Photoreceptor avascular privilege is shielded by soluble VEGF receptor-1
Vascular demarcations in the eye are both striking and vital for vision . The completely avascular photoreceptor layer of the retina lies between the vascular inner retina ( on its apical aspect ) and the highly vascularized and permeable fenestrated choriocapillaris ( Blaauwgeers et al . , 1999 ) ( on its basal aspect ) ( Figure 1—figure supplement 1 ) . The choroid , which has the highest flow of any vascular bed ( Bill et al . , 1983 ) , fulfills the metabolic and oxygen demands of the retinal pigmental epithelium ( RPE ) cells and photoreceptors . In age-related macular degeneration ( AMD ) , angiogenic vessels from the inner retina extend into the photoreceptors ( retinal angiomatous proliferation , or RAP ) , or from choroidal vessels breaching through the complex of the RPE and Bruch’s membrane ( BrM ) into the subretinal space ( choroidal neovascularization , CNV ) . Either can disrupt retinal structure and function , causing irreversible vision loss ( Ambati et al . , 2003b ) . Although AMD is the leading cause of irreversible blindness in the West ( Ambati et al . , 2003b ) , its etiology has not been fully elucidated . Understanding the molecular underpinnings of the physiological vascular zoning ability of the retina has been a challenge for retinal biology for decades and may provide a better understanding of AMD pathogenesis . Vascular endothelial growth factor A ( VEGF-A ) is a potent angiogenic factor with a crucial role in both normal and pathologic vascular growth within the eye ( Saint-Geniez et al . , 2008; Saint-Geniez et al . , 2009; Rajappa et al . , 2010; Carmeliet Jain , 2011; Potente et al . , 2011 ) , and its increased expression is linked to pathologic proliferation of abnormal , highly permeable vessels into the avascular photoreceptor layers ( Amin et al . , 1994; Ambati et al . , 2003b; Rajappa et al . , 2010 ) . A soluble isoform of VEGF receptor-1 ( sVEGFR-1 , also known as sFLT-1 ) acts as a naturally occurring inhibitor of VEGF-A-mediated angiogenesis ( Kendall and Thomas , 1993 ) . sFLT-1 negatively regulates VEGF-A action: first , it binds and sequesters VEGF-A; and second , by heterodimerizing with membrane-bound VEGF-receptor-2 , it prevents VEGF-A occupancy and subsequent signal transduction ( Kendall et al . , 1996 ) . Excess levels of sFLT-1 strongly inhibit CNV in mice and monkeys ( Lai et al . , 2005; Lukason et al . , 2011 ) . In addition , we previously demonstrated that endogenous sFLT-1 is both necessary and sufficient for corneal avascularity ( Ambati et al . , 2006 ) . Therefore , we hypothesized that avascular photoreceptor privilege depends on sFLT-1 and that a deficiency of endogenous sFLT-1 might be involved in CNV or RAP development .
Although the specificity of soluble FLT-1 antibody had been verified , we further confirmed this by ELISA as it is crucial to distinguish it from membrane FLT-1 ( Figure 1—figure supplement 2 ) . We then compared sFLT-1 expression in healthy human eyes and eyes with AMD or RAP ( patient information is shown in Table 1 ) by two different immunostaining methods . Both results demonstrated that soluble FLT-1 was strikingly decreased in the RPE and photoreceptor layers in all AMD ( n = 3 ) and RAP eyes ( n = 3 ) compared with age matched controls , respectively ( Figure 1A , Figure 2—figure supplement 3 ) ; conversely , as has been previously reported , VEGF-A was upregulated significantly in the retina of AMD eyes ( Ambati et al . , 2003b ) . 10 . 7554/eLife . 00324 . 003Table 1 . Demographic information for human globesDOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 003NormalNormalNormalNormalNormalAMD ( CNV ) AMD ( dry ) AMDRAPRAPRAP25/M71/M79/F80/F89/F80/F86/F88/F67/M86/F86/FAll humans are Caucasians . AMD: age-related macular degeneration; CNV: choroidal neovascularization; F: female; M: male; RAP: retinal angiomatous proliferation . 10 . 7554/eLife . 00324 . 004Figure 1 . Soluble FLT-1 expression is reduced in the photoreceptors and RPE from AMD eyes . ( A ) Representative immunostaining images show soluble FLT-1 expression was significantly decreased in an age-related macular degeneration ( AMD ) eye with choroidal neovascularization ( CNV ) ( top , 80 years old , female ) and retinal angiomatous proliferation ( RAP ) ( bottom , 67 years old , female ) compared with aged-matched controls ( 80 years old and 71 years old , respectively , females; black arrow heads point to the photoreceptors; red arrows point to the CNV or RAP lesions ) . ( B ) In situ hybridizations show the localization of sFlt-1 ( purple/blue ) in the photoreceptors and retinal pigment epithelium ( RPE ) layers in Balb/c mice . ( C ) Immunohistochemistry ( IHC ) staining shows sFLT-1 and VEGF expression in wild type mice . Higher relative expression of sFLT-1 to VEGF is observed in the photoreceptors . The magnified images ( bottom ) from the framed area showed that soluble FLT-1 is expressed in the basal side of the RPE layer . ( D ) Representative IHC image shows sFLT-1 is expressed in the basal side of the RPE layer in a young adult healthy human eye ( 25 years old , male ) . CC: choriocapillaris; IN: inner nuclear layer; IS: inner segment layer; ON: outer nuclear layer; OS: outer segment layer . Arrows point to the RPE layer . Scale bar: 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 00410 . 7554/eLife . 00324 . 005Figure 1—figure supplement 1 . Avascularity of the outer retina ( photoreceptors and RPE and BrM ) surrounded by the inner retina with abundant vessels and the highly vascularized choroid in a normal human eye . Red: perlecan staining of vessels; blue: DAPI . Arrow heads point to the vessels . BrM: Bruch's membrane; Ch: choroid; RPE: retinal pigment epithelium; CC: choriocapillaris . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 00510 . 7554/eLife . 00324 . 006Figure 1—figure supplement 2 . sFLT-1 antibody specifically binds to the unique motif of sFLT-1 . Results correspond to an ELISA in which wells are coated with: BLANK , 1% BSA; FLT/FC , a chimeric protein containing the extracellular region of FLT-1 and the human Fc fragment ( R&D Systems , Minneapolis , MN ) ; SF , the peptide utilized to immunize the rabbits to obtain sFLT-1 antibody ( the peptide corresponding to a fragment of the unique C-terminal tail of sFLT-1 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 00610 . 7554/eLife . 00324 . 007Figure 1—figure supplement 3 . sFLT-1 expression is significantly decreased in RPE from an AMD eye with CNV ( 88 years old , female ) compared with the age-matched control ( 89 years old , female ) . The right bottom bright field images shows autofluorescence from degenerated RPE ( green fluorescence blocks the dark pigment of RPE overlying CNV; arrows point to CNV ) . AMD: age-related macular degeneration; Br: Bruch's membrane; Ch: choroid; CNV: choroidal neovascularization; RPE: retinal pigment epithelium . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 00710 . 7554/eLife . 00324 . 008Figure 1—figure supplement 4 . H&E staining images show the histology of two human RAP eyes ( arrows point to the RAP lesion ) . Ch: choroid; RAP: retinal angiomatous proliferation; RPE: retinal pigment epithelium; Ph: Photoreceptor layer . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 008 We next assessed sFLT-1 expression in the retina . In situ hybridization and immunostaining confirmed the presence of sFLT-1 mRNA and protein , respectively , in photoreceptors and RPE , which indicated that both cell types can synthesize and express sFLT-1 ( Figure 1B , C ) . Compared with VEGF-A ( Figure 1C ) , the relative expression pattern of soluble FLT-1 to VEGF is higher in the photoreceptors than in the inner vascular layers of the retina . A theoretical implication is that VEGF is prominent in the inner vascularized retina , the layer which harbors blood vessels and neurons , while sFLT-1 is prominent in the outer avascular retina , consistent with our initial hypothesis . Furthermore , sFLT-1 was principally detected on the basal aspect of the RPE–BrM complex , facing the vascularized choroid ( Figure 1C , D ) . In contrast , in the RPE , VEGF-A localizes on both basal and apical surfaces , consistent with a prior report ( Blaauwgeers et al . , 1999 ) . This polarized distribution is reminiscent of sFLT-1 expression in the cornea , where the highest levels of sFLT-1 are found in the perilimbal region counterposing the vascularized conjunctiva ( Ambati et al . , 2007 ) . This is consistent with VEGF’s vasculotrophic role in the choroid and neurotrophic role in the photoreceptors ( Saint-Geniez et al . , 2008 ) . The different ratio and polarities of VEGF-A and sFLT-1 would likely strike a reasonable balance to maintain a healthy photoreceptor layer and choriocapillaris while preventing vascular invasion of the subretinal space . Given the localization and expression patterns of sFLT-1 , we sought to determine if suppression of subretinal sFLT-1 in mice would induce subretinal angiogenesis or CNV . First , we injected a neutralizing antibody against FLT-1 into the subretinal space in wild type mice . This resulted in increased free VEGF-A levels ( n = 10; p<0 . 