Matrix Metalloproteinase-2 Isoforms Differ within the Aortic Wall of Ascending Aortic Aneurysms Associated with Bicuspid Aortic Valve.

The pathogenesis of ascending thoracic aortic aneurysm (aTAA) is thought to differ between patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV), and one of the causes is different hemodynamics. Influenced by hemodynamics, the tissue levels of proteins associated with aTAA might differ between aTAAs with BAV and TAV and between different localities within the aortic wall. We therefore analyzed aTAA tissue levels of MMP-2 (matrix metalloproteinase-2) isoforms (Pro-MMP-2, active MMP-2, and total MMP-2) and tissue levels of MMP-14, TIMP-2 (tissue inhibitor of metalloproteinase-2), MMP-9, and TIMP-1 in 19 patients with BAV and 23 patients with TAV via gelatin zymography and enzyme-linked immunosorbent assay (ELISA), respectively. TAV and BAV groups' protein levels did not differ significantly. Whereas the TAV group exhibited no significant differences in protein levels between the aneurysm's anterior and posterior parts, the BAV group revealed significantly higher levels of Pro-MMP-2, total MMP-2, and TIMP-2 in the aneurysm's posterior parts (mean Pro-MMP-2 200.52 arbitrary units (AU) versus 161.12 AU, p=0.007; mean total MMP-2 235.22 AU versus 193.68 AU, p=0.002; mean TIMP-2 26.90 ng/ml versus 25.36 ng/ml, p=0.009), whereas the other proteins did not differ significantly within the aortic wall. Thus, MMPs are distributed more heterogeneously within the aortic wall of aTAAs associated with BAV than in those associated with TAV, which is a new aspect for understanding the underlying pathogenesis. This heterogeneous protein level distribution might be attributable to differences in the underlying pathogenesis, especially hemodynamics. This result is important for further studies as it will be essential to specify the location of samples to ensure data comparability regarding the main goals of understanding the pathogenesis of aTAA, optimizing treatments, and establishing a screening method for its potentially deadly complications.

A potential key mechanism in ascending aortic aneurysm development: Detection of a linear relationship between MMP-14/TIMP-2 ratio and active MMP-2.

OBJECTIVES: Elevated matrix metalloproteinase-2 (MMP-2) tissue levels have been associated with ascending thoracic aortic aneurysm (aTAA). As MMP-2 activation is controlled by interactions among matrix metalloproteinase-14 (MMP-14), a tissue inhibitor of metalloproteinases-2 (TIMP-2) and Pro-MMP-2 in cell culture, this activation process might also play a role in aTAA. METHODS: Via gelatin zymography we analyzed tissue levels of MMP-2 isoforms (Pro-MMP-2, active MMP-2, total MMP-2) and via enzyme-linked immunosorbent assay (ELISA,) MMP-14,TIMP-2 and total MMP-2 tissue levels in N = 42 patients with aTAA. As controls, MMP-14 and TIMP-2 aortic tissue levels in N = 9 patients undergoing coronary artery bypass surgery were measured via ELISA, and levels of MMP-2 isoforms in N = 11 patients via gelatin zymography. RESULTS: Active MMP-2 was significantly higher in aTAA than in controls. Patients with aTAA exhibited significantly lower Pro-MMP-2 and TIMP-2 levels. Total MMP-2 and MMP-14 did not differ significantly between groups. Regression analysis revealed a linear relationship between TIMP-2 and the MMP-14/TIMP-2 ratio, as well as active MMP-2 in aTAA. Aneurysmatic tissue can be accurately distinguished from control aortic tissue (AUC = 1) by analyzing the active MMP-2/Pro-MMP-2 ratio with a cutoff value of 0.11, whereas MMP-14 and TIMP-2 roles are negligible in ROC analysis. CONCLUSION: A larger amount of MMP-2 is activated in aTAA than in control aortic tissue-a factor that seems to be a central process in aneurysm development. When active MMP-2 exceeds 10% compared to Pro-MMP-2, we conclude that it originates from aneurysmatic tissue, which we regard as a starting point for further studies of aTAA biomarkers. The tissue's MMP-14/TIMP-2 ratio may regulate the degree of Pro-MMP-2 activation as a determining factor, while the enzymatic activities of MMP-14 and TIMP-2 do not seem to play a key role in aneurysm development.

Characterization of serum matrix metalloproteinase 2/9 levels in patients with ascending aortic aneurysms.

