Endothelial Cell-specific Receptor Tyrosine Kinase Research Articles

The relationship with clinical course and prognosis of serum endothelin-1, angiopoietin-2, and tie-2 levels in Crimean-Congo hemorrhagic fever

Background/aimCrimean-Congo hemorrhagic fever (CCHF) is a serious illness characterized by fever and hemorrhage. Endothelin-1 (ET-1), angiopoietin-2 (Ang-2), and endothelial cell-specific receptor tyrosine kinase (Tie-2) are believed to be important markers of the pathogenesis, clinical course, and prognosis of the disease. The aim of this study was to determine ET-1, Ang-2, and Tie-2 levels in adults with CCHF and investigate the associations between these markers and pathogenesis and disease course.Materials and methodsSixty CCHF patients were included in the study. The patients were classified according to disease severity criteria and Ang-2, Tie-2, and ET-1 levels were compared.ResultsMean serum ET-1 level was 36.62 ± 27.99 pg/mL in the patient group and 3.70 ± 4.71 pg/mL in the control group (P = 0.001). Mean serum Ang-2 levels were 2511.18 ± 1018.64 pg/mL in the patient group and 3570.76 ± 209.52 pg/mL in the control group (P = 0.001). Mean serum Tie-2 levels were 7.35 ± 7.75 ng/mL in the patient group and 0.67 ± 1.26 ng/mL in the control group (P = 0.001). ConclusionElevated ET-1 and Tie-2 levels were associated with more severe disease course, while Ang-2 level was negatively correlated with severity in adult CCHF patients. ET-1, Tie-2, and Ang-2 levels are important prognostic parameters in CCHF and may contribute significantly to treatment and follow-up.

Angiopoietin-2 1087G > A rs3020221 gene polymorphism is associated with in vitro fertilization and embryo transfer outcome

Abstract In in vitro fertilization (IVF) the eggs are fertilized in the laboratory. The success of IVF depends on the angiogenesis. Angiopoetin-2 (ANGPT2) plays a critical role in angiogenesis and acts as a natural antagonist of the endothelial cell–specific receptor tyrosine kinase (Tie-2). A functional single-nucleotide polymorphism (SNP) in the ANGPT2 1087 G > A gene is known to influence in the gene expression in an allele-specific manner. The aim of this project was to study the effect of angiopoietin-2 gene polymorphism on in vitro fertilization and embryo transfer (IVF-ET) in Iran. The prevalence of G/A substitution in exon 4 was determined in 160 infertile patients enrolled who had unsuccessful IVF history and 160 control subjects who had successful IVF history. The genomic DNA was extracted from the whole blood using DNA extraction techniques. Genotype of ANGPT2 1087 G > A was performed using Allele - specific polymerase chain reaction (AS-PCR). The genotype frequencies of GG, AG and AA in the IVF negative were 7.5%, 67.5% and 25%, respectively and in the IVF positive were 15%, 77.5% and 7.5%, respectively. The allele frequencies of G, A in the IVF negative were 41.2%, 58.8%, respectively and in IVF positive were 53.8%, 46.2%, respectively. Statistical analysis showed that there is a significant correlation in the genotype between two groups (OR = 4.11, 95% CI = 2.065–8.18, P = 0.0001). It is concluded that ANGPT2 G/A polymorphism is associated with IVF-ET outcome in a population in the north of Iran.

Abstract A05: The anti-angiogenic activity and pharmacokinetic evaluation of a small molecule JFD-WS in preclinical testing

