Evaluation of Therapeutic Oligonucleotides for Familial Amyloid Polyneuropathy in Patient-Derived Hepatocyte-Like Cells

Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (TTR) gene, predominantly expressed in the liver. Two compounds that knockdown TTR, comprising a small interfering RNA (siRNA; ALN-TTR-02) and an antisense oligonucleotide (ASO; IONIS-TTRRx), are currently being evaluated in clinical trials. Since primary hepatocytes from FAP patients are rarely available for molecular analysis and commercial tissue culture cells or animal models lack the patient-specific genetic background, this study uses primary cells derived from urine of FAP patients. Urine-derived cells were reprogrammed to induced pluripotent stem cells (iPSCs) with high efficiency. Hepatocyte-like cells (HLCs) showing typical hepatic marker expression were obtained from iPSCs of the FAP patients. TTR mRNA expression of FAP HLCs almost reached levels measured in human hepatocytes. To assess TTR knockdown, siTTR1 and TTR-ASO were introduced to HLCs. A significant downregulation (>80%) of TTR mRNA was induced in the HLCs by both oligonucleotides. TTR protein present in the cell culture supernatant of HLCs was similarly downregulated. Gene expression of other hepatic markers was not affected by the therapeutic oligonucleotides. Our data indicate that urine cells (UCs) after reprogramming and hepatic differentiation represent excellent primary human target cells to assess the efficacy and specificity of novel compounds

Moeglichkeiten der Bestimmung des fuer die Durchfuehrung des innerstaedtischen oeffentlichen Personennahverkehrs erforderlichen Aufwandes an Arbeitskraeften

DB Leipzig(101) - Di 1978 B VD 209, T. 1, 2 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Sensory neuropeptides are required for bone and cartilage homeostasis in a murine destabilization-induced osteoarthritis model

Numerous studies identified a role for the sensory neuropeptides substance P (SP) and alpha calcitonin generelated peptide (alpha CGRP) in osteoarthritis (OA) pain behavior. Surprisingly, little attention has been paid on how their trophic effects on cartilage and bone cells might affect structural changes of bone and cartilage in OA pathology. Here, we sought to elucidate sensory neuropeptides influence on structural alterations of bone and cartilage during murine OA pathophysiology. OA was induced by destabilization of the medial meniscus (DMM) in the right knee joint of 12 weeks old male C57B1/6J wildtype (WT) mice and mice either deficient for SP (tachykinin 1 (Tac1) -/-) or alpha CGRP. By OARSI histopathological grading we observed significant cartilage matrix degradation after DMM surgery in alpha CGRP-deficient mice after 4 weeks whereas Tac1 -/- scores were not different to sham mice before 12 weeks after surgery. Indentation-type atomic force microscopy (IT-AFM) identified a strong superficial zone (SZ) cartilage phenotype in Tac1 -/- Sham mice. Opposed to WT and alpha CGRP -/- mice, SZ cartilage of Tac1 -/- mice softened 2 weeks after OA induction. In Tac1 -/- DMM mice, bone volume to total volume ratio (BV/TV) increased significantly compared to the Tac1 -/- Sham group, 2 weeks after surgery. WT mice had reduced BV/TV compared to alpha CGRP -/- and Tac1 -/- mice after 12 weeks. Increased calcified cartilage thickness and medial condyle diameter were detected in the medial tibia of all groups 8 weeks after OA induction by nanoCT analysis. Meniscal ossification occurred in all OA groups, but was significantly stronger in the absence of neuropeptides. Increased serum concentration of the respective non-deleted neuropeptide was observed in both neuropeptide-deficient mice strains. Both neuropeptides protect from age-related bone structural changes under physiological conditions and SP additionally demonstrates an anabolic effect on bone structure preservation in a pathophysiological situation. Both neuropeptide deficient mice display an intrinsic structural cartilage matrix phenotype that might alter progression of cartilage degeneration in OA

Nano-Scale Mechanical Properties of the Articular Cartilage Zones in a Mouse Model of Post-Traumatic Osteoarthritis

Destabilization of the medial meniscus (DMM) surgery in mice is used to elucidate the mechanism of post-traumatic osteoarthritis (PT-OA). The study of cartilage biomechanics in PT-OA is important for understanding the pathophysiology of the condition. We used indentation-type atomic force microscopy (IT-AFM) to assess the nanostiffness of the interterritorial matrix of articular cartilage (AC) zones in the medial and the lateral tibia plateau (MTP and LTP) on native tissue sections 2 and 8 weeks after DMM or Sham surgery. At 2 weeks, pronounced stiffening of the DMM AC was observed compared to Sham, with the most marked changes occurring in the superficial zone and affecting the proteoglycan moiety rather than the collagen network. The LTP cartilage was obviously stiffer than the MTP in DMM, but not in Sham. At 8 weeks, only modest differences in nanostiffness were observed between DMM and Sham. The difference in stiffness between MTP and LTP was reduced, and the proteoglycan and collagen phases changed in a more similar manner. Interestingly, the deep zone was softer in the DMM compared to the Sham. Sham AC showed an increase in stiffness between 2 and 8 weeks, a trend that was counteracted in the DMM group. Collectively, our study demonstrates that nano-scale IT-AFM is a sensitive tool to monitor biomechanical changes during the course of PT-OA

