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Mucins are a class of glycoproteins that serve key roles in tissue protection, hydration, and lubrication. However, their study and application are complicated by their post-translational modifications, heterogeneity, and their propensity to deteriorate upon isolation. As a result, several groups have made strides in synthesizing mucin-mimicking compounds for systematic study and therapeutic applications. In this paper, we review general mucin structure, recent trends in the design and synthesis of mucin mimics, and the ways mucin analogues can outperform native mucins. Specifically, we highlight the structures of lubricin and aggrecan, which are the subject of many biomimetic approaches. Furthermore, we showcase the structural features that allow analogues to mimic mucin functions and categorize them based on their roles as monolayer coatings, gel formers, or adhesion promoters. Lastly, we examine the chemical composition of mucin analogues, emphasizing how modular approaches provide flexibility to repurpose mucin mimics for different applications. Graphical abstract Image 1 Highlights • Mucins are a class of heavily glycosylated glycoproteins. • Mucin use is inhibited by batch heterogeneity, motivating synthesis of mucin mimics. • Mucin mimics can be categorized into motifs by the mucin role they intend to mimic. • In mimics, biological components confer specificity, and synthetic polymers offer structural control. • Modular mimics allow rapid repurposing for different applications and studies. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To determine the regulatory role of interleukin 1 beta (IL-1β) in the Nuclear Factor kappa B (NF-κB) -mediated catabolic effects of the nucleus pulposus cells in human intervertebral disc degeneration under hypoxic conditions. PATIENTS AND METHODS: Human nucleus pulposus cells were cultured and exposed to IL-1β under hypoxic or normoxic environments, with or without NF-κB inhibition. The cell growth was determined using cell counting kit-8; gene and protein expressions were analyzed by Real- time polymerase chain reaction and Western blotting, respectively. RESULTS: Co-treatment w ith IL-1β and hypoxia decreased cell viability in human nucleus pulposus cells. There was a positive effect of IL-1β on human nucleus pulposus cells under hypoxia, which was through the up-regulation of matrix metalloproteinase-3 (MMP-3), MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5. IL-1β-induced expressions of MMP-3, MMP-9, ADAMTS-4, and ADAMTS-5 under hypoxia were accompanied by increased activation of NF-κB. Inhibition of NF- κBp65 by small interfering RNA or specific inhibitor BAY11-7082 blocked IL-1β-dependent gene upregulation of MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 in a hypoxic environment. The gene expression of aggrecan was decreased by IL-1β under hypoxic conditions, which was reversed by either BAY11-7082 or NF-κBp65 knockdown. CONCLUSIONS: IL-1β and hypoxia synergetically contributed to the catabolic effects of the nucleus pulposus cells by upregulating the expression of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 through the activation of NF-κB signaling pathway, indicating that the NF-κB signaling pathway is a key mediator of intervertebral disc degeneration. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (LncRNAs) are involved in the pathogenesis of intervertebral disc degeneration (IDD). However, the biological function and expression of RMRP were still unclear. In our study, we showed that RMRP expression was up‐regulated in degenerated NP tissues compared to normal NP samples, and higher RMRP expression was associated with the disc degeneration grade. Further studies indicated that ectopic expression of RMRP enhanced NP cell growth and also enhanced the expression of ki‐67, PCNA and cyclin D1 in the NP cell. Moreover, overexpression of RMRP promoted the expression of Type II collagen and aggrecan and suppressed the expression of MMP13 and ADAMTS4. In addition, we found that the expression of miR‐206 was down‐regulated in degenerated NP tissues compared to normal NP samples, and lower miR‐206 expression was correlated with the disc degeneration grade. Interestingly, we indicated that miR‐206 expression in NP tissues was negatively correlated with the expression of RMRP. Ectopic expression of miR‐206 suppressed NP cell proliferation and suppressed the expression of Type II collagen and aggrecan and enhanced the expression of MMP13 and ADAMTS4. Furthermore, we demonstrated that overexpression of RMRP increased NP cell growth and regulated ECM expression through targeting miR‐206. These results suggested that lncRNA‐RMRP promoted the progression of IDD through targeting miR‐206, providing an attractive new therapeutic approach for the treatment of IDD disease. [ABSTRACT FROM AUTHOR]

Abstract Osteoarthritis is a painful joint disease characterized by progressive degeneration of the articular cartilage as well as associated changes to the subchondral bone, synovium, and surrounding joint tissues. While the effects of osteoarthritis on the cartilage extracellular matrix (ECM) have been well recognized, it is now becoming apparent that in many cases, the onset of the disease may be initially reflected in the matrix region immediately surrounding the chondrocytes, termed the pericellular matrix (PCM). Growing evidence suggests that the PCM – which along with the enclosed chondrocytes are termed the “chondron” – acts as a critical transducer or “filter” of biochemical and biomechanical signals for the chondrocyte, serving to help regulate the homeostatic balance of chondrocyte metabolic activity in response to environmental signals. Indeed, it appears that alterations in PCM properties and cell-matrix interactions, secondary to genetic, epigenetic, metabolic, or biomechanical stimuli, could in fact serve as initiating or progressive factors for osteoarthritis. Here, we discuss recent advances in the understanding of the role of the PCM, with an emphasis on the reciprocity of changes that occur in this matrix region with disease, as well as how alterations in PCM properties could serve as a driver of ECM-based diseases such as osteoarthritis. Further study of the structure, function, and composition of the PCM in normal and diseased conditions may provide new insights into the understanding of the pathogenesis of osteoarthritis, and presumably new therapeutic approaches for this disease. Highlights • The PCM surrounds all chondrocytes in articular cartilage and regulates their interactions with the environment. • Alterations in PCM properties and composition will influence the chondrocyte's mechanobiologic response to loading. • Some of the earliest biosynthetic and degradative changes in osteoarthritis may initially manifest in the PCM. • Here we review the potential role of PCM pathology as a potential driver, as well as indicator, of osteoarthritis. [ABSTRACT FROM AUTHOR]

