ABSTRACT
Post-mitotic, non-proliferative dermal fibroblasts have crucial functions in maintenance and restoration of tissue homeostasis. They are involved in essential processes such as wound healing, pigmentation and hair growth, but also tumor development and aging-associated diseases. These processes are energetically highly demanding and error prone when mitochondrial damage occurs. However, mitochondrial function in fibroblasts and the influence of mitochondrial dysfunction on fibroblast-specific demands are still unclear. To address these questions, we created a mouse model in which accelerated cell-specific mitochondrial DNA (mtDNA) damage accumulates. We crossed mice carrying a dominant-negative mutant of the mitochondrial replicative helicase Twinkle (RosaSTOP system) with mice that express fibroblast-specific Cre Recombinase (Collagen1A2 CreERT) which can be activated by Tamoxifen (TwinkleFIBRO). Thus, we are able to induce mtDNA deletions and duplications in specific cells, a process which resembles the physiological aging process in humans, where this damage accumulates in all tissues. Upon proliferation in vitro, Tamoxifen induced Twinkle fibroblasts deplete most of their mitochondrial DNA which, although not disturbing the stoichiometry of the respiratory chain complexes, leads to reduced ROS production and mitochondrial membrane potential as well as an anti-inflammatory and anti-fibrotic profile of the cells. In Sodium Azide treated wildtype fibroblasts, without a functioning respiratory chain, we observe the opposite, a rather pro-inflammatory and pro-fibrotic signature. Upon accumulation of mitochondrial DNA mutations in vivo the TwinkleFIBRO mice are protected from fibrosis development induced by intradermal Bleomycin injections. This is due to dampened differentiation of the dermal fibroblasts into α-smooth-muscle-actin positive myofibroblasts in TwinkleFIBRO mice. We thus provide evidence for striking differences of the impact that mtDNA mutations have in contrast to blunted mitochondrial function in dermal fibroblasts and skin homeostasis. These data contribute to improved understanding of mitochondrial function and dysfunction in skin and provide mechanistic insight into potential targets to treat skin fibrosis in the future.
Subject(s)
Bleomycin , Cell Differentiation , DNA, Mitochondrial , Fibrosis , Mutation , Myofibroblasts , Animals , Bleomycin/adverse effects , Bleomycin/toxicity , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Mice , Myofibroblasts/metabolism , Myofibroblasts/pathology , Myofibroblasts/drug effects , Cell Differentiation/drug effects , DNA Helicases/genetics , DNA Helicases/metabolism , Fibroblasts/metabolism , Fibroblasts/drug effects , Fibroblasts/pathology , Tamoxifen/pharmacology , Mitochondria/metabolism , Mitochondria/drug effects , Mitochondria/pathology , Disease Models, Animal , Reactive Oxygen Species/metabolism , Humans , Skin/pathology , Skin/metabolism , Skin/drug effects , Membrane Potential, Mitochondrial/drug effects , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Collagen Type IABSTRACT
Morphological studies of skeletal muscle tissue provide insights into the architecture of muscle fibers, the surrounding cells, and the extracellular matrix (ECM). However, a spatial proteomics analysis of the skeletal muscle including the muscle-tendon transition zone is lacking. Here, we prepare cryotome muscle sections of the mouse soleus muscle and measure each slice using short liquid chromatography-mass spectrometry (LC-MS) gradients. We generate 3,000 high-resolution protein profiles that serve as the basis for a network analysis to reveal the complex architecture of the muscle-tendon junction. Among the protein profiles that increase from muscle to tendon, we find proteins related to neuronal activity, fatty acid biosynthesis, and the renin-angiotensin system (RAS). Blocking the RAS in cultured mouse tenocytes using losartan reduces the ECM synthesis. Overall, our analysis of thin cryotome sections provides a spatial proteome of skeletal muscle and reveals that the RAS acts as an additional regulator of the matrix within muscle-tendon junctions.
