245
Atherosclerosis, 33 (1979) 245-252 0 Elsevier/North-Holland Scientific Publishers,
Ltd.
INCREASED GROWTH OF HUMAN FIBROBLASTS AND ARTERIAL SMOOTH MUSCLE CELLS FROM DIABETIC PATIENTS RELATED TO DIABETIC SERUM FACTORS AND CELL ORIGIN
T. KOSCHINSKY,
C.E. BUNTING,
B. SCHWIPPERT
and F.A. GRIES
Diabetes Research Institute, University of Diisseldorf, Diisseldorf (F.R.G.) (Received 17 November, 1978) (Revised, received 31 January, 1979) (Accepted 31 January, 1979)
summary Fibroblasts from 3 diabetic patients (DF) grew faster, resulting in higher cell counts in the stationary phase than fibroblasts from 3 age-matched healthy volunteers (NF). This difference was apparent when DF or NF were cultured in either diabetic (DS) or normal serum (NS). Diabetic serum increased growth of both DF and NF compared with normal serum. Total protein content per plate paralleled the increase of cell number per plate in relation to cell origin and serum type. DS increased growth and total protein per plate in the arterial smooth muscle cell line from a nondiabetic patient in a way similar to in DF and NF. It is concluded that increased growth of DF in vivo could result in an increased turnover of vascular cells with a shortened replicative lifespan, leading to an accumulation of basal lamina. This effect would be even further accentuated by exposure of DF to DS. Taken together with the increased protein synthesis the accelerated development of diabetic angiopathy could be the final consequence. Key words:
Arterial smooth muscle cells -Cell Growth
culture -Diabetes
mellitus - Fibroblasts -
Introduction Altered metabolism of arterial vascular cells has been implicated in the development of macroangiopathy. The proliferative response of arterial smooth muscle cells which occurs when subendothelial layers of the vascular wall are exposed to serum factors derived from platelets or lipoproteins, has been
246
regarded as a crucial step in the early events of atherogenesis [ 1,2]. Tissue culture studies of arterial smooth muscle cells and fibroblasts obtained from diabetic animals as well as from diabetic patients [3-6] offer evidence that genetic defects in diabetes mellitus can be expressed in vitro and that factors in diabetic serum can change the metabolism of cultured vascular cells. The studies reported here were initiated to quantitate abnormalities in cell growth and protein synthesis in fibroblasts from diabetic patients and to study the effects of human diabetic serum. In addition, the effects of diabetic serum on human arterial smooth muscle cells from a normal subject were studied since this cell type is predominantly involved in the development of atherosclerotic lesions. Materials and Methods The following items were employed: Dulbecco’s modification of Eagle’ medium (DME) (Seromed-GmbH, Munich, F.R.G.), penicillin/streptomycin solution (Seromed-GmbH), trypsin (Bio-Cult/Gibco, Karlsruhe, F.R.G.). [ 6-3H] thymidine (s.a.’ 27 Ci/mmol), and DL-[ l-14C]leucine (specific activity 59 mCi/mmol), (Amersham Buchler, Braunschweig, F.R.G., Human venous blood was collected from 15 badly controlled non-ketotic diabetic patients and from 10 healthy volunteers in the morning after an overnight fast. The blood was placed on ice and then spun at 1500 X g for 15 min at 4°C. The serum was pooled, sterilized by filtration (0.22 cc) and kept frozen for l-3 weeks at -20°C. Characteristic values for 4 different diabetic sera (DS) compared to normal sera (NS) used in the experiments with 3 different cell lines are as follows: glucose, 11.4-13.1 mmol/l vs 4.1-5.2 mmol/l; triglycerides, 1.9-2.1 mmol/l vs 0.9-1.2 mmol/I; cholesterol, 5.1-5.9 mmol/l vs 4.9-5.2 mmol/l; growth hormone, 2.0-3.5 ng/ml vs 1.54.5 ng/ml. It was not possible to measure insulin concentrations in DS as serum insulin antibodies interfere with the insulin assay. Fibroblast cultures were derived from skin biopsies from 2 juvenile-onset diabetics (JOD) (aged 10 and 17 years, insulin therapy, duration of diabetes 1 and 2 years, respectively, without diabetic vascular complications), and from one adult-onset diabetic (AOD) (aged 40 years, on oral sulfonurea treatment). All diabetic patients showed a family history of diabetes among first-degree relatives. Three age-matched, metabolically healthy volunteers with no family history of diabetes served as controls. Informed consent was obtained from all donors. One arterial smooth muscle cell line was derived from the abdominal aorta of a non-diabetic 50-year-old patient with atherosclerosis. This cell line has been established by Dr. Ditschuneit of the Medical Department of the University of Ulm, F.R.G., and further subcultivated at the Diabetes Research Institute using pooled human serum. Each experiment was performed comparing one DF cell line and one NF cell line matched for age and passage number. The cells were between the 5th and 10th passage. Different batches of pooled human sera were used. The effect of DS and NS from single donors did not differ from the effects of pooled DS or
241
NS, respectively (unpublished results). No control parison with the arterial smooth muscle cell line.
