Von Willebrand Factor Staining Research Articles

The impact of von Willebrand factor on fibrin formation and structure unveiled with type 3 von Willebrand disease plasma.

Normally, von Willebrand factor (VWF) remains inactive unless its A1A2 domains undergo a shear stress-triggered conformational change. We demonstrated the capacity of a recombinant A2 domain of VWF to bind and to affect fibrin formation, altering the fibrin clot structure. The data indicated that VWF contains an additional binding site for fibrin in the A2 domain that plays a role in the incorporation of VWF to the polymerizing fibrin. This study is to examine the hypothesis that active plasma VWF directly influence fibrin polymerization and the structure of fibrin clots. The study used healthy and type 3 von Willebrand disease (VWD) plasma, purified plasma VWF, fibrin polymerization assays, confocal microscopy and scanning electron microscopy. The exposed A2 domain in active VWF harbors additional binding sites for fibrinogen, and significantly potentiates fibrin formation (P < 0.02). Antibody against the A2 domain of VWF significantly decreased the initial rate of change of fibrin formation (P < 0.002). Clot analyses revealed a significant difference in porosity between normal and type 3 VWD plasma (P < 0.008), further supported by scanning electron microscopy, which demonstrated thicker fibrin fibers in the presence of plasma VWF (P < 0.0003). Confocal immunofluorescence microscopy showed punctate VWF staining along fibrin fibrils, providing visual evidence of the integration of plasma VWF into the fibrin matrix. The study with type 3 VWD plasma supports the hypothesis that plasma VWF directly influences fibrin polymerization and clot structure. In addition, a conformational change in the A1A2 domains exposes a hidden fibrin(ogen) binding site, indicating that plasma VWF determines the fibrin clot structure.

Overexpressed miR-486 in bone marrow mesenchymal stem cells represses urethral fibrosis and targets Col13a1 in urethral stricture rats.

Urethral stricture (US) is a challenging problem in urology and its pathogenesis of US is closely related to the fibrotic process. Previous evidence has indicated the downregulation of microRNA (miR)-486 in injured urethral specimens of rats. This study aimed to explore the effects of miR-486-overexpressed bone marrow mesenchymal stem cells (BMSCs) on US. BMSCs were identified by detecting their multipotency and surface antigens. Lentivirus virus expressing miR-486 was transduced into rat BMSCs to overexpress miR-486. Transforming growth factor (TGF)-β1 induced fibrotic phenotypes in urethral fibroblasts (UFs) and rat models. Western blotting showed protein levels of collagen I/III and collagen type XIII alpha 1 chain (Col13a1). Real time quantitative polymerase chain reaction was utilized for messenger RNA level evaluation. Hematoxylin-eosin, Masson's trichrome, and Von Willebrand Factor staining were conducted for histopathological analysis. Immunofluorescence staining was employed for detecting alpha smooth muscle actin (α-SMA) expression. Luciferase reporter assay verified the interaction between miR-486 and Col13a1. The results showed that miR-486-overexpressed BMSCs suppressed collagen I/III and α-SMA expression in TGF-β1-stimulated UFs. miR-486-overexpressed BMSCs alleviated urethral fibrosis, collagen deposition, and epithelial injury in the urethral tissue of US rats. miR-486 targeted and negatively regulated Col13a1 in US rats. In conclusion, overexpression of miR-486 in BMSCs targets Col13a1 and attenuates urethral fibrosis in TGF-β1-triggered UFs and US rats.

Decreased Angiopoietin Expression in Underactive Bladder Induced by Long-term Bladder Outlet Obstruction.

Ischemia of the bladder can occur if neovascular formation cannot keep pace with hypoxia induced by chronic bladder outlet obstruction (BOO). The aim of this study was to examine changes in angiogenesis growth factor expression generated by chronic BOO in a rat model of underactive bladder. Twenty female Sprague-Dawley rats aged 6 weeks were assigned to 4 groups (5 rats per group). Group 1 was the control. Group 2 underwent sham surgery. The rats in groups 3 and 4 underwent BOO and were followed up for 1 week and 8 weeks. Cystometry was carried out together with bladder tissue analysis at 1 week and 8 weeks postoperatively. Real-time polymerase chain reaction (PCR) assays were conducted to determine the expression level of angiogenesis-related growth factors. A hypoxia signaling pathway PCR array was additionally carried out. The group that underwent BOO for 8 weeks showed abnormal bladder function, with a diminished intercontraction interval, decreased maximal voiding pressure, and higher volume of residual urine (P<0.05). Hypoxia-inducible factor-1 alpha expression was elevated in this group. The expression levels of vascular endothelial growth factor (VEGF) and VEGF receptor messenger RNA (mRNA) in the BOO group were comparable to those in the control group. However, angiotensin/tie receptor mRNA expression levels increased at 1 week after BOO, but decreased at 8 weeks after BOO. In animals that underwent BOO, fewer blood vessels exhibited positive immunofluorescent staining for von Willebrand factor. Alterations were also seen in the hypoxia signaling pathway PCR array. In a rat model of underactive bladder caused by surgical BOO, reduced angiopoietin expression was demonstrated. This observation might underlie visceral ischemia and fibrosis associated with the procedure. The findings of this study might offer an improved understanding of the disease processes underlying BOO and facilitate selection of the appropriate time to repair the organ in this condition.

