scholarly journals Role of the Tyrosine Phosphatase SHP-2 in Mediating Adrenomedullin Proangiogenic Activity in Solid Tumors

2021 ◽  
Vol 11 ◽  
Author(s):  
Romain Sigaud ◽  
Nadège Dussault ◽  
Caroline Berenguer-Daizé ◽  
Christine Vellutini ◽  
Zohra Benyahia ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tyrosine Phosphatase ◽  
Vascular Endothelial Cell ◽  
Tumor Vasculature ◽  
Cell Junctions ◽  
Cell Permeability ◽  
Tumor Xenograft ◽  
Dependent Manner

VE-cadherin is an essential adhesion molecule in endothelial adherens junctions, and the integrity of these complexes is thought to be regulated by VE-cadherin tyrosine phosphorylation. We have previously shown that adrenomedullin (AM) blockade correlates with elevated levels of phosphorylated VE-cadherin (pVE-cadherinY731) in endothelial cells, associated with impaired barrier function and a persistent increase in vascular endothelial cell permeability. However, the mechanism underlying this effect is unknown. In this article, we demonstrate that the AM-mediated dephosphorylation of pVE-cadherinY731 takes place through activation of the tyrosine phosphatase SHP-2, as judged by the rise of its active fraction phosphorylated at tyrosine 542 (pSHP-2Y542) in HUVECs and glioblastoma-derived-endothelial cells. Both pre-incubation of HUVECs with SHP-2 inhibitors NSC-87877 and SHP099 and SHP-2 silencing hindered AM-induced dephosphorylation of pVE-cadherinY731 in a dose dependent-manner, showing the role of SHP-2 in the regulation of endothelial cell contacts. Furthermore, SHP-2 inhibition impaired AM-induced HUVECs differentiation into cord-like structures in vitro and impeded AM-induced neovascularization in in vivo Matrigel plugs bioassays. Subcutaneously transplanted U87-glioma tumor xenograft mice treated with AM-receptors-blocking antibodies showed a decrease in pSHP-2Y542 associated with VE-cadherin in nascent tumor vasculature when compared to control IgG-treated xenografts.Our findings show that AM acts on VE-cadherin dynamics through pSHP-2Y542 to finally modulate cell-cell junctions in the angiogenesis process, thereby promoting a stable and functional tumor vasculature.

scholarly journals Tyrosine phosphorylation of DEP-1/CD148 as a mechanism controlling Src kinase activation, endothelial cell permeability, invasion, and capillary formation

Blood ◽  
2012 ◽  
Vol 120 (13) ◽  
pp. 2745-2756 ◽  
Author(s):  
Kathleen Spring ◽  
Catherine Chabot ◽  
Simon Langlois ◽  
Line Lapointe ◽  
Nguyen Thu Ngan Trinh ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tyrosine Phosphatase ◽  
Sh2 Domain ◽  
Cell Permeability ◽  
Dependent Manner ◽  
Kinase Activation ◽  
Capillary Formation ◽  
Downstream Signaling ◽  
Src Activation

Abstract DEP-1/CD148 is a receptor-like protein tyrosine phosphatase with antiproliferative and tumor-suppressive functions. Interestingly, it also positively regulates Src family kinases in hematopoietic and endothelial cells, where we showed it promotes VE-cadherin–associated Src activation and endothelial cell survival upon VEGF stimulation. However, the molecular mechanism involved and its biologic functions in endothelial cells remain ill-defined. We demonstrate here that DEP-1 is phosphorylated in a Src- and Fyn-dependent manner on Y1311 and Y1320, which bind the Src SH2 domain. This allows DEP-1–catalyzed dephosphorylation of Src inhibitory Y529 and favors the VEGF-induced phosphorylation of Src substrates VE-cadherin and Cortactin. Accordingly, RNA interference (RNAi)–mediated knockdown of DEP-1 or expression of DEP-1 Y1311F/Y1320F impairs Src-dependent biologic responses mediated by VEGF including permeability, invasion, and branching capillary formation. In addition, our work further reveals that above a threshold expression level, DEP-1 can also dephosphorylate Src Y418 and attenuate downstream signaling and biologic responses, consistent with the quiescent behavior of confluent endothelial cells that express the highest levels of endogenous DEP-1. Collectively, our findings identify the VEGF-dependent phosphorylation of DEP-1 as a novel mechanism controlling Src activation, and show this is essential for the proper regulation of permeability and the promotion of the angiogenic response.

