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1.  Enrichment of calcifying extracellular vesicles using density-based ultracentrifugation protocol 
Journal of Extracellular Vesicles  2014;3:10.3402/jev.v3.25129.
Calcifying extracellular vesicles (EVs) released from cells within atherosclerotic plaques have received increased attention for their role in mediating vascular calcification, a major predictor of cardiovascular morbidity and mortality. However, little is known about the difference between this pathologic vesicle population and other EVs that contribute to physiological cellular processes. One major challenge that hinders research into these differences is the inability to selectively isolate calcifying EVs from other vesicle populations. In this study, we hypothesized that the formation of mineral within calcifying EVs would increase the density of the vesicles such that they would pellet at a faster rate during ultracentrifugation. We show that after 10 min of ultracentrifugation at 100,000×g, calcifying EVs are depleted from the conditioned media of calcifying coronary artery smooth muscle cells and are enriched in the pelleted portion. We utilized mass spectrometry to establish functional proteomic differences between the calcifying EVs enriched in the 10 min ultracentrifugation compared to other vesicle populations preferentially pelleted by longer ultracentrifugation times. The procedures established in this study will allow us to enrich the vesicle population of interest and perform advanced proteomic analyses to find subtle differences between calcifying EVs and other vesicle populations that may be translated into therapeutic targets for vascular calcification. Finally, we will show that the differences in ultracentrifugation times required to pellet the vesicle populations can also be used to estimate physical differences between the vesicles.
PMCID: PMC4261240  PMID: 25491249
calcification; extracellular vesicles; atherosclerosis; ultracentrifugation; isolation
2.  The Role of Extracellular Vesicles in De Novo Mineralization: An Additional Novel Mechanism of Cardiovascular Calcification 
Extracellular vesicles are membrane micro/nanovesicles secreted by many cell types into the circulation and the extracellular milieu in physiological and pathological conditions. Evidence suggests that extracellular vesicles, known as matrix vesicles, play a role in the mineralization of skeletal tissue, but emerging ultrastructural and in vitro studies have demonstrated their contribution to cardiovascular calcification as well. Cells involved in the progression of cardiovascular calcification release active vesicles capable of nucleating hydroxyapatite on their membranes. This review discusses the role of extracellular vesicles in cardiovascular calcification, and elaborates on this additional mechanism of calcification as an alternative pathway to the currently accepted mechanism of biomineralization via osteogenic differentiation.
PMCID: PMC3788633  PMID: 23766262
3.  Macrophage-Derived Matrix Vesicles: An Alternative Novel Mechanism for Microcalcification in Atherosclerotic Plaques 
Circulation research  2013;113(1):72-77.
We previously showed that early calcification of atherosclerotic plaques associates with macrophage accumulation. Chronic renal disease (CRD) and mineral imbalance accelerates calcification and the subsequent release of matrix vesicles (MVs) — precursors of microcalcification.
We tested the hypothesis that macrophage-derived MVs contribute directly to microcalcification.
Methods and Results
Macrophages associated with regions of calcified vesicular structures in human carotid plaques (n=136 patients). In vitro, macrophages released MVs with high calcification and aggregation potential. MVs expressed exosomal markers (CD9 and TSG101), and contained S100A9 and annexin V (Anx5). Silencing S100A9 in vitro and genetic deficiency in S100A9−/− mice reduced MV calcification, while stimulation with S100A9 increased calcification potential. Externalization of phosphatidylserine (PS) after Ca/P stimulation and interaction of S100A9 and Anx5, indicated that a PS-Anx5-S100A9 membrane complex facilitates hydroxyapatite nucleation within the macrophage-derived MV membrane.
Our results support the novel concept that macrophages release calcifying MVs enriched in S100A9 and Anx5, which contribute to accelerated microcalcification in CRD.
PMCID: PMC3703850  PMID: 23616621
Extracellular vesicles; macrophages; calcification; chronic renal disease; atherosclerosis; inflammation; plaque rupture
4.  Myeloperoxidase-targeted imaging of active inflammatory lesions in murine experimental autoimmune encephalomyelitis 
Brain : a journal of neurology  2008;131(0 4):1123-1133.
