Targeted Disruption of TGF-eCSmad3 Signaling Leads to Enhanced Neointimal Hyperplasia With Diminished Matrix Deposition in Response to Vascu
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循环研究杂志 2005年第4期
the Department of Clinical Cell Biology (K.K., K.Y., M.F., H.K., Y.M., S.A., S.M., Y.S.), Chiba University Graduate School of Medicine
Division of Endocrinology and Metabolism (K.Y., Y.S.), Department of Internal Medicine, Chiba University Hospital
Department of Developmental Genetics (K.Y., A.S., T.T.), Chiba University Graduate School of Medicine, Chiba, Japan
Shiraoka Research Station of Biological Science (M.K.), Nissan Chemical Industries, Ltd, Saitama, Japan.
Abstract
The role of transforming growth factor (TGF)- and its signal in atherogenesis is not fully understood. Here, we examined mice lacking Smad3, a major downstream mediator of TGF-, to clarify the precise role of Smad3-dependent signaling in vascular response to injury. Femoral arteries were injured in wild-type and Smad3-null (null) male mice on C57Bl/6 background. Histopathological evaluation of the arteries 1 to 3 weeks after the injury revealed significant enhancement of neointimal hyperplasia in null compared with wild-type mice. Transplantation of null bone marrow to wild-type mice did not enhance neointimal thickening, suggesting that vascular cells in situ play a major role in the response. Null intima contained more proliferating smooth muscle cells (SMC) with less amount of collagen compared with wild-type intima. TGF- caused significant inhibition of cellular proliferation in wild-type aortic SMC, whereas the growth of null SMC was only weakly inhibited by TGF- in vitro, indicating a crucial role of Smad3 in the growth inhibitory function. On the other hand, Smad3-deficiency did not attenuate chemotaxis of SMC toward TGF-. TGF- increased transcript level of 2 type I collagen and tissue inhibitor of metalloproteinases-1, and suppressed expression and activity of matrix metalloproteinases in wild-type SMC. However, these effects of TGF- were diminished in null SMC. Our findings altogether show that the loss of Smad3 pathway causes enhanced neointimal hyperplasia on injury through modulation of growth and matrix regulation in vascular SMC. These results indicate a vasculoprotective role of endogenous Smad3 in response to injury.
Key Words: transforming growth factor- Smad3 atherosclerosis neointimal hyperplasia smooth muscle cells
Introduction
Transforming growth factor (TGF)- is a prototypic member of the TGF- superfamily that exerts a wide range of biological effects on various cell types.1 Well described functions of TGF- including growth inhibition, cell migration, differentiation, extracellular matrix production, and immunomodulation. Abnormality in TGF- signaling may cause pathological conditions such as tumorigenesis, fibrotic disorders, and vascular diseases.2 At present, however, the role of TGF- and its signaling molecules in atherogenesis is not fully understood.
TGF- is often regarded to have proatherosclerotic effect on arteries. For example, TGF- expression is increased in human restenotic lesions as well as in neointimal hyperplasia after balloon injury in animals.3 TGF- facilitates extracellular matrix deposition by stimulating production of procollagen and fibronectin, downregulating the expression of proteases, and upregulating protease inhibitors, such as plasminogen activator inhibitor type I (PAI-I) and tissue inhibitor of metalloproteinase-1 (TIMP-1).4eC8 TGF- transgene into vascular wall causes fibroproliferative intimal thickening in animal models in the presence or absence of vascular injury.9,10 Moreover, TGF- antagonism by antibody, soluble receptor, or ribozyme reduces constrictive remodeling after balloon injury in animals.11eC13
On the other hand, considerable evidence implies antiatherosclerotic effects of TGF-. TGF- has been shown to inhibit proliferation and migration of vascular smooth muscle cells (SMCs) in vitro.14,15 Inhibition of TGF- signal systemically by use of neutralizing antibody and soluble TGF- receptor type (TR)-II or in T-cells by expressing a dominant-negative TR-II results in an unstable plaque phenotype in mouse models of atherosclerosis.16eC18 SMCs obtained from human atherosclerotic plaques were shown to be defective in the TGF- signal pathway and were resistant to TGF-eCmediated growth suppression and apoptosis.19,20 Furthermore, low blood levels of active TGF- were associated with severity of vascular disease in a manner consistent with an antiatherosclerotic effect of TGF-.21
TGF- elicits its effects via signaling through tetramerization of two different receptor serine/threonine kinases, TR-I and TR-II.22,23 Activation of the receptors leads to phosphorylation of cytoplasmic signal transducers Smad2 and Smad3, classified as so-called receptor-activated Smads (R-Smad). The activated R-Smad heteroligomerizes with Smad4, a common mediator Smad, and the complex is transported to the nucleus where it regulates gene expression. In addition, pathways independent of Smads, which involve MAP kinases have also been described.23 In mice lacking TGF- signaling molecules, ie, TR-I and TR-II, Smad2 and Smad4 turned out to be embryonic lethal.24eC26 However, it was recently found that the mice null for Smad3 survive into adulthood.27
We undertook the present study examining Smad3-null mice in vivo and in vitro to elucidate the precise role of Smad3-dependent TGF- signaling in the vascular response to injury.
Materials and Methods
Reagents
Reagents are described in an expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org.
Mice
The generation of Smad3ex8/ex8 null mice by homologous recombination was described previously.27 See expanded Materials and Methods section for details.
Femoral Artery Injury
Mice femoral arteries were injured by use of photochemically-induced thrombosis method.28 See expanded Materials and Methods section for details.
Histological Evaluation
Fixed femoral artery segments were embedded in paraffin and cut into 5-eeCthick serial sections. Six sections per one irradiated segment at 1-mm intervals were stained with hematoxylin and eosin. Neointima was defined as the region between the lumen and the internal elastic lamina. The media was defined as the region between the internal and external elastic lamina. The cross-sectional areas of intima and media were measured using NIH image version 1.62f (National Institutes of Health, USA). The intima-to-media (I/M) ratio was then calculated, and the mean I/M of all 6 sections per one irradiated segments was determined. The sections with intimal hyperplasia were also subjected to Masson’s trichrome staining and immunohistochemistry. Masson’s trichrome-positive intimal area was analyzed using Photoshop version 7.0 (Adobe). All the measurements were made in blinded manner.
