Plasmin Proteolysis of Endothelial Cell and Vessel Wall Associated Tissue Factor Pathway Inhibitor

2001 ◽  
Vol 86 (09) ◽  
pp. 923-928 ◽  
Author(s):  
Paul Stalboerger ◽  
Carmelo Panetta ◽  
Robert Simari ◽  
Noel Caplice
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Cell Surface ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Vessel Wall ◽  
Anticoagulant Activity ◽  
Vascular Cells ◽  
Normal Vessel

SummaryPlasmin is an important protease that mediates clot fibrinolysis and vessel wall extracellular matrix proteolysis. Recently, in vitro studies have suggested that plasmin can cleave and inactivate recombinant TFPI, a major inhibitor of TF-mediated coagulation. We hypothesized that such an interaction may occur in vascular cells expressing TFPI, or in the vessel wall, with implications for thrombolysis. In a series of experiments, we examined the effects of plasmin on cell surface and extracellular matrix (ECM) associated TFPI in endothelial cells (EC) in culture and on EC and smooth muscle cells (SMC) in the vessel wall. Plasmin (0.2 μM) decreased cell surface and matrix associated TFPI activity in cultured endothelial cells by 77 ± 5 % and 69 ± 6% respectively (p < 0.01). Plasminogen, the proenzyme form of plasmin had no such effect on cell surface TFPI or matrix TFPI. Cell surface TFPI antigen measured by fluorescence activated cell sorter (FACS) was also significantly reduced by plasmin. Proteolysis of conditioned medium TFPI was suggested by loss of a ~45kD TFPI on Western Blot analysis following plasmin treatment. Plasmin also proteolysed a ~45kD TFPI protein in the intact ECM of EC, an effect which was inhibited by preincubation with aprotinin, a plasmin inhibitor. Incubation of similar concentrations of plasmin, with homogenates of normal vessel decreased a ~45kD TFPI immunoreactive band on Western blot analysis. Plasmin also decreased surface TFPI activity on frozen sections of normal vessel as measured by an amidolytic assay. Finally, plasmin treatment of atherosclerotic plaque sections caused complete removal of TFPI immunoreactivity associated with luminal EC and intimal SMC, when compared to control treated plaque (n = 3). Together these data suggest that plasmin proteolyses the majority of EC-associated (surface and matrix) TFPI and may remove TFPI from the luminal surface and intima of the vessel wall. TFPI proteolysis in cultured EC was associated with significant reduction in TFPI anticoagulant activity. These data provide evidence that plasmin degradation of TFPI occurs in vascular cells and in the vessel wall and may have implications for rethrombosis following thrombolysis in vivo.

Arsenic Trioxide Affects Early Stage Angiogenesis through Inhibition of CD14 Monocyte Transdifferentiation into Endothelial Cells Induced by Pleiotrophin and mCSF.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1267-1267
Author(s):  
Haiming Chen ◽  
Mingjie Li ◽  
Richard A. Campbell ◽  
Melinda S. Gordon ◽  
Dror Shalitin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Arsenic Trioxide ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Early Stage ◽  
Vascular Endothelial ◽  
Cam Assay ◽  
Rt Pcr ◽  
Vessel Formation

