Background
Tetrahydrobiopterin (BH4) is a cofactor required by the aromatic amino acid hydroxylases enzymes to the phenylalanine metabolism and neurotransmitter biosynthesis [
1]. BH4 is formed de novo from guanosine triphosphate (GTP), by the committing and rate-limiting enzyme GTP cyclohydrolase I (GTPCH I) [
2]. BH4 acts as an essential cofactor for all nitric oxide synthase (NOS) isoforms and as such regulates for nitric oxide (NO) production [
1]. Thus, GTPCH plays a major role in controlling NOS function [
3].
BH4 is a metabolite that serves as a critical cofactor and inhibits superoxide generation from endothelial nitric oxide synthase (eNOS) [
4]. Under normal physiological conditions, eNOS plays an important role in maintaining the healthy state of the endothelium. For instance, eNOs-derived NO plays a key role to maintain the vascular wall in a quiescent state by inhibition of inflammation, cellular proliferation, and thrombosis [
5]. A deficiency in BH4 can cause the uncoupling of eNOS, leading to a reduction in NO bioavailability and increased reactive oxygen species (ROS) production [
6].
Excessive ROS generation caused by BH4 deficiency has been associated with an augmented inflammatory response followed by endothelial dysfunction and tissue injury in a variety of vascular diseases [
7,
8]. Several pro-inflammatory and growth factors have been reported to be associated with excessive oxidative stress generation thus causing deleterious effects on vasculature [
8]. For instance, interleukin-1β (IL-1β), a major mediator of inflammation, has been implicated in vascular repulsion [
9] and capillary degeneration [
10,
11] in the eye. Under several insults including oxidant stress, microglial cells, the phagocytic sentinels in the retina become over-activated and function as a prominent source of IL-1β [
10,
11], as well as another pro-inflammatory factors such as TNF-α, and IL-6 implicated in retinal microvascular injury [
12‐
14].
Thrombospondin-1 (TSP1), a matricellular glycoprotein, is another important molecule secreted in response to injury and stress in the retina [
15]. TSP1 is significantly increased in the vessels of type 2 diabetic rats [
16] and endothelial progenitor cells from
hph-1 mice [
17]. TSP-1 has a potent anti-angiogenic activity [
18,
19], and mice deficient in TSP1 exhibit increased retinal vascular density [
20]; meanwhile, its over expression results in the attenuation of retinal vascular development [
21]. A previous study in our lab showed that trans-arachidonic acids generated in the retina during nitrative stress induce a TSP1-dependent microvascular degeneration [
15]. In contrast, a recent study showed that GTPCH I overexpression, the rate-limiting enzyme of BH4 synthesis, rescues the regenerative capacity of diabetic-impaired endothelial progenitor cells by suppressing oxidative stress and TSP-1 levels [
17].
Clinical evidences suggest potential benefits from BH4 supplementation by improving vascular function in patients with coronary artery diseases, hypertension, hypercholesterolemia, and diabetic vasculopathy [
22‐
25]. Moreover, BH4 supplementation has shown a marked reduction in vascular dysfunction and infiltration of monocytes, T cells, and macrophages in animal models of atherosclerosis [
26,
27].
Up to now, oxidative stress and inflammation have come to be regarded as important mechanisms involved retinal vascular diseases [
11,
28,
29]. However, the effects of BH4 deficiency and inflammation in the eye have been poorly investigated. Recently it was demonstrated that in response to retinal hyperoxia, enhanced eNOS expression led to increased NOS-derived superoxide and dysfunctional NO production, nitrotyrosine accumulation, and exacerbated vessel closure associated with tetrahydrobiopterin (BH4) insufficiency [
30]. In this study, we have used the
hph-1 mouse, which displays 90% deficiency in guanine triphosphate cyclohydrolase (GTP), the rate-limiting enzyme in BH4 synthesis to characterize the integrity of the retinal vessels, and the inflammatory effects of BH4 deficiency in the eye. Our results suggest that, after some days of BH4 deficiency, an excessive number of activated microglia produces an excess of pro-inflammatory factors such as interleukin-1β (IL-1β), NRLP-3, interleukin-6, as well angiostatic factors such as TSP1 and decreased expression of Norrin and its receptor Frizzled-4 (FZD4) that can be associated with retinal microvascular degeneration.
