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The biochemical composition of haemodynamically-stressed vascular tissue
Atherosclerosrs, 60 (1986) 55-59 Elsevier Scientific Publishers Ireland,
55 Ltd.
ATH 03756
The Biochemical Composition of Haemodynamically-Stressed Vascular Tissue Part 2. The Concentrations of Protein and Connective Tissue Components in the Salt Extracts of Experimental Arteriovenous Fistulae Paul F. Davis and William E. Stehbens Wellington Cancer and Medical Research Institute,
Wellington Clinical School of Medrcine, Wellington Hosprtal, Wellington (Nen Zealand)
(Received 28 May, 1985) (Revised, received 11 November, 1985) (Accepted 18 November, 1985)
Summary Arteriovenous fistulae were fashioned between the right external jugular vein and the right common carotid artery in seven experimental sheep, which were then grazed for periods up to 4.25 years. Since the morphological changes in the tissue resemble human atherosclerosis, some of the changes in the extractable protein components in this model were studied. In the experimental venous tissue there was a 1.5-2.5 fold increase in salt-soluble protein. The concentration of extractable collagen was 1.5 times higher in the salt extracts of the experimental veins. The concentration of elastin in the extracts from experimental venous tissue was higher than for the sham-operated veins but the experimental arteries yielded lower levels than the contralateral arteries.
Key words:
Arteriouenous
fistulae
- Collagen - Elastin - Mural damage - Protein
Introduction The structural elastin, constitute
fibrous proteins, a major proportion
collagen and of the blood
Address for correspondence: Dr. Paul F. Davis, Wellington Cancer and Medical Research Institute, Wellington Clinical School of Medicine. Wellington Hospital, Wellington 2, New Zealand. This work was supported financially by the Medical Research Council of New Zealand, the National Heart Foundation of New Zealand and the New Zealand Neurological Foundation. 0021-9150/86/$03.50
0 1986 Elsevier Science Publishers
Ireland.
vessel mass [l]. They are responsible for both the tensile strength and the elastic recoil properties of the vessels [2]. In vascular diseases such as atherosclerosis there are considerable changes to these two components [3-51. Altered haemodynamics within the vascular system can induce morphological and histological changes similar to those seen in atherosclerosis [6-81. To study the relationship of altered flow to the progression of vascular damage, experimental arteriovenous fistulae have been established between the common carotid artery and the external Ltd.
56
chloric acid at 110°C in sealed glass tubes. The hydrolyzates were applied to cellulose minicolumns and the cross-links.were separated from the amino acids according to the method of Skinner [13]. After lyophilization, the water fraction was acetylated with acetic anhydride [14]. The levels of acetylated desmosine were determined by a radioimmunoassay using an antibody to acetylated desmosine that was raised in rabbits and provided by Dr. S.J.M. Skinner. The DNA concentration was determined as previously described [9]. The results were analyzed for statistical significance by the Wilcoxon matched-pairs signed-ranks test.
jugular vein in sheep. We have previously reported on the levels of DNA, calcium and certain lipids in such a model system [9]. In this paper we describe changes in the levels of extractable proteins that are obtained from these experimentally modified tissues. Particularly the soluble collagen and elastin concentrations are noted. Materials and Methods The experimental animals were the same as reported previously [9]. The surgery, the dissection and the tissue extraction are described in that paper. Protein was assayed in the sodium chloride extracts by the method of Lowry et al. [ll] using bovine serum albumin (Sigma) as the standard. Collagen concentrations in the extracts was estimated from the hydroxyproline levels. The extracts were first concentrated at 4°C by ultrafiltration through an Amicon UM05 membrane (nominal molecular weight cuttoff 500). Aliquots were hydrolyzed in constant boiling hydrochloric acid for 24 h at 110°C in sealed glass tubes. After drying the acid and redissolving in water, the hydroxyproline was determined by the method of Woessner [12]. Elastin was determined from the levels of desmosine in the extracts. Aliquots from the extracts concentrated by the Amicon UM05 filter were hydrolyzed for 60 h with constant boiling hydroTABLE
Results Each tissue sample was extracted repetitively in each solvent until the assayable level of protein was negligible. The total protein extracted in each solvent was expressed relative to the dry weight and to the DNA content of the tissue sample. The total soluble protein obtained in the three buffered extractants varied between 6% and 32% of the dry weight of the tissue samples. However, the range of concentrations was mostly between 10% (only 14% of the samples were less than this) and 25% (only 10% were greater than this). Of those below lo%, 6 of the 7 were from sham-operated jugular veins and all those above 25% were from arterial tissue.
