"Role of long non-coing RNAs in the stressed heart". Wisper : a novel cardiac fibroblast-enriched IncRNA that modulate fibrosis in the failing heart

LncRNAs are transcripts greater than 200 nucleotides in length with no apparent coding potential. They exert important regulatory functions in the genome. Their role in cardiac fibrosis is however unexplored. To identify IncRNAs that could modulate cardiac fibrosis, we profiled the long non-coding transcriptome in the infarcted mouse heart, and identified 1500 novel IncRNAs. These IncRNAs have unique characteristics such as high tissue and cell type specificity. Their expression is highly correlated with parameters of cardiac dimensions and function. The majority of these novel IncRNAs are conserved in human. Importantly, human IncRNAs appear to be differentially expressed in heart disease. Using a computational pipeline, we identified a super-enhancer-associated IncRNA, which is dynamically expressed after myocardial infarction. We named this particular transcript Wisper for «Wisp2 super-enhancer- derived IncRNA ». Interestingly, Wisper expression is overexpressed in cardiac fibroblasts as compared to cardiomyocytes or to fibroblasts isolated from other organs than the heart. The importance of Wisper in the biology of fibroblasts was demonstrated in knockdown experiments. Differentiation of cardiac fibroblast into myofibroblasts in vitro is significantly impaired upon Wisper knockdown. Wisper downregulation in cardiac fibroblasts results in a dramatic reduction of fibrotic gene expression, a diminished cell proliferation and an increase in apoptotic cell death. In vivo, depletion of Wisper during the acute phase of the response to infarction is detrimental via increasing the risk of cardiac rupture. On the other hand, Wisper knockdown following infarction in a prevention study reduces fibrosis and preserves cardiac function. Since WISPER is detectable in the human heart, where it is associated with severe cardiac fibrosis, these data suggest that Wisper could represent a novel therapeutic target for limiting the extent of the fibrotic response in the heart. -- Les long ARN non-codants (IncRNAs) sont des ARN de plus de 200 nucléotides qui ne codent pas pour des protéines. Ils exercent d'importantes fonctions dans le génome. Par contre, leur importance dans le développement de la fibrose cardiaque n'a pas été étudiée. Pour identifier des IncRNAs jouant un rôle dans ce processus, le transcriptome non-codant a été étudié dans le coeur de'souris après un infarctus du myocarde. Nous avons découverts 1500 nouveaux IncRNAs. Ces transcrits ont d'uniques caractéristiques. En particulier ils sont extrêmement spécifiques de sous-populations de cellules cardiaques. Par ailleurs, leur expression est remarquablement corrélée avec les paramètres définissant les dimensions du coeur et la fonction cardiaque. La majorité de ces IncRNAs sont conservés chez l'humain. Certains sont modulés dans des pathologies cardiaques. En utilisant une approche bioinformatique, nous avons identifié un IncRNA qui est associé à des séquences amplificatrices et qui est particulièrement enrichi dans les fibroblastes cardiaques. Ce transcrit a été nommé Wisper pour «Wisp2 super-enhancer-derived IncRNA ». L'importance de Wisper dans la biologie des fibroblastes cardiaques est démontrée dans des expériences de déplétion. En l'absence de Wisper, l'expression de protéines impliquées dans le développement de la fibrose est dramatiquement réduite dans les fibroblastes cardiaques. Ceux-ci montrent une prolifération réduite. Le niveau d'apoptose est largement augmenté. In vivo, la déplétion de Wisper pendant la phase aiguë de l'infarctus rehausse le risque de rupture cardiaque. Au contraire, la réduction de l'expression de Wisper pendant la phase chronique diminue la fibrose cardiaque et améliore la fonction du coeur. Puisque Wisper est exprimé dans le coeur humain, ce transcrit représente une nouvelle cible thérapeutique pour limiter la réponse fibrotique dans le coeur.

