CLINICAL PERSPECTIVE

This Article Abstract Full Text (PDF) All Versions of this Article: 114/17/1855    most recent CIRCULATIONAHA.105.601674v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guo, G. Articles by Robinson, P. N. Search for Related Content PubMed PubMed Citation Articles by Guo, G. Articles by Robinson, P. N. Related Collections Animal models of human disease Genetics of cardiovascular disease Other Vascular biology

(Circulation. 2006;114:1855-1862.)
© 2006 American Heart Association, Inc.

Vascular Medicine

Induction of Macrophage Chemotaxis by Aortic Extracts of the mgR Marfan Mouse Model and a GxxPG-Containing Fibrillin-1 Fragment

Gao Guo, DMD; Patrick Booms, PhD; Marc Halushka, MD, PhD; Harry C. Dietz, MD; Andreas Ney, BSc; Sigmar Stricker, PhD; Jochen Hecht, MSc; Stefan Mundlos, MD; Peter N. Robinson, MD, MSc

From the Institute of Medical Genetics, Charité Universitätsmedizin, Humboldt University, Berlin, Germany (G.G., P.B., A.N., S.M., P.N.R.); Department of Pathology (M.H.), Institute of Genetic Medicine, and Howard Hughes Medical Institute, Departments of Pediatrics, Medicine, and Molecular Biology and Genetics (H.C.D.), Johns Hopkins University School of Medicine, Baltimore, Md; and Max Planck Institute for Molecular Genetics, Berlin, Germany (S.S., J.H., S.M.).

Reprint requests to Dr Peter N. Robinson, Institute of Medical Genetics, Charité Universitätsmedizin, Humboldt University, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail peter.robinson{at}charite.de

Received November 14, 2005; revision received June 26, 2006; accepted July 21, 2006.

	Abstract

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Background— The primary cause of early death in untreated Marfan syndrome (MFS) patients is aortic dilatation and dissection.

Methods and Results— We investigated whether ascending aortic samples from the fibrillin-1–underexpressing mgR mouse model for MFS or a recombinant fibrillin-1 fragment containing an elastin-binding protein (EBP) recognition sequence can act as chemotactic stimuli for macrophages. Both the aortic extracts from the mgR/mgR mice and the fibrillin-1 fragment significantly increased macrophage chemotaxis compared with extracts from wild-type mice or buffer controls. The chemotactic response was significantly diminished by pretreatment of macrophages with lactose or with the elastin-derived peptide VGVAPG and by pretreatment of samples with a monoclonal antibody directed against an EBP recognition sequence. Mutation of the EBP recognition sequence in the fibrillin-1 fragment also abolished the chemotactic response. These results indicate the involvement of EBP in mediating the effects. Additionally, investigation of macrophages in aortic specimens of MFS patients demonstrated macrophage infiltration in the tunica media.

Conclusions— Our findings demonstrate that aortic extracts from mgR/mgR mice can stimulate macrophage chemotaxis by interaction with EBP and show that a fibrillin-1 fragment possesses chemotactic stimulatory activity similar to that of elastin degradation peptides. They provide a plausible molecular mechanism for the inflammatory infiltrates observed in the mgR mouse model and suggest that inflammation may represent a component of the complex pathogenesis of MFS.

Key Words: chemotaxis • elastin-binding proteins • fibrillin • Marfan syndrome

	Introduction

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Marfan syndrome (MFS) is a dominantly inherited disorder with highly variable clinical manifestations, including aortic dilatation and dissection, ectopia lentis, and skeletal anomalies.^1,2 The primary cause of death in untreated individuals with MFS is aortic dissection or rupture as a consequence of progressive dilatation of the aortic root. Mutations in the gene for fibrillin-1 (FBN1) cause MFS and a series of related disorders of connective tissue.^3,4 Fibrillin was initially thought to be a purely structural protein in which mutations would exert a dominant negative effect by interfering with the aggregation of wild-type monomers into mature microfibrils. However, subsequent studies on fibrillin-1–underexpressing murine models have emphasized the predominant role of fibrillin-1 in tissue homeostasis.^5,6 More recent results have come together to demonstrate that alterations in transforming growth factor (TGF)–ß signaling play a major role in the pathogenesis of MFS and related disorders.^7–10 In addition, FBN1 mutations have been shown to increase the susceptibility of fibrillin-1 fragments to in vitro proteolysis,^11–13 and fibrillin fragments can themselves upregulate the expression of matrix metalloproteinases (MMPs).^14,15

Clinical Perspective p 1862

Inflammation and elastin fragmentation are prominent characteristics of abdominal aortic aneurysms (AAAs).¹⁶ Although fragmentation of elastic fibers is also found in MFS,^17,18 the inflammatory reaction is much less intense. One previous study has shown small numbers of macrophages surrounding areas of cystic medial necrosis, with the macrophages and smooth muscle cells in these regions being immunopositive for several MMPs, including MMP-2 and MMP-9, that can degrade elastin.¹⁹

Inflammatory infiltrates are an important feature in the mgR MFS mouse model. At birth, the homozygous mgR animals are phenotypically indistinguishable from littermates. However, as early as 8 weeks, monocytes begin to infiltrate the medial layer, followed by adventitial inflammation with fragmentation of the medial elastic network and fibroblast hyperplasia,⁶ suggesting that inflammatory infiltration is relevant to the progression of aortic aneurysm in this MFS mouse model, although the biochemical events that trigger the accumulation of inflammatory cells in the vessel wall have been unclear.

