Faculty Bibliography

Introduction: Damage to the articular cartilage is common, especially through a trauma or injury to the knee joint. Because of the lack of intrinsic capacity to heal, chondral defects remain a major challenge to repair. Current methods used for cartilage regeneration generally result in poorly repaired defects leading to early onset of posttraumatic osteoarthritis (PTOA) and subsequently requiring joint replacement (1). The use of mesenchymal stem cells (MSCs) derived from ones own bone marrow or adipose tissues has been suggested to be used for cartilage repair (2). The transplantation of stem cells for tissue repair requires cell settlement, proliferation and differentiation. The local tissue microenvironment or stem cell niche plays a key role for the successful transplantation of stem cells for tissue repair (3). Very little, however, is known about the stem cell niche required for the successful transplantation of stem cells for cartilage repair. In addition, stem cell settlement and chondrogenesis in cartilage repair has to occur in an unfriendly inflammatory environment in response to injury. In this study, we determined how a novel peptide (NP-0100) that binds to hyaluronan (HA) affects MSC attachment, proliferation and chondrogenic differentiation under normal and inflammatory conditions. Previously we have shown that NP-0100 inhibited catabolic events and stimulated the expression of articular cartilage markers in human articular chondrocytes cultured in an inflammatory environment (4). Therefore, we hypothesized that NP-0100 together with high molecular HA (HMWHA) will enhance cartilage repair by optimizing the stem cell niche for precursor cells to repair cartilage and reduce inflammation. Methods: Chondrogenesis of the multipotential murine C3H/10T1/2 cell line was induced in micromass cultures in the presence of BMP-2 (100ng/ml). In addition, the micromass cultures were treated with NP-0100 or cultured in conditioned media from untreated and IL-1beta-treated articular chondrocytes in the absence or presence of NP-0100. Chondrogenesis was determined by alcian blue staining and real time PCR analysis of chondrocyte marker genes. Cell attachment and proliferation was assessed on tissue culture plates or tissue culture plates with immobilized high molecular HA (HMWHA) or HMWHA together with NP- 0100. Cell attachment was determined by DAPI staining. Cell proliferation was determined using the CCK-8 kit. Results: C3H10T1/2 cells better attached to HMWHA-coated tissue culture plates than to uncoated tissue culture plates. The largest number of cells, however, attached to tissue culture plates that were coated with both HMWHA and NP-0100. In addition, cells showed the highest proliferation rate on HMWHA/NP- 0100-coated plates followed by HMWHA-coated plates. The lowest proliferation rate was detected on uncoated tissue culture plates. Furthermore, NP-0100 stimulated the expression of articular cartilage markers (aggrecan and type II collagen) and Sox-9, a master transcription factor that regulates chondrogenesis. NP-0100-treated C3H/10T1/2 micromass cultures also stained more intensely with Alcian blue, which is indicative of increased levels of sulfated proteoglycans than micromass cultures not treated with NP-0100. Chondrogenesis was markedly inhibited when the micromass cultures were cultured in the presence of conditioned media from IL-1beta-treated human articular chondrocytes compared to conditioned media from untreated human articular chondrocytes. However, conditioned media from human articular chondrocytes treated with IL-1beta in combination with NP-0100 showed a reduced inhibition of chondrogenesis compared to conditioned media from IL-1beta-treated human articular chondrocytes. Discussion: Our findings show that a novel peptide NP-0100 in the presence of HMWHA stimulated attachment, proliferation and chondrogenic differentiation of precursor cells, and this suggests a potential therapeutic role for NP-0100 in promoting cartilage repair. The improved attachment and proliferation of precursor cells on tissue culture plates that were coated with HMWHA and NP-0100 rather than HMWHA alone suggests that NP-0100 stabilizes or crosslinks HMWHA to create a more favorable microenvironment (stem cell niche) for the precursor cells to adhere and proliferate. This notion is supported by a previous study showing that a cross-linked HMWHA is required for the formation of the stem cell niche for precursor cells to repair muscle after injury (5). NP-0100 not only supported the formation of the stem cell niche but also stimulated chondrogenesis of C3H10T1/2 cells in high-density micromass culture. Furthermore, the peptide was able to protect the micromass cultures from an inflammatory environment that otherwise inhibited chondrogenesis. Future studies have to determine the mechanisms by which NP-0100 together with HMWHA stimulates chondrogenesis even in an inflammatory environment

AIM: To assess the feasibility and utility of measuring baseline professional identity formation (PIF) in a theory-based professionalism curriculum for early medical students. METHODS: All 132 entering students completed the professional identity essay (PIE) and the defining issues test (DIT2). Students received score reports with individualized narrative feedback and wrote a structured reflection after a large-group session in which the PIF construct was reviewed. Analysis of PIEs resulted in assignment of a full or transitional PIF stage (1-5). The DIT2 score reflects the proportion of the time students used universal ethical principles to justify a response to 6 moral dilemma cases. Students' reflections were content analyzed. RESULTS: PIF scores were distributed across stage 2/3, stage 3, stage 3/4, and stage 4. No student scores were in stages 1, 2, 4/5, or 5. The mean DIT2 score was 53% (range 9.7?76.5%); the correlation between PIF stage and DIT score was rho = 0.18 (p = 0.03). Students who took an analytic approach to the data and demonstrated both awareness that they are novices and anticipation of continued PIF tended to respond more positively to the feedback. CONCLUSIONS: These PIF scores distributed similarly to novice students in other professions. Developmental-theory based PIF and moral reasoning measures are related. Students reflected on these measures in meaningful ways suggesting utility of measuring PIF scores in medical education.

