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Paracrine secretion of transforming growth factor-β1 in aneurysm healing and stabilization with endovascular smooth muscle cell therapy1
Paracrine secretion of transforming growth factor1 in aneurysm healing and stabilization with endovascular smooth muscle cell therapy Franck Losy, MD,a,c Jianping Dai, MD,aCarine Pages,a Maryvonne Ginat,a Be´atrice Muscatelli-Groux, PhD,a Anne-Marie Guinault,a Elodie Rousselle,a Gianluca Smedile,b Daniel Loisance, MD,a Jean-Pierre Becquemin, MD,a,c and Eric Allaire, MD, PhD,a,b Cre´teil and Paris, France Background: Identification of molecular factors involved in artery wall stabilization after extracellular matrix injury elicited by inflammation and proteolysis has a major role in the development of new therapies for atherosclerosis. A study from our group demonstrated that endovascular seeding of vascular smooth muscle cells (VSMCs) promotes healing and stabilizes experimental aneurysms by downregulating matrix metalloproteinase and upregulating tissue inhibitor of metalloproteinase and collagen gene expression. We analyzed expression of transforming growth factor– beta (TGF-) and its receptors in experimental aneurysms treated with endovascular VSMC therapy. Methods and results: Aneurysms were generated in Fischer 344 rats by 14-day orthotopic implantation of a segment of guinea pig abdominal aorta (xenograft). During an endovascular repeat operation, syngeneic VSMCs were seeded in the aneurysm, always resulting in aneurysm diameter stabilization after 8 weeks, whereas diameter of control aneurysms infused with culture medium further increased. Seven days after repeat operation the intima or thrombus was separated from the aneurysmal wall in the two groups. Reverse transcriptase polymerase chain reaction with the domestic gene 18s as a standard demonstrated that aneurysm stabilization was associated with a statistically significant increase in TGF-1, but not TGF-2 or TGF-3, messenger RNA levels in the intima. Enzyme-linked immunosorbent assay demonstrated increased TGF-1 protein in the aneurysmal wall. mRNA levels of the two serine and threonine kinase TGF- receptors remained unchanged. Conclusions: Healing and stabilization of aneurysms with endovascular cell therapy is associated with a specific pattern of gene expression, resulting in paracrine secretion of TGF-1. Our study provides insight into the molecular mechanisms of arterial aneurysm healing and stabilization. (J Vasc Surg 2003;37:1301-9.)
Once extracellular matrix injury has started, ongoing aneurysmal growth can be interpreted as the consequence of healing failure. In a recent study,1 our group demonstrated that aneurysmal xenograft healing and stabilization can be attained with endovascular seeding of vascular smooth muscle cells (VSMCs). The microscopic appearance of stabilized xenografts suggested to us that the seeded VSMCs trigger a healing process that interrupts aneurysmal wall destruction by inflammation and proteolysis and promotes wall reconstruction.1 Most of the biologic changes induced by endovascular VSMC seeding were observed in the aneurysmal wall itself, whereas the seeded VSMCs remained localized in the intima. Inward From Centre National de la Recherche Scientifique (CNRS) Unite´ Mixte de Recherche (UMR) 7054, Centre de Recherches Chirurgicales, Universite´ Paris XII, UFR de Me´decine, Hoˆpital Henri Mondor,a Service de Chirurgie Thoracique et Vasculaire, Hoˆpital Tenon,b and Service de Chirurgie Vasculaire et Endocrinienne, Hoˆpital Henri Mondor.c Supported by CNRS, La Fondation de l’Avenir pour la Recherche Me´dicale Applique´e (grant ET9-210) and La Fondation de France. Dr Losy was supported by a grant from Assistance Publique–Hoˆpitaux de Paris (APHP), Paris, France. Competition of interest: none. Reprint requests: Eric Allaire, Centre de Recherches Chirurgicales, 8 rue du Ge´ne´ral Sarrail, 94010 Cre´teil, France (e-mail: [email protected]). Copyright © 2003 by The Society for Vascular Surgery and The American Association for Vascular Surgery. 0741-5214/2003/$30.00 ⫹ 0 doi:10.1016/S0741-5214(02)75336-6
localization of seeded cells and outward localization of changes in gene transcription support the view that seeded cells secrete paracrine factors that diffuse centrifugally to reach the aneurysm wall and control inflammation, proteolysis, and wall reconstruction. Transforming growth factor– beta (TGF-) belongs to a family of cytokines with multiple roles in tissue development and healing because they modulate inflammation, proteolysis, extracellular matrix synthesis, cell proliferation, cell survival, and cell migration.2 TGF- activity is regulated by protein concentration, activation by proteolytic cleavage,3 and expression of receptors, particularly the type I and type II serine and threonine protein kinase receptors, which mediate signal transduction to cell nuclei.4 TGF-1 is expressed by VSMCs in the normal artery wall,5 and its production is increased during artery wall repair after mechanical injury.6 Among three identified isoforms, TGF-1 and TGF-2 are involved in healing of cutaneous wounds.7 In abdominal aortic aneurysm in human beings, TGF-1 may be involved in diameter control through production of cystatin C, an inhibitor of cysteine proteases such as elastase cathepsins S and K.8 In the present work we analyzed the expression of the three isoforms of TGF- and its two serine and threonine receptors in experimental aneurysms. To address the role of TGF- in aneurysm growth, we designed a model of already developed aneurysms in rats, in which aneurysm 1301
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Fig 1. Aneurysm healing and stabilization with endovascular VSMC therapy. A, Schema of endovascular infusion in experimental aneurysms attained with 14-day implantation of an aortic xenograft. B, Macroscopic results show stabilization of diameter of aneurysms after VSMC endovascular seeding. C, VSMC staining with anti-␣-actin antibody on cryostatic cross-sections of control (top) and VSMC-seeded (bottom) aneurysms 7 days after endovascular repeat operation. Dashed white line shows separation of tissues during microdissection, enabling separate analysis with RT-PCR and ELISA. Black bar shows media and adventitia thickness. White bar shows intima composed mainly of VSMCs in VSMC-seeded vessels. Th, Thrombus. D, Microscopic appearance of aneurysms 8 weeks after endovascular repeat operation shows a dense collagen network in VSMC-seeded aneurysms (bottom) in comparison with control aneurysms (top). Original magnification ⫻20. *P ⬍ .05; **P ⬍ .01.
diameter stability was attained with endovascular seeding of VSMCs. Our results show that healing and stabilization of already developed aneurysms is associated with a transient increase in TGF-1 expression. We report the first identification of a molecule potentially involved in functional healing of already developed experimental aneurysms. MATERIAL AND METHODS Animals and surgery. Animals housed and cared for according to European Union Standards received analgesia and were anesthetized with 5 mg/100 g body weight of pentobarbital administered intraperitoneally. Hartley guinea pig (Iffa Credo, Lyon, France) abdominal aortas were decellularized with sodium dodecylsulfate, as described.9 The tube of arterial extracellular matrix obtained with this method was grafted orthotopically into male Fischer 344 rats (n ⫽ 50; Iffa Credo) during a first surgical procedure. Syngeneic male Fischer 344 rat VSMCs were isolated from thoracic aortas10 and were grown in RPMI 1640 and medium 199, with L-glutamine and 10% fetal calf serum (FCS; Gibco-BRL, Grand Island, NY). More than 98% of isolated cells grown to 80% confluence were stained positively with an anti-␣-actin antibody (Dakopatt, Copenhagen, Denmark).
