Salah A. Mohamed

Local Generation of C-Reactive Protein in Diseased Coronary Artery Venous Bypass Grafts and Normal Vascular Tissue

Background—Venous coronary artery bypass grafts (CABGs) are prone to accelerated atherosclerosis. In atherosclerotic diseases, serum C-reactive protein (CRP) levels have become an important diagnostic and prognostic marker. The origin of CRP in this setting remains to be elucidated. Methods and Results—Monoclonal anti-CRP identified CRP expression in medial and intimal &agr;-actin–positive smooth muscle cells (SMCs) of diseased CABGs with type V and VI lesions and also of native saphenous veins of atherosclerotic individuals. In addition, patent coronary arteries with type IV and V but not with type I through III lesions exhibited intense SMC staining for CRP. Calcified desobliterates of occluded coronary arteries with end-stage disease did not show SMC staining for CRP and were consistently negative for CRP mRNA, as detected by means of real-time polymerase chain reaction. However, CRP mRNA was expressed in 11 of 15 diseased CABGs and also in 10 of 15 native veins. By contrast, only 3 of 18 internal mammary and 4 of 12 radial arteries with virtually no atherosclerosis were positive for CRP mRNA. Conclusions—CRP is produced by SMCs of atherosclerotic lesions with active disease but not in end-stage plaques. The role of CRP constitutively expressed by normal vascular tissue in vein graft disease has yet to be elucidated.

Mitochondrial DNA deletions and the aging heart.

Mitochondrial DNA (mtDNA) mutations appear to be associated with a wide spectrum of human disorders and proposed to be a potential contributor of aging. However, in an age-dependent increase of the common 4977 bp deletion of human mtDNA still many unanswered questions remain. Comparing mtDNA copy levels in different tissues revealed that cardiac muscle had the highest, while the cortex cerebelli showed the lowest copy number of mtDNA in every donor. Intriguingly, mtDNA copy number showed no changes during aging. In heart tissue, the amount of 4977 bp mtDNA deletion increased in an age-dependent manner showing significant differences at the age of 40 years and older (p<0.005). In vitro studies analyzing human normal cells transfected with telomerase (BJ-T) revealed that oxidative stress (OS)--a well accepted promoter of aging--induced 4977 bp deletion and point mutations as demonstrated by real-time PCR and DHPLC analysis. Interestingly, OS induced apoptosis only in transformed human fibroblasts by activation of the intrinsic (mitochondrial-mediated) signalling pathway as indicated by morphological damage of mitochondria, DNA laddering and increase of the Bax/Bcl-2 ratio. In conclusion, in heart tissue, the amount of the 4977 bp deletion increased in an age-dependent manner and it was more detectable after the 4th decade of life, although there was some scatter in the data. Since, apoptosis was induced by the mitochondria-mediated pathway only in transformed cells, the role for apoptosis in normal tissue of the aging heart remains unclear.

The long non-coding HOTAIR is modulated by cyclic stretch and WNT/β-CATENIN in human aortic valve cells and is a novel repressor of calcification genes.

Aortic valve calcification is a significant and serious clinical problem for which there are no effective medical treatments. Individuals born with bicuspid aortic valves, 1–2% of the population, are at the highest risk of developing aortic valve calcification. Aortic valve calcification involves increased expression of calcification and inflammatory genes. Bicuspid aortic valve leaflets experience increased biomechanical strain as compared to normal tricuspid aortic valves. The molecular pathogenesis involved in the calcification of BAVs are not well understood, especially the molecular response to mechanical stretch. HOTAIR is a long non-coding RNA (lncRNA) that has been implicated with cancer but has not been studied in cardiac disease. We have found that HOTAIR levels are decreased in BAVs and in human aortic interstitial cells (AVICs) exposed to cyclic stretch. Reducing HOTAIR levels via siRNA in AVICs results in increased expression of calcification genes. Our data suggest that β-CATENIN is a stretch responsive signaling pathway that represses HOTAIR. This is the first report demonstrating that HOTAIR is mechanoresponsive and repressed by WNT β-CATENIN signaling. These findings provide novel evidence that HOTAIR is involved in aortic valve calcification.

