The protein expression profile of ACE2 in human tissues

Feria Hikmet; Loren Méar; Mathias Uhlén; Cecilia Lindskog

In order to study the functional role of ACE2 in human tissues, it is necessary to analyze the expression at a cell type-specific level, since smaller subsets of cells may express the receptor that may be below detection limit when mixed with all other cells in a complex tissue sample. Complementing the transcriptomics data with immunohistochemistry not only gives the possibility to define protein localization in different compartments at a single-cell level, but also provides important spatial information in the context of neighboring cells in intact tissue samples. Furthermore, the combination of transcriptomics with proteomics provides an advantageous strategy for studying the functional representation of the human genome. A previous immunohistochemistry study by Hamming et al 11 analyzing ACE2 across a large set of human samples, showed staining in most tissue types examined ( Figure 1 ). Abundant immunoreactivity was displayed in microvilli of small intestine and proximal tubules of kidney, in line with all mRNA expression datasets, but strong positivity was also found in several other organs, including the respiratory system, oral mucosa and skin. While this is partly consistent with transcriptomics data from microarray experiments 5 , the findings are contradictory to all three more recent mRNA expression datasets. In the respiratory system, both alveolar epithelial AT1 and AT2 cells, and epithelial cells of nasopharynx were distinctly stained. The authors also presented a consistent abundant expression of endothelial cells, smooth muscle cells, fibroblasts and adipocytes of all examined tissues, despite the fact that several of the corresponding organs that normally contain high fractions of such stromal and mesenchymal cells had low or absent mRNA levels. The immunohistochemical analysis by Hamming et al was based on a single antibody, quality ensured using three different criteria: 1) incubation of control sections with anti-ACE2 antibody solutions pre-incubated with the synthetic peptide to which the antibody was raised, 2) incubation of control sections with unrelated rabbit polyclonal antibodies, and 3) incubation with PBS instead of primary antibody. All three control sections were negative for immunohistochemistry, however, these methods for validation still do not prove that the antibody is specific for endogenous ACE2 in formalin-fixed, paraffinembedded tissue samples. The antibody thus does not meet the criteria for enhanced validation proposed by the International Working Group for Antibody Validation (IWGAV), formed with representatives from several major academic institutions 12 . IWGAV proposed five different pillars to be used for antibody validation to ensure reproducibility of antibody-based studies and promote stringent strategies for validation. To ensure that an antibody binds to the intended protein target, it is necessary that the validation has been performed in an application-specific manner, using at least one of the strategies suggested by IWGAV.