Four of the selected antibodies (CD244, SH2D1A; RAC1 and RAF1; Table 2) showed no staining even with tissues used as positive controls. Furthermore, double staining with antibodies against CD68 or CD8 together with HCST, TYROBP, PTK2B or PLCG2 revealed that CD68 and CD8 positive cells expressed proteins of the NK-pathway but were not the only inflammatory cells involved in the NK-pathway in the AAA tissue. The results provide strong evidence that this NK Cell Mediated Cytotoxicity Pathway is usually activated in human AAA and useful insight for future studies to dissect the pathogenesis of human AAA. found a significant upregulation of genes and pathways related to immune response and inflammation [10], including genes previously identified in AAA tissue such as and [10] in NVP-BHG712 isomer AAA tissue. To accomplish this, we selected nine members, representing different stages of the activation, of the NK Cell Mediated Cytotoxicity Pathway and carried out histological analysis using tissues samples collected from AAA repair operations and infrarenal aortic samples from age-, sex- and ethnicity-matched controls. As NK cells are present in only low numbers in AAA based on previous studies [14,15], the cellular expression of these proteins in other inflammatory cells was characterized using markers for lymphatic cells (CD8+) and monocytes/macrophages (CD68+). 2. Results 2.1. Immunohistochemical Analysis Demonstrates Staining of Members of NK Cell Mediated Cytotoxicity Pathway in Human AAA Tissue We studied the protein expression of nine different members of the NK Cell Mediated Cytotoxicity Pathway (VAV1, VAV3, PLCG1, PLCG2, HCST, TYROBP, PTK2B, TNFA, and GZMB; Physique 1) in human AAA and compared the results to aortic tissue samples taken from the infrarenal aortae of age- and sex-matched controls (Table 1). Eight (VAV1, VAV3, PLCG2, HCST, TYROBP, PTK2B, TNFA, and GZMB) of the corresponding mRNAs of these proteins had been shown to be significantly elevated and one (PLCG1) significantly decreased in AAA compared to non-aneurysmal aortae (Physique 1). Table 1 Aortic tissue samples used for immunohistochemical staining. [15]. All of these studies provided evidence for the NK pathway activation in AAA. Previous results from a microarray-based mRNA expression study comparing AAA and non-aneurysmal infrarenal aortic tissue showed an increase in 43 of 49 differentially expressed genes from the NK pathway [10]. Similarly, prior work using the apolipoprotein E deficient mouse model of aortic aneurysms found an activation of the same pathway in the aortas of animals treated with angiotensin II as compared with controls [28]. Nine (and in the aneurysm group [28]. This is somewhat surprising since the complete activation of the NK pathway would require these gene products and they were NVP-BHG712 isomer clearly elevated in human AAA based on the current and previous studies [29]. TNFA functions in cell apoptosis and induces MMP expression [27]; GZMB can lyse target SAT1 cells [12,30]. Perforin is also known to be expressed in NK cells and cytotoxic T-cells in human AAA [15,31]. In the current study, the immunostaining of selected members in the NK-pathway confirmed the mRNA expression results around the protein level (Physique 2). The histological investigation suggested NK cells were not the only cell type involved in the activation of the NK pathway because most inflammatory cells showed positive staining (Physique 2). NVP-BHG712 isomer A double staining with GZMB and HCST exhibited that products of the beginning of the activation pathway and the final products were produced in the same cell, providing evidence for the complete activation of the pathway (Physique 3 and Supplementary Physique S1). As the number of NK cells is usually reported to be small in AAA [14,15], it was of interest to see which other cell types have an activated NK pathway. The inflammation in the AAA wall is usually characterized mainly by CD3+ T cells and B cells, a few macrophages and NK cells [14,32]. Macrophages (CD68+) and cytotoxic T cells (CD8+) are thought to play an important role in AAA development by expressing perforin (PRF1) which can damage the membrane of the NVP-BHG712 isomer target cell [15]. The mRNA for was detectable only in AAA tissue, not in the non-aneurysmal aorta. Abundant GZMB expression in human AAA tissue by immunostaining was exhibited in a previously published study [30]. In the apolipoprotein E mouse model, GZMB was shown to NVP-BHG712 isomer act independently of perforin in AAA and absence of GZMB decreased the rate of AAA formation [30]. These results were consistent with the microarray-based expression.