Abstract VEGF signaling through activation of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), an endothelial cell-specific receptor tyrosine kinase is indispensable for developmental angiogenesis and cancer progression. Since VEGFR2 is the dominant angiogenic signaling receptor, it has become an important target in the development of anti-angiogenic therapies. As part of this strategy inducing apoptosis in cancer cells could make the therapeutic agents very effective. The in vitro anti-angiogenic activity of water soluble form of JFD named as JFD-WS was examined by ECMatrixTM gel assay with human umbilical vein endothelial cells (HUVEC) using 0.01-10µM concentrations at different time intervals (0, 4 and 8 hrs). A complete inhibition of in vitro angiogenesis was observed at 8 hrs after treating with 10µM concentration of JFD-WS. Furthermore, the VEGFR2 phosphorylation was inhibited by 38.65% in JFD-WS (1.0µM) treated HUVEC cells. Besides the in vitro anti-angiogenic activity, JFD-WS is also proven as a potent anti-cancer drug in this study by using GI-101A (human breast adenocarcinoma) xenograft implanted athymic nude mice. In our in vivo experiments, the intraperitoneal (i.p.) injection of JFD-WS (at a dose of 100 mg/kg body weight) was able to significantly inhibit the tumor growth compared to the control group. At the end of the treatment period nearly 35.19% inhibition of tumor growth was observed in JFD-WS treated animals along with prolongation of survival. The tumor inhibition was lot more effective when JFD-WS was combined with Taxol (10 mg/kg). Eventually, the serum levels of MUC1 proved a significant regression of the tumor burden in the experimental animals. In addition to the anti-angiogenic ability, the anti-tumor activity of JFD-WS seems to be further enhanced due to induction of apoptosis signals in the xenograft tumor implanted animals. The protein expression levels of key apoptotic signaling molecules such as Bax, Bcl2, cytochrome c, Apaf-1 and cleaved caspase-3 and p53 were analyzed in the tumor samples that were extracted from experimental animals. Interestingly, a significant increase in the expression of pro-apoptotic proteins p53 (3 fold), Bax (10.6 fold) and Apaf-1 (2.2 fold) were observed in JFD-WS treated animals. In support of the induction of apoptotic signals, the expression of anti-apoptotic protein Bcl2 was decreased by 1.8 fold. Consequently, the release of cytochrome c and the cleavage of caspase 3 found in the cytosolic fraction were significantly higher in JFD-WS treated group as compared with the untreated controls. Finally, the pharmacokinetics (PK) property of JFD-WS was analyzed by measuring the concentration in plasma and urine samples of Balb/c mice at different time intervals (Plasma: 2.5, 5, 10, 15, 30, 60, 120, 1440 min; and Urine: 15, 30, 60, 120, 1440 min), using a HPLC method. Our experiments showed that JFD-WS can reach peak plasma concentration after 15 mins and a maximum elimination in urine was observed after 30 mins of i.p. injection. The compound was undetectable in both plasma and urine after 24 hrs. Moreover, a two compartment model of absorption, distribution and elimination for JFD-WS, with the half-life (t1/2) of around 33 mins is suspected. Our results from both in vivo and in vitro experiments confirm the anti-angiogenic and pro-apoptotic effects of JFD-WS in xenograft tumor implanted athymic nude mice. Furthermore, our data clearly indicate that, JFD-WS is highly effective in causing tumor inhibition with minimal toxicity. (This project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida). Citation Format: Thanigaivelan Kanagasabai, Sivanesan Dhandayuthapani, Manasa Subbarao, Janelle Alvarez, Meera Bhalani, Appu Rathinavelu. The anti-angiogenic activity and pharmacokinetic evaluation of a small molecule JFD-WS in preclinical testing. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A05.

Classification and Tie2 mutations in spinal and soft tissue vascular anomalies

Vascular anomalies included hemangiomas and vascular malformations (VMs). VMs are mediated by mutations in the endothelial cell-specific receptor tyrosine kinase Tie2 (TEK),which is essential for angiogenesis and vascular stabilization. We identified five types of Tie2 mutations in 80 patients with soft tissue or spinal VMs by PCR including the previously detected missense mutations 2690A>G (Y897C), 2740C>T (L914F), 2743C>T (R915C), and two nonsense mutations 2763G>A, 2688C>T, we identified Tie2 mutation in primary spinal VMs for the first time. Tie2 mutations were found to be absent in 33 patients with hemangiomas and DNA samples of VMs. In addition, we showed that Tie2 mRNA expression in spinal VMs was similar to soft tissue VMs, but obviously lower than infant hemangiomas (P<0.01). This study provides new insights into spinal VMs, the association of Tie2 and vascular anomalies needs to be further discussed.