cell line was used for com-
Results Representative growth curves of fibroblasts from diabetic patients (DF) compared with age-matched NF are shown in Figs. 1 and 2. Results from the 3rd DF and NF lines are similar and are therefore not shown. DF grew faster and to higher cell densities compared with NF when cultured in either diabetic or normal serum. The various diabetic sera (DS) increased growth in both DF and NF by 30-70% compared with normal serum (NS). Exact adjustment of the glucose concentration in NS to the level of DS did not change the effect of DS. DME plus a mixture of 5% DS and 5% NS stimulated cell growth less in proportion to the dilution of DS, and seemed not to be affected by the addition of an equal amount of normal serum (Fig. 3). Additional data from the experiment documented in Fig. 2A are presented in Figs. 2B-D. Total protein content per plate paralleled the increase in cell number per plate in relation to cell origin and serum type (Fig. 2B). [ 3H] Thymidine incorporation per plate after a 4-h pulse showed significant differences between DF and NF and between DS and NS effects during the log growth phase (3rd day), with the highest incorporation into DF on DS and the
f cells/plate DFI DS
Fig. 1. Effect of diabetic serum (DS) or normal serum (NS) on growth of fibroblasts from a JOD patient and from a healthy volunteer (NF). 1 X lo5 tells/60-mm plate were grown in 3 ml DME supplemented with 10% (v/v) DS or NS at 37’C in a humidified atmosphere of 95% air and 5% CO2. The final Jucoss concentration in the incubation medium with NS was adjusted to that of the experiment with DS: 6.27 mmolfl. & indicates a change of incubation medium. Results are expressed as the mean * SEM of quadruplicete plates. (DF)
248
A
5x105
protein/plate LX105
3x105
2x10:
1x10!
,
F
i
I
L
.
.
l
,
.
&
.
.
1
2
3
4
5
6
3H -Thymidine
A
> ‘h’S
“C-Leucine
t)PM/plate 5x106
0
0 DF/DS q NFlDS B DF/NS
IXIOf
,
I
IUI N/NS
0
DFlDS
pd NFlDS I 6~10~
DFINS
Ill NFI NS
R. 3x10(
2x10(
5,
5,
6. 1x10’
12:L56
dDYS
1
2
1
L
6
days
Fig. 2. Effect of diabetic serum (DS) or normal serum (NS) on (A) growth, (B) protein content per plate, (C) [“HI thymidine incorporation per plate after a 4-h pulse of 10 PC/plate. and (D) [14Clleucine incorporation per plate after a 4-h pulse of 0.6 nC/plate. of fibroblasts from an AOD patient (DF) and from a healthy volunteer (NF). Incubation conditions are the same as in the legend to Fig. 1. Cells were counted from quadruplicate dishes after short trypsfnization using the Coulter-Counter Model ZBI [71. Total protein content per dish was measured from triplicate dishes, after removal of serum Proteins. according to the method of Lowry et al. [El. [3HlThymidine and [ 14CIleucine incorporation per dish was determined from triplicate dishes after removal of extracellular [3Hlthymidine and [ 14Clleucine.
