Complex temporal regulation of capillary morphogenesis by fibroblasts

Abstract

Interactions between endothelial and stromal cells are important for vascularization of regenerating tissue. Fibroblasts (FBs) are responsible for expression of angiogenic growth factors and matrix metalloproteinases, as well as collagen deposition and fibrotic myocardial remodeling. Recently, self-assembling peptide nanofibers were described as a promising environment for cardiac regeneration due to its synthetic nature and control over physiochemical properties. In this study, peptide nanofibers were used as a model system to quantify the dual role of fibroblasts in mediating angiogenesis chemically via expression of angiogenic factors and mechanically via cell-mediated scaffold disruption, extracellular matrix deposition, and remodeling. Human microvascular endothelial cells (ECs), FBs, or cocultures were cultured in three-dimensional nanofibers for up to 6 days. The peptide nanofiber microenvironment supported cell migration, capillary network formation, and cell survival in the absence of detectable scaffold contraction and proteolytic degradation. FBs enhanced early capillary network formation by “assisting” EC migration and increasing vascular endothelial growth factor and Angiopoietin-1 expression in a temporal manner. EC-FB interactions attenuated FB matrix metalloproteinase-2 expression while increasing collagen I deposition, resulting in greater construct stiffness and a more stable microenvironment in cocultures. Whereas FBs are critical for initial steps of angiogenesis in the absence of external angiogenic stimulation, coordinated efforts by ECs and FBs are required for a balance between cell-mediated scaffold disruption, extracellular matrix deposition, and remodeling at later time points. The findings of this study also emphasize the importance of developing a microenvironment that supports cell-cell interactions and cell migration, thus contributing toward an optimal environment for successful cardiac regeneration strategies.

tissue engineering aims to combine the principles of biology and engineering to develop functional tissue substitutes (28). Thus far, one major limitation has been insufficient vascularization of engineered tissues, which is essential for the supply of oxygen, nutrients, and immune cells as well as the removal of cellular by-products and waste. This is particularly important in cardiac tissue engineering approaches, where recent studies have demonstrated the feasibility of engineering functional cardiac muscle in vitro, with promising results after graft implantation in animal models (26, 29, 37, 41, 53). These studies indicate that revascularization of an engineered graft is critical for successful graft implantation and regeneration of ischemic cardiac tissue. However, vascularization due to blood vessel in growth is usually insufficient (37, 41, 51) and scaffold prevascularization (14, 29) results in limited cell survival. Therefore, developing engineering strategies that promote vascularization is one of the key requirements for successful graft implantation and myocardial regeneration.

To develop novel strategies to promote vascularization in engineered cardiac grafts, it is necessary to understand the interactions between the major cardiac cell types. Normal mammalian myocardium has a complex cellular organization, where three major cell types [cardiomyocytes, endothelial cells (ECs), and cardiac fibroblasts (FBs)] are arranged in an intricate spatial network and communicate constantly. The importance of cardiomyocyte-endothelial interactions in angiogenesis has been previously documented (11, 35). However, less is known about interactions between ECs and stromal cells, cardiac FBs in particular.

To study such cell-cell interactions, in vitro coculture assays have been commonly used to represent angiogenesis in vivo (13), with many recent studies applying various three-dimensional systems (including collagen I, fibrin, Matrigel, and scaffold-free approaches) (6, 10, 12, 19, 27, 44, 47). Such in vitro studies have demonstrated that the formation of capillary structures is enhanced by the presence of FBs, with increased endothelial sprouting and migration and decreased endothelial apoptosis (12, 27, 47). Additionally, studies indicate that during capillary assembly, FBs provide chemical signaling via expression of angiogenic factors (22, 23), including vascular endothelial growth factor (VEGF) (5, 18, 47), angiopoietin-1 (33, 47), stromal cell-derived factor-1 (36), hepatocyte growth factor (31, 48), interleukin-8 (1, 39), transforming growth factor β (7, 17), and matrix metalloproteinases (MMPs) (9, 45). Studies also suggest that FBs can regulate angiogenesis by changing the mechanical extracellular microenvironment via matrix deposition and metalloproteinase-mediated extracellular matrix (ECM) remodeling (9, 45). However, in addition to pro-angiogenic effects of ECM remodeling shown in vitro (6, 19, 44), FBs are also the major contributors to excessive fibrotic myocardial remodeling in vivo, which can lead to detrimental collagen matrix deposition and contraction, increased cardiac stiffness, lack of vasculature, and ultimately heart failure (15, 24, 46). Overall, these findings suggest a possible dual role (chemical and mechanical) for FBs during the process of vasculature assembly and remodeling in the environment of healing tissue, such as in the regenerating heart. However, our knowledge of these processes remains incomplete. In particular, the effects of fibroblast-endothelial interactions on the mechanical microenvironment and how these interactions can affect angiogenesis are not well understood. This is partially due to the limitations of the experimental systems used for in vitro studies of angiogenesis. Native systems such as collagen, fibrin, and Matrigel are subject to excessive or uncontrolled scaffold contraction, proteolytic degradation, and often require the addition of external growth factors to promote in vitro angiogenesis, all of which can be hurdles for in vitro studies of FB-mediated angiogenic signaling. Therefore, a three-dimensional culture system, which mimics the native cell environment while uncoupling scaffold-triggered signaling from cell-cell interactions, can provide important insights into the role of endothelial-fibroblast interactions during the angiogenic process.