Inflammatory demyelinating plaques are the pathologic hallmark of active multiple sclerosis and often precede clinical manifestations. Noninvasive early detection of active plaques would thus be crucial in establishing presymptomatic diagnosis and could lead to early preventive treatment strategies. Using murine experimental autoimmune encephalomyelitis as a model of multiple sclerosis, we demonstrate that a prototype paramagnetic myeloperoxidase (MPO) sensor can detect and confirm more, smaller, and earlier active inflammatory lesions in living mice by in vivo magnetic resonance imaging (MRI). We show that MPO expression corresponded with areas of inflammatory cell infiltration and demyelination, and higher MPO activity as detected by MPO imaging, biochemical assays, and histopathological analyses correlated with increased clinical disease severity. Our findings present a potential new translational approach for specific noninvasive inflammatory plaque imaging. This approach could be used in longitudinal studies to identify active demyelinating plaques as well as to more accurately track disease course following treatment in clinical trials.
PMCID: PMC4044727  PMID: 18234693
myeloperoxidase; neuroinflammation; demyelination; targeted imaging; MRI
5.  Visualizing novel concepts of cardiovascular calcification 
Cardiovascular calcification is currently viewed as an active disease process similar to embryonic bone formation. Cardiovascular calcification mainly affects the aortic valve and arteries and is associated with increased mortality risk. Aortic valve and arterial calcification share similar risk factors, including age, gender, diabetes, chronic renal disease, and smoking. However, the exact cellular and molecular mechanism of cardiovascular calcification is unknown. Late-stage cardiovascular calcification can be visualized with conventional imaging modalities such as echocardiography and computed tomography. However, these modalities are limited in their ability to detect the development of early calcification and the progression of calcification until advanced tissue mineralization is apparent. Due to the subsequent late diagnosis of cardiovascular calcification, treatment is usually comprised of invasive interventions such as surgery. The need to understand the process of calcification is therefore warranted and requires new imaging modalities which are able to visualize early cardiovascular calcification. This review focuses on the use of new imaging techniques to visualize novel concepts of cardiovascular calcification.
PMCID: PMC3626075  PMID: 23290463
6.  MicroRNA in cardiovascular calcification: Focus on targets and extracellular vesicle delivery mechanisms 
Circulation research  2013;112(7):1073-1084.
Cardiovascular calcification is a prominent feature of chronic inflammatory disorders — such as chronic kidney disease (CKD), type 2 diabetes (T2D), and atherosclerosis — that associate with significant morbidity and mortality. The concept that similar pathways control both bone remodeling and vascular calcification is widely accepted, but the precise mechanisms of calcification remain largely unknown. The central role of microRNAs (miRNA) as fine-tune regulators in the cardiovascular system and bone biology has gained acceptance and has raised the possibility for novel therapeutic targets. Additionally, circulating miRNAs have been proposed as biomarkers for a wide range of cardiovascular diseases, but knowledge of miRNA biology in cardiovascular calcification is very limited. This review focuses on the role of miRNA in cardiovascular disease, with emphasis on osteogenic processes. Herein, we discuss the current understanding of miRNAs in cardiovascular calcification. Furthermore, we identify a set of miRNAs common to diseases associated with cardiovascular calcification (CKD, T2D, and atherosclerosis), and we hypothesize that these miRNAs may provide a molecular signature for calcification. Finally, we discuss this novel hypothesis with emphasis on known biological and pathological osteogenic processes (e.g. osteogenic differentiation, release of calcifying matrix vesicles). The aim of this review is to provide an organized discussion of the known links between miRNA and calcification that provide emerging concepts for future studies on miRNA biology in cardiovascular calcification, which will be critical for developing new therapeutic strategies.
PMCID: PMC3668680  PMID: 23538277
miRNA; circulating miRNA; cardiovascular calcification; extracellular vesicles; cardiovascular disease; type 2 diabetes mellitus; chronic kidney disease; aortic stenosis; calcification; osteogenic; bone
7.  A Novel Quantitative Approach for Eliminating Sample-To-Sample Variation Using a Hue Saturation Value Analysis Program 
PLoS ONE  2014;9(3):e89627.
As computing technology and image analysis techniques have advanced, the practice of histology has grown from a purely qualitative method to one that is highly quantified. Current image analysis software is imprecise and prone to wide variation due to common artifacts and histological limitations. In order to minimize the impact of these artifacts, a more robust method for quantitative image analysis is required.
