Degeneration Of Choriocapillaris Research Articles

The characterization of a new murine model of central areolar choroidal dystrophy showed that both cones and rods were affected by R195L mutation in peripherin‐2

Aims/Purpose: Central areolar choroidal dystrophy (CACD) is a rare hereditary disease that leads to progressive retinal and choriocapillaris degeneration and vision loss. In Spain, there is a family suffering this disease, since they carry the mutation p.Arg195Leu in PRPH2. Our team have generated a mouse model of CACD carrying the same mutation as the Spanish patients. Our aim was to characterize the progression of CACD in the retina structurally and morphologically in this new murine model.Methods: We studied the model C57BL/6J‐Prph2em1Sal by electroretinography, optomotor test, optical coherence tomography (OCT), immunofluorescence, confocal microscopy, and transmission electron microscopy (TEM) to assess the structural and morphological alteration of the retina during the progression of the disease in Prph2WT/WT, Prph2WT/KI, and Prph2KI/KI mice.Results: A progressive retinal degeneration was found affecting the retina of both Prph2WT/KI and Prph2KI/KI mice models. We observed a significantly decreased retinal function from 3 months of age in Prph2KI/KI and 6 months of age in Prph2WT/KI, that was correlated with a progressive degeneration of the outer nuclear layer (ONL) shown by OCT images. The Prph2KI/KI mice showed a degeneration of photoreceptors with a reduction in the ONL thickness from 3 months of age, and a severe decrease in the number of photoreceptor rows at 6 months of age. The Prph2WT/KI, however, showed a slower decrease in the ONL thickness, starting from 6 months of age. Additionally, we found unstructured and shortened photoreceptor outer segments as evidenced by immunofluorescence against peripherin, rhodopsin and cone‐arrestin. Moreover, TEM images confirmed an abnormal stacking of photoreceptor discs.Conclusions: The generated model is useful for the study of CACD. Moreover, the p.Arg195Leu mutation in PRPH2 causes the alteration of the outer segments first, triggering the degeneration of both cones and rods.

Bioprinted 3D outer retina barrier uncovers RPE-dependent choroidal phenotype in advanced macular degeneration.

Age-related macular degeneration (AMD), a leading cause of blindness, initiates in the outer-blood-retina-barrier (oBRB) formed by the retinal pigment epithelium (RPE), Bruch's membrane, and choriocapillaris. The mechanisms of AMD initiation and progression remain poorly understood owing to the lack of physiologically relevant human oBRB models. To this end, we engineered a native-like three-dimensional (3D) oBRB tissue (3D-oBRB) by bioprinting endothelial cells, pericytes, and fibroblasts on the basal side of a biodegradable scaffold and establishing an RPE monolayer on top. In this 3D-oBRB model, a fully-polarized RPE monolayer provides barrier resistance, induces choriocapillaris fenestration, and supports the formation of Bruch's-membrane-like structure by inducing changes in gene expression in cells of the choroid. Complement activation in the 3D-oBRB triggers dry AMD phenotypes (including subRPE lipid-rich deposits called drusen and choriocapillaris degeneration), and HIF-α stabilization or STAT3 overactivation induce choriocapillaris neovascularization and type-I wet AMD phenotype. The 3D-oBRB provides a physiologically relevant model to studying RPE-choriocapillaris interactions under healthy and diseased conditions.

Choroidal Modifications Preceding the Onset of Macular Neovascularization in Age-Related Macular Degeneration.

IntroductionMacular neovascularization (MNV) is a common complication of age-related macular degeneration (AMD). Although several biomarkers may help to estimate the risk of MNV onset, neovascular complication is difficult to predict. Previous studies showed that the quantitative assessment of choroidal and choriocapillaris changes is useful for the assessment of atrophy expansion. On the other hand, scant data are available regarding the role of this kind of assessment in the setting of MNV. The aim of the study is to analyze choroidal and choriocapillaris changes occurring before the onset of MNV in patients affected by AMD using quantitative optical coherence tomography (OCT) and OCT angiography (OCTA).MethodsThe study was designed as a retrospective case series. Patients affected by AMD, categorized in eyes complicated by MNV and eyes not developing MNV, were retrospectively analyzed for 1 year of follow-up. Choroidal thickness (CT), Sattler layer thickness (SLT) and Haller layer thickness (HLT) were measured on OCT scans. Vessel density (VD) and choriocapillaris (CC) porosity were quantified on OCTA reconstructions. The main outcome measure was the relationship between choroidal and CC parameters, and MNV onset.ResultsWe included 50 eyes of 50 AMD patients (28 male; mean age 74 ± 5 years). Over the 1-year follow-up, 15/50 eyes developed MNV (9 type 1; 3 type 2; 3 mixed type 1–2). Mean best-corrected visual acuity (BCVA) was 0.15 ± 0.15 logMAR at baseline, remaining stable in eyes not developing MNV (0.15 ± 0.12 logMAR; p > 0.05), and worsening to 0.38 ± 0.20 logMAR in eyes developing MNV (p < 0.01). VD values were similar between eyes developing MNV and eyes not complicated by MNV at baseline, with significant worsening detected only in MNV eyes. CC porosity was significantly higher in MNV eyes already before the onset of MNV. Furthermore, SLT was significantly lower in eyes developing MNV. The onset of MNV was preceded by a significant increase in intraretinal hyperreflective foci, whereas choroidal hyperreflective foci showed no evident changes.ConclusionsThe degeneration of CC and the SLT thinning represent early an biomarker of MNV onset in AMD.

