Differential proteome profile, biological pathways, and network relationships of osteogenic proteins in calcified human aortic valves.

Calcific aortic valve disease (CAVD) is the most common heart valve disease requiring intervention. Most research on CAVD has focused on inflammation, ossification, and cellular phenotype transformation. To gain a broader picture into the wide range of cellular and molecular mechanisms involved in this disease, we compared the total protein profiles between calcified and non-calcified areas from 5 human valves resected during surgery. The 1413 positively identified proteins were filtered down to 248 proteins present in both calcified and non-calcified segments of at least 3 of the 5 valves, which were then analyzed using Ingenuity Pathway Analysis. Concurrently, the top 40 differentially abundant proteins were grouped according to their biological functions and shown in interactive networks. Finally, the abundance of selected osteogenic proteins (osteopontin, osteonectin, osteocalcin, osteoprotegerin, and RANK) was quantified using ELISA and/or immunohistochemistry. The top pathways identified were complement system, acute phase response signaling, metabolism, LXR/RXR and FXR/RXR activation, actin cytoskeleton, mineral binding, nucleic acid interaction, structural extracellular matrix (ECM), and angiogenesis. There was a greater abundance of osteopontin, osteonectin, osteocalcin, osteoprotegerin, and RANK in the calcified regions than the non-calcified ones. The osteogenic proteins also formed key connections between the biological signaling pathways in the network model. In conclusion, this proteomic analysis demonstrated the involvement of multiple signaling pathways in CAVD. The interconnectedness of these pathways provides new insights for the treatment of this disease.

Fanconi Anemia DNA Repair Pathway as a New Mechanism to Exploit Cancer Drug Resistance.

Chemotherapy employs anti-cancer drugs to stop the growth of cancerous cells, but one common obstacle to the success is the development of chemoresistance, which leads to failure of the previously effective anti-cancer drugs. Resistance arises from different mechanistic pathways, and in this critical review, we focus on the Fanconi Anemia (FA) pathway in chemoresistance. This pathway has yet to be intensively researched by mainstream cancer researchers. This review aims to inspire a new thrust toward the contribution of the FA pathway to drug resistance in cancer. We believe an indepth understanding of this pathway will open new frontiers to effectively treat drug-resistant cancer.

Morphometric analysis of calcification and fibrous layer thickness in carotid endarterectomy tissues.

BACKGROUND: Advanced atherosclerotic lesions are commonly characterized by the presence of calcification. Several studies indicate that extensive calcification is associated with plaque stability, yet recent studies suggest that calcification morphology and location may adversely affect the mechanical stability of atherosclerotic plaques. The underlying cause of atherosclerotic calcification and the importance of intra-plaque calcium distribution remains poorly understood. METHOD: The goal of this study was the characterization of calcification morphology based on histological features in 20 human carotid endarterectomy (CEA) specimens. Representative frozen sections (10µm thick) were cut from the common, bulb, internal and external segments of CEA tissues and stained with von Kossa׳s reagent for calcium phosphate. The morphology of calcification (calcified patches) and fibrous layer thickness were quantified in 135 histological sections. RESULTS: Intra-plaque calcification was distributed heterogeneously (calcification %-area: bulb segment: 14.2±2.1%; internal segment: 12.9±2.8%; common segment: 4.6±1.1%; p=0.001). Calcified patches were found in 20 CEAs (patch size: <0.1mm(2) to >1.0mm(2)). Calcified patches were most abundant in the bulb and least in the common segment (bulb n=7.30±1.08; internal n=4.81±1.17; common n=2.56±0.56; p=0.0007). Calcified patch circularity decreased with increasing size (<0.1mm(2): 0.77±0.01, 0.1-1mm(2): 0.62±0.01, >1.0mm(2): 0.51±0.02; p=0.0001). A reduced fibrous layer thickness was associated with increased calcium patch size (p<0.0001). CONCLUSIONS: In advanced carotid atherosclerosis, calcification appears to be a heterogeneous and dynamic atherosclerotic plaque component, as indicated by the simultaneous presence of few large stabilizing calcified patches and numerous small calcific patches. Future studies are needed to elucidate the associations of intra-plaque calcification size and distribution with atherothrombotic events.

Differential Aortic and Mitral Valve Interstitial Cell Mineralization and the Induction of Mineralization by Lysophosphatidylcholine In Vitro.

