EXPERIMENTAL
AND
Salt-Soluble KHAY
MOLECULAR
Collagen and Elastin in the Human Aorta and Pulmonary Artery
001,’ M. PAIGE LACY,’
‘Malaghan ‘Medicine,
55, 25-29 (1991)
PATHOLOGY
PAUL AND WILLIAM
Institute of Medical and 4Pathology,
Research Wellington Received
F. DAVIS,’ REINHOLD E. STEHBENS”~
KITTELBERGER,~
and Departments of ‘Obstetrics School of Medicine, Wellington,
& Gynaecology, New Zealand
December
4, 1990
Collagen and elastin, the major structural components of blood vessels, have a very low turnover. In disease, this rate may be increased and an elevation of the tissue concentration of the soluble degradation fragments might be anticipated. In this preliminary study the concentration of extractable collagen and elastin in the aorta and pulmonary artery of eight human subjects postmortem was determined. The proportion of pulmonary artery collagen and elastin that was soluble was generally either equal to or greater than that in the abdominal aorta. The fraction of collagen that was salt extractable was larger than the soluble elastin fraction. 0 1991 Academic PKSS. Inc.
INTRODUCTION The principal extracellular constituents of blood vessels are collagen and elastin (Partridge, 1979). These are essential for the structural integrity of the vessel wall; collagen is chiefly responsible for tensile strength and elastin for elastic recoil (Cox, 1978). In atherosclerosis, alterations in these components occur with a resultant change in physiological function. In particular, a pronounced increase in collagen has been reported (Barnes, 1985) and a loss of elastin has been demonstrated histologically and biochemically (Campa et al., 1987). In this paper the concentrations of salt-extractable collagen and elastin in human aortae and pulmonary arteries are presented. The presence of extractable cross-linked elastin has previously been reported in experimental animal vessels (Davis and Stehbens, 1986) but not in human tissues. These polypeptides are found in both the aorta (a vessel with blood at high pressure and rapid flow) and the pulmonary artery (low-pressure vessel). The soluble collagen as determined by hydroxyproline measurements represents turnover of the molecule but the soluble elastin is measured by desmosine assays and so is a reflection of the degradation of mature fibrous elastin. MATERIALS
AND METHODS
Tissue Samples of abdominal aorta and pulmonary artery were individuals (see Table I for details) at autopsy. Specimens were obtained from two of these individuals. The autopsies than 24 hr after death and the tissues used were free of any tions in which leucocytes might be present.
obtained from eight from the aortic arch were performed less inflammatory condi-
Extraction of Soluble Eiastin and Collagen Loose, perivascular fascia and fat were removed and the tissue samples were washed with cold distilled water to remove blood and then blotted. The tissue was 25 0014-4800191 $3.00 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
26
001
ET AL.
freeze-dried, placed in a liquid nitrogen-cooled mortar, and ground with a pestle. The samples were lyophilized and weighed. The method of extraction was essentially as previously described (Davis and Mackle, 1981) but with the following modifications. Centrifugation following extraction at 4°C was performed for 45 min at 22,000g. Supematant fractions were pooled and concentrated by ultrafiltration on an Amicon YCOS membrane. Protein was determined using the method of Lowry et al. (1951). The final pellet was washed with distilled water, lyophilized, and weighed. All procedures were performed at 4°C unless otherwise stated. Amino Acid Analysis Aliquots of protein were hydrolyzed in vucuo in constant boiling hydrochloric acid for 65 hr at 110°C. The hydrolysate was dried over sodium hydroxide and redissolved in 50 mM hydrochloric acid. Amino acid analysis was performed on a Waters Picotag amino acid analyzer using the procedure of Negro ef al. (1987). Hydroxyproline was assayed using the method of Woessner (1976). Collagen content was determined from the hydroxyproline using a conversion factor of 7.69 (Miller and Gay, 1982) and elastin was calculated from the desmosine and isodesmosine content using a conversion factor of 1087 (Foster, 1982). The total content of both collagen and elastin is calculated by summing the content in the pellet (insoluble) and that in the extract (soluble). Desmosine
