Regulation of neovasculogenesis in co-cultures of aortic adventitial fibroblasts and microvascular endothelial cells by cell-cell interactions and TGF-β/ALK5 signaling

Adventitial fibroblasts (AFs) are critical mediators of vascular remodeling. However, the contributions of AFs towards development of vasculature and the specific mechanisms by which these cells regulate physiological expansion of the vasa vasorum, the specialized microvasculature that supplies nutrients to the vascular wall, are not well understood. To determine the regulatory role of AFs in microvascular endothelial cell (MVEC) neovasculogenesis and to investigate the regulatory pathways utilized for communication between the two cell types, AFs and MVECs were cultured together in poly(ethylene glycol)-based hydrogels. Following preliminary evaluation of a set of cell adhesion peptides (AG10, AG73, A2G78, YIGSR, RGD), 7.5wt% hydrogels containing 3 mM RGD were selected as these substrates did not initiate primitive tubule structures in 3D MVEC monocultures, thus providing a passive platform to study AF-MVEC interaction. The addition of AFs to hydrogels promoted MVEC viability; however, increasing AF density within hydrogels stimulated MVEC proliferation, increased microvessel density and size, and enhanced deposition of basement membrane proteins, collagen IV and laminin. Importantly, AF-MVEC communication through the transforming growth factor beta (TGF-β)/activin receptor-like kinase 5 (ALK5) signaling pathway was observed to mediate microvessel formation, as inhibition of ALK5 significantly decreased MVEC proliferation, microvessel formation, mural cell recruitment, and basement membrane production. These data indicate that AFs regulate MVEC neovasculogenesis and suggest that therapeutics targeting the TGF-β/ALK5 pathway may be useful for regulation of vasculogenic and anti-vasculogenic responses.

comprised 3 mM PEG-SH4, 2 mM PEG-MI4, and 1 mM RGD-MI. Hydrogels without RGD-MI were prepared at a concentration of 5wt%, crosslinked at a thiol to maleimide stoichiometric ratio of 3:2, and comprised 3 mM PEG-SH4 and 2 mM PEG-MI4. To form hydrogels, PEG-SH4, PEG-MI4, and RGD-MI were independently dissolved in buffer (10 mM citrate buffer, pH 4.5) and sterilized by passage through a 0.2 μm nonpyrogenic PVDF filter. Maleimide-and thiol-containing solutions were then mixed together via pipetting. Once mixed via gentle pipetting, 50 L volumes of hydrogel were added to the surface of a 96-well plate, allowed to crosslink for 20 mins at room temperature, and incubated in HBSS for 2 hrs at room temperature. Following removal of HBSS, hydrogels were washed once with EGM2-MV, and MVECs were seeded on the surface of 50 μL of hydrogel in 96-well plates at a density of 5,000 cells per well in 200 μL of medium and incubated at 37°C with 5% CO2. At specific time points, cells were visualized using an EVOS ® FL Auto Imaging System with a 10x UPlanFL 0.3 N.A. objective, controlled by EVOS ® FL Auto software (version 1.6; ThermoFisher), to assess cell attachment.

Assessment of ALK5 Signaling by Cells in 2D Culture:
In studies where TGF-β superfamily pathways were investigated, MVECs or AoAFs were independently seeded at a density of 1x10 4 cells/cm 2 in a 48 well plate (200 L co-culture medium per well) and allowed to adhere for 24 hrs.
Medium was removed and the PrestoBlue® assay was performed, as described above, to establish a baseline of cell numbers. Thereafter, cells were immersed in co-culture medium supplemented with A83-01 (0.5 or 5 M) or DMSO (control) and incubated for an additional 72 hrs at 37°C and 5% CO2. The PrestoBlue® assay was then repeated and the change in signal was used as an indicator of increased cell numbers over time. In a tandem set of experiments, MVECs or AoAFs were seeded and cultured with A83-01 or DMSO as described above, but not subjected to the PrestoBlue® assay. After culture with A83-01 or DMSO for 72 hrs, cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) in preparation for immunostaining.
Immunostaining of 2D cultures: For assessment of 2D monocultures, MVECs and AoAFs were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 30 mins, permeabilized with 0.1% Triton X-100 for 15 min, and blocked with 3% bovine serum albumin in PBS for 30 min. Antibodies are listed in S1 Table. Samples were stained for 1 hr at room temperature with primary antibodies with shaking, washed with PBS, and incubated with secondary antibodies, Phalloidin-568 (1:500), and the nuclear stain Hoechst 33258 (1:1000) for 1 hr at room temperature with shaking. Cells were visualized using an EVOS ® FL Auto Imaging System with a 20x LPlanFL 0.4 N.A. objective, controlled by EVOS ® FL Auto software (version 1.6; ThermoFisher). Negative controls (e.g. cells not exposed to primary antibodies but incubated with secondaries) were used to validate immunofluorescence signals.

MMP-2 Quantification: Conditioned medium was collected and stored at -80°C until analysis.
MMP-2 levels in the medium collected from mono-and co-cultures were quantified via the human MMP-2 Quantikine ELISA kit (R&D Systems, Minneapolis, MN). All assays were used in accordance with the manufacturers' instructions.
To create cell-laden degradable hydrogels, cells were suspended in 10 mM HEPES in HBSS and mixed via gentle pipetting into the solution of hydrogel precursor polymers prior to crosslinking.
For 7.5wt% hydrogels with 1 mM RGD-MI or 15wt% hydrogels with 3 mM RGD-MI, MVECs were encapsulated to achieve 3x10 6 MVECs/mL hydrogel and AoAFs were encapsulated to achieve 3x10 6 AoAFs/mL hydrogel. Once mixed via gentle pipetting, 10 L volumes of hydrogel were added to the surface of an 8 well Nunc TM Lab-Tek TM chambered coverglass and allowed to crosslink for 20 mins at room temperature. Hydrogels were then immersed in co-culture medium (400 L) and incubated at 37°C with 5% CO2. Medium was replaced every 3 days over the duration of these experiments.