Endoglin mutants retained in the endoplasmic reticulum exacerbate loss of function in hereditary hemorrhagic telangiectasia type 1 (HHT1) by exerting dominant negative effects on the wild type allele.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder affecting 1 in 5000-8000 individuals. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is the most common HHT and manifests as diverse vascular malformations ranging from mild symptoms such as epistaxis and mucosal and cutaneous telangiectases to severe arteriovenous malformations (AVMs) in the lungs, brain or liver. HHT1 is caused by heterozygous mutations in the ENG gene, which encodes endoglin, the TGFß homodimeric co-receptor. It was previously shown that some endoglin HHT1-causing variants failed to traffic to the plasma membrane due to their retention in the endoplasmic reticulum (ER) and consequent degradation by ER-associated degradation (ERAD). Endoglin is a homodimer formed in the ER, and we therefore hypothesized that mixed heterodimers might form between ER-retained variants and WT protein, thus hampering its maturation and trafficking to the plasma membrane causing dominant negative effects. Indeed, HA-tagged ER-retained mutants formed heterodimers with Myc-tagged WT endoglin. Moreover, variants L32R, V105D, P165L, I271N and C363Y adversely affected the trafficking of WT endoglin by reducing its maturation and plasma membrane localization. These results strongly suggest dominant negative effects exerted by these ER-retained variants aggravating endoglin loss of function in patients expressing them in the heterozygous state with the WT allele. Moreover, this study may help explain some of the variability observed among HHT1 patients due to the additional loss of function exerted by the dominant negative effects in addition to that due to haploinsufficiency. These findings might also have implications for some of the many conditions impacted by ERAD.

Soluble Endoglin and Syndecan-1 levels predicts the clinical outcome in COVID-19 patients.

Endothelial instability is reported to be involved in the pathogenesis of COVID-19. The mechanism that regulates the endothelial dysfunction and disease virulence is not known. Studies on proteins that are released into circulation by activated endothelial cells may provide some means to understand the disease manifestation. The study investigated the circulating levels of two molecules Endoglin (Eng) and Syndecan-1 (SDC-1) that are presumed to be involved in the maintenance of endothelial integrity and their association with hypercoagulation marker in COVID-19 patients. The serum levels of Eng, SDC-1, D-mer were evaluated using ELISA at the time of admission (DOA) and day 7 post-admission among COVID-19 patients (N = 39 with 17 moderate and 22 severe cases). Compared to the time of admission, there was an increase in sEng and sSDC1 levels in all COVID-19 cases on day 7 post admission. The serum levels of sEng and sSDC-1 was significantly (P ≤ 0.001 & P ≤ 0.01 respectively) elevated in severe cases including the four deceased group compared to moderate cases on day 7 post admission. Further, the study molecules showed a strong positive association (P ≤ 0.001) with the hypercoagulation marker D-mer. The results show an early shedding of the endothelial proteins sEng and sSDC-1 into circulation as a host response to the viral infection during the febrile phase of infection. Increased levels of sEng and sSDC-1 along with D-mer could be beneficial in predicting COVID-19 disease severity.

Umbilical cord mesenchymal stem cells from gestational diabetes show impaired ability to up-regulate paracellular permeability from sub-endothelial niche.

