GDF15 Contributes to the Regulation of the Mechanosensitive Responses of PdL Fibroblasts through the Modulation of IL-37.

During orthodontic tooth movement (OTM), areas of compressive and tensile forces are generated in the periodontal ligament (PdL), a mechanoreactive connective tissue between the teeth and alveolar bone. Mechanically stimulated PdL fibroblasts (PdLFs), the main cell type of PdL, express significantly increased levels of growth differentiation factor 15 (GDF15). In compressed PdL areas, GDF15 plays a fundamental role in modulating relevant OTM processes, including inflammation and osteoclast activation. However, the specific function of this factor in tensile areas has not yet been investigated. Thus, the aim of this study was to investigate the role of GDF15 in the mechanoresponse of human PdLFs (hPdLFs) that were exposed to biaxial tensile forces in vitro. Using siRNA-mediated knockdown experiments, we demonstrated that GDF15 had no impact on the anti-inflammatory force response of elongated hPdLFs. Although the anti-inflammatory markers IL1RN and IL10, as well as the activation of immune cells remained unaffected, we demonstrated an inhibitory role of GDF15 for the IL-37 expression. By analyzing osteogenic markers, including ALPL and RUNX2, along with an assessment of alkaline phosphatase activation, we further showed that the regulation of IL-37 by GDF15 modulates the osteogenic differentiation potential of hPdLFs. Despite bone resorption in tensile areas being rather limited, GDF15 was also found to positively modulate osteoclast activation in those areas, potentially by adjusting the IL-37 levels. In light of our new findings, we hypothesize that GDF15 modulates force-induced processes in tissue and bone remodeling through its various intra- and extracellular signaling pathways as well as interaction partners. Potentially acting as a master regulator, the modulation of GDF15 levels may hold relevance for clinical implications.

GDF15 Promotes the Osteogenic Cell Fate of Periodontal Ligament Fibroblasts, thus Affecting Their Mechanobiological Response.

Periodontal ligament fibroblasts (PdLFs) exert important functions in oral tissue and bone remodeling following mechanical forces, which are specifically applied during orthodontic tooth movement (OTM). Located between the teeth and the alveolar bone, mechanical stress activates the mechanomodulatory functions of PdLFs including regulating local inflammation and activating further bone-remodeling cells. Previous studies suggested growth differentiation factor 15 (GDF15) as an important pro-inflammatory regulator during the PdLF mechanoresponse. GDF15 exerts its effects through both intracrine signaling and receptor binding, possibly even in an autocrine manner. The extent to which PdLFs are susceptible to extracellular GDF15 has not yet been investigated. Thus, our study aims to examine the influence of GDF15 exposure on the cellular properties of PdLFs and their mechanoresponse, which seems particularly relevant regarding disease- and aging-associated elevated GDF15 serum levels. Therefore, in addition to investigating potential GDF15 receptors, we analyzed its impact on the proliferation, survival, senescence, and differentiation of human PdLFs, demonstrating a pro-osteogenic effect upon long-term stimulation. Furthermore, we observed altered force-related inflammation and impaired osteoclast differentiation. Overall, our data suggest a major impact of extracellular GDF15 on PdLF differentiation and their mechanoresponse.

Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation.

In cells, proteins encoded by the same gene do not all behave uniformly but engage in functional subpopulations induced by spatial or temporal segregation. While conventional microscopy has limitations in revealing such spatial and temporal diversity, single-molecule tracking (SMT) microscopy circumvented this problem and allows for high-resolution imaging and quantification of dynamic single-molecule properties. Particularly in the nucleus, SMT has identified specific DNA residence times of transcription factors (TFs), DNA-bound TF fractions and positions of transcriptional hot-spots upon cell stimulation. By contrast to cell stimulation, SMT has not been employed to follow dynamic TF changes along stages of cell differentiation. Herein, we analysed the serum response factor (SRF), a TF involved in the differentiation of many cell types to study nuclear single-molecule dynamics in neuronal differentiation. Our data in living mouse hippocampal neurons show dynamic changes in SRF DNA residence time and SRF DNA-bound fraction between the stages of adhesion, neurite growth and neurite differentiation in axon and dendrites. Using TALM (tracking and localization microscopy), we identified nuclear positions of SRF clusters and observed changes in their numbers and size during differentiation. Furthermore, we show that the SRF cofactor MRTF-A (myocardin-related TF or MKL1) responds to cell activation by enhancing the long-bound DNA fraction. Finally, a first SMT colocalization study of two proteins was performed in living cells showing enhanced SRF/MRTF-A colocalization upon stimulation. In summary, SMT revealed modulation of dynamic TF properties during cell stimulation and differentiation.

