Hypoxia-Mimetic CoCl2 Agent Enhances Pro-Angiogenic Activities in Ovine Amniotic Epithelial Cells-Derived Conditioned Medium

Abstract

Amniotic epithelial stem cells (AECs) are largely studied for their pro-regenerative properties. However, it remains undetermined if low oxygen (O2) levels that AECs experience in vivo can be of value in maintaining their biological properties after isolation. To this aim, the present study has been designed to evaluate the effects of a hypoxia-mimetic agent, cobalt chloride (CoCl2), on AECs’ stemness and angiogenic activities. First, a CoCl2 dose-effect was performed to select the concentration able to induce hypoxia, through HIF-1α stabilization, without promoting any cytotoxicity effect assessed through the analysis of cell vitality, proliferation, and apoptotic-related events. Then, the identified CoCl2 dose was evaluated on the expression and angiogenic properties of AECs’ stemness markers (OCT-4, NANOG, SOX-2) by analysing VEGF expression, angiogenic chemokines’ profiles, and AEC-derived conditioned media activity through an in vitro angiogenic xeno-assay. Results demonstrated that AECs are sensitive to the cytotoxicity effects of CoCl2. The unique concentration leading to HIF-1α stabilization and nuclear translocation was 10 µM, preserving cell viability and proliferation up to 48 h. CoCl2 exposure did not modulate stemness markers in AECs while progressively decreasing VEGF expression. On the contrary, CoCl2 treatment promoted a significant short-term release of angiogenic chemokines in culture media (CM). The enrichment in bio-active factors was confirmed by the ability of CoCl2-derived CM to induce HUVEC growth and the cells’ organization in tubule-like structures. These findings demonstrate that an appropriate dose of CoCl2 can be adopted as a hypoxia-mimetic agent in AECs. The short-term, chemical-induced hypoxic condition can be targeted to enhance AECs’ pro-angiogenic properties by providing a novel approach for stem cell-free therapy protocols.

Tendon 3D Scaffolds Establish a Tailored Microenvironment Instructing Paracrine Mediated Regenerative Amniotic Epithelial Stem Cells Potential

“…Moreover, GCSF was investigated by Ross et al, who found an increase in local cellularity after rotator cuff repair thorough GCSF treatment [ 71 ]. Knowing that TGF-β1 is secreted by AECs [ 19 , 38 ], its release in CM was evaluated by WB, confirming the array results.…”

“…However, TGF-β and IL-8, MCP-1 and IFN-γ ( Figure 8 D) showed lower levels of secretion. Of note, IL-6 and GM-CSF protein contents were not detected in all CM samples probably because antibodies for these two molecules are highly species-specific and thus do not recognize these relative sheep proteins [ 38 ].…”

“…Image Lab software was used to perform densitometric analysis (Image Lab 6.1, Bio-Rad Laboratories, CA, USA), determining the pixel density of each spot by subtracting the background. The signal density of an individual spot was normalized using the signal of positive control detected within each analyzed membrane [ 38 ]. The obtained results were compared by semi-quantitative densitometry of mean pixel density.…”

“…A Western Blot (WB) investigation was carried out to evaluate TGF-β1 protein content in samples’ CM. In detail, equal volume amount (30 µL) of CM collected from CTR, and cells seeded on PLGA fleeces and 3D scaffolds, were processed for WB as previously described [ 38 ]. The total protein amount in CM has been normalized on a Ponceau S stain, according to Sander et al [ 39 ].…”

Highly Sensitive Cyanine Dyes for Rapid Sensing of NAD(P)H in Mitochondria and First-Instar Larvae of Drosophila melanogaster

“…We employed cobalt chloride (CoCl 2 ) to trigger hypoxia and used probe A to determine the NAD­(P)H levels in A549 cells. CoCl 2 is a hypoxia-mimicking agent that stabilizes the HIF-1α subunit by inhibiting its prolyl hydroxylation, which triggers its degradation under normoxic conditions . HIF-1α is a transcription factor activated under low-oxygen circumstances, and its stabilization can induce the upregulation of downstream target genes involved in adaptation to hypoxia, such as those involved in angiogenesis, erythropoiesis, and glycolysis. , CoCl 2 has been widely used as a tool to study cellular responses to hypoxia because of its ease of use and reproducibility .…”