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Human adipose tissue-derived stem cells (hATSC) have been contemplated as reparative cells for cartilage engineering. Chondrogenic differentiation of hATSC can be induced by an enriched culture medium and a three-dimensional environment. Given that bone is vascularized and cartilage is not, oxygen tension has been suggested as a regulatory factor for osteochondrogenic differentiation. Our work aimed at determining whether hypoxia affects the osteochondrogenic potential of hATSC, hATSC were cultured in chondrogenic or osteogenic medium for 28 days, in pellets or monolayers, and under 5% or 20% oxygen tension. Cell differentiation was monitored by real-time PCR (COL2A1, aggrecan, Runx2, and osteocalcin). The chondrogenic differentiation was further evaluated by Alcian blue and immunohistological staining for glycosaminoglycans (GAGs) and type II collagen, respectively. Osteogenic differentiation was also assessed by the staining of mineralized matrix (Alizarin Red) and measurement of alkaline phosphatase (ALP) activity. The expression of chondrogenic markers was upregulated when hATSC were exposed to hypoxia in chondrogenic medium. Conversely, osteocalcin expression, mineralization, and ALP activity were severely reduced under hypoxic conditions even in the presence of osteogenic medium. Our data strongly suggest that hypoxia favors the chondrogenic differentiation of hATSC as evidenced by the expression of the chondrogenic markers, whereas it could alter their osteogenic potential. Our results highlight the differential regulatory role of hypoxia on the chondrogenic and osteogenic differentiation processes of hATSC. These data could help us exploit the potential of tissue engineering and stem cells to replace or restore the function of osteoarticular tissues. adipose tissue-derived stem cells; osteochondrogenesis doi: 10.1152/ajpcell.00398.2009

Cyclooxygenase-1 or -2--which one mediates lipopolysaccharide-induced hypothermia? Am J Physiol Regul Integr Comp Physio1297: R485--R494, 2009. First published June 10, 2009; doi: 10.1152/ajpregu.91026.2008.--Systemic inflammation is associated with either fever or hypothermia. Fever, a response to mild systemic inflammation, is mediated by cyclooxygenase (COX)-2 and not by COX-1. However, it is still disputed whether COX-2, COX-l, neither, or both mediate(s) responses to severe systemic inflammation, and, in particular, the hypothermic response. We compared the effects of SC-236 (COX-2 inhibitor) and SC-560 (COX-1 inhibitor) on the deep body temperature ([T.sub.b]) of rats injected with a lower (10 [micro]g/kg iv) or higher (1,000 [micro]g/kg iv) dose of LPS at different ambient temperatures ([T.sub.a]s). At a neutral [T.sub.a] (30[degrees]C), the rats responded to LPS with a polyphasic fever (lower dose) or a brief hypothermia followed by fever (higher dose). SC-236 (2.5 mg/kg iv) blocked the fever induced by either LPS dose, whereas SC-560 (5 mg/kg iv) altered neither the febrile response to the lower LPS dose nor the fever component of the response to the higher dose. However, SC-560 blocked the initial hypothermia caused by the higher LPS dose. At a subneutral [T.sub.a] (22[degrees]C), the rats responded to LPS with early (70-90 min, nadir) dose-dependent hypothermia. The hypothermic response to either dose was enhanced by SC-236 but blocked by SC-560. The hypothermic response to the higher LPS dose was associated with a fall in arterial blood pressure. This hypotensive response was attenuated by either SC-236 or SC-560. At the onset of LPS-induced hypothermia and hypotension, the functional activity of the COX-1 pathway (COX-1-mediated [PGE.sub.2] synthesis ex vivo) increased in the spleen but not liver, lung, kidney, or brain. The expression of splenic COX-1 was unaffected by LPS. We conclude that COX-1, but not COX-2, mediates LPS hypothermia, and that both COX isoforms are required for LPS hypotension. body temperature; thermoregulation; fever; inflammation

During human embryogenesis, neural crest cells migrate to the anterior chamber of the eye and then differentiate into the inner layers of the cornea, the iridocorneal angle, and the anterior portion of the iris. When proper development does not occur, this causes iridocorneal angle dysgenesis and intraocular pressure (IOP) elevation, which ultimately results in developmental glaucoma. Here, we show that heparan sulfate (HS) deficiency in mouse neural crest cells causes anterior chamber dysgenesis, including corneal endothelium defects, corneal stroma hypoplasia, and iridocorneal angle dysgenesis. These dysfunctions are phenotypes of the human developmental glaucoma, Peters anomaly. In the neural crest cells of mice embryos, disruption of the gene encoding exostosin 1 (Ext1), which is an indispensable enzyme for HS synthesis, resulted in disturbed TGF-[[beta].sub.2] signaling. This led to reduced phosphorylation of Smad2 and downregulated expression of forkhead box C1 (Foxc1) and paired-like homeodomain transcription factor 2 (Pitx2), transcription factors that have been identified as the causative genes for developmental glaucoma. Furthermore, impaired interactions between HS and TGF-[[beta].sub.2] induced developmental glaucoma, which was manifested as an IOP elevation caused by iridocorneal angle dysgenesis. These findings suggest that HS is necessary for neural crest cells to form the anterior chamber via TGF-[[beta].sub.2] signaling. Disturbances of HS synthesis might therefore contribute to the pathology of developmental glaucoma., Introduction Developmental glaucoma is a congenital blinding disease associated with elevated intraocular pressure (IOP) because of anomalies of the drainage structure for the aqueous humor in the eye. The major [...]

