Photoreceptor-Specific Expression of Platelet-Derived Growth Factor-B Results in Traction Retinal Detachment
2000; Elsevier BV; Volume: 157; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64612-3
ISSN1525-2191
AutoresMan Seong Seo, Naoyuki Okamoto, Melissa A. Vinores, Stanley A. Vinores, Sean F. Hackett, Haruhiko Yamada, Eri Yamada, Nancy L. Derevjanik, William J. LaRochelle, Donald J. Zack, Peter A. Campochiaro,
Tópico(s)Retinal Diseases and Treatments
ResumoExpression of platelet-derived growth factor (PDGF)-A and PDGF-B is increased in patients with proliferative retinopathies in which traction retinal detachments occur. Previous studies have demonstrated that increased expression of PDGF-A in the retina of transgenic mice results in retinal gliosis due to proliferation of astrocytes with different retinal phenotypes based on the time of onset and location of the PDGF-A production. In this study, we investigated the effects of PDGF-B in the retina using gain-of-function transgenic mice that express PDGF-B in photoreceptors. These mice show proliferation of astrocytes, pericytes, and, to a lesser extent, endothelial cells, resulting in ectopic cells on the surface and extending into the retina. The sheets of cells exert traction on the retina resulting in traction retinal detachments similar to those seen in humans with proliferative retinopathies. These studies suggest that PDGF-B has more dramatic effects in the retina than PDGF-A, because it acts on additional cell types, in particular on pericytes, which have a highly developed contractile apparatus. These studies in the retina suggest a means that could be used in other tissues throughout the body to achieve graded PDGF effects. They also provide a new model of traction retinal detachment that can be used to investigate new treatments for patients with proliferative retinopathies. Expression of platelet-derived growth factor (PDGF)-A and PDGF-B is increased in patients with proliferative retinopathies in which traction retinal detachments occur. Previous studies have demonstrated that increased expression of PDGF-A in the retina of transgenic mice results in retinal gliosis due to proliferation of astrocytes with different retinal phenotypes based on the time of onset and location of the PDGF-A production. In this study, we investigated the effects of PDGF-B in the retina using gain-of-function transgenic mice that express PDGF-B in photoreceptors. These mice show proliferation of astrocytes, pericytes, and, to a lesser extent, endothelial cells, resulting in ectopic cells on the surface and extending into the retina. The sheets of cells exert traction on the retina resulting in traction retinal detachments similar to those seen in humans with proliferative retinopathies. These studies suggest that PDGF-B has more dramatic effects in the retina than PDGF-A, because it acts on additional cell types, in particular on pericytes, which have a highly developed contractile apparatus. These studies in the retina suggest a means that could be used in other tissues throughout the body to achieve graded PDGF effects. They also provide a new model of traction retinal detachment that can be used to investigate new treatments for patients with proliferative retinopathies. Platelet-derived growth factor (PDGF) was isolated based on its ability to stimulate the proliferation of mesenchymal cells, and it is responsible for a substantial amount of the mitogenic activity in serum.1Ross R Glomset JA Kariya B Harker L A platelet-dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro.Proc Natl Acad Sci USA. 1974; 71: 1207-1210Crossref PubMed Scopus (1352) Google Scholar It is released from aggregated platelets at wound sites and acts as a chemoattractant and mitogen for many cell types that participate in wound repair, including glial cells, smooth muscle cells, pericytes, and fibroblasts.2Heldin CH Westermark B Platelet-derived growth factor: mechanism of action and possible in vivo function.Cell Reg. 1990; 8: 555-566Google Scholar Exogenous PDGF promotes increased wound strength, and neutralizing anti-PDGF antibodies impair wound healing.3Pierce GF Mustoe TA Altrock BW Deuel TF Thomason A Role of platelet-derived growth factor in wound healing.J Cell Biochem. 