a The Framingham Study, Boston University School of Medicine, 73 Mt Wayte Ave Suite 2, Framingham, MA, USA
b Department of Preventive Medicine, Boston University School of Medicine, Boston, MA, USA
c Cardiology Section, Boston University School of Medicine, Boston, MA, USA
d Myocardial Biology Unit, Boston University School of Medicine, Boston, MA, USA
e NHLBI, USA
Received October 15, 2003;
revised May 4, 2004;
accepted May 26, 2004
* Corresponding author. Tel.: +1-508-935-3450; fax: +1-508-626-1262
vasan{at}bu.edu
See page 1475 for the editorial comment on this article1
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Abstract |
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Methods and results We studied 1069 Framingham Heart Study participants (mean age 56 years, 58% women) free of heart failure and previous myocardial infarction. Plasma TIMP-1 was higher in men compared with women, and increased with age, body mass index and total/HDLcholesterol ratio, but decreased with alcohol intake. Plasma TIMP-1 was also directly related to smoking, diabetes and use of anti-hypertensive treatment. Adjusting for age, sex and height, plasma TIMP-1 was positively associated with LV mass, wall thickness, relative wall thickness, end-systolic diameter, and left atrial diameter and the risk of having increased LV end-diastolic diameter or increased wall thickness, and negatively correlated with fractional shortening. Additional adjustment for clinical covariates attenuated the relations of plasma TIMP-1 to most echocardiographic measures.
Conclusions In our cross-sectional investigation, plasma total TIMP-1 was related to major cardiovascular risk factors and to indices of LV hypertrophy and systolic dysfunction. This raises the possibility that cardiovascular risk factors may influence cardiovascular remodelling via extracellular matrix degradation, which may be reflected in plasma TIMP-1 levels.
Key Words: Heart failure Left ventricular hypertrophy Metalloproteinases Remodelling Echocardiography
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Introduction |
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Information on the clinical correlates of plasma TIMP-1 levels in humans is limited. In diabetics, elevated plasma TIMP-1 levels have been observed.12 In hypertensives, plasma TIMP-1 levels were increased and associated with LVH and LV diastolic impairment in some,46 but not all, studies.13 Treatment of hypertension with angiotensin-converting enzyme-inhibitors has been shown to decrease TIMP-1 levels.5 Additionally, plasma TIMP-1 levels are elevated in coronary disease.14,15 Previous studies of small samples of patients with heart failure or LV dilation have yielded inconsistent results, with both increased and decreased levels of myocardial or serum TIMP-1 being reported.2,3,8
Previous clinical studies of plasma TIMP-1 levels are limited by selection bias, and, as noted above, have produced inconsistent results. The factors influencing plasma TIMP-1 levels in the general population are unknown. Furthermore, it is unclear if plasma TIMP-1 levels are related to LV structure and function.
We hypothesised that cardiovascular disease (CVD) risk factors may influence plasma TIMP-1 levels. Additionally, we theorised that remodelling of the cardiac extracellular matrix may be associated with LV dilation and dysfunction, and that this process may be reflected in plasma levels of TIMP-1. Accordingly, we examined the clinical correlates of plasma TIMP-1 and investigated the cross-sectional relations of plasma TIMP-1 to cardiac structure and function in Framingham Heart Study participants.
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Methods |
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We examined the sex-specific distributions of echocardiographic end-diastolic LV internal diameter (LVEDD) and wall thickness (LVWT) among eligible participants (Fig. 1
). We sampled all subjects with measurements of LVEDD and LVWT below median ("referent group", ) and all those with values equal to or exceeding the 90th percentile of LVEDD ("increased LVEDD group",
) or LVWT ("increased LVWT group",
). Thus, we investigated 1044 Offspring cohort participants and 200 Omni cohort participants according to the criteria noted above, in order to maximise statistical and blood sample use efficiency.
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Clinical examination
Participants underwent a standardised medical history and physical examination including measurements of blood pressure; phlebotomy for assessment of CVD risk factors; and a 12-lead electrocardiogram. Diabetes and hypertension were defined according to current guidelines.19,20 Prevalence of CVD, including ischemic heart disease, congestive heart failure, cerebrovascular disease and peripheral vascular disease, were established by a panel of three physicians.21 The Framingham Risk Score was calculated as described previously.18
Plasma total TIMP-1 measurements
Blood samples were drawn from fasting participants in a supine position, centrifuged, and the plasma frozen at 70 °C until assay. Plasma TIMP-1 was measured in duplicate using a two-site sandwich ELISA (Amersham Pharmacia Biotech), which measures free TIMP-1, and TIMP-1 complexed with various MMPs (MMP-1/TIMP-1 complexes, etc.). The intra-assay coefficient of variation for TIMP-1 was <5%.
