The role of O-linked sugars in determining the very low density lipoprotein receptor stability or release from the cell

AbstractThe very low density lipoprotein receptor is a member of the low density lipoprotein receptor supergene family for which two isoforms have been reported, one lacking and the other containing an O-linked sugar domain. In order to gain insight into their functionality, transient and stable transformants separately overexpressing previously cloned bovine variants were analyzed. We report evidence that the variant lacking the O-linked sugar domain presented a rapid cleavage from the cell and that a large amino-terminal very low density lipoprotein receptor fragment was released into the culture medium. As only minor proteolysis was involved in the other very low density lipoprotein receptor variant, the clustered O-linked sugar domain may be responsible for blocking the access to the protease-sensitive site(s). To test this hypothesis, a mutant Chinese hamster ovary cell line, ldlD, with a reversible defect in the protein O-glycosylation, was used. The instability of the O-linked sugar-deficient very low density lipoprotein receptor on the cell surface was comparable to that induced by the proteolysis of the variant lacking the O-linked sugar domain. Moreover, our data suggest that the O-linked sugar domain may also protect the very low density lipoprotein receptor against unspecific proteolysis. Taken together, these results indicate that the presence of the O-linked sugar domain may be required for the stable expression of the very low density lipoprotein receptor on the cell surface and its absence may be required for release of the receptor to the extracellular space. The exclusive expression of the variant lacking the O-linked sugar domain in the bovine aortic endothelium opens new perspectives in the physiological significance of the very low density lipoprotein receptor

Reduction of foveal bulges and other anatomical changes in fellow eyes of patients with unilateral idiopathic macular hole without vitreomacular pathologic changes

Purpose: To compare the foveal characteristics in fellow eyes (FE) of patients with unilateral idiopathic macular hole without vitreomacular pathologic changes with eyes of healthy controls. Methods: Forty-seven FE and 52 eyes of 52 age- and sex-matched healthy controls were studied. Quantitative assessment of the dome-shaped appearance of the hyperreflective lines that represent external limiting membrane (ELM_bulge) and inner outer segment junctions (IS/OS_bulge) were made by optical coherence tomography (OCT) images. Inner retinal complex thickness (IRCT) was quantitatively assessed at 1000 and 2000 µm of the foveal center in nasal and temporal quadrants. Presence of alterations in the inner retinal outer layers and central foveal thickness (CFT) were also analyzed. Results: Significantly lower ELM_bulge (p < 0.0001; Mann-Whitney test) and IS/OS_bulge (p < 0.001; student t test) and higher cases with COST alterations, expressed as a diffuse line (p < 0.006; Chi2 test) were found in FE than control eyes. IRCT were significantly reduced in FE at all the studied locations when comparing to control eyes (p < 0.05; student t test), maintaining anatomical proportionality among locations. Conclusion: FE without pathologic vitreomacular interactions seems to present some central cone alterations that may be related to other causes than vitreomacular traction.Preprin

Reduction of foveal bulges and other anatomical changes in fellow eyes of patients with unilateral idiopathic macular hole without vitreomacular pathologic changes

Purpose: To compare the foveal characteristics in fellow eyes (FE) of patients with unilateral idiopathic macular hole without vitreomacular pathologic changes with eyes of healthy controls. Methods: Forty-seven FE and 52 eyes of 52 age- and sex-matched healthy controls were studied. Quantitative assessment of the dome-shaped appearance of the hyperreflective lines that represent external limiting membrane (ELM_bulge) and inner outer segment junctions (IS/OS_bulge) were made by optical coherence tomography (OCT) images. Inner retinal complex thickness (IRCT) was quantitatively assessed at 1000 and 2000 µm of the foveal center in nasal and temporal quadrants. Presence of alterations in the inner retinal outer layers and central foveal thickness (CFT) were also analyzed. Results: Significantly lower ELM_bulge (p < 0.0001; Mann-Whitney test) and IS/OS_bulge (p < 0.001; student t test) and higher cases with COST alterations, expressed as a diffuse line (p < 0.006; Chi2 test) were found in FE than control eyes. IRCT were significantly reduced in FE at all the studied locations when comparing to control eyes (p < 0.05; student t test), maintaining anatomical proportionality among locations. Conclusion: FE without pathologic vitreomacular interactions seems to present some central cone alterations that may be related to other causes than vitreomacular traction.Preprin

Simultaneous retinal pigment epithelium tear and lamellar macular hole evolving to a full-thickness macular hole after intravitreal therapy

Retinal pigment epithelium (RPE) tear is an uncommoncomplication of neovascular age-related macular degenera-tion (nAMD) with an increasing risk in cases with pigmentepithelium detachment (PED) and may appear sponta-neously or after intravitreal therapy (IVT). Other uncommoncomplications of IVT related to nAMD are lamellar macularhole (LMH) and full-thickness macular hole (FTMH). Here, wepresent a patient with nAMD that developed a RPE tear asso-ciated with LMH that evolved into a FTMH two months laterafter IVT with ranibizumab (Lucentis®). Long-term follow-upwas registered

