Insights from Genetic Model Systems of Retinal Degeneration: Role of Epsins in Retinal Angiogenesis and VEGFR2 Signaling
Yunzhou Dong1,*, Xue Cai2, Yong Wu3, Yanjun Liu3, Lin Deng4, and Hong Chen1,*
1 Vascular Biology Program, Boston Children＊s Hospital, Harvard Medical School, Boston, MA 02115, USA. 2 Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. 3 Department of Internal Medicine, Charles R. Drew University of Medicine & Sciences, University of California School of Medicine, Los Angeles, CA 90059, USA. 4 Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
The retina is a light sensitive tissue that contains specialized photoreceptor cells called rods and cones which process visual signals. These signals are relayed to the brain through interneurons and the fibers of the optic nerve. The retina is susceptible to a variety of degenerative diseases, including age-related macular degeneration (AMD), diabetic retinopathy (DR), retinitis pigmentosa (RP) and other inherited retinal degenerations. In order to reveal the mechanism underlying these diseases and to find methods for the pre-vention/treatment of retinal degeneration, animal models have been generated to mimic human eye diseases. In this paper, several well-characterized and commonly used animal models are reviewed. Of particular interest are the contributions of these models to our understanding of the mechanisms of retinal degeneration and thereby providing novel treatment options including gene therapy, stem cell therapy, nano-medicine, and CRISPR/Cas9 genome editing. Role of newly-identified adaptor protein epsins from our laboratory is discussed in retinal angiogenesis and VEGFR2 signaling.
Retinal Degeneration | CRISPR/Cas9 | Genome Editing Technology | Epsins | VEGFR2 Signaling
It is estimated that more than 15 million people in the world suffer from vision loss due to inherited retinal diseases. Although the wide variety of the causes (mutation of genes and variants leading to vast differences in phenotype), photoreceptor death is a common outcome. While inherited retinal degenerations are currently incurable, animal models provide significantly useful tools to investigate the mechanisms underlying the diseases and thereby produce potential treatments for vision loss.
Two strategies have been commonly used to study the mechanisms of human ocular diseases in animal models: phenotype-driven and genotype-driven. Phenotype-driven studies start with an animal model exhibiting a phenotype of interest and consequently identify the gene(s) responsible for the phenotype. Genotype-driven studies take advantage of genetic engineering approaches, such as the creation of transgenics, knockouts and knockins, to mutate genes known to cause human disease and thereafter examine the associated phenotype . Animal models for retinal degeneration can be categorized into two types: naturally occurring and genetically modified. The former category includes the rodless mouse model (termed rd1) with a defect subsequently identified in the Pde6b (rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta) gene ; rd2 with a defect in retinal degeneration slow (Rds-/-) , rd3 , rd4 , rd5 with a defect in tubby gene (Tub-/-) , rd6 , rd7 (Nr2e3-/-, nuclear receptor subfamily 2, group E, member 3) . Examples of genetically engineered models for inherited retinal degenerations include transgenic mice expressing mutant human rhodopsin (P23H) , transgenic mice with the P23H, V20G and P27L mutations (VPP) in the rhodopsin gene , knockout mice that lack the neural retina leucine zipper gene (Nrl-/-) , the ATP-binding cassette transporter (Abcr-/-) , and the cone-rod homeobox gene (Crx-/-) . The remainder of this review will examine these disease models in more depth.
1. Retinitis pigmentosa (RP)
RP is a heterogeneous group of diseases characterized by loss of night vision followed by progressive loss of peripheral vision (most severely affecting rods). Cones degeneration and loss of central vision usually occurs after the death of most rods . The disease symptoms, electroretinographic (ERG) evaluation and genetic assessment in animal models have been reviewed [15-17]. The three types of RP are categorized by inheritance: autosomal recessive, autosomal dominant, and recessive/ dominant.
1.1. Animal models of recessive RP
1.1.1. Pde6b: PDE6b is a subunit of the rod-specific phosphodiesterase (PDE). PDEs selectively catalyze the hydrolysis of 3' cyclic phosphate bonds in guanine 3', 5' cyclic monophosphate (cGMP). Multiple transcript variants encoding different isoforms have been found for this gene. Mutations in the PDE6b gene result in human RP and are inherited in an autosomal recessive manner. A naturally occurring mutant mouse model (later determined to have a PDE6b mutation) was first described by Keeler in 1924 . The mutant allele carries a nonsense mutation in the Pde6b gene caused by a viral insertion in intron 1 or nonsense mutation in exon 7 C->A transversion in codon 347 (the gene symbol for the mutant is Pde6brd1, formerly referred to as rd1 or rd) [18-20]. Degeneration of the outer retina in this model starts at postnatal day (P) 8 to 10 and processes rapidly with complete loss of rods by P36 . But ¾ cones remains at P17 (whereas only 2% of rods left), and still 1.5% of cones present at 18 months of age . Photoreceptor apoptosis involves co-activation of apoptotic pathways from the mitochondrion and endoplasmic reticulum (ER) and apoptosis-inducing factor (AIF), and caspase 12 and increasing Ca2+ concentration also contribute to this process [22, 23].
