Journal of Vascular Surgery
Volume 48, Issue 5 , Pages 1278-1285, November 2008

Metalloproteinase expression in venous aneurysms

Division of Vascular Surgery, Department of Surgery, University of Texas Medical Branch, Galveston, Tex

Received 9 January 2008; accepted 22 June 2008.

Article Outline

Introduction

Although recognized with increasing frequency, the pathogenesis of venous aneurysms (VA) remains poorly understood. We evaluated 8 patients with 10 VA for the presence, localization and activity of metalloproteinases (MMPs).

Methods

Tissue specimens from VA (n=8), normal saphenous vein (NSV n=7) and varicose veins (VV n=7 were compared by histology and immunohistochemistry (IHC). Histologic sections were stained with H&E, Movats pentachrome and toluidine blue, and IHC specimens with antibodies to CD68, MMP2, MMP9, and MMP13. Protein expression and enzyme activity were determined by Western immunoblotting and zymography.

Results

Three of 4 patients with popliteal VA presented with edema and leg pain and the remaining patient with deep venous thrombosis (DVT) and pulmonary embolism (PE). The 5 popliteal VA were treated by; excision and reanastomosis (n=2) lateral venorrhaphy (n=2) and spiral saphenous vein graft (n=1). The 3 patients with 4 upper extremity VA had discomfort over a compressible mass. Two of the VA were excised and the remaining patients aneurysm ruptured spontaneously. The mesenteric VA, an incidental finding at laparotomy was excised. Thrombus was present in 2 popliteal, 1 upper extremity and in the mesenteric aneurysm. Histologically, VA and VV were characterized by fragmentation of the elastic lamellae, loss of smooth muscle cells (SMCs) and attenuation of the venous wall when compared to NSV. Varicose veins and VA also demonstrated increased expression of MMP-2, MMP-9 and MMP-13 in endothelial cells (ECs), SMCs and adventitial microvessels compared to NSV. Both pro-MMP-2 and pro-MMP-9 were detected by zymography in VA,VV and NSV but only MMP-2 activity was demonstrable.

Conclusions

The structural changes in the venous wall in addition to the increased expression of MMP-2, MMP-9 and MMP-13 in VA compared to NSV and VV suggests a possible causal role for these MMPs in their pathogenesis.

 

Venous aneurysms (VAs) are uncommon focal saccular or fusiform dilations that communicate with the accompanying normal vein by a single channel. The incidence of VAs is unknown. VAs can occur at any age and in either sex with equal frequency. There is usually no history of antecedent trauma, association with an arteriovenous communication or pseudoaneurysm, and the lesion should not be contained within a segment of varicose vein (VV).1, 2

VAs are classified according to their location and have been reported in association with the major veins of the neck, thorax, and abdomen as well as the deep and superficial veins of the upper or lower extremities.3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 The natural history and clinical significance of VAs is related to their location and size. Neck and upper extremity VAs are usually asymptomatic and are most frequently treated because of their size and cosmetic appearance. Thoracic and abdominal VAs are usually asymptomatic and are detected incidentally on computed tomography (CT) or magnetic resonance (MRI) imaging scans.6, 10, 11 In contrast to thoracic VAs, abdominal VAs are more liable to complications such as gastrointestinal hemorrhage and thromboembolism and are treated when discovered.6 Popliteal VAs, the most common of the extremity deep VAs, are usually found during the evaluation of patients with thromboembolic disease or chronic venous insufficiency.1, 5, 16, 18

Although VAs and their potential for complications are well recognized, their pathogenesis remains poorly understood. In this study, we analyzed venous tissue from eight patients with 10 VAs to determine the possible role of matrix metalloproteinases (MMPs) in the pathogenesis of these lesions.

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Materials and methods 

Tissue samples 

Patient demographic data were obtained from review of their medical records and imaging studies. Venous tissue from seven patients with VVs, eight with VAs (5 popliteal, 2 of 4 extremity, 1 mesenteric), and seven with normal saphenous veins (NSVs), used as reference tissue, was analyzed. Each specimen was divided into three segments: one was snap frozen in liquid nitrogen for protein analysis, another was placed in RNALater (Applied Biosystems/Ambion, Austin, Tex) for RNA extraction, and the remaining portion was preserved in Streck Tissue Fixative (STF; Streck Laboratories, Omaha, Neb) or in 10% phosphate-buffered formalin for histology and immunohistochemistry (IHC). Samples were collected in accordance with the requirements of the Institutional Review Board at the University of Texas Medical Branch, Galveston, Texas.

