|Molecular Vision 2004;
Received 4 February 2004 | Accepted 31 March 2004 | Published 2 April 2004
In vivo passage of albumin from the aqueous humor into the lens
Judith R. Sabah,1 Harriet Davidson,2 Eric N.
McConkey,1 Larry Takemoto1
1Division of Biology and 2Department of Clinical Science, Kansas State University, Manhattan, KS
Correspondence to: Larry Takemoto, Ph.D., Division of Biology, Kansas State University, Manhattan, KS, 66506; Phone: (785) 532-6811; FAX: (785) 532-6799; email: email@example.com
Purpose: To determine if albumin, the major protein component of the aqueous humor, passes into the lens in vivo.
Methods: Rat albumin was covalently-labeled with Alexa 488 fluorophore, purified by gel permeation chromatography, then injected into the aqueous chamber of living rats. At 5 min postinjection, lenses were removed and analyzed by HPLC gel permeation chromatography, confocal microscopy, and immunogold electron microscopy.
Results: At 5 min postinjection, HPLC analysis detected measurable amounts of Alexa-labeled albumin in the lens. The results were confirmed by confocal microscopy, which showed passage into epithelial and cortical fiber cells. Immunogold electron microscopy using antibody to the Alexa fluorophore demonstrated intracellular location of the Alexa-albumin complex.
Conclusions: In vivo, significant amounts of albumin pass from the aqueous chamber into cells of the lens, consistent with a possible physiological role for this process involving passage of metabolites into the lens.
As one of the most abundant protein components of the blood, albumin contains high affinity binding sites for many cellular metabolites, including long chain fatty acids, vitamins, hormones, and metal ions . Perhaps the most important of these metabolites are the long chain fatty acids, which are used by cells for both lipid biosynthesis and energy production. It is estimated that over 99% of the long chain fatty acids in blood are bound to albumin . In most mammalian tissues, delivery of the bound metabolites to cells involves passage of albumin from the capillary lumen through the endothelium, into the interstitial space where albumin releases its bound metabolites to cells in a poorly understood mechanism.
In the eye, albumin is also the major protein component of the aqueous humor , which is a filtrate of the plasma and bathes the anterior side of the lens. The serum albumin in this ocular fluid is thought to originate from the blood, after passage through the blood-ocular barrier located primarily in the ciliary body and/or iris root . Serum albumin in the aqueous chamber has been estimated to represent 50% of the total protein of the aqueous humor . Since the anterior side of the lens contains a single layer of metabolically active epithelial cells, it is possible that direct passage of albumin complexes from the aqueous humor through the capsule and into the lens epithelium is very important for proper growth and differentiation of these cells . In addition to epithelial cells, newly differentiated fiber cells also possess significant metabolic activities that would benefit from intralenticular passage of metabolites such as long chain fatty acids, vitamins, hormones, and metal ions.
Besides albumin, the aqueous humor contains many other proteins of physiological importance. Transferrin, an iron binding protein, is thought to act as an anti-oxidant and survival factor in the lens [6,7]. Other growth factors present in lower amounts include fibroblast growth factor , transforming growth factor beta , and SPARC (secreted protein, acidic and rich in cysteine) . Using a similar mechanism as albumin, all these components of the aqueous humor could potentially pass into the lens, where they could have important, if not necessary, roles in the metabolism of cells.
Unlike most tissues of the body, the lens lacks a vascular system, and must rely upon direct passage of metabolites from the aqueous humor into the lens epithelium and multiple layers of fiber cells. While it is clear that small molecular weight components such as amino acids, monosaccharides, and metal ions can readily pass through the capsule and throughout the lens interior using a system of transporters, ion pumps, and gap junctions (for a review, see ), information about the possible entry and intralenticular passage of large molecular weight components of physiological importance such as albumin is completely unknown. In a previous report , we showed that large molecular weight proteins such as the lens crystallins could indeed pass into the intact rat lens in culture. After initial passage into the lens epithelium, these macromolecules could pass from fiber cell to fiber cell in the interior of the lens. This finding suggested that the lens possesses a robust system for the internalization and intralenticular passage of large proteins of physiological importance.
In the following report, we show for the first time that in the living animal, fluorescently-labeled albumin does indeed pass from the aqueous humor into the lens, in a manner consistent with a possible physiological role for this protein involving delivery of metabolites to cells of the lens.
