One million ADSCs were seeded in the T75 flask and incubated at 37 ℃ with 5% CO₂ until reaching 70% confluency. The flasks were refreshed with fresh culture media. For group control (G0), hypoxia (group 3–G3) culture media (ADSCCult I completed medium, Regenemedlab) were used. For group 1 (G1), the completed culture medium with 100 ng/mL of TNF-alpha was used. For group 2 (G2) and group 4 (G4), the basal medium of ADSCCult I (DMEM/F-12) was used. The flasks of G3 and G4 were placed in the 5% oxygen incubators (Binder, Germany). The flasks of other groups were incubated in 20% oxygen, with 5% CO₂, and at 37 ℃. All flasks were incubated for 48 h; then, the supernatants were collected in 50 mL centrifuge tubes. The supernatants were centrifuged at 3,000 g at 4 ℃ for 15 min to remove debris. Finally, the CM were kept in a –86-℃ freezer for further experiments.

This assay was performed using the Angiogenesis Starter Kit (Thermo Fisher, MA). First, the HUVECs included in the kit were expanded using the M200 medium supplemented with LVES (included in the kit). Subsequently, the Geltrex matrix was prepared as instructed and used to cover the surface of wells of 96-well plates. Thereafter, HUVECs were collected and re-suspended in CM (G0: control, G1: TNF-alpha, G2: starvation, G3: hypoxia 5% oxygen, and G4: hypoxia 5% oxygen and starvation) and positive control using M200 medium supplemented with LVES. Next, HUVECs were cultured in CM and M200 medium supplemented with LVES for 48 h at 37 ℃, with 5% CO₂, and in 20% oxygen. Finally, blood vessel formation was observed under an inverted microscope (Carl Zeiss, Germany).

CM were collected and kept at –86 ℃ after removing the cell debris prior to use in this assay. The VEGF concentrations were measured using the VEGF ELISA kits (Abcam). All protocols were performed following the manufacturer’s guidelines.

A serial concentration of TNF-alpha (0, 10, 20, 40, 60, 80, and 100 ng/mL of TNF-alpha) was used to evaluate the effects of TNF-alpha on VEGF secretion of ADSCs. ADSCs were cultured in the 6-well plates with 10⁵ cells per well in the culture medium (ADSCCult I completed medium, Regenmedlab, HCMC, VN) until 70% confluency. The media were removed, and the fresh media supplemented to 0, 10, 20, 40, 60, 80, and 100 ng/mL of TNF-alpha (Thermo Fisher, MA) were used with 2 mL per well. The cells were cultured in an incubator with 5% CO₂, at 37 ℃, and in 20% oxygen for 48 h. The CM were collected, and the cell debris was removed by centrifugation at 3,000 g at 4 ℃ for 15 min. These CM were then used to measure VEGF concentration using an ELISA kit (Abcam).

A serial concentration of TNF-alpha (0, 10, 20, 40, 60, 80, and 100 ng/mL of TNF-alpha) was used to evaluate the effects of TNF-alpha on the apoptosis of HUVECs. First, the HUVECs were seeded on the 6-well plates with 10⁵ cells per well in the culture medium (M200 medium supplemented with LVES, Thermo Fisher) until 70% confluency. The media were removed, and the fresh media M200 (with LVES) supplemented to 0, 10, 20, 40, 60, 80, and 100 ng/mL of TNF-alpha (Thermo Fisher, MA) were used with 2 mL per well. The cells were cultured in an incubator with 5% CO₂, at 37 ℃, and in 20% oxygen for 48 h. The cells were then collected using the detachment reagent (Regenmedlab, HCMC, VN) and then evaluated for the percentage of apoptotic cells using the apoptosis kit, Annexin V/PI (BD Bioscience, MA). All assays were triplicated.

All assays were triplicated. Data were analyzed using the GraphPad Prism 8 software. The statistical analysis was performed using t-test. P-value < 0.05 was considered significant. All data were presented as mean ± standard deviation.