1991; 45: 319-326Crossref PubMed Scopus (459) Google Scholar Thus, PDGF plays an important role in wound repair throughout the body. In the eye, there is low level constitutive expression of PDGFs in perivascular cells, ganglion cells, and in the retinal pigmented epithelium (RPE).4Mudhar HS Pollock RA Wang C Stiles CD Richardson WD PDGF and its receptors in the developing rodent retina and optic nerve.Development. 1993; 118: 539-552PubMed Google Scholar, 5Campochiaro PA Hackett SF Vinores SA Freund J Csaky C La Rochelle W Henderer J Johnson M Rodriguez IR Friedman Z Derevjanik N Dooner J Platelet-derived growth factor is an autocrine growth stimulator in retinal pigmented epithelial cells.J Cell Sci. 1994; 107: 2459-2469PubMed Google Scholar It appears to play a role in recruitment of pericytes and astrocytes into the retina, and may support the survival of these cells in adult retinas.6Lindahl P Johansson BR Leveen P Betsholtz C Pericyte loss and microaneurysm formation in PDGF-B-deficient mice.Science. 1997; 277: 242-245Crossref PubMed Scopus (1797) Google Scholar, 7Benjamin LE Hemo I Keshet E A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B.Development. 1998; 125: 1591-1598Crossref PubMed Google Scholar After retinal detachment there is marked up-regulation of PDGF expression in the RPE.5Campochiaro PA Hackett SF Vinores SA Freund J Csaky C La Rochelle W Henderer J Johnson M Rodriguez IR Friedman Z Derevjanik N Dooner J Platelet-derived growth factor is an autocrine growth stimulator in retinal pigmented epithelial cells.J Cell Sci. 1994; 107: 2459-2469PubMed Google Scholar The retina can be reattached by surgery, but in roughly 10% of cases, scar tissue made up predominantly of RPE and glial cells forms in the vitreous and on the surfaces of the retina and results in traction that redetaches the retina.8Cowley M Conway BP Campochiaro PA Kaiser D Gaskin H Clinical risk factors for proliferative vitreoretinopathy.Arch Ophthalmol. 1989; 107: 1147-1151Crossref PubMed Scopus (177) Google Scholar There is prominent expression of PDGFs in the cells that participate in the scarring. This disease process is called proliferative vitreoretinopathy, the most common cause of failure of retinal reattachment surgery, and PDGFs have been implicated in its pathogenesis.9Vinores SA Henderer JD Mahlow J Chiu C Derevjanik NL LaRochelle W Csaky C Campochiaro PA Isoforms of platelet-derived growth factor and its receptors in epiretinal membranes: immunolocalization to retinal pigmented epithelial cells.Exp Eye Res. 1995; 60: 607-619Crossref PubMed Scopus (66) Google Scholar, 10Campochiaro PA Pathogenic mechanisms in proliferative vitreoretinopathy.Arch Ophthalmol. 1997; 115: 237-241Crossref PubMed Scopus (208) Google Scholar But PDGF also contributes to regulation of development11Mercola M Wang C Kelley J Brownlee C Jackson-Grusby L Stiles C Bowen-Pope D Selective expression of PDGF and its receptor during early mouse embryogenesis.Devel Biol. 1990; 138: 114-122Crossref PubMed Scopus (188) Google Scholar, 12Leveen P Pekny M Gerbre-Medhin S Swolin B Larsson E Betsholtz C Mice deficient for PDGF-B show renal, cardiovascular, and hematological abnormalities.Genes Dev. 1994; 8: 1875-1887Crossref PubMed Scopus (901) Google Scholar, 13Soriano P Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice.Genes Dev. 1994; 8: 1888-1896Crossref PubMed Scopus (820) Google Scholar, 14Schatteman GC Motley ST Effmann EL Bowen-Pope DF Platelet-derived growth factor receptor alpha subunit deleted patch mouse exhibits severe cardiovascular dysmorphogenesis.Teratology. 1995; 51: 351-366Crossref PubMed Scopus (85) Google Scholar and expression of PDGF in neurons suggests possible neurotrophic and/or gliotrophic effects.4Mudhar HS Pollock RA Wang C Stiles CD Richardson WD PDGF and its receptors in the developing rodent retina and optic nerve.Development. 1993; 118: 539-552PubMed Google Scholar, 15Smits A Kato M Westermark B Nister M Heldin CH Funa K Neurotrophic activity of platelet-derived growth factor (PDGF): rat neuronal cells possess functional PDGF beta-type receptors and respond to PDGF.Proc Natl Acad Sci USA. 1991; 88: 8159-8163Crossref PubMed Scopus (212) Google Scholar PDGF is a dimer made up of the products of the PDGF-A and PDGF-B genes, resulting in three isoforms, PDGF-AA, -AB, and -BB. Similarly, two gene products result in three types of dimeric receptors, PDGF receptor (PDGFR)-αα, -αβ, and -ββ.16Hart CE Bowen-Pope DF Platelet-derived growth factor receptor: current views of the two subunit model.J Invest Dermatol. 