1.1.2. Rpe65: RPE65, retinal pigment epithelium-specific 65 kDa protein, is an important component of the visual cycle responsible for regeneration of visual pigment (11-cis-retinal). It is an isomerohydrolase which converts all-trans-retinal to 11-cis-retinol [24-26]. RPE65 has two forms: a soluble form (sRPE65) and a palmitoylated, membrane-bound form (mRPE65). mRPE65 and sRPE65 have different binding specificity and serve as regulatory proteins to control retinoid recycling . Mutation in RPE65 gene causes Leber＊s congenital amaurosis (LCA), a severe form of autosomal recessive RP (ARRP) in humans Rpe65-/- knockout mice were created by replacing the 5＊ flanking region (1.1 kb) containing exons 1, 2 and 3, intervening introns and 0.5 kb of intron c with the PGK (phosphoglycerate kinase I gene promoter)-neo gene cassette . The knockout mice develop a slow retinal degeneration. Retinal anatomy is normal at 7 weeks of age, but some small lipid-like droplets appear in the RPE cells. By 15 weeks of age, rod outer segment (OS) length is shortened . Photoreceptor degeneration is age-dependent  and peaks at 18-24 months . This mouse has nearly undetectable rod ERG responses at a very early age while the cone function remains up to 4 months of age . Rpe65-/- retinas have no 11-cis-retinal and therefore unable to synthesis and recycle rhodopsin; they also lack 11-cis-retinyl esters, and accumulate all-trans-retinyl esters in the RPE cells . A naturally occurring mouse model containing a nonsense mutation in exon of the RPE65 gene (Rpe65rd12) was also identified. The rd12 mutant allele has a C -> T transition, creating a premature stop codon . The retinal phenotype of rd12 mice is similar to Rpe65-/- mice but exhibits small white dots spread throughout the fundus at 5-9 months of age . By 7 months, the OS becomes shorter and the nuclear number of photoreceptors (in the outer nuclear layer-ONL) is reduced to about 30% of wild type . The naturally occurring Swedish Briard dog model contains a 4-nucleotide (AAGA) deletion in the Rpe65 gene at position 487-490 bp resulting in a frameshift and premature termination in exon 5 . The mutant dog exhibits slow progressive retinal degeneration and abnormal ERG (no detectable rod responses and very low cone responses) at 5 weeks of age. At older ages (2.5 years old), large lipid-like inclusions become apparent in the RPE cells, the OSs are disorganized and the number of photoreceptors declines from the periphery to the central retina, although the fundus is normal . Recently, an interesting publication indicated that suppressing thyroid hormone signaling reduces cone death in this mouse .
1.1.3. Nr2e3: Nr2e3 is an orphan nuclear receptor that is a ligand-dependent transcription factor and has dual regulatory function: it activates rod development and suppresses cone development. NR2E3-associated disease in humans is characterized by S cone hyperfunction (enhanced S-cone syndrome), thickened retinal layers, but an otherwise normally structured retina. Lamination is coarse, however, and retinas exhibit a strikingly thick and bulging appearance, localized to an annulus encircling the central fovea . A naturally occurring mutant mouse model, rd7, was determined to have a 380 bp deletion in exons 4 and 5 of the Nr2e3 gene resulting in a frameshift and a premature stop codon. The fundus of these mice exhibits discrete white spots over the entire retina . At early ages, the retinas of rd7 mice contain whorls in OS structures and rosettes in the ONL. The retina has an excess S-cone with slow rod degeneration although normal rod function is detected in young mice. Eventually the mutation causes blindness .
1.1.4. tubby: TUB belongs to the Tubby family of bipartite transcription factors. A splicing mutation in the tub gene caused a G->T transversion at the 3＊ end of exon 11, which results in the replacement of a 44 amino acids at the C-terminal end with an intron-encoded 24 amino acids . This mouse exhibits retinal and cochlear degeneration, maturity onset obesity, insulin resistance and reduced fertilities . It previously was a phenotypic model for human Usher syndrome, but a human ocular disease results from mutation of the TUB gene exhibits the same phenotype seen in the mouse . It has been shown that rapid photoreceptor degeneration starts at P14  and peaks at P19, and at P28, almost half of the photoreceptors are gone . TUB protein is involved in protein trafficking , mutation of TUB protein results in mislocalization of opsins in the retina [39-41]. Cai et al reported that ER stress was correlated with retinal degeneration during tubby retinal development, and the UPR (unfolded protein response) was dysregulated . Moreover, prolonged ER stress condition caused heat shock protein family 70 deficiency, together with the impairment of transportation of the structural proteins to the proper site for building up photoreceptors, caused the failure of the OS formation .