Labeling of albumin
Rat serum albumin (4 mg, Sigma Chemical Co., St. Louis, MO) was covalently coupled with 1.0 mg of the tetrafluorophenyl ester of Alexa 488 (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. The Alexa-albumin complex was purified using a TSK 3000SW column (7.8x300 mm, P. J. Colvert Associates, St. Louis, MO) in buffer containing 0.1 M sodium sulfate and 0.06 M sodium phosphate, pH 7.0, using a flow rate of 1.0 ml/min. The peak corresponding to the monomeric form of rat serum albumin was collected, dialyzed against distilled water, and then lyophilized. The lyophilized powder was redissolved in PBS (0.15 M sodium chloride and 0.010 M sodium phosphate, pH 7.4) and protein concentration determined , using bovine serum albumin as standard. The nature of the monomeric form of Alexa-albumin was ascertained by observing that Alexa-albumin eluted at the exact same time as unlabeled, monomeric albumin (data not shown).
Animals and injection
Male rats (4-6 weeks of age, CD IGS) were purchased from Charles River Laboratories (Wilmington, MD). Animals were anesthetized by a single intraperitoneal injection of Telazol at 40 mg/kg body weight. Once a level plane anesthesia was reached as assessed by monitoring respiration and toe pinch, the animal was placed in lateral recumbancy, and the eye placed under an operating microscope. A 30-gauge needle attached to a 5 μl syringe (Hamilton Company, Reno, NV) was used to inject 3 μl containing 12.1 μg/μl of purified Alexa-albumin in PBS. Injection involved insertion of the needle at the limbus, followed by advancement of the needle into the anterior chamber. All procedures involving anesthesia and injection were done by H. Davidson, who is a board-certified veterinary ophthalmologist. The animal was euthanized 5 min postinjection by carbon dioxide inhalation, and the lens removed from the posterior of the eye. Visualization of the lens using a dissecting microscope indicated no lens opacity or damage to the lens capsule. All procedures were approved by the Institutional Animal Care and Use Committee of Kansas State University and followed the guidelines of the Institute for Laboratory Animal Research.
Quantitation of albumin in the lens
Following its removal, the lens was rinsed twice in 2.0 ml PBS to reduce the risk of proteins adhering to the lens surface. The lens was not decapsulated to avoid possible loss of some or the entire epithelial layer, which we wanted to retain. The lens was homogenized in 1.0 ml of PBS, and then centrifuged at 10,000x g for 10 min in a refrigerated microfuge (Model 5402, Brinkman Instruments Inc., Westbury, NY). Supernatant (25 μl) was injected into an HPLC system attached to a TSK 3000SW column (7.8x300 mm, P. J. Colbert Associates) and eluted at 1.0 ml/min using a buffer containing 0.1 M sodium sulfate and 0.06 M sodium phosphate, pH 7.0. Elution was monitored using a Shimadzu RF-10AXL fluorimeter, with excitation and emission at 495 nm/519 nm, respectively.
Immediately after removal from the eye, the lens was immersed in 4.0 ml of 4% (w/v) freshly prepared paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in PBS. After fixation for 48 h in the dark at room temperature, a vibratome (OTS 4000, Electron Microscopy Sciences) was used to cut 200 μm thick midsagittal sections, parallel to the optical axis. The sections were mounted in glycerol, and viewed using a Nikon Model C1 Eclipse confocal scanning system, equipped with an argon laser and attached to a Nikon TE 2000 inverted microscope. Images were captured using the EZ-C1 software provided by the manufacturer.
