Assessment of Subclinical Atherosclerosis in Asymptomatic People In Vivo: Measurements Suitable for Biomarker and Mendelian Randomization Studies.

BACKGROUND: One strategy to reduce the burden of cardiovascular disease is the early detection and treatment of atherosclerosis. This has led to significant interest in studies of subclinical atherosclerosis, using different phenotypes, not all of which are accurate reflections of the presence of asymptomatic atherosclerotic plaques. The aim of part 2 of this series is to provide a review of the existing literature on purported measures of subclinical disease and recommendations concerning which tests may be appropriate in the prevention of incident cardiovascular disease. METHODS: We conducted a critical review of measurements used to infer the presence of subclinical atherosclerosis in the major conduit arteries and focused on the predictive value of these tests for future cardiovascular events, independent of conventional cardiovascular risk factors, in asymptomatic people. The emphasis was on studies with >10 000 person-years of follow-up, with meta-analysis of results reporting adjusted hazard ratios (HRs) with 95% CIs. The arterial territories were limited to carotid, coronary, aorta, and lower limb arteries. RESULTS: In the carotid arteries, the presence of plaque (8 studies) was independently associated with future stroke (pooled HR, 1.89 [1.04-3.44]) and cardiac events (7 studies), with a pooled HR, 1.77 (1.19-2.62). Increased coronary artery calcium (5 studies) was associated with the risk of coronary heart disease events, pooled HR, 1.54 (1.07-2.07) and increasing severity of calcification (by Agaston score) was associated with escalation of risk (13 studies). An ankle/brachial index (ABI) of <0.9, the pooled HR for cardiovascular death from 7 studies was 2.01 (1.43-2.81). There were insufficient studies of either, thoracic or aortic calcium, aortic diameter, or femoral plaque to synthesize the data based on consistent reporting of these measures. CONCLUSIONS: The presence of carotid plaque, coronary artery calcium, or abnormal ankle pressures seems to be a valid indicator of the presence of subclinical atherosclerosis and may be considered for use in biomarker, Mendelian randomization and similar studies.

Apolipoprotein A-I vascular gene therapy reduces vein-graft atherosclerosis.

Coronary artery venous bypass grafts typically fail because of atherosclerosis driven by lipid and macrophage accumulation. Therapy for vein-graft atherosclerosis is limited to statin drugs, which are only modestly effective. We hypothesized that transduction of vein-graft endothelium of fat-fed rabbits with a helper-dependent adenovirus expressing apolipoprotein AI (HDAdApoAI) would reduce lipid and macrophage accumulation. Fat-fed rabbits received bilateral external jugular vein-to-carotid artery interposition grafts. Four weeks later, one graft per rabbit (n = 23 rabbits) was infused with HDAdApoAI and the contralateral graft with HDAdNull. Grafts were harvested 12 weeks later. Paired analyses of grafts were performed, with vein graft cholesterol, intimal lipid, and macrophage content as the primary endpoints. HDAd genomes were detected in all grafts. APOAI mRNA was median 63-fold higher in HDAdApoAI grafts versus HDAdNull grafts (p < 0.001). HDAdApoAI grafts had a mean 15% lower total cholesterol (by mass spectrometry; p = 0.003); mean 19% lower intimal lipid (by oil red O staining; p = 0.02); and mean 13% lower expression of the macrophage marker CD68 (by reverse transcriptase-mediated quantitative PCR; p = 0.008). In vivo transduction of vein-graft endothelium achieves persistent APOAI expression and reduces vein-graft cholesterol, intimal lipid, and CD68 expression. Vascular gene therapy with APOAI has promise for preventing vein-graft failure caused by atherosclerosis.

Targeting and delivery of microRNA-targeting antisense oligonucleotides in cardiovascular diseases.

