Deletion of CCR2 but not CCR5 or CXCR3 inhibits aortic aneurysm formation

BACKGROUND: Microscopic analysis of abdominal aortic aneurysms (AAAs) demonstrates an abundance of infiltrating leukocytes. The chemokine receptors CCR2, CCR5, and CXCR3 are associated with pathways implicated previously in aneurysm pathogenesis. We hypothesized that genetic deletions of CCR2, CCR5, and CXCR3 would limit leukocyte infiltration and aneurysm formation in a mouse model of AAA. METHODS: CCR2(-/-), CCR5(-/-), CXCR3(-/-), and control mice of the same genetic background were subject to periaortic application of calcium chloride. Aortic diameters were measured before aneurysm induction and at harvest 6 weeks later. Diameters were compared using the Mann-Whitney test. Aortas were stained with H&E and trichrome for histologic analysis. Aortic MMP-2 and MMP-9 activities were measured using zymography. RESULTS: Aneurysm formation was attenuated in CCR2(-/-) mice with the final mean aortic diameter less than that of the control mice (P < .01). Histology revealed preservation of the lamellar architecture and decreased inflammatory cells. Aortic MMP-2 and MMP-9 levels were decreased in CCR2(-/-) mice. CCR5(-/-) and CXCR3(-/-) mice demonstrated no protection from aneurysm formation, which was corroborated by the tissue histology showing similar inflammatory cell infiltration and elastin degradation. CONCLUSIONS: The CCR2 receptor is involved directly in AAA formation, whereas the CCR5 and CXCR3 receptors are not.     

Cigarette smoking increases aortic dilatation without affecting matrix metalloproteinase-9 and -12 expression in a modified mouse model of aneurysm formation

OBJECTIVE: The development of abdominal aortic aneurysms (AAA) is presumed to result from multiple genetic and environmental factors, with exposure to tobacco smoke the single largest known factor predisposing to aneurysm growth. We have attempted to adapt the elastase-perfused animal model to determine whether tobacco exposure can lower the threshold of aortic injury necessary for AAA development. METHODS: Adult C57BL/6 mice underwent transient perfusion of the infrarenal aorta with an active solution of elastase: high-dose (HDE, 0.19 U/mL, n=9), standard-dose (SDE, 0.16 U/mL, n=21) or low-dose (LDE, 0.07 U/mL, n=24). Control animals (n=24) were treated with heat inactivated elastase (HIE). Twenty LDE perfused mice were exposed to cigarette smoke (LDE-S) beginning 2 weeks before perfusion and continuing until aortic harvest. Aortic diameter (AD) was measured preperfusion, postperfusion, and at harvest on day 14. AAA was defined as %DeltaAD>or=100% between preperfusion and harvest. Aortas from each group (except HDE) were analyzed for matrix metalloproteinase-9 (MMP-9) and MMP-12 expression by real-time polymerase chain reaction normalized to glyceraldehyde-3-phosphate dehydrogenase. RESULTS: All SDE mice developed large AAA by %DeltaAD (189.3%+/-16.9%, mean+/-standard error of the mean), but control mice had only a small dilatation (69.7%+/-3.7%, P<.01). Higher doses of elastase did not produce larger aneurysms in HDE mice. In contrast, only 63% of LDE mice showed aneurysmal dilatation, and these were significantly smaller (104.3%+/-4.2%, P<.01). When exposed to cigarette smoke, LDE animals developed significantly larger aneurysms (%DeltaAD, 134.5%+/-7.9%, P=.0021). There was no difference in normalized aortic MMP-9 and MMP-12 expression between elastase doses or between smoke-exposed and unexposed animals. Histologic analysis revealed that smoking increased the extent of aortic elastin degradation when compared with LDE-S animals. CONCLUSION: Aneurysm development in the elastase model is dependent on the quantity of active elastase infused. Exposure of animals to tobacco smoke after a relatively minor aortic elastase injury produces increases in elastin degradation and aneurysm size without affecting MMP-9 or MMP-12 expression. To our knowledge, this is the first demonstration in an animal model that smoking can act as a synergistic factor in AAA development. Further understanding of the relationship between smoking and AAA in this model may help unveil the pathophysiologic pathways involved between cigarette smoke and AAAs.     