1
SOLUBLE
PROTEIN
EXTRACTED
FROM
VASCULAR
TISSUE
Mean + SEM (n = 7). Control artery (A) mg protein /g tissue dry weight 0.2 M Sodium chloride 206.8 j, 19.0 a 0.5 M Sodium chloride 10.2 f 1.2 a 0.167 M EDTA 17.0* 4.1 (B) rng protein /mg DNA 0.2 M Sodium chloride 0.5 M Sodium chloride 0.167 M EDTA Differences a Control b Control ’ Control d Control
56.0* 13.6 acd 3.1 f 0.7 a 4.6+_ 1.5
Proximal artery
Distal artery
Control vein
177.5 * 15.7 9.3* 2.2 12.0* 5.0
167.7 + 20.5 9.8k 2.0 21.9+ 7.5
53.7k8.2 ab 121.2k14.1 b 4.4kl.2 a 5.2+ 2.0 25.7 f 8.2 21.4+ 6.6
34.2 + 15.2 ’ 2.0+ 0.8 2.4? 1.1
that are statistically significant: artery vs control vein (P: 2a < 0.02). vein vs experimental vein (P: 2a < 0.02). artery vs proximal artery (P: 2a < 0.05). artery vs distal artery (P: 2a < 0.02).
22.8 f 1.5* 3.4*
5.9 d 12.6f 3.6 ab 0.4 l.OkO.3 = 0.9 5.3 f 1.4
Proximal vein
35.3 + 12.7 b 0.9f 0.3 4.3* 1.1
Distal vein
Mid-segment of vein
120.0+15.8 b 141.5k15.3 b 3.2+ 1.0 4.9* 2.0 16.0+ 3.7 15.8+_ 7.3
52.5 &-25.1 b 106.4* 39.0 b 1.2+ 0.4 2.9+ 1.1 5.9+ 1.8 6.1 f 1.2
57
For all samples, the majority of the extractable protein was in the 0.2 M sodium chloride solvent. In fact, in 88% of samples extracted, more than 70% of the protein was in this fraction (Table 1). Those with less than 70% of their soluble protein in this solvent were all from veins, with 4 of the 7 sham-operated veins being in this group. When the results are expressed as the soluble protein extracted per mg of DNA the pattern remains similar. The concentrations in the experimental arteries were significantly less than in the control artery (Table 1). However, in all regions of the experimental vein there is an increase in the protein concentration. Collagen concentrations in the salt extracts are based on the hydroxyproline being 13% of the vascular collagen [lo]. Only the buffered 0.2 M sodium chloride extract had detectable levels of hydroxyproline. Less than 1% of the dry weight of tissue was extractable collagen. As a proportion of the tissue dry weight, there is significantly more soluble collagen in the shamoperated artery than in the sham-operated vein (Table 2A). The distal and proximal regions of the experimental vein both have significantly more soluble collagen than does the phlebotomized vein. When soluble collagen is determined relative to the DNA content, there was significantly more in the proximal area and in the mid-segment of the
TABLE
experimental vein compared with the control vein (Table 2B). Salt-soluble collagen constitutes up to 4% of the extracted protein. However, there is no statistical difference for any of the pairings (Table 2C). The presence of elastin was quantified by determining the desmosine concentration in the concentrated 0.2 M sodium chloride extracts by radioimmunoassay. When the level of soluble cross-linked elastin is expressed as a proportion of the tissue dry weight, the experimental venous tissue had higher levels than the contralateral jugular vein (Table 2A). The control artery had higher extractable desmosine than did either region of the experimental artery but none of these differences were significant. However, the sham-operated artery had a higher level of soluble elastin than did the sham vein. When the cross-links were measured relative to the cell content, the proximal region of the anastomosed vein had an elevated level (Table 2B). However, the proximal region of the experimental artery had a significantly lower level of soluble elastin. If the concentration of elastin is related to the total soluble protein, there are statistically significant decreases in the distal region and in the dilated mid-segment of the vein.