Nano-Scale Mechanical Properties of the Articular Cartilage Zones in a Mouse Model of Post-Traumatic Osteoarthritis

Destabilization of the medial meniscus (DMM) surgery in mice is used to elucidate the mechanism of post-traumatic osteoarthritis (PT-OA). The study of cartilage biomechanics in PT-OA is important for understanding the pathophysiology of the condition. We used indentation-type atomic force microscopy (IT-AFM) to assess the nanostiffness of the interterritorial matrix of articular cartilage (AC) zones in the medial and the lateral tibia plateau (MTP and LTP) on native tissue sections 2 and 8 weeks after DMM or Sham surgery. At 2 weeks, pronounced stiffening of the DMM AC was observed compared to Sham, with the most marked changes occurring in the superficial zone and affecting the proteoglycan moiety rather than the collagen network. The LTP cartilage was obviously stiffer than the MTP in DMM, but not in Sham. At 8 weeks, only modest differences in nanostiffness were observed between DMM and Sham. The difference in stiffness between MTP and LTP was reduced, and the proteoglycan and collagen phases changed in a more similar manner. Interestingly, the deep zone was softer in the DMM compared to the Sham. Sham AC showed an increase in stiffness between 2 and 8 weeks, a trend that was counteracted in the DMM group. Collectively, our study demonstrates that nano-scale IT-AFM is a sensitive tool to monitor biomechanical changes during the course of PT-OA

ER Stress in ERp57 Knockout Knee Joint Chondrocytes Induces Osteoarthritic Cartilage Degradation and Osteophyte Formation

Ageing or obesity are risk factors for protein aggregation in the endoplasmic reticulum (ER) of chondrocytes. This condition is called ER stress and leads to induction of the unfolded protein response (UPR), which, depending on the stress level, restores normal cell function or initiates apoptotic cell death. Here the role of ER stress in knee osteoarthritis (OA) was evaluated. It was first tested in vitro and in vivo whether a knockout (KO) of the protein disulfide isomerase ERp57 in chondrocytes induces sufficient ER stress for such analyses. ER stress in ERp57 KO chondrocytes was confirmed by immunofluorescence, immunohistochemistry, and transmission electron microscopy. Knee joints of wildtype (WT) and cartilage-specific ERp57 KO mice (ERp57 cKO) were analyzed by indentation-type atomic force microscopy (IT-AFM), toluidine blue, and immunofluorescence/-histochemical staining. Apoptotic cell death was investigated by a TUNEL assay. Additionally, OA was induced via forced exercise on a treadmill. ER stress in chondrocytes resulted in a reduced compressive stiffness of knee cartilage. With ER stress, 18-month-old mice developed osteoarthritic cartilage degeneration with osteophyte formation in knee joints. These degenerative changes were preceded by apoptotic death in articular chondrocytes. Young mice were not susceptible to OA, even when subjected to forced exercise. This study demonstrates that ER stress induces the development of age-related knee osteoarthritis owing to a decreased protective function of the UPR in chondrocytes with increasing age, while apoptosis increases. Therefore, inhibition of ER stress appears to be an attractive therapeutic target for OA

Mice Lacking the Matrilin Family of Extracellular Matrix Proteins Develop Mild Skeletal Abnormalities and Are Susceptible to Age-Associated Osteoarthritis

Matrilins (MATN1, MATN2, MATN3 and MATN4) are adaptor proteins of the cartilage extracellular matrix (ECM), which bridge the collagen II and proteoglycan networks. In humans, dominant-negative mutations in MATN3 lead to various forms of mild chondrodysplasias. However, single or double matrilin knockout mice generated previously in our laboratory do not show an overt skeletal phenotype, suggesting compensation among the matrilin family members. The aim of our study was to establish a mouse line, which lacks all four matrilins and analyze the consequence of matrilin deficiency on endochondral bone formation and cartilage function. Matn1-4(-/-) mice were viable and fertile, and showed a lumbosacral transition phenotype characterized by the sacralization of the sixth lumbar vertebra. The development of the appendicular skeleton, the structure of the growth plate, chondrocyte differentiation, proliferation, and survival were normal in mutant mice. Biochemical analysis of knee cartilage demonstrated moderate alterations in the extractability of the binding partners of matrilins in Matn1-4(-/-) mice. Atomic force microscopy (AFM) revealed comparable compressive stiffness but higher collagen fiber diameters in the growth plate cartilage of quadruple mutant compared to wild-type mice. Importantly, Matn1-4(-/-) mice developed more severe spontaneous osteoarthritis at the age of 18 months, which was accompanied by changes in the biomechanical properties of the articular cartilage. Interestingly, Matn4(-/-) mice also developed age-associated osteoarthritis suggesting a crucial role of MATN4 in maintaining the stability of the articular cartilage. Collectively, our data provide evidence that matrilins are important to protect articular cartilage from deterioration and are involved in the specification of the vertebral column

Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage

Energy metabolism and extracellular matrix (ECM) function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single-cell RNA-Seq (scRNA-Seq) analysis to uncover the importance of the mitochondrial respiratory chain for ECM homeostasis in mature cartilage. This tissue produces large amounts of a specialized ECM to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of ECM material. scRNASeq analysis identified a cell cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of ECM-related genes in nonarticular chondrocytes. These changes were associated with alterations in ECM composition, a shift in collagen/noncollagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues