RESUMEN
The present study aimed to compare the effect of photobiomodulation with different energy densities on the angiogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and stem cells from human exfoliated deciduous teeth (SHED). Photobiomodulation therapy with a 660 nm diode laser (2.4 J/cm2 and 3.9 J/cm2) on two consecutive days post-culture was applied to two types of stem cells (hPDLSCs and SHED). The Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) test was undertaken to investigate Vascular Endothelial Growth Factor-A (VEGF-A) and Angiopoietin I (ANG-I) genes on days 1, 3, 5, 7, and 10 after the first session of laser application. The 4',6-diamidino-2-phenylindole (DAPI) staining and Methyl Thiazolyl Tetrazolium (MTT) test were conducted on days 1, 3, and 5 after the first session of laser application, to assess the cell viability. The Two-way ANOVA with Tukey post hoc test was used to analyze the outcomes of the MTT and RT-qPCR tests. The results of the MTT and DAPI convergently illustrated that the groups receiving photobiomodulation with 2.4 J/cm2 had higher cell viability compared to 3.9 J/cm2. All experimental groups showed an upregulation of VEGF-A and ANG-I gene expression from day 1 to 5, followed by a downregulation from day 5 to 10. The groups with cultured hPDLSCs and SHED receiving photobiomodulation using 2.4 J/cm2 had the most amounts of VEGF-A and ANG-I gene expression on day 5, respectively. In conclusion, the 660 nm mediated photobiomodulation therapy of cultured SHED and hPDLSCs with 2.4 J/cm2 energy density may be associated with higher angiogenic differentiation (the expression of VEGF-A and ANG-I) as well as higher cell viability compared to the photobiomodulation therapy with 3.9 J/cm2.
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Diferenciación Celular , Terapia por Luz de Baja Intensidad , Ligamento Periodontal , Células Madre , Diente Primario , Factor A de Crecimiento Endotelial Vascular , Humanos , Diferenciación Celular/efectos de la radiación , Terapia por Luz de Baja Intensidad/métodos , Ligamento Periodontal/citología , Ligamento Periodontal/efectos de la radiación , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Madre/efectos de la radiación , Diente Primario/citología , Neovascularización Fisiológica/efectos de la radiación , Reacción en Cadena en Tiempo Real de la Polimerasa , Angiopoyetina 1 , Supervivencia Celular/efectos de la radiación , Láseres de Semiconductores/uso terapéutico , Técnicas In Vitro , Células CultivadasAsunto(s)
Angiopoyetina 1 , Factor A de Crecimiento Endotelial Vascular , Cicatrización de Heridas , Animales , Angiopoyetina 1/genética , Angiopoyetina 1/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas/genética , Ratones , Terapia Genética/métodos , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Técnicas de Transferencia de Gen , HumanosRESUMEN
Mechanical stress regulates various biological processes in cells, tissues, and organs as well as contributes to the pathogenesis of various diseases. The retina is subjected to mechanical stress imposed by intraocular pressure as well as by retinal hemorrhage and edema. Responses to mechanical stress have been studied in retinal pigment epithelial cells and Müller cells of the retina, with the former cells having been found to undergo a stress-induced increase in the expression of vascular endothelial growth factor (VEGF), which plays a key role in physiological and pathological angiogenesis in the retina. We here examined the effects of stretch stimulation on the expression of angiogenic factors in cultured human Müller cells. Reverse transcription and quantitative PCR analysis revealed that expression of the VEGF-A gene was increased by such stimulation in Müller cells, whereas that of the angiopoietin 1 gene was decreased. An enzyme-linked immunosorbent assay showed that stretch stimulation also increased VEGF secretion from these cells. Expression of the transcription factor HIF-1α (hypoxia-inducible factor-1α) was increased at both mRNA and protein levels by stretch stimulation, and the HIF-1α inhibitor CAY10585 prevented the effects of mechanical stress on VEGF-A gene expression and VEGF secretion. Furthermore, RNA-sequencing analysis showed that the expression of angiogenesis-related pathway genes was upregulated by stretch stimulation. Our results thus suggest that mechanical stress induces VEGF production in Müller cells in a manner dependent on HIF-1α, and that HIF-1α is therefore a potential therapeutic target for conditions such as diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion.
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Células Ependimogliales , Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia , Estrés Mecánico , Factor A de Crecimiento Endotelial Vascular , Humanos , Células Ependimogliales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Angiopoyetina 1/metabolismo , Angiopoyetina 1/genética , Western BlottingRESUMEN
Loss of the inner blood-retinal barrier (BRB) integrity is a main feature of ocular diseases such as diabetic macular edema. However, there is a lack of clarity on how inner BRB function is modulated within the diabetic retina. The current study examined whether eucalyptol inhibited inner BRB destruction and aberrant retinal angiogenesis in 33 mM glucose-exposed human retinal microvascular endothelial (RVE) cells and db/db mice. This study further examined the molecular mechanisms underlying endothelial dysfunction including retinal endoplasmic reticulum (ER) stress and angiopoietin (Ang)/Tie axis in conjunction with vascular endothelial growth factor (VEGF). Eucalyptol is a naturally occurring monoterpenoid and an achiral aromatic component of many plants including eucalyptus leaves. Nontoxic eucalyptol reduced the production of amyloid-ß (Aß) protein in glucose-loaded RVE cells and in diabetic mice. This natural compound blocked apoptosis of Aß-exposed RVE cells in diabetic mouse eyes by targeting ER stress via the inhibition of PERK-eIF2α-ATF4-CHOP signaling. Eucalyptol promoted activation of the Ang-1/Tie-2 pathway and dual inhibition of Ang-2/VEGF in Aß-exposed RVE cells and in diabetic eyes. Supply of eucalyptol reversed the induction of junction proteins in glucose/Aß-exposed RVE cells within the retina and reduced permeability. In addition, oral administration of eucalyptol reduced vascular leaks in diabetic retinal vessels. Taken together, these findings clearly show that eucalyptol inhibits glucose-induced Aß-mediated ER stress and manipulates Ang signaling in diabetic retinal vessels, which ultimately blocks abnormal angiogenesis and loss of inner BRB integrity. Therefore, eucalyptol provides new treatment strategies for diabetes-associated RVE defects through modulating diverse therapeutic targets including ER stress, Ang-1/Tie-2 signaling, and Ang-2/VEGF.
