- Introduction
- Materials and Methods
- Materials.
- Preparation of N. americanus larval exsheathing fluid (EF) and excretory/secretory (ES) products.
- Protease activity assay using casein conjugated to fluorescein isothiocyanate (FITC-casein).
- Endothelial cell culture.
- Measuring the integrity of the endothelial monolayer.
- Immunocytochemistry.
- Cytokine quantification by enzyme-linked immunosorbent assays (ELISA).
- Statistical analyses.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Kumar S, Pritchard DI, 1992. Secretion of metalloproteases by living infective larvae of Necator americanus. J Parasitol 78: 917–919.
-
2.
Loukas A, Prociv P, 2001. Immune responses in hookworm infections. Clin Microbiol Rev 14: 689–703.
-
3.
Quinnell RJ, Bethony J, Pritchard DI, 2004. The immunoepidemiology of human hookworm infection. Parasite Immunol 26: 443–454.
-
4.
Boreham PF, 1984. Activation in vitro of some biological systems by extracts of adult worms and microfilariae of Dirofilaria immitis. J Helminthol 58: 207–212.
-
5.
Fallon PG, Teixeira MM, Neice CM, Williams TJ, Hellewell PG, Doenhoff MJ, 1996. Enhancement of Schistosoma mansoni infectivity by intradermal injections of larval extracts: a putative role for larval proteases. J Infect Dis 173: 1460–1466.
-
6.
Ruffer C, Strey A, Janning A, Kim KS, Gerke V, 2004. Cell-cell junctions of dermal microvascular endothelial cells contain tight and adherens junction proteins in spatial proximity. Biochemistry 43: 5360–5369.
-
7.
Stevens T, Garcia JG, Shasby DM, Bhattacharya J, Malik AB, 2000. Mechanisms regulating endothelial cell barrier function. Am J Physiol Lung Cell Mol Physiol 279: L419–L422.
-
8.
Esser S, Lampugnani MG, Corada M, Dejana E, Risau W, 1998. Vascular endothelial growth factor induces VE-cadherin tyrosine phosphorylation in endothelial cells. J Cell Sci 111: 1853–1865.
-
9.
Vogel SM, Easington CR, Minshall RD, Niles WD, Tiruppathi C, Hollenberg SM, Parrillo JE, Malik AB, 2001. Evidence of transcellular permeability pathway in microvessels. Microvasc Res 61: 87–101.
-
10.
Lampugnani MG, Corada M, Caveda L, Breviario F, Ayalon O, Geiger B, Dejana E, 1995. The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin). J Cell Biol 129: 203–217.
-
11.
Imamura T, Potempa J, Pike RN, Travis J, 1995. Dependence of vascular permeability enhancement on cysteine proteinases in vesicles of Porphyromonas gingivalis. Infect Immun 63: 1999–2003.
-
12.
Han ED, MacFarlane RC, Mulligan AN, Scafidi J, Davis AE 3rd, 2002. Increased vascular permeability in C1 inhibitor-deficient mice mediated by the bradykinin type 2 receptor. J Clin Invest 109: 1057–1063.
-
13.
Hansell E, Braschi S, Medzihradszky KF, Sajid M, Debnath M, Ingram J, Lim KC, McKerrow JH, 2008. Proteomic analysis of skin invasion by blood fluke larvae. PLoS Negl Trop Dis 2: e262.
-
14.
Ali MH, Schlidt SA, Chandel NS, Hynes KL, Schumacker PT, Gewertz BL, 1999. Endothelial permeability and IL-6 production during hypoxia: role of ROS in signal transduction. Am J Physiol 277: L1057–L1065.
-
15.
Petreaca ML, Yao M, Liu Y, Defea K, Martins-Green M, 2007. Transactivation of vascular endothelial growth factor receptor-2 by interleukin-8 (IL-8/CXCL8) is required for IL-8/CXCL8-induced endothelial permeability. Mol Biol Cell 18: 5014–5023.
-
16.
Dejana E, Lampugnani MG, Martinez-Estrada O, Bazzoni G, 2000. The molecular organization of endothelial junctions and their functional role in vascular morphogenesis and permeability. Int J Dev Biol 44: 743–748.
-
17.
Firth J, 2002. Endothelial barriers: from hypothetical pores to membrane proteins. J Anat 200: 524–525.
-
18.
Kumar S, Pritchard DI, 1992. The partial characterization of proteases present in the excretory/secretory products and exsheathing fluid of the infective (L3) larva of Necator americanus. Int J Parasitol 22: 563–572.
-
19.
Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, Wheeler CH, Dunn MJ, 2000. A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21: 3666–3672.
-
20.