In vitro studies have shown that Wharton's jelly mesenchymal stem cells (WJ-MSCs) can cross umbilical and uterine endothelial barriers and up-regulate endothelial junctional integrity from sub-endothelial niches. This pericytic behaviour may be lost in pregnancies complicated by gestational diabetes (GDM), where increased vascular permeability and junctional disruption are reported. The aim of the present study was to investigate whether WJ-MSCs isolated from GDM pregnancies displayed any changes in morphology, proliferation, VEGF-A secretion, and their ability to influence paracellular junctional composition and permeability. WJ-MSCs were isolated from human umbilical cords from normal pregnancies (nWJ-MSCs, n=13) and those complicated by GDM (gWJ-MSCs), either diet-controlled (d-GDM, n=13) or metformin-treated (m-GDM, n=9). We recorded that 4-fold more WJ-MSCs migrated from m-GDM, and 2.5-fold from d-GDM cord samples compared with the normal pregnancy. gWJ-MSCs showed a less predominance of spindle-shaped morphology and secreted 3.8-fold more VEGF-A compared with nWJ-MSCs. The number of cells expressing CD105 (Endoglin) was higher in gWJ-MSCs compared with nWJ-MSCs (17%) at P-2. The tracer leakage after 24 h across the HUVEC + gWJ-MSCs bilayer was 22.13% and 11.2% higher in the m-GDM and d-GDM, respectively, HUVEC + nWJ-MSCs. Transfection studies with siRNAs that target Endoglin were performed in n-WJ-MSCs; transfected cells were co-cultured with HUVEC followed by permeability studies and VE-cadherin analyses. Loss of Endoglin also led to increased VEGF-A secretion, increased permeability and affected endothelial stabilization. These results reinforce the pericytic role of nWJ-MSCs to promote vascular repair and the deficient ability of gWJ-MSCs to maintain endothelial barrier integrity.

Angiogenic and vasoactive proteins in the maternal-fetal interface in healthy pregnancies and preeclampsia.

BACKGROUND: Preeclampsia is characterized by maternal endothelial activation and placental dysfunction. Imbalance in maternal angiogenic and vasoactive factors has been linked to the pathophysiology. The contribution of the placenta as a source of these factors remains unclear. Furthermore, little is known about fetal angiogenic and vasoactive proteins and the relation between maternal and fetal levels. OBJECTIVE: We describe placental growth factor, soluble Fms-like tyrosine kinase 1, soluble endoglin, and endothelin 1-3 in 5 vessels in healthy pregnancies, early- and late-onset preeclampsia. Specifically, we aimed to (1) compare protein abundance in vessels at the maternal-fetal interface between early- and late-onset preeclampsia, and healthy pregnancies, (2) describe placental uptake and release of proteins, and (3) describe protein abundance in the maternal vs fetal circulations. STUDY DESIGN: Samples were collected from the maternal radial artery, uterine vein and antecubital vein, and fetal umbilical vein and artery in 75 healthy and 37 preeclamptic mother-fetus pairs (including 19 early-onset preeclampsia and 18 late-onset preeclampsia), during scheduled cesarean delivery. This method allows estimation of placental release and uptake of proteins by calculation of venoarterial differences on each side of the placenta. The microarray-based SomaScan assay quantified the proteins. RESULTS: The abundance of soluble Fms-like tyrosine kinase 1 and endothelin 1 was higher in the maternal vessels in preeclampsia than in healthy pregnancies, with the highest abundance in early-onset preeclampsia. Placental growth factor was lower in the maternal vessels in early-onset preeclampsia than in both healthy and late-onset preeclampsia. Maternal endothelin 2 was higher in preeclampsia, with late-onset preeclampsia having the highest abundance. Our model confirmed placental release of placental growth factor and soluble Fms-like tyrosine kinase 1 to the maternal circulation in all groups. The placenta released soluble Fms-like tyrosine kinase 1 into the fetal circulation in healthy and late-onset preeclampsia pregnancies. Fetal endothelin 1 and soluble Fms-like tyrosine kinase 1 were higher in early-onset preeclampsia, whereas soluble endoglin and endothelin 3 were lower in both preeclampsia groups than healthy controls. Across groups, abundances of placental growth factor, soluble Fms-like tyrosine kinase 1, and endothelin 3 were higher in the maternal artery than the fetal umbilical vein, whereas endothelin 2 was lower. CONCLUSION: An increasing abundance of maternal soluble Fms-like tyrosine kinase 1 and endothelin 1 across the groups healthy, late-onset preeclampsia and early-onset combined with a positive correlation may suggest that these proteins are associated with the pathophysiology and severity of the disease. Elevated endothelin 1 in the fetal circulation in early-onset preeclampsia represents a novel finding. The long-term effects of altered protein abundance in preeclampsia on fetal development and health remain unknown. Further investigation of these proteins' involvement in the pathophysiology and as treatment targets is warranted.

