Expression Of Vascular Endothelial Cadherin Research Articles

Cyclophosphamide Pulse Therapy Normalizes Vascular Abnormalities in a Mouse Model of Systemic Sclerosis Vasculopathy

Intravenous cyclophosphamide pulse, a standard treatment for systemic sclerosis (SSc)-related interstitial lung disease, elicits a disease-modifying effect on SSc vasculopathy, such as fostering microvascular de-remodeling. To investigate the molecular mechanism by which cyclophosphamide mitigates SSc vasculopathy, we employed endothelial cell-specific Fli1 knockout mice that mimic the functional and structural vascular abnormalities characteristic of SSc. Biweekly cyclophosphamide injection improved vascular permeability and structural abnormalities of endothelial cell-specific Fli1 knockout mice in 2 weeks and in 3 months, respectively. In endothelial cell-specific Fli1 knockout mice, a single dose of cyclophosphamide was sufficient to normalize the decreased expression of α-smooth muscle actin in dermal blood vessels and improve the impaired neovascularization in skin-embedded Matrigel plug. Under the same condition, the decreased expression of vascular endothelial cadherin, platelet-derived growth factor B, S1P1, and CCN1 (molecules associated with angiogenesis and/or vasculogenesis) was reversed along with the reversal of endothelial Fli1 expression. In SSc patients, serum CCN1 levels were significantly increased after intravenous cyclophosphamide pulse. Taken together, these results indicate that cyclophosphamide improves Fli1 deficiency-dependent vascular changes by normalizing the expression of angiogenesis- and vasculogenesis-related molecules and endothelial Fli1, which may help to explain the beneficial effect of cyclophosphamide on SSc vasculopathy.

Aturanosides A and B, Glycosylated Anthraquinones with Antiangiogenic Activity from a Soil-Derived Streptomyces Species.

A chemical investigation of a culture extract from a soil-derived Streptomyces sp. RK88-1441 led to the isolation and characterization of two new glycosylated anthraquinones, aturanosides A (1) and B (2), and a new anthraquinone derivative, aturanocin (3). The structures of these compounds were elucidated by detailed NMR and MS spectroscopic analyses. The absolute configurations of the sugar units, based on the magnitudes of the coupling constants, ROESY correlations, and chemical derivatization, from 1 and 2 are 6- O-[ N-acetyl-α-d-glucosamino-(1→2)-α-l-rhamnoside] and 6- O-α-l-rhamnoside, respectively. Compounds 1 and 2 showed no cytotoxicity against human umbilical vein endothelial cells (HUVECs), but significantly suppressed vascular endothelial growth factor (VEGF)-induced tube formation and invasion of HUVECs. The down-regulation of both the phosphorylation of VEGF receptor 2 and the expression of vascular endothelial cadherin at the protein level were also observed.

Dynamic of VE-cadherin-mediated spermatid-Sertoli cell contacts in the mouse seminiferous epithelium.

Spermatids are haploid differentiating cells that, in the meantime they differentiate, translocate along the seminiferous epithelium towards the tubule lumen to be just released as spermatozoa. The success of such a migration depends on dynamic of spermatid-Sertoli cell contacts, the molecular nature of which has not been well defined yet. It was demonstrated that the vascular endothelial cadherin (VEC) is expressed transitorily in the mouse seminiferous epithelium. Here, we evaluated the pattern of VEC expression by immunohistochemistry first in seminiferous tubules at different stages of the epithelial cycle when only unique types of germ cell associations are present. Changes in the pattern of VEC localization according to the step of spermatid differentiation were analysed in detail using testis fragments and spontaneously released germ cells. Utilizing the first wave of spermatogenesis as an in vivo model to have at disposal spermatids at progressive steps of differentiation, we checked for level of looser VEC association with the membrane by performing protein solubilisation under mild detergent conditions and assays through VEC-immunoblotting. Being changes in VEC solubilisation paralleled in changes in phosphotyrosine (pY) content, we evaluated if spermatid VEC undergoes Y658 phosphorylation and if this correlates with VEC solubilisation and spermatid progression in differentiation. Altogether, our study shows a temporally restricted pattern of VEC expression that culminates with the presence of round spermatids to progressively decrease starting from spermatid elongation. Conversely, pY658-VEC signs elongating spermatids; its intracellular polarized compartmentalization suggests a possible involvement of pY658-VEC in the acquisition of spermatid cell polarity.

