Systematic comparison of sequencing-based spatial transcriptomic methods.

Recent developments of sequencing-based spatial transcriptomics (sST) have catalyzed important advancements by facilitating transcriptome-scale spatial gene expression measurement. Despite this progress, efforts to comprehensively benchmark different platforms are currently lacking. The extant variability across technologies and datasets poses challenges in formulating standardized evaluation metrics. In this study, we established a collection of reference tissues and regions characterized by well-defined histological architectures, and used them to generate data to compare 11 sST methods. We highlighted molecular diffusion as a variable parameter across different methods and tissues, significantly affecting the effective resolutions. Furthermore, we observed that spatial transcriptomic data demonstrate unique attributes beyond merely adding a spatial axis to single-cell data, including an enhanced ability to capture patterned rare cell states along with specific markers, albeit being influenced by multiple factors including sequencing depth and resolution. Our study assists biologists in sST platform selection, and helps foster a consensus on evaluation standards and establish a framework for future benchmarking efforts that can be used as a gold standard for the development and benchmarking of computational tools for spatial transcriptomic analysis.

Nr4a1 enhances Wnt4 transcription to promote mesenchymal stem cell osteogenesis and alleviates inflammation-inhibited bone regeneration.

Intense inflammatory response impairs bone marrow mesenchymal stem cell (BMSC)-mediated bone regeneration, with transforming growth factor (TGF)-ß1 being the most highly expressed cytokine. However, how to find effective and safe means to improve bone formation impaired by excessive TGF-ß1 remains unclear. In this study, we found that the expression of orphan nuclear receptor Nr4a1, an endogenous repressor of TGF-ß1, was suppressed directly by TGF-ß1-induced Smad3 and indirectly by Hdac4, respectively. Importantly, Nr4a1 overexpression promoted BMSC osteogenesis and reversed TGF-ß1-mediated osteogenic inhibition and pro-fibrotic effects. Transcriptomic and histologic analyses confirmed that upregulation of Nr4a1 increased the transcription of Wnt family member 4 (Wnt4) and activated Wnt pathway. Mechanistically, Nr4a1 bound to the promoter of Wnt4 and regulated its expression, thereby enhancing the osteogenic capacity of BMSCs. Moreover, treatment with Nr4a1 gene therapy or Nr4a1 agonist Csn-B could promote ectopic bone formation, defect repair, and fracture healing. Finally, we demonstrated the correlation of NR4A1 with osteogenesis and the activation of the WNT4/ß-catenin pathway in human BMSCs and fracture samples. Taken together, these findings uncover the critical role of Nr4a1 in bone formation and alleviation of inflammation-induced bone regeneration disorders, and suggest that Nr4a1 has the potential to be a therapeutic target for accelerating bone healing.

Neuritin accelerates Schwann cell dedifferentiation via PI3K/Akt/mTOR signalling pathway during Wallerian degeneration.

Neuritin, also known as candidate plasticity gene 15 (CPG15), was first identified as one of the activity-dependent gene products in the brain. Previous studies have been reported that Neuritin induces neuritogenesis, neurite arborization, neurite outgrowth and synapse formation, which are involved in the development and functions of the central nervous system. However, the role of Neuritin in peripheral nerve injury is still unknown. Given the importance and necessity of Schwann cell dedifferentiation response to peripheral nerve injury, we aim to investigate the molecular mechanism of Neuritin steering Schwann cell dedifferentiation during Wallerian degeneration (WD) in injured peripheral nerve. Herein, using the explants of sciatic nerve, an ex vivo model of nerve degeneration, we provided evidences indicating that Neuritin vividly accelerates Schwann cell dedifferentiation. Moreover, we found that Neuritin promotes Schwann cell demyelination as well as axonal degeneration, phagocytosis, secretion capacity. In summary, we first described Neuritin acts as a positive regulator for Schwann cell dedifferentiation and WD after peripheral nerve injury.

CRIP1 regulates osteogenic differentiation of bone marrow stromal cells and pre-osteoblasts via the Wnt signaling pathway.

