Inflammatory response of endothelial cells to hepatitis
					C virus recombinant envelope glycoprotein 2 protein exposure

The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.

HCV -; E2 protein -; inflammation -; HUVEC

Authorship

Ana Carolina Urbaczek ⁺+ Corresponding author: anaurba@yahoo.com.br

Laboratório de Imunologia Clínica, Departamento de Análises Clínicas, Escola de Ciências Farmacêuticas, Bauru, SP, BrasilEscola de Ciências FarmacêuticasBrasilBauru, SP, BrasilLaboratório de Imunologia Clínica, Departamento de Análises Clínicas, Escola de Ciências Farmacêuticas, Bauru, SP, Brasil

Lívia Carolina de Abreu Ribeiro

Valdecir Farias Ximenes

Departamento de Química, Faculdade de Ciências, Universidade Estadual Paulista Julio de Mesquita Filho, Bauru, SP, BrasilUniversidade Estadual Paulista Julio de Mesquita FilhoBrasilBauru, SP, BrasilDepartamento de Química, Faculdade de Ciências, Universidade Estadual Paulista Julio de Mesquita Filho, Bauru, SP, Brasil

Ana Afonso

Departamento de Parasitologia Médica, Unidade de Parasitologia Médica e Microbiologia, Instituto de Higiene e Medicina Tropcal, Universidade Nova de Lisboa, Lisboa, PortugalUniversidade Nova de LisboaPortugalLisboa, PortugalDepartamento de Parasitologia Médica, Unidade de Parasitologia Médica e Microbiologia, Instituto de Higiene e Medicina Tropcal, Universidade Nova de Lisboa, Lisboa, Portugal

Departamento de Morfologia e Patologia, Universidade Federal de São Carlos, São Carlos, SP, BrasilUniversidade Federal de São CarlosBrasilSão Carlos, SP, BrasilDepartamento de Morfologia e Patologia, Universidade Federal de São Carlos, São Carlos, SP, Brasil

Grupo de Bioanalítica, Microfabricações e Separações, Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, BrasilUniversidade de São PauloBrasilSão Carlos, SP, BrasilGrupo de Bioanalítica, Microfabricações e Separações, Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brasil

Camila Tita Nogueira

Wesley Cardoso Generoso

Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, BrasilUniversidade Federal de São CarlosBrasilSão Carlos, SP, BrasilDepartamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brasil

Juliana Vieira Alberice

Martina Rudnicki

Escola de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, BrasilUniversidade de São PauloBrasilSão Paulo, SP, BrasilEscola de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil

Renila Ferrer

Luiz Marcos da Fonseca

Paulo Inácio da Costa

+ Corresponding author: anaurba@yahoo.com.br

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (8)

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Fig. 1
: produced envelope glycoprotein 2 (E2) recombinant proteins (sodium dodecyl sulfate polyacrylamide gel electrophoresis 12%). Channel 1: molecular weight marker [BenchMarkTM Protein Ladder (10-220 kDa), Invitrogen]; 2: E2B (approximately 63.5 kDa); 3: E2Y (approximately 50 kDa).

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Fig. 2
: effect of envelope glycoprotein 2 (E2) recombinant proteins on human umbilical vein endothelial cells viability [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay]. Results are expressed as mean and standard deviation of viable cells. The experiments were performed in triplicate. c-: negative control; c+: culture medium RPMI; LPS: lipopolysaccharide (1.0 µg/mL); Sup: culture supernatant Escherichia coli BL21; TNF-α: tumour necrosis factor alpha (10 ng/mL); *: p < 0.05 compared to the negative control; ***: p < 0.001 compared to the negative control.

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Fig. 3
: cytotoxicity of envelope glycoprotein 2 (E2) proteins on human umbilical vein endothelial cells. Results presented as mean and standard deviation of percentage obtained in the assay. In each run, 30,000 cells were analysed and all experiments were performed in triplicate. c-: negative control; early apoptosis: annexin V stained cells; late apoptosis: cells double-positive for annexin V and propidium iodide (PI); LPS: lipopolysaccharide (1.0 µg/mL); necrosis: cells stained with PI; Sup: culture supernatant Escherichia coli BL21; TNF-α: tumour necrosis factor alpha (10 ng/mL); ***: p < 0.001 compared to the negative control.