A second surgical procedure was performed 14 days later, at which time the xenograft diameter had increased more than 50% to comply with aneurysm definition.11 Graft diameter was measured with a grid in the microscope eyepiece immediately after transplantation at repeat operation and before euthanasia at day 7 or week 8 after seeding.12 The aneurysmal xenograft was isolated from blood flow with clamps after gentle dissection. Its lumen was gently rinsed with culture medium through a PE10 catheter introduced at aortotomy performed downstream in the native aorta (Fig 1, A). Passage five to eight VSMCs were resuspended in culture medium with 5% FCS injected into the aneurysmal xenograft lumen (107 cells per xenograft) and allowed to attach by sedimentation during four 90degree rotations (sedimentation duration 2 minutes for each position) for 8 minutes. Previous studies with PKH26 fluorescent dye staining in vivo assessed VSMC intimal location after seeding (data not shown). As control, aneurysmal xenografts were infused with culture medium with 5% FCS without cells. Diameter increase was calculated as: (Diameter at harvest ⫺ Diameter at seeding)/Diameter at seeding. Analysis of mRNA content. TGF-1, TGF-2, TGF3, and TR I and TR II messenger RNA levels were analyzed with reverse transcriptase polymerase chain reac-
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tion (RT-PCR), and compared to the RNA level of domestic gene 18s (Quantum RNA 18s Internal Standards kit; Ambion, Montrouge, France). Intima or thrombus, and media plus adventitia (Fig 1, C) were separated with microdissection and pooled by layers and groups. Total RNA was extracted with TRIzol (Life Technologies, Rockville, Md) and treated with grade I deoxyribonuclease (DNAse; Roche Molecular Biomedicals). RNA was extracted according the same protocol from VSMCs in culture and from normal Fischer 344 rat aorta. RT was performed with random primers, and Superscript II (Life Technologies), deoxyribonucleoside triphosphate (dNTP), dithiothreitol, and ribonuclease inhibitor (Roche Molecular Biochemicals). PCR was performed in a PCR Express thermocycler (Hy baid, Ashford, UK) in the same tube for both the gene of interest (primers: matrix metalloproteinase (MMP)–2; TGF-1, 5'-CG GACTAC TACGCCAAAGAA-3'/5'TCAAAAGACAGCCACTCAGG-3'; TGF-2, 5' CCG CCC ACT TTC TAC AGA CCC 3'/5' GCG CTG GGT GGG AGA TGT TAA 3'; TGF-3, 5' TGC CCA ACC CGA GCT CTA AGC G 3'/ 5' CCT TTG AAT TTG ATC TCC A 3'; TR I, 5'-ACGTTCATGGTTCCGAGAGG-3'/ 5'-TCG CAAAGCTGTCAGCCTAG-3'; TR II, 5'-AAGT CTTGCATGAGCAACTGC-3'/5'-GATGTCAGAGAAGA TGTCC3') (Genset Oligos SA, Paris, France) and 18s. Final PCR conditions were chosen to prevent interference between the two sets of primers. The PCR mix contained Taq polymerase (EurobioTaq; Eurobio, Les Ulis, France) in buffer, dNTP, and magnesium chloride. Negative control assays were performed without Superscript II. Ten microliters of PCR products were run in 2% agarose gel with 5 g/mL of ethidium bromide, visualized under untraviolet light with a video camera. Bands of amplified sequences corresponding to the gene of interest and to 18s were quantified with Gel Analyst (Iconix). Results were expressed as a ratio between signals corresponding to the gene of interest and 18s. ELISA for TGF-1. The sandwich enzyme-linked immunosorbent assay (ELISA; Promega, Madison, Wis) recognizes active, not latent, TGF-1. Quantification was performed according to manufacturer instructions, either after Triton extraction for active form quantification or after acid and ethanol extraction and activation for total TGF-1 quantification.13 Histologic analysis and immunohistochemistry. Cryostat cross sections (5 m) were made from the center of the aneurysmal xenografts. Collagen was evidenced with sirius red staining. Primary antibody for ␣-actin was a mouse monoclonal antibody (Dakopatt), with an alkaline phosphatase–anti-alkaline phosphatase technique (Dakopatt). The anti-TGF-1 polyclonal antibody raised in rabbits was a generous gift from Dr Saez.14 The secondary antibody was a goat anti-rabbit antibody with 3,3'-diaminobenzidine tetrahydrochloride. Control sections were generated by omission of the primary antibody. Statistical analysis. Results are expressed as mean ⫾ SD. Comparisons between two groups were done with the nonparametric Mann-Whitney U test (Statview, version 4.5).
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RESULTS Aneurysm diameter and microscopic appearance VSMC seeding consistently resulted in stabilization of diameter of aneurysmal xenografts.1 Conversely, infusion of culture medium was always followed by a statistically significant increase in aneurysm diameter after 2 months (Fig 1, B). Immunohistochemistry 7 days after endovascular VSMC seeding disclosed a thick intima in aneurysmal xenografts, composed mainly of ␣-actin–positive cells, whereas thrombus covered the luminal aspect of control aneurysmal xenografts (Fig 1, C). In VSMC-seeded aneurysms a dense network of collagen developed in the intima and adventitia (Fig 1, D). Expression of TGF- and its receptors in VSMCs in culture and in experimental aneurysms For all molecules tested, mRNA was detected in cultured cells and in aneurysmal xenografts after 14-day implantation (Fig 2). TGF-2 and TGF-3 were upregulated in aneurysmal xenografts before endovascular infusion. Conversely, TGF-1 mRNA levels remained low in the intima and in the media and adventitia (ie, in the aneurysmal wall itself) but were high in VSMCs in culture. Comparison of TGF-1 mRNA level between native aorta and VSMCs in culture demonstrated that the in vitro phase of VSMCs culture doubled TGF-1 mRNA content. TGF- expression after VSMC therapy TGF- isoform mRNA content. mRNA content encoding for the three isoforms of TGF- was measured 7 days after endovascular reoperation. In the intima or thrombus, TGF-1 mRNA level was higher in the seeded vessels, whereas TGF-2 and TGF-3 mRNA level was similar in the two groups (Table I). In the media plus adventitia, TGF-1 mRNA was increased in the seeded vessels in comparison with control nonseeded vessels, without reaching a statistically significant difference. TGF-2 and TGF-3 mRNA content was not modulated with VSMC seeding (Table I). Kinetics of TGF- mRNA content variations after endovascular VSMC seeding. In aortas harvested 3 and 7 days after endovascular repeat operation, the intima or thrombus and the media plus adventitia were easily separated with microdissection, enabling separate mRNA extraction of the two layers in the two groups. In VSMCseeded aortas harvested at 8 weeks the intima could not be separated from the media plus adventitia. Therefore mRNA analysis at this time was performed from the whole aortic wall in the two groups. As observed at day 7, TGF-1 mRNA content at day 3 was increased in the intima but not in the media plus adventitia after endovascular VSMC seeding. At 8 weeks TGF-1 mRNA content was similar in the two groups (Table II). TGF-2 and TGF-3 mRNA content was similar in the two groups 8 weeks after seeding (data not shown).
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Fig 2. mRNA content in comparison to 18s of TGF- and receptors. Culture of VSMCs strongly induces members of TGF- in comparison with normal rat aorta. TGF-1 mRNA levels are low in 14-day experimental aneurysms. mRNA levels of constitutive MMP-2 are shown for comparison. Data represent results of RT-PCR performed on pools of three experimental aneurysms.
Table I. TGF- isoforms 1, 2, and 3 mRNA content in aneurysmal xenografts 7 days after endovascular repeat operation (TGF-/18s ratio) TGF-1 Intima VSMC ⫺ 0.73 ⫾ 0.10 VSMC ⫹ 1.40 ⫾ 0.36 P .049 Media ⫹ adventitia VSMC ⫺ 0.89 ⫾ 0.04 VSMC ⫹ 1.62 ⫾ 0.88 P .083
TGF-2
TGF-3
0.52 ⫾ 0.58 0.67 ⫾ 0.23 .827
0.74 ⫾ 0.39 0.69 ⫾ 0.08 .513
0.10 ⫾ 0.04 0.11 ⫾ 0.10 .999
0.25 ⫾ 0.25 0.16 ⫾ 0.04 .563
Data represent measurements from three pools of three grafts. VSMC, Vascular smooth muscle cell; ⫺, negative; ⫹, positive.
normal rat aorta the percentage of active over total TGF-1 was low (Fig 3, B). In aneurysmal xenografts just before endovascular repeat operation this percentage was dramatically increased. After endovascular repeat operation and endoluminal infusion, the percentage of active TGF-1 fell to low levels in the intima or thrombus but remained high in the media and adventitia (Fig 3, C). Immunohistochemistry. Immunohistochemistry with anti-TGF-1 antibody evidenced few positive cells in the luminal thrombus and in the medial and adventitial layers 7 days after endoluminal injection of culture medium in the control group. Conversely in the aneurysms 7 days after endoluminal seeding of VSMCs, numerous cells in the intima were strongly positive for anti-TGF-1 staining (Fig 4). Omission of the primary antibody suppressed cell and extracellular matrix staining (data not shown).
TGF-1 protein expression after VSMC therapy
TGF- receptor expression after VSMC therapy
Protein quantification at ELISA. ELISA performed after acid and ethanol extraction was used to measure total, ie, active and latent, TGF-1. Concordant with mRNA levels, total TGF-1 protein was increased in the intima of aneurysmal xenografts 7 days after endovascular seeding, compared with TGF-1 protein in the thrombus of control xenografts 7 days after repeat operation (45.4 ⫾ 9.7 ng/mg in control xenografts, 144.3 ⫾ 30.6 ng/mg in seeded aneurysmal xenografts; P ⬍ .05). In the media plus adventitia, TGF-1 protein was also increased after endovascular VSMC seeding (124.6 ⫾ 5.8 vs 162.9 ⫾ 12.2 ng/mg in control and seeded aneurysmal xenografts, respectively; P ⬍ .05) (Fig 3, A). ELISA performed after extraction with Triton was used to measure active TGF-1. In VSMCs in culture and in
RT-PCR did not evidence significant differences in TR I and TR II mRNA ratio to 18s mRNA levels 7 days after endovascular repeat operation (Intima: TR I, 0.51 ⫾ 0.51 vs 0.86 ⫾ 0.37 [P ⫽ .123, Mann-Whitney U test]); TR II, 0.39 ⫾ 0.25 vs 0.76 ⫾ 0.35 [P ⫽ .275, in aneurysms with no VSMC vs aneurysms with VSMC seeding]. Media plus adventitia: TR I, 0.92 ⫾ 0.56 vs 1.47 ⫾ 1.45 [P ⫽ .827]; TR II, 0.52 ⫾ 0.36 vs 0.73 ⫾ 0.64 [P ⫽ .827, in aneurysms with no VSMC vs aneurysms with VSMC seeding]). DISCUSSION Identification of molecular factors promoting artery wall stabilization after extracellular matrix injury elicited with inflammation and proteolysis is a major issue for new therapies for atherosclerosis.15-19 In vivo manipulations of
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Fig 3. TGF-1 protein content measured with ELISA. A, Seven days after endovascular repeat operation, VSMC seeding is followed by a significant increase in total (latent plus activated) TGF-1, both in the intima and the aneurysm wall. B, Whereas TGF-1 is mostly in its nonactivated form in cultured VSMCs and normal rat aorta, activated TGF-1 is the predominant form of TGF-1 in the inflammatory context of aneurysms. C, Seven days after endovascular repeat operation, TGF-1 is mostly in its activated form in the aneurysmal wall in both groups. Values for total TGF-1 are the average of three measurements on three different grafts in each group. Values for active TGF-1 obtained by pooling values for three grafts. *P ⬍ .05, Mann- Whitney U test.
