Atherosclerosis is a complex, multifactorial disease with both genetic and environmental determinants. Experimental investigation of the effects of these determinants on the development and progression of atherosclerosis has been greatly facilitated by the use of targeted mouse models of the disease, particularly those resulting from the absence of functional genes for apolipoprotein E or the low density lipoprotein receptor (LDLR). This review focuses on the influence on atherosclerosis of combining apoE or LDLR deficiencies with factors affecting atherogenesis, including (1) inflammatory processes, (2) glucose metabolism, (3) blood pressure, and (4) coagulation and fibrinolysis. We also discuss the general problem of using the mouse to test the effects on atherogenesis of human polymorphic variations and future ways of enhancing the usefulness of these mouse models.
apoE; LDL receptor; hypertension; inflammation; diabetes
Natriuretic peptide signaling is important in the regulation of blood pressure as well as in the growth of multiple cell types. To examine the role of natriuretic peptide signaling in atherosclerosis, we crossbred mice that lack natriuretic peptide receptor A (NPRA; Npr1−/−) with atherosclerosis-prone mice that lack apolipoprotein E (apoE; Apoe−/−).
Methods and Results
Doubly deficient Npr1−/− Apoe−/− mice have increased blood pressure relative to Npr1+/+ Apoe−/− mice (118±4 mm Hg compared with 108±2 mm Hg, P<0.05) that is coincident with a 64% greater atherosclerotic lesion size (P<0.005) and more advanced plaque morphology. Additionally, aortic medial thickness is increased by 52% in Npr1−/− Apoe−/− mice relative to Npr1+/+ Apoe−/− mice (P<0.0001). Npr1−/− Apoe−/− mice also have significantly greater cardiac mass (9.0±0.3 mg/g body weight) than either Npr1+/+ Apoe−/− mice (5.8±0.2 mg/g) or Npr1−/− Apoe+/+ mice (7.1±0.2 mg/g), suggesting that the lack of both NPRA and apoE synergistically enhances cardiac hypertrophy.
These data provide evidence that NPR1 is an atherosclerosis susceptibility locus and represents a potential link between atherosclerosis and cardiac hypertrophy. Our results also suggest roles for Npr1 as well as Apoe in regulation of hypertrophic cell growth.
natriuretic peptide receptor A; atherosclerosis; cardiac hypertrophy; vascular smooth muscle cells
Peripheral arterial disease (PAD) is characterized by reduced blood flow to the limbs, usually as a consequence of atherosclerosis, and affects ≈12 million Americans. It is a common cause of cardiovascular morbidity and an independent predictor of cardiovascular mortality. Similar to other atherosclerotic diseases, such as coronary artery disease, PAD is the result of the complex interplay between injurious environmental stimuli and genetic predisposing factors of the host. Genetic susceptibility to PAD is likely contributed by sequence variants in multiple genes, each with modest effects. Although many of these variants probably alter susceptibility both to PAD and to coronary artery disease, it is likely that there exists a set of variants specifically to alter susceptibility to PAD. Despite the prevalence of PAD and its high societal burden, relatively little is known about such genetic variants. This review summarizes our limited present knowledge and gives an overview of recent, more powerful approaches to elucidating the genetic basis of PAD. We discuss the advantages and limitations of genetic studies and highlight the need for collaborative networks of PAD investigators for shedding light on this dark corner of vascular biology.
