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. Author manuscript; available in PMC: 2017 Jan 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2015 Nov 12;36(1):60–68. doi: 10.1161/ATVBAHA.115.306397

Deletion of Periostin Protects Against Atherosclerosis in Mice by Altering Inflammation and Extracellular Matrix Remodeling

Jennifer A Schwanekamp 1,*, Angela Lorts 1,*, Ronald J Vagnozzi 1, Davy Vanhoutte 1, Jeffery D Molkentin 1,2
PMCID: PMC4690815  NIHMSID: NIHMS734587  PMID: 26564821

Abstract

Objective

Periostin is a secreted protein that can alter extracellular matrix remodeling in response to tissue injury. However, the functional role of periostin in the development of atherosclerotic plaques has yet to be described despite its observed induction in diseased vessels and presence in the serum.

Approach and Results

Hyperlipidemic, apolipoprotein E-null mice (ApoE−/−) were crossed with periostin (Postn−/−) gene-deleted mice and placed on a high fat diet for 6 or 14 weeks to induce atherosclerosis. En face analysis of aortas showed significantly decreased lesion areas of ApoE−/− Postn−/− mice compared to ApoE−/− mice, as well as a reduced inflammatory response with less macrophage content. Moreover, diseased aortas from ApoE−/− Postn−/− mice displayed a disorganized extracellular matrix with less collagen cross linking and smaller fibrotic caps, as well as increased matrix metalloprotease-2, matrix metalloprotease-13, and procollagen-lysine, 2-oxoglutarate 5-dioxygenase-1 mRNA expression. Furthermore, loss of periostin was associated with a switch in vascular smooth muscle cells towards a more proliferative and synthetic phenotype. Mechanistically, loss of periostin reduced macrophage recruitment by transforming growth factor-β in cellular migration assays.

Conclusions

These are the first genetic data detailing the function of periostin as a regulator of atherosclerotic lesion formation and progression. The data suggest that periostin could be a therapeutic target for atherosclerotic plaque formation through modulation of the immune response and extracellular matrix remodeling.

Keywords: atherosclerosis, periostin, extracellular matrix, vascular smooth muscle cells

Introduction

Cardiovascular disease remains the leading cause of mortality in the Western world accounting for one third of all deaths, primarily due to myocardial infarction (MI) as a direct result of atherosclerosis.1 Therefore, understanding the biological mechanisms underlying atherosclerotic plaque development is critical towards uncovering new treatment strategies to prevent or delay this disease. Extensive research over the past few decades has shown that imbalances in vascular smooth muscle cell (VSMC) activation, excess extracellular matrix (ECM) production, persistent inflammation, and retention of cholesterol-rich debris can all contribute to plaque formation in atherosclerosis.2, 3 VSMCs are a critical cell type in the vessel wall that are usually quiescent in stable vessels. However, upon injury, VSMCs switch from a contractile, quiescent phenotype to a synthetic, proliferative phenotype, where they produce ECM proteins, such as periostin, thereby contributing to the deposition of a complex and mature cross-linked collagen matrix.2,4,5 Transforming growth factor-β (TGF-β) signaling stimulates VSMCs to proliferate and migrate forming the fibrous cap of the plaque.6–8 Matrix metalloproteases (MMPs) are also expressed to facilitate VSMC migration through the plaque via ECM degradation. Interestingly, several lines of evidence suggest both pro and anti-atherogenic roles for many ECM proteins.6, 9–12 However, both clinical and experimental data agree that excessive MMP expression can contribute to plaque destabilization by favoring ECM degradation, ultimately predisposing plaques to rupture. 13,14

Periostin is a 90-kDa secreted ECM/matricellular protein that contains four fasciclin domains homologous to the insect protein fasciclin I, which is involved in cell adhesion.15, 16 Periostin has been proposed to function upstream and downstream of TGF-β.15, 16 At baseline, low levels of periostin are detected in various adult tissues, but with acute injury periostin expression and secretion are dramatically increased.5, 17 For example, periostin is abundantly re-expressed in the heart following both MI and pressure overload injuries.18–20 In addition, periostin is expressed in embryonic cardiac valves, is re-expressed with valvular injury in the adult, and is known to be secreted by VSMCs.17, 21, 22 Studies have shown that periostin is abundant in degenerated human cardiac valves and the absence of periostin in mice is able to rescue valve degeneration.17 In the vasculature, periostin is up-regulated in vessel walls of rat carotid arteries after balloon injury and it has been detected within the intimal portion of human vessels containing atherosclerotic plaques.22, 23 Additionally, recent work has shown that periostin can act as a chemoattractant for tumor associated macrophage recruitment in a mouse model of glioblastoma. 24 One of the hallmarks of atherosclerotic plaque progression is the migration of monocytes through the vessel wall where these cells differentiate into macrophages and consume excess lipids to form foam cells. Foam cells make up the lipid core and death of these cells eventually results in the development of the necrotic core. 25, 26

