Rat Tail Collagen Research Articles

Collagen Single Fibril Elastic Modulus Measurement Technique

We develop an experimental technique that allows precise measurement of the longitudinal component of the elastic modulus of single fibril in conditions close to in vivo. Using freshly extracted fibrils, avoiding a drying-soaking cycle and keeping deformations below 0.5%, we use a combination of optical tweezers and AFM techniques, and exploit Euler-Bernoulli elasticity theory for data analysis. We found that elastic modulus of rat tail collagen type I cannot be represented by a single quantity but rather is a distribution, significantly broader than the uncertainty of our experimental technique. Additionally, we used AFM to find variations in the fibril diameter. Since bending forces depend on the diameter to the fourth power, this variation is important for estimating the modulus. However, there still exist sources of variation that are not yet accounted for.

Bioactive Chitosan Nanoparticles and Photodynamic Therapy Inhibit Collagen Degradation In Vitro

IntroductionCollagen is the major structural protein of human dentin. Degradation of collagen by bacterial enzymes can facilitate microbial penetration, compromise structural/interfacial integrity, and lower resistance to fracture of dentin. We evaluated the ability of photodynamic therapy (PDT), bioactive chitosan nanoparticles (CSnp), or PDT in combination with CSnp to inhibit bacterial collagenase-mediated degradation of collagen. MethodsRat type 1 fibrillar collagen matrices were untreated or treated with 2.5% glutaraldehyde (GD), 2.5% GD followed by 1% CSnp, 1% CSnp, PDT (rose bengal activated with 540 nm light at 40 J/cm2), or 1% CSnp followed by PDT. Samples, except those used as untreated controls, were exposed to Clostridium histolyticum collagenase (125 CDU/mL) for 24 hours. The soluble digestion products were assessed by hydroxyproline assay, and the remaining adherent collagen was quantified by picrosirius red staining. Fourier transform infrared spectroscopy, immunoblotting, and scanning electron microscopy were used to study the interaction between CSnp/PDT with type 1 collagen. The data were analyzed by 1-way analysis of variance and post hoc Tukey test. ResultsAs assessed by hydroxyproline release into the medium, collagen treated with CSnp, PDT, or a combination of CSnp and PDT exhibited less degradation than untreated controls (3.6-fold, 1.7-fold, and 7.9-fold reduction, respectively; P < .05). Compared with all other treatments, GD-treated collagen was the most resistant to collagenolytic degradation (239.6-fold reduction, P < .05). The abundance of post-treatment residual collagen, as measured by picrosirius red staining, was inversely related to the extent of collagen degradation. Analysis of collagen cross-links with Fourier transform infrared spectroscopy showed that PDT or GD treatments enhanced collagen cross-linking. Immunoblotting of sedimented CSnp indicated that CSnp and collagenase bound with low affinity. However, CSnp-bound collagenase showed a significant reduction in collagenolytic activity compared with controls (P < .05). ConclusionsCombined photochemical cross-linking of rat tail collagen by PDT and binding to CSnp inhibit collagenolytic activity.

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

Cyclical strain modulates metalloprotease and matrix gene expression in human tenocytes via activation of TGFβ

Tendinopathies are a range of diseases characterised by degeneration and chronic tendon pain and represent a significant cause of morbidity. Relatively little is known about the underlying mechanisms; however onset is often associated with physical activity. A number of molecular changes have been documented in tendinopathy such as a decrease in overall collagen content, increased extracellular matrix turnover and protease activity. Metalloproteinases are involved in the homeostasis of the extracellular matrix and expression is regulated by mechanical strain. The aims of this study were to determine the effects of strain upon matrix turnover by measuring metalloproteinase and matrix gene expression and to elucidate the mechanism of action. Primary Human Achilles tenocytes were seeded in type I rat tail collagen gels in a Flexcell™ tissue train system and subjected to 5% cyclic uniaxial strain at 1Hz for 48h. TGFβ1 and TGFβRI inhibitor were added to selected cultures. RNA was measured using qRT-PCR and TGFβ protein levels were determined using a cell based luciferase assay. We observed that mechanical strain regulated the mRNA levels of multiple protease and matrix genes anabolically, and this regulation mirrored that seen with TGFβ stimulation alone. We have also demonstrated that the inhibition of the TGFβ signalling pathway abrogated the strain induced changes in mRNA and that TGFβ activation, rather than gene expression, was increased with mechanical strain. We concluded that TGFβ activation plays an important role in mechanotransduction. Targeting this pathway may have its place in the treatment of tendinopathy.

