Introduction

Thoracic aortic aneurysms (TAAs) are life-threatening diseases defined as a permanent abnormal dilatation of the thoracic aorta. It has been shown that TAAs are associated with dysregulation of TGF-β (transforming growth factor-β) signaling,^1–3 defects in the extracellular matrix,^4–7 and mutations in smooth muscle cell (SMC) contractile genes.^8,9 The anatomic and functional unit connecting SMC and elastic fibers in the aortic wall is called elastin-contractile unit, and the disruption of this unit was shown to cause TAAs because of abnormal mechanosensing of SMCs.¹⁰ The relationship between these defects and aneurysm pathogenesis has begun to be established using various genetic models^11,12; however, the initiation signal and the signaling pathway necessary for sustaining the aneurysmal expansion in each TAA subtype are not fully understood.

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We previously established a mouse model of postnatal TAA by deleting Fbln4 (fibulin-4) gene in vascular SMCs (Fbln4^SMKO, termed SMKO [SMC-specific knockout]). Fbln4 is a secreted glycoprotein and is a component of elastic fibers, where it is localized to microfibrils.¹³ In SMKO aortas, elastic fibers fail to form normal elastic lamina-SMC connections during the early postnatal period, leading to an upregulation of mechanosensitive molecules, such as Egr1 (early growth response 1) and angiotensin-converting enzyme (ACE), resulting in a local elevation of Ang II (angiotensin II) signaling.^7,14 We also showed that an increase in serine/threonine phosphatase Ssh1 (slingshot-1) causes dephosphorylation of cofilin (activated form) and disruption of actin filaments.¹⁰ Furthermore, inhibition of Ang II signaling sufficiently prevented the development of ascending aortic aneurysms and improved elastic fiber integrity and restored actin filaments.¹⁴ However, the molecule responsible for converting the mechanical stress to biochemical signals that result in aneurysm growth has yet to be determined.

Thbs1 (thrombospondin-1) is a homotrimeric glycoprotein secreted by various cells, including vascular SMCs and endothelial cells (ECs). Thbs1 negatively regulates cell adhesion, migration, proliferation, cell-cell interaction, and angiogenesis, whereas positively regulates inflammation and activation of latent TGF-β. This broad spectrum of biological functions is attributable to domain structures with distinct binding sites for various extracellular matrix molecules and cell surface receptors, such as integrins, CD (cluster of differentiation) 36, and CD47.^15–18 Recent studies have shown that Thbs1 contributes to the development of abdominal aortic aneurysm (AAA) through acceleration of vascular inflammation,¹⁹ whereas it was reported that attenuation of Thbs1-directed activation of TGF-β resulted in progression of AAA.²⁰ Thus, the multifunctionality of Thbs1 has complicated the understanding of its role in aortic aneurysms.

Here, we report a dual role for Thbs1 as a mediator of mechanotransduction and critical modulator of arterial wall structure in SMKO mice. We demonstrate that Thbs1 is directly induced by mechanical stretch and Ang II in SMCs and indirectly activates downstream Ssh1-cofilin signaling and leads to actin cytoskeletal remodeling in vivo. Deletion or reduction of Thbs1 restored the disruption of elastic lamina-SMC connections and prevented aneurysm formation, demonstrating a causative role of Thbs1 in aneurysm development in SMKO mice.