Macrophages in foci of inflammation are derived from blood monocytes attracted by chemotactic factors produced in inflammatory foci. Approximately 50 human chemokines have been identified that contribute to monocyte recruitment in vascular and nonvascular diseases.²⁰ However, chemotactic stimulatory activity is also associated with several components of the extracellular matrix, including collagen²¹ and elastin-derived peptides.^22,23 A repeated peptide in elastin, Val-Gly-Val-Ala-Pro-Gly (VGVAPG), is known for its chemotactic activity to fibroblasts and monocytes.²⁴ This effect is mediated by binding of VGVAPG to the 67-kDa elastin binding protein (EBP) present on the surface of mononuclear phagocytes. Elastin-derived peptides released from human AAA tissue can attract mononuclear phagocytes through ligand-receptor reactions with the EBP, which provides a plausible explanation for the inflammatory response that accompanies the development of aneurysms in this disorder.²⁵

We hypothesized that the fragmentation of elastin and fibrillin-rich microfibrils characteristic of human MFS and seen in the mgR mouse model could expose otherwise cryptic EBP motifs that could be responsible for recruiting macrophages to the aorta of the mgR MFS mouse model and thus initiate an inflammatory reaction. In this work, we used a chemotaxis chamber system to show that aortic extracts from mutant mgR/mgR mice and a fibrillin-1 fragment containing an EBP binding sequence both significantly increase monocyte chemotaxis. These results demonstrate that abnormalities of the aorta in the mgR model can have deleterious secondary effects that could significantly contribute to the progression of disease.

	Methods

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Preparation of Murine Aortic Extracts
Heterozygous mgR fibrillin-1–underexpressing mice⁶ were generously provided by Dr Francesco Ramirez. Homozygous (mgR/mgR) mice and their wild-type siblings were included in the study. All animals were genotyped with polymerase chain reaction as described.²⁶ At 2 and 4 months after birth, the mice were killed and the ascending aortas were prepared. Aortic tissue extracts were prepared using a modification of a previously described protocol.²⁵ Briefly, immediately after dissection, tissue specimens were washed 2 times in phosphate-buffered saline (PBS) and weighed. Specimens were then incubated in PBS with 2 mol/L NaCl overnight at 4°C with gentle shaking (20 mL per 1 g tissue). At the end of incubation, aortic tissue was removed, and the incubation solution was centrifuged at 10 000 rpm for 30 minutes to remove particulate debris. Aortic extract solution was stored at 4°C for experiments on the following day (or at –20°C for future repeats).

Cell Culture
Mouse macrophage cell line RAW 264.7 was obtained from American Type Culture Collection. These cells have properties similar to those of mouse resident macrophages and exhibit responsiveness to chemotactic stimuli.²⁷ Macrophages were cultured in medium containing RPMI 1640 (Biochrom AG, Berlin, Germany), 10% FBS, and penicillin/streptomycin mix (final concentration: penicillin, 100 IU/mL; streptomycin, 100 µg/mL). For harvesting the cells, monolayers of RAW 264.7 cells were washed 2 times with PBS with a nonenzymatic cell dissociation solution (Sigma, St. Louis, Mo). After 25 minutes of incubation at room temperature, cells were suspended in PBS, counted, centrifuged, and finally suspended in the chemotaxis buffer consisting of RPMI 1640 supplemented with 1% bovine serum albumin (Sigma) at 1x10⁶ cells/mL.

Recombinant Fibrillin-1 Polypeptides
Two recombinant polypeptides, rFib47^wt and rFib47^mt, were generated as described^11,14,15 to correspond to amino acids I¹⁹²⁹ to E²²⁰⁵. This region encompasses cbEGF-like motifs 29, 30, and 31 and TGFß1bp-like (8-cys) motif 6, followed by cbEGF-like motifs 32 and 33. rFib47^wt contains a putative EBP recognition motif EGFEPG at amino acids 2194 to 2199. In rFib47^mt, the conserved proline residue at position 2198 is mutated to a serine residue (EGFEPGEGFESG), thereby abolishing the EBP consensus sequence.

Chemotaxis Assay
Chemotaxis was assayed in a 48-well chemotaxis chamber (NeuroProbe). The bottom wells were filled with 25 µL of attractant solution. An 8-µm-pore-diameter polyvinylpyrrolidone-free polycarbonate filter (NeuroProbe, Gaithersburg, Md) was placed on the bottom plate. A silicon gasket and the top plate with 48 holes were then mounted, forming the top wells. The cells were added in a volume of 50 µL. In parallel experiments, extraction buffer (PBS plus 2 mol/L NaCl) or chemotaxis buffer was loaded into the bottom wells as a negative control. After 2 hours of incubation at 37°C and 5% CO₂, the filter sheet was removed, and nonmigrated cells were wiped off the top side. The filter was then fixed in 70% methanol for 30 seconds and stained in Wright-Giemsa (Sigma). All assays were done in triplicate during the same experiment. In each well, 4 random fields were chosen with a microscope set at x200 magnification. The fields were observed and stored with the Leica DC viewer program. Fields were scored by 2 observers blinded to the chemotactic stimulus (or control) using an in-house Java program.

Experimental Design
For each time interval, 3 or 4 wild-type and mutant mice were killed, and all aortic extracts were investigated at least 3 times. Polypeptide samples were diluted in chemotaxis buffer to a concentration of 100 µg/mL and were tested for their ability to act as chemotactic stimuli for macrophages. Checkerboard analysis was used to distinguish chemotaxis from chemokinesis.²⁸ To investigate a potential interaction with EBP, cells were exposed to 1 mmol/L lactose or glucose or 0.1 mmol/L VGVAPG for 1 hour incubation at 37°C before the chemotaxis assays were started.^24,29 As a further control, samples were preincubated for 30 minutes at room temperature with the mouse monoclonal antibody (mAb) BA4 derived from immunization against bovine -elastin (Sigma) or with murine IgG (1 mg/mL; Sigma), both at a dilution of 1:1000.

