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The genetic factors of attention deficit hyperactivity disorder (ADHD) are far from fully elucidated. This study aims to get additional insight into the genetic structure of ADHD.First, a transcriptome-wide association study and summary data-based Mendelian randomization analysis were performed to identify ADHD susceptibility genes. Then, genetic variants influencing the expression of the identified susceptibility genes were tested for association with ADHD risk in a sample of Han Chinese children (543 cases and 560 controls). Dual-luciferase reporter gene assays and electrophoretic mobility shift assays were performed to verify the transcriptional regulatory functions of the identified ADHD-associated variants. Additionally, real-time quantitative polymerase chain reaction was applied to quantify the expression levels of target genes in blood samples.Both TIE1 and MED8 were identified as ADHD susceptibility genes. Furthermore, we first found the G allele of rs3768046 was significantly associated with an increased risk of ADHD (recessive model: GG vs AA+AG, OR= 1.659, 95% CI= (1.262, 2.181); additive model: GG vs GA vs AA, OR= 1.493, 95% CI= (1.179, 1.890)). Additionally, in vitro functional experiments revealed that rs3768046 might alter TIE1 expression by affecting the binding sites of transcription factors. Moreover, the expression level of TIE1 in the blood samples of patients was significantly higher than that of controls.Given the moderate statistical power of this study, it is necessary to verify our findings in other larger samples.Together, this study presents the first systematic evidence of TIE1 with potential implications for the genetic basis of ADHD.

Background: Schlemm’s canal (SC) is a large vessel residing in the iridocorneal angle and is required to regulate aqueous humor outflow. Normal SC structure and function is indispensable for maintaining normal intraocular pressure, and elevated intraocular pressure is a risk factor for development of glaucoma. Recent reports have identified a key role of the angiopoietin-Tie2 pathway for SC development and function; however, the role of the orphan receptor Tie1 has not been clarified. Methods: We used Tie1 knock out mice to study the function of Tie1 in SC development and function. Real-time quantitative polymerase chain reaction and Western blot analyses were used to verify Tie1 deletion. High-resolution microscopy of mouse SC whole mount and cross sections were used to study SC morphology. Measurement of intraocular pressure in live mice was used to study the impact of Tie1 on SC function. Results: Tie1 is highly expressed in both human and mouse SC. Tie1 knock out mice display hypomorphic SC and elevated intraocular pressure as a result of attenuated SC development. Conclusions: Tie1 is indispensable for SC development and function, supporting it as a novel target for future SC-targeted glaucoma therapies and a candidate gene for glaucoma in humans.

Anti-angiogenic approaches have significantly advanced the treatment of vascular-related pathologies. The ephemeral outcome and known side effects of the current vascular endothelial growth factor (VEGF)-based anti-angiogenic treatments have intensified research on other growth factors. The angiopoietin/Tie (Ang/Tie) family has an established role in vascular physiology and regulates angiogenesis, vascular permeability, and inflammatory responses. The Ang/Tie family consists of angiopoietins 1-4, their receptors, tie1 and 2 and the vascular endothelial-protein tyrosine phosphatase (VE-PTP). Modulation of Tie2 activation has provided a promising outcome in preclinical models and has led to clinical trials of Ang/Tie-targeting drug candidates for retinal disorders. Although less is known about the role of Ang/Tie in pulmonary disorders, several studies have revealed great potential of the Ang/Tie family members as drug targets for pulmonary vascular disorders as well. In this review, we summarize the functions of the Ang/Tie pathway in retinal and pulmonary vascular physiology and relevant disorders and highlight promising drug candidates targeting this pathway currently being or expected to be under clinical evaluation for retinal and pulmonary vascular disorders.

Preeclampsia (PE) is a huge threat to pregnant women. Our previous study demonstrated that long non-coding RNA (lncRNA) NR_002794 was highly expressed in placentas of PE patients and could regulate the phenotypes of trophoblast cells. However, the downstream regulatory mechanisms of NR_002794 remain unknown. In this text, some potential downstream targets or signaling pathways of NR_002794 were identified through RNA sequencing (RNA-seq) and bioinformatics analysis in SWAN71 trophoblast cells. Western blot assay demonstrated that NR_002794 inactivated protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways and activated cell apoptotic signaling in SWAN71 cells. Both RNA-seq and reverse transcription-quantitative PCR (RT-qPCR) outcomes showed that NR_002794 up-regulation could notably inhibit the expression of C-C motif chemokine ligand 4 like 2 (CCL4L2), interleukin 15 receptor subunit alpha (IL15RA), interleukin 32 (IL32), and tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), while NR_002794 knockdown induced these gene expressions in SWAN71 cells. CCK-8, BrdU, Transwell, wound healing, and flow cytometry analyses showed that NR_002794 inhibited cell proliferation and migration and induced cell apoptosis through down-regulating TIE1 in SWAN71 cells. In conclusion, lncRNA NR_002794 could exert its functions by regulating AKT and ERK1/2 pathways and TIE1 expression in human trophoblast cells.

The human signaling molecules Tie1 and Tie2 receptor tyrosine kinases (RTKs) play important pathophysiological roles in many diseases, including different cancers. The activity of Tie1 is mediated mainly through the downstream angiopoietin-1 (Ang1)-dependent activation of Tie2, rendering both Tie 1 and the Tie1/Tie2/Ang1 axis attractive putative targets for therapeutic intervention. However, the development of inhibitors that target Tie1 and an understanding of their effect on Tie2 and on the Tie1/Tie2/Ang1 axis remain unfulfilled tasks, due, largely, to the facts that Tie1 is an orphan receptor and is difficult to produce and use in the quantities required for immune antibody library screens. In a search for a selective inhibitor of this orphan receptor, we sought to exploit the advantages (e.g., small size that allows binding to hidden epitopes) of non-immune nanobodies and to simultaneously overcome their limitations (i.e., low expression and stability). We thus performed expression, stability, and affinity screens of yeast-surface-displayed naïve and predesigned synthetic (non-immune) nanobody libraries against the Tie1 extracellular domain. The screens yielded a nanobody with high expression and good affinity and specificity for Tie1, thereby yielding preferential binding for Tie1 over Tie2. The stability, selectivity, potency, and therapeutic potential of this synthetic nanobody were profiled using in vitro and cell-based assays. The nanobody triggered Tie1-dependent inhibition of RTK (Tie2, Akt, and Fak) phosphorylation and angiogenesis in endothelial cells, as well as suppression of human glioblastoma cell viability and migration. This study opens the way to developing nanobodies as therapeutics for different cancers associated with Tie1 activation.

INTRODUCTION: Severe hemorrhage (Hem) has been shown to be causal for the development of extra-pulmonary/indirect acute respiratory distress syndrome (iARDS) and is associated with severe endothelial cell (EC) injury. EC growth factors, Angiopoietin (Ang)-1 and -2, maintain vascular homeostasis via tightly regulated competitive interaction with the tyrosine kinase receptor, Tie2, expressed on ECs. OBJECTIVE: Since it has been reported that the orphan receptor, Tie1, may be able to play a role in Ang:Tie2 signaling; we chose to examine Tie1’s capacity to alter the lung Ang:Tie2 interaction in response to the sequential insults of shock/sepsis (cecal ligation and puncture [CLP]), culminating in iARDS. METHODS: Male mice were subjected to Hem alone or sequential Hem followed 24 hours late by CLP that induces iARDS. Changes in lung and/or plasma levels of Tie1, Tie2, Ang-1, Ang-2, various systemic cytokine/chemokines and indices of lung injury/inflammation were then determined. The role of Tie1 was established by intravenous administration of Tie1 specific or control siRNA at 1 hour post-Hem. Alternatively, the contribution of neutrophils was assessed by pre-treating mice with anti-neutrophil antibody depletion 48 hours prior to Hem. RESULTS: Lung tissue levels of Tie1 expression elevated over the 1(st) 6–24 hours post-Hem alone. Subsequently, we found that treatment of Hem/CLP mice with Tie1-specific siRNA not only decreased Tie1 expression in lung tissue compared to control siRNA, but, suppressed the rise in lung inflammatory cytokines, lung MPO and the rise in lung protein leak. Finally, much as we have previously shown that neutrophil interaction with resident pulmonary vascular ECs contribute significantly to Ang-2 release and EC dysfunction, central to the development of iARDS. Here we report that depletion of neutrophils also decreased lung tissue Tie1 expression and increased Tie2 activation in Hem/CLP mice. CONCLUSION: Together, these data imply that shock-induced increased expression of Tie1 can contribute to EC activation by inhibiting Ang:Tie2 interaction, culminating in EC dysfunction and the development of iARDS.