Histologic analysis and IHC 

Paraffin-embedded venous tissue (5-μm) sections from eight VAs were compared by histology and IHC with seven NSVs and seven VVs. Specimens were stained with hematoxylin and eosin and Movat's pentachrome, and selected specimens with toluidine blue. For IHC, sections were stained with specific antibodies to factor V111 antigen, CD-68, and MMP-2, -9, and -13. In brief, specimens were cut and dried overnight in a 60°C oven. Slides were deparaffinized, rehydrated, and processed for IHC on an automated Optimax immunostainer (BioGenex, San Ramon, Calif). Antigen retrieval was performed with BioGenex Antigen Retrieval Citra Plus (pH 6.0) for 10 minutes at 100°C.

The slides were rinsed in distilled water, treated for 5 minutes with 0.3% hydrogen peroxide in 100% methanol to remove endogenous peroxidase activity, and then predigested in 0.0025% protease in phosphate-buffered saline (pH 7.6) for 30 minutes at room temperature. The slides were counterstained with hematoxylin. Endothelial cells were identified with staining for factor VIII antigen and macrophages with CD-68 antibodies (Dako Corp, Carpenteria, Calif). Sections were also stained with antibodies to MMP-2, -9, and -13. Human placenta and pancreatic adenocarcinoma were used as positive controls. The primary antibodies were omitted for negative control specimens. Two to three separate sections from each specimen were examined by two blinded observers.

Western blot analysis 

Antibodies for MMP-2, MMP-9, tissue inhibitor of metalloproteinase-2 (TIMP-2; Oncogene Research Products, Cambridge, Mass), and β-actin (Sigma, St. Louis, Mo) were used for Western immunoblotting. Briefly, tissue for NSVs, VVs, and two VAs were homogenized on ice for 1 minute in radioimmunoprecipitation assay (RIPA) lysis buffer (50 nM Tris-hydrogen chloride, pH 7.5; 150 mM sodium chloride, 1% [wt/vol] sodium deoxycholate, 0.1% sodium dodecyl sulfate, 100 mM sodium orthovanadate σ, 1% [vol/vol] Triton X-100 [Fisher Chemicals, Pittsburgh, Pa]), and complete Mini-EDTA (ethylenediaminetetraacetic acid)-free protease inhibitors (Roche Molecular Biochemicals, Dorval, Quebec, Canada). Tissue homogenates were centrifuged (4°C at 8000 g) for 20 minutes, and supernatants were collected. Polyacrylamide gels were prepared (stacking gel, 4%; separating gel, 10%), and the proteins (50 μg/well) were separated by polyacrylamide gel electrophoresis and then transferred electrophoretically to a 0.45-μm-pore polyvinylidene difluoride membrane.

The blots were washed with Tris-buffered saline with Tween-20 (TBS-T; 150 mM sodium chloride; 20 mM Tris-hydrogen chloride, and 0.1% Tween-20 σ; pH 7.5) and incubated overnight with blocking solution (5% skim milk powder in TBS-T). The blots were incubated with the appropriate primary antibodies at 1:1000 dilution for 1 hour, rinsed three times for 5 minutes each with TBS-T, and incubated with secondary rabbit antiserum conjugated with horseradish peroxidase (1:2000 dilution in blocking solution; 1% milk) for 1 hour.

After incubation with the secondary antibody, the blots were washed three times, 5 minutes each, and the antibody-antigen complex was detected using the ECL Plus detection system (Amersham Biosciences, Piscataway, NJ). The membranes were then exposed to X-OMAT blue film (Kodak Scientific Imaging Products, Rochester, NY), and the intensities of immunoreactive bands were measured by scanning (6200C scanner, Hewlett Packard Canada Ltd, Mississauga, Ontario, Canada). The images were analyzed on a desktop computer using Kodak 1D Image Analysis .3.6 software (Eastman Kodak Co, Rochester, NY). The mean pixel density for each band was analyzed to obtain relative OD units for specific proteins. To standardize for sample loading, the relative OD units for each band were standardized to the relative OD units of the total β-actin.