Palmitate-Triggered COX2/PGE2-Related Hyperinflammation in Dual-Stressed PdL Fibroblasts Is Mediated by Repressive H3K27 Trimethylation.

The interrelationships between periodontal disease, obesity-related hyperlipidemia and mechanical forces and their modulating effects on the epigenetic profile of periodontal ligament (PdL) cells are assumed to be remarkably complex. The PdL serves as a connective tissue between teeth and alveolar bone and is involved in pathogen defense and the inflammatory responses to mechanical stimuli occurring during tooth movement. Altered inflammatory signaling could promote root resorption and tooth loss. Hyperinflammatory COX2/PGE2 signaling was reported for human PdL fibroblasts (HPdLFs) concomitantly stressed with Porphyromonas gingivalis lipopolysaccharides and compressive force after exposure to palmitic acid (PA). The aim of this study was to investigate the extent to which this was modulated by global and gene-specific changes in histone modifications. The expression of key epigenetic players and global H3Kac and H3K27me3 levels were quantitatively evaluated in dual-stressed HPdLFs exposed to PA, revealing a minor force-related reduction in repressive H3K27me3. UNC1999-induced H3K27me3 inhibition reversed the hyperinflammatory responses of dual-stressed PA cultures characterized by increased COX2 expression, PGE2 secretion and THP1 adhesion. The reduced expression of the gene encoding the anti-inflammatory cytokine IL-10 and the increased presence of H3K27me3 at its promoter-associated sites were reversed by inhibitor treatment. Thus, the data highlight an important epigenetic interplay between the different stimuli to which the PdL is exposed.

GDF15 Supports the Inflammatory Response of PdL Fibroblasts Stimulated by P. gingivalis LPS and Concurrent Compression.

Periodontitis is characterized by bacterially induced inflammatory destruction of periodontal tissue. This also affects fibroblasts of the human periodontal ligaments (HPdLF), which play a coordinating role in force-induced tissue and alveolar bone remodeling. Excessive inflammation in the oral tissues has been observed with simultaneous stimulation by pathogens and mechanical forces. Recently, elevated levels of growth differentiation factor 15 (GDF15), an immuno-modulatory member of the transforming growth factor (TGFB) superfamily, were detected under periodontitis-like conditions and in force-stressed PdL cells. In view of the pleiotropic effects of GDF15 in various tissues, this study aims to investigate the role of GDF15 in P. gingivalis-related inflammation of HPdLF and its effect on the excessive inflammatory response to concurrent compressive stress. To this end, the expression and secretion of cytokines (IL6, IL8, COX2/PGE2, TNFα) and the activation of THP1 monocytic cells were analyzed in GDF15 siRNA-treated HPdLF stimulated with P. gingivalis lipopolysaccharides alone and in combination with compressive force. GDF15 knockdown significantly reduced cytokine levels and THP1 activation in LPS-stimulated HPdLF, which was less pronounced with additional compressive stress. Overall, our data suggest a pro-inflammatory role for GDF15 in periodontal disease and demonstrate that GDF15 partially modulates the force-induced excessive inflammatory response of PdLF under these conditions.

Descriptive Proteome Analysis to Investigate Context-Dependent Treatment Responses to OXPHOS Inhibition in Colon Carcinoma Cells Grown as Monolayer and Multicellular Tumor Spheroids.