005 ) and induced CNV ( n = 10 , p<0 . 00005; Figure 2A , B ) , suggesting that sequestration of VEGF-A by FLT-1 maintains subretinal avascularity . Next , we administered FLT-1 neutralizing antibody ( which occupies domains 1–4 of this receptor ) via subretinal injection in Flt-1 tk−/− mice , which are deficient in the tyrosine kinase domain of membrane-bound FLT-1 ( Hiratsuka et al . , 1998 ) . The results demonstrated that the induced CNV in Flt-1 tk−/− mice was accompanied with higher VEGF levels ( n = 10; p<0 . 005; Figure 2C , D ) , strongly suggesting that CNV occurs due to disruption of sFLT-1 , but not of mFLT-1 activity ( which is inactive in these mice ) . This accords with prior studies which have found that soluble but not membrane-bound FLT-1 plays a key role in anti-angiogenic regulation , based on the finding that germline Flt-1 deletion leads to embryonic lethality due to vascular endothelial hyperplasia , while membrane-bound Flt-1 lacking the tyrosine kinase domain is sufficient for normal vascular development in mice ( Fong et al . , 1995; Kappas et al . , 2008 ) . 10 . 7554/eLife . 00324 . 009Figure 2 . Suppression of sFLT-1 by neutralizing antibodies induces CNV while elevating levels of VEGF . VEGF-A levels were tested by ELISA of retinal pigment epithelium ( RPE ) /choroid tissue lysates harvested immediately after CNV images were taken in vivo . ( A and C ) Subretinal injections of FLT-1 antibody induced CNV and increased VEGF-A levels in both wild type and Flt-1 tk−/− mice . ( B and D ) Representative FA ( at a phase of 3 min after fluorescein injection ) and OCT images . Arrows point to CNV . ( E ) ELISA for PlGF showed no significant difference 3 days after subretinal injections of FLT-1 neutralizing antibody and isotype control . CNV: choroidal neovascularization; FA: fluorescein angiography; OCT: optical coherence tomography . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 00910 . 7554/eLife . 00324 . 010Figure 2—figure supplement 1 . Goat anti-mouse FLT-1 neutralizing antibody does not affect ELISA assessment of mouse VEGF-A and mouse PlGF-2 . Goat anti-mouse FLT-1 neutralizing antibody or control goat IgG was added to the standard at 1 ng/ml . ( A ) Mouse VEGF-A ELISA . ( B ) Mouse PlGF-2 ELISA . p Values were calculated by paired Student t test ( each average was paired ) . The presence of anti-FLT-1 antibody did not significantly affect ELISA readings of either cytokine . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 01010 . 7554/eLife . 00324 . 011Figure 2—figure supplement 2 . Excess recombinant FLT-1 inhibits VEGF-A and PlGF-2 detection . Recombinant FLT-1 was added to each concentration of VEGF-A ( A ) or PlGF-2 ( B ) and abolished detection of VEGF-A and PlGF-2 over background levels , indicating that VEGF-A or PlGF-2 that is bound to FLT-1 is not detected by this assay . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 01110 . 7554/eLife . 00324 . 012Figure 2—figure supplement 3 . FLT-1 neutralizing antibody incubation resulted in release of VEGF-A from recombinant FLT-1 in vitro . The design schema is shown on the left . A 2 mg/ml sample of mouse FLT-1-Fc protein ( 471-F1; R&D Systems , Minneapolis , MN ) was immobilized on an ELISA plate . After blocking with 1% BSA , the plate was incubated in 2 ng/ml mouse VEGF-A ( 493-MV-005; R&D ) or mouse PlGF-2 ( 465-PL-010; R&D ) at room temperature ( RT ) for 2 hr . Then , each well was incubated in ( A ) 2 ng/ml mouse VEGF-A , ( B ) 2 ng/ml mouse VEGF-A + 5 mg/ml isotype IgG , ( C ) 2 ng/ml mouse VEGF-A + 5 mg/ml anti-FLT-1 IgG ( AF471; R&D ) , ( D ) 2 ng/ml mouse PlGF-2 , ( E ) 2 ng/ml mouse PlGF-2 + 5 mg/ml isotype IgG , or ( F ) 2 ng/ml mouse PlGF-2 + 5 mg/ml anti-FLT-1 IgG at RT for 6 hr . Using biotinylated anti-VEGF-A ( BAF493; R&D ) or biotinylated anti-PlGF-2 ( BAF465; R&D ) and streptavidin-HRP ( DY998; R&D ) , VEGF-A or PlGF-2 was detected . The results ( row C vs row F ) show that FLT-1 neutralizing antibody desequestered VEGF-A but not PlGF-2 from FLT-1 . *p<0 . 0001 by two-tailed Student t test . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 012 In addition to VEGF-A , sFLT-1 also binds VEGF-B and placental growth factor ( PlGF ) ( Malik et al . , 2006; Fischer et al . , 2008 ) . To determine if PlGF is essential for CNV , we performed subretinal injections of anti-FLT-1 and isotype control antibody in wild type mice and found no significant difference in PlGF expression between both groups at day 3 ( n = 9; Figure 2E ) or at day 10 ( undetectable levels ) . Taken together with the above finding that FLT-1 antibody induced CNV in Flt-1 tk−/− mice ( in which VEGF-B signaling would be expected to be inoperative ) , this indicates that CNV induced by FLT-1 blockade is mediated by desequestration of VEGF-A , not of PlGF or VEGF-B . Moreover , neither PlGF nor VEGF-B are able to compensate for VEGF-A during its blockade , and mice lacking either factor display only minor developmental defects ( Malik et al . , 2006; Zhang et al . , 2009 ) . Furthermore , it is well established that , in contrast to VEGF-A , VEGF-B is neither induced by hypoxia nor essential to angiogenesis ( Hoeben et al . , 2004; Zhang et al . , 2009 ) . Although VEGF-B is dispensable for blood vessel growth , it is critical for blood vessel survival in pathological conditions ( Zhang et al . , 2009 ) . However , our findings do not exclude a role for VEGF-B in RAP or CNV . Knocking down sFLT-1 could increase VEGF-B activity and promote the longevity of CNV lesions after formation rather than promote angiogenesis . However , this pro-survival function is mediated through membrane-bound FLT-1 . Considering the high affinity sFLT-1 has for VEGF , it is crucial to verify that blocking antibodies can displace VEGF from bound sFLT-1 and release free VEGF . Moreover , anti-VEGF antibodies may affect the quantification of VEGF and PlGF , and another consideration is that the particular ELISA used may measure ‘non-free’ VEGF or PlGF ( bound to sFLT-1 ) as well . To clarify these questions , we performed the following series of experiments . First , we determined whether the neutralizing anti-FLT-l antibody affected the measurement of mouse VEGF-A and PlGF-2 , the only isoform of this protein present in mice by ELISA ( Figure 2—figure supplement 1 ) . We did not find any significant difference . Next , to determine whether our ELISA would detect VEGF that is bound to FLT-1 , we examined the effect of excess recombinant FLT-1 protein on the detection of VEGF-A and PlGF-2 by ELISA ( Figure 2—figure supplement 2 ) . In this assay , almost all VEGF-A ( 62 . 5 pg/ml ) would be expected to bind FLT-1 ( 100 ng/ml ) based on an assumed Kd = 10 pM ( free VEGF-A can be estimated to be 1 . 