Endoglin Expression Research Articles

Severe Intrauterine Growth Restriction Pregnancies Have Increased Placental Endoglin Levels: Hypoxic Regulation via Transforming Growth Factor-β3

Endoglin, a co-receptor for transforming growth factor (TGF)-beta 1 and -beta 3 is expressed in the human placenta and plays an important role in the pathogenesis of preeclampsia. Because preeclampsia is associated with hypoxia, and because TGF-beta 3 is overexpressed in preeclamptic pregnancies, we examined the effect of oxygen and TGF-beta 3 on placental endoglin expression and investigated its expression in pathological models of placental hypoxia such as intrauterine growth restriction (IUGR) pregnancies. Endoglin expression was high at 4 to 9 weeks of gestation, when oxygen tension is low, and decreased after 10 weeks, when oxygen tension increases. Exposure of villous explants to low oxygen (3% O2) resulted in elevated expression of both membrane and soluble endoglin compared to standard conditions (20% O2). Moreover, addition of TGF-beta 3 to villous explants under low oxygen conditions increased the expression of endoglin compared to nontreated explants whereas addition of TGF-beta 3-neutralizing antibodies inhibited the low oxygen stimulatory effect on endoglin expression. Endoglin and soluble endoglin expression were significantly increased in placentas of IUGR singletons compared to controls and in the IUGR twin placentas relative to both the control co-twin and the normal twins. These data demonstrate that oxygen regulates the placental expression of endoglin via TGF-beta 3. Reduced placental perfusion leading to placental hypoxia might contribute to the increased expression of endoglin in IUGR pregnancies.

Whole-genome microarray and targeted analysis of angiogenesis-regulating gene expression (ENG, FLT1, VEGF, PlGF) in placentas from pre-eclamptic and small-for-gestational-age pregnancies

Objective. To compare the placental pathology associated with pre-eclampsia (PE) and/or fetal growth restriction, the transcriptomes of placental tissues from PE and small-for-gestational-age (SGA) pregnancies were explored. In addition, a targeted analysis of angiogenesis-regulating gene expression was performed.Methods. Whole-genome microarray analysis was performed on placental tissue from gestational age-matched PE (n = 10), SGA (n = 8) and PE + SGA (n = 10) pregnancies. The expression of genes regulating angiogenesis (endoglin (ENG), fms-related tyrosine kinase 1 (FLT1), vascular endothelial growth factor (VEGF) and placental growth factor (PlGF)) was analyzed by quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR).Results. Microarray analysis did not reveal any significant differences between groups. However, an increased expression of ENG and FLT1 was detected by qRT-PCR in the PE + SGA group.Conclusions. The placental transcriptome did not differ between groups, although an increased anti-angiogenic gene expression in PE + SGA was observed with qRT-PCR analysis. Based on this, we conclude that although microarray technology may represent a powerful tool in generating new hypothesis in complex fields, it may not be sensitive enough to detect subtle changes in gene expression.

A Role for Endoglin as a Suppressor of Malignancy during Mouse Skin Carcinogenesis

Endoglin is a membrane glycoprotein that acts as a coreceptor for transforming growth factor-beta. We and others have previously suggested a function of endoglin as a tumor suppressor in epithelial cancer. Here, we study the expression of endoglin during chemical mouse skin carcinogenesis. We find that shedding of membrane endoglin, allowing the secretion of a soluble endoglin form, is a late event associated with progression from squamous to spindle cell carcinomas. Knockdown of endoglin in transformed keratinocytes activates the Smad2/3 signaling pathway resulting in cell growth arrest, delayed tumor latencies, and a squamous to spindle phenotypic conversion. Forced expression of the long endoglin isoform in spindle carcinoma cells blocks transforming growth factor-beta1 stimulation of Smad2/3 signaling and prevents tumor formation. In contrast, expression of the short endoglin isoform has no effect on spindle cell growth in vitro or in vivo. Our results show that endoglin behaves as a suppressor of malignancy during the late stages of carcinogenesis. Therefore, disruption of membrane endoglin emerges as a crucial event for progression to spindle cell carcinomas.

