American Journal ofPathology, Vol. 136, No.

6, June 1990 Copyright American Association ofPathologists

High Glucose Causes an Increase in Extracellular Matrix Proteins in Cultured Mesangial Cells

Suzanne H. Ayo, Robert A. Radnik, Jo Ann Garoni, William F. Glass 11, and Jeffrey 1. Kreisberg
From the Departments of Pathology and Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, Texas

Diabetic nephropathy is a major cause of the increased morbidity and mortality in insulin-dependent diabetes mellitus. The most significant renal lesion of diabetic nephropathy is expansion of the glomerular mesangium. Thickening of the glomerular basement membrance is also apparent. Mesangial expansion is largely due to the accumulation of extracellular matrix (ECM) proteins such as fibronectin, laminin, and type IV collagen. To determine whether high glucose is responsible for the observed increase in mesangial cell ECM protein accumulation, mesangial cells were grown in tissue culture medium containing 10 mmol/l (millimolar) glucose (normal) or 30 mmol/l glucose (high). The degree of ECM protein accumulation was determined by immunocytochemistry and a solid-phase enzyme-linked immunosorbent assay (ELISA) developed in the laboratory. Mesangial cells cultured for I week contained fibronectin as the most abundant ECM protein, followed by laminin and type IVcollagen. Type IVcollagen was seen only after the cells had piled up into 'hillocks' (approximately 4 weeks of continuous growth without passaging). After 4 weeks in 30 mmol/l glucose, mesangial cells contained increased amounts ofall three matrix proteins. Fibronectin and laminin were increased by approximately 60%, while type IV collagen was increased 50%. Cells subcultured in medium containing 30 mmol/l glucose for 8 months displayed a twofold increase infibronectin and laminin. Thus, high glucose per se can cause changes in mesangial cell ECM. This cell culture model should be useful in elucidating the mechanisms involved. (Am J Pathol 1990, 136:1339-

Diabetes mellitus is a major cause of renal morbidity and mortality.1 Nephropathy in diabetes mellitus follows a period of clinical silence in which only subtle functional alterations can be found. After 15 to 20 years, overt proteinuria, hypertension, and declining glomerular filtration rate (GFR) can be measured in approximately 25 to 40% of patients with insulin-dependent diabetes mellitus.2.3 Semiquantitative light microscopic analysis of renal biopsies demonstrated a correlation between the severity of glomerulosclerosis and loss of renal function.4 Detailed morphologic analysis by Mauer et al5 showed a precise correlation between the severity of mesangial expansion and the clinical manifestations of diabetic nephropathy. The glomerulus undergoes significant structural changes in diabetic nephropathy. The intercapillary glomerulosclerosis or Kimmelstiel-Wilson nodules composed of increased extracellular matrix (ECM) material in the mesangium is virtually pathognomic of diabetes.6 Diabetic humans have increased mesangial staining for fibronectin, laminin, and type IV and V collagens.10 Thus, diabetic glomerulosclerosis appears to be characterized by apparent accumulations of glomerular matrix proteins. Although strong evidence exists in animals and man that the glomerulopathy is related to the diabetic state,11'12 the precise mechanisms of diabetic glomerulosclerosis remain unknown. Because of the insidious course of this disease, the many metabolic alterations, and the lack of suitable animal models, in vivo studies to elucidate the mechanisms of this disease are difficult. With homogeneous cultures, the environment of the mesangial cell, which is involved in ECM turnover, can be precisely controlled. Thus, by manipulating the growth conditions, one can study individual metabolic parameters that might be involved in the pathogenesis of diabetic glomerulosclerosis. In this study we have shown that high glucose per se can cause mesSupported by USPHS, NIH DK 29787 (JIK), the Clayton Foundation (JIK), and the National Kidney Foundation (SHA). Dr. Kreisberg is a Career Scientist, Veterans Administration. Accepted for publication January 30, 1990.
Address reprint requests to Jeffrey-1. Kreisberg, PhD, Department of Pathology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7750.