2
COLLAGEN
AND DESMOSINE
IN 0.2 M SODIUM
CHLORIDE
EXTRACTS
FROM
VASCULAR
TISSUE
Mean + SEM (n = 7). Control artery (A) Weight/g
Distal artery
Control vein
Proximal vein
4.5+ 1.4 115.7+34.1
3.4_+ 0.9 c 273.6 & 135.8
5.0+ 2.1 h 3.2k 1.7 he 104.3k 15.8 ’ 354.0 _+150.1
Distal vein
Mid-segment of vein
4.7+ 2.3 h 160.1 + 76.5
4.8* 2.2 136.9k41.8
tissue dty weight
Collagen (mg) Desmosine (ng) (B) Weight/mg
Proximal artery
4.8+ 1.4 ‘ 349.4 _+141.9 ’
DNA
3.6+ 1.4 Collagen (mg) Desmosine (ng) 238.7 & 92.3 a (C) Weight/mg salt-soluble protein Collagen (pg) Desmosine (ng)
10.85 1.6_+
Differences that are statistically a (P: 2a < 0.02). h (P: 2a < 0.05). c (P: 2u -c 0.10).
2.1 0.5
2.2* 0.8 3.6k 1.5 84.0 f 37.8 = 160.5 + 81.0 11.9* 3.5 0.8_+ 0.2
significant:
12.2+ 2.6+
3.9 1.7
5.6 4.9~ 2.3 h 7.6-t 2.9k 1.5 bc 109.6 + 29.3 ’ 267.5 f 103.6 ’ 183.6 f 82.7 15.4+ 2.6?
4.0 0.8 a
15.0* 3.3+
5.1 1.4
12.6k 1.7k
4.3 0.8 a
6.5_+ 3.6 ’ 156.3i42.4 12.8+ 2.9 1.1 & 0.5 a
58
Discussion An earlier study using experimental arteriovenous fistulae between the jugular vein and the carotid artery in dogs exhibited an increase in the protein that was extractable from the experimental vein compared with the sham-operated vein [15]. In this study sheep have been used as the experimental animals and the haemodynamically injured vein has been subdivided into three regions. The results obtained with the dogs have been confirmed here and it is also shown that there is an increase in extractable protein at all sites with the greatest increase being in the dilated segment of vein opposite the fistula. In contrast neither the proximal nor the distal segments of the experimental artery shows any significant change in the concentrations of saltsoluble protein. Thus it would seem that the altered flow and haemodynamic stress in the venous tissue elicits an increase in soluble protein but there is no overall effect on the experimental artery. As most of the soluble protein is extractable in 0.2 M sodium chloride, it would seem that these proteins may be soluble at physiological pH and salt concentrations. As the DNA concentration in the experimental vein showed no significant change when compared with the phlebotomized vein [9], the increase in protein concentration implies that the cells in the experimental vein have become more synthetically active and so are producing protein at a greater rate. In contrast the cells on the arterial side of the shunt have not been so stimulated. The increase in salt-soluble collagen observed in all three regions of the experimental vein is consistent with the earlier observation using experimental anastomoses of the dog [15]. As with the total protein, the change in soluble collagen in the experimental artery is not of significance. Estimating collagen by hydroxyproline does not distinguish between newly synthesized soluble collagen and solubilized fragments obtained by degradation of fibrous collagen. That there ‘is an increase in total protein in the experimental veins and that the increase in soluble collagen is similar suggests that this increase in hydroxyproline may be due to increased collagen synthesis. Of note is the increased rate of arterial collagen synthesis in
