G protein subunit alpha i2's pivotal role in angiogenesis.

Here we explored the potential role of Gαi2 (G protein subunit alpha i2) in endothelial cell function and angiogenesis. Methods: Genetic methodologies such as shRNA, CRISPR/Cas9, dominant negative mutation, and overexpression were utilized to modify Gαi2 expression or regulate its function. Their effects on endothelial cell functions were assessed in vitro. In vivo, the endothelial-specific Gαi2 shRNA adeno-associated virus (AAV) was utilized to silence Gαi2 expression. The impact of this suppression on retinal angiogenesis in control mice and streptozotocin (STZ)-induced diabetic retinopathy (DR) mice was analyzed. Results: Analysis of single-cell RNA sequencing data revealed Gαi2 (GNAI2) was predominantly expressed in retinal endothelial cells and expression was increased in retinal endothelial cells following oxygen-induced retinopathy (OIR) in mice. Moreover, transcriptome analysis linking Gαi2 to angiogenesis-related processes/pathways, supported by increased Gαi2 expression in experimental OIR mouse retinas, highlighted its possible role in angiogenesis. In various endothelial cell types, shRNA-induced silencing and CRISPR/Cas9-mediated knockout (KO) of Gαi2 resulted in substantial reductions in cell proliferation, migration, invasion, and capillary tube formation. Conversely, Gαi2 over-expression in endothelial cells induced pro-angiogenic activities, enhancing cell proliferation, migration, invasion, and capillary tube formation. Furthermore, our investigation revealed a crucial role of Gαi2 in NFAT (nuclear factor of activated T cells) activation, as evidenced by the down-regulation of NFAT-luciferase reporter activity and pro-angiogenesis NFAT-targeted genes (Egr3, CXCR7, and RND1) in Gαi2-silenced or -KO HUVECs, which were up-regulated in Gαi2-overexpressing endothelial cells. Expression of a dominant negative Gαi2 mutation (S48C) also down-regulated NFAT-targeted genes, slowing proliferation, migration, invasion, and capillary tube formation in HUVECs. Importantly, in vivo experiments revealed that endothelial Gαi2 knockdown inhibited retinal angiogenesis in mice, with a concomitant down-regulation of NFAT-targeted genes in mouse retinal tissue. In contrast, Gαi2 over-expression in endothelial cells enhanced retinal angiogenesis in mice. Single-cell RNA sequencing data confirmed increased levels of Gαi2 specifically in retinal endothelial cells of mice with streptozotocin (STZ)-induced diabetic retinopathy (DR). Importantly, endothelial Gαi2 silencing ameliorated retinal pathological angiogenesis in DR mice. Conclusion: Our study highlights a critical role for Gαi2 in NFAT activation, endothelial cell activation and angiogenesis, offering valuable insights into potential therapeutic strategies for modulating these processes.

Bioinformatics analysis of the key genes in osteosarcoma metastasis and immune invasion.

Background: This study sought to identify potential key genes for osteosarcoma metastasis and analyze their immune infiltration patterns using bioinformatic methods. Methods: We obtained transcriptomic data related to osteosarcoma and osteosarcoma with metastasis from the Therapeutically Applicable Research to Generate Effective Treatment (TARGET) and The Gene Expression Omnibus (GEO) databases and identified the differentially expressed genes (DEGs). We also identified potential key genes for osteosarcoma metastasis by a protein-protein interaction network analysis, and we conducted a Gene Ontology (GO) functional annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify the core genes for prognosis, immune cell infiltration, and drug sensitivity, and the risk prediction and prognosis models of metastasis were constructed. Results: By comparing the transcriptome data of osteosarcomas without metastasis and those with metastasis, a total of 19 core DEGs were identified, and the GO and KEGG analyses revealed an association between these DEGs and the regulation of cell division, secretory granule lumen, the Ras-associated protein 1 (Rap1) signaling pathway, and the mitogen-activated protein kinase (MAPK) signaling pathway. Compared with other immune cells, macrophage infiltration was predominant in osteosarcoma samples with metastatic osteosarcoma, and insulin-like growth factors-1 (IGF1) and myelocytomatosis protein 2 (MYC2) genes were predicted to more than 50 targeted therapeutic agents. A metastasis prediction model with 5 genes [i.e., ecotropic viral integration site 2B (EVI2B), CCAAT/enhancer binding protein (CEBPA), lymphocyte cytosolic protein 2 (LCP2), selectin L (SELL), and Niemann-Pick disease, type C2A (NPC2A)], and a prognostic model with 4 genes [i.e., insulin-like growth factors-2 (IGF2), cathepsin O (CTSO), Niemann-Pick disease, type C2 (NPC2), and amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein (APBB1IP)] were developed. Conclusions: We constructed a metastasis prediction model with 5 genes (i.e., EVI2B, CEBPA, LCP2, SELL, and NPC2A), and a prognostic model with 4 genes (i.e., IGF2, CTSO, NPC2, and APBB1IP) that may be potential biomarkers for osteosarcoma metastasis. Macrophages are the predominant immune infiltrating cells in osteosarcoma metastasis and may provide a new direction for the treatment of osteosarcoma.

