Matrix Metalloproteinase-9 Expression in Calcified Human Aortic Valves: A Histopathologic, Immunohistochemical, and Ultrastructural Study.

The hallmarks of calcific aortic valve disease (CAVD) are the significant quantitative and qualitative changes that occur in the extracellular matrix (ECM), which ultimately lead to increased leaflet stiffness and obstruction of left ventricular outflow. Mounting evidence suggests that ECM remodeling not only contribute to valve cell dysfunction but also alter certain cell signaling pathways responsible for the initiation and progression of the disease state. Matrix metalloproteinases (MMPs), collectively called matrixins, are a family of enzymes known to participate in numerous ECM remodeling events during embryonic development and in disease. The aim of the present study was to evaluate whether changes in MMP-9 expression might be involved in the pathophysiology of CAVD. For this purpose, we have analyzed a total of 19 pathologic valves from patients who underwent aortic valve replacement for calcific aortic stenosis. Microscopically, the cusp tissue showed diffuse fibrosis, neovascularization, and abnormal ECM remodeling with collagen disorganization and mineralization. Western blot and immunohistochemical analyses have been performed on both the areas overlying and remote from the mineral deposits. Protein expression data evidenced a significant upregulation of MMP-9 in the calcified lesion area. Consistent with these observations, immunohistochemistry demonstrated that MMP-9 protein was almost exclusively localized near or around the mineralized nodules, whereas was generally quite weak or absent in areas devoid of any calcification. Our data suggest that MMP-9 may play a key role in CAVD probably by promoting the fibrotic and procalcific remodeling of the ECM.

HIF-1α and VEGF: Immunohistochemical Profile and Possible Function in Human Aortic Valve Stenosis.

Calcific aortic stenosis (CAS) is the most common valvular disease in Western countries. Histological findings in patients with CAS extremely resemble those of atherosclerosis and include accumulation and modification of lipoproteins, inflammation, extracellular matrix remodeling, and calcification. Angiogenesis is another prominent feature of CAS; however, there is only a limited amount of data available regarding the mechanisms behind the pathological neovascularization of a structure that is originally avascular. The present study aims to identify the molecular basis that regulates blood vessel growth in stenotic aortic valves, focusing on the role of HIF-1α and VEGF pathway. A total of 19 native degenerating aortic valves obtained at valve replacement surgery have been processed for Western blot, immunohistochemical, morphometric, and ultrastructural analyses. First, we have demonstrated the adverse ECM remodeling and the significant thickening of the leaflet also showing that HIF-1α and VEGF are significantly upregulated in the stenotic valves, are locally produced and colocalize with angiogenesis and areas of calcification. Next, we have characterized, for the first time to the best of our knowledge, the morphological features of the neovasculature evidencing the presence of intact blood vessels in close proximity to the mineralized zones. These results suggest that the complex structural remodeling of the matrix might reduce oxygen availability in the valve cusp contributing to the stabilization of HIF-1α that in turn induces a metabolic adaptation through the upregulation of VEGF and the formation of new blood vessels not only to overcome the hypoxic state but also to sustain the calcification process.

Expression profile and subcellular localization of GAPDH in the smooth muscle cells of human atherosclerotic plaque: an immunohistochemical and ultrastructural study with biological therapeutic perspectives.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been considered a classical glycolytic enzyme involved exclusively in cytosolic energy production. Several recent studies, however, have demonstrated that GAPDH is a multifunctional protein whose presence and activity can be regulated by disease states and/or experimental manipulation. Expression levels of GAPDH have been shown to be altered in certain tumors as well as in proliferating and differentiating cells. Since dedifferentiation and proliferation of smooth muscle cells (SMCs) are important features of human atherosclerosis, we have characterized the expression profile of GAPDH in the SMCs of atherosclerotic plaques and its putative interrelationship with the synthetic/proliferative status of these cells utilizing the proliferating cell nuclear antigen (PCNA) antibody, a valuable marker of cell proliferation. Western blot data revealed that GAPDH was significantly upregulated in atherosclerotic plaque specimens. Immunohistochemical stains demonstrated that GAPDH accumulated in the nucleus of dedifferentiated SMCs that also showed positive immunoreactivity for PCNA, but remained cytoplasmatic in the contractile SMCs (PCNA-negative), thus reflecting the proliferative, structural and synthetic differences between them. We suggest that, in human atherosclerotic plaque, GAPDH might exert additional functions that are independent of its well-documented glycolytic activity and might play key roles in development of the disease.

Internal mammary artery atherosclerosis: an ultrastructural study of two cases.

