Human Aortic Adventitia Fibroblasts (HAAF)

To study the role of matrix stiffness on human aortic adventitial fibroblast (HAoAF) fate, 3D cell-laden hydrogels were formed under physiological conditions via a Michael-type addition reaction using thiol end-functionalized PEG macromers (PEG-SH4) and bis-maleimide end-functionalized, MMP-sensitive crosslinker peptides (PQ-MI2) to obtain highly elastic hydrogels that accommodated cell-mediated remodeling of the matrix via protease degradation (Fig. 1). As collagen is the predominant structural and cell-adhesive protein found in arterial adventitia and a key mediator of HAoAF activities, 1 mM mono-maleimide pendent RGD peptide (RGD-MI), an adhesive motif found within collagen, was incorporated into the polymer matrix to facilitate HAoAF interactions with the hydrogel network.

Fig. 1. Reaction scheme for enzymatically degradable hydrogel formation (Scott, Rebecca A., et al., 2020).

We next sought to determine whether differences in initial matrix stiffness provoked HAoAF activation just after culture initiation by assessing cell proliferation and secretion of key cytokines, including the angiogenic growth factor vascular endothelial growth factor (VEGF-A), and the inflammatory cytokines interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). Elevated proliferation was observed in soft 5wt% hydrogels in the initial 7 days following encapsulation and proliferation decreased as hydrogel stiffness increased (Fig. 2A), similar to previous reports with fibroblasts in 3D. We further observed that elevated HAoAF proliferation in 5wt% hydrogels corresponded with enhanced production of the angiogenic growth factor, VEGF-A (detected via enzyme-linked immunosorbent assay (ELISA)), compared to 7.5wt% and 10wt% hydrogels (Fig. 3), which is consistent with in vivo reports where increased adventitial cell proliferation corresponded to increased VEGF-A production.

Fig. 2. (A) The proliferation rate of HAoAF encapsulated in 5wt%, 7.5wt%, and 10wt% hydrogels significantly decreased with increasing weight percent over 7 days. (B-C) The percentage of αSMA+ HAoAF was significantly increased in 10wt% hydrogels compared to softer 5wt% and 7.5wt% substrates after 1 day, as confirmed by immunocytochemistry (Scott, Rebecca A., et al., 2020). Blue: Hoechst 33258 (nuclei), Grey: F-actin, Red: αSMA.

Under physiologic and pathophysiologic conditions, alterations to the adventitia are often accompanied by the appearance of proliferative cells expressing α-smooth muscle actin (αSMA), a marker of fibroblast to myofibroblast transition, and increased matrix deposition. Though HAoAF in the 5wt% hydrogels exhibited enhanced proliferation, immunostaining revealed that few cells (<20%) expressed αSMA in 5wt% and 7.5wt% hydrogels 24 hrs post-encapsulation (Fig. 2B-C). On the other hand, ~50% of HAoAF in the stiffer 10wt% hydrogels expressed diffuse αSMA staining after 24 hrs. While our results differ from previous observations in 3D hydrogels, where fibroblast activation decreased with increased stiffness, they are consistent with in vivo observations of pathologic tissue stiffening driving myofibroblast trans-differentiation. Further, αSMA expression in 10wt% hydrogels was accompanied by the secretion of the inflammatory proteins, IL-6 and MCP-1 (measured via ELISA; Fig. 3B-C). Interestingly, significantly increased IL-6 and MCP-1 production by HAoAF encapsulated in 5wt% hydrogels, compared to 7.5wt% hydrogels, suggests that inflammatory cytokine production by activated HAoAF may be regulated through pathways distinct from those driving myofibroblast trans-differentiation.

Fig. 3. HAoAF secretion of (A) VEGF-A, (B) IL-6, and (C) MCP-1, following culture for 1 day in 5wt%, 7.5wt%, and 10wt% hydrogels (Scott, Rebecca A., et al., 2020).

Within native tissues and tissue mimetic substrates, cells actively respond to their surroundings by synthesizing and secreting ECM proteins. Accordingly, we assessed the distribution of collagen I and collagen III, which are the main collagen constituents in the adventitia. Following encapsulation, collagen I and collagen III immunofluorescent staining was primarily localized to the intracellular or pericellular regions in all cultures (Fig. 4D-E). Quantification of collagen levels by corrected total cell fluorescence (CTCF) revealed that collagen I expression was increased among HAoAF in 5wt% hydrogels after 1 day, but then decreased as hydrogel stiffness increased (Fig. 4F). Collagen III expression was also increased in 5wt% hydrogels, compared to cells in 7.5% and 10wt% hydrogels (Fig. 4G). Overall, these results are in agreement with previous findings where substrate stiffness regulates ECM protein production, and support the notion that initial hydrogel conditions strongly impact the composition of the secreted ECM.

Fig. 4. (D-E) HAoAF produced collagen I or collagen III, detected via immunocytochemistry, following encapsulation in 5wt%, 7.5wt%, and 10wt% hydrogels for 1 day. Blue: Hoechst 33258 (nuclei), Grey: F-actin, Green: collagen I or collagen III. (F-G) Expression of collagen I and collagen III expression, measured by corrected total cell fluorescence, decreased with increasing weight percent (Scott, Rebecca A., et al., 2020).