Endothelial Cell-specific Tyrosine Kinase Research Articles

Prospects for the use of biochemical markers in placental in-growth

Placental in-growth is a severe obstetric pathology characterized by invasive placentation and associated with a high-risk of life-threatening hemorrhage. Despite the widespread use of instrumental methods of examination, timely diagnosis of placental in-growth is a challenging issue. Here, we reviewed the existing biochemical markers used for early detection and confirmation of placental in-growth, their specificity and sensitivity, and correlation with gestational age. Significant results were found for the following substances: pregnancy-associated plasma protein A (PAPP-A) in the first trimester, alpha-fetoprotein (AFP) and human beta-chorionic gonadotropin (Beta-hCG) in the second trimester, brain natriuretic peptide, antithrombin III, plasminogen activator inhibitor type I, soluble Tie-2 receptor (endothelial cell-specific tyrosine kinase receptor), and soluble vascular endothelial growth factor receptor-2. Our findings support the use of the aforementioned biomarkers as screening method for placental in-growth in medical practice.

MET somatic activating mutations are responsible for lymphovenous malformation and can be identified using cell-free DNA next generation sequencing liquid biopsy

ObjectiveGermline mutations of either the endothelial cell-specific tyrosine kinase receptor TIE2 or the glomulin (GLMN) gene are responsible for rare inherited venous malformations. Both genes affect the hepatocyte growth factor receptor c-Met, inducing vascular smooth muscle cell migration. Germline mutations of hepatocyte growth factor are responsible for lymphatic malformations, leading to lymphedema. The molecular alteration leading to the abnormal mixed vascular anomaly defined as lymphovenous malformation has remained unknown. MethodsA group of 4 patients with lymphovenous malformations were selected. Plasma was obtained from both peripheral and efferent vein samples at the vascular malformation site for cell-free DNA extraction. When possible, we analyzed tissue biopsy samples from the vascular lesion. ResultsWe have demonstrated that in all four patients, an activating MET mutation was present. In three of the four patients, the same pathogenic activating mutation, T1010I, was identified. The mutation was found at the tissue level for the patient with tissue samples available, confirming its causative role in the lymphovenous malformations. ConclusionsIn the present study, we have demonstrated that cell-free DNA next generation sequencing liquid biopsy is able to identify the MET mutations in affected tissues. Although a wider cohort of patients is necessary to confirm its causative role in lymphovenous malformations, these data suggest that lymphovenous malformations could result from postzygotic somatic mutations in genes that are key regulators of lymphatic development. The noninvasiveness of the method avoids any risk of bleeding and can be easily performed in children. We are confident that the present pioneering results have provided a viable alternative in the future for lymphovenous malformation diagnosis, allowing for subsequent therapy tailored to the genetic defect.

Evaluation of first trimester serum soluble endothelial cell-specific tyrosine kinase receptor in normal and affected pregnancies

Aims: To assess soluble endothelial cell-specific tyrosine kinase receptor (sTie-2) levels in the first trimester of pregnancy and its association with adverse pregnancy outcomes; and examine the predictive accuracy.Study design: In this nested case-control study, serum sTie-2 levels were measured in 2616 women with singleton pregnancies attending first trimester screening in New South Wales, Australia. Multivariate logistic regression models were used to assess the association and predictive accuracy of serum sTie-2 with subsequent adverse pregnancy outcomes.Results: Median (interquartile range) sTie-2 for the total population was 19.6 ng/ml (13.6–26.4). Maternal age, weight, and smoking status significantly affected sTie-2 levels. There was no difference in serum sTie-2 between unaffected and women with adverse pregnancy outcomes. After adjusting for maternal and clinical risk factors, low sTie-2 (<25th centile) was associated with preeclampsia (Adjusted odds ratio: 1.61; 95% CI: 1.01–2.57), however, the accuracy of sTie-2 in predicting preeclampsia was not different from chance (AUC = 0.54; p = 0.08) and does not add valuable predictive information to maternal and clinical risk factors.Conclusions: Our findings suggest that low sTie-2 levels are associated with preeclampsia, however, it does not add valuable information to clinical and maternal risk factor information in predicting preeclampsia or any other adverse pregnancy outcomes.

