The structural and mechanical properties of a surface often play an integral part in the determination of the cell adhesion strength and design parameters for creating a biodegradable electrospun scaffold. Nanofibers composed of the globular proteins bovine serum albumin (BSA) and fibronectin were produced by electrospinning with the electrospun protein scaffold serving as an extracellular matrix to which adhesion interaction will exist with cells via cell surface integrin. This interaction is vital in regulation cell differentiation, growth and migration and cell adhesion. We will demonstrate the ability to manipulate ligand-receptor interaction, the properties of the electrospun fibers, control and the formation of focal adhesions sites in cells cultured on the fibers with the ultimate goal of developing a biomimetric scaffold to investigate how cell adhesion molecules modulate cell behavior in a 3-dimentional culture.

Background: STAT3 is increasingly becoming known for its non-transcriptional regulation of mitochondrial bioenergetic function upon activation of its S727 residue (S727-STAT3). Lengthy mitochondrial dysfunction can lead to cell death. We tested whether an integrin-FAK-STAT3 signaling pathway we recently discovered regulates mitochondrial function and cell survival, and treatments thereof. Methods: Cultured mouse brain bEnd5 endothelial cells were treated with integrin, FAK or STAT3 inhibitors, FAK siRNA, as well as integrin and STAT3 activators. STAT3 null cells were transfected with mutant STAT3 plasmids. Outcome measures included oxygen consumption rate for mitochondrial bioenergetics, Western blotting for protein phosphorylation, mitochondrial membrane potential for mitochondrial integrity, ROS production, and cell counts. Results: Vitronectin-dependent mitochondrial basal respiration, ATP production, and maximum reserve and respiratory capacities were suppressed within 4 h by RGD and αvβ3 integrin antagonist peptides. Conversely, integrin ligands vitronectin, laminin and fibronectin stimulated mitochondrial function. Pharmacological inhibition of FAK completely abolished mitochondrial function within 4 h while FAK siRNA treatments confirmed the specificity of FAK signaling. WT, but not S727A functionally dead mutant STAT3, rescued bioenergetics in cells made null for STAT3 using CRISPR-Cas9. STAT3 inhibition with stattic in whole cells rapidly reduced mitochondrial function and mitochondrial pS727-STAT3. Stattic treatment of isolated mitochondria did not reduce pS727 whereas more was detected upon phosphatase inhibition. This suggests that S727-STAT3 is activated in the cytoplasm and is short-lived upon translocation to the mitochondria. FAK inhibition reduced pS727-STAT3 within mitochondria and reduced mitochondrial function in a non-transcriptional manner, as shown by co-treatment with actinomycin. Treatment with the small molecule bryostatin-1 or hepatocyte growth factor (HGF), which indirectly activate S727-STAT3, preserved mitochondrial function during FAK inhibition, but failed in the presence of the STAT3 inhibitor. FAK inhibition induced loss of mitochondrial membrane potential, which was counteracted by bryostatin, and increased superoxide and hydrogen peroxide production. Bryostatin and HGF reduced the substantial cell death caused by FAK inhibition over a 24 h period. Conclusion: These data suggest that extracellular matrix molecules promote STAT3-dependent mitochondrial function and cell survival through integrin-FAK signaling. We furthermore show a new treatment strategy for cell survival using S727-STAT3 activators.

Stimulation of β-adrenergic receptors (β-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits β-AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with β1 integrins. Herein we tested the hypothesis that MMP-2 impairs β1 integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited β-AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated β-AR-stimulated decreases in mitochondrial membrane potential. Overexpression of β1 integrins using adenoviruses expressing the human β1A-integrin decreased β-AR-stimulated cytochrome c release and apoptosis. Overexpression of β1 integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the β1 integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis.

Vitronectin (VTN) is a glycoprotein in the blood and affects hemostasis. VTN is also present in the extracellular matrix of various organs but little is known about its function in healthy adult tissues. We show, in adult mice, that VTN is uniquely expressed by approximately half of the pericytes of subventricular zone (SVZ) where neurogenesis continues throughout life. Intracerebral VTN antibody injection or VTN knockout reduced neurogenesis as well as expression of pro-neurogenic CNTF, and anti-neurogenic LIF and IL-6. Conversely, injections of VTN, or plasma from VTN+/+, but not VTN−/− mice, increased these cytokines. VTN promoted SVZ neurogenesis when LIF and IL-6 were suppressed by co-administration of a gp130 inhibitor. Unexpectedly, VTN inhibited FAK signaling and VTN−/− mice had increased FAK signaling in the SVZ. Further, an FAK inhibitor or VTN increased CNTF expression, but not in conditional astrocytic FAK knockout mice, suggesting that VTN increases CNTF through FAK inhibition in astrocytes. These results identify a novel role of pericyte-derived VTN in the brain, where it regulates SVZ neurogenesis through co-expression of CNTF, LIF and IL-6. VTN-integrin-FAK and gp130 signaling may provide novel targets to induce neurogenesis for cell replacement therapies.

