Abstract
Background. Increased intima media thickness (IMT) of common carotid arteries (CCAs) and left ventricular mass index (LVMI) are independent risk factors for vascular events and may be related to accumulation of extracellular proteins due to altered metabolism of collagen. Methods. IMT and LVMI were measured ultrasonographically in 50 males with newly diagnosed, untreated, essential hypertension (HTN, 37.7±13.1 years), and 14 controls (C, 32.6±9.7 years). Serum levels of procollagen type I carboxy‐terminal propeptide (PICP), procollagen type III amino‐terminal propeptide (PIIINP), carboxy‐terminal telopeptide (ICTP), matrix metalloproteinase (MMP‐1) and tissue inhibitor of metalloproteinase‐1 (TIMP‐1) were determined using immunoassays. Results. IMT was significantly higher in HTN than in C (0.6±0.1 vs 0.4±0.1 mm, p<0.001) as well as LVMI (119.5±39.9 vs 106.8±18.7 g/m2, p = 0.04) and serum TIMP‐1 (in HNT 691.7±124.6 ng/ml; in C 577.5±70.8 ng/ml, p<0.001). Other parameters did not differ between these groups. The sum of PICP and ICTP was higher in HTN (165.0±46.9 µg/l), than in C (147.1±26.0 µg/l, p = 0.03). TIMP‐1 correlated with IMT (r = 0.33, p = 0.02) in hypertensives. Conclusions. We suggest that the collagenase–anticollagenase system is abnormal in essential hypertension and contributes to cardiovascular remodeling. Increased IMT may be related to the accumulation of extracellular proteins due to altered metabolism of collagen.
Introduction
Essential hypertension significantly increases the risk of cardiovascular complications and continues to be one of leading causes of mortality in populations Citation[1].
Remodeling of the heart and peripheral arteries is one of detrimental effects of elevated blood pressure Citation[2]. This process involves not only vascular smooth muscle cells (VSMC), but extracellular matrix (ECM) as well. Recent investigations indicated that extracellular matrix is involved in various cardiovascular pathologies. It influences numerous functions of cardiac and vascular cells, not only activation, proliferation, but also cell death, leading to complex remodeling of cardiovascular system. Extracellular matrix metabolism is involved not only in hypertension‐related remodeling, but also plays its role in a burden of atherosclerosis, and would help to understand the initiation, progression, distribution and vulnerability of the atherosclerotic lesions Citation[3–6]. Abnormalities in collagen metabolism, the main protein component of ECM, probably have significant role in remodeling. Progressive accumulation of collagen content in the cardiac and vascular wall is thought to occur due to increase in synthesis or due to decreased degradation by matrix metalloproteinases Citation[7,8].
Measurements of specific biomarkers of collagen synthesis and degradation have been used for estimation of extracellular matrix metabolism. Propeptides released from the procollagen molecules are indices of collagen synthesis. Another peptide, cleaved from collagen fibrils serves as an index of collagen degradation Citation[9]. Some experimental and clinical studies indicated altered metabolism of collagen in hypertensives Citation[10–14]. Left ventricular hypertrophy (LVH) is an established cardiovascular risk factor and a parameter of cardiac remodeling in hypertension Citation[15]. Since intima media thickness (IMT) of common carotid arteries could be recognized as an index of vascular remodeling in hypertension Citation[16], we hypothesized that IMT of elastic arteries may be related to accumulation of extracellular proteins due to altered metabolism of collagen Citation[9]. Therefore, we studied potential correlation between the left ventricular mass index (LVMI), collagen parameters and intima media thickness of common carotid arteries (IMT).
