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.

Mutations causing premature termination codons discriminate and generate cellular and clinical variability in HHT.

ABSTRACT: For monogenic diseases caused by pathogenic loss-of-function DNA variants, attention focuses on dysregulated gene-specific pathways, usually considering molecular subtypes together within causal genes. To better understand phenotypic variability in hereditary hemorrhagic telangiectasia (HHT), we subcategorized pathogenic DNA variants in ENG/endoglin, ACVRL1/ALK1, and SMAD4 if they generated premature termination codons (PTCs) subject to nonsense-mediated decay. In 3 patient cohorts, a PTC-based classification system explained some previously puzzling hemorrhage variability. In blood outgrowth endothelial cells (BOECs) derived from patients with ACVRL1+/PTC, ENG+/PTC, and SMAD4+/PTC genotypes, PTC-containing RNA transcripts persisted at low levels (8%-23% expected, varying between replicate cultures); genes differentially expressed to Bonferroni P < .05 in HHT+/PTC BOECs clustered significantly only to generic protein terms (isopeptide-bond/ubiquitin-like conjugation) and pulse-chase experiments detected subtle protein maturation differences but no evidence for PTC-truncated protein. BOECs displaying highest PTC persistence were discriminated in unsupervised hierarchical clustering of near-invariant housekeeper genes, with patterns compatible with higher cellular stress in BOECs with >11% PTC persistence. To test directionality, we used a HeLa reporter system to detect induction of activating transcription factor 4 (ATF4), which controls expression of stress-adaptive genes, and showed that ENG Q436X but not ENG R93X directly induced ATF4. AlphaFold accurately modeled relevant ENG domains, with AlphaMissense suggesting that readthrough substitutions would be benign for ENG R93X and other less rare ENG nonsense variants but more damaging for Q436X. We conclude that PTCs should be distinguished from other loss-of-function variants, PTC transcript levels increase in stressed cells, and readthrough proteins and mechanisms provide promising research avenues.

Novel mathematical approach to accurately quantify 3D endothelial cell morphology and vessel geometry based on fluorescently marked endothelial cell contours: Application to the dorsal aorta of wild-type and Endoglin-deficient zebrafish embryos.

Endothelial cells, which line the lumen of blood vessels, locally sense and respond to blood flow. In response to altered blood flow dynamics during early embryonic development, these cells undergo shape changes that directly affect vessel geometry: In the dorsal aorta of zebrafish embryos, elongation of endothelial cells in the direction of flow between 48 and 72 hours post fertilization (hpf) reduces the vessel's diameter. This remodeling process requires Endoglin; excessive endothelial cell growth in the protein's absence results in vessel diameter increases. To understand how these changes in vessel geometry emerge from morphological changes of individual endothelial cells, we developed a novel mathematical approach that allows 3D reconstruction and quantification of both dorsal aorta geometry and endothelial cell surface morphology. Based on fluorescently marked endothelial cell contours, we inferred cross-sections of the dorsal aorta that accounted for dorsal flattening of the vessel. By projection of endothelial cell contours onto the estimated cross-sections and subsequent triangulation, we finally reconstructed 3D surfaces of the individual cells. By simultaneously reconstructing vessel cross-sections and cell surfaces, we found in an exploratory analysis that morphology varied between endothelial cells located in different sectors of the dorsal aorta in both wild-type and Endoglin-deficient zebrafish embryos: In wild-types, ventral endothelial cells were smaller and more elongated in flow direction than dorsal endothelial cells at both 48 hpf and 72 hpf. Although dorsal and ventral endothelial cells in Endoglin-deficient embryos had similar sizes at 48 hpf, dorsal endothelial cells were much larger at 72 hpf. In Endoglin-deficient embryos, elongation in flow direction increased between 48 hpf and 72 hpf in ventral endothelial cells but hardly changed in dorsal endothelial cells. Hereby, we provide evidence that dorsal endothelial cells contribute most to the disparate changes in dorsal aorta diameter in wild-type and Endoglin-deficient embryos between 48 hpf and 72 hpf.

The EGLN-HIF O2-Sensing System: Multiple Inputs and Feedbacks.

The EGLN (also called PHD) prolyl hydroxylase enzymes and their canonical targets, the HIFα subunits, represent the core of an ancient oxygen-monitoring machinery used by metazoans. In this review, we highlight recent progress in understanding the overlapping versus specific roles of EGLN enzymes and HIF isoforms and discuss how feedback loops based on recently identified noncoding RNAs introduce additional layers of complexity to the hypoxic response. Based on novel interactions identified upstream and downstream of EGLNs, an integrated network connecting oxygen-sensing functions to metabolic and signaling pathways is gradually emerging with broad therapeutic implications.