Immunohistochemistry
Immunohistochemistry is described in the expanded Materials and Methods section.
Bone Marrow Transplantation
Bone marrow transplantation (BMT) was performed principally as described previously.29 Briefly, bone marrow cell suspensions obtained from either Smad3-null or wild-type mice thigh bone were treated with ACK lysis buffer (0.155 mol/L ammonium chloride, 0.1 mol/L disodium EDTA, and 0.01 mol/L potassium bicarbonate) to lyse erythrocytes. The cells were intravenously injected to recipient Smad3-null or wild-type mice (1x106 per body) between the age of 6 and 9 weeks, 3 hours after lethal irradiation (8.5 Gy). Engraftment of the transferred bone marrow was confirmed by polymerase chain reaction (PCR) on peripheral blood DNA according to the protocol by Yang et al.26 Femoral artery injury was performed 6 weeks after the bone marrow transfer.
Cell Culture
Mouse aortic SMCs were obtained and cultured as described by Ohmi et al30 (see expanded Materials and Methods section). Experiments were performed on cells after 5 to 10 passages from the primary culture.
Immunocytochemistry
Immunocytochemical staining using antieC-smooth muscle actin (SMA) and smooth muscle myosin (SMM) antibodies was performed as described by Hasegawa et al31 with some modification (see expanded Materials and Methods section).
Immunoblotting
Immunoblotting was essentially performed as previously described32 (see expanded Materials and Methods section).
Growth Inhibition Assay
Growth inhibition assay was performed as described by Datto et al33 (see expanded Materials and Methods section).
Cell Migration Assay
SMC migration was evaluated by modified Boyden chamber method34 (see expanded Materials and Methods section).
Real-Time Quantitative PCR
Real-time quantitative PCR is described in expanded Materials and Methods section.
Gelatin Zymography
Gelatin zymography is described in the expanded Materials and Methods section.
Statistical Analysis
Results were presented as mean±SEM. Statistical analyses used two-tailed, unpaired student t test.
Results
Mice Lacking Smad3 Show Enhanced Neontimal Hyperplasia in Response to Injury
To evaluate a role of Smad3 in the pathogenesis of neointimal hyperplasia, femoral arteries of wild-type (n=12) and Smad3-null (n=10) male mice were injured by use of the photochemically-induced thrombosis method.28 Histopathological examination of the arteries 1 to 3 weeks after the injury revealed markedly enhanced neointimal thickening in Smad3-null mice compared with wild-type mice (Figure 1A and 1B). As shown in Figure 1C, mean I/M ratios evaluated at 1 and 3 weeks after the injury were significantly higher in Smad3-null arteries (0.193±0.034 at 1 week and 0.541±0.093 at 3 weeks) than those of wild-type arteries (0.059±0.018 at 1 week and 0.115±0.060 at 3 weeks, P<0.01 at each time point).
Immunohistochemical examination showed that both neointimal and medial cells were positive for -SMA (Figure 2A and 2B) but negative for pan-leukocyte marker CD45 (Figure 2C and 2D), indicating that the intima was exclusively composed of SMCs. The same anti-CD45 antibody recognized leukocytes in vasa vasorum (Figure 2D) as well as lymphocytes in the mouse spleen (Figure 2E).
TGF- is well known for its antiinflammatory effect.1,2 To determine whether systemic inflammation due to Smad3 deficiency contributes to enhanced neointimal formation, we injured femoral artery of wild-type and Smad3-null mice after bone marrow transplantation (BMT). Lethally irradiated Smad3-null mice received 1x106 bone marrow cells from a wild-type mouse (BMTwildnull mice). At the same time, irradiated wild-type mice were given bone marrow either from Smad3-null or wild-type mice (BMTnullwild and BMTwildwild mice). Photochemical injury was performed 6 weeks after the bone marrow transfer, and the arterial cross section was analyzed 3 weeks later. As shown in Figure 1D, mean I/M ratio was significantly higher in BMTwildnull arteries (0.353±0.091) than those of BMTnullwild (0.067±0.031, P=0.011) or BMTwildwild (0.073±0.018, P=0.039) arteries. I/M ratios in BMTwildnull and BMTnullwild mice tended to be lower than those of Smad3-null and wild-type mice, respectively, presumably due to the effect of vascular irradiation.35,36 Representative cross sections of BMTnullwild and BMTwildnull femoral arteries are shown in Figure 1E and 1F.
Smad3-Null Intima Is Rich in Proliferating Cells but Contains Low Amounts of Collagen Fibers
Intimal cell proliferation was assessed by immunohistochemical detection of proliferating cell nuclear antigen (PCNA) in the femoral artery sections 1 week after the injury (Figure 3A and 3B). The ratio of the PCNA-positive nuclei to total cell nuclei was higher by 1.8-fold in Smad3-null intima compared with wild-type intima (Figure 3C). The result shows an increased proliferative activity of SMCs in Smad3-null artery at the early stage after injury.
We next evaluated intimal cell density in hematoxylin and eosineCstained arterial sections 3 weeks after the injury. As shown in Figure 4A, the ratio of intimal cell number to total intimal area was 1.6-fold higher in Smad3-null artery (133±8.6) compared with wild-type artery (85.3±7.7, P<0.01), indicating higher cell density relative to extracellular area in Smad3-null intima. Because TGF-/Smad3 signal is implicated in extracellular matrix (ECM) deposition, Masson’s trichrome staining was also performed on a 3-week artery specimen to evaluate the amount of extracellular collagen fibers (Figures 4C and 4D). As summarized in Figure 4B, Smad3-null neointima showed 60% reduction in the ratio of Masson’s trichrome-positive area to total intimal area compared with that of wild-type intima. These results suggest that Smad3 deficiency caused increased SMC number with less collagen deposition in neointima.