atherosclerosis [16]. However, some of the soluble hydroxyproline may still arise from the degradation of fibrous collagen. The presence of soluble elastin was also assayed in these extracts by means of a radioimmunoassay for desmosine. As this marker was one of the elastin cross-links, this elastin arose from the degradation of the fibrous material. Unlike the salt-soluble collagen when measured as a proportion of the tissue weight, there was no statistically significant difference for this elastin between the experimental veins and the control vein. The mean values of the former, however, were higher. Acknowledgements The assistance of Dr. Steve Skinner, Mr. John Manning and Mr. Jean-Claude Schellenberg in the performance of the soluble elastin analyses is much appreciated. References 1 Partridge, SM., Chemistry and structure of the walls of the large arteries. In: W.E. Stehbens (Ed.), Hemodynamics and the Blood Vessel Wall, Charles C. Thomas, Springfield, IL, 1979, 238-293. 2 Cox, R.H., Passive mechanics and connective tissue composition of canine arteries, Amer. J. Physiol., 234 (1978) H533. 3 Rucker, R.B. and Tinker, D., Structure and metabolism of arterial elastin, Int. Rev. Exp. Path., 17 (1978) 1. 4 Derouette, S., Homebeck, W., Loisance, D., Godeau, G., Cachera, J.P. and Robert, L., Studies on elastic tissue of aorta in aortic dissections and Marfan syndrome, Path. Biol., 29 (1981) 539. 5 Olczyk, K., Drozdz, M. and Piwowarczyk, P., Elastin and collagen metabolism in the arterial wall of rats fed an atherogenic diet, Exp. Path., 21 (1982) 221. 6 Stehbens, W.E., Experimental arteriovenous fistulae in normal and cholesterol-fed rabbits, Path., 5 (1979) 311. 7 Stehbens, W.E., Haemodynamic production of lipid deposition, intimal tears, mural dissection and thrombosis in the blood vessel wall, Proc. Roy. Sot. London B, 185 (1974) 357. 8 Stehbens, W.E., The role of hemodynamics in the pathogenesis of atherosclerosis, Progr. Cardiovasc. Dis., 18 (1975) 89. 9 Davis, P.F. and Stehbens, W.E., The biochemical composition of haemodynamically stressed vascular tissue, Part 1 (The lipid, calcium and DNA concentrations in experimental arteriovenous fistulae), Atherosclerosis, 56 (1985) 27. 10 Davis, P.F. and Mackle, Z.M., A simple procedure for the separation of insoluble collagen and elastin, Anal. Biochem., 115 (1981) 11.
59
11 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 12 Woessner, Jr., J.F., Determination of hydroxyproline in connective tissue. In: D.A. Hall (Ed.), The Methodology of Connective Tissue Research, Joynson-Bruwers Ltd., Oxford, 1976, pp. 227-233. 13 Skinner, S.J.M., Rapid method for the purification of the elastin crosslinks, desmosine and isodesmosine, J. Chromatogr., 229 (1982) 200.
14 Skinner, S.J.M., Schellenberg, J.-C. and L&gins, G.C., The estimation of elastin in fetal tissues by radioimmunoassay of isodesmosine, Conn. Tiss. Res., 11 (1983) 113. 15 Smith, R.A., Stehbens, W.E. and Weber, P., Hemodynamitally-induced increase in soluble collagen in the anastomosed vein of experimental arteriovenous fistulae, Atherosclerosis, 23 (1976) 429. 16 McCullagh, K.A. and Ehrhart, L.A., Increased arterial collagen synthesis in experimental canine atherosclerosis. Atherosclerosis, 19 (1974) 13.
