Genomic landscape of retinoblastoma in Rb−/−p130−/− mice resembles human retinoblastoma

Several murine retinoblastoma models have been generated by deleting the genes encoding for retinoblastoma susceptibility protein pRb and one of its family members p107 or p130. In Rb^?/?p107^?/? retinoblastomas, somatic copy number alterations (SCNAs) like Mdm2 amplification or Cdkn2a deletion targeting the p53‐pathway occur, which is uncommon for human retinoblastoma. In our study, we determined SCNAs in retinoblastomas developing in Rb^?/?p130^?/? mice and compared this to murine Rb^?/?p107^?/? tumors and human tumors. Chimeric mice were made by injection of 129/Ola‐derived Rb^?/?p130^?/? embryonic stem cells into wild type C57BL/6 blastocysts. SCNAs of retinoblastoma samples were determined by low‐coverage (～0.5×) whole genome sequencing. In Rb^?/?p130^?/? tumors, SCNAs included gain of chromosomes 1 (3/23 tumors), 8 (1/23 tumors), 10 (1/23 tumors), 11 (2/23 tumors), and 12 (4/23 tumors), which could be mapped to frequently altered chromosomes in human retinoblastomas. While the altered chromosomes in Rb^?/?p130^?/? tumors were similar to those in Rb^?/?p107^?/? tumors, the alteration frequencies were much lower in Rb^?/?p130^?/? tumors. Most of the Rb^?/?p130^?/? tumors (16/23 tumors, 70%) were devoid of SCNAs, in strong contrast to Rb^?/?p107^?/? tumors, which were never (0/15 tumors) SCNA‐devoid. Similarly, to human retinoblastoma, increased age at diagnosis significantly correlated with increased SCNA frequencies. Additionally, focal loss of Cdh11 was observed in one Rb^?/?p130^?/? tumor, which enforces studies in human retinoblastoma that identified CDH11 as a retinoblastoma suppressor. Moreover, based on a comparison of genes altered in human and murine retinoblastoma, we suggest exploring the role of HMGA1 and SRSF3 in retinoblastoma development. © 2016 Wiley Periodicals, Inc.     

Cell type-specific effects of Rb deletion in the murine retina

Certain cells of the human retina are extremely sensitive to loss of function of the retinoblastoma tumor suppressor gene RB. Retinoblastomas develop early in life and at high frequency in individuals heterozygous for a germ-line RB mutation, and sporadic retinoblastomas invariably have somatic mutation in the RB gene. In contrast, retinoblastomas do not develop in Rb+/- mice. Although retinoblastoma is thought to have developmental origins, the function of Rb in retinal development has not been fully characterized. Here we studied the role of Rb in normal retinal development and in retinoblastoma using conditional Rb mutations in the mouse. In late embryogenesis, Rb-deficient retinas exhibited ectopic S-phase and high levels of p53-independent apoptosis, particularly in the differentiating retinal ganglion cell layer. During postnatal retinal development, loss of Rb led to more widespread retinal apoptosis, and adults showed loss of photoreceptors and bipolar cells. Conditional Rb mutation in the retina did not result in retinoblastoma formation even in a p53-mutant background. However, on a p107- or p130-deficient background, Rb mutation in the retina caused retinal dysplasia or retinoblastoma.     

Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum

Medulloblastomas are among the most common malignancies in childhood, and they are associated with substantial mortality and morbidity. The molecular pathogenesis as well as the ontogeny of these neoplasms is still poorly understood. We have generated a mouse model for medulloblastoma by Cre–LoxP-mediated inactivation of Rb and p53 tumor suppressor genes in the cerebellar external granular layer (EGL) cells. GFAP–Cre-mediated recombination was found both in astrocytes and in immature precursor cells of the EGL in the developing cerebellum. GFAP–Cre;Rb^LoxP/LoxP;p53^{−/− or LoxP/LoxP} mice developed highly aggressive embryonal tumors of the cerebellum with typical features of medulloblastoma. These tumors were identified as early as 7 weeks of age on the outer surface of the molecular layer, corresponding to the location of the EGL cells during development. Our results demonstrate that loss of function of RB is essential for medulloblastoma development in the mouse and strongly support the hypothesis that medulloblastomas arise from multipotent precursor cells located in the EGL.     

