Cytokine Profile of Human Abdominal Aortic Aneurysm: Involvement of JAK/STAT Pathway

Abstract

Objective: Abdominal aortic aneurysm (AAA) is characterized by inflammation and destruction of normal tissue architecture. The present study aimed to evaluate the inflammatory signaling cascade by analyzing the cytokines of AAA tissue. Materials and Methods: We analyzed the comprehensive cytokine secretion profiles of 52 cytokines from human AAA in four patients with AAA using fluorescent beads-based multiplex assay. Further, the effect of janus kinase (JAK) inhibition by pyridone 6 on cytokine profiles was also evaluated. Results: Cytokine secretion profiles were found to be similar among the four patients. A high level of JAK/signal transducers and activator of transcription (STAT) pathway activity in AAA tissue in culture was maintained, which may be attributed to the secretion of endogenous JAK-activating cytokines. Inhibition of JAK by pyridone 6 resulted in the suppression of STAT3 phosphorylation and secretion of a subset of chemokines and JAK-activating cytokines. However, the inhibition of JAK had no effect on the secretion of matrix metalloproteinase (MMP)-2, MMP-9, or TGF-β family that is responsible for the metabolism of extracellular matrix. Conclusion: The findings of the present study suggested that AAA tissue exhibits a stereotypical profile of cytokine secretion, where JAK/STAT pathway may play a role in regulating a subset of cytokines. Identification of such a cytokine profile may reveal potential diagnostic markers and therapeutic targets for AAA.

Keywords: JAK/STAT; abdominal aortic aneurysm; cytokines; pyridone 6.

Fig. 1 Human AAA tissue culture and cytokine secretion. (A) Depicted is the procedure of human AAA tissue culture. The anterior aortic wall (approximately 30 mm in length and 15 mm in width) was collected during surgery. The aortic wall was cut into three pieces; proximal, middle, and distal portions of the aortic wall. Each piece was further cut into small pieces (approximately 2 mm×2 mm). Further, the small pieces were transferred to a 12-well tissue culture plate, so that each well received an equal number of pieces from the original proximal, middle, and distal portion of the aortic wall. (B) Culture media was replaced with the fresh DMEM to remove blood clots and tissue debris, 48 h after starting the culture, and further experiments were conducted. After another 48 h of incubating the culture with the indicated concentrations of pyridone 6, the culture media was collected to measure the levels of cytokines secreted into the media. (C) Quantitative analysis of the basal secretion in human AAA tissue culture from four patients is demonstrated for a panel of 49 cytokines. Conditioned media was collected after incubating with human AAA tissue in 12-well culture plates for 48 h, and subjected to the beads-based multiplex assay. Data are expressed as means±standard errors from 4–8 wells of a 12-well culture plate with similar culture conditions.

Fig. 2 Effect of pyridone 6 on the activity of STAT3, MMPs, and TGF-β in human AAA tissue culture. Effect of pyridone 6 on STAT3 activity was evaluated by immunoblotting for activated (phosphorylated) STAT3 (P-STAT3) and total STAT3 after incubation of the AAA tissue with the indicated concentration of pyridone 6 for 48 h. (A) Representative images of immunoblotting for P-STAT3 and STAT3 are shown, and GAPDH was used as the internal control. (B) Expression levels of P-STAT3 normalized to that of GAPDH are represented. (C) Expression levels of STAT3 normalized to that of GAPDH are represented. (D) Secretions of MMP-9 and -2 were evaluated by gelatin zymography of the conditioned media. After collecting the basal secretion for 48 h, AAA tissue culture was treated with or without 100 ng/mL IL-6 and the indicated concentrations of pyridone 6. (E) Basal secretions of TGF-β family are shown in the conditioned media after 48 h of culture. (F) The effect of 10 µM pyridone 6 on the secretions of TGF-β family of cytokines is represented. Data are expressed as means±standard errors. The sample without pyridone 6 treatment was assigned a value of 1. * p<0.05.

Fig. 3 Effect of pyridone 6 on cytokine secretions in human AAA tissue culture. Human AAA tissue was cultured for 48 h without any treatment to measure the basal cytokine secretion (basal secretion). AAA tissue was further cultured with or without 10 µM pyridone 6 for another 48 h (secretion after treatment). The effect of pyridone 6 on cytokine secretion was assessed by the P6/Cont ratio as indicated in Fig. 1. Data are expressed as means±standard errors in the order of the P6/Cont ratio. * p<0.05 for the P6/Cont ratio of the corresponding cytokines.