It has previously been shown that the scaffold made from synthetic RAD16-II peptide nanofibers provides an angiogenic microenvironment that enhances capillary network formation in vitro without the addition of external growth factors and allows for long-term study of cell-cell interactions (34, 42). Importantly, these biocompatible nanofibers can also serve as an angiogenic microenvironment and drug delivery tool for cardiac regeneration in vivo (11, 40) and therefore represent an appropriate in vitro system to study cell-cell interactions during angiogenesis for cardiac tissue engineering applications. Specific properties of this system, including the absence of significant cell-induced scaffold contraction and resistance to proteolysis due to lack of MMP degradation sites (52), may allow for the uncoupling of chemical signaling due to cell-scaffold interactions from cell-cell signaling, thus enabling quantitation of changes in cell behavior and protein expression not masked by scaffold-induced signaling associated with scaffold degradation. Therefore, the goal of this study was to elucidate the mechanisms of temporal regulation of the angiogenic process by FBs using a comprehensive approach and the culture system of RAD16-II nanofibers. We tested the hypothesis that FBs regulate capillary morphogenesis chemically via growth factor expression and mechanically via cell-mediated scaffold disruption, ECM deposition, and remodeling in a temporal manner. Our results suggest that at the early stages of tissue repair process, FBs may play a major role in capillary morphogenesis via both paracrine growth factor signaling and mechanical disruption of ECM to “lead the way” for the formation of EC networks. At the later stages, the role of FBs as regulators of the mechanical microenvironment becomes more prominent. Interestingly, endothelial-fibroblast interactions appeared to help maintain the balance in ECM homeostasis, enhancing both MMP2 and collagen I production and resulting in improved integrity and a stable microenvironment by day 6, as demonstrated by the rheometry analyses.

MATERIALS AND METHODS

Cell culture.

Human microvascular ECs (Cascade Biologics, Portland, OR) and human dermal FBs (Cascade Biologics) were cultured in medium 199 (HyClone, Logan, UT) containing 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), 1% antibiotic-antimycotic (Atlanta Biologicals), 10 μg/ml heparin (Sigma-Aldrich, St. Louis, MO), and 0.2 ng/ml growth supplement (Sigma-Aldrich). Cell cultures were maintained at 37°C in 100% humidified air containing 5% CO₂. Cells of passage 4–12 were used in all experiments.

Experimental groups.

Experimental groups included EC (endothelial cells only), FB (fibroblasts only), and EC+FB (endothelial cells and fibroblasts in a 1:1 ratio).

Sample preparation.

For all experiments, a three-dimensional nanoscaffold made from RAD16-II peptide nanofibers (RARADADARARADADA; SynBioSci, Livermore, CA) served as a controlled microenvironment. In network formation and cell proliferation experiments, cells were surface seeded on 10 mg/ml peptide nanofibers in culture plate inserts (13 mm diameter, 0.4-μm pore size; Millipore, Billerica, MA) at a cell seeding density of 1.0 × 10⁵ cells/cm². For ELISA experiments, cells were embedded in 10 mg/ml peptide nanofibers in culture plate inserts at a density of 5.0 × 10⁶ cells/ml and cultured up to 6 days with daily media changes. For mechanical testing of cell-scaffold constructs using rheometry, cells were embedded in 6 or 10 mg/ml peptide nanofibers at 2.5 × 10⁶ cells/ml and cultured for 1, 3, and 6 days with medium changes every 24 h. For analyses of cell apoptosis, samples were fixed and embedded in paraffin for staining at 3 and 6 days.

Capillary morphogenesis.

Before seeding was completed, ECs and FBs were labeled with CellTracker Dyes (Invitrogen, Carlsbad, CA). Cells were seeded on the nanofibers and cultured for 24 h to allow capillary network formation. After fixation with 2% paraformaldehyde, images were taken (n = 5 per sample) by using an inverted fluorescent microscope (Olympus IX81; Olympus America, Center Valley, PA). Correlation analysis with MATLAB (The MathWorks, Natick, MA) was used to characterize capillary endothelial network size as previously described (34). For three-dimensional network characterization, a built-in integrated three-dimensional imaging system was used (Plus Imaging System, Olympus), which included motorized Z-drive with 10 nanometer step size and imaging/analysis software (ImagePro, Media Cybernetics). Lumens were visualized using Z-stack images of cells stained with EC marker lectin (Vector Labs, Burlingame, CA) with a spacing of 0.8 μm between frames.

Cell proliferation.

CellTiter 96 Aqueous nonradioactive cell proliferation assay (Promega, Madison, WI) was used to assess cell viability and proliferation at days 1, 3, and 6 in culture. Cells were embedded in the nanofibers at a density of 5.0 × 10⁶ cells/ml and cultured with daily medium changes. At each time point, samples were incubated in medium containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)/phenazine methosulfate solution (PMS) solution for 3 h per manufacturer instructions. Media samples from culture inserts were placed in a 96-well plate, and absorbance was measured at 490 nm using an ELISA plate reader. All data were normalized to day 1 EC values for analyses. After testing was completed, MTS/PMS medium was aspirated, and fresh medium was added to the samples.

Cell apoptosis.

Cell-nanofiber constructs were fixed at days 3 and 6 and embedded in paraffin (Fisher Scientific, Pittsburgh, PA). Staining was performed on 5-μm sections. Staining with lectin (fluorescein ulex europaeus agglutinin I; Vector Labs, Burlingame, CA) was performed to identify ECs. Anti-ACTIVE Caspase-3 (Promega, Madison, WI) and DAPI (Invitrogen) staining was performed to assess cell death, and apoptotic cells were counted and compared with total cell numbers.

Sample protein content determination.

