Disc Cells Research Articles

MicroRNA-16 inhibits the lipopolysaccharide-induced inflammatory response in nucleus pulposus cells of the intervertebral disc by targeting TAB3.

IntroductionA variety of inflammatory mediators are produced by the degenerative human intervertebral disc (IVD) tissues spontaneously, suggesting their role in the development of intervertebral disc degeneration (IDD). Our present study was designed to investigate the regulatory effect of microRNA-16 (miR-16) on the lipopolysaccharide (LPS)-induced inflammatory response in nucleus pulposus (NP) cells of the IVD.Material and methodsNP cells were treated with LPS to induce inflammatory responses. The expression of miRNA and genes was determined by qRT-PCR. Western blot and an ELISA kit were used to detect the proteins and protein expression, respectively. A dual luciferase reporter assay was applied to identify the correlation between a miRNA and a gene, and to test nuclear factor-κB (NF-κB) activity.ResultsThe results suggested that miR-16 positively regulated the mRNA and protein expression of extracellular matrix (ECM) genes (including aggrecan and collagen II) in NP cells, while it was negatively correlated with ECM degrading enzymes (including MMP3, MMP13, ADAMTS4, ADAMTS5) and related genes of nitric oxide (NO) reaction. Further studies revealed that miR-16 could oppositely regulate NF-κB and MAPK pathways by directly mediating their upstream gene TAB3.ConclusionsOur study suggested that, in NP cells of the IVD, miR-16 could exert inhibitory effects on the LPS-induced inflammatory response through NF-κB and MAPK pathways by directly mediating TAB3. In this way, miR-16 would play a protective role against LPS-induced IDD and inflammation. Therefore, miR-16 may be a novel therapeutic target for the inhibit of the ECM in the IVD.

Pro-Inflammatory and Neurotrophic Factor Responses of Cells Derived from Degenerative Human Intervertebral Discs to the Opportunistic Pathogen Cutibacterium acnes.

Previously, we proposed the hypothesis that similarities in the inflammatory response observed in acne vulgaris and degenerative disc disease (DDD), especially the central role of interleukin (IL)-1β, may be further evidence of the role of the anaerobic bacterium Cutibacterium (previously Propionibacterium) acnes in the underlying aetiology of disc degeneration. To investigate this, we examined the upregulation of IL-1β, and other known IL-1β-induced inflammatory markers and neurotrophic factors, from nucleus-pulposus-derived disc cells infected in vitro with C. acnes for up to 48 h. Upon infection, significant upregulation of IL-1β, alongside IL-6, IL-8, chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-C motif) ligand 4 (CCL4), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), was observed with cells isolated from the degenerative discs of eight patients versus non-infected controls. Expression levels did, however, depend on gene target, multiplicity and period of infection and, notably, donor response. Pre-treatment of cells with clindamycin prior to infection significantly reduced the production of pro-inflammatory mediators. This study confirms that C. acnes can stimulate the expression of IL-1β and other host molecules previously associated with pathological changes in disc tissue, including neo-innervation. While still controversial, the role of C. acnes in DDD remains biologically credible, and its ability to cause disease likely reflects a combination of factors, particularly individualised response to infection.

Spatiotemporal and functional characterisation of transient receptor potential vanilloid 4 (TRPV4) in the murine intervertebral disc.

The molecular regulators of mechano-transduction in intervertebral disc (IVD) cells are not well understood. The aim of the present study was to characterise the expression and function of the mechano-sensitive ion channel TRPV4 in the IVD. A novel transgenic reporter mouse, in which the endogenous Trpv4 locus drove the expression of LacZ, was used to localise Trpv4 expression at specific stages of spine development [embryonic day (E) 8.5, 12.5, 17.5, postnatal day 1] and time points following skeletal maturity (2.5, 6, 9 and 12 months). The TRPV4-specific agonist GSK1016790A and antagonist GSK2193874 were used to assess the functional response of annulus fibrosus (AF) cells using epifluorescence imaging with Ca2+-sensitive Fura-2 dye and F-actin staining. The effects of TRPV4 agonism and antagonism in mechanically stimulated AF cells were quantified by gene expression analysis. Trpv4 expression was specific to the developing notochord and intervertebral mesenchyme at E12.5. At 2.5, 6 and 9 months, Trpv4 expression was detected in the nucleus pulposus, inner AF, cartilage endplate and vertebral growth plate. AF cells treated with GSK1016790A demonstrated heterogeneity in TRPV4-dependent Ca2+ responses (no response, calcium oscillation or sustained response). TRPV4-induced Ca2+ signalling was associated with Rho/ROCK-dependent actin cytoskeleton remodelling and stress-fibre formation. In AF cells, cyclic-tensile-strain-induced changes in Acan and Prg4 expression were mediated by TRPV4 channel activation. These data establish TRPV4 as an important mechano- sensor regulating IVD mechano-biology.