Serum biomarkers correlated with liver stiffness assessed in a multicenter study of pediatric cholestatic liver disease.

BACKGROUND AND AIMS: Detailed investigation of the biological pathways leading to hepatic fibrosis and identification of liver fibrosis biomarkers may facilitate early interventions for pediatric cholestasis. APPROACH AND RESULTS: A targeted enzyme-linked immunosorbent assay-based panel of nine biomarkers (lysyl oxidase, tissue inhibitor matrix metalloproteinase (MMP) 1, connective tissue growth factor [CTGF], IL-8, endoglin, periostin, Mac-2-binding protein, MMP-3, and MMP-7) was examined in children with biliary atresia (BA; n = 187), alpha-1 antitrypsin deficiency (A1AT; n = 78), and Alagille syndrome (ALGS; n = 65) and correlated with liver stiffness (LSM) and biochemical measures of liver disease. Median age and LSM were 9 years and 9.5 kPa. After adjusting for covariates, there were positive correlations among LSM and endoglin ( p = 0.04) and IL-8 ( p < 0.001) and MMP-7 ( p < 0.001) in participants with BA. The best prediction model for LSM in BA using clinical and lab measurements had an R2 = 0.437; adding IL-8 and MMP-7 improved R2 to 0.523 and 0.526 (both p < 0.0001). In participants with A1AT, CTGF and LSM were negatively correlated ( p = 0.004); adding CTGF to an LSM prediction model improved R2 from 0.524 to 0.577 ( p = 0.0033). Biomarkers did not correlate with LSM in ALGS. A significant number of biomarker/lab correlations were found in participants with BA but not those with A1AT or ALGS. CONCLUSIONS: Endoglin, IL-8, and MMP-7 significantly correlate with increased LSM in children with BA, whereas CTGF inversely correlates with LSM in participants with A1AT; these biomarkers appear to enhance prediction of LSM beyond clinical tests. Future disease-specific investigations of change in these biomarkers over time and as predictors of clinical outcomes will be important.

Functional characterization of variants found in Japanese patients with hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant form of vascular dysplasia. Genetic diagnosis is made by identifying loss-of-function variants in genes, such as ENG and ACVRL1. However, the causal mechanisms of various variants of unknown significance remains unclear. In this study, we analyzed 12 Japanese patients from 11 families who were clinically diagnosed with HHT. Sequencing analysis identified 11 distinct variants in ACVRL1 and ENG. Three of the 11 were truncating variants, leading to a definitive diagnosis, whereas the remaining eight were splice-site and missense variants that required functional analyses. In silico splicing analyses demonstrated that three variants, c.526-3C > G and c.598C > G in ACVRL1, and c.690-1G > A in ENG, caused aberrant splicing, as confirmed by a minigene assay. The five remaining missense variants were p.Arg67Gln, p.Ile256Asn, p.Leu285Pro, and p.Pro424Leu in ACVRL and p.Pro165His in ENG. Nanoluciferase-based bioluminescence analyses demonstrated that these ACVRL1 variants impaired cell membrane trafficking, resulting in the loss of bone morphogenetic protein 9 (BMP9) signal transduction. In contrast, the ENG mutation impaired BMP9 signaling despite normal cell membrane expression. The updated functional analysis methods performed in this study will facilitate effective genetic testing and appropriate medical care for patients with HHT.

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.

Elevated circulating BMP9 aggravates pulmonary angiogenesis in hepatopulmonary syndrome rats through ALK1-Endoglin-Smad1/5/9 signalling.