Suppression of Experimental Aortic Aneurysms: Comparison of Inducible Nitric Oxide Synthase and Cyclooxygenase Inhibitors

The rat model of abdominal aortic aneurysm (AAA) is associated with inflammation, destruction of extracellular matrix, and production of both inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-9 (MMP-9). Indomethacin, a nonselective cyclooxygenase inhibitor, may prevent AAA formation by inhibiting cyclooxygenase-2 (COX-2) activity. We hypothesized that indomethacin, rofecoxib (selective COX-2 inhibitor), and 1400W (selective iNOS activity inhibitor) would decrease aneurysm formation in the rat model. Forty-six male Wistar rats underwent intraaortic elastase infusion in two parallel studies based on medication delivery route. Sixteen rats were randomized to rofecoxib or water by gastric lavage. Thirty rats were randomized to subcutaneous saline, indomethacin, or 1400W. Heart rate, blood pressure and aortic diameters were measured. Western Blot and mRNA analysis for MMP-9 and iNOS was performed on postoperative day 7 aortic segments. Elastin degradation and inflammation were evaluated by immunohistochemistry. Elastase infusion produced AAA in all rats. 1400W significantly limited aneurysm expansion (p=0.01) whereas treatment with indomethacin and rofecoxib did not. Only 1400W significantly increased blood pressure (p < 0.001). Indomethacin alone statistically decreased MMP-9 (p < 0.011). 1400W resulted in greater conservation of aortic elastin than indomethacin (p=0.025). All groups demonstrated statistically similar expression of iNOS. In conclusion, selective iNOS activity inhibitor, 1400W, significantly decreased aneurysm size and preserved aortic elastin without altering MMP-9 levels. Indomethacin significantly decreased MMP-9 expression without decreasing aneurysm size. Rofecoxib did not significantly decrease MMP-9 expression or aneurysm size. Inhibition of iNOS limits aneurysmal expansion by mechanisms other than MMP-9 inhibition. MMP-9 inhibition by indomethacin is not sufficient to limit aneurysm expansion in our model.Presented at the 2002 Lifeline Foundation Research Initiatives in Vascular Disease Conference,Bethesda, MD, April 18-19, 2002.     

MMP-12 has a role in abdominal aortic aneurysms in mice

BACKGROUND: Matrix metalloproteinase (MMP)-12 levels are increased in the abdominal aortic aneurysm (AAA), implicating this protease in AAA pathogenesis. The purpose of this study was to assess the role of MMP-12 in aneurysm formation. METHODS: A murine aneurysm model was generated by periaortic application of 0.25 mol/L calcium chloride (CaCl 2 ) for 15 minutes. Aortic diameters were measured and compared before and 10 weeks after aneurysm induction. Aortic diameter changes for wild type (WT) and MMP-12 knockout (MMP-12 -/- ) mice were determined. MMP-12 production in mouse aorta was analyzed by casein zymography. MMP-2 and MMP-9 expressions were examined by gelatin zymography. Immunohistochemical study was used to measure macrophage infiltration into the aorta. RESULTS: There is an increase of 63 +/- 5% (mean +/- SEM) in aortic diameters of WT mice after CaCl 2 inductions, while MMP-12 -/- mice increased only 26 +/- 14%. Connective tissue staining of aortic sections from WT mice showed disruption and fragmentation of medial elastic fibers, while MMP-12 -/- mice showed only focal elastic lamellae breakdown. MMP-12 levels in WT mice were significantly increased after CaCl 2 treatment, whereas no MMP-12 was detected in MMP-12 -/- mice. There was no difference in the MMP-2 and MMP-9 productions between WT and MMP-12 -/- mice. Immunohistochemical analysis demonstrated that infiltrating macrophages in the aorta of MMP-12 -/- mice were significantly less than WT controls. CONCLUSIONS: MMP-12 deficiency attenuates aneurysm growth, possibly by decreasing macrophage recruitment.     

