Expression Of Von Willebrand Factor Research Articles

CXCR2 is involved in pulmonary intravascular macrophage accumulation and angiogenesis in a rat model of hepatopulmonary syndrome.

Hepatopulmonary syndrome (HPS) is a lung complication in various liver diseases, with high incidence, poor prognosis and no effective non-surgical treatments in patients with hepatocirrhosis. Therefore, assessing HPS pathogenesis to explore proper therapy strategies is clinically relevant. In the present study, male Sprague-Dawley rats underwent sham operation or common bile duct ligation (CBDL). Two weeks post-surgery, the following groups were set up for 2weeks of treatment: sham + normal saline, CBDL + CXCR2 antagonist SB225002, CBDL + tumour necrosis factor α (TNF-α) antagonist PTX and CBDL + normal saline groups. Liver and lung tissues were collected after mean arterial pressure (MAP) and portal venous pressure (PVP) measurements. Haematoxylin and eosin (H&E) staining (lung) and Masson staining (liver) were performed for pathological analyses. Finally, pulmonary tissue RNA and total protein were assessed for target effectors. The mRNA and protein levels of CXCR2 were significantly increased in the pulmonary tissue of CBDL rats. What's more, CXCR2 inhibition by SB225002 reduced the expression of CD68 and von Willebrand factor (vWf) in CBDL rats. Importantly, CXCR2 inhibition suppressed the activation of Akt and extracellular signal-regulated kinase (ERK) in CBDL rats. Antagonization of TNF-α with PTX down-regulated the expression of CXCR2. During HPS pathogenesis in rats, CXCR2 might be involved in the accumulation of pulmonary intravascular macrophages and angiogenesis, possibly by activating Akt and ERK, with additional regulation by TNF-α that enhanced pulmonary angiogenesis by directly acting on the pulmonary tissue. Finally, the present study may provide novel targets for the treatment of HPS.

Differentiation of mesenchymal stem cells from human amniotic fluid to vascular endothelial cells

Endothelial dysfunction is a principle feature of vascular-related disease. Endothelial cells have been acquired for the purposes of the restoration of damaged tissue in therapeutic angiogenesis. However, their use is limited by expansion capacity and the small amount of cells that are obtained. Human amniotic fluid mesenchymal stem cells (hAF-MSCs) are considered an important source for vascular tissue engineering. In this study, hAF-MSCs were characterized and then induced in order to differentiate into the endothelial-like cells. Human amniotic fluid cells (hAFCs) were obtained from amniocentesis at the second trimester of gestation. The cells were characterized as mesenchymal stem cells by flow cytometry. The results showed that the cells were positive for mesenchymal stem cell markers CD44, CD73, CD90 and HLA-ABC, and negative for CD31, Amniotic fluid stem cells marker: CD117, anti-human fibroblasts, HLA-DR and hematopoietic differentiation markers CD34 and CD45. The hAF-MSCs were differentiated into endothelial cells under the induction of vascular endothelial growth factor (VEGF) and analyzed for the expression of the endothelial-specific markers and function. The expression of the endothelial-specific markers was determined by reverse transcriptase-quantitative PCR (RT-qPCR), while immunofluorescent analysis demonstrated that the induced hAF-MSCs expressed von Willebrand factor (vWF), vascular endothelial growth factor receptor 2 (VEGFR2), CD31 and endothelial nitric oxide synthase (eNOS). The network formation assay showed that the induced hAF-MSCs formed partial networks. All results indicated that hAF-MSCs have the potential to be differentiated into endothelial-like cells, while human amniotic fluid might be a suitable source of MSCs for vascularized tissue engineering.

Immunohistochemical Analyses on Angiogenesis in a Porcine Von Willebrand Disease Model

