Extracellular Granzyme K Modulates Angiogenesis by Regulating Soluble VEGFR1 Release From Endothelial Cells

Angiogenesis is crucial for normal development and homeostasis, but also plays a role in many diseases including cardiovascular diseases, autoimmune diseases, and cancer. Granzymes are serine proteases stored in the granules of cytotoxic cells, and have predominantly been studied for their pro-apoptotic role upon delivery in target cells. A growing body of evidence is emerging that granzymes also display extracellular functions, which largely remain unknown. In the present study, we show that extracellular granzyme K (GrK) inhibits angiogenesis and triggers endothelial cells to release soluble VEGFR1 (sVEGFR1), a decoy receptor that inhibits angiogenesis by sequestering VEGF-A. GrK does not cleave off membrane-bound VEGFR1 from the cell surface, does not release potential sVEGFR1 storage pools from endothelial cells, and does not trigger sVEGFR1 release via protease activating receptor-1 (PAR-1) activation. GrK induces de novo sVEGFR1 mRNA and protein expression and subsequent release of sVEGFR1 from endothelial cells. GrK protein is detectable in human colorectal tumor tissue and its levels positively correlate with sVEGFR1 protein levels and negatively correlate with T4 intratumoral angiogenesis and tumor size. In conclusion, extracellular GrK can inhibit angiogenesis via secretion of sVEGFR1 from endothelial cells, thereby sequestering VEGF-A and impairing VEGFR signaling. Our observation that GrK positively correlates with sVEGFR1 and negatively correlates with angiogenesis in colorectal cancer, suggest that the GrK-sVEGFR1-angiogenesis axis may be a valid target for development of novel anti-angiogenic therapies in cancer.

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Novel Treatment of Hypertension by Specifically Targeting E2F for Restoration of Endothelial Dihydrofolate Reductase and eNOS Function Under Oxidative Stress

Hypertension ◽

10.1161/hypertensionaha.118.11643 ◽

2019 ◽

Vol 73 (1) ◽

pp. 179-189 ◽

Cited By ~ 7

Author(s):

Hong Li ◽

Qiang Li ◽

Yixuan Zhang ◽

Wenting Liu ◽

Bo Gu ◽

...

Keyword(s):

Blood Pressure ◽

Endothelial Cells ◽

Protein Expression ◽

Dihydrofolate Reductase ◽

Promoter Activity ◽

Molecular Mechanisms ◽

Therapeutic Option ◽

Protein Levels ◽

Treatment Of Hypertension ◽

Mrna And Protein Expression

We have shown that hydrogen peroxide (H 2 O 2 ) downregulates tetrahydrobiopterin salvage enzyme DHFR (dihydrofolate reductase) to result in eNOS (endothelial NO synthase) uncoupling and elevated blood pressure. Here, we aimed to delineate molecular mechanisms underlying H 2 O 2 downregulation of endothelial DHFR by examining transcriptional pathways hypothesized to modulate DHFR expression and effects on blood pressure regulation of targeting these novel mechanisms. H 2 O 2 dose and time dependently attenuated DHFR mRNA and protein expression and enzymatic activity in endothelial cells. Deletion of E2F-binding sites, but not those of Sp1 (specificity protein 1), abolished H 2 O 2 attenuation of DHFR promoter activity. Overexpression of E2F1/2/3a activated DHFR promoter at baseline and alleviated the inhibitory effect of H 2 O 2 on DHFR promoter activity. H 2 O 2 treatment diminished mRNA and protein expression of E2F1/2/3a, whereas overexpression of E2F isoforms increased DHFR protein levels. Chromatin immunoprecipitation assay indicated direct binding of E2F1/2/3a to the DHFR promoter, which was weakened by H 2 O 2 . E2F1 RNA interference attenuated DHFR protein levels, whereas its overexpression elevated tetrahydrobiopterin levels and tetrahydrobiopterin/dihydrobiopterin ratios in vitro and in vivo. In Ang II (angiotensin II)–infused mice, adenovirus-mediated overexpression of E2F1 markedly abrogated blood pressure to control levels, by restoring endothelial DHFR function to improve NO bioavailability and vasorelaxation. Bioinformatic analyses confirmed a positive correlation between E2F1 and DHFR in human endothelial cells and arteries, and downregulation of both by oxidized phospholipids. In summary, endothelial DHFR is downregulated by H 2 O 2 transcriptionally via an E2F-dependent mechanism, and that specifically targeting E2F1/2/3a to restore DHFR and eNOS function may serve as a novel therapeutic option for the treatment of hypertension.