Since progranulin (PGRN) is a natural ligand of TNF receptors, we assessed whether serum PGRN levels predict and/or reflect responsiveness of RA patients to TNF-antagonist therapy. TNF-antagonist-naive RA patients (N = 35) were started on TNF-antagonist therapy. At baseline and at follow-up visits, DAS28-ESR, DAS28-CRP, and CDAI were calculated, and venous blood was collected for serum PGRN determination. Disease activity and clinical response were based on EULAR criteria. Baseline serum PGRN levels varied considerably and correlated with ESR and CRP. DAS28-ESR, DAS28-CRP, and CDAI were greater in "PGRN-high" than in "PGRN-low". Baseline serum PGRN levels did not predict clinical responsiveness to TNF-antagonist therapy. Nevertheless, changes in serum PGRN levels at 274+ days following initiation of TNF-antagonist therapy correlated with changes in ESR, CRP, DAS28-ESR, DAS28-CRP, and CDAI. At this time, DAS28-ESR, DAS28-CRP, and CDAI in PGRN-high and PGRN-low equalized, but serum PGRN levels remained greater in PGRN-high than in PGRN-low. To our knowledge, the present report is the first prospective study to longitudinally assess changes in serum PGRN levels following initiation of TNF-antagonist therapy. Although pre-treatment serum PGRN levels may not predict clinical responsiveness to TNF-antagonist therapy, changes in serum PGRN levels correlate with changes in disease metrics over time. By inference, administration of PGRN may represent an effective therapeutic option for development in RA patients.

Purpose/Aim of the study: Poor cardiovascular health, including obesity and altered lipid profiles at mid-life, are linked to increased risk of Alzheimer's disease (AD). The biological mechanisms linking cardiovascular health and cognitive function are unclear though are likely to be multifactorial. This study examined the association between various lipoproteins and cognitive functioning in ageing women.

Inflammatory processes are firmly established as central to the development and complications of cardiovascular diseases. Elevated levels of inflammatory markers have been shown to be predictive of future cardiovascular events. The specific targeting of these processes in experimental models has been shown to attenuate myocardial and arterial injury, reduce disease progression, and promote healing. However, the translation of these observations and the demonstration of clear efficacy in clinical practice have been disappointing. A major limitation might be that tools currently used to measure 'inflammation' are insufficiently precise and do not provide information about disease site and activity, or discriminate between functionally important activation pathways. The challenge, therefore, is to make measures of inflammation that are more meaningful, and which can guide specific targeted therapies. In this Review, we consider the roles of inflammatory processes in the related pathologies of atherosclerosis and acute myocardial infarction, by providing an evaluation of the known and emerging inflammatory pathways. We highlight contemporary techniques to characterize and quantify inflammation, and consider how they might be used to guide specific treatments. Finally, we discuss emerging opportunities in the field, including their current limitations and challenges that are the focus of ongoing study.

BACKGROUND: Angiogenesis, the process in which new blood vessels are formed from preexisting ones, is highly dependent on the presence of classical angiogenic factors. Recent evidence suggests that axonal guidance proteins and their receptors can also act as angiogenic regulators. Netrin, a family of laminin-like proteins, specifically Netrin-1 and 4, act via DCC/Neogenin-1 and UNC5 class of receptors to promote or inhibit angiogenesis, depending on the physiological context. METHODS: Mesenchymal stem cells secrete a broad set of classical angiogenic factors. However, little is known about the expression of non-canonical angiogenic factors such as Netrin-1. The aim was to characterize the possible secretion of Netrin ligands by Wharton's jelly-derived mesenchymal stem cells (WJ-MSC). We evaluated if Netrin-1 presence in the conditioned media from these cells was capable of inducing angiogenesis both in vitro and in vivo, using human umbilical vein endothelial cells (HUVEC) and chicken chorioallantoic membrane (CAM), respectively. In addition, we investigated if the RhoA/ROCK pathway is responsible for the integration of Netrin signaling to control vessel formation. RESULTS: The paracrine angiogenic effect of the WJ-MSC-conditioned media is mediated at least in part by Netrin-1 given that pharmacological blockage of Netrin-1 in WJ-MSC resulted in diminished angiogenesis on HUVEC. When HUVEC were stimulated with exogenous Netrin-1 assayed at physiological concentrations (10-200 ng/mL), endothelial vascular migration occurred in a concentration-dependent manner. In line with our determination of Netrin-1 present in WJ-MSC-conditioned media we were able to obtain endothelial tubule formation even in the pg/mL range. Through CAM assays we validated that WJ-MSC-secreted Netrin-1 promotes an increased angiogenesis in vivo. Netrin-1, secreted by WJ-MSC, might mediate its angiogenic effect through specific cell surface receptors on the endothelium, such as UNC5b and/or integrin alpha6beta1, expressed in HUVEC. However, the angiogenic response of Netrin-1 seems not to be mediated through the RhoA/ROCK pathway. CONCLUSIONS: Thus, here we show that stromal production of Netrin-1 is a critical component of the vascular regulatory machinery. This signaling event may have deep implications in the modulation of several processes related to a number of diseases where angiogenesis plays a key role in vascular homeostasis.