55 Ltd.
ATH 03756
The Biochemical Composition of Haemodynamically-Stressed Vascular Tissue Part 2. The Concentrations of Protein and Connective Tissue Components in the Salt Extracts of Experimental Arteriovenous Fistulae Paul F. Davis and William E. Stehbens Wellington Cancer and Medical Research Institute,
Wellington Clinical School of Medrcine, Wellington Hosprtal, Wellington (Nen Zealand)
(Received 28 May, 1985) (Revised, received 11 November, 1985) (Accepted 18 November, 1985)
Summary Arteriovenous fistulae were fashioned between the right external jugular vein and the right common carotid artery in seven experimental sheep, which were then grazed for periods up to 4.25 years. Since the morphological changes in the tissue resemble human atherosclerosis, some of the changes in the extractable protein components in this model were studied. In the experimental venous tissue there was a 1.5-2.5 fold increase in salt-soluble protein. The concentration of extractable collagen was 1.5 times higher in the salt extracts of the experimental veins. The concentration of elastin in the extracts from experimental venous tissue was higher than for the sham-operated veins but the experimental arteries yielded lower levels than the contralateral arteries.
Key words:
Arteriouenous
fistulae
- Collagen - Elastin - Mural damage - Protein
Introduction The structural elastin, constitute
fibrous proteins, a major proportion
collagen and of the blood
Address for correspondence: Dr. Paul F. Davis, Wellington Cancer and Medical Research Institute, Wellington Clinical School of Medicine. Wellington Hospital, Wellington 2, New Zealand. This work was supported financially by the Medical Research Council of New Zealand, the National Heart Foundation of New Zealand and the New Zealand Neurological Foundation. 0021-9150/86/$03.50
0 1986 Elsevier Science Publishers
Ireland.
vessel mass [l]. They are responsible for both the tensile strength and the elastic recoil properties of the vessels [2]. In vascular diseases such as atherosclerosis there are considerable changes to these two components [3-51. Altered haemodynamics within the vascular system can induce morphological and histological changes similar to those seen in atherosclerosis [6-81. To study the relationship of altered flow to the progression of vascular damage, experimental arteriovenous fistulae have been established between the common carotid artery and the external Ltd.
56
chloric acid at 110°C in sealed glass tubes. The hydrolyzates were applied to cellulose minicolumns and the cross-links.were separated from the amino acids according to the method of Skinner [13]. After lyophilization, the water fraction was acetylated with acetic anhydride [14]. The levels of acetylated desmosine were determined by a radioimmunoassay using an antibody to acetylated desmosine that was raised in rabbits and provided by Dr. S.J.M. Skinner. The DNA concentration was determined as previously described [9]. The results were analyzed for statistical significance by the Wilcoxon matched-pairs signed-ranks test.
jugular vein in sheep. We have previously reported on the levels of DNA, calcium and certain lipids in such a model system [9]. In this paper we describe changes in the levels of extractable proteins that are obtained from these experimentally modified tissues. Particularly the soluble collagen and elastin concentrations are noted. Materials and Methods The experimental animals were the same as reported previously [9]. The surgery, the dissection and the tissue extraction are described in that paper. Protein was assayed in the sodium chloride extracts by the method of Lowry et al. [ll] using bovine serum albumin (Sigma) as the standard. Collagen concentrations in the extracts was estimated from the hydroxyproline levels. The extracts were first concentrated at 4°C by ultrafiltration through an Amicon UM05 membrane (nominal molecular weight cuttoff 500). Aliquots were hydrolyzed in constant boiling hydrochloric acid for 24 h at 110°C in sealed glass tubes. After drying the acid and redissolving in water, the hydroxyproline was determined by the method of Woessner [12]. Elastin was determined from the levels of desmosine in the extracts. Aliquots from the extracts concentrated by the Amicon UM05 filter were hydrolyzed for 60 h with constant boiling hydroTABLE
Results Each tissue sample was extracted repetitively in each solvent until the assayable level of protein was negligible. The total protein extracted in each solvent was expressed relative to the dry weight and to the DNA content of the tissue sample. The total soluble protein obtained in the three buffered extractants varied between 6% and 32% of the dry weight of the tissue samples. However, the range of concentrations was mostly between 10% (only 14% of the samples were less than this) and 25% (only 10% were greater than this). Of those below lo%, 6 of the 7 were from sham-operated jugular veins and all those above 25% were from arterial tissue.
