Defective retinal vascular endothelial cell development as a consequence of impaired integrin αVβ8-mediated activation of transforming growth factor-β

Abstract

Deletions of the genes encoding the integrin αVβ8 (Itgav, Itgb8) have been shown to result in abnormal vascular development in the CNS, including prenatal and perinatal hemorrhage. Other work has indicated that a major function of this integrin in vivo is to promote TGFβ activation. In this paper, we show that Itgb8 mRNA is strongly expressed in murine Müller glia and retinal ganglion cells, but not astrocytes. We further show that Itgb8 deletion in the entire retina severely perturbs development of the murine retinal vasculature, elevating vascular branch point density and vascular coverage in the superficial vascular plexus, while severely impairing formation of the deep vascular plexus. The stability of the mutant vasculature is also impaired as assessed by the presence of hemorrhage and vascular basal lamina sleeves lacking endothelial cells. Specific deletion of Itgb8 in Müller glia and neurons, but not deletion in astrocytes, recapitulates the phenotype observed following Itgb8 in the entire retina. Consistent with αVβ8's role in TGFβ1 activation, we show that retinal deletion of Tgfb1 results in very similar retinal vascular abnormalities. The vascular deficits appear to reflect impaired TGFβ signaling in vascular endothelial cells because retinal deletion of Itgb8 reduces phospho-SMAD3 in endothelial cells and endothelial cell-specific deletion of the TGFβRII gene recapitulates the major deficits observed in the Itgb8 and TGFβ1 mutants. Of special interest, the retinal vascular phenotypes observed in each mutant are remarkably similar to those of others following inhibition of neuropilin-1, a receptor previously implicated in TGFβ activation and signaling.

Figure 1.

Absence of integrin αVβ8 perturbs development of the retinal vasculature. A–D, Flatmounts of P6 retinas from control, Itgb8 complete knock-out (β8icr^−/−), nesCre-specific Itgb8 (β8;nesCre), and mGFAPCre-specific (β8;mGFAPCre) mutant retinas. A, Visualization of vasculature (collagen IV) and red blood cells (Ter119) reveals extensive hemorrhage in retinas of β8icr^−/− mutants and substantial, but reduced hemorrhage in β8;nesCre mutants. β8;mGFAPCre retinas are comparatively normal. B, Higher-magnification images of vascular plexuses illustrate perturbed vascular branching and increased vascular density in β8icr^−/− and β8;nesCre mutants compared with β8;mGFAPCre mutants and controls. C, Vascular endothelia (IB4) and collagen IV staining demonstrate elevated numbers of collagen IV sleeves lacking endothelia (arrows) in β8icr^−/− and β8;nesCre mutants compared with β8;mGFAPCre mutants and controls. D, Images of angiogenic front in the control and three mutants reveals no overt differences in tip cell or filopodial morphology. E, Panels from P6 control, Itgb8 complete knock-out (β8icr^−/−) and nesCre-specific Itgb8 (β8;nesCre) mutant retinas colabeled with anti-CD31 (endothelia) and anti-phospho-histone H3 (PH3, proliferating cells). Endothelial proliferation was not analyzed in β8;mGFAPCre mutants. F, Quantifications of vascular branch point density (number per 0.106 mm² field), vessel coverage (percentage of area covered by CD31-expressing endothelial cells), empty collagen IV sleeves as illustrated in C (number per 0.0529 mm² field), and endothelial cell proliferation (number of proliferating (PH3+), CD31+ endothelial cells per field as illustrated in E) demonstrate significant differences between control and β8icr^−/− or β8;nesCre mutants, but no significant difference between control and β8;mGFAPCre mutants [ANOVA p-values < 0.0005; Tukey's subgroup analysis: *p < 0.05, **p < 0.005, ***p < 0.0005, NS = not significant. N = 16 (combined controls), N = 4 (β8icr^−/−, β8;nesCre), N = 3 (β8;mGFAPCre)]. Error bars represent SEM.

Figure 2.

Normal mural cell coverage and arteriovenous differentiation in absence of integrin αVβ8 in P6 retinas. A, Panels illustrate colocalization of endothelial cells (CD31) and pericytes (α-smooth muscle actin, αSMA) in control, complete Itgb8 knock-out (β8icr^−/−) and nesCre-specific Itgb8 (β8;nesCre) mutant retinas. In these images, two veins (v) are depicted on the left and right with an artery (a) present in the middle of each panel. B, Panels present sample images colabeled with CD31 (endothelial cells) and NG2 (pericytes) to visualize pericyte coverage in the control and mutants. C, Panels present representative images of retinas colabeled with CD31(endothelial cells) and desmin (pericytes). Note normal localization of the pericyte markers αSMA, NG2 and desmin in each mutant. D, Panels show representative images of arteries visualized with ephrinB2::eGFP that demonstrate normal arterial differentiation in control and representative β8;nesCre mutant. Scale bars, 100 μm.

Figure 3.

