Initiate Cell Migration Research Articles

Mapping stress inside living cells by atomic force microscopy in response to environmental stimuli

ABSTRACT The response of cells to environmental stimuli, under either physiological or pathological conditions, plays a key role in determining cell fate toward either adaptive survival or controlled death. The efficiency of such a feedback mechanism is closely related to the most challenging human diseases, including cancer. Since cellular responses are implemented through physical forces exerted on intracellular components, more detailed knowledge of force distribution through modern imaging techniques is needed to ensure a mechanistic understanding of these forces. In this work, we mapped these intracellular forces at a whole-cell scale and with submicron resolution to correlate intracellular force distribution to the cytoskeletal structures. Furthermore, we visualized dynamic mechanical responses of the cells adapting to environmental modulations in situ. Such task was achieved by using an informatics-assisted atomic force microscope (AFM) indentation technique where a key step was Markov-chain Monte Carlo optimization to search for both the models used to fit indentation force–displacement curves and probe geometry descriptors. We demonstrated force dynamics within cytoskeleton, as well as nucleoskeleton in living cells which were subjected to mechanical state modulation: myosin motor inhibition, micro-compression stimulation and geometrical confinement manipulation. Our results highlight the alteration in the intracellular prestress to attenuate environmental stimuli; to involve in cellular survival against mechanical signal-initiated death during cancer growth and metastasis; and to initiate cell migration.

Cellular Response to Bone Morphogenetic Proteins-2 and -7 Covalently Bound to Photocrosslinked Heparin-Diazoresin Multilayer.

Despite the plethora of research that exists on recombinant human bone morphogenetic protein-2 and -7 (rhBMP-2 and rhBMP-7) and has been clinically approved, there is still a need to gain information that would allow for their more rational use in bone implantology. The clinical application of supra-physiological dosages of these superactive molecules causes many serious adverse effects. At the cellular level, they play a role in osteogenesis and cellular adhesion, migration, and proliferation around the implant. Therefore, in this work, we investigated the role of the covalent binding of rhBMP-2 and rhBMP-7 separately and in combination with ultrathin multilayers composed of heparin and diazoresin in stem cells. In the first step, we optimized the protein deposition conditions via quartz crystal microbalance (QCM). Then, atomic force microscopy (AFM) and enzyme-linked immunosorbent assay (ELISA) were used to analyze protein-substrate interactions. The effect of the protein binding on the initial cell adhesion, migration, and short-term expression of osteogenesis markers was tested. In the presence of both proteins, cell flattening and adhesion became more prominent, resulting in limited motility. However, the early osteogenic marker expression significantly increased compared to the single protein systems. The presence of single proteins resulted in the elongation of cells, which promoted their migration activity.

Bioinspired Redwood-Like Scaffolds Coordinated by In Situ-Generated Silica-Containing Hybrid Nanocoatings Promote Angiogenesis and Osteogenesis both In Vitro and In Vivo.

Inspired by natural redwood and bone, a biomimetic strategy is presented to develop a highly bioactive redwood-like nanocomposite via radial freeze casting of biocompatible hydrogels followed by the in situ coprecipitation of a Si-containing CaP hybrid nanocoating (SCPN). The engineered material displays radially aligned macrochannels and a porous network structure similar to those of natural redwood. In addition to acting as a mechanical reinforcement, introducing SCPNs into the weak redwood-like scaffold yields not only a nanoroughened surface topography, a low swelling ratio, retarded enzymatic degradation, and enhanced protein absorption abilities but also the sustained sequential release of Si and Ca ions, thereby providing essential biophysical and biochemical cues for effective bone regeneration. Benefiting from the redwood-like structures and bioactive SCPNs, the biomimetic materials create a favorable microenvironment for promoting the initial adhesion, spreading, proliferation, and migration of bone marrow-derived mesenchymal stem cells and human umbilical vein endothelial cells. Furthermore, the in vitro and in vivo data showed that the biocompatible redwood-like scaffold with precipitated SCPN can synergistically promote osteogenesis and angiogenesis in their aligned direction. Collectively, this work presents a novel bioinspired redwood-like material with multifunctional properties that provides new insight into bone defect repair.

Optogenetic control of intracellular flows and cell migration: A comprehensive mathematical analysis with a minimal active gel model.