Subject(s)
Muscle, Skeletal , Proteomics , Tendons , Animals , Proteomics/methods , Muscle, Skeletal/metabolism , Tendons/metabolism , Mice , Extracellular Matrix/metabolism , Male , Mice, Inbred C57BL , Renin-Angiotensin System/physiology , Adaptation, Physiological , Proteome/metabolism , Losartan/pharmacologyABSTRACT
T cells protect tissues from cancer. Although investigations in mice showed that amino acids (AA) critically regulate T cell immunity, this remains poorly understood in humans. Here, we describe the AA composition of interstitial fluids in keratinocyte-derived skin cancers (KDSCs) and study the effect of AA on T cells using models of primary human cells and tissues. Gln contributed to â¼15% of interstitial AAs and promoted interferon gamma (IFN-γ), but not granzyme B (GzB) expression, in CD8+ T cells. Furthermore, the Toll-like receptor 7 agonist imiquimod (IMQ), a common treatment for KDSCs, down-regulated the metabolic gatekeepers c-MYC and mTORC1, as well as the AA transporter ASCT2 and intracellular Gln, Asn, Ala, and Asp in T cells. Reduced proliferation and IFN-γ expression, yet increased GzB, paralleled IMQ effects on AA. Finally, Gln was sufficient to promote IFN-γ-production in IMQ-treated T cells. Our findings indicate that Gln metabolism can be harnessed for treating KDSCs.
ABSTRACT
Degeneration of dopaminergic neurons in the substantia nigra and their striatal axon terminals causes cardinal motor symptoms of Parkinson's disease. In idiopathic cases, high levels of mitochondrial DNA alterations leading to mitochondrial dysfunction are a central feature of these vulnerable neurons. Here we present a mouse model expressing the K320E-variant of the mitochondrial helicase Twinkle in dopaminergic neurons, leading to accelerated mitochondrial DNA mutations. These K320E-TwinkleDaN mice showed normal motor function at 20 months of age, although â¼70% of nigral dopaminergic neurons had perished. Remaining neurons still preserved â¼75% of axon terminals in the dorsal striatum and enabled normal dopamine release. Transcriptome analysis and viral tracing confirmed compensatory axonal sprouting of the surviving neurons. We conclude that a small population of substantia nigra dopaminergic neurons is able to adapt to the accumulation of mitochondrial DNA mutations and maintain motor control.
ABSTRACT
Aiming to address the bone regeneration and cancer therapy functionalities in one single material, in this study, we developed a dual-functional theragenerative three-dimensional (3D) aerogel-based composite scaffold from hybridization of photo-cross-linked silk fibroin (SF) biopolymer with MXene (Ti3C2) two-dimensional (2D) nanosheets. To fabricate the scaffold, we first develop a dual-cross-linked SF-based aerogel scaffold through 3D printing and photo-cross-linking of the self-assembly-driven methacrylate-modified SF (SF-MA) gel with controlled pore size, macroscopic geometry, and mechanical stability. In the next step, to endow a remotely controlled photothermal antiosteosarcoma ablation function to fabricated aerogel scaffold, MXene 2D nanosheets with strong near-infrared (NIR) photon absorption properties were integrated into the 3D-printed scaffolds. While 3D-printed MXene-modified dual-cross-linked SF composite scaffolds can mediate the in vitro growth and proliferation of preosteoblastic cell lines, they also endow a strong photothermal effect upon remote irradiation with NIR laser but also significantly stimulate bone mineral deposition on the scaffold surface. Additionally, besides the local release of the anticancer model drug, the generated heat (45-53 °C) mediated the photothermal ablation of cancer cells. The developed aerogel-based composites and chosen therapeutic techniques are thought to render a significant breakthrough in biomaterials' future clinical applications.
ABSTRACT
The novel coronavirus pandemic, first reported in December 2019, was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In response to viral infections, monocytes are recruited into the lung and subsequently differentiate into dendritic cells (DCs). DCs are critical players in the development of the acute lung inflammation that causes ARDS. Here we focus on the interaction of a specific SARS-CoV-2 open reading frame protein, ORF8, with DCs. We show that ORF8 binds to DCs, causes a pre-maturation of differentiating DCs, and induces the secretion of multiple proinflammatory cytokines by these cells. In addition, we identified DC-SIGN as a possible interaction partner of ORF8 on DCs. Blockade of ORF8 leads to reduced production of IL-1ß, IL-6, IL-12p70, TNF-α, MCP-1 (also named CCL2), and IL-10 by DCs. Therefore, a neutralizing antibody blocking the ORF8-mediated cytokine and chemokine response could be an improved therapeutical strategy against severe SARS-CoV-2.
