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(Circulation. 1996;93:1026-1032.)
© 1996 American Heart Association, Inc.

Articles

Serum Markers of Collagen Type I Metabolism in Spontaneously Hypertensive Rats

Relation to Myocardial Fibrosis

Javier Díez, MD, PhD; Angel Panizo, MD; María J. Gil, DSc; Ignacio Monreal, DSc, MD; Marta Hernández, MD; Javier Pardo Mindán, MD, PhD

From the Vascular Pathophysiology Unit, Department of Internal Medicine, School of Medicine, University of Navarra and Department of Medicine, School of Medicine, University of Zaragoza (J.D., M.H.); the Department of Pathology, University Clinic, University of Navarra (A.P., J.P.M.); and the Department of Clinical Chemistry, University Clinic, University of Navarra, Pamplona, Spain (M.J.G., I.M.).

Correspondence to Javier Díez, MD, PhD, Unidad de Fisiopatología Vascular Departamento de Medicina Interna, Facultad de Medicina, C/ Irunlarrea s/n, 31080 Pamplona, Spain.

	Abstract

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Background The assay of serum peptides of extracellular collagen synthesis and degradation could provide an indirect estimate of the rate of fibrillar turnover. This study was designed to investigate whether serum peptides of collagen type I synthesis and degradation are altered in spontaneously hypertensive rats (SHR) with left ventricular hypertrophy and whether these serum collagen-derived peptides are related to myocardial fibrosis.

Methods and Results We measured serum levels of carboxy-terminal propeptide of procollagen type I (PIP) as a marker of collagen I synthesis and serum levels of the pyridinoline cross-linked telopeptide domain of collagen type I (CITP) as a marker of fibrillar collagen I degradation in ten 36-week-old normotensive Wistar-Kyoto (WKY) rats, ten 36-week-old SHR and, ten 16-week-old SHR treated with the angiotensin-converting enzyme inhibitor quinapril (10 mg/kg body wt per day, orally) for 20 weeks. PIP and CITP were determined by specific radioimmunoassays. Histomorphometric and immunohistochemical studies of the left ventricle were performed in all rats. In untreated SHR compared with WKY rats, we found a more extensive interstitial and perivascular fibrosis, an increased (P<.01) collagen volume fraction, a more marked deposition of collagen type I, an increased (P<.01) serum concentration of PIP, and a similar serum concentration of CITP. In quinapril-treated SHR compared with untreated SHR, we found an absence of left ventricular hypertrophy, a marked decrease of fibrosis, a lower (P<.01) collagen volume fraction, a diminished deposition of collagen type I, a decreased (P<.01) concentration of PIP, and a similar concentration of CITP. A direct correlation was found between the collagen volume fraction and serum PIP (r=.753, P<.05) in untreated SHR.

Conclusions These results suggest that tissue metabolism of collagen type I is abnormal in SHR and can be normalized by treatment with quinapril. On the basis of our findings, we propose that serum PIP may be a marker of collagen type I–dependent myocardial fibrosis in rats with genetic hypertension.

Key Words: collagen • hypertension • peptides • fibrosis

	Introduction

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An exaggerated interstitial and perivascular deposition of fibrillar collagen in the absence of necrosis has been shown in the hypertrophied left ventricle of SHR^{1 2} and humans with essential hypertension.^{3 4} Immunohistochemical and biochemical studies have shown that collagen type I is the major form of collagen in hypertensive myocardial fibrosis.^{4 5} It has been suggested that this reactive fibrosis reflects alterations in myocardial collagen synthesis and degradation induced by humoral factors.⁶ Interestingly, myocardial fibrosis is reversed by ACE inhibition.^{7 8 9 10} More specifically, chronic treatment of SHR⁹ and essential hypertensive patients¹⁰ with an ACE inhibitor has been shown to be associated with a decrease in the amount of collagen type I present in the myocardium.

