- Email: [email protected]
Patterns of regulation of collagen breakdown in articular cartilage
69
OSTEOARTHRITIS SYMPOSIUM
developed Golgi complex, and a few strands of smooth and rough endoplasmic reticulum. Endothelial cells are separated from the growth surface by sparse, finely fibrillar material, which is thought to represent newly synthesized basal lamina. The basal plasma membrane of the endothelial cells is smooth and devoid of microvilli. There are some submembrane accumulations of cytoplasmic filaments along the basal plasma membrane. Heparin, which has been reported to cause elaboration of proteolytic activities by endothelial cells, had no effect on the interaction between endothelial cells and the cartilage matrix. However, heparin significantly increased the number of cytoplasmic filaments and lysosomes. In contrast, endothelial cells grew as contact-inhibited monolayers of flattened cells on the surfaces of extracted cartilage (Fig. I). Cells were separated from the cartilage matrix by abundant basal lamina, which consisted of several discontinuous strands, probably collagenous in nature. There were a few microvilli at the basal plasma membrane, but there was no degradation or penetration of the collagenous matrix of extracted cartilage. However, when endothelial cells were stimulated by heparin, they assumed a polyhe-
dral shape and penetrated the extracted cartilage matrix with numerous microvilli and some cytoplasmic processes (Fig. 2). This penetration of the collagenous matrix was associated with tissue rarefaction and degradation of collagen fibers. This invasion of heparin stimulated endothelial cells was abolished when low concentrations of cartilage-derived AIF was added to the culture medium. AIF also significantly reduced the number of endothelial cells growing on the extracted cartilage matrix. These observations correlated with our findings that AIF inhibited endothelial cell proliferation in vitro. This growth inhibitory activity was accompanied by shape transformation and cell surface alterations. These data provide evidence that the resistance of hyaline cartilage to endothelial cell invasion is regulated in part by tissue derived proteinase inhibitors and an antiproliferative activity directed against endothelial cells. The depletion of components of AIF (e.g., protease inhibitory activity) from diseased cartilage such as osteoarthritic cartilage may explain the susceptibility of this tissue to penetration by activated endothelial cells.
REFERENCES 1. Kuettner KE, Pauli BU. Resistance of cartilage to normal and neoplastic invasion. In: Horton JE, Tarpley TM Jr, Davis WE, eds. Proceedings, Mechanism of Localized Bone loss. Special Supplement to Calcium Tissue Abstracts 1978; 251-78. 2. Eisenstein R, Kuettner KE, Neapolitan C, Soble LW,
Patterns
of regulation
of collagen
Sorgente
N. The resistance
of certain
tissues to invasion;
111.
Cartilage extracts inhibit the growth of fibroblasts and endothelial cells in culture. Am J Pathol 1975;81:337-48. 3. Horton JE, Wezeman FH, Kuettner KE. Inhibition of in vitro bone resorption by a cartilage derived anti-collagenase factor. Science 1978;199: 134244.
breakdown
in articular
cartilage
By E. D. Harris, Jr., C. A. Vater, C. E. Brinckerhofl, R. M. McMillan, and P. Hasselbacher; Dartmouth Medical School, Hanover, New Hampshire
D
IMINISHED joint function in arthritis can be equated with loss of articular cartilage. Collagen is the principal structural protein in cartilage and can be lost by mechanical or enzymic means. Recent studies have shown multiple mechanisms for collagen degradation in cartilage. Several principles are noteworthy. First, factors modulating collagen breakdown are relative, not absolute. For example, although crosslinks among collagen fibrils retard collagenolysis by specific collagenases, this slowing of the reaction is not complete; with sufficient time and high concentrations of enzymes, lysis of even the highly crosslinked collagen fibers in cartilage may occur. Secondly, cells produce enzymes; therefore, cellular
density becomes an important determinant of extracellular matrix destruction. For example, chondrocytes occupy a small colume in cartilage (O.Ol%O.l%), and even after maximal stimulation by mononuclear cell factors chondrocytes produce collagenase at an activity index less than 10% of that produced by rhetrmatoid synovial cells.’ Assuming that superficial lining cells occupy 50% of tissue volume, it is likely
Supported
in part by USPHS
20641 and by grants from Chapter
of the Arthritis
been a postdoctoral fellow
grants AM
the National Foundation.
of the Arthritis
0 1981 by Grune & Stratton, 0049J3172/81/1005-0036$01.00/0
Inc.
