Proteoglycan Accumulation Research Articles

Photobiomodulation inhibits the expression of chondroitin sulfate proteoglycans after spinal cord injury via the Sox9 pathway.

Both glial cells and glia scar greatly affect the development of spinal cord injury and have become hot spots in research on spinal cord injury treatment. The cellular deposition of dense extracellular matrix proteins such as chondroitin sulfate proteoglycans inside and around the glial scar is known to affect axonal growth and be a major obstacle to autogenous repair. These proteins are thus candidate targets for spinal cord injury therapy. Our previous studies demonstrated that 810 nm photobiomodulation inhibited the formation of chondroitin sulfate proteoglycans after spinal cord injury and greatly improved motor function in model animals. However, the specific mechanism and potential targets involved remain to be clarified. In this study, to investigate the therapeutic effect of photobiomodulation, we established a mouse model of spinal cord injury by T9 clamping and irradiated the injury site at a power density of 50 mW/cm2 for 50 minutes once a day for 7 consecutive days. We found that photobiomodulation greatly restored motor function in mice and downregulated chondroitin sulfate proteoglycan expression in the injured spinal cord. Bioinformatics analysis revealed that photobiomodulation inhibited the expression of proteoglycan-related genes induced by spinal cord injury, and versican, a type of proteoglycan, was one of the most markedly changed molecules. Immunofluorescence staining showed that after spinal cord injury, versican was present in astrocytes in spinal cord tissue. The expression of versican in primary astrocytes cultured in vitro increased after inflammation induction, whereas photobiomodulation inhibited the expression of versican. Furthermore, we found that the increased levels of p-Smad3, p-P38 and p-Erk in inflammatory astrocytes were reduced after photobiomodulation treatment and after delivery of inhibitors including FR 180204, (E)-SIS3, and SB 202190. This suggests that Smad3/Sox9 and MAPK/Sox9 pathways may be involved in the effects of photobiomodulation. In summary, our findings show that photobiomodulation modulates the expression of chondroitin sulfate proteoglycans, and versican is one of the key target molecules of photobiomodulation. MAPK/Sox9 and Smad3/Sox9 pathways may play a role in the effects of photobiomodulation on chondroitin sulfate proteoglycan accumulation after spinal cord injury.

A Narrative Review of Morquio Syndrome: Mucopolysaccharidosis (MPS) Type IV

Background: Morquio Syndrome or Mucopolysaccharidosis Type IV (MPS IV) is a rare inherited metabolic disorder characterized by the deficiency of certain lysosomal enzymes involved in the breakdown of mucopolysaccharides. The deficiency results in proteoglycan accumulation, particularly keratan sulfate in various tissues in the body. Morquio Syndrome is classified into two subtypes: Morquio A and Morquio B, which are distinguished by the specific enzyme deficiency involved, with Morquio A being more prevalent. Morquio Syndrome commonly affects the eyes with 46.7% of diagnosed patients experiencing ocular symptoms related to the disorder. Objective: To perform a comprehensive review of the existing literature and summarize the ophthalmological manifestations of Morquio Syndrome. Methods: A methodical literature search was conducted, including papers with abstracts discussing ophthalmology or ocular pathology of “Mucopolysaccharidosis Type IV” or ” Morquio Syndrome.” Twelve relevant articles met the inclusion criteria and were included in the review. Seven of the articles consisted of case reports, collectively reporting on forty-one patients with Morquio Syndrome, primarily Morquio A. The outcome of the narrative review is an overview of the existing literature on ocular presentations of Morquio Syndrome and a summary of case report findings. Results: Forty-one different patients were identified from the included case reports, and forty patients were included as they presented with ocular pathology related to Morquio Syndrome. Corneal opacification was the most common presentation where twenty-seven patients had significant corneal opacification and seven patients had slight corneal opacification. Small, gray, spherical, dust-like opacities that dispersed among the stroma were most commonly seen in cases with corneal opacification. Other ocular presentations included decreased visual acuity, astigmatism, lens opacities, and glaucoma. Conclusion: Morquio syndrome, a rare genetic disorder, exhibits multiple ocular symptoms, with corneal opacification being the most common. While most research has concentrated on Morquio A, the more severe subtype, there's limited information on Morquio B, highlighting a need for more comparative studies. As the syndrome remains incurable, exploring new treatment avenues and understanding the reasons behind these ocular manifestations can significantly improve the quality of life for Morquio patients.

