Keratinocyte-derived S100A9 modulates neutrophil infiltration and affects psoriasis-like skin and joint disease.

OBJECTIVES: S100A9, an alarmin that can form calprotectin (CP) heterodimers with S100A8, is mainly produced by keratinocytes and innate immune cells. The contribution of keratinocyte-derived S100A9 to psoriasis (Ps) and psoriatic arthritis (PsA) was evaluated using mouse models, and the potential usefulness of S100A9 as a Ps/PsA biomarker was assessed in patient samples. METHODS: Conditional S100A9 mice were crossed with DKO* mice, an established psoriasis-like mouse model based on inducible epidermal deletion of c-Jun and JunB to achieve additional epidermal deletion of S100A9 (TKO* mice). Psoriatic skin and joint disease were evaluated in DKO* and TKO* by histology, microCT, RNA and proteomic analyses. Furthermore, S100A9 expression was analysed in skin, serum and synovial fluid samples of patients with Ps and PsA. RESULTS: Compared with DKO* littermates, TKO* mice displayed enhanced skin disease severity, PsA incidence and neutrophil infiltration. Altered epidermal expression of selective pro-inflammatory genes and pathways, increased epidermal phosphorylation of STAT3 and higher circulating TNFα were observed in TKO* mice. In humans, synovial S100A9 levels were higher than the respective serum levels. Importantly, patients with PsA had significantly higher serum concentrations of S100A9, CP, VEGF, IL-6 and TNFα compared with patients with only Ps, but only S100A9 and CP could efficiently discriminate healthy individuals, patients with Ps and patients with PsA. CONCLUSIONS: Keratinocyte-derived S100A9 plays a regulatory role in psoriatic skin and joint disease. In humans, S100A9/CP is a promising marker that could help in identifying patients with Ps at risk of developing PsA.

p38ß and Cancer: The Beginning of the Road.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway is implicated in cancer biology and has been widely studied over the past two decades as a potential therapeutic target. Most of the biological and pathological implications of p38MAPK signaling are often associated with p38α (MAPK14). Recently, several members of the p38 family, including p38γ and p38δ, have been shown to play a crucial role in several pathologies including cancer. However, the specific role of p38ß (MAPK11) in cancer is still elusive, and further investigation is needed. Here, we summarize what is currently known about the role of p38ß in different types of tumors and its putative implication in cancer therapy. All evidence suggests that p38ß might be a key player in cancer development, and could be an important therapeutic target in several pathologies, including cancer.

Oncostatin M binds to extracellular matrix in a bioactive conformation: implications for inflammation and metastasis.

Oncostatin M (OSM) is an interleukin-6-like inflammatory cytokine reported to play a role in a number of pathological processes including cancer. Full-length OSM is expressed as a 26 kDa protein that can be proteolytically processed into 24 kDa and 22 kDa forms via removal of C-terminal peptides. In this study, we examined both the ability of OSM to bind to the extracellular matrix (ECM) and the activity of immobilized OSM on human breast carcinoma cells. OSM was observed to bind to ECM proteins collagen types I and XI, laminin, and fibronectin in a pH-dependent fashion, suggesting a role for electrostatic bonds that involves charged amino acids of both the ECM and OSM. The C-terminal extensions of 24 kDa and 26 kDa OSM, which contains six and thirteen basic amino acids, respectively, enhanced electrostatic binding to ECM at pH 6.5-7.5 when compared to 22 kDa OSM. The highest levels of OSM binding to ECM, though, were observed at acidic pH 5.5, where all forms of OSM bound to ECM proteins to a similar extent. This indicates additional electrostatic binding properties independent of the OSM C-terminal extensions. The reducing agent dithiothreitol also inhibited the binding of OSM to ECM suggesting a role for disulfide bonds in OSM immobilization. OSM immobilized to ECM was protected from cleavage by tumor-associated proteases and maintained activity following incubation at acidic pH for extended periods of time. Importantly, immobilized OSM remained biologically active and was able to induce and sustain the phosphorylation of STAT3 in T47D and ZR-75-1 human breast cancer cells over prolonged periods, as well as increase levels of STAT1 and STAT3 protein expression. Immobilized OSM also induced epithelial-mesenchymal transition-associated morphological changes in T47D cells. Taken together, these data indicate that OSM binds to ECM in a bioactive state that may have important implications for the development of chronic inflammation and tumor metastasis.

Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells.