BACKGROUND: Bone morphogenetic protein 9 (BMP9) is a hepatokine that plays a pivotal role in the progression of liver diseases. Moreover, an increasing number of studies have shown that BMP9 is associated with hepatopulmonary syndrome (HPS), but its role in HPS is unclear. Here, we evaluated the influence of CBDL on BMP9 expression and investigated potential mechanisms of BMP9 signalling in HPS. METHODS: We profiled the circulating BMP9 levels in common bile duct ligation-induced HPS rat model, and then investigated the effects and mechanisms of HPS rat serum on pulmonary vascular endothelial dysfunction in rat model, as well as in primarily cultured rat pulmonary microvascular endothelial cells. RESULTS: Our data revealed that circulating BMP9 levels were significantly increased in the HPS rats compared to control group. Besides, the elevated BMP9 in HPS rat serum was not only crucial for promoting endothelial cell proliferation and tube formation through the activin receptor-like kinase1 (ALK1)-Endoglin-Smad1/5/9 pathway, but also important for accumulation of monocytes. Treatments with ALK1-Fc or silencing ALK1 expression to inhibit the BMP9 signalling pathway effectively eliminated these effects. In agreement with these observations, increased circulating BMP9 was associated with an increase in lung vessel density and accumulation of pro-angiogenic monocytes in the microvasculature in HPS rats. CONCLUSIONS: This study provided evidence that elevated circulating BMP9, secreted from the liver, promote pulmonary angiogenesis in HPS rats via ALK1-Endoglin-Smad1/5/9 pathway. In addition, BMP9-regulated pathways are also involved in accumulation of pro-angiogenic monocytes in the pulmonary microvasculature in HPS rats.

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 imbalance in maternal and cord blood is associated with neonatal birth weight and head circumference in pregnancies with major fetal congenital heart defect.

OBJECTIVES: To ascertain whether abnormalities in neonatal head circumference and/or body weight are associated with levels of angiogenic/antiangiogenic factors in the maternal and cord blood of pregnancies with a congenital heart defect (CHD) and to assess whether the specific type of CHD influences this association. METHODS: This was a multicenter case-control study of women carrying a fetus with major CHD. Recruitment was carried out between June 2010 and July 2018 at four tertiary care hospitals in Spain. Maternal venous blood was drawn at study inclusion and at delivery. Cord blood samples were obtained at birth when possible. Placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) were measured in maternal and cord blood. Biomarker concentrations in the maternal blood were expressed as multiples of the median (MoM). RESULTS: PlGF, sFlt-1 and sEng levels were measured in the maternal blood in 237 cases with CHD and 260 healthy controls, and in the cord blood in 150 cases and 56 controls. Compared with controls, median PlGF MoM in maternal blood was significantly lower in the CHD group (0.959 vs 1.022; P < 0.0001), while median sFlt-1/PlGF ratio MoM was significantly higher (1.032 vs 0.974; P = 0.0085) and no difference was observed in sEng MoM (0.981 vs 1.011; P = 0.4673). Levels of sFlt-1 and sEng were significantly higher in cord blood obtained from fetuses with CHD compared to controls (mean ± standard error of the mean, 447 ± 51 vs 264 ± 20 pg/mL; P = 0.0470 and 8.30 ± 0.92 vs 5.69 ± 0.34 ng/mL; P = 0.0430, respectively). Concentrations of sFlt-1 and the sFlt-1/PlGF ratio in the maternal blood at study inclusion were associated negatively with birth weight and head circumference in the CHD group. The type of CHD anomaly (valvular, conotruncal or left ventricular outflow tract obstruction) did not appear to alter these findings. CONCLUSIONS: Pregnancies with fetal CHD have an antiangiogenic profile in maternal and cord blood. This imbalance is adversely associated with neonatal head circumference and birth weight. © 2023 International Society of Ultrasound in Obstetrics and Gynecology.

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.

Association of cord blood Ang-1 and sCD105 levels with bronchopulmonary dysplasia in preterm infants.

BACKGROUND: To investigate the relationship between cord blood levels of Angiopoietin-1 (Ang-1) and S-endoglin (sCD105) and bronchopulmonary dysplasia (BPD) in preterm infants. METHODS: Sixty-one preterm infants admitted to the neonatal intensive care unit of the study hospital between July 2021 and September 2022 were included. Cord blood was collected after the birth of premature infants. Ang-1 and sCD105 levels were quantified using the vascular endothelial growth factor enzyme-linked immunosorbent assay. Preterm infants were divided into BPD and non-BPD groups, and differences in Ang-1 and sCD105 levels between the two groups were compared. A binary logistic model was used to assess the association between low and high levels Ang-1 and BPD in preterm infants. RESULTS: In the study, there were 20 preterm infants with BPD (32.8%) and 41 preterm infants with non-BPD (67.2%). Ang-1 concentration levels were lower in the BPD group than in the non-BPD group (7105.43 (5617.01-8523.00) pg/ml vs. 10488.03 (7946.19-15962.77) pg/ml, P = 0.027). However, the sCD105 concentration levels were not significantly different between the BPD and non-BPD groups (P = 0.246). A median Ang-1 concentration of 8800.40 pg/ml was calculated. Logistic regression analysis showed that after adjusting for gestational age, birth weight, and maternal prenatal steroid hormone application, the odds ratio (OR) was 8.577 for the risk of BPD in preterm infants with Ang-1 concentrations of ≤ 8800.40 pg/ml compared to those with Ang-1 concentrations of > 8800.40 pg/ml (OR: 8.577, 95% confidence interval: 1.265-58.155, P = 0.028). CONCLUSION: Our study indicated that Ang-1 levels in the cord blood of preterm infants may be associated the risk of BPD. In the future, we will continue to conduct study with large samples.

