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Conceived and designed the experiments: JW AKG JV. Performed the experiments: JW AKG JLS KK MW MJ. Analyzed the data: JW AKG JLS MW MJ. Contributed reagents/materials/analysis tools: QQH MLW CFB JV. Wrote the paper: JW AKG JV.
The nuclear orphan receptor peroxisome proliferator-activated receptor-gamma (PPAR-γ) is expressed in multiple cell types in addition to adipocytes. Upon its activation by natural ligands such as fatty acids and eicosanoids, or by synthetic agonists such as rosiglitazone, PPAR-γ regulates adipogenesis, glucose uptake and inflammatory responses. Recent studies establish a novel role for PPAR-γ signaling as an endogenous mechanism for regulating transforming growth factor-ß (TGF-ß)-dependent fibrogenesis. Here, we sought to characterize PPAR-γ function in the prototypic fibrosing disorder systemic sclerosis (SSc), and delineate the factors governing PPAR-γ expression. We report that PPAR-γ levels were markedly diminished in skin and lung biopsies from patients with SSc, and in fibroblasts explanted from the lesional skin. In normal fibroblasts, treatment with TGF-ß resulted in a time- and dose-dependent down-regulation of PPAR-γ expression. Inhibition occurred at the transcriptional level and was mediated via canonical Smad signal transduction. Genome-wide expression profiling of SSc skin biopsies revealed a marked attenuation of PPAR-γ levels and transcriptional activity in a subset of patients with diffuse cutaneous SSc, which was correlated with the presence of a "TGF-ß responsive gene signature" in these biopsies. Together, these results demonstrate that the expression and function of PPAR-γ are impaired in SSc, and reveal the existence of a reciprocal inhibitory cross-talk between TGF-ß activation and PPAR-γ signaling in the context of fibrogenesis. In light of the potent anti-fibrotic effects attributed to PPAR-γ, these observations lead us to propose that excessive TGF-ß activity in SSc accounts for impaired PPAR-γ function, which in turn contributes to unchecked fibroblast activation and progressive fibrosis.
Systemic sclerosis (SSc) is a complex autoimmune disease of unknown etiology associated with early and transient inflammation and vascular injury, followed by progressive fibrosis affecting the skin and multiple internal organs [1]. Fibrosis, the distinguishing pathological hallmark of SSc, is characterized by overproduction of collagen and other extracellular matrix (ECM) components and accumulation of α-smooth muscle actin (ASMA)-positive myofibroblasts, resulting in excessive connective tissue accumulation and matrix remodeling.
Unchecked fibroblast activation in SSc is the result of a complex series of events orchestrated by transforming growth factor-ß (TGF-ß), a multifunctional cytokine that is produced and secreted by most cell types, and also stored in a latent form in the pericellular matrix [2]. TGF-ß signaling is initiated by ligand binding to the type II TGF-ß receptor (TßRII), which leads to activation of the type I TGF-ß receptor (TßRI). Activated TßRI phosphorylates cytoplasmic Smad2 and Smad3, promoting the formation of Smad heterocomplexes and their nuclear accumulation. Within the nucleus, the Smad complex selectively binds to conserved Smad binding element (SBE) DNA sequences, recruits the histone acetyltransferase p300 and other coactivators, and activates or represses the transcription of target genes such as pro-αI (1) collagen (COL1A1) and collagenase-1, respectively [3], [4], [5], [6].
Peroxisome proliferator-activated receptor-gamma (PPAR-γ) is a nuclear orphan receptor and ligand-inducible transcription factor. Initially identified in adipocytes, PPAR-γ was shown to function as a key regulator of normal adipogenesis [7]. During the past decade, it has become apparent that PPAR-γ is widely expressed in mammalian tissues, and regulates not only genes involved in lipid uptake, synthesis and storage, but also glucose metabolism, cell differentiation, survival and proliferation, and immune and inflammatory responses [8], [9]. Naturally-occurring ligands such as 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2) and lysophosphatidic acid (LPA), as well as synthetic ligands such as pioglitazone and rosiglitazone, and triterpenoids such as 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), directly bind to intracellular PPAR-γ and trigger conformational changes that results in recruitment of the transcriptional coactivators steroid receptor coactivator, retinoid X receptor-alpha and p300/CBP [10]. Within the nucleus, the ligand-PPAR-γ complex binds to PPAR response element (PPRE) DNA sequences of target genes, and positively or negatively regulates their transcription [11].
In addition to its well-established impact on adipogenesis and glucose homeostasis, PPAR-γ appears to have an important novel role in the negative regulation of connective tissue biosynthesis, and is increasingly implicated in both physiologic and pathological matrix remodeling [12]. We have shown that natural and synthetic PPAR-γ ligands, while having little effect on basal rates of collagen synthesis and ASMA expression, effectively blocked the stimulation of these fibrotic responses elicited by TGF-ß [13]. The potent anti-fibrotic effects of the PPAR-γ ligands involved the disruption of Smad-dependent intracellular signal transduction via PPAR-γ-mediated transrepression [14]. Subsequent studies using a variety of cell types have confirmed and expanded on these findings [15], [16], [17]. Moreover, treatment with synthetic PPAR-γ ligands was shown to attenuate experimentally-induced hepatic [18], cardiac [19] and kidney [20] fibrosis, and prevent bleomycin-induced scleroderma [21], in vivo. Additionally, the expression of PPAR-γ is markedly diminished in lesional tissues in rodent models of fibrosis and scleroderma [21], [22], [23], as well as in lesional skin from patients with cicatrical (scarring) alopecia [24]. These observations provide compelling support for a physiological role for PPAR-γ in modulating fibroblast activation and matrix remodeling.
In light of the ability of ligand-activated PPAR-γ to disrupt intracellular TGF-ß signal transduction and prevent excessive fibroblast activation, its levels and biological availability in a particular cell or tissue might be one of the critical factors governing the duration and intensity of profibrotic responses. Indeed, we have shown that augmenting cellular PPAR-γ in normal fibroblasts by ectopic overexpression markedly enhanced their sensitivity to PPAR-γ agonists [13]. However, the mechanisms that regulate the cell-type-specific abundance and activity of PPAR-γ are poorly understood. We therefore sought to investigate the regulation of PPAR-γ expression in SSc, and in explanted lung and dermal fibroblasts. We show here that PPAR-γ expression was markedly diminished in skin and lung biopsies from patients with diffuse cutaneous SSc, as well as in unstimulated fibroblasts explanted from the lesional skin. Incubation of normal fibroblasts with TGF-ß induced a time- and dose-dependent suppression of PPAR-γ gene transcription and activity. Inhibition was mediated via the canonical Smad signal transduction pathway. These results reveal a reciprocal functionally antagonistic relationship between ligand-dependent PPAR-γ signaling that blocks Smad-mediated TGF-ß responses, and Smad-mediated TGF-ß signaling which is shown here to suppress the transcription and activity of PPAR-γ. Unchecked TGF-ß signaling in lesional tissue might account for the impaired local PPAR-γ expression and function in patients with dcSSc. Fibroblasts activated via autocrine or paracrine TGF-ß and consequently defective in PPAR-γ function might be unable to properly modulate TGF-ß signaling, resulting in unchecked fibrogenic stimulation that could contribute to persistence and progression of fibrosis in SSc.
In light of the potent anti-fibrotic activities associated with the PPAR-γ pathway, and the demonstration of diminished PPAR-γ expression in various models of fibrosis, we sought to explore the possibility that deregulated fibrogenesis in SSc was accompanied by tissue-specific alterations in PPAR-γ. To examine genome-wide changes in RNA expression, a previously described skin biopsy-derived microarray dataset was interrogated [25]. Analysis of the data showed that levels of PPAR-γ mRNA in the skin were reduced in biopsies from dcSSc patients compared to healthy controls (Fig. 1A). In contrast, plasminogen activator inhibitor-1 (PAI-1), a TGF-ß-regulated gene that is associated with fibrosis and implicated in its pathogenesis, showed increased expression in SSc biopsies [26], [27]. Intriguingly, expression levels of PAI-1 and PPAR-γ mRNA were inversely correlated in these samples (Fig. 1A).
