W. Gerald Robison

Effects of Chronic Systemic Administration of Basic Fibroblast Growth Factor on Collateral Development in the Canine Heart

BACKGROUND Recently we reported that intracoronary administration of basic fibroblast growth factor (bFGF), a potent angiogenic peptide, increases collateral blood flow in dogs subjected to progressive left circumflex coronary artery (LCx) occlusion. The aim of the present study was to examine the effect of systemically administered bFGF on collateral blood flow and to assess its pharmacokinetics and potential side effects. METHODS AND RESULTS Forty-seven dogs were subjected to progressive ameroid-induced occlusion of the LCx, an intervention known to induce the development of collateral vessels. In phase I of the investigation, dogs were randomized to receive bFGF 1.74 mg/d (n = 10) or saline (n = 9) as a left atrial injection for 4 weeks. Relative collateral blood flow was assessed serially with radiolabeled microspheres in the conscious state during maximal coronary vasodilatation. Initiation of bFGF treatment was temporally associated with a marked acceleration of collateral development; however, collateral flow in control dogs improved toward the end of the study, approaching that of bFGF-treated dogs at the 38-day end point. Phase II of the investigation was a three-armed study of extended duration to determine whether bFGF caused a sustained increase in collateral function. Dogs were randomized to receive bFGF 1.74 mg/d for 9 weeks (n = 7), bFGF 1.74 mg/d for 5 weeks followed by placebo for 4 weeks (n = 11), or placebo for 9 weeks (n = 10). Relative and absolute collateral blood flow were assessed serially with microspheres during maximal coronary vasodilatation. Between the 10th and 17th days after ameroid placement, bFGF-treated dogs exhibited marked improvement in collateral flow such that maximal collateral conductance exceeded that of controls by 24% at the 5-week crossover point. Final collateral conductance was similar in dogs receiving bFGF for 5 and 9 weeks despite withdrawal of treatment in the former group. bFGF administration was associated with a 21% increase in final collateral conductance as well as a 49% increase in collateral zone vascular density. Prolonged bFGF administration was also associated with a decrease in arterial pressure, moderate thrombocytopenia, and moderate, reversible anemia. CONCLUSIONS Systemic administration of bFGF enhanced collateral conductance in dogs with progressive single-vessel coronary occlusion. The beneficial effect of bFGF occurred primarily between the 7th and 14th days of therapy, and regression of collateral development was not noted after withdrawal of treatment. The present investigation provides impetus to the concept that collateral development can be enhanced pharmacologically-specifically by bFGF-raising the possibility that such an intervention might eventually be applied clinically.

Influence of early photoreceptor degeneration on lipofuscin in the retinal pigment epithelium

Experiments were conducted to evaluate the role played by photoreceptor cells in the accumulation of age pigment, or lipofuscin, in the retinal pigment epithelium (RPE). The age-related accumulation of RPE lipofuscin was compared between rats with hereditary photoreceptor degeneration (RDY) and congenic rats with normal retinas. In the RDY animals, the age-related increase in RPE lipofuscin content was substantially less than in normal controls. This suggests that the photoreceptor cells play a significant role in RPE lipofuscin deposition, although they may not be the sole contributors to RPE lipofuscin formation. Evidence that outer-segment components may be converted into lipofuscin fluorophores was provided by the discovery that in young RDY rats, fragments of outer segments from degenerating photoreceptor cells had fluorescence properties similar to those of RPE lipofuscin. Chloroform-methanol extraction of retina-RPE tissue from young normal and dystrophic rats, and analysis of the chloroform fractions by thin-layer chromatography, revealed three distinct fluorescent components associated with the lipofuscin-like fluorescence of the outer-segment fragments in the RDY rats.