The actin cytoskeleton of cells is in continuous motion due to both polymerization of new filaments and their contraction by myosin II molecular motors. Through adhesion to the substrate, such intracellular flow can be converted into cell migration. Recently, optogenetics has emerged as a new powerful experimental method to control both actin polymerization and myosin II contraction. While optogenetic control of polymerization can initiate cell migration by generating protrusion, it is less clear if and how optogenetic control of contraction can also affect cell migration. Here we analyze the latter situation using a minimal variant of active gel theory into which we include optogenetic activation as a spatiotemporally constrained perturbation. The model can describe the symmetrical flow of the actomyosin system observed in optogenetic experiments, but not the long-lasting polarization required for cell migration. Motile solutions become possible if cytoskeletal polymerization is included through the boundary conditions. Optogenetic activation of contraction can then initiate locomotion in a symmetrically spreading cell and strengthen motility in an asymmetrically polymerizing one. If designed appropriately, it can also arrest motility even for protrusive boundaries.

Bifunctional poly (l-lactic acid)/hydrophobic silica nanocomposite layer coated on magnesium stents for enhancing corrosion resistance and endothelial cell responses

Biodegradable magnesium (Mg)-based vascular stents can overcome the limitations of conventional permanent metallic stents, such as late in-stent restenosis and thrombosis, but still have difficulty retarding degradation while providing adequate mechanical support to the blood vessel. We incorporated silica nanoparticles surface-functionalized with hexadecyltrimethoxysilane (mSiNP) into a poly (l-lactic acid) (PLLA) coating as a physical barrier to disturb the penetration of the corrosive medium as well as a bioactive source that releases silicon ions capable of stimulating endothelial cells. The corrosion resistance and biocompatibility of this bifunctional PLLA/mSiNP nanocomposite coating were investigated using different weight ratios of mSiNP. The nanocomposite coating containing more than 10 wt% of the mSiNP (PLLA/10mSiNP and PLLA/20mSiNP) significantly delayed the corrosion of the Mg substrate and exhibited favorable endothelial cell responses, compared to the pure PLLA coating. Specifically, the calculated corrosion rates of PLLA/10mSiNP and PLLA/20mSiNP decreased by half, indicating the durability of the coating after immersion in simulated body fluid for 12 days. Based on the in vitro cellular response, the incorporation of the mSiNPs into the PLLA coating significantly improved the endothelial cell responses to the Mg substrate, showing better initial cell surface coverage, migration, and proliferation rate than those of pure PLLA. These results indicate that the PLLA/mSiNP nanocomposite coatings have significant potential to improve the corrosion resistance and vascular compatibility of biodegradable Mg-based vascular stents.

Using Xenopus Neural Crest Explants to Study Epithelial-Mesenchymal Transition.

The epithelial-mesenchymal transition (EMT) converts coherent epithelial structures into single cells. EMT is a dynamic cellular process that is not systematically completed (not all EMTs lead to single cells) and reversible (cells can re-epithelialize). EMT is orchestrated at multiple levels from transcription, to posttranslational modifications, to protein turnover. It involves remodeling of polarity and adhesion and enhances migratory capabilities. During physiological events such as embryogenesis or wound healing EMT is used to initiate cell migration, but EMT can also occur in pathological settings. In particular, EMT has been linked tofibrosis and cancer. Neural crest (NC) cells, an embryonic stem cell population whose behavior recapitulates the main steps of carcinoma progression, are a great model to study EMT. In this chapter, we provide a fully detailed protocol to extract NC cells from Xenopus embryos and culture them to study the dynamics of cell-cell adhesion, cell motility, and dispersion.

Sequential adaptation of perfusion and transport conditions significantly improves vascular construct recellularization and biomechanics.