Gliomas are highly malignant nervous system tumours. Studies shown that cancer stem cells are one of the main reasons underlying recurrence, metastasis, and poor prognosis in glioma cases. Our previous studies have found that superparamagnetic iron oxide nanoparticles (SPIONs) can act as nucleic acid carriers to drive intracellular overexpression of these nucleic acids. In this study, CD44+/CD133+ glioma stem cells (HuGSCs) were first isolated from surgically resected tissues from patients. qPCR and western blot results showed that Tie1 expression in HuGSCs was significantly higher thanexpression in CD44-/CD133- glioma cells. Bioinformatic analysis and luciferase reporter assays showed that miR-485-5p binds to specific loci on the 3'-UTR of Tie1 mRNA to inhibit Tie1 expression. Subsequently, miR-485-5p/miR-mut and SPION complexes were transfected into HuGSCs. Transmission electron microscopy showed that a highly dense metallic electron cloud is present in HuGSCs. At the same time, in vivo and in vitro studies showed that miR-485-5p@SPIONs can significantly inhibit HuGSC proliferation, invasion, tumourigenicity, and angiogenesis. In-depth analysis showed that Tie1 interacts with neuronal growth factors such as FGF2, BDNF, GDNF, and GFAP. qPCR and western blot results showed that in miR-485-5p@SPIONs-HuGSCs, the expression levels of Tie1 and stem cell markers (Oct4, Sox2, Nanog, CD44, and CD133), and even FGF2, BDNF, GDNF, and GFAP were significantly lower than thelevels in the control group (miR-mut@SPIONs-HuGSCs). Therefore, this study showedthat Tie1 is an important factor that maintains glioma stem cell activity. SPIONs drive miR-485-5p overexpression in cells and inhibit endogenous Tie1 expression to downregulate the protein expression levels of Fgf2/GDNF/GFAP/BDNF and significantly weaken the in vivo and in vitro viability of gliomas.

Objective: To investigate whether serum Tie-1 (sTie-1) is a valuable marker for predicting progression and prognosis of cervical cancer.Methods: Enzyme-linked immunosorbent assay (ELISA) was used to detect serum sTie-1 concentrations in 75 cervical cancer patients, 40 cervical intraepithelial neoplasia (CIN) patients, and 55 healthy controls without cervical lesions, and sTie-1 levels were compared between the groups. Receiver operating characteristic curves was used to evaluate the diagnostic value of sTie-1. The relationship between sTie-1 concentrations in patients with cervical cancer and clinicopathological features and prognosis were analyzed, and the risk factors for postoperative recurrence were determined using univariate and multivariable Cox proportional hazards regression.Results: We found that sTie-1 concentrations gradually increased according to lesion severity (i.e., cancer vs. CIN; p < 0.05) and were significantly elevated in adenocarcinoma compared with healthy controls. sTie-1 levels strongly distinguished between cervical cancer patients and the healthy controls (area under the curve = 0.846; cut-off value = 1,882.64 pg/ml; sensitivity = 74.6%; specificity = 96.4%). Moreover, sTie-1 levels in cervical cancer patients were significantly associated with tumor size, advanced tumor stage, lymph node metastasis, and reduced 4-years progression-free survival. Cervical cancer patients with high sTie-1 concentrations had a 3.123-fold [95% confidence interval (CI): 1.087–8.971, p = 0.034] higher risk for tumor recurrence.Conclusions: Elevated sTie-1 levels in patients with cervical carcinoma were associated with tumor progression and poor prognosis, indicating that sTie-1 may be a valuable marker for predicting progression and prognosis of cervical cancer.

The mechanisms regulating endothelial cell response to hemodynamic forces required for heart valve development, especially valve remodeling, remain elusive. Tie1, an endothelial specific receptor tyrosine kinase, is up-regulated by oscillating shear stress and is required for lymphatic valve development. In this study, we demonstrate that valvular endothelial Tie1 is differentially expressed in a dynamic pattern predicted by disturbed flow during valve remodeling. Following valvular endocardial specific deletion of Tie1 in mice, we observed enlarged aortic valve leaflets, decreased valve stiffness and valvular insufficiency. Valve abnormalities were only detected in late gestation and early postnatal mutant animals and worsened with age. The mutant mice developed perturbed extracellular matrix (ECM) deposition and remodeling characterized by increased glycosaminoglycan and decreased collagen content, as well as increased valve interstitial cell expression of Sox9, a transcription factor essential for normal ECM maturation during heart valve development. This study provides the first evidence that Tie1 is involved in modulation of late valve remodeling and suggests that an important Tie1-Sox9 signaling axis exists through which disturbed flows are converted by endocardial cells to paracrine Sox9 signals to modulate normal matrix remodeling of the aortic valve.

Background Although cigarette smoking is considered one of the key risk factors for lung cancer, 15% of male patients and 53% of female patients with lung cancer are non-smokers. Metabolic changes are critical features of cancer. Therapeutic target identification from a metabolic perspective in non-small cell lung cancer (NSCLC) tissue of female non-smokers has long been ignored. Results Based on microarray data retrieved from Affymetrix expression arrays E-GEOD-19804, we found that the downregulated genes in non-smoking female NSCLC patients tended to participate in protein/amino acid and lipid metabolism, while upregulated genes were more involved in protein/amino acid and carbohydrate metabolism. Combining nutrient metabolic co-expression, protein-protein interaction network construction and overall survival assessment, we identified NR4A1 and TIE1 as potential therapeutic targets for NSCLC in female non-smokers. To accelerate the drug development for non-smoking female NSCLC patients, we identified nilotinib as a potential agonist targeting NR4A1 encoded protein by molecular docking and molecular dynamic stimulation. We also show that nilotinib inhibited proliferation and induced senescence of cells in non-smoking female NSCLC patients in vitro. Conclusions These results not only uncover nutrient metabolic characteristics in non-smoking female NSCLC patients, but also provide a new paradigm for identifying new targets and drugs for novel therapy for such patients.

International audience; Abstract Endothelial–mesenchymal transition (EndMT) is an important source of cancer-associated fibroblasts (CAFs), which facilitates tumour progression. PDAC is characterised by abundant CAFs and tumour necrosis factor-α (TNF-α). Here, we show that TNF-α strongly induces human endothelial cells to undergo EndMT. Interestingly, TNF-α strongly downregulates the expression of the endothelial receptor TIE1, and reciprocally TIE1 overexpression partially prevents TNF-α-induced EndMT, suggesting that TNF-α acts, at least partially, through TIE1 regulation in this process. We also show that TNF-α-induced EndMT is reversible. Furthermore, TNF-α treatment of orthotopic mice resulted in an important increase in the stroma, including CAFs. Finally, secretome analysis identified TNFSF12, as a regulator that is also present in PDAC patients. With the aim of restoring normal angiogenesis and better access to drugs, our results support the development of therapies targeting CAFs or inducing the EndMT reversion process in PDAC.

