PDK1 promotes breast cancer progression by enhancing the stability and transcriptional activity of HIF-1α.

Pyruvate dehydrogenase kinase 1 (PDK1) phosphorylates the pyruvate dehydrogenase complex, which inhibits its activity. Inhibiting pyruvate dehydrogenase complex inhibits the tricarboxylic acid cycle and the reprogramming of tumor cell metabolism to glycolysis, which plays an important role in tumor progression. This study aims to elucidate how PDK1 promotes breast cancer progression. We found that PDK1 was highly expressed in breast cancer tissues, and PDK1 knockdown reduced the proliferation, migration, and tumorigenicity of breast cancer cells and inhibited the HIF-1α (hypoxia-inducible factor 1α) pathway. Further investigation showed that PDK1 promoted the protein stability of HIF-1α by reducing the level of ubiquitination of HIF-1α. The HIF-1α protein levels were dependent on PDK1 kinase activity. Furthermore, HIF-1α phosphorylation at serine 451 was detected in wild-type breast cancer cells but not in PDK1 knockout breast cancer cells. The phosphorylation of HIF-1α at Ser 451 stabilized its protein levels by inhibiting the interaction of HIF-1α with von Hippel-Lindau and prolyl hydroxylase domain. We also found that PDK1 enhanced HIF-1α transcriptional activity. In summary, PDK1 enhances HIF-1α protein stability by phosphorylating HIF-1α at Ser451 and promotes HIF-1α transcriptional activity by enhancing the binding of HIF-1α to P300. PDK1 and HIF-1α form a positive feedback loop to promote breast cancer progression.

Progranulin promotes regulatory T cells plasticity by mitochondrial metabolism through AMPK/PGC-1α pathway in ARDS.

As the aging population increases, the focus on elderly patients with acute respiratory distress syndrome (ARDS) is also increasing. In this article, we found progranulin (PGRN) differential expression in ARDS patients and healthy controls, even in young and old ARDS patients. Its expression strongly correlates with several cytokines in both young and elderly ARDS patients. PGRN has comparable therapeutic effects in young and elderly mice with lipopolysaccharide-induced acute lung injury, manifesting as lung injury, apoptosis, inflammation, and regulatory T cells (Tregs) differentiation. Considering that Tregs differentiation relies on metabolic reprogramming, we discovered that Tregs differentiation was mediated by mitochondrial function, especially in the aged population. Furthermore, we demonstrated that PGRN alleviated the mitochondrial damage during Tregs differentiation through the AMPK/PGC-1α pathway in T cells. Collectively, PGRN may regulate mitochondria function to promote Tregs differentiation through the AMPK/PGC-1α pathway to improve ARDS.

Metabolomic analysis of bone-derived exosomes in osteonecrosis of the femoral head based on UPLC-MS/MS.

INTRODUCTION: Osteonecrosis of the femoral head (ONFH) is a disorder that causes a collapse of the femoral head, requiring subsequent total hip replacement. However, the pathogenesis of ONFH remains largely unclear. Herein, exosome metabolomics analyses were conducted to explore the pathophysiology of ONFH. OBJECTIVES: This study aimed to conduct metabolic profiling of bone-derived exosomes of ONFH. METHODS: 30 ONFH patients and 30 femoral neck fracture (FNF) patients were included in this study. Exosomes were harvested from the femoral head by using ultracentrifugation. Ultraperformance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was performed in combination with multivariate statistical analysis to reveal and provided new insight into identify the global metabolic profile of ONFH. RESULTS: The results of transmission electron microscope (TEM), nanoparticle tracking analysis (NTA), and Western blots indicated that the microvesicles isolated from the femoral head were exosomes. Several compounds were identified, including lipids and lipid-like molecules, amino acids, peptides, organooxygen compounds. 44 differential metabolites were screened between ONFH and FNF patients. The up-and down-regulation of Riboflavin metabolism, Pantothenate and CoA biosynthesis, Glycerophospholipid metabolism, and Sphingolipid metabolism were associated with ONFH pathophysiology. CONCLUSION: Our results suggest that metabolomics has huge prospects for elucidating pathophysiology of ONFH.

Vitamin B2 Prevents Glucocorticoid-Caused Damage of Blood Vessels in Osteonecrosis of the Femoral Head.

