Vascular Expression of Transient Receptor Potential Vanilloid 1 (TRPV1)

Dear Editor, In the February 2014 issue of the Journal of Histochemistry & Cytochemistry, Tόth and colleagues published a report on Transient Receptor Potential Vanilloid 1 (TRPV1) expression and function in the rat vascular system (Tόth et al. 2014). The authors found that several commercially available anti-TRPV1 antibodies were not selective for TRPV1, and identified two that were considered to be individually selective for either neuronal or vascular—specifically smooth muscle—TRPV1. We have carried out similar studies on the role of TRPV1 in the mouse vascular system (which shares 95% nucleotide sequence homology with the rat), focusing specifically on endothelial cells. Like Tόth and colleagues, we found several commercially available anti-TRPV1 antibodies lacked specificity for TRPV1, highlighting the importance of conducting functional analysis of TRPV1 expression and activity. We found no evidence of functional TRPV1 expression in isolated murine endothelial cells and smooth muscle cells, despite numerous previous reports to the contrary. These data call into question much of what has been published on TRPV1 expression in the vasculature. The role—and indeed the very presence—of TRPV1 in vascular tissue remain highly contentious. While several groups have reported evidence of endothelial TRPV1 expression (Bratz et al. 2008; Fantozzi et al. 2003; Yang et al. 2010), others have failed to reproduce these findings (Cavanaugh et al. 2011; Marrelli et al. 2007). Many studies have relied on mRNA expression alone, and protein quantification has often been conducted in the absence of appropriate controls, or using antibodies that have not been validated in TRPV1 knockout (KO) tissue. We aimed to clarify these discrepancies using a combination of biochemical and functional analysis in a number of different endothelial cell lines. Using reverse transcription and PCR amplification of vascular cDNA, we found evidence— consistent with the observations of Tόth and colleagues—of TRPV1 mRNA expression in aortic lysates of wild type (WT), but not TRPV1 KO, mice and in freshly isolated and immortalized endothelial cells from three different species (Fig. 1A). TRPV4 expression, here used as a positive control, was clearly evident in all tissues (Fig. 1A), consistent with published reports (Baylie and Brayden 2011). In order to confirm protein expression, we used ACC-030 (Alomone Labs; Jerusalem, Israel), a widely used anti-TRPV1 antibody that, in previous reports, showed no immunoreactivity in samples from TRPV1 KO mice (Yang et al. 2010); albeit, this was in the absence of a protein loading control. We observed clear and distinct bands in aortic and dorsal root ganglia lysates of KO mice of identical origins to those used by Yang et al. (Fig. 1B), suggesting— in line with the results of Tόth and colleagues—that this antibody is not a suitable indicator of TRPV1 protein expression. Furthermore, these bands were observed at approximately 75 kDa, which is 20 kDa smaller than the predicted molecular weight of TRPV1 (Caterina et al. 1997); this is further evidence of non-selective immunoreactivity. We additionally tested three other commercially available antibodies (Table 1) that were similarly non-selective for TRPV1 (Fig. 1C–1E). Although TRPV1 KO mice are functional knockouts and retain the C-terminus of the TRPV1 gene against which most TRPV1 antibodies are raised, sequencing of TRPV1 KO cDNA 581014 JHCXXX10.1369/0022155415581014Sand et al.Vascular expression of TRPV1 research-article2015