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von Willebrand factor biosynthesis and partitioning between constitutive and regulated pathways of secretion after thrombin stimulation

Blood ◽

10.1182/blood.v73.3.706.706 ◽

1989 ◽

Vol 73 (3) ◽

pp. 706-711 ◽

Cited By ~ 32

Author(s):

T Mayadas ◽

DD Wagner ◽

PJ Simpson

Keyword(s):

Endothelial Cells ◽

Von Willebrand Factor ◽

De Novo ◽

Umbilical Vein ◽

Protein Levels ◽

Storage Pool ◽

Umbilical Vein Endothelial Cells ◽

Von Willebrand ◽

Willebrand Factor ◽

Specific Mrna

Abstract The major part of von Willebrand factor (vWf) synthesized in cultured endothelial cells is secreted constitutively without stimulation and consists of all multimeric forms of vWf. In contrast, stimulation with secretagogues such as thrombin results in the release of vWf from the storage pool, the Weibel-Palade bodies which contain only the largest, most biologically potent multimeric forms of vWf. We wished to determine whether the signal for release of vWf might also function as a signal for replenishment of the vWf by enhancing de novo biosynthesis and if replenishment of the vWf storage pool involved a diversion of newly synthesized vWf from the constitutive pathway to the regulated pathway. vWf mRNA and protein levels in unstimulated human umbilical vein endothelial cells were compared with cells that were briefly stimulated with 1 U/mL thrombin for 15 minutes and then incubated without thrombin for periods up to 72 hours. A comparison was also made between unstimulated cells and cells continuously exposed to thrombin for up to 48 hours. Thrombin stimulation, brief or continuous, had no significant effect on subsequent biosynthesis of vWf protein or vWf- specific mRNA. Since thrombin releases vWf only from the storage pool, we examined the possibility of diversion of newly synthesized vWf from the constitutive pathway to the regulated pathway. Cells were pulse- labeled, incubated for 15 minutes with and without thrombin, chased for various periods in unlabeled media, and briefly restimulated with thrombin. No significant redistribution of vWf between the two pathways was observed as a result of thrombin stimulation for the time periods tested.

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Regulation of Interleukin-8 Expression in Human Endometrial Endothelial Cells: A Potential Mechanism for the Pathogenesis of Endometriosis

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2004-1813 ◽

2005 ◽

Vol 90 (3) ◽

pp. 1805-1811 ◽

Cited By ~ 37

Author(s):

Janelle Luk ◽

Yasemin Seval ◽

Umit A. Kayisli ◽

Murat Ulukus ◽

Cagnur E. Ulukus ◽

...

Keyword(s):

Endothelial Cells ◽

Sex Steroids ◽

Cycle Phase ◽

Eutopic Endometrium ◽

Protein Levels ◽

Cytokine Levels ◽

Mrna And Protein Expression ◽

The Relationship

The elevation of the proinflammatory chemoattractant cytokine levels in ectopic and eutopic endometrium of endometriosis implies an inflammatory basis for this disease. The relationship between endothelial cells and leukocytes is likely to be important in the regulation of inflammatory mediators of endometriosis. The aim of this study was to describe the temporal and spatial expression of IL-8 in human endometrial endothelial cells (HEEC) in vivo and to compare the in vitro regulation of IL-8 expression by sex steroids in HEEC from women with or without endometriosis. Eutopic endometrial tissues and endometriosis implants were grouped according to menstrual cycle phase and examined by immunohistochemistry for IL-8 expression. Endothelial cells of endometriotic implants expressed higher IL-8 immunoreactivity compared with endothelial cells of eutopic endometrium from women with or without endometriosis (P < 0.02). For in vitro studies, HEEC were isolated from women with or without endometriosis and grown to preconfluence. The purity of cultured HEEC (90–95%) was confirmed by immunocytochemistry using endothelium-specific markers, CD31 and CD146. The effects of estradiol (5 × 10−8m), progesterone (10−7m), or both on IL-8 mRNA and protein levels were analyzed by RT-PCR and ELISA, respectively. Sex steroids reduced the expression of IL-8 mRNA and protein in HEEC from women without endometriosis. In contrast, both estradiol and progesterone stimulated IL-8 mRNA and protein expression in HEEC from women with endometriosis. We postulate that the stimulation of chemokine expression by sex steroids in HEEC of women with endometriosis may play a role in the inflammatory aspect of this disease.