1990; 94: 535-575Crossref Scopus (56) Google Scholar PDGFR-α binds both PDGF-A and -B, while PDGFR-β binds only PDGF-B. Therefore, PDGF-A is more selective than PDGF-B and, theoretically, it might affect fewer cell types in a given tissue than PDGF-B depending on the expression of α and β receptors on various cell types. At least two cell types appear to have very selective expression of PDGFR isoforms. Glial precursor O2-A progenitor cells from the optic nerve express PDGFR-α, but not PDGFR-β,17Barres BA Raff MC Control of oligodendrocyte number in the developing rat optic nerve.Neuron. 1994; 12: 935-942Abstract Full Text PDF PubMed Scopus (355) Google Scholar while pericytes express PDGFR-β, but not PDGFR-α.6Lindahl P Johansson BR Leveen P Betsholtz C Pericyte loss and microaneurysm formation in PDGF-B-deficient mice.Science. 1997; 277: 242-245Crossref PubMed Scopus (1797) Google Scholar Also, it has been suggested, based on in vitro data, that endothelial cells participating in angiogenesis may express PDGFR-β.18Battegay EJ Rupp J Iruela-Arispe L Sage EH Pech M PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF beta-receptors.J Cell Biol. 1994; 125: 917-928Crossref PubMed Scopus (342) Google Scholar Because each of these cell types is located in the retina, the retina provides a good model system to explore similarities and differences in the activities of PDGF isoforms. Recently, we coupled the cDNA of human PDGF-A to the bovine rhodopsin promoter and generated transgenic mice (rho/PDGF mice) with photoreceptor-specific expression of PDGF-A.19Yamada H Yamada E Ando A Seo M-S Esumi N Okamoto N Vinores M LaRochelle W Zack DJ Campochiaro PA Platelet-derived growth factor-A-induced retinal gliosis protects against ischemic retinopathy.Am J Pathol. 2000; 156: 477-487Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar These mice developed a fairly subtle phenotype due to glial infiltration of the inner retina. The ectopic glial cells conferred a striking resistance to oxygen-induced retinal vascular nonperfusion and prevented the development of neovascularization. In this study, we used gain-of-function transgenic mice that express PDGF-B in photoreceptors to compare the effects of PDGF-B in the retina to those of PDGF-A and to determine whether these mice would exhibit a phenotype like that seen in patients with proliferative retinopathies. A 907-bp cDNA fragment of human PDGF-B, including 117 bp of 3′ untranslated sequence, the entire coding region, and 68 bp of untranslated 5′ sequence, was cloned into a plasmid containing the 2.2 kb Hin dIII/NaeI fragment from the bovine rhodopsin promoter.20Zack DJ Bennett J Wang Y Davenport C Klaunberg B Gearhart J Nathans J Unusual topography of bovine rhodopsin promoter-lac z fusion gene expression in transgenic mouse retinas.Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (188) Google Scholar The plasmid also contained an intron and a polyA addition site derived from the mouse protamine gene and a eukaryotic consensus ribosomal binding site. After transformation, a clone with correct orientation was selected. DNA was double CsCl purified and cut with Eco RI to provide a 3579-bp fusion gene (Figure 1). The fusion gene was purified and transgenic mice were generated using established techniques as previously described.20Zack DJ Bennett J Wang Y Davenport C Klaunberg B Gearhart J Nathans J Unusual topography of bovine rhodopsin promoter-lac z fusion gene expression in transgenic mouse retinas.Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (188) Google Scholar The fusion gene was injected into the pronuclei of B6AF1 (female) × C57BL/6J (male). All offspring were backcrossed with C57BL/6J mice. Mice were screened for the presence of the transgene by either Southern blot analysis or polymerase chain reaction (PCR) of tail DNA.20Zack DJ Bennett J Wang Y Davenport C Klaunberg B Gearhart J Nathans J Unusual topography of bovine rhodopsin promoter-lac z fusion gene expression in transgenic mouse retinas.Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (188) Google Scholar Tail pieces were digested overnight at 55°C in 50 mmol/L Tris (pH 7.5), 100 mmol/L EDTA, 400 mmol/L NaCl, and 0.5% sodium dodecyl sulfate containing 0.6 μg/μl proteinase K. PCR was done at an annealing temperature of 58°C, with primers that amplify 589 bp of transgene-specific sequence, P1 (5′-GTCCAGCCGGAGCCCCGTG-3′) and P2 (5′-CCGCACAATCTCGATCTTTCTCACC-3′) (Figure 1). At appropriate time points, mice were sacrificed, eyes were removed, and retinas were dissected. Retinal RNA was isolated using the guanidine isothiocyanate method as described by Chomczynski and Sacchi.21Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (66740) Google Scholar Reverse transcription was carried out with ∼0.5 μg of total RNA, reverse transcriptase (SuperScript II, Life Technologies, Gaithersburg, MD), and 5.0 μM oligo d(T) primer. Aliquots of the cDNAs were used for PCR amplification with primers for the hPDGFB/mP1 fusion gene that amplify across an intron-exon border, P3 (5′-ATAGACCGCACCAACGCCAACTTC-3′) and P4 (5′-TGTGGCGAGATGCTCTTGAAGTCTGGTA-3′) (Figure 1). The expected PCR products for the hPDGFB/mP1 fusion gene fragment from genomic DNA and mRNA are 787 bp and 693 bp, respectively. Titrations were performed to ensure that PCR reactions were carried out in the linear range of amplification. Mouse S16 ribosomal protein primers (5′-CACTGCAAACGGGGAAATGG-3′ and 5′-TGAGATGGACTGTCGGATGG-3′) were used to provide an internal control for the amount of template in the PCR reactions. RNA blot hybridization analysis was done as previously described5Campochiaro PA Hackett SF Vinores SA Freund J Csaky C La Rochelle W Henderer J Johnson M Rodriguez IR Friedman Z Derevjanik N Dooner J Platelet-derived growth factor is an autocrine growth stimulator in retinal pigmented epithelial cells.J Cell Sci. 1994; 107: 2459-2469PubMed Google Scholar using 10 μg of total retinal RNA. The 842 bp Bam HI fragment of hPDGFB or the 598-bp Bam HI fragment of hVEGF were labeled with32P by hexanucleotide random priming and used as cDNA probes. Hybridization temperature was 65°C and the membrane was washed twice for 60 minutes at room temperature in 2× SSC, 0.1% SDS, followed by a 15 minute wash at 58°C in 1× SSC, 0.1% SDS, and a final 15-minute wash at 65°C in 0.5× SSC, 0.1% SDS. Transgene-positive and littermate control mice were sacrificed at various time points, and their eyes were removed and frozen in optimal cutting temperature medium (OCT, Miles Diagnostics, Elkhart, IN). Ten-micron sections were cut and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS). The sections were washed and immunohistochemical staining was performed as previously described5Campochiaro PA Hackett SF Vinores SA Freund J Csaky C La Rochelle W Henderer J Johnson M Rodriguez IR Friedman Z Derevjanik N Dooner J Platelet-derived growth factor is an autocrine growth stimulator in retinal pigmented epithelial cells.J Cell Sci. 1994; 107: 2459-2469PubMed Google Scholar with a 1:50 dilution of a monoclonal antibody that recognizes PDGF-B.22LaRochelle WJ Robbins KC Aaronson SA Immunochemical localizations of the epitope for a monoclonal antibody that neutralizes human platelet-derived growth factor mitogenic activity.Mol Cell Biol. 1989; 9: 3538-3542Crossref PubMed Scopus (29) Google Scholar Specificity of staining was assessed by substitution of nonimmune serum for primary antibody and by preabsorption of primary antibody with antigenic peptide as previously described.23Vinores SA Youssri AI Luna JD Chen Y-S Bhargave S Vinores MA Schoenfeld C-L Peng B Chan C-C LaRochelle W Green WR Campochiaro PA Upregulation of vascular endothelial growth factor in ischemic and non-ischemic human and experimental retinal disease.Histol Histopathol. 