The role of telomerase reverse transcriptase in the mitochondrial protective functions of Angiotensin-(1-7) in diabetic CD34+ cells.

Angiotensin (Ang)-(1-7) stimulates vasoprotective functions of diabetic (DB) CD34+ hematopoietic stem/progenitor cells partly by decreasing reactive oxygen species (ROS), increasing nitric oxide (NO) levels and decreasing TGFß1 secretion. Telomerase reverse transcriptase (TERT) translocates to mitochondria and regulates ROS generation. Alternative splicing of TERT results in variants α-, ß- and α-ß-TERT, which may oppose functions of full-length (FL) TERT. This study tested if the protective functions of Ang-(1-7) or TGFß1-silencing are mediated by mitoTERT and that diabetes decreases FL-TERT expression by inducing splicing. CD34+ cells were isolated from the peripheral blood mononuclear cells of nondiabetic (ND, n = 68) or DB (n = 74) subjects. NO and mitoROS levels were evaluated by flow cytometry. TERT splice variants and mitoDNA-lesions were characterized by qPCR. TRAP assay was used for telomerase activity. Decoy peptide was used to block mitochondrial translocation (mitoXTERT). TERT inhibitor or mitoXTERT prevented the effects of Ang-(1-7) on NO or mitoROS levels in DB-CD34+ cells. FL-TERT expression and telomerase activity were lower and mitoDNA-lesions were higher in DB cells compared to ND and were reversed by Ang-(1-7) or TGFß1-silencing. The prevalence of TERT splice variants, with predominant ß-TERT expression, was higher and the expression of FL-TERT was lower in DB cells (n = 25) compared to ND (n = 30). Ang-(1-7) or TGFß1-silencing decreased TERT-splicing and increased FL-TERT. Blocking of ß-splicing increased FL-TERT and protected mitoDNA in DB-cells. The findings suggest that diabetes induces TERT-splicing in CD34+ cells and that ß-TERT splice variant largely contributes to the mitoDNA oxidative damage.

ACE2 gene transfer ameliorates vasoreparative dysfunction in CD34+ cells derived from diabetic older adults.

Diabetes increases the risk for ischemic vascular diseases, which is further elevated in older adults. Bone marrow-derived hematopoietic CD34+ stem/progenitor cells have the potential of revascularization; however, diabetes attenuates vasoreparative functions. Angiotensin-converting enzyme 2 (ACE2) is the vasoprotective enzyme of renin-angiotensin system in contrast with the canonical angiotensin-converting enzyme (ACE). The present study tested the hypothesis that diabetic dysfunction is associated with ACE2/ACE imbalance in hematopoietic stem/progenitor cells (HSPCs) and that increasing ACE2 expression would restore reparative functions. Blood samples from male and female diabetic (n=71) or nondiabetic (n=62) individuals were obtained and CD34+ cells were enumerated by flow cytometry. ACE and ACE2 enzyme activities were determined in cell lysates. Lentiviral (LV) approach was used to increase the expression of soluble ACE2 protein. Cells from diabetic older adults (DB) or nondiabetic individuals (Control) were evaluated for their ability to stimulate revascularization in a mouse model of hindlimb ischemia (HLI). DB cells attenuated the recovery of blood flow to ischemic areas in nondiabetic mice compared with that observed with Control cells. Administration of DB cells modified with LV-ACE2 resulted in complete restoration of blood flow. HLI in diabetic mice resulted in poor recovery with amputations, which was not reversed by either Control or DB cells. LV-ACE2 modification of Control or DB cells resulted in blood flow recovery in diabetic mice. In vitro treatment with Ang-(1-7) modified paracrine profile in diabetic CD34+ cells. The present study suggests that vasoreparative dysfunction in CD34+ cells from diabetic older adults is associated with ACE2/ACE imbalance and that increased ACE2 expression enhances the revascularization potential.

A Preclinical Safety Study of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells for Macular Degeneration.