249
NF
1.106
cells/plate
3.105
Lx105
2x105
l#,
1
***
1
2
3
_,
L
1
,)
5
6
days
Fig. 3. Growth of fibroblasts from a healthy volunteer (NF) in DIME supplemented with 10% diabetic serum (DS), or 10% normal serum (NW. or a combination of 6% DS + 5% NS (DNS). Incubation conditions are the same es in the legend to Fig. 1.
lowest into NF, on NS. These differences disappeared at confluency (6th day). At this time [ “klleucine incorporation increased 2-2.5-fold, differences now being observed between DF and NF and DS versus NS effect. In Figs. 4A and B one representative experiment out of 5 with 4 different batches of DS and NS is shown. DS increased growth and total protein per plate in the arterial smooth muscle cell line from a nondiabetic patient in a way similar to in DF and NF. Discussion The mechanisms underlying the changes in arterial vascular tissues leading to diabetic micro-and macroangiopathy are poorly understood. On the basis of in vitro studies of plating efficiency and replicative lifespan of fibroblasts from diabetic, prediabetic and normal donors [ 5,9], it was suggested that the diabetic state is associated with cellular characteristics of accelerated aging. Abnormalities in protein and collagen metabolism of fibroblasts from juvenileonset and adult-onset diabetic donors have also been described [lo]. Besides the expression of genetic abnormalities in the fibroblasts of
250
diabetics, effects of a prolonged exposure to an abnormal metabolic environment have been suspected as pathogenic factors for diabetic angiopathy [ 3,11-141. The relevance of the results derived from animal cell lines or using animal sera might, however, be questioned. Recent information uncovered different growth effects of normal human serum compared to the fetal bovine serum most widely used in cell culture experiments [ 151. Therefore it seems to be necessary to use homologous systems, that is human cell lines with human serum. Using this homologous in vitro system the growth-stimulating effects of DS could be confirmed in man and extended to non-diabetic human smooth muscle cells. For the first time an increase growth capacity of cells from 3 diabetics as compared to 3 metabolically normal subjects has been established by 4 lines of evidence: the cell number per plate, the [ 3H]thymidine incorporation per plate during log-phase growth, the protein content per plate and the [‘4C]leucine incorporation per plate at confluency. The difference in cell proliferation was independent of the culture medium. Increased growth in the
25n106
20d06
15x106
lDd06
sp.106
m106
4 1
2
3
L
4 5
6
c
4 7
6
9
VI
(1
Flg.4A.
121 &YS
251
period of 5-7 days was evident in cells from both IOD and AOD patients. These results are suggestive of some common metabolic changes in vascular cells from both types of diabetes. This possibly represents an in vitro expression of a genetic defect. The final glucose, triglyceride, cholesterol or growth hormone concentrations in the incubation medium were fairly similar and exact adjustment of the glucose concentration in normal serum to the level of diabetic serum did not change the response of the cells. Therefore these factors seem not to be responsible for the markedly different effects of DS and NS. The replicative lifespan of cultured fibroblasts is agedependent [5]. Its decrease in diabetics could be the result of an increased cell turnover compared to age-matched normal controls. As far as these in vitro results are representative of the in vivo situation, chronic exposure of vascular cells from diabetics to diabetic serum could result in an increased growth and cell turnover, with more cells dying and being A pgprotein/plate 900
G9
SK
600
I
1 1
2
3
4
1
1 5
6
7
8
1 9
10
11
12
.s
13 &YS
Fig. 4B.
Fig. 4. Effect of diabetic serum (DS) or normal serum (NS) on (A) growth, and (B) protein content per plate, of arterial smooth muscle cells from a non-diabetic patient. Results are expressed as the mean f SEM of quadruplicate plates. J indicates a change of incubation medium.
