Molecular and Functional Cargo of Plasma-Derived Exosomes in Patients with Hereditary Hemorrhagic Telangiectasia.

Background: Hereditary Hemorrhagic Telangiectasia (HHT) is a genetic disorder leading to frequent bleeding in several organs. As HHT diagnosis is demanding and depends on clinical criteria, liquid biopsy would be beneficial. Exosomes from biofluids are nano-sized vesicles for intercellular communication. Their cargo and characteristics represent biomarkers for many diseases. Here, exosomes of HHT patients were examined regarding their biosignature. Methods: Exosomes were isolated from the plasma of 20 HHT patients and 17 healthy donors (HDs). The total exosomal protein was quantified, and specific proteins were analyzed using Western blot and antibody arrays. Human umbilical vein endothelial cells (HUVECs) co-incubated with exosomes were functionally examined via immunofluorescence, proliferation, and scratch assay. Results: The levels of the angiogenesis-regulating protein Thrombospondin-1 were significantly higher in HHT compared to HD exosomes. Among HHT, but not HD exosomes, a negative correlation between total exosomal protein and soluble Endoglin (sENG) levels was found. Other exosomal proteins (ALK1, ALK5) and the particle concentration significantly correlated with disease severity parameters (total consultations/interventions, epistaxis severity score) in HHT patients. Functionally, HUVECs were able to internalize both HD and HHT exosomes, inducing a similar change in the F-Actin structure and a reduction in migration and proliferation. Conclusions: This study provided first insights into the protein cargo and function of HHT-derived exosomes. The data indicate changes in sENG secretion via exosomes and reveal exosomal Thrombospondin-1 as a potential biomarker for HHT. Several exosomal characteristics were pointed out as potential liquid biomarkers for disease severity, revealing a possible new way of diagnosis and prognosis of HHT.

Generation of an Isogenic Hereditary Hemorrhagic Telangiectasia Model via Prime Editing in Human Induced Pluripotent Stem Cells.

Prime editing (PE) is a recently developed genome-editing technique that enables versatile editing. Despite its flexibility and potential, applying PE in human induced pluripotent stem cells (hiPSCs) has not been extensively addressed. Genetic disease models using patient-derived hiPSCs have been used to study mechanisms and drug efficacy. However, genetic differences between patient and control cells have been attributed to the inaccuracy of the disease model, highlighting the significance of isogenic hiPSC models. Hereditary hemorrhagic telangiectasia 1 (HHT1) is a genetic disorder caused by an autosomal dominant mutation in endoglin (ENG). Although previous HHT models using mice and HUVEC have been used, these models did not sufficiently elucidate the relationship between the genotype and disease phenotype in HHT, demanding more clinically relevant models that reflect human genetics. Therefore, in this study, we used PE to propose a method for establishing an isogenic hiPSC line. Clinically reported target mutation in ENG was selected, and a strategy for PE was designed. After cloning the ENGineered PE guide RNA, hiPSCs were nucleofected along with PEmax and hMLH1dn plasmids. As a result, hiPSC clones with the intended mutation were obtained, which showed no changes in pluripotency or genetic integrity. Furthermore, introducing the ENG mutation increased the expression of proangiogenic markers during endothelial organoid differentiation. Consequently, our results suggest the potential of PE as a toolkit for establishing isogenic lines, enabling disease modeling based on hiPSC-derived disease-related cells or organoids. This approach is expected to stimulate mechanistic and therapeutic studies on genetic diseases.a.

ENG is a Biomarker of Prognosis and Angiogenesis in Liver Cancer, and Promotes the Differentiation of Tumor Cells into Vascular ECs.

BACKGROUND: Liver cancer is a highly lethal malignancy with frequent recurrence, widespread metastasis, and low survival rates. The aim of this study was to explore the role of Endoglin (ENG) in liver cancer progression, as well as its impacts on angiogenesis, immune cell infiltration, and the therapeutic efficacy of sorafenib. METHODS: A comprehensive evaluation was conducted using online databases Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA), 76 pairs of clinical specimens of tumor and adjacent non-tumor liver tissue, and tissue samples from 32 hepatocellular carcinoma (HCC) patients treated with sorafenib. ENG expression levels were evaluated using quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), Western blot, and immunohistochemical analysis. Cox regression analysis, Spearman rank correlation analysis, and survival analysis were used to assess the results. Functional experiments included Transwell migration assays and tube formation assays with Human Umbilical Vein Endothelial Cells (HUVECs). RESULTS: Tumor cells exhibited retro-differentiation into endothelial-like cells, with a significant increase in ENG expression in these tumor-derived endothelial cells (TDECs). High expression of ENG was associated with more aggressive cancer characteristics and worse patient prognosis. Pathway enrichment and functional analyses identified ENG as a key regulator of immune responses and angiogenesis in liver cancer. Further studies confirmed that ENG increases the expression of Collagen type Iα1 (COL1A1), thereby promoting angiogenesis in liver cancer. Additionally, HCC patients with elevated ENG levels responded well to sorafenib treatment. CONCLUSIONS: This study found that ENG is an important biomarker of prognosis in liver cancer. Moreover, ENG is associated with endothelial cell differentiation in liver cancer and plays a crucial role in formation of the tumor vasculature. The assessment of ENG expression could be a promising strategy to identify liver cancer patients who might benefit from targeted immunotherapies.