Tie1 is a receptor tyrosine kinase expressed in endothelial cells, where it modulates Angiopoietin/Tie2 signaling. Previous studies have shown that mouse Tie1 mutants exhibit severe cardiovascular defects; however, much remains to be learned about the role of Tie1, especially during cardiac development. To further understand Tie1 function, we generated a zebrafish tie1 mutant line. Homozygous mutant embryos display reduced endothelial and endocardial cell numbers and reduced heart size. Live imaging and ultrastructural analyses at embryonic stages revealed increased cardiac jelly thickness as well as cardiomyocyte defects, including a loss of sarcomere organization and altered cell shape. Transcriptomic profiling of embryonic hearts uncovered the downregulation of tll1, which encodes a Tolloid-like protease, in tie1-/- compared with wild-type siblings. Using mRNA injections into one-cell stage embryos, we found that tll1 overexpression could partially rescue the tie1 mutant cardiac phenotypes including the endocardial and myocardial cell numbers as well as the cardiac jelly thickness. Altogether, our results indicate the importance of a Tie1-Tolloid-like 1 axis in paracrine signaling during cardiac development.

Platinum resistance is one of the most challenging problems in ovarian cancer treatment. High-throughput functional siRNA screening identified tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE-1) as a gene that confers cells resistant to cisplatin. Conversely enforced over-expression of TIE-1 was validated to decrease cisplatin sensitivity in multiple ovarian cancer cell lines and up-regulation of TIE-1 was correlated with poor prognosis and cisplatin resistance in patients with ovarian cancer. Mechanistically, TIE-1 up-regulates the nucleotide excision repair (NER) system mediated by xeroderma pigmentosum complementation group C (XPC), thereby leading to decreased susceptibility to cisplatin-induced cell death without affecting cisplatin uptake and excretion. Importantly potentiation of therapeutic efficacy by TIE-1 inhibition was selective to DNA-adduct-type chemotherapeutic platinum reagents. Therefore, TIE-1 is suggested to promote XPC-dependent NER, rendering ovarian cancer cells resistant to platinum. Accompanied with novel findings, TIE-1 could represent as a novel therapeutic target for platinum-resistant ovarian cancer.

Supplemental Digital Content is available in the text., Objective— Tie1 (tyrosine kinase containing immunoglobulin and epidermal growth factor homology 1), an endothelial and hematopoietic cell–specific receptor tyrosine kinase, is an important regulator of angiogenesis and critical for maintaining vascular integrity. The post-transcriptional regulation of tie1 mRNA expression is not understood, but it might partly explain Tie1’s differential expression pattern in endothelium. Following up on our previous work that identified natural antisense transcripts from the tie1 locus—tie1 antisense (tie1AS), which regulates tie1 mRNA levels in zebrafish—we attempted to identify the mechanism of this regulation. Approach and Results— Through in vitro and in vivo ribonucleoprotein binding studies, we demonstrated that tie1AS long noncoding RNA interacts with an RNA binding protein—embryonic lethal and abnormal vision Drosophila-like 1 (Elavl1)—that regulates tie1 mRNA levels. When we disrupted the interaction between tie1AS and Elavl1 by using constitutively active antisense morpholino oligonucleotides or photoactivatable morpholino oligonucleotides, tie1 mRNA levels increased between 26 and 31 hours post-fertilization, particularly in the head. This increase correlated with dilation of primordial midbrain channels, smaller eyes, and reduced ventricular space. We also observed these phenotypes when we used CRISPR (clustered regularly interspaced short palindromic repeats)–mediated CRISPRi (CRISPR-mediated interference) to knock down tie1AS. Treatment of the morpholino oligonucleotide–injected embryos with a small molecule that decreased tie1 mRNA levels rescued all 3 abnormal phenotypes. Conclusions— We identified a novel mode of temporal and spatial post-transcriptional regulation of tie1 mRNA. It involves long noncoding RNA, tie1AS, and Elavl1 (an interactor of tie1AS).

The endothelial tyrosine kinase receptor Tie1 remains poorly characterized, largely owing to its orphan receptor status. Global Tie1 inactivation causes late embryonic lethality, thereby reflecting its importance during development. Tie1 also plays pivotal roles during pathologies such as atherosclerosis and tumorigenesis. In order to study the contribution of Tie1 to tumor progression and metastasis, we conditionally deleted Tie1 in endothelial cells at different stages of tumor growth and metastatic dissemination. Tie1 deletion during primary tumor growth in mice led to a decrease in microvessel density and an increase in mural cell coverage with improved vessel perfusion. Reduced angiogenesis and enhanced vascular normalization resulted in a progressive increase of intratumoral necrosis that caused a growth delay only at later stages of tumor progression. Concomitantly, surgical removal of the primary tumor decreased the number of circulating tumor cells, reduced metastasis, and prolonged overall survival. Additionally, Tie1 deletion in experimental murine metastasis models prevented extravasation of tumor cells into the lungs and reduced metastatic foci. Taken together, the data support Tie1 as a therapeutic target by defining its regulatory functions during angiogenesis and vascular abnormalization and identifying its role during metastasis.

Diabetes mellitus type 2 predisposes patients to various microvascular complications. In the current experiment, the potent role of diabetes mellitus was investigated on the content of VEGFR-1, -2, Tie-1 and -2, and Akt in human endothelial progenitor cells. The gene expression profile of mTOR and Hedgehog signaling pathways were measured by PCR array. The possible crosstalk between RTKs, mTOR and Hedgehog signaling was also studied by bioinformatic analysis. Endothelial progenitor cells were incubated with serum from normal and diabetic for 7days. Compared to non-treated cells, diabetic serum-induced cell apoptosis (~2-fold) and prohibited cell migration toward bFGF (p0.001). ELISA analysis showed that diabetes exposed cells had increased abundance of Tie-1, -2 and VEGFR-2 and reduced amount of VEGFR-1 (p0.0001) in diabetic cells. Western blotting showed a marked reduction in the protein level of Akt after cells exposure to serum from diabetic subjects (p0.0001). PCR array revealed a significant stimulation of both mTOR and Hedgehog signaling pathways in diabetic cells (p0.05). According to data from bioinformatic datasets, we showed VEGFR-1, -2 and Tie-2, but not Tie-1, are master regulators of angiogenesis. There is a crosstalk between RTKs and mTOR signaling by involving P62, GABARAPL1, and HTT genes. It seems that physical interaction and co-expression of Akt decreased the level of VEGFR-1 in diabetic cells. Regarding data from the present experiment, diabetic serum contributed to uncontrolled induction of both mTOR and Hedgehog signaling in endothelial progenitor cells. Diabetes mellitus induces mTOR pathway by involving receptor tyrosine kinases while Hedgehog stimulation is independent of these receptors.

Angiopoietins and their TIE receptors are important regulators of vascular stability and remodeling. These molecules are involved not only in the normal development of kidney glomeruli, but also in disease, thus making them promising targets for therapies. Although TIE receptors are mainly found in endothelial cells, some reports observed TIE2 expression in glomerular podocytes as well. This suggests a role of angiopoietins in the regulation of podocytes. In the present study, we aimed to map the subcellular localization of TIE receptors in metanephric glomeruli of fetal pigs using high-resolution immunogold electron microscopy and the relative labeling index stereological approach. TIE1 and TIE2 antibody labeling was detected on the abluminal side of endothelial cell membranes. In endothelial cells, 4.5% of TIE2 was observed close to cell-cell contacts and 11.9% of TIE2 was found in closely associated pairs, which suggests the presence of homodimers. Interestingly, both receptors were also expressed in podocyte foot processes indicating that TIE1 and TIE2 may play a similar role in podocytes as in endothelial cells.

Purpose To investigate whole transcriptional differences between proliferative diabetic retinopathy (PDR) neovascular membranes (NVMs) and retinas, and the regulatory genes participating in retinal neovascularization in PDR. Methods We used high-throughput sequencing technology to capture the whole-genome gene expression levels of all participants, including 23 patients with PDR or branch retinal vein occlusion (BRVO), 3 normal retinal samples, and 2 retinal samples from type II diabetic (T2D) eyes by donation, followed by analyses of expression patterns using bioinformatics methods, then validation of the data by in situ hybridization and Western blotting. Results We showed that transcriptional profiles of the NVMs were distinct from those of the retinas. Angiogenesis growth factors VEGFC, ANGPT1, ANGPT2, and EFNB2, and their receptors FLT4, TIE1, TIE2, and EPHB4, respectively, were overexpressed. Expression of VEGFA was highly upregulated in T2D retina, but low in the NVMs, while angiogenesis transcription factors, including ETS1 and ERG, were coordinately upregulated in NVMs. Conclusions This study described a PDR neovascularization model in which pathological retina-secreted vascular endothelial growth factor A (VEGFA) enhanced the expression of a set of angiogenesis transcription factors and growth factors, to cooperatively induce the retinal neovascularization. Based on these results, novel potential therapeutic targets and biomarkers for PDR treatment and diagnosis are suggested.