pRb2/p130 protein expression and RBL2 mutation analysis in Burkitt lymphoma from Uganda

Background  

The members of the retinoblastoma protein family, pRb, p107 and pRb2 (p130), are central players in controlling the cell cycle. Whereas disturbed function of pRb is commonly seen in human cancers, it is still an open question whether pRb2 is involved in tumorigenic processes. However, altered subcellular localization of pRb2 and mutations in the pRb2-encoding gene RBL2 have been described for some tumours, including Burkitt lymphomas (BL).     

Overcoming cellular senescence in human cancer pathogenesis

Elevation of p16, the CDKN2/p16 tumor suppressor gene (TSG) product, occurs at senescence in normal human uroepithelial cells (HUC). Immortal HUCs and bladder cancer cell lines show either alteration of p16 or pRb, the product of the retinoblastoma (RB) TSG. In addition, many human cancers show p16 or pRb alteration along with other genetic alterations that we associated with immortalization, including +20q and −3p. These observations led us to hypothesize that p16 elevation plays a critical role in senescence cell cycle arrest and that overcoming this block is an important step in tumorigenesis in vivo, as well as immortalization in vitro. Using a novel approach, we tested these hypotheses in the present study by examining p16 and pRb status in primary culture (P0) and after passage in vitro of transitional cell carcinoma (TCC) biopsies that represented both superficial bladder tumors and invasive bladder cancers. We demonstrated that all superficial TCCs showed elevated p16 after limited passage in vitro and then senesced, like normal HUCs. In contrast, all muscle invasive TCCs contained either a p16 or a pRb alteration at P0 and all spontaneously bypassed senescence (P=0.001). Comparative genomic hybridization (CGH) was used to identify regions of chromosome loss or gain in all TCC samples. The application of a statistical model to the CGH data showed a high probability of elevated alteration rates of +20q11−q12 (0.99) and +8p22−pter (0.94) in the immortal muscle invasive TCCs, and of −9q (0.99) in the superficial TCCs. Three myoinvasive TCCs lost 3p13−p14. In this study, four of six myoinvasive TCCs also showed TP53 mutation that associated well with genome instability (P=0.001), as previously hypothesized. Notably, TP53 mutation, which has been used as a marker of tumor progression in many human cancers, was less significant in associating with progression in this study (P=0.04) than was p16 or pRb alteration (P=0.001). Thus, these data support a new model in which overcoming senescence plays a critical role in human cancer pathogenesis and requires at least two genetic changes that occur in several combinations that can include either p16 or pRb loss and at least one additional alteration, such as +20q11−q12, −3p13−p14, or −8p21−pter.     

Tissue-specific tumor suppressor activity of retinoblastoma gene homologs p107 and p130

The retinoblastoma gene family consists of three genes: RB, p107, and p130. While loss of pRB causes retinoblastoma in humans and pituitary gland tumors in mice, tumorigenesis in other tissues may be suppressed by p107 and p130. To test this hypothesis, we have generated chimeric mice from embryonic stem cells carrying compound loss-of-function mutations in the Rb gene family. We found that Rb/p107- and Rb/p130-deficient mice were highly cancer prone. We conclude that in a variety of tissues tumor development by loss of pRB is suppressed by its homologs p107 and p130. The redundancy of the retinoblastoma proteins in vivo is reflected by the behavior of Rb-family-defective mouse embryonic fibroblasts in vitro.     

Expression of a human polyomavirus oncoprotein and tumour suppressor proteins in medulloblastomas.

