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Type I and type III collagen synthesis and composition in the valve matrix in aortic valve stenosis
Atherosclerosis 189 (2006) 91–98
Type I and type III collagen synthesis and composition in the valve matrix in aortic valve stenosis Heidi A. Eriksen a , Jari Satta b , Juha Risteli a,e , Mikko Veijola c , P¨aivi V¨are d , Ylermi Soini d,∗ a
d
Department of Clinical Chemistry, University of Oulu, Oulu, Finland b Cardiovascular Surgery, University of Oulu, Oulu, Finland c Forensic Medicine, University of Oulu, Oulu, Finland Department of Pathology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu, Finland e Department of Clinical Chemistry, Kuopio University Hospital, Kuopio, Finland
Received 17 October 2005; received in revised form 18 November 2005; accepted 21 November 2005 Available online 6 January 2006
Abstract Changes in the collagenous matrix may contribute to the pathogenesis and progression of human aortic valve stenosis (AS). To evaluate the significance of collagen I and III in the pathogenesis of AS, we studied their synthesis in diseased valves. Type I and type III collagen mRNA expression and the immunohistochemical localization of the collagen antigens were studied from 36 AS and 2 normal aortic valves. The concentrations of propeptides and telopeptide structure of type I (PINP, PICP, and ICTP) and those of III collagens (PIIINP and IIINTP) were measured by radioimmunoassays in soluble tissue extracts and trypsin-solubilized calcified and non-calcified matrices of 11 AS and 24 healthy aortic valves of different ages. The synthesis of type I collagen, localized in the myofibroblasts adjacent to calcified nodules, was two- to three-fold in the AS samples compared to the controls. The proportion of collagen in the total protein fraction was 90% in the healthy valves, 50% in the non-calcified matrix, and 10% in the calcified matrix of AS valves. In the calcified valves, the ICTP content was six-fold compared to the age-matched controls and two-fold compared to the young control group. In the controls, the amount of ICTP in type I collagen decreased with age (r = −0.908, p < 0.001) and was replaced by other cross-linked C-telopeptide structure. The concentration of type III collagen decreased during aging (r = −0.753, p < 0.001). The decrease in total collagen content, despite the increase in type I collagen synthesis indicates an increase in collagen turnover in AS. The calcification of the aortic valves is accompanied by increased amount of ICTP in type I collagen. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Collagen; Matrix; Valve; Heart; Degeneration
1. Introduction Ectopic calcification may lead to devastating clinical consequences when present in heart valves. Despite the intense interest, the molecular pathological mechanism of aortic valve stenosis (AS) still remains poorly understood. Recent evidence suggests an active process resembling atherosclerosis in many ways [1], sharing features with skeletal ∗
Corresponding author. Tel.: +358 8 5375948; fax: +358 8 35375953. E-mail address: [email protected] (Y. Soini).
0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.11.034
bone formation, such as chondrocyte and osteoblast differentiation, mineralization, bone matrix deposition, and bone resorption [2]. However, other lines of evidence suggest that, in fact, calcification process is a passive phenomenon and that specific gene products are there not to favor it, but to prevent it [3]. However, ECM is of paramount importance in the formation of calcified vascular structures, as evidenced by experiments showing that a matrix of appropriate composition and organization even in the absence of cells, can become calcified [4].
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The cardiovascular extracellular matrix (ECM) is a complex mixture of collagens, elastin, glycoproteins, and proteoglycans, which provide mechanical stability and comprise a variety of insoluble ligands important in cell signaling. The collagen matrix of healthy aortic valves is predominantly type I (70%), with significant amounts of type III (25%) [4]. Type I and type III collagen are synthesized as procollagens containing propeptide extensions at both ends of the molecule. After secretion into the ECM, the propeptides are removed and the collagen molecules aggregate to form the fibril. They are stabilized by intra- and intermolecular cross-linking. The main sites for cross-linking are the lysine (Lys) and hydroxylysine (Hyl) residues in the telopeptide regions at both ends of the molecule. Initially, a telopeptide combines with certain helical regions of another molecule to form a divalent immature cross-link, which may, over time, mature into a higher valence of cross-link, the nature of which varies within different tissues [5]. As an essential part in forming the framework convenient for ectopic tissue calcification the aim of the present study was to compare the collagenous matrix of healthy and calcified human aortic valves using in situ hybridization, biochemical measurements of the different collagen antigens, and immunohistochemical staining. The calcified and noncalcified matrices of the AS samples were studied separately for the type I and type III collagen composition.
2. Methods 2.1. Patient samples For in situ hybridization and immunohistochemical staining, 36 consecutive calcified and 2 control aortic valves were collected from the files of the Department of Pathology, Oulu University Hospital (15 men, 23 women, mean (S.D.) age 69 (8) years, range 45–84 years). All tissues had been fixed in 10% buffered formalin and embedded in paraffin. The histological diagnosis in each individual valve was assessed by two authors (Y.S. and P.V.) from routinely stained hematoxylin–eosin slides using a semiquantitative evaluation. For the biochemical analyses, 11 consecutive calcified aortic valves were collected from patients undergoing valve replacement operations (six men, five women, mean age 67 (9) years, range 45–78 years). The control group consisted of 24 macroscopically normal, non-calcified, smooth, and pliable tricuspid aortic valves collected from cadavers. In order to evaluate possible age-related changes, this group was stratified into two subgroups: those under 50 years old (n = 10, nine men, one woman, mean age 33 (9) years, range 18–44 years) (YOUNG) and those over 50 years old (n = 14, 12 men, 2 women, mean age 65 (9) years, range 52–86 years) (OLD). The study was approved by the Research Ethics Committee of Oulu University Hospital. The investigation conforms with the principles outlined in the Declaration of Helsinki.