Abstract We have discovered a novel mechanism leading to blood vessel formation involving transdifferentiation of monocytes into endothelial cells by tumor cell production of pleiotrophin (PTN), a protein highly produced by myeloma (H. Chen et al, Blood, 2005; Yeh et al BJH, 2006). Arsenic trioxide (ATO) induces apoptosis of cancer cells directly through a number of mechanisms, and this drug has also been shown to inhibit angiogenesis. However, it remains unknown whether ATO affects the earliest stages of angiogenesis and vasculogenesis important in tumor development. We purified human monocytes (CD14+) and cultured these cells on collagen I-coated dishes. mCSF was added to the cells after 1 hour of culture. PTN was added twice to the culture, once after 24 hours and again after 5 days with or without ATO or bortezomib. FLK-1 expression (VEGFR-2) showed that the cells incubated on collagen I without drugs formed tube-like structures in the presence of PTN and mCSF. However, the tube-like structures disappeared after adding either the IC50 (5x10−6M) dose or low (5x10−7M) dose of ATO. FLK-1 staining remains in the tube-like structures with low doses (3x10−12M) of bortezomib. In order to examine whether ATO or bortezomib affects endothelial gene expression when monocytes are induced to transdifferentiate in the presence of these cytokines, we also examined expression using RT-PCR on endothelial cell genes (vascular endothelial growth factor receptor-2 (Flk-1), Tie-2 and von Willebrand factor (vWF)) and Western blot analysis for protein expression. The results of both RT-PCR and Western blot analysis showed that the expression of endothelial markers was blocked at both the higher (5x10−6M) and lower (5x10−7M) doses of ATO. In contrast, the expression of endothelial markers was not reduced by adding low dose bortezomib (3x10−12M). We further examined the effects of ATO and bortezomib on early stage angiogenesis in vivo using the chorioallantoic membrane (CAM) assay. Fertilized chick eggs were incubated horizontally at 38°C in a humidified incubator, windowed by day 3 of incubation and processed by day 8. The tested micro-sponge with ATO (5x10−6M) or bortezomib (3x10−11M) or control reagents was implanted on the CAM. The eggs were sealed with adhesive tape and returned to the incubator for 48 hours. The assay scored positive when two independent observers reported a significant reduction of vessels in the treated area. The results of the CAM assay showed that compared to saline, ATO significantly reduced new macroscopic and microscopic vessel formation. In contrast, bortezomib did not affect angiogenesis in the CAM assay. These experiments define a previously unrecognized novel mechanism by which ATO may have anti-angiogenetic effects in cancer patients-preventing the transdifferentiation of monocytes into endothelial cells by PTN. They also suggest ATO as a potential new specific agent to inhibit angiogenesis resulting from transdifferentiation of monocytes into vascular endothelial cells driven by pleiotrophin and mCSF. These results suggest a novel way by which anti-cancer agents may impact angiogenesis.

Phenotypic, Genotypic, and Functional Characterization Of AML-Derived Endothelial Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1411-1411
Author(s):  
Russell J Pizzo ◽  
Myra Coppage ◽  
Karen Rosell ◽  
Kimberly Morse ◽  
Jane L. Liesveld
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Leukemia Cell ◽  
Normal Subjects ◽  
Rt Pcr ◽  
Altered Expression ◽  
Normal Subject