Portosinusoidal Vascular Disorder: A Heretofore Unrecognized Manifestation of Sickle Cell Disease?

Portosinusoidal vascular disorder (PSVD) is a recently proposed histopathologic entity that encompasses a spectrum of often subtle hepatic microvascular lesions and related microarchitectural abnormalities. Clinical manifestations may arise years after histologic diagnosis and include extrahepatic portal vein thrombosis and portal hypertension. While the histopathologic features of PSVD have been associated with numerous clinical conditions, most notably prothrombotic/vasculopathic disorders, PSVD has not yet been described in sickle cell disease. This gap is striking given the central role of microvascular dysfunction in sickle cell disease and well-described patterns of hepatic injury and dysfunction in this population. This case series is the first to explore the prevalence and pathogenesis of PSVD in sickle cell disease. Forty-one diagnostically adequate liver biopsies from patients with sickle cell disease were identified across the archives of 5 tertiary medical centers. All biopsies exhibited at least 1 histopathologic feature associated with PSVD (mean 3.8 features/case). Overall, 90.2% of patients met the criteria for a diagnosis of PSVD based on the presence of specific histopathologic and/or clinical findings. Immunohistochemical stains for von Willebrand factor, CD34, and glutamine synthetase were performed on 36 cases (87.8%). Aberrant (centrilobular sinusoidal) CD34 and von Willebrand factor staining was present in 97.2% and 86.1% of cases, respectively. Glutamine synthetase reactivity was at least mildly decreased in zone 3 hepatocytes in 52.8% of cases. We posit that chronic erythrocyte sickling results in dysfunction and remodeling of the portal microvasculature, culminating in regression of zone 3 hepatocytes. The presence of PSVD may explain, at least in part, the hepatic dysfunction observed in this patient population. These patients may also benefit from extended clinical surveillance for portal hypertension and other complications. While subtle and prone to overdiagnosis, the features of PSVD should be carefully considered when interpreting liver biopsies from patients with sickle cell disease.

Protective effect of Kaempferol on endothelial cell injury in glucocorticoid induced osteonecrosis of the femoral head

To explore the effect of Kaempferol on bone microvascular endothelial cells (BMECs) in glucocorticoid induced osteonecrosis of the femoral head (GIONFH) in vitro. BMECs were isolated from cancellous bone of femoral head or femoral neck donated voluntarily by patients with femoral neck fracture. BMECs were identified by von Willebrand factor and CD31 immunofluorescence staining and tube formation assay. The cell counting kit 8 (CCK-8) assay was used to screen the optimal concentration and the time point of dexamethasone (Dex) to inhibit the cell activity and the optimal concentration of Kaempferol to improve the inhibition of Dex. Then the BMECs were divided into 4 groups, namely, the cell group (group A), the cells treated with optimal concentration of Dex group (group B), the cells treated with optimal concentration of Dex+1 μmol/L Kaempferol group (group C), and the cells treated with optimal concentration of Dex+5 μmol/L Kaempferol group (group D). EdU assay, in vitro tube formation assay, TUNEL staining assay, Annexin Ⅴ/propidium iodide (PI) staining assay, Transwell migration assay, scratch healing assay, and Western blot assay were used to detect the effect of Kaempferol on the proliferation, tube formation, apoptosis, migration, and protein expression of BMECs treated with Dex. The cultured cells were identified as BMECs. CCK-8 assay showed that the optimal concentration and the time point of Dex to inhibit cell activity was 300 μmol/L for 24 hours, and the optimal concentration of Kaempferol to improve the inhibitory activity of Dex was 1 μmol/L. EdU and tube formation assays showed that the cell proliferation rate, tube length, and number of branch points were significantly lower in groups B-D than in group A, and in groups B and D than in group C ( P<0.05). TUNEL and Annexin V/PI staining assays showed that the rates of TUNEL positive cells and apoptotic cells were significantly higher in groups B-D than in group A, and in groups B and D than in group C ( P<0.05). Scratch healing assay and Transwell migration assay showed that the scratch healing rate and the number of migration cells were significantly lower in groups B-D than in group A, and in groups B and D than in group C ( P<0.05). Western blot assay demonstrated that the relative expressions of Cleaved Caspase-3 and Bax proteins were significantly higher in groups B-D than in group A, and in groups B and D than in group C ( P<0.05); the relative expressions of matrix metalloproteinase 2, Cyclin D1, Cyclin E1, VEGFA, and Bcl2 proteins were significantly lower in groups B-D than in group A, and in groups B and D than in group C ( P<0.05). Kaempferol can alleviate the damage and dysfunction of BMECs in GIONFH.