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Diabetes Mellitus Experimental , Retinopatía Diabética , Estrés del Retículo Endoplásmico , Eucaliptol , Transducción de Señal , Animales , Estrés del Retículo Endoplásmico/efectos de los fármacos , Eucaliptol/farmacología , Ratones , Retinopatía Diabética/metabolismo , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/patología , Transducción de Señal/efectos de los fármacos , Humanos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Barrera Hematorretinal/metabolismo , Barrera Hematorretinal/efectos de los fármacos , Masculino , Apoptosis/efectos de los fármacos , Angiopoyetina 1/metabolismo , Ratones Endogámicos C57BL , Vasos Retinianos/metabolismo , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/patologíaRESUMEN
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.
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Angiopoyetina 1 , Displasia Broncopulmonar , Endoglina , Sangre Fetal , Recien Nacido Prematuro , Humanos , Displasia Broncopulmonar/sangre , Recién Nacido , Endoglina/sangre , Recien Nacido Prematuro/sangre , Sangre Fetal/química , Sangre Fetal/metabolismo , Femenino , Masculino , Angiopoyetina 1/sangre , Biomarcadores/sangre , Modelos LogísticosRESUMEN
BACKGROUND: In addition to their fundamental roles in preserving vascular integrity, platelets also contribute to tumor angiogenesis and metastasis. However, despite being a reservoir for angiogenic and metastatic cytokines, platelets also harbor negative regulators of tumor progression. Angpt1 (angiopoietin-1) is a cytokine essential for developmental angiogenesis that also protects against tumor cell metastasis through an undefined mechanism. Although activated platelets release Angpt1 from α-granules into circulation, the contributions of platelet Angpt1 to tumor growth, angiogenesis, and metastasis have not been investigated. METHODS: Using cytokine arrays and ELISAs, we first compared platelet Angpt1 levels in breast and melanoma mouse tumor models to tumor-free controls. We then assessed tumor growth and metastasis in mice lacking megakaryocyte and platelet Angpt1 (Angpt1Plt KO). The spontaneous metastasis of mammary-injected tumor cells to the lungs was quantified using RT-PCR (reverse transcription-polymerase chain reaction). The lung colonization of intravenously injected tumor cells and tumor cell extravasation were determined using fluorescent microscopy and flow cytometry. RESULTS: Platelet Angpt1 is selectively upregulated in the PyMT (polyoma middle tumor antigen) breast cancer mouse model, and platelets are the principal source of Angpt1 in blood circulation. While primary tumor growth and angiogenesis were unaffected, Angpt1Plt KO mice had both increased spontaneous lung metastasis and tumor cell lung colonization following mammary or intravenous injection, respectively. Although platelet Angpt1 did not affect initial tumor cell entrapment in the lungs, Angpt1Plt KO mice had increased tumor cell retention and extravasation. Serum from Angpt1Plt KO mice increased endothelial permeability and reduced VE (vascular endothelial)-cadherin expression at endothelial junctions compared with serum from control mice (Angpt1WT). CONCLUSIONS: Platelets provide an intravascular source of Angpt1 that restrains tumor metastasis by preserving the lung microvasculature to limit tumor cell extravasation.