Abstract Background In addition to participation in homing, egress, and transmigration of hematopoietic cells, marrow endothelium also contributes to regulation of hematopoiesis with effects on cell proliferation and survival. Characteristics of marrow—derived endothelial cells from normal subjects have been described (Blood 1994; 84: 10-19), but characterization of endothelial cells in leukemia states is incomplete. Angiogenesis is known to be increased in AML marrows, and circulating endothelial progenitors are increased and correlate with disease status and response to treatment. Furthermore, cytokines secreted by endothelial cells such as vascular endothelial growth factor (VEGF) have been found to serve as growth factors for leukemia, sometimes in a paracrine or autocrine fashion. Despite these findings, inhibition of VEGF with agents such as bevacizumab has not demonstrated clinical anti-leukemia activity. Since our group and others have shown that endothelial cells from multiple vascular beds (human umbilical vein endothelial cells—HUVECs), human microvascular endothelial cells derived from skin (HMEC-1 cell line), and normal subject—derived endothelial cells are able to prevent spontaneous or therapy-induced apoptosis in AML blasts, it is important to understand the phenotype and characteristics of endothelial cells isolated from AML patients to understand their functional roles and to see if they might have an angiogenic gene expression profile as has been described in multiple myeloma (Clin Cancer Res 2009 15:5369). Methods Endothelial cells were purified from marrow aspirates obtained with consent from normal subjects or from newly diagnosed AML patients. Cells were isolated using anti-CD105-PE (BD Bioscience) followed by anti-PE microbead selection (Miltenyi™) or after disruption of marrow spicules with subsequent selection for endothelial cells in endothelial cell selective medium (EGM-2, Lonza). Cells between 2nd and 4th passage were utilized for analysis. Protein expression was determined by flow cytometry, Western blotting, or RT-PCR. Matrigel™ tubule formation and acetyl-LDL expression were determined as per previously published methods, as were adhesion, CFU-L, and transmigration assays. RNASeq was performed by the Functional Genomics Core at the University of Rochester after extraction of polyadenylated RNA from purified total RNA. Conversion to cDNA occurred with the Illumina TruSeq™ preparation kit, and sequencing was accomplished with the Illumina Genome Analyzer IIx. CASAVA software was utilized for analysis. Results Marrow derived endothelial cells from normal and AML subjects express CD105 (endoglin), CD31(PECAM), CD106 (VCAM), CD146 (MCAM), CD54 (ICAM), and CD34. They do not express CD14 nor CD45, and they demonstrate low level expression of CD144 (VE-cadherin). By RT-PCR, they express Tie-2, VEGF, and eNOS (endothelial nitric oxide synthase). They express acetyl-LDL and form tubular structures in Matrigel™. Phosphorylated components of the mTOR and PI3K/Akt pathways were also expressed by Western blot analysis. Culture of AML cells with endothelial cells from both normal and AML subjects supported adhesion, transmigration, and CFU-L outgrowth, but no significant differences were noted in these functions between normal and AML—derived endothelial cells in vitro assays. RNASeq analysis revealed 130 genes significantly up—or down—regulated in AML derived endothelial cells as compared with those derived from normal marrow. Endothelial cells from both sources had a distinct signature from marrow—derived fibroblasts. The genes differentially expressed (p<0.001) were included in biological function categories involving cancer, cell development, cell growth and proliferation, cell signaling, inflammatory response, and cell death and survival. Further pathway analysis revealed upregulation of c-Fos, and this upregulation in AML vs. normal subject derived endothelial cells was confirmed by Western blot analysis. Genes involved in chemotaxis such as CXCL16 were also upregulated. Conclusions AML—derived endothelial cells exhibit similar phenotype and function as their normal marrow—derived counterparts, but genomic analysis suggests a differential signature with altered expression of genes which could play a role in leukemogenesis or leukemia cell maintenance in the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Novel Protein Encoded by C6orf105 Regulates Tissue Factor Pathway Inhibitor Expression and Function In Human Endothelial Cells In Normal Conditions and During Androgen Stimulation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 348-348
Author(s):  
Cristina Lupu ◽  
Hua Zhu ◽  
Jonathan Wren ◽  
Florea Lupu
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Western Blot ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Anticoagulant Activity ◽  
Uncharacterized Protein ◽  
Tissue Factor Pathway ◽  
Anticoagulant Function ◽  
And Function