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Fig. 4
: PARP cleavage in recombinant envelope glycoprotein 2 (E2)-induced apoptosis in human umbilical vein endothelial cells. Cells were pre-treated (C, D) or no (A, B) with N-acetylcysteine (NAC) for 1 h and incubated with recombinant E2 proteins, E2Y (A, C) and E2B (B, D) in different concentrations. The experiments were performed in triplicate. Line 2: 7.81 µg/mL; 3: 15.63 µg/mL; 4: 31.25 µg/mL; 5: 62.5 µg/mL; 6: 125 µg/mL; 7: 250 µg/mL; A-D1: lysate of untreated cells; E1: cells without pre-treatment with NAC and treated with tumour necrosis factor alpha (TNF-α); E2: cells pre-treated with NAC and treated with TNF-α; E3: cells without pre-treatment with NAC and treated with lipopolysaccharide (LPS); E4: cells pre-treated with NAC and treated with LPS; F1-4: actin.

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Fig. 5
: nitric oxide (NO) production by envelope glycoprotein 2 (E2)-stimulated human umbilical vein endothelial cells. Results are expressed as mean and standard deviation of NO produced in µM. The experiments were performed in triplicate. c-: NO spontaneous production, cells and culture medium; LPS: lipopolysaccharide (1.0 µg/mL); NAC: N-acetylcysteine; Sup: culture supernatant Escherichia coli BL21; TNF-α: tumour necrosis factor alpha (10 ng/mL); ***: p < 0.001 compared to the negative control.

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Fig. 6
: hydrogen peroxide production by envelope glycoprotein 2 (E2)-stimulated human umbilical vein endothelial cells. Results presented as mean and standard deviation of the mean fluorescence intensity (MFI). The experiments were performed in triplicate. c-: control fluorescence spontaneous, cells without stimulation; LPS: lipopolysaccharide (1.0 µg/mL); NAC: N-acetylcysteine; Sup: culture supernatant Escherichia coli BL21; TNF-α: tumour necrosis factor alpha (10 ng/mL);***: p < 0.001 in relation to negative control.

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Fig. 7
: interleukin-8 (IL-8) production by envelope glycoprotein 2 (E2)-stimulated human umbilical vein endothelial cells. Results presented as mean and standard deviation. The experiments were performed in triplicate. c-: cells and medium and phosphate-buffered saline (pH 7.2). LPS: lipopolysaccharide (1.0 µg/mL); Sup: culture supernatant Escherichia coli BL21; TNF-α: tumour necrosis factor alpha (10 ng/mL); ***: p < 0.001 compared to negative control; *: p < 0.05 compared to negative control.

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Fig. 8
: vascular endothelial growth factor A (VEGF-A) production by envelope glycoprotein 2 (E2)-stimulated human umbilical vein endothelial cells. Results presented as mean and standard deviation. The experiments were performed in triplicate. LPS: lipopolysaccharide (1.0 µg/mL); TNF-α: tumour necrosis factor alpha (10 ng/mL); Sup: culture supernatant Escherichia coli BL21; c-: cells and medium and phosphate-buffered saline (pH 7.2); **: p < 0.01 compared to the negative control; ***: p < 0.001 compared to the negative control.

Fig. 1 : produced envelope glycoprotein 2 (E2) recombinant proteins (sodium dodecyl sulfate polyacrylamide gel electrophoresis 12%). Channel 1: molecular weight marker [BenchMarkTM Protein Ladder (10-220 kDa), Invitrogen]; 2: E2B (approximately 63.5 kDa); 3: E2Y (approximately 50 kDa).

Fig. 2 : effect of envelope glycoprotein 2 (E2) recombinant proteins on human umbilical vein endothelial cells viability [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay]. Results are expressed as mean and standard deviation of viable cells. The experiments were performed in triplicate. c-: negative control; c+: culture medium RPMI; LPS: lipopolysaccharide (1.0 µg/mL); Sup: culture supernatant Escherichia coli BL21; TNF-α: tumour necrosis factor alpha (10 ng/mL); *: p < 0.05 compared to the negative control; ***: p < 0.001 compared to the negative control.