Table II. Kinetics of TGF-1 mRNA content evolution in aneurysmal xenografts after endovascular repeat operation (TGF-1/18s ratio); pools of three grafts.
Intima VSMC ⫺ VSMC ⫹ P Media ⫹ adventitia VSMC ⫺ VSMC ⫹ P
Day 14 ⫹ 3
Day 14 ⫹ 7
Day 14 ⫹ 8 wk
0.61 ⫾ 0.13 1.26 ⫾ 0.43 .049
0.73 ⫾ 0.10 1.40 ⫾ 0.36 .049
VSMC ⫺ 0.84 ⫾ 0.32 VSMC ⫹ 0.96 ⫾ 0.15
0.72 ⫾ 0.26 0.43 ⫾ 0.31 .127
0.89 ⫾ 0.04 1.62 ⫾ 0.88 .083
.999
Data represent measurements from three pools. VSMC, Vascular smooth muscle cell; ⫺, negative; ⫹, positive.
arteries constantly resulting in wall stability or instability enable development of predictive biology because tissue analysis can be performed before major wall remodeling occurs. VSMC endovascular seeding in already formed aneurysms offers a unique opportunity to understand which cellular and molecular mechanisms may trigger aneurysm stabilization and healing. Our data demonstrate a
correlation between TGF-1 and aneurysm stabilization, with TGF-1 expression electively increased in aneurysmal aortic xenografts after VSMC endovascular seeding leading to control of diameter, a decisional clinical end point for aortic aneurysms.20 Most TGF-1 appears to be synthesized in the intimal layer where VSMCs have been seeded, whereas the level of activated protein is increased in the
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Fig 4. TGF-1 immunostaining of aneurysms 7 days after endovascular repeat operation. In the control group (VSMC-), few TGF-1–positive cells (long arrows) are observed, either in luminal thrombus or adventitia. In VSMC-seeded vessels, numerous cells stained with anti-TGF-1 antibody, mainly in the intima. In both groups, patchy areas of extracellular matrix stained with anti-TGF-1 antibody (open arrow). L, Lumen.
entire wall thickness of the VSMC-treated aneurysm. The increase in TGF-1 mRNA is transient, and disappears 2 months after cell therapy. Since TR I and TR II are expressed in aneurysmal xenografts, we provide strong evidence that TGF-1 is a paracrine factor secreted at the initial phase of aneurysmal xenograft stabilization attained with endovascular VSMC therapy. Differences between groups for TGF-1 expression are related to differences in the most endoluminal layer of the wall, ie, thrombus versus intimal accumulation of VSMCs. In our study, TFG-1 mRNA levels exceeded levels of the two other isoforms in VSMCs in vitro before seeding and in aneurysmal xenografts in vivo after VSMC seeding. Moreover, among the three isoforms of TGF-, only TGF-1 mRNA levels were correlated with aneurysmal xenograft stabilization with endovascular VSMC therapy. In atherosclerotic lesions in human beings, Bobik et al5 observed a correlation between atherosclerosis severity and expression of TGF-1 but not TGF-3. Whereas TGF-1 gene polymorphism is associated with severity of coronary disease after heart transplantation,21 the polymorphism of TGF-2 does not influence early ischemic heart disease.22 Involvement of TGF-1 in structural changes in arteries in adults is further suggested by the efficiency of TGF-1
overexpression in changing normal wall structure23,24 and is now evidenced in the setting of experimental aneurysm expansion and healing. Most TGF-1 isolated in aneurysmal xenografts before and after endovascular repeat operation was the active form. Our data also show that in the normal aortic wall and in cultured cells, TGF-1 mostly is in its inactive proform. MMP-2, MMP-9,25 and plasmin26 can activate latent TGF-1. We have shown previously that MMP-2 and MMP-9 are upregulated in aneurysmal xenografts12 and that the plasmin pathway is strongly stimulated in and is essential to aneurysmal degeneration of aortic xenografts.27 In normal rat aorta and in VSMCs in culture, MMP-2 is inactive and MMP-9 is not expressed. This suggests that activation of TGF-1 is triggered by the proteolytic context of xenograft aneurysmal degeneration and expansion. Stabilization with endovascular VSMC seeding is associated with both an increase in total TGF-1 and a high proportion of activated TGF-1 in the aneurysmal wall. After release and activation by proteolytic cleavage, TGF-1 activity in tissues is conditioned by local expression of receptors. Among TGF- receptors identified on the basis of their affinity for TGF- family members, TR I and TR II in particular have a cytoplasmic domain with serine
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and threonine kinase activity and signal to cell nucleus through the Smad cascade.4,28 Our results suggest that aneurysmal xenografts express these receptors and therefore can respond to TGF-. TR I and TR II expression was not modified at mRNA level by VSMC endovascular seeding, demonstrating that TGF-1 expression is the only member of TGF family and receptors induced with cell therapy. Data from the present study demonstrate that the bulk of TGF-1 is synthesized in the intima, where VSMCs have been seeded, whereas TGF-1 protein is distributed throughout the aneurysm wall after seeding. These divergent localizations of synthesis and location of protein in the wall suggest that TGF-1 is an intimal paracrine factor that diffuses centrifugally to the aneurysmal lesion. The healing process leading to aneurysmal xenograft stabilization with endovascular seeding of VSMCs is initiated by changes in the aneurysm itself: reduced content in mRNA encoding for MMP-1, MMP-3, MMP-9, and MMP-12 and increased content in tissue inhibitor metalloproteinase (TIMP)–1, TIMP-3, and collagen types I and III mRNA.1 TGF-1 exerts paracrine control on gene expression in cells.29 Under defined experimental conditions, TGF-1 shifts the MMP-dependant proteolytic balance toward inhibition by downregulating expression of various MMPs and upregulating TIMP expression at mRNA levels.30,31 As another contribution to proteolysis regulation, TGF-1 induces plasminogen-activator inhibitor (PAI)–1 expression.32 PAI-1 overexpression suppresses aneurysmal degeneration in the xenograft model by inhibiting MMP-9 activation.27 Apart from decreasing proteolysis, TGF-1 increases collagen production in various cell types, including fibroblasts and VSMCs,33 and lysyl oxidase critical for collagen cross-linking and organization into fibers.34 TGF-1 is induced during healing after arterial balloon injury, and its perfusion stimulates VSMC proliferation in balloon-injured rat carotid arteries.6 Taken together, these data designate TGF-1 as a paracrine factor mediating decreased wall destruction and increased wall reconstruction after endovascular VSMC seeding in aneurysmal xenografts. Since many cell types respond to stress by increasing TGF-1 synthesis, and since TGF-1 may exert opposite effects depending on cell phenotype, clarifying the role of TGF-1 in arterial stability is not easy.35 In nonseeded aneurysmal xenografts, sources of TGF-1 may be activated inflammatory cells, as observed in atherosclerotic lesions in human beings5,36 and in allograft arteriosclerosis.37 The luminal thrombus observed in aneurysmal xenografts may also provide TGF-1 through platelet recruitment and degranulation.38 After endoluminal seeding, a thick intimal layer mainly composed of VSMCs develops, with scattered monocytes and macrophages. We cannot rule out a possible role of VSMC–inflammatory cell crosstalk in regulation of TGF-1 production or activation within the intima. However, the bulk of mRNA extracted from this neointima is derived from VSMCs, the main cell type in this layer. Hence VSMCs accumulating after endo-
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vascular seeding appear to be the main source of TGF-1. The healing process leading to aneurysmal xenograft stabilization with endovascular seeding of VSMCs is associated with induction of TIMP-1 and TIMP-3, collagen types I and III in the intima.14 TGF-1 exerts autocrine control of gene expression in cells.39 In addition to exerting a paracrine effect in the aneurysmal wall, TGF1 may participate in endovascular aortic reconstruction after VSMC endovascular seeding. VSMC stress may be an important determinant of TGF-1 induction in our study. Isolation and culture strongly induced TGF-1 at the transcriptional level in VSMCs before seeding, with mostly the inactive proform being produced. Unlike endovascular prostheses,40 endovascular seeding does not exclude the aneurysmal wall from arterial hemodynamic radial strain.14 Mechanical strain induces production of TGF-1, and to a lesser extent TGF3.41,42 TGF-1 is responsible for VSMC collagen synthesis in response to sustained mechanical strain.42,43 By reestablishing a significant population of VSMCs in the wall, endovascular seeding may correct aneurysmal wall healing deficiency by increasing the capability to synthesize matrix in response to hemodynamical strain through augmented TGF-1 synthesis. Limitations and perspectives. Involvement of TGF-1 in atherosclerotic aneurysms in human beings has not been explored. Aortic aneurysmal degeneration of aortic