Jay D. Potts

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.

Organization of fibroblasts in the heart.

Cardiac fibroblasts are organized into a three‐dimensional network in the heart. This organization follows the endomysial weave network that surrounds groups of myocytes. Reverse transcriptase‐polymerase chain reaction, Western blots, and immunohistochemistry were used to show that discoidin domain receptor 2 (DDR2) was specific for cardiac fibroblasts and not expressed on endothelial cells, smooth muscle cells, or cardiac myocytes. DDR2 is expressed early in development and in the adult heart. High voltage electron microscopy (HVEM), scanning electron microscopy, and laser scanning confocal microscopy document the three‐dimensional organization of fibroblasts in the heart. Antibodies against connexin 43 and 45 showed different patterns but confirmed, along with HVEM, that fibroblasts are connected to each other as well as cardiac myocytes. The implications of this arrangement of fibroblasts can be important to cardiac function. The signaling of DDR2 and the expression of matrix metalloproteinase 2 in relation to collagen turnover and remodeling is discussed. Developmental Dynamics 230:787–794, 2004.

Regulation of Osteogenic Differentiation of Rat Bone Marrow Stromal Cells on 2D Nanorod Substrates

Bone marrow stromal cells (BMSCs) possess multi-lineage differentiation potential and can be induced to undergo differentiation into various cell types with the correct combination of chemical and environmental factors. Although, they have shown great prospects in therapeutic and medical applications, less is known about their behavior on nanosurfaces mimicking the extra cellular matrix (ECM). In this report we have employed 2D substrates coated with tobacco mosaic virus (TMV) nanorods to study the differentiation process of BMSCs into osteoblast like cells. TMV is a rod-shaped plant virus with an average length of 300 nm and diameter of 18 nm. The osteogenic differentiation of BMSCs on TMV was studied over time points of 7, 14 and 21 days. We examined the temporal gene expression changes during these time points by real-time quantitative PCR (RT-qPCR) analysis. As expected, osteo-specific genes (osteocalcin, osteopontin and osteonectin) were upregulated and showed a maximum change in expression on TMV at 14 days which was 7 days earlier than on tissue culture plastic (TCP). Based on the genes expression profile generated by RT-qPCR experiments, we proposed that the early interaction of cells with TMV triggers on signaling pathways which regulate speedy expression of osteocalcin in turn, resulting in early mineralization of the cells. To further investigate these regulating factors we studied global changes in gene expression (DNA microarray analyses) during osteogenic differentiation on the nanosubstrate. Multitudes of genes were affected by culturing cells on nanorod substrate, which corroborated our initial PCR findings. Microarray analysis further revealed additional targets influenced by the presence of nanorods on the surface, of which, the expression of bone morphogenetic protein 2 (BMP2) was of particular interests. Further investigation into the temporal change of BMP2, revealed that it acts as a major promoter in signaling the early regulation of osteocalcin on TMV coated substrates.

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.

The promotion of osteoblastic differentiation of rat bone marrow stromal cells by a polyvalent plant mosaic virus

To investigate the role that the micro/nano-environment plays on the differentiation pathway of bone marrow stromal cells (BMSCs) into osteoblasts, we employed a 2D substrate coated with turnip yellow mosaic virus (TYMV) particles. TYMV is a non-enveloped icosahedral plant virus which has an average diameter 28 nm and the protein cage structure consists of 180 identical subunits. The temporal effect of TYMV coated substrate on the adhesion and differentiation capacity of the BMSCs was monitored for selected time periods of 7, 14 and 21 days. We examined the gene expression profile of BMSCs cultured in primary media (undifferentiated cells) and cells induced to osteoblast lineage by real time PCR analysis. To further corroborate our findings, we investigated the expression of osteogenic markers using immunohistochemistry and cytochemical staining. As expected, the genes involved in the process of osteogenic differentiation were activated more during the growth of cells under osteogenic media. In addition, we found that the BMSCs induced to undergo osteogenic differentiation on TYMV coated substrates formed fully mineralized nodules comprising of osteoblast-like cells around day 14. Comparing the gene expression pattern of BMSCs induced to osteogenic differentiation under standard culture conditions with the cells induced on TYMV substrates, we found significant differences in the temporal expression and level of expression of several key genes. Our findings indicate that TYMV, as a biogenic nanoparticle, can be employed as a model to modulate the nano-environment of the substrates in order to gain an insight into the role that the micro/nano-environment has in regulating adhesion, growth and differentiation of BMSCs towards osteogenic lineage, which will be vital for designing compatible biomaterials for tissue engineering purposes.

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.

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.

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.

A 3-D cardiac muscle construct for exploring adult marrow stem cell based myocardial regeneration.

Adult bone marrow stromal cells (BMSCs) are capable of differentiating into cardiomyocyte-like cells in vitro and contribute to myocardial regeneration in vivo. Consequently, BMSCs may potentially play a vital role in cardiac repair and regeneration. However, this concept has been limited by inadequate and inconsistent differentiation of BMSCs into cardiomyocytes along with poor survival and integration of neo-cardiomyocytes after implantation into ischemic myocardium. In order to overcome these barriers and to explore adult stem cell based myocardial regeneration, we have developed an in vitro model of three-dimensional (3-D) cardiac muscle using rat ventricular embryonic cardiomyocytes (ECMs) and BMSCs. When ECMs and BMSCs were seeded sequentially onto a 3-D tubular scaffold engineered from topographically aligned type I collagen-fibers and cultured in basal medium for 7, 14, 21, or 28 days, the maturation and co-differentiation into a cardiomyocyte lineage was observed. Phenotypic induction was characterized at morphological, immunological, biochemical and molecular levels. The observed expression of transcripts coding for cardiomyocyte phenotypic markers and the immunolocalization of cardiomyogenic lineage-associated proteins revealed typical expression patterns of neo-cardiomyogenesis. At the biochemical level differentiating cells exhibited appropriate metabolic activity and at the ultrastructural level myofibrillar and sarcomeric organization were indicative of an immature phenotype. Our 3-D co-culture system sustains the ECMs in vitro continuum of differentiation process and simultaneously induces the maturation and differentiation of BMSCs into cardiomyocyte-like cells. Thus, this novel 3-D co-culture system provides a useful in vitro model to investigate the functional role and interplay of developing ECMs and BMSCs during cardiomyogenic differentiation.