Von Willebrand disease (VWD), a blood coagulation disorder, is also known to cause angiodysplasia. Hitherto, no animal model has been found with angiodysplasia that can be studied in vivo. In addition, VWD patients tend to have a higher incidence of miscarriages for reasons unknown. Thus, we aimed to examine the influence of von Willebrand factor (VWF) on the female reproductive tract histology and the expression and distribution of angiogenic factors in a porcine model for VWD types 1 and 3. The disease-causing tandem duplication within the VWF gene occurred naturally in these pigs, making them a rare and valuable model. Reproductive organs of 6 animals (2 of each mutant genotype and 2 wildtype (WT) animals) were harvested. Genotype plus phenotype were confirmed. Several angiogenic factors were chosen for possible connections to VWF and analyzed alongside VWF by immunohistochemistry and quantitative gene expression studies. VWD type 3 animals showed angiodysplasia in the uterus and shifting of integrin αVβ3 from the apical membrane of uterine epithelium to the cytoplasm accompanied by increased vascular endothelial growth factor (VEGF) expression. Varying staining patterns for angiopoietin (Ang)-2 were observed among the genotypes. As compared with WT, the ovaries of the VWD type 3 animals showed decreased gene expression of ANG2 and increased gene expression of TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) 2, with some differences in the ANG/TIE-system among the mutant genotypes. In conclusion, severely reduced VWF seems to evoke angiodysplasia in the porcine uterus. Varying distribution and expression of angiogenic factors suggest that this large animal model is promising for investigation of influence of VWF on angiogenesis in larger groups.

Background Angiopoietin growth factors (Angs) regulate angiogenesis and lymphangiogenesis by binding to the endothelial Tie2 receptor. Ang2 expression is elevated in tissue hypoxia and inflammation, which also induce cleavage of the extracellular domain of the orphan Tie1 receptor. Here we have examined if the concentrations of Ang2 and the soluble extracellular domain of Tie1 in patient plasma are associated with the prognosis of patients with metastatic breast cancer. Methods Plasma Tie1 and Ang2 levels were measured in metastatic breast cancer patients treated in a phase II trial with a taxane-bevacizumab combination chemotherapy in the first-line treatment setting. They were analyzed before treatment, after 6 weeks and 6 months of treatment, and at the final study visit. Using the median concentrations as cutoffs, Tie1 and Ang2 data were dichotomized into low and high concentration groups. Additionally, we analyzed Tie1 concentrations in plasma from 10 healthy women participating in a breast cancer primary prevention study. Results Plasma samples were available from 58 (89%) of the 65 patients treated in the trial. The baseline Tie1 levels of the healthy controls were significantly lower than those of the metastatic patients (p

Angiopoietins 1–4 (Ang1–4) represent an important family of growth factors, whose activities are mediated through the tyrosine kinase receptors, Tie1 and Tie2. The best characterized are angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang1 is a potent angiogenic growth factor signaling through Tie2, whereas Ang2 was initially identified as a vascular disruptive agent with antagonistic activity through the same receptor. Recent data demonstrates that Ang2 has context-dependent agonist activities. Ang2 plays important roles in physiological processes and the deregulation of its expression is characteristic of several diseases. In this review, we summarize the activity of Ang2 on blood and lymphatic endothelial cells, its significance in human physiology and disease, and provide a current view of the molecular signaling pathways regulated by Ang2 in endothelial cells.

Background Ovarian cancer is the most lethal gynecologic malignancy due to the tumor's acquisition of chemoresistance to platinum-based chemotherapy. To solve this problem, we conducted RNAi-based large-scale screening and determined that tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE-1) is a key molecule involved in the platinum resistance of ovarian cancer cells. Recently, a variety of studies have investigated that small extracellular vesicles (sEVs) contribute to the communication between cancer cells, including the development of chemoresistance in ovarian cancer. The purpose of our study is to determine if sEVs-derived TIE-1 is involved in the chemoresistance of ovarian cancer cells. Materials and Methods TIE-1-overexpressed TOV112D cells, termed TOV112DTIE-1 cells, were established, and sEVs were isolated from TOV112DTIE-1 cells supernatants by ultracentrifugation. We assessed cisplatin sensitivity in recipient cells with TOV112DTIE-1-derived sEVs by cell-Titer Glo kit. We also asked whether sEV-derived TIE-1 suppressed the DNA damage response in recipient cells and evaluated the DNA damage response by counting cells positive for DNA damage foci. Results TIE-1 was contained within sEVTIE-1 derived from the cellular supernatant of TOV112DTIE-1. We showed that sEV-derived TIE-1 decreased chemosensitivity to cisplatin by suppressing the DNA damage response in recipient cells. Conclusion Our findings suggest that sEV-derived TIE-1 could be a new therapeutic target for refractory ovarian cancer.

Endothelial cells that form the inner layer of blood and lymphatic vessels are important regulators of vascular functions and centrally involved in the pathogenesis of vascular diseases. In addition to the vascular endothelial growth factor (VEGF) receptor pathway, the angiopoietin (Ang)–Tie system is a second endothelial cell specific ligand–receptor signalling system necessary for embryonic cardiovascular and lymphatic development. The Ang–Tie system also regulates postnatal angiogenesis, vessel remodelling, vascular permeability and inflammation to maintain vascular homoeostasis in adult physiology. This system is implicated in numerous diseases where the vasculature has an important contribution, such as cancer, sepsis, diabetes, atherosclerosis and ocular diseases. Furthermore, mutations in the TIE2 signalling pathway cause defects in vascular morphogenesis, resulting in venous malformations and primary congenital glaucoma. Here, we review recent advances in the understanding of the Ang–Tie signalling system, including cross-talk with the vascular endothelial protein tyrosine phosphatase (VE-PTP) and the integrin cell adhesion receptors, focusing on the Ang–Tie system in vascular development and pathogenesis of vascular diseases.

In order to investigate the promoting effect of low-intensity treadmill exercise on rat dorsal wound healing and the mechanism, 20 Sprague-Dawley rats were randomly divided into two groups: exercise group (Ex) and non-exercise group (non-ex). The rats in Ex group were given treadmill exercise for one month, and those in non-ex group raised on the same conditions without treadmill exercise. Both groups received dorsal wound operation with free access to food and water. By two-week continuous observation and recording of the wound area, the healing rate was analyzed. The blood sample was collected at day 14 post-operation via cardiac puncture for determination of the number of endothelial progenitor cells (EPCs) by flow cytometry, and the concentrations of relevant cytokines such as basic fibroblast growth factor (bFGF), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were measured by ELISA. The skin tissue around the wound was dissected to observe the vascular density under the microscope after HE staining, to detect the mRNA level of VEGFR2 and angiopoietin-1 (Ang-1) receptor using RT-qPCR, and protein expression of a-smooth muscle actin (αSMA) and type III collagen (ColIII) using Western blotting. It was found that the wound area in Ex group was smaller at the same time point than in non-ex group. The number of circulating EPCs was greater and the concentrations of vasoactive factors such as VEGF, eNOS and bFGF were higher in Ex group than in non-ex group. HE staining displayed a higher vessel density in Ex group than in non-ex group. Moreover, the mRNA expression of VEGFR2 and Ang-1 detected in the wound tissue in Ex group was higher than in non-ex group. Meanwhile, the protein expression of αSMA and ColIII was more abundant in Ex group than in non-ex group. Conclusively, the above results demonstrate Ex rats had a higher wound healing rate, suggesting low-intensity treadmill exercise accelerates wound healing. The present work may provide some hint for future study of treating refractory wound.

