High levels of serum prolactin protect against diabetic retinopathy by increasing ocular vasoinhibins

Abstract

Objective: Increased retinal vasopermeability (RVP) occurs early in diabetes and is crucial for the development of sight-threatening proliferative diabetic retinopathy (DR). The hormone prolactin (PRL) is proteolytically processed to vasoinhibins, a family of peptides that inhibit the excessive RVP related to DR. Here, we investigate the circulating levels of PRL in association with DR in men and test whether increased circulating PRL, by serving as a source of ocular vasoinhibins, can reduce the pathological RVP in diabetes.

Research design and methods: Serum PRL was evaluated in 40 nondiabetic and 181 diabetic men at various stages of DR. Retinal vasoinhibins were measured in rats rendered hyperprolactinemic by placing two anterior pituitary grafts under the kidney capsule and in PRL receptor-null mice. RVP was determined in hyperprolactinemic rats subjected to the intraocular injection of vascular endothelial growth factor (VEGF) or made diabetic with streptozotocin.

Results: The circulating levels of PRL increased in diabetes and were higher in diabetic patients without retinopathy than in those with proliferative DR. In rodents, hyperprolactinemia led to vasoinhibin accumulation within the retina; genetic deletion of the PRL receptor prevented this effect, indicating receptor-mediated incorporation of systemic PRL into the eye. Hyperprolactinemia reduced both VEGF-induced and diabetes-induced increase of RVP. This reduction was blocked by bromocriptine, an inhibitor of pituitary PRL secretion, which lowers the levels of circulating PRL and retinal vasoinhibins.

Conclusions: Circulating PRL influences the progression of DR after its intraocular conversion to vasoinhibins. Inducing hyperprolactinemia may represent a novel therapy against DR.

Figures

FIG. 1.

Circulating levels of PRL are higher in diabetic patients than in nondiabetic control subjects and higher in diabetic subjects with no retinopathy than in those with PDR. A–C: Serum PRL levels measured by ELISA in the whole cohort of nondiabetic control subjects and diabetic patients with NDR, NPDR, or PDR (A); control subjects and patients having type 1 or type 2 diabetes (B); diabetic patients and control subjects having prehypertension (≥120/80 mmHg) or hypertension (≥140/90 mmHg) (C). D: Diabetic groups matched for diabetes duration (NDR, 14.1 ± 2.3 years; NPDR, 14.1 ± 1.3 years; and PDR, 15 ± 1.4 years), glycemia (NDR, 161.2 ± 20.5 mg/dl; NPDR, 176.3 ± 18.7 mg/dl; and PDR, 210.5 ± 34.7 mg/dl), glycosylated hemoglobin (NDR, 8.1 ± 0.5%; NPDR, 9.8 ± 0.8%; and PDR, 10.2 ± 1.05%), cholesterol (NDR, 176.9 ± 16.1 mg/dl; NPDR, 181.8 ± 9.3 mg/dl; and PDR, 208.8 ± 10.5 mg/dl), creatinine (NDR, 1 ± 0.04 mg/dl; NPDR, 0.9 ± 0.05 mg/dl; and PDR, 1.1 ± 0.1 mg/dl), and systolic blood pressure (NDR, 128.2 ± 4.7 mmHg; NPDR, 122.5 ± 3.5 mmHg; and PDR, 130 ± 4.8 mmHg). Data are means ± SEM. Numbers inside bars correspond to n values. *P ≤ 0.001 vs. control; #P < 0.05 vs. NDR. C, control subjects; D, diabetic subjects.

FIG. 2.

Hyperprolactinemia results in higher levels of retinal vasoinhibins via PRL receptor–mediated PRL internalization into the eye. A–C: Rats implanted (AP) or not (Sham) with two APs under the renal capsule for 15 days were injected (Bromo) or not with bromocriptine. A: Serum PRL levels as measured by the Nb2 cell bioassay. *P < 0.05 vs. Sham (n = 20 rats per group). Western blot analysis of vasoinhibin levels in the retina (B) and corresponding densitometric analysis normalized to β-tubulin (C). *P < 0.05 vs. Sham (n = 3, each a pool of four retinas). D: Representative immunohistochemistry of rat ciliary body sections stained with monoclonal anti-PRL receptor (PRLR) antibody or without primary antibody (left) (n = 3 independent experiments). Scale bar, 50 μm. E: Serum PRL levels assessed with the Nb2 cell bioassay in wild-type (wt) and PRLR-null (PRL−/−) mice. *P < 0.05 vs. wild-type mice (n = 8 mice per group). Representative Western blot analysis of retinal vasoinhibins in wild-type and PRLR−/− mice (different lanes from the same gel) (F) and corresponding evaluation of retinal vasoinhibins by densitometry normalized to β-tubulin (G) (n = 3, each a pool of six retinas). Data in A, C, E, and G are means ± SEM. Vi, vasoinhibins.

FIG. 3.

Hyperprolactinemia mitigates excessive retinal vasopermeability in VEGF-treated or diabetic rats. A–C: Rats implanted (AP) or not with two APs under the kidney capsule were injected intravitreally with PBS or 9 μmol/l VEGF and examined 24 h later. A: Representative images of fluorescein-labeled retinas. B: Quantification of vascular area from 10 flat-mounted retinas in each group. Scale bar = 500 μm. C: Retinal vasopermeability determined by the Evans blue assay. *P < 0.05 vs. PBS, #P < 0.05 vs. VEGF-treated nonimplanted rats (n = 3 independent experiments). D–F: Nonimplanted and AP-implanted rats made diabetic for 75 days by streptozotocin were injected (Bromo) or not with bromocriptine and evaluated for serum PRL levels using the Nb2 cell bioassay (*P < 0.05 vs. nondiabetic control and nonimplanted control and diabetic rats; n = 4 rats per group) (D), retinal vasoinhibins by Western blot (pool from four retinas) (E), and retinal vasopermeability by the Evans blue method (n = 4 rats per group) (F). *P < 0.05 vs. nondiabetic control, #P < 0.05 vs. diabetic nonimplanted control, and ##P < 0.05 vs. AP diabetic without bromocriptine. Data in B–D and F are means ± SEM. Vi, vasoinhibins.