Polyol effects on growth factors and MAPK signaling in rat retinal capillary cells

Abstract

Purpose: Recent studies report that growth factor and signaling changes in rat lenses do not directly result from the presence of diabetes or sorbitol/galactitol (polyol) formation/accumulation, but from secondary osmotic changes associated with the aldose reductase (AR) catalyzed polyol formation. AR is also present in rat retinal pericyte and endothelial cells; however, significant polyol formation only occurs in pericytes and this does not appear to be linked to osmotic changes. The purpose of this study was to determine whether polyol formation and AR activity are similarly linked to growth factor and signaling changes in the rat capillary cells despite the apparent absence of osmotic stress.

Methods: Conditionally immortalized rat retinal pericyte (TR-rPCT) and endothelial (TR-iBRB) cell lines were cultured on collagen type 1-coated dishes in the DMEM containing 5.5 mM glucose. After 24 h of initial culture, the medium was replaced with a serum-free medium containing 5.5, 25, or 50 mM glucose or galactose with/without the aldose reductase inhibitors (ARIs) AL1576 or tolrestat for periods of up to 48 h. Growth factors and transduction pathways were measured by Western blots using the antibodies against basic FGF, IGF-1, TGF-β, P-ERK1/2, P-SAPK/JNK, and P-Akt.

Results: Sorbitol accumulation was only observed in pericytes, while galactitol was present in both pericytes and endothelial cells. Pericytes cultured in high glucose showed increased expression of the growth factors basic FGF, IGF-1, TGF-β, and signaling in P-Akt, P-ERK1/2, and P-SAPK/JNK compared with those cultured in 5.5 mM glucose and these expressions were normalized by the presence of ARIs. Similar results were observed with galactose media. In contrast, endothelial cells cultured in high glucose media showed neither growth factor or signaling changes. In galactose media, endothelial cells showed increased expression of basic FGF, IGF-1, TGF-β, P-ERK1/2, and P-SAPK/JNK, which were only partially reduced by ARIs.

Conclusion: Growth factor and MAPK signaling expression in pericytes are linked to the presence of polyols. Pericytes, which readily accumulate sorbitol/galactitol that is inhibited by ARIs, show expression changes similar to those observed in rat lenses. In contrast, endothelial cells only show partial expression changes that are linked to galactitol accumulation.

FIG. 1.

Comparison of growth factor expression levels normalized to GAPDH of rat retinal pericytes (TR-rPCT) and endothelial (TR-iBRB) cells cultured for 48 h in media containing up to 50 mM glucose with/without the presence of 10 μM of the aldose reductase inhibitors (ARIs) AL1576 or Tolrestat. Panels (A, C, E) show changes in pericytes of bFGF, IGF-1, and TGF-β levels, respectively, while (B, D, F) show changes in endothelial cell levels of bFGF, IGF-1, and TGF-β, respectively. No expression changes were observed in endothelial cells, while pericytes showed expression changes in high glucose that were normalized by the presence of ARIs. Mean±standard error of mean (SEM); n=3–5. *Significantly different (P≤0.05) compared with 5 mM glucose control.

FIG. 2.

Comparison of expression levels normalized to GAPDH of rat retinal pericytes (TR-rPCT) and endothelial (TR-iBRB) cells cultured for 48 h in media containing up to 50 mM glucose with/without the presence of 10 μM of the ARIs AL1576 or Tolrestat. Panels (A, C, E) show signaling changes in pericytes of Akt, Erk1/2 (44/42 MAPK)m or JNK (SAPK/JNK) levels, respectively, while (B, D, F) show changes in endothelial cell levels of Akt, Erk1/2, or JNK, respectively. No expression changes in response to glucose were observed in endothelial cells. Mean±SEM; n=3–5. *Significantly different (P≤0.05) compared with 5 mM glucose control.

FIG. 3.

Comparison of growth factor expression levels normalized to GAPDH of rat retinal pericytes (TR-rPCT) and endothelial (TR-iBRB) cells cultured for 48 h in media containing up to 50 mM galactose with/without the presence of 10 μM of the ARIs AL1576 or Tolrestat. Panels (A, C, E) show changes in pericytes of bFGF, IGF-1, and TGF-β levels, respectively, while (B, D, F) show changes in endothelial cell levels of bFGF, IGF-1, and TGF-β, respectively. Similar expression changes in response to high galactose were observed in both pericytes and endothelial cells and these changes were reduced by the presence of ARIs. Mean±SEM; n=3–5. *Significantly different (P≤0.05) compared with 5 mM galactose control.

FIG. 4.

Comparison of expression levels normalized to GAPDH of rat retinal pericytes (TR-rPCT) and endothelial (TR-iBRB) cells cultured for 48 h in media containing up to 50 mM galactose with/without the presence of 10 μM of the ARIs AL1576 or Tolrestat. Panels (A, C, E) show signaling changes in pericytes of Akt, Erk1/2 (44/42 MAPK), or JNK (SAPK/JNK) levels, respectively, while (B, D, F) show changes in endothelial cell levels of Akt, Erk1/2, or JNK, respectively. Mean±SEM; n=3–5. *Significantly different (P≤0.05) compared with 5 mM galactose control.