Investigating the Role of Hypoxia-Inducible Factor-1 Activation in the Vascularization of Modular Tissue Engineered Constructs

Vascularization is crucial to the fabrication of engineered tissues of therapeutically relevant size and cell loads. While prolonged hypoxia is detrimental to cell survival in these constructs, it is also a necessary driver of angiogenesis, exerting many of its effects via the hypoxia inducible factor (HIF)-1 pathway^1,2.

Modular tissue engineered constructs are formed by random assembly of micrometer-scale cylindrical collagen pieces (“modules”) embedded with adipose-derived mesenchymal stromal cells (adMSC) and enveloped with endothelial cells (EC); injected modules remodel in vivo to form a vascularized organoid^3,4. In a recent study, varying cell densities, module diameter and implant volume affected graft-derived vascularization. Vessel densities were greatest (p<0.01) in implants with pronounced nuclear expression of HIF-1α at day 7, as with “large” volume implants (0.10 mL, 1.5×10⁷ adMSC/mL, 3.9×10⁴ EC/mL) and “small” volume implants with the highest cell density (0.01 mL, 1.5×10⁸ adMSC/mL, 9.8×10⁴ EC/mL)⁵. HIF-1 is hypothesized to govern: 1) the survival of graft-derived cells immediately following implantation, and 2) subsequent recruitment of angiogenic myeloid cells to the hypoxic site.

Here, the role of HIF-1 on module-induced vascularization was elucidated using pharmacological and genetic methods. SCID-bg mice were provided daily injections of 2 mg/kg digoxin or saline starting 2 days prior to implantation of modules (in vitro preconditioned in 100 nM digoxin or saline for 6 hours), and continuing on to 7 days post-implantation. Digoxin-treated implants showed reduced graft-derived and total vessel formation compared with saline-treated controls at day 7 (19±1.9 vs. 44±9.9 UEA1⁺ graft-derived vessels/mm² and 19±3.0 vs. 46±10 CD31⁺ total vessels/mm²; p<0.01; n=5). Effects of HIF-1 inhibition correlated with reduced recruitment of endothelial progenitor cells (CD45^- CD117⁺ VEGFR2⁺) to module implants 3 days post-surgery (6.0±1.0 vs. 18±2.0 cells/mg implant; p<0.01; n=5).

In other experiments, HIF-1 was inhibited by lentiviral-mediated transduction of HUVEC with short hairpin RNA (shRNA) targeting HIF-1α. Upon implantation in modules, knockdown of HIF-1α in HUVEC reduced total vessel densities relative to targeting a scramble sequence at day 7 (13±4.0 vs. 26±4.0 CD31⁺ total vessels/mm²; p<0.05; n=5). However, no effect was seen at day 14, likely due to compensatory effects of HIF-2α upregulation at day 7 (135±8.0 vs. 54±13 HIF-2α⁺ nuclei/mm²; p<0.01; n=5).

Results from pharmacological and genetic methods of inhibition highlight HIF-1 as an early driver of module-induced vascularization, potentially exerting its effects by modulating recruitment of angiogenic EPC to the implant. Currently, we are examining ways of incorporating a HIF stabilizing small molecule, desferrioxamine, into modular tissue engineering to increase the rate of mature vessel formation. Knowledge gained from these studies may be harnessed to better apply modular tissue engineering to advance cell therapy.

References

1. Zhang, X. et al. (2011) Wound Repair Regen 18(2): 193-201.
2. Semenza, G.L. (2014) Annu Rev Pathol 9: 47-71.
3. McGuigan, A. and Sefton, M.V. (2006) PNAS 103(31): 11461-66.
4. Butler, M.J. and Sefton, M.V. (2012) Tissue Eng Part A 18(15- 16):1628-41.
5. Lam, G.C. and Sefton, M.V. (2015) Tissue Eng Part A 21(3-4): 803-16.