ABSTRACT
Collagen XII, belonging to the fibril-associated collagens, is a homotrimeric secreted extracellular matrix (ECM) protein encoded by the COL12A1 gene. Mutations in the human COL12A1 gene cause an Ehlers-Danlos/myopathy overlap syndrome leading to skeletal abnormalities and muscle weakness. Here, we studied the role of collagen XII in joint pathophysiology by analyzing collagen XII deficient mice and human patients. We found that collagen XII is widely expressed across multiple connective tissue of the developing joint. Lack of collagen XII in mice destabilizes tendons and the femoral trochlear groove to induce patellar subluxation in the patellofemoral joint. These changes are associated with an ECM damage response in tendon and secondary quadriceps muscle degeneration. Moreover, patellar subluxation was also identified as a clinical feature of human patients with collagen XII deficiency. The results provide an explanation for joint hyperlaxity in mice and human patients with collagen XII deficiency.
ABSTRACT
BACKGROUND: The olfactory bulb has a key role for nasal delivery of drugs to the brain by its access from the nasal mucosa and its connection to the subventricular zone. The aim of this study was to investigate the neuromodulatory capacity of human milk of premature infants on the olfactory bulb. METHODS: Olfactory bulbs from P1 mice were embedded in a collagen I gel and incubated with DMEM supplemented with the aqueous phase of human colostrum (Col) of five mothers after very preterm birth, mature milk (Mat) of the same mothers or without supplement (Ctrl). After 7 days, the neurite outgrowth was quantified. Proteome analysis of the milk samples was performed using unlabeled mass spectrometry. RESULTS: Outgrowth increased significantly in bulbs exposed to Col but not when exposed to Mat. Mass spectrometry revealed profound differences in the proteome of Col versus Mat. Among 21 upregulated proteins in Col were proteins involved in neurite outgrowth, axon guidance, neuromodulation and longevity. CONCLUSIONS: A high bioactivity of human preterm colostrum on murine neonatal neurogenic tissue is demonstrated to be associated with a proteome profoundly differing from mature milk. IMPACT: The hypothesis has been raised that neonatal brain damage in a preterm infant could potentially be ameliorated by intranasal application of maternal breast milk. In an in-vitro model using neonatal murine olfactory bulb explants a significant stimulatory effect by human preterm colostrum is observed. Proteomics reveals upregulated neuroactive proteins in human colostrum compared to mature milk. A confirmation of this exploratory study would indicate that preterm colostrum stimulates neurogenic tissue. Early intranasal colostrum application might attenuate perinatal loss of neurogenic tissue thereby contributing to reducing complications such as cerebral palsy.
Subject(s)
Colostrum , Premature Birth , Infant , Pregnancy , Female , Humans , Infant, Newborn , Animals , Mice , Colostrum/chemistry , Infant, Premature , Premature Birth/metabolism , Proteome , Milk, Human/chemistryABSTRACT
MicroRNAs (miRNAs) post-transcriptionally regulate cartilage and bone development and function, however, only few miRNAs have been described to play a role for cartilage to bone transition in vivo. Previously, we showed that cartilage-specific deletion of the Mirc24 cluster in newborn male mice leads to impaired growth plate cartilage development due to increased RAF/MEK/ERK signaling and affects the stability of the cartilage extracellular matrix on account of decreased SOX6 and SOX9 and increased MMP13 levels. Here, we studied how Mirc24 cluster inactivation in cartilage and osteoblasts leads to an increased bone density associated with defects in collagen remodeling in trabecular bone. No changes in osteoblast distribution were observed, whereas the number of osteoclasts was reduced and TRAP activity in osteoclasts decreased. Surprisingly, an increased level of cluster-encoded miR-322 or miR-503 raises Rankl gene expression and inactivation of the cluster in chondrocytes reduces Rankl expression. These results suggest that the Mirc24 cluster regulates Rankl expression in chondrocytes at the chondro-osseous border, where the cluster is mainly expressed to modulate osteoclast formation, bone remodeling and bone integrity.
Subject(s)
MicroRNAs , Animals , Bone Remodeling/genetics , Cartilage/metabolism , Male , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Osteoblasts , Osteoclasts/metabolismABSTRACT
LTBP1 is a large extracellular matrix protein and an associated ligand of fibrillin-microfibrils. Knowledge of LTBP1 functions is largely limited to its role in targeting and sequestering TGFß growth factors within the extracellular matrix, thereby regulating their bioavailability. However, the recent description of a wide spectrum of phenotypes in multiple tissues in patients harboring LTBP1 pathogenic variants suggests a multifaceted role of the protein in the homeostasis of connective tissues. To better understand the human pathology caused by LTBP1 deficiency it is important to investigate its functional role in extracellular matrix formation. In this study, we show that LTBP1 coordinates the incorporation of fibrillin-1 and -2 into the extracellular matrix in vitro. We also demonstrate that this function is differentially exerted by the two isoforms, the short and long forms of LTBP1. Thereby our findings uncover a novel TGFß-independent LTBP1 function potentially contributing to the development of connective tissue disorders.
