Adult equine bone marrow stromal cells produce a cartilage-like ECM mechanically superior to animal-matched adult chondrocytes.

Our objective was to evaluate the age-dependent mechanical phenotype of bone marrow stromal cell- (BMSC-) and chondrocyte-produced cartilage-like neo-tissue and to elucidate the matrix-associated mechanisms which generate this phenotype. Cells from both immature (2-4 month-old foals) and skeletally-mature (2-5 year-old adults) mixed-breed horses were isolated from animal-matched bone marrow and cartilage tissue, encapsulated in self-assembling-peptide hydrogels, and cultured with and without TGF-beta1 supplementation. BMSCs and chondrocytes from both donor ages were encapsulated with high viability. BMSCs from both ages produced neo-tissue with higher mechanical stiffness than that produced by either young or adult chondrocytes. Young, but not adult, chondrocytes proliferated in response to TGF-beta1 while BMSCs from both age groups proliferated with TGF-beta1. Young chondrocytes stimulated by TGF-beta1 accumulated ECM with 10-fold higher sulfated-glycosaminoglycan content than adult chondrocytes and 2-3-fold higher than BMSCs of either age. The opposite trend was observed for hydroxyproline content, with BMSCs accumulating 2-3-fold more than chondrocytes, independent of age. Size-exclusion chromatography of extracted proteoglycans showed that an aggrecan-like peak was the predominant sulfated proteoglycan for all cell types. Direct measurement of aggrecan core protein length and chondroitin sulfate chain length by single molecule atomic force microscopy imaging revealed that, independent of age, BMSCs produced longer core protein and longer chondroitin sulfate chains, and fewer short core protein molecules than chondrocytes, suggesting that the BMSC-produced aggrecan has a phenotype more characteristic of young tissue than chondrocyte-produced aggrecan. Aggrecan ultrastructure, ECM composition, and cellular proliferation combine to suggest a mechanism by which BMSCs produce a superior cartilage-like neo-tissue than either young or adult chondrocytes.

Caprine articular, meniscus and intervertebral disc cartilage: an integral analysis of collagen network and chondrocytes.

Cartilage is a tissue with only limited reparative capacities. A small part of its volume is composed of cells, the remaining part being the hydrated extracellular matrix (ECM) with collagens and proteoglycans as its main constituents. The functioning of cartilage depends heavily on its ECM. Although it is known that the various (fibro)cartilaginous tissues (articular cartilage, annulus fibrosus, nucleus pulposus, and meniscus) differ from one each other with respect to their molecular make-up, remarkable little quantitative information is available with respect to its biochemical constituents, such as collagen content, or the various posttranslational modifications of collagen. Furthermore, we have noticed that tissue-engineering strategies to replace cartilaginous tissues pay in general little attention to the biochemical differences of the tissues or the phenotypical differences of the (fibro)chondrocytes under consideration. The goal of this paper is therefore to provide quantitative biochemical data from these tissues as a reference for further studies. We have chosen the goat as the source of these tissues, as this animal is widely accepted as an animal model in orthopaedic studies, e.g. in the field of cartilage degeneration and tissue engineering. Furthermore, we provide data on mRNA levels (from genes encoding proteins/enzymes involved in the synthesis and degradation of the ECM) from (fibro)chondrocytes that are freshly isolated from these tissues and from the same (fibro)chondrocytes that are cultured for 18 days in alginate beads. Expression levels of genes involved in the cross-linking of collagen were different between cells isolated from various cartilaginous tissues. This opens the possibility to include more markers than the commonly used chondrogenic markers type II collagen and aggrecan for cartilage tissue-engineering applications.

An experimental study on collagen content and biomechanical properties of sclera after posterior sclera reinforcement.