Methods and Results
Here we present a novel image analysis software, based on the hue saturation value color space, to be applied to a wide variety of histological stains and tissue types. By using hue, saturation, and value variables instead of the more common red, green, and blue variables, our software offers some distinct advantages over other commercially available programs. We tested the program by analyzing several common histological stains, performed on tissue sections that ranged from 4 µm to 10 µm in thickness, using both a red green blue color space and a hue saturation value color space.
We demonstrated that our new software is a simple method for quantitative analysis of histological sections, which is highly robust to variations in section thickness, sectioning artifacts, and stain quality, eliminating sample-to-sample variation.
PMCID: PMC3940696  PMID: 24595280
9.  Medial and Intimal Calcification in Chronic Kidney Disease: Stressing the Contributions 
PMCID: PMC3835265  PMID: 24060959
Editorials; atherosclerosis; chronic kidney disease; endoplasmic reticulum stress; inflammation; intimal and medial calcification
10.  Inhibition of bone morphogenetic protein signaling reduces vascular calcification and atherosclerosis 
Arteriosclerosis, thrombosis, and vascular biology  2012;32(3):10.1161/ATVBAHA.111.242594.
The expression of bone morphogenetic proteins (BMPs) is enhanced in human atherosclerotic and calcific vascular lesions. While genetic gain- and loss-of-function experiments in mice have supported a causal role of BMP signaling in atherosclerosis and vascular calcification, it remains uncertain whether BMP signaling might be targeted pharmacologically to ameliorate both of these processes.
Methods and Results
We tested the impact of pharmacologic BMP inhibition upon atherosclerosis and calcification in low density lipoprotein receptor-deficient (LDLR−/−) mice. LDLR−/− mice fed a high-fat diet developed abundant vascular calcification within twenty weeks. Prolonged treatment of LDLR−/− mice with the small molecule BMP inhibitor LDN-193189 was well-tolerated and potently inhibited development of atheroma, as well as associated vascular inflammation, osteogenic activity, and calcification. Administration of recombinant BMP antagonist ALK3-Fc replicated the anti-atherosclerotic and anti-inflammatory effects of LDN-193189. Treatment of human aortic endothelial cells with LDN-193189 or ALK3-Fc abrogated the production of reactive oxygen species (ROS) induced by oxidized LDL, a known early event in atherogenesis. Unexpectedly, treatment of mice with LDN-193189 lowered LDL serum cholesterol by 35% and markedly decreased hepatosteatosis without inhibiting HMG-CoA reductase activity. Treatment with BMP2 increased, whereas LDN-193189 or ALK3-Fc inhibited apolipoprotein B100 secretion in HepG2 cells, suggesting that BMP signaling contributes to the regulation of cholesterol biosynthesis.
These results definitively implicate BMP signaling in atherosclerosis and calcification, while uncovering a previously unidentified role for BMP signaling in LDL cholesterol metabolism. BMP inhibition may be helpful in the treatment of atherosclerosis and associated vascular calcification.
PMCID: PMC3679546  PMID: 22223731
atherosclerosis; inflammation; lipoproteins; hypercholesterolemia
11.  Elastogenesis at the onset of human cardiac valve development 
Development (Cambridge, England)  2013;140(11):2345-2353.
Semilunar valve leaflets have a well-described trilaminar histoarchitecture, with a sophisticated elastic fiber network. It was previously proposed that elastin-containing fibers play a subordinate role in early human cardiac valve development; however, this assumption was based on data obtained from mouse models and human second and third trimester tissues. Here, we systematically analyzed tissues from human fetal first (4-12 weeks) and second (13-18 weeks) trimester, adolescent (14-19 years) and adult (50-55 years) hearts to monitor the temporal and spatial distribution of elastic fibers, focusing on semilunar valves. Global expression analyses revealed that the transcription of genes essential for elastic fiber formation starts early within the first trimester. These data were confirmed by quantitative PCR and immunohistochemistry employing antibodies that recognize fibronectin, fibrillin 1, 2 and 3, EMILIN1 and fibulin 4 and 5, which were all expressed at the onset of cardiac cushion formation (~week 4 of development). Tropoelastin/elastin protein expression was first detectable in leaflets of 7-week hearts. We revealed that immature elastic fibers are organized in early human cardiovascular development and that mature elastin-containing fibers first evolve in semilunar valves when blood pressure and heartbeat accelerate. Our findings provide a conceptual framework with the potential to offer novel insights into human cardiac valve development and disease.