1.2. Animal models of dominant RP
1.2.1. Rds: Retinal degeneration slow (RDS, peripherin/rds or peripherin 2) is a tetraspanin glycoprotein required for the morphogenesis and structural stability of photoreceptors. It is also necessary for proper OS orientation and disc renewal. Mutations in this gene cause dominant RP and dominant macular degeneration (MD) in humans. The naturally occurring rds mutant mouse contains an insertion of 9.5 kb of foreign DNA into exon 2 of the Rds gene and results in large transcripts but no functional RDS protein . The phenotype of rds+/- is a typical autosomal dominant RP (ADRP) while the phenotype of rds-/- is a much severe form of RP. The rds-/- homozygous mouse completely lacks OS, has no detectable ERG, accompanying by slow photoreceptor degeneration . The heterozygous mouse exhibits a haploinsufficiency RP phenotype characterized by progressive, early onset rod degeneration followed by late onset cone degeneration [44, 45]. Photoreceptors start dying at P14, and the complete loss of the cells takes almost a year. There are several other mouse models with mutation in Rds. The P216L mutation causes ADRP in humans, and transgenic mice carrying P216L-RDS exhibit faster photoreceptor degeneration than non-transgenic littermates and its phenotype is similar to that seen in patients . Transgenic mice carrying an ADRP-causing mutation (C214S) and one copy of wild type RDS exhibit a phenotype similar to the rds+/- mice . Nystuen et al reported that a novel Rds gene mutation, nmf193 mutation, was caused by a single base change that causes aberrant splicing between exons 1 and 2, and exhibited OS defects and progressive retinal degeneration. Its histological features of photoreceptor degeneration were similar to that of Nr2e3 (rd7/rd7) .
1.2.2. Nrl: Nrl (neural retinal leucine zipper), a basic motif-leucine zipper transcription factor, is a critical intrinsic regulator of photoreceptor development and function. Mutations in this gene have been identified associated with dominant RP [49, 50]. The lack of NRL in the genetically engineered knockout mice leads to a complete absence of rods , and conversion of rods to S-cones [11, 51]. The retina exhibits progressive degeneration in adulthood. Cone function is supernormal at 1 month of age and decreases by more than two-thirds between 2 to 7 months of age . The adult retina contains rosettes throughout the entire ONL .
1.2.3. Crx: Crx (cone-rod homeobox) is a photoreceptor-specific transcription factor which plays a critical role in the differentiation of photoreceptor cells. This homeodomain protein is necessary for the differentiation and maintenance of normal cones and rods. Mutations in human Crx gene are associated with RP and LCA . Crx knockout mice were created by the replacement of exon 3 and part of exon 4 with the PGK-neo cassette . At all ages, Crx-/- retinas lack OSs thus abrogates the phototransduction pathway in photoreceptors. Degeneration of photoreceptor cells starts at P21 and is almost complete at 6 months. The expression of photoreceptor specific genes is also reduced . Transmission and scanning electron microscopy observation of the photoreceptor development demonstrated that numerous small vesicles were scattered in the ※future OS§ area. Structural analysis indicates that OS morphogenesis was blocked at the elongation stage, likely due to decreased expression of required genes .
1.3. Animal models of recessive/dominant RP
1.3.1. Rho: Rhodopsin, a G-protein coupled receptor, is the protein required for the formation of functional retinal pigment (rhodopsin + 11-cis-retinal) in rods. Rhodopsin-mediated signaling starts the phototransduction cascade and is required for rod-based vision and the OS formation. Mutations in this gene cause ADRP and ARRP in humans. One of the most common dominant rhodopsin-disease causing mutations is P23H. Another most commonly used mouse (the VPP transgenic model) was created by introducing three amino acid substitutions (G->V, H->P & L->P) at the N-terminus of mouse opsin gene . Rho knockout mouse develops a normal complement of rods but without OS and lost photoreceptor cells within 3 months. The visual function was mediated by cones only at the age of 8 weeks . ERG assessment of VPP mice demonstrated that the rod amplitude declined by 1 month and gradually decreased to 30% of normal ERG . Machida et al  created P23H transgenic rat models carrying mutant P23H mouse opsin which exhibited different degeneration rate and progressive shortening rod OS and photoreceptor cell loss with age. S334ter, another rhodopsin mutation associated with RP in patients is caused by a premature termination codon at residue 334. Transgenic mice, expressing S334ter truncated mutant protein lacking the last 15 amino acids of the C-terminal, exhibit mislocalization of the truncated opsin to inner segment and ONL besides the OS, and progressive retinal degeneration [57, 58]. Recently, ER stress induced by aggregation and mislocalization of mutant protein is suggested to be involved in photoreceptor degeneration in P23H and S334ter mice [59, 60].
Taken together, RP, a genetically heterogeneous group of disorders, is classically characterized by 1) impaired rod function/night vision defects, 2) progressive retinal degeneration beginning in the midperiphery, and 3) accumulation of retinal deposits. RP usually spares the central retina in the early stages, but eventually most RP patients lose both rod and cone function.
