Biomarkers of Connective Tissue Aging: Biosynthesis of Fibronectin, Collagen Type 111, and Elastase J. LABAT-ROBERT, P. KERN, AND L. ROBERT Lahoratoire de Biologie du Tissu Conjonctif URA CNRS 1460 FaculrP de MPdecine UniversitP Paris XII 94010 CrPteil, France
INTRODUCTION Aging of connective tissues is a result of (a) the aging of mesenchymal cells; (b) the aging of the postsynthetic matrix; and (c) consecutive modifications of cellmatrix interactions. These modifications of connective tissues are often accompanied by the occurrence of age-dependent diseases that further strongly modify the structure and function of these tissues. Such diseases are the cardiovascular (atheroarteriosclerosis) and osteoarticular (osteoporosis, osteoarthritis) diseases, to mention only the most frequent ones. The progressive modification of the skin is also among the consequences of the above-mentioned modifications. During our studies on these pathologies, we tried to define some parameters that would enable us to quantify the modifications of the aging of connective tissues in order to compare apparently “programmed” age-dependent modifications with pathological modifications, “accelerated” aging, and in general to compare biological aging with photoaging. We shall summarize here some of these recent results. CELLULAR AGING IN CONNECTIVE TISSUES Most of the mesenchymal cells involved in the biosynthesis of extracellular matrix (ECM) are mitotic cells, and as such they are obeying the “Hayflick rule.”’ Their limited number of population doublings was well documented. It is, however, not yet clear what mechanisms determine this in vitro aging phenomenon. Most importantly, is their in vivo aging entirely or partially dependent on this exhaustion of their proliferation potential? We could show that arterial smooth muscle cells in explant cultures exhibited an age-dependent decrease of their matrix biosynthetic activity, independently of their division, which was minimized during the 24-h incubation period with the radioactive precursors.* When mitotic mesenchymal cells are cultured in a three-dimensional collagen matrix, their rate of proliferation is severely ~ u r t a i l e dWe . ~ also studied the regulation of matrix production during in vitro proliferative aging of cells according to the Hayflick model. When porcine skin fibroblasts were serially passaged, it appeared that their rate of collagen biosynthesis and accumulation decreased earlier than their proliferation ~ a p a c i t y . ~ More recent studies by V. Renaud-Salis’ have shown that in a serially passaged human skin fibroblast culture the biosynthesis of collagen was up-regulated during 16
LABAT-ROBERT ef al.: CONNECTIVE TISSUE AGING
17
1.0
0.5
I
.
. 10
.
.
.
.
0
.
.
10
CELL PASSAW
AG6 A
B
1
I
'* Cell passages
C
O
I 8-17
14-15
-. *.
Weeks
D
FIGURE 1. Progressive increase of elastase-type endopeptidase activity in (A) human aorta extracts; (B) porcine aorta smooth muscle cell cultures during serial passages; (C) human skin fibroblast cultures during serial passages; and (D) skin extracts from mice, in uiuo aging. [Reprinted by permission from (A, B) Robert ef a/.,'*(C) Homsy er a/.,'3and (D) Boyer ef n1.141
1
0
L 2
12
16
22
(mOiilh8)
FIGURE 2. Progressive increase with age of fibronectin biosynthesis in skin explant cultures of mice. (Reprinted by permission from Boyer ef a/.'6)
ANNALS NEW YORK ACADEMY OF SCIENCES
18
PROPORTION OF COLLAGEN TYPE 111
2-3
12-13
FIGURE 3. Progressive increase of collagen III/I (Reprinted by permission from Boyer ef a/.'6)
16-17
22
Ag.(monW
+ 111 ratio in mouse skin explant cultures.
the decline of cell proliferation. We also could show that fibronectin biosynthesis was progressively up-regulated during serial passages of human skin fibroblasts and vascular smooth muscle cell^.^*^ It appears, therefore, that cell proliferation and matrix biosynthesis may well be independently regulated. Our studies of the elastin receptor suggested that cell death in matrix-rich organs could well be due more to apoptosis produced by progressive increase in intracellular calcium and loss of its homeostatic regulation than to exhaustion of their division potential.' Because of these considerations, the study of cells alone can hardly be used for the characterization of the gradual aging of connective tissues.
