Alberto Smith

Novel Candidate Genes in Unstable Areas of Human Atherosclerotic Plaques

Objective—Comparison of gene expression in stable versus unstable atherosclerotic plaque may be confounded by interpatient variability. The aim of this study was to identify differences in gene expression between stable and unstable segments of plaque obtained from the same patient. Methods and Results—Human carotid endarterectomy specimens were segmented and macroscopically classified using a morphological classification system. Two analytical methods, an intraplaque and an interplaque analysis, revealed 170 and 1916 differentially expressed genes, respectively using Affymetrix gene chip analysis. A total of 115 genes were identified from both analyses. The differential expression of 27 genes was also confirmed using quantitative-polymerase chain reaction on a larger panel of samples. Eighteen of these genes have not been associated previously with plaque instability, including the metalloproteinase, ADAMDEC1 (≈37-fold), retinoic acid receptor responder-1 (≈5-fold), and cysteine protease legumain (≈3-fold). Matrix metalloproteinase-9 (MMP-9), cathepsin B, and a novel gene, legumain, a potential activator of MMPs and cathepsins, were also confirmed at the protein level. Conclusions—The differential expression of 18 genes not previously associated with plaque rupture has been confirmed in stable and unstable regions of the same atherosclerotic plaque. These genes may represent novel targets for the treatment of unstable plaque or useful diagnostic markers of plaque instability.

Extracellular Matrix Composition and Remodeling in Human Abdominal Aortic Aneurysms: A Proteomics Approach

Abdominal aortic aneurysms (AAA) are characterized by pathological remodeling of the aortic extracellular matrix (ECM). However, besides the well-characterized elastolysis and collagenolysis little is known about changes in other ECM proteins. Previous proteomics studies on AAA focused on cellular changes without emphasis on the ECM. In the present study, ECM proteins and their degradation products were selectively extracted from aneurysmal and control aortas using a solubility-based subfractionation methodology and analyzed by gel-liquid chromatography-tandem MS and label-free quantitation. The proteomics analysis revealed novel changes in the ECM of AAA, including increased expression as well as degradation of collagen XII, thrombospondin 2, aortic carboxypeptidase-like protein, periostin, fibronectin and tenascin. Proteomics also confirmed the accumulation of macrophage metalloelastase (MMP-12). Incubation of control aortic tissue with recombinant MMP-12 resulted in the extensive fragmentation of these glycoproteins, most of which are novel substrates of MMP-12. In conclusion, our proteomics methodology allowed the first detailed analysis of the ECM in AAA and identified markers of pathological ECM remodeling related to MMP-12 activity.

Mutations in FOXC2 Are Strongly Associated With Primary Valve Failure in Veins of the Lower Limb

Background— Mutations in the FOXC2 gene cause lymphedema distichiasis, an inherited primary lymphedema in which a significant number of patients have varicose veins. Because lymphedema distichiasis is believed to be caused by lymphatic valve failure (reflux), and FOXC2 is highly expressed on venous valves in mouse embryos, we tested the hypothesis that FOXC2 mutations may be linked to venous valve failure and reflux. Methods and Results— The venous system of the leg was investigated with Duplex ultrasound. Pathological reflux was recorded by color Duplex ultrasound in all 18 participants with a FOXC2 mutation, including 3 without lymphedema. Every participant with a mutation in FOXC2 showed reflux in the great saphenous vein (n=18), compared with only 1 of 12 referents (including 10 family members; P<0.0001, Fisher exact test). Deep vein reflux was recorded in 14 of 18 participants. Conclusions— FOXC2 is the first gene in which mutations have been strongly associated with primary venous valve failure in both the superficial and deep veins in the lower limb. This gene appears to be important for the normal development and maintenance of venous and lymphatic valves.

Failure of Thrombus to Resolve in Urokinase-Type Plasminogen Activator Gene–Knockout Mice Rescue by Normal Bone Marrow–Derived Cells

Background—Monocytes may have an important role in the resolution of venous thrombosis. Increased expression of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) is associated with an ingress of monocytes into the thrombus. This study was designed to evaluate the importance of these activators in thrombus resolution. Methods and Results—Inferior caval vein thrombosis was induced in cohorts of adult wild-type, uPA gene-knockout (uPA−/−), and tPA gene-knockout (tPA−/−) mice in a flow model. Thrombi were harvested from wild-type and uPA−/− mice (n=60 per group) between 1 and 60 days. Thrombi were also obtained from groups of wild-type and tPA−/− mice (n=24 per group) between 1 and 28 days. Thrombus size and macrophage content were measured by computer-assisted image analysis. Thrombus resolution was significantly impaired in the uPA−/− mice compared with wild-type controls (P <0.0001) but was unaffected in tPA−/− mice. Monocyte content in wild-type mice was highest at 14 days after thrombus induction and was ≈4 times greater than in uPA−/− mice (P =0.0043). Thrombus size in uPA−/− mice transplanted with wild-type marrow (0.29±0.06 mm2) was significantly smaller than in uPA−/− mice given uPA−/− bone marrow (3.9±1.1 mm2) (P =0.0022). Donor bone marrow–derived cells expressing LacZ were present in the thrombus after transplantation. Conclusions—The resolution of experimental venous thrombus is dependent on uPA but is unaffected by the absence of tPA. Absence of uPA is also associated with delayed monocyte recruitment into the thrombus. Transplanting wild-type bone marrow restores thrombus resolution in uPA−/− animals, suggesting an important role for bone marrow–derived cells in this process.