scholarly journals VE-PTP maintains the endothelial barrier via plakoglobin and becomes dissociated from VE-cadherin by leukocytes and by VEGF

2008 ◽  
Vol 205 (12) ◽  
pp. 2929-2945 ◽  
Author(s):  
Astrid F. Nottebaum ◽  
Giuseppe Cagna ◽  
Mark Winderlich ◽  
Alexander C. Gamp ◽  
Ruth Linnepe ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tyrosine Phosphatase ◽  
Cell Permeability ◽  
Cell Contact ◽  
Vascular Endothelial ◽  
General Role ◽  
Cell Contacts ◽  
Factor Α ◽  
Endothelial Growth Factor

We have shown recently that vascular endothelial protein tyrosine phosphatase (VE-PTP), an endothelial-specific membrane protein, associates with vascular endothelial (VE)–cadherin and enhances VE-cadherin function in transfected cells (Nawroth, R., G. Poell, A. Ranft, U. Samulowitz, G. Fachinger, M. Golding, D.T. Shima, U. Deutsch, and D. Vestweber. 2002. EMBO J. 21:4885–4895). We show that VE-PTP is indeed required for endothelial cell contact integrity, because down-regulation of its expression enhanced endothelial cell permeability, augmented leukocyte transmigration, and inhibited VE-cadherin–mediated adhesion. Binding of neutrophils as well as lymphocytes to endothelial cells triggered rapid (5 min) dissociation of VE-PTP from VE-cadherin. This dissociation was only seen with tumor necrosis factor α–activated, but not resting, endothelial cells. Besides leukocytes, vascular endothelial growth factor also rapidly dissociated VE-PTP from VE-cadherin, indicative of a more general role of VE-PTP in the regulation of endothelial cell contacts. Dissociation of VE-PTP and VE-cadherin in endothelial cells was accompanied by tyrosine phoshorylation of VE-cadherin, β-catenin, and plakoglobin. Surprisingly, only plakoglobin but not β-catenin was necessary for VE-PTP to support VE-cadherin adhesion in endothelial cells. In addition, inhibiting the expression of VE-PTP preferentially increased tyrosine phosphorylation of plakoglobin but not β-catenin. In conclusion, leukocytes interacting with endothelial cells rapidly dissociate VE-PTP from VE-cadherin, weakening endothelial cell contacts via a mechanism that requires plakoglobin but not β-catenin.

Junctional Adhesion Molecule-1 Is Involved in bFGF-Induced Angiogenesis In Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 840-840
Author(s):  
Vesselina G. Cooke ◽  
Meghna U. Naik ◽  
William Skarnes ◽  
Ulhas P. Naik
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Blood Vessels ◽  
Adhesion Molecule ◽  
The Body ◽  
Cell Junctions ◽  
Fluorescein Angiogram ◽  
And Migration

