Research ArticleMATERIALS SCIENCE

Evolutionarily conserved sequence motif analysis guides development of chemically defined hydrogels for therapeutic vascularization

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Science Advances  08 Jul 2020:
Vol. 6, no. 28, eaaz5894
DOI: 10.1126/sciadv.aaz5894
  • Fig. 1 Evolutionary conservation and motif analysis enable identification of LM-derived RGD peptides for microarray construction.

    (A) LM structural diagram with location of all RGD-containing domains. (B) Schematic diagram showing the species of Euarchontoglires used for bioinformatic alignment screening (*) in context of their phylogenetic relationship. Different colors indicate each subfamily from Mammalia. (C) Bitmap plots of motif analysis of the highly conserved sequences among species in Euarchontoglires. Dashed-line boxes from bitmap plots determine the highly conserved positions around/at RGD (80% of max bit score set as the filter criteria). The most repeated amino acid (signified by size of letter) from each conserved peptide position was used as the representative amino acid for synthesis. (D) The list of identified consensus LM-RGD sequences from motif analysis and their correspondent origin domains (Fn-RGD and Vn-RGD included as the positive controls) for LM-RGD microarray screening. Glycine in red as the linker for the peptides from LM-α1 was optimized by following experiments.

  • Fig. 2 Hydrogel microarrays facilitate discovery of LM-derived peptide α1 for improved EC attachment.

    (A and B) Heat map of HUVEC attachment and quantification of saturated attachment (from sigmoidal plots of peptide concentration versus attached cell number) on the LM-RGD–derived hydrogel microarray; n ≥ 10. * indicates significantly more HUVEC attachment than on RDG, α1#-, γ3#-, and α5-1–modified hydrogels; P < 0.05. ** indicates significantly more HUVEC attachment than remaining groups; P < 0.05. *** indicates significantly more HUVEC attachment than on β4-modified hydrogels; P < 0.05. (C) Library of glycine-linker optimization for the peptides from LM-α1. (D and E) Heat map of HUVEC attachment and quantification of saturated attachment on the hydrogel microarray for glycine-linker optimization; n ≥ 10. * indicates significant differences between groups. n.s. indicates no significant difference. (F) Library of reported α1-similar sequences or α1-correspondent sequences from the species out of Euarchontoglires. (G and H) Heat map of HUVEC attachment and quantification of the saturated attachment on the α1-based hydrogel microarray; n ≥ 10. * indicates significantly more HUVEC attachment than on hydrogels modified with the correspondent motifs from Aves and Sus scrofa; P < 0.05. ** indicates significantly more HUVEC attachment on α1-modified poly(ethylene glycol) (PEG) hydrogels than on PA26- (22) and A99-modified (22) hydrogels; P < 0.05. All plots represent means ± SD.

  • Fig. 3 α1-modified hydrogel binds to both Fn- and LM-binding integrins of ECs.

    (A) Quantifications of normalized attached HUVEC (with/without integrin blocking) number on α1-/RGDS-/β4-modified alginate hydrogels; n = 6. * indicates significant difference compared to no integrin blocking; P < 0.05. (B) The library of selected non-RGD, LM-binding-integrin targeted peptides for hydrogel microarray screening. (C) Heat map of HUVEC attachment on the hydrogel microarray of the non-RGD, LM-binding-integrin targeted peptides with/without additional 3 mM RGDS. α1-modified PEG hydrogel set as the positive control. (D) Quantification of the saturated attached HUVEC number based on the sigmoidal plots of peptide concentration versus attached cell number from the microarray of LM-binding-integrin targeted peptides with/without additional 3 mM RGDS; n ≥ 10. * indicates significant difference compared to “without 3 mM RGDS”; P < 0.05. (E) Quantification of the average attached HUVEC number at 3 mM LM-binding-integrin targeted, non-RGD peptides with additional 3 mM RGDS; n ≥ 10. α1-modified PEG hydrogel set (6 mM) acts as the positive control as the total peptide concentration is fixed at 6 mM. * indicates significant difference compared to rest of groups; P < 0.05. All plots represent means ± SD/SEM.

  • Fig. 4 RNA-seq analyses to characterize vasculogenic material-cell interaction in α1-modified hydrogel.

