The DPP4 Inhibitor Linagliptin Protects from Experimental Diabetic Retinopathy

Abstract

Background/aims: Dipeptidyl peptidase 4 (DPP4) inhibitors improve glycemic control in type 2 diabetes, however, their influence on the retinal neurovascular unit remains unclear.

Methods: Vasculo- and neuroprotective effects were assessed in experimental diabetic retinopathy and high glucose-cultivated C. elegans, respectively. In STZ-diabetic Wistar rats (diabetes duration of 24 weeks), DPP4 activity (fluorometric assay), GLP-1 (ELISA), methylglyoxal (LC-MS/MS), acellular capillaries and pericytes (quantitative retinal morphometry), SDF-1a and heme oxygenase-1 (ELISA), HMGB-1, Iba1 and Thy1.1 (immunohistochemistry), nuclei in the ganglion cell layer, GFAP (western blot), and IL-1beta, Icam1, Cxcr4, catalase and beta-actin (quantitative RT-PCR) were determined. In C. elegans, neuronal function was determined using worm tracking software.

Results: Linagliptin decreased DPP4 activity by 77% and resulted in an 11.5-fold increase in active GLP-1. Blood glucose and HbA1c were reduced by 13% and 14% and retinal methylglyoxal by 66%. The increase in acellular capillaries was diminished by 70% and linagliptin prevented the loss of pericytes and retinal ganglion cells. The rise in Iba-1 positive microglia was reduced by 73% with linagliptin. In addition, the increase in retinal Il1b expression was decreased by 65%. As a functional correlate, impairment of motility (body bending frequency) was significantly prevented in C. elegans.

Conclusion: Our data suggest that linagliptin has a protective effect on the microvasculature of the diabetic retina, most likely due to a combination of neuroprotective and antioxidative effects of linagliptin on the neurovascular unit.

Fig 1. Pharmacological activity of linagliptin.

(A) Plasma DPP4 activity was quantified by spectrophotometric monitoring in nondiabetic [N], diabetic [D] and linagliptin-treated diabetic animals [D+Lina], (B) total plasma GLP-1 and (C) active plasma GLP-1 were measured by ELISA. Data are expressed as mean ± SD. *P < 0.05, ***P < 0.001, (n = 22 for all parameters).

Fig 2. Metabolic effects of linagliptin.

(A) Blood glucose (n = 22), (B) body weight (n = 22), (C) HbA_1c (measured by affinity chromatography, n = 22), and (D) retinal MG (measured by derivatization with 1,2-diamino-4,5-dimethoxybenzene using HPLC, n = 5) were determined. Data are expressed as mean ± SD. **P < 0.01, ***P < 0.001.

Fig 3. Effect of linagliptin on experimental diabetic retinopathy.

(A) Representative images of PAS-stained retinal digest preparations. Arrowheads indicate pericytes [P] and arrows indicate acellular capillaries [AC]. (B) Numbers of acellular capillaries, and (C) pericyte numbers in capillary areas were determined by quantitative retinal morphometry (n = 7). (D) Representative images of PAS-stained paraffin sections (3 μm), (E) numbers of cell nuclei in the ganglion cell layer (GCL) (n = 5). Data are expressed as mean ± SD. **P < 0.01, ***P < 0.001.

Fig 4. Quantification of glial and microglial activation.

(A) Glial activation was quantified by Western blotting using glial fibrillary acidic protein (GFAP) as a marker (n = 5). (B) Representative blot for GFAP and β-Tubulin as loading control.(C) Assessment of microglial activation by quantification of ionized calcium-binding adapter molecule 1 (Iba-1) positive cells (n = 5), and (D) representative immunohistochemistry of the superficial layer in whole mount preparations. Data are expressed as mean ± SD. **P < 0.01, ***P < 0.001.

Fig 5. Assessment of inflammatory and angiogenic genes.

Expression of pro-inflammatory, (A) Il1b and (B) Icam1, and pro-angiogenic, (C) Cxcr4, markers were determined using by quantitative RT-PCR. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, (n = 7 for all parameters).