Methods
Animal care
hph-1 mice were bred in-house and genotyped to confirm homozygous, dominantly inherited GTPCH1 gene deficiency (data not shown). C57BL/6 mice (Charles River, ON, Canada) were utilized as wild type (WT) controls, since this is the background of the hph-1 mice utilized in this study. All experiments were conducted in accordance with ARVO statement regarding the use of animals in ophthalmic and vision research and approved by the Animal Care Committee of the Hôpital Maisonneuve-Rosemont and The Hospital for Sick Children in accordance with guidelines established by the Canadian Council on Animal Care.
Histological analysis of the eyes from WT and hph-1 mice
Eyes enucleated from WT and hph-1 mice at postnatal day 1 (P1), P7, P14, and P22 were immediately immersed in a mixture of 2.5% glutaraldehyde and 4% paraformaldehyde for 24 h. Eyes were dehydrated and embedded in paraffin, and then, serial sections were cut at 10 μm thickness with a microtome and examined to determine the center of optic nerve. Sections were stained with H&E and images obtained in an epifluorescence microscope (Zeiss AxioObserver.Z1) at 10×. Images were merged using the MosiaX option in the AxioVision 4.6.5 software.
Real time quantitative PCR analysis.
Eyes were enucleated from WT and hph-1 mice at postnatal day 7, 14, and 22, and retinas were rapidly dissected and processed for RNA using RiboZol (Amresco, N580, OH, USA). Total cellular RNA was isolated by acidic phenol/chloroform extraction followed by treatment with DNase I (Roche Diagnostics, Mannheim, Germany) to remove any contaminating genomic DNA. 1 μg of total RNA was reverse-transcribed into cDNA using iScript™ RT Supermix (BioRad) as described by manufacturer’s instructions. cDNA was analyzed by Quantitative real-time PCR using iTaq™ Universal SYBR® Green Supermix (BioRad) with primers targeting for "?>IL-1β; (5′- CTGGTACATCAGCACCTCACA-3′ and 5′-GAGCTCCTTAACATGCCCTG-3′), NRLP3; (5′-AGCCAGAGTGGAATGACACG-3′ and 5′-CGTGTAGCGACTGTTGAGGT-3′), IL-6; (5′-CACTTCACAAGTCGGAGGCT-3′ and 5′-CTGCAAGTGCATCATCGTTGT-3′), TSP-1; (5′- GCTGCCAATCATAACCAGCG-3′ and 5′-TTCGTTAAAGGCCGAGTGCT-3′), Norrin; (5′-CGCTGCATGAGACACCATTAT-3′ and 5′-CTCAGAGCGTGATGCCTGG-3′), Frizzled-4; (5′-TTCCTTTGTTCGGTTTATGTGCC-3′ and 5′- CTCTCAGGACTGGTTCACAGC-3′), Sema3A; (5′-GCTCCTGCTCCGTAGCCTGC-3′ and 5′- TCGGCGTTGCTTTCGGTCCC-3′) and Iba-1; (5′-CTGAGGAGCCATGAGCCAAA-3′ and 5′-CCAGCATTCGCTTCAAGGAC-3′). Primers were designed using Primer Bank and NCBI Primer Blast software. Quantitative analysis of gene expression was generated by using an ABI Prism 7500 Sequence Detection System and calculated relative to 18S universal primer pair (Ambion) expression using the ΔcT method.
Retinal flat-mounts
Eyes were enucleated from WT mice and
hph-1 mice at P1, P7, P14, and P22 and fixed in 4% paraformaldehyde for 1 hour at room temperature and then stored in PBS at 4 °C until used. The cornea and lens were removed, and the retina was gently separated from the underlying choroid and sclera under a dissecting microscope. Then, the retinas were incubated overnight at 4 °C in 1% Triton X-100-1 mM CaCl
2/phosphate-buffered saline (PBS) with the TRITC-conjugated lectin endothelial cell marker
Bandeiraea simplicifolia (1:100; Sigma-Aldrich, St. Louis, MO), the specific microglia marker Iba-1 (1:500; Wako Chemicals USA, Inc], or the mouse monoclonal TSP-1 antibody (1:200; EMD Millipore). Iba-1 or TSP-1 antibodies were labeled for 2 h with Alexa-488-conjugated goat anti-rabbit (1:1000) or Alexa-594-conjugated goat anti-mouse (1:500), respectively, obtained from Molecular Probes (Eugene, OR). Retinas were washed in PBS and mounted on microscope slides (Bio Nuclear Diagnostics Inc., Toronto, ON) under cover slips with Fluoro-Gel® (Electron Microscopy Sciences, Hatfield, PA) as the mounting media. Retinas were photographed under a confocal microscope (Olympus, Richmond Hill, Canada) at 30× or an epifluorescence microscope at 10× using a Zeiss AxioObserver.Z1. Images were merged into a single file using the MosiaX option in the AxioVision 4.6.5 software (Zeiss). Vascular density was calculated for the full retina surface by using the software AngioTool as previously described [
31]. AngioTool computes several morphological and spatial parameters including vascular density by assessing the variation in foreground and background pixel mass densities across an image [
31].