Several genes involved in the interaction between Azospirillum brasilense Sp7 and plants are located on the pRhico plasmid. Here we report the characterization of an Sp7 mutant strain with impairment of the pRhico-located gene wzm. This gene encodes an inner-membrane component of an ATP-binding cassette (ABC) transporter with similarity to transporters involved in surface polysaccharide export. Indeed, SDS-PAGE revealed that LPS synthesis is affected in the wzm mutant. No significant differences were observed between wild-type and mutant strains in exopolysaccharide (EPS) amount; however, several differences were observed between them in EPS monosaccharide composition, and only wild-type colonies stained positively with Congo red. Microscopy revealed that wzm mutant cells are longer and thinner, and exhibit several differences in their cell surface relative to the wild-type. The wzm mutant was more resistant to oxidative stress, starvation, desiccation, heat and osmotic shock than the wild-type. In contrast, the mutant was more susceptible than the wild-type to UV radiation and saline stress. The strains also differed in their susceptibility to different antibiotics. Differences between the strains were also observed in their outer-membrane protein composition. No differences were observed between strains in their ability to attach to sweet corn roots and seeds, and to promote growth under the tested conditions. As LPS plays an important role in cell envelope structural integrity, we propose that the pleiotropic phenotypic changes observed in the wzm mutant are due to its altered LPS relative to the wild-type.

Glycosaminoglycan storage begins in prenatal life in patients with mucopolysaccharidosis (MPS). In fact, prenatal hydrops is a common manifestation of MPS VII because of [beta]-glucuronidase (GUS) deficiency. One way to address prenatal storage might be to deliver the missing enzyme across the placenta into the fetal circulation. Maternal IgG is transported across the placenta by the neonatal Fc receptor (FcRn), which recognizes the Fc domain of IgG and mediates transcytosis from maternal to fetal circulation. We hypothesized that we could exploit this process to deliver corrective enzyme to the fetus. To test this hypothesis, the C-terminal fusion protein, GUS-Fc, was compared with native, untagged, recombinant GUS for clearance from the maternal circulation, delivery to the fetus, and reduction of lysosomal storage in offspring of MPS VII mice. We observed that GUS-Fc, infused into pregnant mothers on embryonic days 17 and 18, was transported across the placenta. Similarly infused untagged GUS was not delivered to the fetus. GUS-Fc plasma enzyme activity in newborn MPS VII mice was 1,000 times that seen after administration of untagged GUS and [approximately equal to] 100 times that of untreated WT newborns. Reduced lysosomal storage in heart valves, liver, and spleen provided evidence that in utero enzyme replacement therapy with GUS-Fc targeted sites of storage in the MPS VII fetus. We hypothesize that this noninvasive approach could deliver the missing lysosomal enzyme to a fetus with any lysosomal storage disease. It might also provide a method for inducing immune tolerance to the missing enzyme or another foreign protein. enzyme replacement therapy | lysosomal storage disease | receptor-mediated endocytosis | transplacental delivery

The structure of an intact glycosaminoglycan (GAG) chain of the bikunin proteoglycan (PG) is studied by using a combined top-down and bottom-up sequencing strategy. Electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry analysis of the size-uniform fraction of bikunin GAG mixture has determined the chain length and number of sulfo groups in the intact GAGs.

Glycosaminogycans (GAGs) are involved in numerous vital functions in the human body. Mapping the GAG concentration in vivo is desirable for the diagnosis and monitoring of a number of diseases such as osteoarthritis, which affects millions of individuals. GAG loss in cartilage is typically an initiating event in osteoarthritis. Another widespread pathology related to GAG is intervertebral disk degeneration. Currently existing techniques for GAG monitoring, such as delayed gadolinium-enhanced MRI contrast (dGEMRIC), [T.sub.1p], and [sup.23]Na MRI, have some practical limitations. We show that by exploiting the exchangeable protons of GAG one may directly measure the localized GAG concentration in vivo with high sensitivity and therefore obtain a powerful diagnostic MRI method. cartilage | MRI | osteoarthritis | NOE proteoglycan

