# Hypoxic Preconditioned Nanofat at 1% O2 for 24 h Loses Its Regenerative In Vivo Vascularization Capacity

## TL;DR

Hypoxic preconditioning of nanofat increases pro-angiogenic proteins but reduces its ability to support blood vessel growth in living tissue.

## Contribution

This study shows that 24-hour hypoxic preconditioning of nanofat does not improve its in vivo vascularization despite a pro-angiogenic protein profile.

## Key findings

- Hypoxic preconditioning increases HIF-1α and SDF-1 expression in nanofat without affecting cell viability.
- Preconditioned nanofat has a pro-angiogenic protein profile but reduces functional microvessel density in vivo.
- Milder hypoxic protocols may be more effective for enhancing nanofat vascularization.

## Abstract

What are the main findings?
Hypoxic preconditioning at 1% O2 for 24 h activates nanofat and shifts its protein expression profile towards a pro-angiogenic phenotype without affecting its viability.The applied hypoxic preconditioning protocol does not improve the in vivo vascularization capacity of nanofat after its seeding on implanted dermal substitutes.

Hypoxic preconditioning at 1% O2 for 24 h activates nanofat and shifts its protein expression profile towards a pro-angiogenic phenotype without affecting its viability.

The applied hypoxic preconditioning protocol does not improve the in vivo vascularization capacity of nanofat after its seeding on implanted dermal substitutes.

What are the implications of the main findings?
Hypoxic preconditioning at 1% O2 for 24 h may be too stressful for nanofat that is subsequently exposed to prolonged in vivo hypoxia.Milder preconditioning protocols should be alternatively tested in future studies.

Hypoxic preconditioning at 1% O2 for 24 h may be too stressful for nanofat that is subsequently exposed to prolonged in vivo hypoxia.

Milder preconditioning protocols should be alternatively tested in future studies.

Hypoxic preconditioning is increasingly explored to enhance the survival and vascularization of fat grafts. In this study, nanofat from donor mice was exposed to hypoxia (1% O2) for 24 h to investigate the effects of this preconditioning protocol on the viability, gene expression and vascularization capacity of this mechanically processed fat derivative. Ex vivo analyses revealed that hypoxic preconditioning does neither affect apoptotic nor necrotic cell death within nanofat but significantly upregulates the expression of hypoxia-inducible factor (HIF)-1α and stromal cell-derived factor (SDF)-1 compared to non-preconditioned nanofat. Moreover, preconditioned nanofat exhibited a pro-angiogenic protein expression profile. For in vivo analyses, dermal substitutes were either seeded with preconditioned or non-preconditioned nanofat and transferred into dorsal skinfold chambers of mice to assess their vascularization by intravital fluorescence microscopy. Unexpectedly, implants seeded with preconditioned nanofat exhibited a significantly reduced functional microvessel density when compared to non-preconditioned controls. Immunohistochemical analyses also confirmed a lower microvessel density within the implants of the preconditioned group. These findings suggest that hypoxic preconditioning at 1% O2 for 24 h cannot be recommended for enhancing the regenerative in vivo vascularization capacity of nanofat. Therefore, milder preconditioning protocols with shorter periods of hypoxia or higher oxygen levels should be alternatively tested in future studies.