We read with interest both the recent paper by Dunzendorfer et al1 and the accompanying commentary. 2 We wish to clarify some key points. Dunzendorfer et al report that under antiatherogenic conditions of laminar flow, human coronary artery endothelial cells (ECs) express low levels of Toll-like receptor 2 (TLR2) mRNA and protein. Dunzendorfer et al also reported that TLR2 mRNA and protein expression was increased under conditions of low shear stress and that laminar flow resulted in protein kinase CK2 phosphorylation of SP1, which inhibited binding of SP1 to the TLR2 promoter. These results conflict in part with data published 2 years previously by Liang et al, 3 although the authors did not cite this work. That study showed low levels of TLR2 mRNA expression in ECs under laminar flow. There was no change in TLR2 expression but increased TLR4 expression and nuclear factor κB activation with low shear stress in ECs.

Moreover, the totality of data reported to date do not unequivocally demonstrate TLR2 expression by ECs that is functionally relevant and, instead, support a much more prominent role for TLR4-mediated signaling in ECs in diseases such as atherosclerosis. It is conceivable that coronary artery ECs, which are exposed to somewhat different hemodynamic forces from microvessel or venous ECs, might express a different repertoire of TLRs. However, human aortic ECs are exposed to similar hemodynamic stresses as coronary artery ECs, and Walton et al were unable to detect transcripts encoding TLR2 in these cells. 4 Furthermore, in our hands, human coronary artery ECs, like human dermal microvessel ECs, do not express TLR2 mRNA and are unresponsive to a variety of TLR2 ligands (KSM and MA, unpublished data, 2004). Dunzendorfer and colleagues used immunostaining in combination with fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis to show TLR2 protein expression in coronary artery ECs. Identical monoclonal antibody was used for both studies. The images depicted in Figure 2B of the article1 show weak spotty TLR2 immunofluorescence. From these images, it is impossible to determine the subcellular localization of immunofluorescence signals. They might be localized to the cytoplasm, membrane, nucleus, or some combination of these. This staining pattern is commonly found with nonspecific binding of primary antibody. The study does not show or mention control experiments to exclude this possibility. Control experiments with blocking peptide and isotype control antibody staining would resolve this question. Colocalization studies using confocal microscopy would clarify the subcellular localization of TLR2 protein in ECs. Specificity problems of the TLR2 antibodies used also raise doubts about the FACS analyses. Scatter plots from FACS analysis would indicate the quality of the data, but were not included. Western blotting would establish the significance of TLR2 protein expression in ECs and would corroborate immunofluorescent microscopy and FACS. In the absence of controls, and given the uncertainties in the data presented, it is difficult to evaluate the validity of the TLR2 protein expression results in coronary artery ECs.