peripheral vascular disease; genetics; epidemiology; atherosclerosis
LRP1 is a large endocytic and signaling receptor that is widely expressed. In the liver, LRP1 plays an important role in regulating the plasma levels of blood coagulation factor VIII (fVIII) by mediating its uptake and subsequent degradation. fVIII is a key plasma protein that is deficient in Hemophilia A, and circulates in complex with von Willebrand factor (vWf). Since vWf blocks binding of fVIII to LRP1, questions remain regarding the molecule mechanisms by which LRP1 removes fVIII from the circulation. LRP1 also regulates cell surface levels of tissue factor (TF), a component of the extrinsic blood coagulation pathway. This occurs when tissue factor pathway inhibitor (TFPI) bridges the fVII/TF complex to LRP1, resulting in rapid LRP1-mediated internalization and down regulation of coagulant activity. In the vasculature LRP1 also plays protective role from the development of aneurysms. Mice in which the lrp1 gene is selectively deleted in vascular smooth muscle cells develop a phenotype similar to the progression of aneurysm formation in human patient, revealing that these mice are ideal for investigating molecular mechanisms associated with aneurysm formation. Studies suggest that LRP1 protects against elastin fiber fragmentation by reducing excess protease activity in the vessel wall. These proteases include high-temperature requirement factor A1 (HtrA1), MMP2, MMP9 and MT1-MMP. In addition LRP1 regulates matrix deposition, in part by modulating levels of connective tissue growth factor. Defining pathways modulated by LRP1 that lead to aneurysm formation and defining its role in thrombosis may allow for more effective intervention in patients.
Recent evidence suggests G-protein–coupled receptor-2–interacting protein-1 (GIT1) overexpression in several human metastatic tumors, including breast, lung, and prostate. Tumor metastasis is associated with an increase in angiogenesis. We have showed previously that GIT1 is required for postnatal angiogenesis during lung development. However, the functional role of GIT1 in pathological angiogenesis during tumor growth is unknown.
Approach and Results
In the present study, we show inhibition of angiogenesis in matrigel implants as well as reduced tumor angiogenesis and melanoma tumor growth in GIT1-knockout mice. We demonstrate that this is a result of impaired directional migration of GIT1-depleted endothelial cells toward a vascular endothelial growth factor gradient. Cortactin-mediated lamellipodia formation in the leading edge is critical for directional migration. We observed a significant reduction in cortactin localization and lamellipodia formation in the leading edge of GIT1-depleted endothelial cells. We specifically identified that the Spa homology domain (aa 250–420) of GIT1 is required for GIT1–cortactin complex localization to the leading edge. The mechanisms involved extracellular signal-regulated kinases 1 and 2–mediated Cortactin-S405 phosphorylation and activation of Rac1/Cdc42. Finally, using gain of function studies, we show that a constitutively active mutant of cortactin restored directional migration of GIT1-depleted cells.
Our data demonstrated that a GIT1–cortactin association through GIT1-Spa homology domain is required for cortactin localization to the leading edge and is essential for endothelial cell directional migration and tumor angiogenesis.
cortactin; endothelial cells; G-protein–coupled receptor kinase interacting protein-1; tumor angiogenesis
Cardiovascular disease is a complex disorder influenced by interactions of genetic variants with environmental factors. However, there is no information from large community-based studies examining the relationship of circulating cell–specific RNA to inflammatory proteins. In light of the associations among inflammatory biomarkers, obesity, platelet function, and cardiovascular disease, we sought to examine the relationships of C-reactive protein (CRP) and interleukin-6 (IL-6) to the expression of key inflammatory transcripts in platelets.
Approach and Results
We quantified circulating levels of CRP and IL-6 in 1625 participants of the Framingham Heart Study (FHS) Offspring cohort examination 8 (mean age, 66.6±6.6 years; 46% men). We measured the expression of 15 relevant genes by high-throughput quantitative reverse transcriptase polymerase chain reaction from platelet-derived RNA and used multivariable regression to relate serum concentrations of CRP and IL-6 with gene expression. Levels of CRP and IL-6 were associated with 10 of the 15 platelet-derived inflammatory transcripts, ALOX5, CRP, IFIT1, IL6, PTGER2, S100A9, SELENBP1, TLR2, TLR4, and TNFRSF1B (P<0.001). Associations between platelet mRNA expression with CRP and IL-6 persisted after multivariable adjustment for potentially confounding factors. Six genes positively associated with CRP or IL-6 in the FHS sample were also upregulated in megakaryocytes in response to CRP or IL-6 exposure.
