American Journal of Pathology, Vol. 138, No. 6, June 1991 Copyright X American Association of Pathologist

In Vitro Analysis of Extracellular Matrix Production by Porcine Glomerular Mesangial and Vascular Smooth Muscle Cells

Yung-Yen Chiang,*t Shigeo Takebayashi, and Terry D. Oberley*t From the Pathology Section, William S. Middleton Memorial

Veterans Hospital,* and the Department of Pathology, University of Wisconsin, Madison, Wisconsin; and the Second Department of Pathology,* University of Fukuoka, Fukuoka, Japan

nine vasopressin has been demonstrated.1 2 In the present studies, we attempted to compare extracellular matrix production by mesangial cells and vascular smooth muscle cells. The similarity between these cells types was investigated in tissue cultures using light microscopy, immunohistochemical techniques, and biochemical analysis of extracellular collagen, fibronectin, and laminin.

Proliferation potential and extracellular matrix production were compared in cultured porcine glomerular mesangial cells and arterial and venous smooth muscle cells. Mesangial and arterial smooth muscle cells proliferated more rapidly than venous smooth muscle cells. In immunofluorescence studies, mesangial and arterial smooth muscle cells stained strongly for collagen types I, III, and V; venous smooth muscle showed weaker staining for collagens III and V Total collagen synthesis in cultured mesangial and arterial smooth muscle cells was lower than in venous smooth muscle cells. Electrophoretic analysis showed type I collagen predominated in all cell types, although levels were highest in mesangial and arterial smooth muscle cells. Collagen V (a3) occurred only in venous smooth muscle cells. Mesangial and arterial smooth muscle cells showed cellbound fibronectin and laminin, which also were secreted into the medium Venous smooth muscle cells secreted fibronectin, but all laminin was cell bound The findings suggest a strong similarity between mesangial and arterial smooth muscle cells. (Am J Pathol 1991, 138:1349-1358)

Kidney glomerular mesangial cells are morphologically similar to vascular smooth muscle cells. The close resemblance between mesangial cells and arterial smooth muscle with respect to response to various growth factors, intracellular cyclic adenosine monophosphate (cAMP) content, and dose-dependent increase of cAMP after hormonal stimulation with prostaglandin 12 and argi-

Materials and Methods Isolation and Culture of Mesangial and Vascular Cells Isolation of Mesangial Cells

Kidneys were obtained from young pigs. The dissected cortex was thinly sliced and glomeruli were isolated with sieves of 60 mesh (250 ,u), 150 mesh (105 ,u), and 200 mesh (74 p).3 Glomeruli adhered to the tissue culture plastic substrate within 3 to 5 days, and then the cells began to grow from the glomeruli. Three weeks later, the cell layer became confluent. To select for mesangial cells, primary explants (composed of both epithelial and mesangial cells) were treated with bacterial collagenase (750 U/ml of type IV bacterial collagenase from Sigma Chemical Co., St. Louis, MO) for 30 minutes at 37°C. Spindle cells (mesangial cells) were separated from epithelial cells by suction of detached cells with a Pasteur pipette and placing them into new culture flasks. Our experiments were performed on passages 3 and 8; very few epithelial cells remain viable even at passage 3 (unpublished observations, this study, and reference 4). Cells from passages 3 and 8 were negative for factor VIII Supported by the Medical Research Service, Department of Veterans Affairs.

Accepted for publication January 31, 1991. Address reprint requests to Dr. Yung-Yen Chiang, Pathology Section, William S. Middleton Memorial Veterans Hospital, 2500 Overlook Terrace, Madison, WI 53705.

