Proangiogenic Hypoxia-Mimicking Agents Attenuate Osteogenic Potential of Adipose Stem/Stromal Cells

Abstract

BACKGROUND: Insufficient vascularization hampers bone tissue engineering strategies for reconstructing large bone defects. Delivery of prolyl-hydroxylase inhibitors (PHIs) is an interesting approach to upregulate vascular endothelial growth factor (VEGF) by mimicking hypoxic stabilization of hypoxia-inducible factor-1alpha (HIF-1a). This study assessed two PHIs: dimethyloxalylglycine (DMOG) and baicalein for their effects on human adipose tissue-derived mesenchymal stem/stromal cells (AT-MSCs). METHODS: Isolated AT-MSCs were characterized and treated with PHIs to assess the cellular proliferation response. Immunostaining and western-blots served to verify the HIF-1a stabilization response. The optimized concentrations for long-term treatment were tested for their effects on the cell cycle, apoptosis, cytokine secretion, and osteogenic differentiation of AT-MSCs. Gene expression levels were evaluated for alkaline phosphatase (ALPL), bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor A (VEGFA), secreted phosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). In addition, stemness-related genes Kruppel-like factor 4 (KLF4), Nanog homeobox (NANOG), and octamer-binding transcription factor 4 (OCT4) were assessed. RESULTS: PHIs stabilized HIF-1a in a dose-dependent manner and showed evident dose-and time dependent antiproliferative effects. With doses maintaining proliferation, DMOG and baicalein diminished the effect of osteogenic induction on the expression of RUNX2, ALPL, and COL1A1, and suppressed the formation of mineralized matrix. Suppressed osteogenic response of AT-MSCs was accompanied by an upregulation of stemness-related genes. CONCLUSION: PHIs significantly reduced the osteogenic differentiation of AT-MSCs and rather upregulated stemnessrelated genes. PHIs proangiogenic potential should be weighed against their longterm direct inhibitory effects on the osteogenic differentiation of AT-MSCs. Keywords Dimethyloxalylglycine Á Baicalein Á Hypoxia-inducible factor-1alpha Á Adipose tissue-derived mesenchymal stem/stromal cells Á Osteogenesis Riitta Seppänen-Kaijansinkko and Bettina Mannerström contributed equally.

Investigation of conditioned medium properties obtained from human umbilical cord mesenchymal stem/stromal cells preconditioned with dimethyloxalylglycine in a correlation with ultrastructural changes

“…Two studies that analyzed HIF‐1α expressions due to preconditioning bone marrow MSCs with DMOG stated that the optimal DMOG dose was 750 and 1000 μM, respectively (Esmaeilzade et al, 2019; Liang et al, 2019). However, another study showed that 500 μM DMOG could attenuate cell viability of adipose tissue‐derived MSCs following 24 h of treatment, even reducing cell viability significantly following 3 days long treatment (Abu‐Shahba et al, 2020). At this point, MSCs were preconditioned with different doses of DMOG to determine at which dose DMOG would be most effective.…”

Advances in Anti-Cancer Activities of Flavonoids in Scutellariae radix: Perspectives on Mechanism

“…They indicated that Wnt/β-catenin was a key pathway involved, and the feature protein β-catenin, glycogen synthase kinase 3β (GSK3β), and BAX were identified as the potential targets [ 39 ]. Baicalein also exhibited a potential inhibitory effect on the growth and development of cervical carcinoma via negatively regulating Wnt/β-catenin signaling, suppressing the transcriptional activity of β-catenin on pro-tumor genes like high-mobility-group box-2 (Sox-2), matrix metalloproteinases (MMP-2), and Nanog homeobox [ 40 ]. Moreover, investigations on the relationship between decidual protein induced by progesterone (DEPP) and tumor cell death have gradually become a hot spot [ 41 ].…”

HIF-1α Regulates Bone Homeostasis and Angiogenesis, Participating in the Occurrence of Bone Metabolic Diseases

“…IOX2 and dimethyloxalylglycine (DMOG), as inhibitors of PHD, reduce the degradation of HIF-1α, regulate the expression of downstream target genes of HIF-1α, prompting angiogenesis and the proliferation, migration, and osteogenic differentiation of BMSCs, as well as inhibiting osteoblast apoptosis [ 17 , 113 , 114 , 115 , 116 , 117 , 118 ]. However, the long-term duration activation of HIF-1α by DMOG and baicalein inhibits the osteogenic differentiation and enhances angiogenesis of adipose-derived stem cells (ASCs), while the stem cell characteristics are preserved, which still shows superiority in repairing rat skull defects [ 119 , 120 ] ( Figure 3 ).…”