We have previously identified selective upregulation of the mevalonate pathway genes upon inhibition of oxidative phosphorylation (OXPHOS) in quiescent cancer cells. Using mass spectrometry-based proteomics, we here investigated whether these responses are corroborated on the protein level and whether proteomics could yield unique insights into context-dependent biology. HCT116 colon carcinoma cells were cultured as monolayer cultures, proliferative multicellular tumor spheroids (P-MCTS), or quiescent (Q-MCTS) multicellular tumor spheroids and exposed to OXPHOS inhibitors: nitazoxanide, FCCP, oligomycin, and salinomycin or the HMG-CoA-reductase inhibitor simvastatin at two different doses for 6 and 24 h. Samples were processed using an in-depth bottom-up proteomics workflow resulting in a total of 9286 identified protein groups. Gene set enrichment analysis showed profound differences between the three cell systems and confirmed differential enrichment of hypoxia, OXPHOS, and cell cycle progression-related protein responses in P-MCTS and Q-MCTS. Treatment experiments showed that the observed drug-induced alterations in gene expression of metabolically challenged cells are not translated directly to the protein level, but the results reaffirmed OXPHOS as a selective vulnerability of quiescent cancer cells. This work provides rationale for the use of deep proteome profiling to identify context-dependent treatment responses and encourages further studies investigating metabolic processes that could be co-targeted together with OXPHOS to eradicate quiescent cancer cells.

Establishment of an in vitro 3D model for neuroblastoma enables preclinical investigation of combined tumor-stroma drug targeting.

The majority of anti-cancer therapies target the proliferating tumor cells, while the tumor stroma, principally unaffected, survives, and provide a niche for surviving tumor cells. Combining tumor cell and stroma-targeting therapies thus have a potential to improve patient outcome. The neuroblastoma stroma contains cancer-associated fibroblasts expressing microsomal prostaglandin E synthase-1 (mPGES-1). mPGES-1-derived prostaglandin E2 (PGE2 ) is known to promote tumor growth through increased proliferation and survival of tumor cells, immune suppression, angiogenesis, and therapy resistance, and we, therefore, hypothesize that mPGES-1 constitutes an interesting stromal target. Here, we aimed to develop a relevant in vitro model to study combination therapies. Co-culturing of neuroblastoma and fibroblast cells in 3D tumor spheroids mimic neuroblastoma tumors with regard to the cyclooxygenase/mPGES-1/PGE2 pathway. Using the spheroid model, we show that the inhibition of fibroblast-derived mPGES-1 enhanced the cytotoxic effect of doxorubicin and vincristine and significantly reduced tumor cell viability and spheroid growth. Cyclic treatment with vincristine in combination with an mPGES-1 inhibitor abrogated cell repopulation. Moreover, inhibition of mPGES-1 potentiated the cytotoxic effect of vincristine on established neuroblastoma allografts in mice. In conclusion, we established a 3D neuroblastoma model, highlighting the potential of combining stromal targeting of mPGES-1 with tumor cell targeting drugs like vincristine.

Nrf2 in early vascular ageing: Calcification, senescence and therapy.

Under normal physiological conditions, free radical generation and antioxidant defences are balanced, and reactive oxygen species (ROS) usually act as secondary messengers in a plethora of biological processes. However, when this balance is impaired, oxidative stress develops due to imbalanced redox homeostasis resulting in cellular damage. Oxidative stress is now recognized as a trigger of cellular senescence, which is associated with multiple chronic 'burden of lifestyle' diseases, including atherosclerosis, type-2 diabetes, chronic kidney disease and vascular calcification; all of which possess signs of early vascular ageing. Nuclear factor erythroid 2-related factor 2 (Nrf2), termed the master regulator of antioxidant responses, is a transcription factor found to be frequently dysregulated in conditions characterized by oxidative stress and inflammation. Recent evidence suggests that activation of Nrf2 may be beneficial in protecting against vascular senescence and calcification. Both natural and synthetic Nrf2 agonists have been introduced as promising drug classes in different phases of clinical trials. However, overexpression of the Nrf2 pathway has also been linked to tumorigenesis, which highlights the requirement for further understanding of pathways involving Nrf2 activity, especially in the context of cellular senescence and vascular calcification. Therefore, comprehensive translational pre-clinical and clinical studies addressing the targeting capabilities of Nrf2 agonists are urgently required. The present review discusses the impact of Nrf2 in senescence and calcification in early vascular ageing, with focus on the potential clinical implications of Nrf2 agonists and non-pharmacological Nrf2 therapeutics.

