Immobilization of Cell-Adhesive Laminin Peptides in Degradable PEGDA Hydrogels Influences Endothelial Cell Tubulogenesis

Attachment, spreading, and organization of endothelial cells into tubule networks are mediated by interactions between cells in the extracellular microenvironment. Laminins are key extracellular matrix components and regulators of cell adhesion, migration, and proliferation. In this study, laminin-derived peptides were conjugated to poly(ethylene glycol) (PEG) monoacrylate and covalently incorporated into degradable PEG diacrylate (PEGDA) hydrogels to investigate the influence of these peptides on endothelial cellular adhesion and function in organizing into tubule networks. Degradable PEGDA hydrogels were synthesized by incorporating a matrix metalloproteinase (MMP)-sensitive peptide, GGGPQGIWGQGK (abbreviated PQ), into the polymer backbone. The secretion ofMMP-2 and MMP-9 by endothelial cells promotes polymer degradation and consequently cell migration. We demonstrate the formation of extensive networks of tubule-like structures by encapsulated human umbilical vein endothelial cells in hydrogels with immobilized synthetic peptides. The resulting structures were stabilized by pericyte precursor cells (10T1/2s) in vitro. During tubule formation and stabilization, extracellular matrix proteins such as collagen IV and laminin were deposited. Tubules formed in the matrix of metalloproteinase sensitive hydrogels were visualized from 7 days to 4 weeks in response to different combination of peptides. Moreover, hydrogels functionalized with laminin peptides and transplanted in a mouse cornea supported the ingrowth and attachment of endothelial cells to the hydrogel during angiogenesis. Results of this study illustrate the use of laminin-derived peptides as potential candidates for modification of biomaterials to support angiogenesis

A Synthetic Matrix with Independently Tunable Biochemistry and Mechanical Properties to Study Epithelial Morphogenesis and EMT in a Lung Adenocarcinoma Model

Better understanding of the biophysical and biochemical cues of the tumor extracellular matrix environment that influence metastasis may have important implications for new cancer therapeutics. Initial exploration into this question has used naturally derived protein matrices that suffer from variability, poor control over matrix biochemistry, and inability to modify the matrix biochemistry and mechanics. Here, we report the use of a synthetic polymer-based scaffold composed primarily of poly(ethylene glycol), or PEG, modified with bioactive peptides to study murine models of lung adenocarcinoma. In this study, we focus on matrix-derived influences on epithelial morphogenesis of a metastatic cell line (344SQ) that harbors mutations in Kras and p53 (trp53) and is prone to a microRNA-200 (miR-200)–dependent epithelial–mesenchymal transition (EMT) and metastasis. The modified PEG hydrogels feature biospecific cell adhesion and cell-mediated proteolytic degradation with independently adjustable matrix stiffness. 344SQ encapsulated in bioactive peptide-modified, matrix metalloproteinase–degradable PEG hydrogels formed lumenized epithelial spheres comparable to that seen with three-dimensional culture in Matrigel. Altering both matrix stiffness and the concentration of cell-adhesive ligand significantly influenced epithelial morphogenesis as manifest by differences in the extent of lumenization, in patterns of intrasphere apoptosis and proliferation, and in expression of epithelial polarity markers. Regardless of matrix composition, exposure to TGF-β induced a loss of epithelial morphologic features, shift in expression of EMT marker genes, and decrease in mir-200 levels consistent with EMT. Our findings help illuminate matrix-derived cues that influence epithelial morphogenesis and highlight the potential utility that this synthetic matrix-mimetic tool has for cancer biology

Improved Angiogenesis in Response to Localized Delivery of Macrophage-Recruiting Molecules