With the aging of the global demographic, the prevention and treatment of osteoporosis are becoming crucial issues. The gradual loss of self-renewal and osteogenic differentiation capabilities in bone marrow stromal cells (BMSCs) is one of the key factors contributing to osteoporosis. To explore the regulatory mechanisms of BMSCs differentiation, we collected bone marrow cells of femoral heads from patients undergoing total hip arthroplasty for single-cell RNA sequencing analysis. Single-cell RNA sequencing revealed significantly reduced CRIP1 (Cysteine-Rich Intestinal Protein 1) expression and osteogenic capacity in the BMSCs of osteoporosis patients compared to non-osteoporosis group. CRIP1 is a gene that encodes a member of the LIM/double zinc finger protein family, which is involved in the regulation of various cellular processes including cell growth, development, and differentiation. CRIP1 knockdown resulted in decreased alkaline phosphatase activity, mineralization and expression of osteogenic markers, indicating impaired osteogenic differentiation. Conversely, CRIP1 overexpression, both in vitro and in vivo, enhanced osteogenic differentiation and rescued bone mass reduction in ovariectomy-induced osteoporosis mice model. The study further established CRIP1's modulation of osteogenesis through the Wnt signaling pathway, suggesting that targeting CRIP1 could offer a novel approach for osteoporosis treatment by promoting bone formation and preventing bone loss.

2D capsid formation within an oscillatory energy landscape: orderly self-assembly depends on the interplay between a dynamic potential and intrinsic relaxation times.

Multiple dissipative self-assembly protocols designed to create novel structures or to reduce kinetic traps have recently emerged. Specifically, temporal oscillations of particle interactions have been shown effective at both aims, but investigations thus far have focused on systems of simple colloids or their binary mixtures. In this work, we expand our understanding of the effect of temporally oscillating interactions to a two-dimensional coarse-grained viral capsid-like model that undergoes a self-limited assembly. This model includes multiple intrinsic relaxation times due to the internal structure of the capsid subunits and, under certain interaction regimes, proceeds via a two-step nucleation mechanism. We find that oscillations much faster than the local intrinsic relaxation times can be described via a time averaged inter-particle potential across a wide range of interaction strengths, while oscillations much slower than these relaxation times result in structures that adapt to the attraction strength of the current half-cycle. Interestingly, oscillation periods similar to these relaxation times shift the interaction window over which orderly assembly occurs by enabling error correction during the half-cycles with weaker attractions. Our results provide fundamental insights to non-equilibrium self-assembly on temporally variant energy landscapes.

Stochastic distinguishability of Markovian trajectories.

The ability to distinguish between stochastic systems based on their trajectories is crucial in thermodynamics, chemistry, and biophysics. The Kullback-Leibler (KL) divergence, DKLAB(0,τ), quantifies the distinguishability between the two ensembles of length-τ trajectories from Markov processes A and B. However, evaluating DKLAB(0,τ) from histograms of trajectories faces sufficient sampling difficulties, and no theory explicitly reveals what dynamical features contribute to the distinguishability. This work provides a general formula that decomposes DKLAB(0,τ) in space and time for any Markov processes, arbitrarily far from equilibrium or steady state. It circumvents the sampling difficulty of evaluating DKLAB(0,τ). Furthermore, it explicitly connects trajectory KL divergence with individual transition events and their waiting time statistics. The results provide insights into understanding distinguishability between Markov processes, leading to new theoretical frameworks for designing biological sensors and optimizing signal transduction.

Identifying Candidate Genes Associated with Sporadic Amyotrophic Lateral Sclerosis via Integrative Analysis of Transcriptome-Wide Association Study and Messenger RNA Expression Profile.

