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Influence of subretinal fluid in advanced stage retinopathy of prematurity on proangiogenic response and cell proliferation.

Ma J, Mehta M, Lam G, Cyr D, Ng TF, Hirose T, Tawansy KA, Taylor AW, Lashkari K - Mol. Vis. (2014)

Bottom Line: The angiogenic potential of SRF from ROP eyes was measured using a combination of capillary cord formation in a fibrin clot assay, and its proliferative effect was tested with a DNA synthesis of human retinal microvascular endothelial cells.SRF from ROP eyes supported cell proliferation and endothelial cord formation while SRF from RD eyes had inhibitory effects.The cytokine profile and biologic properties of SRF in ROP promote a proangiogenic environment, which supports the maintenance and proliferation of fibrous membranes associated with advanced stages of ROP.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Mass. Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA.

ABSTRACT

Purpose: The clinical phenotype of advanced stage retinopathy of prematurity (ROP, stages 4 and 5) cannot be replicated in an animal model. To dissect the molecular events that can lead up to advanced ROP, we examined subretinal fluid (SRF) and surgically dissected retrolental membranes from patients with advanced ROP to evaluate its influences on cell proliferation, angiogenic properties, and macrophage polarity.

Methods: We compared our findings to SRF collected from patients with uncomplicated rhegmatogenous retinal detachment (RD) without proliferative vitreoretinopathy and surgically dissected epiretinal membrane from eyes with macular pucker. All subretinal fluid samples were equalized for protein. The angiogenic potential of SRF from ROP eyes was measured using a combination of capillary cord formation in a fibrin clot assay, and its proliferative effect was tested with a DNA synthesis of human retinal microvascular endothelial cells. Findings were compared with SRF collected from participants with uncomplicated rhegmatogenous RD without proliferative vitreoretinopathy. The ability of SRF to induce nitric oxide production was measured in vitro using murine J774A.1 macrophages. Cytokine profiles of SRF from ROP and RD eyes were measured using a multienzyme-linked immunosorbent assay (ELISA). Fluorescent immunohistochemistry of retrolental membranes from ROP was performed to detect the presence of leukocytes and the composition of tissue macrophages using markers for M1 and M2 differentiation.

Results: The cytokine composition in SRF revealed that in ROP, not only were several proangiogenic factors were preferentially elevated but also the profile of proinflammatory factors was also increased compared to the RD eyes. SRF from ROP eyes supported cell proliferation and endothelial cord formation while SRF from RD eyes had inhibitory effects. SRF from eyes with ROP but not RD robustly induced nitric oxide production in macrophages. Furthermore, fluorescent immunostaining revealed a preponderance of M1 over M2 macrophages in retrolental fibrous membranes from ROP eyes. The cytokine profile and biologic properties of SRF in ROP promote a proangiogenic environment, which supports the maintenance and proliferation of fibrous membranes associated with advanced stages of ROP. In contrast, SRF from RD eyes exhibits a suppressive environment for endothelial cell proliferation and angiogenesis.

Conclusions: Our investigation demonstrates that the microenvironment in advanced ROP eyes is proangiogenic and proinflammatory. These findings suggest that management of advanced ROP should not be limited to the surgical removal of the fibrovascular membranes and antiangiogenic therapy but also directed to anti-inflammatory therapy and to promote M2 activation over M1 activity.

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Nitrite production in cultured macrophages incubated with subretinal fluid from eyes with retinopathy of prematurity and retinal detachment. A: Subretinal fluid (SRF) from eyes with retinopathy of prematurity (ROP) induced robust production of nitrite at levels similar to lipopolysaccharide (LPS; positive control; Wilcoxon signed-rank test, - p ≥ 0.05, *p < 0.05, **p < 0.001). SRF from eyes with retinal detachment (RD) did not have any measurable effects. B: Detailed comparison of nitrite production between every two different conditions, for instance, p > 0.05 between macrophage and medium, p < macrophage + LPS 1 μg/ml versus medium.
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f3: Nitrite production in cultured macrophages incubated with subretinal fluid from eyes with retinopathy of prematurity and retinal detachment. A: Subretinal fluid (SRF) from eyes with retinopathy of prematurity (ROP) induced robust production of nitrite at levels similar to lipopolysaccharide (LPS; positive control; Wilcoxon signed-rank test, - p ≥ 0.05, *p < 0.05, **p < 0.001). SRF from eyes with retinal detachment (RD) did not have any measurable effects. B: Detailed comparison of nitrite production between every two different conditions, for instance, p > 0.05 between macrophage and medium, p < macrophage + LPS 1 μg/ml versus medium.

Mentions: To determine whether SRF from ROP eyes activates macrophages and potentially promotes inflammation, the ability of SRF to induce nitric oxide production in macrophages was assessed. The nitrite levels in the condition media relative to the nitric oxide levels generated by the macrophages was significantly higher in the cultures of macrophages treated with SFR from ROP eyes and at the same level of macrophages stimulated with LPS (Figure 3A). In contrast, SRF from RD eyes stimulated no more nitrate than the macrophages at rest (Figure 3A). Statistical significance was measured between every two culture conditions on nitric oxide production, and all p values are listed in Figure 3B. This demonstrates that along with the proangiogenic activity there is proinflammatory support by the SRF of ROP eyes as suggested by their cytokine profile.