A role for periostin in the regulation of ECM production and maturation has been defined in many tissues including the heart and skin, where loss of periostin results in decreased collagen cross-linking and degradation of the ECM. 4, 5, 18 Periostin is also known to bind to collagen I in the heart, and loss of periostin results in decreased collagen V deposition after MI with altered ventricular remodeling.4, 5, 18 Although periostin has been linked to the pathogenesis of many cardiovascular diseases, its specific role during atherosclerotic plaque formation and progression remains undefined.

Materials and Methods

Materials and methods are available in the online-only supplement

Results

Periostin is induced in atherosclerotic plaques

To determine if periostin is upregulated during the development of atherosclerosis, ApoE−/− mice were fed a western, high fat diet (HFD) and their aortas and serum were assayed for periostin. Indeed, periostin protein was significantly increased in the aortic intima and plaques of ApoE−/− mice after 14 weeks on HFD (Figure 1A). As a control, we also assessed the aortic valves, which are known areas of continuous periostin expression.21, 27, 28 Serum levels of periostin were also increased in ApoE−/− mice compared to controls after 6 weeks of HFD (Figure 1B). Following either 6 or 14 weeks of HFD, no differences in body weight, triglyceride or cholesterol levels between ApoE−/− and ApoE−/− Postn−/− mice were detected (Supplementary Table I). Additionally, total cholesterol levels in both ApoE−/− and ApoE−/− Postn−/− mice were significantly higher than Wildtype (WT) and Postn−/− mice indicating that HFD did result in hypercholesterolemia in mice deficient for ApoE (data not shown).

Figure 1.

Figure 1

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Periostin expression is altered during atherosclerotic development. A. Representative images of WT and ApoE−/− aortic root sections stained for periostin (green), DAPI (blue) and αSMA (red). Scale = 100 μM. Arrows show the aortic valves as a control. Arrowheads show induction of periostin protein in the diseased aorta and intima. B. Periostin ELISA performed on serum harvested from the indicated genotypes of mice after 6 weeks of HFD. Number of mice used is shown in the graph. *P<0.05 vs WT

Loss of periostin decreases atherosclerotic plaque formation

En face analysis of the aorta was performed to assess the progression of atherosclerotic disease in ApoE−/− versus ApoE−/− Postn−/− mice. WT and Postn−/− displayed no development of atherosclerotic lesions after 6 and 14 weeks on HFD (data not shown). Plaque burden in the aortic arch and the abdominal aorta of ApoE−/− Postn−/− mice was significantly reduced compared to ApoE−/− after both 6 and 14 weeks of HFD (Table 1). Additionally, the abdominal aortas of ApoE−/− Postn−/− mice displayed reduced plaque burden after 6 and 14 wks on HFD versus ApoE−/− mice (Figure 2A). Furthermore, the aortic roots of ApoE−/− Postn−/− mice showed significantly reduced lipids by oil red O staining compared with ApoE−/− controls, indicating that loss of periostin decreases lesion size (Figures 2B and 2C). Taken together these data suggest that expression of periostin during plaque formation and maturation permits disease progression.

Table 1.

Aortic atherosclerotic lesion area as a percent of total aorta area in ApoE−/− and ApoE−/−Postn−/− mice

6 weeks 14 weeks
ApoE−/− ApoE−/−Postn−/− ApoE−/− ApoE−/−Postn−/−
Aortic Arch 3.51 ± 0.58 1.46 ± 0.22* 22.17 ± 2.08 9.45 ± 1.25*
Thoracic Aorta 0.73 ± 0.12 0.35 ± 0.07* 8.27 ± 0.80 3.83 ± 0.60*
Abdominal Aorta 1.03 ± 0.29 0.42 ± 0.13 9.89 ± 0.74 7.87 ± 1.96*
Total 1.34 ± 0.15 0.59 ± 0.08* 11.02 ± 0.72 5.85 ± 0.44*
n 10 7 11 8
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Data are represented as mean ± SEM.