Abstract Neovascularization is a multistep process that occurs in the body in both physiological and pathological conditions. We have recently shown that Junctional Adhesion Molecule-1 (JAM-1), a member of the Ig superfamily of molecules, is involved in endothelial cell adhesion and migration, leading to angiogenesis. In quiescent endothelial cells, JAM-1 is located at the cell-cell junctions where it forms a complex with integrin αvβ3. Upon treatment of the cells with growth factors, such as bFGF, JAM-1 dissociates from its complex with αvβ3 and redistributes to the cell surface. Blockage of the extracellular domain of JAM-1 inhibits bFGF-induced endothelial cell morphology, proliferation and angiogenesis. Additionally, functional knock-down of JAM-1 using the RNAi technique in endothelial cells showed decreased adhesion and migration of these cells, indicating a possible role for JAM-1 in angiogenesis. In this report, we show that JAM-1 has an important role in bFGF-induced angiogenesis in vivo. Here we present for the first time the generation JAM-1 knock-out mice, using the gene trap strategy. We have successfully confirmed the JAM-1 −/− genotype via Southern, Northern, and Western blot analyses. JAM-1 −/− mice are viable and do not seem to have any external abnormalities, except that they appear to be smaller in size. Retinal fluorescein angiogram revealed no evidence for morphological defects in the vasculature of JAM-1 −/− mice. To evaluate the role of JAM-1 in angiogenesis, we performed an aortic ring assay with both wild type and JAM-1−/− mice. Mouse thoracic aortas were harvested, cross-sectioned into rings of 1-mm thickness, and cultured in a three-dimensional Matrigel supplied with 50 ng/ml bFGF. Vascular sproutings were counted every other day for a period of 7 days at which time they were stained with crystal violet and photographed. Aortic rings from WT mice treated with bFGF showed a 2.8-fold increase in microvessel growth, compared to WT controls with no supplementation of bFGF. In contrast, microvessel sproutings in bFGF treated aortic rings from JAM-1 −/− mice were no more than the vessels in the WT control mice. These results suggest that JAM-1 may be important for bFGF induced angiogenesis. To further confirm the role of JAM-1 in angiogenesis, WT and JAM-1 −/− mice were injected in their flank region with Matrigel containing 80 ng/ml bFGF and 60 U/ml heparin. Two weeks after injection, Matrigel plugs were excised, embedded in paraffin, and the presence of blood vessels was visualized by H&E staining. Matrigel plugs from control WT mice that were not treated with bFGF showed no vascularization, while bFGF supplied Matrigel plugs from WT mice showed a robust vessel growth. Interestingly, bFGF-treated Matrigel plugs form JAM-1−/− mice failed to produce any blood vessels. These ex vivo and in vivo studies using JAM-1−/− mice suggest that JAM-1 has a unique and essential role in bFGF-induced angiogenesis.

The role of caveolin-1 in PCB77-induced eNOS phosphorylation in human-derived endothelial cells

2007 ◽  
Vol 293 (6) ◽  
pp. H3340-H3347 ◽  
Author(s):  
Eun Jin Lim ◽  
Eric J. Smart ◽  
Michal Toborek ◽  
Bernhard Hennig
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Dna Binding ◽  
Vascular Endothelial Cell ◽  
Vascular Endothelial ◽  
Human Vascular Endothelial Cell ◽  
Caveolin 1 ◽  
Enos Phosphorylation

Polychlorinated biphenyls (PCBs) may contribute to the pathology of atherosclerosis by activating inflammatory responses in vascular endothelial cells. Endothelial nitric oxide synthase (eNOS) is colocalized with caveolae and is a critical regulator of vascular homeostasis. PCBs may be proatherogenic by causing dysfunctional eNOS signaling. The objective of this study was to investigate the role of caveolin-1 in PCB-induced endothelial dysfunction with a focus on mechanisms associated with eNOS signaling. Cells derived from an immortalized human vascular endothelial cell line were treated with PCB77 to study nitrotyrosine formation through eNOS signaling. Phosphorylation studies of eNOS, caveolin-1, and kinases, such as Src, phosphatidylinositol 3-kinase (PI3K), and Akt, were conducted in cells containing either functional or small-interfering RNA-silenced caveolin-1 protein. We also investigated caveolin-1-regulated mechanisms associated with PCB-induced markers of peroxynitrite formation and DNA binding of NF-κB. Cellular exposure to PCB77 increased eNOS phosphorylation and nitric oxide production, as well as peroxynitrite levels. A subsequent PCB-induced increase in NF-κB DNA binding may have implications in oxidative stress-mediated inflammatory mechanisms. The activation of eNOS by PCB77 treatment was blocked by inhibitors of the Src/PI3K/Akt pathway. PCB77 also increased phosphorylation of caveolin-1, indicating caveolae-dependent endocytosis. Caveolin-1 silencing abolished both the PCB-stimulated Akt and eNOS phosphorylation, suggesting a regulatory role of caveolae in PCB-induced eNOS signaling. These findings suggest that PCB77 induces eNOS phosphorylation in endothelial cells through a Src/PI3K/Akt-dependent mechanism, events regulated by functional caveolin-1. Our data provide evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and toxicity induced by persistent environmental pollutants such as coplanar PCBs.

scholarly journals Low Concentration of S100A8/9 Promotes Angiogenesis-Related Activity of Vascular Endothelial Cells: Bridges among Inflammation, Angiogenesis, and Tumorigenesis?