Osteonecrosis of the femoral head (ONFH) is a disorder that can cause collapse of the femoral head. The damage and dysfunction of femoral head microvascular endothelial cells are related to the pathogenesis of glucocorticoid-induced ONFH. Reports suggest that vitamin B2 can promote osteoblast differentiation and prevent low bone mineral density and prevent reperfusion oxidative injury. To explore the effect and possible molecular mechanism of vitamin B2 on the ONFH and Human Umbilical Vein Endothelial Cells (HUVECs), we performed a rat model of ONFH by dexamethasone. The rats were randomly divided into four groups: control group, vitamin B2 group, dexamethasone group, and dexamethasone combined with vitamin B2 treatment group. HUVECs were used to further prove the role and mechanism of vitamin B2 in vitro. In patients, according to immunohistochemical and qRT-PCR of the femoral head, the angiogenic capacity of the ONFH femoral head is compromised. In vivo, it showed that vitamin B2 could inhibit glucocorticoid-induced ONFH-like changes in rats by suppressing cell apoptosis, promoting the regeneration of blood vessels, and increasing bone mass. According to in vitro results, vitamin B2 could induce the migration of HUVECs, enhance the expression of angiogenesis-related factors, and inhibit glucocorticoid-induced apoptosis. The underlying mechanism may be that vitamin B2 activates the PI3K signaling pathway. Vitamin B2 alleviated dexamethasone-induced ONFH, and vitamin B2 could promote the proliferation and migration of HUVECs and inhibit their apoptosis by activating the PI3K/Akt signaling pathway. Vitamin B2 may be a potentially effective treatment for ONFH.

Dual-aptamer-engineered M1 macrophage with enhanced specific targeting and checkpoint blocking for solid-tumor immunotherapy.

Chimeric antigen receptor T (CAR-T) cell therapy has faced a series of challenges and has shown very little efficacy in solid tumors to date. Although genetically engineered macrophages have achieved definite therapeutic effect in solid tumors, heterogeneous expression of engineered proteins and the potential for toxicity limit further applications. Herein, we propose a nongenetic and simple macrophage cell engineering strategy through glycan metabolic labeling and click reaction for the treatment of solid tumors. The aptamer-engineered M1 macrophage (ApEn-M1) showed enhanced active targeting ability for tumor cells in vitro and in vivo, resulting in significant cytotoxicity effects. Moreover, ApEn-M1 exhibited superior antitumor efficacy in a breast cancer xenograft mouse model and a lung metastasis mouse model of breast cancer. Interestingly, the ApEn-M1 could reprogram the immunity microenvironment by increasing T cell infiltration and enhancing T cell activity in the tumor region. Additionally, the administration of ApEn-M1 showed no obvious systemic side effects. With glycan metabolic labeling, the macrophages could be efficiently labeled with aptamers on the cell surface via click reaction without genetic alteration or cell damage. Hence, this study serves as a proof of concept for cell-surface anchor engineering and expands the range of nongenetic macrophage cell engineering strategies.

[miR-100-5p is involved in non-traumatic osteonecrosis of the femoral head by inhibiting the migration and osteogenic differentiation of BMSCs].

Objective To investigate the role of miR-100-5p in the pathogenesis of non-traumatic osteonecrosis of the femoral head (NONFH). Methods The miRNA expression in patients with NONFH was detected by real-time quantitative PCR, the high expression of miR-100-5p in femoral head tissues of the patients determined. Rat bone marrow mesenchymal stem cells (rBMSCs) were cultured and divided into 5 groups: blank control group, dexamethasone treatment group (treated with dexamethasone for 3 days), miR-NC group (transfected with miR-NC), agomiR-100-5p group (overexpression of miR-100-5p), and antagomiR-100-5p group (transfected with miR-100-5p antagonist). The mRNA expression levels of miR-100-5p, alkaline phosphatase (ALP), Runt-associated transcription factor 2 (RUNX2), and collagen type I (Col1) were detected by real-time quantitative PCR. The protein expressions of ALP, RUNX2, Col1, and bone morphogenetic protein receptor 2 (BMPR2) were detected by Western blotting. The effect of miR-100-5p on the migration ability of rBMSCs was evaluated by scratch healing assay. And the effect of miR-100-5p on osteogenic differentiation ability of rBMSCs was investigated by alizarin red staining. Results miR-100-5p was significantly upregulated in the femoral head bone tissue of NONFH patients compared with normal femoral head bone tissue. Compared with those in the normal rBMSCs, the expression of miR-100-5p in rBMSCs treated with 20 µmol/L of dexamethasone was up-regulated. The upregulation of miR-100-5p in rBMSCs reduced the expressions of ALP, RUNX2, Col1, and BMPR2, and inhibited the osteogenic differentiation and migration abilities of rBMSCs. Conclusion The expression of miR-100-5p is elevated in bone tissues of NONFH patients and in rBMSCs treated with 20 µmol/L of dexamethasone. The up-regulated miR-100-5p may be involved in the pathogenesis of NONFH by inhibiting the migration and osteogenic differentiation of rBMSCs.