Growth Inhibition by TGF- Is Attenuated in SMCs Lacking Smad3
To identify the mechanisms by which Smad3 deficiency caused exaggerated intimal hyperplasia, biological responses of the aortic SMCs obtained from wild-type and Smad3-null mice were examined in vitro. The cells were positive for both -SMA and SMM (Figure 5A and 5B) as examined by immunocytochemistry. They also exhibited the classic "hills and valley" appearance, a feature characteristic of confluent cultured vascular SMCs. No morphological differences were observed between wild-type and Smad3-null SMCs (data not shown). It was confirmed by immunoblotting that SMCs derived from Smad3-null mice lacked expression of Smad3, whereas Smad2 level was similar in both cells (Figure 5C).
The SMCs were first tested for proliferation. As shown in Figure 6A, TGF- dose-dependently inhibited FBS-stimulated DNA synthesis in wild-type SMCs with the maximal inhibition of 70% at 1 ng/mL and higher doses. In contrast, growth of Smad3-null SMCs was only weakly (<30%) inhibited by TGF-. In addition, the basal growth rate of the null cells was 1.4-fold higher than that of the wild-type. Similar results were obtained for two additional cell lines of each genotype. The results firmly establish an essential role for Smad3 in TGF-eCmediated inhibition of cellular proliferation in vascular SMCs.
Smad3 Deficiency Does Not Attenuate TGF-eCMediated Migratory Response in SMCs
The cells were next examined for migration, another function crucial to neointimal formation. Aschcroft et al37 previously reported that Smad3-null monocytes and neutrophils were unable to migrate toward TGF-, suggesting Smad3 is required for migration signal downstream of TGF-. As shown in Figure 6B, Smad3-null SMCs dose-dependently migrated toward TGF- at least to a similar extent as wild-type SMCs in a modified Boyden chamber assay. Moreover, Smad3-null cells showed a higher migratory capacity (P<0.05) than wild-type cells at 10 ng/mL TGF-. The result suggests that Smad3-dependent signal is not essential for TGF-eCinduced chemotaxis in murine vascular SMCs.
SMCs Require Smad3 for the Regulation of Type I Collagen, Matrix Metalloproteinases, and TIMP-1 by TGF-
Previous studies suggested that migration of medial SMCs to intima involves extracellular matrix degradation.38,39 Because TGF- is implicated in extracellular matrix metabolism through transcriptional regulation of collagens, matrix metalloproteinases (MMPs), and TIMP-1,7,8 we examined the ability of TGF- to regulate mRNA expression of these components in wild-type and Smad3-null SMC. Transcript levels of COL1A2, membrane-type matrix metalloproteinase 1 (MT1-MMP), and TIMP-1 were evaluated by real-time quantitative PCR. As shown in Figure 7A, TGF- time-dependently upregulated mRNA level of COL1A2 in wild-type SMCs with a maximal increase of 3-fold. Induction of COL1A2 by TGF- was significantly less in Smad3-null SMCs compared with wild-type cells at all time points. TGF- suppressed mRNA expression of MT1-MMP, an activator of proeCMMP-2,40 to 64% of the basal level in wild-type SMCs (Figure 7B). However, MT1-MMP level was not affected by TGF- in Smad3-null SMCs. Moreover, TGF- increased TIMP-1 expression by 5-fold over the basal level in wild-type SMCs (Figure 7C), whereas no significant induction was observed in Smad3-null SMCs. Finally, the effect of TGF- on MMP activity in SMC culture media was examined by gelatin zymography (Figure 7D). The basal gelatinolytic activity of MMP-2 in a serum-free conditioned media was similar for wild-type and Smad3-null SMCs. TGF- time-dependently suppressed MMP-2 activity in wild-type cells with the maximal suppression of 29% at 24 hours, but it did not show significant effect in Smad3-null SMCs. These results suggest that Smad3 plays an essential role in TGF-eCmediated regulation of type I collagen, MMPs, and TIMP-1 in vascular SMCs.
Discussion
We report six novel findings in this article. First, mice lacking Smad3 showed a significant enhancement of neointimal hyperplasia on endothelial injury compared with corresponding wild-type mice. Second, neointima of Smad3-null mouse after injury contained a larger number of PCNA-positive cells compared with wild-type, indicating an increased proliferative activity of Smad3-null SMCs in vivo. Third, Smad3-null neointima showed higher cell density with reduced collagen area. Fourth, TGF-eCinduced growth inhibition was diminished in Smad3-null SMCs in vitro. Fifth, Smad3-null SMCs retained migratory activity toward TGF-. And finally, Smad3-null SMCs were impaired in induction of type I collagen and TIMP-1 as well as in suppression of MMPs by TGF-. These results confirm a regulatory role of endogenous Smad3 in vascular remodeling in response to injury.
Enhanced neointimal hyperplasia in Smad3-null mice (Figure 1) lend support to previous reports describing the association of low TGF- activity either at the ligand or receptor levels with intimal lesion formation. Grainger et al41 showed that transgenic expression of apolipoprotein(a) promoted SMC proliferation and subsequent development of early vascular lesions by inhibiting proteolytic activation of TGF-. Conversely, treatment with the antiestrogen tamoxifen increased serum TGF-1 levels and suppressed the formation of aortic lesions in mice42; a similar effect was also observed in human subjects.43 McCaffrey et al19 reported that reduced TR-II activity due to genomic mutations led to SMC expansion in human atherosclerosis. Moreover, inhibition of TGF- by use of a soluble type II receptor or a neutralizing antibody accelerated atherosclerosis and induced an unstable plaque phenotype in apoE-deficient mice.17,18 And our present findings, for the first time, demonstrate a direct evidence that attenuation of TGF- signal at the postreceptor level results in enhanced neointimal formation on injury.
