Deficient Endothelial Cells Research Articles

Effect of Ulinastatin on Syndecan-2-Mediated Vascular Damage in IDH2-Deficient Endothelial Cells.

Syndecan-2 (SDC2), a cell-surface heparin sulfate proteoglycan of the glycocalyx, is mainly expressed in endothelial cells. Although oxidative stress and inflammatory mediators have been shown to mediate dysfunction of the glycocalyx, little is known about their role in vascular endothelial cells. In this study, we aimed to identify the mechanism that regulates SDC2 expression in isocitrate dehydrogenase 2 (IDH2)-deficient endothelial cells, and to investigate the effect of ulinastatin (UTI) on this mechanism. We showed that knockdown of IDH2 induced SDC2 expression in human umbilical vein endothelial cells (HUVECs). Matrix metalloproteinase 7 (MMP7) influences SDC2 expression. When IDH2 was downregulated, MMP7 expression was increased, as was TGF-β signaling, which regulates MMP7. Inhibition of MMP7 activity using MMP inhibitor II significantly reduced SDC2, suggesting that IDH2 mediated SDC2 expression via MMP7. Moreover, expression of SDC2 and MMP7, as well as TGF-β signaling, increased in response to IDH2 deficiency, and treatment with UTI reversed this increase. Similarly, the increase in SDC2, MMP7, and TGF-β signaling in the aorta of IDH2 knockout mice was reversed by UTI treatment. These findings suggest that IDH2 deficiency induces SDC2 expression via TGF-β and MMP7 signaling in endothelial cells.

Local complement factor H protects kidney endothelial cell structure and function

Factor H (FH) is a critical regulator of the alternative complement pathway and its deficiency or mutation underlie kidney diseases such as dense deposit disease. Since vascular dysfunction is an important facet of kidney disease, maintaining optimal function of the lining endothelial cells is important for vascular health. To investigate the molecular mechanisms that are regulated by FH in endothelial cells, FH deficient and sufficient mouse kidney endothelial cell cultures were established. Endothelial FH deficiency resulted in cytoskeletal remodeling, increased angiogenic potential, loss of cellular layer integrity and increased cell proliferation. FH reconstitution prevented these FH-dependent proliferative changes. Respiratory flux analysis showed reduced basal mitochondrial respiration, ATP production and maximal respiratory capacity in FH deficient endothelial cells, while proton leak remained unaltered. Similar changes were observed in FH deficient human glomerular endothelial cells indicating the translational potential of these studies. Gene expression analysis revealed that the FH-dependent gene changes in mouse kidney endothelial cells include significant upregulation of genes involved in inflammation and the complement system. The transcription factor nuclear factor-kB, that regulates many biological processes, was translocated from the cytoplasm to the nucleus in the absence of FH. Thus, our studies show the functional relevance of intrinsic FH in kidney endothelial cells in man and mouse.

High intraluminal pressure promotes vascular inflammation via caveolin-1

The aetiology and progression of hypertension involves various endogenous systems, such as the renin angiotensin system, the sympathetic nervous system, and endothelial dysfunction. Recent data suggest that vascular inflammation may also play a key role in the pathogenesis of hypertension. This study sought to determine whether high intraluminal pressure results in vascular inflammation. Leukocyte adhesion was assessed in rat carotid arteries exposed to 1 h of high intraluminal pressure. The effect of intraluminal pressure on signaling mechanisms including reactive oxygen species production (ROS), arginase expression, and NFĸB translocation was monitored. 1 h exposure to high intraluminal pressure (120 mmHg) resulted in increased leukocyte adhesion and inflammatory gene expression in rat carotid arteries. High intraluminal pressure also resulted in a downstream signaling cascade of ROS production, arginase expression, and NFĸB translocation. This process was found to be angiotensin II-independent and mediated by the mechanosensor caveolae, as caveolin-1 (Cav1)-deficient endothelial cells and mice were protected from pressure-induced vascular inflammatory signaling and leukocyte adhesion. Cav1 deficiency also resulted in a reduction in pressure-induced glomerular macrophage infiltration in vivo. These findings demonstrate Cav1 is an important mechanosensor in pressure-induced vascular and renal inflammation.