Amyotrophic lateral sclerosis, a fatal neurodegeneration disease affecting motor neurons in the brain and spinal cord, is difficult to diagnose and treat. The objective of this study is to identify novel candidate genes related to ALS. Transcriptome-wide association study of ALS was conducted by integrating the genome-wide association study summary data (including 1234 ALS patients and 2850 controls) and pre-computed gene expression weights of different tissues. The ALS-associated genes identified by TWAS were further compared with the differentially expressed genes detected by the mRNA expression profiles of the sporadic ALS. Functional enrichment and annotation analysis of identified genes were performed by an R package and the functional mapping and annotation software. TWAS identified 761 significant genes (PTWAS < 0.05), 627 Gene ontology terms, and 8 Kyoto Encyclopedia of Genes and Genomes pathways for ALS, such as C9orf72, with three expression quantitative trait loci were found significantly: rs2453554 (PTWAS CBRS = 4.68 × 10-10, PTWAS CBRS = 2.54 × 10-9), rs10967976 (PTWAS CBRS = 7.85 × 10-10, PTWAS CBRS = 8.91 × 10-9, PTWAS CBRS = 1.49 × 10-7, PTWAS CBRS = 5.59 × 10-7), rs3849946 (PTWAS CBRS = 7.69 × 10-4, PTWAS YBL = 4.02 × 10-2), Mitochondrion (Padj = 4.22 × 10-16), and Cell cycle (Padj = 2.04 × 10-3). Moreover, 107 common genes, 4 KEGG pathways and 41 GO terms were detected by integrating mRNA expression profiles of sALS, such as CPVL (FC = 2.06, PmRNA = 6.99 × 10-6, PTWAS CBR = 2.88 × 10-2, PTWAS CBR = 4.37 × 10-2), Pyrimidine Metabolism (Padj = 2.43 × 10-2), and Cell Activation (Padj = 5.54 × 10-3). Multiple candidate genes and pathways were detected for ALS. Our findings may provide novel clues for understanding the genetic mechanism of ALS.

miR-10396b-3p inhibits mechanical stress-induced ligamentum flavum hypertrophy by targeting IL-11.

Ligamentum flavum hypertrophy (LFH) leads to lumbar spinal stenosis (LSS) caused by LF tissue inflammation and fibrosis. Emerging evidence has indicated that dysregulated microRNAs (miRNAs) have an important role in inflammation and fibrosis. Mechanical stress (MS) has been explored as an initiating step in LFH pathology progression; the inflammation-related miRNAs induced after mechanical stress have been implicated in fibrosis pathology. However, the pathophysiological mechanism of MS-miRNAs-LFH remains to be elucidated. Using miRNAs sequencing analysis and subsequent confirmation with qRT-PCR assays, we identified the decreased expression of miR-10396b-3p and increased expression of IL-11 (interleukin-11) as responses to the development of LSS in hypertrophied LF tissues. We also found that IL-11 is positively correlated with fibrosis indicators of collagen I and collagen III. The up-regulation of miR-10396b-3p significantly decreased the level of IL-11 expression, whereas miR-10396b-3p down-regulation increased IL-11 expression in vitro. Luciferase reporter assay indicates that IL-11 is a direct target of miR-10396b-3p. Furthermore, cyclic mechanical stress inhibits miR-10396b-3p and induces IL-11, collagen I, and collagen III in vitro. Our results showed that overexpression of miR-10396b-3p suppresses MS-induced LFH by inhibiting collagen I and III via the inhibition of IL-11. These data suggest that the MS-miR-10396b-3p-IL-11 axis plays a key role in the pathological progression of LFH.

EGF Contributes to Hypertrophy of Human Ligamentum Flavum via the TGF-ß1/Smad3 Signaling Pathway.

Background: The most common spinal disorder in elderly is lumbar spinal canal stenosis (LSCS). Previous studies showed that ligamentum flavum hypertrophy (LFH) with fibrosis as the main pathological change is one of the pathogenic factors leading to LSCS. Epidermal Growth Factor (EGF) is known to have an intimate relationship with fibrosis in various tissues. Nevertheless, currently, there are few studies regarding EGF in LFH. The effect of EGF on the development of LFH is unknown, and the underlying pathomechanism remains unclear. In this study, we investigated the role of EGF in LFH and its potential molecular mechanism. Methods: First, the expression levels of EGF, phosphorylation of EGF receptor (pEGFR), Transforming growth factor-ß1 (TGF-ß1), Phosphorylated Smad3 (pSmad3), collagen I and collagen III were examined via immunohistochemistry and Western blot in LF tissues from patients with LSCS or Non-LSCS. Second, primary LF cells were isolated from adults with normal LF thickness and were cultured with different concentrations of exogenous EGF with or without erlotinib/TGF-ß1-neutralizing antibody. Results: The results showed that EGF, pEGFR, TGF-ß1, pSmad3, collagen I and collagen III protein expression in the LSCS group was significantly higher than that in the Non-LSCS group. Meanwhile, pEGFR, TGF-ß1, pSmad3, collagen I and collagen III protein expression was significantly enhanced in LF cells after exogenous EGF exposure, which can be notably blocked by erlotinib. In addition, pSmad3, collagen I and collagen III protein expression was blocked by TGF-ß1-neutralizing antibody. Conclusions: EGF promotes the synthesis of collagen I and collagen III via the TGF-ß1/Smad3 signaling pathway, which eventually contributes to LFH.