Collagen I is synthesized as procollagen with a small amino terminal and a larger carboxy-terminal propeptide. Once secreted into the extracellular space, the propeptides are removed by specific endopeptidases, thus allowing integration of the rigid collagen triple helix into the growing fibril.¹¹ The 100-kD PIP formed during this process is released into the blood. A stoichiometric ratio of 1:1 exists between the number of collagen molecules produced and that of PIP released.¹² Therefore, the serum concentration of PIP has been proposed as a useful marker of collagen type I synthesis.^{13 14}

CITP is a 12-kD peptide produced, together with other peptides, when collagen fibrils undergo resorption.¹⁵ CITP is constituted by the carboxy-terminal telopeptide parts of two ₁ chains of one collagen molecule and one ₁- or ₂-derived helical chain of another collagen molecule, cross-linked by a pyridinoline ring. This peptide is found in an immunochemically intact form in blood, where it appears to be derived from tissues.¹⁶ In recent studies,^{17 18} serum concentration of CITP was found to be related to the intensity of the degradation of collagen type I fibrils.

Recently, we have found that serum concentrations of PIP were abnormally increased in patients with essential hypertension and became normalized after treatment with an ACE inhibitor.¹⁹ Furthermore, serum PIP was related to several anatomic and functional alterations of the left ventricle in hypertensive patients.¹⁹ However, because no cardiac biopsies were performed in this study, the cardiac origin of the peptide remains speculative. In addition, by measuring serum PIP, we assessed the formation of collagen type I but not its degradation.

Therefore, in the present study we compared PIP and CITP as markers, respectively, of extracellular collagen type I synthesis and degradation, with histomorphometric and immunohistochemical parameters of myocardial fibrosis in SHR with established left ventricular hypertrophy. In addition, the impact of the ACE inhibitor quinapril on the above two collagen type I–related peptides was also investigated.

	Methods

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Animals
The investigations performed were in accordance with institutional guidelines. Sixteen-week-old male WKY rats (n=10) and 16-week-old SHR (n=10) (WKY and SHR groups, respectively) were observed in a colony for 20 weeks and killed by decapitation for biochemical and morphological studies. In addition, 16-week-old SHR (n=10) were treated with oral quinapril (10 mg/kg body wt per day) for 20 weeks and killed at 36 weeks (group Q) as previously reported.⁹ The drug was dissolved in drinking water, and the concentration was adjusted for the daily water intake and body weight to obtain an average daily dose of 10 mg/kg body wt. All rats were housed in individual cages and fed with standard rat chow and tap water ad libitum; they were kept in a quiet room at constant temperature (20°C to 22°C) and humidity (50% to 60%).

Before the animals were killed, they were anesthetized (methohexital 50 mg/kg IP), and blood was obtained from the rat's eye by venipuncture. Once the animals had been killed by decapitation, the heart was removed en bloc, and cardiac dimensions were measured. The left ventricle was dissected and washed thoroughly with normal saline to remove any contaminating blood and then immediately snap-frozen in liquid NO₂ and stored at -80°C for later histomorphologic and immunohistochemical studies.

Systolic blood pressure was measured every 2 weeks in all the animals by the standard tail-cuff method using an LE 5007 pressure computer (LETICA Scientific Instruments).

Serum Peptides
Serum samples to determine PIP and CITP were taken at the time of venipuncture and stored at -40°C for up to 6 months.

Serum PIP was determined by the radioimmunoassay method described by Melkko et al,²⁰ using antisera specifically directed against the carboxy-terminal peptide procollagen type I (Orion Diagnostica). We incubated 100-µL aliquots of standard or serum samples with 200 µL of the tracer solution (¹²⁵I-labeled PIP, about 50 000 cpm) and 200 µL of diluted antiserum against PIP (rabbit) for 2 hours at 37°C. We then added 500 µL of the solid-phase second-antibody suspension (goat anti-rabbit) to each tube and vortex-mixed. After 30 minutes at room temperature, the bound fraction was separated by centrifugation (2000g, 15 minutes, 4°C). The supernatant containing the unbound tracer was decantated, and the radioactivity of the precipitate containing the bound tracer was counted with a Gammachem 9612 counter (Serono Diagnostics).

The mean recovery of four serum samples with different concentrations of PIP (3 to 30 µg/L) and mixed in different ratios was 97%. The interassay and intra-assay variations for determining PIP were 7% and 3%, respectively. The sensitivity (lower detection limit) was 0.5 µg of PIP/L.