Senescence in the pathogenesis of age-related macular degeneration.

Age-related macular degeneration (AMD) is a complex eye disease underlined by the death of photoreceptors and degeneration of retinal pigment epithelium (RPE) and choriocapillaris (CC). The mechanism(s) responsible for massive and progressive retinal degeneration is not completely known. Senescence, a state of permanent inhibition of cell growth, may be induced by many factors important for AMD pathogenesis and results in senescence-associated secretory phenotype (SASP) that releases growth factors, cytokines, chemokines, proteases and other molecules inducing inflammation and other AMD-related effects. These effects can be induced in the affected cell and neighboring cells, leading to progression of AMD phenotype. Senescent cells also release reactive oxygen species that increase SASP propagation. Many other pathways of senescence-related AMD pathogenesis, including autophagy, the cGAS-STING signaling, degeneration of CC by membrane attack complex, can be considered. A2E, a fluorophore present in lipofuscin, amyloid-beta peptide and humanin, a mitochondria-derived peptide, may link AMD with senescence. Further studies on senescence in AMD pathogenesis to check the possibility of opening a perspective of the use of drugs killing senescent cells (senolytics) and terminating SASP bystander effects (senostatics) might be beneficial for AMD that at present is an incurable disease.

TOXIC EFFECTS OF HYDROXYCHLOROQUINE ON THE CHOROID: Evidence From Multimodal Imaging.

To investigate the choroidal changes that occur in hydroxychloroquine (HCQ) retinopathy using multimodal imaging including fluorescein angiography, indocyanine green angiography, and optical coherence tomography (OCT) angiography and to correlate these changes with retinal findings obtained using OCT and fundus autofluorescence. In 20 patients (n = 40 eyes) with systemic lupus erythematosus or rheumatoid arthritis diagnosed to have HCQ retinopathy, imaging modalities including swept-source OCT, fundus autofluorescence, fluorescein angiography, indocyanine green angiography, and OCT angiography were used to evaluate retinal and choroidal changes associated with retinopathy. The assessments included specific findings such as presence of hyperfluorescent or hypofluorescent lesions on angiography and signal void zones on OCT angiography, their frequencies, and the correlations among the retinal and choroidal findings. These findings were also correlated with the severity of retinopathy. Retinopathy progression was defined using fundus autofluorescence and OCT and correlated with the retinal/choroidal findings. Fluorescein angiography demonstrated a hyperfluorescent area, which reflects a defective retinal pigment epithelium, with multiple tiny dark spots within the area. Indocyanine green angiography showed a hypofluorescent area with dark spots, which was matched to the hypoautofluorescent area on fundus autofluorescence. Although there were no specific morphologic abnormalities in the choroid layers using en face choroidal imaging, OCT angiography demonstrated signal void areas on the choriocapillaris in the areas of the retinal pigment epithelium defect. Even after cessation of HCQ, there was progression of retinopathy in eyes with choroidal involvement, particularly on the area of choroidal findings. Multimodal imaging demonstrates choriocapillaris degeneration in eyes with HCQ retinopathy, particularly those with severe retinopathy. The choroidal change was associated with outer retinal toxicity of HCQ.

An Acute Injury Model for the Phenotypic Characteristics of Geographic Atrophy.

