Expression Of Von Willebrand Factor Research Articles

Shiga toxin down-regulates ERG protein in endothelial cells and impairs angiogenesis

BackgroundShiga toxin (Stx) can activate inflammatory signaling, leading to vascular dysfunction and promotion of a pro-thrombotic tissue microenvironment. Stx can trigger the development of the enterohemorrhagic (childhood) hemolytic uremic syndrome (eHUS), a triad of thrombocytopenia, hemolytic anemia, and acute kidney injury, often requiring dialysis. Additional features may include damage to other organs, including the gastrointestinal tract, pancreas, brain and cardiovascular system; death occurs in 2–5 %. eHUS is a thrombotic microangiopathy; thus, endothelial cell (EC) injury and platelet fibrin thrombus formation in glomerular arterioles and in the arterioles of other affected organs are likely. To elucidate mechanisms of this microangiopathy, we examined in human ECs the regulation of the platelet adhesion proteins P-selectin and von Willebrand factor (VWF), along with the downregulation of erythroblast-transformation-specific transcription factor (ERG) a key regulator of angiogenesis and megakaryocyte development. MethodsVWF, P-selectin, and ERG levels were determined using immunofluorescence and Western blot in human umbilical endothelial cells (HUVECs). HUVECs were treated with tumor necrosis factor-alpha (TNF-α), Stx-1 or both, versus normal controls. Capillary morphogenesis on Matrigel was performed using HUVECs treated, for 22 h with TNF-α, Stx-1, or both, or treated 4 h with Stx-1 alone or in combination with TNF-α for 22 h. ResultsStx-1 significantly reduced ERG and VWF expression on HUVECs, but upregulated P-selectin expression. ERG levels decreased with Stx-1 alone or in combination with TNF-α, in the nuclear, perinuclear and cytoplasmatic regions. Stx-1 reduced capillary morphogenesis, while Stx-1-TNF-α combined treatment reduced capillary morphogenesis still further. ConclusionsIn the presence of Stx-1 or TNF-α or both treatments, ECs were activated, expressing higher levels of P-selectin and lower levels of VWF. Our findings, further, provide evidence that Stx-1 downregulates ERG, repressing angiogenesis in vitro.

Effect of Xuming Decoction in Records of Proved Prescriptions, Ancient a nd Modern on cerebral ischemic injury and angiogenesis in rat model of acute cerebral infarction

This paper aims to explore the effect of Xuming Decoction in the Records of Proved Prescriptions, Ancient and Modern on cerebral ischemic injury and angiogenesis in the rat model of acute cerebral infarction. SD rats were randomized into 6 groups: sham group, model group, low-, medium-, and high-dose(5.13, 10.26, and 20.52 g·kg~(-1), respectively) Xuming Decoction groups, and butylphthalide(0.06 g·kg~(-1)) group. After the successful establishment of the rat model by middle cerebral artery occlusion(MCAO), rats in the sham and model groups were administrated with distilled water and those in other groups with corresponding drugs for 7 consecutive days. After the neurological function was scored, all the rats were sacrificed, and the brain tissue samples were collected. The degree of cerebral ischemic injury was assessed by the neurological deficit score and staining with 2,3,5-triphenyltetrazolium chloride. Hematoxylin-eosin staining was performed to observe the pathological changes in the brain. Transmission electron microscopy was employed to observe the ultrastructures of neurons and microvascular endothelial cells(ECs) on the ischemic side of the brain tissue. Immunofluorescence assay was employed to detect the expression of von Willebrand factor(vWF) and hematopoietic progenitor cell antigen CD34(CD34) in the ischemic brain tissue. Real-time PCR and Western blot were employed to determine the mRNA and protein levels, respectively, of Runt-related transcription factor 1(RUNX1), vascular endothelial growth factor(VEGF), angiopoietin-1(Ang-1), angiopoietin-2(Ang-2), and VEGF receptor 2(VEGFR2) in the ischemic brain tissue. The results showed that compared with the sham group, the model group showed increased neurological deficit score and cerebral infarction area(P<0.01), pathological changes, and damaged ultrastructure of neurons and microvascular ECs in the ischemic brain tissue. Furthermore, the modeling up-regulated the mRNA levels of RUNX1, VEGF, Ang-1, Ang-2, and VEGFR2(P<0.01) and the protein levels of vWF, CD34, RUNX1, VEGF, Ang-1, Ang-2, and VEGFR2(P<0.05 or P<0.01). Compared with the model group, high-dose Xuming Decoction and butylphthalide decreased the neurological deficit score and cerebral infarction area(P<0.01) and alleviated the pathological changes and damage of the ultrastructure of neurons and microvascular ECs in the ischemic brain tissue. Moreover, they up-regulated the mRNA levels of RUNX1, VEGF, Ang-1, Ang-2, and VEGFR2(P<0.01) and the protein levels of vWF, CD34, RUNX1, VEGF, Ang-1, Ang-2, and VEGFR2(P<0.01). The results suggest that Xuming Decoction in the Records of Proved Prescriptions, Ancient and Modern can promote the angiogenesis and collateral circulation establishment to alleviate neurological dysfunction of the ischemic brain tissue in MCAO rats by regulating the RUNX1/VEGF pathway.