Methods

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Results

Upregulation of Thbs1 in the Aneurysmal Wall

As we reported previously, the aortic wall begins to expand in SMKO aortas at postnatal day (P) 7, and the aneurysm is established by P30.^10,14 We defined this period as a critical therapeutic time window in which aneurysm formation could be prevented by inhibiting Ang II-mediated signaling caused by local upregulation of ACE.¹⁴ Transcript level of Thbs1 in SMKO aortas was already increased at P1, before the aneurysm formation, and markedly increased at P30 (Figure 1A). Western blot analysis comparing the expression of Thbs1 in the ascending and descending aortas of CTRL (control) and SMKO mice at P30 showed a marked upregulation of Thbs1 specifically in the ascending aortas of SMKO mice (Figure 1B). Consistent with Western blot data, immunostaining showed strong expression of Thbs1 in the SMKO ascending aortas, including the aortic root compared with that of CTRL aortas (Figure 1C and 1D). Thbs1 was highly expressed in both EC and SMC layers, which was apparent at P9 SMKO aortas (Online Figure I). Interestingly, although phosphorylated cofilin (pCofilin) was decreased (activated) in SMCs of SMKO aortas compared with CTRL, cofilin phosphorylation was maintained in ECs. These data indicated that Thbs1 was highly expressed in the aneurysmal wall before and during aneurysmal expansion in SMKO aortas.

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Fbln4 Deficiency Increases the Response to Ang II and Mechanical Stretch and Upregulates Thbs1 in SMCs

To understand the upstream signals for Thbs1 induction in SMCs, we turned to an in vitro system and performed small interfering RNA (siRNA)-mediated KD (knockdown) of Fbln4. Fbln4 or Scr (as CTRL) was administered to rat SMCs, and the efficiency of KD was confirmed by quantitative polymerase chain reaction (qPCR; Figure 2A). We first examined the response of Fbln4 KD cells to Ang II in the absence of serum (Figure 2B). Ang II induced rapid phosphorylation of ERK (extracellular signal-regulated kinase) in 10 minutes in both Fbln4 KD and CTRL cells. Consistent with previous reports,^21,22 Egr1 was induced by Ang II in both groups, but the level of Egr1 was significantly higher in Fbln4 KD compared with CTRL. Thbs1 was increased at 60 minutes in Fbln4 KD cells, whereas Thbs1 upregulation was observed at 180 minutes after Ang II stimulation in CTRL cells. Similar results on the induction of Thbs1 by Ang II were obtained using primary SMCs isolated from P30 SMKO aortas (Online Figure II), as well as in Fbln4 knockout rat SMCs generated by CRISPR-Cas9 genome editing technology (Online Figure III). However, neither condition was sufficient to affect the phosphorylation status of cofilin.

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Next, to examine the effects of mechanical stimuli on Thbs1 expression, Scr and Fbln4 KD cells were subjected to cyclic stretch (1 Hz; 20% strain) for 16 hours. Fbln4 KD responded to cyclic stretch and upregulated Egr1 and Thbs1 after 1 hour of stimulation, and the amplitude of expression was significantly higher in Fbln4 KD cells compared with CTRL cells (Figure 2C). Upregulation of phosphorylated ERK (pERK) peaked at 8 hours in both Fbln4 KD and CTRL cells, and the levels were comparable between the 2 cells, indicating that ERK activation at the late phase was not affected by Fbln4 KD. Because SMCs in SMKO aortas were shown to be less differentiated compared with CTRL SMCs⁷ and the responses to Ang II and mechanical stretch can be influenced by the differentiation status of SMCs,^23,24 it is plausible that Fbln4 deficiency exacerbated the response to Ang II and mechanical stretch, leading to the increased expression of Egr1 and Thbs1.

Egr1 Is Required for Upregulation of Thbs1

Ang II induces ERK activation by 2 distinct pathways downstream of Agtr1a (angiotensin II receptor, type 1a), mediated by G protein (heterotrimeric guanine nucleotide-binding protein) and Arrb2 (β-arrestin 2).^25,26 To examine the involvement of these pathways in Ang II-induced Thbs1 induction, KD for Agtr1a, Agtr2, and Arrb2 was performed. The KD efficiency was confirmed by qPCR (Figure 3A). Cells were cultured in serum-free media overnight and stimulated with Ang II (100 nmol/L) for 3 hours. In Scr-treated CTRL cells, upregulation of Thbs1 and ERK phosphorylation were reproducibly observed (Figure 3B). Agtr1a KD inhibited ERK phosphorylation and abolished Thbs1 expression, whereas Agtr2 KD showed no effects. Arrb2 KD also showed reduced ERK phosphorylation and suppressed Thbs1 expression.