Abstract 4492: Blockade of angiopoietin-2 or Tie2 is equally effective at inhibiting tumor growth and reducing tumor vessel density in most human tumor xenograft models

Abstract Angiopoietin-1 (Ang1) and -2 (Ang2) regulate angiogenesis via the endothelial cell-specific receptor tyrosine kinase Tie2. Blocking Ang2 binding to Tie2 decreases vessel density and inhibits tumor growth in various human xenograft models. However, not all tumors respond to Ang2 blockade, which could be due to high Ang1 levels acting as another ligand for Tie2, to Ang2 activities independent of Tie2, or to a weak role for Ang-2/Tie2 signaling in these tumors. To elucidate if Ang1 plays an integral role in tumor angiogenesis and growth, we compared the effects of blocking Ang2 or Tie2 in various tumor xenografts with differing levels of Ang1 expression. We found that in six out of seven tumor models, antibodies that specifically bind Ang2 (REGN910, 10 mg/kg 2x/wk) or that bind Tie2 and block binding of both Ang1 and Ang2 (REGN1376, 10 mg/kg 2x/wk) were equally efficacious at inhibiting tumor growth and decreasing vessel density. In only one tumor model (Calu-6), blockade of Tie2 with REGN1376 was more effective than blockade of Ang2 (REGN910) at reducing tumor growth and decreasing tumor vessel density. Although Calu-6 tumors express high Ang1 levels, other tumors tested (Lox, LS174T) have comparable or higher Ang1 levels and respond similarly to Ang2 and Tie2 blockade, thus the levels of Ang1 do not account for the differential response of Calu-6 tumors. Taken together, our data suggest that Ang2 is the dominant ligand for Tie2 in the microenvironment of most tumors, and further, that the effects of Ang2 are mediated primarily via Tie2. These findings support the approach to specifically block the binding of Ang2 to Tie2 as an anti-angiogenic therapy. Citation Format: Alexander P. Adler, Christopher Daly, Asma A. Parveen, Thomas Nevins, Jing Shan, Jeanette Fairhurst, Tammy Huang, Joel Martin, Nicholas Papadopoulos, George D. Yancopoulos, Gavin Thurston, Gavin Thurston, Alexandra Eichten. Blockade of angiopoietin-2 or Tie2 is equally effective at inhibiting tumor growth and reducing tumor vessel density in most human tumor xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4492. doi:10.1158/1538-7445.AM2014-4492

AB0206 Altered Expression of Angiopoietins and Tie-2 in Ischemic Lesions of UCD-206 Chickens, an Animal Model for Systemic Sclerosis

BackgroundSystemic sclerosis (SSc) is an autoimmune connective tissue disease affecting skin and internal organs. It is characterized by vascular damage, inflammation, and fibrosis. Microvascular injury and insufficient angiogenesis lead to...

Angiopoietin-2 Functions as a Tie2 Agonist in Tumor Models, Where It Limits the Effects of VEGF Inhibition

The angiopoietins Ang1 (ANGPT1) and Ang2 (ANGPT2) are secreted factors that bind to the endothelial cell-specific receptor tyrosine kinase Tie2 (TEK) and regulate angiogenesis. Ang1 activates Tie2 to promote blood vessel maturation and stabilization. In contrast, Ang2, which is highly expressed by tumor endothelial cells, is thought to inhibit Tie2 activity and destabilize blood vessels, thereby facilitating VEGF-dependent vessel growth. Here, we show that the inhibition of tumor xenograft growth caused by an Ang2-specific antibody (REGN910) is reversed by systemic administration of the Tie2 agonist Ang1. These results indicate that Ang2 blockade inhibits tumor growth by decreasing Tie2 activity, showing that Ang2 is a Tie2 activator. REGN910 treatment of tumors resulted in increased expression of genes that are repressed by Tie2 activation, providing further evidence that REGN910 inhibits Tie2 signaling. Combination treatment with REGN910 plus the VEGF blocker aflibercept reduced tumor vascularity and tumor perfusion more dramatically than either single agent, resulting in more extensive tumor cell death and more potent inhibition of tumor growth. Challenging the prevailing model of Ang2 as a destabilizing factor, our findings indicate that Ang2 plays a protective role in tumor endothelial cells by activating Tie2, thereby limiting the antivascular effects of VEGF inhibition. Thus, blockade of Ang2 might enhance the clinical benefits currently provided by anti-VEGF agents. .