Self-assembled tetrahedral framework nucleic acid mediates tumor-associated macrophage reprogramming and restores antitumor immunity.

There is increasing interest in depleting or repolarizing tumor-associated macrophages (TAMs) to generate a proinflammatory effect. However, TAMs usually display an immunosuppressive M2-like phenotype in the tumor microenvironment. Apparently, developing a macrophage-targeting delivery system with immunomodulatory agents is urgent. In this study, an efficient siRNA and CpG ODNs delivery system (CpG-siRNA-tFNA) was prepared with nucleic acid stepwise self-assembled. The tFNA composed of CpG ODNs and siRNA showed a higher stability and an enhanced cellular uptake efficiency. Moreover, the CpG-siRNA-tFNA effectively reprogrammed TAMs toward M1 phenotype polarization with increased proinflammatory cytokine secretion and NF-κB signal pathway activation, which triggers dramatic antitumor immune responses. Additionally, the CpG-siRNA-tFNA exhibited superior antitumor efficacy in a breast cancer xenograft mouse model without obvious systemic side effects. Taken together, CpG-siRNA-tFNA displayed greatly antitumor effect by facilitating TAM polarization toward M1 phenotypes in favor of immunotherapy. Hence, we have developed an efficient therapeutic strategy with immunomodulatory agents for clinical applications.

[Lipid metabolomic analysis in exosomes of osteonecrosis of the femoral head based on ultra performance liquid chromatography-tandem mass spectrometry].

Osteonecrosis of the femoral head (ONFH) can lead to its collapse which requires total hip arthroplasty. Exosomes, which are important for intercellular communication are involved in a series of physiological and pathological processes, and therefore play a unique role in disease diagnosis and treatment. In this study, untargeted metabolomics was used to investigate the metabolic characteristics of lipids in exosomes of femoral head tissue with osteonecrosis and to explain the metabolic changes that occur in the body during this disease. Ultracentrifugation was used to separate and enrich exosomes from femoral head tissue with osteonecrosis. Exosomes were identified using dynamic light scattering (DLS), Western blotting, and transmission electron microscopy (TEM). Gradient elution was performed with ultrapure water and acetonitrile as mobile phases using a Kinetex XB-C18 column (100 mm×2.1 mm, 2.6 µm). The column oven temperature, flow rate of the mobile phase, and duration were 30 ℃, 300 µL/min, and 15 min, respectively. A triple TOF 4600 high resolution mass spectrometry system was used, and the mass scan range of m/z was set at 100 -1000. Other conditions were as follows: sheath gas, 380 kPa; auxiliary gas, 380 kPa; curtain gas, 170 kPa; and atomization temperature, 600 ℃. Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) combined with multivariate statistical analysis was used to identify the lipid metabolic profile of ONFH-derived exosomes. The exosome metabolites were characterized in detail, which enables their identification and provided a reliable method for quality evaluation. After transforming the obtained original data using MarkView software, peak identification, peak alignment, subtraction of solvent peak, impurity peak, noise filtering, and other treatments, a three-dimensional matrix was obtained from the exported data table. Principal component analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA) in the SIMCA-P14.1 software were used for multivariate statistical analysis of differentially expressed exosome lipid metabolites. This strategy was validated using lipid metabolites from patients with ONFH and healthy controls. The correlation distribution was shown according to the point dispersion of the PCA score plot, and lipid metabolites from the same disease showed ideal clustering. This result indicates a small difference between the groups. A good clustering effect is also obtained using OPLS-DA, and the statistical model has high reliability. A total of 18 significantly altered lipid metabolites were detected in the exosomes, including acrylolipids, fatty acid esters, glycerides, and their derivatives. The pathway analysis was conducted with MetaboAnalyst (https://www.metaboanalyst.ca/) via database source including the HMDB (http://www.hmdb.ca/) and MMCD (http://mmcd.nmrfam.wisc.edu/) for confirming the impacted metabolic pathways and visualization. Metabolic pathway analysis showed that glycerophospholipid and sphingolipid metabolism were the most significantly altered in exosomes. An imbalance between sphingolipids and glycerophospholipids leads to lipotoxic damage, which is implicated in the pathophysiology of common metabolic diseases. Furthermore, glycerophospholipids are correlated with cell proliferation, differentiation, and apoptosis, and the change in glycerophospholipid ratio can reflect the disturbance in lipid metabolism. The metabolic changes in exosomes may reflect the metabolic changes in ONFH. In this study, lipid metabolomics analysis based on UPLC-MS/MS was used to determine metabolic differences between exosomes extracted from ONFN and femoral neck fracture (FNF). Metabolomic analysis of necrotic femoral head tissue-derived exosomes can help explore the most relevant pathways for assessing the changes in exosome metabolism that affect exosome metabolism in necrotic bone tissue.