2.2. In situ hybridization Four hundred base pairs of cDNA fragments from the carboxyterminal propeptide domain of the ␣1-chain of human type I and type III procollagen [6,7] were subcloned into the polylinker site of the pGEM1 vector (Promega, Madison, WI) and used as in situ probes. The procedure has been described previously [8]. The results were assessed independently by two of the authors (Y.S.and P.V.) (kappa coefficient of 0.717 (p > 0.001)) on a semiquantitative scale as follows: (−) no signals present, (+) only weak, (++) moderate, and (+++) strong mRNA signals present. 2.3. Tissue preparation for biochemical analyses The calcified (weight 466–1763 mg) and the control valves (weight 114–313 mg) were cut into pieces. The possible necrotic regions in the diseased valves were removed (one case). The samples were suspended into PBS–Tween 20 (0.4%), pH 7.2, to a concentration of 20 mg/ml, homogenized by sonication (4 × 15 s) in an ice bath, incubated in ice for 30 min, and centrifuged (15,000 × g, 30 min). The supernatants corresponding to the soluble tissue extracts were collected for the type I and type III procollagen propeptide analyses and the insoluble residues were lyophilized. The lyophilized pellets were reduced with NaBH4 as described earlier [10]. The samples were washed several times with distilled water, centrifuged (15,000 × g, 30 min), decanted, and lyophilized. The diseased valves were processed in order to separate the soft connective tissue outside the mineral (SOFT) from the calcified matrix (CALC). The SOFT fraction was removed by trypsin digestion as described earlier [10]. The samples were centrifuged (15,000 × g, 30 min) and the supernatants collected, and stored frozen for biochemical analyses. The remaining mineral-protected CALC fraction was washed several times with distilled water and demineralized with 0.5 M EDTA, 50 mM Tris–HCl, pH 7.4, as described earlier [11]. The exposed collagen was heat-denatured and digested with TPCK-treated trypsin as described above. The healthy control valves were digested with trypsin as described for the SOFT fraction. 2.4. Measurement of total protein and collagen content The total protein content was measured by the Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Hydroxyproline was analyzed in the insoluble residues as described earlier [12], assuming that hydroxyproline accounts for 12.4% (w/w) of total collagens. The results are expressed as per total protein content of the samples. 2.5. Immunoassays for type I and type III collagens Procollagen synthesis was assessed in the soluble tissue extracts using radioimmunoassays (RIA) for the amino-
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(PINP) and carboxyterminal (PICP) propeptides of type I collagen and for the aminoterminal propeptide (PIIINP) of type III collagen (Orion Diagnostica, Oulunsalo, Finland). The results are expressed as per total protein content of the sample. Type I collagen in the insoluble tissue digests was analyzed with the RIA for trivalently cross-linked carboxyterminal telopeptide structure, ICTP (Orion Diagnostica), and with an in-house SP 4 RIA (synthetic peptide SAGFDFSFLPQPPQEKY, Neosystem Laboratories, Strasbourg, France) [10,13]. The trivalently pyridinoline crosslinked aminoterminal telopeptide of type III collagen, IIINTP, was analyzed by an in-house RIA described earlier [11,14,15]. These results were expressed as moles per mole of collagen. 2.6. Size exclusion chromatography Amounts equal to 8 g of ICTP of the trypsin digests of the insoluble matrix of one control (male, age 61 years) and the SOFT and CALC fractions of one AS sample (male, 60 years) were applied to the column (S-100 HR, Pharmacia, Uppsala, Sweden). Fractions of 2 ml were collected at a flow rate of 6 ml/h. ICTP and synthetic peptide SP 4 was analyzed from the fractions. 2.7. Immunohistochemistry
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Linear regression was used for the correlation analysis. The data are expressed as means with standard deviations (S.D.).
3. Results 3.1. Type I and type III procollagen synthesis In situ hybridization revealed expression of type I collagen mRNA in all stenotic valves (Table 1). The mRNA signals were especially concentrated in and around the calcified nodules in fibroblastic/myofibroblastic cells and also in endothelial cells (Fig. 1). For type III collagen mRNA, only weak signals could be detected in 13 of the 30 cases studied. The distribution of signals was similar to that seen in type I collagen. The control valves expressed no signals for type I or type III collagen transcripts. The type I procollagen synthesis was increased also at protein level. Biochemical assays showed the type I collagen propeptides, PINP, and PICP, to be significantly increased in the calcified aortic valves compared to the controls (Table 2). However, soluble PIIINP levels did not differ between the groups. There were no age-related changes in type I or type III procollagen synthesis in the control group. 3.2. Type I and type III collagen telopeptide structures in the insoluble matrix
The avidin–biotin-immunoperoxidase technique was used for the immunohistochemical staining as described in detail earlier [14,15]. The sections were pretreated with trypsin by incubating in a solution containing 0.14 g trypsin in 100 ml of 0.1% CaCl2 pH 7.8 for 40 min at +37 ◦ C. Specific rabbit anti-human antibodies against PINP, PICP, ICTP, PIIINP, and IIINTP were applied [14]. As a negative control, PBS was substituted for the primary antibody in the staining reaction. Staining was assessed semiquantitatively and divided in four groups as follows: (−) no staining, (+) 0–10%, (++) 10–20%, and (+++) >20% of valvular area positive. 2.8. Statistical analysis Statistical analysis was performed using the SPSS software (SPSS Inc., Chicago, IL, USA). The independentsamples or paired-samples, as appropriate, t-test was used to assess the statistical significance of the differences.