xenografts reproduces some structural, cellular, and enzymatic aspects of aneurysms in human beings, ie, extensive injury of the extracellular matrix,9 inflammation in the media and adventitia,44 depletion in VSMCs,45 luminal thrombus, adventitial neovessels, and upregulation of MMPs and enzymes of the plasmin pathway.12,27 Unlike other animal models, aneurysmal degeneration of aortic xenografts is slow enough to allow endovascular repeat operation to stabilize degenerated vessels with severe injury of the extracellular matrix. Although the origins of aneurysmal degeneration of xenografts and atherosclerotic aortas are clearly different, the terminal cellular and enzymatic effectors are similar, offering a unique opportunity to test new therapies using the aneurysmal vessel as a scaffold to form a new, stable vessel from spontaneously nonhealing vessels. Whether TGF-1 may downregulate proteolysis and promote wall reconstruction in aneurysms in human beings will need to be studied with organ culture. Our data and those from other groups show that the increase in TGF-1 levels after VSMC seeding or mechanical stimulation42 is transient. Our results raise the possibility that short overexpression of TGF-1 may trigger healing in aneurysmal xenografts. We are developing a gene transfer approach to evaluate the effect of TGF-1 overexpression in an attempt to stabilize aneurysmal xenografts. We thank P. Druelle, J. Magnard, and P. Mario for animal care; A. Maurette and N. Sauvant for administrative and secretarial assistance; and Doctor J. Saez, INSERM
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U369, Lyon, France, for providing the anti-rat TGF-1 antibody. REFERENCES 1. Allaire E, Muscatelli-Groux B, Guinault A-M, Page`s C, Goussard A, Mandet C, et al. Vascular smooth muscle cell endovascular therapy stabilizes dilated aortas injured by inflammation and proteolysis in a xenograft model. Ann Surg. In Press 2003. 2. Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor beta in human disease. N Engl J Med 2000;342:1350-8. 3. Munger JS, Harpel JG, Gleizes PE, Mazzieri R, Nunes I, Rifkin DB. Latent transforming growth factor-beta: Structural features and mechanisms of activation. Kidney Int 1997;51:1376-82. 4. Derynck R, Zhang Y, Feng XH. Smads: Transcriptional activators of TGF-beta responses. Cell 1998;95:737-40. 5. Bobik A, Agrotis A, Kanellakis P, Dilley R, Krushinsky A, Smirnov V, et al. Distinct patterns of transforming growth factor-beta isoform and receptor expression in human atherosclerotic lesions: Colocalization implicates TGF-beta in fibrofatty lesion development. Circulation 1999;99:2883-91. 6. Majesky MW, Lindner V, Twardzik DR, Schwartz SM, Reidy MA. Production of transforming growth factor -1 during repair of arterial injury. J Clin Invest 1991;88:904-10. 7. Shah M, Foreman DM, Ferguson MW. Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci 1995;108:985-1002. 8. Shi GP, Sukhova GK, Grubb A, Ducharme A, Rhode LH, Lee RT, et al. Cystatin C deficiency in human atherosclerosis and aortic aneurysms. J Clin Invest 1999;104:1191-97. 9. Allaire E, Guettier C, Michel JB. Cell-free arterial grafts: Morphologic characteristics of aortic isografts, allografts, and xenografts in rats. J Vasc Surg 1994;19:446-56. 10. Battle T, Arnal JF, Challah M, Michel JB. Selective isolation of rat aortic wall layers and their cell types in culture: Application to converting enzyme activity measurement. Tissue Cell 1994;26:943-55. 11. Ernst CB. Abdominal aortic aneurysm. N Engl J Med 1993;328:116772. 12. Allaire E, Forough R, Clowes MM, Starcher B, Clowes AW. Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model. J Clin Invest 1998;102:1413-20. 13. Roberts AB, Lamb LC, Newton DL, Sporn MB, De Larco JE, Todd GJ. Transforming growth factors: Isolation of polypeptides from virally and chemically transformed cells by acid/ethanol extraction. Proc Natl Acad Sci U S A 1980;77:3494-8. 14. Gautier C, Levacher C, Avallet O, Vigier M, Rouiller-Fabre V, Lecerf L, et al. Immunohistochemical localization of transforming growth factorbeta 1 in the fetal and neonatal rat testis. Mol Cell Endocrinol 1994; 99:55-61. 15. Ross R. The pathogenesis of atherosclerosis: A perspective for the 1990s. Nature 1993;362:801-9. 16. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med 1992;326:242-50. 17. Golledge J, Greenhalgh RM, Davies AH. The symptomatic carotid plaque. Stroke 2000;31:774-81. 18. Shah PK. Inflammation, metalloproteinases, and increased proteolysis: An emerging pathophysiological paradigm in aortic aneurysm. Circulation 1997;96:2115-7. 19. Carmeliet P. Proteinases in cardiovascular aneurysms and rupture: Targets for therapy. J Clin Invest 2000;105:1519-20. 20. Mortality results for randomised controlled trial of early elective surgery or ultrasonographic surveillance for small abdominal aortic aneurysms. The UK Small Aneurysm Trial Participants. Lancet 1998;352:1649-55. 21. Holweg CT, Baan CC, Balk AH, Niesters HG, Maat AP, Mulder PM, et al. The transforming growth factor-beta1 codon 10 gene polymorphism and accelerated graft vascular disease after clinical heart transplantation. Transplantation 2001;71:1463-67.
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22. Biggart S, Chin D, Fauchon M, Cardew G, du FL, Harker N, et al. Association of genetic polymorphisms in the ACE, ApoE, and TGF beta genes with early onset ischemic heart disease. Clin Cardiol 1998;21: 831-6. 23. Schulick AH, Taylor AJ, Zuo W, Qiu CB, Dong G, Woodward RN, et al. Overexpression of transforming growth factor beta1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia. Proc Natl Acad Sci U S A 1998;95:6983-8. 24. Nabel EG, Shum L, Pompili VJ, Yang ZY, San H, Shu HB, et al. Direct transfer of transforming growth factor beta 1 gene into arteries stimulates fibrocellular hyperplasia. Proc Natl Acad Sci U S A 1993;90: 10759-63. 25. Yu Q, Stamenkovic I. Cell surface–localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes Dev 2000;14:163-76. 26. Lyons RM, Gentry LE, Purchio AF, Moses HL. Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin. J Cell Biol 1990;110:1361-7. 27. Allaire E, Hasenstab D, Kenagy R, Clowes MM, Starcher B, Clowes AW. Overpression of plasminogen activator-inhibitor prevents arterial extracellular matrix degradation by blocking matrix metalloproteinase activity. Circulation 1998;98:249-55. 28. Larsson J, Goumans MJ, Sjostrand LJ, van Rooijen MA, Ward D, Leveen P, et al. Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice. EMBO J 2001;20: 1663-73. 29. Farrehi PM, Ozaki CK, Carmeliet P, Fay WP. Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice. Circulation 1998;97:1002-8. 30. Overall CM, Wrana JL, Sodek J. Independant regulation of collagenase, 72-kD progelatinase, and metalloendoproteinase inhibitor expression in human fibroblasts by transforming growth factor-. J Biol Chem 1989;264:1860-9. 31. Vaday GG, Schor H, Rahat MA, Lahat N, Lider O. Transforming growth factor-beta suppresses tumor necrosis factor alpha-induced matrix metalloproteinase-9 expression in monocytes. J Leukoc Biol 2001; 69:613-21. 32. Lund LR, Riccio A, Andreasen PA, Nielsen LS, Kristensen P, Laiho M, et al. Transforming growth factor-beta is a strong and fast acting positive regulator of the level of type-1 plasminogen activator inhibitor mRNA in WI-38 human lung fibroblasts. EMBO J 1987;6: 1281-6. 33. Letterio JJ, Roberts AB. Regulation of immune responses by TGF-beta. Annu Rev Immunol 1998;16:137-61. 34. Shanley CJ, Gharaee-Kermani M, Sarkar R, Welling TH, Kriegel A, Ford JW, et al. Transforming growth factor-beta 1 increases lysyl oxidase enzyme activity and mRNA in rat aortic smooth muscle cells. J Vasc Surg 1997;25:446-52. 35. Kulkarni AB, Huh CG, Becker D, Geiser A, Lyght M, Flanders KC, et al. Transforming growth factor b1 null mutation in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci U S A 1993;90:770-4. 36. Frostegard J, Ulfgren AK, Nyberg P, Hedin U, Swedenborg J, Andersson U, et al. Cytokine expression in advanced human atherosclerotic plaques: Dominance of pro-inflammatory (Th1) and macrophage-stimulating cytokines. Atherosclerosis 1999;145:33-43. 37. Koglin J, Glysing-Jensen T, Raisanen-Sokolowski A, Russell ME. Immune sources of transforming growth factor-beta1 reduce transplant arteriosclerosis: Insight derived from a knockout mouse model. Circ Res 1998;83:652-60. 38. Grainger DJ, Wakefield L, Bethell HW, Farndale RW, Metcalfe JC. Release and activation of platelet latent TGF-beta in blood clots during dissolution with plasmin. Natl Med 1995;1:932-7. 39. Sudarshan C, Yaswen L, Kulkarni A, Raghow R. Phenotypic consequences of transforming growth factor beta1 gene ablation in murine embryonic fibroblasts: Autocrine control of cell proliferation and extracellular matrix biosynthesis. J Cell Physiol 1998;176:67-75. 40. Parodi JC, Palmaz JC, Barone HD. Transfemoral intraluminal graft implantation for abdominal aortic aneurysms. Ann Vasc Surg 1991;5: 491-9.
