The role of renin angiotensin system in retinal inflammation

Purpose: Retinopathy of prematurity (ROP) is the main cause of vision loss and blindness in children, and is replicated and intensively studied in rodent models of oxygen-induced retinopathy (OIR). One signature feature of ROP is retinal neovascularization, which is also present in patients with proliferative diabetic retinopathy (PDR). Inflammation is another feature in ROP and PDR. In both diseases, the renin angiotensin system (RAS) is dysregulated, and blockade of RAS via angiotensin II (Ang II) type-1 receptor (AT1R) blockers (ARB) and angiotensin-converting enzyme (ACE) inhibitors have shown successful reduction of the incidence and progression of retinopathy. These treatments, however, do not fully attenuate the retinal pathology. This may be due to additional mechanisms beyond the canonical RAS, which influence retinopathy in addition to RAS. Recent studies have been done to show a contribution of the innate immune system to ROP, with the involvement of macrophage and increases expression of cytokines (1-3). However, the role of the adaptive immune system to the development of ROP has not been comprehensively studied. Another consideration is the involvement of pathways additional to the traditional RAS, such as the alternative pathway of prorenin working independently of Ang II and AT1R. Clinical study identified plasma prorenin as an early marker for retinopathy in patients with diabetic mellitus, as it was at an elevated level in patients with retinopathy (4). Blocking prorenin binding to its putative receptor, the (pro)renin receptor [(P)RR] using a decoy peptide to the prorenin binding region reduced the pathological neovascularization and the expression of associated pro-angiogenic factors without affecting the physiological neovascularization in a rodent model of ROP (5). However, other studies failed to show an effect of HRP inhibiting (pro)renin signaling independent of Ang II (6, 7). In this study we aimed to elucidate (1) the role of adaptive immune system to the development of ROP, and whether RAS is involved in this process, using ARB, and (2) the role of prorenin in the inflammation and angiogenesis processes, both of which are involved in the development of ROP. Methods: To address Aim 1, mouse model of OIR was induced in C57BL/6J mice by hyperoxic treatment with 75% O₂ from postnatal (P) days 7 to 11, followed by room air from P12 to P18 for the development of neovascularization. Treatment with a common ARB, valsartan (40mg⁻¹kg⁻¹ per day) was administrated from P12 to P18. Retinal obliteration and pathological vascularization was evaluated using retinal wholemounts stained with FITC-isolectin. Flow cytometry was used to characterize adaptive immune profiles, in particular, CD4⁺ T cells, in control, untreated OIR, and valsartan treated OIR mice in secondary lymphoid organs. Quantitative real-time PCR was utilized to measure the retinal expression of several pro-inflammatory and pro-angiogenic factors, and cytokines influencing T lymphocyte differentiation and proliferation, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-10 (IL-10), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF) and Forkhead box P3 (Foxp3). Retinal mRNA expression of the master transcription factors controlling TH cell differentiation signal transducer and activator of transcription 3 and 5 (STAT3 and STAT5) were also examined by quantitative real-time PCR. In vitro study on cultured CD4⁺ T lymphocytes was used to further investigate the involvement of RAS and the effect of hypoxia on CD4⁺ T cell proliferation, using common methods such as flow cytometry, quantitative real-time PCR, Western Blotting and ELISA. To address Aim 2, rat retinal microglia cells were obtained from neonatal Sprague-Dawley (SD) rats and cultured. Treatment with prorenin was administrated on microglia pre-incubated with a common ARB candesartan to investigate Ang II independent effect of prorenin on retinal microglia. Quantitative real-time PCR was used to examine the expression of pro-inflammatory and pro-angiogenic factors IL-1β, IL-6, TNF-α, monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1) and VEGF, as well as anti-inflammatory factor IL-10. Duo set ELISA was utilized to examine TNF-α and VEGF secretion in response to prorenin treatment to retinal microglia cells. Additional experiment was performed on bovine retinal endothelial cells (BREC) to investigate the effect of prorenin on retinal endothelial cells. A MTT proliferation assay was performed to examine the effect of prorenin on BREC proliferation, and quantitative real-time PCR was used to examine expression of pro-inflammatory and pro-angiogenic factors in response to prorenin treatment. VEGF, angiopoietin-1 and -2, Tie2, MCP-1 and ICAM-1 were all examined. Whether prorenin acts through binding to the (pro)renin receptor [(P)RR] was also investigated, in an attempt to knock down (P)RR gene in rat retinal microglia, however, was not successful to date. Results: In mouse model of OIR, ARB treatment significantly reduced the vascular pathology associated with OIR at P18. This reduction is associated with a reduction in the expression of IL-6, TNF-α, IFN-γ and VEGF in the retina. In vitro study confirmed that CD4⁺ T lymphocytes expressed a functional RAS on the cells, with increased mRNA expression RAS components on Foxp3⁺ Tregs than on conventional CD4⁺ T cells. ARB treated OIR animals showed altered activation profile of T lymphocytes in the secondary lymphoid organs, more interestingly, showed a significant proliferation of Foxp3⁺ Tregs in pooled lymph nodes and spleen between P13 and P18, whereas untreated OIR mice only had transient proliferation of Foxp3⁺ Tregs at P13. The proliferation of Foxp3⁺ Tregs in ARB treated OIR mice were associated with an upregulation of Foxp3 mRNA expression in the retina at P18, together with a change in TGF-β mRNA level. A direct evidence of a proliferation of Foxp3⁺ Tregs came from retinal wholemounts of Foxp3-RFP mice, that ARB treated OIR mice showed more Foxp3⁺ Tregs in the retinal vasculature at P18. A dramatic increase in STAT5: STAT3 mRNA expression in the retina in ARB treated animals revealed the mechanism by which ARB benefits OIR through modulation of Foxp3⁺ Treg population. Prorenin induced inflammation in rat retinal microglial cells, with upregulation of pro-inflammatory genes IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1, together with an increase in cytokine secretion of TNF-α, none of which were reduced by pre-incubation with ARB. Prorenin also promoted proliferation and tubulogenesis of BREC, and caused upregulation of pro-inflammatory genes MCP-1, ICAM-1, and pro-angiogenic factors VEGF, angiopoietin-1, and its receptor Tie2. Conclusions: Results from these studies suggest that the adaptive immune system, especially Foxp3⁺ Tregs, play an important role in the development of ROP, and ARB benefits the vasculopathy partially through the proliferation of Foxp3⁺ Tregs. Prorenin induced inflammation in retinal microglia independent of Ang II effect. It also caused proliferation and increased the gene expression of pro-angiogenic and pro-inflammatory factors in retinal endothelial cells. These results suggest an additional mechanism by which prorenin affects inflammation and angiogenesis process on retinal cell populations, which may have an effect on the development of ROP.