Objectives: Women with von Willebrand Disease (VWD) type 3 may develop miscarriage during pregnancy. There is evidence that besides its role in coagulation von Willebrand Factor (VWF) is also involved in angiogenesis and defects of this pathway could play a role in miscarriage. For this reason, VWF as well as proteins with known functions in angiogenesis were analysed by immunohistochemistry of several tissues in a pig model comprising all VWD type 3 genotypes (VWD, heterozygous carriers, wildtype).Methods: Uterus tissue samples including the endometrium were collected from a total of 6 pigs. Of these, two were affected by VWD type 3, two were heterozygous carriers, and the remaining two were wildtype individuals, which served as controls. Hematoxylin-eosin-staining was implemented for morphological evaluation of the tissues, especially with regard to possible differences in structure and pattern of blood vessels. In a second step immunohistochemical analyses comparing the expression of angiogenic factors including VWF were performed in VWD-animals, genetic carriers and wildtype pigs.Results: Hematoxylin-eosin-staining of histological sections of the porcine uteri comprising the different genotypes revealed differences in the vascular architecture within the lamina propria. Compared to non pregnant wild type pigs where blood vessels in the lamina propria of the endometrium usually are of smaller caliber (mostly categorized as arterioles, venules and capillaries), the lamina propria of VWD type 3 animals contained multiple large arteries and veins (of the type usually found in the uterine wall). The immunohistochemical analysis for VWF showed almost no VWF expression in the pigs affected by VWD. In wildtype and heterozygous pigs, expression was obvious in the endothelium. A narrow band of VWF expression apically on the epithel cells as well as partial expression in glands was seen only in wildtype pigs, but not in heterozygous pigs. For some, but not all further angiogenic factors analysed, expression differences were present among the porcine VWD genotypes.Conclusion: The results of our study confirm effects of VWF on the uterine vascular architecture and on the expression of specific angiogenic factors. Pigs affected by VWD showed almost no expression of VWF and the blood vessels within the uterine lamina propria had a surprisingly high percentage of large arteries and veins indicating a crucial role of vWF for uterine angiogenesis. Expression differences among the genotypes were obvious for specific angiogenic factors. DisclosuresVon Depka:Octapharma: Other: Study Investigator, Speakers Bureau.

Type 3 Von Willebrand Disease: A Case Report of a Rare Entity

Background:Von Willebrand Factor (VWF) is essential for proper hemostasis. It serves as a carrier protein for factor VIII and also aids in platelet adherence to damaged endothelium. Von Willebrand Disease (VWD) may result from congenital absence of VWF, structural or functional abnormalities or mutations leading to mild decreases in expression of VWF. Type 3 VWD is an autosomal recessive disorder, which accounts for only 1% of patients with VWD and is characterized by a virtual absence of VWF and low levels of Factor VIII.Objective:To describe a case of Type 3 VWD including the presentation, diagnosis and clinical course.Design/Method: Case Report.A 4 month old female presented with a history of 8 hours of persistent bleeding from an injection site following vaccination administration. At that time, the patient’s platelet count and PT/PTT were normal. She had a few intermittent episodes of persistent bleeding with minor abrasions and had labs repeated at the age of 4 months which were significant for a prolonged PTT of 59.5 seconds with normal PT and platelet count. Factor VIII level was <1% and VWF activity was <15%.Results:A complete Von Willebrand panel was sent to Mayo Medical Labs and showed a Factor VIII level of 2%, VWF antigen 6%, VWF activity of 8, and an absence of VWF multimers. Confirmatory VWF full gene sequence analysis testing was sent to Blood Center of Wisconsin and showed a heterozygous sequence variant, c.2435delC, in the VWF gene consistent with a diagnosis of VWD Type 3. VWF full gene sequence analysis was also sent on both parents. Mother's results showed that she is a carrier of the same VWF gene sequence variant. Father's results were negative. Given the patient only has a heterozygous sequence variant yet still has expression of VWD, gene duplication testing was sent to Blood Center of Wisconsin to look for possible duplication of this area. Results of this testing showed that the patient had two pathogenic variants, one on each allele: heterozygous c.243delC in exon 18 and a deletion involving exon 6 in the VWF gene.Conclusion:This case represents the clinical presentation and diagnosis of a patient with Type 3 VWD. While VWD is not uncommon, diagnosis of VWD Type 3 is quite rare. VWD Type 3 is inherited in an autosomal recessive fashion. In the case of our patient, only one parent was identified as a carrier of the same gene sequence mutation. Therefore, the second pathogenic variant resulting in VWD Type 3 was found to be a de novo deletion, further adding to the uniqueness of this case. DisclosuresNo relevant conflicts of interest to declare.

Expression of Sequence Variants Identified in Type 1 VWD Subjects in the Zimmerman Program Study Reveals Defects in VWF Secretion and Multimerization