Elucidation of the Phenotypic, Functional, and Molecular Topography of a Myeloerythroid Progenitor Cell Hierarchy

The major myeloid blood cell lineages are generated from hematopoietic stem cells by differentiation through a series of increasingly committed progenitor cells. Precise characterization of intermediate progenitors is important for understanding fundamental differentiation processes and a variety of disease states, including leukemia. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin, and CD41. Our studies revealed a hierarchy of myeloerythroid progenitors with distinct lineage potentials. The global gene expression signatures of these subsets were consistent with their functional capacities, and hierarchical clustering analysis suggested likely lineage relationships. These studies provide valuable tools for understanding myeloid lineage commitment, including isolation of an early erythroid-restricted precursor, and add to existing models of hematopoietic differentiation by suggesting that progenitors of the innate and adaptive immune system can separate late, following the divergence of megakaryocytic/erythroid potential.

Transforming Growth Factor-β Binding Receptor Endoglin (CD105) Expression in Esophageal Cancer and in Adjacent Nontumorous Esophagus as Prognostic Predictor of Recurrence

We hypothesized that the potent neovascularization marker endoglin (CD105), by differentially highlighting a subset of microvessels (MV) in esophageal cancer (EC), could provide better prognostic/therapeutic information than the panendothelial marker CD34, which also highlights MV. Endoglin messenger ribonucleic acid (mRNA) expression in normal, malignant, and adjacent nontumorous esophagus tissue was quantified by real-time reverse-transcription polymerase chain reaction (RT-PCR). Sections of formalin-fixed, paraffin-embedded tissues were analyzed immunohistochemically for CD105 and CD34. MV density was counted following a standard protocol. Circulating soluble endoglin levels were determined in patient and control sera, and compared with clinical outcome. CD105 mRNA was upregulated by a median factor of 2.89 in ECs versus controls. In 28% of patients, CD105 mRNA was upregulated by a median factor of 2.65 in adjacent non-tumorous versus normal tissue. In tumor tissues, CD105 was stained negatively or positively only in a subset of MV. CD34 always showed positive extensive MV staining. In adjacent nontumorous esophagus, CD105 rarely showed diffuse MV staining, while CD34 stained blood-vessel endothelial cells in all non-neoplastic tissue. CD105 expression was high in residual highly dysplastic Barrett's-type mucosa associated with some adenocarcinomas. No statistically significant difference in endoglin serum levels appeared between patients and normal subjects. Correlation with clinicopathological data showed higher intra-tumor MV-CD105+ scores at more-advanced clinical stages. High-scoring MV-CD105+ patients had significantly shorter disease-free and overall survival; MV-CD34+ density was not survival related. Diffuse CD105 expression in adjacent nontumorous esophagus predicted poorer disease-free and overall survival. Our findings could help identify EC patients who may benefit from targeted anti-angiogenic therapies.

Endoglin Is Not Critical for Hematopoietic Stem Cell Engraftment and Reconstitution but Regulates Adult Erythroid Development

Endoglin is a transforming growth factor-beta (TGF-beta) accessory receptor recently identified as being highly expressed on long-term repopulating hematopoietic stem cells (HSC). However, little is known regarding its function in these cells. We have used two complementary approaches toward understanding endoglin's role in HSC biology: one that efficiently knocks down expression via lentiviral-driven short hairpin RNA and another that uses retroviral-mediated overexpression. Altering endoglin expression had functional consequences for hematopoietic progenitors in vitro such that endoglin-suppressed myeloid progenitors (colony-forming unit-granulocyte macrophage) displayed a higher degree of sensitivity to TGF-beta-mediated growth inhibition, whereas endoglin-overexpressing cells were partially resistant. However, transplantation of transduced bone marrow enriched in primitive hematopoietic stem and progenitor cells revealed that neither endoglin suppression nor endoglin overexpression affected the ability of stem cells to short-term or long-term repopulate recipient marrow. Furthermore, transplantation of cells altered in endoglin expression yielded normal white blood cell proportions and peripheral blood platelets. Interestingly, decreasing endoglin expression increased the clonogenic capacity of early blast-forming unit-erythroid progenitors, whereas overexpression compromised erythroid differentiation at the basophilic erythroblast phase, suggesting a pivotal role for endoglin at key stages of adult erythropoietic development.

