The astrocyte-expressed integrin αvβ8 governs blood vessel sprouting in the developing retina

Abstract

The mouse retina is vascularized after birth when angiogenic blood vessels grow and sprout along a pre-formed latticework of astrocytes. How astrocyte-derived cues control patterns of blood vessel growth and sprouting, however, remains enigmatic. Here, we have used molecular genetic strategies in mice to demonstrate that αvβ8 integrin expressed in astrocytes is essential for neovascularization of the developing retina. Selective ablation of αv or β8 integrin gene expression in astrocytes leads to impaired blood vessel sprouting and intraretinal hemorrhage, particularly during formation of the secondary vascular plexus. These pathologies correlate, in part, with diminished αvβ8 integrin-mediated activation of extracellular matrix-bound latent transforming growth factor βs (TGFβs) and defective TGFβ signaling in vascular endothelial cells, but not astrocytes. Collectively, our data demonstrate that αvβ8 integrin is a component of a paracrine signaling axis that links astrocytes to blood vessels and is essential for proper regulation of retinal angiogenesis.

Fig. 1.

αvβ8 integrin is expressed in mouse neonatal retinal astrocytes. (A,B) Retinal astrocytes were cultured from wild-type (A) or β8^–/– (B) P5 littermates. Immunofluorescence reveals that the majority of cells (>95%) express GFAP. (C) Detergent-soluble astrocyte lysates were immunoblotted with anti-αv and anti-β8 integrin antibodies. Note the robust αvβ8 integrin expression in wild-type cells, whereas there is complete loss of β8 integrin protein expression in β8^–/– cells owing to integrin gene ablation. (D) Detergent-soluble lysates were prepared from P3, P7 and P14 wild-type retinas and then immunoblotted with anti-αv and anti-β8 integrin antibodies. Note the robust expression of αv and β8 integrin proteins at the various developmental ages.

Fig. 2.

β8^–/– mice develop retinal vascular pathologies. (A,B) Retinas from P5 wild-type (A) or β8^–/– (B) littermates were stained with anti-CD31 (green) and anti-NG2 (red) to visualize vascular endothelial cells and pericytes, respectively. Unlike the endothelial tip cells extending well-defined filopodia in wild-type retinas (arrows in A), note the blunted tip cell morphologies in β8^–/– retinas (arrows in B). Note that endothelial cells are associated with pericytes in both control and β8^–/– samples. (C,D) Retinas from P5 wild-type (C) or β8^–/– (D) littermates were stained with anti-CD31 (green) and anti-GFAP (red) to visualize vascular endothelial cells and astrocytes, respectively. Note that endothelial cells in wild-type and β8^–/– retinas are associated with a normal latticework of astrocytes. (E) Quantification of CD31⁺ filopodia per endothelial tip cell in retinas of wild-type and β8^–/– mice. (F) Quantification of filopodial sprouts per 100 μm retinal blood vessel length were quantified in wild-type and β8^–/– mice. ^*P<0.001. Error bars represent s.d. Images in A-D are shown at 200× magnification.

Fig. 3.

Impaired development of the secondary retinal vascular plexus in β8^–/– mice. (A) Images of whole retinas isolated from P14 wild-type (upper panel) or β8^–/– (lower panel) littermates. Note the severe intraretinal hemorrhage in the β8^–/– sample (arrows). (B,C) Retinas from P14 wild-type (B) or β8^–/– (C) littermates were labeled with anti-CD31 and anti-NG2 antibodies to reveal vascular endothelial cells and pericytes, respectively. Note that in β8^–/– mice vascular endothelial cells display impaired sprouting into deeper retinal layers and form glomeruloid-like tufts that contain NG2-positive pericytes (arrows in C). (D,E) Retinas from P14 wild-type (D) or β8^–/– (E) littermates were labeled with anti-CD31 and anti-GFAP antibodies to reveal vascular endothelial cells and astrocytes, respectively. Although β8^–/– mice blood vessels form abnormal glomeruloid-like structures, the GFAP-expressing astrocyte network underlying the primary plexus appears normal (arrows in E). (F) Coronal sections through wild-type (upper panel) and β8^–/– (lower panel) retinas were stained with anti-CD31 to reveal vascular endothelial cells. Unlike the elaborate vascular sprouts in wild-type samples (arrowheads in upper panel), note the diminished blood vessel sprouting into the outer nuclear layer (ONL) and inner nuclear layer (INL) in β8^–/– mice (arrows in lower panel). GCL, ganglion cell layer. (G) Quantification of integrin-dependent formation of the secondary vascular plexus determined by quantifying blood vessels in the INL and ONL of wild-type and β8^–/– mice (n=3 sections per genotype). ^*P<0.001. Error bars represent s.d. Images in B-F are shown at 200× magnification.

Fig. 4.

