Smad Expression in Human Atherosclerotic Lesions
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《动脉硬化血栓血管生物学》
From the Baker Medical Research Institute (N.K., A.A., A.B.), Alfred Hospital, Melbourne, Victoria, Australia; and the Institute of Experimental Cardiology (N.K., Y.A., O.I., V.S., E.T.), Russian Cardiology Research Industrial Complex, Moscow, Russia.
ABSTRACT
Objective— Transforming growth factor-beta (TGF-?) has been implicated in the pathogenesis of human atherosclerosis but its actions during lesion progression are poorly understood. Smad2, Smad3, and Smad4 proteins are signaling molecules by which TGF-? modulates gene transcription. Our objective was to define the actions of TGF-? during lesion progression in humans by examining the expression of Smads in relation to TGF-?–mediated responses.
Methods and Results— Immunohistochemistry and reverse-transcription polymerase chain reaction demonstrated Smad2, Smad3, and Smad4 expression in macrophages of fibrofatty lesions and their upregulation after differentiation of monocytes to macrophages. The major Smad splice variants expressed by the macrophages were those that are transcriptionally most active. Macrophages also expressed cyclin inhibitors whose expression is induced via Smad proteins. The cytoplasmic location of p21Waf1 suggests it may protect macrophages from apoptosis. Smooth muscle cells (SMCs) within the fibrofatty lesions did not express the Smad proteins or the cyclin inhibitors. SMCs of fibrous plaques expressed all 3 Smad proteins.
Conclusions— In human atherosclerotic lesions, the actions of TGF-? appear restricted to SMCs in fibrous plaques and macrophages in fatty streaks/fibrofatty lesions. The lack of key TGF-? signaling components in SMCs of fibrofatty lesions indicates impaired ability of these cells to initiate TGF-?–mediated Smad-dependent transcriptional responses.
The actions of TGF-? in human atherosclerotic lesions were defined by examining the expression of Smad proteins in relation to TGF-?–mediated responses. Expression of Smad proteins was restricted to macrophages of fibrofatty lesions and SMCs of fibrous plaques. Smad-dependent TGF-? signaling appeared to be impaired SMCs of fibrofatty lesions.
Key Words: Smad ? p15INK4B ? p21Waf1 ? collagen ? human atherosclerotic lesions
Introduction
Transforming growth factor-beta (TGF-?) is a multifunctional cytokine/growth factor capable of regulating the growth, differentiation, and functions of immune and nonimmune cells.1 Although TGF-? is highly expressed in human atherosclerotic lesions, its contribution to lesion development and progression is still unclear.2,3 Expression of TGF-? isoforms and signaling receptors is high in macrophages and smooth muscle cells (SMCs) of aortic fibrofatty lesions.2 Expression is also apparent within the collagen-rich fibrous caps, but unaffected intima does not express the necessary signaling receptors.2 Recent studies in ApoE knockout mice examining the significance of TGF-? in the development of atherosclerosis using antibodies that neutralize TGF-? isoforms4 or a recombinant dominant-negative type II TGF-? receptor to prevent the actions of TGF-?,5 indicate roles in promoting fibrosis and reducing lesion inflammation. However, the significance of the findings for human atherosclerosis is unclear, because removal of whole-body TGF-? augments systemic inflammation.3 A TGF-?1 null mutation in mice causes excessive inflammatory responses and early death.6 Also, abrogation of TGF-? signaling in T cells leads to spontaneous T-cell differentiation and autoimmune disease,7 whereas disruption of TGF-? signaling in bone marrow-derived cells leads to dramatic expansion of myeloid cells, primarily monocytes and macrophages, and is associated with cachexia and mortality.8
TGF-? has the potential to initiate important effects in vessels that can influence the development of atherosclerosis and requires the phosphorylation of 2 intracellular transcription mediators, Smad2 and Smad3. The phosphorylated forms of Smad2 and Smad3 form heteromeric complexes with Smad4, then translocate to the nucleus to participate in regulating gene expression.1 Genes known to be activated by TGF-?/Smad signaling that have the potential to affect lesion characteristics include the 1 chain of type I collagen, the 2 chain of type I collagen, the 1 chain of type III collagen,9,10 and connective tissue growth factor.11 Repression of collagenase-1 by TGF-?/Smad-dependent mechanisms12 and stimulated increases in plasminogen activator inhibitor type 1 gene activity can also affect the characteristics of developing atherosclerotic lesions.13,14 The TGF-?/Smad signaling system has the potential to promote lipid accumulation by stimulating the expression of biglycan,15 a small leucine-rich proteoglycan that binds low-density lipoprotein and high-density lipoprotein.16 TGF-?/Smad signaling also elevates the cyclin-dependent kinase inhibitors,17,18 p15Ink4B and p21Waf1/Cip1, affecting cell proliferative responses and the sensitivity of cells to apoptosis.
Because of the potential importance of the TGF-?/Smad signaling in the development of human atherosclerotic lesions, we examined the expression of Smads that mediate TGF-? responses in normal human aorta and aorta containing atherosclerotic lesions of varying severity. We demonstrate that expression of Smads is highly upregulated in macrophages of fatty streaks/fibrofatty lesions and SMCs in fibrous plaques, but their absence in SMCs within fibrofatty lesions also suggests local impaired TGF-?/Smad signaling, which would contribute to low interstitial collagen content in such lesions. Unlike macrophages in fibrofatty lesions, these SMCs do not express p21Waf1, making them potentially more susceptible to apoptosis.
Methods
Collection of Tissues and Vessel Pathology
Thoracic aortas and coronary arteries were collected during autopsy, not later than 6 hours after death, from 27 male and female individuals, aged 19 to 67 years, at the Russian Cardiology Research Industrial Complex, Moscow. All subjects died of sudden death, not from infectious or other disorders, which could have potentially affected the expression of cellular proteins (please see http://atvb.ahajournals.org for specific details in online supplementary data and online Table I). The severity of atherosclerotic lesions were classified according to AHA guidelines after staining with Oil Red O, hematoxylin and eosin, and/or trichrome.2,19
Cleaned vessel segments were frozen in OCT (Miles Inc, Elkart, Ind) in liquid nitrogen and stored at –80°C. Vessel segments designated for RNA analysis by reverse-transcription polymerase chain reaction (RT-PCR) were dissected free of adventitia and the intima was peeled off from the media before freezing in liquid nitrogen. Histological studies were performed on 20 nonatherosclerotic segments, 25 segments containing fatty streaks/fibrofatty lesions, and 7 segments of fibrous plaques. RNA was extracted from 8 intimas possessing fatty streaks/fibrofatty lesions. The procedures for collecting tissues for the studies were approved by the Russian Cardiology Research Centre’s Human Ethics Experimentation Committee.
Antibodies
Please see http://atvb.ahajournals.org for specific details in online supplementary data.
Immunohistochemistry
The expression of Smad2, Smad3, and Smad4 was examined in frozen 6-μm cross-sections and simultaneously colocalized with CD68 antigen and -SM actin by double immunostaining as described previously (please see online supplementary data at http://atvb.ahajournals.org for details).20
Staining for Collagens Type I and III by Picrosirius Red
Frozen sections from fibrofatty streaks and fibrous plaques were incubated for 90 minutes in 0.1% Sirius red F3BA (Polyscience Inc) in saturated picric acid. After rinsing twice in 0.01N HCl and in distilled water, sections were briefly dehydrated with 70% ethanol and put under coverslips. Staining with Sirius red was analyzed by light microscopy.21
RNA Extraction and Analysis of Smad Splice Variants
RNA was extracted from fibrofatty lesions, fibrous plaques, and cultured cells, then subjected to RT-PCR for analysis of expression of Smad splice variants (please see specific details in online supplementary data at http://atvb.ahajournals.org).
Cell Cultures
The human promonocyte (THP-1) cell line (American Cell Type Collection) was cultured in RPMI-1640 medium containing 10% fetal calf serum and ?-mercaptoethanol. Differentiation to macrophages was induced by 4-day exposure to TGF-?1 (6 ng/mL)21 and the activity of macrophage nonspecific acetate esterase was measured to confirm differentiation to macrophages using a nonspecific acetate esterase kit (Sigma Chemical Corp) according to the manufacturer’s instructions.
Intimal SMCs were isolated from fibrofatty lesions and fibrous plaques of human aortas and subcultured as previously described.20
Statistics
Cell counts were performed by 2 independent observers. Ratios of positive cells were analyzed using Kruskal–Wallis 1-way test on ranks. Differences with P<0.05 were considered as statistically significant. Data are expressed as mean±SEM.
Results
Smads in Nonatherosclerotic Aorta
Immunoreactive Smads known to mediate TGF-? responses, Smad2, Smad3, and Smad4, were not detectable in SMCs of nonatherosclerotic aortas, either in the intima (Figure 1, top) or the media (not shown).
Figure 1. Expression of Smad proteins in human aorta. Top panels, Lack of Smad2, Smad3, and Smad4 expression in the normal SMC-rich intima. Middle panels, Smad2, Smad3, and Smad4 expression (black) in fibrofatty lesions is restricted to CD68-positive macrophages/macrophage-derived foam cells (red, see arrows). Bottom panels, -Actin–positive SMCs (red) within fibrofatty lesions do not express the Smad proteins (black). Original magnification x160.
Expression of Smads in Fatty Streaks/Fibrofatty Lesions
Fibrofatty lesions were defined using previously published criteria2,19,20 using oil red O staining of extracellular and intracellular lipids, hematoxylin and eosin, and/or trichrome (not shown); macrophages expressing Smads were differentiated from SMCs by CD68 antigen expression (Figure 1, middle).2,20 Approximately 90% of the macrophages and macrophage-derived foam cells within fatty streaks/fibrofatty lesions of aorta expressed high concentrations of Smad2, Smad3, and Smad4 (Figure 1, middle). In the macrophages, Smad 2 expression was apparent in 91.9±2.5% of the macrophage population, whereas Smad3- and Smad4-expressing macrophages represented 89.8±5.3% and 85.7±1.1% of the population, respectively. TGF-? appears to contribute to the elevated Smad expression in the macrophages, because during TGF-?–induced differentiation of THP-1 monocytes to macrophages, Smad2, Smad3, and Smad4 increased markedly (Figure 2A). This expression was accompanied by large increases in esterase activity, confirming differentiation (Figure 2B). In contrast, no SMCs, identified as -SM actin-positive cells located in the macrophage-rich regions expressed the 3 Smads (Figure 1, lower), despite previous reports, indicating high expression of TGF-? isoforms and signaling receptors.2 This pattern of Smad expression was also observed in fibrofatty lesions of coronary arteries (Figure I, available online at http://atvb.ahajournals.org). A lack of staining with rabbit and mouse control IgGs confirmed the specificities of the Smad stainings (Figures I and II, available online at http://atvb.ahajournals.org). Because alternative splicing of hn-RNA encoding, Smad4 and Smad2 result in peptides that differ in their DNA binding properties and ability to initiate gene transcription,22,23 we also examined the extent to which the splice variants contribute to immunoreactive Smads in the fibrofatty lesions. RT-PCR of mRNA from fibrofatty lesions using Smad4 oligonucleotide primers, covering most of its MH1 domain, the linker domain, and part of the N-terminus of the MH2 domain, resulted in 3 cDNA fragments (Figure 3A and 3B). The largest fragment accounted for nearly 90% of the PCR product and encoded the MH1, the linker, and MH2 domains. Cycle sequencing of the 587-bp cDNA fragment that accounted for 10% of the PCR products indicated deletions in exons 5 and 6 from the linker regions.23 The minor Smad4 PCR product of 369 bp was the consequence of deletions in exons 4, 5, and 6. Studies in THP-1 promonocytes/macrophages indicated an essentially similar pattern of expression to that observed in the fatty streaks/fibrofatty lesions (Figure 3C). RT-PCR using oligonucleotides for Smad3 mRNA indicated the presence of a major band (584 bp) and minor product (450 bp). The minor PCR product encodes Smad3 mRNA with an exon 3 deletion, which involves the carboxy-terminal part of the MH1 domain and part of the linker domain (Genbank Accession number E33804). RT-PCR of Smad2 mRNA produced 2 cDNA fragments 786-bp (major) and 696-bp (minor) in size. The minor fragment could be accounted for by deletion of exon 3, resulting in a splice variant that binds directly to DNA, in a manner similar to Smad3.22 Thus the major splice variants of Smad2, 3, and 4 expressed in fibrofatty lesions and THP-1 macrophages represented those most active in TGF-?–mediated gene transcription.