The human auditory brainstem, especially the cochlear nucleus (CN) and the superior olivary complex (SOC) are characterized by a high density of neurons associated with perineuronal nets (PNs). PNs build a specific form of extracellular matrix surrounding the neuronal somata, proximal dendrites and axon initial segments. They restrict synaptic plasticity and control high-frequency synaptic activity, a prominent characteristic of neurons of the auditory brainstem. The distribution of PNs within the auditory brainstem has been investigated in a number of mammalian species. However, much less is known regarding PNs in the human auditory brainstem. The present study aimed at the immunohistochemical identification of PNs in the cochlear nucleus (CN) and superior olivary complex (SOC) in the human brainstem. We focused on the complex nature and molecular variability of PNs in the CN and SOC by using specific antibodies against the main PN components (aggrecan, brevican, neurocan and hyaluronan and proteoglycan link protein 1). Virtually all subnuclei within the ventral CN and SOC were found to be associated with PNs. Direct comparison between gerbil and human yielded similar fine structure of PNs and confirmed the typical tight interdigitation of PNs with synaptic terminals in both species. Noticeably, an elaborate combination of immunohistochemical labelings clearly supports the still debated existence of the medial nucleus of trapezoid body (MNTB) in the human brain. In conclusion, the present study demonstrates that PNs form a prominent extracellular structure on CN and SOC neurons in the human brain, potentially stabilizing synaptic contacts, which is in agreement with many other mammalian species. [ABSTRACT FROM AUTHOR]

Highlights • OX1R is expressed in human primary cell culture of chondrocytes. • Orexin A ameliorated IL-1β- induced degradation of type II collagen and aggrecan. • Orexin A reduced the expression of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5. • Orexin A prevents the activation of the IKK/IκBα/ NF-κB signaling pathway. Abstract Excessive production of pro-inflammatory cytokines such as interleukin-1β (IL-1β) plays a key role in the pathophysiological development of osteoarthritis (OA). Orexin A is an important peptide of hypothalamic origin, which has displayed its multiple biological functions in several chronic diseases via activation of its specific G-protein coupled receptors, orexin-1 receptor (OX1R) and orexin-2 receptor (OX2R). In this study, we aimed to characterize the protective effects of orexin A against IL-1β-induced degradation of articular cartilage matrixes in human chondrocytes. Our results indicate that OX1R but not OX2R was expressed in human chondrocytes. We also found that the expression of OX1R was significantly lower in chondrocytes from OA patients, and that treatment with IL-1β decreased the expression of OX1R in a dose-dependent manner. The presence of orexin A ameliorated IL-1β-induced degradation of type II collagen and aggrecan, the two major components of articular cartilage matrixes. Our results also show that orexin A prevented IL-1β-induced expression of catabolic enzymes such as MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5. Mechanistically, orexin A treatment abolished activation of the transcriptional factor NF-κB via inhibition of KKα/IκB-α phosphorylation and IκB-α degradation. These findings suggest that orexin A might act as an effective therapeutic agent for the treatment of OA. [ABSTRACT FROM AUTHOR]

Aggrecan is a high molecular weight proteoglycan that plays a critical role in cartilage structure and the function of joints, providing intervertebral disc and cartilage with the ability to resist compressive loads. Aggrecan degradation in articular cartilage is a significant event in early-stage osteoarthritis (OA). Currently, no effective treatment exists for OA other than pain relief. Dextrose (D-glucose) prolotherapy has shown promising activity in the treatment of different musculoskeletal disorders, including OA. However, little is known about the molecular mechanism of the glucose effect in OA and on the regulation of chondrogenesis. We show for the first time that glucose upregulates aggrecan expression and subsequent chondrogenesis in ATDC5 cells. Moreover, we found that glucose-induced aggrecan expression is mediated through the protein kinase Cα (PKCα)- and p38-dependent pathway. As demonstrated by microRNA (miR) and luciferase analyses, the glucose-induced PKCα/p38 signaling axis is responsible for downregulating miR141-3p which targets to the 3′untranslated region of aggrecan. In summary, we show that glucose enhances chondrogenesis by upregulating aggrecan expression via the PKCα-p38-miR141-3p signaling pathway. This result provides new insights into the mechanism of glucose on chondrogenesis. [ABSTRACT FROM AUTHOR]

Delta‐like 2 (Dlk2), a glycoprotein highly homologous to Dlk1, belongs to the Notch/Delta/Serrata family. Dlk2 has been shown to be an important regulator of adipogenesis; however, its role in other cellular differentiation processes is still unknown. Therefore, in this study, we aimed to determine the role of Dlk2 in chondrogenic differentiation. We found that Dlk2 overexpression promoted the growth of ATDC5 cells but inhibited insulin‐induced ATDC5 chondrogenic differentiation, as supported by the reduction in cartilage matrix formation and gene expression of aggrecan (acan), collagentype II (col2a1) and X (col10a1). In contrast, Dlk2 silencing inhibited the proliferation of ATDC5 cells but enhanced their chondrogenic differentiation. We then evaluated the roles of mitogen‐activated protein kinases (MAPKs), which are activated by insulin during the chondrogenesis of ATDC5 cells. Overexpression of Dlk2 protein strongly promoted the activation of p38, but not extracellular signal‐regulated kinase (ERK) 1/2 and c‐Jun N‐terminal kinase (JNK). Moreover, as expected, Dlk2 silencing inhibited the activation of p38, but had no effect on the ERK1/2 and JNK pathways. Finally, we also detected the expression of Dlk2 in mouse epiphyseal cartilage during embryo development. The expression of the Dlk2 protein in the limb bud could be detected at embryonic day 11.5; additionally, it was found to decrease in the superficial zones, but remained unchanged in the deep/hypertrophic zones. In conclusion, our results suggested that Dlk2 acted as an important regulator of chondrogenesis through the p38 pathway. These findings may lead to strategies for the treatment of cartilage‐related diseases such as osteoarthritis. [ABSTRACT FROM AUTHOR]