miRNA-429 suppresses osteogenic differentiation of human adipose-derived mesenchymal stem cells under oxidative stress via targeting SCD-1.

[This retracts the article DOI: 10.3892/etm.2019.8246.].

Carfilzomib alleviated osteoporosis by targeting PSME1/2 to activate Wnt/ß-catenin signaling.

Osteoporosis (OP) is characterized by decreased bone mineral density and impaired bone strength. Carfilzomib (CFZ) is a new-generation proteasome inhibitor and has been found to affect bone metabolism. However, the effect and mechanism of CFZ on OP has not been investigated systematically. In this study, we found that protein levels of proteasome activator subunit 1/2 (PSME1/2) increased in OP, and accumulated mostly in osteoblasts and osteoclasts. Treatment with PSME1/2 recombinant protein inhibited osteogenesis and promoted osteoclast formation in vitro. Also, PSME1/2 inhibited the expression of ß-catenin protein, resulting in limitation of Wnt/ß-catenin signaling. CFZ inhibited PSME1 and PSME2 proteasome activities and increased ß-catenin protein level, resulting in the translocation of ß-catenin to the nucleus and activation of canonical Wnt/ß-catenin signaling, further promoting osteogenesis and inhibiting osteoclastic differentiation. In vivo, we conducted ovariectomy (OVX) to create a model of OVX-induced postmenopausal OP in mice. When analyzed by micro-CT scanning, enhancement of bone mineral density, bone volume, trabecular number, and thickness was seen in the CFZ-treated mice. Also, we noticed increased osteogenesis and decreased osteoclastogenesis, diminished expression of PSME1 and PSME2 and activated Wnt/ß-catenin signaling in bone sections from OP mice treated with CFZ. Overall, our data indicated that PSME1/2 may serve as new targets for the treatment of OP, and targeting PSME1/2 with CFZ provides a candidate therapeutic molecule for postmenopausal OP.

HA15 alleviates bone loss in ovariectomy-induced osteoporosis by targeting HSPA5.

The imbalance between osteogenesis and adipogenesis in the bone marrow is the main characteristic of osteoporosis (OP). Thus, exploring regulation of the differentiation of bone marrow stromal cells (BMSCs) into osteoblasts and adipocytes is important to identify novel targets for the treatment of OP. In the present study, the master regulator of endoplasmic reticulum (ER) stress, heat shock protein family A (Hsp70) member 5 (HSPA5) was shown to significantly accumulate in osteoblasts and adipocytes, but not in osteoclasts in bone sections from aged and postmenopausal OP mice. In vitro study revealed that HSPA5 negatively modulated osteogenic differentiation and positively promoted adipogenic differentiation, and that targeting HSPA5 with its inhibitor HA15 enhanced osteogenic differentiation and inhibited adipogenic differentiation. Also, HA15 treatment induces ER stress and autophagy, and decreases apoptosis in cells. We constructed a postmenopausal OP model in mice with ovariectomy surgery, and treated the mice with HA15. The results showed that HA15 treatment induced appropriate ER stress, activated autophagy and decreased apoptosis in osteoblasts, thereby alleviating bone loss in vivo. Our results indicated that HSPA5 participated in OP pathogenesis by regulating the differentiation of BMSCs. HSPA5 may serve as a new target for the treatment of OP, and targeting HSPA5 with HA15 prevents the progression of OP and provides a candidate therapeutic molecule for postmenopausal OP.