36 pg/ml by Michaelis–Menten kinetics ) . The ELISA showed less detection of VEGF-A and PlGF after saturation with excess recombinant FLT-1 , that is , it did not detect non-free VEGF-A and non-free PlGF-2 . These data demonstrate that non-free VEGF or non-free PlGF-2 is not being detected at significant levels by our ELISA technique . Finally , we determined whether anti-FLT-1 neutralizing antibody released VEGF-A but not PlGF-2 from recombinant FLT-1 ( Figure 2—figure supplement 3 ) . After coating ELISA plates with FLT-1 and then incubating with VEGF-A or PlGF-2 , Flt-1 neutralizing antibody was added . The results demonstrate that FLT-1 neutralizing antibody released VEGF-A but not PlGF-2 from recombinant FLT-1 . Although the exact reason for this is unknown , it is possible that FLT-1 neutralizing antibody may change the FLT-1 conformation , affecting binding of FLT-1 to VEGF-A but not PlGF-2 . Our second strategy was a post-transcriptional selective knockdown of sFLT-1 via subretinal injection of adeno-associated viral ( AAV ) delivery of a short hairpin RNA ( shRNA ) targeting sFLT-1 ( AAV . shRNA . sFlt-1 . Gfp ) . We have previously shown that this shRNA is selective to sFLT-1 and does not affect membrane-bound FLT-1 ( Ambati et al . , 2006 ) . We further confirmed this by western blot ( Figure 3—figure supplement 1 ) . As controls , shRNA ( AAV . shRNA . Gfp ) , viral vector expressing non-specific cargo ( AAV . Gfp ) , or phosphate buffered saline ( PBS ) was injected into the subretinal space . Subretinal delivery of AAV . shRNA . sFlt-1 . Gfp demonstrated efficient transduction of the RPE and the photoreceptor layer ( Figure 3—figure supplement 2 ) . We analyzed CNV and measured VEGF-A at 4 weeks after subretinal injection instead of 10 days as above , as AAV-mediated subretinal gene delivery usually takes a longer time before expression ( Shao et al . , 2012 ) . Compared to controls , treatment with AAV . shRNA . sFlt-1 . Gfp resulted in downregulation of sFLT-1 ( Figure 3A ) , increased free VEGF-A ( n = 10; p<0 . 05; Figure 3B ) , and induced CNV ( n = 10; p<0 . 05; Figure 3B , C , Figure 3—figure supplement 3 ) . Furthermore , as we reported recently ( Luo et al . , 2013 ) , AAV . shRNA . sFlt-1 induced primary CNV ( defined as a CNV that initially occurred at the injection site ) , as well as secondary CNV ( defined as a lesion that is more than ½ a disc diameter from the primary CNV lesion , and that shows no leakage on fluorescein angiography ( FA ) at least 2 weeks after injection ) 4 weeks after injection . This phenomenon was not found in the controls , although insignificant CNV developed initially due to injury . The length of time required for secondary CNV occurrence might derive from the time to and long duration of AAV-mediated gene expression in the mouse eye which has previously been demonstrated ( Schlichtenbrede et al . , 2003; Kjellstrom et al . , 2007; Shao et al . , 2012 ) . To further determine if CNV induced by sFLT-1 inhibition is attributable to VEGF-A , we repeated the above set of AAV-mediated sFLT-1 knockdown studies in Vegfalox/lox mice ( Gerber et al . , 1999 ) . The reduction of VEGF protein in RPE cells via subretinal delivery of a plasmid encoding Cre recombinase ( pCre ) in Vegfaloxp/loxp mice was demonstrated by immunohistochemistry ( IHC ) staining ( Figure 3—figure supplement 4 ) . Cre expression was verified by western blot ( Figure 3—figure supplement 5 ) . Increased VEGF-A expression in the RPE/choroid ( n = 10; p<0 . 0005 ) and CNV ( n = 10; p<0 . 05 ) in Vegfalox/lox mice treated with subretinal AAV . shRNA . sFlt-1 ( Figure 3D ) was suppressed by subretinal delivery of pCre . This confirms that the release of sequestered VEGF-A is requisite for CNV development following sFLT-1 antagonism . We observed that the sFLT-1 fluorescence signal became stronger after subretinal injection in treatment or control ( Figure 3A ) eyes compared to normal eyes without injury ( Figure 1C ) . The higher sFLT-1 expression might be due to the inflammatory reaction following injury . 10 . 7554/eLife . 00324 . 013Figure 3 . Subretinal adeno-associated viral delivery of short hairpin RNA targeting sFLT-1 induces VEGF-A and CNV . VEGF-A levels from retinal pigment epithelium ( RPE ) /choroid tissues were tested by ELISA from tissue lysates harvested immediately after choroidal neovascularization ( CNV ) images were taken in vivo . ( A ) Western blot ( top ) and immunohistochemistry ( IHC ) staining of sFLT-1 results confirmed that sFLT-1 was knocked down by AAV . shRNA . sFlt-1 . Gfp but not in controls at 4 weeks . ( B ) Subretinal AAV . shRNA . sFlt-1 . Gfp induced CNV and VEGF-A compared to controls in wild type mice ( 4 weeks ) . CNV was observed in controls due to disruption of Bruch’s membrane by subretinal injection . ( C ) Representative fluorescein angiography ( FA ) and H&E staining images ( 40× ) ( arrows point to leakage or neovessels ) show CNV is more extensive after AAV . shRNA . sFlt-1 . Gfp treatment compared to controls . ( D ) CNV was not induced by subretinal AAV . shRNA . sFlt-1 when VEGF-A release was suppressed ( 4 weeks ) by pCre in Vegfalox/lox mice compared with pNull control in which VEGF levels rose and CNV was induced . ( E ) IFN-γ levels increased in AAV . shRNA . sFlt-1 . Gfp and AAV . shRNA . Gfp ( 4 weeks ) compared with AAV . Gfp and phosphate buffered saline ( PBS ) in C57BL/6J mice . No significant difference was found among groups in the Tlr3−/− mice ( 4 weeks ) . ( F ) Subretinal AAV . shRNA . sFlt-1 . Gfp induced VEGF-A and CNV ( 4 weeks ) in Tlr3−/− mice . Representative FA images show the leakage in each group . VEGF-A levels were tested by ELISA using RPE/choroid complex samples . *p<0 . 05; **p<0 . 005; ***p<0 . 0005 . NS: no significant difference . Scale bar: 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 01310 . 7554/eLife . 00324 . 014Figure 3—figure supplement 1 . Western blot shows that AAV . shRNA . sFlt-1 . Gfp specifically affects sFLT-1 but not mFLT-1 . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 01410 . 7554/eLife . 00324 . 015Figure 3—figure supplement 2 . AAV . shRNA . sFlt-1 . Gfp ( A ) transfected more than 3 out of 4 quadrants and targeted the photoreceptors , RPE , and BrM by subretinal injection ( 2 weeks ) . ( B ) Sham control . The fundi are imaged by a Spectralis imager in vivo ( top ) . Retinal cross-sections ( bottom ) are imaged by confocal microscopy . Green is GFP , blue is DAPI . Scale bar: 10 µm . BrM: Bruch's membrane; GFP: green fluorescent protein; IN: inner nuclear layer; ON: outer nuclear layer; Ph: photoreceptors; RPE: retinal pigment epithelium . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 01510 . 7554/eLife . 00324 . 016Figure 3—figure supplement 3 . Representative images show different sizes of CNV in AAV . shRNA . sFlt-1 . Gfp ( A ) and controls ( B ) at 4 weeks after subretinal injection . FA and ICG images were taken in vivo . Representative immunohistochemistry staining on flat-mounts ( 4× ) and cryosections ( 20× ) and transmission electron microscopy images showed abundant neovessels ( arrows ) in AAV . shRNA . sFlt-1 . Gfp but not control injection . Scale bar: 10 μm . CNV: choroidal neovascularization; FA: fluorescein angiography; ICG: indocyanine green . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 01610 . 7554/eLife . 00324 . 017Figure 3—figure supplement 4 . IHC staining of VEGF indicates that the VEGF decreased in the area of injection ( arrows ) but not in the non-injected area ( arrow heads ) . Top: VEGF; middle: VEGF + bright field; bottom: isotype control of VEGF . Scale bar: 10 µm . IHC: immunohistochemistry; RPE: retinal pigment epithelium . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 01710 . 7554/eLife . 00324 . 018Figure 3—figure supplement 5 . Western blot confirms Cre expression in Vegfalox/lox mice 10 days after pCre subretinal delivery ( lane 1: pCre , lane 2: pNull ) . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 01810 . 7554/eLife . 00324 . 019Figure 3—figure supplement 6 . IL-12 levels increased in both AAV . shRNA . sFlt-1 . Gfp and AAV . shRNA . Gfp treated mice ( 4 weeks ) compared to AAV . Gfp and PBS control in the C57BL/6J mice . No significant difference was found between each group of Tlr3−/− mice ( 4 weeks ) . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 019 Treatment with siRNAs can affect several cytokines , such as interferon-gamma ( IFN-γ ) and interleukin 12 ( IL-12 ) , via activation of toll-like receptor 3 ( TLR3 ) , and suppress CNV as a general siRNA class effect ( Kleinman et al . , 2008 ) . We found that AAV . shRNA , targeting either sFlt-1 or a non-specific sequence , up-regulated IFN-γ ( n = 10; p<0 . 01 ) and IL-12 ( n = 10; p<0 . 001; Figure 3E and Figure 3—figure supplement 6 ) , both of which are induced by TLR3 . However , non-specific AAV . shRNA . Gfp failed to induce CNV in wild type mice ( n = 10; Figure 3B ) , suggesting that CNV formation in AAV . shRNA . sFlt-1 . Gfp treated mice is due not to non-specific immune activation but rather to sFLT-1 suppression . To confirm this , we repeated the described experiments in Tlr3-knockout mice ( Tlr3–/– mice ) . Subretinal injection of AAV . shRNA . sFlt-1 . Gfp ( n = 10 ) but not non-specific AAV . shRNA . Gfp ( n = 10 ) increased VEGF-A levels ( p<0 . 