Qualitative Histopathological Classification of Primary Bone Tumors Using Deep Learning: A Pilot Study.

BACKGROUND: Histopathological diagnosis of bone tumors is challenging for pathologists. We aim to classify bone tumors histopathologically in terms of aggressiveness using deep learning (DL) and compare performance with pathologists. METHODS: A total of 427 pathological slides of bone tumors were produced and scanned as whole slide imaging (WSI). Tumor area of WSI was annotated by pathologists and cropped into 716,838 image patches of 256 × 256 pixels for training. After six DL models were trained and validated in patch level, performance was evaluated on testing dataset for binary classification (benign vs. non-benign) and ternary classification (benign vs. intermediate vs. malignant) in patch-level and slide-level prediction. The performance of four pathologists with different experiences was compared to the best-performing models. The gradient-weighted class activation mapping was used to visualize patch's important area. RESULTS: VGG-16 and Inception V3 performed better than other models in patch-level binary and ternary classification. For slide-level prediction, VGG-16 and Inception V3 had area under curve of 0.962 and 0.971 for binary classification and Cohen's kappa score (CKS) of 0.731 and 0.802 for ternary classification. The senior pathologist had CKS of 0.685 comparable to both models (p = 0.688 and p = 0.287) while attending and junior pathologists showed lower CKS than the best model (each p < 0.05). Visualization showed that the DL model depended on pathological features to make predictions. CONCLUSION: DL can effectively classify bone tumors histopathologically in terms of aggressiveness with performance similar to senior pathologists. Our results are promising and would help expedite the future application of DL-assisted histopathological diagnosis for bone tumors.

Exosomal miR-100-5p inhibits osteogenesis of hBMSCs and angiogenesis of HUVECs by suppressing the BMPR2/Smad1/5/9 signalling pathway.

BACKGROUND: Nontraumatic osteonecrosis of the femoral head (NONFH) is a common, progressive, and refractory orthopaedic disease. Decreased osteogenesis and angiogenesis are considered the main factors in the pathogenesis of NONFH. We aimed to figure out whether exosomes and exosomal miRNA from necrotic bone tissues of patients with NONFH are involved in the pathogenesis of NONFH and reveal the underlying mechanisms. METHODS: RT-PCR and western blotting (WB) were used to detect the expression of osteogenic, adipogenic, and angiogenic markers. ALP staining and Alizarin Red S (ARS) staining were used to evaluate osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Oil Red O staining was performed to assess the adipocyte deposition. A tube formation assay was used to study angiogenesis of human umbilical vascular endothelial cells (HUVECs). H&E staining and immunohistochemistry (IHC) staining were used to detect the effect of the NONFH exosomes in vivo. MicroRNA sequencing was conducted to identify potential regulators in the NONFH exosomes. The target relationship between miR-100-5p and BMPR2 was predicted and confirmed by a dual luciferase reporter assay and WB. RESULTS: The NONFH exosomes reduced the osteogenic differentiation of hBMSCs and angiogenesis of HUVECs. In addition, the injection of the NONFH exosomes caused thinning and disruption of bone trabeculae in the femoral heads of rats. MiR-100-5p expression was upregulated in the NONFH exosomes and inhibited the osteogenesis of hBMSCs and angiogenesis of HUVECs by targeting BMPR2 and suppressing the BMPR2/SMAD1/5/9 signalling pathway. Silencing miR-100-5p expression rescued the reduction in osteogenesis and angiogenesis caused by the NONFH exosomes by activating the BMPR2/SMAD1/5/9 signalling pathway. CONCLUSION: The NONFH exosomal miR-100-5p can lead to NONFH-like damage by targeting BMPR2 and suppressing the BMPR2/SMAD1/5/9 signalling pathway, which may be involved in the pathophysiological mechanisms of nontraumatic osteonecrosis of the femoral head (NONFH).

Malalignment sign on knee magnetic resonance imaging: a new predictor for excessive femoral anteversion in patients with patellar dislocation.

