Endothelial Aggregation Research Articles

"Prognosis and Complications of Hyperuricemia Patients Undergoing Isolated Carotid Endarterectomy"

Hyperuricemia is associated with deleterious effects on endothelial dysfunction, oxidative metabolism, platelet adhesiveness, hemorheology, and aggregation

Endothelial protection and anti-platelet aggregation activities of Ficus tsiangii extracts

Endothelial protection and anti-platelet aggregation activities of Ficus tsiangii extracts

Abstract 511: Mef2 Transcription Factors are Essential Regulators of Endothelial Morphology and Functions

Background: Laminar shear stress on the endothelium promotes atheroprotecive gene expression. In vitro experiments support a model of laminar shear stress activating the Mef2 transcription factors that in turn induce transcription of Klf2 and Klf4, which regulate many anti-inflammatory and anti-thrombotic genes. However, this model has not been tested in vivo. Three Mef2 transcription factors (Mef2a, -c, and -d) are expressed in the endothelium. To understand their functions and test this model, we generated mice with inducible, endothelial-specific deletions of Mef2c, Mef2a/c, and Mef2a/c/d. Methods & Results: We previously reported that endothelial Mef2c inhibits the formation of endothelial actin stress fibers and migration of smooth muscle cells across the internal elastic lamina into the intima. Combined deletion of endothelial Mef2a/c produced a similar phenotype. Neither of these deletions affected survival or altered the levels of Klf2 or Klf4. However, combined deletions of Mef2a/c/d led to death 10-14 days after induction. Pulmonary hemorrhage was consistently observed as was variable amounts in other organs. En face imaging of the aorta and vena cava revealed a substantial increase in endothelial cell density and proliferation. The aortic endothelium displayed extensive actin stress fibers but the overall organization was not substantially changed. However, the vena cava was disorganized with endothelial cell aggregation. Transcriptome analysis showed ≥ 2-fold alterations in expression of 894 genes, with many important for endothelial function. Notably, Klf2 and Klf4 were both decreased in Mef2a/c/d deficient aortic endothelium to a similar extent as the Mef2s. This is consistent with the phenotypic similarity of Klf2/4 and Mef2a/c/d deletions. Comparison of genes altered by Klf2/4 and Mef2a/c/d deletions revealed that 37% of Mef2-dependent genes are also Klf2/4-dependent. Conclusions: Together, these data support a model in which Mef2 transcription factors redundantly regulate endothelial expression of Klf2 and Klf4 in response to shear stress to promote atheroprotective gene expression. They further regulate expression of many genes important for endothelial function independently of Klf2/4.

Abstract 4252: Establishment of a multicellular 3D cell culture model for tumor - endothelial cell interaction

Abstract Introduction: Due to the increasing understanding of the mechanisms relevant to the genesis of cancer, we are experiencing a transition from disease to target-oriented therapy. One major hurdle for the development of these targeted therapeutic regimens, however, is the limited availability of predictive in vitro models. The critical challenge is to develop culture models better reflecting in vivo conditions. We present data that highlights the differences of RNA expression of in vivo like 3D microtissues consisting of tumour cells, fibroblasts and two different endothelial cell lines compared to normal 2D cell culture conditions. Methods: 96-well hanging drop microtiter plates (InSphero AG, Zürich, Switzerland) were applied for the production of 3D mono-, co- and tri-cultures including the human lung cancer cell lines A549 or Colo699 alone or in combination with a human lung fibroblast cell line (SV-80) and either a human umbilical vein endothelial cell line (HUVEC) or the primary human lung microvascular endothelial cell line (HMVEC-L). In addition, to conventional histology (H&E), tumour endothelial spheroid aggregation was displayed immunohistochemically (IHC) by protein expression of e-cadherin, CD31, von Willebrand factor (vWF) and α-muscle actin (α-SMA). RNA expression profiling by Affymetrix chip analysis was performed for multicellular 3D microtissues and 2D cultured cell lines. Results: Endothelial cells aggregated in coherent tube like structures preferentially in the fibroblast consisting core of all microtissues. Furthermore, endothelial cells expressed α-SMA only in microtissues that consisted both of fibroblasts and tumour cells indicating an interaction between these two cell types. RNA expression profiles revealed a high number of regulated genes in tri-cultures when compared to microtissues only consisting of mono- or co-cultures or to traditional 2D cultivated cells. Regulated genes played an important role either in cell cycle, organelle fission, wound healing and DNA packing. Interestingly, no difference in the RNA expression was displayed in microtissues containing either immortalized or primary endothelial cells, Finally, a relation of RNA expression between our cell culture model and patient data was identified. Conclusion: We demonstrate that cultivation of cells as multicellular microtissues in a 3D environment led not only to a difference in RNA but also in protein expression due to cell - cell interactions. Our data support the importance of performing complex co-culture for investigating tumour stroma interactions. Citation Format: Arno Amann, Marit Zwierzina, Johann Kern, Gabriele Gamerith, Stefan Koeck, Edith Lorenz, Johannes Rainer, Heinz Zwierzina. Establishment of a multicellular 3D cell culture model for tumor - endothelial cell interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4252.

