Inflammation and TGF-ß Signaling Differ between Abdominal Aneurysms and Occlusive Disease.

Abdominal aortic aneurysms (AAA), are usually asymptomatic until rupture causes fatal bleeding, posing a major vascular health problem. AAAs are associated with advanced age, male gender, and cardiovascular risk factors (e.g. hypertension and smoking). Strikingly, AAA and AOD (arterial occlusive disease) patients have a similar atherosclerotic burden, yet develop either arterial dilatation or occlusion, respectively. The molecular mechanisms underlying this diversion are yet unknown. As this knowledge could improve AAA treatment strategies, we aimed to identify genes and signaling pathways involved. We compared RNA expression profiles of abdominal aortic AAA and AOD patient samples. Based on differential gene expression profiles, we selected a gene set that could serve as blood biomarker or as pharmacological intervention target for AAA. In this AAA gene list we identified previously AAA-associated genes COL11A1, ADIPOQ, and LPL, thus validating our approach as well as novel genes; CXCL13, SLC7A5, FDC-SP not previously linked to aneurysmal disease. Pathway analysis revealed overrepresentation of significantly altered immune-related pathways between AAA and AOD. Additionally, we found bone morphogenetic protein (BMP) signaling inhibition simultaneous with activation of transforming growth factor ß (TGF-ß) signaling associated with AAA. Concluding our gene expression profiling approach identifies novel genes and an interplay between BMP and TGF-ß signaling regulation specifically for AAA.

Defective Connective Tissue Remodeling in Smad3 Mice Leads to Accelerated Aneurysmal Growth Through Disturbed Downstream TGF-ß Signaling.

Aneurysm-osteoarthritis syndrome characterized by unpredictable aortic aneurysm formation, is caused by SMAD3 mutations. SMAD3 is part of the SMAD2/3/4 transcription factor, essential for TGF-ß-activated transcription. Although TGF-ß-related gene mutations result in aneurysms, the underlying mechanism is unknown. Here, we examined aneurysm formation and progression in Smad3-/- animals. Smad3-/- animals developed aortic aneurysms rapidly, resulting in premature death. Aortic wall immunohistochemistry showed no increase in extracellular matrix and collagen accumulation, nor loss of vascular smooth muscle cells (VSMCs) but instead revealed medial elastin disruption and adventitial inflammation. Remarkably, matrix metalloproteases (MMPs) were not activated in VSMCs, but rather specifically in inflammatory areas. Although Smad3-/- aortas showed increased nuclear pSmad2 and pErk, indicating TGF-ß receptor activation, downstream TGF-ß-activated target genes were not upregulated. Increased pSmad2 and pErk staining in pre-aneurysmal Smad3-/- aortas implied that aortic damage and TGF-ß receptor-activated signaling precede aortic inflammation. Finally, impaired downstream TGF-ß activated transcription resulted in increased Smad3-/- VSMC proliferation. Smad3 deficiency leads to imbalanced activation of downstream genes, no activation of MMPs in VSMCs, and immune responses resulting in rapid aortic wall dilatation and rupture. Our findings uncover new possibilities for treatment of SMAD3 patients; instead of targeting TGF-ß signaling, immune suppression may be more beneficial.

Fibulin-4 deficiency increases TGF-ß signalling in aortic smooth muscle cells due to elevated TGF-ß2 levels.

Fibulins are extracellular matrix proteins associated with elastic fibres. Homozygous Fibulin-4 mutations lead to life-threatening abnormalities such as aortic aneurysms. Aortic aneurysms in Fibulin-4 mutant mice were associated with upregulation of TGF-ß signalling. How Fibulin-4 deficiency leads to deregulation of the TGF-ß pathway is largely unknown. Isolated aortic smooth muscle cells (SMCs) from Fibulin-4 deficient mice showed reduced growth, which could be reversed by treatment with TGF-ß neutralizing antibodies. In Fibulin-4 deficient SMCs increased TGF-ß signalling was detected using a transcriptional reporter assay and by increased SMAD2 phosphorylation. Next, we investigated if the increased activity was due to increased levels of the three TGF-ß isoforms. These data revealed slightly increased TGF-ß1 and markedly increased TGF-ß2 levels. Significantly increased TGF-ß2 levels were also detectable in plasma from homozygous Fibulin-4(R/R) mice, not in wild type mice. TGF-ß2 levels were reduced after losartan treatment, an angiotensin-II type-1 receptor blocker, known to prevent aortic aneurysm formation. In conclusion, we have shown increased TGF-ß signalling in isolated SMCs from Fibulin-4 deficient mouse aortas, not only caused by increased levels of TGF-ß1, but especially TGF-ß2. These data provide new insights in the molecular interaction between Fibulin-4 and TGF-ß pathway regulation in the pathogenesis of aortic aneurysms.