1
SOLUBLE
PROTEIN
EXTRACTED
FROM
VASCULAR
TISSUE
Mean + SEM (n = 7). Control artery (A) mg protein /g tissue dry weight 0.2 M Sodium chloride 206.8 j, 19.0 a 0.5 M Sodium chloride 10.2 f 1.2 a 0.167 M EDTA 17.0* 4.1 (B) rng protein /mg DNA 0.2 M Sodium chloride 0.5 M Sodium chloride 0.167 M EDTA Differences a Control b Control ’ Control d Control
56.0* 13.6 acd 3.1 f 0.7 a 4.6+_ 1.5
Proximal artery
Distal artery
Control vein
177.5 * 15.7 9.3* 2.2 12.0* 5.0
167.7 + 20.5 9.8k 2.0 21.9+ 7.5
53.7k8.2 ab 121.2k14.1 b 4.4kl.2 a 5.2+ 2.0 25.7 f 8.2 21.4+ 6.6
34.2 + 15.2 ’ 2.0+ 0.8 2.4? 1.1
that are statistically significant: artery vs control vein (P: 2a < 0.02). vein vs experimental vein (P: 2a < 0.02). artery vs proximal artery (P: 2a < 0.05). artery vs distal artery (P: 2a < 0.02).
22.8 f 1.5* 3.4*
5.9 d 12.6f 3.6 ab 0.4 l.OkO.3 = 0.9 5.3 f 1.4
Proximal vein
35.3 + 12.7 b 0.9f 0.3 4.3* 1.1
Distal vein
Mid-segment of vein
120.0+15.8 b 141.5k15.3 b 3.2+ 1.0 4.9* 2.0 16.0+ 3.7 15.8+_ 7.3
52.5 &-25.1 b 106.4* 39.0 b 1.2+ 0.4 2.9+ 1.1 5.9+ 1.8 6.1 f 1.2
57
For all samples, the majority of the extractable protein was in the 0.2 M sodium chloride solvent. In fact, in 88% of samples extracted, more than 70% of the protein was in this fraction (Table 1). Those with less than 70% of their soluble protein in this solvent were all from veins, with 4 of the 7 sham-operated veins being in this group. When the results are expressed as the soluble protein extracted per mg of DNA the pattern remains similar. The concentrations in the experimental arteries were significantly less than in the control artery (Table 1). However, in all regions of the experimental vein there is an increase in the protein concentration. Collagen concentrations in the salt extracts are based on the hydroxyproline being 13% of the vascular collagen [lo]. Only the buffered 0.2 M sodium chloride extract had detectable levels of hydroxyproline. Less than 1% of the dry weight of tissue was extractable collagen. As a proportion of the tissue dry weight, there is significantly more soluble collagen in the shamoperated artery than in the sham-operated vein (Table 2A). The distal and proximal regions of the experimental vein both have significantly more soluble collagen than does the phlebotomized vein. When soluble collagen is determined relative to the DNA content, there was significantly more in the proximal area and in the mid-segment of the
TABLE
experimental vein compared with the control vein (Table 2B). Salt-soluble collagen constitutes up to 4% of the extracted protein. However, there is no statistical difference for any of the pairings (Table 2C). The presence of elastin was quantified by determining the desmosine concentration in the concentrated 0.2 M sodium chloride extracts by radioimmunoassay. When the level of soluble cross-linked elastin is expressed as a proportion of the tissue dry weight, the experimental venous tissue had higher levels than the contralateral jugular vein (Table 2A). The control artery had higher extractable desmosine than did either region of the experimental artery but none of these differences were significant. However, the sham-operated artery had a higher level of soluble elastin than did the sham vein. When the cross-links were measured relative to the cell content, the proximal region of the anastomosed vein had an elevated level (Table 2B). However, the proximal region of the experimental artery had a significantly lower level of soluble elastin. If the concentration of elastin is related to the total soluble protein, there are statistically significant decreases in the distal region and in the dilated mid-segment of the vein.