Increasing Endoglin Deletion in Endothelial Cells Exacerbates the Severity of Brain Arteriovenous Malformation in Mouse.

Endoglin (ENG) mutation causes type 1 hereditary hemorrhagic telangiectasia (HHT1). HHT1 patients have arteriovenous malformations (AVMs) in multiple organs, including the brain. In mice, Eng deletion induced by R26RCreER or SM22αCre leads to AVM development in the brain and other organs. We hypothesized that an increase in Eng- negative ECs will enhance AVM severity. To increase EC Eng deletion, we used a codon-improved cre (icre), which is more potent in recombination of the floxed alleles than the wild-type (WT) cre. R26RCreER;Engf/f mice that have a Rosa promoter driving and tamoxifen (TM)-inducible WT cre expression globally, and PdgfbiCreER;Engf/f mice that have a Pdgfb promoter driving and TM-inducible icre expression in ECs were treated with three intra-peritoneal injections of TM (2.5 mg/25 g of body weight) to delete Eng globally or in the ECs. AAV-VEGF was stereotactically injected into the brain to induce brain focal angiogenesis and brain AVM. We found that icre caused more Eng deletion in the brain, indicated by a lower level of Eng proteins (p < 0.001) and fewer Eng-positive ECs (p = 0.01) than mice with WT cre. Mice with icre-mediated Eng deletion have more abnormal vessels (p = 0.02), CD68+ macrophages (p = 0.002), and hemorrhage (p = 0.04) and less vascular pericyte and smooth muscle coverage than mice with WT cre. In addition, arteriovenous shunts were detected in the intestines of icre mice, a phenotype that has not been detected in WT cre mice before. RNA-seq analysis showed that 8 out of the 10 top upregulated pathways identified by gene ontology (GO) analysis are related to inflammation. Therefore, the increase in Eng deletion in ECs exacerbates AVM severity, which is associated with enhanced inflammation. Strategies that can reduce Eng-negative ECs could be used to develop new therapies to reduce AVM severity for HHT1 patients.

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.

Employment of diverse in vitro systems for analyzing multiple aspects of disease, hereditary hemorrhagic telangiectasia (HHT).

BACKGROUND: In vitro disease modeling enables translational research by providing insight into disease pathophysiology and molecular mechanisms, leading to the development of novel therapeutics. Nevertheless, in vitro systems have limitations for recapitulating the complexity of tissues, and a single model system is insufficient to gain a comprehensive understanding of a disease. RESULTS: Here we explored the potential of using several models in combination to provide mechanistic insight into hereditary hemorrhagic telangiectasia (HHT), a genetic vascular disorder. Genome editing was performed to establish hPSCs (H9) with ENG haploinsufficiency and several in vitro models were used to recapitulate the functional aspects of the cells that constitute blood vessels. In a 2D culture system, endothelial cells showed early senescence, reduced viability, and heightened susceptibility to apoptotic insults, and smooth muscle cells (SMCs) exhibited similar behavior to their wild-type counterparts. Features of HHT were evident in 3D blood-vessel organoid systems, including thickening of capillary structures, decreased interaction between ECs and surrounding SMCs, and reduced cell viability. Features of ENG haploinsufficiency were observed in arterial and venous EC subtypes, with arterial ECs showing significant impairments. Molecular biological approaches confirmed the significant downregulation of Notch signaling in HHT-ECs. CONCLUSIONS: Overall, we demonstrated refined research strategies to enhance our comprehension of HHT, providing valuable insights for pathogenic analysis and the exploration of innovative therapeutic interventions. Additionally, these results underscore the importance of employing diverse in vitro systems to assess multiple aspects of disease, which is challenging using a single in vitro system.

