Ординатура / Офтальмология / Английские материалы / Age-Related Changes of the Human Eye_Cavallotti, Cerulli_2008

density, shape, and size are distributed randomly in the cytoplasm of RPE, even among the lipofuscin granules.

Bruch’s Membrane Changes

Most of the mitochondria show focal loss of cristae, and a decrease in matrix density. Numerous peroxisomes and single membrane-bound, empty vacuoles can also be seen. In the upper part of the RPE, accumulation of lipofuscin and melanolipofuscin can be observed. The basement membrane of RPE is slightly thickened. Electrodens fibrillo-granular material and several small profiles of vacuoles can be seen in the Bruch’s membrane.

Age-related Changes in the Macular Region

The Drusen is located just beneath the RPE basement membrane, it contains electrodense granules, various forms of membrane-like structures, and amorphous material. A few electrodense granules can also be seen in the collagenous layers of the Brush’s membrane. The RPE cell contains some small mitochondria with normal appearance, but some of them show a seriously altered structure.

Basement Membrane of RPE

The basement membrane shows several focal, or worth-like thickenings with electrodense areas. The inner collagenous layer of Bruch’s membrane contains numerous profiles of vacuoles and patches of fibrillo-granular material. The elastic layer is interrupted, while the outer collagenous layer shows normal appearance in this place. Cytoplasm of the RPE contains numerous abnormal mitochon- dria—characterized by loss of cristae and translucent matrix. Several microsomes and peroxisomes can also be seen.

Thicking of Capillary Basement

Thickening of the capillary basement was due to the addition of mostly homogeneous material, a few fibrils, and electrodense granular material

Peroxisomal Contribution to Basement Membrane Thickening of RPE

Typical thickening of the Bruch’s membrane due to the addition of abnormal basement membrane material to the basement membrane of RPE. The inner collagenous layer of Bruch’s membrane contains numerous small profiles of vacuoles, and patches of

fibrillo-granular material. Almost all mitochondria of the RPE show focal electrontranslucent areas of the matrix and loss of cristae. Elastic layer, interposed between inner and outer collagenous layers, shows various thickness and electron density, as well as several interruptions. In a transversal section, the abnormal basement membrane forms numerous worth-like deposits in which cytoplasmic processes of the basal cell membrane can be seen. In an oblique section of the Brach’s membrane, numerous peroxisomes can be seen in the basal laminal deposits and in the RPE. Cytoplasmic processes of RPE form labyrinths between basal laminal deposits. Filamentary structure of abnormal basement membrane can be observed in longitudinal section and/or cross-section of abnormal basement membrane with exocytosis of vesicles.

Histochemical Composition of Bruch’s Membrane

PAS positive staining of Bruch’s membrane. In some places the pericapillary connective tissue also shows strong Anisotropy of the collagen fibrils in the Bruch’s membrane as shown by phenol reaction: basement membrane of the choriocapillaries shows more intensive anisotropy compared to RPE. Anisotropy of the lipids in the Bruch’s membrane and in the pericapillary wall are bright in the dark background. The basement membrane of RPE and choriocapillaries show intensive anisotropy after ABT reaction due to presence of vi.cinal OH groups in linear order.

Statistical Analysis of Anisotropy

Statistical analysis showed that aging and AMD affect basement membrane composition differently. The averaged values (in nm) of the anisotropy of collagen, GFs, and lipids are summarized in the following table, along with the statistical significance of comparisons between controls and patients with AMD (see Table 15.1). The relationships between age and the anisotropy of the ECM components were analyzed using the simple linear regression model. The estimated equations are reported in the following table in which the coefficients of determination R2 and the correlation coefficients r between age and the anisotropy of the observed substances are also indicated (see Table 15.2). Using the Fisher’s transformation, the correlation coefficients calculated in the two groups were compared. As concerns the anisotropy of

AMD Group (n = 26); CONTROL Group (n = 26).

AMD Group (n = 26); CONTROL Group (n = 26).

collagen and GFs, no statistical significance was found. On the other hand, the coefficients of correlation between lipids and age were found significantly different (P < 0.001) in the two groups—they were higher in the AMD group.