05 ) and induced CNV ( p<0 . 005 ) in Tlr3–/– mice ( Figure 3F ) . Levels of IFN-γ and IL-12 were not significantly different in either group ( n = 10; Figure 3E and Figure 3—figure supplement 6 ) , suggesting that CNV is due to sFLT-1 knockdown rather than non-specific immune activation . The third strategy was genomic deletion . We suppressed sFLT-1 by a Cre/lox-mediated conditional gene ablation because Flt-1 deletion is lethal ( Fong et al . , 1995 ) . Subretinal injection of pCre in Flt-1lox/lox mice reduced sFLT-1 , induced CNV ( n = 10; p<0 . 01 ) , and increased free VEGF-A in the RPE/choroid ( n = 8; p<0 . 01 ) ( Figure 4A ) . 10 . 7554/eLife . 00324 . 020Figure 4 . Suppression of soluble Flt-1 by conditional Cre/lox-mediated Flt-1 gene ablation induced CNV . ( A ) Western blot from the retinal pigment epithelium ( RPE ) /choroid tissue confirmed sFLT-1 knockdown by pCre . Subretinal injection of pCre induced CNV and increased VEGF-A levels in Flt-1lox/lox mice ( 2 weeks; arrows point to CNV ) . Representative FA images showed CNV lesions in both the pCre and pNull groups ( left bottom ) . ( B ) Deletion of tomato fluorescence showed that Cre expression ( mosaic pattern ) was restricted to RPE in transgenic mT/mg vmd2-cre+ mice ( at 4 weeks of age ) and was restricted to photoreceptors in transgenic mT/mG iCre-75+ mice ( at 4 weeks of age ) , respectively ( arrows point to the location of Cre expression ) . ( C ) Representative FA and OCT ( bottom ) images showed in vivo that a homozygous RPE-specific Flt-1 knockout ( Vmd2-cre+flt-1lox/lox ) mouse ( 48 days old ) developed CNV spontaneously without surgical injury , and a homozygous photoreceptor-specific Flt-1 knockout mouse ( iCre-75+flt-1lox/lox ) ( 30 days old ) developed retinal angiomatous proliferation ( RAP ) spontaneously without surgical injury . The littermate controls without loxp were normal . ( D ) Representative IHC images ( top panels ) showed the retinal neovessels in the above Vmd2-cre+flt-1lox/lox ( CNV ) mouse at 3 months of age . The littermate controls without loxp were normal ( red stained vessels ) . In situ hybridization ( middle panels , blue stained sFlt-1 ) and IHC images ( bottom panels , red stained sFLT-1 ) showed decreased expression of sFLT-1 RNA and protein in the RPE ( Vmd2-cre+flt-1lox/lox ) ( arrows ) . ( E ) Representative IHC images ( top panels ) showed the retinal neovessels in the above iCre-75+flt-1lox/lox mouse ( RAP ) at 3 months of age . The littermate controls without loxp were normal ( red stained vessels ) . In situ hybridization ( middle panels , blue stained sFlt-1 ) and IHC images ( bottom panels , red stained sFLT-1 ) showed decreased expression of sFLT-1 RNA and protein in the photoreceptors ( iCre-75+flt-1lox/lox ) ( arrows ) . Scale bar: 100 µm . CNV: choroidal neovascularization; FA: fluorescein angiography; IHC: immunohistochemistry; IN: inner nuclear layer; OCT: optical coherence tomography; ON: outer nuclear layer; OS: outer segment layer . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 02010 . 7554/eLife . 00324 . 021Figure 4—figure supplement 1 . Genotyping of RPE-specific Flt-1 knockout mice ( obtained by cross-breeding with Vmd2-cre+ mice and Flt-1lox/lox mice ) with PCR . Lanes 1–3: heterozygous ( Flt-1lox/+ ) , homozygous ( Flt-1lox/lox ) , and wild type ( Flt-1+/+ ) , respectively . Lanes 4 and 5: cre− and cre+ , respectively . Lanes 6 and 7: rtTA− and rtTA+ , respectively . RPE: retinal pigment epithelium . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 02110 . 7554/eLife . 00324 . 022Figure 4—figure supplement 2 . Representative RT-PCR shows Cre expression in a Vmd2-cre+ flt-1+/+ mouse ( lane 2 ) . No Cre expression was observed in wild type mice ( lane 1 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 02210 . 7554/eLife . 00324 . 023Figure 4—figure supplement 3 . CNV progression in homozygous RPE-specific Flt-1 knockout ( Vmd2-cre+flt-1lox/lox ) mice . Representative FA images ( at same time after fluorescence injection ) from a 38 day old mouse demonstrate that CNV lesions appear 8 days after initial imaging in previously non-fluorescent areas , indicating progression of CNV . CNV: choroidal neovascularization; FA: fluorescein angiography; RPE: retinal pigment epithelium . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 02310 . 7554/eLife . 00324 . 024Figure 4—figure supplement 4 . Representative toluidine blue staining images show CNV in a homozygous conditional Flt-1 knockout mouse ( Vmd2-cre+flt-1lox/lox , 3 months old ) compared to normal retinal morphology in a littermate control . CNV: choroidal neovascularization; IS: inner segment layer; ON: outer nuclear layer; OS: outer segment layer; RPE: retinal pigment epithelium . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 02410 . 7554/eLife . 00324 . 025Figure 4—figure supplement 5 . Representative TEM images show CNV in a homozygous conditional Flt-1 knockout mouse ( Vmd2-cre+flt-1lox/lox ) compared to normal retinal architecture in a littermate control . CC: choriocapillaris; CNV: choroidal neovascularization; TEM: transmission electron microscopy; RPE: retinal pigment epithelium . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 02510 . 7554/eLife . 00324 . 026Figure 4—figure supplement 6 . Representative western blot shows increased VEGF-A levels in the RPE/choroid of a Vmd2-cre+flt-1lox/+ mouse with CNV ( lane 2 ) compared to a littermate control ( Vmd2-cre+flt-1+/+ ) without CNV ( lane 1 ) . CNV: choroidal neovascularization; RPE; retinal pigment epithelium . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 02610 . 7554/eLife . 00324 . 027Figure 4—figure supplement 7 . Early vascular changes with normal retinal structures occur in homozygous conditional Flt-1 knockout mice ( Vmd2-cre+flt-1lox/lox ) . Top panels: Toluidine blue staining images show the morphology of the retina . There are no significant changes in the retina structure . There is a bump ( frame ) in the RPE layer in a homozygous conditional Flt-1 knockout mouse ( Vmd2-cre+flt-1lox/lox ) compared to littermate control . Middle and bottom panels ( magnified transmission electron microscopy images of the corresponding frames ) : There is an elongated profile ( asterisk ) within Bruch's membrane that is consistent with a tongue of endothelial cell cytoplasm by its electron density . There is a nucleus on the external side of the choriocapillary , and it is separated from the endothelial lining by a basement membrane . Bruch's membrane is intact . This is consistent with nascent choroidal endothelial changes preceding the development of frank CNV breaching Bruch's membrane . Br: Bruch's membrane; Ch: choroid; CNV: choroidal neovascularization; OS: outer segment layer; RBC: red blood cell; RPE: retinal pigment epithelium . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 02710 . 7554/eLife . 00324 . 028Figure 4—figure supplement 8 . FA images show that no CNV has developed in homozygous conditional Flt-1 knockout mice ( Vmd2-cre+flt-1lox/lox ) at p21 . CNV: choroidal neovascularization; FA: fluorescein angiography . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 02810 . 7554/eLife . 00324 . 029Figure 4—figure supplement 9 . Toluidine blue staining images show the morphology of the retina at p28 . Most areas of retina are normal in Vmd2-cre+flt-1lox/lox mice . In a CNV lesion , the overlying neuronal outer nuclear layer is mainly intact . Scale bar: 10 µm . CNV: choroidal neovascularization . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 029 To avoid the effects of mechanical disruption of the retina by subretinal injection ( as shown in PBS or sham controls above ) , we performed conditional ablation of Flt-1 in the RPE or photoreceptors to determine whether CNV or RAP could be induced without injury . We interbred transgenic Vmd2-cre+ mice ( Cre recombinase expressed specifically in RPE ) ( Le et al . , 2008 ) or iCre-75+ mice ( Cre recombinase expressed specifically in photoreceptors ) ( Le et al . , 2008 ) with floxed Flt-1 mice . Cre expression in the two Cre lines was identified by interbreeding with mT/mG mice , which express tomato/EGFP in all tissues ( Figure 4B ) . The results showed that Cre expression , as indicated by tomato deletion resulting in exclusive Egfp fluorescence , was restricted to the RPE in floxed mT/mG mice bred with Vmd2-cre+ and to the photoreceptors in floxed mT/mG mice crossed with the iCre-75+ line ( Figure 4B ) . At 1–3 months of age , nearly all homozygous RPE-specific Flt-1 knockout mice ( Vmd2-cre+flt-1lox/lox ) ( 18/22 eyes , 82%; p<0 . 