Promotion of osteogenesis in BMSC under hypoxia by ATF4 via the PERK-eIF2α signaling pathway.

Mandibular distraction osteogenesis (MDO) is an endogenous tissue engineering technology in which bone marrow mesenchymal stem cells (BMSC) play a key role in MDO-related osteogenesis. Activating transcription factor 4 (ATF4) is involved in osteogenesis through activation of PERK (Protein kinase R-like endoplasmic reticulum kinase) in endoplasmic reticulum stress (ERS) condition under hypoxia. However, the specific role of ATF4 in MDO with BMSC remains unknown. The aim of this study was to explore the effects of ATF4 in MDO with BMSC under hypoxia. Briefly, canine BMSCs were cultured in a hypoxic chamber, and effects of hypoxia were evaluated using cell migration assay and Alizarin Red S staining. Expression levels of protein kinase R-like endoplasmic reticulum kinase, eukaryotic translation initiation factor 2α, ATF4, osteocalcin, and bone sialoprotein were evaluated using quantitative polymerase chain reaction and western blotting. BMSCs were transduced with the ATF4-small interfering RNA lentivirus. The effects were evaluated using all the aforementioned experiments. The results showed that hypoxia promoted migration, osteoblast differentiation, and ATF4 expression in BMSC. ATF4 knockdown in BMSC significantly inhibited migration and osteoblast differentiation abilities, while hypoxia reversed these effects to some extent. In addition, the molecular mechanism partly depended on the ERS signaling pathway, with ATF4 as the key factor. In summary, we presented a novel mechanism of ATF4-mediated regulation of BMSC under hypoxia.

Hsp20 Promotes Endothelial Progenitor Cell Angiogenesis via Activation of PI3K/Akt Signaling Pathway under Hypoxia.

BACKGROUND: Mandibular distraction osteogenesis (MDO) is a kind of endogenous tissue engineering technology that lengthens the jaw and opens airway so that a patient can breathe safely and comfortably on his or her own. Endothelial progenitor cells (EPCs) are crucial for MDO-related angiogenesis. Moreover, emerging evidence suggests that heat shock protein 20 (Hsp20) modulates angiogenesis under hypoxic conditions. However, the specific role of Hsp20 in EPCs, in the context of MDO, is not yet known. The aim of this study was to explore the expression of Hsp20 during MDO and the effects of Hsp20 on EPCs under hypoxia. METHODS: Mandibular distraction osteogenesis and mandibular bone defect (MBD) canine model were established. The expression of CD34, CD133, HIF-1α, and Hsp20 in callus was detected by immunofluorescence on day 14 after surgery. Canine bone marrow EPCs were cultured, with or without optimal cobalt chloride (CoCl2) concentration. Hypoxic effects, caused by CoCl2, were evaluated by means of the cell cycle, cell apoptosis, transwell cell migration, and tube formation assays. The Hsp20/KDR/PI3K/Akt expression levels were evaluated via immunofluorescence, RT-qPCR, and western blot. Next, EPCs were incorporated with either Hsp20-overexpression or Hsp20-siRNA lentivirus. The resulting effects were evaluated as described above. RESULTS: CD34, CD133, HIF-1α, and Hsp20 were displayed more positive in the callus of MDO compared with MBD. In addition, hypoxic conditions, generated by 0.1 mM CoCl2, in canine EPCs, accelerated cell proliferation, migration, tube formation, and Hsp20 expression. Hsp20 overexpression in EPCs significantly stimulated cell proliferation, migration, and tube formation, whereas Hsp20 inhibition produced the opposite effect. Additionally, the molecular mechanism was partly dependent on the KDR/PI3K/Akt pathway. CONCLUSION: In summary, herein, we present a novel mechanism of Hsp20-mediated regulation of canine EPCs via Akt activation in a hypoxic microenvironment.

Hypoxia improved vasculogenesis in distraction osteogenesis through Mesenchymal-Epithelial transition (MET), Wnt/ß-catenin signaling pathway, and autophagy.

INTRODUCTION: The osteogenesis rate of distraction osteogenesis is 4-6 times faster than that of infants, far beyond fracture healing. However, the osteogenesis mechanism of DO is complicated and inconclusive owing to two significant elements: mechanical tension which is well explored and trauma caused by bone fracture. Vasculogenesis and EPCs are critical for successful bone regeneration during DO. Thus, this study aimed to explore the effects of hypoxia caused by trauma or CoCl2 on the vasculogenesis of DO and EPCs. MATERIAL AND METHODS: Mandibular DO and BF models were generated using 6 beagle dogs with a distraction rate of 1 mm per day for 7 days or acute lengthening for 7 mm. The vasculogenesis in DO-gap or BF-gap were assessed via histological analyses, qRT-PCR and immunofluorescence staining. Dog bone marrow EPCs were isolated and cultured with or without 0.1 mM CoCl2. The effect of hypoxia caused by CoCl2 were subsequently valuated via in vitro assays including Cell Counting Kit-8, transwell assay, qRT-PCR, western blot, and immunofluorescence staining. RESULTS: Histological analyses, qRT-PCR and immunofluorescence staining revealed that vasculogenesis markedly accelerated in DO-gap compared with BF-gap, and the DO-gap displayed more positive to CD133, CD34, HIF-1α, E-cadherin, beclin1, ß-catenin, VEGF, bFGF, and less positive to ZEB1 than BF-gap. In addition, in vitro analyses revealed CoCl2 treatment enhanced EPCs proliferation and migration, and the levels of HIF-1α, E-cadherin, ß-catenin, beclin1, VEGF, bFGF of EPCs were increased, but the level of ZEB1 was decreased. CONCLUSION: Our studies showed that hypoxia promoted vasculogenesis in DO and EPCs, and the mechanism may involve autophagy, Wnt/ß-catenin signaling pathway, and Mesenchymal-Epithelial transition (MET).