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Angiopoyetina 1 , Plaquetas , Neoplasias Pulmonares , Ratones Noqueados , Neovascularización Patológica , Animales , Angiopoyetina 1/genética , Angiopoyetina 1/metabolismo , Angiopoyetina 1/sangre , Plaquetas/metabolismo , Plaquetas/patología , Femenino , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/prevención & control , Ratones Endogámicos C57BL , Melanoma Experimental/patología , Melanoma Experimental/metabolismo , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/sangre , Melanoma Experimental/secundario , Melanoma Experimental/genética , Línea Celular Tumoral , Ratones , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/sangre , Carga Tumoral , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: It has been proposed that anti-angiogenesis therapy could induce tumor "vascular normalization" and further enhance the efficacy of chemotherapy, radiotherapy, target therapy, and immunotherapy for nearly twenty years. However, the detailed molecular mechanism of this phenomenon is still obscure. METHOD: Overexpression and knockout of CCL28 in human lung adenocarcinoma cell line A549 and murine lung adenocarcinoma cell line LLC, respectively, were utilized to establish mouse models. Single-cell sequencing was performed to analyze the proportion of different cell clusters and metabolic changes in the tumor microenvironment (TME). Immunofluorescence and multiplex immunohistochemistry were conducted in murine tumor tissues and clinical biopsy samples to assess the percentage of pericytes coverage. Primary pericytes were isolated from lung adenocarcinoma tumor tissues using magnetic-activated cell sorting (MACS). These pericytes were then treated with recombinant human CCL28 protein, followed by transwell migration assays and RNA sequencing analysis. Changes in the secretome and metabolome were examined, and verification of retinoic acid metabolism alterations in pericytes was conducted using quantitative real-time PCR, western blotting, and LC-MS technology. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was employed to validate the transcriptional regulatory ability and affinity of RXRα to specific sites at the ANGPT1 promoter. RESULTS: Our study showed that after undergoing anti-angiogenesis treatment, the tumor exhibited a state of ischemia and hypoxia, leading to an upregulation in the expression of CCL28 in hypoxic lung adenocarcinoma cells by the hypoxia-sensitive transcription factor CEBPB. Increased CCL28 could promote tumor vascular normalization through recruiting and metabolic reprogramming pericytes in the tumor microenvironment. Mechanistically, CCL28 modified the retinoic acid (RA) metabolism and increased ANGPT1 expression via RXRα in pericytes, thereby enhancing the stability of endothelial cells. CONCLUSION: We reported the details of the molecular mechanisms of "vascular normalization" after anti-angiogenesis therapy for the first time. Our work might provide a prospective molecular marker for guiding the clinical arrangement of combination therapy between anti-angiogenesis treatment and other therapies.
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Adenocarcinoma del Pulmón , Angiopoyetina 1 , Quimiocinas CC , Neoplasias Pulmonares , Pericitos , Pericitos/metabolismo , Ratones , Humanos , Animales , Angiopoyetina 1/metabolismo , Angiopoyetina 1/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Quimiocinas CC/metabolismo , Quimiocinas CC/genética , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Microambiente Tumoral , Neovascularización Patológica/metabolismo , Línea Celular TumoralRESUMEN
BACKGROUND: Currently, clinical indicators for evaluating endothelial permeability in sepsis are unavailable. Endothelium-derived extracellular vesicles (EDEVs) are emerging as biomarkers of endothelial injury. Platelet endothelial cell adhesion molecule (PECAM) and vascular endothelial (VE)-cadherin are constitutively expressed endothelial intercellular adhesion molecules that regulate intercellular adhesion and permeability. Herein, we investigated the possible association between EDEVs expressing intercellular adhesion molecules (PECAM+ or VE-cadherin+ EDEVs) and endothelial permeability and sepsis severity. METHODS: Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor alpha (TNF-α) directly or after pretreatment with permeability-modifying reagents such as angiopoietin-1, prostacyclin, or vascular endothelial growth factor (VEGF) to alter TNF-α-induced endothelial hyperpermeability. Endothelial permeability was measured using the dextran assay or transendothelial electrical resistance. Additionally, a prospective cross-sectional observational study was conducted to analyze circulating EDEV levels in patients with sepsis. EDEVs were examined in HUVEC culture supernatants or patient plasma (nonsepsis, n = 30; sepsis, n = 30; septic shock, n = 42) using flow cytometry. The Wilcoxon rank-sum test was used for comparisons between 2 groups. Comparisons among 3 or more groups were performed using the Steel-Dwass test. Spearman's test was used for correlation analysis. Statistical significance was set at P < .05. RESULTS: TNF-α stimulation of HUVECs significantly increased EDEV release and endothelial permeability. Pretreatment with angiopoietin-1 or prostacyclin suppressed the TNF-α-induced increase in endothelial permeability and inhibited the release of PECAM+ and VE-cadherin+ EDEVs. In contrast, pretreatment with VEGF increased TNF-α-induced endothelial permeability and the release of PECAM+ and VE-cadherin+ EDEVs. However, pretreatment with permeability-modifying reagents did not affect the release of EDEVs expressing inflammatory stimulus-inducible endothelial adhesion molecules such as E-selectin, intracellular adhesion molecule-1, or vascular cell adhesion molecule-1. The number of PECAM+ EDEVs on admission in the septic-shock group (232 [124, 590]/µL) was significantly higher (P = .043) than that in the sepsis group (138 [77,267]/µL), with an average treatment effect of 98/µL (95% confidence interval [CI], 2-270/µL), and the number of VE-cadherin+ EDEVs in the septic-shock group (173 [76,339]/µL) was also significantly higher (P = .004) than that in the sepsis group (81 [42,159]/µL), with an average treatment effect (ATE) of 79/µL (95% CI, 19-171/µL); these EDEV levels remained elevated until day 5. CONCLUSIONS: EDEVs expressing intercellular adhesion molecules (PECAM+ or VE-cadherin+ EDEVs) may reflect increased endothelial permeability and could be valuable diagnostic and prognostic markers for sepsis.
