Matrix Proteoglycans Research Articles

Longitudinal Imaging of the Skull Base Synchondroses Demonstrate Prevention of a Premature Ossification After Recifercept Treatment in Mouse Model of Achondroplasia.

ABSTRACTAchondroplasia is the most common form of short‐limb dwarfism. In this disorder, endochondral ossification is impaired due to gain‐of‐function mutation in the Fibroblast Growth Factor Receptor 3 (FGFR3) gene. In addition to short limbs, cranial base bones are also affected leading to shortening of the skull base and to serious neurological complications associated with foramen magnum stenosis. These complications are thought to be due to the delay or premature arrest of skull base growth, caused by an accelerated ossification of the sphenooccipital (SOS) and the intraoccipital (IOS) synchondroses. Skull synchondroses consist of two opposite growth plates sharing a common reserve zone of chondrocytes. In this study, we first characterized the skull base synchondroses ossification in a mouse model of achondroplasia carrying the human G380R mutation (Fgfr3 ach/+ ). We then addressed whether Recifercept, a soluble FGFR3, could prevent premature closure of these synchondroses. Postnatal radiological observations revealed the presence of bony bridge structures in one or more synchondroses in Fgfr3 ach/+ mice as early as postnatal day 3 in the most severe cases. The presence of early ossification correlated with the severity of the disease as it was associated with an arrest of the cranial base bone growth. Histological analyses indicated changes in the synchondroses structure and matrix proteoglycan contents confirming a process of ossification. Treatment of Fgfr3 ach/+ mice with Recifercept compared with vehicle prevented premature synchondrosis ossification and the transition to bone, resulting in improved skull shape and cranium ratio. Given the impact of Recifercept on synchondrosis inactivation, it is possible that it could prevent one of the most severe complication of achondroplasia if used early enough during bone development. These data support the clinical development of Recifercept for achondroplasia, and suggests that early treatment may be required to best address impaired endochondral bone growth. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Non-contact acoustic micro-tapping optical coherence elastography (AμT-OCE) in the cornea: Mechanical model, ex vivo and in vivo results, and direct comparison with mechanical tests

The cornea is one of the primary determinants of visual performance. Its highly pressurized anisotropic structure of collagen fibrils embedded in a hydrated proteoglycan matrix forms the clear refractive primary lens of the eye. Despite recent success in corneal topography, there are no clinical tools to predict shape changes from vision-correction therapies such as LASIK and CXL due to the absence of non-contact methods that can map corneal elasticity in vivo. Here we review our newest results in quantification of corneal elasticity. Recently, we produced a technology combining non-contact excitation of sub-mm wavelength mechanical waves in the cornea using air-coupled acoustic micro-tapping (AμT), with 1.6 MHz scan-rate phase-sensitive OCT to track their propagation three-dimensionally in real time. However, corneal moduli reconstruction from measured wavefields requires an anisotropic elastic model and cornea’s bounded, non-flat geometry must be taken into account. Thus, a nearly incompressible transversally isotropic (NITI) model of corneal elasticity was developed. We have performed multiple ex vivo and in vivo studies in rabbit, porcine and human corneas and compared AμT-OCE results directly with conventional mechanical tests (tensile extensometry and shear rheometry). These studies demonstrate that in-plane Young’s and out-of-plane shear moduli can be accurately mapped with non-contact, non-invasive AμT-OCE.

Role of Lipid Accumulation and Inflammation in Atherosclerosis: Focus on Molecular and Cellular Mechanisms.

Atherosclerosis is a chronic lipid-driven and maladaptive inflammatory disease of arterial intima. It is characterized by the dysfunction of lipid homeostasis and signaling pathways that control the inflammation. This article reviews the role of inflammation and lipid accumulation, especially low-density lipoprotein (LDL), in the pathogenesis of atherosclerosis, with more emphasis on cellular mechanisms. Furthermore, this review will briefly highlight the role of medicinal plants, long non-coding RNA (lncRNA), and microRNAs in the pathophysiology, treatment, and prevention of atherosclerosis. Lipid homeostasis at various levels, including receptor-mediated uptake, synthesis, storage, metabolism, efflux, and its impairments are important for the development of atherosclerosis. The major source of cholesterol and lipid accumulation in the arterial wall is proatherogenic modified low-density lipoprotein (mLDL). Modified lipoproteins, such as oxidized low-density lipoprotein (ox-LDL) and LDL binding with proteoglycans of the extracellular matrix in the intima of blood vessels, cause aggregation of lipoprotein particles, endothelial damage, leukocyte recruitment, foam cell formation, and inflammation. Inflammation is the key contributor to atherosclerosis and participates in all phases of atherosclerosis. Also, several studies have shown that microRNAs and lncRNAs have appeared as key regulators of several physiological and pathophysiological processes in atherosclerosis, including regulation of HDL biogenesis, cholesterol efflux, lipid metabolism, regulating of smooth muscle proliferation, and controlling of inflammation. Thus, both lipid homeostasis and the inflammatory immune response are closely linked, and their cellular and molecular pathways interact with each other.