A novel frameshift mutation of the endoglin(ENG) gene causes hereditary hemorrhagic telangiectasia in a Chinese family.

PURPOSE: Hereditary hemorrhagic telangiectasia (HHT) is a dominantly inherited disorder that involves epistaxis, mucocutaneous telangiectases, and visceral arteriovenous malformations (AVMs). This study aims to investigate the genetic causes in a Chinese family with HHT. METHODS: HHT was confirmed according to Curaçao's diagnostic criteria. Three patients diagnosed with HHT and healthy members were recruited. Whole-exome sequencing (WES) and sanger sequencing were performed to define the patient's genetically pathogenic factor. RESULTS: The proband presented with recurrent epistaxis, hepatopulmonary arteriovenous malformation, and adenocarcinoma. A novel frameshift mutation (c.1376_1377delAC, p.H459Lfs*41) of the ENG gene was revealed in affected individuals by WES. There was no report of this variant and predicted to be highly damaging by causing truncation of the ENG protein. CONCLUSION: We report a novel variant in the ENG gene in Chinese that extends the mutational and phenotypic spectra of the ENG gene, and also demonstrates the feasibility of WES in the application of genetic diagnosis of HHT.

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.

Investigation of the Genetic Determinants of Telangiectasia and Solid Organ Arteriovenous Malformation Formation in Hereditary Hemorrhagic Telangiectasia (HHT).

Telangiectases and arteriovenous malformations (AVMs) are the characteristic lesions of Hereditary Hemorrhagic Telangiectasia (HHT). Somatic second-hit loss-of-function variations in the HHT causative genes, ENG and ACVRL1, have been described in dermal telangiectasias. It is unclear if somatic second-hit mutations also cause the formation of AVMs and nasal telangiectasias in HHT. To investigate the genetic mechanism of AVM formation in HHT, we evaluated multiple affected tissues from fourteen individuals. DNA was extracted from fresh/frozen tissue of 15 nasal telangiectasia, 4 dermal telangiectasia, and 9 normal control tissue biopsies, from nine unrelated individuals with HHT. DNA from six formalin-fixed paraffin-embedded (FFPE) AVM tissues (brain, lung, liver, and gallbladder) from five individuals was evaluated. A 736 vascular malformation and cancer gene next-generation sequencing (NGS) panel was used to evaluate these tissues down to 1% somatic mosaicism. Somatic second-hit mutations were identified in three in four AVM biopsies (75%) or half of the FFPE (50%) samples, including the loss of heterozygosity in ENG in one brain AVM sample, in which the germline mutation occurred in a different allele than a nearby somatic mutation (both are loss-of-function mutations). Eight of nine (88.9%) patients in whom telangiectasia tissues were evaluated had a somatic mutation ranging from 0.68 to 1.96% in the same gene with the germline mutation. Six of fifteen (40%) nasal and two of four (50%) dermal telangiectasia had a detectable somatic second hit. Additional low-level somatic mutations in other genes were identified in several telangiectasias. This is the first report that nasal telangiectasias and solid organ AVMs in HHT are caused by very-low-level somatic biallelic second-hit mutations.

Evaluation of Microvascular Density in Glioblastomas in Relation to p53 and Ki67 Immunoexpression.

