V Nacher

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.

Carbohydrate characterization of quail primordial germ cells during migration and gonadal differentiation

A selection of lectins were used to study changes in the distribution of sugar residues in primordial germ cells (PGCs) during gonadal colonization and differentiation. Although the cytochemical characteristics of PGCs have been described in the chick, little is known about such characteristics in other avian species of interest to experimental biology. Therefore, we studied embryos of Japanese quail (Coturnix coturnix japonica) by light and laser confocal scanning microscopy, using the QH1 antibody as a tool to identify PGCs in both sexes and at all stages. LEA, WGA and RCA‐I bound to PGCs in a similar way to QH1. LEA was the best marker for all stages. The presence of acid phosphatase and the intense reaction of LEA, WGA, RCA‐I and WFA at the cell surface were shown to be a useful tool in the study of the migratory PGCs of the quail. Quails were sexed histologically in younger embryos than in chick; results were confirmed by PCR. The lectin‐binding pattern changed drastically in differentiated gonads. Cell surface reactivity was almost entirely lost. Quail differentiating oogonia showed a characteristic binding pattern to QH1 and to the lectins WGA, RCA‐I and WFA. Binding was observed in intense spots in the Golgi complex, and there was a specific PNA reaction. These results suggest that some selective sugar binding sites on the PGCs play a significant role in their migration, colonization and maturation.

β-Catenin expression during vascular development and degeneration of avian mesonephros

β‐Catenin is a structural component of adherens junctions, a regulator of the Wnt signalling pathway and a transcriptional co‐activator with a key role in vascular patterning. The avian mesonephros is a transitory embryonic kidney that is used in the study of vascular development and degeneration. Here we examine β‐catenin expression in this model during vascular development and degeneration. Quail embryos with developing or degenerating mesonephros were studied, on day 6 (30HH) or day 11 of incubation (40HH), respectively. QH1 whole mounts of developing mesonephros revealed numerous angioblast‐like cells situated in the paramesonephric duct that seem to invade the mesonephros. Although these cells did not express β‐catenin, the surrounding periductal mesenchymal cells translocated high levels of β‐catenin into the nucleus. In contrast, degenerating mesonephros were devoid of angioblast‐like cells and β‐catenin was lower than in the developing mesonephros. β‐Catenin was significantly reduced in the glomerular capillary tuffs, indicating that it was particularly down‐regulated in the vascular system. No sex‐related differences in β‐catenin expression were observed in degenerating mesonephros. Furthermore, two special populations of glomerular and peritubular endothelial cells were observed in degenerating mesonephros: one translocating β‐catenin into the nucleus and the other in apoptosis that did not translocate it. In conclusion, our results indicate that the paramesonephric duct is a potential new vasculogenetic pathway, and suggest that β‐catenin plays a role in the fate of mesonephric endothelial cells.

Cold ischaemia, innate immunity and deterioration of the glomerular filtration barrier in antibody-mediated acute rejection

BACKGROUND In renal transplantation, cold ischaemia (CI) determines acute rejection through innate immunity among others. Acute rejection episodes are a risk factor for late allograft dysfunction and proteinuria. This implies some alteration of the glomerular filtration barrier (GFB). Besides its effects on acute rejection, we hypothesized that CI might somehow damage the GFB being directly responsible for late proteinuria. METHODS On rat kidney allografts suffering from antibody-mediated acute rejection with or without CI and compared with syngeneic grafts, we quantified the gene expression of innate and adaptive immune mediators and assessed the capillary glomerular basement membranes (CapBM) by immunostaining collagen-IV (ColIV). ColIV was also assessed in equivalent groups from a previous chronic study followed up for 24 weeks. RESULTS CI up-regulated enzymes critical in the stabilization of collagen chains, increasing ColIV deposition and thickening the CapBM. CI increased the C4d and IgG deposits within grafts, amplified innate immunity (heat shock protein 70, fibronectin, Toll-like-receptor-4 and MyD88) and synergized with alloreactivity in triggering adaptive response through CD40. CONCLUSIONS Initial CI increased the ColIV deposition in CapBM, damaging the GFB and being responsible for part of the proteinuria associated with late allograft dysfunction. This deterioration of the GFB is related to the early innate immunity activation and subsequent up-regulation of CD40 in acute rejected grafts. In chronic rejected allografts, thickened CapBM may be a consequence of an unresolved immune-inflammatory response worsened by CI.

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.

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.

Endothelial Cell Transduction in Primary Cultures from Regressing Mesonephros

Loss of renal function during normal aging is associated with vascular alterations. Consequently, new therapeutic approaches, including gene therapy, to protect renal endothelial cells are expected to be greatly beneficial. Quail mesonephros is a transitory embryonic kidney that has been used for the study of vascular development and involution. Vascular alterations in regressing mesonephros are similar to those observed in aging kidney. In the present study, we examined adenovirus-mediated gene transfer to endothelial cells in primary cultures from developing and regressing quail mesonephros. Quail embryos with developing and regressing mesonephros were examined on day 6 (30HH) and day 11 (40HH) of incubation, respectively. The senescence markers, associated β-galactosidase activity and p16INK4a, were examined in whole mesonephros. Quail embryos were injected intracardiacally with adenoviral vectors (rAd-CMV-LacZ) and endothelial cell transduction examined. In addition, primary cell cultures from mesonephros were exposed to adenoviral vectors. Endothelial cells in primary cultures were identified as QH1(+), LEP100(–) and acidic phosphatase(–) cells and adenovirus-transduced cells were those positive for bacterial-associated β-galactosidase activity. We report that endothelial cells in the whole regressing mesonephros and primary cell cultures expressed senescence markers. In addition, we observed that adenoviral vectors were able to transduce endothelial cells in the whole regressing mesonephros, and that cultured endothelial and macrophagic cells from the regressing mesonephros were more efficiently transduced than those derived from the developing mesonephros. Our results suggest that quail mesonephros provides a practical model to assay gene transfer to endothelial cells in regressing/senescent vessels.