Clinical significance of serum soluble Tie1 levels in patients with systemic sclerosis

Tie1 is an endothelial cell-specific tyrosine kinase receptor, which maintains vascular integrity and regulates angiogenesis via modulating angiopoietin/Tie2 signaling. Since the altered angiogenesis is closely related to the developmental process of systemic sclerosis (SSc), we herein investigated the clinical significance of serum soluble Tie1 (sTie1) levels and the expression levels of Tie1 in dermal microvascular endothelial cells (DMECs) in patients with SSc. Although serum sTie1 levels were comparable among total SSc, diffuse cutaneous SSc (dcSSc), limited cutaneous SSc (lcSSc), and healthy controls, SSc patients with decreased serum sTie1 levels had significantly shorter disease duration than those with serum sTie1 levels not decreased. In SSc patients with disease duration of >6 years, the prevalence of clinical symptoms associated with proliferative vasculopathy, such as digital ulcers, scleroderma renal crisis, and elevated right ventricular systolic pressure, was significantly higher in patients with decreased serum sTie1 levels than in those with serum sTie1 levels not decreased. In immunohistochemistry, Tie1 expression was reduced in DMECs of SSc patients with disease duration of <3 years compared with those of healthy controls. Collectively, in SSc patients with short disease duration, decreased serum sTie1 levels may reflect the down-regulation of Tie1 in DMECs. The decrease in serum sTie1 levels may serve as a marker of proliferative vasculopathy in SSc with disease duration of >6 years.

Significant decrease in maternal serum concentrations of angiopoietin‐1 and ‐2 after delivery

To characterize the physiological distribution of angiopoietins (Ang)-1 and Ang-2 and soluble endothelial cell-specific tyrosine kinase receptor-2 (Tie-2) at term and following delivery. A prospective, descriptive study. Helsinki University Central Hospital. Twenty healthy term pregnant women undergoing elective cesarean delivery and their newborns. The concentrations were analysed by enzyme-linked immunosorbent assay in maternal antepartum and the first postpartum day sera, umbilical serum, amniotic fluid and maternal and newborn urine. Concentrations of Ang-1, Ang-1 and Tie-2. Results. Concentrations of maternal serum Ang-1 and Ang-2 decreased after delivery {[median (range)]: Ang-1, from 33 (25-51) to 30 (18-49) ng/mL, p= 0.017; and Ang-2, from 5.4 (1.8-18) to 1.4 (0.7-4.6) ng/mL, p < 0.0001}, whereas Tie-2 concentrations remained stable [23 (13-41) vs. 25 (14-29) ng/mL, p= 0.107]. Compared with maternal antepartum serum, umbilical serum concentrations of Ang-1 [46 (28-59) ng/mL, p < 0.0001] and Tie-2 [45 (21-71) ng/mL, p < 0.0001] were higher and those of Ang-2 similar [5.4 (1.8-18) vs. 4.2 (2.9-6.0) ng/mL; p= 0.067]. Low concentrations of Ang-1 [1.2 (0.1-2.2) ng/mL], Ang-2 [1.1 (0.3-4.1) ng/mL] and Tie-2 [0.4 (0.08-0.9) ng/mL] were observed in amniotic fluid, but they were undetectable in newborn urine and in most of the maternal urine samples. Maternal Ang-1 and Ang-2 concentrations decreased following delivery. Umbilical concentrations of Ang-1 and Tie-2 were higher than the maternal concentrations.

The Expression of Angiopoietin-1 and −2 in the Endometrium of Women with Abnormal Bleeding Induced by an Intra-Uterine Device

This study identified differences in expression of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in the endometrium of 33 women with abnormal bleeding induced by an intra-uterine device (IUD; 23 proliferative phase, 10 secretory phase) compared with 28 control samples from normal endometria in women without an IUD fitted (12 proliferative phase, 16 secretory phase). Expression of Ang-1, Ang-2 and endothelial cell-specific tyrosine kinase receptor-2 (Tie-2) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), and Ang-1 and Ang-2 protein levels were detected by immunohistochemistry. The RT-PCR results showed significantly decreased Ang-1 expression in the endometrium from IUD patients during both phases compared with the control women. Expression of Tie-2 mRNA and the Ang-1:Ang-2 mRNA ratio were also significantly decreased in endometria from IUD patients in the secretory phase compared with the control women. Immunohistochemical analysis showed elevated Ang-2 protein levels in secretory phase endometrium from IUD patients compared with the control women. These results suggest that the angiopoietin/Tie-2 system promotes vascular remodelling in the endometrium and that changes in the expression of Ang-1, Ang-2 and Tie-2 may contribute to abnormal uterine bleeding in some IUD users.