001 ) , and about half of the heterozygous conditional Flt-1 knockout mice ( Vmd2-cre+flt-1lox/+ ) ( 17/42 eyes , 40%; p=0 . 009 ) developed CNV , which progressed over time , compared with 18% ( 2/22 eyes , 9% ) of littermate controls ( Vmd2-cre+flt-1+/+ ) . Similarly , at 1–3 months of age , both homozygous ( iCre-75+flt-1lox/lox , 8/14 eyes , 57%; p<0 . 05 ) and heterozygous ( 13/24 eyes , 54%; p<0 . 05 ) photoreceptor-specific Flt-1 knockout mice ( iCre-75+flt-1lox/+ ) developed RAP , compared with 10% ( 1/10 eyes ) of littermate controls ( iCre-75+flt-1+/+ ) . The number of lesions with leakages on FA ranged from one to four per eye in the floxed animals , whereas in littermate controls , there was no more than one neovascular lesion per eye . RAP lesions started from the inner retina ( arising from retinal vessels ) at an early stage and invaded the photoreceptor layer , eventually anastomosing with the choroidal neovasculature ( Figure 4C–E , Figure 4—figure supplements 1–5 and Tables 2 and 3 ) . 10 . 7554/eLife . 00324 . 030Table 2 . Transgenic mice with Cre/lox-mediated conditional gene ablation of Flt-1 in the retinal pigment epithelium ( RPE ) developed choroidal neovascularization ( CNV ) ( <3 months old ) DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 030homozygous ( Vmd2-cre+ flt-1lox/lox ) heterozygous ( Vmd2-cre+ flt-1lox/+ ) littermate control ( Vmd2-cre+ flt-1+/+ ) Litter 11/1 mice , 2/2 eyesNANALitter 22/2 mice , 3/4 eyes5/7 mice , 9/14 eyes0/1 mouse 0/2 eyesLitter 31/1 mouse , 2/2 eyes1/2 mice , 1/4 eyesNALitter 41/1 mouse , 2/2 eyes2/3 mice , 2/6 eyesNALitter 52/2 mice , 4/4 eyes1/2 mice , 1/4 eyes1/2 mice , 1/4 eyesLitter 63/3 mice , 4/6 eyes1/2 mice , 1/4 eyes1/5 mice , 1/10 eyesLitter 71/1 mouse , 1/2 eyes2/5 mice , 3/10 eyes0/3 mice , 1/6 eyesTotal11/11 mice ( 100% ) 12/21 mice ( 57% ) 2/11 mice ( 18% ) 18/22 eyes ( 82% , p= 1 . 3E-6 ) 17/42 ( 40% , p=0 . 009 ) 2/22 eyes ( 9% ) 10 . 7554/eLife . 00324 . 031Table 3 . Transgenic mice with Cre/lox-mediated conditional gene ablation of Flt-1 in the photoreceptors developed retinal angiomatous proliferation ( RAP ) ( <3 months old ) DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 031homozygous ( iCre-75+ flt-1lox/lox ) heterozygous ( iCre-75+ flt-1lox/+ ) littermate control ( iCre-75+ flt-1+/+ ) Litter 12/4 mice , 3/8 eyesNA0/1 mouse 0/2 eyesLitter 21/1 mice , 1/2 eyes3/4 mice , 6/8 eyesNALitter 31/1 mouse , 2/2 eyes2/4 mice , 4/8 eyes0/1 mouse , 0/2 eyesLitter 41/1 mice , 2/2 eyes1/2 mice , 1/4 eyes1/2 mice , 1/4 eyesLitter 5NA1/1 mouse , 2/2 eyesNALitter 6NA0/1 mouse , 0/2 eyes0/1 mouse , 0/2 eyesTotal6/7 mice ( 86% ) 7/12 mice ( 58% ) 1/5 mice ( 20% ) 8 /14 eyes ( 57% , p<0 . 05 ) 13/24 ( 54% , p<0 . 05 ) 1/10 eyes ( 10% ) We observed rare CNV occurrences in the littermate controls . These results may be accounted for by Cre ‘leakiness’ or toxicity , which has been reported in several transgenic models using the Cre-lox system ( Editorial , 2007; Schmidt-Supprian and Rajewsky , 2007 ) . We further confirmed that sFLT-1 was down-regulated by in situ hybridization and IHC , while VEGF-A was up-regulated by western blotting in the conditional Flt-1 knockout mice , compared with littermate controls ( Figure 4D , E , Figure 4—figure supplement 6 ) , suggesting CNV/RAP occurrence is due to sFLT-1 knockdown in the RPE or photoreceptors . We observed no changes in sFLT-1 expression in the inner retinae of the two conditional Flt-1 knockouts . However , sFLT-1 expression was somewhat decreased in the inner retina following subretinal injection with AAV . shRNA . sFlt-1 . This may be due to diffusion and leakage of the injection fluid along the injection track . Although there are several inherited retinal degeneration models with changes in RPE/photoreceptors where CNV does not develop , it is theoretically possible that pathological disturbances in RPE or photoreceptors due to Flt-1 knockout may themselves contribute to the development of CNV . Furthermore , CNV by its nature is anatomically disruptive to the RPE which in turn leads to photoreceptor disruption . To determine whether the CNV/RAP lesions we observed were due to preceding retinal changes , we performed IHC staining on sections of VMD-Crefltloxp/loxp mice at p7 , p14 , and p21 . No CNV lesions were seen on retinal sections at these timepoints . However , we found evidence of nascent choroidal endothelial changes abutting Bruch’s membrane at p21 ( Figure 4—figure supplement 8 ) . At p21 , the earliest feasible timepoint for angiography , we observed no CNV on FA ( Figure 4—figure supplement 9 ) . The earliest timepoint that we detected CNV angiographically was at p28 ( Figure 4—figure supplement 9 ) . Taken together , these data indicate early choroidal vascular changes without changes in the overlying neural retina . It is important to note that we cannot exclude sFLT-1 production originating from proteolytic cleavage . To the best of our knowledge , ADAM10 is the only described protease that cleaves membrane-bound FLT-1 to the soluble isoform ( Zhao et al . , 2010 ) . We found that ADAM10 is expressed in the inner retinal layers ( Figure 5 ) , but not in the RPE or photoreceptors where we found that knockdown of sFLT-1 breached photoreceptor avascular privilege . Hence , a proteolytic cleavage-mediated origin for outer retinal sFLT-1 is unlikely . 10 . 7554/eLife . 00324 . 032Figure 5 . Representative IHC staining images show the ADAM10 expression in the inner layer of the retina . IHC: immunohistochemistry; IN: inner nuclear layer; IP: inner plexiform layer; ON: outer nuclear layer; RPE: retinal pigment epithelium . DOI: http://dx . doi . org/10 . 7554/eLife . 00324 . 032
The neovascular processes we observed , either of CNV or RAP phenotypes in RPE/photoreceptor-specific Flt-1 knockout mice , recapitulate many of the characteristics of human CNV or RAP ( different subtypes of AMD ) ( Donati et al . , 2006 ) . In contrast to the transgenic mice which develop CNV in senility ( Ambati et al . , 2003a; Imamura et al . , 2006; Zhao et al . , 2011 ) or a model of RAP in the systemic VLDLR−/− mouse ( Heckenlively et al . , 2003 ) , the mouse models presented herein exhibit CNV or RAP in heterozygous transgenic mice as well and within a rapid timeframe , indicating the vital role of sFLT-1 in maintaining the integrity of photoreceptor avascular privilege . Moreover , previous studies found that transgenic mice which overexpress VEGF-A in either RPE or photoreceptors developed intrachoroidal neovascularization or retinal neovascularization but not CNV or RAP ( Okamoto et al . , 1997; Schwesinger et al . , 2001 ) . These phenomena suggest that overexpression of VEGF-A in the retina or RPE is not sufficient to overcome the subretinal vascular barrier to induce CNV . On the other hand , in our study , mice with reduced sFLT-1 expression in the RPE developed CNV , indicating that sFLT-1 down-regulation is sufficient to cause CNV . Hence , the ratio of soluble FLT-1 to VEGF is likely critical to the maintenance of the subretinal vascular barrier . We believe physiologic sFLT-1 is not disruptive to normal neural function due to both a critical balance and the polarity of sFLT-1 expression by RPE . Further , the models presented herein more closely mimic human AMD , rendering these transgenic models quite useful for further studies and translational applications . While diffusion of sFLT-1 between the RPE and photoreceptors is theoretically possible , the intervening interphotoreceptor matrix could trap sFLT-1 , preventing free diffusion . In addition , sFLT-1 secretion by the RPE is polarized basally towards the choroid; therefore , VEGF secretion from the RPE can elicit choroidal neovascularization . We observed less CNV in the injection site after AAV . shRNA . sFlt-1 delivery than after antibody or genomic knockdown; this may be due to persistence of preformed sFLT-1 , as the turnover of this protein in the RPE is , to our knowledge , unknown . Another interesting observation is that lower levels of both