Richard L. Goodwin

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.

A Novel Tubular Scaffold for Cardiovascular Tissue Engineering

We have developed a counter rotating cone extrusion device to produce the next generation of three-dimensional collagen scaffold for tissue engineering. The device can produce a continuously varying fibril angle from the lumen to the outside of a 5-mm-diameter collagen tube, similar to the pattern of heart muscle cells in the intact heart. Our scaffold is a novel, oriented, type I collagen, tubular scaffold. We selected collagen because we believe there are important signals from the collagen both geometrically and biochemically that elicit the in vivo -like phenotypic response from the cardiomyocytes. We have shown that cardiomyocytes can be cultured in these tubes and resemble an in vivo phenotype. This new model system will provide important information leading to the design and construction of a functional, biologically based assist device.

A three-dimensional model of vasculogenesis

Postnatal bone marrow contains various subpopulations of resident and circulating stem cells (HSCs, BMSCs/MSCs) and progenitor cells (MAPCs, EPCs) that are capable of differentiating into one or more of the cellular components of the vascular bed in vitro as well as contribute to postnatal neo-vascularization in vivo. When rat BMSCs were seeded onto a three-dimensional (3-D) tubular scaffold engineered from topographically aligned type I collagen fibers and cultured either in vasculogenic or non-vasculogenic media for 7, 14, 21 or 28 days, the maturation and co-differentiation into endothelial and/or smooth muscle cell lineages were observed. Phenotypic induction of these substrate-grown cells was assayed at transcript level by real-time PCR and at protein level by confocal microscopy. In the present study, the observed upregulation of transcripts coding for vascular phenotypic markers is reminiscent of an in vivo expression pattern. Immunolocalization of vasculogenic lineage-associated markers revealed typical expression patterns of vascular endothelial and smooth muscle cells. These endothelial cells exhibited high metabolism of acetylated low-density lipoprotein. In addition to the induced monolayers of endothelial cells, the presence of numerous microvascular capillary-like structures was observed throughout the construct. At the level of scanning electron microscopy, smooth-walled cylindrical tube-like structures with smooth muscle cells and/or pericytes attached to its surface were elucidated. Our 3-D culture system not only induces the maturation and differentiation of BMSCs into vascular cell lineages but also supports microvessel morphogenesis. Thus, this unique in vitro model provides an excellent platform to study the temporal and spatial regulation of postnatal de novo vasculogenesis, as well as attack the lingering limit in developing engineered tissues, that is perfusion.

PERIOSTIN PROMOTES A FIBROBLASTIC LINEAGE PATHWAY IN ATRIOVENTRICULAR VALVE PROGENITOR CELLS

Differentiation of prevalvular mesenchyme into valve fibroblasts is an integral step towards the development of functionally mature cardiac valves. Although clinically relevant, little is known regarding the molecular and cellular mechanisms by which this process proceeds. Genes that are regulated in a spatio‐temporal pattern during valve remodeling are candidates for affecting this differentiation process. Based on its expression pattern, we have focused our studies on the role of the matricellular gene, periostin, in regulating the differentiation of cushion mesenchymal cells into valve fibroblasts. Herein, we demonstrate that periostin expression is coincident with and regulates type I collagen protein production, a major component of mature valve tissue. Adenoviral‐mediated knock‐down of periostin in atrioventricular mesenchyme resulted in a decrease in collagen I protein expression and aberrant induction of myocyte markers indicating an alteration in AV mesenchyme differentiation. In vitro analyses using a novel “cardiotube” assay further demonstrated that expression of periostin regulates lineage commitment of valve precursor cells. In these cells, expression of periostin and collagen I are regulated, in part, by TGFβ‐3. We further demonstrate that TGFβ‐3, through a periostin/collagen pathway, enhances the viscoelastic properties of AV cushion tissue surface tension and plays a crucial role in regulating valve remodeling. Thus, data presented here demonstrate that periostin, a TGFβ‐3 responsive gene, functions as a crucial mediator of chick AV valve maturation via promoting mesenchymal‐to‐fibroblast differentiation while blocking differentiation of alternative cell types (myocytes). Developmental Dynamics 238:1052–1063, 2009.