1997; 12: 99-109PubMed Google Scholar At various time points, mice were sacrificed and eyes were snap-frozen in OCT or fixed in 10% buffered formalin and embedded in paraffin. Frozen or paraffin sections were stained with hematoxylin and eosin (H&E), histochemically stained with biotinylated Griffonia simplicifolia isolectin-B4 (GSA; Vector Laboratories, Burlingame, CA), or immunohistochemically stained with a 1:1000 dilution of a rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; Dako, Santa Barbara, CA), a 1:50 dilution of rabbit polyclonal antibodies to α-smooth muscle actin (Biogenix, San Ramon, CA), a 1:40 dilution of rabbit polyclonal antibody to vascular endothelial growth factor (VEGF; Santa Cruz Biotechnology, Santa Cruz, CA), or a 1:200 dilution of a rabbit polyclonal antibody directed against platelet-endothelial cell adhesion molecule (PECAM; Santa Cruz Biotechnology). PDGF-B was visualized with HistoMark Red (Gaithersburg, MD) according to the manufacturer's instructions and other antigens were visualized with diaminobenzidine (Research Genetics, Huntsville, AL). Staining with biotinylated GSA (Vector Laboratories), which selectively binds to vascular cells, was done as previously described.24Ozaki H Okamoto N Ortega S Chang M Ozaki K Sadda S Vinores MA Derevjanik N Zack DJ Basilico C Campochiaro PA Basic fibroblast growth factor is neither necessary nor sufficient for the development of retinal neovascularization.Am J Pathol. 1998; 153: 757-765Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Slides were incubated in methanol/H2O2 for 30 minutes at 4°C, washed with 0.05 mol/L Tris-buffered saline (TBS), pH 7.4, and incubated for 30 minutes in 10% normal porcine serum. Slides were rinsed with 0.05 Mol/L TBS and incubated 1 hour at 37°C with biotinylated lectin. After being rinsed with 0.05 Mol/L TBS, slides were incubated with avidin coupled to peroxidase (Vector Laboratories) for 45 minutes at room temperature. After being washed for 10 minutes with 0.05 mol/L Tris buffer, pH 7.6, slides were incubated with diaminobenzidine (Research Genetics) to give a brown reaction product, and mounted with Cytoseal (Stephens Scientific, Riverdale, NJ). Six independent lines that incorporated the rhodopsin promoter/PDGFB fusion gene (Figure 1) were obtained (designated rho/PDGFB1–6). The founders were backcrossed with C57BL/6J mice to establish transgenic lines. Mice that were heterozygous at the transgene locus were used in all analyses. At postnatal day 14 (P14), RT-PCR, using total retinal RNA as template and primers specific for transgene mRNA, showed good expression in lines rho/PDGFB1–4, barely detectable expression in line 5, and no detectable expression in line 6 (Figure 2A). A time course in mice from line 1 showed transgene mRNA was first detectable at P5 or P6, increased to a steady state level by about P10, and was maintained through at least 3 months after birth, the longest time point examined (Figure 2B). Northern analysis also suggested that steady state levels of mRNA were reached by day 10, and there appeared to be similar levels of expression in lines 1, 2, and 3 (Figure 2C). Immunohistochemistry with an antibody that specifically recognizes PDGF-B showed reaction product in photoreceptors, with an intense band of staining in the region of photoreceptor terminals (Figure 3). Similar localization was seen for PDGF-A in several lines of rho/PDGFA mice,19Yamada H Yamada E Ando A Seo M-S Esumi N Okamoto N Vinores M LaRochelle W Zack DJ Campochiaro PA Platelet-derived growth factor-A-induced retinal gliosis protects against ischemic retinopathy.Am J Pathol. 2000; 156: 477-487Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar suggesting the possibility of polarized secretion. Although rho/PDGFB mice show the greatest staining for PDGF-B in photoreceptors, there is also increased staining for PDGF-B in the inner retina compared to wild-type mice, suggesting that PDGF-BB diffuses through the retina and is accessible to cells in the inner retina. For each of the 6 transgenic lines, histopathological evaluation was done between 3 and 9 months of age on 8 to 10 mice selected from 3 generations. All mice from lines 1–4 showed traction detachments of the retinas in both eyes, whereas mice from lines 5 and 6 had normal appearing retinas with no detachments, presumably due to the low expression levels of transgene-derived PDGF-B in the latter two lines. In many instances, the retina became adherent to the back of the lens, forming a retrolental mass. There was striking degeneration of the detached retinas and prominent epiretinal and subretinal cellular membranes (Figure 4). The traction detachments in rho/PDGFB