Toward individualized management of the ascending aorta in bicuspid aortic valve surgery: The role of valve phenotype in 1362 patients

OBJECTIVE Decision making regarding the management of the ascending aorta (AA) in patients with a bicuspid aortic valve (BAV) undergoing valve surgery has hardly been individualized and remains controversial. We analyzed our individualized, multifactorial approach, focusing on the BAV phenotype. METHODS In 1362 patients (1044 men) undergoing aortic valve surgery, the BAV phenotypes were intraoperatively classified and retrospectively analyzed. The mean follow-up was 5.4±3.6 years (range, 0-14; 7334 patient-years), and the data were 96.5% complete. The individualized AA management decision process mainly included the AA diameter, age, body surface area, macroscopic AA configuration, and the perceived tissue strength of the aortic wall resulting in 3 AA treatment groups: no intervention, aortoplasty (AoP), and AA replacement (AAR). RESULTS In 906 patients (66.5%), no intervention was performed and 172 (12.6%) and 284 (20.9%) underwent AoP and AAR, respectively. The hospital mortality was 1.1% for no intervention, 0.6% for AoP, and 0.4% for AAR (P=.4). The 10-year survival was similar for all 3 groups and comparable to that of the general population. Five reoperations on the AA occurred, 4 in the no intervention and 1 in the AoP group. BAV type 2/unicuspid patients were younger and more had undergone AAR in absolute numbers and after allowing for the AA diameter. Also, in patients with BAV type 1 LR and regurgitation, AAR was performed more often. CONCLUSIONS The individualized, multifactorial management of AA in patients with BAV during aortic valve surgery leads to excellent results. The threshold AA diameter for intervention (AoP or AAR) varied from 34 to 51 mm (mean, 43.9). BAV type 2/unicuspid and BAV type 1 LR with regurgitation emerged as determinants for more liberal AAR in our practice. Longer term follow-up is necessary to confirm our conclusions.

The fate of the bicuspid valve aortopathy after aortic valve replacement.

OBJECTIVES The fate of the aortic dimensions in patients with a bicuspid aortic valve (BAV) after aortic valve replacement (AVR) is unclear. We investigated the evolution of aortic root and ascending aorta dimensions in patients with a BAV after AVR. To neutralize the effect of pathological transvalvular haemodynamics on aortic dimensions, we evaluated our hypotheses in patients with normal transvalvular haemodynamics after a subcoronary autograft procedure, which preserves intact the native aortic wall. METHODS We excluded patients operated on for endocarditis; who developed autograft insufficiency > trivial and who required autograft reoperation during the follow-up. We included 448 patients (361 with BAV; 340 males; 44.6 ± 11.4 years; mean follow-up: 7.5 ± 3.9 years). Valve phenotype was determined during surgery. Annual echocardiographic examinations (n = 3336) were performed (follow-up completeness: 98%). To allow for somatometric, gender and age influences, z-values of measurements were calculated from the general population (GP) and analysed using longitudinal methods. RESULTS The increase in ascending aorta did not differ from that expected in the GP (0.04 z-values/year; P = 0.06). No difference could be observed in diameter increase rates between BAV and tricuspid aortic valve patients (TAV) (0.04 vs 0.06 z-values/year; P = 0.3), as well as between BAV phenotypes. The sinus increase did not differ from that expected in the GP (0.03 z-values/year; P = 0.1), and no significant differences could be observed between BAV phenotypes. In patients undergoing aortoplasty (n = 70), no significant difference in the rates of ascending aorta and sinus increase could be observed, compared with the GP. CONCLUSION For the time period of this study and in patients with normal aortic root haemodynamics after AVR, ascending aorta dimensions over time are similar to that of the matched GP. Patients with a BAV did not exhibit higher rates of ascending aorta dilatation after AVR than patients with TAV. At least for the first postoperative decade, transvalvular haemodynamics appear to exhibit a greater influence than the genetic component of BAV on the development of the BAV aortopathy.