AGE-DEPENDENT MODIFICATIONS OF THE ECM The age-dependent modifications of the macromolecules of ECM can be divided into modifications (qualitative and quantitative) of their rate of synthesis and postsynthetic modifications of matrix macromolecules. Such postsynthetic modifications are the progressive cross-linking of collagen fibers described by Verzafl and attributed by the team of Cerami and Vlassara to the Maillard r e a ~ t i o n Another .~ example is the progressive accumulation of lipids and calcium in elastic fibers.lO-'lThe proteolytic degradation of matrix macromolecules is another example of postsynthetic modification. The age-dependent up-regulation of the elastase-type proteases in vivo and in vitro is one of the measurable factors underlying this process. As shown in FIGURE 1, this up-regulation of
LABAT-ROBERT et al.: CONNECTIVE TISSUE AGING
19
B FIGURE 4. Skin biopsy from a Werner patient (A), as compared to a normal age-matched control (B).Immunofluorescence for fibronectin.’’
20
ANNALS NEW YORK ACADEMY OF SCIENCES
elastase-type endopeptidases could be confirmed both for a membrane-bound smooth muscle cell serine-protease12 and for a metalloendopeptidase of human skin fibr0b1asts.l~ More recently this could also be shown for a mouse skin elastase-type end~peptidase.'~ Therefore, this is one of the parameters that can be used for the quantitation of the in vivo aging of connective tissues. These findings were recently confirmed in our laboratory by V. Rena~d-Salis.~ As far as the modifications of the matrix biosynthetic processes are concerned, we could show that the above-mentioned parallelism between in uitro and in uiuo aging existed for at least two such biosynthetic processes: the progressive increase in fibronectin biosynthesis (FIG.2)I5*I6and the progressive increase of the collagen type III/I I11 r a t i ~ . ' ~ As , ~ ' shown in FIGURE 3, this increase could be confirmed in experiments on cell cultures and on ex vivo skin explant cultures. It appears, therefore, that these two independent parameters can be used for the characterization of in uitro and in uiuo aging of connective tissues such as the skin. Because of measurable differences in the age-dependent decline of the different physiological functions of connective tissues, it is conceivable that these parameters might show also different rates of decline in different connective tissues. This could, however, become a further asset in their use as comparative parameters in aging studies.
+
RELATIONSHIP BETWEEN AGING OF CONNECTIVE TISSUES AND THEIR AGE-DEPENDENT PATHOLOGIES When the above-mentioned parameters were studied in age-dependent diseases, it could be shown that their progression was accelerated in the same direction as during nonpathological aging. A strongly accelerated increase of fibronectin, collagen type III/I ratio could be demonstrated in diabete~''.'~and UV induced "photoaging of the skin."20 A Werner skin biopsy also revealed a strongly increased fibronectin immunofluorescence (FIG.4). Similarly, an increased elastasetype protease activity could be demonstrated in aorta extracts of rabbits with experimentally induced atherosclerosis.2' The above-mentioned age-dependent increase of human aorta elastase activity may be the result of its age-dependent up-regulation and the acceleration of this increase by progressive atherosclerosis.
DISCUSSION, CONCLUSIONS It appears from the above-summarized results that the determination in skin biopsy explant cultures of the rate of fibronectin and collagen type III/I + 111 synthesis together with elastase-type endopeptidase determination yield a reliable measure of in uivo biological aging. These parameters satisfy the prerequisites of changing progressively in the same direction in in vitro and in uiuo aging. The
LABAT-ROBERT et al.: CONNECTIVE TISSUE AGING
21
acceleration of t h e increase of these parameters in age-dependent diseases such as diabetes and Werner syndrome and in photo-aging confirms their value as valid parameters of connective tissue aging.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10.
1I.
12. 13. 14. 15. 16. 17. 18. 19.