Genetic Therapy for Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration can cause chronic lower back pain (LBP), leading to disability. Despite significant advances in the treatment of discogenic LBP, the limitations of current treatments have sparked interest in biological approaches, including growth factor and stem cell injection, as new treatment options for patients with chronic LBP due to IVD degeneration (IVDD). Gene therapy represents exciting new possibilities for IVDD treatment, but treatment is still in its infancy. Literature searches were conducted using PubMed and Google Scholar to provide an overview of the principles and current state of gene therapy for IVDD. Gene transfer to degenerated disc cells in vitro and in animal models is reviewed. In addition, this review describes the use of gene silencing by RNA interference (RNAi) and gene editing by the clustered regularly interspaced short palindromic repeats (CRISPR) system, as well as the mammalian target of rapamycin (mTOR) signaling in vitro and in animal models. Significant technological advances in recent years have opened the door to a new generation of intradiscal gene therapy for the treatment of chronic discogenic LBP.

Integrated transcriptome and proteome analyses identify novel regulatory network of nucleus pulposus cells in intervertebral disc degeneration

BackgroundDegeneration of intervertebral disc is a major cause of lower back pain and neck pain. Studies have tried to unveil the regulatory network using either transcriptomic or proteomic analysis. However, neither have fully elucidated the exact mechanism of degeneration process. Since post-transcriptional regulation may affect gene expression by modulating the translational process of mRNA to protein product, a combined transcriptomic and proteomic study may provide more insight into the key regulatory network of Intervertebral disc degeneration.MethodsIn order to obtain the proteomic and transcriptomic data, we performed label-free proteome analysis on freshly isolated nucleus pulposus cells and obtained transcriptome profiling data from the Gene Expression Omnibus repository. To identify the key regulatory network of intervertebral disc degeneration in nucleus pulposus cells, we performed bioinformatic analyses and established a protein-RNA interacting network. To validate the candidate genes, we performed in vitro experimentation and immunochemistry labeling to identify their potential function during nucleus pulposus degeneration.ResultsThe label-free proteome analysis identified altogether 656 proteins, and 503 of which were differentially expressed between nucleus pulposus cells from degenerated or normal disc cells. Using the existing nucleus pulposus transcriptomic profiling data, we integrated the proteomic and transcriptomic data of nucleus pulposus cells, and established a protein-RNA interacting network to show the combined regulatory network of intervertebral disc degeneration. In the network, we found 9 genes showed significant changes, and 6 of which (CHI3L1, KRT19, COL6A2, DPT, TNFAIP6 and COL11A2) showed concordant changes in both protein and mRNA level. Further functional analysis showed these candidates can significantly affect the degeneration of the nucleus pulposus cell when altering their expression.ConclusionsThis study is the first to use combined analysis of proteomic and transcriptomic profiling data to identify novel regulatory network of nucleus pulposus cells in intervertebral disc degeneration. Our established protein-RNA interacting network demonstrated novel regulatory mechanisms and key genes that may play vital roles in the pathogenesis of intervertebral disc degeneration.