A Novel Choroidal Endothelial Cell Line Has a Decreased Affinity for the Age-Related Macular Degeneration–Associated Complement Factor H Variant 402H

Choroidal endothelial cells play a central role in the pathogenesis of age-related macular degeneration (AMD). Protocols for isolating primary choroidal endothelial cells have been described but require access to human donor eyes, which is a limiting factor. Therefore, a conditionally immortalized choroidal endothelial cell (ciChEnC) line has been established. Choroidal endothelial cells were selected by magnetic-activated cell sorting and conditionally immortalized using temperature-sensitive simian virus 40 large T antigen and human telomerase. The cell line obtained was characterized based on expression of endothelial marker proteins and endothelial cell-specific responses to various stimuli. Binding of AMD-associated and non-AMD variants of complement factor H in the context of a recombinant CCP6-8 (complement control protein domains 6-8) construct was determined using ELISA. ciChEnCs maintained morphology and von Willebrand factor and vascular endothelial cadherin expression for up to 27 passages. The cells internalized acetylated low-density lipoprotein, formed tubes on Matrigel, and increased intercellular adhesion molecule-1 expression in response to tumor necrosis factor-α. Cells grew into dense monolayers with barrier function and showed characteristics of choriocapillary cells, such as expression of plasmalemma vesicle-associated protein, human leukocyte antigen ABC, carbonic anhydrase IV, and membrane indentations reflecting fenestrations. ciChEnCs synthesized glycosaminoglycans chondroitin sulfate and the complement factor H ligand heparan sulfate. Interestingly, binding of the AMD-associated 402H variant of factor H to ciChEnC was significantly decreased compared to the 402Y variant. A novel ciChEnC cell line with choriocapillary characteristics has been established and should greatly facilitate investigation of the pathogenesis of AMD in the context of the choriocapillary microenvironment.

The Expression and Functional Significance of Vascular Endothelial-Cadherin, CD44, and Vimentin in Oral Squamous Cell Carcinoma.

Objectives:Ninety percent of head and neck cancers are squamous cell carcinoma which develops in the oral cavity. Metastasis is the main causative factor for death in 90% of all cancer-related deaths and begins with the invasion of tumor cells through the walls of small blood vessels or lymph vessels. A growing body of evidence has shown that vasculogenic mimicry (VM) facilitates tumor growth and cancer metastasis. The current study aimed to present the role of vascular endothelial (VE)-cadherin, CD44, and vimentin in inducing VM and epithelial-mesenchymal transition (EMT) and to identify the cancer stem cell (CSC) niche in different grades of oral squamous cell carcinoma (OSCC).Materials and Methods:A total of 63 OSCC samples (21 samples each grade) were collected from the archive of Pathology Department of Besat educational hospital, Hamadan, Iran, from 2000 to 2015. VE-cadherin, CD44, and vimentin/periodic acid–Schiff (PAS) double-staining were used to validate VM. VM was identified by the detection of PAS-positive loops surrounded by tumor cells. Chi-square test was used to examine the differences between the variables. Significant level was set at 0.05. Pearson's correlation was used to assess the co-localization of the markers.Results:There were statistically significant differences between tumor grade and the expression levels of VE-cadherin, CD44, and vimentin (P = 0.000). In addition, significant differences were found between tumor grade and microvessel density (P = 0.000) and between tumor grade and VM (P = 0.000).Conclusion:Our results may disclose a definite relationship between VE-cadherin, CD44 and vimentin expression levels, VM formation, EMT, CSCs, and microvessel count in OSCC samples. For this reason, it is suggested that VE-cadherin, CD44, and vimentin are related to angiogenesis and VM formation in OSCC, therefore, in tumor progression and metastasis. Recently, antitumor angiogenic therapies have been challenged. The presence of VM may explain the failure of antiangiogenic treatments.