Immunohistochemistry of Human Aortic Specimens
Human aortic segments were obtained from the surgical pathology records of the Johns Hopkins Hospital. Samples from 28 persons with MFS and from 4 control subjects (3 heart donors 2, 9, and 24 years of age and 1 sample from a 30-year-old man who had died after a motor vehicle accident) were analyzed. Slides were cut from paraffin-embedded, formalin-fixed tissues. After deparaffinization, antigen retrieval was performed in a Tris buffer (pH 9.5) for 38 minutes. Slides were incubated with CD68 (KP-1) antibody (a macrophage marker³⁰) for 32 minutes at room temperature. Slides were then blocked from endogenous peroxidase for 4 minutes with inhibitor solution (Ventana Medical Systems Inc) before secondary antibody and diaminobenzidine incubations of 8 minutes, respectively. Slides were then counterstained with hematoxylin, and a coverslip was placed. All steps were performed on a Ventana BenchMark XT automated IHC stainer using standard reagents (Ventana Medical Systems Inc, Tucson, Ariz).

CD68⁺ cells were counted only in the tunica media. CD68⁺ cells naturally occur in the underlying adventitia without relation to vascular disease and were not counted. CD68⁺ cells along the intima were not included because atherosclerotic plaques or fatty streaks could potentially influence the cell counts.

Statistical Analysis
Data are expressed as mean±SE. The value for the mean macrophage chemotactic activity (number of migrated cells) of each group was compared using the Student’s t test. Statistical significance was accepted at P<0.05. The bar charts show mean and SEs.

The authors had full access to the data and take full responsibility for their integrity. All authors have read and agree to the manuscript as written.

	Results

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Aortic Extracts Induce Macrophage Migration
We investigated whether aortic specimens from homozygous mutant mgR mice can act as a chemotactic stimulus for macrophages. The mgR mouse is a well-characterized mouse model for MFS.⁶ Two- and 4-month-old homozygous mgR/mgR mice were compared with age-matched wild-type littermates. As previously described,^6,31 aortic specimens from 2-month-old mice exhibited greatly increased intimal hyperplasia, disorganization of elastin, and cellular infiltration compared with wild-type specimens (data not shown). We tested aortic extracts from 2- and 4-month-old mice for their ability to induce chemotaxis using a chemotaxis chamber system and RAW 264.7 murine monocytes. Counts of migrated cells were determined in a randomized, blinded fashion. This showed that mgR/mgR aortic samples stimulated monocyte chemotaxis 3 to 4 times more than extracts from wild-type mice or controls. The difference was statistically significant. The mgR/mgR samples from both 2-month-old (Figure 1A) and 4-month-old (data not shown) mice showed increased chemotaxis, but the difference between both age groups was not statistically significant. The above experiments were repeated on a total of four 2-month-old mice and four 4-month-old mgR/mgR mice with similar results.

View larger version (12K): [in this window] [in a new window]   Figure 1. mgR/mgR aortic extracts induce chemotaxis. A, Aortic extracts of 2-month-old mutant (mut) and wild-type (wt) mice were added to the lower wells of chemotaxis chamber separately. Extraction buffer was used as a negative control (–). Chemotaxis is expressed as the number of migrated RAW 264.7 macrophages. B, Reduction of chemotactic effect by lactose. Macrophages were exposed to 1 mmol/L lactose for 1 hour, and aortic extracts of 2-month-old mutant mice were added to the lower wells of the chemotaxis chamber. Without L indicates no lactose preincubation; with L, lactose preincubation. Extraction buffer was used as negative control (–). Results are mean±SE. n=12 per group (4 fields each for 3 wells during the same experiment). *P<0.05, **P<0.001, ***P<0.0001 vs control.

We hypothesized that the increase in chemotactic activity could be related to activation of the elastin-laminin receptor resulting from binding of elastin degradation products to the EBP. It has been previously described that extracts from surgical AAA specimens and elastin degradation products, including the hexapeptide VGVAPG, increase macrophage chemotaxis.²⁵ We confirmed that the chemotaxis by RAW 264.7 cells is also stimulated by VGVAPG. Stimulation with 10^–5 mol/L VGVAPG led to an increase in migrated cells from 8.0±1.4 to 50.4±2.2 cells per field.

Incubation of macrophages in lactose causes them to shed the EBP, and this can be used to demonstrate specificity of a chemotactic stimulus on ligand-receptor interactions involving the EBP.^25,29 We therefore repeated the experiments with preincubation of macrophages in lactose for 1 hour. This treatment significantly reduced chemotaxis in 2-month-old mutant mice (Figure 1B). A similar difference was observed for 4-month-old mice (data not shown). No inhibitory effect was observed after pretreatment with glucose (data not shown), as has been previously described for stimulation with AAA extracts.²⁵

We performed checkerboard analysis²⁸ to determine whether the macrophage migration was related to directed chemotaxis or undirected chemokinesis. Macrophages showed migration only in the presence of mutant aortic extracts in the lower well; adding mutant aortic extracts to the upper well greatly reduced the observed cell counts (see the Table). This result indicates that the observed cell migration is caused by chemotaxis rather than by (undirected) chemokinesis.

View this table: [in this window] [in a new window]   Checkerboard Analysis of RAW 264.7 Macrophage Migration Stimulated by Aortic Extracts

A Recombinant Fibrillin-1 Fragment Containing a Putative EBP Binding Site Acts as a Chemotactic Stimulus
We have recently shown that incubation with a recombinant fibrillin-1 fragment (rFib47^wt) increases MMP-1 expression and production 7- to 9-fold; the effect was abrogated by introducing a mutation of the EBP recognition sequence (GxxPGGxxSG).¹⁵ We therefore investigated whether this fibrillin fragment could also affect macrophage chemotaxis. The fibrillin-1 fragment rFib47^wt increased chemotaxis 4-fold compared with control (buffer) samples (Figure 2). The corresponding mutant construct was not significantly different from control (Figure 2A), indicating specificity of the reaction for EBP. Additionally, preincubating macrophages with lactose also reduced the chemotactic effects of the GxxPG polypeptide (Figure 2B). In a separate experiment, macrophages were pretreated with 0.1 mmol/L VGVAPG hexapeptide for 1 hour, and then the chemotaxis experiment was performed as above. Preincubation with VGVAPG blocked the chemotactic response, reducing the mean number of migrated cells from 10.8±2.0 to 1.7±0.3 cells per field. These results indicate that a fibrillin-1 fragment containing a putative EBP ligand GxxPG sequence can act as a chemotactic stimulus and that its effects are specific to the interaction with EBP.