Elastic, Dynamic Viscoelastic and Model-Derived Fibril-Reinforced Poroelastic Mechanical Properties of Normal and Osteoarthritic Human Femoral Condyle Cartilage

Osteoarthritis (OA) degrades articular cartilage and weakens its function. Modern fibril-reinforced poroelastic (FRPE) computational models can distinguish the mechanical properties of main cartilage constituents, namely collagen, proteoglycans, and fluid, thus, they can precisely characterize the complex mechanical behavior of the tissue. However, these properties are not known for human femoral condyle cartilage. Therefore, we aimed to characterize them from human subjects undergoing knee replacement and from deceased donors without known OA. Multi-step stress-relaxation measurements coupled with sample-specific finite element analyses were conducted to obtain the FRPE material properties. Samples were graded using OARSI scoring to determine the severity of histopathological cartilage degradation. The results suggest that alterations in the FRPE properties are not evident in the moderate stages of cartilage degradation (OARSI 2-3) as compared with normal tissue (OARSI 0-1). Drastic deterioration of the FRPE properties was observed in severely degraded cartilage (OARSI 4). We also found that the FRPE properties of femoral condyle cartilage related to the collagen network (initial fibril-network modulus) and proteoglycan matrix (non-fibrillar matrix modulus) were greater compared to tibial and patellar cartilage in OA. These findings may inform cartilage tissue-engineering efforts and help to improve the accuracy of cartilage representations in computational knee joint models.

In vitro detection of structural changes in chondrogenic mesenchymal stem cell pellets at the nano-scale using nano-sensitive optical coherance tomography

In vitro detection of structural changes in chondrogenic mesenchymal stem cell pellets at the nano-scale using nano-sensitive optical coherance tomography

Proteomic analysis of lacrimal gland adenoid cystic carcinoma with high-grade transformation

Objective: To analyze the protein expression differences of lacrimal gland adenoid cystic carcinoma (LACC) with high-grade transformation (HGT). Methods: Experimental study. A total of 8 paraffin tissue samples were collected in Tianjin Medical University Eye Hospital from December 2012 to January 2019. According to pathological examination, the samples were divided into the LACC group and the LACC-HGT group, with 4 cases in each group. The LACC group included 2 male samples and 2 female samples, with an average age of 53 years. The LACC-HGT group included 2 male samples and 2 female samples, with an average age of 44 years. Primary cells were cultured from fresh tumor tissue. Isobaric tags for relative and absolute quantification techniques were used to screen the differentially expressed proteins between the two groups, and bioinformatics analysis was conducted for the differentially expressed proteins. Microarray was used to screen differentially expressed mRNAs between LACC and LACC-HGT primary cells. The mass spectrum data were intersected with mRNA microarray data, and quantitative real-time (qRT) PCR was performed to verify the results. Proteomics and microarray data were compared using the independent sample t test. The qRT-PCR data were compared pairwise by one-way analysis of variance. Results: A total of 105 HGT-related differential proteins were detected in this study, including 50 up-regulated proteins and 55 down-regulated proteins. The significantly up-regulated proteins included hemoglobin subunit beta, hemoglobin subunit alpha 1, and collagen type Ⅵ alpha 2 chain; the significantly down-regulated proteins included Cereblon, adenosylhomocysteinase like 2, and ribosomal protein L39 pseudogene 5. Gene ontology analysis results showed that the LACC-HGT differential proteins were mainly located in the cytoplasm, vesicle cavity, and extracellular matrix, had organic acid binding and molecular carrier activity, and participated in the regulation of extracellular matrix composition, immunity, inflammation, apoptosis, and other biological processes. Pathway analysis showed that the LACC-HGT differential proteins were mainly involved in signal pathways such as mitogen-activated protein kinase signal pathway and extracellular matrix proteoglycans and glycan metabolism signal pathway. Protein complex prediction analysis screened out 4 up-regulated protein complexes and 1 down-regulated protein complex. There were 15 LACC-HGT differential proteins that overlapped with mRNA chip differential genes, of which 6 were tumor-related proteins including collagen type XIV alpha 1 chain (COL14A1), EMAP like 4 (EML4), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), NDRG family member 2 (NDRG2), osteoglycin (OGN) an Ras homolog family member C (RhoC). The main function was the movement and migration of tumor cells. The qRT-PCR results showed that the relative expression levels of COL14A1, EML4, ITIH4, NDRG2, OGN, and RhoC in primary LACC-1, LACC-2, LACC-HGT-1, and LACC-HGT-2 cells were significantly different (F=1 675.98, 38.53, 27.37, 16.47, 13.38, 25.22, all P<0.01). For example, the relative expression of COL14A1 in primary LACC-HGT-1 (16.09±0.51) and LACC-HGT-2 (9.96±0.34) cells was significantly higher than that in primary LACC-1 (1.00±0.13) and LACC-2 (0.67±0.08) cells (all P<0.05). Conclusion: There are differentially expressed proteins between LACC-HGT and LACC, among which COL14A1, EML4, ITIH4, NDRG2, OGN, and RhoC may play an important role in LACC-HGT and can be used as potential targets of LACC-HGT in further study. (Chin J Ophthalmol, 2021, 57: 531-539).