CoQ10 Deficient Endothelial Cell Culture Model for the Investigation of CoQ10 Blood-Brain Barrier Transport.

Primary coenzyme Q10 (CoQ10) deficiency is unique among mitochondrial respiratory chain disorders in that it is potentially treatable if high-dose CoQ10 supplements are given in the early stages of the disease. While supplements improve peripheral abnormalities, neurological symptoms are only partially or temporarily ameliorated. The reasons for this refractory response to CoQ10 supplementation are unclear, however, a contributory factor may be the poor transfer of CoQ10 across the blood–brain barrier (BBB). The aim of this study was to investigate mechanisms of CoQ10 transport across the BBB, using normal and pathophysiological (CoQ10 deficient) cell culture models. The study identifies lipoprotein-associated CoQ10 transcytosis in both directions across the in vitro BBB. Uptake via SR-B1 (Scavenger Receptor) and RAGE (Receptor for Advanced Glycation Endproducts), is matched by efflux via LDLR (Low Density Lipoprotein Receptor) transporters, resulting in no “net” transport across the BBB. In the CoQ10 deficient model, BBB tight junctions were disrupted and CoQ10 “net” transport to the brain side increased. The addition of anti-oxidants did not improve CoQ10 uptake to the brain side. This study is the first to generate in vitro BBB endothelial cell models of CoQ10 deficiency, and the first to identify lipoprotein-associated uptake and efflux mechanisms regulating CoQ10 distribution across the BBB. The results imply that the uptake of exogenous CoQ10 into the brain might be improved by the administration of LDLR inhibitors, or by interventions to stimulate luminal activity of SR-B1 transporters.

Endothelial β-Catenin Signaling Supports Postnatal Brain and Retinal Angiogenesis by Promoting Sprouting, Tip Cell Formation, and VEGFR (Vascular Endothelial Growth Factor Receptor) 2 Expression.

Activation of endothelial β-catenin signaling by neural cell-derived Norrin or Wnt ligands is vital for the vascularization of the retina and brain. Mutations in members of the Norrin/β-catenin pathway contribute to inherited blinding disorders because of defective vascular development and dysfunctional blood-retina barrier. Despite a vital role for endothelial β-catenin signaling in central nervous system health and disease, its contribution to central nervous system angiogenesis and its interactions with downstream signaling cascades remains incompletely understood. Approach and Results: Here, using genetically modified mouse models, we show that impaired endothelial β-catenin signaling caused hypovascularization of the postnatal retina and brain because of deficient endothelial cell proliferation and sprouting. Mosaic genetic analysis demonstrated that endothelial β-catenin promotes but is not required for tip cell formation. In addition, pharmacological treatment revealed that angiogenesis under conditions of inhibited Notch signaling depends upon endothelial β-catenin. Importantly, impaired endothelial β-catenin signaling abrogated the expression of the VEGFR (vascular endothelial growth factor receptor)-2 and VEGFR3 in brain microvessels but not in the lung endothelium. Our study identifies molecular crosstalk between the Wnt/β-catenin and the Notch and VEGF-A signaling pathways and strongly suggest that endothelial β-catenin signaling supports central nervous system angiogenesis by promoting endothelial cell sprouting, tip cell formation, and VEGF-A/VEGFR2 signaling.