0593. Reelin mediates the human neutrophil peptide-induced endothelial dysfunction and platelet aggregation

Atherosclerosis is an inflammatory disease with fundamental primitive events including endothelial dysfunction and platelet aggregation [1]. Human neutrophil peptides (HNP) are the most abundant cationic proteins in neutrophils and are released into the extracellular milieu upon activation [2]. HNP have previously been detected in atherosclerotic lesions, and are in high concentrations in blood of patients with acute coronary syndrome [2]. We have previously demonstrated that HNP can induce foam cell formation, platelet aggregation and leukocyte recruitment through the LRP8 signaling pathway [3], but there is no direct interaction between HNP and LRP8.

Histidine-rich glycoprotein prevents septic lethality through neutrophil regulation

Although there have been many clinical trials for treatment of sepsis, no drugs are available at present. Sepsis is thought to be complex disorders; activation/lesion of vascular endothelial cells, accelerated coagulation and platelet aggregation, leukocyte activation or paralysis, and hypercytokinemia. To treat the pathological status of sepsis, it must be necessary to control these plural disorders simultaneously or sequentially. In the present study, we identified and characterized a plasma protein histidine-rich glycoprotein (HRG) as a factor that decreases dramatically in septic condition and maintains neutrophil's shape and functional quiescence. We clarified the involvement of HRG in septic pathogenesis and propose a novel therapy for sepsis based on that.

Endothelial Differentiation Gene-1, a New Downstream Gene Is Involved in RTEF-1 Induced Angiogenesis in Endothelial Cells

Related Transcriptional Enhancer Factor-1 (RTEF-1) has been suggested to induce angiogenesis through regulating target genes. Whether RTEF-1 has a direct role in angiogenesis and what specific genes are involved in RTEF-1 driven angiogenisis have not been elucidated. We found that over-expressing RTEF-1 in Human dermal microvascular endothelial cells-1 (HMEC-1) significantly increased endothelial cell aggregation, growth and migration while the processes were inhibited by siRNA of RTEF-1. In addition, we observed that Endothelial differentiation gene-1 (Edg-1) expression was up-regulated by RTEF-1 at the transcriptional level. RTEF-1 could bind to Edg-1 promoter and subsequently induce its activity. Edg-1 siRNA significantly blocked RTEF-1-driven increases in endothelial cell aggregation in a Matrigel assay and retarded RTEF-1-induced endothelial cell growth and migration. Pertussis Toxin (PTX), a Gi/Go protein sensitive inhibitor, was found to inhibit RTEF-1 driven endothelial cell aggregation and migration. Our data demonstrates that Edg-1 is a potential target gene of RTEF-1 and is involved in RTEF-1-induced angiogenesis in endothelial cells. Gi/Go protein coupled receptor pathway plays a role in RTEF-1 driven angiogenesis in endothelial cells.

Top Differential Diagnosis Should Be Microscopic Polyangiitis in ANCA-Positive Patient with Diffuse Pulmonary Hemorrhage and Hemosiderosis.

A rat model of antineutrophil cytoplasmic antibody (ANCA) associated vasculitides reveals crescentic glomerulonephritis as seen in human renal biopsies and diffuse lung hemorrhage that is not well documented in human lung biopsies. A 64-year-old male, with shortness of breath and mild elevation of serum creatinine, was found to have a positive serum test for ANCA, but negative antiglomerular basement membrane antibody. A renal biopsy showed pauci-immune type of crescentic glomerulonephritis and focal arteritis. The prior lung wedge biopsy was retrospectively reviewed to show diffuse hemorrhage and hemosiderosis with focal giant cells. In addition, small arteries revealed subtle neutrophil aggregation, and margination along vascular endothelium, but no definitive vasculitis. The pathology of ANCA associated vasculitides results from activated neutrophils by ANCA and subsequent activation of the alternative complement cascade with endothelial injury, neutrophil aggregation and margination. Our findings, after the correlation between lung biopsy and renal biopsy, imply that the top differential diagnosis in the lung biopsy should be microscopic polyangiitis when diffuse pulmonary hemorrhage and hemosiderosis are present in this ANCA-positive patient.