Discussion

Photoreceptor cells have a very high turnover rate, probably the highest among neuronal cells. It comprises a continuous shedding of the apical plasma membranes (discs) of the photoreceptors’ outer segment (POS) and their renewal in the cell body. Each RPE cell opposes around 24-44 POS, and 10-15 percent of each outer segment is renewed daily—thus, each RPE cell consumes and degrades about 20,004,000 discs per day.21 This phagocytosis is mediated by CD36 receptors of RPE that have developed adequate cellular systems to metabolize this enormous quantity of membranes.22 Lipid components of disc membranes may have two distinct metabolic fates—retinol and docosahexaenoic acids are recycled to photoreceptor cells through the interphotoreceptor matrix.23 The rest of the lipids are further catabolized by mitochondrial beta oxidation. Recent studies show that RPE cells may also uptake low-density lipoprotein (LDL) from the choriocapillaries using a similar receptor mediated pathway.24 These plasma lipids enter the POS turnover and are either used for the renewal of disc membranes or catabolized by mitochondrial beta oxidation.25 An accumulation of residual bodies or lipofuscin granules in the cytoplasm of RPE cells are common findings, and it is thought to be a sign of incomplete catabolism of POS disc membranes. Lipofuscin is a heterogeneous material composed of a mixture of lipids low-density lipoprotein (LDL) particularly lipid peroxides, proteins, and different fluorescent compounds derived mainly from vitamin A. Lipofuscin granules appear at the early26 age of 18 months, and until the age of 80, their amount increases approximately twenty fold.27 However, recent studies have shown that lipofuscin accumulation is not an end stage of failed lipid metabolism—instead, it is a manifestation of the current balance between production and elimination of lipid peroxides.28 There is a considerable body of evidence that well-established detoxifying mechanisms exist in RPE to eliminate lipid peroxides. Besides the common cytosolic antioxidant substances (glutathion, ascorbat, and alpha tocopherol), certain subcellular structures (mitochondria, peroxisomes, and melanosomes) and mechanism (exocytosis) serve to maintain RPE homeostasis.

First, accumulation of lipofuscin in RPE cells greatly reduces their phagocytic capacity in vitro.29. If this negative feedback works in vivo, it may be an important protective mechanism to prevent lipid overload of RPE. Accumulation of empty vacuoles in the Bruch’s membrane, which are cholesterols and cholesterol esters, may be a sign of arrested lipid uptake from choriocapillaries.9

Second, melanosomes of the RPE are connected to the phagolysosomal degradation pathway and are involved in many important functions, such as protection from photo-stress and detoxification of peroxides. The lipofuscin content of RPE seems to be inversely related to the melanin content, which suggests that melanin may have a protective role in preventing lipofuscin formation.

Third, through radiation and by directly scavenging free radicals,30 early histological studies show that RPE appears to discharge partially degraded membrane material from their basal region toward Bruch’s membrane and choriocapillaries. This mechanism seems to be part of the lipid transportation into the choriocapillaris, as shown by experimental studies.5,31 Finally, mitochondrial beta oxidation is considered to be the major pathway metabolizing fatty acids.32 High mitochondrial enzyme-activity was found in the RPE.33 Currently, no in vivo date on the saturation threshold of mitochondrial beta oxidation in RPE. However, the unmetabolized lipid peroxides certainly can compromise mitochondrial functions. Particularly inner membranes, where electron transport enzymes and mitochondrial DNA (mtDNA) are located, are sensible for oxidative damage.34 Lipofuscin granules of the RPE cells, which are toxic to these cells, probably act through mitochondrial damage, thus supporting a role for lipofuscin in aging and AMD.35 Our observations, the first time, showed morphological alterations of mitochondria in early AMD. Focal loss of cristae, associated with focal decrease of electron density of matrix, were common findings. Sometimes, more extensive alterations of cristae and matrix were seen, but no early apoptotic alterations (swelling, blebs of external membrane) were found. We considered these mitochondrial abnormalities, besides infraand extracellular accumulation of partially metabolized lipids, to be morphological manifestations of saturated lipid catabolism in RPE—due to lipid overload either from photoreceptor shedding or from the plasma.