Abstract 2734: Impact of the mutation profile and versican status on lymphocyte infiltration in early age onset colorectal cancer

Abstract Background: The incidence of colorectal cancer (CRC) in those of pre-screening age continues to rise. A deeper understanding of the differences in tumor biology in early age onset (EAO) cancers is needed. Differences in the tumor microenvironment have been understudied in this setting. We previously have identified versican (VCAN), a large matrix proteoglycan, as an important factor in lymphocyte exclusion in CRC. Here we explore the impact of the mutation profile and VCAN status on the infiltration of CD8+ and CD4+ lymphocytes in EAO CRC. Methods: Cancer tissues from 153 patients with CRC were stained via immunohistochemistry for CD4, CD8, and VCAN. CD4 and CD8 stains were quantified by number of positive-staining tumor-infiltrating lymphocytes per high powered field (TILs/HPF). VCAN stains were quantified on an intensity scale from 0-3+. 121 of these patients had tissue that was sequenced using the Qiagen Comprehensive Cancer targeted sequencing panel, and variations were called using Strelka. Patients were split by age into early-onset (EAO; age at diagnosis &amp;lt;50) and later-onset (LAO; age at diagnosis 50 or later). Results: Mutations in BRAF and APC were significantly more common in LAO cancers (p&amp;lt;0.05 for both). TP53 mutations were correlated with significantly lower CD4+ and CD8+ TILs/HPF in the LAO cohort (12.6 CD4+ TILs/HPF for TP53-wild type [WT] vs 3.5 for TP53-mutant, p&amp;lt;0.001; 10.0 CD8+ TILs/HPF for WT vs 3.2 for mutant, p&amp;lt;0.01), but trended opposite, though not statistically significant, in the EAO group (2.4 vs 5.9 CD4+ TILs/HPF; 3.0 vs 5.9 CD8+ TILs/HPF). PIK3CA mutations were correlated with an increase in CD8+ TILs in the LAO cohort (5.3 vs 11.9 CD8+ TILs/HPF; p&amp;lt;0.05) but a decrease in CD4+ TILs in the EAO cohort (4.9 vs 0.8 CD4+ TILs/HPF; p&amp;lt;0.05). TP53-WT tumors are disproportionately low in VCAN in the LAO cohort (p&amp;lt;0.01). Differences in VCAN accumulation could account for the changes in CD8+ and CD4+ T lymphocyte infiltration across the age cohorts and TP53 mutation status. Conclusions: The impacts of TP53 and PIK3CA mutations on immune infiltration in CRC differ in EAO compared to later onset cancers, indicating that the processes controlling immune infiltration vary between age cohorts. Such differences in immune infiltration may be linked to changes in microenvironmental factors such as VCAN. These data warrant further investigation into the relationship between tumor microenvironment and mutation profiles in EAO CRC. Citation Format: Katherine A. Johnson, Philip B. Emmerich, Anna L. Lippert, Cheri A. Pasch, Linda Clipson, Wei Zhang, Kristina A. Matkowskyj, Dustin A. Deming. Impact of the mutation profile and versican status on lymphocyte infiltration in early age onset colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2734.

Versican (VCAN) Proteolysis Predicts T-Cell Infiltration in Myeloma Bone Marrow Post- Autologous Stem Cell Transplant (ASCT)