Histone-Induced Lung Injury and Coagulation Activation Are Reduced By C-Reactive Protein In The Acute Phase Response

Whilst histones are essential building blocks within cells in forming the basis of chromatin, their extracellular effects can be extremely toxic. We have shown in in vivo models with translational relevance to patients with severe trauma that circulating histones increase mortality through acute lung injury with multifocal alveolar hemorrhage, thrombi formation and severe lung edema. from patients within 4 hours of trauma exhibited histone-specific endothelial cell toxicity when histone levels reached 50 µg/ml. threshold was associated with increased interleukin (IL)-6 levels (r=0.55, p < 0.01) and thrombin-antithrombin (TAT) levels. However, serum from trauma patients at timepoints beyond 6 hours no longer exhibited histone-specific toxicity despite elevated histone levels. These samples were found to have increasing levels of C reactive protein (CRP), which were low until 4-6 hours post trauma. To examine if CRP could be neutralising the toxicity of circulating histones, in vitro culture systems demonstrate that CRP release is linked to extracellular histones as histones induce leucocytes to release pre-synthesized IL-6, which in turn induces the release of CRP by hepatocytes. a biosensor and gel overlay assays, CRP interacts with all individual histones and forms. Functionally, CRP (100 µg/ml) significantly reduces histone-induced endothelial cell damage, permeability increase and platelet aggregation by ∼50% (p < 0.05), which is confirmed in vivo as CRP (10 mg/kg) inhibits endothelial damage [reduced circulating sTM by 1 fold (p < 0.05)], vascular permeability [reduced Lung wet/dry weight ratio to control levels (p < 0.05)], coagulation activation [reduced circulating TAT by 1 fold (p < 0.05)] and thrombocytopenia [increased platelet/HCT ratio by 2 folds (p < 0.05)]. Histological examination showed that CRP-infusion reduced lung edema, hemorrhage and thrombosis in mice challenged by a lethal dose of histones (75 mg/kg) to rescue these mice., induction of the acute phase response using casein reduced histone-induced pathological changes and protected mice challenged with a lethal dose of histones. trauma patients, CRP–histone complexes could be detected and was responsible for the loss of histone-specific cytotoxicity as addition of exogenous CRP to clinical samples with high histone but low CRP levels abolished the cytotoxic effects. The histone-specific manner was similar to the effect of incubating the patient samples with an anti-histone antibody. In conclusion, elevation in circulating histones following extensive cell death results in an acute phase reaction that involves CRP in neutralising further histone-induced cytotoxicity., this is a new specific role described for human CRP amidst its properties in generic host defence. Clinically, this CRP response lags behind the histone surge in patients and a time-critical period therefore exists for potential clinical interventions using anti-histone reagents. Disclosures: No relevant conflicts of interest to declare.

Efficacy of Anagrelide Compared with Hydroxyurea in Patients with Essential Thrombocythemia.

The essential thrombocythemia (ET) is characterized by thrombohemorrhagic risk. ET patients receiving HU and anti-aggregants show normal platelets but present platelet coagulant and endothelial aggregation. Anagrelide (ANA) is a platelet-lowering drug that inhibits platelet aggregation. Therefore, we evaluated platelets, platelet factor 4 (PF4), prothrombin fragment 1+2 (F1+2), thrombin antithrombin complex (TAT) and d-dimer (DD) and von Willebrand factor (vWF) as markers of platelet coagulant and endothelial aggregation, respectively, in ET patients treated either with HU or ANA. All measurements were performed before treatment and when complete response, defined as platelet < 400 x109/L for more than 1 month, was achieved. Prolonged thrombocytosis causes thrombohemorrhagic risk. Hence, we investigated the cytoreduction time. The study comprised 36 patients (19 men, 17 women; mean age 65 years) who fulfilled PVSG and WHO. The mean duration of disease was 6 years. 17 patients received HU and 19 patients were on ANA. The average dose of HU was 0.5 g/d. ANA was initially administered at a dose of 0.5 mg/d. The dose was increased by 0.5 mg/d every week until the platelets had decreased to below 400 x 109/L. The average maintenance was 1.7 mg/d. All patients were on aspirin at dose of 100 mg/d and 50 mg/d in HU and ANA group, respectively. None of the patients had acquired or inherited thrombotic risk factors or had inherited coagulopathy. Eleven out of 17 HU patients (5 men, 6 women; mean age 68.6 years) developed thrombosis including six transient ischemic attack (TIA), three myocardial infarct (MI) and two deep venous thrombosis (DVT). The thrombosis occurred at a median time from diagnosis of 2 years. The ANA group did not experience thrombosis. Platelets were determined by automated analyser. PF4, F1+2, TAT and DD and vWF were assayed by ELISA and immunoturbidimetric assay, respectively. Before treatment, all patients had high platelets (1027±297 x 109/L) and elevated PF4, F1+2, TAT and DD (134±48 IU/mL vs 6±2 IU/mL, 3±2 nmol/L vs 0.6±0.2 nmol/L, 25±34 μg/L vs 2.7±1 μg/L, 224±172 μg/L vs 197±43 μg/L, respectively) (p< 0.0001, p<0.0001, p<0.0001 and p<0.0001, respectively) and low vWF (23±9% vs 102±30%) (p<0.0001). After treatment all patients had platelets <400 x 109/L (364 ±77 x 109/L), whereas PF4, F1+2, TAT and DD were higher (147±45 IU/mL, 3±2 nmol/L, 24±41 μg/L and 264±291 μg/L, respectively) and vWF was lower (34±9.9%) in HU group than controls (p<0.0001, p<0.0001, p<0.0001, p=0.001 and p<0.0001, respectively) and normal in ANA group (7±3 IU/mL, 1.1±0.8 nmol/L, 2.4±1.1 μg/L, 121±84 μg/L and 95±37%, respectively). The mean cytoreduction time was 3.2 and 1.8 months in HU and ANA group, respectively. A correlation there was between PF4 and F1+2 and TAT and DD and vWF (p=0.004, p=0.005, p=0.022 and p<0.0001, respectively). vWF did not correlate with F1+2, TAT and DD. Platelets did not correlate with thrombosis, whereas a correlation there was between PF4, F1+2, TAT and DD and thrombosis (p<0.0001, p=0.015, p=0.005 and p=0.034, respectively). A difference was observed in cytoreduction time between HU and ANA (p<0.0001). These results might support the superiority of ANA as antithrombotic drug besides its platelet-lowering effect.