Injectable Tissue-Specific Hydrogel System for Pulp–Dentin Regeneration

The quest for finding a suitable scaffold system that supports cell survival and function and, ultimately, the regeneration of the pulp–dentin complex remains challenging. Herein, we hypothesized that dental pulp stem cells (DPSCs) encapsulated in a collagen-based hydrogel with varying stiffness would regenerate functional dental pulp and dentin when concentrically injected into the tooth slices. Collagen hydrogels with concentrations of 3 mg/mL (Col3) and 10 mg/mL (Col10) were prepared, and their stiffness and microstructure were assessed using a rheometer and scanning electron microscopy, respectively. DPSCs were then encapsulated in the hydrogels, and their viability and differentiation capacity toward endothelial and odontogenic lineages were evaluated using live/dead assay and quantitative real-time polymerase chain reaction. For in vivo experiments, DPSC-encapsulated collagen hydrogels with different stiffness, with or without growth factors, were injected into pulp chambers of dentin tooth slices and implanted subcutaneously in severe combined immunodeficient (SCID) mice. Specifically, vascular endothelial growth factor (VEGF [50 ng/mL]) was loaded into Col3 and bone morphogenetic protein (BMP2 [50 ng/mL]) into Col10. Pulp–dentin regeneration was evaluated by histological and immunofluorescence staining. Data were analyzed using 1-way or 2-way analysis of variance accordingly (α = 0.05). Rheology and microscopy data revealed that Col10 had a stiffness of 8,142 Pa with a more condensed and less porous structure, whereas Col3 had a stiffness of 735 Pa with a loose microstructure. Furthermore, both Col3 and Col10 supported DPSCs’ survival. Quantitative polymerase chain reaction showed Col3 promoted significantly higher von Willebrand factor (VWF) and CD31 expression after 7 and 14 d under endothelial differentiation conditions (P < 0.05), whereas Col10 enhanced the expression of dentin sialophosphoprotein (DSPP), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and collagen 1 (Col1) after 7, 14, and 21 d of odontogenic differentiation (P < 0.05). Hematoxylin and eosin and immunofluorescence (CD31 and vWF) staining revealed Col10+Col3+DPSCs+GFs enhanced pulp–dentin tissue regeneration. In conclusion, the collagen-based concentric construct modified by growth factors guided the specific lineage differentiation of DPSCs and promoted pulp–dentin tissue regeneration in vivo.

Approaches to induce the maturation process of human induced pluripotent stem cell derived-endothelial cells to generate a robust model.

Endothelial cells generated from induced pluripotent stem cells (hiPSC-ECs) show the majority of endothelial cell characteristics and markers, such as cobblestone morphology and the expression of VEGF and VE-cadherin. However, these cells are failing to show a mature endothelial cell phenotype, which is represented by the low expression and production of von Willebrand Factor (VWF) leading to the round morphology of the Weibel Palade Bodies (WPBs). The aim of this study was to improve the maturation process of hiPSC-ECs and to increase the levels of VWF. hiPSC-ECs were differentiated by a standard differentiation protocol from hiPSCs generated from healthy control donors. To induce maturation, the main focus was to increase the expression and/or production of VWF by the adjustment of potential parameters influencing differentiation and maturation. We also compared alternative differentiation protocols. Cells were analyzed for the expression of endothelial cell markers, WPB structure, and the production and secretion of VWF by flow cytometry, confocal microscopy and ELISA. The generated hiPSC-ECs have typical endothelial cell surface expression profiles, with low expression levels of non-endothelial markers as expected. Co-culture with pericytes, varying concentrations and timing of differentiation factors, applying some level of flow, and the addition of HDAC inhibitors did not substantially improve maturation of hiPSC-ECs. Transfection with the transcription factor ETV2 to induce a faster hiPSC-EC differentiation process resulted in a limited increase in VWF production, secretion, and elongation of WPB structure. Alternative differentiation protocols had limited effect. hiPSCs-ECs have the potential to show a more mature endothelial phenotype with elongated WPBs after >30 days in culture. However, this comes with limitations as there are very few cells detected, and cells are deteriorating after being in culture for extended periods of time.

Coronary microthrombi in the failing human heart: the role of von Willebrand factor and PECAM-1.

The recognition of microthrombi in the heart microcirculation has recently emerged from studies in COVID-19 decedents. The present study investigated the ultrastructure of coronary microthrombi in heart failure (HF) due to cardiomyopathies that are unrelated to COVID-19 infection. In addition, we have investigated the role of von Willebrand factor (VWF) and PECAM-1 in microthrombus formation. We used electron microscopy to investigate the occurrence of microthrombi in patients with HF due to dilated (DCM, n = 7), inflammatory (MYO, n = 6) and ischemic (ICM, n = 7) cardiomyopathy and 4 control patients. VWF and PECAM-1 was studied by quantitative immunohistochemistry and Western blot. In comparison to control, the number of microthrombi was increased 7-9 times in HF. This was associated with a 3.5-fold increase in the number of Weibel-Palade bodies (WPb) in DCM and MYO compared to control. A fivefold increase in WPb in ICM was significantly different from control, DCM and MYO. In Western blot, VWF was increased twofold in DCM and MYO, and more than threefold in ICM. The difference between ICM and DCM and MYO was statistically significant. These results were confirmed by quantitative immunohistochemistry. Compared to control, PECAM-1 was by approximatively threefold increased in all groups of patients. This is the first study to demonstrate the occurrence of microthrombi in the failing human heart. The occurrence of microthrombi is associated with increased expression of VWF and the number of WPb, being more pronounced in ICM. These changes are likely not compensated by increases in PECAM-1 expression.