Immunoassay
Covalent linkage of desmosine to gelatin was performed following the procedure by Robins (1982). The conjugate contained 63-70 nM desmosine/mg gelatin as determined by amino acid analysis. The immunoassay was adapted from the procedure described by Gunja-Smith (1985) and performed at room temperature. Briefly, 60 wells (inner wells only) of the microtiter plates were coated with 100 ~1 per well of desmosine-gelatin conjugate (4-8 mg/ml) dissolved in 0.02% sodium azide in phosphate-buffered saline (PBS-AZ). After incubation for 18 hr the plates were washed with 0.05% Tween 20 in PBS (PBS-Tw) and stored desiccated. One hundred-microliter aliquots of antidesmosine (1:30,000 in 1% bovine serum albumin in PBS-AZ (PBS-BSA-AZ) were incubated with 100 p1 of sample or standard for 2 hr. Fifty microliters were transferred to the coated wells and incubated for 30 min. After washing three times with PBS-Tw, 100 ~1 of biotinylated antirabbit antibody (I:1000 in PBSBSA-AZ) was added to each well. After 30 min of incubation, the plates were washed. Streptavidin-biotin-peroxidase complex (1: 1000 in PBS-BSA-AZ) (100 pl) was added and incubated a further 30 min. Following washing, 100 pl of o-phenylenediamine substrate (Wolters et al., 1976) was added and color was developed for 5-10 min. After stopping the reaction with 100 pl 2.5 M sulphuric acid, absorbance was measured at 490 mm. RESULTS
AND DISCUSSION
The concentrations of collagen and elastin present in the saline extract from the human aorta and pulmonary artery samples are shown in Table I. The rate of degradation of the fibrous forms of these proteins is normally low but in diseases such as atherosclerosis this rate increases (Robert and Robert, 1980; Campa et al.,
SALT-SOLUBLE Salt-Extractable
COLLAGEN
AND ELASTIN
27
TABLE I Collagen and Elastin from Human Aorta and Pulmonary Artery Soluble collagen (X 103)/ Total collagen
Soluble elastin (X 103)/ Total elastin
Male (33 years) Abdominal aorta Pulmonary artery
9.75 9.42
1.45 6.29
Male (52 years) Aortic arch Abdominal aorta Pulmonary artery
8.53 5.17 5.78
0.93 3.12 1.55
Male (60 years) Abdominal aorta Pulmonary artery
1.44 21.01
Male (65 years) Abdominal aorta Pulmonary artery
3.66 4.62
Male (70 years) Abdominal aorta Pulmonary artery
25.06 20.22
0 0
Male (75 years) Abdominal aorta Pulmonary artery
5.42 32.1
0 3.84
Female (75 years) Aortic arch Abdominal aorta Pulmonary artery
2.49 2.19 14.79
0.31 0.54 0.74
Female (77 years) Abdominal aorta Pulmonary artery
3.72 13.14
0.49 1.51
0 0 2.07 0.72
Note. There is a significant difference (P:2 a cO.05) for the soluble collagen content of abdominal aorta and pulmonary artery (Mann-Whitney test).
1987). Elevated blood pressure and flow are known to influence the turnover rate of both proteins in blood vessels (Wolinsky, 1970; Ooshima et al., 1974; Davis and Stehbens, 1986; Keeley and Johnson, 1986; Ito et al., 1987; Ryan et al., 1988). These parameters are different in the aorta and the pulmonary artery and so may contribute to a differing rate of degradation. There are a number of factors that may influence the data obtained and the subsequent interpretation. After death, the tissues undergo considerable degradation. In order to restrict these undesirable influences in this work to a minimum, the vessels were obtained as soon as possible postmortem. The soluble collagen as a fraction of the total collagen in the pulmonary artery is higher than that in the aorta. However, in some individuals the concentrations were similar. This may be a reflection of an actual difference in the turnover rate or it could also be due to differences in architecture or in the insoluble collagen content of the vessels. Another factor that will influence these proportions is the rate of removal of these saline-soluble peptides. The role of diffusion through the tissue into the blood or into the lymphatic vessels is unknown. This property may