252
replenished at an accelerated rate [9,16]. Taken together with the increased synthesis of abnormal collagen [lo] and proteins, an excessive thickening of the vascular wall in diabetics could be easily explained. At the moment this model offers the opportunity to study in more detail diabetes-specific changes in cell metabolism that are possibly related to diabetic angiopathy under controlled in vitro conditions. References 1 Ross, F. and Glomset, J.A.. Atherosclerosis and the arterial smooth muscle cell, Science, 180 (1973) 1332. 2 Colwell, J.A.. Sagel. J.. Crook, L., Chambers, A. and Laimins. M., Correlation of platelet aggregation plasma factor activity and megathrombocytes in diabetic subjects with and without vascular disease, Metabolism, 26 (1977) 207. 3 Ledet. T.. FischepDzoga. K. and Wissler. R.W., Growth of rabbit aortic smooth-muscle cells cultured in media containing diabetic and hyperlipemic serum, Diabetes, 25 (1976) 207. 4 Rowe, D.W.. Starman. B.J.. Fujhnoto. W.Y. and Williams. R.H.. Abnormalities in proliferation and protein synthesis in skin fibroblasts cultures from patients with diabetes mellitus. Diabetes, 26 (1977) 284. and 5 Soeldner, S.. Moermann, E.J.. Soeldner. J.S., Gleason, R.E. and Barnett, D.M.. Chronologic physiologic age affect replicative life-span of fibroblasts from diabetic, prediabetic and normal donors, Science, 199 (1978) 781. 6 Koschinsky. T.. Biinting, C.E., Schwippert, B. and Gries. F.A.. Changes in lipoprotein metabolism and growth of fibroblasts from diabetic patients related to diabetic serum, Dfabetologia, 15 (1978) 247 (Abstract). 7 Harris, M.. Kruse. P.F. and Patterson, M.K.. Electronic enumeration and sizing of cells. In: P.F. Kruse and M.K. Patterson (Eds.), Tissue Culture -Methods and Applications, Academic Press, New York, 1973. PP. 400-406. 8 Lowry. O.M., Rosebrough. N.J.. Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 9 Vracko. R., Basal lamina layering in diabetes mellitus. Diabetes, 23 (1973) 94. 10 Kohn. R.R. and Henssc. S., Abnormal collagen in cultures of fibroblasts from human being with diabetes mellitus, Biochem. Biophys. Res. Commun.. 76 (1977) 765. 11 Turner. J.L. and Biermann. E.L., Effects of glucose and sorbitol on proliferation of cultured human skin fibroblasts and arterial smooth-muscle cells. Diabetes, 27 (1978) 583. 12 More& B. and Froesch. E.R.. Fibroblasts as an experimental tool in metabolic and hormone studies, Europ. J. Clin. Invest., 3 (1973) 119. 13 Ledet. T.. Growth hormone.antiserum suppresses the growth effect of diabetic serum, Diabetes. 26 (1977) 789. 14 Ledet. 1011.
T.. Growth
hormone
stimulates the growth of arterial medial cells in vitro, Diabetes, 25 (1976)
16 Cooper, J.T. and Goldstein, S., Comparative studies on human skin fibroblasts - Life span and lipid metabolism in medium containing fetal bovine or human serum, In Vitro, 12 (1977) 473. 16 Vracko. R. and Benditt, E.P.. Restricted replicative life-span of diabetic fibroblasts in vitro -Its relation to microangiopathy, Fed. Proc.. 34 (1975) 68.
Atherosclerosis, 33 (1979) 245-252 0 Elsevier/North-Holland Scientific Publishers,
Ltd.
INCREASED GROWTH OF HUMAN FIBROBLASTS AND ARTERIAL SMOOTH MUSCLE CELLS FROM DIABETIC PATIENTS RELATED TO DIABETIC SERUM FACTORS AND CELL ORIGIN
T. KOSCHINSKY,
C.E. BUNTING,
B. SCHWIPPERT
and F.A. GRIES
Diabetes Research Institute, University of Diisseldorf, Diisseldorf (F.R.G.) (Received 17 November, 1978) (Revised, received 31 January, 1979) (Accepted 31 January, 1979)
summary Fibroblasts from 3 diabetic patients (DF) grew faster, resulting in higher cell counts in the stationary phase than fibroblasts from 3 age-matched healthy volunteers (NF). This difference was apparent when DF or NF were cultured in either diabetic (DS) or normal serum (NS). Diabetic serum increased growth of both DF and NF compared with normal serum. Total protein content per plate paralleled the increase of cell number per plate in relation to cell origin and serum type. DS increased growth and total protein per plate in the arterial smooth muscle cell line from a nondiabetic patient in a way similar to in DF and NF. It is concluded that increased growth of DF in vivo could result in an increased turnover of vascular cells with a shortened replicative lifespan, leading to an accumulation of basal lamina. This effect would be even further accentuated by exposure of DF to DS. Taken together with the increased protein synthesis the accelerated development of diabetic angiopathy could be the final consequence. Key words:
Arterial smooth muscle cells -Cell Growth
culture -Diabetes
mellitus - Fibroblasts -
Introduction Altered metabolism of arterial vascular cells has been implicated in the development of macroangiopathy. The proliferative response of arterial smooth muscle cells which occurs when subendothelial layers of the vascular wall are exposed to serum factors derived from platelets or lipoproteins, has been
246