ISSLS prize in basic science 2021: a novel inducible system to regulate transgene expression of TIMP1

PurposeInflammatory and oxidative stress upregulates matrix metalloproteinase (MMP) activity, leading to intervertebral disc degeneration (IDD). Gene therapy using human tissue inhibitor of metalloproteinase 1 (hTIMP1) has effectively treated IDD in animal models. However, persistent unregulated transgene expression may have negative side effects. We developed a recombinant adeno-associated viral (AAV) gene vector, AAV-NFκB-hTIMP1, that only expresses the hTIMP1 transgene under conditions of stress.MethodsRabbit disc cells were transfected or transduced with AAV-CMV-hTIMP1, which constitutively expresses hTIMP1, or AAV-NFκB-hTIMP1. Disc cells were selectively treated with IL-1β. NFκB activation was verified by nuclear translocation. hTIMP1 mRNA and protein expression were measured by RT-PCR and ELISA, respectively. MMP activity was measured by following cleavage of a fluorogenic substrate.ResultsIL-1β stimulation activated NFκB demonstrating that IL-1β was a surrogate for inflammatory stress. Stimulating AAV-NFκB-hTIMP1 cells with IL-1β increased hTIMP1 expression compared to unstimulated cells. AAV-CMV-hTIMP1 cells demonstrated high levels of hTIMP1 expression regardless of IL-1β stimulation. hTIMP1 expression was comparable between IL-1β stimulated AAV-NFκB-hTIMP1 cells and AAV-CMV-hTIMP1 cells. MMP activity was decreased in AAV-NFκB-hTIMP1 cells compared to baseline levels or cells exposed to IL-1β.ConclusionAAV-NFκB-hTIMP1 is a novel inducible transgene delivery system. NFκB regulatory elements ensure that hTIMP1 expression occurs only with inflammation, which is central to IDD development. Unlike previous inducible systems, the AAV-NFκB-hTIMP1 construct is dependent on endogenous factors, which minimizes potential side effects caused by constitutive transgene overexpression. It also prevents the unnecessary production of transgene products in cells that do not require therapy.

Uncovering the secretome of mesenchymal stromal cells exposed to healthy, traumatic, and degenerative intervertebral discs: a proteomic analysis

BackgroundMesenchymal stromal cells (MSCs) have been introduced as promising cell source for regenerative medicine. Besides their multilineage differentiation capacity, MSCs release a wide spectrum of bioactive factors. This secretome holds immunomodulatory and regenerative capacities. In intervertebral disc (IVD) cells, application of MSC secretome has been shown to decrease the apoptosis rate, induce proliferation, and promote production of extracellular matrix (ECM). For clinical translation of secretome-based treatment, characterization of the secretome composition is needed to better understand the induced biological processes and identify potentially effective secretomes.MethodsThis study aimed to investigate the proteome released by bone marrow-derived MSCs following exposure to a healthy, traumatic, or degenerative human IVD environment by mass spectroscopy and quantitative immunoassay analyses. Exposure of MSCs to the proinflammatory stimulus interleukin 1β (IL-1β) was used as control.ResultsCompared to MSC baseline secretome, there were 224 significantly up- or downregulated proteins following healthy, 179 following traumatic, 223 following degenerative IVD, and 160 proteins following IL-1β stimulus. Stimulation of MSCs with IVD conditioned media induced a more complex MSC secretome, involving more biological processes, compared to stimulation with IL-1β. The MSC response to stimulation with IVD conditioned medium was dependent on their pathological status.ConclusionsThe MSC secretome seemed to match the primary need of the IVD: homeostasis maintenance in the case of healthy IVDs, versus immunomodulation, adjustment of ECM synthesis and degradation disbalance, and ECM (re) organization in the case of traumatic and degenerative IVDs. These findings highlight the importance of cell preconditioning in the development of tailored secretome therapies.Graphical abstractThe secretome of human bone marrow-derived mesenchymal stromal cells (MSCs) stimulated with intervertebral disc (IVD) conditioned medium was analyzed by proteomic profiling. Depending on the pathological state of the IVD, the MSC secretome protein composition indicated immunomodulatory or anabolic activity of the secretome. These findings may have implications for tailored secretome therapy for the IVD and other tissues.

Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative disc cells following hyperbaric oxygen treatment

BackgroundMicroRNA (miRNA) plays a vital role in the intervertebral disc (IVD) degeneration. The expression level of miR-573 was downregulated whereas Bax was upregulated notably in human degenerative nucleus pulposus cells. In this study, we aimed to investigate the role of miR-573 in human degenerative nucleus pulposus (NP) cells following hyperbaric oxygen (HBO) treatment.MethodsNP cells were separated from human degenerated IVD tissues. The control cells were maintained in 5% CO2/95% air and the hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The mRNA and protein levels of Bax were measured. The proliferation of NPCs was detected using MTT assay. The protein expression levels of Bax, cleaved caspase 9, cleaved caspase 3, pro-caspase 9, and pro-caspase 3 were examined.ResultsBioinformatics analysis indicated that the 3′ untranslated region (UTR) of the Bax mRNA contained the “seed-matched-sequence” for hsa-miR-573, which was validated via reporter assays. MiR-573 was induced by HBO and simultaneous suppression of Bax was observed in NP cells. Knockdown of miR-573 resulted in upregulation of Bax expression in HBO-treated cells. In addition, overexpression of miR-573 by HBO increased cell proliferation and coupled with inhibition of cell apoptosis. The cleavage of pro-caspase 9 and pro-caspase 3 was suppressed while the levels of cleaved caspase 9 and caspase 3 were decreased in HBO-treated cells. Transfection with anti-miR-573 partly suppressed the effects of HBO.ConclusionMir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative NP cells following HBO treatment.

Studies of Myc super-competition and clonal growth in Drosophila males and females.

Cell competition is a cell selection process that arises in growing tissues as a result of interactions between cells of different fitness. This behavior is also observed in Myc super-competition, where healthy wild type cells in growing wing discs of Drosophila are outcompeted by nearby cells that express higher levels of the Myc oncogene. Most work on Myc super-competition has examined it in mixed populations of male and female larvae. However, as physiological and genetic differences between Drosophila males and females could affect the competitive behavior of cells, we have investigated whether sex differences affect the process. Here we show that both male and female wing disc cells are subject to Myc super-competition. Female disc cells appear to be more sensitive to competitive elimination than male cells, potentially due to differences in baseline cellular Myc levels between the sexes. We also report sexual dimorphism of cell size and number between male and female growing wing discs that is independent of competition; wing discs and wing pouches from females are larger than males’ due to larger cell size and cell number. We suggest that separately examining male and female tissues in cell competition assays could enhance our understanding of the effects of sex-specific pathways on cell and super-competition.

Small molecule-based treatment approaches for intervertebral disc degeneration: Current options and future directions.

Low back pain (LBP) is a major reason for disability, and symptomatic intervertebral disc (IVD) degeneration (IDD) contributes to roughly 40% of all LBP cases. Current treatment modalities for IDD include conservative and surgical strategies. Unfortunately, there is a significant number of patients in which conventional therapies fail with the result that these patients remain suffering from chronic pain and disability. Furthermore, none of the current therapies successfully address the underlying biological problem - the symptomatic degenerated disc. Both spinal fusion as well as total disc replacement devices reduce spinal motion and are associated with adjacent segment disease. Thus, there is an unmet need for novel and stage-adjusted therapies to combat IDD. Several new treatment options aiming to regenerate the IVD are currently under investigation. The most common approaches include tissue engineering, growth factor therapy, gene therapy, and cell-based treatments according to the stage of degeneration. Recently, the regenerative activity of small molecules (low molecular weight organic compounds with less than 900 daltons) on IDD was demonstrated. However, small molecule-based therapy in IDD is still in its infancy due to limited knowledge about the mechanisms that control different cell signaling pathways of IVD homeostasis. Small molecules can act as anti-inflammatory, anti-apoptotic, anti-oxidative, and anabolic agents, which can prevent further degeneration of disc cells and enhance their regeneration. This review pursues to give a comprehensive overview of small molecules, focusing on low molecular weight organic compounds, and their potential utilization in patients with IDD based on recent in vitro, in vivo, and pre-clinical studies.

