Ricardo A. Moreno-Rodriguez

Periostin regulates collagen fibrillogenesis and the biomechanical properties of connective tissues

Periostin is predominantly expressed in collagen‐rich fibrous connective tissues that are subjected to constant mechanical stresses including: heart valves, tendons, perichondrium, cornea, and the periodontal ligament (PDL). Based on these data we hypothesize that periostin can regulate collagen I fibrillogenesis and thereby affect the biomechanical properties of connective tissues. Immunoprecipitation and immunogold transmission electron microscopy experiments demonstrate that periostin is capable of directly interacting with collagen I. To analyze the potential role of periostin in collagen I fibrillogenesis, gene targeted mice were generated. Transmission electron microscopy and morphometric analyses demonstrated reduced collagen fibril diameters in skin dermis of periostin knockout mice, an indication of aberrant collagen I fibrillogenesis. In addition, differential scanning calorimetry (DSC) demonstrated a lower collagen denaturing temperature in periostin knockout mice, reflecting a reduced level of collagen cross‐linking. Functional biomechanical properties of periostin null skin specimens and atrioventricular (AV) valve explant experiments provided direct evidence of the role that periostin plays in regulating the viscoelastic properties of connective tissues. Collectively, these data demonstrate for the first time that periostin can regulate collagen I fibrillogenesis and thereby serves as an important mediator of the biomechanical properties of fibrous connective tissues. J. Cell. Biochem. 101: 695–711, 2007.

Periostin Is Required for Maturation and Extracellular Matrix Stabilization of Noncardiomyocyte Lineages of the Heart

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 (perilacZ) knockout mice exhibit viable valve disease, with neonatal lethality in a minority and latent disease with leaflet abnormalities in the viable majority. Surviving perilacZ-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-&bgr; signaling. Neonatal perilacZ 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-&bgr; 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-&bgr;–dependent pathway during establishment of the mature heart. Thus, perilacZ knockouts provide a new model of viable latent valve disease.

Endocardial Cells Form the Coronary Arteries by Angiogenesis through Myocardial-Endocardial VEGF Signaling

The origins and developmental mechanisms of coronary arteries are incompletely understood. We show here by fate mapping, clonal analysis, and immunohistochemistry that endocardial cells generate the endothelium of coronary arteries. Dye tracking, live imaging, and tissue transplantation also revealed that ventricular endocardial cells are not terminally differentiated; instead, they are angiogenic and form coronary endothelial networks. Myocardial Vegf-a or endocardial Vegfr-2 deletion inhibited coronary angiogenesis and arterial formation by ventricular endocardial cells. In contrast, lineage and knockout studies showed that endocardial cells make a small contribution to the coronary veins, the formation of which is independent of myocardial-to-endocardial Vegf signaling. Thus, contrary to the current view of a common source for the coronary vessels, our findings indicate that the coronary arteries and veins have distinct origins and are formed by different mechanisms. This information may help develop better cell therapies for coronary artery disease.

Developmental basis of adult cardiovascular diseases: valvular heart diseases.

In this chapter, we review the working hypothesis that the roots of adult valvular heart disease (VHD) lie in embryonic development. Valvulogenesis is a complex process in which growth factors signal the process of endocardium‐to‐mesenchyme transformation (EMT) resulting in formation of prevalvular “cushions.” The post‐EMT processes, whereby cushions are morphogenetically remolded into valve leaflets, are less well understood, but they require periostin. Mice with targeted deletion of periostin develop degenerative changes similar to human forms of VHD. Mitral valves are also abnormally elongated in hypertrophic cardiomyopathy (HCM), which plays an important role in clinical disease expression. However, the mechanism for this is unclear, but correlates with enhanced expression of periostin in a specific population of ventricular cells derived from the embryonic proepicardial organ, which accumulate at sites where valvular endocardial EMT is reactivated. Collectively, these findings suggest that developmental mechanisms underlie adult valve responses to genetic mutations in degenerative VHD and HCM.

The many facets of the matricelluar protein periostin during cardiac development, remodeling, and pathophysiology

Periostin is a member of a growing family of matricellular proteins, defined by their ability to interact with components of the extracellular milieu, and with receptors at the cell surface. Through these interactions, periostin has been shown to play a crucial role as a profibrogenic molecule during tissue morphogenesis. Tissues destined to become fibrous structures are dependent on cooperative interactions between periostin and its binding partners, whereas in its absence, these structures either totally or partially fail to become mature fibrous entities. Within the heart, fibrogenic differentiation is required for normal tissue maturation, remodeling and function, as well as in response to a pathological myocardial insult. In this review, aspects related to the function of periostin during cardiac morphogenesis, remodeling and pathology are summarized.

