Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Mar 20:2022:6795274.
doi: 10.1155/2022/6795274. eCollection 2022.

Implantation of Hypoxia-Induced Mesenchymal Stem Cell Advances Therapeutic Angiogenesis

Affiliations

Implantation of Hypoxia-Induced Mesenchymal Stem Cell Advances Therapeutic Angiogenesis

Farina Mohamad Yusoff et al. Stem Cells Int. .

Abstract

Hypoxia preconditioning enhances the paracrine abilities of mesenchymal stem cells (MSCs) for vascular regeneration and tissue healing. Implantation of hypoxia-induced mesenchymal stem cells (hi-MSCs) may further improve limb perfusion in a murine model of hindlimb ischemia. This study is aimed at determining whether implantation of hi-MSCs is an effective modality for improving outcomes of treatment of ischemic artery diseases. We evaluated the effects of human bone marrow-derived MSC implantation on limb blood flow in an ischemic hindlimb model. hi-MSCs were prepared by cell culture under 1% oxygen for 24 hours prior to implantation. A total of 1 × 105 MSCs and hi-MSCs and phosphate-buffered saline (PBS) were intramuscularly implanted into ischemic muscles at 36 hours after surgery. Restoration of blood flow and muscle perfusion was evaluated by laser Doppler perfusion imaging. Blood perfusion recovery, enhanced vessel densities, and improvement of function of the ischemia limb were significantly greater in the hi-MSC group than in the MSC or PBS group. Immunochemistry revealed that hi-MSCs had higher expression levels of hypoxia-inducible factor-1 alpha and vascular endothelial growth factor A than those in MSCs. In addition, an endothelial cell-inducing medium showed high expression levels of vascular endothelial growth factor, platelet endothelial cell adhesion molecule-1, and von Willebrand factor in hi-MSCs compared to those in MSCs. These findings suggest that pretreatment of MSCs with a hypoxia condition and implantation of hi-MSCs advances neovascularization capability with enhanced therapeutic angiogenic effects in a murine hindlimb ischemia model.

PubMed Disclaimer

Conflict of interest statement

The Department of Stem Cell Biology and Medicine, Graduate School of Biomedical & Health Sciences, Hiroshima University, is a collaborative research laboratory funded by TWOCELLS Company, Limited. All remaining authors have declared that no conflicts of interest exist.

Figures

Figure 1
Figure 1
Schedule of the study and hindlimb ischemic model. (a) Protocol schedule for the in vivo disease model, mesenchymal stem cell (MSC) implantations, follow-up, and acquisition of samples. (b) Hindlimb ischemic model and phosphate-buffered saline (PBS)/MSC/hypoxia-induced MSC (hi-MSC) injection sites (stars).
Figure 2
Figure 2
Protocol schedule for in vitro endothelial cell (EC) differentiation challenge in mesenchymal stem cells (MSCs) vs. hypoxia-induced MSC (hi-MSC) models, follow-up, and acquisition of samples at day 3 and day 7 after application of EC-induction medium.
Figure 3
Figure 3
Cell viability and proliferation assays. The absorbance value of hypoxia-induced MSCs (hi-MSCs) was more significantly increased than that of mesenchymal stem cells (MSCs) in a time-dependent manner. The absorbance value of MSCs (n = 5) and hi-MSCs (n = 5) was assessed at each time point (0, 12, and 24 hours).
Figure 4
Figure 4
Serial laser Doppler perfusion images (LDPIs) before and after making the ischemic hindlimb model. (a) Representative images of LDPI studies. (b) LDPI index studies in phosphate-buffered saline (PBS; n = 6), mesenchymal stem cell (MSC; n = 6), and hypoxia-induced MSC (hi-MSC; n = 6) implantation groups.
Figure 5
Figure 5
Mean cluster of differentiation 31 (CD31) intensity at day 28 after surgical induction of ischemia. (a) Mean CD31 intensity in phosphate-buffered saline (PBS; n = 6), mesenchymal stem cell (MSC; n = 6), and hypoxia-induced MSC (hi-MSC; n = 6) injection groups. (b) Representative images of CD31 immunostaining. Scale bar: 50 μm.
Figure 6
Figure 6
Expression of angiogenic cytokines in cultured mesenchymal stem cells (MSCs) and hypoxia-induced MSCs (hi-MSCs) prior to implantation. hi-MSCs showed significantly higher expression levels of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor A (VEGF-A) than those in MSCs. The expression level of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) was slightly lower in the hi-MSC group than in the MSC group.
Figure 7
Figure 7
Endothelial cell (EC) differentiation challenge of mesenchymal stem cells (MSCs) and hypoxia-induced MSCs (hi-MSCs) with analysis through TaqMan and SYBR Gene Expression Assays at day 3 and day 7 after cells were cultured with EC-induction medium. (a) Analysis of vascular endothelial growth factor (VEGF) gene expression. (b) Analysis of platelet endothelial cell adhesion molecule-1 (PECAM-1) gene expression. (c) Analysis of von Willebrand factor (vWF) gene expression. hi-MSC population of cells may have added potential for EC differentiation compared to the MSC population of cells.
Figure 8
Figure 8
In vitro cell migration assays of mesenchymal stem cells (MSCs), hypoxia-induced MSCs (hi-MSCs), coculture of MSCs with human umbilical vein endothelial cells (HUVECs) (MSC coculture), and coculture of hi-MSCs with HUVECs (hi-MSC coculture) at each time point of the 3rd hour (3 hr), 6th hour (6 hr), and 10th hour (10 hr) with (a, c) 3 μm pore-sized inserts and (b, d) 8 μm pore-sized. The hi-MSC population of cells may have added potential for coculture migration capacity compared to the MSC population of cells. Scale bar: 50 μm.

Similar articles

Cited by

References

    1. Farber A. Chronic limb-threatening ischemia. New England Journal of Medicine . 2018;379(2):171–180. doi: 10.1056/NEJMcp1709326. - DOI - PubMed
    1. Chevalier J., Yin H., Arpino J. M., et al. Obstruction of Small Arterioles in Patients with Critical Limb Ischemia due to Partial Endothelial-to-Mesenchymal Transition. Iscience . 2020;23(6, article 101251) doi: 10.1016/j.isci.2020.101251. - DOI - PMC - PubMed
    1. Masi S., Rizzoni D., Taddei S., et al. Assessment and pathophysiology of microvascular disease: recent progress and clinical implications. European Heart Journal . 2021;42(26):2590–2604. doi: 10.1093/eurheartj/ehaa857. - DOI - PMC - PubMed
    1. Behroozian A., Beckman J. A. Microvascular disease increases amputation in patients with peripheral artery disease. Arteriosclerosis, Thrombosis, and Vascular Biology . 2020;40(3):534–540. doi: 10.1161/ATVBAHA.119.312859. - DOI - PubMed
    1. Duff S., Mafilios M. S., Bhounsule P., Hasegawa J. T. The burden of critical limb ischemia: a review of recent literature. Vascular Health and Risk Management . 2019;Volume 15:187–208. doi: 10.2147/VHRM.S209241. - DOI - PMC - PubMed

LinkOut - more resources