Stimulated emission depletion (STED) microscopy provides a new opportunity to study fine sub-cellular structures and highly dynamic cellular processes, which are challenging to observe using conventional optical microscopy. Using actin as an example, we explored the feasibility of using a continuous wave (CW)-STED microscope to study the fine structure and dynamics in fixed and live cells. Actin plays an important role in cellular processes, whose functioning involves dynamic formation and reorganization of fine structures of actin filaments. Frequently used confocal fluorescence and STED microscopy dyes were employed to image fixed PC-12 cells (dyed with phalloidin- fluorescein isothiocyante) and live rat chondrosarcoma cells (RCS) transfected with actin-green fluorescent protein (GFP). Compared to conventional confocal fluorescence microscopy, CW-STED microscopy shows improved spatial resolution in both fixed and live cells. We were able to monitor cell morphology changes continuously; however, the number of repetitive analyses were limited primarily by the dyes used in these experiments and could be improved with the use of dyes less susceptible to photobleaching. In conclusion, CW-STED may disclose new information for biological systems with a proper characteristic length scale. The challenges of using CW-STED microscopy to study cell structures are discussed.

Intracellular domain fragment of CD44 alters CD44 function in chondrocytes.

The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44.

Optimal 3D culture of primary articular chondrocytes for use in the rotating wall vessel bioreactor.

INTRODUCTION: Reliable culturing methods for primary articular chondrocytes are essential to study the effects of loading and unloading on joint tissue at the cellular level. Due to the limited proliferation capacity of primary chondrocytes and their tendency to dedifferentiate in conventional culture conditions, long-term culturing conditions of primary chondrocytes can be challenging. The goal of this study was to develop a suspension culturing technique that not only would retain the cellular morphology, but also maintain the gene expression characteristics of primary articular chondrocytes. METHODS: Three-dimensional culturing methods were compared and optimized for primary articular chondrocytes in the rotating wall vessel bioreactor, which changes the mechanical culture conditions to provide a form of suspension culture optimized for low shear and turbulence. We performed gene expression analysis and morphological characterization of cells cultured in alginate beads, Cytopore-2 microcarriers, primary monolayer culture, and passaged monolayer cultures using reverse transcription-PCR and laser scanning confocal microscopy. RESULTS: Primary chondrocytes grown on Cytopore-2 microcarriers maintained the phenotypical morphology and gene expression pattern observed in primary bovine articular chondrocytes, and retained these characteristics for up to 9 d. DISCUSSION: Our results provide a novel and alternative culturing technique for primary chondrocytes suitable for studies that require suspension such as those using the rotating wall vessel bioreactor. In addition, we provide an alternative culturing technique for primary chondrocytes that can impact future mechanistic studies of osteoarthritis progression, treatments for cartilage damage and repair, and cartilage tissue engineering.

Induction of CD44 cleavage in articular chondrocytes.

OBJECTIVE: The hyaluronan receptor CD44 provides chondrocytes with a mechanism for sensing and responding to changes in the extracellular matrix. The purpose of this study was to document the fragmentation and loss of CD44 and to determine the likely mechanisms involved. METHODS: A polyclonal anti-CD44 cytotail antibody was generated to detect CD44 fragmentation by Western blot analysis. Chondrocytes were isolated from human or bovine articular cartilage. Primary articular chondrocytes were treated with interleukin-1beta (IL-1beta), hyaluronan oligosaccharides, or phorbol myristate acetate or were passaged and subcultured in monolayer to induce dedifferentiation. Conditions that altered the capacity of CD44 to transit into lipid rafts, or pharmacologic inhibitors of metalloproteinase or gamma-secretase activity were used to define the mechanism of fragmentation of CD44. RESULTS: Chondrocytes from osteoarthritic cartilage exhibited CD44 fragmentation as low molecular mass bands, corresponding to the CD44-EXT and CD44-ICD bands. Following dedifferentiation of chondrocytes or treatment of primary chondrocytes with hyaluronan oligosaccharides, IL-1beta, or phorbol myristate acetate, CD44 fragmentation was enhanced. Subsequent culture of the dedifferentiated chondrocytes in 3-dimensional alginate beads rescued the chondrocyte phenotype and diminished the fragmentation of CD44. Fragmentation of CD44 in chondrocytes was blocked in the presence of the metalloproteinase inhibitor GM6001 and the gamma-secretase inhibitor DAPT. CONCLUSION: CD44 fragmentation, consistent with a signature pattern reported for sequential metalloproteinase/gamma-secretase cleavage of CD44, is a common metabolic feature of chondrocytes that have undergone dedifferentiation in vitro and osteoarthritic chondrocytes. Transit of CD44 into lipid rafts may be required for its fragmentation.