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angial cells to accumulate increased amounts of ECM proteins. This should be a useful model to study the mechanisms involved in this disease process.

the range reported for streptozotocin diabetic rats (6

.UU/Ml).16
DNA and Cellular Protein Determinations

Materials and Methods Reagents

RPMI 1640, fetal calf serum (FCS), Hank's balanced salt solution, antibiotic, and antimycotic agent were from Irvine Scientific (Santa Ana, CA). Trypsin-EDTA (ethylenediaminetetraacetic acid), rabbit anti-laminin, and HEPES were from GIBCO Laboratories (Grand Island, NY). Fibronectin, laminin, rabbit anti-goat IgG, goat and rabbit peroxidase-anti-peroxidase (PAP), 3',3'-diaminobenzidine, 4-6diamidino-2-phenylindole, and DNA were from Sigma Chemical Co. (St. Louis, MO). Goat anti-type IV collagen was from Southern Biotechnology Assoc., Inc. (Birmingham, AL). Type IV collagen was from Nuitta Gelatindo, Ltd. (St. Petersburg, FL). Rabbit anti-fibronectin and goat anti-rabbit IgG was from Cappel (West Chester, PA). HRP color development reagent was from Bio-Rad Laboratories (Richmond, CA). Cell Culture

Mesangial cells were plated in 6-well multiwell (35-mm) dishes for 1, 2, and 4 weeks. The media was aspirated and the cells were rinsed 3 times in ice-cold phosphatebuffered saline (PBS) (0.02 mol/l [molar], pH 7.3). Cells were scraped with 1 ml of cold PBS 4 times. The cell suspension was sonicated for 8 to 10 seconds while in an ice bath. One hundred microliters of the samples and DNA standards were pipetted in 2.5 ml of DAPI (4-6-diamidino2-phenylindole) reagent and vortexed immediately.17 All incubations were done in the dark at 250C for 20 minutes minimum and 120 minutes maximum. Relative fluorescence was measured in a fluorescence spectrophotometer at 360 nm excitation and 450 nm emission. For protein determinations, cells were extracted in 2.5% sodium dodecyl sulfate sample buffer and proteins quantified by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL).18

Immunocytochemistry
Mesangial cells were grown in chamber slides (4-well) (Lab-Tek, Naperville, IL) under varying conditions and stained by the peroxidase anti-peroxidase method of Sternberger.'9 They were first rinsed three times in 20 mmol/l PBS, pH 7.5 and then fixed with 3.7% paraformaldehyde for 10 minutes at 250C. After fixation, they were rinsed three times with PBS and permeabilized with 0.01% Triton for 3 minutes. Cells were preincubated with normal blocking serum, which was prepared from the species in which the secondary antibody was made, washed, and incubated for 1 hour at 370C with primary antibodies. These antibodies were used at the dilution of 1:500 to 1 :1000 in PBS. Our antibodies to fibronectin, laminin, and type IV collagen were obtained commercially. Although the manufacturer claims specificity, we tested each of the antisera by Western blot and dot blot immunoassay. We confirmed the manufacturer's claims that each antiserum was specific. Unbound antibody was aspirated and the cells were rinsed three times with PBS. Cells were incubated with a 1:25 dilution of appropriate secondary antibody (either goat anti-rabbit or rabbit anti-goat) for 1 hour at 370C and rinsed three times with PBS. Next, cells were incubated with appropriate peroxidase anti-peroxidase complex (prepared in rabbit or goat) for 1 hour at 370C and washed three times with PBS. They then were stained with 3'3-diaminobenzidine (0.3 ug/ml) and 0.005% H202 in 50 mmol/I TRIS-HCI (pH 7.5) for 5 minutes at 250C and rinsed three times with PBS. The cells were counterstained with Harris hematoxylin for 30 seconds,

Isolation and Culture of Glomerular Mesangial Cells

Glomeruli were isolated from 200-g male SpragueDawley rats (Harlan Sprague-Dawley, Houston, TX) using a graded-sieve technique and were plated for culture in RPMI 1640 tissue culture medium with 20% FCS plus penicillin, streptomycin, and Fungizone (E. R. Squibb & Sons, Princeton, NJ) for explant growth of glomerular cells.13'14 Positive identification of mesangial cells was obtained by ultrastructural examination and shape change in response to cyclic adenosine monophosphate (cAMP)-elevating agents.'5 Mesangial cells were grown for varying periods under normal (10 mmol/I [millimolar]) and high (30 mmol/l) glucose conditions. The osmolality of the two types of media were within two standard deviations as measured by the freeze point method (10 mmol/l glucose = 277 + 8.1 mOsm/kg H20; 30 mmol/l glucose = 287 11.4 mOsm/ kg H20). Although 10 mmol/I glucose is high compared with normal blood levels of glucose (5 mmol/1), this is the amount necessary for mesangial cells to grow well in culture (J. 1. Kreisberg, personal observation). Also, because tissue culture medium contains 20% FCS, the possibility exists that there could be a considerable amount of insulin present. However, when the insulin concentration was measured by radioimmunoassay (RIA) (Smith Kline BioScience Laboratories, Houston, TX), we found it to be in

