Long-Term Treatment of Azathioprine in Rats Induces Vessel Mineralization

Abstract

Medial vascular calcification (mVC) is closely related to cardiovascular disease, especially in patients suffering from chronic kidney disease (CKD). Even after successful kidney transplantation, cardiovascular mortality remains increased. There is evidence that immunosuppressive drugs might influence pathophysiological mechanisms in the vessel wall. Previously, we have shown in vitro that mVC is induced in vascular smooth muscle cells (VSMCs) upon treatment with azathioprine (AZA). This effect was confirmed in the current study in an in vivo rat model treated with AZA for 24 weeks. The calcium content increased in the aortic tissue upon AZA treatment. The pathophysiologic mechanisms involve AZA catabolism to 6-thiouracil via xanthine oxidase (XO) with subsequent induction of oxidative stress. Proinflammatory cytokines, such as interleukin (IL)-1ß and IL-6, increase upon AZA treatment, both systemically and in the aortic tissue. Further, VSMCs show an increased expression of core-binding factor α-1, alkaline phosphatase and osteopontin. As the AZA effect could be decreased in NLRP3−/− aortic rings in an ex vivo experiment, the signaling pathway might be, at least in part, dependent on the NLRP3 inflammasome. Although human studies are necessary to confirm the harmful effects of AZA on vascular stiffening, these results provide further evidence of induction of VSMC calcification under AZA treatment and its effects on vessel structure.

Stressor-Induced “Inflammaging” of Vascular Smooth Muscle Cells via Nlrp3-Mediated Pro-inflammatory Auto-Loop

“…This could be confirmed in the ex vivo setting with Nlrp3 −/− mice, where the calcification induction by DOX is also inhibited compared to control mice. This finding is comparable to our previous results regarding the stressor azathioprine, whose calcifying effect could also no longer be seen in aortic rings from Nlrp3 −/− mice ( 35 ). Therefore, Il-1β appears to be a very important effector cytokine, especially for the maintenance of a SASP after primary initiation of the SASP by the induction of “inflammaging” due to a cellular stressor.…”

“…A microarray study has shown that senescent VSMCs reveal differential regulation of Matrix Gla Protein, Bmp-2, Osteoprotegerin, and Il-1β ( 34 ). In our own previous work, we demonstrated that plasma concentrations of Il-1β and Il-6 were significantly increased in rats treated with azathioprine, another known cellular stressor and ROS inducer, over a 24-weeks period of treatment ( 35 ). Increased expression of Il-1β and Il-6 was also detected in the aortas of treated rats, which were associated with increased expression of the osteogenic proteins Bmp-2, Alp and Cbfa1 ( 35 ).…”

“…In our own previous work, we demonstrated that plasma concentrations of Il-1β and Il-6 were significantly increased in rats treated with azathioprine, another known cellular stressor and ROS inducer, over a 24-weeks period of treatment ( 35 ). Increased expression of Il-1β and Il-6 was also detected in the aortas of treated rats, which were associated with increased expression of the osteogenic proteins Bmp-2, Alp and Cbfa1 ( 35 ). Therefore, we examined the effect of DOX on the expression of Il-1β and Il-6.…”

Biomechanical Properties of the Aortic Wall: Changes during Vascular Calcification

“…The current study aims to establish and validate a method to investigate these biomechanical properties in the process of vascular calcification using an ex vivo setting. For the induction of vascular calcification, two inducers known from in vitro and in vivo settings were used: high phosphate medium and azathioprine (AZA) [7,8]. The results clearly demonstrate that the biomechanical properties were distinguishable between control and stimulation, and therefore provide novel markers to describe vessel functionality during calcification progression.…”

Endoplasmic Reticulum Stress and Pathogenesis of Vascular Calcification

“…The NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is an essential component of the innate immune system and can induce the secretion of the proinflammatory cytokine interleukin 1 beta (IL-1β) in a caspase-1-dependent manner ( 82 , 83 ). Moreover, IL-1β can activate the secretion of the receptor activator of NF-κB ligand, which promotes the formation of osteoclasts, leading to VC ( 84 , 85 ).…”