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Changyou Li ◽  
Siyuan Li ◽  
Changkai Jia ◽  
Lingling Yang ◽  
Zicheng Song ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial Cell ◽  
Tumor Development ◽  
Inflammatory Cells ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Low Concentration ◽  
Huvec Cells

Previous studies showed that several members of the S100A family are involved in neovascularization and tumor development. This study checked whether low concentrations of S100A8 or S100A9 has any effect on the behaviour of vascular endothelial cells. A human umbilical vascular endothelial cell (HUVEC) line was used to measure vascular endothelial cell bioactivity related to angiogenesis, such as cell proliferation, migration, and vessel formation. In the low concentration range up to 10 μg/mL, either each alone or in combination, S100A8 and S100A9 proteins promoted proliferation of HUVEC cells in a dose-dependent manner. The presence of both proteins in culture showed additive effects over each single protein. Both proteins enhanced HUVEC cells to migrate across the transwell membrane and to form tube-like structures on the Matrigel surface. When mixed in Matrigel and injected subcutaneously in Balb/c mice, both proteins increased vessel development in the gel plugs. Microarray assay of HUVEC cells treated with 10 μg/mL S100A8 revealed that ribosome pathway, pathogenicEscherichia coliinfection pathway, apoptosis, and stress response genes were modulated by S100A8 treatment. We propose that S100A8 and S100A9 proteins from either infiltrating inflammatory cells or tumor cells play an important role in the interplay among inflammation, angiogenesis, and tumorigenesis.

scholarly journals Role of protein kinase C in tumor necrosis factor induction of endothelial cell urokinase-type plasminogen activator

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2608-2617 ◽  
Author(s):  
MJ Niedbala ◽  
M Stein-Picarella
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Plasminogen Activator ◽  
Matrix Remodeling ◽  
Dependent Manner ◽  
Pkc Inhibitors ◽  
Tnf Induction ◽  
Dose Dependent ◽  
Pai 1

Tumor necrosis factor (TNF) can promote endothelial cell transcription, synthesis, and secretion of urokinase plasminogen activator (uPA) augmenting extracellular matrix remodeling and influencing cellular differentiation. In this report, the role of the protein kinase C (PKC) pathway in mediating TNF induction of uPA in human umbilical vein endothelial cells is described. The PKC inhibitors (H-7, staurosporine, and calphostin C), but not HA-1004, inhibited TNF-induced uPA expression, synthesis, and secretion in a dose-dependent manner. Analysis of cell-free conditioned medium obtained from PKC inhibitor- treated cultures by micro-enzyme-linked immunosorbent assay methodologies using uPA- and plasminogen activator inhibitor type 1 (PAI-1)-specific monoclonal antibodies indicate that the decrease in uPA activity observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography was a direct result of decreased extracellular uPA antigen and not a consequence of increased PAI-1 antigen. The effect of PKC inhibitors was specific for TNF-mediated increased uPA expression because cytokine induction of PAI-1 was not influenced by these agents. Northern blot analyses also showed that PKC inhibitor treatment of endothelial cells resulted in a decreased steady- state level of uPA mRNA with no measurable change in PAI-1 mRNA in cultures incubated with TNF. Downregulation of cellular PKC by 18 hours of phorbol myristate acetate (PMA) pretreatment of endothelial cell cultures abolished TNF-mediated extracellular uPA induction. This effect was specific for PMA because 4-alpha PMA pretreatment of cells, which does not stimulate PKC, was ineffective in altering TNF induction of endothelial cell uPA. Induction of PKC directly with PMA, mezerein, and (-)-octylindolactam V increased endothelial cell levels of extracellular uPA in a time- and dose-dependent manner. In addition, this increase in endothelial cell extracellular uPA activity mediated by PKC agonists could be inhibited with PKC inhibitors. Endothelial cells treated with TNF acquire the ability to invade extracellular matrix and reorganize into tube-like structures when grown on Matrigel- coated culture dishes, a behavior blocked by H-7, but not by HA 1004. In summary, these data implicate a role for the PKC pathway in the TNF- mediated induction of uPA expression, subsequent matrix remodeling, and the formation of tube-like structures, a process important in neovascularization, wound healing, and leukocyte extravasation.