The evaluation of a multiphasic 3D-bioplotted scaffold seeded with adipose derived stem cells to repair osteochondral defects in a porcine model.

There is a need for the development of effective treatments for focal articular cartilage injuries. We previously developed a multiphasic 3D-bioplotted osteochondral scaffold design that can drive site-specific tissue formation when seeded with adipose-derived stem cells (ASC). The objective of this study was to evaluate this scaffold in a large animal model. Osteochondral defects were generated in the trochlear groove of Yucatan minipigs and repaired with scaffolds that either contained or lacked an electrospun tidemark and were either unseeded or seeded with ASC. Implants were monitored via computed tomography (CT) over the course of 4 months of in vivo implantation and compared to both open lesions and autologous explants. ICRS II evaluation indicated that defects with ASC-seeded scaffolds had healing that most closely resembled the aulogous explant. Scaffold-facilitated subchondral bone repair mimicked the structure of native bone tissue, but cartilage matrix staining was not apparent within the scaffold. The open lesions had the highest volumetric infill detected using CT analysis (p < 0.05), but the repair tissue was largely disorganized. The acellular scaffold without a tidemark had significantly more volumetric filling than either the acellular or ASC seeded groups containing a tidemark (p < 0.05), suggesting that the tidemark limited cell infiltration into the cartilage portion of the scaffold. Overall, scaffold groups repaired the defect more successfully than an open lesion but achieved limited repair in the cartilage region. With further optimization, this approach holds potential to treat focal cartilage lesions in a highly personalized manner using a human patient's own ASC cells.

Investigation of multiphasic 3D-bioplotted scaffolds for site-specific chondrogenic and osteogenic differentiation of human adipose-derived stem cells for osteochondral tissue engineering applications.

Osteoarthritis is a degenerative joint disease that limits mobility of the affected joint due to the degradation of articular cartilage and subchondral bone. The limited regenerative capacity of cartilage presents significant challenges when attempting to repair or reverse the effects of cartilage degradation. Tissue engineered medical products are a promising alternative to treat osteochondral degeneration due to their potential to integrate into the patient's existing tissue. The goal of this study was to create a scaffold that would induce site-specific osteogenic and chondrogenic differentiation of human adipose-derived stem cells (hASC) to generate a full osteochondral implant. Scaffolds were fabricated using 3D-bioplotting of biodegradable polycraprolactone (PCL) with either ß-tricalcium phosphate (TCP) or decellularized bovine cartilage extracellular matrix (dECM) to drive site-specific hASC osteogenesis and chondrogenesis, respectively. PCL-dECM scaffolds demonstrated elevated matrix deposition and organization in scaffolds seeded with hASC as well as a reduction in collagen I gene expression. 3D-bioplotted PCL scaffolds with 20% TCP demonstrated elevated calcium deposition, endogenous alkaline phosphatase activity, and osteopontin gene expression. Osteochondral scaffolds comprised of hASC-seeded 3D-bioplotted PCL-TCP, electrospun PCL, and 3D-bioplotted PCL-dECM phases were evaluated and demonstrated site-specific osteochondral tissue characteristics. This technique holds great promise as cartilage morbidity is minimized since autologous cartilage harvest is not required, tissue rejection is minimized via use of an abundant and accessible source of autologous stem cells, and biofabrication techniques allow for a precise, customizable methodology to rapidly produce the scaffold.

LRP receptors in chondrocytes are modulated by simulated microgravity and cyclic hydrostatic pressure.

Mechanical loading is essential for the maintenance of musculoskeletal homeostasis. Cartilage has been demonstrated to be highly mechanoresponsive, but the mechanisms by which chondrocytes respond to mechanical stimuli are not clearly understood. The goal of the study was to determine how LRP4, LRP5, and LRP6 within canonical Wnt-signaling are regulated in simulated microgravity and cyclic hydrostatic pressure, and to investigate the potential role of LRP 4/5/6 in cartilage degeneration. Rat chondrosacroma cell (RCS) pellets were stimulated using either cyclic hydrostatic pressure (1Hz, 7.5 MPa, 4hr/day) or simulated microgravity in a rotating wall vessel (RWV) bioreactor (11RPM, 24hr/day). LRP4/5/6 mRNA expression was assessed by RT-qPCR and LRP5 protein expression was determined by fluorescent immunostaining. To further evaluate our in vitro findings in vivo, mice were subjected to hindlimb suspension for 14 days and the femoral heads stained for LRP5 expression. We found that, in vitro, LRP4/5/6 mRNA expression is modulated in a time-dependent manner by mechanical stimulation. Additionally, LRP5 protein expression is upregulated in response to both simulated microgravity and cyclic hydrostatic pressure. LRP5 is also upregulated in vivo in the articular cartilage of hindlimb suspended mice. This is the first study to examine how LRP4/5/6, critical receptors within musculoskeletal biology, respond to mechanical stimulation. Further elucidation of this mechanism could provide significant clinical benefit for the identification of pharmaceutical targets for the maintenance of cartilage health.