VEGF-A and CNV were observed in Vegfalox/lox mice compared with wild type mice in AAV-mediated sFlt-1 knockdown studies . It is possible that the insertion of lox sites flanking the VEGF-A gene can affect gene expression in conditions of injury . This could be due to several mechanisms including disruption of cis-regulatory elements , interruption of unknown miRNA binding sites , and effects on mRNA stability by the palindromic nature of loxp sites . Loxp insertion has previously been reported to affect the transcription of genes in cis in selected circumstances ( Sun and Storb , 2001; Meier et al . , 2010 ) . Nonetheless , taken together , our findings demonstrate that protein blockade , transcriptional silencing , or genomic ablation of sFLT-1 can induce CNV/RAP , showing that sFLT-1 , through sequestration of VEGF-A , is a key molecular defender of vision-critical photoreceptor avascular privilege . Our transgenic models would not be expected to directly affect Flt-1 expression in retinal or choroidal vessels . However , the knockdown of Flt-1 in photoreceptors in our iCre-75+flt-1lox/lox mice may contribute to focal areas of photoreceptor degeneration due to the depletion of Flt-1 compromising Wnt signaling . Reduced VEGF-A expression has been observed in CNV specimens surgically obtained from patients with persistent CNV not responsive to anti-VEGF-A therapy . This reduction may explain why some patients stop responding to anti-VEGF-A therapy after a period of time ( Tatar et al . , 2009 ) . Endothelial sFLT-1 expression is regulated by VEGF , acts as an autocrine regulator of endothelial cell function , and controls angiogenesis ( Ahmad et al . , 2011 ) . An important area of future investigation will be to determine , based on stage of CNV development , how anti-VEGF-A therapy can be complemented both with approaches to suppress underlying hypoxia and inflammation and with strategies to maintain or increase sFLT-1 expression , perhaps revitalizing normal vascular barriers . The milieu in the photoreceptor/RPE/Bruch’s membrane/choriocapillaris is complicated . Some endogenous angiogenesis inhibitors ( e . g . , pigment epithelium-derived factor ) in the RPE/Bruch’s membrane/choriocapillaris complex have been reported to be reduced in AMD and may contribute to the barrier against choroidal neovascular invasion ( Bhutto et al . , 2008 ) . Nevertheless , the findings in this study offer greater insight into the molecular mechanisms responsible for retinal vascular demarcations and CNV/RAP , and the described molecular advances have broader ramifications . They impact testing of anti-angiogenic pharmaceuticals , may illuminate the mechanistic basis of angiogenesis in other diseases affecting over a billion people ( Potente et al . , 2011 ) , hold translational import for a key cause of blindness , and support the ongoing clinical trial of AAV . sFlt-1 for the treatment of neovascular AMD ( clinical trial NCT01024998 at www . clinicaltrials . gov ) . Further , they highlight a potential use of sFLT-1 as a therapeutic target in situations where angiogenesis is desirable , as well as a new role for sFLT-1 , which has recently been found to protect renal podocytes ( Jin et al . , 2012 ) , and the genetic models of AMD we describe are the first which exhibit CNV or RAP spontaneously at an early age , rendering them original non-invasive platform technologies with a variety of applications .
C57BL/6J mice ( 6–8 weeks; The Jackson Laboratory , Bar Harbor , ME ) , Balb/cJ mice ( 6 weeks; The Jackson Laboratory ) , Vegfalox/lox mice ( Gerber et al . , 1999 ) ( 6–8 weeks , C57BL/6 background; gift of Genentech , San Francisco , CA ) , Tlr3−/− mice ( 6–8 weeks , B6;129S1-Tlr3tm1Flv/J; The Jackson Laboratory ) , Flt-1lox/lox mice ( Baldwin et al . , 2004 ) ( 6–8 weeks , gift of Genentech ) , Vmd2-cre mice ( Le et al . , 2008 ) ( 8–10 weeks , FVB/N mice background , heterozygotes do not have any RD1 mutation; Cre expression in RPE continues from embryonic day 9 to postnatal day 60; gift of Dr Yun Zheng Le , University of Oklahoma Health Sciences Center ) , iCre-75+ mice ( Li et al . , 2005 ) ( 14–20 weeks , C57BL/6 × SJL background; Cre expression in photoreceptors begins earlier than postnatal day 11; gift of Dr Ching-Kang Chen , University of Utah ) , mT/mG mice ( 14–20 weeks; STOCK Gt ( ROSA ) 26Sortm4 ( ACTB-tdTomato , -EGFP ) Luo/J; The Jackson Laboratory ) and Flt-1Tk−/− mice ( 14–20 weeks ) ( Hiratsuka et al . , 1998 ) were used . Experimental groups were age and sex matched . To identify Cre expression in the RPE layer of Vmd2-cre+ ( Le et al . , 2008 ) or photoreceptor layer of iCre-75+ mice ( Li et al . , 2005 ) , the mT/mG mice which express tomato/EGFP in all tissues were interbred with the above lines , respectively and the deletion of tomato fluorescence was checked at the age of 4 weeks . To generate RPE ( or photoreceptor ) specific Flt-1 knockout mice , Vmd2-cre+ ( or iCre-75+ ) mice and Flt-1lox/lox mice were cross-bred to obtain the homozygous ( Vmd2-creflt-1lox/lox or iCre-75+ flt-1lox/lox ) , heterozygous ( Vmd2-cre+ flt-1lox/+ or iCre-75+ flt-1lox/+ ) , and littermate controls ( Vmd2-cre+flt-1+/+ or iCre-75+flt-1+/+ ) . These mice were identified with genotyping as described ( Li et al . , 2005; Le et al . , 2008 ) . All mice were age and sex matched ( mice used for subretinal injection were 6–8 weeks old and male ) and fed with autoclaved 8656 Teklad Sterilizable Rodent diet provided by Harlan Laboratories ( Indianapolis , IN ) . Cages were routinely changed once every 2 weeks throughout the course of this study . Experiments were approved by the University of Utah Institutional Animal Care and Use Committee and conformed to the Association for Research in Vision and Ophthalmology Statement on Animal Research . Normal human fresh globes were obtained within 4 hr of death from the Utah Lions Eye Bank , and sections were prepared as described below . Cryosection slides ( 12 µm or 5 µm thickness ) of human ocular AMD and normal control tissue were obtained from the Ambati Laboratory at the University of Kentucky . AMD tissue and normal human tissue was matched for gender and age in each case . Specimens were taken from the eyes of AMD patients who had never received anti-VEGF antibody ( Lucentis or Avastin ) therapy . AAV . shRNA . sFlt-1 was developed as previously described ( Ambati et al . , 2006 ) . In brief , short hairpin ( sh ) RNA expression cassettes ( SECs ) were developed by in vitro PCR amplification . SECs are PCR products that consist of promoter and terminator sequences flanking a hairpin siRNA template . Multiple target sequences along with different combinations of promoters were screened to identify the most effective siRNA capable of gene knockdown in vitro and in vivo . We used pAAV-MCS plasmids ( AAV Helper-Free System; Agilent Technologies , Santa Clara , CA ) containing inverted terminal repeats ( Line et al . , 2005 ) . We obtained AcGfp cDNA from pIRES2-AcGfp1 ( Clontech Laboratories , Mountain View , CA ) , and generated pAAV-AcGfp to insert AcGfp cDNA into the multi-cloning site of pAAV-MCS . shFlt and shNegative cDNA with an H1 promoter were inserted into pAAV-AcGfp using a PstI site to obtain pAAV-AcGfp-shFlt and pAAV-AcGfp-shNegative . Serotype 2 AAV vector containing plasmids were produced by the Viral Vector Core at the University of Massachusetts Medical School . The target ( sFLT-1 ) sequence is aatgattgtaccacacaaagt . A target for shRNA in identical regions of human and mouse soluble FLT-1 unique intron 13 derived tail was selected , since the intron 13 derived tail of 31 amino acids is only present in soluble FLT-1 and not in membrane FLT-1 , so soluble FLT-1 was specifically abrogated . A 21 base DNA oligonucleotide with 30% locked nucleic acid ( LNA; Exiqon , Woburn , MA ) substitutions was used as an anti-sense probe against sFLT-1 mRNA . The sequence of the LNA probe was actttgtgtggtacaatcatt . The calculated RNA Tm value is 46°C . A 21-mer sFLT-1-sense-LNA probe with a sequence aatgattgtaccacacaaagtc ( predicted RNA Tm value of 46°C ) was used as control . Both LNA probes were digoxigenin ( Kaneko et al . , 2011 ) labeled at the 5′ and 3′ ends . Mice were placed under general anesthesia with an intraperitoneal injection of tribromoethanol ( 289 mg/kg ) . Topical 0 . 