scholarly journals Heat Shock Protein A12B Protects Vascular Endothelial Cells Against Sepsis-Induced Acute Lung Injury in Mice

2017 ◽  
Vol 42 (1) ◽  
pp. 156-168 ◽  
Author(s):  
Yi Chen ◽  
Lei Wang ◽  
Qiuxiang Kang ◽  
Xu Zhang ◽  
Guifang Yu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Caspase 3 ◽  
Myeloperoxidase Activity ◽  
Expression Levels ◽  
Erk Phosphorylation ◽  
Tnf Α ◽  
Nasal Inhalation ◽  
Group A

Background: Pulmonary endothelial injury is a critical process in the pathogenesis of acute lung injury (ALI) during sepsis. Heat shock protein A12B (HSPA12B) is mainly expressed in endothelial cells and protects against several harmful factors. However, the effects of HSPA12B in sepsis-induced ALI and its potential mechanisms of action remain unclear. Methods: For in vivo experiments, C57BL/6 mice were randomly divided into four groups (n=15): a sham operation group, a cecal ligation and puncture (CLP) group, a HSPA12B siRNA-CLP group and a negative control (NC) siRNA-CLP group. The mice were treated by nasal inhalation of 2-OMe-modified HSPA12B siRNA or NC siRNA. Sepsis was induced by CLP. Samples were harvested 24 and 48 hours post-CLP surgery. Pathological changes and scoring of lung tissue samples were monitored using hematoxylin and eosin staining. Levels of pro-inflammatory cytokines (e.g., interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6) and myeloperoxidase activity in bronchoalveolar lavage fluid were analyzed by ELISA. Pulmonary edema was assessed using a wet-to-dry weight ratio. Neutrophils and alveolar macrophages were counted using flow cytometry. Pulmonary endothelial cell apoptosis was detected by TUNEL staining. Expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blot analysis. In addition, 7-day survival was recorded. For in vitro experiments, human umbilical vein endothelial cells were pre-transfected with HSPA12B siRNA or pIRES2-EGFP-HSPA12B-Flag plasmid and treated with lipopolysaccharide; subsequently, the expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blotting. Results: Nasal inhalation of nano-polymer-encapsulated HSPA12B siRNA specifically downregulated mRNA and protein expression levels of HSPA12B in lung tissues. The administration of HSPA12B siRNA aggravated lung pathological injury, upregulated pro-inflammatory cytokine (e.g., IL-1β, TNF-α, and IL-6) expression, and increased myeloperoxidase activity, neutrophil infiltration, pulmonary edema, and pulmonary endothelial cell apoptosis. Additionally, HSPA12B knockdown worsened survival after CLP surgery. The potential protective mechanisms of HSPA12B may involve the inhibition of ERK phosphorylation and caspase-3 activation in vivo and in vitro. Conclusion: HSPA12B protected against sepsis-induced ALI. The potential mechanism may be partly due to the inhibition of ERK phosphorylation and caspase-3 activation. These findings provide a potential therapeutic target for treating sepsis.

scholarly journals Anti-breast cancer effect and mechanism of ethyl acetate extract of persimmon leaves

2020 ◽  
Author(s):  
Fang Zhuge ◽  
Xiusha Wei ◽  
Yani Wu ◽  
Guining Wei ◽  
Ming Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Ethyl Acetate ◽  
Caspase 3 ◽  
Ethyl Acetate Extract ◽  
Tumor Inhibition ◽  
Expression Levels ◽  
Tnf Α ◽  
Tumor Inhibition Rate

