Collagen I hydrogel microstructure and composition conjointly regulate vascular network formation

Abstract

Neovascularization is a hallmark of physiological and pathological tissue remodeling that is regulated in part by the extracellular matrix (ECM). Collagen I hydrogels or Matrigel are frequently used to study vascular network formation; however, in isolation these materials do not typically mimic the integrated effects of ECM structure and composition that may influence endothelial cells in vivo. Here, we have utilized microfabricated 3D culture models to control collagen I microstructure in the presence and absence of Matrigel and tested the effect of these variations on vascular network formation by human cerebral microvascular endothelial cells (hCMECs). Varied collagen microarchitecture was achieved by adjusting the gelation temperature and subsequently confirmed by structural analysis. Casting at colder temperature increased collagen fiber thickness and length, and inclusion of Matrigel further pronounced these differences. Interestingly, the presence of Matrigel affected vascular network formation by modulating hCMEC growth, whereas altered collagen fiber structure impacted the morphology and maturity of the developed vascular network. These differences were related to substrate-dependent changes in interleukin-8 (IL-8) secretion and were functionally relevant as vascular networks preformed in more fibrillar, Matrigel-containing hydrogels promoted angiogenic sprouting. Our studies indicate that collagen hydrogel microstructure and composition conjointly regulate vascular network formation with implications for translational and basic science approaches.

Statement of significance: Neovascularization is a hallmark of both tissue homeostasis and disease and is in part regulated by cell remodeling that occurs in the extracellular matrix (ECM). The use of bio-mimetic hydrogel cell culture systems has been used to study the effects of the ECM on cell behavior. Here, we employ a hydrogel system that enables control over both the structure and composition of the ECM and subsequently investigated the effects that these have on blood vessel dynamics. Finally, we linked these differences to changes in protein secretion and the implications that this may play in scientific translation.

Keywords: Endothelial cells; Extracellular matrix; Interleukin-8; Microenvironment; Vascularization.

Figure 1. Experimental design

a) Microwells were fabricated by polymerizing poly(dimethylsiloxane) (PDMS) on photolithographically etched wafers and were individually punched out and transferred into conventional 24-well culture plates. b) Type-1 collagen was cast into the microwells with (M) and without (NM) Matrigel and allowed to crosslink either in an ice bath (cold cast [CC], 4°C) or in an incubator (warm cast [WC], 37°C). For all cell experiments, human endothelial cells were suspended in the different hydrogels prior to gelation, while characterization experiments were performed with cell-free gels.

Figure 2. Hydrogel composition and casting conditions impact collagen fibrillar structure

a) Confocal Reflectance and b) Scanning Electron Microscopy micrographs of cell-free hydrogels prepared by cold (CC) or warm (WC) casting in the presence (M) or absence (NM) of Matrigel. Scale bars are 50 μm and 1 μm, respectively. Image analysis of SEM micrographs indicated that collagen fiber c) length and d) diameter increases with decreased casting temperature; effects were enhanced in Matrigel-containing hydrogels. e) Changes in matrix architecture due to varied hydrogel composition and casting conditions were maintained in the presence of hCMECs. Blue is DAPI and red is phalloidin. Scale bars are 50 μm. f) Quantification of cell number by image analysis revealed that presence of Matrigel enhances hCMEC growth in hydrogels more prominently than altered microstructure. P-values less than 0.001 were labeled with *** when comparing CC vs. WC or °°° when comparing M vs. NM.

Figure 3. Collagen hydrogel structure and composition influence 3D endothelial network assembly

a) Confocal micrographs of hCMEC cultures suggest that hydrogel composition and casting conditions influence vasculogenic network assembly. Blue is DAPI and red is phalloidin. Scale bars are 50 μm. b) Image analysis of confocal micrographs indicates that vascular branches are thicker in Matrigel-containing hydrogels and that gels prepared by cold-casting further increase this effect. c) Orthographic re-slicing of confocal micrographs demonstrates that thick vascular branches contain lumens. Blue is DAPI and red is phalloidin. Scale bar is 50μm. d) Image analysis reveals that vascular cords in Matrigel-containing, cold-cast hydrogels are more branched than in all other conditions. e) Similar differences in morphology and f) vascular branch cross sectional area were observed with HUVECs. Blue is DAPI and red is phalloidin. Scale bars are 50 μm. P-values less than 0.05 and 0.001 were labeled with * and *** when comparing CC vs. WC and p-values less than 0.05 and 0.001 were labeled with ° and °°° when comparing M vs. NM.