Serum CITP was measured by radioimmunoassay according to Risteli et al,¹⁶ using antisera specifically directed against the carboxy-terminal telopeptide of collagen type I (Orion Diagnostica). We incubated 100-µL aliquots of standard or serum samples with 200 µL of the tracer solution (¹²⁵I-labeled CITP, about 50 000 cpm) and 200 µL of diluted antiserum against CITP (rabbit) for 2 hours at 37°C. We then added 500 µL of the solid-phase second-antibody suspension (goat anti-rabbit) to each tube and vortex-mixed. After 30 minutes at room temperature, the bound fraction was separated by centrifugation (2000g, 30 minutes, 4°C). The supernatant containing the unbound tracer was decantated, and the radioactivity of the precipitate containing the bound tracer was counted with a Gammachem 9612 counter (Serono Diagnostics).

The mean recovery of four serum samples with different concentrations of CITP (1.5 to 15 µg/L) and mixed in different ratios was 98%. The interassay and intra-assay variations for measuring CITP were 8% and 6%, respectively. The sensitivity was 0.5 µg CITP/L.

Histomorphological and Immunohistochemical Studies
Coronal sections of the left ventricle were obtained from its equator. The equator was selected as representative of the whole left ventricle. Interventricular septal thickness was measured as the maximal distance between the subendocardium and the subepicardium. Tissue samples also were obtained from the liver, the left lung, and the cremaster muscle in each rat.

The collagen-specific stain Masson's trichrome was used on 5-µm-thick, paraffin-embedded sections. To evaluate the extension of collagen deposits, collagen volume fraction was determined with an automatic image analyzer (Microm IP 1.6) and calculated as the sum of the surface of the connective tissue of the section divided by the total surface of the section.

For immunohistochemical study, the avidin-biotin complex method was used. The primary antibody used was collagen type I (Biogenex) at a dilution of 1:50. A semiquantitative scale was developed to measure the amount of interstitial and perivascular collagen type I seen at low power (x10). The amount of collagen type I was graded on a scale of 0 to 3+, with 0 representing the absence of collagen type I; 1+ being mild deposits; 2+ corresponding to moderate deposits; and 3+ being severe deposits.

Statistical Analysis
Data are expressed as mean±SEM. A multiple comparison test (Scheffé's method) was performed to compare mean values of measured parameters in the three different experimental groups. The correlation between continuously distributed variables was tested by univariate regression analysis. The significance level was assumed at P<.05.

	Results

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Blood Pressure
At the beginning of the experiment, systolic blood pressure was significantly increased in SHR compared with WKY rats (data not shown). Although systolic blood pressure remained elevated at hypertensive levels in SHR throughout the experimental 20-week period, blood pressure decreased to values close to those seen in normotensive WKY rats in all rats from group Q, and it remained at these levels throughout the experiment. Therefore, at 36 weeks of age, systolic blood pressure was higher (P<.01) in SHR (247±7 mm Hg) compared with WKY rats (153±3 mm Hg) and Q rats (166±5 mm Hg).

Left Ventricular Hypertrophy
SHR had left ventricular hypertrophy when expressed as the increase of cardiac weight normalized to body weight (data not shown) and the increase of interventricular septal thickness (5.30±0.19 versus 3.87±0.11 mm, P<.01) (Fig 1). Cardiac dimensions in Q rats exhibited values close to those of WKY rats and lower (P<.01) than those of SHR (interventricular septal thickness, 4.31±0.14 mm) (Fig 1).

View larger version (9K): [in this window] [in a new window]   Figure 1. Data points show interventricular septal thickness (IST) in normotensive WKY rats, untreated SHR, and SHR treated with quinapril (Q).

Myocardial Fibrosis and Collagen
The myocardial collagen volume fraction of the left ventricle was increased (P<.01) in SHR compared with WKY rats (5.65±0.10% versus 3.54±0.13%) (Fig 2). After 20 weeks of oral administration of quinapril, collagen volume fraction was normalized in Q rats to values seen in WKY rats (3.39±0.10%) (Fig 2), this value being lower (P<.01) than the value measured in SHR.

View larger version (9K): [in this window] [in a new window]   Figure 2. Data points show collagen volume fraction (CVF) in normotensive WKY rats, untreated SHR, and SHR treated with quinapril (Q).

