Polydopamine-based biofunctional substrate coating promotes mesenchymal stem cell migration

Rapid migration of mesenchymal stem cells (MSCs) on device surfaces could support in vivo tissue integration and might facilitate in vitro organoid formation. Here, polydopamine (PDA) is explored as a biofunctional coating to effectively promote MSC motility. It is hypothesized that PDA stimulates fibronectin deposition and in this way enhances integrin-mediated migration capability. The random and directional cell migration was investigated by time-lapse microscopy and gap closure assay respectively, and analysed with softwares as computational tools. A higher amount of deposited fibronectin was observed on PDA substrate, compared to the non-coated substrate. The integrin β1 activation and focal adhesion kinase (FAK) phosphorylation at Y397 were enhanced on PDA substrate, but the F-actin cytoskeleton was not altered, suggesting MSC migration on PDA was regulated by integrin initiated FAK signalling. This study strengthens the biofunctionality of PDA coating for regulating stem cells and offering a way of facilitating tissue integration of devices. Graphic abstract Polydopamine-coated substrate induces increased fibronectin deposition of mesenchymal stem cells, and promotes cell migration via integrin-initiated FAK signaling, compared to non-coated polystyrene-based standard tissue culture surface. In this way, multifunctional PDA coating could support in vivo tissue integration on implant surface and promote in vitro organoid formation.

Fibronectin-mediated inflammatory signaling through integrin α5 in vascular remodeling

Adhesion of vascular endothelial cells (ECs) to the underlying basement membrane potently modulates EC inflammatory activation. The normal basement membrane proteins laminin and collagen IV attenuate inflammatory signaling in part through integrin α2β1. In contrast, fibronectin, the provisional matrix protein found in injured, remodeling or inflamed vessels, sensitizes ECs to inflammatory stimuli through integrins α5β1and and αvβ3. A chimeric integrin in which the cytoplasmic domain of α5 is replaced with that of α2 pairs with β1 and binds fibronectin but signals like α2β1. Here, we examined mice in which integrin α5 is replaced with the α5/2 chimera, using the transverse aortic constriction (TAC) and partial carotid ligation (PCL) models of vessel remodeling. Following TAC and PCL surgery, WT mice showed increased fibronectin deposition and expression of inflammatory markers, which were strongly attenuated in a5/2 mice. α5/2 mice also showed reduced artery wall hypertrophy in the TAC model and diminished inward remodeling in the PCL model. Acute atherosclerosis after PCL in hyperlipidemic ApoE-/- mice on a high fat diet was dramatically decreased in α5/2 mice. These results underscore the key role for integrin α5 signaling in pathological vascular remodeling and support its potential as a therapeutic target.

Hyaluronic acid drives mesenchymal stromal cell-derived extracellular matrix assembly by promoting fibronectin fibrillogenesis

Hyaluronic acid (HA) is present at sites of ongoing fibronectin fibrillogenesis (fibrillar adhesions) and necessary for efficient fibronectin fibrillogenesis. As a result, fibronectin deposition can be enhanced by exogenous HA.

Tumor cell–organized fibronectin maintenance of a dormant breast cancer population

Tumors can undergo long periods of dormancy, with cancer cells entering a largely quiescent, nonproliferative state before reactivation and outgrowth. To understand the role of the extracellular matrix (ECM) in regulating tumor dormancy, we created an in vitro cell culture system with carefully controlled ECM substrates to observe entrance into and exit from dormancy with live imaging. We saw that cell populations capable of surviving entrance into long-term dormancy were heterogeneous, containing quiescent, cell cycle–arrested, and actively proliferating cells. Cell populations capable of entering dormancy formed an organized, fibrillar fibronectin matrix via αvβ3 and α5β1 integrin adhesion, ROCK-generated tension, and TGFβ2 stimulation, and cancer cell outgrowth after dormancy required MMP-2–mediated fibronectin degradation. We propose this approach as a useful, in vitro method to study factors important in regulating dormancy, and we used it here to elucidate a role for fibronectin deposition and MMP activation.

Integrin Affinity Modulation Critically Regulates Atherogenic Endothelial Activation in vitro and in vivo

While vital to platelet and leukocyte adhesion, the role of integrin affinity modulation in adherent cells remains controversial. In endothelial cells, atheroprone hemodynamics and oxidized lipoproteins drive an increase in the high affinity conformation of α5β1 integrins in endothelial cells in vitro, and α5β1 integrin inhibitors reduce proinflammatory endothelial activation to these stimuli in vitro and in vivo. However, the importance of α5β1 integrin affinity modulation to endothelial phenotype remains unknown. We now show that endothelial cells (talin1 L325R) unable to induce high affinity integrins initially adhere and spread, but show significant defects in nascent adhesion formation. In contrast, overall focal adhesion number, area, and composition in stably adherent cells are similar between talin1 wildtype and talin1 L325R endothelial cells. However, talin1 L325R endothelial cells fail to induce high affinity α5β1 integrins, fibronectin deposition, and proinflammatory responses to atheroprone hemodynamics and oxidized lipoproteins. Inducing the high affinity conformation of α5β1 integrins in talin1 L325R cells partially restores fibronectin deposition, whereas NF-κB activation and maximal fibronectin deposition require both integrin activation and other integrin-independent signaling. In endothelial-specific talin1 L325R mice, atheroprone hemodynamics fail to promote inflammation and macrophage recruitment, demonstrating a vital role for integrin activation in regulating endothelial phenotype.

Tumor cell-organized fibronectin is required to maintain a dormant breast cancer population

Tumors can undergo long periods of dormancy, with cancer cells entering a largely quiescent, non-proliferative state before reactivation and outgrowth. For a patient, these post-remission tumors are often drug resistant and highly aggressive, resulting in poor prognosis. To understand the role of the extracellular matrix (ECM) in regulating tumor dormancy, we created anin vitrocell culture system that combines carefully controlled ECM substrates with nutrient deprivation to observe entranceintoand exitfromdormancy with live imaging. We saw that cell populations capable of surviving entrance into long-term dormancy were heterogeneous, containing quiescent, cell cycle arrested, and actively proliferating cells. Cell populations that endured extended periods of serum-deprivation-induced dormancy formed an organized, fibrillar fibronectin matrix via αvβ3and α5β1integrin adhesion, ROCK-generated tension, and TGFβ2 stimulation. We surmised that the fibronectin matrix was primarily a mediator of cell survival, not proliferation, during the serum-deprivation stress, bacause cancer cell outgrowth after dormancy required MMP-2-mediated fibronectin degradation. Given the difficulty of animal models in observing entrance and exit from dormancy in real-time, we propose this approach as a new,in vitromethod to study factors important in regulating dormancy, and we used it here to elucidate a role for fibronectin deposition and MMP activation.