Figure 1

The isolated ADSCs exhibited full characteristics of MSCs suggested by ISCT regarding minimal criteria of MSCs. They displayed the fibroblast-like shape when adhered to the surface of plastic flasks. They also revealed that the marker profiles of MSCs were positive with CD44 (100%), CD73 (100%), CD90 (100%), and CD105 (98.56%) and negative with CD14 (0 %), CD34 (0 %), CD45 (0 %), and HLA-DR (0 %) (Figure 1). These cells were successfully induced to osteoblasts that could be positive with Alizarin Red staining (Figure 2), chondroblasts that could be positive with Alcian blue staining (Figure 2), and adipocytes that could be positive with Oil Red O staining (Figure 2).

The CM collected from different groups indicated different concentrations of VEGF. The highest concentration of VEGF was recorded in G3 in which the cells were treated in a hypoxia condition, and the lowest concentration of VEGF was recorded in G2 in which the cells were treated in TNF-alpha stress. The VEGF concentrations measured were 6,116 ± 527.9 pg/mL, 1,536 ± 432.1 pg/mL, 1,569 ± 710 pg/mL, 10,829 ± 640 pg/mL, and 3,574 ± 505 pg/mL in the supernatants of G0, G1, G2, G3, and G4, respectively (Figure 3).

The results revealed that in a normal culture condition (G0, control), ADSCs could produce a high concentration of VEGF in the CM; however, this concentration could be increased in a hypoxia condition (6,116 ± 527.9 pg/mL vs. 10,829 ± 640 pg/mL, respectively, for normoxia vs. hypoxia condition; p <0.05). In the conditions of TNF-alpha treatment (G1), starvation (G2), or a combination of hypoxia and starvation (G4), the concentrations of VEGF in CM significantly reduced compared to the control (G0; p < 0.05). In conditions of TNF-alpha treatment (G1) and starvation (G2), the concentrations of VEGF were too low with a non-significant difference between these groups (1,536 ± 432.1 pg/mL vs. 1,569 ± 710 pg/mL, respectively, for G1 vs. G2; p > 0.05).

Figure 4

Figure 5

Figure 6

TNF-alpha could cause apoptosis in HUVECs. At a high concentration of TNF-alpha (100 ng/mL), about 30% of HUVECs underwent apoptosis. *p < 0.05.

The effects of TNF-alpha on VEGF secretion of ADSCs were investigated in different doses (10, 20, 40, 60, 80, and 100 ng/mL). The results revealed that VEGF secretion of ADSCs depends on the concentrations of TNF-alpha (Figure 5). The VEGF concentrations recorded in the CM at treatments of 10 ng/mL and 20 ng/mL of TNF-alpha were higher than that in the CM of control (0 ng/mL of TNF-alpha; 6,600 ± 420 pg/mL and 8,410 ± 502 pg/mL in 10 ng/mL and 20 ng/mL, respectively, of TNF-alpha treatments compared to 5,105 ± 346 pg/mL in control; p < 0.05). However, when TNF-alpha concentrations increased from 40 ng/mL to 100 ng/mL, the VEGF concentrations in the CM gradually reduced. At concentrations of TNF-alpha 80 ng/mL and 100 ng/mL, the VEGF concentrations were lower than that in control (3014 ± 221 pg/mL and 1343 ± 453 pg/mL, respectively, in groups of 80 ng/mL and 100 ng/mL of TNF-alpha treatments compared to 5,105 ± 346 pg/mL in control; p < 0.05).

To evaluate the effects of TNF-alpha in CM on HUVECs, HUVECs were treated with CM collected from culture media supplemented with various concentrations of TNF-alpha. The results revealed that percentages of apoptotic HUVECs gradually increased in response to increased TNF-alpha concentrations (Figure 6). At 0 ng/mL of TNF-alpha, there were 5.50 ± 1.42% apoptotic HUVECs, while this ratio increased to 10.42 ± 2.89 in 10 ng/mL, 14.31 ± 2.31 % in 20 ng/mL, 16.77 ± 3.45 % in 40 ng/mL, 23.67 ± 3.49 % in 80 ng/mL, and 29.32 ± 5.68 % in 100 ng/mL of TNF-alpha (p < 0.05).