Hotez PJ, Narasimhan S, Haggerty J, Milstone L, Bhopale V, Schad GA, Richards FF, 1992. Hyaluronidase from infective Ancylostoma hookworm larvae and its possible function as a virulence factor in tissue invasion and in cutaneous larva migrans. Infect Immun 60: 1018–1023.
-
21.
Brown A, Girod N, Billett EE, Pritchard DI, 1999. Necator americanus (human hookworm) aspartyl proteinases and digestion of skin macromolecules during skin penetration. Am J Trop Med Hyg 60: 840–847.
-
22.
Jaffe EA, Nachman RL, Becker CG, Minick CR, 1973. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52: 2745–2756.
-
23.
Kim EA, Kim JA, Park MH, Jung SC, Suh SH, Pang MG, Kim YJ, 2009. Lysophosphatidylcholine induces endothelial cell injury by nitric oxide production through oxidative stress. J Matern Fetal Neonatal Med 22: 325–331.
-
24.
Williamson AL, Lecchi P, Turk BE, Choe Y, Hotez PJ, McKerrow JH, Cantley LC, Sajid M, Craik CS, Loukas A, 2004. A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms. J Biol Chem 279: 35950–35957.
-
25.
Matthews BE, 1975. Mechanism of skin penetration by Ancylostoma tubaeforme larvae. Parasitology 70: 25–38.
-
26.
Yosipovitch G, Tur E, Morduchowicz G, Boner G, 1993. Skin surface pH, moisture, and pruritus in haemodialysis patients. Nephrol Dial Transplant 8: 1129–1132.
-
27.
Lafleur MA, Hollenberg MD, Atkinson SJ, Knauper V, Murphy G, Edwards DR, 2001. Activation of pro-(matrix metalloproteinase-2) (pro-MMP-2) by thrombin is membrane-type-MMP-dependent in human umbilical vein endothelial cells and generates a distinct 63 kDa active species. Biochem J 357: 107–115.
-
28.
Alexander J, 2002. Extracellular matrix, junctional integrity and matrix metalloproteinase interactions. J Anat 200: 561–574.
-
29.
Johannsson E, Henriksen T, Iversen PO, 2007. Increase in matrix metalloproteinases from endothelial cells exposed to umbilical cord plasma from high birth weight newborns. Am J Physiol Regul Integr Comp Physiol 292: R1563–R1568.
-
30.
Blum MS, Toninelli E, Anderson JM, Balda MS, Zhou J, O'Donnell L, Pardi R, Bender JR, 1997. Cytoskeletal rearrangement mediates human microvascular endothelial tight junction modulation by cytokines. Am J Physiol 273: H286–H294.
-
31.
Nwariaku FE, Liu Z, Zhu X, Nahari D, Ingle C, Wu RF, Gu Y, Sarosi G, Terada LS, 2004. NADPH oxidase mediates vascular endothelial cadherin phosphorylation and endothelial dysfunction. Blood 104: 3214–3220.
-
32.
Hordijk PL, Anthony E, Mul FP, Rientsma R, Oomen LC, Roos D, 1999. Vascular-endothelial-cadherin modulates endothelial monolayer permeability. J Cell Sci 112: 1915–1923.
-
33.
Budworth RA, Anderson M, Clothier RH, Leach L, 1999. Histamine-induced changes in the actin cytoskeleton of the human microvascular endothelial cell line HMEC-1. Toxicol In Vitro 13: 789–795.
-
34.
Leach L, Eaton BM, Westcott ED, Firth JA, 1995. Effect of histamine on endothelial permeability and structure and adhesion molecules of the paracellular junctions of perfused human placental microvessels. Microvasc Res 50: 323–337.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 646 | 549 | 25 |
| Full Text Views | 224 | 6 | 0 |
| PDF Downloads | 87 | 9 | 0 |
Hookworm (Necator americanus) Larval Enzymes Disrupt Human Vascular Endothelium
Nahed Souadkia
Nahed Souadkia
Division of Molecular and Cellular Science, Immune Modulation, Boots Science Building, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom; School of Biomedical Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham Medical School, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom
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Alan Brown
Alan Brown
Division of Molecular and Cellular Science, Immune Modulation, Boots Science Building, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom; School of Biomedical Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham Medical School, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom
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Lopa Leach
Lopa Leach
Division of Molecular and Cellular Science, Immune Modulation, Boots Science Building, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom; School of Biomedical Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham Medical School, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom
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David I. Pritchard
David I. Pritchard
Division of Molecular and Cellular Science, Immune Modulation, Boots Science Building, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom; School of Biomedical Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham Medical School, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom
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Knowledge of the molecular mechanisms used by Necator americanus larvae to penetrate the human skin and the vasculature would aid the development of effective vaccines against this important pathogen. In this work, the impact of N. americanus exsheathing fluid (EF) and excretory/secretory products (ES) on the endothelial barrier was examined using human umbilical vein endothelial cells (HUVEC). Cellular responses were assessed by investigating molecular changes at cell–cell junctions and by determining levels of secreted IL-6, IL-8, and vascular endothelial growth factor (VEGF) in the culture medium. It would appear that a repertoire of larval proteases caused a dose-related increase in endothelial permeability as characterized by a decrease in monolayer resistance with increased permeation of tracer-albumin. These barrier changes were associated with disruption of junctional vascular endothelial cadherin (VE-cadherin) and F-actin and an increase in endothelial secretion of IL-6 and IL-8. Our data suggest that larval proteases play an important role in negotiating the endothelium.
