Enhancement of Endothelialization by Topographical Features Is Mediated by PTP1B-Dependent Endothelial Adherens Junctions Remodeling

Abstract

Endothelial Cells (ECs) form cohesive cellular lining of the vasculature and play essential roles in both developmental processes and pathological conditions. Collective migration and proliferation of endothelial cells (ECs) are key processes underlying endothelialization of vessels as well as vascular graft, but the complex interplay of mechanical and biochemical signals regulating these processes are still not fully elucidated. While surface topography and biochemical modifications have been used to enhance endothelialization in vitro, thus far such single-modality modifications have met with limited success. As combination therapy that utilizes multiple modalities has shown improvement in addressing various intractable and complex biomedical conditions, here, we explore a combined strategy that utilizes topographical features in conjunction with pharmacological perturbations. We characterized EC behaviors in response to micrometer-scale grating topography in concert with pharmacological perturbations of endothelial adherens junctions (EAJ) regulators. We found that the protein tyrosine phosphatase, PTP1B, serves as a potent regulator of EAJ stability, with PTP1B inhibition synergizing with grating topographies to modulate EAJ rearrangement, thereby augmenting global EC monolayer sheet orientation, proliferation, connectivity, and collective cell migration. Our data delineates the crosstalk between cell−ECM topography sensing and cell−cell junction integrity maintenance and suggests that the combined use of grating topography and PTP1B inhibitor could be a promising strategy for promoting collective EC migration and proliferation.

Anisotropic topographies restore endothelial monolayer integrity and promote the proliferation of senescent endothelial cells

“…As it has been observed that micro-structured surfaces with gratings can enhance EC alignment and improve the integrity of cell-to-cell junctions ( 15 ), we produced surfaces with an anisotropic topography with gratings of 1 μm width and height ( Supplementary Figure 4 ) and tested under static conditions whether these topographies have a positive impact on the biomechanical properties of both non-senescent and senescent HUVECs. At first, we investigated the cell anchoring to the surface (flat vs. grating) by imaging paxillin and its phosphorylated form as important regulators of FA assembly and disassembly ( Figures 3A,B ) ( 30 ).…”

“…For example, Gorji et al showed that ECs cultivated under static conditions on a micro-scale grating topography respond similarly to the AJs and actin cytoskeleton remodeling that occurs under laminar flow conditions. In particular, EC cultivation on gratings promote their shape elongation due to F-actin stress fibers and the formation of linear VEC AJs, as shown by the increase in EC aspect ratio and tortuosity index, respectively ( 46 ). In line with these results, Franco et al demonstrated a 40% increase in endothelial spreading on gratings with groove width and depth of 1 μm due to focal adhesion kinase (FAK) activation, as well as enhanced FA maturation and myosin-II dependent cell contractility ( 47 ).…”

“…The tortuosity was then computed as the ratio of L / D . Tortuosity index values close to 1 indicate a straight junction, while higher values indicate highly convoluted junction topology ( 15 ). A measure of average cell orientation was obtained from the VEC trace or the F-actin signal using the freehand selection tool of ImageJ.…”

“…Biochemical changes during the transition from proliferating to senescent ECs are well characterized, but associated biomechanical changes are relatively unknown. As it has been observed that micro-structured surfaces with gratings can enhance EC alignment and improve the integrity of cell-to-cell junctions ( 15 ), we hypothesized that surfaces with gratings may not only improve the biomechanical properties of senescent ECs, but also increase their replication capacity.…”

Self-supervised classification of subcellular morphometric phenotypes reveals extracellular matrix-specific morphological responses

“…All in all, given that the number of morphological clusters observed are in the range of several, it is hopeful that the underlying biochemical signalling differences that underlie such heterogeneity will be tractable. In parallel to dissecting the origin of morphological heterogeneity, further experimentation could also explore the contribution of ECM density, substrate rigidity or viscoelastic properties, as well as spatial organization both at the micro-and nano-scale, in contributing to the morphological profiles of the cells 50,51 .…”