Suppression of experimental abdominal aortic aneurysms by systemic treatment with a hydroxamate-based matrix metalloproteinase inhibitor (RS 132908)

BACKGROUND: Abdominal aortic aneurysms (AAAs) are associated with chronic inflammation, disruption of medial elastin, and increased local production of elastolytic matrix metalloproteinases (MMPs). The purpose of this study was to investigate how treatment with a hydroxamate-based MMP antagonist (RS 132908) might affect the development of experimental AAAs. METHODS: Male Wistar rats underwent intraluminal perfusion of the abdominal aorta with 50 units of porcine pancreatic elastase followed by treatment for 14 days with RS 132908 (100 mg/kg/day subcutaneously; n = 8) or with vehicle alone (n = 6). The external aortic diameter (AD) was measured in millimeters before elastase perfusion and at death, with AAA defined as an increase in AD (DeltaAD) of at least 100%. Aortic wall elastin and collagen concentrations were measured with assays for desmosine and hydroxyproline, and fixed aortic tissues were examined by light microscopy. RESULTS: AAAs developed in all vehicle-treated rats, with a mean AD (+/- SE) that increased from 1.60 +/- 0.03 mm before perfusion to 5.98 +/- 1.02 mm on day 14 (DeltaAD = 276.4 +/- 67.7%). AAAs developed in only five of eight animals (62.5%) after MMP inhibition, with a mean AD that increased from 1.56 +/- 0.05 mm to 3.59 +/- 0.34 mm (DeltaAD = 128.1 +/- 18.7%; P <.05, vs vehicle). The overall inhibition of aortic dilatation attributable to RS 132908 was 53.6 +/- 6.8%. Aortic wall desmosine fell by 85.4% in the vehicle-treated rats (1210.6 +/- 87.8 pmol/sample to 176.7 +/- 33.4 pmol/sample; P <.05) but only by 65.6% in the animals treated with RS 312908 (416.2 +/- 120.5 pmol/sample). In contrast, hydroxyproline was not significantly affected by either elastase perfusion or drug treatment. Microscopic examination revealed the preservation of pericellular elastin and a greater degree of fibrocollagenous wall thickening after MMP inhibition, with no detectable difference in the extent of inflammation. CONCLUSIONS: Systemic MMP inhibition suppresses aneurysmal dilatation in the elastase-induced rodent model of AAA. Consistent with its direct inhibitory effect on various MMPs, RS 132908 promotes the preservation of aortic elastin and appears to enhance a profibrotic response within the aortic wall. Hydroxamate-based MMP antagonists may therefore be useful in the development of pharmacologic approaches to the suppression of AAAs.     

四环素抑制实验性大鼠腹主动脉瘤形成的初步研究

目的；研究四环素对实验性大鼠腹主动脉瘤形成的作用。方法：建立大鼠腹主动脉瘤弹力蛋白酶灌注模型，随机分为实验组（10只）皮下注射四环素（25mg/d)，对照组（10只）皮下注射生理盐水，观察两组动脉瘤形成率和动脉瘤组织学改变，并应用免疫组织化学和分子原位杂交技术检测基质金属蛋白酶－2和9的表达。结果：灌注2周后实验组大鼠腹主动脉瘤形成率10％，对照组大鼠动脉瘤形成率为10％，两组差异有显著意义（P＜0．05），实验组大鼠动脉壁组织MMP－2，MMP－9蛋白表达为弱阳性或阴性，对照组动物瘤组织中两者表达明显增高，两位杂交检测结果表明两组mRNA表达强度无明显差异。结论：四环素能够降低基质金属蛋白酶－2和9的表达，从而减低动脉壁组织弹力纤维的降解，抑制实验性大鼠腹主动脉瘤的形成。     

Aminoterminal propeptide of type III procollagen and matrix metalloproteinases-2 and -9 failed to serve as serum markers for abdominal aortic aneurysm.

OBJECTIVES: Matrix-metalloproteinase (MMP)-2 and -9 and aminoterminal propeptide of type III collagen (NIIINP) have been reported to be elevated in patients with abdominal aortic aneurysm (AAA). The aim of our study was to test NIIINP, MMP-2 and -9 as potential serum markers for AAA in a large population group at risk for AAA. METHODS: Fifty-five to 70 year old men were screened for AAA by abdominal ultrasound. Simultaneously, blood samples were taken and the patients were interviewed for known risk factors for AAA. Patients with a dilatation of the infrarenal aorta of > or =25mm (Group 1, n=76) were compared to randomly assigned patients with normal aortic diameters (Group 2, n=83). A third group consisted of patients scheduled for operation of AAA (n=19). RESULTS: A total of 987 men were investigated with ultrasound. Seventy-six (7.7%) had an aortic dilatation > or =25mm. Aortic dilatation was correlated with age (P=0.0001). However, serum levels of NIIINP and MMP 2 were not different between the three groups of patients. For MMP-9 there was a weak inverse correlation with lower serum levels in patients with aortic dilatation (P=0.043). CONCLUSIONS: Both MMP-2 and -9 and NIIINP failed to show relevance as serum markers for aortic dilatation. Our results are, therefore, in contradiction to previous published results. AAAs cannot be diagnosed with a simple blood test.     

