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

[Figure: see text].

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.

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

OBJECTIVE: Prenatal deletion of the type II transforming growth factor-ß (TGF-ß) 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-ß signaling, and blockade of TGF-ß 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-CreER(T2)) alleles and homozygous floxed Tgfbr2 alleles. Mice were injected with tamoxifen, and their aortas examined 4 and 14 weeks later. Both SMC-CreER(T2) 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-ß 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-ß signaling, acutely alters SMC gene expression, and rapidly results in severe and durable aortopathy. These results suggest that pharmacological blockade of TGF-ß signaling in humans could cause aortic disease rather than prevent it.

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.

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-[beta]1 limits plaque growth, stabilizes plaque structure, and prevents aortic dilation in apolipoprotein E-null mice.

OBJECTIVE: Impairment of transforming growth factor (TGF)-beta1 signaling accelerates atherosclerosis in experimental mice. However, it is uncertain whether increased TGF-beta1 expression would retard atherosclerosis. The role of TGF-beta1 in aneurysm formation is also controversial. We tested whether overexpression of active TGF-beta1 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-beta1 in their hearts. Compared to littermate controls, these mice had elevated cardiac and plasma TGF-beta1, less aortic root atherosclerosis (P< or =0.002), fewer lesions in the thoracic and abdominal aortae (P< or =0.01), less aortic root dilation (P<0.001), and fewer pseudoaneurysms (P=0.02). Mechanistic studies revealed no effect of TGF-beta1 overexpression on plasma lipids or cytokines, or on peripheral lymphoid organ cells. However, aortae of TGF-beta1-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-beta1 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.

Systematic RNAi studies on the role of Sp/KLF factors in globin gene expression and erythroid differentiation.

Sp/KLF family of factors regulates gene expression by binding to the CACCC/GC/GT boxes in the DNA through their highly conserved three zinc finger domains. To investigate the role of this family of factors in erythroid differentiation and globin gene expression, we first measured the expression levels of selected Sp/KLF factors in primary cells of fetal and adult stages of erythroid development. This quantitative analysis revealed that their expression levels vary significantly in cells of either stages of the erythroid development. Significant difference in their expression levels was observed between fetal and adult erythroid cells for some Sp/KLF factors. Functional studies using RNA interference revealed that the silencing of Sp1 and KLF8 resulted in elevated level of gamma globin expression in K562 cells. In addition, K562 cells become visibly red after Sp1 knockdown. Benzidine staining revealed significant hemoglobinization of these cells, indicating erythroid differentiation. Moreover, the expression of PU.1, ETS1 and Notch1 is significantly down-regulated in the cells that underwent erythroid differentiation following Sp1 knockdown. Overexpression of PU.1 or ETS1 efficiently blocked the erythroid differentiation caused by Sp1 knockdown in K562 cells. The expression of c-Kit, however, was significantly up-regulated. These data indicate that Sp1 may play an important role in erythroid differentiation.

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.

Reduction of mouse atherosclerosis by urokinase inhibition or with a limited-spectrum matrix metalloproteinase inhibitor.

AIMS: Elevated activity of urokinase plasminogen activator (uPA) and MMPs in human arteries is associated with accelerated atherosclerosis, aneurysms, and plaque rupture. We used Apoe-null mice with macrophage-specific uPA overexpression (SR-uPA mice; a well-characterized model of protease-accelerated atherosclerosis) to investigate whether systemic inhibition of proteolytic activity of uPA or a subset of MMPs can reduce protease-induced atherosclerosis and aortic dilation. METHODS AND RESULTS: SR-uPA mice were fed a high-fat diet for 10 weeks and treated either with an antibody inhibiting mouse uPA (mU1) or a control antibody. mU1-treated mice were also compared with PBS-treated non-uPA-overexpressing Apoe-null mice. Other SR-uPA mice were treated with one of three doses of a limited-spectrum synthetic MMP inhibitor (XL784) or vehicle. mU1 reduced aortic root intimal lesion area (20%; P = 0.05) and aortic root circumference (12%; P = 0.01). All XL784 doses reduced aortic root intimal lesion area (22-29%) and oil-red-O-positive lesion area (36-42%; P < 0.05 for all doses and both end points), with trends towards reduced aortic root circumference (6-10%). Neither mU1 nor XL784 significantly altered percent aortic surface lesion coverage. Several lines of evidence identified MMP-13 as a mediator of uPA-induced aortic MMP activity. CONCLUSIONS: Pharmacological inhibition of either uPA or selected MMPs decreased atherosclerosis in SR-uPA mice. uPA inhibition decreased aortic dilation. Differential effects of both agents on aortic root vs. distal aortic atherosclerosis suggest prevention of atherosclerosis progression vs. initiation. Systemic inhibition of uPA or a subset of MMPs shows promise for treating atherosclerosis.

Lumbar motoneuron fate in a mouse model of amyotrophic lateral sclerosis.

Onuf's nucleus, a collection of motoneurons within the spinal cord, is often spared in the neurodegenerative disorder amyotrophic lateral sclerosis. To assess whether these cells survive in a rodent model of this disease, motoneurons were counted in the spinal nucleus of the bulbocavernosus (an homologous structure to Onuf's), as well as in two other cell groups at the same level of the spinal cord, the dorsolateral nucleus and the retrodorsolateral nucleus. In mice displaying signs of neurodegeneration, both the dorsolateral and retrodorsolateral nuclei displayed significant motoneuron loss compared to controls; this cell loss was particularly exaggerated in the retrodorsolateral nucleus of animals displaying a rapid disease progression. However, no significant decline in motoneuron number was observed in the spinal nucleus of the bulbocavernosus, and the perineal muscle bulbocavernosus, which is innervated by this nucleus, appeared to be unaffected. This was in stark contrast to the thigh muscles, which displayed significant atrophy. Overall, these data indicate that the spinal nucleus of the bulbocavernosus is spared from degeneration in an animal model of amyotrophic lateral sclerosis, paralleling observations in patients suffering from this disease. Further study of this resistance to motoneuron loss may provide useful insights into the pathophysiology of the degenerative process.