Biological characterization of new inhibitors of microsomal PGE synthase-1 in preclinical models of inflammation and vascular tone.

BACKGROUND AND PURPOSE: Microsomal PGE synthase-1 (mPGES-1), the inducible synthase that catalyses the terminal step in PGE2 biosynthesis, is of high interest as therapeutic target to treat inflammation. Inhibition of mPGES-1 is suggested to be safer than traditional NSAIDs, and recent data demonstrate anti-constrictive effects on vascular tone, indicating new therapeutic opportunities. However, there is a lack of potent mPGES-1 inhibitors lacking interspecies differences for conducting in vivo studies in relevant preclinical disease models. EXPERIMENTAL APPROACH: Potency was determined based on the reduction of PGE2 formation in recombinant enzyme assays, cellular assay, human whole blood assay, and air pouch mouse model. Anti-inflammatory properties were assessed by acute paw swelling in a paw oedema rat model. Effect on vascular tone was determined with human ex vivo wire myography. KEY RESULTS: We report five new mPGES-1 inhibitors (named 934, 117, 118, 322, and 323) that selectively inhibit recombinant human and rat mPGES-1 with IC50 values of 10-29 and 67-250 nM respectively. The compounds inhibited PGE2 production in a cellular assay (IC50 values 0.15-0.82 µM) and in a human whole blood assay (IC50 values 3.3-8.7 µM). Moreover, the compounds blocked PGE2 formation in an air pouch mouse model and reduced acute paw swelling in a paw oedema rat model. Human ex vivo wire myography analysis showed reduced adrenergic vasoconstriction after incubation with the compounds. CONCLUSION AND IMPLICATIONS: These mPGES-1 inhibitors can be used as refined tools in further investigations of the role of mPGES-1 in inflammation and microvascular disease.

Spatial clusters of life expectancy and association with cardiovascular disease mortality and cancer mortality in the contiguous United States: 1980-2014.

The average life expectancy varies greatly from county to county in USA and there are also spatial variations in the county mortality rates for cardiovascular disease (CVD) and cancer, the top two causes of death. An association between these two groups of diseases has not been identified by cluster analysis previously. The main objective in this study was to investigate and quantify the associations between mortality due to CVD, cancer mortality and life expectancy based on US county data between 1980 and 2014. Regression analysis was used to adjust life expectancy for the mortality due to CVD and that due to cancer. In addition to the spatial life expectancy trends, we also studied existing trends over time with the software JOINPOINT to see how life expectancy is influenced by changes in mortality due to CVD and cancer mortality. The study setting was the 48 contiguous US states, while participants were 3,100 counties and their populations of all ages during the period 1980-2014. The main outcomes are spatial clusters of unusually low or high levels of life expectancy in addition to identifying which county level life expectancy locations were significantly associated with mortality due to CVD and/or cancer. Life expectancy has been improving steadily from 1980 to 2014, but the rate of increase per year (indicated by variation of the trend slope) changed significantly at five joinpoints, the latest of which occurred in 2010 when the slope changed from 0.29 (1980-1982) to 0.03 (2010-2014). Our results reveal that there are significant, purely spatial clusters in some geographical areas where life expectancy rates are significantly higher (or lower) than in the rest of the contiguous US. It is also shown that there is a significant association between the life expectancy level and the corresponding CVD mortality, and there is also a significant association between life expectancy level and the corresponding overall cancer mortality. The general trends (regression slopes) over time for the USA in life expectancy mortality, CVD mortality and cancer mortality have changed significantly after 2009-2010.