HAYFLICK. L. 1977. The cellular basis for biological aging. In Handbook of the Biology of Aging. C. E. Finch & L. Haytlick, Eds.: 159-186. van Nostrand Reinhold. New York, NY. Y. COURTOIS & L. ROBERT.1976. Age-dependence of the MOCZAR, M., J. OUZILOU, biosynthesis of intercellular matrix macromolecules of rabbit aorta in organ culture and cell culture. Gerontology 22: 461-472. COULOMB, B. & L. DUBERTRET. 1988. Reconstruction cutanCe et communications tissulaires. Medecine/Sciences 4: 101- 108. EL NABOUT, R., M. MARTIN.J . REMY.P. KERN,L. ROBERT& C. LAFUMA.1989. Collagen synthesis and deposition in cultured fibrolasts from subcutaneous radiationinduced fibrosis. Modification as function of ageing. Matrix 9: 41 1-420. RENAUD-SALIS, V. 1991. Alteration fonctionnelle induite par irradiation gamma ( 1 . 2 et 3 gray) du fibroblaste de derme humain au cours du vieillissement cellulaire in vitro. Th&e Dr. es Sci.. UniversitC Paris-Sud. F. CRECHET & J. REMY.1990. Fibronectin MARTIN. M., R. EL NABOUT,C. LAFUMA, and collagen gene expression during in vitro aging of pig skin fibroblasts. Exp. Cell Res. 191: 8-13. ROBERT,L., M. P. JACOB& J. LABAT-ROBERT. 1992. Cell-matrix interactions in the genesis of arteriosclerosis and atheroma: Effect of aging. Ann. N.Y. Acad. Sci. This volume. VERZAR. F. 1964. Aging of the collagen fiber. In International Review of Connective Tissue Research, Vol. 2. D. A. Hall Ed.: 243-300. Academic Press. New York, NY. VLASSARA, H. 1990. Advanced nonenzymatic tissue glycosylation: Cell-mediated interactions implicated in the complications associated with diabetes and aging. Blood Purif. 8: 223-232. ROBERT.L. & A. M. ROBERT.1980. Elastin, elastase and arteriosclerosis. In Frontiers of Matrix Biology, Vol. 8. A. M. Robert 19L. Robert, Eds.: 130-173. S. Karger. Basel. ROBERT.L. & W. HORNEBECK. 1989. Elastin and Elastases, Vols. 1-2. CRC Press. Boca Raton, FL. ROBERT.L.. J. LABAT-ROBERT & W. HORNEBECK. 1986. Aging and atherosclerosis. In Atherosclerosis Reviews, Vol. 14. A. M. Cotto & R. Paoletti, Eds.: 143-170. Raven Press. New York, NY. HOMSY,R., P. PELLETIER-LEBON, J. M. T I X I E R , G. GODEAU,L. ROBERT& W. HORNEBECK. 1988. Characterization of human skin fibroblast elastase activity. J . Invest. Dermatol. 91: 472-477. & J. LABAT-ROBERT. 1992. Age-dependent increase of BOYER,B.. A. FOURTANIER elastase-type endopeptidase activity of mouse skin. Gerontology. Submitted for publication. J., J. P. POTAZMAN, J . C. DEROUETTE & L. ROBERT.1981. AgeLABAT-ROBERT, dependent increase of human plasma fibronectin. Cell Biol. Int. Rep. 5: 969-973. BOYER.B., P. KERN,A. FOURTANIER & J. LABAT-ROBERT. 1991. Age-dependent variations of the biosynthesis of fibronectin and fibrous collagens in mouse skin. Exp. Gerontol. 26 375-383. KERN,P., B. SEBERT& L. ROBERT.1986. Increased type Ill/type I collagen ratios in diabetic human conjunctival biopsies. Clin. Physiol. Biochem. 4 113-1 19. LABAT-ROBERT J. & L. ROBERT.1990. Modulation of collagens and fibronectin expression during ageing and age-related diseases. In The Theoretical Basis of Aging Research, Vol. 2. L. Robert & G. Hofecker, Eds.: 163-168. Vienna Aging Series. 1990. Modifications of the relative rates KERN,P., C. ASSELOT& J. LABAT-ROBERT.
22
ANNALS NEW YORK ACADEMY OF SCIENCES
of biosynthesis of collagens and fibronectin in diabetes. I n Structure, Molecular Biology, and Pathology of Collagen. Ann. N.Y. Acad. Sci. 580: 576-578. 20. BOYER,B., A. FOURTANIER, P. KERN& J. LABAT-ROBERT. 1992. UVA and UVB induced changes in collagen and fibronectin biosynthesis in the skin of hairless mice. J. Photochem. Photobiol. 14: 247-259. 1982. Variation of 21. JACOB, M. P., D. BRECHEMIER, L. ROBERT62 W. HORNEBECK. elastase-type protease activity and elastin biosynthesis in rabbit aorta induced by cholesterol diet. Artery 1 0 310-316.