To evaluate the levels of PPAR protein in the skin, biopsies from three patients with SSc and three healthy controls were examined by immunohistochemistry. Low levels of PPAR-γ could be detected in the normal dermis, consistent with prior reports [28]. Detectable PPAR-γ in these skin biopsies was localized mostly to the cells of the epidermis, with scattered dermal cells showing positive immunostaining (Fig. 1B). Lesional skin biopsies from patients with dcSSc consistently showed markedly reduced PPAR-γ immunostaining in the epidermis, and a complete absence of PPAR-γ in the dermis.
Because pulmonary fibrosis commonly complicates SSc, we next examined the expression of PPAR-γ in the lungs. For this purpose, lung biopsies from five SSc patients with advanced pulmonary fibrosis and five individuals with no pulmonary fibrosis were examined by immunohistochemistry. Strong immunostaining was evident in control lungs, with prominent PPAR-γ expression seen in interstitial fibroblasts, alveolar lining cells and macrophages, and displaying a predominantly nuclear distribution (Fig. 1C). In striking contrast, we found that PPAR-γ was virtually undetectable at fibrotic loci in lung biopsies from all five patients with SSc. Immunostaining was specific for PPAR-γ, as substitution of the primary antibody with non-specific mouse IgG resulted in complete absence of staining (not shown).
To further characterize PPAR-γ expression in SSc, we examined explanted lesional skin fibroblasts. The clinical characteristics of the patients studied are shown in Table 1. Total RNA was isolated from confluent low-passage SSc and healthy control skin fibroblasts and examined in parallel by real-time qPCR. The results showed substantially diminished levels of PPAR-γ mRNA in lesional fibroblasts from six SSc patients (Fig. 2A). The mean reduction in PPAR-γ mRNA levels was >70% (p=0.0006) compared to healthy controls. These results were reproducible and consistent over serial passage of the fibroblasts. To compare the cellular abundance of PPAR-γ protein, nuclear fractions were subjected to Western analysis. All three SSc fibroblast lines showed dramatically reduced levels of nuclear PPAR-γ compared to healthy control fibroblasts (Fig. 2B). Taken together, these results demonstrate deficient PPAR-γ mRNA and protein expression in skin and lung biopsies and in explanted dermal fibroblasts from patients with dcSSc.
In light of the central role that TGF-ß plays in mediating the pathogenesis of fibrosis, we sought to examine the possibility that it might modulate the expression of PPAR-γ, and that activated TGF-ß signaling could account for impaired PPAR-γ expression seen in SSc. Previous studies have described the effects of TGF-ß on gene expression in normal dermal fibroblasts using DNA microarray analysis [29]. Analysis of the data showed that TGF-ß treatment of two independent lines of dermal fibroblasts was associated with a time-dependent decrease in PPAR-γ mRNA expression (data not shown) that was accompanied by a similar inhibition of MMP-1 and Smad3, as we had described previously [3], [6]. This decrease was mirrored by an increase in the expression of fibrosis-related genes such as ASMA, SERPINE1, CTGF and COMP1. In separate experiments, normal lung fibroblasts incubated with TGF-ß showed an identical decrease in PPAR-γ mRNA expression (data not shown).
To further explore the regulation of PPAR-γ mRNA expression, confluent normal dermal fibroblasts were incubated with 10 ng/ml TGF-ß for various periods. Under the experimental conditions employed, there was no detectable cell toxicity due to TGF-β (data not shown). The results of real-time qPCR demonstrated a progressive time- and dose-dependent decrease in the levels of PPAR-γ1 and PPAR-γ2 mRNA, with a maximal >70% inhibition after 24 h (Fig. 3A and data not shown). Essentially identical results were obtained when normal lung fibroblasts were incubated with TGF-ß (data not shown). Suppression of PPAR-γ mRNA in TGF-ß-treated fibroblasts was further confirmed by Northern analysis (Fig. 3B). Remarkably, we noted that the negative regulation of PPAR-γ by TGF-ß was cell type-specific. For instance, in sharp contrast to fibroblasts, incubation of monocyte-derived macrophages with TGF-ß caused stimulation of both PPAR-γ1 and PPAR-γ2 mRNA (Fig. 3C and data not shown). Furthermore, we found that the Th2 cytokine IL-4 stimulated PPAR-γ mRNA in macrophages, whereas it had no effect in fibroblasts.
To evaluate the functional consequences of inhibition by TGF-ß, we examined the regulation PPAR-γ transcriptional activity. Fibroblasts were transfected with PPRE-luc and, 18 h later, rosiglitazone and TGF-ß were added to the cultures. Following a further 48 h incubation, cultures were harvested and whole cell lysates were assayed for their luciferase activities. The results showed that while rosiglitazone induced a >2-fold increase in the activity of the PPRE promoter, TGF-ß significantly attenuated PPRE-driven luc activity both in the presence and absence of rosiglitazone (Fig. 4A). As expected, the cellular levels of PPAR-γ showed a marked reduction at 48 h in TGF-ß-treated fibroblasts (Fig. 4B). These results indicate that inhibition of PPAR-γ gene expression by TGF-ß was accompanied by reduced transcriptional activity both in the absence and presence of PPAR-γ ligand.
A series of experiments were performed in order to characterize the cellular mechanisms underlying the potent inhibitory effect of TGF-ß on PPAR-γ expression. First, we used selective protein kinase inhibitors to identify intracellular signaling pathways potentially mediating the TGF-ß effect. These studies showed that neither U0126, SB203580 nor SP600125, specific inhibitors of the MEK1/2, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, respectively, abrogated the TGF-ß-induced suppression of PPAR-γ1 and PPAR-γ2 mRNA (Fig. 5A and data not shown). In contrast, the ALK5 inhibitor SB431542, which blocks TßRI-mediated Smad2/3 phosphorylation, almost completely prevented the inhibitory effects. Interestingly, ALK5 blockade by itself caused a >2-fold increase in PPAR-γ mRNA levels, indicating that constitutive TGF-ß signaling causes basal repression of PPAR-γ mRNA in normal fibroblasts. Furthermore, while TGF-ß caused a dose-dependent inhibition of PPAR-γ-luc activity in transiently transfected normal dermal fibroblasts, pretreatment of the cultures with SB431542 completely abrogated this effect (data not shown). These results suggest that TGF-ß-induced inhibition of PPAR-γ occurred at the level of transcription and was mediated via the canonical Smad pathway.
Additional studies were pursued to further explore the role of the Smad signaling in PPAR-γ regulation. First, constitutively active (TßRIca) or non-functional (TßRIL45) mutants of TßRI were ectopically expressed in normal fibroblasts. As shown in Fig. 5B, Ad-TßRIca induced a dose-dependent suppression of PPAR-γ1 mRNA in the absence of exogenous TGF-ß, whereas the non-functional TßRIL45 had no effect. Moreover, adenoviral expression of Smad7, an inhibitory SMAD that forms stable association with the TGF-ß receptor complex and prevents phosphorylation of pathway-restricted Smads, blocked the inhibition of PPAR-γ mRNA by TGF-ß (Fig. 5C). As would be expected, Smad7 blocked TGF-ß stimulation of collagen synthesis in infected fibroblasts (Fig. 5C, right panel). Because Smad7 can abrogate both Smad2- and Smad3-dependent responses, we sought to clarify the specific role of cellular Smad3 in PPAR-γ suppression using mouse fibroblasts deficient in Smad3 [30]. The results showed that in contrast to wildtype fibroblasts treated with TGF-ß, in Smad3−/− fibroblasts TGF-ß failed to down-regulate PPAR-γ mRNA (Fig. 5D). As expected, TGF-ß stimulation of Type I collagen gene expression was also lost in Smad3-deficient cells. On the other hand, transient overexpression of Smad3 in normal human dermal fibroblasts was by itself sufficient to repress the activity of the PPAR-γ promoter (Fig. 5E). Taken together, these results firmly establish the critical roles of the canonical Smad signaling pathway involving TßRI and Smad3 as both necessary and sufficient for driving transcriptional suppression of PPAR-γ in response to TGF-ß.