EphrinB-mediated reverse signalling controls junctional integrity and pro-inflammatory differentiation of endothelial cells

2014 ◽  
Vol 112 (07) ◽  
pp. 151-163 ◽  
Author(s):  
Hui Liu ◽  
Kavi Devraj ◽  
Kerstin Möller ◽  
Stefan Liebner ◽  
Markus Hecker ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Junctions ◽  
Cell Permeability ◽  
Endothelial Surface ◽  
Inflammatory Phenotype ◽  
Src Homology ◽  
Leukocyte Populations ◽  
The Impact

SummaryThe EphB/ephrinB receptor-ligand system is pivotal for the development of the embryonic vasculature and for angiogenesis in the adult organism. We observed that (i) the expression of ephrinB2 and ephrinB1 is up-regulated in capillaries during inflammation, that (ii) these ligands are localised on the luminal endothelial surface, and that (iii) they interact with the ephrinB-receptor EphB2 on monocyte/macrophages. This study delineates the impact of ephrinB-mediated reverse signalling on the integrity and proinflammatory differentiation of the endothelium. To this end, in vitro analyses with human cultured endothelial cells reveal that knockdown of ephrinB2 or ephrinB1 impairs monocyte transmigration through the endothelium. While ephrinB2 but not ephrinB1 interacts with PECAM-1 (CD31) in this context, reverse signalling by ephrinB1 but not ephrinB2 elicits a c-Jun N-terminal kinase (JNK)-dependent up-regulation of E-selectin expression. Furthermore, treatment of endothelial cells with soluble EphB2 receptor bodies or EphB2-overexpressing mouse myeloma cells links ephrinB2 to PECAM-1 and induces its Src-dependent phosphorylation while diminishing Src homology phosphotyrosyl phosphatase-2 (SHP-2) activity and increasing endothelial cell permeability. We conclude that extravasation of EphB2 positive leukocyte populations is facilitated by lowering the integrity of endothelial cell junctions and enhancing the pro-inflammatory phenotype of the endothelium through activation of ephrinB ligands.

Thrombin mediates mitogenesis and survival of human endothelial cells through distinct mechanisms

2008 ◽  
Vol 294 (5) ◽  
pp. C1215-C1226 ◽  
Author(s):  
Panagiota Zania ◽  
Matthew Papaconstantinou ◽  
Christodoulos S. Flordellis ◽  
Michael E. Maragoudakis ◽  
Nikos E. Tsopanoglou
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Endothelial Cell Proliferation ◽  
Dependent Manner ◽  
Human Endothelial Cells ◽  
Vascular Protection ◽  
Concentration Dependent Manner ◽  
Poor Growth

Thrombin has been reported to play a pivotal role in the initiation of angiogenesis by indirectly regulating and organizing a network of angiogenic molecules. In addition, it has been proposed that thrombin can directly activate endothelial cell proliferation. However, in this report it was shown that thrombin is a poor growth factor for human endothelial cells, and its modest mitogenic activity is mediated indirectly by the release of heparin-binding epidermal growth factor, subsequent to proteinase-activated receptor 1 (PAR1) activation. On the other hand, it was demonstrated that thrombin is a potent anti-apoptotic factor for endothelial cells, pointing to a novel role of thrombin in vascular protection. Analysis by annexin V-propidium iodide double staining revealed that thrombin, specifically, promoted survival of serum-starved endothelial cells in a concentration-dependent manner. In contrast to its mitogenic effect, the anti-apoptotic effect of thrombin was largely independent of its catalytic activity and was mediated through interaction with ανβ3 and α5β1 integrins, whereas the involvement of PAR1 was limited. These results provide new insights in understanding the role of thrombin in endothelial cell signaling and vascular biology.

scholarly journals Vascular Endothelial Integrity Affects the Severity of Enterovirus-Mediated Cardiomyopathy

2021 ◽  
Vol 22 (6) ◽  
pp. 3053
Author(s):  
Jin-Ho Park ◽  
Ha-Hyeon Shin ◽  
Hyun-Seung Rhyu ◽  
So-Hee Kim ◽  
Eun-Seok Jeon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Vascular Permeability ◽  
Germ Line ◽  
Vascular Endothelial Cell ◽  
Cell Junctions ◽  
Blue Dye ◽  
Vascular Endothelial ◽  
Knock Out ◽  
Endothelial Junction

