Nucleolin promotes angiogenesis and endothelial metabolism along the oncofetal axis in the human brain vasculature.

Glioblastomas are among the deadliest human cancers and are highly vascularized. Angiogenesis is dynamic during brain development, almost quiescent in the adult brain but reactivated in vascular-dependent CNS pathologies, including brain tumors. The oncofetal axis describes the reactivation of fetal programs in tumors, but its relevance in endothelial and perivascular cells of the human brain vasculature in glial brain tumors is unexplored. Nucleolin is a regulator of cell proliferation and angiogenesis, but its roles in the brain vasculature remain unknown. Here, we studied the expression of Nucleolin in the neurovascular unit in human fetal brains, adult brains, and human gliomas in vivo as well as its effects on sprouting angiogenesis and endothelial metabolism in vitro. Nucleolin is highly expressed in endothelial and perivascular cells during brain development, downregulated in the adult brain, and upregulated in glioma. Moreover, Nucleolin expression correlated with glioma malignancy in vivo. In culture, siRNA-mediated Nucleolin knockdown reduced human brain endothelial cell (HCMEC) and HUVEC sprouting angiogenesis, proliferation, filopodia extension, and glucose metabolism. Furthermore, inhibition of Nucleolin with the aptamer AS1411 decreased brain endothelial cell proliferation in vitro. Mechanistically, Nucleolin knockdown in HCMECs and HUVECs uncovered regulation of angiogenesis involving VEGFR2 and of endothelial glycolysis. These findings identify Nucleolin as a neurodevelopmental factor reactivated in glioma that promotes sprouting angiogenesis and endothelial metabolism, characterizing Nucleolin as an oncofetal protein. Our findings have potential implications in the therapeutic targeting of glioma.

Nanoconfinement of microvilli alters gene expression and boosts T cell activation.

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.

Phosphorylated fibronectin enhances cell attachment and upregulates mechanical cell functions.

A large number of extracellular matrix proteins have been found in phosphorylated states, yet little is known about how the phosphorylation of extracellular matrix proteins might affect cell functions. We thus tested the hypothesis whether the phosphorylation of fibronectin, a major adhesion protein, affects cell behavior. Controlled in vitro phosphorylation of fibronectin by a casein kinase II (CKII) significantly upregulated cell traction forces and total strain energy generated by fibroblasts on nanopillar arrays, and consequently other elementary cell functions including cell spreading and metabolic activity. Mass spectrometry of plasma fibronectin from healthy human donors then identified a constitutively phosphorylated site in the C-terminus, and numerous other residues that became phosphorylated by the CKII kinase in vitro. Our findings open up novel strategies for translational applications including targeting diseased ECM, or to develop assays that probe the phosphorylation state of the ECM or blood as potential cancer markers.

Nanopillar force measurements reveal actin-cap-mediated YAP mechanotransduction.

A robust nanopillar platform with increased spatial resolution reveals that perinuclear forces, originating from stress fibres spanning the nucleus of fibroblasts, are significantly higher on these nanostructured substrates than the forces acting on peripheral adhesions. Many perinuclear adhesions embrace several nanopillars at once, pulling them into ß1-integrin- and zyxin-rich clusters, which are able to translocate in the direction of cell motion without losing their tensile strength. The high perinuclear forces are greatly reduced upon inhibition of cell contractility or actin polymerization and disruption of the actin cap by KASH dominant-negative mutant expression. LMNA null fibroblasts have higher peripheral versus perinuclear forces, impaired perinuclear ß1-integrin recruitment, as well as YAP nuclear translocation, functional alterations that can be rescued by lamin A expression. These highly tensed actin-cap fibres are required for YAP nuclear signalling and thus play far more important roles in sensing nanotopographies and mechanochemical signal conversion than previously thought.

Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis.

Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. 'compact and adsorbed to collagen' versus 'extended and fibrillar' fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin's contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen decoration of single fibronectin fibers enhanced the non-persistent migration of both Fnf/f and Fn-/- MEFs, the migration speed was increased for Fn-/- MEFs on plasma fibronectin fibers compared to Fnf/f MEFs. In contrast, the average speed was the same for all cells on collagen-coated Fn fibers. A Fn-FRET sensor revealed that fibronectin on average was more extended on the microtissue surface compared to fibronectin in the core. Gradients of collagen-to-fibronectin ratios and of the fraction of collagen-adsorbed to stretched fibrillar fibronectin conformations might thereby provide critical cell migration cues. This study highlights a dominant role for fibronectin in tissue morphogenesis and the development of tissue heterogeneities.