New applanation tonometer for myopic patients after laser refractive surgery

Ajuts: MI received a personal research grant from the Catalonian Ophthalmology Society 2016-2018 Barcelona, Spain. This society played no role in the design or conduct of this research.This study assesses the agreement between intraocular pressure (IOP) measurements taken with the Goldmann applanation tonometer (GAT) and a new experimental applanation tonometer with a convexly shaped apex (CT) after laser myopic refractive surgery. Two different CT radii (CT1 and CT2) were designed with a finite element analyser, and a prospective double masked study on 102 eyes from 102 patients was carried out. A Bland-Altman plot and intra-class correlation coefficient (ICC) were calculated to assess the agreement between GAT measurements and the measurements of both CT1 and CT2 before and after myopic laser assisted in situ keratomileusis (LASIK; n = 73) and photorefractive keratectomy (PRK; n = 29). We evaluated a subset of two subgroups (n = 36 each) for intra and inter-observer (IA/IE) error. From the whole cohort, the best IOP agreement was observed between GATpre and CT1post surgery: 16.09 ± 2.92 vs 16.42 ± 2.87 (p 0.8 (95% CI) in all cases. CT1 proved more accurate in the LASIK subgroup. In conclusion, our new version of GAT could be used with post-surgery LASIK patients as a more accurate measurement device compared to the current reference tonometer

Cord blood and amniotic membrane extract eye drop preparations display immune-suppressive and regenerative properties

long term consequences for their quality of life. Treatment modalities that can address the delicate balance of tissue regeneration, inflammation and maintenance of corneal transparency are therefore needed. We have recently formulated two novel eye drops from placental tissues: cord blood platelet lysate (CBED) and amniotic membrane extract eye drops (AMED), which can be used to treat severe ocular disorders. Here we characterise these two preparations by measuring: (a) growth factors (GF) and cytokines composition, (b) promotion of human corneal epithelial cell (HCEC) growth and (c) effects on immune cells in a lymphocyte culture assay. Finally, their bioavailability was assayed in an ex vivo porcine corneal model. We show that both preparations contain GF and cytokines that were able to promote the in vitro growth of HCEC and support repair in an in vitro scratch test. When assessed in a lymphocyte culture, both favoured immune suppression reducing the cellular expression of NKG2D and CD107a as well as the production of interferon gamma (IFN-γ) in natural killer, NKT and T cells. Regarding bioavailability, CBED active molecules were found mainly in the pre-corneal fraction with some penetration into the corneal fraction, in an ex vivo model. In summary, both placentalderived allogeneic preparations, CBED and AMED, display regenerative and immunomodulatory capabilities. These results will help define mechanisms of action and the best indications and doses of each product for use in a particular patient and support the development of off-the-shelf therapies for ocular surface pathologies in which wound healing defects and inflammatory events are contributing factors

Priming human adipose-derived mesenchymal stem cells for corneal surface regeneration.

Limbal stem cells (LSC) maintain the transparency of the corneal epithelium. Chemical burns lead the loss of LSC inducing an up-regulation of pro-inflammatory and pro-angiogenic factors, triggering corneal neovascularization and blindness. Adipose tissue-derived mesenchymal stem cells (AT-MSC) have shown promise in animal models to treat LSC deficiency (LSCD), but there are not studies showing their efficacy when primed with different media before transplantation. We cultured AT-MSC with standard medium and media used to culture LSC for clinical application. We demonstrated that different media changed the AT-MSC paracrine secretion showing different paracrine effector functions in an in vivo model of chemical burn and in response to a novel in vitro model of corneal inflammation by alkali induction. Treatment of LSCD with AT-MSC changed the angiogenic and inflammatory cytokine profile of mice corneas. AT-MSC cultured with the medium that improved their cytokine secretion, enhanced the anti-angiogenic and anti-inflammatory profile of the treated corneas. Those corneas also presented better outcome in terms of corneal transparency, neovascularization and histologic reconstruction. Priming human AT-MSC with LSC specific medium can potentiate their ability to improve corneal wound healing, decrease neovascularization and inflammation modulating paracrine effector functions in an in vivo optimized rat model of LSCD

Novel association of high C-reactive protein levels and A69S at risk alleles in wet age-related macular degeneration women