Pro-inflammatory and neurotrophic-factor response of degenerative human intervertebral disc cells to the opportunistic pathogen Cutibacterium acnes

Pro-inflammatory and neurotrophic-factor response of degenerative human intervertebral disc cells to the opportunistic pathogen Cutibacterium acnes

Crosstalk between Autophagy and Apoptosis in Intervertebral Disc Degeneration

Objective: To describe the relationship between autophagy and apoptosis and the possible signaling pathways involved in degenerative lumbar intervertebral disc. Summary of Background Data: Autophagy and apoptosis are regulatory cellular mechanisms that determine many pathologies, including degenerative intervertebral disc disease. The interactions between these events in the damage or protection of intervertebral disc cells and in cellular homeostasis remain controversial. Methods: The sample size was twenty patients who underwent lumbar spine surgery for symptomatic disc herniation or spondylolisthesis. Intervertebral discs were classified by magnetic resonance as Pfirrmann grade IV and grade V. Six patients were operated on two levels, resulting in twenty-six intervertebral discs that were submitted to immunohistochemistry to verify the protein expression of autophagy and apoptosis markers. Results: The autophagic markers had greater protein expression in the human intervertebral disc (Pfirrmann Grades IV and V). Under these conditions, autophagy and apoptosis showed a negative correlation. Regarding apoptosis, caspase 8 presented the highest protein expression, which allows inferring the preference for the extrinsic pathway in cell death. Conclusions: Autophagy had the greatest protein expression negative profile compared to apoptosis. Caspase 8 had the highest protein expression in apoptosis.

AAV6 as an effective gene delivery vector for prolonged transgene expression in intervertebral disc cells in vivo

Intervertebral disc degeneration is the main contributor to low back pain, now the leading cause of disability worldwide. Gene transfer, either in a therapeutic attempt or in basic research to understand the mechanisms of disc degeneration, is a fascinating and promising tool to manipulate the complex physiology of the disc. Viral vectors based on the adeno-associated virus (AAV) have emerged as powerful transgene delivery vehicles yet a systematic investigation into their respective tropism, transduction efficiency, and relative toxicity have not yet been performed in the disc in vivo. Herein, we used in vivo bioluminescence imaging to systematically compare multiple AAV serotypes, injection volumes, titers, promoters, and luciferase reporters to determine which result in high transduction efficiency of murine nucleus pulposus (NP) cells in vivo. We find that AAV6 using a CAG promoter to drive transgene expression, delivered into the NP of murine caudal discs at a titer of 1011 GC/mL, provides excellent transduction efficiency/kinetics and low toxicity in vivo. We also show, for the first time, that the transduction of NP cells can be significantly boosted in vivo by the use of small cell permeabilization peptides. Finally, to our knowledge, we are the first to demonstrate the use of optical tissue clearing and three-dimensional lightsheet microscopy in the disc, which was used to visualize fine details of tissue and cell architecture in whole intact discs following AAV6 delivery. Taken together, these data will contribute to the success of using AAV-mediated gene delivery for basic and translational studies of the IVD.

Novel biomarkers of intervertebral disc cells and evidence of stem cells in the intervertebral disc

Rat intervertebral disc (IVD) is one of the most commonly used and cost-effective alternative models for human IVD. Many IVD related clinical studies need to be pre-tested on rat IVDs. However, studies on the heterogeneous cell clusters of the rat IVD are inadequate, and a further understanding of the marker genes and cell phenotypes of healthy mature IVD cells is essential. In this study, we used the 10X Genomics technology to analyze the single-cell transcriptome of purified wild-type rat IVDs. We identified potentially new gene markers of IVDs via single-cell sequencing. Based on the unsupervised cluster analysis of 13,578 single-cell transcripts, 3 known IVD cell types were identified. We provided a complete single-cell gene expression map of the IVD. Immunohistochemical and immunofluorescence images of rat disc sections confirmed the new marker genes of all cell types. One group of heterologous cell groups expressed multi-functional stem cell (MSC)-specific genes, indicating the stem cell potential of IVD cells. We provided the phenotype and marker genes of IVD cells at the single-cell level, reconfirmed existing data, and proposed new marker genes, including MSC marker genes. By identifying more accurate target cells and genes, our results pave the way for further study of the response of individual disc cells to disease states and provide the basis for future disc regeneration therapies.

Simulating intervertebral disc cell behaviour within 3D multifactorial environments.

