Catheter-based retrograde coronary sinus infusion is a practical delivery technique for introducing biological molecules into the cardiac system.

OBJECTIVES: To demonstrate coronary sinus (CS) retrograde catheterization as a practicable technique for delivering biologics into the heart. BACKGROUND: There are many options to deliver biologics into the heart. However, there is no single optimal technique when considering safety, biologic retention, and reproducibility. Retrograde delivery has the potential to address many of these concerns. This study evaluated retrograde CS infusion of luciferase-expressing plasmid in a porcine model using the Advance® CS Coronary Sinus Infusion Catheter and bioluminescence imaging to track the expression of the infused biological markers. METHODS: Plasmid was delivered retrograde into the CS in one of three infusion volumes. Twenty-four hours post-infusion, hearts were excised and underwent bioluminescence imaging to characterize the expression of the infusates. Heart and lung biopsies were also assessed for luciferase expression using RT-qPCR. RESULTS: Retrograde infusion was safe and successful in all nine test subjects. Luciferase detection was inconsistent in the low volume group. Bioluminescence was confined predominantly along the posterolateral left ventricle for medium volume infusions and was more broadly dispersed along the anterior side of the heart for high volume infusions. Tissue mRNA analysis corroborated the bioluminescence results, with the highest concentration of luciferase expression localized in the left ventricle. CONCLUSIONS: Retrograde CS infusion is a promising technique for delivering biological molecules to the heart. Specifically, this study demonstrated that the low pressure coronary venous system accommodates a wide range of infusion volumes and that biological infusates can be maintained in situ following the resumption of coronary venous flow.

A novel role for thrombopoietin in regulating osteoclast development in humans and mice.

Emerging data suggest that megakaryocytes (MKs) play a significant role in skeletal homeostasis. Indeed, osteosclerosis observed in several MK-related disorders may be a result of increased numbers of MKs. In support of this idea, we have previously demonstrated that MKs increase osteoblast (OB) proliferation by a direct cell-cell contact mechanism and that MKs also inhibit osteoclast (OC) formation. As MKs and OCs are derived from the same hematopoietic precursor, in these osteoclastogenesis studies we examined the role of the main MK growth factor, thrombopoietin (TPO) on OC formation and bone resorption. Here we show that TPO directly increases OC formation and differentiation in vitro. Specifically, we demonstrate the TPO receptor (c-mpl or CD110) is expressed on cells of the OC lineage, c-mpl is required for TPO to enhance OC formation in vitro, and TPO activates the mitogen-activated protein kinases, Janus kinase/signal transducer and activator of transcription, and nuclear factor-kappaB signaling pathways, but does not activate the PI3K/AKT pathway. Further, we found TPO enhances OC resorption in CD14+CD110+ human OC progenitors derived from peripheral blood mononuclear cells, and further separating OC progenitors based on CD110 expression enriches for mature OC development. The regulation of OCs by TPO highlights a novel therapeutic target for bone loss diseases and may be important to consider in the numerous hematologic disorders associated with alterations in TPO/c-mpl signaling as well as in patients suffering from bone disorders.

Fluid flow assays.

Mechanical signals are major regulators of skeletal homeostasis as the addition of exogenous load is followed by enhanced bone formation and the removal of normal loads is followed by net bone loss. The mechanism by which bone cells perceive and respond to changes in their biophysical environment are still poorly understood, but it is widely accepted that the detection of interstitial fluid flow is an initiating cue. In this chapter, we describe two in vitro systems designed to examine the effects of fluid flow on bone cell behavior and to elucidate the signaling cascades activated by this stimulus. The first utilizes a parallel plate flow chamber designed to stimulate a single bone cell type grown on glass slides. The second employs a rotating disk fluid flow apparatus. Commercially-available cell culture inserts allow one type of bone cell to be exposed to fluid flow and signals to be communicated to a second bone cell model not exposed to fluid flow.

ATP release mediates fluid flow-induced proliferation of human bone marrow stromal cells.