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Because it has been reported that G protein-mediated ERK phosphorylation requires the activation of PKC (protein kinase C),^25,26 we tested the effect of a PKC inhibitor Ro-31–8425 on Ang II-induced Thbs1 expression (Figure 3C). Ro-31–8425 suppressed Thbs1 expression in a dose-dependent manner; however, it had no effects on ERK phosphorylation. In contrast, pretreatment with PD0325901—a MEK (mitogen-activated protein kinase) inhibitor—completely blocked pERK and suppressed Thbs1 expression. These data suggested that there were pERK-dependent and independent pathways downstream of G proteins that regulate expression of Thbs1. Because it has been reported that Ang II-induced ERK activation is mediated by Egr1,²⁷ and Egr1 is a known activator of Thbs1,²⁸ we tested whether Ang II-induced Thbs1 expression was dependent on Egr1 in rat SMCs. Egr1 was knocked down by 2 sets of siRNA (Figure 3D), and Ang II was administered to induce Thbs1 expression. Egr1 KD abolished phosphorylation of ERK and Ang II-induced Thbs1 expression for both sets of siRNA (Figure 3E). These results indicated that Egr1 was an essential mediator of Thbs1 downstream of Ang II-Agtr1a.

We also examined the regulation of Thbs1 by mechanical stretch in SMCs. We used Agtr1a, Agrt2, Arrbs2, and Egr1 KD cells and subjected them to cyclic stretch (1 Hz; 20% strain) for 8 hours. As Figure 3F shows, Thbs1 was induced by cyclic stretch in Scr-treated cells with upregulation of Egr1 and pERK. Agtr1a KD modestly suppressed the increase in Thbs1 expression with stretch, without affecting Egr1. Agtr2 KD or Arrb2 KD did not change the effect of stretch on Thbs1 expression or Egr1 expression. In contrast, Egr1 KD completely suppressed Thbs1 expression and pERK upregulation in response to stretch, indicating that Egr1 is an essential regulator of Thbs1 expression downstream of mechanical stress.

Deletion of Thbs1 Attenuates Aneurysm Formation in SMKO Aortas

To test whether Thbs1 contributed to aneurysm formation in SMKO mice, we generated SMKO mice on a Thbs1-null background DKO (SMKO;Thbs1 knockout). qPCR confirmed the absence of Thbs1 mRNA in DKO aortas (Figure 4A). The aneurysms were examined at 3 months of age and compared with respective CTRL littermates. Aneurysms were mostly prevented in DKO mice (30 of 38; 78.9%), and some of SMKO;Thbs1^+/− mice (12 of 25; 48%) also showed amelioration of the aneurysm (Figure 4B; Table 1). Morphology of SMCs and collagen deposition in rescued DKO aortas were comparable with that of CTRL aortas, and DKO aortas contained intact elastic fibers that were visualized by Hart staining (Figure 4C). The internal elastic lamina perimeter and outer perimeter were smaller in DKO compared with SMKO aortas; however, the aortic wall was thicker in DKO aortas compared with CTRL (Figure 4D). We hypothesized that the deletion of Thbs1 enhanced elastic fiber maturation and prevented aneurysm formation. To test this hypothesis, we performed in vitro elastogenesis assays using neonatal human dermal fibroblasts²⁹ and examined the function of Thbs1 in elastic fiber assembly by siRNA (Online Figure IVA). Scr siRNA-treated cells (CTRL) did not affect elastic fiber formation, whereas FBLN4 siRNA suppressed the deposition of elastin onto the fibrillin-1–positive microfibrils (Online Figure IVB). THBS1 siRNA-treated cells were indistinguishable from Scr-treated cells, and the combination of THBS1 and FBLN4 siRNA did not improve elastic fiber assembly. These results showed that Thbs1 was not directly involved in elastic fiber assembly in vitro.

