Collagen Fibril Organization Research Articles

Kathon Induces Fibrotic Inflammation in Lungs: The First Animal Study Revealing a Causal Relationship between Humidifier Disinfectant Exposure and Eosinophil and Th2-Mediated Fibrosis Induction.

Currently available toxicity data on humidifier disinfectants are primarily limited to polyhexamethylene guanidine phosphate-induced lung fibrosis. We, therefore, investigated whether the sterilizer component Kathon, which is a mixture of chloromethylisothiazolinone and methylisothiazolinone, induces fibrotic lung injury following direct lung exposure in an animal model. Mice were intratracheally instilled with either the vehicle or Kathon. Differential cell counts, cytokine analysis, and histological analysis of lung tissue were then performed to characterize the injury features, and we investigated whether Kathon altered fibrosis-related gene expression in lung tissues via RNA-Seq and bioinformatics. Cell counting showed that Kathon exposure increased the proportion of macrophages, eosinophils, and neutrophils. Moreover, T helper 2 (Th2) cytokine levels in the bronchoalveolar lavage were significantly increased in the Kathon groups. Histopathological analysis revealed increased perivascular/alveolar inflammation, eosinophilic cells, mucous cell hyperplasia, and pulmonary fibrosis following Kathon exposure. Additionally, Kathon exposure modulated the expression of genes related to fibrotic inflammation, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, extracellular signal regulated kinase (ERK)1 and ERK2 cascade, extracellular matrix (ECM)-receptor interaction pathway, transforming growth factor beta receptor signaling pathway, cellular response to tumor necrosis factor, and collagen fibril organization. Our results suggest that Kathon exposure is associated with fibrotic lung injury via a Th2-dependent pathway and is thus a possible risk factor for fibrosis.

Collagen XI regulates the acquisition of collagen fibril structure, organization and functional properties in tendon

Collagen XI is a fibril-forming collagen that regulates collagen fibrillogenesis. Collagen XI is normally associated with collagen II-containing tissues such as cartilage, but it also is expressed broadly during development in collagen I-containing tissues, including tendons. The goals of this study are to define the roles of collagen XI in regulation of tendon fibrillar structure and the relationship to function. A conditional Col11a1-null mouse model was created to permit the spatial and temporal manipulation of Col11a1 expression. We hypothesize that collagen XI functions to regulate fibril assembly, organization and, therefore, tendon function. Previous work using cho mice with ablated Col11a1 alleles supported roles for collagen XI in tendon fibril assembly. Homozygous cho/cho mice have a perinatal lethal phenotype that limited the studies. To circumvent this, a conditional Col11a1flox/flox mouse model was created where exon 3 was flanked with loxP sites. Breeding with Scleraxis-Cre (Scx-Cre) mice yielded a tendon-specific Col11a1-null mouse line, Col11a1Δten/Δten. Col11a1flox/flox mice had no phenotype compared to wild type C57BL/6 mice and other control mice, e.g., Col11a1flox/flox and Scx-Cre. Col11a1flox/flox mice expressed Col11a1 mRNA at levels comparable to wild type and Scx-Cre mice. In contrast, in Col11a1Δten/Δten mice, Col11a1 mRNA expression decreased to baseline in flexor digitorum longus tendons (FDL). Collagen XI protein expression was absent in Col11a1Δten/Δten FDLs, and at ~50% in Col11a1+/Δten compared to controls. Phenotypically, Col11a1Δten/Δten mice had significantly decreased body weights (p < 0.001), grip strengths (p < 0.001), and with age developed gait impairment becoming hypomobile. In the absence of Col11a1, the tendon collagen fibrillar matrix was abnormal when analyzed using transmission electron microscopy. Reducing Col11a1 and, therefore collagen XI content, resulted in abnormal fibril structure, loss of normal fibril diameter control with a significant shift to small diameters and disrupted parallel alignment of fibrils. These alterations in matrix structure were observed in developing (day 4), maturing (day 30) and mature (day 60) mice. Altering the time of knockdown using inducible I-Col11a1−/− mice indicated that the primary regulatory foci for collagen XI was in development. In mature Col11a1Δten/Δten FDLs a significant decrease in the biomechanical properties was observed. The decrease in maximum stress and modulus suggest that fundamental differences in the material properties in the absence of Col11a1 expression underlie the mechanical deficiencies. These data demonstrate an essential role for collagen XI in regulation of tendon fibril assembly and organization occurring primarily during development.