In healthy valves, collagen accounted 90% of the total protein content, whereas the relative contents in calcified valves were 40% in the SOFT fraction and only 10% in the CALC fraction (Fig. 2A). Both the SOFT and the CALC fraction of AS valves had significantly increased ICTP contents compared to the controls (Fig. 2B). The SP4 assay, which has broader immunoreactivity detecting all differently crosslinked and non-cross-linked C-terminal telopeptide structures of type I collagen, showed no difference between the YOUNG, SOFT, and CALC groups, whereas the OLD group contained significantly less SP4 reactive material than the other groups (p < 0.001). The ratio of SP4 to ICTP was significantly higher in the healthy controls (YOUNG 4.48 (1.13), OLD 5.40 (0.73)) than in either the SOFT (1.73 (0.16)), or the CALC (1.84 (0.28)) fractions of calcified valves (both p < 0.001). The amount of IIINTP did not differ between the YOUNG, SOFT, and CALC groups, but the OLD group had signifi-
Table 1 Results of in situ hybridization experiments and immunohistochemical stainings (PINP, PICP, PIIINP, and IIINTP antibodies) with data of the calcification level of the diseased valves Scale
Type I collagen mRNA
Type III collagen mRNA
PINP
PICP
PIIINP
IIINTP
Calcification
– + ++ +++
0 1 7 22
17 13 0 0
0 15 19 2
0 5 16 13
0 0 10 25
0 11 11 10
1 3 11 21
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Fig. 1. In situ hybridization of aortic valves. (A) With a type I collagen antisense probe, clear signals (arrows) can be detected in fusiform cells around the calcified nodules (asterisk) of a calcified valve. (B) A negative control with a sense probe shows no signals in a calcified valve. (C) In a normal valve, no detectable signals are present for collagen type I mRNA. (D) For type III collagen mRNA, weak signals (arrows) can be seen in cells adjacent to degenerative calcified tissue (asterisk).
cantly less IIINTP than the other groups (Fig. 2C). Type III collagen accounted for about 30–40% of total collagen based on the finding that the IIINTP content was approximately 0.3–0.4 mol/mol of collagen and that a major proportion of type III collagen is cross-linked by a structure detectable with IIINTP assay [15]. 3.3. Qualitative analysis of the size of type I collagen C-telopeptides In the control sample, SP 4 assay detected a C-telopeptide structure larger than the ICTP antigen, which eluted as a lefthanded shoulder in the peak containing the ICTP antigen (Fig. 3A). This was absent in the AS samples (Fig. 3B and C). The control valve had also a right-handed shoulder in the ICTP-containing peak (Fig. 3A) that did not correspond to the divalently cross-linked forms. The AS samples contained immature divalent and non-cross-linked C-telopeptide forms detectable with SP 4 assay. These telopeptide structures have
been characterized previously from bone (H. Eriksen, 2003, unpublished observations). 3.4. Collagen composition with age The concentration of ICTP decreased significantly with age in healthy aortic valves (Fig. 4A). At the same time, there was an increase in the ratio of SP4 to ICTP, indicating a change in the nature of collagen cross-links in the carboxyterminal telopeptide (Fig. 4B). In healthy aortic valves, there was a negative correlation between age and IIINTP (Fig. 4C), suggesting a decrease in the amount of type III collagen during ageing. 3.5. Immunohistochemistry PINP, PICP, ICTP, PIIINP, and IIINTP staining was found in all diseased valves (Table 1). PINP and PICP staining was frequently concentrated around calcified areas in the valvular
Table 2 Type I and type III procollagen propeptides in soluble tissue extracts PINP (g/g)
PICP (g/g)
PIIINP (g/g)
Healthy valves (n = 24) YOUNG (n = 10) OLD (n = 14)
6.43 (3.98) 5.78 (2.41)
41.21 (27.93) 60.45 (23.27)
41.72 (28.74) 45.80 (18.25)
Calcified valves (n = 11)
13.53 (6.21)*
147.48 (65.05)*
40.25 (25.03)
The results are expressed as weight per weight of total protein. Values are expressed as means with standard deviations in parentheses. * p < 0.001 calcified valves vs. healthy valves (both age <50 and >50 years).
H.A. Eriksen et al. / Atherosclerosis 189 (2006) 91–98
Fig. 2. Type I and type III collagen structures and total collagen content of the insoluble matrix. (A) The total collagen content assessed by hydroxyproline is expressed as per total protein content. (B) The amount of ICTP is expressed as moles per mole of collagen and (C) the amount of IIINTP as moles per mole of collagen. * p < 0.001, † p < 0.01.
structures (Fig. 5). Similar staining was seen with PIIINP. On the other hand, IIINTP showed mainly linear staining in the valves. With ICTP, mainly linear moderate or strong staining was seen in all of the cases studied, but the staining in diseased valves was irregularly distributed in the calcified areas. In normal valves, no PINP, PICP, or PIIINP staining could be seen, but linear immunoreactivity was observed with IIINTP. The degree of calcification in diseased valves was not associated with either the synthetic level or the presence of type I or type III collagens (Table 1).
4. Discussion Despite the two- to three-fold increased synthesis of type I procollagen, the total collagen content was considerably
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Fig. 3. Size exclusion chromatography of (A) one control, (B) the SOFT, and (C) the CALC fraction of one AS sample. Trivalently cross-linked ICTP is indicated by open circles, and the elution position is marked with bars. The relative SP4 immunoreactivity (closed circles) at the position of ICTP elution, was about two-fold in the healthy sample compared to the SOFT or CALC fractions. The SP4 assay revealed a shoulder in the ICTP peak (big arrow) that was absent in both the mineralized and the non-mineralized fractions of the AS sample. The divalently cross-linked and non-cross-linked C-terminal telopeptide structures present in both SOFT and CALC fraction of AS sample are indicated by small arrow.