RNA isolation 

Venous tissue was preserved in RNALater and total RNA extracted with RNAzol using the Ultraspec RNA Isolation System according to the manufacturer's protocol (Biotex, Houston, Tex). The quality of total RNA was controlled by agarose gel electrophoresis as demonstrated by the presence of intact ribosomal RNA (28S and 18S).

One-step real-time reverse transcription-polymerase chain reaction procedure, primers, and probes 

We used Applied Biosystems assays-on-demand 20× assay mix of primers and TaqMan MGB probes (FAM dye-labeled) for our target gene and predeveloped 18S rRNA (VIC-dye labeled probe) TaqMan assay reagent (P/N 4319413E) for endogenous control.

Relative quantitation of gene expression 

Separate tubes (singleplex) one-step reverse transcription-polymerase chain reaction (RT-PCR) was performed with 40 ng RNA for both target genes and endogenous control. The reagent we used was TaqMan one-step RT-PCR master mix reagent kit (P/N 4309169). The cycling parameters for one-step RT-PCR was: reverse transcription 48°C for 30 minutes, AmpliTaq activation 95°C for 10 minutes, denaturation 95°C for 15 seconds, and annealing/extension 60°C for 1 minute (repeat 40 times) on the ABI7000 (Applied Biosystems). Duplicate CT values were analyzed in Excel software (Microsoft Corp, Redmond, Wash) using the comparative CT (ΔΔCT) method as described by the manufacturer (Applied Biosystems). The amount of target (2ΔΔCT) was obtained by normalizing to an endogenous reference (18s) and relative to a calibrator (one of the experimental samples).

Zymography 

Protein was mixed (1:1) with Novex tris-glycerine-sodium dodecylsulfate buffer (Invitrogen, Carlsbad, Calif) and incubated at room temperature for 10 minutes. The samples were loaded into a Novex 10% Zymogram gelatin gel (Invitrogen) and electrophoresis was initiated. The gel was stained with Coomassie blue R-250 for 30 minutes and then destained with Coomassie R250 destaining solution.

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Results 

Patient demographics are summarized in the Table. VAs were detected in seven men and one woman. Popliteal VAs (n = 5) were the most common aneurysms seen in this study. The diagnosis was made by ultrasonography, MRI, or phlebography (Fig 1, Fig 2). In two patients, there was sufficient redundancy to allow excision of the VA and end–end anastomosis. The patient with bilateral popliteal VAs, who presented with DVT and pulmonary embolism, had excision of both VAs and repair by lateral venorrhaphy. Thrombus in a popliteal VA and the completed lateral repair is shown in Fig 3. The remaining patient had excision of the VA, and the defect was reconstituted with a spiral saphenous vein graft. The graft subsequently occluded despite the patient being fully anticoagulated with warfarin sodium. The remaining four popliteal VA repairs remained patent by duplex ultrasound scanning between 9 and 84 months after their surgery. The patient with the occluded vein graft repair has minimal pedal edema controlled with support hose.

Table. Patient demographics
AgeSexSiteMedical historySymptomsSize, cmIntervention
56FPoplitealRight lower extremity DVT, pulmonary hypertensionRight leg pain2×2Excision, reanastomosis
58MPoplitealNoneLeft knee pain4×3Spiral vein graft
46MBilateralPulmonary embolismDyspnea syncope4×4Bilateral
Excision
PoplitealSameSame4×2Venorrhaphy
71MPoplitealCAD, ETOH abuseLeft leg pain1.7×2.5Excision, reanastomosis
26MForearmNoneRight arm pain2×2Ligation/excision
32MHandNoneRight hand pain2.5×2.5Ligation/excision
82MBilateral forearmHypertensionMass2×2Spontaneous rupture
65MMesentericCirrhosisIncidental6×4Ligation/excision

CAD, Coronary artery disease; DVT, deep vein thrombosis; ETOH, ethyl alcohol.

Two of four extremity VAs were excised without recurrence in the follow-up period (Fig 4). The most recent patient seen with an upper extremity VA had a similar lesion excised 40 years previously from the left antecubital fossa. The VA on his right forearm ruptured spontaneously while awaiting excision and is presently partially thrombosed. The mesenteric VA was an incidental finding at laparotomy and excised. The patient died during the postoperative period from unrelated causes.