One of the key physiologic functions of PPAR-γ is the regulation of adipogenesis. Ectopic PPAR-γ is both necessary and sufficient to induce PPAR-γ-dependent adipogenic differentiation in normal mesenchymal cells [7], [31]. The potent inhibitory effect of TGF-ß on the expression and activity of PPAR-γ prompted us to examine the modulation of adipogenesis by TGF-ß. For this purpose, human subcutaneous preadipocytes and normal adult dermal fibroblasts were incubated in adipogenic differentiation media. After three cycles of adipogenic induction, prominent intracellular lipid accumulation was evident in both preadipocytes as well as in dermal fibroblasts (Fig. 6). That these cells underwent adipogenic differentiation was further confirmed by the marked up-regulation of the adipocyte-specific marker FABP4. Inclusion of TGF-ß in the differentiation media resulted in a substantial decrease in PPAR-γ mRNA. Importantly, TGF-ß completely prevented adipogenic differentiation in both subcutaneous preadipocytes and dermal fibroblasts.
By causing a substantial reduction in cellular PPAR-γ abundance, TGF-ß might be expected to attenuate the cell's ability to respond to PPAR-γ ligands. To examine this possibility, the regulation of FABP4, a cardinal endogenous target for activated PPAR-γ was studied. Confluent dermal fibroblasts were incubated with TGF-ß, with rosiglitazone added to the cultures after 24 h. Following a further 24 h incubation, cultures were harvested. As shown in Fig. 7A, in the presence of TGF-ß, rosiglitazone failed to induce full stimulation of FABP4. These results indicate that TGF-ß exposure resulted in partial loss of PPAR-γ–dependent adipogenic effects of rosiglitazone, presumably due to the marked decrease in cellular PPAR-γ abundance induced by exposure to TGF-ß. In contrast, rosiglitazone was capable of partially reducing the stimulation of collagen and ASMA expression when given 24 h following TGF-ß treatment (Fig. 7A–C). These results indicate that TGF-ß markedly attenuated the adipogenic activities of the synthetic PPAR-γ ligand, while only partially attenuating its anti-fibrotic activities, suggesting that the latter might also involve some PPAR-γ-independent mechanisms.
In a previous report using microarray-based genome-wide expression profiling of scleroderma skin biopsies, a pattern of gene expression consistent with active TGF-ß signaling was observed in subsets of SSc patients with diffuse cutaneous disease [29]. In light of the potent inhibitory effects of TGF-ß on PPAR-γ demonstrated in our studies, we sought to evaluate the expression of PPAR-γ-regulated genes in the subset of dcSSc patients whose skin biopsies showed a prominent "TGF-ß activated signature". A list of 87 putative PPAR-γ target genes was compiled from an extensive literature review. The expression of these genes was then examined in a gene expression microarray dataset from a well-characterized cohort of SSc patients [25]. None of these patients were receiving thiazolidinediones at the time of the biopsies. The data, organized by hierarchical clustering based on the expression level of TGF-ß-responsive genes as demonstrated previously [29], are shown as TGF-ß-activated (red) and TGF-ß-not activated (black) clusters. PPAR-γ target genes compiled from literature review showed decreased expression in biopsies from the TGF-ß-activated dcSSc subset compared to the TGF-ß-not activated SSc subset, or to healthy controls (Fig. 8). The significance of differential regulation for each gene was determined using Significance Analysis of Microarrays (SAM). There were forty-one putative PPAR-γ target genes with significant differential expression in the TGF-ß-activated versus TGF-ß-not activated SSc subsets (FDR <0.78%). Thirty-three of these PPAR-γ target genes (80%) showed reduced expression in the TGF-ß-activated group, and elevated expression in the TGF-ß-not-activated group (Fig. 8).
Excessive extracellular matrix accumulation and tissue contraction, the hallmarks of fibrosis, are mediated by fibroblasts and myofibroblasts activated by TGF-ß [1]. Blocking TGF-ß activity therefore represents an appealing approach to fibrosis therapy, and there is intense interest in identifying the intracellular pathways that positively or negatively modulate the fibrotic responses. Recent research findings suggest that PPAR-γ signaling is a novel cell-intrinsic mechanism to negatively regulate profibrotic responses.
PPAR-γ exerts potent antifibrotic effects in vitro and in vivo. We showed previously that PPAR-γ activation by natural or synthetic ligands abrogated collagen synthesis, cell migration and myofibroblast transdifferentiation induced by TGF-ß [21]. While the precise mechanisms underlying abrogation of TGF-ß signaling remain to be fully delineated, we found that PPAR-γ blocked Smad-dependent transcriptional responses without interfering with Smad phosphorylation or nuclear accumulation. Instead, PPAR-γ appears to compete with ligand-inducible transcription factors for binding to limiting p300, squelching Smad/p300 complex formation in TGF-ß-stimulated fibroblasts [14]. Furthermore, in animal models of TGF-ß-dependent fibrosis, rosiglitazone treatment attenuated the development of dermal thickness and collagen accumulation in vivo [21], [32]. Because the biological activities of PPAR-γ ligands depend largely on their interaction with PPAR-γ, the relative levels or biologically availability of PPAR-γ are important factors governing the intensity of ligand-induced cellular responses. Surprisingly, little is known about the expression of PPAR-γ in fibroblasts, and its regulation during inflammation, tissue repair and fibrosis. In the present studies we found that the levels of PPAR-γ protein and mRNA were substantially lower in SSc lesional tissue and explanted skin fibroblasts than in healthy controls. Moreover, genome-wide expression analysis using microarrays demonstrated that SSc skin biopsies with evidence of active TGF-ß signaling had low expression of PPAR-γ-regulated genes, revealing an inverse relationship between TGF-ß activity and PPAR-γ-dependent responses in fibrotic tissues.
We examined the regulation of PPAR-γ by TGF-ß in normal fibroblasts. Despite its importance in determining the intensity of ligand-induced responses, the factors involved in the regulation of PPAR-γ expression are not well understood. Our results demonstrated that TGF-ß caused a potent time- and dose-dependent and cell-type specific down-regulation of PPAR-γ transcription. This inhibitory response was mediated through the canonical Smad signal transduction pathway. A 2.7 kb fragment of the PPAR-γ gene promoter harboring two putative Smad binding elements was sufficient for inhibition by TGF-ß. Taken together with previous findings, the present results indicate that TGF-ß and PPAR-γ are engaged in a mutually antagonistic reciprocal cross-talk, whereby the PPAR-γ pathway abrogates cellular responses induced by TGF-ß, while TGF-ß-Smad inhibits the expression of PPAR-γ and its target genes.