UNLABELLED: Oscillatory fluid flow induced the vesicular release of ATP from human BMSCs that directly contributes to the induction of BMSC proliferation. Degrading extracellular nucleotides prevents fluid flow-induced increases in intracellular calcium concentration, the activation of calcineurin, and the nuclear translocation of NFAT. INTRODUCTION: Regulation of bone cell activity by autocrine/paracrine factors is a well-established mechanism by which skeletal homeostasis is regulated by mechanical signals. The release of extracellular nucleotides in particular has been shown to induce many of the responses thought to be necessary for load-induced bone formation. In these studies, we examined the effect of oscillatory fluid flow on the release of ATP from bone marrow stromal cells (BMSCs) and the effect of ATP release on BMSC proliferation and intracellular calcium signaling pathways. MATERIALS AND METHODS: BMSCs were exposed to oscillatory fluid flow, and the concentration of ATP in conditioned media samples was determined using a luciferin:luciferase-based reaction. Western blot analysis was used to examine the expression of purinergic receptors. Using pharmacological antagonists of gap junction hemichannels and vesicular trafficking, we studied the mechanism of ATP release from BMSCs. Apyrase was used to study the effect of extracellular nucleotides on intracellular calcium concentration, calcineurin activity, and nuclear factor of activated T cells (NFAT) nuclear translocation. RESULTS AND CONCLUSIONS: Fluid flow exposure induced the flow rate-dependent release of ATP from BMSCs that was attenuated by treatment with monensin and N-ethylmaleimide, suggesting a vesicular mechanism. Treating BMSCs with ATP, but not other nucleotides, increased cellular proliferation. Moreover, extracellular ATP was a prerequisite for fluid flow-induced increases in intracellular calcium concentration, activation of calcineurin, the nuclear translocation of NFATc1, and proliferation. These data indicate that ATP regulates not only osteoblastic and osteocytic cell behavior but also that of mesenchymal precursors and support our hypothesis that similar mechanotransduction mechanisms are activated by fluid flow in these cell types.

Integrin expression and osteopontin regulation in human fetal osteoblastic cells mediated by substratum surface characteristics.

Integrin-mediated adhesion of anchorage-dependent cells to scaffolds is a critical component of tissue engineering. We investigated integrin expression by the human fetal osteoblastic cell line, hFOB 1.19 (hFOB), as a function of substratum surface wettability. The influence of surface wettability on bone cell phenotype was also examined. Plasma-treated quartz (PTQ) and glass (PTG) (hydrophilic, contact angles of 0 degrees), octadecyltrichlorosilane-treated quartz (STQ) and glass (STG) (hydrophobic, contact angles above about 100 degrees), and tissue culture polystyrene were used for cell culture. hFOB cells cultured on hydrophilic substrata displayed well-developed actin stress fibers relative to cells on hydrophobic substrata. Western blot analysis revealed that hFOB cells cultured on hydrophobic substrata (STQ or STG) express lower levels of alphav and beta3 integrin subunits than do cells on hydrophilic substrata (PTQ or PTG). This effect was more pronounced in cells on STQ than on STG. These variations in integrin expression were lessened by extended culture time. Double- labeled integrin/actin immunofluorescence confirmed Western blot results, that is, cells cultured on PTQ displayed distinct, large plaques of alphav and beta3 subunits and integrin alphavbeta3, as well as their colocalization with actin stress fiber ends, whereas cells on STQ did not display integrin plaques after 24 h and displayed only minimal plaque formation after 3 days. Vinculin, a focal adhesion protein that mediates binding between the integrin and actin cytoskeleton, appeared in Western blots to mimic the variations of alphav and beta3 expression with respect to surface wettability. Interestingly, real-time RT-PCR analysis showed that hFOB cultured on hydrophobic substrata, which have downregulated alphav and beta3 integrin subunits, displayed greater steady state mRNA levels of osteopontin, an extracellular matrix (ECM) protein containing the Arg-Gly-Asp (RGD) integrin recognition sequence, than did cells cultured on hydrophilic substrata. Our results imply that substratum surface wettability regulates integrin-mediated bone cell adhesion and further influences the expression of bone cell-ECM complexes.