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rinsed, allowed to air dry, and coverslipped using Permount (Fisher Scientific, New York, NY).

lose because the inclusion of up to 10 jg of bovine serum albumin with the protein standard does not effect the results.

Dot Blot Immunoassay (Solid Phase ELISA)

Mesangial cells were plated in 6-well multiwell (35-mm) dishes (Corning, Corning, NY) for 1, 2, and 4 weeks. The media was aspirated and the cells were rinsed three times with Hank's balanced salt solution with Ca", Mg++ (HBSS++). Solubilization of whole cell matrix proteins was accomplished by a procedure developed by Madri et al.20 Briefly, 2 mol/l urea in 20 mmol/l TRIS, pH 7.5, was added, and the cells were scraped and allowed to agitate overnight at 40C. Samples were vortexed and microfuged for 1 minute. To be certain we were recovering proteins with 2 mol/l urea, we coated culture dishes with all three ECM proteins, extracted with 2 mol/l urea, and measured our recovery by the dot blot immunoassay. We were able to recover 100% of the added proteins. The supernatant and matrix protein standards were diluted in 2 mol/l urea in TRIS before application to nitrocellulose membranes (0.2 ,um pore, Hoefer Scientific Instruments, San Francisco, CA). The membranes were pre-wetted in TRISbuffered saline (TBS) (20 mmol/l TRIS, 500 mmol/l NaCI, pH 7.5), and assembled with the apparatus (Bio-Rad, Richmond, CA). Samples (100 to 300 1l) were applied to the wells with vacuum filtration and the wells were rinsed with TBS. The membrane was removed, rinsed in TBS, and incubated for 1 hour at 25C with 5% nonfat dry milk in TBS and gentle rocking. The milk solution was removed and replaced by the appropriate primary antibody diluted into the 5% milk-TBS solution (1:500 to 1 :1000) and incubated for 1 hour at 25C with rocking. The unbound antibody was removed and the membranes washed three times with TBS. The blots were stained by the peroxidase anti-peroxidase method. Appropriate secondary antibodies (1:50) and PAP (1:400) were each diluted in 5% milkTBS and incubated for 30 minutes at 25C with gentle rocking. Blots were rinsed three times with TBS between each application. Blots were developed in TBS:methanol (5:1) containing 0.15% H202 and 0.05% 4-chloro-1-naphthol until the lowest standard became faintly visible (approximately 2 minutes). Standards ranging from 1 to 100 ng were applied in quadruplicate. Relative absorbencies were determined using an LKB 2202 ultrascan laser densitometer with an HP 3390A integrator in peak height mode. Each experiment has its own standard curve. Standard data from laser-scanning densitometry was fitted to linear and second- and third-order polynomial equations. The data is linear from 1 to 10 ng. Beyond this range there is a nonlinear increase in absorbance with increased antigen. However, application of a polynomial equation allows useful information to be obtained. The observed nonlinearity is not the result of saturation of the nitrocellu-