Matrix metalloproteinase-specific inhibition of Ca2+ entry mechanisms of vascular contraction

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a common disease with as yet unclear cause. Increased matrix metalloproteinase (MMP) levels in the plasma and aorta are a consistent finding in AAA. Although the role of MMPs in AAA has largely been attributed to degradation of the extracellular matrix proteins, the effects of MMPs on the mechanisms of aortic contraction are unclear. The purpose of this study was to test the hypothesis that MMPs promote aortic dilation by inhibiting the Ca2+ mobilization mechanisms of smooth muscle contraction. METHODS: Isometric contraction and 45Ca2+ influx were measured in aortic strips isolated from male Sprague-Dawley rats treated or not treated with MMP-2 and MMP-9. RESULTS: In normal Krebs solution (2.5 mmol/L Ca2+ ) phenylephrine (10-5 mol/L) caused contraction of the aortic strips, which was significantly inhibited (P < .05) by MMP-2 (maximum, 48.9% +/- 5.0%) and to a greater extent by MMP-9 (maximum, 69.8% +/- 6.2%). The MMP-induced inhibition of phenylephrine contraction depended on concentration and time. The inhibitory effects of MMPs on phenylephrine contraction were reversible. In Ca2+ -free (2 mmol/L ethylene glycol bis[beta-aminoethyl ether]-N,N,N',N'-tetraacetic acid) Krebs solution phenylephrine caused a small contraction that was not inhibited by MMP-2 or MMP-9, which suggests that MMPs do not inhibit Ca2+ release from the intracellular stores. Membrane depolarization with 96 mmol/L of potassium chloride, which stimulates Ca2+ entry from the extracellular space, caused a time-dependent and reversible contraction, which was inhibited by MMP-2 and MMP-9. Histologic studies of MMP-treated tissues stained with hematoxylin-eosin or Verhoeff stain for elastin confirmed the absence of degradation of the extracellular matrix. MMP-2 and MMP-9 also caused significant inhibition of 45Ca2+ influx induced by phenylephrine and potassium chloride. CONCLUSIONS: These data suggest that MMP-2 and MMP-9 promote aortic dilation by inhibiting the Ca2+ entry mechanism of vascular smooth muscle contraction. CLINICAL RELEVANCE: Abdominal aortic aneurysm (AAA) is a slow and progressive disease. The late stages of AAA are characterized by degenerative changes in the extracellular matrix and smooth muscle components of the aortic wall. The present study describes novel inhibitory effects of matrix metalloproteinase (MMP) on the Ca2+ entry mechanisms of aortic smooth muscle contraction, even in the absence of extracellular matrix degradation. The MMP-induced inhibition of aortic contraction may further explain the role of increased MMP activity particularly during the early development of AAA. Chronic exposure to MMPs may lead to protracted inhibition of aortic contraction, progressive aortic dilation, and aneurysm formation. MMP-9 is a more potent inhibitor of aortic contraction than MMP-2, consistent with a more dominant role in AAA. Restoration and preservation of smooth muscle contractile function by specific inhibitors of MMPs may represent a new strategy in preventing the progression of small AAA.     

Effects of tissue inhibitor of metalloproteinase 2 deficiency on aneurysm formation