Purpose: Age-related macular degeneration (AMD) is a common disease trending towards epidemic proportions and is a leading cause of irreversible vision loss in people over the age of 65. A pathomechanism of AMD is death and/or dysfunction of retinal pigment epithelial (RPE) cells; RPE loss invariably results in photoreceptor atrophy. Treatment options for AMD are very limited, and include vitamin supplements and lifestyle changes. An exciting potential therapy currently being tested in clinical trials is transplantation of stem cell-derived RPE. Methods: We developed a NIH-registered embryonic stem line (CR-4), and in this study set out to determine if CR4-RPE are tolerated in normal mice and in murine models of retinal degeneration by injecting a bolus of CR4-RPE cells in the subretinal space of immunosuppressed wild-type, Mer mutant (Merkd), and Elovl4 deficient mice. Results: Mice with CR-RPE grafts were monitored daily, were examined routinely using OCT, and histology was prepared and examined at terminal end-points. Based on the parameters of the study, none of the animals with CR-RPE grafts (n=36) experienced any obvious adverse reactions. Conclusions: We conclude that transplanted CR-4 hES-derived RPE cells are well tolerated in immunosuppressed healthy and dystrophic murine retinas.

Complement Receptor 1 (CR1/CD35)-expressing retinal pigment epithelial cells as a potential therapy for age-related macular degeneration.

The purpose of this study was to identify a membrane-bound complement inhibitor that could be overexpressed on retinal pigment epithelial cells (RPE) providing a potential therapy for age-related macular degeneration (AMD). This type of therapy may allow replacement of damaged RPE with cells that are able to limit complement activation in the retina. Complement Receptor 1 (CR1) is a membrane-bound complement inhibitor commonly found on erythrocytes and immune cells. In this study, QPCR and flow cytometry data demonstrated that CR1 is not well-expressed by RPE, indicating that its overexpression may provide extra protection from complement activation. To screen CR1 for this ability, a stable CR1-expressing ARPE19 line was created using a combination of antibiotic selection and FACS. Cell-based assays were used to demonstrate that addition of CR1 inhibited deposition of complement proteins C3b and C6 on the transfected line. In the end, this study identifies CR1 as a complement inhibitor that may be overexpressed on stem cell-derived RPE to create a potential "enhanced" cell therapy for AMD. A combination cell/complement therapy may create transplantable RPE better suited to avoid complement-mediated lysis and limit chronic inflammation in the retina.

Derivation and characterization of the human embryonic stem cell line CR-4: Differentiation to human retinal pigment epithelial cells.

The CR-4 human embryonic stem cell line was derived from the inner cell mass of a developing blastocyst. This cell line has been adapted to grow in feeder-free conditions and is especially well-suited for differentiation to retinal pigment epithelium. The line demonstrates a normal human 46,XX female karyotype. Pluripotency was assessed through qRT-PCR for expression of NANOG, OCT-4, and SOX-2. A teratoma assay was performed and results were positive for all three germ layers. Testing for Mycoplasma was negative.

North Carolina Macular Dystrophy Is Caused by Dysregulation of the Retinal Transcription Factor PRDM13.

PURPOSE: To identify specific mutations causing North Carolina macular dystrophy (NCMD). DESIGN: Whole-genome sequencing coupled with reverse transcription polymerase chain reaction (RT-PCR) analysis of gene expression in human retinal cells. PARTICIPANTS: A total of 141 members of 12 families with NCMD and 261 unrelated control individuals. METHODS: Genome sequencing was performed on 8 affected individuals from 3 families affected with chromosome 6-linked NCMD (MCDR1) and 2 individuals affected with chromosome 5-linked NCMD (MCDR3). Variants observed in the MCDR1 locus with frequencies <1% in published databases were confirmed using Sanger sequencing. Confirmed variants absent from all published databases were sought in 8 additional MCDR1 families and 261 controls. The RT-PCR analysis of selected genes was performed in stem cell-derived human retinal cells. MAIN OUTCOME MEASURES: Co-segregation of rare genetic variants with disease phenotype. RESULTS: Five sequenced individuals with MCDR1-linked NCMD shared a haplotype of 14 rare variants spanning 1 Mb of the disease-causing allele. One of these variants (V1) was absent from all published databases and all 261 controls, but was found in 5 additional NCMD kindreds. This variant lies in a DNase 1 hypersensitivity site (DHS) upstream of both the PRDM13 and CCNC genes. Sanger sequencing of 1 kb centered on V1 was performed in the remaining 4 NCMD probands, and 2 additional novel single nucleotide variants (V2 in 3 families and V3 in 1 family) were identified in the DHS within 134 bp of the location of V1. A complete duplication of the PRDM13 gene was also discovered in a single family (V4). The RT-PCR analysis of PRDM13 expression in developing retinal cells revealed marked developmental regulation. Next-generation sequencing of 2 individuals with MCDR3-linked NCMD revealed a 900-kb duplication that included the entire IRX1 gene (V5). The 5 mutations V1 to V5 segregated perfectly in the 102 affected and 39 unaffected members of the 12 NCMD families. CONCLUSIONS: We identified 5 rare mutations, each capable of arresting human macular development. Four of these strongly implicate the involvement of PRDM13 in macular development, whereas the pathophysiologic mechanism of the fifth remains unknown but may involve the developmental dysregulation of IRX1.

