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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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Immunohistochemical localization for macrophages in retrolental membranes from eyes with retinopathy of prematurity. A: Expression of CD40 (representing M1 polarity; arrows) and CD206 markers (representing M2 polarity; arrowheads) in tissue macrophages associated with retinopathy of prematurity (ROP) tissue. B–C: Antihuman inducible nitric oxide synthase (iNOS) and arginase antibodies were also used to detect M1 and M2 macrophages, respectively. D: Distribution of M1 and M2 macrophages in retrolental membranes from eyes with retinopathy of prematurity (ROP); the predominant macrophage is the M1 type, and there is only a limited number of M2 type macrophages (** p<0.001). Scale bar = 20 µm.
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f4: Immunohistochemical localization for macrophages in retrolental membranes from eyes with retinopathy of prematurity. A: Expression of CD40 (representing M1 polarity; arrows) and CD206 markers (representing M2 polarity; arrowheads) in tissue macrophages associated with retinopathy of prematurity (ROP) tissue. B–C: Antihuman inducible nitric oxide synthase (iNOS) and arginase antibodies were also used to detect M1 and M2 macrophages, respectively. D: Distribution of M1 and M2 macrophages in retrolental membranes from eyes with retinopathy of prematurity (ROP); the predominant macrophage is the M1 type, and there is only a limited number of M2 type macrophages (** p<0.001). Scale bar = 20 µm.

Mentions: Results demonstrate that the SRF from ROP eyes potentially promotes angiogenesis and inflammation. This suggests that monocytes that migrate to the fibrovascular membranes would be polarized to into either M1 macrophages to mediate inflammation or to M2 macrophages that would contribute to the proangiogenic state of the SRF. Immunostaining of these membranes for CD40 and CD206 (representing M1 and M2 polarity, respectively) showed a preponderance of CD40 over CD206 markers (Figure 4A). Expression of the M1 intracellular enzyme iNOS and the M2 intracellular enzyme arginase 1 was sporadic (Figure 4B–C). Overall, the expression of M1 over M2 markers in retrolental membranes from ROP eyes increased eightfold (88.8±7.0% versus 11.2±7.0%, p<0.001, Figure 4D). This demonstrates that these proangiogenic and proinflammatory cytokines mediate development of M1 and M2 macrophage polarity in the fibrous membranes of eyes with ROP.


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)

Immunohistochemical localization for macrophages in retrolental membranes from eyes with retinopathy of prematurity. A: Expression of CD40 (representing M1 polarity; arrows) and CD206 markers (representing M2 polarity; arrowheads) in tissue macrophages associated with retinopathy of prematurity (ROP) tissue. B–C: Antihuman inducible nitric oxide synthase (iNOS) and arginase antibodies were also used to detect M1 and M2 macrophages, respectively. D: Distribution of M1 and M2 macrophages in retrolental membranes from eyes with retinopathy of prematurity (ROP); the predominant macrophage is the M1 type, and there is only a limited number of M2 type macrophages (** p<0.001). Scale bar = 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Immunohistochemical localization for macrophages in retrolental membranes from eyes with retinopathy of prematurity. A: Expression of CD40 (representing M1 polarity; arrows) and CD206 markers (representing M2 polarity; arrowheads) in tissue macrophages associated with retinopathy of prematurity (ROP) tissue. B–C: Antihuman inducible nitric oxide synthase (iNOS) and arginase antibodies were also used to detect M1 and M2 macrophages, respectively. D: Distribution of M1 and M2 macrophages in retrolental membranes from eyes with retinopathy of prematurity (ROP); the predominant macrophage is the M1 type, and there is only a limited number of M2 type macrophages (** p<0.001). Scale bar = 20 µm.
Mentions: Results demonstrate that the SRF from ROP eyes potentially promotes angiogenesis and inflammation. This suggests that monocytes that migrate to the fibrovascular membranes would be polarized to into either M1 macrophages to mediate inflammation or to M2 macrophages that would contribute to the proangiogenic state of the SRF. Immunostaining of these membranes for CD40 and CD206 (representing M1 and M2 polarity, respectively) showed a preponderance of CD40 over CD206 markers (Figure 4A). Expression of the M1 intracellular enzyme iNOS and the M2 intracellular enzyme arginase 1 was sporadic (Figure 4B–C). Overall, the expression of M1 over M2 markers in retrolental membranes from ROP eyes increased eightfold (88.8±7.0% versus 11.2±7.0%, p<0.001, Figure 4D). This demonstrates that these proangiogenic and proinflammatory cytokines mediate development of M1 and M2 macrophage polarity in the fibrous membranes of eyes with ROP.

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