AIMS: Although the aetiology of medulloblastoma remains elusive, several lines of evidence suggest an association with the human neurotropic polyomavirus JC and its oncoprotein T antigen. The tumour forming properties of JC virus T antigen are the result, at least in part, of its ability to bind and inactivate tumour suppressor/cell cycle regulatory proteins, such as p53 and the retinoblastoma family of proteins. METHODS: To examine potential relations between these factors, immunohistochemistry was used to determine associations between the T antigen and the expression of p53 and the retinoblastoma proteins pRb, p107, and Rb2/p130 in eight medulloblastomas. RESULTS: Only the three medulloblastomas with T antigen expression also showed nuclear positivity with antibodies to p53. Although immunohistochemistry detected nuclear labelling for pRb in five of the cases, the three that were positive for T antigen showed the highest pRb labelling. The retinoblastoma related proteins p107 and Rb2/p130 were also immunopositive in most T antigen positive medulloblastomas. Double label immunohistochemistry also demonstrated p53 and pRb positivity in the same cells that were T antigen positive. CONCLUSIONS: These correlations suggest that associations between T antigen and p53 and/or T antigen and pRb occur in some of these tumours. These data provide indirect evidence that JC virus, acting through T antigen, might be involved in the formation and progression of medulloblastoma.     

Phosphorylation of p27(KIP1) in the developing retina and retinoblastoma

Cellular distribution of the p27(KIP1) protein and its phosphorylation on threonine (T) 187 in mouse retinas from three stages of development, and retinoblastoma were examined. Retinas in C57Bl6 mice at embryonic day (E) 14, postnatal day (P) 1 and P11 were analyzed using immunohistochemistry with anti-p27(KIP1), threonine-187-phosphorylated p27(KIP1) (T187-phospho-p27), bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA) antibodies, and phosphorylated histon H3 (pHiston H3), which is a marker for cells in M phase. p27(KIP1) knockout (-/-) mice and human retinoblastoma were also analyzed. T187-phospho-p27 was detected in the outermost layer of the retina, whereas several neuroblastic cells expressed p27(KIP1) at E14. Many neuroblastic cells expressed BrdU in the middle layer. At P1, p27(KIP1) was detected in the ganglion cell layer and neuroblastic layer. T187-phospho-p27 was detected in the outermost layer, and that was localized in mitotic cells that also showed pHiston H3-positive. At P11, p27(KIP1) was detected in the inner nuclear layer, whereas T187-phospho-p27-positive or mitotic cells were not. BrdU positive nuclei were not detected in wild-type but were noted in the inner nuclear layer and the outer nuclear layer of the p27(KIP1) -/- mice retina at P11. In retinoblastoma, tumor cells formed numerous rosettes with Flexner-Wintersteiner rosettes. Several pHiston H3 -positive nuclei were noted in the tumor cells forming Flexner-Wintersteiner rosettes. Several T187-phospho-p27-positive nuclei were also detected in the mitotic cells forming Flexner-Wintersteiner rosettes. PCNA was expressed in rosette-forming cells. In conclusion, T187-phospho-p27(KIP1) was correlated with M phase of the cell cycle in the developing retina and retinoblastoma.     

Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes

Caspases are fundamental components of the mammalian apoptotic machinery, but the precise contribution of individual caspases is controversial. CPP32 (caspase 3) is a prototypical caspase that becomes activated during apoptosis. In this study, we took a comprehensive approach to examining the role of CPP32 in apoptosis using mice, embryonic stem (ES) cells, and mouse embryonic fibroblasts (MEFs) deficient for CPP32. CPP32^ex3−/− mice have reduced viability and, consistent with an earlier report, display defective neuronal apoptosis and neurological defects. Inactivation of CPP32 dramatically reduces apoptosis in diverse settings, including activation-induced cell death (AICD) of peripheral T cells, as well as chemotherapy-induced apoptosis of oncogenically transformed CPP32^−/− MEFs. As well, the requirement for CPP32 can be remarkably stimulus-dependent: In ES cells, CPP32 is necessary for efficient apoptosis following UV- but not γ-irradiation. Conversely, the same stimulus can show a tissue-specific dependence on CPP32: Hence, TNFα treatment induces normal levels of apoptosis in CPP32 deficient thymocytes, but defective apoptosis in oncogenically transformed MEFs. Finally, in some settings, CPP32 is required for certain apoptotic events but not others: Select CPP32^ex3−/− cell types undergoing cell death are incapable of chromatin condensation and DNA degradation, but display other hallmarks of apoptosis. Together, these results indicate that CPP32 is an essential component in apoptotic events that is remarkably system- and stimulus-dependent. Consequently, drugs that inhibit CPP32 may preferentially disrupt specific forms of cell death.     