195: Differential expression of tunica internal endothelial cell kinase in the center and periphery of normal and growth restricted placentas

Aberrant placental angiogenesis has a role in fetal growth restriction (FGR). Angiogenic factors have been found to differ between the center and periphery of normal placentas. Tunica Internal Endothelial Cell Kinase (Tie2) is an endothelial cell-specific receptor tyrosine kinase, whose activation is positively and negatively modulated by angiopoietin family. Angiopoietin-mediated modulation of Tie2 activation contributes to normal blood vessel development, maturation, maintenance, remodeling and stability.

Angiopoietin 2 Mediates the Differentiation and Migration of Neural Progenitor Cells in the Subventricular Zone after Stroke

Ischemic stroke stimulates neurogenesis in the adult rodent brain. The molecules underlying stroke-induced neurogenesis have not been fully investigated. Using real-time reverse transcription-PCR, we found that stroke substantially up-regulated angiopoietin 2 (ANG2), a proangiogenic gene, expression in subventricular zone neural progenitor cells. Incubation of neural progenitor cells with recombinant human ANG2 significantly increased the number of beta-III tubulin-positive cells, a marker of immature neurons, but did not alter the number of glial fibrillary acidic protein (GFAP)-positive cells, a marker of astrocytes, suggesting that ANG2 promotes neuronal differentiation. Blockage of the ANG2 receptor, Tie2, with small interference RNA (siRNA)-Tie2 attenuated recombinant human ANG2 (rhANG2)-increased beta-III tubulin mRNA levels compared with levels in the progenitor cells transfected with control siRNA. Chromatin immunoprecipitation analysis revealed that CCAAT/enhancer-binding protein (C/EBP beta) up-regulated by rhANG2 bound to beta-III tubulin, which is consistent with published data that there are several C/EBP beta binding sites in the promoter of beta-III tubulin gene. In addition, rhANG2 enhanced migration of neural progenitor cells measured by single neurosphere assay. Blockage of Tie2 with siRNA-Tie2 and a Tie2-neutralizing antibody did not suppress ANG2-enhanced migration. However, inhibition of matrix metalloproteinases with GM6001 blocked ANG2-enhanced migration. Collectively, our data suggest that interaction of ANG2, a proangiogenic factor, with its receptor Tie2 promotes neural progenitor cell differentiation into neuronal lineage cells, whereas ANG2 regulates neural progenitor cell migration through matrix metalloproteinases, which do not require its receptor Tie2.

Endogenous Erythropoietin Signaling Is Required for Normal Neural Progenitor Cell Proliferation

Erythropoietin (Epo) and its receptor (EpoR), critical for erythropoiesis, are expressed in the nervous system. Prior to death in utero because of severe anemia EpoR-null mice have fewer neural progenitor cells, and differentiated neurons are markedly sensitive to hypoxia, suggesting that during development Epo stimulates neural cell proliferation and prevents neuron apoptosis by promoting oxygen delivery to brain or by direct interaction with neural cells. Here we present evidence that neural progenitor cells express EpoR at higher levels compared with mature neurons; that Epo stimulates proliferation of embryonic neural progenitor cells; and that endogenous Epo contributes to neural progenitor cell proliferation and maintenance. EpoR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue but not in brain. Although these mice exhibited normal hematopoiesis and erythrocyte production and survived to adulthood, neural cell proliferation and viability were affected. Embryonic brain exhibited increased neural cell apoptosis, and neural cell proliferation was reduced in the adult hippocampus and subventricular zone. Neural cells from these animals were more sensitive to hypoxia/glutamate neurotoxicity than normal neurons in culture and in vivo. These observations demonstrate that endogenous Epo/EpoR signaling promotes cell survival in embryonic brain and contributes to neural cell proliferation in adult brain in regions associated with neurogenesis. Therefore, Epo exerts extra-hematopoietic function and contributes directly to brain development, maintenance, and repair by promoting cell survival and proliferation independent of insult, injury, or ischemia.