Results
To test the effect of glucose on mesangial ECM protein accumulation, we plated cells onto chamber slides and grew them for 1, 2, and 4 weeks, without passaging, in RPMI 1640 tissue culture medium containing 20% FCS with either 10 mmol/l or 30 mmol/l glucose. Although 10 mmol/l glucose is above plasma values (5 mmol/I), this is the amount necessary to sustain mesangial cell growth. The insulin concentration in the medium was tested by RIA (Smith Kline Bio-Science Laboratories, Houston, TX) and found to be in the range reported for steptozotocin diabetic animals (6 OU/ml).16 Thus, these experiments were indeed studying the effect of glucose on ECM protein accumulation. After 1, 2, and 4 weeks of growth, the cells were rinsed, fixed, and processed for immunocytochemical demonstration of fibronectin, laminin, and type IV collagen. We found that cultures of rat mesangial cells contained fibronectin as the most abundant ECM protein, followed by laminin and type IV collagen. Fibronectin and laminin showed a predominately extracellular fibrillar staining pattern in confluent cells (Figure 1A, B). In nonconfluent cells, cytoplasmic granular staining was also seen similar to that reported by Ishimura et al.2' The staining was more dense at areas of cell-to-cell contact. In contrast, type IV collagen staining was predominately observed in areas where the cells had piled up and formed what has been referred to as 'hillocks'22 (Figure 1 C). This was rarely observed before 4 weeks of growth. The staining was predominately coarsely granular and in the areas of cell-to-cell contact. After 4 weeks of 30 mmol/l glucose, all three ECM proteins examined were increased in amount compared with cells grown in 10 mmol/l glucose (Figures 2, 3, and 4). The increase in ECM deposition was not due to an increase in the number of cells and not accompanied by an increase in total cellular protein. Before 4 weeks, the staining intensity was similar in both 10 mmol/l and 30 mmol/l glucose cells. To quantitate these observations, we developed a solid-phase ELISA (see Materials and Methods). The results obtained by this ELISA confirmed our immunocytochemical findings (Table 1). Namely, 1) mesangial cells contain fibronectin as the most abundant ECM protein, followed by laminin and type IV collagen, 2) type IV collagen was measurable only after 4 weeks of growth, when mesangial cell hillocks had formed, and 3) after 4 weeks of growth in 30 mmol/l glucose, all three ECM proteins were significantly increased in amount as compared with 10 mmol/l glucose. Fibronectin and laminin were in-

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Figure 1. A: Immunoperoxidase staining for fibronectin in mesangial cells cultured in 10 mmol/l glucose. Note the fibrillar sta ining pattern. Cells were fixed and stained 1 week after platitng. (X 100). B: Immunoperoxidase stainingfor laminin in mesangial cells cultured in 10 mmol/l glucose. Similar to the fibronectin-stained cells, laminin also shows a fibrillar stainingpattern (arrows). Cells werefixed and stained 1 week after plating, (X 100). C: Immunoperoxidase stainingfor type IVcollagen in cells cultured in 10 mmol/l glucose. To demonstrate type IV collagen, it was necessary to grow the cells past confluence to allow the formation of mesangial cell hillocks (these cells were grown for 4 weeks without passaging). Note that the staining is predominately in the cells that have piled up (arrows) (X 100).

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Figure 2. A: Immunoperoxidase staining for fbronectin from mesangial cells grown for 4 weeks witbout passaging in 10 mmol/l glucose. Note the)fibrillar staining pattern and that the degree ofstaining is greater in the hillocks (arrows). B: Mesangial cells grown for 4i weeks in .30 mmol/l glucose) anld stainedfurftbrunectin. Note that the pattern of staining is similar to 10 mmol/l glucose cells but the) staining intentsity is much grevatrer (X5O).

creased approximately 60%, whereas type IV collagen increased by 50% in cells grown in 30 mmol/l glucose. Because diabetic glomerulosclerosis takes many years to develop, we were interested in examining the ECM content after many months of growth in 30 mmol/l versus 10 mmol/l glucose. To do such experiments, it was necessary to subculture the cells (The cells begin to peel off the dishes if they are allowed to grow on the substratum for longer than 4 weeks without passaging.). We used cells that were cultured for 8 months. Cells were subcultured twice weekly. These cells responded in a similar manner as younger mesangial cells to both isoproterenol and vasopressin (ie, shape change in response to cAMP-elevating agents and inhibition of shape change with addition of vasopressin13,15), indicating that they were functionally like earlier passaged cells. After 6 weeks of growth in 30 mmol/l glucose, there was approximately a 40% increase in fibronectin and laminin content compared with the 10 mmol/l glucose cells. Both proteins were increased in 30 mmol/l glucose by twofold at 8 months (Table 2). Type IV collagen was not measurable, as these cells were not allowed to pile up and form hillocks where type IV collagen was previously

seen. Similar results were found by immunocytochemistry for fibronectin and laminin (Figures 5, 6).

