Periostin is required for maturation and extracellular matrix stabilization of noncardiomyocyte lineages of the heart - PubMed (original) (raw)

. 2008 Apr 11;102(7):752-60.

doi: 10.1161/CIRCRESAHA.107.159517. Epub 2008 Feb 22.

Robert B Hinton, Ricardo A Moreno-Rodriguez, Jian Wang, Rhonda Rogers, Andrew Lindsley, Fang Li, David A Ingram, Donald Menick, Loren Field, Anthony B Firulli, Jeffery D Molkentin, Roger Markwald, Simon J Conway

Affiliations

Periostin is required for maturation and extracellular matrix stabilization of noncardiomyocyte lineages of the heart

Paige Snider et al. Circ Res. 2008.

Abstract

The secreted periostin protein, which marks mesenchymal cells in endocardial cushions following epithelial-mesenchymal transformation and in mature valves following remodeling, is a putative valvulogenesis target molecule. Indeed, periostin is expressed throughout cardiovascular morphogenesis and in all 4 adult mice valves (annulus and leaflets). Additionally, periostin is expressed throughout the fibrous cardiac skeleton and endocardial cushions in the developing heart but is absent from both normal and/or pathological mouse cardiomyocytes. Periostin (peri(lacZ)) knockout mice exhibit viable valve disease, with neonatal lethality in a minority and latent disease with leaflet abnormalities in the viable majority. Surviving peri(lacZ)-null leaflets are truncated, contain ectopic cardiomyocytes and smooth muscle, misexpress the cartilage proteoglycan aggrecan, demonstrate disorganized matrix stratification, and exhibit reduced transforming growth factor-beta signaling. Neonatal peri(lacZ) nulls that die (14%) display additional defects, including leaflet discontinuities, delamination defects, and deposition of acellular extracellular matrix. Assessment of collagen production, 3D lattice formation ability, and transforming growth factor-beta responsiveness indicate periostin-deficient fibroblasts are unable to support normal valvular remodeling and establishment of a mature cardiac skeleton. Furthermore, pediatric stenotic bicuspid aortic valves that have lost normal extracellular matrix trilaminar stratification have greatly reduced periostin. This suggests that loss of periostin results in inappropriate differentiation of mesenchymal cushion cells and valvular abnormalities via a transforming growth factor-beta-dependent pathway during establishment of the mature heart. Thus, peri(lacZ) knockouts provide a new model of viable latent valve disease.

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Figures

Figure 1

Figure 1

Periostin in developing mouse heart. A through C, In situ reveals periostin (peri) (white grains in darkfield) is expressed throughout the developing cardiac skeleton and endocardial cushions but is absent from endothelium (red arrowhead) (C) and cardiomyocytes. D through F, Immunohistochemistry similarly shows periostin (brown diaminobenzidine substrate) is expressed throughout cardiac skeleton, epicardium (red arrowheads) (D), and most valvular cushion cells but is absent from endothelial cells and cardiomyocytes. In E15 valves (D), periostin is predominately localized to inner cushion regions abutting cardiomyocytes (arrows in D). Robust expression is seen in cardiac fibroblasts and developing annulus (asterisk in F). Neonatal expression is restricted to fibrous (f) rib of mature leaflet and largely absent from ventricularis (V) layer (E and F). G, Western reveals relative levels of periostin increase as the heart undergoes maturation. In E11 hearts (n=4 pooled hearts), only 3 isoforms are weakly present (largest is ≈90 kDa), but all 4 isoforms (≈90, 87, 85, and 82 kDa) are readily detectable in newborns (Nb) (n=3 pooled hearts). Note actin (≈42 kDa) is equally expressed and serves as a loading control. Scale bars, 200 µm (A and D). lv, left ventricle; mit, mitral; neo, neonatal; ra, right atria; rv, right ventricle; tri, tricuspid.