*

indicates p<0.05 compared to ApoE−/−.

Figure 2.

Figure 2

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Periostin contributes to plaque development in atherosclerotic mice. A. Quantification of total aortic plaque content as a percentage of the total aortic area after 6 weeks (left) and 14 weeks (right) of HFD in the indicated groups of mice. *P<0.01 vs ApoE−/−. B. Representative 10x magnification images of oil red O stained aortic root sections after 14 weeks of HFD. C. Quantification of the percent oil red O staining in the aortic root sections of the indicated genotypes after 14 weeks of HFD. *P<0.05 vs ApoE−/−

Plaque architecture is altered upon loss of periostin

To investigate how the loss of periostin alters plaque formation and organization, we performed detailed histological analysis on the aortic root of ApoE−/− versus ApoE−/− Postn−/− mice following 14 weeks of HFD. ApoE−/− Postn−/− plaques had a smaller necrotic core and fibrous cap (Figure 3A, arrows). We quantified the percent of the total plaque occupied by fibrous cap, which was significantly smaller in ApoE−/− Postn−/− mice compared to ApoE−/− controls (Figure 3B). Additionally, the plaques within ApoE−/− mice had more cholesterol clefts when compared to the plaques within ApoE−/− Postn−/− mice (Figure 3C). Together, these data suggest that periostin contributes to plaque development and that loss of this gene appears to delay disease progression.

Figure 3.

Figure 3

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Loss of periostin reduces fibrous cap and cholesterol cleft formation. A. Representative images of H&E stained aortic root sections at 10x magnification for the indicated genotypes at the indicated times. The arrows on the Postn−/− ApoE−/− sections show a smaller necrotic core versus the core areas from ApoE−/− aortas at each time point. B. Quantification of the percent of the plaque taken up by the fibrous cap after 14 weeks of HFD. C. Quantification of the number of cholesterol clefts normalized to plaque area after 14 weeks of HFD. The number of mice used is shown within each of the panels. *P<0.05 vs ApoE−/−.

Collagen disorganization in ApoE−/− Postn−/− plaques

Based on previous findings that loss of periostin alters the ECM in the heart, skin, and skeletal muscle after injury, we hypothesized that deletion of Postn would also affect the ECM in aortic vasculature, which could impact atherosclerosis susceptibility and progression. Sirius red staining showed no difference in total collagen content normalized to plaque area in histological sections of aortas from ApoE−/− and ApoE−/−Postn−/− HFD- fed mice (Figures 4A and 4B). However when visualized under polarized light to assess collagen fiber maturity, the plaque region of ApoE−/− mice contained significantly more orange-red, tightly packed and well-organized collagen fibers, whereas ApoE−/− Postn−/− plaques displayed significantly greater yellow-green, loosely packed and less cross-linked collagen fibers (Figures 4A and 4C). To determine whether this was due to a defect in collagen production and/or MMP-mediated ECM breakdown in ApoE−/− Postn−/− mice, we analyzed mRNA expression of several collagens, MMPs, and other ECM remodeling genes in thoracic aortas from all 4 groups of mice not subjected to HFD. It is important to note that even on normal diet, ApoE−/− mice develop hypercholesterolemia by 6 weeks of age and early plaque development can be observed by 10 months of age.29 Therefore, to exclude any potential alterations in mRNA expression due to the increased plaque burden in the ApoE−/− mice on HFD, these analyses were performed on mice fed normal chow. Additionally, vessels early in disease might also suggest a more mechanistically proximal array of gene expression changes that contributes to disease initiation and progression. In early disease aortas we observed that expression of collagens 1α1, 1α2, and 3α1 were all significantly increased in aortas from both Postn−/− and ApoE−/− Postn−/− mice compared with WT and ApoE−/− (Figures 4D, 4E, and 4F). However, the expression of collagen 5α3 was significantly increased only in ApoE−/− Postn−/− aortas compared to the three other experimental groups (Figure 4G).

Figure 4.