Immunogold transmission electron microscopy
Upon removal, lenses were fixed overnight at 4 °C in freshly prepared 4% (w/v) paraformaldehyde (Electron Microscopy Sciences), 4% (w/v) sucrose in 0.1 M sodium cacodylate buffer (pH 7.4). Then lenses were washed several times first with 0.1 M sodium cacodylate buffer at 4 °C and then with distilled water at room temperature. Next, dehydration was started in a 50% (v/v) ethanol solution for 15 min at room temperature under constant rotation (RT, CR). Subsequently, 3 dehydration steps were performed in solutions containing 70% (v/v) ethanol and LR White resin (London Resin Co., Reading, UK) at the following ratios of 2:1 and 1:1, for 2 h each. Next, lenses were infiltrated overnight (RT, CR) using 100% LR White resin. The following day the LR White was replaced twice with fresh resin for 1 h each time (RT, CR). Finally, lenses were placed in fresh LR White resin and polymerized at 45 °C for 24 h and at 60 °C for another 24 h. Sections were cut with a Reichter-Jung Ultracut E ultramicrotome. Semi-thick sections (0.5 μm) of the entire lens were obtained using a diamond knife, stained with 1% (w/v) toluidine blue, and viewed using an optical microscope to orientate the tissue and prepare ulthrathin sections of regions of interest. Ultrathin sections (85-90 nanometers) were cut using a diamond ultraknife, and placed on 200 mesh nickel grids (Ted Pella, Redding, CA). Grids were then prepared for immunolabeling. The whole immunolabeling process was performed in a humidified chamber. Lens sections were blocked with 1% (w/v) bovine serum albumin (BSA) in PBS for 30 min. The primary antibody was an anti-Alexa 488 antibody made in rabbit (Nanoprobe, Yaplanck, NY) at dilution 1:100 in 1% (w/v) BSA in PBS. Grids were floated on drops containing the primary antibody for 1 h. Then, the grids were washed 3 times for 3 min in PBS. The secondary antibody, a goat anti-rabbit IgG labeled with 1.4 nanometer gold particles (Nanoprobe, Yaplanck, NY), was prepared at a dilution of 1:100 in 1% (w/v) BSA in PBS. Grids were incubated on drops containing the secondary antibody for 1 h. Then the grids were washed 3 times for 5 min in PBS, and 3 times for 5 min in distilled water. The 1.4 nanometer gold particles were enlarged using the manufacturer's enhancement procedure (Nanoprobe). Grids were stained with 2% (w/v) uranyl acetate and 2% (w/v) lead citrate prior to visualization using a Hitachi H-300 electron microscope. Control grids were made by omitting the primary antibody, and by immunolabeling sections from lenses of eyes that had not been treated with Alexa-albumin.
Figure 1 shows confocal scanning microscopy of a midsagittal section, cut parallel to the optical axis, from an animal injected with Alexa-rat albumin. The short, 5 min postinjection time was chosen to minimize drainage of the labeled protein from the aqueous chamber before its possible passage into the lens. After injection into the aqueous chamber (Figure 1A), Alexa-albumin passed into the lens, and was found predominantly at the anterior side of the lens. Injection of an equal volume of PBS containing no Alexa-albumin showed no detectable fluorescence (Figure 1B). The boxed image in Figure 1A at higher magnification (Figure 1C) showed that the Alexa-albumin definitely entered the lens on the anterior side, to a depth of at least 60-100 μm. Since the total thickness of the capsule plus epithelium in this region of the rat lens is approximately 25-30 μm (W.-K. Loo, personal communication), the results clearly showed that Alexa-albumin had penetrated into the epithelium and fiber cells of the cortex.
To ascertain passage into the lens, as well as to quantitate the amount of Alexa-albumin that passed into the lens, the lens was homogenized, and after centrifugation the supernatant fraction was analyzed by an HPLC gel permeation column attached to a fluorimeter. The elution profile in Figure 2 shows a major peak of fluorescence at 9.3 min (solid arrow), which corresponds to the elution time of the monomeric form of rat albumin. Also present was a minor peak eluting with the void volume (4.9 min, open arrow), suggesting that a small percentage of the internalized albumin aggregated with itself and/or with other lens components. A standard curve of Alexa-albumin was derived from chromatography of known amounts of Alexa-albumin. For each known amount, a peak eluted at about 9 min. The area under the peak was weighted and plotted on semi-log paper, with the x axis representing the concentration of albumin, and the y axis representing the weight of the area of the corresponding peak. We obtained a linear relationship, which allowed us to calculate the amount of albumin internalized into the lens by weighing the area of the 9.3 min peak. We found that 0.79% of total injected albumin was internalized into the lens at 5 min postinjection.
To determine the localization of Alexa-albumin relative to epithelial and fiber cells in more detail, thin sections of the lens were probed with antibody to the Alexa 488 fluorophore, followed by treatment with secondary antibody linked to gold particles. Figure 3 shows an image containing the capsule, epithelium, and cortical fiber cells. In the epithelium and fiber cell regions, enhanced gold particles of approximately 5-30 nm diameter were uniformly distributed throughout the intracellular space of the capsule, epithelium, and cortical fiber cells, strongly suggesting that passage of Alexa-albumin into the lens occurs at least in large part via intracellular passage through epithelial and cortical fiber cells.