2. Age related macular degeneration (AMD)
AMD is another heterogeneous set of diseases characterized by the presence of soft and hard drusen, hyperpigmentation and hypopigmentation of the RPE, RPE atrophy, geographic atrophy, choroidal neovascularization (CNV) and later fibrous scarring. AMD is the major cause of blindness among the elderly in the developed country (review ). AMD is usually classified as two forms: ※dry§ AMD which involves the accumulation of debris and deposits in the outer retina, and ※wet§ AMD which involves angiogenesis and neovascular changes (see 3. Retinal neovascularization). Because of the complicated etiology which involves genetic, environmental, and unknown factors, there is no animal models that truly mimic AMD phenotype, although several animal models have been generated that shed light on various aspects of macular degenerative processes. Models with features of dry AMD include Abcr-/- and ELOVL4-/- (elongation of very long-chain fatty acid-like 4). Both Abcr-/ and ELOVL4-/- cause Stargardt macular dystrophy (STGD) and share some pathological features with AMD, but ELOVL4-/- occurs at younger age than Abcr-/. The common biomarker for both AMD and STGD is accumulation of high levels of lysosomal bodies (lipofuscin) in the RPE cells, which precedes RPE atrophy and degeneration of the photoreceptors in the macula (review ). Models with features of wet AMD can be generated by either surgical or genetic (such as endothelial growth factor (VEGF) overexpression) and exhibit CNV, and ultimately functional impairment occurs.
2.1. Abcr: ABCR, ATP-binding cassette transporter sub-family A member 4 (ABCA4), is a multi-spanning membrane-bound protein exclusively localized in the rim region of OS discs. ABCR is hypothesized to function as an outward-directed flippase for removing all-trans-RAL and its derivatives from the OS disc lumen to the cytoplasmic side [12, 62]. Mutations in the Abcr gene cause a wide spectrum of retinal degenerative diseases including AMD and recessive RP in humans. The Abcr-/- mouse was created by replacing 4.0 kb of the promoter region plus exon 1 by a PGK-neo cassette . These mice exhibit normal photoreceptor structure with very slow degeneration . Abcr-/- have delayed rod recovery and abnormal clearance of all-trans-retinal from the OS, which results in the accumulation of N-retinylidene phosphatidylethanolamine (N-ret-PE) and all-trans-RAL in the OSs . It is hypothesized that accumulation of these abnormal retinoid compounds leads to the eventual formation of high levels of small, electron dense lipofuscin granules (at 44 weeks of age), lipofusion fluorophores, A2E and A2PE-H2, in RPE cells .
2.2. ELOVL4: ELOVL4, a membrane-bound protein residing on the ER of the retina, participates in the biosynthesis of very long-chain fatty acids and docosahexaenoic acid (DHA) . Mutations in this gene are associated with Stargardt-like macular dystrophy (STGD3) and autosomal dominant Stargardt-like macular dystrophy (ADMD), also referred to as autosomal dominant atrophic macular degeneration in humans. The transgenic mice were created by introducing 5-bp deletion of nucleotides (deletion of AACTT at 790每794 bp) of the human wild type ELOVL4 gene . In this mouse model, high A2E levels (compared to wild type) in the RPE were detected at 2 months of age even in lines that express very low amounts of transgenic protein and lipofuscin accumulates in the RPE at 7 months of age . In lines that express higher levels of the transgene, photoreceptor cells fail to form normal OS structures. The loss of 50% of photoreceptors occurs at 6 weeks, 16 weeks and 18 months in higher, middle, and lower ELOVL4-expressing lines, respectively . Decline in retinal function correlates with the expression level of mutant ELOVL4 . ELOVL 4 belongs to the ELO (elongation of long chain fatty acid) family and possesses biochemical features and therefore participation in reduction reactions during fatty acid elongation . Further studies demonstrated that ELOVL4 is required for the synthesis of C28 and C30 saturated fatty acids (VLC-FA) and of C28-C38 very long chain polyunsaturated fatty acids (VLC-PUFA). Mutation of this gene causes the failure of protein target to the ER, the site of fatty acid biosynthesis .
2.3. Cathepsin D: Cathepsin D is a lysosomal aspartyl protease which is a member of the peptidase C1 family. Mutations in this gene cause early childhood blindness and progressive psychomotor disabilities . Transgenic mice expressing a mutant form of cathepsin D (mcd) in RPE cells manifest many features of ※dry§ AMD [69, 70], including increased levels of autofluorescent debris and increased basal laminar deposits (BLD, is the accumulation of granular material with wide-spaced collagen located between the plasma membrane and the basal lamina of the RPE). Mcd mice exhibit photoreceptor degeneration, OS shortening, and accumulation of immunoreactive photoreceptor breakdown products in RPE cells. Accelerated photoreceptor cell death was detected in 12-month-old mcd/mcd mice .
