Response of rat retinal capillary pericytes and endothelial cells to glucose

Abstract

Aim: The purpose of this study was to investigate the effects of hyperglycemia, its fluctuations, and glucose starvation on the expression of glucose-regulated protein 78/binding immunoglobulin protein (GRP78/BiP), one of the most commonly used markers of endoplasmic reticulum stress, in rat capillary pericytes and endothelial cells cultured separately and together.

Methods: Conditionally immortalized rat retinal pericyte and endothelial cell lines were cultured in dishes coated with collagen type I in Dulbecco's modified Eagle's medium containing 5.5 mM glucose. For cocultures, pericytes and endothelial cells were seeded together on rat tail collagen type I-coated cell culture plates. After 24 h of initial culture, the medium was replaced with serum-free medium containing 0-100 mM glucose for periods of up to 72 h. GRP78/BiP, caspase-3, and nuclear factor-κB expression were investigated using western blots.

Results: No significant increase in GRP78/BiP expression was observed when pericytes, endothelial cells, or cocultures were exposed to either 25, 50, or 100 mM glucose for 48 h compared with the control level of 5.5 mM glucose. Similarly, no change in expression of GRP78/BiP was observed when media glucose levels were reduced from either 5.5 or 25 to 1 mM. GRP78/BiP expression significantly increased when cells were cultured for 24 h in glucose-deprived medium. This was accompanied by a time-dependent increase in the expression of caspase-3 and nuclear factor-κB.

Conclusion: In diabetic retinopathy, hyperglycemia has been reported to induce apoptosis in retinal capillary vascular cells, but these studies suggest that the apoptosis is not linked to the expression of GRP78/BiP, one of the most commonly used markers of endoplasmic reticulum stress. However, GRP78/BiP-linked apoptosis may play a role in vascular changes associated with retinal ischemia/reperfusion.

FIG. 1.

Outline of glucose concentrations and their variations utilized with pericytes, endothelial cells, and their cocultures. Color images available online at www.liebertonline.com/jop.

FIG. 2.

Effect of glucose and its fluctuations on GRP78/BiP expression normalized to α-tubulin in (A) pericytes and (B) endothelial cells cultured for 72 h. Mean ± SD; n = 3; *significant (P = 0.05) increases compared with similar cells cultured for 72 h with 5.5 mM glucose. GRP78/BiP, glucose-regulated protein 78/binding immunoglobulin protein; SD, standard deviation. Color images available online at www.liebertonline.com/jop.

FIG. 3.

Effect of increased glucose concentrations on the expression of GRP78/BiP normalized to α-tubulin in (A) pericytes and (B) endothelial cells cultured for 24 h. Mean ± SD; n = 3; *significant (P = 0.05) increases compared with the initial 5.5 mM glucose.

FIG. 4.

Time-dependent effect of glucose deprivation on the expression of GRP78/BiP (A, B), caspase 3 (C, D), and NF-κB (E, F) normalized to α-tubulin in pericytes and endothelial cells, respectively, which were cultured for 24 h in glucose-absent medium. Mean ± SD; n = 3; *significant (P = 0.05) increases compared with the initial 0 time point. NF-κB, nuclear factor-κB.

FIG. 5.

(A) Effect of glucose and its fluctuations on GRP78/BiP expression normalized with α-tubulin in cocultures of pericytes and endothelial cells cultured for 72 h. (B) Effect of glucose deprivation on GRP78/BiP expression normalized with α-tubulin in cocultures of pericytes and endothelial cells cultured for 24 h in glucose-absent media. (C) Effect of increased glucose concentrations on the expression of GRP78/BiP normalized with α-tubulin in cocultures of pericytes and endothelial cells cultured for 24 h. Mean ± SD; n = 3. *In A and C, significant (P = 0.05) increases compared with similar cells cultured for 72 h with 5.5 mM glucose; in B, significant (P = 0.05) increases compared with the initial 0 time point. Color images available online at www.liebertonline.com/jop.