Increased intraocular insulin-like growth factor-I triggers blood-retinal barrier breakdown

Abstract

Blood-retinal barrier (BRB) breakdown is a key event in diabetic retinopathy and other ocular disorders that leads to increased retinal vascular permeability. This causes edema and tissue damage resulting in visual impairment. Insulin-like growth factor-I (IGF-I) is involved in these processes, although the relative contribution of increased systemic versus intraocular IGF-I remains controversial. Here, to elucidate the role of this factor in BRB breakdown, transgenic mice with either local or systemic elevations of IGF-I have been examined. High intraocular IGF-I, resulting from overexpression of IGF-I in the retina, increased IGF-I receptor content and signaling and led to accumulation of vascular endothelial growth factor. This was parallel to up-regulation of vascular Intercellular adhesion molecule I and retinal infiltration by bone marrow-derived microglial cells. These alterations resulted in increased vessel paracellular permeability to both low and high molecular weight compounds in IGF-I-overexpressing retinas and agreed with the loss of vascular tight junction integrity observed by electron microscopy and the altered junctional protein content. In contrast, mice with chronically elevated serum IGF-I did not show alterations in the retinal vasculature structure and permeability, indicating that circulating IGF-I cannot initiate BRB breakdown. Consistent with a key role of IGF-I signaling in retinal diseases, a strong up-regulation of the IGF-I receptor in human retinas with marked gliosis was also observed. Thus, this study demonstrates that intraocular IGF-I, but not systemic IGF-I, is sufficient to trigger processes leading to BRB breakdown and increased retinal vascular permeability. Therefore, therapeutic interventions designed to counteract local IGF-I effects may prove successful to prevent BRB disruption.

FIGURE 1.

Increased IGF-I signaling in mice overexpressing IGF-I in the retina. A, IGF-I was detected in the aqueous humor (AH) of transgenic but not WT eyes (n = 4). WT and TgIGF-I showed similar IGF-I circulating levels. IGF-I levels were measured by enzyme-linked immunosorbent assay. B, IGF-IR levels in retinas from 3-month-old mice. Tubulin (Tub) was used as a loading control. A representative immunoblot and corresponding densitometric analysis are shown (n = 4 pools, 2 mice/pool). C, the immunohistochemistry for IGF-IR in retinas of 3-month-old mice showed strong signal in cells of the ganglion cell layer and in radial processes of Müller cells (arrowheads). Endothelial cells were positive for IGF-IR in both WT and TgIGF-I (insets, arrows). D, Akt activation in whole-eye extracts. Representative immunoblot and densitometric analysis showing increased phospho-Akt/total Akt proportion in 3-month-old TgIGF-I (n = 4). E, representative immunoblot for VEGF in 10 μl of aqueous humor. Scale bars, 25 μm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

FIGURE 2.

BRB breakdown in transgenic mice with increased intraocular IGF-I. A, retinal vascular permeability to mannitol. Results are normalized to 1 for WT (n = 5). B, in vivo assessment of BRB integrity in 6-month-old mice after intravenous injection of Cy5.5 and scanning with an optical molecular imager. Right, intensity scale. C, permeability of the retinal vasculature to circulating HRP after intravenous injection in 6-month-old mice. In TgIGF-I, an HRP reaction product was detected in cells of the vessel wall and of the retinal parenchyma (right panels, insets). Non-injected TgIGF-I were used as negative controls showing the endogenous peroxidase activity of erythrocytes. Original magnification, 1000×. Nu, nuclei.

FIGURE 3.

Paracellular permeability and TJ integrity in TgIGF-I. A, retinas from 4-month-old mice were analyzed after perfusion with Fluo-ConA to reveal sites with increased paracellular permeability. Only weak binding of Fluo-ConA to venules was observed in WT mice, presumably to the small number of receptors present on the luminal side of endothelial cells. In TgIGF-I mice, strong staining was observed, predominantly in venules (arrows), but also in post-capillary venules (arrowheads) and some capillary areas. B, staining of Fluo-ConA perfused retinas with collagen type IV antibody showed that the Fluo-ConA signal was juxtaposed to that of the basement membrane. Original magnification: 100× in A (left panels), 400× in A (right panels), 200× in B (top panel). Scale bar: 0.5 μm in B (bottom panel). C, transmission electron microscopy of retinal vessels of 6-month-old animals after intravenous HRP injection (n = 3). In WT mice, TJs showed normal appearance, and HRP could not be detected between adjacent endothelial cells (EC). In TgIGF-I, HRP was detected along the interendothelial clefts, indicating disruption of the corresponding TJ. D, details of tight junctions from transgenic vessels in which HRP is detected in the interendothelial space. E, HRP accumulation was also observed in the cytoplasm of pericytes of transgenic vessels. In C and D, arrows indicate cell-to-cell contact areas. In E, arrows indicate HRP reaction product accumulation. BM, basement membrane; L, vessel lumen; P, pericyte. Scale bars: 1 μm in C top panels, 0.2 μm in C bottom panels and D, 0.5 μm in E.