Evidence of cell loss from the rat retina during senescence

In order to determine whether neuronal cell death in the retina accompanies senescence, retinas from pigmented ACI rats of various ages were analysed for changes in dimensions, cell densities, DNA content, and rhodopsin levels. Between the ages of 4 and 30 months, there were significant declines in the densities of nuclei in all three nuclear layers of the retina. Over this age span, nuclear density in the outer nuclear layer (ONL) declined by 32-41%, depending on the region of the retina examined, while inner nuclear layer (INL) nuclear density decreased by 25-34%. Over the same age range, the nuclear density in the ganglion cell layer (GCL) declined by 25-28%. The eye increased in size over the 4-30 month age span, particularly between 4 and 12 months. The retinal stretching that resulted from this growth could only partially account for the age-related decline in retinal cell densities. Depending on the region of the retina examined, decreases in nuclear density of 17-26% for the ONL, 9-17% for the INL, and 16-20% for the GCL were apparently due to cell death. Measurements of retinal DNA content support the conclusion that significant cell loss from the retina occurs during senescence; between 4 and 30 months of age retinal DNA content decreased by about 13%. Age-related changes in retinal rhodopsin content did not parallel apparent changes in photoreceptor number. Between 4 and 11 months of age, rhodopsin levels rose by about 10%, and then remained constant up to 26 months. Over this same age span, there was an apparent decrease in photoreceptor number of about 15%.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-glycated albumin therapy ameliorates early retinal microvascular pathology in db/db mice.

Hyperglycemia plays a primary causal role in the early vascular damage leading to diabetic retinopathy, but the intermediate biochemical mechanisms involved are not known. Because albumin modified by Amadori glucose adducts has been implicated in the pathogenesis of diabetic nephropathy, we investigated whether or not glycated albumin plays a similar role in diabetic retinopathy. We observed basement membrane thickening and an accumulation of basement membrane material in the capillaries of the outer plexiform layer of retinae from diabetic db/db mice compared with their nondiabetic db/m littermates. Both of these abnormalities were ameliorated by chronic (8 week) treatment with monoclonal antibodies that specifically recognize Amadori-modified glycated albumin (and not other glucose-modified or advanced glycation endproducts-modified proteins), despite the fact that the administration of these antibodies did not alter the glycemic status of the diabetic animals. Thus, albumin containing Amadori glucose adducts contributes to the pathogenesis of diabetic retinal vascular disease, and agents that neutralize or prevent the formation of excess glycated albumin in diabetes may offer prophylaxis against the early changes of diabetic retinopathy.

Age-related changes in the retinal pigment epithelium of pigmented rats

A number of significant age-related changes were found to occur in the retinal pigment epithelium (RPE) of normal pigmented ACI rats. Age-related morphological changes in the RPE included: changes in RPE cell height, an increase in lipofuscin content, alterations in the apical microvilli and basal infoldings, and an apparent thickening of the RPE basal lamina. Unusual banded structures were often seen within the basal infoldings of the RPE in senescent but not in young adult rats. Lipid droplets were relatively common in the RPE of senescent rats, but were quite rare in 4- and 11-month-old animals. In addition, contact between the RPE apical microvilli and the photoreceptor outer segments appeared to become less intimate with age. Phagocytosis of rod outer segment discs appeared to be reduced between 4 and 32 months of age. This reduction was greater than could be accounted for solely by a concomitant age-related decrease in the number of photoreceptors per unit retinal length. RPE-choroid acid phosphatase activity (a lysosomal marker), on the other hand, did not appear to change with age. The molar ratio of vitamin A-palmitate to vitamin A-stearate in the PRE-choroid rose significantly with age, from about 1.85 at both 4 and 11 months, to over 2.5 at 28 months. At each age, the palmitate- to stearate-ester ratio was the same in the dark-adapted and light-adapted eyes.

Lipofuscin autofluorescence: evidence for vitamin A involvement in the retina.