PMCID: PMC3912871  PMID: 23637335
Heart; Elastin; Elastic fibers; Extracellular matrix; Heart valves
13.  Vesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation 
PLoS Biology  2012;10(12):e1001450.
Vesiclepedia is a community-annotated compendium of molecular data on extracellular vesicles.
Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.
PMCID: PMC3525526  PMID: 23271954
14.  Selective inhibition of matrix metalloproteinase 13 (MMP-13) increases collagen content of established mouse atheromata 
Evidence has linked collagen loss with the onset of acute coronary events.
This study tested the hypothesis that selective MMP-13 collagenase inhibition increases collagen content in already established and nascent mouse atheromata.
Methods and Results
In vitro and in situ experiments documented the selectivity and efficacy of an orally available MMP-13 inhibitor (MMP13i-A). In vivo observations monitored macrophage accumulation and MMP-13 activity using molecular imaging. After 10 weeks of MMP13i-A treatment, apoE-/- mice with evolving or established lesions exhibited reduced MMP-13 activity without affecting macrophage content, measured either by intravital microscopy or fluorescence reflectance imaging. Histological analysis indicated that MMP13-iA did not affect plaque size, or macrophage or smooth-muscle cell accumulation. Administration of MMP13i-A to mice with evolving or established atheromata substantially increased plaque interstitial collagen content in the intima and locally in the fibrous cap, compared to vehicle-treated controls. Analysis of collagen revealed thicker collagen fibers within the plaques of treated groups.
Pharmacological MMP-13 inhibition yields collagen accumulation in plaques (a feature associated in humans with resistance to rupture), even in established plaques. This study of considerable clinical relevance furnishes new mechanistic insight into regulation of the plaque's extracellular matrix, and validates molecular imaging for studying plaque biology.
PMCID: PMC3200308  PMID: 21903941
atherosclerosis; MMP-13; inhibitor; collagen; molecular imaging
16.  Statins suppress apolipoprotein CIII-induced vascular endothelial cell activation and monocyte adhesion 
European Heart Journal  2012;34(8):615-624.
Activation of vascular endothelial cells (ECs) contributes importantly to inflammation and atherogenesis. We previously reported that apolipoprotein CIII (apoCIII), found abundantly on circulating triglyceride-rich lipoproteins, enhances adhesion of human monocytes to ECs in vitro. Statins may exert lipid-independent anti-inflammatory effects. The present study examined whether statins suppress apoCIII-induced EC activation in vitro and in vivo.
Methods and results
Physiologically relevant concentrations of purified human apoCIII enhanced attachment of the monocyte-like cell line THP-1 to human saphenous vein ECs (HSVECs) or human coronary artery ECs (HCAECs) under both static and laminar shear stress conditions. This process mainly depends on vascular cell adhesion molecule-1 (VCAM-1), as a blocking VCAM-1 antibody abolished apoCIII-induced monocyte adhesion. ApoCIII significantly increased VCAM-1 expression in HSVECs and HCAECs. Pre-treatment with statins suppressed apoCIII-induced VCAM-1 expression and monocyte adhesion, with two lipophilic statins (pitavastatin and atorvastatin) exhibiting inhibitory effects at lower concentration than those of hydrophilic pravastatin. Nuclear factor κB (NF-κB) mediated apoCIII-induced VCAM-1 expression, as demonstrated via loss-of-function experiments, and pitavastatin treatment suppressed NF-κB activation. Furthermore, in the aorta of hypercholesterolaemic Ldlr−/− mice, pitavastatin administration in vivo suppressed VCAM-1 mRNA and protein, induced by apoCIII bolus injection. Similarly, in a subcutaneous dorsal air pouch mouse model of leucocyte recruitment, apoCIII injection induced F4/80+ monocyte and macrophage accumulation, whereas pitavastatin administration reduced this effect.
These findings further establish the direct role of apoCIII in atherogenesis and suggest that anti-inflammatory effects of statins could improve vascular disease in the population with elevated plasma apoCIII.