Antagonizing CD105 and androgen receptor to target stromal-epithelial interactions for clinical benefit.

Androgen receptor signaling inhibitors (ARSIs) are standard of care for advanced prostate cancer (PCa) patients. Eventual resistance to ARSIs can include the expression of androgen receptor (AR) splice variant, AR-V7, expression as a recognized means of ligand-independent androgen signaling. We demonstrated that interleukin (IL)-6-mediated AR-V7 expression requires bone morphogenic protein (BMP) and CD105 receptor activity in both PCa and associated fibroblasts. Chromatin immunoprecipitation supported CD105-dependent ID1- and E2F-mediated expression of RBM38. Further, RNA immune precipitation demonstrated RBM38 binds the AR-cryptic exon 3 to enable AR-V7 generation. The forced expression of AR-V7 by primary prostatic fibroblasts diminished PCa sensitivity to ARSI. Conversely, downregulation of AR-V7 expression in cancer epithelia and associated fibroblasts was achieved by a CD105-neutralizing antibody, carotuximab. These compelling pre-clinical findings initiated an interventional study in PCa patients developing ARSI resistance. The combination of carotuximab and ARSI (i.e., enzalutamide or abiraterone) provided disease stabilization in four of nine assessable ARSI-refractory patients. Circulating tumor cell evaluation showed AR-V7 downregulation in the responsive subjects on combination treatment and revealed a three-gene panel that was predictive of response. The systemic antagonism of BMP/CD105 signaling can support ARSI re-sensitization in pre-clinical models and subjects that have otherwise developed resistance due to AR-V7 expression.

MerTK Drives Proliferation and Metastatic Potential in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by the absence of the estrogen receptor, progesterone receptor, and receptor tyrosine kinase HER2 expression. Due to the limited number of FDA-approved targeted therapies for TNBC, there is an ongoing need to understand the molecular underpinnings of TNBC for the development of novel combinatorial treatment strategies. This study evaluated the role of the MerTK receptor tyrosine kinase on proliferation and invasion/metastatic potential in TNBC. Immunohistochemical analysis demonstrated MerTK expression in 58% of patient-derived TNBC xenografts. The stable overexpression of MerTK in human TNBC cell lines induced an increase in proliferation rates, robust in vivo tumor growth, heightened migration/invasion potential, and enhanced lung metastases. NanoString nCounter analysis of MerTK-overexpressing SUM102 cells (SUM102-MerTK) revealed upregulation of several signaling pathways, which ultimately drive cell cycle progression, reduce apoptosis, and enhance cell survival. Proteomic profiling indicated increased endoglin (ENG) production in SUM102-MerTK clones, suggesting that MerTK creates a conducive environment for increased proliferative and metastatic activity via elevated ENG expression. To determine ENG's role in increasing proliferation and/or metastatic potential, we knocked out ENG in a SUM102-MerTK clone with CRISPR technology. Although this ENG knockout clone exhibited similar in vivo growth to the parental SUM102-MerTK clone, lung metastasis numbers were significantly decreased ~4-fold, indicating that MerTK enhances invasion and metastasis through ENG. Our data suggest that MerTK regulates a unique proliferative signature in TNBC, promoting robust tumor growth and increased metastatic potential through ENG upregulation. Targeting MerTK and ENG simultaneously may provide a novel therapeutic approach for TNBC patients.