Discussion
The results of this study demonstrate that glomerular mesangial cells cultured under high glucose conditions accumulate increased amounts of fibronectin, laminin, and type IV collagen similar to the diabetic mesangium in vivo. The mechanisms that lead to this accumulation are not as yet understood, but clearly must involve increased synthesis and/or decreased degradation of ECM proteins by mesangial cells. In this regard, preliminary studies show that mesangial cell fibronectin synthesis and mRNA are significantly elevated after 1 week of growth in 30 mmol/l glucose.23 Recent studies indicate that there is a cell surface site distinct from the cell adhesion receptor that mediates the assembly of fibronectin into fibrillar disulfidebonded structures of the cell surface.2425 Cells apparently assemble the fibronectin matrix by first binding the fibronectin and subsequently transferring it to the matrix. Thus, fibronectin deposition into the ECM may be

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Figure 3. A: Immutnoperoxidase staininlg for laminzini in cells grown for 4 weeks without passaginig in 10 mmol/l glucose. Note the predominately fibrillar stainitng patternt. B: Cells grown for 4 weeks in 30 mmol/l glucose show increased staininig in both hillocks anid valleys (X200).

affected not only by its rate of synthesis and degradation but also by its binding and rate of incorporation into the matrix. The accumulation of mesangial ECM material in diabetes mellitus and in cultures of mesangial cells grown in 30 mmol/l glucose may be the result of increased ECM protein biosynthesis. Protein synthesis is regulated at multiple levels, any or all of which could be affected by elevated glucose concentrations. These include mRNA transcription, processing, degradation, and translation into protein. In this regard, studies on whole glomeruli isolated from rats made diabetic with streptozotocin have demonstrated increased glomerular basement membrane (GBM) synthesis26 as well as increased activity of the enzymes involved in basement membrane synthesis.27 Poulsom et al28 have demonstrated increased expression of mRNA coding for the B1 chain of laminin but not for type IV collagen in whole kidney RNA isolated from streptozotocin diabetic rats of 11 to 28 weeks duration, consistent with increased synthesis of this protein. Human umbilical vein endothelial cells grown in 30 mmol/l glucose for 11 days showed increased expression of mRNA coding for fibronectin and type IV collagen.29 This change in gene expression was maintained through multiple pas-

sages, independent of the proliferative state of the cells and of the substratum on which they were grown; however, shorter periods of exposure to high glucose (ie, 24 or 48 hours) did not have an effect on the transcripts. In further support of increased synthesis as the mechanism involved in ECM accumulation in diabetes, Phan-Thanh et a130 showed in pulse chase studies that fibroblasts isolated from the genetically diabetic KK mouse skin synthesized fibronectin at an increased rate when compared with normal fibroblasts. Additionally, the rate of incorporation of synthesized fibronectin into the ECM was increased. Accumulation of ECM in the diabetic mesangium may be due to decreased activity of degradative enzymes or excess secretion of enzyme inhibitory proteins. The mesangial cell contains highly specialized systems for the normal degradation or catabolism of matrix proteins.31'32 Cultures of mesangial cells secrete a neutral metalloendopeptidase with degradative activity specifically against type IV collagen of isolated GBM.3' In addition to secreting a metalloendopeptidase, mesangial cells secrete appreciable amounts of a functional metalloprotease inhibitor, which controls metalloendopeptidase activity.33 This inhibitor was found to be antigenically related to the tissue

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Figure 4. A: Immunioperoxidase staining for tpe IV collagen in mesangial cells grown for 4 weeks without passaging in 10 mmol/l glucose. Note that the hillock shows most of the staining (arrows). B: Cells grown for 4 weeks in 30 mmol/l glucose show increased stainiing in the hillock for tjpe IV collagen. Note also that the staining seems to outline the cells. In some areas the staining pattern appears globular (arrows) (X 100).

inhibitor of metalloendopeptidase. In addition to secreting metalloprotease, cultures of mesangial cells produce the urokinase-type plasminogen activator (uPA)32 as well as the type inhibitor of uPA (Kreisberg, unpublished observations). Plasminogen activators are serine proteases that catalyze the conversion of the proenzyme, plasminogen, to the active enzyme plasmin, a wide-spectrum protease capable of degrading most glycoprotein components of the ECM.34 Glass et a135 recently reported that
a

shape change in mesangial cells treated with cAMP-elevating agents was associated with release of fibronectin into the medium. Both shape change and fibronectin release were inhibited by pretreatment with an antibody to uPA. Thus, uPA seems to be important for ECM turnover and cell function (ie, shape change) in cultures of mesangial cells. Alterations to these proteases or their inhibitors could result in decreased degradation of ECM proteins. In support of such a mechanism, Cohen, Surma and Wu36