mice are similar to the traction retinal detachments seen in humans with proliferative vitreoretinopathy, in which there are tractional membranes made up primarily of retinal pigmented epithelial (RPE) cells and retinal glial cells, or to the traction retinal detachments seen in retinopathy of prematurity, in which there is a prominent vasoproliferative component in addition to proliferation of RPE and glia. To explore why overexpression of PDGF-B in the retina leads to traction retinal detachment, a detailed investigation of the cellular involvement and sequence of events was performed primarily in lines 1 and 2. Astrocytes are small glial cells that in adult mouse retinas are normally found in the nerve fiber layer and adjacent to retinal blood vessels. Muller cells are large glial cells extending from the inner to the outer surface of the retina. Developing astrocytes are known to have PDGFR-αs and are very responsive to PDGFs for at least 2 weeks after birth.25Reneker LW Overbeek PA Lens-specific expression of PDGF-A in transgenic mice results in retinal astrocytic hamartomas.Invest Ophthalmol Vis Sci. 1996; 37: 2455-2466PubMed Google Scholar They migrate from the optic nerve into the posterior portion of the inner retina and then spread anteriorly until they populate the entire nerve fiber layer. They associate with blood vessels in the nerve fiber layer and are visualized by immunohistochemical staining for glial fibrillary acidic protein (GFAP). At P6, which is soon after transgene expression begins, rho/PDGFB1 or -2 and wild-type mice showed identical patterns of sparse GFAP-positive astrocytes at the inner surface of the retina (Figure 5, P6+ and P6−), but by P7, GFAP staining along the inner surface of the retina was increased in transgenics compared to wild-type mice. The difference was more pronounced at P8 when there was clearly a multilayered collection of astrocytes in transgenics. At P9 and P10, in addition to continued sparse staining at the retinal surface, wild-type mice showed focal areas of GFAP staining in the inner nuclear layer, presumably due to migration of astrocytes into the retina along with penetrating retinal vessels. In contrast, rho/PDGFB1 and -2 mice showed a thick carpet of astrocytes on the surface of the retina and cords of invading cells extending into the inner nuclear layer that were quite prominent by P10. At P12 and beyond, there was often evidence of retinal folding with focal areas of retinal detachment that progressed to total retinal detachment at later time points. Perturbations of the retina, including detachment, often result in increased expression of GFAP in Muller cells; this is illustrated by the radial streaks of staining seen at P14 and beyond in transgenics (Figure 5; P14, P21, and P28). The carpet of astrocytes along the retinal surface became more compact and less prominent at later time points. Pericytes have PDGFR-βs,4Mudhar HS Pollock RA Wang C Stiles CD Richardson WD PDGF and its receptors in the developing rodent retina and optic nerve.Development. 1993; 118: 539-552PubMed Google Scholar, 6Lindahl P Johansson BR Leveen P Betsholtz C Pericyte loss and microaneurysm formation in PDGF-B-deficient mice.Science. 1997; 277: 242-245Crossref PubMed Scopus (1797) Google Scholar and PDGF-BB stimulates their proliferation.26Hirschi KK Rohovsky SA Beck LH Smith SR D'Amore PA Endothelial cells modulate the proliferation of mural cell precursors via platelet-derived growth factor-BB and heterotypic cell contact.Circ Res. 1999; 84: 298-305Crossref PubMed Scopus (293) Google Scholar Also, there is some suggestion that endothelial cells may express PDGFR-βs under certain conditions.18Battegay EJ Rupp J Iruela-Arispe L Sage EH Pech M PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF beta-receptors.J Cell Biol. 1994; 125: 917-928Crossref PubMed Scopus (342) Google Scholar Therefore, we sought to determine whether pericytes and endothelial cells participate in the proliferative response in rho/PDGFB mice. GSA lectin selectively binds vascular cells and has been suggested to be an endothelial cell-specific marker,27Sahagun G Moore SA Fabry Z Shelper RL Hart MN Purification of murine endothelial cell cultures by flow cytometry using fluorescein-labeled Griffonia simplicifolia agglutinin.Amer J Pathol. 