Nogo-A is a negative regulator of CNS angiogenesis.

Nogo-A is an important axonal growth inhibitor in the adult and developing CNS. In vitro, Nogo-A has been shown to inhibit migration and cell spreading of neuronal and nonneuronal cell types. Here, we studied in vivo and in vitro effects of Nogo-A on vascular endothelial cells during angiogenesis of the early postnatal brain and retina in which Nogo-A is expressed by many types of neurons. Genetic ablation or virus-mediated knock down of Nogo-A or neutralization of Nogo-A with an antibody caused a marked increase in the blood vessel density in vivo. In culture, Nogo-A inhibited spreading, migration, and sprouting of primary brain microvascular endothelial cells (MVECs) in a dose-dependent manner and induced the retraction of MVEC lamellipodia and filopodia. Mechanistically, we show that only the Nogo-A-specific Delta 20 domain exerts inhibitory effects on MVECs, but the Nogo-66 fragment, an inhibitory domain common to Nogo-A, -B, and -C, does not. Furthermore, the action of Nogo-A Delta 20 on MVECs required the intracellular activation of the Ras homolog gene family, member A (Rho-A)-associated, coiled-coil containing protein kinase (ROCK)-Myosin II pathway. The inhibitory effects of early postnatal brain membranes or cultured neurons on MVECs were relieved significantly by anti-Nogo-A antibodies. These findings identify Nogo-A as an important negative regulator of developmental angiogenesis in the CNS. They may have important implications in CNS pathologies involving angiogenesis such as stroke, brain tumors, and retinopathies.

Investigation of the growth of focal adhesions using protein nanoarrays fabricated by nanocontact printing using size tunable polymeric nanopillars.

Here we describe a simple approach to create various sizes of protein nanoarrays for the investigation of cell adhesion. Using a combination of nanosphere lithography, oxygen plasma treatment, deep etching and nanomolding processes, well-ordered polymeric nanopillar arrays have been fabricated with diameters in the range of 50-600 nm. These nanopillar arrays were used as stamps for nanocontact printing to create fibronectin nanoarrays, which were used to study the size dependent formation of focal adhesion. It was found that cells can adhere and spread on fibronectin nanoarrays with a fibronectin pattern as small as 50 nm. It was also found that the average size of focal adhesion decreased as the size of the fibronectin pattern was reduced.

Polymeric nanopillar arrays for cell traction force measurements.

This paper reports the development of a novel force measurement device based on polymeric nanopillar arrays. The device was fabricated by a process combining nanosphere lithography, oxygen plasma treatment, deep etching and nano-molding. Well-ordered polymeric nanopillar arrays with various diameters and aspect ratios were fabricated and used as cell culture substrates. Cell traction forces were measured by the deflection of the nanopillars. Since the location of the nanopillars can be monitored at all times, this device allows for the measurement of the evolution of adhesion forces over time.

Observation of enhanced cell adhesion and transfection efficiency on superhydrophobic surfaces.

It was found that cells attached preferentially on the roughened area of patterned superhydrophobic surfaces allowing the formation of cell microarrays with the advantages of improved cell adhesion, natural separation of colonies and enhanced transfection efficiency.

Revealing the spatial distribution of the site enhancement for the surface enhanced Raman scattering on the regular nanoparticle arrays.

The spatial distribution of the site enhancement for the surface-enhanced Raman scattering (SERS) on the regular nanoparticle arrays has been investigated by the confocal Raman microscopy. It was found that the spatial distribution of the Raman signals on the well-ordered nanoparticle arrays was very inhomogeneous and concentrated on the defects of the nanoparticle arrays. The SERS signals were also observed to depend on the thickness of silver film and the defect density. It has been demonstrated that the number of SERS active sites can be increased ten folds by trimming the size of nanoparticles using oxygen plasma.

Behavior of single DNA molecules in the well-ordered nanopores.