Long Non-coding RNAs Are Differentially Expressed After Different Exercise Training Programs.

BackgroundMolecular regulation related to the health benefits of different exercise modes remains unclear. Long non-coding RNAs (lncRNAs) have emerged as an RNA class with regulatory functions in health and diseases. Here, we analyzed the expression of lncRNAs after different exercise training programs and their possible modes of action related to physical exercise adaptations.MethodsPublic high-throughput RNA-seq data (skeletal muscle biopsies) were downloaded, and bioinformatics analysis was performed. We primarily analyzed data reports of 12 weeks of resistance training (RT), high-intensity interval training (HIIT), and combined (CT) exercise training. In addition, we analyzed data from 8 weeks of endurance training (ET). Differential expression analysis of lncRNAs was performed, and an adjusted P-value < 0.1 and log2 (fold change) ≥0.5 or ≤−0.5 were set as the cutoff values to identify differentially expressed lncRNAs (DELs).ResultsWe identified 204 DELs after 12 weeks of HIIT, 43 DELs after RT, and 15 DELs after CT. Moreover, 52 lncRNAs were differentially expressed after 8 weeks of ET. The lncRNA expression pattern after physical exercise was very specific, with distinct expression profiles for the different training programs, where few lncRNAs were common among the exercise types. LncRNAs may regulate molecular responses to exercise, such as collagen fibril organization, extracellular matrix organization, myoblast and plasma membrane fusion, skeletal muscle contraction, synaptic transmission, PI3K and TORC regulation, autophagy, and angiogenesis.ConclusionFor the first time, we show that lncRNAs are differentially expressed in skeletal muscle after different physical exercise programs, and these lncRNAs may act in various biological processes related to physical activity adaptations.

Integrated Bioinformatics Analysis of Hub Genes and Pathways Associated with a Compression Model of Spinal Cord Injury in Rats.

BackgroundSpinal cord injury (SCI) is a serious nervous system condition that can cause lifelong disability. The aim of this study was to identify potential molecular mechanisms and therapeutic targets for SCI.Material/MethodsWe constructed a weighted gene coexpression network and predicted which hub genes are involved in SCI. A compression model of SCI was established in 45 Sprague-Dawley rats, which were divided into 5 groups (n=9 per group): a sham operation group, and 1, 3, 5, and 7 days post-SCI groups. The spinal cord tissue on the injured site was harvested on 1, 3, 5, and 7 days after SCI and 3 days after surgery in the sham operation group. High-throughput sequencing was applied to investigate the expression profile of the mRNA in all samples. Differentially expressed genes were screened and included in weighted gene coexpression network analysis (WGCNA). Co-expressed modules and hub genes were identified by WGCNA. The biological functions of each module were investigated using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases.ResultsAccording to the RNA-seq data, a total of 1965 differentially expressed genes were screened, and WGCNA identified 10 coexpression modules and 5 hub genes. Module function analysis revealed that SCI was associated with immune response, cell division, neuron projection development, and collagen fibril organization.ConclusionsOur study revealed dynamic changes in a variety of biological processes following SCI and identified 5 hub genes via WGCNA. These results provide insights into the molecular mechanisms and therapeutic targets of SCI.

Ellipsoidal mesoscale mineralization pattern in human cortical bone revealed in 3D by plasma focused ion beam serial sectioning