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To gain more insights into the ultrastructure of DKO aortas, we performed electron microscopy, compared with CTRL and SMKO aortas. Consistent with the previous findings, SMKO aorta showed severe disruption of elastic fibers and a loss of connections between the elastic laminae and SMCs—connections that were evident in CTRL aortas (arrows in CTRL; Figure 4E). Surprisingly, the elastic laminae in rescued DKO aortas were thicker and much more organized compared with SMKO aortas and contained numerous short elastin extensions extending toward the SMCs (arrowheads in DKO; Figure 4E). These findings suggested that the deletion of Thbs1 promoted the organization of elastic fibers in vivo. To examine the impact of improved elastic fiber organization on biomechanics of the aorta, we performed pressure-diameter experiments for DKO aortas and compared with CTRL and SMKO aortas.^30,31 The outer diameter of DKO aortas exhibited pressure-dependent dilatation, and significant differences were not found at any pressures compared with CTRL (Figure 4Fa). Compliance—the inverse of stiffness—was higher in SMKO aortas between 0 and 50 mm Hg and became significantly lower above physiological pressures (>100 mm Hg; Figure 4Fb). DKO aortas showed lower compliance than SMKO aortas between 0 and 50 mm Hg similar to CTRL; however, it was significantly decreased compared with CTRL between 125 and 170 mm Hg and behaved similar to SMKO aortas (Figure 4Fb). We then examined the material properties of DKO aortas by calculating tangent modulus using stress-stretch relationship (Figure 4Fc and 4Fd). Tangent modulus of DKO aortas was significantly lower than SMKO aortas but did not differ from that of CTRL at any pressure, indicating the restoration of the material properties in DKO aortas. Taken together, prevention of aneurysm development was supported by normalization of material properties and improved quality of elastic fibers.

Deletion of Thbs1 Suppresses Abnormal Mechanotransduction in the Aneurysmal Wall

To determine which signaling pathways were affected in DKO aortas, we performed Western blot analyses to compare the signaling molecules dysregulated in SMKO, including ACE, Thbs1, Egr1, pSmad2 (phosphorylated mothers against decapentaplegic homolog 2), pERK, pCofilin, and Ssh1. Although ACE and pERK remained high in DKO aortas, Ssh1 and Egr1 were significantly decreased, and pCofilin was increased in DKO aortas (Figure 5A). Because Thbs1 is known to activate latent TGF-β, the rescue might have been achieved through inhibition of TGF-β. However, pSmad2 levels were unchanged in DKO aortas (Figure 5A), indicating that the rescue was independent of canonical TGF-β signaling and that activation of latent TGF-β by Thbs1 was not extensive in SMKO animals. Immunostaining results confirmed that pCofilin levels were restored to CTRL levels in DKO aortas (Figure 5B). Accordingly, actin filaments in DKO aortas were continuous compared with those of SMKO aortas (Figure 5C). Interestingly, qPCR analysis revealed that expressions of SMC contractile genes were unchanged compared with SMKO aorta except upregulation of Acta2 (α-smooth muscle actin; Figure 5D). Because Acta2 is the major actin isotype in SMCs, Acta2 upregulation may have been contributed to the increased actin filaments.

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Next, to test the possibility that Thbs1 directly inhibited phosphorylation of cofilin in SMCs in the absence of Fbln4, we used Fbln4KO rat SMCs (described in Online Figure III) and primary SMCs isolated from SMKO aortas (described in Online Figure II) and treated with rThbs1 (recombinant Thbs1) for 6 hours followed by Western blot analysis. The phosphorylation status of cofilin was not affected by addition of rThbs1 in Fbln4KO rat SMCs (Online Figure VA) or primary SMKO SMCs (Online Figure VB). It has been reported that Thbs1 inhibits pCofilin in macrophage,³² which cell type is frequently observed in experimental aortic aneurysms.³³ Therefore, we evaluated the presence of macrophages in the aneurysmal wall of CTRL, SMKO, and DKO mice (Online Figure VI). qPCR analysis showed an increase in Cd68 and Mcp1 transcripts in SMKO but not in DKO aortas (Online Figure VIA). However, immunostaining for CD68 in SMKO or DKO aortas showed no accumulation of CD68-positive macrophages (Online Figure VIB), consistent with our previous findings.⁷