Our data highlight the strong connection between the circulating inflammatory biomarkers CRP and IL-6 and platelet gene expression, adjusting for cardiovascular disease risk factors. Our results also suggest that body weight may directly influence these associations.
cardiovascular disease; inflammation; mRNA platelets
Cholesterol efflux relates to cardiovascular disease but cannot predict cellular cholesterol mass changes. We asked whether influx and net flux assays provide additional insights.
Approach and Results
Adapt a bidirectional flux assay to cells where efflux has clinical correlates and examine the association of influx, efflux, and net flux to serum triglycerides (TGs). Apolipoprotein B–depleted (high-density lipoprotein-fraction) serum from individuals with unfavorable lipids (median [interquartile range]; high-density lipoprotein-cholesterol=39 [32–42], low-density lipoprotein-cholesterol=109 [97–137], TGs=258 [184–335] mg/dL; n=13) promoted greater ATP-binding cassette transporter A1–mediated [1,2-3H] cholesterol efflux (3.8±0.3%/4 hour versus 1.2±0.4%/4 hour; P<0.0001) from cyclic 3’,5’-amp(CTP-amp)-treated J774 macrophages than from individuals with favorable lipids (high-density lipoprotein-cholesterol=72 [58–88], low-density lipoprotein-cholesterol=111 [97–131], TGs=65 [56–69] mg/dL; n=10). Thus, high TGs associated with more ATP-binding cassette transporter A1 acceptors. Efflux of cholesterol mass (µg free cholesterol/mg cell protein per 8 hour) to serum was also higher (7.06±0.33 versus 5.83±0.48; P=0.04). However, whole sera from individuals with unfavorable lipids promoted more influx (5.14±0.65 versus 2.48±0.85; P=0.02) and lower net release of cholesterol mass (1.93±0.46 versus 3.36±0.47; P=0.04). The pattern differed when mass flux was measured using apolipoprotein B–depleted serum rather than serum. Although individuals with favorable lipids tended to have greater influx than those with unfavorable lipids, efflux to apolipoprotein B–depleted serum was markedly higher (6.81±0.04 versus 2.62±0.14; P<0.0001), resulting in an efflux:influx ratio of ≈3-fold. Thus both serum and apolipoprotein B–depleted serum from individuals with favorable lipids promoted greater net cholesterol mass release despite increased ATP-binding cassette transporter A1–mediated efflux in samples of individuals with high TGs/unfavorable lipids.
When considering the efficiency of serum specimens to modulate cell cholesterol content, both influx and efflux need to be measured.
coronary artery disease; high-density lipoprotein-1
Tissue factor pathway inhibitor (TFPI) is produced in 2 isoforms: TFPIα, a soluble protein in plasma, platelets, and endothelial cells, and TFPIβ, a glycosylphosphatidylinositol-anchored protein on endothelium. Protein S (PS) functions as a cofactor for TFPIα, enhancing the inhibition of factor Xa. However, PS does not alter the inhibition of prothrombinase by TFPIα, and PS interactions with TFPIβ are undescribed. Thus, the physiological role and scope of the PS–TFPI system remain unclear.
Approach and Results
Here, the cofactor activity of PS toward platelet and endothelial TFPIα and endothelial TFPIβ was quantified. PS enhanced the inhibition of factor Xa by TFPIα from platelets and endothelial cells and stabilized the TFPIα/factor Xa inhibitory complex, delaying thrombin generation by prothrombinase. By contrast, PS did not enhance the inhibitory activity of TFPIβ or a membrane-anchored form of TFPI containing the PS-binding third Kunitz domain (K1K2K3) although PS did function as a cofactor for K1K2K3 enzymatically released from the cell surface.