Enhancement of human adipose-derived stem cell expansion and stability for clinical use

Co-culture techniques associating both dermal fibroblasts and epidermal keratinocytes have shown to have better clinical outcome than keratinocyte culture alone for the treatment of severe burns. Since fat grafting has been shown to improve scar remodelling, new techniques such as cell-therapy-assisted surgical reconstruction with isolated and expanded autologous adipose-derived stem cells (ASCs) would be of benefit to increase graft acceptation. Therefore, integrating ASCs into standardized procedures for cultured skin grafting could be of benefit for the patient if cell quality and quantity could be maintained. The purpose of this study was to evaluate ASC processing from adult tissue with simple isolation (without enzymatic steps), expansion (low density of 325-3,000 cells/cm2) and storage conditions to assure methods to enhance the cellular resistance when transferred back to the patient. Co-culture with cell-banked skin progenitor cells (FE002-SK2) showed an increase of 40-50% ASCs yield at high passages alongside with a better preservation of morphology, proper adipogenic and osteogenic differentiation and efficient biocompatibility with 3D collagen scaffolds. ASCs can be considered as a valuable additional cell source to be delivered in biological bandages to the patient in a need of tissue reconstruction such as burn patients.

Thermoplastic bioactive composite – with special reference to dissolution behaviour and tissue response

Bioactive glasses are surface-active ceramic materials which support and accelerate bone growth in the body. During the healing of a bone fracture or a large bone defect, fixation is often needed. The aim of this thesis was to determine the dissolution behaviour and biocompatibility of a composite consisting of poly(ε-caprolactone-co-DL-lactide) and bioactive glass (S53P4). In addition the applicability as an injectable material straight to a bone defect was assessed. In in vitro tests the dissolution behaviour of plain copolymer and composites containing bioactive glass granules was evaluated, as well as surface reactivity and the material’s capability to form apatite in simulated body fluid (SBF). The human fibroblast proliferation was tested on materials in cell culture. In in vivo experiments, toxicological tests, material degradation and tissue reactions were tested both in subcutaneous space and in experimental bone defects. The composites containing bioactive glass formed a unified layer of apatite on their surface in SBF. The size and amount of glass granules affected the degradation of polymer matrix, as well the material’s surface reactivity. In cell culture on the test materials the human gingival fibroblasts proliferated and matured faster compared with control materials. In in vitro tests a connective tissue capsule was formed around the specimens, and became thinner in the course of time. Foreign body cell reactions in toxicological tests were mild. In experimental bone defects the specimens with a high concentration of small bioactive glass granules (<45 μm) formed a dense apatite surface layer that restricted the bone ingrowth to material. The range of large glass granules (90-315 μm) with high concentrations formed the best bonding with bone, but slow degradation on the copolymer restricted the bone growth only in the superficial layers. In these studies, the handling properties of the material proved to be good and tissue reactions were mild. The reactivity of bioactive glass was retained inside the copolymer matrix, thus enabling bone conductivity with composites. However, the copolymer was noticed to degradate too slowly compared with the bone healing. Therefore, the porosity of the material should be increased in order to improve tissue healing.

Lamellarin D bioconjugates II: synthesis and cellular internalization of dendrimer and nuclear location signal derivatives

The design and synthesis of Lamellarin D conjugates with a nuclear localization signal peptide and a poly(ethylene glycol)-based dendrimer are described. Conjugates 1-4 were obtained in 8-84% overall yields from the corresponding protected Lamellarin D. Conjugates 1 and 4 are 1.4 to 3.3-fold more cytotoxic than the parent compound against three human tumor cell lines(MDA-MB-231 breast, A-549 lung, and HT-29 colon). Besides, conjugates 3, 4 showed a decrease in activity potency in BJ skin fibroblasts, a normal cell culture. Cellular internalization was analyzed and nuclear distribution pattern was observed for 4, which contains a nuclear localization signalling sequence.

Cyclin-dependent kinases 4 and 6 control tumor progression and direct glucose oxidation in the pentose cycle

Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G1 phase. Aberrant expression of CDK4 and CDK6 is a hall- mark of cancer, which would suggest that CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G1 phase. The metabolic effects of calcein AM (the calcein acetoxymethyl-ester) on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phos-phate pathway was significantly altered. To elucidate whe-ther these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors.