Successful engineering of complex organs requires improved methods to promote rapid and stable vascularization of artificial tissue scaffolds. Toward this goal, tissue engineering strategies utilize the release of pro-angiogenic growth factors, alone or in combination, from biomaterials to induce angiogenesis. In this study we have used intravital microscopy to define key, dynamic cellular changes induced by the release of pro-angiogenic factors from polyethylene glycol diacrylate hydrogels transplanted in vivo. Our data show robust macrophage recruitment when the potent and synergistic angiogenic factors, PDGFBB and FGF2 were used as compared with VEGF alone and intravital imaging suggested roles for macrophages in endothelial tip cell migration and anastomosis, as well as pericyte-like behavior. Further data from in vivo experiments show that delivery of CSF1 with VEGF can dramatically improve the poor angiogenic response seen with VEGF alone. These studies show that incorporating macrophage-recruiting factors into the design of pro-angiogenic biomaterial scaffolds is a key strategy likely to be necessary for stable vascularization and survival of implanted artificial tissues

An increased number of Csf1r-EGFP+ cells appear in the cornea ahead of sprouting vessels and pericyte recruitment.

<p>At 10 days post-implantation, both VEGF- (a) and PDGFBB/FGF2- induced (d) vessel beds exhibit clear NG2-DsRed+ pericyte investment, but the pericytes in VEGF sample appeared less elongated indicating a possible difference in vascular coverage. There were qualitatively fewer Csf1r-EGFP+ cells in the VEGF (b) samples as compared to PDGFBB/FGF2 (e), which also showed more structural heterogeneity. DsRed+ EGFP+ double positive cells were never observed (c and f). Csf1r-EGFP+ cells were examined at 3 days post-implantation. Compared to blank (g), VEGF- (h), PDGFBB- (i) and FGF2-releasing gels (j), the PDGFBB/FGF2-releasing gels (k) exhibited a greater density of Csf1r-EGFP+ cells in the region occupied by the hydrogels (l, n = 3, * <i>P</i><0.05, *** <i>P</i><0.001).</p

CSF1-mediated macrophage recruitment enhances VEGF-induced angiogenesis.

<p>PEGDA hydrogels encapsulated with VEGF (320 ng), CSF1 (320 ng), VEGF (320 ng) + CSF1 (320 ng) or PDGFBB (320 ng) + FGF2 (80 ng) were implanted in <i>Csf1r-EGFP</i>^<i>+/tg</i> or <i>Flk1-myr</i>::<i>mCherry</i>^<i>+/tg</i> mice. Quantification of confocal images (a-d) indicated that the density of Csf1r-EGFP+ cells recruited by either CSF1 or CSF1/VEGF at day 3 was significantly higher than VEGF alone and similar to the density of PDGDBB/FGF2 (i). When comparing vessel density and total vessel length at day 10 (e-h), CSF1 alone was non-angiogenic. However, the combination of CSF1/VEGF induced a more robust angiogenic response compared to VEGF alone that was comparable to PDGFBB/FGF2 (j and k). When CSF1 was co-delivered with VEGF, the recruited macrophages not only improved the angiogenic response, but also enhanced pericyte investment on VEGF-induced vessels (o-q), similar to PDGFBB/FGF2 induced vessels (l-n). Abbreviations: VEGF (V); CSF1 (C); VEGF+CSF1 (V+C); PDGFBB+FGF2 (P+F).</p

PDGFBB/FGF2-stimulated directional migration of Csf1r-EGFP+ phagocytes.

<p>Time-lapse, <i>in vivo</i> imaging of <i>Csf1r-EGFP</i>^<i>+/tg</i> corneas, starting at 3 hour post implantation, showed enhanced Csf1r-EGFP+ cell motility in response to PDGFBB/FGF2-releasing hydrogels as compared to blank or VEGF (a-c). From these movies (3 separate movies from 3 individual mice and each condition), Csf1r-EGFP+ cell density (d), total displacement (e) and migration velocity (f) were calculated and shown to be greater for the PDGFBB/FGF-implanted corneas. Time lapse imaging of directional migration of the Csf1r-EGFP+ phagocytes was performed at 3.5 hour post implantation (g) and analyzed by Imaris (h). The displacement and angle of displacement of each Csf1r-EGFP+ cell was plotted on a rose plot (i) and indicated that the Csf1r-EGFP+ cells stimulated by PDGFBB/FGF2 migrated toward implanted hydrogels.</p

Corneal tissues surrounding the implanted PDGFBB/FGF2-releasing hydrogels were better perfused than corneas containing VEGF hydrogels.

