The online version of this article (https://doi.org/10.1186/s12974-017-0955-x) contains supplementary material, which is available to authorized users.
Tetrahydrobiopterin (BH4) is an essential cofactor in multiple metabolic processes and plays an essential role in maintaining the inflammatory and neurovascular homeostasis. In this study, we have investigated the deleterious effects of BH4 deficiency on retinal vasculature during development.
hph-1 mice, which display deficiency in BH4 synthesis, were used to characterize the inflammatory effects and the integrity of retinal microvasculature. BH4 levels in retinas from hph-1 and wild type (WT) mice were measured by LC-MS/MS. Retinal microvascular area and microglial cells number were quantified in hph-1 and WT mice at different ages. Retinal expression of pro-inflammatory, anti-angiogenic, and neuronal-derived factors was analyzed by qPCR. BH4 supplementation was evaluated in vitro, ex-vivo, and in vivo models.
Our findings demonstrated that BH4 levels in the retina from hph-1 mice were significantly lower by ~ 90% at all ages analyzed compared to WT mice. Juvenile hph-1 mice showed iris atrophy, persistent fetal vasculature, significant increase in the number of microglial cells (p < 0.01), as well as a marked degeneration of the retinal microvasculature. Retinal microvascular alterations in juvenile hph-1 mice were associated with a decreased expression in Norrin (0.2-fold) and its receptor Frizzled-4 (FZD4; 0.51-fold), as well as with an augmented expression of pro-inflammatory factors such as IL-6 (3.2-fold), NRLP-3 (4.4-fold), IL-1β (8.6-fold), and the anti-angiogenic factor thrombospondin-1 (TSP-1; 17.5-fold). We found that TSP-1 derived from activated microglial cells is a factor responsible of inducing microvascular degeneration, but BH4 supplementation markedly prevented hyperoxia-induced microglial activation in vitro and microvascular injury in an ex-vivo model of microvascular angiogenesis and an in vivo model of oxygen-induced retinopathy (OIR).
Our findings reveal that BH4 is a key cofactor in regulating the expression of inflammatory and anti-angiogenic factors that play an important function in the maintenance of retinal microvasculature.
Additional file 1: Figure S1. Morphology of the eyes from hph-1 and WT mice. (A) Representative images of the eyeballs from wild type (WT) and hph-1 mice analyzed by H&E staining at postnatal day 1, 7, and 14. The pictures show a reduction in the size, as well as, appearance of hypertrophy in the iris and persistence of fetal hyaloid vasculature in the eyes of hph-1 mice. (PDF 297 kb)12974_2017_955_MOESM1_ESM.pdf
Additional file 2: Figure S2. Tetrahydrobiopterin (BH4) content in hph-1 mice at early ages. BH4 content (ng/mg of tissue) in retinas from control (WT) and hph-1 mice at postnatal day 7 and 14 (P7 and P14) was measured by using liquid chromatography tandem mass spectrometry (LC-MS/MS). The levels of BH4 were significantly decreased by ~ 90% in the retinas of hph-1 at P7 and P14 compared with WT mice. Results showed in the histograms are expressed as means ± SEM of n = 5 mice for each group. **p < 0.005 and ***p < 0.0001 compared to control. (PDF 109 kb)12974_2017_955_MOESM2_ESM.pdf
Additional file 3: Figure S3. Retinal flat-mounts from hph-1 and WT mice at different ages. Representative images of whole-mounted retinas labeled with TRITC-conjugated lectin endothelial cell marker Bandeiraea simplicifolia showing retinal vasculature from controls (WT) and hph-1 mice at different postnatal ages (P1, P7, P14, and P22). Note that at P1, the vessels in the retina are mainly represented by the hyaloid vasculature. (PDF 263 kb)12974_2017_955_MOESM3_ESM.pdf
Additional file 4: Figure S4. Vascular density in hph-1 and WT mice at early ages. The quantification of the vascular density in both superficial and deep retinal vascular complexes was not significant in hph-1 mice compared to the WT control at P1, P7, and P14. Results showed in the histograms are expressed as means ± SEM of n = 3–4 retinas for each group. ns = not significant compared to the control. (PDF 193 kb)12974_2017_955_MOESM4_ESM.pdf
Additional file 5: Figure S5. Tetrahydrobiopterin (BH4) deficiency is associated with upregulated expression of pro-inflammatory mediators at P14. Quantitative real-time PCR analysis was performed on whole retinas at P7 and P14 from control (WT) and hph-1 animals; control values were set at 1. A significant increase in retinal mRNA expression of IL-1β (p < 0.01), NLRP3 (p < 0.03), and TSP-1 (p < 0.005), but not on IL-6 (p < 0.04) was detected in hph-1 mice at P14. Decrease on Norrin (p < 0.01) and its receptor Frizzled 4 (FZD4; p < 0.001) were detected in hph-1 retinas compared with the control at P14. Norrin was significantly augmented in hph-1 mice at P7. Values are mean ± SEM of n = 9–10 animals per group. The fold changes were normalized to 18S as internal control. Significant differences (p value) in the mRNA levels between control and hph-1 mice are shown in the graphs; *p < 0.05 and **p < 0.001 compared to the control. (PDF 252 kb)12974_2017_955_MOESM5_ESM.pdf
Additional file 6: Figure S6. Interleukin-1β (IL-1β) is localized on microglial cells and Semaphorin 3A (Sema3A) is decreased in the retinas from BH4 deficient mice. (A) Representative confocal images showing immunoreactivity for IL-1β (green) and Iba-1 (red) merged with DAPI (blue and yellow) in retinal cryosections from control (WT) and hph-1 mice at 22-day-old (n = 3 per group). Co-staining of IL-1β with Iba-1 was detected on microglial cells localized in the ganglion cell layer (GCL) and deep plexiform layer (IPL) in retinas from hph-1 mice but not in WT mice. Scale bar = 50 μm. (B) Quantitative real-time PCR analysis was performed on whole retinas at P22 from control (WT) and hph-1 animals; control values were set at 1. A significant decrease in retinal mRNA expression of Sema3A (***p < 0.0001; n = 10), was detected in hph-1 retinas compared with the control. Values are mean ± SEM. The fold changes were normalized to 18S as internal control. (PDF 333 kb)12974_2017_955_MOESM6_ESM.pdf
Additional file 7: Figure S7. Intraocular supplementation of tetrahydrobiopterin (BH4) prevents retinal vasoobliteration in mice exposed to oxygen-induced retinopathy (OIR). Representative images of flat-mounted retinas labeled with fluorescein-labeled Griffonia Simplicifolia Lectin 1 (GSL 1), isolectin B4 to examine vasoobliteration in animals exposed to 75% oxygen from P7 until P9. Animals were intravitreally injected at P7 with 100 μM of BH4 or vehicle (PBS steril 1×) and retinas analyzed at P9. Significant differences (p value) in the vasoobliterated area between vehicle and BH4 treatment after 48 h of hyperoxia are shown in the graphs; ***p < 0.0002 compared to hyperoxia. (PDF 292 kb)12974_2017_955_MOESM7_ESM.pdf
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- Tetrahydrobiopterin (BH4) deficiency is associated with augmented inflammation and microvascular degeneration in the retina
José Carlos Rivera
- BioMed Central
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