Cell-nanofiber constructs were cultured in no growth factor medium (cell culture medium without additional growth factor supplementation) and collected at day 6 and stored in TriReagent (Molecular Research Center, Cincinnati, OH) at −80°C until testing. Protein isolation was performed per the manufacturer's protocol. Total protein content in the samples was determined using Coomassie Plus Assay Kit (Thermo Fisher Scientific, Rockford, IL).

Protein content using enzyme-linked immunosorbent assay.

For all enzyme-linked immunosorbent assay (ELISA) experiments, cell culture medium (M199 with 10% FBS, 1% antibiotic-antimycotic, and 10 μg/ml heparin) without growth supplement was used with daily medium changes. Medium and matrix samples were collected at days 1, 3, and 6 and stored at −80°C until testing. ELISA kits (R&D Systems, Minneapolis, MN) were used per manufacturer's protocol to determine protein concentrations in medium samples (human VEGF, angiopoietin-1, total MMP9, and MMP2/TIMP2) and matrix samples (human VEGF and angiopoietin-1). Collagen I expression was quantified by using an ELISA for human collagen I in both medium and matrix samples, as described in Ref. 32 (human collagen I, antibodies, and substrates from Southern Biotechnology, Birmingham, AL). Protein expression in matrix samples was normalized by using total protein content. For all ELISAs, additional controls of the cell culture medium alone (containing 10% serum) were included to confirm that growth factors in the serum would not affect protein expression and detection, with no differences observed between medium samples and the 0 pg/ml standard.

Mechanical testing of cell-scaffold constructs using rheometry.

Elastic moduli (G′) of peptide nanofibers (6 or 10 mg/ml) with living cells or nanofibers alone (controls) at days 1, 3, and 6 were measured with a parallel-plate rheometer (Bohlin Instruments, East Brunswick, NJ). With the use of molds, circular constructs of 8-mm diameter and ∼500-μm height were formed on glass slides. The glass slides were covered with cell culture medium and cultured within an incubator with daily medium changes. For testing, glass slides were transferred and secured to the bottom plate of the rheometer. The top parallel plate was lowered to a gap height that ensured complete contact with the sample and a constant strain amplitude (γ = 0.01) frequency sweep (f = 0.1–10 Hz) was performed. The elastic modulus (G′) served as an indicator of overall cell-seeded construct stiffness, and moduli values measured at 0.1 Hz are reported in the text.

Statistical analysis.

The results are reported as means ± SD. Statistical comparisons between experimental groups were performed by using either two-way ANOVA or Student's t-test as appropriate. Additionally, multifactor ANOVA with post hoc tests with Bonferroni corrections (SPSS, SPSS, Chicago, IL) were used to test the effects of cell type, peptide concentration, and time in culture on the material properties of cell-nanofiber constructs. Results were considered statistically significant at P < 0.05. All experiments were performed in triplicates and repeated at least two times.

RESULTS

Endothelial-fibroblast cell migration regulates capillary morphogenesis.

Immediately after seeding was completed, both ECs and FBs attached and spread uniformly across the peptide nanofibers (Fig. 1A). By 24 h, coordinated migration of ECs and FBs was seen, with FBs surrounding the nascent endothelial networks and a distinct lack of cells outside of these networks (Fig. 1B). The appearance of the endothelial networks (Fig. 1B, green) in EC+FB cocultures was similar to the capillary morphogenesis in this material by ECs alone, as reported previously by Sieminski et al. (42) and by our group (34). Consistent with previous results (34, 35), Z-stack images of endothelial structures clearly show formation of hollow lumens (Fig. 1C). Interestingly, noticeable scaffold disruption likely resulting from extensive cell migration was observed in FB-containing constructs, with a lesser effect in EC-only samples. Correlation analysis of capillary networks (34) demonstrated that networks formed faster and were significantly larger (Fig. 1D) when ECs were cultured in the presence of FBs at all time points (P < 0.001). As early as 3.5 h, EC+FB cocultures were significantly larger than EC cultures (20.4 ± 4.7 vs. 12.7 ± 0.4 μm, respectively, P < 0.001). This trend continued and by 24 h, EC+FB networks remained significantly larger (42.6 ± 10.4 μm) when compared with EC-only networks (24.7 ± 4.5 μm, P < 0.001).

Fig. 1.

Capillary morphogenesis in endothelial and endothelial-fibroblast cultures on peptide nanofibers. A: uniform distribution of endothelial cells (ECs) (lectin, green) and fibroblasts (FBs) (CellTracker, orange) immediately (1 h) after cell seeding. Scale bar = 50 μm. B: at 24 h after cell seeding, coordinated cell migration and formation of multicell networks is seen in cocultures, with FBs (CellTracker, orange) surrounding the nascent EC (lectin, green) networks. Scale bar = 50 μm. C: 3-D reconstruction of Z-stack images of single endothelial structure from sample in B was used to visualize lumen formation. Hollow endothelial lumens were observed in (xy) plane (along the structure, bottom left) and through (xz) plane (through the structure, bottom right). Scale bar = 10 μm. D: characteristic size (microns) of capillary networks is significantly larger in endothelial-fibroblast cocultures (EC+FB) compared with EC cultures at 3.5, 6, 9, and 24 h. *P < 0.001 between EC and EC+FB at all time points.

The peptide nanofibers provide a stable microenvironment that supports long-term cell survival.

