The FAT1 Cadherin Drives Vascular Smooth Muscle Cell Migration.

Vascular smooth muscle cells (VSMCs) are normally quiescent and non-migratory, regulating the contraction and relaxation of blood vessels to control the vascular tone. In response to arterial injury, these cells become active; they proliferate, secrete matrix proteins, and migrate, and thereby contribute importantly to the progression of several cardiovascular diseases. VSMC migration specifically supports atherosclerosis, restenosis after catheter-based intervention, transplant vasculopathy, and vascular remodeling during the formation of aneurysms. The atypical cadherin FAT1 is expressed robustly in activated VSMCs and promotes their migration. A positive role of FAT1 in the migration of other cell types, including neurons, fibroblasts, podocytes, and astrocyte progenitors, has also been described. In cancer biology, however, the effect of FAT1 on migration depends on the cancer type or context, as FAT1 either suppresses or enhances cancer cell migration and invasion. With this review, we describe what is known about FAT1's effects on cell migration as well as the factors that influence FAT1-dependent migration. In VSMCs, these factors include angiotensin II, which activates FAT1 expression and cell migration, and proteins of the Atrophin family: Atrophin-1 and the short isoform of Atrophin-2, which promote VSMC migration, and the long isoform of Atrophin-2, which exerts negative effects on FAT1-dependent VSMC migration.

The Atypical Cadherin FAT1 Limits Mitochondrial Respiration and Proliferation of Vascular Smooth Muscle Cells.

Smooth muscle cells contribute to cardiovascular disease, the leading cause of death worldwide. The capacity of these cells to undergo phenotypic switching in mature arteries of the systemic circulation underlies their pathogenic role in atherosclerosis and restenosis, among other vascular diseases. Growth factors and cytokines, extracellular matrix components, regulation of gene expression, neuronal influences, and mechanical forces contribute to smooth muscle cell phenotypic switching. Comparatively little is known about cell metabolism in this process. Studies of cancer and endothelial cell biology have highlighted the importance of cellular metabolic processes for phenotypic transitions that accompany tumor growth and angiogenesis. However, the understanding of cell metabolism during smooth muscle cell phenotypic modulation is incipient. Studies of the atypical cadherin FAT1, which is strongly upregulated in smooth muscle cells in response to arterial injury, suggest that it has important and distinctive functions in this context, mediating control of both smooth muscle cell mitochondrial metabolism and cell proliferation. Here we review the progress made in understanding how FAT1 affects the smooth muscle cell phenotype, highlighting the significance of FAT1 as a processed protein and unexpected regulator of mitochondrial respiration. These mechanisms suggest how a transmembrane protein may relay signals from the extracellular milieu to mitochondria to control metabolic activity during smooth muscle cell phenotypic switching.

Allograft inflammatory factor-1 supports macrophage survival and efferocytosis and limits necrosis in atherosclerotic plaques.

BACKGROUND AND AIMS: Allograft inflammatory factor-1 (AIF1) has been characterized as a pro-inflammatory molecule expressed primarily in the monocyte/macrophage (MP) lineage and positively associated with various forms of vascular disease, including atherosclerosis. Studies of AIF1 in atherosclerosis have relied on mouse models in which AIF1 was overexpressed in either myeloid or smooth muscle cells, resulting in increased atherosclerotic plaque burden. How physiologic expression of AIF1 contributes to MP biology in atherogenesis is not known. METHODS: Effects of global AIF1 deficiency on atherosclerosis were assessed by crossing Aif1-/- and ApoE-/- mice, and provoking hyperlipidemia with high fat diet feeding. Atherosclerotic plaques were studied en face and in cross section. Bone marrow-derived MPs (BMDMs) were isolated from Aif1-/- mice for study in culture. RESULTS: Atherosclerotic plaques in Aif1-/-;ApoE-/- mice showed larger necrotic cores compared to those in ApoE-/- animals, without change in overall lesion burden. In vitro, lack of AIF1 reduced BMDM survival, phagocytosis, and efferocytosis. Mechanistically, AIF1 supported activation of the NF-κB pathway and expression of related target genes involved in stress response, inflammation, and apoptosis. Consistent with this in vitro BMDM phenotype, AIF1 deficiency reduced NF-κB pathway activity in vivo and increased apoptotic cell number in atherosclerotic lesions from Aif1-/-;ApoE-/- mice. CONCLUSIONS: These findings characterize AIF1 as a positive regulator of the NF-κB pathway that supports MP functions such as survival and efferocytosis. In inflammatory settings such as atherosclerosis, these AIF1-dependent activities serve to clear cellular and other debris and limit necrotic core expansion, and may oppose lesion destabilization.