View larger version (14K): [in this window] [in a new window]   Figure 2. Chemotactic response of RAW 264.7 macrophage to recombinant fibrillin-1 polypeptides. A, Two recombinant polypeptides, rFib47wt and rFib47mut (100 µg/mL), were added to lower wells. RAW 264.7 macrophages were added to upper wells. B, The wild-type recombinant polypeptide rFib47wt was used as a chemotactic stimulant for untreated macrophages (rFib47/alone) and macrophages that had been exposed to 1 mmol/L lactose for 1 hour (rFib47/lactose). Chemotaxis buffer was used as negative control. Results are mean±SE. n=12 per group (4 fields each for 3 wells during the same experiment). ***P<0.0001 vs control.

BA4 Inhibits Chemotaxis to mgR/mgR Aortic Extracts or the GxxPG Fibrillin-1 Fragment
The monoclonal antibody BA4 was raised against bovine -elastin but has been shown to react against VGVAPG and several other xGxxPG hexapeptides that interact with EBP.³² Pretreatment of mgR/mgR aortic extracts from 4-month-old mice or rFib47^wt with BA4 significantly reduced the chemotactic response (Figure 3).

View larger version (13K): [in this window] [in a new window]   Figure 3. Chemotactic response of RAW 264.7 macrophages is inhibited by pretreatment with monoclonal antibody BA4. A, The 4-month-old mgR/mgR aortic extract was analyzed in isolation, with mAb BA4, or with IgG. Extraction buffer was used as negative control. Probability values are for comparison with mgR/mgR sample. B, Recombinant rFib47wt construct was analyzed in isolation, with mAb BA4, or with IgG. Chemotaxis buffer was performed as negative control. Results are mean±SE. n=12 per group (4 fields each for 3 wells during the same experiment). *P<0.05, **P<0.001 vs rFib47wt.

Macrophage Infiltration Is Observed in Surgical Aortic Specimens of Marfan Patients
To investigate a potential role of macrophage infiltration and inflammation in human MFS, we have investigated surgical ascending aorta specimens from 28 persons with MFS who underwent aortic surgery and 4 control samples. Four MFS patients had identified FBN1 mutations; the remainder all met clinical diagnostic criteria for MFS. The presence of macrophages was assessed with immunostaining with an anti-CD68 antibody. Monocytes and macrophages are not typically seen in the intimal and medial layers of the arterial wall except in the setting of an inflammatory process such as atherosclerosis, vasculitis, or trauma. Monocytes and macrophages can be seen in the underlying adventitia of vessels in both MFS and control samples in a nonspecific fashion. A collection of macrophages was noted in the tunica intima of a single MFS patient, which may be related to the disease or likely to a fatty streak; otherwise, no CD68⁺ cells were noted in the tunica intima of any other sample.

Investigation of the 4 control samples confirmed this finding, showing an average of only 0.25±0.14 CD68⁺ cells per x200 microscopic field. In contrast, the samples from all MFS patients showed an average of 3.7±0.6 CD68⁺ cells per field (mean±SE; range, 0 to 15). The difference was statistically significant. There was a tendency for samples from MFS patients >25 years of age to have more CD68⁺ cells that did not reach statistical significance. Figure 4 summarizes our results. Figure 5 presents a representative case study.

View larger version (15K): [in this window] [in a new window]   Figure 4. Ascending aortic medial inflammation in surgical specimens. CD68+ cells per x200 microscopic field. Samples from 4 controls and 28 persons with MFS. Eleven persons with MFS were <20 years of age; 17 were >25 years of age. Data represent mean±SE. *P<0.05, **P<0.001, ***P<0.0001 vs control.

View larger version (75K): [in this window] [in a new window]   Figure 5. Surgical ascending aortic specimen from a 10-year-old black male with MFS who presented for elective aortic root replacement for a >5.5-cm ascending aortic aneurysm. The resected tissue showed multiple areas of cystic medial degeneration throughout the tunica media. A macrophage infiltrate (CD68+ cells) was present in the tunica media in areas of cystic medial degeneration and along the vasa vasorum. Macrophages also were noted in the adventitia as a nonspecific finding but were not present in the tunica intima. A, Typical histological image of a severely affected aorta in an MFS patient with cystic medial degeneration at risk for dissection (hematoxylin and eosin, x40). B–D, CD68 immunohistochemical stains at magnifications of x40, x100, and x200, respectively. Note the presence of macrophages surrounding foci of cystic medial degeneration and along the vasa vasorum (C, top). This case represents one of the more significant CD68 infiltrates noted within the cohort.

	Discussion

Top Abstract Introduction Methods Results Discussion Conclusions References

 
In this work, we have demonstrated that aortic extracts of the mgR MFS mouse model induce chemotaxis and that this effect is mediated by EBP. Additionally, we have shown that a recombinant fibrillin-1 fragment containing a GxxPG EBP recognition sequence is able to induce chemotaxis in a comparable fashion. Macrophage infiltration and inflammation play a well-known role in AAAs^33,34 but have not previously been investigated in MFS. In AAAs, soluble elastin-derived peptides released from AAA tissue act as chemoattractants for mononuclear phagocytes,²⁵ providing a plausible explanation for the intense inflammatory response seen in that disorder. Macrophage-released proteases, including MMP-12, could be responsible for further destruction of elastin-rich tissues in the aorta.³⁵