Targeting the Tie2/Tek Receptor in Astrocytomas

Tie2 is an endothelial cell-specific receptor tyrosine kinase, whose activation is positively and negatively modulated by angiopoietin-1 and angiopoietin-2, respectively. Angiopoietin-mediated modulation of Tie2 activation contributes to normal vessel development and stability, however, its role in tumor angiogenesis is not well known. We investigated the role of Tie2 activation in malignant astrocytomas, a common and highly vascularized primary human brain tumor. We found that Tie2 expression and activation increases with increasing malignancy grade of astrocytomas. Inhibition of Tie2, using a kinase-deficient Tie2 construct, decreases growth of malignant human astrocytoma subcutaneous and intracranial xenografts. Tie2 inactivation disrupted the tumor vascularity, with a decrease in microvascular density, increased presence of abnormally dilated vessels, and loss of interaction between endothelial cells and surrounding smooth muscle cells, all collectively resulting in increased tumor cell apoptosis. Overall, these findings strongly suggest that Tie2 activation contributes significantly to astrocytoma tumor angiogenesis and growth. We postulate that targeting Tie2 activation, either independently or in conjunction with other anti-angiogenic therapies, such as against vascular endothelial growth factor, is of potential clinical interest.

Ets motifs are necessary for endothelial cell-specific expression of a 723-bp Tie-2 promoter/enhancer in Hprt targeted transgenic mice.

Tie-2 is an endothelial cell-specific receptor tyrosine kinase that is involved in the remodeling of blood vessels and angiogenesis. Our goal was to characterize Tie-2 promoter function as a means of providing insight into the mechanisms of endothelial cell-specific gene regulation. When targeted to the Hprt locus of mice, a small Tie-2 promoter fragment (containing a 300-bp intronic enhancer coupled upstream to a 423-bp core promoter) (T-short) directed widespread endothelial cell expression in vivo. The T-short promoter contains 2 clusters of Ets sites, one in the first exon, the other in the intronic enhancer. In cultured endothelial cells, a combined mutation of the Ets motifs resulted in a significant reduction in promoter activity. Consistent with these results, the same Ets mutations resulted in a loss of detectable expression of the T-short promoter in all vascular beds with the notable exception of the brain. These results suggest that the T-short promoter contains information for widespread expression in the vascular tree, Ets sites are necessary for in vivo promoter activity, and the shorter Tie-2 fragment may be useful as a tool to direct heterologous gene expression within the intact endothelium.

Differential expression of angiopoietins 1 and 2 and their receptor Tie-2 in human endometrium

Angiogenesis, the growth of new capillaries from pre-existing blood vessels, is a physiological process involved in both normal menstrual cycling and implantation of the embryo. So far, very little is known about the expression of angiopoietins, growth factors involved in angiogenesis, in human endometrium. Both angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are ligands for the endothelial cell-specific receptor tyrosine kinase Tie-2. In this study we determined the mRNA expression of Ang-1, Ang-2 and Tie-2 by quantitative competitive RT/(QC)-PCR (including specifically designed competitor cDNA) in biopsied human endometrium throughout the menstrual cycle. We detected the mRNA for the angiopoietins in 30 out of 32 endometrial biopsies (94%), covering early proliferative (n = 4), mid proliferative (n = 12), late proliferative (n = 3), early secretory (n = 3), mid secretory (n = 5) and late secretory (n = 3) phases. Analysis of the target/competitor ratios (QC-PCR) revealed that Ang-1 mRNA expression was significantly up-regulated (P = 0.027) during the secretory phase of the menstrual cycle. In contrast, the expression levels of both Ang-2 mRNA and Tie-2 mRNA showed only minor variations at different cycle stages. These findings were confirmed by the relative expression ratio of Ang-1 versus Ang-2 in a multiplex PCR. The expression of Ang-1, Ang-2 and Tie-2 mRNA was detected in both isolated endometrial epithelial and stromal cell fractions. Immunohistochemical localization of the proteins revealed qualitative differences in both cell type and cycle stage expression. In conclusion, the enhanced Ang-1 expression during the secretory phase might serve to stabilize the newly developed blood vessels.

Loss of responsiveness to chemotactic factors by deletion of the C-terminal protein interaction site of angiomotin.