Abstract Abstract 348 Cardiovascular disease (CVD) and thrombotic complications (deep vein thrombosis/venous thromboembolism, DVT/VTE) represent major health problems, with men having higher rates of clinical events than women. Tissue Factor Pathway Inhibitor (TFPI) is the key natural inhibitor of coagulation: it neutralizes factor Xa (FXa) and inhibits tissue factor-factor VIIa (TF-FVIIa) in the presence of FXa. In vivo most of TFPI is in endothelial cells (EC), reversibly bound to yet unidentified receptors, and glycosyl phosphatidylinositol-floated in caveolae and/or lipid raft microdomains. Intravascular thrombosis occurs frequently in older people, especially associated with cancer, diabetes, or CVD. TF is directly involved in tumor hypercoagulability, angiogenesis and metastasis. Cell-associated TFPI is the most physiologically significant inhibitor of the TF-FVIIa- triggered coagulation pathway; nevertheless, very few mechanisms/factors that could regulate the natural expression of TFPI have been identified so far. Here we describe androgen treatment of EC as a novel way to preserve and/or enhance a healthy vascular function, particularly related to the regulation of TFPI-dependent anticoagulant function of the endothelium. Our hypothesis is that a yet uncharacterized protein encoded by C6orf105 is a novel regulator of TFPI expression and function in EC, both in native conditions and during androgen stimulation. “In silico” data mining using global meta-analysis of publicly available NCBI's Gene Expression Omnibus 2-channel human microarray datasets identified C6orf105 as highly co-expressed with TFPI and following a parallel co-regulation. The uncharacterized protein has 230-aa, Mr ∼27 kDa, 5–6 predicted transmembrane domains and has sequence similarities with members of the androgen-inducible genes family. We tentatively named it TFPI-Regulating Factor (TFPI-RF). Real-time qPCR and western blot confirmed robust expression of TFPI-RF in EC in culture (HUVEC and EA.hy926 hybrid cell line). By immunofluorescence (IMF) TFPI-RF appears both on the cell surface and intracellularly co-localizing with TFPI and caveolin-1 (cav-1). Post-transcriptional (siRNA) down-regulation of TFPI-RF decreased TFPI, both as protein (∼2-times) and as anticoagulant activity (∼3-fold), apparently by reducing the co-localization of the TF-FVIIa-FXa-TFPI complex with cav-1. Over-expression of TFPI-RF in HUVEC and EA.hy926 led to enhanced co-localization of TFPI-RF with TFPI, and increased TFPI mRNA and anticoagulant activity (∼2-times). Western blot of cellular fractions after extraction with Triton X-114 and temperature-induced phase separation revealed the presence of TFPI and TFPI-RF in detergent-insoluble fractions, which suggests predominant lipid raft association. IMF illustrates TFPI-RF co-clustering with TFPI and cav-1 or GM1 (raft marker) in live EC incubated with anti-TFPI antibody or Cholera Toxin-B, respectively. The effect of androgens was studied by incubating EC with 30 nM dehydrotestosterone (DHT) or equivalent testosterone-BSA (cell-impermeable). 1-h incubation led to 2-times enhanced TFPI activity, increased co-localization of the quaternary complex with cav-1 and TFPI-RF, and enhanced exposure of TFPI and TFPI-RF on the cell surface. 24-h treatment with DHT up-regulates the expression of both TFPI (2-fold) and TFPI-RF (3-fold), as well as the TFPI inhibitory activity against FXa. DHT failed to enhance TFPI activity in TFPI-RF siRNA EC. Our results reveal a novel mechanism of up-regulation of the anticoagulant activity of endogenous TFPI in response to physiological levels of androgen. While the precise role of androgens in the ageing process is unclear, it is believed that androgen replacement could have beneficial influence on the declining functions in the elderly. Our data could expand on the effects of androgens on the haemostatic function of the endothelium and discover new roles for novel proteins like C6orf105/TFPI-RF in enhancing the endothelial anticoagulant function. These may open possibilities to manipulate the cellular endogenous TFPI and/or other intrinsic factors to counteract pro-thrombotic states associated with CVD, DVT/VTE, sepsis and cancer. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of Thrombin-Cleaved Osteopontin and Integrin α9 and β1 Signaling Pathway Molecules in Chronic Subdural Hematomas

2021 ◽  
Author(s):  
Koji Osuka ◽  
Yusuke Ohmichi ◽  
Mika Ohmichi ◽  
Chiharu Suzuki ◽  
Masahiro Aoyama ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Outer Membrane ◽  
Signaling Pathway ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Matrix Protein ◽  
Chronic Subdural Hematoma ◽  
Extracellular Matrix Protein

Abstract Chronic subdural hematoma (CSDH) is considered to be an inflammatory and angiogenic disease. Osteopontin is an extracellular matrix protein. Osteopontin is cleaved by thrombin, resulting in N-half osteopontin, which is more prominent in integrin signal transduction. We examined the expression of N-half osteopontin in the CSDH fluid and the expression of integrin α9 and β1 and the downstream components of the angiogenic signaling pathways in the outer membrane of CSDHs. Twenty samples of CSDH fluid and 8 samples of CSDH outer membrane were included. The concentrations of N-half osteopontin in the CSDH fluid were measured using ELISA kits. The expression of integrin α9 and β1, vinculin, talin-1, focal adhesion kinase (FAK), paxillin, α-actin, Src and β-actin was examined by western blot analysis. The expression of integrin α9 and β1, FAK and paxillin was also examined by immunohistochemistry. We investigated whether CSDH fluid could activate FAK in cultured endothelial cells in vitro. The concentration of N-half osteopontin in CSDH fluid was significantly higher than that in the serum. Western blot analysis revealed above-mentioned molecules. In addition, integrin α9 and β1, FAK and paxillin were localized in the endothelial cells of vessels within the CSDH outer membrane. FAK was significantly phosphorylated immediately after treatment with CSDH fluid. Our data suggest that N-half osteopontin in CSDH fluid promotes neovascularization in endothelial cells through integrins α9 and β1. The N-half osteopontin and integrin signaling pathway might be a useful therapeutic target for treating the growth of refractory CSDH.