Increased PCNA-positive intimal cells in vivo (Figure 3) and defect in TGF-eCinduced growth suppression in vitro (Figure 6A) suggest that increased proliferative activity of SMCs contributes to the prominent neointimal formation in Smad3-null mice. Importance of Smad3 in TGF-eCmediated growth inhibition has well been described in other cell types such as CD-stimulated primary splenocytes and embryonic fibroblasts.33 Our results verify that Smad3, also in vascular SMCs, plays a major role in growth inhibitory function of TGF-. It is to be noted that lack of Smad3 did not eliminate TGF-eCinduced growth suppression in SMCs (Figure 6A). The residual growth inhibitory activity is likely to depend on another mediator downstream of TGF- receptors, possibly Smad2.
Ashcroft et al37 reported that Smad3 is required for TGF-eCinduced migration of monocytes, leukocytes, and keratinocytes. Unexpectedly, Smad3-null SMCs were able to migrate toward TGF- (Figure 6B). The finding suggests that, in contrast to the growth inhibitory function, Smad3-dependent signal is not essential for chemotaxis by TGF- in murine vascular SMCs. It is therefore likely that the ability of medial SMCs to migrate into intima is preserved in Smad3-null arteries. The signaling pathway responsible for TGF-eCinduced SMC motility remains to be elucidated.
TGF- is known as a potent inducer of ECM deposition. It has been demonstrated that overexpression and intravenous administration of TGF- caused arterial intimal thickening largely consisted of increased ECM.10,44 TGF- exerts fibrogenic activity through enhancement of ECM synthesis as well as inhibition of ECM degradation by downregulating MMP expression and upregulating MMP inhibitors.6eC8 Previous studies, mainly performed on dermal fibroblasts, showed that TGF-eCmediated regulation of many ECM-related genes, such as type I, III, V, and VI collagens, TIMP-1 and MMP-1 was Smad3-dependent.45eC47 In this study, we reported that Smad3-null neointima was rich in SMCs with relatively less matrix-deposition compared with wild-type intima, as evaluated by intimal cell density and Masson’s trichrome staining (Figure 4), confirming a crucial role of Smad3-dependent signals in vascular ECM regulation. Moreover, TGF- was unable to enhance mRNA expression of COL1A2 and TIMP-1 or suppress MT1-MMP expression in Smad3-null SMCs (Figure 7), establishing Smad3-dependency of these genes in vascular SMCs. Regulation of MMP-2 or gelatinase also seems to depend on Smad3-pathway in SMCs, because TGF- attenuated MMP-2 activity in the culture media of wild-type but not in Smad3-null SMCs. Because degradation of matrix scaffold by MMPs enables cell movement and general tissue reorganization,38,39 inability of TGF- to suppress MMPs in Smad3-null SMCs may facilitate cell migration from media to intima in vivo.48 Our in vitro finding that Smad3-null SMCs show a higher migration than wild-type at 10 ng/mL TGF- (Figure 6B) may support this idea. MMP activity uninhibited by TGF- as well as decreased matrix deposition might also have contributed to enhancement of intimal thickening in Smad3-null mice.
There have been reports on injury models suggesting that TGF- promotes intimal thickening.3,9eC13,49 The present result that Smad3 deficiency accelerates intimal response to injury appears inconsistent with these results. However, we do not think that our findings contradict other reports on TGF- transgene or antagonism. Our model differs from any other previous models in the point it specifically lacks Smad3 signal but not other TGF- signal components, eg, Smad2 and MAP kinases. Smad3 not only transduces signal downstream of TGF-, but also plays a major role in signaling of activins,22,23 other members of the TGF- superfamily. Activin A is expressed in atherosclerotic lesion50 and promotes the contractile or nonproliferative phenotype of SMCs,51 playing a role in stabilization of atherosclerotic plaque. Adenovirus-mediated overexpression of activin A suppresses neointimal formation.51 Although we have not examined the involvement of activin A in the present study, it is assumable that the defect in activin A signal in addition to TGF- accounts for the drastic neointimal hyperplasia in Smad3-null mice. It is of interest to determine whether specific activation of Smad3 in arterial SMCs in vivo attenuates neointimal hyperplasia. As another possibility, proinflammatory status caused by systemic Smad3 deficiency27 might have influenced neointimal response. Although our BMT results (Figure 2D through 2F) show that the degree of intimal hyperplasia mainly depends on the origin of blood vessels and not of bone marrow cells, further investigation is needed to elucidate the entire role of inflammation in Smad3-null vascular response.
Finally, overactivation of TGF-eCSmad3 pathway is implicated in various fibrotic diseases involving organs such as skin, lung, liver, and kidney. Molecular agents that block Smad3-dependent TGF- signal are anticipated as an ideal therapeutic option for these disorders.46 However, our present results lead us to surmise that systemic suppression of Smad3 signaling can cause undesirable effects in the arteries by facilitating proliferative intimal lesions. Therefore, selective drug-delivery to the affected organs as well as careful monitoring of possible vascular lesions should be considered on clinical application of Smad3 inhibitors for fibrotic diseases.
In conclusion, mice lacking Smad3 developed marked neointimal hyperplasia on injury accompanying modulation of growth and matrix regulation in vascular SMCs. This study documents direct evidence and novel information on the functional significance: a vasculoprotective role of Smad3-dependent TGF- signaling in response to injury.
Acknowledgments
This study is supported by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Health, Labor and Welfare, Japan Heart Foundation, grants from Mitsui Sumitomo Welfare Foundation and NOVARTIS Foundation for Gerontological Research to Koutaro Yokote. We thank Drs A. Roberts and C. Deng (National Institutes of Health, USA) for providing us with heterozygous mice for Smad3 disruption, Drs K. Harigaya and M. Higashi for valuable advice on histological examination, Drs K. Sonezaki and T. Tokuyama for fruitful discussion, and A. Takada for technical assistance.
