Endocrine Abstracts

Changes in the composition of the extracellular matrix (ECM) induce different cell signaling in normal tissues and in pathological conditions. ECM alteration in the adrenal microenvironment may contribute to both the development and function of adrenal gland. The adrenal gland consists of a cortex, with three concentric zones, glomerulosa (ZG), fasciculata (ZF) and reticularis (ZR), and the medulla (M), surrounded by a capsule. Here, we describe the differences among the ECM signatures of fractions obtained from rat adrenal glands, the outer fraction (OF), the inner fraction (IF) and the medulla (M). Sprague-Dawley rats (250 ±30g, n=6) were euthanized by decapitation and adrenal glands were removed and decapsulated to separate the capsule/zona glomerulosa (OF) from the zona fasciculata/reticularis (IF) followed by removal of medulla (M). The samples were frozen in liquid nitrogen and stored at -80°C. ECM enrichment through decellularization was achieved through incubation in 1% Triton-X for 2h30 min at room temperature following incubation in 4.0% deoxycholate until complete decellularization. The absence of remaining intact cells was certified by DNA quantification and microscopy analysis of decellularized tissues stained with DAPI. Pellets containing ECM proteins were treated as described in Trombetta-Lima et al. (J. Proteome Res. 2021, 20, 4693-4707). We compared the proteomic profiles of OF, IF and M fractions samples (n= 6). Raw files of LC-MS/MS were processed into MaxQuant software version 2.1.4 using Andromeda search engine against SwissProt Ratus norvegicus and Mus musculus combined databases (54,778 and 17,121 entries, respectively, downloaded from Uniprot.org November 2022). The total identified and regulated proteins were compared with MatrisomeDB 2.0. Our analysis revealed that 69, 144 and 121 total proteins were detected in OF, IF and M decellularized fractions, respectively. Ninety-two proteins were identified as common to the OF, IF and M fractions, 23 of which are ECM proteins. Among the total proteins from adrenal fractions analyzed, 89 were regulated proteins, 22 of which are ECM proteins. Four proteins were differentially expressed in the OF, 22 in the IF and 8 in the M. Only the OF showed 3 differentially expressed ECM proteins, which are Fibrillin 2 (Fbn2), Collagen XIV alfa1 (Col14a1) and Collagen IV alfa3 (Col4a3) which will be validated by immunoblotting and immunohistochemistry. Therefore, we report differences in ECM composition of the outermost zone of rat adrenal that may influence adrenal pathophysiology.