BMP10 functions independently from BMP9 for the development of a proper arteriovenous network.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic vascular disorder characterized by the presence of arteriovenous malformation (AVM) in multiple organs. HHT is caused by mutations in genes encoding major constituents for transforming growth factor-ß (TGF-ß) family signaling: endoglin (ENG), activin receptor-like kinase 1 (ALK1), and SMAD4. The identity of physiological ligands for this ENG-ALK1 signaling pertinent to AVM formation has yet to be clearly determined. To investigate whether bone morphogenetic protein 9 (BMP9), BMP10, or both are physiological ligands of ENG-ALK1 signaling involved in arteriovenous network formation, we generated a novel Bmp10 conditional knockout mouse strain. We examined whether global Bmp10-inducible knockout (iKO) mice develop AVMs at neonatal and adult stages in comparison with control, Bmp9-KO, and Bmp9/10-double KO (dKO) mice. Bmp10-iKO and Bmp9/10-dKO mice showed AVMs in developing retina, postnatal brain, and adult wounded skin, while Bmp9-KO did not display any noticeable vascular defects. Bmp10 deficiency resulted in increased proliferation and size of endothelial cells in AVM vessels. The impaired neurovascular integrity in the brain and retina of Bmp10-iKO and Bmp9/10-dKO mice was detected. Bmp9/10-dKO mice exhibited the lethality and vascular malformation similar to Bmp10-iKO mice, but their phenotypes were more pronounced. Administration of BMP10 protein, but not BMP9 protein, prevented retinal AVM in Bmp9/10-dKO and endothelial-specific Eng-iKO mice. These data indicate that BMP10 is indispensable for the development of a proper arteriovenous network, whereas BMP9 has limited compensatory functions for the loss of BMP10. We suggest that BMP10 is the most relevant physiological ligand of the ENG-ALK1 signaling pathway pertinent to HHT pathogenesis.

Executive summary of the 14th HHT international scientific conference.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by small, dilated clustered vessels (telangiectasias) and by larger visceral arteriovenous malformations (AVMs), which directly connect the feeding arteries with the draining veins. These lesions are fragile, prone to rupture, and lead to recurrent epistaxis and/or internal hemorrhage among other complications. Germline heterozygous loss-of-function (LOF) mutations in Bone Morphogenic Protein 9 (BMP9) and BMP10 signaling pathway genes (endoglin-ENG, activin like kinase 1 ACVRL1 aka ALK1, and SMAD4) cause different subtypes of HHT (HHT1, HHT2 and HHT-juvenile polyposis (JP)) and have a worldwide combined incidence of about 1:5000. Expert clinicians and international scientists gathered in Cascais, Portugal from September 29th to October 2nd, 2022 to present the latest scientific research in the HHT field and novel treatment strategies for people living with HHT. During the largest HHT scientific conference yet, participants included 293 in person and 46 virtually. An impressive 209 abstracts were accepted to the meeting and 59 were selected for oral presentations. The remaining 150 abstracts were presented during judged poster sessions. This review article summarizes the basic and clinical abstracts selected as oral presentations with their new observations and discoveries as well as surrounding discussion and debate. Two discussion-based workshops were also held during the conference, each focusing on mechanisms and clinical perspectives in either AVM formation and progression or current and future therapies for HHT. Our hope is that this paper will represent the current progress and the remaining unanswered questions surrounding HHT, in order to serve as an update for those within the field and an invitation to those scientists and clinicians as yet outside of the field of HHT.

Tumoral CD105 promotes immunosuppression, metastasis, and angiogenesis in renal cell carcinoma.

CD105 (endoglin) is a transmembrane protein that functions as a TGF-beta coreceptor and is highly expressed on endothelial cells. Unsurprisingly, preclinical and clinical evidence strongly suggests that CD105 is an important contributor to tumor angiogenesis and tumor progression. Emerging evidence suggests that CD105 is also expressed by tumor cells themselves in certain cancers such as renal cell carcinoma (RCC). In human RCC tumor cells, CD105 expression is associated with stem cell-like properties and contributes to the malignant phenotype in vitro and in xenograft models. However, as a regulator of TGF-beta signaling, there is a striking lack of evidence for the role of tumor-expressed CD105 in the anti-tumor immune response and the tumor microenvironment. In this study, we report that tumor cell-expressed CD105 potentiates both the in vitro and in vivo tumorigenic potential of RCC in a syngeneic murine RCC tumor model. Importantly, we find that tumor cell-expressed CD105 sculpts the tumor microenvironment by enhancing the recruitment of immunosuppressive cell types and inhibiting the polyfunctionality of tumor-infiltrating CD4+ and CD8+ T cells. Finally, while CD105 expression by endothelial cells is a well-established contributor to tumor angiogenesis, we also find that tumor cell-expressed CD105 significantly contributes to tumor angiogenesis in RCC.

