Single‐Cell Transcriptomic Census of Endothelial Changes Induced by Matrix Stiffness and the Association with Atherosclerosis

Abstract

Vascular endothelial cell (EC) plasticity plays a critical role in the progression of atherosclerosis by giving rise to mesenchymal phenotypes in the plaque lesion. Despite the evidence for arterial stiffening as a major contributor to atherosclerosis, the complex interplay among atherogenic stimuli in vivo has hindered attempts to determine the effects of extracellular matrix (ECM) stiffness on endothelial‐mesenchymal transition (EndMT). To study the regulatory effects of ECM stiffness on EndMT, an in vitro model is developed in which human coronary artery ECs are cultured on physiological or pathological stiffness substrates. Leveraging single‐cell RNA sequencing, cell clusters with mesenchymal transcriptional features are identified to be more prevalent on pathological substrates than physiological substrates. Trajectory inference analyses reveal a novel mesenchymal‐to‐endothelial reverse transition, which is blocked by pathological stiffness substrates, in addition to the expected EndMT trajectory. ECs pushed to a mesenchymal character by pathological stiffness substrates are enriched in transcriptional signatures of atherosclerotic ECs from human and murine plaques. This study characterizes at single‐cell resolution the transcriptional programs that underpin EC plasticity in both physiological or pathological milieus, and thus serves as a valuable resource for more precisely defining EndMT and the transcriptional programs contributing to atherosclerosis.

Elastin‐like protein hydrogels with controllable stress relaxation rate and stiffness modulate endothelial cell function

“…With these rheological properties in mind, we created a family of ELP‐PEG hydrogels, which we refer to as hydrogels of low‐ (~500 Pa), medium‐ (~1.2 kPa), and high‐ (~3.3 kPa) stiffness (Figure 2C,D). This range in stiffness values was consistent with the range of reported values for EC stiffness (~0.5–4 kPa) 6,34–37 . To achieve stiffness‐matched hydrogel conditions, we modified the ELP concentration for the gels containing PEG‐ALD and the gels containing PEG‐BZA.…”

“…To quantify the degradation of ELP-PEG hydrogels in vivo, fluorescently labeled protein-engineered hydrogels fabricated from ELP-HYD modified with tyrosine selective 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-dione azide (PTAD-AZ) 31 were conjugated with sulfonatedcyanine 5 (Cy5) via click-chemistry to yield ELP-HYD-Cy5. The fluorescent ELP was mixed at a 1:10 ratio with nonfluorescent ELP-HYD before encapsulating 10 6 HUVECs and HDNFs at a 1:1 ratio and then injecting subcutaneously in the abdomen of SCID mice.…”

“…This range in stiffness values was consistent with the range of reported values for EC stiffness ($0.5-4 kPa). 6,[34][35][36][37] To achieve stiffness-matched hydrogel conditions, we modified the ELP concentration for the gels containing PEG-ALD and the gels containing PEG-BZA. Due to differences in equilibrium constants for the respective crosslinking reactions, 38 it was necessary to modify the concentration of ELP to achieve the same elastic modulus when substituting the aliphatic aldehyde for the benzaldehyde (Figure S4).…”

“…The ECM microenvironment mediates the response of vascular ECs to form new vasculature through biomolecular and physical cues. Previous studies from our group and others have demonstrated that cell‐adhesive ligands and mechanical properties differentially regulate cellular responses such as cell migration, proliferation, differentiation, dedifferentiation, and apoptosis 3–13 . ECMs within the human body are viscoelastic rather than purely elastic, and cells respond to matrix viscoelasticity 14–16 .…”

“…Previous studies from our group and others have demonstrated that cell-adhesive ligands and mechanical properties differentially regulate cellular responses such as cell migration, proliferation, differentiation, dedifferentiation, and apoptosis. [3][4][5][6][7][8][9][10][11][12][13] ECMs within the human body are viscoelastic rather than purely elastic, and cells respond to matrix viscoelasticity. [14][15][16] Naturally derived ECMs exhibit stress relaxation, in which a cell-applied force results in matrix remodeling, leading to the relaxation of cell-imposed stress.…”