Subject(s)
Extracellular Matrix , Latent TGF-beta Binding Proteins , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/metabolism , Fibrillin-1/genetics , Fibrillin-1/metabolism , Fibrillin-2/genetics , Fibrillin-2/metabolism , Fibrillins/metabolism , Humans , Latent TGF-beta Binding Proteins/genetics , Latent TGF-beta Binding Proteins/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolismABSTRACT
Energy metabolism and extracellular matrix (ECM) function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single-cell RNA-Seq (scRNA-Seq) analysis to uncover the importance of the mitochondrial respiratory chain for ECM homeostasis in mature cartilage. This tissue produces large amounts of a specialized ECM to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of ECM material. scRNA-Seq analysis identified a cell cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of ECM-related genes in nonarticular chondrocytes. These changes were associated with alterations in ECM composition, a shift in collagen/noncollagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues.
Subject(s)
Cartilage/metabolism , Extracellular Matrix/metabolism , Femur/metabolism , RNA-Seq , Single-Cell Analysis , Animals , Electron Transport , Extracellular Matrix/genetics , Mice , Mice, TransgenicABSTRACT
NEW FINDINGS: What is the central question of this study? While muscle fibre atrophy in response to immobilisation has been extensively examined, intramuscular connective tissue, particularly endomysium, has been largely neglected: does endomysium content of the soleus muscle increase during bed rest? What is the main finding and its importance? Absolute endomysium content did not change, and previous studies reporting an increase are explicable by muscle fibre atrophy. It must be expected that even a relative connective tissue accumulation will lead to an increase in muscle stiffness. ABSTRACT: Muscle fibres atrophy during conditions of disuse. Whilst animal data suggest an increase in endomysium content with disuse, that information is not available for humans. We hypothesised that endomysium content increases during immobilisation. To test this hypothesis, biopsy samples of the soleus muscle obtained from 21 volunteers who underwent 60 days of bed rest were analysed using immunofluorescence-labelled laminin γ-1 to delineate individual muscle fibres as well as the endomysium space. The endomysium-to-fibre-area ratio (EFAr, as a percentage) was assessed as a measure related to stiffness, and the endomysium-to-fibre-number ratio (EFNr) was calculated to determine whether any increase in EFAr was absolute, or could be attributed to muscle fibre shrinkage. As expected, we found muscle fibre atrophy (P = 0.0031) that amounted to shrinkage by 16.6% (SD 28.2%) on day 55 of bed rest. ENAr increased on day 55 of bed rest (P < 0.001). However, when analysing EFNr, no effect of bed rest was found (P = 0.62). These results demonstrate that an increase in EFAr is likely to be a direct effect of muscle fibre atrophy. Based on the assumption that the total number of muscle fibres remains unchanged during 55 days of bed rest, this implies that the absolute amount of connective tissue in the soleus muscle remained unchanged. The increased relative endomysium content, however, could be functionally related to an increase in muscle stiffness.