CM from MSCs is considered a promising cell-free agent for regenerative medicine. As more publications showed that the bio-effects of MSCs are mostly related to their secretomes, CM are of significant interest to researchers and pharmaceutical companies. CM are a combination of various factors such as soluble cytokines, EVs produced by MSCs, and culture media compositions. Under certain stress conditions, MSCs can change the gene expression and production of some cytokines and EVs that cause changes in their bioeffects on target cells. We proposed that when cultured in hypoxia, starvation, or a combination of hypoxia and starvation and TNF-alpha treatment, ADSCs can change the composition of their CM which in turn changes their angiogenic potential. Therefore, in this study, we treated ADSCs in four stress conditions: hypoxia, starvation, a combination of hypoxia and starvation, and TNF-alpha treatment and the assessment of CM on angiogenesis in vitro.

In the first experiment, we confirmed that isolated ADSCs express the MSC phenotype as suggested by Dominici et al. (2006)28. Indeed, these cells expressed that the marker profile of MSCs was positive with CD44, CD73, CD90, and CD105 and negative with hematopoietic cells CD14, CD34, CD45, and HLA-DR. Moreover, they could also be induced to mesenchymal cells, including osteoblasts, chondrocytes, and adipocytes in vitro.

In the subsequent experiment, the VEGF concentrations in CM collected under various stress conditions were measured. The results showed that ADSCs over-expressed VEGF production only in hypoxia, while in starvation, hypoxia and starvation, and TNF-alpha treatment, ADSC down-regulated VEGF production. Hypoxia can increase VEGF expression at mRNA and protein levels, which was also recorded in some published articles29, 30, 31, 32. Liu et al. (2013) preconditioned ADSCs in 1% oxygen and demonstrated that in this condition, ADSCs significantly increased the expression of angiogenic factors including VEGF and b-FGF33. This observation was similar to MSCs from the bone marrow34. Lagonda et al. (2018) also showed that at a 3% oxygen level, MSCs from umbilical cord tissue, bone marrow, and adipose tissue significantly increased VEGF synthesis and secretion compared to normoxia35. The mechanism under this observation is related to the activation of HIF-1 alpha caused by hypoxia36. The roles of HIF-1 alpha in angiogenesis are demonstrated in several studies37, 38, 39, 40. Indeed, HIF-1 alpha is a transcription factor regulating the expression of angiogenesis, preventing apoptosis, and inducing migration of cells to ischemia. Hypoxia is known as a condition that induces the expression of HIF-1 alpha38, 39. Besides the effects on VEGF production, hypoxia also causes universal effects on cell proliferation, cell metabolism, cell differentiation, cell apoptosis, and cell migration. In some studies, MSCs from the umbilical cord41, adipose tissue32, and urine stem cells42 could increase cell proliferation in hypoxic conditions (1.5% — 5% oxygen)41, while in other studies, MSCs could reduce their proliferation in hypoxic condition43.

Starvation is also a common kind of stress that can change MSCs’ characteristics. In this study, we found that starvation reduced VEGF production. This observation was also reported by Chua et al. (2013)23 and Rezaie et al. (2018)44. The reason for this property has not been explained in the previous publications. This study also has not figured out why VEGF production reduced in starvation. We proposed that this characteristic could relate to cell proliferation that significantly reduces the stress of starvation conditions. The considerable decrease in VEGF production in starvation could cause a significant reduction of VEGF when ADSCs are treated with both the stresses of hypoxia and starvation.

The effects of TNF-alpha on the angiogenic potential of MSCs are also controversial. A few studies suggested that TNF-alpha could stimulate VEGF production in MSCs45, 46, 47, while others demonstrated that TNF-alpha is an inhibitor of VEGF production in MSCs48. In this study, the effects of TNF-alpha depended on its dose. At a low dose (below 40 ng/mL), TNF-alpha could stimulate ADSCs to produce VEGF; however, at a higher dose, TNF-alpha inhibited VEGF production.