Echocardiographic methods
All participants underwent routine transthoracic echocardiography with Doppler colour flow imaging. M-mode measurements of LV dimensions were obtained using the leading edge-to-leading edge technique.22 The interventricular septum thickness (IVS), posterior LV wall thickness (PW) and LVEDD were measured at end-diastole. Left atrial diameter was measured at end-systole, as was the LV internal diameter (LVESD). LVWT was calculated as IVS+PW, and LV relative wall thickness was calculated as (IVS+PW)/LVEDD. LVM was calculated as 0.8[1.04(IVS+LVEDD+PW)3(LVEDD)3]+0.6 g.23 Endocardial LV fractional shortening (LVFS) was calculated as (LVEDD-LVESD)/LVEDD. Mid-wall LVFS, calculated as previously described,24 was used in additional analyses. Valve disease was defined as >mild degree of stenosis or regurgitation of the aortic or mitral valves on Doppler echocardiography. Reproducibility of echocardiographic measurements was excellent.25
Statistical analyses
We used graphical analysis and descriptive statistics to assess the distributional properties of each of our analytical variables (including means, standard deviations, medians, quartiles and ranges). We used natural logarithmic transformation for variables with skewed distributions (TIMP-1, LVM, LVWT, relative wall thickness, LVEDD, LVESD, left atrial diameter and alcoholic drinks/week).
Step-wise multiple linear regression models were used to investigate the relation of select clinical variables (age, sex, ethnicity, body mass index, smoking, alcohol intake, diabetes, total/HDLcholesterol ratio, systolic blood pressure, anti-hypertensive treatment, and heart rate) to plasma TIMP-1 levels [log-transformed, ln(TIMP-1)] in the total sample, and in the referent group. We evaluated clinical correlates in both groups to understand whether determinants of TIMP-1 in a "healthier" subset of individuals with normal LV dimensions differed from that observed in the larger sample. A p value of <0.05 was used for retaining variables in the stepwise models. Self-reported ethnicity was used as a dichotomous variable (Caucasian vs. non-Caucasian). In secondary analyses, we forced LVWT and LVEDD into the models evaluating clinical correlates of TIMP-1.
We evaluated the relations of ln(TIMP-1) to the Framingham Risk Score (modelled as a continuous variable) in multivariable models adjusting for age, sex, body mass index, and other correlates identified above. We also used analysis of variance to examine trends in ln(TIMP-1) across sex-specific quartiles of the Framingham Risk Score.
We used multiple logistic regression to examine the associations of ln(TIMP-1) with increased LVEDD or increased LVWT (separate models for each). Subjects with both increased LVEDD and increased LVWT were included in the increased LVEDD and increased LVWT groups in the separate models. Two models were examined in a hierarchical fashion:
We used analysis of covariance to examine trends in covariate-adjusted means of echocardiographic variables across sex-specific quartiles of plasma TIMP-1 levels. In the multivariable-adjusted models, relations between plasma TIMP-1 and left atrial diameter were also adjusted for the presence of atrial fibrillation and valve disease. We initially tested for statistical interactions between plasma TIMP-1 levels and sex, but because no interaction with sex was noted, sex-pooled analyses are presented.
Regression models using log-transformed echocardiographic (dependent) variables better met the assumptions of homoscedasticity, independence, and normality of residuals with the exception of fractional shortening, for which the untransformed residuals were preferable. TIMP-1 and continuous covariates were log transformed to reduce skewness and kurtosis that would otherwise influence the slope of the relationship with the dependent variables. Although log-transformed echocardiographic variables (with the exception of fractional shortening) were used as dependent variables in regression models, least squares means were computed in original units by back-transforming log-adjusted means into the original units.
Because the analyses of echocardiographic measurements as continuous variables pooled data for individuals in the three LV groups, we performed several additional analyses to: (a) test the hypotheses that the slope of the ln(TIMP-1) and LV relations were similar in the referent group versus the group with increased LV measurements; and (b) examine effect modification by group status by incorporating interaction terms [LV groupxln(TIMP-1)] in the multivariable models.
A two-sided p value <0.05 was considered statistically significant for all analyses.
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Results |
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Echocardiographic correlates of plasma total TIMP-1 in entire sample
TIMP-1 levels were significantly higher in the increased LVEDD and increased LVWT groups compared to the referent group adjusting for age, sex and height, but not in multivariable-adjusted models. The odds of having increased LVEDD or increased LVWT increased significantly by 2028% per SD increase in ln(TIMP-1) or a quartile increase in TIMP-1 in age-, sex- and height-adjusted models, but not in multivariable-adjusted ones (Table 3
). The relations of TIMP-1 to LV measures did not change when analyses were repeated for subgroups of individuals receiving and not using
anti-hypertensive treatment.
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Discussion |
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Plasma total TIMP-1 and risk factors for atherosclerotic disease
The direct relations of plasma TIMP-1 levels to all major CVD risk factors is a striking finding of the present study. The observation that plasma TIMP-1 was increased in diabetics and participants using anti-hypertensive treatment is in agreement with previous studies using smaller samples.46,12 To our knowledge, the positive associations of plasma TIMP-1 with obesity and dyslipidaemia have not been reported previously. The increase in plasma TIMP-1 levels with age, male gender and smoking is also noteworthy. The associations of clinical risk factors to TIMP-1 levels were unaltered upon additional adjustment for LV measures.