In contrast to significant cell-seeded construct contraction reported previously for collagen-containing gels, the peptide nanofibers experienced limited scaffold contraction in vitro with <15% contraction at day 7. This is in agreement with the values for RAD16-II peptide contraction reported previously (43) when compared with ∼70% for collagen I gel (43). To determine whether this stable microenvironment supports long-term cell survival, an MTS-based cell viability assay was performed on cells embedded in nanofibers and cultured up to 6 days. Results demonstrated that the viable cell number (Fig. 2A) remained at 80% or greater of the 225,000 total cells initially embedded within the peptide nanofibers, in culture medium containing 10% FBS with no additional growth factors. The percentage of viable cells was not significantly different between the experimental groups at days 3 and 6 (92 ± 3% and 93 ± 5% of EC+FB cocultures vs. 82 ± 6% and 80 ± 5% of ECs vs. 87 ± 11% and 97 ± 2% of FBs at days 3 and 6, respectively). Staining for caspase-3 showed no significant difference in cell apoptosis levels (Fig. 2B) in cells embedded in the peptide nanofibers (21 ± 13% and 24 ± 3% of EC+FB cocultures vs. 22 ± 7% and 20 ± 8% of ECs vs. 19 ± 4% and 18 ± 2% of FBs at days 3 and 6, respectively). Overall, these results indicate the balance between cell proliferation and apoptosis and an overall stability in the peptide nanofiber microenvironment.

Fig. 2.

Long-term cell viability and apoptosis levels in peptide nanofiber cultures. A: cell viability was measured using MTS-based viability assay. The number of viable cells in culture remained at 80% or greater of the 225,000 total cells initially seeded for all experimental groups up at days 3 and 6. B: cell apoptosis was measured by staining for caspase-3 positive cells. The percentage of apoptotic cells was 20–30% for all experimental groups at days 3 and 6.

FBs mediate capillary morphogenesis via modulating temporal profiles of VEGF and angiopoietin-1 expression.

VEGF is a major angiogenic stimulus and activation signal for endothelial migration and sprouting (23). Cellular VEGF expression in the sample medium was measured using ELISA (Fig. 3A). The results showed that VEGF levels in the medium were largely determined by FBs. At day 1, VEGF expression was significantly higher in EC+FB and FB cultures (362 ± 75 and 995 ± 153 pg/ml, respectively) compared with EC cultures (109 ± 38 pg/ml, P < 0.05). At day 3, VEGF expression in EC+FB and FB cultures (226 ± 58 and 470 ± 100 pg/ml, respectively) was significantly reduced from day 1 (P < 0.05) but still remained higher than expression by ECs (119 ± 42 pg/ml, P < 0.05). Beyond day 3, protein expression of soluble VEGF remained low in all experimental groups. Matrix-bound VEGF expression was also measured using ELISA (Fig. 3B). In EC cultures, matrix-bound VEGF levels at day 1 (0.142 ± 0.040 pg VEGF/μg total protein) and day 3 (0.109 ± 0.048 pg VEGF/μg total protein) were significantly higher than at day 6 (0.055 ± 0.021 pg VEGF/μg total protein, P < 0.05). Similarly, protein levels in FB cultures at day 1 (0.103 ± 0.005 pg VEGF/μg total protein) and day 3 (0.109 ± 0.019 pg VEGF/μg total protein) were significantly greater than levels at day 6 (0.057 ± 0.006 pg VEGF/μg total protein, P < 0.05). In EC+FB cocultures, a significant decrease in matrix-bound VEGF was observed from day 1 (0.111 ± 0.025 pg VEGF/μg total) to both day 3 (0.067 ± 0.024 pg VEGF/μg total protein, P < 0.05) and day 6 (0.055 ± 0.010 pg VEGF/μg total protein, P < 0.05). No significant difference in matrix-bound VEGF was observed among EC+FB, EC, and FB cultures at any time point.

Fig. 3.

Temporal profiles of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) expression in the medium and matrix bound. ELISA was performed on samples collected from cell-nanofiber constructs at days 1, 3, and 6 to determine concentration of VEGF (A and B) and Ang-1 (C and D). A: VEGF expression in the medium was significantly higher in FB cultures and EC+FB cultures compared with EC controls. Temporal decreases in VEGF expression in FB and EC+FB cultures were also observed. B: no significant difference in matrix-bound VEGF was observed among EC+FB, EC, and FB cultures at any time point. However, a temporal decrease in matrix-bound VEGF expression was observed in all experimental groups. C: Ang-1 protein expression in the medium was significantly higher in FB cultures (all time points) and EC+FB cultures (days 3 and 6) compared with EC controls. A temporal increase was also observed in Ang-1 expression in EC+FB, but not EC- or FB-only cultures. D: expression of matrix-bound Ang-1 was significantly lower at day 1 in FB and EC+FB cultures compared with EC controls; however, no significant differences were observed between the experimental groups at later time points. A temporal decrease in matrix-bound Ang-1 expression was observed in all experimental groups. *P < 0.05 compared with EC samples; +P < 0.05 compared with FB samples; #P < 0.05 compared with day 1 samples of same experimental group; ^P < 0.05 compared with day 3 samples of same experimental group.