Allograft Inflammatory Factor-1 Links T-Cell Activation, Interferon Response, and Macrophage Activation in Chronic Kawasaki Disease Arteritis.

BACKGROUND: Kawasaki disease (KD) is widely viewed as an acute arteritis. However, our pathologic studies show that chronic coronary arteritis can persist long after disease onset and is closely linked with arterial stenosis. Transcriptome profiling of acute KD arteritis tissues revealed upregulation of T lymphocyte, type I interferon, and allograft inflammatory factor-1 (AIF1) genes. We determined whether these immune responses persist in chronic KD arteritis, and we investigated the role of AIF1 in these responses. METHODS: Gene expression in chronic KD and childhood control arteries was determined by real-time reverse-transcriptase polymerase chain reaction, and arterial protein expression was determined by immunohistochemistry and immunofluorescence. Allograft inflammatory factor-1 small-interfering ribonucleic acid macrophage treatment was performed to investigate the role of AIF1 in macrophage and T lymphocyte activation. RESULTS: Allograft inflammatory factor-1 protein was highly expressed in stenotic KD arteries and colocalized with the macrophage marker CD68. T lymphocyte and interferon pathway genes were significantly upregulated in chronic KD coronary artery tissues. Alpha interferon-induced macrophage expression of CD80 and major histocompatibility complex class II was dependent on AIF1, and macrophage expression of AIF1 was required for antigen-specific T lymphocyte activation. CONCLUSIONS: Allograft inflammatory factor-1, originally identified in posttransplant arterial stenosis, is markedly upregulated in KD stenotic arterial tissues. T lymphocyte and type I interferon responses persist in chronic KD arteritis. Allograft inflammatory factor-1 may play multiple roles linking type I interferon response, macrophage activation, and antigen-specific T lymphocyte activation. These results suggest the likely importance of lymphocyte-myeloid cell cross-talk in the pathogenesis of KD arteritis and can inform selection of new immunotherapies for clinical trials in high-risk KD children.

Control of mitochondrial function and cell growth by the atypical cadherin Fat1.

Mitochondrial products such as ATP, reactive oxygen species, and aspartate are key regulators of cellular metabolism and growth. Abnormal mitochondrial function compromises integrated growth-related processes such as development and tissue repair, as well as homeostatic mechanisms that counteract ageing and neurodegeneration, cardiovascular disease, and cancer. Physiologic mechanisms that control mitochondrial activity in such settings remain incompletely understood. Here we show that the atypical Fat1 cadherin acts as a molecular 'brake' on mitochondrial respiration that regulates vascular smooth muscle cell (SMC) proliferation after arterial injury. Fragments of Fat1 accumulate in SMC mitochondria, and the Fat1 intracellular domain interacts with multiple mitochondrial proteins, including critical factors associated with the inner mitochondrial membrane. SMCs lacking Fat1 (Fat1KO) grow faster, consume more oxygen for ATP production, and contain more aspartate. Notably, expression in Fat1KO cells of a modified Fat1 intracellular domain that localizes exclusively to mitochondria largely normalizes oxygen consumption, and the growth advantage of these cells can be suppressed by inhibition of mitochondrial respiration, which suggest that a Fat1-mediated growth control mechanism is intrinsic to mitochondria. Consistent with this idea, Fat1 species associate with multiple respiratory complexes, and Fat1 deletion both increases the activity of complexes I and II and promotes the formation of complex-I-containing supercomplexes. In vivo, Fat1 is expressed in injured human and mouse arteries, and inactivation of SMC Fat1 in mice potentiates the response to vascular damage, with markedly increased medial hyperplasia and neointimal growth, and evidence of higher SMC mitochondrial respiration. These studies suggest that Fat1 controls mitochondrial activity to restrain cell growth during the reparative, proliferative state induced by vascular injury. Given recent reports linking Fat1 to cancer, abnormal kidney and muscle development, and neuropsychiatric disease, this Fat1 function may have importance in other settings of altered cell growth and metabolism.