5% proparacaine solution and 1% tropicamide ophthalmic solution obtained from the Moran Eye Center pharmacy were applied to the cornea as an anesthetic and dilator , respectively . Using an Olympus stereomicroscope for magnification , a small incision was made behind the limbus with a 30 . 5 gauge needle . To deliver 1 μl of each treatment , a custom 33 gauge , blunt tipped microsyringe ( Hamilton , Reno , NV ) was inserted through the incision , vitreous , and retina into the subretinal space in the posterior pole of the retina , taking care to avoid the lens . Fundus examinations during the injection procedure showed partial retinal detachment , confirming successful subretinal delivery . To inhibit sFLT-1 by shRNA in C57BL/6J mice , 1 µl of AAV . shRNA . sFlt-1 . Gfp ( 1 . 4 × 1011 genomic copies/ml ) was injected subretinally , with AAV . shRNA . Gfp ( 1 . 4 × 1011 genomic copies/ml ) , AAV . Gfp ( 1 . 4 × 1011 genomic copies/ml ) or PBS injected as controls . For genomic deletion in Flt-1lox/lox mice , a plasmid encoding Cre recombinase ( pCre; 1 µg , gift of RK Nordeen , University of Colorado ) with 10% NeuroPORTER Transfection Reagent ( Genlantis , San Diego , CA ) as a plasmid transfection enhancer ( Kachi et al . , 2005 ) was injected into the subretinal space . Analogous empty plasmid ( pNull , 1 µg ) with 10% NeuroPORTER was injected as a control . For genomic deletion in Vegfalox/lox mice , 1 µl of 50% pCre , 10% NeuroPORTER , and 40% AAV . shRNA . sFlt-1 . Gfp was injected into the subretinal space . As controls , 1 µl of the following vectors were injected into the subretinal space of Vegfalox/lox mice: ( 1 ) 50% pNull , 10% NeuroPORTER , and 40% AAV . shRNA . sFlt-1 . Gfp; ( 2 ) 50% pCre , 10% NeuroPORTER , and 40% AAV . shRNA . Gfp; ( 3 ) 50% pCre , 10% NeuroPORTER , and 40% AAV . Gfp; or ( 4 ) 50% pCre , 10% NeuroPORTER , and 40% 1× PBS . A 1 µl ( 0 . 2 µg ) sample of goat FLT-1 antibody ( mouse myeloma cell line NSO derived recombinant mouse VEGFR1 , Ser27Glu759; R&D Systems , Minneapolis , MN ) or equivalent goat isotype IgG ( Jackson ImmunoResearch , West Grove , PA ) as control were subretinally injected into C57BL/6J , Flt-1tk−/− , or Tlr3−/− mice , respectively . Mice were anesthetized with tribromoethanol ( 289 mg/kg ) and the pupils were dilated with topical 1% tropicamide ophthalmic solution ( Bausch & Lomb , Rochester , NY ) . All in vivo imaging described was performed using a Spectralis confocal ophthalmoscope ( Heidelberg Engineering , Vista , CA ) . HRA-OCT imaging of mouse fundi was performed 2 , 4 , 6 , and 10 weeks after subretinal injections with AAV . shRNA . sFlt-1 , or at 21 days , 1 , 2 , and 3 months of age in the transgenic mice . Green fluorescent protein ( GFP ) expression was observed with a red free or autofluorescence filter . Mice were intraperitoneally injected with 0 . 1 ml of 10% sodium fluorescein ( Akorn , Lake Forest , IL ) or 0 . 2 ml of 8 mg/ml indocyanine green ( ICG , Akorn ) . CNV was evaluated by experienced retinal specialists using a combination of images obtained by FA , optical coherence tomography ( OCT ) , ICG , or a MICRON II small animal retinal imaging microscope ( Phoenix Research Laboratories , San Ramon , CA ) . Quantification of lesions in vivo was performed by using OCT images according to established procedures ( Schmucker and Schaeffel , 2004; Joeres et al . , 2007; Furino et al . , 2009; Ruggeri et al . , 2009; Giani et al . , 2011; Luo et al . , 2013 ) . This method has been widely used for estimating CNV volumes in vivo , especially in AMD patients , and has proved to be a highly reproducible quantitative measurement ( Joeres et al . , 2007; Giani et al . , 2011; Luo et al . , 2013 ) . B-scans were exported from Heidelberg Eye Explorer ( HEE ) , Heidelberg’s image analysis software , in . jpeg format and imported as complete stacks into Seg3D software . Seg3D is a segmentation processing and analyzing tool developed by the University of Utah Scientific Computing and Imaging Institute and the NIH/NCRR Center for Integrative Biomedical Computing ( Volumetric Image Segmentation and Visualization , Scientific Computing and Imaging Institute [Furino et al . , 2009] , available at http://www . seg3d . org ) . Boundaries of the CNV lesion and deformations of the RPE were manually outlined using a polyline tool by a masked and experienced technician ( Luo et al . , 2013; Yang et al . , 2013 ) . Seg3D masked the region enclosed within the polyline for each consecutive b-scan in pixels squared . An automated feature of Seg3D calculated two-dimensional lesion area , summing the masked b-scans for each stack . Scaling factors obtained from HEE were used to convert pixel2 to μm2 . After adjusting for murine corneal curvature to 1 . 4 mm ( Ahlers et al . , 2008 ) , the scaling factors for both x- and z-axes were 1 . 45 ± 2 and 3 . 87 μm/pixel , respectively . To calculate lesion volume ( μm3 ) , the lesional area on each scan line was multiplied by the distance between scans ( 52 ± 2 μm; provided by HEE software ) , and then summated . Finally , estimated volumes were converted to mm3 and reported . CNV was quantified by FA ( at 3–5 min after fluorescein injection ) and OCT 10 days after antibody injection and 4 weeks after injection of AAV . shRNA . sFlt-1 . The eyes were enucleated , fixed in 4% paraformaldehyde ( PFA ) ( Electron Microscopy Sciences , Hatfield , PA ) for 2 hr at 4°C , cryoprotected in 30% sucrose overnight , and embedded in Tissue-Tek O . C . T . Compound ( Sakura Finetek USA , Torrance , CA ) . Sections ( 12 µm ) were cut on a cryostat . Rabbit sFLT-1 primary antibody ( Orecchia et al . , 2003 ) ( 0 . 03 µg/µl , rabbit anti‐C‐terminal of sFLT-1 ) was provided by Drs Lacal and Orecchia , Instituto Dermopatico dell’Immacolata , Instituto di Ricovero e Cura a Carattere Scientifico ( IDI-IRCCS ) , Rome , Italy . The same concentrations of rabbit IgG ( Invitrogen , Carlsbad , CA ) were used as isotype controls . The Vector M . O . M . ( Mouse on Mouse ) immunodetection kit ( Vector Laboratories , Burlingame , CA ) was used to detect mouse antibody on mouse tissue according to the manufacturer’s instructions . To quench autofluorescence , 0 . 3% Sudan Black ( wt/vol ) ( Sigma-Aldrich , St . Louis , MO ) in 70% EtOH ( vol/vol ) was applied to slides; Dylight 405 ( 1:100; Jackson ImmunoResearch ) was used as a secondary antibody to avoid RPE autofluorescence without Sudan Black application . To determine sFLT-1 and/or VEGF expression in mice , sections were blocked in 10% goat serum ( Abcam , Cambridge , MA ) diluted in IHC buffer ( 1% BSA , 1% FBS , and 0 . 3% Triton X-100 in PBS pH 7 . 4 ) for 1 hr followed by overnight incubation with rabbit sFLT-1 primary antibody and/or mouse anti-VEGF monoclonal antibody ( 1:100; Abcam ) diluted in blocking solution . The Vector M . O . M . immunodetection kit was used to detect mouse antibody on mouse tissue according to the manufacturer’s instructions . After primary antibody incubation , slides were washed and incubated in secondary antibody Alexa Fluor 488 goat anti-rabbit ( 1:400; Invitrogen ) and/or Alexa Fluor 546 goat anti-mouse ( 1:400; Invitrogen ) for 1 hr at room temperature ( RT ) . After washing three times with PBS , VECTASHIELD Mounting Medium with DAPI ( Vector Laboratories ) was applied and slides were coverslipped . The same concentrations of rabbit IgG were used as isotype controls . To determine sFLT-1 and/or VEGF expression in human tissue , the same primary secondary antibodies were used as above . After secondary antibody application , 0 . 3% Sudan Black ( wt/vol ) was applied to slides for 10 min to quench autofluorescence . Slides were rinsed quickly with PBS eight times and mounted . Dylight 405 was used instead of the above secondary antibody to avoid the RPE autofluorescence without Sudan Black application . To demonstrate vessels in the human sections , rat anti-heparan sulfate proteoglycan ( perlecan ) monoclonal antibody ( 1:200 , clone A7L6; Millipore , Billerica , MA ) was used as primary antibody , and anti-rat IgG-FITC ( 1:1000 , F6258; Sigma-Aldrich ) was used as secondary antibody , followed by Sudan Black staining as describe above . VECTASHIELD Mounting Medium with DAPI was used and the slides were coverslipped . Hematoxylin and eosin ( H&E ) staining , toluidine blue staining , and transmission electron microscopy followed the protocols ( http://prometheus . med . utah . edu/~marclab/marclab_09_tools-protocols . html ) ( Luo et al . , 2013 ) . Samples were sectioned at 1 μm for H&E staining or toluidine blue staining . The following materials were used: 10% neutral buffered formalin ( EMD Chemicals USA , Gibbstown , NJ ) , hematoxylin solution ( Sigma-Aldrich ) , acid alcohol ( Thermo Scientific , Waltham , MA ) , Bluing Reagent ( 0 . 