Subject(s)
Muscle Fibers, Skeletal , Muscle, Skeletal , Animals , Bed Rest , Humans , Muscle Fibers, Skeletal/physiology , Muscle, Skeletal/pathology , MyocardiumABSTRACT
Scaffold-mediated tissue engineering has become a golden solution for the regeneration of damaged bone tissues that lack self-regeneration capability. A successful scaffold in bone tissue engineering comprises a multitude of suitable biological, microarchitectural, and mechanical properties acting as different signaling cues for the cells to mediate the new tissue formation. Therefore, careful design of bioactive scaffold macro- and microstructures in multiple length scales and biophysical properties fulfilling the tissue repair demands are necessary yet challenging to achieve. Herein, we have developed an antibacterial and biocompatible silica-silk fibroin (SF) gel-based ink through novel yet simple chemical approaches of sol-gel and self-assembly followed by processing the obtained gels as three-dimensional (3D) hybrid aerogel-based scaffolds exploiting the advanced materials design approaches of micro-extrusion-based 3D printing, and directional freeze-casting/drying approaches. As the main constituent of the hybrid biocompatible scaffold of this study, we used the SF extracted from Bombyx mori silkworm cocoon. However, to increase the cell responsivity and bactericidal efficiency of the final scaffold, thiol-ended antimicrobial and cell adhesive peptide sequence (SH-CM-RGD) was conjugated to silica-SF hybrid gels via covalent attachment using a spacer molecule through either preprint (prior to sol-gel) or during the post-printing steps on the previously printed silica-SF gel. In the next step, the hybrid Silica-SF-CM-RGD hydrogel ink was 3D-printed into the construct with interconnected porous structure with hierarchically organized porosity and a combination of several promising properties. Namely, due to the covalent linkage of the antibacterial peptide to the SF, the scaffold shows potent bactericidal efficiency toward Gram-positive and Gram-negative bacteria. Moreover, nanostructured silica components in the 3D-printed composites could intertwine with SF-CM-RGD to support the mechanical properties in the final scaffold and the final osteoconductivity of the scaffold. This study supports the promising properties of 3D-printed silica-SF-based hybrid aerogels constructs for repairing bone defect.
Subject(s)
Fibroins , Nanostructures , Anti-Bacterial Agents/pharmacology , Biomimetics , Gram-Negative Bacteria , Gram-Positive Bacteria , Hydrogels , Peptides , Porosity , Printing, Three-Dimensional , Silicon Dioxide , Tissue ScaffoldsABSTRACT
During wound healing, fibroblasts differentiate into nonproliferative contractile myofibroblasts, contribute to skin repair, and eventually undergo apoptosis or become senescent. MicroRNAs are post-transcriptional regulators of gene expression networks that control cell fate and survival and may also regulate senescence. In this study, we determined the regulated microRNAs in myofibroblasts isolated from wounds and analyzed their role in senescent myofibroblast formation. Transcriptome profiling showed that a 200 kilobase pair region of the Dlk1-Dio3âimprinted domain on mouse chromosome 12 encodes for most of the upregulated microRNAs in the entire genome of mouse myofibroblasts. Among those, miR-127-3p induced a myofibroblast-like phenotype associated with a block in proliferation. Molecular analysis revealed that miR-127-3p induced a prolonged cell cycle arrest with unique molecular features of senescence, including the activation of the senescence-associated ß-galactosidase, increase in p53 and p21 levels, inhibition of lamin B1, proliferation factors, and the production of senescence-associated inflammatory and extracellular matrixâremodeling components. Hence, miR-127-3p emerges as an epigenetic activator regulating the transition from repair to remodeling during skin wound healing but may also induce age-related defects, pathological scarring, and fibrosis, all linked to myofibroblast senescence.
Subject(s)
Cellular Senescence/genetics , MicroRNAs/metabolism , Myofibroblasts/pathology , Skin/injuries , Wound Healing/genetics , Animals , Calcium-Binding Proteins/genetics , Cell Differentiation/genetics , Chromosomes, Mammalian/genetics , Disease Models, Animal , Epigenesis, Genetic , Gene Expression Profiling , Humans , Iodide Peroxidase/genetics , Mice , Skin/pathologyABSTRACT
GnRH enhances the expression of annexin A5 (ANXA5) in pituitary gonadotropes, and ANXA5 enhances gonadotropin secretion. However, the impact of ANXA5 regulation on the expression of pituitary hormone genes remains unclear. Here, using quantitative PCR, we demonstrated that ANXA5 deficiency in female mice reduced the expression of Fshb and Gh in their pituitary glands. Transcriptome analysis confirmed a specific increase in Nr4a3 mRNA expression in addition to lower levels of Fshb expression in ANXA5-deficient female pituitary glands. This gene was then found to be a GnRH-inducible immediate early gene, and its increased expression caused protein to accumulate in the nucleus after administration of a GnRH agonist in LßT2 cells, which are an in vitro pituitary gonadotrope model. The increase in ANXA5 protein levels in LßT2 cells clearly suppressed Nr4a3 expression. siRNA-mediated inhibition of Nr4a3 expression increased Fshb expression. The results revealed that GnRH stimulates Nr4a3 and Anxa5 sequentially. NR4A3 suppression of Fshb may be necessary for later massive secretion of FSH by GnRH in gonadotropes, and Nr4a3 would be negatively regulated by ANXA5 to increase FSH secretion.