CDKN2A Gene Deletions and Loss of p16 Expression Occur in Osteosarcomas That Lack RB Alterations

Osteosarcomas often suffer mutations of the RB (retinoblastoma) gene, with resultant inactivation of the pRb protein. pRb is one component in a cell-cycle control pathway that includes the p16 (encoded by the CDKN2A gene) and cyclin-dependent kinase 4 (cdk4, encoded by the CDK4 gene) proteins. We therefore sought to determine whether the CDKN2A and CDK4 genes were altered in those osteosarcomas that lacked RB inactivation. Twenty-one osteosarcomas (2 low-grade and 19 high-grade) were evaluated for homozygous deletion of the CDKN2A gene, CDK4 amplification, and allelic loss of the RB gene, as well as for expression of p16 and pRb proteins. Five high-grade osteosarcomas showed loss of p16 expression; four of these had homozygous CDKN2A deletions, and the fifth had a probable deletion obscured by numerous nonneoplastic, p16-immunopositive multinucleated giant cells. Thus, p16 immunohistochemistry may provide a sensitive means for assessing CDKN2A status. Twelve tumors (including the two low-grade osteosarcomas) were immunopositive for pRb, and nine tumors were immunonegative for pRb. Of the five cases with CDKN2A/p16 alterations, none had allelic loss of the RB gene and all expressed pRb, suggesting that each of these tumors had an intact RB gene. None of the tumors showed CDK4 amplification. No alterations were detected in the two low-grade osteosarcomas. This study suggests that CDKN2A is a tumor suppressor inactivated in osteosarcomas that lack RB mutations and that the p16-pRb cell-cycle control pathway is deregulated in a large number of high-grade osteosarcomas.     

Functional link between retinoblastoma family of proteins and the Wnt signaling pathway in mouse epidermis.

The retinoblastoma family of proteins (pRb, p107, and p130) modulates cell cycle progression and differentiation of several tissues. We have demonstrated recently that p107 and p130 regulate keratinocyte terminal differentiation and hair follicle morphogenesis and development in vivo. This last aspect appears to be mediated by defective signaling from the mesenchyme and is associated with altered bone morphogenetic protein-4 (BMP4) -dependent signaling. However, many alterations were also found in the epithelial compartment. Given the importance of betacatenin in hair biology and in BMP signaling, we studied its expression in p107/p130-deficient skin. Although normal expression of betacatenin was found in p107/p130-deficient hair follicles, we found increased nuclear accumulation of betacatenin in the basal keratinocytes of the p107/p130-deficient mice skin. Biochemical analysis revealed that such an increase in betacatenin was due to the disruption of Axin/GSK3beta/betacatenin complexes promoted by the increased expression of Frat, the mouse homologue of GSK3betabinding protein (GBP), in epidermis, precluding the degradation of betacatenin. Collectively, these data represent the first evidence that retinoblastoma family and Wnt signaling pathways might be interconnected by functional links in skin.     

Loss of Rb proteins causes genomic instability in the absence of mitogenic signaling

Loss of G1/S control is a hallmark of cancer, and is often caused by inactivation of the retinoblastoma pathway. However, mouse embryonic fibroblasts lacking the retinoblastoma genes RB1, p107, and p130 (TKO MEFs) are still subject to cell cycle control: Upon mitogen deprivation, they enter and complete S phase, but then firmly arrest in G2. We now show that G2-arrested TKO MEFs have accumulated DNA damage. Upon mitogen readdition, cells resume proliferation, although only part of the damage is repaired. As a result, mitotic cells show chromatid breaks and chromatid cohesion defects. These aberrations lead to aneuploidy in the descendent cell population. Thus, our results demonstrate that unfavorable growth conditions can cause genomic instability in cells lacking G1/S control. This mechanism may allow premalignant tumor cells to acquire additional genetic alterations that promote tumorigenesis.     

