovirus transfection, we verified that moesin has an effect on VE-cadherin expression in HUVECs, while transcription factor KLF4 may participate in this process. In addition, treatment of advanced glycation end products (AGEs) induced abnormal distribution of VE-cadherin in retinal microvessels from C57BL/6 wild type mice, and in vitro studies indicated that moesin Thr558 phosphorylation had a critical role in AGE-induced VE-cadherin internalization from cytomembrane to cytoplasm. Further investigation demonstrated that the inhibition of F-actin polymerization with cytochalasin D could abolish AGE- and Thr558 phosphor-moesin-mediated VE-cadherin internalization. ConclusionThis study suggests that moesin regulates VE-cadherin expression through KLF4 and the state of moesin phosphorylation at Thr558 affects the integrity of VE-cadherin-based AJs. Thr558 phosphor-moesin mediates AGE-induced VE-cadherin internalization through cytoskeleton reassembling.

Heterocomplexes between the atypical chemokine MIF and the CXC-motif chemokine CXCL4L1 regulate inflammation and thrombus formation

To fulfil its orchestration of immune cell trafficking, a network of chemokines and receptors developed that capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within this network. Moreover, some inflammatory mediators, not structurally linked to classical chemokines, bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified macrophage migration inhibitory factor (MIF) as an ACK that binds to chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine–chemokine interactions extend to ACKs and that MIF forms heterocomplexes with classical chemokines. We tested this hypothesis by using an unbiased chemokine protein array. Platelet chemokine CXCL4L1 (but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12) was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, also establishing high-affinity binding. We next determined whether heterocomplex formation modulates inflammatory/atherogenic activities of MIF. Complex formation was observed to inhibit MIF-elicited T-cell chemotaxis as assessed by transwell migration assay and in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ, as well as MIF-mediated monocyte adhesion on aortic endothelial cell monolayers under flow stress conditions. Of note, CXCL4L1 blocked binding of Alexa-MIF to a soluble surrogate of CXCR4 and co-incubation with CXCL4L1 attenuated MIF responses in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. Because MIF and CXCL4L1 are platelet-derived products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and in clinical human thrombus sections obtained from peripheral artery disease (PAD) in patients undergoing thrombectomy. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction that adds to the complexity of the chemokine interactome and chemokine/receptor-network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be target structures that can be exploited to modulate inflammation and thrombosis.

Monoclonal anti-endoglin antibody TRC105 (carotuximab) prevents hypercholesterolemia and hyperglycemia-induced endothelial dysfunction in human aortic endothelial cells.

Endoglin (Eng) is a co-receptor of the transforming growth factor β superfamily playing an important role in endothelial dysfunction. TRC105 (carotuximab) is a monoclonal antibody that blocks Eng and its downstream Smad signaling pathway. Here we have investigated for the first time the effects of TRC105 treatment on the development of endothelial dysfunction induced by 7-ketocholesterol (7K) or high glucose (HG), focusing on Eng expression, signaling, and function. In the hypercholesterolemia study, human aortic endothelial cells (HAoECs) were treated with TRC105 (300 μg/ml) for 1 h, followed by the addition of 7K (10 μg/ml) for another 12 h. In the hyperglycemia study, HAoECs were exposed to HG (45 mM) for 60 h, followed by the addition of TRC105 for another 12 h, and cells treated with 5mM glucose and 40 mM mannitol served as control. Protein levels, adhesion, and transmigration of monocytes were assessed by flow cytometry, mRNA expression was measured by qRT-PCR. 7K and HG treatment increased protein levels of NF-κB and Eng and adhesion and transmigration of monocytes through HAoECs monolayer. TRC105 pretreatment reduced the 7K- or HG-induced Eng protein levels and pSmad1/5 and pSmad2/3 signaling. Despite increased protein levels of P-selectin and VCAM-1, TRC105 mediated blockage of Eng prevented 7K- and HG-induced adhesion and transmigration of monocytes through endothelial monolayers. These results suggest that TRC105-mediated Eng blockage can counteract the hypercholesterolemia- and hyperglycemia-induced endothelial dysfunction in HAoECs, suggesting that Eng might be a potential therapeutic target in disorders associated with elevated cholesterol and glucose levels.