ABSTRACT
Plasticity of the proteome is critical to adapt to varying conditions. Control of mitochondrial protein import contributes to this plasticity. Here, we identified a pathway that regulates mitochondrial protein import by regulated N-terminal processing. We demonstrate that dipeptidyl peptidases 8/9 (DPP8/9) mediate the N-terminal processing of adenylate kinase 2 (AK2) en route to mitochondria. We show that AK2 is a substrate of the mitochondrial disulfide relay, thus lacking an N-terminal mitochondrial targeting sequence and undergoing comparatively slow import. DPP9-mediated processing of AK2 induces its rapid proteasomal degradation and prevents cytosolic accumulation of enzymatically active AK2. Besides AK2, we identify more than 100 mitochondrial proteins with putative DPP8/9 recognition sites and demonstrate that DPP8/9 influence the cellular levels of a number of these proteins. Collectively, we provide in this study a conceptual framework on how regulated cytosolic processing controls levels of mitochondrial proteins as well as their dual localization to mitochondria and other compartments.
Subject(s)
Adenylate Kinase/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Mitochondrial Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis , HEK293 Cells , HeLa Cells , Humans , Protein TransportABSTRACT
MicroRNAs (miRNAs) regulate cartilage differentiation and contribute to the onset and progression of joint degeneration. These small RNA molecules may affect extracellular matrix organization (ECM) in cartilage, but for only a few miRNAs has this role been defined in vivo. Previously, we showed that cartilage-specific genetic ablation of the Mirc24 cluster in mice leads to impaired cartilage development due to increased RAF/MEK/ERK pathway activation. Here, we studied the expression of the cluster in cartilage by LacZ reporter gene assays and determined its role for extracellular matrix homeostasis by proteome and immunoblot analysis. The cluster is expressed in prehypertrophic/hypertrophic chondrocytes of the growth plate and we now show that the cluster is also highly expressed in articular cartilage. Cartilage-specific loss of the cluster leads to increased proteoglycan 4 and matrix metallopeptidase 13 levels and decreased aggrecan and collagen X levels in epiphyseal cartilage. Interestingly, these changes are linked to a decrease in SRY-related HMG box-containing (SOX) transcription factors 6 and 9, which regulate ECM production in chondrocytes. Our data suggests that the Mirc24 cluster is important for ECM homoeostasis and the expression of transcriptional regulators of matrix production in cartilage.
Subject(s)
Cartilage, Articular/metabolism , Extracellular Matrix Proteins/genetics , MicroRNAs/genetics , Osteoarthritis/genetics , Animals , Cartilage, Articular/physiology , Collagen Type II/genetics , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation , Growth Plate/chemistry , Male , Matrix Metalloproteinase 13/genetics , Matrix Metalloproteinase 13/metabolism , Mice, Transgenic , Multigene Family , Proteoglycans/genetics , Proteoglycans/metabolism , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , SOXD Transcription Factors/genetics , SOXD Transcription Factors/metabolismABSTRACT
With rising demand for cartilage tissue repair and replacement, the differentiation of mesenchymal stem cells (BMSCs) into cartilage tissue forming cells provides a promising solution. Often, the BMSC-derived cartilage does not remain stable and continues maturing to bone through the process of endochondral ossification in vivo. Similar to the growth plate, invasion of blood vessels is an early hallmark of endochondral ossification and a necessary step for completion of ossification. This invasion originates from preexisting vessels that expand via angiogenesis, induced by secreted factors produced by the cartilage graft. In this study, we aimed to identify factors secreted by chondrogenically differentiated bone marrow-derived human BMSCs to modulate angiogenesis. The secretome of chondrogenic pellets at day 21 of the differentiation program was collected and tested for angiogenic capacity using in vitro endothelial migration and proliferation assays as well as the chick chorioallantoic membrane (CAM) assay. Taken together, these assays confirmed the pro-angiogenic potential of the secretome. Putative secreted angiogenic factors present in this medium were identified by comparative global transcriptome analysis between murine growth plate cartilage, human chondrogenic BMSC pellets and human neonatal articular cartilage. We then verified by PCR eight candidate angiogenesis modulating factors secreted by differentiated BMSCs. Among those, Serpin E1 and Indian Hedgehog (IHH) had a higher level of expression in BMSC-derived cartilage compared to articular chondrocyte derived cartilage. To understand the role of these factors in the pro-angiogenic secretome, we used neutralizing antibodies to functionally block them in the conditioned medium. Here, we observed a 1.4-fold increase of endothelial cell proliferation when blocking IHH and 1.5-fold by Serpin E1 blocking compared to unblocked control conditioned medium. Furthermore, endothelial migration was increased 1.9-fold by Serpin E1 blocking and 2.7-fold by IHH blocking. This suggests that the pro-angiogenic potential of chondrogenically differentiated BMSC secretome could be further augmented through inhibition of specific factors such as IHH and Serpin E1 identified as anti-angiogenic factors.
