Proteins In Endothelium Research Articles

Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity.

[Figure: see text].

Abstract 723: A Nitric Oxide-donor Based Vasodilatory Therapy Mitigates Hypoxia-induced Brain Injury on a Zebrafish Model of Hypoxic Ischemic Encephalopathy

Hypoxic ischemic encephalopathy (HIE), a brain injury caused by cerebral hypoxia due to perinatal asphyxia, has high mortality, high morbidity and severe sequelae, but therapeutic options remain limited. Cerebral vasoconstriction has been known to contribute to neuronal damage of HIE. Here we report a zebrafish model of brain injury induced by hypoxia and reoxygenation to mimic HIE, and explore the therapeutic potential of a vasodilatory agent to mitigate neuronal damage. Larval zebrafish (6 dpf) were immersed in a hypoxic medium (dissolved O2 below 0.2 ppm) for 15 min to induce brain injury, and then a normoxic medium to mimic resuscitation. The vascular width, blood flow and cerebral cell death were assessed with confocal imaging on transgenic fish that express fluorescent proteins in endothelium, red blood cells (RBCs) and cranial motor neurons, respectively. Neurological function was determined with a modified scoring system based on the movement, coordination and response of zebrafish to stimuli. Larvae survived from the hypoxic assault were categorized according to the severity of impaired cerebral hemodynamics. The serious group was featured with greater cerebral vasoconstriction (32% vs. 6%) and lower blood flow (82 vs. 728 RBCs/min) relative to the moderate group. These two groups showed notably different outcome measured 2 d post hypoxia with the serious group having a significantly higher cerebral cell death (34% vs. 4%), a lower neurological score (2.5 vs. 6) and survival rate (60% vs. 89%) relative to the moderate group. The strong correlation between the post-hypoxia cerebral hemodynamics and the outcome indicates that a vasodilatory therapy might be a strategy to ameliorate hypoxia induced brain injury. A treatment of s-nitrosoglutathione (GSNO, 15 μM), a nitric oxide donor, effectively alleviated vasoconstriction (7.4% vs. 14.3%), improved the neurological score (3.5 vs. 2.5) and increased the survival rate (63% vs. 54%) relative to the untreated control. In conclusion, a zebrafish model of HIE has been developed. We show that cerebral vasoconstriction may contribute the brain injury associated with HIE, and vasodilatory therapies possess a potential for the remedy of HIE.

Abstract 16: BcrKinase is a Novel Akt Kinase That Modulates Scavenger Receptor BI- and PDZK1-dependent Actions of HDL in Endothelium

High density lipoprotein cholesterol (HDL) has direct atheroprotective actions on endothelium. These are mediated by scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1, and they entail the activation of Akt kinase, which phosphorylates and thereby stimulates endothelial nitric oxide synthase (eNOS). In the present work we sought to determine how PDZK1 couples HDL/SR-BI to Akt and eNOS to modulate endothelial function. Using tandem affinity purification (TAP) following the infection of the human endothelial cell line EAhy926 with adenovirus expressing TAP-tagged PDZK1, we identified Breakpoint Cluster Region (Bcr) kinase as a PDZK1 interacting protein in endothelium. Whereas Bcr is well-known as a component of the Bcr-Abl fusion protein that results from translocation of the Philadelphia chromosome in chronic myelogenous leukemia, little is known of its function in endothelial cells. Bcr contains several distinctive domains including a C-terminal PDZ binding motif and a serine/threonine protein kinase domain. In primary human aortic endothelial cells (HAEC), we determined that endogenous Bcr interacts with PDZK1 in an HDL-dependent manner, and that Bcr is required for HDL-induced activation of eNOS and HDL stimulation of endothelial cell migration, which underlies the ability of the lipoprotein to promote endothelial monolayer integrity. Studies of mutant forms of Bcr with disruption of PDZK1 binding or kinase activity introduced into endothelial cells further revealed that Bcr-PDZK1 interaction and its kinase function are required for HDL activation of Akt and eNOS. Using a novel kinase assay that we recently developed that employs time-resolved Forster resonance energy transfer, we found that via SR-BI and PDZK1, HDL stimulates Bcr kinase activity in endothelial cells more than 20-fold. In addition, using Akt-based peptides in studies of the two known kinases for Akt, mTOR and PDK1, we determined that HDL activates Bcr kinase to directly phosphorylate Akt-Ser473 in an mTOR independent manner, and that Akt-Thr308 is a direct substrate of PDK1. These collective findings have identified Bcr to be a novel kinase for Akt, and they have revealed that Bcr is critically involved in HDL modulation of endothelial cell phenotype.

120 Blood flow is required for endothelial repair in a novel in vivo model using zebrafish embryos

IntroductionEndothelial repair is essential for vascular homeostasis but difficult to study in vivo using existing animal models. The role of blood flow in the repair response remains unclear. We therefore...