Although serum albumin plays an important role in delivery of long chain fatty acids, vitamins, and hormones to cells in many tissues of the body, its possible role in the physiology of the lens has yet to be determined. Since serum albumin is the major component of the aqueous humor, and since the aqueous humor is thought to provide important metabolites to be used by the rapidly dividing lens epithelium, it is possible that serum albumin in the aqueous humor may provide the same functional role in the lens as it does in other parts of the body.
As a first step in defining a possible role in lens metabolism, it is necessary to demonstrate that albumin passes into the lens, where in a mechanism analogous to other tissues of the body, the albumin molecule can release its bound metabolite for use by the cell. Although it has been thought for many years that large molecular weight proteins such as albumin could not pass into the lens, recently we have shown that large proteins such as native α-crystallin (oligomeric molecular weight 800,000) can readily pass into the cultured rat lens, followed by internalization into both epithelial and fiber cells . Once internalized into lens cells, passage of large macromolecules from fiber cell to fiber cell in the interior of the lens is consistent with studies showing cell-cell passage of GFP-tagged proteins deep within the nucleus of the chick lens .
Consistent with a possible physiological role in the lens, in this report we have found that Alexa-labeled rat albumin can pass into the lens of the living animal. Based upon the results of Figure 1, even after a relatively short period of 5 min postinjection, Alexa-albumin was present in both the epithelium and cortical fiber cells of the lens to a depth of at least 60 μm. As expected following injection into the aqueous chamber, most of Alexa-albumin was present at the anterior side of the lens. Detectable amounts were also present at the posterior end. Passage of Alexa-albumin towards the posterior end of the lens could have arisen by initial entry at the equatorial regions, followed by extracellular passage within the lens as seen after intravitreal injection of the marker protein horseradish peroxidase . Alternatively, some of the Alexa-albumin injected into the aqueous chamber could have passed into the vitreous chamber, in a retrograde direction from normal aqueous flow, followed by direct internalization into fiber cells at the posterior side of the lens.
Lenses were analyzed 5 min postinjection, to minimize the drainage of Alexa-albumin injected into the aqueous chamber. The only study of protein drainage from the aqueous chamber of the rat eye reported a value of 2.23% of total protein cleared per min . Hence the actual value of albumin uptake into the lens is probably larger than that reported in Figure 2, since some of the injected albumin should have already been drained before it could enter the lens.
Based upon the results of Figure 3, which showed uniform distribution of enhanced gold particles throughout the epithelial cells and fiber cells, a major route of albumin passage into the lens involves intracellular passage. This conclusion is consistent with a previous report using confocal microscopy, which characterized the passage of lens crystallins into rat lenses in culture . In contrast, studies using the marker protein horseradish peroxidase have shown that passage of this protein into the rat lens occurs predominantly by an extracellular route, although intercellular localization was also observed [15,17,18]. The reasons for the differences in localization between the two proteins are not presently known, but could be related to the known mechanism of albumin internalization into cells of other tissues such as the capillary endothelium, which involves receptor mediated internalization into caveolae and transcytosis across the cell into the interstitial space [1,19].
As an avascular tissue, lens cells in the interior of the lens must rely upon passage of metabolites through multiple layers of cells. For low molecular weight metabolites, passage can be facilitated via a well-characterized system of gap junctions . For large molecular weight proteins such as albumin, the mechanism of passage is completely unknown. Based upon the results of this report, as well as characterization of albumin passage in other tissues, it is possible that passage of this physiologically important macromolecule from the aqueous humor into the lens, followed by passage from lens cell to lens cell, could at least in part involve a transcytotic route of passage that would facilitate delivery of important metabolites known to be transported by this protein. In this manner, it would be possible for cells in the lens interior to obtain components such as long chain fatty acids, vitamins, and hormones necessary for proper metabolism and differentiation.
We wish to acknowledge K. Albin for assistance in the animal injections, and B. Fenwick and L. Willard for their assistance in the immunogold transmission electron microscopy. This study was supported by a grant from the National Eye Institute to LT.
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