Faculty Bibliography

Pulsed electromagnetic fields (PEMFs) have been documented to promote bone fracture healing in nonunions and increase lumbar spinal fusion rates. However, the molecular mechanisms by which PEMF stimulates differentiation of human bone marrow stromal cells (hBMSCs) into osteoblasts are not well understood. In this study the PEMF effects on hBMSCs were studied by microarray analysis. PEMF stimulation of hBMSCs' cell numbers mainly affected genes of cell cycle regulation, cell structure, and growth receptors or kinase pathways. In the differentiation and mineralization stages, PEMF regulated preosteoblast gene expression and notably, the transforming growth factor-beta (TGF-beta) signaling pathway and microRNA 21 (miR21) were most highly regulated. PEMF stimulated activation of Smad2 and miR21-5p expression in differentiated osteoblasts, and TGF-beta signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF-beta signaling pathway, was found to be miR21-5p's putative target gene and PEMF caused a decrease in Smad7 expression. Expression of Runx2 was increased by PEMF treatment and the miR21-5p inhibitor prevented the PEMF stimulation of Runx2 expression in differentiating cells. Thus, PEMF could mediate its effects on bone metabolism by activation of the TGF-beta signaling pathway and stimulation of expression of miR21-5p in hBMSCs.

Sonic Hedgehog (Shh) signaling regulates mesenchymal proliferation and differentiation during embryonic lung development. In the adult lung, Shh signaling maintains mesenchymal quiescence and is dysregulated in diseases such as IPF and COPD. Our previous data implicated a role for Shh in postnatal lung development. Here we report a detailed analysis of Shh signaling during murine postnatal lung development. We show that Shh pathway expression and activity during alveolarization (P0-P14) are distinct from those during maturation (P14-P24). This biphasic pattern is paralleled by the transient presence of Gli1+;alpha-smooth muscle actin (aSMA)+ myofibroblasts in the growing alveolar septal tips. Carefully-timed inhibition of Hedgehog (Hh) signaling during alveolarization defined mechanisms by which Shh influences the mesenchymal compartment. First, interruption of Hh signaling at earlier time points results in increased lung compliance and wall structure defects of increasing severity, ranging from moderately enlarged alveolar airspaces to markedly enlarged airspaces and fewer secondary septa. Second, Shh signaling is required for myofibroblast differentiation: Hh inhibition during early alveolarization almost completely eliminates Gli1+;aSMA+ cells at the septal tips, and Gli1-lineage tracing revealed that Gli1+ cells do not undergo apoptosis after Hh inhibition, but remain in the alveolar septa and are unable to express aSMA. Third, Shh signaling is vital to mesenchymal proliferation during alveolarization, as Hh inhibition decreased proliferation of Gli1+ cells and their progeny. Our study establishes Shh as a new alveolarization promoting factor that might be affected in perinatal lung diseases that are associated with impaired alveolarization.

Latent-transforming growth factor beta-binding protein 3 (LTBP-3) is important for craniofacial morphogenesis and hard tissue mineralization, as it is essential for activation of transforming growth factor-beta (TGF-beta). To investigate the role of LTBP-3 in tooth formation we performed micro-computed tomography (micro-CT), histology, and scanning electron microscopy analyses of adult Ltbp3-/- mice. The Ltbp3-/- mutants presented with unique craniofacial malformations and reductions in enamel formation that began at the matrix formation stage. Organization of maturation-stage ameloblasts was severely disrupted. The lateral side of the incisor was affected most. Reduced enamel mineralization, modification of the enamel prism pattern, and enamel nodules were observed throughout the incisors, as revealed by scanning electron microscopy. Molar roots had internal irregular bulbous-like formations. The cementum thickness was reduced, and microscopic dentinal tubules showed minor nanostructural changes. Thus, LTBP-3 is required for ameloblast differentiation and for the formation of decussating enamel prisms, to prevent enamel nodule formation, and for proper root morphogenesis. Also, and consistent with the role of TGF-beta signaling during mineralization, almost all craniofacial bone components were affected in Ltbp3-/- mice, especially those involving the upper jaw and snout. This mouse model demonstrates phenotypic overlap with Verloes Bourguignon syndrome, also caused by mutation of LTBP3, which is hallmarked by craniofacial anomalies and amelogenesis imperfecta phenotypes.

Recent developments in tissue clearing methods have provided investigators with an invaluable tool for visualizing and mapping three dimensional macromolecular structures and processes. By implementing a routine protocol, based on the passive clarity technique (PACT) method, for tissue clearing, the Research Histopathology Core at NYU Langone Medical Center seeks to provide investigators with a reliable, customizable service in conjunction with the immunohistochemistry (IHC) and Microscopy Cores in order to produce results in the most efficient way possible for both the investigators and the cores. The PACT method of clearing allows visualization of endogenous fluorescence and immunofluorescence labeling performed by the Core, or both. Stabilization through transparent hydrogel cross-linking, followed by delipidation in an sodium dodecyl sulfate buffer, results in a clear tissue sample that remains structurally sound with proteins, nucleic acids, and any associated labels in place. The clearing buffer can also be modified to allow simultaneous decalcification of bone specimens. Final clearing is achieved in a refractive index matching solution (RIMS buffer) which also serves as the microscopy medium. Cleared and labeled tissue can then be imaged on the Microscopy Core's Zeiss lightsheet microscope, allowing multichannel fluorescence from a range of angles and Z-stacking. The lightsheet microscope excites and detects only one thin optical section of the specimen at a time, making three dimensional imaging exceptionally light efficient. By honing proficiency in tissue clearing via the PACT method and working in close collaboration with neighboring core labs, the Histopathology Core can increase its breadth of expertise while relieving investigators of the time and cost intensive burden of protocol development and training.

Introduction: Post-ablation scarring is used as a method to uncouple and/or silence pro-arrhythmic circuits. It has been previously suggested that circulating bone marrow derived cells (BMDC) are capable of homing into myocardial infarction scars. It is possible that intercellular junctions form between myocytes and BMDCs and may contribute to ablation failure and recurrence of arrhythmias. Methods: We tested whether BMDCs populate an ablation scars and contribute to functional coupling between the scar and surrounding myocardium. Wild type C57BI/6 mice (n=17) underwent radiation-induced myeloablation and subsequent transplantation with bone marrow progenitors obtained from fetal Cx43 WT (bmcWT) or Cx43 deficient (bmcKO) mice. Results: All donor cells constitutively expressed mCherry protein. Right ventricular ablation was carried out thirty days post transplantation and hearts were studied 30 day post ablation. Cells expressing mCherry and vimentin were observed throughout the scar suggesting donor cells differentiated into a mesenchymal lineage. Coupling between the uninjured myocardium and the scar was assayed with optical mapping. Suction electrode was placed on uninjured myocardium next to the scar to deliver current pulses. Conclusions: Changes in membrane voltage were measured optically at three different sites: Uninjured myocardium, Scar and Remote area (see figure). These data demonstrate that BMDCs can couple to the surrounding myocardium and contribute to the electrophysiological properties of ablation scar tissue. Delivery of modified BMDCs could be used to modifythe post-ablation scar electrophysiological properties. (Figure Presented)