Background Antiangiogenic therapy, although part of standard treatment in ovarian cancer, has variable efficacy. Furthermore, little is known about the prognostic biomarkers and factors influencing angiogenesis in cancer tissue. We evaluated the expression of angiopoietin-2 and two endothelial tyrosine kinase receptors, Tie-1 and Tie-2, and assessed their value in the prediction of survival in patients with malignant epithelial ovarian cancer. We also compared the expression of these factors between primary high grade serous tumors and their distant metastasis. Materials and methods We evaluated 86 women with primary epithelial ovarian cancer. Matched distal omental metastasis were investigated in 18.6% cases (N = 16). The expression levels of angiogenic factors were evaluated by immunohistochemistry in 306 specimens and by qRT-PCR in 111 samples. Results A high epithelial expression level of Tie-2 is a significant prognostic factor in primary high grade serous ovarian cancer. It predicted significantly shorter overall survival both in univariate (p Conclusions The angiogenic factor, angiopoietin-2, and its receptor Tie-2 seem to be significant prognostic factors in primary epithelial ovarian cancer. Their expression levels are also increased in metastatic lesions in comparison with primary tumors.

The root of Croton crassifolius Geisel is traditionally used in China for the treatment of snake bites, stomach ache, sternalgia, joint pain, pharyngitis, jaundice and rheumatoid arthritis, while in Thailand, it has been used as an anticancer herbal medicine by the indigenous people. Yet, its pharmacological studies are still limited, especially towards its anticancer property. Anti-angiogenesis is a promising therapeutic strategy in the anti-cancer treatment. Previous studies have shown strong anti-angiogenic activity in the low polar fraction of the herb. Nevertheless, the potent compound which is responsible for the anti-angiogenesis, and its molecular mechanism have never been reported.To determine the potent anti-angiogenic component in C. crassifolius and its molecular mechanism of action.C. crassifolius was extracted using supercritical fluid extraction and steam distillation. The anti-angiogenic activities of the two extracts were evaluated in the zebrafish model by quantitative endogenous alkaline phosphatase assay. The chemical compounds in the active extract were isolated using chromatographic methods, and their structures were elucidated using different spectroscopic techniques. The content/quantity of the active compounds in this extract was determined with HPLC analysis. The molecular mechanism of the most active compound was further studied using the real-time PCR assay. Besides, its cytotoxicity on various cancer and normal cell lines was evaluated using the cell-counting kit.Supercritical fluid extract (SFE) of C. crassifolius showed better anti-angiogenic activity than that of steam distillation extract (SDE). Three sesquiterpenes, namely, cyperenoic acid, 8-hydroxy-α-guaiene and (+)-guaia-l(10),ll-dien-9-one, were isolated and identified in the SFE. Among them, cyperenoic acid displayed the strongest anti-angiogenic activity by 51.7% of the control at 10μM, while the others showed little effect. HPLC results showed that cyperenoic acid was the major component in the SFE with 9.97% (w/w). Results of the real-time PCR assay suggested that the cyperenoic acid affected multiple molecular targets related to angiogenesis including vascular endothelial growth factor (Vegfa), angpiopoietin (Angpt), and their receptors. Cytotoxicity assay showed cyperenoic acid possessed little toxicity toward cancer and normal cells.Cyperenoic acid is an important anti-angiogenic component present in C. crassifolius and serve as a potent inhibitor in the angiogenesis in the zebrafish embryo model. The anti-angiogenic property, but not the cytotoxicity, of C. crassifolius provides a scientific basis for its traditional use in cancer treatment.

Tie1 is a receptor tyrosine kinase with broad expression in embryonic endothelium. Reduction of Tie1 levels in mouse embryos with a hypomorphic Tie1 allele resulted in abnormal lymphatic patterning and architecture, decreased lymphatic draining efficiency, and ultimately, embryonic demise. Here we report that Tie1 is present uniformly throughout the lymphatics and from late embryonic/early postnatal stages, becomes more restricted to lymphatic valve regions. To investigate later events of lymphatic development, we employed Cre-loxP recombination utilizing a floxed Tie1 allele and an Nfatc1Cre line, to provide loxP excision predominantly in lymphatic endothelium and developing valves. Interestingly, unlike the early prenatal defects previously described by ubiquitous endothelial deletion, excision of Tie1 with Nfatc1Cre resulted in abnormal lymphatic defects in postnatal mice and was characterized by agenesis of lymphatic valves and a deficiency of collecting lymphatic vessels. Attenuation of Tie1 signaling in lymphatic endothelium prevented initiation of lymphatic valve specification by Prox1 high expression lymphatic endothelial cells that is associated with the onset of turbulent flow in the lymphatic circulation. Our findings reveal a fundamental role for Tie1 signaling during lymphatic vessel remodeling and valve morphogenesis and implicate it as a candidate gene involved in primary lymphedema.

Pervasive transcription is a feature of the human genome that requires better understanding. Over the last decade or so, RNA species longer than 200 nucleotides-dubbed long non-coding RNA (lncRNAs)-had been found in sense or anti-sense orientation within or outside of genes that encode proteins. Importantly, lncRNA-mediated gene regulation and the elements that control lncRNA expression are a source of fascination among molecular biologists. In vascular biology, a dozen or so lncRNAs had been identified, and progress occurs each day. In this review, we highlighted our laboratories' contribution to the lncRNA field by discussing lessons learned from two lncRNAs in the tyrosine kinase containing immunoglobulin and epidermal growth factor homology1 (Tie1) and delta-like 4 (Dll4) loci. These genes are responsible for basic vascular patterning and pathophysiological remodeling in angiogenesis.

Functional cross-talk between Tie2 and Integrin signaling pathways is essential to coordinate endothelial cell adhesion and migration in response to the extracellular matrix, yet the mechanisms behind this phenomenon are unclear. Here, we examine the possibility that receptor cross-talk is driven through uncharacterized Tie-integrin interactions on the endothelial surface. Using a live cell FRET-based proximity assay, we monitor Tie-integrin receptor recognition and demonstrate that both Tie1 and Tie2 readily associate with integrins α5ß1 and αVß3 through their respective ectodomains. Although not required, Tie2-integrin association is significantly enhanced in the presence of the extracellular component and integrin ligand fibronectin. In vitro binding assays with purified components reveal that Tie-integrin recognition is direct, and further demonstrate that the receptor binding domain of the Tie2 ligand Ang-1, but not the receptor binding domain of Ang-2, can independently associate with α5ß1 or αVß3. Finally, we reveal that cooperative Tie/integrin interactions selectively stimulate ERK/MAPK signaling in the presence of both Ang-1 and fibronectin, suggesting a molecular mechanism to sensitize Tie2 to extracellular matrix. We provide a mechanistic model highlighting the role of receptor localization and association in regulating distinct signaling cascades and in turn, the angiogenic switch.