Thrombus was present in two popliteal VAs, one upper extremity VA, and the mesenteric VA. Histologic examination demonstrated significant structural differences between the three types of specimens. The intima of NSVs was characterized by a single layer of endothelial cells (ECs) with occasional foci of intimal thickening and a well-formed medial layer consisting of elastic lamellae, collagen, smooth muscle cell (SMC) bundles, and proteoglycans. The adventitial layer was composed of muscle fibers, collagen, fibroblasts, SMCs, and vaso vasorum. The endothelial layer of the diseased vein segments was discontinuous interspersed with areas of intimal thickening. The walls of both VVs and VAs were thinner than NSVs due to fragmentation and attenuation of the elastic fibers, loss of SMCs, and an increase in collagen content.

In VV specimens, the thinning of the venous was asymmetric and involved part or the circumference of the wall in different venous segments. Occasional CD68-positively staining inflammatory cells were detected by IHC in the specimens examined. Mast cells were demonstrated in all three specimens, with a greater number of these cells in VVs. Medial calcification was noted in the wall of the mesenteric aneurysm. MMP-2, -9, and -13 were expressed in ECs, SMCs, and adventitial microvessels in VAs, and only rarely in NSVs or VVs (Fig 5). MMP-2 and MMP-9 were detected in all three types by Western immunoblotting, but the differences between the types of tissue were variable and not statistically different. TIMPS-2 was also detected in all three types of tissue (Fig 6), with one specimen in each group showing greater expression of the inhibitor than the others. Zymography showed expression of both pro-MMP-2 and pro-MMP-9. However, only MMP-2 activity could be demonstrated (Fig 7). RT-PCR of NSVs, VVs, and VAs, as well as DNA sequencing of one VA, showed no evidence of fibrillin.

  • View full-size image.
  • Fig 5. 

    Histology and immunohistochemistry comparing representative sections of normal vein, varicose vein, and venous aneurysm (original magnification ×200) demonstrating attenuation of the venous wall in varicose vein and venous aneurysm. The expression of matrix metalloproteinase (MMP) -2, -9, and -13 is increased in venous aneurysms compared with normal vein and varicose vein. H&E, Hematoxylin and eosin.

  • View full-size image.
  • Fig 6. 

    Western immunoblot demonstrating variable expression of matrix metalloproteinase (MMP) -2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-2 in varicose vein (VV), normal saphenous vein (NSV), and venous aneurysm (VA). One specimen in each group demonstrated increased expression of TIMP-2.

  • View full-size image.
  • Fig 7. 

    Gelatin zymography showing expression of pro-matrix metalloproteinase (MMP)-2 and pro-MMP-9. Pro-MMP-2 was detected in all specimens with variable activity. NSV, Normal saphenous vein; VA, venous aneurysm; VV, varicose vein.

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Discussion 

The degradation and remodeling of the extracellular matrix by MMPs has been implicated in the pathogenesis of abdominal aortic aneurysms (AAAs) and VVs. Because of the association of MMPs with AAAs (MMP-2, MMP-9) and VVs (MMPs-1, -2, -3, -9, -13), we postulated that MMPs may also play a role in the pathogenesis of VAs.19, 20, 21, 22, 23 In this study, we analyzed and compared tissue samples from patients with VAs, VVs, and NSVs to determine if there were any differences in the expression and localization of MMP-2, -9, and -13. We selected MMP-13 for this analysis because of its wide spread specificity for fibrillar collagens type I, II, III, XI, laminin, fibronectin, tenascin, and gelatin and its ability to activate pro-MMP-9.24

Histologically, several morphologic differences were evident between VAs, VVs, and NSVs. Focal areas of endothelial denudation, fragmentation, and attenuation of the elastic lamellae, loss of SMCs, and medial fibrosis in areas of thrombus adherence were characteristic findings in VA and to a lesser extent in VV specimens. The morphologic changes in both VVs and VAs may only involve part of the circumference of the venous wall. The asymmetric distribution of the disease process explains the feasibility of using lateral venorrhaphy to repair VAs. Fragmentation of elastic tissue is a characteristic feature of both arterial and venous aneurysms. Dobrin and Mrkvicka25 have shown in experimental studies that the dissolution of elastic tissue in arterial segments leads to dilatation of the vessel wall. Whether fragmentation of the elastic tissue lamellae is a precursor to dilatation in the venous wall has not been determined.

