Carlie J.M. de Vries

Strong Induction of Members of the Chitinase Family of Proteins in Atherosclerosis Chitotriosidase and Human Cartilage gp-39 Expressed in Lesion Macrophages

Atherosclerosis is initiated by the infiltration of monocytes into the subendothelial space of the vessel wall and subsequent lipid accumulation of the activated macrophages. The molecular mechanisms involved in the anomalous behavior of macrophages in atherogenesis have only partially been disclosed. Chitotriosidase and human cartilage gp-39 (HC gp-39) are members of the chitinase family of proteins and are expressed in lipid-laden macrophages accumulated in various organs during Gaucher disease. In addition, as shown in this study, chitotriosidase and HC gp-39 can be induced with distinct kinetics in cultured macrophages. We investigated the expression of these chitinase-like genes in the human atherosclerotic vessel wall by in situ hybridizations on atherosclerotic specimens derived from femoral artery (4 specimens), aorta (4 specimens), iliac artery (3 specimens), carotid artery (4 specimens), and coronary artery (1 specimen), as well as 5 specimens derived from apparently normal vascular tissue. We show for the first time that chitotriosidase and HC gp-39 expression was strongly upregulated in distinct subsets of macrophages in the atherosclerotic plaque. The expression patterns of chitotriosidase and HC gp-39 were compared and shown to be different from the patterns observed for the extracellular matrix protein osteopontin and the macrophage marker tartrate-resistant acid phosphatase. Our data emphasize the remarkable phenotypic variation among macrophages present in the atherosclerotic lesion. Furthermore, chitotriosidase enzyme activity was shown to be elevated up to 55-fold in extracts of atherosclerotic tissue. Although a function for chitotriosidase and HC gp-39 has not been identified, we hypothesize a role in cell migration and tissue remodeling during atherogenesis.

Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes.

Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4- to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15- and 36-fold at shear stresses greater than approximately 5 dyne/cm2, whereas cyclic stretch had no effect. Prolonged LKLF induction in the absence of flow changed the expression of angiotensin-converting enzyme, endothelin-1, adrenomedullin, and endothelial nitric oxide synthase to levels similar to those observed under prolonged flow. LKLF repression by siRNA suppressed the flow response of endothelin-1, adrenomedullin, and endothelial nitric oxide synthase (P < 0.05). Thus, we demonstrate that endothelial LKLF is regulated by flow in vivo and is a transcriptional regulator of several endothelial genes that control vascular tone in response to flow.

Follistatins neutralize activin bioactivity by inhibition of activin binding to its type II receptors.

Follistatin is an activin-binding protein, which inhibits activin bioactivity in several biological systems. In the present study it is demonstrated that preincubation of iodinated activin A with follistatin, purified from porcine follicular fluid, completely abolished the binding of activin to activin type IIA, IIB2 and IIB4 receptors, and consequently to activin type IB receptor, transiently transfected in COS cells. Binding of activin A to membrane proteins on the activin-responsive P19 embryonal carcinoma cells was also prevented by this follistatin preparation. The same results were obtained with a carboxy-terminally truncated form of follistatin (FS-288), which is only present in minor amounts in the purified follistatin preparation. Since FS-288 has a high affinity for heparan sulfate proteoglycans on the cell surface, we tested whether membrane-bound FS-288 presents activin A to the different activin receptors, thereby facilitating activin binding. FS-288 did bind to the cell surface of transfected COS cells, but inhibited the binding of activin A to its receptors IIA, IIB2 and IIB4. Furthermore, after addition of FS-288 to K562 erythroleukemia cells, the total binding of activin via cell surface-bound FS-288 was increased, whereas the binding of activin A to activin type II and type I receptors present on these cells was inhibited. These findings reveal that different forms of follistatin can neutralize activin bioactivity by interference with binding of activin to all known activin type II receptors, rather than that they inhibit the binding of the type I receptor to the activin/activin type II receptor complex. In addition, our studies indicate that cell surface-associated follistatin cannot present ligand to signalling receptors.

Nuclear Receptors Nur77, Nurr1, and NOR-1 Expressed in Atherosclerotic Lesion Macrophages Reduce Lipid Loading and Inflammatory Responses

Objective—Atherosclerosis is an inflammatory disease in which macrophage activation and lipid loading play a crucial role. In this study, we investigated expression and function of the NR4A nuclear receptor family, comprising Nur77 (NR4A1, TR3), Nurr1 (NR4A2), and NOR-1 (NR4A3) in human macrophages. Methods and Results—Nur77, Nurr1, and NOR-1 are expressed in early and advanced human atherosclerotic lesion macrophages primarily in areas of plaque activation/progression as detected by in situ-hybridization and immunohistochemistry. Protein expression localizes to the nucleus. Primary and THP-1 macrophages transiently express NR4A-factors in response to lipopolysaccharide and tumor necrosis factor α. Lentiviral overexpression of Nur77, Nurr1, or NOR-1 reduces expression and production of interleukin (IL)-1β and IL-6 proinflammatory cytokines and IL-8, macrophage inflammatory protein-1α and -1β and monocyte chemoattractant protein-1 chemokines. In addition, NR4A-factors reduce oxidized–low-density lipoprotein uptake, consistent with downregulation of scavenger receptor-A, CD36, and CD11b macrophage marker genes. Knockdown of Nur77 or NOR-1 with gene-specific lentiviral short-hairpin RNAs resulted in enhanced cytokine and chemokine synthesis, increased lipid loading, and augmented CD11b expression, demonstrating endogenous NR4A-factors to inhibit macrophage activation, foam-cell formation, and differentiation. Conclusion—NR4A-factors are expressed in human atherosclerotic lesion macrophages and reduce human macrophage lipid loading and inflammatory responses, providing further evidence for a protective role of NR4A-factors in atherogenesis.