The lymphatic system comprises a network of interconnected and blind-ended vessels whose central function is to maintain fluid homeostasis.1 Similar to the vessels of the circulatory system, the lymphatic system consists of a hierarchical network of small and large caliber vessels, which function together to allow fluid drainage. Lymphatic capillaries are lined by endothelial cells that are highly discontinuous, making them permeable to surrounding fluid to facilitate resorption. By contrast, larger lymphatic collecting ducts possess a thin smooth muscle layer and valves to promote unidirectional transport of fluid toward connection points with the circulatory system at the subclavian veins. In addition to fluid homeostasis, the lymphatic system and the constituent lymph nodes are a portal for lymphocytes, as well as antigen-presenting cells, while also functioning in lipid absorption in the digestive system.1 Given their wide-ranging importance in these normal physiological processes, it is not surprising that the lymphatic system is implicated in several pathological conditions. Indeed, inflammation is intimately associated with lymphatic function and is a central cause of lymphedema in a wide range of acquired and congenital diseases.1 Notably, congenital forms of lymphedema are associated with mutations in several genes important for lymphatic vessel formation, including those encoding the receptor tyrosine kinase FLT4 and its ligand, VEGFC.2 Articles, see p 1263 and 1276 The lymphatic system is also essential for cardiac homeostasis and has been increasingly implicated in related diseases. Importantly, lymphatic vessels can play a central role to facilitate repair of cardiac tissue after myocardial infarction.3 Indeed, injection of VEGFC after myocardial infarction can lead to sustained improvement in cardiac function, including increased ejection fraction, and reduction in both end-systolic volume and hypertrophy. In these cases, the presence of additional lymphatic vessels induced by VEGFC is thought to better resolve post-myocardial infarction edema …

Blood flow influences atherosclerosis by generating wall shear stress, which alters endothelial cell (EC) physiology. Low shear stress induces dedifferentiation of EC through a process termed endothelial-to-mesenchymal transition (EndMT). The mechanisms underlying shear stress-regulation of EndMT are uncertain. Here we investigated the role of the transcription factor Snail in low shear stress-induced EndMT. Studies of cultured EC exposed to flow revealed that low shear stress induced Snail expression. Using gene silencing it was demonstrated that Snail positively regulated the expression of EndMT markers (Slug, N-cadherin, α-SMA) in EC exposed to low shear stress. Gene silencing also revealed that Snail enhanced the permeability of endothelial monolayers to macromolecules by promoting EC proliferation and migration. En face staining of the murine aorta or carotid arteries modified with flow-altering cuffs demonstrated that Snail was expressed preferentially at low shear stress sites that are predisposed to atherosclerosis. Snail was also expressed in EC overlying atherosclerotic plaques in coronary arteries from patients with ischemic heart disease implying a role in human arterial disease. We conclude that Snail is an essential driver of EndMT under low shear stress conditions and may promote early atherogenesis by enhancing vascular permeability.

Objective— The genetic program underlying lymphatic development is still incompletely understood. This study aims to dissect the role of receptor tyrosine kinase with immunoglobulin-like and EGF (epidermal growth factor)-like domains 1 (Tie1) and Tie2 in lymphatic formation using genetically modified mouse models. Approach and Results— We generated conditional knockout mouse models targeting Tie1, Tie2, and angiopoietin-2 in this study. Tie1 Δ ICD /Δ ICD mice, with its intracellular domain targeted, appeared normal at E10.5 but displayed subcutaneous edema by E13.5. Lymph sac formation occurred in Tie1 Δ ICD /Δ ICD mice, but they had defects with the remodeling of primary lymphatic network to form collecting vessels and valvulogenesis. Consistently, induced deletion of Tie1-ICD postnatally using a ubiquitous Cre deleter led to abnormal lymphangiogenesis and valve formation in Tie1-ICD iUCKO/ − mice. In comparison with the lymphatic phenotype of Tie1 mutants, we found that the diameter of lymphatic capillaries was significantly less in mice deficient of angiopoietin-2, besides the disruption of collecting lymphatic vessel formation as previously reported. There was also no lymphedema observed in Ang2 −/− mice during embryonic development, which differs from that of Tie1 Δ ICD /Δ ICD mice. We further investigated whether Tie1 exerted its function via Tie2 during lymphatic development. To our surprise, genetic deletion of Tie2 ( Tie2 iUCKO/ − ) in neonate mice did not affect lymphatic vessel growth and maturation. Conclusions— In contrast to the important role of Tie2 in the regulation of blood vascular development, Tie1 is crucial in the process of lymphatic remodeling and maturation, which is independent of Tie2.

Objective: The aim of this study was to determine the characteristic factors for vascular development and maintenance levels as well as correlation between Tie-1 receptors, Tie-2 receptors and the corresponding ligands - angiopoietins - in systemic sclerosis (SSc) patients. Materials and Methods: Serum levels of Tie-1, Tie-2, Ang-1 and Ang-2 were measured in 25 SSc patients and healthy controls. Results: There was a statistically significant difference in serum Tie-1 (p = 0.009) and Ang-2 (p = 0.001) levels in SSc patients compared with healthy controls. Significant correlations between Tie-1 and Tie-2 (ρ = 0.70, p = 0.0001) and between Tie-1 and Ang-2 (ρ = -0.92, p = 0.002) were found in the SSc group. Serum levels of Tie-2 were positively associated with esophagus changes (U = 2.03, p = 0.041) and Ang-1 was negatively correlated with duration of Raynaud's phenomenon (ρ = -0.75, p = 0.00008). Conclusion: The increase in serum concentration of Tie-1 and Ang-2 in patients with SSc may confirm a molecular imbalance between receptor tyrosine kinases Tie and their ligands.

BACKGROUND The use of a transgenic pig for xenotransplantation and as a cardiovascular disease model has caught much attention in the past decades. The vascular endothelial cell is the primary modification target for the application of genetically modified pigs in this field. However, the powerful porcine endothelial cell-specific promoter is still so rare that the mouse and human promoters are commonly used. In the study, the porcine Tie1 (sTie1) promoter was identified and characterized as a potential endothelial cell-specific promoter to generate a cardiovascular disease model. METHODS Tie1 promoters with different lengths of 5'-regulatory regions were cloned, and major putative DNA-binding motifs were mutated by site-directed mutagenesis. All fragments were ligated into the luciferase reporter system and were transiently transfected into endothelial cells to identify luciferase activity using a dual luciferase reporter assay. RESULTS The luciferase activities of sTie1 promoters with different lengths of the 5'-regulatory region were tested. Results showed that the luciferase activity of the 1234-bp sTie1 fragment was the strongest compared with that of others (P

Despite the altered expression of Tie receptors and angiopoietin ligands during hypoxic conditions, the effect of hypoxia on Tie-mediated endothelial responses has not been elucidated. In this study, we found that hypoxia increased Tie receptor expression but attenuated angiopoietin-1 (Ang1)-induced Tie2 activity, including Tie2 phosphorylation, Tie2 downstream signaling activation, and endothelial cell tube formation. However, Ang1 binding to endothelial cells was increased during hypoxic conditions. We demonstrated that Tie1 suppression restored the Tie2 activity and that Tie1-mediated Tie2 suppression was independent of tyrosine phosphatase activity. These results suggest that under hypoxic conditions, Tie1 is critical for reducing Ang1-induced Tie2 activity and angiogenesis.

金沢大学医薬保健研究域医学系, Tie2 is a receptor tyrosine kinase expressed on vascular endothelial cells (ECs). It has dual roles in promoting angiogenesis and stabilizing blood vessels, and it has been suggested that Tie2 forms dimers and/or oligomers in the absence of angiopoietin-1 (Ang1); however, the mechanism of ligand-independent dimerization of Tie2 and its biological significance have not been clarified. Using a bimolecular fluorescence complementation assay and a kinase-inactive Tie2 mutant, we show here that ligand-independent Tie2 dimerization is induced without Tie2 phosphorylation. Moreover, based on the fact that Tie1 never forms heterodimers with Tie2 in the absence of Ang1 despite having high amino acid sequence homology with Tie2, we searched for ligand-independent dimerization domains of Tie2 by reference to the difference with Tie1. We found that the YIA sequence of the intracellular domain of Tie2 corresponding to the LAS sequence in Tie1 is essential for this dimerization. When the YIA sequence was replaced by LAS in Tie2 (Tie2YIA/LAS), ligand-independent dimer was not formed in the absence of Ang1. When activation of Tie2YIA/LAS was induced by a high dose of Ang1, phosphorylation of Tie2 was limited compared with wild-type Tie2, resulting in retardation of activation of Erk downstream of Tie2. Therefore, these data suggest that ligand-independent dimerization of Tie2 is essential for a strong response upon stimulation with high dose Ang1. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Angiogenesis is a complex cellular process involving multiple regulatory growth factors and growth factor receptors. Among them, the ligands for the endothelial-specific tunica intima endothelial receptor tyrosine kinase 2 (Tie2) receptor kinase, angiopoietin-1 (Ang1) and Ang2, play essential roles in balancing vessel stability and regression during both developmental and tumor-induced angiogenesis. Despite possessing a high degree of sequence identity, Ang1 and Ang2 have distinct functional roles and cell-signaling characteristics. Here, we present the crystal structures of Ang1 both unbound and in complex with the Tie2 ectodomain. Comparison of the Ang1-containing structures with their Ang2-containing counterparts provide insight into the mechanism of receptor activation and reveal molecular surfaces important for interactions with Tie2 coreceptors and associated signaling proteins. Using structure-based mutagenesis, we identify a loop within the angiopoietin P domain, adjacent to the receptor-binding interface, which confers the specific agonist/antagonist properties of the molecule. We demonstrate using cell-based assays that an Ang2 chimera containing the Ang1 loop sequence behaves functionally similarly to Ang1 as a constitutive Tie2 agonist, able to efficiently dissociate the inhibitory Tie1/Tie2 complex and elicit Tie2 clustering and downstream signaling.