Low back pain is responsible for more global disability than any other condition. Its incidence is closely related to intervertebral disc (IVD) failure, which is likely caused by an accumulation of microtrauma within the IVD. Crucial factors in microtrauma development are not entirely known yet, probably because their exploration in vivo or in vitro remains tremendously challenging. In silico modelling is, therefore, definitively appealing, and shall include approaches to integrate influences of multiple cell stimuli at the microscale. Accordingly, this study introduces a hybrid Agent-based (AB) model in IVD research and exploits network modelling solutions in systems biology to mimic the cellular behaviour of Nucleus Pulposus cells exposed to a 3D multifactorial biochemical environment, based on mathematical integrations of existing experimental knowledge. Cellular activity reflected by mRNA expression of Aggrecan, Collagen type I, Collagen type II, MMP-3 and ADAMTS were calculated for inflamed and non-inflamed cells. mRNA expression over long periods of time is additionally determined including cell viability estimations. Model predictions were eventually validated with independent experimental data. As it combines experimental data to simulate cell behaviour exposed to a multifactorial environment, the present methodology was able to reproduce cell death within 3 days under glucose deprivation and a 50% decrease in cell viability after 7 days in an acidic environment. Cellular mRNA expression under non-inflamed conditions simulated a quantifiable catabolic shift under an adverse cell environment, and model predictions of mRNA expression of inflamed cells provide new explanation possibilities for unexpected results achieved in experimental research. The AB model as well as used mathematical functions were built with open source software. Final functions implemented in the AB model and complete AB model parameters are provided as Supplementary Material. Experimental input and validation data were provided through referenced, published papers. The code corresponding to the model can be shared upon request and shall be reused after proper training. Supplementary data are available at Bioinformatics online.

Investigation of fluid flow velocity within the intervertebral disc

Enhancing fluid flow velocity within the intervertebral disc may allow to increase solute transportation rates and improve disc nutrition as the sufficient supply of the nutrients to cells of intervertebral disc is a key factor in order to avoid or delay processes of disc degeneration. Poroelastic finite element model of lumbar intervertebral disc is used to calculate fluid flow velocity with the intervertebral disc due to flexion, extension, lateral bending and axial rotation. When comparing the average of flow velocity, the highest values are calculated when flexion and extension moments were applied to the disc, but lateral bending induces the highest value of velocity component in Y direction in nucleus pulposus, yet the assumption that lateral bending could be more beneficial in order to improve nutrients supply to intervertebral disc cannot be substantiated only by this data, as calculated values of velocity component Y are significantly lower that total velocity values.

Efficacy of hyaluronic acid on intervertebral disc inflammation: An in vitro study using notochordal cell lines and human disc cells.

Hyaluronic acid (HA) is widely recognized as a therapeutic target and currently used in medicine. However, HA metabolism during intervertebral disc degeneration (IVDD) has not been completely elucidated. This study aimed to evaluate the efficacy of HA on intervertebral disc (IVD) inflammation and identify the main molecules modulating HA degradation in IVDs. To assess HA function in IVD cells in vitro, we treated human disc cells and U-CH1-N cells, a notochordal nucleus pulposus cell line, with HA or hyaluronidase. Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis showed that tumor necrosis factor alpha (TNF-α)-mediated induction of the expression of TNF-α and cyclooxygenase-2 (COX2) was clearly neutralized by HA treatment, andthe expression of TNF-α and COX2 was significantly induced by hyaluronidase treatment in both cell types. Additionally, Western blot analysis showed that hyaluronidase-induced phosphorylation of p38 and Erk1/2, and that TNF-α-mediated phosphorylation of p38 and Erk1/2 was clearly reduced by HA addition. In degenerating human IVD samples, immunohistochemistry for hyaluronidase showed that the expression of hyaluronidases including HYAL1, HYAL2, and cell migration-inducing protein (CEMIP) tended to increase in accordance with IVDD. In particular, HYAL1 showed statistically significant differences. In vitro study also confirmed asimilar phenomenon that TNF-α treatment increased both messenger RNA and protein expression in both cell types. Our results demonstrated that HA could potentially suppress IVDD by regulating p38 and Erk1/2 pathways, and that the expression of HYAL1 was correlated with IVDD progression. These findings indicated that HYAL1 would be a potential molecular target for suppressing IVDD by controlling HA metabolism.