Materials and methods
Subject population and study design
We studied 50 males with newly diagnosed, untreated, uncomplicated, stage I or II essential hypertension without any coexisting diseases (HTN, 37.7±13.1 years), and 14 healthy age‐matched controls who did not receive any medications (C, 32.6±9.7 years). Exclusion criteria included conditions potentially affecting collagen metabolism such as neoplasms, rheumatic diseases, bone diseases, diabetes mellitus, liver or kidney diseases, smoking, trauma/surgery in the year before the study. None of the subjects was treated with antihypertensive, hypolipemic medications or steroids. All of the subjects had clinical examination performed by a physician, basic laboratory studies with standard methods, 12‐lead EKG, ambulatory blood pressure monitoring and echocardiography. Secondary forms of hypertension were excluded in HTN based on daily urine for metanephrines, abdominal ultrasonography and Doppler ultrasonography of the renal arteries. Subjects enrolled into the study had additional laboratory tests of collagen and bone metabolism, large artery studies like intima media thickness of the common carotid arteries (IMT), and VHS recordings of the echocardiographic studies. Blood pressure was measured with standard, office method corresponding to WHO guidelines Citation[17] and with ambulatory blood pressure monitoring (ABPM).
The investigation conforms to the principles outlined in the Declaration of Helsinki. The protocol was accepted by the Institutional Review Board and all subjects gave their written informed consents to be included in the study.
Carotid ultrasound
IMT was measured with ultrasound imaging system (SONOS 4500, Philips, Andover, USA), using a 7.5‐MHz linear vascular probe. Measurements were performed, with ECG gating, on the two‐dimensional longitudinal lateral view of both common carotid arteries (CCAs), 2 cm below bifurcation. Measurements were done of the leading edges of the blood–intima and media–adventitia interfaces defining IMT on the far wall of the common carotid arteries in diastole. A mean value of 12 (six for each CCA) consecutive measurements was used. Analysis was performed offline, manually, on the recorded video image by an experienced sonographer blinded to the clinical characteristics of the subjects.
Echocardiographic studies
Examinations were performed with SONOS 4500 (Philips, Andover, USA) imaging system using a 2.5‐MHz cardiac phased array probe. Two‐dimensional harmonic and M‐mode measurements were made with ECG gating related to standard recommendations Citation[18]. Left ventricular mass (LVM) was calculated according to Devereux formula and adjusted for body surface area (BSA) to obtain LVM index (LVMI) Citation[19]. LVH was diagnosed when LVMI⩾116 g/m2Citation[20].
Ambulatory blood pressure measurements
Ambulatory blood pressure measurements were performed with Space Labs 90207 monitors, (SpaceLabs, Redmont, Washington, USA). Readings were obtained every 15 min during the day (06.00–23.00 h) and every 20 min at night (23.00–06.00 h).
Serum parameters of collagen metabolism
Blood samples were collected from antecubital veins. Samples were centrifuged and serum was frozen in −70°C till assayed. To assess collagen synthesis we used serum levels of procollagen type I carboxy‐terminal propeptide (PICP) and procollagen type III amino‐terminal propeptide (PIIINP), which are released from procollagens during collagen synthesis. To estimate collagen degradation we determined the serum levels of carboxy‐terminal telopeptide (ICTP), which is released during collagen cleavage Citation[21]. All measurements were done by radioimmunoassays (Type I Procollagen PICP Radioimmunoassay Kit, Type III Procollagen PIIINP Radioimmunoassay Kit, Type I Telopeptide ICTP Radioimmunoassay Kit, Orion Diagnostica, Espoo, Finland). Serum levels of matrix metalloproteinase (collagenase, MMP‐1), the main extracellular enzyme responsible for initiation of collagen degradation, as well as serum levels of the tissue inhibitor of metalloproteinase‐1 (TIMP‐1) were measured. These two parameters were assayed with immunoenzymatic method (ELISA), (Biotrak MMP‐1 human ELISA system, Biotrak TIMP‐1 human ELISA system, Amersham Pharmacia Biotech UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, UK). All measurements were performed in duplicate.