Integrin αVβ8 is required for the development of the outer deep vascular plexus. A, B, Diagrams illustrate schematically the development of the deep retinal vascular plexus. By P9, vessels have sprouted from the superficial vascular plexus (SVP; green) and establish the outer deep vascular plexus (oDVP; red). Subsequently, the oDVP remodels, and the inner deep vascular pleuxus (iDVP) is established (B) by P14. Below diagrams: A, Flat-mounted retinas at P9 were stained for red blood cells (anti-Ter119) and vessels [anti-collagen IV (col IV), or anti-CD31]. Left panels demonstrate vascular instability and retinal hemorrhage in P9 β8icr^−/− mutants. Middle and right panels are representative confocal optical slices taken from the superficial vascular plexus (SVP; green), and the outer deep vascular plexus (oDVP; red) within flat-mounted P9 retinas stained with the endothelial cell marker, anti-CD31. Middle panels are overlays of the SVP and DVP. Right panels in A are higher-magnification insets of outer DVP from boxed areas in middle panels. In contrast to control, β8icr^−/− mutants do not establish a deep vascular plexus. B, Optical slices from flat mounted P14 retinas stained with IB4 illustrate perturbed remodeling of superficial vascular plexus (SVP; green), abnormally thickened vessels in the inner deep vascular plexus (iDVP; blue), and continued lack of an outer deep vascular plexus (oDVP; red) in β8icr^−/− mutants compared with control. Scale bars, 100 μm.

Figure 4.

Itgb8 expression in Müller glia and retinal ganglion cells, but not retinal astrocytes. A, In situ hybridization for Itgb8 mRNA in P9 wild-type retina (top), and β8;nesCre mutant retinas (bottom). Itgb8-expressing Müller glia span the full thickness of the retina with heavy expression in the inner nuclear layer (INL), where Müller glia cell bodies reside, and in the nerve fiber layer (NFL) where Müller glial endfeet and astrocytes normally reside. There is staining for Itgb8 mRNA in the ganglion cell layer (GCL), indicating that retinal ganglion cells, in addition to Müller glia, express Itgb8. Note that all staining for Itgb8 mRNA is lost in the β8;nesCre mutant. The dark brown dots in the β8;nesCre mutant are red blood cells from retinal hemorrhage in this mutant. B–D, NesCre recombines Müller glia and retinal ganglion cells, but not retinal astrocytes, while mGFAPCre specifically recombines retinal astrocytes. B, Images depict nesCre-directed Rosa26RtdTomato;AI14 reporter expression (red), colabeled with Müller glia markers, nestin (green), Cellular retinaldehyde-binding protein (Cralp; green), and glutamine synthetase (GS; blue). NesCre directs recombination of Müller glia, retinal ganglion cells, and other neural cells of the retina. Arrowheads point to heavy expression of endogenous nestin on recombined Müller glia next to vessels penetrating into the deep retina. C, D, RosaR26-eYFP reporter expression (green) with endogenous nestin or GFAP (red), optical sections from flat mount retinas taken at the level of the NFL (C) or INL (D). C, Panels depict overlap of endogenous nestin with that of the nesCre-eYFP reporter. Arrowheads point to recombined Müller glial endfeet wrapping around blood vessels (unstained). Note absence of overlap of endogenous GFAP present in retinal astrocytes with the eYFP reporter. Arrow points to an exemplary astrocyte cell body labeled by GFAP, but lacking eYFP recombination marker. Note that because nesCre promotes recombination in progenitors of many retinal cells in addition to Müller Glia, there is extensive expression of the eYFP reporter in cells that do not express nestin. Right panels depict overlap of endogenous GFAP (red) with mGFAPCre (mGfap-Cre73.12)-mediated recombination as visualized using the RosaR26-eYFP reporter (green). Arrow points to an astrocyte cell body labeled by GFAP with strong overlapping eYFP recombination marker. D, Fibers of Müller glia extend into the deeper retina and are recombined by nesCre, whereas there are no GFAP-staining astrocyte fibers in this layer, and no recombination from the mGFAPcre transgene in this layer. Scale bars, 50 μm.

Figure 5.

Phospho-SMAD3 signaling is reduced, in endothelial cells of integrin β8 mutants. A, Top, Representative thin sections from P9 retinas illustrating phospho-SMAD3 (pSMAD3, red) colocalization with endothelial cell-specific-CD31 (green) in control and itgb8 complete knock-out retinas. Bottom are rainbow spectrum intensity maps of phospho-SMAD3 staining delimited by CD31 staining from boxed regions in panels above (red, most intense staining; blue, least intense staining). Whereas most endothelial cells in the superficial and deep vascular plexuses of control mice are strongly positive (arrows) and few weakly positive (arrowheads) for pSMAD3 staining, relatively few endothelial cells are strongly positive and most are weakly positive for pSMAD3 in mutants. Note the thickened superficial vascular plexus (SVP) and lack of a deep vascular plexus (DVP) in the mutant versus control sections. B, Quantification of vascular nuclei per 100 μm length of the vasculature documents a significant increase in the density of vasculature-associated endothelial cells in the mutant. The intensity of phospho-SMAD3 labeling of each endothelial nucleus was quantified in B in arbitrary units. The intensity of labeling in individual nuclei as well as the mean leveling densities presented in this graph documents a significant reduction in endothelial cell-specific phospho-SMAD3 levels in mutants compared with controls. p-values from Student's t test: *p = 0.019, ***p < 0.0001; N = 3 (controls), N = 3 (β8icr^−/− mutants). Error bars in all graphs represent SEM. Scale bars: (A), 100 μm; (C), 50 μm.