While more animals exhibited low grades of deposition of collagen type I in the WKY group, more animals exhibited high grades in the SHR group (Table 1). After administration of quinapril, the distribution of treated SHR was deplaced to values seen in WKY rats (Table 1).

View this table: [in this window] [in a new window]   Table 1. Grade of Collagen Type I Deposits

Fig 3 shows a representative picture of collagen type I deposition in the myocardium of the three groups of rats. Collagen type I stained large strands or thin waves around isolated cardiomyocytes and a reticular network of fibrotic tissue through the myocardium of SHR. In contrast, collagen type I reacted very slightly between cardiomyocytes in both WKY rats and Q rats (Fig 3).

View larger version (388K): [in this window] [in a new window]   Figure 3. Left ventricle from WKY rats (A), untreated SHR (B), and SHR treated with quinapril (C). Sections were immunolabeled with anticollagen type I. The accumulation of collagen type I mainly in the perimysium and perivascular region can be seen as large strands irregular in size and shape in SHR. In contrast, very small amounts of collagen type I are present in WKY rats and quinapril-treated rats. Magnification x200.

Collagen in Other Organs
As shown in Table 2, the collagen volume fraction of several organs (ie, liver, lung, skeletal muscle) was similar in WKY rats and SHR. Thus, fibrosis of these organs can be excluded in SHR.

View this table: [in this window] [in a new window]   Table 2. Collagen Volume Fraction in Several Organs

Serum Peptides
As shown in Fig 4, serum concentration of PIP in WKY rats ranged from 4.70 to 10.20 µg/L (mean, 8.54±0.56 µg/L). Serum concentration of PIP was higher (P<.01) in SHR (13.13±0.94 µg/L) than in WKY rats (Fig 4). Nine SHR exhibited values of PIP above the upper end of this parameter seen in WKY rats. After treatment, PIP values in Q rats were similar to those measured in WKY rats (8.88±0.62 µg/L) and lower (P<.01) than values measured in SHR (Fig 4). Three Q rats exhibited values of PIP above the upper normal limit.

View larger version (9K): [in this window] [in a new window]   Figure 4. Data points show serum concentration of PIP in normotensive WKY rats, untreated SHR, and SHR treated with quinapril (Q).

A direct correlation was found between collagen volume fraction and serum PIP (y=6.54x-23.82, r=.753, P<.05) in SHR (Fig 5). No correlations between collagen volume fraction and serum PIP were found in WKY rats or Q rats.

View larger version (8K): [in this window] [in a new window]   Figure 5. Scatterplot shows direct correlation (y=6.54x-23.82) between collagen volume fraction (CVF) and serum concentrations of PIP in SHR.

Serum concentration of CITP in WKY rats ranged from 4.50 to 5.90 µg/L (mean, 5.24±0.17 µg/L, Fig 6). Although SHR did tend to exhibit a higher serum CITP concentration (5.86±0.19 µg/L) than WKY rats, the difference was not statistically significant (Fig 6). Four SHR exhibited values of CITP above the upper end in WKY rats. The serum concentration of CITP measured in Q rats (5.79±0.16 µg/L) was similar to that measured in SHR (Fig 6). Although five Q rats exhibited values of CITP above the upper end in WKY rats, mean values of the peptide measured in the two groups were not statistically different.

View larger version (8K): [in this window] [in a new window]   Figure 6. Data points show serum concentration of CITP in normotensive WKY rats, untreated SHR, and SHR treated with quinapril (Q).

No significant correlations were found between CITP and collagen volume fraction or between PIP and CITP in this study.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The main finding of the current study is that an increased serum concentration of PIP is present in SHR compared with WKY rats. In addition, we show that an association exists between abnormally high serum PIP and myocardial fibrosis in SHR. Neither of the two alterations are present in SHR submitted to chronic treatment with the ACE inhibitor quinapril.

The rate of extracellular synthesis of collagen type I can be assessed by measuring the serum concentration of PIP, which is freed during the extracellular processing of procollagen type I before the collagen molecules form collagen fibers.¹¹ This peptide appears to be eliminated from the blood by the liver.²¹ Taking into account that hepatobiliary function has been found to be preserved in SHR,²² it can be proposed that an elevated serum concentration of PIP present in SHR represents an increased production of the peptide.