Longitudinal Assessment of Curaçao Criteria in Children with Hereditary Hemorrhagic Telangiectasia.

OBJECTIVE: To assess the utility of the Curaçao criteria by age over time in children with hereditary hemorrhagic telangiectasia (HHT). STUDY DESIGN: This was a single-center, retrospective analysis of patients attending the HHT clinic at the Hospital for Sick Children (Toronto, Canada) between 2000 and 2019. The evaluation of the Curaçao criteria was completed during initial and follow-up visits. Screening for pulmonary and brain arteriovenous malformations was completed at 5 yearly intervals. RESULTS: A total of 116 patients with genetic confirmation of HHT were included in the analysis. At initial screening at a median (IQR) age of 8.4 (2.8, 12.9) years, 41% met criteria for a definite clinical diagnosis (≥3 criteria). In children <6 years at presentation, only 23% fulfilled at least 3 criteria initially. In longitudinal follow-up, 63% reached a definite clinical diagnosis, with a median (IQR) follow-up duration of 5.2 (3.2, 7.9) years (P = .005). Specifically, more patients met the epistaxis and telangiectasia criteria at last visit compared with initial (79% vs 60%; P = .006; 47% vs 30%; P = .02) but not for the arteriovenous malformation criterion (59% vs 57%; P = .65). CONCLUSIONS: In the pediatric population, most patients do not meet definite clinical criteria of HHT at initial presentation. Although the number of diagnostic criteria met increased over time, mainly due to new onset of epistaxis and telangiectasia, accuracy remained low during follow-up visits. Relying solely on clinical criteria may lead to underdiagnosis of HHT in children.

Protein Phosphatase 2A Activation Via ApoER2 in Trophoblasts Drives Preeclampsia in a Mouse Model of the Antiphospholipid Syndrome.

[Figure: see text].

Microcirculation surrounding end-stage human chronic skin wounds is associated with endoglin/CD146/ALK-1 expression, endothelial cell proliferation and an absence of p16Ink4a.

Angiogenesis is an essential part of normal skin healing, re-establishing blood flow in developing granulation tissue. Non-healing skin wounds are associated with impaired angiogenesis and although the role of re-establishing macroscopic blood flow to limbs to prevent wound chronicity is well investigated, less is known about vascular alterations at the microcirculatory level. We hypothesised that significant phenotypic changes would be evident in blood vessels surrounding chronic skin wounds. Wound edge tissue, proximal to wound (2 cm from wound edge) and non-involved skin (>10 cm from wound edge) was harvested under informed consent from 20 patients undergoing elective amputation due to critical limb ischemia. To assess blood vessel structure and viability, tissue was prepared for histological analysis and labelled with antibodies specific for PECAM-1 (CD31), CD146, endoglin, ALK-1, ALK-5, and p16Ink4a as a marker of cellular senescence. Density of microvasculature was significantly increased in wound edge dermis, which was concomitant with increased labelling for endoglin and CD146. The number of CD31 positive vessel density was unchanged in wound edge tissue relative to non-involved tissue. Co-labelling of endoglin with the transforming growth factor receptor ALK-1, and to a lesser extent ALK-5, demonstrated activation of endothelial cells which correlated with PCNA labelling indicative of proliferation. Analysis of p16Ink4a staining showed a complete lack of immunoreactivity in the vasculature and dermis, although staining was evident in sub-populations of keratinocytes. We conclude that the endoglin-ALK-1-endothelial proliferation axis is active in the vasculature at the edge of chronic skin wounds and is not associated with p16Ink4a mediated senescence. This information could be further used to guide treatment of chronic skin wounds and optimise debridement protocols.

Cord blood transforming growth factor-ß-induced as predictive biomarker of retinopathy of prematurity in preterm infants.