Endothelial progenitor cells control remodeling of uterine spiral arteries for the establishment of utero-placental circulation.

Placental ischemia, resulting from inadequate remodeling of uterine spiral arteries, is a factor in the development of preeclampsia. However, the effect of endothelial progenitor cells that play a role in the vascular injury-repair program is largely unexplored during remodeling. Here, we observe that preeclampsia-afflicted uterine spiral arteries transition to a synthetic phenotype in vascular smooth muscle cells and characterize the regulatory axis in endothelial progenitor cells during remodeling in human decidua basalis. Excessive sEng, secreted by AMP-activated protein kinase (AMPK)-deficient endothelial progenitor cells through the inhibition of HO-1, damages residual endothelium and leads to the accumulation of extracellular matrix produced by vascular smooth muscle cells during remodeling, which is further confirmed by animal models. Collectively, our findings suggest that the impaired functionality of endothelial progenitor cells contributes to the narrowing of remodeled uterine spiral arteries, leading to reduced utero-placental perfusion. This mechanism holds promise in elucidating the pathogenesis of preeclampsia.

Antisense oligonucleotide-mediated terminal intron retention of endoglin: A potential strategy to inhibit renal interstitial fibrosis.

TGF-ß is considered an important cytokine in the development of interstitial fibrosis in chronic kidney disease. The TGF-ß co-receptor endoglin (ENG) tends to be upregulated in kidney fibrosis. ENG has two membrane bound isoforms generated via alternative splicing. Long-ENG was shown to enhance the extent of renal fibrosis in an unilateral ureteral obstruction mouse model, while short-ENG inhibited renal fibrosis. Here we aimed to achieve terminal intron retention of endoglin using antisense-oligo nucleotides (ASOs), thereby shifting the ratio towards short-ENG to inhibit the TGF-ß1-mediated pro-fibrotic response. We isolated mRNA from kidney biopsies of patients with chronic allograft disease (CAD) (n = 12) and measured total ENG and short-ENG mRNA levels. ENG mRNA was upregulated 2.3 fold (p < 0.05) in kidneys of CAD patients compared to controls, while the percentage short-ENG of the total ENG mRNA was significantly lower (1.8 fold; p < 0.05). Transfection of ASOs that target splicing regulatory sites of ENG into TK173 fibroblasts led to higher levels of short-ENG (2 fold; p < 0.05). In addition, we stimulated these cells with TGF-ß1 and measured a decrease in upregulation of ACTA2, COL1A1 and FN1 mRNA levels, and protein expression of αSMA, collagen type I, and fibronectin. These results show a potential for ENG ASOs as a therapy to reduce interstitial fibrosis in CKD.

HDAC6 Enhances Endoglin Expression through Deacetylation of Transcription Factor SP1, Potentiating BMP9-Induced Angiogenesis.

Histone deacetylase 6 (HDAC6) plays a crucial role in the acetylation of non-histone proteins and is notably implicated in angiogenesis, though its underlying mechanisms were previously not fully understood. This study conducted transcriptomic and proteomic analyses on vascular endothelial cells with HDAC6 knockdown, identifying endoglin (ENG) as a key downstream protein regulated by HDAC6. This protein is vital for maintaining vascular integrity and plays a complex role in angiogenesis, particularly in its interaction with bone morphogenetic protein 9 (BMP9). In experiments using human umbilical vein endothelial cells (HUVECs), the pro-angiogenic effects of BMP9 were observed, which diminished following the knockdown of HDAC6 and ENG. Western blot analysis revealed that BMP9 treatment increased SMAD1/5/9 phosphorylation, a process hindered by HDAC6 knockdown, correlating with reduced ENG expression. Mechanistically, our study indicates that HDAC6 modulates ENG transcription by influencing promoter activity, leading to increased acetylation of transcription factor SP1 and consequently altering its transcriptional activity. Additionally, the study delves into the structural role of HDAC6, particularly its CD2 domain, in regulating SP1 acetylation and subsequently ENG expression. In conclusion, the present study underscores the critical function of HDAC6 in modulating SP1 acetylation and ENG expression, thereby significantly affecting BMP9-mediated angiogenesis. This finding highlights the potential of HDAC6 as a therapeutic target in angiogenesis-related processes.

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.

The ENG/VEGFα Pathway Is Likely Affected by a Nonsense Variant of Endoglin (ENG)/CD105, Causing Hereditary Hemorrhagic Telangiectasia Type 1 (HHT1) in a Chinese Family.