Recellularization of ex vivo-derived scaffolds remains a significant hurdle primarily due to the scaffolds subcellular pore size that restricts initial cell seeding to the scaffolds periphery and inhibits migration over time. With the aim to improve cell migration, repopulation, and graft mechanics, the effects of a four-step culture approach were assessed. Using an ex vivo-derived vein as a model scaffold, human smooth muscle cells were first seeded onto its ablumen (Step 1: 3 hr) and an aggressive 0-100% nutrient gradient (lumenal flow under hypotensive pressure) was created to initiate cell migration across the scaffold (Step 2: Day 0 to 19). The effects of a prolonged aggressive nutrient gradient created by this single lumenal flow was then compared with a dual flow (lumenal and ablumenal) in Step 3 (Day 20 to 30). Analyses showed that a single lumenal flow maintained for 30 days resulted in a higher proportion of cells migrating across the scaffold toward the vessel lumen (nutrient source), with improved distribution. In Step 4 (Day 31 to 45), the transition from hypotensive pressure (12/8 mmHg) to normotensive (arterial-like) pressure (120/80 mmHg) was assessed. It demonstrated that recellularized scaffolds exposed to arterial pressures have increased glycosaminoglycan deposition, physiological modulus, and Young's modulus. By using this stepwise conditioning, the challenging recellularization of a vein-based scaffold and its positive remodeling toward arterial biomechanics were obtained.

Macrophage-derived exosomes accelerate wound healing through their anti-inflammation effects in a diabetic rat model

Chronic, subclinical inflammation was often observed in the diabetic wound area, causing inadequate and delayed wound-healing effects by failing to initiate cell migration, proliferation, and extracellular matrix deposition. Therefore, we presented macrophage-derived exosomes (Exos) and explored their potential for inhibiting inflammation and accelerating diabetic wound healing in a skin defect, diabetic rat model. A thorough investigation demonstrated that Exos exerted anti-inflammatory effects by inhibiting the secretion of pro-inflammatory enzymes and cytokines. Furthermore, they accelerated the wound-healing process by inducing endothelial cell proliferation and migration to improve angiogenesis and re-epithelialization in diabetic wounds.

YAP and TAZ limit cytoskeletal and focal adhesion maturation to enable persistent cell motility.

Cell migration initiates by traction generation through reciprocal actomyosin tension and focal adhesion reinforcement, but continued motility requires adaptive cytoskeletal remodeling and adhesion release. Here, we asked whether de novo gene expression contributes to this cytoskeletal feedback. We found that global inhibition of transcription or translation does not impair initial cell polarization or migration initiation, but causes eventual migratory arrest through excessive cytoskeletal tension and over-maturation of focal adhesions, tethering cells to their matrix. The transcriptional coactivators YAP and TAZ mediate this feedback response, modulating cell mechanics by limiting cytoskeletal and focal adhesion maturation to enable persistent cell motility and 3D vasculogenesis. Motile arrest after YAP/TAZ ablation was partially rescued by depletion of the YAP/TAZ-dependent myosin phosphatase regulator, NUAK2, or by inhibition of Rho-ROCK-myosin II. Together, these data establish a transcriptional feedback axis necessary to maintain a responsive cytoskeletal equilibrium and persistent migration.

The vesicular transfer of CLIC1 from glioblastoma to microvascular endothelial cells requires TRPM7.

Chloride intracellular channel 1 (CLIC1) is highly expressed and secreted by human glioblastoma cells and cell lines such as U87, initiating cell migration and tumor growth. Here, we examined whether CLIC1 could be transferred to human primary microvascular endothelial cells (HMEC). We previously reported that the oncogenic microRNA, miR-5096, increased the release of extracellular vesicles (EVs) by which it increased its own transfer from U87 to surrounding cells. Thus, we also examined its effect on the CLIC1 transfer. In homotypic cultures, miR-5096 did not increase the expression of CLIC1 in U87 nor in HMEC. However, the endothelial CLIC1 level increased after exposure to EVs released by U87, and even more by miR-5096-loaded U87. The EVs-transferred CLIC1 was active in HMEC, promoting endothelial sprouting in matrigel. Cell exposure to EVs induced cytosolic Ca2+ spikes which were dependent on the transient receptor potential melastatin member 7 (TRPM7). TRPM7 silencing prevented Ca2+ spikes and the subsequent CLIC1 delivery into HMEC. Our data suggest that the vesicular transfer of CLIC1 between cells requires TRMP7 expression in recipient endothelial cells. How the vesicular transfer of CLIC1 is modulated in cancer therapy is a future challenge.