PURPOSE: To evaluate the clinical relevance of the newly identified malalignment sign in predicting excessive femoral anteversion in patients with patellar dislocations. METHODS: A total of 55 patients with patellar dislocation who underwent surgical treatment between 2016 and 2019 were included in this study. Femoral anteversion, tibial torsion, and the femorotibial index were measured via a CT scan. The malalignment sign on the knee MRI was defined as a malalignment between the lateral side of the intercondylar fossa of the femur and the lateral intercondylar eminence of the tibial plateau. RESULTS: A positive malalignment sign was observed in 36 of the 55 patients. Increased femoral anteversion was significantly correlated with the number of frames with a positive malalignment sign (r = 0.511, P < 0.001). The value of femoral anteversion was significantly greater in the group with a positive malalignment sign (P = 0.02). For a femoral anteversion value of 32°, the sensitivity and specificity of the malalignment sign reached the maximal level of 89.5% and 47.2%, respectively. CONCLUSION: Increased femoral anteversion correlated significantly with a positive malalignment sign on knee MRI. However, tibial torsion did not affect the malalignment sign. A positive malalignment sign is evidence for femoral derotation osteotomy. LEVEL OF EVIDENCE: Level IV.

CD41-deficient exosomes from non-traumatic femoral head necrosis tissues impair osteogenic differentiation and migration of mesenchymal stem cells.

Non-traumatic osteonecrosis of the femoral head (ONFH) is clinically a devastating and progressive disease without an effective treatment. Mesenchymal stem cells (MSCs) transplantation has been used to treat ONFH in early stage, but the failure rate of this therapy is high due to the reduced osteogenic differentiation and migration of the transplanted MSCs related with pathological bone tissues. However, the mechanism responsible for this decrease is still unclear. Therefore, we assume that the implanted MSCs might be influenced by signals delivered from pathological bone tissue, where the exosomes might play a critical role in this delivery. This study showed that exosomes from ONFH bone tissues (ONFH-exos) were able to induce GC-induced ONFH-like damage, in vivo and impair osteogenic differentiation and migration of MSCs, in vitro. Then, we analyzed the differentially expressed proteins (DEPs) in ONFH-exos using proteomic technology and identified 842 differentially expressed proteins (DEPs). On the basis of gene ontology (GO) enrichment analysis of DEPs, fold-changes and previous report, cell adhesion-related CD41 (integrin α2b) was selected for further investigation. Our study showed that the CD41 (integrin α2b) was distinctly decreased in ONFH-exos, compared to NOR-exos, and downregulation of CD41 could impair osteogenic differentiation and migration of the MSCs, where CD41-integrin ß3-FAK-Akt-Runx2 pathway was involved. Finally, our study further suggested that CD41-affluent NOR-exos could restore the glucocorticoid-induced decline of osteogenic differentiation and migration in MSCs, and prevent GC-induced ONFH-like damage in rat models. Taken together, our study results revealed that in the progress of ONFH, exosomes from the pathological bone brought about the failure of MSCs repairing the necrotic bone for lack of some critical proteins, like integrin CD41, and prompted the progression of experimentally induced ONFH-like status in the rat. CD41 could be considered as the target of early diagnosis and therapy in ONFH.

[Progranulin aggravates postmenopausal osteoporosis in ovariectomized mice].

Objective To investigate the effect of progranulin (PGRN) on osteoporosis in ovariectomized mice. Methods PGRN-knockout (PGRN-/-) and wild-type mice were ovariectomized to induce postmenopausal osteoporosis models. Next, the bone tissues in all mice were scanned by Micro-CT and three-dimensional reconstruction was performed to detect the micro-structure, followed by trabecula data analysis. The morphology and osteoclasts in the bone tissues of PGRN-/- and wild-type mice were observed by HE staining and TRAP staining, respectively. The expression of receptor activator for nuclear factor-κB ligand (RANKL), tumor necrosis factor α (TNF-α) and P65 were detected by immunohistochemistry. The expression of TRAP mRNA in the mice was measured using fluorescence quantitative PCR and the protein expression of MMP9, MMP14, P65 was detected by Western blot analysis. Results Bone mineral density (BMD), bone volume fraction (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) in the PGRN-/- group were significantly higher than those in the wild-type group, while the trabecular separation (Tb.S) in the PGRN-/- group was in the contrary. The degree of osteoporosis was less severe and number of osteoclasts in the PGRN-/- mice were reduced, likewise, RANKL, TNF-α, MMP9, MMP14 and P65 as well as TRAP mRNA were down-regulated in the PGRN-/- group compared with the wild-type group. Conclusion PGRN aggravates the postmenopausal osteoporosis in ovariectomized mice.