Clusterin knockdown has effects on intracellular and secreted von Willebrand factor in human umbilical vein endothelial cells.

Alterations in von Willebrand factor (VWF) have an important role in human health and disease. Deficiency of VWF is associated with symptoms of bleeding and excesses of VWF are associated with thrombotic outcomes. Understanding the mechanisms that drive VWF regulation can lead to a better understanding of modulation of VWF levels in humans. We identified clusterin (CLU) as a potential candidate regulator of VWF based on a single cell RNA sequencing (scRNA-seq) analysis in control endothelial cells (ECs) and von Willebrand disease (VWD) endothelial colony-forming-cells (ECFCs). We found that patients with deficiencies of VWF (von Willebrand disease, VWD) had decreased CLU expression and ECs with low VWF expression also had low CLU expression. Based on these findings, we sought to evaluate the role of CLU in the regulation of VWF, specifically as it relates to VWD. As CLU is primarily thought to be a golgi protein involved in protein chaperoning, we hypothesized that knockdown of CLU would lead to decreases in VWF and alterations in Weibel-Palade bodies (WPBs). We used both siRNA- and CRISPR-Cas9-based approaches to modulate CLU in human umbilical vein endothelial cells (HUVECs) and evaluated VWF protein levels, VWF mRNA copy number, and WPB quantity and size. We demonstrated that siRNA-based knockdown of CLU resulted in decreases in VWF content in cellular lysates and supernatants, but no significant change in WPB quantity or size. A CRISPR-Cas9-based knockdown of CLU demonstrated similar findings of decreases in intracellular VWF content but no significant change in WPB quantity or size. Our data suggests that CLU knockdown is associated with decreases in cellular VWF content but does not affect VWF mRNA levels or WPB quantity or size.

Mechanism of astragaloside Ⅳ combined with Panax notoginseng saponins in regulating angiogenesis to treat cerebral ischemia based on network pharmacology and experimental verification

Network pharmacology and animal and cell experiments were employed to explore the mechanism of astragaloside Ⅳ(AST Ⅳ) combined with Panax notoginseng saponins(PNS) in regulating angiogenesis to treat cerebral ischemia. The method of network pharmacology was used to predict the possible mechanisms of AST Ⅳ and PNS in treating cerebral ischemia by mediating angiogenesis. In vivo experiment: SD rats were randomized into sham, model, and AST Ⅳ(10 mg·kg~(-1)) + PNS(25 mg·kg~(-1)) groups, and the model of cerebral ischemia was established with middle cerebral artery occlusion(MCAO) method. AST Ⅳ and PNS were administered by gavage twice a day. the Longa method was employed to measure the neurological deficits. The brain tissue was stained with hematoxylin-eosin(HE) to reveal the pathological damage. Immunohistochemical assay was employed to measure the expression of von Willebrand factor(vWF), and immunofluorescence assay to measure the expression of vascular endothelial growth factor A(VEGFA). Western blot was employed to determine the protein levels of vascular endothelial growth factor receptor 2(VEGFR2), VEGFA, phosphorylated phosphatidylinositol 3-kinase(p-PI3K), and phosphorylated protein kinase B(p-AKT) in the brain tissue. In vitro experiment: the primary generation of rat brain microvascular endothelial cells(rBEMCs) was cultured and identified. The third-generation rBMECs were assigned into control, model, AST Ⅳ(50 μmol·L~(-1)) + PNS(30 μmol·L~(-1)), LY294002(PI3K/AKT signaling pathway inhibitor), 740Y-P(PI3K/AKT signaling pathway agonist), AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P groups. Oxygen glucose deprivation/re-oxygenation(OGD/R) was employed to establish the cell model of cerebral ischemia-reperfusion injury. The cell counting kit-8(CCK-8) and scratch assay were employed to examine the survival and migration of rBEMCs, respectively. Matrigel was used to evaluate the tube formation from rBEMCs. The Transwell assay was employed to examine endothelial cell permeability. Western blot was employed to determine the expression of VEGFR2, VEGFA, p-PI3K, and p-AKT in rBEMCs. The results of network pharmacology analysis showed that AST Ⅳ and PNS regulated 21 targets including VEGFA and AKT1 of angiogenesis in cerebral infarction. Most of these 21 targets were involved in the PI3K/AKT signaling pathway. The in vivo experiments showed that compared with the model group, AST Ⅳ + PNS reduced the neurological deficit score(P&lt;0.05) and the cell damage rate in the brain tissue(P&lt;0.05), promoted the expression of vWF and VEGFA(P&lt;0.01) and angiogenesis, and up-regulated the expression of proteins in the PI3K/AKT pathway(P&lt;0.05, P&lt;0.01). The in vitro experiments showed that compared with the model group, the AST Ⅳ + PNS, 740Y-P, AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P improved the survival of rBEMCs after OGD/R, enhanced the migration of rBEMCs, increased the tubes formed by rBEMCs, up-regulated the expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P&lt;0.05, P&lt;0.01). Compared with the LY294002 group, the AST Ⅳ + PNS + LY294002 group showed increased survival rate, migration rate, and number of tubes, up-regulated expression of proteins in the PI3K/AKT pathway, and decreased endothelial cell permeability(P&lt;0.05,P&lt;0.01). Compared with the AST Ⅳ + PNS and 740Y-P groups, the AST Ⅳ + PNS + 740Y-P group presented increased survival rate, migration rate, and number of tubes and up-regulated expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P&lt;0.01). This study indicates that AST Ⅳ and PNS can promote angiogenesis after cerebral ischemia by activating the PI3K/AKT signaling pathway.