2
COLLAGEN
AND DESMOSINE
IN 0.2 M SODIUM
CHLORIDE
EXTRACTS
FROM
VASCULAR
TISSUE
Mean + SEM (n = 7). Control artery (A) Weight/g
Distal artery
Control vein
Proximal vein
4.5+ 1.4 115.7+34.1
3.4_+ 0.9 c 273.6 & 135.8
5.0+ 2.1 h 3.2k 1.7 he 104.3k 15.8 ’ 354.0 _+150.1
Distal vein
Mid-segment of vein
4.7+ 2.3 h 160.1 + 76.5
4.8* 2.2 136.9k41.8
tissue dty weight
Collagen (mg) Desmosine (ng) (B) Weight/mg
Proximal artery
4.8+ 1.4 ‘ 349.4 _+141.9 ’
DNA
3.6+ 1.4 Collagen (mg) Desmosine (ng) 238.7 & 92.3 a (C) Weight/mg salt-soluble protein Collagen (pg) Desmosine (ng)
10.85 1.6_+
Differences that are statistically a (P: 2a < 0.02). h (P: 2a < 0.05). c (P: 2u -c 0.10).
2.1 0.5
2.2* 0.8 3.6k 1.5 84.0 f 37.8 = 160.5 + 81.0 11.9* 3.5 0.8_+ 0.2
significant:
12.2+ 2.6+
3.9 1.7
5.6 4.9~ 2.3 h 7.6-t 2.9k 1.5 bc 109.6 + 29.3 ’ 267.5 f 103.6 ’ 183.6 f 82.7 15.4+ 2.6?
4.0 0.8 a
15.0* 3.3+
5.1 1.4
12.6k 1.7k
4.3 0.8 a
6.5_+ 3.6 ’ 156.3i42.4 12.8+ 2.9 1.1 & 0.5 a
58
Discussion An earlier study using experimental arteriovenous fistulae between the jugular vein and the carotid artery in dogs exhibited an increase in the protein that was extractable from the experimental vein compared with the sham-operated vein [15]. In this study sheep have been used as the experimental animals and the haemodynamically injured vein has been subdivided into three regions. The results obtained with the dogs have been confirmed here and it is also shown that there is an increase in extractable protein at all sites with the greatest increase being in the dilated segment of vein opposite the fistula. In contrast neither the proximal nor the distal segments of the experimental artery shows any significant change in the concentrations of saltsoluble protein. Thus it would seem that the altered flow and haemodynamic stress in the venous tissue elicits an increase in soluble protein but there is no overall effect on the experimental artery. As most of the soluble protein is extractable in 0.2 M sodium chloride, it would seem that these proteins may be soluble at physiological pH and salt concentrations. As the DNA concentration in the experimental vein showed no significant change when compared with the phlebotomized vein [9], the increase in protein concentration implies that the cells in the experimental vein have become more synthetically active and so are producing protein at a greater rate. In contrast the cells on the arterial side of the shunt have not been so stimulated. The increase in salt-soluble collagen observed in all three regions of the experimental vein is consistent with the earlier observation using experimental anastomoses of the dog [15]. As with the total protein, the change in soluble collagen in the experimental artery is not of significance. Estimating collagen by hydroxyproline does not distinguish between newly synthesized soluble collagen and solubilized fragments obtained by degradation of fibrous collagen. That there ‘is an increase in total protein in the experimental veins and that the increase in soluble collagen is similar suggests that this increase in hydroxyproline may be due to increased collagen synthesis. Of note is the increased rate of arterial collagen synthesis in
