Glycogen synthase kinase-3α limits ischemic injury, cardiac rupture, post-myocardial infarction remodeling and death.

BACKGROUND: The molecular pathways that regulate the extent of ischemic injury and post-myocardial infarction (MI) remodeling are not well understood. We recently demonstrated that glycogen synthase kinase-3α (GSK-3α) is critical to the heart's response to pressure overload. However, the role, if any, of GSK-3α in regulating ischemic injury and its consequences is not known. METHODS AND RESULTS: MI was induced in wild-type (WT) versus GSK-3α((-/-)) (KO) littermates by left anterior descending coronary artery ligation. Pre-MI, WT, and KO hearts had comparable chamber dimensions and ventricular function, but as early as 1 week post-MI, KO mice had significantly more left ventricular dilatation and dysfunction than WT mice. KO mice also had increased mortality during the first 10 days post-MI (43% versus 22%; P=0.04), and postmortem examination confirmed cardiac rupture as the cause of most of the deaths. In the mice that survived the first 10 days, left ventricular dilatation and dysfunction remained worse in the KO mice throughout the study (8 weeks). Hypertrophy, fibrosis, and heart failure were all increased in the KO mice. Given the early deaths due to rupture and the significant reduction in left ventricular function evident as early as 1 week post-MI, we examined infarct size following a 48-hour coronary artery ligation and found it to be increased in the KO mice. This was accompanied by increased apoptosis in the border zone of the MI. This increased susceptibility to ischemic injury-induced apoptosis was also seen in cardiomyocytes isolated from the KO mice that were exposed to hypoxia. Finally, Bax translocation to the mitochondria and cytochrome C release into the cytosol were increased in the KO mice. CONCLUSION: GSK-3α confers resistance to ischemic injury, at least in part, via limiting apoptosis. Loss of GSK-3α promotes ischemic injury, increases risk of cardiac rupture, accentuates post-MI remodeling and left ventricular dysfunction, and increases the progression to heart failure. These findings are in striking contrast to multiple previous reports in which deletion or inhibition of GSK-3ß is protective.

Phosphate and calcium are required for TGFbeta-mediated stimulation of ANK expression and function during chondrogenesis.

The expression of ANK, a key player in biomineralization, is stimulated by treatment with TGFbeta. The purpose of this study was to determine whether TGFbeta stimulation of ANK expression during chondrogenesis was dependent upon the influx of calcium and phosphate into cells. Treatment of ATDC5 cells with TGFbeta increased ANK expression during all phases of chondrogenic differentiation, particularly at day 14 (proliferation) and day 32 (mineralizing hypertrophy) of culture. Phosphate uptake studies in the presence and absence of phosphonoformic acid (PFA), a competitive inhibitor of the type III Na(+)/Pi channels Pit-1 and Pit-2, indicated that the stimulation of ANK expression by TGFbeta required the influx of phosphate, specifically by the Pit-1 transporter, at all phases of differentiation. At hypertrophy, when alkaline phosphatase is highly expressed, inhibition of its activity with levamisole also abrogated the stimulatory effect of TGFbeta on ANK expression, further illustrating that Pi availability and uptake by the cells is necessary for stimulation of ANK expression in response to TGFbeta. Since previous studies of endochondral ossification in the growth plate have shown that L-type calcium channels are essential for chondrogenesis, we investigated their role in the TGFbeta-stimulated ANK response in ATDC5 cells. Treatment with nifedipine to inhibit calcium influx via the L-type channel Cav1.2 (alpha(1C)) inhibited the TGFbeta stimulated increase in ANK expression at all phases of chondrogenesis. Our findings indicate that TGFbeta stimulation of ANK expression is dependent upon the influx of phosphate and calcium into ATDC5 cells at all stages of differentiation.

Modulation of Cell Attachment, Proliferation, and Angiogenesis by Decellularized, Dehydrated Human Amniotic Membrane in In Vitro Models.