Visualizing bone mineralization and collagen fibril organization at intermediate scales between the nanometer and the hundreds of microns range, is still an important challenge. Similarly, visualizing cellular components which locally affect the tissue structure requires a precision of a few tens of nanometers at maximum while spanning several tens of micrometers. In the last decade, gallium focused ion beam (FIB) equipped with a scanning electron microscope (SEM) proved to be an extremely valuable structural tool to meet those ends. In this study, we assess the capability of a recent plasma FIB-SEM technology which provides a potential increase in measurement speed over gallium FIB-SEM, thus paving the way to larger volume analysis. Nanometer-scale layers of demineralized and mineralized unstained human femoral lamellar bone were sequentially sectioned over volumes of 6–16,000 μm3. Analysis of mineralized tissue revealed prolate ellipsoidal mineral clusters measuring approximately 1.1 µm in length by 700 nm at their maximum diameter. Those features, suggested by others in high resolution studies, appear here as a ubiquitous motif in mineralized lamellar bone over thousands of microns cubed, suggesting a heterogeneous and yet regular pattern of mineral deposition past the single collagen fibril level. This large scale view retained sufficient resolution to visualize the collagen fibrils while also partly visualizing the lacuno-canalicular network in three-dimensions. These findings are strong evidence for suitability of PFIB as a bone analysis tool and the need to revisit bone mineralization over multi-length scales with mineralized tissue.

Identification of PLOD Family Genes as Novel Prognostic Biomarkers for Hepatocellular Carcinoma.

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies with rising incidence and persistently high mortality. Previous researches have demonstrated that some PLOD family members are associated with tumor progression and metastasis in most human cancers. However, the prognostic and biological roles of PLODs in HCC remain largely unknown.MethodsONCOMINE, HPA, UALCAN, GEPIA, cBioPortal, GeneMANIA, NetworkAnalyst, Metascape, DAVID 6.8, and TIMER were used to determine the prognostic values and biological function of PLOD family members in HCC.ResultsThe mRNA and protein expression patterns of PLOD family members were noticeably upregulated in HCC compared to normal tissue. The high expression levels of PLOD1 and PLOD2 genes were significantly correlated with higher tumor grades in HCC patients. In addition, the high expression levels of PLOD1–3 were remarkably associated with poor overall survival in HCC patients, while high PLOD1 and PLOD3 expression were markedly associated with worse disease-free survival. In the co-expression gene analysis, 20 genes were primarily associated with the differentially expressed PLOD family members in HCC cases. Through functional enrichment analysis, the biological functions of PLODs in HCC were mainly involved in collagen fibril organization, lysine degradation, collagen biosynthesis, and modifying enzymes. Furthermore, the expression levels of PLOD1–3 were positively correlated with the activities of tumor-infiltrating immune cells, including macrophages, neutrophils, CD4+ T cells, and dendritic cells. Besides, the expression levels of PLOD2 and PLOD3 were positively correlated with the infiltrating levels of B cells.ConclusionThe findings of this study could provide novel insights into the identification of prognostic biomarkers for HCC patients.

Analysis of key genes and pathways associated with the pathogenesis of intervertebral disc degeneration

BackgroundIntervertebral disc degeneration (IDD) is widely known as the main contributor to low back pain which has a negative socioeconomic impact worldwide. However, the underlying mechanism remains unclear. This study aims to analyze the dataset GSE23130 using bioinformatics methods to identify the pivotal genes and pathways associated with IDD.Material/methodsThe gene expression data of GSE23130 was downloaded, and differentially expressed genes (DEGs) were extracted from 8 samples and 15 controls. GO and KEGG pathway enrichment analyses were performed. Also, protein–protein interaction (PPI) network was constructed and visualized, followed by identification of hub genes and key module.ResultsA total of 30 downregulated and 79 upregulated genes were identified. The DEGs were mainly enriched in the regulation of protein catabolic process, extracellular matrix organization, collagen fibril organization, and extracellular structure organization. Meanwhile, we found that most DEGs were primarily enriched in the PI3K-Akt signaling pathway. The top 10 hub genes were FN1, COL1A2, SPARC, COL3A1, CTGF, LUM, TIMP1, THBS2, COL5A2, and TGFB1.ConclusionsIn summary, key candidate genes and pathways were identified by using integrated bioinformatics analysis, which may provide insights into the underlying mechanisms and offer potential target genes for the treatment of IDD.

Collagen ultrastructural symmetry and its malignant alterations in human breast cancer revealed by polarization-resolved second-harmonic generation microscopy.

Several specific alterations of the extracellular matrix can be considered a distinctive hallmark of cancer. In particular, a different morphology of the collagen scaffold is frequently found within the peritumoural environment. In this study, we report about a significant difference in the ultrastructural organization of collagen at the supra-molecular level between the perilesional scaffold and the tumour area in human breast carcinoma samples. In particular, we demonstrated that polarization-resolved second-harmonic generation (P-SHG) microscopy is able to link the altered collagen architecture at the ultrastructural level found in perilesional tissue with a different organization of collagen fibrils at the molecular level.