The basic biochemical mechanisms underlying many heritable human polycystic diseases are unknown despite evidence that most cases are caused by mutations in members of several protein families, the most prominent being the polycystin gene family, whose products are found on the primary cilia, or due to mutations in posttranslational processing and transport. Inherited polycystic kidney disease, the most prevalent polycystic disease, currently affects [approximately equal to]500,000 people in the United States. Decreases in proteoglycans (PGs) have been found in tissues and cultured cells from patients who suffer from autosomal dominant polycystic kidney disease, and this PG decrease has been hypothesized to be responsible for cystogenesis. This is possible because alterations in PG concentrations would be predicted to disrupt many homeostatic mechanisms of growth, development, and metabolism. To test this hypothesis, we have generated mice lacking xylosyltransferase 2 (XylT2), an enzyme involved in PG biosynthesis. Here we show that inactivation of XylT2 results in a substantial reduction in PGs and a phenotype characteristic of many aspects of polycystic liver and kidney disease, including biliary epithelial cysts, renal tubule dilation, organ fibrosis, and basement membrane abnormalities. Our findings demonstrate that alterations in PG concentrations can occur due to loss of XylT2, and that reduced PGs can induce cyst development. animal model | extracellular matrix | genetics | proteoglycans |I cyst

Mutation in the prion gene PRNP accounts for 10-15% of human prion diseases. However, little is known about the mechanisms by which mutant prion proteins (PrPs) cause disease. Here we investigated the effects of 10 different pathogenic mutations on the conformation and ligand-binding activity of recombinant human PrP (rPrP). We found that mutant rPrPs react more strongly with N terminus-specific antibodies, indicative of a more exposed N terminus. The N terminus of PrP contains a glycosaminoglycan (GAG)-binding motif. Binding of GAG is important in prion disease. Accordingly, all mutant rPrPs bind more GAG, and GAG promotes the aggregation of mutant rPrPs more efficiently than wild-type recombinant normal cellular PrP ([rPrp.sup.C]). Furthermore, point mutations in PRNP also cause conformational changes in the region between residues 109 and 136, resulting in the exposure of a second, normally buried, GAG-binding motif. Importantly, brain-derived PrP from transgenic mice, which express a pathogenic mutant with nine extra octapeptide repeats, also binds more strongly to GAG than wild-type [PrP.sup.C]. Thus, several rPrPs with distinct pathogenic mutations have common conformational changes, which enhance binding to GAG. These changes may contribute to the pathogenesis of inherited prion diseases. genetics | structure

The idea that an osmotically inactive [Na.sup.+] storage pool exists that can be varied to accommodate states of [Na.sup.+] retention and/or [Na.sup.+] loss is controversial. We speculated that considerable amounts of osmotically inactive Na+ are lost with growth and that additional dietary salt excess or salt deficit alters the polyanionic character of extracellular glycosaminoglycans in osmotically inactive [Na.sup.+] reservoirs. Six-week-old Sprague-Dawley rats were fed low-salt (0.1%; LS) or high-salt (8%; HS) diets for 1 or 4 wk. At their death, we separated the tissues and determined their [Na.sup.+], [K.sup.+], and water content. Three weeks of growth reduced the total body [Na.sup.+] content relative to dry weight (rTB[Na.sup.+]) by 23%. This 'growth-programmed' [Na.sup.+] loss originated from the bone and the completely skinned and bone-removed carcasses. The [Na.sup.+] loss was osmotically inactive (45-50%) or osmotically active (50-55%). In rats aged 10 wk, compared with HS, 4 wk of LS reduced rTB[Na.sup.+] by 9%. This dietary-induced [Na.sup.+] loss was osmotically inactive ([approximately equal to] 50%) and originated largely from the skin, while [approximately equal to] 50% was osmotically active. LS for 1 wk did not reduce skin [Na.sup.+] content. The mobilization of osmotically inactive skin [Na.sup.+] with long-term salt deprivation was associated with decreased negatively charged skin glycosaminoglycan content and thereby a decreased water-free [Na.sup.+] binding capacity in the extracellular matrix. Our data not only serve to explain discrepant results in salt balance studies but also show that glycosaminoglycans may provide an actively regulated interstitial cation exchange mechanism that participates in volume and blood pressure homeostasis. extrarenal sodium balance; hypertension; volume; sodium reservoirs; glycosaminoglycans doi:10.1152/ajprenal.00300.2006

The effect of glycosaminoglycans of varying degrees of sulfation was examined on the kinetics of protein C activation by factor Xa (f.Xa) in the presence of [Ca.sup.2+] and phospahtidylcholine-phosphatidylserine vesicles (PVPS). The findings suggest that a high affinity glycosaminoglycan binding site is exposed when f.Xa binds to PCPS.