Background Context: Endothelin-1 (ET-1) is an inflammatory mediator associated with cartilage end plate (CEP) degeneration in the intervertebral disc (IVD). SOX9 is downregulated during CEP degeneration, along with its targets, collagen II and aggrecan. Wnt/β-catenin signaling is associated with CEP degeneration and a downstream target of SOX9; however, the precise mechanism of CEP degeneration and the role of ET-1 are largely unknown.Purpose: The purpose of the study was to evaluate the influence of the endothelin-A receptor antagonist, BQ-123, on ET-1-induced effects on cartilaginous end plate cells (CECs) associated with CEP degeneration via the Wnt/β-catenin signaling pathway.Study Design/setting: The influence of ET-1 on the expression levels of collagen II, aggrecan, and SOX9 in CECs and the effect of BQ-123 in this context were investigated.Methods: To establish a model for CEP degeneration, three lumbar discs (L3-L4, L4-L5, and L5-L6 levels) in New Zealand white rabbits were punctured close to the vertebral end plate using a 14G needle. Intervertebral disc degeneration was evaluated by magnetic resonance imaging 4 weeks after vertebral end plate injury. CECs were then isolated from the degenerated CEPs to allow evaluation of the role of ET-1 and BQ-123 and to investigate their effects on the Wnt/β-catenin signaling pathway. The expression of ET-1 in CECs from degenerated CEPs was analyzed by immunofluorescent staining. Changes in the levels of collagen II, aggrecan, and SOX9 were evaluated in CECs by real-time polymerase chain reaction and by Western blotting. The Wnt/β-catenin signaling pathway was also investigated by Western blotting.Results: After 4 weeks, IVDs with vertebral end plate injury exhibited clear signs of disc degeneration. Immunofluorescent staining showed that ET-1 was expressed in the cytoplasm of CECs. Endothelin-1 stimulation significantly inhibited the expression of collagen II, aggrecan, and SOX9 in CECs, whereas BQ-123 increased the levels of these three molecules. In addition, ET-1 stimulation increased the expression of β-catenin, cyclin D1, and Dvl1 in the Wnt/β-catenin signaling pathway of CECs from degenerated discs and reduced the expression of GSK-3β, whereas BQ-123 had the opposite effect.Conclusions: Endothelin-1 can reduce levels of collagen II, aggrecan, and SOX9 in CECs through activation of the Wnt/β-catenin signaling pathway, whereas BQ-123 attenuates these negative effects, highlighting a new molecular mechanism with potential for exploitation for treatment of CEP degeneration. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is the most common form of arthritis and is caused by the breakdown of joint cartilage. The present study aimed to investigate an effective method for the treatment of OA. It was demonstrated that, compared with other patients, patients with OA exhibited lower mRNA expression levels of SMAD family member 2 (SMAD2). MicroRNA (miR)-486-5p was predicted to bind with SMAD2, which was verified by dual-luciferase reporter assay. Compared withcontrol patients who had no known history of OA or rheumatoid arthritis, patients with OA exhibited higher miR-486-5p expression level. Treatment with miR-486-5p mimics inhibited proliferation and migration of CHON-001 human chondrocytes, and also inhibited the expression levels of type II collagen and aggrecan. However, treatment with a miR-486-5p inhibitor promoted proliferation and migration, and the expression of type II collagen and aggrecan. Short interfering RNA-directed silencing of SMAD2 reversed the upregulated proliferation and migration and the expression level of type II collagen and aggrecan induced by the miR-486-5p inhibitor. In conclusion, the results of the present study indicated that miR-486-5p was upregulated in OA and may inhibit chondrocyte proliferation and migration by suppressing SMAD2. [ABSTRACT FROM AUTHOR]

Summary: Objective: Mutations in the aggrecan gene (ACAN) have been identified in two autosomal dominant skeletal dysplasias, spondyloepiphyseal dysplasia, Kimberley type (SEDK), and osteochondritis dissecans, as well as in a severe recessive dysplasia, spondyloepimetaphyseal dysplasia, aggrecan type. Next‐generation sequencing (NGS) has aided the identification of heterozygous ACAN mutations in individuals with short stature, minor skeletal defects and mild facial dysmorphisms, some of whom have advanced bone age (BA), poor pubertal spurt and early growth cessation as well as precocious osteoarthritis. Design and methods: This study involves clinical and genetic characterization of 16 probands with heterozygous ACAN variants, 14 with short stature and mild skeletal defects (group 1) and two with SEDK (group 2). Subsequently, we reviewed the literature to determine the frequency of the different clinical characteristics in ACAN‐positive individuals. Results: A total of 16 ACAN variants were located throughout the gene, six pathogenic mutations and 10 variants of unknown significance (VUS). Interestingly, brachydactyly was observed in all probands. Probands from group 1 with a pathogenic mutation tended to be shorter, and 60% had an advanced BA compared to 0% in those with a VUS. A higher incidence of coxa valga was observed in individuals with a VUS (37% vs 0%). Nevertheless, other features were present at similar frequencies. Conclusions: ACAN should be considered as a candidate gene in patients with short stature and minor skeletal defects, particularly those with brachydactyly, and in patients with spondyloepiphyseal dysplasia. It is also important to note that advanced BA and osteoarticular complications are not obligatory conditions for aggrecanopathies/aggrecan‐associated dysplasias. [ABSTRACT FROM AUTHOR]