A lipofuscin-like autofluorescence develops in the degenerating photoreceptor cells of the RCS rat, a strain with inherited retinal dystrophy. In animals with normal retinas, age-related lipofuscin accumulation in the eye is restricted to the retinal pigment epithelium (RPE). Previous investigations have established that RPE lipofuscin accumulation in the normal rat retina can be reduced by dietary vitamin A deficiency. In order to determine whether the photoreceptor-derived fluorescence in the RCS rat retina is related to RPE lipofuscin fluorescence, the influence of dietary vitamin A on the fluorophore content of the RCS rat retina was studied. Vitamin A deficiency substantially reduced the autofluorescence associated with degenerating photoreceptor cells of the RCS rat retina. A specific vitamin A-dependent fluorophore was isolated from these retinas using thin-layer chromatography (TLC). The mobility of this fluorophore on TLC differs from that of the major age-dependent fluorophore isolated from the RPE of normal rats. Thus, if the vitamin A-dependent fluorophores of the photoreceptors and RPE are related, it appears that the fluorophore generated in the photoreceptor cells must undergo chemical modification once it has been taken up by the RPE. The fact that both the RPE- and photoreceptor-associated fluorophores are vitamin A-dependent suggests that such a relationship between them is likely. These experiments indicate that the RPE is somewhat different from other lipofuscin-accumulating tissues in that a major precursor of RPE lipofuscin fluorophores originates in another cell type and enters the RPE via phagocytosis.

What is lipofuscin? Defining characteristics and differentiation from other autofluorescent lysosomal storage bodies

Lipofuscins, also known as age-pigments, have three defining characteristics: (1) they consist of intracellular secondary lysosomes; (2) they have a yellow autofluorescent emission when excited by near ultraviolet or blue light; and (3) they accumulate during normal senescence. Lysosomal storage bodies with similar fluorescence properties accumulate in various cell types as a result of specific pathological conditions or experimental manipulations. As a class, the latter are often referred to as ceroid pigments. In general, the mechanisms involved in the formation of ceroid pigments cannot be assumed to be closely similar to those involved in lipofuscin formation. In fact, the mechanisms of formation almost certainly differ, not only between lipofuscins and ceroids, but also among different lipofuscins and different ceroids. Presently, the most detailed knowledge about the mechanisms involved in lipofuscin formation come from studies on the retinal pigment epithelium (RPE) of the eye. These studies indicate that at least the autofluorescent constituents of RPE lipofuscin are generated from derivatives of vitamin A that occur in the retina. Oxidative stress to the retina appears to promote the formation of these RPE fluorophores. Whether similar mechanisms are involved in the formation of the lipofuscins that occur in other tissues remains to be determined. The mechanisms involved in RPE lipofuscin fluorophore formation are closely related to metabolic pathways that are specific to the retina. Thus, it appears likely that the mechanisms by which lipofuscins form in other tissues differ fundamentally from those that underlie RPE lipofuscin formation.

Relationship between dietary retinol and lipofuscin in the retinal pigment epithelium

A variety of evidence suggests that autoxidation of cellular components probably plays a significant role in the age-related accumulation of lipofuscin, or age-pigment, in the mammalian retinal pigment epithelium (RPE). Among the likely candidates for conversion into RPE lipofuscin fluorophores via autoxidative mechanisms are vitamin A compounds, which are present in the retina and RPE in high concentrations. Vitamin E, an important lipid antioxidant, is likely to inhibit vitamin A autoxidation. Experiments were conducted to evaluate the significance of vitamin A autoxidation in the deposition of lipofuscin in the RPE. Albino rats were fed diets either supplemented with or lacking vitamin E. Each of these two groups of animals was further subdivided into three groups which were fed different levels of vitamin A palmitate: none, 14.0 mumol/kg diet, and 80.5 mumol/kg diet. After 26 weeks, the animals were killed and the RPE lipofuscin contents were determined by both fluorescence measurements and quantitative ultrastructural morphometry. Vitamin A palmitate deficiency led to significant reductions in RPE lipofuscin deposition, relative to the amounts of this pigment present in the groups receiving vitamin A palmitate in their diets. The relative magnitude of the vitamin A effect was greater in the vitamin E-supplemented groups than in the groups fed the diets deficient in vitamin E. This finding suggests that vitamin E interacts with vitamin A ester metabolites in vivo in a more complex manner than simply acting as an antioxidant protectant. Rats fed the diets containing the higher level of vitamin A palmitate failed to display elevated RPE lipofuscin contents relative to those in the rats fed 14.0 mumol of vitamin A palmitate/kg diet. Failure of high vitamin A intake to enhance RPE lipofuscin deposition may have been due to the fact that intake of vitamin A above normal levels did not lead to an elevation in vitamin A content of the retinal tissue. Establishing an effect of vitamin A deficiency on RPE lipofuscin deposition and characterization of the interactions between vitamins E and A are important steps toward defining precisely the molecular and cellular mechanisms underlying age-pigment accumulation in the RPE.