Protective Effect of the Pearl Extract from Pinctada fucata martensii Dunker on UV-Induced Photoaging in Mice.

Although the effect of pearl powder has been recognized for more than a thousand years from healthcare to beauty care, there has yet to be an in-depth understanding of its anti-photoaging effect. In the present study, the protective effect of pearl extract (PE) on UV-induced photoaging in mice was evaluated. First, the amino acid analysis of PE was carried out. Then, different dosages of pearl extract gel (PEG) were applied topically on the shaved dorsal skins regions of mice before UV irradiation. Skin physiological and histological analysis, antioxidant enzymes and inflammatory factor test were used to evaluate the anti-photoaging effect of PEG. The results showed that PEG contained 14 amino acids, and could inhibit UV-irritated skin wrinkles, laxity, thickness, and dryness. Moreover, PEG upregulated the activities of CAT, GSH-Px, SOD and decreased MDA level, and suppressed the production of IL-1ß, IL-6, PGE2 , TNF-α, and COX-2 in UV-irradiated mice. The therapeutic effect in high dose PEG group was superior to those of positive control (Vitamin E). This study demonstrated the underlying mechanisms of PEG against UV-irritated photoaging. And PEG possesses a potential use in photoprotective medicines and cosmetics.

Hydroxychloroquine/chloroquine and the risk of acute kidney injury in COVID-19 patients: a systematic review and meta-analysis.

OBJECTIVES: Hydroxychloroquine/chloroquine has been widely used as part of the standard treatment for patients with coronavirus disease 2019 (COVID-19). We conducted a systematic review and meta-analysis to determine whether hydroxychloroquine/chloroquine increases the risk of acute kidney injury (AKI) in COVID-19 patients. METHODS: PubMed and Embase were searched for related publications from inception to Dec 31, 2021, including randomized controlled trials (RCTs) and non-randomized studies of interventions (NRSIs) comparing the risk of AKI and/or increased creatinine in COVID-19 patients receiving hydroxychloroquine/chloroquine and other controls (active treatment and placebo). We conducted separate meta-analyses for RCTs and NRSIs based on fixed-effect model, with odds ratios (ORs) being considered as effect sizes. RESULTS: We included 21 studies in the analysis, with 12 were RCTs. Based on the RCTs, compared to placebo, the OR was 1.19 (95% confidence interval [CI]: 0.86, 1.64; p = .30, n = 4, moderate quality) for AKI and 1.00 (95%CI: 0.64, 1.56; p = .99, n = 5, moderate quality) for increased creatinine for patients received hydroxychloroquine/chloroquine treatment; compared to active treatment, the odds was 1.28 (95%CI: 0.65, 2.53; p = .47, n = 2, low quality) for AKI and 0.64 (95%CI: 0.13, 3.20; p = .59, n = 1, low quality) for increased creatine. Evidence from NRSIs showed slightly increased odds of AKI, with low quality. CONCLUSION: Based on current available studies which were graded as low to moderate quality, there is insufficient evidence to conclude that hydroxychloroquine/chloroquine use is associated with increased risk of AKI or raised creatinine. Abbreviations: AKI: acute kidney injury; COVID-19: Coronavirus Disease 2019; RCT: randomized controlled trials; NRSI: non-randomized studies of interventions; OR: odds ratios; ROBIS-I: Risk Of Bias In Non-randomized Studies - of Interventions.

Macrophage-derived neurotrophin-3 promotes heterotopic ossification in rats.