Abstract TP146: The Effect Of Statins On Thrombus Composition Retrieved By Mechanical Thrombectomy In Acute Ischemic Stroke

Introduction: Ample evidence exists in support of the anti-inflammatory properties of statins which in turn could affect arterial thrombosis. Compositional analysis of thrombi retrieved by MT may provide us with knowledge useful for evaluating the effect of current Statin therapy on thrombus composition. Methods: Consecutive AIS-LVO patients were included. Histopathological evaluation of thrombi retrieved during MT was performed using Martius Scarlet Blue staining (MSB) and immunohistochemistry for von Willebrand Factor (vWF) and anti-citrulinated H3 (CitH3; NETs marker) and the composition of thrombi was determined using image analysis software. Thrombus was considered rich in a given composition if that composition was higher than its overall mean. We reviewed patient’s medication records and cataloged any statin therapy prior to MT. Results: 117 patients were included in this study. The mean density of red blood cells (RBCs), white blood cells (WBCs), platelets (PLT), fibrin (FIB), vWF and NETs (CitH3) in thrombi was 43.4%, 3.7%, 23.6%, 29.3%, 14.3% and 21.1% respectively. Twenty-seven patients were taking Statins at the time of MT. Statins were not associated with pre-thrombectomy IV-tPA, and stroke etiology (p values: &gt;0.05). There was no association between Statins and thrombi composition and type (p values: &gt;0.05 for all thrombus components and type). Additionally, Statins were not associated with MT outcomes including number of device passes and final mTICI score. Conclusion: Pre-stroke Statin therapy was not associated with differences in thrombus composition.

Differences in Vasa Vasorum Distribution in Human Aortic Aneurysms and Atheromas.

The pathophysiological difference between aortic atheromas and aneurysms is unknown. We focused on the vasa vasorum (VV), which play a critical role in maintaining aortic homeostasis and are also involved in vascular diseases. We investigated the differences in VV between the atheromas and aneurysms. Human abdominal aortic samples were obtained from patients with abdominal aortic aneurysm during surgery or autopsy cases. Autopsy cases were divided into 2 groups according to atheromas. The VV were evaluated using immunohistochemical staining for von Willebrand factor. Intimal VV increased in both the atheroma and aneurysm groups, medial VV increased, and adventitial VV decreased only in the aneurysm group. We also observed that the medial VV were connected to the adventitial VV in the atheroma group and to intimal VV in the aneurysm group. We suggest the outside-in VV or inside-out VV theories. Atheroma induces hypoxia of aortic walls, and angiogenic factors might induce an increase of intimal VV derived from adventitial VV (outside-in VV). However, adventitial VV decrease induces hypoxia of aortic walls, and angiogenic factors might induce an increase of intimal VV derived from aortic lumen (inside-out VV). These differences of VV may contribute in elucidating the pathophysiology of aortic diseases.

The Role of Von Willebrand Factor in the Pathogenesis of Pulmonary Vascular Thrombosis in COVID-19.

The increased plasma levels of von Willebrand factor (VWF) in patients with COVID-19 was reported in many studies, and its correlation with disease severity and mortality suggest its important role in the pathogenesis of thrombosis in COVID-19. We performed histological and immunohistochemical studies of the lungs of 29 patients who died from COVID-19. We found a significant increase in the intensity of immunohistochemical reaction for VWF in the pulmonary vascular endothelium when the disease duration was more than 10 days. In the patients who had thrombotic complications, the VWF immunostaining in the pulmonary vascular endothelium was significantly more intense than in nonsurvivors without thrombotic complications. Duration of disease and thrombotic complications were found to be independent predictors of increased VWF immunostaining in the endothelium of pulmonary vessels. We also revealed that bacterial pneumonia was associated with increased VWF staining intensity in pulmonary arterial, arteriolar, and venular endothelium, while lung ventilation was an independent predictor of increased VWF immunostaining in arterial endothelium. The results of the study demonstrated an important role of endothelial VWF in the pathogenesis of thrombus formation in COVID-19.

Correlation of Neutrophil to Lymphocyte Ratio with Expression of Neutrophil Extracellular Traps Within Stroke Emboli.