BACKGROUND: Decellularized, dehydrated human amniotic membrane (DDHAM) is an extracellular matrix devoid of cells, cell debris, and growth factors. This study examines the effect of cell attachment to the DDHAM and the induced cellular responses. MATERIALS AND METHODS: The cell types employed in this study were human dermal fibroblasts (HDF), human epithelial keratinocytes (HEK), and human dermal microvascular endothelial cells (HDMEC), all of which play critical roles in the wound healing process. Further, the DDHAM was compared to a dehydrated human amnion/chorion membrane (dHACM), which contains and releases biological entities including growth factors and cytokines. The HDF and HEK were cultured on the DDHAM and the dHACM, and cell imaging and proliferation assays were performed to evaluate cell attachment to and the ability to proliferate on the DDHAM relative to the dHACM. In addition, the effect of soluble factors released by the DDHAM and the dHACM on cell survival, attachment, and proliferation were examined. The authors also evaluated the effect of soluble factors produced by culturing cells on the DDHAM in in vitro functional assays, including cell survival and endothelial cell migration in a wound closure angiogenesis assay. RESULTS: The HDF and HEK cells readily attached to and proliferated on the DDHAM, while the dHACM did not support cell attachment and proliferation when cultured under the same conditions. Soluble factors secreted when HDF were cultured on the DDHAM enhanced both endothelial cell and keratinocyte survival and endothelial cell migration in a wound closure assay. CONCLUSIONS: Although DDHAM is only an extracellular matrix and serves primarily as a scaffold, it has sufficient cues to allow for cell attachment and proliferation. Further, the biological entities released as a consequence of cell attachment promote cell survival and migration.

The pleiotropic Arabidopsis frd mutation with altered coordination of chloroplast biogenesis, cell size and differentiation, organ size and number.

In higher plants, plastid development must be tightly coordinated with cell and organ development. In this paper, a novel T-DNA-mutagenized Arabidopsis line showing chlorotic leaves and minute stature was identified in a genetic screen for altered chloroplast development. The mutation corresponded to a single locus on chromosome IV and was associated with insertion of the T-DNA. This locus was named FARFADET and resulted in pleiotropic effects on chloroplast biogenesis, cell size and differentiation, organ size and number. Thus, in contrast with previously described chlorotic mutants, frd mutants were affected not only in chloroplast development and chlorophyll accumulation, but also in cell and organ development. Alteration of differentiation affected different cell types such as leaf epidermal cells, trichomes, mesophyll cells, and columella cells. A major effect on mesophyll cell differentiation was the lack of palisadic parenchyma and absence of grana stacks. Moreover, meristem size and lateral meristem initiation were affected. Genetic and molecular characterisation showed that the T-DNA insertion generated 41 bp deletion in a potential miRNA precursor. The predicted miRNA target genes were involved in plant development and stress. It is therefore hypothesized that the frd mutation had affected coordination of cell developmental span and the control of the division-differentiation balance.

Cytosolic phospholipase A(2)α protects against ischemia/reperfusion injury in the heart.

Studies with sPLA(2) Group X, and cPLA(2) α gene-targeted mice suggest that absence of sPLA(2) Group X results in protection from ischemia/reperfusion (I/R) injury in the heart, and absence of cPLA(2) α Group IV is protective in the brain. Although latter studies might suggest a similar deleterious role for cPLA(2) α in I/R injury in the heart, the pathophysiology of stroke is intricately related to excitotoxicity and cannot necessarily be extrapolated to the heart. We report here that unlike findings in the brain, cPLA(2) α((-/-)) mice have exaggerated injury following I/R in vivo. In contrast, there is no difference in injury induced by simulated ischemia in cardiomyocytes isolated from cPLA(2) α((-/-)) versus cPLA(2) α((+/+)) mice. This suggests that cPLA(2) α does not have an important cardiomyocyte autonomous effect on ischemic injury. Prostaglandin E(2) (PGE(2) ) levels are significantly reduced in the hearts of the cPLA(2) α((-/-)) mice, and the enhanced injury is ameliorated by treatment with the PGE analog, misoprostol. We demonstrate that cPLA(2) α is cardioprotective in vivo, and this is likely via cPLA(2) α-mediated production of cardioprotective eicosanoids. These studies are the first to identify a protective role for cPLA(2) in I/R injury in any organ and raise concerns over long-term inhibition of cPLA(2).

Oxygen tension regulates the expression of ANK (progressive ankylosis) in an HIF-1-dependent manner in growth plate chondrocytes.