Construction of Self-defensive Antibacterial and Osteogenic AgNPs/Gentamicin Coatings with Chitosan as Nanovalves for Controlled release

To solve the Ti implants-associated infection and poor osseointegration problems, we have constructed the AgNPs/gentamicin (Gen)-loaded silk fibroin (SF) coating with acceptable antibacterial and osteogenic aptitude. Nevertheless, due to uncontrollably sustained drug release, this bactericidal coating encountered some tricky problems, such as local high Ag concentration, short life-span and potential cytotoxicity. In this work, a chitosan (CS) barrier layer was constructed to prebuilt the SF-based film by two means, dip-coating (DCS) and spin-coating (SCS). Intriguingly, the CS barrier layer constructed by spin-coating highly improved the hydrophilic and protein-absorbed performances. As verified in the release profile, both coatings showed a prolonged and pH-dependent pattern of Ag+ with an accelerated release in acidic condition. Also, the multilayer coating with a SCS barrier layer showed an apparent bacteria-trigged antibacterial and biofilm-inhibited performances, whereas the improvements of antibacterial abilities of DCS coating were limited. The mechanisms could be explained that the pH decrease induced by the attachment and proliferation of bacteria triggered collapse of CS barrier layer, accelerating the release of bactericides. Moreover, benefitted from pH-dependent release behavior of Ag and bioactive SCS layer, functional coatings highly enhanced the initial adhesion, migration and proliferation of preosteoblast MC3T3-E1 cells, and subsequently accelerated osteoblast differentiation (alkaline phosphatase production). A relevant aspect of this work was to demonstrate the essential effect of reasonable construction of self-defensive barrier layer in achieving the balance between the high-efficiency bacterial killing and osteogenic activity, and highlighted its excellent potential in clinical applications.

Mechanistic role of transglutaminase-2 in focal adhesions

Transglutaminase (TG)-2 interacts with matrix proteins and integrins, forming focal adhesions (FA) to initiate cell migration, thus playing a vital role in wound healing. Previously we showed that TG-2 influenced phosphorylation of paxillin and other FA proteins. Here, we aimed to investigate the molecular mechanism of TG-2 regulation of paxillin. Human corneal epithelial cells expressing shRNA against TG-2 (shTG) and scrambled sequence control (shRNA) were cultured. TG-2 was pulled down by anti-paxillin antibody, but not MAP3K12. Cell-free interaction assay with immobilized paxillin shows that TG-2 bind to paxillin directly. JNK was the strongest kinase for paxillin phosphorylation in the in-vitro kinase screen, but TG-2 could not phosphorylate paxillin directly. Increasing TG-2 concentrations did not increase the amount of JNK in the TG-2/paxillin complex. Immunofluoresent staining shows that TG-2 colocalises with vinculin and paxillin in FA of migrating cells. TG-2 binds to paxillin and JNK-containing FA but does not recruit JNK directly. Taken together with previous findings, TG-2 binds paxillin non-covalently, and JNK can phosphorylate paxillin, these processes critically regulate corneal epithelial adhesion and migration.

Molecular genetic mechanisms of ovarian organization and development

A review of the literature devoted to the analysis of conditions and development of the female reproductive gland. From the moment of the formation of urogenital scallops to the formation of a full gonad, at the different stages of ontogeny, a huge number of genes, factors and proteins are expressed, since in them the primary sex cells and in their surroundings render folliculogenesis and determine the reproductive health of a woman. Aberrant production of these factors can be the cause of ovarian dysfunction and disorders. Different points of view on the emergence of the problem of migration and colonization of germ cells in female gonads, as well as directions for further fundamental and practical research. The development and differentiation of the follicle is a succession of events that are tightly regulated by endocrine factors, intraocular regulators and intercellular interactions. The initial migration of germ cells and their multiplication within the urogenital scallop require regulation using integrated signals such as growth factors, transcription and differentiation, secreted oocytes, transforming growth factors, growth factor and differentiation-9, bone morphogenetic proteins, stem cell factor, basic growth factor of fibroblasts, tumor factor of Williams transcription, gene steroidogenesis, antimulylerovy hormone, meiosis-regulating genes and many others, as well as a contact these interactions of germ cells with extracellular matrix proteins and cellular substrates attract the developing gonads. Recent studies indicate the possibility of obtaining human gametes from the culture of stem cells, a distinct molecular genetic mechanism of the origin, migration and colonization of the sex cells. Consequently, the understanding that all the subtleties and molecular mechanisms at each stage of the bookmarking and development of the ovaries, germ cells and their environment, the death of gametes, can help in finding regulators and preventing pathological follicular depletion.