Here we describe a simple approach to fabricate robust three-dimensional periodic porous nanostructures inside the microchannels. In this approach, the colloidal crystals were first grown inside the microchannel using an evaporation-assisted self-assembly process. Then the void spaces among the colloidal crystals were filled with epoxy-based negative tone photoresist. After subsequent development and nanoparticle removal, the well-ordered nanoporous structures inside the microchannel could be fabricated. Depending on the size of the colloidal nanoparticles, periodic porous nanostructures inside the microchannels with cavity size of 330 and 570 nm have been obtained. The dimensions of interconnecting pores for these cavities were around 40 and 64 nm, respectively. The behavior of single lambda-phage DNA molecules in these nanoporous structures was studied using fluorescence microscopy. It was found that the length of DNA molecules oscillated in the nanoporous structures. The measured length for lambda-phage DNA was larger in the 330 nm cavity than those measured in the 570 nm cavity.

Nanofluidic system for the studies of single DNA molecules.

Here, we describe a simple and low-cost lithographic technique to fabricate size-controllable nanopillar arrays inside the microfluidic channels for the studies of single DNA molecules. In this approach, nanosphere lithography has been employed to grow a single layer of well-ordered close-packed colloidal crystals inside the microfluidic channels. The size of the polymeric colloidal nanoparticles could be trimmed by oxygen plasma treatment. These size-trimmed colloidal nanoparticles were then used as the etching mask in a deep etching process. As a result, well-ordered size-controllable nanopillar arrays could be fabricated inside the microfluidic channels. The gap distance between the nanopillars could be tuned between 20 and 80 nm allowing the formation of nanofluidic system where the behavior of a single lambda-phage DNA molecule has been investigated. It was found that the lambda-phage DNA molecule could be fully stretched in the nanofluidic system formed by nanopillars with 50 nm gap distance at a field of 50 V/cm.

Morphological control of CuPc and its application in organic solar cells.

We have prepared organic photovoltaic (OPV) cells possessing an ideal bulk heterojunction (BHJ) structure using the self-assembly of copper phthalocyanine (CuPc) as the donor material and fullerene (C(60)) as the acceptor. The variable self-assembly behavior of CuPc on a diverse range of substrates (surface energies) allowed us to control the morphology of the interface and the degree of carrier transportation within the active layer. We observed rod-like CuPc structures on indium-tin oxide (ITO), poly(3,4-ethylenedioxythiophene)-poly(4-styrenesulfonate) (PEDOT:PSS) and Au substrates. Accordingly, the interfaces and continuing transport path between CuPc and fullerene domains could be greatly improved due to the ideal BHJ structure. In this paper, we discuss the mechanisms of producing CuPc rod-like films on ITO, PEDOT:PSS and Au. The OPV cell performance was greatly enhanced when a mixture of horizontal and vertical CuPc rods was present on the PEDOT:PSS surfaces, i.e. the power conversion efficiency was 50 times greater than that of the corresponding device featuring a planar CuPc structure.

Monolithic integration of well-ordered nanoporous structures in the microfluidic channels for bioseparation.

Gel electrophoresis and capillary gel electrophoresis are widely used for the separation of biomolecules. With increasing demand in the miniaturized devices such as lab-on-a-chip, it is necessary to integrate such a separation component into a chip format. Here, we describe a simple approach to fabricate robust three-dimensional periodic porous nanostructures inside the microchannels for the separation of DNA molecules. In our approach, the colloidal crystals were first grown inside the microchannel using evaporation assisted self-assembly process. Then the void spaces among the colloidal crystals were filled with epoxy-based negative tone photoresist (SU-8). UV radiation was used to cure the photoresist at the desired area inside the microchannel. After subsequent development and nanoparticle removal, the well-ordered nanoporous structures inside the microchannel were obtained. Our results indicated that it was possible to construct periodic porous nanostructures inside the microchannels with cavity size around 300 nm and interconnecting pores around 30 nm. The mobility of large DNA molecules with different sizes was measured as a function of the applied electric field in the nanoporous materials. It was also demonstrated that 1 kilo-base pair (kbp) DNA ladders could be separated in such an integrated system within 10 min under moderate electric field.

Actively controlled self-assembly of colloidal crystals in microfluidic networks by electrocapillary forces.

We report a simple approach to actively control the formation of the self-assembled colloidal crystals in the microfluidic networks using a combination of electrocapillary forces and evaporation-induced self-assembly. Using this scheme, we can not only selectively fabricate the colloidal crystals in the desired channels, but we can also build colloidal crystals with different optical properties in different channels or in the same channel.