Figure 4. Collagen hydrogel structure and composition affect collagen IV deposition

a) Confocal micrographs and corresponding heat maps as well as b) image analysis suggest that vascular structures formed in cold-cast, Matrigel-containing hydrogels deposit more type-IV collagen relative to all other conditions. Scale bars are 50 μm. P-values less than 0.05 and 0.0001 were labeled with * and **** when comparing CC vs. WC and p-values less than 0.05, 0.001, and 0.0001 were labeled with or °, °°°, and °°°° when comparing M vs. NM.

Figure 5. Hydrogel-dependent changes of vasculogenesis impact subsequent endothelial cell invasion and anastomosis

a) Schematic depicting the experimental design of invasion experiments in blank and pre-vascularized collagen hydrogels. A monolayer of mCherry-labeled hCMECs was seeded on top of hydrogels that were either pre-vascularized with unlabeled hCMECs for 11 days or were left blank. b) Confocal micrographs show both individually invading endothelial cells (labeled with *) and anastomosis between labeled and unlabeled endothelial cells (labeled with arrow). Blue is DAPI, red is mCherry, and green is phalloidin. Scale bars are 50 μm. c) Confocal re-slices suggest that invasion of mCherry+ endothelial cells into the pre-vascularized vs. blank bulk is significantly enhanced in the presence of Matrigel. Blue is DAPI, red is mCherry, and green is type-IV collagen. Scale bar is 50 μm. d) Confocal image analysis confirmed that invasion from the endothelial monolayer was increased in pre-vascularized cultures, irrespective of condition. P-values less than 0.001 and 0.0001 were labeled with *** and ****. e) Quantification of the number of invaded mCherry+ endothelial cells and f) mCherry+ fluorescence intensity distribution supports these findings and further suggests that vascular invasion into pre-vascularized cultures is enhanced in cold-cast gels. P-values less than 0.05 were labeled with * when comparing CC vs. WC and p-values less than 0.01 and 0.001 were labeled with °° and °°° when comparing M vs. NM.

Figure 6. Hydrogel structure and composition influence vasculogenesis and subsequent anastomosis through varying IL-8 secretion

a) ELISA indicates that hCMECs secrete enhanced levels of IL-8 in the presence of Matrigel, and that cold-casting enhances IL-8 secretion in collagen gels without Matrigel. ELISA also confirms that Matrigel alone contains no significant levels of IL-8. P-values less than 0.05 were labeled with * when comparing CC vs. WC and p-values less than 0.01 and 0.001 were labeled with °° and °°° when comparing M vs. NM. b) Inhibition of IL-8 with a function-blocking antibody decreased vascular cross sectional area in Matrigel conditions. P-values less than 0.05 and 0.0001 were labeled with * and **** when comparing Control vs. Anti-IL-8. c) Confocal micrographs indicate that IL-8 inhibition completely disrupts vascular network assembly in Matrigel-containing, cold-cast hydrogels, while having a diminished effect in all other conditions. Blue is DAPI and red is phalloidin. Scale bars are 50 μm. d) Inhibition of IL-8 reduces vascular branching in Matrigel-containing, cold-cast hydrogels to similar levels as in Matrigel-containing, warm-cast hydrogels. P-values less than 0.001 were labeled with *** when comparing Control vs. Anti-IL-8.

Figure 7. Inhibition of IL-8 decreases endothelial cell invasion and anastomosis

a) Analysis of endothelial cell invasion according to Fig. 5a reveals that blockade of IL-8 during vasculogenic network assembly significantly reduces subsequent endothelial cell invasion from an adjacent endothelial monolayer; this effect was more pronounced in Matrigel-containing vs. Matrigel-free, cold-cast hydrogels. P-values less than 0.001 and 0.0001 were labeled with *** and **** when comparing Pre-vascularized, Control vs. Pre-vascularized, Anti-IL-8. b) IL-8 inhibition also diminishes integration of mCherry+ into the preformed vascular networks regardless of condition. Blue is DAPI, red is mCherry, and green is phalloidin. Scale bars are 100 μm.