Hypoxia preconditioned DPSC-derived exosomes regulate angiogenesis via transferring LOXL2

Mesenchymal stem cells (MSCs) play a crucial regulatory role in angiogenesis and tissue regeneration through exosome secretion. Dental pulp stem cells (DPSCs) are mesenchymal stem cells with great application prospects. Exosomes derived from DPSCs (DPSC-Exos) have also been shown to promote angiogenesis [1]. DPSCs are easier to isolate and culture than other types of MSCs; however, a recent study showed that DPSC-EVs are less potent in endothelial cell chemotaxis, an important step in angiogenesis, than extracellular vesicles of bone marrow-derived mesenchymal stromal cells (BMSC-EVs) [2]. Therefore, there is an urgent need to discover ways to improve the angiogenic potential of DPSC-Exos.

Since exosomal cargo reflect the characteristics of their parental cells, stimulating MSCs under different conditions or subjecting them to genetic modification are feasible methods to alter the cargo and improve the biological functions of their exosomes [3,4]. As reported, stem cells are located in low oxygen tension microenvironments in vivo [5], while DPSCs cultured under hypoxic conditions in vitro possess more favorable biological characteristics and thus stronger potential for angiogenesis [[6], [7], [8]]. Moreover, our previous study confirmed that hypoxia enhances the angiogenic potential of DPSC-Exos [9], suggesting that hypoxia preconditioning is an effective method to improve the angiogenic capacity of DPSC-Exos. The mechanism, however, remains unclear. Sánchez-Sánchez et al. [10] found that a hypoxia-regulated microRNA in DPSC-EVs, miR-4732–3p, stimulates angiogenesis and exerts cardioprotective actions against ischemic insult. However, there was no direct evidence that hypoxia exerted such effects through regulating exosomal miR-4732–3p in their study. Studies have shown that hypoxia preconditioning of MSCs activates angiogenesis-related signaling pathways in target cells via the upregulation of proteins or miRNAs in exosomes. In Han's study, exosomes of hypoxia-treated adipose-derived mesenchymal stem cells (ADSCs) were shown to enhance angiogenesis via regulation of VEGF/VEGF-R signaling [11]. Gao et al. [12] found that hypoxia induces the expression of HMGB1 in BMSC-derived exosomes and exosomal HMGB1 promotes angiogenesis via JNK/HIF-1α signaling. Additionally, Li et al. [13] observed that small extracellular vesicles (sEVs) from hypoxia-preconditioned BMSCs had increased vascular density, reduced infarct size and improved cardiac function in mice with myocardial infarction through upregulation of miR-486–5p. MiR-126 was also found to mediate the angiogenic effects of exosomes derived from hypoxic human umbilical cord MSCs (HucMSCs) during bone fracture healing [14]. Stem cells from human exfoliated deciduous teeth (SHEDs) have also been extensively studied as dental stem cells, and exosomes derived from hypoxia-preconditioned SHED cells possess a higher capacity to promote the tube formation of endothelial cells through transfer of both let-7f-5p and miR-210–3p [15]. Nonetheless, the identity of the molecule in hypoxia-preconditioned DPSC-Exos responsible for the enhancement of angiogenesis has been unclear prior to our report.

In view of the complex cargo of exosomes, we previously conducted comparative proteomic analysis of normoxia and hypoxia groups of DPSC-Exos to identify the key proteins in hypoxic DPSC-Exos responsible for mediating and promoting angiogenesis [9], which provided us with clues for this study. The results of the proteomic analysis revealed that angiogenesis-associated proteins such as lysyl oxidase-like 2 (LOXL2), matrix metallopeptidase 2 (MMP-2), transforming growth factor beta induced (TGFBI), Thrombospondin 1 (THBS1), nucleolin (NCL), ATP synthase subunit beta (ATP5B), and heparan sulfate proteoglycan 2 (HSPG2) were upregulated in the hypoxia group of DPSC-Exos. Since LOXL2 is linked with several proteins involved in collagen remodeling and has been shown to take part in the biological process of angiogenesis, it attracted our attention.

LOXL2 belongs to the lysyl oxidase family [16]. Originally discovered as an enzyme in the extracellular matrix, LOXL2 can catalyze the deamination of lysines and hydroxylysines and promote the crosslinking of elastin or collagen [17,18]. Studies have demonstrated that LOXL2 is an important regulator of physiological angiogenesis. LOXL2 expression is elevated during ischemic angiogenesis, while LOXL2 knockdown in zebrafish embryos results in absence of intersegmental vessel circulation [19]. LOXL2 deletion in mice led to developmental defects of the cardiovascular system and perinatal death [20,21]. In vitro experiments support the role of LOXL2 in regulating capillary formation via the modulation of endothelial cell migration and proliferation as well as collagen IV assembly in the vascular basement membrane [19]. Hypoxia inducible factor 1 (HIF-1) can directly bind to LOXL2-HRE1 and induce LOXL2 expression [22,23].

In our previous study, we observed that hypoxia increased LOXL2 expression in both DPSCs and DPSC-Exos, and further enhance the angiogenic potential of DPSC-Exos. Considering that LOXL2 is involved in angiogenesis, we hypothesized that LOXL2 is one of the key molecules in hypoxia-DPSC-derived exosomes (Hypo-Exos) that mediates the enhancement of angiogenesis. In this study, we silenced LOXL2 in Hypo-Exos and carried out a series of in vitro experiments to verify this hypothesis. The results of this study will reveal the possible mechanism through which hypoxia enhances the angiogenic potential of DPSC-Exos and could therefore promote the application of DPSC-Exos in regenerative medicine.

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