The type I activin receptor ActRIB is required for egg cylinder organization and gastrulation in the mouse

ActRIB is a type I transmembrane serine/threonine kinase receptor that has been shown to form heteromeric complexes with the type II activin receptors to mediate activin signal. To investigate the function of ActRIB in mammalian development, we generated ActRIB-deficient ES cell lines and mice by gene targeting. Analysis of the ActRIB^−/− embryos showed that the epiblast and the extraembryonic ectoderm were disorganized, resulting in disruption and developmental arrest of the egg cylinder before gastrulation. To assess the function of ActRIB in mesoderm formation and gastrulation, chimera analysis was conducted. We found that ActRIB^−/− ES cells injected into wild-type blastocysts were able to contribute to the mesoderm in chimeric embryos, suggesting that ActRIB is not required for mesoderm formation. Primitive streak formation, however, was impaired in chimeras when ActRIB^−/− cells contributed highly to the epiblast. Further, chimeras generated by injection of wild-type ES cells into ActRIB^−/− blastocysts formed relatively normal extraembryonic tissues, but the embryo proper developed poorly probably resulting from severe gastrulation defect. These results provide genetic evidence that ActRIB functions in both epiblast and extraembryonic cells to mediate signals that are required for egg cylinder organization and gastrulation.     

p53-Regulated Apoptosis Is Differentiation Dependent in Ultraviolet B-Irradiated Mouse Keratinocytes

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (−/−) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53−/− mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m² UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53−/− cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53−/− cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction.     

Prevention of hepatic apoptosis and embryonic lethality in RelA/TNFR-1 double knockout mice

Mice deficient in the nuclear factor κB (NF-κB)-transactivating gene RelA (p65) die at embryonic days 14–15 with massive liver apoptosis. In the adult liver, activation of the NF-κB heterodimer RelA/p50 can cause hepatocyte proliferation, apoptosis, or the induction of acute-phase response genes. We examined, during wild-type fetal liver development, the expression of the Rel family member proteins, as well as other proteins known to be important for NF-κB activation. We found these proteins and active NF-κB complexes in the developing liver from at least 2 days before the onset of lethality observed in RelA knockouts. This suggests that the timing of NF-κB activation is not related to the timing of lethality. We therefore hypothesized that, in the absence of RelA, embryos were sensitized to tumor necrosis factor (TNF) receptor 1 (TNFR-1)-mediated apoptosis. Thus, we generated mice that were deficient in both RelA and TNFR-1 to determine whether apoptotic signaling through TNFR-1 was responsible for the lethal phenotype. RelA/TNFR-1 double knockout mice survived embryonic development and were born with normal livers without evidence of increased hepatocyte apoptosis. These animals became runted shortly after birth and survived an average of 10 days, dying from acute hepatitis with an extensive hepatic infiltration of immature neutrophils. We conclude that neither RelA nor TNFR-1 is required for liver development and that RelA protects the embryonic liver from TNFR-1-mediated apoptotic signals. However, the absence of both TNFR-1 signaling and RelA activity in newborn mice makes these animals susceptible to endogenous hepatic infection.     

Stage-specific apoptosis, developmental delay, and embryonic lethality in mice homozygous for a targeted disruption in the murine Bloom’s syndrome gene

Bloom’s syndrome is a human autosomal genetic disorder characterized at the cellular level by genome instability and increased sister chomatid exchanges (SCEs). Clinical features of the disease include proportional dwarfism and a predisposition to develop a wide variety of malignancies. The human BLM gene has been cloned recently and encodes a DNA helicase. Mouse embryos homozygous for a targeted mutation in the murine Bloom’s syndrome gene (Blm) are developmentally delayed and die by embryonic day 13.5. The fact that the interrupted gene is the homolog of the human BLM gene was confirmed by its homologous sequence, its chromosomal location, and by demonstrating high numbers of SCEs in cultured murine Blm^−/− fibroblasts. The proportional dwarfism seen in the human is consistent with the small size and developmental delay (12–24 hr) seen during mid-gestation in murine Blm^−/− embryos. Interestingly, the growth retardation in mutant embryos can be accounted for by a wave of increased apoptosis in the epiblast restricted to early post-implantation embryogenesis. Mutant embryos do not survive past day 13.5, and at this time exhibit severe anemia. Red blood cells and their precursors from Blm^−/− embryos are heterogeneous in appearance and have increased numbers of macrocytes and micronuclei. Both the apoptotic wave and the appearance of micronuclei in red blood cells are likely cellular consequences of damaged DNA caused by effects on replicating or segregating chromosomes.     