The roles of vitamin E and unsaturated fatty acids in the visual process.

Relatively high proportions of long-chain, polyunsaturated fatty acids seem to be required in rod photoreceptor membranes in order to provide the precise microenvironment for the proper function of the visual pigment rhodopsin. At the same time, such high levels of lipid unsaturation put the photoreceptor membranes at a high risk for autoxidation. The antioxidant vitamin E which can minimize autoxidation of polyunsaturated fatty acids is found in rather high concentrations in the outer segment membranes. Dietary deficiency in vitamin E induces disintegration of rod outer segment membranes, probably by increasing autoxidation. Also, it greatly accelerates the accumulation of aging pigments in the retinal pigment epithelium, probably because these lipofuscin granules do indeed represent the end products of lipid peroxidation. Vitamin E supplements, up to threefold normal levels, appear to provide no significant protection of the retina from light damage produced either by short but acute or by long-term, low level exposures to light. This is not consistent with current theories which implicate lipid peroxidation in the destruction of rod outer segments in light damaged retinas; more work is needed before any relation between retinal light damage and vitamin E levels can be assessed. Surprisingly, the amount of lipofuscin granule accumulation in the retinal pigment epithelium is influenced dramatically by dietary levels of vitamin A. Even retinas lacking a source of polyunsaturated fatty acids from rod outer segments still may accumulate massive lipofuscin if dietary vitamin A is provided. Perhaps vitamin A, which has such a dynamic relationship with the retinal pigment epithelium, becomes oxidized, and then contributes to the formation of a lipofuscin-like pigment. Centrophenoxine, a drug claimed to be effective in reversing the accumulation of age-related lipofuscin in the central nervous system, has no obvious effect in the eye or uterus in removing the lipofuscin granules induced by vitamin E deficiency. Microperoxisomes are abundant in the retinal pigment epithelium, and may be associated with rapid lipid turnover and/or utilization of lipid soluble vitamins. Their potential roles, however, need further documentation and clarification. Recently developed techniques and new discoveries in lipid research open the way for many fruitful studies on the interactions and precise roles of lipids and lipid-soluble vitamins in vision.

The role of lens epithelium in sugar cataract formation

Previous evidence has shown clearly that sugar cataract formation results from unusually high intracellular levels of polyol. Documentation of polyol-related histological changes in the cortical fiber cells of the equatorial zone has been extensive. However, little attention has been given to the early changes in the lens epithelial cells, in spite of the fact that the highest level of aldose reductase is found in this layer of the lens. Also, cultured lens epithelial cells exposed to high sugar levels exhibit rapid accumulation of polyol and show ultrastructural alterations. Therefore, a study was designed to evaluate the role of the lens epithelium in sugar cataract formation. Specifically, an attempt was made to localize the earliest fine structural lesions in intact lenses of galactose-fed rats and to test their relation to aldose reductase. Rats were fed either a normal diet or a 50% galactose diet with or without sorbinil, an aldose reductase inhibitor. Rats were killed at varying periods of time ranging from 6 to 96 hr, and the eyes were processed for light and electron microscopy. The first detectable abnormalities occurred after 36 hr of galactose feeding, and were limited to the central lens epithelium. Cell edema, apparent dilution of cytoplasm, rounding of nuclei, aberrant intracellular vacuoles, and loss of normal tortuosity of cell boundaries were the salient lesions. No changes were detectable in the equatorial zone until 48 hr, and no deviation from the control structure was found in any of the rats treated with an aldose reductase inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)