<p>3D reconstructed SV-OCT images of the mouse corneas implanted with VEGF (a) and PDGFBB/FGF2 (c) hydrogel for 10 days. SV-OCT analysis showed a greater number of perfused vessels in the corneas implanted with PDGFBB/FGF2-releasing (d) hydrogels when compared to corneas implanted with VEGF-releasing hydrogels (b). Vessel density measurement by calculating the ratio of the volume of SV signal to the tissue volume from the OCT signal showed that PDGFBB/FGF2 induced higher density of perfused vessel than VEGF in the corneal tissues (e, n = 3, *<i>P</i><0.05).</p

PDGFBB/FGF2-responding Csf1r-EGFP+ cells are comprised of different macrophage subtypes.

<p>qrtPCR analysis was performed on corneas implanted with blank, VEGF-, and PDGFBB/FGF2-releasing hydrogels 5 days post-implantation (a). The phagocytes marker <i>Csf1r</i> was significantly upregulated in PDGFBB/FGF2-implanted corneas reflecting the increase in Csf1r-GFP+ cells. The neutrophil marker <i>Gsr</i> (Ly6g) showed no difference among all three groups whereas <i>Emr1</i> (F4/80) expression was significantly up-regulated in the PDGFBB/FGF2-implanted corneas indicating an enrichment of mature macrophages. F4/80 immunofluorescence and co-localization with Csf1r-EGFP, at 10 days post-implantation, further confirmed the presence of macrophages among the Flk1-myr::mCherry+ vessels within PDGFBB/FGF2-implanted corneas (b). qrtPCR indicated that both M1 (<i>Nos2</i> and <i>Tnfa</i>) and M2 (<i>Arg1</i> and <i>Chi3l3</i>) macrophage marker genes were significantly up regulated in the PDGFBB/FGF2 implanted corneas compared to blank or VEGF hydrogels (c). Abbreviations: Blank (B); VEGF (V); PDGFBB/FGF2 (P/F).</p

The combination of PDGFBB and FGF2 induces robust angiogenesis with well-organized vascular structure.

<p>A schematic diagram of the cornea micropocket assay (a-d). A hydrogel with releasable pro-angiogenic factor is implanted into the cornea micropocket (a) and induced vessels grow toward implanted hydrogel from the limbal region (b). For analysis, the cornea is dissected from the rest of the eye (c) and flat mounted as four quadrants (d). Flk1-myr::mCherry+ vessels induced by VEGF (e) or PDGFBB/FGF2 (f) were examined at 10 days post-implantation. Vessel density induced by PDGFBB/FGF2 was significantly higher than VEGF (i, n = 3, ***<i>P</i><0.001), as well as the total vessel length (j, n = 3, **<i>P</i><0.01). Vessel structure was compared between VEGF- (g) and PDGFBB/FGF2- (h) induced vessels in regions that have similar vessel coverage on the surface of hydrogel. VEGF–induced vessels appeared as disorganized with extensive sprouting while PDGFBB/FGF2-induced vessels appeared more established and lumenized. Lacunarity parameter measurements indicated that PDGFBB/FGF2-induced vessels have larger spaces between vessels compared to VEGF-induce vessels (k, n = 3, *<i>P</i><0.05). Values are presented as mean ± SEM.</p

Bone marrow derived macrophages (BMDMs) increase HUVEC cord length and number in 3D collagen gels.

<p>After 24 and 72 hours, cord structures formed by HUVECs (a) or HUVECs + BMDMs at 5:1 ratio (d) were fixed and immunofluorescence stained with PECAM, imaged with confocal microscopy and skeletonized by using open snake tracing algorithm in FARSIGHT software (b and e). In the BMDMs/HUVECs co-cultures, the number of cords over 40 um (c, n = 6, ***<i>P</i><0.001, One-way ANOVA) and the distribution of cord lengths (f, n = 6, ***<i>P</i><0.001, Kruspal-Wallis) were significantly increased at 72 hours. Panel g shows an example of a macrophage physically associating with HUVECs and 3D rendering of the z-stack at different angles indicated that the macrophage was bridging the junction between two separate cord structures (arrows in h and i).</p