14780 and AM
and New Hampshire Dr.
McMillan
Foundation.
has
OSTEOARTHRITIS SYMPOSIUM
70
Table
1.
Rabbit
Articular
Chondrocytes Acid
and Synovial
on Collagenase
Fibroblasts.
and Collagenase
Effects Inhibitor
of Phorbolesters
(PMA)
and Retinoic
Production. Flbroblasts
Chondrocytes
Collagenase
Collagena%? Collagenase*
Untreated
19 k 4
PMAllO~‘M) Retinoic
acid
(10 A
PMA and retinoic
lCollagenase
M)
444
15 t 3
414 f 126
84 ? 63
f 28
960
285 i 22
10
1 /.~g collagen
10
11 k2 acid
units:
58i
degraded/hr/mg
cell protein
at
lnhibltort
Collagenase*
InhlbWxt
228 f 228
+ 84
264 i 6
7 r 0.7
390 k 162
208 t 64
336 + 102
37°C.
tlnhibitor: units of trypsin-activated collagenase inhibited/hr/mg cell protem at 37 “C
that the synovial enzymes exert SO-500-fold greater impact on certain areas of cartilage than do cartilage enzymes. As an illustration of inherent differences in cell types in response to agents stimulating collagenase and collagenase inhibitors, examine the data in Table I from chondrocytes and synovial cells in the same normal rabbit in matrix-free monolayer cultures at confluence. The third principle is that multiple different types of stimuli are sufficient to stimulate collagenase production (presumably by induction of mRNA synthesis, although other factors may play a role). Factors that stimlate collagenase production by synovial cells in monolayer culture include mononuclear cell factor (Dayer, Krane). phagocytosis (Werb,
Table
2.
Release
Synovial
of Collagenase
Fibroblasts Urate
and PGE,
Treated
Crystals
with
(units
+
from
SD) I-PGE,
COllagi?llaSe
MSUM irsimll
W/mg cell protelnl
0
0
50
0
150
Rabbit
Monosodium
(nglmg cell protein) 24 + 4
Mainardi, et al.), proteinase treatment (Werb), fusogens (Brinckerhoff et al.), and synthetic agents including phorbolesters (Brinckerhoff et al.) and cytochalasin B (Harris et al.). It has been our observation that a common denominator in stimulation of collagenase production in homogeneous fibroblast cultures may be a significant perturbation of cell membrane. To test this, we incubated rabbit synovial tibroblasts with monosodium urate (MSUM) crystals2 We have no evidence that these crystals are phagocytosed by these cells under conditions of serum-free culture. but an effect on cell membranes can be predicted by the highly charged nature of these crystals. A dose-dependent production of latent collagenase and prostaglandin Ez was found, as shown in Table. 2. Approximately 6 x IO5 cells in 60-min culture dishes were incubated at 37°C with 3-ml DMEM-0.2% lactalbumin hydrolysate with variable MSUM concentrations. After three days, the culture medium was removed and the levels of collagenase and immunoreactive PGE, were determined. Latent collagenase was activated using l.7-mM aminophenylmercuric acetate.
16 k 5
2.97
+ 0.77
In addition
to providing
a model for the study of
274 -f 36
enzyme induction
828 + 163
mechanism to explain progressive joint destruction
200
6.42
+ 1.35
300
12.57
+ 2.32
3,444
t 918
500
16.00
? 1.25
7.761
f 1,719
the inflammatory
in synovial cells, these data offer a and proliferative
by
lesion in chronic
tophaceous gout.
REFERENCES I.
Harris
D Jr, Brinckerhoff
CE,
Vater
A.
collagenase from synovial tissues in rheumatoid
Reiease of arthritis.
Panagi GS, Johnson PM, eds. lmmunopathogenesis matoid arthritis.
Chertsey (Surrey):
In:
of rheu-
Reedbooks, 40: 147-54,
RM.
Vater
CA.
Hasselbacher
P. Induction
synthesis in synovial tibro-
blasts treated with monosodium urate crystals (in press). 3. Brinckerhoff ED Jr. Inhibition in rheumatoid
979. 2. McMillan
of collagenase and prostaglanding
36.
CE,
McMillan
RM.
Dayer
J-M.