Angiopoietin-1 (Ang-1) is an angiogenic factor that is implicated in stabilization of nascent capillaries (23). Cellular Ang-1 expression in the sample medium was measured using ELISA (Fig. 3C). Similar to the results for VEGF expression, the presence of FBs resulted in higher protein levels of Ang-1 when compared with endothelial-only constructs. However, the temporal profiles of Ang-1 expression were different from those for VEGF, consistent with different functions for VEGF and Ang-1 during angiogenesis as an angiogenic activation signal and capillary stabilization signal, respectively. At day 1, Ang-1 expression in FB cultures (828 ± 241 pg/ml) was significantly higher than expression in EC+FB cultures (375 ± 178 pg/ml) and EC cultures (322 ± 201 pg/ml, P < 0.05). Ang-1 expression increased from day 1 to day 3 and expression in FB and EC+FB cultures (2,965 ± 674 and 1,393 ± 312 pg/ml, respectively) remained significantly higher than expression in EC cultures (625 ± 211 pg/ml, P < 0.05). At later time points, Ang-1 expression was reduced in FB cultures and increased in EC+FB cultures, though both remained higher than EC cultures. ELISA was also used to measure matrix-bound Ang-1 levels (Fig. 3D). In EC cultures, no difference was observed in matrix-bound Ang-1 levels from day 1 (14.3 ± 2.0 pg Ang-1/μg total protein) to day 3 (14.2 ± 5.3 pg Ang-1/μg total protein); however, a significant decrease was observed from day 1 to day 6 (9.4 ± 1.8 pg Ang-1/μg total protein, P < 0.05). Similarly, no difference was observed in protein levels in FB cultures from day 1 (11.3 ± 0.4 pg Ang-1/μg total protein) to day 3 (12.0 ± 0.7 pg Ang-1/μg total protein), whereas levels at day 6 (8.7 ± 0.3 pg Ang-1/μg total protein, P < 0.05) were significantly decreased from day 1. The same trend was seen in EC+FB cocultures, where no difference was seen in matrix-bound Ang-1 levels from day 1 (10.6 ± 1.8 pg Ang-1/μg total protein) to day 3 (13.0 ± 6.1 pg Ang-1/μg total protein, P < 0.05). However, matrix-bound Ang-1 was significantly decreased from day 1 to day 6 (8.0 ± 0.6 pg Ang-1/μg total protein). Additionally, at day 1 EC cultures had significantly greater matrix-bound Ang-1 levels than both FB and EC+FB cultures (P < 0.05); however, no significant differences were observed among EC+FB, EC, and FB cultures at later time points.

Endothelial-fibroblast interactions play a role in maintaining the balance for ECM turnover.

To characterize ECM turnover, protein expression of MMP2/TIMP2 and MMP9 [proteases that participate in the degradation and remodeling of the ECM (45)], and collagen I (Col I, a major component of the ECM) was measured using ELISA (Figs. 4 and 5, respectively). At early time points, no significant differences in MMP2/TIMP2 expression exist between the experimental groups. Expression of MMP2/TIMP2 at day 3 (Fig. 4A) in EC+FB and FB cultures (47 ± 6 and 62 ± 3 ng/ml, respectively) was significantly higher compared with EC samples (30 ± 0.3 ng/ml, P < 0.05). Expression of MMP2/TIMP2 at day 6 in EC+FB and FB cultures (37 ± 3 and 56 ± 5 ng/ml, respectively) was significantly higher when compared with EC samples (1.2 ± 0.3 ng/ml, P < 0.05). Interestingly, MMP2/TIMP2 levels in EC+FB cocultures were more than twice the values for EC cultures at day 6, suggesting that this difference is not simply due to half of the cells in EC+FB constructs being FBs and that endothelial-fibroblast interactions may play a role in regulation of extracellular MMP2 levels. In contrast to MMP2/TIMP2 expression, the protein levels for MMP9 were low, and no significant differences between cell types were observed (Fig. 4B).

Fig. 4.

Protein expression of matrix metalloproteinase-2(MMP) and -9. ELISA was performed on medium samples collected from cell-nanofiber constructs at days 3 and 6 to determine concentration of MMP2/TIMP2 (A) and MMP9 (B). A: at each time point, MMP2/TIMP2 protein expression was significantly higher in FB cultures and EC+FB cocultures compared with EC cultures. There was a temporal decrease observed in MMP2/TIMP2 protein expression observed in EC+FB and EC cultures. B: there were no differences observed in MMP9 expression. *P < 0.05 compared with EC samples; +P < 0.05 compared with FB samples; ^P < 0.05 compared with day 3 samples of same experimental group.

Fig. 5.

Collagen I deposition. ELISA was performed on cell-nanofiber matrix samples (A) and medium samples (B) at days 3 and 6 to determine protein levels of collagen I. A: protein levels of collagen I in the matrix are significantly higher in EC+FB cocultures in the peptide nanofiber microenvironment at days 3 and 6 in culture compared with EC-only cultures. A temporal decrease in collagen I protein levels was also observed in EC+FB cultures. B: protein levels of collagen I in the medium were low, with no significant differences detected between experimental groups at either day 3 or day 6. Expression levels were normalized using total protein content as measured using Bradford assay. *P < 0.05 compared with EC samples; +P < 0.05 compared with FB samples; ^P < 0.05 compared with day 3 samples of same experimental group.

Similar to MMP2/TIMP2 data, the results for collagen I expression gave evidence of the role of endothelial-fibroblast interactions in regulation of ECM deposition (Fig. 5A). Early expression of Col I in the matrix at day 3 demonstrated significantly higher levels in EC+FB cocultures (179 ± 71 pg Col I/μg total protein) compared with EC and FB cultures (49 ± 27 and 77 ± 58 pg Col I/μg total protein, respectively, P < 0.05). By day 6, EC+FB cocultures Col I levels in the matrix had significantly decreased from day 3 (62 ± 12 pg Col I/μg total protein, P < 0.05) and were similar to FB levels (62 ± 15 pg Col I/μg total protein), with both significantly higher than EC levels (34 ± 9 pg Col I/μg total protein, P < 0.05). These observed levels for EC+FB cocultures suggest the “nonlinear” effect of EC-FB interactions on Col I deposition, because in the absence of these interactions the expected values of Col I protein in EC+FB cocultures would be half-way between EC and FB levels. Low Col I levels in the medium were observed (Fig. 5B), with no significant differences detected between experimental groups (EC+FB, EC, FB) at either day 3 (1.06 ± 0.70, 1.04 ± 0.59, and 1.28 ± 0.34 ng/ml, respectively) or day 6 (0.87 ± 0.56, 1.12 ± 0.49, 1.19 ± 0.89 ng/ml, respectively).