Purpose: To explore the relationship between plasma C-reactive protein (CRP) levels, the main ARMS2 gene single nucleotide polymorphism (SNP), and gender in patients with neovascular age-related macular degeneration (wet AMD). Methods: Our study included 131 patients with wetAMD [age-related eye disease study (AREDS) category 4] and 153 control participants (AREDS category 1) from two Spanish retinal units. CRP levels were determined on blood samples by high-sensitivity ELISA assay. According to their CRP level, subjects were categorized into three well-established CRP categories: low (3.00 mg/L, H-CRP). Genomic DNA was extracted from oral swabs using QIAcube (Qiagen, Hilden, Germany) and the A69S; rs10490924 of ARMS2 gene was genotyped by allelic discrimination with validated TaqMan assays (Applied Biosystems, Foster City, CA, USA). Univariate and multivariate logistic regression adjusted for age was used to analyze the genomic frequencies and to calculate odds ratio (OR) using SNPStats software. Results: Considering CRP risk categories, H-CRP group showed a significant [OR 4.0 (1.9-8.3)] association with wetAMD compared to L-CRP group. The risk genotypes of A69S (TT) SNPs showed an association with wetAMD risk [OR 14.0 (4.8-40.8)]. Interestingly, the gender stratification of the CRP categories showed a significant increase in CRP levels in wetAMD women compared with control women [OR 6.9 (2.2-22.3)] and with wetAMD men [OR 4.6 (1.3-16.9)]. In addition, the subgroup analysis of CRP within A69S genotype and gender showed a link in women between the A69S and CRP levels in the AMD group compared to controls [OR 4.2 (1.4-12.6)]. Conclusion: Our study shows, for the first time, that a different genetic association related with gender could contribute to AMD risk. As a consequence, the risk of female gender in the different CRP levels and A69S SNP frequencies could be taken into consideration to the established risk relationship of high levels of CRP and its association with risk A69S genotype

Age-Related Macular Degeneration: Clinical Findings, Histopathology and Imaging Techniques

Age-related macular degeneration (AMD) is the most common cause of blindness among people over age 55 years in industrialized countries. Known major risk factors for AMD include: age >55 years, history of smoking, white race, and mutations in various components of the complement system. Early AMD is characterized by the presence of drusen and pigmentary abnormalities. Late AMD is associated with central visual loss and is characterized by the presence of choroidal neovascularization and/or geographic atrophy. Early AMD is associated with a number of biochemical abnormalities including oxidative damage to retinal pigment epithelium (RPE) cells, complement deposition in the RPE-Bruch's membrane-choriocapillaris complex, lipidization of Bruch's membrane, and extracellular matrix abnormalities (e.g. collagen crosslinking, advanced glycation end product formation). Antiangiogenic drugs block the vascular leakage associated with choroidal new vessels, thus reducing retinal edema and stabilizing or restoring vision. At this time, there are no proven effective treatments for the nonexudative complications of AMD. Modern ocular imaging technologies (including spectral domain and phase variance optical coherence tomography, short- and long-wavelength fundus autofluorescence, adaptive optics-scanning laser ophthalmoscopy, and near-infrared reflectance) enable one to follow changes in the RPE, photoreceptors, and choriocapillaris quantitatively as the disease progresses. In addition, one can quantitatively assess the volume of drusen and areas of atrophy. These data, when correlated with the known histopathology of AMD, may provide useful measures of treatment efficacy that are likely to be more sensitive and reproducible than conventional end points such as visual acuity and rate of enlargement of geographic atrophy. As a result, these imaging technologies may be valuable in assessing the effects of cell-based therapy for patients with AMD

Scleral Fixation of Posteriorly Dislocated Intraocular Lenses by 23-Gauge Vitrectomy without Anterior Segment Approach

Background. To evaluate visual outcomes, corneal changes, intraocular lens (IOL) stability, and complications after repositioning posteriorly dislocated IOLs and sulcus fixation with polyester sutures. Design. Prospective consecutive case series. Setting. Institut Universitari Barraquer. Participants. 25 eyes of 25 patients with posteriorly dislocated IOL. Methods. The patients underwent 23-gauge vitrectomy via the sulcus to rescue dislocated IOLs and fix them to the scleral wall with a previously looped nonabsorbable polyester suture. Main Outcome Measures. Best corrected visual acuity (BCVA) LogMAR, corneal astigmatism, endothelial cell count, IOL stability, and postoperative complications. Results. Mean follow-up time was 18.8 ± 10.9 months. Mean surgery time was 33 ± 2 minutes. Mean BCVA improved from 0.30 ± 0.48 before surgery to 0.18 ± 0.60 (p = 0.015) at 1 month, which persisted to 12 months (0.18 ± 0.60). Neither corneal astigmatism nor endothelial cell count showed alterations 1 year after surgery. Complications included IOL subluxation in 1 eye (4%), vitreous hemorrhage in 2 eyes (8%), transient hypotony in 2 eyes (8%), and cystic macular edema in 1 eye (4%). No patients presented retinal detachment. Conclusion. This surgical technique proved successful in the management of dislocated IOL. Functional results were good and the complications were easily resolved