Abstract TMP112: Von Willebrand Factor (VWF) Modulates Smooth Muscle Cell Proliferation During the Outward Remodeling of Leptomeningeal Collateral Arterioles Following Stroke

Introduction: The leptomeningeal collaterals (LMCs) undergo outward remodeling after stroke to increase rescue flow toward the occluded region. We previously showed increased VWF in remodeled LMCs and others have shown that VWF can act as a smooth muscle cell (SMC) mitogen. Here we test the hypothesis that VWF modulates vascular remodeling of LMCs in the post-stroke brain. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) was performed in C57BL6 WT mice (cohort 1; 4 mos), aged (cohort 2; 24 mos) or VWF KO and WT male mice (cohort 3; 4 mos). At 3, 7, and 14 days after pdMCAO, the pial vessels were processed for VWF/Ki67/SMA immunofluorescence and morphometric analysis. Lastly, distribution of VWF was evaluated in LMCs from human and pig brains. Results: During the 3-14 days post-stroke, WT mice of cohort 1 demonstrated significantly increased VWF in ipsilateral LMCs as well as increased Ki67 positive cells. In ECs, Ki67-positive cells were 10.68 ± 1.48 and 10.73 ± 1.26 per mm of vessel at 3 and 7 days, respectively. In SMCs, Ki67-positive cells were 0.049 ± 0.22 and 4.546 ± 0.95 per mm vessel at 3 and 7 days, respectively. By 14 days, LMC no longer showed Ki67-positive ECs or SMCs. In aged cohort 2, along with attenuated VWF expression and remodeling, the Ki67-positive ECs and SMCs were significantly decreased in the aged mice compared to young mice (Ki67-positive ECs were reduced by 40% and 50% at 3 and 7 days, respectively; Ki67-positive SMCs were reduced by 75% at 7 days). In cohort 3, Ki67-positive SMCs were absent in the VWF-KOs at 3 days post-stroke. However, by 7 days, Ki67-positive SMCs were equivalent between WT and VWF-KOs (4.8-5.0 per mm vessel). Lastly, the presence of VWF was detected in the smooth muscle layer of LMCs of human and pig brains. Conclusions: Our study revealed increased VWF in the mural layer of remodeled LMCs and both EC and SMC proliferation. The remodeling response, VWF expression, and number of proliferative cells were attenuated in LMCs from aged brains. Knockout of VWF resulted in reduced LMC remodeling and delayed SMC proliferation. Our findings also provide evidence for VWF within the smooth muscle layer of pig and human brain arterioles, supporting the broader potential role of VWF as a molecular player in vascular remodeling.

Functional Assessment of VWD Type 2N Variants Using Mammalian Cell Displayed Surface-Tethered Von Willebrand Factor

Background von Willebrand factor (VWF) is a large plasma protein critical in hemostasis and thrombosis. Defects in VWF cause the inherited autosomal bleeding disorder von Willebrand disease (VWD). One critical function of VWF is to bind circulating coagulation factor VIII (FVIII) and protect it from rapid clearance. Type 2N VWD is a recessive form of VWD characterized by low FVIII levels due to impairment in VWF-FVIII binding. Type 2N VWD clinically is similar to mild-to-moderate hemophilia A, an X-linked bleeding disorder caused by deficiency in FVIII. It is critical to distinguish type 2N VWD from hemophilia A because the treatments are different. VWF genotyping can inform accurate diagnoses, treatment, and testing in relatives. With the rapid expansion of DNA sequencing, a growing number of DNA variants are being identified in the VWF gene. However, the interpretation of significance of these variants is often challenged by insufficient evidence to inform confident classification regarding pathogenicity. In vitro functional assays provide an avenue to develop the evidence needed to accurately assign functional significance to DNA variants. We adapted multiplexed surface tethering of extracellular proteins (MultiSTEP), our mammalian cell surface display system for secreted proteins, to display VWF and test the functional impact of VWF variants on VWF-FVIII binding. Methods We constructed a VWF cDNA of common VWF coding sequences by mutating rare variants to the more common nucleotides. The regions encoding the VWF propeptide were deleted (∆Pro-VWF). ∆Pro-VWF was cloned into our MultiSTEP cassette that provides a C-terminal flexible linker, Strep II tag, and CD28 transmembrane domain. Three VWF missense variants implicated in type 2N VWD (Table 1) that had varying levels of evidence to support classification of pathogenicity were chosen for study - VWF c.2561 G&amp;gt;A (p.R854Q), c.2572 T&amp;gt;A (p.C858S), and c.2635 G&amp;gt;A (p.D879N). Variants were subcloned into our ∆Pro-VWF cassette and then recombined into the genomically integrated MultiSTEP landing pad in HEK293 Freestyle cells. Following induction of expression, VWF-displaying cells were incubated with recombinant FVIII. VWF expression was measured using a polyclonal anti-VWF antibody, and VWF-FVIII binding was detected with two different monoclonal antibodies to FVIII. Cells were incubated with fluorescently-conjugated secondary antibodies and cell surface VWF and FVIII abundance determined by flow cytometry. Results We show that ∆Pro-VWF robustly displays in our MultiSTEP system and binds FVIII. All three VWF missense variants express VWF at levels similar to ∆Pro-VWF. Relative to ∆Pro-VWF all three variants show marked impairment in VWF-FVIII binding (Figure 1), providing strong in vitro evidence for pathogenicity in type 2N VWD useful in variant reinterpretation (bold column in Table 1). Conclusions We have developed an in vitro mammalian cell surface display system for the interrogation of VWF variants and adapted it to study VWF-FVIII binding. The Pathogenic variant p.R854Q shows decreased VWF-FVIII binding in this assay, as expected. The Likely Pathogenic variant p.D879N and the Variant of Uncertain Significance p.C858S both exhibit a profound loss of VWF-FVIII binding in this assay. Our results provide evidence for reclassification of p.C858S to Likely Pathogenic for type 2N VWD. We expect our system can be used at scale to similarly interrogate the functional significance of a multiplexed library of VWF variants and improve variant interpretation in VWD.