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Ashcroft GS, Yang X, Glick AB, Weinstein M, Letterio JL, Mizel DE, Anzano M, Greenwell-Wild T, Wahl SM, Deng C, Roberts AB. Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nat Cell Biol. 1999; 1: 260eC266.
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Engelse MA, Neele JM, van Achterberg TA, van Aken BE, van Schaik RH, Pannekoek H, de Vries CJ. Human activin-A is expressed in the atherosclerotic lesion and promotes the contractile phenotype of smooth muscle cells. Circ Res. 1999; 85: 931eC939.(Kazuki Kobayashi, Koutaro)
Division of Endocrinology and Metabolism (K.Y., Y.S.), Department of Internal Medicine, Chiba University Hospital
Department of Developmental Genetics (K.Y., A.S., T.T.), Chiba University Graduate School of Medicine, Chiba, Japan
Shiraoka Research Station of Biological Science (M.K.), Nissan Chemical Industries, Ltd, Saitama, Japan.
Abstract
The role of transforming growth factor (TGF)- and its signal in atherogenesis is not fully understood. Here, we examined mice lacking Smad3, a major downstream mediator of TGF-, to clarify the precise role of Smad3-dependent signaling in vascular response to injury. Femoral arteries were injured in wild-type and Smad3-null (null) male mice on C57Bl/6 background. Histopathological evaluation of the arteries 1 to 3 weeks after the injury revealed significant enhancement of neointimal hyperplasia in null compared with wild-type mice. Transplantation of null bone marrow to wild-type mice did not enhance neointimal thickening, suggesting that vascular cells in situ play a major role in the response. Null intima contained more proliferating smooth muscle cells (SMC) with less amount of collagen compared with wild-type intima. TGF- caused significant inhibition of cellular proliferation in wild-type aortic SMC, whereas the growth of null SMC was only weakly inhibited by TGF- in vitro, indicating a crucial role of Smad3 in the growth inhibitory function. On the other hand, Smad3-deficiency did not attenuate chemotaxis of SMC toward TGF-. TGF- increased transcript level of 2 type I collagen and tissue inhibitor of metalloproteinases-1, and suppressed expression and activity of matrix metalloproteinases in wild-type SMC. However, these effects of TGF- were diminished in null SMC. Our findings altogether show that the loss of Smad3 pathway causes enhanced neointimal hyperplasia on injury through modulation of growth and matrix regulation in vascular SMC. These results indicate a vasculoprotective role of endogenous Smad3 in response to injury.
Key Words: transforming growth factor- Smad3 atherosclerosis neointimal hyperplasia smooth muscle cells
Introduction
Transforming growth factor (TGF)- is a prototypic member of the TGF- superfamily that exerts a wide range of biological effects on various cell types.1 Well described functions of TGF- including growth inhibition, cell migration, differentiation, extracellular matrix production, and immunomodulation. Abnormality in TGF- signaling may cause pathological conditions such as tumorigenesis, fibrotic disorders, and vascular diseases.2 At present, however, the role of TGF- and its signaling molecules in atherogenesis is not fully understood.
TGF- is often regarded to have proatherosclerotic effect on arteries. For example, TGF- expression is increased in human restenotic lesions as well as in neointimal hyperplasia after balloon injury in animals.3 TGF- facilitates extracellular matrix deposition by stimulating production of procollagen and fibronectin, downregulating the expression of proteases, and upregulating protease inhibitors, such as plasminogen activator inhibitor type I (PAI-I) and tissue inhibitor of metalloproteinase-1 (TIMP-1).4eC8 TGF- transgene into vascular wall causes fibroproliferative intimal thickening in animal models in the presence or absence of vascular injury.9,10 Moreover, TGF- antagonism by antibody, soluble receptor, or ribozyme reduces constrictive remodeling after balloon injury in animals.11eC13
On the other hand, considerable evidence implies antiatherosclerotic effects of TGF-. TGF- has been shown to inhibit proliferation and migration of vascular smooth muscle cells (SMCs) in vitro.14,15 Inhibition of TGF- signal systemically by use of neutralizing antibody and soluble TGF- receptor type (TR)-II or in T-cells by expressing a dominant-negative TR-II results in an unstable plaque phenotype in mouse models of atherosclerosis.16eC18 SMCs obtained from human atherosclerotic plaques were shown to be defective in the TGF- signal pathway and were resistant to TGF-eCmediated growth suppression and apoptosis.19,20 Furthermore, low blood levels of active TGF- were associated with severity of vascular disease in a manner consistent with an antiatherosclerotic effect of TGF-.21
TGF- elicits its effects via signaling through tetramerization of two different receptor serine/threonine kinases, TR-I and TR-II.22,23 Activation of the receptors leads to phosphorylation of cytoplasmic signal transducers Smad2 and Smad3, classified as so-called receptor-activated Smads (R-Smad). The activated R-Smad heteroligomerizes with Smad4, a common mediator Smad, and the complex is transported to the nucleus where it regulates gene expression. In addition, pathways independent of Smads, which involve MAP kinases have also been described.23 In mice lacking TGF- signaling molecules, ie, TR-I and TR-II, Smad2 and Smad4 turned out to be embryonic lethal.24eC26 However, it was recently found that the mice null for Smad3 survive into adulthood.27
We undertook the present study examining Smad3-null mice in vivo and in vitro to elucidate the precise role of Smad3-dependent TGF- signaling in the vascular response to injury.
Materials and Methods
Reagents
Reagents are described in an expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org.
Mice
The generation of Smad3ex8/ex8 null mice by homologous recombination was described previously.27 See expanded Materials and Methods section for details.
Femoral Artery Injury
Mice femoral arteries were injured by use of photochemically-induced thrombosis method.28 See expanded Materials and Methods section for details.