Immunofluorescence in retinal cryosections
Eyes were enucleated from WT and hph-1 mice at P22 and then fixed in 4% paraformaldehyde at room temperature for 2 h. Eyes were incubated overnight at 4 °C in a 30% sucrose solution prior to embedding in OCT compound (TissueTek®). Coronal sections of the eyes were cut at a thickness of 10 μm by using a Cryostat (Leica). Sections were subsequently washed with PBS, blocked and permeabilized for 1 h at room temperature and subsequently incubated overnight with Lectin from Bandeiraea simplicifolia TRITC conjugate (Sigma; 1:100) for retinal vasculature and/or antibodies to rabbit Iba-1 (Wako Chemicals USA, Inc.; 1:400), goat IL-1β (R&D systems; 1:300), or rabbit anti-CD36 antibody (Abcam; 1:200). The primary antibodies were labeled for 2 h with Alexa-488 goat anti-rabbit or donkey anti-goat IgG obtained from Molecular Probes (Eugene, OR) and used at dilutions of 1:1000. Samples were visualized using 30× objective with an IX81 confocal microscope (Olympus), and images were obtained with Fluoview 3.1 software (Olympus).
Tetrahydrobiopterin (BH4) content in retinal tissues
Retinal tissues (pool of five retinas per sample,
n = 3 per group) were collected from WT mice and
hph-1 mice at postnatal day 7, 14, and 22. Determination of BH4 in retinal tissues was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS) as previously described [
32]. Briefly, analysis was performed on an AB Sciex 5500QTRAP mass spectrometer (Foster City, CA, USA) coupled with a Shimadzu Nexera ultrahigh pressure liquid chromatograph system (Kyoto, Japan). Pterins were separated by a binary gradient using reversed-phase HPLC on an EZfaast 250 × 2 mm 4 μm AAA-MS column, with a 4 × 2 mm SecurityGuard column. A calibration curve was prepared in water containing 0.2% dithiothreitol (DTT) (Sigma Aldrich, Oakville, ON) over the range of 25–1600 nmol/L for BH4. A deproteinization solution containing internal standards for
15N–BH4 was prepared in 0.1 M perchloric acid (Sigma Aldrich) containing 0.2% DTT at a final concentration of 1000 nmol/L each. Retinal tissue samples were deproteinized and then centrifuged at 14800 rpm at 4 °C for 10 min. Thirty microliter of supernatant was combined with 120 μl of water containing 0.2% DTT. Processed supernatants were transferred to a microtiter plate, and 10 μL was injected for analysis.
Preparation of microglia condition media (MGCM)
Microglia cell line (SIM-A9) was used and cultured as previously reported [
33]. Briefly, microglial cells (800, 000 cells per well) were cultured in 6-well plates (Sarstedt, Inc., Newton, NC, USA) with DMEM/F12 (1:1) supplemented with 10% fetal bovine serum (FBS), 5% of horse serum (HS), and 1% penicillin/streptomycin. After 24 h, the cells were starved with DMEM/F12 (1:1) free of FBS and HS for 6 h. Then, microglial cells cultures in presence or absence of 100 μM of (6R)-5,6,7,8-Tetrahydrobiopterin dihydrochloride (BH4; Sigma, Cat. T4425) were exposed to hyperoxia (75% oxygen and 25% nitrogen; Hyp-MGCM) in a modular incubator chamber (Billups-Rothenberg, Inc) and maintained in a humidified CO
2 incubator at 37 °C for 24 h. Microglial cells in matching controls (Nor-MGCM) were incubated at 37 °C in an incubator with 95% air and 5% CO
2 and collected at the same time point. Cell lysates were quickly processed for RNA using RiboZol (Amresco, N580, OH, USA). The conditioned media was stored at −80 and later used in choroidal explant assay.