Vascular smooth muscle-specific YAP/TAZ deletion triggers aneurysm development in mouse aorta.

Inadequate adaption to mechanical forces, including blood pressure, contributes to development of arterial aneurysms. Recent studies have pointed to a mechanoprotective role of YAP and TAZ in vascular smooth muscle cells (SMCs). Here, we identified reduced expression of YAP1 in human aortic aneurysms. Vascular SMC-specific knockouts (KOs) of YAP/TAZ were thus generated using the integrin α8-Cre (Itga8-Cre) mouse model (i8-YT-KO). i8-YT-KO mice spontaneously developed aneurysms in the abdominal aorta within 2 weeks of KO induction and in smaller arteries at later times. The vascular specificity of Itga8-Cre circumvented gastrointestinal effects. Aortic aneurysms were characterized by elastin disarray, SMC apoptosis, and accumulation of proteoglycans and immune cell populations. RNA sequencing, proteomics, and myography demonstrated decreased contractile differentiation of SMCs and impaired vascular contractility. This associated with partial loss of myocardin expression, reduced blood pressure, and edema. Mediators in the inflammatory cGAS/STING pathway were increased. A sizeable increase in SOX9, along with several direct target genes, including aggrecan (Acan), contributed to proteoglycan accumulation. This was the earliest detectable change, occurring 3 days after KO induction and before the proinflammatory transition. In conclusion, Itga8-Cre deletion of YAP and TAZ represents a rapid and spontaneous aneurysm model that recapitulates features of human abdominal aortic aneurysms.

The Role of ADAMTS Proteoglycanases in Thoracic Aortic Disease.

Thoracic aortic aneurysm and dissection (TAAD) are complex disease states with high morbidity and mortality that pose significant challenges to early diagnosis. Patients with an aneurysm are asymptomatic and typically present to the emergency department only after the development of a dissection. The extracellular matrix (ECM) plays a crucial role in regulating the aortic structure and function. The histopathologic hallmark termed medial degeneration is characterised by smooth muscle cell (SMC) loss, the degradation of elastic and collagen fibres and proteoglycan (PG) accumulation. Covalently attached to the protein core of PGs are a number of glycosaminoglycan chains, negatively charged molecules that provide flexibility, compressibility, and viscoelasticity to the aorta. PG pooling in the media can produce discontinuities in the aortic wall leading to increased local stress. The accumulation of PGs is likely due to an imbalance between their synthesis by SMCs and decreased proteolysis by A Disintegrin-like and Metalloproteinase with Thrombospondin motifs (ADAMTS) proteoglycanases in the ECM. Mouse models of TAAD indicated that these proteases exert a crucial, albeit complex and not fully elucidated, role in this disease. This has led to a mounting interest in utilising ADAMTS proteoglycanases as biomarkers of TAAD. In this review, we discuss the role of ADAMTSs in thoracic aortic disease and their potential use in facilitating the clinical diagnosis of TAAD and disease progression.

Understanding of arthrofibrosis: New explorative insights into extracellular matrix remodeling of synovial fibroblasts.

Arthrofibrosis following total knee arthroplasty is a fibroproliferative joint disorder marked by dysregulated biosynthesis of extracellular matrix proteins, such as collagens and proteoglycans. The underlying cellular events remain incompletely understood. Myofibroblasts are highly contractile matrix-producing cells characterized by increased alpha-smooth muscle actin expression and xylosyltransferase-I (XT-I) secretion. Human XT-I has been identified as a key mediator of arthrofibrotic remodeling. Primary fibroblasts from patients with arthrofibrosis provide a useful in vitro model to identify and characterize disease regulators and potential therapeutic targets. This study aims at characterizing primary synovial fibroblasts from arthrofibrotic tissues (AFib) regarding their molecular and cellular phenotype by utilizing myofibroblast cell culture models. Compared to synovial control fibroblasts (CF), AFib are marked by enhanced cell contractility and a higher XT secretion rate, demonstrating an increased fibroblast-to-myofibroblast transition rate during arthrofibrosis. Histochemical assays and quantitative gene expression analysis confirmed higher collagen and proteoglycan expression and accumulation in AFib compared to CF. Furthermore, fibrosis-based gene expression profiling identified novel modifier genes in the context of arthrofibrosis remodeling. In summary, this study revealed a unique profibrotic phenotype in AFib that resembles some traits of other fibroproliferative diseases and can be used for the future development of therapeutic interventions.

Biomechanical and transcriptional evidence that smooth muscle cell death drives an osteochondrogenic phenotype and severe proximal vascular disease in progeria.