PMCID: PMC3578265  PMID: 22927557
Apolipoprotein CIII; Vascular endothelial cells; Monocytes; HMG-CoA reductase inhibitors; Atherosclerosis
17.  Cardiovascular Inflammation 
PMCID: PMC3317089  PMID: 22518345
18.  In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation 
Nature medicine  2011;17(9):1142-1146.
Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where (together with fibrin and platelets) it initiates the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin detected S. aureus in endocarditic vegetations via noninvasive fluorescence or PET imaging. These prothrombin derivatives bound to staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.
PMCID: PMC3169740  PMID: 21857652
endocarditis; staphylocoagulase; prothrombin; noninvasive imaging; von Willebrand factor binding protein
19.  Mitral Valve Endothelial Cells With Osteogenic Differentiation Potential 
Cardiac valvular endothelium is unique in its ability to undergo endothelial-to-mesenchymal transformation, a differentiation process that is essential for valve development and has been proposed as mechanism for replenishing the interstitial cells of mature valves. We hypothesized that the valvular endothelium contains endothelial cells that are direct precursors to osteoblastic valvular interstitial cells (VICs).
Methods and Results
Clonal cell populations from ovine mitral valve leaflets were isolated by single cell plating. Mitral valvular endothelial and mesenchymal clones were tested for osteogenic, adipogenic, and chondrogenic differentiation, determined by the expression of lineage-specific markers. Mitral valvular endothelial clones showed a propensity for osteogenic, as well as chondrogenic differentiation that was comparable to a mitral valvular VIC clone and to bone marrow–derived mesenchymal stem cells. Osteogenic differentiation was not detected in nonvalvular endothelial cells. Regions of osteocalcin expression, a marker of osteoblastic differentiation, were detected along the endothelium of mitral valves that had been subjected in vivo to mechanical stretch.
Mitral valve leaflets contain endothelial cells with multilineage mesenchymal differentiation potential, including osteogenic differentiation. This unique feature suggests that postnatal mitral valvular endothelium harbors a reserve of progenitor cells that can contribute to osteogenic and chondrogenic VICs.
PMCID: PMC3210435  PMID: 21164078
endothelium; heart valves; vascular biology; EMT; endothelial cells; mitral valve; osteogenic differentiation
20.  Indocyanine Green Enables Near-Infrared Fluorescence Imaging of Lipid-Rich, Inflamed Atherosclerotic Plaques 
Science translational medicine  2011;3(84):84ra45.
New high-resolution molecular and structural imaging strategies are needed to visualize high-risk plaques that are likely to cause acute myocardial infarction, because current diagnostic methods do not reliably identify at-risk subjects. While molecular imaging agents are available for lower-resolution detection of atherosclerosis in large arteries, a lack of imaging agents coupled to high-resolution modalities has limited molecular imaging of atherosclerosis in the smaller coronary arteries [AU: ok? YES]. Here, we have demonstrated that indocyanine green (ICG), an FDA-approved near-infrared fluorescence (NIRF) emitting compound, targets atheromas within 20 minutes of injection and provides sufficient signal enhancement for in vivo detection of lipid-rich, inflamed, coronary-sized plaques in atherosclerotic rabbits. In vivo NIRF sensing was achieved with an intravascular wire in the aortae, a vessel of comparable caliber to human coronary arteries. Ex vivo fluorescence reflectance imaging studies showed high plaque target-to-background ratios in atheroma-bearing rabbits injected with ICG, compared to atheroma-bearing rabbits injected with saline. In vitro studies using human macrophages established that ICG preferentially targets lipid-loaded macrophages. In an early clinical study of human atheroma specimens from four patients, we found that ICG colocalized with plaque macrophages and lipids. The atheroma-targeting capability of ICG has the potential to accelerate the clinical development of NIRF molecular imaging of high-risk plaques in humans.
PMCID: PMC3112179  PMID: 21613624
21.  Pioglitazone Suppresses Inflammation In Vivo In Murine Carotid Atherosclerosis: Novel Detection by Dual-Target Fluorescence Molecular Imaging 
Anti-inflammatory actions of peroxisome proliferator-activated receptor (PPAR)-γ agonists such as pioglitazone (PIO) may underlie their reported but incompletely understood repression of atherosclerosis. This molecular imaging study investigated the effects of pioglitazone on plaque matrix metalloproteinase (MMP) and macrophage responses in vivo.
