Topography-driven alterations in endothelial cell phenotype and contact guidance.
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ABSTRACT:
SUBMITTER: Almonacid Suarez AM
PROVIDER: S-EPMC7330714 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mammalian cells detect and respond to topographical cues presented in natural and synthetic biomaterials both in vivo and in vitro. Micro- and nano-structures influence the adhesion, morphology, proliferation, migration, and differentiation of many phenotypes. Although the mechanisms that underpin cell-topography interactions remain elusive, synthetic substrates with well-defined micro- and nano-structures are important tools to elucidate the origin of these responses. Substrates with reconfigurable topography are desirable because programmable cues can be harmonized with dynamic cellular responses. Here we present a lithography-free fabrication technique that can reversibly present topographical cues using an actuation mechanism that minimizes the confounding effects of applied stimuli. This method utilizes strain-induced buckling instabilities in bilayer substrate materials with rigid uniform silicon oxide membranes that are thermally deposited on elastomeric substrates. The resulting surfaces are capable of reversible of substrates between three distinct states: flat substrates (A = 1.53 ± 0.55 nm; Rms = 0.317 ± 0.048 nm); parallel wavy grating arrays (A∥= 483.6 ± 7.8 nm; λ∥= 4.78 ± 0.16 μm); perpendicular wavy grating arrays (A⊥= 429.3 ± 5.8 nm; λ⊥= 4.95 ± 0.36 μm). The cytoskeleton dynamics of 3T3 fibroblasts in response to these surfaces was measured using optical microscopy. Fibroblasts cultured on dynamic substrates that are switched from flat to topographic features (FLAT-WAVY) exhibit a robust and rapid change in gross morphology as measured by a reduction in circularity from 0.30 ± 0.13 to 0.15 ± 0.08 after 5 min. Conversely, dynamic substrate sequences of FLAT-WAVY-FLAT do not significantly alter the gross steady-state morphology. Taken together, substrates that present topographic structures reversibly can elucidate dynamic aspects of cell-topography interactions.
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Project description:Cancer cell migration is essential for metastasis, during which cancer cells move through the tumor and reach the blood vessels. In vivo, cancer cells are exposed to contact guidance and chemotactic cues. Depending on the strength of such cues, cells will migrate in a random or directed manner. While similar cues may also stimulate cell proliferation, it is not clear whether cell cycle progression affects migration of cancer cells and whether this effect is different in random versus directed migration. In this study, we tested the effect of cell cycle progression on contact guided migration in 2D and 3D environments, in the breast carcinoma cell line, FUCCI-MDA-MB-231. The results were quantified from live cell microscopy images using the open source lineage editing and validation image analysis tools (LEVER). In 2D, cells were placed inside 10 ?m-wide microchannels to stimulate contact guidance, with or without an additional chemotactic gradient of the soluble epidermal growth factor. In 3D, contact guidance was modeled by aligned collagen fibers. In both 2D and 3D, contact guidance was cell cycle-dependent, while the addition of the chemo-attractant gradient in 2D increased cell velocity and persistence in directionally migrating cells, regardless of their cell cycle phases. In both 2D and 3D contact guidance, cells in the G1 phase of the cell cycle outperformed cells in the S/G2 phase in terms of migration persistence and instantaneous velocity. These data suggest that in the presence of contact guidance cues in vivo, breast carcinoma cells in the G1 phase of the cell cycle may be more efficient in reaching the neighboring vasculature.
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Project description:Directed migration of groups of cells is a critical aspect of tissue morphogenesis that ensures proper tissue organization and, consequently, function. Cells moving in groups, unlike single cells, must coordinate their migratory behavior to maintain tissue integrity. During directed migration, cells are guided by a combination of mechanical and chemical cues presented by neighboring cells and the surrounding extracellular matrix. One important class of signals that guide cell migration includes topographic cues. Although the contact guidance response of individual cells to topographic cues has been extensively characterized, little is known about the response of groups of cells to topographic cues, the impact of such cues on cell-cell coordination within groups, and the transmission of nonautonomous contact guidance information between neighboring cells. Here, we explore these phenomena by quantifying the migratory response of confluent monolayers of epithelial and fibroblast cells to contact guidance cues provided by grooved topography. We show that, in both sparse clusters and confluent sheets, individual cells are contact-guided by grooves and show more coordinated behavior on grooved versus flat substrates. Furthermore, we demonstrate both in vitro and in silico that the guidance signal provided by a groove can propagate between neighboring cells in a confluent monolayer, and that the distance over which signal propagation occurs is not significantly influenced by the strength of cell-cell junctions but is an emergent property, similar to cellular streaming, triggered by mechanical exclusion interactions within the collective system.
