Figure 9 shows the differentially expressed proteins participating in the APR for the RHC-to-control ischemic response comparison (C3), again alongside a companion heat map showing how each protein in the pathway was affected across all three experimental conditions and comparisons. Also provided in tabular form are the fold changes and respective
P values for the expression differences of all of the proteins in the canonical, sorted by comparison. Note the robust downregulation of almost all of the major proteins comprising this particular response to ischemia as a result of parental RHC, including a 3.1- to 3.8-fold downregulation of the A1, A2, and H apolipoprotein transporters; a 4.4-fold downregulation in transthyretin expression (which, among other functions, transports retinol, or vitamin A, in the plasma by associating with retinol binding-proteins); a 1.9- to 5.2-fold downregulation in the levels of the plasma protease regulators alpha-2-HS-glycoprotein, alpha-2-macroglobulin, and hemopexin [HPX], a 3.5- to 4.8-fold downregulation in selected complement factors (CFB, C3, and C9); and a 1.7- to 4.2-fold downregulation in selected coagulation and extracellular matrix regulators, including fibronectin (FN1), plasminogen, prothrombin, and the alpha, beta, and gamma subunits of fibrinogen. Of note, FN1, which serves as a ligand for integrin membrane receptors that modulate actin cytoskeleton signaling and remodeling, in addition to playing a role in protein chaperoning, binding, and cell activation in the plasma, differed in expression nearly fourfold between C1 and C3. Additionally, we found a 2.4- to 7.9-fold downregulation in the selected serine protease inhibitors α1-anti-trypsin (SERPINA1), α1-antichymotrypsin (SERPINA3), and heparin cofactor 2 (SERPIND1). Conversely, the expression of MnSOD, the mitochondrial enzyme that clears superoxide and thus plays a central role combating ischemia-related oxidative stress and a proinflammatory cytokine burden, was upregulated in the F1-*RHC ischemic retina. These changes contrast with those defining the control ischemic response for this pathway (C1,
Supplementary Fig. S4, upper panel), which is characterized by the robust, 2- to 10-fold, significant upregulation of all of these same pathway proteins (except SERPINA1), as well as others in this canonical, secondary to increases in upstream glucocorticoid receptor activation, p38MAPK, and Rac. In turn, the effect of parental RHC (C2) is so robust with regard to inhibiting this overall response systemically that, bioinformatically speaking, the acute phase response does not register as activated (
Supplementary Fig. 4, lower panel), and none of the APR pathway proteins exhibited significant changes in these retinae at 10 days postischemia. The following specific molecular example underscores how robustly parental RHC dampened the APR when using C3 as a reference. The proinflammatory cytokine TGF-β1, which was identified as one of the top-ranking upstream regulators in C1 with a
z-score of 5.3, was expressed at nearly sevenfold lower levels in C3 (
z-score of –1.8), a difference that was also confirmed by our MS results with respect to protein abundance. Additional differences in immune-related protein expression between C1 and C3 are also evident as heat map differences for a number of biological functions listed in
Figure 9, including cellular mobilization, cell invasion, and endocytosis/engulfment of cells.