Modulating membrane fluidity corrects Batten disease phenotypes in vitro and in vivo

The neuronal ceroid lipofuscinoses are a class of inherited neurodegenerative diseases characterized by the accumulation of autofluorescent storage material. The most common neuronal ceroid lipofuscinosis has juvenile onset with rapid onset blindness and progressive degeneration of cognitive processes. The juvenile form is caused by mutations in the CLN3 gene, which encodes the protein CLN3. While mouse models of Cln3 deficiency show mild disease phenotypes, it is apparent from patient tissue- and cell-based studies that its loss impacts many cellular processes. Using Cln3 deficient mice, we previously described defects in mouse brain endothelial cells and blood-brain barrier (BBB) permeability. Here we expand on this to other components of the BBB and show that Cln3 deficient mice have increased astrocyte endfeet area. Interestingly, this phenotype is corrected by treatment with a commonly used GAP junction inhibitor, carbenoxolone (CBX). In addition to its action on GAP junctions, CBX has also been proposed to alter lipid microdomains. In this work, we show that CBX modifies lipid microdomains and corrects membrane fluidity alterations in Cln3 deficient endothelial cells, which in turn improves defects in endocytosis, caveolin-1 distribution at the plasma membrane, and Cdc42 activity. In further work using the NIH Library of Integrated Network-based Cellular Signatures (LINCS), we discovered other small molecules whose impact was similar to CBX in that they improved Cln3-deficient cell phenotypes. Moreover, Cln3 deficient mice treated orally with CBX exhibited recovery of impaired BBB responses and reduced autofluorescence. CBX and the compounds identified by LINCS, many of which have been used in humans or approved for other indications, may find therapeutic benefit in children suffering from CLN3 deficiency through mechanisms independent of their original intended use.

Defective autophagy is a key feature of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is a major cerebrovascular disease affecting approximately 0.3–0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss-of-function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss-of-function activates the mTOR-ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole-cular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2-silenced human endothelial cells and in both cells and tissues from an endothelial-specific CCM3-knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial-to-mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions.

Destabilization of endothelial cell‐cell contacts modifies inflammatory responses

Changes in the stability of endothelial cell‐cell contacts are commonly associated with chronic and acute vascular inflammation. However, it remains unclear how changes in endothelial cell‐cell contact impact the progress of vascular inflammation. KRIT1‐deficient animals lack the adherens junction accessory protein KRIT1, and exhibit a loss of cell‐cell contact stability. Krit1+/‐ mice exhibit increased baseline microvessel permeability, but maintain normal leukocyte responses. Stimulation of Krit1+/‐ mice with various inflammatory mediators enhanced edema formation but not leukocyte activation. Krit1+/‐ mice exhibited relatively normal leukocyte responses to TNF‐α, IL‐1β, and histamine, but, notably, Krit1+/‐ vessels did not become more permeable following TNF‐a treatment, contrary to what was seen in littermate controls. Though TNF‐α signaling in KRIT1 deficient endothelial cells appeared normal, these cells displayed increased oxidative stress, and treatment with TNF‐α failed to enhance reactive oxygen species (ROS) levels. In vivo, targeted inhibition of endothelial ROS was sufficient to restore microvascular permeability in Krit1+/‐ mice to WT levels. However, antioxidant treatment of TNF‐α‐treated WT and Krit1+/‐ animals reversed only venular permeability. KRIT1 depletion‐induced oxidative stress could be reversed by treatment with an NADPH‐oxidase inhibitor, suggesting that TNF‐α driven induction of NADPH oxidase may be inoperative in the absence of KRIT1. Thus, destabilization of endothelial adherens junctions following depletion of KRIT1 plays a vascular bed‐dependent role in modifying the vascular response to inflammation.