1349

1350

Chiang, Takebayashi, and Oberley

AlPJune 1991, Vol. 138, No. 6

by the avidin-biotin-peroxidase immunoperoxidase method. Isolation of Smooth Muscle Cells from the Aorta Smooth muscle cells were obtained by the method of Ross.5 The medium and adventitia were carefully removed from the extirpated swine thoracic aorta, and the inner layer of the aortic intima was harvested. It was cut into small pieces (1 mm3), which were placed intima side down and allowed to adhere to the culture dish. Cell growth began within 5 to 10 days, after which the cells were subcultured. These cells were confluent in 2 to 3 weeks. Isolation of Smooth Muscle Cells from the Vena Cava Swine vena cava was extirpated and washed several times in physiologic saline. The smooth muscle cells were isolated and cultured using the same methodology as for aortic smooth muscle cells. Cell Culture All cells were cultured at 37°C in an incubator containing 5% CO2 and 95% air. The medium was Dulbecco's modified Eagle minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Grand Island New York) and 100 ,ug/ml kanamycin sulfate. Medium was changed every 4 days. After the cells reached confluence, they were subcultured with 2% ethylenediaminetetra-acetic acid (EDTA) and 0.25% trypsin. Smooth muscle cells and mesangial cells used in analyses reported here were from passage 3. Studies on passage 8 cells gave similar results (data not shown).

Analysis of Cell Growth Cells (1 x 1 05) were propagated in 35-mm plastic petri dishes containing 2 ml DMEM with 10% fetal bovine serum. Daily for the first 7 days, cells from 3 Petri dishes were detached by EDTA-trypsin treatment and counted in an automated blood cell counter (Sysmex Microcellcounter F-300, Sakura, Inc., Tokyo, Japan). Cells from each dish were counted four times and the counts were

averaged.

Immunofluorescent Antibody Technique Cultured cells were fixed with ethanol at 4°C for 10 minutes and treated with 10% normal animal serum. They

then were allowed to react for 90 minutes with primary antibody: rabbit anti-human collagen 1, 111, IV, or V (Southern Biotechnology Assoc., Inc., Birmingham, AL), goat anti-mouse fibronectin, or anti-laminin antibodies (Transformation Research, Inc., Framingham, MA) (1:10). Secondary antibodies used in the indirect immunofluorescent antibody stain6'7 for collagen were fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit gamma G immunoglobulin (IgG; H + L) antiserum (Zymed Laboratories, Inc., San Francisco, CA), and for fibronectin and laminin were FITC-labeled rabbit anti-goat IgG antiserum (Bioprocessing, Inc., Durham, UK). The average intensity of staining of a flask of cells was rated on an arbitrary scale of 0 to 4 + by a pathologist (TDO) who was blinded to the treatment conditions.

Collagen Production Cells were incubated for 24 hours with 50 ,uCi/ml of L[2,3-3H]proline (Amersham), 0.1 mmol/l (millimolar) sodium ascorbate, and 0.5 mmol/l W3-aminopropionitrile fumaric acid. Cells then were harvested. Samples of cells and media were boiled for 5 minutes and neutral protease inhibitors (0.25 mol/l [molar] EDTA, 10 mmol/I phenylmethylsulfonyl fluoride [PMSF], 0.1 mol/l Nethylmaleimide) were added. These sample were used for quantitative analyses.

Quantitative Analysis Collagen was determined by the digestion method of Peterkofsky et al.8 Samples were dialyzed and lyophilized, then redissolved in 0.04 mol/l NaOH and neutralized by TRIS buffer, pH 7.4. After treatment with collagenase at 37°C for 2 hours, 10% trichloroacetic acid and 0.5% tannic acid were added. Collagenous proteins were separated from noncollagenous proteins by centrifugation. Both fractions were dissolved in a liquid scintillator ACS 11 and radioactivity was measured in a liquid scintillation counter (Packard 300 CD, Downers Grove, IL). Qualitative Analysis The Hata method9 was used. Samples were precipitated by adding ammonium sulfate (176 mg/ml), dissolved in 0.5 mol/I acetic acid, and digested with pepsin at 40C for 6 hours. They were neutralized with 5 N NaOH, lyophilized, and used for electrophoresis. The concentrating gel for electrophoresis was 3% acrylamide (pH 6.8), and the separation gel was 5% acrylamide (pH 8.9).10