Hutchinson-Gilford Progeria Syndrome results in rapid aging and severe cardiovascular sequelae that accelerate near end-of-life. We found a progressive disease process in proximal elastic arteries that was less evident in distal muscular arteries. Changes in aortic structure and function were then associated with changes in transcriptomics assessed via both bulk and single cell RNA sequencing, which suggested a novel sequence of progressive aortic disease: adverse extracellular matrix remodeling followed by mechanical stress-induced smooth muscle cell death, leading a subset of remnant smooth muscle cells to an osteochondrogenic phenotype that results in an accumulation of proteoglycans that thickens the aortic wall and increases pulse wave velocity, with late calcification exacerbating these effects. Increased central artery pulse wave velocity is known to drive left ventricular diastolic dysfunction, the primary diagnosis in progeria children. It appears that mechanical stresses above ~ 80kPa initiate this progressive aortic disease process, explaining why elastic lamellar structures that are organized early in development under low wall stresses appear to be nearly normal whereas other medial constituents worsen progressively in adulthood. Mitigating early mechanical stress-driven smooth muscle cell loss/phenotypic modulation promises to have important cardiovascular implications in progeria patients.

Gua Lou Er Chen decoction attenuates atherosclerosis by reducing proteoglycans accumulation and inflammation

BackgroundProteoglycans (PGs) accumulation and inflammation are two interactional pathological processes of atherosclerosis (AS). Up to now, there is no ideal drug for decreasing these pathological changes. Gua Lou Er Chen decoction (GED) has been used to treat AS for several years. However, if GED could treat AS through reducing PGs accumulation and inflammation remains unknown. PurposeThis study was designed to illustrate whether GED could attenuate AS by reducing chondroitin sulphate proteoglycan (CSPG) expressions and alleviating inflammation. MethodsIn vivo study, apolipoprotein E-deficient mice were fed a high-fat diet to induce AS. In vitro study, oxidised low-density lipoprotein (ox-LDL) and tumour necrosis factor (TNF)-α were used to induce proteoglycans accumulation and inflammation changes of vascular smooth muscle cells (VSMCs) and RAW264.7 macrophages. Oil Red O was used to stain mouse aortic lipid plaque. Haematoxylin eosin staining was used to assess the pathological changes of aortic valve and thoracic aorta. Specialised kits were used to identify blood lipids and sGAGs. Immunofluorescence and immunohistochemistry was used to identify aortic valve CSPG and versican. Western blotting, enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction were used to measure versican, interleukin (IL)-6, TNF-α, and chondroitin sulphate (CS) synthetase expressions. CCK-8 was used to measure the cells proliferation. ResultsIn vivo experiments revealed that GED significantly improved hyperlipidemia, lowered lipid plaque deposition in the aorta, and increased plaque stability of AS mice. In addition, further studies revealed that GED lowered the sGAGs, CSPG, and versican levels and down-regulated CS synthetase and inflammatory factor expressions. In vitro experiments revealed that GED decreased TNF-α expression in the RAW264.7 macrophage supernatant stimulated by ox-LDL; decreased versican, CS-related synthetase, and IL-6 expressions; reduced VSMC proliferation stimulated by ox-LDL; down-regulated sGAG and versican expressions of VSMCs stimulated by TNF-α. ConclusionOur results demonstrated that GED could attenuate AS by reducing hyperlipidemia, hyper-expression of CSPG, and inflammation. This study might provide a novel insight into the development of innovative drug for AS.

Inhibition of Astrocytic Carbohydrate Sulfotransferase 15 Promotes Nerve Repair After Spinal Cord Injury via Mitigation of CSPG Mediated Axonal Inhibition.

Nerve tissue regeneration is a significant problem. After neural diseases and damage such as spinal cord injury (SCI), the accumulation of chondroitin sulfate proteoglycans (CSPG) comprising axonal inhibitory glycosaminoglycan chains in the microenvironment is a major barrier that obstructs nerve repair. Interfering with the production of glycosaminoglycans, especially the critical inhibitory chains, could be a potential therapeutic strategy for SCI, which is, however, poorly defined. This study identifies Chst15, the chondroitin sulfotransferase controlling the generation of axonal inhibitory chondroitin sulfate-E, as a therapeutic target of SCI. Using a recently reported small molecular Chst15 inhibitor, this study investigates the effects of Chst15 inhibition on astrocyte behaviors and the associated consequences of in vivo disruption of the inhibitory microenvironment. Deposition of CSPGs in the extracellular matrix and migration of astrocytes are both significantly impaired by Chst15 inhibition. Administration of the inhibitor in transected spinal cord tissues of rats effectively promotes motor functional restoration and nerve tissue regeneration by a mechanism related to the attenuation of inhibitory CSPGs, glial scar formation and inflammatory responses. This study highlights the role of Chst15 in the CSPG-mediated inhibition of neural recovery after SCI and proposes an effective neuroregenerative therapeutic strategy that uses Chst15 as a potential target.