Long Non-Coding RNA HOXA-AS2 Regulates Malignant Glioma Behaviors and Vasculogenic Mimicry Formation via the MiR-373/EGFR Axis

Background/Aims: Vasculogenic mimicry (VM) has been reported to be a novel glioma neovascularization process. Anti-VM therapy provides new insight into glioma clinical management. In this study, we revealed the role of the long non-coding RNA HOXA cluster antisense RNA 2 (HOXA-AS2) in malignant glioma behaviors and VM formation. Methods: Quantitative real-time PCR was performed to determine the expression levels of HOXA-AS2 in glioma samples and glioblastoma cell lines. CD34-periodic acid-Schiff dual-staining was performed to assess VM in glioma samples. CCK-8, transwell, and Matrigel tube formation assays were performed to measure the effects of HOXA-AS2 knockdown on cell viability, migration, invasion, and VM tube formation, respectively. RNA immunoprecipitation, dual-luciferase reporter and Western blot assays were performed to explore the molecular mechanisms underlying the functions of HOXS-AS2 in glioblastoma cells. A nude mouse xenograft model was used to investigate the role of HOXA-AS2 in xenograft glioma growth and VM density. Student’s t-tests, one-way ANOVAs followed by Bonferroni posthoc tests, and chi-square tests were used for the statistical analyses. Results: HOXA-AS2 was upregulated in glioma samples and cell lines and was positively correlated with VM. HOXA-AS2 knockdown attenuated cell viability, migration, invasion, and VM formation in glioma cells and inhibited the expression of vascular endothelial-cadherin (VE-cadherin), as well as the expression and activity of matrix metalloproteinase matrix metalloproteinase (MMP)-2 and MMP-9. miR-373 was downregulated in glioma samples and cell lines and suppressed malignancy in glioblastoma cells. HOXA-AS2 bound to miR-373 and negatively regulated its expression. Epidermal growth factor receptor (EGFR), a target of miR-373, increased the expression levels of VE-cadherin, as well as the expression and activity levels of MMP-2 and MMP-9, via activating phosphatidylinositol 3-kinase/serine/threonine kinase pathways. HOXA-AS2 knockdown combined with miR-373 overexpression yielded optimal tumor suppressive effects and the lowest VM density in vivo. Conclusion: HOXA-AS2 knockdown inhibited malignant glioma behaviors and VM formation via the miR-373/EGFR axis.

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression.

The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.

The role and mechanism of mesenchymal stem cell in modulating human pulmonary microvascule endothelial cell permeability via paracrine hepatocyte growth factor

Objective: To explore the role and mechanism of mesenchymal stem cell (MSC) in modulating human pulmonary microvascular endothelial cell (HPMECs) permeability via hepatocyte growth factor (HGF). Methods: The study introduced a co-cultured model between HPMECs and human mesenchymal stem cell conditioned media (MSC-CM) collected from MSCs after 24 h hypoxia culture, and meanwhile HGF was neutralized in MSC-CM by anti-HGF antibody respectively, followed by lipopolysaccharide (LPS) stimulation. Finally, the following measurements were performed: the permeability of HPMECs, the protein expression of vascular endothelial cadherin (VE-cadherin), Occludin in HPMECs by Western blot, HPMECs apoptosis by Annexin V-FITC/PI and HPMECs proliferation by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide(MTT). Results: Compared to LPS group (4.15±0.88), MSC-CM reduced endothelial paracellular permeability injured by LPS(1.56±0.36, P<0.01), however, the MSC-CM effect was significantly blocked by anti-HGF antibody(3.11±0.74, P<0.05). Furthermore MSC-CM significantly increased the expression of VE-cadherin(0.71±0.05 vs. 0.38±0.19, P<0.05)and Occludin protein(0.96±0.05 vs. 0.51±0.02, P<0.05) in HPMECs, which was significantly blocked by anti-HGF antibody (P<0.05). MSC-CM significantly reduced the number of early apoptotic cells (6.82±1.80 vs. 17.09±1.89, P<0.05). However, the effect of MSC-CM was significantly blocked by neutralizing HGF (12.07±0.98, P<0.01). The cell viability results by MTT assay confirmed that MSC-CM(6.82±1.80, P<0.05)restored cell viability to a greater extent than LPS stimulation only(0.47±0.09), and meanwhile the MSC-CM effect was significantly inhibited by neutralizing HGF from MSC-CM with anti-HGF antibody (0.69±0.29, P<0.05). Conclusion: HGF secreted by MSCs reduces endothelial cell paracelluar permeability induced by LPS, and the possible mechanisms include remodelling of endothelial intercellular adherence junction, promoting endothelial cell proliferation and restraining endothelial cell apoptosis.

Novel function of E26 transformation-specific domain-containing protein ELK3 in lymphatic endothelial cells.