Figure 4

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Loss of periostin alters the ECM of atherosclerotic plaques and vasculature. A. Representative images of sirius red stained aortic root sections under bright field (top) and polarized light (bottom). B. Percent collagen content quantified from sirius red stained sections under bright field. C. Quantification of the orange red (OR) and yellow green (YG) birefringence under polarized light normalized to plaque area. *P<0.05 vs ApoE−/−. D-H. Quantitative real time PCR for mRNA levels of non-diseased pooled thoracic aortas (n≥4) of the indicated genotypes for the indicated genes. I,J. MMP3-specific zymography (blue gel, Zym.) and quantitation from the indicated genotype of mice (N=3 in I). The 2 panels below in “J” are Western blots for MMP3 (M3) and GAPDH control (con.). K-O. Quantitative real time PCR for mRNA levels of the indicated gene from non-diseased pooled thoracic aortas (n≥4) of the indicated genotypes of mice. P<0.05 vs WT (#), vs ApoE−/− (*), vs Postn−/− (†).

Several MMPs have been shown to be involved in atherosclerotic plaque development and maintenance 30, 31 The expression (mRNA and protein) and enzymatic activity of MMP3 was significantly increased in ApoE−/− Postn−/− aortas compared to the 3 other groups (Figures 4H–J). MMP9 expression remained unchanged (Figures 4K). MMP12 has been shown to correlate with advanced atherosclerotic lesions in humans and promote macrophage invasion. 30 We found decreased mRNA levels of MMP12 in ApoE−/− Postn−/− versus ApoE−/− aortas, suggesting reduced disease propensity (Figure 4L). Additionally, we found significantly increased expression of procollagen-lysine, 2-oxoglutarate 5-dioxygenase-1 (Plod1) and MMP13, genes that are involved in collagen degradation and maturation, in pre-diseased ApoE−/− Postn−/− aortas compared to ApoE−/− aortas (Figures 4M and 4N).31, 32 We also observed a significant increase in lysyl oxidase (Lox) expression, which is required for plaque stability and collagen crosslinking in atherosclerosis, in pre-diseased ApoE−/− Postn−/− aortas (Figure 4O). These data suggest that loss of Postn in the ApoE−/− background does not impair collagen production, but that it likely affects collagen organization and/or maturation due to decreased cross-linking and MMP-mediated degradation, which could impact the initiation and progression of atherosclerosis. Taken together, loss of the Postn gene appears to lead to select defects in vascular collagen homeostasis early in disease, which alters atherosclerotic progression in ApoE−/− mice.

Altered cellularity in ApoE−/− Postn−/− atherosclerotic plaques

Inflammatory events in the vessels typically lead to VSMC phenotype changes, which we analyzed here. A significant decrease was observed in the number of smooth muscle α-actin (α-SMA) positive cells, a marker of the contractile phenotype of VSMCs, in the aortas of ApoE−/− Postn−/− mice compared to ApoE−/− mice, suggesting a more synthetic phenotype (Figure 5A). We confirmed and expanded these results by performing qPCR for gene expression changes that are associated with the synthetic phenotype of VSMCs on thoracic aortas prior to disease manifestation (Figures 5B–D). We observed decreases in α-SMA and smooth muscle myosin heavy chain (SM-MHC) mRNA in ApoE−/−, and ApoE−/−Postn−/− compared to WT suggesting that VSMCs in hypercholesterolemic aortas may switch phenotype early in disease progression (Figure 5B). Unexpectedly, loss of Postn alone resulted in a significant reduction in α-SMA and SM-MHC mRNA levels in pre-diseased aortas which could be associated with an underlying defect in the ECM environment of these vessels (Figure 5B and 5C). Osteopontin, another marker of the synthetic cellular phenotype, was also dramatically induced in ApoE−/− aortas at disease initiation, but this effect was significantly reduced in double null aortas suggesting a potential delay in disease progression (Figure 5D).

Figure 5.

Figure 5

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Loss of periostin alters plaque cellular content, proliferation and VSMC gene expression. A, Quantification of αSMA positive cells normalized to plaque area. *P<0.05 vs ApoE−/−. B–D, qPCR for the indicated mRNA from aortas of the indicated mice on 14w HFD. *P<0.05 vs WT; #P<0.05 vs Postn−/− †P<0.05 vs ApoE−/−. E. Representative images of EdU positive nuclei (green) after 14w of HFD, scale = 20 μm. F, G. Quantification of EdU positive nuclei normalized to plaque area on HFD for the time shown. *P<0.05 vs ApoE−/−. H-J. qPCR for mRNA levels of the indicated genes from isolated WT and Postn−/− VSMCs. *P<0.05 vs WT.