Author Notes
Financial support: We acknowledge the Algerian government for funding this work.
Authors' addresses: Nahed Souadkia, Alan Brown, and David I. Pritchard, School of Pharmacy, Nottingham University, University Park, Nottingham, United Kingdom, E-mail: paxns@nottingham.ac.uk. Lopa Leach, School of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Queen's Medical Centre, United Kingdom.
Reprint requests: Nahed Souadkia, School of Pharmacy, Nottingham University, University Park, Nottingham, NG7 2RD, United Kingdom.
- Introduction
- Materials and Methods
- Materials.
- Preparation of N. americanus larval exsheathing fluid (EF) and excretory/secretory (ES) products.
- Protease activity assay using casein conjugated to fluorescein isothiocyanate (FITC-casein).
- Endothelial cell culture.
- Measuring the integrity of the endothelial monolayer.
- Immunocytochemistry.
- Cytokine quantification by enzyme-linked immunosorbent assays (ELISA).
- Statistical analyses.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Kumar S, Pritchard DI, 1992. Secretion of metalloproteases by living infective larvae of Necator americanus. J Parasitol 78: 917–919.
-
2.
Loukas A, Prociv P, 2001. Immune responses in hookworm infections. Clin Microbiol Rev 14: 689–703.
-
3.
Quinnell RJ, Bethony J, Pritchard DI, 2004. The immunoepidemiology of human hookworm infection. Parasite Immunol 26: 443–454.
-
4.
Boreham PF, 1984. Activation in vitro of some biological systems by extracts of adult worms and microfilariae of Dirofilaria immitis. J Helminthol 58: 207–212.
-
5.
Fallon PG, Teixeira MM, Neice CM, Williams TJ, Hellewell PG, Doenhoff MJ, 1996. Enhancement of Schistosoma mansoni infectivity by intradermal injections of larval extracts: a putative role for larval proteases. J Infect Dis 173: 1460–1466.
-
6.
Ruffer C, Strey A, Janning A, Kim KS, Gerke V, 2004. Cell-cell junctions of dermal microvascular endothelial cells contain tight and adherens junction proteins in spatial proximity. Biochemistry 43: 5360–5369.
-
7.
Stevens T, Garcia JG, Shasby DM, Bhattacharya J, Malik AB, 2000. Mechanisms regulating endothelial cell barrier function. Am J Physiol Lung Cell Mol Physiol 279: L419–L422.
-
8.
Esser S, Lampugnani MG, Corada M, Dejana E, Risau W, 1998. Vascular endothelial growth factor induces VE-cadherin tyrosine phosphorylation in endothelial cells. J Cell Sci 111: 1853–1865.
-
9.
Vogel SM, Easington CR, Minshall RD, Niles WD, Tiruppathi C, Hollenberg SM, Parrillo JE, Malik AB, 2001. Evidence of transcellular permeability pathway in microvessels. Microvasc Res 61: 87–101.
-
10.
Lampugnani MG, Corada M, Caveda L, Breviario F, Ayalon O, Geiger B, Dejana E, 1995. The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin). J Cell Biol 129: 203–217.
-
11.
Imamura T, Potempa J, Pike RN, Travis J, 1995. Dependence of vascular permeability enhancement on cysteine proteinases in vesicles of Porphyromonas gingivalis. Infect Immun 63: 1999–2003.
-
12.
Han ED, MacFarlane RC, Mulligan AN, Scafidi J, Davis AE 3rd, 2002. Increased vascular permeability in C1 inhibitor-deficient mice mediated by the bradykinin type 2 receptor. J Clin Invest 109: 1057–1063.
-
13.
Hansell E, Braschi S, Medzihradszky KF, Sajid M, Debnath M, Ingram J, Lim KC, McKerrow JH, 2008. Proteomic analysis of skin invasion by blood fluke larvae. PLoS Negl Trop Dis 2: e262.
-
14.
Ali MH, Schlidt SA, Chandel NS, Hynes KL, Schumacker PT, Gewertz BL, 1999. Endothelial permeability and IL-6 production during hypoxia: role of ROS in signal transduction. Am J Physiol 277: L1057–L1065.
-
15.