BACKGROUND: The development of pathological myopia is associated with reduced scleral collagen accumulation, scleral thinning, and loss of scleral tissue, in both humans and animal models. Posterior scleral reinforcement (PSR) was considered as an effective way for treating pathological myopia. Yet it is not well understood the possible role of collagen on the sclera reinforcement mechanisms in the PSR surgery. METHODS: PSR surgery was performed on the normal adult New Zealand white rabbits eyes. Human sclera was used as reinforcement materials. At 1, 2, 3, 6, 9 months after the PSR surgery, scleral hydroxyproline (Hyp) synthesis and collagen fibers arrangement were determined by enzymolysic hydrolysis assay and histological morphology technique. An Instron test machine was used to investigate the elastic modulus of sclera. FINDINGS: It was found that the elastic modulus and Hyp content of reinforced sclera were lower at first month after surgery, and then gradually up to physiological level in the following months. Those two indexes were close to that of the normal control groups at 9 months. INTERPRETATION: These findings indicate that sclera elastic modulus was associated with both change of Hyp content and collagen fibers arrangement after PSR. The therapeutic effect of PSR surgery was confirmed not only from biological but also biomechanical aspects.

Role of hydroxyprolines in the in vitro oxidative folding and biological activity of conotoxins.

Hydroxylation of proline residue occurs in specific peptides and proteins derived from plants and animals, but the functional role of this modification has been characterized primarily in collagen. Marine cone snails produce disulfide-rich peptides that have undergone a plethora of posttranslational modifications, including proline hydroxylation. Although Conus snails extensively utilize proline hydroxylation, the consequences of this modification remain largely unexplored. In this work, we investigated the function of 4-hydroxyproline (Hyp) in conotoxins from three distinct gene families: mu-, omega-, and alpha-conotoxins. Analogues of mu-GIIIA, omega-MVIIC, alpha-GI, and alpha-ImI were synthesized with either Pro or Hyp, and their in vitro oxidative folding and biological activity were characterized. For GIIIA, which naturally contains three Hyp residues, the modifications improved the ability to block NaV1.4 sodium channels but did not affect folding. In contrast, the presence of Hyp in MVIIC had a significant impact on the oxidative folding but not on the biological activity. The folding yields for the MVIIC[Pro7Hyp] analogue were approximately 2-fold higher than for MVIIC under a variety of optimized oxidation conditions. For alpha-conotoxins ImI and GI, the hydroxylation of the conserved Pro residue improved their folding but impaired their activities against target receptors. Since prolyl-4-hydroxylase and protein disulfide isomerase coexist as a heterotetramer in the ER, we discuss the effects of Hyp on the folding of conotoxins in the context of cis-trans isomerization of Pro and Hyp. Taken together, our data suggest that proline hydroxylation is important for both in vitro oxidative folding and the bioactivity of conotoxins.

Biochemical markers of the mechanical quality of engineered hyaline cartilage.

The aim of this study was to determine whether or not biochemical markers can be used as surrogate measures for the mechanical quality of tissue engineered cartilage. The biochemical composition of tissue engineered cartilage constructs were altered by varying either (i) the initial cell seeding density of the scaffold (seeding density protocol) or (ii) the length of time the engineered tissue was cultured (culture period protocol). The aggregate or Young's moduli of the constructs were measured (by confined or unconfined compression respectively), and compared with the composition of the extracellular matrix by quantitative measurement of the glycosaminoglycan (GAG), hydroxyproline, collagen I and collagen II and collagen cross-links. The aggregate modulus correlated positively with both GAG and collagen II content, but not with collagen I content. Young's modulus correlated positively with GAG, collagen II and collagen I content, and the ratio of mature to immature cross-links. There was no significant correlation of Young's Modulus with total collagen measured as hydroxyproline content. These results suggested that hydroxyproline determination may be an unreliable indicator of mechanical quality of tissue engineered cartilage, and that a measure of collagen II and GAG content is required to predict the biomechanical quality of tissue engineered cartilage.

Unhydroxylated triple helical collagen I produced in transgenic plants provides new clues on the role of hydroxyproline in collagen folding and fibril formation.

Human unhydroxylated homotrimeric triple-helical collagen I produced in transgenic plants was used as an experimental model to provide insights into the role of hydroxyproline in molecular folding and fibril formation. By using chemically cross-linked molecules, we show here that the absence of hydroxyproline residues does not prevent correct folding of the recombinant collagen although it markedly slows down the propagation rate compared with bovine fully hydroxylated homotrimeric collagen I. Relatively slow cis-trans-isomerization in the absence of hydroxyproline likely represents the rate-limiting factor in the propagation of the unhydroxylated collagen helix. Because of the lack of hydroxylation, recombinant collagen molecules showed increased flexibility as well as a reduced melting temperature compared with native homotrimers and heterotrimers, whereas the distribution of charged amino acids was unchanged. However, unlike with bovine collagen I, the recombinant collagen did not self-assemble into banded fibrils in physiological ionic strength buffer at 20 degrees C. Striated fibrils were only obtained with low ionic strength buffer. We propose that, under physiological ionic strength conditions, the hydroxyl groups in the native molecule retain water more efficiently thus favoring correct fibril formation. The importance of hydroxyproline in collagen self-assembly suggested by others from the crystal structures of collagen model peptides is thus confirmed experimentally on the entire collagen molecule.