Persistent unilateral central serous chorioretinopathy in a breastfeeding woman.

A 40-year-old woman presented with blurred vision and floaters in her right eye for the past 7 months. The patient was in the sixth month of pregnancy at onset of the ocular symptoms and had persistent ocular disturbances postpartum. Her medical and ocular history were unremarkable except for LASIK surgery. Examination revealed an uncorrected visual acuity of 20/30 and 20/20 in the right and left eye, respectively. Fundus examination showed a round, subretinal exudate involving the foveal centre. Central serous chorioretinopathy was diagnosed by fluorescein angiogram and optical coherence tomography (OCT) showing foveal neurosensory detachment and treated with intravitreal bevacizumab. At her 4-month follow-up (8 month postpartum), OCT continued to show persistent foveal subretinal fluid. Patient declined further treatment and on follow-up 1 year later, still showed a persistent neurosensory detachment on OCT testing.

A dominant mutation in hexokinase 1 (HK1) causes retinitis pigmentosa.

PURPOSE: To identify the cause of retinitis pigmentosa (RP) in UTAD003, a large, six-generation Louisiana family with autosomal dominant retinitis pigmentosa (adRP). METHODS: A series of strategies, including candidate gene screening, linkage exclusion, genome-wide linkage mapping, and whole-exome next-generation sequencing, was used to identify a mutation in a novel disease gene on chromosome 10q22.1. Probands from an additional 404 retinal degeneration families were subsequently screened for mutations in this gene. RESULTS: Exome sequencing in UTAD003 led to identification of a single, novel coding variant (c.2539G>A, p.Glu847Lys) in hexokinase 1 (HK1) present in all affected individuals and absent from normal controls. One affected family member carries two copies of the mutation and has an unusually severe form of disease, consistent with homozygosity for this mutation. Screening of additional adRP probands identified four other families (American, Canadian, and Sicilian) with the same mutation and a similar range of phenotypes. The families share a rare 450-kilobase haplotype containing the mutation, suggesting a founder mutation among otherwise unrelated families. CONCLUSIONS: We identified an HK1 mutation in five adRP families. Hexokinase 1 catalyzes phosphorylation of glucose to glucose-6-phosphate. HK1 is expressed in retina, with two abundant isoforms expressed at similar levels. The Glu847Lys mutation is located at a highly conserved position in the protein, outside the catalytic domains. We hypothesize that the effect of this mutation is limited to the retina, as no systemic abnormalities in glycolysis were detected. Prevalence of the HK1 mutation in our cohort of RP families is 1%.

Central serous chorioretinopathy after blunt trauma.

The authors report a case of a 46-year-old Hispanic male with central serous chorioretinopathy (CSC) following blunt trauma to the left eye. The patient presented with a complaint of throbbing headache and blurry vision in left eye. The patient was diagnosed with diabetes mellitus 1 year previous to the event. On examination, uncorrected visual acuity was 20/20 OD, 20/200 OS. No anisocoria or afferent pupillary defect was present. Intraocular pressure was normal. Subconjunctival haemorrhage and lid ecchymosis were present in OS and fundus examination showed serous macular detachment and central retinal pigment epithelium detachment, and no evidence of diabetic retinopathy. Optical coherence tomography OS showed subretinal fluid and fluorescein angiography demonstrated the typical 'smokestack' pattern of leakage into the subretinal space. The patient received observational therapy for 4 months and the CSC spontaneously resolved with visual acuity of 20/20 in left eye.

Comparison of the modified Early Treatment Diabetic Retinopathy Study and mild macular grid laser photocoagulation strategies for diabetic macular edema.

