Protease nexin-1 regulates retinal vascular development

Abstract

We recently identified protease nexin-1 (PN-1) or serpinE2, as a possibly underestimated player in maintaining angiogenic balance. Here, we used the well-characterized postnatal vascular development of newborn mouse retina to further investigate the role and the mechanism of action of PN-1 in physiological angiogenesis. The development of retinal vasculature was analysed by endothelial cell staining with isolectin B4. PN-1-deficient (PN-1(-/-)) retina displayed increased vascularization in the postnatal period, with elevated capillary thickness and density, compared to their wild-type littermate (WT). Moreover, PN-1(-/-) retina presented more veins/arteries than WT retina. The kinetics of retinal vasculature development, retinal VEGF expression and overall retinal structure were similar in WT and PN-1(-/-) mice, but we observed a hyperproliferation of vascular cells in PN-1(-/-) retina. Expression of PN-1 was analysed by immunoblotting and X-Gal staining of retinas from mice expressing beta-galactosidase under a PN-1 promoter. PN-1 was highly expressed in the first week following birth and then progressively decreased to a low level in adult retina where it localized on the retinal arteries. PCR arrays performed on mouse retinal RNA identified two angiogenesis-related factors, midkine and Smad5, that were overexpressed in PN-1(-/-) newborn mice and this was confirmed by RT-PCR. Both the higher vascularization and the overexpression of midkine and Smad5 mRNA were also observed in gastrocnemius muscle of PN-1(-/-) mice, suggesting that PN-1 interferes with these pathways. Together, our results demonstrate that PN-1 strongly limits physiological angiogenesis and suggest that modulation of PN-1 expression could represent a new way to regulate angiogenesis.

Keywords: Angiogenesis; PN-1; Retina; Serpin; SerpinE2.

Fig. 1

Vascularization of the retina of 4- and 6-day-old pups (P4 and P6, respectively) was analysed by endothelial cell staining with isolectin B4 (in red). Flat-mount retina from wild-type (WT) and PN-1-deficient mice (PN-1^−/−) are shown. a Total retina, scale bar 1 mm. The vascular radius indicated by arrows was measured, and the results were expressed as the percentage of retinal radius (3–4 sections per retina, n ≥ 4 retinas from different animals). b Magnification, scale bar 150 µm at P4, 100 µm at P6. Representative images are shown. Capillary density was analysed after magnification of the 100 × 300 µm areas indicated by rectangles and quantified as the percentage of area covered by vessels (6–8 sections per retina, n ≥ 4 retinas from different animals)

Fig. 2

PN-1 deficiency is associated with an increased retinal vascularization. Quantification of capillary thickness, length, number of junctions and of intercapillary spaces was performed on the areas indicated in Fig. 1b using the Biologic Analyser Software (6–8 sections per retina, n ≥ 4 retinas from different animals). *p < 0.05, **p < 0.01, ***p < 0.0001 vs. WT

Fig. 3

PN-1 deficiency is associated with an increased retinal vascularization—quantification of retinal veins and arteries. a Whole-mount WT and PN-1^−/− P7 retinas were stained with isolectin B4; rectangles bordering the central optic nerve area are shown in higher magnification in b. c Vascularization of adult retina. Flat-mount retinas were immunostained with antibody to smooth muscle alpha actin (in green) to reveal mainly retinal arteries. Representative images from at least three different animals are shown. d The number of retinal veins and arteries was counted manually. It ranged from 4 to 7 in WT and PN-1^−/− retina (mice from P4 to adult age were pooled, with n = 40 for each genotype). For each genotype, the number of retinas having four, five, six or seven veins and arteries was compiled, and the results were expressed as the percentage of the total number of retinas

Fig. 4

PN-1 expression in mouse retina. a PN-1 expression in retinal lysates was analysed by immunoblotting at different postnatal ages, and results were quantified by densitometric analysis. A representative blot out of three blots performed on three different sets of retina is shown on the left, and on the right; the corresponding results (analysis of 6 retinas per age) are expressed as the percentage of the maximal expression, observed at P3. X-gal staining of whole-mount PN-1/LacZ retina. b Whole retina. c Magnification of the optic nerve area of the image shown in b. Circles represent the optic nerve head region. No staining was observed in retinas from WT mice. d X-gal staining of PN-1/LacZ retinal cross sections. INL inner nuclear layer, GCL ganglion cell layer, RPE retinal pigment epithelium. Representative images are shown out of at least three different animals. Scale bars 1 mm in b, 200 µm in c, 50 µm in d

Fig. 5

Retinal structure, pericyte coverage and astrocyte template are not modified by PN-1 deficiency. a Histological sections of historesin-embedded eyes from adult WT and PN-1^−/− mice. GCL ganglion cells layer, INL inner cells layer, ONL outer cell layer. b In green, pericyte coverage was visualized by NG2 immunostaining of P4 retinas, together with endothelial cells staining by isolectin in red. Scale bars 30 µm. c Astrocyte distribution was visualized by PDGFRα and GFAP immunostaining of P4 retinas, scale bars 50 µm. PDGFRα and GFAP positive astrocytes were analysed in avascular and vascularized areas, respectively. Representative images are shown. Quantifications indicate no difference in any of the markers analysed between WT and PN-1^−/− retinas (4–6 fields par retina from at least 3 different animals)

Fig. 6

PN-1 deficiency does not modify retinal tip cells, but increases endothelial cell proliferation. a Vessels were visualized by isolectin B4 staining of P5 WT and PN-1^−/− retinas. Images of angiogenic front and quantification of filopodia in WT and KO retinas reveal no difference in tip cells. Representative images are shown (4 fields par retina, ≥3 different animals), scale bar 30 µm. b P5 retinas were stained with isolectin B4 (Isl, red) and an antibody against KI67 (green). Scale bar 50 µm. Representative images (4 fields per retina, 4 retinas from different animal of each genotype) and quantification of proliferating (Ki67+) endothelial cells (Isl+) are shown

Fig. 7

PN-1 deficiency does not modify VEGF level, but increases midkine and Smad5 expression in the retina. a VEGF was quantified by ELISA in retinal lysates (n = 3–5 from different animals). b Gene expression analysis by PCR-based micro array and conventional RT-PCR on retinal mRNA revealed increased expression of midkine and Smad 5 (n ≥ 3 experiments). c Representative immunoblots of P4 retinal whole lysates with antibodies to midkine or Smad5 (n ≥ 3 immunoblots performed on different sets of retina) and densitometric analysis of the blots, expressed as mean intensity (midkine or Smad5/actin ratio) relative to WT, determined for each immunoblot. *p < 0.05, **p < 0.001, ***p < 0.0001 vs. WT, ns non-significant

Fig. 8

In gastrocnemius muscle, PN-1 deficiency is also associated with increased vascularization and increased expression of midkine and Smad5. Muscle sections were stained with isolectin B4 (a) or with smooth muscle alpha actin (SMA) (b). Representative images are shown from at least four different animals. Vascular coverage was quantified by densitometric analysis of isolectin images and arterioles were counted manually on SMA images. **p < 0.01, ***p < 0.001 vs. WT. c Midkine and Smad5 mRNA levels were compared in WT and PN-1^−/− tibialis muscles by RT-PCR (n = 26)