Harris
by retinoic acid of collagenase production
synovial cells. N Engl J Med
1980;303:432-
OSTEOARTHRITIS SYMPOSIUM
developed Golgi complex, and a few strands of smooth and rough endoplasmic reticulum. Endothelial cells are separated from the growth surface by sparse, finely fibrillar material, which is thought to represent newly synthesized basal lamina. The basal plasma membrane of the endothelial cells is smooth and devoid of microvilli. There are some submembrane accumulations of cytoplasmic filaments along the basal plasma membrane. Heparin, which has been reported to cause elaboration of proteolytic activities by endothelial cells, had no effect on the interaction between endothelial cells and the cartilage matrix. However, heparin significantly increased the number of cytoplasmic filaments and lysosomes. In contrast, endothelial cells grew as contact-inhibited monolayers of flattened cells on the surfaces of extracted cartilage (Fig. I). Cells were separated from the cartilage matrix by abundant basal lamina, which consisted of several discontinuous strands, probably collagenous in nature. There were a few microvilli at the basal plasma membrane, but there was no degradation or penetration of the collagenous matrix of extracted cartilage. However, when endothelial cells were stimulated by heparin, they assumed a polyhe-
dral shape and penetrated the extracted cartilage matrix with numerous microvilli and some cytoplasmic processes (Fig. 2). This penetration of the collagenous matrix was associated with tissue rarefaction and degradation of collagen fibers. This invasion of heparin stimulated endothelial cells was abolished when low concentrations of cartilage-derived AIF was added to the culture medium. AIF also significantly reduced the number of endothelial cells growing on the extracted cartilage matrix. These observations correlated with our findings that AIF inhibited endothelial cell proliferation in vitro. This growth inhibitory activity was accompanied by shape transformation and cell surface alterations. These data provide evidence that the resistance of hyaline cartilage to endothelial cell invasion is regulated in part by tissue derived proteinase inhibitors and an antiproliferative activity directed against endothelial cells. The depletion of components of AIF (e.g., protease inhibitory activity) from diseased cartilage such as osteoarthritic cartilage may explain the susceptibility of this tissue to penetration by activated endothelial cells.
REFERENCES 1. Kuettner KE, Pauli BU. Resistance of cartilage to normal and neoplastic invasion. In: Horton JE, Tarpley TM Jr, Davis WE, eds. Proceedings, Mechanism of Localized Bone loss. Special Supplement to Calcium Tissue Abstracts 1978; 251-78. 2. Eisenstein R, Kuettner KE, Neapolitan C, Soble LW,
Patterns
of regulation
of collagen
Sorgente
N. The resistance
of certain
tissues to invasion;
111.
Cartilage extracts inhibit the growth of fibroblasts and endothelial cells in culture. Am J Pathol 1975;81:337-48. 3. Horton JE, Wezeman FH, Kuettner KE. Inhibition of in vitro bone resorption by a cartilage derived anti-collagenase factor. Science 1978;199: 134244.
breakdown
in articular
cartilage
By E. D. Harris, Jr., C. A. Vater, C. E. Brinckerhofl, R. M. McMillan, and P. Hasselbacher; Dartmouth Medical School, Hanover, New Hampshire
D
IMINISHED joint function in arthritis can be equated with loss of articular cartilage. Collagen is the principal structural protein in cartilage and can be lost by mechanical or enzymic means. Recent studies have shown multiple mechanisms for collagen degradation in cartilage. Several principles are noteworthy. First, factors modulating collagen breakdown are relative, not absolute. For example, although crosslinks among collagen fibrils retard collagenolysis by specific collagenases, this slowing of the reaction is not complete; with sufficient time and high concentrations of enzymes, lysis of even the highly crosslinked collagen fibers in cartilage may occur. Secondly, cells produce enzymes; therefore, cellular
density becomes an important determinant of extracellular matrix destruction. For example, chondrocytes occupy a small colume in cartilage (O.Ol%O.l%), and even after maximal stimulation by mononuclear cell factors chondrocytes produce collagenase at an activity index less than 10% of that produced by rhetrmatoid synovial cells.’ Assuming that superficial lining cells occupy 50% of tissue volume, it is likely
Supported
in part by USPHS
20641 and by grants from Chapter
of the Arthritis
been a postdoctoral fellow
grants AM
the National Foundation.
of the Arthritis
0 1981 by Grune & Stratton, 0049J3172/81/1005-0036$01.00/0
Inc.
