Atherosclerosis and Lipoproteins |
From the Department of Physiology (M.W.M., A.D.); the Division of Pharmaceutical Science, College of Pharmacy (L.A.C.); and the Division of Cardiovascular Medicine, Gill Heart Institute (A.D.), University of Kentucky, Lexington.
Correspondence to Alan Daugherty, PhD, Division of Cardiovascular Medicine, Sanders Brown, Room 424, Gill Heart Institute, University of Kentucky, Lexington, KY 40536-0230. E-mail adaugh{at}uky.edu
| Abstract |
|---|
|
|
|---|
Methods and Results Mice were placed on a high-fat diet for 1 week before infusion with either saline or AngII (1000 ng · kg-1 · min-1) via osmotic pumps for 28 days. Doxycycline (30 mg · kg-1 · d-1) was administered in the drinking water to both saline- and AngII-infused mice. Administration of doxycycline did not significantly influence systolic blood pressure, serum cholesterol concentrations, or lipoprotein-cholesterol distribution. Doxycycline had no effect on the extent of atherosclerosis in saline- or AngII-infused mice. In contrast, doxycycline markedly reduced the incidence of AAA formation (86% vs 35%, AngII vs AngII+doxycycline, respectively; P<0.05), in addition to reducing aneurysm severity.
Conclusions These data do not imply a role for MMPs in AngII-induced atherosclerosis but provide evidence consistent with a role in AngII-induced AAA formation.
Key Words: angiotensin aneurysms atherosclerosis doxycycline matrix metalloproteinases
| Introduction |
|---|
|
|
|---|
The vascular pathologies of atherosclerosis and AAA are both associated with a dynamic state of deposition and degradation of the extracellular matrix. Degradation of lesional extracellular matrix is likely to occur through 1 or more of the class of matrix metalloproteinases (MMPs). A role for MMPs in the atherogenic process may be inferred through the detection of MMP-1, -2, -3, -9, -11, and -14 in lesions.714 Based on their role in the degradation of the extracellular matrix, enhanced activity of MMPs has been considered to increase the development of atherosclerosis.15 However, recent studies with MMP-3 or tissue inhibitor of matrix metalloproteinases (TIMP)-1deficient mice and MMP-1overexpressing mice have provided evidence to the contrary.1618
The MMP class of enzymes has also been implicated in the development of AAAs. This disease is defined by the medial degeneration that results from the destruction of both elastin and collagen. Several MMPs have been detected in AAAs, including 4 that degrade elastic fibers (MMP-2, -7, -9, and -12), several that degrade interstitial collagens (MMP -1, -2, -8, -13, and -14), and others that degrade denatured collagen (MMP-2 and -9).1925 Evidence supporting a role for MMPs in animal models of AAAs includes data demonstrating resistance to AAAs in mice lacking MMP-2 or -9.26 27 In addition, a deficiency of urokinase plasminogen activator in apoE-/- mice reduced micro-AAAs, which was attributed to decreased activation of MMP-12.28
The limited literature suggests that AngII exhibits cell-specific regulation of MMPs. In cardiac myocytes29 and pericytes,30 AngII promoted the synthesis of MMP-2 but decreased its presence in vascular smooth muscle cells.31 AngII directly promoted the synthesis of MMP-9 in cardiac myocytes.32 Administration of the angiotensin type 1 (AT1) receptor antagonist losartan to cardiomyopathic hamsters reduced cardiac mRNA expression of several MMPs.33 In addition, administration of losartan to cholesterol-fed rabbits reduced the mRNA expression of MMP-1 in the aorta and was suggested to contribute to the antiatherogenic effects of AT1 receptor blockade.34 These data collectively suggest that AngII may regulate MMPs; however, the role of MMPs in AngII-induced vascular disease is not known.