2.4. ApoE: Apolipoprotein E deficiency produced hypercho-lesterol and also developed thickened lipid-rich Bruch＊s membrane and increased levels of autofluorescent debris . BLD appears in the ApoE-/- mice at 2 months of age . Besides developing a multiple pathological characters associated with human AMD, ApoE4 deficient mice also develop marked CNV .
2.5. XLJR: X-linked juvenile retinoschisis is an early-onset and a common form of macular degeneration in male. It is caused by a mutation in the retinoschisin (RS1) gene . The retinoschisin-null mice, Rsh1 (homolog of human RS1 gene) deficient mice, exhibit normal scotopic a-wave but scotopic b-wave and photopic b-wave are significantly reduced. The disease is a panretinal disorder which effects the organization of the retinal cell layers and the retinal synapse structure .
2.6. Rds: One form of Rds mutation (R172W) also causes AMD. R172W transgenic mouse model exhibits significantly diminished structure and function of both cones and rods in wild type background, indicating a dominant-negative, cone每rod defect. Whereas in the rds+/- background, the mice maintained the normal cone function at early ages but cone responses were diminished to 41% of the wild type level. Conversely a significant rescue of rod function and improvement of rod OS structure were shown signifying a preferential damaging effect of the mutation on cones. Although rds-/- mice have no detectable rod or cone responses, R172W animals in rds-/- background retained 30% of wild type structure and rod function, but no significant rescue of cone function was detected at 1 month of age .
2.7. Ccl2/Ccr2: CCL2 (chemokine (C-C motif) ligand 2), also known as monocyte chemotactic protein-1 (MCP-1), is a small cytokine belonging to the CC chemokine family. CCL2 recruits monocytes, memory T cells, and dendritic cells to sites of tissue injury and infection. CCL2 and the cell surface receptor that binds CCL2, CCR2, are associated with macrophage mobilization. It has been reported that single nucleotide polymorphisms in Ccl2/Cx3Cr1 are associated with AMD , but no mutations have been identified in human eyes. Ccl2 and Ccr2 knockout mice were generated by targeted gene disruption and exhibit defects in monocyte recruitment to sites of inflammation [77, 78]. This model exhibits features of both ※dry§ and ※wet§ AMD, including drusen, lipofuscin (start at 9 months of age), Bruch＊s membrane thickening and disruption, RPE degeneration, ONL loss, as well as CNV and blood vessel leakage .
In summary, AMD is the leading cause of vision loss and blindness among adults over age 65 years old. Macular degeneration is diagnosed as either dry (non-neovascular) or wet (neovascular) form. AMD begins with characteristic yellow deposits (drusen) in the macula. People with drusen could develop advanced AMD.
3. Retinal neovascularization
Retinal neovascularization (RNV) is characterized with a disturbed vascular bed, formation of capillary microaneurysms, increased vascular permeability, and proliferation of the new vessels and fibrous tissues [80, 81]. Neovascularization (NV) is associated with a variety of conditions including vascular occlusion, sickle cell diseases, and sarcoidosis. The animal models used to study NV include models of DR, retinopathy of prematurity (ROP) and exudative (wet) AMD, in which growth of new capillaries from preexisting blood vessels in the eye causes CNV or RNV. Studies suggest that angiogenic and angiostatic factors such as VEGF  and Insulin-like growth factor (IGF)  play pivotal roles in the pathogenesis of ocular neovascularization. Animal models for NV include vldlr-/- (the very low density lipoprotein receptor knockout), laser photocoagulation-induced CNV, oxygen induced retinopathy (OIR) (also termed as ROP) . Here we only focus on the animal models caused by gene defects.
3.1. Ins2 (Akita mice): Insulin is a hormone that is central to regulating the energy and glucose metabolism in the body. Mutations in the insulin gene lead to diabetic retinopathy in human beings. A naturally occurring diabetic mouse model for type I diabetes (Ins2Akita) has a point mutation (a single amino acid substitution) in the Insulin 2 gene causing misfolding of the insulin protein and leading to rapid onset of hyperglycemia and hypoinsulinemia in heterozygous mice by ~4 weeks of age . Increased retinal vascular permeability occurred at ~16 weeks, an increase in cellular capillaries, decrease in the thickness of the whole retina and the inner and outer nuclear layers were also observed at 3 months of age [86-88]. In addition, severe impairment of synaptic connectivity in the outer plexiform layer (OPL) and significant loss of amacrine and ganglion cells were reported in this mouse model [87, 88].
