Published online before print September 25, 2008, doi: 10.1161/CIRCRESAHA.108.185645
© 2008 American Heart Association, Inc.
Molecular Medicine |
Thrombospondin-1 Deficiency Accelerates Atherosclerotic Plaque Maturation in ApoE–/– Mice
From the Center for Molecular and Vascular Biology (R.M., P.V., S.V.k., M.F.H.); Center for Transgene Technology and Gene Therapy (M.T.); and Atherosclerosis and Metabolism Unit (P.H.), Department of Cardiovascular Diseases, University of Leuven, Belgium.
Correspondence to Marc Hoylaerts, PhD, Center for Molecular and Vascular Biology, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium. E-mail Marc.Hoylaerts{at}med.kuleuven.be
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Abstract |
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Thrombospondin (TSP)1 is implicated in various inflammatory processes, but its role in atherosclerotic plaque formation and progression is unclear. Therefore, the development of atherosclerosis was compared in ApoE–/– and Tsp1–/–ApoE–/– mice kept on a normocholesterolemic diet. At 6 months, morphometric analysis of the aortic root of both mouse genotypes showed comparable lesion areas. Even when plaque burden increased
5-fold in ApoE–/– and 10-fold in Tsp1–/–ApoE–/– mice, during the subsequent 3 months, total plaque areas were comparable at 9 months. In contrast, plaque composition differed substantially between genotypes: smooth muscle cell areas, mostly located in the fibrous cap of ApoE–/– plaques, both at 6 and 9 months, were 3-fold smaller in Tsp1–/–ApoE–/– plaques, which, in addition, were also more fibrotic. Moreover, inflammation by macrophages was twice as high in Tsp1–/–ApoE–/– plaques. This correlated with a 30-fold elevated incidence of elastic lamina degradation, with matrix metalloproteinase-9 accumulation, underneath plaques and manifestation of ectasia, exclusively in Tsp1–/–ApoE–/– mice. At 9 months, the necrotic core was 1.4-fold larger and 4-fold higher numbers of undigested disintegrated apoptotic cells were found in Tsp1–/–ApoE–/– plaques. Phagocytosis of platelets by cultured Tsp1–/– macrophages revealed the instrumental role of TSP1 in phagocytosis, corroborating the defective intraplaque phagocytosis of apoptotic cells. Hence, the altered smooth muscle cell phenotype in Tsp1–/–ApoE–/– mice has limited quantitative impact on atherosclerosis, but defective TSP1-mediated phagocytosis enhanced plaque necrotic core formation, accelerating inflammation and macrophage-induced elastin degradation by metalloproteinases, speeding up plaque maturation and vessel wall degeneration.
Key Words: atherosclerosis • matricellular proteins • transgenic mice • vascular inflammation
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Introduction |
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Thrombospondin (TSP)1 is a matricellular multidomain protein, interacting with several receptors, ligands, and matrix components.1 Functional studies in Tsp1–/– mice have shown that TSP1 inhibits angiogenesis,2 promotes the healing of excisional wounds,3 contributes to pulmonary homeostasis,4 and limits the inflammatory response and fibrotic remodeling after myocardial infarction.4 TSP1 also plays a role in platelet and thrombus adhesion to injured blood vessels5 and limits nitric oxide (NO)-mediated vasodilation and tissue perfusion in experimental models of ischemic injury.6