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von Willebrand factor biosynthesis and partitioning between constitutive and regulated pathways of secretion after thrombin stimulation

Blood ◽

10.1182/blood.v73.3.706.bloodjournal733706 ◽

1989 ◽

Vol 73 (3) ◽

pp. 706-711 ◽

Cited By ~ 1

Author(s):

T Mayadas ◽

DD Wagner ◽

PJ Simpson

Keyword(s):

Endothelial Cells ◽

Von Willebrand Factor ◽

De Novo ◽

Umbilical Vein ◽

Protein Levels ◽

Storage Pool ◽

Umbilical Vein Endothelial Cells ◽

Von Willebrand ◽

Willebrand Factor ◽

Specific Mrna

The major part of von Willebrand factor (vWf) synthesized in cultured endothelial cells is secreted constitutively without stimulation and consists of all multimeric forms of vWf. In contrast, stimulation with secretagogues such as thrombin results in the release of vWf from the storage pool, the Weibel-Palade bodies which contain only the largest, most biologically potent multimeric forms of vWf. We wished to determine whether the signal for release of vWf might also function as a signal for replenishment of the vWf by enhancing de novo biosynthesis and if replenishment of the vWf storage pool involved a diversion of newly synthesized vWf from the constitutive pathway to the regulated pathway. vWf mRNA and protein levels in unstimulated human umbilical vein endothelial cells were compared with cells that were briefly stimulated with 1 U/mL thrombin for 15 minutes and then incubated without thrombin for periods up to 72 hours. A comparison was also made between unstimulated cells and cells continuously exposed to thrombin for up to 48 hours. Thrombin stimulation, brief or continuous, had no significant effect on subsequent biosynthesis of vWf protein or vWf- specific mRNA. Since thrombin releases vWf only from the storage pool, we examined the possibility of diversion of newly synthesized vWf from the constitutive pathway to the regulated pathway. Cells were pulse- labeled, incubated for 15 minutes with and without thrombin, chased for various periods in unlabeled media, and briefly restimulated with thrombin. No significant redistribution of vWf between the two pathways was observed as a result of thrombin stimulation for the time periods tested.

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The Increased Sensitivity of Platelets to Prostacyclin in Marathon Runners

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661107 ◽

1984 ◽

Vol 51 (03) ◽

pp. 385-387 ◽

Cited By ~ 8

Author(s):

Clive J Dix ◽

David G Hassall ◽

K Richard Bruckdorfer

Keyword(s):

Cardiovascular Disease ◽

Endothelial Cells ◽

Platelet Aggregation ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

Platelet Rich Plasma ◽

Marathon Runners ◽

Inhibition Of Platelet Aggregation ◽

Increased Sensitivity ◽

Membrane Bound

SummaryPlatelet-rich plasma was obtained 24 hr after the race ended from athletes who ran in the London marathon. The platelets were only marginally less sensitive to adrenaline than were those of non-runners using conventional aggregation tests. However, the runners’ platelets were much more sensitive to inhibition by prostacyclin, a prostaglandin synthesized by endothelial cells. It appeared that this effect was due to a greater activity in the platelets of the membrane-bound adenylate cyclase enzyme which generates intracellular cyclic AMP. Cyclic AMP production is known to be stimulated by prostacyclin and to cause the inhibition of platelet aggregation. The results indicate another possible protective effect of exercise against cardiovascular disease which is independent of the known changes in lipoprotein concentrations previously observed in athletes.