To the best of our knowledge, no previous study has specifically focused on the role of macrophages in MFS, although the presence of macrophages was noted in surrounding areas of cystic medial necrosis in a study about changes in MMP immunohistochemistry in aortic specimens from MFS patients.¹⁹ In the present study, we have identified a statistically significant increase in the number of macrophages in the tunica media of 28 persons with MFS. We note that although histological analysis of patient specimens can provide us with important information, the results of such an analysis need to be interpreted with caution for 2 reasons. First, a surgical specimen is a snapshot taken years after the initiation of aortic disease, and it is not possible to know whether the initiating factors are still present or visible in the specimen. Multiple stages of aortic disease have been recognized in AAAs, each of which shows significant differences in parameters such as MMP-12 activity.³⁵ Surgical specimens obtained during prophylactic or acute aortic root replacement in patients with MFS are likely to show the result of long-term aortic disease and will not necessarily reflect the initiating processes. Second, it is not possible to differentiate causative factors and epiphenomena on the basis of the appearance of a histological analysis. Nonetheless, we have presented definitive evidence that macrophage infiltration can be observed in aortic specimens of MFS patients and have presented the first specific demonstration of CD68⁺ macrophages in aortic specimens of MFS patients. Together with our experimental results, these findings support the notion that inflammation could play a role in the pathogenesis of MFS. We are planning further investigations to address the question of whether EBP ligands are present in increased concentrations in samples from MFS patients.

Although intense inflammatory changes are not routinely observed in patients with MFS, loss of elastin, disorganization of tissue architecture, and fragmentation of fibrillin-rich microfibrils are well-known characteristics of the disorder.^17,18,36 Fragmentation and disarray of elastic fibers is a focal and acquired event that occurs in association with abnormal production of matrix-degrading enzymes by resident vascular smooth muscle cells and ultimately vessel wall inflammation in the mgR mouse model.^6,31 Fragments of extracellular matrix may have signaling activities that the corresponding intact molecules do not have, so fragments may alter feedback regulation and contribute to progression of tissue damage.³⁷

It is therefore possible that early alterations in the extracellular matrix of the mgR mouse themselves have secondary deleterious effects. For instance, fragmentation of the aortic media could release soluble elastin degradation products or fibrillin-1 fragments that recruit macrophages into the aorta; if these macrophages release proteolytic enzymes that cause further fragmentation of tissues, a vicious cycle could ensue. A similar model has been proposed for cigarette smoke–induced emphysema in a mouse model; cigarette smoke could induce macrophages to produce macrophage elastase, which cleaves elastic tissue, producing elastin-derived peptides that are chemoattractant to monocytes. Thus, a positive feedback loop could perpetuate macrophage accumulation and lung destruction.^38,39 Indeed, the paradigm of limited elastic fiber degradation progressing to intense elastolysis in association with inflammatory infiltration of the vessel wall and increased expression of MMPs is emerging as a common theme in the pathogenesis of abdominal aortic aneurysms.^16,40,41

The EBP is 1 of the 3 protein subunits that make up the elastin-laminin receptor.⁴² This protein has previously been implicated in a variety of signaling processes mediated by binding to elastin-derived peptides, such as mechanotransduction,⁴³ induction of MMP expression,⁴⁴ and chemotaxis.²⁴ We have demonstrated specificity for EBP of the chemotactic response toward mgR/mgR aortic extracts and rFib47^wt by showing inhibition of the response by pretreatment of macrophages by lactose, which is known to cause shedding of the EBP from the cell surface,⁴² and by pretreatment with BA4 or VGVAPG. Additionally, in vitro mutation of the EBP consensus site in rFib47^wt from EGFEPG to EGFESG severely inhibited the chemotactic effect. This mutation was introduced to demonstrate the specificity for the EBP and does not represent a mutation found in MFS.

Although elastin-derived fragments are presumably quantitatively more important than fibrillin in inducing chemotaxis in the mgR/mgR mouse, our finding that a GxxPG-carrying fibrillin-1 fragment can also induce chemotaxis shows that chemotactic effects are not limited to elastin. We have provided evidence that the effects of the fibrillin-1 fragment are mediated by interaction with EBP. The fact that the same fibrillin-1 fragment can both induce increased MMP-1 expression and act as a chemotactic stimulus is consistent with the finding that elastin-derived peptides have both properties.^24,44 We note that the present experiments were not designed to differentiate between elastin- or fibrillin-derived EBP ligands in the mgR/mgR aortic extracts. Fragmentation of aortic tissue could potentially affect both elastin and fibrillin-1. Further work is required to determine the relative importance of elastin- or fibrillin-derived EBP ligands.

	Conclusions

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Our results highlight the possibility that the fragmentation of the ECM seen in the mgR MFS mouse model is itself an important contributory factor toward the initiation of the inflammatory reaction in the aortic wall. Although it is not possible to conclude that inflammation plays a role in initiation or progression of aortic dilatation in MFS, we have provided evidence that increased numbers of macrophages are found in surgical aortic specimens from MFS patients. This suggests the possibility that recruitment of inflammatory cells by elastin-derived peptides and fibrillin fragments may contribute to the complex pathogenesis of MFS. Because suppression of immune signaling by agents such as rapamycin⁴⁵ and of MMP production by doxycyclin⁴⁶ effectively prevents the occurrence of AAAs in animal models, future research along these lines could be fruitful with respect to the development of novel therapies for human MFS.

	Acknowledgments

 
We thank Ludger Hartmann and Janine Wetzel for excellent animal care.

Sources of Funding

This work was supported by Marfan Hilfe (Deutschland) eV. Other support was provided by the National Marfan Foundation (US), the Howard Hughes Medical Institute (US), and NIH (US) grants AR41135 and AR049698.

Disclosures

None.

	References

Top Abstract Introduction Methods Results Discussion Conclusions References

 
1. Robinson PN, Arteaga-Solis E, Baldock C, Collod-Beroud G, Booms P, De Paepe A, Dietz HC, Guo G, Handford PA, Judge DP, Kielty CM, Loeys B, Milewicz DM, Ney A, Ramirez F, Reinhardt DP, Tiedemann K, Whiteman P, Godfrey M. The molecular genetics of Marfan syndrome and related disorders. J Med Genet. 2006; 43: 769–787.[Abstract/Free Full Text]

2. Robinson PN, Godfrey M. Marfan Syndrome: A Primer for Clinicians and Scientists. New York, NY: Kluwer Academic/Plenum Publishers; 2004.