It has been hypothesized that circulating neutrophils have a direct correlation with the composition of emboli in acute ischemic stroke (AIS). The aim of this study is to evaluate the association between neutrophil-lymphocyte ratio (NLR) in peripheral blood and the expression of neutrophil extracellular traps (NETs) within stroke emboli. Consecutive patients with acute ischemic stroke (AIS) due to large vessel occlusion (LVO) that underwent mechanical thrombectomy (MT) were included. Patients were divided into two groups based on NLR median value. Retrieved thrombi were histologically analyzed using Martius Scarlett Blue (MSB) for main thrombus components including red blood cells (RBCs), white blood cells (WBCs), fibrin and platelet. Immunohistochemistry staining for von Willebrand Factor (vWF) and anti-citrullinated H3 (H3Cit; NETs marker) was also performed. Samples from a total of 84 patients were included. The average percentage of RBCs, WBCs, fibrin, platelet, H3Cit, and vWF components in thrombi were 45.1%, 3.5%, 21.8%, 29.6%, 19.7% and 14.8% respectively. When stratifying by NLR group [low (≤3.94) versus high (>3.95)], high NLR group had significantly more WBCs (4.5%), fibrin (24.2%), H3Cit (22.7%) and vWF (17.1%) thrombus fractions compared to low NLR group. Additionally, RBC content (38.8%) was lower in the high NLR group. NLR is correlated with the amounts of WBCs, fibrin, NETs and vWF within the thrombi retrieved from AIS patients due to LVO.

Diverse thrombus composition in thrombectomy stroke patients with longer time to recanalization

Background and purposeDelayed time to recanalization is associated with reduced recanalization success of mechanical thrombectomy (MT) and thrombolysis in acute ischemic stroke (AIS). The reasons for this are unclear. We hypothesized that alterations in thrombus structure and composition could be responsible for this. MethodsRetrieved thrombi from AIS patients who underwent MT less than 8 h from symptom onset to groin puncture (SOGP) were evaluated. Patients were divided into early (≤4 h.) vs delayed (> 4 h) groups based SOGP timing. Thrombi were histologically analysed using Martius Scarlett Blue and immunohistochemistry staining for von Willebrand Factor (vWF), anti-citrullinated H3 (H3Cit; NETs [neutrophil extracellular traps] marker). We used inferential statistics including, t-test, artificial neural network (ANN) to interpret the data. ResultsA total of 137 thrombi were collected. The overall average percentage of red blood cells (RBC), white blood cells (WBC), platelet, fibrin, H3Cit, and vWF components in thrombi was 45.83%, 3.58%, 22.23%, 28.27%, 19.97% and 16.23% respectively. Delayed group had higher WBCs, (p = 0.02), fibrin (p = 0.02), H3Cit (p = 0.04) and vWF (p = 0.03) thrombus fractions compared to early group. Based on ANN model, the most important factors for predicting the number of passes required for successful recanalization are fibrin and RBC contents of the thrombus followed by vWF and H3Cit contents. ConclusionsLonger time to recanalization was associated with increased WBCs, fibrin, H3Cit and vWF fractions of thrombi reflecting possible in situ maturation of thrombus components. Increased fibrin, NETs and vWF composition may reduce likelihood of revascularization by altering thrombus mechanical properties.

Characterizing the host response to rhPDGF-BB in a rat spinal arthrodesis model.

BackgroundDue to the constraints surrounding autograft bone, surgeons have turned to osteoinductive agents to augment spinal fusion. Reports of complications and questionable efficacy slowed the adoption of these alternatives. Recombinant human platelet‐derived growth factor B homodimer (rhPDGF‐BB) has been Food and Drug Administration (FDA)‐approved (Augment) to promote fusion in other areas of orthopedics, but its characterization in spine fusion has not yet been tested. The purpose of this study is to characterize the host response to PDGF‐BB in vivo.MethodsEighty female Fischer rats underwent L4‐5 posterolateral fusion using one of four implant types: (a) iliac crest syngeneic allograft harvested from syngeneic donors, (b) β‐TCP/bovine collagen matrix (β‐TCP/Col) with sodium acetate buffer, (c) β‐TCP/Col with 0.3 mg/mL “low dose,” or (d) β‐TCP/Col with 3.0 mg/mL “high dose” of rhPDGF‐BB. Animals underwent magnetic resonance imaging (MRI) and serum cytokine quantification at 4, 7, 10, and 21 days, postoperatively. Tissues were processed for immunofluorescence staining for Ki67 and von Willebrand factor (vWF) to assess neovascularization.ResultsMRI demonstrated no differences in fluid accumulation among the four treatment groups at any of the time points. Serum cytokine analysis showed no clinically significant differences between treatment groups in 20 of the 27 cytokines. Inflammatory cytokines IFN‐γ, IL‐1β, IL‐18, MCP‐1, MIP‐1α, TNF‐α were not induced by rhPDGF‐BB. Histology showed no differences in cell infiltration, and Ki67 and vWF immunofluorescence staining was similar among groups.ConclusionsrhPDGF‐BB delivered with a β‐TCP/Col matrix exerts no exaggerated systemic or local host inflammatory response when compared to iliac crest syngeneic allograft bone or the control carrier. rhPDGF‐BB mixed with a β‐TCP/Col matrix could be a viable and safe biologic alternative to syngeneic allograft in spine fusion. Further studies need to be performed to evaluate efficacy in this setting.