Role of bulge epidermal stem cells and TSLP signaling in psoriasis.

Psoriasis is a common inflammatory skin disease involving a cross-talk between epidermal and immune cells. The role of specific epidermal stem cell populations, including hair follicle stem cells (HF-SCs) in psoriasis is not well defined. Here, we show reduced expression of c-JUN and JUNB in bulge HF-SCs in patients with scalp psoriasis. Using lineage tracing in mouse models of skin inflammation with inducible deletion of c-Jun and JunB, we found that mutant bulge HF-SCs initiate epidermal hyperplasia and skin inflammation. Mechanistically, thymic stromal lymphopoietin (TSLP) was identified in mutant cells as a paracrine factor stimulating proliferation of neighboring non-mutant epidermal cells, while mutant inter-follicular epidermal (IFE) cells are lost over time. Blocking TSLP in psoriasis-like mice reduced skin inflammation and decreased epidermal proliferation, VEGFα expression, and STAT5 activation. These findings unravel distinct roles of HF-SCs and IFE cells in inflammatory skin disease and provide novel mechanistic insights into epidermal cell interactions in inflammation.

OSM-induced CD44 contributes to breast cancer metastatic potential through cell detachment but not epithelial-mesenchymal transition.

BACKGROUND: Hormone receptor status in human breast cancer cells is a strong indicator of the aggressiveness of a tumor. Triple negative breast cancers (TNBC) are aggressive, difficult to treat, and contribute to high incidences of metastasis by possessing characteristics such as increased tumor cell migration and a large presence of the transmembrane protein, cluster of differentiation 44 (CD44) on the cell membrane. Estrogen receptor-positive (ER+) cells are less aggressive and do not migrate until undergoing an epithelial-mesenchymal transition (EMT). METHODS: The relationship between EMT and CD44 during metastatic events is assessed by observing changes in EMT markers, tumor cell detachment, and migration following cytokine treatment on both parental and CD44 knockdown human breast tumor cells. RESULTS: ER+ T47D and MCF-7 human breast cancer cells treated with OSM demonstrate increased CD44 expression and CD44 cleavage. Conversely, ER- MDA-MB-231 human breast cancer cells do not show a change in CD44 expression nor undergo EMT in the presence of OSM. In ER+ cells, knockdown expression of CD44 by shRNA did not prevent EMT but did change metastatic processes such as cellular detachment and migration. OSM-induced migration was decreased in both ER+ and ER- cells with shCD44 cells compared to control cells, while the promotion of tumor cell detachment by OSM was decreased in ER+ MCF7-shCD44 cells, as compared to control cells. Interestingly, OSM-induced detachment in ER- MDA-MB-231-shCD44 cells that normally don't detach at significant rates. CONCLUSION: OSM promotes both EMT and tumor cell detachment in ER+ breast cancer cells. Yet, CD44 knockdown did not affect OSM-induced EMT in these cells, while independently decreasing OSM-induced cell detachment. These results suggest that regulation of CD44 by OSM is important for at least part of the metastatic cascade in ER+ breast cancer.

Comparison of Simulated Microgravity and Hydrostatic Pressure for Chondrogenesis of hASC.