It is of note that Thbs1 was highly expressed in ECs of SMKO aortas from P9 (Online Figure I); however, phosphorylation of cofilin was unaffected. To examine whether Thbs1 in ECs contributed to the formation of aneurysms via acting on their receptors, we generated double knockout mice for SMKO and Cd36 (SMKO;Cd36^−/−), as well as SMKO and Cd47 (SMKO;Cd47^−/−), both of which are known Thbs1 receptors expressed in ECs.^34,35 Both double knockout mice were evaluated for the presence of aneurysms by gross observations at P30 (Online Figure VIIA). A large aneurysm was observed in the ascending aortas of SMKO;Cd36^−/− and SMKO; Cd47^−/− animals with severe tortuous descending aortas. Histological observations revealed thick aortic wall and disrupted elastic fibers in SMKO, as well as SMKO;Cd36^−/− and SMKO; Cd47^−/− animals (Online Figure VIIB). Morphometric analysis showed that internal elastic lamina perimeter, outer perimeter, total vessel area, and wall thickness were largely comparable among SMKO, SMKO;Cd36^−/− and SMKO;Cd47^−/− mice (Online Figure VIIC). These data indicate that Thbs1 does not directly alter phosphorylation of cofilin in SMCs, and neither CD36 nor CD47 is involved in the aneurysm formation in SMKO mice.

Thbs1 Is Increased in Human TAAs

Finally, to evaluate whether the THBS1-mediated pathway was involved in the pathogenesis of TAA in humans, we examined the expression of THBS1 and pERK levels in TAA samples from nonsyndromic sporadic patients with TAA that underwent surgery. Aortic wall punch biopsies obtained from patients undergoing coronary artery bypass surgery were designated as nonaneurysmal CTRL. There were no differences in sex, age, metabolic rate, blood pressure, and cardiac functions between CTRL and TAA (Table 2). Western blot analysis revealed a significant increase in THBS1 levels in aneurysmal tissues compared with CTRL (Figure 6A). In contrast, pERK levels were comparable between CTRL and TAA samples. Histological analysis of TAA with high THBS1 (n=6) and low THBS1 (n=4) both showed elastic fiber fragmentation, and the degree of fragmentation seems comparable between the 2 groups (Figure 6B). THBS1 levels did not correlate with clinical features (Online Figure VIII). Electron microscopy images from TAA with high THBS1 exhibited the same loss of elastic lamina-SMC connections that was observed in SMKO aortas (Figure 6C). Taken together, the data demonstrated that THBS1 elevation was associated with nonsyndromic sporadic TAA in humans.

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Discussion

In this study, we reported the upregulation of Thbs1 in TAAs of mice and humans and showed that genetic deletion of Thbs1 prevented aneurysms in SMKO mice. In SMCs, Thbs1 expression was markedly induced by mechanical stretch and Ang II, and Egr1 was required for the induction of Thbs1. Deletion of Fbln4 in vitro enhanced the responses to these stimuli and led to the increased expression of Egr1 and Thbs1, further contributing to the transduction of mechanical stress into biochemical signals in SMCs. Aortas from DKO mice showed restoration of elastic lamina-SMC connections with numerous short elastin extensions and suppression of Ssh1-cofilin signaling, revealing the unrecognized role of Thbs1 as a modulator of arterial wall integrity.

Novel Role of Thbs1 in Mediating Mechanical Stress in the Aneurysmal Wall

Thbs1 is maintained at low levels in the postnatal vessels and elevated in various vascular diseases, including pulmonary arterial hypertension,³⁶ aortic aneurysms,³⁷ atherosclerosis,³⁸ cerebral cavernous malformations,³⁹ and ischemia reperfusion injury.⁴⁰ The role of Thbs1 in these conditions seem to be highly context dependent but is largely associated with activation of TGF-β or inflammatory cytokine production. For example, in AAAs, Thbs1 promotes macrophage recruitment and contributes to adventitial inflammation during aneurysm formation,¹⁹ whereas blockade of Thbs1-directed TGF-β activation or Thbs1 deficiency promotes inflammation and accelerates AAA.^20,38 Because central pathogenesis of TAA is different from that of AAA and a reduced contribution of inflammation has been observed,⁴¹ we speculated that the mechanistic role of Thbs1 in TAA might be distinct from that of AAA. Our current study shows that pathological Thbs1 contributes to abnormal mechanosensing of SMCs and is essential for the development of TAAs in SMKO mice.

