Creation, validation, and quantitative analysis of protein expression in vascular tissue microarrays

Background: Tissue microarrays (TMAs) are collections of multiple tissue cores placed in parallel in a single acceptor block and traditionally used to investigate protein expression in neoplastic tissues. We validated the use of TMAs to investigate protein expression in vascular segments. Methods: Vascular tissues were collected from 100 adult subjects undergoing autopsy. A diverse set of vessels were harvested and arrayed over 17 TMAs. A total of 1377 unique tissues, each with a 1.5-mm feature size, were analyzed using histochemical and immunohistochemical (IHC) diaminobenzidine (DAB) methods. Results: Histomorphometric analysis of vascular disease demonstrated the TMA features captured the majority of the vascular alterations (intimal hyperplasia and atherosclerosis) seen in the original blood vessel section. Measurements of IHC staining intensity based on color deconvolution were used to quantify antigen abundance in defined regions of interest (ROI). Validation was performed using antibodies to connective tissue growth factor (CTGF), receptor for advanced glycation end products (AGER/RAGE), and matrix metalloproteinase 3 (MMP-3). IHC staining was highly correlated between duplicate features from the same vascular site over these three proteins. Conclusion: This study validates the use of TMA technology to investigate the vascular wall utilizing staining intensity data.