After injury, TSP1 is rapidly upregulated to modulate cell–cell and cell–matrix interactions, as part of normal tissue repair,7,8 but chronic vascular injury is also associated with TSP1 upregulation, eg, in diabetic rats9 and during hyperplasia.10 Mutations in TSP1, TSP2, and TSP4 have further been linked to increased risk for myocardial infarction.11 Nevertheless, the role of TSP1 in the progression of cardiovascular diseases is poorly documented,12 and recent analyses question the link between several TSP1 polymorphisms and myocardial infarction.13 Yet, TSP1 has been implicated in atherosclerosis, because of its strong upregulation in atherosclerotic plaques of hypercholesterolemic animals.10,14
Development of atherosclerosis leads to smooth muscle cell (SMC) activation and migration, as well as to enhanced vascular infiltration of macrophages and differentiation into foam cells. TSP1 facilitates proliferation and migration of vascular SMCs during neointima formation.15 It seems equally important in inflammation, because Tsp1–/– mice show enhanced leukocyte infiltration, pneumonia with macrophages and neutrophils, and mild pancreatic inflammation.16 TSP1 is secreted by and adheres to neutrophils, monocytes, macrophages, and T cells, and several TSP1 receptors are present on inflammatory cells.17 Indeed, CD36, CD47, and integrin 
vβ3 are TSP1 receptors, capable of downregulating proinflammatory cytokine release by dendritic cells (DC).18 Furthermore, TSP1-CD47 interactions selectively downregulate IL12 production by monocytes, inhibit DC maturation and contribute to refractoriness of mature DCs.19 This action limits further activation and advocates a role for TSP1 in the inflammatory response resolution. Although the proinflammatory phenotype in the absence of TSP1 has been attributed to deficient conversion of latent to active transforming growth factor (TGF)β1,20 the physiological relevance of TGFβ1 activation by TSP1 is a subject of controversy, because TGFβ1 can also be activated via other pathways and TGFβ1-independent effects of TSP1 on inflammatory cells have been described.
Maturation of atherosclerotic lesions into complex plaques with necrotic core critically depends on the degree of intraplaque phagocytosis, a process that occurs with poor efficiency in mature plaques.21 When, in advanced atherosclerotic plaques, apoptotic cells are no longer rapidly removed by macrophages, the resulting secondary necrosis elicits additional inflammatory responses, which further enhance plaque progression. TSP1 mediates phagocytosis of apoptotic cells via an vβ3/CD36/TSP1 ligand bridge between macrophages and apoptotic cells,22 suggestive of an additional role for TSP1 in atherogenesis.
Little is known on how this multitude of TSP1-dependent cellular interactions may contribute to atherosclerosis. We have, therefore, intercrossed Tsp1–/– and ApoE–/– mice, generating double gene-deficient mice. ApoE–/– and Tsp1–/–ApoE–/– mice were fed a normal chow and lesion development was studied in the aortic root, primarily via immunohistochemistry and semiquantitative microscopic morphometry.
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Materials and Methods |
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Mouse Model of Atherosclerosis
Tsp1–/– mice were kindly provided by Dr J. Lawler (Harvard Medical School, Boston, Mass). C57Bl6 Tsp1–/– mice were intercrossed with C57Bl6 ApoE–/– mice; Tsp1–/–ApoE–/– and littermate control ApoE–/– were generated and fed a normal chow ad libitum. At 3, 6, 9, and 12 months of age, female mice were anesthetized with Nembutal (60 mg/kg) and transcardially perfused with 1% paraformaldehyde under physiological pressure, after which tissue was removed for histological processing. All animal experiments were reviewed and approved by the Institutional Review Board of the University of Leuven and were performed in compliance with the guidelines of the International Society on Thrombosis and Hemostasis.23 Blood and lipid analysis are detailed in the online data supplement, available http://circres.ahajournals.org.