Abstract 447: Activation of IL-1β-Producing Inflammasomes Triggered by RNA Receptor RIG-I in Mouse Endothelial Cells

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Yang Zhang ◽  
Xiang Li ◽  
Xiao-Xue Li ◽  
Ashley L Pitzer ◽  
Pin-Lan Li
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammasome Activation ◽  
Type I ◽  
Mrna Levels ◽  
Cholesterol Crystals ◽  
Double Stranded Rna ◽  
Caspase 1

Retinoic acid-inducible gene-I (RIG-I) is a putative RNA helicase and recently identified as a cytosolic RNA receptor in mammalian cells. The role of RIG-I in the regulation of vascular function under physiological and pathological conditions is unknown. The present study tested whether RIG-I activation triggers inflammasome formation, turning on inflammation in mouse endothelial cells (EOMA cell line). By real time RT-PCR and Western blot analysis, transfection of mouse ECs with RIG-I specific agonist, 5’-triphosphate double-stranded RNA (3pRNA, 0.5 mg/L) increased RIG-I mRNA level by 106% and protein level by 81% compared to those in control double-stranded RNA (dsRNA) transfected ECs. ELISA analyses showed that 3pRNA significantly increased release of type I IFN alpha by 31 folds and IL-1 beta (a prototype cytokine from inflammasome activation) by 8 folds in these ECs. Proatherogenic stimulation of mouse ECs with cholesterol crystals or 7-ketocholesterol also markedly increased protein expression of RIG-I, but had no effect on RIG-I mRNA levels. Measurements of active caspase-1, an inflammasome activation marker using FLICA fluorescent probe that specifically binds to cleaved caspase-1, demonstrated that 3pRNA doubled FLICA positive cells compared to that in control dsRNA transfected ECs. Interestingly, cholesterol crystals significantly increased FLICA positive cells by 3 folds. This activation of caspase-1 in ECs by cholesterol crystals was further confirmed by increase in cleaved caspase-1 (p10) using Western blot analysis and by enhanced IL-1 beta release as detected by ELISA. In the presence of 3pRNA, cholesterol crystal-induced inflammasome activation was not further augmented. These data indicate that increased expression and activity of RIG-I activate IL-1 beta producing inflammasomes in ECs, which may represent an early molecular mechanism mediating vascular inflammation or injury upon atherogenic stimulations.

scholarly journals The Therapeutic Effect of DL-3-n-Butylphthalide in Rats with Chronic Cerebral Hypoperfusion through Downregulation of Amyloid Precursor Protein and Matrix Metalloproteinase-2

2012 ◽  
Vol 40 (3) ◽  
pp. 967-975 ◽  
Author(s):  
W Wei ◽  
W Zhang ◽  
Y Huang ◽  
Y Li ◽  
G Zhu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cerebral Cortex ◽  
Matrix Metalloproteinase ◽  
Western Blot ◽  
Amyloid Precursor Protein ◽  
Therapeutic Effect ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Chronic Cerebral Hypoperfusion ◽  
Cerebral Hypoperfusion

OBJECTIVE: To investigate the therapeutic effect of DL-3- n-butylphthalide (DL-NBP) in rats with chronic cerebral hypoperfusion. METHODS: Chronic cerebral hypoperfusion was modelled by bilateral permanent occlusion of common carotid arteries in Wistar rats. The therapeutic effect of DL-NBP in hypoperfused rats was evaluated using the Morris water maze task. The levels and deposition of matrix metalloproteinase (MMP) and the amyloid precursor protein β-amyloid 40 (Aβ40) were measured by Western blot analysis and immunohistochemistry in the cerebral cortex and hippocampus. RESULTS: Treatment with DL-NBP significantly improved the learning and memory ability of hypoperfused rats. Western blot analysis indicated that, in comparison with the sham-operated control group, protein levels of Aβ40 and MMP-2 were significantly increased in the cerebral cortex of hypoperfused rats, and treatment with DL-NBP prevented this hypoperfusion-induced increase in Aβ40 and MMP-2. Immunohistochemical analysis showed that Aβ40 and MMP-2 were deposited in venous endothelial cells at day 3 and in arterial endothelial cells at day 14 after hypoperfusion. CONCLUSION: This study indicated that DL-NBP has therapeutic effects on chronic cerebral hypoperfusion and provided a useful insight into the potential molecular mechanisms underlying the therapeutic effect of DL-NBP in chronic cerebral hypoperfusion.