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41. Riser BL, Cortes P, Heilig C, Grondin J, Ladson-Wofford S, Patterson D, et al. Cyclic stretching force selectively up-regulates transforming growth factor-beta isoforms in cultured rat mesangial cells. Am J Pathol 1996;148:1915-23. 42. O’Callaghan CJ, Williams B. Mechanical strain-induced extracellular matrix production by human vascular smooth muscle cells: Role of TGF-beta(1). Hypertension 2000;36:319-24. 43. Li Q, Muragaki Y, Hatamura I, Ueno H, Ooshima A. Stretch-induced collagen synthesis in cultured smooth muscle cells from rabbit aortic
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media and a possible involvement of angiotensin II and transforming growth factor-beta. J Vasc Res 1998;35:93-103. 44. Allaire E, Mandet C, Bruneval P, Bensenane S, Becquemin JP, Michel JB. Cell and extracellular matrix rejection in arterial concordant and discordant xenografts in rat. Transplantation 1996;62:794-803. 45. Allaire E, Muscatelli-Groux B, Mandet C, Guinault A-M, Bruneval P, Desgranges P, et al. Paracrine effect of vascular smooth muscle cells in the prevention of aortic aneurysm formation. J Vasc Surg 2002;36:1018-26. Submitted Jul 18, 2002; accepted Nov 14, 2002.
AVAILABILITY OF JOURNAL BACK ISSUES As a service to our subscribers, copies of back issues of Journal of Vascular Surgery for the preceding 5 years are maintained and are available for purchase from Mosby until inventory is depleted. Please write to Mosby, Subscription Customer Service, 6277 Sea Harbor Dr, Orlando, FL 32887, or call 800-6542452 or 407-345-4000 for information on availability of particular issues and prices.
Once extracellular matrix injury has started, ongoing aneurysmal growth can be interpreted as the consequence of healing failure. In a recent study,1 our group demonstrated that aneurysmal xenograft healing and stabilization can be attained with endovascular seeding of vascular smooth muscle cells (VSMCs). The microscopic appearance of stabilized xenografts suggested to us that the seeded VSMCs trigger a healing process that interrupts aneurysmal wall destruction by inflammation and proteolysis and promotes wall reconstruction.1 Most of the biologic changes induced by endovascular VSMC seeding were observed in the aneurysmal wall itself, whereas the seeded VSMCs remained localized in the intima. Inward From Centre National de la Recherche Scientifique (CNRS) Unite´ Mixte de Recherche (UMR) 7054, Centre de Recherches Chirurgicales, Universite´ Paris XII, UFR de Me´decine, Hoˆpital Henri Mondor,a Service de Chirurgie Thoracique et Vasculaire, Hoˆpital Tenon,b and Service de Chirurgie Vasculaire et Endocrinienne, Hoˆpital Henri Mondor.c Supported by CNRS, La Fondation de l’Avenir pour la Recherche Me´dicale Applique´e (grant ET9-210) and La Fondation de France. Dr Losy was supported by a grant from Assistance Publique–Hoˆpitaux de Paris (APHP), Paris, France. Competition of interest: none. Reprint requests: Eric Allaire, Centre de Recherches Chirurgicales, 8 rue du Ge´ne´ral Sarrail, 94010 Cre´teil, France (e-mail: [email protected]). Copyright © 2003 by The Society for Vascular Surgery and The American Association for Vascular Surgery. 0741-5214/2003/$30.00 ⫹ 0 doi:10.1016/S0741-5214(02)75336-6
localization of seeded cells and outward localization of changes in gene transcription support the view that seeded cells secrete paracrine factors that diffuse centrifugally to reach the aneurysm wall and control inflammation, proteolysis, and wall reconstruction. Transforming growth factor– beta (TGF-) belongs to a family of cytokines with multiple roles in tissue development and healing because they modulate inflammation, proteolysis, extracellular matrix synthesis, cell proliferation, cell survival, and cell migration.2 TGF- activity is regulated by protein concentration, activation by proteolytic cleavage,3 and expression of receptors, particularly the type I and type II serine and threonine protein kinase receptors, which mediate signal transduction to cell nuclei.4 TGF-1 is expressed by VSMCs in the normal artery wall,5 and its production is increased during artery wall repair after mechanical injury.6 Among three identified isoforms, TGF-1 and TGF-2 are involved in healing of cutaneous wounds.7 In abdominal aortic aneurysm in human beings, TGF-1 may be involved in diameter control through production of cystatin C, an inhibitor of cysteine proteases such as elastase cathepsins S and K.8 In the present work we analyzed the expression of the three isoforms of TGF- and its two serine and threonine receptors in experimental aneurysms. To address the role of TGF- in aneurysm growth, we designed a model of already developed aneurysms in rats, in which aneurysm 1301
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Fig 1. Aneurysm healing and stabilization with endovascular VSMC therapy. A, Schema of endovascular infusion in experimental aneurysms attained with 14-day implantation of an aortic xenograft. B, Macroscopic results show stabilization of diameter of aneurysms after VSMC endovascular seeding. C, VSMC staining with anti-␣-actin antibody on cryostatic cross-sections of control (top) and VSMC-seeded (bottom) aneurysms 7 days after endovascular repeat operation. Dashed white line shows separation of tissues during microdissection, enabling separate analysis with RT-PCR and ELISA. Black bar shows media and adventitia thickness. White bar shows intima composed mainly of VSMCs in VSMC-seeded vessels. Th, Thrombus. D, Microscopic appearance of aneurysms 8 weeks after endovascular repeat operation shows a dense collagen network in VSMC-seeded aneurysms (bottom) in comparison with control aneurysms (top). Original magnification ⫻20. *P ⬍ .05; **P ⬍ .01.
diameter stability was attained with endovascular seeding of VSMCs. Our results show that healing and stabilization of already developed aneurysms is associated with a transient increase in TGF-1 expression. We report the first identification of a molecule potentially involved in functional healing of already developed experimental aneurysms. MATERIAL AND METHODS Animals and surgery. Animals housed and cared for according to European Union Standards received analgesia and were anesthetized with 5 mg/100 g body weight of pentobarbital administered intraperitoneally. Hartley guinea pig (Iffa Credo, Lyon, France) abdominal aortas were decellularized with sodium dodecylsulfate, as described.9 The tube of arterial extracellular matrix obtained with this method was grafted orthotopically into male Fischer 344 rats (n ⫽ 50; Iffa Credo) during a first surgical procedure. Syngeneic male Fischer 344 rat VSMCs were isolated from thoracic aortas10 and were grown in RPMI 1640 and medium 199, with L-glutamine and 10% fetal calf serum (FCS; Gibco-BRL, Grand Island, NY). More than 98% of isolated cells grown to 80% confluence were stained positively with an anti-␣-actin antibody (Dakopatt, Copenhagen, Denmark).