OBJECTIVE: Matrix metalloproteinase (MMP)-2 has been shown to play a pivotal role in aortic aneurysm formation. Its activation requires formation of a trimolecular complex of MMP-2, tissue inhibitor of metalloproteinase-2 (TIMP-2), and membrane type 1 (MT1)-MMP, which is attached to the cell surface. At higher concentrations, TIMP-2 becomes an inhibitor of MMP-2. Thus, TIMP-2 could both augment and inhibit matrix degradation. This study was undertaken to define the net effect of TIMP-2 on matrix destruction and aneurysm formation. METHODS: The abdominal aortas of wild-type and TIMP-2-deficient (TIMP-2 -/-) mice were exposed to 0.25 mol/L CaCl2 or 0.9% NaCl for 15 minutes after laparotomy. Aortic diameters were measured before treatment and 6 weeks after aneurysm induction. In addition, aortic tissues were studied for MMP-2 activation by zymography, and matrix structure was studied by connective tissue staining. RESULTS: The aortic diameter increased in both wild-type and TIMP-2-/- mice. The increase in the TIMP-2 -/- mice was significantly smaller after CaCl2 treatment (51% +/- 3%) compared with the diameter of wild-type mice (67% +/- 4%). Connective staining of aortic sections from the CaCl2-treated mice revealed disruption and fragmentation of the medial elastic lamellae in both wild-type and TIMP-2 -/- mice. Zymographic analysis showed that active MMP-2 levels were decreased in TIMP-2 -/- aortas compared with wild-type mice. CONCLUSIONS: Targeted deletion of TIMP-2 results in attenuation of aneurysm development. Despite its name as an inhibitor of MMPs, TIMP-2 promotes aortic enlargement in vivo, presumably through its role as a cofactor in the activation of MMP-2. CLINICAL RELEVANCE: Abdominal aortic aneurysmal (AAA) disease is a potentially fatal disorder that screening studies have detected in 2% to 9% of the general population. Medical therapy designed to inhibit the progression of small aneurysms includes control of hypertension and smoking cessation; neither of these measures is of proven benefit. Effective and directed medical treatments for small AAAs await elucidation of key etiologic factors. Understanding precisely which molecules mediate AAA development, and blocking the activity of these molecules, could lead to important new therapies. Through our research, we have found that tissue inhibitor of metalloproteinase (TIMP)-2 has a role in this process in an experimental model of aortic aneurysms. We believe that TIMP-2 promotes aortic enlargement in vivo by activating matrix metalloproteinase 2.     

Matrix metalloproteinase expressions in arteriosclerotic aneurysmal disease

Medial degeneration of extracellular matrix (ECM) proteins in the wall of abdominal aortas results in smooth muscle cell destruction, a loss of architectural integrity, and abdominal aortic aneurysm (AAA) formation. It has been theorized that an imbalance between proteinases and their naturally occurring inhibitors is the cause of these observed histologic abnormalities. Therefore, the purpose of this investigation was to determine if differences in the matrix metalloproteinase (MMP) -2 and -9, tissue inhibitor of metalloproteinase-1 (TIMP-1), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA) protein and activity levels existed between infrarenal AAA and normal abdominal aortic tissue specimens. Between November 1995 and January 1997, 10 patients undergoing elective infrarenal AAA repair had a portion of their aneurysm walls snap frozen in liquid nitrogen and processed for subsequent western blot or zymographic analysis. Tissue specimens from 6 normal abdominal aortas obtained from fresh cadaver specimens were similarly processed and served as controls. Protein levels for MMP-2, MMP-9, TIMP-1, uPA, and tPA were analyzed by western blotting. The degree of MMP-2 and MMP-9 gelatinolytic activity was analyzed by zymography. Detection and immunolocalization for MMP-2, MMP-9 and CD68 was performed on tissue sections of AAA and normal infrarenal abdominal aortas fixed in 10% formalin. MMP-9 and tPA protein levels were increased in AAAs compared to controls by western blotting. However, uPA levels were slightly increased in controls. No differences in TIMP-1 protein levels were identified. Similarly, zymography demonstrated increased MMP-2 and MMP-9 gelatinolytic activity in AAAs compared to controls (p < or = 0.05). CD68-positive cells (macrophages) in the adventitia and media demonstrated immunoreactivity to MMP-9. This investigation demonstrated increased MMP-9 proteinase activity and tPA protein levels in the walls of AAAs, as well as inflammatory leukocyte invasion of the adventitia and media compared to controls. These data suggest that leukocyte-derived MMP-9 is associated with aortic wall degeneration and aneurysm formation. Furthermore, activation of MMP-9 may be caused by increased tPA levels in the walls of AAAs.     