In Vitro Extracellular Matrix Production 1351 AJPJune 1991, Vol. 138, No. 6

After electrophoresis the gel was fixed for 1 hour, dipped in EN3HANCE (Dupont, Wilmington, DE) for 1 hour, and dried. Hyperfilm (Amersham) then was exposed to the dried gels for 10 days at - 800C, and the concentrations of the collagen bands on the film were measured with a densitometer. Purified collagen types 1, Ill, IV, and V (Medical Koken, Tokyo, Japan) were used as markers in electrophoresis.

apoprotinin were added and the samples were centrifuged. The supernatants were used to determine fibronectin and laminin by immunoprecipitation as follows. Each sample was incubated with normal goat serum at 40C for 1 hour, then shaken with prewashed Pansorbin (Staphylococcus aureus cells; Calbiochem, La Jolla, CA) at 40C for 1 hour. After centrifugation, the supernatant was incubated with antisera against rabbit fibronectin or laminin at 40C for 4 hours. It then was mixed with 100 ,ul of prewashed Pansorbin and shaken overnight. The Pansorbin-immune complex mixture obtained after centrifugation was washed five times in solution A (10 mmoVI TRIS-HCI, pH 7.4/0.7% Triton X-1 00/0.7% deoxycholate/ 0.2% SDS/0.7% bovine serum albumin/i 50 mmolA NaCI). This was followed by two washings each in 1 moVI NaCI in solution A, 0.5 mol/l NaCI in solution A, 0.2% SDS in solution A, and 10 mmol/l TRIS-HCI, pH 7.4/0.1% SDS/2 mmolA EDTA.12'13 Laemmli's buffer was added and the washed Pansorbin-immune complex mixtures were boiled for 3 minutes. After centrifugation, the supematants were analyzed for fibronectin and laminin by SDSPAGE and fluorography.

Immunoblotting Samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Towbin.11 Bands were transferred from the gel to a nitrocellulose membrane (0.45-, pore size) with a BIO-RAD transfer apparatus for 3 hours at 0.1 A, and incubated with normal rat serum overnight. The membrane was washed in TRIS-HCVNaCI/Triton X-100 buffer, incubated for 3 hours with the primary antibody (anti-human collagen IV antiserum), then incubated for 1 hour with the secondary antibody and stained with 0.02% 4-chloro-1-naphthol/0.01% H202/50 mmol/l TRIS-HCI pH 7.5.

Results

Immunoprecipitation Cells were cultured in methionine-free medium at 370C for 30 minutes, then labeled with 50 ,uCi of [35S]methionine for 15 hours. The labeled cells were washed in PBS and lysis buffer Dulbecco's phosphatebuffered saline containing 1% Triton X-1 00, 0.5% sodium deoxycholate, and 0.1% SDS was added. After complete lysis by ultrasonication, the protein inhibitors PMSF and Figure 1. Growth of mesangial cells (MC), arterial smooth muscle cells (ASMC), and venous smooth muscle cells (VSMC). Cells were counted daily for the first 7 days of culture. Cells on three petri dishes were counted four times each, using an automated blood cell counter, and the average was calculated.

Cell Morphology and Growth Under light microscopy, mesangial cells were oval or spindlelike, often with dendritic processes. Active pinocytosis was observed in the first week of culture. After passage, cells formed a layer with hills and valleys. This was seen also in cultures of arterial smooth muscle cells (ar-

Cell Growth Curve ._-

"Ion 0 C) 4.'

1

2

3

4

days in culture

5

6

7

1352

Chiang, Takebayashi, and Oberley

AJPJune 1991, Vol. 138, No. 6

Similar results were obtained in three separate experiments.

terial cells). Venous smooth muscle cells (venous cells) were slender and spindlelike, somewhat smaller than mesangial and arterial cells. The layer of venous cells also displayed hills and valleys. Growth curves of all cell types showed rapid proliferation from day 0 to day 2 (Figure 1). From day 3 to day 7, mesangial and arterial cells grew continuously; growth of venous cells was significantly slower. The growth curve shown in Figure 1 is from a representative experiment.

Immunofluorescent Antibody Studies Mesangial (Figure 2) and arterial (Figure 3) cells showed similar immunofluorescence staining (Table 1). Immunostaining was intense for collagens 1, 111, and V and fi-

in

Ir. $

.d .