Lymphatic endothelial cells (LEC) are major components of the tumor microenvironment and, due to the relative leakiness of lymphatic vessels compared with blood vessels, are essential for tumor dissemination and metastasis. In the present study, small interfering RNA-mediated suppression of E26 transformation-specific domain-containing protein Elk-3 (ELK3) inhibited the proliferation, migration and tube-forming ability of LEC. Suppression of ELK3 decreased vascular endothelial-cadherin expression levels and increased the phosphorylation of β-catenin. Furthermore, vascular endothelial growth factor receptor-3 (VEGFR-3) mRNA and protein expression levels were suppressed by the transfection of LEC with siELK3. As VEGFR-3 serves a major role in lymphangiogenesis, ELK3 may be a novel therapeutic target to inhibit tumor dissemination through the lymphatic system.

Identification of Antiangiogenic Potential and Cellular Mechanisms of Napyradiomycin A1 Isolated from the Marine-Derived Streptomyces sp. YP127

Angiogenesis is the process of new blood vessel formation. Excessive angiogenesis is a critical factor in the progression of cancer, macular degeneration, and other chronic inflammatory diseases. When investigating the effects of crude extracts of cultured marine microorganisms, an extract of the cultured Streptomyces sp. YP127 strain was found to inhibit human umbilical vein endothelial cell (HUVEC) tube formation. Bioassay-guided fractionation and spectroscopic data analyses led to the identification of napyradiomycin A1 (1) as an antiangiogenic component of the extract. Compound 1 inhibited HUVEC tube formation in a concentration-dependent manner. It inhibited endothelial cell proliferation but did not affect human dermal fibroblast proliferation. Compound 1 also suppressed migration and invasion of vascular endothelial cells. In addition, compound 1 suppressed vascular endothelial cadherin expression and increased the permeability of the endothelial cell membrane. These results suggested that compound 1 modulates cell permeability and inhibits the angiogenesis of endothelial cells.

SFLT-1 inhibits proliferation, migration, and invasion of colorectal cancer SW480 cells through vascular mimicry formation suppression

To investigate the effects of soluble fms-like tyrosine kinase-1 on the vascular mimicry formation, proliferation, migration, and invasion of colorectal cancer SW480 cells. The recombinant plasmid pBLAST49-sFLT-1 or pBLAST49 control plasmid was transfected into SW480 cells to obtain hsFLT-1-SW480 or Ctrl-SW480 cells. The three-dimensional model culture, sulforhodamine B assay, scratch assay, and Transwell assay were performed to detect the vascular mimicry formation, proliferation, migration, and invasion of colorectal cancer SW480 cells, respectively. Western blotting was used to detect the expression of vascular endothelial-cadherin protein. Compared with Ctrl-SW480 cells, vascular mimicry formation ((0.85 ± 0.04) vs (7.40 ± 0.69), p < 0.05) and vascular endothelial-cadherin expression ((1.25 ± 0.08) vs (1.89 ± 0.03), p < 0.05) were significantly decreased, and the growth rate was also significantly decreased in hsFLT-1-SW480 cells ((32.54 ± 5.12) vs (88.13 ± 11.52), p < 0.05). Moreover, the migration ((0.46 ± 0.08) vs (0.94 ± 0.03), p < 0.05) and invasion capacity ((59.14 ± 3.64) vs (134.85 ± 10.16), p < 0.05) of SW480 cells were significantly inhibited upon soluble fms-like tyrosine kinase-1 transfection. soluble fms-like tyrosine kinase-1 inhibits cell proliferation, migration, and invasion of colorectal cancer SW480 cells through suppression of vascular mimicry formation, which provides a good basis for the development of new drugs for the treatment of colorectal cancer by targeting both angiogenesis and vascular mimicry formation.

Neurons secrete miR-132-containing exosomes to regulate brain vascular integrity

Vascular integrity helps maintain brain microenvironment homeostasis, which is critical for the normal development and function of the central nervous system. It is known that neural cells can regulate brain vascular integrity. However, due to the high complexity of neurovascular interactions involved, understanding of the neural regulation of brain vascular integrity is still rudimentary. Using intact zebrafish larvae and cultured rodent brain cells, we find that neurons transfer miR-132, a highly conserved and neuron-enriched microRNA, via secreting exosomes to endothelial cells (ECs) to maintain brain vascular integrity. Following translocation to ECs through exosome internalization, miR-132 regulates the expression of vascular endothelial cadherin (VE-cadherin), an important adherens junction protein, by directly targeting eukaryotic elongation factor2kinase (eef2k). Disruption of neuronal miR-132 expression or exosome secretion, or overexpression of vascular eef2k impairs VE-cadherin expression and brain vascular integrity. Our study indicates that miR-132 acts as an intercellular signal mediating neural regulation of the brain vascular integrity and suggests that the neuronal exosome is a novel avenue for neurovascular communication.

