scholarly journals Prolyl 3-Hydroxylase 2 Is a Molecular Player of Angiogenesis

2021 ◽  
Vol 22 (8) ◽  
pp. 3896
Author(s):  
Paola Pignata ◽  
Ivana Apicella ◽  
Valeria Cicatiello ◽  
Caterina Puglisi ◽  
Sara Magliacane Magliacane Trotta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Collagen Iv ◽  
Mitogen Activated Protein Kinase ◽  
Vegf Receptor ◽  
Loss Of Function ◽  
Type Iv ◽  
Umbilical Vein Endothelial Cells

Prolyl 3-hydroxylase 2 (P3H2) catalyzes the post-translational formation of 3-hydroxyproline on collagens, mainly on type IV. Its activity has never been directly associated to angiogenesis. Here, we identified P3H2 gene through a deep-sequencing transcriptome analysis of human umbilical vein endothelial cells (HUVECs) stimulated with vascular endothelial growth factor A (VEGF-A). Differently from many previous studies we carried out the stimulation not on starved HUVECs, but on cells grown to maintain the best condition for their in vitro survival and propagation. We showed that P3H2 is induced by VEGF-A in two primary human endothelial cell lines and that its transcription is modulated by VEGF-A/VEGF receptor 2 (VEGFR-2) signaling pathway through p38 mitogen-activated protein kinase (MAPK). Then, we demonstrated that P3H2, through its activity on type IV Collagen, is essential for angiogenesis properties of endothelial cells in vitro by performing experiments of gain- and loss-of-function. Immunofluorescence studies showed that the overexpression of P3H2 induced a more condensed status of Collagen IV, accompanied by an alignment of the cells along the Collagen IV bundles, so towards an evident pro-angiogenic status. Finally, we found that P3h2 knockdown prevents pathological angiogenesis in vivo, in the model of laser-induced choroid neovascularization. Together these findings reveal that P3H2 is a new molecular player involved in new vessels formation and could be considered as a potential target for anti-angiogenesis therapy.

scholarly journals Generation of a Novel In Vitro Model to Study Endothelial Dysfunction from Atherothrombotic Specimens

2021 ◽  
Author(s):  
Susan Gallogly ◽  
Takeshi Fujisawa ◽  
John D. Hung ◽  
Mairi Brittan ◽  
Elizabeth M. Skinner ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
In Vitro Model ◽  
Umbilical Vein ◽  
Coronary Syndrome ◽  
Coronary Endothelial Cells ◽  
Vitro Model ◽  
Umbilical Vein Endothelial Cells

Abstract Purpose Endothelial dysfunction is central to the pathogenesis of acute coronary syndrome. The study of diseased endothelium is very challenging due to inherent difficulties in isolating endothelial cells from the coronary vascular bed. We sought to isolate and characterise coronary endothelial cells from patients undergoing thrombectomy for myocardial infarction to develop a patient-specific in vitro model of endothelial dysfunction. Methods In a prospective cohort study, 49 patients underwent percutaneous coronary intervention with thrombus aspiration. Specimens were cultured, and coronary endothelial outgrowth (CEO) cells were isolated. CEO cells, endothelial cells isolated from peripheral blood, explanted coronary arteries, and umbilical veins were phenotyped and assessed functionally in vitro and in vivo. Results CEO cells were obtained from 27/37 (73%) atherothrombotic specimens and gave rise to cells with cobblestone morphology expressing CD146 (94 ± 6%), CD31 (87 ± 14%), and von Willebrand factor (100 ± 1%). Proliferation of CEO cells was impaired compared to both coronary artery and umbilical vein endothelial cells (population doubling time, 2.5 ± 1.0 versus 1.6 ± 0.3 and 1.2 ± 0.3 days, respectively). Cell migration was also reduced compared to umbilical vein endothelial cells (29 ± 20% versus 85±19%). Importantly, unlike control endothelial cells, dysfunctional CEO cells did not incorporate into new vessels or promote angiogenesis in vivo. Conclusions CEO cells can be reliably isolated and cultured from thrombectomy specimens in patients with acute coronary syndrome. Compared to controls, patient-derived coronary endothelial cells had impaired capacity to proliferate, migrate, and contribute to angiogenesis. CEO cells could be used to identify novel therapeutic targets to enhance endothelial function and prevent acute coronary syndromes.

scholarly journals Study of pro-angiogenic activity of astilbin on human umbilical vein endothelial cells in vitro and zebrafish in vivo

RSC Advances ◽  
2019 ◽  
Vol 9 (40) ◽  
pp. 22921-22930 ◽  
Author(s):  
Kongpeng Lv ◽  
Qin Ren ◽  
Xingyan Zhang ◽  
Keda Zhang ◽  
Jia Fei ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Angiogenic Activity ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

Pro-angiogenic activity of astilbin on endothelial cells in vitro and zebrafish in vivo.

