Effects Of Urocortin On The Development Of Vasculitis & COX-2 Expression In Thromboangiitis Obliterans Rat Model And Rat Aortic Endothelial Cells

Thromboangiitis obliterans (TAO) is a nonatherosclerotic, segmental, inflammatory disease that most commonly affects the small and medium-sized arteries, veins and nerves of the extremities. In the characteristic acute phase lesion, in association with occlusive cellular thrombosis, the acute inflammation which involves all layers of the vessel wall leads TAO to be classified as a vasculitis. Cold, tobacco and sexual hormone are reported to have strong associations with TAO. However, the exact etiology for TAO has not been well defined. Furthermore, treatments of TAO are diverse while curative effects of TAO are controversial. Taken together, it is greatly necessary and meaningful to explore the mechanisms of TAO.Urocortin (Ucn), a 40 aa corticotropin-releasing factor (CRF) family peptide, has been demonstrated to be widely expressed in peripheral tissues including cardiovascular system, gastrointestinal tract, immune system, et al. Recently, Ucn has been considered to be a potent locally expressed autocrine or paracrine pro-inflammatory factor in a series of inflammatory diseases, such as rheumatoid arthritis & osteoarthritis and ulcerative colitis. Moreover, it can stimulate the release of pro-inflammatory mediators under inflammatory conditions. In addition, Ucn may have a negative impact on the normal function of ECs by suppressing vascularization.COX-2, the rate-limiting enzyme in metabolism of arachidonic acid, has been abundantly reported to take part in inflammatory diseases including artherosclerosis and TAO. In case-control studies, prostacycline derivatives have shown to be more effective than placebo for the therapy of. Moreover, blockade COX-2 could ameliorate capability of endothelium-dependent vasodilation of brachial arteroy in TAO patients. On the other hand, CRF family peptides could induce COX-2 expression under a series of inflammatory conditions, such as human arthritis, vasculitis and so on. Furthermore, COX-2 pathway is involved in regulation of Ucn-mediated pro-inflammatory cytokine productions, such as TNF-α, IL-6 and so on. COX-2 inhibitor application could abolish the above effects induced by Ucn. Taken together, CRF family peptides may participate in the pathophysiology of many inflammatory conditions via COX-2 pathway.Taken together, it is reasonable to believe that Ucn, which can be synthesized and secreted by systemic vasculature, may be a potent locally expressed pro-inflammatory factor in TAO, which is classified as a vasculitis with endothelial dysfunction. However, Ucn's function on peripheral vasculitis and potential role in modulating TAO progression has rarely been illustrated. In this study, we investigated and first reported the role of Ucn in sodium laurate induced TAO rat model and COX-2 expression. Furthermore, we examined the effects of Ucn on COX-2 expression in lipopolysaccharide (LPS)-induced rat aortic endothelial cells (RAECs) and explore the relevant mechanisms.Part 1 Effects of urocortin on the development of vasculitis and COX-2 expression in thromboangiitis obliterans rat modelBackground and purpose: The present study was performed to examine the effects of Ucn on sodium laurate induced peripheral arterial occlusive disease in rats, exploring the mechanisms of thromboangiitis obliterans (TAO).Experimental approach: Sodium laurate induced peripheral vasculitic rat model was made. The degree of the disease on the 12th day was macroscopically graded. Histopathology and transmission electron microscopy observation were used to investigate the histological changes of rat femoral arteries. The parameters of blood routine, blood rheology, blood coagulation and plasma Ucn, TXB2 and PGE2 levels were measured. The expressions of Ucn, CRFR1/2 and COX-2 at both mRNA and protein levels were determined by RT-PCR and Western blotting.Key results: Most rats showed TAO signs and symptoms on the 12th day after sodium laurate injection. In model group, the blood was in a hypercoagulable state; plasma Ucn and PGE2 levels were elevated; and the expressions of Ucn, CRFR1, CRFR1αsubtype and COX-2 from rat femoral arteries were markedly increased. Ucn application aggravated the hypercoagulable state and augmented COX-2 expression. Furthermore, these effects could be abolished by CRFR1 antagonist, NBI-27914, or nonselective CRFRs antagonist, astressin, while CRFR2 antagonist, antisauvagine-30, could not influence these effects of Ucn. Conclusion and implications: These findings suggest that Ucn can aggravate the hypercoagulable state and vasculitis in sodium laurate induced TAO model rats via CRFR1. COX-2 may participate in this aggravating process.Part 2 Urocortin induced COX-2 expression via corticotrophin releasing factor type 2 receptor in rat aortic endothelial cellsBackground and purpose: The present study was performed to examine the effects of Ucn on COX-2 expression in lipopolysaccharide (LPS)-induced rat aortic endothelial cells (RAECs) and explore the relevant mechanisms.Experimental approach: RAECs were isolated from adult male Wistar rats and identified at the first passage. Experiments were performed on cells at passages 5 through 8 from primary culture. The expression of COX-2 at both mRNA and protein levels were determined by semi-quantitative RT-PCR and Western blotting analysis. Levels of PGE2 in culture medium were measured by ELISA. Furthermore, phosphorylation status of p38MAPK, ERK1/2, JNK, Akt and NF-κB was analyzed by Western blotting; nuclear translocation of NF-κB was observed by immunofluorescence.Key results: Ucn augmented LPS-induced RAECs COX-2 expression in a time- and concentration-dependent manner. Ucn application increased PGE2 levels. These effects could be abolished by CRFR2 antagonist, antisauvagine-30, but not by CRFR1 antagonist, NBI-27914. Moreover, Ucn2 application activated p38MAPK and augmented NF-κB nuclear translocation while ERK1/2, JNK and Akt pathways were not involved in this process.Conclusions and implications: These findings suggest that Ucn exerted a pro-inflammatory function by augmenting LPS-induced RAECs COX-2 expression via CRFR2, in which p38MAPK and NF-κB pathways were involved.The major contributions of the present study lie in:1. The in vivo study suggests that Ucn can aggravate the hypercoagulable state and vasculitis in sodium laurate induced TAO model rats via CRFR1. COX-2 may participate in this aggravating process. These works provide a novel mechanism for the development of TAO.2. The in vitro study suggests that Ucn exerts a pro-inflammatory function by augmenting LPS-induced RAECs COX-2 expression via CRFR2, in which p38MAPK and NF-κB pathways were involved. Targeting CRFR2 may be a novel approach to treat vasculitis. 3. Taken together, the in vivo and in vitro studies strongly indicate that COX-2 is one of the important target of Ucn'participating in vasculitis, which provides a therapeutic approach for vasculitis.