Mimicking Loading-Induced Cartilage Self-Heating in Vitro Promotes Matrix Formation in Chondrocyte-Laden Constructs with Different Mechanical Properties.

Articular cartilage presents a mechanically sensitive tissue. Chondrocytes, the sole cell type residing in the tissue, perceive and react to physical cues as signals that significantly modulate their behavior. Hyaline cartilage is a connective tissue with high dissipative capabilities, able to increase its temperature during daily activities, thus providing a dynamic thermal milieu for the residing chondrocytes. This condition, self-heating, which is still chiefly ignored among the scientific community, adds a new thermal dimension in cartilage mechanobiology. Motivated by the lack of studies exploring this dynamic temperature increase as a potential stimulus in cartilage-engineered constructs, we aimed to elucidate whether loading-induced evolved temperature serves as an independent or complementary regulatory cue for chondrocyte function. In particular, we evaluated the chondrocytes' response to thermal and/or mechanical stimulation in two types of scaffolds exhibiting dissipation levels close to healthy and degenerated articular cartilage. It was found, in both scaffold groups, that the combination of dynamic thermal and mechanical stimuli induced superior effects in the expression of major chondrogenic genes, such as SOX9 and LOXL2, compared to either signal alone. Similar effects were also observed in proteoglycan accumulation over time, along with increased mRNA transcription and synthesis of TRPV4, and for the first time demonstrated in chondrocytes, TREK1 ion channels. Conversely, the chondrogenic response of cells to isolated thermal or mechanical cues was generally scaffold-type dependent. Nonetheless, the significance of thermal stimulus as a chondro-inductive signal was better supported in both studied groups. Our data indicates that the temperature evolution is necessary for chondrocytes to more effectively perceive and translate applied mechanical loading.

Adipose and lipoma stem cells: A donor-matched comparison.

Lipomas are slow growing benign fat tumors that develop in soft tissues of the mesoderm. Thus, the specific (dys-)function of mesenchymal stem cells (MSCs) has been suggested in the development of lipomas, but details of the tumor pathogenesis remain unclear. Existing studies comparing stem cells from native adipose (adipose stem cells [ASCs]) and lipomatous tissues (LSCs) have reported contradicting findings. However, harvesting ASCs and LSCs from different individuals might have influenced proper comparison. Therefore, we aimed to characterize donor-matched ASCs and LSCs to investigate metabolic activity, proliferation, capability for tri-linear differentiation (chondrogenesis, adipogenesis, osteogenesis), and the secretome of mature adipocytes and lipomacytes. Both stem cell types did not differ in metabolic activity, but ASCs demonstrated stronger proliferation than LSCs. While there was no difference in proteoglycan accumulation during chondrogenic differentiation, adipogenesis was higher in ASCs, with more lipid vacuole formation. Conversely, LSCs showed increased osteogenesis by higher calcium deposition. Lipomacytes showed stronger secretory activity and released higher levels of certain adipokines. Our findings indicated that LSCs possessed important characteristics of MSCs, including ASCs. However, LSCs' low proliferation and adipogenic differentiation behavior did not appear to account for enhanced tissue proliferation, but the secretome of lipomacytes could contribute to lipomatous neoplasm.

A Placebo‐Controlled Phase I Study in Healthy Subjects Assessing NVG‐291, which Targets CNS Receptor Protein Tyrosine Phosphatase Sigma (PTPσ)