Figure 6.

Absence of Tgfb1, and inhibition of TGFβ signaling in retinal endothelial cells recapitulate the vascular abnormalities observed in the Itgb8 mutants. A, Representative images of retina flat-mounts of control, Tgfb1^−/− and endothelial-specific Tgfbr2 mutant (TGFβRII;iΔEC) P6 retinas. TGFβRII;iΔEC mutant retinas were generated using animals homozygous for the Tgfbr2-flox allele bearing also a Pdgfb promoter-regulated Cre-ER-T2 transgene. Cre recombination was induced by intragastric tamoxifen injections (50 μg) at P1, P2, and P3. Loss of TGFβ1 or TGFβRII results in hemorrhage at P6 as illustrated by extravascular red blood cells (Ter119) in the mutant retinas (left panels). Endothelial cells, visualized by endothelial cell-specific expression of tdTomato (Pdgfb-CreER^TM2-mediated expression of recombination marker tdTomato from Rosa26RtdTomato;AI14 allele) and/or CD31 labeling illustrate enhanced vascular branching density in P6 Tgfb1^−/− and TGFβRII;iΔEC mutant retinas. Localization of vascular endothelia (IB4) and collagen IV illustrate increased presence of collagen IV sleeve segments lacking endothelial cells (IB4-negative; arrows) in Tgfb1^−/− and TGFβRII;iΔEC mutants. The relative distribution of α-smooth muscle actin (SMA) and NG2-expressing pericytes compared with CD31-expressing endothelial cells in control compared with mutants suggests that pericyte coverage is normal in the mutants. The same panels document reasonably normal arterial (a) and venous (v) differentiation in mutants. Colabeling with anti-CD31 and the tdTomato recombination reporter (to colabel endothelial cells) and phosphohistone3 (PH3) illustrate an increased density of proliferating endothelial cells in P6 Tgfb1^−/− and TGFβRII;iΔEC mutant retinas. Images of the angiogenic front in control and mutant P6 retinas indicate that the morphologies and numbers of endothelial tip cells and their filopodia are not obviously perturbed in the Tgfb1^−/− or endothelial-specific Tgfbr2 (TGFβRII;iΔEC) mutants. B, Panels present quantification of changes in retinal vascular development at P6 as a result of Tgfb1^−/− deletion or endothelial cell-specific deletion of Tgfbr2. Results demonstrate that as a result of loss of TGFβ1 or impaired TGFβRII function, there are increases in vascular branch point density, the area of retina covered by vascular endothelial cells, the number of collagen IV sleeve segments lacking endothelial cell coverage, and the number of proliferating endothelial cells. ANOVA p-values: branch points per field, p < 0.0056; vessel coverage (%), Col IV sleeves per field, and PH3⁺/CD31⁺ cells per field, p < 0.0001. Tukey's subgroup analysis: *p < 0.05, **p < 0.005, ***p < 0.0005, NS = not significant; N = 9 (combined controls), N = 3 (Tgfb1^−/−, TGFβRII;iΔEC). Error bars represent SEM. C, Representative images of flat-mounts of control, Tgfb1^−/− and endothelial-specific Tgfbr2 (TGFβRII;iΔEC) mutant P9 and P14 retinas. Endothelial-specific Tgfbr2 mutant retinas were analyzed following tamoxifen administration (50 μg) on P5 and P6, retinal vascular phenotypes were analyzed on P9 by immunostaining vessels (IB4; green) and red blood cells (Ter119). Confocal optical slices were taken from the superficial vascular plexus (SVP, green), the inner deep vascular plexus (iDVP; blue), and the outer deep vascular plexus (oDVP, red) as illustrated in diagram in Figure 1. At P9, absence of TGFβ1 or acute loss of TGFβRII from endothelial cells leads to disruption of the SVP with corresponding leakage of red blood cells. At P9 formation of the outer DVP is dramatically inhibited in the absence of TGFβ1 or TGFβ receptor 2 in retinal endothelial cells. Right panels in P9 retina image sets are higher-magnification insets of outer DVP from boxed areas in middle panels. At P14 absence of TGFβ results in continued failure of formation of the outer deep vascular plexus. The inner deep vascular plexus does form in the mutant with abnormally thickened vessels, similar to the phenotypes in the Itgb8 mutant. At P14, TGFβRII;iΔEC mutants were sickly and obviously smaller than littermate controls, and so were not analyzed. Scale bars, 100 μm.