Imaging of the human Glisson's capsule by two-photon excitation microscopy and mechanical characterisation by uniaxial tensile tests

Liver mechanical properties have been well investigated through many studies at the macroscopic level. These studies allowed elaborating more or less sophisticated models providing material data. The rupture phenomenon has been studied as well, for example by Brunon et al. [Brunon et al. 2011] to collect data on how the liver, and more precisely the Glisson's capsule, responds to mechanical loading. With the emergence of multi-modal microscopy, these phenomenon have been considered to a whole different scale, as we can now investigate the behaviour of these materials at the microscopic level. This way, we can have a better understanding of damage and rupture mechanisms of these tissues, linking them to microstructure organisation, and thus develop realistic models based on microstructure. Recent studies have therefore focused on imaging the microstructure organisation of biological materials during or after loading. For example Goulam Houssen et al. [Goulam Houssen et al., 2011] have monitored the rat tail collagen fibers behaviour during uniaxial loading using Second-Harmonic Generation (SHG) from TwoPhoton Excitation Microscopy (TPE). Keyes et al. [Keyes et al., 2012] used SHG and autofluorescence from TPE to image pressurised porcine coronary arteries and observed fibers realignment. Thus, the aim of this study is to observe how the different constitutive fibers of the Glisson's capsule are organized and how they react when subjected to uniaxial tensile loading, in order to understand how microstructure organisation impacts macro mechanical properties. In a first step, we worked on the imaging of the Glisson's capsule by TPE to enforce our knowledge of its components and their state before loading. In a second step, we performed preliminary tensile tests using an in situ micro tensile stage to estimate mechanical parameters such as apparent modulus, ultimate stress and ultimate strain. 2. Methods

Superparamagnetic nanoparticles targeting cancer cells invading the extracellular matrix.

3083 Background: Metastatic cancer cells secrete matrix metalloproteinases (MMP), which allow cancer cells to burrow their way to the nearby vasculature. Therefore, MMPs are potential targets for anticancer treatment. Nanoparticles (NPs) can be crafted to adhere to the ECM and subsequently be released in response to advance of invasive cells. The objective of the study is to elucidate the interaction of cancer cells in a three-dimensional culture, with functionalized SPIONS targeted to the extracellular matrix around them. Methods: SPIONS, 15-20 nm in size were functionalized using different coatings: ficoll 400, sucrose, lysine-arginine, dextran50,000. Cellular uptake studies using cervical adenocarcinoma HeLa cell lines were performed to determine the optimum formulation taken up by the cells, using electron microscopy and Prussian blue staining for optical microscopy. Two types of extracellular matrices were used: Rat-tail Collagen type I and ECM gel from Engelbreth-Holm-Swarm murine sarcoma with NPs embedded in them. To assess the capability of cells to invade the matrix, cells were grown on the surface of the matrices for 1 week To evaluate the ability to grow, expand and migrate inside the matrix, cells were embedded within the matrix and left for 14 days. Comparison was made in the presence or absence of SPIONS. Results: Sucrose-coated SPIONS were taken up the best by HeLa cell lines as evaluated by MRI. MMP-1 Secretion allowed HeLa cell invasion of collagen type-1 matrix unidirectionally. Cells could adhere, proliferate, differentiate and migrate in the absence of SPIONS. Cells positive for MMP-9 invaded ECM gel from Engelbreth-Hol-Swarm murine sarcoma matrix also only in the absence of SPIONS. Cells that were found engulfing SPIONS showed morphological features of apoptosis as nuclear pyknosis and karryorhexis. Conclusions: Targeting NPs to the ECM surrounding cancer cells that have developed a metastatic potential represents an attractive platform for cancer therapeutics. The findings show a great promise for development of new theranostic agents, that can be directed to the tumor environment using external magnetic fields, with subsequent suppression of invasion and even destroying malignant cells.

