Jie Hong Hu

TGF-β1 Limits Plaque Growth, Stabilizes Plaque Structure, and Prevents Aortic Dilation in Apolipoprotein E-Null Mice

Objective—Impairment of transforming growth factor (TGF)-&bgr;1 signaling accelerates atherosclerosis in experimental mice. However, it is uncertain whether increased TGF-&bgr;1 expression would retard atherosclerosis. The role of TGF-&bgr;1 in aneurysm formation is also controversial. We tested whether overexpression of active TGF-&bgr;1 in hyperlipidemic mice affects atherogenesis and aortic dilation. Methods and Results—We generated apolipoprotein E–null mice with transgenes that allow regulated overexpression of active TGF-&bgr;1 in their hearts. Compared to littermate controls, these mice had elevated cardiac and plasma TGF-&bgr;1, less aortic root atherosclerosis (P≤0.002), fewer lesions in the thoracic and abdominal aortae (P≤0.01), less aortic root dilation (P<0.001), and fewer pseudoaneurysms (P=0.02). Mechanistic studies revealed no effect of TGF-&bgr;1 overexpression on plasma lipids or cytokines, or on peripheral lymphoid organ cells. However, aortae of TGF-&bgr;1–overexpressing mice had fewer T-lymphocytes, more collagen, less lipid, lower expression of inflammatory cytokines and matrix metalloproteinase-13, and higher expression of tissue inhibitor of metalloproteinase-2. Conclusions—When overexpressed in the heart and plasma, TGF-&bgr;1 is an antiatherogenic, vasculoprotective cytokine that limits atherosclerosis and prevents aortic dilation. These actions are associated with significant changes in cellularity, collagen and lipid accumulation, and gene expression in the artery wall.

Mechanisms of Urokinase Plasminogen Activator (uPA)-mediated Atherosclerosis ROLE OF THE uPA RECEPTOR AND S100A8/A9 PROTEINS

Data from clinical studies, cell culture, and animal models implicate the urokinase plasminogen activator (uPA)/uPA receptor (uPAR)/plasminogen system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/uPAR/plasminogen stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression and mice genetically deficient in uPAR to elucidate mechanisms of uPA/uPAR/plasminogen-accelerated atherosclerosis and aneurysm formation. We found that macrophage-specific uPA overexpression accelerates atherosclerosis and causes aortic root dilation in fat-fed Ldlr−/− mice (as we previously reported in Apoe−/− mice). Macrophage-expressed uPA accelerates atherosclerosis by stimulation of lesion progression rather than initiation and causes disproportionate lipid accumulation in early lesions. uPA-accelerated atherosclerosis and aortic dilation are largely, if not completely, independent of uPAR. In the absence of uPA overexpression, however, uPAR contributes modestly to both atherosclerosis and aortic dilation. Microarray studies identified S100A8 and S100A9 mRNA as the most highly up-regulated transcripts in uPA-overexpressing macrophages; up-regulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also up-regulated in the aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is up-regulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Macrophage microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm in a second animal model that macrophage-expressed uPA stimulates atherosclerosis and aortic dilation. They also reveal uPAR independence of these actions and implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease.

Overexpression of Urokinase by Plaque Macrophages Causes Histological Features of Plaque Rupture and Increases Vascular Matrix Metalloproteinase Activity in Aged Apolipoprotein E–Null Mice

Background— The mechanisms of atherosclerotic plaque rupture are poorly understood. Urokinase-type plasminogen activator (uPA) is expressed at elevated levels by macrophages in advanced human plaques. Patients with evidence of increased plasminogen activation have an elevated risk of major cardiovascular events. We used atherosclerotic mice to test the hypothesis that increased macrophage uPA expression in advanced plaques would cause histological features similar to those in ruptured human plaques. Methods and Results— Bone marrow from transgenic mice with increased macrophage uPA expression or nontransgenic controls (all apolipoprotein E-null [Apoe−/−]) was transplanted into 35-week-old Apoe−/− recipients, and innominate lesions and aortas were examined 8 to 13 weeks later. Donor macrophages accumulated in innominate lesions adjacent to plaque caps and in aortas, increasing uPA expression at both sites. Recipients of uPA-overexpressing macrophages had an increased prevalence of intraplaque hemorrhage (61% versus 13%; P=0.002) as well as increased lesion fibrin staining and fibrous cap disruption (P=0.06 for both). Transplantation of uPA-overexpressing macrophages increased aortic matrix metalloproteinase activity (40%; P=0.02). This increase was independent of matrix metalloproteinase-9. Conclusions— In advanced plaques of Apoe−/− mice, macrophage uPA overexpression causes intraplaque hemorrhage and fibrous cap disruption, features associated with human plaque rupture. uPA overexpression also increases vascular matrix metalloproteinase activity. These data provide a mechanism that connects macrophage uPA expression, matrix metalloproteinase activity, and plaque rupture features in mice. The data also suggest that elevated plaque plasminogen activator expression and plasminogen activation in humans may be causally linked to plaque rupture and cardiovascular events.