Podoplanin requires sialylated O-glycans for stable expression on lymphatic endothelial cells and for interaction with platelets

AbstractO-glycosylation of podoplanin (PDPN) on lymphatic endothelial cells is critical for the separation of blood and lymphatic systems by interacting with platelet C-type lectin-like receptor 2 during development. However, how O-glycosylation controls endothelial PDPN function and expression remains unclear. In this study, we report that core 1 O-glycan–deficient or desialylated PDPN was highly susceptible to proteolytic degradation by various proteases, including metalloproteinases (MMP)-2/9. We found that the lymph contained activated MMP-2/9 and incubation of the lymph reduced surface levels of PDPN on core 1 O-glycan–deficient endothelial cells, but not on wild-type ECs. The lymph from mice with sepsis induced by cecal ligation and puncture, which contained bacteria-derived sialidase, reduced PDPN levels on wild-type ECs. The MMP inhibitor, GM6001, rescued these reductions. Additionally, GM6001 treatment rescued the reduction of PDPN level on lymphatic endothelial cells in mice lacking endothelial core 1 O-glycan or cecal ligation and puncture-treated mice. Furthermore, core 1 O-glycan–deficient or desialylated PDPN impaired platelet interaction under physiological flow. These data indicate that sialylated O-glycans of PDPN are essential for platelet adhesion and prevent PDPN from proteolytic degradation primarily mediated by MMPs in the lymph.

Management of Cerebral Cavernous Malformations: Molecular Insight and Clinical Considerations

Introduction: Cavernous malformations (cavernomas) occur in 0.4% - 0.9% of the population. Clinical symptoms are mainly caused by recurrent hemorrhages and/or their progressive growth. The neurological deficit depends on their exact localization. Most common symptoms are epileptic seizures. Particularly, in deep seated lesions (brain stem, basal ganglia, etc.) a well elaborated preoperative imaging is critical in ordert to select the most favorable approach for every patient. Usually, cavernomas occur as single lesions, but in some individuals (10-20%) more than one cavernoma might be diagnosed. Particularly in these patients a treatment strategy is critically dependant on the understanding of the biology of multiple (and familial?) lesions. Aim: Therefore, our recent research aimed at (1) unshadowing the biological activity and ageressivenes of cavernomas (2) understanding their human genetics and (3) increasing the imaging quality of cavernomas and associated venous malfomations. Material and Methods: Endothelial cells were isolated and purified from the fresh operative specimens of sporadic CCMs with single lesions (CCM-ECs). The expression profile of VEGF and its receptors as well as CCM1-3 genes were detected by real-time PCR. Endothelial proliferation, migration, sprouting and tube formation were compared in the absence and the presence of angiogenic stimuli and after CCM1-3 siRNA in CCM-ECs and in different types of healthy endothelial cells. Furthermore, a strong correlation with VEGF related neoangiogenesis as well as the influence of a PTEN deficiency was studied. Patients with multiple lesions were evalutated for germ line mutations of the CCM1-3 genes. By using susceptibility weighted imaging (SWI) at 7T high field strength MRI both patients with single and multiple lesions were evaluated for cavernomas and associated venous malformations (VM). Results: A highly activated VEGF system was revealed in CCM-ECs. Accordingly, CCM-ECs exhibited marked growth potential under normal culture conditions and a significantly high proliferation activity in response to various angiogenic stimuli including hypoxia, fetal calf serum and VEGF treatment. Furthermore, a significantly higher mobility, spontaneous growth of sprouts and extensively branched tubes were exclusively detected in CCM-ECs. In comparison to healthy endothelia, CCM-ECs resisted apoptotic stimuli and showed distinct responses toward to activating angiogenesis after CCM1 silencing. Endothelila proliferation was conducted by the VEGF related pathways. PTEN deficiency in endothelial cavernoma cells seems to play a role particularly in patients suffering from multiple cavernomas. Tyrosin-kinase receptor inhibitors suppresses neoangiogenetic activity in CCM1 deficient endothelial cells in an animal model. In patients with multiple cavernomas, a germ line mutation of the CCM1-3 genes can be found in up to 80%. Patients with CCM1 mutations might additionally display a cutaneous nevus. In the patients harbouring single cavernomas, a solitary or multiple venous drainage was found in all lesions using susceptibility weighted imaging (SWI) at 7T high field strength MRI. A typical so called developmental anomaly (DVA) was found in 30% of the lesions. In the other cases, associated VM were depicted as well, although showing a different phenotype when compared with DVAs. Conclusion: Cavernomas are growing and active lesions similar to benign tumors with also similar underlying molecular growth mechanisms. The highly activated VEGF system and the distinct angiogenic characteristics of CCM-ECs may serve as an endothelia-pathomechanism synergistically acting together with genetic and other factors for the development of vascular lesions in the human brain. Particularly, the surgical resection of lesions with a CCM1-3 gene defects (familial cavernomas) should be evaluated very carefully. Ultra-high-field magnetic resonance imaging at 7T improves the detection of cavernomas. The improved depiction of small cavernomas might possibly help to clarify cryptogenic seizures, which represent 30% of all seizures. We believe, that the additional information gained will have an impact on treatment plans for deep seated lesions in the future. Furthermore, our data support previous assumptions of CCMs being notoriously associated with local venous malformation (VM and DVA) involving larger outflow vessels.