Ascending aorta dilation in association with bicuspid aortic valve: A maturation defect of the aortic wall

OBJECTIVE Patients with a bicuspid aortic valve have increased susceptibility to the development of ascending aortic dilation and dissection compared with persons with a tricuspid valve. To unravel a possible different mechanism underlying dilation in bicuspidy and tricuspidy, a comparison of the structure of the aortic wall was made. METHODS Ascending aortic wall biopsies were divided into 4 groups: bicuspid (n=36) and tricuspid (n=23) without and with dilation. The expression of vascular smooth muscle cell maturation markers including lamin A/C, which plays a pivotal role in smooth muscle cell differentiation, and its splicing variant progerin indicative of aging, were studied immunohistochemically. Attention was also paid to the inflammatory status. RESULTS There is a significant difference in the structure and maturation of the aortic wall in bicuspidy, persisting in the dilated aortic wall, presenting with a thinner intima, lower expression of α smooth muscle actin, smooth muscle 22α, calponin, and almost absent expression of smoothelin. We show for the first time significantly lowered lamin A/C expression in bicuspidy. Progerin was found to be significantly increased in the media of the dilated wall in tricuspidy, also showing increased periaortic inflammation. CONCLUSIONS The structure of the nondilated and dilated aortic wall in bicuspidy and tricuspidy are intrinsically different, with the latter having more aspects of aging. In bicuspidy there is a defective smooth muscle cell differentiation possibly linked to lowered lamin A/C expression. Based on this vessel wall immaturity and increased susceptibility to dilation, different diagnostic and therapeutic approaches are warranted.

Locally Different Endothelial Nitric Oxide Synthase Protein Levels in Ascending Aortic Aneurysms of Bicuspid and Tricuspid Aortic Valve

Aims. Dysregulated expression of the endothelial nitric oxide synthase (eNOS) is observed in aortic aneurysms associated with bicuspid aortic valve (BAV). We determined eNOS protein levels in various areas in ascending aortic aneurysms. Methods and Results. Aneurysmal specimens were collected from 19 patients, 14 with BAV and 5 with tricuspid aortic valve (TAV). ENOS protein levels were measured in the outer curve (convexity), the opposite side (concavity), the distal and above the sinotubular junction (proximal) aneurysm. Cultured aortic cells were treated with NO synthesis inhibitor L-NAME and the amounts of 35 apoptosis-related proteins were determined. In patients with BAV, eNOS levels were significantly lower in the proximal aorta than in the concavity and distal aorta. ENOS protein levels were also lower in the convexity than in the concavity. While the convexity and distal aorta showed similar eNOS protein levels in BAV and TAV patients, levels were higher in TAV proximal aorta. Inhibition of NO synthesis in aneurysmal aortic cells by L-NAME led to a cytosolic increase in the levels of mitochondrial serine protease HTRA2/Omi. Conclusion. ENOS protein levels were varied at different areas of the aneurysmal aorta. The dysregulation of nitric oxide can lead to an increase in proapoptotic HTRA2/Omi.

The stretch responsive microRNA miR-148a-3p is a novel repressor of IKBKB, NF-κB signaling, and inflammatory gene expression in human aortic valve cells

Bicuspid aortic valves calcify at a significantly higher rate than normal aortic valves, a process that involves increased inflammation. Because we have previously found that bicuspid aortic valve experience greater stretch, we investigated the potential connection between stretch and inflammation in human aortic valve interstitial cells (AVICs). Microarray, quantitative PCR (qPCR), and protein assays performed on AVICs exposed to cyclic stretch showed that stretch was sufficient to increase expression of interleukin and metalloproteinase family members by more than 1.5‐fold. Conditioned medium from stretched AVICs was sufficient to activate leukocytes. microRNA sequencing and qPCR experiments demonstrated that miR‐148a‐3p was repressed in both stretched AVICs (43% repression) and, as a clinical correlate, human bicuspid aortic valves (63% reduction). miR‐148a‐3p was found to be a novel repressor of IKBKB based on data from qPCR, luciferase, and Western blot experiments. Furthermore, increasing miR‐148a‐3p levels in AVICs was sufficient to decrease NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells) signaling and NF‐κB target gene expression. Our data demonstrate that stretch‐mediated activation of inflammatory pathways is at least partly the result of stretch‐repression of miR‐148a‐3p and a consequent failure to repress IKBKB. To our knowledge, we are the first to report that cyclic stretch of human AVICs activates inflammatory genes in a tissue‐autonomous manner via a microRNA that regulates a central inflammatory pathway.—Patel, V., Carrion, K., Hollands, A., Hinton, A., Gallegos, T., Dyo, J., Sasik, R., Leire, E., Hardiman, G., Mohamed, S. A., Nigam, S., King, C. C., Nizet, V., Nigam V. The stretch responsive microRNA miR‐148a‐3p is a novel repressor of IKBKB, NF‐κB signaling, and inflammatory gene expression in human aortic valve cells. FASEB J. 29, 1859‐1868 (2015). www.fasebj.org