Anisotropic and viscoelastic tensile mechanical properties of aponeurosis: Experimentation, modeling, and tissue microstructure

Aponeuroses are stiff sheath-like components of the muscle-tendon unit that play a vital role in force transmission and thus locomotion. There is clear importance of the aponeurosis in musculoskeletal function, but there have been relatively few studies of aponeurosis material properties to date. The goals of this work were to: 1) perform tensile stress-relaxation tests, 2) perform planar biaxial tests, 3) employ computational modeling to the data from 1 to 2, and 4) perform scanning electron microscopy to determine collagen fibril organization for aponeurosis tissue. Viscoelastic modeling and statistical analysis of stress-relaxation data showed that while relaxation rate differed statistically between strain levels (p = 0.044), functionally the relaxation behavior was nearly the same. Biaxial testing and associated modeling highlighted the nonlinear (toe region of ~2–3% strain) and anisotropic (longitudinal direction linear modulus ~50 MPa, transverse ~2.5 MPa) tensile mechanical behavior of aponeurosis tissue. Comparisons of various constitutive formulations showed that a transversely isotropic Ogden approach balanced strong fitting (goodness of fit 0.984) with a limited number of parameters (five), while damage modeling parameters were also provided. Scanning electron microscopy showed a composite structure of highly aligned, partially wavy collagen fibrils with more random collagen cables for aponeurosis microstructure. Future work to expand microstructural analysis and use these data to inform computational modeling would benefit this work and the field.

How Does Diabetes Impair Penile Tissues during Erectile Dysfunction?

Erectile dysfunction (ED) is a significant but underestimated complication during diabetes mellitus (DM). Currently, few special treatments are available clinically due to the lack of specific therapeutic targets. Genomic analysis can be helpful to find potential targets. In this study, the gene expression under diabetic ED condition was analyzed using a gene array, and the significance of the outcomes was evaluated through clinical data. The expressions of 15923 genes were analyzed using R software. Differential expression genes (DEGs) were identified through the constructed volcano plot. The function enrichment of Gene Ontology (GO) and KEGG was screened with the DAVID online tool. The interaction between these DEGs was revealed through constructing a protein-protein interaction network and the hub genes were uncovered using the STRING and Cytoscape tool. Lastly, the data of diabetic ED patients were applied to verify the bioinformatics findings. The study showed that 75 genes in the rat penile tissues were upregulated, while 97 genes were downregulated on the diabetic ED condition. These genes were mainly involved in extracellular matrix composition, collagen fibril organization, as well as protein digestion & absorption. Additionally, insulin-related signaling pathways were affected. The clinical analysis indicated that insulin resistance was associated with the diabetic ED severity. Notably, the bioinformatics analysis also suggested that ferroptosis pathway was probably activated under the diabetic ED condition. The impaired protein synthesis induced by deficient insulin signaling is an important cause of the diabetic ED. The improvement of protein synthesis through restoring insulin function may be potentially useful for diabetic ED therapy.

Intrastromal Injection of Hyaluronidase Alters the Structural and Biomechanical Properties of the Corneal Stroma

PurposeGlycosaminoglycans (GAGs) are important components of the corneal stroma, and their spatiotemporal arrangement regulates the organization of collagen fibrils and maintains corneal transparency. This study was undertaken to determine the consequences of hyaluronidase (HAse) injected into the corneal stroma on stromal stiffness and ultrastructure.MethodsEqual volumes of HAse or balanced salt solution (vehicle) were injected intrastromally into the corneas of New Zealand white rabbits. Ophthalmic examination and multimodal imaging techniques, including Fourier-domain optical coherence tomography and in vivo confocal microscopy (IVCM), were performed at multiple time points to evaluate the impact of HAse treatment in vivo. Atomic force microscopy and transmission electron microscopy (TEM) were used to measure corneal stiffness and collagen's interfibrillar spacing, respectively.ResultsCentral corneal thickness progressively decreased after HAse injection, reaching its lowest value at day 7, and then returned to normal by day 42. The HAse did not impact the corneal endothelium but transiently altered keratocyte morphology at days 1 and 7, as measured by IVCM. HAse-injected corneas became stiffer by day 1 postinjection, were stiffest at day 7, and returned to preinjection values by day 90. Changes in stromal stiffness correlated with decreased interfibrillar spacing as measured by TEM.ConclusionsDegradation of GAGs by HAse decreases the corneal thickness and increases stromal stiffness through increased packing of the collagen fibrils in a time-dependent manner.Translational RelevanceIntrastromal HAse injection appears relatively safe in the normal cornea, but its impact on corneal biomechanics and structure under pathologic conditions requires further study.