Abstract Aim: The anti-breast cancer effect and mechanism of ethyl acetate extract of persimmon leaves (PLE) were determined. Methods: Persimmon leaves were extracted by reflux at 80°C with 80% ethanol as the solvent. The total extracts of persimmon leaves were extracted with ethyl acetate, and the yield was calculated by weighing. The mouse breast cancer cell line 4T1 was cultured in vitro, and different concentrations of PLE were added. At the same time, the effects of PLE at different concentrations (25, 50, 100 µg/ml) on cell apoptosis ability were detected by Acridine Orange and Ethidium Bromide (AOEB) and flow cytometry experiments. In addition, real-time quantitative PCR (real-time PCR, RT-PCR) was used to test the expression of the Bax, Bcl-2, ERK1/2, MEK1/2 and RAF genes. In vivo tumor-bearing mouse model: A breast cancer transplant tumor model was established with BALB/c mice. The doses of PLE were 30, 60 and 120 mg/kg body weight/d, and the dose of CTX was 20 mg/kg body weight/d. The tumor inhibition rate and the effects of PLE on immune organs in tumor-bearing mice with 4T1 breast cancer were determined. The expression levels of IL-6, TNF-α, TGF-β and VEGFA in the serum of mice were detected by ELISA. The expression of the Bax, Bcl2, ERK1/2, MEK1/2 and RAF genes was determined by RT-PCR. The protein expression levels of Bax, Bcl-2, Caspase-3, p-MEK, p-JNK and p-P38 in tumor tissues were detected by immunohistochemistry. In addition, the protein expression levels of MAPK pathway components were assessed through Western blotting.Results: A total of 119.34 g ethyl acetate extract was obtained from 3 kg persimmon leaves with a yield of 3.98%. In vitro: MTT results indicated a strong antiproliferative effect of PLE on breast cancer cell lines. AOEB and flow cytometry assays showed that PLE promoted the apoptosis of breast cancer cells. PCR results showed that PLE could inhibit Bcl-2, promote Bax expression, and downregulate ERK1/2, MEK1/2, and RAF gene expression. In vivo: PLE had a significant inhibitory effect on breast cancer, and the tumor inhibition rates were 11.65%, 33.71% and 47.24% from low dose to high dose, respectively, showing a concentration dependence. The tumor inhibition rate of CTX was 57.74%. Meanwhile, PLE can increase the spleen and thymus index of 4T1 mice and decrease the liver index of 4T1 mice. Compared with the model group, PLE significantly reduced the expression levels of IL-6, TNF-α, TGF-β and VEGFA in the serum of mice. PCR results showed that PLE could inhibit Bcl-2, promote Bax expression, and downregulate ERK1/2, MEK1/2, and RAF gene expression. Immunohistochemical results showed that the PLE group and CTX group significantly promoted the expression of Bax and Caspase-3 proteins and downregulated the expression of Bcl-2, p-MEK, p-JNK and p-P38 proteins. WB results showed that PLE regulated the expression of proteins in the MAPK pathway.Conclusion: PLE enhances immunity, inhibits angiogenesis, inhibits 4T1 cell proliferation and induces apoptosis. Its apoptosis mechanism is related to the regulation of Bax/Bcl-2/Caspase-3 protein and the phosphorylation of regulatory proteins related to the MAPK signaling pathway.

scholarly journals Methylprednisolone Protects SAP and Pancreatitis-Associated ALI in Mice by Inhibiting NLRP3 Inflammasome Through NF-κB

2020 ◽  
Author(s):  
Chuan-jiang Liu ◽  
Qiang Fu ◽  
Wenjing Zhou ◽  
Xu Zhang ◽  
Rui Chen ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Nlrp3 Inflammasome ◽  
Antioxidant Properties ◽  
Underlying Mechanism ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Expression Levels ◽  
Tnf Α

Abstract Background: Methylprednisolone (MP) is a synthetic corticosteroid with potent anti-inflammatory and antioxidant properties used as therapy for a variety of diseases. The underlying mechanism of MP to reduce acute pancreatitis still needs to be elucidated.Methods: Twenty-four male C57BL/6 mice (6-8 weeks) were used to establish SAP mouse model by administering an intraperitoneal injection of Cae and LPS. Amylase expression levels of serum and PLF were measured with an amylase assay kit. The concentrations of IL-1β and TNF-α in the serum and PLF were detected by ELISA. The level of pancreatic and lung tissue damage and inflammation was assessed by H&E staining and immunofluorescence staining. Western blot and qPCR were used to detect the expression levels of NLRP3, IL-1β and TNF-αin vivo and in vitro.Results: In this study, we found MP, used in the early phase of SAP, decreased the levels of IL-1β and TNF-α in serum and peritoneal lavage fluids (PLF), reduced the level of serum amylase and the expression of MPO in lung tissue, attenuated the pathological injury of the pancreas and lungs in a dose-dependent manner. The expression of NLRP3 and IL-1β in pancreas and lungs was down-regulated significantly depending on the MP concentration. In vitro, MP reduced the levels of IL-1β and TNF-α by down-regulating the expression of NLRP3, IL-1β and p-NF-κB in isolated peritoneal macrophages. Conclusion: MP can attenuate the injury of pancreas and lungs, and the inflammatory response in SAP mice by down-regulating the activation of NF-κB and the NLRP3 inflammasome.