In addition to TGF-ß, IL-13, Wnt, CCN2, as well as hypoxia, have all been shown to inhibit PPAR-γ expression or activity [33], [34], [35], [36], [37]. Particularly interesting is lysophosphatidic acid (LPA), a bioactive lipid that potently inhibits the expression of PPAR-γ in a variety of cell types [38], while also acting as a PPAR-γ agonist [39]. The signaling mechanisms mediating down-regulation of PPAR-γ in response to these cues are not well understood. It is noteworthy that CCN2 and Wnt are known to be up-regulated in the fibrotic cellular milieu in SSc, and chronic tissue hypoxia due to microvascular insufficiency is prominent [1], [40]. Thus, multiple factors implicated in the pathogenesis of fibrosis might contribute to the profound down-regulation of PPAR-γ in SSc. We and others have also observed that PPAR-γ protein and mRNA levels were persistently reduced in explanted SSc fibroblasts [17]. At present, it is unclear whether autonomous PPAR-γ down-regulation represents a cell-inherent abnormality in gene regulation, such as epigenetic modifications at the PPAR-γ promoter, or results from constitutive autocrine TGF-ß stimulation. The latter mechanism is plausible in light of the finding that in normal fibroblasts, constitutive basal TGF-ß signaling maintains tonic suppression of PPAR-γ (Fig. 5A). Epigenetic modification represents an appealing possibility to account for low level of PPAR-γ gene expression, since methylation-mediated silencing of inhibitory transcription factors such as Fli1 or Smad7 has been implicated as a mechanism for autonomous activation of SSc fibroblasts [41],[42]. While we cannot fully exclude the possibility that hypermethylation of the regulatory elements of the PPAR-γ gene result in gene silencing in SSc fibroblasts, in preliminary studies the methylation inhibitor 5-azacytidine failed to normalize PPAR-γ expression (data not shown).
Analysis of microarray data from SSc skin biopsies previously identified a patient subset with a "TGF-ß-responsive gene signature" [25]. This pattern of gene expression was only seen in biopsies from patients with dcSSc, and was not found in biopsies from patients with other forms of scleroderma, or from healthy controls. The "TGF-ß responsive signature" identifies a subset of scleroderma patients with extensive skin involvement and propensity to pulmonary fibrosis [29]. Remarkably we observed that the expression of a majority of differentially regulated PPAR-γ target genes (80%) was diminished in the "TGF-ß responsive gene signature" subset biopsies. These observations reveal an inverse relationship in the tissue expression of TGF-ß-inducible genes and PPAR-γ inducible genes, a result not unexpected in view of our present findings that TGF-ß suppresses PPAR-γ expression and transcriptional activity in fibroblasts, and our previous demonstration that PPAR-γ directly inhibits Smad-mediated TGF-ß responses. The reciprocal relationship between the expressions of these two sets of genes is consistent with a mutually antagonistic cross-talk of the TGF-ß and PPAR-γ pathways. In this context it is remarkable that diminished PPAP-γ expression or function is a recurring theme in human fibrotic conditions and animal models of fibrosis. For example, intracellular PPAR-γ levels were diminished in alveolar macrophages from patients with pulmonary fibrosis [43], and in the fibrotic lesions in cicatrical alopecia [24]. In addition to the association of low PPAR-γ with fibrosis in various human disorders, evidence for a causal relationship comes from animal models. Tissue-specific down-regulation of PPAR-γ, whether due to loss-of-function mutations [44], genetic targeting [45], [46], or overexpression of PPAR-γ inhibitors such as TGF-ß or Wnt [47], [48], is invariably accompanied by exuberant local fibrosis. It is intriguing that in SSc, skin fibrosis is commonly accompanied by a reduction in the subcutaneous fat layer, and in some biopsies, complete adipose tissue atrophy [49]. We speculate that in these cases active TGF-ß signaling in the dermis suppresses PPAR-γ in situ with consequent disruption of adipogenesis, loss of adipocytes and lipoatrophy. These observations highlight the importance of a functional antagonism between profibrotic TGF-ß signaling on the one hand, and the anti-fibrotic PPAR-γ pathway on the other, in the pathogenesis of SSc.
One of the critical mechanisms employed by TGF-β in the promotion of fibrosis is epithelial-mesenchymal transition (EMT) [50]. In this study, we observed reduced levels of epidermal PPAR-γ in SSc skin biopsies, whereas normal skin biopsies showed strong immunostaining in the basal epidermis (Figs 1B). High level of TGF-ß and ASMA expression in the basal epidermis have been reported previously [51]. Because PPAR-γ signaling potently inhibits TGF-β-induced EMT in vitro [52], these observations raise the possibility that an important physiologic role of PPAR-γ in the epidermis is to prevent deregulated EMT, and its loss in SSc may contribute to promotion of EMT and fibrosis.
Since a primary biological function of PPAR-γ is to serve as an intracellular sensor for changes in fatty acid or prostaglandin levels, its abundance and availability are important in determining the intensity of ligand responses. Cellular responses elicited by endogenous ligands, such as adipogenic differentiation of mesenchymal progenitor cells, are dependent on PPAR-γ signaling [31]. We have previously shown that augmenting levels of PPAR-γ by means of ectopic overexpression enhanced the cellular responsiveness to a PPAR-γ ligand [13], [53]. In light of the potent down-regulation of PPAR-γ observed in TGF-ß-treated cells, we anticipated that TGF-ß might reduce cellular sensitivity to PPAR-γ ligands. We found however that fibroblast activation was blunted by rosiglitazone even when it was added to the cultures 24 h after TGF-ß. Therefore, synthetic PPAR-γ ligands are capable of abrogating profibrotic responses even in the face of low PPAR-γ abundance. Currently approved PPAR-γ ligands such as rosiglitazone have been linked to side effects, including fluid retention, heart failure, and other cardiovascular events, raising questions regarding their safety [54]. Novel PPAR-γ agonists with selective activity and potentially improved safety are under active investigation [55].
In summary, these results reveal that PPAR-γ expression and activity are impaired in SSc, which is correlated with active TGF-ß signaling and fibrosis in lesional tissues. There exists a reciprocal relation between the expression of TGF-ß-activated genes and PPAR-γ-regulated genes in lesional tissue and in explanted SSc fibroblasts. PPAR-γ plays a physiologic role in tissue remodeling as a negative regulator of fibroblast activation and differentiation, which is highlighted by the link between loss of PPAR-γ and spontaneous emergence of fibrosis. Reduced PPAR-γ in SSc, resulting from its suppression by TGF-ß and related cytokines, hypoxia and other mechanisms might contribute to unchecked fibroblast activation and persistent fibrogenesis.
The protocols for human tissue collection were approved by the Institutional Review Boards for Human Studies at Northwestern University and University of Pittsburgh. A written informed consent was obtained from all participants.
Primary cultures of dermal fibroblasts were established by explantation from biopsies of neonatal foreskins, or from affected dorsal forearm of seven patients with SSc and four healthy controls [3], [56]. Only patients with diffuse cutaneous SSc (dcSSc) who were not receiving treatment with corticosteroids were studied. Primary cell cultures were also established from the dorsal skin of newborn Smad3-null (Smad3–/–) mice or wildtype (Smad3+/+) littermates [30]. Unless otherwise indicated, all fibroblasts were maintained at 37°C in an atmosphere of 5% CO2 in Eagle's Minimum Essential Medium (EMEM) or Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% vitamins, 1% penicillin/streptomycin, and 2 mM L-glutamine (all from BioWhittaker, Walkersville, MD), and studied between passages 4 and 8. When fibroblasts reached early confluence, fresh media containing TGF-ß (Genzyme, Framingham, MA) were added to the cultures. In selected experiments, cultures were pretreated with 15d-PGJ2 (BioMol, Plymouth Meeting, PA), troglitazone (GlaxoSmithKline, King of Prussia, PA), rosiglitazone (Cayman Chemical, Ann Arbor, MI), interleukin-4 (IL-4) or platelet-derived growth factor (PDGF) (both from Sigma, St. Louis, MO) for indicated periods. In other experiments, the protein kinase inhibitors U0126 (Cell Signaling Technology, Beverly, MA), SB431542 or SB203580 (both from Sigma) at 10 µM, or SP600125 (Sigma) at 20 µM, were added to the cultures 60 min prior to TGF-ß. In experiments focusing on profibrotic responses in fibroblasts, TGF-β at 10 ng/ml, a concentration shown to be maximally effective and non-toxic in prior studies, was used. Cell viability was determined by Trypan blue dye exclusion.