Endothelial expression of endoglin in normocholesterolemic and hypercholesterolemic C57BL/6J mice before and after atorvastatin treatment

Endoglin (CD105) is a homodimeric transmembrane glycoprotein strongly related to transforming growth factor (TGF)-beta signaling and many pathological states. In this study, we wanted to evaluate whether endoglin is expressed in normocholesterolemic and hypercholesterolemic C57BL/6J mice as well as whether it is affected by atorvastatin treatment in these mice. C57BL/6J mice were fed with chow diet or an atherogenic diet for 12 weeks after weaning. In 2 atorvastatin-treated groups, mice were fed the same diets (chow or atherogenic) as described above except atorvastatin was added at the dosage of 10 mg x kg(-1) x day(-1) for the last 8 weeks before euthanasia. Biochemical analysis of blood samples revealed that administration of atherogenic diet significantly increased levels of total cholesterol, VLDL, LDL, and decreased levels of HDL. Atorvastatin treatment resulted in a significant decrease in total cholesterol and VLDL only in mice fed by atherogenic diet. Quantitative stereological analysis revealed that atorvastatin significantly decreased endothelial expression of endoglin in C57BL/6J mice fed the atherogenic diet. In conclusion, we demonstrated that endothelial expression of endoglin is upregulated by hypercholesterolemia and decreased by the hypolipidemic effect of atorvastatin in C57BL/6J mice, suggesting that endoglin expression could be involved in atherogenesis.

Endoglin is required for myogenic differentiation potential of neural crest stem cells

Genetic studies show that TGFβ signaling is essential for vascular development, although the mechanism through which this pathway operates is incompletely understood. Here we demonstrate that the TGFβ auxiliary coreceptor endoglin ( eng, CD105) is expressed in a subset of neural crest stem cells (NCSCs) in vivo and is required for their myogenic differentiation. Overexpression of endoglin in the neural crest caused pericardial hemorrhaging, correlating with altered vascular smooth muscle cell investment in the walls of major vessels and upregulation of smooth muscle α-actin protein levels. Clonogenic differentiation assay of NCSCs derived from neural tube explants demonstrated that only NCSC expressing high levels of endoglin (NCSC CD105+) had myogenic differentiation potential. Furthermore, myogenic potential was deficient in NCSCs obtained from endoglin null embryos. Expression of endoglin in NCSCs declined with age, coinciding with a reduction in both smooth muscle differentiation potential and TGFβ1 responsiveness. These findings demonstrate a cell autonomous role for endoglin in smooth muscle cell specification contributing to vascular integrity.

Coculture with Prostate Cancer Cells Alters Endoglin Expression and Attenuates Transforming Growth Factor-β Signaling in Reactive Bone Marrow Stromal Cells

A dynamic interplay between prostate cancer cells and reactive bone stroma modulates growth of metastases within bone. We used microarray analysis to screen for changes in gene expression in bone marrow stromal cells cocultured with prostate cancer cells and found reduced expression of endoglin, a transmembrane glycoprotein that functions as an auxiliary coreceptor for members of the transforming growth factor beta (TGF-beta) family of cytokines. The downstream TGF-beta/bone morphogenetic protein signaling pathway including Smad1 and Smad2/3 also was attenuated, as was Smad-dependent gene transcription. Smad1/5/8-dependent inhibitor of DNA binding 1 expression and Smad2/3-dependent plasminogen activator inhibitor I expression both were decreased and were accompanied by decreased cell proliferation. Small interfering RNA-mediated knockdown of endoglin in HS-5 cells verified that the effects on signaling were a direct result of the attenuation of endoglin. These data illustrate that endoglin acts as a positive regulator of both activin receptor-like kinase 1-induced Smad1/5/8 activation and activin receptor-like kinase 5-induced Smad2/3 activation in bone marrow stromal cells. In addition, the data illustrate that one early event of metastasis upon the arrival of prostate cancer cells into the bone stroma is attenuated endoglin expression in the stromal cells, which subsequently alters Smad signaling and cell proliferation. We hypothesize that coculture of bone marrow stromal cells with prostate cancer cells alters TGF-beta signaling in the stromal cells, ultimately facilitating growth of the cancer cells in the bone compartment. Collectively, these studies suggest that prostate cancer cells modulate TGF-beta responsiveness of bone marrow stroma as one means of facilitating their own growth in bone.

