To date, this is the first study that compares the differential transcriptosome of MVECs isolated from the skin of normal subjects and from the lesional skin of SSc patients affected by the diffuse form of the disease in the avascular phase.
The number of 6,724 genes that passed the filtering criteria is in agreement with those previously obtained in other human cell lines by microarray analysis [9
] and by massively parallel signature sequencing [32
]. We have observed that the majority of genes are expressed at similar levels in N-MVECs and SSc-MVECs and that, interestingly, 3.2% of the total filtered transcripts (199 genes) were differentially expressed. Considering the correctness of comparing data limited to transcripts, we show that, in SSc-MVECs, the dysregulation involves only a small number of genes controlling a large number of processes that are critical to the biology of endothelial cells.
Surprisingly, we have found that SSc-MVECs exhibit a pro-angiogenic gene expression pattern (Table ). In SSc, the angiogenic process is severely impaired in the late phases of the disease [33
], independent of the increased levels of circulating Vascular Endothelial Growth Factor (VEGF) and FGF2 [34
]. Therefore, it is likely that some critical checkpoints in the control of angiogenesis are altered at the MVEC level. Our data indicate down-regulation of PLXNB1, a receptor for semaphorin that tracks the pathway to migrating endothelial cells by activation of the MET oncogene and by stimulating Rho-initiated pathways [13
], of tissue KLK9, KLK11 and KLK12, shown to be required for MVEC migration and proliferation [12
], and of DSG2, which is positively involved in homophilic cell-cell interaction [15
]. At the same time, PTX3, an angiogenesis inhibitor that acts by binding FGF2 [14
], is up-regulated (Table ). We propose to interpret these data as a stabilization of a pro-angiogenic pattern dictated by angiogenesis factors such as VEGF and FGF2 that is blocked or rendered ineffective by the strong down-regulation of the critical adhesion/invasion/proliferation systems and by up-regulation of the angiogenesis inhibitor PTX3 (Table ). Due to a common technical problem during the spotting procedure, microarray data were not available for matrix metallo-protease-12 (MMP12), which in a previous study on the same SSc-MVECs was found to be up-regulated and responsible for urokinase-type plasminogen activator receptor (uPAR) truncation and subsequent angiogenesis impairment [18
]. In this previous work we suggested the possibility that the alterations we observed in SSc-MVECs may initially be stimulated by environment factors and then become the product of the hypoxia-induced selection of an endothelial cell population more suitable to survive in the hypoxic micro-environment typical of the disease. Still, unexpectedly, in SSc-MVECs we have observed an overall up-regulation of many components of several transduction systems. The large majority of such alterations (Table , Additional file 5
) deal with transduction by small GTPase proteins, which couple signals from ECM molecules to the cell cytoskeleton, inducing alternating states of cell contraction/relaxation [39
]. However, up-regulation of Rho GDP dissociation inhibitor beta (ARHGDIB) is likely to provide a critical 'bottle-neck' to GTPase protein transductions by impairing the substitution of GDP with GTP, thus preventing the receptor to enter its activation state [40
]. Also, PLXNB1 down-regulation inhibits semaphorin-directed MVEC migration by blunting Rho-initiated pathways [36
]. Further, an overall impairment to cell proliferation in SSc-MVECs must also be related to down-regulation of MAP4K1, a serine/threonine kinase involved in a variety of cellular signaling cascades [41
Provided that all the observed alterations in gene expression may be important in the pathogenesis of vascular damage in SSc, our results allow the identification of some genes that may block a correct angiogenesis program in SSc-MVECs. Of particular interest may be genes involved in: MVEC migration, proliferation and adhesion (down-regulated PLXNB1, KLKs, DSG2); inhibition of angiogenesis (up-regulated PTX3); and alteration of signal transduction pathways (up-regulated ARHGDIB, down-regulated PLXNB1, down-regulated MAP4K1) (Figure ). For six of such genes (PLXNB1, PTX3, KLK9, KLK11, KLK12, DSG2), we have provided evidence of a critical role in the angiogenic process (this work and [12
Figure 5 Impairment of angiogenesis in systemic sclerosis. Genes differentially expressed in microvascular endothelial cells (MVECs) from patients with systemic sclerosis (SSc-MVECs) are grouped according to Table 3. Upward and downward arrows indicate up-regulation (more ...)
Over-expression of genes of proteasome and ubiquitin pathways suggests the possibility that the observed gene alterations are the effect of a cell adaptation to a particularly hostile environment. It is interesting to underline the induction of 3 metallothionein genes in SSc-MVECs. Metallothioneins belong to a family of stress-induced proteins that regulate Zn and Cu availability and also modulate the amount and activity of NF-K
B, a transcription factor for genes involved in apoptosis, immune response and inflammation [42
Since RNA harvested from MVECs was obtained from cells between the fourth and seventh passage, this could raise concerns about MVEC stability and/or possible selection of cells more likely to survive in culture. However, we previously showed that RT-PCR, performed with RNA isolates of cells from single patients at early and late culture passages to validate tissue KLK expression, gave similar results, in agreement with microarray data obtained with pooled RNA [12
]. Moreover, the demonstration of down-regulation of KLK9, KLK11, KLK12 and DSG2 in skin biopsies of SSc patients by immunocytochemistry suggests that at least some of the reported alterations pre-exist to the isolation and culture propagation techniques. Nonetheless, a possible selection bias, responsible for some of the reported differences, cannot be ruled out. There is evidence that inference from most genes is not adversely affected by pooling, such that pooling is recommended when fewer than three arrays are used in each condition [43
]. Additionally, due to the small number of replicates, we decided to apply the dye swap design to minimize the gene-specific dye bias [44
], which is the major source of experimental variability between replicates. In the present study we have applied markedly stringent criteria for feature extraction and data normalization, which could blunt the identification of differentially expressed genes. Nevertheless, we believe that our findings, related to single genes, or classes of genes, may provide hints for future research and are worthy of a more in-depth study to identify possible ways to correct some critical molecular defects and to recover, at least partially, the angiogenic attitude of SSc-MVECs.