von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder. Type 1 is the most common form of VWD and results in a partial quantitative deficiency of von Willebrand Factor (VWF). The mechanisms underlying type 1 VWD are still not very well understood although reduced VWF secretion and increased VWF clearance have been implicated in causing VWD. We aimed to characterize novel sequence variants (SV) identified in the VWF gene in type 1 VWD patients recruited through the Zimmerman Program for the Molecular and Clinical Biology of VWD in order to define the underlying mechanism and explore if SV in a particular domain are mechanistically similar.We utilized homozygous expression in human embryonic kidney cells (HEK-293T) to study the effect of VWF SV on VWF secretion, intracellular retention, multimerization, and function. Novel SV have been identified throughout the entire VWF protein. We introduced the following variants into a VWF-mycHis plasmid vector: V86M, W199X, C524Y, M947V, R960P, G994D, C996W, R1204W, Q1353X, E1660X, R1763Q, C2199Y, Q2256H, T2282I, P2524L, A2569E, C2693F, C2701Y, and C2754Y. Sequence variants were confirmed by Sanger sequencing. Variant VWF cDNA is transfected homozygously into HEK-293T cells. The supernatants and cell lysates from 3 independent transfections are collected and analyzed by ELISA for VWF:Ag and VWF binding to collagen type III (VWF:CB). VWF multimer structure is analyzed by SDS-agarose gel electrophoresis and western blotting. The genotype-phenotype patient data is correlated with the data from expression studies to explore a model to predict the impact of the SV on the VWD phenotype.Variants V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F demonstrated secretion comparable to that of wild type (WT)-VWF. In contrast, VWF variants R960P and C2701Y showed reduced VWF secretion (<50% of WT) with increased VWF in the cell lysate. VWF variants W199X, C524Y, G994D, C996W, Q1353X, E1660X, C2199Y, and C2754Y demonstrated a complete absence of secreted VWF. Not unexpectedly, homozygous expression of stop codon variants W199X, Q1353X, and E1660X demonstrated no VWF in the cell lysate. However, non-secreted VWF variants C524Y, G994D, C996W, C2199Y, and C2754Y showed intracellular retention with detectable VWF in the cell lysate. SV occurring at cysteine residues (C524Y, C996W, C2199Y, C2701Y, and C2754Y) all had reduced secretion and increased intracellular retention, consistent with altered conformation leading to increased intracellular chaperone interaction and proteasomal degradation. VWF binding to collagen is dependent on the presence of high molecular weight multimers (HMWM). VWF:CB/VWF:Ag is used to predict multimer structure with VWF:CB/VWF:Ag < 0.7 indicative of loss of the HMWM. VWF variants V86M, M947V, R1763Q, Q2256H, P2524L, C2701Y had VWF:CB/VWF:Ag ≥ 0.7 consistent with normal multimer structure, while variants R960P, R1204W, T2282I, A2569E, and C2693F had VWF:CB/VWF:Ag < 0.7 indicating abnormal multimer structure.47.3% of the 19 VWF variants studied had normal VWF secretion, 10.5% had reduced secretion with increased intracellular retention, and 26.3% revealed absent secretion with intracellular retention. Variants with a premature stop codon did not synthesize VWF at all. Some SV had normal secretion and multimerization (V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F) implying that the VWD phenotype in these patients results from yet unidentified mechanisms and may not be associated with these SV. Reduced plasma survival is unlikely as these patients had normal VWFpp/VWF:Ag level consistent with normal VWF clearance. Among the VWF variants with normal or decreased secretion, 45.4% had reduced VWF:CB/VWF:Ag consistent with abnormal multimer structure. Heterozygous expression, as observed in the patient, is expected to normalize these multimerization defects. The decreased or absent secretion observed for 52.7% of the variants studied correlates with the patient phenotype, indicating reduced secretion is the mechanism underlying these patients' type 1 VWD phenotype. No domain-specific correlation of VWF secretion or multimer abnormality was observed.In summary, VWF expression studies confirmed the causative nature of many, but not all of the novel sequence variants identified in type 1 VWD subjects in the Zimmerman Program. DisclosuresNo relevant conflicts of interest to declare.

Differentiation of Genome Edited Human iPSCs into Endothelial Progenitor Cells for Hemophilia Α Therapy