Metal Material, Properties and Design Methods of Porous Biomedical Scaffolds for Additive Manufacturing: A Review.

Design an implant similar to the human bone is one of the critical problems in bone tissue engineering. Metal porous scaffolds have good prospects in bone tissue replacement due to their matching elastic modulus, better strength, and biocompatibility. However, traditional processing methods are challenging to fabricate scaffolds with a porous structure, limiting the development of porous scaffolds. With the advancement of additive manufacturing (AM) and computer-aided technologies, the development of porous metal scaffolds also ushers in unprecedented opportunities. In recent years, many new metal materials and innovative design methods are used to fabricate porous scaffolds with excellent mechanical properties and biocompatibility. This article reviews the research progress of porous metal scaffolds, and introduces the AM technologies used in porous metal scaffolds. Then the applications of different metal materials in bone scaffolds are summarized, and the advantages and limitations of various scaffold design methods are discussed. Finally, we look forward to the development prospects of AM in porous metal scaffolds.

miRNA-429 suppresses osteogenic differentiation of human adipose-derived mesenchymal stem cells under oxidative stress via targeting SCD-1.

Role of microRNA-429 (miRNA-429) in osteogenic differentiation of hADMSCs was elucidated to explore the potential mechanism. Serum level of miRNA-429 in osteoporosis patients and controls was determined by quantitative real-time polymerase chain reaction (qRT-PCR). After H2O2 induction in hADMSCs, cell viability and reactive oxygen species (ROS) level were determined by cell-counting kit (CCK-8) assay and flow cytometry, respectively. Alkaline phosphatase (ALP) activity in H2O2-induced hADMSCs was also detected. The binding condition between miRNA-429 and SCD-1 was verified by dual-luciferase reporter gene assay. Relative levels of osteogenesis-related genes influenced by SCD-1 and miRNA-429 were detected by qRT-PCR. Furthermore, regulatory effects of SCD-1 and miRNA-429 on ALP activity and calcification ability of hADMSCs were evaluated. miRNA-429 was significantly upregulated in serum of osteoporosis patients. During the process of osteogenesis differentiation, H2O2 induction gradually upregulated miRNA-429 in hADMSCs. Overexpression of miRNA-429 markedly reduced ALP activity. Subsequent dual-luciferase reporter gene assay verified that miRNA-429 could bind to SCD-1 and negatively regulated its protein level in hADMSCs. SCD-1 was obviously downregulated in the osteogenesis differentiation of hADMSCs under oxidative stress. Moreover, silencing of SCD-1 suppressed expression of osteogenesis-related gene, ALP activity and calcification ability. Notably, SCD-1 knockdown partially reversed the regulatory effect of miRNA-429 on the osteogenic differentiation of hADMSCs. miRNA-429 suppresses the osteogenic differentiation of hADMSCs under oxidative stress via downregulating SCD-1.

Association between functional polymorphisms in IL-33/ST2 pathway and risk of osteosarcoma.

