Vasomotor Dysfunction in the Mesenteric Artery after Organ Culture with Cyclosporin A

Abstract

Cyclosporin A (CsA) is a widely used immunosuppressive agent that also causes hypertension. However, the molecular basis for CsA-induced hypertension remains elusive. In this study, we established an in vitro model for CsA-induced pathological changes in vasculature. Rat mesenteric arteries were incubated with CsA for 24 hr in an organ culture system. Both vasocontraction and vasorelaxation in mesenteric artery were studied using myograph technology, and mRNA expressions of the contractile receptors were determined by real-time PCR. The results showed that CsA increased the mRNA expression and contractile function of several G-protein-coupled receptors (GPCRs), such as endothelin receptor type B (ET B ), 5-hydroxytryptamine type 1B (5-HT 1B ) and 1D (5-HT 1D ), in vascular smooth muscle cells. In addition, both nitric oxide (NO)-mediated vasorelaxation and endothelium-independent relaxation were impaired by CsA. In summary, this study showed the enhanced GPCRs-mediated contraction and reduced vasorelaxation in mesenteric artery after organ culture with CsA, which mimicked the CsA-induced pathological changes in the vascular system in vivo. Furthermore, this organ culture system would be an ideal in vitro tool to study the molecular mechanisms of CsA-induced hypertension.Cyclosporin A (CsA) is an immunosuppressive drug most widely used in organ transplantation to prevent organ rejection due to its selectivity and effectiveness. However, the usage of CsA is accompanied by mild to severe side effects, and the clinically most important are nephrotoxicity and hypertension [1]. There is definitive proof for cyclosporine to increase blood pressure in human beings and animals [2], and the underlying mechanisms by which CsA causes arterial hypertension after kidney transplantation are related to the activation of the sympathetic nervous system [3,4], the increased vasoconstriction [5,6] and the impaired vasodilatation [7,8]. Although the molecular mechanisms for CsA-induced hypertension are still under active investigation, it is hampered by the lack of suitable in vitro models to study the roles of specific signalling pathway in CsA-induced vascular pathogenesis.Organ culture of arteries is an in vitro model for vascular endothelium dysfunction [9], phenotypic modulation of vascular smooth muscle cells (VSMC) [10] and enhanced GPCRsmediated contraction [11][12][13], all of which mimic the pathological changes in vascular diseases. Furthermore, involvement of various intracellular signal transduction pathways is conveniently to be determined with this model using specific inhibitors to co-incubate with arteries.This study aimed to establish an in vitro model, organ culture of rat mesenteric arteries with CsA for 24 hr and to examine the effects of CsA on the vasomotor function including contractile receptor-mediated vasoconstriction and vasorelaxation. Our results showed that the enhanced vasocontraction and impaired vasorelaxation in arteries may contribute to the CsA-induced hypertension. MethodsChemicals and...

Cyclosporin A upregulates ETB receptor in vascular smooth muscle via activation of mitogen-activating protein kinases and NF-κB pathways

“…Our previous study showed the ET B -mediated vasoconstriction was enhanced after organ culture with CsA in a dose-dependent manner (Zheng et al, 2013). The present data showed mRNA expression of ET B receptor was augmented by CsA treatment in the dose-and time-dependent manner (Fig.…”

“…1) and protein expression (Fig. 2), and ET B receptor-mediated vasoconstriction (Zheng et al, 2013); and these changes were attenuated by transcription inhibitor AcD (Figs. 3 and 4), suggesting the de novo transcriptional mechanism contributed to upregulation of ET B receptor induced by CsA.…”

“…In the organ culture system, CsA 10 À6 M increased ET B mRNA expression (Fig. 1) but failed to induce the upregulation of ET B -mediated vasoconstriction (Zheng et al, 2013), which might be due to the fact that contractions induced by S6c (ET B -mediated) were more enhanced in small arteries than in larger arteries (Adner et al, 1998). The arteries used in the present study were the superior mesenteric arteries, where CsA 10 À6 M had no effect on ET B -mediated vasoconstriction.…”

“…The ET B receptor-mediated contraction was measured by using 620 M myograph system (DMT, Aarhus N, Denmark) as described previously (Zheng et al, 2013). Briefly, incubated segments were immersed in temperature-controlled myograph bath, and mounted for continuous recording of isometric tension with the Chart software from ADInstruments (Bella Vista, NSW, Australia).…”

“…Although numerous factors have been implicated in the development of CsA-induced hypertension (Hoorn et al, 2012;Niehof and Borlak, 2011), the molecular mechanisms remain obscure. Our previous study with organ culture system showed that CsA upregulated both mRNA expression and contractile function of endothelin B receptor (ET B ) in vasculature (Zheng et al, 2013), but the underlying mechanisms were not completely understood.…”