Discovered three decades ago, microRNAs (miRNAs) are now recognized as key players in the pathophysiology of multiple human diseases, including those affecting the cardiovascular system. As such, miRNAs have emerged as promising therapeutic targets for preventing the onset and/or progression of several cardiovascular diseases. Anti-miRNA antisense oligonucleotides or "antagomirs" precisely block the activity of specific miRNAs and are therefore a promising therapeutic strategy to repress pathological miRNAs. In this review, we describe advancements in antisense oligonucleotide chemistry that have significantly improved efficacy and safety. Moreover, we summarize recent approaches for the targeted delivery of antagomirs to cardiovascular tissues, highlighting major advantages as well as limitations of viral (i.e., adenovirus, adeno-associated virus, and lentivirus) and non-viral (i.e., liposomes, extracellular vesicles, and polymer nanoparticles) delivery systems. We discuss recent preclinical studies that use targeted antagomir delivery systems to treat three major cardiovascular diseases (atherosclerosis, myocardial infarction, and cardiac hypertrophy, including hypertrophy caused by hypertension), highlighting therapeutic results and discussing challenges that limit clinical applicability.

Novel expression cassettes for increasing apolipoprotein AI transgene expression in vascular endothelial cells.

Transduction of endothelial cells (EC) with a vector that expresses apolipoprotein A-I (APOAI) reduces atherosclerosis in arteries of fat-fed rabbits. However, the effects on atherosclerosis are partial and might be enhanced if APOAI expression could be increased. With a goal of developing an expression cassette that generates higher levels of APOAI mRNA in EC, we tested 4 strategies, largely in vitro: addition of 2 types of enhancers, addition of computationally identified EC-specific cis-regulatory modules (CRM), and insertion of the rabbit APOAI gene at the transcription start site (TSS) of sequences cloned from genes that are highly expressed in cultured EC. Addition of a shear stress-responsive enhancer did not increase APOAI expression. Addition of 2 copies of a Mef2c enhancer increased APOAI expression from a moderately active promoter/enhancer but decreased APOAI expression from a highly active promoter/enhancer. Of the 11 CRMs, 3 increased APOAI expression from a moderately active promoter (2-7-fold; P < 0.05); none increased expression from a highly active promoter/enhancer. Insertion of the APOAI gene into the TSS of highly expressed EC genes did not increase expression above levels obtained with a moderately active promoter/enhancer. New strategies are needed to further increase APOAI transgene expression in EC.

Blockade of TGF-ß (Transforming Growth Factor Beta) Signaling by Deletion of Tgfbr2 in Smooth Muscle Cells of 11-Month-Old Mice Alters Aortic Structure and Causes Vasomotor Dysfunction-Brief Report.

BACKGROUND: To test the hypothesis that smooth muscle cell (SMC) TGF-ß (transforming growth factor beta) signaling contributes to maintenance of aortic structure and function beyond the early postnatal period. METHODS: We deleted the TBR2 (type 2 TGF-ß receptor) in SMC of 11-month-old mice (genotype Acta2-CreERT2+/0Tgfbr2f/f, termed TBR2SMΔ) and compared their ascending aorta structure and vasomotor function to controls (Acta2-CreERT20/0Tgfbr2f/f, termed TBR2f/f). RESULTS: We confirmed loss of aortic SMC TBR2 by immunoblotting. Four weeks after SMC TBR2 loss, TBR2SMΔ mice did not have aortic rupture, ulceration, dissection, dilation, or evidence of medial hemorrhage. However, aortic medial area of TBR2SMΔ mice was increased by 27% (0.14±0.01 versus 0.11±0.01 mm2; P=0.01) and medial thickness was increased by 23% (40±1.9 versus 33±1.3 µm; P=0.004) compared with littermate controls. Wire myography performed on ascending aortic rings showed hypercontractility of TBR2SMΔ aortas to phenylephrine (Emax, 15.9±1.2 versus 10.8±0.7 mN; P=0.0003) and reduced relaxation and sensitivity to acetylcholine (Emax, 64±14% versus 96±2%; P=0.001; -logEC50, 6.9±0.1 versus 7.7±0.1; P=0.0001). Neither maximal relaxation nor sensitivity to sodium nitroprusside differed (Emax, 102±0.3% versus 101±0.3%; -logEC50, 8.0±0.04 versus 7.9±0.08; P>0.4 for both). CONCLUSIONS: Loss of TGF-ß signaling in aortic SMC of 1-year-old mice does not cause early severe aortopathy or death; however, it causes mild structural and substantial physiological abnormalities. SMC TGF-ß signaling plays an important role in maintaining aortic homeostasis in older mice. This role should be considered in the design of clinical studies that aim to prevent aortopathy by blocking SMC TGF-ß signaling.