I

I _w~~~~~~k- ;x, 1:..4-F'Ir-W*#feM3i

.W-4v~'i

;,.

.ft, >e

Figure 2. Immunofluorescence staining in mesangial cells of collagen I (a), III (b), IV (c), and V (d), fibronectin (e), and laminin (f) (x 180).

In Vitro Extracellular Matrix Production

1353

AJP June 1991, Vol. 138, No. 6

-

Figure 3. Immunofluorescence staining in arterial smnooth muscle cells of collagen I (a), III (b), IV (c), and V (d), flbronectin (e), and laminin (f) (x 180).

bronectin. In contrast, venous cells immunostained only weakly for collagens Ill and V (Figure 4, Table 1). Staining of laminin and collagen IV was weak in all three cell types.

Collagen Synthesis Total Collagen Synthesis Table 2 shows the total collagen synthesis per cell, amount of noncollagen protein synthesis, and ratio of col-

lagen synthesis to total protein synthesis. In terms of radioactivity, collagen synthesis by mesangial cells was 0.5 dprm/cell. This was comparable to that in arterial cells (0.4 dprm/cell) but less than in venous cells (1.4 dprn/cell). The amount of noncollagenous protein produced by mesangial cells was 0.4 dpm/cell, which also was lower than in venous cells. According to the equation of Peterkofsky et al,8 the ratio of collagen synthesis to protein synthesis was 0.231 in venous cells, 0.171 in mesangial cells, and 0.177 in arterial cells.

1354

Chiang, Takebayashi, and Oberley

AJP June 1991, Vol. 138, No. 6

Table 1. Immunofluorescence Staining for Extracellular MatrixMolecules Collagen type ll

IV

V

Fibronectin

Laminin

+++

++

+

++

++

Trace

+++

++

Trace

++

++

Trace

++++

+

Trace

Trace

Cell type

Mesangial Arterial smooth muscle Venous smooth muscle

+

+

Graded on scale of 0 (negative) to 4 + (strongly positive). The average intensity of a flask of cells was evaluated by a pathologist (TDO) who was blinded to the treatment conditions.

*Bi

Figure 4. Immunofluorescence staining in venous smooth muscle cells of collagen I (a), laminin (f) (x 180).

HII (b), IV (c), and V (d), fibronectin

(e), and

In Vitro Extracellular Matrix Production

1355

AJP June 1991, Vol. 138, No. 6

Table 2. Incotporation of[3H]Proline into Collagen and Noncollagen Protein Ratio of

Cell type Mesangial Arterial smooth muscle Venous smooth muscle

collagen to total

Collagen protein (dpm/cell) 0.5

Noncollagen protein (dpm/cell) 0.4

17.1

0.4

0.3

17.7

1.4

0.8

23.1

protein* (%)

Values are the mean of triplicate determinations in a representative experiment. * Calculated from the Peterkovsky equation, based on the relative amounts of proline in collagen protein and noncollagen protein: dpm in collagen 5.4 x dpm in noncollagen + dpm in collagen x 10

Type of Collagen Table 3 shows the relative amounts of each type of collagen synthesized by the three types of cells. Type collagen dominated in all cells, ranging from 88.2% to 91.0%. The proportion of collagen was higher in mesangial and arterial cells than in venous cells. Collagen V content ranged from 4.2% to 7.4%, and was highest in venous cells. A remarkable observation was that collagen V (a3) was found only in venous smooth muscle cells (Figure 5, lanes 4-6). Type Ill collagen constituted 4.3% to 5.1% of the total collagen. There was no significant difference between cell types. Because only a trace of collagen IV was detected, this analysis was done by immunoblotting. Mesangial and arterial cells synthesized more type IV collagen than did venous cells (Figure 5, lanes 7-9).