The proximal promoter region of ANK, a gene that codes for a protein that regulates the transport of inorganic pyrophosphate, contains two hypoxia responsive elements (HREs); therefore, we studied the expression and function of ANK at different oxygen tensions. ATDC5 and N1511 clonal chondrocytic cells were cultured in either hypoxia (2% O(2)) or normoxia (21% O(2)). Transcript and protein levels of ANK were depressed in hypoxic conditions, as were levels of extracellular pyrophosphate (ePPi). To determine whether HIF-1 was involved in the oxemic response, Hif-1alpha knockdown cells were exposed to varying oxygen conditions and ANK expression was assessed. Knockdown of Hif-1alpha resulted in low levels of expression of ANK in hypoxia and normoxia. Chromatin immunoprecipitation (ChIP) assays explored the binding of Hif-1alpha to ANK HREs and showed that Hif-1alpha is able to bind to the HREs of ANK more avidly in normoxia than in hypoxia. Furthermore, functional studies of Hif-1alpha activity using luciferase reporter assays of wildtype and mutagenized HREs showed that only HRE-1 binds Hif-1alpha in normoxia. Expression of ANK in growth plate and articular cartilage was low in hypoxic regions of the tissues, and higher levels of ANK expression were observed in the synovium and meniscus in regions that have a normally higher oxygen tension. The data suggest that ANK expression and function in vitro and in vivo are repressed in hypoxic environments and that the effect is regulated by HIF-1.

Transforming growth factor-beta1 regulates fibronectin isoform expression and splicing factor SRp40 expression during ATDC5 chondrogenic maturation.

Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that includes the type II repeat extra domain B (EDB) but excludes extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for the progression of chondrogenesis. To test this, we treated the pre-chondrogenic cell line ATDC5 with transforming growth factor-beta1, which has been shown to modulate expression of the EDA and EDB exons, as well as the late markers of chondrocyte maturation; it also slightly accelerates the early acquisition of a sulfated proteoglycan matrix without affecting cell proliferation. When chondrocytes are treated with TGF-beta1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with the regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. To determine if overexpression of the SRp40 isoforms altered FN and FN EDA organization, cDNAs encoding these isoforms were overexpressed in ATDC5 cells. Overexpression of the long-form of SRp40 yielded an FN organization similar to TGF-beta1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-beta1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression.

New developments in the epidemiology and genetics of gout.

The prevalence of gout appears to be rapidly increasing worldwide and is no longer a disorder suffered primarily by over-fed alcohol consumers. Emerging risk factors include longevity, metabolic syndrome, and new classes of pharmacologic agents. In some ethnic populations, no obvious risk factors can explain the high incidence of hyperuricemia and gout, suggesting a genetic liability. Studies to identify genes associated with gout have included families with defects in purine metabolism, as well as families in whom the occurrence of gout is secondary to renal disorders such as juvenile hyperuricemic nephropathy and medullary cystic kidney disease. Case-control studies of isolated aboriginal cohorts suffering from primary gout have revealed several chromosomal loci that may harbor genes that are important to the development and/or progression of gout.

Role of the progressive ankylosis gene in cartilage mineralization.

PURPOSE OF REVIEW: Among the myriad of players in the calcification of cartilage, ANK is a relatively new entrant. It is a multipass transmembrane protein that regulates the transport of inorganic pyrophosphate between the cell and the extracellular space. Mutations in ANK result in two distinct calcification disorders: craniometaphyseal dysplasia and familial calcium pyrophosphate dihydrate deposition disease. The purpose of this review is to highlight recent work on the role of ANK in physiological and pathological calcification of articular and growth plate cartilage. RECENT FINDINGS: New information on the function of ANK suggests that the protein is part of a constellation of critical components that interact to regulate the elaboration of inorganic pyrophosphate. In addition to ANK, these components include alkaline phosphatase, the ectoenzyme PC-1, and osteopontin. ANK expression is also regulated by a variety of growth factors and cytokines that may further affect the transport of inorganic pyrophosphate and may be particularly relevant to the increased levels of expression of ANK in cartilage from chondrocalcinosis and osteoarthritis patients. SUMMARY: Additional studies will be required to understand the contribution of ANK in shaping the fine balance of components necessary for crystal deposition in degenerating articular cartilage. Furthermore, the precise role of inherited mutations in ANK on the elaboration of inorganic pyrophosphate, and the ultimate deposition of either basic calcium phosphate or calcium pyrophosphate dihydrate crystals, remains unclear.

