The induction of pro‐angiogenic processes within a collagen scaffold via exogenous estradiol and endometrial epithelial cells

Abstract

Nutrient transport remains a major limitation in the design of biomaterials. One approach to overcome this constraint is to incorporate features to induce angiogenesis-mediated microvasculature formation. Angiogenesis requires a temporal presentation of both pro- and anti-angiogenic factors to achieve stable vasculature, leading to increasingly complex biomaterial design scheme. The endometrium, the lining of the uterus and site of embryo implantation, exemplifies a non-pathological model of rapid growth, shedding, and re-growth of dense vascular networks regulated by the dynamic actions of estradiol and progesterone. In this study, we examined the individual and combined response of endometrial epithelial cells and human umbilical vein endothelial cells to exogenous estradiol within a three-dimensional collagen scaffold. While endothelial cells did not respond to exogenous estradiol, estradiol directly stimulated endometrial epithelial cell transduction pathways and resulted in dose-dependent increases in endogenous VEGF production. Co-culture experiments using conditioned media demonstrated estradiol stimulation of endometrial epithelial cells can induce functional changes in endothelial cells within the collagen biomaterial. We also report the effect of direct endometrial epithelial and endothelial co-culture as well as covalent immobilization of estradiol within the collagen biomaterial. These efforts establish the suitability of an endometrial-inspired model for promoting pro-angiogenic events within regenerative medicine applications. These results also suggest the potential for developing biomaterial-based models of the endometrium.

Proangiogenic Activity of Endometrial Epithelial and Stromal Cells in Response to Estradiol in Gelatin Hydrogels

“…This project builds on previous effort by our laboratories where we reported the effects of estradiol on proangiogenic signaling on cocultures of endometrial epithelial and nonendometrial endothelial cells in a model collagen scaffold. [13] While vessel supporting cells direct angiogenesis through many means, we chose to focus primarily on how hormones affect angiogenic gene expression and VEGF production to parallel the angiogenic factor supplementation used in other angiogenic biomaterial research. [18] A primary motivator of the transition from a collagen-based scaffold with pores of order 150 μm in size to a (gelatin-based) hydrogel was the ability to rapidly visualize de novo formation of vessel networks (as an early marker of angiogenesis) within the hydrogel (4–7 d), while similar studies in porous collagen scaffolds can take significantly longer (order weeks).…”

“…It is possible that the nuanced effects of E2 and P4 may have been overwhelmed by other proangiogenic signals, specifically heightened levels of hypoxia within the hydrogel due to both its three-dimensional nature [23] as well as the stromal cell-mediated hydrogel contraction which could heightened regional levels of hypoxia. Though E2 supplementation was sufficient to induce a response in open-cell collagen scaffolds, [13] ongoing efforts are exploring an expanded dose range using hydrogels more resistant to contraction-based remodeling.…”

“…[25] Additionally, we also previously reported that E2 supplementation of endometrial adenocarcinoma cells increased VEGF production in a collagen scaffolds. [13] The potential that hydrogel-induced changes in hypoxia may mask the effects of hormone signals at the dosages tested suggests a need to further optimize the GelMA hydrogel for endometrial cell culture such as changes in macromere and photoinitiator concentration in order to test a wider library of hydrogel environments. [14a,b] Given the known significance of hypoxia on vascular network formation as well as our demonstration of SHBG-based approaches to increase the bioavailability of estradiol within the hydrogel, ongoing efforts are exploring the coordinated effect of hypoxia (1% O 2 ) and the presence of enhanced E2 availability on proangiogenic signals, notably proteases and secreted biomolecules.…”

“…We had previously shown that endoEpCs in 3D biomaterial culture produce endogenous VEGF in response to E2 that was sufficient to enhance the activity of endothelial cultures. [13] While endoEpCs were found to alter angiogenic gene expression to hormone supplementation, the addition of endoEpCs to generate tricultures with endothelial cells and endoSCs led to diminished metrics of vessel network formation. These findings suggest that while endoEpCs are capable of generating proangiogenic factors in response to exogenous E2 stimulation, direct coculture of endometrial epithelial cells and endothelial cells is detrimental.…”

“…Recent efforts in our lab showed that soluble estradiol was sufficient to induce VEGF secretion by endometrial epithelial cells at a level sufficient to induce changes in human umbilical vein endothelial cells (HUVECs) metabolic activity in a model collagen scaffold. [13] The open pore architecture of the collagen scaffold made it difficult to monitor subsequent vessel formation events. Here, we describe the use of estradiol and progesterone to alter the individual and combined proangiogenic activities of endometrial epithelial and stromal cells (endoEpCs and endoSCs, respectively), as well as subsequent endothelial cell vessel formation, within a methacrylamide-functionalized gelatin (GelMA) hydrogel recently described by our laboratory.…”