Bovine preimplantation embryo development is affected by the stiffness of the culture substrate

A recent study (Kolahi et al., 2012) demonstrated that mouse embryo development in vitro may be affected by the stiffness of the substrate on which the embryos are placed during culture. Those results showed that embryos had a developmental advantage, and more of them hatched from their zona pellucidae when cultured on a collagen gel matrix (stiffness = 1 kPa—the expected stiffness of uterine epithelium) compared to those cultured on polystyrene Petri dishes (stiffness = 1 GPa; Kolahi et al., 2012). For our study, we hypothesized that the use of a collagen gel substrate during culture would be advantageous for bovine embryo development when compared to the commonly used polystyrene substrate. On three separate occasions, bovine oocytes were matured for ∼22 hr. After maturation, cumulus oocyte complexes were co-cultured with Percoll gradient-purified spermatozoa (a mixture from three bulls) for 18 hr (Rivera et al., 2003). Putative zygotes were stripped of their cumulus cells by vortexing, and then cultured in KSOM-BE1 (Rivera et al., 2003) at 38.5°C in a humidified atmosphere containing 5% O2, 5% CO2, and 90% N2. After 24 hr of culture, all embryos at or beyond the two-cell stage (cleavage = 82 ± 2.1%) were harvested and pooled. Embryos were equally divided and cultured on either polystyrene Petri dishes or collagen gels at a density of 30 embryos/50 µl microdrop, 90 embryos per treatment/day. The collagen gel (rat tail collagen, high concentration type-1; BD biosciences, San Jose, CA) was prepared to a final concentration of 3 mg/ml following the manufacturer's specifications (i.e., Alternate Gelation Procedure for BD™ Collagen I, Rat tail) and used to coat the Petri dish surface. Gelled collagen was rinsed three times with KSOM-BE1 prior to embryo culture. Embryos were cultured for 9 days post-fertilization, and development was assessed on Days 7–9. Embryos were divided into six categories, namely: (1) early blastocyst (embryos without an obvious inner cell mass, but with discernible pockets of fluids within); (2) blastocyst (embryo containing a blastocoelic cavity and an obvious inner cell mass, but of similar size as an oocyte); (3) expanding blastocyst (enlarging blastocyst with a thinning zona pellucida); (4) expanded blastocyst (large blastocyst with an extremely thin, yet intact, zona pellucida); (5) hatching blastocysts (blastocyst escaping the zona pellucida); or (6) hatched blastocysts (embryos without a zona pellucida). ANOVA was used to determine differences between treatments. Our results show that more of the embryos cultured on the collagen gel developed to the blastocyst stage when compared to their polystyrene-cultured counterparts (P < 0.001; Fig. 1). In addition, blastocysts were more developmentally advanced in the collagen gel-cultured group than in the polystyrene-cultured group (P < 0.006; Fig. 1). Lastly, embryos tended to have an increased hatching frequency (P = 0.08) when cultured in collagen (i.e., 0 ± 0, 2.6 ± 1.3, 10 ± 1.3 vs. 0 ± 0, 0.74 ± 0.7, 5.2 ± 1.3, Days 7–9 for collagen or polystyrene substrates, respectively). Thus, similar to murine embryos, bovine embryos benefit from being cultured on a collagen gel when compared to the commonly used polystyrene Petri dish. Bovine embryo development to the blastocyst stage. Embryos were randomly selected at the two- to four-cell stage, and cultured on either a collagen gel (1 kPa surface stiffness) or the commonly used polystyrene Petri dish (1 GPa surface stiffness). The experiment was repeated on three different days. Development was assessed on Days 7–9 after fertilization. n = 270 embryos/treatment. Data are represented as mean ± standard error mean.

Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device

Organs-on-chips are microengineered in vitro tissue structures that can be used as platforms for physiological and pathological research. They provide tissue-like microenvironments in which different cell types can be co-cultured in a controlled manner to create synthetic organ mimics. Blood vessels are an integral part of all tissues in the human body. Development of vascular structures is therefore an important research topic for advancing the field of organs-on-chips since generated tissues will require a blood or nutrient supply. Here, we have engineered three-dimensional constructs of vascular tissue inside microchannels by injecting a mixture of human umbilical vein endothelial cells, human embryonic stem cell-derived pericytes (the precursors of vascular smooth muscle cells) and rat tail collagen I into a polydimethylsiloxane microfluidic channel with dimensions 500 μm × 120 μm × 1 cm (w × h × l). Over the course of 12 h, the cells organized themselves into a single long tube resembling a blood vessel that followed the contours of the channel. Detailed examination of tube morphology by confocal microscopy revealed a mature endothelial monolayer with complete PECAM-1 staining at cell-cell contacts and pericytes incorporated inside the tubular structures. We also demonstrated that tube formation was disrupted in the presence of a neutralizing antibody against transforming growth factor-beta (TGF-β). The TGF-β signaling pathway is essential for normal vascular development; deletion of any of its components in mouse development results in defective vasculogenesis and angiogenesis and mutations in humans have been linked to multiple vascular genetic diseases. In the engineered microvessels, inhibition of TGF-β signaling resulted in tubes with smaller diameters and higher tortuosity, highly reminiscent of the abnormal vessels observed in patients with one particular vascular disease known as hereditary hemorrhagic telangiectasia (HHT). In summary, we have developed microengineered three-dimensional vascular structures that can be used as a model to test the effects of drugs and study the interaction between different human vascular cell types. In the future, the model may be integrated into larger tissue constructs to advance the development of organs-on-chips.

Colostrogenesis: candidate genes for IgG1 transcytosis mechanisms in primary bovine mammary epithelial cells

Bovine colostrogenesis is distinguished by the specific transfer of IgG1 from the blood to mammary secretions. The process has been shown to be initiated by hormones and occurs during the last weeks of pregnancy when steroid concentrations of estradiol (E2 ) and progesterone (P4 ) are highly elevated. Rodent intestinal uptake of immunoglobulin G is mediated by a receptor termed Fc fragment of IgG, Receptor, Transporter, alpha (FcGRT) and supported by light chain Beta-2-Microglobulin (β2M). We hypothesized that steroid hormone treatments (E2 and P4 ) of bovine mammary epithelial cells in vitro would induce up-regulation of IgG1 transcytosis candidate gene mRNA expression suggesting involvement in IgG1 transcytosis. Two different primary bovine mammary epithelial cell cultures were cultured on plastic and rat tail collagen and treated with hormonal combinations (steroids/lactogenic hormones). Evaluated mRNA components were bLactoferrin (bLf: a control), bFcGRT, β2M, and various small GTPases; the latter components are reported to direct endosomal movements in eukaryotic cells. All tested transcytosis components showed strong expression of mRNA in the cells. Expression of bFcGRT, bRab25 and bRhoB were significantly up-regulated (p<0.05) by steroid hormones. bRab25 and bRhoB showed increased expression by steroid treatments, but also with lactogenic hormones. Analysis for the oestrogen receptor (ER) mRNA was mostly negative, but 25% of the cultures tested exhibited weak expression, while the progesterone receptor (PR) mRNA was always detected. bRab25 and bRhoB and likely bFcGRT are potential candidate genes for IgG1 transcytosis in bovine mammary cells.

