MicroRNA-432-5p regulates sprouting and intussusceptive angiogenesis in osteosarcoma microenvironment by targeting PDGFB.

Osteosarcoma (OS) is a type of bone tumor conferred with high metastatic potential. Attainable growth of tumors necessitates functional vasculature mediated by sprouting angiogenesis (SA) and intussusceptive angiogenesis (IA). However, the regulation of IA and SA is still unclear in OS. To understand the mechanisms adopted by OS to induce angiogenesis, initially, we assessed the expression profile of a set of miRNAs' in both OS cells (SaOS2 and MG63) and normal bone cells. Amongst them, miR-432-5p was found to be highly downregulated in OS. The functional role of miR-432-5p in OS was further analyzed using miR-432-5p mimic/inhibitor. Platelet-derived growth factor-B (PDGFB) was found to be a putative target of miR-432-5p and it was further confirmed that the PDGFB 3'UTR is directly targeted by miR-432-5p using the luciferase reporter gene system. PDGFB was found to be secreted by OS to regulate angiogenesis by targeting the cells in its microenvironment. The conditioned medium obtained from miR-432-5p mimic transfected MG63 and SaOS2 cells decreased cell viability, proliferation, migration, and aorta ring formation in endothelial cells. The miRNA mimic/inhibitor transfected MG63 and SaOS2 cells were placed on SA (day 6) and IA (day 9) phase of CAM development to analyze SA and IA mechanisms. It was found that miR-432-5p mimic transfection in OS promotes the transition of SA to IA which was documented by the angiogenic parameters and SA and IA-associated gene expression. Interestingly, this outcome was also supported by the zebrafish tumor xenograft model. Corroborating these results, it is clear that miR-432-5p expression in OS cells regulates SA and IA by targeting PDGFB genes. We conclude that targeting miR-432-5p/PDGFB signaling can be a potential therapeutic strategy to treat OS along with other existing strategies.

Melatonin regulates tumor angiogenesis via miR-424-5p/VEGFA signaling pathway in osteosarcoma.

Melatonin is recognized as an anti-angiogenic agent, but its function in the tumor microenvironment especially in osteosarcoma remains uncertain. Among the selected miRNAs, miR-205, miR-424, miR-140, miR-106, and miR-519 were upregulated by melatonin in osteosarcoma cells. The functional role of miR-424-5p in osteosarcoma was further analyzed using miR-424-5p mimic/inhibitor. VEGFA mRNA and protein expression were altered by miR-424-5p mimic/inhibitor transfection with and without melatonin treatment and it was further identified that the VEGFA 3'UTR is directly targeted by miR-424-5p using the luciferase reporter gene system. The conditioned medium from SaOS2 and MG63 cells treated with melatonin and/or transfected with miR-424-5p mimic/inhibitor was exposed to endothelial cells, and cell proliferation and migration was analyzed. MG-63 and SaOS2 cells are also transfected with miR-424-5p inhibitors and positioned on CAM vascular bed to study the angiogenic activity at both morphological and molecular level under melatonin treatment. Our observations demonstrate for the first time that, melatonin upregulated the expression of miR-424-5p in osteosarcoma inhibiting VEGFA. Furthermore, it suppresses tumor angiogenesis, modulating surrounding endothelial cell proliferation and migration as well as the morphology of blood vessels, and angiogenic growth factors. These findings suggest that melatonin could play a pivotal role in tumor suppression via miR-424-5p/VEGFA axis.

Models to investigate intussusceptive angiogenesis: A special note on CRISPR/Cas9 based system in zebrafish.

Angiogenesis is a distinct process which follows sprouting angiogenesis (SA) and intussusceptive angiogenesis (IA) forming the basis for various physiological and pathological scenarios. Angiogenesis is a double edged sword exerting both desirable and discernible effects owing to the referred microenvironment. Therapeutic interventions to promote angiogenesis in regenerative medicine is essential to achieve functional syncytium of tissue constructs while, angiogenic inhibition is a key therapeutic target to suppress tumor growth. In the recent years, clustered regularly interspaced short palindromic repeats associated 9 (CRISPR-Cas9) based gene editing approaches have been gaining considerable attention in the field of biomedical research owing to its ease in tailoring targeted genome in living organisms. The Zebrafish model, with adequately high-throughput fitness, is a likely option for genome editing and angiogenesis research. In this review, we focus on the implication of Zebrafish as a model to study IA and furthermore enumerate CRISPR/Cas9 based genome editing in Zebrafish as a candidate for modeling different types of angiogenesis and support its candidature as a model organism.

Chitosan based thermoresponsive hydrogel containing graphene oxide for bone tissue repair.

Chitosan (CS), glycerophosphate (GP) based injectable hydrogels are explored for its implications in bone defect healing and regeneration. Both acellular and cell laden CS based hydrogels are widely investigated and improved through the inclusion of various nanoparticles, polymers and bioactive molecules. In order to improve its applicability for bone tissue repair, we developed an injectable, thermosensitive CS hydrogel containing graphene oxide (GO) and investigated its properties. The hydrogels were investigated for its porous architecture using scanning electron microscopy (SEM), swelling property, protein adsorption ability, degradation rate and exogenous biomineralization. GO addition improved the physico-chemical properties with notable betterment. The CS/GP/GO hydrogel was biocompatible to mesenchymal stem cells and they were metabolically active upon encapsulation. The hydrogel promoted osteogenic differentiation of mouse mesenchymal stem cells by upregualtion of Runt-related transcription factor 2 (Runx2), Alkaline phosphatase (ALP), Type -1 collagen (COL-1), and osteocalcin (OC) under osteogenic conditions. The hydrogel proves to be an amenable platform for carrying cells and exhibited suitable properties to be a potential candidate for bone tissue regeneration.

Synthesis and characterization of silibinin/phenanthroline/neocuproine copper(II) complexes for augmenting bone tissue regeneration: an in vitro analysis.

In the recent decades, flavonoid metal complexes have been widely investigated for their multifaceted role in enabling osteoblast differentiation and bone formation. Silibinin complexed with copper(II) ion has been synthesized along with two mixed ligand complexes, namely copper(II) silibinin-phenanthroline and neocuproine as co-ligands, and their positive role in promoting neovacularization and osteoblast differentiation was investigated. Silibinin mono complex [Cu(sil)(H2O)2] and [Cu(sil)(phen)] showed similar UV-visible absorption in the region of 315 and 222 nm, whereas Cu(silibinin)(neocuproine) [Cu(sil)(neo)] showed a blueshift in the 320 nm transition. The involvement of carbonyl group present in the C-ring in metal ion chelation was identified by FT-IR analysis. Thermal gravimetric analysis (TGA) depicted that [Cu(sil)(neo)] has higher thermal stability when compared with the control silibinin and Cu-silibinin mono, and phen complexes. Cu-silibinin complexes were found to be non-toxic to human MG-63 cells and mouse mesenchymal stem cells (MSCs). Our investigations identified the positive role of these complexes in promoting osteoblast differentiation by enhancing calcium deposition and alkaline phosphatase (ALP) activity at the cellular level and stimulation of osteoblastic marker genes such as Runx2, ALP, type 1 collagen, and OCN mRNAs expression at the molecular level. These complexes also supported angiogenesis by upregulation of VEGF and Ang 1 expression in mouse MSCs. Hence, our results suggest that the potential of these metal complexes along with mixed ligand complexes promoted osteoblast differentiation, thus warranting its candidature for bone tissue regeneration application.