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Platelets Stimulate Thromboplastin Synthesis in Human Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657323 ◽

1983 ◽

Vol 49 (02) ◽

pp. 069-072 ◽

Cited By ~ 20

Author(s):

U L H Johnsen ◽

T Lyberg ◽

K S Galdal ◽

H Prydz

Keyword(s):

Endothelial Cells ◽

De Novo ◽

Umbilical Vein ◽

Time Dependent ◽

De Novo Synthesis ◽

Human Endothelial Cells ◽

Tumor Promotor ◽

Coagulation Assay ◽

Platelet Aggregates ◽

Umbilical Vein Endothelial Cells

SummaryHuman umbilical vein endothelial cells in culture synthesize thromboplastin upon stimulation with phytohaemagglutinin (PHA) or the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The thromboplastin activity is further strongly enhanced in a time dependent reaction by the presence of gel-filtered platelets or platelet aggregates. This effect was demonstrable at platelet concentrations lower than those normally found in plasma, it may thus be of pathophysiological relevance. The thromboplastin activity increased with increasing number of platelets added. Cycloheximide inhibited the increase, suggesting that de novo synthesis of the protein component of thromboplastin, apoprotein III, is necessary.When care was taken to remove monocytes no thromboplastin activity and no apoprotein HI antigen could be demonstrated in suspensions of gel-filtered platelets, platelets aggregated with thrombin or homogenized platelets when studied with a coagulation assay and an antibody neutralization technique.

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Delivery of Therapeutic Agents to the Central Nervous System and the Promise of Extracellular Vesicles

Pharmaceutics ◽

10.3390/pharmaceutics13040492 ◽

2021 ◽

Vol 13 (4) ◽

pp. 492

Author(s):

Charlotte A. René ◽

Robin J. Parks

Keyword(s):

Central Nervous System ◽

Endothelial Cells ◽

Nervous System ◽

Blood Brain Barrier ◽

Extracellular Vesicles ◽

Therapeutic Agents ◽

Target Cells ◽

Significant Barrier ◽

The Central Nervous System ◽

Delivery Vehicles

The central nervous system (CNS) is surrounded by the blood–brain barrier (BBB), a semipermeable border of endothelial cells that prevents pathogens, solutes and most molecules from non-selectively crossing into the CNS. Thus, the BBB acts to protect the CNS from potentially deleterious insults. Unfortunately, the BBB also frequently presents a significant barrier to therapies, impeding passage of drugs and biologicals to target cells within the CNS. This review provides an overview of different approaches to deliver therapeutics across the BBB, with an emphasis in extracellular vesicles as delivery vehicles to the CNS.

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The “Vesicular Intelligence” Strategy of Blood Cancers

Genes ◽

10.3390/genes12030416 ◽

2021 ◽

Vol 12 (3) ◽

pp. 416

Author(s):

Dorian Forte ◽

Martina Barone ◽

Francesca Palandri ◽

Lucia Catani

Keyword(s):

Clonal Evolution ◽

Biological Properties ◽

Hemopoietic Stem Cell ◽

Target Cells ◽

Cancer Cell Growth ◽

Cell Compartment ◽

Blood Cancer ◽

Non Invasive ◽

Membrane Bound ◽

Immune Potential

Blood cancers are a heterogeneous group of disorders including leukemia, multiple myeloma, and lymphoma. They may derive from the clonal evolution of the hemopoietic stem cell compartment or from the transformation of progenitors with immune potential. Extracellular vesicles (EVs) are membrane-bound nanovesicles which are released by cells into body fluids with a role in intercellular communication in physiology and pathology, including cancer. EV cargos are enriched in nucleic acids, proteins, and lipids, and these molecules can be delivered to target cells to influence their biological properties and modify surrounding or distant targets. In this review, we will describe the “smart strategy” on how blood cancer-derived EVs modulate tumor cell development and maintenance. Moreover, we will also depict the function of microenvironment-derived EVs in blood cancers and discuss how the interplay between tumor and microenvironment affects blood cancer cell growth and spreading, immune response, angiogenesis, thrombogenicity, and drug resistance. The potential of EVs as non-invasive biomarkers will be also discussed. Lastly, we discuss the clinical application viewpoint of EVs in blood cancers. Overall, blood cancers apply a ‘vesicular intelligence’ strategy to spread signals over their microenvironment, promoting the development and/or maintenance of the malignant clone.