OC15.06: *Endothelial damage and inflammation cell signalling triggered by pre‐eclampsia versus COVID‐19 in pregnancy

To study endothelial damage and alteration in signalling pathways in endothelial cells exposed to sera from pregnant women with PE and COVID-19 in vitro. Sera samples were obtained from pregnant women with COVID-19 infection classified into mild (n=10) or severe (n=9) in addition to normotensive pregnancies as controls (n=10) and patients with PE (n=13). Endothelial cells were obtained from the human dermal microvascular endothelial cell line. Nuclei and deposits of ICAM-1and Von Willebrand factor (VWF) staining was performed, and micrographs were captured by fluorescent microscopy. The area covered by fluorescent ICAM-1 or VWF labelling was calculated and expressed as the average fold increase compared to controls. The effect of sera samples on the inflammation cell signalling pathways was assessed by the quantification of phospho-P38MAPK. Both COVID-19 and PE induced an overexpression of ICAM-1 and VWF on endothelial cells although the effect of pre-eclampsia was less pronounced than the one triggered by severe COVID-19 (p < 0.05). Exposure of endothelial cells to the sera of severe COVID-19 and PE had the capacity to activate inflammatory signalling pathways. Immunoblots showed an increase in the degree of phosphorylation of p38MAPK being both severe COVID-19 and PE statistically different from controls (p < 0.05) (figure 1). Both COVID-19 and PE trigger endothelial damage and induce the activation of inflammation cell signalling pathways in endothelial cells. The effect on cell adhesion and prothrombosis of severe COVID-19 is more pronounced than PE. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Mitochondrial Fission-Mediated Lung Development in Newborn Rats With Hyperoxia-Induced Bronchopulmonary Dysplasia With Pulmonary Hypertension.

Background: Bronchopulmonary dysplasia (BPD) is the most common chronic respiratory disease in premature infants. Oxygen inhalation and mechanical ventilation are common treatments, which can cause hyperoxia-induced lung injury, but the underlying mechanism is not yet understood. Mitochondrial fission is essential for mitochondrial homeostasis. The objective of this study was to determine whether mitochondrial fission (dynamin-related protein 1, Drp1) is an important mediator of hyperoxia lung injury in rats.Methods: The animal model of BPD was induced with high oxygen (80–85% O2). Pulmonary histological changes were observed by hematoxylin-eosin (HE) staining. Pulmonary microvessels were observed by immunofluorescence staining of von Willebrand Factor (vWF). Protein expression levels of Drp1 and p-Drp1 (Ser616) were observed using Western Blot. We used echocardiography to measure pulmonary artery acceleration time (PAT), pulmonary vascular resistance index (PVRi), peak flow velocity of the pulmonary artery (PFVP), pulmonary arteriovenous diameter, and pulmonary vein peak velocity. Mitochondrial division inhibitor-1 (Mdivi-1) was used as an inhibitor of Drp1, and administered through intraperitoneal injection (25 mg/kg).Results: Pulmonary artery resistance of the hyperoxide-induced neonatal rat model of BPD increased after it entered normoxic convalescence. During the critical stage of alveolar development in neonatal rats exposed to high oxygen levels for an extended period, the expression and phosphorylation of Drp1 increased in lung tissues. When Drp1 expression was inhibited, small pulmonary vessel development improved and PH was relieved.Conclusion: Our study shows that excessive mitochondrial fission is an important mediator of hyperoxia-induced pulmonary vascular injury, and inhibition of mitochondrial fission may be a useful treatment for hyperoxia-induced related pulmonary diseases.

MicroRNA Sequencing of CD34+ Sorted Adipose Stem Cells Undergoing Endotheliogenesis.