Comprehensive bioinformatics analysis of the role of VWF in the tumor microenvironment of malignant mesothelioma.

To explore the influence and effect of tumor microenvironment on the development of malignant mesothelioma using machine learning methods. 87 open cases were downloaded from the Cancer Genome Atlas database including transcriptome data, clinical data, and mutation data. The immune, stromal, and estimate scores were calculated for each case by using the ESTIMATE algorithm, and then the cases were grouped according to high and low stromal scores to predict all-cause survival in malignant mesothelioma cases. Their mutation data were analyzed to reveal the differences in mutated genes between the 2 groups, and then the von Willebrand factor (VWF) and FCRL3 genes were identified according to the intersection of DEGs and high-frequency mutated genes. Lastly, the correlation between VWF and the immune checkpoint of 22 kinds of immune cells was analyzed by using the CIBERSORT package of R software. A significant difference was found in the survival time of patients between the high and low stromal score groups. High expression of the VWF gene was negatively correlated with the prognosis of malignant mesothelioma, and the expression of VWF was positively correlated with naive B cells and activated CD4 memory T cells and negatively correlated with NK cells. The results revealed that high expression of VWF may involve in the development of malignant mesothelioma, and the anti-CTLA4 immune checkpoint treatment may have certain efficacy.

OC 13.2 Improved Maturation of Human Pluripotent Stem Cell-Derived Endothelial Cells to Study Von Willebrand Factor Expression and Function

OC 13.2 Improved Maturation of Human Pluripotent Stem Cell-Derived Endothelial Cells to Study Von Willebrand Factor Expression and Function

Aging Is Associated With Organ-Specific Alterations in the Level and Expression Pattern of von Willebrand Factor.

VWF (von Willebrand factor) is an endothelial-specific procoagulant protein with a major role in thrombosis. Aging is associated with increased circulating levels of VWF, which presents a risk factor for thrombus formation. Circulating plasma, cellular protein, and mRNA levels of VWF were determined and compared in young and aged mice. Major organs were subjected to immunofluorescence analyses to determine the vascular pattern of VWF expression and the presence of platelet aggregates. An in vitro model of aging, using extended culture time of endothelial cells, was used to explore the mechanism of age-associated increased VWF levels. Increased circulating plasma levels of VWF with elevated levels of larger multimers, indicative of VWF functional activity, were observed in aged mice. VWF mRNA and cellular protein levels were significantly increased in the brains, lungs, and livers but not in the kidneys and hearts of aged mice. Higher proportion of small vessels in brains, lungs, and livers of aged mice exhibited VWF expression compared with young, and this was concomitant with increased platelet aggregate formation. Prolonged culture of endothelial cells resulted in increased cell senescence that correlated with increased VWF expression; VWF expression was specifically detected in senescent cultured endothelial cells and abolished in response to p53 knockdown. A significantly higher proportion of VWF expressing endothelial cells in vivo exhibited senescence markers SA-β-Gal (senescence-associated β-galactosidase) and p53 in aged mouse brains compared with that of the young. Aging elicits a heterogenic response in endothelial cells with regard to VWF expression, leading to organ-specific increase in VWF levels and alterations in vascular tree pattern of expression. This is concomitant with increased platelet aggregate formation. The age-associated increase in VWF expression may be modulated through the process of cell senescence, and p53 transcription factor contributes to its regulation.