Procalcitonin Impairs Endothelial Cell Function and Viability.

Procalcitonin is used as a diagnostic tool for the identification and risk stratification of septic patients. Procalcitonin plasma concentrations tightly correlate with the severity of the ongoing inflammatory reaction and can rise up to 10,000-fold. Impairment of endothelial cell function plays an important role in the pathogenesis of hypotension and disturbed organ perfusion during sepsis. We investigated the possible effects of procalcitonin itself on endothelial cell function and viability. Human endothelial cells were exposed to 0.01 to 100 ng/mL procalcitonin and investigated for endothelial permeability using transwells, migration in a scratch wound assay and new capillary formation on extracellular matrix in vitro. Tumor necrosis factor-α and vascular endothelial growth factor served as positive controls. Procalcitonin's impact on the response of endothelial cells toward ischemia was investigated in vivo in the murine model of unilateral femoral artery ligation. Procalcitonin-exposed endothelial cells were subjected to immunoblot for the investigation of vascular endothelial-cadherin expression and angiogenic signaling pathways. Flow cytometry was used for the detection of inflammatory activation and viability, and genomic analysis was performed. Data are presented as difference in means and 95% confidence intervals; statistical analyses were performed using analysis of variance/Bonferroni, and P values are reported as adjusted for multiple comparisons (Padjust). Tumor necrosis factor-α and 0.1 ng/mL procalcitonin induced endothelial barrier disruption after incubation of endothelial monolayers for 6 hours (-2.53 [-4.16 to -0.89], P = .0008 and -2.09 [-3.73 to -0.45], Padjust = .0064 compared with vehicle-treated control, respectively). Procalcitonin beginning at concentrations of 0.02 ng/mL reduced endothelial cell migration (0.26 [0.06 to 0.47], Padjust = .0069) and new capillary formation in vitro (0.47 [0.28 to 0.66], Padjust < .0001) contrasting the proangiogenic action of vascular endothelial growth factor. Left ventricular injection of procalcitonin in mice on postoperative day 1, 3, and 5 after induction of ischemia impaired new capillary formation and recovery of hindlimb perfusion in vivo (number of capillaries/mm in the ischemic leg of vehicle-treated versus procalcitonin-treated mice, 852.6 [383.4-1322], Padjust = .0002). Twenty-four-hour incubation with procalcitonin reduced the expression of vascular endothelial-cadherin at 100 ng/mL (0.39 [0.06-0.71], Padjust = .0167) and induced endothelial cell death (apoptosis, -5.4 [-10.67 to -0.13], Padjust = .0431). No alteration in the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 or extracellular signal-regulated kinase 1/2, and AKT signaling pathways was observed. Genomic analysis revealed regulation of a variety of genes involved in inflammation, angiogenesis, and cell growth. This study found that procalcitonin itself impaired several aspects of endothelial cell function. Procalcitonin-induced loss of endothelial barrier function may contribute to capillary leakage and therapy-refractory hypotension during sepsis. Anti-angiogenic properties of procalcitonin at low concentrations could also identify procalcitonin as a mediator of vascular disease associated with the metabolic syndrome. Future studies are needed to further test procalcitonin as a potential therapeutic target for preserving vascular dysfunction during acute and chronic inflammatory disorders.

Expression of Vascular Endothelial-Cadherin in Mucoepidermoid Carcinoma: Role in Cancer Development.

