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.
20Symmetry breaking of protein distribution and cytoskeleton organization is an 21 essential aspect for development of apico--basal polarity. In embryonic cells this 22 process is largely cell autonomous, while differentiated epithelial cells collectively 23 polarize during epithelium formation. We report here that the de novo polarization 24 of mature hepatocytes is a cell autonomous process. Single hepatocytes developed 25 bona fide secretory hemi--apical lumens upon adhesion to finely tuned substrates 26 bio--functionalized with cadherin and extra cellular matrix. The creation of this single 27 cell liver allows unprecedented control and imaging resolution of the lumenogenesis 28 process. We demonstrate that the density and localization of cadherins along the 29 initial cell--cell contact acted as a key factor triggering the reorganization from lateral 30 to apical actin cortex. Consequently, we established why hepatocytes could form 31 asymmetric lumens in heterotypic doublets involving another ectopic epithelial cell 32 originating from kidney, breast, or colon. 33 34 stimulate the creation of a border brush with a partial localization of the polarity 50 complexes (17).The question then arises whether de novo establishment of epithelial 51 polarity is a cell autonomous response triggered by external cues or a collective 52 response associated to the concomitant development of polarity in neighboring 53 cells. Usual epithelial models (tissue, cell monolayer, or cysts) inherently fail to 54 address this question. Considering a cell polarizing in these multi--cellular contexts, 55 the concomitant reorganization of cell--cell contacts of the neighboring cells can act 56 as a polarization cue and as a response to its polarization establishment. 57This study presents a novel model where single primary hepatocytes develop 58 independent bona fide secretory apical poles when grown in synthetic 59 microenvironments. In this context, we demonstrate for the first time that de novo 60 apical lumen development is a genetically controlled cell autonomous process. It 61 depends mainly on actin cortex rearrangements triggered by the biophysical 62properties of the cadherin--mediated adherens junction along the initial lateral cell--63 cell contact. Subsequently, we demonstrate that, hepatocytes can form functional 64 lumen with a whole variety of epithelial cells. In particular, we show that mature 65 hepatocytes can polarize with immature hepatocytes during the differentiation 66 process. 67 68 Results: 69We investigated the de novo lumenogenesis of bile canaliculi to test if the apical 70 polarity development is a cell autonomous process triggered by simple cues sensed 71 by the cell along the non--polarized initial cell--cell contact. We used hepatocytes at 72 different maturation stages during differentiation. Mature hepatocytes develop 73 intercellular secretory apical lumen called bile canaliculi, connecting the hepatocytes 74 to the biliary system. They consist of small, elongated tubules (2 μm in diameter) 75 sealed...
Rationale:Failure of small synthetic vascular grafts is largely due to late endothelialization and has been an ongoing challenge in the treatment of cardiovascular diseases. Objective:Previous strategies developed to promote graft endothelialization include surface topographical modulation and biochemical modifications. However, these have been met with limited success. Importantly, although the integrity of Endothelial Cell (EC) monolayer is crucial for endothelialization, the crosstalk between surface topography and cell-cell connectivity is still not well understood. Here we explored a combined strategy that utilizes both topographical features and pharmacological perturbations. Methods and result:We characterized EC behaviors in response to micron-scale grating topography in conjunction with pharmacological perturbations of endothelial adherens junctions (EAJ) regulators. We studied the EA.hy 926 cell-cell junctions and monolayer integrity using the junctional markers upon the inhibitory effect of EAJ regulator on both planar and grating topographies substrates.We identified a protein tyrosine phosphatase, PTP1B, as a potent regulator of EAJ stability. Next, we studied the physiologically relevant behaviors of EC using primary human coronary arterial endothelial cells (HCAEC). Our results showed that PTP1B inhibition synergized with grating topographies to modulate EAJ rearrangement, thereby controlling global EC monolayer sheet orientation, connectivity and collective cell migration to promote endothelialization.Our results showed that PTP1B inhibition synergized with grating topographies to modulate EAJ rearrangement, thereby controlling global EC monolayer sheet orientation, connectivity and collective cell migration and proliferation. Conclusion:The synergistic effect of PTP1B inhibition and grating topographies could be useful for the promotion of endothelialization by enhancing EC migration and proliferation. IntroductionHealthy blood vessels are covered by a contiguous monolayer of endothelial cells (ECs), which maintain vasculature hemostasis and serve as a blood-compatible interface. Injured arteries and the associated EC malfunctions result in inflammatory responses, subsequent vessel thickening due to the migration and proliferation of smooth muscle cells, plaque formation, and partial or complete occlusion of the vessels [1]. Such atherosclerotic processes underlie peripheral and cardiovascular diseases, stroke, and myocardial infarction which are among the most prevalent causes of death worldwide [2,3]. Among the available treatment options, the replacement of the occluded vessel with either an autologous vein or a synthetic vascular graft has been widely practiced. However, while the implantation of large vessels has been a standard procedure, synthetic small vascular graft (diameter <6 mm) has remained problematic. The primary cause of small graft failure has been attributed to the lack of proper endothelialization [4] [5] [6]. Thus, there remains an unmet clinical need for a small vascular gra...
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