PURPOSE: Calcific aortic valve disease (CAVD) is a serious condition with vast uncertainty regarding the precise mechanism leading to valve calcification. This study was undertaken to examine the role of the lipid lysophosphatidylcholine (LPC) in a comparison of aortic and mitral valve cellular mineralization. METHODS: The proportion of LPC in differentially calcified regions of diseased aortic valves was determined using thin layer chromatography (TLC). Next, porcine valvular interstitial cells (pVICs) from the aortic (paVICs) and mitral valve (pmVICs) were cultured with LPC (10-1 - 105 nM) and analyzed for cellular mineralization, alkaline phosphatase activity (ALPa), proliferation, and apoptosis. RESULTS: TLC showed a higher percentage of LPC in calcified regions of tissue compared to non-calcified regions. In pVIC cultures, with the exception of 105 nM LPC, increasing concentrations of LPC led to an increase in phosphate mineralization. Increased levels of calcium content were exhibited at 104 nm LPC application compared to baseline controls. Compared to pmVIC cultures, paVIC cultures had greater total phosphate mineralization, ALPa, calcium content, and apoptosis, under both a baseline control and LPC-treated conditions. CONCLUSIONS: This study showed that LPC has the capacity to promote pVIC calcification. Also, paVICs have a greater propensity for mineralization than pmVICs. LPC may be a key factor in the transition of the aortic valve from a healthy to diseased state. In addition, there are intrinsic differences that exist between VICs from different valves that may play a key role in heart valve pathology.

Distribution of calcification in carotid endarterectomy tissues: comparison of micro-computed tomography imaging with histology.

BACKGROUND: Calcification in atherosclerotic plaques has been viewed as a marker of plaque stability, but whether calcification accumulates in specific anatomic sites in the carotid artery is unknown. We determined the burden and distribution of calcified plaque in carotid endarterectomy (CEA) tissues. METHODS: A total of 22 CEA tissues were imaged with high-resolution micro-computed tomography (micro-CT). Total plaque burden and total calcium score using the Agatston method were quantified. The Agatston score (AS) was also normalized for tissue size. Plaque and calcium distribution were analyzed separately for three CEA regions: common segment (CS), bulb segment (BS), and internal/external segments (IES). RESULTS: The average CEA tissue length was 40.83 (interquartile range [IQR] 33.31-42.41) mm with total plaque burden of 103.45 (IQR: 78.84-156.81) mm(3) and total AS of 38.58 (IQR 11.59-89.97). Total plaque volume was 21.02 (IQR: 14.47-25.42) mm(3) in the CS, 37.89 (22.59-48.32) mm(3) in the BS, and 54.05 (36.87-74.52) mm(3) in the IES. Of the 22 tissues, 15 had no calcium in the CS compared with three in the bulb and two in the IES. Normalized calcified plaque was most prevalent in the BS, the IES and was least prevalent in the CS. The overall correlation of calcification between histology sections and matched micro-CT images was 0.86 (p<0.001). CONCLUSIONS: Calcified plaque is heterogeneously distributed in CEA tissues with most in the bulb and IES regions. The amount of calcification in micro-CT slices shows a high correlation with matched histology sections.

Differentiating the aging of the mitral valve from human and canine myxomatous degeneration.

During the course of both canine and human aging, the mitral valve remodels in generally predictable ways. The connection between these aging changes and the morbidity and mortality that accompany pathologic conditions has not been made clear. By exploring work that has investigated the specific valvular changes in both age and disease, with respect to the cells and the extracellular matrix found within the mitral valve, heretofore unexplored connections between age and myxomatous valve disease can be found. This review addresses several studies that have been conducted to explore such age and disease related changes in extracellular matrix, valvular endothelial and interstitial cells, and valve innervation, and also reviews attempts to correlate aging and myxomatous disease. Such connections can highlight avenues for future research and help provide insight as to when an individual diverts from an aging pattern into a diseased pathway. Recognizing these patterns and opportunities could result in earlier intervention and the hope of reduced morbidity and mortality for patients.

Structural and cellular changes in canine myxomatous mitral valve disease: an image analysis study.

BACKGROUND AND AIM OF THE STUDY: Myxomatous mitral valve disease (MMVD) is the single most common cardiac disease of the dog, and bears close similarities to chronic degenerative mitral valve disease in humans. However, limited quantitative data are available on cellular and morphological changes in both species. The study aim was to use an image analysis system to examine various morphological changes associated with MMVD, and in particular to measure changes in cell numbers in overtly myxomatous areas of the distal portion of the valve. METHODS: Mitral valve complexes were collected from normal dogs and dogs with varying severity of myxomatous mitral valve disease (veterinary Whitney grades 1-4; a measure of disease severity and age-related disease progression in the dog). An image analysis technique (ImageJ; National Institutes of Health, USA) was used to measure valve leaflet length, thickness, connective tissue content and density, glycosaminoglycan (GAG) content, cell number and shape in normal and myxomatous areas of diseased valves. RESULTS: There was a change in the valve leaflet anterior/posterior length ratio in the diseased valves, suggestive of valve lengthening. Distinct and statistically significant (p < 0.01) changes occurred in the valve thickness ratio for both anterior and posterior leaflets as the disease progressed, and the posterior leaflet thickness ratios were consistently higher than for the anterior leaflets. There was a statistically significant decrease in cell numbers in overtly myxomatous areas of the distal portion of affected valves compared to similar locations in normal valves, but there was no difference between the different grades of disease. The majority of cells in both diseased and normal valves had a circularity score typical of a spindle (elongated) shape. Connective tissue derangement was clearly seen in the myxomatous areas, and this was associated with a significant reduction in connective tissue density. The reduction in connective tissue density was associated with advancing disease severity (age). There was an increase in GAG expression with disease severity, as shown by the level of Alcian blue staining, but this could not be quantified with ImageJ. CONCLUSION: Mitral valve myxomatous degeneration in the dog is associated with lengthening and thickening of valve leaflets, a loss of connective tissue, and a decrease in cell numbers in selected myxomatous areas, but no change in cell circularity. Some of these changes were age- (disease severity-) related.