Histological and Morphometric Analysis
Immunohistochemical Staining
Tissue preparation and histochemical stainings are described in the online data supplement Before immunohistochemical staining, in general, antigen retrieval was performed by incubation for 20 minutes in Dako Target Retrieval Solution (Dako, Carpinteria, Calif) at 90°C. Endogenous peroxidase activity was blocked by incubation for 20 minutes in methanol, containing 0.3% H2O2, followed by incubation with the appropriate serum to mask nonspecific binding sites. Sections were then incubated overnight with antibodies against SMC actin (1/500 Dako SMA Mo851), heavy chain myosin (1/500 Abcam 685), CD31 (1/500 BD Pharmingen 557355), CD45 (1/100 BD 553076), MAC3 (1/50 Pharmingen Rat anti-MAC3), proliferating cell nuclear antigen (PCNA) (Novocastra NCL-PCNA), matrix metalloproteinase (MMP)9 (1/50 R&D Systems AF 909), and TSP1 (5 µg/mL in-house rabbit anti-human TSP1 biotinylated, preadsorbed with liver extract from Tsp1–/– mice, which procedure strongly reduced interfering TSP2 signals). With smooth muscle actin (SMA), CD31, CD45, MAC3, MMP9, and PCNA, appropriate peroxidase-labeled secondary antibodies were used and peroxidase activity was visualized using 0.06% 3,3'-diaminobenzidine (DAB)/0.01% H2O2. Apoptotic cells were detected by TUNEL staining (ApopTag Peroxidase in situ, Chemicon S7100). The anti-myosin antibody was detected via an Alexa fluor-conjugated secondary antibody. Nucleus 2',6'-diamidino-2-phenylindole (DAPI) counterstaining was performed with Vectashield+DAPI mounting medium.
Apoptotic Cell Assessment
Apoptosis was quantified as the percentage of the total number of TUNEL-stained cells. TUNEL-positive cells were further divided into intact and disintegrated (both with a fragmented nucleus or further degraded) cells, according to their degree of nuclear fragmentation.
Quantification of Ectasia (Pseudoaneurysm Formation)
Rupture of elastic laminas, leading to thinning of the media, outward protrusion, or opening of the vessel wall was interpreted as ectasia. The number of events was counted in independent sections to avoid double enumeration of the same event.
ELISA Measurements and Western Blotting
TGFβ1 and tumor necrosis factor (TNF) were quantified in plasma and tissue extracts via commercially available kits (R&D systems). Aortas were pulverized in liquid nitrogen and dissolved in 10 mmol/L phosphate buffer, pH 7.2, containing 150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS, 0.5% Na-deoxycholate and 0.2% Na-azide. Extracts were vortexed and shaken for 1 hour at 4°C. To extract matrix-bound TGFβ1, an additional extract was made with 8 mol/L urea, added to the buffer. After centrifugation at 12,000 g of the latter fraction, both fractions were mixed at equal volumes. TGFβ1 was measured with and without acidification, to quantify total and active TGFβ1, respectively. TSP1 was dosed in EDTA-plasma in a home-made ELISA. To this end, microtiter plate wells were coated with the in-house monoclonal antibody 36A8, raised in Tsp1–/– mice against human TSP1, purified as described.5 After blocking and washing, plasma samples or purified human TSP15 were incubated for 1 hour. Then, bound TSP1 was revealed via a rabbit anti-human TSP1 biotinylated antibody and Vectastain ABC staining (Vector Laboratories Inc, Burlingame, Calif), according to standard procedures. Extracts from vascular extracts from plaque containing areas and plaque-free areas were subjected to SDS-PAGE via loading 20 µg of protein per lane everywhere. After electrophoresis and blotting onto nitrocellulose, phospho-Smad was detected with the anti–phospho-Smad antibody p-Smad2/3 (Ser423/425; Santa Cruz Biotechnology, sc-11769).
In Vitro Foam Cell Assay and Phagocytosis
Peritoneal macrophages were harvested from ApoE–/– and Tsp1–/–ApoE–/– mice 3 days after intraperitoneal injection of 4% thioglycolate. Cells were washed with PBS, counted and plated at a density of 106 cells per 3.5-cm well in Optimem medium with 0.02% lactalbumine hydrolysate and 0.01% FBS in the absence or presence of human TSP1 (0 to 10 µg/mL), purified as reported.5 Cells were allowed to attach overnight; nonadherent cells were removed by washing with PBS and cells were stimulated with 30 mg/mL Cu–oxidized (ox) LDL. In the Tsp1–/– macrophage cultures, human TSP1 was added (0, 5, or 20 µg/mL). Cellular uptake of oxLDL was measured after 24 hours after washing, fixation with gluteraldehyde, and staining with oil red O. TNF levels were determined in culture supernatants, collected at 72 hours, and frozen until assayed.