To further investigate the synthetic VSMC phenotype, mice were injected with EdU after both 6 and 14 weeks of HFD. ApoE−/− Postn−/− mice on diet for both time-points had significantly more cellular proliferation in the aortas as measured by EdU positive nuclei compared to ApoE−/− mice (Figure 5E–G). Additionally, the majority of the EdU positive nuclei were also positive for vimentin staining, a marker of synthetic VSMCs (Fig. 5E).33, 34 Furthermore, qPCR performed on isolated WT and Postn−/− VSMCs showed that loss of periostin resulted in significantly decreased mRNA expression of Neuropeptide Y (NPY), Macrophage scavenger receptor 1 (MSR1), and β2 integrin (Figure 5H–J). Several studies have shown these genes to be upregulated in the development of atherosclerosis.35–37 Taken together, these data indicate that the VSMC phenotype switch characteristic of atherosclerosis is intact in ApoE−/−Postn−/− mice.

Loss of Periostin affects the infiltration of inflammatory cells into the plaque

Periostin is known to be involved in the inflammatory response; therefore, we surveyed the macrophage content (Mac 3) of aortic plaques in ApoE−/− versus ApoE−/− Postn−/− mice. Immunohistochemical analysis showed significantly fewer cells positive for Mac 3 in diseased aortas when normalized to plaque area in ApoE−/− Postn−/− mice compared to ApoE−/− mice (Figure 6A, B).17, 38 To understand the mechanism behind decreased macrophage content in ApoE−/− Postn−/− plaques we analyzed isolated macrophages. Bone marrow derived macrophages cultured for five days in media containing 100 U/mL of macrophage colony stimulating factor (MCSF) were analyzed by flow cytometry and found to be 96% (WT) and 94% (Postn−/−) CD11b positive, indicative of differentiated macrophages (Supplementary Figure I). A previous study showed that periostin can act as a chemoattractant for macrophages in a model of glioblastoma.24 Indeed, similar to TGF-β, recombinant periostin protein caused a significant increase in the migration of WT macrophages but not Postn−/− macrophages (Figure 6C). We also investigated whether periostin produced and secreted by VSMCs could be responsible for macrophage recruitment to atherosclerotic plaques. However, we observed no difference in the ability of WT macrophages to migrate towards WT or Postn−/− VSMCs, either with or without co-stimulation from TGF-β, suggesting that factors other than periostin secreted from VSMCs were part of the migratory effect (Figure 6D). Macrophages deleted for Postn−/− were unable to migrate in response to TGF-β, yet they still responded to other general factors secreted from both WT and Postn−/− VSMCs (Figure 6D). These results show a fundamental defect in the ability of Postn−/− macrophages to migrate towards TGF-β. We observed no differences in the ability of WT or Postn−/− macrophages to adhere to WT or Postn−/− VSMC (Supplementary Figure II). Taken together, these data suggest that one possible reason for decreased inflammation in the plaques of ApoE−/− Postn−/− mice is due to impairment in the ability of Postn−/− macrophages to migrate into the plaques (Figure 6E). This, in turn, results in a decrease in the number of macrophages available to differentiate into foam cells that will eventually form the lipid core, resulting in a smaller plaque (Figure 6F).

Figure 6.

Figure 6

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Loss of periostin impairs macrophage (mΦ) migration into atherosclerotic plaques. A. Representative images of Mac3 staining of the aortic root of the indicated genotypes, scale = 50 μm. B. Quantification of Mac3 positive cells normalized to plaque area. Number of mice used is shown in the graph. *P<0.05 vs ApoE−/−. C. WT and Postn−/− mΦ migration in response to TGF-β or recombinant periostin (rPn). *P<0.05 vs WT media. D. WT or Postn−/− macrophage migration towards WT and Postn−/− VSMCs with or without TGF-β. N=3 experiments, #P<0.05 vs WT no TGF-β; *P<0.05 vs WT with TGF-β. E-F. Diagrams showing upon TGF-β or periostin (Pn) stimulation, circulating WT macrophages are able to migrate towards the vessel wall. However, macrophages deficient for Postn migrate less efficiently (E) contributing to smaller plaques, less organized collagen and less inflammation (F).