Human peripheral blood macrophages were isolated from buffy coats from healthy donors (Lifesource, Glenview, IL) by Histopaque (Sigma) gradient centrifugation followed by countercurrent centrifugal elutriation (JE-6B, Beckman Coulter, Palo Alto, CA). Isolated monocytes were ≥90% pure as determined by morphology, non-specific esterase staining and CD14 expression by flow cytometry. Following adherence to plates for 60 min, monocytes differentiated into macrophages for seven days in Roswell Park Memorial Institute (RPMI) media containing 20% heat-inactivated FBS, L-glutamine, and penicillin and streptomycin, and used in experiments.
Human subcutaneous preadipocytes (Zen-Bio Inc, Research Triangle Park, NC) or low-passage adult dermal fibroblasts were induced to undergo in vitro adipogenic differentiation as previously described [57]. Briefly, when cells reached confluence, cultures were incubated in DM-2 adipogenic induction media for three days, followed by AM-1 adipogenic maintenance media for a further three days (both from Zen-Bio). This cycle was then repeated three times. Following the final DM-AM cycle, incubation in AM was continued for up to a further 28 days prior to harvesting.
Total RNA was isolated from confluent fibroblasts using TRIzol Reagent (Invitrogen, Carlsbad, CA), and levels of mRNA were determined by Northern analysis or real-time quantitative PCR (qPCR). Northern analysis was performed using α-32P-labeled human PPAR-γ 1 cDNA probes [13]. Following washing of the nitrocellulose membranes, the RNA-cDNA hybrids were visualized by autoradiography. For real-time quantitative qPCR, reverse transcription were performed using SuperScript First-strand synthesis system (Invitrogen) according to the manufacturer's protocol. For real-time qPCR, reactions were performed on ABI-Prism 7300 sequence detection PCR machine (Applied Biosystem, Forster City, CA) according to the manufacturer's protocol [58]. The primers used for qPCR are listed in Table 2. Relative mRNA expression levels were normalized with ß-actin mRNA or 18S RNA levels and determined by calculating ΔΔCt.
The PPAR-γ–luc plasmid contains 2.7 kb of the 5′ flanking region of the human PPAR-γ gene promoter linked to the luciferase reporter gene [59], [60]. The PPRE-luc plasmid contains 3 tandem copies of the PPAR response element (PPRE) sequence of the acyl-coenzyme A oxidase gene linked to thymidine kinase and luciferase genes [61]. A Smad3 expression vector has been previously described [62]. Replication-incompetent adenoviral vectors expressing constitutively active human TßRI (Ad-TßRIca), L45 loop-mutated TßRI that lacks Smad binding ability (Ad-TßRIL45), and control adenovirus (Ad-null) were from Vesa Kaartinen [63].
Fibroblasts (105 cells/well) were seeded in 12-well plates, and at early confluence, transiently cotransfected with reporter constructs and expression vectors or appropriate empty vectors using Superfect Reagent (Qiagen, Valencia, CA), or infected with adenoviruses. Following incubation with TGF-ß for 24 h, cultures were harvested and whole cell lysates were assayed for their luciferase activities [13]. The pRL-TK Renilla luciferase (pRL-TK-Luc) plasmid was used in each transfection experiment as an internal control. Experiments were performed in triplicate.
At the end of the incubation periods, cultures were harvested, whole cell lysates or nuclear or cytosolic fractions were prepared, and aliquots containing equal amounts of proteins (10–15 µg) were subjected to Western analysis [13]. Membranes were immunobloted with primary antibodies to human PPAR-γ (1250 dilution) [64], Smad1/2/3, actin, TßRI, Smad7 (1200 dilution, all from Santa Cruz Biotechnology, Santa Cruz, CA), Smad3 (1250 dilution; Zymed), phospho-Smad2 (11000 dilution; Cell Signaling Technology, Danvers, MA) or Type I collagen (1400 dilution; Southern Biotechnology, Birmingham, AL). Membranes were then washed and incubated with appropriate secondary antibodies for 45 min, and antigen–antibody complexes were visualized by chemiluminescence (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. The results were normalized against the intensity of the ß-actin in each sample.
Fibroblasts were seeded on cover slides in a 6-well chamber and incubated in serum-free EMEM containing 0.1% BSA, TGF-ß (10 ng/ml) and rosiglitazone (10 µM). At the end of the incubation periods, cells were fixed and processed for immunocytochemistry [65]. The slides were incubated with primary antibodies against PPAR-γ or α-smooth muscle actin for 120 min, followed by Alexa® 488-conjugated chicken-anti-rabbit IgG or chicken-anti-mouse IgG (Invitrogen) for 60 min. Nuclei were identified with 4′-6-Diamidino-2-phenylindole (DAPI). Non-immunized IgG was used in each experiment as a negative control. Following stringent washing, slides were examined using Zeiss UV Meta 510 confocal microscope (Carl Zeiss, Jena, Germany). Each experiment was repeated at least three times with consistent results.
Lung tissues were obtained from five patients with SSc-associated pulmonary fibrosis who were undergoing lung transplant surgery and from unused normal lungs from five donors. Sections (5 µm) were deparaffinized, rehydrated and immersed in TBS-T buffer (Tris-buffered saline −0.1%Tween 20), treated with target retrieval solution (DAKO, Carpinteria, CA) at 95°C for 10 min and incubated with primary antibody against PPAR-γ (1100 dilution, Santa Cruz). Bound antibodies were detected using secondary antibodies and System-HRP (DAB) (Dako, Glostrup, Denmark). Substitution of the primary antibody with isotype-matched irrelevant IgG served as negative controls. Sections were counterstained with hematoxylin, mounted with Permount (Fisher Scientific, Pittsburgh, PA) and viewed under an Axioskop microscope (Carl Zeiss). Pictures were taken and modified by Nuance Multiple Spectra CCD with Nuance 2.10 software. To quantify PPAR-γ expression, cells in five microscopic fields in each sample were scored as clearly immunopositive or negative for PPAR-γ by two independent examiners, and the ratio of positive cells to total cells in each field was calculated.
The generation and analysis of the microarray dataset using skin biopsies from patients with SSc and normal controls was described previously [25]. The results of microarray analysis were ordered according to their "TGF-ß-responsive gene signature" expression [29]. To interrogate the expression of PPAR-γ target genes in the dataset, 87 genes known from the literature to be regulated by PPAR-γ were extracted from the microarray data. Those genes showing statistically significant differences in expression between "TGF-ß activated" and "TGF-ß not activated" subsets were selected for examination.
The data are presented as means ± SD of multiple determinations. The significance of differences between experimental and control groups was determined by Student's t-test using GraphPad t-test calculator. A value of p<0.05 was considered statistically significant. For analysis of microarray data, Significance Analysis of Microarrays (SAM) using the Student's t-test function and data randomization to establish a False Discovery Rate (FDR) was used. Forty-one genes were selected with an FDR (q-value) of ≤0.78%.
We are grateful to Anping Chen (St. Louis University, St. Louis, MO) for PPAR-γ-luc plasmids, Christopher Glass (University of California, San Diego, CA) for PPRE-luc plasmid, Vesa Kaartinen (University of Southern California, Los Angeles, CA) for TβRI adenovirus, Jan Reddy (Northwestern University, Chicago, IL) for murine PPAR-γ1 adenovirus, Yaacov Barak (The Jackson Laboratory, Bar Harbor ME) for PPAR-γ antibodies, and Bashar Kahaleh (University of Toledo, Toledo OH) and Richard Pope (Northwestern University) for helpful discussions.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The work was supported by grants from the National Institutes of Health (AR-42309 and AR-49025) and the Scleroderma Research Foundation. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Time: 7 p.m.; Venue: The Q; TV/Radio: FS Ohio/WAKR
Cavs probables: PG Ramon Sessions, SG Anthony Parker, SF Alonzo Gee, PF J.J. Hickson, C Ryan Hollins
Nets probables: PG Jordan Farmar, SG Anthony Morrow, SF Stephen Graham, PF Kris Humphries, C Brook Lopez
Officials: Marc Davis, Josh Tiven, Sean Wright
Injury report: Cavs -- Anderson Varejao (ankle) out, Semih Erden (groin) doubtful, F Antawn Jamison (finger) out, Baron Davis (back) doubtful; Nets -- Deron Williams (wrist) out; F Damion Jones (foot) out; G-F Quinton Ross (back) out.