28
001 ET AL.
differ between the two vessels and also temporally, individually, and from area to area. It may also be affected by hypertension. The salt-soluble elastin in these same vessels is lower than the corresponding collagen concentration. In several samples none could be detected. The elastin concentration in body fluids such as urine is also less than that of collagen (McIntosh et al., 1989). As with the collagen, the soluble elastin concentration tends to be higher in the pulmonary artery than in the aorta although once again there are exceptions. In this determination, the specific elastin crosslink desmosine is assayed and so the soluble elastin is that which has resulted from the degradation of the fibrous polymer. Differences in the rate of degradation and the rate of removal of the soluble elastin from the vessel wall may be partially responsible for the variations observed here. Desmosine, which is unevenly distributed in elastin, has been used as the marker. If the rate of removal of crosslinked peptides differs from that of hydrophobic fragments, then the measurement of desmosine in tissue extracts would not be indicative of elastin solubilization. Severity of degeneration is expected to be variable in different regions of the vasculature, in different individuals, and at different ages. The presence of soluble degraded fragments of elastin within human vascular tissue has not been reported previously. In experimental arteriovenous tistulae there is a proliferative lesion in the venous tissue manifesting many of the features of human atherosclerosis (Stehbens, 1974). In these, there is also increased extractable collagen and elastin (Smith et al., 1976; Davis and Stehbens, 1986). This preliminary study has shown that soluble collagen and elastin are present and can be quantified in blood vessels. The presence of these, particularly the crosslinked elastin fragments, is suggestive of increased vascular fragility. The actual concentration in the tissue is likely to be influenced by a number of contributory parameters. The determination of the rate of degradation requires the development of technology so that it can be monitored continuously. ACKNOWLEDGMENTS This work was supported by grants from the Wellington Medical Research Foundation, the Medical Research Council of New Zealand, the National Heart Foundation of New Zealand, the New Zealand Dairy Board, and the New Zealand Meat Research and Development Council.
REFERENCES M. J. (1985). Collagens in atherosclerosis. Collagen Relat. Res. 5, 65-97. CAMPA, J. S., GREENHALGH, R. M., and POWELL, J. T. (1987). Elastin degradation in abdominal aortic aneurysms. Atherosclerosis 65, 13-21. Cox, R. H. (1978). Passive mechanics and connective tissue composition of canine arteries. Am. J. Physiol. 234, H533-H54 1. DAVIS, P. F., and MACKLE, Z. M. (1981). A simple procedure for the separation of insoluble collagen and elastin. Anal. Biochem. 115, 11-17. DAVIS, P. F., and STEHBENS, W. E. (1986). The biochemical composition of haemodynamically stressed vascular tissue. 2. The concentrations of protein and connective tissue components in the salt extracts of experimental arteriovenous tistulae. Afherosclerosis 60, 55-59. FOSTER, J. A. (1982). Elastin structure and biosynthesis: An overview. In “Methods in Enzymology” (L. W. Cunningham and D. W. Frederiksen, Eds.), Vol. 82, pp. 559-570. Academic Press, San Diego. GUNJA-SMITH, Z. (1985). An enzyme-linked immunosorbent assay to quantitate the elastin crosslink desmosine in tissue and urine samples. Anal. Biochem. 147, 258-264. BARNES,
SALT-SOLUBLE
COLLAGEN
AND
ELASTIN
29
H., KWAN, C. Y., and DANIEL, E. E. (1987). Elastin and elastase-like enzyme changes in aorta of rat with malignant hypertension. Exp. Mol. Pathol. 47, 26-36. KEELEY, F. W., and JOHNSON, D. J. (1986). The effect of developing hypertension on the synthesis and accumulation of elastin in the aorta of the rat. Biochem. Cell Biol. 64, 38-43. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MCINTOSH, C. J., DAVIS, P. F., RYAN, P. A., and STEHBENS, W. E. (1989). Urinary excretion of connective tissue protein markers in arterial disease. Angiology 40, 814-817. MILLER, E. J., and GAY, S. (1982). Collagen: An overview. In “Methods in Enzymology” (L. W. Cunningham and D. W. Frederiksen, Eds.), Vol. 82, pp. 3-32. Academic Press, New York. NEGRO, A., GARBISA, S., GOTTE, L., and SPINA, M. (1987). The use of reverse-phase highperformance liquid chromatography and precolumn derivatization with dansyl chloride for quantitation of specific amino acids in collagen and elastin. Anal. Biochem. 