One approach to the elucidation of the role of MMPs in a disease process is the use of pharmacological inhibitors. A widely used compound is doxycycline, which, though commonly known for its antibiotic properties, exerts therapeutic effects through inhibition of MMPs.35 An advantage of using doxycycline to study the role of MMPs in a disease process is the broad-spectrum inhibition of MMPs. We hypothesized that AngII induces the vascular pathologies of atherosclerosis and AAAs in hyperlipidemic mice by augmenting MMPs. To test this hypothesis, we determined the effect of doxycycline on the development of AngII-induced atherosclerosis and AAA formation in LDL receptor-/- mice. Results from this study provide evidence that is consistent with a role for MMPs in the development of AngII-induced AAAs and provide further support for defining the therapeutic benefit of this compound in humans afflicted with the disease.3638
| Methods |
|---|
|
|
|---|
Doxycycline Administration
Doxycycline (Sigma) was administered daily in the drinking water at an approximate dose of 30 mg · kg-1 · d-1 (based on the average daily water consumption). This dose has been shown previously to block MMP activity in vivo.26 Doxycycline treatment was begun 1 week before pump implantation and continued throughout the study. The drug was protected from light, and a fresh drug solution was provided every other day.
Blood Pressure Measurements
Systolic blood pressures were obtained from conscious mice by using a computerized tail-cuff method (BP-2000 Visitech Systems). Mice were acclimated to the instrument for at least 1 week before implantation of the osmotic pumps. Measurements were recorded at the same time of day throughout the study. Individual mice received 10 initial pressure readings to acclimate them to the procedure, and then 10 additional cycles were measured to obtain the daily mean systolic pressure. The criterion for acceptance of measurements was at least 5 recorded pressures per run that had a standard deviation of <30 mm Hg per animal.
Serum Lipids and Lipoprotein Concentrations
Serum total cholesterol concentrations were determined with enzymatic assay kits (Wako Chemical Co). Lipoprotein cholesterol distributions were evaluated in individual serum samples (50 µL) from 8 mice in each group after fractionation by size-exclusion chromatography on a single Superose 6 column.2 Fractions were collected, and cholesterol concentrations were determined with enzymatic kits.
Quantification of Atherosclerosis and Characterization of Aneurysms
Atherosclerotic lesions were quantified in the arch and thoracic aorta as described previously.2,39 The abdominal aorta was excluded from analysis owing to the presence of large AAAs. Data are expressed as percent of the intimal surface covered by grossly discernible atherosclerotic lesions. Determination of the presence of an AAA was made by 2 independent observers. The severity of AAA was based on a previously described classification in which AAAs were assigned to a group dependent on the gross appearance of the tissue.3,4
Statistics
For each parameter, the mean and SEM were calculated. Blood pressure data were analyzed by 3-way repeated-measures ANOVA followed by Tukeys post hoc test (SAS statistical by a 2-way ANOVA followed by Tukeys post hoc test for all pairwise comparisons. Incidence of aneurysm formation was examined by using Fishers exact test. Values of P<0.05 were considered statistically significant.
| Results |
|---|
|
|
|---|
|
Doxycycline Did Not Alter the Development of Hypertension in Response to AngII
LDL receptor-/- mice infused with AngII developed moderate increases (
25 mm Hg) in systolic blood pressure during the 4 weeks of the study. We did not demonstrate an increase in arterial blood pressure at this dose during AngII infusion in previous studies; however, those were performed in anesthetized mice.2 This elevation in blood pressure was apparent within 3 days after pump implantation and was maintained throughout the course of AngII infusion. Doxycycline did not affect systolic blood pressure in either AngII- or saline-infused mice (Figure 1).
|
Doxycycline Did Not Alter the Distribution of Lipoprotein Cholesterol
To determine whether doxycycline administration had any effect on lipid metabolism, plasma cholesterol concentrations were measured, and the distribution of lipoprotein cholesterol was determined by size-exclusion chromatography. Serum total cholesterol concentrations were unchanged by AngII or doxycycline administration (Table 1). Also, there were no effects of AngII or doxycycline administration on the distribution of cholesterol among lipoprotein fractions (Figure 2).
|
Doxycycline Did Not Alter the Extent of AngII-Induced Atherosclerosis
The extent of atherosclerosis was measured by an en face method and expressed as percent lesion covering the intimal area. Doxycycline had no effect on lesion area in saline-infused mice. As reported previously,2 AngII infusions produced significant increases in lesion area in the thoracic aorta (Figure 3). Increases in lesion area in the aorta in response to AngII were not influenced by doxycycline (19±1% vs 17±1%, AngII alone vs AngII+doxycycline, respectively).
