Faculty Bibliography

INTRODUCTION: Osteoarthritis (OA) is a degenerative disease of the entire joint.¹ In healthy joints the articulating surfaces are encased in a smooth layer of hyaline cartilage and are further protected by the surrounding synovial fluid. High molecular weight hyaluronan (HMWHA) is present at relatively high levels in the synovial fluid and cartilage matrix.'²' HMWHA provides viscoelastic protection and lubrication of the cartilage surfaces, and has also been shown to have important anti-inflammatory properties.'³-⁵' During inflammation hyaluronan (HA) can be degraded to lower molecular weight HA (LMWHA) by the increased levels of free radicals and endogenous hyaluronidases present in the inflamed joint.'⁶' Interestingly, LMWHA, which can signal through a number of different receptors (TLR-2 and 4, CD44 and RHAMM), was suggested to act as potent inflammatory mediator in the joint.'⁷' Our collaborators identified a 15-mer peptide that binds to LMWHA and reduced inflammation and fibrogenesis in excisional skin wounds.^<8' In this study we hypothesized that LMWHA generated in the OA joint is a key mediator for stimulating catabolic and inflammatory events in joint cells and that interfering with LMWHA signaling using the novel peptide inhibitor will attenuate joint inflammation. Methods: Human articular chondrocytes were isolated from articular cartilage samples obtained from patients (donor age range 48 - 67) undergoing total knee replacement surgery at NYU Hospital for Joint Diseases. Knee cartilage was harvested from regions with no macroscopically evident degeneration. The collection of tissue from patients undergoing knee replacement surgery was approved by the Institutional Regulatory Board at NYU School of Medicine. Human chondrocytes were isolated from these cartilage samples and cultured as described previously.'⁹' Cultured chondrocyte were switched to serum-free medium for 24 h and then treated with inflammatory stimuli (interleukin-lbeta (IL-lp), LMWHA; average molecular weight of lOkDa). In addition, serum-starved cells were treated with the 15-mer peptide at various concentrations. A synovial fibroblast cell line (SW982) was also used and was cultured as described previously.'¹⁰' The analysis of HA concentrations in the culture medium was done as described by us previously.'¹¹' Western blotting and real time PCR analysis was performed as described by us.^<12) Results: In the present study we used human articular chondrocytes and a synovial fibroblast cell line (SW982) to determine the involvement of LMWHA promoting inflammation in the joint cavity. We show that LMWHA stimulated catabolic markers (Cox-2, IL-6, iNOS, MMP-13) and inhibited the expression of articular cartilage markers (aggrecan, type II collagen) in human articular chondrocytes and SW982 synovial cells. LMWHA treatment stimulated the NF-KB and the MAP kinase (ERK, JNK, p38) signaling pathways in human chondrocytes similar to IL-lp treatment. Whereas the stimulation of these signaling pathways occurred within the first hour of treatment with IL-lp, the stimulation of these pathways by LMWHA occurred at later time points (6h and 24h). The novel peptide inhibitor markedly reduced the activation of these signaling pathways in LMWHA-treated chondrocytes. In addition, it markedly decreased the expression of catabolic markers and increased the expression of articular cartilage markers in LMWHA-treated chondrocytes. The peptide also inhibited the expression of catabolic markers in IL-1 p-treated human articular chondrocytes and SW982 cells and increased articular cartilage marker expression in IL-1 p-treaded chondrocytes. Treatment of human articular chondrocytes with IL-lp resulted in a marked increase of HA released into the culture medium over time. This increase in HA release correlated with an increase in catabolic markers and a reduction in anabolic markers over time, similar to the effects of the LMWHA treatment. Discussion: Our findings demonstrate that LMWHA stimulates inflammatory and catabolic events in joint cells via activation of NF-Kb and ERK, JNK and p38 signaling pathways. In addition, we demonstrate that a novel peptide that binds to LMWHA and interferes with LMWHA signaling attenuates inflammatory and catabolic events in joint cells mediated by LMWHA and IL-lp. More specifically, we have shown that the novel peptide inhibitor dramatically reduced IL-6 and Cox-2 levels in articular chondrocytes and synovial fibroblasts. Both of these catabolic markers have been previously shown to be associated with increased pain levels in patients with OA.'¹³' These findings suggest that LMWHA plays a key role in mediating inflammation in joint cells during OA pathology, and that a novel peptide inhibitor of LMWHA signaling may act as a novel compound to specifically reduce inflammation and pain in the joint and ultimately slow down cartilage degradation during OA. SIGNIFICANCE: This study identified LMWHA as a key mediator of inflammatory and catabolic events in joint cells. In addition, we determined that interfering with LMWHA signaling using a novel peptide that binds LMWHA may provide a novel therapeutic strategy to reduce inflammation in the OA joint and ultimately slow down cartilage degradation during OA

[This corrects the article DOI: 10.1371/journal.pone.0150697.].