decreased in AS compared to normal aortic valves. This indicates an increased turnover of type I collagen in AS, the degradation exceeding the synthesis. The increased turnover seemed to be particularly located around the calcified nodules. Type I collagen accounted for approximately 60–70% of total collagen based on the finding that, in all the studied valves, the proportion of type III collagen was 30–40% of total collagen. Although the turnover had increased the relative proportions of type I and type III collagens remained the same between the groups. However, the trivalent ICTP levels were very low, only 5–13% of type I collagen being cross-linked in the control valves and 30% in the calcified valves by a telopeptide structure detectable with ICTP assay. The ICTP assay has been shown to detect trivalent Ctelopeptide structures that contain two ␣1(I)-telopeptide domains attached together with a helical part of either ␣1-
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Fig. 4. Effect of age in healthy control samples. (A) ICTP decreased over age (r = −0.908, p < 0.001). (B) The SP 4/ICTP ratio correlated positively with age (r = 0.538, p < 0.01). (C) IIINTP had correlated negatively with age (r = −0.753, p < 0.001).
or ␣2-chain, such as trivalent pyridinolines and pyrroles. This precludes the divalently cross-linked, non-cross-linked telopeptides and the trivalent skin-like HHL cross-linked structures [13,16]. In contrast to ICTP, the SP 4 assay detects, with small divergence, all telopeptide structures containing at least one C-terminal ␣1(I)-telopeptide. In addition to the divalently cross-linked and non-crosslinked structures, these include trivalent ICTP and HHL structures. The appropriate molecular packing of collagen, characterized by specific intermolecular cross-linking pattern, has been suggested to be necessary for mineralization to occur [9,17]. The level of telopeptidyl lysine hydroxylation by specific lysyl hydroxylase (LH) isoenzymes is thought to play a role in tissue specificities of different cross-link formation. In soft connective tissues, such as skin and cornea, the Lys aldehyde-derived cross-linking pathway predominates,
whereas the Hyl aldehyde pathway prevails in tissues prone to high mechanical forces, such as bone, ligament, and tendon. The mature cross-linking structure of the former pathway is trivalent histidinohydroxylysinonorleucine (HHL) and of the latter trivalent pyridinolines and pyrroles. In aortic valve it is unclear which cross-linking pathway predominates, but it is likely that to some extent both cross-linking pathways are functional. In normal physiological ageing, the ICTP proportion decreased with a relative increase in SP 4-detectable telopeptide structures, suggesting a change in the nature of mature cross-links in the C-terminal telopeptide, as has also been observed in human Achilles tendons [10]. This could be caused by the decrease in the activity LH isoenzyme responsible for the hydroxylation of telopeptidyl lysines (telopeptidyl LH) and changing the cross-linking towards Lys aldehyde pathway. Thus, in normal aging, the change could be from, for instance, pyridinoline toward skinlike HHL or even as yet uncharacterized cross-linking [16]. In the calcified AS valves the SP4 assay results were at the same level as in the young healthy controls, indicating that elevated ICTP concentrations are not due to an increased proportion of type I collagen but rather to a change in the nature of C-terminal cross-linking. This was supported by the size exclusion chromatography finding of a larger cross-linked C-telopeptide structure than ICTP antigen in the healthy valve, which was absent in the AS fractions. If there is an actual change in the cross-linking pathway, where the mature structure would be detectable by ICTP assay, this would require the increase in the activity of telopeptidyl LH. Other explanation for the ICTP increase in AS may be selective degradation, the other crosslinked C-terminal telopeptide forms being possibly more susceptible to enzymatic degradation leaving the collagenous matrix containing ICTP-like structures intact. The appearance of divalent and non-cross-linked C-telopeptide structures in AS valves may also indicate increased turnover in the AS matrix, where the immature cross-links do not have time to mature before degraded. It can also be related to the presence of mineral, as has been seen in bone [18]. The aminoterminal telopeptide of type III collagen has been shown to be cross-linked only by hydroxylysylpyridinoline, the structure being detectable with IIINTP assay [15]. Thus, the IIINTP decrease suggests a decrease in type III collagen during ageing. In conclusion, we found considerably increased synthesis of type I collagen, but only slight upregulation of type III collagen in calcified valves. Despite this, considerably decreased collagen content was observed in calcified valves, indicating an increased collagen turnover. Our results imply active remodeling and change in the organization of the collagenous matrix, represented by a change in the cross-linking of telopeptides toward ICTP-like structures containing at least two ␣1(I) C-telopeptides.
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Fig. 5. Immunohistochemical stainings of the aortic valves. (A) In a calcified valve, PINP immunoreactivity (arrows) can be detected adjacent to the calcified nodules (asterisk). (B) In a control valve, no PINP immunoreactivity is present. (C) In ICTP staining of a calcified valve, strong immunoreactivity (arrows) is present around the calcific nodules (asterisk). Immunoreactivity is linear and irregularly present. (D) In a normal valve, linear immunostaining is seen with ICTP. (E) Strong immunoreactivity (arrows) for PIIINP can be detected in areas adjacent to the calcific nodules (asterisk), suggesting either synthesis of type III collagen or presence of type III pN-collagen in the diseased valve. (F) In IIINTP immunostaining of calcified valves, immunoreactivity is linear and not so clearly concentrated around the calcified nodules (asterisk).
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[8] Soini Y, Satta J, Maatta M, et al. Expression of MMP2, MMP9, MT1MMP, TIMP1, and TIMP2 mRNA in valvular lesions of the heart. J Pathol 2001;194:225–31. [9] Knott L, Tarlton JF, Bailey AJ. Chemistry of collagen cross-linking: biochemical changes in collagen during the partial mineralization of turkey leg tendon. Biochem J 1997;322:535–42. [10] Eriksen HA, Pajala A, Leppilahti J, et al. Increased content of type III collagen at the rupture site of human Achilles tendon. J Orthopaed Res 2002;20:1352–7. [11] Bode MK, Mosorin M, Satta J, et al. Complete processing of type III collagen in atherosclerotic plaques. Arterioscler Thromb Vasc Biol 1999;19:1506–11. [12] Brown SJ, Worsfold M, Sharp CA. Microplate assay for the measurement of hydroxyproline in acid-hydrolyzed tissue samples. Biotechiques 2001;30:38–42. [13] Sassi ML, Eriksen HA, Risteli L, et al. Immunochemical characterization of assay for carboxyterminal telopeptide of human type I collagen: loss of antigenicity by treatment with cathepsin K. Bone 2000;26:367–73. [14] Bode MK, Soini Y, Melkko J, et al. Increased amount of type III pNcollagen in human abdominal aortic aneurysms: evidence for impaired type III collagen fibrillogenesis. J Vasc Surg 2000;32:1201–7.
