Hypoxic Preconditioned ADSC Exosomes Enhance Vaginal Wound Healing via Accelerated Keratinocyte Proliferation and Migration through Akt/HIF-1α Axis Activation

Obstetrics and Gynecology Medicine Life Sciences Bioengineering Cell Biology
Adipose Mesenchymal Stem Cells Exosomes Vaginal Injury Akt HIF-1α

Vaginal wound healing is a critical issue in gynecological surgery, especially following vaginal delivery or pelvic reconstructive surgery. Infection and poor healing of vaginal wounds can lead to severe complications such as chronic pain, infection, and dysfunction. Therefore, accelerating vaginal wound healing has been a focal point of clinical research. In recent years, the role of mesenchymal stem cells (MSCs) and their exosomes in wound healing has garnered significant attention. Exosomes are nanoscale vesicles secreted by cells that can transmit biological information molecules, promoting cell proliferation, migration, and angiogenesis. Particularly, hypoxia-preconditioned exosomes, due to their enhanced bioactivity, are considered to have potential therapeutic value in wound healing.

This study aimed to investigate whether hypoxia-preconditioned adipose-derived stem cell (ADSC) exosomes (Hypoxic ADSC Exosomes, Hexo) could accelerate vaginal wound healing by activating the Akt/HIF-1α axis and to elucidate the underlying molecular mechanisms.

Source of the Paper

The research was conducted by Xiaoyun Yang, Shasha Zhang, and other authors from Tongji Hospital, Tongji University School of Medicine. The findings were published online on September 4, 2024, in the journal Cellular and Molecular Bioengineering.

Research Process

1. Isolation and Identification of ADSCs

Researchers isolated ADSCs from adipose tissue of healthy subjects and rats. The expression of surface markers (CD90, CD29, CD105, CD44) was detected by immunofluorescence staining to confirm their stem cell characteristics. Additionally, the multipotent differentiation potential of ADSCs was verified through Oil Red O staining and alkaline phosphatase staining.

2. Extraction and Analysis of Exosomes

ADSCs were cultured under hypoxic conditions (1% O2) for 24 hours, and the cell supernatant was collected. Exosomes were extracted via ultracentrifugation. The size and morphology of the exosomes were analyzed using nanoparticle tracking analysis (Nanosight LM10) and transmission electron microscopy (TEM), confirming their typical cup-shaped or spherical structure.

3. Treatment of HaCaT Cells

HaCaT cells (human keratinocytes) were co-cultured with exosomes at different concentrations (50 μg/mL). Interventions were performed using the Akt inhibitor (LY294002) and HIF-1α inhibitor (PX478). The expression levels of Akt, p-Akt, and HIF-1α were detected by Western blot and RT-PCR.

4. In Vitro Experiments: Cell Proliferation and Migration

The effects of exosomes on HaCaT cell proliferation and migration were evaluated using the CCK-8 assay and scratch assay. Results showed that Hexo treatment significantly promoted HaCaT cell proliferation and migration, and this effect was reversed by Akt and HIF-1α inhibitors.

5. In Vivo Experiments: Vaginal Wound Healing Model

A rat vaginal injury model was constructed, and Hexo or PBS was injected into the wound site. The expression of inflammatory factors (TNF-α, IL-1β, IL-6) was detected by ELISA, and the levels of reactive oxygen species (ROS) and angiogenesis marker (CD31) were assessed by immunofluorescence staining. Results showed that Hexo treatment significantly reduced the levels of inflammatory factors and ROS while promoting angiogenesis.

Key Findings

  1. Hexo Promotes HaCaT Cell Proliferation and Migration: Hexo treatment significantly increased the proliferation rate and migration ability of HaCaT cells, and this effect depended on the activation of the Akt/HIF-1α axis.
  2. Hexo Accelerates Vaginal Wound Healing: In the vaginal injury model, Hexo treatment significantly reduced the levels of inflammatory factors and ROS while promoting angiogenesis, thereby accelerating wound healing.
  3. Critical Role of the Akt/HIF-1α Axis: Inhibition of Akt or HIF-1α reversed the promoting effect of Hexo on wound healing, indicating that the Akt/HIF-1α axis plays a key role in Hexo-mediated wound healing.

Conclusion

This study found that hypoxia-preconditioned ADSC exosomes significantly accelerated vaginal wound healing by activating the Akt/HIF-1α axis. Hexo not only inhibited inflammatory responses and ROS accumulation but also promoted angiogenesis and the proliferation and migration of keratinocytes. This research provides a new therapeutic strategy for vaginal wound healing and holds significant clinical application value.

Highlights of the Research

  1. Unique Advantages of Hypoxia-Preconditioned Exosomes: Hypoxia preconditioning enhanced the bioactivity of exosomes, resulting in stronger therapeutic effects in wound healing.
  2. Critical Role of the Akt/HIF-1α Axis: The study first revealed the key role of the Akt/HIF-1α axis in Hexo-mediated wound healing, providing new molecular targets for future research.
  3. Potential for Clinical Applications: As a novel therapeutic approach, Hexo holds promise for use in gynecological surgery and chronic wound treatment.

Additional Valuable Information

The research was funded by the National Key Research and Development Program of China (2023YFC2411205) and the National Natural Science Foundation of China (82071630, 81771560, 81702745), among others. All authors declared no conflicts of interest, and the research data are available from the corresponding author upon reasonable request.

Through this study, we not only gained a deeper understanding of the mechanisms of hypoxia-preconditioned exosomes but also provided new therapeutic insights for vaginal wound healing, holding significant scientific and clinical implications.