Atherosclerosis of the internal mammary artery (IMA) is generally regarded as a rare (but existent) pathological entity with only a few cases reported in the most recent literature. The only study which to our knowledge has investigated the ultrastructural features of IMA atherosclerosis, demonstrate the presence of endothelial cells loss, defects of internal elastic lamina with no evidence of lipid accumulation. In the present study, we describe two cases of IMA atherosclerosis in which ultrastructural analysis revealed the presence of a typical atherosclerotic plaque morphology with infiltration of inflammatory cells, formation of intraplaque lipid pools, and accumulation of lipid-laden foam cells throughout the thickened intima, never described in this rare lesion before. Microscopically, the lesions were also characterized by intimal thickening, invagination of endothelial cells, migration of smooth muscle cells with splitting, fenestration and/or fragmentation of the elastic sheets. Our observations add new data to the scarce and contradictory literature and to this largely understudied vascular disorder.

MnSOD expression in human atherosclerotic plaques: an immunohistochemical and ultrastructural study.

BACKGROUND: Over the last decades, the role of oxidative stress in atherosclerosis has become well established, and considerable effort has been made to understand the mechanism of action of free radicals within the cardiovascular system. Conversely, relatively little attention has been directed towards the characterization of the antioxidant status of the arterial wall under disease state. Among the antioxidant enzymes, the manganese-dependent and mitochondria-specific isoform of SOD (MnSOD) represents the first line of defense against superoxide radicals attack. To date, the pathological significance of MnSOD in atherosclerosis is still unclear with conflicting data published. METHODS: In the present study, we used immunohistochemical techniques at the light and electron microscopy level in combination with biochemical assays to localize and characterize the activity and expression profiles of MnSOD in healthy and atherosclerotic human aorta. RESULTS: MnSOD has been found to be highly expressed in the atherosclerotic plaques where specifically localized to the foam cells of the lipid-rich regions but not to other (nonfoamy) cell types. No ultrastructural evidence of apoptosis, such as chromatin condensation and membrane blebbing, has been observed in MnSOD-expressing cells. The up-regulation of MnSOD at the protein level has been associated with a parallel, significant increase of its catalytic activity. CONCLUSIONS: Our data demonstrate that MnSOD is not negatively regulated by the prooxidative and proinflammatory environment of the plaque and evidence a regional and cellular selectivity of MnSOD protein expression under disease state. We suggest that MnSOD induction might represent a protective response against the cytotoxic effect of oxidized low-density lipoprotein.

Effect of bradykinin on nitric oxide production, urea synthesis and viability of rat hepatocyte cultures.

BACKGROUND: It is well known that cytotoxic factors, such as lipopolysaccharides, derange nitrogen metabolism in hepatocytes and nitric oxide (NO) is involved among the other factors regulating this metabolic pathway. Hepatocytes have been shown to express large levels of NO following exposure to endotoxins, such as bacterial lipopolysaccharide and/or cytokines, such as tumour necrosis factor-alpha (TNFalpha), interleukin-1. The control role of arginine in both urea and NO biosynthesis is well known, when NO is synthesized from arginine, by the NOS reaction, citrulline is produced. Thus, the urea cycle is bypassed by the NOS reaction. Many authors demonstrated in other cellular types, like cardiomyocytes, that bradykinin caused the increase in reactive oxygen species (ROS) generation. The simultaneous increase of NO and ROS levels could cause peroxynitrite synthesis, inducing damage and reducing cell viability. The aim of this research is to study the effect of bradykinin, a proinflammatory mediator, on cell viability and on urea production in cultures of rat hepatocytes. RESULTS: Hepatocytes were treated with bradykinin, that stimulates nitric oxide synthase (NOS). NO release was determined using 4,5 diaminofluorescein diacetate (DAF-2DA), as fluorescent indicator of NO. Addition of the NOS inhibitor, Ng-nitro-L-arginine methyl ester (L-NAME), to the culture medium inhibited the increase of NO production. Exposure of hepatocytes to bradykinin 0,1 mM for 2 hours resulted in a significant decrease of urea synthesis. Cell viability, instead, showed a significant decrease 24 hours after the end of bradykinin treatment as determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium (MTT) assay. L-NAME addition recovered urea production and cell viability at control values. CONCLUSION: The findings suggest that the cell toxicity, after bradykinin treatment, effectively depends upon exposure to increased NO levels and the effects are prevented by L-NAME. The results show also that the increased NO synthesis induces a reduced urea production, that is another index of cell damage.