The pathway(s) involved in Thbs1 upregulation has been investigated in various cell types, and Ang II is generally known as an upstream regulator of Thbs1.^42,43 TGF-β by forming a positive feedback loop also induces Thbs1 expression in a Smad2-dependent manner,⁴⁴ and this pathway can be amplified by Ang II because TGF-β is a transcriptional target of Ang II.⁴⁵ In addition, recent studies have shown that physical factors, such as hypoxia and disturbed flow, induce Thbs1.^36,46 In SMKO aortas, Thbs1 is highly upregulated in ECs and inner portions of SMCs in the ascending aortas, supporting the relationship between physical factors and Thbs1 expression. Thbs1 is more robustly upregulated in Fbln4 KD SMCs than CTRL in response to mechanical stretch, indicating that SMKO SMCs may be exquisitely sensitive to changes in mechanical stimuli, such as increased pulse pressure,¹⁴ decreased compliance, and increased elastic modulus observed in SMKO aorta. The unique and complex mechanical environment of the ascending aorta⁴⁷ in combination with altered SMC mechanosensing may contribute to localized Thbs1 signaling. Whereas Ang II quickly activates ERK via Agtr1a and upregulates Thbs1 in isolated SMCs, mechanical stretch induces Thbs1 at 8 hours with delayed ERK activation. Intriguingly, in both cases, Egr1 is essential for Thbs1 induction. Taken together, our results suggest that Thbs1 is a mechanosensitive matricellular protein upregulated by Egr1 that activates ERK, which in turn induces Egr1 and produces Ang II via upregulation of ACE, generating a feed forward loop for aneurysm expansion in the ascending aorta.

Deletion of Thbs1 Prevents Aneurysm Formation With Restored Elastic Lamina-SMC Connections in SMKO Aorta

Thbs1 is a matricellular protein known to mediate cell-matrix interactions with a limited role in tissue morphogenesis. Based on our results using DKO mice, the deletion of Thbs1 is sufficient to prevent aneurysm formation in SMKO mice. Surprisingly, the DKO aorta showed better quality of elastic fibers with restored connections between elastic lamina and SMCs, and actin cytoskeletal abnormality is corrected. This suggests that Thbs1 is a negative regulator of elastic fiber organization. Although we failed to make a direct link between Thbs1 and inhibition of elastic fiber assembly in vitro, there are possible explanations for the potential contribution of Thbs1 in elastic fiber formation. First, electron microscopy analysis showed numerous elastic extensions between elastic laminae and SMCs, indicating the restoration of the elastin-contractile units, thus the vessel wall was stabilized in DKO mice. The absence of antiadhesive activity of Thbs1 may contribute to the increased formation of elastic lamina-SMC connections in DKO mice. This is the first demonstration that a loss of Thbs1 improves the elastic fiber organization in the structurally compromised aortas. Second, it has previously been shown that inhibition of versican, which is a large extracellular matrix proteoglycan, promotes tropoelastin synthesis and elastic fiber formation in human leiomyosarcoma SMCs.⁴⁸ Because versican forms a functional complex with Thbs1 and is colocalized on microfibrils,⁴⁹ deletion of Thbs1 may have disrupted the inhibitory complex and facilitated elastic fiber formation in DKO aortas.