Although regeneration via the reprogramming of one cell lineage to another occurs in fish and amphibians, it is not observed in mammals. We discovered in mouse that during wound healing adipocytes regenerate from myofibroblasts, a cell type thought to be differentiated and non-adipogenic. Myofibroblast reprogramming required neogenic hair follicles, which triggered BMP signaling and then activation of adipocyte transcription factors expressed during development. Overexpression of the BMP antagonist, noggin, in hair follicles or deletion of the BMP receptor in myofibroblasts prevented adipocyte formation. Adipocytes formed from human keloid fibroblasts when treated with either BMP or when placed with human hair follicles in vitro. Thus, we identify the myofibroblast as a plastic cell type that may be manipulated to treat scars in humans.

Skeletal myogenesis is regulated by signal transduction, but the factors and mechanisms involved are not well understood. The group I Paks Pak1 and Pak2 are related protein kinases and direct effectors of Cdc42 and Rac1. Group I Paks are ubiquitously expressed and specifically required for myoblast fusion in Drosophila We report that both Pak1 and Pak2 are activated during mammalian myoblast differentiation. One pathway of activation is initiated by N-cadherin ligation and involves the cadherin coreceptor Cdo with its downstream effector, Cdc42. Individual genetic deletion of Pak1 and Pak2 in mice has no overt effect on skeletal muscle development or regeneration. However, combined muscle-specific deletion of Pak1 and Pak2 results in reduced muscle mass and a higher proportion of myofibers with a smaller cross-sectional area. This phenotype is exacerbated after repair to acute injury. Furthermore, primary myoblasts lacking Pak1 and Pak2 display delayed expression of myogenic differentiation markers and myotube formation. These results identify Pak1 and Pak2 as redundant regulators of myoblast differentiation in vitro and in vivo and as components of the promyogenic Ncad/Cdo/Cdc42 signaling pathway.

Neogenin-1 (NEO1) is a transmembrane receptor involved in axonal guidance, angiogenesis, neuronal cell migration and cell death, during both embryonic development and adult homeostasis. It has been described as a dependence receptor, because it promotes cell death in the absence of its ligands (Netrin and Repulsive Guidance Molecule (RGM) families) and cell survival when they are present. Although NEO1 and its ligands are involved in tumor progression, their precise role in tumor cell survival and migration remain unclear. Public databases contain extensive information regarding the expression of NEO1 and its ligands Netrin-1 (NTN1) and Netrin-4 (NTN4) in primary neuroblastoma (NB) tumors. Analysis of this data revealed that patients with high expression levels of both NEO1 and NTN4 have a poor survival rate. Accordingly, our analyses in NB cell lines with different genetic backgrounds revealed that knocking-down NEO1 reduces cell migration, whereas silencing of endogenous NTN4 induced cell death. Conversely, overexpression of NEO1 resulted in higher cell migration in the presence of NTN4, and increased apoptosis in the absence of ligand. Increased apoptosis was prevented when utilizing physiological concentrations of exogenous Netrin-4. Likewise, cell death induced after NTN4 knock-down was rescued when NEO1 was transiently silenced, thus revealing an important role for NEO1 in NB cell survival. In vivo analysis, using the chicken embryo chorioallantoic membrane (CAM) model, showed that NEO1 and endogenous NTN4 are involved in tumor extravasation and metastasis. Our data collectively demonstrate that endogenous NTN4/NEO1 maintain NB growth via both pro-survival and pro-migratory molecular signaling.

Serotonin is an evolutionarily ancient molecule that functions in generating and modulating many behavioral states. Although much is known about how serotonin acts on its cellular targets, how serotonin release is regulated in vivo remains poorly understood. In the nematode C. elegans, serotonin neurons that drive female reproductive behavior are directly modulated by inhibitory neuropeptides. Here, we report the isolation of mutants in which inhibitory neuropeptides fail to properly modulate serotonin neurons and the behavior they mediate. The corresponding mutations affect the T-type calcium channel CCA-1 and symmetrically re-tune its voltage-dependencies of activation and inactivation towards more hyperpolarized potentials. This shift in voltage dependency strongly and specifically bypasses the behavioral and cell physiological effects of peptidergic inhibition on serotonin neurons. Our results indicate that T-type calcium channels are critical regulators of a C. elegans serotonergic circuit and demonstrate a mechanism in which T-type channels functionally gate inhibitory modulation in vivo.