Resveratrol prevents embryonic oxidative stress and apoptosis associated with diabetic embryopathy, and improves glucose and lipid profile of diabetic dam

SCOPE Diabetic embryopathy, a consequence of diabetic pregnancy, is associated with increase in embryonic oxidative stress and apoptosis, which lead to severe embryonic damage at early stage of organogenesis. METHODS AND RESULTS This study investigated if resveratrol, found in red grapes and blue-berries, may prevent diabetes-induced oxidative stress and apoptosis in embryos and have beneficial effects in diabetic dams. A rodent model of diabetic embryopathy was used. Diabetes was associated with lowered reduced glutathione levels (26.98%), increased total thiol (100.47%) and lipid peroxidation (124.73%) in embryos, and increased blood sugar (384.03%), cholesterol (98.39%) and triglyceride (1025.35%) in diabetic dams. Increased apoptosis (272.20%) was also observed in the embryos of diabetic dams. Administration of resveratrol (100 mg/kg body weight (b.w.)) during pregnancy prevented both oxidative stress and apoptosis in embryos. Resveratrol reduced embryonic maldevelopment by improving embryo weight (41.23%), crown rump length (16.50%) and somite number (11.22%). It further improved the glucose (33.32%) and lipid (cholesterol 41.74%, triglyceride 60.64%) profile of the diabetic dams, which also represents the protective role of resveratrol in diabetes. CONCLUSION Resveratrol was found to prevent embryonic oxidative stress and apoptosis. It also improved glucose and lipid profile of diabetic dams, indicating the beneficial effects in diabetic pregnancy.

The Connexin43 Carboxyl-Terminal Peptide ACT1 Modulates the Biological Response to Silicone Implants

Background: The implantation of a biomedical device elicits a wound-healing response that progresses through the three phases of wound healing: inflammation, cellular proliferation, and matrix remodeling. This response culminates in a fibrous collagen encapsulation of the implant. Subsequent contraction of this “scar-like” tissue can lead to physical disfigurement, implant extrusion, or impairment of implant function, necessitating surgical revision or removal. ACT1 is a synthetic peptide derived from the carboxyl-terminal sequence of the cellular gap junction protein connexin43. This novel peptide has recently been shown to modulate cutaneous wound healing, reduce scarring, and promote regenerative repair of the skin following injury. In this study, the authors investigated the ability of the ACT1 peptide to modulate the wound-healing response to biomedical device implantation. Methods: Silicone disks coated with either vehicle control or ACT1 peptide were implanted submuscularly into male Sprague-Dawley rats. Capsulectomies were performed on days 1, 2, 3, 14, and 28. The implant capsules and surrounding tissue were analyzed histologically and biochemically. Results: ACT1 modulated the wound-healing response to silicone implants by attenuating neutrophil infiltration, increasing vascularity of the capsule tissue, reducing type I collagen deposition around the implant, and reducing the continued presence of contractile myofibroblasts. Conclusion: ACT1 may provide an enabling technology for modulating the wound-healing response to implants, promoting integration of implanted materials and tissue-engineered devices in the human body.

Fluid flow forces and rhoA regulate fibrous development of the atrioventricular valves

Fibrous development of the extracellular matrix (ECM) of cardiac valves is necessary for proper heart function. Pathological remodeling of valve ECM is observed in both pediatric and adult cardiac disorders. It is well established that intracardiac hemodynamics play a significant role in the morphogenesis of cardiovascular tissues. However, the mechanisms that transduce mechanical forces into morphogenetic processes are not well understood. Here, we report the development of a three-dimensional, in vitro culture system that allows for culture of embryonic valve tissue under specific pulsatile flow conditions. This system was used to investigate the role that fluid flow plays in fibrous ECM expression during valve formation and to test the underlying cellular mechanisms that regulate this mechanotransduction. When cultured under pulsatile flow, developing valve tissues upregulated fibrous ECM expression at both the transcript and protein levels in comparison to no-flow controls. Flow-cultured valve tissues also underwent morphological development, as cushions elongated into leaflet-like structures that were absent in no-flow controls. Furthermore, rhoA, a member of the cytoskeletal actin-regulating GTPase family of proteins, was upregulated and activated by flow culture. Inhibition of the downstream rhoA effector kinase, ROCK, blocked flow-driven fibrous ECM accumulation and tissue stiffening, while the addition of lysophosphatidic acid (LPA), a rhoA activator, stimulated fibrous ECM deposition and tissue stiffening. These results support a prominent role for the rhoA pathway in the mechanotransduction of hemodynamic forces during fibrous remodeling of developing valve tissue. Our results also point to a potential link between regulation of the actinomyosin cytoskeleton and fibrous ECM synthesis in cardiovascular tissues.