Loss of Transforming Growth Factor Beta Signaling in Aortic Smooth Muscle Cells Causes Endothelial Dysfunction and Aortic Hypercontractility.

[Figure: see text].

Jugular Vein Injection of High-Titer Lentiviral Vectors Does Not Transduce the Aorta-Brief Report.

OBJECTIVE: Efficient gene transfer to the vascular wall via intravenous vector injection would be useful for experimental vascular biology and gene therapy. Initial studies of lentiviral vector tropism suggested that intravenously injected vectors do not transduce murine vascular tissue; however, there are also reports of highly efficient aortic transduction after jugular vein injection of high-titer lentiviral vectors. We sought to reproduce these results. Approach and Results: We injected high-titer preparations of GFP (green fluorescent protein)-expressing lentiviral vector into jugular veins of 8 mice; 6 mice received vehicle only. Four days later, samples of aorta (thoracic and abdominal), liver, spleen, and other tissues were harvested and processed for quantitative polymerase chain reaction detection of vector DNA and immunohistochemical detection of GFP. Our vector DNA assay did not detect transduction of any of the 16 aortic segments. This finding excludes an aortic transduction efficiency of >0.02 vector copies per cell. In contrast, vector DNA was detected in all 8 spleen and liver extracts (median, 0.8 and 0.1 vector copies per cell, respectively; P<0.001 versus vehicle controls). Quantitative polymerase chain reaction signals from DNA extracted from heart, lung, kidney, skeletal muscle, and femoral artery did not differ from background polymerase chain reaction signals from DNA extracted from tissues of vehicle-injected mice (P≥0.7 for all). Immunohistochemistry revealed GFP in scattered cells in spleen and liver, not in aorta. CONCLUSIONS: Injection of high-titer lentiviral vectors via the jugular vein transduces cells in the spleen and liver but does not efficiently transduce the aorta. Graphic Abstract: A graphic abstract is available for this article.

Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture.

RATIONALE: Plaque rupture is the proximate cause of most myocardial infarctions and many strokes. However, the molecular mechanisms that precipitate plaque rupture are unknown. OBJECTIVE: By applying proteomic and bioinformatic approaches in mouse models of protease-induced plaque rupture and in ruptured human plaques, we aimed to illuminate biochemical pathways through which proteolysis causes plaque rupture and identify substrates that are cleaved in ruptured plaques. METHODS AND RESULTS: We performed shotgun proteomics analyses of aortas of transgenic mice with macrophage-specific overexpression of urokinase (SR-uPA+/0 mice) and of SR-uPA+/0 bone marrow transplant recipients, and we used bioinformatic tools to evaluate protein abundance and functional category enrichment in these aortas. In parallel, we performed shotgun proteomics and bioinformatics studies on extracts of ruptured and stable areas of freshly harvested human carotid plaques. We also applied a separate protein-analysis method (protein topography and migration analysis platform) to attempt to identify substrates and proteolytic fragments in mouse and human plaque extracts. Approximately 10% of extracted aortic proteins were reproducibly altered in SR-uPA+/0 aortas. Proteases, inflammatory signaling molecules, as well as proteins involved with cell adhesion, the cytoskeleton, and apoptosis, were increased. ECM (Extracellular matrix) proteins, including basement-membrane proteins, were decreased. Approximately 40% of proteins were altered in ruptured versus stable areas of human carotid plaques, including many of the same functional categories that were altered in SR-uPA+/0 aortas. Collagens were minimally altered in SR-uPA+/0 aortas and ruptured human plaques; however, several basement-membrane proteins were reduced in both SR-uPA+/0 aortas and ruptured human plaques. Protein topography and migration analysis platform did not detect robust increases in proteolytic fragments of ECM proteins in either setting. CONCLUSIONS: Parallel studies of SR-uPA+/0 mouse aortas and human plaques identify mechanisms that connect proteolysis with plaque rupture, including inflammation, basement-membrane protein loss, and apoptosis. Basement-membrane protein loss is a prominent feature of ruptured human plaques, suggesting a major role for basement-membrane proteins in maintaining plaque stability.