Matrix permissiveness for cell migration is a regulator of extracellular mechanical environment.

A parallel-plate rheometer (Fig. 6A) was used to measure the mechanical properties of cell-nanofiber constructs (10 mg/ml peptide nanofibers) at days 1, 3, and 6 (Fig. 6B), with elastic modulus (G′) serving as an indicator of construct stiffness. All cell-nanofiber constructs (EC+FB, EC, FB) had higher stiffness values than nanofiber-only controls (1.97 ± 0.19 kPa at day 1), indicating the contribution of cell stiffness to overall stiffness. At day 1, only the highly migratory FBs appeared able to easily move through the peptide nanofibers, resulting in scaffold disruption and a significantly lower FB construct stiffness (2.16 ± 0.38 kPa) compared with EC and EC+FB constructs (4.29 ± 0.11 and 4.28 ± 0.59 kPa, respectively, P < 0.05). At day 3, the stiffness values are influenced by cell migration and subsequent peptide nanofiber disruption, with lower stiffness values measured for the FB and EC+FB constructs (2.91 ± 0.65 and 2.95 ± 0.70 kPa, respectively) compared with the less migratory EC constructs (3.63 ± 0.25 kPa). At day 6, EC+FB coculture stiffness values (6.02 ± 0.71 kPa) are significantly increased from days 1 and 3 (P < 0.05) and are higher than ECs (4.14 ± 0.22 kPa) and significantly higher than more migratory FBs (2.63 ± 0.61 kPa, P < 0.05).

Fig. 6.

Stiffness of cell-nanofiber constructs measured using rheometry. A: schematic of parallel plate rheometer setup, showing living cells embedded in the peptide-nanofiber construct for testing. B: elastic moduli (G′) values for cell-nanofiber constructs (10 mg/ml peptide nanofibers). FBs play a more significant role in the denser concentration, as FB only constructs displayed lowest stiffness values at all time points. EC+FB cocultures have significantly greater stiffness values at day 6 compared with other experimental groups as well as previous time points. C: G′ values for cell-nanofiber constructs (6 mg/ml peptide nanofibers). The more compliant peptide nanofibers result in increased construct stiffness in EC+FB cocultures at day 6 compared with previous time points, similar to the denser concentration. G′ (kPa) is reported at frequency of 0.1 Hz. *P < 0.05 compared with EC samples; +P < 0.05 compared with FB samples; #P < 0.05 compared with day 1 samples of same experimental group; ^P < 0.05 compared with day 3 samples of same experimental group.

A more compliant nanofiber environment results in long-term maintenance of greater construct stiffness in EC+FB cocultures.

Recent studies indicate that more compliant matrices promote in vitro network formation and cell migration (43, 50). Therefore, the effect of the scaffold stiffness on cell mechanical microenvironment was investigated in rheometry experiments using both a compliant nanofibers scaffold (6 mg/ml peptide concentration) and the stiffer peptide nanofibers scaffold (10 mg/ml peptide concentration), which better represents the stiff fibrotic microenvironment of the healing heart.

In the compliant nanofiber concentration (Fig. 6C), again all cell-nanofiber constructs (EC+FB, EC, FB) had higher stiffness values than nanofiber-only controls (0.62 ± 0.02 kPa at day 1). Coordinated EC and FB migration and peptide nanofiber disruption in the EC+FB constructs led to lower stiffness (0.64 ± 0.13 kPa) compared with EC and FB constructs (0.97 ± 0.11 and 0.77 ± 0.19 kPa, respectively) at day 1. The stiffness values at day 3 of all cell-nanofiber constructs (EC+FB, EC, FB) are similar (0.71 ± 0.03, 0.72 ± 0.10, and 0.70 ± 0.03 kPa, respectively), indicating that the effects of cell proliferation, cell migration, and ECM remodeling are roughly equivalent at this time. By day 6, EC+FB cocultures have significantly increased stiffness values (0.94 ± 0.09 kPa) compared with days 1 and 3 (P < 0.05). Also at day 6, EC+FB stiffness is higher than ECs (0.87 ± 0.02 kPa) and significantly higher than more migratory FBs (0.64 ± 0.05 kPa, P < 0.05).

Statistical analyses showed that cell composition, time in culture, and peptide nanofiber concentration were all significant factors in the stiffness of cell-nanofiber constructs (P < 0.001 for each factor, ANOVA). Further analysis revealed that significant differences existed in the stiffness values between days 1 and 6 and days 3 and 6 (P < 0.001, Bonferroni post hoc t-test). In the cell composition, significant stiffness differences existed between FBs cultures and both EC cultures and EC+FB cocultures (P < 0.001, Bonferroni post hoc t-test).