BACKGROUND: Cartilage tissue engineering is a growing field due to the lack of regenerative capacity of native tissue. The use of bioreactors for cartilage tissue engineering is common, but the results are controversial. Some studies suggest that microgravity bioreactors are ideal for chondrogenesis, while others show that mimicking hydrostatic pressure is crucial for cartilage formation. A parallel study comparing the effects of loading and unloading on chondrogenesis has not been performed. METHODS: The goal of this study was to evaluate chondrogenesis of human adipose-derived stem cells (hASC) under two different mechanical stimuli relative to static culture: microgravity and cyclic hydrostatic pressure (CHP). Pellets of hASC were cultured for 14 d under simulated microgravity using a rotating wall vessel bioreactor or under CHP (7.5 MPa, 1 Hz, 4 h · d-1) using a hydrostatic pressure vessel. RESULTS: We found that CHP increased mRNA expression of Aggrecan, Sox9, and Collagen II, caused a threefold increase in sulfated glycosaminoglycan production, and resulted in stronger vimentin staining intensity and organization relative to microgravity. In addition, Wnt-signaling patterns were altered in a manner that suggests that simulated microgravity decreases chondrogenic differentiation when compared to CHP. DISCUSSION: Our goal was to compare chondrogenic differentiation of hASC using a microgravity bioreactor and a hydrostatic pressure vessel, two commonly used bioreactors in cartilage tissue engineering. Our results indicate that CHP promotes hASC chondrogenesis and that microgravity may inhibit hASC chondrogenesis. Our findings further suggest that cartilage formation and regeneration might be compromised in space due to the lack of mechanical loading.Mellor LF, Steward AJ, Nordberg RC, Taylor MA, Loboa EG. Comparison of simulated microgravity and hydrostatic pressure for chondrogenesis of hASC. Aerosp Med Hum Perform. 2017; 88(4):377-384.

Fabrication and Evaluation of Electrospun, 3D-Bioplotted, and Combination of Electrospun/3D-Bioplotted Scaffolds for Tissue Engineering Applications.

Electrospun scaffolds provide a dense framework of nanofibers with pore sizes and fiber diameters that closely resemble the architecture of native extracellular matrix. However, it generates limited three-dimensional structures of relevant physiological thicknesses. 3D printing allows digitally controlled fabrication of three-dimensional single/multimaterial constructs with precisely ordered fiber and pore architecture in a single build. However, this approach generally lacks the ability to achieve submicron resolution features to mimic native tissue. The goal of this study was to fabricate and evaluate 3D printed, electrospun, and combination of 3D printed/electrospun scaffolds to mimic the native architecture of heterogeneous tissue. We assessed their ability to support viability and proliferation of human adipose derived stem cells (hASC). Cells had increased proliferation and high viability over 21 days on all scaffolds. We further tested implantation of stacked-electrospun scaffold versus combined electrospun/3D scaffold on a cadaveric pig knee model and found that stacked-electrospun scaffold easily delaminated during implantation while the combined scaffold was easier to implant. Our approach combining these two commonly used scaffold fabrication technologies allows for the creation of a scaffold with more close resemblance to heterogeneous tissue architecture, holding great potential for tissue engineering and regenerative medicine applications of osteochondral tissue and other heterogeneous tissues.

Extracellular Calcium Modulates Chondrogenic and Osteogenic Differentiation of Human Adipose-Derived Stem Cells: A Novel Approach for Osteochondral Tissue Engineering Using a Single Stem Cell Source.

We have previously shown that elevating extracellular calcium from a concentration of 1.8 to 8 mM accelerates and increases human adipose-derived stem cell (hASC) osteogenic differentiation and cell-mediated calcium accretion, even in the absence of any other soluble osteogenic factors in the culture medium. However, the effects of elevated calcium on hASC chondrogenic differentiation have not been reported. The goal of this study was to determine the effects of varied calcium concentrations on chondrogenic differentiation of hASC. We hypothesized that exposure to elevated extracellular calcium (8 mM concentration) in a chondrogenic differentiation medium (CDM) would inhibit chondrogenesis of hASC when compared to basal calcium (1.8 mM concentration) controls. We further hypothesized that a full osteochondral construct could be engineered by controlling local release of calcium to induce site-specific chondrogenesis and osteogenesis using only hASC as the cell source. Human ASC was cultured as micromass pellets in CDM containing transforming growth factor-ß1 and bone morphogenetic protein 6 for 28 days at extracellular calcium concentrations of either 1.8 mM (basal) or 8 mM (elevated). Our findings indicated that elevated calcium induced osteogenesis and inhibited chondrogenesis in hASC. Based on these findings, stacked polylactic acid nanofibrous scaffolds containing either 0% or 20% tricalcium phosphate (TCP) nanoparticles were electrospun and tested for site-specific chondrogenesis and osteogenesis. Histological assays confirmed that human ASC differentiated locally to generate calcified tissue in layers containing 20% TCP, and cartilage in the layers with no TCP when cultured in CDM. This is the first study to report the effects of elevated calcium on chondrogenic differentiation of hASC, and to develop osteochondral nanofibrous scaffolds using a single cell source and controlled calcium release to induce site-specific differentiation. This approach holds great promise for osteochondral tissue engineering using a single cell source (hASC) and single scaffold.