Bone tissue metabolism
Since several studies showed an influence of bone tissue diseases on collagen‐related blood parameters Citation[22], we used following laboratory indices of bone metabolism to exclude possible differences between the groups. To assess bone metabolism, we measured several serum parameters reflecting bone tissue turnover, such as calcitonin (CT‐U.S.‐IRMA, Bio Source, Nivelles, Belgium), osteocalcin (OSTEO‐RIACT, CIS Bio‐International, Gif‐sur‐Yvette, France), parathormone (ELSA‐PTH, CIS Bio‐International, Gif‐sur‐Yvette, France), vitamin D3 (1,25(OH)2‐Vit.D‐RIA‐CT, Bio Source, Nivelles, Belgium) and ionized calcium in the full blood (ABL 700 Series, Radiometer Copenhagen, Denmark).
Statistics
Statistical analysis was performed with SAS package, SAS Institute Inc, Cary, USA. Normal distribution of variables was estimated with Shapiro–Wilk test. Parameters with normal distribution were showed as mean±SD, otherwise median and range were presented. Differences between two groups were tested by Student's t‐test for unpaired data of normal distribution. Correlation between variables was tested by Spearman test separately for each group. p<0.05 was considered statistically significant.
Results
Characteristics of the HTN and C are presented in . Both groups did not differ in age, weight, height or body surface area (BSA). Body mass index was significantly higher in HTN (27.6±3.6 vs 24.9±3.5, p = 0.01). HTN had significantly higher blood pressure values both in office and ambulatory measurements.
Table I. Characteristics of the subjects.
Properties of the large arteries and echocardiographic studies
Intima media thickness (IMT) was significantly higher in HTN than in C (0.6±0.1 vs 0.4±0.1 mm, p<0.001). Diameters of the CCAs were practically identical in both groups (6.6±0.1 mm vs 6.6±0.1 mm, p = 0.66).
HTN had significantly higher LVMI than C (119.5±39.9 vs 106.8±18.7 g/m2, p = 0.04) (). LVH was diagnosed in 21 hypertensives but in none of the C. The subjects presented without global or regional wall motion abnormalities and without significant valve disease. We did not find correlations between LVMI and IMT (r = 0.127, p = 0.43).
Table II. Echocardiographic parameters.
Biochemical markers of collagen metabolism
Concentrations of PICP were non‐significantly higher in the HTN vs C (161.0±46.0 µg/l vs 143.5±25.8 µg/l, respectively, p = 0.07). Similarly, serum levels of PIIINP and ICTP were non‐significantly higher in the HTN (2.6±0.9 and 4.1±2.0 μg/l, respectively) than in C (2.4±0.5 µg/l, p = 0.29, and 3.6±1.2 µg/l, p = 0.22).
To analyze collagen metabolism, we implemented a parameter used by Diez & Laviades, a ratio of serum levels of PICP and ICTP, which was proposed as an index of coupling between the synthesis and degradation of collagen type I Citation[21]. We did not observe any significant difference between the HTN and C regarding this index (44.6±16.2 µg/l, and 44.8±16.0 µg/l, respectively, p = 0.95). However, we found a significant difference between a sum of serum levels of PICP and ICTP in the HTN (165.0±46.9 µg/l) and in the C (147.1±26.0 µg/l, p = 0.03) (). Serum levels of PICP correlated with levels of PIIINP (r = 0.42, p<0.01) and ICTP (r = 0.52, p<0.001) in hypertensives. Serum levels of PIIINP also correlated with ICTP (r = 0.49, p<0.001). There were no anticipated correlations between LVM, LVMI or ambulatory blood pressure and the indices of collagen metabolism.
Table III. Parameters of collagen metabolism in the studied subjects.
Collagenase (MMP‐1) and its inhibitor (TIMP‐1) serum levels
Serum concentrations of TIMP‐1 were significantly higher in hypertensives (691.7±124.6 ng/ml) than in C (577.5±70.8 ng/ml, p<0.001), whereas serum levels of collagenase (median 0.7, range 0.00–73.5 vs median 0.2, range 0.00–6.0 ng/ml, p = 0.09) did not differ between these groups.