3.2. Vldlr: VLDLR is a lipoprotein receptor which plays an important role in VLDL-triglyceride metabolism and the reelin signaling pathway. Mutations in this gene cause VLDLR-associated cerebellar hypoplasia. Genetic association analysis has linked VLDLR to AMD . The VLDLR knockout mouse (vldlr-/-) is characterized with progressive NV [90, 91], in which new blood vessels originate from the inner retina (the OPL) and extend through the ONL to the subretinal space and choroid, and caused RPE disruption, exposure of Bruch＊s membrane, photoreceptor death, and significant fibrosis . This model represents a distinct form of AMD, called RAP (retinal angiomatous proliferation). At two months of age, the vldlr-/- mice exhibit reduced mRNA levels for genes required for the phototransduction cascade (rhodopsin and cone-opsin) , but retinal degeneration and ERG decline occurred very late (4 months to 1 year) . Recent studies suggest that inflammation, Wnt and the ASK1-P38/JNK-NF-百B signaling pathway play a pathogenic role in vldlr -/- mouse [91, 94, 95].
3.3. VEGF: Overexpression of VEGF in the eye often stimulates NV, which originates from the vascular bed closest to the ganglion cell layer. Genetic association analysis suggests excessive VEGF level is linked to AMD . Several transgenic mouse models were generated by introducing different VEGF isoforms such as human VEGF165 [96, 97] or murine VEGF  to the animals. Almost all the animal models display extensive and rapid NV which results in severe retinal damage .
3.4. IGF-1: IGF-1 is also known as somatomedin C or mechano growth factor. IGF-1 is a hormone similar in molecular structure to insulin. It plays an important role in childhood growth and continues to have anabolic effects in adults. Transgenic mice overexpressing IGF-1 in the retina develop vascular alterations such as pericyte loss, thickened capillary basement membrane, intraretinal microvascular abnormalities, as well as proliferative retinopathy and retinal detachment .
In summary, NV is a significant complication of several ocular disorders. Common to all the proliferative retinopathies is the initial development of retinal vascular closure. Diabetic retinopathy induces most NV events. Animal models open new avenues to investigate the underlying pathogenesis of NV and methods of controlling this important ocular complication.
Perspectives of therapeutic treatment of retinal degenerative diseases
Animal models have been widely used in preclinical drug screening for human diseases, including eye diseases. Historically, gene therapies are virus-based and exhibit high transduction efficiency (review [100-102]). In recent years, rAAV (recombinant adeno-associated virus) has been safely and successfully employed to deliver RPE65 gene to animal models including Rpe65-/- mice, Rpe65rd12 mice, and Rpe65-/- dogs (with functional rescue last 1 year and half ). Moreover, the present of subretinally delivered rAAV vectors in the retinas of dogs and primates last for several years . These experiments have been sufficiently successful in terms of restoration of visual function, improvements in chromophore and rhodopsin levels, retardation of the photoreceptor degeneration, and visual acuity [105-107]. These breakthroughs have prompted the successful initiation of multiple human clinical trials (review ). Clinical trials involving 12 patients (NCT00643747) [109, 110], 15 patients (NCT00481546) , and 5 patients (NCT-00516477) [112, 113] showed improvement of retinal sensitivity up to 3 years. Significant improvement in visual function, visual acuity, and visual field up to 3 years was documented [111, 113]. Since many degenerative eye diseases are caused by gain-of-function mutations, treatments often have to include both a knockdown and a gene replacement component. For example, RNA interference每based suppression of mutant rhodopsin, together with gene replacement significant improved retinal function in a Rho-adRP mouse model . For loss-of-function mutations, gene replacement strategies have been the most common. Several gene replacement studies using RDS have been undertaken using AAV vector [115, 116]. After delivery of AAV containing RDS, rods form stable OS discs  and retinal function is improved . Rescue of rd1 mice is difficult because of too fast retinal degeneration. So far, only partial structural preservation and minimal function rescue were achieved in this mouse model. Ali group reported that AAV-mediated gene replacement therapy with the removal of Gpr179 mutation results in robust structural (80% of wild type), functional (60% of wild type) and behavioral rescue (appears as normal visual acuity) in rd1 mice . Combinatorial delivery of the neurotrophic factor GDNF (glial cell line-derived neurotrophic factor) and Rds gene by AAV to the rds-/- retina, resulted in significantly improved retinal function when compared to a Rds-replacement therapy alone . In spite of these successes, rAAV-based treatments have traditionally been limited by the capacity of the vector. However, a recent study demonstrated that modified rAAV 2/5 can package the 6.8 kb mouse Abcr gene (much larger than the initial AAV capacity of 4.8 kb)  and when delivered to the Abcr-/- mouse retina resulted in reduced A2E levels in RPE cells and increased ABCR protein expression. In an AAV-mediated secretion gene therapy (AAV.sFlt-1) in diseased mice and monkey, the expression of the transgene lasted for up to 8 months and the reduction of CNV in all treated eyes was observed . Furthermore, rAAV.sFLT-1 clinical trial for treatment of ※wet§ AMD patient was carried out and 9 patients were enrolled for the subretinal injection (NTC01494805). Clinical assessment demonstrated that rAAV.sFLT-1 was safe and well-tolerated . In addition, AAV has also been employed to deliver neuroprotective factors alone such as PEDF (pigment epithelium-derived factor) to a mouse CNV model , GDNF to S334ter rats with rhodopsin mutation  and CNTF (ciliary neurotrophic factor) to rds-/- mice , P23H and S334ter rats , and rd1 mice , and resulted in effective inhibition of CNV, retardation of photoreceptor apoptosis and delayed retinal degeneration.