The PS–TFPI anticoagulant system is limited to plasma TFPIα and TFPIα released from platelets and endothelial cells. PS likely functions to localize solution-phase TFPIα to the cell surface, where factor Xa is bound. PS does not alter the activity of membrane-associated TFPI. Because activated platelets release TFPIα and PS, the PS–TFPIα anticoagulant system may act physiologically to dampen thrombin generation at the platelet surface.
blood coagulation; hemorrhage; thromboplastin; thrombosis
Tissue factor pathway inhibitor (TFPI) blocks the initiation of coagulation by inhibiting TF-activated factor VII, activated factor X, and early prothrombinase. Humans produce two 3′ splice variants, TFPIα and TFPIβ, which are differentially expressed in endothelial cells and platelets and possess distinct structural features affecting their inhibitory function. TFPI also undergoes alternative splicing of exon 2 within its 5′ untranslated region. The role of exon 2 splicing in translational regulation of human TFPI isoform expression is investigated.
Approach and Results
Exon 2 splicing occurs in TFPIα and TFPIβ transcripts. Human tissue mRNA analysis uncovered a wide variability of exon 2 expression. Polysome analysis revealed a repressive effect of exon 2 on TFPIβ translation but not on TFPIα. Luciferase reporter assays further exposed strong translational repression of TFPIβ (90%) but not TFPIα. Use of a Morpholino to remove exon 2 from TFPI mRNA increased cell surface expression of endogenous TFPIβ. Exon 2 also repressed luciferase production (80% to 90%) when paired with the β-actin 3′ untranslated region, suggesting that it is a general translational negative element whose effects are overcome by the TFPIα 3′ untranslated region.
Exon 2 is a molecular switch that prevents translation of TFPIβ. This is the first demonstration of a 5′ untranslated region alternative splicing event that alters translation of isoforms produced via independent 3′ splicing events within the same gene. Therefore, it represents a previously unrecognized mechanism for translational control of protein expression. Differential expression of exon 2 denotes a mechanism to provide temporal and tissue-specific regulation of TFPIβ-mediated anticoagulant activity.
alternative splicing; gene expression regulation; tissue factor pathway inhibitor
Proteasome inhibitors are in use to treat hematologic cancers, but also reduce thrombosis. Whether the proteasome participates in platelet activation or function is opaque since little is known of the proteasome in these terminally differentiated cells.
Approach and Results
Platelets displayed all three primary proteasome protease activities, which MG132 and bortezomib (Velcade®) inhibited. Proteasome substrates are marked by ubiquitin, and platelets contained a functional ubiquitination system that modified the proteome by mono- and poly-ubiquitination. Systemic MG132 strongly suppressed formation of occlusive, platelet-rich thrombi in FeCl3-damaged carotid arteries. Transfusion of platelets treated ex vivo with MG132 and washed prior to transfusion into thrombocytopenic mice also reduced carotid artery thrombosis. Proteasome inhibition reduced platelet aggregation by low thrombin concentrations and ristocetin-stimulated agglutination through the GPIb-IX-V complex. This receptor was not appropriately internalized after proteasome inhibition in stimulated platelets, and spreading and clot retraction after MG132 exposure also were decreased. The effects of proteasome inhibitors were not confined to a single receptor as MG132 suppressed thrombin-, ADP-, and LPS-stimulated microparticle shedding. Proteasome inhibition increased ubiquitin decoration of cytoplasmic proteins, including the cytoskeletal proteins Filamin A and Talin-1. Mass spectrometry revealed a single MG132-sensitive tryptic cleavage after R1745 in an extended Filamin A loop, which would separate its actin-binding domain from its carboxy terminal GPIbα binding domain.
Platelets contain a ubiquitin/proteasome system that marks cytoskeletal proteins for proteolytic modification to promote productive platelet-platelet and platelet-wall interactions.