INTRODUCTION Aging of connective tissues is a result of (a) the aging of mesenchymal cells; (b) the aging of the postsynthetic matrix; and (c) consecutive modifications of cellmatrix interactions. These modifications of connective tissues are often accompanied by the occurrence of age-dependent diseases that further strongly modify the structure and function of these tissues. Such diseases are the cardiovascular (atheroarteriosclerosis) and osteoarticular (osteoporosis, osteoarthritis) diseases, to mention only the most frequent ones. The progressive modification of the skin is also among the consequences of the above-mentioned modifications. During our studies on these pathologies, we tried to define some parameters that would enable us to quantify the modifications of the aging of connective tissues in order to compare apparently “programmed” age-dependent modifications with pathological modifications, “accelerated” aging, and in general to compare biological aging with photoaging. We shall summarize here some of these recent results. CELLULAR AGING IN CONNECTIVE TISSUES Most of the mesenchymal cells involved in the biosynthesis of extracellular matrix (ECM) are mitotic cells, and as such they are obeying the “Hayflick rule.”’ Their limited number of population doublings was well documented. It is, however, not yet clear what mechanisms determine this in vitro aging phenomenon. Most importantly, is their in vivo aging entirely or partially dependent on this exhaustion of their proliferation potential? We could show that arterial smooth muscle cells in explant cultures exhibited an age-dependent decrease of their matrix biosynthetic activity, independently of their division, which was minimized during the 24-h incubation period with the radioactive precursors.* When mitotic mesenchymal cells are cultured in a three-dimensional collagen matrix, their rate of proliferation is severely ~ u r t a i l e dWe . ~ also studied the regulation of matrix production during in vitro proliferative aging of cells according to the Hayflick model. When porcine skin fibroblasts were serially passaged, it appeared that their rate of collagen biosynthesis and accumulation decreased earlier than their proliferation ~ a p a c i t y . ~ More recent studies by V. Renaud-Salis’ have shown that in a serially passaged human skin fibroblast culture the biosynthesis of collagen was up-regulated during 16
LABAT-ROBERT ef al.: CONNECTIVE TISSUE AGING
17
1.0
0.5
I
.
. 10
.
.
.
.
0
.
.
10
CELL PASSAW
AG6 A
B
1
I
'* Cell passages
C
O
I 8-17
14-15
-. *.
Weeks
D
FIGURE 1. Progressive increase of elastase-type endopeptidase activity in (A) human aorta extracts; (B) porcine aorta smooth muscle cell cultures during serial passages; (C) human skin fibroblast cultures during serial passages; and (D) skin extracts from mice, in uiuo aging. [Reprinted by permission from (A, B) Robert ef a/.,'*(C) Homsy er a/.,'3and (D) Boyer ef n1.141
1
0
L 2
12
16
22
(mOiilh8)
FIGURE 2. Progressive increase with age of fibronectin biosynthesis in skin explant cultures of mice. (Reprinted by permission from Boyer ef a/.'6)
ANNALS NEW YORK ACADEMY OF SCIENCES
18
PROPORTION OF COLLAGEN TYPE 111
2-3
12-13
FIGURE 3. Progressive increase of collagen III/I (Reprinted by permission from Boyer ef a/.'6)
16-17
22
Ag.(monW
+ 111 ratio in mouse skin explant cultures.
the decline of cell proliferation. We also could show that fibronectin biosynthesis was progressively up-regulated during serial passages of human skin fibroblasts and vascular smooth muscle cell^.^*^ It appears, therefore, that cell proliferation and matrix biosynthesis may well be independently regulated. Our studies of the elastin receptor suggested that cell death in matrix-rich organs could well be due more to apoptosis produced by progressive increase in intracellular calcium and loss of its homeostatic regulation than to exhaustion of their division potential.' Because of these considerations, the study of cells alone can hardly be used for the characterization of the gradual aging of connective tissues.