Who's hot/Who's not: J.J. Hickson has averaged 23 points in his last two games. ... Small forward Alonzo Gee is shooting 30 percent (6 of 20) and averaging 5.7 points in his last three games. ... Nets center Brook Lopez is averaging 24.2 points and 7.4 rebounds over his last 11 games. ... Kris Humphries has grabbed double figures in rebounds in 11 straight games and 14 of his last 17.
* Ryan Hollins will start at center tonight, with J.J. Hickson shifting to power forward and Samardo Samuels moving to the bench. Scott simply wanted more size against the Nets, who start 7-foot center Brook Lopez. Hollins is also 7 feet tall, while Hickson and Samuels are both 6-9.
* Hollins and Lopez have a little history. Well, sort of. The Cavs were trying to break that dreadful losing streak in late January and had a chance to win at New Jersey. Hollins was supposed to foul Lopez late, but as Lopez was backing him down, Hollins kept slapping Lopez on the back, which sparked this gem of a line from Scott: "I have no idea what Ryan was doing."
* Scott said the coaches got a good laugh out of it Wednesday morning, but that they wouldn't address it too much with Hollins for fear he'd go the other way and get a flagrant foul. Hollins three flagrant fouls tie him with Philadelphia's Elton Brand for the most this season. Hollins is the only player in the league with two flagrant-2 fouls.
* As expected, Baron Davis won't play tonight. Coach Byron Scott is sticking with his plan of giving Davis a full practice to test his back before playing him again. If all goes well at practice tomorrow, Davis should be available for Friday's game against the Pistons.
* The Nets will be without star point guard Deron Williams, who could miss the rest of the season with strained tendons in his wrist (no one is happier about that than the Utah Jazz, who own the Nets' first-round pick this summer as part of the deal). Jordan Farmar will start again in place of Williams. He has started the last two games and had a career-high 17 assists Sunday at Washington.
* Speaking of Williams, Scott loved the Nets' ability to land him at the trade deadline this season. Scott thought he was the best point guard in the league last season and, like most others around the league, believe you begin building teams with a big and a point guard. The Nets now have both in Lopez and Williams. "Nowadays with the way NBA games are played, if you have an elite point guard, you have a chance to be really good," Scott said. "I'm sure (Nets coach) Avery Johnson is pretty happy right now."
* It will be easy to tell what kind of game Hickson has at forward tonight just by looking at his rebounding and free throw totals. If he doesn't get to the line and he doesn't have a high number of rebounds, then he likely spent too much time around the perimeter and not enough time in the paint. Hickson is at his best when he's aggressive and attacking the basket, not shooting 14-foot jumpers.
* One of the reasons Scott wanted to keep Hickson at the center position was because of how effective he has been there, even against bigger bodies. But the combination of the 6-9 Hickson and the 6-9 Samuels was apparently too small for his liking.


text:
Editorial: Main Avenue will be revitalized before we know it
Very soon we'll be looking at a brand new Main street and revitalized downtown Park Rapids. The anticipation is building and it finally seems within reach.
However, first we'll have months of construction that might try our patience at times. We'll have to continue thinking about the final outcome.
Water leaks and sink holes have been popping up this summer on Main, illustrating the need to replace the infrastructure now. Construction can't come soon enough. Reconstruction is slated to begin Aug. 2, with some preliminary work starting the last week in July.
To kick off the construction, the Downtown Business Association is sponsoring a free community picnic from 5 to 8 p.m. Thursday, July 22 on the first four blocks of Main to generate support during the renewal project.
Aang, from the television series Avatar and the new film, "The Last Airbender," will greet fans at Pioneer Park, courtesy of Arvig Communication Systems.
Entertainment at Pioneer Park will include the following: Fran Gack, Ben Ranson, Northern Light Wind Quintet, Alabaster Falls and Northern Light Opera Company.
Nate Luetgers will do caricature drawings and the Hubbard County Sheriff's Office will be doing Kids IDs. There will be live radio coverage as well.
The first 200 kids who eat at the picnic will receive a sand pail and shovel they can use to dig for prizes in a giant sand pile. Kids through age 12 are asked to sign up from 5 to 6 p.m. and the dig will start at 6 p.m.
Hot dogs and hamburgers, chips, cookies and soft drinks are on the menu. Other beverages and treats will be available from downtown restaurants.
A brief groundbreaking ceremony is planned at 4:45 p.m.
Bring a camera to capture before and after picture of historic Main street. Visitors are invited to pick up the new Positively Park Rapids Destination Guide, including a map of the area featuring the best places to shop, dine and play.
We hope everyone will attend.
Things are looking up. Business owners are optimistic. This promotion is a great way to bring the community downtown. We can't wait to see what downtown will be like once work is completed.
The end is nearing. It will be a long time before construction is needed again. Let's go into this with a positive attitude. We'll be pleased with the outcome.


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» Spanish in Guate
Spanish in Guate
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January 14th 2013
Published: April 15th 2013
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SSTT snowboardsurfstaveltrain
So I haven't kept up with my blog at all this trip so I am going to try and catch it up. I apologize this is going to be all over the place and probably the worst grammar I'm just writing things as I remember them.
Jan 12
.... Guatemala
Mom and dad drive me to Hoboken to have dinner at an awesome cuban restaurant and then crash at Terry and Katie lennons house for the night. Well we didnt really sleep though we staying up drinking and hanging out. I took about a 45 min nap then caught my taxi to the airport. When I landed in Guate city I was greeted by a guy that had a sign with my name on it as well as a few others. So we had to wait. On the way to Antigua I found out I was the only one in the minivan headed to my school, little odd. I got dropped off and walked by the drivers girlfriend to a house where Margarita my guate mother met me and gave me the rules and tour of the house and my room. Shortly afterwards I met Magnus a young Norwegian kid that was going to be taking classes as well. Since it was
Sunday dinner
wasn't included so he and I walked around and found a traditional restaurant, which was hard to do because Antigua is super super touristy. The prices are close to what they are in the states it's crazy. I really didn't like the scene there of that many gringos walking around. It wasnt going to help my spanish much. Especially talking with Magnus all the time although we tried talking in spanish it was just easier an faster to speak in English. First week of school went pretty well. I had 5 hours of classes in the am then 2 in the afternoon. My morning teacher was a nice girl with many years experience. She was friendly and fun I wound up having here the next 3 weeks. My afternoon teacher was pretty quite and reserved we just talked and didnt do much grammar work so I changed her for another one in the afternoons. The school had activities most days from spanish movies to city tours or ruin tours. The first week I didn't do anything because I had classes in the afternoons and tried just studying and whatnot. I felt pretty comfortable last year when I left Honduras but I was struggling when I got back; 2 steps forward one step back. Anyway that weekend Magnus and I walked around and hit the local market for a meal. Well we both paid for that the following days though. I rarely get sick from street food or market food even to the point I didn't have dad renew my cypro prescription. My morning teacher and I walked to a pharmacy early in the week and got me sorted. The next week I learned I could do the activities and have my class before and after the activity. One day we went to some local ruins. I should explain the ruins are of buildings built by the spanish when they were conquering here which were later destroyed by earthquakes and or war. One day we went to a jade factor where the workers were hand making jewelry and sculptures apparently certain types of jade are worth millions. I bought a cool little necklace that has a Mayan symbol on it which means "the ending of an era and the begins of a new one" Let see one of the other tours was of a nut farm I think it was macadamia it was pretty interesting rather than sell the nuts as food the guy could extract the oil and use it to make beauty products and make heaps more money. We all got quick facial massages with the oil. I remember when we were leaving we had to wait for a bus to get back into town. I tried hitch hiking with the group and Magnus and I were poking fun at each other, everyone had a good laugh. I recall later meeting an older couple at one of the
Thursday night
dinners, we got to talking and the big "why spanish" I explained I wanted to own a hostel or be a tour guide and they both said I would be a great tour guide. They were really sold on it. At first I didn't think I even met them before but later realized they were on the trip and saw me joking around with Magnus and my teachers and I guess how I carried myself in school as well. It was nice to hear from them since I hadn't really spent time with them or anything. It's one thing if good friends or others travelers say it but an older couple who just observed it was pretty neat. I also did a coffee tour where I learned that espresso because its roasted longer had less caffeine and it is acidity, don't know why people drink it. I can't wait to have a normal cup of coffee in the states. I cant remember any other tours right now if any. But Magnus and i did meet a guy named mike who I later caught up with in nicaragua when Christopher was here. We also went to watch Le miseras one night because we were trying to hit on these 2 Aussie girls but sadly the girls didn't even go to the movie. We also tried getting them to climb a volcano one weekend but that failed as well. Haha. As for the volcano though it was a decent time. It was a massive group and a super slow pace but still very pretty views. We were told if we were lucky we could see magma at night. It was a beautifully day leading up to it but as we neared the highest point we were allowed to go the peak got socked in. We did however, get to cook some marshmallows in thermal vents and some German guys even cooked some steaks. I didn't even know there was going to be active vents. We hiked down after sunset luckily I brought my head lamp, as I lead the pack back down to the bus. The other super important part about this trip was I met a Canadian girl who told me about the tour she was doing. 40 days over land from Nicaragua to Costa to Panama learning to surf and cultural site seeing. Sounds like a great company for me to work for. I have been emailing them but they won't hire me without going on a tour. They want to see how I interact with other travelers and the tour leader. I totally should have gotten the Canadian girls info to follow up with her about the trip plus she was super cute but I failed there. Haha The Aussie girls did finally agree to drinks their last night in town so we couldn't have repulsed them that much. HahaLets see my last Saturday class my teacher and I hit a bunch of ruins and sites I had missed in town and we also hiked up to the big mirador Jesus statue. It was a great way to spend my last class and day in Antigua. The next day I was suppose to be picked up by
or so but the guys couldn't find my house luckily I have a local sim and called them, I had to walk a couple blocks to a big church because I couldn't describe where my house was. Feb 3rd I thinkI arrive in Xela or QuetzaltenangoThe school cels maya who Kirk (a guy i met kitesurfing in costa with bob s.) told me about. He recommended a house and a teacher. Sadly I didn't get either so the first week, I was a bit bummed. My family was pretty nice the father was friendly but the food was pretty terrible. I did however join a gym there and was hitting it every weekday at least once a day if not twice. I had to get in shape for Utah and surfing in Nica. My first week I had 5 hours of spanish in the am and 3 hours in the avo. My am teacher although pretty wasn't the best teacher and my pm teacher and I just talked or he managed to talk for the whole time. But at least I understood him. Haha. The following week I had Miriam the teacher Kirk told me about wow she was great. She teachers to DELE standards which is recognized by Spain you can take a test and get certified depending on the level you are. Sadly I was only able to have her for one week because she had people who had reserved her months in advance. So I got pawned off to ... Can't remember her name she also taught to DELE standards but I still liked Miriam a bit better. Actually I think I liked my afternoon teacher the best I don't think she was a DELE teacher but she was super strict and made me read little kids books and write summaries and things. Her name is Ingrid. The third week I finally asked if I could change my house luckily the house that Kirk recommended had an opening. It was a boarding house they call it a hospedaje which is house for students. She had a few travelers that went to the spanish school then she had a few kids that were from the country side that would stay with her for the week so yet could go to school in the city. It turned out to be the best home stay yet. Sandra the mother was so involved and interesting. She would always engage us in conversations. It was great. I even went on a trip with the family to the local "6flags" water park it was a really neat experience. Although 6flags isn't really my cup of tea it was nice to hang out with them. The son in law and I hung out the most because he wanted to go on bigger slides and his kids didn't want to. Lets see this school was one of the most professional and informative schools. The first Monday we had a meeting for about 30 mins to go over rules the home stays city info ect. It had some 70 students most were from colleges some were just travelers. It also offered tons of activities however it wasn't like the other school if you did the activities you missed class. So I didn't bother doing any of the activities even the weekend ones because normally it was the full weekend and I had class on Saturdays. I went to 2 of the night movies but they were super boring and terrible. I also went to one of the speakers who was also boring for me at least haha. My Saturday classes were pretty good though we would talk a bit, do a tiny bit of grammar then teachers would teach me how to make a local dish for lunch. They turned out amazing I can only hope I can recreate them at home. My first Saturday class I convinced my cute teacher to take me up to the mirador we had to drive across the city on her scooter then hike up for an hour or so. Obviously again it had a beautiful view the city, it was much bigger and spread out than I thought. So I just looked through my photos to see if I could find anything else I didn't remember or write about. Sadly no I think I used my photo to take some photos in Xela but new iPhone software kept coming out and I think I lost them. Oh well my time in Xela was mostly class and the gym anyway. I kept trying to get to the pool but the main office was closed every time I went and no one knew the actually open swim hours. I even went to Walmart to buy googles which I broke trying to make them into Sweds haha. I spent most weekends waking the city passing time in coffe shops studying or watching some of the shows Magnus gave me. I spent a lot of time in the second hand markets. They import tons of donated cloths from the states and possible Canada it's crazy. The open air dirt floor market was nuts. It's full of everything shirts, pants, skirts, shoes, crocs bags, ext. it was insane but it would take you years to go through it all. There were 2 actual stores called MegaPack. These stores had all second hand stuff but things were sorted and organized. I wound up buying some t shirts for around $1. I even bought a pair of fake uggs for less than 3 bucks, which I brought to Utah and later re donated there. Haha. Well lets see guess that pretty much brings me to Utah. I had to take a bus from Xela back to guate city where I spent the night at a guest house very near the airport in a heavily gated community. It was a nice place but nothing to do there as it was a neighborhood. But I was fortunate enough to leave my board there while I was gone. Utah was awesome, great time with the family it was a nice break from traveling life. I got to eat some buffalo wings and some other amazing food as well as go riding with the fam. It was pretty awesome thanks so much mom and dad. We could have used some more snow but ken and I managed to find some freshies in the trees. We also got to hit a wailers concert in park city which was a lot of fun. Mom and dad even held on till about 11 although they missed the much better second act. Haha My flight was hours later than everyone else's so I got to drive to check out the promenade and the actual salt lake. It was a pretty massive lake with a beautiful mountain range behind it. Anyway I finally caught my plane back to guate city where I picked up my board and had to transfer to another hostel more in the city because the guest house was booked. I ended up doing a city tour that day and was thoroughly impressed. You always hear terrible things about guate city but it was actually very pretty and tranquil. Of course there are neighborhoods you don't want to be in day or night but that's a city for you right. It was a Sunday and they close down one of the major lanes in the city so people can walk, bike or skate around. It was really neat idea. We did some other site seeing buildings and markets and what not. Then myself and the other 2 guys ended up going to Hard Rock Cafe for dinner since there wasn't anything else open. My burger was delicious it had some awesome BBQ sauce on it. After dinner I headed back to reorganize and get ready for my 20hr bus ride to Nicaragua.
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SSTT snowboardsurfstaveltrain
Im 25 I love the outdoors and traveling is my passion. Im trying to figure out how to make a career out of the 2 so my parents will be happy and get out of their house haha. But for now i work as a waiter at the best restaurant in the world and i lifeguard on the greatest beach in the world as well. When im not saving money im out Traveling Surfing Spearfishing, Snowboarding, anything crazy/fun, and Sightseeing along the way...