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[15] Bode MK, Mosorin M, Satta J, et al. Increased amount of type III pNcollagen in AAA when compared with AOD. Eur J Vasc Endovasc Surg 2002;23:413–20. [16] Sassi ML, Jukkola A, Riekki R, et al. Type I collagen turnover and cross-linking are increased in irradiated skin of breast cancer patients. Radiother Oncol 2001;58:317–23. [17] Wassen MH, Lammens J, Tekoppele JM, et al. Collagen structure regulates fibril mineralization in osteogenesis as revealed by
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Type I and type III collagen synthesis and composition in the valve matrix in aortic valve stenosis Heidi A. Eriksen a , Jari Satta b , Juha Risteli a,e , Mikko Veijola c , P¨aivi V¨are d , Ylermi Soini d,∗ a
d
Department of Clinical Chemistry, University of Oulu, Oulu, Finland b Cardiovascular Surgery, University of Oulu, Oulu, Finland c Forensic Medicine, University of Oulu, Oulu, Finland Department of Pathology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu, Finland e Department of Clinical Chemistry, Kuopio University Hospital, Kuopio, Finland
Received 17 October 2005; received in revised form 18 November 2005; accepted 21 November 2005 Available online 6 January 2006
Abstract Changes in the collagenous matrix may contribute to the pathogenesis and progression of human aortic valve stenosis (AS). To evaluate the significance of collagen I and III in the pathogenesis of AS, we studied their synthesis in diseased valves. Type I and type III collagen mRNA expression and the immunohistochemical localization of the collagen antigens were studied from 36 AS and 2 normal aortic valves. The concentrations of propeptides and telopeptide structure of type I (PINP, PICP, and ICTP) and those of III collagens (PIIINP and IIINTP) were measured by radioimmunoassays in soluble tissue extracts and trypsin-solubilized calcified and non-calcified matrices of 11 AS and 24 healthy aortic valves of different ages. The synthesis of type I collagen, localized in the myofibroblasts adjacent to calcified nodules, was two- to three-fold in the AS samples compared to the controls. The proportion of collagen in the total protein fraction was 90% in the healthy valves, 50% in the non-calcified matrix, and 10% in the calcified matrix of AS valves. In the calcified valves, the ICTP content was six-fold compared to the age-matched controls and two-fold compared to the young control group. In the controls, the amount of ICTP in type I collagen decreased with age (r = −0.908, p < 0.001) and was replaced by other cross-linked C-telopeptide structure. The concentration of type III collagen decreased during aging (r = −0.753, p < 0.001). The decrease in total collagen content, despite the increase in type I collagen synthesis indicates an increase in collagen turnover in AS. The calcification of the aortic valves is accompanied by increased amount of ICTP in type I collagen. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Collagen; Matrix; Valve; Heart; Degeneration
1. Introduction Ectopic calcification may lead to devastating clinical consequences when present in heart valves. Despite the intense interest, the molecular pathological mechanism of aortic valve stenosis (AS) still remains poorly understood. Recent evidence suggests an active process resembling atherosclerosis in many ways [1], sharing features with skeletal ∗
Corresponding author. Tel.: +358 8 5375948; fax: +358 8 35375953. E-mail address: [email protected] (Y. Soini).
0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.11.034
bone formation, such as chondrocyte and osteoblast differentiation, mineralization, bone matrix deposition, and bone resorption [2]. However, other lines of evidence suggest that, in fact, calcification process is a passive phenomenon and that specific gene products are there not to favor it, but to prevent it [3]. However, ECM is of paramount importance in the formation of calcified vascular structures, as evidenced by experiments showing that a matrix of appropriate composition and organization even in the absence of cells, can become calcified [4].
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The cardiovascular extracellular matrix (ECM) is a complex mixture of collagens, elastin, glycoproteins, and proteoglycans, which provide mechanical stability and comprise a variety of insoluble ligands important in cell signaling. The collagen matrix of healthy aortic valves is predominantly type I (70%), with significant amounts of type III (25%) [4]. Type I and type III collagen are synthesized as procollagens containing propeptide extensions at both ends of the molecule. After secretion into the ECM, the propeptides are removed and the collagen molecules aggregate to form the fibril. They are stabilized by intra- and intermolecular cross-linking. The main sites for cross-linking are the lysine (Lys) and hydroxylysine (Hyl) residues in the telopeptide regions at both ends of the molecule. Initially, a telopeptide combines with certain helical regions of another molecule to form a divalent immature cross-link, which may, over time, mature into a higher valence of cross-link, the nature of which varies within different tissues [5]. As an essential part in forming the framework convenient for ectopic tissue calcification the aim of the present study was to compare the collagenous matrix of healthy and calcified human aortic valves using in situ hybridization, biochemical measurements of the different collagen antigens, and immunohistochemical staining. The calcified and noncalcified matrices of the AS samples were studied separately for the type I and type III collagen composition.