AGE-DEPENDENT MODIFICATIONS OF THE ECM The age-dependent modifications of the macromolecules of ECM can be divided into modifications (qualitative and quantitative) of their rate of synthesis and postsynthetic modifications of matrix macromolecules. Such postsynthetic modifications are the progressive cross-linking of collagen fibers described by Verzafl and attributed by the team of Cerami and Vlassara to the Maillard r e a ~ t i o n Another .~ example is the progressive accumulation of lipids and calcium in elastic fibers.lO-'lThe proteolytic degradation of matrix macromolecules is another example of postsynthetic modification. The age-dependent up-regulation of the elastase-type proteases in vivo and in vitro is one of the measurable factors underlying this process. As shown in FIGURE 1, this up-regulation of
LABAT-ROBERT et al.: CONNECTIVE TISSUE AGING
19
B FIGURE 4. Skin biopsy from a Werner patient (A), as compared to a normal age-matched control (B).Immunofluorescence for fibronectin.’’
20
ANNALS NEW YORK ACADEMY OF SCIENCES
elastase-type endopeptidases could be confirmed both for a membrane-bound smooth muscle cell serine-protease12 and for a metalloendopeptidase of human skin fibr0b1asts.l~ More recently this could also be shown for a mouse skin elastase-type end~peptidase.'~ Therefore, this is one of the parameters that can be used for the quantitation of the in vivo aging of connective tissues. These findings were recently confirmed in our laboratory by V. Rena~d-Salis.~ As far as the modifications of the matrix biosynthetic processes are concerned, we could show that the above-mentioned parallelism between in uitro and in uiuo aging existed for at least two such biosynthetic processes: the progressive increase in fibronectin biosynthesis (FIG.2)I5*I6and the progressive increase of the collagen type III/I I11 r a t i ~ . ' ~ As , ~ ' shown in FIGURE 3, this increase could be confirmed in experiments on cell cultures and on ex vivo skin explant cultures. It appears, therefore, that these two independent parameters can be used for the characterization of in uitro and in uiuo aging of connective tissues such as the skin. Because of measurable differences in the age-dependent decline of the different physiological functions of connective tissues, it is conceivable that these parameters might show also different rates of decline in different connective tissues. This could, however, become a further asset in their use as comparative parameters in aging studies.
+
RELATIONSHIP BETWEEN AGING OF CONNECTIVE TISSUES AND THEIR AGE-DEPENDENT PATHOLOGIES When the above-mentioned parameters were studied in age-dependent diseases, it could be shown that their progression was accelerated in the same direction as during nonpathological aging. A strongly accelerated increase of fibronectin, collagen type III/I ratio could be demonstrated in diabete~''.'~and UV induced "photoaging of the skin."20 A Werner skin biopsy also revealed a strongly increased fibronectin immunofluorescence (FIG.4). Similarly, an increased elastasetype protease activity could be demonstrated in aorta extracts of rabbits with experimentally induced atherosclerosis.2' The above-mentioned age-dependent increase of human aorta elastase activity may be the result of its age-dependent up-regulation and the acceleration of this increase by progressive atherosclerosis.
DISCUSSION, CONCLUSIONS It appears from the above-summarized results that the determination in skin biopsy explant cultures of the rate of fibronectin and collagen type III/I + 111 synthesis together with elastase-type endopeptidase determination yield a reliable measure of in uivo biological aging. These parameters satisfy the prerequisites of changing progressively in the same direction in in vitro and in uiuo aging. The
LABAT-ROBERT et al.: CONNECTIVE TISSUE AGING
21
acceleration of t h e increase of these parameters in age-dependent diseases such as diabetes and Werner syndrome and in photo-aging confirms their value as valid parameters of connective tissue aging.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10.
1I.
12. 13. 14. 15. 16. 17. 18. 19.