Suppression of experimental abdominal aortic aneurysms in the rat by treatment with angiotensin-converting enzyme inhibitors

PURPOSE: Pathologic remodeling of the extracellular matrix is a critical mechanism in the development and progression of abdominal aortic aneurysms (AAAs). Although angiotensin-converting enzyme (ACE) inhibitors are known to alter vascular wall remodeling in other conditions, their effects on AAAs are unknown. In this study we assessed the effect of ACE inhibitors in a rodent model of aneurysm development. METHODS: Male Wistar rats underwent transient aortic perfusion with porcine pancreatic elastase, followed by treatment with one of three ACE inhibitors (captopril [CP], lisinopril [LP], or enalapril [EP]), an angiotensin (AT)1 receptor antagonist (losartan [LOS]), or water alone (9 rats in each group). Blood pressure and aortic diameter (AD) were measured before elastase perfusion and on day 14, with an AAA defined as an increase in AD (DeltaAD) of more than 100%. The structural features of the aortic wall were examined by means of light microscopy. RESULTS: Aneurysmal dilatation consistently developed within 14 days of elastase perfusion in untreated rats, coinciding with the development of a transmural inflammatory response and destruction of the elastic media (mean DeltaAD, 223% +/- 28%). All three ACE inhibitors prevented AAA development (mean DeltaAD: CP, 67% +/- 4%; LP, 18% +/- 12%; and EP, 14% +/- 3%; each P <.05 vs controls). ACE inhibitors also attenuated the degradation of medial elastin without diminishing the inflammatory response. Surprisingly, the aneurysm-suppressing effects of ACE inhibitors were dissociated from their effects on systemic hemodynamics, and LOS had no significant effect on aneurysm development compared with untreated controls (mean DeltaAD, 186% +/- 19%). CONCLUSION: Treatment with ACE inhibitors suppresses the development of elastase-induced AAAs in the rat. Although this is associated with the preservation of medial elastin, the mechanisms underlying these effects appear to be distinct from hemodynamic alterations alone or events mediated solely by AT1 receptors. Further studies are needed to elucidate how ACE inhibitors influence aortic wall matrix remodeling during aneurysmal degeneration.     

Elastase is not sufficient to induce experimental abdominal aortic aneurysms

PURPOSE: Research investigating abdominal aortic aneurysms (AAAs) commonly uses a rat model dependent on aortic infusion of porcine pancreatic elastase to initiate AAA formation. Unfortunately, the sizes of AAAs generated by this model have varied widely among published studies. This may reflect lot-to-lot variations in commercial elastase preparations. This study was undertaken to investigate the ability of different lots of elastase to induce AAAs and explain the variability identified. METHODS: Four lots of elastase were evaluated in the standard rat AAA model. Saline solution was used as a control. Additional groups of rats were treated with higher concentrations of elastase with or without the macrophage activator thioglycollate medium. Aortic diameters were measured in all rats. Inflammation and elastin degradation was examined histologically. Elastase activity and purity were evaluated for all lots. RESULTS: Of the four lots tested, only one was able to consistently generate AAAs at the standard dose (P <.05). Increasing the amount of elastase infused produced AAAs in some ineffective lots. Infusion of thioglycollate medium in combination with otherwise ineffective elastase produced AAAs (P =.02). However, the elastase with the highest purity failed to generate AAAs, even at the highest dose tested or in combination with thyioglycollate medium. Thioglycollate medium alone failed to result in AAA formation. All elastase lots displayed elastolytic activity in vitro and produced elastin degradation in vivo. Elastin degradation did not correlate with AAA size in elastase-treated rats (P = NS). Aneurysm size correlated with extent of inflammation (P =.005). CONCLUSION: Induction of AAAs does not correlate with elastolytic activity. Infusion of pure elastase alone is not sufficient to induce AAA formation in spite of evidence of elastin degradation. Presumed inflammatory modifiers, which contaminate some elastase preparations, enhance AAA formation. Future use of this rat model will need to take the variability of elastase preparations into account with controls for each new elastase lot.     