Biosynthesis of Fibronectin and Laminin Confluent cultures were labeled with [35S]methionine, and newly synthesized fibronectin and laminin were assayed in the cells and the media by immunoprecipitation, SDS-PAGE, and fluorography (Figure 6). In all three cell types, SDS-PAGE under reducing conditions showed fibronectin as a molecule of 240 kd. The membrane-

bound form of fibronectin was found in all cell types, but at a much lower level in venous cells. In contrast, the amount of fibronectin secreted into the tissue culture fluid was similar for all cell types. Amounts of membranebound and secreted fibronectin were similar for mesangial and arterial cells, but most of the fibronectin produced by venous cells was secreted into the culture medium. An unidentified 115-kd protein also was immunoprecipitated from mesangial and arterial cells by anti-fibronectin antiserum (Figure 6). All three cell types synthesized laminin, as shown by assay under reducing conditions (Figure 7: 230- and 280-kd bands correspond to subunits Bi and B2). The three cell types contained similar amounts of membranebound laminin, but very small amounts were secreted by venous cells. This result is the opposite of that for fibronectin. It indicates that 1) in cultured confluent venous smooth muscle cells, laminin is synthesized and retained by the cell, whereas fibronectin is synthesized and secreted; and 2) the mode of extracellular matrix synthesis of venous cells is different from that in mesangial and arterial cells. An unidentified protein of 93 kd was immunoprecipitated in mesangial and arterial cells by antilaminin antiserum.

Discussion It is known that mesangial cells closely resemble vascular smooth muscle cells morphologically,' 14 and it has been demonstrated that arterial and venous smooth muscle cells differ remarkably in their cell growth potential."5,6 The present study confirms that porcine arterial smooth muscle cells have a greater growth capability than venous smooth muscle cells. Extracellular matrix synthesis by mesangial cells morphologically resembles that of arterial smooth muscle cells, and production of extracellular matrix is of the interstitial tissue type. Collagens and Ill and fibronectin are reported as being particularly high.1718 In mesangial cells, with fluorescent antibody techniques, we confirmed previous findings,1920 indicating resemblance to arterial smooth muscle cells. In venous smooth muscle cells, however, only small amounts of fibronectin were de-

Table 3. Relative Amounts of Collagen Types I, III IV, and V Collagen type (%) ll IV Cell type 0. 4.7 91.0 Mesangial Arterial smooth 0 5.1 90.3 muscle Venous smooth 0 4.3 88.2 muscle Values are averages of duplicate densitometer absorption readings.

Ratio of types

V 4.2

111:1

V:l

0.051

0.046

4.5

0.056

0.048

7.4

0.048

0.083

1356

Chiang, Takebayashi, and Oberley

AJPJune 1991, Vol. 138, No. 6

a

4 5 M A

1 2 3 M A V

6 V

TOP-mm

7 8 9 M A V [111 IV

lV--s ai (IV) .*-al ai

Figure 5. Fluorograms of lH3Hproline-labeled collagen chains elaborated by mesangial cells (M), arterial smooth muscle cells (A), and venous smooth muscle cells (V) under reducing and nonreducing conditions, and immunoblot of collagen IV after SDS-PAGE under nonreducing conditions. Under nonreducing conditions, type V (a3) collagen was found only in venous smooth muscle cells (lane 6). Arrows indicate positions of marker collagens (run on a separate gel).

alaia(

)-

--ai( I 4a2 (1)

;i m.

Reduced

Non reduced

bt.

.

;.^.,

'.

Immunoblotting

Collagen IV is specifically localized in basement membranes and putatively interacts with laminin.23 Very low levels were synthesized and there was no significant difference between the cell types we studied. Because fluorescent antibody techniques showed only weak immunostaining, relative amounts of collagen IV synthesis were estimated by immunoblotting. Mesangial and arterial smooth muscle cells showed more collagen IV than venous smooth muscle cells. Fibronectin participates in cell adhesion24 25 and has a crucial role in cell growth.26 Fujikawa et a127 reported that fibronectin is involved in wound healing. In the present study, fibronectin biosynthesis was analyzed in the three cell types. We found that synthesis of the secreted form was the same in the different cell types, but that there was very little cell-associated fibronectin in venous smooth muscle cells. Venous cells rapidly secreted newly synthesized fibronectin into the culture fluid. This observation was confirmed by immunofluorescent antibody techniques. A possible explanation for this difference is that fibronectin readily binds on mesangial and arterial smooth muscle cell surfaces, but not on venous