Platelet factor 4 enhances generation of activated protein C in vitro and in vivo

Blood ◽  
2003 ◽  
Vol 102 (1) ◽  
pp. 146-151 ◽  
Author(s):  
Arne Slungaard ◽  
Jose A. Fernandez ◽  
John H. Griffin ◽  
Nigel S. Key ◽  
Janel R. Long ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Platelet Factor 4 ◽  
In Vivo Model ◽  
Platelet Factor ◽  
Umbilical Vein Endothelial Cells

Abstract Platelet factor 4 (PF4), an abundant platelet α-granule protein, accelerates in vitro generation of activated protein C (APC) by soluble thrombin/thrombomodulin (TM) complexes up to 25-fold. To test the hypothesis that PF4 similarly stimulates endothelium-associated TM, we assessed the influence of human PF4 on thrombin-dependent APC generation by cultured endothelial monolayers. APC generated in the presence of 1 to 100 μg PF4 was up to 5-fold higher than baseline for human umbilical vein endothelial cells, 10-fold higher for microvascular endothelial cells, and unaltered for blood outgrowth endothelial cells. In an in vivo model, cynomolgus monkeys (n = 6, each serving as its own control) were infused with either PF4 (7.5 mg/kg) or vehicle buffer, then with human thrombin (1.0 μg/kg/min) for 10 minutes. Circulating APC levels (baseline 3 ng/mL) peaked at 10 minutes, when PF4-treated and vehicle-treated animals had APC levels of 67 ± 5 ng/mL and 39 ± 2 ng/mL, respectively (P < .001). The activated partial thromboplastin time (APTT; baseline, 28 seconds) increased maximally by 27 ± 6 seconds in PF4-treated animals and by 9 ± 1 seconds in control animals at 30 minutes (P < .001). PF4-dependent increases in circulating APC and APTT persisted more than 2-fold greater than that of control's from 10 through 120 minutes (P ≤ .04). All APTT prolongations were essentially reversed by monoclonal antibody C3, which blocks APC activity. Thus, physiologically relevant concentrations of PF4 stimulate thrombin-dependent APC generation both in vitro by cultured endothelial cells and in vivo in a primate thrombin infusion model. These findings suggest that PF4 may play a previously unsuspected physiologic role in enhancing APC generation. (Blood. 2003;102:146-151)

Effect of hypoxia on adherence of granulocytes to endothelial cells in vitro

1994 ◽  
Vol 267 (3) ◽  
pp. H874-H879 ◽  
Author(s):  
A. Pietersma ◽  
N. De Jong ◽  
J. F. Koster ◽  
W. Sluiter
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Calcium Ionophore ◽  
Intercellular Adhesion Molecule ◽  
Protective Mechanism ◽  
Hypoxic Conditions ◽  
Umbilical Vein Endothelial Cells ◽  
Stimulation Of

The objective of this study was to investigate the effect of hypoxia on the adhesiveness of endothelial cells for granulocytes. Human umbilical vein endothelial cells (HUVEC) were exposed to a PO2 of 7.5 mmHg (1.0 kPa), and the adherence of granulocytes was assessed under continuous hypoxia by means of a hypoxic incubator room. After 2 h of hypoxia the adherence of granulocytes decreased to 50% of the normoxic control, which was not due to a decreased viability of the endothelial cells nor to an increased generation of the antiadhesive factors nitric oxide, prostacyclin, and adenosine. Hypoxia also had no effect on the expression of intercellular adhesion molecule (ICAM)-1 or ICAM-2 on the endothelium. Although the mechanism of the action of hypoxia on the adhesiveness of endothelial cells remains unclear as yet, our data suggest that HUVEC possess a protective mechanism that prevents granulocyte adherence to endothelial cells under extreme hypoxic conditions. The decreased adherence seems paradoxical to the in vivo situation for which the increased margination of granulocytes within the vascular compartment of the ischemic tissue has been observed. However, hypoxia did not impair the potential adhesiveness of HUVEC, since stimulation of endothelial cells under hypoxic conditions with calcium ionophore or lipopolysaccharide increased the adherence of granulocytes in a similar fashion as under normoxic conditions. We therefore conclude that the increased margination of granulocytes during ischemia may be accomplished by the additional stimulation of hypoxic endothelial cells.

scholarly journals Characterization of the biological effects of a novel protein kinase D inhibitor in endothelial cells

2010 ◽  
Vol 429 (3) ◽  
pp. 565-572 ◽  
Author(s):  
Ian M. Evans ◽  
Azadeh Bagherzadeh ◽  
Mark Charles ◽  
Tony Raynham ◽  
Chris Ireson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Umbilical Vein ◽  
Biological Effects ◽  
Camp Response Element Binding ◽  
Mitogen Activated Protein Kinase ◽  
Protein Kinase D ◽  
Vegf Receptor

VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.

