L Mendes-Jorge

Scavenger function of resident autofluorescent perivascular macrophages and their contribution to the maintenance of the blood-retinal barrier.

PURPOSE The retina contains two distinct populations of monocyte-derived cells: perivascular macrophages, and microglia. The present study was undertaken to evaluate the presence and function in mouse and human retinas of a subtype of resident perivascular macrophages with scavenger function, different from microglia, in physiological conditions and during retinopathy. METHODS Perivascular macrophages were characterized by means of confocal microscopy, electron microscopy, and flow cytometry analyses. Two murine models of blood-retinal barrier breakdown and photoreceptor degeneration were used to analyze the role of these macrophages during retinopathy. RESULTS The macrophages analyzed constituted a small population of resident perivascular cells different from microglia, since they were Iba-1 negative. Although these cells expressed F4/80 and CD11b antigens in common with microglia, they also expressed BM8 and MOMA-2 epitopes, which are macrophagic markers not expressed by microglia. Perivascular macrophages emitted autofluorescence due to cytoplasmic inclusions containing protein-bound oxidized lipids. They constitutively expressed the scavenger receptor class A and moved along blood vessels, providing an additional coating to thinner areas of the basement membrane. Moreover, they accumulated blood-borne horseradish peroxidase and acetylated low-density lipoprotein in healthy retinas. In addition, during blood-retinal barrier breakdown and photoreceptor degeneration, these cells migrated to the lesion site. CONCLUSIONS All these morphologic and functional features are consistent with those described for brain Mato cells. Thus, this study showed the presence of autofluorescent perivascular macrophages, different from microglia, with a scavenger function that may contribute to the maintenance of the blood-retinal barrier in healthy conditions and that are also involved in retinopathy.

Morphological characterization of pecteneal hyalocytes in the developing quail retina

The periphery of the vitreous body contains a population of cells termed hyalocytes. Despite the existence for more than one century of publications devoted to the pecten oculi, a convoluted coil of blood vessels that seems to be the primary source of nutrients for the avian avascular retina, little information can be found concerning the pecteneal hyalocytes. These cells are situated on the inner limiting membrane in close relationship with the convolute blood vessels. To characterize the origin and macrophagic activity of pecteneal hyalocytes, we have analysed two different stages of quail eye development using histochemistry and immunohistochemistry. Pecteneal hyalocytes express the QH1 epitope and cKit, confirming that these cells belong to the haematopoietic system. They also express vimentin, an intermediate filament protein present in cells of mesenchymal origin and very important for differentiation of fully active macrophages. However, similarly as described in porcine hyalocytes, pecteneal hyalocytes express the glial fibrillary acidic protein, a recognized neuroglial marker. Pecteneal hyalocytes did not express other neuroglial markers, such as glutamine synthetase or S100. Acidic phosphatase was activated and Lep100 was found in secondary lysosomes, confirming phagocytic activity of pecteneal hyalocytes during ocular development. Pecteneal hyalocytes strongly react with RCA‐I, WFA, WGA, PNA, SNA, LEA and SBA lectins, whereas other avian macrophages from thymus and the bursa of Fabricius did not bind PNA, SNA and LEA lectins. Interestingly, WGA lectin reacts with all kinds of avian macrophages, including pecteneal hyalocytes, probably reflecting the specific binding of WGA to components of the phagocytic and endocytic pathways. In conclusion, pecteneal hyalocytes are a special subtype of blood‐borne macrophages that express markers not specifically associated with the haematopoietic system.

L-Ferritin Binding to Scara5: A New Iron Traffic Pathway Potentially Implicated in Retinopathy

Iron is essential in the retina because the heme-containing enzyme guanylate cyclase modulates phototransduction in rods and cones. Transferrin endocytosis is the classical pathway for obtaining iron from the blood circulation in the retina. However, the iron storage protein ferritin has been also recently proposed as an iron carrier. In this study, the presence of Scara5 and its binding to L-ferritin was investigated in the retina. Our results showed that Scara5, the specific receptor for L-ferritin, was expressed in mouse and human retinas in many cell types, including endothelial cells. Furthermore, we showed that intravenously injected ferritin crossed the blood retinal barrier through L-ferritin binding to Scara5 in endothelial cells. Thus, suggesting the existence of a new pathway for iron delivery and trafficking in the retina. In a murine model of photoreceptor degeneration, Scara5 was downregulated, pointing out this receptor as a potential player implicated in retinopathy and also as a possible therapeutic target.

Imaging of cellular aging in human retinal blood vessels.

To date two main aging vascular lesions have been reported in elderly human retinas: acellular capillaries and microaneurysms. However, their exact mechanism of formation remains unclear. Using high resolution microscopy techniques we revise cellular alterations observed in aged human retinal vessels, such as lipofuscin accumulation, caveolae malfunction, blood basement membrane disruption and enhanced apoptosis that could trigger the development of these aging vascular lesions. Moreover, we have generated a set of original images comparing retinal vasculature between middle and old aged healthy humans to show in a comprehensive manner the main structural and ultrastructural alterations occurred during age in retinal blood vessels.