RAGE controls activation and anti-inflammatory signalling of protein C.

AimsThe receptor for advanced glycation endproducts, RAGE, is a multiligand receptor and NF-κB activator leading to perpetuation of inflammation. We investigated whether and how RAGE is involved in mediation of anti-inflammatory properties of protein C.Methods and ResultsWe analyzed the effect of protein C on leukocyte adhesion and transmigration in WT- and RAGE-deficient mice using intravital microscopy of cremaster muscle venules during trauma- and TNFα-induced inflammation. Both, protein C (PC, Ceprotin, 100 U/kg) and activated protein C (aPC, 24 µg/kg/h) treatment significantly inhibited leukocyte adhesion in WT mice in these inflammation models. The impaired leukocyte adhesion after trauma-induced inflammation in RAGE knockout mice could not be further reduced by PC and aPC. After TNFα-stimulation, however, aPC but not PC treatment effectively blocked leukocyte adhesion in these mice. Consequently, we asked whether RAGE is involved in PC activation. Since RAGE-deficient mice and endothelial cells showed insufficient PC activation, and since thrombomodulin (TM) and endothelial protein C receptor (EPCR) are reduced on the mRNA and protein level in RAGE deficient endothelial cells, an involvement of RAGE in TM-EPCR-dependent PC activation is likely. Moreover, TNFα-induced activation of MAPK and upregulation of ICAM-1 and VCAM-1 are reduced both in response to aPC treatment and in the absence of RAGE. Thus, there seems to be interplay of the RAGE and the PC pathway in inflammation.ConclusionRAGE controls anti-inflammatory properties and activation of PC, which might involve EPCR and TM.

Abstract C3: Tumor-derived soluble uPAR regulates tumor angiogenesis via PTEN.

Abstract The urokinase receptor uPAR (CD87), a GPI-anchored protein, is frequently over-expressed in tumor cells. uPAR provokes numerous cellular functions by focusing proteolytic activity on the cell surface, but also by inducing intracellular signal transduction via transmembrane interaction partners, thus inducing cell migration and invasion as well as cell survival. Soluble tumor-derived uPAR has recently been described to activate endothelial cells and was suggested to promote angiogenesis. Consistently, high uPAR expression in tumor cells or high serum levels of circulating uPAR predict worse prognosis of breast, lung or colorectal cancer patients. In this study, we found that tumor-derived soluble uPAR is a key regulator of PTEN expression in endothelial cells. HEK cells deficient in endogenous uPAR or uPAR deficient endothelial cells derived from uPAR -/- mice had high PTEN mRNA as well as protein levels, while reconstitution of endogenous as well as addition of soluble exogenous uPAR decreased PTEN levels. uPAR, thereby, led to an integrin-dependent activation of the NFkB pathway, a known transcription pathway inhibitor of PTEN. As a consequence of PTEN downregulation, the downstream PKB/Akt signalling pathway became activated, which led to an enhanced migratory as well as cell survival activity in endothelial cells. Cross-breeding of uPAR -/- mice with endothelial cell-specific PTEN +/- mice, thereby led to a rescue of the high migratory phenotype of PTEN heterozygous endothelial cells in vitro and angiogenesis formation in a directed in vivo angiogenesis assay (DIVAA). From our date we conclude that tumor-derived uPAR is a major regulator of PTEN expression in endothelial cells, which leads to the induction of the PI3K/Akt-pathway and an enhanced angiogenic phenotype. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C3. Citation Format: Gerald W. Prager, Matthias Unseld, Marina Poettler, Christoph Zielinski. Tumor-derived soluble uPAR regulates tumor angiogenesis via PTEN. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C3.