Discussion

The data presented here provide the first genetic evidence supporting the role of periostin in the pathogenesis of atherosclerosis. We observed a decrease in plaque size and overall plaque burden in the ApoE−/−Postn−/− mice compared to ApoE−/− mice, suggesting that loss of periostin is able to delay disease progression. This is not without precedence, since periostin is a known ECM cellular adhesion protein that, in other disease models such as muscular dystrophy and MI, has been shown to alter and even reduce the disease course.18, 38 In agreement with those studies, we found that the absence of periostin significantly alters ECM remodeling within the atherosclerotic plaque. Specifically, we observed that the deletion of Postn resulted in decreased collagen maturation and cross-linking and reduced the size of the fibrous cap; associated with increased gene expression of MMP3, MMP13 and Plod1. We found that periostin also affects the cellular composition of the plaque, marked by less inflammation and a pronounced phenotypic change of the inhabitant VSMC population towards a more synthetic phenotype. Finally, we observed that macrophages generated from WT bone marrow are more competent to migrate in response to inflammatory stimuli compared with those from Postn−/− mice. Although our data are unique in describing the role of periostin deletion in reducing the progression of atherosclerosis, our results are consistent with previous studies of both cardiac valve degeneration and arterial injury models where mice deficient for periostin were better protected from injury.17, 22

In agreement with many models of acute and chronic injury, periostin was absent in the vessel wall at baseline, but strongly re-expressed in the intima during atherosclerotic plaque formation.18, 22, 38 In fact, periostin was previously detected in the intima of human calcified atherosclerotic lesions, while absent in the vessel wall at baseline, indicating that periostin is re-expressed during vascular injury.17, 23 Furthermore, a proteomic screen performed with human coronary arteries identified periostin as highly expressed in coronary atherosclerotic plaques.23 In addition, a recent genomic study associated common variants of periostin with arterial lesions in young adults with premature development of atherosclerotic plaques.39 Taken together, these data suggest a prominent role for periostin in the pathogenesis of atherosclerotic plaque formation and disease progression.

Previous work strongly suggests a direct interaction between periostin and other known ECM proteins, such as fibronectin, tenascin C, collagens I and V, heparin, and the MMPs.4, 40 Periostin is known to be expressed in fibrous tissues such as tendons, skin, and valves where collagen is important to maintain biomechanical properties. Collagen I fibril maturation is critical to form a mature, cross-linked matrix. Loss of periostin disrupts this process resulting in defective collagen maturation indicating a prominent role of periostin in collagen fibrillogenesis.4 In addition, previous studies in skin and during muscular dystrophy revealed that the maturity of collagen in the ECM is reduced in mice deficient for Postn, resulting in altered wound healing marked by incomplete or delayed scar formation, which also holds true in the heart.18, 19, 38, 41 Consistent with these data, we detected impaired collagen fibrillogenesis in ApoE−/− mice deficient for Postn as indicated by decreased collagen maturation in the plaques.

Given these observations we suggest that the decreased plaque burden observed in the Postn−/− mice could be due to alterations in pathologic ECM remodeling, where lack of periostin directly alters the abundance, assembly, and/or biological properties of the collagen matrix in the plaque microenvironment. On the other hand, consistent with the pathology of human atherosclerotic lesions, we observed increased MMP3 expression in the aortas of ApoE−/− Postn−/− mice.14 Interestingly, models of atherosclerosis that are genetically deficient for MMP3 have larger plaques with an unusual, defective ECM, associated with more collagen and fewer VSMCs.13, 42 Thus, besides impaired collagen cross-linking and maturation in the absence of periostin, one must consider that the immature collagen matrix noted in ApoE−/− Postn−/− plaques could be due to increased matrix degradation, which is supported by an increase in MMP3 activity. This is also supported by the increase in MMP13 and Plod1 levels we observed in double-deficient mice compared to ApoE−/− mice. Studies have shown that MMP13 also contributes to collagen degradation and blocking MMP13 results in increased fibrous cap size.31 Furthermore, Plod1 is involved in collagen cross-linking and collagen maturation suggesting that the double null mouse may be attempting to upregulate collagen crosslinking genes to compensate for this inherent defect in the Postn−/− mouse prior to disease onset. 32