Methods and Results
In vitro, pioglitazone suppressed MMP-9 mRNA expression in murine peritoneal macrophages (P<0.05). To assess pioglitazone's effects on plaque inflammation, nondiabetic apoE−/− mice on high-cholesterol diet (HCD) received a MMP-activatable fluorescence imaging agent and a spectrally-distinct macrophage-avid fluorescent nanoparticle. After 24 hours, mice underwent survival dual-target intravital fluorescence microscopy (IVFM) of carotid arterial plaques. These mice were then randomized to HCD or HCD+PIO 0.012% for 8 weeks, followed by a second IVFM study of the same carotid plaque. In the HCD group, in vivo MMP and macrophage target-to-background ratios (TBRs) increased similarly (P<0.01 vs. baseline). In contrast, pioglitazone reduced MMP and macrophage TBRs (P<0.01 vs. HCD). Changes in MMP and macrophage signals correlated strongly (r-values≥0.75). Microscopy demonstrated MMP and macrophage reductions in pioglitazone-treated mice, as well as a PIO-modulated increase in plaque collagen.
Serial optical molecular imaging demonstrates that plaque MMP and macrophage activity in vivo intensify with hypercholesterolemia and are reduced by pioglitazone therapy.
PMCID: PMC3030475  PMID: 20689078
atherosclerosis; pioglitazone; inflammation; molecular imaging; fluorescence
22.  Molecular Imaging Insights into Early Inflammatory Stages of Arterial and Aortic Valve Calcification 
Circulation research  2011;108(11):1381-1391.
Traditional imaging modalities such as computed tomography, although perfectly adept at identifying and quantifying advanced calcification, cannot detect the early stages of this disorder and offer limited insight into the mechanisms of mineral dysregulation. This review presents optical molecular imaging as a promising tool that simultaneously detects pathobiological processes associated with inflammation and early stages of calcification in vivo at the (sub)cellular levels.
Research into treatment of cardiovascular calcification is lacking, as shown by clinical trials that have failed to demonstrate the reduction of calcific aortic stenosis. Hence the need to elucidate the pathways that contribute to cardiovascular calcification and to develop new therapeutic strategies to prevent or reverse calcification has driven investigations into the use of molecular imaging. This review discusses studies that have used molecular imaging methods to advance knowledge of cardiovascular calcification, focusing in particular on the inflammation-dependent mechanisms of arterial and aortic valve calcification.
PMCID: PMC3139950  PMID: 21617135
Aortic valve; atherosclerosis; inflammation; calcification; molecular imaging
23.  Fibroblast activation protein is induced by inflammation and degrades type I collagen in thin-cap fibroatheromata 
European Heart Journal  2011;32(21):2713-2722.
Collagen degradation in atherosclerotic plaques with thin fibrous caps renders them more prone to rupture. Fibroblast activation protein (FAP) plays a role in arthritis and tumour formation through its collagenase activity. However, the significance of FAP in thin-cap human fibroatheromata remains unknown.
Methods and results
We detected enhanced FAP expression in type IV–V human aortic atheromata (n = 12), compared with type II–III lesions (n = 9; P < 0.01) and healthy aortae (n = 8; P < 0.01) by immunostaining and western blot analyses. Fibroblast activation protein was also increased in thin-cap (<65 µm) vs. thick-cap (≥65 µm) human coronary fibroatheromata (n = 12; P < 0.01). Fibroblast activation protein was expressed by human aortic smooth muscle cells (HASMC) as shown by colocalization on immunofluorescent aortic plaque stainings (n = 10; P < 0.01) and by flow cytometry in cell culture. Although macrophages did not express FAP, macrophage burden in human aortic plaques correlated with FAP expression (n = 12; R2= 0.763; P < 0.05). Enzyme-linked immunosorbent assays showed a time- and dose-dependent up-regulation of FAP in response to human tumour necrosis factor α (TNFα) in HASMC (n = 6; P < 0.01). Moreover, supernatants from peripheral blood-derived macrophages induced FAP expression in cultured HASMC (n = 6; P < 0.01), an effect abolished by blocking TNFα (n = 6; P < 0.01). Fibroblast activation protein associated with collagen-poor regions in human coronary fibrous caps and digested type I collagen and gelatin in vitro (n = 6; P < 0.01). Zymography revealed that FAP-mediated collagenase activity was neutralized by an antibody directed against the FAP catalytic domain both in HASMC (n = 6; P < 0.01) and in fibrous caps of atherosclerotic plaques (n = 10; P < 0.01).