Excessive endoplasmic reticulum (ER) stress leads to cell loss in many diseases, e.g., contributing to endothelial cell loss after spinal cord injury. Here, we determined whether ER stress-induced mitochondrial dysfunction could be explained by interruption of the focal adhesion kinase (FAK)-mitochondrial STAT3 pathway we recently discovered. ER stress was induced in brain-derived mouse bEnd5 endothelial cells by thapsigargin or tunicamycin and caused apoptotic cell death over a 72 h period. In concert, ER stress caused mitochondrial dysfunction as shown by reduced bioenergetic function, loss of mitochondrial membrane potential and increased mitophagy. ER stress caused a reduction in mitochondrial phosphorylated S727-STAT3, known to be important for maintaining mitochondrial function. Normal activation or phosphorylation of the upstream cytoplasmic FAK was also reduced, through mechanisms that involve tyrosine phosphatases and calcium signaling, as shown by pharmacological inhibitors, bisperoxovanadium (bpV) and 2-aminoethoxydiphenylborane (APB), respectively. APB mitigated the reduction in FAK and STAT3 phosphorylation, and improved endothelial cell survival caused by ER stress. Transfection of cells rendered null for STAT3 using CRISPR technology with STAT3 mutants confirmed the specific involvement of S727-STAT3 inhibition in ER stress-mediated cell loss. These data suggest that loss of FAK signaling during ER stress causes mitochondrial dysfunction by reducing the protective effects of mitochondrial STAT3, leading to endothelial cell death. We propose that stimulation of the FAK-STAT3 pathway is a novel therapeutic approach against pathological ER stress.

Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of many studies, the effects that environmental changes have on this form of movement are poorly understood. In certain pathologies where the cornea exists in a chronic hypoxic state, wound healing is delayed. The goal of this work is to examine the changes in corneal structure in hypoxic corneas that may affect migration and to determine the effects that changes in basement membrane stiffness and focal adhesion turnover have on epithelial cell migration. We analyzed migration after injury in organ cultures and determined that hypoxic corneas exhibited alterations in leading edge morphology. Under hypoxia, fibronectin localization to the apical epithelium and anterior stroma was reduced. Investigators have suggested that alterations in basal lamina composition may increase the stiffness of the membrane. To examine the effect that increased stiffness has on collective migration we performed traction force microscopy. Using multi-layered corneal epithelial sheets, we developed a novel method to analyze the generation of cellular traction forces and the directionality of sheet movement on polyacrylamide gels. We determined that the leading edges of corneal epithelial sheets undergo contraction prior to migration. Alterations in stiffness affected the amount of force exerted by cells at the leading edge. On stiffer surfaces, individual cells within sheets exhibited greater movement compared to cells on softer substrates. To further assess sheet dynamics, we examined the activation of focal adhesion proteins in hypoxic corneas and in human corneal limbal epithelial (HCLE) cells seeded onto soft and rigid substrates. Wounded hypoxic corneas displayed alterations in the localization of the focal adhesion proteins paxillin and vinculin. In HCLE cells cultured on stiff substrates, there was an elevation in vinculin pY1065 phosphorylation after injury, a reduction in vinculin-positive focal adhesions, and decreased actin bundle thickness. Our results demonstrate that changes in membrane stiffness may affect cellular tractions and vinculin dynamics, possibly contributing to the delayed healing response associated with certain corneal pathologies.

The objectives of this research were to better characterize the protein signaling complexes that form in response to spermatozoa binding to the bovine oocyte vitelline membrane and to elucidate their potential involvement in oocyte activation. Integrins located on the vitelline membrane of bovine oocytes have been implicated in mediating the sperm-oocyte interaction. Anti-integrin function blocking antibodies and immunofluorescence were utilized in order to reveal that the αV and β1 integrin subunits are essential for fertilization in the bovine and could form the integrin heterodimer involved in the sperm-oocyte interaction. Focal adhesion kinase is localized to focal adhesions and is a key component of signal transduction pathways mediated by integrins. The presence of focal adhesion kinase in bovine oocytes was verified by real-time polymerase chain reaction and immunoprecipitation and the localization of focal adhesion kinase at the site of sperm binding to the oocyte plasma membrane was verified using immunohistochemistry. The inhibition of focal adhesion kinase resulted in fewer cleaved embryos in addition to a reduction in the number of oocytes responding with calcium transients. Phospholipase C isoforms regulate the release of calcium from the endoplasmic reticulum and are known to interact with integrins and focal adhesion kinase. The experiments reported in this dissertation explored the involvement of phospholipase C isoforms endogenous to the oocyte in mediating the calcium release associated with fertilization. Reduction in phospholipase C messenger ribonucleic acid levels for the phospholipase C isoforms γ1 and γ2 resulted in significantly lower cleavage rates compared to the controls. Interestingly, the reduction in messenger ribonucleic acid levels for phospholipase ζ failed to impact cleavage. Maximizing protein levels for the phospholipase C isoforms ζ and γ2 resulted in a significantly higher number of oocytes reaching the 2-cell stage compared to all other treatment groups and not significantly different than the activation control. Together these data illustrate the involvement of the αV and β1 integrin subunits, focal adhesion kinase, and the potential involvement of multiple endogenous phospholipase C isoforms (γ1 and γ2) in bovine oocyte activation. A more complete understanding of the molecular players involved in fertilization could have beneficial impacts for human fertility, assisted reproduction, and improved efficiency of animal somatic cell nuclear transfer.