The expression of MMP-2, -9, and -13 was localized to SMCs, ECS, and microvascular ECs lining adventitial microvessels in VAs by IHC. These MMPs were only infrequently detected in VVs or NSVs. MMP expression in patients with AAAs has been correlated with the intensity of the inflammatory cell infiltrate.19 An unexpected finding in the specimens we examined was the infrequent occurrence of inflammatory cells. Only occasional positively staining CD-68 cells were detected in the media and adventitia of the three types of venous specimen. Mast cells, another source of proteases, were also observed in these two regions of the vessel wall, but most often in VV specimens.

Although SMCs and ECS are known to express MMPs and other proteases, the significance of MMP expression by the cells and the factors inducing their expression remain uncertain. Recent human and experimental studies suggest that the expression of MMPs by SMCs and ECs may influence the relaxation/constriction properties of the venous wall and contribute to the development of VVs. Also, the loss of endothelial integrity may expose the components of the vessel wall to degradation by MMPs.21, 26 The quantity of MMPs produced by SMCs and ECs, compared with that produced by macrophages—the predominant source of MMPs—appears to be insufficient to account for the morphologic changes in the venous wall. The infrequent detection of positively staining CD68 cells in our study contrasts with the observations of Sayer and Smith,27 who found macrophages and mast cells widely distributed throughout all the layers of the venous wall. Possible explanations for the differences between these two studies may relate to regional differences in the intensity of the inflammatory response and MMP expression, specimen sampling bias, or the stage of the disease. The absence of regional differences in mast cell tryptase or chymase messenger RNA or protein expression suggests that these cells only play a limited role in the matrix changes seen in VVs.23

The expression of MMP-2 and MMP-9 by Western immunoblotting was quite variable and did not correlate with the IHC findings. Similarly TIMP-2 was detected in all three types of tissue, but the levels of expression did not correlate with that of MMP-2. The lack of correlation between the IHC findings and Western immunoblotting may relate to the asymmetric distribution of the vessel wall changes, the levels of protein expression, or differences in the epitopes used. Although both pro-MMP-2 and pro-MMP-9 were present, only MMP-2 activity was demonstrable by zymography.

Increased tissue expression and plasma levels of MMPs 1, -2, -3, -9, and -13 has been demonstrated in patients with VVs, and MMPs-2, -9, and -13 in tissue of patients with VAs in the present study. The relevance of these findings to the pathogenesis of VAs is presently unknown. Whether the presence of these proteases is merely a manifestation of ongoing inflammation or contributes to the venous dilatation and other structural changes characteristic of VVs and VAs remains to be determined.23, 25, 27

Because fibrillin-rich microfibrils are essential to the formation and integrity of elastin and fragmentation of the elastic lamellae as a prominent feature of VVs and VAs, we evaluated all three specimen types with RT-PCR but were unable to demonstrate any fibrillin in the tissues examined.28

An interesting observation is the absence of reports in the literature of the concurrent detection of arterial and venous aneurysms in the same individual, which suggests that the local and systemic factors mediating aneurysmal degeneration in arterial and venous tissue may be different.

This study has several limitations. Evaluation of human VAs represents end-stage disease and may not reflect the factors that initiate or contribute to the pathogenesis of these lesions. The amount of tissue available for analysis remains a problem. Because of their rarity, it is unlikely that prospective studies of VAs will be undertaken. A multicenter registry and prospective collection of tissue samples may provide some of the answers. Ultimately, what is lacking is the availability of animal models of venous disease.

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Conclusion 

In this preliminary study we have demonstrated increased expression of MMP-2, -9, and -13 in VAs compared with NSVs and VVs. This observation in conjunction with the morphologic changes in the extracellular matrix of venous aneurysmal lesions suggests a possible causal role for MMPs in their pathogenesis.

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Author contributions 


Conception and design: AS, GH

Analysis and interpretation: AS, GH, LK

Data collection: CI, MG, QZ

Writing the article: CI, MG, GH

Critical revision of the article: AS, LK, GH

Final approval of the article: GH

Statistical analysis: Not applicable

Obtained funding: Not applicable

Overall responsibility: GH

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 Competition of interest: none.

PII: S0741-5214(08)01094-X

doi:10.1016/j.jvs.2008.06.056

Journal of Vascular Surgery
Volume 48, Issue 5 , Pages 1278-1285, November 2008

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