tected, whereas laminin was found in greater abundance. These findings suggest that mesangial cells are phenotypically closer to arterial than to venous smooth muscle cells. The ratio of collagen to total protein was significantly higher in venous smooth muscle cells than in the other cell types (Table 2). When analyzed by SDS-PAGE under reducing conditions, collagen (al, a2) predominated in all cells; similar, but much smaller, amounts of collagens Ill, IV, and V were synthesized in all cells. With electrophoresis under nonreducing conditions, however, collagen V (a3) was barely detectable in mesangial and arterial cells but was clearly present in venous cells. Collagen V is localized in the basement membrane and the interstitium,21 but its function is obscure. In fluorescent microscopy, the collagen V signal in venous smooth muscle cells was weak, contradicting the synthetic product analysis. A possible reason for this is that degradation by proteases may occur in the venous cell interstitium. Conversely Voss and Rayterberg22 pointed out that the epitope of collagen V may be modified by other collagens and thus cannot be detected. Figure 6. SDS-PAGE (5% gel) andfluorography of samples from cell layer and culture medium of mesangial cells (M), arterial smooth muscle cells (A), and venous smooth muscle cells (V) labeled with [35S]-methionine. Immunoprecipitation was performed with antiserum against fibronectin. Fibronectin bands are indicated by arrowheads. Arrows indicate molecular weight makers.

V)

u-a3 V -*-a2 V

Mo

Cell M A

V

Medium M A V

M A V

M

A V

TOE .4.

200

-

116

93-

Reduced

Non reduced

Reduced

Non reduced

In Vitro Extracellular Matrix Production 1357 AJP June 1991, Vol. 138, No. 6

Cell M A V

Medium

M A V

M A V

M A V

TO Pa-up,

200

Figure 7. SDS-PAGE (5% gel) andfluorography of samples from cell layer and culture medium of mesangial cells (M),* arterial smooth muscle cells (A), and venous smooth muscle cells (V) labeled with [35S]-methionine. Immunoprecipita- * tion was performed with antiserum against laminin. Laminin A and B chain bands are indicated by arrowheads. Arrows indicate molecular weight markers.

smooth muscle cell surfaces. Although the mechanism is unknown, our experimental results indicate that fibronectin accumulates in mesangial and arterial smooth muscle cells after confluence. Membrane-bound and secreted laminin was similarly synthesized in mesangial and arterial smooth muscle cells, but only the membrane-bound form was present in venous smooth muscle cells. Laminin localizes in basement membranes of various tissues and is assumed to participate in cell adhesion,2829 proliferation,3031 and maintenance of morphology.32 Reports that laminin accelerates elongation of the neurite even in the absence of nerve growth factors in certain nerve cells suggests the possibility that it is involved in differentiation.33i36 Structurally it is a cruciform molecule consisting of one chain each of A, B1, and B2 polypeptide.37 In our study, B1 and B2 chains were detected in all cells, but A chain was not. This may be explained either by the lack of crossreactivity of the antibody we used or by failure of A-chain synthesis by these three cell types in culture. Recent reports support either possibility.38'39 Further investigation is necessary to resolve this issue. The present study shows that mesangial cells closely resemble arterial smooth muscle cells in laminin synthesis. It is interesting that the laminin newly synthesized in confluent cultures of venous smooth muscle cells was mostly cell associated, whereas most fibronectin was secreted. All data in the present paper are from cells at passage 3. Similar results, however, were obtained from passage 8 cells (unpublished). It is possible that different results would have been obtained from primary or late-passage mesangial cells, but we have not performed these studies. By analyzing patterns of distribution and synthesis of extracellular matrix components in cultured mesangial, arterial smooth muscle, and venous smooth muscle cells, we have demonstrated that mesangial and arterial cells

6

-

Reduced

Non reduced

Reduced

Non reduced

are similar. Further studies are necessary to elucidate the respective roles of these cell types in glomerular disease and arteriosclerosis.

Acknowledgments The authors thank Drs. Y. Joney and T. Noboru for technical assistance and Dr. Carol Steinhart for editorial review of the manuscript.