Correction

Correction

Optimized multi-axial loading to enhance chondrogenesis and limit proteoglycan loss in cartilage explants during bioreactor experimentation

Purpose: Osteoarthritis (OA) is a disease of the whole joint, with associated changes in the subchondral bone, synovium, bone marrow, muscles and ligaments, but with cartilage degeneration being the hallmark of OA. Mechanical loading is a key control signal in the regulation of the metabolic activity (i.e. homeostasis) of the articular chondrocytes, the unique cell population found in cartilage, embedded in their self-produced extra-cellular matrix (ECM). To study the effects of mechanical loading on the biological response of cartilage, explants can be loaded in bioreactors. Multi-axial loading bioreactors that combine compressive with torsional loading can mimic the loading of articular cartilage. This study aims at informing bioreactor protocol development to define mechanical parameters that optimize the biological response of cartilage explants during multi-directional loading experiments. Indeed, the local biological response may vary from chondrogenesis in some parts to proteoglycan loss in others depending on the mechanical parameters. This study uses a finite element method to investigate the effects of multi-axial loading characteristics on the local mechanical environment (stresses and strains) of the explants while accounting for material properties of the non-fibrillar matrix of cartilage explants. The mechanical environment is then related to chondrogenesis (based on maximum compressive principal strain (MCPS) - Zahedmanesh et al., 2014) and proteoglycan loss (based on the fluid velocity (FV) - Orozco et al., 2018). Methods: The cartilage explant was modelled as a 3D cylinder of 8mm diameter and equal height in Abaqus. A fiber-reinforced poroelastic (FRPE) constitutive model (Ebrahimi et al., 2019) was used to define the material, in which the non-fibrillar matrix of the cartilage explant was modelled as a porous hyperelastic (Neohookean) material saturated with fluid (water) and the collagen fiber network was modelled as nonlinear elastic material that can only withstand tensile loading. Four families of organized collagen fibrils (primary fibrils) and 13 families of randomly oriented fibrils (secondary fibrils) formed the collagen network. Ratio of density of the primary to the secondary fibrils was set to 12.16. The bottom surface of the explant was fully constrained and sinusoidal (frequency of 1Hz) unconfined compression and torsion loading were applied to the top surface. Free fluid flow was allowed through the lateral surface. The initial and strain-dependent fibril network moduli were respectively set to 0.41 and 15.42 MPa and the constant for displacement dependence of permeability in the non-fibrillar matrix was set to 3.36 (Ebrahimi et al., 2019). The sensitivity of the maximum values of FV and MCPS was studied at 9 key locations (see figure a), whereby the material parameters of the non-fibrillar matrix and loading range were varied. This resulted in five input parameters: elastic modulus (E=[0.15,0.6]MPa), Poisson’s ratio (nu=[0.1,0.45]), initial permeability (k0=[0.4,1.6]×10-15m4/Ns), compressive strain (Comp=[5,20]%) and torsion loading (Tor=[5,20]°). A full-factorial design of experiment method was used and a first-order polynomial surface including the interactions fitted the responses. Results: MCPS varied between 8.4% and 36.5% and is independent of the material properties of non-fibrillar matrix (E, nu and k0) but has a high and moderate dependency on Compressive (Comp) and torsion loading (Tor) magnitudes, respectively (figure b). The maximum value of MCPS always occurs at the bottom edge, i.e. at the distal cartilage bone interface (point 7 in figure a). FV varies between 1.2×10-7 mm/sec and 4.8×10-5 mm/sec and dominantly depends on initial permeability (k0) and compressive loading (Comp), while being independent of Poisson’s ratio (nu) and slightly dependent on elastic modulus (E) and torsion loading (Tor) (figure c). The maximum value of FV always occurs at the center of the side surface (point 4 in figure a). Conclusions: The parameter sensitivity analysis Results show that by increasing compression and torsion loading within the bioreactor the chondrogenesis (which depends on MCPS) may increase, independent of the material properties studied. Therefore, within the selected ranges studied here, a decrease in material stiffness (softening of the cartilage) due to OA won’t affect the chondrogenesis in cartilage explants. Based on these results, if enough high-quality chondrocyte cells are provided in a cell-seeded implant-cartilage unit, chondrogenesis will increase by increasing the compression and torsion loadings applied within the bioreactor up to at least 20% and 20°, respectively. On the other hand, increasing compression loading will increase FV, thereby degenerating the extracellular matrix through proteoglycan loss. Also, increasing initial permeability (k0) will increase FV under loading, while FV is independent of elastic modulus (E), Poisson’s ration (nu) and torsion loading (Tor). Therefore, to limit proteoglycan loss and enhance chondrogenesis in cartilage explants during bioreactor experiments, compression loading might be decreased (to control FV ) but torsion loading increased (to increase MCPS). In future, follow-up biological studies are needed to test the added value of these model-informed bioreactor protocols.

