The clinical heterogeneity in AOS patients makes it difficul

The clinical heterogeneity in AOS patients makes it difficult to study SMAD3 mutational effects on aneurysm formation. Fortunately, due to the homogenous genetic background, genetically engineered mouse models are useful in pinpointing the specific molecular mechanism leading to disease. Smad3 knockout animals present with skeletal abnormalities and osteoarthritis (OA) and as such, they have been used as a model to study OA (Yang and Cao, 2001; Li et al., 2009). A cardiovascular phenotype in these animals was overlooked until the recent link of human SMAD3 mutations and aortic aneurysms was established (Regalado et al., 2011; van de Laar et al., 2011; Ye et al., 2013). Here we describe the cardiovascular phenotype of the Smad3 knockout mice and reveal the underlying mechanism of aneurysm growth caused by a SMAD3 deficiency.
Materials and Methods
Results
Discussion In humans, SMAD3 mutations cause a syndrome with cardiovascular, craniofacial, cutaneous and skeletal anomalies. Aortic aneurysms and osteoarthritis characterize this syndrome known as AOS or LDS3. Although an increasing number of SMAD3 mutations have recently been reported (Aubart et al., 2014; Fitzgerald et al., 2014; Hilhorst-Hofstee et al., 2013; Zhang et al., 1996), the functional effect of these mutations remains to be explored. The molecular mechanism underlying AOS, resulting from SMAD3 mutations is largely unknown. In this study we investigated the vascular phenotype in Smad3 mutant mice and explored its effects on the TGF-β signaling cascade. As the functional consequence of heterozygous SMAD3 patient mutations at the protein level is unclear; it might for example lead to a null or non-functional protein (e.g. not able to bind SMAD2), we decided to study the effect of Smad3 Nanaomycin A in Smad3−/− mice to better understand its role in aneurysm formation. In Smad3−/− mice, Smad3 deficiency leads to aortic aneurysms, which mainly affect the aortic root and ascending aorta, but also extends to other major arteries. Aneurysm formation is highly dependent on the age of the animals, as has been observed in AOS patients with SMAD3 mutations, which display an age-dependent penetrance. As shown here, male Smad3−/− animals suffered a more severe aortic phenotype (both dilatation and elongation) and consequently, died earlier than female Smad3−/− animals. The resulting phenotype due to Smad3 deficiency in mice is very similar to the vascular phenotype described in patients with SMAD3 mutations offering an excellent opportunity for disease modeling. Despite the homogeneous genetic background aneurysms still present irregularly in the Smad3−/− mice, which suggests that other external factors, like for example blood pressure and subtle intrinsic variations in transcriptional activation can also determine the variability in the resulting phenotype. Strikingly, histology of the aortic wall shows that, unlike other models for aneurysmal disease, there is no increase in ECM accumulation, nor excessive collagen staining, or loss of vascular smooth muscle cells. However, we observe increased aortic pSmad2 and pERK activation. Moreover, this activation is already apparent before Smad3−/− animals present with aneurysms, showing that this activation precedes aneurysm formation. Importantly, these changes in the Smad3−/− aortic wall are distinct from what was previously observed for Fibulin-4 animals, which have reduced expression of the ECM protein Fibulin-4, and as a result also show aneurysm formation. Instead, Fibulin-4 aortas show increased ECM remodeling, and increased collagen and elastin structures (Hanada et al., 2007; Moltzer et al., 2011). Yet, they also show increased pSmad2 and pERK activation. A comparison of the Smad3−/− and Fibulin-4 changes in aneurysm formation is shown in Fig. 8. These findings led us to hypothesize that TGF-β signaling downstream of Smad3 might be deregulated due to Smad3 deficiency. While AOS and LDS patients have heterozygous loss of function mutations affecting genes from the TGF-β signaling pathway (TGFΒR1/2, SMAD3, TGFB2, TGFB3), aortic tissues of these patients show a signature of upregulated TGF-β pathway signaling, as indicated by the overexpression of pSMAD2, pERK1/2 and CTGF (Loeys et al., 2005; van de Laar et al., 2011; Lindsay et al., 2012). This phenomenon is known as the TGF-β paradox (for comprehensive review see (Massague, 2012)). We therefore examined TGF-β receptor activation in the aortic wall of Smad3−/− animals by investigating pSmad2 expression. We observed that while lacking Smad3, Smad2 can still be phosphorylated and transported to the nucleus. Thus, lack of Smad3 might alter downstream transcriptional activation in the nucleus (also see Fig. 8). This lack of Smad3 might also shift TGF-β activated signaling via Smad2, which has similar but not overlapping functions (Moustakas and Heldin, 2002; Zhang et al., 1996), also resulting in an altered transcriptional response. We reasoned that these changes lead to reduction in the downstream transcription of genes such as ECM components and MMPs. Indeed, we demonstrate here that transcriptional activation of multiple genes downstream of the TGF-β signaling cascade is absent. Again, this is in contrast to what was previously found for Fibulin-4 aortas and VSMCs where downstream TGF-β-induced transcription was activated (Ramnath et al., 2015). Moreover, we found that the increased proliferation of Smad3−/− VSMCs is not due to increased pErk, but rather due to the lack of downstream TGF-β induced transcriptional activation. Moreover, it shows that Smad3 normally plays a role in TGF-β mediated growth inhibition.