regarded as a crucial step in the early events of atherogenesis [ 1,2]. Tissue culture studies of arterial smooth muscle cells and fibroblasts obtained from diabetic animals as well as from diabetic patients [3-6] offer evidence that genetic defects in diabetes mellitus can be expressed in vitro and that factors in diabetic serum can change the metabolism of cultured vascular cells. The studies reported here were initiated to quantitate abnormalities in cell growth and protein synthesis in fibroblasts from diabetic patients and to study the effects of human diabetic serum. In addition, the effects of diabetic serum on human arterial smooth muscle cells from a normal subject were studied since this cell type is predominantly involved in the development of atherosclerotic lesions. Materials and Methods The following items were employed: Dulbecco’s modification of Eagle’ medium (DME) (Seromed-GmbH, Munich, F.R.G.), penicillin/streptomycin solution (Seromed-GmbH), trypsin (Bio-Cult/Gibco, Karlsruhe, F.R.G.). [ 6-3H] thymidine (s.a.’ 27 Ci/mmol), and DL-[ l-14C]leucine (specific activity 59 mCi/mmol), (Amersham Buchler, Braunschweig, F.R.G., Human venous blood was collected from 15 badly controlled non-ketotic diabetic patients and from 10 healthy volunteers in the morning after an overnight fast. The blood was placed on ice and then spun at 1500 X g for 15 min at 4°C. The serum was pooled, sterilized by filtration (0.22 cc) and kept frozen for l-3 weeks at -20°C. Characteristic values for 4 different diabetic sera (DS) compared to normal sera (NS) used in the experiments with 3 different cell lines are as follows: glucose, 11.4-13.1 mmol/l vs 4.1-5.2 mmol/l; triglycerides, 1.9-2.1 mmol/l vs 0.9-1.2 mmol/I; cholesterol, 5.1-5.9 mmol/l vs 4.9-5.2 mmol/l; growth hormone, 2.0-3.5 ng/ml vs 1.54.5 ng/ml. It was not possible to measure insulin concentrations in DS as serum insulin antibodies interfere with the insulin assay. Fibroblast cultures were derived from skin biopsies from 2 juvenile-onset diabetics (JOD) (aged 10 and 17 years, insulin therapy, duration of diabetes 1 and 2 years, respectively, without diabetic vascular complications), and from one adult-onset diabetic (AOD) (aged 40 years, on oral sulfonurea treatment). All diabetic patients showed a family history of diabetes among first-degree relatives. Three age-matched, metabolically healthy volunteers with no family history of diabetes served as controls. Informed consent was obtained from all donors. One arterial smooth muscle cell line was derived from the abdominal aorta of a non-diabetic 50-year-old patient with atherosclerosis. This cell line has been established by Dr. Ditschuneit of the Medical Department of the University of Ulm, F.R.G., and further subcultivated at the Diabetes Research Institute using pooled human serum. Each experiment was performed comparing one DF cell line and one NF cell line matched for age and passage number. The cells were between the 5th and 10th passage. Different batches of pooled human sera were used. The effect of DS and NS from single donors did not differ from the effects of pooled DS or
241
NS, respectively (unpublished results). No control parison with the arterial smooth muscle cell line.
cell line was used for com-
Results Representative growth curves of fibroblasts from diabetic patients (DF) compared with age-matched NF are shown in Figs. 1 and 2. Results from the 3rd DF and NF lines are similar and are therefore not shown. DF grew faster and to higher cell densities compared with NF when cultured in either diabetic or normal serum. The various diabetic sera (DS) increased growth in both DF and NF by 30-70% compared with normal serum (NS). Exact adjustment of the glucose concentration in NS to the level of DS did not change the effect of DS. DME plus a mixture of 5% DS and 5% NS stimulated cell growth less in proportion to the dilution of DS, and seemed not to be affected by the addition of an equal amount of normal serum (Fig. 3). Additional data from the experiment documented in Fig. 2A are presented in Figs. 2B-D. Total protein content per plate paralleled the increase in cell number per plate in relation to cell origin and serum type (Fig. 2B). [ 3H] Thymidine incorporation per plate after a 4-h pulse showed significant differences between DF and NF and between DS and NS effects during the log growth phase (3rd day), with the highest incorporation into DF on DS and the
f cells/plate DFI DS
Fig. 1. Effect of diabetic serum (DS) or normal serum (NS) on growth of fibroblasts from a JOD patient and from a healthy volunteer (NF). 1 X lo5 tells/60-mm plate were grown in 3 ml DME supplemented with 10% (v/v) DS or NS at 37’C in a humidified atmosphere of 95% air and 5% CO2. The final Jucoss concentration in the incubation medium with NS was adjusted to that of the experiment with DS: 6.27 mmolfl. & indicates a change of incubation medium. Results are expressed as the mean * SEM of quadruplicete plates. (DF)
248
A
5x105
protein/plate LX105
3x105
2x10:
1x10!