Surprisingly, despite a decrease in mature collagen, the VSMCs in the aortas of the ApoE−/− Postn−/− mice do robustly switch from a contractile to synthetic phenotype, evident by increased proliferation, decreased α-SMA, decreased SM-MHC expression, increased osteopontin expression, and increased vimentin staining.33, 34 This is consistent with a previous study that detected a decrease in the concentration of α-SMA positive cells at the site of injury in a Postn deficient model of MI.19 Therefore, the decrease in the size of the fibrous caps observed in ApoE−/− Postn−/− mice is not due to changes in the characteristics of the VSMCs, as they still undergo a phenotypic switch, but rather related to changes in the ECM microenvironment. The decrease in fibrous cap size may be potentiated by the inability of the VSMCs to migrate through the altered matrix. This is supported by previous work demonstrating that periostin directly affects cellular migration through known interactions with integrins.43 Similar observations were made in other disease models where periostin was deleted, such as muscular dystrophy, where there was a decrease in inflammatory cells.38 Additionally, the plaques of ApoE−/− Postn−/− deficient mice display a decrease in macrophage content. Macrophages that are deficient for periostin are unable to migrate in response to inflammatory stimuli, such as TGF-β. These data are supported by previous work showing that deficiency or inhibition of periostin prevents the development or progression of many allergic inflammatory diseases including allergic dermatitis, asthma and chronic rhinosinusitis.44, 45 The presence of periostin in an inflammatory or injured environment appears to impart competency for macrophages to infiltrate and further impact disease.

While loss of Postn appears beneficial/protective with preserved vessel lumen and delayed disease progression, our model does not provide insights into the long-term ramifications of atherosclerosis, including plaque rupture, embolization, and subsequent tissue ischemia. Both human and animal studies have described plaques with a small fibrous cap as less stable and more prone to rupture, indicating that the atherosclerotic plaques in the ApoE−/− Postn−/− mice might be more disease predisposing.46–50 However, the decrease in activated macrophages in the ApoE−/− Postn−/− mice may be beneficial, as inflammatory activity in human plaques is associated with a higher incidence of plaque rupture with subsequent thromboembolism leading to ischemic neurological and cardiovascular events.51 It is well established that plaques in the atherosclerotic ApoE−/− mouse model are more resistant to rupture making it difficult to conclude whether the benefit of a decrease in plaque burden and inflammation outweighs the described benefit of a large stable fibrous cap.52

The data presented here demonstrate that the loss of periostin in a mouse model of atherosclerosis decreases mature collagen production, inflammatory cell activation and migration, and alters VSMC behavior. Taken together, we can conclude that loss of periostin delays atherosclerotic disease progression via alterations in the ECM and modification of the migratory potential of macrophages, a hallmark of atherosclerosis. Our results suggest periostin as a potential therapeutic target to slow the progression of disease associated with atherosclerosis.

Supplementary Material

Methods and Meterial
Supplementary Material

Significance.

Heart disease is the leading cause of death in the Western world. Atherosclerosis, the buildup of plaques within the arteries, is the major contributor to mortality associated with heart disease. Therefore, understanding the underlying mechanism by which atherosclerosis develops is crucial to establishing new treatments. Our study is the first to examine a genetic model of periostin deletion in an atherosclerotic mouse background. Periostin is an extracellular matrix protein known to be involved in wound healing and tissue remodeling, however, the role of periostin in the development of atherosclerosis has not, until now, been examined. We show that loss of periostin alters the extracellular matrix of the circulatory vessels and delays atherosclerosis.

Acknowledgments

None

Funding Source

This work was supported by grants from the National Institutes of Health (J.D.M.) and the Howard Hughes Medical Institute (J.D.M.). A.L. was supported by an NIH K-12 award J.A.S. was supported by an NIH training grant (T32-HL007752).

Non-standard Abbreviations and Acronyms

ApoE

apolipoprotein E

α-SMA

smooth muscle alpha actin

ECM

extracellular matrix

EdU

5-ethynyl-2′-deoxyuridine

HFD

high fat diet

Lox

lysyl oxidase

MI

myocardial infarction

MMPs

matrix metalloproteases

MSR1

macrophage scavenger receptor 1,

NPY

neuropeptide Y

Plod1

procollagen-lysine 2-oxoglutarate 5-dioxygenase

qPCR

quantitative polymerase chain reaction

SM-MHC

smooth muscle myosin heavy chain

TGF-β

transforming growth factor β

VSMC

vascular smooth muscle cell

WT

wildtype

Footnotes

Disclosures

None

References

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Supplementary Materials

Methods and Meterial
NIHMS734587-supplement-Methods_and_Meterial.pdf (139.5KB, pdf)
Supplementary Material
NIHMS734587-supplement-Supplementary_Material.pdf (264.7KB, pdf)

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