The HtrA family of chaperones and serine proteases is important for regulating stress responses and controlling protein quality in the periplasm of bacteria. HtrA is also associated with infectious diseases since inactivation of htrA genes results in significantly reduced virulence properties by various bacterial pathogens. These virulence features of HtrA can be attributed to reduced fitness of the bacteria, higher susceptibility to environmental stress and/or diminished secretion of virulence factors. In some Gram-negative and Gram-positive pathogens, HtrA itself can be exposed to the extracellular environment promoting bacterial colonisation and invasion of host tissues. Most of our knowledge on the function of exported HtrAs stems from research on Helicobacter pylori, Campylobacter jejuni, Borrelia burgdorferi, Bacillus anthracis, and Chlamydia species. Here, we discuss recent progress showing that extracellular HtrAs are able to cleave cell-to-cell junction factors including E-cadherin, occludin, and claudin-8, as well as extracellular matrix proteins such as fibronectin, aggrecan, and proteoglycans, disrupting the epithelial barrier and producing substantial host cell damage. We propose that the export of HtrAs is a newly discovered strategy, also applied by additional bacterial pathogens. Consequently, exported HtrA proteases represent highly attractive targets for antibacterial treatment by inhibiting their proteolytic activity or application in vaccine development. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) constitutes a proper micro-environment for cell proliferation, migration and differentiation, as well as playing pivotal roles in developmental processes including endochondral ossification. Cartilage ECM is mainly composed of fibrous proteins, including collagen, proteoglycan, and hyaluronan. Because almost all ECM components are transported by intracellular vesicular transport systems, molecules that mediate vesicle transport are also important for endochondral ossification. Giantin, encoded by the Golgb1 gene, is a tethering factor for coatomer 1 (COPI) vesicles and functions in the cis-medial Golgi compartments. An insertion mutation in the Golgb1 gene, resulting in a lack of giantin protein expression, has been detected in ocd / ocd rats that exhibit a pleiotropic phenotype including osteochondrodysplasia. To reveal the function of giantin in chondrogenesis, the present study assessed the effects of loss of giantin expression on cartilage ECM and Golgi morphology. Giantin was expressed in normal, but not in ocd/ocd , chondrocytes in the epiphyseal areas of embryonic femurs, whereas GM130 was expressed in both normal and ocd/ocd chondrocytes. The staining intensities of safranin O and azan (aniline blue) were reduced and enhanced, respectively, in epiphyseal cartilage of ocd/ocd femurs. Immunostaining showed that levels of type II collagen and fibronectin were comparable in normal and ocd/ocd cartilage. Levels of type XI collagen were higher, while levels of aggrecan, link protein and hyaluronan were lower, in ocd/ocd than in normal cartilage, although semi-quantitative RT-PCR showed similar levels of type XI collagen, aggrecan and link protein mRNAs in normal and ocd / ocd cartilage. Isolated chondrocytes of ocd / ocd and normal rats showed similar immunostaining patterns for cis -, medial-, and trans -Golgi marker proteins, whereas monolayers of ocd/ocd chondrocytes showed reduced levels of aggrecan and link protein and increased level of type XI collagen in spite of similar transcripts levels. These findings suggest that giantin plays a pivotal role in coordinated production of aggrecan, link protein and type XI collagen in chondrocytes, and that loss of giantin causes osteochondrodysplasia with disturbance of these ECM components. [ABSTRACT FROM AUTHOR]

Background: Nowadays, Human adipocyte-derived stem cells (hADSCs) has been widely used in tissue engineering because of its unique features such as extraction from more sources, more easily and non-invasive extraction methods. In order to increase cell-cell interactions, similar to embryonic pre-cartilage condensation, the use of three dimensional (3D) high-density cell culture systems such as Pellet and Micromass that simulates optimal condensation in chondrogenesis in vivo is necessary. Also, these culture systems provide the proper diffusion of nutrients. Aggrecan is a proteoglycan and one of the important components of extracellular matrix of cartilage tissue that plays an important role in the organization of the extracellular matrix. The high concentrations of aggrecan produces the osmotic properties that is necessary to normal tissue function of cartilage. In current study, Aggrecan gene expression was investigated during chondrogenesis of hADSCs in two Pellet and Micromass culture systems. Methods: This experimental study was done in Department of Anatomical Sciences Department of Faculty Medical in Isfahan University of Medical Sciences, Iran, from April 2013 to January 2015. First, the abdominal adipose tissue was obtained from three patients after obtaining written consent during their liposuction surgeries. ADSCs were extracted by mechanical and enzymatic methods and were cultured in monolayer culture. Then, in order to induction of chondrogenic differentiation, 5×105 cells of third passage (P3) were transferred to three-dimensional culture systems Pellet and Micromass containing chondrogenic mediums in experimental groups of 7 and 14 days. The evaluation of aggrecan gene expression was performed by real-time PCR technique. Results: Gene expression analysis revealed that aggrecan was significantly increased in micromass culture at day 14 compared to Pellet culture at days 14 and 7 (P≤0.01). Also, aggrecan was significantly increased in Micromass culture at day 7 compared to Pellet culture at day 7 (P≤0.05). Conclusion: Due to higher expression of aggrecan gene in Micromass culture compared to Pellet culture, this system may be more efficient than Pellet culture in synthesis of aggrecan in chondrogenic differentiation of ADSCs. [ABSTRACT FROM AUTHOR]

Intervertebral disc (IVD) degeneration may cause many diseases and pain. Stem cell migration toward the site of IVD degeneration is a key factor for IVD regeneration. In the current study, we prepared albumin/heparin nanoparticles (BHNPs) as injectable carriers of stromal cell-derived factor-1α (SDF-1α, also known as C-X-C motif chemokine 12), a powerful chemoattractant for the homing of bone marrow resident mesenchymal stem cells (MSCs), for protection of the molecule against degradation for a sustained release. The NPs have relatively uniform small size, with a diameter of about 110 nm. The NPs possess a high loading capacity of SDF-1α with a sustained release profile. The bioactivity of the obtained BHNPs/SDF was then studied in vitro and in vivo. The BHNPs/SDF can induce migration of MSCs in a dose-dependent manner in vitro. After injected into the damaged disc, BHNPs/SDF induce much better regeneration of annulus fibrosus and nucleus pulposus, compared to SDF-1α and BHNPs alone, evidenced with better histological grade scores and higher expression of SOX9, Aggrecan, and Collagen type II at the level of both mRNA and protein. This study provides a simple nanoplatform to load SDF-1α and protect it against degradation, with potential application in inductive tissue regeneration in vivo. Statement of significance Stem cell migration toward the site of IVD degeneration is a key event to promote IVD regeneration. In the current study, we prepared albumin/heparin nanoparticles (BHNPs) as injectable carriers to protect SDF-1α against degradation and for the sustained release of the molecule. After injected into the damaged disc, BHNPs/SDF induced much better regeneration of IVD, compared to SDF-1α and BHNPs alone. This study provides a simple nanoplatform to load SDF-1α and protect it from degradation, with potential application in inductive tissue regeneration in vivo. [ABSTRACT FROM AUTHOR]