,
F
i
I
L
.
.
l
,
.
&
.
.
1
2
3
4
5
6
3H -Thymidine
A
> ‘h’S
“C-Leucine
t)PM/plate 5x106
0
0 DF/DS q NFlDS B DF/NS
IXIOf
,
I
IUI N/NS
0
DFlDS
pd NFlDS I 6~10~
DFINS
Ill NFI NS
R. 3x10(
2x10(
5,
5,
6. 1x10’
12:L56
dDYS
1
2
1
L
6
days
Fig. 2. Effect of diabetic serum (DS) or normal serum (NS) on (A) growth, (B) protein content per plate, (C) [“HI thymidine incorporation per plate after a 4-h pulse of 10 PC/plate. and (D) [14Clleucine incorporation per plate after a 4-h pulse of 0.6 nC/plate. of fibroblasts from an AOD patient (DF) and from a healthy volunteer (NF). Incubation conditions are the same as in the legend to Fig. 1. Cells were counted from quadruplicate dishes after short trypsfnization using the Coulter-Counter Model ZBI [71. Total protein content per dish was measured from triplicate dishes, after removal of serum Proteins. according to the method of Lowry et al. [El. [3HlThymidine and [ 14CIleucine incorporation per dish was determined from triplicate dishes after removal of extracellular [3Hlthymidine and [ 14Clleucine.
249
NF
1.106
cells/plate
3.105
Lx105
2x105
l#,
1
***
1
2
3
_,
L
1
,)
5
6
days
Fig. 3. Growth of fibroblasts from a healthy volunteer (NF) in DIME supplemented with 10% diabetic serum (DS), or 10% normal serum (NW. or a combination of 6% DS + 5% NS (DNS). Incubation conditions are the same es in the legend to Fig. 1.
lowest into NF, on NS. These differences disappeared at confluency (6th day). At this time [ “klleucine incorporation increased 2-2.5-fold, differences now being observed between DF and NF and DS versus NS effect. In Figs. 4A and B one representative experiment out of 5 with 4 different batches of DS and NS is shown. DS increased growth and total protein per plate in the arterial smooth muscle cell line from a nondiabetic patient in a way similar to in DF and NF. Discussion The mechanisms underlying the changes in arterial vascular tissues leading to diabetic micro-and macroangiopathy are poorly understood. On the basis of in vitro studies of plating efficiency and replicative lifespan of fibroblasts from diabetic, prediabetic and normal donors [ 5,9], it was suggested that the diabetic state is associated with cellular characteristics of accelerated aging. Abnormalities in protein and collagen metabolism of fibroblasts from juvenileonset and adult-onset diabetic donors have also been described [lo]. Besides the expression of genetic abnormalities in the fibroblasts of
250
diabetics, effects of a prolonged exposure to an abnormal metabolic environment have been suspected as pathogenic factors for diabetic angiopathy [ 3,11-141. The relevance of the results derived from animal cell lines or using animal sera might, however, be questioned. Recent information uncovered different growth effects of normal human serum compared to the fetal bovine serum most widely used in cell culture experiments [ 151. Therefore it seems to be necessary to use homologous systems, that is human cell lines with human serum. Using this homologous in vitro system the growth-stimulating effects of DS could be confirmed in man and extended to non-diabetic human smooth muscle cells. For the first time an increase growth capacity of cells from 3 diabetics as compared to 3 metabolically normal subjects has been established by 4 lines of evidence: the cell number per plate, the [ 3H]thymidine incorporation per plate during log-phase growth, the protein content per plate and the [‘4C]leucine incorporation per plate at confluency. The difference in cell proliferation was independent of the culture medium. Increased growth in the
25n106
20d06
15x106
lDd06
sp.106
m106
4 1
2
3
L
4 5
6
c
4 7
6
9
VI
(1
Flg.4A.