Repellent and Attractant Guidance Cues Initiate Cell Migration by Distinct Rear-Driven and Front-Driven Cytoskeletal Mechanisms

SummaryAttractive and repulsive cell guidance is essential for animal life and important in disease. Cell migration toward attractants dominates studies [1, 2, 3, 4, 5, 6, 7, 8], but migration away from repellents is important in biology yet relatively little studied [5, 9, 10]. It is widely held that cells initiate migration by protrusion of their front [11, 12, 13, 14, 15], yet this has not been explicitly tested for cell guidance because cell margin displacement at opposite ends of the cell has not been distinguished for any cue. We argue that protrusion of the front, retraction of the rear, or both together could in principle break cell symmetry and start migration in response to guidance cues [16]. Here, we find in the Dictyostelium model [6] that an attractant—cAMP—breaks symmetry by causing protrusion of the front of the cell, whereas its repellent analog—8CPT—breaks symmetry by causing retraction of the rear. Protrusion of the front of these cells in response to cAMP starts with local actin filament assembly, while the delayed retraction of the rear is independent of both myosin II polarization and of motor-based contractility. On the contrary, myosin II accumulates locally in the rear of the cell in response to 8CPT, anticipating retraction and required for it, while local actin assembly is delayed and couples to delayed protrusion at the front. These data reveal an important new concept in the understanding of cell guidance.

Tenascin-C induces migration and invasion through JNK/c-Jun signalling in pancreatic cancer

Tenascin-C (TNC), a large extracellular matrix glycoprotein, has been reported to be associated with metastasis and poor prognosis in pancreatic cancer. However, the effects and mechanisms of TNC in pancreatic cancer metastasis largely remain unclear. We performed Transwell assays to investigate the effects of TNC on Capan-2, AsPC-1 and PANC-1 cells. In addition, western blot and RT-qPCR assays were used to examine potential TNC metastasis-associated targets, such as JNK/c-Jun, Paxillin/FAK, E-cadherin, N-cadherin, Vimentin, and MMP9/2. Lastly, we utilized a variety of methods, such as immunofluorescence, gelatin zymography and immunoprecipitation, to determine the molecular mechanisms of TNC in pancreatic cancer cell motility. The present study showed that TNC induced migration and invasion in pancreatic cancer cells and regulated a number of metastasis-associated proteins, including the EMT markers, MMP9 and Paxillin. Moreover, our data showed that TNC induced pancreatic cancer cells to generate an EMT phenotype and acquire motility potential through the activation of JNK/c-Jun signalling. In addition, TNC increased the DNA binding activity of c-Jun to the MMP9 promoter, an action likely resulting in increased MMP9 expression and activity. TNC/JNK also markedly induced the phosphorylation of Paxillin on serine 178, which is critical for the association between FAK and Paxillin and promoted the formation of focal adhesions. TNC/JNK initiates cell migration and invasion of pancreatic cancer cells through the promotion of EMT, the transactivation of MMP9 and the phosphorylation of Paxillin on serine 178. TNC may be a potential therapeutic target for treating pancreatic cancer metastasis.

Strontium (Sr) and silver (Ag) loaded nanotubular structures with combined osteoinductive and antimicrobial activities