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Antígenos CD , Cadherinas , Permeabilidad Capilar , Vesículas Extracelulares , Células Endoteliales de la Vena Umbilical Humana , Sepsis , Índice de Severidad de la Enfermedad , Humanos , Vesículas Extracelulares/metabolismo , Sepsis/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Masculino , Estudios Prospectivos , Antígenos CD/metabolismo , Femenino , Persona de Mediana Edad , Cadherinas/metabolismo , Anciano , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Estudios Transversales , Células Cultivadas , Angiopoyetina 1/metabolismo , Biomarcadores/metabolismo , Biomarcadores/sangre , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Endotelio Vascular/metabolismo , Epoprostenol/metabolismoRESUMEN
INTRODUCTION: Our objective was to examine the factors associated with the serum angiopoietin-2/angiopoietin-1 (Angpt-2/Angpt-1) ratio in peritoneal dialysis (PD) patients and to investigate the association between Angpt-2/Angpt-1 ratio and cardiovascular and all-cause mortality. METHODS: Patients on PD who were prevalent between January 2014 and April 2015 in the center of Renji Hospital were enrolled. At the time of enrollment, serum and dialysate samples were collected to detect biochemical parameters, serum angiopoietin-2 and angiopoietin-1 levels. Patients were dichotomized into two groups according to a median of Angpt-2/Angpt-1 ratio and followed up prospectively until the end of the study. RESULTS: A total of 325 patients were enrolled, including 168 males (51.7%) with a mean age of 56.9 ± 14.2 years and a median PD duration of 32.4 (9.8-55.9) months. Multiple linear regression showed pulse pressure (ß = 0.206, p < .001) and high-sensitivity C-reactive protein (hs-CRP) (ß = 0.149, p = .011) were positively correlated with serum Angpt-2/Angpt-1 ratio, while residual renal function (RRF) (ß= -0.219, p < .001) was negatively correlated with serum Angpt-2/Angpt-1 ratio. Multivariate Cox regression analysis showed the high serum Angpt-2/Angpt-1 ratio was an independent predictor of cardiovascular mortality (hazard ratio (HR)=2.467, 95% confidence interval (CI) 1.243-4.895, p = .010) and all-cause mortality (HR = 1.486, 95%CI 1.038-2.127, p = .031). In further subgroup analysis by gender, a significant association was shown in high Angpt-2/Angpt-1 ratio with all-cause mortality in male (p < .05), but not in female patients (p>.05). CONCLUSIONS: High Angpt-2/Angpt-1 ratio is an independent risk factor for cardiovascular and all-cause mortality in PD patients.
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Angiopoyetina 1 , Angiopoyetina 2 , Enfermedades Cardiovasculares , Diálisis Peritoneal , Humanos , Masculino , Angiopoyetina 2/sangre , Femenino , Persona de Mediana Edad , Diálisis Peritoneal/mortalidad , Estudios Prospectivos , Enfermedades Cardiovasculares/mortalidad , Enfermedades Cardiovasculares/sangre , Anciano , Angiopoyetina 1/sangre , Adulto , Fallo Renal Crónico/terapia , Fallo Renal Crónico/sangre , Fallo Renal Crónico/mortalidad , Proteína C-Reactiva/análisis , Biomarcadores/sangre , Causas de Muerte , Factores de Riesgo , Modelos de Riesgos ProporcionalesRESUMEN
Atherosclerosis represents the major cause of mortality worldwide and triggers higher risk of acute cardiovascular events. Pericytes-endothelial cells (ECs) communication is orchestrated by ligand-receptor interaction generating a microenvironment which results in intraplaque neovascularization, that is closely associated with atherosclerotic plaque instability. Notoginsenoside R1 (R1) exhibits anti-atherosclerotic bioactivity, but its effect on angiogenesis in atherosclerotic plaque remains elusive. The aim of our study is to explore the therapeutic effect of R1 on vulnerable plaque and investigate its potential mechanism against intraplaque neovascularization. The impacts of R1 on plaque stability and intraplaque neovascularization were assessed in ApoE-/- mice induced by high-fat diet. Pericytes-ECs direct or non-direct contact co-cultured with VEGF-A stimulation were used as the in vitro angiogenesis models. Overexpressing Ang1 in pericytes was performed to investigate the underlying mechanism. In vivo experiments, R1 treatment reversed atherosclerotic plaque vulnerability and decreased the presence of neovessels in ApoE-/- mice. Additionally, R1 reduced the expression of Ang1 in pericytes. In vitro experiments demonstrated that R1 suppressed pro-angiogenic behavior of ECs induced by pericytes cultured with VEGF-A. Mechanistic studies revealed that the anti-angiogenic effect of R1 was dependent on the inhibition of Ang1 and Tie2 expression, as the effects were partially reversed after Ang1 overexpressing in pericytes. Our study demonstrated that R1 treatment inhibited intraplaque neovascularization by governing pericyte-EC association via suppressing Ang1-Tie2/PI3K-AKT paracrine signaling pathway. R1 represents a novel therapeutic strategy for atherosclerotic vulnerable plaques in clinical application.
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Angiopoyetina 1 , Aterosclerosis , Células Endoteliales , Ginsenósidos , Neovascularización Patológica , Pericitos , Placa Aterosclerótica , Receptor TIE-2 , Animales , Ginsenósidos/farmacología , Ratones , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Aterosclerosis/tratamiento farmacológico , Placa Aterosclerótica/tratamiento farmacológico , Receptor TIE-2/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Angiopoyetina 1/metabolismo , Ratones Endogámicos C57BL , Masculino , Comunicación Celular/efectos de los fármacos , Humanos , Transducción de Señal/efectos de los fármacos , Apolipoproteínas E , Dieta Alta en Grasa , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese herbal medicine Panax japonicus C.A. Meyer has a long history in clinical treatment of rheumatoid arthritis (RA). Total saponins of Panax japonicus C.A. Meyer (TSPJs) were extracted from the root of Panax japonicus C.A. Meyer, and its anti-rheumatism mechanism is still unclear. AIM OF THE STUDY: To investigate whether TSPJs attenuated synovial angiogenesis in RA and explore the potential mechanisms. MATERIALS AND METHODS: Potential TSPJs targets involving gene function were predicted by network pharmacology related databases. Bioinformatics analysis and molecular docking technology were used to predict the mechanism of TSPJs in the treatment of RA. The predicted results were validated by cell experiments and a collagen-induced arthritis (CIA) mouse model. RESULTS: Bioinformatics analysis results showed that TSPJs may inhibit RA-related angiogenesis through the hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) pathways. In vitro, different doses of TSPJs showed a good inhibitory effect on the tube formation of EA.hy926 cells. The results of the cellular thermal shift assay indicated that TSPJs can bind to the HIF-1α, VEGFA, and angiopoietin-1 (ANG-1) proteins. In vivo, the administration of TSPJs alleviated the symptoms of CIA mice, including the arthritis index, hind paw thickness, and swollen joint count. The histological results demonstrated that TSPJs inhibited inflammation, angiogenesis, bone damage, and cartilage destruction. Furthermore, TSPJs decreased the number of vessels and the expression level of CD31. The mechanistic results revealed that TSPJs decreased the expression of HIF-1α, VEGFA, and ANG-1 in the serum or synovial tissues of CIA mice. CONCLUSION: These results suggest that TSPJs effectively inhibit angiogenesis in RA, and the mechanism may be related to inhibiting the HIF-1α/VEGF/ANG-1 axis.
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Inhibidores de la Angiogénesis , Angiopoyetina 1 , Artritis Experimental , Artritis Reumatoide , Subunidad alfa del Factor 1 Inducible por Hipoxia , Panax , Saponinas , Factor A de Crecimiento Endotelial Vascular , Animales , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Saponinas/farmacología , Saponinas/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/metabolismo , Panax/química , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/patología , Humanos , Inhibidores de la Angiogénesis/farmacología , Masculino , Ratones , Angiopoyetina 1/metabolismo , Simulación del Acoplamiento Molecular , Ratones Endogámicos DBA , Neovascularización Patológica/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Raíces de Plantas/químicaRESUMEN
Development of the vascular system is regulated by multiple signaling pathways mediated by receptor tyrosine kinases. Among them, angiopoietin (Ang)/Tie signaling regulates lymphatic and blood vessel development in mammals. Of the two Tie receptors, Tie2 is well known as a key mediator of Ang/Tie signaling, but, unexpectedly, recent studies have revealed that the Tie2 locus has been lost in many vertebrate species, whereas the Tie1 gene is more commonly present. However, Tie1-driven signaling pathways, including ligands and cellular functions, are not well understood. Here, we performed comprehensive mutant analyses of angiopoietins and Tie receptors in zebrafish and found that only angpt1 and tie1 mutants show defects in trunk lymphatic vessel development. Among zebrafish angiopoietins, only Angpt1 binds to Tie1 as a ligand. We indirectly monitored Ang1/Tie1 signaling and detected Tie1 activation in sprouting endothelial cells, where Tie1 inhibits nuclear import of EGFP-Foxo1a. Angpt1/Tie1 signaling functions in endothelial cell migration and proliferation, and in lymphatic specification during early lymphangiogenesis, at least in part by modulating Vegfc/Vegfr3 signaling. Thus, we show that Angpt1/Tie1 signaling constitutes an essential signaling pathway for lymphatic development in zebrafish.
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Angiopoyetina 1 , Linfangiogénesis , Receptor TIE-1 , Transducción de Señal , Proteínas de Pez Cebra , Pez Cebra , Animales , Angiopoyetina 1/metabolismo , Angiopoyetina 1/genética , Movimiento Celular , Proliferación Celular , Células Endoteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Linfangiogénesis/genética , Vasos Linfáticos/metabolismo , Vasos Linfáticos/embriología , Mutación/genética , Unión Proteica , Receptor TIE-1/metabolismo , Receptor TIE-1/genética , Factor C de Crecimiento Endotelial Vascular/metabolismo , Factor C de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Pez Cebra/embriología , Pez Cebra/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
The activation of the angiopoietin (Angpt)-Tie system is linked to endothelial dysfunction during sepsis. Bacterial quorum-sensing molecules function as pathogen-associated molecular patterns. However, their impact on the endothelium and the Angpt-Tie system remains unclear. Therefore, this study investigated whether treatment with N-3-oxododecanoyl homoserine lactone (3OC12-HSL), a quorum-sensing molecule derived from Pseudomonas aeruginosa, impaired endothelial function in human umbilical vein endothelial cells. 3OC12-HSL treatment impaired tube formation even at sublethal concentrations, and immunocytochemistry analysis revealed that it seemed to reduce vascular endothelial-cadherin expression at the cell-cell interface. Upon assessing the mRNA expression patterns of genes associated with the Angpt-Tie axis, the expressions of Angpt2, Forkhead box protein O1, Tie1, and vascular endothelial growth factor 2 were found to be upregulated in the 3OC12-HSL-treated cells. Moreover, western blot analysis revealed that 3OC12-HSL treatment increased Angpt2 expression. A co-immunoprecipitation assay was conducted to assess the effect of 3OC12-HSL on the IQ motif containing GTPase activating protein 1 (IQGAP1) and Rac1 complex and the interaction between these proteins was consistently maintained regardless of 3OC12-HSL treatment. Next, recombinant human (rh)-Angpt1 was added to assess whether it modulated the effects of 3OC12-HSL treatment. rh-Angpt1 addition increased cellular viability, improved endothelial function, and reversed the overall patterns of mRNA and protein expression in endothelial cells treated with 3OC12-HSL. Additionally, it was related to the increased expression of phospho-Akt and the IQGAP1 and Rac1 complex. Collectively, our findings indicated that 3OC12-HSL from Pseudomonas aeruginosa can impair endothelial integrity via the activation of the Angpt-Tie axis, which appeared to be reversed by rh-Angpt1 treatment.