Hereditary hemorrhagic telangiectasia (HHT), also called Rendu-Osler syndrome, is a group of rare genetic diseases characterized by autosomal dominance, multisystemic vascular dysplasia, and age-related penetrance. This includes arteriovenous malformations (AVMs) in the skin, brain, lung, liver, and mucous membranes. The correlations between the phenotype and genotype for HHT are not clear. An HHT Chinese pedigree was recruited. Whole exome sequencing (WES) analysis, Sanger verification, and co-segregation were conducted. Western blotting was performed for monitoring ENG/VEGFα signaling. As a result, a nonsense, heterozygous variant for ENG/CD105: c.G1169A:p. Trp390Ter of the proband with hereditary hemorrhagic telangiectasia type 1 (HHT1) was identified, which co-segregated with the disease in the M666 pedigree. Western blotting found that, compared with the normal levels associated with non-carrier family members, the ENG protein levels in the proband showed approximately a one-half decrease (47.4% decrease), while levels of the VEGFα protein, in the proband, showed approximately a one-quarter decrease (25.6% decrease), implying that ENG haploinsufficiency, displayed in the carrier of this variant, may affect VEGFα expression downregulation. Pearson and Spearman correlation analyses further supported TGFß/ENG/VEGFα signaling, implying ENG regulation in the blood vessels. Thus, next-generation sequencing including WES should provide an accurate strategy for gene diagnosis, therapy, genetic counseling, and clinical management for rare genetic diseases including that in HHT1 patients.

High-throughput Analysis of Capillary Density in Skeletal Muscle Cross Sections.

Capillary density in skeletal muscles is key to estimate exercise capacity in healthy individuals, athletes, and those with muscle-related pathologies. Here, we present a step-by-step, high-throughput semi-automated method for quantifying capillary density from whole human skeletal muscle cross-sections, in areas of the muscle occupied by myofibers. We provide a detailed protocol for immunofluorescence staining, image acquisition, processing, and quantification. Image processing is performed in ImageJ, and data analysis is conducted in R. The provided protocol allows high-throughput quantification of capillary density. Key features • This protocol builds upon the method and results described in Abbassi-Daloii et al. (2023b). • It includes step-by-step details on image acquisition and image processing of the entire muscle section. • It enables high-throughput and semi-automated image quantification of capillary density. • It provides a robust analysis for determining capillary density over the entire muscle cross section.

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.

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.

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.

Effects of physical activity on placental analytes in nulliparous persons.

INTRODUCTION: Physical activity during pregnancy has long been investigated for its role in preeclampsia prevention. The mechanism of this relationship is unknown, although some studies suggest physical activity may affect placental analytes throughout pregnancy. The objective of this study was to determine the effect of physical activity on preeclampsia-associated placental analytes using a prospective cohort of pregnant nulliparous patients. METHODS: This was a secondary analysis of the Nulliparous Pregnancy Outcomes Study: Monitoring Mothers-to-Be. Frequency and duration of up to three leisure activities was reported in the first and second trimesters and was analyzed, with participants either meeting or not meeting the recommended exercise of 150 min per week. Levels of the following placental analytes, placental growth factor, soluble endoglin, and soluble fms-like tyrosine kinase-1 (sFLT1), were analyzed stratified by the physical activity level. RESULTS: A total of 1,956 participants were included in the analysis. The level of sFLT1 in the first trimester was lower in the group that had ≥ 150 min per week of physical activity, compared to the group that had < 150 min (846.3 [821.6, 871,8] versus 893.0 [864.5,922.5], p = 0.017). There were no significant sFLT1 changes in the second trimester based on physical activity. After controlling for maternal demographic and clinical factors, sFLT1 levels in the second trimester were significantly lower (p = 0.049) in participants that had ≥ 150 min of physical activity per week. DISCUSSION: Our findings of decreased sFLT1 levels suggest this could be the mechanism explaining the association between PA in pregnancy and lower risk of preeclampsia.

Endoglin and soluble endoglin in liver sinusoidal endothelial dysfunction in vivo.

