Margaret J. Springett

Human Aortic Valve Calcification Is Associated With an Osteoblast Phenotype

Background—Calcific aortic stenosis is the third most common cardiovascular disease in the United States. We hypothesized that the mechanism for aortic valve calcification is similar to skeletal bone formation and that this process is mediated by an osteoblast-like phenotype. Methods and Results—To test this hypothesis, we examined calcified human aortic valves replaced at surgery (n=22) and normal human valves (n=20) removed at time of cardiac transplantation. Contact microradiography and micro-computerized tomography were used to assess the 2-dimensional and 3-dimensional extent of mineralization. Mineralization borders were identified with von Kossa and Goldner’s stains. Electron microscopy and energy-dispersive spectroscopy were performed for identification of bone ultrastructure and CaPO4 composition. To analyze for the osteoblast and bone markers, reverse transcriptase–polymerase chain reaction was performed on calcified versus normal human valves for osteopontin, bone sialoprotein, osteocalcin, alkaline phosphatase, and the osteoblast-specific transcription factor Cbfa1. Microradiography and micro-computerized tomography confirmed the presence of calcification in the valve. Special stains for hydroxyapatite and CaPO4 were positive in calcification margins. Electron microscopy identified mineralization, whereas energy-dispersive spectroscopy confirmed the presence of elemental CaPO4. Reverse transcriptase–polymerase chain reaction revealed increased mRNA levels of osteopontin, bone sialoprotein, osteocalcin, and Cbfa1 in the calcified valves. There was no change in alkaline phosphatase mRNA level but an increase in the protein expression in the diseased valves. Conclusions—These findings support the concept that aortic valve calcification is not a random degenerative process but an active regulated process associated with an osteoblast-like phenotype.

Atorvastatin Inhibits Hypercholesterolemia-Induced Cellular Proliferation and Bone Matrix Production in the Rabbit Aortic Valve

Background—Despite the common occurrence of aortic stenosis, the cellular causes of the disorder are unknown, in part because of the absence of experimental models. We hypothesized that atherosclerosis and early bone matrix expression in the aortic valve occurs secondary to experimental hypercholesterolemia and that treatment with atorvastatin modifies this transformation. Methods and Results—To test this hypothesis, we developed an experimental hypercholesterolemic rabbit model. New Zealand White rabbits (n=48) were studied: group 1 (n=16), normal diet; group 2 (n=16), 1% (wt/wt) cholesterol diet; and group 3 (n=16), 1% (wt/wt) cholesterol diet plus atorvastatin (3 mg/kg per day). The aortic valves were examined with hematoxylin and eosin stain, Masson trichrome, macrophage (RAM 11), proliferation cell nuclear antigen (PCNA), and osteopontin immunostains. Cholesterol and highly sensitive C-reactive protein (hsCRP) serum levels were obtained by standard assays. Computerized morphometry and digital image analysis were performed for quantifying PCNA (% area). Electron microscopy and immunogold labeling were performed for osteopontin. Semiquantitative RT-PCR was performed for the osteoblast bone markers [alkaline phosphatase, osteopontin, and osteoblast lineage-specific transcription factor (Cbfa-1)]. There was an increase in cholesterol, hsCRP, PCNA, RAM 11, and osteopontin and osteoblast gene markers (alkaline phosphatase, osteopontin, and Cbfa-1) in the cholesterol-fed rabbits compared with control rabbits. All markers except hsCRP were reduced by atorvastatin. Conclusions—These findings of increased macrophages, PCNA levels, and bone matrix proteins in the aortic valve during experimental hypercholesterolemia provide evidence of a proliferative atherosclerosis–like process in the aortic valve associated with the transformation to an osteoblast-like phenotype that is inhibited by atorvastatin.