Stem cell therapy is considered an optimistic approach to replace current treatments for cartilage defects. Recently, human urine-derived stem cells (hUSCs), which are isolated from the urine, are studied as a promising candidate for many tissue engineering therapies due to their multipotency and sufficient proliferation activities. However, it has not yet been reported whether hUSCs can be employed in cartilage defects. In this study, we revealed that induced hUSCs expressed chondrogenic-related proteins, including aggrecan and collagen II, and their gene expression levels were upregulated in vitro. Moreover, we combined hUSCs with hyaluronic acid (HA) and injected hUSCs-HA into a rabbit knee joint with cartilage defect. Twelve weeks after the injection, the histologic analyses (HE, toluidine blue, and Masson trichrome staining), immunohistochemistry (aggrecan and collagen II), and histologic grade of the sample indicated that hUSCs-HA could stimulate much more neocartilage formation compared with hUSCs alone, pure HA, and saline, which only induced the modest cartilage regeneration. In this study, we demonstrated that hUSCs could be a potential cell source for stem cell therapies to treat cartilage-related defects in the future. [ABSTRACT FROM AUTHOR]

Lumbar disc disease (LDD) is common in aged populations, and it is primarily caused by intervertebral disc degeneration (IDD). Cartilage intermediate layer protein (CILP), which is specifically expressed in intervertebral discs (IVDs), is suspected to be associated with IDD. However, it remains unclear whether CILP contributes to IDD in humans. Furthermore, the regulation of CILP in human IVDs is poorly understood, especially by mechanical stimuli, which are regarded as primary factors promoting IDD. To address these issues, the present study collected nucleus pulposus (NP) cells from patients undergoing lumbar spinal surgery for degenerative disc disease (DDD). Subsequently, CILP expression was measured in human NP cells in response to mechanical stimuli, including cyclic compressive stress and cyclic tensile strain (CTS), by reverse transcription‑quantitative polymerase chain reaction and western blotting. Aggrecan and collagen II, which are the main components of the extracellular matrix (ECM) and traditional degenerative markers for IDD, were detected following the treatment with CILP small interfering (si)RNA or recombinant human CILP (rhCILP) at various concentrations to determine whether CILP contributes to IDD by negatively regulating expression of the ECM. The results revealed that CILP expression in loaded NP cells was significantly increased compared with that in non‑loaded cells under compressive loading, and that it was markedly decreased in cells under tensile loading, in contrast with the expression of aggrecan and collagen II in response to the same stimuli. Furthermore, CILP siRNA effectively inhibited CILP expression and significantly increased the expression of aggrecan and collagen II. In addition, treatment of NP cells with a high concentration of rhCILP resulted in significantly decreased expression of aggrecan and collagen II. In conclusion, these results demonstrated for the first time, to the best of our knowledge, that in human NP cells, CILP is regulated by mechanical stress and that its expression affects ECM synthesis. Therefore, CILP represents a promising therapeutic target for preventing loss of the matrix during IDD as a novel treatment strategy. [ABSTRACT FROM AUTHOR]

Background/Aims: Low back pain has become one of the most common musculoskeletal diseases in the world. Studies have shown that intervertebral disc degeneration (IDD) is an important factor leading to low back pain, but the mechanisms underlying IDD remain largely unknown. Research over the past decade has suggested critical roles for microRNAs (miRNAs) in natural growth and disease progression. However, it remains poorly understood whether circular RNAs participate in IDD. Methods: Clinical IDD samples were collected from 20 patients who underwent discectomy. Weighted gene co-expression network analysis was used to identify the co-expression miRNA network modules (highly co-expressed clusters of miRNAs) that were associated with IDD grade. Results: miR-3150a-3p was the most significantly up-regulated miRNA in module “Blue.” Notably, aggrecan (ACAN) was identified as a direct target gene of miR-3150a-3p and ACAN expression was regulated by miR-3150a-3p. Overexpression of miR-3150a-3p decreased ACAN expression in nucleus pulposus cells, whereas inhibition of miR-3150a-3p increased ACAN expression. In addition, ACAN expression was negatively correlated with IDD grade. Conclusion: Our study suggests that the reduction of ACAN expression induced by the upregulation of miR-3150a-3p might participate in the development of IDD. [ABSTRACT FROM AUTHOR]