Harnsäure, ein kardiovaskulärer Risikofaktor?

Serum uric acid represents an important, independent risk factor for cardiovascular and renal disease in patients with hypertension, heart failure, or diabetes. Elevated serum uric acid is highly predictive of mortality in patients with heart failure or coronary artery disease and of cardiovascular events in patients with diabetes. Although the mechanism(s) by which uric acid may play a pathogenetic role in cardiovascular disease is unclear, hyperuricemia is associated with deleterious effects on endothelial dysfunction, oxidative metabolism, platelet adhesiveness, hemrheology, and aggregation. Whether a reduction in uric acid impacts CV and renal disease remains to be determined. However, recent findings from LIFE in hypertensive patients with LVH suggest the possibility that a treatment-induced decrease in serum uric acid may indeed attenuate cardiovascular risk. Almost one third of the treatment benefit of a losartan-based versus atenolol-based therapy on the composite endpoint (death, myocardial infarction, or stroke) may be ascribed to differences in achieved serum uric acid levels. Clearly, randomized clinical trials are needed to investigate further the long-term cardioprotective benefits issue of reducing hyperuricemia in hypertensive patients.

Fibrin Plugging as a Cause of Microcirculatory Occlusion During Photodynamic Therapy

An experimental system that allows the white light observation of rapid changes in vessels without disturbance by red laser light was used. Mice were injected with mono-L-aspartyl chlorin-e-6 (Npe-6) i.v. via the tail vein and were immediately exposed to laser light. White emboli were observed forming on the inside of the vessel walls within seconds after commencement of light exposure. Emboli adhered to vessel walls and caused vascular obstruction. Light microscopy of the exposed material using fibrin staining was performed. Electron microscopy on the same material was also carried out. The embolisation time was influenced by both drug dose and laser power. With low laser power, it took a long time to stop the blood flow. Fibrin staining revealed the white emboli to be composed of fibrin. Electron microscopy findings revealed damage to endothelial cells and platelet aggregation. This study suggests that two main mechanisms (direct cellular damage and vascular shut-down ) might actually be complementary and synergistic in the production of vascular lesions using photodynamic therapy.

Trypsin-induced aggregation of bovine pulmonary artery endothelial cells cultured on microcarriers

We studied adherence between ‘luminal’ surfaces of pulmonary artery endothelial cells by standard aggregometry techniques, widely used for measuring aggregation of platelets and granulocytes. Using suspensions of bovine pulmonary artery endothelial cells cultured on microcarrier beads, in an aggregometer, we found that trypsin caused endothelial aggregation. The aggregation response occurred at trypsin concentrations as low as 0.001%. The degree of trypsin-induced aggregation indicated by the magnitude of the change in light transmission through the endothelial suspensions was related to the trypsin concentration, reaching a maximum level at trypsin concentrations of 0.01%. We conclude that trypsin, even in very low concentrations, causes adherence between ‘luminal’ surfaces of pulmonary endothelial cells probably because the enzyme destroys cell surface proteins which are necessary to prevent intercellular adherence. The method we describe may be useful for studying cell-cell interactions of endothelium.