Differentiation of human embryonic stem cells to endothelial cells via improved three-dimension approach

To establish an improved three-dimension (3D) and serum-free approach to differentiate human embryonic stem cells (hESCs) into endothelial cells, and detect the endothelial functions of the obtained cells. We cultured undifferentiated H9 human embryonic stem cell line in low-adhesion dishes to form embryonic bodies (EBs). After 12 days, EBs were harvested, re-suspended into rat tail collagen type I, and put into the incubator (37℃). After 30 minutes, EGM-2 culture medium was added to the solidified collagen, and the EBs were cultured for another 3 days to form embryonic body-sproutings (EB-sproutings). EB-sproutings were digested with 0.25% collagenase I and 0.56 U/ml Liberase Blendzyme for 20 minutes respectively, and the CD31(+) cells were sorted by FACS. The endothelial functions were tested by Dil-ac-LDL uptake assay and tube formation assay. This approach raised the efficiency of endothelial differentiation to 18%, and also avoided the contamination with animal materials. The obtained hESC-derived endothelial cells (hESC-ECs) had the similar pattern of surface biomarkers as human umbilical vein endothelial cells (HUVECs), and their endothelial functions were confirmed by the uptake of Dil-ac-LDL and the tube formation on Matrigel. The improved 3D approach can enhance the efficiency of differentiation from hESCs into endothelial cells. Furthermore, serum free differentiation system may be applied in future hESC-based therapies for various ischemic diseases.

S-Adenosylmethionine-dependent Protein Methylation Is Required for Expression of Selenoprotein P and Gluconeogenic Enzymes in HepG2 Human Hepatocytes

Cellular methylation processes enable expression of gluconeogenic enzymes and metabolism of the nutrient selenium. Selenium status has been proposed to relate to type II diabetes risk, and plasma levels of selenoprotein P (SEPP1) have been positively correlated with insulin resistance. Increased expression of gluconeogenic enzymes glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase 1 (PCK1) has negative consequences for blood glucose management in type II diabetics. Transcriptional regulation of SEPP1 is directed by the same transcription factors that control the expression of G6PC and PCK1, and these factors are activated by methylation of arginine residues. We sought to determine whether expression of SEPP1 and the aforementioned glucoconeogenic enzymes are regulated by protein methylation, the levels of which are reliant upon adequate S-adenosylmethionine (SAM) and inhibited by S-adenosylhomocysteine (SAH). We treated a human hepatocyte cell line, HepG2, with inhibitors of adenosylhomocysteine hydrolase (AHCY) known to increase concentration of SAH before analysis of G6PC, PCK1, and SEPP1 expression. Increasing SAH decreased 1) the SAM/SAH ratio, 2) protein-arginine methylation, and 3) expression of SEPP1, G6PC, and PCK1 transcripts. Furthermore, hormone-dependent induction of gluconeogenic enzymes was reduced by inhibition of protein methylation. When protein-arginine methyltransferase 1 expression was reduced by siRNA treatment, G6PC expression was inhibited. These findings demonstrate that hepatocellular SAM-dependent protein methylation is required for both SEPP1 and gluconeogenic enzyme expression and that inhibition of protein arginine methylation might provide a route to therapeutic interventions in type II diabetes.

Hyaluronan Anchored to Activated CD44 on Central Nervous System Vascular Endothelial Cells Promotes Lymphocyte Extravasation in Experimental Autoimmune Encephalomyelitis

The extravasation of lymphocytes across central nervous system (CNS) vascular endothelium is a key step in inflammatory demyelinating diseases including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The glycosaminoglycan hyaluronan (HA) and its receptor, CD44, have been implicated in this process but their precise roles are unclear. We find that CD44(-/-) mice have a delayed onset of EAE compared with wild type animals. Using an in vitro lymphocyte rolling assay, we find that fewer slow rolling (<1 μm/s) wild type (WT) activated lymphocytes interact with CD44(-/-) brain vascular endothelial cells (ECs) than with WT ECs. We also find that CD44(-/-) ECs fail to anchor HA to their surfaces, and that slow rolling lymphocyte interactions with WT ECs are inhibited when the ECs are treated with a pegylated form of the PH20 hyaluronidase (PEG-PH20). Subcutaneous injection of PEG-PH20 delays the onset of EAE symptoms by ~1 day and transiently ameliorates symptoms for 2 days following disease onset. These improved symptoms correspond histologically to degradation of HA in the lumen of CNS blood vessels, decreased demyelination, and impaired CD4(+) T-cell extravasation. Collectively these data suggest that HA tethered to CD44 on CNS ECs is critical for the extravasation of activated T cells into the CNS providing new insight into the mechanisms promoting inflammatory demyelinating disease.

Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth

Olfm1, a secreted highly conserved glycoprotein, is detected in peripheral and central nervous tissues and participates in neural progenitor maintenance, cell death in brain, and optic nerve arborization. In this study, we identified Olfm1 as a molecule promoting axon growth through interaction with the Nogo A receptor (NgR1) complex. Olfm1 is coexpressed with NgR1 in dorsal root ganglia and retinal ganglion cells in embryonic and postnatal mice. Olfm1 specifically binds to NgR1, as judged by alkaline phosphatase assay and coimmunoprecipitation. The addition of Olfm1 inhibited the growth cone collapse of dorsal root ganglia neurons induced by myelin-associated inhibitors, indicating that Olfm1 attenuates the NgR1 receptor functions. Olfm1 caused the inhibition of NgR1 signaling by interfering with interaction between NgR1 and its coreceptors p75NTR or LINGO-1. In zebrafish, inhibition of optic nerve extension by olfm1 morpholino oligonucleotides was partially rescued by dominant negative ngr1 or lingo-1. These data introduce Olfm1 as a novel NgR1 ligand that may modulate the functions of the NgR1 complex in axonal growth.

Breast epithelial tissue morphology is affected in 3D cultures by species‐specific collagen‐based extracellular matrix

Collagen-based gels have been widely used to determine the factors that regulate branching morphogenesis in the mammary gland. The patterns of biomechanical gradients and collagen reorganization influence the shape and orientation of epithelial structures in three-dimensional (3D) conditions. We explored in greater detail whether collagen type I fibers with distinct biomechanical and fiber-assembling properties, isolated from either bovine or rat tail tendon, differentially affected the epithelial phenotype in a tissue culture model of the human breast. Rat tail collagen fibers were densely packed into significantly longer and thicker bundles compared to those of the bovine type (average fascicle length 7.35 and 2.29 μm, respectively; p = 0.0001), indicating increased fiber alignment and biomechanical enablement in the former. MCF10A epithelial cells formed elaborated branched tubular structures in bovine but only nonbranched ducts and acini in rat tail collagen matrices. Ductal branching in bovine collagen was associated with interactions between neighboring structures mediated through packed collagen fibers; these fiber-mediated interactions were absent in rat tail collagen gels. Normal breast fibroblasts increased the final size and number of ducts only in rat tail collagen gels while not affecting branching. Our results suggest that the species of origin of collagen used in organotypic cultures may influence epithelial differentiation into alveolar or ductal structures and the patterns of epithelial branching. These observations underscore the importance of considering the species of origin and fiber alignment properties of collagen when engineering branching organs in 3D matrices and interpreting their role in the tissue phenotype.

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum(1). Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra(2). The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions(3,4). These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or βIII tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Altered Angiogenesis in Caveolin-1 Gene–Deficient Mice Is Restored by Ablation of Endothelial Nitric Oxide Synthase