atherosclerosis [16]. However, some of the soluble hydroxyproline may still arise from the degradation of fibrous collagen. The presence of soluble elastin was also assayed in these extracts by means of a radioimmunoassay for desmosine. As this marker was one of the elastin cross-links, this elastin arose from the degradation of the fibrous material. Unlike the salt-soluble collagen when measured as a proportion of the tissue weight, there was no statistically significant difference for this elastin between the experimental veins and the control vein. The mean values of the former, however, were higher. Acknowledgements The assistance of Dr. Steve Skinner, Mr. John Manning and Mr. Jean-Claude Schellenberg in the performance of the soluble elastin analyses is much appreciated. References 1 Partridge, SM., Chemistry and structure of the walls of the large arteries. In: W.E. Stehbens (Ed.), Hemodynamics and the Blood Vessel Wall, Charles C. Thomas, Springfield, IL, 1979, 238-293. 2 Cox, R.H., Passive mechanics and connective tissue composition of canine arteries, Amer. J. Physiol., 234 (1978) H533. 3 Rucker, R.B. and Tinker, D., Structure and metabolism of arterial elastin, Int. Rev. Exp. Path., 17 (1978) 1. 4 Derouette, S., Homebeck, W., Loisance, D., Godeau, G., Cachera, J.P. and Robert, L., Studies on elastic tissue of aorta in aortic dissections and Marfan syndrome, Path. Biol., 29 (1981) 539. 5 Olczyk, K., Drozdz, M. and Piwowarczyk, P., Elastin and collagen metabolism in the arterial wall of rats fed an atherogenic diet, Exp. Path., 21 (1982) 221. 6 Stehbens, W.E., Experimental arteriovenous fistulae in normal and cholesterol-fed rabbits, Path., 5 (1979) 311. 7 Stehbens, W.E., Haemodynamic production of lipid deposition, intimal tears, mural dissection and thrombosis in the blood vessel wall, Proc. Roy. Sot. London B, 185 (1974) 357. 8 Stehbens, W.E., The role of hemodynamics in the pathogenesis of atherosclerosis, Progr. Cardiovasc. Dis., 18 (1975) 89. 9 Davis, P.F. and Stehbens, W.E., The biochemical composition of haemodynamically stressed vascular tissue, Part 1 (The lipid, calcium and DNA concentrations in experimental arteriovenous fistulae), Atherosclerosis, 56 (1985) 27. 10 Davis, P.F. and Mackle, Z.M., A simple procedure for the separation of insoluble collagen and elastin, Anal. Biochem., 115 (1981) 11.
59
11 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 12 Woessner, Jr., J.F., Determination of hydroxyproline in connective tissue. In: D.A. Hall (Ed.), The Methodology of Connective Tissue Research, Joynson-Bruwers Ltd., Oxford, 1976, pp. 227-233. 13 Skinner, S.J.M., Rapid method for the purification of the elastin crosslinks, desmosine and isodesmosine, J. Chromatogr., 229 (1982) 200.
14 Skinner, S.J.M., Schellenberg, J.-C. and L&gins, G.C., The estimation of elastin in fetal tissues by radioimmunoassay of isodesmosine, Conn. Tiss. Res., 11 (1983) 113. 15 Smith, R.A., Stehbens, W.E. and Weber, P., Hemodynamitally-induced increase in soluble collagen in the anastomosed vein of experimental arteriovenous fistulae, Atherosclerosis, 23 (1976) 429. 16 McCullagh, K.A. and Ehrhart, L.A., Increased arterial collagen synthesis in experimental canine atherosclerosis. Atherosclerosis, 19 (1974) 13.