Alternatively, adhered macrophages were incubated with 5 µmol/L Cell Tracker Orange CMTMR (Molecular Probes) for 45 minutes. On removal of free dye, washed murine platelets were added, at 10 platelets per macrophage. Platelets were labeled with 10 µmol/L Cell Tracker Green CMFDA for 30 minutes. On incubation for 20 hours in Optimem medium, cell cultures were washed with medium and fixed for 60 minutes with 1% paraformaldehyde in PBS. Phagocytosis was then quantitated via confocal microscopy on a Zeiss Axioskop20 microscope (Zeiss, Jena, Germany) in superposed green and red images, as described.24
Statistical Analysis
Data are represented as the means±SEM. Significance of differences was analyzed using a 2-tailed unpaired t test with Welch correction, allowing for populations with different SDs. Numbers of phagocytosed platelets were analyzed via 2x2 contingency tables. A value of P<0.05 was considered statistically significant.
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Results |
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TSP1 Expression During Atherogenesis and Hypercholesterolemia
The expression of TSP1 increases during hypercholesterolemia.10,14 Therefore, TSP1 was measured, by ELISA, in plasma of ApoE–/– mice at 3, 6, and 9 months. TSP1 was not upregulated over time in ApoE–/– plasma. Moreover, there was no difference between ApoE–/– and wild-type (WT) mice at 9 months (1.2±0.8 µg/mL in ApoE–/– versus 1.8±0.9 µg/mL in WT mice). In addition, we analyzed the TSP1 localization in the atherosclerotic vessel wall in the aortic root of ApoE–/– mice. Both at 6 and 9 months, weak TSP1 staining was found in the adventitia and media (Figure 1); strong staining was found in SMCs in the fibrous cap, inflammatory cells in the shoulder of the plaque and foam cells (Figure 1B and 1C). At 9 months, the necrotic core also stained for TSP1 (Figure 1). Together, these results suggested a vascular, rather than systemic, role for TSP1 in atherosclerosis development.
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TSP1 Deficiency Delays Only the Initial Plaque Formation
At 3 months, no clear evidence of plaque formation was observed, neither in ApoE–/– nor in Tsp1–/–ApoE–/– mice. At 6 months, both genotypes had atherosclerotic lesions in the aortic root. At 12 months, oil red O staining revealed the presence of lesions throughout the entire aorta (Figure 2A), most pronounced in the aortic arch for each genotype. Therefore, these sites were further investigated via microscopic morphometry of aortic root cross-sections. Individual plaques microscopically appeared very similar in both genotypes (Figure 2B and 2C). Over the next 3 months, the mean plaque areas (not shown) and total plaque areas (Figure 2D) increased 5-fold in ApoE–/– mice and 10-fold in Tsp1–/–ApoE–/– mice but remained comparable at 9 months, for both genotypes. During this 3-month interval, the cross-sectional luminal aortic root area approximately doubled, resulting in comparable fractions of the intra–elastic–lamina area, occupied by plaque, at 9 months (27.7±9.7% [n=24] in ApoE–/– mice versus 32.7±8.5% [n=28] in Tsp1–/–ApoE–/– mice). Thus, quantitatively, atherosclerotic lesion development was comparable in ApoE–/– and Tsp1–/–ApoE–/– aortic roots, over the first 9 months of their lifespan. Lesion development in the abdominal aorta (Figure 2A) was not further investigated.