HIF-1alpha binding to VHL is regulated by stimulus-sensitive proline hydroxylation.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a global transcriptional regulator of the hypoxic response. Under normoxic conditions, HIF-1alpha is recognized by the von Hippel-Lindau tumor-suppressor protein (VHL), a component of an E3 ubiquitin ligase complex. This interaction thereby promotes the rapid degradation of HIF-1alpha. Under hypoxic conditions, HIF-1alpha is stabilized. We have previously shown that VHL binds in a hypoxia-sensitive manner to a 27-aa segment of HIF-1alpha, and that this regulation depends on a posttranslational modification of HIF-1alpha. Through a combination of in vivo coimmunoprecipitation assays using VHL and a panel of point mutants of HIF-1alpha in this region, as well as MS and in vitro binding assays, we now provide evidence that this modification, which occurs under normoxic conditions, is hydroxylation of Pro-564 of HIF-1alpha. The data furthermore show that this proline hydroxylation is the primary regulator of VHL binding.

Endurecimento de feijäo (Phaseolus vulgaris L.): proteínas da parede celular ricas em hidroxiprolina / Hardening of bean (Phaseolus vulgaris L.): cell wall proteins rich in hydroxiprolin

O feijão Phaseolus vulgaris é uma leguminosa muito consumida no Brasil e faz parte da alimentação diária da população da América Latina. Entretanto, dependendo das condições e tempo de armazenamento, o feijão sofre o fenômeno de endurecimento, o qual se caracteriza pelo maior tempo gasto em sua cocção. As causas do endurecimento podem estar relacionadas, entre outras, com mudanças na parede celular dos cotilédones do feijão. Componentes da parede celular como polissacarídeos, compostos fenólicos e glicoproteínas podem sofrer ligações cruzadas entre si, tornando a parede mais rígida e resistente à cocção. As glicoproteínas da parede celular são ricas em hidroxiprolina e são denominadas extensivas. O mecanismo pelo qual as extensinas participam do endurecimento do feijão ainda não foi estabelecido...

Inhibition of tissue repair by spironolactone: role of mineralocorticoids in fibrous tissue formation.

Mineralocorticoids have been implicated in promoting fibrous tissue formation in various organs. In the present study, we sought to address the potential contribution of mineralocorticoids to fibrous tissue formation using a skin pouch model which has proved valuable for the analysis of inflammatory and wound healing responses. Skin pouches were induced in rats by administration of a phorbol ester, croton oil (0.5 ml of a 1% solution). After 2 weeks, rats were killed and intact pouch tissue collected. Pouch weights of control and aldosterone-treated (0.75 microg/h via osmotic minipump) rats were similar (3.33 +/- 0.44 g vs. 3.70 +/- 0.28 g respectively). However, pouch weights were reduced by more than 50% in spironolactone-treated (25 mg/day powdered in food) animals (1.62 +/- 0.22 g and 1.27 +/- 0.23 g respectively in aldosterone and spironolactone alone groups). To ascertain the effects of different treatments on collagen accumulation, hydroxyproline concentration was measured. Compared with controls, hydroxyproline concentration was significantly reduced following spironolactone treatment (17.1 +/- 0.08 vs. 7.5 +/- 2.0 microg/mg dry wt, respectively, p < 0.01). This response to spironolactone was negated by coadministration of aldosterone (hydroxyproline concentration was 18.6 +/- 2.1 microg/mg dry wt). Following bilateral adrenalectomy, spironolactone reduced pouch weight and hydroxyproline concentration, which was not the case for adrenalectomy alone. Two week aldosterone administration in uninephrectomized rats on high salt diet was deemed ineffective in modulating pouch development (pouch wet wts were 3.48 +/- 0.4 g vs. 3.00 +/- 0.19 g in controls and aldosterone-treated rats, respectively). Mineralocorticoid receptor expression in pouch tissue was demonstrated by RT/PCR. Furthermore, NADP+-dependent 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) activity was detected in pouch tissue, together with lower levels of NAD+-dependent 11beta-HSD2. Spironolactone (p < 0.05) significantly reduced 11beta-HSD1 activity compared with controls. Thus, fibrous tissue possesses requisite components of MC action, and antagonism of mineralocorticoid receptors by spironolactone attenuates its formation. Pouch formation is under the influence of circulating MC and, we would like to propose, is also mediated through corticosteroids generated de novo at the site of tissue repair.