We have recently identified a novel protein, named angiomotin, by its ability to bind the angiogenesis inhibitor angiostatin in the yeast two-hybrid system. Angiomotin belongs to a family with two other members, AmotL-1 and -2 characterized by coiled-coil and C-terminal PDZ binding domains. Here we show that the putative PDZ binding motif of angiomotin serves as a protein recognition site and that deletion of three amino acids in this site results in inhibition of chemotaxis. Furthermore, endothelial cells expressing mutant angiomotin failed to migrate and form tubes in an in vitro tube formation assay. To study the effect of angiomotin on embryonic angiogenesis, we generated transgenic mice expressing wild-type angiomotin and the C-terminal deletion mutant driven by the endothelial cell-specific receptor tyrosine kinase (TIE) promoter. Expression of mutant angiomotin in endothelial cells inhibited migration into the neuroectoderm and intersomitic regions resulting in death at embryonic day 9.5. In contrast, mice expressing wild-type angiomotin developed normally and were fertile. These results suggest that the putative PDZ binding motif of angiomotin plays a critical role in regulating the responsiveness of endothelial cells to chemotactic cues.

Active Immunization Against the Vascular Endothelial Growth Factor Receptor flk1 Inhibits Tumor Angiogenesis and Metastasis

The vascular endothelial growth factor (VEGF) receptor fetal liver kinase 1 (flk1; VEGFR-2, KDR) is an endothelial cell–specific receptor tyrosine kinase that mediates physiological and pathological angiogenesis. We hypothesized that an active immunotherapy approach targeting flk1 may inhibit tumor angiogenesis and metastasis. To test this hypothesis, we first evaluated whether immune responses to flk1 could be elicited in mice by immunization with dendritic cells pulsed with a soluble flk1 protein (DC-flk1). This immunization generated flk1-specific neutralizing antibody and CD8+ cytotoxic T cell responses, breaking tolerance to self-flk1 antigen. Tumor-induced angiogenesis was suppressed in immunized mice as measured in an alginate bead assay. Development of pulmonary metastases was strongly inhibited in DC-flk1–immunized mice challenged with B16 melanoma or Lewis lung carcinoma cells. DC-flk1 immunization also significantly prolonged the survival of mice challenged with Lewis lung tumors. Thus, an active immunization strategy that targets an angiogenesis-related antigen on endothelium can inhibit angiogenesis and may be a useful approach for treating angiogenesis-related diseases.

Tie-2 and angiopoietin-1 expression in human thyroid tumors.

Tie-2 is an endothelial cell-specific receptor tyrosine kinase involved in vascular maturation and remodeling. Although its expression is considered to be restricted to vascular endothelial cells and hematopoietic progenitors, our immunohistochemical and in situ hybridization studies showed that Tie-2 and its ligand, angiopoietin (Ang)-l were expressed not only in benign and malignant human thyroid tumor cells but also in hyperplastic regions of adenomatous goiter. To confirm the expression in these tissues further, we used a laser capture microdissection system to isolate epithelial tumor cells from tissue specimens selectively, and demonstrated the expression of Tie-2 and Ang-1 mRNAs in tumor cells by RT-PCR analysis. Furthermore, Tie-2 and Ang-1 mRNAs and proteins were also detected in rat thyroid cell lines, FRTL-5 and PCCL-3. Our results suggest that Ang-1/Tie-2 signaling may be involved in the proliferation of thyroid epithelial cells.

A Gene for Inherited Cutaneous Venous Anomalies (“Glomangiomas”) Localizes to Chromosome 1p21-22

Venous malformations (VMs) are localized defects of vascular morphogenesis. They can occur in every organ system, most commonly in skin and muscle. They can cause pain and bleeding, and in some critical locations they can be life threatening. Usually venous anomalies occur sporadically, but families with dominant inheritance have been identified. Using linkage analysis, we have established in earlier reports that some families with inherited VMs show linkage to chromosome 9p21; the mutation causes ligand-independent activation of an endothelial cell-specific receptor tyrosine kinase, TIE-2. Here we show that VMs with glomus cells (known as "glomangiomas"), inherited as an autosomal dominant trait in five families, are not linked to 9p21 but, instead, link to a new locus, on 1p21-p22, called "VMGLOM" (LOD score 12.70 at recombination fraction.00). We exclude three known positional candidate genes, DR1 (depressor of transcription 1), TGFBR3 (transforming growth factor-beta receptor, type 3), and TFA (tissue factor). We hypothesize that cutaneous venous anomalies (i.e., glomangiomas) are caused by mutations in a novel gene that may act to regulate angiogenesis, in concert with the TIE-2 signaling pathway.