AbstractBackgroundSubstantial data from animal models of spinal cord injury (SCI), multiple sclerosis (MS) and Alzheimer’s disease (AD) indicate that accumulation of chondroitin sulfate proteoglycans (CSPGs) at sites of CNS damage can lead to inhibition of CNS repair. A CSPG receptor termed protein tyrosine phosphatase sigma (PTPσ) has been implicated in mediating the inhibitory effect of CSPGs. NervGen is developing a first‐in‐class PTPσ modulator NVG‐291, a systemically administered therapeutic peptide derived from the cytoplasmic domain of PTPs, for treatment of nervous system damage. A rodent analogue of NVG‐291 was found to improve functional outcomes, axonal regeneration, remyelination and neuroplasticity in animal models of CNS damage.MethodPart 1 (SAD portion) enrolled 37 subjects randomly assigned into 6 dose cohorts of placebo or NVG‐291. The doses tested exceed the human equivalent doses that showed efficacy in several animal models including SCI and MS. Part 2 (MAD portion) will dose up to 18 subjects randomly assigned into 3 dose cohorts to receive NVG‐291 or placebo once‐daily for 14 days. CSF will be collected in additional subjects for analysis of NVG‐291 concentration and biomarker exploration.ResultNVG‐291 has been safe and well‐tolerated through the 6 SAD cohorts (Part 1). Part 2 will complete in 2022. Part 1/2 results will be presented.ConclusionThis Phase 1 study will establish the safety, tolerability, and pharmacokinetics of NVG‐291 to support advancement to a clinical trial in patients with AD in 2022.

Metformin improves tendon degeneration by blocking translocation of HMGB1 and suppressing tendon inflammation and senescence in aging mice.

This study aimed to characterize aging-induced tendinopathy in mouse Achilles tendon and also to assess the treatment effects of metformin (Met) on aging tendon. We showed that compared to young tendon, aging tendon was in an inflammatory and senescent state as shown by increased expression of inflammatory disulfide HMGB1 (dsHMGB1), inflammatory macrophage marker CD68, and senescent cell markers SA-β-gal, p53, and p16. Moreover, aging tendon was degenerated marked by accumulation of proteoglycans and lipids in its interior. However, treatment of aging tendon by intraperitoneal (IP) injection of Met, a specific inhibitor of HMGB1, reduced dsHMGB1 levels, decreased the expression of CD68, SA-β-gal, CCN1, and p16 in vitro and in vivo. Furthermore, Met treatment also increased the number of NS, SSEA-1, and CD73 positive stem cells in culture and improved the tendon structure in aging mouse. These findings of this study indicate that Met exerts anti-inflammatory and anti-senescent effects on aging tendon.

Plasma and Urine Levels of Glycosaminoglycans in Patients with Systemic Sclerosis and Their Relationship to Selected Interleukins and Marker of Early Kidney Injury.

Systemic sclerosis (SSc) is a chronic connective tissue disease characterized by immune system dysfunction, vasculopathy, and progressive fibrosis of the skin and internal organs, resulting from excessive accumulation of extracellular matrix (ECM) elements, including collagen and proteoglycans (PGs). An uncontrolled PG proliferation, caused by disturbances in their metabolism in tissues, is most likely reflected in the quantitative changes of their components, i.e., glycosaminoglycans (GAGs), in body fluids. Therefore, the aim of this study was to quantify the different types of GAGs in the blood and urine of systemic sclerosis patients. Chondroitin/dermatan sulfates (CS/DS) and heparan sulfates/heparin (HS/H) were quantified by hexuronic acid assay and electrophoretic fractionation, while hyaluronic acid (HA) and keratan sulfates were evaluated using ELISA tests. In turn, individual urinary GAGs were determined using the Blyscan™ Sulfated Glycosaminoglycan Assay Kit. Our results showed that the plasma concentrations of CS/DS, HS/H, HA, and KS in systemic sclerosis patients were significantly higher compared with those in healthy subjects. In the case of urine measurements, we have found that in SSc patients, CS/DC concentrations were significantly higher, while HA concentrations were significantly lower compared with the values observed in the urine of healthy subjects. Importantly, the found by us correlations between plasma keratan sulfate levels and both the duration of the disease and the severity of skin lesions, as expressed by the Rodnan scale, seems to suggest this GAG as a potential marker in assessing disease progression and activity. In addition, a level of urinary excretion of all types of GAGs due to their high positive correlation with uACR, may be a valuable complementary test in the diagnosis of early renal dysfunction in the course of SSc.

Multimodality imaging and transcriptomics to phenotype mitral valve dystrophy in a unique knock-in Filamin-A rat model.