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4-Butirolactona , Angiopoyetina 2 , Homoserina , Células Endoteliales de la Vena Umbilical Humana , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Angiopoyetina 2/metabolismo , Angiopoyetina 2/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Homoserina/análogos & derivados , Homoserina/farmacología , Homoserina/metabolismo , Cadherinas/metabolismo , Cadherinas/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Transducción de Señal/efectos de los fármacos , Antígenos CD/metabolismo , Antígenos CD/genética , Angiopoyetina 1/metabolismo , Angiopoyetina 1/genéticaRESUMEN
Abnormal angiogenesis leads to tumor progression and metastasis in colorectal cancer (CRC). This study aimed to elucidate the association between angiogenesis-related genes, including VEGF-A, ANGPT-1, and ANGPT-2 with both metastatic and microsatellite alterations at selected tetranucleotide repeats (EMAST) subtypes of CRC. We conducted a thorough assessment of the ANGPT-1, ANGPT-2, and VEGF-A gene expression utilizing publicly available RNA sequencing and microarray datasets. Then, the experimental validation was performed in 122 CRC patients, considering their disease metastasis and EMAST+/- profile by using reverse transcription polymerase chain reaction (RT-PCR). Subsequently, a competing endogenous RNA (ceRNA) network associated with these angiogenesis-related genes was constructed and analyzed. The expression level of VEGF-A and ANGPT-2 genes were significantly higher in tumor tissues as compared with normal adjacent tissues (P-value < 0.001). Nevertheless, ANGPT-1 had a significantly lower expression in tumor samples than in normal colon tissue (P-value < 0.01). We identified a significantly increased VEGF-A (P-value = 0.002) and decreased ANGPT-1 (P-value = 0.04) expression in EMAST+ colorectal tumors. Regarding metastasis, a significantly increased VEGF-A and ANGPT-2 expression (P-value = 0.001) and decreased ANGPT-1 expression (P-value < 0.05) were established in metastatic CRC patients. Remarkably, co-expression analysis also showed a strong correlation between ANGPT-2 and VEGF-A gene expressions. The ceRNA network was constructed by ANGPT-1, ANGPT-2, VEGF-A, and experimentally validated miRNAs (hsa-miR-190a-3p, hsa-miR-374c-5p, hsa-miR-452-5p, and hsa-miR-889-3p), lncRNAs (AFAP1-AS1, KCNQ1OT1 and MALAT1), and TFs (Sp1, E2F1, and STAT3). Network analysis revealed that colorectal cancer is amongst the 82 significant pathways. We demonstrated a significant differential expression of VEGF-A and ANGPT-1 in colorectal cancer patients exhibiting the EMAST+ phenotype. This finding provides novel insights into the molecular pathogenesis of colorectal cancer, specifically in EMAST subtypes. Yet, the generalization of in silico findings to EMAST+ colorectal cancer warrants future experimental investigations. In the end, this study proposes that the EMAST biomarker could serve as an additional perspective on CMS4 biology which is well-defined by activated angiogenesis and worse overall survival.
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Angiopoyetina 1 , Angiopoyetina 2 , Neoplasias Colorrectales , Regulación Neoplásica de la Expresión Génica , Neovascularización Patológica , Factor A de Crecimiento Endotelial Vascular , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Angiopoyetina 1/genética , Angiopoyetina 1/metabolismo , Angiopoyetina 2/genética , Angiopoyetina 2/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Metástasis de la Neoplasia , Anciano , Repeticiones de Microsatélite/genética , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , AngiogénesisRESUMEN
BACKGROUND: Obstructive sleep apnea (OSA) is increasingly recognized as a common condition in the general population and causes significant OSA-associated morbidities including cardiovascular and cerebrovascular events such as cerebral small vessel disease (CSVD) and stroke. METHODS: In this study, using sensitive ELISA immunoassays, we measured subset of endothelial/vascular and inflammatory biomarkers as well as neurofilament light chain (NfL), a sensitive marker for neuroaxonal injury, using plasma from OSA patients post-stroke (Acute Cerebral Infarction (ACI), N = 26) to determine their usefulness as potential prognostic markers in disease progression. RESULTS: Our results showed significantly increased plasma TNFα and NfL concentrations and decreased concentrations of platelet derived growth factor (PDGF-AA) in post-stroke OSA patients with more severe white matter hyperintensities (WMHs). And after separating the patients based on sex, compared to females, male post-stroke OSA patients with severe WMHs have increased circulating levels of inflammatory chemokine CXCL10 and cytokine Interleukin-10 (IL-10) and significantly decreased levels of Angiopoietin-1 (Ang-1) an important protein responsible for endothelial/vascular integrity functions. Importantly, in a subset of newly diagnosed OSA patients (without prior history of stroke), significantly increased plasma CXCL10 levels and decreased plasma Ang-1 levels were also readily observed when compared to healthy controls, indicating possible altered endothelial integrity and ongoing vascular inflammation in these newly diagnosed OSA patients. CONCLUSIONS: In summary, our study has identified a novel set of plasma biomarkers including PDGF-AA, CXCL10 and Ang-1 for their potential prognostic value for disease outcomes pre- and post-stroke in OSA patients and use as surrogate markers to measure efficacy of treatment modalities.