ABSTRACT
This study was performed to identify transcriptional alterations in male intrauterine growth restricted (IUGR) rats during and at the end of nephrogenesis in order to generate hypotheses which molecular mechanisms contribute to adverse kidney programming. IUGR was induced by low protein (LP) diet throughout pregnancy, bilateral uterine vessel ligation (LIG), or intrauterine stress (IUS) by sham operation. Offspring of unimpaired dams served as controls. Significant acute kidney damage was ruled out by negative results for proteins indicative of ER-stress, autophagy, apoptosis, or infiltration with macrophages. Renal gene expression was examined by transcriptome microarrays, demonstrating 53 (LP, n = 12; LIG, n = 32; IUS, n = 9) and 134 (LP, n = 10; LIG, n = 41; IUS, n = 83) differentially expressed transcripts on postnatal days (PND) 1 and 7, respectively. Reduced Pilra (all IUGR groups, PND 7), Nupr1 (LP and LIG, PND 7), and Kap (LIG, PND 1) as well as increased Ccl20, S100a8/a9 (LIG, PND 1), Ifna4, and Ltb4r2 (IUS, PND 7) indicated that inflammation-related molecular dysregulation could be a "common" feature after IUGR of different origins. Network analyses of transcripts and predicted upstream regulators hinted at proinflammatory adaptions mainly in LIG (arachidonic acid-binding, neutrophil aggregation, toll-like-receptor, NF-kappa B, and TNF signaling) and dysregulation of AMPK and PPAR signaling in LP pups. The latter may increase susceptibility towards obesity-associated kidney damage. Western blots of the most prominent predicted upstream regulators confirmed significant dysregulation of RICTOR in LP (PND 7) and LIG pups (PND 1), suggesting that mTOR-related processes could further modulate kidney programming in these groups of IUGR pups. KEY MESSAGES: Inflammation-related transcripts are dysregulated in neonatal IUGR rat kidneys. Upstream analyses indicate renal metabolic dysregulation after low protein diet. RICTOR is dysregulated after low protein diet and uterine vessel ligation.
Subject(s)
Fetal Growth Retardation/genetics , Kidney/metabolism , Animals , Animals, Newborn , Kidney/growth & development , Male , Organ Size , Rats, Wistar , TranscriptomeABSTRACT
The continuously growing mouse incisor provides a fascinating model for studying stem cell regulation and organ renewal. In the incisor, epithelial and mesenchymal stem cells assure lifelong tooth growth. The epithelial stem cells reside in a niche known as the cervical loop. Mesenchymal stem cells are located in the nearby apical neurovascular bundle and in the neural plexus. So far, little is known about extracellular cues that are controlling incisor stem cell renewal and guidance. The extracellular matrix protein tenascin-W, also known as tenascin-N (TNN), is expressed in the mesenchyme of the pulp and of the periodontal ligament of the incisor, and is closely associated with collagen 3 fibers. Here, we report for the first time the phenotype of tenascin-W/TNN deficient mice, which in a C57BL/6N background exhibit a reduced body weight and lifespan. We found major defects in the alveolar bone and periodontal ligament of the growing rodent incisors, whereas molars were not affected. The alveolar bone around the incisor was replaced by a dense scar-like connective tissue, enriched with newly formed nerve fibers likely leading to periodontal pain, less food intake and reduced body weight. Using soft food to reduce mechanical load on the incisor partially rescued the phenotype. In situ hybridization and Gli1 reporter mouse experiments revealed decreased hedgehog signaling in the incisor mesenchymal stem cell compartment, which coordinates the development of mesenchymal stem cell niche. These results indicate that TNN deficiency in mice affects periodontal remodeling and increases nerve fiber branching. Through periodontal pain the food intake is reduced and the incisor renewal and the neurovascular sonic hedgehog secretion rate are reduced. In conclusion, tenascin-W/TNN seems to have a primary function in rapid periodontal tissue remodeling and a secondary function in mechanosensation.