Figure 2. Regulation of Smad2, Smad3, and Smad4 expression in THP-1 monocytes during differentiation to macrophages, stimulated by 4-day exposure to TGF-?1. A, Smad2, Smad3, and Smad4 peptides (brown stains) are shown before (control) and after differentiation to macrophages. B, Marked elevation in nonspecific acetate esterase activity after exposure to TGF-?1. Original magnifications x400.
Figure 3. RT-PCR analysis of Smad splice variants in fibrofatty lesions and THP-1 macrophages. RT-PCR products of Smad4 mRNAs (A, B, C) depicted as a major 756-bp fragment and a minor 587-bp fragment with deletions of exons 5 and 6. C, cDNA fragments have been purified and re-amplified to show more clearly their expression in fibrofatty lesions. The 369-bp product is the consequence of deletion of exons 4, 5, and 6. All deletions in Smad4 shorten its linker domain. RT-PCR of Smad3 mRNAs from fibrofatty lesions and THP-1 macrophages (D, F) depicted as 584-bp and 450-bp cDNA fragments; the 450-bp fragment represents mRNA with exon 3 deleted, shortening the MH1 domain and the linker domain. E, Restriction enzyme digestion of Smad3 cDNA fragments by Sau3AI and NcoI yields fragments of the predicted size. The RT-PCR products of Smad2 mRNAs isolated from fibrofatty lesions and THP-1 macrophages (G, I) are depicted as 786-bp and 696-bp cDNA fragments. The smaller 696-bp fragment has a deletion of exon 3 (within the MH1 domain), enabling its product to directly interact with DNA. H, Restriction enzyme digestion of Smad2 cDNA with RsaI yields fragments of the predicted size. Fatty streaks/fibrofatty lesions and THP-1 macrophages all express the same Smad2, Smad3, and Smad4 splice variants, (A and C, D and F, G and I, respectively).
p15Ink4B and p21Waf1,Cip1 in Fibrofatty Lesions
Two cyclin-dependent kinase inhibitors (CKIs) based on sequence homology and targets of CKI members, p15Ink4B from the INK4 family and p21Waf1 from the CIP/KIP family, can be activated by TGF-? in many cell types17,18,24 and can function to exit cells from the cell cycle,25,26 influence differentiation,27,28 and prevent apoptosis.29 Because their expression by TGF-? is dependent on Smad2, 3, and 4, we next examined their expression in the fibrofatty lesions. Expression of p15Ink4B in fibrofatty lesions was restricted to macrophages and most apparent in cytoplasm. Expression was present in 74.7±8.6% of the macrophage population. It was absent in SMCs within fibrofatty lesions (Figure 4A). Similarly, p21Waf1 was expressed by the macrophages, in 65.3±8.5% of the population and absent in the SMCs, consistent with the pattern of Smad expression. In cultured monocytes, elevations in the expression of p15INK4B and p21WAF1 induced by TGF-? were apparent 24 hours after exposure and maintained during the ensuing 3 days, when the monocytes differentiated into macrophages (Figure 4B). The lack of staining with a control mouse IgG and p15INK4B blocking peptides confirmed the specificity of staining for p21Waf1 and p15INK4B (Figures II and III, available online at http://atvb.ahajournals.org).
Figure 4. Expression of p15Ink4B and p21Waf1 in aorta and macrophages. A, p15Ink4B and p21Waf1 expression (red) in macrophages (black; double-stained cells indicated by arrows) but not in SMC of fibrofatty lesions or normal intima. B, TGF-?1–mediated elevations in p15Ink4B and p21Waf1 expression in THP-1 monocytes/macrophages. Original magnifications x400.
Expression of Smads in Fibrous Plaques
In aortic fibrous plaques, none of the cells expressed CD68 antigen, consistent with mostly SMCs populating this type of plaque. In contrast to the SMCs in the macrophage-rich regions of lesions, SMCs within such collagen-rich fibrous plaques expressed all 3 immunoreactive Smad peptides (Figure 5). On average, 80% of the SMCs in fibrous plaques expressed Smad2, Smad3, and Smad4; Smad 2 was expressed in 81.4±0.4% of the population whereas Smad3 and Smad4 were expressed by 80.8±3.3% and 82.2±3.7% of the population. SMCs comprising fibrous plaques of coronary arteries also expressed Smad proteins (not shown). Splice variants of mRNAs encoding Smad4 and Smad2 were expressed by cultured intimal SMCs in similar pattern to that in cultured THP-1 macrophages (Figure 3). Smad3 mRNA in the cultured intimal SMCs was represented by the single RT-PCR-generated cDNA fragment, 584-bp in size.
Figure 5. Smad2, Smad3, and Smad4 expression (black stain) in SMCs of fibrous plaques (see arrows). Original magnifications x160.
Because Smad expression is critical for TGF-?–mediated collagen gene expression,30 we also compared collagen expression in fibrous plaques and fibrofatty lesions. On average, collagen content in fibrous plaques was 3-fold greater than in fibrofatty lesions (Figure IV, available online at http://atvb.ahajournals.org), consistent with the differential expression of Smads in SMCs of the 2 types of lesions.