160, 39-46. OOSHIMA, A., FULLER, G. C., CARDINALE, C. J., SPECTOR, S., and UDENFRIEND, S. (1974). Increased collagen synthesis in blood vessels of hypertensive rats and its reversal by antihypertensive agents. Proc. Natl. Acad. Sci. 71, 3019-3023. PARTRIDGE, S. M. (1979). Chemistry and structure of the walls of the large arteries. In “Hemodynamics and the Blood Vessel Wall” (W. E. Stehbens, Ed.), pp. 238-293. Thomas, Springfield, IL. ROBERT, L., and ROBERT, A. M. (Eds.) (1980). Elastin, elastase and arteriosclerosis. In “Frontiers of Matrix Biology,” pp. 130-173. Karger, Basel. ROBINS, S. P. (1982). An enzyme-linked immunoassay for the collagen cross-link pyridinoline. Biothem. J. 207, 617-620. RYAN, P. A., DAVIS, P. F., and STEHBENS, W. E. (1988). The biochemical composition of haemodynamically stressed vascular tissue. 3. The collagen composition of experimental arteriovenous fistulae. Atherosclerosis 71, 157-163. SMITH, R. A., STEHBENS, W. E., and WEBER, P. (1976). Hemodynamically-induced increase in soluble collagen in the anastomosed vein of experimental arteriovenous fistulae. Atherosclerosis 23, ITO,
429-436. STEHBENS,
W. E. (1974). Haemodynamic production of lipid deposition, intimal tears, mural dissection and thrombosis in the blood vessel wall. Proc. R. Sot. London B. 185, 357-373. WOESSNER, J. F., JR. (1976). Determination of hydroxyproline in connective tissue. In “The Methodology of Connective Tissue Research” (D. A. Hall, Ed.), pp. 227-233. Joynson-Bruvvers, Oxford. WOLINSKY, H. (1970). Response of the rat aortic media to hypertension. Circ. Res. 26, 507-522. WOLTERS, G., KUUPERS, L., KACAKI, J., and SCHUURS,A. (1976). Solid-phase enzyme-immunoassay for detection of hepatitis B surface antigen. J. Clin. Pathol. 29, 873-879.
AND
Salt-Soluble KHAY
MOLECULAR
Collagen and Elastin in the Human Aorta and Pulmonary Artery
001,’ M. PAIGE LACY,’
‘Malaghan ‘Medicine,
55, 25-29 (1991)
PATHOLOGY
PAUL AND WILLIAM
Institute of Medical and 4Pathology,
Research Wellington Received
F. DAVIS,’ REINHOLD E. STEHBENS”~
KITTELBERGER,~
and Departments of ‘Obstetrics School of Medicine, Wellington,
& Gynaecology, New Zealand
December
4, 1990
Collagen and elastin, the major structural components of blood vessels, have a very low turnover. In disease, this rate may be increased and an elevation of the tissue concentration of the soluble degradation fragments might be anticipated. In this preliminary study the concentration of extractable collagen and elastin in the aorta and pulmonary artery of eight human subjects postmortem was determined. The proportion of pulmonary artery collagen and elastin that was soluble was generally either equal to or greater than that in the abdominal aorta. The fraction of collagen that was salt extractable was larger than the soluble elastin fraction. 0 1991 Academic PKSS. Inc.
INTRODUCTION The principal extracellular constituents of blood vessels are collagen and elastin (Partridge, 1979). These are essential for the structural integrity of the vessel wall; collagen is chiefly responsible for tensile strength and elastin for elastic recoil (Cox, 1978). In atherosclerosis, alterations in these components occur with a resultant change in physiological function. In particular, a pronounced increase in collagen has been reported (Barnes, 1985) and a loss of elastin has been demonstrated histologically and biochemically (Campa et al., 1987). In this paper the concentrations of salt-extractable collagen and elastin in human aortae and pulmonary arteries are presented. The presence of extractable cross-linked elastin has previously been reported in experimental animal vessels (Davis and Stehbens, 1986) but not in human tissues. These polypeptides are found in both the aorta (a vessel with blood at high pressure and rapid flow) and the pulmonary artery (low-pressure vessel). The soluble collagen as determined by hydroxyproline measurements represents turnover of the molecule but the soluble elastin is measured by desmosine assays and so is a reflection of the degradation of mature fibrous elastin. MATERIALS
AND METHODS
Tissue Samples of abdominal aorta and pulmonary artery were individuals (see Table I for details) at autopsy. Specimens were obtained from two of these individuals. The autopsies than 24 hr after death and the tissues used were free of any tions in which leucocytes might be present.