PurposeGeographic atrophy (GA) is the late stage of non-neovascular age-related macular degeneration. A lack of animal models for GA has hampered treatment efforts. Presented herein is a rat model for GA using subretinal injection of sodium iodate (NaIO3).MethodsRats were given subretinal injections of NaIO3 (5 μg/μL) using a pico-injector. Fundus photographs and spectral domain optical coherent tomography scans were collected at 1, 3, 7, 14, and 28 days after injection, at which time rats were euthanized and eyes were enucleated. Eyes were either cryopreserved or dissected into retinal and choroidal flatmounts. Fluorescence immunohistochemistry was performed for retinal glial fibrillary acidic protein (activated Müller cells and astrocytes) and vimentin (Müller cells), as well as peanut agglutin lectin (photoreceptors) labeling. RPE/choroids were labeled for RPE65 and CD34. Images were collected on Zeiss confocal microscopes.ResultsFundus photos, spectral domain optical coherent tomography, and RPE65 staining revealed well-demarcated areas with focal loss of RPE and photoreceptors in NaIO3-treated rats. At 1 day after injection, RPE cells appeared normal. By 3 days, there was patchy RPE and photoreceptor loss in the injected area. RPE and photoreceptors were completely degenerated in the injected area by 7 days. A large subretinal glial membrane occupied the degenerated area. Choriocapillaris was highly attenuated in the injected area at 14 and 28 days.ConclusionsThe rat model reported herein mimics the photoreceptor cell loss, RPE atrophy, glial membrane formation, and choriocapillaris degeneration seen in GA. This model will be valuable for developing and testing drugs and progenitor cell regenerative therapies for GA.

Molecular genetics ‎characterization and homology modeling of the CHM gene mutation: A study on its association with choroideremia.

Choroideremia (CHM) is a rare form of X-linked chorioretinal dystrophy that is caused by mutations in the CHM gene. Mutations in the Rab escort protein-1 (REP-1), an ubiquitously encoded protein of the CHM gene, lead to prenylation and vesicle trafficking deficiency in the protein, resulting in the progressive degeneration of choriocapillaris, retinal pigment epithelium (RPE), and photoreceptors. Despite previous studies concerning this disease, no effective diagnostic tests or established therapeutic interventions currently exist for CHM. In this paper, we reviewed ‎the pathogenic ‎effects of synonymous hotspot mutation in the CHM gene and the genotypic-phenotypic associations in families with CHM. In addition, we employed a combination of molecular dynamics simulations and principal component analysis to gain insight into the underlying molecular basis of these deleterious and disease-causing hotspot mutation ‎analogs. These computer predictions provide strong evidence that the C > T nonsynonymous hotspot mutations of CHM spectrum contribute to overall RPE retinopathy. These findings increase our understanding of the CHM ‎pathogenesis, which may potentially define a new approach in developing novel symbiotic strategies for genetic diagnosis and specific treatment of inherited retinal diseases.

VEGF regulates local inhibitory complement proteins in the eye and kidney.

Outer retinal and renal glomerular functions rely on specialized vasculature maintained by VEGF that is produced by neighboring epithelial cells, the retinal pigment epithelium (RPE) and podocytes, respectively. Dysregulation of RPE- and podocyte-derived VEGF is associated with neovascularization in wet age-related macular degeneration (ARMD), choriocapillaris degeneration, and glomerular thrombotic microangiopathy (TMA). Since complement activation and genetic variants in inhibitory complement factor H (CFH) are also features of both ARMD and TMA, we hypothesized that VEGF and CFH interact. Here, we demonstrated that VEGF inhibition decreases local CFH and other complement regulators in the eye and kidney through reduced VEGFR2/PKC-α/CREB signaling. Patient podocytes and RPE cells carrying disease-associated CFH genetic variants had more alternative complement pathway deposits than controls. These deposits were increased by VEGF antagonism, a common wet ARMD treatment, suggesting that VEGF inhibition could reduce cellular complement regulatory capacity. VEGF antagonism also increased markers of endothelial cell activation, which was partially reduced by genetic complement inhibition. Together, these results suggest that VEGF protects the retinal and glomerular microvasculature, not only through VEGFR2-mediated vasculotrophism, but also through modulation of local complement proteins that could protect against complement-mediated damage. Though further study is warranted, these findings could be relevant for patients receiving VEGF antagonists.

Senescence Increases Choroidal Endothelial Stiffness and Susceptibility to Complement Injury: Implications for Choriocapillaris Loss in AMD.

