On-demand hypoxic hydrogels reveal acute regulation of angiogenesis

Introduction: The lack of oxygen (O2), or hypoxia (O2 < 5%), is a well-recognized biochemical signal that plays a key role in both physiological and disease states. Reactive oxygen species (ROS) production is elevated under hypoxia, allowing the transcription of hypoxia inducible factors (HIFs) that orchestrate numerous cellular processes, including cell proliferation, metabolism, and angiogenesis. Chronic hypoxia is common in cancer and cardiovascular disease, while acute hypoxia, typically lasting only a few hours, poses a threat in stressed environments even without an underlying condition. However, the lack of appropriate models to recapitulate acute hypoxia has hindered our understanding of its long-term impact. Lining the inner blood vessels, endothelial cells are the first to encounter changes in O2 concentrations, including rapid drop leading to acute hypoxia. Current in vitro models focus on how individual endothelial cells or endothelial monolayers respond to low O2 environments generated by conventional hypoxia chambers. Yet, due to gas diffusion and cell-dependent factors, the user-defined O2 level does not predict the O2 concentrations in the cellular microenvironment. Angiogenesis, the formation of new vascular sprouts from pre-existing blood vessels, is essential for efficient O2 and nutrient delivery to tissues. Therefore, hypoxia impacts sprouting from the already established 3D vessels, rather than individual ECs. Here, we report the fabrication of a novel hydrogel system that can introduce acute hypoxia “on-demand” in already established endothelial sprouts. The work developed here lay the foundation for uncovering the mechanism by which established vascular sprouts respond to acutely induced hypoxia.

Materials and

Methods: The “on-demand” hypoxic hydrogel (Gel-Dex) is fabricated by using gelatin conjugated with ferulic acid (Gel-FA) with multi-aldehyde modified dextran (Dex-CHO) (Figure 1A). Gel-FA is synthesized using EDC-NHS coupling, while Dex-CHO is synthesized through sodium periodate oxidation. Polymer precursor solutions (4 wt% Gel-FA, 0.5 wt% Dex-CHO) are mixed at room temperature and Gel-Dex is formed through imine bonding between the free amine groups of the gelatin backbone and the aldehyde groups of Dex-CHO. Laccase is introduced to the cell culture medium at 2.5 U ml-1, crosslinking the FA moieties and generating moderate hypoxia “on-demand”. We monitor the O2 levels noninvasively using patch sensors at the bottom of the Gel-Dex hydrogel (PreSens). We confirm that shear modulus does not change upon laccase crosslinking using shear rheology (TA instruments). Spheroids from endothelial colony forming cells are generated using the hanging drop method and mixed with the hydrogel precursor solution and cell culture medium supplemented with 50 ng ml-1 VEGF and 25 ng ml-1 FGF. Laccase is introduced to the system on day 2 for 6 hours and then removed from the cell culture medium. Gene knockdown of HIF1α and/or HIF2α was performed using small interfering RNA (siRNA) prior to spheroid formation (Horizon Discovery). Small-molecule inhibitors are used to study the role of ROS (DPI) and matrix metalloproteases (GM6001) in mediating the enhanced angiogenic response under acute hypoxia. The hydrogels are fixed and stained on day 3. Sprouting quantification is performed using AQuTAS (Matlab algorithm) and Imaris (Oxford Instruments).

Results, Conclusions, and Discussions: Endothelial spheroids are encapsulated in the Gel-Dex hydrogels and cultured for 2 days to establish sprouts. On day 2, laccase is added to the cell culture medium, introducing hypoxia “on-demand”. Continuous noninvasive measurements at the bottom of Gel-Dex show that moderate hypoxia (O2~5%) is achieved approximately within 6 hours of laccase incubation (Figure 1B). After 6 hours laccase is removed and the system returns to nonhypoxic (> 7.5%) O2 levels. Shear rheology confirmed that laccase addition did not increase hydrogel stiffness immediately after the 6-hour incubation period or 24 hours after removing laccase (Figure 1C). The 6-hour timepoint was chosen as it allows to investigate how short-term moderate hypoxia impacts vascular sprouts, while minimizing potential hydrogel stiffening due to excess FA crosslinking. On day 3 (Figure 1D), an increase in the sprouting (Figure 1E) is observed in the “on-demand” hypoxic hydrogels compared to their nonhypoxic counterparts. This indicates that short-term exposure to moderate hypoxia on day 2, enhances angiogenesis from the established sprouts. Gene knockdown of HIF1α and/or HIF2α does not decrease the sprouting (Figure 1F), showing that the underlying mechanism is independent of HIFs. This is most likely due to the moderate hypoxic levels (O2 ~ 5%) achieved by the hydrogel platform since HIFs are proteolytically degraded between 6% and atmospheric O2. Mitochondrial ROS start to produce at ~5% O2 and have been proposed to orchestrate the initial response to acute hypoxia. Small-molecule inhibition of ROS decreased sprouting, indicating that ROS mediates the angiogenic response (Figure 1G). Inhibition of matrix metalloproteinases following induction of acute hypoxia decreased endothelial sprouts (Figure 1H), suggesting that matrix degradation facilitates angiogenesis following acute hypoxia. To summarize, we developed Gel-Dex hydrogels that support 3D sprouting from endothelial spheroids and introduce acute moderate hypoxia “on-demand” on the established vascular sprouts. We found that acute exposure to moderate hypoxia is sufficient to rapidly increases ROS production and subsequently activates MMPs. Elevated MMP expression facilitates efficient matrix degradation thus enhancing angiogenesis from the established sprouts (Figure 1I). On-going studies focus on delineating ROS-dependent protein targets that contribute to angiogenesis in response to acute hypoxia.

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