Study Design: An in vitro study using human degenerated nucleus pulposus cells.Objective: To determine the effect of osmolality and different osmolytes on the regeneration by human nucleus pulposus cells through gene expression and extracellular matrix production.Summary Of Background Data: Intervertebral disc (IVD) degeneration is a major problem in developed countries. Regeneration of the IVD can prevent pain and costs due to diminished work absence and health care, and improve quality of life. The osmotic value of a disc decreases during degeneration due to loss of proteoglycans and might increase degeneration. It is known that gene expression of matrix genes of nucleus pulposus (NP) cells increases when cultured in hyperosmotic medium. Thus, increasing the osmolality of the disc might be beneficial for disc regeneration.Methods: In the current study, isolated degenerated human NP cells were used in regeneration culture with medium of different osmolalities, adjusted with different osmolytes. NaCl, urea and sucrose. The cells were cultured for 28 days and expression of matrix genes and production of glycosaminoglycans and collagen II were measured.Results: Gene expression for both collagen II and aggrecan increased with increasing osmolality using NaCl or sucrose, but not urea. Protein production however, was not affected by increasing osmolality and was decreased when using urea and sucrose. Expression of genes for Col1A1, MMP13, and MMP14 decreased with increasing osmolality, whereas expression of LOXL2 and LOXL3 increased. Transient expression of TonEBP was found 6 hours after the start of culture, but not at later time points.Conclusion: Although expression of matrix genes is upregulated, hyperosmolality does not enhance matrix production by nucleus pulposus cells. Raising osmolality can potentially increase matrix production, but in itself is not sufficient to accomplish regeneration in the current in vitro culture system.Level Of Evidence: N /A. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is the most common form of arthritis worldwide. Excessive production of pro-inflammatory cytokines such as interleukin-1β (IL-1β) plays a key role in the pathogenesis of OA. OA is generally characterized by degradation of extracellular matrixes such as type II collagen and aggrecans mediated by matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS). Ghrelin is a secreted peptide hormone regulating appetite and the distribution and rate of use of energy. However, the physiological and pharmacological roles of Ghrelin on the pathological progression of OA haven’t been reported before. In the current study, our results indicate that Ghrelin reduced IL-1β-induced expression of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 in a concentration-dependent manner. Notably, Ghrelin ameliorated IL-1β-induced degradation of type II collagen and aggrecan. Mechanistically, Ghrelin is able to inhibit the expression of IRF-1 mediated by inactivating the JAK2/STAT3 pathway. However, Ghrelin didn’t have any impact on IL-1β induced activation of p38. Taken together, our findings identify a novel function of Ghrelin on inhibiting the degradation of type II collagen and aggrecan. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to analyze serum a disintegrin-like and metalloproteinase with thrombospondin-type motifs-1 (ADAMTS-1) and aggrecan levels in adolescents and younger-aged females with polycystic ovary syndrome (PCOS) compared with ovulatory controls to determine whether these are potential markers for the prediction of PCOS diagnosis. We also aimed to determine whether they could predict the development of clinical implications associated with PCOS.Method: PCOS (n = 49) and ovulatory age-matched controls (n = 41) (mean age, 18.6 ± 2.5) were recruited. Anthropometric measurements were recorded and biochemical parameters were analyzed. Serum ADAMTS-1 and aggrecan levels were determined with enzyme-linked immunosorbent assay. The predictive effects of ADAMTS-1 and aggrecan on the diagnosis of PCOS and for the development of cardiovascular disease (CVD) risk and insulin resistance (IR) were evaluated. The correlation between investigated markers and anthropometric, biochemical, and hormonal parameters were also investigated.Results: Mean serum ADAMTS-1 level was increased in adolescents and younger-aged females with PCOS compared to ovulatory controls. An elevated ADAMTS-1 level was positive predictive of the diagnosis of PCOS with the best cut-off value of 2.5 ng/ml (sensitivity 69% and specificity 78%). A positive predictive role of ADAMTS-1 on the development of CVD risk and IR was found among all patients. Serum ADAMTS-1 and aggrecan levels were significantly and positively correlated with each other.Conclusion: Increased levels of ADAMTS-1 could be a potential marker for the etiopathogenesis of PCOS in adolescents and younger-aged females and predict the development of CVD risk and IR among all patients with the same age. [ABSTRACT FROM AUTHOR]

Aggrecan, a chondroitin sulfate (CS) proteoglycan, forms lattice-like extracellular matrix structures called perineuronal nets (PNNs). Neocortical PNNs primarily ensheath parvalbumin-expressing inhibitory neurons (parvalbumin, PV cells) late in brain development. Emerging evidence indicates that PNNs promote the maturation of PV cells by enhancing the incorporation of homeobox protein Otx2 and regulating experience-dependent neural plasticity. Wisteria floribunda agglutinin (WFA), an N-acetylgalactosamine-specific plant lectin, binds to the CS chains of aggrecan and has been widely used to visualize PNNs. Although PNNs show substantial molecular heterogeneity, the importance of this heterogeneity in neural plasticity remains unknown. Here, in addition to WFA lectin, we used the two monoclonal antibodies Cat315 and Cat316, both of which recognize the glycan structures of aggrecan, to investigate the molecular heterogeneity of PNNs. WFA detected the highest number of PNNs in all cortical layers, whereas Cat315 and Cat316 labeled only a subset of PNNs. WFAC, Cat315+, and Cat316+ PNNs showed different laminar distributions in the adult visual cortex. WFA, Cat315 and Cat316 detected distinct, but partially overlapping, populations of PNNs. Based on the reactivities of these probes, we categorized PNNs into four groups. We found that two subpopulation of PNNs, one with higher and one with lower WFA-staining are differentially labeled by Cat316 and Cat315, respectively. CS chains recognized by Cat316 were diminished in mice deficient in an enzyme involved in the initiation of CS-biosynthesis. Furthermore, WFA+ and Cat316+ aggrecan were spatially segregated and formed microdomains in a single PNN. Otx2 co-localized with Cat316+ but not with WFAC aggrecan in PNNs. Our results suggest that the heterogeneity of PNNs around PV cells may affect the functional maturation of these cells. [ABSTRACT FROM AUTHOR]