Smad Expression in Cultured SMCs From Fibrous Plaques and Fibrofatty Lesions
To determine whether the differential expression of Smads by SMC within fibrofatty lesions and fibrous plaques were the consequence of environmental factors in the lesions, we examined the expression of Smads in cells isolated and cultured from the 2 lesions. SMCs cultured from both lesions expressed all 3 Smads to a similar extent (Figure V, available online at http://atvb.ahajournals.org), suggesting that environmental factors in fibrofatty lesions were most likely responsible for the impaired expression of Smads in these cells.
Discussion
Our findings indicate a differential pattern of expression of the TGF-?/Smad signaling system within human atherosclerotic lesions, suggesting that TGF-? does not affect all cell types. Smad2, Smad3, and Smad4 are highly expressed in macrophages and macrophage-derived foam cells of fatty streaks/fibrofatty lesions, apparently enhanced after monocyte–macrophage differentiation. In contrast, SMCs in these lesions do not express Smads, indicating impaired transcriptional responsiveness to TGF-? in these cells. This impairment is likely to contribute to the low interstitial collagen in fibrofatty lesions and an increased susceptibility of these cells to apoptosis. In contrast, SMCs in fibrous plaques express high levels of all 3 Smads, consistent with TGF-?/Smad signaling contributing to collagen gene expression and fibrous lesion development.
Although several signaling mechanisms participate in TGF-?–mediated cell effects, including activated extracellular signal-regulated kinases,31 c-Jun N-terminal kinases32 and p38 kinases, Smad signaling plays a central role in nearly all transcriptional responses. In monocytes, Smad3 is required for TGF-?–induced chemotaxis and TGF-? expression.33 Mice lacking Smad3 also exhibit an impaired local inflammatory response during wound healing.33 Because expression of TGF-? isoforms by SMCs in the fatty streaks is high,2 TGF-?/Smad signaling in monocytes could contribute to monocyte accumulation by enhancing chemotaxis, thereby contributing to fibrofatty lesion development. It may also lead to elevations in TGF-? production by monocytes/macrophages within the lesions because TGF-? autoinduction is Smad3-dependent.33 Although we did not examine the expression of the antagonistic Smad, Smad7, we used expression of cyclin inhibitors as an indicator of Smad signaling activity. Our observations that macrophages within fatty streaks/fibrofatty lesions express p15Ink4B and p21Cip1/Waf1 suggest that Smad expression in these cells and also Smad-dependent TGF-?–mediated gene transcription is one mechanism that can contribute to their elevation. Expression of cyclin inhibitors has been associated with arrest of cells in G1 in differentiation and protection from apoptosis. Although the functional significance of p15Ink4B expression in the macrophages is yet to be defined,28 cytoplasmic p21Cip1/Waf1 has been associated with apoptosis-resistant monocytes.29 Possibly cytoplasmic p21Cip1/Waf1 in macrophages is protective against oxidative stress-induced apoptosis.
The apparent lack of expression of Smads by SMCs within the macrophage-rich regions of fatty streaks/fibrofatty lesions is surprising, considering cultured human aortic intimal SMCs from fibrofatty lesions and fibrous plaques express Smad2, Smad3, and Smad4 peptides (see Results) and mRNAs (Kalinina, unpublished). It is possible that the Smads are present, but the levels are below the detection limits of the antibodies. Such low expression would markedly limit the transcriptional effects of Smads stimulated by TGF-?.34 This lack of Smad expression makes it unlikely that the elevations in the low-density lipoprotein-binding proteoglycan, biglycan, within atherosclerotic lesions is a consequence of TGF-? action, despite its dependence on Smad3.15 In marked contrast, SMCs within fibrous plaques express high levels of all 3 Smads, suggesting a functionally intact TGF-?/Smad signaling system that may contribute to collagen accumulation. Although the mechanisms responsible for regulating Smad expression in SMCs are yet to be determined, our finding that Smad expression became apparent when the SMCs from the fibrofatty lesions were placed into cell culture suggests that factors within the fibrofatty lesions may be responsible for their lack of expression. Their re-expression when the cells were cultured prevented us from critically examining the extent to which TGF-? mediated Smad-dependent transcriptional responses important in collagen accumulation, collagen gene, and PAI-1 gene induction were affected. Recently, Smad2/3 expression and Smad4 expression have been shown to be upregulated in cirrhotic liver compared with normal liver;35 coordinate regulation has also been observed in T cells36 and during folliculogenesis.37
Recent studies using neutralizing antibodies or soluble type II truncated TGF-? receptors in ApoE–/– mice indicate roles for TGF-? in fibrosis and reducing inflammation of atherosclerotic lesions.4,5 However, interpretation of these results is somewhat complicated by findings that removal of whole-body TGF-? augments systemic inflammation.3,6,7 The high expression of Smads in macrophages of human fatty streaks/fibrofatty lesions suggests that TGF-? influences the functions of macrophages within atherosclerotic lesions, increasing their resistance to apoptosis and possibly also regulating their responses to inflammatory cytokines. The lack of expression of Smads in SMCs of fibrofatty lesions suggests that collagen production stimulated by TGF-? may be impaired in these cells, contrasting with those in fibrous plaques. This supports previous observations indicating a decreased number of procollagen expressing SMC in fibrofatty lesions.38 Collagen degradation by matrix metalloproteinases in macrophage-rich regions of atherosclerotic lesions is an important mechanism limiting collagen accumulation in such regions and contributes to the development of unstable lesions.39 Our study for the first time to our knowledge provides evidence for impaired TGF-?/Smad signaling in SMCs within fibrofatty lesions because of the lack of Smad2, Smad3, and Smad4 expression. Understanding the factors/mechanisms that regulate Smad expression in SMCs of fibrofatty lesions may lead to novel approaches to restore TGF-? responsiveness. Such approaches may lead to stabilization of atherosclerotic lesions via TGF-?–mediating elevations in collagen accumulation.