Petreaca ML, Yao M, Liu Y, Defea K, Martins-Green M, 2007. Transactivation of vascular endothelial growth factor receptor-2 by interleukin-8 (IL-8/CXCL8) is required for IL-8/CXCL8-induced endothelial permeability. Mol Biol Cell 18: 5014–5023.
-
16.
Dejana E, Lampugnani MG, Martinez-Estrada O, Bazzoni G, 2000. The molecular organization of endothelial junctions and their functional role in vascular morphogenesis and permeability. Int J Dev Biol 44: 743–748.
-
17.
Firth J, 2002. Endothelial barriers: from hypothetical pores to membrane proteins. J Anat 200: 524–525.
-
18.
Kumar S, Pritchard DI, 1992. The partial characterization of proteases present in the excretory/secretory products and exsheathing fluid of the infective (L3) larva of Necator americanus. Int J Parasitol 22: 563–572.
-
19.
Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, Wheeler CH, Dunn MJ, 2000. A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21: 3666–3672.
-
20.
Hotez PJ, Narasimhan S, Haggerty J, Milstone L, Bhopale V, Schad GA, Richards FF, 1992. Hyaluronidase from infective Ancylostoma hookworm larvae and its possible function as a virulence factor in tissue invasion and in cutaneous larva migrans. Infect Immun 60: 1018–1023.
-
21.
Brown A, Girod N, Billett EE, Pritchard DI, 1999. Necator americanus (human hookworm) aspartyl proteinases and digestion of skin macromolecules during skin penetration. Am J Trop Med Hyg 60: 840–847.
-
22.
Jaffe EA, Nachman RL, Becker CG, Minick CR, 1973. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52: 2745–2756.
-
23.
Kim EA, Kim JA, Park MH, Jung SC, Suh SH, Pang MG, Kim YJ, 2009. Lysophosphatidylcholine induces endothelial cell injury by nitric oxide production through oxidative stress. J Matern Fetal Neonatal Med 22: 325–331.
-
24.
Williamson AL, Lecchi P, Turk BE, Choe Y, Hotez PJ, McKerrow JH, Cantley LC, Sajid M, Craik CS, Loukas A, 2004. A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms. J Biol Chem 279: 35950–35957.
-
25.
Matthews BE, 1975. Mechanism of skin penetration by Ancylostoma tubaeforme larvae. Parasitology 70: 25–38.
-
26.
Yosipovitch G, Tur E, Morduchowicz G, Boner G, 1993. Skin surface pH, moisture, and pruritus in haemodialysis patients. Nephrol Dial Transplant 8: 1129–1132.
-
27.
Lafleur MA, Hollenberg MD, Atkinson SJ, Knauper V, Murphy G, Edwards DR, 2001. Activation of pro-(matrix metalloproteinase-2) (pro-MMP-2) by thrombin is membrane-type-MMP-dependent in human umbilical vein endothelial cells and generates a distinct 63 kDa active species. Biochem J 357: 107–115.
-
28.
Alexander J, 2002. Extracellular matrix, junctional integrity and matrix metalloproteinase interactions. J Anat 200: 561–574.
-
29.
Johannsson E, Henriksen T, Iversen PO, 2007. Increase in matrix metalloproteinases from endothelial cells exposed to umbilical cord plasma from high birth weight newborns. Am J Physiol Regul Integr Comp Physiol 292: R1563–R1568.
-
30.
Blum MS, Toninelli E, Anderson JM, Balda MS, Zhou J, O'Donnell L, Pardi R, Bender JR, 1997. Cytoskeletal rearrangement mediates human microvascular endothelial tight junction modulation by cytokines. Am J Physiol 273: H286–H294.
-
31.
Nwariaku FE, Liu Z, Zhu X, Nahari D, Ingle C, Wu RF, Gu Y, Sarosi G, Terada LS, 2004. NADPH oxidase mediates vascular endothelial cadherin phosphorylation and endothelial dysfunction. Blood 104: 3214–3220.
-
32.
Hordijk PL, Anthony E, Mul FP, Rientsma R, Oomen LC, Roos D, 1999. Vascular-endothelial-cadherin modulates endothelial monolayer permeability. J Cell Sci 112: 1915–1923.
-
33.
Budworth RA, Anderson M, Clothier RH, Leach L, 1999. Histamine-induced changes in the actin cytoskeleton of the human microvascular endothelial cell line HMEC-1. Toxicol In Vitro 13: 789–795.
-
34.
Leach L, Eaton BM, Westcott ED, Firth JA, 1995. Effect of histamine on endothelial permeability and structure and adhesion molecules of the paracellular junctions of perfused human placental microvessels. Microvasc Res 50: 323–337.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 646 | 549 | 25 |
| Full Text Views | 224 | 6 | 0 |
| PDF Downloads | 87 | 9 | 0 |