Intercapillary bridging cells: Immunocytochemical characteristics of cells that connect blood vessels in the retina

Intervascular bridges are fibrous strands that connect neighboring capillaries. These strands present associated cells, intervascular bridging cells (IBCs), whose nature and functional significance remains controversial. The aim of this study was to characterize the immunophenotype of IBCs, and contribute to understand their mechanical and intercellular communication properties in the retina. Quantification and retinal distribution of IBCs were also determined. For this purpose, C57BL/6N and nestin-GFP transgenic mice, as well as human retinas, were used. Whole-mount retinas were studied by means of immunohistochemistry and cytochemistry, and isolation of retinal vasculature was achieved by trypsin/pepsin digest technique. PAS reaction and the immunolabeling with anti-collagen IV and laminin antibodies revealed that IBCs were completely surrounded by a basement membrane, connecting two or more neighboring capillaries. IBCs were scarce and their number decreased with age. They were preferentially localized in the deep vascular plexus. In a murine model of experimental glaucoma, methylcellulose injected eyes showed retinal neovascularization and increased number of IBCs in the deep vascular plexus. IBCs were marked with anti-NG2, anti-PDGFR-β and anti-CD34 antibodies, and with tomato lectin, and were negative for PECAM-1. IBCs expressed nestin and filamentous actin, but desmin and α-smooth muscle actin were not detected. Moreover, these cells expressed the gap junction protein connexin 43. These results showed that IBCs had a pericytic nature since they expressed NG2 and the receptor for PDGF-B, and they were negative for PECAM-1. However, they were marked with CD34 and the tomato lectin, suggesting that they constitute a special subtype of pericytes, sharing characteristics with endothelial cells. IBCs presumably present mechanical functions due to the presence of filamentous actin. Connexin 43 was found in IBCs, suggesting that these cells allow intercellular communication between adjacent capillaries. This may represent an advantage for vasomotor tone integration and coordination in blood vessels without innervation, such as those of the retina.

Blood Vessel Basement Membrane Alterations in Human Retinal Microaneurysms During Aging

Purpose Microaneurysms, considered a hallmark of retinal vascular disease, are present in aged retinas. Here, the basement membrane of human retinal microaneurysms has been analyzed during aging. Methods Retinas were obtained from 17 nondiabetic donors. Whole mount retinas and paraffin sections were marked immunohistochemically with antibodies against the main components of the blood basement membrane. Trypsin digestion and transmission electron microscopy also were performed. Results Small microaneurysms presented increased expression of collagen IV, laminin, fibronectin, nidogen, and perlecan, along with basement membrane thickening. Unexpectedly, crosslinked fibrils of collagen III, a type of collagen absent in retinal capillaries, were found specifically in small microaneurysms. This was parallel to enhanced lysyl oxidase-like (LOXL) 2 and 4 expression. Large microaneurysms showed diminution of protein content, as well as disorganization, in their basement membrane. This was concomitant with an increased expression of matrix-metalloproteinase (MMP)-9 and plasminogen activator inhibitor (PAI)-1. Pericyte coverage declined between small and large microaneurysms. Conclusions Thickening of the basement membrane in small microaneurysms by accumulation of matrix proteins probably produced by recruited pericytes, together with the appearance of crosslinked collagen III fibrils probably due to the action of LOXL2 and LOXL4, could be considered as compensatory mechanisms to strengthen the vascular wall in the early phase of microaneurysm formation. Later, increased activity of MMP-9 and PAI-I, which produce disruption of the blood basement membrane and expansion of microthrombi respectively, and loss of pericytes, which produces weakening of the vascular wall, could explain the wall dilation observed in the late phase of microaneurysm formation.

The Use of Confocal Laser Microscopy to Analyze Mouse Retinal Blood Vessels

In the retina there is a compromise between transparency and optimal oxygenation [3]. Thus, retinal vasculature must show special characteristics in order to minimize their interference with the light path. Retinal capillaries are sparse and small [4], representing only 5% of the total retinal mass [5]. Hence, retinal blood volume is relatively low [6]. This feature, together with an extremely active cellular metabolism, 10% of resting body energy expenditure is consumed by retinal tissue [7], makes retina very susceptible to hypoxia.

Cellular senescence is associated with human retinal microaneurysm formation during aging

Purpose Microaneurysms are present in healthy old-age human retinas. However, to date, no age-related pathogenic mechanism has been implicated in their formation. Here, cellular senescence, a hallmark of aging and several age-related diseases, has been analyzed in the old-age human retina and in the retina of a progeric mouse. Methods Retinas were obtained from 17 nondiabetic donors and from mice deficient in Bmi1. Cellular senescence was analyzed by immunohistochemistry, senescent-associated β-galactosidase activity assay, Sudan black B staining, conventional transmission electron microscopy, and immunoelectronmicroscopy. Results Neurons, but not neuroglia, and blood vessels undergo cellular senescence in the old-age human retina. The canonical senescence markers p16, p53, and p21 were up-regulated and coexisted with apoptosis in old-age human microaneurysms. Senescent endothelial cells were discontinuously covered by fibronectin, and p16 colocalized with the β1 subunit of fibronectin receptor α5β1 integrin under the endothelial cellular membrane, suggesting anoikis as a mechanism involved in endothelial cell apoptosis. In a progeric mouse model deficient in Bmi1, where p21 was overexpressed, the retinal blood vessels displayed an aging phenotype characterized by enlarged caveolae and lipofuscin accumulation. Although mouse retina is not prone to develop microaneurysms, Bmi1-deficient mice presented abundant retinal microaneurysms. Conclusions Together, these results uncover cellular senescence as a player during the formation of microaneurysms in old-age human retinas.

Correction: L-Ferritin Binding to Scara5: A New Iron Traffic Pathway Potentially Implicated in Retinopathy

[This corrects the article DOI: 10.1371/journal.pone.0106974.].