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ABSTRACT
Objective— Transforming growth factor-beta (TGF-?) has been implicated in the pathogenesis of human atherosclerosis but its actions during lesion progression are poorly understood. Smad2, Smad3, and Smad4 proteins are signaling molecules by which TGF-? modulates gene transcription. Our objective was to define the actions of TGF-? during lesion progression in humans by examining the expression of Smads in relation to TGF-?–mediated responses.
Methods and Results— Immunohistochemistry and reverse-transcription polymerase chain reaction demonstrated Smad2, Smad3, and Smad4 expression in macrophages of fibrofatty lesions and their upregulation after differentiation of monocytes to macrophages. The major Smad splice variants expressed by the macrophages were those that are transcriptionally most active. Macrophages also expressed cyclin inhibitors whose expression is induced via Smad proteins. The cytoplasmic location of p21Waf1 suggests it may protect macrophages from apoptosis. Smooth muscle cells (SMCs) within the fibrofatty lesions did not express the Smad proteins or the cyclin inhibitors. SMCs of fibrous plaques expressed all 3 Smad proteins.
Conclusions— In human atherosclerotic lesions, the actions of TGF-? appear restricted to SMCs in fibrous plaques and macrophages in fatty streaks/fibrofatty lesions. The lack of key TGF-? signaling components in SMCs of fibrofatty lesions indicates impaired ability of these cells to initiate TGF-?–mediated Smad-dependent transcriptional responses.
The actions of TGF-? in human atherosclerotic lesions were defined by examining the expression of Smad proteins in relation to TGF-?–mediated responses. Expression of Smad proteins was restricted to macrophages of fibrofatty lesions and SMCs of fibrous plaques. Smad-dependent TGF-? signaling appeared to be impaired SMCs of fibrofatty lesions.
Key Words: Smad ? p15INK4B ? p21Waf1 ? collagen ? human atherosclerotic lesions
Introduction
Transforming growth factor-beta (TGF-?) is a multifunctional cytokine/growth factor capable of regulating the growth, differentiation, and functions of immune and nonimmune cells.1 Although TGF-? is highly expressed in human atherosclerotic lesions, its contribution to lesion development and progression is still unclear.2,3 Expression of TGF-? isoforms and signaling receptors is high in macrophages and smooth muscle cells (SMCs) of aortic fibrofatty lesions.2 Expression is also apparent within the collagen-rich fibrous caps, but unaffected intima does not express the necessary signaling receptors.2 Recent studies in ApoE knockout mice examining the significance of TGF-? in the development of atherosclerosis using antibodies that neutralize TGF-? isoforms4 or a recombinant dominant-negative type II TGF-? receptor to prevent the actions of TGF-?,5 indicate roles in promoting fibrosis and reducing lesion inflammation. However, the significance of the findings for human atherosclerosis is unclear, because removal of whole-body TGF-? augments systemic inflammation.3 A TGF-?1 null mutation in mice causes excessive inflammatory responses and early death.6 Also, abrogation of TGF-? signaling in T cells leads to spontaneous T-cell differentiation and autoimmune disease,7 whereas disruption of TGF-? signaling in bone marrow-derived cells leads to dramatic expansion of myeloid cells, primarily monocytes and macrophages, and is associated with cachexia and mortality.8
TGF-? has the potential to initiate important effects in vessels that can influence the development of atherosclerosis and requires the phosphorylation of 2 intracellular transcription mediators, Smad2 and Smad3. The phosphorylated forms of Smad2 and Smad3 form heteromeric complexes with Smad4, then translocate to the nucleus to participate in regulating gene expression.1 Genes known to be activated by TGF-?/Smad signaling that have the potential to affect lesion characteristics include the 1 chain of type I collagen, the 2 chain of type I collagen, the 1 chain of type III collagen,9,10 and connective tissue growth factor.11 Repression of collagenase-1 by TGF-?/Smad-dependent mechanisms12 and stimulated increases in plasminogen activator inhibitor type 1 gene activity can also affect the characteristics of developing atherosclerotic lesions.13,14 The TGF-?/Smad signaling system has the potential to promote lipid accumulation by stimulating the expression of biglycan,15 a small leucine-rich proteoglycan that binds low-density lipoprotein and high-density lipoprotein.16 TGF-?/Smad signaling also elevates the cyclin-dependent kinase inhibitors,17,18 p15Ink4B and p21Waf1/Cip1, affecting cell proliferative responses and the sensitivity of cells to apoptosis.
Because of the potential importance of the TGF-?/Smad signaling in the development of human atherosclerotic lesions, we examined the expression of Smads that mediate TGF-? responses in normal human aorta and aorta containing atherosclerotic lesions of varying severity. We demonstrate that expression of Smads is highly upregulated in macrophages of fatty streaks/fibrofatty lesions and SMCs in fibrous plaques, but their absence in SMCs within fibrofatty lesions also suggests local impaired TGF-?/Smad signaling, which would contribute to low interstitial collagen content in such lesions. Unlike macrophages in fibrofatty lesions, these SMCs do not express p21Waf1, making them potentially more susceptible to apoptosis.