    (A to C) Visualization of all (max 350 terms) significant (P < 0.05) GO terms of DE genes (fold change > 1.5 or < −1.5, adjusted P < 0.05) from HUVECs in α1- versus RGDS-functionalized hydrogels. GO terms of CC, MF, and BP are organized in semantic similarity–based scatterplots using REViGO that combines redundant terms into a single, representative term based on a simple clustering algorithm relying on semantic similarity measures. (D and E) The Venn diagram of up-regulated (D) and down-regulated (E) genes from transcriptomic assay of α1/RGDS and Matrigel/RGDS in the vasculature development GO-BP term. (F) Heat map of DE genes assembled from the GO-BP vasculature development term (GO:0001944) gene list showing the gene expression profile of α1-modified hydrogels over RGDS-modified hydrogels.

  • Fig. 5 The vasculogenesis assay on RGDS-, A99-, PA26-, β4-, and α1-modified hydrogels revealed the capacity of α1-modified hydrogel to promote vasculogenic network formation.

    (A) Representative images of HUVEC vascular network in RGDS-, A99-, PA26-, β4-, and α1-modified hydrogels and Matrigel (scale bar, 100 μm). Green, CD31; blue, 4′,6-diamidino-2-phenylindole (DAPI). Sprouting HUVEC clusters were found with elongated HUVECs in β4- and α1-modified hydrogels and Matrigel. (B to D) Quantification of total network length, branches, and branch points of EC network in 3D hydrogel culture (RGDS-, A99-, PA26-, β4-, and α1-modified hydrogels and Matrigel); n ≥ 11 for each group. * indicates significant difference; P < 0.05. All plots represent means ± SD.

  • Fig. 6 Enhanced recovery of blood perfusion after ischemic hindlimb injury using α1-formulated hydrogel.

    (A) Representative images of laser Doppler perfusion imaging on the ischemic hindlimb after injecting the different formulations (medium, α1 only, and α1 + MMPQK) of alginate hydrogels for serial analysis (left leg, normal; right leg, ischemic). (B) Quantification of blood flow recovery percentage of ischemic limb to normal limb (n = 4 to 5). * indicates significance difference; P < 0.05. N.S. indicates no significant difference between the groups; P > 0.05. # indicates improved blood flow of the α1 hydrogel–treated group compared to medium-treated group at day 28; P = 0.053. ## indicates the improved blood flow of α1 + MMPQK hydrogel–treated group compared to α1-treated group at day 28; P = 0.054. (C) Representative images of laser Doppler perfusion imaging on the ischemic hindlimb after injecting the formulations of the second batch (groups shown in fig. S9B) alginate hydrogels for serial analysis (left leg, normal; right leg, ischemic). (D) Quantification of blood flow recovery percentage of ischemic limb to normal limb (n = 4 to 5). * indicates significance difference; P < 0.05. # indicates the improved blood flow of α1 + MMPQK hydrogel–treated ischemic legs compared to RGDS + MMPQK hydrogel ischemic legs at day 14; P = 0.052.

  • Fig. 7 Improved functional and morphological recovery from ischemic hindlimb injury using α1-formulated hydrogel.

    (A) Gait analysis of the stride length on ischemic hindlimb (n = 4 to 5). # indicates improved stride length of 2×(α1 + MMPQK) hydrogel–treated ischemic legs compared to medium groups (significant functional improvement) at day 14; P = 0.053. (B to D) Quantification of fibrotic areas (%) from Masson’s trichrome (MT) analysis (B), anti–smooth muscle actin–positive (α-SMA+) arteries (C), and vWF-positive (vWF+) capillaries (D) from treated ischemic muscle tissue sections and controls at 4 weeks after surgery, n ≥ 20 for the quantification. * indicates significance difference; P < 0.05. ** indicates significantly more α-SMA+ arteries and vWF+ capillaries of 2×(α1 + MMPQK)–treated ischemic legs compared to remaining groups at day 28; P < 0.05. The significant analyses of all these histological assays are shown in fig. S9 (C to E). (E) Representative images of hematoxylin and eosin (H&E), MT, and immunohistochemical staining (α-SMA and vWF) of treated ischemic muscle tissue sections and controls at 4 weeks after surgery (scale bars, 100 μm). All plots represent means ± SD/SEM.

Supplementary Materials

  • Supplementary Materials

    Evolutionarily conserved sequence motif analysis guides development of chemically defined hydrogels for therapeutic vascularization

    Jia Jia, Eun Je Jeon, Mei Li, Dylan J. Richards, Soojin Lee, Youngmee Jung, Ryan W. Barrs, Robert Coyle, Xiaoyang Li, James C. Chou, Michael J. Yost, Sharon Gerecht, Seung-Woo Cho, Ying Mei

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