Research Paper Volume 16, Issue 19 pp 12726—12768

Figure 2. Integrated analysis of GWAS BMD hits with baseline proteomics and RNA-seq identified eight targets including Asrgl1 and Timp2. (A) Intersection of baseline proteomics DEPs, RNA-seq DEGs, and a GWAS database [39] revealed 8 targets. Of these, Sost, Asrgl1 and Timp2 were further evaluated histologically. (B) SOST expression was validated using IHC in tibias of young-adult and old mice (transverse sections cut at the proximal diaphysis). SOST expression was restricted to osteocytes. (C) Fewer SOST-positive and (D) total osteocytes (per 600 mm x 600 mm ROI) were present in old bone. (E) The percentage of SOST-positive osteocytes and (F) the areal density of SOST-positive osteocytes were not lower in old bone. (G) Asrgl1 expression was characterized using RNA ISH. (H) Most of the periosteal surface was covered by cells expressing Asrgl1, and this percentage of cells decreased with age. (I) Periosteal Asrgl1 expression was lower in old bone. (J) Slightly less than half of osteocytes (OCY) within cortical bone expressed Asrgl1, and this percentage of OCY was not lower in old bone. (K) The areal density of Asrgl1-positive osteocytes trended to be lower in old bone but did not reach significance. (L) Timp2 expression was characterized using RNA ISH. (M) Most of the periosteal surface was covered by cells expressing Timp2, and this percentage of cells trended to decrease with age. (N) Periosteal Timp2 expression was two times lower in old bone. (O) Most osteocytes within cortical bone expressed Timp2, and this percentage of OCY decreased with age. (P) The areal density of Timp2-positive osteocytes decreased with age. Abbreviations: OCY: Osteocyte; CB: Cortical bone; Ma: Marrow; Mu: Muscle; Pos: Positive; Ps: Periosteal; Surf: Surface). Data shown as mean +/−SD. p-values calculated by unpaired, 2-tailed t-test; n = 6–7 mice per age.