Treatment response in the PIVENS trial is associated with decreased Hedgehog pathway activity

Abstract

Hedgehog (Hh) ligand production by ballooned hepatocytes drives nonalcoholic steatohepatitis (NASH) progression in mice. The NIDDK-sponsored PIVENS trial (NCT00063622) showed that vitamin E (VitE) improved NASH. We investigated whether VitE treatment and improvement in NASH were associated with changes in Hh pathway activity. Immunohistochemistry (IHC) was performed on both pre- and posttreatment liver biopsies of 59 PIVENS patients randomized to VitE (n = 30) or placebo (n = 29). Sonic Hh (Shh) ligand-producing cells and Shh-responsive cells were quantified. The latter was accomplished by triple IHC for gli2+ (marker of Hh signaling), sox-9 (progenitor marker), and α-smooth muscle actin (α-SMA; myofibroblast marker). Ballooned hepatocytes were quantified by keratin 8/18 and ubiquitin (K8/18/Ub) staining. IHC results were correlated with primary clinical and histologic PIVENS data. Pretreatment clinical, histologic, and IHC parameters did not differ significantly in the two treatment groups. Regardless of treatment arm, the number of Shh+ hepatocytes correlated with K8/18/Ub foci (r(2) = 0.47, P < 0.001) and aspartate aminotransferase (AST) (r(2) = 0.15, P = 0.002). Treatment-related changes in the numbers of Shh+ hepatocytes correlated with changes in serum AST (partial r(2) = 0.75, P < 0.0001), hepatocyte ballooning (P = 0.004), the ductular reaction (i.e., numbers of gli2+/sox9+ cells; P = 0.03 and α-SMA+ cells; P = 0.10), and fibrosis stage (P = 0.02). Treatment response was associated with a greater decrease in Shh+ hepatocytes than nonresponse (P = 0.007). The VitE group demonstrated a greater reduction in K8/18/Ub+ foci (P < 0.08) and Shh+ hepatocytes (P < 0.05) than the placebo group, effects that became more significant after correction for baseline differences and multiple linear regression analysis.

Conclusion: During PIVENS, treatment response correlated with loss of Shh+ hepatocytes and improvement in Hh-regulated processes that promote NASH progression.

FIGURE 1

The plot shows results from simple linear regression analysis between the changes in Shh+ ballooned hepatocytes (horizontal axis) and the changes in K8/18-negative hepatocytes/ubiquitin-positive foci (vertical axis). There was significant positive correlation (p<0.0001).

FIGURE 2

The plot shows simple linear regression between the changes in Shh+ ballooned hepatocytes (horizontal axis) and the changes in serum AST (vertical axis).There was significant positive correlation (p=0.002).

FIGURE 3

Representative photomicrographs of a VitE responder pre- and post-treatment show decreased numbers of Shh+ hepatocytes (A and B, arrows, Shh is brown), decreased numbers of K8/18-negative hepatocytes/Ub+ foci (C and D, arrows, K8/18 is brown, Ub is red), and decreased ductular reaction (E and F, arrows, α-SMA is brown, gli-2 is red, and sox-9 is blue), with marked pericellular fibrosis (inset E) in the pre-treatment biopsy. All photos are at 400× magnification.

FIGURE 4

Model of Hh mediated, deregulated fibrogenic repair in NASH.