Objective To investigate the abnormal expressions of Tie1 on the valves of great saphenous varicose vein, and to discuss the relationship between the phenomenon and pathogenesis of varicose vein of lower extremity. Methods Varicose veins group 18 samples, normal control group 14 samples. Immunohistochemistry staining has investigated the expression of CD31 and Tie1 in the first valves of great saphenous veins. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) has checked mRNA expression of Tie1. Western blot has checked the expression of Tie1 protein in venous valves. Results In normal control group valves, there was no difference between proximal and distal sides endothelium, which expressing CD31 in both valvar basement and valve cusp (positive endothelial cells [ECs] percentage: P > 0.05, P > 0.05). However, the endothelium of the proximal side demonstrates Tie1 stronger than distal side in valvar basement (positive ECs percentage: P < 0.05), which was not found at valve cusp (positive ECs percentage: P > 0.05). In varicose veins group, the endothelium of proximal side cells expresses CD31 weaker than distal side at both valvar basement and valve cusp (positive ECs percentage: P < 0.05, P < 0.05) besides the morphological alteration of valves. Moreover, it expresses Tie1 much weaker than diatal side (positive ECs percentage: P < 0.01). Semi-quantitative RT-PCR showed that valves of varicose veins group expressed Tie1 much weaker than the normal control group ( P < 0.01). Western blot could not detect the expression of Tie1 in venous valves. Conclusion The decreasing expression of Tie1 may play an important role in the pathogenesis of primary lower extremity varicose veins.

Background Recent studies of hepatic regeneration have mainly focused on the growth of parenchymal cells. However, remodeling of liver vessels seems to be crucial during hepatic regeneration. In this study, we investigated the influence of antiangiogenesis on hepatic regeneration using sFlt-1, a soluble receptor for vascular endothelial growth factor that acts as a dominant negative receptor, and the hepatocyte growth factor antagonist NK4. Methods A sFlt-1–expressing adenoviral vector, an NK4-expressing adenoviral vector, or both combined were infected into C57BL6 mice via the tail vein. A 70% partial hepatectomy was performed on all of the mice 48 hours after infection. The remnants of the liver were removed after the partial hepatectomy, and hepatic regeneration was assessed by measuring the remnant liver weight and hepatocyte mitosis, bromodeoxyuridine staining, immunohistochemical staining with anti–platelet endothelial cell adhesion molecule-1 antibodies, and real-time polymerase chain reaction studies for angiogenic factors. Results The immunohistochemical staining for CD31 showed suppression of sinusoidal endothelial cells growth in sFlt-1–expressing adenoviral vector–and NK4-expressing adenoviral vector–infected mice. Increases in the remnant hepatic weight were significantly lower in the sFlt-1–expressing adenoviral vector–infected mice. The bromodeoxyuridine index and mitotic cell results revealed a significant decrease in hepatic regeneration in the sFlt-1–expressing adenoviral vector–and NK4-expressing adenoviral vector–infected mice. The suppressive effects on hepatic regeneration were significantly enhanced by combined sFlt-1–expressing adenoviral vector and NK4-expressing adenoviral vector infection. Real-time polymerase chain reaction results revealed the significant suppression of angiogenic growth factor receptors Tie-1 and Tie-2. Conclusion The angiogenesis inhibitor significantly suppressed hepatic regeneration. These results suggest that hepatic regeneration after hepatectomy closely correlates with angiogenesis.

The Tie receptors 1 and 2 (Tie1/2) play crucial roles in embryonic angiogenesis. Recent studies suggest enhanced expression of Tie1 in several types of cancer and negative correlations between Tie1 levels and clinical outcome. These observations suggest important functions of Tie1 not only for vascular formation but also in tumorigenesis. Ligands for Tie2, that is angiopoietins 1‐4, have been identified, but not for Tie1. To determine the molecular functions of Tie1, its detailed characterization in tumors would be helpful. Herein, we report that Tie1 is up‐regulated in colorectal cancer. Detailed analysis using tumor‐bearing models and immunohistochemistry combined with Flow cytometric analysis and cell sorting (FACS) revealed that Tie1 protein was expressed in a small population of malignant tumor cells. Intriguingly, Tie1 expression was observed and could be maintained only in vivo. Further analysis using sphere‐formation culture revealed that Tie1‐positive cells are enriched within the population of tumor cells with cancer stemness properties. Indeed, Tie1‐positive tumor cells derived from a murine model overexpressed Lgr5, a typical stemness marker for colorectal cancer. Our results provide a novel insight into Tie1 function in tumorigenesis and suggest clinical applications to target cancer stem cells.

The angiopoietin/Tie (ANG/Tie) receptor system controls developmental and tumor angiogenesis, inflammatory vascular remodeling, and vessel leakage. ANG1 is a Tie2 agonist that promotes vascular stabilization in inflammation and sepsis, whereas ANG2 is a context-dependent Tie2 agonist or antagonist. A limited understanding of ANG signaling mechanisms and the orphan receptor Tie1 has hindered development of ANG/Tie-targeted therapeutics. Here, we determined that both ANG1 and ANG2 binding to Tie2 increases Tie1-Tie2 interactions in a β1 integrin–dependent manner and that Tie1 regulates ANG-induced Tie2 trafficking in endothelial cells. Endothelial Tie1 was essential for the agonist activity of ANG1 and autocrine ANG2. Deletion of endothelial Tie1 in mice reduced Tie2 phosphorylation and downstream Akt activation, increased FOXO1 nuclear localization and transcriptional activation, and prevented ANG1- and ANG2-induced capillary-to-venous remodeling. However, in acute endotoxemia, the Tie1 ectodomain that is responsible for interaction with Tie2 was rapidly cleaved, ANG1 agonist activity was decreased, and autocrine ANG2 agonist activity was lost, which led to suppression of Tie2 signaling. Tie1 cleavage also occurred in patients with hantavirus infection. These results support a model in which Tie1 directly interacts with Tie2 to promote ANG-induced vascular responses under noninflammatory conditions, whereas in inflammation, Tie1 cleavage contributes to loss of ANG2 agonist activity and vascular stability.