Assessment of atherosclerotic plaque burden with an elastin-specific magnetic resonance contrast agent

Atherosclerosis and its consequences remain the main cause of mortality in industrialized and developing nations. Plaque burden and progression have been shown to be independent predictors for future cardiac events by intravascular ultrasound. Routine prospective imaging is hampered by the invasive nature of intravascular ultrasound. A noninvasive technique would therefore be more suitable for screening of atherosclerosis in large populations. Here we introduce an elastin-specific magnetic resonance contrast agent (ESMA) for noninvasive quantification of plaque burden in a mouse model of atherosclerosis. The strong signal provided by ESMA allows for imaging with high spatial resolution, resulting in accurate assessment of plaque burden. Additionally, plaque characterization by quantifying intraplaque elastin content using signal intensity measurements is possible. Changes in elastin content and the high abundance of elastin during plaque development, in combination with the imaging properties of ESMA, provide potential for noninvasive assessment of plaque burden by molecular magnetic resonance imaging (MRI).

Genes regulating lymphangiogenesis control venous valve formation and maintenance in mice

Chronic venous disease and venous hypertension are common consequences of valve insufficiency, yet the molecular mechanisms regulating the formation and maintenance of venous valves have not been studied. Here, we provide what we believe to be the first description of venous valve morphogenesis and identify signaling pathways required for the process. The initial stages of valve development were found to involve induction of ephrin-B2, a key marker of arterial identity, by venous endothelial cells. Intriguingly, developing and mature venous valves also expressed a repertoire of proteins, including prospero-related homeobox 1 (Prox1), Vegfr3, and integrin-α9, previously characterized as specific and critical regulators of lymphangiogenesis. Using global and venous valve-selective knockout mice, we further demonstrate the requirement of ephrin-B2 and integrin-α9 signaling for the development and maintenance of venous valves. Our findings therefore identified molecular regulators of venous valve development and maintenance and highlighted the involvement of common morphogenetic processes and signaling pathways in controlling valve formation in veins and lymphatic vessels. Unexpectedly, we found that venous valve endothelial cells closely resemble lymphatic (valve) endothelia at the molecular level, suggesting plasticity in the ability of a terminally differentiated endothelial cell to take on a different phenotypic identity.

Leukocytes and the natural history of deep vein thrombosis: current concepts and future directions

Observational studies have shown that inflammatory cells accumulate within the thrombus and surrounding vein wall during the natural history of venous thrombosis. More recent studies have begun to unravel the mechanisms that regulate this interaction and have confirmed that thrombosis and inflammation are intimately linked. This review outlines our current knowledge of the complex relationship between inflammatory cell activity and venous thrombosis and highlights new areas of research in this field. A better understanding of this relationship could lead to the development of novel therapeutic targets that inhibit thrombus formation or promote its resolution.

Monocyte chemotactic protein-1 (MCP-1) accelerates the organization and resolution of venous thrombi