Background/ Hypothesis:Versican (VCAN), a large matrix proteoglycan, accumulates in myeloma lesions (Hope et al., Blood 123(21): 3305-3315, 2014). VCAN sustains tumor-promoting inflammation but also generates immunosuppression through IL-6 and IL-10-mediated antigen-presenting cell dysfunction (Tang et al., Cell Rep 13(12): 2851-2864, 2015). VCAN proteolysis generates bioactive fragments (VCAN-derived matrikines, e.g., versikine) that oppose the functions of the parent macromolecule, intact VCAN (Hope et al., Blood 128(5): 680-685, 2016). VCAN-matrikines promote “T-cell inflammation” through co-ordinated effects on antigen-presenting and myeloid regulatory cells. In particular, we have shown that VCAN-matrikines promote Batf3-lineage dendritic cell (DC) differentiation from Flt3L-mobilized bone marrow progenitors and induce IL-12 but not IL-10 production by myeloid cells (Hope et al., J Immunol, in press, 2017). Batf3-DC constitute an intratumorally sparse DC subset with critical roles in tumor antigen cross-presentation and effector T-cell homing. Recently, VCAN-producing macrophages were shown to expand post-ASCT in myeloma and correlate with minimal residual disease (MRD) persistence (Arana, Zabaleta et al., Blood 128(22): 482-482, 2016). However, it is unknown whether VCAN proteolysis predicts T-cell infiltration post-ASCT for myeloma at a timepoint (Day 90-100 post-ASCT) when MRD is routinely quantitated and immunotherapies to control MRD are often instituted.Specific Aims:1.To detect VCAN proteolysis in paraffin-embedded bone marrow tissue obtained post-ASCT for myeloma2.To investigate the relationship between T-cell infiltration and VCAN proteolysis in post-ASCT myeloma bone marrowStudy Design:Bone marrow core biopsies from 40 patients who underwent ASCT for myeloma were analyzed at day 90-100 post-ASCT. ADAMTS protease-mediated VCAN proteolysis was analyzed by immunohistochemistry (IHC) through detection of neo-epitope DPEAAE as previously shown (Hope et al., J Immunol, in press, 2017). DPEAAE staining intensity was scored in the following scale: 0=none, +=patchy, ++= diffuse /weak, +++= diffuse/ strong. T-cell infiltration was scored in the following scale: rare, dispersed and intense. Clusters of T-cells (>5 CD8+ cells clustered together) were independently scored. Granzyme B staining status by IHC was scored using a scale similar to CD8+ status.Results:We found that prior de-calcification procedures did not prevent optimal evaluation and scoring of VCAN proteolysis status by IHC. Intense CD8+ T-cell infiltration and/or CD8+ clustering were only seen in the presence of intense (++ or +++) VCAN proteolysis. 14/14 marrows classified as displaying intense CD8+ infiltration plus CD8+ clusters were also scored as VCAN proteolysis ++/+++. 17/17 marrows with intense CD8+ infiltration were scored as VCAN-proteolysis ++/+++. 18/18 marrows with at least one CD8+ cluster detected in the specimen were VCAN proteolysis ++/+++. 67% of marrows (n=12/18) with +++ VCAN proteolysis had intense CD8+ infiltration. 83% (n=15/18) of marrows with +++ VCAN proteolysis had at least one CD8+ cluster detected. Intense staining for granzyme B was only seen in the presence of ++/+++ VCAN proteolysis (n=10/10).Conclusions/ Future implications:Intense VCAN proteolysis correlated with evidence of adaptive immune activation post-ASCT. T-cell infiltration in myeloma bone marrow post-ASCT was exclusively seen in the presence of intense VCAN proteolysis. Mechanistically, we hypothesize that T-cell infiltration is the result of enhanced Batf3-DC density in VCAN proteolysis-rich myeloma tumor microenvironments. We are currently investigating VCAN proteolysis-detection as an immune biomarker to predict efficacy of anti- myeloma immunotherapies as well as in relationship with MRD status post ASCT. There is a dearth of immune biomarkers that can be analyzed on standard de-calcified, paraffin-embedded bone marrow core specimens. For example, PD-L1 detection is problematic using de-calcified bone marrow specimens. By contrast, detection of VCAN proteolysis was unaffected by the de-calcification process. VCAN-matrikines (e.g., versikine) can also be therapeutically used to boost Batf3-DC whose paucity is often the limiting factor for efficacy of checkpoint inhibition and other immunotherapies. DisclosuresDhakal:Celgene: Honoraria.

In vitro and in vivo effect of polycaprolactone nanofiber coating on polyethylene glycol diacrylate scaffolds for intervertebral disc repair

Polyethylene glycol diacrylate (PEGDA) is an important class of photosensitive polymer with many tissue engineering applications. This study compared PEGDA and polycaprolactone (PCL) nanofiber matrix (NFM) coated PEGDA, referred to as PCL-PEGDA, scaffolds for their application in multiple tissue repair such as articular cartilage, nucleus pulposus of the intervertebral disc (IVD). We examined each scaffold morphology, porosity, swelling ratio, degradation, mechanical strength, and in vitro cytocompatibility properties. A defect was created in Sprague Dawley rat tail IVD by scraping native cartilage tissue and disc space, then implanting the scaffolds in the disc space for 4 weeks to evaluate in vivo efficacy of multi-tissue repair. Maintenance of disc height and creation of a new cell matrix was assessed to evaluate each scaffold’s ability to repair the tissue defect. Although both PEGDA and PCL-PEGDA scaffolds showed similar porosity ∼73%, we observed distinct topographical characteristics and a higher effect of degradation on the water-absorbing capacity for PEGDA compared to PCL-PEGDA. Mechanical tests showed higher compressive strength and modulus of PCL-PEGDA compared to PEGDA. In vitro cell studies show that the PCL NFM layer covering PEGDA improved osteoblast cell adhesion, proliferation, and migration into the PEGDA layer. In vivo studies concluded that the PEGDA scaffold alone was not ideal for implantation in rat caudal disc space without PCL nanofiber coating due to low compressive strength and modulus. In vivo results confirm that the PCL-PEGDA scaffold-maintained disc space and created a proteoglycan and collagen-rich new tissue matrix in the defect site after 4 weeks of scaffold implantation. We concluded that our developed PCL-PEGDA has the potential to be used in multi-tissue defect site repair.