FIGURE 4.

Altered tight junction protein profile in TgIGF-I retinas. A, confocal microscopy of ZO-1 immunolocalization in 6-month-old mice. Clear staining of EC borders was observed in WT mice (upper panels). Under identical scanning conditions, segments of transgenic vessels showed weak or no ZO-1 signal (bottom panels). The reflection mode of the microscope was used to confirm the presence of the vessel (right bottom panel). B, representative immunoblots for retinal occludin (65 kDa), claudin-1 (23 kDa), and claudin-5 (23 and 46 kDa) at 3 months of age (n = 4 pools, 2 mice/pool). C, adherent junction proteins in TgIGF-I retinas. VE-cadherin content in pooled retinas (2 mice/pool, n = 4 pools) of 3-month-old WT and TgIGF-I mice is shown. Tubulin attests to equal loading. Scale bars: 25 μm.

FIGURE 5.

Contribution of BM-derived cells to retinal inflammation in TgIGF-I. A, immunoblot for retinal ICAM-1 (2 mice/pool, n = 4 pools) in 3-month-old mice. Tubulin was used as a loading control. B, TgIGF-I mice showed strong ICAM-1 immunoreactivity on retinal endothelial cells. Collagen type IV immunostaining was used to highlight vascular structures. C, experimental design for the transplantation of donor GFP-expressing BM cells to lethally irradiated 2-month-old mice to determine the contribution of BM-derived cells to retinal inflammation in TgIGF-I. D, GFP⁺ cells of microglial morphology (inset) spread throughout the retinal parenchyma of transplanted mice. Scale bars: B, 25 μm; C, 10 μm.

FIGURE 6.

Effects of chronically elevated serum IGF-I on the eye and retina. A, increased serum IGF-I levels in TgbGH mice in both fed and overnight fasted conditions (n = 10). The expression of bGH is under the control of the phosphoenolpyruvate carboxykinase promoter, which has increased activity during fasting. B, no external macroscopic alterations were observed in the eyes of TgbGH. C, left panel, no histological abnormalities of the cornea, iris, lens, or retina were found in hematoxylin/eosin-stained sections. Right panel, higher magnification of a TgbGH retina showing normal distribution and thickness of retinal layers. D, scanning laser ophthalmoscope images after injection of indocyanine green to evaluate the retinal vessels. E, retinal angiographies showing the similarity in the capillary network between WT and TgbGH retinas. Original magnification: 100×. No differences were found in the leakage of mannitol (F) or Cy5.5 (G), confirming the integrity of the BRB in TgbGH (n = 4). H, no differences in GFAP and VEGF content were observed by Western blot (n = 3, 1 eye/mouse, 2 mice/pool). Tubulin (Tub) documents equal loading. I, GFAP was confined to the retinal surface in TgbGH eyes, discarding the presence of gliosis. A normal pattern of VEGF immunostaining was also observed. Scale bars: 25 μm. All studies were performed in 6-month-old mice. C, cornea; I, iris; L, lenses. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

FIGURE 7.

Up-regulation of IGF-IR in human retinas with gliosis. Immunofluorescent detection of GFAP was used as an indicator of retinal gliosis and ongoing retinal disease. Donors 1 and 2 showed preserved retinal structure with GFAP signal confined to the ganglion cell layer and around blood vessels, indicating the absence of retinopathy. Donors 3, 4, and 5 showed abnormal retinal structure and clear up-regulation of GFAP. IGF-IR was strongly up-regulated in serial sections from donors that also showed marked up-regulation of GFAP (D3, D4, D5). GFAP and IGF-IR showed similar pattern of immunostaining. D, human donor. Scale bars: 25 μm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.