Introduction Despite the advent of biological therapies for the treatment of rheumatoid arthritis, there is a compelling need to develop alternative therapeutic targets for nonresponders to existing treatments. Soluble receptors occur naturally in vivo, such as the splice variant of the cell surface receptor for vascular endothelial growth factor (VEGF) – a key regulator of angiogenesis in rheumatoid arthritis. Bioinformatics analyses predict that the majority of human genes undergo alternative splicing, generating proteins – many of which may have regulatory functions. The objective of the present study was to identify alternative splice variants (ASV) from cell surface receptor genes, and to determine whether the novel proteins encoded exert therapeutic activity in an in vivo model of arthritis. Methods To identify novel splice variants, we performed RT-PCR using an mRNA pool representing major human tissue types and tumors. Novel ASV were identified by alignment of each cloned sequence to its respective genomic sequence in comparison with full-length transcripts. To test whether these ASV have biologic activity, we characterized a subset of them for ligand binding, and for efficacy in an animal model of arthritis. The in vivo study was accomplished using adenoviruses expressing secreted ASV. Results We cloned 60 novel human ASV from 21 genes, encoding cell surface receptors – many of which are known to be important in the regulation of angiogenesis. The ASV were characterized by exon extension, intron retention and alternative exon utilization. Efficient expression and secretion of selected ASV – corresponding to VEGF receptor type 1, VEGF receptor type 2, VEGF receptor type 3, angiopoietin receptor Tie1, Met (receptor for hepatocyte growth factor), colony-stimulating factor 1 receptor, platelet-derived growth factor receptor beta, fibroblast growth factor receptor 1, Kit, and RAGE – was demonstrated, together with binding to their cognate ligands. Importantly, ASV derived from VEGF receptor type 1 and Tie1, and to a lesser extent from VEGF receptor type 2 and fibroblast growth factor receptor 1, reduced clinical signs of arthritis in vivo. The reduction was paralleled by decreased joint inflammation and destruction. Conclusion The present study shows that unique ASV derived from receptors that play key roles in angiogenesis – namely, VEGF receptor type 1 and, for the first time, Tie1 – can markedly reduce arthritis severity. More broadly, our results demonstrate that ASV are a source of novel proteins with therapeutic potential in diseases in which angiogenesis and cellular hyperplasia play a central role, such as rheumatoid arthritis.

Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1 + CD31 dim vascular endothelial (VE)-cadherin − CD45 − precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1 + precursor cell, but not the VE-cadherin + endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1 + precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1 + local precursor to VSMC in a spatiotemporal fashion across all vascular beds.

Endocardial cells play a critical role in cardiac development and function, forming the innermost layer of the early (tubular) heart, separated from the myocardium by extracellular matrix (ECM). However, knowledge is limited regarding the interactions of cardiac progenitors and surrounding ECM during dramatic tissue rearrangements and concomitant cellular repositioning events that underlie endocardial morphogenesis. By analyzing the movements of immunolabeled ECM components (fibronectin, fibrillin-2) and TIE1 positive endocardial progenitors in time-lapse recordings of quail embryonic development, we demonstrate that the transformation of the primary heart field within the anterior lateral plate mesoderm (LPM) into a tubular heart involves the precise co-movement of primordial endocardial cells with the surrounding ECM. Thus, the ECM of the tubular heart contains filaments that were associated with the anterior LPM at earlier developmental stages. Moreover, endocardial cells exhibit surprisingly little directed active motility, that is, sustained directed movements relative to the surrounding ECM microenvironment. These findings point to the importance of large-scale tissue movements that convect cells to the appropriate positions during cardiac organogenesis.

Tie-1 and Tie-2 tyrosine kinase receptors are expressed specifically on vascular endothelial cells and on a certain subtype of macrophages implicated in angiogenesis, thus, they have been a major focus of angiogenesis research. Tie-1 and Tie-2 are essential for vascular maturation during developmental, physiological and pathological angiogenesis. Angiopoietin 1-4 (Ang-1-4) have been identified as bona fide ligands of the Tie-2 receptor, while Tie-1 remains an orphan receptor which is able to heterodimerize with Tie-2 and to modulate Tie-2 signal transduction. The most exhaustively studied angiopoietins are Ang-1 and Ang-2. Ang-1 is a critical player in vessel maturation and it mediates migration, adhesion and survival of endothelial cells. Ang-2 disrupts the connections between the endothelium and perivascular cells and promotes cell death and vascular regression. Yet, in conjunction with VEGF, Ang-2 promotes neo-vascularization. Hence, angiopoietins exert crucial roles in the angiogenic switch during tumor progression, and increased expression of Ang-2 relative to Ang-1 in tumors correlates with poor prognosis. Its central role in the regulation of physiological and pathological angiogenesis makes the angiopoietin/Tie signaling pathway a therapeutically attractive target for the treatment of vascular disease and cancer.

Atherosclerotic plaques develop in a non-random manner along the vasculature following a hemodynamically determined distribution profile. The pathogenesis of shear stress-induced inflammation and atherosclerotic lesion formation has led to discussions about personalized strategies in prevention and treatment. Recent discoveries involving the tyrosine kinase receptor, Tie1 in (1) mechanotransduction, (2) inflammation and (3) neovascularization has invigorated these efforts. In this review, we will present the current understanding on Tie1 and its role in these key components of atherogenesis.

Although the response of endothelial cells to the disturbed blood flow in the vicinity of atherosclerotic lesions is known to be distinct from that elicited by nonatherogenic laminar flow, the mechanisms involved are poorly understood. Our initial studies confirmed that expression of the endothelial receptor tyrosine kinase Tie1 was evident at regions of atherogenic flow in mature animals. We therefore hypothesized that Tie1 plays a role in the endothelial response to atherogenic shear stress. Consistent with this, we found that Tie1+/- mice bred to the apoE-deficient background displayed a 35% reduction in atherosclerosis relative to Tie1+/+;Apoe-/- mice. Since deletion of Tie1 results in embryonic lethality secondary to vascular dysfunction, we used conditional and inducible mutagenesis to study the effect of endothelial-specific Tie1 attenuation on atherogenesis in Apoe-/- mice and found a dose-dependent decrease in atherosclerotic lesions. Analysis of primary aortic endothelial cells indicated that atheroprotective laminar flow decreased Tie1 expression in vitro. Attenuation of Tie1 was associated with an increase in eNOS expression and Tie2 phosphorylation. In addition, Tie1 attenuation increased IkBα expression while decreasing ICAM levels. In summary, we have found that shear stress conditions that modulate atherogenic events also regulate Tie1 expression. Therefore, Tie1 may play a novel proinflammatory role in atherosclerosis.

Previous studies have shown that oxidized products of the phospholipid PAPC (Ox-PAPC) are strong activators of aortic endothelial cells and play an important role in atherosclerosis and other inflammatory diseases. We and others have demonstrated that Ox-PAPC activates specific signaling pathways and regulates a large number of genes. Using a phosphoproteomic approach based on phosphopeptide enrichment and mass spectrometry analysis, we identified candidate changes in Ox-PAPC-induced protein phosphorylation of 228 proteins. Functional annotation of these proteins showed an enrichment of the regulation of cytoskeleton, junctional components, and tyrosine kinases, all of which may contribute to the phenotypic and molecular changes observed in endothelial cells treated with Ox-PAPC. Many changes in protein phosphorylation induced by Ox-PAPC are reported here for the first time and provide new insights into the mechanism of activation by oxidized lipids, including phosphorylation-based signal transduction.

Endothelin-1 (ET-1) is up-regulated in the endothelial cells and astrocytes under ischemia. Transgenic mice with astrocytic ET-1 over-expression (GET-1) showed more severe neurological deficit and larger infarct after transient middle cerebral artery occlusion (MCAO). Here, the significance of endothelial ET-1 in ischemic brain injury was investigated using transgenic mice with the endothelial ET-1 over-expression (TET-1). Increased ET-1 level was observed in the TET-1 brain infarct core after transient MCAO. ET(A) receptor expression was induced in the penumbra and ET(A) antagonist (A-147627) partially normalized the infarct volume and neurological deficit. In the infarct core of TET-1 brain, superoxide, nitrotyrosine, and gp91(phox) levels were increased. TET-1 brain displayed increased matrix metalloproteinase-2 expression, water content, immunoglobulin leakage and decreased occludin level in the ipsilateral hemisphere indicative of BBB breakdown and hemispheric edema. Interestingly, AQP-4 expression was increased in the penumbra of TET-1 brain following transient MCAO leading to the water accumulation. Taken together, endothelial ET-1 over-expression and ETA receptor activation contributes to the increased oxidative stress, water accumulation and BBB breakdown after transient MCAO leading to more severe neurological deficit and increased infarct.