3. Dietz HC, Cutting CR, Pyeritz RE, Maslen CL, Sakai LY, Corson GM, Puffenberger EG, Hamosh A, Nanthakumar EJ, Curristin SM, Stetten G, Meyers DA, Francomano CA. Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene. Nature. 1991; 352: 337–339.[CrossRef][Medline] [Order article via Infotrieve]

4. Robinson PN, Booms P, Katzke S, Ladewig M, Neumann L, Palz M, Pregla R, Tiecke F, Rosenberg T. Mutations of FBN1 and genotype-phenotype correlations in Marfan syndrome and related fibrillinopathies. Hum Mutat. 2002; 20: 153–161.[CrossRef][Medline] [Order article via Infotrieve]

5. Pereira L, Andrikopoulos K, Tian J, Lee SY, Keene DR, Ono R, Reinhardt DP, Sakai LY, Biery NJ, Bunton T, Dietz HC, Ramirez F. Targetting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome. Nat Genet. 1997; 17: 218–222.[CrossRef][Medline] [Order article via Infotrieve]

6. Pereira L, Lee SY, Gayraud B, Andrikopoulos K, Shapiro SD, Bunton T, Biery NJ, Dietz HC, Sakai LY, Ramirez F. Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1. Proc Natl Acad Sci U S A. 1999; 96: 3819–3823.[Abstract/Free Full Text]

7. Neptune ER, Frischmeyer PA, Arking DE, Myers L, Bunton TE, Gayraud B, Ramirez F, Sakai LY, Dietz HC. Dysregulation of TGF-beta activation contributes to pathogenesis in Marfan syndrome. Nat Genet. 2003; 33: 407–411.[CrossRef][Medline] [Order article via Infotrieve]

8. Ng CM, Cheng A, Myers LA, Martinez-Murillo F, Jie C, Bedja D, Gabrielson KL, Hausladen JM, Mecham RP, Judge DP, Dietz HC. TGF-ß-dependent pathogenesis of mitral valve prolapse in a mouse model of Marfan syndrome. J Clin Invest. 2004; 114: 1586–1592.[CrossRef][Medline] [Order article via Infotrieve]

9. Mizuguchi T, Collod-Beroud G, Akiyama T, Abifadel M, Harada N, Morisaki T, Allard D, Varret M, Claustres M, Morisaki H, Ihara M, Kinoshita A, Yoshiura K, Junien C, Kajii T, Jondeau G, Ohta T, Kishino T, Furukawa Y, Nakamura Y, Niikawa N, Boileau C, Matsumoto N. Heterozygous TGFBR2 mutations in Marfan syndrome. Nat Genet. 2004; 36: 855–860.[CrossRef][Medline] [Order article via Infotrieve]

10. Loeys BL, Chen J, Neptune ER, Judge DP, Podowski M, Holm T, Meyers J, Leitch CC, Katsanis N, Sharifi N, Xu FL, Myers LA, Spevak PJ, Cameron DE, De Backer J, Hellemans J, Chen Y, Davis EC, Webb CL, Kress W, Coucke P, Rifkin DB, De Paepe AM, Dietz HC. A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2. Nat Genet. 2005; 37: 275–281.[CrossRef][Medline] [Order article via Infotrieve]

11. Booms P, Tiecke F, Rosenberg T, Hagemeier C, Robinson PN. Differential effect of FBN1 mutations on in vitro proteolysis of recombinant fibrillin-1 fragments. Hum Genet. 2000; 107: 216–224.[CrossRef][Medline] [Order article via Infotrieve]

12. McGettrick AJ, Knott V, Willis A, Handford PA. Molecular effects of calcium binding mutations in Marfan syndrome depend on domain context. Hum Mol Genet. 2000; 9: 1987–1994.[Abstract/Free Full Text]

13. Reinhardt DP, Ono RN, Notbohm H, Muller PK, Bachinger HP, Sakai LY. Mutations in calcium-binding epidermal growth factor modules render fibrillin-1 susceptible to proteolysis: a potential disease-causing mechanism in Marfan syndrome. J Biol Chem. 2000; 275: 12339–12345.[Abstract/Free Full Text]

14. Booms P, Pregla R, Ney A, Barthel F, Reinhardt DP, Pletschacher A, Mundlos S, Robinson PN. RGD-containing fibrillin-1 fragments upregulate matrix metalloproteinase expression in cell culture: a potential factor in the pathogenesis of the Marfan syndrome. Hum Genet. 2005; 116: 51–61.[CrossRef][Medline] [Order article via Infotrieve]

15. Booms P, Ney A, Barthel F, Moroy G, Counsell D, Gille C, Guo G, Pregla R, Mundlos S, Alix AJP, Robinson PN. A fibrillin-1-fragment containing the elastin-binding-protein GxxPG consensus sequence upregulates matrix metalloproteinase-1: biochemical and computational analysis. J Mol Cell Cardiol. 2006; 40: 234–246.[CrossRef][Medline] [Order article via Infotrieve]

16. Ailawadi G, Eliason JL, Upchurch GR Jr. Current concepts in the pathogenesis of abdominal aortic aneurysm. J Vasc Surg. 2003; 38: 584–588.[CrossRef][Medline] [Order article via Infotrieve]

17. Fleischer KJ, Nousari HC, Anhalt GJ, Stone CD, Laschinger JC. Immunohistochemical abnormalities of fibrillin in cardiovascular tissues in Marfan’s syndrome. Ann Thorac Surg. 1997; 63: 1012–1017.[Abstract/Free Full Text]

18. Roberts WC, Honig HS. The spectrum of cardiovascular disease in the Marfan syndrome: a clinico-morphologic study of 18 necropsy patients and comparison to 151 previously reported necropsy patients. Am Heart J. 1982; 104: 115–135.[CrossRef][Medline] [Order article via Infotrieve]