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TSP1 Deficiency Modulates the Intraplaque Composition
SMCs in Tsp1–/– mice proliferate and migrate less and synthesize more collagen than WT SMCs in a mouse model of neointima formation.15 Therefore, the amount of contractile SMCs (via SMA staining) and collagen (via Sirius red staining) were quantified in the plaques. The SMA-positive area was significantly reduced in Tsp1–/–ApoE–/– plaques at 6 and 9 months (Figure 3A through 3C). The SMA-positive cells were typically located in the media and in the fibrous cap in both genotypes (Figure 3A and 3B and insets). The plaque content of collagen, detected via Sirius red staining, strongly increased between 6 and 9 months in both genotypes (Figure 3D through 3F). Furthermore, Tsp1–/–ApoE–/– plaques contained more collagen than ApoE–/– plaques (Figure 3F), both at 6 and at 9 months, but the relative differences were larger at 6 than at 9 months. Plaque collagen was generally more abundant in the fibrous cap, progressively diminishing toward the inside of the plaque. Analysis of cellular proliferation (via PCNA staining) showed that proliferating cells were mostly associated with inflammatory cells (not shown) and rarely with SMCs. This is consistent with previous reports.25,26 The large variability in the degree of cell proliferation in different plaques made it impossible to reliably compare both genotypes quantitatively. Additional staining of SMCs for myosin (see Figure I in the online data supplement) confirmed the presence of SMCs in the plaques of both genotypes. MSB staining of the lesion of both genotypes (see supplemental Figure II) illustrated more extensive collagen accumulation inside plaques of Tsp1–/–ApoE–/– than of ApoE–/– mice.
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TSP1 Deficiency Leads to High Plaque Macrophage Numbers
Because of the fast lesion expansion between 6 and 9 months, plaque composition was studied at both these time-points. At 6 months, the plaque content of leukocytes (CD45 staining) (Figure 4A and 4B) and macrophages (MAC3 staining) (Figure 4D and 4E) did not differ between genotypes, ie, vascular inflammation was not different (Figure 4C and 4F). However, at 9 months, vascular inflammation of the plaque, measured via CD45 staining, not only doubled in Tsp1–/–ApoE–/– mice compared to 6 months (Figure 4C), but it also exceeded the inflammation found in ApoE–/– plaques. Macrophage numbers increased 4-fold in ApoE–/– mice from 6 to 9 months but rose 10-fold in Tsp1–/–ApoE–/– mice, to the point where most inflammatory cells in Tsp1–/–ApoE–/– mice appeared to be macrophages, 2-fold higher in number than in 9 month old ApoE–/– mouse plaques (Figure 4F).
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Cytokine analysis for this proinflammatory vascular milieu at 9 months, via quantification of TNF in aortic extracts, showed 2-fold higher concentrations in vessel extracts of Tsp1–/–ApoE–/– (17.7±10 pg/mg total protein, n=11) than ApoE–/– vessels (9.5±2.2 pg/mg total protein, n=11, P=0.018). To investigate whether absent activation of TGFβ, an important antiinflammatory agent activated by TSP1, was responsible for the enhanced inflammation in Tsp1–/–ApoE–/– mice, we also measured TGFβ1 levels in these vascular extracts. We found the levels of total (ApoE–/–: 143±180 pg/mg total protein [n=10]; versus Tsp1–/–ApoE–/–: 121±119 pg/mg total protein [n=10]) and active (ApoE–/–: 124±180 pg/mg total protein [n=10]; versus Tsp1–/–ApoE–/–: 92±110 pg/mg total protein [n=10]) TGFβ to be comparable for both genotypes, suggesting that the increased inflammation in the absence of TSP1 was not the result of reduced TGFβ activation. Correspondingly, we found no differences in the degree of Smad phosphorylation in both genotypes, by analyzing the plaque-free environment around the plaques (see supplemental Figure III), for both genotypes. Only weak Smad phosphorylation was observed in the plaque area of each genotype, further excluding functional coupling between TSP1 and TGFβ1 activation in the present model of atherogenesis.