OBJECTIVES: The objectives of this study were to identify interrelations between matrix metalloproteinase-2 (MMP2)/MMP9 levels and clinical variables in patients with aortic root/ascending aortic aneurysms and to describe comorbidities as possible biasing factors in the widely discussed correlation of serum MMP levels and aortic diameter. METHODS: Serum MMP9 and MMP2 levels were quantified in 32 consecutive patients with ascending aortic and/or aortic root aneurysms (>45 mm) at the Heart Center University of Freiburg from May 2013 to January 2014. The influence of comorbidities and medication on serum MMP2 and MMP9 levels was studied. We took into account ascending aortic diameter (aAD), aortic valve configuration, hypertension, age and hyperlipidaemia as factors possibly altering serum MMP levels. The relation between serum MMP levels and aAD was examined by a correlation test based on ranks. RESULTS: Serum MMP2 levels and aAD correlated positively. Correlation was increased in patients without hyperlipidaemia (Spearman's ρ = 0.62, P = 0.008 in patients without hyperlipidaemia; ρ = 0.409, P = 0.020 in all patients). Serum MMP9 levels did not correlate with aAD and showed greater variation. Serum MMP9 levels were significantly associated with hyperlipidaemia (P = 0.037). CONCLUSIONS: The distinct correlation patterns in patients with and without hyperlipidaemia have to be considered when defining the potential of MMP2 as a biomarker in future studies. The relation between MMP9 and hyperlipidaemia has to be further investigated.

MMP-2 Isoforms in Aortic Tissue and Serum of Patients with Ascending Aortic Aneurysms and Aortic Root Aneurysms.

OBJECTIVE: The need for biological markers of aortic wall stress and risk of rupture or dissection of ascending aortic aneurysms is obvious. To date, wall stress cannot be related to a certain biological marker. We analyzed aortic tissue and serum for the presence of different MMP-2 isoforms to find a connection between serum and tissue MMP-2 and to evaluate the potential of different MMP-2 isoforms as markers of high wall stress. METHODS: Serum and aortic tissue from n = 24 patients and serum from n = 19 healthy controls was analyzed by ELISA and gelatin zymography. 24 patients had ascending aortic aneurysms, 10 of them also had aortic root aneurysms. Three patients had normally functioning valves, 12 had regurgitation alone, eight had regurgitation and stenosis and one had only stenosis. Patients had bicuspid and tricuspid aortic valves (9/15). Serum samples were taken preoperatively, and the aortic wall specimen collected during surgical aortic repair. RESULTS: Pro-MMP-2 was identified in all serum and tissue samples. Pro-MMP-2 was detected in all tissue and serum samples from patients with ascending aortic/aortic root aneurysms, irrespective of valve morphology or other clinical parameters and in serum from healthy controls. We also identified active MMP-2 in all tissue samples from patients with ascending aortic/aortic root aneurysms. None of the analyzed serum samples revealed signals relatable to active MMP-2. No correlation between aortic tissue total MMP-2 or tissue pro-MMP-2 or tissue active MMP-2 and serum MMP-2 was found and tissue MMP-2/pro-MMP-2/active MMP-2 did not correlate with aortic diameter. This evidence shows that pro-MMP-2 is the predominant MMP-2 species in serum of patients and healthy individuals and in aneurysmatic aortic tissue, irrespective of aortic valve configuration. Active MMP-2 species are either not released into systemic circulation or not detectable in serum. There is no reliable connection between aortic tissue-and serum MMP-2 isoforms, nor any indication that pro-MMP-2 functions as a common marker of high aortic wall stress.

Four-dimensional magnetic resonance imaging-derived ascending aortic flow eccentricity and flow compression are linked to aneurysm morphology†.

OBJECTIVES: The impact of specific blood flow patterns within ascending aortic and/or aortic root aneurysms on aortic morphology is unknown. We investigated the interrelation of ascending aortic flow compression/peripheralization and aneurysm morphology with respect to sinotubuar junction (STJ) definition. METHODS: Thirty-one patients (aortic root/ascending aortic aneurysm >45 mm) underwent flow-sensitive 4D magnetic resonance thoracic aortic flow measurement at 3 Tesla (Siemens, Germany) at two different institutions (Freiburg, Germany, and San Francisco, CA, USA). Time-resolved image data post-processing and visualization of mid-systolic, mid-ascending aortic flow were performed using local vector fields. The Flow Compression Index (FCI) was calculated individually as a fraction of the area of high-velocity mid-systolic flow over the complete cross-sectional ascending aortic area. According to aortic aneurysm morphology, patients were grouped as (i) small root, eccentric ascending aortic aneurysm (STJ definition) and (ii) enlarged aortic root, non-eccentric ascending aortic aneurysm with diffuse root and tubular enlargement. RESULTS: The mean FCI over all patients was 0.47 ± 0.5 (0.37-0.99). High levels of flow compression/peripheralization (FCI <0.6) were linked to eccentric aneurysm morphology (Group A, n = 11), while low levels or absence of aortic flow compression/peripheralization (FCI >0.8) occurred more often in Group B (n = 20). The FCI was 0.48 ± 0.05 in Group A and 0.78 ± 0.14 in Group B (P < 0.001). Distribution of bicuspid aortic valve (P = 0.6) and type of valve dysfunction (P = 0.22 for aortic stenosis) was not found to be different between groups. CONCLUSIONS: Irrespective of aortic valve morphology and function, ascending aortic blood flow patterns are linked to distinct patterns of ascending aortic aneurysm morphology. Implementation of quantitative local blood flow analyses might help to improve aneurysm risk stratification in the future.