DISCUSSION

In this study, both chemical and mechanical regulation of capillary morphogenesis by FBs were for the first time investigated simultaneously in a temporal manner. Recent studies have established the effect of the mechanical environment on angiogenesis in vitro by showing that extracellular matrix stiffness modulates formation of endothelial networks via altering traction forces exerted by the cells (25, 43). Whereas there is literature regarding FB roles in capillary morphogenesis either as a source of angiogenic growth factors (47) or as a substrate for endothelial culture and migration in scaffold-free in vitro cultures (27), the possible effects of FBs on angiogenesis via modulation of the mechanical environment and the interplay between mechanical and chemical signaling have not been studied in the same system. Therefore, the goal of this study was to elucidate the mechanisms of FB regulation of angiogenic process by using a comprehensive approach and a recently described culture system. Scaffold made from RAD16-II peptide nanofibers was used to mimic the in vivo three-dimensional matrix architecture and support cell functions and interactions (30). Importantly, cell viability was stable within the nanofiber culture system, indicating that the system supports cell survival and that the observed results do not directly stem from significant cell proliferation and/or death. The obtained results demonstrate that FBs provide a complex time-dependent regulation of in vitro capillary morphogenesis and overall cell-scaffold homeostasis, as seen in the proposed temporal schematic (Fig. 7). The results show that secretion of soluble growth factors (VEGF, Ang-1) by and migration of FBs and corresponding scaffold disruption promote capillary morphogenesis and stabilization at early time points. At the later stages, the role of FBs as regulators of mechanical microenvironment becomes more prominent via maintaining the balance in ECM turnover via MMP2 and collagen I production, which resulted in improved integrity and a stable microenvironment.

Fig. 7.

Proposed schematic of angiogenic regulation by FBs in endothelial-fibroblast cocultures in vitro. Arrows denote the relative levels of protein expression and stiffness in the cocultures at days 1, 3, and 6. The results show that FBs mediate angiogenic process in vitro via two major mechanisms: direct regulation of capillary morphogenesis via expression of angiogenic factors, and indirect regulation by altering mechanical microenvironment via matrix disruption, deposition, and remodeling. Initially, the process is regulated by chemical signaling from FBs (a sharp increase in VEGF levels, compared with EC-only cultures) and EC-FB co-migration at day 1. Gradually, this initial response is replaced by stabilizing factors (increases in Ang-1 and decreases in VEGF levels) and signaling via alterations in mechanical microenvironment, where endothelial-fibroblast interactions appeared to help maintain the balance in ECM homeostasis, enhancing both MMP2 and collagen I production.

Interestingly, the effect of FBs on the protein expression of two major angiogenic factors, VEGF and Ang-1, in the coculture with ECs in the nanofiber culture system appears to be precisely orchestrated to parallel stages of angiogenic response in vivo. Indeed, the angiogenic process in vivo (49) starts with VEGF-induced activation of ECs and capillary sprouting. The elevated levels of VEGF gradually decrease, when sprouting is replaced by capillary stabilization, which is largely mediated by pericyte-produced Ang-1 and is accompanied by increased levels of this protein. Consistent with this view, the results of this study show that in EC+FB cocultures, VEGF levels are highest at day 1 and then gradually decrease. Interestingly, VEGF levels in EC+FB cocultures are slightly lower at all time points than expected based on cell numbers alone, suggesting that in coculture cellular VEGF production may be reduced, although this difference is not pronounced. In contrast to the observed VEGF temporal pattern, Ang-1 expression steadily rises from day 1 to day 6, implying reduced endothelial migration and stabilization of newly formed endothelial networks. In fact, Ang-1 levels in coculture at day 6 are much higher than what would be expected based on cell numbers alone, suggesting that in coculture Ang-1 production is enhanced. Importantly, this pattern of Ang-1 expression does not occur in either EC- or FB-only cultures and has not been observed previously in other in vitro systems, where addition of angiogenic factors is usually required to induce angiogenesis, precluding quantitation of endogenous (cell-induced) Ang-1 levels.

A new finding of this study is the role that FBs play in facilitating EC migration during active stages of capillary morphogenesis. Indeed, the significantly larger and faster forming networks seen in EC-FB cocultures indicate that migratory cells such as FBs promote EC organization and assembly into larger networks in this system. Interestingly, the peptide nanofibers lack MMP degradation sites by design (52), so a mechanism other than proteolytic migration (25) is responsible for cell migration within the nanofiber matrix. Amoeboid migration driven both by cells squeezing through the matrix pores and through deformation of the ECM network (16) (allowing for circumnavigation rather than degradation of ECM barriers) is the likely mechanism of cell migration within the nanofiber scaffold. Thus these results suggest a novel mechanism for FBs to regulate network formation by ECs via mechanical disruption of the scaffold and thus “leading the way” for endothelial networks to spread. Recent studies demonstrated that ECM mechanical environment affects angiogenesis via regulating contractile forces generated by the ECs (25). Therefore, our finding that migratory FBs promote capillary morphogenesis via mechanical ECM disruption may have an important implication for cardiac regeneration, where ECs need to overcome the “stiffness barrier” of fibrotic scarring to penetrate the ischemic area.