Maternal Serum Angiopoietin-1 and -2 and Tie-2 in Early Pregnancy Ending in Preeclampsia or Intrauterine Growth Retardation

The antiangiogenic growth factor angiopoietin-2 (Ang-2) antagonizes, whereas angiopoietin-1 (Ang-1) activates the endothelial cell-specific tyrosine kinase receptor-2 (Tie-2). In preeclampsia, circulating concentrations of Ang-1 are increased and those of Ang-2 and Tie-2 are decreased. We wanted to study whether maternal serum concentrations of Ang-1, Ang-2, and Tie-2 are altered at gestational wk 12-15 or 16-20 in women with subsequent preeclampsia or intrauterine growth retardation (IUGR). This was a case-control study. The study was conducted in Helsinki University Central Hospital, a tertiary referral center. This study comprised 124 pregnant women, of whom 49 developed preeclampsia and 16 gave birth to infants with IUGR, and 59 healthy women served as controls. Serum concentrations of Ang-1, Ang-2, and Tie-2 were assessed by ELISA. Data were combined with our earlier data on soluble VEGF receptor (sVEGFR)-1. At gestational wk 12-15, the median concentrations of Ang-1, Ang-2, or Tie-2 were all similar between the study groups. At 16-20 wk, Ang-2 concentrations were higher in women with subsequent preeclampsia [25.0 ng/ml, 19.3-39.5 ng/ml; median, interquartile range (IQR)] than in the controls (17.7 ng/ml, 10.8-27.4 ng/ml, P = 0.006). The odds ratio of high Ang-2 concentrations for subsequent preeclampsia was 4.2 (95% confidence interval 1.4-12.6; P = 0.011) and high Ang-2 combined with high sVEGFR-1, 6.4 (95% confidence interval 2.2-18.7; P = 0.001). Maternal serum Ang-2 concentrations are increased prior to preeclampsia. High concentrations of both Ang-2 and sVEGFR-1 indicate subsequent disease.

Transcriptome of Angiopoietin 1–Activated Human Umbilical Vein Endothelial Cells

Angiopoietin 1 (Ang-1) is the main ligand for endothelial cell-specific tyrosine kinase (Tie-2) receptors and it promotes migration and proliferation and inhibits apoptosis and vascular leakage. The exact mechanisms through which the Ang-1 exerts these effects remain unclear. The authors exposed human umbilical vein endothelial cells (HUVECs) to Ang-1 (300 ng/mL) for 4 h and conducted gene expression profiling using oligonucleotide microarrays. Real-time polymerase chain reaction (PCR) was also conducted to verify several of the genes that were regulated by Ang-1. Exposure to Ang-1 resulted in induction of 86 genes that are involved in endothelial cell (EC) proliferation, differentiation, migration, and survival. Thirty-six of these genes, including stanniocalcin, cyclin D1, vascular endothelial growth factor C, fms-related tyrosine kinase 1, interleukin 8, and CXCR4 have previously been shown to be induced by vascular endothelial growth factor (VEGF), suggesting significant similarities between VEGF and Ang-1 pathways. Ang-1 exposure also inhibited mRNA expressions of 49 genes, most of which are involved in cell cycle arrest, apoptosis, and suppression of transcription. These results indicate that Ang-1 triggers coordinated responses in endothelial cells designed to inhibit the expression of proapoptotic and antiproliferative genes and up-regulate proproliferative, proangiogenic, and antiapoptotic pathways. Moreover, we also found that the Erk1/2, phosphatidylinositol (PI) 3-kinase, and the mTOR pathways are involved in Ang-1-induced gene expression in HUVECs.