Apoptosis of leukocytes triggered by acute DNA damage promotes lymphoma formation

Apoptosis triggered by p53 upon DNA damage secures removal of cells with compromised genomes, and is thought to prevent tumorigenesis. In contrast, we provide evidence that p53-induced apoptosis can actively drive tumor formation. Mice defective in p53-induced apoptosis due to loss of its proapoptotic target gene, puma, resist γ-irradiation (IR)-induced lymphomagenesis. In wild-type animals, repeated irradiation injury-induced expansion of hematopoietic stem/progenitor cells (HSCs) leads to lymphoma formation. Puma^−/− HSCs, protected from IR-induced cell death, show reduced compensatory proliferation and replication stress-associated DNA damage, and fail to form thymic lymphomas, demonstrating that the maintenance of stem/progenitor cell homeostasis is critical to prevent IR-induced tumorigenesis.     

Alterations of Retinal Vasculature in Cystathionine–β-Synthase Heterozygous Mice: A Model of Mild to Moderate Hyperhomocysteinemia

Mild to moderate hyperhomocysteinemia is prevalent in humans and is implicated in neurovascular diseases, including recently in certain retinal diseases. Herein, we used hyperhomocysteinemic mice deficient in the Cbs gene encoding cystathionine–β-synthase (Cbs^+/−) to evaluate retinal vascular integrity. The Cbs^+/+ (wild type) and Cbs^+/− (heterozygous) mice (aged 16 to 52 weeks) were subjected to fluorescein angiography and optical coherence tomography to assess vasculature in vivo. Retinas harvested for cryosectioning or flat mount preparations were subjected to immunofluorescence microscopy to detect blood vessels (isolectin-B4), angiogenesis [anti-vascular endothelial growth factor (VEGF) and anti-CD105], gliosis [anti-glial fibrillary acidic protein (GFAP)], pericytes (anti-neural/glial antigen 2), blood-retinal barrier [anti–zonula occludens protein 1 (ZO-1) and anti-occludin], and hypoxia [anti–pimonidazole hydrochloride (Hypoxyprobe-1)]. Levels of VEGF, GFAP, ZO-1, and occludin were determined by immunoblotting. Results of these analyses showed a mild vascular phenotype in young mice, which progressed with age. Fluorescein angiography revealed progressive neovascularization and vascular leakage in Cbs^+/− mice; optical coherence tomography confirmed new vessels in the vitreous by 1 year. Immunofluorescence microscopy demonstrated vascular patterns consistent with ischemia, including a capillary-free zone centrally and new vessels with capillary tufts midperipherally in older mice. This was associated with increased VEGF, CD105, and GFAP and decreased ZO-1/occludin levels in the Cbs^+/− retinas. Retinal vein occlusion was observed in some Cbs^+/− mouse retinas. We conclude that mild to moderate elevation of homocysteine in Cbs^+/− mice is accompanied by progressive alterations in retinal vasculature characterized by ischemia, neovascularization, incompetent blood-retinal barrier, and vascular occlusion.Homocysteine (Hcy), a sulfur-containing amino acid, is an intermediate in methionine metabolism. It is converted to either methionine, via the remethylation pathway, or cysteine for permanent disposal via the trans-sulfuration pathway. Hyperhomocysteinemia (HHcy) results from increased Hcy dietary loading (eg, diet rich in methionine), decreased rates of Hcy metabolism due to deficiency of vitamin cofactors (folic acid and vitamins B₆ and B₁₂), or genetic mutations of enzymes involved in remethylation or trans-sulfuration pathways. Mild to moderate HHcy is prevalent in 5% to 10% of the general population, up to 90% of patients undergoing hemodialysis, and approximately 40% of patients with peripheral vascular disease.^1,2 HHcy is a risk factor for human cardiovascular diseases (eg, stroke and venous thrombosis) and neurodegenerative diseases.