Background: Extracellular matrix (ECM) remodeling contributes to in-stent restenosis and thrombosis. Despite its important clinical implications, little is known about ECM changes post-stent implantation.Methods: Bare-metal and drug-eluting stents were implanted in pig coronary arteries with an overstretch under optical coherence tomography guidance. Stented segments were harvested 1, 3, 7, 14, and 28 days post-stenting for proteomics analysis of the media and neointima.Results: A total of 151 ECM and ECM-associated proteins were identified by mass spectrometry. After stent implantation, proteins involved in regulating calcification were upregulated in the neointima of drug-eluting stents. The earliest changes in the media were proteins involved in inflammation and thrombosis, followed by changes in regulatory ECM proteins. By day 28, basement membrane proteins were reduced in drug-eluting stents in comparison with bare-metal stents. In contrast, the large aggregating proteoglycan aggrecan was increased. Aggrecanases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family contribute to the catabolism of vascular proteoglycans. An increase in ADAMTS-specific aggrecan fragments was accompanied by a notable shift from ADAMTS1 and ADAMTS5 to ADAMTS4 gene expression after stent implantation. Immunostaining in human stented coronary arteries confirmed the presence of aggrecan and aggrecan fragments, in particular, at the contacts of the stent struts with the artery. Further investigation of aggrecan presence in the human vasculature revealed that aggrecan and aggrecan cleavage were more abundant in human arteries than in human veins. In addition, aggrecan synthesis was induced on grafting a vein into the arterial circulation, suggesting an important role for aggrecan in vascular plasticity. Finally, lack of ADAMTS-5 activity in mice resulted in an accumulation of aggrecan and a dilation of the thoracic aorta, confirming that aggrecanase activity regulates aggrecan abundance in the arterial wall and contributes to vascular remodeling.Conclusions: Significant differences were identified by proteomics in the ECM of coronary arteries after bare-metal and drug-eluting stent implantation, most notably an upregulation of aggrecan, a major ECM component of cartilaginous tissues that confers resistance to compression. The accumulation of aggrecan coincided with a shift in ADAMTS gene expression. This study provides the first evidence implicating aggrecan and aggrecanases in the vascular injury response after stenting. [ABSTRACT FROM AUTHOR]

The inflammatory, degenerative and rheumatic diseases are triggered and evolving at osteoarticular level due to the disruption of functionality of constitutive cells and extracellular microenvironment correlated with systemic responses as synthesis of progressively degradative cytokines. Objectives. The in vitro bioactive efficacy of standardized small sea fish extract has therapeutic relevance on the strength of optimal physiological and structural biologic system rendered by primary cells isolated from human cartilage (HCH, PromoCell) unlike standardized cell lines that may have genetic and functional changes and beside the primary cells directly isolated from animal tissue that do not reproduce an authentic biological response because of metabolic stress adaptation to growing conditions in vitro. The main therapeutic targets in osteoarticular disorders are regeneration of affected cartilage and attenuation of local inflammatory processes. Materials and methods. The in vitro articular matrix reconstruction action was evaluated by molecular tests with relative quantification of gene expression (qPCR) for aggrecan, the predominant component of human cartilage, and for the enzyme responsible for degradation of aggrecan, aggrecanase (ADAMTS4) whose activities were phenotypically measured by ELISA (enzyme-linked immunosorbent assay) test. The anti-inflammatory profile was proved by techniques of quantitative evaluation of mRNA level and detection of extracellular soluble proteins by flow-cytometry (BDTM Cytometric Bead Array) for pro-inflammatory cytokines IL-6, IL-8 and gene expression for nuclear factor NF-κB. Results and discussion. The results prove the matrix regenerator effect of standardized small fish extract by stimulation at gene expression level of aggrecan synthesis in early stages of chondrocytes differentiation, directing cells to a mature functional status, as well as by inhibition of aggrecan degradation by action on gene expression and extracellular activity of ADAMTS4. The standardized small sea fish extract has an anti-inflammatory effect proven by extracellular and transcriptional inhibition of pro-inflammatory cytokines, IL-6, IL-8 and the nuclear factor NF-κB gene. Conclusions. The in vitro anti-inflammatory and regenerative action of standardized small fish extract reflects a significant therapeutic potential for osteoarticular disease sustained both through the effects at gene and phenotypic expression levels and through the reproducible characteristics of dynamics of in vivo chondrocytes evolution evidenced by primary cell lines. [ABSTRACT FROM AUTHOR]

Currently, umbilical cord mesenchymal stem cells have the potential to be used as treatment options for any cartilage lesion. This research aimed to evaluate the effects of conditioned medium from umbilical cord mesenchymal stem cells (UC-MSC) on damaged cartilage through the expression of proteins TGF-β1, SOX-9, type II collagen and aggrecan, which are known to be related to chondrogenesis. UC-MSC were isolated from 19-days-pregnant Wistar mice and were cultured using the standard procedure to obtain 80% confluence. Subsequently, the culture was confirmed through a microscopic examination that was driven to be an embryoid body to obtain a pre-condition medium. This research utilized 3-month-old male Wistar mice and was categorized into 6 groups (3 control and 3 treatment groups). Each animal had surgery performed to create a femur condyle cartilage defect. The treatment groups were administered a dose of stem cells at 1 mL/kg. Next, immunohistochemical (IHC) staining was performed to examine the expression of TGF-β1, SOX-9, type II collagen and aggrecan in the 2nd, 3rd, and 4th month of evaluation. The results were analyzed statistically using ANOVA test. For each of the treatment groups, there was increased expression (p < 0.05) in all proteins TGF-β1, SOX-9, type II collagen and aggrecan when compared with control groups at the 2nd, 3rd, and 4th month of evaluation. Pre-conditioned medium from UC-MSC potentially increases the expression of TGF-β1, SOX-9, type II collagen and aggrecan in the damaged cartilage of Wistar mice. [ABSTRACT FROM AUTHOR]

Hemodynamic forces have been known for a long time to regulate cardiogenic processes such as cardiac valve development. During embryonic development in vertebrates, the outflow tract (OFT) adjacent to the ventricle comes under increasing hemodynamic load as cardiogenesis proceeds. Consequently, extracellular matrix components are produced in this region as the cardiac cushions form which will eventually give rise to the aortic valves. The proteoglycan AGGRECAN is a key component of the aortic valves and is frequently found to be deregulated in a variety of aortic valve diseases. Here we demonstrate that aggrecan expression in the OFT of developing zebrafish embryos is hemodynamically dependent, a process presumably mediated by mechanosensitive channels. Furthermore, knockdown or knockout of aggrecan leads to failure of the OFT to develop resulting in stenosis. Based on these findings we analysed the expression of AGGRECAN in human bicuspid aortic valves (BAV). We found that in type 0 BAV there was a significant reduction in the expression of AGGRECAN . Our data indicate that aggrecan is required for OFT development and when its expression is reduced this is associated with BAV in humans. [ABSTRACT FROM AUTHOR]

