Fibromuscular dysplasia (FMD) is a rare, nonatherosclerotic arterial disease for which the molecular basis is unknown. We comprehensively studied 47 subjects with FMD, including physical examination, spine magnetic resonance imaging, bone densitometry, and brain magnetic resonance angiography. Inflammatory biomarkers in plasma and transforming growth factor beta (TGF-beta) cytokines in patient-derived dermal fibroblasts were measured by ELISA. Arterial pathology other than medial fibrodysplasia with multifocal stenosis included cerebral aneurysm, found in 12.8% of subjects. Extra-arterial pathology included low bone density (P< 0.001); early onset degenerative spine disease (95.7%); increased incidence of Chiari I malformation (6.4%) and dural ectasia (42.6%); and physical examination findings of a mild connective tissue dysplasia (95.7%). Screening for mutations causing known genetically mediated arteriopathies was unrevealing. We found elevated plasma TGF-beta 1 (P = 0.009), TGF-beta 2 (P = 0.004) and additional inflammatory markers, and increased TGF-beta 1 (P = 0.0009) and TGF-beta 2 (P = 0.0001) secretion in dermal fibroblast cell lines from subjects with FMD compared to age-and gender-matched controls. Detailed phenotyping of patients with FMD allowed us to demonstrate that FMD is a systemic disease with alterations in common with the spectrum of genetic syndromes that involve altered TGF-beta signaling and offers TGF-beta as a marker of FMD.

Background—Vascular Ehlers–Danlos syndrome (VEDS) causes reduced life expectancy because of arterial dissections/rupture and hollow organ rupture. Although the causative gene,COL3A1, was identified >20 years ago, there has been limited progress in understanding the disease mechanisms or identifying treatments.Methods and Results—We studied inflammatory and transforming growth factor-β (TGF-β) signaling biomarkers in plasma and from dermal fibroblasts from patients with VEDS. Analyses were done in terms of clinical disease severity, genotype–phenotype correlations, and body composition and fat deposition alterations. VEDS subjects had increased circulating TGF-β1, TGF-β2, monocyte chemotactic protein-1, C-reactive protein, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and leptin and decreased interleukin-8 versus controls. VEDS dermal fibroblasts secreted more TGF-β2, whereas downstream canonical/noncanonical TGF-β signaling was not different. Patients withCOL3A1exon skipping mutations had higher plasma intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and VEDS probands had abnormally high plasma C-reactive protein versus affected patients identified through family members before any disease manifestations. Patients with VEDS had higher mean platelet volumes, suggesting increased platelet turnover because of ongoing vascular damage, as well as increased regional truncal adiposity.Conclusions—These findings suggest that VEDS is a systemic disease with a major inflammatory component. C-reactive protein is linked to disease state and may be a disease activity marker. No changes in downstream TGF-β signaling and increased platelet turnover suggest that chronic vascular damage may partially explain increased plasma TGF-β1. Finally, we found a novel role for dysregulated TGF-β2, as well as adipocyte dysfunction, as demonstrated through reduced interleukin-8 and elevated leptin in VEDS.

The vascular type of the Ehlers-Danlos syndrome (vEDS) is caused by dominant-negative mutations in the procollagen type III (COL3A1) gene. Patients with this autosomal dominant disorder have a shortened life expectancy due to complications from ruptured vessels or hollow organs. We tested the effectiveness of allele-specific RNA interference (RNAi) to reduce the mutated phenotype in fibroblasts. Small-interfering RNAs (siRNAs) discriminating between wild-type and mutant COL3A1 allele were identified by a luciferase reporter gene assay and in primary fibroblasts from a normal donor and a patient with vEDS. The best discriminative siRNA with the mutation at position 10 resulted in >90% silencing of the mutant allele without affecting the wild-type allele. Transmission and immunogold electron microscopy of extracted extracellular matrices from untreated fibroblasts of the patient with vEDS revealed structurally abnormal fibrils. After siRNA treatment, collagen fibrils became similar to fibrils from fibroblasts of normal and COL3A1 haploinsufficient donors. In addition, it was shown that expression of mutated COL3A1 activates the unfolded protein response and that reduction of the amount of mutated protein by siRNA reduces cellular stress. Taken together, the results provide evidence that allele-specific siRNAs are able to reduce negative effects of mutated COL3A1 proteins. Thus, the application of allele-specific RNAi may be a promising direction for future personalized therapies to reduce the severity of vEDS.-Muller, G. A., Hansen, U., Xu, Z., Griswold, B., Talan, M. I., McDonnell, N. B., Briest, W. Allele-specific siRNA knockdown as a personalized treatment strategy for vascular Ehlers-Danlos syndrome in human fibroblasts. FASEB J. 26, 668-677 (2012). www.fasebj.org