▪Gene and cell therapy for hemophilia A requires the use of the appropriate target cell for genetic modification and, given the advances in genome editing, an approach that can be applied universally for the wide variety of genetic mutations in the coagulation factor VIII gene (F8) responsible for hemophilia A. Recent studies using two different conditional knockout mouse models showed that the principal, and possibly exclusive, source for FVIII in the circulation are endothelial cells (Everett LA et al. Blood 2014.123: 3697; Fahs SA et al. Blood 2014.123: 3706). Since endothelial cells are present in all the major organs previously thought to produce coagulation factor VIII (FVIII), these studies provide the basis for the earlier reports indicating different tissues (liver, lung, spleen, lymphatic tissue) as sources of FVIII. ECs conveniently express von Willebrand factor (vWF) that is essential for the stability of FVIII. The precursor of ECs, endothelial progenitor cells (EPCs), have been isolated from adult human peripheral blood and cord blood. EPCs can readily integrate into existing vascular system upon intravenous injection. EPCs are quite rare in peripheral blood (about 20 colony forming cells per 100 mL of blood). Moreover, EPCs derived from adult peripheral blood have lower proliferative potential than those obtained from cord blood (Ingram DA. Blood. 2004. 104: 2752). In contrast, induced pluripotent stem cells (iPSCs), that are more amenable for expansion, can be readily differentiated into EPCs. Studies have also shown that iPSC-derived EPCs when injected intrahepatically in mice integrated into liver sinusoids, resulted in therapeutic levels of FVIII production (Xu D et al. PNAS 2009. 106: 808).Here we describe an optimized in vitro differentiation protocol for derivation of EPCs from iPSCs. We have previously reported the generation and characterization of human iPSCs from lung fibroblasts (Srinivasakumar et al. PeerJ. 2013;1:e224). In this study we used human iPSCs generated from adult dermal fibroblasts using Yamanaka’s non-integrating Epstein-Barr based episomal vectors. We used a step-wise differentiation protocol for obtaining EPCs that was a combination of a method for differentiation of iPSCs into hematopoietic progenitors (Fig A, Steps 1 & 2) to generate hemangioblasts (Niwa A et al. PLoS ONE 6(7): e22261), and a protocol for obtaining EPCs from peripheral blood (Step 3) (Mead LE et al. Current Protocols in Stem Cell Biol. 2C.1.1-2C.1.27). A sorting step after differentiation into hemangioblasts followed by a final round of sorting after step 3 yielded >90% pure population of EPC that exhibited the canonical cell surface markers: CD31 and CD34, and absence of CD45 (Fig B). The cells also took up fluorophore-conjugated acetylated LDL (acLDL-A488) that was inhibited with 50x excess of unlabeled acLDL (Fig C). Immunofluorescence staining for vWF revealed characteristic staining reminiscent of Weibel-Palade bodies in the cytoplasm (Fig D). The cells exhibited the typical tube formation ability in Matrigel (Fig E). Additional studies are needed to determine the proliferative potential of these cells and their ability to integrate into vasculature.To address the myriad mutations shown to be responsible for hemophilia A, we have designed high efficiency dimeric guide RNAs (as part of a separate study) (Zaboikin M et al. Manuscript in preparation) for use with the CRISPR/dCas9-Fok1 system (Tsai SQ et al. Nat Biotechnol. 2014. 32:569) for precise modification at the F8 locus downstream of the first coding exon. We also showed in that study the replacement of target sequence at the site with that of a donor template sequence with desired attributes. We hypothesize that using a donor template that encodes the F8 promoter driving a functional F8 cDNA for homology directed repair at the target double stranded break site will provide an universal solution for the large variety of mutations observed in hemophilia A. Results of genome editing of iPSCs using the above mentioned CRISPR/dCas9-Fok1 system (together with the donor template) followed by the differentiation of genetically modified iPSCs into EPCs will be presented. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Screening of differentially expressed protein kinases in bone marrow endothelial cells and the protective effects of the p38a inhibitor SB203580 on bone marrow in liver fibrosis.

Hematological abnormalities are frequently observed in patients with liver cirrhosis (LC). A previous study demonstrated that the apoptosis and damage of endothelial cells could cause the hematological abnormalities in LC. Protein kinases are one of the most important factors that regulate cell behavior, and are potential therapeutic targets for the treatment of a number of diseases. In a previous study, whole genome profiling was used to identify differentially expressed genes in human bone marrow endothelial cells treated with serum from 26 patients with LC. From this data set, the present study identified 14 differentially expressed kinase genes in human bone marrow endothelial cells in LC from the microarray data, including p38a, AKT1 and PDK1. Pathway analysis revealed that these kinase genes were enriched in certain important LC-associated pathways (e.g. MAPK and WNT signaling pathway). Literature mining revealed that p38a was associated with bone marrow apoptosis; therefore, p38a and its inhibitor, SB203580, were selected as potential therapeutic targets in the present study. The results of hematoxylin-eosin and Masson's trichrome staining of livers from a rat model of liver fibrosis (LF) that underwent ligation of the bile duct demonstrated that SB203580 reduced the degree of LF. In addition, SB203580-treated rats with LF demonstrated a significantly higher number of platelets when compared with the untreated group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis indicated that apoptosis of bone marrow tissue in rats with LF was inhibited by SB203580. In addition, the results from the immunohistochemical analysis demonstrated that SB203580 reduced the expression of von Willebrand factor and caspase 3 in the bone marrow of rats with LF. In conclusion, the results from the present study indicate that the p38a kinase inhibitor, SB203580, may exhibit a protective effect on bone marrow tissues in rats with LF. This suggests that protein kinases and their inhibitors may present novel therapeutic strategies for the treatment of hematological abnormalities in patients with LC.

Endothelial damage in major depression patients is modulated by SSRI treatment, as demonstrated by circulating biomarkers and an in vitro cell model.