The WNT-TCF signaling pathway participates in adult tissue homeostasis and repair, and is hyperactive in a number of human diseases including cancers of the colon. Whereas to date there are no antagonists approved for patient use, a potential problem for their sustained use is the blockade of WNT signaling in healthy tissues, thus provoking potentially serious co-lateral damage. Here we have screened a library of plant and microorganism small molecules for novel WNT signaling antagonists and describe withanolide F as a potent WNT-TCF response blocker. This steroidal lactone inhibits TCF-dependent colon cancer xenograft growth and mimics the effects of genetic blockade of TCF and of ivermectin, a previously reported WNT-TCF blocker. However, withanolide F is unique in that it imposes a long-lasting repression of tumor growth, WNT-TCF targets and cancer stem cell clonogenicity after drug treatment. These findings are paralleled by its modulation of chromatin regulators and its alteration of overall H3K4me1 levels. Our results open up the possibility to permanently repress essential signaling responses in cancer cells through limited treatments with small molecules.

Amyloid beta (Abeta) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Several lines of investigation support the notion that synaptic pathology, one of the strongest correlates to cognitive impairment, is related to the progressive accumulation of neurotoxic Abeta oligomers. Since the process of oligomerization/fibrillization is concentration-dependent, it is highly reliant on the homeostatic mechanisms that regulate the steady state levels of Abeta influencing the delicate balance between rate of synthesis, dynamics of aggregation, and clearance kinetics. Emerging new data suggest that reduced Abeta clearance, particularly in the aging brain, plays a critical role in the process of amyloid formation and AD pathogenesis. Using well-defined monomeric and low molecular mass oligomeric Abeta1-40 species stereotaxically injected into the brain of C57BL/6 wild-type mice in combination with biochemical and mass spectrometric analyses in CSF, our data clearly demonstrate that Abeta physiologic removal is extremely fast and involves local proteolytic degradation leading to the generation of heterogeneous C-terminally cleaved proteolytic products, while providing clear indication of the detrimental role of oligomerization for brain Abeta efflux. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Abeta. The findings indicate that clearance from brain interstitial fluid follows local and systemic paths and that in addition to the blood-brain barrier, local enzymatic degradation and the bulk flow transport through the choroid plexus into the CSF play significant roles. Our studies highlight the diverse factors influencing brain clearance and the participation of various routes of elimination opening up new research opportunities for the understanding of altered mechanisms triggering AD pathology and for the potential design of combined therapeutic strategies.

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.

We investigated the contribution of blood vessel formation and neuronal excitability to the development of functional neural circuitry in larval zebrafish by analyzing oculomotor performance in response to visual and vestibular stimuli. To address the dependence of neuronal function on the presence of blood vessels, we compared wild type embryos to reck and cloche mutants that lacked intracerebral blood vessels. To test how neuronal excitability impacts neuronal development and intracerebral vascularization, we blocked neural activity using Tetraodotoxin (TTX) and Tricaine. In reck mutants, we found both slow phase horizontal tracking and fast phase resets with only a slightly reduced amplitude and bandwidth. Spontaneous saccades, eye position holding and vestibular gravitoinertial induced eye rotation were also present. All of these behaviors except for visual tracking were observed in cloche mutants that lacked any head vasculature. Thus, numerous oculomotor neuronal circuits spanning the forebrain, midbrain and hindbrain compartments, ending in motor innervations of the eye muscles, were correctly formed and generated appropriate oculomotor behaviors without blood vessels. However, our observations indicate that beginning at approximately six days, circulation was required for sustained behavioral performance. We further found that blocking neuronal excitability with either TTX or Tricaine up to 4-5 days post fertilization did not noticeably interfere with intracerebral blood vessel formation in wild type larvae. After removal of drug treatments, the oculomotor behaviors returned within hours. Thus, development of neuronal circuits that drive oculomotor performance does not require neuronal spiking or activity. Together these findings demonstrate that neither vascularization nor neuronal excitability are essential for the formation of numerous oculomotor nuclei with intricately designed connectivity and signal processing. We conclude that a genetic blueprint specifies early larval structural and physiological features, and this developmental strategy may be viewed as a unique adaptation required for early survival.