³ It has been implicated in eye-related diseases, including ectopia lentis,⁴ glaucoma (primary and secondary open-angle glaucoma, exfoliation glaucoma, and pigmentary glaucoma),^5–7 macular degeneration, maculopathy, retinal degeneration,^8–10 diabetic retinopathy,^11,12 and retinal vascular diseases (ie, central retinal vein occlusion, branch retinal vein occlusion, and central retinal artery occlusion^13–16), although its role in these diseases is inconsistent.Our laboratory and others have used in vitro and in vivo experimental models to understand the mechanisms by which HHcy affects retina. Apoptotic neuronal cell death was induced in primary mouse retinal ganglion cells incubated with elevated, but physiologically relevant, levels of Hcy (50 μmol/L).¹⁷ In vivo studies showed that intravitreal injection of high dosages of d,l-Hcy-thiolactone led to marked ganglion cell loss and disruption of the inner retina in mice within 5 days of injection.¹⁸ Lower-dosage d,l-Hcy-thiolactone intravitreal injections led to marked loss of photoreceptor cells within 15 days and ablation of the outer retinal nuclear layer within 90 days.¹⁹ Mice deficient or lacking the gene encoding cystathionine–β-synthase (CBS)²⁰ have proved useful for analysis of the effects of mild to severe endogenous elevation of Hcy on several tissues, including retina.^21–26 CBS is a key enzyme in the trans-sulfuration pathway, and its deficiency is the most common cause of inherited homocystinuria.⁴The Cbs^−/− mice have a 30- to 40-fold increase in plasma Hcy²⁰ and a shortened life span of 3 to 5 weeks. Retinal Hcy levels are increased by approximately sevenfold in Cbs^−/− mice compared with age-matched wild-type littermates.²² Functional studies of the visual system in these mice reveal significantly reduced amplitudes of the dark- and light-adapted electroretinogram (ERG) and a marked delay in the N1 implicit time of the visual-evoked potential, which reflects signal transmission to the visual cortex.²⁵ Comprehensive histological assessment of Cbs^−/− retinas demonstrates profound loss of cells in the retinal ganglion cell layer, marked disruption of the inner/outer nuclear retinal layers, and hypertrophy of the retinal pigment epithelial cell layer.²²Functional studies of retinas of Cbs^+/− mice, which have a much milder HHcy with an approximately fourfold to sevenfold increase in plasma Hcy (and a twofold increase in retinal Hcy), reveal a gradual reduction of the light and dark amplitudes of the ERG, reduced direct-coupled ERG light peak, and a slight increase in the implicit times of the visual-evoked potential.²⁵ Histological analyses of Cbs^+/− mouse retinas show a much milder retinal phenotype than Cbs^−/− mice, characterized by moderate cell loss in the ganglion cell layer and decreased thickness of the inner plexiform and inner and outer nuclear layers.²² Altered mitochondria of the nerve fiber layer and increased expression of the mitochondrial proteins, Opa1 and Fis1, accompanied the retinal ganglion cell loss.²⁴ The functional deficits and histological alterations in the Cbs^+/− mouse become evident by approximately 30 weeks of age.^22,25Recently, we analyzed the retinal vasculature of Cbs^−/− mice to determine whether the profound neuronal degeneration of the retina was accompanied by a vascular phenotype.²⁶ The study was restricted to mice aged 3 weeks owing to the short life span of the homozygous (Cbs^−/−) mice. Angiography revealed considerable vascular leakage in Cbs^−/− retinas, and the immunohistochemical analysis revealed vascular patterns consistent with ischemia. Vascular endothelial growth factor (VEGF), a marker of new blood vessels, was increased in Cbs^−/− retinas, and the blood-retinal barrier was compromised. In this same study, the vasculature of the heterozygous (Cbs^+/−) mice at 3 weeks showed minimal evidence of vasculopathy, but raised the question as to whether retinal vascular alterations would be detectable with advancing age. Because mild HHcy is common in the human population, the present study investigated the retinal vasculature of the Cbs^+/− mice as a function of age. Our data demonstrate subtle vascular changes early that become more prominent by 4 months of age and progress to ischemia and neovascularization by 6 to 12 months of age.