There is a link between depression, cardiovascular events and inflammation. We have explored this connection through endothelial dysfunction, using in vivo and in vitro approaches. We evaluated circulating biomarkers of endothelial dysfunction in patients with major depression at their diagnosis (MD-0) and during antidepressant treatment with the selective serotonin reuptake inhibitor escitalopram, for 8 and 24 weeks (MD-8 and MD-24). Results were always compared with matched healthy controls (CON). We measured in vivo circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) in blood samples, and assessed plasma levels of soluble von Willebrand factor (VWF) and vascular cell adhesion molecule-1 (VCAM-1). CEC counts, soluble VWF and VCAM-1 were statistically elevated in MD-0 (P<0.01 versus CON) and gradually decreased during treatment. Conversely, EPC levels were lower in MD-0, tending to increase throughout treatment. In vitro studies were performed in human endothelial cells cultured in the presence of sera from each study group. Elevated expression of the inflammation marker intercellular adhesion molecule-1 and oxidative stress, with lower presence of endothelial nitric oxide synthase and higher reactive oxygen species production, were found in cells exposed to MD-0 sera (P<0.05 versus CON). These results were normalized in cells exposed to MD-24 sera. Thrombogenicity of extracellular matrices generated by these cells, measured as expression of VWF, tissue factor and platelet reactivity, showed non-significant differences. We provide a model of cultured endothelial cells reproducing endothelial dysfunction in naive patients with major depression, demonstrating endothelial damage and inflammation at diagnosis, and recovering with selective serotonin reuptake inhibitor treatment for 24 weeks.

Development of an Atlantic salmon heart endothelial cell line (ASHe) that responds to lysophosphatidic acid (LPA).

As diseases and abnormalities of the heart can interfere with the aquaculture of Atlantic salmon, the heart was investigated as a source of cell lines that could be used to study the cellular basis of these conditions. An Atlantic salmon heart endothelial cell line, ASHe, was developed and characterized for growth properties, endothelial cell characteristics, and responsiveness to lysophosphatidic acid (LPA). AHSe cells stained negative for senescence associated ß-galactosidase and grew well in 10 and 20% FBS/L15 at high cell density, but not in L15 medium supplemented with calf serum. It displayed many endothelial cell-like characteristics including a cobblestone morphology, capillary-like structures formation on Matrigel, and expression of von Willebrand factor and endothelial cell-related tight junction proteins ZO-1, claudin 3, and claudin 5. ASHe cells responded to the cardiovascular modulator, LPA, in two contrasting ways. LPA at 5 and 25μM inhibited the ability of ASHe cells to heal a wound but stimulated their proliferation, especially as evaluated by colony formation in low-density cultures. The enhancement of proliferation by LPA parallels what has been observed previously in mammalian endothelial cell cultures exposed to LPA, whereas the LPA slowing of ASHe cell migration contrasted with the LPA-enhanced migration of some mammalian cells. Therefore, this cell line is a potentially useful model for future comparative studies on piscine and mammalian cardiovascular cell biology and for studies on diseases of Atlantic salmon in aquaculture.

Induction of endothelial cell proliferation and von Willebrand factor expression and secretion by leukemic plasma of patients with chronic lymphocytic leukemia before and after inhibition of NF-κB.

Although certain evidence has indicated a role for angiogenesis in the pathophysiology of hematopoietic malignancies, its role in chronic lymphocytic leukemia (CLL) prognosis is yet to be defined. To our knowledge, the effects of CLL plasma on cell culture have not been addressed. Therefore, we investigated the effects of CLL plasma on cell cycle regulation and von Willebrand factor (vWF) secretion, and expression in human umbilical vein endothelial cell cultures (HUVECs). Since nuclear factor-kappa B (NF-κB) transcription factor has been a therapeutic target for treatment of cancer, we inhibited NF-κB using small interfering RNA to clarify if there is a role for this factor in probable effects. The cells were treated with the plasma of patients with CLL. Subsequently, cell cycle phase distribution, vWF secretion, expression, and storage were detected using ELISA, flow cytometry, and immunohistochemical staining. In addition, NF-κB was inhibited using small interfering RNA. Plasma treatment promoted cell cycle progression by decreasing the cell number in G1 phase, while increasing the cell number in S phase and G2M phase. A significant increase of vWF expression, secretion, and storage was found, associated with the vWF levels of patients' plasma. We found that induction of cell cycle promotion, but not vWF expression and secretion, was partially suppressed by this inhibition. We found that endothelial cell cycle and vWF expression and secretion affected by CLL plasma and NF-κB play a role in the former. These findings would be useful for understanding the prognostic importance of plasma angiogenic factor levels in CLL.