Active involvement of Robo1 and Robo4 in filopodia formation and endothelial cell motility mediated via WASP and other actin nucleation‐promoting factors

This study aimed to further elucidate the function of Roundabout proteins in endothelium. We show that both Robo1 and Robo4 are present in human umbilical vein endothelial cells (HUVECs) and have knocked expression down using small interfering RNA (siRNA) technology. Roundabout knockout endothelial cells were then studied in a variety of in vitro assays. We also performed a yeast 2-hybrid analysis using the intracellular domain of Robo4 as bait to identify interacting proteins and downstream signaling. Both Robo1 and Robo4 siRNA knockdown and transfection of Robo4-green fluorescent protein inhibited endothelial cell movement and disrupted tube formation on Matrigel. Consistent with a role in regulating cell movement, yeast 2-hybrid and glutathione-S-transferase pulldown analyses show Robo4 binding to a Wiskott-Aldrich syndrome protein (WASP), neural Wiskott-Aldrich syndrome protein, and WASP-interacting protein actin-nucleating complex. We have further shown that Robo1 forms a heterodimeric complex with Robo4, and that transfection of Robo4GFP into HUVECs induces filopodia formation. We finally show using Robo1 knockdown cells that Robo1 is essential for Robo4-mediated filopodia induction. Our results favor a model whereby Slit2 binding to a Robo1/Robo4 heterodimer activates actin nucleation-promoting factors to promote endothelial cell migration.

Adenovirus-Mediated Heme Oxygenase Transfer Inhibits Graft Arteriosclerosis in Rat Aortic Transplants

Purpose Heme oxygenase-1 (HO-1) has been implicated in graft protection because its induction is associated with long-surviving allografts. The aim of this study was to analyze the efficacy of adenovirus-mediated HO-1 overexpression to inhibit graft arteriosclerosis and estimate whether the protective role correlated with nuclear factor κB (NF = κB) expression and grafts apoptosis. Methods Aortic transplantation was performed using inbred Brown-Norway (BN) rats (RT 1n male) as donors and Lewis rats (RT 1l male) as recipients. The experiments were divided into four groups: group A, Lewis–Lewis ( n = 6), no treatment; group B, BN–Lewis ( n = 6), no treatment; group C, BN–Lewis ( n = 6), donor aorta perfused with buffer containing 4 × 10 10 pfu Ad-Null (the noncoding adenoviral vector); and group D, BN–Lewis ( n = 6), donor aorta perfused with buffer containing 4 × 10 10 pfu Ad-HO-1 (adenoviral vector coding for HO-1). All grafts were harvested at 60 days after transplantation. Reverse transcriptase polymerase chain reaction was performed to detect the expression of HO-1 gene. Hematoxylin and eosin staining was used to detect the morphology and cytology of the transplanted vessels. Intimal thickening was detected by Masson staining. Immunohistochemistry was used to detect the localization and expression of HO-1 and NF-κB and TUNEL assays to detect the apoptosis of the grafts. Results Gene transfer of HO-1 grafts using an adenoviral vector resulted in the expression of HO-1 protein in endothelium and adventitia. We observed that the intimal thickness in Ad-HO-1–treated aortas (11.11 ± 0.92 μm) was significantly thinner compared with untreated (85.20 ± 6.90 μm) or Ad-null treated (87.20 ± 6.20 μm) aortas ( P < .01). Immunohistology showed that treatment with Ad-HO-1 resulted in a significant reduction in leukocyte infiltration and a decreased number of vascular smooth muscle cells in the intima, compared with Ad-null–treated aortas. The levels of NF-κB and the number of apoptotic cells in Ad-HO-1–treated aortas showed significantly lower compared with Ad-null–treated arotas ( P < .05). Conclusion HO-1 prevented the development of graft arteriosclerosis in the rat aortic transplant model. The protective role of HO-1 in chronic rejection lesions seemed to correlate with downregulation of the expression of NF-κB and inhibition of apoptosis in the grafts.

Differential expression of vascular endothelial growth factor-A (VEGF-A) and VEGF-B after brain injury.

Our previous study demonstrated that vascular endothelial growth factor (VEGF), now referred to as VEGF-A, plays a significant role in blood-brain barrier (BBB) breakdown and angiogenesis after brain injury. In this study, VEGF-A expression was compared with that of VEGF-B in the rat cortical cold injury model over a period of 6 hours to 6 days post-injury. VEGF-A and VEGF-B mRNA were detected by in situ hybridization and their protein was detected by immunohistochemistry. The presence of VEGF-A and VEGF-B proteins in endothelium of lesion vessels was related to BBB breakdown by double labeling for either of these growth factors and fibronectin, which was used as a marker of BBB breakdown. Significant induction of both VEGF-A and VEGF-B mRNA occurred at the lesion site during the period of maximal endothelial proliferation. VEGF-A mRNA levels peaked at 3 and 4 days post-injury and returned to basal expression by day 6, while VEGF-B mRNA levels remained elevated up to day 6. VEGF-B protein was constitutively expressed in endothelium of all cerebral vessels. After brain injury, there was increased immunoreactivity for VEGF-B at the lesion site, this protein being present in the endothelium and vascular smooth muscle cells of pial vessels, in inflammatory cells, and later in proliferating endothelial cells, endothelium of neovessels, and astrocytes. Lesion vessels showing BBB breakdown to fibronectin showed endothelial VEGF-A protein but not VEGF-B protein. Constitutive expression of VEGF-B in normal endothelium suggests that it may have a role in maintenance of the BBB in steady states, while its induction at both the gene and protein level post-injury indicates that it has an essential role in angiogenesis and the repair processes after brain injury.