Lactate oxidative phosphorylation by annulus fibrosus cells: evidence for lactate-dependent metabolic symbiosis in intervertebral discs

BackgroundIntervertebral disc degeneration contributes to low back pain. The avascular intervertebral disc consists of a central hypoxic nucleus pulpous (NP) surrounded by the more oxygenated annulus fibrosus (AF). Lactic acid, an abundant end-product of NP glycolysis, has long been viewed as a harmful waste that acidifies disc tissue and decreases cell viability and function. As lactic acid is readily converted into lactate in disc tissue, the objective of this study was to determine whether lactate could be used by AF cells as a carbon source rather than being removed from disc tissue as a waste byproduct.MethodsImport and conversion of lactate to tricarboxylic acid (TCA) cycle intermediates and amino acids in rabbit AF cells were measured by heavy-isotope (13C-lactate) tracing experiments using mass spectrometry. Levels of protein expression of lactate converting enzymes, lactate importer and exporter in NP and AF tissues were quantified by Western blots. Effects of lactate on proteoglycan (35S-sulfate) and collagen (3H-proline) matrix protein synthesis and oxidative phosphorylation (Seahorse XFe96 Extracellular Flux Analyzer) in AF cells were assessed.ResultsHeavy-isotope tracing experiments revealed that AF cells imported and converted lactate into TCA cycle intermediates and amino acids using in vitro cell culture and in vivo models. Addition of exogenous lactate (4 mM) in culture media induced expression of the lactate importer MCT1 and increased oxygen consumption rate by 50%, mitochondrial ATP-linked respiration by 30%, and collagen synthesis by 50% in AF cell cultures grown under physiologic oxygen (2-5% O2) and glucose concentration (1-5 mM). AF tissue highly expresses MCT1, LDH-H, an enzyme that preferentially converts lactate to pyruvate, and PDH, an enzyme that converts pyruvate to acetyl-coA. In contrast, NP tissue highly expresses MCT4, a lactate exporter, and LDH-M, an enzyme that preferentially converts pyruvate to lactate.ConclusionsThese findings support disc lactate-dependent metabolic symbiosis in which lactate produced by the hypoxic, glycolytic NP cells is utilized by the more oxygenated AF cells via oxidative phosphorylation for energy and matrix production, thus shifting the current research paradigm of viewing disc lactate as a waste product to considering it as an important biofuel. These scientifically impactful results suggest novel therapeutic targets in disc metabolism and degeneration.

Clinical Relevance of Elastin in the Structure and Function of Skin.

Elastin is the main component of elastic fibers, which provide stretch, recoil, and elasticity to the skin. Normal levels of elastic fiber production, organization, and integration with other cutaneous extracellular matrix proteins, proteoglycans, and glycosaminoglycans are integral to maintaining healthy skin structure, function, and youthful appearance. Although elastin has very low turnover, its production decreases after individuals reach maturity and it is susceptible to damage from many factors. With advancing age and exposure to environmental insults, elastic fibers degrade. This degradation contributes to the loss of the skin’s structural integrity; combined with subcutaneous fat loss, this results in looser, sagging skin, causing undesirable changes in appearance. The most dramatic changes occur in chronically sun-exposed skin, which displays sharply altered amounts and arrangements of cutaneous elastic fibers, decreased fine elastic fibers in the superficial dermis connecting to the epidermis, and replacement of the normal collagen-rich superficial dermis with abnormal clumps of solar elastosis material. Disruption of elastic fiber networks also leads to undesirable characteristics in wound healing, and the worsening structure and appearance of scars and stretch marks. Identifying ways to replenish elastin and elastic fibers should improve the skin’s appearance, texture, resiliency, and wound-healing capabilities. However, few therapies are capable of repairing elastic fibers or substantially reorganizing the elastin/microfibril network. This review describes the clinical relevance of elastin in the context of the structure and function of healthy and aging skin, wound healing, and scars and introduces new approaches being developed to target elastin production and elastic fiber formation.

