Vadadustat, a HIF Prolyl Hydroxylase Inhibitor, Improves Immunomodulatory Properties of Human Mesenchymal Stromal Cells

Abstract

The therapeutic potential of mesenchymal stromal cells (MSCs) is largely attributed to their immunomodulatory properties, which can be further improved by hypoxia priming. In this study, we investigated the immunomodulatory properties of MSCs preconditioned with hypoxia-mimetic Vadadustat (AKB-6548, Akebia). Gene expression analysis of immunomodulatory factors was performed by real-time polymerase chain reaction (real-time PCR) on RNA isolated from six human bone-marrow derived MSCs populations preconditioned for 6 h with 40 μM Vadadustat compared to control MSCs. The effect of Vadadustat preconditioning on MSCs secretome was determined using Proteome Profiler and Luminex, while their immunomodulatory activity was assessed by mixed lymphocyte reaction (MLR) and Culturex transwell migration assays. Real-time PCR revealed that Vadadustat downregulated genes related to immune system: IL24, IL1B, CXCL8, PDCD1LG1, PDCD1LG2, HIF1A, CCL2 and IL6, and upregulated IL17RD, CCL28 and LEP. Vadadustat caused a marked decrease in the secretion of IL6 (by 51%), HGF (by 47%), CCL7 (MCP3) (by 42%) and CXCL8 (by 40%). Vadadustat potentiated the inhibitory effect of MSCs on the proliferation of alloactivated human peripheral blood mononuclear cells (PBMCs), and reduced monocytes-enriched PBMCs chemotaxis towards the MSCs secretome. Preconditioning with Vadadustat may constitute a valuable approach to improve the therapeutic properties of MSCs.

Gene Expression Profile of Human Mesenchymal Stromal Cells Exposed to Hypoxic and Pseudohypoxic Preconditioning—An Analysis by RNA Sequencing

“…They adhered to the plastic culture surface and formed colonies, showed expression of the surface markers CD73 (mean 99.5%), CD90 (mean 99.0%), and CD105 (mean 99.4%) and the absence of CD45, CD34, CD11b, CD19, and HLA-DR antigens in 98.3% of the cells, and demonstrated the ability to differentiate into cartilage, bone, and adipose tissue cells. Detailed results of identification analyses of the six BM-MSCs populations used in this study have been published previously in Zielniok et al (2020) ( Supplementary Tables S1–S3 , donors 1–6 in [ 11 ]).…”

“…Many studies to date have demonstrated the extensive role that hypoxia and its associated transcriptional changes play in maintaining the therapeutic properties of MSCs [ 7 , 23 , 24 , 25 , 26 ]. And the fact that the transcription factor HIF-1α is a principal mediator of these changes is supported by positive results from studies involving its pharmacological stabilization [ 11 , 27 , 28 , 29 ]. However, in the present study, we showed that hypoxic preconditioning and pharmacological stabilization of HIF-1α by Vadadustat are markedly distinct in their effect at the gene expression level in MSCs.…”

“…The mean age of the BM-MSCs donors was 50.5 years and the median was 44 years. All procedures were performed as we previously described [ 11 , 38 ]. The bone marrow samples were initially mechanically disassociated, then washed, centrifuged and suspended and seeded on a plastic culture dish (BD Primaria™, BD Biosciences, San Jose, CA, USA) in growth medium (low glucose DMEM, Biowest, Riverside, MO, USA) supplemented with 10% fetal bovine serum (Biowest, Riverside, MO, USA) and antibiotic-antimycotic solution (1% penicillin-streptomycin; 0.5% amphotericin B, invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).…”

“…The methods used to identify BM-MSCs were described in detail in our previous work [ 11 ]. We performed phenotyping of BM-MSCs on a flow cytometer (BD FACS Canto II using BD FACS Diva Software (BD Biosciences, San Jose, CA, USA)) using the BD Stemflow™ hMSC Analysis Kit (BD Biosciences).…”

“…Furthermore, exposure to hypoxic or hypoxia-like conditions has been shown to improve migration, regenerative and proangiogenic potential as well as stemness of MSCs [ 7 , 8 , 9 , 10 ]. In our recent study, we demonstrated that inhibition of hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) by Vadadustat (AKB-6548) regulated the immunomodulatory potential of human bone marrow-derived MSCs [ 11 ]. PHD2 is one of three isoforms of oxygen-dependent dioxygenases responsible for hydroxylation of two proline residues in Hypoxia-inducible transcription factors alpha (HIFα).…”