Atorvastatin inhibits calcification and enhances nitric oxide synthase production in the hypercholesterolaemic aortic valve

Objective: To study in a rabbit model the expression of endothelial nitric oxide synthase (eNOS) in association with the development of calcification of the aortic valve, and to assess the effects of atorvastatin on eNOS expression, nitrite concentration, and aortic valve calcification. Methods: Rabbits (n  =  48) were treated for three months: 16, forming a control group, were fed a normal diet; 16 were fed a 0.5% (wt/wt) high cholesterol diet; and 16 were fed a 0.5% (wt/wt) cholesterol diet plus atorvastatin (2.5 mg/kg/day). The aortic valves were examined with eNOS immunostains and western blotting. Cholesterol and high sensitivity C reactive protein (hsCRP) concentrations were determined by standard assays. Serum nitrite concentrations were measured with a nitric oxide analyser. eNOS was localised by electron microscopy and immunogold labelling. Calcification in the aortic valve was evaluated by micro-computed tomography (CT). Results: Cholesterol, hsCRP, and aortic valve calcification were increased in the cholesterol fed compared with control animals. Atorvastatin inhibited calcification in the aortic valve as assessed by micro-CT. eNOS protein concentrations were unchanged in the control and cholesterol groups but increased in the atorvastatin treated group. Serum nitrite concentrations were decreased in the hypercholesterolaemic animals and increased in the group treated with atorvastatin. Conclusion: These data provide evidence that chronic experimental hypercholesterolaemia produces bone mineralisation in the aortic valve, which is inhibited by atorvastatin.

Disruption of the FG nucleoporin NUP98 causes selective changes in nuclear pore complex stoichiometry and function

The NUP98 gene encodes precursor proteins that generate two nucleoplasmically oriented nucleoporins, NUP98 and NUP96. By using gene targeting, we have selectively disrupted the murine NUP98 protein, leaving intact the expression and localization of NUP96. We show that NUP98 is essential for mouse gastrulation, a developmental stage that is associated with rapid cell proliferation, but dispensable for basal cell growth. NUP98−/− cells had an intact nuclear envelope with a normal number of embedded nuclear pore complexes. Typically, NUP98-deficient cells contained on average approximately 5-fold more cytoplasmic annulate lamellae than control cells. We found that a set of cytoplasmically oriented nucleoporins, including NUP358, NUP214, NUP88, and p62, assembled inefficiently into nuclear pores of NUP98−/− cells. Instead, these nucleoporins were prominently associated with the annulate lamellae. By contrast, a group of nucleoplasmically oriented nucleoporins, including NUP153, NUP50, NUP96, and NUP93, had no affinity for annulate lamellae and assembled normally into nuclear pores. Mutant pores were significantly impaired in transport receptor-mediated docking of proteins with a nuclear localization signal or M9 import signal and showed weak nuclear import of such substrates. In contrast, the ability of mutant pores to import ribosomal protein L23a and spliceosome protein U1A appeared intact. These observations show that NUP98 disruption selectively impairs discrete protein import pathways and support the idea that transport of distinct import complexes through the nuclear pore complex is mediated by specific subsets of nucleoporins.

Ultrastructural Localization of Collagen IV, Fibronectin, and Laminin in the Trabecular Meshwork of Normal and Glaucomatous Eyes

Purpose: To determine whether differences in the ultrastructural characteristics or composition of the basement membranes of the trabecular lamellae and Schlemm’s canal exist in normal eyes and eyes with primary open-angle glaucoma (POAG). Basement membranes play key roles in the attachment of the overlying trabecular cells and Schlemm’s canal cells. Methods: Electron microscopy used in conjunction with immunogold labeling was used to examine the ultrastructure of the basement membranes in the trabecular meshwork and to determine the presence of collagen IV, laminin, and fibronectin in 6 normal eyes and 6 eyes with POAG. To determine which cells in the meshwork synthesized these molecules in situ hybridization was studied in an additional 8 normal eyes. Results: No distinctive ultrastructural changes were found in the basement membranes of glaucomatous eyes, whether early or advanced disease, when compared with normal eyes. Label for all three proteins was present in the basement membranes of the trabecular lamellae, Schlemm’s canal, and in scattered patches within the juxtacanalicular tissue. Laminin and fibronectin were most abundant in the periphery of the sheath material surrounding the elastic tendons in the juxtacanalicular tissue. In contrast to previously published light microscopic studies, no increase in fibronectin was found in glaucoma. Regions of the basement membrane of the canal underlying giant vacuoles were similar to regions without giant vacuoles in both appearance and labeling. In situ hybridization revealed that mRNA for all three proteins was present in most trabecular cells throughout the meshwork; no regional differences in cellular labeling within were observed. Conclusion: The ultrastructural characteristics and immunogold labeling of basement membranes were similar in normal and glaucomatous eyes; no additional structures were labeled in POAG eyes that were not also labeled in normal eyes. Label of the patches of amorphous fibrogranular material within the juxtacanalicular tissue suggests it is basement membrane in origin, while the sheath material which is known to accumulate in POAG was not heavily labeled and does not appear to be basement membrane in origin.

