Spatial patterning of endothelium modulates cell morphology, adhesiveness and transcriptional signature

Abstract

Microscale and nanoscale structures can spatially pattern endothelial cells (ECs) into parallel-aligned organization, mimicking their cellular alignment in blood vessels exposed to laminar shear stress. However, the effects of spatial patterning on the function and global transcriptome of ECs are incompletely characterized. We used both parallel-aligned micropatterned and nanopatterned biomaterials to evaluate the effects of spatial patterning on the phenotype of ECs, based on gene expression profiling, functional characterization of monocyte adhesion, and quantification of cellular morphology. We demonstrate that both micropatterned and aligned nanofibrillar biomaterials could effectively guide EC organization along the direction of the micropatterned channels or nanofibrils, respectively. The ability of ECs to sense spatial patterning cues were abrogated in the presence of cytoskeletal disruption agents. Moreover, both micropatterned and aligned nanofibrillar substrates promoted an athero-resistant EC phenotype by reducing endothelial adhesiveness for monocytes and platelets, as well as by downregulating the expression of adhesion proteins and chemokines. We further found that micropatterned ECs have a transcriptional signature that is unique from non-patterned ECs, as well as from ECs aligned by shear stress. These findings highlight the importance of spatial patterning cues in guiding EC organization and function, which may have clinical relevance in the development of vascular grafts that promote patency.

Fig. 1. Micropatterned ECs have spatially oriented alignment and morphology

A. ECs organize their F-actin assembly along the direction of the microgrooves. Comparative immunofluorescence staining of FAK (B) and paxillin (C) on micropatterned or non-patterned control PDMS substrates. Corresponding F-actin staining are shown for reference. D-E. Quantification of cellular alignment by the angle of orientation (D) and order parameter (E). F. Quantification of cellular elongation by the cell shape index. Arrows indicate direction of microgrooves. Scale bar: 100 μm (A); 50 μm (B-C). ** P<0.0001 (n=3), *P<0.01 (n=3)

Fig. 2. Effect of micropatterning on endothelial adhesiveness

A. Shown are immunofluorescently labeled monocytes adhered onto micropatterned ECs. B. Quantification of monocyte adhesion (**P<0.0001, n=6). C. Gene expression of adhesion-related markers and chemokines (**P<0.0075, n=4). Arrow denotes direction of microgrooves. Scale bar: 250 μm.

Fig. 3. Aligned nanofibrillar collagen modulates EC alignment and morphology

A. SEM of randomly oriented and aligned collagen. B. ECs organize their F-actin assembly along the direction of the aligned nanofibrils. Comparative immunofluorescence staining of FAK (C) and paxillin (D) on randomly oriented and aligned collagen. Quantification of EC alignment based on the angle of orientation (E) and order parameter (F). Quantification of cell elongation based on the cell shape index (G). Arrows indicate direction of nanofibrils. Scale bar: 600 nm (A); 100 μm (B); 50 μm (C-D). ** P<0.0001 (n=6)

Fig. 4. Disruption of cytoskeletal proteins abrogate nanofibrillar collagen-mediated EC alignment and elongation

The addition of cytochalasin D (cyto D) or nocodazole (noco) inhibited F-actin assembly on aligned collagen substrates, based on immunofluorescence staining of F-actin or microtubules (A). Quantification of the angle of orientation (B) and cell shape index (C) in the presence of cytoskeleton disruption agents. Arrow denotes orientation of nanofibrils. Scale bar: 100 μm (A). **P<0.01 (n=3), *P<0.05 (n=3)

Fig 5. Effect of aligned nanofibrillar collagen on endothelial adhesiveness

A. Shown are immunofluorescently labeled monocytes or platelets adhered onto aligned nanofibrillar ECs. B. Quantification of monocyte adhesion (*P<0.05, n=4). C. Quantification of platelet adhesion (*P<0.05, n=6); D. Gene expression of adhesion related markers and chemokines (**P<0.01, *P<0.05, n=4). Arrow denotes direction of nanofibrils. Scale bar: 250 μm.

Fig 6. Transcriptional profiling of micropatterning-induced EC alignment

A. Heat map and clustering analysis of the top 100 upregulated and downregulated genes. Red indicates log2 intensity>0 and Green indicates log2<0. All Array data available from GEO (accession number GSE42596). B-C. Venn diagrams depict significantly upregulated (B) and downregulated (C) genes in common, when comparing ECs alignmed by by either micropatterning or shear stress.