Heterotopic ossification (HO) is a debilitating condition that results from traumatic injuries or genetic diseases, for which the underlying mechanisms remain unclear. Recently, we have demonstrated the expression of neurotrophin-3 (NT-3) and its role in promoting HO formation via mediating endothelial-mesenchymal transition (EndMT) of vascular endothelial cells. The current study investigated the role of NT-3 on the surrounding mesenchymal cells and its potential origin throughout HO formation at injured Achilles tendons in rats. We used an Achilles tenotomy to induce HO formation in vivo and cultured primary tendon-derived stem cells (TDSCs) to investigate the underlying mechanisms mediating the osteogenesis in vitro. Furthermore, RAW264.7 cells were employed to identify the origin of NT-3. The mRNA levels of NGF, BDNF, NT-3, and NT-4 and their tyrosine protein kinase (Trk) receptors as well as p75 receptor were elevated at injury sites. NT-3 and TrkC showed the highest induction. Neutralization of the NT-3-induced effects by the pan-Trk inhibitor GNF5837 reduced the expression of bone/cartilage-related genes while injection of NT-3 promoted HO formation with elevated mRNA of bone/cartilage-related markers at injured sites. In vitro, NT-3 accelerated osteogenic differentiation and mineralization of TDSCs through activation of the ERK1/2 and PI3K/Akt signaling pathways. Moreover, the colocalization of NT-3 and macrophages, including M1 and M2 macrophages, was observed in injured sites throughout HO formation, and in vitro studies demonstrated that activated macrophages mediated the secretion of NT-3. In addition, an increasing concentration of serum or supernatant NT-3 was observed both in vivo and in vitro. Depletion of macrophages with clodronate-loaded liposomes reduced HO formation as well as secretion and mRNA expression of NT-3. Our study suggests that macrophage-derived NT-3 may promote HO formation and osteogenesis of TDSCs via the ERK1/2 and PI3K/Akt signaling pathways, which may provide new insights for the therapeutic directions of HO in the future.

Neurotrophin-3 acts on the endothelial-mesenchymal transition of heterotopic ossification in rats.

Despite the fact that extensive studies have focused on heterotopic ossification (HO), its molecular mechanism remains unclear. The endothelial-mesenchymal transition (EndMT), which may be partially modulated by neuroendocrine cytokines is thought to play a major role in HO. Neurotrophin-3 (NT-3), which has neuroendocrine characteristics is believed to promote skeletal remodeling. Herein, we suggest that that NT-3 may promote HO formation through regulation of EndMT. Here, we used an in vivo model of HO and an in vitro model of EndMT induction to elucidate the effect and underlying mechanism of NT-3 on EndMT in HO. Our results showed that heterotopic bone and cartilage arose from EndMT and NT-3 promoted HO formation in vivo. Our in vitro results showed that NT-3 up-regulated mesenchymal markers (FSP-1, α-SMA and N-cadherin) and mesenchymal stem cell (MSC) markers (STRO-1, CD44 and CD90) and down-regulated endothelial markers (Tie-1, VE-cadherin and CD31). Moreover, NT-3 enhanced a chondrogenesis marker (Sox9) and osteogenesis markers (OCN and Runx2) via activation of EndMT. However, both EndMT specific inhibitor and tropomyosin-related kinase C (TrkC) specific inhibitor rescued NT-3-induced HO formation and EndMT induction in vivo and in vitro. In conclusion, our findings demonstrate that NT-3 promotes HO formation via modulation of EndMT both in vivo and in vitro, which offers a new potential target for the prevention and therapy of HO.

Identification of upstream miRNAs of SNAI2 and their influence on the metastasis of gastrointestinal stromal tumors.