HAYFLICK. L. 1977. The cellular basis for biological aging. In Handbook of the Biology of Aging. C. E. Finch & L. Haytlick, Eds.: 159-186. van Nostrand Reinhold. New York, NY. Y. COURTOIS & L. ROBERT.1976. Age-dependence of the MOCZAR, M., J. OUZILOU, biosynthesis of intercellular matrix macromolecules of rabbit aorta in organ culture and cell culture. Gerontology 22: 461-472. COULOMB, B. & L. DUBERTRET. 1988. Reconstruction cutanCe et communications tissulaires. Medecine/Sciences 4: 101- 108. EL NABOUT, R., M. MARTIN.J . REMY.P. KERN,L. ROBERT& C. LAFUMA.1989. Collagen synthesis and deposition in cultured fibrolasts from subcutaneous radiationinduced fibrosis. Modification as function of ageing. Matrix 9: 41 1-420. RENAUD-SALIS, V. 1991. Alteration fonctionnelle induite par irradiation gamma ( 1 . 2 et 3 gray) du fibroblaste de derme humain au cours du vieillissement cellulaire in vitro. Th&e Dr. es Sci.. UniversitC Paris-Sud. F. CRECHET & J. REMY.1990. Fibronectin MARTIN. M., R. EL NABOUT,C. LAFUMA, and collagen gene expression during in vitro aging of pig skin fibroblasts. Exp. Cell Res. 191: 8-13. ROBERT,L., M. P. JACOB& J. LABAT-ROBERT. 1992. Cell-matrix interactions in the genesis of arteriosclerosis and atheroma: Effect of aging. Ann. N.Y. Acad. Sci. This volume. VERZAR. F. 1964. Aging of the collagen fiber. In International Review of Connective Tissue Research, Vol. 2. D. A. Hall Ed.: 243-300. Academic Press. New York, NY. VLASSARA, H. 1990. Advanced nonenzymatic tissue glycosylation: Cell-mediated interactions implicated in the complications associated with diabetes and aging. Blood Purif. 8: 223-232. ROBERT.L. & A. M. ROBERT.1980. Elastin, elastase and arteriosclerosis. In Frontiers of Matrix Biology, Vol. 8. A. M. Robert 19L. Robert, Eds.: 130-173. S. Karger. Basel. ROBERT.L. & W. HORNEBECK. 1989. Elastin and Elastases, Vols. 1-2. CRC Press. Boca Raton, FL. ROBERT.L.. J. LABAT-ROBERT & W. HORNEBECK. 1986. Aging and atherosclerosis. In Atherosclerosis Reviews, Vol. 14. A. M. Cotto & R. Paoletti, Eds.: 143-170. Raven Press. New York, NY. HOMSY,R., P. PELLETIER-LEBON, J. M. T I X I E R , G. GODEAU,L. ROBERT& W. HORNEBECK. 1988. Characterization of human skin fibroblast elastase activity. J . Invest. Dermatol. 91: 472-477. & J. LABAT-ROBERT. 1992. Age-dependent increase of BOYER,B.. A. FOURTANIER elastase-type endopeptidase activity of mouse skin. Gerontology. Submitted for publication. J., J. P. POTAZMAN, J . C. DEROUETTE & L. ROBERT.1981. AgeLABAT-ROBERT, dependent increase of human plasma fibronectin. Cell Biol. Int. Rep. 5: 969-973. BOYER.B., P. KERN,A. FOURTANIER & J. LABAT-ROBERT. 1991. Age-dependent variations of the biosynthesis of fibronectin and fibrous collagens in mouse skin. Exp. Gerontol. 26 375-383. KERN,P., B. SEBERT& L. ROBERT.1986. Increased type Ill/type I collagen ratios in diabetic human conjunctival biopsies. Clin. Physiol. Biochem. 4 113-1 19. LABAT-ROBERT J. & L. ROBERT.1990. Modulation of collagens and fibronectin expression during ageing and age-related diseases. In The Theoretical Basis of Aging Research, Vol. 2. L. Robert & G. Hofecker, Eds.: 163-168. Vienna Aging Series. 1990. Modifications of the relative rates KERN,P., C. ASSELOT& J. LABAT-ROBERT.
22
ANNALS NEW YORK ACADEMY OF SCIENCES
of biosynthesis of collagens and fibronectin in diabetes. I n Structure, Molecular Biology, and Pathology of Collagen. Ann. N.Y. Acad. Sci. 580: 576-578. 20. BOYER,B., A. FOURTANIER, P. KERN& J. LABAT-ROBERT. 1992. UVA and UVB induced changes in collagen and fibronectin biosynthesis in the skin of hairless mice. J. Photochem. Photobiol. 14: 247-259. 1982. Variation of 21. JACOB, M. P., D. BRECHEMIER, L. ROBERT62 W. HORNEBECK. elastase-type protease activity and elastin biosynthesis in rabbit aorta induced by cholesterol diet. Artery 1 0 310-316.