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Biomarcadores , Apnea Obstructiva del Sueño , Accidente Cerebrovascular , Humanos , Apnea Obstructiva del Sueño/sangre , Apnea Obstructiva del Sueño/complicaciones , Masculino , Biomarcadores/sangre , Femenino , Persona de Mediana Edad , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/etiología , Anciano , Factor de Crecimiento Derivado de Plaquetas/análisis , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas de Neurofilamentos/sangre , Factor de Necrosis Tumoral alfa/sangre , Quimiocina CXCL10/sangre , Angiopoyetina 1/sangre , Inflamación/sangre , Interleucina-10/sangreRESUMEN
Neonatal sepsis is a major cause of childhood mortality. Limited diagnostic tools and mechanistic insights have hampered our abilities to develop prophylactic or therapeutic interventions. Biomarkers in human neonatal sepsis have been repeatedly identified as associated with dysregulation of angiopoietin signaling and altered arachidonic acid metabolism. We here provide the mechanistic evidence in support of the relevance for these observations. Angiopoetin-1 (Ang-1), which promotes vascular integrity, was decreased in blood plasma of human and murine septic newborns. In preclinical models, administration of Ang-1 provided prophylactic protection from septic death. Arachidonic acid metabolism appears to be functionally connected to Ang-1 via reactive oxygen species (ROS) with a direct role of nitric oxide (NO). Strengthening this intersection via oral administration of arachidonic acid and/or the NO donor L-arginine provided prophylactic as well as therapeutic protection from septic death while also increasing plasma Ang-1 levels among septic newborns. Our data highlight that targeting angiogenesis-associated pathways with interventions that increase Ang-1 activity directly or indirectly through ROS/eNOS provide promising avenues to prevent and/or treat severe neonatal sepsis.
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Angiopoyetina 1 , Sepsis Neonatal , Óxido Nítrico , Especies Reactivas de Oxígeno , Humanos , Animales , Recién Nacido , Angiopoyetina 1/sangre , Angiopoyetina 1/metabolismo , Ratones , Especies Reactivas de Oxígeno/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico/sangre , Ácido Araquidónico/metabolismo , Ácido Araquidónico/sangre , Femenino , Masculino , Arginina/sangre , Arginina/metabolismo , Transducción de Señal , Óxido Nítrico Sintasa de Tipo III/metabolismo , Neovascularización Patológica/metabolismo , Biomarcadores/sangre , Modelos Animales de Enfermedad , Animales Recién Nacidos , AngiogénesisRESUMEN
Glioblastoma multiforme (GBM) acquires resistance to bevacizumab (Bev) treatment. Bev affects angiogenic factors other than vascular endothelial growth factor (VEGF), which are poorly understood. We investigated changes in angiogenic factors under and after Bev therapy, including angiopoietin-1 (ANGPT1), angiopoietin-2 (ANGPT2), placental growth factor (PLGF), fibroblast growth factor 2, and ephrin A2 (EphA2). Fifty-four GBM tissues, including 28 specimens from 14 cases as paired specimens from the same patient obtained in three settings: initial tumor resection (naïve Bev), tumors resected following Bev therapy (effective Bev), and recurrent tumors after Bev therapy (refractory Bev). Immunohistochemistry assessed their expressions in tumor vessels and its correlation with recurrent MRI patterns. PLGF expression was higher in the effective Bev group than in the naïve Bev group (p = 0.024) and remained high in the refractory Bev group. ANGPT2 and EphA2 expressions were higher in the refractory Bev group than in the naïve Bev group (p = 0.047 and 0.028, respectively). PLGF expression was higher in the refractory Bev group compared with the naïve Bev group for paired specimens (p = 0.036). PLGF was more abundant in T2 diffuse/circumscribe patterns (p = 0.046). This is the first study to evaluate angiogenic factors other than VEGF during effective and refractory Bev therapy in patient-derived specimens.