Therapeutic Interventions to Reduce Radiation Induced Dermal Injury in a Murine Model of Tissue Expander Based Breast Reconstruction.

Radiation therapy (XRT) induced dermal injury disrupts type I collagen architecture. This impairs cutaneous viscoelasticity, which may contribute to the high rate of complications in expander-based breast reconstruction with adjuvant XRT. The objective of this study was to further elucidate the mechanism of radiation-induced dermal injury and to determine if amifostine (AMF) or deferoxamine (DFO) mitigates type I collagen injury in an irradiated murine model of expander-based breast reconstruction. Female Lewis rats (n = 20) were grouped: expander (control), expander-XRT (XRT), expander-XRT-AMF (AMF), and expander-XRT-DFO (DFO). Expanders were surgically placed. All XRT groups received 28 Gy of XRT. The AMF group received AMF 30 minutes before XRT, and the DFO group used a patch for delivery 5 days post-XRT. After a 20-day recovery period, skin was harvested. Atomic force microscopy and Raman spectroscopy were performed to evaluate type I collagen sheet organization and tissue compositional properties, respectively. Type I collagen fibril disorganization was significantly increased in the XRT group compared with the control (83.8% vs 22.4%; P = 0.001). Collagen/matrix ratios were greatly reduced in the XRT group compared with the control group (0.49 ± 0.09 vs 0.66 ± 0.09; P = 0.017). Prophylactic AMF demonstrated a marked reduction in type I collagen fibril disorganization on atomic force microscopy (15.9% vs 83.8%; P = 0.001). In fact, AMF normalized type I collagen organization in irradiated tissues to the level of the nonirradiated control (P = 0.122). Based on Raman spectroscopy, both AMF and DFO demonstrated significant differential protective effects on expanded-irradiated tissues. Collagen/matrix ratios were significantly preserved in the AMF group compared with the XRT group (0.49 ± 0.09 vs 0.69 ± 0.10; P = 0.010). β-Sheet/α-helix ratios were significantly increased in the DFO group compared with the XRT group (1.76 ± 0.03 vs 1.86 ± 0.06; P = 0.038). Amifostine resulted in a significant improvement in type I collagen fibril organization and collagen synthesis, whereas DFO mitigated abnormal changes in collagen secondary structure in an irradiated murine model of expander-based breast reconstruction. These therapeutics offer the ability to retain the native microarchitecture of type I collagen after radiation. Amifostine and DFO may offer clinical utility to reduce radiation induced dermal injury, potentially decreasing the high complication rate of expander-based breast reconstruction with adjuvant XRT and improving surgical outcomes.