OBJECTIVE: To compare 2 laser photocoagulation techniques for treatment of diabetic macular edema: the modified Early Treatment Diabetic Retinopathy Study (ETDRS) direct/grid photocoagulation technique and a potentially milder (but potentially more extensive) mild macular grid (MMG) laser technique in which microaneurysms are not treated directly and small mild burns are placed throughout the macula, whether or not edema is present. METHODS: Two hundred sixty-three subjects (mean age, 59 years) with previously untreated diabetic macular edema were randomly assigned to receive laser photocoagulation by either the modified ETDRS (162 eyes) or MMG (161 eyes) technique. Visual acuity, fundus photographs, and optical coherence tomography measurements were obtained at baseline and at 3.5, 8, and 12 months. Treatment was repeated if diabetic macular edema persisted. MAIN OUTCOME MEASURE: Change in optical coherence tomography measurements at 12-month follow-up. RESULTS: Among eyes with a baseline central subfield thickness of 250 microm or greater, central subfield thickening decreased by an average of 88 microm in the modified ETDRS group and by 49 microm in the MMG group at 12-month follow-up (adjusted mean difference, 33 microm; 95% confidence interval, 5-61 microm; P = .02). Weighted inner zone thickening by optical coherence tomography decreased by 42 microm in the modified ETDRS group and by 28 microm in the MMG group (adjusted mean difference, 14 microm; 95% confidence interval, 1-27 microm; P = .04); maximum retinal thickening (maximum thickening of the central and 4 inner subfields) decreased by 66 and 39 microm, respectively (adjusted mean difference, 27 microm; 95% confidence interval, 6-47 microm; P = .01), and retinal volume decreased by 0.8 and 0.4 mm3, respectively (adjusted mean difference, 0.3 mm3; 95% confidence interval, 0.02-0.53 mm3; P = .03). At 12 months, the mean change in visual acuity was 0 letters in the modified ETDRS group and 2 letters worse in the MMG group (adjusted mean difference, 2 letters; 95% confidence interval, -0.5 to 5 letters; P = .10). CONCLUSIONS: At 12 months after treatment, the MMG technique was less effective at reducing optical coherence tomography-measured retinal thickening than the more extensively evaluated current modified ETDRS laser photocoagulation approach. However, the visual acuity outcome with both approaches is not substantially different. Given these findings, a larger long-term trial of the MMG technique is not justified. APPLICATION TO CLINICAL PRACTICE: Modified ETDRS focal photocoagulation should continue to be a standard approach for treating diabetic macular edema. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00071773.

Squalamine lactate for exudative age-related macular degeneration.

Squalamine lactate inhibits angiogenesis by a long-lived, intracellular mechanism of action. The drug is taken up into activated endothelial cells through caveolae, small invaginations in the cellular membrane. Subsequently, the drug binds to and "chaperones" calmodulin to an intracellular membrane compartment and blocks angiogenesis at several levels. A series of basic investigations, preclinical studies, and human clinical trials have begun to establish the proof of concept, efficacy, and safety parameters for use of squalamine lactate as a therapeutic agent for exudative age-related macular degeneration and several types of malignancies.

Prevalence of disease-causing mutations in families with autosomal dominant retinitis pigmentosa: a screen of known genes in 200 families.

PURPOSE: To survey families with clinical evidence of autosomal dominant retinitis pigmentosa (adRP) for mutations in genes known to cause adRP. METHODS: Two hundred adRP families, drawn from a cohort of more than 400 potential families, were selected by analysis of pedigrees. Minimum criteria for inclusion in the adRP cohort included either evidence of at least three generations of affected individuals or two generations with evidence of male-to-male transmission. Probands from each family were screened for mutations in 13 genes known to cause adRP: CA4, CRX, FSCN2, IMPDH1, NRL, PRPF3 (RP18), PRPF8 (RP13), PRPF31 (RP11), RDS, RHO, ROM1, RP1, and RP9. Families without mutations in autosomal genes and in which an X-linked mode of inheritance could not be excluded were tested for mutations in ORF 15 of X-linked RPGR. Potentially pathogenic variants were evaluated based on a variety of genetic and computational criteria, to confirm or exclude pathogenicity. RESULTS: A total of 82 distinct, rare (nonpolymorphic) variants were detected among the genes tested. Of these, 57 are clearly pathogenic based on multiple criteria, 10 are probably pathogenic, and 15 are probably benign. In the cohort of 200 families, 94 (47%) have one of the clearly pathogenic variants and 10 (5%) have one of the probably pathogenic variants. One family (0.5%) has digenic RDS-ROM1 mutations. Two families (1%) have a pathogenic RPGR mutation, indicating that families with apparent autosomal transmission of RP may actually have X-linked genetic disease. Thus, 107 families (53.5%) have mutations in known genes, leaving 93 whose underlying cause is still unknown. CONCLUSIONS: Together, the known adRP genes account for retinal disease in approximately half of the families in this survey, mostly Americans of European origin. Among the adRP genes, IMPDH1, PRPF8, PRPF31, RDS, RHO, and RP1 each accounts for more than 2% of the total; CRX, PRPF3, and RPGR each accounts for roughly 1%. Disease-causing mutations were not found in CA4, FSCN2, NRL, or RP9. Because some mutations are frequent and some regions are more likely to harbor mutations than others, more than two thirds of the detected mutations can be found by screening less than 10% of the total gene sequences. Among the remaining families, mutations may lie in regions of known genes that were not tested, mutations may not be detectable by PCR-based sequencing, or other loci may be involved.

