Journal of Ophthalmology & Clinical Research Category: Clinical Type: Research Article
Deletion of SPARC Enhances Retinal Vaso-Obliteration in Mouse Model of Oxygen-Induced Retinopathy
- Doaa Sobeih1, Khaled A Hussein2, Neveen Said3, Kouros Mohamed4, Mohamed Al-Shabrawey5*
- 1 Ophthalmology, Oral Biology/Anatomy, Medical College Of Georgia (MCG), Georgia Regents University (GRU), GRU, United States
- 2 Ophthalmology, Oral Biology/anatomy, Medical College Of Georgia (mcg), Georgia Regents University (GRU), Gru, United States
- 3 Department Of Radiation Oncology, University Of Virginia School Of Medicine, United States
- 4 Department Of Opthalmology, Igdrasol, Irivin, CA, United States
- 5 Department Of Oral Biology, College Of Dental Medicine, 1120 15th Street, Augusta, GA 30912, United States
*Corresponding Author:Mohamed Al-Shabrawey
Department Of Oral Biology, College Of Dental Medicine, 1120 15th Street, Augusta, GA 30912, United States
Received Date: Jun 01, 2014 Accepted Date: Aug 08, 2014 Published Date: Aug 22, 2014
It is well known that VEGF angiogenic effects occur through activation of VEGFR-2 (KDR); which is considered the main angiogenic receptor of VEGF. Meanwhile, the role of VEGFR-1(FLT1) is still unclear. Prior reports showed that it negatively affects the pathological NV and counteracts the mitogenic effect of VEGFR-2 [11,12]. Bussolati and his colleagues demonstrated that VEGFR-1 releases Nitric Oxide (NO) that acts as a molecular switch to limit the mitogenic activity of VEGFR-2 and promotes the differentiation of the cells into vascular tubes .
This was confirmed by earlier studies that showed that absence of VEGFR-1 is lethal to mice as a result of abnormal vascular development . Moreover, studies by Rahimi et al., on pig aortic endothelial cells demonstrated that activation of VEGFR-1 halts angiogenesis by antagonizing VEGFR-2 responses . On the other hand, recent studies showed that selective stimulation of VEGFR-1, using its specific agonist (Placenta Growth Factor-1; PlGF-1) was critical to vessel survival by preventing vaso-obliteration in retina during the hyperoxic stage of OIR (p7-p12) without affecting vaso-proliferation .
The Secreted Protein Acidic and Rich in Cysteine (SPARC) is a matricellular protein with contradictory role in angiogenesis. SPARC was heavily studied in tumor angiogenesis [17-19]. Several studies demonstrated SPARC as angiostatic molecule as a result of the negative correlation with VEGF expression in cancer colon , and glioma . The regulatory role of SPARC in angiogenesis has been linked to both VEGF and metalloproteinase (MMP) signaling. In particular, it has the ability to inhibit the tyrosine phosphorylation of FLT1 , regulating VEGF expression as well as the activity of MMP7 and 9 [22,23]. In contrast, SPARC over-expression was associated with increased angiogenesis in posterior uveal melanoma  and was found to be highly expressed in renal cell carcinoma, a highly vascular tumor overexpressing VEGF . On the other hand, the role of SPARC in RNV has not been yet elucidated. Recently, SPARC overexpression by subretinal injection of recombinant adenovirus has been shown to induce preretinal NV and growth of new vessels in the inner plexiform layer in the rat eye. This was associated with a noticeable increase in the permeability and blood vessels proliferation with simultaneous overexpression of SPARC and VEGF when compared with VEGF overexpression . On the other hand, inhibition of SPARC switch the angiogenic effect of VEGF-A to angiostatic effect and decreased the size of laser induced Choroidal NV (CNV), and this effect was inhibited after intravitreal injection of rhSPARC. Moreover, injection of anti-SPARC L-peptide the day before CNV laser induction was able to decrease the size of CNV .