121 &YS
251
period of 5-7 days was evident in cells from both IOD and AOD patients. These results are suggestive of some common metabolic changes in vascular cells from both types of diabetes. This possibly represents an in vitro expression of a genetic defect. The final glucose, triglyceride, cholesterol or growth hormone concentrations in the incubation medium were fairly similar and exact adjustment of the glucose concentration in normal serum to the level of diabetic serum did not change the response of the cells. Therefore these factors seem not to be responsible for the markedly different effects of DS and NS. The replicative lifespan of cultured fibroblasts is agedependent [5]. Its decrease in diabetics could be the result of an increased cell turnover compared to age-matched normal controls. As far as these in vitro results are representative of the in vivo situation, chronic exposure of vascular cells from diabetics to diabetic serum could result in an increased growth and cell turnover, with more cells dying and being A pgprotein/plate 900
G9
SK
600
I
1 1
2
3
4
1
1 5
6
7
8
1 9
10
11
12
.s
13 &YS
Fig. 4B.
Fig. 4. Effect of diabetic serum (DS) or normal serum (NS) on (A) growth, and (B) protein content per plate, of arterial smooth muscle cells from a non-diabetic patient. Results are expressed as the mean f SEM of quadruplicate plates. J indicates a change of incubation medium.
252
replenished at an accelerated rate [9,16]. Taken together with the increased synthesis of abnormal collagen [lo] and proteins, an excessive thickening of the vascular wall in diabetics could be easily explained. At the moment this model offers the opportunity to study in more detail diabetes-specific changes in cell metabolism that are possibly related to diabetic angiopathy under controlled in vitro conditions. References 1 Ross, F. and Glomset, J.A.. Atherosclerosis and the arterial smooth muscle cell, Science, 180 (1973) 1332. 2 Colwell, J.A.. Sagel. J.. Crook, L., Chambers, A. and Laimins. M., Correlation of platelet aggregation plasma factor activity and megathrombocytes in diabetic subjects with and without vascular disease, Metabolism, 26 (1977) 207. 3 Ledet. T.. FischepDzoga. K. and Wissler. R.W., Growth of rabbit aortic smooth-muscle cells cultured in media containing diabetic and hyperlipemic serum, Diabetes, 25 (1976) 207. 4 Rowe, D.W.. Starman. B.J.. Fujhnoto. W.Y. and Williams. R.H.. Abnormalities in proliferation and protein synthesis in skin fibroblasts cultures from patients with diabetes mellitus. Diabetes, 26 (1977) 284. and 5 Soeldner, S.. Moermann, E.J.. Soeldner. J.S., Gleason, R.E. and Barnett, D.M.. Chronologic physiologic age affect replicative life-span of fibroblasts from diabetic, prediabetic and normal donors, Science, 199 (1978) 781. 6 Koschinsky. T.. Biinting, C.E., Schwippert, B. and Gries. F.A.. Changes in lipoprotein metabolism and growth of fibroblasts from diabetic patients related to diabetic serum, Dfabetologia, 15 (1978) 247 (Abstract). 7 Harris, M.. Kruse. P.F. and Patterson, M.K.. Electronic enumeration and sizing of cells. In: P.F. Kruse and M.K. Patterson (Eds.), Tissue Culture -Methods and Applications, Academic Press, New York, 1973. PP. 400-406. 8 Lowry. O.M., Rosebrough. N.J.. Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 9 Vracko. R., Basal lamina layering in diabetes mellitus. Diabetes, 23 (1973) 94. 10 Kohn. R.R. and Henssc. S., Abnormal collagen in cultures of fibroblasts from human being with diabetes mellitus, Biochem. Biophys. Res. Commun.. 76 (1977) 765. 11 Turner. J.L. and Biermann. E.L., Effects of glucose and sorbitol on proliferation of cultured human skin fibroblasts and arterial smooth-muscle cells. Diabetes, 27 (1978) 583. 12 More& B. and Froesch. E.R.. Fibroblasts as an experimental tool in metabolic and hormone studies, Europ. J. Clin. Invest., 3 (1973) 119. 13 Ledet. T.. Growth hormone.antiserum suppresses the growth effect of diabetic serum, Diabetes. 26 (1977) 789. 14 Ledet. 1011.
T.. Growth
hormone
stimulates the growth of arterial medial cells in vitro, Diabetes, 25 (1976)
16 Cooper, J.T. and Goldstein, S., Comparative studies on human skin fibroblasts - Life span and lipid metabolism in medium containing fetal bovine or human serum, In Vitro, 12 (1977) 473. 16 Vracko. R. and Benditt, E.P.. Restricted replicative life-span of diabetic fibroblasts in vitro -Its relation to microangiopathy, Fed. Proc.. 34 (1975) 68.
