Differential protein kinase C isoform abundance in ascending aortic aneurysms from patients with bicuspid versus tricuspid aortic valves.

BACKGROUND: It is recognized that different events contribute to the initiation of ascending thoracic aortic aneurysms (ATAAs) in patients with bicuspid aortic valves (BAV) versus patients with tricuspid aortic valves (TAV), but the molecular signaling pathways driving aneurysm formation remain unclear. Protein kinase C (PKC) is a superfamily of kinases which differentially mediate signaling events that lead to altered gene expression and cellular function, and may regulate downstream mediators of vascular remodeling. The present study tested the hypothesis that ATAA development in patients with BAV versus TAV proceeds by independent signaling pathways involving differential PKC signaling. METHODS AND RESULTS: ATAA samples were collected from BAV (n=57) and TAV (n=55) patients and assessed for 10 different PKC isoforms by immunoblotting. Results were expressed as a percent change in abundance (mean+/-SEM) from a nonaneurysmal control group (100%, n=21). Correlation analysis was performed, and relationships between PKC and matrix metalloproteinase abundance were reported. In the BAV group, classic and novel PKC isoforms (PKC-alpha, betaI, gamma, epsilon, theta) were increased, whereas PKC-eta and atypical PKC-zeta were decreased. In the TAV group, classic and novel isoforms were decreased and atypical PKC-zeta was elevated. Positive correlations between PKC and matrix metalloproteinase abundance were identified. CONCLUSIONS: Differential PKC isoform abundance was observed in ATAA samples from patients with BAV versus TAV, suggesting independent molecular signaling pathways may be operative. Induction of independent transcriptional programs may result and may provide a mechanistic foundation for developing selective diagnostic/therapeutic strategies for patients with ATAAs secondary to BAV or TAV.

Expression of matrix metalloproteinases and endogenous inhibitors within ascending aortic aneurysms of patients with bicuspid or tricuspid aortic valves.

OBJECTIVE: The mechanisms contributing to ascending thoracic aortic aneurysms associated with bicuspid aortic valves may differ from ascending thoracic aortic aneurysms with tricuspid aortic valves. Matrix metalloproteinases and their endogenous inhibitors have been causally linked to ascending thoracic aortic aneurysm formation. This study tested the hypothesis that specific and different matrix metalloproteinase and tissue inhibitors of metalloproteinase profiles would be observed in ascending thoracic aortic aneurysm samples from patients with bicuspid aortic valves versus tricuspid aortic valves. METHODS: Ascending thoracic aortic aneurysm samples taken from patients with bicuspid aortic valve (n = 53) and patients with tricuspid aortic valve (n = 46) were assessed for representative subtypes of all matrix metalloproteinase classes and all 4 known tissue inhibitors of metalloproteinases. Levels were compared [optical density units, median (interquartile range)] both to reference control ascending aortic samples (n = 26) and within each valve group by aneurysm diameter (< or =3.9 cm, 4.0-5.9 cm and > or =6.0 cm). RESULTS: Different and specific matrix metalloproteinase and tissue inhibitors of metalloproteinase profiles were observed in the ascending thoracic aortic aneurysm groups. In bicuspid aortic valves, matrix metalloproteinase-2 increased by 34% when compared with either tricuspid aortic valves or control (P < .05), and matrix metalloproteinase-14 decreased by 59% compared with tricuspid aortic valves (P < .05). In tricuspid aortic valve samples, tissue inhibitors of metalloproteinase-2 decreased by 35% when compared with either tricuspid aortic valves or control (P < .05), and matrix metalloproteinase-13 increased by 140% in the 4.0- to 5.9-cm diameter range (P < .05). CONCLUSIONS: A unique matrix metalloproteinase and tissue inhibitor of metalloproteinase portfolio was observed in ascending thoracic aortic aneurysms from patients with bicuspid aortic valve compared with patients with tricuspid aortic valve. These differences, suggesting disparate mechanisms of extracellular matrix remodeling, may provide unique biochemical targets for ascending thoracic aortic aneurysm prognostication and treatment in these 2 groups of patients.

Protein kinase C isoform activation and endothelin-1 mediated defects in myocyte contractility after cardioplegic arrest and reperfusion.