Histological Evaluation
Fixed femoral artery segments were embedded in paraffin and cut into 5-eeCthick serial sections. Six sections per one irradiated segment at 1-mm intervals were stained with hematoxylin and eosin. Neointima was defined as the region between the lumen and the internal elastic lamina. The media was defined as the region between the internal and external elastic lamina. The cross-sectional areas of intima and media were measured using NIH image version 1.62f (National Institutes of Health, USA). The intima-to-media (I/M) ratio was then calculated, and the mean I/M of all 6 sections per one irradiated segments was determined. The sections with intimal hyperplasia were also subjected to Masson’s trichrome staining and immunohistochemistry. Masson’s trichrome-positive intimal area was analyzed using Photoshop version 7.0 (Adobe). All the measurements were made in blinded manner.
Immunohistochemistry
Immunohistochemistry is described in the expanded Materials and Methods section.
Bone Marrow Transplantation
Bone marrow transplantation (BMT) was performed principally as described previously.29 Briefly, bone marrow cell suspensions obtained from either Smad3-null or wild-type mice thigh bone were treated with ACK lysis buffer (0.155 mol/L ammonium chloride, 0.1 mol/L disodium EDTA, and 0.01 mol/L potassium bicarbonate) to lyse erythrocytes. The cells were intravenously injected to recipient Smad3-null or wild-type mice (1x106 per body) between the age of 6 and 9 weeks, 3 hours after lethal irradiation (8.5 Gy). Engraftment of the transferred bone marrow was confirmed by polymerase chain reaction (PCR) on peripheral blood DNA according to the protocol by Yang et al.26 Femoral artery injury was performed 6 weeks after the bone marrow transfer.
Cell Culture
Mouse aortic SMCs were obtained and cultured as described by Ohmi et al30 (see expanded Materials and Methods section). Experiments were performed on cells after 5 to 10 passages from the primary culture.
Immunocytochemistry
Immunocytochemical staining using antieC-smooth muscle actin (SMA) and smooth muscle myosin (SMM) antibodies was performed as described by Hasegawa et al31 with some modification (see expanded Materials and Methods section).
Immunoblotting
Immunoblotting was essentially performed as previously described32 (see expanded Materials and Methods section).
Growth Inhibition Assay
Growth inhibition assay was performed as described by Datto et al33 (see expanded Materials and Methods section).
Cell Migration Assay
SMC migration was evaluated by modified Boyden chamber method34 (see expanded Materials and Methods section).
Real-Time Quantitative PCR
Real-time quantitative PCR is described in expanded Materials and Methods section.
Gelatin Zymography
Gelatin zymography is described in the expanded Materials and Methods section.
Statistical Analysis
Results were presented as mean±SEM. Statistical analyses used two-tailed, unpaired student t test.
Results
Mice Lacking Smad3 Show Enhanced Neontimal Hyperplasia in Response to Injury
To evaluate a role of Smad3 in the pathogenesis of neointimal hyperplasia, femoral arteries of wild-type (n=12) and Smad3-null (n=10) male mice were injured by use of the photochemically-induced thrombosis method.28 Histopathological examination of the arteries 1 to 3 weeks after the injury revealed markedly enhanced neointimal thickening in Smad3-null mice compared with wild-type mice (Figure 1A and 1B). As shown in Figure 1C, mean I/M ratios evaluated at 1 and 3 weeks after the injury were significantly higher in Smad3-null arteries (0.193±0.034 at 1 week and 0.541±0.093 at 3 weeks) than those of wild-type arteries (0.059±0.018 at 1 week and 0.115±0.060 at 3 weeks, P<0.01 at each time point).
Immunohistochemical examination showed that both neointimal and medial cells were positive for -SMA (Figure 2A and 2B) but negative for pan-leukocyte marker CD45 (Figure 2C and 2D), indicating that the intima was exclusively composed of SMCs. The same anti-CD45 antibody recognized leukocytes in vasa vasorum (Figure 2D) as well as lymphocytes in the mouse spleen (Figure 2E).
TGF- is well known for its antiinflammatory effect.1,2 To determine whether systemic inflammation due to Smad3 deficiency contributes to enhanced neointimal formation, we injured femoral artery of wild-type and Smad3-null mice after bone marrow transplantation (BMT). Lethally irradiated Smad3-null mice received 1x106 bone marrow cells from a wild-type mouse (BMTwildnull mice). At the same time, irradiated wild-type mice were given bone marrow either from Smad3-null or wild-type mice (BMTnullwild and BMTwildwild mice). Photochemical injury was performed 6 weeks after the bone marrow transfer, and the arterial cross section was analyzed 3 weeks later. As shown in Figure 1D, mean I/M ratio was significantly higher in BMTwildnull arteries (0.353±0.091) than those of BMTnullwild (0.067±0.031, P=0.011) or BMTwildwild (0.073±0.018, P=0.039) arteries. I/M ratios in BMTwildnull and BMTnullwild mice tended to be lower than those of Smad3-null and wild-type mice, respectively, presumably due to the effect of vascular irradiation.35,36 Representative cross sections of BMTnullwild and BMTwildnull femoral arteries are shown in Figure 1E and 1F.
Smad3-Null Intima Is Rich in Proliferating Cells but Contains Low Amounts of Collagen Fibers
Intimal cell proliferation was assessed by immunohistochemical detection of proliferating cell nuclear antigen (PCNA) in the femoral artery sections 1 week after the injury (Figure 3A and 3B). The ratio of the PCNA-positive nuclei to total cell nuclei was higher by 1.8-fold in Smad3-null intima compared with wild-type intima (Figure 3C). The result shows an increased proliferative activity of SMCs in Smad3-null artery at the early stage after injury.
We next evaluated intimal cell density in hematoxylin and eosineCstained arterial sections 3 weeks after the injury. As shown in Figure 4A, the ratio of intimal cell number to total intimal area was 1.6-fold higher in Smad3-null artery (133±8.6) compared with wild-type artery (85.3±7.7, P<0.01), indicating higher cell density relative to extracellular area in Smad3-null intima. Because TGF-/Smad3 signal is implicated in extracellular matrix (ECM) deposition, Masson’s trichrome staining was also performed on a 3-week artery specimen to evaluate the amount of extracellular collagen fibers (Figures 4C and 4D). As summarized in Figure 4B, Smad3-null neointima showed 60% reduction in the ratio of Masson’s trichrome-positive area to total intimal area compared with that of wild-type intima. These results suggest that Smad3 deficiency caused increased SMC number with less collagen deposition in neointima.
