(B-54) “On-Demand” Hypoxic Hydrogels Reveal the Role of Mitochondria in Angiogenesis

Introduction:: Changes in oxygen (O₂) tension, mainly hypoxia (< 5% O₂), regulate cell differentiation and tissue morphogenesis. Indeed, most studies focus on understanding how cells respond to different O₂ microenvironments and how those impact differentiation and tissue formation. Mitochondria are subcellular organelles that play a pivotal role in a myriad of fundamental cellular processes. They undergo coordinated cycles of fusion and fission to maintain their morphology and distribution. In endothelial cells, mitochondria are crucial for cell signaling, dictating the vascular response to changes in the surrounding microenvironment, including hypoxia. However, hypoxia occurs with disease development such as cancer and atherosclerosis, impacting blood vessel phenotype and function. Moreover, occasions of rapid drop in oxygen, such as in extreme environments or elevation, could have devastating effects on tissue function and viability. Thus, it is essential to understand how hypoxia affects tissues rather than individual cells. In vitro systems that can impose controlled hypoxia on the 3D tissue are lacking. Here, we report the fabrication of a hydrogel system that can introduce “on-demand” hypoxia in already established tissue, the vasculature. The use of this “on-demand” hypoxic hydrogel could lay the foundation to uncover the molecular mechanisms by which the vasculature senses and responds to hypoxia.

Materials and Methods:: The “on-demand” hypoxic hydrogel is fabricated by mixing catechol-modified hyaluronic acid with collagen I (Col-HA). Catechol-modified hyaluronic acid is synthesized using a two-step process. We initially introduce aldehyde groups on the HA backbone by using sodium periodate oxidation overnight. Following this, dopamine is conjugated on the di-aldehyde groups of oxidized HA through a Schiff base reaction. The degree of substitution of catechol groups of HA was ~20% as determined by ¹H NMR. Endothelial colony-forming cells (ECFCs) are encapsulated in Col-HA and cultured in ECGM media supplemented with VEGF for 3 days. Once vasculature is established, we introduce laccase to the cell media, which dimerizes the catechol molecules through an oxygen-consuming reaction. We monitor the partial pressure of oxygen continuously at the bottom of the hydrogels using non-invasive sensor patches. We track and analyze the live mitochondria in the engineered microvasculature using the Mitometer algorithm. Mitochondria are stained prior to ECFC encapsulation and tracked on day 3, before and after the hypoxic exposure. The vascular networks are fixed and analyzed on day 4. For network analysis, z-stacks of 200 μm are taken and analyzed using the Imaris Filament tracer software.


Results, Conclusions, and Discussions:: Results and Discussion: We allowed ECFCs to assemble 3D vascular networks for 3 days (Fig. 1A) while continuously monitoring the dissolved O₂ levels in Col-HA using non-invasive sensor patches. We introduced laccase to the system, exposing the established vasculature to a rapidly generated hypoxic microenvironment for 6 hours (Fig. 1B). Networks were fixed, stained, and analyzed on day 4. We observed an increase in average and total vessel length and volume (Fig. 1C), suggesting that the acute decrease in O₂ promotes angiogenesis in the pre-existing capillaries. We then hypothesized that mitochondria sense the rapid change in O₂ and guide the formation of new vessels. We found that the “on-demand” hypoxic microenvironment triggered a change in mitochondrial morphology, promoting rounder mitochondria (Fig. 1D). We further observed that acute hypoxia reduced the frequency of mitochondrial fusion-to-fission events in the engineered microvessels (Fig. 1E), suggesting that mitochondrial dynamics may have an oxygen-sensing role in the vasculature.



Conclusions: We engineered an in vitro hydrogel system capable of supporting 3D tissue and imposing “on-demand” hypoxia. Our hydrogel platform initially facilitates the formation of vascular networks, followed by the controlled consumption of oxygen, leading to hypoxic microenvironment around the established vascular networks. Using the Col-HA system, we show that short-term hypoxia impacts the vascular phenotype, promoting an angiogenic response. We employed live-cell imaging and tracking of mitochondria to decipher their role in the oxygen-sensing machinery of the vasculature. Our results indicate that mitochondria may act as mediators of the increased angiogenesis observed under acute hypoxia. On-going studies focus on scrutinizing the role of mitochondria as potential oxygen sensors in the vasculature.


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