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. 2017 Apr:123:142-154.
doi: 10.1016/j.biomaterials.2017.01.037. Epub 2017 Jan 30.

Perivascular extracellular matrix hydrogels mimic native matrix microarchitecture and promote angiogenesis via basic fibroblast growth factor

George R Fercana #  1 , Saigopalakrishna Yerneni #  1 , Marie Billaud  1 , Jennifer C Hill  1 , Paul VanRyzin  1 , Tara D Richards  1 , Brian M Sicari  1 , Scott A Johnson  1 , Stephen F Badylak  1 , Phil G Campbell  1 , Thomas G Gleason  1 , Julie A Phillippi  1
Affiliations

Perivascular extracellular matrix hydrogels mimic native matrix microarchitecture and promote angiogenesis via basic fibroblast growth factor

George R Fercana et al. Biomaterials. 2017 Apr.

Abstract

Extracellular matrix (ECM)-derived bioscaffolds have been shown to elicit tissue repair through retention of bioactive signals. Given that the adventitia of large blood vessels is a richly vascularized microenvironment, we hypothesized that perivascular ECM contains bioactive signals that influence cells of blood vessel lineages. ECM bioscaffolds were derived from decellularized human and porcine aortic adventitia (hAdv and pAdv, respectively) and then shown have minimal DNA content and retain elastin and collagen proteins. Hydrogel formulations of hAdv and pAdv ECM bioscaffolds exhibited gelation kinetics similar to ECM hydrogels derived from porcine small intestinal submucosa (pSIS). hAdv and pAdv ECM hydrogels displayed thinner, less undulated, and fibrous microarchitecture reminiscent of native adventitia, with slight differences in ultrastructure visible in comparison to pSIS ECM hydrogels. Pepsin-digested pAdv and pSIS ECM bioscaffolds increased proliferation of human adventitia-derived endothelial cells and this effect was mediated in part by basic fibroblast growth factor (FGF2). Human endothelial cells cultured on Matrigel substrates formed more numerous and longer tube-like structures when supplemented with pAdv ECM bioscaffolds, and FGF2 mediated this matrix signaling. ECM bioscaffolds derived from pAdv promoted FGF2-dependent in vivo angiogenesis in the chick chorioallantoic membrane model. Using an angiogenesis-focused protein array, we detected 55 angiogenesis-related proteins, including FGF2 in hAdv, pAdv and pSIS ECMs. Interestingly, 19 of these factors were less abundant in ECMs bioscaffolds derived from aneurysmal specimens of human aorta when compared with non-aneurysmal (normal) specimens. This study reveals that Adv ECM hydrogels recapitulate matrix fiber microarchitecture of native adventitia, and retain angiogenesis-related actors and bioactive properties such as FGF2 signaling capable of influencing processes important for angiogenesis. This work supports the use of Adv ECM bioscaffolds for both discovery biology and potential translation towards microvascular regeneration in clinical applications.

Keywords: Adventitia; Aneurysm; Angiogenesis; Endothelial cell; Extracellular matrix; Hydrogel; Tube formation.