scholarly journals L-Selectin–Dependent Leukocyte Adhesion to Microvascular But Not to Macrovascular Endothelial Cells of the Human Coronary System

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3228-3235 ◽  
Author(s):  
A. Zakrzewicz ◽  
M. Gräfe ◽  
D. Terbeek ◽  
M. Bongrazio ◽  
W. Auch-Schwelk ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Time Course ◽  
Leukocyte Adhesion ◽  
Flow Chamber ◽  
Shear Stresses ◽  
Selectin Ligands ◽  
Tnf Α ◽  
Factor Α

Abstract To characterize L-selectin–dependent cell adhesion to human vascular endothelium, human cardiac microvascular endothelial cells (HCMEC) and human coronary endothelial cells (HCEC) were isolated from explanted human hearts. The adhesion behavior of human (NALM-6) and mouse (300.19) pre-B cells transfected with cDNA encoding for human L-selectin was compared with that of the respective nontransfected cells in a flow chamber in vitro. More than 80% of the adhesion to tumor necrosis factor-α (TNF-α)–stimulated HCMEC at shear stresses <2 dyne/cm2 was L-selectin dependent and could be equally well blocked by an anti–L-selectin antibody or a L-selectin-IgG-chimera. No L-selectin dependent adhesion to HCEC could be shown. The L-selectin dependent adhesion to HCMEC was insensitive to neuraminidase, but greatly inhibited by addition of NaClO3 , which inhibits posttranslational sulfation and remained elevated for at least 24 hours of stimulation. E-selectin dependent adhesion of HL60 cells to HCMEC was blocked by neuraminidase, but not by NaClO3 and returned to control levels within 18 hours of HCMEC stimulation. It is concluded that microvascular, but not macrovascular endothelial cells express TNF-α–inducible sulfated ligand(s) for L-selectin, which differ from known L-selectin ligands, because sialylation is not required. The prolonged time course of L-selectin dependent adhesion suggests a role in sustained leukocyte recruitment into inflammatory sites in vivo.

scholarly journals Genetic alteration of endothelial heparan sulfate selectively inhibits tumor angiogenesis

2007 ◽  
Vol 177 (3) ◽  
pp. 539-549 ◽  
Author(s):  
Mark M. Fuster ◽  
Lianchun Wang ◽  
Janice Castagnola ◽  
Lyudmila Sikora ◽  
Krisanavane Reddi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Heparan Sulfate ◽  
Tumor Angiogenesis ◽  
Wound Repair ◽  
Reproductive Capacity ◽  
Erk Phosphorylation ◽  
Tumor Endothelium ◽  
A Cell

To examine the role of endothelial heparan sulfate during angiogenesis, we generated mice bearing an endothelial-targeted deletion in the biosynthetic enzyme N-acetylglucosamine N-deacetylase/N-sulfotransferase 1 (Ndst1). Physiological angiogenesis during cutaneous wound repair was unaffected, as was growth and reproductive capacity of the mice. In contrast, pathological angiogenesis in experimental tumors was altered, resulting in smaller tumors and reduced microvascular density and branching. To simulate the angiogenic environment of the tumor, endothelial cells were isolated and propagated in vitro with proangiogenic growth factors. Binding of FGF-2 and VEGF164 to cells and to purified heparan sulfate was dramatically reduced. Mutant endothelial cells also exhibited altered sprouting responses to FGF-2 and VEGF164, reduced Erk phosphorylation, and an increase in apoptosis in branching assays. Corresponding changes in growth factor binding to tumor endothelium and apoptosis were also observed in vivo. These findings demonstrate a cell-autonomous effect of heparan sulfate on endothelial cell growth in the context of tumor angiogenesis.

scholarly journals Amlexanox As a Possible Breakthrough Drug for MLL/AF4-Positive Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1590-1590
Author(s):  
Hayato Tamai ◽  
Hiroki Yamaguchi ◽  
Koichi Miyake ◽  
Miyuki Takatori ◽  
Tomoaki Kitano ◽  
...  
Keyword(s):  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Control Diet ◽  
Hematopoietic Stem ◽  
Tnf Α ◽  
Breakthrough Drug