Sodium tanshinone IIA sulfonate ameliorates neointima by protecting endothelial progenitor cells in diabetic mice

BackgroundEndothelial progenitor cells (EPCs) transplantation is one of the effective therapies for neointima associated with endothelial injury. Diabetes impairs the function of EPCs and cumbers neointima prevention of EPC transplantation with an ambiguous mechanism. Sodium Tanshinone IIA Sulfonate (STS) is an endothelium-protective drug but whether STS protects EPCs in diabetes is still unknown.MethodsEPCs were treated with High Glucose (HG), STS, and Nucleotide-binding Domain-(NOD) like Receptor 3 (NLRP3), caspase-1, the Receptor of Advanced Glycation End products (AGEs) (RAGE) inhibitors, Thioredoxin-Interacting Protein (TXNIP) siRNA, and EPC proliferation, differentiation functions, and senescence were detected. The treated EPCs were transplanted into db/db mice with the wire-injured Common Carotid Artery (CCA), and the CD31 expression and neointima were detected in the CCA inner wall.ResultsWe found that STS inhibited HG-induced expression of NLRP3, the production of active caspase-1 (p20) and mature IL-1β, the expression of catalase (CAT) cleavage, γ-H2AX, and p21 in EPCs. STS restored the expression of Ki67, CD31 and von Willebrand Factor (vWF) in EPCs; AGEs were found in the HG-treated EPCs supernatant, and RAGE blocking inhibited the expression of TXNIP and the production of p20, which was mimicked by STS. STS recovered the expression of CD31 in the wire-injured CCA inner wall and the prevention of neointima in diabetic mice with EPCs transplantation.ConclusionSTS inhibits the aggravated neointima hyperplasia by protecting the proliferation and differentiation functions of EPC and inhibiting EPC senescence in diabetic mice. The mechanism is related to the preservation of CAT activity by inhibiting the RAGE-TXNIP-NLRP3 inflammasome pathway.

The Gonadal Maturity and Gene Expressions of Female Giant Freshwater Prawn (Macrobrachium rosenbergii) after Dietary Administration of Medroxyprogesterone Acetate

Abstract One of the problems in giant prawn cultivation is female giant prawns laying eggs during rearing. They will incubate their eggs for three weeks so that the energy from the expected feed for growth is used for egg development and other reproductive activities. Giving MPA hormone to giant prawns either by injection or oral could inhibit gonadal maturation and increase growth rate. The maturation process of the prawn ovary consists of the activation of some complex cellular mechanisms involving genes that regulate the stages of oocyte development. This study aimed to evaluate the response of gonadal maturity and the expression of the MrvWD-Kazal gene in giant prawns fed with a diet containing MPA hormone. The design used in this study was a completely randomized design with four treatments and five replications. The treatments were feeding a diet added with MPA with a concentration of 0 mg.kg-1 feed as control (K), 50 mg.kg-1 feed (P1), 100 mg.kg-1 feed (P2), and 150 mg.kg-1 feed (P3). MPA hormone at a concentration of 50-150 mg/kg feed could inhibit the gonadal maturation of female prawns. In 100 mg/kg of feed concentration showed the lowest gene expression level, indicating an inhibition of gonadal maturation molecularly. The administration of MPA hormone through the feed is a recommended method of aquaculture because it is more applicable and effective than by injection, even though it has a non-uniform impact on each individual. Highlight Research Administration of MPA hormone through the feed to female giant freshwater prawns can inhibit the level of gonad maturity. Expression of von Willebrand factor D (vWD) – Kazal gene were analyzed in different levels of prawn maturity Gonadosomatic index and gonadal histology confirmed that different levels of administration of MPA hormone through the feed could affect the gonad development. The administration of MPA hormone through the feed is a recommended method for aquaculture because it is more applicable and effective to inhibit early maturation of prawns.

ELABELA/APJ Axis Prevents Diabetic Glomerular Endothelial Injury by Regulating AMPK/NLRP3 Pathway

ELABELA (ELA), a recently discovered peptide, is highly expressed in adult kidneys and the endothelium system. It has been identified as a novel endogenous ligand for the apelin receptor (APJ). This study aims to investigate the role of ELA in diabetic glomerular endothelial pyroptosis and its underlying mechanism. Initially, a significant decrease in ELA mRNA levels was observed in the renal cortex of db/db mice and high glucose–treated glomerular endothelial cells (GECs). It was also found that ELA deficiency in ELA+/− mice significantly accelerated diabetic glomerular injury, as shown by exacerbated glomerular morphological damage, increased serum creatine and blood urea nitrogen, and elevated 24-h urinary albumin excretion. In addition, in vivo overexpression of ELA prevented diabetic glomerular injury, reduced von Willebrand factor expression, restored endothelial marker CD31 expression, and attenuated the production of adhesive molecules such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. Furthermore, in vitro studies confirmed that treatment with ELA inhibited GEC injury by regulating the NOD-like receptor protein 3 (NLRP3) inflammasome, as indicated by blocking NLRP3 inflammasome formation, decreasing cleaved Caspase-1 production, and inhibiting interleukin-1β and interleukin-18 production. Moreover, in vitro experiments demonstrated that the protective effects of ELA in GECs during hyperglycemia were diminished by inhibiting adenosine monophosphate–activated protein kinase (AMPK) using Compound C or by APJ deficiency. Taken together, this study provides the first evidence that ELA treatment could prevent diabetic glomerular endothelial injury, which is partly mediated by the regulation of the AMPK/NLRP3 signaling pathway. Therefore, pharmacologically targeting ELA may serve as a novel therapeutic strategy for diabetic kidney disease.