Growth Inhibition by TGF- Is Attenuated in SMCs Lacking Smad3
To identify the mechanisms by which Smad3 deficiency caused exaggerated intimal hyperplasia, biological responses of the aortic SMCs obtained from wild-type and Smad3-null mice were examined in vitro. The cells were positive for both -SMA and SMM (Figure 5A and 5B) as examined by immunocytochemistry. They also exhibited the classic "hills and valley" appearance, a feature characteristic of confluent cultured vascular SMCs. No morphological differences were observed between wild-type and Smad3-null SMCs (data not shown). It was confirmed by immunoblotting that SMCs derived from Smad3-null mice lacked expression of Smad3, whereas Smad2 level was similar in both cells (Figure 5C).
The SMCs were first tested for proliferation. As shown in Figure 6A, TGF- dose-dependently inhibited FBS-stimulated DNA synthesis in wild-type SMCs with the maximal inhibition of 70% at 1 ng/mL and higher doses. In contrast, growth of Smad3-null SMCs was only weakly (<30%) inhibited by TGF-. In addition, the basal growth rate of the null cells was 1.4-fold higher than that of the wild-type. Similar results were obtained for two additional cell lines of each genotype. The results firmly establish an essential role for Smad3 in TGF-eCmediated inhibition of cellular proliferation in vascular SMCs.
Smad3 Deficiency Does Not Attenuate TGF-eCMediated Migratory Response in SMCs
The cells were next examined for migration, another function crucial to neointimal formation. Aschcroft et al37 previously reported that Smad3-null monocytes and neutrophils were unable to migrate toward TGF-, suggesting Smad3 is required for migration signal downstream of TGF-. As shown in Figure 6B, Smad3-null SMCs dose-dependently migrated toward TGF- at least to a similar extent as wild-type SMCs in a modified Boyden chamber assay. Moreover, Smad3-null cells showed a higher migratory capacity (P<0.05) than wild-type cells at 10 ng/mL TGF-. The result suggests that Smad3-dependent signal is not essential for TGF-eCinduced chemotaxis in murine vascular SMCs.
SMCs Require Smad3 for the Regulation of Type I Collagen, Matrix Metalloproteinases, and TIMP-1 by TGF-
Previous studies suggested that migration of medial SMCs to intima involves extracellular matrix degradation.38,39 Because TGF- is implicated in extracellular matrix metabolism through transcriptional regulation of collagens, matrix metalloproteinases (MMPs), and TIMP-1,7,8 we examined the ability of TGF- to regulate mRNA expression of these components in wild-type and Smad3-null SMC. Transcript levels of COL1A2, membrane-type matrix metalloproteinase 1 (MT1-MMP), and TIMP-1 were evaluated by real-time quantitative PCR. As shown in Figure 7A, TGF- time-dependently upregulated mRNA level of COL1A2 in wild-type SMCs with a maximal increase of 3-fold. Induction of COL1A2 by TGF- was significantly less in Smad3-null SMCs compared with wild-type cells at all time points. TGF- suppressed mRNA expression of MT1-MMP, an activator of proeCMMP-2,40 to 64% of the basal level in wild-type SMCs (Figure 7B). However, MT1-MMP level was not affected by TGF- in Smad3-null SMCs. Moreover, TGF- increased TIMP-1 expression by 5-fold over the basal level in wild-type SMCs (Figure 7C), whereas no significant induction was observed in Smad3-null SMCs. Finally, the effect of TGF- on MMP activity in SMC culture media was examined by gelatin zymography (Figure 7D). The basal gelatinolytic activity of MMP-2 in a serum-free conditioned media was similar for wild-type and Smad3-null SMCs. TGF- time-dependently suppressed MMP-2 activity in wild-type cells with the maximal suppression of 29% at 24 hours, but it did not show significant effect in Smad3-null SMCs. These results suggest that Smad3 plays an essential role in TGF-eCmediated regulation of type I collagen, MMPs, and TIMP-1 in vascular SMCs.
Discussion
We report six novel findings in this article. First, mice lacking Smad3 showed a significant enhancement of neointimal hyperplasia on endothelial injury compared with corresponding wild-type mice. Second, neointima of Smad3-null mouse after injury contained a larger number of PCNA-positive cells compared with wild-type, indicating an increased proliferative activity of Smad3-null SMCs in vivo. Third, Smad3-null neointima showed higher cell density with reduced collagen area. Fourth, TGF-eCinduced growth inhibition was diminished in Smad3-null SMCs in vitro. Fifth, Smad3-null SMCs retained migratory activity toward TGF-. And finally, Smad3-null SMCs were impaired in induction of type I collagen and TIMP-1 as well as in suppression of MMPs by TGF-. These results confirm a regulatory role of endogenous Smad3 in vascular remodeling in response to injury.
Enhanced neointimal hyperplasia in Smad3-null mice (Figure 1) lend support to previous reports describing the association of low TGF- activity either at the ligand or receptor levels with intimal lesion formation. Grainger et al41 showed that transgenic expression of apolipoprotein(a) promoted SMC proliferation and subsequent development of early vascular lesions by inhibiting proteolytic activation of TGF-. Conversely, treatment with the antiestrogen tamoxifen increased serum TGF-1 levels and suppressed the formation of aortic lesions in mice42; a similar effect was also observed in human subjects.43 McCaffrey et al19 reported that reduced TR-II activity due to genomic mutations led to SMC expansion in human atherosclerosis. Moreover, inhibition of TGF- by use of a soluble type II receptor or a neutralizing antibody accelerated atherosclerosis and induced an unstable plaque phenotype in apoE-deficient mice.17,18 And our present findings, for the first time, demonstrate a direct evidence that attenuation of TGF- signal at the postreceptor level results in enhanced neointimal formation on injury.
