Constitutive high pSMAD2/3 could be due to the fact that these cells were expanded in serum containing medium where endogenous TGFb levels could have been elevated. Presence of the transcripts for the receptor subunits ALK1 and ALK5 in both serum-starved DN and KC fibroblasts indicates that the cells have the means to operate both VE-821 signal axes. Immunohistochemistry of corneal sections also detected pSMAD2/3 and pSMAD1/5/8 in the stroma associated with keratocytes. The pSMAD1/5/8 staining appeared stronger in the KC corneas, although this remains to be tested in a larger set of DN and KC corneas. By IHC staining we had also detected increased staining of TGFb2 and pSMAD2/3 in the epithelial layers of keratoconus corneas, but had not tested pSMAD1/ 5/8 in that study. It is not clear at the moment as to how increased phosphorylated intermediates may relate to overall TGFb signal propagation. The TGFb network is complex, and increased ligand or increased pSMAD signal intermediates do not necessarily correlate with increased signal propagation. Moreover, how these signaling axes regulate gene expression programs and contribute to reduced ECM proteins as seen in our proteomic study of KC are not well understood. Interestingly, a group of heritable connective tissue diseases which include syndromic aortic aneurisms, including Marfan syndrome, Loeys-Dietz syndrome, Shprintzen-Goldberg syndrome, all associated with vessel wall weakening, have been traced back to genetic variants in multiple components of the TGFb signal network. Keratoconus may share certain molecular pathways with these diseases, opening new avenues to research its pathogenesis. A body of earlier work on bovine corneas have shown that primary stromal cells plated in serum-free DMEM: F12 with small amounts of platelet-poor horse serum or ITS displayed a dendritic morphology and synthesized keratocan, the corneal keratan sulfate proteoglycan considered to be a marker for keratocytes. Similarly, we recently showed that primary cells extracted from donor human corneas could be plated overnight in growth factor and serum-free DMEM: F12 for attachment and FG-4592 subsequently grown in the presence of ITS and phosphoascorbic acid expressed keratocan.