Aberrant association between vascular endothelial growth factor receptor-2 and VE-cadherin in response to vascular endothelial growth factor-a in Shb-deficient lung endothelial cells

Vascular permeability is a hallmark response to the main angiogenic factor VEGF-A and we have previously described a reduction of this response in Shb knockout mice. To characterize the molecular mechanisms responsible for this effect, endothelial cells were isolated from lungs and analyzed in vitro. Shb deficient endothelial cells exhibited less migration in a scratch wound-healing assay both under basal conditions and after vascular endothelial growth factor-A (VEGF-A) stimulation, suggesting a functional impairment of these cells in vitro. Staining for VE-cadherin and vascular endothelial growth factor receptor-2 (VEGFR-2) showed co-localization in adherens junctions and in intracellular sites such as the perinuclear region in wild-type and Shb knockout cells. VEGF-A decreased the VE-cadherin/VEGFR-2 co-localization in membrane structures resembling adherens junctions in wild-type cells whereas no such response was noted in the Shb knockout cells. VE-cadherin/VEGFR-2 co-localization was also recorded using spinning-disk confocal microscopy and VEGF-A caused a reduced association in the wild-type cells whereas the opposite pattern was observed in the Shb knockout cells. The latter expressed slightly more of cell surface VEGFR-2. VEGF-A stimulated extracellular-signal regulated kinase, Akt and Rac1 activities in the wild-type cells whereas no such responses were noted in the knockout cells. We conclude that aberrant signaling characteristics with respect to ERK, Akt and Rac1 are likely explanations for the observed altered pattern of VE-cadherin/VEGFR-2 association. The latter is important for understanding the reduced in vivo vascular permeability response in Shb knockout mice, a phenomenon that has patho-physiological relevance.

Th17 lymphocyte adhesion to endothelium is highly dependent on E‐selectin and ICAM‐1 mediated interactions

T cell subset specific migration to inflammatory sites is tightly regulated involving interaction of the T cells with the endothelium. T helper 17 (Th17) cells often appear at the same sites of inflammation than T helper 1 (Th1) cells but not simultaneously, sometimes they appear at different sites and sometimes co‐existence does occur. However, Th17 cells adhesion to vascular endothelium and migration into tissues and organs has not been well studied. We examined the adhesion of mouse Th17 cells to endothelial adhesion molecules and endothelium under flow in vitro and to microvessels in vivo, and characterized their migratory phenotype by flow cytometry and qRT‐PCR. More Th17 than Th1 cells interacted with E‐selectin and fewer bound to TNF‐α activated E‐selectin deficient endothelial cells. Intravital microscopy demonstrated that Th17 cells engage in more rolling interactions with TNF‐α treated microvessels than Th1 cells in wild type mice but not in E‐selectin deficient mice. Th17 adhesion to ICAM‐1 was dependent on integrin activation by CCL20. In an air pouch model of inflammation, CCL20 triggered recruitment of Th17 but not Th1 cells. Our data provide evidence that Th17 cells adhesion with the endothelium is highly dependent on E‐selectin and ICAM‐1 and is quantitatively different from Th1 cells. Funded by NIH: K99‐HL097406 (PA); HL36028 (FWL, AHL); K08‐HL086672 (KJC); HL53993 (FWL).