While several microRNAs (miRNAs) that regulate the endotheliogenesis and further promote angiogenesis have been identified in various cancers, the identification of miRNAs that can drive the differentiation of adipose derived stromal/stem cells (ASCs) into the endothelial lineage has been largely unexplored. In this study, CD34+ ASCs sorted using magnetic bead separation were induced to differentiate along the endothelial pathway. miRNA sequencing of ASCs at day 3, 9, and 14 of endothelial differentiation was performed on Ion Proton sequencing system. The data obtained by this high-throughput method were aligned to the human genome HG38, and the differentially expressed miRNAs during endothelial differentiation at various time points (day 3, 9, and 14) were identified. The gene targets of the identified miRNAs were obtained through miRWalk database. The network-pathway analysis of miRNAs and their targets was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatic tools to determine the potential candidate miRNAs that promote endothelial differentiation. Based on these analyses, six upregulated miRNAs (miR-181a-5p, miR-330-5p, miR-335-3p, miR-15b-5p, miR-99a-5p, and miR-199a-5p) and six downregulated miRNAs (miR-145-5p, miR-155-5p, miR-193a-3p, miR-125a-5p, miR-221-5p, and miR-222-3p) were chosen for further studies. In vitro evaluation of these miRNAs to induce endothelial differentiation when transfected into CD34+ sorted ASCs was studied using Von Willebrand Factor (VWF) staining and quantitative real time-polymerase chain reaction (qRT-PCR). Our results suggest that miRNAs: 335-5p, 330-5p, 181a-5p and anti-miRNAs: 125a-5p, 145-5p can likely induce endothelial differentiation in CD34+ sorted ASCs. Further studies are clearly required to elucidate the specific mechanisms on how miRNAs or anti-miRNAs identified through bioinformatics approach can induce the endotheliogenesis in ASCs.

Mitomycin C induces pulmonary vascular endothelial-to-mesenchymal transition and pulmonary veno-occlusive disease via Smad3-dependent pathway in rats.

Pulmonary veno-occlusive disease (PVOD) is a rare disease characterized by the obstruction of small pulmonary veins leading to pulmonary hypertension. However, the mechanisms underlying pulmonary vessel occlusion remain largely unclear. A mitomycin C (MMC)-induced PVOD rat model was used as in vivo animal model, and primarily cultured rat pulmonary microvascular endothelial cells (PMVECs) were used as in vitro cell model. Our data suggested an endothelial-to-mesenchymal transition (EndoMT) may be present in the pulmonary microvessels isolated from either PVOD patients or MMC-induced PVOD rats. In comparison to the control vessels, vessels from both PVOD patients and PVOD rats had co-localized staining of specific endothelial marker von Willebrand factor (vWF) and mesenchymal marker α-smooth muscle actin (α-SMA), suggesting the presence of cells that co-express endothelial and mesenchymal markers. In both the lung tissues of MMC-induced PVOD rats and MMC-treated rat PMVECs there were decreased levels of endothelial markers (e.g. VE-cadherin and CD31) and increased mesenchymal markers (e.g. vimentin, fibronectin and α-SMA) were detected indicating EndoMT. Moreover, MMC-induced activation of the TGFβ/Smad3/Snail axis, while blocking this pathway with either selective Smad3 inhibitor (SIS3) or small interfering RNA (siRNA) against Smad3, dramatically abolished the MMC-induced EndoMT. Notably, treatment with SIS3 remarkably prevented the pathogenesis of MMC-induced PVOD in rats. Our data indicated that targeted inhibition of Smad3 leads to a potential, novel strategy for PVOD therapy, likely by inhibiting the EndoMT in pulmonary microvasculature.

Drug-eluting Vein Graft with Acetylsalicylic Acid-Ticagrelor-Unfractionated Heparin Complex Inhibits Early Graft Thrombosis

Background:Bypass graft surgery remains to be an important treatment option for left main and multivessel coronary artery disease. Approximately 2% of saphenous vein grafts are lost immediately after the coronary artery bypass graft operations and 12% in the first month due to thrombosis.Aims:To administer one anticoagulant and two antiplatelet agents in a way that locally affects the vein graft before the bypass operation and to thereby analyse their effects on early graft thrombosis.Study Design:Animal experimentation.Methods:Since ticagrelor was used locally for the first time in this study, its efficacy in combination with other drugs (acetylsalicylic acid, acetylsalicylic acid and ticagrelor, and acetylsalicylic acid + ticagrelor + unfractionated heparin) was examined on rats including control (untreated) and sham (pluronic gel) group (n=14 for each group). Before the tunica adventitia layer of the femoral veins was bypassed to the femoral artery, it was coated with the drug-eluting pluronic F-127 gel. The presence or absence of thrombus in the vein graft samples was recorded under light microscopy. In vein graft preparations where thrombus was detected, the thrombus area (μm2) was calculated using the Axiovision software. Immunohistochemical staining was performed with the anti-rat von Willebrand factor polyclonal antibody kit.Results:The number of preparations containing thrombus was significantly lower in the acetylsalicylic acid + ticagrelor + unfractionated heparin group than in the acetylsalicylic acid, control, and sham groups, according to the comparisons made on the 1st and 3rd days (p=0.001 and 0.02, respectively). von Willebrand factor staining was significantly lower in the acetylsalicylic acid + ticagrelor + unfractionated heparin group than in the other groups on the 3rd day (p=0.005).Conclusion:Locally effective acetylsalicylic acid-ticagrelor-unfractionated heparin complex has been shown to significantly reduce thrombus formation in vein grafts in this experimental model. Local administration of these drugs, which are routinely administered orally just before stent implantations, on the vein graft before the bypass is performed can prevent the loss of vein grafts due to thrombus, thereby reducing the mortality and morbidity of these patients.