Differential requirement of NPHP1 for compartmentalized protein localization during photoreceptor outer segment development and maintenance.

Nephrocystin (NPHP1) is a ciliary transition zone protein and its ablation causes nephronophthisis (NPHP) with partially penetrant retinal dystrophy. However, the precise requirements of NPHP1 in photoreceptors are not well understood. Here, we characterize retinal degeneration in a mouse model of NPHP1 and show that NPHP1 is required to prevent infiltration of inner segment plasma membrane proteins into the outer segment during the photoreceptor maturation. We demonstrate that Nphp1 gene-trap mutant mice, which were previously described as null, are likely hypomorphs due to the production of a small quantity of functional mRNAs derived from nonsense-associated altered splicing and skipping of two exons including the one harboring the gene-trap. In homozygous mutant animals, inner segment plasma membrane proteins such as syntaxin-3 (STX3), synaptosomal-associated protein 25 (SNAP25), and interphotoreceptor matrix proteoglycan 2 (IMPG2) accumulate in the outer segment when outer segments are actively elongating. This phenotype, however, is spontaneously ameliorated after the outer segment elongation is completed. Consistent with this, some photoreceptor cell loss (~30%) occurs during the photoreceptor maturation period but it stops afterward. We further show that Nphp1 genetically interacts with Cep290, another NPHP gene, and that a reduction of Cep290 gene dose results in retinal degeneration that continues until adulthood in Nphp1 mutant mice. These findings demonstrate that NPHP1 is required for the confinement of inner segment plasma membrane proteins during the outer segment development, but its requirement diminishes as photoreceptors mature. Our study also suggests that additional mutations in other NPHP genes may influence the penetrance of retinopathy in human NPHP1 patients.

Role of ECM in the Brain‐Gut Connection

We sought to determine the role of extracellular matrix (ECM) proteoglycans on regulating brain function and cell metabolism. In the first series of studies, we used dietary fructose to perturbate metabolism based on evidence that fructose is a major contributor to the epidemic of diabetes and obesity. Our transcriptomic studies (Meng et al, EBiomed, 157(7), 2016) showed that the gene for the proteoglycan biglycan (Bgn) occupies a central position in a network of genes receptive to the action of fructose in the hypothalamus (main brain center of metabolic control). We pursued studies further and exposed male biglycan knockout mice (Bgn0/-) to fructose for 7 weeks, and results showed that the Bgn0/- gene perturbation compensated for a decrement in learning and memory performance in mice exposed to fructose (Ying et al., BBADIS, 1864(12) 2018). Learning and memory performance was consistent with an attenuation of the action of fructose on hippocampal plasticity markers CREB and BDNF. Bgn siRNA treatment abolished the effects of fructose on CREB and BDNF levels, in conjunction with reducing a fructose-related increase in Bgn protein. In addition, fructose consumption perturbed the systemic metabolism of glucose and lipids, that were also altered in the Bgn0/ mice. Transcriptomic profiling of hypothalamus, hippocampus, and liver supported the regulatory action of Bgn on key molecular pathways involved in metabolism, immune response, and neuronal plasticity. Overall results underscore the tissue-specific role of the extracellular matrix in the regulation of metabolism and brain function, and support Bgn as a key modulator of the impact of fructose across body and brain. In separate studies, we found that the gene for the proteoglycan fibromodulin (Fmod) controls a network of genes responsive to the action of brain trauma (EBiomed, 184(16) 2017). The action of Fmod (42-80 KDa) on the brain is poorly understood and rather known for affecting fibrillogenesis, cell adhesion, cytokine activity in the periphery. We found that deletion of Fmod gene mitigated the TBI-related increase in the pro-inflammatory marker toll like receptor 4 (TLR4), and attenuated a reduction in cell metabolic markers PGC1alpha and cytochrome oxidase II (COII), in the mouse hypothalamus after TBI. The latter suggests that Fmod deletion can protect against the cell metabolic crisis observed after TBI. In addition, given the regulatory role of the hypothalamus on peripheral metabolism, we sought to determine the effects of Fmod genetic deletion on the homeostasis of lipids in the liver on mice exposed to TBI. Results showed that the deletion of the Fmod gene protected against the action of brain trauma on key lipids and the levels of fatty acid synthase (FAS), a major lipogenic enzyme commonly involved in fatty acid processing. These results suggest a potential effect of Fmod on brain trauma outcome in brain and periphery.

Dynamic Tuning of Viscoelastic Hydrogels with Carbonyl Iron Microparticles Reveals the Rapid Response of Cells to Three-Dimensional Substrate Mechanics.

