Can the TLR-4-Mediated Signaling Pathway Be “A Key Inflammatory Promoter for Sporadic TAA”?

Thoracic aorta shows with advancing age various changes and a progressive deterioration in structure and function. As a result, vascular remodeling (VR) and medial degeneration (MD) occur as pathological entities responsible principally for the sporadic TAA onset. Little is known about their genetic, molecular, and cellular mechanisms. Recent evidence is proposing the strong role of a chronic immune/inflammatory process in their evocation and progression. Thus, we evaluated the potential role of Toll like receptor- (TLR-) 4-mediated signaling pathway and its polymorphisms in sporadic TAA. Genetic, immunohistochemical, and biochemical analyses were assessed. Interestingly, the rs4986790 TLR4 polymorphism confers a higher susceptibility for sporadic TAA (OR = 14.4, P = 0.0008) and it represents, together with rs1799752 ACE, rs3918242 MMP-9, and rs2285053 MMP-2 SNPs, an independent sporadic TAA risk factor. In consistency with these data, a significant association was observed between their combined risk genotype and sporadic TAA. Cases bearing this risk genotype showed higher systemic inflammatory mediator levels, significant inflammatory/immune infiltrate, a typical MD phenotype, lower telomere length, and positive correlations with histopatological abnormalities, hypertension, smoking, and ageing. Thus, TLR4 pathway should seem to have a key role in sporadic TAA. It might represent a potential useful tool for preventing and monitoring sporadic TAA and developing personalized treatments.

evaluating those of aortic arch from suprasternal view; detecting other measurements perpendicular to aorta long axis using leading edge to leading edge method [1S] Color Doppler was used to assess presence and severity of aortic regurgitation, and aortic stenosis was excluded both by pulsed-wave and continuous-wave Doppler. Furthermore, aortic root and ascending aorta diameter sizes were carried out similarly to helical CT image analysis technique, described by Hager et al [2S]. The aorta was, thus, sized at 3 levels: level 1, aortic sinuses; level 2, sinotubular junction; level 3, right main pulmonary artery. Slices were adjusted for each level to obtain an oblique axial plane perpendicular to aorta course. Internal aorta diameter was then measured with electronic calipers in 2 directions at 90° to one another. The mean of these values was deemed diameter to be used for calculations.

Aortic specimens, histopathological assays and identification of three phenotypes
Because of histological condition of some patient aortas, full aortic segments were unfortunately collected from resected aortic wall of only 100 S-TAA patients at surgical time. They were fixed in 10% neutral buffered formalin for 24 hours and then processed for routine paraffin embedding.
Surgical specimens subsequently were photographer and measured (maximum transverse diameter).
For microscopic examinations, multiple histological sections from each sample were prepared and stained with hematoxylin-eosin, Weigert-van Gieson, Alcian-PAS (see Figure 1S.) Using the grading systems defined by Matthias Bechtel and colleagues [31] (see Figure 1S and 2S in online Supplementary Material) and previously described in our recent studies [16,[27][28][29][30][31], we detected three morphological MD phenotypes. They were characterized to have a different quantitative relationship of cystic MD, fibrosis, apoptosis and amount of MMP-9 (see Figure 1S and 2S), described as follows and previously reported in a previous study [30] Phenotype I: cystic medial degeneration balanced by a substitutive fibrosis, in absence of medial apoptosis and with a low MMP-9 concentration; Phenotype II: higher cystic medial degeneration than substitutive fibrosis, with focal medial apoptosis, and with mainly a modest MMP-9 amount; Phenotype III: elevated cystic medial degeneration, without substitutive fibrosis, with plurifocal medial apoptosis, and with an elevated MMP-9 concentration.
Specimens were then incubated with TdT and fluorescein-labeled dUTP in a humidified atmosphere for 1 h at 37°C. In situ apoptosis staining was revealed by using an AP converter. DNA strand breaks were detected by using the 5-bromo-4chloro-3-indolyl-phosphate (BCIP/NBT, Dako, Italy) substrate chromogen. Tissues were subsequently counterstained with eosin under light microscopy (see Figure 2S) .

Genotyping
DNA samples of 128 matched controls and 161 S-TAA cases were extracted from peripheral blood samples collected in tripotassium EDTA and purified by using a QIAamp Blood DNA Maxi kit (Qiagen, Dusseldorf, Germany). Samples were genotyped for ten SNPs located in promoter and coding regions of selected genes (see Table S1). Two procedures were utilised, such as Restriction

Inflammatory plasma molecule measurements
Venous blood samples were collected from all patients and control subjects in a fasting state (more than 8 h without food administration from onset). In patients who underwent surgical treatment, blood samples were collected before surgery. Plasma samples were obtained after a centrifugation of 3500 rpm at 4°C for 15 min immediately after collection and then stored at −80 °C for further analysis. Plasma IL-6, TNF-α, MMP-2 and MMP-9 were measured by using ELISA technique, with the R&D Systems (Minneapolis, MN, USA), according to the manufacturer's instructions. CRP was determined by a high-sensitivity assay using a BN II nephelometer (Dade Behring, Marburg, Germany). Detection limits were 0.7 pg/ml, 0.5 pg/ml, 0.154 ng/ml, 0.156 ng/ml and 0.17mg/l for IL-6, TNF-α, MMP-2, MMP-9 and CRP respectively. All assays were run in duplicate.

Assessment of mean terminal restriction fragment length
Genomic DNA was extracted from leukocytes of both the two cohorts and the mean terminal restriction fragment (TRF) length, a marker of telomere length, was measured by a chemiluminesence technique using TAGGG telomere length assay kits (Roche-Applied Science, Germany) [9S]. In the present study, we examined the mean TRF length in 30 patients and 30 controls selected randomly, but having the same age and gender. Accordingly, the same number of males and females (20 and 10, respectively) characterised both the two cohorts studied. Legends of figures Figure 1S. Control aortas and histo-pathological abnormalities in aorta tissues of S-TAA patients. Normal aorta (a, a1, a2, a3). Cystic medial changes of grade I (b), II (c) and III (d); Elastic fragmentation of grade I (b1), II (c1) and III (d1). Medial fibrosis of grade I (b2), II (c2) and III (d2). Medial necrosis of grade I (b3), II (c3) and III (d3). Figure 2S. Medial apoptosis and MMP-9 amounts in tissue samples. In a2 and a3 images of control aorta wall conditions. In b2 and c2 images of focal and plurifocal medial apoptosis, respectively, in patient tissues. In b3 (low), c3 (moderate ) and d3 (elevated) levels of MMP-9 in patient samples