A second surgical procedure was performed 14 days later, at which time the xenograft diameter had increased more than 50% to comply with aneurysm definition.11 Graft diameter was measured with a grid in the microscope eyepiece immediately after transplantation at repeat operation and before euthanasia at day 7 or week 8 after seeding.12 The aneurysmal xenograft was isolated from blood flow with clamps after gentle dissection. Its lumen was gently rinsed with culture medium through a PE10 catheter introduced at aortotomy performed downstream in the native aorta (Fig 1, A). Passage five to eight VSMCs were resuspended in culture medium with 5% FCS injected into the aneurysmal xenograft lumen (107 cells per xenograft) and allowed to attach by sedimentation during four 90degree rotations (sedimentation duration 2 minutes for each position) for 8 minutes. Previous studies with PKH26 fluorescent dye staining in vivo assessed VSMC intimal location after seeding (data not shown). As control, aneurysmal xenografts were infused with culture medium with 5% FCS without cells. Diameter increase was calculated as: (Diameter at harvest ⫺ Diameter at seeding)/Diameter at seeding. Analysis of mRNA content. TGF-1, TGF-2, TGF3, and TR I and TR II messenger RNA levels were analyzed with reverse transcriptase polymerase chain reac-
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tion (RT-PCR), and compared to the RNA level of domestic gene 18s (Quantum RNA 18s Internal Standards kit; Ambion, Montrouge, France). Intima or thrombus, and media plus adventitia (Fig 1, C) were separated with microdissection and pooled by layers and groups. Total RNA was extracted with TRIzol (Life Technologies, Rockville, Md) and treated with grade I deoxyribonuclease (DNAse; Roche Molecular Biomedicals). RNA was extracted according the same protocol from VSMCs in culture and from normal Fischer 344 rat aorta. RT was performed with random primers, and Superscript II (Life Technologies), deoxyribonucleoside triphosphate (dNTP), dithiothreitol, and ribonuclease inhibitor (Roche Molecular Biochemicals). PCR was performed in a PCR Express thermocycler (Hy baid, Ashford, UK) in the same tube for both the gene of interest (primers: matrix metalloproteinase (MMP)–2; TGF-1, 5'-CG GACTAC TACGCCAAAGAA-3'/5'TCAAAAGACAGCCACTCAGG-3'; TGF-2, 5' CCG CCC ACT TTC TAC AGA CCC 3'/5' GCG CTG GGT GGG AGA TGT TAA 3'; TGF-3, 5' TGC CCA ACC CGA GCT CTA AGC G 3'/ 5' CCT TTG AAT TTG ATC TCC A 3'; TR I, 5'-ACGTTCATGGTTCCGAGAGG-3'/ 5'-TCG CAAAGCTGTCAGCCTAG-3'; TR II, 5'-AAGT CTTGCATGAGCAACTGC-3'/5'-GATGTCAGAGAAGA TGTCC3') (Genset Oligos SA, Paris, France) and 18s. Final PCR conditions were chosen to prevent interference between the two sets of primers. The PCR mix contained Taq polymerase (EurobioTaq; Eurobio, Les Ulis, France) in buffer, dNTP, and magnesium chloride. Negative control assays were performed without Superscript II. Ten microliters of PCR products were run in 2% agarose gel with 5 g/mL of ethidium bromide, visualized under untraviolet light with a video camera. Bands of amplified sequences corresponding to the gene of interest and to 18s were quantified with Gel Analyst (Iconix). Results were expressed as a ratio between signals corresponding to the gene of interest and 18s. ELISA for TGF-1. The sandwich enzyme-linked immunosorbent assay (ELISA; Promega, Madison, Wis) recognizes active, not latent, TGF-1. Quantification was performed according to manufacturer instructions, either after Triton extraction for active form quantification or after acid and ethanol extraction and activation for total TGF-1 quantification.13 Histologic analysis and immunohistochemistry. Cryostat cross sections (5 m) were made from the center of the aneurysmal xenografts. Collagen was evidenced with sirius red staining. Primary antibody for ␣-actin was a mouse monoclonal antibody (Dakopatt), with an alkaline phosphatase–anti-alkaline phosphatase technique (Dakopatt). The anti-TGF-1 polyclonal antibody raised in rabbits was a generous gift from Dr Saez.14 The secondary antibody was a goat anti-rabbit antibody with 3,3'-diaminobenzidine tetrahydrochloride. Control sections were generated by omission of the primary antibody. Statistical analysis. Results are expressed as mean ⫾ SD. Comparisons between two groups were done with the nonparametric Mann-Whitney U test (Statview, version 4.5).
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RESULTS Aneurysm diameter and microscopic appearance VSMC seeding consistently resulted in stabilization of diameter of aneurysmal xenografts.1 Conversely, infusion of culture medium was always followed by a statistically significant increase in aneurysm diameter after 2 months (Fig 1, B). Immunohistochemistry 7 days after endovascular VSMC seeding disclosed a thick intima in aneurysmal xenografts, composed mainly of ␣-actin–positive cells, whereas thrombus covered the luminal aspect of control aneurysmal xenografts (Fig 1, C). In VSMC-seeded aneurysms a dense network of collagen developed in the intima and adventitia (Fig 1, D). Expression of TGF- and its receptors in VSMCs in culture and in experimental aneurysms For all molecules tested, mRNA was detected in cultured cells and in aneurysmal xenografts after 14-day implantation (Fig 2). TGF-2 and TGF-3 were upregulated in aneurysmal xenografts before endovascular infusion. Conversely, TGF-1 mRNA levels remained low in the intima and in the media and adventitia (ie, in the aneurysmal wall itself) but were high in VSMCs in culture. Comparison of TGF-1 mRNA level between native aorta and VSMCs in culture demonstrated that the in vitro phase of VSMCs culture doubled TGF-1 mRNA content. TGF- expression after VSMC therapy TGF- isoform mRNA content. mRNA content encoding for the three isoforms of TGF- was measured 7 days after endovascular reoperation. In the intima or thrombus, TGF-1 mRNA level was higher in the seeded vessels, whereas TGF-2 and TGF-3 mRNA level was similar in the two groups (Table I). In the media plus adventitia, TGF-1 mRNA was increased in the seeded vessels in comparison with control nonseeded vessels, without reaching a statistically significant difference. TGF-2 and TGF-3 mRNA content was not modulated with VSMC seeding (Table I). Kinetics of TGF- mRNA content variations after endovascular VSMC seeding. In aortas harvested 3 and 7 days after endovascular repeat operation, the intima or thrombus and the media plus adventitia were easily separated with microdissection, enabling separate mRNA extraction of the two layers in the two groups. In VSMCseeded aortas harvested at 8 weeks the intima could not be separated from the media plus adventitia. Therefore mRNA analysis at this time was performed from the whole aortic wall in the two groups. As observed at day 7, TGF-1 mRNA content at day 3 was increased in the intima but not in the media plus adventitia after endovascular VSMC seeding. At 8 weeks TGF-1 mRNA content was similar in the two groups (Table II). TGF-2 and TGF-3 mRNA content was similar in the two groups 8 weeks after seeding (data not shown).
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Fig 2. mRNA content in comparison to 18s of TGF- and receptors. Culture of VSMCs strongly induces members of TGF- in comparison with normal rat aorta. TGF-1 mRNA levels are low in 14-day experimental aneurysms. mRNA levels of constitutive MMP-2 are shown for comparison. Data represent results of RT-PCR performed on pools of three experimental aneurysms.
Table I. TGF- isoforms 1, 2, and 3 mRNA content in aneurysmal xenografts 7 days after endovascular repeat operation (TGF-/18s ratio) TGF-1 Intima VSMC ⫺ 0.73 ⫾ 0.10 VSMC ⫹ 1.40 ⫾ 0.36 P .049 Media ⫹ adventitia VSMC ⫺ 0.89 ⫾ 0.04 VSMC ⫹ 1.62 ⫾ 0.88 P .083
TGF-2
TGF-3
0.52 ⫾ 0.58 0.67 ⫾ 0.23 .827
0.74 ⫾ 0.39 0.69 ⫾ 0.08 .513
0.10 ⫾ 0.04 0.11 ⫾ 0.10 .999
0.25 ⫾ 0.25 0.16 ⫾ 0.04 .563
Data represent measurements from three pools of three grafts. VSMC, Vascular smooth muscle cell; ⫺, negative; ⫹, positive.
normal rat aorta the percentage of active over total TGF-1 was low (Fig 3, B). In aneurysmal xenografts just before endovascular repeat operation this percentage was dramatically increased. After endovascular repeat operation and endoluminal infusion, the percentage of active TGF-1 fell to low levels in the intima or thrombus but remained high in the media and adventitia (Fig 3, C). Immunohistochemistry. Immunohistochemistry with anti-TGF-1 antibody evidenced few positive cells in the luminal thrombus and in the medial and adventitial layers 7 days after endoluminal injection of culture medium in the control group. Conversely in the aneurysms 7 days after endoluminal seeding of VSMCs, numerous cells in the intima were strongly positive for anti-TGF-1 staining (Fig 4). Omission of the primary antibody suppressed cell and extracellular matrix staining (data not shown).