Transplantation of hypoxia-inducible factor-1α gene modified neural stem cells increases cell survival and angiogenesis after cerebral ischemia

Previous studies have indicated the beneficial effects of transplanted neural stem cells (NSCs) on cerebral ischemia. Hypoxia-inducible factor-1α (HIF-1α) is a master transcription factor of cellular hypoxic gene expression, and its signal pathway might be the primary mechanism through which hypoxia promotes the growth of NSCs. To test the hypothesis that HIF-1α contributes to the therapeutic effect of NSCs transplantation in cerebral ischemia, we compared the efficacy of transplanting PBS, NSCs infected with recombinant adenovirus, HIF-1α gene and NSCs infected with a recombinant adenovirus vector with HIF-1α gene (HIF-1α-NSCs). A transient middle cerebral artery occlusion (tMCAO) was used in this study. PBS, HIF-1α gene, NSCs and HIF-1α-NSCs were respectively injected into cortical peri-infarction of the rat brain at 24 h after MCAO. Neurological deficits were assessed using the modified neurological severity score (NSS). Immunohistochemistry for BrdU, VEGF, Von Willebrand Factor and Nissl staining were performed. Compared with other groups HIF-1α-NSCs showed better behavioral recovery at 7, 14, 21and 28 days, and lesser degree of brain atrophy in cortex and hemisphere. More BrdU-positive cells in HIF-1α-NSCs group than that in NSCs group. Expression of VEGF and Von Willebrand Factor are both higher in HIF-1α-NSCs than those in HIF-1α or in NSCs group. Thus, we concluded that during the early period after transplantation HIF-1α infected NSCs expressed gene products，which reduce brain injury by improving the survival of NSCs and protecting the vascular system．

First trimester infection with Listeria monocytogenes is characterized by decidual and placental inflammation in pregnant cynomolgus macaques

First trimester infection with Listeria monocytogenes is characterized by decidual and placental inflammation in pregnant cynomolgus macaques

Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation.

Hypoxia-inducible factor-1α (HIF-1α) can control a transcriptional factor forkhead box P3 (Foxp3) protein expression in T lymphocyte differentiation through proteasome-mediated degradation. In this study, we unveil a reverse regulatory mechanism contributing to bladder cancer progression; Foxp3 expression attenuates HIF-1α degradation. We first demonstrated that Foxp3 expression positively correlates with the metastatic potential in T24 cells and can increase the expression of HIF-1α-target genes, such as vascular endothelial growth factor (VEGF) and glucose transporter (GLUT). Foxp3 protein can bind with HIF-1α, particularly under hypoxia. In vivo ubiquination assay demonstrated that Foxp3 can decrease HIF-1α degradation in a dose-dependent manner. Knocking-down of Foxp3 expression blocks in vivo tumor growth in mice and prolongs mice's survival, which is associated with von Willebrand factor expression. Thirty-three of 145 (22.8 %) bladder tumors exhibit Foxp3 expression. Foxp3 expression is an independent predictor for disease progression in superficial bladder cancer patients (p = 0.032), associated with less number of intratumoral CD8+ lymphocyte. The metaanalysis from 2 published datasets showed Foxp3 expression is positively associated with GLUT−4, −9, and VEGF-A, B-, D expression. This reverse post-translational regulation of HIF-1α protein by Foxp3 provides a new potential target for developing new therapeutic strategy for bladder cancer.

Changes, and the Relevance Thereof, in Mitochondrial Morphology during Differentiation into Endothelial Cells.

The roles of mitochondria in various physiological functions of vascular endothelial cells have been investigated extensively. Morphological studies in relation to physiological functions have been performed. However, there have been few reports of morphological investigations related to stem cell differentiation. This was the first morphological study of mitochondria in relation to endothelial differentiation and focused on quantitative analysis of changes in mitochondrial morphology, number, area, and length during differentiation of human mesenchymal stem cells (hMSCs) into endothelial-like cells. To induce differentiation, we engaged vascular endothelial growth factors and flow-induced shear stress. Cells were classified according to the expression of von Willebrand factor as hMSCs, differentiating cells, and almost fully differentiated cells. Based on imaging analysis, we investigated changes in mitochondrial number, area, and length. In addition, mitochondrial networks were quantified on a single-mitochondrion basis by introducing a branch form factor. The data indicated that the mitochondrial number, area per cell, and length were decreased with differentiation. The mitochondrial morphology became simpler with progression of differentiation. These findings could be explained in view of energy level during differentiation; a higher level of energy is needed during differentiation, with larger numbers of mitochondria with branches. Application of this method to differentiation into other lineages will explain the energy levels required to control stem cell differentiation.

Sinusoidal endothelial cell and hepatic stellate cell phenotype correlates with stage of fibrosis in chronic liver disease in dogs.