Plasminogen mediates the atherogenic effects of macrophage-expressed urokinase and accelerates atherosclerosis in apoE-knockout mice.

Urokinase-type plasminogen activator (uPA) is expressed at elevated levels in atherosclerotic human arteries, primarily in macrophages. Plasminogen (Plg), the primary physiologic substrate of uPA, is present at significant levels in blood and interstitial fluid. Both uPA and Plg have activities that could affect atherosclerosis progression. Moreover, correlations between increased Plg activation and accelerated atherosclerosis are reported in several human studies. However, a coherent picture of the role of the uPA/Plg system in atherogenesis has not yet emerged, with at least one animal study suggesting that Plg is atheroprotective. We used a transgenic mouse model of macrophage-targeted uPA overexpression in apolipoprotein E-deficient mice to investigate the roles of uPA and Plg in atherosclerosis. We found that macrophage-expressed uPA accelerated atherosclerotic plaque growth and promoted aortic root dilation through Plg-dependent pathways. These pathways appeared to affect lesion progression rather than initiation and to include actions that disproportionately increase lipid accumulation in the artery wall. In addition, loss of Plg was protective against atherosclerosis both in the presence and absence of uPA overexpression. Transgenic mice with macrophage-targeted uPA overexpression reveal atherogenic roles for both uPA and Plg and are a useful experimental setting for investigating the molecular mechanisms that underlie clinically established relationships between uPA expression, Plg activation, and atherosclerosis progression.

Postnatal Deletion of the Type II Transforming Growth Factor-β Receptor in Smooth Muscle Cells Causes Severe Aortopathy in Mice

Objective—Prenatal deletion of the type II transforming growth factor-&bgr; (TGF-&bgr;) receptor (TBRII) prevents normal vascular morphogenesis and smooth muscle cell (SMC) differentiation, causing embryonic death. The role of TBRII in adult SMC is less well studied. Clarification of this role has important clinical implications because TBRII deletion should ablate TGF-&bgr; signaling, and blockade of TGF-&bgr; signaling is envisioned as a treatment for human aortopathies. We hypothesized that postnatal loss of SMC TBRII would cause aortopathy. Approach and Results—We generated mice with either of 2 tamoxifen-inducible SMC-specific Cre (SMC-CreERT2) alleles and homozygous floxed Tgfbr2 alleles. Mice were injected with tamoxifen, and their aortas examined 4 and 14 weeks later. Both SMC-CreERT2 alleles efficiently and specifically rearranged a floxed reporter gene and efficiently rearranged a floxed Tgfbr2 allele, resulting in loss of aortic medial TBRII protein. Loss of SMC TBRII caused severe aortopathy, including hemorrhage, ulceration, dissection, dilation, accumulation of macrophage markers, elastolysis, abnormal proteoglycan accumulation, and aberrant SMC gene expression. All areas of the aorta were affected, with the most severe pathology in the ascending aorta. Cre-mediated loss of SMC TBRII in vitro ablated both canonical and noncanonical TGF-&bgr; signaling and reproduced some of the gene expression abnormalities detected in vivo. Conclusions—SMC TBRII plays a critical role in maintaining postnatal aortic homeostasis. Loss of SMC TBRII disrupts TGF-&bgr; signaling, acutely alters SMC gene expression, and rapidly results in severe and durable aortopathy. These results suggest that pharmacological blockade of TGF-&bgr; signaling in humans could cause aortic disease rather than prevent it.

Aortopathy in a Mouse Model of Marfan Syndrome Is Not Mediated by Altered Transforming Growth Factor β 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‐β (TGF‐β) signaling in aortic medial smooth muscle cells (SMC). However, there are many reasons to doubt that TGF‐β signaling drives MFS‐associated aortopathy. We used a mouse model to test whether SMC TGF‐β signaling is perturbed by a fibrillin‐1 variant that causes MFS and whether blockade of SMC TGF‐β signaling prevents MFS‐associated aortopathy. Methods and Results MFS mice (Fbn1 C1039G/+ genotype) were genetically modified to allow postnatal SMC‐specific deletion of the type II TGF‐β receptor (TBRII; essential for physiologic TGF‐β signaling). In young MFS mice with and without superimposed deletion of SMC‐TBRII, we measured aortic dimensions, histopathology, activation of aortic SMC TGF‐β signaling pathways, and changes in aortic SMC gene expression. Young Fbn1 C1039G/+ 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‐β signaling was unaltered in aortic SMC of young MFS mice; however, SMC‐specific deletion of TBRII in Fbn1 C1039G/+ mice significantly decreased activation of SMC TGF‐β signaling pathways. Conclusions In young Fbn1 C1039G/+ mice, aortopathy develops in the absence of detectable alterations in SMC TGF‐β signaling. Loss of physiologic SMC TGF‐β signaling exacerbates MFS‐associated aortopathy. Our data support a protective role for SMC TGF‐β signaling during early development of MFS‐associated aortopathy.