Two frequent problems are associated with the titanium surfaces of bone/dental implants: lack of native tissue integration and associated infection. These problems have prompted a significant body of research regarding the modification of these surfaces. The present study describes a hydrothermal treatment for the fabrication of strontium (Sr) and silver (Ag) loaded nanotubular structures with different tube diameters on titanium surfaces. The Sr loading from a Sr(OH)2 solution was regulated by the size of the inner diameter of the titanium nanotubes (NT) (30nm or 80nm, formed at 10V or 40V, respectively). The quantity of Ag was adjusted by immersing the samples in 1.5 or 2.0M AgNO3 solutions. Sr and Ag were released in a controllable and prolonged matter from the NT–Ag.Sr samples, with negligible cytotoxicity. Prominent antibacterial activity was observed due to the release of Ag. Sr incorporation enhanced the initial cell adhesion, migration, and proliferation of preosteoblast MC3T3-E1 cells. Sr release also up-regulated the expression of osteogenic genes and induced mineralization, as suggested by the presence of more mineralized calcium nodules in cells cultured on NT–Ag.Sr surfaces. In vivo experiments showed that the Sr-loaded samples accelerated the formation of new bone in both osteoporosis and bone defect models, as confirmed by X-ray, Micro-CT evaluation, and histomorphometric analysis of rats implanted with NT–Ag.Sr samples. The antibacterial activity and outstanding osteogenic properties of NT–Ag.Sr samples highlight their excellent potential for use in clinical applications. Statement of SignificanceTwo frequent problems associated with Ti surfaces, widely used in orthopedic and dental arenas, are their lack of native tissue integration and risk of infection.We describe a novel approach for the fabrication of strontium (Sr) and silver (Ag) loaded nanotubular structures on titanium surfaces. A relevant aspect of this work is the demonstration of long-lasting and controllable Ag release, leading to excellent antibacterial and anti-adherent properties against methicillin-resistant Staphylococcus aureus (MRSA), and Gram-negative bacteria such as Escherichia coli. The extended release of Sr accelerates the filling of bone defects by improving the repair of damaged cortical bone and increasing trabecular bone microarchitecture. Our results highlight the potential of Sr and Ag loaded nanotubular structures for use in clinical applications.

Abstract 5217: Microenvironmental influences on glioma stem cell migration

Abstract Glioblastoma (GBM) is the most aggressive of the primary brain tumors and carries a poor prognosis due to its highly infiltrative nature and tendency to progress. Glioma stem cells are a small tumor-initiating population in GBM whose properties are incompletely understood. Autocrine and paracrine factors associated with specific microenvironmental niches, such as the perivascular space and hypoxic regions, are thought to attract and support this stem cell component. Precise mechanisms guiding the homing to these niches have not been defined. Glioma stem cells express the receptor CXCR4, which initiates cell migration in the presence of the chemokine, stromal cell-derived factor-1α (SDF-1α). Both hypoxic and vascular regions in the brain express SDF-1α. Therefore, we hypothesize that glioma stem cells transplanted into the brain are driven by SDF-1α chemoattraction towards vascular and hypoxic niches. Cells were sorted for the stem cell marker CD133 using MACS for two explanted human GBM cell lines grown as neurospheres (N08-74 and N08-30). Sorted cells were compared to unsorted cells by Western blot for expression of CD133, hypoxia inducible factor-1α (HIF-1α), CXCR4 and stem cell markers under normoxia and hypoxia. Two million CD133-sorted cells suspended in PBS were delivered into the nasal mucosa of NOD/SCID mice to test cellular migration into the brain via intranasal transplantation. Coronal sections of brain tissue were analyzed at 12 hours after intranasal cell delivery. In both GBM cell lines (N08-30 and N08-74), we confirmed that sorted cells expressed more CD133 than unsorted cells and that both cell lines expressed CXCR4. Further, CXCR4 expression was significantly greater in the CD133-sorted N08-30 cells with a trend in an increase of c-Myc expression compared to unsorted cells. Hypoxic exposure of N08-74 CD133-sorted cells showed a trend of increased HIF-1α and c-Myc expression. In mice, we demonstrated that CD133-sorted cells can migrate into the brain when delivered intranasally. To show the potential for migration towards the vasculature, we show that SDF-1α is expressed in the brain vasculature in vivo. These data demonstrate that GBM cells can be sorted for CD133-positive glioma stem cells, respond to hypoxia, and express the SDF-1α receptor, CXCR4. In one cell line, CXCR4 expression increased after CD133 cell sorting. With intranasal delivery, CD133-sorted cells can migrate into the brain. Understanding the migration behavior of glioma stem cells relative to the diverse microenvironments has implications in treatment for targeting certain migratory populations or microenvironments. These data prepare the conditions to test the migratory potential of glioma stem cells in hypoxic and vascular environments in the brain. Citation Format: Monica J. Chau, Myles R. McCrary, Subhas Mukherjee, Daniel J. Brat. Microenvironmental influences on glioma stem cell migration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5217. doi:10.1158/1538-7445.AM2015-5217

Corneal Regeneration by Deep Anterior Lamellar Keratoplasty (DALK) Using Decellularized Corneal Matrix.

