ABCB5+ mesenchymal stromal cells therapy protects from hypoxia by restoring Ca2+ homeostasis in vitro and in vivo

Abstract

Background: Hypoxia in ischemic disease impairs Ca²⁺ homeostasis and may promote angiogenesis. The therapeutic efficacy of mesenchymal stromal cells (MSCs) in peripheral arterial occlusive disease is well established, yet its influence on cellular Ca²⁺ homeostasis remains to be elucidated. We addressed the influence of ATP-binding cassette subfamily B member 5 positive mesenchymal stromal cells (ABCB5⁺ MSCs) on Ca²⁺ homeostasis in hypoxic human umbilical vein endothelial cells (HUVECs) in vitro and in vivo.

Methods: Hypoxia was induced in HUVECs by Cobalt (II) chloride (CoCl₂) or Deferoxamine (DFO). Dynamic changes in the cytosolic- and endoplasmic reticulum (ER) Ca²⁺ and changes in reactive oxygen species were assessed by appropriate fluorescence-based sensors. Metabolic activity, cell migration, and tube formation were assessed by standard assays. Acute-on-chronic ischemia in Apolipoprotein E knock-out (ApoE^-/-) mice was performed by double ligation of the right femoral artery (DFLA). ABCB5⁺ MSC cells were injected into the ischemic limb. Functional recovery after DFLA and histology of gastrocnemius and aorta were assessed.

Results: Hypoxia-induced impairment of cytosolic and ER Ca²⁺ were restored by ABCB5⁺ MSCs or their conditioned medium. Similar was found for changes in intracellular ROS production, metabolic activity, migratory ability and tube formation. The restoration was paralleled by an increased expression of the Ca²⁺ transporter Sarco-/endoplasmic reticulum ATPase 2a (SERCA2a) and the phosphorylation of Phospholamban (PLN). In acute-on-chronic ischemia, ABCB5⁺ MSCs treated mice showed a higher microvascular density, increased SERCA2a expression and PLN phosphorylation relative to untreated controls.

Conclusions: ABCB5⁺ MSCs therapy can restore cellular Ca²⁺ homeostasis, which may beneficially affect the angiogenic function of endothelial cells under hypoxia in vitro and in vivo.

Keywords: Angiogenesis; Calcium ion; Endothelial cells; Hypoxia; Mesenchymal stromal cells; Peripheral artery disease; Stem cell therapy.