Objectives:Mucoepidermoid carcinoma (MEC) accounts for 35% of all malignant salivary gland tumors. Previous investigations have shown that vasculogenic mimicry (VM) exists in many cancers which can be used as a prognostic factor of poor prognosis. Elevated expression level of vascular endothelial (VE)-cadherin has been implicated in cancer neovascularization, growth, and progression. The current study aimed to study the presence of VE-cadherin in VM channels and tumor cells in different grades of MEC.Materials and Methods:A total of 63 MEC samples (21 samples in each grade) were collected from the archive of pathology department of Besat Educational Hospital, Hamadan, Iran, from 2002 to 2016. Hematoxylin and eosin staining was performed to confirm the previous diagnosis. The specimens were then processed for immunohistochemistry analysis. Then, periodic acid–Schiff staining was performed. Analyses were conducted through SPSS software version 22.0 (SPSS, Inc., Chicago, IL, USA). Chi-square test was used to examine the differences between categorical variables. Significance level was set at 0.05. Pearson's correlation was used to assess the co-localization of the marker.Results:A total of 63 samples (35 men; 55.6%, and 28 women; 44.4%) were used for immunohistochemical study. There were statistically significant differences between tumor grade and the expression levels of VE-cadherin (P = 0.000), between tumor grade and VM formation (P = 0.000), and also between tumor grade and microvessel density (MVD) (P = 0.000). Additionally, there was a strong positive correlation between tumor grade and VE-cadherin expression level (Pearson's r = 0.875, P < 0.000).Conclusions:Our results may disclose a definite relationship between VE-cadherin expression level, VM, epithelial–mesenchymal transition, cancer stem cells, and MVD in MEC samples. Thus, it is reasonable to suggest that VE-cadherin is related to angiogenesis and VM formation in MECs.

Effects of diabetes and cardiopulmonary bypass on expression of adherens junction proteins in human peripheral tissue

We investigated the changes in adherens junction proteins, such as vascular endothelial-cadherin and β-catenin, of skeletal muscle and vessels in patients with or without diabetes in the setting of cardiopulmonary bypass and cardiac operation. Skeletal muscle tissue samples were harvested pre- and post-cardiopulmonary bypass from nondiabetic (hemoglobin A1c: 5.4±0.1), controlled diabetic (hemoglobin A1c: 6.3±0.1), and uncontrolled diabetic patients (hemoglobin A1c: 9.6±0.3) undergoing coronary artery bypass grafting operation (n=8 per group). The expression/phosphorylation of adherens junction proteins vascular endothelial-cadherin and β-catenin were assessed by immunoblotting and immuno-histochemistry. Endothelial function of skeletal muscle arterioles was determined by videomicroscopy in response to the vasodilator substance P. The protein expression of total vascular endothelial-cadherin was not changed at baseline or between pre-and post-cardiopulmonary bypass among groups. The pre-cardiopulmonary bypass level of phospho-vascular endothelial-cadherin was found to be significantly increased in the uncontrolled diabetic patients group compared with the nondiabetic or controlled diabetic groups (P<.05). The post-cardiopulmonary bypass levels of phospho-vascular endothelial-cadherin were significantly increased compared with pre-cardiopulmonary bypass in all groups (P<.05 each), and this increase was greater in the uncontrolled diabetic patients group than that of the nondiabetic or controlled diabetic groups (P<.05). Expression of basal β-catenin protein in the uncontrolled diabetic group was decreased compared with nondiabetic or controlled diabetic groups (P<.05). There were significant decreases in the β-catenin protein expression between pre- and post-cardiopulmonary bypass in all 3 groups (P<.05 each), and this decrease was greater in the uncontrolled diabetic patients group than the nondiabetic group (P<.05). There were decreases in the relaxation response of skeletal muscle arterioles to substance P after cardiopulmonary bypass in all 3 groups (P<.05), and this alteration was more pronounced in the uncontrolled diabetic patients (P<.05). Uncontrolled diabetes causes inactivation and reduction in the expression of endothelial adherens junction proteins in the arterioles of skeletal muscle early after cardiopulmonary bypass. The enhanced phosphorylation of vascular endothelial-cadherin and degradation of β-catenin indicate deterioration of these proteins and damage of the cell-cell endothelial junctions, specifically in the diabetic peripheral vessels. These alterations may contribute to the increases in peripheral vascular permeability and endothelial dysfunction.

Tumour cell-derived WNT5B modulates in vitro lymphangiogenesis via induction of partial endothelial-mesenchymal transition of lymphatic endothelial cells