PURPOSE Organization, recanalization, and contraction are common to wound healing and thrombus resolution. Monocytes are essential to wound healing and are also found in venous thrombi. We measured endogenous levels of the monocyte chemotactic protein-1 (MCP-1) in naturally resolving venous thrombi and determined the effect of injecting MCP-1 into newly formed thrombus. METHODS Endogenous MCP-1 levels were estimated in rat blood, thrombi, and the adjacent vessel wall after thrombus formation, in cohorts of eight animals at 1, 7, and 14 days. In another group (n = 10), 1 microgram of MCP-1 was injected into newly formed thrombi. Carrier was injected into the thrombi of control animals (n = 10). Thrombi and adjacent vein walls were obtained for histology at 7 days. Thrombi were given an arbitrary organization score based on erythrocyte and extracellular matrix content, which was assessed by means of computerized and observer analysis. Specimen weight, thrombus area, and cellular and monocyte content were measured. RESULTS Endogenous MCP-1 increased between days 1 and 7 in the thrombus (1-day median, 1.1 ng/g wet wt; 1-day range, 0.8 to 1.4 ng/g wet wt; 7-day median, 5.4 ng/g wet wt; 7-day range, 1.5 to 7.4 ng/g wet wt; P <.0001) and vein wall (1-day median, 1.5 ng/g wet wt; 1-day range, 0.8 to 4.3 ng/g wet wt; 7-day median, 3.3 ng/g wet wt; 7-day range, 2.7 to 8.3 ng/g wet wt; P <. 05). At 14 days, thrombus was incorporated in the vein wall, and total MCP-1 levels remained high (median, 3.9 ng/g wet wt; range, 1.1 to 7.4 ng/g wet wt). Less MCP-1 was found in the thrombus than the adjacent vessel wall at day 1 (P <.05), but there was no difference at day 7. MCP-1 could not be detected in the blood. MCP-1 injection into thrombus increased the computer (P =.016) and observer (P =.004) organization scores, reduced the thrombus area (from median, 3. 4 mm(2), and range, 1.5 to 5.7 mm(2), to median, 0.2 mm(2), and range, 0.02 to 2.6 mm(2); P =.048), and increased the surrounding vessel wall monocyte content (P =.008). Specimen weights of treated animals were lower than those of control animals (P <.02). CONCLUSION Venous thrombus MCP-1 levels increase during natural resolution. MCP-1 treatment increased the organization and resolution of thrombi. MCP-1 may therefore be of therapeutic use.

Galectin-3 Is an Amplifier of Inflammation in Atherosclerotic Plaque Progression Through Macrophage Activation And Monocyte Chemoattraction

Objective—Galectin-3 (Gal-3) is a 26-kDa lectin known to regulate many aspects of inflammatory cell behavior. We assessed the hypothesis that increased levels of Gal-3 contribute to atherosclerotic plaque progression by enhancing monocyte chemoattraction through macrophage activation. Methods and Results—Gal-3 was found to be upregulated in unstable plaque regions of carotid endarterectomy (CEA) specimens compared with stable regions from the same patient (3.2-fold, P<0.05) at the mRNA (n=12) and (2.3-fold, P<0.01) at the protein level (n=9). Analysis of aortic tissue from ApoE−/− mice on a high fat diet (n=14) and wild-type controls (n=9) showed that Gal-3 mRNA and protein levels are elevated by 16.3-fold (P<0.001) and 12.2-fold (P<0.01) and that Gal-3 staining colocalizes with macrophages. In vitro, conditioned media from Gal-3–treated human macrophages induced an up to 6-fold increase in human monocyte chemotaxis (P<0.01, ANOVA), an effect that was reduced by 66 and 60% by Pertussis Toxin (PTX) and the Vaccinia virus protein 35K, respectively. Microarray analysis of human macrophages and subsequent qPCR validation confirmed the upregulation of CC chemokines in response to Gal-3 treatment. Conclusions—Our data suggest that Gal-3 is both a marker of atherosclerotic plaque progression and a central contributor to the pathology by amplification of key proinflammatory molecules.

Electrospray ionization mass spectrometry identifies substrates and products of lipoprotein-associated phospholipase A2 in oxidized human low density lipoprotein

There is increasing evidence that modified phospholipid products of low density lipoprotein (LDL) oxidation mediate inflammatory processes within vulnerable atherosclerotic lesions. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is present in vulnerable plaque regions where it acts on phospholipid oxidation products to generate the pro-inflammatory lysophsopholipids and oxidized non-esterified fatty acids. This association together with identification of circulating Lp-PLA2 levels as an independent predictor of cardiovascular disease provides a rationale for development of Lp-PLA2 inhibitors as therapy for atherosclerosis. Here we report a systematic analysis of the effects of in vitro oxidation in the absence and presence of an Lp-PLA2 inhibitor on the phosphatidylcholine (PC) composition of human LDL. Mass spectrometry identifies three classes of PC whose concentration is significantly enhanced during LDL oxidation. Of these, a series of molecules, represented by peaks in the m/z range 594-666 and identified as truncated PC oxidation products by accurate mass measurements using an LTQ Orbitrap mass spectrometer, are the predominant substrates for Lp-PLA2. A second series of oxidation products, represented by peaks in the m/z range 746-830 and identified by LTQ Orbitrap analysis as non-truncated oxidized PCs, are quantitatively more abundant but are less efficient Lp-PLA2 substrates. The major PC products of Lp-PLA2, saturated and mono-unsaturated lyso-PC, constitute the third class. Mass spectrometric analysis confirms the presence of many of these PCs within human atherosclerotic lesions, suggesting that they could potentially be used as in vivo markers of atherosclerotic disease progression and response to Lp-PLA2 inhibitor therapy.