Improved viability of murine skin flaps using a gelatin hydrogel sheet impregnated with bFGF.

Basic fibroblast growth factor (bFGF) promotes epithelial cell proliferation and angiogenesis but its clinical applications are limited by its short half-life and low retention. Recently developed gelatin hydrogel sheets able to release physiologically active substances in a controlled manner have the potential to overcome these issues. In this study, the effects of gelatin hydrogel sheets impregnated with bFGF on flap survival and angiogenesis were examined in a murine skin flap model. A flap of 1 × 3cm was generated on the backs of 60 C57BL/6 mice. The mice were divided into five groups (n = 12/group): Group I, untreated; Group II, treated with a gelatin hydrogel sheet impregnated with saline; Group III, treated with bFGF (50µg) without sheets; Groups IV and V, treated with gelatin hydrogel sheets impregnated with 50 and 100µg of bFGF, respectively. On the seventh day after surgery, the flap survival area and vascular network were examined and hematoxylin and eosin and von Willebrand factor staining were used for histological examinations. The flap survival areas were significantly larger in Groups IV and V than in other groups. The area of new vessels was significantly larger in Group IV than in the other groups. In the murine skin flap model, gelatin hydrogel sheets impregnated with bFGF promoted angiogenesis and improved flap survival. These findings support the use of bFGF-impregnated gelatin hydrogel sheets for improving ischemic flap survival in clinical settings.

Efficacy of Recombinant Human-Soluble Thrombomodulin for Severe Acute Pancreatitis in a Rat Experimental Model.

Early death in severe acute pancreatitis (SAP) is caused by pancreatic necrosis and multiple-organ failure due to microcirculation disorder. The aim of this study was to prove that recombinant human-soluble thrombomodulin (rTM) has therapeutic effects on SAP by preventing pancreatic necrosis and organ failure. Male Wister rats were used. Cerulein was administered intraperitoneally 4 times every 1 hour, and lipopolysaccharide was administered intraperitoneally 3 hours after. One hour after administration of lipopolysaccharide, rTM was injected intravenously. Rats were observed for 24 hours after starting the experiment, and the survival rate was evaluated. All surviving rats were killed, and the blood sample, liver, and pancreas were excised. Serum amylase, aspartate aminotransferase, alanine aminotransferase, and high mobility group box 1 were measured, and the liver and pancreas were examined histologically. For the evaluation of microcirculation, von Willebrand factor staining was performed. Serum amylase, aspartate aminotransferase, and alanine aminotransferase were significantly decreased. The survival rate was significantly improved to 100%. Moreover, serum high mobility group box 1 was decreased. Liver injury and pancreatic necrosis became less severe, and microcirculation was preserved histologically. Early administration of rTM prevents organ failure by maintenance of microcirculation and improves prognoses of SAP.

Release of endothelial activation markers in lungs of patients with malaria-associated acute respiratory distress syndrome

BackgroundMalaria-associated acute respiratory distress syndrome (MA-ARDS) is an understudied complication of malaria and is characterized by pulmonary inflammation and disruption of the alveolar-capillary membrane. Its pathogenesis remains poorly understood. Since endothelial activation plays an important role in other malarial complications, the expression of two endothelial activation markers, von Willebrand factor (VWF) and angiopoietin-2 (ANG-2), was investigated in the lungs of patients with MA-ARDS.MethodsPost-mortem lung sections of Plasmodium falciparum-infected patients without alveolar oedema (NA), P. falciparum-infected patients with alveolar oedema (MA-ARDS), and uninfected people who died accidentally with no pathological changes to the lungs (CON) were immunohistochemically stained for VWF and ANG-2, and were evaluated with semi-quantitative analysis.ResultsAlveolar oedematous VWF and ANG-2 and intravascular VWF staining were significantly increased in patients with MA-ARDS versus infected and uninfected control groups. The levels of VWF in the alveolar septa and endothelial lining of large blood vessels of patients with MA-ARDS was significantly decreased compared to controls. ANG-2 expression was increased in the alveolar septa of malaria patients without alveolar oedema versus control patients, while ANG-2+ leukocytes were increased in the alveoli in both infected patient groups.ConclusionsThis study documents a high level of VWF and ANG-2, two endothelial activation markers in the oedematous alveoli of post-mortem lung sections of Thai patients with MA-ARDS. Decreased detection of VWF in the endothelial lining of blood vessels, in parallel with an increased presence of intravascular VWF staining suggests marked endothelial activation and Weibel–Palade body release in the lungs of patients with MA-ARDS.