Degenerative mitral valve dystrophy (MVD) leading to mitral valve prolapse is the most frequent form of MV disease, and there is currently no pharmacological treatment available. The limited understanding of the pathophysiological mechanisms leading to MVD limits our ability to identify therapeutic targets. This study aimed to reveal the main pathophysiological pathways involved in MVD via the multimodality imaging and transcriptomic analysis of the new and unique knock-in (KI) rat model for the FilaminA-P637Q (FlnA-P637Q) mutation associated-MVD. Wild-type (WT) and KI rats were evaluated morphologically, functionally, and histologically between 3-week-old and 3-to-6-month-old based on Doppler echocardiography, 3D micro-computed tomography (microCT), and standard histology. RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC-seq) were performed on 3-week-old WT and KI mitral valves and valvular cells, respectively, to highlight the main signalling pathways associated with MVD. Echocardiographic exploration confirmed MV elongation (2.0 ± 0.1 mm vs. 1.8 ± 0.1, P = 0.001), as well as MV thickening and prolapse in KI animals compared to WT at 3 weeks. 3D MV volume quantified by microCT was significantly increased in KI animals (+58% vs. WT, P = 0.02). Histological analyses revealed a myxomatous remodelling in KI MV characterized by proteoglycans accumulation. A persistent phenotype was observed in adult KI rats. Signalling pathways related to extracellular matrix homeostasis, response to molecular stress, epithelial cell migration, endothelial to mesenchymal transition, chemotaxis and immune cell migration, were identified based on RNA-seq analysis. ATAC-seq analysis points to the critical role of transforming growth factor-β and inflammation in the disease. The KI FlnA-P637Q rat model mimics human myxomatous MVD, offering a unique opportunity to decipher pathophysiological mechanisms related to this disease. Extracellular matrix organization, epithelial cell migration, response to mechanical stress, and a central contribution of immune cells are highlighted as the main signalling pathways leading to myxomatous MVD. Our findings pave the road to decipher underlying molecular mechanisms and the specific role of distinct cell populations in this context.

Cell Surface Accumulation of Intracellular Leucine Proline-Enriched Proteoglycan 1 Enhances Odontogenic Potential of Human Dental Pulp Stem Cells.

Primary dental pulp cells can be differentiated into odontoblast-like cells, which are responsible for dentin formation and mineralization. Successful differentiation of primary dental pulp cells can be verified using a few markers. However, odontoblast-specific cell surface markers have not been fully studied yet. LEucine PRoline-Enriched Proteoglycan 1 (LEPRE1) is a basement membrane-associated proteoglycan. LEPRE1 protein levels are increased during odontoblastic differentiation of human dental pulp cells (hDPCs). Intracellular and cell surface accumulation of this protein completely disappeared during dentin maturation and mineralization. Cell surface binding of an anti-LEPRE1 monoclonal antibody that could recognize an extracellular region was gradually increased in the odontoblastic stage. Overexpression and knockdown experiments showed that accumulation of intracellular LEPRE1 could lead to inefficient odontoblastic differentiation and that the movement of LEPRE1 from intracellular region to the cell surface was required for odontoblastic differentiation. Indeed, when LEPRE1 already located on the cell surface was blocked by the anti-LEPRE1 monoclonal antibody, odontoblastic differentiation of hDPCs was inhibited. In this study, we looked at other aspects of LEPRE1 function as a cell surface molecule rather than its known intracellular hydroxylase activity. Our results indicate that this protein has potential as a specific cell surface marker in odontoblastic differentiation.

First study on the effect of transforming growth factor beta 1 and insulin-like growth factor 1 on the chondrogenesis of elephant articular chondrocytes in a scaffold-based 3D culture model.

Background and Aim:Osteoarthritis (OA) is recognized as a degenerative joint disease that leads to chronic pain and low quality of life in animals. Captive elephants, the largest land mammals with a long lifespan, are more prone to develop OA due to restricted spaces and insufficient physical activity. This study aimed to investigate the effect of transforming growth factor-β1 (TGF-β1) and insulin-like growth factor 1 (IGF-1) on elephant chondrogenesis in a scaffold culture of articular chondrocytes.Materials and Methods:Elephant chondrocytes-seeded gelatin scaffolds were cultured in chondrogenic media with or without 10 ng/mL of TGF-β1 or IGF-1 alone or 5–10 ng/mL of their combination for up to 21 days. The mRNA expression of cartilage-specific anabolic genes, ACAN and COL2A1, was analyzed using a real-time reverse transcription-polymerase chain reaction. The amounts of sulfated glycosaminoglycans (sGAGs) in conditioned media and contents in cultured scaffolds were determined through dimethylmethylene blue assay. Cell morphology, accumulation of proteoglycans, and details of the cultured scaffolds were determined using hematoxylin-eosin staining, safranin O staining, and scanning electron microscopy (SEM), respectively.Results:TGF-β1 alone significantly upregulated ACAN gene expression but not COL2A1, while IGF-1 alone did not enhance both ACAN and COL2A1 genes. The combination significantly upregulated both mRNA expression levels of ACAN and COL2A1 gene at day 14. The sGAGs accumulation and contents in the treatment groups, except IGF-1 tended to be higher than the controls, concomitantly with the production of the extracellular matrix, showed the formation of a cartilage-like tissue through histological and SEM analyses.Conclusion:Together, our results suggest that the single treatment of TGF-β1 has a selective effect on ACAN gene, while the combined growth factors seem to be an advantage on elephant chondrogenesis. This three-dimensional culture model is probably helpful for developing cartilage regeneration in vitro and is further applied in tissue engineering for OA treatment in vivo.