2. Methods 2.1. Patient samples For in situ hybridization and immunohistochemical staining, 36 consecutive calcified and 2 control aortic valves were collected from the files of the Department of Pathology, Oulu University Hospital (15 men, 23 women, mean (S.D.) age 69 (8) years, range 45–84 years). All tissues had been fixed in 10% buffered formalin and embedded in paraffin. The histological diagnosis in each individual valve was assessed by two authors (Y.S. and P.V.) from routinely stained hematoxylin–eosin slides using a semiquantitative evaluation. For the biochemical analyses, 11 consecutive calcified aortic valves were collected from patients undergoing valve replacement operations (six men, five women, mean age 67 (9) years, range 45–78 years). The control group consisted of 24 macroscopically normal, non-calcified, smooth, and pliable tricuspid aortic valves collected from cadavers. In order to evaluate possible age-related changes, this group was stratified into two subgroups: those under 50 years old (n = 10, nine men, one woman, mean age 33 (9) years, range 18–44 years) (YOUNG) and those over 50 years old (n = 14, 12 men, 2 women, mean age 65 (9) years, range 52–86 years) (OLD). The study was approved by the Research Ethics Committee of Oulu University Hospital. The investigation conforms with the principles outlined in the Declaration of Helsinki.
2.2. In situ hybridization Four hundred base pairs of cDNA fragments from the carboxyterminal propeptide domain of the ␣1-chain of human type I and type III procollagen [6,7] were subcloned into the polylinker site of the pGEM1 vector (Promega, Madison, WI) and used as in situ probes. The procedure has been described previously [8]. The results were assessed independently by two of the authors (Y.S.and P.V.) (kappa coefficient of 0.717 (p > 0.001)) on a semiquantitative scale as follows: (−) no signals present, (+) only weak, (++) moderate, and (+++) strong mRNA signals present. 2.3. Tissue preparation for biochemical analyses The calcified (weight 466–1763 mg) and the control valves (weight 114–313 mg) were cut into pieces. The possible necrotic regions in the diseased valves were removed (one case). The samples were suspended into PBS–Tween 20 (0.4%), pH 7.2, to a concentration of 20 mg/ml, homogenized by sonication (4 × 15 s) in an ice bath, incubated in ice for 30 min, and centrifuged (15,000 × g, 30 min). The supernatants corresponding to the soluble tissue extracts were collected for the type I and type III procollagen propeptide analyses and the insoluble residues were lyophilized. The lyophilized pellets were reduced with NaBH4 as described earlier [10]. The samples were washed several times with distilled water, centrifuged (15,000 × g, 30 min), decanted, and lyophilized. The diseased valves were processed in order to separate the soft connective tissue outside the mineral (SOFT) from the calcified matrix (CALC). The SOFT fraction was removed by trypsin digestion as described earlier [10]. The samples were centrifuged (15,000 × g, 30 min) and the supernatants collected, and stored frozen for biochemical analyses. The remaining mineral-protected CALC fraction was washed several times with distilled water and demineralized with 0.5 M EDTA, 50 mM Tris–HCl, pH 7.4, as described earlier [11]. The exposed collagen was heat-denatured and digested with TPCK-treated trypsin as described above. The healthy control valves were digested with trypsin as described for the SOFT fraction. 2.4. Measurement of total protein and collagen content The total protein content was measured by the Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Hydroxyproline was analyzed in the insoluble residues as described earlier [12], assuming that hydroxyproline accounts for 12.4% (w/w) of total collagens. The results are expressed as per total protein content of the samples. 2.5. Immunoassays for type I and type III collagens Procollagen synthesis was assessed in the soluble tissue extracts using radioimmunoassays (RIA) for the amino-
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(PINP) and carboxyterminal (PICP) propeptides of type I collagen and for the aminoterminal propeptide (PIIINP) of type III collagen (Orion Diagnostica, Oulunsalo, Finland). The results are expressed as per total protein content of the sample. Type I collagen in the insoluble tissue digests was analyzed with the RIA for trivalently cross-linked carboxyterminal telopeptide structure, ICTP (Orion Diagnostica), and with an in-house SP 4 RIA (synthetic peptide SAGFDFSFLPQPPQEKY, Neosystem Laboratories, Strasbourg, France) [10,13]. The trivalently pyridinoline crosslinked aminoterminal telopeptide of type III collagen, IIINTP, was analyzed by an in-house RIA described earlier [11,14,15]. These results were expressed as moles per mole of collagen. 2.6. Size exclusion chromatography Amounts equal to 8 g of ICTP of the trypsin digests of the insoluble matrix of one control (male, age 61 years) and the SOFT and CALC fractions of one AS sample (male, 60 years) were applied to the column (S-100 HR, Pharmacia, Uppsala, Sweden). Fractions of 2 ml were collected at a flow rate of 6 ml/h. ICTP and synthetic peptide SP 4 was analyzed from the fractions. 2.7. Immunohistochemistry
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Linear regression was used for the correlation analysis. The data are expressed as means with standard deviations (S.D.).
3. Results 3.1. Type I and type III procollagen synthesis In situ hybridization revealed expression of type I collagen mRNA in all stenotic valves (Table 1). The mRNA signals were especially concentrated in and around the calcified nodules in fibroblastic/myofibroblastic cells and also in endothelial cells (Fig. 1). For type III collagen mRNA, only weak signals could be detected in 13 of the 30 cases studied. The distribution of signals was similar to that seen in type I collagen. The control valves expressed no signals for type I or type III collagen transcripts. The type I procollagen synthesis was increased also at protein level. Biochemical assays showed the type I collagen propeptides, PINP, and PICP, to be significantly increased in the calcified aortic valves compared to the controls (Table 2). However, soluble PIIINP levels did not differ between the groups. There were no age-related changes in type I or type III procollagen synthesis in the control group. 3.2. Type I and type III collagen telopeptide structures in the insoluble matrix
The avidin–biotin-immunoperoxidase technique was used for the immunohistochemical staining as described in detail earlier [14,15]. The sections were pretreated with trypsin by incubating in a solution containing 0.14 g trypsin in 100 ml of 0.1% CaCl2 pH 7.8 for 40 min at +37 ◦ C. Specific rabbit anti-human antibodies against PINP, PICP, ICTP, PIIINP, and IIINTP were applied [14]. As a negative control, PBS was substituted for the primary antibody in the staining reaction. Staining was assessed semiquantitatively and divided in four groups as follows: (−) no staining, (+) 0–10%, (++) 10–20%, and (+++) >20% of valvular area positive. 2.8. Statistical analysis Statistical analysis was performed using the SPSS software (SPSS Inc., Chicago, IL, USA). The independentsamples or paired-samples, as appropriate, t-test was used to assess the statistical significance of the differences.
In healthy valves, collagen accounted 90% of the total protein content, whereas the relative contents in calcified valves were 40% in the SOFT fraction and only 10% in the CALC fraction (Fig. 2A). Both the SOFT and the CALC fraction of AS valves had significantly increased ICTP contents compared to the controls (Fig. 2B). The SP4 assay, which has broader immunoreactivity detecting all differently crosslinked and non-cross-linked C-terminal telopeptide structures of type I collagen, showed no difference between the YOUNG, SOFT, and CALC groups, whereas the OLD group contained significantly less SP4 reactive material than the other groups (p < 0.001). The ratio of SP4 to ICTP was significantly higher in the healthy controls (YOUNG 4.48 (1.13), OLD 5.40 (0.73)) than in either the SOFT (1.73 (0.16)), or the CALC (1.84 (0.28)) fractions of calcified valves (both p < 0.001). The amount of IIINTP did not differ between the YOUNG, SOFT, and CALC groups, but the OLD group had signifi-
Table 1 Results of in situ hybridization experiments and immunohistochemical stainings (PINP, PICP, PIIINP, and IIINTP antibodies) with data of the calcification level of the diseased valves Scale
Type I collagen mRNA
Type III collagen mRNA
PINP
PICP
PIIINP
IIINTP
Calcification
– + ++ +++
0 1 7 22
17 13 0 0
0 15 19 2
0 5 16 13
0 0 10 25
0 11 11 10
1 3 11 21
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Fig. 1. In situ hybridization of aortic valves. (A) With a type I collagen antisense probe, clear signals (arrows) can be detected in fusiform cells around the calcified nodules (asterisk) of a calcified valve. (B) A negative control with a sense probe shows no signals in a calcified valve. (C) In a normal valve, no detectable signals are present for collagen type I mRNA. (D) For type III collagen mRNA, weak signals (arrows) can be seen in cells adjacent to degenerative calcified tissue (asterisk).
cantly less IIINTP than the other groups (Fig. 2C). Type III collagen accounted for about 30–40% of total collagen based on the finding that the IIINTP content was approximately 0.3–0.4 mol/mol of collagen and that a major proportion of type III collagen is cross-linked by a structure detectable with IIINTP assay [15]. 3.3. Qualitative analysis of the size of type I collagen C-telopeptides In the control sample, SP 4 assay detected a C-telopeptide structure larger than the ICTP antigen, which eluted as a lefthanded shoulder in the peak containing the ICTP antigen (Fig. 3A). This was absent in the AS samples (Fig. 3B and C). The control valve had also a right-handed shoulder in the ICTP-containing peak (Fig. 3A) that did not correspond to the divalently cross-linked forms. The AS samples contained immature divalent and non-cross-linked C-telopeptide forms detectable with SP 4 assay. These telopeptide structures have
been characterized previously from bone (H. Eriksen, 2003, unpublished observations). 3.4. Collagen composition with age The concentration of ICTP decreased significantly with age in healthy aortic valves (Fig. 4A). At the same time, there was an increase in the ratio of SP4 to ICTP, indicating a change in the nature of collagen cross-links in the carboxyterminal telopeptide (Fig. 4B). In healthy aortic valves, there was a negative correlation between age and IIINTP (Fig. 4C), suggesting a decrease in the amount of type III collagen during ageing. 3.5. Immunohistochemistry PINP, PICP, ICTP, PIIINP, and IIINTP staining was found in all diseased valves (Table 1). PINP and PICP staining was frequently concentrated around calcified areas in the valvular
Table 2 Type I and type III procollagen propeptides in soluble tissue extracts PINP (g/g)
PICP (g/g)
PIIINP (g/g)
Healthy valves (n = 24) YOUNG (n = 10) OLD (n = 14)
6.43 (3.98) 5.78 (2.41)
41.21 (27.93) 60.45 (23.27)
41.72 (28.74) 45.80 (18.25)
Calcified valves (n = 11)
13.53 (6.21)*
147.48 (65.05)*
40.25 (25.03)
The results are expressed as weight per weight of total protein. Values are expressed as means with standard deviations in parentheses. * p < 0.001 calcified valves vs. healthy valves (both age <50 and >50 years).
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Fig. 2. Type I and type III collagen structures and total collagen content of the insoluble matrix. (A) The total collagen content assessed by hydroxyproline is expressed as per total protein content. (B) The amount of ICTP is expressed as moles per mole of collagen and (C) the amount of IIINTP as moles per mole of collagen. * p < 0.001, † p < 0.01.
structures (Fig. 5). Similar staining was seen with PIIINP. On the other hand, IIINTP showed mainly linear staining in the valves. With ICTP, mainly linear moderate or strong staining was seen in all of the cases studied, but the staining in diseased valves was irregularly distributed in the calcified areas. In normal valves, no PINP, PICP, or PIIINP staining could be seen, but linear immunoreactivity was observed with IIINTP. The degree of calcification in diseased valves was not associated with either the synthetic level or the presence of type I or type III collagens (Table 1).