We evaluated the extent of hepatic fibrosis in chronic liver disease of dogs using a modification of Ishak's staging criteria for human chronic liver disease, and examined the association of stage of fibrosis with immunophenotypic markers of transdifferentiation of hepatic sinusoidal endothelial cells and hepatic stellate cells. Formalin-fixed, paraffin-embedded, hematoxylin and eosin-stained liver biopsy specimens from 45 case dogs with chronic liver disease and 55 healthy control dogs were scored for the presence and extent of fibrosis. This stage score for fibrosis strongly correlated with upregulated von Willebrand factor (vWF) expression in lobular sinusoidal endothelial cells (Spearman correlation coefficient [SCC] = 0.57, p < 0.05). Immunoreactivity for vWF factor was identified in 68.9% of case biopsies, varying in distribution from periportal to diffuse, whereas vWF immunoreactivity was identified in only 14.5% of control specimens, and was restricted to the immediate periportal sinusoids. The majority of both case and control biopsies exhibited similar prominent lobular perisinusoidal expression of alpha-smooth muscle actin (α-SMA). A minority of specimens (17.8% of case biopsies, 1.8% of control biopsies) exhibited low perisinoidal α-SMA expression, and there was a weak negative correlation between α-SMA expression and stage of fibrosis (SCC = -0.29, p = 0.0037). These results document a method for staging the severity of fibrosis in canine liver biopsies, and show a strong association between fibrosis and increased expression of vWF in hepatic sinusoidal endothelial cells.

Prenatal stress-induced increases in hippocampal von Willebrand factor expression are prevented by concurrent prenatal escitalopram

Prenatal stress has been linked to deficits in neurological function including deficient social behavior, alterations in learning and memory, impaired stress regulation, and susceptibility to adult disease. In addition, prenatal environment is known to alter cardiovascular health; however, limited information is available regarding the cerebrovascular consequences of prenatal stress exposure. Vascular disturbances late in life may lead to cerebral hypoperfusion which is linked to a variety of neurodegenerative and psychiatric diseases. The known impact of cerebrovascular compromise on neuronal function and behavior highlights the importance of characterizing the impact of stress on not just neurons and glia, but also cerebrovasculature. Von Willebrand factor has previously been shown to be impacted by prenatal stress and is predictive of cerebrovascular health. Here we assess the impact of prenatal stress on von Willebrand factor and related angiogenic factors. Furthermore, we assess the potential protective effects of concurrent anti-depressant treatment during in utero stress exposure on the assessed cerebrovascular endpoints. Prenatal stress augmented expression of von Willebrand factor which was prevented by concurrent in utero escitalopram treatment. The functional implications of this increase in von Willebrand factor remain elusive, but the presented data demonstrate that although prenatal stress did not independently impact total vascularization, exposure to chronic stress in adulthood decreased blood vessel length. In addition, the current study demonstrates that production of reactive oxygen species in the hippocampus is decreased by prenatal exposure to escitalopram. Collectively, these findings demonstrate that the prenatal experience can cause complex changes in adult cerebral vascular structure and function.

Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo

AbstractCirculating factor VIII (FVIII) is derived from liver and from extrahepatic sources probably of endothelial origin, but the vascular sites of FVIII production remain unclear. Among organs profiled, only liver and lymph nodes (LNs) show abundant expression of F8 messenger RNA (mRNA). Transcriptomic profiling of subsets of stromal cells, including endothelial cells (ECs) from mouse LNs and other tissues, showed that F8 mRNA is expressed by lymphatic ECs (LECs) but not by capillary ECs (capECs), fibroblastic reticular cells, or hematopoietic cells. Among blood ECs profiled, F8 expression was seen only in fenestrated ECs (liver sinusoidal and renal glomerular ECs) and some high endothelial venules. In contrast, von Willebrand factor mRNA was expressed in capECs but not in LECs; it was coexpressed with F8 mRNA in postcapillary high endothelial venules. Purified LECs and liver sinusoidal ECs but not capECs from LNs secrete active FVIII in culture, and human and mouse lymph contained substantial FVIII:C activity. Our results revealed localized vascular expression of FVIII and von Willebrand factor and identified LECs as a major cellular source of FVIII in extrahepatic tissues.

An integrative microfluidically supported in vitro model of an endothelial barrier combined with cortical spheroids simulates effects of neuroinflammation in neocortex development.

The development of therapeutic substances to treat diseases of the central nervous system is hampered by the tightness and selectivity of the blood-brain barrier. Moreover, testing of potential drugs is time-consuming and cost-intensive. Here, we established a new microfluidically supported, biochip-based model of the brain endothelial barrier in combination with brain cortical spheroids suitable to detect effects of neuroinflammation upon disruption of the endothelial layer in response to inflammatory signals. Unilateral perfusion of the endothelial cell layer with a cytokine mix comprising tumor necrosis factor, IL-1β, IFNγ, and lipopolysaccharide resulted in a loss of endothelial von Willebrand factor and VE-cadherin expression accompanied with an increased leakage of the endothelial layer and diminished endothelial cell viability. In addition, cytokine treatment caused a loss of neocortex differentiation markers Tbr1, Tbr2, and Pax6 in the cortical spheroids concomitant with reduced cell viability and spheroid integrity. From these observations, we conclude that our endothelial barrier/cortex model is suitable to specifically reflect cytokine-induced effects on barrier integrity and to uncover damage and impairment of cortical tissue development and viability. With all its limitations, the model represents a novel tool to study cross-communication between the brain endothelial barrier and underlying cortical tissue that can be utilized for toxicity and drug screening studies focusing on inflammation and neocortex formation.