Protein phosphorylation networks in motor neuron death.

The disorder amyotrophic lateral sclerosis (ALS) is characterized by the death of specific groups of neurons, especially motor neurons, which innervate skeletal muscle, and neurons connecting the cerebral cortex with motor neurons, such as corticospinal tract neurons. There have been numerous attempts to elucidate why there is selective involvement of motor neurons in ALS. Recent observations have demonstrated altered activities and protein levels of diverse kinases in the brain and spinal cord of transgenic mice that overexpress a mutant superoxide dismutase (mSOD) gene that is found in patients with the familial form of ALS, as well as in patients who have died with ALS. These results suggest that the alteration of protein phosphorylation may be involved in the pathogenesis of ALS. The changes in protein kinase and phosphatase expression and activity can affect the activation of important neuronal neurotransmitter receptors such as NMDA receptors or other signaling proteins and can trigger, or modify, the process producing neuronal loss in ALS. These various kinases, phosphatases and signaling proteins are involved in many signaling pathways; however, they have close interactions with each other. Therefore, an understanding of the role of protein kinases and protein phosphatases and the molecular organization of protein phosphorylation networks are useful to determine the mechanisms of selective motor neuron death.

Sources of cells that contribute to atherosclerotic intimal calcification: an in vivo genetic fate mapping study.

AIMS: Vascular cartilaginous metaplasia and calcification are common in patients with atherosclerosis. However, sources of cells contributing to the development of this complication are currently unknown. In this study, we ascertained the origin of cells that give rise to cartilaginous and bony elements in atherosclerotic vessels. METHODS AND RESULTS: We utilized genetic fate mapping strategies to trace cells of smooth muscle (SM) origin via SM22α-Cre recombinase and Rosa26-LacZ Cre reporter alleles. In animals expressing both transgenes, co-existence within a single cell of ß-galactosidase [marking cells originally derived from SM cells (SMCs)] with osteochondrogenic (Runx2/Cbfa1) or chondrocytic (Sox9, type II collagen) markers, along with simultaneous loss of SM lineage proteins, provides a strong evidence supporting reprogramming of SMCs towards osteochondrogenic or chondrocytic differentiation. Using this technique, we found that vascular SMCs accounted for ~80% of Runx2/Cbfa1-positive cells and almost all of type II collagen-positive cells (~98%) in atherosclerotic vessels of LDLr-/- and ApoE-/- mice. We also assessed contribution from bone marrow (BM)-derived cells via analysing vessels dissected from chimerical ApoE-/- mice transplanted with green fluorescence protein-expressing BM. Marrow-derived cells were found to account for ~20% of Runx2/Cbfa1-positive cells in calcified atherosclerotic vessels of ApoE-/- mice. CONCLUSION: Our results are the first to definitively identify cell sources attributable to atherosclerotic intimal calcification. SMCs were found to be a major contributor that reprogrammed its lineage towards osteochondrogenesis. Marrow-derived cells from the circulation also contributed significantly to the early osteochondrogenic differentiation in atherosclerotic vessels.

Protein kinase and protein phosphatase expression in the central nervous system of G93A mSOD over-expressing mice.

The expressions of 78 protein kinases, 24 protein phosphatases and 31 phosphoproteins were investigated by Kinetworks trade mark analysis in brain and spinal cord tissue of transgenic mice over-expressing G93A mutant superoxide dismutase (mSOD), a murine model of amyotrophic lateral sclerosis (ALS). In the brains of affected mSOD mice, we observed increased expression of cAMP-dependent protein kinase (PKA, 111% increase compared with control), and protein phosphatase 2B Aalpha-catalytic subunit (calcineurin, 109% increase), and reductions in the levels of PAK3 (76% decrease) and protein phosphatase 2C Cbeta-subunit (32% decrease). Increased Ser259 phosphorylation of Raf1 (126% increase) in mSOD mice correlated with higher expression of p73 Raf1 (147% increase). There was also increased p73 Raf1 (69% increase) and Ser259 phosphorylation (45% increase) in the spinal cords of mSOD mice. While adducin underwent enhanced phosphorylation (alphaS724, 90% increase; gammaS662, 290% increase) in mSOD brain, its phosphorylation was lower in the mSOD spinal cord (alphaS724, 53% decrease; gammaS662, 46% decrease). In spinal cords of affected mSOD mice, we also observed elevated expression of casein kinase 1delta (CK1delta, 157% increase), JAK2 (84% increase), PKA (183% increase), protein kinase C (PKC) delta (123% increase), p124 PKC micro (142% increase), and RhoA kinase (221% increase), and enhanced phosphorylation of extracellular regulated kinases 1 (ERK1, T202/Y204, 90% increase), and 2 (ERK2, T185/Y187, 73% increase), p38 MAP kinase (T180/Y182, 1570% increase), and PKBalpha (T308, 154% increase; S473, 61% increase). There was also reduced phosphorylation of RB (S780, 45% decrease; S807/S811, 65% decrease), Src (Y418, 63% decrease) and p40 SAPK/JNKbeta (T183/Y185, 43% decrease). Variability in the expression of kinases, phosphatases and phosphorylation of their substrates was observed even in mutant animals having a similar phenotype. The expression and phosphorylation differences between mSOD and control mice were dissimilar to those between ALS patients and controls. This finding indicates that the activation of protein kinases and phosphoproteins is different with neuron loss in the mSOD mouse compared with that seen in patients with the sporadic form of ALS.