Figure 2

Figure 2

Periostin in cardiac fibroblast lineage. A through C, Periostin (A), αMHC-EGFP (B), and 4′,6-diamidino-2-phenylindole (DAPI) (C) expression in the same section through adult αMHC-EGFP heart. Periostin is restricted to vascular smooth muscle cells around coronaries (arrow in A) and cardiac fibroblasts (arrowheads in A) but absent from EGFP cardiomyocytes. D, Neonatal heart double-stained with anti-periostin (red) and cardiomyocyte-specific MF20 (green) antibodies (Ab). Fibroblasts (arrows) are periostin-positive, whereas cardiomyocytes are negative. E, Disassociated suspension of 4-day neonatal mouse hearts cocultured for 48 hours on collagen and stained for periostin (red) and sarcomeric actin (green). Note only fibroblasts are red (appear rounded as takes ≈5 days to spread on collagen). F, E13.5 right atria and ventricle double-stained with anti-periostin and MF20 antibodies. Note there is no overlap (ie, yellow), and fibrillar-like periostin expression is present only in noncardiomyocyte lineages. G and H, Immunohistochemistry on cultured E14 cardiac fibroblasts reveals both periostin (G) and collagen type I (H) are present in cytoplasm. Negative controls lacking primary and/or secondary did not produce staining. (I) Western blotting showed that E14 cardiac fibroblasts express mainly ≈82-kDa periostin isoform in cells and secrete larger ≈90- and ≈87-kDa isoforms. Equal loading of isolated cells and supernatant (supern) was verified via amido-black staining. J, Whereas E14 and newborn fibroblasts express periostin, cardiomyocytes lack expression following RT-PCR analysis. When treated with 1 ng/mL TGFβ overnight, periostin upregulation (7X) is observed in only fibroblasts. Note GAPDH is equally expressed in all samples (n=4 per genotype/age). Scale bars: 10 µm (A and G).

Figure 3

Figure 3

Periostin knockout mouse hearts. a, Whole-mount lacZ staining of peri lacZ+/− E13, newborn, and 4-month-old (Ad) hearts. Note perilacZ in E13 cushions (front and back). Histology reveals perilacZ is confined to aortic, pulmonary (p), mitral, and tricuspid valves and cardiac fibroblasts but is absent from cardiomyocytes. b, Analysis of viable adult _perilacZ_-null hearts (n=68; range 4 to 65 weeks). _perilacZ_-null hearts (−/−) are dilated around the OFT (arrow). Histology revealed 100% of null leaflets were short and thickened. Scale in b, 100 µm. c, In nonviable nulls (≈14%), neonatal mutant hearts are abnormally shaped (bulging right ventricle indicated by the asterisk), being rounded and lacking a distinct apex (A). Eosin counterstaining of transverse sections of 12-day neonatal _perilacZ_-null hearts. Whereas viable _perilacZ_-null mitral valves are normal (B), nonviable perilacZ heart (C) has its mitral septal leaflet tethered to the septum (n=8 abnormally shaped perilacZ nulls examined). αSMA coimmunostaining of _lacZ_-stained 2-week nonviable _perilacZ_-null hearts revealed deposition of amorphous ECM in some leaflets (arrow in D) when compared with heterozygotes and viable perilacZ nulls, and this amorphous ECM was acellular and negative for lacZ and αSMA.

Figure 4

Figure 4

Characterization of surviving adult knockout heart phenotypes. Phase (A and C) and fluorescein isothiocyanate∓MF20 (B and D) images of isolated and immunostained adult _perilacZ_-null (A and B) and wild-type (C and D) leaflets. Note ectopic MF20-positive myocyte clusters in null valves (arrow in B) and papillary muscles (pap) appropriately express MF20. E, RT-PCR analysis of isolated E13 hearts and rests (whole embryo minus heart) and adult hearts and isolated rib cDNA samples (n=8 isolated E13 hearts/rest and 4 adult hearts/ribs from each respective genotype were pooled). Periostin mRNA is absent in perilacZ nulls and ≈50% reduced in heterozygotes. Levels of βigH3, alkaline phosphatase (alp), fibulin-1c/1d are unaltered, but aggrecan is misexpressed in nulls (large*). Aggrecan expression is also dose-dependently upregulated in adult heterozygotes (small*). Significantly, aggrecan (mature and developmentally regulated isoforms) is expressed normally in E13 embryos. F and G, In situ revealed surviving adult _perilacZ_-null valves (G) continue to express aggrecan, whereas normal littermates have switched it off (n=4).