Adult and fetal human mesangial cells interact with specific laminin domains

1991 ◽  
Vol 261 (4) ◽  
pp. F688-F695
Author(s):  
B. S. Weeks ◽  
J. B. Kopp ◽  
S. Horikoshi ◽  
F. B. Cannon ◽  
M. Garrett ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Cell Attachment ◽  
Binding Protein ◽  
Laminin Binding Protein ◽  
Laminin Binding

Mesangial cells are centrally located pericytes in the renal glomerulus. They are surrounded by an extracellular matrix and directly contact the glomerular basement membrane in vivo. Because these interactions are critical for renal development and function, we have studied human mesangial cell interactions with laminin, a major adhesive component of basement membranes present in the extracellular matrix of the mesangium. Human fetal and adult mesangial cell attachment was stimulated by both laminin and the laminin-derived synthetic peptides YIGSR-NH2, CQAGTFALRGDNPQG-NH2, and CIKVAVS-NH2. Furthermore, mesangial cells spread on laminin as well as on both the RGD-containing and CIKVAVS peptides. When added in solution, all three peptides inhibited mesangial cell attachment to laminin, and the latter two peptides inhibited mesangial cell spreading on laminin. Laminin affinity column chromatography demonstrated several low-molecular-mass laminin-binding proteins ranging from between 35 and 42 kDa, which predominated in fetal mesangial cells, whereas a higher molecular mass laminin-binding protein of 65 kDa was predominant in adult mesangial cells. Western blot analysis with an anti-32-kDa laminin-binding protein antibody showed increased expression of both 31- and 42-kDa proteins in fetal mesangial cells when compared with the adult. The antisera to the 32-kDa laminin-binding protein also inhibited fetal mesangial spreading on the CIKVAVS peptide. Western blot analysis with an anti-67-kDa laminin-binding protein antibody revealed a 110-kDa protein in adult mesangial cells that was not present in fetal mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals HtrA1 Is Specifically Up-Regulated in Active Keloid Lesions and Stimulates Keloids Development

2017 ◽  
Author(s):  
Satoko Yamawaki ◽  
Motoko Naitoh ◽  
Hiroshi Kubota ◽  
Rino Aya ◽  
Yasuhiro Katayama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Cell Proliferation ◽  
In Situ Hybridization ◽  
Cell Surface ◽  
Western Blot ◽  
Blot Analysis ◽  
Normal Skin ◽  
Immunohistochemical Analysis

1) Background: Keloids occur after the failure during the wound-healing process, persist the inflammation and are refractory to various treatments. The pathogenesis of keloids is still unclear. We previously analyzed the gene expression profiles in keloid tissue using microarray and Northern blot analysis and found that HtrA1 was markedly upregulated in the keloid lesions. HtrA1 is a member of the HtrA family of serine protease, has been suggested to play a role in the pathogenesis of various diseases including age-related macular degeneration and osteoarthritis by modulating proteins in extracellular matrix or cell surface. We focused on HtrA1, analyzed the localization and the role in keloid pathogenesis. 2) Methods: Twenty seven keloid patients and seven unrelated patients were enrolled in this study. We performed in situ hybridization analysis, immunohistochemical analysis, western blot analysis and cell proliferation assay. 3) Results: First, the fibroblast-like cells expressed HtrA1 higher in the active keloid lesions than in the surrounding lesions in situ hybridization. Second, the proportion of HtrA1-positive cells in keloid was higher than that of in normal skin significantly in immunohistochemical analysis. Third, HtrA1 protein was up-regulated, relative to normal skin tissue samples in western blot analysis. Finally, silencing of HtrA1 gene expression suppressed the cell proliferation significantly. 4) Conclusion: HtrA1 was highly expressed in keloid tissues and the suppression of HtrA1 gene inhibited the proliferation of keloid-derived fibroblasts. HtrA1 may promote keloid development through accelerating cell proliferation and remodeling keloid-specific extracellular matrix or cell surface molecules. HtrA1 is suggested to have an important role in keloid pathogenesis.