Abstract Background: MLL/AF4-positive acute lymphoblastic leukemia (ALL) is associated with poor prognosis even after allogeneic hematopoietic stem cell transplantation. Previously, we reported that this ALL shows resistance to TNF-α, which is the factor involved in the graft versus leukemia (GVL) effect or tumor immunity, by upregulation of S100A6 expression followed by interference with the p53-caspase pathway. Amlexanox, an anti-allergic drug, was reported to inhibit the translocation pathway of endogenous S100A6 in endothelial cells. Aims: This study was performed to examine the effects of Amlexanox on MLL/AF4-positive ALL. Methods: In vitro analysis, cell growth of MLL/AF4-positive ALL cell lines ( SEM and RS4;11) were analyzed with TNF-α (10 ng/mL) and Amlexanox (0, 10, and 100 µg/mL).The effect of Amlexanox on S100A6 and p53-caspase pathways were examined by Western blotting (WB) analysis. In vivo analysis MLL/AF4-positive transgenic mice model, which show CD45R/B220+leukemia by 12 months of age we established and human peripheral blood mononuclear cell (Hu-PBMC) NOD/SCID mice transplanted with SEM-Luc were examined to compare mice fed diet containing Amlexanox (0.02%) with mice fed control diet. Results: There were no significant differences between the growth of SEM or RS4;11 cells in the absence or presence of 10 µg/mL of Amlexanox in vitro under 10 ng/mL of TNF-α. However, both cells showed significant growth inhibition by 10 ng/mL of TNF-α in the presence of 100 µg/mL of Amlexanox (P = 0.0085 for SEM, P = 0.0196 for RS4;11) WB analysis showed that S100A6 was activated in the presence of 10 ng/mL TNF-α, and activated S100A6 was decreased and both acetyl-p53/p53 ratio and cleaved caspase 3/caspase 3 ratio were increased in cells treated with 100 µg/mL of Amlexanox under 10 ng/mL of TNF-α in the MLL/AF4-positive human ALL cell lines. In vivo, MLL/AF4-positive transgenic mice fed a diet containing Amlexanox (0.02%) developed significantly less volume of CD45R/B220+ leukemia at the age of 1 year in comparison with mice fed control diet (P<0.001 for BM and .P<0.001 for spleen). Hu-PBMC NOD/SCID mice transplanted with SEM-Luc in the Amlexanox group showed significantly longer survival than those in the control group (P < 0.014). Conclusions: Amlexanox may be a breakthrough drug for MLL/AF4-positive ALL because it inhibits the resistance of MLL/AF4-positive ALL to TNF-α by downregulating S100A6 expression followed by upregulating the p53-caspase pathway.Specifically, allogeneic hematopoietic stem cell transplantation is expected to show beneficial effects in combination with Amlexanox. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dephosphorylation Targets Bcl-2 for Ubiquitin-dependent Degradation: A Link between the Apoptosome and the Proteasome Pathway

1999 ◽  
Vol 189 (11) ◽  
pp. 1815-1822 ◽  
Author(s):  
Stefanie Dimmeler ◽  
Kristin Breitschopf ◽  
Judith Haendeler ◽  
Andreas M. Zeiher
Keyword(s):  
Endothelial Cells ◽  
Map Kinase ◽  
Inflammatory Diseases ◽  
Apoptotic Cell ◽  
Proteasome Inhibitors ◽  
Inhibitory Function ◽  
Tnf Α ◽  
Proteasome Pathway

Injury of the endothelial cells by the induction of apoptotic cell death may play an important role in the pathophysiology of atherosclerosis and the progression of inflammatory diseases. Here, we demonstrate an essential role for the ubiquitin-dependent proteasome complex in stimulus-induced degradation of the antiapoptotic protein Bcl-2. Bcl-2 is specifically degraded after stimulation of human endothelial cells with tumor necrosis factor (TNF)-α in a process that is inhibited by specific proteasome inhibitors. In addition, the mutation of the potential ubiquitin-acceptor amino acids of Bcl-2 provides protection against TNF-α– and staurosporine-induced degradation in vitro and in vivo. Moreover, mimicking phosphorylation of the putative mitogen-activated protein (MAP) kinase sites of the Bcl-2 protein (Thr 56, Thr 74, and Ser 87) abolishes its degradation, suggesting a link between the MAP kinase pathway to the proteasome pathway. Finally, inhibition of Bcl-2 degradation either by suppressing ubiquitin-dependent proteasomal degradation or by mimicking continuous phosphorylation of the putative MAP kinase sites in the Bcl-2 protein confers resistance against induction of apoptosis. Thus, the degradation of Bcl-2 may unleash the inhibitory function of Bcl-2 over the apoptosome and may thereby amplify the activation of the caspase cascade.