Selective ablation of αv integrin gene expression in astrocytes causes retinal angiogenesis pathologies. (A) Confirmation of Nestin-Cre-mediated deletion of αvflox gene in P14 retinas (r) but not matched tail snips (t). Genomic DNA was analyzed by PCR-based methods. Note that in the retinal sample there is a reduction in the ∼350 bp band owing to Cre-mediated recombination of the αvflox allele. (B) Detergent-soluble retinal lysates from P14 Nestin-Cre;αvflox/+ control or Nestin-Cre;αvflox/flox mice were immunoblotted with anti-αv antibodies. Note the diminished levels of αv integrin protein in mutant samples. (C) Images of retinas isolated from P14 Nestin-Cre;αvflox/+ control (left panel) or Nestin-Cre;αvflox/flox mutant mice (right panel) revealing intraretinal hemorrhage in mutant samples (arrows). (D,E) Retinas from P14 Nestin-Cre;αvflox/+ control (D) or Nestin-Cre;αvflox/flox mutant (E) mice were immunolabeled with anti-CD31 and anti-NG2 antibodies to visualize vascular endothelial cells and pericytes, respectively. Note the abnormal glomeruloid-like blood vessels comprising endothelial cells and pericytes in mutant samples (arrows in E). (F,G) Retinas from P14 Nestin-Cre;αvflox/+ control (F) or Nestin-Cre;αvflox/flox mutant (G) mice were immunolabeled with anti-CD31 and anti-GFAP antibodies to visualize vascular endothelial cells and astrocytes, respectively. Note that the abnormal vascular structures in mutant retinas (arrows in G) are associated with an apparently normal astrocyte network, although there is increased GFAP expression probably due to reactive astrogliosis resulting from intraretinal hemorrhage. Images in D-G are shown at 200× magnification.

Fig. 5.

Selective ablation of β8 integrin gene expression in astrocytes causes retinal angiogenesis pathologies. (A) Nestin-Cre-mediated deletion of β8flox gene in genomic DNA isolated from P14 retinas (r) but not in that isolated from matched tail snips (t). In the retina there is a reduction in the ∼350 bp band owing to Cre-mediated recombination of the β8flox allele. (B) Detergent-soluble retinal lysates from P14 Nestin-Cre;αvflox/+ control or Nestin-Cre;β8flox/flox mice were immunoblotted with anti-β8 integrin antibodies. Note the diminished levels of β8 integrin protein in mutant samples. (C) Images of retinas isolated from P14 Nestin-Cre;β8flox/+ control (left panel) or Nestin-Cre;β8flox/flox mutant mice (right panel) revealing intraretinal hemorrhage in mutant samples (arrows). (D,E) Retinas from P14 Nestin-Cre;β8flox/+ control (D) or Nestin-Cre;β8flox/flox mutant (E) mice were immunolabeled with anti-CD31 and anti-NG2 antibodies to visualize vascular endothelial cells and pericytes, respectively. Note the abnormal glomeruloid-like vascular tufts comprising endothelial cells and pericytes in mutant samples (arrows in E). (F,G) Retinas from P14 Nestin-Cre;β8flox/+ control (F) or Nestin-Cre;β8flox/flox mutant (G) mice were immunolabeled with anti-CD31 and anti-GFAP antibodies to visualize vascular endothelial cells and astrocytes, respectively. Note that the abnormal morphologies of blood vessels in conditional knockout retinas (arrows in G), although there is an apparently normal astrocyte latticework. Images in D-G are shown at 200× magnification.

Fig. 6.

Retinal angiogenesis is not perturbed in mice lacking Tgfbr2 gene expression in astrocytes. (A) Nestin-Cre-mediated deletion of the Tgfbr2flox allele in genomic DNA isolated from the retina (r) but not matched tail snips (t) as confirmed by PCR analysis. In the retina there is a reduction in the ∼500 bp band owing to Cre-mediated recombination of the Tgfbr2flox allele. (B) Images of retinas isolated from P14 Nestin-Cre;Tgfbr2flox/+ control (left panel) or Nestin-Cre;Tgfbr2flox/flox mutant mice (right panel). Note the absence of hemorrhage in the mutant retinas. (C,D) Retinas from P14 control (C) and Nestin-Cre;Tgfbr2flox/flox mutant (D) mice were immunolabeled with anti-CD31 and anti-NG2 antibodies to visualize vascular endothelial cells and pericytes, respectively. Note the normal blood vessel cytoarchitecture containing vascular endothelial cells and pericytes in control and mutant samples. (E,F) Retinas from P14 control (E) and Nestin-Cre;Tgfbr2flox/flox mutant (F) mice were immunolabeled with anti-CD31 and anti-GFAP antibodies to visualize vascular endothelial cells and astrocytes, respectively. Note the normal endothelial cell and astrocyte interactions in both control and mutant samples. Images in C-F are shown at 200× magnification.

Fig. 7.

Anti-TGFβ neutralizing antibodies induce acute retinal angiogenesis defects. (A) Primary brain microvascular endothelial cells were treated for varying times (0, 30 or 60 minutes) with 1 ng/ml TGFβ1 (+TGFβ) or retinal astrocyte conditioned media (CM) containing anti-TGFβ neutralizing antibodies (+anti-TGFβ) or control IgGs (+IgGs). Note that treatment of endothelial cell cultures with TGFβ1 or CM+IgG leads to a time-dependent increase in Smad3 phosphorylation. By contrast, anti-TGFβ neutralizing antibodies inhibit Smad3 phosphorylation in endothelial cells. (B,C) P9 mouse pups were anesthetized and injected intraocularly with anti-TGFβ neutralizing antibodies (B) or control IgGs (C). Retinas were fluorescently labeled with IsoB4-Alexa488 to visualize blood vessels. Note the abnormal blood vessel morphologies in P9 mice injected with anti-TGFβ blocking antibodies (arrows in B). Images are shown at 200× magnification in the upper panels and at 400× magnification in lower panels. (D) Quantification of retinal angiogenesis pathologies in mice intraocularly injected with control IgGs or anti-TGFβ antibodies. ^*P<0.001. Error bars represent s.d.