Caveolin-1 is an essential structural protein of caveolae, specialized plasma membrane organelles highly abundant in endothelial cells, where they regulate multiple functions including angiogenesis. Caveolin-1 exerts a tonic inhibition of endothelial nitric oxide synthase (eNOS) activity. Accordingly, caveolin-1 gene-disrupted mice have enhanced eNOS activity as well as increased systemic nitric oxide (NO) levels. We hypothesized that excess eNOS activity, secondary to caveolin deficiency, would mediate the decreased angiogenesis observed in caveolin-1 gene-disrupted mice. We tested tumor angiogenesis in mice lacking either one or both proteins, using in vitro, ex vivo, and in vivo assays. We show that endothelial cell migration, tube formation, cell sprouting from aortic rings, tumor growth, and angiogenesis are all significantly impaired in both caveolin-1-null and eNOS-null mice. We further show that these parameters were either partially or fully restored in double knockout mice that lack both caveolin-1 and eNOS. Furthermore, the effects of genetic ablation of eNOS are mimicked by the administration of the NOS inhibitor N-nitro-L-arginine methyl ester hydrochloride (L-NAME), including the reversal of the caveolin-1-null mouse angiogenic phenotype. This study is the first to demonstrate the detrimental effects of unregulated eNOS activity on angiogenesis, and shows that impaired tumor angiogenesis in caveolin-1-null mice is, at least in part, the result of enhanced eNOS activity.

Corneal stromal cells use both high- and low-contractility migration mechanisms in 3-D collagen matrices

Corneal keratocyte migration can impact both corneal clarity and refractive outcome following injury or refractive surgery. In this study, we investigated how culture conditions, ECM properties, and Rho kinase activity regulate the mechanics of keratocyte migration, using a nested collagen matrix model. Time-lapse imaging demonstrated that both serum and PDGF stimulate keratocyte migration into the outer matrix. Although the velocity of cell migration was similar, cells in serum were bipolar and induced significant matrix deformation during migration, whereas PDGF induced extension of branching dendritic processes with smaller, more localized force generation. These differences in cell-induced matrix reorganization were verified with a global matrix contraction assay and confocal reflection imaging, using both bovine and rat tail collagen. When constructs were detached from the substrate to lower the effective stiffness, migration was significantly reduced in serum; but was unchanged in PDGF. These differences in migration mechanics were mediated, in part, by Rho kinase. Overall, corneal keratocytes can effectively migrate through collagen matrices using varying degrees of cellular force generation. Low-contractility migration may facilitate keratocyte repopulation of the stroma following surgery or injury, without altering the structural and mechanical properties that are critical to maintaining corneal transparency.

Hypoxia-Inducible Factor Regulates Expression of Surfactant Protein in Alveolar Type II Cells In Vitro

Alveolar type II (ATII) cells cultured at an air-liquid (A/L) interface maintain differentiation, but they lose these properties when they are submerged. Others showed that an oxygen tension gradient develops in the culture medium as ATII cells consume oxygen. Therefore, we wondered whether hypoxia inducible factor (HIF) signaling could explain differences in the phenotypes of ATII cells cultured under A/L interface or submerged conditions. ATII cells were isolated from male Sprague-Dawley rats and cultured on inserts coated with a mixture of rat-tail collagen and Matrigel, in medium including 5% rat serum and 10 ng/ml keratinocyte growth factor, with their apical surfaces either exposed to air or submerged. The A/L interface condition maintained the expression of surfactant proteins, whereas that expression was down-regulated under the submerged condition, and the effect was rapid and reversible. Under submerged conditions, there was an increase in HIF1α and HIF2α in nuclear extracts, mRNA levels of HIF inducible genes, vascular endothelial growth factor, glucose transporter-1 (GLUT1), and the protein level of pyruvate dehydrogenase kinase isozyme-1. The expression of surfactant proteins was suppressed and GLUT1 mRNA levels were induced when cells were cultured with 1 mM dimethyloxalyl glycine. The expression of surfactant proteins was restored under submerged conditions with supplemented 60% oxygen. HIF signaling and oxygen tension at the surface of cells appears to be important in regulating the phenotype of rat ATII cells.