Peroxisomes are small, round or oval single membrane-bound organelles, and are found in almost all eukaryota cells. Peroxisomes derive from endoplasmic reticulum, and their functions are related to lipid metabolism. They contribute to the beta oxidation of fatty acids. This pathway is essential for the catabolism of a variety of substances that are not oxidized by mitochondria. Peroxisomes are also involved in the metabolism of hydrogen peroxide. Finally, peroxisomes catalyze the initial steps in the biosynthesis of some lipids, such as plasmalogens. A peculiar characteristic of peroxisomes is their inducibility by a variety of compounds, which can activate a new member of the nuclear receptor superfamily called peroxisome proliferator-activated receptors (PPARs). The ligand-activated PPARs heterodimerize with the 9-cis retinoic acid receptor (RXR) and the complex bind-to-response elements located in the regulatory region of target genes. Intracellular elevation of naturally occurring fatty acids and eicosanoids activate PPARs, resulting in peroxisome proliferation and activation of lipid metabolism. PPAR alpha up-regulates genes of

lipid catabolism, while PPAR gamma up-regulates genes of lipogenesis and enhances intracellular lipid storage.36 Lipid peroxides, particularly oxidized LDL, can also activate37 PPAR gamma. Our observations confirmed the abundance of peroxisomes in the RPE in both normal aging and in AMD. The functional interpretation of these finding suggested that peroxisomes may be involved in the lipid turnover of POS. We suppose that presence of peroxisomes in RPE may be a morphologic manifestation of the activation of an alternative pathway for lipid degradation. This hypothesis is supported by observations that the induction of peroxisome proliferation is associated with a strong stimulation of the enzymes involved in peroxisomal beta oxidation.36 Recently, similar results were described in the RPE indicating peroxisomal contribution to POS turnover.38

Basal laminar deposits appear as diffuse or worth-like thickening of the basal membrane of RPE and choriocapillaries. In contrast to the normal basement membrane, these thickenings usually show fibrillary structure with focal electrodensity.2,39 Immunohisto-chemical studies show that BLD contains type IV collagen, heparan sulfate proteoglycans, and laminin and it is now considered to be an accumulation of an abnormal basement membrane.14 Our electron and polarization microscopic studies confirmed both fibrillary structures and the highly glycated nature of BLD, and we suggested that these structures be denominated as glycated fibrils(GFs) to distinguish from the normal basement membrane constituents, which usually do not show fibrillary appearance. In addition, polarization microscopy clearly showed a lipid layer just beneath the RPE and in the wall of choriocapillaries, suggesting that lipids are also added to the excessive basement membrane. Furthermore, these lipids, at least in part, are in organized form as structure lipids. Their hydrocarbon chains are oriented perpendicular to the length of basement membrane. Similar deposition of lipids were found in the adult human Descemet’s membrane, which is the basement membrane of the corneal endothelium.17 This phenomena was particularly evident at the periphery of aged corneas.40 All these findings justify an assumption that aging of the basement membrane is associated with deposition of structure lipids in it.

Our observations showed an unexpected topographic correlation between peroxisomes and basement membrane alterations in AMD. At the basal region of RPE cells, peroxisomes seem to fuse with basal plasma membranes and extrude their content into basement membrane. These peroxisome-derived materials showed a high degree of electron density similar to those of the lipid peroxides in the peroxisomes. It was recently demonstrated that Bruch’s membranes from white donors aged 40-78 years contain significant amounts of lipids and lipid peroxides.7,8 These authors suggested that lipid peroxides may accumulate within Bruch’s membrane by a combination of both discharge from RPE cells and from oxidized LDL coming from the plasma. Our observations showed a third possibility that lipid peroxides may also come from peroxisomes of the RPE cells. These observations suggested that proliferation of peroxisomes may also be associated with deposition of lipids or lipid peroxides in the ECM. This may be a new feature of peroxisome functions.