Figure 5

Figure 5

Null valvular pathology. Wild-type E12.5 (A) and _perilacZ_-null (B) sagittal sections following alcian blue staining (pH 2.5) reveals increase in glycosaminoglycans in knockout OFT and AV cushions (n=5 per genotype). C and D, Platelet endothelial cell adhesion molecule immunostaining of wild-type and knockout leaflets indicates endothelium is intact. E and F, Resorcin-Fuchsin/van Gieson collagen/elastin staining shows null attachment sites are significantly undersized (arrow in F; n=7). G and H, Representative high-power views of hematoxylin/ eosin-stained viable +/+ adult (G) and peri _lacZ_−/−(H) mitral leaflets illustrating disorganization in nulls. Note that the defined, elongated pink collagen layer is absent in perilacZ null (arrow in H). Scale: 10 µm (G and H).

Figure 6

Figure 6

Periostin is responsive to TGFβ but can also mediate TGFβ responsiveness. A, In situ demonstrated periostin is downregulated in E11.5 Foxc1 (c1) knockouts and upregulated in E14 Smad6 nulls. Note reduced periostin in _Foxc1_-null cushions, mandibular arch (m), and umbilical (top images) and upregulation in hyperplastic _Smad6_-null OFT cushions (bottom images). Hematoxylin/eosin staining of transverse sections (middle images) illustrate enlarged OFT cushions and the presence of persistent truncus arterious (*) when inhibitory Smad6 is removed. B, Western blot analysis revealed elevated periostin (9-fold) in 2- and 10-week αMHC-TGFβ1–expressing transgenic ventricles, compared with nontransgenic littermates. C, 3H-proline incorporation assays indicate collagen production is reduced in null E14 MEFs when compared with wild types (+/+=8663±81 vs −/−=5674±94), and they fail to upregulate collagen synthesis with the addition of 1 ng/mL TGFβ (+/+ = 11 138±77 vs −/−=5286±124). Data are expressed as counts per minute of [3H]proline in 10 cells (n=3 independent lines/genotype). D, Three-dimensional collagen lattice contraction after 5 days in response to 0.1 ng/mL TGFβ was compared in 2 independent perilacZ nulls vs +/+ littermate lines. Note the reduced contraction (≈18%) in both nulls, despite the presence of equal cell numbers (10 cells/well). E and F, pSmad2,3 is attenuated in E14 _perilacZ_-null leaflets (F) when compared with +/+ littermate controls (E) (n=4 per genotype).

Figure 7

Figure 7

Periostin expression and hematoxylin/eosin analysis in pediatric semilunar valves. A and B, Normal pediatric aortic valves demonstrate diffuse trilaminar periostin expression with increased expression in fibrosa (f) and spongiosa (s) relative to ventricularis (v). ECM fibrils (probably collagen) are elongated and arranged in a regular manner to provide a robust support structure. C and D, Stenosed aortic valves demonstrate overall markedly decreased periostin expression with an absence of expression in expanded fibrosa (bar in C) and disorganized ECM, as evidenced by increased valvular interstitial cells and disorganized collagen (arrows in D). Median age of samples is 6 years (range, 10 months to 15 years; n=9) compared with a median age of 9 years for controls (range, 3 to 14 years; n=6). A and C and B and D are viewed at similar magnifications. Scale bars: 1 mm (A); 50 µm (B).

Figure 8

Figure 8

Diagram of the proposed function of periostin. In response to TGFβ-mediated upstream signaling via phosphorylation of TGFβ-responsive receptors and Smad activation, periostin is upregulated (in addition to other established valvular remodeling factors, collagen, elastin, matrix metalloproteinases/ tissue inhibitors of metalloproteinase [MMPs/TIMPs], fibrillin-1). Both matrix-bound and secreted periostin acts to suppress nonvalvular phenotypes and promote valve mesenchymal differentiation and maturation by influencing ECM assembly, collagen realignment, and establishment of the valvular trilaminar structure in a TGFβ superfamily-dependent fashion.

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