Cell-substrate interactions are important to study for development of accurate in vitro research platforms.  Recently it has been demonstrated that physical microenvironment of cells directly affects cellular motility and cytoskeletal arrangement.  Specifically, previous studies have explored the role of material stiffness (Young's modulus: N/m2) on cell behavior including attachment, spreading, migration, cytoskeleton arrangement (stress fiber and focal adhesion distribution) and differentiation. In this study using our recently described non-electrospinning fiber manufacturing platform, customized scaffolds of suspended nanofibers are developed to study single cell behavior in a tunable structural stiffness (N/m) environment.  Suspended fibers of three different diameters (400, 700 and 1200 nm) are deposited in aligned configurations in two lengths of 1 and 2 mm using the previously described STEP (Spinneret based Tunable Engineered Parameters) platform.  These fibers present a gradient of structural stiffness to the cells at constant material stiffness.   Single cells attached to fibers are constrained to move along the fiber axis and with increase in structural stiffness are observed to spread to longer lengths, put out longer focal adhesions, have elongated nucleus with decreased migration rates. Furthermore, more than 60% of cell population is observed to migrate from areas of low to high structural stiffness. Additionally dividing cells are observed to round up and daughter cells are observed to migrate away from each other after division. Interestingly, dividing rounded cells are found to be anchored to the fibers through thin protrusions emanating from the focal adhesion sites. These results indicate a substrate stiffness sensing mechanism that goes beyond the traditionally accepted modulus sensing that cells have been shown to respond to previously.  From this work, the importance of structural stiffness in cellular mechanosensing at the single cell-nanofiber scaled warrants consideration of the above factors in accurate design of scaffolds in future. / Master of Science

Indiana University-Purdue University Indianapolis (IUPUI) / Mesenchymal stem cells (MSCs) are responsive to mechanical stimuli that play an essential role in directing their differentiation to the chondrogenic lineage. A better understanding of the mechanisms that allow MSCs to respond to mechanical stimuli is important to improving cartilage tissue engineering and regenerative medicine. Hydrostatic pressure (HP) in particular is known to be a primary mechanical force in joints. However, little is known about the underlying mechanisms that facilitate HP mechanotransduction. Understanding the signaling pathways in MSCs in transducing HP to a beneficial biologic response and their interrelationship were the focus of this thesis. Studies used porcine marrow-derived MSCs seeded in agarose gel. Calcium ion Ca++ signaling, focal adhesion kinase (FAK) involvement, and sirtuin1 activity were investigated in conjunction with HP application. Intracellular Ca++ concentration was previously shown to be changed with HP application. In our study a bioreactor was used to apply a single application of HP to the MSC-seeded gel structures and observe Ca++ signaling via live imaging of a fluorescent calcium indicator in cells. However, no fluctuations in Ca++ concentrations were observed with 10 minutes loading of HP. Additionally a problem with the biore actor design was discovered. First the gel was floating around in the bioreactor even without loading. After stabilizing the gel and stopping it from floating, there were still about 16 µm of movement and deformation in the system. The movement and deformation was analyzed for the gel structure and different parts of the bioreactor. Furthermore, we investigated the role of FAK in early and late chondrogenesis and also its involvement in HP mechanotransduction. A FAK inhibitor was used on MSCs from day 1 to 21 and showed a dose-dependent suppression of chondrogenesis. However, when low doses of FAK inhibitor added to the MSC culture from day 21 to 42, chondrogenesis was not inhibited. With 4 hour cyclic HP, FAK phosphorylation increased. The beneficial effect of HP was suppressed with overnight addition of the FAK inhibitor to MSC medium, suggesting FAK involvement in HP mechanotransd ucation by MSCs. Moreover, sirtuin1 participation in MSC chondrogenesis and mechanotransduc tion was also explored. The results indicated that overnight sirtuin1 inhibition in creased chondrogenic gene expression (Agc, Col2, and Sox9) in MSCs. Additionally, the activity of sirtuin1 was decreased with both 4 hour cyclic hydrostatic pressure and inhibitor application. These two together demonstrated that sirtuin1 inhibition enhances chondrogenesis. In this research we have investigated the role of Ca++ signaling, FAK involvement, and sirtuin1 activity in the mechanotransduction of HP in MSCs. These understand ings about the mechanisms regulating the chondrogenesis with respect to HP could have important implications for cartilage tissue engineering and regenerative studies.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21159号 / 農博第2285号 / 新制||農||1060(附属図書館) / 学位論文||H30||N5133(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 阪井 康能, 教授 矢﨑 一史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM