SSc Skin Grafting Stimulates Angiogenesis in SCID Mice

<p>Macroscopic analysis of the vessels in the underside of normal (A and B) and SSc (C and D) skin grafts reveals more pronounced angiogenesis in SSc grafts; paclitaxel (Taxol) treatment has no effect on angiogenesis at the macroscopic level (B and D versus A and C). Microscopic analysis shows that the number of vessels in three high-power fields of the SSc grafts is greater than that of normal skin grafts; paclitaxel has no effect on angiogenesis at the microscopic level (E). TRT-PCR for mouse <i>PECAM-1</i> demonstrates a substantial increase in mouse <i>PECAM-1</i> mRNA expression in SSc and normal skin grafts regardless of paclitaxel treatment; the amplitude of the increase is significantly greater in SSc than in normal skin grafts (F). Human <i>PECAM-1</i> is expressed in much lower levels in SSc than in normal skin tissues, indicative of vascular deficiency in SSc; the lack of difference in the expression level of human <i>PECAM-1</i> in SSc and normal skin tissues before and after transplantation indicates that the neovascular cells are derived from the recipients (G).</p

Paclitaxel Treatment Suppresses Smad2 Phosphorylation

<p>Strong nuclear staining for phospho-Smad2 (A) and total Smad2 (B) is observed in an SSc skin graft. Smad2 phosphorylation (C) is rare in normal graft, which expresses abundant Smad2 (D). Smad2 phosphorylation is suppressed with paclitaxel (Taxol) treatment (E), without affecting total Smad2 (F) in SSc skin graft. Tx, transplantation.</p

Paclitaxel Decreases COLA2 mRNA Expression in SSc Grafts

<p>TRT-PCR analysis shows that the expression of <i>COL1A2</i> is reduced 4.5-fold with paclitaxel (Taxol) treatment in SSC grafts, reaching a level equivalent to that of normal skin grafts. By contrast, paclitaxel has no effects on <i>COL1A2</i> expression in normal skin grafts.</p

Paclitaxel Decreases Collagen Deposition in SSc Grafts

<p>VVM staining shows abundant, thick collagen bundles in the SSc graft (A). The amount of collagen is markedly reduced, and the collagen fibers become finer with paclitaxel (Taxol) treatment (B), comparable to that seen in the normal skin graft (C). Similarly, IHC staining demonstrates intense collagen-1 staining in the SSc graft (D), which is substantially decreased with paclitaxel treatment (E) to a level similar to that seen in the normal skin graft (F).</p

Gene Expression Barcode for EDS signature genes.

<p>The heat map is derived from individual barcode scores from zero (black) to one (red) with red indicating an increased likelihood that the gene is present in all samples in the experiment from which the tissue specific expression estimate is derived (see McCall et al. for complete description of the barcode algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034951#pone.0034951-McCall1" target="_blank">[21]</a>). As shown, the genes for the EDS signature were overrepresented for three tissues, in particular (cord blood reticulocytes, adult blood reticulocytes, and bone marrow), and their respective number of barcode expression calls are as indicated in the barchart directly below the corresponding areas of the barcode heatmap.</p

Heat map illustration of the distribution of gene expression among all samples for genes selected by pathways.

<p>A) TLR (Toll-like Receptor) Signaling, B) TCR (T cell receptor) Pathway, Ca²⁺/NFAT Signaling. Red indicates increase and green indicates decrease in relative gene expression for each gene calculated individually across all samples.</p

ALAS2 gene expression positively correlates with increasing disease severity in IPAH patients.

<p>EDS gene expression for 12 IPAH patients for whom hemodynamic measurements were available within four months of the date of blood draw. The clinical parameters of right atrial pressure (RAmean), cardiac index (CI), pulmonary vascular resistance index (PVRI), and pulmonary artery saturation (PA sat) were used. Disease severity in IPAH patients as indicated by increasing RAmean and PVRI are strongly correlated with increasing ALAS2 gene expression and negatively correlated with measures of healthy lung and coronary function such as PA saturation and Cardiac Index (CI).</p

Results from gene set analysis using the EDS as the query gene list. Enrichment scores are as described above.

<p>Results from gene set analysis using the EDS as the query gene list. Enrichment scores are as described above.</p

Statistically significant differential expression of erythroid CD71+ specific genes.

<p>A) ALAS2 and B) ERAF/AHSP genes in hypertension groups (SSc-PH-ILD, IPAH, and SSc-PAH) versus healthy controls and scleroderma (SSc). The box plots give the mean (horizontal black line), sample values (short blue lines), and 84% confidence interval (CI) (red lines) for each group, (non-overlap of the 84% CIs of two groups is an approximate indicator of significant difference between their means at the 0.05 level of significance). C) Individual ALAS2 and ERAF gene expression microarray results were validated by RT-PCR for high and low EDS patients across all hypertension classes (SSc-PAH, IPAH, SSc-PH-ILD).</p> <p>Further inspection of the EDS gene list also showed the inclusion of genes for both the GATA1 and KLF transcription factors which are both essential for erythroid development [28,29]. A test by gene set analysis of the entire dataset comparing each PH group directly versus the SSc group as the baseline group showed a significant and specific enrichment among genes which contain three different GATA transcription factor binding sites in their upstream promoter regions (TRANSFAC [30]) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034951#pone-0034951-g007" target="_blank">Figure 7A</a>). Interestingly, the genes up-regulated in each of the GATA transcription factor binding site gene sets were mostly non-overlapping either with each other (an average of 59–62% unique genes for each gene list) or with the EDS gene expression signature itself (95% unique non-overlapping genes) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034951#pone-0034951-g007" target="_blank">Figure 7</a>B) indicating that the effects of elevated GATA1 gene expression are both pervasive in the PH groups and are supplemental to the EDS gene expression signature itself. The observation that downstream regulatory events are related directly to EDS elevation (through the up-regulation of the GATA1 transcription factor gene) and are associated with PH groups, taken together with the previously demonstrated strong association of the EDS with reticulocyte maturation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034951#pone-0034951-g005" target="_blank">Figure 5</a>), led us to the identification of this signature as the Erythroid Development Signature (EDS).</p

Heat map of unsupervised clustering (genes only) of 296 genes selected for both high variance (from a set of 500 most variant genes) and also as representative of six major patterns of distinctly correlated genes across the dataset.

<p>These patterns included an Immune Response signature (up- or down-regulated between controls and disease: IR-UR and IR-DR, respectively), the Erythroid Differentiation Signature (EDS) a Platelet specific signature (Pl), and an Immature Neutrophil Signature (INS). The gender specific cluster acts as a positive control, the specific signatures are discussed in detail in the text, particularly that of the EDS.</p