Increased PCNA-positive intimal cells in vivo (Figure 3) and defect in TGF-eCinduced growth suppression in vitro (Figure 6A) suggest that increased proliferative activity of SMCs contributes to the prominent neointimal formation in Smad3-null mice. Importance of Smad3 in TGF-eCmediated growth inhibition has well been described in other cell types such as CD-stimulated primary splenocytes and embryonic fibroblasts.33 Our results verify that Smad3, also in vascular SMCs, plays a major role in growth inhibitory function of TGF-. It is to be noted that lack of Smad3 did not eliminate TGF-eCinduced growth suppression in SMCs (Figure 6A). The residual growth inhibitory activity is likely to depend on another mediator downstream of TGF- receptors, possibly Smad2.
Ashcroft et al37 reported that Smad3 is required for TGF-eCinduced migration of monocytes, leukocytes, and keratinocytes. Unexpectedly, Smad3-null SMCs were able to migrate toward TGF- (Figure 6B). The finding suggests that, in contrast to the growth inhibitory function, Smad3-dependent signal is not essential for chemotaxis by TGF- in murine vascular SMCs. It is therefore likely that the ability of medial SMCs to migrate into intima is preserved in Smad3-null arteries. The signaling pathway responsible for TGF-eCinduced SMC motility remains to be elucidated.
TGF- is known as a potent inducer of ECM deposition. It has been demonstrated that overexpression and intravenous administration of TGF- caused arterial intimal thickening largely consisted of increased ECM.10,44 TGF- exerts fibrogenic activity through enhancement of ECM synthesis as well as inhibition of ECM degradation by downregulating MMP expression and upregulating MMP inhibitors.6eC8 Previous studies, mainly performed on dermal fibroblasts, showed that TGF-eCmediated regulation of many ECM-related genes, such as type I, III, V, and VI collagens, TIMP-1 and MMP-1 was Smad3-dependent.45eC47 In this study, we reported that Smad3-null neointima was rich in SMCs with relatively less matrix-deposition compared with wild-type intima, as evaluated by intimal cell density and Masson’s trichrome staining (Figure 4), confirming a crucial role of Smad3-dependent signals in vascular ECM regulation. Moreover, TGF- was unable to enhance mRNA expression of COL1A2 and TIMP-1 or suppress MT1-MMP expression in Smad3-null SMCs (Figure 7), establishing Smad3-dependency of these genes in vascular SMCs. Regulation of MMP-2 or gelatinase also seems to depend on Smad3-pathway in SMCs, because TGF- attenuated MMP-2 activity in the culture media of wild-type but not in Smad3-null SMCs. Because degradation of matrix scaffold by MMPs enables cell movement and general tissue reorganization,38,39 inability of TGF- to suppress MMPs in Smad3-null SMCs may facilitate cell migration from media to intima in vivo.48 Our in vitro finding that Smad3-null SMCs show a higher migration than wild-type at 10 ng/mL TGF- (Figure 6B) may support this idea. MMP activity uninhibited by TGF- as well as decreased matrix deposition might also have contributed to enhancement of intimal thickening in Smad3-null mice.
There have been reports on injury models suggesting that TGF- promotes intimal thickening.3,9eC13,49 The present result that Smad3 deficiency accelerates intimal response to injury appears inconsistent with these results. However, we do not think that our findings contradict other reports on TGF- transgene or antagonism. Our model differs from any other previous models in the point it specifically lacks Smad3 signal but not other TGF- signal components, eg, Smad2 and MAP kinases. Smad3 not only transduces signal downstream of TGF-, but also plays a major role in signaling of activins,22,23 other members of the TGF- superfamily. Activin A is expressed in atherosclerotic lesion50 and promotes the contractile or nonproliferative phenotype of SMCs,51 playing a role in stabilization of atherosclerotic plaque. Adenovirus-mediated overexpression of activin A suppresses neointimal formation.51 Although we have not examined the involvement of activin A in the present study, it is assumable that the defect in activin A signal in addition to TGF- accounts for the drastic neointimal hyperplasia in Smad3-null mice. It is of interest to determine whether specific activation of Smad3 in arterial SMCs in vivo attenuates neointimal hyperplasia. As another possibility, proinflammatory status caused by systemic Smad3 deficiency27 might have influenced neointimal response. Although our BMT results (Figure 2D through 2F) show that the degree of intimal hyperplasia mainly depends on the origin of blood vessels and not of bone marrow cells, further investigation is needed to elucidate the entire role of inflammation in Smad3-null vascular response.
Finally, overactivation of TGF-eCSmad3 pathway is implicated in various fibrotic diseases involving organs such as skin, lung, liver, and kidney. Molecular agents that block Smad3-dependent TGF- signal are anticipated as an ideal therapeutic option for these disorders.46 However, our present results lead us to surmise that systemic suppression of Smad3 signaling can cause undesirable effects in the arteries by facilitating proliferative intimal lesions. Therefore, selective drug-delivery to the affected organs as well as careful monitoring of possible vascular lesions should be considered on clinical application of Smad3 inhibitors for fibrotic diseases.
In conclusion, mice lacking Smad3 developed marked neointimal hyperplasia on injury accompanying modulation of growth and matrix regulation in vascular SMCs. This study documents direct evidence and novel information on the functional significance: a vasculoprotective role of Smad3-dependent TGF- signaling in response to injury.
Acknowledgments
This study is supported by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Health, Labor and Welfare, Japan Heart Foundation, grants from Mitsui Sumitomo Welfare Foundation and NOVARTIS Foundation for Gerontological Research to Koutaro Yokote. We thank Drs A. Roberts and C. Deng (National Institutes of Health, USA) for providing us with heterozygous mice for Smad3 disruption, Drs K. Harigaya and M. Higashi for valuable advice on histological examination, Drs K. Sonezaki and T. Tokuyama for fruitful discussion, and A. Takada for technical assistance.
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