Macrophages in the plaque produce metalloproteinases that degrade the vascular wall.27 In accordance with the high macrophage content of Tsp1–/–ApoE–/– plaques, ectasia or pseudoaneurysm formation was found in bulging plaques, as illustrated in Figure 5A. Ectasia was 30-fold more abundant in cross-sections of Tsp1–/–ApoE–/– than ApoE–/– mice (Figure 5D), in which it was rarely encountered. Figure 5C (and inset) show that the macrophage-derived metalloproteinase MMP9 concentrated on the elastic membranes and in the vicinity of the 9-month-old media of Tsp1–/–ApoE–/– mice. These findings are compatible with inflammatory cell– derived elastin degradation in the elastic membranes by MMP9 (Figure 5B). The high plaque inflammation in Tsp1–/–ApoE–/– mice at 9 months, therefore, seems to trigger metalloproteinase-mediated elastin degradation and ectasia.
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Plaque Maturation in Tsp1–/–ApoE–/– Mice
Formation of a necrotic (lipid) core in advanced plaques starts with enhanced macrophage uptake of oxLDL via scavenger receptors, then, foam cell formation and death, followed by inadequate debris removal by phagocytosis.28 In accordance with the higher inflammation of macrophages (Figure 4), the area of the lipid core, expressed as a percentage of the total plaque area, was significantly larger in 9 month old Tsp1–/–ApoE–/– (52.4±17%, n=20) than in ApoE–/– (37.2±15%, n=19, P=0.007) mice. TSP1 has previously been implicated in phagocytosis of apoptotic cells.22 Because CD36 is a TSP1 receptor and the crucial scavenger receptor for oxLDL on macrophages,29 we have assessed the role of TSP1 in macrophage activation and in the uptake of Cu-oxLDL by macrophages. First, we observed that thioglycollate-elicited peritoneal macrophages from Tsp1–/– mice showed a reduced survival after 24 hours, compared to C57Bl/6 control macrophages, a defect that was dose-dependently restored by adding human TSP1 to the cultures (Figure 6A). The addition of a standardized concentration of Cu-oxLDL both activated peritoneal macrophages from C57Bl/6 control and Tsp1–/– mice, as judged from the TNF levels in the supernatants of these cultures (Figure 6B). Tsp1–/– macrophages were activated less, but addition of TSP1 largely corrected the differences between both genotypes, in agreement with its protective phenotype on Tsp1–/– macrophages (Figure 6A). Accordingly, oil red O–stained WT (26±14%) and Tsp1–/– macrophages, without (29±13%) or cultured with purified TSP1 (24±13%), revealed a comparable proportion of total cells having taken up lipids.
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The role of TSP1 in phagocytosis, therefore, was investigated in these cultures. Baseline phagocytosis was comparable in WT and Tsp1–/– macrophages after 20 hours of coculture with WT or Tsp1–/– platelets, respectively. However, a dose-dependent increase in phagocytosis efficiency was found on addition of TSP1 (Figure 7A). Figure 7B illustrates the interactions between green platelets and red macrophages, with phagocytosed platelets showing as yellow spots. We, therefore, have analyzed the degree of intraplaque phagocytosis at 9 months, via the identification of nonphagocytosed apoptotic cells. These cells were identified via their TUNEL positivity and loss of integrity. TUNEL-stained cells were not abundant and were not present in all sections (ie, when abundant noncellular staining in the necrotic core was excluded from analysis). The total number of apoptotic cells was not different between genotypes (Figure 7C). However, the number of disintegrated TUNEL-positive cells was 4-fold higher in Tsp1–/–ApoE–/– than in ApoE–/– plaques, constituting more than 50% of the TUNEL-positive cells (Figure 7C), suggestive of defective phagocytosis of apoptotic cells in the absence of TSP1. Figure 7D illustrates the morphological appearance of these different TUNEL-positive cells in detail.