Level of Macrophage uPA Expression Is an Important Determinant of Atherosclerotic Lesion Growth in Apoe −/− Mice

Objective—Enhanced plasminogen activation, mediated by overexpression of urokinase-type plasminogen activator (uPA), accelerates atherosclerosis in apolipoprotein E–null mice. However, the mechanisms through which uPA acts remain unclear. In addition, although elevated uPA expression can accelerate murine atherosclerosis, there is not yet any evidence that decreased uPA expression would retard atherosclerosis. Methods and Results—We used a bone marrow transplant (BMT) approach and apolipoprotein E–deficient (Apoe−/−) mice to investigate cellular mechanisms of uPA-accelerated atherosclerosis, aortic dilation, and sudden death. We also used BMT to determine whether postnatal loss of uPA expression in macrophages retards atherosclerosis. BMT from uPA-overexpressing mice yielded recipients with macrophage-specific uPA overexpression; whereas BMT from uPA knockout mice yielded recipients with macrophage-specific loss of uPA expression. Recipients of uPA-overexpressing BM acquired all the vascular phenotypes (accelerated atherosclerosis, aortic medial destruction and dilation, severe coronary stenoses) as well as the sudden death phenotype of uPA-overexpressing mice. Moreover, fat-fed 37-week-old recipients of uPA-null BM had significantly less atherosclerosis than recipients of uPA wild-type marrow (40% less aortic surface lesion area; P=0.03). Conclusions—The level of uPA expression by macrophages—over a broad range—is an important determinant of atherosclerotic lesion growth in Apoe−/− mice.

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

Objective— The role of TGF-&bgr; (transforming growth factor-&bgr;) signaling in abdominal aortic aneurysm (AAA) formation is controversial. Others reported that systemic blockade of TGF-&bgr; by neutralizing antibodies accelerated AAA development in angiotensin II-infused mice. This result is consistent with other studies suggesting that TGF-&bgr; signaling prevents AAA. Development of a therapy for AAA that exploits the protective actions of TGF-&bgr; would be facilitated by identification of the mechanisms through which TGF-&bgr; prevents AAA. We hypothesized that TGF-&bgr; 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-&bgr; blockade), mice with antibody-mediated systemic neutralization of TGF-&bgr;, and mice with genetically based smooth muscle–specific loss of TGF-&bgr; signaling. Surprisingly, we found that systemic—but not smooth muscle–specific—TGF-&bgr; 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-&bgr; 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-&bgr; neutralization—significantly accelerated development of aortic pathology, including increased prevalence of intramural hematomas, medial thinning, and adventitial thickening. Conclusion— Our results suggest that TGF-&bgr; 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.

What’s the Skinny on Elastin Deficiency and Supravalvular Aortic Stenosis?

Supravalvular aortic stenosis (SVAS), a localized narrowing of the aorta just distal to the aortic valve, has been encountered sporadically for over 130 years.1,2 In 1961, Williams reported 4 patients with SVAS who shared syndromic features, including short stature, mental retardation, peripheral pulmonary artery stenoses, and a characteristic facies.3 Beuren et al4 reported additional cases, and this syndrome is now known both as Williams syndrome (WS) and Williams–Beuren syndrome. Familial clustering of nonsyndromic SVAS5,6 and WS7 was later identified, bolstering the notion that both WS and SVAS are genetically based. See accompanying article on page 930 Williams hypothesized that SVAS was caused by a combination of medial hypertrophy and aortic wall constriction, but proposed no underlying mechanisms. Others proposed intimal fibrous thickening, due to hyperplasia and excessive collagen deposition (Figure [A]), as potential pathogeneses.9,10 However, the mechanisms that might account for cell proliferation and fibrosis remained uncertain. In 1993, Keating’s group discovered that (1) individuals with both familial and sporadic WS were hemizygous at the elastin locus11 and (2) members of a family with SVAS had a balanced translocation that disrupted the elastin gene and cosegregated with the SVAS phenotype.12 Because elastin is a major component of the aortic wall, comprising ≈50% of total protein,13 and because aortas of individuals with SVAS have decreased elastin content and abnormal elastin architecture,10 elastin deficiency seemed the obvious underlying cause of SVAS. This conclusion was solidified by detection of elastin point mutations in individuals with SVAS.14 Keating’s group speculated that SVAS associated with elastin deficiency was caused by decreased aortic elasticity leading to endothelial dysfunction, and that this nondenuding endothelial injury led to intimal proliferation, fibrosis, and stenosis (Figure [B]).12 Figure. Models of pathogenesis of aortic …