Serum levels of PIIINP correlated with TIMP‐1 levels (r = 0.33, p<0.05). We did not find correlation between serum levels of collagenase (MMP‐1) and other parameters of collagen metabolism. Interestingly, serum TIMP‐1 concentrations were related to IMT (r = 0.34, p<0.05) in HTN. There were no correlations between TIMP‐1 and LVMI (r = 0.005, p = 0.97).
Bone tissue metabolism indices
Both groups had similar serum levels of ionized calcium and such bone turnover equivalents as ionized calcium, and serum levels of calcitonin, osteocalcin, vitamin D3, parathormone, which practically exclude any significant metabolic bone disease from influencing the results of the study ().
Table IV. Parameters of bone tissue turnover.
Discussion
Patients with newly diagnosed, untreated, essential hypertension had significantly higher IMT when compared with healthy, age‐matched controls. However, both groups presented with IMT within normal range for this age group Citation[24]. Altered collagen metabolism in hypertension influences IMT of the common carotid arteries (CCAs), which are usually relatively free from atherosclerotic plaques. IMT of the CCAs could be considered a marker of early arterial wall remodeling, rather than a surrogate for atherosclerosis. The common carotid arteries are less likely to develop intrusive plaques than bifurcation or internal segments, therefore they are useful in studies directed more towards remodeling than atherosclerosis Citation[16]. We found significant correlation between IMT of the carotid arteries and serum TIMP‐1 levels. Moreover, serum TIMP‐1 was significantly increased in hypertensives when compared to controls. It is, along with collagenase (MMP‐1), a key enzyme controlling collagen metabolism. While MMP‐1 cleaves ICTP from collagen molecule and diminishes collagen content, TIMP‐1 may facilitate fibrosis by direct inhibition of collagenase. Our results of increased TIMP‐1 levels in hypertensive patients correspond with several previous studies, which also detected elevated levels of TIMP in essential hypertension Citation[25–27]. Sundström et al. recently presented data from the Framingham Study, linked TIMP‐1 levels to the cardiovascular risk factors. These authors found additionally a significant correlation between TIMP‐1 levels and LVH Citation[26,27]. Although we did not observe such relation in our study, we found a significant correlation between TIMP‐1 and IMT of the carotid arteries. We suggest that TIMP‐1 level may be a biomarker of the hypertensive remodeling not only of the left ventricle of the heart but the entire cardiovascular system Citation[28]. We observed no statistically significant difference in MMP‐1 levels between studied groups. Therefore, we could not state whether elevated TIMP‐1 causing inhibition of MMP‐1 would lead to an increase in collagen content in tissues as would be expected Citation[29,30]. Some authors suggested that TIMP‐1 may be elevated as a consequence of feedback regulation compensating for an increased MMP‐1 activity and collagen I degradation Citation[31]. However, it can also be assumed that an increase in TIMP‐1 may cause a decrease in MMP‐1, which results in an increase in collagen content in ECM. It would end up with increased collagen volume fraction in the cardiovascular system in hypertensives Citation[10],Citation[23],Citation[32].
Although hypertensives had higher serum levels of the peptides reflecting collagen I and III synthesis, these differences did not reach statistical significance. Similarly, serum levels of a telopeptide from a cleaved collagen molecule were not significantly higher in hypertensives, when compared with controls. Previously, Diez et al. found higher ratio of PICP and ICTP levels in hypertensives than in controls Citation[32–34] and suggested that elevated PICP to ICTP ratio may indicate disordered balance of collagen metabolism, resulting in progressive fibrosis of the cardiovascular system. However, we did not find any significant difference. Therefore, we tried other ratios between collagen markers of synthesis and degradation. Among them, we added PICP and ICTP, as we wanted to improve probability of discovering potentially increased turnover of collagen in studied subjects, and we found a significant difference between a sum of serum levels of PICP and ICTP in the HTN when compared to the controls. This observation may suggest an increased collagen turnover. Significant correlation between PICP, PIIINP and ICTP, may further confirm that an enhanced collagen synthesis exists together with its increased degradation, but seemingly not efficient to avoid subtle but progressive fibrosis. We did not see difference in PIIINP levels between two groups, despite the fact that this parameter is also interpreted as collagen type III turnover index Citation[35].
