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 α‐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 α‐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.

Activated macrophages are critical cellular participants in inflammatory disease states. Transforming growth factor (TGF)-beta1 is a growth factor with pleiotropic effects including inhibition of immune cell activation. Although the pathway of gene activation by TGF-beta1 via Smad proteins has recently been elucidated, suppression of gene expression by TGF-beta1 remains poorly understood. We found that of Smad1-Smad7, Smad3 alone was able to inhibit expression of markers of macrophage activation (inducible nitric-oxide synthase and matrix metalloproteinase-12) following lipopolysaccharide treatment in gene reporter assays. Transient and constitutive overexpression of a dominant negative Smad3 opposed the inhibitory effect of TGF-beta1. Domain swapping experiments suggest that both the Smad MH-1 and MH-2 domains are required for inhibition. Mutation of a critical amino acid residue required for DNA binding in the MH-1 of Smad3 (R74A) resulted in the loss of inhibition. Transient overexpression of p300, an interactor of the Smad MH-2 domain, partially alleviated the inhibition by TGF-beta1/Smad3, suggesting that inhibition of gene expression may be due to increased competition for limiting amounts of this coactivator. Our results have implications for the understanding of gene suppression by TGF-beta1 and for the regulation of activated macrophages by TGF-beta1.

Homocysteine is an important and independent risk factor for arteriosclerosis. We showed previously that homocysteine stimulates vascular smooth muscle cell proliferation, a hallmark of arteriosclerosis. We show here that homocysteine and serum increased DNA synthesis synergistically in both human and rat aortic smooth muscle cells (RASMCs). Treatment of quiescent RASMCs with 1 mM homocysteine or 2% calf serum for 36 h increased cyclin A mRNA levels by 8- and 14-fold, respectively, whereas homocysteine plus serum increased cyclin A mRNA levels by 40-fold, indicating a synergistic induction of cyclin A mRNA. Homocysteine did not increase the half-life of cyclin A mRNA (2.9 h), but it did increase the transcriptional rate of the cyclin A gene in nuclear run-on experiments. The positive effect of homocysteine on cyclin A gene transcription was confirmed by our finding that homocysteine increased cyclin A promoter activity and ATF-binding protein levels in RASMCs. Finally, 1 mM homocysteine increased cyclin A protein levels and cyclin A-associated kinase activity by threefold. This homocysteine-induced expression lesions by promoting proliferation of vascular smooth muscle cells.

Matrix metalloproteinases (MMP) have been identified in vulnerable areas of atherosclerotic plaques and may contribute to plaque instability through extracellular matrix degradation. Human metalloelastase (MMP-12) is a macrophage-specific MMP with broad substrate specificity and is capable of degrading proteins found in the extracellular matrix of atheromas. Despite its potential importance, little is known about the regulation of MMP-12 expression in the context of atherosclerosis. In this study, we report that in human peripheral blood-derived macrophages, MMP-12 mRNA was markedly up-regulated by several pro-atherosclerotic cytokines and growth factors including interleukin-1beta, tumor necrosis factor-alpha, macrophage colony-stimulating factor, vascular endothelial growth factor, and platelet-derived growth factor-BB. In contrast, the pleiotropic anti-inflammatory growth factor transforming growth factor-beta1 (TGF-beta1) inhibited cytokine-mediated induction of MMP-12 mRNA, protein, and enzymatic activity. Analyses of MMP-12 promoter through transient transfections and electrophoretic mobility shift assays indicated that both its induction by cytokines and its inhibition by TGF-beta1 depended on signaling through an AP-1 site at -81 base pairs. Moreover, the inhibitory effect of TGF-beta1 on MMP-12 was dependent on Smad3. Taken together, MMP-12 is induced by several factors implicated in atherosclerosis. The inhibition of MMP-12 expression by TGF-beta1 suggests that TGF-beta1, acting via Smad3, may promote plaque stability.

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.