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Inhibidores de la Angiogénesis , Angiopoyetina 2 , Bevacizumab , Neoplasias Encefálicas , Glioblastoma , Neovascularización Patológica , Humanos , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/cirugía , Bevacizumab/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Masculino , Femenino , Persona de Mediana Edad , Anciano , Neovascularización Patológica/tratamiento farmacológico , Adulto , Angiopoyetina 2/metabolismo , Inhibidores de la Angiogénesis/uso terapéutico , Factor de Crecimiento Placentario/metabolismo , Antineoplásicos Inmunológicos/uso terapéutico , Angiopoyetina 1/metabolismo , Recurrencia Local de NeoplasiaRESUMEN
While several effective therapies for critically ill patients with COVID-19 have been identified in large, well-conducted trials, the mechanisms underlying these therapies have not been investigated in depth. Our aim is to investigate the association between various immunosuppressive therapies (corticosteroids, tocilizumab and anakinra) and the change in endothelial host response over time in critically ill COVID-19 patients. We conducted a pre-specified multicenter post-hoc analysis in a Dutch cohort of COVID-19 patients admitted to the ICU between March 2020 and September 2021 due to hypoxemic respiratory failure. A panel of 18 immune response biomarkers in the complement, coagulation and endothelial function domains were measured using ELISA or Luminex. Biomarkers were measured on day 0-1, day 2-4 and day 6-8 after start of COVID-19 treatment. Patients were categorized into four treatment groups: no immunomodulatory treatment, corticosteroids, anakinra plus corticosteroids, or tocilizumab plus corticosteroids. The association between treatment group and the change in concentrations of biomarkers was estimated with linear mixed-effects models, using no immunomodulatory treatment as reference group. 109 patients with a median age of 62 years [IQR 54-70] of whom 72% (n = 78) was male, were included in this analysis. Both anakinra plus corticosteroids (n = 22) and tocilizumab plus corticosteroids (n = 38) were associated with an increase in angiopoietin-1 compared to no immune modulator (n = 23) (beta of 0.033 [0.002-0.064] and 0.041 [0.013-0.070] per day, respectively). These treatments, as well as corticosteroids alone (n = 26), were further associated with a decrease in the ratio of angiopoietin-2/angiopoietin-1 (beta of 0.071 [0.034-0.107], 0.060 [0.030-0.091] and 0.043 [0.001-0.085] per day, respectively). Anakinra plus corticosteroids and tocilizumab plus corticosteroids were associated with a decrease in concentrations of complement complex 5b-9 compared to no immunomodulatory treatment (0.038 [0.006-0.071] and 0.023 [0.000-0.047], respectively). Currently established treatments for critically ill COVID-19 patients are associated with a change in biomarkers of the angiopoietin and complement pathways, possibly indicating a role for stability of the endothelium. These results increase the understanding of the mechanisms of interventions and are possibly useful for stratification of patients with other inflammatory conditions which may potentially benefit from these treatments.
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COVID-19 , Humanos , Masculino , Persona de Mediana Edad , Anciano , Angiopoyetina 1 , SARS-CoV-2 , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Enfermedad Crítica/terapia , Tratamiento Farmacológico de COVID-19 , Corticoesteroides/uso terapéutico , Terapia de Inmunosupresión , BiomarcadoresRESUMEN
OBJECTIVE: To evaluate the relationships between the severity of myxomatous mitral valve disease (MMVD) and pulmonary hypertension (PH) and serum angiopoietin (Ang)-1 and Ang-2 concentrations in dogs with MMVD. ANIMALS: 74 dogs (control, n = 12; MMVD, n = 62) were included. METHODS: Serum Ang-1 and Ang-2 concentrations were estimated using the canine-specific ELISA kit. The concentrations were compared between dogs with MMVD and healthy dogs, and they were analyzed according to the severity of MMVD and PH. RESULTS: The median serum Ang-1 concentration did not differ among the study groups. The median serum Ang-2 concentration was higher in dogs with stage B2 MMVD (P = .041) and acute congestive heart failure (P = .002) than in control dogs. In addition, the median serum Ang-2 concentration was higher in MMVD dogs with PH than in those without PH (P = .031). Serum Ang-2 concentration was correlated with vertebral heart score (rs = 0.36, P = .004) and vertebral left atrial score (r = 0.50, P < .001) in dogs with MMVD, and correlated with vertebral heart score (r = 0.63, P = .01), maximum E wave amplitude of the diastolic transmitral flow (rs = 0.61, P = .018), ejection fraction (rs = -0.77, P < .001) and fractional shortening (rs = -0.56, P = .032) in dogs with acute congestive heart failure. CLINICAL RELEVANCE: Circulating Ang-2 levels increase in dogs with the severity of MMVD and the presence of PH.
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Angiopoyetina 2 , Enfermedades de los Perros , Hipertensión Pulmonar , Animales , Perros , Enfermedades de los Perros/sangre , Hipertensión Pulmonar/veterinaria , Hipertensión Pulmonar/sangre , Angiopoyetina 2/sangre , Masculino , Femenino , Insuficiencia de la Válvula Mitral/veterinaria , Insuficiencia de la Válvula Mitral/sangre , Angiopoyetina 1/sangre , Estudios de Casos y Controles , Enfermedades de las Válvulas Cardíacas/veterinaria , Enfermedades de las Válvulas Cardíacas/sangreRESUMEN
The poor remodeling of placental spiral arteries seen in preeclampsia is also discussed to contribute to recurrent pregnancy loss (RPL) preceded by abnormal angiogenesis and excessive complement activation. Low levels of Mannose-binding-lectin (MBL), a pattern recognition molecule (PRM) of the lectin pathway, have been found in women with RPL. We propose that pregnancy loss is connected to defective angiogenesis with reperfusion damage in the placenta and decreased levels of PRM in the lectin pathway in women with RPL. In this cohort study, we investigate the angiogenic factors and the lectin complement pathway in early pregnancy and their time-dependent relationship with pregnancy outcomes in 76 women with secondary RPL (sRPL) who have at least four prior pregnancy losses and a live birth. We evaluated levels of Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Vascular Endothelial Growth Factor (VEGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and the PRMs, MBL, ficolin-1, -2, -3 and an additional soluble PRM, Pentraxin-3, during the 5th, 6th, and 7th gestational weeks. Our results showed that, compared to live births, pregnancies that ended in loss were associated with elevated VEGF levels and decreased levels of the Ang-2/Ang-1 ratio. Also, increasing levels of ficolin-2 were significantly associated with pregnancy loss, with MBL showing no association. Our research suggests that women with sRPL may have inadequate placentation with impaired angiogenesis in pregnancies ending in a loss.