Endoglin Differentially Modulates Antagonistic Transforming Growth Factor-β1 and BMP-7 Signaling

Transforming growth factor-beta1 (TGF-beta1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-beta type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-beta/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-beta1 signaling and the role of endoglin for both pathways in endoglin-deficient L(6)E(9) cells. In this myoblastic cell line, TGF-beta1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-beta1) are two examples. TGF-beta1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-beta1-induced collagen I expression and (CAGA)(12)-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-beta1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.

P142 Expression of Ki-67 and endoglin (CD 105) proteins in stages I and II squamous cell carcinoma of oral tongue and floor of the mouth: association with demographic, clinical and histopathological factors prognostic analysis

P142 Expression of Ki-67 and endoglin (CD 105) proteins in stages I and II squamous cell carcinoma of oral tongue and floor of the mouth: association with demographic, clinical and histopathological factors prognostic analysis

Endoglin (CD105) and Vascular Endothelial Growth Factor as Prognostic Markers in Prostatic Adenocarcinoma

We studied endoglin and vascular endothelial growth factor (VEGF) expression as prognostic markers in prostatic adenocarcinoma in 50 radical prostatectomy specimens. Cases were further categorized by Gleason score as follows: 8 to 10, 9 cases; 7(4 + 3), 9 cases; 7 (3 + 4), 14 cases; 6, 13 cases; and 4 or 5, 5 cases. All cases were immunostained for endoglin, CD31, and VEGF. Positively stained microvessels were counted in densely vascular foci in a x 400 field. VEGF staining intensity was scored on a 2-tiered scale. Results were correlated with survival and other parameters. Endoglin demonstrated significantly more microvessels than did CD31 (mean +/- SD, 37 +/- 15 vs 22 +/- 17; P < .001). VEGF expression was low in 21 cases (42%) and high in 29 (58%). Endoglin correlated positively with Gleason score, lymph node metastases, tumor stage, and preoperative prostate-specific antigen level (P < .05) but not with CD31. VEGF correlated significantly with angiolymphatic invasion and Gleason score (P < .05). A high endoglin microvessel count and VEGF expression correlated with shorter survival. Endoglin is a more specific and sensitive marker for tumor angiogenesis than CD31 and may serve as a prognostic marker for prostatic adenocarcinoma.

Endoglin (CD105) and Vascular Endothelial Growth Factor as Prognostic Markers in Prostatic Adenocarcinoma

We studied endoglin and vascular endothelial growth factor (VEGF) expression as prognostic markers in prostatic adenocarcinoma in 50 radical prostatectomy specimens. Cases were further categorized by Gleason score as follows: 8 to 10, 9 cases; 7 (4 + 3), 9 cases; 7 (3 + 4), 14 cases; 6, 13 cases; and 4 or 5, 5 cases. All cases were immunostained for endoglin, CD31, and VEGF. Positively stained microvessels were counted in densely vascular foci in a ·400 field. VEGF staining intensity was scored on a 2-tiered scale. Results were correlated with survival and other parameters. Endoglin demonstrated significantly more microvessels than did CD31 (mean ± SD, 37 ± 15 vs 22 ± 17; P < .001). VEGF expression was low in 21 cases (42%) and high in 29 (58%). Endoglin correlated positively with Gleason score, lymph node metastases, tumor stage, and preoperative prostate-specific antigen level (P < .05) but not with CD31. VEGF correlated significantly with angiolymphatic invasion and Gleason score (P < .05). A high endoglin microvessel count and VEGF expression correlated with shorter survival. Endoglin is a more specific and sensitive marker for tumor angiogenesis than CD31 and may serve as a prognostic marker for prostatic adenocarcinoma.