Lack of periostin leads to suppression of Notch1 signaling and calcific aortic valve disease

The Postn gene encodes protein periostin. During embryonic development, it is highly expressed in the outflow tract (OFT) endocardial cushions of the developing heart, which give rise to several structures of the mature heart including the aortic valve. Periostin was previously implicated in osteoblast differentiation, cancer metastasis, and tooth and bone development, but its role in cardiac OFT development is unclear. To elucidate the role that periostin plays in the developing heart we analyzed cardiac OFT phenotype in mice after deletion of the Postn gene. We found that lack of periostin in the embryonic OFT leads to ectopic expression of the proosteogenic growth factor pleiotrophin (Ptn) and overexpression of delta-like 1 homolog (Dlk1), a negative regulator of Notch1, in the distal (prevalvular) cushions of the OFT. This resulted in suppression of Notch1 signaling, strong induction of the central transcriptional regulator of osteoblast cell fate Runx2, upregulation of osteopontin and osteocalcin expression, and subsequent calcification of the aortic valve. Our data suggest that periostin represses a default osteogenic program in the OFT cushion mesenchyme and promotes differentiation along a fibrogenic lineage. Lack of periostin causes derepression of the osteogenic potential of OFT mesenchymal cells, calcium deposition, and calcific aortic valve disease. These results establish periostin as a key regulator of OFT endocardial cushion mesenchymal cell fate during embryonic development.

Primitive Interventricular Septum, Its Primordium, and Its Contribution in the Definitive Interventricular Septum: In Vivo Labelling Study in the Chick Embryo Heart

Because the studies on the embryological development of the primitive interventricular septum have been done with postmortem material, we do not know the site within the cardiac tube and the developmental stage at which the primordium appears and its anatomical manifestation in the mature heart. Consequently, we do not know its real contribution to the constitution of the definitive interventricular septum.

Bidirectional fusion of the heart-forming fields in the developing chick embryo

It is generally thought that the early pre‐tubular chick heart is formed by fusion of the anterior or cephalic limits of the paired cardiogenic fields. However, this study shows that the heart fields initially fuse at their midpoint to form a transitory “butterfly”‐shaped, cardiogenic structure. Fusion then progresses bi‐directionally along the longitudinal axis in both cranial and caudal directions. Using in vivo labeling, we demonstrate that cells along the ventral fusion line are highly motile, crossing future primitive segments. We found that mesoderm cells migrated cephalically from the unfused tips of the anterior/cephalic wings into the head mesenchyme in the region that has been called the secondary heart field. Perturbing the anterior/cranial fusion results in formation of a bi‐conal heart. A theoretical role of the ventral fusion line acting as a “heart organizer” and its role in cardia bifida is discussed. Developmental Dynamics 235:191–202, 2006.

Expression of the familial cardiac valvular dystrophy gene, filamin‐A, during heart morphogenesis

Myxoid degeneration of the cardiac valves is a common feature in a heterogeneous group of disorders that includes Marfan syndrome and isolated valvular diseases. Mitral valve prolapse is the most common outcome of these and remains one of the most common indications for valvular surgery. While the etiology of the disease is unknown, recent genetic studies have demonstrated that an X‐linked form of familial cardiac valvular dystrophy can be attributed to mutations in the Filamin‐A gene. Since these inheritable mutations are present from conception, we hypothesize that filamin‐A mutations present at the time of valve morphogenesis lead to dysfunction that progresses postnatally to clinically relevant disease. Therefore, by carefully evaluating genetic factors (such as filamin‐A) that play a substantial role in MVP, we can elucidate relevant developmental pathways that contribute to its pathogenesis. In order to understand how developmental expression of a mutant protein can lead to valve disease, the spatio‐temporal distribution of filamin‐A during cardiac morphogenesis must first be characterized. Although previously thought of as a ubiquitously expressed gene, we demonstrate that filamin‐A is robustly expressed in non‐myocyte cells throughout cardiac morphogenesis including epicardial and endocardial cells, and mesenchymal cells derived by EMT from these two epithelia, as well as mesenchyme of neural crest origin. In postnatal hearts, expression of filamin‐A is significantly decreased in the atrioventricular and outflow tract valve leaflets and their suspensory apparatus. Characterization of the temporal and spatial expression pattern of filamin‐A during cardiac morphogenesis is a crucial first step in our understanding of how mutations in filamin‐A result in clinically relevant valve disease. Developmental Dynamics 239:2118–2127, 2010

Formation and Septation of the Tubular Heart: Integrating the Dynamics of Morphology With Emerging Molecular Concepts

In the present chapter, we seek to integrate the progressive and dynamic changes in structure that transform a bent, hollow tube into a mature, fully defined, four-chambered heart with emerging concepts of molecular regulation.