Metastasis of the cervical lymph nodes frequently leads to poor survival of patients with oral squamous cell carcinoma (OSCC). The underlying mechanisms of lymph node metastasis are unclear. Wingless-type MMTV integration site family, member 5B (WNT5B), one component of the WNT signal pathway, was markedly up-regulated in OSCC sublines with high potential of lymphatic metastasis compared to that in OSCC cells with low nodal metastasis. Increased WNT5B mRNA was demonstrated in human OSCC tissues in comparison with adjacent non-tumorous tissues. Interestingly, the high level of WNT5B protein in serum was associated with lymph node metastasis in OSCC patients. Knockdown of WNT5B expression in OSCC sublines did not affect tumour growth but impaired lymph node metastasis and tumour lymphangiogenesis of orthotopic transplantation. Conditioned medium from WNT5B knockdown cells reduced the tube formation of lymphatic endothelial cells (LECs). In contrast, recombinant WNT5B enhanced the tube formation, permeability and migration of LECs. In LECs stained with phalloidin, the morphology of those treated with recombinant WNT5B changed from flat to spindle-like. Recombinant WNT5B also increased α-smooth muscle actin and inhibited the expression of vascular endothelial-cadherin but retained characteristics of endothelial cells. The results suggest that WNT5B functions in the partial endothelial-mesenchymal transition (EndoMT). Furthermore, WNT5B-induced tube formation was impaired in the LECs following the knockdown of EndoMT-related transcription factor, SNAIL or SLUG. The WNT5B-induced expression of Snail or Slug was abolished by IWR-1-endo and Rac1 inhibitors, which are involved in the WNT/β-catenin and planar cell polarity pathways, respectively. Collectively, the data suggest that WNT5B induces tube formation by regulating the expression of Snail and Slug proteins through activation of canonical and non-canonical WNT signalling pathways.

Protective Effects of Imatinib on Ischemia/Reperfusion Injury in Rat Lung

Ischemia/reperfusion injury (IRI) remains a significant complication after lung transplantation. Endothelial damage and inflammation contribute to its development. Imatinib has been reported to regulate vascular permeability by maintaining endothelial junctions and showing antiinflammatory effects through inhibition of the Abl kinases. We hypothesized that imatinib could have a protective effect against IRI. Male Lewis rats were heparinized and underwent left thoracotomy, and the left hilum was clamped for 90 minutes followed by reperfusion for 120 minutes. Imatinib mesylate (50 mg/kg) and a solvent were administered intraperitoneally 20 minutes before ischemia in the imatinib group and the vehicle group, respectively (n= 7 in each group). After reperfusion, lung function, lung wet to dry weight (W/D) ratio, and histologic findings were obtained. The expression of vascular endothelial cadherin (VEC), the phosphorylation level of CrkL (pCrkL) (an exclusive target of Abl kinases), and the cytokine level were evaluated using lung tissue lysate. The imatinib concentrations of plasma and lungs after reperfusion were measured in this hilar clamp model (n= 7). In the imatinib group, lung function was improved with a lower W/D ratio. Perivascular edema and neutrophil infiltration were ameliorated. The imatinib group demonstrated maintained expression of VEC, inhibition of pCrkL, and a significantly higher level of interleukin (IL)-10. The imatinib concentration in both lungs showed a strong correlation with plasma concentration. In a rat IRI model, imatinib attenuated lung injury by an antipermeability and antiinflammatory effect. The delivery and function of imatinib in the lung was also confirmed in this model.

Role of β-arrestin-1 in inhibition of endoxin-induced activation of MAPK signaling pathway in pulmonary microvascular endothelial cells by penehyclidine hydrochloride

Objective To investigate the role of β-arrestin-1 in inhibition of endoxin-induced activation of MAPK signaling pathway in pulmonary microvascular endothelial cells (PMVECs) by penehyclidine hydrochloride (PHC). Methods Human PMVECs were seeded in 6-well plates (2 ml/well) or in culture flasks (4 ml/flask) at the density of 1×105 cells/ml, and randomly divided into 5 groups (n=20 each) using a random number table: empty plasmid transfection group (group C), lipopolysaccharide (LPS) + empty plasmid transfection group (LPS group), PHC+ LPS+ empty plasmid transfection group (P+ LPS group), LPS+ β-arrestin-1 short hairpin RNA (shRNA) transfection group (LPS+ shRNA group), and PHC + LPS+ β-arrestin-1 shRNA transfection group (P+ LPS+ shRNA group). After the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA, the cells were incubated for 24 h. At 24 h of incubation, LPS with the final concentration of 0.1 μg/ml was added, and the cells were then incubated for 1 h in LPS and LPS+ shRNA groups.In P+ LPS and P+ LPS+ shRNA groups, PHC with the final concentration of 2 μg/ml was added, and the cells were incubated for 1 h, and then LPS with the final concentration of 0.1 μg/ml was added, and the cells were incubated for 1 h. The expression of filamentous actin (F-actin) was detected by flow cytometry.The expression of myosin light chain kinase (MLCK) and vascular endothelial-cadherin (VE-cadherin) was detected by immunofluorescence.The expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated c-Jun N-terminal kinase (p-JNK) was determined by Western blot.The expression of β-arrestin-1 mRNA was determined by real-time polymerase chain reaction. Results Compared with group C, the expression of F-actin, VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated, and the expression of MLCK, p-ERK1/2 and p-JNK was up-regulated in group LPS, and the expression of p-ERK1/2 and p-JNK was significantly up-regulated (P 0.05). Compared with group LPS, the expression of F-actin, VE-cadherin and β-arrestin-1 mRNA was significantly up-regulated, and the expression of MLCK, p-ERK1/2 and p-JNK was down-regulated in group P+ LPS, and the expression of F-actin, VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated, and the expression of MLCK and p-JNK was up-regulated in group LPS+ shRNA (P<0.05). Compared with group P+ LPS, the expression of F-actin, VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated, and the expression of MLCK, p-ERK1/2 and p-JNK was up-regulated in group P+ LPS+ shRNA (P<0.05). Conclusion The mechanism by which PHC inhibits endoxin-induced activation of MAPK signaling pathway in PMVECs is partially related to up-regulation of β-arrestin-1 expression. Key words: Arrestins; Cholinergic antagonists; Endotoxins; Lung; Capillaries; Endothelial cells; Extracellular signal-regulated MAP kinases; JNK mitogen-activated protein kinases