Thbs1 is hardly expressed in CTRL aortas, whereas it is highly expressed in ECs and SMCs of SMKO aortas. Our knockout study using Cd36^−/− and Cd47^−/− mice did not lead to the rescue of aneurysms in SMKO, indicating that the function of Thbs1 is not mediated via these receptors. However, we failed to determine whether the function of Thbs1 was cell autonomous in SMCs or EC-derived Thbs1 acted on neighboring SMCs. A recent study showed that deletion of an Agtr1a in ECs attenuates the development of ascending aneurysms in Marfan model mice, where pathological changes are usually seen in SMCs.⁵⁰ Therefore, it is possible in SMKO mice that an Agtr1a drives Thbs1 production in ECs, which is delivered and acts on SMC in a noncell autonomous manner. It will be informative to determine the cell type responsible for production and execution of the deleterious effect of Thbs1 during aneurysm formation.

As we have shown previously, the activity of cofilin, which is regulated by its phosphatase Ssh1, causes abnormal actin cytoskeletal remodeling in SMKO aortas.¹⁰ Although the expression of ACE and pERK remained unchanged, Ssh1 levels are decreased, and phosphorylation of cofilin is increased in DKO aortas. As a result, actin filaments are continuously compared with those of SMKO aortas. It is likely that restoration of elastic fibers in the absence of Thbs1 indirectly suppresses the Ssh1-cofilin pathway in DKO aortas. Alternatively, because Thbs1 has been shown to bind the actin cytoskeleton via its cell surface receptors⁵¹ or to increase phosphorylation of FAK (focal adhesion kinase) in SMCs,⁵² the absence of Thbs1 may have altered the actin filaments via the alteration of focal adhesions in SMCs.

Application to Human Aortic Aneurysms

Imaging techniques, such as computed tomography, magnetic resonance imaging, and ultrasound, are currently used for routine diagnosis and monitoring of aneurysms. However, these imaging tools cannot be used to accurately determine the preclinical status and predict the timing for dissection or rupture of aneurysms.⁵³ It has been reported that aortic size does not always provide an accurate prediction for type A or type B aortic dissection.^54–56 Therefore, there is a pressing need for the discovery of blood-borne biomarkers that enable us to assess the dissection or rupture risk in a timely manner. Recent analysis of differentially expressed proteins in aortic aneurysms in human patients showed that Thbs1 is increased in TAAs.^37,57 Although the association of Thbs1 and aortic aneurysms is not fully established in humans, it was suggested that a high serum concentration of Thbs1 was associated with slower growth of AAA in humans.⁵⁸ Intra-arterial thrombus secretome analysis showed a negative association between Thbs1 and AAA.⁵⁹ Our mouse study demonstrates that Thbs1 is directly involved in TAA formation, and an increase in Thbs1 transcripts is already evident before developing a distinct aneurysm. Although our model fails to address the relevance of Thbs1 in aortic dissection, temporal analysis reveals that it could potentially predict the preclinical status of the aneurysm. Second application is to develop anti-THBS1 therapy for prevention/control of aneurysm growth. Ang II receptor blockers have been suggested as a new drug to treat aneurysm growth in patients with Marfan syndrome⁴⁵; however, large clinical trials comparing the effects between losartan and β-blocker failed to establish the advantage of losartan over atenolol.⁶⁰ Directly targeting Thbs1 and inhibiting maladaptive upregulation of Thbs1 may be effective in controlling the aneurysm growth, especially those with a high level of THBS1 at the early clinical stage of aneurysm. Further analysis with human serum Thbs1 levels and its association with imaging data will provide a basis for Thbs1-targeted therapy as a potential treatment for TAA.

Acknowledgments

We thank G. Urquhart, K. Matsumura, M. Masuda, J.V. Alves, and S. Sato for technical assistance, N.A. Abumrad for providing Cd36-null mice, P.A. Oldenborg for providing Cd47-null mice, and Y. Jinzenji and L.J. Ringuette for assistance with electron microscopy. We acknowledge technical support from the Facility for Electron Microscopy Research at McGill University and the Histology Core Laboratory at UT Southwestern Medical Center. We appreciate R. Ann Word for critical reading of the manuscript.

Footnotes