Methods
Collection of Tissues and Vessel Pathology
Thoracic aortas and coronary arteries were collected during autopsy, not later than 6 hours after death, from 27 male and female individuals, aged 19 to 67 years, at the Russian Cardiology Research Industrial Complex, Moscow. All subjects died of sudden death, not from infectious or other disorders, which could have potentially affected the expression of cellular proteins (please see http://atvb.ahajournals.org for specific details in online supplementary data and online Table I). The severity of atherosclerotic lesions were classified according to AHA guidelines after staining with Oil Red O, hematoxylin and eosin, and/or trichrome.2,19
Cleaned vessel segments were frozen in OCT (Miles Inc, Elkart, Ind) in liquid nitrogen and stored at –80°C. Vessel segments designated for RNA analysis by reverse-transcription polymerase chain reaction (RT-PCR) were dissected free of adventitia and the intima was peeled off from the media before freezing in liquid nitrogen. Histological studies were performed on 20 nonatherosclerotic segments, 25 segments containing fatty streaks/fibrofatty lesions, and 7 segments of fibrous plaques. RNA was extracted from 8 intimas possessing fatty streaks/fibrofatty lesions. The procedures for collecting tissues for the studies were approved by the Russian Cardiology Research Centre’s Human Ethics Experimentation Committee.
Antibodies
Please see http://atvb.ahajournals.org for specific details in online supplementary data.
Immunohistochemistry
The expression of Smad2, Smad3, and Smad4 was examined in frozen 6-μm cross-sections and simultaneously colocalized with CD68 antigen and -SM actin by double immunostaining as described previously (please see online supplementary data at http://atvb.ahajournals.org for details).20
Staining for Collagens Type I and III by Picrosirius Red
Frozen sections from fibrofatty streaks and fibrous plaques were incubated for 90 minutes in 0.1% Sirius red F3BA (Polyscience Inc) in saturated picric acid. After rinsing twice in 0.01N HCl and in distilled water, sections were briefly dehydrated with 70% ethanol and put under coverslips. Staining with Sirius red was analyzed by light microscopy.21
RNA Extraction and Analysis of Smad Splice Variants
RNA was extracted from fibrofatty lesions, fibrous plaques, and cultured cells, then subjected to RT-PCR for analysis of expression of Smad splice variants (please see specific details in online supplementary data at http://atvb.ahajournals.org).
Cell Cultures
The human promonocyte (THP-1) cell line (American Cell Type Collection) was cultured in RPMI-1640 medium containing 10% fetal calf serum and ?-mercaptoethanol. Differentiation to macrophages was induced by 4-day exposure to TGF-?1 (6 ng/mL)21 and the activity of macrophage nonspecific acetate esterase was measured to confirm differentiation to macrophages using a nonspecific acetate esterase kit (Sigma Chemical Corp) according to the manufacturer’s instructions.
Intimal SMCs were isolated from fibrofatty lesions and fibrous plaques of human aortas and subcultured as previously described.20
Statistics
Cell counts were performed by 2 independent observers. Ratios of positive cells were analyzed using Kruskal–Wallis 1-way test on ranks. Differences with P<0.05 were considered as statistically significant. Data are expressed as mean±SEM.
Results
Smads in Nonatherosclerotic Aorta
Immunoreactive Smads known to mediate TGF-? responses, Smad2, Smad3, and Smad4, were not detectable in SMCs of nonatherosclerotic aortas, either in the intima (Figure 1, top) or the media (not shown).
Figure 1. Expression of Smad proteins in human aorta. Top panels, Lack of Smad2, Smad3, and Smad4 expression in the normal SMC-rich intima. Middle panels, Smad2, Smad3, and Smad4 expression (black) in fibrofatty lesions is restricted to CD68-positive macrophages/macrophage-derived foam cells (red, see arrows). Bottom panels, -Actin–positive SMCs (red) within fibrofatty lesions do not express the Smad proteins (black). Original magnification x160.
Expression of Smads in Fatty Streaks/Fibrofatty Lesions
Fibrofatty lesions were defined using previously published criteria2,19,20 using oil red O staining of extracellular and intracellular lipids, hematoxylin and eosin, and/or trichrome (not shown); macrophages expressing Smads were differentiated from SMCs by CD68 antigen expression (Figure 1, middle).2,20 Approximately 90% of the macrophages and macrophage-derived foam cells within fatty streaks/fibrofatty lesions of aorta expressed high concentrations of Smad2, Smad3, and Smad4 (Figure 1, middle). In the macrophages, Smad 2 expression was apparent in 91.9±2.5% of the macrophage population, whereas Smad3- and Smad4-expressing macrophages represented 89.8±5.3% and 85.7±1.1% of the population, respectively. TGF-? appears to contribute to the elevated Smad expression in the macrophages, because during TGF-?–induced differentiation of THP-1 monocytes to macrophages, Smad2, Smad3, and Smad4 increased markedly (Figure 2A). This expression was accompanied by large increases in esterase activity, confirming differentiation (Figure 2B). In contrast, no SMCs, identified as -SM actin-positive cells located in the macrophage-rich regions expressed the 3 Smads (Figure 1, lower), despite previous reports, indicating high expression of TGF-? isoforms and signaling receptors.2 This pattern of Smad expression was also observed in fibrofatty lesions of coronary arteries (Figure I, available online at http://atvb.ahajournals.org). A lack of staining with rabbit and mouse control IgGs confirmed the specificities of the Smad stainings (Figures I and II, available online at http://atvb.ahajournals.org). Because alternative splicing of hn-RNA encoding, Smad4 and Smad2 result in peptides that differ in their DNA binding properties and ability to initiate gene transcription,22,23 we also examined the extent to which the splice variants contribute to immunoreactive Smads in the fibrofatty lesions. RT-PCR of mRNA from fibrofatty lesions using Smad4 oligonucleotide primers, covering most of its MH1 domain, the linker domain, and part of the N-terminus of the MH2 domain, resulted in 3 cDNA fragments (Figure 3A and 3B). The largest fragment accounted for nearly 90% of the PCR product and encoded the MH1, the linker, and MH2 domains. Cycle sequencing of the 587-bp cDNA fragment that accounted for 10% of the PCR products indicated deletions in exons 5 and 6 from the linker regions.23 The minor Smad4 PCR product of 369 bp was the consequence of deletions in exons 4, 5, and 6. Studies in THP-1 promonocytes/macrophages indicated an essentially similar pattern of expression to that observed in the fatty streaks/fibrofatty lesions (Figure 3C). RT-PCR using oligonucleotides for Smad3 mRNA indicated the presence of a major band (584 bp) and minor product (450 bp). The minor PCR product encodes Smad3 mRNA with an exon 3 deletion, which involves the carboxy-terminal part of the MH1 domain and part of the linker domain (Genbank Accession number E33804). RT-PCR of Smad2 mRNA produced 2 cDNA fragments 786-bp (major) and 696-bp (minor) in size. The minor fragment could be accounted for by deletion of exon 3, resulting in a splice variant that binds directly to DNA, in a manner similar to Smad3.22 Thus the major splice variants of Smad2, 3, and 4 expressed in fibrofatty lesions and THP-1 macrophages represented those most active in TGF-?–mediated gene transcription.