Several observations have led to the proposal that increased production of PIP is a useful marker of stimulated fibrogenesis.^{13 14} Accordingly, the finding of elevated serum concentration of PIP in SHR is in agreement with our previous finding in essential hypertensive patients¹⁹ and reinforces the idea that arterial hypertension represents a condition characterized by fibrogenic hyperactivity.

As previously found by others⁵ and by ourselves,⁹ we did observe a significant increase in fibrillar collagen type I in the left ventricle of SHR. Furthermore, we found that serum PIP correlates with myocardial collagen volume fraction in SHR. Therefore, it is tempting to hypothesize that increased serum PIP present in SHR may reflect an increased ventricular synthesis of fibrillar collagen type I.

The question of how changes in the cardiac compartment of collagen type I can alter concentrations of PIP in the circulation deserves some comments. McAnulty and Laurent²³ have shown that mean cardiac collagen content of the rat is approximately 5.3x10³ µg/g wet wt. When the cardiac weight of rats studied here was taken into account (data not shown), the calculated mean values of cardiac collagen were 8.05x10³ µg in WKY rats, 9.48x10³ µg in untreated SHR, and 7.05x10³ µg in treated SHR. Since in the adult rat more than 80% of total myocardial collagen is type I²⁴ and the molecular weight of collagen type I is of 407 000, the calculated numbers of molecules of this substance were 9.60x10¹⁸ in WKY rats, 11.36x10¹⁸ in untreated SHR, and 8.40x10¹⁸ in treated SHR. Because a stoichiometric ratio of 1:1 exists between the number of collagen type I molecules and that of PIP released,¹² the same number of molecules of the peptide can be calculated in the three experimental groups of rats. On the other hand, assuming that mean total blood volume of the rat is approximately 64.1 mL/kg body wt,²⁵ the calculated mean values of total circulating PIP were 0.308 µg in WKY rats, 0.515 µg in untreated SHR, and 0.233 µg in treated SHR. When the molecular weight of PIP (100 000) was taken into account, the calculated numbers of circulating molecules were 1.85x10¹⁵ in WKY rats, 3.09x10¹⁵ in untreated SHR, and 1.40x10¹⁵ in treated SHR. Since the mean daily collagen synthesis rate in the rat heart has been shown to be 5.2%,²³ it seems reasonable to propose that under a quantitative point of view, changes in the cardiac formation of collagen type I are able to modify serum levels of PIP in the rat.

The possibility that an increase in the cardiac synthesis of collagen type I may increase serum PIP in SHR is further supported when considering that other extracardiac sources (for example, the liver¹³ ) able to elevate the serum peptide can be excluded in SHR of this study (see Table 2). On the other hand, although increased serum concentration of PIP has been reported in hyperthyroidism, a disease characterized by an increase of bone turnover,¹⁴ neither abnormal blood levels of thyroid hormones²² nor bone remodeling²⁶ have been found in SHR. In addition, serum CITP, which is also increased in those alterations of the bone characterized by increased release of PIP,²⁷ was found to be normal in untreated SHR of this study. Finally, since vascular remodeling by an excess of collagen proteins occurs in systemic hypertension,²⁸ the vascular wall might be another source of PIP in SHR. However, as previously shown by Bashey et al,²⁹ the proportion of collagen type I is lower, and the proportion of collagen V higher, in the aorta of SHR compared with WKY rats.

Due to its pharmacological properties,³⁰ quinapril seems to be unable to influence the hepatobiliary elimination of PIP. Thus, the association of a normal serum concentration of PIP with a normal amount of collagen in the myocardium of SHR treated with quinapril suggests that this ACE inhibitor normalizes serum PIP by diminishing the production of the peptide via a decrease of the myocardial synthesis of collagen type I.

Hypertensive myocardial fibrosis appears to be induced primarily by nonhemodynamic mechanisms.⁶ In vitro, angiotensin II and aldosterone have been shown to directly enhance the collagen synthesis of rat cardiac fibroblasts.³¹ Therefore, it is likely that a decreased systemic and/or local production of effector hormones of the renin-angiotensin-aldosterone system is involved in the ability of quinapril to depress myocardial synthesis of collagen type I in SHR. Nevertheless, other alternative mechanisms, for example, a decrease in blood pressure, increases in prostaglandin E₂, bradykinin, and nitric oxide, also deserve consideration.