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In the present study, the role of TSP1 in atherogenic vessel degeneration has been dissected in the well-established ApoE–/– mouse model of spontaneous atherosclerosis.30 The absence of TSP1 affected onset of atherosclerotic lesion formation and plaque growth in the aortic root only minimally. In contrast, Tsp1–/–ApoE–/– mice manifested attenuated SMC migration and proliferation, associated with more fibrotic lesions. In more advanced stages of plaque maturation, they also showed accelerated necrotic core formation. This proinflammatory milieu was found to coincide with enhanced inflammation in the plaques of 9 month old Tsp1–/–ApoE–/– mice and enhanced presence of MMP9, coinciding with degradation of vascular elastic laminae, leading to ectasia.
General morphological inspection of ApoE–/– and Tsp1–/–ApoE–/– plaques revealed no gross differences. Yet, gene deficiency of TSP1 is associated with defective SMC proliferation and migration, and a more fibrotic SMC, in a model of carotid artery ligation.15 Throughout the entire atherogenesis period studied, contractile SMC numbers were lower in Tsp1–/–ApoE–/– than in ApoE–/– plaques, in agreement with the reported role for TSP1 in SMC activation and migration. Correspondingly, the fibrous cap in Tsp1–/–ApoE–/– plaques contained fewer SMCs. Because SMA primarily stains contractile SMCs, we also have stained for myosin (see the online data supplement), primarily revealing SMCs inside plaques of both genotypes but less in media and fibrous cap, albeit to a highly variable degree. Yet, this staining confirmed the presence of noncontractile SMCs inside plaques. In agreement with the fibrotic phenotype of Tsp1–/– SMCs,15 more collagen was, indeed, deposited in lesions of Tsp1–/–ApoE–/– than of ApoE–/– plaques, findings corroborated by Sirius red and MSB plaque staining (see the online data supplement). Hence, the potentially higher vulnerability to plaque rupture in mature Tsp1–/–ApoE–/– plaques, resulting from lower fibrous cap SMC numbers, was counteracted by the larger collagen content in these plaques, adding to their stability. This dual role for TSP1 in the control of plaque SMCs may explain why, in the presence of high macrophage numbers in the mature plaque, ectasia is observed, but not plaque rupture, even though both processes depend on MMPs derived from macrophages also present in the shoulder of the plaque. It is not clear whether further plaque progression in Tsp1–/–ApoE–/– mice would eventually cause plaque rupture, known to be rare in mice.31
Monocytes, which adhere to activated endothelium, infiltrate in the vessel wall and differentiate into macrophages. Plaque progression is associated with foam cell formation and ultimately leads to the formation of a necrotic lipid core, largely as a result of progressively failing phagocytosis of apoptotic and necrotic cells. Despite a reported role for TSP1 in the regulation of the expression of cell adhesion molecules and promotion of monocyte binding to endothelium,32 we found no genotypic differences in inflammatory cell infiltration in the vessel wall at 6 months, indicating that endothelium was not differently activated in ApoE–/– and Tsp1–/–ApoE–/– mice. From 6 to 9 months, extensive plaque progression was accompanied by 4-fold versus 10-fold elevation of macrophage numbers in ApoE–/– and Tsp1–/–ApoE–/– plaques, respectively. Although the overall plaque burden was comparable between genotypes, plaque maturation was accelerated in Tsp1–/–ApoE–/– mice. Hence, the increased macrophage inflammation at 9 months cannot be explained by upregulation of endothelial adhesion molecules in this interval but appears to relate to the presence of the larger necrotic core in Tsp1–/–ApoE–/– mice.