Vascular endothelial growth factor and endoglin (CD-105) in gastric cancer

Vascular endothelial growth factor (VEGF) overexpression has been associated with advanced stage and poor survival in several cancers. Additionally, CD-105 (endoglin) was proposed as a marker of neovascularization in solid malignancies. The aim of the present study was to (1) evaluate the VEGF and CD-105 expression in gastric carcinoma, (2) determine the role of VEGF gene sequence variations in VEGF expression in gastric carcinoma, and (3) correlate the results of VEGF and CD-105 expression with other standard prognostic parameters, such as size, grade, stage of the disease, metastases, and patient survival. VEGF and CD-105 expression were evaluated in 100 unrelated gastric cancer patients using immunohistochemistry. For the genotyping, DNA was isolated from the blood of the gastric cancer patients and from 100 healthy individuals. The genotyping was performed by polymerase chain-restriction fragment length polymorphism analysis. VEGF protein was strongly expressed in the cytoplasm of 36% of the gastric carcinoma samples tested. In all cases, high VEGF expression was accompanied with high endoglin expression. Our results revealed no statistical significant association of any VEGF gene polymorphism with the VEGF and endoglin expression. The correlation of VEGF/CD-105 expression with the clinicopathological parameters of gastric cancer showed that the high expression of VEGF/CD015 was correlated only with lymph node metastasis (P = 0.028). The Kaplan-Meier survival curves have shown a clear association of overall survival after diagnosis of gastric cancer with high VEGF, as well as high CD-105 expression. Our results support that VEGF and CD-105 are closely relevant to lymph node metastasis and act as two valuable indicators of prognosis.

EXPRESSION OF HYPOXIA-INDUCING FACTOR-1α AND ENDOGLIN IN INTIMAL HYPERPLASIA OF THE MIDDLE CEREBRAL ARTERY OF PATIENTS WITH MOYAMOYA DISEASE

Moyamoya disease (MMD) is a cerebrovascular occlusive disease characterized by progressive stenosis or occlusion at the distal ends of the bilateral internal arteries. In MMD, intimal hyperplasia was previously reported to be found in autopsy samples. In this study focusing on the mechanism of remodeling of the intracranial arterial walls of patients with MMD, we surgically collected tiny pieces of the wall of the middle cerebral artery (MCA) from patients with MMD and analyzed them using histological and immunohistochemical methods. Twelve patients underwent surgical procedures for treatment of standard indications of MMD at Kyoto University Hospital. Specimens of MCA were obtained from MMD patients during the surgical procedures. Nine MCA samples were also obtained in the same way from control patients. The samples were analyzed by immunohistochemical methods. MCA specimens from MMD patients had a thicker intima than those from the control group. In MMD samples, the immunoreactivity indicating hypoxia-inducing factor-1alpha was higher in the endothelium and intima; endoglin expression was also higher in the endothelium. No vascular endothelial growth factor immunoreactivity was detectable in the MMD samples. In addition, transforming growth factor-beta3 immunoreactivity was also detected and was co-localized with that of hypoxia-inducing factor-1alpha and endoglin, mainly in the endothelium. Our results indicate that the MCA specimens from MMD patients had thicker intimal walls than the specimens from control patients. In addition, hypoxia-inducing factor-1alpha and endoglin were overexpressed in the intima of the MCA of MMD patients.