obtained from eight from the aortic arch were performed less inflammatory condi-
Extraction of Soluble Eiastin and Collagen Loose, perivascular fascia and fat were removed and the tissue samples were washed with cold distilled water to remove blood and then blotted. The tissue was 25 0014-4800191 $3.00 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
26
001
ET AL.
freeze-dried, placed in a liquid nitrogen-cooled mortar, and ground with a pestle. The samples were lyophilized and weighed. The method of extraction was essentially as previously described (Davis and Mackle, 1981) but with the following modifications. Centrifugation following extraction at 4°C was performed for 45 min at 22,000g. Supematant fractions were pooled and concentrated by ultrafiltration on an Amicon YCOS membrane. Protein was determined using the method of Lowry et al. (1951). The final pellet was washed with distilled water, lyophilized, and weighed. All procedures were performed at 4°C unless otherwise stated. Amino Acid Analysis Aliquots of protein were hydrolyzed in vucuo in constant boiling hydrochloric acid for 65 hr at 110°C. The hydrolysate was dried over sodium hydroxide and redissolved in 50 mM hydrochloric acid. Amino acid analysis was performed on a Waters Picotag amino acid analyzer using the procedure of Negro ef al. (1987). Hydroxyproline was assayed using the method of Woessner (1976). Collagen content was determined from the hydroxyproline using a conversion factor of 7.69 (Miller and Gay, 1982) and elastin was calculated from the desmosine and isodesmosine content using a conversion factor of 1087 (Foster, 1982). The total content of both collagen and elastin is calculated by summing the content in the pellet (insoluble) and that in the extract (soluble). Desmosine
Immunoassay
Covalent linkage of desmosine to gelatin was performed following the procedure by Robins (1982). The conjugate contained 63-70 nM desmosine/mg gelatin as determined by amino acid analysis. The immunoassay was adapted from the procedure described by Gunja-Smith (1985) and performed at room temperature. Briefly, 60 wells (inner wells only) of the microtiter plates were coated with 100 ~1 per well of desmosine-gelatin conjugate (4-8 mg/ml) dissolved in 0.02% sodium azide in phosphate-buffered saline (PBS-AZ). After incubation for 18 hr the plates were washed with 0.05% Tween 20 in PBS (PBS-Tw) and stored desiccated. One hundred-microliter aliquots of antidesmosine (1:30,000 in 1% bovine serum albumin in PBS-AZ (PBS-BSA-AZ) were incubated with 100 p1 of sample or standard for 2 hr. Fifty microliters were transferred to the coated wells and incubated for 30 min. After washing three times with PBS-Tw, 100 ~1 of biotinylated antirabbit antibody (I:1000 in PBSBSA-AZ) was added to each well. After 30 min of incubation, the plates were washed. Streptavidin-biotin-peroxidase complex (1: 1000 in PBS-BSA-AZ) (100 pl) was added and incubated a further 30 min. Following washing, 100 pl of o-phenylenediamine substrate (Wolters et al., 1976) was added and color was developed for 5-10 min. After stopping the reaction with 100 pl 2.5 M sulphuric acid, absorbance was measured at 490 mm. RESULTS
AND DISCUSSION
The concentrations of collagen and elastin present in the saline extract from the human aorta and pulmonary artery samples are shown in Table I. The rate of degradation of the fibrous forms of these proteins is normally low but in diseases such as atherosclerosis this rate increases (Robert and Robert, 1980; Campa et al.,
SALT-SOLUBLE Salt-Extractable
COLLAGEN
AND ELASTIN
27
TABLE I Collagen and Elastin from Human Aorta and Pulmonary Artery Soluble collagen (X 103)/ Total collagen
Soluble elastin (X 103)/ Total elastin
Male (33 years) Abdominal aorta Pulmonary artery
9.75 9.42
1.45 6.29
Male (52 years) Aortic arch Abdominal aorta Pulmonary artery
8.53 5.17 5.78
0.93 3.12 1.55
Male (60 years) Abdominal aorta Pulmonary artery
1.44 21.01
Male (65 years) Abdominal aorta Pulmonary artery
3.66 4.62
Male (70 years) Abdominal aorta Pulmonary artery
25.06 20.22
0 0
Male (75 years) Abdominal aorta Pulmonary artery
5.42 32.1
0 3.84
Female (75 years) Aortic arch Abdominal aorta Pulmonary artery
2.49 2.19 14.79
0.31 0.54 0.74
Female (77 years) Abdominal aorta Pulmonary artery
3.72 13.14
0.49 1.51
0 0 2.07 0.72
Note. There is a significant difference (P:2 a cO.05) for the soluble collagen content of abdominal aorta and pulmonary artery (Mann-Whitney test).