25 NH4 solution; Richard-Allan Scientific , Kalamazoo , MI ) , Eosin solution ( Sigma-Aldrich ) , and Cytoseal XYL ( Fisher Scientific , Pittsburgh , PA ) . Cryosections ( 12 µm ) of BALB/cJ mouse eye tissue were prepared as described above . Sections were fixed in 4% PFA for 10 min followed by 5 µg/ml proteinase-K treatment in DEPC treated water at RT for 10 min . Pre-hybridization was performed in hybridization buffer ( 50% formamide , 5× SSC , 5× Denhardt’s , 250 µg/ml yeast RNA , 500 µg/ml SSD , 2% Roche blocking reagents , and 0 . 2% Tween 20 ) at RT for 4 hr , followed by hybridization with 20 nM anti-sense sFLT-1 LNA probe . Hybridization was performed at 55°C . After hybridization , stringent washes were performed at 60°C with 5× SSC for 30 min , 2× SSC for 30 min , 1× SSC for 30 min , and 0 . 2× SSC for 1 hr . Procedures to avoid RNase contamination were used during the following steps . Slides were blocked in Tris buffer ( 0 . 1 M Tris pH 7 . 5/0 . 15 M NaCl ) containing 10% sheep serum for 1 hr and then incubated in alkaline phosphatase-conjugated anti-digoxigenin antibody ( 1:2000; Roche Applied Science , Indianapolis , IN ) overnight at 4°C . Enzymatic development was performed using 4-nitroblue tetrazolium ( NBT ) and 5-brom-4-chloro-30-indolylphosphate ( BCIP ) substrate ( Roche Applied Science ) , forming a dark blue NBT-formazan precipitate within 2–3 hr . Images were obtained with an Axiovert 200 inverted microscope ( Carl Zeiss Microscopy ) . In order to avoid the interruption from dark RPE , white mice lines were used . Mouse RPE and choroid were harvested , placed in 10% RIPA buffer ( Sigma-Aldrich ) and sonicated 3–5 times . Samples were spun at 14 , 000×g for 15 min and the supernatant , containing total protein extract , was transferred to a clean labeled tube . Tissue supernatant was used to perform all protein assays . Total protein was quantified using a Pierce BCA Protein Assay Kit ( ThermoFisher Scientific , Rockford , IL ) according to the manufacturer’s protocol . Mouse VEGF-A ( BMS619; eBioscience , San Diego , CA ) , mouse IL-12 ( KMC0121; Invitrogen ) , mouse IFN-γ ( KMC4021; Invitrogen ) , and mouse placental growth factor ( E0114m; Uscn Life , Wuhan , China ) ELISAs were performed according to the manufacturer’s instructions to quantify VEGF , IL-12 and IFN-γ , and PlGF , respectively . All assays were read using an ELx800 Absorbance Microplate Reader and Gene5 software ( BioTek , Winooski , VT ) . AAVs or plasmid subretinal injections are described above . Two weeks after the injection , we examined sFLT-1 protein reduction by AAV . shRNA . sFlt-1 using western blot from the RPE/choroid . After the mice were euthanized , the RPE/choroid complex was separated from the sclera under a stereomicroscope and placed in 120 μl of RIPA buffer ( Sigma-Aldrich ) containing protease inhibitor cocktail ( Roche Applied Science ) . Samples were homogenized with a sonic dismembrator ( ThermoFisher Scientific ) , and total protein concentration was determined using 10 µl of sample and a Pierce BCA assay kit ( ThermoFisher Scientific ) . The proteins were eluted with Laemmli buffer ( Bio-Rad Laboratories , Hercules , CA ) on ice for 5 min , boiled for 5 min , and centrifuged at 14 , 000×g for 5 min . Then 10 µg of protein were run through a 10% acrylamide gel under reducing conditions . After SDS-PAGE , proteins were transferred to a nitrocellulose membrane and incubated for 1 hr with blocking buffer ( 3% BSA and 0 . 05% Tween 20 in TBS for FLT-1 or 5% non-fat milk and 0 . 05% Tween 20 in TBS for other proteins ) then incubated with anti-FLT-1 antibody targeting D1–D5 of FLT-1 ( 1:1000 , ab9540; Abcam ) which recognize sFLT-1 and mFLT-1 , or Novagen anti-CRE antibody ( 1:5000 , 69050; EMD Millipore , Billerica , MA ) overnight at 4°C . The membrane was washed once in TBST ( 0 . 05% Tween 20/TBS ) and twice in TBS . Finally , the membrane was incubated with the appropriate secondary HRP-linked antibody in blocking buffer for 30 min at RT . After washing once with TBST and three times with TBS , the membrane was developed using an Amersham ECL Plus Western Blotting Detection Kit ( GE Healthcare Bio-Sciences , Piscataway , NJ ) . Chemiluminescence was detected by a FOTO/Analyst Electronic Imaging System ( FOTODYNE , Hartland , WI ) . As an internal control , a western blot detecting β-actin was performed as follows: the membrane was incubated in Pierce Restore Plus Western Blot Stripping Buffer ( ThermoFisher Scientific ) followed by blocking , primary antibody incubation , washing , HRP conjugated secondary antibody incubation , washing , and development as described above . Mouse monoclonal anti-β-actin ( 1:5000 , ab6276; Abcam ) was used as the primary antibody . Differences in CNV volumes and mean level of protein or mRNA were compared using the Student t test or Mann–Whitney U test with Bonferroni correction for multiple comparisons . Differences in CNV occurrence in transgenic mice were compared with the χ2 test . Statistical significant was set at p<0 . 05 . Data are presented as mean ± SEM .
Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid . Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization , two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration ( AMD ) , the leading cause of irreversible blindness in industrialized nations . Here we show that sFLT-1 , an endogenous inhibitor of vascular endothelial growth factor A ( VEGF-A ) , is synthesized by photoreceptors and retinal pigment epithelium ( RPE ) , and is decreased in human AMD . Suppression of sFLT-1 by antibodies , adeno-associated virus-mediated RNA interference , or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity . These findings help explain the vascular zoning of the retina , which is critical for vision , and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life .
The inner surface of the vertebrate eye is lined with a multilayered structure known as the retina . The bottom layer of the retina is composed of rods and cones—neurons that are directly sensitive to light—and is called the photoreceptor layer . Rods function primarily in dim light and provide black-and-white vision , while cones support daytime vision and are responsible for colour perception . Unlike the upper layers of the retina , the photoreceptor layer does not contain blood vessels: oxygen and nutrients are instead provided by a structure just underneath the retina called the choroid . The eye relies on the rods and cones converting light into electrical signals , and the photoreceptor layer must remain free of blood vessels for this process to work properly . If blood vessels extend into the photoreceptor layer from rest of the retina ( which is above it ) or the choroid ( below ) , they can disrupt the retina and give rise to a condition called age-related macular degeneration , which is a leading cause of irreversible blindness in adults . Within the eye , the development of new blood vessels from pre-existing vessels is stimulated by a protein known as vascular endothelial growth factor A ( VEGF-A ) , while an inhibitor protein called sFLT-1 prevents the growth of new blood vessels in the other tissues of the eye like the cornea . However , it has not been clear what keeps the photoreceptor layer ( and also the cells that support the photoreceptor layer ) free of blood vessels , and what happens to disrupt this process of vascular demarcation in age-related macular degeneration . Now , Luo et al . reveal that cells in the photoreceptor layer produce sFLT-1 , and that the levels of this protein are indeed reduced in people with age-related macular degeneration . Using genetic and pharmacological methods , they show that a reduction in sFLT-1 triggers blood vessels to grow into the photoreceptor layer from above or below . Luo et al . also report two new genetic mouse models in which blood vessels form spontaneously in the photoreceptor layer at an early age , which should prove useful for further research into age-related macular degeneration .