Efeito da associaçäo da dexametasona e cisplatina intraperitoneais, na cicatrizaçäo de anastomose cólica experimental, em ratos / Effect of the association of dexametasone and intraperitoneal cisplatin in cicatrization of experimental colonic anastomosis in rats

INTRODUÇÄO: Quimioterapia adjuvante com cisplatina está em uso crescente no tratamento do câncer do cólon. Todavia, a cisplatina e outras drogas usadas para tratar os efeitos adversos podem interferir com a cicatrizaçäo de anastomose após ressecçäo cólica. O objetivo deste estudo foi avaliar o efeito da associaçäo cisplatina e dexametasona, sobre a cicatrizaçäo de anastomose cólica em rato. MATERIAL E MÉTODO: 72 ratos foram divididos em três grupos de 24 animais. Foi administrada a todos injeçäo intraperitoneal de soluçäo salina equivalente a 20 porcento de seu peso corporal, no dia prévio à realizaçäo de anastomose cólica. Ao grupo controle näo foi aplicado tratamento exceto solução salina. No grupo cisplatina foi injetada cisplatina intraperitoneal (I.P.) (3 mg/kg de peso). No grupo cisplatina/dexametasona, foi adcionada dexametasona I.P. (0,28 mg/kg de peso na véspera e no dia da operação) à igual dose da cisplatina. Uma anastomose término-terminal no cólon proximal foi realizada em todos os grupos, usando sutura com pontos separados, de fio de mononylon Ethilon 6-0, em um plano. Um segmento de 1 cm do cólon proximal foi ressecado e os ratos sacrificados após 7, 14 e 21 dias. Fizeram-se hemogramas na véspera da operaçäo e no dia do sacrifício. Pressäo de ruptura foi obtida, no dia do sacrifício, de cada segmento anastomótico pela sua articulaçäo a um sistema dotado de um manômetro, sendo o segmento submetido à insuflaçäo com ar. Foram comparadas as pressöes de ruptura em grupos e subgrupos de ratos e verificadas eventuais deiscências. Fragmentos das anastomoses foram obtidos durante o sacrificio para determinaçäo de hidroxiprolina e análise histológica. RESULTADOS: No 7§ dia, observou-se que a pressäo de ruptura no grupo cisplatina/dexametasona foi mais baixa do que no grupo controle e grupo cisplantina respectivamente (Mann-Whitney, p=0,01 e p=0,23). No grupo cisplantina, a pressäo de ruptura foi mais baixa (Mann-Whitney, p=0,07) que no controle. Nas comparaçöes no 14§ dia näo houve diferença estatisticamente significante. No 21§ dia, a pressäo foi mais baixa nos grupos cisplatina e cisplatina/dexametasona com diferença estatisticamente significante (Kruskal-Wallis, p=0,03) na comparaçäo entre os três grupos. Este efeito foi significante (Mann-Whitney p=0,01) somente na comparaçäo entre os grupos controle e cisplatina/dexametasona...

The mechanism of uptake of ascorbic acid into osteoblasts and leukocytes.

Ascorbic acid is taken up into osteoblast cells by a saturable, stereospecific, Na(+)-dependent transporter, accumulating ascorbic acid to a level 100-fold that in the medium. The ascorbic acid uptake rate correlated with intracellular hydroxyproline synthesis. A second, distinct mechanism has also been described for accumulation of ascorbic acid into neutrophils and myeloid leukemia cells. This appears to be Na(+)-independent and relies on the glucose transporter GLUT1 to ferry dehydroascorbic acid (DHA) into cells and then to trap it as ascorbic acid to a high concentration.