1989; 134: 1227-1232PubMed Google Scholar, 28Schulte BA Spicer SS Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates.Amer J Anat. 1983; 168: 345-362Crossref PubMed Scopus (107) Google Scholar but we have recently demonstrated that it binds to both endothelial cells and pericytes.29Vinores SA Derevjanik NL Ozaki H Okamoto N Campochiaro PA Cellular mechanisms of blood-retinal barrier dysfunction in macular edema.Doc Ophthalmol. 1999; 97: 217-228Crossref PubMed Google Scholar There was identical staining for GSA on the surface of the retina in wild-type and transgenic mice at P6, which is around the time of onset of PDGF-B expression in transgenics (Figure 6). This represents the developing retinal vessels on the surface of the retina. By P7 there was more GSA staining along the inner surface of the retina in transgenics compared to wild-type mice, and the difference was more pronounced at P8 when there was clearly a multilayered collection of vascular cells in transgenics. At P9, wild-type mice show penetrating retinal vessels that form the intermediate and deep capillary beds that assume the appearance seen in normal adults between P10 and P12. In transgenics, the formation of the deep capillary beds was impaired. On P9, there were many more vascular cells on the surface of the retina in transgenic compared to wild-type mice, but fewer vascular cells had migrated into the retina. At P10, there were thick, finger-like projections extending into the inner retina from a multilayered carpet of cells on the retinal surface. The invading cords of vascular cells did not appear to be organized into vessels. At P12, much of the inner retina was infiltrated and replaced by GSA-positive cells. At P14 and later time points, there appeared to be some involution and contraction of the vascular cells, resulting in focal areas of retinal detachment that progressed and resulted in total retinal detachment. Staining for GSA was compared in retinas of P12 mice from each of the rho/PDGFB lines, except line 6, which was lost because mice from this line reproduced poorly. Mice from lines 1–4 showed similar abnormalities in each (Figure 7). Mice from line 5, which had barely detectable transgene expression, showed the mildest phenotype with some development of the deep capillary bed and minimal increase in GSA-positive cells in the inner retina. A deficiency of normally perfused retinal vessels was confirmed in whole mounts of fluorescein-labeled dextran-perfused retinas from line 1 (Figure 8). The capillary network was much less dense in transgenics compared to wild type mice, and there were several moderate-sized areas of nonperfusion (Figure 8B, asterisks). As expected from the disorganized appearance of the vascular cell proliferation seen in retinal sections, whole mounts of perfused retinas failed to show definite neovascularization, but did show occasional vascular malformations (Figure 8B, arrows).Figure 8Retinal whole mounts of fluorescein dextran-perfused transgene-positive (+) and -negative (−)rho/PDGFB1 mice show that transgene-positive mice have decreased retinal capillary density, vascular malformations, and no neovascularization. Mice were perfused with fluorescein-labeled dextran at P21 and retinal whole mounts were examined by fluorescence microscopy. Transgene-positive mice showed an irregular capillary bed that was less dense than that in transgene-negative mice, and showed moderate sized areas of nonperfused retina (asterisks). No neovascularization was identified, but there were vascular malformations (arrows). B and C are higher power views of the same retina shown in A, and E and F are higher power views of the same retina shown in D.View Large Image Figure ViewerDownload (PPT) Anti-PECAM binds endothelial cells and not pericytes,30Schimmenti LA Yan HC Madri JA Albelda SM Platelet endothelial cell adhesion molecule, PECAM-1, modulates cell migration.J Cell Physiol. 1992; 153: 417-428Crossref PubMed Scopus (115) Google Scholar, 31Romer LH McLean NV Yan HC Daise M Sun J DeLisser HM INF-gamma and TNF-alpha induce redistribution of PECAM-1 (CD 31) on human endothelial cells.J Immunol. 1995; 154: 6582-
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