PURPOSE: To determine whether 14 inflammation-, angiogenesis-, and adhesion-related proteins in cord blood (CB), alone or in combination with conventional perinatal factors, could predict retinopathy of prematurity (ROP) in preterm infants. METHODS: Data from 111 preterm infants (born at ≤ 32.0 weeks) were retrospectively reviewed. The levels of endoglin, E-selectin, HSP70, IGFBP-3/4, LBP, lipocaline-2, M-CSFR, MIP-1α, pentraxin 3, P-selectin, TGFBI, TGF-ß1, and TNFR2 were assessed in stored CB samples collected at birth using ELISA kits. The primary endpoints included severe ROP (≥ stage 3) and type 1 ROP requiring treatment. RESULTS: ROP was diagnosed in 29 infants (26.1%), among whom 14 (12.6%) had severe ROP and seven (6.3%) had type 1 ROP. Multivariate logistic regression showed that decreased CB TGFBI levels were significantly associated with severe ROP and type 1 ROP after adjusting for gestational age at birth. Stepwise regression analysis allowed to design prediction models with good accuracy, which comprised low CB TGFBI levels and low birth weight (BW) as predictors for severe ROP (area under the curve [AUC] = 0.888), and low CB endoglin levels and low BW as predictors for type 1 ROP (AUC = 0.950). None of the other CB proteins evaluated were found to be associated with severe ROP or type 1 ROP. CONCLUSIONS: Low CB TGFBI levels are associated with severe ROP and type 1 ROP, independently of gestational age. Moreover, combined predictive models based on CB TGFBI and endoglin levels, along with BW data, may act as good indicators at birth for the neonatal risk of ROP progression.

Increased soluble endoglin levels in newly-diagnosed type 2 diabetic patients are associated with endothelial dysfunction.

Endothelial dysfunction (ED) contributes to the pathologic process underlying macrovascular complications, a common complication of type 2 diabetes mellitus (T2DM). Soluble endoglin (sEng) shed from the extracellular domain of the entire endoglin molecule blocks endothelial protection mediated by transforming growth factor-beta 1 (TGF-ß1). The reactive hyperemia index (RHI), which is determined by reactive hyperemia peripheral arterial tonometry (RH-PAT), is a new index with which to evaluate ED. This study determined the changes in serum sEng levels in newly-diagnosed (untreated) T2DM patients and the correlation with the RHI. The T2DM group included 34 newly-diagnosed T2DM patients, while the control group included 53 healthy adults. The clinical data from the two groups were evaluated retrospectively. The intima-media thickness (IMT) of the common carotid artery (CCA) and the ankle-brachial index (ABI) of both legs were used to assess structural vascular changes. The serum sEng level was determined using an ELISA kit. Endothelial function was assessed using RH-PAT and the RHI was computed. The serum sEng level in the T2DM group was significantly greater than the control group, although the RHI was significantly lower in the T2DM group (p < 0.05). The serum sEng level was negatively correlated with the RHI in T2DM patents (r = 0.354, p = 0.041). The serum sEng level, CCA-IMT, and ABI were not significantly correlated with T2DM (p > 0.05). In summary, among newly-diagnosed T2DM patients, the serum sEng levels were inversely correlated with the RHI, and an elevated sEng level may be associated with ED.

Correlation Between Endoglin and Malignant Phenotype in Human Melanoma Cells: Analysis of hsa-mir-214 and hsa-mir-370 in Cells and Their Extracellular Vesicles.