Interleukin (IL)-33/ST2 pathway plays crucial roles in tumour growth and metastasis. The aim of this study was to investigate the association of two functional polymorphisms (IL-33 rs7025417 and ST2 rs3821204) with osteosarcoma (OS) risk. The rs7025417 and rs3821204 were genotyped by Taqman assay. IL-33mRNA and protein levels were measured by real-time PCR or enzyme-linked immunosorbent assay. The luciferase activity was measured by a dual luciferase reporter gene assay. The allele-specific transcription factor binding for rs7025417 was examined by ChIP-seq. The IL-33 rs7025417 CC genotype was significantly associated with a decreased risk of OS (CC vs TT: OR = 0.59, 95% CI, 0.41-0.85; recessive model: OR = 0.68, 95% CI, 0.49-0.94; C vs T: OR = 0.76, 95% CI, 0.63-0.91). Combined analysis showed that the IL-33 rs7025417CT/CC-ST2 rs3821204CG/CC and the IL-33 rs7025417CT/CC-ST2 rs3821204GG genotypes also had a decreased risk of OS. IL-33mRNA and protein levels in OS patients were significantly higher than controls. Patients with the rs7025417 CC genotype exhibited lower levels of IL-33 (P = .03). The rs7025417 C allele presented a lower transcriptional activity by disrupting the binding site to c-Myb (P < .01). Moreover, the rs3821204 G/C influences the transcriptional activity and ST2mRNA expression by altering the binding site of miR-202-3p. These findings suggest that the rs7025417 and rs3821204 may have a combined effect to protect against the development of OS by decreasing the expression levels of IL-33 or ST2.

Association of interleukin 16 gene polymorphisms and plasma IL16 level with osteosarcoma risk.

Interleukin (IL) 16 plays a key role in inflammatory diseases as well as in tumorigenesis of osteosarcoma (OS). The aim of this study was to investigate the association of IL16 polymorphisms and plasma IL16 level with OS risk in a Chinese population. We genotyped IL16 rs4778889, rs11556218, and rs4072111 in 358 patients with OS and 402 controls using a polymerase chain reaction-restriction fragment length polymorphism assay. Plasma IL16 level was measured by enzyme-linked immunosorbent assay. Rs11556218 was associated with an increased risk of OS in heterozygote comparison (adjusted OR = 1.65, 95% CI, 1.23-2.21, P < 0.001), dominant model (adjusted OR = 1.66, 95% CI, 1.24-2.21, P < 0.001), and allele comparison (adjusted OR = 1.44, 95% CI, 1.14-1.81, P = 0.002). Moreover, rs11556218 TG/GG genotypes were associated with higher levels of IL16 as compared to TT genotype (P = 0.03). However, no significant association of rs4778889 and rs4072111 and OS was found. These findings suggest that rs11556218 TG/GG genotypes may be associated with increased susceptibility to OS, probably by increasing the production of IL16 level.

Associations of IL-27 polymorphisms and serum IL-27p28 levels with osteosarcoma risk.

Interleukin (IL)-27 is a novel cytokine secreted by stimulation of antigen-presenting cells. No previous studies currently reported the role of IL-27 in the carcinogenesis of osteosarcoma. We aimed to investigate the association of IL-27 polymorphisms and serum IL-27p28 with osteosarcoma risk in a Chinese population.One hundred and sixty osteosarcoma patients and 250 health controls were selected. IL-27 gene -964 A/G, 2905 T/G, and 4730 T/C polymorphisms were determined by using polymerase chain reaction-restriction fragment length polymorphism. Enzyme-linked immunosorbent assay were used to detect serum IL-27p28 levels.The serum IL-27p28 levels were significantly lower in osteosarcoma patients compared with those in controls (P < 0.01). Serum IL-27p28 levels in stages III-IV were lower than those in stages I-II of osteosarcoma (P < 0.05); similar results were also found in patients with metastasis, that is, patients with metastasis have higher IL-27p28 levels than those without metastasis (P < 0.05). There were no associations between genotype and allele frequencies of IL-27 -964 A/G, 2905 T/G, 4730 T/C, and the risk of osteosarcoma (P > 0.05). Stratification analysis also failed to show the associations between -964 A/G, 2905 T/G, and 4730 T/C polymorphisms and the clinical stage and metastasis of osteosarcoma (P > 0.05). Three possible haplotypes (ATT, GTT, and GGC) were identified, but no associations were found between them and the osteosarcoma risk (P > 0.05).This study indicates that the lower serum IL-27p28 levels may be associated with development and progression of osteosarcoma, but IL-27 gene -964 A/G, 2905 T/G, and 4730 T/C polymorphisms and their haplotypes are not associated with osteosarcoma risk.