However, the reduction of VEGF in the CM did not directly affect the failure of CM from TNF-alpha-stressed ADSC culture in the blood vessel formation of HUVECs. Indeed, the results indicate that in hypoxia (G2) and hypoxia combined with starvation (G4), the levels of VEGF were similar to that of TNF-alpha treatment (G1), but their CM also supported blood vessel formation of HUVECs in vitro. Based on this finding, we propose that the remaining TNF-alpha in CM had a direct negative effect on the HUVECs and inhibited blood vessel formation. Under a microscope, we found that almost all HUVECs could enter an apoptotic phase. Therefore we propose that TNF-alpha could cause apoptosis in HUVECs in the next experiment.

To demonstrate that CM from TNF-alpha-stressed ADSC culture depends on reducing VEGF and that the percentage of apoptotic HUVECs directly depends on the concentration of TNF-alpha, CM derived from 100 ng/mL of TNF-alpha culture medium caused to 29.32 ± 5.68 % apoptotic HUVECs after 48 h of treatment. Hence, the effects of TNF-alpha in culture medium affect both ADSCs and HUVECs. Furthermore, although it can enhance VEGF production at low doses, TNF-alpha directly drives HUVECs to apoptosis; the blood vessel formation assay confirmed this analysis.

Blood vessels could be formed in vitro from HUVECs in CM from control (without stress), hypoxia, starvation, and starvation and hypoxia but not in the TNF-alpha stress group. The VEGF concentration can affect the angiogenic potential of these CM but does not depend on their concentration. In the starvation stress or starvation combined with hypoxia conditions, the VEGF concentration was also low compared to that in TNF-alpha stress. However, the blood vessel matrix could be formed in vitro. This implies that extra TNF-alpha in the CM of TNF-alpha stress ADSC culture inhibited the blood vessel formation of HUVECs. Moreover, TNF-alpha also changed the EV synthesis and production. Ahn et al. (2018) suggested that MSCs can produce EVs to carry and transfer VEGF to injured cells in models of neonatal hyperoxic lung injuries49.

CM from preconditioned MSC cultures are considered new components of regenerative medicine. CM can be used as free-cell therapy; they open up new opportunities for tissue healing. In this study, we found that CM from various culture conditions can directly affect VEGF production of ADSCs which in turn can change the angiogenic potential of CM. In hypoxic culture conditions, ADSCs strongly produce and secrete VEGF in the CM, while in starvation culture conditions as well as TNF-alpha treatment, ADSCs significantly reduced VEGF production. However, it seems that not only did VEGF in CM affect blood vessel formation of HUVECs but also had other effects. The extra TNF-alpha in CM of TNF-alpha treatment negatively affects the blood vessel formation by promoting apoptosis of HUVECs. Other mechanisms underlying this observation that can relate to the EVs and exosome synthesis and production of ADSCs treated with TNF-alpha have not been evaluated in this study.

ADSC: Adipose derived stem cell

CM: Conditioned medium

HGF: Hepatic growth factor

HUVEC: Human umbilical vein endothelial cell

IGF: Insulin-like growth factor

IL: Interleukin

MCP: Monocyte chemotactic factor

MSC: Mesenchymal stem cell

SDF: Stromal cell-derived factor

TNF: Tissue necrosis factor

VCAM: Vascular cell adhesion protein

VEGF: Vascular endothelial growth factor

Authors thank Van Hanh General Hospital, Ho Chi Minh City, Viet Nam approved to use the adipose tissues from donors for the study.

Phuc Van Pham contributed to the conceptualization and design of the study, the acquisition, analysis and interpretation of data. Ngoc Bich Vu, Binh Thanh Vu and Khanh Nha Nguyen were for drafting the article and revising the article critically for important intellectual content. All authors read and approved the final manuscript.

This work is supported by Vietnam National University Ho Chi Minh City, No.TX2019-18-02.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.

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