Aging-Associated Changes in Cognition, Expression and Epigenetic Regulation of Chondroitin 6-Sulfotransferase Chst3.

Understanding changes in the expression of genes involved in regulating various components of the neural extracellular matrix (ECM) during aging can provide an insight into aging-associated decline in synaptic and cognitive functions. Hence, in this study, we compared the expression levels of ECM-related genes in the hippocampus of young, aged and very aged mice. ECM gene expression was downregulated, despite the accumulation of ECM proteoglycans during aging. The most robustly downregulated gene was carbohydrate sulfotransferase 3 (Chst3), the enzyme responsible for the chondroitin 6-sulfation (C6S) of proteoglycans. Further analysis of epigenetic mechanisms revealed a decrease in H3K4me3, three methyl groups at the lysine 4 on the histone H3 proteins, associated with the promoter region of the Chst3 gene, resulting in the downregulation of Chst3 expression in non-neuronal cells. Cluster analysis revealed that the expression of lecticans—substrates of CHST3—is tightly co-regulated with this enzyme. These changes in ECM-related genes were accompanied by an age-confounded decline in cognitive performance. Despite the co-directional impairment in cognitive function and average Chst3 expression in the studied age groups, at the individual level we found a negative correlation between mRNA levels of Chst3 and cognitive performance within the very aged group. An analysis of correlations between the expression of ECM-related genes and cognitive performance in novel object versus novel location recognition tasks revealed an apparent trade-off in the positive gene effects in one task at the expense of another. Further analysis revealed that, despite the reduction in the Chst3 mRNA, the expression of CHST3 protein is increased in glial cells but not in neurons, which, however, does not lead to changes in the absolute level of C6S and even results in the decrease in C6S in perineuronal, perisynaptic and periaxonal ECM relative to the elevated expression of its protein carrier versican.

Congested Heart Failure in Small Breeds Dogs

Mitral valve disease, commonly known as MVD, is the most common heart disease in dogs, accounting for nearly 75% of all dogs affected. The characteristic of this predominant disease includes collagen degradation, elastic fiber fragmentation and proteoglycan accumulation, leading to deterioration of the mitral valve and ultimately, congestive heart failure (CHF). Treatment to reverse or even delay the damaging effects of MVD has not yet been discovered due to a lack of awareness of the underlying processes. Furthermore, this disease in dogs has many similarities with the human MVD and may potentially serve as a translational model for studying MVD further. Therefore, it is imperative to identify the causes and underlying mechanisms associated with canine MVD in order to treat these patients more efficiently at the earliest detectable stage. MicroRNAs (miRNAs) are small, 21‐25 nucleotides in length, non‐coding RNAs that can down‐regulate the expression of protein coding genes by binding to the 3’UTR sites in their messenger RNAs (mRNA), causing either degradation or gene silencing. Prior studies have shown that one miRNA may regulate multiple mRNAs, subsequently effecting the downstream levels of gene expression. Furthermore, if miRNAs prove to be a useful biomarker of heart disease, not only may they serve as detectors of early disease onset, but they may also be useful as therapeutic agents. Given this recent data, we propose to test the hypothesis that circulating miRNAs may be useful biomarkers for detection of CHF, secondary to MVD. Liquid biopsies (blood samples) were collected in EDTA tubes from 7 healthy dogs with no prior history of heart disease and 7 dogs being treated for CHF secondary to MVD. Total serum RNA was isolated using miRNeasy Serum/Plasma Kit (Qiagen). Isolated RNA was then reverse transcribed using miScript II RT Kit (Qiagen) and lastly, miRNA was quantified using miScript SYBR Green PCR Kit, along with miScript miRNA PCR Arrays (Qiagen).