Choroidal sprouting assay
WT mice pups were sacrificed on P22, and eyes immediately enucleated and kept in ice-cold EBM-2 medium before dissection. Cornea and lens were removed from the anterior of the eye and choroid-scleral complex was separated from the retina and cut into approximately 1 mm × 1 mm. Choroid/sclera fragments were placed in 30 μL of growth factor-reduced BD Matrigel TM (BD Biosciences, Cat. 354,230) seeded in 24-well plates. After seeding the choroid, plates were incubated at 37 °C in a cell culture incubator without medium for 15 min for the BD MatrigelTM to solidify. Five hundred microliters of EBM-2 medium (LONZA, Cat. CC-3156) supplemented with endothelium growth medium (EGM) kit (Lonza, Cat. CC-4147) and 50 units/mL of Penicillin/Streptomycin (GIBCO, Cat. 15,142) was then added to each well and incubated at 37 °C with 95% air and 5% CO2 for 48 h before any treatment. After this time, the media was removed and choroidal explants were incubated with Nor-MGCM or Hyp-MGCM in presence or absence of a TSP-1 neutralizing antibody (1.2 μg/ml; EMD Millipore) for 48 h. Phase contrast photos of individual explants were taken at days 4 and 6 using a ZEISS AxioOberver microscope, and the microvascular sprouting area was quantified with the computer software ImageJ 1.46r (National Institute of Health).
Oxygen-induced retinopathy
Mice pups were exposed with their mothers in a 75% oxygen environment from postnatal day 7 to P9 using BioSpherix oxycycler (Model #A84XOV) to induce retinal vaso-obliteration (VO) as previously described [
34,
35]. Animals were anesthetized in 3% isoflurane in oxygen and injected intravitreally at P7 with 100 μM of (6R)-5,6,7,8-Tetrahydrobiopterin dihydrochloride (BH4; Sigma, Cat. T4425) or vehicle (sterile PBS 1×) using a Hamilton syringe equipped with 50-gauge glass capillary. At P9, mice pups were sacrificed and retinas were dissected and stained overnight at 4 °C with fluorescein-labeled
Griffonia Simplicifolia Lectin 1 (GSL 1), isolectin B4 (FL 1201, Vector Labs; 1:100) with 1 mM CaCl
2 in PBS as described above. Quantification of VO was assessed using the computer software ImageJ as previously described [
36].
Statistical analysis
Results are expressed as mean ± SEM. Two-tailed independent Student t tests was used to analyze data after confirming that the data are normally distributed. Comparisons between groups were made using one-way ANOVA followed by the post hoc Bonferroni’s multiple comparison test. Statistical significance was set at p < 0.05.
Discussion
Tetrahydrobiopterin (BH4) is an essential cofactor present in all tissues in the body and plays a key role in a number of biological processes including endothelial cell survival and immune response [
47‐
49]. In this study, BH4-deficient mice showed a series of morphological and functional changes in the eye including a decremented reduction in the globe size, hypertrophy iris, persistence of fetal hyaloid vasculature and microvascular degeneration. We focus our study particularly on retinal microvascular injury based in the fact that BH4 deficiency has been associated with an augmented inflammatory response followed by endothelial dysfunction and tissue injury observed in a variety of experimental and clinical vascular diseases [
7,
8]. Similar to a previous report [
45], we have not detected major differences in vascular development between wild-type and
hph-1 mice in both superficial and deep vascular plexuses until postnatal day 14.
Surprisingly, in retinas of BH4-deficient mice evaluated at P22, we detected a dramatic microvascular degeneration in the superficial and deep vascular plexuses associated with an exacerbated inflammatory response characterized by an augmented number of microglial cells, as well as, a high production of pro-inflammatory factors. Therefore, we propose that in the absence of BH4, microglial cells could become activated and secrete factors that play a key role in the microvascular degeneration in the eye of these animals. Microglial cells have been reported to contribute significantly in retinal vascular development [
50,
51]; however, their participation in microvascular degeneration has also been demonstrated [
11,
39].