Characterization of the gap junction protein connexin37 in murine endothelium, respiratory epithelium, and after transfection in human HeLa cells

Affinity-purified antibodies to oligopeptides derived from two different regions of the carboxyterminus and cytoplasmic loop or to the last 103 C-terminal amino acids of mouse connexin37 (Cx37) were used to characterize expression of this gap junctional protein in endothelium of several murine tissues. Cx37 was expressed in endothelium of large blood vessels in brain, liver, kidney, spleen, heart, and lung, but not in capillaries. In addition, weak Cx37 immuno-signals were observed in lung respiratory epithelium of small bronchi and in alveolar epithelial cells of bronchioli. The ratios of Cx37 protein to Cx37 mRNA in adult and embryonic kidney as well as skin were 29-303-fold larger than in lung, suggesting that Cx37 mRNA was translated at different efficiencies in kidney and skin versus lung. Cx37 protein was more abundant in embryonic kidney and lung than in the corresponding adult tissues. After differential centrifugation of plasma membrane fractions in sucrose gradients, we found that Cx37-containing gap junctions in lung were much smaller than Cx32 and Cx26 aggregates from liver. HeLa cells were transfected with mouse Cx37 cDNA. In these cells, mouse Cx37 protein was phosphorylated mainly at serine, less at tyrosine, and very little at threonine residues. Three conductance states were resolved at 110, 240, and 315 pS.

The unique role of interferon-gamma in the regulation of MHC expression on arterial endothelium.

We examined the expression of MHC class I and II in the arterial endothelium of interferon-gamma (IFN-gamma, GKO) and IFN-gamma-R (IFN-gamma-R, GRKO) gene knockout mice in comparison with mice with intact IFN-gamma and IFN-gamma-R genes, BALB/c and 129Sv/J wild-type, respectively. The GKO and GRKO were produced by gene targeting. MHC class I and II expression was assessed by mAb binding to frozen tissue (kidney, spleen, heart, liver) sections by immunoperoxidase staining in the basal state and after various stimuli: allogeneic cells, oxazolone skin sensitization, LPS, and rIFN-gamma. As controls, we also examined the expression of two other IFN-gamma inducible genes present in the endothelium, Ly-6 and ICAM-1. We found that basal class I expression was present in the small arteries and arterioles of BALB/c and 129Sv/J wild-type mice but absent from arterial endothelium of GKO and GRKO mice. Class I was induced in the endothelium of BALB/c and 129Sv/J wild-type mice by three in vivo stimuli: allogeneic, LPS, and oxazolone, whereas class II was only induced after allogeneic stimulus. Administration of rIFN-gamma induced class I in the endothelium of GKO and BALB/c wild-type mice. The basal expression of Ly-6 and ICAM-1 was similar in the arteries of GKO and BALB/c wild-type mice, indicating that, the basal expression of these proteins in endothelium is IFN-gamma independent, unlike class I. In summary, basal class I expression in arterial endothelium is not constitutive as previously believed, but is dependent on basal IFN-gamma production. IFN-gamma has an essential role in the induction of class I and II expression in arterial endothelium. The fact that MHC class I is induced in endothelium may be useful therapeutically for reduction of immune recognition in transplantation.

Characterization of the ZO-1 protein in endothelial and other cell lines

A high molecular weight tight junction-associated protein, ZO-1, has been demonstrated in liver (hepatocytes) and in both epithelium and endothelium. We carried out studies to examine the presence of the protein in vascular endothelial cell cultures and several other types of cultured cells, and the relationship between the ZO-1 protein content and confluency of endothelial cell monolayers. Immunofluorescence labelling of endothelial monolayers and two types of epithelial monolayers, IEC-6 and MDCK, with monoclonal antibody against ZO-1 protein localized the protein to the cell peripheries. Its association with the cell periphery only occurred when cells had contact with one another as demonstrated in endothelial cells. We have been able to show a positive correlation between the ZO-1 content of the cells and the extent of monolayer confluency in the endothelial cells by immunoblotting. The protein is much less expressed in nonconfluent endothelial cell monolayers and totally absent from mouse myeloma cultures. The presence and confluence-related expression of the protein in endothelium give support to the hypothesis that tight junctions exist in confluent endothelial cells and that the ZO-1 protein is expressed under the conditions where tight junction interactions occur.