Developmental Basis for Filamin-A Associated Myxomatous Mitral Valve Disease

AIMS We hypothesized that the structure and function of the mature valves is largely dependent upon how these tissues are built during development, and defects in how the valves are built can lead to the pathological progression of a disease phenotype. Thus, we sought to uncover potential developmental origins and mechanistic underpinnings causal to myxomatous mitral valve disease. We focus on how filamin-A, a cytoskeletal binding protein with strong links to human myxomatous valve disease, can function as a regulatory interface to control proper mitral valve development. METHODS AND RESULTS Filamin-A-deficient mice exhibit abnormally enlarged mitral valves during foetal life, which progresses to a myxomatous phenotype by 2 months of age. Through expression studies, in silico modelling, 3D morphometry, biochemical studies, and 3D matrix assays, we demonstrate that the inception of the valve disease occurs during foetal life and can be attributed, in part, to a deficiency of interstitial cells to efficiently organize the extracellular matrix (ECM). This ECM organization during foetal valve gestation is due, in part, to molecular interactions between filamin-A, serotonin, and the cross-linking enzyme, transglutaminase-2 (TG2). Pharmacological and genetic perturbations that inhibit serotonin-TG2-filamin-A interactions lead to impaired ECM remodelling and engender progression to a myxomatous valve phenotype. CONCLUSIONS These findings illustrate a molecular mechanism by which valve interstitial cells, through a serotonin, TG, and filamin-A pathway, regulate matrix organization during foetal valve development. Additionally, these data indicate that disrupting key regulatory interactions during valve development can set the stage for the generation of postnatal myxomatous valve disease.

Epicardial Development in the Rat: A New Perspective

Development of the epicardium is critical to proper heart formation. It provides all of the precursor cells that form the coronary system and supplies signals that stimulate cardiac myocyte proliferation. The epicardium forms from mesothelial cells associated with the septum transversum and is referred to as the proepicardium (PE). Two different methods by which these PE cells colonize the developing heart have been described. In avians, PE cells form a bridge to the heart over which PE cells migrate onto the heart. In fish and mammals, PE cells form vesicles of cells that detach from the mesothelium, float through the pericardial cavity, and attach to the heart. A previous study of rat PE development investigated this process at the histological level. Protein markers have been developed since this study. Thus, we investigated this important developmental process coupled with these new markers using other visualization techniques such as scanning electron microscopy (SEM) and confocal microscopy. Finally, a novel, three-dimensional (3-D) culture system was used to confirm the identity of the PE cells. In this study, we found convincing evidence that the rat PE cells directly attach to the heart in a manner similar to that observed in avians.

A three-dimensional tubular scaffold that modulates the osteogenic and vasculogenic differentiation of rat bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) or mesenchymal stem cells (MSCs) are a heterogeneous population of cells that are multipotent. When rat BMSCs were seeded onto a 3-dimensional (3-D) tubular scaffold engineered from aligned type I collagen strands and cultured in osteogenic medium, they simultaneously matured and differentiated into osteoblastic and vascular cell lineages. In addition, these osteoblasts produced mineralized matricellular deposits. BMSCs were seeded at a density of 2 x 10(6) cells/15 mm tube and cultured in basal or osteogenic medium for 3, 6, and 9 days. These cells were subsequently processed for real-time reverse-transcriptase polymerase chain reaction (RT-qPCR), immunohistochemical, cytochemical, and biochemical analyses. Immunolocalization of lineage-specific proteins was visualized using confocal microscopy. In the present study, the expression pattern of key osteogenic markers significantly differed in response to basal and osteogenic media. Alkaline phosphatase activity and calcium content increased significantly over the observed period of time in osteogenic medium. The observed up-regulation of transcripts coding for osteoblastic phenotypic markers is reminiscent of in vivo expression patterns. Abundant sheets of Pecam (CD31) -, Flk-1 (vascular endothelial growth factor receptor-2) -, CD34-, tomato lectin-, and alpha-smooth muscle actin-positive cells were observed in these tube cultures. Moreover, nascent capillary-like vessels were also seen amid the osteoblasts in osteogenic cultures. Our 3-D culture system augmented the maturation and differentiation of BMSCs into osteoblasts. Thus, our in vitro model provides an excellent opportunity to study the concurrent temporal and spatial regulation of osteogenesis and vasculogenesis during bone development.