Endoglin and its involvement in transdifferentiation of hepatic stellate cells

Aims: Endoglin, a transmembrane accessory receptor for TGF-beta was recently found to be highly expressed on the surface of hepatic stellate cells. Up to now endoglin was mainly known as a TGF-beta receptor on endothelial cells, in which mutations causes the vascular disorder, hereditary haemorrhagic telangiectasia (HHT1). In the current study we tried to evaluate the role of endoglin in liver fibrosis modulating the classical TGF-beta pathway, which is mainly responsible for an increased extracellular matrix protein (ECM) synthesis during liver fibrosis. Methods: The expression of Endoglin in rat hepatic stellate cells (HSC) was specifically down regulated by siRNA. The knock down efficiency was verified by Western-Blotting. The consequence of downregulating endoglin was evaluated by (CAGA)12MLP-Luc reporter assay and by Western-Blotting using pSmad specific antibodies as well as antibodies against Collagen I, Fibronectin and alpha-sm-actin. Morphological changes in transdifferentiation were monitored by light microscopy. Results: After transfection of 1 day old rat HSC with siRNA against endoglin, the protein level was reduced at day 3 to an extent of approx. 95%. This knock down level was maintained for the following 10 days. Within this time-period we stimulated the cells with TGF-beta1 and analyzed the stimulation by (CAGA)12MLP-Luc reporter assay and Western-Blotting as a read out system. Thus we could demonstrate, that reduction of endoglin expression leads to an increased Smad3 phosphorylation as well as an enhanced (CAGA)12MLP-Luc reporter activity. Furthermore, we were able to show differences in transdifferentiation kinetic by monitoring morphological changes of culture-activated HSC in a time course of 12 days after siRNA transfection. Conclusion: It was possible to show, that endoglin is involved in the TGF-beta pathway in HSC, since reduction of endoglin leads to significant changes in Smad phosphorylation. Furthermore, the reduction of endoglin suppresses trans-differentiation from HSC to myofibroblastic phenotype cells (MFB) while overexpression of endoglin ends up in enhanced transdifferentiation.

Expression of vascular endothelial growth factor and assessment of microvascular density with CD 34 and endoglin in proliferative endometrium, endometrial hyperplasia, and endometrial carcinoma

The aim of this study was to compare vascular endothelial growth factor (VEGF), CD 34, and endoglin expressions as markers of angiogenesis in proliferative endometrium (PE), endometrial hyperplasia (EH), and endometrial carcinoma (EC) and to find the possible impact of angiogenesis on malign transformation. Formalin-fixed, paraffin-embedded tissues from 12 patients with PE, 23 patients with simple EH and complex EH with atypia, and 31 patients with EC were included. A semiquantitative scoring system was used to assess the intensity and degree of staining of VEGF. Microvessel density (MVD) was assessed with endoglin and anti-CD 34 in most vascular areas. VEGF expression was significantly higher in EC and EH than PE, but there was no difference between EC and EH. According to CD 34 staining, there were no differences in MVD between groups. However, mean MVD counts assessed by endoglin were significantly higher in EC than PE and EH. Although VEGF expression in EC was significantly higher, it did not correlate with other measures of angiogenesis. MVD using endoglin seemed to reflect neoplastic angiogenesis better than CD 34.

Therapeutic action of tranexamic acid in hereditary haemorrhagic telangiectasia (HHT): Regulation of ALK-1/endoglin pathway in endothelial cells

Recurrent epistaxis is the most frequent clinical manifestation of hereditary haemorrhagic telangiectasia (HHT). Its treatment is difficult. Our objective was to assess the use of tranexamic acid (TA), an antifibrinolytic drug, for the treatment of epistaxis in HHT patients and to investigate in vitro the effects of TA over endoglin and ALK-1 expression and activity in endothelial cells. A prospective study was carried out on patients with epistaxis treated with oral TA in the HHT Unit of Sierrallana Hospital (Cantabria, Spain). Primary cultures of endothelial cells were treated with TA to measure the levels of endoglin and ALK-1 at the cell surface by flow cytometry. RNA levels were also measured by real-time PCR, and the transcriptional effects of TA on reporters for endoglin, ALK-1 and the endoglin/ALK-1 TGF-beta pathway were assessed. The results showed that the fourteen HHT patients treated orally with TA improved, and the frequency and severity of their epistaxis were decreased. No complications derived from the treatment were observed. Cultured endothelial cells incubated with TA exhibited increased levels of endoglin and ALK-1 at the protein and mRNA levels, enhanced TGF-beta signaling, and improved endothelial cell functions like tubulogenesis and migration. In summary, oral administration of TA proved beneficial for epistaxis treatment in selected patients with HHT. In addition to its already reported antifibrinolytic effects, TA stimulates the expression ofALK-1 and endoglin, as well as the activity of the ALK-1/endoglin pathway.