ß-Catenin C-terminal signals suppress p53 and are essential for artery formation.

Increased activity of the tumour suppressor p53 is incompatible with embryogenesis, but how p53 is controlled is not fully understood. Differential requirements for p53 inhibitors Mdm2 and Mdm4 during development suggest that these control mechanisms are context-dependent. Artery formation requires investment of nascent endothelial tubes by smooth muscle cells (SMCs). Here, we find that embryos lacking SMC ß-catenin suffer impaired arterial maturation and die by E12.5, with increased vascular wall p53 activity. ß-Catenin-deficient SMCs show no change in p53 levels, but greater p53 acetylation and activity, plus impaired growth and survival. In vivo, SMC p53 inactivation suppresses phenotypes caused by loss of ß-catenin. Mechanistically, ß-catenin C-terminal interactions inhibit Creb-binding protein-dependent p53 acetylation and p53 transcriptional activity, and are required for artery formation. Thus in SMCs, the ß-catenin C-terminus indirectly represses p53, and this function is essential for embryogenesis. These findings have implications for angiogenesis, tissue engineering and vascular disease.

FAT1 mutations cause a glomerulotubular nephropathy.

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease (CKD). Here we show that recessive mutations in FAT1 cause a distinct renal disease entity in four families with a combination of SRNS, tubular ectasia, haematuria and facultative neurological involvement. Loss of FAT1 results in decreased cell adhesion and migration in fibroblasts and podocytes and the decreased migration is partially reversed by a RAC1/CDC42 activator. Podocyte-specific deletion of Fat1 in mice induces abnormal glomerular filtration barrier development, leading to podocyte foot process effacement. Knockdown of Fat1 in renal tubular cells reduces migration, decreases active RAC1 and CDC42, and induces defects in lumen formation. Knockdown of fat1 in zebrafish causes pronephric cysts, which is partially rescued by RAC1/CDC42 activators, confirming a role of the two small GTPases in the pathogenesis. These findings provide new insights into the pathogenesis of SRNS and tubulopathy, linking FAT1 and RAC1/CDC42 to podocyte and tubular cell function.

Donor and recipient cell surface colony stimulating factor-1 promote neointimal formation in transplant-associated arteriosclerosis.

OBJECTIVE: Transplant-associated arteriosclerosis manifests as progressive vascular neointimal expansion throughout the arterial system of allografted solid organs, and eventually compromises graft perfusion and function. Allografts placed in colony stimulating factor (CSF)-1-deficient osteopetrotic (Csf1(op)/Csf1(op)) mice develop very little neointima, a finding attributed to impaired recipient macrophage function. We examined how CSF-1 affects neointima-derived vascular smooth muscle cells, tested the significance of CSF-1 expressed in donor tissue, and evaluated the contribution of secreted versus cell surface CSF-1 isoforms in transplant-associated arteriosclerosis. METHODS AND RESULTS: CSF-1 activated specific signaling pathways to promote migration, survival, and proliferation of cultured vascular smooth muscle cells. Tumor necrosis factor-α addition increased CSF-1 and CSF-1 receptor expression, and tumor necrosis factor-α-driven proliferation was blocked by anti-CSF-1 antibody. In a mouse vascular allograft model, lack of recipient or donor CSF-1 impaired neointima formation; the latter suggests local CSF-1 function within the allograft. Moreover, reconstitution of donor or recipient cell surface CSF-1, without secreted CSF-1, restored neointimal formation. CONCLUSIONS: Vascular smooth muscle cells activation, including that mediated by tumor necrosis factor-α, can be driven in an autocrine/juxtacrine manner by CSF-1. These studies provide evidence for local function of CSF-1 in neointimal expansion, and identify CSF-1 signaling in vascular smooth muscle cells, particularly cell surface CSF-1 signaling, as a target for therapeutic strategies in transplant-associated arteriosclerosis.