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Project description:Background Posttranslational protein modification with O-linked N-acetylglucosamine (O-GlcNAc) is linked to high glucose levels in type 2 diabetes mellitus (T2DM) and may alter cellular function. We sought to elucidate the involvement of O-GlcNAc modification in endothelial dysfunction in patients with T2DM. Methods and Results Freshly isolated endothelial cells obtained by J-wire biopsy from a forearm vein of patients with T2DM (n=18) was compared with controls (n=10). Endothelial O-GlcNAc levels were 1.8-ford higher in T2DM patients than in nondiabetic controls (P=0.003). Higher endothelial O-GlcNAc levels correlated with serum fasting blood glucose level (r=0.433, P=0.024) and hemoglobin A1c (r=0.418, P=0.042). In endothelial cells from patients with T2DM, normal glucose conditions (24 hours at 5 mmol/L) lowered O-GlcNAc levels and restored insulin-mediated activation of endothelial nitric oxide synthase, whereas high glucose conditions (30 mmol/L) maintained both O-GlcNAc levels and impaired insulin action. Treatment of endothelial cells with Thiamet G, an O-GlcNAcase inhibitor, increased O-GlcNAc levels and blunted the improvement of insulin-mediated endothelial nitric oxide synthase phosphorylation by glucose normalization. Conclusions Taken together, our findings indicate a role for O-GlcNAc modification in the dynamic, glucose-induced impairment of endothelial nitric oxide synthase activation in endothelial cells from patients with T2DM. O-GlcNAc protein modification may be a treatment target for vascular dysfunction in T2DM.
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Project description:Adult stem cells such as mesenchymal stem cells (MSC) are known to possess the ability to augment neovascularization processes and are thus widely popular as an autologous source of progenitor cells. However there is a huge gap in our current knowledge of mechanisms involved in differentiating MSC into endothelial cells (EC), essential for lining engineered blood vessels. To fill up this gap, we attempted to differentiate human MSC into EC, by culturing the former onto chemically fixed layers of EC or its ECM, respectively. We expected direct contact of MSC when cultured atop fixed EC or its ECM, would coax the former to differentiate into EC. Results showed that human MSC cultured atop chemically fixed EC or its ECM using EC-medium showed enhanced expression of CD31, a marker for EC, compared to other cases. Further in all human MSC cultured using EC-medium, typically characteristic cobble stone shaped morphologies were noted in comparison to cells cultured using MSC medium, implying that the differentiated cells were sensitive to soluble VEGF supplementation present in the EC-medium. Results will enhance and affect therapies utilizing autologous MSC as a cell source for generating vascular cells to be used in a variety of tissue engineering applications.
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Project description:Mature Schwann cells (SCs) retain dedifferentiation potential throughout adulthood. Still, how dedifferentiation occurs remains uncertain. Results from a variety of cell-based assays using in vitro cultured cAMP-differentiated and myelinating SCs revealed the existence of a novel dedifferentiating activity expressed on the surface of dorsal root ganglion (DRG) axons. This activity had the capacity to prevent SC differentiation and elicit dedifferentiation through direct SC-axon contact. Evidence is provided showing that a rapid loss of myelinating SC markers concomitant to proliferation occurred even in the presence of elevated cAMP, a signal that is required to drive and maintain a differentiated state. The dedifferentiating activity was a membrane-bound protein found exclusively in DRG neurons, as judged by its subcellular partitioning, sensitivity to proteolytic degradation and cell-type specificity, and remained active even after disruption of cellular organization. It differed from the membrane-anchored neuregulin-1 isoforms that are responsible for axon contact-induced SC proliferation and exerted its action independently of mitogenic signaling emanating from receptor tyrosine kinases and mitogen-activated protein kinases such as ERK and JNK. Interestingly, dedifferentiation occurred without concomitant changes in the expression of Krox-20, a transcriptional enhancer of myelination, and c-Jun, an inhibitor of myelination. In sum, our data indicated the existence of cell surface axon-derived signals that override pro-differentiating cues, drive dedifferentiation and allow SCs to proliferate in response to axonal mitogens. This axonal signal may negatively regulate myelination at the onset or reversal of the differentiated state. GLIA 2017;65:851-863.