Taken together previous reports about the regulatory role of SPARC in angiogenesis, we hypothesized that SPARC might be a crucial factor in regulating RNV. Thus, the aim of the current study was to characterize the changes in the levels of retinal SPARC in OIR mouse model and to test the effect of its deletion on RNV and vascular regeneration during OIR. We also investigated the effect of hypoxia on SPARC expression and the mutual regulatory effect between SPARC and VEGF in cultured Human Retinal Endothelial Cells (HRECs).
MATERIALS AND METHODS
SPARC knockout mice were prepared and backcrossed with C57Bl/6 mice before use as described before [28,29]. The study was strictly adhered with the ARVO statement for the use of animals in ophthalmology and vision research and approved by animal care committee at Georgia Regents University. Both wild type and SPARC knockout OIR mouse model were prepared as we described previously [9,30,31]. Briefly, all newborn pups (n=10) were kept at room air during the first 7 post natal days (p7) then they were transferred to high oxygen (75% O2) chamber (Biospherix, Lacona, NY) for 5 days (p7 - p12) before they were returned to room air (oxygen 21%). Sacrifice was carried out at p14 (n=5) and p17 (n=5). Age matching mice were kept at room air and used as control (n=5). One retina of each animal was homogenized using RIPA buffer supplemented with protease and phosphatase inhibitor cocktail to be used for Western blot analysis and the other eye was processed as frozen sections or retinal flat-mount as previously described by us for immunofluorescence studies [30,31].
To investigate the effect of SPARC on VEGF expression, HRECs were treated for 24 hours with or without different concentrations of SPARC (100ng/ml, 200ng/ml, and 300ng/ml). Meanwhile, to test the effect of VEGF on SPARC secretion, HRECs were treated with or without different concentrations of VEGF (10ng/ml, 50ng/ml, and 100ng/ml). At the end of the experiment the Conditioned Media (CM) was collected and analyzed by ELISA and the cells were homogenized in RIPA buffer then cell lysate was collected for Western blotting assay.
Immunofluorescence studies of retinal vessels and SPARC expression
To determine the changes in the levels and localization of SPARC during OIR, we used immunofluorescence staining of retinal frozen sections (10µ thick). The expression of SPARC in mouse model of OIR and normal mice was detected by immunofluorescence using 1/200 rabbit anti-SPARC antibody (Cell Signaling, Carlsbad, CA) and isolectin-B4 (15µg/ml). Frozen sections were fixed in paraformaldehyde (4%) for 10 minutes, washed with PBS (pH 7.5) 3 times/ 10 minutes, and permeabilized with Triton X (0.2%) before they were incubated in normal goat serum (30%) for 30 minutes. The sections were then incubated with anti SPARC primary antibody overnight in 4°C. To label the blood vessels and identify SPARC expression, sections then were washed with 1x PBS 3 times/ 10 minutes and TX 1% and incubated with (25µg/ml) Texas red - conjugated Avidin D (Vector Laboratories, Burlingame, CA) and 1/500 Oregon green-goat secondary anti-Rabbit (Life Technologies, Grand Island, NY) and protected from light for one hour at room temperature. Immuno-stained slides were covered using 4'6-Diamidino-2-Phenylindole (DAPI) mounting medium (Vector Laboratories, Burlingame, CA) and images were obtained using Axiovision; Carl Zeiss Meditec, Inc., Dublin, CA.
Oxygen induced retinopathy is associated with low levels of SPARC
Immunofluorescence (Figure 1b) showed diffuse SPARC immunoreactivity in different layers of normal retina including nerve fiber, ganglion cells, inner and outer nuclear layers. The most noticeable immunoreactivity was localized in glial cell processes, perivascular in relation to the inner retinal vessels and in ganglion cells. On the other hand, SPARC immunoreactivity was much less in OIR compared to the control retina and was localized mainly in nerve fiber and ganglion cell layers, inner nuclear layer and RPE.