Pathway Analysis of Differentially Expressed Genes in Patients with Acute Aortic Dissection

Background Acute aortic dissection (AAD) is a life-threatening condition with high mortality and a relatively unclarified pathophysiological mechanism. Although differentially expressed genes in AAD have been recognized, interactions between these genes remain poorly defined. This study was conducted to gain a better understanding of the molecular mechanisms underlying AAD and to support the future development of a clinical test for monitoring patients at high risk. Materials and Methods Aortic tissue was collected from 19 patients with AAD (mean age 61.7 ± 13.1 years), and from eight other patients (mean age 32.9 ± 12.2 years) who carried the mutated gene for Marfan syndrome (MS). Six patients (mean age 56.7 ± 12.3 years) served as the control group. The PIQOR™ Immunology microarray with 1076 probes in quadruplicates was utilized; the differentially expressed genes were analysed in a MedScan search using PathwayAssist software. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and protein analysis were performed. Results Interactions of MS fibrillin-1 (FBN1) in the MedScan pathway analysis showed four genes, fibulin-1 (FBLN1), fibulin-2 (FBLN2), decorin (DCN) and microfibrillar associated protein 5 (MFAP5), which were differentially expressed in all tissue from AAD. The validation of these genes by qRT-PCR revealed a minimum of three-fold downregulation of FBLN1 (0.5 ± 0.4 vs. 6.1 ± 2.3 fold, p = 0.003) and of DCN (2.5 ± 1.0 vs. 8.5 ± 4.7 fold, p = 0.04) in AAD compared to MS and control samples. Conclusions Downregulation of fibrillin-1 (FBN1) may weaken extracellular components in the aorta and/or interfer with the transmission of cellular signals and eventually cause AAD. Additional research on these four identified genes can be a starting point to develop a diagnostic tool.

Bicuspid aortic valve: phosphorylation of c-Kit and downstream targets are prognostic for future aortopathy

OBJECTIVES The clinical course of many patients with a bicuspid aortic valve (BAV) is complicated by ascending aortic dilatation. Currently, the indication for aortic surgery is solely based on the aortic diameter and subsequently only a small proportion of BAV patients undergoing valve surgery require concomitant ascending aortic replacement based on these recommendations. Unfortunately, a substantial number of BAV patients still develop aortic dilatation in the future and would potentially benefit from a more aggressive approach towards ascending aortic replacement. We, therefore, designed this study to identify molecular biological markers in the aortic wall predictive of aortopathy in BAV. METHODS Ascending aortic wall specimen of BAV (n = 36) and tricuspid aortic valve (TAV) (n = 23), both without and with (>44 mm) dilatation were investigated histologically and immunohistochemically for the expression of markers for vascular remodelling [transforming growth factor (TGF)-β, phosphorylated Smad2, matrix metalloproteinase 9 (MMP9)], cellular differentiation [c-Kit, phosphorylated-c-Kit, hypoxia-inducable factor-1 alpha (HIF1α)] and haemodynamic influences on the aortic wall [endothelial nitric oxide (eNOS)]. RESULTS All BAV patients showed significantly less inflammation (P < 0.001) and an altered intima/media ratio when compared with TAV patients. The expression of markers of a signalling pathway characteristic for cellular dedifferentiation, as exemplified by the marked expression of c-Kit, phosphorylated c-Kit and HIF1α; in the dilated BAV group was however completely comparable with only a subgroup of the non-dilated BAV (BAb), whereas the remainder of the non-dilated BAV group (BAa) was significantly distinct. This difference between the dilated BAV and BAa was further confirmed in the expression of TGF-β, phosphorylated Smad2, MMP9 and eNOS. Besides the expression pattern, similarity in the dilated BAV and BAb was also noted clinically in the most common variant of commissure position and conjoined raphe of the BAV. Based on these observations, we consider the BAb group a likely candidate for future dilatation as opposed to the BAa group. CONCLUSIONS Using a panel of molecular tissue markers, the non-dilated BAV patients can be divided into groups susceptible and non-susceptible to aortopathy.