14780 and AM
and New Hampshire Dr.
McMillan
Foundation.
has
OSTEOARTHRITIS SYMPOSIUM
70
Table
1.
Rabbit
Articular
Chondrocytes Acid
and Synovial
on Collagenase
Fibroblasts.
and Collagenase
Effects Inhibitor
of Phorbolesters
(PMA)
and Retinoic
Production. Flbroblasts
Chondrocytes
Collagenase
Collagena%? Collagenase*
Untreated
19 k 4
PMAllO~‘M) Retinoic
acid
(10 A
PMA and retinoic
lCollagenase
M)
444
15 t 3
414 f 126
84 ? 63
f 28
960
285 i 22
10
1 /.~g collagen
10
11 k2 acid
units:
58i
degraded/hr/mg
cell protein
at
lnhibltort
Collagenase*
InhlbWxt
228 f 228
+ 84
264 i 6
7 r 0.7
390 k 162
208 t 64
336 + 102
37°C.
tlnhibitor: units of trypsin-activated collagenase inhibited/hr/mg cell protem at 37 “C
that the synovial enzymes exert SO-500-fold greater impact on certain areas of cartilage than do cartilage enzymes. As an illustration of inherent differences in cell types in response to agents stimulating collagenase and collagenase inhibitors, examine the data in Table I from chondrocytes and synovial cells in the same normal rabbit in matrix-free monolayer cultures at confluence. The third principle is that multiple different types of stimuli are sufficient to stimulate collagenase production (presumably by induction of mRNA synthesis, although other factors may play a role). Factors that stimlate collagenase production by synovial cells in monolayer culture include mononuclear cell factor (Dayer, Krane). phagocytosis (Werb,
Table
2.
Release
Synovial
of Collagenase
Fibroblasts Urate
and PGE,
Treated
Crystals
with
(units
+
from
SD) I-PGE,
COllagi?llaSe
MSUM irsimll
W/mg cell protelnl
0
0
50
0
150
Rabbit
Monosodium
(nglmg cell protein) 24 + 4
Mainardi, et al.), proteinase treatment (Werb), fusogens (Brinckerhoff et al.), and synthetic agents including phorbolesters (Brinckerhoff et al.) and cytochalasin B (Harris et al.). It has been our observation that a common denominator in stimulation of collagenase production in homogeneous fibroblast cultures may be a significant perturbation of cell membrane. To test this, we incubated rabbit synovial tibroblasts with monosodium urate (MSUM) crystals2 We have no evidence that these crystals are phagocytosed by these cells under conditions of serum-free culture. but an effect on cell membranes can be predicted by the highly charged nature of these crystals. A dose-dependent production of latent collagenase and prostaglandin Ez was found, as shown in Table. 2. Approximately 6 x IO5 cells in 60-min culture dishes were incubated at 37°C with 3-ml DMEM-0.2% lactalbumin hydrolysate with variable MSUM concentrations. After three days, the culture medium was removed and the levels of collagenase and immunoreactive PGE, were determined. Latent collagenase was activated using l.7-mM aminophenylmercuric acetate.
16 k 5
2.97
+ 0.77
In addition
to providing
a model for the study of
274 -f 36
enzyme induction
828 + 163
mechanism to explain progressive joint destruction
200
6.42
+ 1.35
300
12.57
+ 2.32
3,444
t 918
500
16.00
? 1.25
7.761
f 1,719
the inflammatory
in synovial cells, these data offer a and proliferative
by
lesion in chronic
tophaceous gout.
REFERENCES I.
Harris
D Jr, Brinckerhoff
CE,
Vater
A.
collagenase from synovial tissues in rheumatoid
Reiease of arthritis.
Panagi GS, Johnson PM, eds. lmmunopathogenesis matoid arthritis.
Chertsey (Surrey):
In:
of rheu-
Reedbooks, 40: 147-54,
RM.
Vater
CA.
Hasselbacher
P. Induction
synthesis in synovial tibro-
blasts treated with monosodium urate crystals (in press). 3. Brinckerhoff ED Jr. Inhibition in rheumatoid
979. 2. McMillan
of collagenase and prostaglanding
36.
CE,
McMillan
RM.
Dayer
J-M.
Harris
by retinoic acid of collagenase production
synovial cells. N Engl J Med
1980;303:432-