scholarly journals Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice

2006 ◽  
Vol 290 (4) ◽  
pp. H1713-H1720 ◽  
Author(s):  
Bunyen Teng ◽  
Habib R. Ansari ◽  
Peter J. Oldenburg ◽  
J. Schnermann ◽  
S. Jamal Mustafa
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Western Blot ◽  
Smooth Muscle Cells ◽  
Knockout Mice ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Muscle Cells ◽  
Positive Staining

Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A1 receptor (A1AR)-knockout (A1KO) mice and their wild-type (A1WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiotics) through the aortic arch. The efflux was collected at 30-, 60-, and 90-min intervals. The cells were centrifuged, and the pellets were mixed with medium [medium 199-F-12 medium with 10% FBS and 2% antibiotics (for endothelial cells) and advanced DMEM with 10% FBS, 10% mouse serum, 2% GlutaMax, and 2% antibiotics (for smooth muscle cells)] and plated. Endothelial cells were characterized by a cobblestone appearance and positive staining with acetylated LDL labeled with 1,1′-dioctadecyl-3,3,3′,3-tetramethylindocarbocyanine perchlorate. Smooth muscle cells were characterized by positive staining of smooth muscle α-actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was ∼91%. Western blot analysis showed expression of smoothelin in the cells from passages 3, 7, and 11 in A1WT and A1KO mice. Furthermore, the A1AR was characterized by Western blot analysis using an A1AR-specific antibody. To our knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system.

scholarly journals Glycosylation of human proteinase-activated receptor-2 (hPAR2): role in cell surface expression and signalling

2002 ◽  
Vol 368 (2) ◽  
pp. 495-505 ◽  
Author(s):  
Steven J. COMPTON ◽  
Sabrina SANDHU ◽  
Suranga J. WIJESURIYA ◽  
Morley D. HOLLENBERG
Keyword(s):  
Cell Surface ◽  
Western Blot ◽  
Molecular Mass ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mutant Cell ◽  
Surface Expression ◽  
Receptor Expression ◽  
Cell Surface Expression

We have analysed the role of N-linked glycosylation in regulating human proteinase-activated receptor-2 (hPAR2) expression and function. Epitope-tagged wild-type hPAR2 (wt-hPAR2) or hPAR2 that lacked glycosylation sequons (following site-directed mutagenesis) in either the N-terminus [hPAR2N30A (Asn30→Ala)], extracellular loop 2 [ECL2; hPAR2N222Q (Asn222→Gln) or hPAR2N222A (Asn222→Ala)] or both (hPAR2N30A,N222A or hPAR2N30A,N222Q) were expressed in the Chinese-hamster ovary (CHO) fibroblast cell line, Pro5. Western blot analysis of wt-hPAR2 showed mature wt-hPAR2 to have a molecular mass of 55—100kDa, and 33—48kDa following N-glycosidase F deglycosylation. FACS analysis and immunocytochemistry of the wt-hPAR2 and PAR2 mutant cell lines revealed that removal of both glycosylation sequons decreases (50% of wt-hPAR2) cell surface expression. Western blot analysis indicated that both N-linked sites are glycosylated. In functional studies, hPAR2N30A displayed a selective and significant increase in sensitivity towards tryptase. Interestingly, hPAR2N222A displayed a loss in sensitivity towards all PAR2 agonists tested. However, further analysis revealed receptor sensitivity to alanine mutations in this domain, as the more conservative substitution hPAR2N222Q displayed no change in response to PAR2 agonists. hPAR2N30A,N222Q displayed increased sensitivity towards tryptase, but a loss in sensitivity towards trypsin and the synthetic peptide SLIGRL-NH2, although this loss in sensitivity towards trypsin and SLIGRL-NH2 was secondary to changes in cell-surface expression. Finally, expression of sialic-acid-deficient wt-hPAR2 in the CHO Lec2 glycosylation-deficient mutant cell line, showed a 40kDa loss in molecular mass, in addition to a marked and selective increase in sensitivity towards tryptase. We conclude that hPAR2 N-linked glycosylation and sialylation regulates receptor expression and/or signalling.

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