Age-related macular degeneration (AMD) commonly causes blindness in the elderly. Yet, it is untreatable in the large fraction of all AMD patients that develop the early dry form. Dry AMD is marked by the deposition of membrane attack complex (MAC) on choriocapillaris (CC), which is implicated in CC degeneration and subsequent atrophy of overlying retinal pigment epithelium. Since MAC is also found on the CC of young eyes, here we investigated whether and how aging increases choroidal endothelial susceptibility to MAC injury. Monkey chorioretinal endothelial cells (ECs, RF/6A) were cultured to high passages (>P60) to achieve replicative senescence. We treated ECs with complement-competent human serum to promote MAC deposition and injury, which were assessed by flow cytometry and trypan blue exclusion assay, respectively. Stiffness of EC was measured by atomic force microscopy indentation while Rho GTPase activity was quantified by Rho G-LISA assay. Our findings reveal that senescent ECs are significantly stiffer than their normal counterparts, which correlates with higher cytoskeletal Rho activity in these cells and their greater susceptibility to complement (MAC) injury. Importantly, inhibition of Rho activity in senescent ECs significantly reduced cell stiffness and MAC-induced lysis. By revealing an important role of senescence-associated choroidal EC stiffening in complement injury, these findings implicate CC stiffening as an important determinant of age-related CC atrophy seen in dry AMD. Future studies are needed to validate these findings in appropriate animal models so new therapeutic targets can be identified for treatment of dry AMD.

Structural and molecular changes in the aging choroid: implications for age-related macular degeneration

Age-related macular degeneration (AMD) is a devastating disease-causing vision loss in millions of people around the world. In advanced stages of disease, death of photoreceptor cells, retinal pigment epithelial cells, and choroidal endothelial cells (CECs) are common. Loss of endothelial cells of the choriocapillaris is one of the earliest detectable events in AMD, and, because the outer retina relies on the choriocapillaris for metabolic support, this loss may be the trigger for progression to more advanced stages. Here we highlight evidence for loss of CECs, including changes to vascular density within the choriocapillaris, altered abundance of CEC markers, and changes to overall thickness of the choroid. Furthermore, we review the key components and functions of the choroid, as well as Bruch's membrane, both of which are vital for healthy vision. We discuss changes to the structure and molecular composition of these tissues, many of which develop with age and may contribute to AMD pathogenesis. For example, a crucial event that occurs in the aging choriocapillaris is accumulation of the membrane attack complex, which may result in complement-mediated CEC lysis, and may be a primary cause for AMD-associated choriocapillaris degeneration. The actions of elevated monomeric C-reactive protein in the choriocapillaris in at-risk individuals may also contribute to the inflammatory environment in the choroid and promote disease progression. Finally, we discuss the progress that has been made in the development of AMD therapies, with a focus on cell replacement.

Is the age-related macular degeneration (AMD) vascular disease, part of vasculopathy, respectively? Novel considerations on AMD arising from the newest pathophysiological, clinical and clinical- pharmacological observations (Preliminary Communication)

Is the age-related macular degeneration (AMD) vascular disease, part of vasculopathy, respectively? Novel considerations on AMD arising from the newest pathophysiological, clinical and clinical- pharmacological observations (Preliminary Communication)

Choroideremia: New Findings from Ocular Pathology and Review of Recent Literature

Histopathology of young individuals affected by choroideremia is rarely available to allow correlation with the clinical presentation. A 30-year-old man with choroideremia died in a motor vehicle accident and one eye was subjected to histopathological examination. Immunoblot analysis of protein derived from white blood cells of a living brother, also affected with choroideremia, confirmed the absence of Rab escort protein-1, the normal CHM gene product. Direct sequencing of the coding region and adjacent splice sites of the CHM gene was undertaken on genomic DNA from the living brother and revealed a transition mutation, C to T, in exon 6 (R253X) which resulted in a stop codon and was predicted to truncate the protein product. Histopathological examination of the eye of the deceased brother showed relative independent degeneration of choriocapillaris, retinal pigment epithelium, and retina, similar to observations in the mouse model of choroideremia. In addition, mild T-lymphocytic infiltration was found within the choroid. The ophthalmic features and the pathology of choroideremia are discussed in light of new findings in the current case.

Relationship between RPE and Choriocapillaris in Age-Related Macular Degeneration