The colocalization of GFs and structure lipids remains an intriguing question. Two mechanisms may be involved in this procedure. There is accumulating evidence that intracellular lipid peroxides may also influence production of proteoglycans

and their extracellular deposition. High-cholesterol diets for rats resulted in thickening of the endothelial basement membrane of the choriocapillaries.41,42 Exposure of human arterial smooth muscle cells to linoleic acid increased 2-10 times the expression of mRNA for the core proteins of proteoglycans (versican, decorin, and syndecan) compared to control cells. Darglitazone—a PPARgamma ligand—neutralizes the linoleic acid induction of the decorin gene. This suggests that some of the linoleic acid effects are mediated by PPAR gamma.43 Intracellular accumulation of lipid peroxides may also enhance production of advanced glycation end products (AGE).44,45 Three stages can be distinguished during the glycation process: 1) nonenzymatic glycation of proteins (formation of Amadori product); 2) glycoxidation reactions; and 3) fibril formation of AGEs. Recent data46 suggests that lipid peroxides play an essential role in glycoxidation in aging, atherosclerosis, and diabetes . Biochemical and immunohisto-chemical studies show a linear age-dependent increase in AGEs in human Bruch’s membranes, basal membranes, and Drusen; in choroidal extracellular tissue in both diabetic and nondiabetic eyes, and in surgically removed subretinal membranes of patients with AMD.47,48,49 Vitronectin—a multifunctional glycoprotein and a major constituent of Bruch’ membrane thickenings—is synthesized by RPE.50 It is of interest that AGEs colocalizing with vitronectin were detected in diabetic retina,51 suggesting that GFs (seen in basement membranes) may also contain both glycoprotein and AGEs. However, colocalization of lipid peroxides and GFs, in the abnormal basement membrane of RPE and choriocapillaries, may represent a convergence between structure lipids and GFs synthesis—both of them are regulated by nuclear receptor activation and performed by endoplasmic reticulum-derived organelles. Whether the extracellular deposition of structure lipids and GFs represents a glycolipid and/or glycoxilipid formation, and whether these structures are formed intaor extracellularly, remains to be determined.

Polarization microscopy of Bruch’s membrane revealed that all basement membrane components (collagen, GFs, and lipid) increased with age in both control and AMD retinas. The anisotropy of each components was higher in AMD compared to the control group (see Table 15.1). The simple linear model is adequate to explain the dependence of anisotropy on age in both groups. Aging influences collagen and GFs in the same manner—in both AMD and the control group—while anisotropy of lipids in AMD increases at a much higher rate (see Table 15.2, correlation coefficient 0.98 vs 0172). It is of interest that the difference between aging and AMD was also significant for GFs, but over 75 years saturation or even decline of GF-induced anisotropy was found in AMD but not in the control group. Based on these observations, we may conclude that one of the significant differences between aging and AMD is manifested by more marked deposition of lipids and GFs in the ECM

Current concepts on aging and age-related diseases asssigned a primary role to mutation of mtDNA due to cumulative effect of reactive oxigen species.52 An impaired mitochondrial function results in further generation of reactive species and subsequent oxidative damage to the mitochondria itself and to other cellular or extracellular constituents. Besides reduced ATP generation, which affects almost all cell functions, activation of caspase cascade for cell death may take place.18

Taking our electron microscopic and polarization microscopic findings together, we suppose that mitochondria may be involved in the activation of an alternative pathway for ECM pathology. By-products of mitochondrial lipid metabolism (lipid peroxides, oxiLDL, and eicosanoids), in addition to the ortodox peroxisomal functions, may also activate peroxisomes to deposit abnormal basement membrane material. Thus, the peroxisomal contribution to basement membrane deposition may be one of the pathophysiologic links between mitochondrial and ECM pathology. Both are common in aging, in age-related diseases, and in several other diseases. Further studies are certainly needed to verify this hypothesis.

Conclusions

Morphological evidences suggested that a) mitochondria and peroxisomes of RPE contribute to the abnormal lipid metabolism of photoreceptor outer segments, and b) peroxisomes may be involved in the deposition of abnormal basement membrane material. Thus, extracellular matrix alterations, besides being due to entrapped abnormal metabolites (lipid peroxides, free radicals), may also come from activation of peroxisomes. Contribution of peroxisomes to the extracellular matrix formation, this new function of peroxisomes, may be a pathophysiologic link between mitochondrial and extracellular matrix abnormalities seen in aging, age-related neurodegenerative diseases, atherosclerosis, and diabetic microangiopathy.

Acknowledgements The authors thank Ida Bozso for her excellent technical assistance, and Livia Feher and Alessandro Mariani for their contribution to this chapter.

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