|
Doxycyline Markedly Attenuated AngII-Induced AAAs
The incidence of AAAs in the suprarenal aorta of AngII-infused mice was 85%. Orally administered doxycycline decreased the incidence (85% vs 35%, AngII alone vs AngII+doxycycline, respectively; P<0.003; Table 2) and the severity of aneurysms formed by AngII (Figure 4). Both thrombus formation and the extensive dilation of the aorta seen in AngII-infused mice (Figure 4A) were inhibited in mice administered doxycycline. In the few AAAs that formed in AngII-infused mice given doxycycline, there was slight hypertrophy of the surrounding adventitial tissue, yet markedly less than that seen in AngII-infused mice. By using a previously described classification scheme to provide an index of pathological severity, we ascertained that AAAs formed in mice treated with doxycycline were much less advanced than those observed in mice infused with AngII alone (Figure 4B).3,4
|
|
| Discussion |
|---|
|
|
|---|
Inhibition of MMPs by Doxycycline
Doxycycline has several pharmacological properties, but its therapeutic utility in reducing AAAs in experimental mice and humans has been ascribed to inhibition of MMPs.26 In the present study, the dose of doxycycline administered to inhibit MMPs was based on previous reports.26,40,41 Doxycycline inhibits a broad range of MMPs by proposed mechanisms that include transcriptional inhibition and a direct effect by coordination with the catalytic site.35 In preliminary studies, we found that the dose of doxycycline used in the present study inhibited surgically induced elevations in serum MMP-9 (authors unpublished observations). However, serum concentrations may not be indicative of the induction of augmented local proteolytic activity in discrete regions of arteries. We propose inhibition of local MMPs as the mechanism of doxycyclines attenuation of AAA formation. This assertion is based on the drugs known inhibition of this class of enzymes and the demonstrated role of specific MMPs in the disease process. Further studies with structural analogues of the drug may assist in establishing this mechanism.
Lack of Effect of Doxycycline on AngII-Induced Atherosclerotic Lesions
Several MMPs are present in atherosclerotic lesions, as indicated earlier. However, the mere presence of MMPs in tissue is not an indication of the extent of activity, because this is dependent on complex regulatory steps of enzyme activation and attenuation by endogenous inhibitors.15 In situ gel zymography has demonstrated that MMP activity in atherosclerotic lesions is enhanced, particularly at shoulder regions.7,42 Although MMP activity is commonly considered detrimental to the development of atherosclerotic lesions, this notion has been challenged by recent studies in mice in which MMP-1 was overexpressed in a macrophage-specific manner or in which MMP-3 was deleted.17,18 Overexpression of MMP-1, which is not normally expressed in mice, unexpectedly decreased the extent of atherosclerosis,18 whereas deletion of MMP-3 had no effect on lesion size.17 The effect of TIMP-1mediated inhibition of MMPs is unclear, because both overexpression and deletion studies have demonstrated reduced atherosclerosis in apoE-/- mice.16,43 Further studies are needed to determine whether prolonged administration of doxycycline influences lesion formation in LDL receptor-/- mice.
This is the first study in which the effects of doxycycline on AngII-induced atherosclerosis have been defined. Doxycycline was used as a broad inhibitor of MMPs to effectively inhibit multiple MMPs. At the dosage used, there was no effect of doxycycline on AngII-induced atherosclerosis. These results indicate that MMPs do not contribute to the rapidly formed atherosclerotic lesions that develop in response to 1 month of AngII infusion. However, this does not discount a role for MMPs during more protracted intervals of AngII administration or a hyperlipidemic diet. Also, it does not negate a role for MMPs in advanced stages of the disease, in which their activity may be a determinant of plaque rupture.44
Doxycycline Inhibits AngII-Induced AAA Formation
MMPs are hypothesized to be important mediators in the development of AAAs, although other elastolytic enzymes, such as cathepsins S and K, have also been speculated to be involved in the disease process.45 Consistent with the hypothesis that MMPs are involved in AAA formation, administration of doxycycline significantly reduced both the incidence and severity of AngII-induced AAA formation. AngII infusion into hyperlipidemic mice leads to medial degeneration that presumably underlies the development of AAAs.1 Medial degeneration may be a primary event that leads to inflammation due to the chemotactic properties of elastin degradation products. Alternatively, medial degeneration may be caused by the inflammatory process. Although the sequence of biochemical and cellular events in evolving AAAs has not been defined, MMP inhibition could potentially contribute to attenuation of AAA formation either by inhibiting MMPs within the arterial wall or in the infiltrating leukocytes.