Inhibition of Tie-2 Signaling Induces Endothelial Cell Apoptosis, Decreases Akt Signaling and Induces Endothelial Cell Expression of the Endogenous Anti-Angiogenic Molecule, Thrombospondin-1

Small molecule inhibitors of endothelial cell specific tyrosine kinases are currently under investigation as potential means to block tumor angiogenesis. We have investigated the utility of blocking Tie-2 signaling in endothelial cells as a potential anti-angiogenic strategy. We have found that interruption of Tie-2 signaling either via RNAi or overexpression of a kinase-dead Tie-2 led to loss of endothelial cell viability, even in the presence of serum. Mechanistically, this is linked to a block in Akt signaling and increased thrombospondin expression. Thrombospondins are endogenous anti-angiogenic matricellular proteins known to regulate tumor growth and angiogenesis. We observed that both Tie-2 and subsequent PI3Kinase signaling regulates thrombospondin-1 expression. These data have lead to the model that Angiopoietin signaling through Tie-2 activates PI3Kinase/Akt, which represses thrombospondin expression. Thus, targeting Tie-2 with small molecules maybe efficacious as an anti-angiogenic therapy.

Measurement of the soluble angiopoietin receptor tie-2 in patients with coronary artery disease: development and application of an immunoassay.

The angiopoietin family has emerged as a group of crucial growth factors to normal angiogenesis. They are essential to the development of the mature vessel wall and interact with the endothelium via endothelial cell-specific tyrosine kinase receptors, tie-1 and tie-2. The role of the tie-2 receptor has been extensively examined in neovascularization associated with malignancy, but little is known about the role it may play in atherosclerosis, a condition whose pathophysiology also involves angiogenesis. Soluble tie-2 has been detected in the plasma of healthy controls, but this has yet to be applied to patients in the clinical setting. We developed an ELISA to detect plasma tie-2 levels and applied these to a clinical setting. The intra- and interassay coefficients of variation for the assay were 4.7% and 9.6%, respectively. We then measured levels of tie-2, vascular endothelial growth factor (VEGF), another factor associated with angiogenesis, and the soluble VEGF receptor Flt-1 (sFlt-1) in 75 patients with coronary artery disease [25 with acute myocardial infarction (AMI), 25 with acute coronary syndromes (ACS) and 25 with stable angina] and 25 healthy controls. Median [IQR, interquartile range] levels of tie-2 were significantly higher in the coronary artery disease patients (AMI 12 [10-17] ng mL-1, ACS 10 [9-14] ng mL-1, stable angina 9 [3-11] ng mL-1) when compared with the controls (7.5 [7-9] ng mL-1P = 0.004). As expected, levels of VEGF and sFlt were significantly different from those in the healthy controls (P = 0.011 and P < 0.001, respectively). Significant correlations were found between levels of tie-2 and VEGF (Spearman r = 0.59, P < 0.001), tie-2 and sFlt-1 (r = 0.45, P < 0.001) and VEGF and sFlt-1 (r = 0.56, P < 0.001) in the whole study group. We suggest that tie-2 may be potentially used as a marker of angiogenesis in atherosclerosis and may help elucidate the role of the angiopoietin/tie-2 system in atherogenesis.

Lymphangiogenic growth factors, receptors and therapies.

The lymphatic vasculature is essential for the maintenance of normal fluid balance and for the immune responses, but it is also involved in a variety of diseases. Hypoplasia or dysfunction of the lymphatic vessels can lead to lymphedema, whereas hyperplasia or abnormal growth of these vessels are associated with lymphangiomas and lymphangiosarcomas. Lymphatic vessels are also involved in lymph node and systemic metastasis of cancer cells. Recent novel findings on the molecular mechanisms involved in lymphatic vessel development and regulation allow the modulation of the lymphangiogenic process and specific targeting of the lymphatic endothelium. Recent results show that the homeodomain transcription factor Prox-1 is an important lymphatic endothelial cell (LEC) fate-determining factor which can induce LEC-specific gene transcription even in blood vascular endothelial cells (BECs). This suggests that the distinct phenotypes of cells in the adult vascular endothelium are plastic and sensitive to transcriptional reprogramming, which might be useful for future therapeutic applications involving endothelial cells. Vascular endothelial growth factor-C (VEGF-C) and VEGF-D are peptide growth factors capable of inducing the growth of new lymphatic vessels in vivo in a process called lymphangiogenesis. They belong to the larger family which also includes VEGF, placenta growth factor (PlGF) and VEGF-B, VEGF-C and VEGF-D are ligands for the endothelial cell specific tyrosine kinase receptors VEGFR-2 and VEGFR-3. In adult human as well as mouse tissues VEGFR-3 is expressed predominantly in lymphatic endothelial cells which line the inner surface of lymphatic vessels. While VEGFR-2 is thought to be the main mediator of angiogenesis, VEGFR-3 signaling is crucial for the development of the lymphatic vessels. Heterozygous inactivation of the VEGFR-3 tyrosine kinase leads to primary lymphedema due to defective lymphatic drainage in the limbs. Other factors that seem to be involved in lymphangiogenesis include the Tie/angiopoietin system, neuropilin-2 and integrin alpha 9. VEGF-C induces lymphatic vessel growth, but high levels of VEGF-C also resulted in blood vessel leakiness and growth. The VEGFR-3-specific mutant form of VEGF-C called VEGF-C156S lacks blood vascular side effects but is sufficient for therapeutic lymphangiogenesis in a mouse model of lymphedema. As VEGF-C156S is a specific lymphatic endothelial growth factor in the skin, it provides an attractive molecule for pro-lymphangiogenic therapy.