Adenovirus-Mediated Gene Transfer to Human Cerebral Arteries

Gene therapy is being investigated as a putative treatment option for cardiovascular diseases, including cerebral vasospasm. Because there is presently no information regarding gene transfer to human cerebral arteries, the principal objective of this study was to characterize adenovirus-mediated expression and function of recombinant endothelial nitric oxide synthase (eNOS) gene in human pial arteries. Pial arteries (outer diameter 500 to 1000 μm) were isolated from 30 patients undergoing temporal lobectomy for intractable seizures and were studied using histologic staining, histochemistry, electron microscopy, and isometric force recording. Gene transfer experiments were performed ex vivo using adenoviral vectors encoding genes for bovine eNOS (AdCMVeNOS) and Escherichia coli β-galactosidase (AdCMVLacZ). In transduced arteries, studied 24 hours after exposure to vectors, expression of recombinant β-galactosidase and eNOS was detected by histochemistry, localizing mainly to the adventitia (n = 4). Immunoelectron microscopy localized recombinant eNOS in adventitial fibroblasts. During contractions to U46619, bradykinin-induced relaxations were significantly augmented in AdCMVeNOS-transduced rings compared with control and AdCMVLacZ-transduced rings (P < 0.01; n = 6). The NOS inhibitor L-nitroarginine methylester (L-NAME) caused significantly greater contraction in AdCMVeNOS-transduced rings (P < 0.001; n = 4) and inhibited bradykinin-induced relaxations in control and transduced rings (P < 0.001; n = 6). The current findings suggest that in AdCMVeNOS-transduced human pial arteries, expression of recombinant eNOS occurs mainly in adventitial fibroblasts where it augments relaxations to NO-dependent agonists such as bradykinin. Findings from the current study might be beneficial in future clinical applications of gene therapy for the treatment or prevention of cerebral vasospasm.

Localization of Caveolin 1 in Aortic Valve Endothelial Cells Using Antigen Retrieval

Ultrastructural analysis of aortic valve endothelial cells subjected to growth arrest revealed many vesicles defined as caveolae by the localization of caveolin. Translocation of caveolin after exposure to oxidized LDL suggests that the localization of caveolin may be a valuable tool to study models of early atherogenesis. In this study, several antigen retrieval protocols were tested in osmium-fixed and Spurr-embedded cells to determine the optimal method of antigen retrieval in our model system. SDS produced the most consistent labeling pattern. A quantitative evaluation revealed that SDS significantly increased the labeling density in Spurr-embedded cells. The labeling pattern appeared as clusters of gold particles, 15–40 nm in diameter, that were associated with membranes of a similar size which may represent the neck region of the caveolae.

Antigen Retrieval of Basement Membrane Proteins from Archival Eye Tissues

Antigen retrieval (AR) methods can unmask tissue antigens that have been altered by fixation, processing, storage, or resin interactions. This is particularly important in the study of archival tissues, because primary fixatives and storage times may vary among specimens. We performed an electron microscopic study of basement membrane components of the aqueous humor drainage pathways from archival eye tissue. AR (heated citrate buffer, pH 6.0, LR White resin) increased the amount of label of collagen IV and fibronectin in tissue fixed in four different fixatives, including those containing glutaraldehyde. Labeling density was approximately doubled after AR for most fixatives, with the largest increase for tissues fixed in 4% paraformaldehyde/2% glutaraldehyde. Duration of storage time for archival tissues did not affect AR results. AR did not change the components of the extracellular matrix labeled; no “new” components were labeled after AR. We conclude that AR in citrate buffer can be used on selected extracellular matrix antigens to enhance label that would otherwise be lost due to fixation and storage.