Hypoxia promotes adipose-derived stem cell proliferation via VEGF Hypoxia promotes adipose-derived stem cell proliferation via VEGF

— Adipose-derived stem cells (ADSCs) are a promising mesenchymal stem cell source with therapeutic applications. Recent studies have shown that ADSCs could be expanded in vitro without phenotype changes. This study aimed to evaluate the effect of hypoxia on ADSC proliferation in vitro and to determine the role of vascular endothelial growth factor (VEGF) in ADSC proliferation. ADSCs were selectively cultured from the stromal vascular fraction obtained from adipose tissue in DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic. ADSCs were cultured under two conditions: hypoxia (5% O 2 ) and normal oxygen (21% O 2 ). The effects of the oxygen concentration on cell proliferation were examined by cell cycle and doubling time. The expression of VEGF was evaluated by the ELISA assay. The role of VEGF in ADSC proliferation was studied by neutralizing VEGF with anti-VEGF monoclonal antibodies. We found that the ADSC proliferation rate was significantly higher under hypoxia compared with normoxia. In hypoxia, ADSCs also triggered VEGF expression. However, neutralizing VEGF with anti-VEGF monoclonal antibodies significantly reduced the proliferation rate. These results suggest that hypoxia stimulated ADSC proliferation in association with oxygen. On days 2, 4, 6, 8, 10, 12, 14 and 16, the cells were lysed in200 μ L 0.02% sodium dodecyl sul-phate (SDS) inDNase-free water, after which the cell number in thesamples was determined using a Pico-GreendsDNAquantitation kit (Invitrogen). The number of cells was calculatedusing a theoretical value of 6.6 pg DNA/cell. The experiment was performed three times, each in

This study aimed to identify another mechanism of hypoxia that stimulates ADSC proliferation. Our findings provide new insight into controlling ADSCs in research and medical applications.

Isolation of stromal vascular fractions (SVFs)
Adipose tissues were collected from the abdomen by aspiration with a suitable needle from donors who signed a consent form. Approximately 50-100 mL of lipoaspirate were collected in two 50 mL sterile syringes. The syringes were stored in a sterile box at 2-8°C and were immediately transferred to the laboratory.
The SVF was isolated from adipose tissues using a Cell Extraction Kit (BioMedFactory, Ho Chi Minh, Vietnam) according to the manufacturer's instructions. Briefly, adipose tissue was mixed with an enzyme solution containing collagenase at 37°C for 15 min. Then, the cell suspension obtained was centrifuged at 3000 g for 10 min, and the SVF was obtained as the pellet. The pellet was washed with PBS to remove any residual enzymes, and resuspended in PBS to determine cell quantity and viability using an automatic cell counter (NucleoCounter, Chemometec, Denmark).

SVF culture
SVF samples were cultured in MSCCult medium (BioMedFactory) containing DMEM/F12 supplemented with antibiotic-antimycotic and 10% fetal bovine serum (FBS). The cells were plated at 5 × 10 4 cells/mL in T-75 flasks (Corning) and incubated at 37°C with 5% CO2. After 3 days of incubation, 6 mL of fresh medium was added to each flask. After 7 days, the medium was replaced with 12 mL of fresh media. The medium was subsequently replaced every 3 days until the cells reached 70-80% confluence, and then they were subcultured.

ADSC phenotyping and characterization
Cell markers were analyzed following a previously published protocol. Briefly, cells were washed twice with PBS containing 1% bovine serum albumin (BSA). The cells were then stained with anti-CD14-FITC, anti-CD44-PE, anti-CD45-FITC, anti-CD105-FITC, anti-CD90-PE and anti-HLA-DR-FITC antibodies (all purchased from BD Biosciences, San Jose, CA, USA). Stained cells were analyzed by a FACSCalibur flow cytometer (BD Biosciences). Isotype controls were used in all analyses.

Normoxia and hypoxia culture
To determine the effect of hypoxia on cell proliferation, the ADSC were seeded at a density of 1000 cells/wellin 96-well plates (Costar, Acton, MA, USA) and culturedat 5% or 20% oxygen. On days 2, 4, 6, 8, 10, 12, 14 and 16, the cells were lysed in200 μL 0.02% sodium dodecyl sulphate (SDS) inDNase-free water, after which the cell number in thesamples was determined using a Pico-GreendsDNAquantitation kit (Invitrogen). The number of cells was calculatedusing a theoretical value of 6.6 pg DNA/cell. The experiment was performed three times, each in quadruplicate.
Similarly, this procedure was repeated in a tri-gas incubator in which the oxygen concentration was controlled to 5%.

Cell cycle
Cell cycle analysis was carried out according to the following protocols. Cells from each group were washed twice with PBS and fixed in cold 70% ethanol for at least 3 h at 4°C. Cells were then washed twice with PBS and stained with 1 mL of propidium iodide (PI; 20 μg/mL). RNase A (10 μg/mL) was added to the samples and incubated for 3 h at 4°C. Stained cells were analyzed by flow cytometry using CellQuest Pro software (BD Biosciences, Franklin Lakes, NJ, USA).  . 2), nd HLA-DR. d into three cytes, which blasts, which d chondrob -Fig 2).  AD He et al., 2015;.

Effects of neutralizing VEGF
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ized that ther ADSC prolifer hypoxia: the first one is VEGF-dependent, and the second is VEGF-independent. In fact, the effects of hypoxia on ADSC proliferation have been demonstrated to involve activation of the HIF-1ɑ pathway (Kakudo et al., 2015). Using ERK and Akt inhibitors, Kakudoet al. (2015) have determined that the effects of hypoxia on ADSC proliferation could be blocked, and that HIF-1α knockdown by siRNA partially inhibited the hypoxia-induced FGF-2 expression in ADSCs (Kakudo et al., 2015). In the second pathway, we speculated that VEGF affects ADSCs via receptors other than VEGFR-1 and VEGR-2. Thus, we evaluated PDGFRβ expression in hypoxic ADSCs and showed that ADSCs highly expressed this receptor. In previous reports, PDGFRβ has been demonstrated as a facultative receptor for VEGF (Ball et al., 2007;Mabry et al., 2010;Pfister et al., 2012). Ball et al. (2007) have shown that VEGF-A could bind to both PDGFRα and PDGFRβ, inducing tyrosine phosphorylation. When inhibited, VEGF-A-induced MSC migration and proliferation were suppressed (Ball et al., 2007).
In combination with previous studies, we propose that hypoxia can stimulate ADSC proliferation via two mechanisms: hypoxia-stimulated expression of HIF-1 activates this signaling pathway, which induces VEGF secretion into the conditioned medium. The autocrine VEGF effect on ADSCs significantly triggers ADSC proliferation by efficiently reducing the proportion of S phase cells and reducing the doubling time.

CONCLUSION
ADSCs are promising stem cells for clinical applications. Hypoxia treatment was used to trigger ADSC proliferation. This study showed that hypoxia strongly stimulated ADSC proliferation. Under hypoxia, ADSCs were stimulated to produce VEGF, however, VEGF may have an autocrine effect on ADSCs. Secreted VEGF stimulated ADSC proliferation via PDGFRβ. These findings will contribute to understanding stem cell proliferation under hypoxia as well as application of this mechanism to control stem cell proliferation and differentiation.