Fibroblast activation protein expression in HASMC is induced by macrophage-derived TNFα. Fibroblast activation protein associates with thin-cap human coronary fibroatheromata and contributes to type I collagen breakdown in fibrous caps.
PMCID: PMC3205479  PMID: 21292680
Atherosclerosis; Antibodies; Collagen; Inflammation; Smooth muscle cells
Biomaterials  2009;31(6):1114.
We have previously shown that combined flexure and flow (CFF) augment engineered heart valve tissue formation using bone marrow-derived mesenchymal stem cells (MSC) seeded on polyglycolic acid (PGA)/poly-L-lactic acid (PLLA) blend nonwoven fibrous scaffolds (Engelmayr, et al., Biomaterials 2006; vol. 27 pp. 6083–95). In the present study, we sought to determine if these phenomena were reproducible at the organ level in a functional tri-leaflet valve. Tissue engineered valve constructs (TEVC) were fabricated using PGA/PLLA nonwoven fibrous scaffolds then seeded with MSCs. Tissue formation rates using both standard and augmented (using basic fibroblast growth factor [bFGF] and ascorbic acid-2-phosphate [AA2P]) media to enhance the overall production of collagen were evaluated, along with their relation to the local fluid flow fields. The resulting TEVCs were statically cultured for 3 weeks, followed by a 3 week dynamic culture period using our organ level bioreactor (Hildebrand et al., ABME, Vol. 32, pp. 1039–49, 2004) under approximated pulmonary artery conditions. Results indicated that supplemented media accelerated collagen formation (~185% increase in collagen mass/MSC compared to standard media), as well as increasing collagen mass production from 3.90 to 4.43 pg/cell/week from 3 to 6 weeks. Using augmented media, dynamic conditioning increased collagen mass production rate from 7.23 to 13.65 pg/cell/week (88.8%) during the dynamic culture period, along with greater preservation of net DNA. Moreover, when compared to our previous CFF study, organ level conditioning increased the collagen production rate from 4.76 to 6.42 pg/cell/week (35%). Newly conducted CFD studies of the CFF specimen flow patterns suggested that oscillatory surface shear stresses were surprisingly similar to a tri-leaflet valve. Overall, we found that the use of simulated pulmonary artery conditions resulted substantially large collagen mass production levels and rates found in our earlier CFF study. Moreover, given the fact that the scaffolds underwent modest strains (~7% max) during either CFF or physiological conditioning, the oscillatory surface shear stresses estimated in both studies may play a substantial role in eliciting MSC collagen production in the highly dynamic engineered heart valve fluid mechanical environment.
PMCID: PMC2813971  PMID: 19944458
25.  Endothelial Progenitor Cells as a Sole Source for Ex Vivo Seeding of Tissue-Engineered Heart Valves 
Tissue Engineering. Part A  2009;16(1):257-267.
Purposes: We investigated whether circulating endothelial progenitor cells (EPCs) can be used as a cell source for the creation of a tissue-engineered heart valve (TEHV). Methods: Trileaflet valved conduits were fabricated using nonwoven polyglycolic acid/poly-4-hydroxybutyrate polymer. Ovine peripheral blood EPCs were dynamically seeded onto a valved conduit and incubated for 7, 14, and 21 days. Results: Before seeding, EPCs were shown to express CD31+, eNOS+, and VE-Cadherin+ but not α-smooth muscle actin. Histological analysis demonstrated relatively homogenous cellular ingrowth throughout the valved conduit. TEHV constructs revealed the presence of endothelial cell (EC) markers and α-smooth muscle actin+ cells comparable with native valves. Protein levels were comparable with native valves and exceeded those in unseeded controls. EPC-TEHV demonstrated a temporal pattern of matrix metalloproteinases-2/9 expression and tissue inhibitors of metalloproteinase activities comparable to that of native valves. Mechanical properties of EPC-TEHV demonstrated significantly greater stiffness than that of the unseeded scaffolds and native valves. Conclusions: Circulating EPC appears to have the potential to provide both interstitial and endothelial functions and could potentially serve as a single-cell source for construction of autologous heart valves.
PMCID: PMC2811057  PMID: 19698056

Results 1-25 (50)