The purpose of this study is to demonstrate the feasibility of DALK using a decellularized corneal matrix obtained by HHP methodology. Porcine corneas were hydrostatically pressurized at 980 MPa at 10°C for 10 minutes to destroy the cells, followed by washing with EGM-2 medium to remove the cell debris. The HHP-treated corneas were stained with H-E to assess the efficacy of decellularization. The decellularized corneal matrix of 300 μm thickness and 6.0 mm diameter was transplanted onto a 6.0 mm diameter keratectomy wound. The time course of regeneration on the decellularized corneal matrix was evaluated by haze grading score, fluorescein staining, and immunohistochemistry. H-E staining revealed that no cell nuclei were observed in the decellularized corneal matrix. The decellularized corneal matrices were opaque immediately after transplantation, but became completely transparent after 4 months. Fluorescein staining revealed that initial migration of epithelial cells over the grafts was slow, taking 3 months to completely cover the implant. Histological sections revealed that the implanted decellularized corneal matrix was completely integrated with the receptive rabbit cornea, and keratocytes infiltrated into the decellularized corneal matrix 6 months after transplantation. No inflammatory cells such as macrophages, or neovascularization, were observed during the implantation period. The decellularized corneal matrix improved corneal transparency, and remodelled the graft after being transplanted, demonstrating that the matrix obtained by HHP was a useful graft for corneal tissue regeneration.

Functional analysis of Hairy genes in Xenopus neural crest initial specification and cell migration.

Neural crest formation is one of the fundamental processes in the early stages of embryonic development in vertebrates. This transient and multipotent embryonic cell population is able to generate a variety of tissues and cell types in the adult body. hairy genes are transcription factors that contain a basic helix-loop-helix domain which binds to DNA. In Xenopus three hairy genes are known: hairy1, hairy2a, and hairy2b. The requirement of hairy genes was explored in early neural crest development although the late requirements of these genes during neural crest maintenance, migration and derivatives formation are still unknown. In this work, we extended the analysis of Xenopus hairy genes expression patterns and described new domains of expression. Functional analysis showed that hairy genes are required for the induction and migration of the neural crest and for the control of apoptosis. Moreover, we showed that hairy genes function as transcriptional repressors and that they are down-regulated by bone morphogenetic protein-Smad signaling and positively regulated by the Notch/Delta-Su(h) pathway. Our results indicate that hairy genes have a functional equivalence between them and that they are required for multiple processes during neural crest development.

Rho Kinase Inhibitor Fasudil Suppresses the Vasculogenic Mimicry of B16 Mouse Melanoma Cells Both In Vitro and In Vivo.

The aim of this study was to investigate the biologic role of the Rho kinase inhibitor fasudil in the vasculogenic mimicry (VM) of B16 mouse melanoma cells. It was previously reported that RhoA plays a critical role in angiogenesis by coordinating endothelial cell cytoskeleton remodeling and promoting endothelial cell motility. Although RhoA has been implicated in the regulation of angiogenesis, little has been described regarding its control of these tumor cell-lined channels. In this study, we established an in vitro model of VM using 3-dimensional cell culturing of mouse B16 melanoma cells and studied VM in vivo by transplanting B16 cells into C57/BL mice. Next, we explored the effect of RhoA and Rho-associated, coiled-coil containing protein kinase (ROCK) on VM formation using the Rho kinase inhibitor fasudil. We provide direct evidence that fasudil leads to reduced vascular-like channels in Matrigel. Additional experiments suggested that fasudil prevents both initial cellular architecture changes and cell migration in vitro. Finally, we provide in-depth evidence for the underlying mechanisms of fasudil-induced VM destruction using the Rho-GTPase agonist lysophosphatidic acid. In vivo studies revealed that fasudil reduced B16 melanoma cell xenograft tumor growth without causing significant toxicity in mice. Fasudil-treated tumors also displayed fewer VM channels. These results suggest that fasudil may be an emerging therapeutic option for targeting cancer VM.