Exosome-Mediated Transfer of Anti-miR-33a-5p from Transduced Endothelial Cells Enhances Macrophage and Vascular Smooth Muscle Cell Cholesterol Efflux.

Atherosclerosis is a disease of large- and medium-sized arteries that is caused by cholesterol accumulation in arterial intimal cells, including macrophages and smooth muscle cells (SMC). Cholesterol accumulation in these cells can be prevented or reversed in preclinical models-and atherosclerosis reduced-by transgenesis that increases expression of molecules that control cholesterol efflux, including apolipoprotein AI (apoAI) and ATP-binding cassette subfamily A, member 1 (ABCA1). In a previous work, we showed that transduction of arterial endothelial cells (EC)-with a helper-dependent adenovirus (HDAd) expressing apoAI-enhanced EC cholesterol efflux in vitro and decreased atherosclerosis in vivo. Similarly, overexpression of ABCA1 in cultured EC increased cholesterol efflux and decreased inflammatory gene expression. These EC-targeted gene-therapy strategies might be improved by concurrent upregulation of cholesterol-efflux pathways in other intimal cell types. Here, we report modification of this strategy to enable delivery of therapeutic nucleic acids to cells of the sub-endothelium. We constructed an HDAd (HDAdXMoAntimiR33a5p) that expresses an antagomiR directed at miR-33a-5p (a microRNA that suppresses cholesterol efflux by silencing ABCA1). HDAdXMoAntimiR33a5p contains a sequence motif that enhances uptake of anti-miR-33a-5p into exosomes. Cultured EC release exosomes containing small RNA, including miR-33a-5p. After transduction with HDAdXMoAntimiR33a5p, EC-derived exosomes containing anti-miR-33a-5p accumulate in conditioned medium (CM). When this CM is added to macrophages or SMC, anti-miR-33a-5p is detected in these target cells. Exosome-mediated transfer of anti-miR-33a-5p reduces miR-33a-5p by ∼65-80%, increases ABCA1 protein by 1.6-2.2-fold, and increases apoAI-mediated cholesterol efflux by 1.4-1.6-fold (all p ≤ 0.01). These effects were absent in macrophages and SMC incubated in exosome-depleted CM. EC transduced with HDAdXMoAntimiR33a5p release exosomes that can transfer anti-miR-33a-5p to other intimal cell types, upregulating cholesterol efflux from these cells. This strategy provides a platform for genetic modification of intimal and medial cells, using a vector that transduces only EC.

ABCA1 Overexpression in Endothelial Cells In Vitro Enhances ApoAI-Mediated Cholesterol Efflux and Decreases Inflammation.

Atherosclerosis, a disease of blood vessels, is driven by cholesterol accumulation and inflammation. Gene therapy that removes cholesterol from blood vessels and decreases inflammation is a promising approach for prevention and treatment of atherosclerosis. In previous work, we reported that helper-dependent adenoviral (HDAd) overexpression of apolipoprotein A-I (apoAI) in endothelial cells (ECs) increases cholesterol efflux in vitro and reduces atherosclerosis in vivo. However, the effect of HDAdApoAI on atherosclerosis is partial. To improve this therapy, we considered concurrent overexpression of ATP-binding cassette subfamily A, member 1 (ABCA1), a protein that is required for apoAI-mediated cholesterol efflux. Before attempting combined apoAI/ABCA1 gene therapy, we tested whether an HDAd that expresses ABCA1 (HDAdABCA1) increases EC cholesterol efflux, whether increased cholesterol efflux alters normal EC physiology, and whether ABCA1 overexpression in ECs has anti-inflammatory effects. HDAdABCA1 increased EC ABCA1 protein (∼3-fold; p < 0.001) and apoAI-mediated cholesterol efflux (2.3-fold; p = 0.007). Under basal culture conditions, ABCA1 overexpression did not alter EC proliferation, metabolism, migration, apoptosis, nitric oxide production, or inflammatory gene expression. However, in serum-starved, apoAI-treated EC, ABCA1 overexpression had anti-inflammatory effects: decreased inflammatory gene expression (∼50%; p ≤ 0.02 for interleukin [IL]-6, tumor necrosis factor [TNF]-α, and vascular cell adhesion protein-1); reduced lipid-raft Toll-like receptor 4 (80%; p = 0.001); and a trend towards increased nitric oxide production (∼55%; p = 0.1). In ECs stimulated with lipopolysaccharide, ABCA1 overexpression markedly decreased inflammatory gene expression (∼90% for IL-6 and TNF-α; p < 0.001). Therefore, EC ABCA1 overexpression has no toxic effects and counteracts the two key drivers of atherosclerosis: cholesterol accumulation and inflammation. In vivo testing of HDAdABCA1 is warranted.

