Background/Purpose: Progressive functional and structural vascular disorder is one of the hallmark features of Systemic Sclerosis (Scleroderma, SSc). Vascular dysfunction leads to dysregulated vascular tone control and activation of vascular smooth muscle cells (vSMCs) resulting in enhanced vasospasm and intimal hyperplasia. We previously reported downregulation of the prostacyclin receptor (IP) in SSc skin and vSMCs. In this study we examined IP signaling in SSc and control vSMCs using an IP-specific agonist. Also, it is now clear that in conditions where IP expression is repressed, as in Pulmonary Arterial Hypertension (PAH) or in experimental IP knockdown, the prostacyclin analog treprostinil may engage other receptors including PPARs, EP1, EP2, EP4, or DP1 to mediate its effects. Thus, we examined the role of these receptors in mediating treprostinil effects.

Methods: vSMCs were isolated from involved SSc skin (no=5) matched healthy subjects. cAMP levels were measured by ELISA after the addition of treprostinil or the specific IP agonist MRE 269, before and after the addition of the epigenetic inhibitors 5-Aza 2 deoxycytidine (Aza) and trichostatin (TSA). Cell proliferation was measured by MTT assay. Treprostinil inhibition of proliferation was tested after the addition of specific antagonists to EP1, EP2, EP4, DP1, PPAR α, δ, and ɣ.

Results: Treprostinil stimulated cAMP expression in SSc and control cells to the same degree, while MRE 269 stimulated cAMP in control cells but failed to stimulate SSc cells. The addition of epigenetic inhibitors Aza and TSA restored MRE 269 ability to stimulate cAMP in SSc cells to a level equal to control values. Treprostinil inhibited vSMCs proliferation in a dose-dependent fashion. The addition of the specific antagonist to EP1, EP4, DP1, PPAR α, and δ to SSc and control vSMCs did not influence the degree of treprostinil mediated inhibition of vSMCs proliferation. The addition of PPAR ɣ antagonist had modest effects on treprostinil mediated inhibition of SSc vSMCs proliferation, while the addition of EP2 antagonist significantly diminished treprostinil inhibition of SSc vSMCs proliferation. The expression levels of EP2 were upregulated 9 folds in SSc vSMCs and PPARɣ 3 folds over levels in control cells. There was no difference in the expression levels of PPAR α, β/ δ, EP1, and EP4 between SSc and control vSMCs and DP1 expression level was significantly repressed in SSc cells.

Conclusion: We identified defective IP receptor signaling in SSc cells that were reversed by the addition of the epigenetic inhibitors confirming the involvement of epigenetics in the repression of IP expression in SSc cells. Moreover, treprostinil stimulated cAMP in SSc and normal cells equally suggesting that treprostinil uses other receptors to mediate its effects. Using specific antagonists, we show that treprostinil engages mainly EP2 in it is effects and to a much less extent PPARɣ. mRNA expression data showed a significant increase expression of EP4 and PPAR- γ in SSc cells when compared to normal cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/upregulation-of-prostanoid-ep2-receptors-and-meditation-of-treprostinil-anti-proliferative-effects-in-scleroderma-smooth-muscle-cells/