Atrial tissue expression of receptor for advanced glycation end-products (RAGE) and atrial fibrosis in patients with mitral valve disease

BackgroundIt has been reported that receptor for advanced glycation end-products (RAGE) plays a significant role in cardiac fibrosis. Nonetheless, the precise relationship between the RAGE and atrial fibrosis has never been studied in humans. The aim of this study was to determine whether degree of atrial fibrosis was associated with atrial tissue expression of RAGE in patients with mitral valve disease (MVD). MethodsWe collected human left atrial (LA) appendage tissue from 25 patients who underwent mitral valve surgery. We quantified the expression of RAGE and other protein markers by Western blotting and compared these levels with histological evaluations. ResultsRAGE expression in the LA appendage tissue was significantly correlated with atrial fibrosis (r=0.681, p=0.001). RAGE expression (regression coefficient [B] 9.49, 95% confidence interval [CI] 4.76–14.2, p<0.001) and LA diameter (B 0.43, 95% CI 0.13–0.74, p=0.008) were independently associated with degree of atrial fibrosis in multiple linear regression analysis. RAGE expression was significantly correlated with protein expression of von Willebrand factor (r=0.659, p<0.001), vascular endothelial cadherin (r=0.757, p<0.001), ICAM-1 (r=0.568, p=0.003), and PECAM-1 (r=0.423, p=0.035) in the LA appendage tissue. In addition, patients with severe mitral stenosis (MS) had higher atrial RAGE expression than those with no, mild, or moderate MS (p=0.013). Patients with MVD and atrial fibrillation (AF) had more severe atrial fibrosis (p=0.024) and higher RAGE expression (p=0.047) than those who remained in sinus rhythm. ConclusionsAtrial tissue expression of RAGE was significantly associated with atrial fibrosis, severe MS, and AF rhythm in patients with MVD.

Human Umbilical Cord-Derived Mesenchymal Stromal Cells Improve Left Ventricular Function, Perfusion, and Remodeling in a Porcine Model of Chronic Myocardial Ischemia.

: Stem cell therapy has emerged as a new strategy for treatment of ischemic heart disease. Although umbilical cord-derived mesenchymal stromal cells (UC-MSCs) have been used preferentially in the acute ischemia model, data for the chronic ischemia model are lacking. In this study, we investigated the effect of UC-MSCs originated from Wharton's jelly in the treatment of chronic myocardial ischemia in a porcine model induced by ameroid constrictor. Four weeks after ameroid constrictor placement, the surviving animals were divided randomly into two groups to undergo saline injection (n = 6) or UC-MSC transplantation (n = 6) through the left main coronary artery. Two additional intravenous administrations of UC-MSCs were performed in the following 2 weeks to enhance therapeutic effect. Cardiac function and perfusion were examined just before and at 4 weeks after intracoronary transplantation. The results showed that pigs with UC-MSC transplantation exhibited significantly greater left ventricular ejection fraction compared with control animals (61.3% ± 1.3% vs. 50.3% ± 2.0%, p < .05). The systolic thickening fraction in the infarcted left ventricular wall was also improved (41.2% ± 3.3% vs. 46.2% ± 2.3%, p < .01). Additionally, the administration of UC-MSCs promoted collateral development and myocardial perfusion. The indices of fibrosis and apoptosis were also significantly reduced. Immunofluorescence staining showed clusters of CM-DiI-labeled cells in the border zone, some of which expressed von Willebrand factor. These results suggest that UC-MSC treatment improves left ventricular function, perfusion, and remodeling in a porcine model with chronic myocardial ischemia. Ischemic heart disease is the leading cause of death worldwide. Many patients with chronic myocardial ischemia are not suitable for surgery and have no effective drug treatment; they are called "no-option" patients. This study finds that umbilical cord-derived mesenchymal stromal cells transplanted by intracoronary delivery combined with two intravenous administrations was safe and could significantly improve left ventricular function, perfusion, and remodeling in a large-animal model of chronic myocardial ischemia, which provides a new choice for the no-option patients. In addition, this study used clinical-grade mesenchymal stem cells with delivery and assessment methods commonly used clinically to facilitate further clinical transformation.