TGF-1 protein expression after VSMC therapy
TGF- receptor expression after VSMC therapy
Protein quantification at ELISA. ELISA performed after acid and ethanol extraction was used to measure total, ie, active and latent, TGF-1. Concordant with mRNA levels, total TGF-1 protein was increased in the intima of aneurysmal xenografts 7 days after endovascular seeding, compared with TGF-1 protein in the thrombus of control xenografts 7 days after repeat operation (45.4 ⫾ 9.7 ng/mg in control xenografts, 144.3 ⫾ 30.6 ng/mg in seeded aneurysmal xenografts; P ⬍ .05). In the media plus adventitia, TGF-1 protein was also increased after endovascular VSMC seeding (124.6 ⫾ 5.8 vs 162.9 ⫾ 12.2 ng/mg in control and seeded aneurysmal xenografts, respectively; P ⬍ .05) (Fig 3, A). ELISA performed after extraction with Triton was used to measure active TGF-1. In VSMCs in culture and in
RT-PCR did not evidence significant differences in TR I and TR II mRNA ratio to 18s mRNA levels 7 days after endovascular repeat operation (Intima: TR I, 0.51 ⫾ 0.51 vs 0.86 ⫾ 0.37 [P ⫽ .123, Mann-Whitney U test]); TR II, 0.39 ⫾ 0.25 vs 0.76 ⫾ 0.35 [P ⫽ .275, in aneurysms with no VSMC vs aneurysms with VSMC seeding]. Media plus adventitia: TR I, 0.92 ⫾ 0.56 vs 1.47 ⫾ 1.45 [P ⫽ .827]; TR II, 0.52 ⫾ 0.36 vs 0.73 ⫾ 0.64 [P ⫽ .827, in aneurysms with no VSMC vs aneurysms with VSMC seeding]). DISCUSSION Identification of molecular factors promoting artery wall stabilization after extracellular matrix injury elicited with inflammation and proteolysis is a major issue for new therapies for atherosclerosis.15-19 In vivo manipulations of
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Fig 3. TGF-1 protein content measured with ELISA. A, Seven days after endovascular repeat operation, VSMC seeding is followed by a significant increase in total (latent plus activated) TGF-1, both in the intima and the aneurysm wall. B, Whereas TGF-1 is mostly in its nonactivated form in cultured VSMCs and normal rat aorta, activated TGF-1 is the predominant form of TGF-1 in the inflammatory context of aneurysms. C, Seven days after endovascular repeat operation, TGF-1 is mostly in its activated form in the aneurysmal wall in both groups. Values for total TGF-1 are the average of three measurements on three different grafts in each group. Values for active TGF-1 obtained by pooling values for three grafts. *P ⬍ .05, Mann- Whitney U test.
Table II. Kinetics of TGF-1 mRNA content evolution in aneurysmal xenografts after endovascular repeat operation (TGF-1/18s ratio); pools of three grafts.
Intima VSMC ⫺ VSMC ⫹ P Media ⫹ adventitia VSMC ⫺ VSMC ⫹ P
Day 14 ⫹ 3
Day 14 ⫹ 7
Day 14 ⫹ 8 wk
0.61 ⫾ 0.13 1.26 ⫾ 0.43 .049
0.73 ⫾ 0.10 1.40 ⫾ 0.36 .049
VSMC ⫺ 0.84 ⫾ 0.32 VSMC ⫹ 0.96 ⫾ 0.15
0.72 ⫾ 0.26 0.43 ⫾ 0.31 .127
0.89 ⫾ 0.04 1.62 ⫾ 0.88 .083
.999
Data represent measurements from three pools. VSMC, Vascular smooth muscle cell; ⫺, negative; ⫹, positive.
arteries constantly resulting in wall stability or instability enable development of predictive biology because tissue analysis can be performed before major wall remodeling occurs. VSMC endovascular seeding in already formed aneurysms offers a unique opportunity to understand which cellular and molecular mechanisms may trigger aneurysm stabilization and healing. Our data demonstrate a
correlation between TGF-1 and aneurysm stabilization, with TGF-1 expression electively increased in aneurysmal aortic xenografts after VSMC endovascular seeding leading to control of diameter, a decisional clinical end point for aortic aneurysms.20 Most TGF-1 appears to be synthesized in the intimal layer where VSMCs have been seeded, whereas the level of activated protein is increased in the
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Fig 4. TGF-1 immunostaining of aneurysms 7 days after endovascular repeat operation. In the control group (VSMC-), few TGF-1–positive cells (long arrows) are observed, either in luminal thrombus or adventitia. In VSMC-seeded vessels, numerous cells stained with anti-TGF-1 antibody, mainly in the intima. In both groups, patchy areas of extracellular matrix stained with anti-TGF-1 antibody (open arrow). L, Lumen.
entire wall thickness of the VSMC-treated aneurysm. The increase in TGF-1 mRNA is transient, and disappears 2 months after cell therapy. Since TR I and TR II are expressed in aneurysmal xenografts, we provide strong evidence that TGF-1 is a paracrine factor secreted at the initial phase of aneurysmal xenograft stabilization attained with endovascular VSMC therapy. Differences between groups for TGF-1 expression are related to differences in the most endoluminal layer of the wall, ie, thrombus versus intimal accumulation of VSMCs. In our study, TFG-1 mRNA levels exceeded levels of the two other isoforms in VSMCs in vitro before seeding and in aneurysmal xenografts in vivo after VSMC seeding. Moreover, among the three isoforms of TGF-, only TGF-1 mRNA levels were correlated with aneurysmal xenograft stabilization with endovascular VSMC therapy. In atherosclerotic lesions in human beings, Bobik et al5 observed a correlation between atherosclerosis severity and expression of TGF-1 but not TGF-3. Whereas TGF-1 gene polymorphism is associated with severity of coronary disease after heart transplantation,21 the polymorphism of TGF-2 does not influence early ischemic heart disease.22 Involvement of TGF-1 in structural changes in arteries in adults is further suggested by the efficiency of TGF-1
overexpression in changing normal wall structure23,24 and is now evidenced in the setting of experimental aneurysm expansion and healing. Most TGF-1 isolated in aneurysmal xenografts before and after endovascular repeat operation was the active form. Our data also show that in the normal aortic wall and in cultured cells, TGF-1 mostly is in its inactive proform. MMP-2, MMP-9,25 and plasmin26 can activate latent TGF-1. We have shown previously that MMP-2 and MMP-9 are upregulated in aneurysmal xenografts12 and that the plasmin pathway is strongly stimulated in and is essential to aneurysmal degeneration of aortic xenografts.27 In normal rat aorta and in VSMCs in culture, MMP-2 is inactive and MMP-9 is not expressed. This suggests that activation of TGF-1 is triggered by the proteolytic context of xenograft aneurysmal degeneration and expansion. Stabilization with endovascular VSMC seeding is associated with both an increase in total TGF-1 and a high proportion of activated TGF-1 in the aneurysmal wall. After release and activation by proteolytic cleavage, TGF-1 activity in tissues is conditioned by local expression of receptors. Among TGF- receptors identified on the basis of their affinity for TGF- family members, TR I and TR II in particular have a cytoplasmic domain with serine
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and threonine kinase activity and signal to cell nucleus through the Smad cascade.4,28 Our results suggest that aneurysmal xenografts express these receptors and therefore can respond to TGF-. TR I and TR II expression was not modified at mRNA level by VSMC endovascular seeding, demonstrating that TGF-1 expression is the only member of TGF family and receptors induced with cell therapy. Data from the present study demonstrate that the bulk of TGF-1 is synthesized in the intima, where VSMCs have been seeded, whereas TGF-1 protein is distributed throughout the aneurysm wall after seeding. These divergent localizations of synthesis and location of protein in the wall suggest that TGF-1 is an intimal paracrine factor that diffuses centrifugally to the aneurysmal lesion. The healing process leading to aneurysmal xenograft stabilization with endovascular seeding of VSMCs is initiated by changes in the aneurysm itself: reduced content in mRNA encoding for MMP-1, MMP-3, MMP-9, and MMP-12 and increased content in tissue inhibitor metalloproteinase (TIMP)–1, TIMP-3, and collagen types I and III mRNA.1 TGF-1 exerts paracrine control on gene expression in cells.29 Under defined experimental conditions, TGF-1 shifts the MMP-dependant proteolytic balance toward inhibition by downregulating expression of various MMPs and upregulating TIMP expression at mRNA levels.30,31 As another contribution to proteolysis regulation, TGF-1 induces plasminogen-activator inhibitor (PAI)–1 expression.32 PAI-1 overexpression suppresses aneurysmal degeneration in the xenograft model by inhibiting MMP-9 activation.27 Apart from decreasing proteolysis, TGF-1 increases collagen production in various cell types, including fibroblasts and VSMCs,33 and lysyl oxidase critical for collagen cross-linking and organization into fibers.34 TGF-1 is induced during healing after arterial balloon injury, and its perfusion stimulates VSMC proliferation in balloon-injured rat carotid arteries.6 Taken together, these data designate TGF-1 as a paracrine factor mediating decreased wall destruction and increased wall reconstruction after endovascular VSMC seeding in aneurysmal xenografts. Since many cell types respond to stress by increasing TGF-1 synthesis, and since TGF-1 may exert opposite effects depending on cell phenotype, clarifying the role of TGF-1 in arterial stability is not easy.35 In nonseeded aneurysmal xenografts, sources of TGF-1 may be activated inflammatory cells, as observed in atherosclerotic lesions in human beings5,36 and in allograft arteriosclerosis.37 The luminal thrombus observed in aneurysmal xenografts may also provide TGF-1 through platelet recruitment and degranulation.38 After endoluminal seeding, a thick intimal layer mainly composed of VSMCs develops, with scattered monocytes and macrophages. We cannot rule out a possible role of VSMC–inflammatory cell crosstalk in regulation of TGF-1 production or activation within the intima. However, the bulk of mRNA extracted from this neointima is derived from VSMCs, the main cell type in this layer. Hence VSMCs accumulating after endo-