oxLDL is a potent agonist of macrophage activation via CD36.33 Accordingly, we found that oxLDL potently induced TNF-expression in isolated peritoneal macrophages, both of ApoE–/– and Tsp1–/–ApoE–/– mice. Absence of TSP1 diminished the oxLDL-induced TNF production, but this defect appeared to be attributable to the reduced survival of Tsp1–/– macrophages in vitro and was restored by adding human TSP1. These findings allowed us to exclude functionally relevant competition between oxLDL and TSP1 for the same CD36 receptor,29 in agreement with the existence of different binding sites on CD36 for oxLDL and TSP1.34 Hence, these cell culture experiments ruled out that the absence of TSP1 in ApoE–/– mice would directly be responsible for enhanced CD36 mediated macrophage activation by oxLDL and for accumulation of macrophages in the maturing plaque. Furthermore, although interactions between oxLDL and TSP1 have been shown to suppress the TSP1-dependent activation of TGFβ,35 no differences were detected in total or active TGFβ1 between ApoE–/– and Tsp1–/–ApoE–/– mice, in opposition to a central role for TSP1 in the control of TGFβ function inside atherosclerotic plaques. This is corroborated by the occurrence of comparable Smad phosphorylation in vascular segments lining the plaques, despite low phospho-Smad levels inside lesions (see the online data supplement). Also, the increased collagen content of Tsp1–/–ApoE–/– plaques argues against a functional deficiency in TGFβ activation, because defective TGFβ activation would, instead, lead to a drop in collagen synthesis.36
oxLDL can induce apoptosis of macrophages via CD36,37 and TSP1 can inhibit this process.37 We have investigated apoptosis in plaques of ApoE–/– and Tsp1–/–ApoE–/– mice at 9 months. The total number of TUNEL-positive cells was comparable between genotypes, excluding a prominent role for TSP1 in the control of apoptosis, inside maturing plaques. New findings suggest an important role for regulatory T cells in atherosclerotic lesion formation.38 TSP1 promotes generation of regulatory T cells through its receptor CD47, present on the surface of T cells.39 TSP1/CD47 signaling mediates apoptosis of T cells, downregulating the inflammatory response and limiting collateral damage.40 In view of the comparable degree of apoptosis in ApoE–/– and Tsp1–/–ApoE–/– mice, we did not investigate in larger detail whether the increased inflammation at 9 months was related to a different degree of regulatory T-cell activation during apoptosis.
Our findings confirm the instrumental role of TSP1 in macrophage-mediated phagocytosis22 of platelets in cell cultures. Therefore, the 4-fold elevated presence of nonremoved disintegrated cells in the plaques of Tsp1–/–ApoE–/– mice to over half of the TUNEL-positive cells points to defective phagocytosis in the absence of TSP1. This finding supports the interpretation made above that not cell apoptosis is elevated in Tsp1–/–ApoE–/– plaques. Rather, the accumulation of necrotic material, as a result of defective phagocytosis, appears to accelerate necrotic core development. This core, in turn, may trigger additional inflammation and macrophage infiltration.
This work has uncovered a role for TSP1 in plaque maturation, but additional work will have to address the molecular pathways driven by TSP1 inside plaques to understand how TSP1 modulates atherosclerotic plaque advancement. Such work will provide understanding of its role in phagocytosis inside plaques, in necrotic core progression, and in inflammatory vascular degeneration.
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Acknowledgments |
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We thank Michele Landeloos for help with the lipid isolation, Katrien Cludts for help with the mice, and Sven Terclavers for the assistance during microscopical analysis.
Sources of Funding
This work was supported by Katholieke Universiteit Leuven grant GOA/2004/09 and by the Fonds voor Wetenschappelijk Onderzoek–Vlaanderen (project no G.0569.05). The Center for Molecular and Vascular Biology is supported by the Excellentie financiering KULeuven (EF/05/013). M.T. is a research fellow of the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie-Vlaanderen (Belgium).
Disclosures
None.
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Footnotes |
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Original received December 3, 2007; resubmission received August 18, 2008; accepted September 11, 2008.
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References |
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