Morphoimmunohistochemical changes in the coronary arteries early after stenting

Introduction: Modern methods of surgical treatment for coronary heart disease include stenting and bypass surgery. In this regard, a morphoimmunohistochemical study of the state of the coronary arteries in the early stages after stenting will make it possible to predict postoperative complications. Materials and methods: The protocols of autopsies of 20 deceased were studied (10 men, mean age 64.7 years, 10 women, mean age 76.9 years). In 85 %, progressive stenosing atherosclerosis of the mouth of the left coronary artery (CA) and the anterior interventricular branch was noted. Taking into account the development of complications after stenting, three chronological groups were identified: 1) up to 3 days; 2) 37 days; 3) 715 days. The computer program ImageJ (US National Institutes of Health, USA) was used for CA morphometry. Statistical processing of the obtained results was carried out on a personal computer using the statistical tools of the Statistica 10 program. Result : It was shown that during the first 3 days after stenting of coronary arteries, acute damage to endothelial cells in the form of desquamation, dystrophic and necrobiotic processes, intramural hemorrhages in the areas of atheromas localization are observed in their walls. After 37 days and later, 715 days after stenting of the coronary artery, parietal mixed (fibrin-leukocytic), fibrin thrombi with signs of organization are observed in the lumens of the coronary artery, which is combined with the phenomena of neoangiogenesis of the artery wall. Conclusions: Damage to endothelial cells and related disturbances in the rheological properties of blood due to the expression of von Willebrand factor can initiate endothelial dysfunction with the development of thrombosis, thromboembolism of small coronary branches, which can cause myocardial perfusion disorders with the development of acute infarction.

Dihydroartemisinin ameliorates cerebral I/R injury in rats via regulating VWF and autophagy-mediated SIRT1/FOXO1 pathway.

Dihydroartemisinin (DHA) has been found to inhibit the expression of von Willebrand factor (VWF), a marker of endothelial cell injury, but its mechanism in cerebral ischemia/reperfusion (I/R) injury remains obscure. In this study, I/R model was constructed through middle cerebral artery occlusion (MCAO) in rats, followed by DHA administration. The effect of DHA on rat cerebral I/R injury was investigated by 2,3,5-triphenyltetrazolium chloride staining, hematoxylin and eosin staining, TUNEL staining, and Western blot. Brain microvascular endothelial cells (BMVECs) isolated from newborn rats were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R), and then treated with DHA. The results showed that MCAO treatment induced infarction, nerve cell apoptosis, and brain tissue impairment in rats, which was mitigated by DHA. OGD/R inhibited viability and accelerated apoptosis of BMVECs, which was alleviated by DHA. I/R procedures or OGD/R up-regulated expressions of VWF, ATG7, Beclin1, and LC3-II/LC3-I ratio, while down-regulating Occludin, Claudin-5, ZO-1, P62, SIRT1, and FOXO1 expressions in vivo and in vitro; however, these effects of I/R procedures or OGD/R were offset by DHA. VWF overexpression reversed the above effects of DHA on OGD/R-induced BMVECs. In summary, DHA ameliorates cerebral I/R injury in rats by reducing VWF level and activating autophagy-mediated SIRT1/FOXO1 signaling pathway.

#4000 ROLE OF ENDOTHELIAL CELLS IN VASCULAR CALCIFICATION

Abstract Background and Aims Vascular calcification (VC) represents a common pathological feature of cardiovascular disease and VC caused by hyperphosphatemia is one of the hallmarks of chronic kidney disease (CKD). Therefore, elucidating the pathogenesis of VC may have significant clinical benefits for CKD patients. Vascular smooth muscle cells challenged with high-phosphate (Pi) actively participate to VC by trans-differentiating into simil-osteoblastic cells that acquire the capability to deposit hydroxyapatite crystals in the extracellular matrix of tunica media in arteries. The role of endothelial cells (ECs) in high-Pi calcification has been poorly investigated until now. Nonetheless, following particular stimuli ECs can lose their endothelial characteristics and acquire several different phenotypes transdifferentiating with a process known as endothelial-to-mesenchymal transition (EndMT). Given the central role of VC for CKD patients wellbeing we decided to investigate whether ECs may have a role in VC process. Method We set up an in vitro calcification model with human aortic ECs (HAECs) challenged with 2, 2.5, 3, 3.5 mM high-Pi for 7 days. Calcium deposition, cell viability and cell trans-differentiation were evaluated. Results In our model we observed deposition of calcium phosphate crystals induced by high-Pi that resulted significant from 3 up to 3.5 mM Pi (Ctr 0.43 ± 0.01; 3 mM Pi 6.72 ± 1.15; 3.5 mM Pi 17.31 ± 1.88; micromol Ca++/mg protein; P &amp;lt; .01; Fig 1A-B). We found that calcification is mediated by Pi-influx, as demonstrated by the inhibition of calcium deposition by Phosphonoformic acid (PFA), an inhibitor of Na/Pi cotransporter Pit-1 (3.5 mM Pi 15.52 ± 1.97; 1 mM PFA 0.6 ± 0.05; micromol Ca++/mg protein; P &amp;lt; .0001; Fig 1A, C). In addition, in our model high-Pi did not show any significant toxic effect at every concentration tested compared to control cells. In Pi-treated ECs we observed a clear change in cell-morphology from cobblestone to spindle shaped, a recognized sign of EndMT. Moreover, we observed a change in extracellular matrix composition more similar to osteoblastic matrix. In facts, ECs treated with Pi were Alcian Blue positive, a staining that binds glycosaminoglycans contained in bone matrix, with blue granules of calcium-phosphate in the cytoplasm of treated cells. Starting from day 5 up to day 7 the transition was confirmed by an up-regulation of SNAIL and N-cadherin mRNA expressions, one of the master genes and one the markers of EndMT process, respectively. A modification of endothelial phenotype was also supported by a progressive decrease in protein expression of Von Willebrand factor and VE-Cadherin observed at day 9 of calcification (−53.7% and −32.6% respectively). Moreover, from day 5 till day 7 we found some osteoblastic differentiation signs such as a progressive increase of RUNX2 and BMP2 mRNA expression and an increase of protective MGP mRNA levels. Conclusion Our data demonstrate that HAECs are able to calcify. High-Pi induces trans-differentiation in simil-osteoblastic cells and a progressive loss of endothelial characteristics through the EndMT process. More studies are needed to better elucidate the role for ECs in CKD VC process.