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vascular seeding appear to be the main source of TGF-1. The healing process leading to aneurysmal xenograft stabilization with endovascular seeding of VSMCs is associated with induction of TIMP-1 and TIMP-3, collagen types I and III in the intima.14 TGF-1 exerts autocrine control of gene expression in cells.39 In addition to exerting a paracrine effect in the aneurysmal wall, TGF1 may participate in endovascular aortic reconstruction after VSMC endovascular seeding. VSMC stress may be an important determinant of TGF-1 induction in our study. Isolation and culture strongly induced TGF-1 at the transcriptional level in VSMCs before seeding, with mostly the inactive proform being produced. Unlike endovascular prostheses,40 endovascular seeding does not exclude the aneurysmal wall from arterial hemodynamic radial strain.14 Mechanical strain induces production of TGF-1, and to a lesser extent TGF3.41,42 TGF-1 is responsible for VSMC collagen synthesis in response to sustained mechanical strain.42,43 By reestablishing a significant population of VSMCs in the wall, endovascular seeding may correct aneurysmal wall healing deficiency by increasing the capability to synthesize matrix in response to hemodynamical strain through augmented TGF-1 synthesis. Limitations and perspectives. Involvement of TGF-1 in atherosclerotic aneurysms in human beings has not been explored. Aortic aneurysmal degeneration of aortic xenografts reproduces some structural, cellular, and enzymatic aspects of aneurysms in human beings, ie, extensive injury of the extracellular matrix,9 inflammation in the media and adventitia,44 depletion in VSMCs,45 luminal thrombus, adventitial neovessels, and upregulation of MMPs and enzymes of the plasmin pathway.12,27 Unlike other animal models, aneurysmal degeneration of aortic xenografts is slow enough to allow endovascular repeat operation to stabilize degenerated vessels with severe injury of the extracellular matrix. Although the origins of aneurysmal degeneration of xenografts and atherosclerotic aortas are clearly different, the terminal cellular and enzymatic effectors are similar, offering a unique opportunity to test new therapies using the aneurysmal vessel as a scaffold to form a new, stable vessel from spontaneously nonhealing vessels. Whether TGF-1 may downregulate proteolysis and promote wall reconstruction in aneurysms in human beings will need to be studied with organ culture. Our data and those from other groups show that the increase in TGF-1 levels after VSMC seeding or mechanical stimulation42 is transient. Our results raise the possibility that short overexpression of TGF-1 may trigger healing in aneurysmal xenografts. We are developing a gene transfer approach to evaluate the effect of TGF-1 overexpression in an attempt to stabilize aneurysmal xenografts. We thank P. Druelle, J. Magnard, and P. Mario for animal care; A. Maurette and N. Sauvant for administrative and secretarial assistance; and Doctor J. Saez, INSERM
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22. Biggart S, Chin D, Fauchon M, Cardew G, du FL, Harker N, et al. Association of genetic polymorphisms in the ACE, ApoE, and TGF beta genes with early onset ischemic heart disease. Clin Cardiol 1998;21: 831-6. 23. Schulick AH, Taylor AJ, Zuo W, Qiu CB, Dong G, Woodward RN, et al. Overexpression of transforming growth factor beta1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia. Proc Natl Acad Sci U S A 1998;95:6983-8. 24. Nabel EG, Shum L, Pompili VJ, Yang ZY, San H, Shu HB, et al. Direct transfer of transforming growth factor beta 1 gene into arteries stimulates fibrocellular hyperplasia. Proc Natl Acad Sci U S A 1993;90: 10759-63. 25. Yu Q, Stamenkovic I. Cell surface–localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes Dev 2000;14:163-76. 26. Lyons RM, Gentry LE, Purchio AF, Moses HL. Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin. J Cell Biol 1990;110:1361-7. 27. Allaire E, Hasenstab D, Kenagy R, Clowes MM, Starcher B, Clowes AW. Overpression of plasminogen activator-inhibitor prevents arterial extracellular matrix degradation by blocking matrix metalloproteinase activity. Circulation 1998;98:249-55. 28. Larsson J, Goumans MJ, Sjostrand LJ, van Rooijen MA, Ward D, Leveen P, et al. Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice. EMBO J 2001;20: 1663-73. 29. Farrehi PM, Ozaki CK, Carmeliet P, Fay WP. Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice. Circulation 1998;97:1002-8. 30. Overall CM, Wrana JL, Sodek J. Independant regulation of collagenase, 72-kD progelatinase, and metalloendoproteinase inhibitor expression in human fibroblasts by transforming growth factor-. J Biol Chem 1989;264:1860-9. 31. Vaday GG, Schor H, Rahat MA, Lahat N, Lider O. Transforming growth factor-beta suppresses tumor necrosis factor alpha-induced matrix metalloproteinase-9 expression in monocytes. J Leukoc Biol 2001; 69:613-21. 32. Lund LR, Riccio A, Andreasen PA, Nielsen LS, Kristensen P, Laiho M, et al. Transforming growth factor-beta is a strong and fast acting positive regulator of the level of type-1 plasminogen activator inhibitor mRNA in WI-38 human lung fibroblasts. EMBO J 1987;6: 1281-6. 33. Letterio JJ, Roberts AB. Regulation of immune responses by TGF-beta. Annu Rev Immunol 1998;16:137-61. 34. Shanley CJ, Gharaee-Kermani M, Sarkar R, Welling TH, Kriegel A, Ford JW, et al. Transforming growth factor-beta 1 increases lysyl oxidase enzyme activity and mRNA in rat aortic smooth muscle cells. J Vasc Surg 1997;25:446-52. 35. Kulkarni AB, Huh CG, Becker D, Geiser A, Lyght M, Flanders KC, et al. Transforming growth factor b1 null mutation in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci U S A 1993;90:770-4. 36. Frostegard J, Ulfgren AK, Nyberg P, Hedin U, Swedenborg J, Andersson U, et al. Cytokine expression in advanced human atherosclerotic plaques: Dominance of pro-inflammatory (Th1) and macrophage-stimulating cytokines. Atherosclerosis 1999;145:33-43. 37. Koglin J, Glysing-Jensen T, Raisanen-Sokolowski A, Russell ME. Immune sources of transforming growth factor-beta1 reduce transplant arteriosclerosis: Insight derived from a knockout mouse model. Circ Res 1998;83:652-60. 38. Grainger DJ, Wakefield L, Bethell HW, Farndale RW, Metcalfe JC. Release and activation of platelet latent TGF-beta in blood clots during dissolution with plasmin. Natl Med 1995;1:932-7. 39. Sudarshan C, Yaswen L, Kulkarni A, Raghow R. Phenotypic consequences of transforming growth factor beta1 gene ablation in murine embryonic fibroblasts: Autocrine control of cell proliferation and extracellular matrix biosynthesis. J Cell Physiol 1998;176:67-75. 40. Parodi JC, Palmaz JC, Barone HD. Transfemoral intraluminal graft implantation for abdominal aortic aneurysms. Ann Vasc Surg 1991;5: 491-9.
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41. Riser BL, Cortes P, Heilig C, Grondin J, Ladson-Wofford S, Patterson D, et al. Cyclic stretching force selectively up-regulates transforming growth factor-beta isoforms in cultured rat mesangial cells. Am J Pathol 1996;148:1915-23. 42. O’Callaghan CJ, Williams B. Mechanical strain-induced extracellular matrix production by human vascular smooth muscle cells: Role of TGF-beta(1). Hypertension 2000;36:319-24. 43. Li Q, Muragaki Y, Hatamura I, Ueno H, Ooshima A. Stretch-induced collagen synthesis in cultured smooth muscle cells from rabbit aortic
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media and a possible involvement of angiotensin II and transforming growth factor-beta. J Vasc Res 1998;35:93-103. 44. Allaire E, Mandet C, Bruneval P, Bensenane S, Becquemin JP, Michel JB. Cell and extracellular matrix rejection in arterial concordant and discordant xenografts in rat. Transplantation 1996;62:794-803. 45. Allaire E, Muscatelli-Groux B, Mandet C, Guinault A-M, Bruneval P, Desgranges P, et al. Paracrine effect of vascular smooth muscle cells in the prevention of aortic aneurysm formation. J Vasc Surg 2002;36:1018-26. Submitted Jul 18, 2002; accepted Nov 14, 2002.
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