19. Segura AM, Luna RE, Horiba K, Stetler-Stevenson WG, McAllister HA Jr, Willerson JT, Ferrans VJ. Immunohistochemistry of matrix metalloproteinases and their inhibitors in thoracic aortic aneurysms and aortic valves of patients with Marfan’s syndrome. Circulation. 1998; 98: II-331–II-337.[Medline] [Order article via Infotrieve]

20. Charo IF, Taubman MB. Chemokines in the pathogenesis of vascular disease. Circ Res. 2004; 95: 858–866.[Abstract/Free Full Text]

21. Postlethwaite AE, Kang AH. Collagen-and collagen peptide-induced chemotaxis of human blood monocytes. J Exp Med. 1976; 143: 1299–1307.[Abstract/Free Full Text]

22. Hunninghake GW, Davidson JM, Rennard S, Szapiel S, Gadek JE, Crystal RG. Elastin fragments attract macrophage precursors to diseased sites in pulmonary emphysema. Science. 1981; 212: 925–927.[Abstract/Free Full Text]

23. Senior RM, Griffin GL, Mecham RP. Chemotactic activity of elastin-derived peptides. J Clin Invest. 1980; 66: 859–862.[Medline] [Order article via Infotrieve]

24. Senior RM, Griffin GL, Mecham RP, Wrenn DS, Prasad KU, Urry DW. Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibroblasts and monocytes. J Cell Biol. 1984; 99: 870–874.[Abstract/Free Full Text]

25. Hance KA, Tataria M, Ziporin SJ, Lee JK, Thompson RW. Monocyte chemotactic activity in human abdominal aortic aneurysms: role of elastin degradation peptides and the 67-kD cell surface elastin receptor. J Vasc Surg. 2002; 35: 254–261.[CrossRef][Medline] [Order article via Infotrieve]

26. Hartner A, Eifert T, Haas CS, Tuysuz C, Hilgers KF, Reinhardt DP, Amann K. Characterization of the renal phenotype in a mouse model of Marfan syndrome. Virchows Arch. 2004; 445: 382–388.[CrossRef][Medline] [Order article via Infotrieve]

27. Aksamit RR, Falk W, Leonard EJ. Chemotaxis by mouse macrophage cell lines. J Immunol. 1981; 126: 2194–2199.[Abstract]

28. Zigmond SH, Hirsch JG. Leukocyte locomotion and chemotaxis: new methods for evaluation, and demonstration of a cell-derived chemotactic factor. J Exp Med. 1973; 137: 387–410.[Abstract]

29. Hinek A, Wrenn DS, Mecham RP, Barondes SH. The elastin receptor: a galactoside-binding protein. Science. 1988; 239: 1539–1541.[Abstract/Free Full Text]

30. Pulford KA, Sipos A, Cordell JL, Stross WP, Mason DY. Distribution of the CD68 macrophage/myeloid associated antigen. Int Immunol. 1990; 2: 973–980.[Abstract/Free Full Text]

31. Bunton TE, Biery NJ, Myers L, Gayraud B, Ramirez F, Dietz HC. Phenotypic alteration of vascular smooth muscle cells precedes elastolysis in a mouse model of Marfan syndrome. Circ Res. 2001; 88: 37–43.[Abstract/Free Full Text]

32. Grosso LE, Scott M. Peptide sequences selected by BA4, a tropoelastin-specific monoclonal antibody, are ligands for the 67-kilodalton bovine elastin receptor. Biochemistry. 1993; 32: 13369–13374.[CrossRef][Medline] [Order article via Infotrieve]

33. Rijbroek A, Moll FL, von Dijk HA, Meijer R, Jansen JW. Inflammation of the abdominal aortic aneurysm wall. Eur J Vasc Surg. 1994; 8: 41–46.[CrossRef][Medline] [Order article via Infotrieve]

34. Brophy CM, Reilly JM, Smith GJ, Tilson MD. The role of inflammation in nonspecific abdominal aortic aneurysm disease. Ann Vasc Surg. 1991; 5: 229–233.[CrossRef][Medline] [Order article via Infotrieve]

35. Curci JA, Liao S, Huffman MD, Shapiro SD, Thompson RW. Expression and localization of macrophage elastase (matrix metalloproteinase-12) in abdominal aortic aneurysms. J Clin Invest. 1998; 102: 1900–1910.[Medline] [Order article via Infotrieve]

36. Gott VL, Laschinger JC, Cameron DE, Dietz HC, Greene PS, Gillinov AM, Pyeritz RE, Alejo DE, Fleischer KJ, Anhalt GJ, Stone CD, McKusick VA. The Marfan syndrome and the cardiovascular surgeon. Eur J Cardiothorac Surg. 1996; 10: 149–158.[Abstract]

37. Homandberg GA. Potential regulation of cartilage metabolism in osteoarthritis by fibronectin fragments. Front Biosci. 1999; 4: D713–D730.[Medline] [Order article via Infotrieve]

38. Shapiro SD. Diverse roles of macrophage matrix metalloproteinases in tissue destruction and tumor growth. Thromb Haemost. 1999; 82: 846–849.[Medline] [Order article via Infotrieve]

39. Houghton AM, Quintero PA, Perkins DL, Kobayashi DK, Kelley DG, Marconcini LA, Mecham RP, Senior RM, Shapiro SD. Elastin fragments drive disease progression in a murine model of emphysema. J Clin Invest. 2006; 116: 753–759.[CrossRef][Medline] [Order article via Infotrieve]

40. Freestone T, Turner RJ, Coady A, Higman DJ, Greenhalgh RM, Powell JT. Inflammation and matrix metalloproteinases in the enlarging abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol. 1995; 15: 1145–1151.[Abstract/Free Full Text]