Difference in Th1 and Th17 Lymphocyte Adhesion to Endothelium

T cell subset-specific migration to inflammatory sites is tightly regulated and involves interaction of the T cells with the endothelium. Th17 cells often appear at different inflammatory sites than Th1 cells, or both subsets appear at the same sites but at different times. Differences in T cell subset adhesion to endothelium may contribute to subset-specific migratory behavior, but this possibility has not been well studied. We examined the adhesion of mouse Th17 cells to endothelial adhesion molecules and endothelium under flow in vitro and to microvessels in vivo and we characterized their migratory phenotype by flow cytometry and quantitative RT-PCR. More Th17 than Th1 cells interacted with E-selectin. Fewer Th17 than Th1 cells bound to TNF-α-activated E-selectin-deficient endothelial cells, and intravital microscopy studies demonstrated that Th17 cells engage in more rolling interactions with TNF-α-treated microvessels than Th1 cells in wild-type mice but not in E-selectin-deficient mice. Th17 adhesion to ICAM-1 was dependent on integrin activation by CCL20, the ligand for CCR6, which is highly expressed by Th17 cells. In an air pouch model of inflammation, CCL20 triggered recruitment of Th17 but not Th1 cells. These data provide evidence that E-selectin- and ICAM-1-dependent adhesion of Th17 and Th1 cells with endothelium are quantitatively different.

Radiotherapy Suppresses Angiogenesis in Mice through TGF-βRI/ALK5-Dependent Inhibition of Endothelial Cell Sprouting

BackgroundRadiotherapy is widely used to treat cancer. While rapidly dividing cancer cells are naturally considered the main target of radiotherapy, emerging evidence indicates that radiotherapy also affects endothelial cell functions, and possibly also their angiogenic capacity. In spite of its clinical relevance, such putative anti-angiogenic effect of radiotherapy has not been thoroughly characterized. We have investigated the effect of ionizing radiation on angiogenesis using in vivo, ex vivo and in vitro experimental models in combination with genetic and pharmacological interventions.Principal FindingsHere we show that high doses ionizing radiation locally suppressed VEGF- and FGF-2-induced Matrigel plug angiogenesis in mice in vivo and prevented endothelial cell sprouting from mouse aortic rings following in vivo or ex vivo irradiation. Quiescent human endothelial cells exposed to ionizing radiation in vitro resisted apoptosis, demonstrated reduced sprouting, migration and proliferation capacities, showed enhanced adhesion to matrix proteins, and underwent premature senescence. Irradiation induced the expression of P53 and P21 proteins in endothelial cells, but p53 or p21 deficiency and P21 silencing did not prevent radiation-induced inhibition of sprouting or proliferation. Radiation induced Smad-2 phosphorylation in skin in vivo and in endothelial cells in vitro. Inhibition of the TGF-β type I receptor ALK5 rescued deficient endothelial cell sprouting and migration but not proliferation in vitro and restored defective Matrigel plug angiogenesis in irradiated mice in vivo. ALK5 inhibition, however, did not rescue deficient proliferation. Notch signaling, known to hinder angiogenesis, was activated by radiation but its inhibition, alone or in combination with ALK5 inhibition, did not rescue suppressed proliferation.ConclusionsThese results demonstrate that irradiation of quiescent endothelial cells suppresses subsequent angiogenesis and that ALK5 is a critical mediator of this suppression. These results extend our understanding of radiotherapy-induced endothelial dysfunctions, relevant to both therapeutic and unwanted effects of radiotherapy.