Potentiation of angiogenic response by ischemic and hypoxic preconditioning of the heart.

This review is intended to discuss the newly discovered role of preconditioning which should make it an attractive therapeutic stimulus for repairing the injured myocardium. We recently found that apart from rendering the myocardium tolerant to ischemic reperfusion injury, preconditioning also potentiates angiogenesis. Our study demonstrated for the first time that both ischemic and hypoxic preconditioning triggered myocardial angiogenesis at the capillary and arteriolar levels which nicely corroborated with the improved myocardial contractile function. Hypoxic preconditioning resulted in the stimulation of VEGF, the most potent angiogenic factor known to date. In concert, endothelial cell specific tyrosine kinase receptors, Tie 1, Tie 2 and Flt-1 and Flk-1 were also significantly enhanced in the preconditioned myocardium. The redox-regulated transcription factor NF kappa B was found to play an essential role in the preconditioning regulation of angiogenesis.

Vascular endothelial growth factor receptor-3.

Cell-cell communication during vascular development and tumour angiogenesis seems to involve at least five endothelial cell-specific tyrosine kinase receptors belonging to two distinct subclasses: two receptors of the Tie family, and three vascular endothelial cell growth factor receptors, VEGFR-1, -2 and -3, originally named Fltl (Fms-like tyrosine kinase), KDR/Flk-1 (Kinase insert-domain containing receptor or fetal-liver kinase-1) and Flt4, respectively. VEGFRs are subclass-III receptor tyrosine kinases, homologous to the platelet-derived growth factor (PDGF)-receptor family, having seven immunoglobulin homology domains in the extracellular domain, and a tyrosine kinase intracellular domain split by a kinase insert sequence (for recent reviews, see Klagsbrun and D’Amore 1996; Folkman and D’Amore 1996; Mustonen and Alitalo 1995; Korpelainen and Alitalo, 1998, Claesson-WELSH, this book).

Role of vascular endothelial growth factor in the response to vessel injury

In the post-embryonic life, physiological angiogenesis is tightly controlled. Angiogenesis also occurs in pathological circumstances such as tumor vessel proliferation, retinal neovascularization and ischemia. The development of collateral circulation is not only not deleterious, but life saving. Other cases such as neoplastic neovascularization are the basis of the continuous growth of tumors and metastases, and therefore constitute a target of therapeutical efforts. Among a list of molecules able to control angiogenesis, we emphasize the pivotal role of vascular endothelial growth factor (VEGF). VEGF is a potent mitogen for endothelial cells, but is devoid of mitogenic activity for other cell types. VEGF is a polypeptide with four main different isoforms that are remarkably different in terms of solubility and affinity for matrix proteins. VEGF interacts with two endothelial cell-specific tyrosine kinase receptors. The main interest of its study lies in VEGF's role in pathological angiogenic processes, where an increase in the VEGF mRNA expression has been consistently observed. An interesting example is the up-regulation of VEGF's and VEGF receptors' mRNA in a considerable number of human tumors and retina, where they have a critical role in the development of neovascularization. In recent work in our laboratory, we have found further potential interactions of VEGF with pathophysiological mechanisms, namely, the increase in VEGF gene expression under exposure to reactive oxygen species and the positive interaction between VEGF and erythropoietin. VEGF has outstanding possibilities for therapeutic applications aimed at inhibiting or favoring the development of new vessels.