The overall effect of endothelial-fibroblast interactions on the extracellular mechanical environment was studied by employing a rheometry approach to measure the mechanical stiffness of three-dimensional cell-scaffold constructs. The G′ served as an indicator of the cell-construct stiffness, reflecting the balance between scaffold disruption due to cell (mostly FB) migration and extracellular matrix remodeling due to deposition and proteolysis of new matrix components (such as Col I). To the best of our knowledge, this is the first time that rheometry has been used to measure the material properties of constructs seeded with live cells. The rheometry results showed that trends seen in network formation and protein expression were accompanied by the corresponding changes in stiffness values of cell-seeded nanofiber constructs. The results suggest that cell migration and associated scaffold disruption play the major role in overall stability of mechanical microenvironment, and that this effect depends on time in culture and the cell type. For highly migratory FBs, stiffness values do not change significantly throughout the experimental duration and are the lowest of all three groups, with no significant differences in Col I and MMP2 levels between the time points. In endothelial-only constructs, there is initial EC activation, enhanced migration, and associated scaffold disruption, resulting in decreased stiffness at day 3 compared with day 1. At later times, slightly increased levels of Ang-1 and capillary stabilization are associated with less migration, with reduced Col I concentration and MMP2 remodeling, resulting in overall higher values of stiffness at day 6. However, in EC+FB constructs, endothelial-fibroblast interactions result in a significantly different temporal pattern for alterations in the mechanical environment. Thus the results show that initially (from day 1 to day 3), in the stiffer scaffold, mechanical stiffness of the EC+FB constructs is significantly decreasing, even though the Col I concentration is the highest at day 3. This sharp decrease in stiffness between day 1 and day 3 does not occur in more compliant scaffold, where there is less resistance for cell migration, suggesting that migration-induced scaffold disruption is mostly responsible for the decreased mechanical stiffness at day 3. Additionally, these high Col I levels (more than expected based on cell numbers alone) are paralleled by high MMP2/TIMP2 levels in EC+FB cocultures at day 3, consistent with increased MMP2 production by ECs in Col I-rich environment (20). Interestingly, Col I has also been shown to enhance endothelial production of MT1-MMP and subsequently MMP2 activation (2, 20), which may explain observed decreases in both MMP2/TIMP2 and collagen I levels at day 6. However, increasing levels of Ang-1 and stabilization of endothelial networks due to endothelial-fibroblast interactions from day 3 on are associated with significant increases in stiffness of EC+FB constructs at day 6 in both stiff and compliant scaffolds, even compared with day 3. These results suggest that a combination of ECM deposition and remodeling overcomes the structural nanofiber disruption caused by cell migration, resulting in greater stiffness values, better structural integrity, and a stabilization of the overall microenvironment at day 6 when compared with earlier time points.

In this study, the effects of matrix concentration on cell-cell interactions and mechanical regulation of the microenvironment were studied by comparing stiffness values for cell-seeded constructs made using two peptide nanofiber concentrations: a stiffer nanoscaffold (10 mg/ml peptide nanofibers), which better represents the microenvironment of the healing heart, and a more compliant nanoscaffold (6 mg/ml peptide nanofibers) to investigate previous observations that more compliant matrices promote in vitro network formation and cell migration (43, 50). In these studies, the compliant nanofibers allowed for coordinated EC and FB migration and scaffold disruption. In contrast, in the stiff nanofibers, only the highly migratory FBs appeared able to easily move through the peptide, resulting in lower stiffness in the FB constructs. These results are in agreement with previously reported enhanced capillary morphogenesis in more compliant RAD16-II scaffolds (21). In the compliant nanofibers, there was a steady increase in cell-seeded construct stiffness from day 1 to day 3, in contrast to sharp decrease and subsequent increase in modulus of the stiffer nanofibers. Therefore, the data confirm that in this culture system, similar to others (19, 43, 50), matrix stiffness does affect cell behavior, with FBs playing a more significant role in mechanical regulation in denser nanofibers. Physiologically, this is more representative of the scar environment in the healing heart where FBs often play a role in aberrant matrix remodeling. In contrast, our results suggest that more compliant nanofibers better support migration of both ECs and FBs, allowing for a better balance between cell migration and ECM remodeling and may ultimately be better suited for improved angiogenesis and long-term stability.

To accurately represent the process of cardiac tissue revascularization and remodeling in vitro, an ideal system would include all three major cardiac cell types (cardiomyocytes, ECs and cardiac FBs) from the same organism. In this study, we chose to use human ECs and FBs in a 1:1 ratio to represent cell behavior in healing human cardiac tissue (4, 8). Whereas human microvascular ECs are readily available, human cardiac FBs are not. Therefore, human dermal FBs were used, which may be a limitation of the study. However, given similarities that exist between cardiac and dermal wound healing (38), we believe that this study provides important findings that reflect phenotypic FB behavior and are representative of endothelial-fibroblast interactions during cardiac tissue revascularization.

In summary, this study demonstrates that FBs mediate angiogenic process in vitro via two major mechanisms: direct regulation of capillary morphogenesis via expression of angiogenic factors and indirect regulation by altering mechanical microenvironment via matrix disruption, deposition, and remodeling. The results show that both of these mechanisms are time dependent, and that at each time point, EC behavior is likely to be controlled by a combination of factors, such as concentration of angiogenic growth factors, ECM composition, stiffness, and remodeling, all of which are in turn influenced by the presence of FBs. Thus our findings provide insight into the complex intercellular signaling occurring between FBs and ECs in the context of angiogenesis and cardiac regeneration. The use of peptide nanofibers in this study effectively allowed for the uncoupling of cell-cell interactions from cell-scaffold interactions and allowed for the simultaneous investigation of both the chemical and mechanical roles of FBs in angiogenesis in an in vitro system. The results of this study, along with recent in vivo studies in mouse cardiac tissue (11, 21) and during wound healing in diabetic mice (3), suggest that peptide nanofiber environment may be uniquely suited as a tissue engineering substrate for regeneration of vascular tissues. The findings of this study emphasize the importance of creating the microenvironment that supports cell-cell interactions and is permissive for cell migration, thus contributing toward creating the optimal environment for cardiac tissue engineering applications.

GRANTS

This project was supported by an American Heart Association Beginning Grant-In-Aid (BGIA-0765425B) and NIH/NDDK (1R21DK07881-01A1) to D. A. Narmoneva, American Heart Association predoctoral fellowships for J. R. Hurley (GRA09PRE2150073) and S. Balaji (0815371D) and a National Science Foundation Integrative Graduate Education and Research Traineeship (NSF-IGERT 0333377) to J. R. Hurley.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).