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Project description:Tissue engineering techniques for the regeneration of large bone defects require sufficient vascularisation of the applied constructs to ensure a sufficient supply of oxygen and nutrients. In our previous work, prevascularised 3D scaffolds have been successfully established by coculture of bone marrow derived stem cells (MSCs) and endothelial progenitor cells (EPCs). We identified stabilising pericytes (PCs) as part of newly formed capillary-like structures. In the present study, we report preliminary data on the interactions between MSCs and EPCs, leading to the differentiation of pericyte-like cells. MSCs and EPCs were seeded in transwell cultures, direct cocultures, and single cultures. Cells were cultured for 10 days in IMDM 10% FCS or IMDM 5% FCS 5% platelet lysate medium. Gene expression of PC markers, CD146, NG2, αSMA, and PDGFR-β, was analysed using RT-PCR at days 0, 3, 7, and 10. The upregulation of CD146, NG2, and αSMA in MSCs in direct coculture with EPCs advocates the MSCs' differentiation towards a pericyte-like phenotype in vitro. These results suggest that pericyte-like cells derive from MSCs and that cell-cell contact with EPCs is an important factor for this differentiation process. These findings emphasise the concept of coculture strategies to promote angiogenesis for cell-based tissue engineered bone grafts.
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Project description:The natural topographical microchannels in human skin have recently been shown to be capable of guiding propagating cracks. In this article we examine the ability to guide fracture by incorporating similar topographical features into both single, and dual layer elastomer membranes that exhibit uniform thickness. In single layer membranes, crack guidance is achieved by minimizing the nadir thickness of incorporated v-shaped channels, maximizing the release of localized strain energy. In dual layer membranes, crack guidance along embedded channels is achieved via interfacial delamination, which requires less energy to create a new surface than molecular debonding. In both membrane types, guided crack growth is only temporary. However, utilizing multiple embedded channels, non-contiguous crack control can be maintained at angles up to 45° from the mode I fracture condition. The ability to control and deflect fracture holds great potential for improving the robustness and lifespan of flexible electronics and stretchable sensors.
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Project description:Despite the ubiquitous importance of cell contact guidance, the signal-inducing contact guidance of mammalian cells in an aligned fibril network has defied elucidation. This is due to multiple interdependent signals that an aligned fibril network presents to cells, including, at least, anisotropy of adhesion, porosity, and mechanical resistance. By forming aligned fibrin gels with the same alignment strength, but cross-linked to different extents, the anisotropic mechanical resistance hypothesis of contact guidance was tested for human dermal fibroblasts. The cross-linking was shown to increase the mechanical resistance anisotropy, without detectable change in network microstructure and without change in cell adhesion to the cross-linked fibrin gel. This methodology thus isolated anisotropic mechanical resistance as a variable for fixed anisotropy of adhesion and porosity. The mechanical resistance anisotropy |Y*| -1 - |X*| -1 increased over fourfold in terms of the Fourier magnitudes of microbead displacement |X*| and |Y*| at the drive frequency with respect to alignment direction Y obtained by optical forces in active microrheology. Cells were found to exhibit stronger contact guidance in the cross-linked gels possessing greater mechanical resistance anisotropy: the cell anisotropy index based on the tensor of cell orientation, which has a range 0 to 1, increased by 18% with the fourfold increase in mechanical resistance anisotropy. We also show that modulation of adhesion via function-blocking antibodies can modulate the guidance response, suggesting a concomitant role of cell adhesion. These results indicate that fibroblasts can exhibit contact guidance in aligned fibril networks by sensing anisotropy of network mechanical resistance.
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Project description:The path created by aligned Schwann cells (SCs) after nerve injury underlies peripheral nerve regeneration. We developed geometric bioinspired substrates to extract key information needed for axon guidance by deconstructing the topographical cues presented by SCs. We have previously reported materials that directly replicate SC topography with micro- and nanoscale resolution, but a detailed explanation of the means of directed axon extension on SC topography has not yet been described. Here, using neurite tracing and time-lapse microscopy, we analyzed the SC features that influence axon guidance. Novel poly(dimethylsiloxane) materials, fabricated via photolithography, incorporated bioinspired topographical components with the shapes and sizes of aligned SCs, namely somas and processes, where the lengths of the processes were varied but the soma geometry and dimensions were kept constant. Rat dorsal root ganglia neurites aligned to all materials presenting bioinspired topography after 5days in culture and aligned to bioinspired materials presenting soma and process features after only 17h in culture. The key findings of this study were: neurite response to underlying bioinspired topographical features was time dependent, with neurites aligned most strongly to materials presenting combinations of soma and process features at 5days, with higher than average density of either process or soma features, but at 17h they aligned more strongly to materials presenting average densities of soma and process features and to materials presenting process features only. These studies elucidate the influence of SC topography on axon guidance in a time-dependent setting and have implications for the optimization of nerve regeneration strategies.
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