Current methods to dynamically tune three-dimensional hydrogel mechanics require specific chemistries and substrates that make modest, slow, and often irreversible changes in their mechanical properties, exclude the use of protein-based scaffolds, or alter the hydrogel microstructure and pore size. Here, we rapidly and reversibly alter the mechanical properties of hydrogels consisting of extracellular matrix proteins and proteoglycans by adding carbonyl iron microparticles (MPs) and applying external magnetic fields. This approach drastically alters hydrogel mechanics: rheology reveals that application of a 4000 Oe magnetic field to a 5 mg/mL collagen hydrogel containing 10 wt % MPs increases the storage modulus from approximately 1.5 to 30 kPa. Cell morphology experiments show that cells embedded within these hydrogels rapidly sense the magnetically induced changes in ECM stiffness. Ca2+ transients are altered within seconds of stiffening or subsequent softening, and slower but still dynamic changes occur in YAP nuclear translocation in response to time-dependent application of a magnetic field. The near instantaneous change in hydrogel mechanics provides new insight into the effect of changing extracellular stiffness on both acute and chronic changes in diverse cell types embedded in protein-based scaffolds. Due to its flexibility, this method is broadly applicable to future studies interrogating cell mechanotransduction in three-dimensional substrates.

Mechanical response of composite fiber networks subjected to local contractile deformation

The extracellular matrix (ECM), forming the cell microenvironment, is a random fiber network whose heterogeneous microstructure serves a vital role in cellular functions such as migration, proliferation, and differentiation. Although prior computational studies used a network of randomly crosslinked fibers for investigating the ECM mechanical response due to the cell contraction, we hereby investigate the cell-induced deformation by considering the interplay between the collagen fibers and soft proteoglycan matrix domain. For this purpose, we represent the complex ECM microstructure by a double network model in which a random network of relatively stiff fibers (representing collagens) is embedded in a homogeneous network (representing the matrix domain). We demonstrate that the interaction between the network of stiff fibers and the network of matrix fibers plays a significant role in the propagation of cell-induced displacements. In particular, as the stiffness of the matrix domain increases, the long-range displacement propagation becomes suppressed. Furthermore, the degree of inhomogeneity in the displacement propagation is controlled by the relative stiffness of fiber and matrix networks. The influence of matrix stiffness becomes prominent for composite networks with random networks having a low average network connectivity and/or composed of fibers with low bending rigidity.

Mitoprotective therapy reduces inflammation and prevents cartilage turnover in an in vivo model of posttraumatic osteoarthritis

Mitoprotective therapy reduces inflammation and prevents cartilage turnover in an in vivo model of posttraumatic osteoarthritis

LncRNA-CRNDE regulates BMSC chondrogenic differentiation and promotes cartilage repair in osteoarthritis through SIRT1/SOX9.

Osteoarthritis (OA) is the most common chronic and degenerative joint disease. Although traditional OA medications can partially relieve pain, these medications cannot completely cure OA. Therefore, it is particularly important to find an effective treatment for OA. This study explored the function of long non-coding RNA (lncRNA)-colorectal neoplasia differentially expressed gene (CRNDE) in the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and the underlying molecular mechanism, aiming to develop a new treatment method for osteoarthritis. BMSCs were isolated from rat bone marrow using the gradient centrifugation method. And BMSC chondrogenic differentiation was induced with chondrogenic medium. The expression of lncRNA-CRNDE was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Silent information regulator factor 2-related enzyme 1(SIRT1) and cartilage marker genes Aggrecan and collagen 2 (α1) protein expression were researched using western blot. Alcian blue staining was employed to examine the content of cartilage matrix proteoglycan glycosaminoglycan (GAG). The interaction between lncRNA-CRNDE and SIRT1 was detected by RNA pull-down and RNA immunoprecipitation (RIP) assay. Ubiquitination experiments were performed to measure the ubiquitination level of SIRT1. The combination between SMAD ubiquitination regulatory factor 2 (SMURF2) and SIRT1, as well as SRY-related high-mobility-group box 9 (SOX9) and collagen 2 (α1) promoter, was detected by Co-immunoprecipitation or ChIP. With the prolongation of induction time, the expression of lncRNA-CRNDE, SIRT1, cartilage marker genes Aggrecan and collagen 2 (α1) in BMSC osteogenic differentiation was gradually increased. Also, the content of cartilage matrix proteoglycan GAG was gradually elevated with the extension of the induction time. Further increase in the expression of SIRT1, cartilage marker genes Aggrecan and collagen 2 (α1) by overexpression of lncRNA-CRNDE also indicated elevated GAG content. RNA pull-down and RIP assay confirmed the binding between lncRNA-CRNDE and SIRT1. qRT-PCR and western blot showed that interference with lncRNA-CRNDE significantly inhibited the protein expression of SIRT1. BMSCs transfected with si-CRNDE increased ubiquitination levels of SIRT1 mediated by the E3 ligase SMURF2, leading to the reduced protein stability of SIRT1. However, overexpression of lncRNA-CRNDE increased the binding ability of SOX9 and collagen 2 (α1) promoter, which was reversed by the simultaneous transfection ofCRNDE overexpression (pcDNA-CRNDE) and SIRT1small interferingRNA (si-SIRT1). lncRNA-CRNDE regulates BMSC chondrogenic differentiation to promote cartilage repair in osteoarthritis through SIRT1/SOX9.