Characterization of Gastrin-Induced Proangiogenic Effects In vivo in Orthotopic U373 Experimental Human Glioblastomas and In vitro in Human Umbilical Vein Endothelial Cells

2004 ◽  
Vol 10 (24) ◽  
pp. 8250-8265 ◽  
Author(s):  
Florence Lefranc ◽  
Tatjana Mijatovic ◽  
Véronique Mathieu ◽  
Sandrine Rorive ◽  
Christine Decaestecker ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

scholarly journals Flagellar Hook Protein FlgE Induces Microvascular Hyperpermeability via Ectopic ATP Synthase β on Endothelial Surface

2021 ◽  
Vol 11 ◽  
Author(s):  
Yuanyuan Li ◽  
Ying Shen ◽  
Yudan Zheng ◽  
Shundong Ji ◽  
Mengru Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Abdominal Cavity ◽  
Tube Formation ◽  
Atp Production ◽  
Endothelial Surface ◽  
Umbilical Vein Endothelial Cells

We previously demonstrated the immunostimulatory efficacy of Pseudomonas aeruginosa flagellar hook protein FlgE on epithelial cells, presumably via ectopic ATP synthases or subunits ATP5B on cell membranes. Here, by using recombinant wild-type FlgE, mutant FlgE (FlgEM; bearing mutations on two postulated critical epitopes B and F), and a FlgE analog in pull-down assay, Western blotting, flow cytometry, and ELISA, actual bindings of FlgE proteins or epitope B/F peptides with ATP5B were all confirmed. Upon treatment with FlgE proteins, human umbilical vein endothelial cells (HUVECs) and SV40-immortalized murine vascular endothelial cells manifested decreased proliferation, migration, tube formation, and surface ATP production and increased apoptosis. FlgE proteins increased the permeability of HUVEC monolayers to soluble large molecules like dextran as well as to neutrophils. Immunofluorescence showed that FlgE induced clustering and conjugation of F-actin in HUVECs. In Balb/c-nude mice bearing transplanted solid tumors, FlgE proteins induced a microvascular hyperpermeability in pinna, lungs, tumor mass, and abdominal cavity. All effects observed in FlgE proteins were partially or completely impaired in FlgEM proteins or blocked by pretreatment with anti-ATP5B antibodies. Upon coculture of bacteria with HUVECs, FlgE was detectable in the membrane and cytosol of HUVECs. It was concluded that FlgE posed a pathogenic ligand of ectopic ATP5B that, upon FlgE–ATP5B coupling on endothelial cells, modulated properties and increased permeability of endothelial layers both in vitro and in vivo. The FlgE-ectopic ATP5B duo might contribute to the pathogenesis of disorders associated with bacterial infection or ectopic ATP5B-positive cells.

Size-dependent intracellular immunotargeting of therapeutic cargoes into endothelial cells

Blood ◽  
2002 ◽  
Vol 99 (3) ◽  
pp. 912-922 ◽  
Author(s):  
Rainer Wiewrodt ◽  
Anu P. Thomas ◽  
Luca Cipelletti ◽  
Melpo Christofidou-Solomidou ◽  
David A. Weitz ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Practical Significance ◽  
Target Cells ◽  
Gene Therapies ◽  
Intracellular Targeting ◽  
Pulmonary Uptake ◽  
Umbilical Vein Endothelial Cells

Abstract Cell-selective intracellular targeting is a key element of more specific and safe enzyme, toxin, and gene therapies. Endothelium poorly internalizes certain candidate carriers for vascular immunotargeting, such as antibodies to platelet endothelial cell adhesion molecule 1 (PECAM–1). Conjugation of poorly internalizable antibodies with streptavidin (SA) facilitates the intracellular uptake. Although both small and large (100-nm versus 1000-nm diameter) anti-PECAM/SA–beta galactosidase (SA–β-gal) conjugates bound selectively to PECAM-expressing cells, only small conjugates showed intracellular accumulation of active β-gal. To study whether size of the conjugates controls the uptake, a series of anti-PECAM/SA and anti-PECAM/bead conjugates ranging from 80 nm to 5 μm in diameter were produced. Human umbilical vein endothelial cells and PECAM-transfected mesothelioma cells internalized 80- to 350-nm anti-PECAM conjugates, but not conjugates larger than 500 nm. Further, size controls intracellular targeting of active therapeutic cargoes in vitro and in vivo. Small anti-PECAM/DNA conjugates transfected target cells in culture 5-fold more effectively than their large counterpart (350- versus 4200-nm diameter). To evaluate the practical significance of the size-controlled subcellular addressing, we coupled glucose oxidase (GOX) to anti-PECAM and antithrombomodulin. Both types of conjugates had equally high pulmonary uptake after intravenous injection in mice, yet only small (200- to 250-nm), not large (600- to 700-nm), GOX conjugates caused profound oxidative vascular injury in the lungs, presumably owing to intracellular generation of H2O2. Thus, engineering of affinity carriers of specific size permits intracellular delivery of active cargoes to endothelium in vitro and in vivo, a paradigm useful for the targeting of drugs, genes, and toxins.

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