Biphasic effect of pioglitazone on isolated human endothelial progenitor cells: Involvement of peroxisome proliferator-activated receptor-γ and transforming growth factor-β1

Endothelial progenitor cells (EPCs) have been implicated in vascular repair and found to be functionally impaired in patients with diabetes. We evaluated the effects of the anti-diabetic drug pioglitazone on human EPC function and the involvement of PPAR-gamma and TGF-beta1. EPCs in culture were characterized at day 7 by the development of colony-forming units (CFUs) and flow cytometry assessment of differentiation marker (DiI-ac-LDL/lectin, KDR and CD31). Adhesion on fibronectin and fibrinogen in flow was analyzed as functional parameter. Treatment with pioglitazone for 72 hours increased the number of EPC-CFUs, DiI-ac-LDL(+)/lectin(+), CD31(+) and KDR(+) EPCs at 1 microM but not at 10 microM. Since pioglitazone did not significantly alter proliferation and apoptosis in cultured EPCs, the increase in EPC number was most likely attributable to augmented adhesion and differentiation. Indeed, pioglitazone increased EPC adhesion in flow at 1 microM, an effect prevented by PPAR-gamma and beta2-integrin blockade. In contrast, pioglitazone did not promote EPC adhesion at 10 microM; however, increased adhesion became evident by co-incubation with a blocking TGF-beta1 antibody. As determined by ELISA, pioglitazone induced a persistent increase in TGF-beta1 secretion only at 10 microM when a significantly elevated expression of endoglin, the accessory receptor for TGF-beta1, was also observed. Taken together, pioglitazone exerts biphasic effects on the function of isolated EPCs, causing a PPAR-gamma-dependent stimulation at 1 microM and a TGF-beta1-mediated suppression at 10 microM. These results may help to define optimal therapeutic doses of pioglitazone for improving endothelial dysfunction.

Endoglin and Alk5 regulate epithelial–mesenchymal transformation during cardiac valve formation

Endoglin is an accessory receptor for TGFβ and can associate with Alk5 or Alk2. Although prior studies indicated that endoglin and Alk5 were not directly involved in epithelial–mesenchymal transformation (EMT) in the heart, the expression pattern of endoglin prompted a re-examination. We here show that loss of endoglin expression mediated by either antisense DNA or siRNA results in a direct perturbation of EMT and reduced expression of EMT markers including slug, runx2, RhoA, and latrophilin-2. An examination of BrdU incorporation shows that, while endoglin regulates proliferation at an early stage, reduced endothelial cell proliferation does not account for the loss of mesenchyme. As Alk5 interacts with endoglin, we utilized siRNA and a specific inhibitor, HTS466284 (HTS), to perturb this receptor as well. Alk5 inhibition produced similar effects to the inhibition of endoglin. There was a reduction in mesenchymal cell formation and loss of EMT marker expression similar to that seen with endoglin. Alk5 kinase inhibition produced a similar loss of EMT marker expression but showed a contrasting upregulation of the proliferation and remodeling markers, Cyclin B2 and β-catenin. Alk5 and endoglin both mediate endothelial cell proliferation in younger explants but, by stage 16, loss of endoglin no longer alters proliferation rates. These data show that both Alk5 and endoglin are directly involved in the process of EMT, that they interact with both TGFβ-regulated activation and invasion pathways and that the roles of these receptors change during cardiac development.