Figure 2. Regulation of Smad2, Smad3, and Smad4 expression in THP-1 monocytes during differentiation to macrophages, stimulated by 4-day exposure to TGF-?1. A, Smad2, Smad3, and Smad4 peptides (brown stains) are shown before (control) and after differentiation to macrophages. B, Marked elevation in nonspecific acetate esterase activity after exposure to TGF-?1. Original magnifications x400.
Figure 3. RT-PCR analysis of Smad splice variants in fibrofatty lesions and THP-1 macrophages. RT-PCR products of Smad4 mRNAs (A, B, C) depicted as a major 756-bp fragment and a minor 587-bp fragment with deletions of exons 5 and 6. C, cDNA fragments have been purified and re-amplified to show more clearly their expression in fibrofatty lesions. The 369-bp product is the consequence of deletion of exons 4, 5, and 6. All deletions in Smad4 shorten its linker domain. RT-PCR of Smad3 mRNAs from fibrofatty lesions and THP-1 macrophages (D, F) depicted as 584-bp and 450-bp cDNA fragments; the 450-bp fragment represents mRNA with exon 3 deleted, shortening the MH1 domain and the linker domain. E, Restriction enzyme digestion of Smad3 cDNA fragments by Sau3AI and NcoI yields fragments of the predicted size. The RT-PCR products of Smad2 mRNAs isolated from fibrofatty lesions and THP-1 macrophages (G, I) are depicted as 786-bp and 696-bp cDNA fragments. The smaller 696-bp fragment has a deletion of exon 3 (within the MH1 domain), enabling its product to directly interact with DNA. H, Restriction enzyme digestion of Smad2 cDNA with RsaI yields fragments of the predicted size. Fatty streaks/fibrofatty lesions and THP-1 macrophages all express the same Smad2, Smad3, and Smad4 splice variants, (A and C, D and F, G and I, respectively).
p15Ink4B and p21Waf1,Cip1 in Fibrofatty Lesions
Two cyclin-dependent kinase inhibitors (CKIs) based on sequence homology and targets of CKI members, p15Ink4B from the INK4 family and p21Waf1 from the CIP/KIP family, can be activated by TGF-? in many cell types17,18,24 and can function to exit cells from the cell cycle,25,26 influence differentiation,27,28 and prevent apoptosis.29 Because their expression by TGF-? is dependent on Smad2, 3, and 4, we next examined their expression in the fibrofatty lesions. Expression of p15Ink4B in fibrofatty lesions was restricted to macrophages and most apparent in cytoplasm. Expression was present in 74.7±8.6% of the macrophage population. It was absent in SMCs within fibrofatty lesions (Figure 4A). Similarly, p21Waf1 was expressed by the macrophages, in 65.3±8.5% of the population and absent in the SMCs, consistent with the pattern of Smad expression. In cultured monocytes, elevations in the expression of p15INK4B and p21WAF1 induced by TGF-? were apparent 24 hours after exposure and maintained during the ensuing 3 days, when the monocytes differentiated into macrophages (Figure 4B). The lack of staining with a control mouse IgG and p15INK4B blocking peptides confirmed the specificity of staining for p21Waf1 and p15INK4B (Figures II and III, available online at http://atvb.ahajournals.org).
Figure 4. Expression of p15Ink4B and p21Waf1 in aorta and macrophages. A, p15Ink4B and p21Waf1 expression (red) in macrophages (black; double-stained cells indicated by arrows) but not in SMC of fibrofatty lesions or normal intima. B, TGF-?1–mediated elevations in p15Ink4B and p21Waf1 expression in THP-1 monocytes/macrophages. Original magnifications x400.
Expression of Smads in Fibrous Plaques
In aortic fibrous plaques, none of the cells expressed CD68 antigen, consistent with mostly SMCs populating this type of plaque. In contrast to the SMCs in the macrophage-rich regions of lesions, SMCs within such collagen-rich fibrous plaques expressed all 3 immunoreactive Smad peptides (Figure 5). On average, 80% of the SMCs in fibrous plaques expressed Smad2, Smad3, and Smad4; Smad 2 was expressed in 81.4±0.4% of the population whereas Smad3 and Smad4 were expressed by 80.8±3.3% and 82.2±3.7% of the population. SMCs comprising fibrous plaques of coronary arteries also expressed Smad proteins (not shown). Splice variants of mRNAs encoding Smad4 and Smad2 were expressed by cultured intimal SMCs in similar pattern to that in cultured THP-1 macrophages (Figure 3). Smad3 mRNA in the cultured intimal SMCs was represented by the single RT-PCR-generated cDNA fragment, 584-bp in size.
Figure 5. Smad2, Smad3, and Smad4 expression (black stain) in SMCs of fibrous plaques (see arrows). Original magnifications x160.
Because Smad expression is critical for TGF-?–mediated collagen gene expression,30 we also compared collagen expression in fibrous plaques and fibrofatty lesions. On average, collagen content in fibrous plaques was 3-fold greater than in fibrofatty lesions (Figure IV, available online at http://atvb.ahajournals.org), consistent with the differential expression of Smads in SMCs of the 2 types of lesions.