1987). Elevated blood pressure and flow are known to influence the turnover rate of both proteins in blood vessels (Wolinsky, 1970; Ooshima et al., 1974; Davis and Stehbens, 1986; Keeley and Johnson, 1986; Ito et al., 1987; Ryan et al., 1988). These parameters are different in the aorta and the pulmonary artery and so may contribute to a differing rate of degradation. There are a number of factors that may influence the data obtained and the subsequent interpretation. After death, the tissues undergo considerable degradation. In order to restrict these undesirable influences in this work to a minimum, the vessels were obtained as soon as possible postmortem. The soluble collagen as a fraction of the total collagen in the pulmonary artery is higher than that in the aorta. However, in some individuals the concentrations were similar. This may be a reflection of an actual difference in the turnover rate or it could also be due to differences in architecture or in the insoluble collagen content of the vessels. Another factor that will influence these proportions is the rate of removal of these saline-soluble peptides. The role of diffusion through the tissue into the blood or into the lymphatic vessels is unknown. This property may
28
001 ET AL.
differ between the two vessels and also temporally, individually, and from area to area. It may also be affected by hypertension. The salt-soluble elastin in these same vessels is lower than the corresponding collagen concentration. In several samples none could be detected. The elastin concentration in body fluids such as urine is also less than that of collagen (McIntosh et al., 1989). As with the collagen, the soluble elastin concentration tends to be higher in the pulmonary artery than in the aorta although once again there are exceptions. In this determination, the specific elastin crosslink desmosine is assayed and so the soluble elastin is that which has resulted from the degradation of the fibrous polymer. Differences in the rate of degradation and the rate of removal of the soluble elastin from the vessel wall may be partially responsible for the variations observed here. Desmosine, which is unevenly distributed in elastin, has been used as the marker. If the rate of removal of crosslinked peptides differs from that of hydrophobic fragments, then the measurement of desmosine in tissue extracts would not be indicative of elastin solubilization. Severity of degeneration is expected to be variable in different regions of the vasculature, in different individuals, and at different ages. The presence of soluble degraded fragments of elastin within human vascular tissue has not been reported previously. In experimental arteriovenous tistulae there is a proliferative lesion in the venous tissue manifesting many of the features of human atherosclerosis (Stehbens, 1974). In these, there is also increased extractable collagen and elastin (Smith et al., 1976; Davis and Stehbens, 1986). This preliminary study has shown that soluble collagen and elastin are present and can be quantified in blood vessels. The presence of these, particularly the crosslinked elastin fragments, is suggestive of increased vascular fragility. The actual concentration in the tissue is likely to be influenced by a number of contributory parameters. The determination of the rate of degradation requires the development of technology so that it can be monitored continuously. ACKNOWLEDGMENTS This work was supported by grants from the Wellington Medical Research Foundation, the Medical Research Council of New Zealand, the National Heart Foundation of New Zealand, the New Zealand Dairy Board, and the New Zealand Meat Research and Development Council.