Although some papers discussed an impact of collagen metabolism on cardiac anatomy and function in essential hypertension, in our database search before the start of our study, there were no articles, listed in the PubMed Library, on possible association between collagen and intima media thickness of carotid arteries. However, recently, Olsen et al. have published a LIFE substudy, relating markers of collagen synthesis to blood pressure and vascular hypertrophy Citation[36]. They suggested that cardiovascular fibrosis, assessed by collagen markers, is related to hemodynamic load as previously showed in animal studies Citation[37,38].
However, that would be true mostly for primary hypertension, as in our group of patients. In some types of secondary forms of hypertension, humoral factors could play also an important role Citation[38,39]. That is one of the reasons why we decided to include only untreated, newly diagnosed hypertensives. We tried meticulously to select subjects to represent essential hypertensives without previous treatment that could influence collagen metabolism. We studied males to exclude potential influence of changes in estrogen/progesterone levels on ECM. Because of our inclusion criteria, our subjects were young when compared to the general population with cardiovascular diseases. They had hypertension stage I–II according to JNC VII, which means that we were observing changes in ECM metabolism in the very early stages of hypertensive disease. We showed that our patients, despite a normal range of IMT and their lack of other overt hypertension‐related target organ damage, had higher values of IMT than the controls. Elevated TIMP‐1 would have lead to an augmentation in collagen deposition in the walls of the heart and blood vessels even in our relatively young subjects
Nevertheless, we have not found correlation between collagen indices and LVMI, similarly to Olsen et al., who studied patients with previous treatment Citation[36].
Additionally to parameters related directly to the cardiovascular system, we tried to broaden the scope of the study and used several tests for indices of bone tissue metabolism to assess their possible relationship with collagen, but we did not find any significant changes between studied groups.
Limitations of the study
We assessed collagenase serum levels, not activity, which was less convenient and more expensive at the beginning of the study, but potentially might be more accurate for the assessment of metalloproteinase metabolism. We studied only MMP‐1, as a dominant metalloproteinase, first in the complex chain of degradation of collagen molecules. However, several different types of metalloproteinases, mostly MMP‐2 and MMP‐9 have grown in scientific importance. However, even in the papers with all these three enzymes studied, their activities were in fact in a linear relationship Citation[30].
It is not that easy definitely to relate particular serum particles, such as PICP, ICTP and others used in the studies, to specific stages of collagen metabolism. Additionally, analyzing the results, it is difficult to differentiate whether there is an increased turnover or an abnormal metabolism of collagen. One of potential limitations is that measuring serum levels of peptides such as PICP, PIIINP and ICTP, we indicate a more likely metabolism of collagen than its content in tissues. The ultimate way of studying collagen fraction in any tissue is of course histopathology following invasive procedures. In relatively rare histopathological studies, the authors found increased collagen volume fraction in hypertensive patients Citation[10],Citation[26]. Because of the invasive nature of these methods, and our design of the study with a control group, we did not perform them on our subjects.
Performing several measurements, from clinical ones, through biochemical, ultrasonographical, to echocardiographical, on a selected group of newly diagnosed, untreated hypertensives, we were only able to study relatively small number of subjects; therefore, statistical value of comparisons has been limited. However, most of the studies with collagen metabolism involve rather small numbers of patients.
Conclusions
In conclusion, we suggest that the collagenase–anticollagenase system is abnormal in essential hypertension and contributes to cardiovascular remodeling. Increased IMT can be related to the accumulation of extracellular proteins because of altered metabolism of collagen. TIMP‐1 level may be a biomarker of the hypertensive remodeling not only of the left ventricle of the heart but the entire cardiovascular system.
Acknowledgements
This work was supported by grant 3PO5B08322 from The Ministry of Scientific Research and Information Technology.
Dr Szmigielski received the Fellowship Award for Young Scientists from The Foundation for the Polish Science in the year 2003 and 2004. No conflicts of interest exist.
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