Because of the cross-sectional design of the study, we cannot definitively elucidate the temporal sequence behind the observation that major CVD risk factors were related to plasma TIMP-1 levels. One possibility is that CVD risk factors may operate partly by modulating cardiac and vascular structure via TIMP-1 levels. Recent experimental data suggests that TIMP-1 in certain settings may be pro-atherosclerotic,26 which would support such an interpretation. An alternative explanation is that an increased plasma TIMP-1 level may be an epiphenomenon or an adaptive response to an increase in MMP activity, the latter being promoted by CVD risk factors. Taken together, the plasma TIMP-1 level may be a marker of cardiovascular extracellular matrix remodelling, a process that may increase with rising cardiovascular risk as measured by the Framingham Risk Score. Future longitudinal studies should clarify whether measurement of extracellular matrix markers such as plasma TIMP-1 has a place in the global CVD risk assessment.
Plasma total TIMP-1 and cardiac structure and function
We observed a direct relationship between plasma TIMP-1 levels and LVM, LVWT, relative wall thickness and odds for increased LVWT, consistent with most previous experimental9 and small human4,5 studies. Additionally, plasma TIMP-1 levels were related to LVESD and risk of increased LVEDD. These associations were attenuated in multivariate models. The relations between TIMP-1 and left atrial diameter may in part be explained by the close relation between left atrial size and LVM.27
Several traditional CVD risk factors are known to predict LVH,28,29 and we noted their associations with plasma TIMP-1 levels. Therefore, we performed additional analyses to examine whether plasma TIMP-1 was related to LV measures after adjusting for these known risk factors as potential confounding factors. The finding that relations between TIMP-1 and LVH indices were attenuated by adjustment for traditional CVD risk factors is consistent with the hypothesis that the risk factors may influence LV structure through effects on TIMP-1 levels. Alternatively, it is possible that the CVD risk factors influence LV remodelling directly, and TIMP-1 levels are a marker of the remodelling process. A third possibility is that TIMP-1 is a marker of vascular remodelling, which may result in increased LV afterload and consequent LVH.
The association of plasma TIMP-1 with increased LVWT merits comment. The major biological role of TIMP-1 appears to be to balance the activity of MMPs. In this context, one might predict that increased TIMP-1 levels would inhibit myocardial MMP activity, leading to increased myocardial collagen deposition and increased LVWT, as we have seen here. TIMP-1 also has growth-promoting effects independent of its MMP inhibitory effects,30 and this may also play a role in our observations.
The relations between plasma TIMP-1 and systolic function expressed as endocardial and mid-wall LVFS and their close correlate LVESD were independent of traditional risk factors, which could support an effect of TIMP-1 above and beyond the effects of these risk factors on LV systolic function. Demonstration of an association between TIMP-1 and LVFS within the normal range is an intriguing finding from a mechanistic point of view. One possible explanation for this observation is that the increased total TIMP-1 measured is indicative of increased MMP activity and increased matrix degradation, which may result in a disrupted extracellular matrix architecture, and associated LV dysfunction.2,3,8 Another is that higher TIMP-1 levels may indicate an increased collagen content of the cardiac extracellular matrix, and such an increase may adversely impact LV systolic function.
Strengths and limitations
Some differences in findings between the present and previous studies may be attributed to differences in study subjects (our sample was relatively healthy) and to the sample sizes studied (we used a large community-based sample with greater statistical power to detect modest associations with risk factors). We also assessed the associations of TIMP-1 and LV measurements in relation to known CVD risk factors in hierarchical statistical models, which has not been done previously.46
Several limitations of our study merit description. We did not assess LV diastolic function, arterial compliance or arrhythmias. A comprehensive assessment of these characteristics would have provided valuable additional insights. We also had few non-Caucasian participants in this study and hence limited power to examine the impact of ethnicity on TIMP-1 levels or LV effects. We examined plasma total TIMP-1, which gives limited information on myocardial TIMP-1 levels and also combines information on both free TIMP-1, and TIMP-1 complexed with various MMPs. The interpretation might be aided by measures of MMP activity, MMP/TIMP-1 complexes and protocollagen peptides. Plasma TIMP-1 arises from several organ systems, including but not limited to cardiovascular tissues. Some caution in the interpretation of clinical correlates of TIMP-1 may be warranted due to the sampling strategy based on the distribution of LV measurements. Lastly, it is important to note that we examined the relations of several echocardiographic measurements with plasma TIMP-1. While we did not adjust for multiple comparisons, it is important to note that all analyses were specified a priori. We believe it is unlikely that the association of plasma TIMP-1 with fractional shortening is solely due to chance as a result of multiple testing given the degree of statistical significance observed in our study (, Table 4), the consistency of results in age- and sex-adjusted and multivariable models, and biological plausibility.
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Conclusions |
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Appendix A |
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Acknowledgments |
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Footnotes |
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References |
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