References 1. Tsuru N, Jimi S, Takebayashi S: A study on function and morphology of mesangial cells as arteral smooth muscle cells. Jpn J Nephrol 1987; 6:615-622 (in Japanese) 2. Takebayashi S: The function and morphology of mesangial cells as arterial smooth muscle cells-role of cyclic nucleotides. Kouseishyo Special Disease, Japan, 1985, 59:139 (in Japanese) 3. Striker GE, Striker U: Biology of disease: Glomerular cell culture. Lab Invest 1985; 53:122-131 4. Oberley TD, Yang AH, Gould-Kostka J: Selection of kidney cell types in primary glomerular explant outgrowths by in vitro culture conditions. J Cell Sci 1986; 84:69-92 5. Ross R: The smooth muscle cell 11. J Cell Biol 1971; 50:172186 6. Bruno V, Jurgen R: Localization of collagen types 1, 111, IV, V, fibronectin and laminin in human arteries by the indirect immunofluorescence method. Pathol Res Pract 986; 181:568575 7. Yoshikatsu 0, Shogo K, Yutaka M: Altered synthesis of collagen types in cultured arterial smooth muscle cells during phenotypic modulation by dimethyl sulfoxide. Acta Pathol Jpn 1989; 39:15-22 8. Peterkofsky B, Chojkier M, Bateman J: Determination of collagen synthesis in tissue and cell culture systems, Immunochemistry of the Extracellular Matrix. Vol II. Applications.

1358

Chiang, Takebayashi, and Oberley

AJPJune 1991, Vol. 138, No. 6

1st edition. Edited by H Furthmayr. Boca Raton, FL, CRC Press, 1982, pp 19-47 9. Hata R, Ninomiya Y, Nagai Y, Tsukada Y: Biosynthesis of interstitial types of collagen by albumin-producing rat liver parenchymal cell (hepatocyte) clones in culture. Biochemistry 1980; 16:169-176. 10. Weber K, Osbom M: Proteins and sodium dodecyl sulfate: Molecular weight determination on polyacrylamide gel and procedures. The Proteins. Vol 1. 3rd edition. Edited by H Neurath, RL Hill, CL Boeder. New York, Academic Press, 1975, pp 179-223 11. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979, 76:4350-4354 12. Noboru T, Yoshio M, Motomu K, Yuji M, Yukio I: Evidence for carboxyl terminal processing and glycolipid anchoring of human carcinoembryonic antigen. J Biol Chem 1988, 263:12716-12720. 13. Gospodarowicz D, Greenburg G, Foidart JM, Savion N: The production and localization of laminin in cultured vascular and corneal endothelial cells. J Cell Physiol 1981, 197:171183 14. Scheinman JI: Human glomerular smooth muscle (mesangial) cells in culture. Lab Invest 1976, 34:150-156 15. Takebayashi S, Kamio A, Shiraishi K, Shitama K: Arterial smooth muscle cells in atherogenesis, as compared with veins. J Jpn Coll Angiol 1978, 18:429-437 (in Japanese) 16. Takebayashi S: The effect of developing atherosclerosis on vascular smooth muscle. Clin Sci 1977, 13:1142-1148 (in Japanese) 17. Ishimura E, Sterzel RB, Budde K, Kashgarian M: Formation of extracellular matrix by cultured rat mesangial cells. Am J Pathol 1989, 134:843-855 18. Haralson MA, Jacobson HR, Hoover RH: Collagen polymorphism in cultured rat kidney mesangial cells. Lab Invest 1987, 57:513-523 19. Scheinman JI, Foidart JM, Michael AF: The immunohistology of glomerular antigens V. The collagenous antigens of the glomerulus. Lab Invest 1980, 43:373-381 20. Sterzel RB, Lovett DH, Foellmer HG, Perfetto M: Mesangial cell hillocks-Nodular foci of exaggerated growth of cells and matrix in prolonged culture. Am J Pathol 1986, 125:130-1 40 21. Nagai Y, Fujimoto D (eds): Collagen metabolism and disease. Kodansya 1989, 126-129 (in Japanese) 22. Voss B, Rayterberg J: Localization of collagen type 1, 111, IV, V, fibronectin and laminin in human arteries by the indirect immunofluorescence method. Pathol Res Pract 1986, 187:568-575 23. Toshihiko H: Macromolecular composition and role of extracellular matrix. Cell 1989, 21:3-7 24. Hedin U, Thyberg J: Plasma fibronectin promotes modulation of arterial smooth muscle cells from contractile to synthetic phenotype. Differentiation 1987, 33:239-246 25. Perris R, Johansson S: Amphibian neural crest cell migration on purified extracellular matrix components: A chondroitin