Synthesis of geranylhydroquinone derivatives with potencial cytotoxic activity

Natural geranylhydroquinone 1 and geranyl-p-methoxyphenol 2 were prepared by Electrophilic Aromatic Substitution (EAS) reactions between geraniol and 1,4-hydroquinone or p-methoxyphenol respectively, using BF3∙Et2O as a catalyst. Furthermore, natural geranylquinone 3, geranyl-1,4-dimethoxyquinone 4 and the new geranyl-4-methoxyphenyl acetate 5 were obtained by chemical transformations of 1 and 2. The compounds were evaluated for their in vitro cytotoxicity activities against cultured human cancer cells of PC-3 human prostate cancer, MCF-7 and MDA-MB-231 breast carcinoma, and Dermal Human Fibroblasts DHF. IC50 values were in the µM range.

Tolerância de fungos ectomicorrízicos e plantas associadas a níveis tóxicos de metais

As ectomicorrizas são associações entre raízes de plantas e fungos, de grande importância para a sobrevivência e crescimento vegetal, por aumentar o aporte nutricional e a proteção contra fatores adversos. Os fungos ectomicorrízicos podem quelar íons de metais tóxicos, retirando-os do solo e diminuindo os danos ao hospedeiro. Quatro isolados de fungos ectomicorrízicos foram utilizados neste estudo: UFSC-Sc68 (Scleroderma sp.); UFSC-Ch163 (Chondrogaster angustisporus); UFSC-Pt116 e UFSC-Pt188 (Pisolithus microcarpus). Mudas de Eucalyptus dunnii foram testadas quanto à sua tolerância aos metais alumínio (Al), cádmio (Cd), cromo (Cr) e manganês (Mn) misturados ao substrato de plantio, em dois experimentos em casa de vegetação. No primeiro, em ausência dos fungos e, no segundo, após inoculação com esses organismos. No experimento sem inoculação, o metal mais tóxico foi o Cr. Nenhuma planta sobreviveu nas doses 1000 e 10000 mg.kg-1 desse metal. Para os outros metais, a matéria seca diminuiu com o aumento da dose. Já no estudo com plantas inoculadas, o Cd foi o metal mais tóxico, que reduziu significativamente a sobrevivência e o crescimento das plantas. Os isolados fúngicos UFSC-Pt188 e UFSC-Ch163 foram os mais eficientes na proteção contra os metais, melhorando a sobrevivência e a produção de matéria seca das plantas. O isolado UFSC-Ch163, em particular, foi o mais eficiente na tolerância das plantas, o que indica seu potencial de utilização em solos contaminados para favorecer o estabelecimento e o crescimento das plantas.

In vitro EVALUATION OF EUCALYPTUS ECTOMYCORRHIZAE ON SUBSTRATE WITH PHOSPHORUS DOSES FOR FUNGAL PRE-SELECTION

The benefit promoted by ectomycorrhizal depends on the interaction between symbionts and phosphorus (P) contents. Phosphorus effect on ectomycorrhizal formation and the effectiveness of these in promoting plant growth for fungal pre-selection were assessed under in vitro conditions. For P effect evaluation, Eucalyptus urophylla seedlings inoculated with four Pisolithus sp. isolates and others non-inoculated were grown on substrate containing 0.87, 1.16 and 1.72 mg P per plant. For evaluation of effectiveness and fungal pre-selection, other 30 isolates of Pisolithus sp., Pisolithus microcarpus ITA06 isolate, Amanita muscaria AM16 isolate, Scleroderma areolatum SC129 isolate were studied. D26 isolate promoted the highest plant heights for the three P doses, D51 at the lower dose and D72 at the intermediate dose. P doses did not influenced shoot fresh weight and fungal colonization. In the pre-selection of fungi, 14 isolates of Pisolithus sp., P. microcarpus ITA06 isolate and S. areolatum SC129isolate increased plant height and fresh weight. D82 isolate of Pisolithus sp. had effect singly on plant height while D17 and D58 on fresh weight. Of these, only D15, D17, D58 and ITA06 had typical ectomycorrhizae. The cultivation in vitro has shown adequate for pre-selection of ectomycorrhizal fungi. Colonization and benefits depend on species and isolate. D15, D17 and D58 of Pisolithus sp. and P. microcarpus isolate ITA06 are the most promising for nursery studies.