Vascular Endothelial-Cadherin Expression After Reperfusion Correlates With Lung Injury in Rat Lung Transplantation

Vascular endothelial-cadherin (VEC), composing the adherens junction of endothelial cells, has been shown to regulate vascular permeability. The aim of this study was to investigate VEC expression during cold ischemia (CI) and reperfusion and whether a dibutyryl cyclic adenosine 3',5'-monophosphate (db-cAMP) and nitroglycerin (NTG) additive to preservation solution can maintain VEC. Rat left lung transplantation after CI was performed, and reperfusion was done for 2 hours. The experimental groups were Control (n= 5), CI3 (n= 5), CI6 (n= 6), and CI6+AMP/NTG (n= 6) with minimum ischemic period, 3 hours and 6 hours of CI, respectively, and 6 hours of CI with db-cAMP and NTG additive to preservation solution, respectively. Lung mechanics, lung wet-to-dry weight ratio (W/D), and the histologic findings of the graft were evaluated. The expression of VEC was evaluated by Western blot analysis of the lung tissue lysates. The CI3 group showed a decrease in dynamic compliance with perivascular edema. Dynamic compliance, graft oxygenation, and W/D were substantially deteriorated, and intraalveolar edema with neutrophil infiltration was recognized in the CI6 group. They were improved in the CI6+AMP/NTG group. VEC expression was maintained during CI. After reperfusion, it reduced substantially in the CI6 group and was maintained in the CI6+AMP/NTG group. VEC expression was maintained during CI; however, it reduced early after reperfusion after 6 hours of CI, correlating with severe intraalveolar edema. db-cAMP/NTG additive to the preservation solution contributed to the maintenance of VEC expression after reperfusion.

Effects of NOX1 on fibroblastic changes of endothelial cells in radiation‑induced pulmonary fibrosis.

Lung fibrosis is a major complication in radiation-induced lung damage following thoracic radiotherapy, while the underlying mechanism has remained to be elucidated. The present study performed immunofluorescence and immunoblot assays on irradiated human pulmonary artery endothelial cells (HPAECs) with or without pre-treatment with VAS2870, a novel NADPH oxidase (NOX) inhibitor, or small hairpin (sh)RNA against NOX1, -2 or -4. VAS2870 reduced the cellular reactive oxygen species content induced by 5 Gy radiation in HPAECs and inhibited phenotypic changes in fibrotic cells, including increased alpha smooth muscle actin and vimentin, and decreased CD31 and vascular endothelial cadherin expression. These fibrotic changes were significantly inhibited by treatment with NOX1 shRNA, but not by NOX2 or NOX4 shRNA. Next, the role of NOX1 in pulmonary fibrosis development was assessed in the lung tissues of C57BL/6J mice following thoracic irradiation using trichrome staining. Administration of an NOX1-specific inhibitor suppressed radiation-induced collagen deposition and fibroblastic changes in the endothelial cells (ECs) of these mice. The results suggested that radiation-induced pulmonary fibrosis may be efficiently reduced by specific inhibition of NOX1, an effect mediated by reduction of fibrotic changes of ECs.