BACKGROUND: Endothelin-1 (ET-1) is released after hyperkalemic cardioplegic arrest (CA) and reperfusion and may contribute to contractile dysfunction. ET-1 receptor transduction causes activation of protein kinase C (PKC) isoforms, which can cause differential intracellular events. The goal of this study was to determine which PKC isoforms contribute to myocyte contractile dysfunction with ET-1 and CA. METHODS AND RESULTS: Percent shortening (PERSHORT) and the time to 50% relaxation (T50) were measured in porcine (n =22) left ventricular myocytes, randomized (minimum: 30 cells/group) to normothermia: (cell media for 2 hours/37 degrees C), and CA: (2 hours/4 degrees C, 24 mEq K+ solution followed by reperfusion in cell media), ET-1/CA: (100 pM ET-1 during CA). Studies were performed in the presence and absence of PKC inhibitors (500 nM) against the classical (Beta-I, Beta-II, Gamma) and novel (Epsilon, Eta) isoforms (myocytes from a minimum of 3 pigs per inhibitor). CA reduced PERSHORT by approximately 35% from normothermia (P<0.05), which was further reduced with ET-1. PKC-Beta-II or PKC-Gamma inhibition increased PERSHORT from ET-1/CA as well as CA only (P<0.05). CA prolonged T50 by approximately 19% from normothermia (P<0.05) and was further prolonged with ET-1. Inhibition of the classical PKC isoforms reduced T50 from ET-1/CA (P<0.05). Inhibition of novel PKC isoforms did not yield similar effects on either PERSHORT or T50 with ET-1/CA. CONCLUSIONS: Inhibition of the classical PKC isoforms relieved the negative inotropic and lusitropic effects of ET-1 after CA. These findings provide mechanistic support for developing targeted inhibitory strategies with respect to ET-1 signaling and myocyte contractile dysfunction with cardioplegic arrest and reperfusion.

Expression of matrix metalloproteinases and endogenous inhibitors within ascending aortic aneurysms of patients with Marfan syndrome.

BACKGROUND: Marfan syndrome (MFS) is known to cause ascending thoracic aortic aneurysms (ATAAs). Transforming growth factor beta (TGF-beta) has recently been implicated in this process. Imbalances between the matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) have also been shown to contribute to aneurysm formation. Whether and to what degree MMP, TIMP, and TGF-beta signaling profiles are altered in ATAAs in MFS compared with non-MFS patients remains unknown. METHODS AND RESULTS: ATAA samples taken during aortic replacement from age-matched MFS (n=9) and non-MFS (n=18) patients were assessed for representative subtypes of all MMP classes, all 4 known TIMPs, and type 2 TGF-beta receptors (TGFBR2). Results were expressed as a percentage (mean+/-SEM) of reference control samples (100%; n=18) obtained from patients without ATAA. In MFS, decreased MMP-2 (76+/-7; P<0.05 versus control), increased MMP-12 (161+/-27% versus control; P<0.05), and increased MT1-MMP (248+/-64% versus 91+/-21 non-MFS and control; P<0.05) were observed. TIMP-3 (74+/-23%) was reduced compared with control values (P<0.05) and TIMP-2 was elevated (128+/-31%) compared with non-MFS (73+/-19%; P<0.05). In non-MFS samples, MMP-1 (70+/-16%), MMP-3 (77+/-18%), MMP-8 (75+/-11%), MMP-9 (69+/-14%), and MMP-12 (85+/-15%) were decreased compared with control (P<0.05). TIMPs 1 to 3 were reduced in non-MFS compared with control values (P<0.05). TGFBR2 were increased in MFS (193+/-32%) compared with non-MFS (95+/-16%) and controls (P<0.05). CONCLUSIONS: A unique MMP and TIMP portfolio was observed in ATAAs from MFS compared with non-MFS patients. In addition, MFS samples showed evidence of increased TGF-beta signaling. These differences suggest disparate mechanisms of extracellular matrix remodeling between these 2 groups of patients.

Alterations in cultured myocardial fibroblast function following the development of left ventricular failure.

A structural event in the progression of left ventricular (LV) failure is myocardial extracellular matrix (ECM) remodeling. The myocardial fibroblast is a major cell type influencing the ECM, but whether and to what degree specific phenotypic differences in myocardial fibroblasts can be demonstrated to occur in culture with the development of LV failure remains unclear. Adult pigs (25 kg) were used for control myocardial fibroblast preparations (N=5) or following pacing-induced LV failure (N=5; 240 bpm, 3 weeks). LV remodeling occurred with pacing as evidenced by increased LV end diastolic volume (132+/-11 vs. 60+/-4 mL for control; P<0.05). Functional parameters including migration, adhesion, collagen and matrix metalloproteinase release were assessed in fibroblast cultures from passages 1-4. The following findings were consistent with each passage and the results were analyzed with control values set to 100%. Migration of LV failure fibroblasts increased by over 170% (P<0.05). Adhesion to collagen I, laminin and fibronectin was increased by over 160% in LV failure fibroblasts (P<0.05). beta(1) integrin density decreased by 50% in LV failure fibroblasts (P<0.05). Fibrillar collagen release increased by over 130% and matrix metalloproteinase-2 increased by 140% in LV failure fibroblasts (P<0.05). The unique findings of this study are two-fold. First, after a pathological stimulus in-vivo, adult myocardial fibroblasts maintain a consistent phenotype through early passages in-vivo. Second, a differential release of, and response to ECM components occurred in LV failure fibroblasts. Thus, a phenotypic transformation of the myocardial fibroblast occurs with the development of LV failure, which in turn may contribute to matrix remodeling and presents as a potential cellular therapeutic target.