The regulation and function of collagenase-3 (MMP-13) in cutaneous wound healing and squamous cell carcinoma

Matrix metalloproteinase-13 (MMP-13) is a potent proteolytic enzyme, whose expression has been previously associated with fetal bone development and postnatal bone remodeling and with adult gingival wound healing. MMP-13 is also known to be involved in the growth and invasion of various cancers including squamous cell carcinoma (SCC) of the skin. The aim of this study was to further elucidate the function and regulation of MMP-13 in wound repair and cancer. In this study, it was shown that fetal skin fibroblasts express MMP-13 in response to transforming growth factor-β in a p38 MAP kinase dependent manner. In addition, MMP-13 was found to be expressed in vivo by wound fibroblasts in human fetal skin grafted on SCID mice. Adenovirally delivered expression of MMP-13 enhanced collagen matrix contraction by fibroblasts in vitro in association with altered cytoskeletal structure, enhanced proliferation and survival. These results indicate that MMP-13 is involved in cell-mediated collagen matrix remodeling and suggest a role for MMP-13 in superior matrix remodeling and scarless healing of fetal skin wounds. Using an MMP-13 deficient mouse strain, it was shown that MMP-13 is essential for the normal development of experimental granulation tissue in mice. MMP-13 was implicated in the regulation of myofibroblast function and angiogenesis and the expression of genes involved in cellular proliferation and movement, immune response, angiogenesis and proteolysis. Finally, epidermal mitogen, keratinocyte growth factor (KGF) was shown to suppress the malignant properties of skin SCC cells by downregulating the expression of several target genes with potential cancer promoting properties, including MMP-13, and by reducing SCC cell invasion. These results provide evidence that MMP-13 potently regulates cell viability, myofibroblast function and angiogenesis associated with wound healing and cancer. In addition, fibroblasts expressing MMP-13 show high collagen reorganization capacity. Moreover, the results suggest that KGF mediates the anti-cancer effects on skin SCC

Morphological aspects of buffaloes (Bubalus bubalis) umbilical cord

Buffalo is an important livestock resource, with a great participation in agricultural systems, providing milk, meat, and work power. Umbilical cord is responsible for maternal-fetal nutrients exchange during pregnancy, and its alterations can compromise the fetal development. We investigated ten pregnant uteruses collected from cross-bread buffaloes in different stages of gestation. Pregnancy and fetal age was determined by measuring the apex sacral length and development period was calculated by previously published formula. Umbilical cords were measured for length determination. Umbilical cord vascular net and anastomosis were observed by injection of Neoprene latex. Histological sections of the umbilical cord were studied after stain with HE, picrossirius, toluidine blue, orceine, and PAS reaction. Buffaloes' umbilical cord was formed by two central arteries, an allantois duct and two peripheral veins. The artery wall was composed by large quantity of collagen, elastic fibers, fibroblasts and large number of vasa vasorum. The allantois duct was located between the arteries and presented a great number of small nourishing vessels. Small nourishing vessels should be carefully considered to avoid to be mistaken to the arterials and veins vasa vasorum. Medium length of umbilical cord from buffalos was 11.8cm (minimum of 6.8cm and maximum of 17.4cm).

