Abstract Body: Background: Limb ischemia presents a significant public health challenge, involving abnormal proliferation and angiogenesis of vascular endothelial cells. Recent studies suggest that adipose-derived stem cells (ADSCs) offer a promising therapeutic option. Also, hyperbaric oxygen (HBO) therapy has been shown to enhance blood flow and angiogenesis in ischemic limbs but how HBO pre-conditions ADSCs and its EVs to enhance the capability of angiogenesis in ischemic limbs remains unknown.
Methods: Conditioned medium of ADSCs (ADSCs CM) and HBO-boosted conditioned medium of ADSCs (HBO-ADSCs CM) were obtained from ADSCs with or without HBO treatment, respectively. Following limb ischemia surgery, we evaluated the angiogenic capacity in C57BL/6J mice treated with HBO-ADSCs CM compared to those treated with ADSCs CM. Additionally, in vitro experiments assessed the effects of HBO-ADSCs CM on human umbilical vein endothelial cells (HUVECs) following oxygen-glucose deprivation (OGD) injury through proliferation, migration, and tube formation assays. Proteomic analysis identified the angiogenic regulator in HBO-ADSC EVs and we further validated its impact on endothelial functions.
Results: Assessment through laser Doppler perfusion imaging and CD31 staining revealed that mice receiving HBO-ADSCs CM after limb ischemia surgery exhibited significantly enhanced angiogenic capability but the benefit diminished in EV depleted CM. In vitro analysis demonstrated that HBO-ADSCs CM treatment rescued impaired proliferation, migration, and tube formation of HUVECs under OGD injury. Proteomic analysis identified 93 upregulated proteins in ADSC-EVs following HBO treatment. Among them, Proteasome 20S Subunit Beta 6 (PSMB6), a key regulator of proteolytic degradation, was validated with the most highly expression in ADSC-EVs post-HBO treatment. Suppression of PSMB6 expression in HUVECs was also observed to impaired their proliferation, migration, and tube formation under OGD injury.
Conclusion: Our results indicate that HBO-ADSCs CM treatment enhances angiogenic capacity both in vivo and in vitro models of limb ischemia. Proteomic analysis highlights PSMB6 with in HBO-ADSCs EVs as a crucial factor promoting angiogenesis and perfusion recovery post-limb ischemia. Hence, HBO-ADSCs CM therapy emerges as a promising treatment option for limb ischemia, potentially benefiting patients unable to undergo high-risk surgical procedures.