Coxsackievirus and adenovirus receptor (CAR) is present in epithelial and vascular endothelial cell junctions. We have previously shown a hemorrhagic phenotype in germ-line CAR knock-out mouse embryos; we have also found that CAR interacts with ZO-1 and β-catenin. However, the role of CAR in vascular endothelial junction permeability has not been proven. To understand the roles of CAR in the vascular endothelial junctions, we generated endothelium-specific CAR knockout (CAR-eKO) mice. In the absence of CAR, the endothelial cell layer showed an increase in transmembrane electrical resistance (TER, Ω) and coxsackievirus permeability. Evans blue dye and 70 kDa dextran-FITC were delivered by tail vein injection. We observed increased vascular permeability in the hearts of adult CAR-eKO mice compare with wild-type (WT) mice. There was a marked increase in monocyte and macrophage penetration into the peritoneal cavity caused by thioglycolate-induced peritonitis. We found that CAR ablation in endothelial cells was not significantly increased coxsackievirus B3 (CVB3) induced myocarditis in murine model. However, tissue virus titers were significantly higher in CAR-eKO mice compared with WT. Moreover, CVB3 was detected in the brain of CAR-eKO mice. Endothelial CAR deletion affects the expression of major endothelial junction proteins, such as cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1) in the cultured endothelial cells as well as liver vessel. We suggest that CAR expression is required for normal vascular permeability and endothelial tight junction homeostasis. Furthermore, CVB3 organ penetration and myocarditis severities were dependent on the endothelial CAR level.

scholarly journals Role of protein kinase C in tumor necrosis factor induction of endothelial cell urokinase-type plasminogen activator

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2608-2617 ◽  
Author(s):  
MJ Niedbala ◽  
M Stein-Picarella
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Plasminogen Activator ◽  
Matrix Remodeling ◽  
Dependent Manner ◽  
Pkc Inhibitors ◽  
Tnf Induction ◽  
Dose Dependent ◽  
Pai 1

Abstract Tumor necrosis factor (TNF) can promote endothelial cell transcription, synthesis, and secretion of urokinase plasminogen activator (uPA) augmenting extracellular matrix remodeling and influencing cellular differentiation. In this report, the role of the protein kinase C (PKC) pathway in mediating TNF induction of uPA in human umbilical vein endothelial cells is described. The PKC inhibitors (H-7, staurosporine, and calphostin C), but not HA-1004, inhibited TNF-induced uPA expression, synthesis, and secretion in a dose-dependent manner. Analysis of cell-free conditioned medium obtained from PKC inhibitor- treated cultures by micro-enzyme-linked immunosorbent assay methodologies using uPA- and plasminogen activator inhibitor type 1 (PAI-1)-specific monoclonal antibodies indicate that the decrease in uPA activity observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography was a direct result of decreased extracellular uPA antigen and not a consequence of increased PAI-1 antigen. The effect of PKC inhibitors was specific for TNF-mediated increased uPA expression because cytokine induction of PAI-1 was not influenced by these agents. Northern blot analyses also showed that PKC inhibitor treatment of endothelial cells resulted in a decreased steady- state level of uPA mRNA with no measurable change in PAI-1 mRNA in cultures incubated with TNF. Downregulation of cellular PKC by 18 hours of phorbol myristate acetate (PMA) pretreatment of endothelial cell cultures abolished TNF-mediated extracellular uPA induction. This effect was specific for PMA because 4-alpha PMA pretreatment of cells, which does not stimulate PKC, was ineffective in altering TNF induction of endothelial cell uPA. Induction of PKC directly with PMA, mezerein, and (-)-octylindolactam V increased endothelial cell levels of extracellular uPA in a time- and dose-dependent manner. In addition, this increase in endothelial cell extracellular uPA activity mediated by PKC agonists could be inhibited with PKC inhibitors. Endothelial cells treated with TNF acquire the ability to invade extracellular matrix and reorganize into tube-like structures when grown on Matrigel- coated culture dishes, a behavior blocked by H-7, but not by HA 1004. In summary, these data implicate a role for the PKC pathway in the TNF- mediated induction of uPA expression, subsequent matrix remodeling, and the formation of tube-like structures, a process important in neovascularization, wound healing, and leukocyte extravasation.

Sign in / Sign up

Export Citation Format

Share Document