Nanomedicine arises as a new attractive approach for treatment of retinal degenerative diseases in the last decade. Using polyethylene glycol (PEG) compacted DNA nanoparticles, Cai et al successfully achieved significantly structural and functional improvement of the rds+/- mouse model after subretinal delivery of the wild type Rds gene at P5 and P22 [126, 127]. In addition, Cai et al using catalytic and direct antioxidants cerium oxide nanoparticles (nanoceria) to treat tubby and vldlr-/- young adult mice at P28 by intravitreal injection resulted in sustained protection against retinal degeneration in tubby mice  and regression of existing neovascularization in vldlr-/- mice .
In recent years, the use of stem cells (SCs) and regenerative medicine have attracted great attention in both the scientific community and the public. Regenerative therapy for retinal degeneration in animal models holds a promise to restore vision in the diseased eye. Induced pluripotent SCs (iPSCs), with capacity to differentiate into specialized cells, are the most promising candidates for clinical application . Despite encouraging results, iPSCs will likely need to be used in combination with up-to-date tissue engineering to achieve ultimate success . Several recent reports claim successful improvement of vision in different animal models such as Rpe65-/- mice , Royal College Surgeons rat  and Crx-/- mice . Human embryonic SCs (HESCs)-derived retina tissues were transplanted into two primate models of photoreceptor degeneration, a range of retinal cell types was differentiated with rods and cones developed structured ONL . Two clinical trials enrollment of 9 patients each (NCT01345006 for Stargardt＊s macular dystrophy, and NCT01344993 for ※dry§ AMD) for subretinal transplantation of hESC-derived RPE, up to 3 year follow up demonstrated best-correction of visual acuity which was significantly improved and remained stable for 3 years. In addition, increased subretinal pigment was present at the border of the atrophic area, suggesting successful cellular engraftment. No adverse side effects were observed [133, 134]. Although much progress has been made, plentiful challenges remain to be solved. A major obstacle to developing stem cell-based therapies for degenerative retinal disorders is the poor integration and differentiation of retinal stem cells transplanted into the recipient retinas. The current challenge is to understand the developmental processes that guide embryonic or adult stem cells towards to differentiation to photoreceptor, so that large numbers of these cells might be transplanted at the optimal stage. Future advances in reproductive cloning technology could lead to the successful generation of stem cells from adult somatic cells, thereby facilitating auto-transplantation of genetically identical cells in patients requiring photoreceptor replacement . For additional information, readers are referred to these excellent review articles [135-141].
Epsins are adaptor proteins in endocytosis for ubiquitylated receptors on plasma membrane . In animal models, we have identified the functions of epsins in embryogenesis via Notch signaling was by severely reducing Notch primary target genes in DKO embryos , in vascular angiogenesis, lymphatic angiogenesis  and tumor angiogenesis [145-148] via VEGFR2 or VEGFR3 signaling, in colon cancer via Wnt signaling . Treatment of pups (P3-P6) of wild type C57BL/6 by an epsin ubiquitin interacting motif (UIM) memetic peptide UPI leads to overwhelming vessels in the retina . Loss of endothelial epsins (EC-iDKO) in mice produced heightening VEGFR2 signaling and increased postnatal retinal angiogenesis , strongly suggesting that epsins are critical for physiological retinal angiogenesis. Interestingly, physiological angiogenesis is increased by administration of UIM peptide, but not UIM E3, 4, 5A mutant peptide . Whether epsins play a role in retinal degeneration and diabetic retinopathy warrants further investigation. Under pathological conditions, administration of siRNA of epsins by liposome or UPI peptide may have therapeutic outcomes.
CRISPR/Cas9 genome editing technology is emerging and develops very quickly in recent years [150-156]. Using this new technology, several groups have obtained promising results to correct (or repair) mutant gene sequences from patient-derived iPSCs to treat inherited ocular diseases including RP, AMD, and other retinal diseases (review ). Recently, CRISPR/Cas9 system was used to identify the disease-causative gene in the rd1 mice and genome editing-generated CRISPR-repaired mice showed robust rescue of the neurofunction . Bakondi B. et al reported that disruption of a dominantly genetic mutation S334ter of rhodopsin gene (RhoS334) by CRISPR/Cas9 in a rat ADRP model prevent retinal degeneration and improve visual function . Furthermore, Bassuk AG et al generated iPSCs from a patient with a mutation in the retinitis pigmentosa GTPase regulator (RPGR) gene, which causes an aggressive, X-linked variant of RP (XLRP) . About 13% of RPGR gene copies showed mutation correction and conversion to the wild type allele. This is the first report using CRISPR to correct a pathogenic mutation in iPSCs derived from a patient with photoreceptor degeneration. This important proof-of-concept finding supports the development of personalized iPSC-based transplantation therapies for retinal diseases . These reports strongly support that CRISPR/Cas9 genome editing technology holds great promising to correct genetic mutations of retinal degeneration.