Photoconversion and nuclear trafficking cycles determine phytochrome A's response profile to far-red light.

Phytochrome A (phyA) is the only photoreceptor in plants, initiating responses in far-red light and, as such, essential for survival in canopy shade. Although the absorption and the ratio of active versus total phyA are maximal in red light, far-red light is the most efficient trigger of phyA-dependent responses. Using a joint experimental-theoretical approach, we unravel the mechanism underlying this shift of the phyA action peak from red to far-red light and show that it relies on specific molecular interactions rather than on intrinsic changes to phyA's spectral properties. According to our model, the dissociation rate of the phyA-FHY1/FHL nuclear import complex is a principle determinant of the phyA action peak. The findings suggest how higher plants acquired the ability to sense far-red light from an ancestral photoreceptor tuned to respond to red light.

FHY1 mediates nuclear import of the light-activated phytochrome A photoreceptor.

The phytochrome (phy) family of photoreceptors is of crucial importance throughout the life cycle of higher plants. Light-induced nuclear import is required for most phytochrome responses. Nuclear accumulation of phyA is dependent on two related proteins called FHY1 (Far-red elongated HYpocotyl 1) and FHL (FHY1 Like), with FHY1 playing the predominant function. The transcription of FHY1 and FHL are controlled by FHY3 (Far-red elongated HYpocotyl 3) and FAR1 (FAr-red impaired Response 1), a related pair of transcription factors, which thus indirectly control phyA nuclear accumulation. FHY1 and FHL preferentially interact with the light-activated form of phyA, but the mechanism by which they enable photoreceptor accumulation in the nucleus remains unsolved. Sequence comparison of numerous FHY1-related proteins indicates that only the NLS located at the N-terminus and the phyA-interaction domain located at the C-terminus are conserved. We demonstrate that these two parts of FHY1 are sufficient for FHY1 function. phyA nuclear accumulation is inhibited in the presence of high levels of FHY1 variants unable to enter the nucleus. Furthermore, nuclear accumulation of phyA becomes light- and FHY1-independent when an NLS sequence is fused to phyA, strongly suggesting that FHY1 mediates nuclear import of light-activated phyA. In accordance with this idea, FHY1 and FHY3 become functionally dispensable in seedlings expressing a constitutively nuclear version of phyA. Our data suggest that the mechanism uncovered in Arabidopsis is conserved in higher plants. Moreover, this mechanism allows us to propose a model explaining why phyA needs a specific nuclear import pathway.

FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome A photoreceptor.

The phytochrome family of red/far-red photoreceptors is involved in the regulation of a wide range of developmental responses in plants. The Arabidopsis genome contains five phytochromes (phyA-E), among which phyA and phyB play the most important roles. Phytochromes localize to the cytosol in the dark and accumulate in the nucleus under light conditions, inducing specific phytochrome-mediated responses. Light-regulated nuclear accumulation of the phytochrome photoreceptors is therefore considered a key regulatory step of these pathways. In fact, one of the most severe phyA signaling mutants, fhy1 (far red elongated hypocotyl 1), is strongly affected in nuclear accumulation of phyA. The fhy1 fhl (fhy1 like) double mutant, lacking both FHY1 and its only close homolog FHL, is virtually blind to far-red light like phyA null seedlings. Here we show that FHL accounts for residual amounts of phyA in the nucleus in a fhy1 background and that nuclear accumulation of phyA is completely inhibited in an fhy1 FHL RNAi knock-down line. Moreover, we demonstrate that FHL and phyA interact with each other in a light-dependent manner and that they co-localize in light-induced nuclear speckles. We also identify a phyA-binding site at the C-terminus of FHY1 and FHL, and show that the N-terminal 406 amino acids of phyA are sufficient for the interaction with FHY1/FHL.

Nuclear accumulation of the phytochrome A photoreceptor requires FHY1.

The phytochrome family of red/far-red (R/FR)-responsive photoreceptors plays a key role throughout the life cycle of plants . Arabidopsis has five phytochromes, phyA-phyE, among which phyA and phyB play the most predominant functions . Light-regulated nuclear accumulation of the phytochromes is an important regulatory step of this pathway, but to this date no factor specifically required for this event has been identified . Among all phyA signaling mutants, fhy1 and fhy3 (far-red elongated hypocotyl 1 and 3) have the most severe hyposensitive phenotype, indicating that they play particularly important roles . FHY1 is a small plant-specific protein of unknown function localized both in the nucleus and the cytoplasm . Here we show that FHY1 is specifically required for the light-regulated nuclear accumulation of phyA but not phyB. Moreover, phyA accumulation is only slightly affected in fhy3, indicating that the diminished nuclear accumulation of phyA observed in fhy1 seedlings is not simply a general consequence of reduced phyA signaling. By in vitro pull-down and yeast two-hybrid analyses, we demonstrate that FHY1 physically interacts with phyA, preferentially in its active Pfr form. Furthermore, FHY1 and phyA colocalize in planta. We therefore identify the first component required for light-regulated phytochrome nuclear accumulation.