BACKGROUND: SNAI2, a member of the snail zinc finger protein family, plays an important role in the metastasis of several types of carcinoma. OBJECTIVE: This study aims to investigate the upstream miRNAs of SNAI2 and their influence on the metastasis of gastrointestinal stromal tumors (GISTs). METHODS: The expression levels of SNAI2, CDH1, and CDH2 in GISTs were determined by immunohistochemistry, and the correlations with their clinicopathologic characteristics were analyzed. Subsequently, the miRNAs involved in regulating SNAI2 expression were predicted by bioinformatics technique, screened by miRNA microarray tests, and verified by real-time PCR, dual luciferase reporter assay, and invasion assay. The influence of SNAI2 and miRNAs on the invasive ability of the GIST cells and the related mechanism were detected. OUTCOMES: SNAI2 expression significantly increased and CDH1 expression markedly decreased in the cases of GISTs with distant metastasis. Silencing of the SNAI2 gene impaired the invasiveness of GIST cells in vitro. MiR-200b-3p, miR-30c-1-3P, and miR-363-3P were verified as the upstream metastasis-associated miRNAs of SNAI2 in GISTs by miRNA microarray, real-time PCR, dual luciferase reporter assay, and invasion assay. They bound to the 3'-UTR of SNAI2, downregulated SNAI2 expression, and inhibited the invasiveness of GIST cells. SNAI2 targetedly bound to the promoter of the CDH1 gene, downregulated the expression of CDH1, and contributed to the metastasis of GISTs. CONCLUSION: SNAI2 and CDH1 correlated with the metastasis of GISTs, and silencing of the SNAI2 gene impaired the invasiveness of GIST cells. MiR-200b-3p, miR-30c-1-3P, and miR-363-3P contribute to the metastasis of GISTs in vitro by mediating the SNAI2/CDH1 axis. SNAI2 may be a potential target for the treatment of GISTs in the future.

Development and Characterization of a Novel Bipedal Standing Mouse Model of Intervertebral Disc and Facet Joint Degeneration.

BACKGROUND: Intervertebral disc degeneration is a major cause of chronic low back pain, and excessive loading contributes to intervertebral disc degeneration. However, the lack of an effective bipedal in vivo animal model limits research about this condition. QUESTIONS/PURPOSES: To evaluate the utility of a new type of bipedal standing mouse model for intervertebral disc degeneration, we asked: (1) Are there spinal degeneration changes in bipedal mice as determined by lumbar disc height, histologic features, and immunohistochemistry measures compared with control mice? (2) Are the bipedal mice comparable to aged mice for simulating the spinal degeneration caused by increased stress? METHODS: Thirty-two 8-week-old male C57BL/6 mice were divided into experimental and control groups. Based on their hydrophobia, mice in the experimental group were placed in a limited water-containing space (5 mm deep) and were thereby induced to actively take a bipedal standing posture. This was conducted twice a day for a total of 6 hours a day, 7 days a week. Control mice were similarly placed in a limited but water-free space. Video surveillance was used to calculate the percentage of time spent in the bipedal stance for the two groups of mice. Compared with the control group, the percentage of time standing on both feet in the experimental group was higher (48% ± 5%, 95% confidence interval [CI], 42%-54% versus 95% ± 1%, 95% CI, 92%-97%; p < 0.001). Eight mice from both groups were then randomly euthanized at either 6 or 10 weeks and lumbar spine specimens (L3-L6) were collected. The lumbar disc height index (DHI%) of the two groups was compared using micro-CT measurements, and the extent of disc degeneration was assessed based on histologic staining (cartilage endplate height, disc degeneration score) and by immunohistochemistry (Col2a1,CollagenX, matrix metalloprotease-13 [MMP-13], osteocalcin [OCN]). In addition, the histopathologic features of spinal degeneration were compared with 12- and 18-month-old mice. A p value < 0.05 indicated a significant difference. RESULTS: Lumbar disc degeneration was aggravated after 10 weeks with the DHI% decreasing (5.0% ± 0.4%; 95% CI, 4.6%-5.5% versus 4.6 ± 0.3%; 95% CI, 4.3%-4.9%; p = 0.011). Histologically, the cartilage endplate height of the experimental group was decreased compared with the control group (30 ± 6 µm; 95% CI, 24-37 µm versus 70 ± 7 µm; 95% CI, 63-79 µm; p < 0.001), and the disc degeneration score was increased (5 ± 1; 95% CI, 4-6 versus 1 ± 1; 95% CI, 0-2; p < 0.001). Expression of Col2a1, vimentin, and aggrecan in the experimental group was decreased compared with the control group, whereas the expressions of collagen X (60% ± 2%; 95% CI, 55%-66% versus 19% ± 3%; 95% CI, 17%-24%; p < 0.001), MMP-13 (54% ± 8%; 95% CI, 49%-61% versus 1% ± 1%; 95% CI, 1%-2%; p < 0.001), and OCN (41% ± 3%; 95% CI, 34%-49% versus 5% ± 1%; 95% CI, 2%-7%, p < 0.001) were increased. The spine degeneration caused by this model was primarily manifested in the degeneration of the annulus fibrosus and facet joints compared with aged mice, whereas the degree of degeneration in the nucleus pulposus tissue and cartilage endplates was mild. CONCLUSIONS: We believe we have established a noninvasive and effective in vivo bipedal mouse model for studying disc degeneration and biologic signal transduction comparable to that seen in intervertebral disc degeneration. CLINICAL RELEVANCE: This in vivo mouse model of intervertebral disc degeneration can simulate the pathogenesis of spinal degeneration caused by increased stress and this can be used to study questions such as disc herniation in young adults.