4. Discussion Despite the two- to three-fold increased synthesis of type I procollagen, the total collagen content was considerably
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Fig. 3. Size exclusion chromatography of (A) one control, (B) the SOFT, and (C) the CALC fraction of one AS sample. Trivalently cross-linked ICTP is indicated by open circles, and the elution position is marked with bars. The relative SP4 immunoreactivity (closed circles) at the position of ICTP elution, was about two-fold in the healthy sample compared to the SOFT or CALC fractions. The SP4 assay revealed a shoulder in the ICTP peak (big arrow) that was absent in both the mineralized and the non-mineralized fractions of the AS sample. The divalently cross-linked and non-cross-linked C-terminal telopeptide structures present in both SOFT and CALC fraction of AS sample are indicated by small arrow.
decreased in AS compared to normal aortic valves. This indicates an increased turnover of type I collagen in AS, the degradation exceeding the synthesis. The increased turnover seemed to be particularly located around the calcified nodules. Type I collagen accounted for approximately 60–70% of total collagen based on the finding that, in all the studied valves, the proportion of type III collagen was 30–40% of total collagen. Although the turnover had increased the relative proportions of type I and type III collagens remained the same between the groups. However, the trivalent ICTP levels were very low, only 5–13% of type I collagen being cross-linked in the control valves and 30% in the calcified valves by a telopeptide structure detectable with ICTP assay. The ICTP assay has been shown to detect trivalent Ctelopeptide structures that contain two ␣1(I)-telopeptide domains attached together with a helical part of either ␣1-
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Fig. 4. Effect of age in healthy control samples. (A) ICTP decreased over age (r = −0.908, p < 0.001). (B) The SP 4/ICTP ratio correlated positively with age (r = 0.538, p < 0.01). (C) IIINTP had correlated negatively with age (r = −0.753, p < 0.001).
or ␣2-chain, such as trivalent pyridinolines and pyrroles. This precludes the divalently cross-linked, non-cross-linked telopeptides and the trivalent skin-like HHL cross-linked structures [13,16]. In contrast to ICTP, the SP 4 assay detects, with small divergence, all telopeptide structures containing at least one C-terminal ␣1(I)-telopeptide. In addition to the divalently cross-linked and non-crosslinked structures, these include trivalent ICTP and HHL structures. The appropriate molecular packing of collagen, characterized by specific intermolecular cross-linking pattern, has been suggested to be necessary for mineralization to occur [9,17]. The level of telopeptidyl lysine hydroxylation by specific lysyl hydroxylase (LH) isoenzymes is thought to play a role in tissue specificities of different cross-link formation. In soft connective tissues, such as skin and cornea, the Lys aldehyde-derived cross-linking pathway predominates,
whereas the Hyl aldehyde pathway prevails in tissues prone to high mechanical forces, such as bone, ligament, and tendon. The mature cross-linking structure of the former pathway is trivalent histidinohydroxylysinonorleucine (HHL) and of the latter trivalent pyridinolines and pyrroles. In aortic valve it is unclear which cross-linking pathway predominates, but it is likely that to some extent both cross-linking pathways are functional. In normal physiological ageing, the ICTP proportion decreased with a relative increase in SP 4-detectable telopeptide structures, suggesting a change in the nature of mature cross-links in the C-terminal telopeptide, as has also been observed in human Achilles tendons [10]. This could be caused by the decrease in the activity LH isoenzyme responsible for the hydroxylation of telopeptidyl lysines (telopeptidyl LH) and changing the cross-linking towards Lys aldehyde pathway. Thus, in normal aging, the change could be from, for instance, pyridinoline toward skinlike HHL or even as yet uncharacterized cross-linking [16]. In the calcified AS valves the SP4 assay results were at the same level as in the young healthy controls, indicating that elevated ICTP concentrations are not due to an increased proportion of type I collagen but rather to a change in the nature of C-terminal cross-linking. This was supported by the size exclusion chromatography finding of a larger cross-linked C-telopeptide structure than ICTP antigen in the healthy valve, which was absent in the AS fractions. If there is an actual change in the cross-linking pathway, where the mature structure would be detectable by ICTP assay, this would require the increase in the activity of telopeptidyl LH. Other explanation for the ICTP increase in AS may be selective degradation, the other crosslinked C-terminal telopeptide forms being possibly more susceptible to enzymatic degradation leaving the collagenous matrix containing ICTP-like structures intact. The appearance of divalent and non-cross-linked C-telopeptide structures in AS valves may also indicate increased turnover in the AS matrix, where the immature cross-links do not have time to mature before degraded. It can also be related to the presence of mineral, as has been seen in bone [18]. The aminoterminal telopeptide of type III collagen has been shown to be cross-linked only by hydroxylysylpyridinoline, the structure being detectable with IIINTP assay [15]. Thus, the IIINTP decrease suggests a decrease in type III collagen during ageing. In conclusion, we found considerably increased synthesis of type I collagen, but only slight upregulation of type III collagen in calcified valves. Despite this, considerably decreased collagen content was observed in calcified valves, indicating an increased collagen turnover. Our results imply active remodeling and change in the organization of the collagenous matrix, represented by a change in the cross-linking of telopeptides toward ICTP-like structures containing at least two ␣1(I) C-telopeptides.
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Fig. 5. Immunohistochemical stainings of the aortic valves. (A) In a calcified valve, PINP immunoreactivity (arrows) can be detected adjacent to the calcified nodules (asterisk). (B) In a control valve, no PINP immunoreactivity is present. (C) In ICTP staining of a calcified valve, strong immunoreactivity (arrows) is present around the calcific nodules (asterisk). Immunoreactivity is linear and irregularly present. (D) In a normal valve, linear immunostaining is seen with ICTP. (E) Strong immunoreactivity (arrows) for PIIINP can be detected in areas adjacent to the calcific nodules (asterisk), suggesting either synthesis of type III collagen or presence of type III pN-collagen in the diseased valve. (F) In IIINTP immunostaining of calcified valves, immunoreactivity is linear and not so clearly concentrated around the calcified nodules (asterisk).
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