α2,6-Sialic Acid on Platelet Endothelial Cell Adhesion Molecule (PECAM) Regulates Its Homophilic Interactions and Downstream Antiapoptotic Signaling

Antiangiogenesis therapies are now part of the standard repertoire of cancer therapies, but the mechanisms for the proliferation and survival of endothelial cells are not fully understood. Although endothelial cells are covered with a glycocalyx, little is known about how endothelial glycosylation regulates endothelial functions. Here, we show that α2,6-sialic acid is necessary for the cell-surface residency of platelet endothelial cell adhesion molecule (PECAM), a member of the immunoglobulin superfamily that plays multiple roles in cell adhesion, mechanical stress sensing, antiapoptosis, and angiogenesis. As a possible underlying mechanism, we found that the homophilic interactions of PECAM in endothelial cells were dependent on α2,6-sialic acid. We also found that the absence of α2,6-sialic acid down-regulated the tyrosine phosphorylation of PECAM and recruitment of Src homology 2 domain-containing protein-tyrosine phosphatase 2 and rendered the cells more prone to mitochondrion-dependent apoptosis, as evaluated using PECAM- deficient endothelial cells. The present findings open up a new possibility that modulation of glycosylation could be one of the promising strategies for regulating angiogenesis.

Cilia‐mediated heat shock protein 27 (hsp27) suppression modulates actin polymerization and organization in wild type mouse and oak ridge polycystic kidney (orpk) cells

Primary cilia play a pivotal role in the development of polycystic kidney disease (PKD), a state with an enhanced propensity for hypertension. Cilia, characterized as mechano/chemo‐sensing organelles, are uniquely situated to communicate extracellular signals to intracellular responses. Because cytoskeletal remodeling plays a significant role in cellular adaptation, we postulated that stress‐induced hsp27, which associates with actin to stabilize the cytoskeleton, could mediate this response. To that end, we evaluated shear and oxidative stress on hsp27 levels and actin polymerization in cilia deficient endothelial cells isolated from the orpk model of PKD. By immunoblotting orpk had a basal suppression of hsp27 compared to wild‐type (wt) cells. Basal suppression could be overcome by oxidative stress but not shear stress. Basal actin levels were consistently elevated in orpk when compared to wt cells and stress had a minimal effect on these levels. Actin organization was inversely correlated with hsp27 levels. Our observations provide evidence that disruption of cilia formation suppresses basal hsp27 levels by an undetermined mechanism and increases the unorganized state of actin. Results suggest primary cilia have a role in regulating actin organization and in providing cytoskeletal adaptation to stress potentially through hsp27. (AHA‐0630257N, NIH‐HL084451)

Alterations in cell number and cell morphology in copper deficient endothelial cells

Alterations in cell number and cell morphology in copper deficient endothelial cells

Endothelial intercellular adhesion molecule (ICAM)–2 regulates angiogenesis

AbstractEndothelial junctions maintain endothelial integrity and vascular homeostasis. They modulate cell trafficking into tissues, mediate cell-cell contact and regulate endothelial survival and apoptosis. Junctional adhesion molecules such as vascular endothelial (VE)–cadherin and CD31/platelet endothelial cell adhesion molecule (PECAM) mediate contact between adjacent endothelial cells and regulate leukocyte transmigration and angiogenesis. The leukocyte adhesion molecule intercellular adhesion molecule 2 (ICAM-2) is expressed at the endothelial junctions. In this study we demonstrate that endothelial ICAM-2 also mediates angiogenesis. Using ICAM-2–deficient mice and ICAM-2–deficient endothelial cells, we show that the lack of ICAM-2 expression results in impaired angiogenesis both in vitro and in vivo. We show that ICAM-2 supports homophilic interaction, and that this may be involved in tube formation. ICAM-2–deficient cells show defective in vitro migration, as well as increased apoptosis in response to serum deprivation, anti-Fas antibody, or staurosporine. ICAM-2 signaling in human umbilical vein endothelial cells (HUVECs) was found to activate the small guanosine triphosphatase (GTPase) Rac, which is required for endothelial tube formation and migration. These data indicate that ICAM-2 may regulate angiogenesis via several mechanisms including survival, cell migration, and Rac activation. Our findings identify a novel pathway regulating angiogenesis through ICAM-2 and a novel mechanism for Rac activation during angiogenesis.