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Aspirin Action in Endothelial Cells: Different Patterns of Response Between Chemokine CX3CL1/CX3CR1 and TNF-α/TNFR1 Signaling Pathways.

Szukiewicz D, Wojciechowska M, Bilska A, Stangret A, Szewczyk G, Mittal TK, Watroba M, Kochanowski J - Cardiovasc Drugs Ther (2015)

Bottom Line: We examined the effects of aspirin on CX3CL1 and TNF-α production, as well as CX3CR1 and TNFR1 expression.Aspirin significantly (p <  .05) decreased CX3CL1 production, and the mean decrease in CX3CL1 production was inversely proportional to increased (p < 0.05) expression of CX3CR1.Autoregulation between CX3CL1 and CX3CR1 may explain overexpression of CX3CR1 as the compensatory effect in aspirin-treated HUVECs.

View Article: PubMed Central - PubMed

Affiliation: Department of General & Experimental Pathology with Centre for Preclinical Research and Technology (CEPT), Medical University of Warsaw, ul. Pawinskiego 3C, 02-106, Warsaw, Poland, Dszukiewicz@hotmail.com.

ABSTRACT

Purpose: TNF-α induces fractalkine (CX3CL1) and its receptor CX3CR1 in endothelial cells through NF-қB activation. NF-қB inhibitors may reduce the expression of CX3CL1, and modulation of the CX3CL1/CX3CR1 signaling was proposed as a new target for aspirin. We examined the effects of aspirin on CX3CL1 and TNF-α production, as well as CX3CR1 and TNFR1 expression.

Methods: HUVECs isolated after term pregnancies (N = 28) were cultured in vitro. Lipopolysaccharide (1 μg/ml) was used as CX3CL1 inducer. HUVECs were exposed to six different concentrations of aspirin (between 1.0 and 6.0 mM) during 7 days. The levels of CX3CL1 and TNF-α in the culture media were measured using ELISA. After termination of the cultures, mean expressions of CX3CR1 and TNFR1 were examined in the immunostained paraffin sections using quantitative immunohistochemistry.

Results: Aspirin significantly (p <  .05) decreased CX3CL1 production, and the mean decrease in CX3CL1 production was inversely proportional to increased (p < 0.05) expression of CX3CR1. The combined mean CX3CL1 concentrations, including all time points, equaled 782.18 ± 74.4 pg/ml in aspirin treated HUVECs compared to a total concentration of 2467.53 ± 127.5 pg/ml combined from the respective time points in the controls. An inhibition of TNF-α production in HUVECs after pretreatment with aspirin was observed. Unlike in the case of CX3CR1 expression, there were no signs of TNFR1 upregulation.

Conclusions: Autoregulation between CX3CL1 and CX3CR1 may explain overexpression of CX3CR1 as the compensatory effect in aspirin-treated HUVECs. Inhibition of CX3CR1 could prevent thrombotic complications in the early period after discontinuation of aspirin.

No MeSH data available.


Related in: MedlinePlus

a. Immunohistochemical visualization of the receptor TNFR1 (TNFRSF1A) in HUVECs culture (the tones of pink-purple color; the image captured through optical microscope was digitally transformed for morphometric purposes); b. Mean final expressions (Ef) of TNFRSF1A in HUVECs cultures exposured to different doses of aspirin (groups I to VI), including control group VII. The mean initial value of TNFRSF1A expression (Ei) was taken as 100 % for each group. Twenty four visual fields were analysed within each group I–VII (12 at time point Ei and 12 at Ef, respectively)
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Fig5: a. Immunohistochemical visualization of the receptor TNFR1 (TNFRSF1A) in HUVECs culture (the tones of pink-purple color; the image captured through optical microscope was digitally transformed for morphometric purposes); b. Mean final expressions (Ef) of TNFRSF1A in HUVECs cultures exposured to different doses of aspirin (groups I to VI), including control group VII. The mean initial value of TNFRSF1A expression (Ei) was taken as 100 % for each group. Twenty four visual fields were analysed within each group I–VII (12 at time point Ei and 12 at Ef, respectively)

Mentions: The methodological correctness of the immunostaining procedure was confirmed, and visualization of the TNFR1 was adequate for its expression assessment using quantitative immunohistochemistry (Fig. 5a). Unlike in the case of CX3CR1 expression, there were no signs indicating upregulation of TNFR1. The mean expression of the receptor after 7 days of culture (Ef) did not significantly differ from the starting point before aspirin treatment (Ei) but were significantly lower (p < 0.05) in groups V and VI (Fig. 5b). However, the results in the two latter groups may reflect augmented cytotoxicity due to higher doses of aspirin (5.0 and 6.0 mM, respectively). Nonetheless, despite the aspirin-related decreased production of TNF-α in cultured HUVECs, the mean expression of its main receptor TNFR1(TNFRSF1A) remained unchanged or was slightly (p > 0.05) reduced (which should be interpreted with caution, considering cytotoxicity) compared to the initial values (Ei) obtained without pretreatment with aspirin. The difference between the final and initial expression of CX3CR1 in the control (aspirin-free) group VII did not reach statistical significance (p > 0.05).Fig. 5


Aspirin Action in Endothelial Cells: Different Patterns of Response Between Chemokine CX3CL1/CX3CR1 and TNF-α/TNFR1 Signaling Pathways.