Accidental and experimental Closantel intoxication in Uruguayan sheep

Abstract An outbreak of Closantel intoxication in sheep in Uruguay is described. The outbreak occurred in a group of 1300 weaning lambs treated orally with a 10% solution of Closantel. One hundred forty eight lambs showed clinical signs of intoxication and 14 died. The clinical signs included mydriasis, nystagmus, and negative pupillary reflex, bilateral blindness, bump into objects, and lateral movement of the head. No macroscopic lesions were observed. The histological lesions of the retina were cytoplasmic vacuolization in ganglion cells and in cells of the inner and outer nuclear layers with different degrees of atrophy. Vacuolization and axonal degeneration were observed in the optic nerve, with multifocal areas of fibrosis and infiltration by lymphocytes and Gitter cells. To reproduce the intoxication, four sheep were given two, four and 10 times the therapeutic dose of Closantel (0.1g/kg of BW). Only the animals receiving 10 times the recommended dose showed clinical signs. The histological examination of the lesions in experimental sheep showed similar results to those described in the accidental outbreak, except for the absence of optic nerve fibrosis and inflammation, characterizing an acute phase. Axonal myelin sheaths loss, fibroblasts and collagen fibers were observed in the ultrastructural study of the optic nerve of accidental intoxicated animals. The optic nerve of experimentally intoxicated animals had vacuoles that separated the myelin sheaths of axons. To prevent outbreaks it is suggested to weigh the animals before Closantel administration to avoid errors in dose calculation.

Fluorescence-based imaging of cellular defect in lysinuric protein intolerance (LPI)

Fluoresenssiperusteiset kuvantamismenetelmät lysinurisen proteiini-intoleranssin (LPI) soluhäiriön tutkimuksessa Lysinurinen proteiini-intoleranssi on suomalaiseen tautiperintöön kuuluva autosomaalisesti peit¬tyvästi periytyvä sairaus, jonka aiheuttaa kationisten aminohappojen kuljetushäiriö munuaisten ja ohutsuolen epiteelisolujen basolateraalikalvolla. Aminohappojen kuljetushäiriö johtaa moniin oirei¬siin, kuten kasvuhäiriöön, osteoporoosiin, immuunijärjestelmän häiriöihin, oksenteluun ja runsaspro¬teiinisen ravinnon nauttimisen jälkeiseen hyperammonemiaan. LPI-geeni SLC7A7 (solute carrier family 7 member 7) koodaa y+LAT1 proteiinia, joka on basolateraali¬nen kationisten ja neutraalien aminohappojen kuljettimen kevyt ketju, joka muodostaa heterodimee¬rin raskaan alayksikön 4F2hc:n kanssa. Tällä hetkellä SLC7A7-geenistä tunnetaan yli 50 LPI:n aiheut¬tavaa mutaatiota. Tässä tutkimuksessa erityyppisiä y+LAT1:n LPI-mutaatiota sekä yhdeksän C-terminaalista polypep¬tidiä lyhentävää deleetiota kuvannettiin nisäkässoluissa y+LAT1:n GFP (green fluorescent protein) -fuusioproteiineina. Tulokset vahvistivat muissa soluissa tehdyt havainnot siitä, että 4F2hc on edel¬lytyksenä y+LAT1:n solukalvokuljetukselle, G54V-pistemutantti sijaitsee solukalvolla samoin kuin vil¬lityyppinen proteiini, mutta lukukehystä muuttavia ja proteiinia lyhentäviä mutantteja ei kuljeteta solukalvoon. Lisäksi havaittiin, että poikkeuksena tästä säännöstä ovat y+LAT1-deleetioproteiinit, joista puuttui korkeintaan 50 C-terminaalista aminohappoa. Nämä lyhentyneet kuljettimet sijaitsevat solukalvolla kuten villityyppiset ja LPI-pistemutanttiproteiinit. Dimerisaation osuutta kuljetushäiriön synnyssä tutkittiin käyttämällä fluorescence resonance energy transfer (FRET) menetelmää. Heterodimeerin alayksiköistä kloonattiin ECFP (cyan) ja EYFP (yellow) fuusioproteiinit, joita ilmennettiin nisäkässoluissa, ja FRET mitattiin virtaussytometri-FRET -menetel¬mällä (FACS-FRET). Tutkimuksissa kaikkien mutanttien havaittiin dimerisoituvan yhtä tehokkaasti. Kul¬jetushäiriön syynä ei siten ole alayksiköiden dimerisaation estyminen mutaation seurauksena. Tutkimuksessa havaittiin, että kaikki mutantti-y+LAT1-transfektiot tuottavat vähemmän transfektoi¬tuneita soluja kuin villityyppisen y+LAT1:n transfektiot. Solupopulaatioissa, joihin oli tranfektoitu lu¬kukehystä muuttava tai stop-kodonin tuottava mutaatio havaittiin suurempi kuolleisuus kuin saman näytteen transfektoitumattomissa soluissa, kun taas villityyppistä tai G54V-pistemutanttia tuottavas¬sa solupopulaatiossa oli pienempi kuolleisuus kuin saman näytteen fuusioproteiinia ilmentämättö¬missä soluissa. Tulos osoittaa mutanttiproteiinien erilaiset vaikutukset niitä ilmentäviin soluihin, joko suoraan y+LAT1:n tai 4F2hc:n kautta aiheutuneina. LPIFin SLC7A7 lähetti-RNA:n määrä ei merkittävästi poikennut villityyppisen määrästä fibroblasteissa ja lymfoblasteissa. SLC7A7:n promoottorianalyysissä oli osoitettavissa säätelyalueita geenin 5’ ei-koo¬daavalla alueella sekä ensimmäisten kahden intronin alueella. LPI-taudin tautimekanismin kannalta keskeisin tekijä on kuitenkin aminohappokuljetuksen häiriö, jonka vaikutuksesta näistä aminohapoista riippuvaiset prosessit elimistössä eivät toimi normaalisti. Havaittu virheellinen y+LAT1/4F2hc kuljetuskompleksin sijainti edellyttää lisätutkimuksia sen mahdol¬lisen kliinisen merkityksen selvittämiseksi.