SAXS-CT: a nanostructure resolving microscopy for macroscopic biologic specimens

SAXS-CT is an emerging powerful imaging technique which bridges the gap between information retrieved from high-resolution local techniques and information from low-resolution, large field-of-view imaging, to determine the nanostructure characteristics of well-ordered tissues, e.g., mineralized collagen in bone. However, in the case of soft tissues, features such as poor nanostructural organization and high susceptibility to radiation-induced damage limit the use of SAXS-CT. Here, by combining the freeze-drying the specimen, preceded by formalin fixation, with the nanostructure survey we identified and monitored alterations on the hierarchical arrangement of triglycerides and collagen fibrils three-dimensionally in breast tumor specimens without requiring sample staining. A high density of aligned collagen was observed precisely on the invasion front of the breast carcinoma, showing the direction of cancer spread, whereas substantial content of triglycerides was identified, where the healthy tissue was located. Finally, the approach developed here provides a path to high-resolution nanostructural probing with a large field-of-view, which was demonstrated through the visualization of characteristic nanostructural arrangement and quantification of content and degree of organization of collagen fibrils in normal, benign and malignant human breast tissue.

Elucidating Decorin\u2019s role in the preovulatory follicle

BackgroundDCN (decorin) is a proteoglycan known to be involved in regulating cell proliferation, collagen fibril organization and migration. In our global transcriptome RNA-sequencing approach to systematically identify new ovulation-associated genes, DCN was identified as one of the highly regulated genes. We therefore hypothesize that DCN may have a role in ovulatory processes such as cell migration and proliferation.AimTo characterize the expression, regulation and function of the proteoglycan DCN in the human ovarian follicles during the preovulatory period.MethodsThe in-vivo expression of DCN mRNA in mural (MGCs) and cumulus (CGCs) granulosa cells was characterized using quantitative RT-PCR and western blot.A signaling study was performed by treating human MGCs cultures with gonadotropins and different stimulators and inhibitors to determine their effect on DCN expression by qRT- PCR and elucidate the pathways regulating these proteins.In a functional study, KGN granulosa cell line was used to study cell migration with a scratch assay.ResultsDCN mRNA expression was significantly higher in MGCs compared to CGCs. DCN mRNA was significantly higher in CGCs surrounding mature metaphase II (MII) oocytes compared to CGCs of germinal vesicle (GV) and metaphase I (MI) oocytes.hCG significantly increased DCN mRNA and protein expression levels in cultured MGCs. Using signal transduction activators and inhibitors, we demonstrated that DCN induction by LH/hCG is carried out via PKA, PKC, ERK/MEK, and PI3K pathways.We showed that DCN expression is also induced in high-density cell cultures, in a dose-dependent pattern. In addition, progesterone induced a significant increase in DCN secretion to the media. MGCs from follicles of endometriosis patients exhibited reduced (about 20% of) mRNA transcriptions levels compared to MGCs follicles of control patients. More significantly, we found that DCN has an inhibiting effect on KGN cell migration.ConclusionsOur study indicates that DCN is a unique ovulatory gene.Our findings support the hypothesis that DCN plays an important new role during the preovulatory period and ovulation, and stress its involvement in endometriosis infertility. A better understanding of DCN role in ovulation and endometriosis may provide treatment for some types of infertility.

The Biointegration of a Porcine Acellular Dermal Matrix in a Novel Radiated Breast Reconstruction Model.

Ideal acellular dermal matrices (ADM) for breast reconstruction exhibit native extracellular matrix (ECM) structure to allow rapid biointegration and appropriate mechanical properties for desired clinical outcomes. In a novel in vivo model of irradiated breast reconstruction, we describe the cellular and vascular ingrowth of Artia, a porcine product chemically prepared to mimic the biomechanics of human ADM, with retained natural ECM structure to encourage cellular ingrowth. Utilizing the murine dorsal skinfold model, Artia was implanted into 16 C57bl/6 mice. Eight of the mice received a single dose 35 Gy radiation to the skin, followed by 12 weeks to produce radiation fibrosis and 8 mice served as nonradiated controls. Real-time photoacoustic microscopy of vascular integration and oxygen saturation within the ADM were made over 14 days. At 21 days, vascular ingrowth (CD31), fibroblast scar tissue formation (alpha smooth-muscle actin α-SMA, vimentin), and macrophage function (M2/M1 ratio) were evaluated. Scanning electron microscopy images of Artia were produced to help interpret the potential orientation of cellular and vascular ingrowth. Repeated photoacoustic microscopy imaging demonstrated vascular ingrowth increasing over 14 days, with a commensurate increase in oxygen saturation within both radiated and nonradiated ADM-albeit at an insignificantly lower rate in the radiated group. By day 21, robust CD31 staining was seen that was insignificantly greater in the nonradiated group. Of the fibroblast markers, vimentin expression was significantly greater in the radiated group (P < 0.05). Macrophage lineage phenotype was consistent with remodeling physiology in both radiated and nonradiated groups. Scanning electron microscopy demonstrated transversely organized collagen fibrils with natural porous ECM structure to allow cellular ingrowth. Artia demonstrates appropriate biointegration, with increased oxygen saturation by 14 days, consistent with the performance of other collagen substrates in this model. Radiation fibrosis resulted in higher vimentin expression yet did not impact macrophage phenotype while only modestly decreasing Artia biointegration suggesting that ADM may have a role in reconstructive efforts in a radiated setting. Taken together with its enhanced biomechanics, this porcine ADM product is well poised to be clinically applicable to breast reconstruction.