REFERENCES M. J. (1985). Collagens in atherosclerosis. Collagen Relat. Res. 5, 65-97. CAMPA, J. S., GREENHALGH, R. M., and POWELL, J. T. (1987). Elastin degradation in abdominal aortic aneurysms. Atherosclerosis 65, 13-21. Cox, R. H. (1978). Passive mechanics and connective tissue composition of canine arteries. Am. J. Physiol. 234, H533-H54 1. DAVIS, P. F., and MACKLE, Z. M. (1981). A simple procedure for the separation of insoluble collagen and elastin. Anal. Biochem. 115, 11-17. DAVIS, P. F., and STEHBENS, W. E. (1986). The biochemical composition of haemodynamically stressed vascular tissue. 2. The concentrations of protein and connective tissue components in the salt extracts of experimental arteriovenous tistulae. Afherosclerosis 60, 55-59. FOSTER, J. A. (1982). Elastin structure and biosynthesis: An overview. In “Methods in Enzymology” (L. W. Cunningham and D. W. Frederiksen, Eds.), Vol. 82, pp. 559-570. Academic Press, San Diego. GUNJA-SMITH, Z. (1985). An enzyme-linked immunosorbent assay to quantitate the elastin crosslink desmosine in tissue and urine samples. Anal. Biochem. 147, 258-264. BARNES,
SALT-SOLUBLE
COLLAGEN
AND
ELASTIN
29
H., KWAN, C. Y., and DANIEL, E. E. (1987). Elastin and elastase-like enzyme changes in aorta of rat with malignant hypertension. Exp. Mol. Pathol. 47, 26-36. KEELEY, F. W., and JOHNSON, D. J. (1986). The effect of developing hypertension on the synthesis and accumulation of elastin in the aorta of the rat. Biochem. Cell Biol. 64, 38-43. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MCINTOSH, C. J., DAVIS, P. F., RYAN, P. A., and STEHBENS, W. E. (1989). Urinary excretion of connective tissue protein markers in arterial disease. Angiology 40, 814-817. MILLER, E. J., and GAY, S. (1982). Collagen: An overview. In “Methods in Enzymology” (L. W. Cunningham and D. W. Frederiksen, Eds.), Vol. 82, pp. 3-32. Academic Press, New York. NEGRO, A., GARBISA, S., GOTTE, L., and SPINA, M. (1987). The use of reverse-phase highperformance liquid chromatography and precolumn derivatization with dansyl chloride for quantitation of specific amino acids in collagen and elastin. Anal. Biochem. 160, 39-46. OOSHIMA, A., FULLER, G. C., CARDINALE, C. J., SPECTOR, S., and UDENFRIEND, S. (1974). Increased collagen synthesis in blood vessels of hypertensive rats and its reversal by antihypertensive agents. Proc. Natl. Acad. Sci. 71, 3019-3023. PARTRIDGE, S. M. (1979). Chemistry and structure of the walls of the large arteries. In “Hemodynamics and the Blood Vessel Wall” (W. E. Stehbens, Ed.), pp. 238-293. Thomas, Springfield, IL. ROBERT, L., and ROBERT, A. M. (Eds.) (1980). Elastin, elastase and arteriosclerosis. In “Frontiers of Matrix Biology,” pp. 130-173. Karger, Basel. ROBINS, S. P. (1982). An enzyme-linked immunoassay for the collagen cross-link pyridinoline. Biothem. J. 207, 617-620. RYAN, P. A., DAVIS, P. F., and STEHBENS, W. E. (1988). The biochemical composition of haemodynamically stressed vascular tissue. 3. The collagen composition of experimental arteriovenous fistulae. Atherosclerosis 71, 157-163. SMITH, R. A., STEHBENS, W. E., and WEBER, P. (1976). Hemodynamically-induced increase in soluble collagen in the anastomosed vein of experimental arteriovenous fistulae. Atherosclerosis 23, ITO,
429-436. STEHBENS,
W. E. (1974). Haemodynamic production of lipid deposition, intimal tears, mural dissection and thrombosis in the blood vessel wall. Proc. R. Sot. London B. 185, 357-373. WOESSNER, J. F., JR. (1976). Determination of hydroxyproline in connective tissue. In “The Methodology of Connective Tissue Research” (D. A. Hall, Ed.), pp. 227-233. Joynson-Bruvvers, Oxford. WOLINSKY, H. (1970). Response of the rat aortic media to hypertension. Circ. Res. 26, 507-522. WOLTERS, G., KUUPERS, L., KACAKI, J., and SCHUURS,A. (1976). Solid-phase enzyme-immunoassay for detection of hepatitis B surface antigen. J. Clin. Pathol. 29, 873-879.