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That was a nice article on content Tourism Portal, really got to learning the detailed structure of a tourism content. Really a nice post, thanks for sharing this information.
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C. 1940 Vintage 2.07 Carat Sapphire and 1.20 ct. t.w. Diamond Ring in 18kt Yellow Gold. Size 7.75
Item#: 883122
C. 1940. This stunning Retro-era sapphire and diamond ring from our Estate collection has head-turning power! At the center, a glorious 2.07 carat oval sapphire beams a brilliant blue amid a halo of 1.20 ct. t.w. diamond rounds, set on a polished 18kt yellow gold shank. Diamond and sapphire ring. Exclusive, one-of-a-kind Estate Jewelry.
Product Details (for C. 1940 Vintage 2.07 Carat Sapphire and 1.20 ct. t.w. Diamond Ring in 18kt Yellow Gold. Size 7.75)
5/8 inch
18kt close 18KT: Karat (abbreviated kt or K) is a unit that indicates the purity of gold in the metal. 18kt is 75% gold. In Europe, 18K yellow gold is most favored and used in jewelry. Yellow Gold close YELLOW GOLD: Gold is a precious metal with a natural yellow color. It is quite soft and often alloyed with other metals to increase its strength. The purity of the metal is indicated by a unit called a "karat," with 24kt having the highest percentage of natural gold.
Polished close POLISHED: A buffed-out finish used to produce a mirror-like shine.
Gemstone 1:
Diamond close Diamond: The hardest and most brilliant of all gemstones, a diamond can make the heart race with its pure and fiery spark. An ancient symbol of love, a diamond is the preferred gemstone for tokens of affection, such as rings, earrings, necklaces and bracelets. Diamond is the birthstone for April. (Quantity: 8, Shape & Cutting Style: Round Old Mine Cut close OLD MINE CUT: An antique gemstone cut dating back to the 1700s. Features a rounded square silhouette similar to the modern cushion-cut, but conforms to the shape of the rough gemstone in order to preserve the weight. The culet is usually large enough to be easily seen through the table of the diamond. , Color Grade: I-J close Color Grade I-J: Near-colorless , Clarity: SI1-SI2 close CLARITY SI1-SI2: Slightly Included. Inclusions are noticeable under 10x magnification. , Weight: 1.20 ct. t.w.)
Center Gemstone:
Sapphire close SAPPHIRE: A favorite gemstone of ancient priests and royalty, the sapphire radiates sophistication and grace. This prized gem comes in every hue of blue, plus the more unexpected shades of pink, yellow, green and orange, and is September's birthstone. (Color: Blue, Quantity: 1, Shape & Cutting Style: Oval close OVAL: An elliptical shape that resembles an egg. , Weight: 2.07 carats, Size: 8.5X6.5 mm)
All weights and measures are approximate.
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By Doug Ferguson, AP Golf Writer
No other golf tournament in the world attracts more viewers than the Masters. They just didn't get to see very much.
It wasn't long ago when the only live video from Augusta National was 10 1/2 hours of television spread over four rounds, and the biggest chunk was three hours devoted to the final round on CBS Sports. This was 1998, one year after Tiger Woods won his first green jacket, earning the highest rating (14.1) for a golf tournament in the cable era.
The front nine was rarely shown, and former chairman Jack Stephens was asked one year for an update on extended television coverage. He said that progress was slow, which led one reporter to ask him to elaborate.
"Well, progress is slow because we don't want it to happen," Stephens replied.
Ten years later, the modern era of media has arrived at the gates of Magnolia Lane.
"We need to be very much on the leading edge, as opposed to following," Masters chairman Billy Payne said. "We have a long list of 'firsts' in terms of putting on a golf tournament."
ESPN has signed on as a cable partner to broadcast the first two rounds, giving the Masters more international coverage than it had before. And this year, coverage starts Wednesday when the Par 3 Tournament is televised for the first time on ESPN.
Network coverage expanded in 2002 when CBS Sports was given the go-ahead to broadcast the entire final round, and now there are 14 hours of TV coverage.
But that's only a start.
For the second straight year, "Masters Extra" will be available on the tournament Web site (www.masters.org), featuring one hour of streaming video before televisions coverage begins. There also is "Amen Corner Live," streaming video from perhaps the most famous stretch of golf at Augusta National. This year, the club as added video from the par-5 15th and par-3 16th holes.
There's also a Masters blog and video games.
"What we have done in the last eight to 10 years is researched and become aware that our fans and sports fans in general have a dramatically changed appetite for the way they want information delivered," Payne said. "This is a beginning. We'll see where it takes us."
It didn't happen overnight.
Just as past chairman Hootie Johnson said changes to the golf course had been in the works for years, Payne said Johnson was the one who identified a fan base that was going digital.
"It seemed to us - and I credit this importantly to Hootie - that in order to continue to deliver entertainment and help grow the game of golf, we needed to make much of our content available to people who wanted it - in a way they wanted it delivered."
There has been little change to the golf course for the 72nd Masters, which starts Thursday. A few pine trees were removed from the right of the 11th fairway. The first tee was moved up about 10 yards to create more space between the practice green. A massive viewing area was created on the hill left of the 16th green.
The real change for this Masters is found over the airwaves.
"There's a lot of tradition at Augusta National, but there are new elements, which is exciting," said Sean McManus, president of CBS News and Sports, which has televised every Masters since 1956. "It's been pretty gradual. Billy likes to challenge us all. His entire motivation is centered around quality. He wants everything to do with television and the Internet to be the best in the business."
CBS produces the coverage for streaming video from Amen Corner and Nos. 15 and 16. There is always a camera trained on every shot at any time of the day. Now, more people can see it over the Internet.
It is a significant change from even a decade ago, and there was concern that saturated coverage was not in the Masters' best interest.
"I would disagree that any changes have oversaturated the appetite or demand for Masters coverage and information," Payne said. "As it relates to television, we still have a fairly limited number of hours of coverage compared with any of the other majors."
That much is true, especially on the weekdays.
The U.S. Open is televised on ESPN from 10 a.m. to 7 p.m. the first two rounds, and its weekend coverage can be as long as 14 hours, nearly as much as the Masters has all week. The PGA Championship has twice as much TV coverage as the Masters, going from 2 p.m. to 8 p.m. on the weekdays, and 11 a.m. to 7 p.m. on the weekend.
The Masters still offers TV in small doses, complemented by its Web site.
"With Amen Corner and then 15 and 16, on a list of the most thrilling things that happen in golf, those are the top," Payne said. "Those were natural. In large measure, they have the effect of being entertaining, but they tease the upcoming broadcast.
"People see a little, and they want to see more."
In his nearly two years as chairman, Payne has made several changes to broaden the appeal of the Masters. He said when he took over that it was a "mandate" to reach people around the world, and he would be exploring every avenue.
One step was bringing in ESPN and its worldwide audience, something the PGA Tour lost when it signed a 15-year deal with The Golf Channel to televise its first two rounds and some early-season tournaments.
One criticism of the PGA Tour's deal is that The Golf Channel reaches only golf fans; ESPN reaches sports fans.
"They have a great international reach," Payne said, noting that the Masters reaches nearly 200 countries. "And I think we're going to see a larger weekday audience."
The biggest difference is how the coverage is delivered - not only on television, but on the Internet.
"I have a lot of people say to me, 'There goes another hour of work on Thursday and Friday,"' Payne said. "I just laugh. Because that's probably what I'll be doing, too."
The Associated Press


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Dr Vranjes
Rosso Nobile Room Fragrance
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Created by Italian perfumer Paolo Vranjes, these fragrances are presented in custom-designed glass bottles inspired by the lines of the famous "Duomo", Florence's Cathedral of Santa Maria di Fiori.
The favourite in Italy. This rich, deep fragrance is based on a fine dessert wine blended with essential oils, herbs and spices. Its fruity sweet heady notes sit well in a study, office or den. An excellent choice for the men in your life.
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