The aim of this study was to characterize structural and property modifications of intramuscular connective tissue (IMCT) during extended aging. Longissimus lumborum (LL), Gluteus medius (GM), and Gastrocnemius (GT) muscles were collected from 10 USDA choice carcasses, fabricated and assigned to one of four aging periods: 3, 21, 42, or 63 days (n = 120). As expected, tenderness improved, and IMCT texture weakened after 21 days of postmortem aging (dpm; P < 0.05). In addition, transition temperature of collagen decreased (P < 0.01) after 42 dpm. It is interesting to note the collagen structure was also altered where relative % of γ chain decreased after 42 dpm (P < 0.05), and the α1 chain % increased at 63 days (P < 0.01). Finally, The LL and GT had a decrease in the 75 kDa aggrecan fragments from 3 to 21 to 42 dpm (P < 0.05). This study provided evidence that IMCT weakens during postmortem aging due to the modifications of IMCT components such as collagen and proteoglycan. [ABSTRACT FROM AUTHOR]

As part of the extracellular matrix (ECM), perineuronal nets (PNs) are polyanionic, chondroitin sulfate proteoglycan (CSPG)-rich coatings of certain neurons, known to be affected in various neural diseases. Although these structures are considered as important parts of the neurovascular unit (NVU), their role during evolution of acute ischemic stroke and subsequent tissue damage is poorly understood and only a few preclinical studies analyzed PNs after acute ischemic stroke. By employing three models of experimental focal cerebral ischemia, this study was focused on histopathological alterations of PNs and concomitant vascular, glial and neuronal changes according to the NVU concept. We analyzed brain tissues obtained 1 day after ischemia onset from: (a) mice after filament-based permanent middle cerebral artery occlusion (pMCAO); (b) rats subjected to thromboembolic MACO; and (c) sheep at 14 days after electrosurgically induced focal cerebral ischemia. Multiple fluorescence labeling was applied to explore simultaneous alterations of NVU and ECM. Serial mouse sections labeled with the net marker Wisteria floribunda agglutinin (WFA) displayed largely decomposed and nearly erased PNs in infarcted neocortical areas that were demarcated by up-regulated immunoreactivity for vascular collagen IV (Coll IV). Subsequent semi-quantitative analyses in mice confirmed significantly decreased WFA-staining along the ischemic border zone and a relative decrease in the directly ischemia-affected neocortex. Triple fluorescence labeling throughout the three animal models revealed up-regulated Coll IV and decomposed PNs accompanied by activated astroglia and altered immunoreactivity for parvalbumin, a calcium-binding protein in fast-firing GABAergic neurons which are predominantly surrounded by neocortical PNs. Furthermore, ischemic neocortical areas in rodents simultaneously displayed less intense staining of WFA, aggrecan, the net components neurocan, versican and the cartilage link protein (CRTL) as well as markers in net-bearing neurons such as the potassium channel subunit Kv3.1b and neuronal nuclei (NeuN). In summary, theconsistent observations based on three different stroke models confirmed that PNs are highly sensitive constituents of the NVU along with impaired associated GABAergic neurons. These results suggest that PNs could be promising targets of future stroke treatment, and further studies should address their reorganization and plasticity in both stabilizing the acute stroke as well as supportive effects during the chronic phase of stroke.

Background: Sex hormones play important role in proliferation, differentiation, maturation and the scheduled death of chondrocytes. Although, some studies report the regulatory role of estrogen in the development and progression of cartilage, some of the mechanisms still remain unclear, including the role of estrogen in the expression of cartilage-specific genes in chondrogenesis process. We studied this role in the present study. Methods: We used adipose-derived stem cells, which were previously differentiated into cartilage tissue in pellet culture system in the control (without estrogen in culture medium) and experimental (with estrogen in culture medium) groups. Production of chondrogenesis markers, type II collagen and aggrecan were evaluated in experimental and control groups via immunohistochemical (IHC) technique. Then, the results were evaluated with Image-J software and statistical analysis were done. Findings: Estrogen led to decrease of type II collagen and increase of aggrecan production. Conclusion: This study showed that chondrogenesis could be affected by estrogen.

Analysis of both the aggregated and non-aggregated fractions of aggrecan isolated from adult human intervertebral disc using immunoblotting with antibodies specific for the different domains constituting the aggrecan core protein or atomic force microscopy revealed that many components contained the G1 domain. However, little of the disc aggrecan was able to reform aggregates with hyaluronan, as determined by gel filtration chromatography, suggesting that the G1 domains had been rendered non-functional. Since previous studies have shown that disc aggrecan undergoes non-enzymatic glycation with age, the functional effect of such modification was investigated in vitro using bovine aggrecan isolated from young animals. Incubation of monomeric aggrecan with ribose to induce glycation rendered it unable to form complexes with hyaluronan stable to agarose gel electrophoresis or gel filtration chromatography. Similarly, extended treatment of intact proteoglycan aggregate with ribose resulted in destabilisation of the complex with separation of the aggrecan from the hyaluronan. Although it is clear that proteolysis occurs in the intervertebral disc and gives rise to some non-aggregating molecules, a different mechanism is required to explain the presence of many non-aggregating molecules bearing the G1 domain. The products of non-enzymatic glycation of the globular domains of aggrecan would account for this phenomenon and explain why some of the non-aggregating molecules are still large proteoglycans. While such molecules may be retained in the nucleus pulposus, they may be able to diffuse within it, reducing the ability of the tissue to resist compression under asymmetric loading such as bending and ultimately contributing to disc degeneration.