Allelic and locus heterogeneity in inherited venous malformations

Venous malformations are low-flow vascular lesions consisting of disorganized thin-walled vascular channels. These can occur sporadically but also as an autosomal dominant condition termed venous malformations, cutaneous and mucosal (VMCM; OMIM 600195). In two large unrelated kindreds mapping to chromosome 9, the identical R849W missense mutation was identified in the first kinase domain of Tie2, an endothelial cell-specific receptor tyrosine kinase. We report here the identification of four new kindreds with inherited venous malformations. Unlike the initial two families described, these four families demonstrate allelic and locus heterogeneity. In one of these families, the R849W mutation co-segregates with the disease phenotype. Three other families with venous malformations lack this mutation. One of these families is linked to markers near TIE2 on chromosome 9. In this family, we identified a novel mutation within the first kinase domain of Tie2 resulting in a Y897S change. Results from COS-1 cell transfections using expression constructs containing either the R849W or the Y897S mutation suggest that the receptors containing either mutation show ligand-independent hyperphosphorylation. These results suggest a gain-of-function mechanism for development of venous malformations in these families. Of the two remaining families, one excludes linkage to the TIE2 locus, establishing the existence of at least one additional locus for dominantly inherited venous malformations.

The Definition of Cell Type

What do we mean by “cell type?” Two articles in this issue of Circulation Research address this question in different ways. The first study, by Dube et al,1 uses very well-defined tools to define endothelial differentiation at the level of transcriptional control. The second study, by Kowal et al,2 looks for novel genes that distinguish two cell types. These studies, taken together, illustrate a major change in how we define cell type, a change that is about to be accelerated by the power of systematic genomics. Dube et al1 use in vitro systems to explore the promoter structure for the endothelial cell-specific receptor tyrosine kinase Tie2. Tie2 is a receptor for both angiopoetin-1 and angiopoetin-2. Like vascular endothelial growth factor, angiopoetin-1 is essential for normal vascular development whereas angiopoetin-2 is a naturally occurring antagonist for angiotensin I and Tie2. Thus, regulation of expression of the Tie2 receptor is likely a key issue in the formation of blood vessels.3 4 Dube et al1 identify a putative Ets transcription site in the Tie2 promoter. One of the proteins binding to this site, NERF2, can be shown to promote transcription in endothelial cells but not in the other cells studied. Related NERFs and other Ets binding factors either did not have this activity or were not found in the cultured cells used by these authors. Although mechanistically solid for transfected endothelial cell lines in vitro, the studies presented by Dube et al1 provide no evidence that NERF2 is expressed in endothelial cells in vivo, and the in vitro studies only use Chinese hamster ovary, 293, and CV-1 cell lines as comparison cells. These aneuploid, heterogeneously derived cell lines may or may not provide a representative comparison with the endothelial cell strains used in the present study, much …

The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R.

Tek/Tie2 is an endothelial cell-specific receptor tyrosine kinase that has been shown to play a role in vascular development of the mouse. Targeted mutagenesis of both Tek and its agonistic ligand, Angiopoietin-1, result in embryonic lethality, demonstrating that the signal transduction pathway(s) mediated by this receptor are crucial for normal embryonic development. In an attempt to identify downstream signaling partners of the Tek receptor, we have used the yeast two-hybrid system to identify phosphotyrosine-dependent interactions. Using this approach, we have identified a novel docking molecule called Dok-R, which has sequence and structural homology to p62dok and IRS-3. Mapping of the phosphotyrosine-interaction domain within Dok-R shows that Dok-R interacts with Tek through a PTB domain. Dok-R is coexpressed with Tek in a number of endothelial cell lines. We show that coexpression of Dok-R with activated Tek results in tyrosine phosphorylation of Dok-R and that rasGAP and Nck coimmunoprecipitate with phosphorylated Dok-R. Furthermore, Dok-R is constitutively bound to Crk presumably through the proline rich tail of Dok-R. The cloning of Dok-R represents the first downstream substrate of the activated Tek receptor, and suggests that Tek can signal through a multitude of pathways.