41. Buckley C, Wyble CW, Borhani M, Ennis TL, Kobayashi DK, Curci JA, Shapiro SD, Thompson RW. Accelerated enlargement of experimental abdominal aortic aneurysms in a mouse model of chronic cigarette smoke exposure. J Am Coll Surg. 2004; 199: 896–903.[CrossRef][Medline] [Order article via Infotrieve]

42. Mecham RP, Hinek A, Entwistle R, Wrenn DS, Griffin GL, Senior RM. Elastin binds to a multifunctional 67-kilodalton peripheral membrane protein. Biochemistry. 1989; 28: 3716–3722.[CrossRef][Medline] [Order article via Infotrieve]

43. Spofford CM, Chilian WM. Mechanotransduction via the elastin-laminin receptor (ELR) in resistance arteries. J Biomech. 2003; 36: 645–652.[CrossRef][Medline] [Order article via Infotrieve]

44. Brassart B, Fuchs P, Huet E, Alix AJP, Wallach J, Tamburro AM, Delacoux F, Haye B, Emonard H, Hornebeck W, Debelle L. Conformational dependence of collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in cultured fibroblasts. J Biol Chem. 2001; 276: 5222–5227.[Abstract/Free Full Text]

45. Lawrence DM, Singh RS, Franklin DP, Carey DJ, Elmore JR. Rapamycin suppresses experimental aortic aneurysm growth. J Vasc Surg. 2004; 40: 334–338.[CrossRef][Medline] [Order article via Infotrieve]

46. Petrinec D, Liao S, Holmes DR, Reilly JM, Parks WC, Thompson RW. 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. J Vasc Surg. 1996; 23: 336–346.[CrossRef][Medline] [Order article via Infotrieve]

---

 

CLINICAL PERSPECTIVE

Marfan syndrome (MFS) is a common inherited disorder of connected tissue often associated with progressive dilatation of the ascending aorta, leading to acute aortic dissection. The molecular pathogenesis of MFS is complex, with alterations in transforming growth factor-ß metabolism and a number of other factors thought to play some role in the initiation and progression of disease. In this work, we have shown that aortic extracts of the mgR mouse model for MFS elicit macrophage chemotaxis. The effects were inhibited by pretreatment with lactose or with a monoclonal antibody directed against an elastin-binding protein (EBP) interaction sequence in elastin, indicating involvement of EBP in mediating the effects. We have also shown that a recombinant fibrillin-1 fragment containing an EBP interaction sequence can elicit chemotaxis of macrophages by interaction with EBP. In a clinical study of aortic specimens from 28 persons with MFS, we demonstrate a significant increase in CD68⁺ macrophage counts in the tunica media compared with control subjects. Although this study was not designed to investigate the role of elastin or fibrillin-1 fragments in human MFS, our clinical and experimental findings support the notion that inflammation induced by elastin or fibrillin-1 fragments could play some role in the pathogenesis of MFS. This raises the possibility of novel therapeutic options, such as matrix metalloproteinase inhibitors to block fibrillin/elastin degradation or antiinflammatory agents to inhibit invasion of macrophages into aortic tissue.

This article has been cited by other articles:

				F. Ramirez and H. C. Dietz Extracellular Microfibrils in Vertebrate Development and Disease Processes J. Biol. Chem., May 29, 2009; 284(22): 14677 - 14681. [Full Text] [PDF]

				C. K. Wieslander, D. D. Rahn, D. D. McIntire, J. F. Acevedo, P. G. Drewes, H. Yanagisawa, and R. A. Word Quantification of Pelvic Organ Prolapse in Mice: Vaginal Protease Activity Precedes Increased MOPQ Scores in Fibulin 5 Knockout Mice Biol Reprod, March 1, 2009; 80(3): 407 - 414. [Abstract] [Full Text] [PDF]

				Z. Touat, L. Lepage, V. Ollivier, P. Nataf, U. Hvass, J. Labreuche, M. Jandrot-Perrus, J.-B. Michel, and G. Jondeau Dilation-Dependent Activation of Platelets and Prothrombin in Human Thoracic Ascending Aortic Aneurysm Arterioscler. Thromb. Vasc. Biol., May 1, 2008; 28(5): 940 - 946. [Abstract] [Full Text] [PDF]

				P. Matt, J. Habashi, T. Carrel, D. E. Cameron, J. E. Van Eyk, and H. C. Dietz Recent advances in understanding Marfan syndrome: should we now treat surgical patients with losartan? J. Thorac. Cardiovasc. Surg., February 1, 2008; 135(2): 389 - 394. [Abstract] [Full Text] [PDF]

				E. Spiekerkoetter, C. M. Alvira, Y.-M. Kim, A. Bruneau, K. L. Pricola, L. Wang, N. Ambartsumian, and M. Rabinovitch Reactivation of {gamma}HV68 induces neointimal lesions in pulmonary arteries of S100A4/Mts1-overexpressing mice in association with degradation of elastin Am J Physiol Lung Cell Mol Physiol, February 1, 2008; 294(2): L276 - L289. [Abstract] [Full Text] [PDF]

				A. W.Y. Chung, K. Au Yeung, G. G.S. Sandor, D. P. Judge, H. C. Dietz, and C. van Breemen Loss of Elastic Fiber Integrity and Reduction of Vascular Smooth Muscle Contraction Resulting From the Upregulated Activities of Matrix Metalloproteinase-2 and -9 in the Thoracic Aortic Aneurysm in Marfan Syndrome Circ. Res., August 31, 2007; 101(5): 512 - 522. [Abstract] [Full Text] [PDF]

				Y. von Kodolitsch and P. N Robinson Marfan syndrome: an update of genetics, medical and surgical management Heart, June 1, 2007; 93(6): 755 - 760. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 114/17/1855    most recent CIRCULATIONAHA.105.601674v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guo, G. Articles by Robinson, P. N. Search for Related Content PubMed PubMed Citation Articles by Guo, G. Articles by Robinson, P. N. Related Collections Animal models of human disease Genetics of cardiovascular disease Other Vascular biology