Smad Expression in Cultured SMCs From Fibrous Plaques and Fibrofatty Lesions
To determine whether the differential expression of Smads by SMC within fibrofatty lesions and fibrous plaques were the consequence of environmental factors in the lesions, we examined the expression of Smads in cells isolated and cultured from the 2 lesions. SMCs cultured from both lesions expressed all 3 Smads to a similar extent (Figure V, available online at http://atvb.ahajournals.org), suggesting that environmental factors in fibrofatty lesions were most likely responsible for the impaired expression of Smads in these cells.
Discussion
Our findings indicate a differential pattern of expression of the TGF-?/Smad signaling system within human atherosclerotic lesions, suggesting that TGF-? does not affect all cell types. Smad2, Smad3, and Smad4 are highly expressed in macrophages and macrophage-derived foam cells of fatty streaks/fibrofatty lesions, apparently enhanced after monocyte–macrophage differentiation. In contrast, SMCs in these lesions do not express Smads, indicating impaired transcriptional responsiveness to TGF-? in these cells. This impairment is likely to contribute to the low interstitial collagen in fibrofatty lesions and an increased susceptibility of these cells to apoptosis. In contrast, SMCs in fibrous plaques express high levels of all 3 Smads, consistent with TGF-?/Smad signaling contributing to collagen gene expression and fibrous lesion development.
Although several signaling mechanisms participate in TGF-?–mediated cell effects, including activated extracellular signal-regulated kinases,31 c-Jun N-terminal kinases32 and p38 kinases, Smad signaling plays a central role in nearly all transcriptional responses. In monocytes, Smad3 is required for TGF-?–induced chemotaxis and TGF-? expression.33 Mice lacking Smad3 also exhibit an impaired local inflammatory response during wound healing.33 Because expression of TGF-? isoforms by SMCs in the fatty streaks is high,2 TGF-?/Smad signaling in monocytes could contribute to monocyte accumulation by enhancing chemotaxis, thereby contributing to fibrofatty lesion development. It may also lead to elevations in TGF-? production by monocytes/macrophages within the lesions because TGF-? autoinduction is Smad3-dependent.33 Although we did not examine the expression of the antagonistic Smad, Smad7, we used expression of cyclin inhibitors as an indicator of Smad signaling activity. Our observations that macrophages within fatty streaks/fibrofatty lesions express p15Ink4B and p21Cip1/Waf1 suggest that Smad expression in these cells and also Smad-dependent TGF-?–mediated gene transcription is one mechanism that can contribute to their elevation. Expression of cyclin inhibitors has been associated with arrest of cells in G1 in differentiation and protection from apoptosis. Although the functional significance of p15Ink4B expression in the macrophages is yet to be defined,28 cytoplasmic p21Cip1/Waf1 has been associated with apoptosis-resistant monocytes.29 Possibly cytoplasmic p21Cip1/Waf1 in macrophages is protective against oxidative stress-induced apoptosis.
The apparent lack of expression of Smads by SMCs within the macrophage-rich regions of fatty streaks/fibrofatty lesions is surprising, considering cultured human aortic intimal SMCs from fibrofatty lesions and fibrous plaques express Smad2, Smad3, and Smad4 peptides (see Results) and mRNAs (Kalinina, unpublished). It is possible that the Smads are present, but the levels are below the detection limits of the antibodies. Such low expression would markedly limit the transcriptional effects of Smads stimulated by TGF-?.34 This lack of Smad expression makes it unlikely that the elevations in the low-density lipoprotein-binding proteoglycan, biglycan, within atherosclerotic lesions is a consequence of TGF-? action, despite its dependence on Smad3.15 In marked contrast, SMCs within fibrous plaques express high levels of all 3 Smads, suggesting a functionally intact TGF-?/Smad signaling system that may contribute to collagen accumulation. Although the mechanisms responsible for regulating Smad expression in SMCs are yet to be determined, our finding that Smad expression became apparent when the SMCs from the fibrofatty lesions were placed into cell culture suggests that factors within the fibrofatty lesions may be responsible for their lack of expression. Their re-expression when the cells were cultured prevented us from critically examining the extent to which TGF-? mediated Smad-dependent transcriptional responses important in collagen accumulation, collagen gene, and PAI-1 gene induction were affected. Recently, Smad2/3 expression and Smad4 expression have been shown to be upregulated in cirrhotic liver compared with normal liver;35 coordinate regulation has also been observed in T cells36 and during folliculogenesis.37
Recent studies using neutralizing antibodies or soluble type II truncated TGF-? receptors in ApoE–/– mice indicate roles for TGF-? in fibrosis and reducing inflammation of atherosclerotic lesions.4,5 However, interpretation of these results is somewhat complicated by findings that removal of whole-body TGF-? augments systemic inflammation.3,6,7 The high expression of Smads in macrophages of human fatty streaks/fibrofatty lesions suggests that TGF-? influences the functions of macrophages within atherosclerotic lesions, increasing their resistance to apoptosis and possibly also regulating their responses to inflammatory cytokines. The lack of expression of Smads in SMCs of fibrofatty lesions suggests that collagen production stimulated by TGF-? may be impaired in these cells, contrasting with those in fibrous plaques. This supports previous observations indicating a decreased number of procollagen expressing SMC in fibrofatty lesions.38 Collagen degradation by matrix metalloproteinases in macrophage-rich regions of atherosclerotic lesions is an important mechanism limiting collagen accumulation in such regions and contributes to the development of unstable lesions.39 Our study for the first time to our knowledge provides evidence for impaired TGF-?/Smad signaling in SMCs within fibrofatty lesions because of the lack of Smad2, Smad3, and Smad4 expression. Understanding the factors/mechanisms that regulate Smad expression in SMCs of fibrofatty lesions may lead to novel approaches to restore TGF-? responsiveness. Such approaches may lead to stabilization of atherosclerotic lesions via TGF-?–mediating elevations in collagen accumulation.
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