Rapamycin suppresses experimental aortic aneurysm growth

OBJECTIVE: Inflammatory modulators are important in the pathogenesis of aneurysmal disease. Gene expression profiling of experimental abdominal aortic aneurysm (AAA) tissue demonstrated upregulation of the FK506BP12 (rapamycin binding protein) gene product. Rapamycin is a potent immunosuppressor that prevents recurrent stenosis. However, its effect on aneurysm formation has not been studied. We therefore examined the effect of rapamycin in an experimental rat AAA model. METHODS: Adult male Wistar rats underwent elastase infusion into isolated infrarenal aortas to create experimental aneurysms. Rats were randomized to receive either rapamycin or placebo via gastric lavage daily starting on the day of surgery. On postoperative day 7 the aneurysm was measured, the infrarenal aorta was harvested, and the rats were euthanized. NF kappa B was measured with Western blotting as a marker of inflammation. Matrix metalloproteinase (MMP)-9 protein levels were measured. Hematoxylin-eosin and elastin staining were used to examine tissue inflammation and elastin preservation. RESULTS: Aneurysms were significantly smaller in diameter in the rapamycin-treated group (3.3 +/- 0.7 mm vs 4.5 +/- 0.5 mm; P <.0001). NF kappa B levels were significantly reduced by 64% +/- 14% in rapamycin-treated aortas (P =.023). MMP-9 was reduced in rapamycin-treated aortas by 54% +/- 22% (P =.043). Hematoxylin-eosin and elastin staining showed no changes in inflammatory infiltrate or degradation of elastin fibers in elastase-infused aortic segments in rapamycin-treated rats. CONCLUSION: Rapamycin significantly reduces the rate of aneurysm expansion in the experimental AAA rat model by 40%. Biochemical evidence suggests that this is related to suppression of inflammatory signaling and decreased MMP-9 levels. Rapamycin could provide a new treatment for small aneurysms. CLINICAL RELEVANCE: Human aortic aneurysms are characterized histologically by an inflammatory infiltrate with severe proteolytic destruction. Rapamycin is an immunosuppressive agent commonly used to control transplant rejection and intimal hyperplasia by modulating the inflammatory cascade. In this experimental model rapamycin suppressed aneurysm expansion, decreased NF kappa B activation (a marker of inflammation), and decreased matrix metalloproteinase-9 levels. It is hoped that rapamycin or other similar anti-inflammatory drugs will one day be able to control aneurysm expansion in patients     

Mechanism of inhibition of matrix metalloproteinase-2 expression by doxycycline in human aortic smooth muscle cells

Degradation of the extracellular matrix components elastin and collagen has been implicated in vascular diseases, including abdominal aortic aneurysm (AAA) and atherosclerotic plaque rupture. Increased expression of matrix metalloproteinases (MMPs) is involved in these disease processes. Our previous studies have demonstrated that MMP-2 derived from mesenchymal cells is required for aneurysm development in a murine model. Doxycycline is a nonspecific inhibitor of MMPs. In the present study, the mechanisms of the inhibitory effects of doxycycline on MMP-2 expression from cultured human aortic smooth muscle cells (SMCs) and human aortic aneurysm tissue explants were studied. Doxycycline inhibited MMP-2 expression from cultured SMCs in a concentration-dependent manner (5-40 microg/mL; inhibitory concentration of 50%, 6.5 microg/mL). At normal therapeutic serum concentration (5 microg/mL) doxycycline significantly reduced MMP-2 production from SMCs (37%; P <.05), which were stimulated with conditioned media from macrophage or lymphocyte co-culture simulating the inflammatory milieu of AAA tissue. This correlated with a decrease in MMP-2 mRNA half-life, from 49 hours to 28 hours, which suggests that doxycycline inhibits SMC MMP-2 production in part by reducing MMP-2 mRNA stability. When AAA tissue was cultured for 10 days with doxycycline at concentrations of 2.5 to 40 microg/mL, the media exhibited a concentration-dependent decrease in both active and latent forms of MMP-2 and MMP-9. Doxycycline at a concentration of 5 microg/mL reduced active and latent MMP-2 secreted from cultured AAA tissue by 50% and 30%, respectively (P <.05). These study findings demonstrate that doxycycline at standard therapeutic serum concentrations inhibits MMP-2 expression from cultured human aortic SMCs and AAA tissue explants. Inasmuch as MMP activity contributes to extracellular matrix degradation in AAAs and atherosclerotic plaque, doxycycline may have potential value in treating these diseases.     

Effects of Calcium Channel Blocker Azelnidipine on Experimental Abdominal Aortic Aneurysms