Absence of heme oxygenase-1 exacerbates atherosclerotic lesion formation and vascular remodeling.

To examine the role of heme oxygenase (HO)-1 in the pathophysiology of vascular diseases, we generated mice deficient in both HO-1 and apolipoprotein E (HO-1-/-apoE-/-). Despite similar total plasma cholesterol levels in response to hypercholesterolemia, HO-1-/-apoE-/- mice, in comparison with HO-1+/+apoE-/- mice, had an accelerated and more advanced atherosclerotic lesion formation. In addition to greater lipid accumulation, these advanced lesions from HO-1-/-apoE-/- mice contained macrophages and smooth muscle alpha-actin-positive cells. We further tested the role of HO-1 on neointimal formation in a mouse model of vein graft stenosis. Autologous vein grafts in HO-1-/- mice showed robust neointima consisting of alpha-actin-positive vascular smooth muscle cells (VSMC) 10 days after surgery in comparison to the smaller neointima formed in autologous vein grafts in HO-1+/+ mice. However, at 14 days after surgery, the neointima from composite vessels of HO-1-/- mice was composed mainly of acellular material, indicative of substantial VSMC death. VSMC isolated from HO-1-/- mice were susceptible to oxidant stress, leading to cell death. Our data demonstrate that HO-1 plays an essential protective role in the pathophysiology of atherosclerosis and vein graft stenosis.

Macrophage-restricted and interferon gamma-inducible expression of the allograft inflammatory factor-1 gene requires Pu.1.

Expression of allograft inflammatory factor-1 (Aif-1), a 17-kDa protein bearing an EF-hand Ca(2+) binding motif, increases markedly in monocytes and macrophages participating in allo- and autoimmune reactions, including the perivascular inflammation in transplanted hearts, microglial infiltrates in experimental autoimmune neuritis, and the inflamed pancreas of prediabetic BB rats. To investigate the mechanism of this regulation, we isolated the mouse aif-1 gene and determined its genomic organization. The gene has six exons distributed over 1.6 kilobases, an interferon gamma-inducible DNase I-hypersensitive site near -900, and flanking sequences on either side predicted to associate with nuclear matrix. Reporter gene analyses identified sequences between -902 and -789, including consensus Ets and interferon regulatory factor elements, required for macrophage-specific and interferon gamma-inducible transcriptional activity. Pu.1 bound to the Ets site in electromobility shift assay and forced expression of Pu.1 activated the aif-1 promoter in 3T3 fibroblasts, in which it is normally inactive. However, the transcriptional activity of a concatamer of the Ets site alone did not increase with interferon gamma treatment. Cooperation between Pu.1 and proteins binding to the interferon regulatory factor element appears to be necessary for both macrophage-specific and interferon gamma-inducible expression of the aif-1 gene.

Cyclin A transcriptional suppression is the major mechanism mediating homocysteine-induced endothelial cell growth inhibition.

Previously, it was reported that homocysteine (Hcy) specifically inhibits the growth of endothelial cells (ECs), suppresses Ras/mitogen-activated protein (MAP) signaling, and arrests cell growth at the G(1)/S transition of the cell cycle. The present study investigated the molecular mechanisms underlying this cell-cycle effect. Results showed that clinically relevant concentrations (50 microM) of Hcy significantly inhibited the expression of cyclin A messenger RNA (mRNA) in ECs in a dose- and time-dependent manner. G(1)/S-associated molecules that might account for this block were not changed, because Hcy did not affect mRNA and protein expression of cyclin D1 and cyclin E. Cyclin D1- and E-associated kinase activities were unchanged. In contrast, cyclin A-associated kinase activity and CDK2 kinase activity were markedly suppressed. Nuclear run-on assay demonstrated that Hcy decreased the transcription rate of the cyclin A gene but had no effect on the half-life of cyclin A mRNA. In transient transfection experiments, Hcy significantly inhibited cyclin A promoter activity in endothelial cells, but not in vascular smooth muscle cells. Finally, adenovirus-transduced cyclin A expression restored EC growth inhibition and overcame the S phase block imposed by Hcy. Taken together, these findings indicate that cyclin A is a critical functional target of Hcy-mediated EC growth inhibition.