Autoradiography reveals regional metabolic differences in the endometrium of pregnant and nonpregnant mice

The rodent endometrium undergoes remarkable modifications during pregnancy, resulting from a redifferentiation of its fibroblasts. During this modification (decidualization), the fibroblasts transform into large, polyhedral cells that establish intercellular junctions. Decidualization proceeds from the subepithelial stroma towards the deep stroma situated next to the myometrium and creates regions composed of cells in different stages of differentiation. We studied by autoradiography whether cells of these different regions have different levels of macromolecular synthesis. Radioactive amino acids or radioactive sulfate were administered to mice during estrus or on different days of pregnancy. The animals were killed 30 min after injection of the precursors and the uteri were processed for light microscope autoradiography. Silver grains were counted over cells of different regions of the endometrium and are reported as the number of silver grains per area. Higher levels of incorporation of amino acids were found in pregnant animals as compared to animals in estrus. In pregnant animals, the region of decidual cells or the region of fibroblasts transforming into decidual cells showed the highest levels of synthesis. Radioactive sulfate incorporation, on the other hand, was generally higher in nonpregnant animals. Animals without decidual cell transformation (nonpregnant and 4th day of pregnancy) showed a differential incorporation by subepithelial and deep stroma fibroblasts. This study shows that regional differences in synthetic activity exist in cells that are in different stages of transformation into decidual cells as well as in different regions of the endometrium of nonpregnant mice

Cellular and matrix interactions during the development of T lymphocytes

The thymus contains an extensive extracellular matrix. Although thymocytes express integrins capable of binding to matrix molecules, the functional significance of the matrix for T cell development is uncertain. We have shown that the matrix is associated with thymic fibroblasts which are required for the CD44+ CD25+ stage of double negative (CD4-8-) thymocyte development. The survival of cells at this stage is dependent on IL-7 and we propose that the role of fibroblasts is to present, via the matrix, IL-7 to developing T cells.

The hematopoietic stroma

All blood cells are derived from a small common pool of totipotent cells, called hematopoietic stem cells. The process is strictly regulated by the hematopoietic microenvironment, which includes stromal cells, extracellular matrix molecules and soluble regulatory factors. Several experimental in vitro assays have been developed for the study of hematopoietic differentiation, and have provided valuable information on the stroma, which includes, among other cell types, macrophages, fibroblasts, adipocytes, and endothelial cells. The composition, ontogeny, and function in physiological as well as pathological conditions of stroma are discussed.