Table 1. Effect of Glucose on Accumulation ofExtracellular Matrix Proteins by Rat Mesangial Cells in Culture Duration of growth Fibronectin (ng) Laminin (ng) Type IV collagen (ng) in glucose Glucose concentration DNA (jig) DNA (Mg) DNA (,g) (nonpassaged) 10mmol/l 1 wk 1107+ 74.1 NM 554.5 30 mmol/l 1 wk 1251 50.4 NM 45 3.0 10 mmol/I 2 wk 3006 261.0 44 3.5 NM 30 mmol/I 2 wk 3368 245.2 NM 55 3.5 10 mmol/l 4 wk 4632 234.9 55 1.5 650 65.0 4 wk 30 mmol/l 7584 529.3* 85 5.5* 1005 39.5*
Results are means SEM of the mean values from at least 10 samples for each condition. * P < 0.05 30 mmol/l vs. 10 mmol/l glucose (Student-Newman-Keuls test) NM, not measurable.

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Table 2. Effect of Glucose on Accumulation ofExtracellularMatrix Proteins by RatMesangial Cells in Culture ng Fibronectin ng Laminin Duration of growth in glucose (subcultured) Glucose concentration Ag DNA zg DNA 3.6 110 1966 97.3 6 weeks 10 mmol/l 160 10.6* 2713110.3* 6weeks 30mmol/l 106.6 5.0 1603 172.4 8 months 1 0 mmol/l 223.6 21.6* 3603 104.4* 8 months 30 mmol/l
*

Results are means SEM of the mean values from at least five samples for each condition. P < 0.05 30 mmol/l vs. 10 mmol/l glucose (Student-Newman-Keuls test)

showed with pulse chase studies in streptozotocin diabetic rats prolongation of GBM turnover suggestive that diminished degradation may also be a contributing factor in GBM accumulation. Glucose may be exerting its effect on mesangial cell matrix turnover in many ways. Metabolic factors attributable to high glucose and/or changes in medium tonicity may cause alterations in matrix turnover. However, one of the major pathophysiologic consequences of hyperglycemia is the excessive chemical interaction of glucose with proteins, leading to its attachment to these proteins without the aid of enzymes.37 Some of these early glycosylation end products of long-lived proteins (such as those in

the mesangium) do not dissociate, but instead undergo a complex series of chemical rearrangements to form irreversible advanced glycosylation end products. These do not return to normal when the hyperglycemia is corrected, but accumulate over the lifetime of the protein. It has been shown that glycosylation of proteins renders them resistant to degradation by proteases. For example, the susceptibility of glycosylated GBM to digestion by proteases such as papain, pepsin, and trypsin is considerably reduced.' Thus, another mechanism of excessive ECM accumulation in cultures of rat mesangial cells grown in medium containing 30 mmol/l glucose may be resistance of glycosylated ECM proteins to degradation.

Figure 5. Immunoperoxidase staining forfibronectin after 8 months of growth in 10 mmol/l (A) and 30 mmol/l (B) glucose. Cells were fixed and stained after 1 week ofplating Note the intcreased degree ofstaining in the 30 mmoll Iglulcose cells (B), (X 100).

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Figure 6. Immunoperoxidase stainiing for laminin after 8 months of growth in 10 mmoo/ (A) and 30 mmol/l (B) glucose. Cells were fixed anidstainied after 1 week ofplating. Similar to thefJibrontectin results in Figure 5, laminin showed increased staining in the 30 mmol/lglucose cells (B), (X200).

In conclusion, this model of increased ECM deposition under conditions in which the cells are grown in high-glucose-containing medium should help to elucidate the mechanisms involved and perhaps aid in establishing therapeutic modalities to combat this condition.

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Acknowledgments
The authors thank Diann Smith and Sharon Cloer for typing the manuscript, and Dr. T. Prihoda for his help with the statistical analyses.