Enhancing chondrogenic potential via mesenchymal stem cell sheet multilayering.

Advanced tissue engineering approaches for direct articular cartilage replacement in vivo employ mesenchymal stem cell (MSC) sources, exploiting innate chondrogenic potential to fabricate hyaline-like constructs in vitro within three-dimensional (3D) culture conditions. Cell sheet technology represents one such advanced 3D scaffold-free cell culture platform, and previous work has shown that 3D MSC sheets are capable of in vitro hyaline-like chondrogenic differentiation. The present study aims to build upon this understanding and elucidate the effects of an established cell sheet manipulation technique, cell sheet multilayering, on fabrication of MSC-derived hyaline-like cartilage 3D layered constructs in vitro. To achieve this goal, multilayered MSC sheets are prepared and assessed for structural and biochemical transitions throughout chondrogenesis. Results support MSC multilayering as a means of increasing construct thickness and 3D cellular interactions related to in vitro chondrogenesis, including N-cadherin, connexin 43, and integrin β-1. Data indicate that increasing construct thickness from 14 μm (1-layer construct) to 25 μm (2-layer construct) increases these cellular interactions and subsequent in vitro MSC chondrogenesis. However, a clear initial thickness threshold (33 μm - 3-layer construct) is evident that decreases the rate and extent of in vitro chondrogenesis, specifically chondrogenic gene expressions (Sox9, aggrecan, type II collagen) and sulfated proteoglycan accumulation in deposited extracellular matrix (ECM). Together, these data support the utility of cell sheet multilayering as a platform for tailoring construct thickness and subsequent MSC chondrogenesis for future articular cartilage regeneration applications.

Functional, structural and molecular characterization of a new mitral valve prolapse animal model: the FLNA-P637Q KI rat

Abstract Introduction Mitral Valve Prolapse (MVP) affects 3% of the population and is characterized by a heterogeneous mitral leaflet remodeling. The pathophysiological mechanisms involved in MVP development are not fully understood, the only therapeutic option remains the surgical valve replacement. We previously identified FLNA as the first gene causing MVP and recently generated a unique knock-in rat model for the FLNA-P637Q mutation that now paves the road to study the molecular mechanisms involved in MVP development. Purpose The aim of our study was to characterize the morphological, functional and molecular expression of the valvular disease in our unique KI rat model. Methods 5 wild-type (WT) and 10 KI rats were evaluated at 3, 6 and 13 weeks. Comprehensive 2D echocardiography was performed to determine valve function and morphology. 3D quantitative analysis of the mitral valve (MV) remodelling was done using micro computed tomography (microCT). MV tissue composition was analysed based on histological and immunohistochemistry. Transcriptomic comparison was performed using RNA-sequencing approach. Results Based on the qualitative echocardiographic assessment of the valve, a high genotype-phenotype concordance was established for WT and KI animals (100%, 93% and 100% matching for each time points). The anterior leaflet was longer in KI comparatively to WT rats (+12 to +14% increase at all time points (p<0.01)). Increased lengths corroborated the increased leaflets volume assessed by microCT analysis (+20 to +58% in KI compared to WT all time points (p<0.05)). Histological and immunohistological analyses (leaflet's thickening, hypercellularity, proteoglycans accumulation without calcification) pointed out towards a myxomatous valve disease. The differential gene expression profile established by RNAseq analysis revealed that inflammation, epithelial cell migration or mechanical transduction pathways were specifically activated in KI valves. Genes such as Itgb2 (+1.30x), Ccl12 (+2.44x), Ccl2 (+1.79x), Ccl28 (+1.53x), Ccl7 (+2.95x), S100a8 (+8.40x) or S100a9 (+2.67x) were significantly upregulated in the GO:0060326 “cell chemotaxis”, p=2.31x10–5. In the GO:0043542 “endothelial cell migration”, p=1.59x10–6, Klf4 (+1.31x) and Tgfbr1 (+1.21x) were upregulated. Genes part of the GO:0048771 “tissue remodelling” (p=5.52x10–5) were also found upregulated. Conclusion These results establish that our unique KI FLNA-P637Q rat develops a myxomatous MV dystrophy comparable to the one described in MVP patients and thus constitutes a pertinent model to study the pathophysiological molecular mechanisms associated with MVP development. Our results point to molecular pathways including inflammation and epithelial activation, which constitute potential therapeutic targets. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Connect Talent