Biological and Molecular Evidences for the Antitumor Potential of EGb 761 in Glioma Cells In Vitro and In Vivo

2021 ◽  
Vol 15 (5) ◽  
pp. 685-692
Author(s):  
Xin Sui ◽  
Jia Zhou ◽  
Yan Xu ◽  
Yuchen Wang ◽  
Guangfu Lv ◽  
...  
Keyword(s):  
Growth Factor ◽  
Caspase 3 ◽  
Glioma Cells ◽  
Signalling Pathway ◽  
Migration And Invasion ◽  
Egb 761 ◽  
Western Blots ◽  
Expression Levels

EGb 761, the standardized extract from the Ginkgo biloba leaves, has therapeutic effect on many diseases. However, its mechanisms on glioma remain to be fully established. This study aims to investigate the possible effects of EGb 761 on glioma cells, to explore its potential mechanism. The glioma cells SHG44 and U251 were used as materials, the proliferation, migration and invasion were assessed by the MTT, the scratch-wound and Transwell assays were performed respectively. Levels of insulin-like growth factor-1, Bcl-2, p53, Smad2/3, Bax, cleaved caspase-3 and p-Smad2/3 were determined by western blots. The development and progression of U251 glioma cell were measured in vivo, and the apoptosis was evaluated. The results showed that EGb 761 could inhibit the proliferation, migration, and invasion of SHG-44 and U251 cells in vitro. Meanwhile, the expression levels of IGF-1 and Bcl-2, and the transforming growth factor-β (TGF-β) signaling were inhibited. In contrast, the expression levels of p53, Bax, and cleaved caspase-3 were increased significantly. In conclusion, this study suggested that EGb 761 could suppress the growth of glioma cells in vitro and in vivo, possibly by inhibiting the TGF-β signalling pathway and activating the p53 signalling pathway.

Carcinoembryonic Antigen (CEA) Regulates Tumor-Angiogenesis in Colon Carcinomas.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1897-1897
Author(s):  
Kira Braemswig ◽  
Marina Poettler ◽  
Wazlawa Kalinowska ◽  
Christoph Zielinski ◽  
Gerald W Prager
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tumor Angiogenesis ◽  
Endothelial Cell Proliferation ◽  
Erk Phosphorylation ◽  
Dose Dependent Increase ◽  
Dose Dependent

Abstract Human carcinoembryonic antigen (CEA) is a cell surface adhesion molecule member of the Immunoglobulin Superfamily (IgSF). Aberrant upregulation and secretion of soluble CEA is a common feature found in a wide variety of human cancers such as colon, breast and lung. Previous in vitro and in vivo results have demonstrated that CEA can affect tumor cell behavior including the inhibition of cell differentiation and apoptosis. However, any functional effects on angiogenic endothelial cell behavior are so far unknown. In the present work we found that in endothelial cells exogenous CEA led to a time and dose dependent increase in ERK phosphorylation, which was inhibited by the specific MEK inhibitor U0126. Thereby, the observed CEA effect was comparable in time and intense with the canonical angiogenic growth factor VEGF. The CEA-induced ERK phosphorylation was not affected by the blockage of VEGFR-2 / flk-1 using a specific inhibiting peptide (CBO-P11), which indicates a VEGF-independent mechanism. Furthermore, co-stimulation of endothelial cells with VEGF and CEA shows synergistic effects on ERK phosphorylation. While in endothelial cells no endogenous expression of CEA is detected, its putative receptor, the CEA receptor (CEAR), is highly expressed as shown by immunohistochemical staining of paraffin-embedded colon carcinoma sections as well as in biochemical analyses. When an activating antibody against CEAR was used, CEA-induced ERK phosphorylation was mimicked, while downregulation of CEAR by siRNA diminished CEA-induced signal transduction, significantly. To test a biological relevance of our findings, we first measured endothelial cell proliferation: CEA led to a dose dependent increase in endothelial cell proliferation in vitro, which again revealed a synergistic effect with VEGF. Thereby, CEA-induced endothelial cell proliferation was again independent of VEGFR-2 / flk-1. A biological role of CEA in tumor-angiogenesis was reflected by an in vivo model using CEA Mimotope immunized BALB/c mice, which were transplanted with MethA/CEA overexpressing tumor cells. Immunohistological analyses of these tumors revealed a significantly reduced vascular density, which was accompanied with diminished tumor growth. Our data provide first evidence of CEA as a novel pro-angiogenic activator of endothelial cells, which results in an increase in endothelial cell proliferation, independent of VEGFR-2. Furthermore, by targeting CEA in an in vivo mouse model, tumor-angiogenesis was markley reduced, indicating a potential therapeutic target in cancer.