Decorin-mediated suppression of tumorigenesis, invasion, and metastasis in inflammatory breast cancer

Inflammatory breast cancer (IBC) is a clinically distinct and highly aggressive form of breast cancer with rapid onset and a strong propensity to metastasize. The molecular mechanisms underlying the aggressiveness and metastatic propensity of IBC are largely unknown. Herein, we report that decorin (DCN), a small leucine-rich extracellular matrix proteoglycan, is downregulated in tumors from patients with IBC. Overexpression of DCN in IBC cells markedly decreased migration, invasion, and cancer stem cells in vitro and inhibited tumor growth and metastasis in IBC xenograft mouse models. Mechanistically, DCN functioned as a suppressor of invasion and tumor growth in IBC by destabilizing E-cadherin and inhibiting EGFR/ERK signaling. DCN physically binds E-cadherin in IBC cells and accelerates its degradation through an autophagy-linked lysosomal pathway. We established that DCN inhibits tumorigenesis and metastasis in IBC cells by negatively regulating the E-cadherin/EGFR/ERK axis. Our findings offer a potential therapeutic strategy for IBC, and provide a novel mechanism for IBC pathobiology.

Glabridin inhibits osteoarthritis development by protecting chondrocytes against oxidative stress, apoptosis and promoting mTOR mediated autophagy

Osteoarthritis (OA) is a common chronic degenerative disease that affects the elderly. Thus far, no pharmacological therapy approved by regulators has shown a convincing effect on OA. Glabridin, a small molecule, is a well-known and powerful natural antioxidant, which has a strong scavenging effect on free radicals. This study attempted to explore the role and underlying mechanisms of Glabridin on OA both in vitro and in vivo. In the in vitro study, Glabridin was found to increase the expression levels of extracellular matrix (ECM) related genes, Collagen II, Aggrecan (ACAN), SRY-box 9 (SOX9) and proteoglycan 4 (PRG4). Moreover, Glabridin was observed to significantly reduce the level of oxidative stress in OA chondrocytes while effectively reducing the apoptosis of chondrocytes. Glabridin was also found to significantly increase the autophagy of human OA chondrocytes. During the in vivo study, intraarticular injection of Glabridin was observed to alleviate OA progression and protect chondrocytes against apoptosis following anterior cruciate ligament transection (ACLT) in rats. Furthermore, the mammalian target of rapamycin (mTOR) mediated autophagy was identified as one of the potential mediators of Glabridin activity. Overall, Glabridin protects articular cartilage from damage in rats with OA by protecting chondrocytes against oxidative stress, apoptosis and promoting mTOR mediated autophagy.

Brevican and Neurocan Peptides as Potential Cerebrospinal Fluid Biomarkers for Differentiation Between Vascular Dementia and Alzheimer's Disease.

Brevican and neurocan are central nervous system-specific extracellular matrix proteoglycans. They are degraded by extracellular enzymes, such as metalloproteinases. However, their degradation profile is largely unexplored in cerebrospinal fluid (CSF). The study aim was to quantify proteolytic peptides derived from brevican and neurocan in human CSF of patients with Alzheimer's disease (AD) and vascular dementia (VaD) compared with controls. The first cohort consisted of 75 individuals including 25 patients with AD, 7 with mild cognitive impairment (MCI) diagnosed with AD upon follow-up, 10 patients with VaD or MCI diagnosed with VaD upon follow-up, and 33 healthy controls and cognitively stable MCI patients. In the second cohort, 31 individuals were included (5 AD patients, 14 VaD patients and 12 healthy controls). Twenty proteolytic peptides derived from brevican (n = 9) and neurocan (n = 11) were quantified using high-resolution parallel reaction monitoring mass spectrometry. In the first cohort, the majority of CSF concentrations of brevican and neurocan peptides were significantly decreased inVaDas compared withADpatients (AUC=0.83.0.93, p≤0.05) and as compared with the control group (AUC=0.79.0.87, p≤0.05). In the second cohort, CSF concentrations of two brevican peptides (B87, B156) were significantly decreased in VaD compared with AD (AUC=0.86.0.91, p≤0.05) and to controls (AUC=0.80.0.82, p≤0.05), while other brevican and neurocan peptides showed a clear trend to be decreased in VaD compared with AD (AUC=0.64.80, p>0.05). No peptides differed between AD and controls. Brevican and neurocan peptides are potential diagnostic biomarkers for VaD, with ability to separate VaD from AD.