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EXTH-51. ANTI-INVASIVE EFFICACY AND SURVIVAL BENEFIT OF THE YAP-TEAD INHIBITOR VERTEPORFIN IN PRECLINICAL GLIOBLASTOMA MODELS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.405 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii98-ii98

Author(s):

Anne Marie Barrette ◽

Alexandros Bouras ◽

German Nudelman ◽

Zarmeen Mussa ◽

Elena Zaslavsky ◽

...

Keyword(s):

Survival Benefit ◽

De Novo ◽

Epithelial Mesenchymal Transition ◽

Intraperitoneal Administration ◽

Standard Of Care ◽

Tumor Burden ◽

Mesenchymal Transition ◽

Protein Levels

Abstract Glioblastoma (GBM) remains an incurable disease, in large part due to its malignant infiltrative spread, and current clinical therapy fails to target the invasive nature of tumor cells in disease progression and recurrence. Here, we use the YAP-TEAD inhibitor Verteporfin to target a convergence point for regulating tumor invasion/metastasis and establish the robust anti-invasive therapeutic efficacy of this FDA-approved drug and its survival benefit across several preclinical glioma models. Using patient-derived GBM cells and orthotopic xenograft models (PDX), we show that Verteporfin treatment disrupts YAP/TAZ-TEAD activity and processes related to cell adhesion, migration and epithelial-mesenchymal transition. In-vitro, Verteporfin impairs tumor migration, invasion and motility dynamics. In-vivo, intraperitoneal administration of Verteporfin in mice with orthotopic PDX tumors shows consistent drug accumulation within the brain and decreased infiltrative tumor burden, across three independent experiments. Interestingly, PDX tumors with impaired invasion after Verteporfin treatment downregulate CDH2 and ITGB1 adhesion protein levels within the tumor microenvironment. Finally, Verteporfin treatment confers survival benefit in two independent PDX models: as monotherapy in de-novo GBM and in combination with standard-of-care chemoradiation in recurrent GBM. These findings indicate potential therapeutic value of this FDA-approved drug if repurposed for GBM patients.

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Cyclooxygenase-2 Glycosylation Is Affected by Peroxynitrite in Endothelial Cells: Impact on Enzyme Activity and Degradation

Antioxidants ◽

10.3390/antiox10030496 ◽

2021 ◽

Vol 10 (3) ◽

pp. 496

Author(s):

Sonia Eligini ◽

Susanna Colli ◽

Aida Habib ◽

Giancarlo Aldini ◽

Alessandra Altomare ◽

...

Keyword(s):

Endothelial Cells ◽

De Novo ◽

Cyclooxygenase 2 ◽

Nitric Oxide Donors ◽

Metabolic Labeling ◽

Dependent Manner ◽

De Novo Synthesis ◽

Human Endothelial Cells ◽

Prolonged Incubation ◽

Cox 2

The exposure of human endothelial cells to 3-morpholinosydnonimine (SIN-1) induced the expression of cyclooxygenase-2 (COX-2) in a dose- and time-dependent manner. Interestingly, after a prolonged incubation (>8 h) several proteoforms were visualized by Western blot, corresponding to different states of glycosylation of the protein. This effect was specific for SIN-1 that generates peroxynitrite and it was not detected with other nitric oxide-donors. Metabolic labeling experiments using 35S or cycloheximide suggested that the formation of hypoglycosylated COX-2 was dependent on de novo synthesis of the protein rather than the deglycosylation of the native protein. Moreover, SIN-1 reduced the activity of the hexokinase, the enzyme responsible for the first step of glycolysis. The hypoglycosylated COX-2 induced by SIN-1 showed a reduced capacity to generate prostaglandins and the activity was only partially recovered after immunoprecipitation. Finally, hypoglycosylated COX-2 showed a more rapid rate of degradation compared to COX-2 induced by IL-1α and an alteration in the localization with an accumulation mainly detected in the nuclear membrane. Our results have important implication to understand the effect of peroxynitrite on COX-2 expression and activity, and they may help to identify new pharmacological tools direct to increase COX-2 degradation or to inhibit its activity.

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