Transcriptome Profile of Human Fibroblasts in an Ex Vivo Culture.

Implantation of autologous fibroblasts is a method used to correct age-related changes in facial skin. The aim of this study was to establish the optimal population of cultured human fibroblasts according to the organization of the extracellular matrix in the dermis. Transcriptome profile analysis of cells derived from three consecutive passages indicated that fibroblasts after the second passage were the population with the greatest number of upregulated genes encoding the critical biological processes responsible for skin regeneration, such as extracellular matrix organization, collagen fibril organization, and cell adhesion. Furthermore, genes encoding proteinases responsible for the degradation of dermal extracellular matrix proteins were noticeably downregulated at this stage of culture. Autologous fibroblasts seem to be an optimal and safe biological filler for the renewal of all skin structures.

DNA Methylation-based Diagnostic and Prognostic Biomarkers for Glioblastoma

Glioblastomas are the most common primary central nervous system malignancy tumor in adults. Glioblastoma patients have poor prognosis, with an average survival period of approximately 14 mo after diagnosis. To date, there are a limited number of effective treatment methods for glioblastoma, and its molecular mechanisms remain elusive. In this article, we analyzed the key biomarkers and pathways in glioblastoma patients based on gene expression and DNA methylation datasets. The 60 hypomethylated/upregulated genes and 110 hypermethylated/downregulated genes were identified in GSE50923, GSE50161, and GSE116520 microarrays. Functional enrichment analyses indicated that these methylated-differentially expressed genes were primarily involved in collagen fibril organization, chemical synaptic transmission, extracellular matrix-receptor interaction, and GABAergic synapse. The hub genes were screened from a protein–protein interaction network; in selected genes, increased NMB mRNA level was associated with favorable overall survival, while elevated CHI3L1, POSTN, S100A4, LOX, S100A11, IGFBP2, SLC12A5, VSNL1, and RGS4 mRNA levels were associated with poor overall survival in glioblastoma patients. Additionally, CHI3L1, S100A4, LOX, and S100A11 expressions were negatively correlated with their corresponding methylation status. Furthermore, the receiver-operator characteristic curve analysis indicated that CHI3L1, S100A4, LOX, and S100A11 can also serve as highly specific and sensitive diagnostic biomarkers for glioblastoma patients. Collectively, our study revealed the possible methylated-differentially expressed genes and associated pathways in glioblastoma and identified four DNA methylation-based biomarkers of glioblastoma. These results may provide insight on diagnostic and prognostic biomarkers, and therapeutic targets in glioblastoma.

Corneal extracellular matrix decellularization.

Decellularized corneal scaffolds have the potential to be used as alternatives to donor corneas during keratoplasty. Here a decellularization technique is described that involves the use of sodium dodecyl sulfate, Triton-X100, DNAse and RNAse to remove cells and cellular constituents. We have previously found that this combination of chemicals and enzymes to be effective at removing cells while retaining extracellular matrix proteins. In addition, different methods for assessing if the decellularization process has been successful are discussed. These include techniques to identify and quantify the presence of cells, DNA and extracellular matrix components as well as methods to examine the collagen fibril organization and scaffold transparency.