Deletion of SPARC enhanced vaso-obliteration in OIR
Hypoxia down-regulates SPARC expression and secretion in HRECs
VEGF induces SPARC secretion in HRECs
SPARC up-regulates VEGF expression in HRECs
- Significant decrease in retinal levels of SPARC protein during early stage of OIR (p14)
- Enhanced vaso-obliteration and mild increase in RNV in SPARC-deficient mice during OIR
- Down-regulation of SPARC production in HRECs by hypoxia
- Positive feedback between SPARC and VEGF treatment in HRECs
Hypoxia treatment of HRECs showed significant reduction in the levels of SPARC expression and secretion. Whether this decrease in SPARC levels by hypoxia is required for endothelial survival via maintaining constitutively active VEGFR-1 needs further investigation.
Our data from wild type OIR animal model showed significant decrease in SPARC level by p14 which was restored back to normal levels by p17. The decrease in SPARC during the early stage of relative hypoxia (p14) in OIR may contribute to the persistence of the central retinal capillary degeneration (vaso-obliteration) via switching VEGF signaling to VEGFR-1. However restoration of SPARC normal levels by p17 may enhance the angiogenic effect of VEGF retinal pool through suppression of VEGFR-1 tyrosine phosphorylation and thus switching VEGF signaling to VEGFR-2 mitogenic activity. In support of our data, overexpression of SPARC in rat's eye was able to induce subretinal NV by itself and aggravated the inflammatory and angiogenic effect of VEGF when combined overexpression of VEGF and SPARC . Our in vitro studies performed on HRECs revealed that VEGF elicits regulatory effect on retinal SPARC. VEGF increased SPARC expression/secretion in HRECs suggesting that VEGF might induce its angiogenic effect in part through up-regulation of SPARC to suppress VEGFR-1 phosphorylation and subsequently eliminate its suppressive effect on VEGFR-2 . Our in vitro data are supported by an earlier report that showed increased SPARC secretion by VEGF treatment in cultured Human Umbilical Vein Endothelial Cells (HUVECs). On the other hand, SPARC promoted VEGF expression in HRECs. Our in vitro data demonstrated positive feedback between the VEGF and SPARC treatment in HRECs suggesting that SPARC might play apro-angiogenic role not only through modulating the VEGFR-1 activity but also via regulating VEGF expression.
In conclusion, our data suggest that decreased SPARC production during OIR and in response to hypoxia might play a role in the development of RNV via enhancing central retinal vaso-obliteration. The underlying mechanism needs further investigation. However the regulatory role of SPARC on VEGF signaling through inhibition of VEGFR-1 tyrosine phosphorylation and regulation of VEGF expression might provide a clue. Taken together our current data and previous reports, we conclude that decreased SPARC production in wild type mice during OIR and lack of SPARC in the knockout mice enhances the activity of VEGFR-1 causing attenuation of vascular regeneration in the central retina during OIR (Figure 6). This also might explain the mild increase in RNV in SPARC-deficient mice which could be linked to the increased vaso-obliteration area and subsequent hypoxia and further increase in VEGF expression in the retina of this animal model. Our in vivo studies were generated from OIR animal model which mimic the retinopathy of prematurity in humans so further studies are needed to investigate whether the SPARC deletion and expression demonstrate similar pattern in other models of ocular neovasculrization. Our data suggest SPARC as a novel therapeutic target to prevent development of RNV during ischemic retinopathy. The use of SPARC during early stage of ischemic retinopathy might be beneficial in preventing capillary degeneration, the driving force for VEGF signaling and subsequent RNV during ischemic retinopathy via switching the activity of VEGF signaling to be mainly through VEGFR-2.
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Citation:Sobeih D, Hussein KA, Said N, Mohamed K Al-Shabrawey M (2014) Deletion of SPARC Enhances Retinal Vaso-Obliteration in Mouse Model of Oxygen-Induced Retinopathy. J Ophthalmic Clin Res 1: 002.
Copyright: © 2014 Doaa Sobeih, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.