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts.

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium.

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

Apo A-I (Apolipoprotein A-I) Vascular Gene Therapy Provides Durable Protection Against Atherosclerosis in Hyperlipidemic Rabbits.

OBJECTIVE: Gene therapy that expresses apo A-I (apolipoprotein A-I) from vascular wall cells has promise for preventing and reversing atherosclerosis. Previously, we reported that transduction of carotid artery endothelial cells with a helper-dependent adenoviral (HDAd) vector expressing apo A-I reduced early (4 weeks) fatty streak development in fat-fed rabbits. Here, we tested whether the same HDAd could provide long-term protection against development of more complex lesions. APPROACH AND RESULTS: Fat-fed rabbits (n=25) underwent bilateral carotid artery gene transfer, with their left and right common carotids randomized to receive either a control vector (HDAdNull) or an apo A-I-expressing vector (HDAdApoAI). Twenty-four additional weeks of high-fat diet yielded complex intimal lesions containing lipid-rich macrophages as well as smooth muscle cells, often in a lesion cap. Twenty-four weeks after gene transfer, high levels of apo A-I mRNA (median ≥250-fold above background) were present in all HDAdApoAI-treated arteries. Compared with paired control HDAdNull-treated arteries in the same rabbit, HDAdApoAI-treated arteries had 30% less median intimal lesion volume (P=0.03), with concomitant reductions (23%-32%) in intimal lipid, macrophage, and smooth muscle cell content (P≤0.05 for all). HDAdApoAI-treated arteries also had decreased intimal inflammatory markers. VCAM-1 (vascular cell adhesion molecule-1)-stained area was reduced by 36% (P=0.03), with trends toward lower expression of ICAM-1 (intercellular adhesion molecule-1), MCP-1 (monocyte chemoattractant protein 1), and TNF-α (tumor necrosis factor-α; 13%-39% less; P=0.06-0.1). CONCLUSIONS: In rabbits with severe hyperlipidemia, transduction of vascular endothelial cells with an apo A-I-expressing HDAd yields at least 24 weeks of local apo A-I expression that durably reduces atherosclerotic lesion growth and intimal inflammation.

Plasminogen promotes cholesterol efflux by the ABCA1 pathway.

Using genetic and biochemical approaches, we investigated proteins that regulate macrophage cholesterol efflux capacity (CEC) and ABCA1-specific CEC (ABCA1 CEC), 2 functional assays that predict cardiovascular disease (CVD). Macrophage CEC and the concentration of HDL particles were markedly reduced in mice deficient in apolipoprotein A-I (APOA1) or apolipoprotein E (APOE) but not apolipoprotein A-IV (APOA4). ABCA1 CEC was markedly reduced in APOA1-deficient mice but was barely affected in mice deficient in APOE or APOA4. High-resolution size-exclusion chromatography of plasma produced 2 major peaks of ABCA1 CEC activity. The early-eluting peak, which coeluted with HDL, was markedly reduced in APOA1- or APOE-deficient mice. The late-eluting peak was modestly reduced in APOA1-deficient mice but little affected in APOE- or APOA4-deficient mice. Ion-exchange chromatography and shotgun proteomics suggested that plasminogen (PLG) accounted for a substantial fraction of the ABCA1 CEC activity in the peak not associated with HDL. Human PLG promoted cholesterol efflux by the ABCA1 pathway, and PLG-dependent efflux was inhibited by lipoprotein(a) [Lp(a)]. Our observations identify APOA1, APOE, and PLG as key determinants of CEC. Because PLG and Lp(a) associate with human CVD risk, interplay among the proteins might affect atherosclerosis by regulating cholesterol efflux from macrophages.