Evidence for Regulated Interleukin-4 Expression in Chondrocyte-Scaffolds under In Vitro Inflammatory Conditions

ObjectiveTo elucidate the anti-inflammatory and anabolic effects of regulated expression of IL-4 in chondrocyte-scaffolds under in vitro inflammatory conditions.MethodsMature articular chondrocytes from dogs (n = 3) were conditioned through transient transfection using pcDNA3.1.cIL-4 (constitutive) or pCOX-2.cIL-4 (cytokine-responsive) plasmids. Conditioned cells were seeded in alginate microspheres and rat-tail collagen type I matrix (CaReS®) to generate two types of tissue-engineered 3-dimensional scaffolds. Inflammatory arthritis was simulated in the packed chondrocytes through exogenous addition of recombinant canine (rc) IL-1β (100 ng/ml) plus rcTNFα (50 ng/ml) in culture media for 96 hours. Harvested cells and culture media were analyzed by various assays to monitor the anti-inflammatory and regenerative (anabolic) properties of cIL-4.ResultscIL-4 was expressed from COX-2 promoter exclusively on the addition of rcIL-1β and rcTNFα while its expression from CMV promoter was constitutive. The expressed cIL-4 downregulated the mRNA expression of IL-1β, TNFα, IL-6, iNOS and COX-2 in the cells and inhibited the production of NO and PGE2 in culture media. At the same time, it up-regulated the expression of IGF-1, IL-1ra, COL2a1 and aggrecan in conditioned chondrocytes in both scaffolds along with a diminished release of total collagen and sGAG into the culture media. An increased amount of cIL-4 protein was detected both in chondrocyte cell lysate and in concentrated culture media. Neutralizing anti-cIL-4 antibody assay confirmed that the anti-inflammatory and regenerative effects seen are exclusively driven by cIL-4. There was a restricted expression of IL-4 under COX-2 promoter possibly due to negative feedback loop while it was over-expressed under CMV promoter (undesirable). Furthermore, the anti-inflammatory /anabolic outcomes from both scaffolds were reproducible and the therapeutic effects of cIL-4 were both scaffold- and promoter-independent.ConclusionsRegulated expression of therapeutic candidate gene(s) coupled with suitable scaffold(s) could potentially serve as a useful tissue-engineering tool to devise future treatment strategies for osteoarthritis.

Consensus Substrate Sequence for Protein-tyrosine Phosphatase Receptor Type Z

Protein-tyrosine phosphatase receptor type Z (Ptprz) has multiple substrate proteins, including G protein-coupled receptor kinase-interactor 1 (Git1), membrane-associated guanylate kinase, WW and PDZ domain-containing 1 (Magi1), and GTPase-activating protein for Rho GTPase (p190RhoGAP). We have identified a dephosphorylation site at Tyr-1105 of p190RhoGAP; however, the structural determinants employed for substrate recognition of Ptprz have not been fully defined. In the present study, we revealed that Ptprz selectively dephosphorylates Git1 at Tyr-554, and Magi1 at Tyr-373 and Tyr-858 by in vitro and cell-based assays. Of note, the dephosphorylation of the Magi1 Tyr-858 site required PDZ domain-mediated interaction between Magi1 and Ptprz in the cellular context. Alignment of the primary sequences surrounding the target phosphotyrosine residue in these three substrates showed considerable similarity, suggesting a consensus motif for recognition by Ptprz. We then estimated the contribution of surrounding individual amino acid side chains to the catalytic efficiency by using fluorescent peptides based on the Git1 Tyr-554 sequence in vitro. The typical substrate motif for the catalytic domain of Ptprz was deduced to be Glu/Asp-Glu/Asp-Glu/Asp-Xaa-Ile/Val-Tyr(P)-Xaa (Xaa is not an acidic residue). Intriguingly, a G854D substitution of the Magi1 Tyr-858 site matching better to the motif sequence turned this site to be susceptible to dephosphorylation by Ptprz independent of the PDZ domain-mediated interaction in cells. Furthermore, we found by database screening that the substrate motif is present in several proteins, including paxillin at Tyr-118, its major phosphorylation site. Expectedly, we verified that Ptprz efficiently dephosphorylates paxillin at this site in cells. Our study thus provides key insights into the molecular basis for the substrate recognition of Ptprz.