Integration of magnetic resonance imaging and protein and metabolite CSF measurements to enable early diagnosis of secondary progressive multiple sclerosis.

Molecular networks in neurological diseases are complex. Despite this fact, contemporary biomarkers are in most cases interpreted in isolation, leading to a significant loss of information and power. We present an analytical approach to scrutinize and combine information from biomarkers originating from multiple sources with the aim of discovering a condensed set of biomarkers that in combination could distinguish the progressive degenerative phenotype of multiple sclerosis (SPMS) from the relapsing-remitting phenotype (RRMS). Methods: Clinical and magnetic resonance imaging (MRI) data were integrated with data from protein and metabolite measurements of cerebrospinal fluid, and a method was developed to sift through all the variables to establish a small set of highly informative measurements. This prospective study included 16 SPMS patients, 30 RRMS patients and 10 controls. Protein concentrations were quantitated with multiplexed fluorescent bead-based immunoassays and ELISA. The metabolome was recorded using liquid chromatography-mass spectrometry. Clinical follow-up data of the SPMS patients were used to assess disease progression and development of disability. Results: Eleven variables were in combination able to distinguish SPMS from RRMS patients with high confidence superior to any single measurement. The identified variables consisted of three MRI variables: the size of the spinal cord and the third ventricle and the total number of T1 hypointense lesions; six proteins: galectin-9, monocyte chemoattractant protein-1 (MCP-1), transforming growth factor alpha (TGF-α), tumor necrosis factor alpha (TNF-α), soluble CD40L (sCD40L) and platelet-derived growth factor AA (PDGF-AA); and two metabolites: 20ß-dihydrocortisol (20ß-DHF) and indolepyruvate. The proteins myelin basic protein (MBP) and macrophage-derived chemokine (MDC), as well as the metabolites 20ß-DHF and 5,6-dihydroxyprostaglandin F1a (5,6-DH-PGF1), were identified as potential biomarkers of disability progression. Conclusion: Our study demonstrates, in a limited but well-defined and data-rich cohort, the importance and value of combining multiple biomarkers to aid diagnostics and track disease progression.

Impact of amyloid ß aggregate maturation on antibody treatment in APP23 mice.

INTRODUCTION: The deposition of the amyloid ß protein (Aß) in the brain is a hallmark of Alzheimer's disease (AD). Removal of Aß by Aß-antibody treatment has been developed as a potential treatment strategy against AD. First clinical trials showed neither a stop nor a reduction of disease progression. Recently, we have shown that the formation of soluble and insoluble Aß aggregates in the human brain follows a hierarchical sequence of three biochemical maturation stages (B-Aß stages). To test the impact of the B-Aß stage on Aß immunotherapy, we treated transgenic mice expressing human amyloid precursor protein (APP) carrying the Swedish mutation (KM670/671NL; APP23) with the Aß-antibody ß1 or phosphate-buffered saline (PBS) beginning 1) at 3 months, before the onset of dendrite degeneration and plaque deposition, and 2) at 7 months, after the start of Aß plaque deposition and dendrite degeneration. RESULTS: At 5 months of age, first Aß aggregates in APP23 brain consisted of non-modified Aß (representing B-Aß stage 1) whereas mature Aß-aggregates containing N-terminal truncated, pyroglutamate-modified AßN3pE and phosphorylated Aß (representing B-Aß stage 3) were found at 11 months of age in both ß1- and PBS-treated animals. Protective effects on commissural neurons with highly ramified dendritic trees were observed only in 3-month-old ß1-treated animals sacrificed at 5 months. When treatment started at 7 months of age, no differences in the numbers of healthy commissural neurons were observed between ß1- and PBS-treated APP23 mice sacrificed with 11 months. CONCLUSIONS: Aß antibody treatment was capable of protecting neurons from dendritic degeneration as long as Aß aggregation was absent or represented B-Aß stage 1 but had no protective or curative effect in later stages with mature Aß aggregates (B-Aß stage 3). These data indicate that the maturation stage of Aß aggregates has impact on potential treatment effects in APP23 mice.