Leptin accelerates the pathogenesis of heterotopic ossification in rat tendon tissues via mTORC1 signaling.

Leptin, an adipocyte-derived cytokine associated with bone metabolism, is believed to play a critical role in the pathogenesis of heterotopic ossification (HO). The effect and underlying action mechanism of leptin were investigated on osteogenic differentiation of tendon-derived stem cells (TDSCs) in vitro and the HO formation in rat tendons. Isolated rat TDSCs were treated with various concentrations of leptin in the presence or absence of mTORC1 signaling specific inhibitor rapamycin in vitro. A rat model with Achilles tenotomy was employed to evaluate the effect of leptin on HO formation together with or without rapamycin treatment. In vitro studies with TDSCs showed that leptin increased the expression of osteogenic biomarkers (alkaline phosphatase, runt-related transcription factor 2, osterix, osteocalcin) and enhanced mineralization of TDSCs via activating the mTORC1 signal pathway (as indicated by phosphorylation of p70 ribosomal S6 kinase 1 and p70 ribosomal S6). However, mTORC1 signaling blockade with rapamycin treatment suppressed leptin-induced osteogenic differentiation and mineralization. In vivo studies showed that leptin promoted HO formation in the Achilles tendon after tenotomy, and rapamycin treatment blocked leptin-induced HO formation. In conclusion, leptin can promote TDSC osteogenic differentiation and heterotopic bone formation via mTORC1 signaling in both vitro and vivo model, which provides a new potential therapeutic target for HO prevention.

Locally Produced IGF-1 Promotes Hypertrophy of the Ligamentum Flavum via the mTORC1 Signaling Pathway.

BACKGROUND/AIMS: Narrowing of the lumbar spinal canal is a condition called lumbar spinal stenosis (LSS) and is a high-morbidity problem in the elderly. LSS is commonly caused by hypertrophy of the ligamentum flavum (HLF). Previous studies showed that fibrosis of the ligamentum flavum (LF) largely contributed to HLF. However, the underlying pathomechanism remains unclear. Insulin-like growth factor-1 (IGF-1) is known to have an intimate relationship with fibrosis in various tissues. Nevertheless, currently, there are few studies regarding IGF-1 in HLF. In this study, we investigated the role of IGF-1 in HLF and its potential molecular mechanism of action. METHODS: First, the IGF-1, phosphorylation of IGF-1 receptor (pIGF-1R), phosphorylation of AKT (pAKT), phosphorylation of S6(pS6), collagen I and collagen III expression levels were examined via immunohistochemistry and Western blotting in LF tissues from patients with LSS or Non-LSS. Second, primary LF cells were isolated from adults with a normal LF thickness and were cultured with different concentrations of IGF-1 with or without NVP-AEW541/rapamycin. RESULTS: The results showed that IGF-1, pIGF-1R, pAKT, pS6, collagen I and collagen III protein expression in the LSS group was significantly higher than that in the Non-LSS group. Meanwhile, pIGF-1R, pAKT, pS6, collagen I and collagen III protein expression was significantly enhanced in LF cells after IGF-1 exposure, which can be notably blocked by NVP-AEW541. In addition, pS6, collagen I and collagen III protein expression was blocked by rapamycin. CONCLUSIONS: Enhanced IGF-1 promotes the synthesis of collagen I and collagen III via the mTORC1 signaling pathway, which eventually contributes to hypertrophy of the ligamentum flavum.

Characterization of a Novel Calcific Achilles Tendinopathy Model in Mice: Contralateral Tendinopathy Induced by Unilateral Tenotomy.