A critical role of RUNX1 in governing megakaryocyte-primed hematopoietic stem cell differentiation.

As a transcription factor in the RUNT domain core-binding factor family, RUNX1 is crucial in multiple stages of hematopoiesis, and its mutation can cause familial platelet disorder with a predisposition to acute myeloid leukemia. Previous work has established that RUNX1 is involved in the maturation of megakaryocytes (MKs) and the production of platelets. Recent studies have shown that there exists a subpopulation of hematopoietic stem cells (HSCs) with relatively high expression of von Willebrand factor and CD41 at the apex of the HSC hierarchy, termed MK-HSCs, which can give rise to MKs without going through the traditional differentiation trajectory from HSC via MPP (multipotent progenitors) and MEP (megakaryocyte-erythroid progenitor). Here, by using Runx1F/FMx1-Cre mouse model, we discovered that the MK-HSC to MK direct differentiation can occur within 1 cell division, and RUNX1 is an important regulator in the process. Runx1 knockout results in a drastic decrease in platelet counts and a severe defect in the differentiation from MK-HSCs to MKs. Single cell RNA sequencing (RNAseq) analysis shows that MK-HSCs have a distinct gene expression signature compared with non-MK-HSCs, and Runx1 deletion alters the platelet and MK-related gene expression in MK-HSCs. Furthermore, bulk RNAseq and Cut&Run analyses show that RUNX1 binds to multiple essential MK or platelet developmental genes, such as Spi1, Selp, and Itga2b and regulates their expressions in MK-HSCs. Thus, by modulating the expression of MK-related genes, RUNX1 governs the direct differentiation from MK-HSCs to MKs and platelets.

A Systematic Study of Traditional Chinese Medicine for the Treatment of Lung Adenocarcinoma Using a Reverse Network of Key Targets Based on Bioinformatics and Molecular Docking: Curcumin and Trans-Resveratrol as Potential Drug Candidates for Lung Adenocarcinoma

Background Lung adenocarcinoma (LUAD) is a dominant tumor with high morbidity and mortality. In spite of innovations in surgery, chemotherapy, and targeted therapies, the prognosis for patients with LUAD remains unsatisfactory. Therefore, it is particularly important to study the biological markers of the occurrence, development, and prognosis of this disease. Methods In this study, 3 datasets were selected from the Gene Expression Omnibus (GEO) database to screen differentially expressed genes (DEGs). Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using Database Annotation, Visualization and Integrated Discovery (DAVID). In addition, a protein-protein interaction network was performed and 10 key genes were analyzed. Gene and protein expression were analyzed using The Cancer Genome Atlas (TCGA) and The Human Protein Atlas (HPA) databases, respectively. Then, overall survival (OS) rate was analyzed using Kaplan-Meier plotter database. The chemical components were downloaded from the HERB database. Finally, the chemical constituents were docked with 10 targets by molecular docking. By using in vitro studies, we evaluated the effects of curcumin and trans-resveratrol on the proliferation of LUAD cells. Results A total of 122 upregulated and 434 downregulated genes were identified. Then, 10 key genes (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), interleukin-1 beta (IL1B), von Willebrand factor (VWF), matrix metalloproteinase-9 (MMP9), CD44 antigen (CD44), transcription factor Jun (JUN), platelet endothelial cell adhesion molecule (PECAM1), hematopoietic progenitor cell antigen CD34 (CD34), peroxisome proliferator-activated receptor gamma (PPARG), and collagen alpha-1(I) chain (COL1A1)) were obtained. TCGA results showed that GAPDH, MMP9, and COL1A1 were upregulated, while IL-1B, VWF, CD44, JUN, PECAM1, CD34, and PPARG were downregulated in cancer tissues. Kaplan-Meier plotter analysis also showed that high expression of GAPDH, PPARG, and COL1A1, and low expression of VWF, PECAM1, CD34, PPARG, and COL1A1 were associated with poor prognosis in LUAD. Combined with gene expression, staging, immunohistochemistry, and OS prognostic analysis, the results showed that COL1A1 was a potential target for the occurrence and prognosis of LUAD. In addition, 2 key components (curcumin and trans-resveratrol) had good docking effect with COL1A1. We found that curcumin and trans-resveratrol significantly inhibited the proliferation of LUAD cells and significantly decreased the expression of COL1A1 and MMP9. Conclusion In conclusion, our study identified the potential target of LUAD and the main traditional Chinese medicine components (curcumin and trans-resveratrol) for LUAD treatment. This study provides potential therapeutic targets and treatment strategies for the treatment of LUAD.