Platelet proteasome; Low dose thrombin; Microparticles and Cytoskeletal proteins
T cells, particularly CD8+ T cells, are major participants in obesity-linked adipose tissue (AT) inflammation. We examined the mechanisms of CD8+ T-cell accumulation and activation in AT and the role of CD11a, a β2 integrin.
Approach and Results
CD8+ T cells in AT of obese mice showed activated phenotypes with increased proliferation and interferon-γ expression. In vitro, CD8+ T cells from mouse AT displayed increased interferon-γ expression and proliferation to stimulation with interleukin-12 and interleukin-18, which were increased in obese AT. CD11a was upregulated in CD8+ T cells in obese mice. Ablation of CD11a in obese mice dramatically reduced T-cell accumulation, activation, and proliferation in AT. Adoptive transfer showed that CD8+ T cells from wild-type mice, but not from CD11a-deficient mice, infiltrated into AT of recipient obese wild-type mice. CD11a deficiency also reduced tumor necrosis factor-α–producing and interleukin-12–producing macrophages in AT and improved insulin resistance.
Combined action of cytokines in obese AT induces proliferative response of CD8+ T cells locally, which, along with increased infiltration, contributes to CD8+ T-cell accumulation and activation in AT. CD11a plays a crucial role in AT inflammation by participating in T-cell infiltration and activation.
adipose tissue; inflammation; insulin resistance; obesity
Intermediate and small conductance KCa channels IK1 (KCa3.1) and SK3 (KCa2.3) are primary targets of endothelial Ca2+ signals in the arterial vasculature and their ablation results in increased arterial tone and hypertension. Activation of IK1 channels by local Ca2+ transients from internal stores or plasma membrane channels promotes arterial hyperpolarization and vasodilation. Here, we assess arteries from genetically altered IK1 knockout mice (IK1−/−) to determine whether IK1 channels exert a positive feedback influence on endothelial Ca2+ dynamics.
Approach and Results
Using confocal imaging and custom data analysis software we found that while the occurrence of basal endothelial Ca2+ dynamics was not different between IK1−/− and wild-type (WT) mice (p > 0.05), the frequency of acetylcholine (ACh 2 µM)-stimulated Ca2+ dynamics was greatly depressed in IK1−/− endothelium (515 ± 153 vs. 1860 ± 319 events; p < 0.01). In IK1−/−/SK3T/T mice, ancillary suppression (+Dox) or overexpression (−Dox) of SK3 channels had little additional impact on the occurrence of events under basal or ACh-stimulated conditions. SK3 overexpression did, however, restore the depressed event amplitudes. Removal of extracellular Ca2+ reduced ACh-induced Ca2+ dynamics to the same level in WT and IK1−/− arteries. Blockade of IK1 and SK3 with the combination of charybdotoxin (0.1 µM) and apamin (0.5 µM) or TRPV4 channels with HC-067047 (1 µM) reduced ACh Ca2+ dynamics in WT arteries to the level of IK1−/−/SK3T/T+Dox arteries. These drug effects were not additive.
IK1, and to some extent SK3 channels, exert a substantial positive feedback influence on endothelial Ca2+ dynamics.
Endothelium; Calcium; IK1; SK3; TRPV4
Inducible Degrader Of the Low-density lipoprotein receptor (IDOL) is an E3 ubiquitin ligase that mediates the ubiquitination and degradation of the low-density lipoprotein receptor (LDLR). IDOL expression is controlled at the transcriptional level by the cholesterol-sensing nuclear receptor LXR. In response to rising cellular sterol levels, activated LXR induces IDOL production, thereby limiting further uptake of exogenous cholesterol through the LDLR pathway. The LXR–IDOL–LDLR mechanism for feedback inhibition of cholesterol uptake is independent of and complementary to the SREBP pathway. Since the initial description of the LXR–IDOL pathway, biochemical studies have helped to define the structural basis for both IDOL target recognition and LDLR ubiquitin transfer. Recent work has also suggested links between IDOL and human lipid metabolism.