Purpose Azelnidipine has recently been recognized in vascular remodeling. However, the effects of azelnidipine on aneurysmal disease have not yet been studied. The aim of this study was to evaluate whether azelnidipine can inhibit a further expansion of aneurysmal disease. Methods Experimental abdominal aortic aneurysms (AAAs) were created in a rat model by perfusing elastase. The rats in the first group received no treatment (n = 10). In the second group (n = 10) azelnidipine (2 mg/kg) was administered to the animals from 3 days before perfusion. The aortic diameter (AD) was measured at the time of initial surgery and death on postoperative day 14. The production of matrix metalloproteinases (MMP)-2 and -9 was analyzed by gelatin zymography. Results The aortic diameter was smaller in the azelnidipine group than in the control (7.875 ± 1.454 vs 10.745 ± 0.551 mm, P < 0.01). the active MMP-2 and MMP-9 levels decreased in the azelnidipine group. Hematoxylin–eosin and elastin staining revealed fewer changes in the inflammatory infiltrate and degradation of elastin in the azelnidipine group. Conclusion Azelnidipine reduced the expansion of experimental AAAs. Azelnidipine therefore appears to influence the inflammatory oxidative response seen in AAAs while also decreasing the MMP-2 and MMP-9 levels. In addition, azelnidipine inhibited aortic dilatation.     

Doxycycline inhibition of aneurysmal degeneration in an elastase-induced rat model of abdominal aortic aneurysm: Preservation of aortic elastin associated with suppressed production of 92 kD gelatinase

Purpose: Increased local production of matrix metalloproteinases (MMPs) is a potential mechanism underlying structural protein degradation in abdominal aortic aneurysms (AAA). With an elastase-induced rodent model of AAA, we determined whether pharmacologic treatment with an MMP-inhibiting tetracycline might limit the development of experimental AAA in vivo.Methods: Forty-eight Wistar rats underwent a 2-hour perfusion of the abdominal aorta with 50 U porcine pancreatic elastase and were then treated with either subcutaneous doxycycline (25 mg/day; n = 24) or saline solution vehicle (n = 24). Aortic diameter was measured before and after elastase perfusion was performed and before the rats were killed at 0, 2, 7, or 14 days, and AAAs were defined as an increase in aortic diameter to at least twice that before perfusion. At death the aortic tissues were either perfusion-fixed for histologic evaluation or extracted for substrate zymographic evaluation.Results: Aortic diameter was not different between groups at 0 or 2 days, but it was significantly less in animals treated with doxycycline at both 7 and 14 days (mean ± SEM, p < 0.01). After day 2 the incidence of AAA was reduced from 83% (10 of 12 rats treated with saline solution) to 8% (1 of 12 animals treated with doxycycline). By histologic assessment doxycycline prevented the structural deterioration of aortic elastin without decreasing the influx of inflammatory cells. Increased aortic wall production of 92 kD gelatinase observed in a saline solution-treated control group was markedly suppressed in animals treated with doxycycline.Conclusions: Treatment with an MMP-inhibiting tetracycline inhibits the development of experimental AAA in vivo. This inhibition may be due to selective blockade of elastolytic MMP expression in infiltrating inflammatory cells. Additional experiments, however, are necessitated to fully delineate this process. (J VASC SURG 1996;23:336-46.)     

Matrix metalloproteinase 8 (neutrophil collagenase) in the pathogenesis of abdominal aortic aneurysm

BACKGROUND: Loss of elastin is the initiating event in abdominal aortic aneurysm (AAA) formation, whereas loss of collagen is required for continued expansion. The elastolytic matrix metalloproteinases (MMPs) 2 and 9 are well described, but the source of excessive collagenolysis remains undefined. The aim of this study was to determine the expression of MMP-8, a potent type I collagenase, in normal aorta and AAA. METHODS: Infrarenal aortic biopsies were taken from 40 AAA and ten age-matched normal aortas. The concentrations of MMP-8 protein and its inhibitors, tissue inhibitor of metalloproteinase (TIMP) 1 and TIMP-2, were quantified by enzyme-linked immunosorbent assay. Immunohistochemistry was used to localize MMP-8 expression. RESULTS: MMP-8 concentrations were significantly raised in AAA compared with normal aorta (active MMP-8: 4.5 versus 0.5 ng per mg protein, P < 0.001; total MMP-8: 16.6 versus 2.8 ng per mg protein, P < 0.001). Levels of TIMP-1 and TIMP-2 were significantly lower in AAA than in normal aortic samples (TIMP-1: 142.2 versus 302.8 ng per mg protein; P = 0.010; TIMP-2: 9.2 versus 33.1 ng per mg protein, P < 0.001). Immunohistochemistry localized MMP-8 to mesenchymal cells within the adventitia of the aortic wall. CONCLUSION: The high concentration of MMP-8 in aortic aneurysms represents a potent pathway for collagen degradation, and hence aneurysm formation and expansion.