sulfate proteoglycan inhibits locomotion on fibronectin substrates. J Cell Biol 1987, 105:2511-2521 26. Oberley TD, Steinert BW, Yang A-H, Anderson PJ: Kidney glomerular explants in serum-free media. Virchows Arch 1985, 50:209-235 27. Fujikawa LS, Foster CS, Gipson IK, Colvin RB: Basement membrane components in healing rabbit comeal epithelial wounds: Immunofluorescence and ultrastructural studies. J Cell Biol 1984, 98:128-138 28. Couchman JR, Hook M, Rees DA, Timpl R: Adhesion, growth, and matrix production by fibroblasts on laminin substrates. J Cell Biol 1983, 96:171-183 29. Vlodavasky I, Gospodarowicz D: Respective roles of laminin and fibronectin in adhesion of human carcinoma and sarcoma cells. Nature 1981, 289:304-306 30. Kleinman HK, McGarvey ML, Hassell JR, Martin GR, BaronVan Evercooren A, Dubois-Dalcq M: The role of laminin in basement membranes and in the growth, adhesion, and differentiation of cells, The Role of Extracellular Matrix in Development. Edited by RL Trelstad. New York, Alan R. Liss, 1984, pp 123-143 31. Terranova VP, Williams JE, Liotta LA, Martin GR: Modulation of the metastatic activity of melanoma cells by laminin and fibronectin. Science 1984, 226:982-985 32. Sugre SP, Hay ED: Response of basal epithelial cell surface and cytoskeleton to solubilized extracellular matrix molecules. J Cell Biol 1981, 91:45-54 33. Baron-Van Evercooren A, Kleinman HK, Ohno S, Maranogos P, Schwartz JP, Dubois-Dalcq ME: Nerve growth factor, laminin and fibronectin promote neurite growth in human fetal sensory ganglion cultures. J Neurosci Res 1982, 8:179-193 34. Manthorpe M, Engvall E, Ruoslahti E, Longo M, Davis GE, Varon S: Laminin promotes neuritic regeneration from cultured peripheral and central neurons. J Cell Biol 1983, 97:1882-1990 35. Rogers SL, Letourneau PC, Palm SL, McCarthy J, Furcht LT: Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin. Dev Biol 1983, 98:212-220 36. Yoshii S, Yamamuro T, Ito S, Hayashi M: In vivo guidance of regenerating nerve by laminin-coated filaments. Brain Res 1987, 20:107-119 37. Rao CN, Margulies IM, Tralka TS, Terranova VP, Madri JA, Liotta LA: Isolation of a subunit of laminin and its role in molecular structure and tumor cell attachment. J Biol Chem 1982, 257:9740-9744 38. Abrahamson DR, Irwin MH, John PS, Perry EW, Accavitti MA, Heck LW, Couchman JR: Selective immunoreactivities of kidney basement membranes to monoclonal antibodies against laminin: Localization of the end of the long arm and the short arms to discrete microdomains. J Cell Biol 1989, 109:3477-3491 39. Ekblom M, Klein G, Mugrauer G, Fecker L, Deutzmann R, Timpl R, Ekblom P: Transient and locally restricted expression of laminin A chain mRNA by developing epithelial cells during kidney organogenesis. Cell 1990, 60:337-346

In vitro analysis of extracellular matrix production by porcine glomerular mesangial and vascular smooth muscle cells.

Proliferation potential and extracellular matrix production were compared in cultured porcine glomerular mesangial cells and arterial and venous smoot...
3MB Sizes 0 Downloads 0 Views