Liver sinusoidal endothelial cells (LSECs) play a crucial role in regulating the hepatic function. Endoglin (ENG), a transmembrane glycoprotein, was shown to be related to the development of endothelial dysfunction. In this study, we hypothesized the relationship between changes in ENG expression and markers of liver sinusoidal endothelial dysfunction (LSED) during liver impairment. Male C57BL/6J mice aged 9-12 weeks were fed with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet (intrahepatic cholestasis) or choline-deficient l-amino acid defined high-fat diet (CDAA-HFD) (non-alcoholic steatohepatitis (NASH)). Significant increases in liver enzymes, fibrosis, and inflammation biomarkers were observed in both cholestasis and NASH. Decreased p-eNOS/eNOS and VE-cadherin protein expression and a significant increase in VCAM-1 and ICAM-1 expression were detected, indicating LSED in both mouse models of liver damage. A significant reduction of ENG in the DDC-fed mice, while a significant increase of ENG in the CDAA-HFD group was observed. Both DDC and CDAA-HFD-fed mice showed a significant increase in MMP-14 protein expression, which is related to significantly increased levels of soluble endoglin (sENG) in the plasma. In conclusion, we demonstrated that intrahepatic cholestasis and NASH result in an altered ENG expression, predominantly in LSECs, suggesting a critical role of ENG expression for the proper function of liver sinusoids. Both pathologies resulted in elevated sENG levels, cleaved by MMP-14 expressed predominantly from LSECs, indicating sENG as a liver injury biomarker.

VEGFR2 Expression Correlates with Postnatal Development of Brain Arteriovenous Malformations in a Mouse Model of Type I Hereditary Hemorrhagic Telangiectasia.

Brain arteriovenous malformations (BAVMs) are a critical concern in hereditary hemorrhagic telangiectasia (HHT) patients, carrying the risk of life-threatening intracranial hemorrhage. While traditionally seen as congenital, the debate continues due to documented de novo cases. Our primary goal was to identify the precise postnatal window in which deletion of the HHT gene Endoglin (Eng) triggers BAVM development. We employed SclCreER(+);Eng2f/2f mice, enabling timed Eng gene deletion in endothelial cells via tamoxifen. Tamoxifen was given during four postnatal periods: P1-3, P8-10, P15-17, and P22-24. BAVM development was assessed at 2-3 months using latex dye perfusion. We examined the angiogenic activity by assessing vascular endothelial growth factor receptor 2 (VEGFR2) expression via Western blotting and Flk1-LacZ reporter mice. Longitudinal magnetic resonance angiography (MRA) was conducted up to 9 months. BAVMs emerged in 88% (P1-3), 86% (P8-10), and 55% (P15-17) of cases, with varying localization. Notably, the P22-24 group did not develop BAVMs but exhibited skin AVMs. VEGFR2 expression peaked in the initial 2 postnatal weeks, coinciding with BAVM onset. These findings support the "second hit" theory, highlighting the role of early postnatal angiogenesis in initiating BAVM development in HHT type I mice.

Low Plasma Levels of Soluble Endoglin and Cardiovascular Events in Patients Undergoing Coronary Angiography.

TGF-ß is recognized as playing a protective role against atherosclerosis. Endoglin is a receptor for TGF-ß, and its expression is upregulated in atherosclerotic plaques. Endoglin is secreted from the cell membrane into the circulation as a soluble form (sEng). We previously reported that plasma sEng levels were low in patients with coronary artery disease (CAD). However, the prognostic value of sEng levels has not been clarified. We investigated the association between plasma sEng levels and cardiovascular events in 403 patients who had an elective coronary angiography and were then followed up. Cardiovascular events were defined as cardiovascular death, myocardial infarction, unstable angina, heart failure, stroke, or coronary revascularization. Of the 403 patients, 209 (52%) had CAD. Plasma sEng levels were lower in patients with CAD than in those without CAD (median 4.26 vs. 4.41 ng/mL, p < 0.025). During a mean follow-up period of 7.5 ± 4.5 years, cardiovascular events occurred in 79 patients. Compared with 324 patients without events, 79 with events had lower sEng levels (3.95 vs. 4.39 ng/mL) and more often had an sEng level < 3.9 ng/mL (47% vs. 28%) (p < 0.02). A Kaplan-Meier analysis showed lower event-free survival in patients with sEng < 3.9 ng/mL than in those with ≥3.9 ng/mL (p < 0.02). In a multivariate Cox proportional hazards analysis, the sEng level (<3.9 ng/mL) was an independent predictor of cardiovascular events (hazard ratio: 1.59; 95%CI: 1.01-2.49). Furthermore, only among the 209 patients with CAD, the sEng level was also a predictor of further cardiovascular events (hazard ratio: 2.07; 95%CI: 1.24-3.45). Thus, low plasma sEng levels were found to be associated with an increased risk of cardiovascular events in patients with CAD and patients undergoing coronary angiography.