Achilles tendinopathy is a significant clinical disease characterized by activity-related pain, focal movement limitation, and intratendinous imaging changes. However, treatment of Achilles tendinopathy has been based mainly on theoretical rationale and clinical experience because of its unclear underlying pathogenesis and mechanism. The purpose of the study was to develop a simple but reproducible overuse-induced animal model of Achilles tendinopathy in mice to better understand the underlying mechanism and prevent calcific Achilles tendinopathy. A total of 80 C57/B6 mice (8 or 9 weeks old) were employed and randomly divided into control and experimental groups. Unilateral Achilles tenotomy was performed on the right hind limbs in the experiment group. 12 weeks after unilateral Achilles tenotomy, the onset of Achilles tendinopathy in the contralateral Achilles tendon was determined by radiological assessment, histologic analysis, electron microscopy observation, and biomechanical test. The onset of calcific Achilles tendinopathy in contralateral Achilles tendon was confirmed after 12 weeks of unilateral tenotomy. The contralateral Achilles tendon in the experimental group was characterized as hypercellularity, neovascularization, and fused collagen fiber disarrangement, compared with the control group. Importantly, intra-tendon endochondral ossification and calcaneus deformity were featured in contralateral Achilles tendon. In addition, poor biomechanical properties in the contralateral Achilles tendon revealed the incidence of Achilles tendinopathy. We hereby introduce a novel, simple, but reproducible spontaneous contralateral calcific Achilles tendinopathy model in mice, which represents overuse conditions during tendinopathy development in humans. It should be a useful tool to further study the underlying pathogenesis of calcific Achilles tendinopathy.

Preparation and evaluation of anti-renal fibrosis activity of novel truncated TGF-ß receptor type II.

Production of excessive transforming growth factor-beta 1 (TGF-ß1) with elevated TGF-ß1 activity has been implicated in renal fibrosis via renal epithelial cells activation and collagen deposition. As such, attenuating the binding of TGF-ß1 to its receptor TGF-beta receptor type II (TGF-ßRII) in TGF-ß1-dependent signaling is an attractive target for the control of renal fibrosis. Here, we verified the interaction between novel truncated human TGF-ßRII (thTßRII, Thr23-Gln166) and TGF-ß1, prepared thTßRII in Escherichia coli, and assessed the effects of thTßRII on TGF-ß1-induced human kidney epithelial cells (HK-2) and unilateral ureteral obstruction (UUO) model of renal fibrosis. Our data showed that thTßRII accounted for up to 20% of the total protein and 40% of the inclusion bodies of whole cell lysates under the optimal conditions (0.8 mM IPTG and 25°C for 6 H). Most of the expressed protein in inclusion body was refolded by dialysis refolding procedures and purified by Ni2+ -IDA affinity chromatography. Furthermore, thTßRII decreased type I collagen and α-smooth muscle actin protein expression in TGF-ß1-induced HK-2 cells, and ameliorated kidney morphology and fibrotic responses in fibrosis animal. These findings indicate that thTßRII holds great promise for developing new treatments for renal fibrosis.

mTORC1 Prevents Preosteoblast Differentiation through the Notch Signaling Pathway.

The mechanistic target of rapamycin (mTOR) integrates both intracellular and extracellular signals to regulate cell growth and metabolism. However, the role of mTOR signaling in osteoblast differentiation and bone formation is undefined, and the underlying mechanisms have not been elucidated. Here, we report that activation of mTOR complex 1 (mTORC1) is required for preosteoblast proliferation; however, inactivation of mTORC1 is essential for their differentiation and maturation. Inhibition of mTORC1 prevented preosteoblast proliferation, but enhanced their differentiation in vitro and in mice. Activation of mTORC1 by deletion of tuberous sclerosis 1 (Tsc1) in preosteoblasts produced immature woven bone in mice due to excess proliferation but impaired differentiation and maturation of the cells. The mTORC1-specific inhibitor, rapamycin, restored these in vitro and in vivo phenotypic changes. Mechanistically, mTORC1 prevented osteoblast maturation through activation of the STAT3/p63/Jagged/Notch pathway and downregulation of Runx2. Preosteoblasts with hyperactive mTORC1 reacquired the capacity to fully differentiate and maturate when subjected to inhibition of the Notch pathway. Together, these findings identified the role of mTORC1 in osteoblast formation and established that mTORC1 prevents preosteoblast differentiation and maturation through activation of the Notch pathway.