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Figures

Figure 1
Figure 1
Preparation and characterization of ECM bioscaffolds. A) ECM bioscaffold as a lyophilized ground powder. B) Hydrogel formation from pH-neutralized pepsin-digested ECM bioscaffolds after 1 hr at 37°C. C) DNA extracts from 1.2 mg total tissue weight were qualitatively analyzed using ethidium bromide-containing agarose gel electrophoresis.
Figure 2
Figure 2
Scanning electron microscopy of adventitial ECM hydrogel. Decellularized tissue and ECM hydrogels were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy. Representative micrographs showing decellularized human adventitia (Adv) (A-B), human Adv hydrogel (C-D), porcine Adv hydrogel (E-F) and porcine small intestinal submucosa (SIS) hydrogel (G-H) at 5,000X (A, C, E) and 10,000X (B, D, F) magnifications. All scale bars=1μm.
Figure 3
Figure 3
Turbidimetric gelation kinetics of ECM hydrogels. Gelation of pH-neutralized ECM digests was monitored using optical density (O.D.) readings at 405 nm at 37°C for 90 min. A) Porcine adventitia (Adv) (4, 8 and 16 mg/mL). B) Normalized turbidimetric gelation kinetics of porcine SIS (8 mg/mL), human Adv and porcine Adv (16 mg/mL).
Figure 4
Figure 4
FGF2-mediated stimulation of primary endothelial cell proliferation by ECMs. Primary human adventitia-derived endothelial cells were cultured in the presence of 10 μg/mL porcine adventitial (pAdv, solid bars) or porcine small intestinal submucosa (pSIS, gray bars) ECM. Cells in their basal culture medium, FGF2 inhibitor alone (100 nM PD173074 in DMSO), or an equivalent volume of DMSO and digestion buffer (1 mg/mL pepsin in 0.01 N HCl) served as controls (open bars). Quantification of MTS conversion was performed after 72 hr of exposure to the above conditions using a commercial assay and results were expressed as percent change of untreated cells. One representative of three independent experiments is displayed. Bars represent mean of four assay replicates ± standard deviation. * indicates p<0.05 when compared with untreated condition, **indicates p<0.02, and # indicates p<0.005.
Figure 5
Figure 5
Effect of ECM bioscaffolds on network formation of tube-like structures in vitro. Human adventitia-derived endothelial cells were cultured on growth factor reduced-Matrigel substrates: A) DMSO, 0.05% (v/v), B) Digestion buffer (1% (w/v) Pepsin in 0.1 N HCl), C) pAdv ECM, and D) pSIS ECM. FGF2 inhibitor PD173074 (100 nM) was added to the culture medium of above treatments shown in parallel wells (E-H). A-H: One representative 10X field is shown, selected from one of three replicates of two independent experiments. All scale bars= 50 μm for A-H. Quantification of the number (I) and total length (J) of tube-like structures from 5×5 stitched fields captured at 10X for non-ECM-supplemented (open bars), pAdv (solid bars) and pSIS (gray bars) ECM-supplemented substrates in the absence and presence of PD173074. Bars represent mean of three assay replicates ± standard deviation. Images and graphs represent data from one of two independent experiments. *Significant from pepsin HCl, p<0.02; #Significant from pAdv ECM-treated cells in the absence of PD173074, p<0.03.
Figure 6
Figure 6
Effect of ECM bioscaffolds on angiogenesis in vivo. A) Representative bright field images of scaffolds before (Time 0) and after (72 hr) incubation on the chorioallantoic membrane (CAM) of the chick embryo. The pro-angiogenic response to pSIS and pAdv ECM-containing fibrin scaffolds (250 μg/mL) is revealed by the spoke-wheel pattern along the perimeter of the scaffolds. There was no appreciable angiogenic response detected around scaffolds loaded with digestion buffer (1% (w/v) pepsin in 0.1 N HCl) or DMSO. Addition of the FGF2 inhibitor PD173074 (100 nM) abrogated the angiogenic response to pAdv ECM. Addition of the inhibitor vehicle only (DMSO) did not alter the angiogenic response to pAdv ECM. All scale bars for panel A = 5mm. B) Representative histological cross-sections of CAM assay scaffolds. The CAM vasculature was visualized using injected tomato lectin-Dylight® 650 (red) and nuclei are labeled with Hoechst dye (blue). A dashed white line denotes the scaffold/CAM interface. Scaffolds loaded with digestion buffer alone exhibited no vessel invasion. pSIS ECM (250 μg/mL) stimulated invasion of new vasculature (denoted by arrowheads) toward the scaffold as did pAdv ECM in a dose-dependent manner for concentrations 50-250 μg/mL. The maximum tested dose of pAdv ECM (500 μg/mL) inhibited invasion of blood vessels into the scaffold. C) Addition of DMSO did not alter pAdv ECM-induced invasion of blood vessels and FGF2 inhibitor PD173074 blocked the effect of pAdv ECM-loaded scaffolds. All scale bars in panels B and C= 500 μm. *Avascular zone comprised of lectin-negative cells. D). Representative histological cross-sections showing chemoattraction of lectin-negative cells in an avascular zone (*) adjacent to invading lectin-positive cells (arrowheads) in pAdv ECM-loaded fibrin scaffold (250 μg/mL) (i) and inhibition of invasion of lectin-positive cells in 500 μg/ml pAdv ECM-loaded fibrin scaffold (ii). (*) avascular zone comprised of lectin-negative cells, (arrowheads) vascular zone of lectin-positive cells. All scale bars for panel D = 20 μm.
Figure 7
Figure 7
Protein array-based profile of angiogenesis-related proteins. Lyophilized ECM bioscaffolds (300 μg total protein) were evaluated for the presence of 55 angiogenesis-related proteins in duplicate using the Human Angiogenesis Proteome Profiler Array. Densitometric values are provided in Table 3. Images for porcine and human ECM blots reflect exposure times of 20 min and 10 min respectively. Dashed line boxes=positive control reference spots. Dotted line boxes=negative control reference spots.
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