Background/Purpose: Cold exposure induces vasospasm and incites reperfusion injury in SSc. The mechanisms responsible for enhanced cold sensitivity in SSc are poorly understood. Transient receptor potential melastatine8 (TRPM8) is a well characterized cold sensing cation channel. To date, TRPM8 expression has not been described in human microvascular endothelial cells (MVEC). In this study we thought to search for TRPM8 expression in SSc MVEC and skin. We also investigated the effects of TRPM8 activation on MVEC gene expression. 

Methods: MVEC were isolated from involved SSc skin and from matched healthy control subjects. The expression of TRPM8 was determined by RT-PCR, immunohistochemistry and western blotting.  TRPM8 activation in MVEC was triggered by the TRPM8 agonist menthol or by exposure of cells to cold temperature (18C°). The intracellular calcium concentration was determined by Ca²⁺ microfluorometry using the prototypical TRPM8 agonist menthol.  The effects of TRPM8 activation on the mRNA expression levels of ET1, NOS3 and PTGIS and the expression levels of TRPM8 in SSc-MVEC and SSc skin biopsies were quantitated by real time PCR.

Results: TRPM8 gene and protein expression in HMVEC are demonstrated by RT-PCR, Western blotting and immunohistochemistry.  In Ca²⁺ microfluorometry studies, we showed increased MVEC intracellular calcium ([Ca2⁺]_i) in response to the TRPM8 agonist menthol. The activation of the TRPM8 in HMVEC stimulated by cold significantly increased expression of ET1 (2.39 folds ± 0.21) and decreased NOS3 (62% ± 5.1 reduction) and PTGIS (61% ± 4.8) expression levels. These effects were attenuated by the addition of the TRPM8 antagonist capsazepine. Similar results were obtained with menthol treated HMVEC. The TRPM8 mRNA expression levels were significantly increased in SSc-MVEC (2.59 fold ± 0.22 vs. control MVEC) and SSc-skin biopsies (25.52 fold ± 2.28 vs. control skin biopsies).

Conclusion: The study demonstrates that human MVEC express functional TRPM8 and that there is increased expression of TRPM8 in SSc skin and in SSc-MVEC. TRPM8 may be involved in cold-induced vascular dysfunction through increase ET1, and decrease NOS3 and PTGIS mRNA expression. The increased expression levels of TRPM8 in SSc-MVECs and SSc skin may be involved in enhanced cold sensitivity in SSc.  The bulk of TRPM8 skin expression in SSc seems to be extravascular and possibly neuronal.

These results suggest that the blockade of TRPM8 activation could be an effective therapeutic strategy in SSc vasculopathy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/enhanced-expression-of-the-cold-sensing-receptor-trpm8-in-scleroderma-endothelial-cells-and-skin-and-endothelial-dysfunction-following-trpm8-activation/