Increased lateral tension is sufficient for epithelial folding in Drosophila.

The folding of epithelial sheets is important for tissues, organs and embryos to attain their proper shapes. Epithelial folding requires subcellular modulations of mechanical forces in cells. Fold formation has mainly been attributed to mechanical force generation at apical cell sides, but several studies indicate a role of mechanical tension at lateral cell sides in this process. However, whether lateral tension increase is sufficient to drive epithelial folding remains unclear. Here, we have used optogenetics to locally increase mechanical force generation at apical, lateral or basal sides of epithelial Drosophila wing disc cells, an important model for studying morphogenesis. We show that optogenetic recruitment of RhoGEF2 to apical, lateral or basal cell sides leads to local accumulation of F-actin and increase in mechanical tension. Increased lateral tension, but not increased apical or basal tension, results in sizeable fold formation. Our results stress the diversification of folding mechanisms between different tissues and highlight the importance of lateral tension increase for epithelial folding.

In vitro model of distinct catabolic and inflammatory response patterns of endothelial cells to intervertebral disc cell degeneration

To evaluate dominant cell-to-cell paracrine interactions, including those of human annulus fibrosus (AF), nucleus pulposus (NP), and endothelial cells (ECs), in the production of inflammatory mediators and catabolic enzymes, ECs was cultured in soluble factors derived from AF or NP cells (AFCM or NPCM, respectively) and vice versa. We analysed IL-6 and -8, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and -3, nerve growth factor (NGF)-β, and brain-derived neurotrophic factors (BDNFs) with qRT-PCR and ELISA. We implement a microfluidic platform to analyse migration properties of AF and NP cells and ECs in 3D cultures. Our results show that IL-1β-stimulated AF cells produced significantly higher levels of IL-6 and -8, VEGF, and MMP-1 than IL-1β-stimulated NP cells. However, production of IL-6 and -8, VEGF, and MMP-3 was significantly higher in NP cells than in AF cells, under the presence of ECs conditioned medium. We observed considerable migration of NP cells co-cultured with ECs through the microfluidic platform. These results suggest that AF cells may play a major role in the initial degeneration of intervertebral disc. Furthermore, it was found that interactions between NP cells and ECs may play a significant role in the development or progression of diseases.

Placenta Creta: A Spectrum of Lesions Associated with Shallow Placental Implantation.

Background On placental histology, placenta creta (PC) ranges from clinical placenta percreta through placenta increta and accreta (clinical and occult) to myometrial fibers with intervening decidua. This retrospective study aimed to investigate the clinicopathologic correlations of these lesions. Methods A total of 169 recent consecutive cases with PC (group 1) were compared with 1661 cases without PC examined during the same period (group 2). The frequencies of 25 independent clinical and 40 placental phenotypes were statistically compared between the groups using chi-square test or analysis of variance where appropriate. Results Group 1 placentas, as compared with group 2 placentas, were statistically significantly (p < 0.05) associated with caesarean sections (11.2% vs. 7.5%), antepartum hemorrhage (17.7% vs 11.6.%), gestational hypertension (11.2% vs 4.3%), preeclampsia (11.8% vs 2.6%), complicated third stage of labor (18.9% vs 6.4%), villous infarction (14.2% vs 8.9%), chronic hypoxic patterns of placental injury, particularly the uterine pattern (14.8%, vs 9.6%), massive perivillous fibrin deposition (9.5% vs 5.3%), chorionic disc chorionic microcysts (21.9% vs 15.9%), clusters of maternal floor multinucleate trophoblasts (27.8% vs 21.2%), excessive trophoblasts of chorionic disc (24.3% vs 17.3%), segmental fetal vascular malperfusion (27.8% vs 19.9%), and fetal vascular ectasia (26.2% vs 15.2%). Conclusion Because of the association of PC with gestational hypertensive diseases, acute and chronic placental hypoxic lesions, increased extravillous trophoblasts in the chorionic disc, chorionic microcysts, and maternal floor trophoblastic giant cells, PC should be regarded as a lesion of abnormal placental implantation and abnormal trophoblast invasion rather than decidual deficiency only.