Szukiewicz D, Wojciechowska M, Bilska A, Stangret A, Szewczyk G, Mittal TK, Watroba M, Kochanowski J - Cardiovasc Drugs Ther (2015)

a. Immunohistochemical visualization of the receptor TNFR1 (TNFRSF1A) in HUVECs culture (the tones of pink-purple color; the image captured through optical microscope was digitally transformed for morphometric purposes); b. Mean final expressions (Ef) of TNFRSF1A in HUVECs cultures exposured to different doses of aspirin (groups I to VI), including control group VII. The mean initial value of TNFRSF1A expression (Ei) was taken as 100 % for each group. Twenty four visual fields were analysed within each group I–VII (12 at time point Ei and 12 at Ef, respectively)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4522030&req=5

Fig5: a. Immunohistochemical visualization of the receptor TNFR1 (TNFRSF1A) in HUVECs culture (the tones of pink-purple color; the image captured through optical microscope was digitally transformed for morphometric purposes); b. Mean final expressions (Ef) of TNFRSF1A in HUVECs cultures exposured to different doses of aspirin (groups I to VI), including control group VII. The mean initial value of TNFRSF1A expression (Ei) was taken as 100 % for each group. Twenty four visual fields were analysed within each group I–VII (12 at time point Ei and 12 at Ef, respectively)
Mentions: The methodological correctness of the immunostaining procedure was confirmed, and visualization of the TNFR1 was adequate for its expression assessment using quantitative immunohistochemistry (Fig. 5a). Unlike in the case of CX3CR1 expression, there were no signs indicating upregulation of TNFR1. The mean expression of the receptor after 7 days of culture (Ef) did not significantly differ from the starting point before aspirin treatment (Ei) but were significantly lower (p < 0.05) in groups V and VI (Fig. 5b). However, the results in the two latter groups may reflect augmented cytotoxicity due to higher doses of aspirin (5.0 and 6.0 mM, respectively). Nonetheless, despite the aspirin-related decreased production of TNF-α in cultured HUVECs, the mean expression of its main receptor TNFR1(TNFRSF1A) remained unchanged or was slightly (p > 0.05) reduced (which should be interpreted with caution, considering cytotoxicity) compared to the initial values (Ei) obtained without pretreatment with aspirin. The difference between the final and initial expression of CX3CR1 in the control (aspirin-free) group VII did not reach statistical significance (p > 0.05).Fig. 5

Bottom Line: We examined the effects of aspirin on CX3CL1 and TNF-α production, as well as CX3CR1 and TNFR1 expression.Aspirin significantly (p <  .05) decreased CX3CL1 production, and the mean decrease in CX3CL1 production was inversely proportional to increased (p < 0.05) expression of CX3CR1.Autoregulation between CX3CL1 and CX3CR1 may explain overexpression of CX3CR1 as the compensatory effect in aspirin-treated HUVECs.

View Article: PubMed Central - PubMed

Affiliation: Department of General & Experimental Pathology with Centre for Preclinical Research and Technology (CEPT), Medical University of Warsaw, ul. Pawinskiego 3C, 02-106, Warsaw, Poland, Dszukiewicz@hotmail.com.

ABSTRACT

Purpose: TNF-α induces fractalkine (CX3CL1) and its receptor CX3CR1 in endothelial cells through NF-қB activation. NF-қB inhibitors may reduce the expression of CX3CL1, and modulation of the CX3CL1/CX3CR1 signaling was proposed as a new target for aspirin. We examined the effects of aspirin on CX3CL1 and TNF-α production, as well as CX3CR1 and TNFR1 expression.

Methods: HUVECs isolated after term pregnancies (N = 28) were cultured in vitro. Lipopolysaccharide (1 μg/ml) was used as CX3CL1 inducer. HUVECs were exposed to six different concentrations of aspirin (between 1.0 and 6.0 mM) during 7 days. The levels of CX3CL1 and TNF-α in the culture media were measured using ELISA. After termination of the cultures, mean expressions of CX3CR1 and TNFR1 were examined in the immunostained paraffin sections using quantitative immunohistochemistry.

Results: Aspirin significantly (p <  .05) decreased CX3CL1 production, and the mean decrease in CX3CL1 production was inversely proportional to increased (p < 0.05) expression of CX3CR1. The combined mean CX3CL1 concentrations, including all time points, equaled 782.18 ± 74.4 pg/ml in aspirin treated HUVECs compared to a total concentration of 2467.53 ± 127.5 pg/ml combined from the respective time points in the controls. An inhibition of TNF-α production in HUVECs after pretreatment with aspirin was observed. Unlike in the case of CX3CR1 expression, there were no signs of TNFR1 upregulation.

Conclusions: Autoregulation between CX3CL1 and CX3CR1 may explain overexpression of CX3CR1 as the compensatory effect in aspirin-treated HUVECs. Inhibition of CX3CR1 could prevent thrombotic complications in the early period after discontinuation of aspirin.

No MeSH data available.


Related in: MedlinePlus