Membrane-type matrix metalloproteinase-mediated angiogenesis in a fibrin-collagen matrix

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1810-1817 ◽  
Author(s):  
Annemie Collen ◽  
Roeland Hanemaaijer ◽  
Florea Lupu ◽  
Paul H. A. Quax ◽  
Natascha van Lent ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Matrix Metalloproteinase ◽  
Collagen Matrix ◽  
Tubular Structures ◽  
Tubule Formation ◽  
Tnf Α ◽  
Membrane Type

Adult angiogenesis, associated with pathologic conditions, is often accompanied by the formation of a fibrinous exudate. This temporary matrix consists mainly of fibrin but is intermingled with plasma proteins and collagen fibers. The formation of capillary structures in a fibrinous matrix in vivo was mimicked by an in vitro model, in which human microvascular endothelial cells (hMVECs) seeded on top of a fibrin-10% collagen matrix form capillarylike tubular structures after stimulation with basic fibroblast growth factor/tumor necrosis factor α (bFGF/TNF-α) or vascular endothelial growth factor (VEGF)/TNF-α. In the fibrin-collagen matrix the metalloproteinase inhibitor BB94 inhibited tubule formation by 70% to 80%. Simultaneous inhibition of plasmin and metalloproteinases by aprotinin and BB94 caused a nearly complete inhibition of tubule formation. Adenoviral transduction of tissue inhibitor of metalloproteinases 1 (TIMP-1) and TIMP-3 into endothelial cells revealed that TIMP-3 markedly inhibited angiogenesis, whereas TIMP-1 had only a minor effect. Immunohistochemical analysis showed the presence of matrix metalloproteinase 1 (MMP-1), MMP-2, and membrane-type 1 (MT1)–MMP, whereas MMP-9 was absent. The endothelial production of these MMPs was confirmed by antigen assays and real-time polymerase chain reaction (PCR). MT1-MMP mRNA was markedly increased in endothelial cells under conditions that induced tubular structures. The presence of MMP-1, MMP-2, and MT1-MMP was also demonstrated in vivo in the newly formed vessels of a recanalized arterial mural thrombus. These data suggest that MMPs, in particular MT-MMPs, play a pivotal role in the formation of capillarylike tubular structures in a collagen-containing fibrin matrix in vitro and may be involved in angiogenesis in a fibrinous exudate in vivo.

Protease-activated receptors 1 and 4 mediate thrombin signaling in endothelial cells

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3224-3231 ◽  
Author(s):  
Hiroshi Kataoka ◽  
Justin R. Hamilton ◽  
David D. McKemy ◽  
Eric Camerer ◽  
Yao-Wu Zheng ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Wild Type ◽  
Protease Activated Receptors ◽  
Erk Phosphorylation ◽  
Low Concentrations ◽  
Extracellular Signal ◽  
High Concentrations ◽  
Mouse Aorta

AbstractDefining the relative importance of protease-activated receptors (PARs) for thrombin signaling in mouse endothelial cells is critical for a basic understanding of thrombin signaling in these cells and for the rational use of knockout mice to probe the roles of thrombin's actions on endothelial cells in vivo. We examined thrombin- and PAR agonist–induced increases in cytoplasmic calcium, phosphoinositide hydrolysis, extracellular signal-regulated kinase (ERK) phosphorylation, and gene expression in endothelial cells from wild-type and PAR-deficient mice. PAR1 and PAR4 agonists triggered responses in wild-type but not in Par1–/– and Par4–/– endothelial cells, respectively. Calcium imaging confirmed that a substantial fraction of individual endothelial cells responded to both agonists. Compared with wild-type cells, Par1–/– endothelial cells showed markedly decreased responses to low concentrations of thrombin, and cells that lacked both PAR1 and PAR4 showed no responses to even high concentrations of thrombin. Similar results were obtained when endothelial-dependent vasorelaxation of freshly isolated mouse aorta was used as an index of signaling in native endothelial cells. Thus PAR1 is the major thrombin receptor in mouse endothelial cells, but PAR4 also contributes. These receptors serve at least partially redundant roles in endothelial cells in vitro and in vivo and together are necessary for the thrombin responses measured.

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