Evaluation of innervation of the mitral valves and the effects of myxomatous degeneration in dogs.

OBJECTIVE: To map aspects of the innervation of the mitral valve complex and determine any association with the development or progression of myxomatous mitral valve disease (MMVD) in dogs. SAMPLE POPULATION: Septal mitral valve leaflets from 11 dogs aged 6 months to > 10 years. PROCEDURES: Expression of protein gene product 9.5 (general neuronal marker), tyrosine hydroxylase (adrenergic innervation marker), vasoactive intestinal peptide (parasympathetic innervation marker), and calcitonin gene-related peptide (sensory innervation marker) was assessed by use of a standard immunohistochemical technique. Innervation was assessed qualitatively and semiquantitatively. Differences between valvular zones and between groups were analyzed statistically. RESULTS: MMVD was present in leaflets of all dogs > or = 5 years of age. Innervation was confirmed in all leaflets but was markedly reduced in leaflets of dogs > 10 years of age. Innervation was most dense at the base of valves and mainly associated with the epimysial, perimysial, and endomysial layers of the muscle and blood vessels within the valve. Innervation was reduced within the middle zone of the valve and lacking at the free edge. Innervation was not identified at the tip of the leaflet, the free edge, or the chordae. Nerve fibers were mostly sympathetic, with the remainder being parasympathetic or sensory. Existence of MMVD did not alter the pattern or density of innervation. CONCLUSIONS AND CLINICAL RELEVANCE: Mitral valve leaflets in the study dogs were innervated, with most of the nerve fibers associated with the myocardium in the valve base. Development of MMVD appeared to precede the reduction of innervation associated with advancing age.

Distribution of myofibroblasts, smooth muscle-like cells, macrophages, and mast cells in mitral valve leaflets of dogs with myxomatous mitral valve disease.

OBJECTIVE: To map the cellular distribution and phenotypic alteration of the predominant stromal cell population throughout the entire valve length of dogs with myxomatous mitral valve disease (MMVD). SAMPLE POPULATION: 31 mitral valve complexes (ie, mitral valve leaflets) collected from 4 clinically normal dogs and 27 dogs with MMVD of varying severity. PROCEDURES: A combination of standard histologic and immunohistochemical techniques was used to identify pathologic changes, the presence of mast cells, and the density and distribution of cells expressing vimentin, desmin, alpha-smooth muscle actin (alpha-SMA), smooth muscle myosin, and the macrophage marker MAC387. RESULTS: Vimentin-positive cells predominated in the mitral valve leaflets from clinically normal dogs and were located throughout the leaflet, but cell density was appreciably decreased with disease progression, and minimal cell numbers were found in distinct myxomatous areas. Cells that were positive for alpha-SMA were uncommon in the mitral valve leaflets from clinically normal dogs and only seen in appreciable numbers in mitral valves of dogs with severe late-stage disease, in which cells were typically located close to the ventricularis valve surface. A slight increase in mast cell numbers was observed in the distal zone of affected leaflets. CONCLUSIONS AND CLINICAL RELEVANCE: Activated-myofibroblasts (alpha-SMA-positive cells) were increased and inactive-myofibroblasts (vimentin-positive cells) were reduced in mitral valve leaflets of dogs with MMVD, compared with that of clinically normal dogs. Impact on Human Medicine-This is the first description of spatial and temporal alterations in mitral valve cells of any species with MMVD and has clinical importance in the understanding of disease development in dogs and humans.

Collagen organization in canine myxomatous mitral valve disease: an x-ray diffraction study.

Collagen fibrils, a major component of mitral valve leaflets, play an important role in defining shape and providing mechanical strength and flexibility. Histopathological studies show that collagen fibrils undergo dramatic changes in the course of myxomatous mitral valve disease in both dogs and humans. However, little is known about the detailed organization of collagen in this disease. This study was designed to analyze and compare collagen fibril organization in healthy and lesional areas of myxomatous mitral valves of dogs, using synchrotron small-angle x-ray diffraction. The orientation, density, and alignment of collagen fibrils were mapped across six different valves. The findings reveal a preferred collagen alignment in the main body of the leaflets between two commissures. Qualitative and quantitative analysis of the data showed significant differences between affected and lesion-free areas in terms of collagen content, fibril alignment, and total tissue volume. Regression analysis of the amount of collagen compared to the total tissue content at each point revealed a significant relationship between these two parameters in lesion-free but not in affected areas. This is the first time this technique has been used to map collagen fibrils in cardiac tissue; the findings have important applications to human cardiology.