P5L mutation in Ank results in an increase in extracellular inorganic pyrophosphate during proliferation and nonmineralizing hypertrophy in stably transduced ATDC5 cells.

Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank, result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank as well as with familial chondrocalcinosis-causing Ank mutations. We evaluated the elaboration of inorganic pyrophosphate, the activity of pyrophosphate-modulating enzymes, and the mineralization in the transduced cells. Expression of transduced protein was confirmed by quantitative real-time PCR and by ELISA. Levels of inorganic pyrophosphate were measured, as were the activities of nucleotide pyrophosphatase phosphodiesterase and alkaline phosphatase. We also evaluated the expression of markers of chondrocyte maturation and the nature of the mineralization phase elaborated by transduced cells. The cell line expressing the proline to leucine mutation at position 5 (P5L) consistently displayed higher levels of extracellular inorganic pyrophosphate and higher phosphodiesterase activity than the other transduced lines. During hypertrophy, however, extracellular inorganic pyrophosphate levels were modulated by alkaline phosphatase activity in this cell system, resulting in the deposition of basic calcium phosphate crystals only in all transduced cell lines. Cells overexpressing wild-type Ank displayed a higher level of expression of type X collagen than cells transduced with mutant Ank. Other markers of hypertrophy and terminal differentiation, such as alkaline phosphatase, osteopontin, and runx2, were not significantly different in cells expressing wild-type or mutant Ank in comparison with cells transduced with an empty vector or with untransduced cells. These results suggest that the P5L Ank mutant is capable of demonstrating a gain-of-function with respect to extracellular inorganic pyrophosphate elaboration, but this effect is modified by high levels of expression of alkaline phosphatase in ATDC5 cells during hypertrophy and terminal differentiation, resulting in the deposition of basic calcium phosphate crystals.

Genetics of chondrocalcinosis.

Rapid developments in genetic analysis have enabled the dissection of a variety of arthropathies that are inherited in a Mendelian manner. These disorders include calcium crystal arthropathies such as calcium pyrophosphate dihydrate deposition (CPPD) disease and hydroxyapatite deposition disease. In CPPD disease, mutations in a recently discovered gene, ANKH, have been demonstrated in five affected families and may also be associated with the idiopathic deposition of calcium pyrophosphate dihydrate crystals. The product of ANKH appears to be involved in cellular transport of inorganic pyrophosphate (PPi) and mutations in ANKH have been shown to have a significant impact on the regulation of intra- and extracellular levels of PPi. In families with hydroxyapatite deposition disease, no gene locus has yet been linked to the disorder.

Transforming growth factor-beta1 (TGF-beta1) regulates ATDC5 chondrogenic differentiation and fibronectin isoform expression.

Regulated splicing of fibronectin (FN) occurs during the mesenchymal to chondrocyte transition and ultimately results in the relative enrichment of an extra domain B (EDB) exon-containing FN isoform with the suggestion that FN isoforms may play a functional role in chondrogenesis. Promotion of chondrogenesis can also be achieved by treatment with transforming growth factor-beta (TGF-beta), which also regulates FN isoform expression. We have examined the effects of TGF-beta treatment on the assumption of the chondrogenic phenotype in the teratoma-derived cell line ATDC5 and tested whether these effects on chondrogenesis are paralleled by appropriate changes in FN isoform expression. ATDC5 cells were maintained in a pre-chondrogenic state and, in this state, treated with 10 ng/ml TGF-beta. The cells started to elaborate a matrix rich in sulfated proteoglycans, such that within the first 12 days of culture, TGF-beta1 treatment appeared to slightly accelerate early acquisition of an Alcian blue-stained matrix, and caused a dose- and time-dependent decrease in collagen type I expression; changes in collagen type II expression were variable. At later times, cells treated with TGF-beta became indistinguishable from those of the controls. Interestingly, TGF-beta treatment caused a significant dose- and time-dependent decrease in the proportion of FN containing the extra domain A (EDA) and the EDB exons. These data suggest that TGF-beta induces the early stages of chondrogenic maturation in this pre-chondrogenic line and that TGF-beta treatment increases expression of FN isoforms that lack the EDA and EDB exons.