Fig. 3 : cytotoxicity of envelope glycoprotein 2 (E2) proteins on human umbilical vein endothelial cells. Results presented as mean and standard deviation of percentage obtained in the assay. In each run, 30,000 cells were analysed and all experiments were performed in triplicate. c-: negative control; early apoptosis: annexin V stained cells; late apoptosis: cells double-positive for annexin V and propidium iodide (PI); LPS: lipopolysaccharide (1.0 µg/mL); necrosis: cells stained with PI; Sup: culture supernatant Escherichia coli BL21; TNF-α: tumour necrosis factor alpha (10 ng/mL); ***: p < 0.001 compared to the negative control.

Fig. 4 : PARP cleavage in recombinant envelope glycoprotein 2 (E2)-induced apoptosis in human umbilical vein endothelial cells. Cells were pre-treated (C, D) or no (A, B) with N-acetylcysteine (NAC) for 1 h and incubated with recombinant E2 proteins, E2Y (A, C) and E2B (B, D) in different concentrations. The experiments were performed in triplicate. Line 2: 7.81 µg/mL; 3: 15.63 µg/mL; 4: 31.25 µg/mL; 5: 62.5 µg/mL; 6: 125 µg/mL; 7: 250 µg/mL; A-D1: lysate of untreated cells; E1: cells without pre-treatment with NAC and treated with tumour necrosis factor alpha (TNF-α); E2: cells pre-treated with NAC and treated with TNF-α; E3: cells without pre-treatment with NAC and treated with lipopolysaccharide (LPS); E4: cells pre-treated with NAC and treated with LPS; F1-4: actin.

Fig. 5 : nitric oxide (NO) production by envelope glycoprotein 2 (E2)-stimulated human umbilical vein endothelial cells. Results are expressed as mean and standard deviation of NO produced in µM. The experiments were performed in triplicate. c-: NO spontaneous production, cells and culture medium; LPS: lipopolysaccharide (1.0 µg/mL); NAC: N-acetylcysteine; Sup: culture supernatant Escherichia coli BL21; TNF-α: tumour necrosis factor alpha (10 ng/mL); ***: p < 0.001 compared to the negative control.

Fig. 6 : hydrogen peroxide production by envelope glycoprotein 2 (E2)-stimulated human umbilical vein endothelial cells. Results presented as mean and standard deviation of the mean fluorescence intensity (MFI). The experiments were performed in triplicate. c-: control fluorescence spontaneous, cells without stimulation; LPS: lipopolysaccharide (1.0 µg/mL); NAC: N-acetylcysteine; Sup: culture supernatant Escherichia coli BL21; TNF-α: tumour necrosis factor alpha (10 ng/mL);***: p < 0.001 in relation to negative control.

Fig. 7 : interleukin-8 (IL-8) production by envelope glycoprotein 2 (E2)-stimulated human umbilical vein endothelial cells. Results presented as mean and standard deviation. The experiments were performed in triplicate. c-: cells and medium and phosphate-buffered saline (pH 7.2). LPS: lipopolysaccharide (1.0 µg/mL); Sup: culture supernatant Escherichia coli BL21; TNF-α: tumour necrosis factor alpha (10 ng/mL); ***: p < 0.001 compared to negative control; *: p < 0.05 compared to negative control.

Fig. 8 : vascular endothelial growth factor A (VEGF-A) production by envelope glycoprotein 2 (E2)-stimulated human umbilical vein endothelial cells. Results presented as mean and standard deviation. The experiments were performed in triplicate. LPS: lipopolysaccharide (1.0 µg/mL); TNF-α: tumour necrosis factor alpha (10 ng/mL); Sup: culture supernatant Escherichia coli BL21; c-: cells and medium and phosphate-buffered saline (pH 7.2); : p < 0.01 compared to the negative control; *: p < 0.001 compared to the negative control.

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Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

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[1] + Corresponding author: anaurba@yahoo.com.br