Influence of subretinal fluid in advanced stage retinopathy of prematurity on proangiogenic response and cell proliferation.

Ma J, Mehta M, Lam G, Cyr D, Ng TF, Hirose T, Tawansy KA, Taylor AW, Lashkari K - Mol. Vis. (2014)

Nitrite production in cultured macrophages incubated with subretinal fluid from eyes with retinopathy of prematurity and retinal detachment. A: Subretinal fluid (SRF) from eyes with retinopathy of prematurity (ROP) induced robust production of nitrite at levels similar to lipopolysaccharide (LPS; positive control; Wilcoxon signed-rank test, - p ≥ 0.05, *p < 0.05, **p < 0.001). SRF from eyes with retinal detachment (RD) did not have any measurable effects. B: Detailed comparison of nitrite production between every two different conditions, for instance, p > 0.05 between macrophage and medium, p < macrophage + LPS 1 μg/ml versus medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4067231&req=5

f3: Nitrite production in cultured macrophages incubated with subretinal fluid from eyes with retinopathy of prematurity and retinal detachment. A: Subretinal fluid (SRF) from eyes with retinopathy of prematurity (ROP) induced robust production of nitrite at levels similar to lipopolysaccharide (LPS; positive control; Wilcoxon signed-rank test, - p ≥ 0.05, *p < 0.05, **p < 0.001). SRF from eyes with retinal detachment (RD) did not have any measurable effects. B: Detailed comparison of nitrite production between every two different conditions, for instance, p > 0.05 between macrophage and medium, p < macrophage + LPS 1 μg/ml versus medium.
Mentions: To determine whether SRF from ROP eyes activates macrophages and potentially promotes inflammation, the ability of SRF to induce nitric oxide production in macrophages was assessed. The nitrite levels in the condition media relative to the nitric oxide levels generated by the macrophages was significantly higher in the cultures of macrophages treated with SFR from ROP eyes and at the same level of macrophages stimulated with LPS (Figure 3A). In contrast, SRF from RD eyes stimulated no more nitrate than the macrophages at rest (Figure 3A). Statistical significance was measured between every two culture conditions on nitric oxide production, and all p values are listed in Figure 3B. This demonstrates that along with the proangiogenic activity there is proinflammatory support by the SRF of ROP eyes as suggested by their cytokine profile.

Bottom Line: The angiogenic potential of SRF from ROP eyes was measured using a combination of capillary cord formation in a fibrin clot assay, and its proliferative effect was tested with a DNA synthesis of human retinal microvascular endothelial cells.SRF from ROP eyes supported cell proliferation and endothelial cord formation while SRF from RD eyes had inhibitory effects.The cytokine profile and biologic properties of SRF in ROP promote a proangiogenic environment, which supports the maintenance and proliferation of fibrous membranes associated with advanced stages of ROP.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Mass. Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA.

ABSTRACT

Purpose: The clinical phenotype of advanced stage retinopathy of prematurity (ROP, stages 4 and 5) cannot be replicated in an animal model. To dissect the molecular events that can lead up to advanced ROP, we examined subretinal fluid (SRF) and surgically dissected retrolental membranes from patients with advanced ROP to evaluate its influences on cell proliferation, angiogenic properties, and macrophage polarity.

Methods: We compared our findings to SRF collected from patients with uncomplicated rhegmatogenous retinal detachment (RD) without proliferative vitreoretinopathy and surgically dissected epiretinal membrane from eyes with macular pucker. All subretinal fluid samples were equalized for protein. The angiogenic potential of SRF from ROP eyes was measured using a combination of capillary cord formation in a fibrin clot assay, and its proliferative effect was tested with a DNA synthesis of human retinal microvascular endothelial cells. Findings were compared with SRF collected from participants with uncomplicated rhegmatogenous RD without proliferative vitreoretinopathy. The ability of SRF to induce nitric oxide production was measured in vitro using murine J774A.1 macrophages. Cytokine profiles of SRF from ROP and RD eyes were measured using a multienzyme-linked immunosorbent assay (ELISA). Fluorescent immunohistochemistry of retrolental membranes from ROP was performed to detect the presence of leukocytes and the composition of tissue macrophages using markers for M1 and M2 differentiation.

Results: The cytokine composition in SRF revealed that in ROP, not only were several proangiogenic factors were preferentially elevated but also the profile of proinflammatory factors was also increased compared to the RD eyes. SRF from ROP eyes supported cell proliferation and endothelial cord formation while SRF from RD eyes had inhibitory effects. SRF from eyes with ROP but not RD robustly induced nitric oxide production in macrophages. Furthermore, fluorescent immunostaining revealed a preponderance of M1 over M2 macrophages in retrolental fibrous membranes from ROP eyes. The cytokine profile and biologic properties of SRF in ROP promote a proangiogenic environment, which supports the maintenance and proliferation of fibrous membranes associated with advanced stages of ROP. In contrast, SRF from RD eyes exhibits a suppressive environment for endothelial cell proliferation and angiogenesis.

Conclusions: Our investigation demonstrates that the microenvironment in advanced ROP eyes is proangiogenic and proinflammatory. These findings suggest that management of advanced ROP should not be limited to the surgical removal of the fibrovascular membranes and antiangiogenic therapy but also directed to anti-inflammatory therapy and to promote M2 activation over M1 activity.

Show MeSH
Related in: MedlinePlus