The purpose of this study was to examine the relationships between choriocapillaris (CC) and retinal pigment epithelial changes in age-related macular degeneration (AMD). Morphologic changes in the retinal pigment epithelium (RPE)/choriocapillaris complex were quantified in dry and wet forms of AMD, and the results were compared with those in aged control eyes without maculopathy. Postmortem choroids from three aged control subjects, five subjects with geographic atrophy (GA), and three subjects with wet AMD were analyzed using a semiquantitative computer-assisted morphometric technique developed to measure the percentages of retinal pigment epithelial and CC areas in choroidal wholemounts incubated for alkaline phosphatase activity. The tissues were subsequently embedded in methacrylate and were sectioned so that structural changes could be examined. There was a linear relationship between the loss of RPE and CC in GA. A 50% reduction in vascular area was found in regions of complete retinal pigment epithelial atrophy. Extreme constriction of remaining viable capillaries was found in areas devoid of RPE. Adjacent to active choroidal neovascularization (CNV) in wet AMD, CC dropout was evident in the absence of retinal pigment epithelial atrophy, resulting in a 50% decrease in vascular area. Lumenal diameters of the remaining capillaries in wet AMD eyes were similar to those in control eyes. The primary insult in GA appears to be at the level of the RPE, and there is an intimate relationship between retinal pigment epithelial atrophy and secondary CC degeneration. CC degeneration occurs in the presence of viable RPE in wet AMD. The RPE in regions of vascular dropout are presumably hypoxic, which may result in an increase in VEGF production by the RPE and stimulation of CNV.

Morphological Changes of Retinal Pigment Epithelium and Choroid in rd-mice

The sequence of degenerative changes in the retinal pigment epithelium (RPE) and the choroid of retinal degeneration (rd)-mice was studied in correlation with photoreceptor changes. Three weeks to 26-month-old animals were investigated using light and transmission electron microscopy, enzyme histochemistry and quantitative morphology. Changes in the choriocapillaris (CC) were additionally studied by scanning electron microscopy of corrosion cast preparations. In 3-week-old mice, in which most of the outer segments of photoreceptors in the central portion of the retina had disappeared but remnants of the cells were still present, the RPE was enlarged and showed elongated microvilli. In 8-week-old animals, the photoreceptors were completely absent in large areas of the posterior pole region. In these areas the RPE was also completely lost. Quantitative evaluation performed in histological serial sections showed that loss of RPE measured as length of RPE-free Bruch's membrane, continuously increased up to the age of 20 months. In 8-week-old animals, CC adjacent to degenerating RPE showed loss of fenestration. In 10-week-old animals, the CC disappeared in those areas where the RPE was already lacking. The loss of CC increased with increasing age and in 20-month-old animals 5–10% of the entire CC was lacking. Loss of the related arterioles and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d)-positive nerve fibers occurred only in ∼2-year-old rd-mice. Compared to other animal models, RPE and CC defects in rd-mice are relatively large. The rd-mice might therefore provide a good tool to study factors involved in CC degeneration.

Choriocapillaris degeneration and related pathologic changes in human diabetic eyes.

To measure the extent of choriocapillaris degeneration (CCD) in diabetic choroids and to study the association of CCD with choroidal neovascularization and pathologic changes in Bruch's membrane-like basal laminar deposits. Human choroids from 10 postmortem subjects (diabetic, 5 [group 1]; nondiabetic, 5 [group 2]) were incubated for the histochemical demonstration of alkaline phosphatase and nonspecific esterase activities, permitting analysis of the choroidal vasculature and polymorphonuclear leukocytes, respectively. The tissue was then flat embedded and sectioned for structural analysis. Areas of CCD were measured in the flat perspective by computer-assisted image analysis and verified in cross-sections of flat-embedded tissue. The CCD in choroids from subjects with diabetes (group 1) appeared in 2 patterns: diffuse (partial loss of alkaline phosphatase activity in a poorly defined area, ie, degeneration of some capillary segments) and focal (complete degeneration of choriocapillaris or loss of alkaline phosphatase activity in a relatively well-defined area). The mean+/-SD percentage of the choroid with focal CCD in group 1 was 5.08%+/-1.13% of the total choroidal area vs 1. 16%+/-0.35% in group 2 (P<.001). Focal CCD in group 1 was more prominent in the posterior pole than in the peripheral choroid. Choroidal neovascularization was associated with some areas of diffuse CCD in group 1. Pathologic changes in Bruch's membrane-like basal laminar deposits were often associated with CCD; the thickness of the deposits was greater in group 1 than in group 2 and greater in areas with focal CCD than in areas with diffuse or no CCD. The percentage of choroid with focal CCD in group 1 choroids was more than 4-fold greater than that in nondiabetic choroids. The presence of CCD was related to basal laminar deposits and, in some cases, to choroidal neovascularization.

Decreased choriocapillaris perfusion following surgical excision of choroidal neovascular membranes in age-related macular degeneration

AIMS/BACKGROUNDTo evaluate macular changes following surgical excision of subfoveal choroidal neovascular membranes (CNVs) in age-related macular degeneration (AMD).METHODSThe clinical records, fluorescein angiograms, and CNV histopathology of 12 patients with AMD...