Biomimetic bone mechanotransduction modeling in neonatal rat femur organ cultures: structural verification of proof of concept.

The goal of this work was to develop and validate a whole bone organ culture model to be utilized in biomimetic mechanotransduction research. Femurs harvested from 2-day-old neonatal rat pups were maintained in culture for 1 week post-harvest and assessed for growth and viability. For stimulation studies, femurs were physiologically stimulated for 350 cycles 24 h post-harvest then maintained in culture for 1 week at which time structural tests were conducted. Comparing 1 and 8 days in culture, bones grew significantly in size over the 7-day culture period. In addition, histology supported adequate diffusion and organ viability at 2 weeks in culture. For stimulation studies, 350 cycles of physiologic loading 24 h post-harvest resulted in increased bone strength over the 7-day culture period. In this work, structural proof of concept was established for the use of whole bone organ cultures as mechanotransduction models. Specifically, this work established that these cultures grow and remain viable in culture, are adequately nourished via diffusion and are capable of responding to a brief bout of mechanical stimulation with an increase in strength.

The role of gap junctions in megakaryocyte-mediated osteoblast proliferation and differentiation.

Gap junctions (GJs) are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules (e.g. calcium ions, inositol phosphates, and cyclic nucleotides) to pass from cell to cell. Over the past two decades, many studies have described a role for GJ intercellular communication (GJIC) in the proliferation and differentiation of many cells, including bone cells. Recently, we reported that megakaryocytes (MKs) enhance osteoblast (OB) proliferation by a juxtacrine signaling mechanism. Here we determine whether this response is facilitated by GJIC. First we demonstrate that MKs express connexin 43 (Cx43), the predominant GJ protein expressed by bone cells, including OBs. Next, we provide data showing that MKs can communicate with OBs via GJIC, and that the addition of two distinct GJ uncouplers, 18alpha-glycyrrhetinic acid (alphaGA) or oleamide, inhibits this communication. We then demonstrate that inhibiting MK-mediated GJIC further enhances the ability of MKs to stimulate OB proliferation. Finally, we show that while culturing MKs with OBs reduces gene expression of several differentiation markers/matrix proteins (type I collagen, osteocalcin, and alkaline phosphatase), reduces alkaline phosphatase enzymatic activity, and decreases mineralization in OBs, blocking GJIC does not result in MK-induced reductions in OB gene expression, enzymatic levels, or mineralized nodule formation. Overall, these data provide evidence that GJIC between MKs and OBs is functional, and that inhibiting GJIC in MK-OB cultures enhances OB proliferation without apparently altering differentiation when compared to similarly treated OB cultures. Thus, these observations regarding MK-OB GJIC inhibition may provide insight regarding potential novel targets for anabolic bone formation.

MAP kinase and calcium signaling mediate fluid flow-induced human mesenchymal stem cell proliferation.

Mechanical signals are important regulators of skeletal homeostasis, and strain-induced oscillatory fluid flow is a potent mechanical stimulus. Although the mechanisms by which osteoblasts and osteocytes respond to fluid flow are being elucidated, little is known about the mechanisms by which bone marrow-derived mesenchymal stem cells respond to such stimuli. Here we show that the intracellular signaling cascades activated in human mesenchymal stem cells by fluid flow are similar to those activated in osteoblastic cells. Oscillatory fluid flow inducing shear stresses of 5, 10, and 20 dyn/cm(2) triggered rapid, flow rate-dependent increases in intracellular calcium that pharmacological studies suggest are inositol trisphosphate mediated. The application of fluid flow also induced the phosphorylation of extracellular signal-regulated kinase-1 and -2 as well as the activation of the calcium-sensitive protein phosphatase calcineurin in mesenchymal stem cells. Activation of these signaling pathways combined to induce a robust increase in cellular proliferation. These data suggest that mechanically induced fluid flow regulates not only osteoblastic behavior but also that of mesenchymal precursors, implying that the observed osteogenic response to mechanical loading may be mediated by alterations in the cellular behavior of multiple members of the osteoblast lineage, perhaps by a common signaling pathway.