Another finding of this study is that serum concentration of CITP is normal in both untreated SHR and SHR treated with quinapril.

Collagen degradation may occur either intracellularly or extracellularly.³² The extent of intracellular degradation can be assessed with the use of radiolabeled proline. However, a problem that limits the use of this technique is that of reutilization of the radiolabeled amino acid.³³ On the other hand, the rate of extracellular collagen type I degradation usually has been estimated by assays of urinary imino acid 4-hydroxyproline and assays of urinary pyridoline and deoxipyridoline cross-links. The main limitation of these assays is that they are not specific for type I collagen.^{34 35} Measurement of CITP antigen concentration is more specific to assessing extracellular degradation because CITP is collagen type I–specific and, because it is cross-linked, known to be derived from molecules that have been incorporated into collagen fibrils.¹⁶

From previous observations, serum CITP has been proposed as a useful marker of extracellular collagen type I degradation.^{16 17 18} Thus, the finding of normal serum concentration of CITP in SHR suggests that extracellular degradation of collagen type I is unaltered in this model of genetic hypertension. Furthermore, the association of normal serum CITP with increased myocardial collagen content in SHR suggests that a depressed extracellular degradation of collagen type I does not account for the accumulation of this fiber in the myocardium of SHR. Nevertheless, because a direct measurement of collagenolytic activity of myocardial tissue was not performed in this study, a compromised degradation of collagen type I cannot be excluded completely in SHR.

On the other hand, since more than half of the collagen synthesized in the heart is degraded intracellularly,²³ the possibility exists that intracellular degradation of collagen type I is altered in SHR. Two arguments support this possibility: (1) using histochemical methods, Doering et al³⁶ found indirect evidence of increased collagen fiber degradation in the left ventricle of rats with pressure-overload hypertrophy, and (2) on the basis of studies on the turnover of cardiac muscle protein, Laurent et al³⁷ proposed that increments in both collagen synthesis and degradation are simultaneously operative in pressure-overload hypertrophy.

Angiotensin II has been shown to decrease collagenase activity in cultured adult rat cardiac fibroblasts.³¹ On the other hand, an increase of myocardial collagenase activity has been found in SHR receiving the ACE inhibitor lisinopril.³⁸ Thus, it appears that the fibrolytic response of the myocardium of SHR to ACE inhibitors may be due in part to enhanced extracellular collagen degradation by activation of tissue collagenase.

These observations are contrary to our finding that despite the regression of myocardial fibrosis observed in SHR treated with quinapril, CITP is not altered in these rats. Several explanations may account for such a discrepancy. First, as previously mentioned, we did not measure directly the collagenolytic activity of the myocardial tissue of treated SHR. Thus, the possibility remains that this activity is enhanced after treatment with quinapril, as shown with other ACE inhibitors.³⁸ An alternative explanation is that serum concentration of CITP does not increase in SHR treated with quinapril because renal elimination of CITP is stimulated by this compound. This possibility is further sustained because CITP appears to be cleared from the circulation via glomerular filtration¹⁶ and because quinapril increases the glomerular filtration rate in rats.³⁰

Summary
Our findings show an abnormal increase of serum concentration of PIP and a normal serum concentration of CITP in SHR. The effects of quinapril on serum concentrations of the two peptides suggest that the renin-angiotensin-aldosterone system may participate in the excessive conversion of procollagen type I to collagen type I in SHR. On the other hand, the relation observed between serum PIP and collagen type I content of the left ventricle permits us to propose that this circulating procollagen I–derived peptide may reflect ongoing myocardial fibrosis in SHR. This adds support to our previous work in essential hypertension,¹⁹ thus providing a potential noninvasive method to detect the presence of myocardial fibrosis in hypertensive patients.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme CITP = collagen type I carboxy-terminal cross-linked telopeptide PIP = procollagen type I carboxy-terminal peptide SHR = spontaneously hypertensive rats WKY = Wistar-Kyoto

Received October 2, 1995; accepted October 4, 1995.

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