Retinal lesions as clues to disseminated bacterial and candidal infections: frequency, natural history, and etiology.

Invasive bacterial and candidal infections are known to involve the retina, but the natural history of the retinal lesions and the utility of ophthalmologic consultation in the critical care setting as a diagnostic tool are not well understood. We 1) performed weekly funduscopic examinations on 77 medical and surgical patients in intensive care units (ICUs), 2) analyzed results of serial ocular examinations in 180 non-neutropenic patients with candidemia, and 3) reviewed the English literature on the association of retinal lesions with disseminated bacterial or candidal infection (DBCI). We found that 15 (19%) of the ICU patients had retinal lesions consistent with DBCI. Of these 15, 1 had clearly sepsis-related retinal lesions, while 13 (87%) had 1 or more systemic disease that could have explained their retinal findings (6 diabetic retinopathy; 2 human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) retinopathy; 2 hypertensive retinopathy; 1 hemolytic uremic syndrome, and 1 leukemia). Multivariate analysis revealed that systemic disease (odds ratio 8.37, 95% confidence intervals: 3.24-21.56) independently correlated with the presence of retinal lesions while DBCI, trauma, hyperalimentation, and transfusion of blood products were not independently predictive in any analysis. Twenty of the 180 (15%) candidemic patients had retinal lesions. Two (1%) had classic 3-dimensional white lesions with vitreal extension, and 5 (2.7%) had chorioretinal lesions without vitreal haziness. Notably, 10% of patients had superficial retinal hemorrhages and/or cotton wool spots that could have been due to either candidemia or a systemic disease (diabetes, hypertension, renal failure, closed head trauma). Concurrent bacteremia occurred in 3 of the 27 patients with eye lesions. Retinal lesions resolved in a mean of 33 days. None of the patients had symptoms at the time of the retinal finding. We found 3 studies that prospectively assessed retinal lesions in bacteremic patients. The frequency of retinal lesions in these series varied from 12% to 26%, with the most common lesions being cotton wool spots followed by superficial retinal hemorrhages. White-centered hemorrhages were seen in about 15% +/- 2 of bacteremic patients. Five studies prospectively evaluated candidemic patients for Candida endophthalmitis. These studies observed rates from 0% to 78% for lesions consistent with candidal endophthalmitis. Most studies performed recently found that nonspecific lesions such as cotton wool spots or superficial retinal hemorrhages occurred with a frequency of 11% to 20%. The availability of less toxic antifungal agents, more frequent use of empirical therapy, and the trend to early treatment may be altering the frequency of this complication. Observation of a classic 3-dimensional retina-based vitreal inflammatory process is virtually diagnostic of endogenous endophthalmitis due to Candida spp., but such lesions are relatively uncommon. Conversely, nonspecific lesions that could be due to bacterial or candidal endophthalmitis (cotton wool spots, retinal hemorrhages, and Roth spots) are seen frequently. These lesions are most often due to an underlying systemic disease rather than an infection. Serial examinations provide the best evidence that a given lesion is due to an intercurrent infection. The current low rate of vitreal extension of retinal process appears to be due to the high rate of empirical or therapeutic use of antifungal agents in high-risk patient groups. Ophthalmoscopy should be performed in patients with known candidemia. However, ophthalmoscopic examination seems to have little value in assisting with the discovery of occult disseminated candidiasis or bacterial infection.