TGF-ß (Transforming Growth Factor-ß) Signaling Protects the Thoracic and Abdominal Aorta From Angiotensin II-Induced Pathology by Distinct Mechanisms.

OBJECTIVE: The role of TGF-ß (transforming growth factor-ß) signaling in abdominal aortic aneurysm (AAA) formation is controversial. Others reported that systemic blockade of TGF-ß by neutralizing antibodies accelerated AAA development in angiotensin II-infused mice. This result is consistent with other studies suggesting that TGF-ß signaling prevents AAA. Development of a therapy for AAA that exploits the protective actions of TGF-ß would be facilitated by identification of the mechanisms through which TGF-ß prevents AAA. We hypothesized that TGF-ß signaling prevents AAA by its actions on aortic medial smooth muscle cells. APPROACH AND RESULTS: We compared the prevalence, severity, and histopathology of angiotensin II-induced AAA among control mice (no TGF-ß blockade), mice with antibody-mediated systemic neutralization of TGF-ß, and mice with genetically based smooth muscle-specific loss of TGF-ß signaling. Surprisingly, we found that systemic-but not smooth muscle-specific-TGF-ß blockade significantly increased the prevalence of AAA and tended to increase AAA severity, adventitial thickening, and aortic wall macrophage accumulation. In contrast, abdominal aortas of mice with smooth muscle-specific loss of TGF-ß signaling differed from controls only in having a thinner media. We examined thoracic aortas of the same mice. Here we found that smooth muscle-specific loss of Tgfbr2-but not systemic TGF-ß neutralization-significantly accelerated development of aortic pathology, including increased prevalence of intramural hematomas, medial thinning, and adventitial thickening. CONCLUSION: Our results suggest that TGF-ß signaling prevents both abdominal and thoracic aneurysmal disease but does so by distinct mechanisms. Smooth muscle extrinsic signaling protects the abdominal aorta and smooth muscle intrinsic signaling protects the thoracic aorta.

Aortopathy in a Mouse Model of Marfan Syndrome Is Not Mediated by Altered Transforming Growth Factor ß Signaling.

BACKGROUND: Marfan syndrome (MFS) is caused by mutations in the gene encoding fibrillin-1 (FBN1); however, the mechanisms through which fibrillin-1 deficiency causes MFS-associated aortopathy are uncertain. Recently, attention was focused on the hypothesis that MFS-associated aortopathy is caused by increased transforming growth factor-ß (TGF-ß) signaling in aortic medial smooth muscle cells (SMC). However, there are many reasons to doubt that TGF-ß signaling drives MFS-associated aortopathy. We used a mouse model to test whether SMC TGF-ß signaling is perturbed by a fibrillin-1 variant that causes MFS and whether blockade of SMC TGF-ß signaling prevents MFS-associated aortopathy. METHODS AND RESULTS: MFS mice (Fbn1C1039G/+ genotype) were genetically modified to allow postnatal SMC-specific deletion of the type II TGF-ß receptor (TBRII; essential for physiologic TGF-ß signaling). In young MFS mice with and without superimposed deletion of SMC-TBRII, we measured aortic dimensions, histopathology, activation of aortic SMC TGF-ß signaling pathways, and changes in aortic SMC gene expression. Young Fbn1C1039G/+ mice had ascending aortic dilation and significant disruption of aortic medial architecture. Both aortic dilation and disrupted medial architecture were exacerbated by superimposed deletion of TBRII. TGF-ß signaling was unaltered in aortic SMC of young MFS mice; however, SMC-specific deletion of TBRII in Fbn1C1039G/+ mice significantly decreased activation of SMC TGF-ß signaling pathways. CONCLUSIONS: In young Fbn1C1039G/+ mice, aortopathy develops in the absence of detectable alterations in SMC TGF-ß signaling. Loss of physiologic SMC TGF-ß signaling exacerbates MFS-associated aortopathy. Our data support a protective role for SMC TGF-ß signaling during early development of MFS-associated aortopathy.