Under severe insults, microglia become overactivated and functions as a prominent source of cytotoxic oxidant stress and pro-inflammatory factors implicated in microvascular alterations in several ocular diseases in humans [
52] and animal models [
14,
53,
54]. In this regard, high levels of IL-6, NLRP3, IL-1β, and TSP-1 detected in the retinas from
hph-1 mice were considered as possible candidates involved in microvascular degeneration. We focus our attention on IL-1β and TSP-1, based on the fact that both molecules are largely implicated in retinal microvascular degeneration [
11,
15,
20]. For instance, a previous study in our laboratory showed that overactivated microglial cells become the main source of IL-1β, which is implicated in retinal microvascular degeneration not directly, but through the release of pro-apoptotic/repulsive factor Semaphorin 3A (Sema3A) from adjacent neurons [
11]. Surprisingly, in the present study, the levels of Sema3A were found to be decrease, suggesting that IL-1β/Sema3A signaling is probably not implicated in microvascular degeneration in
hph-1 mice.
On the other hand, TSP-1, a matricelllular glycoprotein has also proven to have a potent anti-angiogenic activity in the eye. For intance, mice deficient in TSP-1 have shown an increased retinal vascular density [
20], whereas overexpression of TSP-1 in the eye results in the attenuation of the retinal vascular development [
55]. Previous reports have shown that under oxidative stress, TSP-1 is secreted and responsible to induce retinal microvascular degeneration [
15,
55] by acting through its receptor CD36, localized on endothelium [
56,
57]. In this study, we have detected augmented levels of TSP-1 on activated microglial cells, as well as, its CD36 receptor localized on injured retinal microvasculature from BH4-deficient mice. Per these findings, we suggested that in BH-deficient mice, retinal microglia activated is an important source of TSP-1, which is directly responsible to induce retinal microvascular degeneration in the superficial and deep vascular plexuses.
To demonstrate this hypothesis, microglial cells were in vitro cultured under hyperoxic stress conditions to suppress BH4 levels as previously reported [
45]. We showed that in response to hyperoxic stress, microglial cells become activated and secreted high levels of TSP-1, which in turn caused choroidal microvascular degeneration on the ex-vivo choroidal sprouting assay. This angiostatic effect of microglia-derived TSP-1 was suppressed by BH4 supplementation and/or by using a specific neutralizing TSP-1 antibody. Importantly, these findings were consistent with previous studies demonstrating that BH4 is critical to preserve the function and regenerative capacity on endothelial progenitor cells, at least in part, by suppressing oxidative stress and TSP-1 [
17]. Additional to this, we also have shown that BH4 supplementation decreased microglial activation and TSP-1 release in vitro and significantly diminished oxygen-induced microvascular injury in vivo. This protective effect of BH4 on the retinal vasculature in vivo could be explained in part due to a possible inhibition in the expression of TSP-1, which has been shown to be augmented during the vascular degeneration period that occurs approximately after 24 h of hyperoxia in the oxygen-induced retinopathy (OIR) model [
15]. Likewise, TSP-1-deficient mice are less susceptible to oxygen-induced degeneration [
20].
Besides this, our findings do not fully exclude the contribution of other factors implicated on microvascular damage. For instance, retinal alterations in absence of BH4 were associated with a decreased expression of protective factors such as Wnt ligand Norrin and its receptor FZD4 that play an important role in the maintenance of the deep retinal vasculature. Norrin is a small protein secreted exclusively in Müller cells and is critical in the formation of the deep capillary network during retinal development [
40]. Norrin exerts its angiogenic effects by binding to the Wnt receptor FZD4. That, in cooperation with its co-receptors Lrp5 and the TSPAN12, activates the canonical Wnt/β-catenin signaling pathway in Müller cells or microvascular endothelial cells and triggers the expression of targets genes involved in vascular growth, remodeling, and maintenance [
40,
58]. Accordingly, a deficiency in Norrin, FZD4, Lrp5/6, or TSPAN12 leads to a marked deficiency in intraretinal vascular development [
40]. Moreover, canonical Wnt signaling is also implicated in regression of embryonic hyaloid vessels in the eye [
59]. Mice deficient in Wnt ligand norrin or Wnt co-receptor Lrp5 have persistent embryonic hyaloid vessels in the eye [
60,
61]. These findings can in part explain the association between low levels of norrin and the presence of hyaloid vessels in young adult
hph-1 mice at P22. Therefore, based on all these previous facts, we propose that in addition to TSP-1, the decreased expression of Norrin and FZD4 might be part of the mechanism implicated in the detrimental effect of the deep microvasculature and the persistence of hyaloid vessels in the retina from
hph-1 mice, but this needs to be further investigated.