Collagen synthesis inhibition reduces clustering of heparan sulfate proteoglycan and acetylcholine receptors but not agrin or p65, at neuromuscular contacts in vitro.

We have studied presynaptic and postsynaptic differentiation at neuromuscular junctions in vitro by examining the localization of synapse-specific proteins. In nerve-muscle co-cultures, the synaptic vesicle protein synaptotagmin (p65) accumulated in the nerve terminal overlying myotubes in association with postsynaptic clusters of acetylcholine receptors (AChRs), heparan sulfate proteoglycan (HSPG), laminin, and agrin. Inhibition of collagen synthesis with cis-hydroxyproline decreased the nerve-induced clustering of AChRs in muscle cells as well as that caused by exogenous agrin in muscle-only cultures. Moreover, accumulation of HSPG at contacts was also inhibited in cis-hydroxyproline-treated cultures. However, accumulation of p65 in nerve fibers at sites of muscle contact, a sign of presynaptic differentiation, was unaffected by cis-hydroxyproline treatment. In addition, even in cis-hydroxyproline-inhibited cultures, agrin was evident at more than 90% of contacts showing accumulation of p65 in the nerve terminal. Therefore, a mechanism exists to maintain agrin concentrations at nerve-muscle contacts, even when at least some extracellular matrix (ECM) proteins are disrupted. Our results suggest that HSPG is not required for the induction of nerve terminal differentiation but are consistent with the idea that HSPG or other ECM proteins are important in both nerve- and agrin-induced AChR clustering. In particular, agrin accumulation at sites of nerve-muscle contact is not sufficient to induce AChR clusters when the ECM at these contacts is disrupted.

Tumor necrosis factor-alpha inhibits in vivo collagen synthesis.

In this study we sought to determine the in vivo role of tumor necrosis factor-alpha (TNF-alpha) at the wound-healing site. In vivo abrogation of endogenous TNF-alpha activity in experimental wounds by administration of anti-murine TNF-alpha rabbit serum resulted in a significant 77.5% increase in wound collagen deposition, as assessed by wound sponge granuloma hydroxyproline content. Administration of pharmacologic doses of recombinant murine TNF-alpha into subcutaneously inserted polyvinyl alcohol sponges resulted in an increase in collagen deposition (1594 +/- 117 vs 1014 +/- 49 and 1588 +/- 135 vs 1014 +/- 49 micrograms/100 mg sponge, for TNF-alpha in situ administration at a dose of 0.05 and 0.5 micrograms, respectively). This effect could be abolished by the simultaneous systemic treatment of the animals with the antiinflammatory drug indomethacin. The data suggest that the enhanced collagen deposition after TNF-alpha administration is a consequence of a nonspecific inflammatory activity that indirectly promotes collagen synthesis. The data also support the hypothesis that endogenous wound TNF-alpha down-regulates collagen synthesis during normal wound healing.

Across-fiber patterns may contain a sensory code for stimulus intensity.

Various models for sensory coding have used statistical approaches based on the assumption that the stimulus intensity parameter is represented in the afferent neurons as mean firing frequency. In this paper we question the assumption that this is the only code for intensity. We show that in lobster olfactory receptors narrowly tuned to hydroxyproline, an across-fiber pattern (AFP) code distinguished more concentration levels over a 5 log step range than a response magnitude code and, unlike the latter, was unaffected by response summation time. AFP discrimination of stimulus intensity appears to be based on high inter-cell response variability and low intra-cell response variability.

Mechanical behavior of articular cartilage quantitative changes with enzymatic alteration of the proteoglycan fraction.

The in-vitro viscoelastic mechanical response of normal rabbit articular cartilage is strongly dependent on the quantity and integrity of the proteoglycan fraction of the tissue matrix. Experimental results demonstrate that specific functional relationships exist between shear moduli, retardation time spectra, and proteoglycan content. Quantitative enzymolysis of the proteoglycan fraction of the tissue alters the form of these relationships in a fashion consistent with the altered physiochemical make-up of the tissue. The observed changes in mechanical behavior with controlled enzymolysis are similar to those associated with the early stages of osteoarthritis, rheumatoid arthritis, joint sepsis, and synovitis in animal models.