Endoglin (CD105) is an auxiliary receptor of transforming growth factor (TGF)-ß family members that is expressed in human melanomas. It is heterogeneously expressed by primary and metastatic melanoma cells, and endoglin targeting as a therapeutic strategy for melanoma tumors is currently been explored. However, its involvement in tumor development and malignancy is not fully understood. Here, we find that endoglin expression correlates with malignancy of primary melanomas and cultured melanoma cell lines. Next, we have analyzed the effect of ectopic endoglin expression on two miRNAs (hsa-mir-214 and hsa-mir-370), both involved in melanoma tumor progression and endoglin regulation. We show that compared with control cells, overexpression of endoglin in the WM-164 melanoma cell line induces; (i) a significant increase of hsa-mir-214 levels in small extracellular vesicles (EVs) as well as an increased trend in cells; and (ii) significantly lower levels of hsa-mir-370 in the EVs fractions, whereas no significant differences were found in cells. As hsa-mir-214 and hsa-mir-370 are not just involved in melanoma tumor progression, but they can also target endoglin-expressing endothelial cells in the tumor vasculature, these results suggest a complex and differential regulatory mechanism involving the intracellular and extracellular signaling of hsa-mir-214 and hsa-mir-370 in melanoma development and progression.

Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes.

BACKGROUND: Fluid shear stress enhances endothelial SMAD1/5 signaling via the BMP9-bound ALK1 receptor complex supported by the co-receptor Endoglin. While moderate SMAD1/5 activation is required to maintain endothelial quiescence, excessive SMAD1/5 signaling promotes endothelial dysfunction. Increased BMP signaling participates in endothelial-to-mesenchymal transition and inflammation culminating in vascular diseases such as atherosclerosis. While the function of Endoglin has so far been described under picomolar concentrations of BMP9 and short-term shear application, we investigated Endoglin under physiological BMP9 and long-term pathophysiological shear conditions. RESULTS: We report here that knock-down of Endoglin leads to exacerbated SMAD1/5 phosphorylation and atheroprone gene expression profile in HUVECs sheared for 24 h. Making use of the ligand-trap ALK1-Fc, we furthermore show that this increase is dependent on BMP9/10. Mechanistically, we reveal that long-term exposure of ECs to low laminar shear stress leads to enhanced Endoglin expression and endocytosis of Endoglin in Caveolin-1-positive early endosomes. In these endosomes, we could localize the ALK1-Endoglin complex, labeled BMP9 as well as SMAD1, highlighting Caveolin-1 vesicles as a SMAD signaling compartment in cells exposed to low atheroprone laminar shear stress. CONCLUSIONS: We identified Endoglin to be essential in preventing excessive activation of SMAD1/5 under physiological flow conditions and Caveolin-1-positive early endosomes as a new flow-regulated signaling compartment for BMP9-ALK1-Endoglin signaling axis in atheroprone flow conditions.

Prognostic significance of a panel of two biomarkers (E-cadherin and CD105) in laryngeal cancer.

This study aimed to evaluate the clinicopathologic significance of the combined immunohistochemical expression of the epithelial-mesenchymal transition marker E-cadherin and the angiogenesis marker CD105 in laryngeal squamous cell carcinoma and assess correlation of their expression. Eighty-five patients who underwent complete resection as primary treatment were selected for this study. E-cadherin and CD105 expression levels were determined by immunohistochemistry. The receiver operating curve approach was applied to determine the cut-off value and separate patients with high and low expression of markers. The high-risk group ("CD105 high" and "E-cadherin low" expression) showed statistically significant correlations with age less than 66 years (p = 0.039), advanced T-status (T3-4) (p = 0.046), aggressive TNM stage (stage III-IV) (p = 0.003) and locoregional recurrence of disease (p = 0.004). In the Kaplan-Meier analyses, the high-risk group had significantly worse prognoses than other risk groups (log-rank test 2 = 9.415, p = 0.024). Spearman's correlation coefficient analysis showed a nonsignificant negative correlation between the expression of E-cadherin and CD105 (rho = -0.073, p = 0.505). Simultaneous consideration of E-cadherin and CD105 is a simple panel of markers to determine aggressive tumour phenotype with a higher risk of disease recurrence in patients with laryngeal cancer.

The Potential Role of MiRs-139-5p and -454-3p in Endoglin-Knockdown-Induced Angiogenic Dysfunction in HUVECs.

Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic disease characterized by aberrant angiogenesis and vascular malformations. Mutations in the transforming growth factor beta co-receptor, endoglin (ENG), account for approximately half of known HHT cases and cause abnormal angiogenic activity in endothelial cells (ECs). To date, how ENG deficiency contributes to EC dysfunction remains to be fully understood. MicroRNAs (miRNAs) regulate virtually every cellular process. We hypothesized that ENG depletion results in miRNA dysregulation that plays an important role in mediating EC dysfunction. Our goal was to test the hypothesis by identifying dysregulated miRNAs in ENG-knockdown human umbilical vein endothelial cells (HUVECs) and characterizing their potential role in EC function. We identified 32 potentially downregulated miRNAs in ENG-knockdown HUVECs with a TaqMan miRNA microarray. MiRs-139-5p and -454-3p were found to be significantly downregulated after RT-qPCR validation. While the inhibition of miR-139-5p or miR-454-3p had no effect on HUVEC viability, proliferation or apoptosis, angiogenic capacity was significantly compromised as determined by a tube formation assay. Most notably, the overexpression of miRs-139-5p and -454-3p rescued impaired tube formation in HUVECs with ENG knockdown. To our knowledge, we are the first to demonstrate miRNA alterations after the knockdown of ENG in HUVECs. Our results indicate a potential role of miRs-139-5p and -454-3p in ENG-deficiency-induced angiogenic dysfunction in ECs. Further study to examine the involvement of miRs-139-5p and -454-3p in HHT pathogenesis is warranted.

Similar Pro- and Antiangiogenic Profiles Close to Delivery in Different Clinical Presentations of Two Pregnancy Syndromes: Preeclampsia and Fetal Growth Restriction.

The purpose of this study was to evaluate serum levels of anti- and pro-angiogenic substances measured using enzyme-linked immunosorbent assays and their ratios in pregnancies complicated by different clinical subsets of placental ischemic syndrome: preeclampsia and/or fetal growth restriction. A prospective case-control study was performed consisting of 77 singleton pregnancies complicated by preeclampsia, preeclampsia with concurrent fetal growth restriction (FGR), and isolated normotensive FGR pairwise matched by gestational age with healthy pregnancies. The entire study cohort was analyzed with respect to adverse pregnancy outcomes that occurred. In all investigated subgroups, placental growth factor (PlGF) was lower and soluble endoglin (sEng), the soluble fms-like tyrosine kinase-1-sFlt-1/PlGF and sFlt-1*sEng/PlGF ratios were higher than in the control group. The differences were most strongly pronounced in the PE with concurrent FGR group and in the sFlt-1/PlGF ratio. The highest sFlt-1 values in preeclamptic patients suggest that this substance may be responsible for reaching the threshold needed for PE to develop as a maternal manifestation of ischemic placental disease. The FGR is characterized by an elevated maternal sFlt-1/PlGF ratio, which boosts at the moment of indicated delivery due to fetal risk. We concluded that angiogenic imbalance is reflective of placental disease regardless of its clinical manifestation in the mother, and may be used as support for the diagnosis and prognosis of FGR.

TGF-ß superfamily co-receptors in cancer.

Transforming growth factor-ß (TGF-ß) superfamily signaling via their cognate receptors is frequently modified by TGF-ß superfamily co-receptors. Signaling through SMAD-mediated pathways may be enhanced or depressed depending on the specific co-receptor and cell context. This dynamic effect on signaling is further modified by the release of many of the co-receptors from the membrane to generate soluble forms that are often antagonistic to the membrane-bound receptors. The co-receptors discussed here include TßRIII (betaglycan), endoglin, BAMBI, CD109, SCUBE proteins, neuropilins, Cripto-1, MuSK, and RGMs. Dysregulation of these co-receptors can lead to altered TGF-ß superfamily signaling that contributes to the pathophysiology of many cancers through regulation of growth, metastatic potential, and the tumor microenvironment. Here we describe the role of several TGF-ß superfamily co-receptors on TGF-ß superfamily signaling and the impact on cellular and physiological functions with a particular focus on cancer, including a discussion on recent pharmacological advances and potential clinical applications targeting these co-receptors.