Glioblastoma is the most aggressive tumor in the central nervous system, with a survival rate of less than 15 months despite multimodal therapy. Tumor recurrence frequently occurs after removal. Tumoral angiogenesis, the formation of neovessels, has a positive impact on tumor progression and invasion, although there are controversial results in the specialized literature regarding its impact on survival. This study aims to correlate the immunoexpression of angiogenesis markers (CD34, CD105) with the proliferation index Ki67 and p53 in primary and secondary glioblastomas. This retrospective study included 54 patients diagnosed with glioblastoma at the Pathology Department of County Emergency Clinical Hospital Târgu Mureș. Microvascular density was determined using CD34 and CD105 antibodies, and the results were correlated with the immunoexpression of p53, IDH1, ATRX and Ki67. The number of neoformed blood vessels varied among cases, characterized by different shapes and calibers, with endothelial cells showing modified morphology and moderate to marked pleomorphism. Neovessels with a glomeruloid aspect, associated with intense positivity for CD34 or CD105 in endothelial cells, were observed, characteristic of glioblastomas. Mean microvascular density values were higher for the CD34 marker in all cases, though there were no statistically significant differences compared to CD105. Mutant IDH1 and ATRX glioblastomas, wild-type p53 glioblastomas, and those with a Ki67 index above 20% showed a more abundant microvascular density, with statistical correlations not reaching significance. This study highlighted a variety of percentage intervals of microvascular density in primary and secondary glioblastomas using immunohistochemical markers CD34 and CD105, respectively, with no statistically significant correlation between evaluated microvascular density and p53 or Ki67.

EPDR1 is a noncanonical effector of insulin-mediated angiogenesis regulated by an endothelial-specific TGF-ß receptor complex.

Insulin signaling in blood vessels primarily functions to stimulate angiogenesis and maintain vascular homeostasis through the canonical PI3K and MAPK signaling pathways. However, angiogenesis is a complex process coordinated by multiple other signaling events. Here, we report a distinct crosstalk between the insulin receptor and endoglin/activin receptor-like kinase 1 (ALK1), an endothelial cell-specific TGF-ß receptor complex essential for angiogenesis. While the endoglin-ALK1 complex normally binds to TGF-ß or bone morphogenetic protein 9 (BMP9) to promote gene regulation via transcription factors Smad1/5, we show that insulin drives insulin receptor oligomerization with endoglin-ALK1 at the cell surface to trigger rapid Smad1/5 activation. Through quantitative proteomic analysis, we identify ependymin-related protein 1 (EPDR1) as a major Smad1/5 gene target induced by insulin but not by TGF-ß or BMP9. We found endothelial EPDR1 expression is minimal at the basal state but is markedly enhanced upon prolonged insulin treatment to promote cell migration and formation of capillary tubules. Conversely, we demonstrate EPDR1 depletion strongly abrogates these angiogenic effects, indicating that EPDR1 is a crucial mediator of insulin-induced angiogenesis. Taken together, these results suggest important therapeutic implications for EPDR1 and the TGF-ß pathways in pathologic angiogenesis during hyperinsulinemia and insulin resistance.

External validation of genetically predicted protein biomarkers for pancreatic cancer risk using aptamer-based plasma levels: A prospective analysis in the Atherosclerosis Risk in Communities Study.

Genetically predicted proteins have been associated with pancreatic cancer risk previously. We aimed to externally validate the associations of 53 candidate proteins with pancreatic cancer risk using directly measured, prediagnostic levels. We conducted a prospective cohort study of 10 355 US Black and White men and women in the Atherosclerosis Risk in Communities (ARIC) study. Aptamer-based plasma proteomic profiling was previously performed using blood collected in 1993 to 1995, from which the proteins were selected. By 2015 (median: 20 years), 93 incident pancreatic cancer cases were ascertained. Cox regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for protein tertiles, and adjust for age, race, and known risk factors. Of the 53 proteins, three were statistically significantly, positively associated with risk-GLCE (tertile 3 vs 1: HR = 1.88, 95% CI: 1.12-3.13; P-trend = 0.01), GOLM1 (aptamer 1: HR = 1.98, 95% CI: 1.16-3.37; P-trend = 0.01; aptamer 2: HR = 1.86, 95% CI: 1.07-3.24; P-trend = 0.05), and QSOX2 (HR = 1.96, 95% CI: 1.09-3.58; P-trend = 0.05); two were inversely associated-F177A (HR = 0.59, 95% CI: 0.35-1.00; P-trend = 0.05) and LIFsR (HR = 0.55, 95% CI: 0.32-0.93; P-trend = 0.03); and one showed a statistically significant lower risk in the middle tertile-endoglin (HR = 0.50, 95% CI: 0.29-0.86); by chance, we expected significant associations for 2.65 proteins. FAM3D, IP10, sTie-1 (positive); SEM6A and JAG1 (inverse) were suggestively associated with risk. Of these 11, 10 proteins-endoglin, FAM3D, F177A, GLCE, GOLM1, JAG1, LIFsR, QSOX2, SEM6A and sTie-1-were consistent in direction of association with the discovery studies. This prospective study validated or supports 10 proteins as associated with pancreatic cancer risk.

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.