Disease Phenotype and Clonal Amplification in Calreticulin del52 and ins5 Knock-in Mice Are Dependent on the Type of Mutations and Gene Dosage

Introduction BCR-ABL-negative myeloproliferative neoplasms (MPNs) result from the transformation of a hematopoietic stem cell (HSC). Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). All CALR mutations induce a frameshift to the same alternative reading frame generating a new C-terminal tail. The two most frequent CALR mutations are a 52 bp deletion (del52) or type 1 and a 5 bp insertion (ins5) or type 2. In patients, del52 and ins5 are equally found in ET but del52 is more frequent in PMF. In mouse retroviral model, del52 mice progress from ET to myelofibrosis (MF) while ins5 mice remain mostly with an ET. Methods In order to study the effect of endogenous levels of del52 and ins5 in hematopoiesis, we generated conditional knock-in (KI) mice expressing the murine CALR del52 or ins5 with the human mutated C-terminal tail under the control of a Scl-driven tamoxifen-inducible Cre recombinase (Scl-CreERT). We have also used Ubi-GFP transgenic mice to perform competitive engraftments. Results After tamoxifen-induction, both del52 and ins5 KI mice developed a rapid thrombocytosis, more severe in the homozygous than the heterozygous setting. In contrast, leukocytosis was observed only in homozygous setting. At similar zygosity, del52 induced a higher thrombocytosis compared to ins5. After 10 months of induction, both the bone marrow (BM) and the spleen of homozygous del52 KI mice and, to a much lower extent of homozygous ins5 KI mice, presented a significant increase in megakaryocytes (MKs) and in MK progenitors by flow cytometry. Von Willebrand factor staining showed that both del52 and ins5 homozygous mice displayed giant polylobulated MKs, associated with a similar increase in ploidy (mean ploidy 32N-33N). Heterozygous del52 presented also an increase ploidy of MK (mean of 25N) compared to controls (mean of 17N), whereas the MK ploidy of heterozygous ins5 mice was similar to control mice. The increase in number and size of MKs in homozygous del52 mice partially explained the significant decrease in BM cellularity and the splenomegaly. Moreover, we observed a decrease in BM erythroblasts and, in spleen, an increase in both erythroblast and granulocytic precursors together with a decrease in lymphocytes associated with a major disorganization of white pulp territories. Thus, the del52 homozygous KI mice developed features of a MF-like disease further illustrated by the presence of reticulin fibers stained with silver, mainly in spleen. Presence of fibrosis was not as pronounced in heterozygous del52 mice and more rarely observed in spleen of homozygous ins5 KI mice. In homozygous del52 KI mice, there was a significant amplification of the HSC compartment in both BM and spleen that was stronger than in homozygous ins5 mice. To study whether del52 and ins5 could provide a competitive advantage to HSCs, we performed BM transplantation with increasing percentages of non-induced homozygous del52 or ins5 cells with wild-type GFP+ cells into lethally-irradiated recipient mice. The homozygous del52 BM cells strongly competed wild-type hematopoiesis from an initial engraftment as low as 10% of mutated clones, reaching 100% in both blood myeloid cells and BM HSC compartments at 4 months. In contrast, out-competition of wild-type hematopoiesis by homozygous ins5 cells was slower, especially when less than 50% of mutated cells were initially engrafted suggesting that del52 provides a stronger advantage to the HSCs than ins5. Conclusion In conclusion, these results demonstrate that modeling CALRdel52 and ins5 mutations in mice can successfully recapitulate the differences in phenotype observed in patients, i.e del52 KI mice recapitulate an ET progressing to MF while ins5 KI mice only mimic an ET. This might be explained by a more profound effect of del52 than ins5 at the level of HSC. These KI mice offer solid in vivo models to investigate the mechanism of action of both types of mutations on HSCs and MKs and will be used to test new therapeutic approaches. Disclosures: No relevant conflicts of interest to declare. Disclosures Constantinescu: AlsaTech: Other: Co-Founde; Wiley &amp; Sons: Other: Editor in Chief, Journal of Cellular and Molecular Medicine; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AgenDix GmbH: Other: Co-Founder, MyeloPro Research and Diagnostics.