The continued presence of some aneurysmal formation may have been due to incomplete MMP inhibition at the doxycycline dose used. However, the AAAs in doxycycline-administered mice were minor expansions of the suprarenal aorta that lacked a significant thrombotic component. The ability of doxycycline to inhibit formation of AAAs in this and other animal models of the disease indicates the importance of this group of enzymes to the disease process.40,41,46
Divergence of Effects on Atherosclerosis and AAAs
It has been commonly considered that AAAs are a consequence of the atherosclerotic process, although this viewpoint has been challenged.47,48 The present study demonstrated a divergent effect of doxycycline on the extent of atherosclerosis and the severity of AAAs. A differential effect of manipulation of MMPs on atherosclerosis versus aortic aneurysm has been noted in mouse studies with other MMP inhibitors49 and in mice with deficiencies of either MMP-3 or TIMP-1.16,17 The AAAs that form in response to AngII are highly localized to the suprarenal artery, a region of the aorta that does not typically exhibit atherosclerosis in mice of this young age.1,2,50 Moreover, in earlier studies, we have not observed atherosclerotic lesions in the region of AAA formation.2 However, previous results from our laboratory demonstrate that a hyperlipidemic environment markedly augments the formation of AAAs by AngII in female mice.2 Therefore, although hyperlipidemia promotes AngII-induced AAAs, the divergent effects of doxycycline on these vascular pathologies provide further evidence that atherosclerosis may not be responsible for the formation of AngII-induced AAAs.
Conclusions
In summary, results from this study demonstrate that doxycycline has negligible effects on the development of AngII-induced atherosclerosis in LDL receptor-/- mice but significantly attenuates the development of AAAs. The proposed mechanism for the effect of doxycycline on AngII-induced AAAs is inhibition of MMPs elaborated from cells at the vascular lesion and a reduction in degradation of the extracellular matrix. Studies to further define the role of MMPs in AngII-induced AAA formation will require selective MMP inhibitors and mice with deficiencies of a specific MMP.
| Acknowledgments |
|---|
Received December 11, 2002; accepted January 15, 2003.
| References |
|---|
|
|
|---|
B activation in cultured neonatal rat cardiomyocytes through protein kinase C signaling pathway. J Mol Cell Cardiol. 2000; 32: 17671778.[CrossRef][Medline]
[Order article via Infotrieve]
B by losartan in hypercholesterolemic rabbits. J Cardiovasc Pharmacol. 2002; 39: 332339.[CrossRef][Medline]
[Order article via Infotrieve]
This article has been cited by other articles:
![]() |
P. A. Murphy, M. T. Y. Lam, X. Wu, T. N. Kim, S. M. Vartanian, A. W. Bollen, T. R. Carlson, and R. A. Wang Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice PNAS, August 5, 2008; 105(31): 10901 - 10906. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. W.Y. Chung, H. H. C. Yang, M. W. Radomski, and C. van Breemen Long-Term Doxycycline Is More Effective Than Atenolol to Prevent Thoracic Aortic Aneurysm in Marfan Syndrome Through the Inhibition of Matrix Metalloproteinase-2 and -9 Circ. Res., April 25, 2008; 102(8): e73 - e85. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Wang, E. Lee, W. Song, E. Ricciotti, D. J. Rader, J. A. Lawson, E. Pure, and G. A. FitzGerald Microsomal Prostaglandin E Synthase-1 Deletion Suppresses Oxidative Stress and Angiotensin II-Induced Abdominal Aortic Aneurysm Formation Circulation, March 11, 2008; 117(10): 1302 - 1309. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. B. Morris, A. R. Olzinski, R. E. Bernard, K. Aravindhan, R. C. Mirabile, R. Boyce, R. N. Willette, and B. M. Jucker p38 MAPK Inhibition Reduces Aortic Ultrasmall Superparamagnetic Iron Oxide Uptake in a Mouse Model of Atherosclerosis: MRI Assessment Arterioscler. Thromb. Vasc. Biol., February 1, 2008; 28(2): 265 - 271. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. Heeneman, J. C. Sluimer, and M. J.A.P. Daemen Angiotensin-Converting Enzyme and Vascular Remodeling Circ. Res., August 31, 2007; 101(5): 441 - 454. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. L. McCormick, D. Gavrila, and N. L. Weintraub Role of Oxidative Stress in the Pathogenesis of Abdominal Aortic Aneurysms Arterioscler. Thromb. Vasc. Biol., March 1, 2007; 27(3): 461 - 469. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Golledge, J. Muller, A. Daugherty, and P. Norman Abdominal Aortic Aneurysm: Pathogenesis and Implications for Management Arterioscler. Thromb. Vasc. Biol., December 1, 2006; 26(12): 2605 - 2613. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. DAUGHERTY, D. L RATERI, and L. A CASSIS Role of the Renin-Angiotensin System in the Development of Abdominal Aortic Aneurysms in Animals and Humans Ann. N.Y. Acad. Sci., November 1, 2006; 1085(1): 82 - 91. [Abstract] [Full Text] [PDF] |
||||
![]() |
G. M. Buga, J. S. Frank, G. A. Mottino, M. Hendizadeh, A. Hakhamian, J. H. Tillisch, S. T. Reddy, M. Navab, G. M. Anantharamaiah, L. J. Ignarro, et al. D-4F decreases brain arteriole inflammation and improves cognitive performance in LDL receptor-null mice on a Western diet J. Lipid Res., October 1, 2006; 47(10): 2148 - 2160. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. L. Johnson, R. Fritsche-Danielson, M. Behrendt, A. Westin-Eriksson, H. Wennbo, M. Herslof, M. Elebring, S. J. George, W. L. McPheat, and C. L. Jackson Effect of broad-spectrum matrix metalloproteinase inhibition on atherosclerotic plaque stability Cardiovasc Res, August 1, 2006; 71(3): 586 - 595. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Thomas, D. Gavrila, M. L. McCormick, F. J. Miller Jr, A. Daugherty, L. A. Cassis, K. C. Dellsperger, and N. L. Weintraub Deletion of p47phox Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E-Deficient Mice Circulation, August 1, 2006; 114(5): 404 - 413. [Abstract] [Full Text] [PDF] |
||||
![]() |
G. Stoll and M. Bendszus Inflammation and Atherosclerosis: Novel Insights Into Plaque Formation and Destabilization Stroke, July 1, 2006; 37(7): 1923 - 1932. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. Franco, B. Ho, D. Mulholland, G. Hou, M. Islam, K. Donaldson, and M. P. Bendeck Doxycycline Alters Vascular Smooth Muscle Cell Adhesion, Migration, and Reorganization of Fibrillar Collagen Matrices Am. J. Pathol., May 1, 2006; 168(5): 1697 - 1709. [Abstract] [Full Text] [PDF] |
||||
![]() |
V. L. King, D. B. Trivedi, J. M. Gitlin, and C. D. Loftin Selective Cyclooxygenase-2 Inhibition With Celecoxib Decreases Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Mice Arterioscler. Thromb. Vasc. Biol., May 1, 2006; 26(5): 1137 - 1143. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. M. Dollery and P. Libby Atherosclerosis and proteinase activation Cardiovasc Res, February 15, 2006; 69(3): 625 - 635. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. L. Johnson, S. J. George, A. C. Newby, and C. L. Jackson Divergent effects of matrix metalloproteinases 3, 7, 9, and 12 on atherosclerotic plaque stability in mouse brachiocephalic arteries PNAS, October 25, 2005; 102(43): 15575 - 15580. [Abstract] [Full Text] [PDF] |
||||
![]() |
Y.-X. Wang, B. Martin-McNulty, V. da Cunha, J. Vincelette, X. Lu, Q. Feng, M. Halks-Miller, M. Mahmoudi, M. Schroeder, B. Subramanyam, et al. Fasudil, a Rho-Kinase Inhibitor, Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm in Apolipoprotein E-Deficient Mice by Inhibiting Apoptosis and Proteolysis Circulation, May 3, 2005; 111(17): 2219 - 2226. [Abstract] [Full Text] [PDF] |
||||
![]() |
R. M. Touyz, C. Mercure, Y. He, D. Javeshghani, G. Yao, G. E. Callera, A. Yogi, N. Lochard, and T. L. Reudelhuber Angiotensin II-Dependent Chronic Hypertension and Cardiac Hypertrophy Are Unaffected by gp91phox-Containing NADPH Oxidase Hypertension, April 1, 2005; 45(4): 530 - 537. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. C. Newby Dual Role of Matrix Metalloproteinases (Matrixins) in Intimal Thickening and Atherosclerotic Plaque Rupture Physiol Rev, January 1, 2005; 85(1): 1 - 31. [Abstract] [Full Text] [PDF] |