The extensive search for naturally occurring retinal degenerative models has been long but successful. Genetic alteration of genes has significantly increased the pool of animal models. The study of physiology and pathology in these animal models has accumulated abundant knowledge about the genetics, biochemistry, signaling, metabolism, as well as structure and function underlying the pathology of retinal degenerations, especially through inflammation-mediated signaling pathways. These animal models provide a platform for scientists to uncover the underlying molecular mechanisms and hold promise in preclinical drug screening and potential gene therapy or stem cell therapy for the treatment of human ocular diseases. However, it should be noted that each animal model could not completely reproduce human retinal degeneration diseases. Drug screening from animal models needs further validation using other approach. Besides, animal model of retinal degeneration per se may contain other complications. For example, tubby mice are obese and hard to reproduce offspring. Therefore, each animal model has its limitations in the identification of molecular mechanism or in preclinical drug screening. Targeting retinal degeneration-associated proteins including epsins by siRNA-liposome technology or by CRISPR/Cas9 genome editing is a perspective to correct or repair genetic mutation-induced retinal degeneration.
ABCR: ATP-binding cassette transporter; ADRP: autosomal dominant retinitis pigmentosa; AMD: age-related macular degeneration; ADMD: autosomal dominant Stargardt-like macular dystrophy; ARRP: autosomal recessive retinitis pigmentosa; ARVO: The Association for Research in Vision and Ophthalmology; BLD: basal laminar deposit; Cas-9: CRISPR associated protein 9; ccl2: CC-cytokin ligand 2; ccr2: CC-cytokine receptor 2; CNTF: ciliary neurotrophic factor; CNV: choroidal neovascularization; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeat; Crx: cone每rod homeobox; DHA: docosahexaenoic acid; DR: diabetic retinopathy; ELO: elongation of long chain fatty acid; ELOVL4: elongation of very long chain fatty acid protein 4; ER: endoplasmic reticulum; ERG: Electroretinography; GDNF: glial cell line-derived neurotrophic factor; HESCs: human embryonic stem cells; IGF: Insulin-like growth factor; iPSCs: induced pluripotent stem cells; LCA: Leber＊s congenital amaurosis; Mcd: mutation form of cathepsin D; Nrl: neural retina leucine zipper; Nr2e3: nuclear receptor subfamily 2, group E, member 3; OIR: oxygen induced retinopathy; ONL: outer nuclear layer; OPL: outer plexiform layer; OS: outer segment; Pde6b: rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta; PEDF: pigment epithelium-derived factor; PGK: phosphoglycerate kinase I; rAAV: recombinant adeno-associated virus; RAP: retinal angiomatous proliferation; rd: retinal degeneration; Rds: retinal degeneration slow; Rho: rhodopsin; RNV: retinal neovascularization; ROP: retinopathy of prematurity; RP: retinitis pigmentosa; RPE: retinal pigment epithelium; RPE65: Retinal pigment epithelium-specific 65 kDa protein; RPGR: retinitis pigmentosa GTPase regulator; RS1: retinoschisin; SCs: Stem cells; STGD: Stargardt macular dystrophy; VEGF: vascular endothelial growth factor; VEGFR2: vascular endothelial growth factor receptor 2; VLDLr: very low density lipoprotein receptor; XLJR: linked juvenile retinoschisis.
All authors read and approved the manuscript. YD and XC initiated the topic and co-wrote the manuscript. YW, YL, LD, and HC contributed to discuss and edit the article.
This work was supported in part by National Institutes of Health (NIH) grants R01HL-093242, R01 HL118676, R01HL-130845, P20 RR018758, AHA Established Investigator Award, a National Scientific Development Grant from the American Heart Association (0835544N), and a grant from Department of Defense W81XWH-11-1-00226 to H. Chen; by OCAST grants AR11-043, HR14-056 and American Heart Association AHA-SDG grant 12SDG8760002 to Y. Dong; This work was also supported in part by Accelerating Excellence in Translational Science Pilot Grants G0812D05 and NIH/NCI SC1CA200517 to Y. Wu., and a NIH grant SC1DK104821 to Y. L.
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Conflict of Interest: No conflicts declared.
YD and XC contribute equally to this work.
* Corresponding Author: Yunzhou Dong and Hong Chen.
Vascular Biology Program, Boston Children＊s Hospital,
Harvard Medical School, Boston, MA 02115, USA.
Tel: 617-919-6304; Fax: 617-730-0232
© 2017 by the Authors | Journal of Nature and Science (JNSCI).