Background—Marfan syndrome (MFS) is caused by mutations in the fibrillin-1 gene and dysregulation of transforming growth factor (TGF). Recent evidence suggests that losartan, an AT1 blocker that blunts TGF activation, may be an effective treatment for MFS. We hypothesized that dysregulation of TGF might be mirrored in circulating TGF concentrations. Methods and Results—Serum obtained from MFS mutant mice (Fbn1 C1039G/+ ) treated with losartan was analyzed for circulating TGF1 concentrations, and compared to those from placebo treated and wild-type mice. Aortic root size was measured by echocardiography. Data was validated in patients with MFS and healthy individuals. In mice, circulating total TGF1 concentrations increased with age and were elevated in older untreated Fbn1 C1039G/+ mice compared to wild-type

Objective: TGFβ signaling has been shown to play a major role in the pathogenesis of Marfan syndrome (MFS), leading to a novel treatment strategy through TGFβ inhibition using losartan. We hypothesized that circulating TGFβ levels may be elevated in disorders that disrupt arterial wall extracellular matrix, such as the vascular form of Ehlers-Danlos syndrome (VEDS). Patients and Methods: Circulating levels of TGFβ-1 were measured in EDTA plasma of 141 patients with MFS (n=21, mean age 43±16y), VEDS (n=41, 37±13y) and controls (n=79, 48±13y) using an electrochemoluminescence platform (LLOQ 4±2.6pg/ml). Results: In keeping with prior work, this study revealed increased circulating TGFβ in patients with MFS compared to control patients (9.2±1.2ng/ml vs. 2.5±0.4ng/ml, p<0.0001). Remarkably, the levels seen in patients with VEDS were also significantly higher than controls (8.0±1.0ng/ml vs. 2.5±0.4ng/ml, p<0.0001) and statistically indistinguishable from those seen in MFS (9.2±1.2ng/ml vs. 8.0±1.0ng/ml, p=0.5). In a bivariate logistic regression model, the odds ratio was 7.5 (p=0.02, CI 1.3– 43.3) when comparing quartiles of TGFβ values in VEDS patients with age, sex and BMI matched controls. There was a non-statistically significant trend for higher TGFβ levels in patients without cardiovascular medication (MFS, 11.4±3.0ng/ml; VEDS, 8.5±1.3ng/ml) compared to patients treated with ARBs and/or ACE-Is (MFS, 6.1±1.0ng/ml; VEDS, 4.6±0.8ng/ml), whereas β-blockers appeared to have some impact on TGFβ levels in MFS (9.0±1.7ng/ml) but not in VEDS patients (8.3±1.8ng/ml). The influence of age on TGFβ levels in the controls is statistically significant but weak (r=0.3, p=0.007). In the disease groups, TGFβ is inversely related to age (MFS, r=−0.7, p<0.0001; VEDS, r=−0.1, p=0.6), which is most likely due to ascertainment bias towards a less severe phenotype in the older patients and longer duration of medical therapy, especially in the MFS population. Conclusion: Further investigation is warranted to establish the role of TGFβ signaling in the pathogenesis of VEDS. The current results may help to provide a rationale for the use of TGFβ lowering drug therapy in this patient population as it is emerging in the MFS patient population.