Protective function of transcription factor TR3 orphan receptor in atherogenesis: decreased lesion formation in carotid artery ligation model in TR3 transgenic mice.

Background—Smooth muscle cells (SMCs) play a key role in intimal thickening in atherosclerosis and restenosis. The precise signaling pathways by which the proliferation of SMCs is regulated are largely unknown. The TR3 orphan receptor, the mitogen-induced nuclear orphan receptor (MINOR), and the nuclear receptor of T cells (NOT) are a subfamily of transcription factors belonging to the nuclear receptor superfamily and are induced in activated SMCs. In this study, we investigated the role of these transcription factors in SMC proliferation in atherogenesis. Methods and Results—Multiple human vascular specimens at distinct stages of atherosclerosis (lesion types II to V by American Heart Association classification) derived from 14 different individuals were studied for expression of these transcription factors. We observed expression of TR3, MINOR, and NOT in neointimal SMCs, whereas no expression was detected in medial SMCs. Adenovirus-mediated expression of a dominant-negative variant of TR3, which suppresses the transcriptional activity of each subfamily member, increases DNA synthesis and decreases p27Kip1 protein expression in cultured SMCs. We generated transgenic mice that express this dominant-negative variant or full-length TR3 under control of a vascular SMC-specific promoter. Carotid artery ligation of transgenic mice that express the dominant-negative variant of TR3 in arterial SMCs, compared with lesions formed in wild-type mice, results in a 3-fold increase in neointimal formation, whereas neointimal formation is inhibited 5-fold in transgenic mice expressing full-length TR3. Conclusions—Our results reveal that TR3 and possibly other members of this transcription factor subfamily inhibit vascular lesion formation. These transcription factors could serve as novel targets in the treatment of vascular disease.

The orphan nuclear receptor NGFIB regulates transcription of 3β-hydroxysteroid dehydrogenase: Implications for the control of adrenal functional zonation

3β-Hydroxysteroid dehydrogenase type 2 (HSD3B2) is a steroid-metabolizing enzyme that is essential for adrenal production of mineralocorticoids and glucocorticoids. Thus, HSD3B2 is expressed at high levels in the glomerulosa and fasciculata, where these steroids are produced. In contrast, the production of dehydroepiandrosterone (DHEA) and DHEA sulfate in the adrenal reticularis is inversely correlated with the expression of HSD3B2. The reasons for the zonal expression of HSD3B2 are not known but represent an important aspect in the biochemical zonation of the adrenal. Using microarray, real time reverse transcriptase-PCR, immunohistochemistry, and HSD3B2 promoter analysis, we demonstrate that the NGFIB family of nuclear hormone receptors plays a critical part in the regulation of HSD3B2 transcription and may play an important role in the functional zonation of the adrenal gland. Microarray analysis of cortisol- versus DHEA sulfate-producing adrenal tissue demonstrated that NGIFB paralleled expression of HSD3B2 with expression much higher in cortisol-producing adrenal tissue; this observation was also demonstrated using real time reverse transcriptase-PCR analysis. In addition, immunohistochemistry confirmed that within adult and fetal adrenal gland NGFIB expression paralleled expression of HSD3B2. Transient transfections into H295R adrenal cells demonstrated that NGFIB family members enhanced HSD3B2 reporter activity but had no effect on a 17α-hydroxylase (CYP17) promoter construct. Deletion and mutational analyses of the 5′-flanking region of the HSD3B2 gene identified a consensus NGFIB response element that bound NGFIB in mobility shift assays. Infection of cultured human adrenal cells with adenovirus-containing NGFIB increased cortisol production by 8-fold and increased expression of HSD3B2 mRNA 26-fold over that observed in mock-infected cells. In primary cultures of adrenal cells, ACTH, an activator of HSD3B2, rapidly induced expression of NGFIB. These results suggest that NGFIB plays a crucial role in adrenal zonation by regulating HSD3B2 gene transcription.

Vascular calcification: expression patterns of the osteoblast-specific gene core binding factor α-1 and the protective factor matrix gla protein in human atherogenesis

OBJECTIVE Increasing evidence suggests that vascular calcification is a regulated process. We studied the vascular expression pattern of a key factor in mineralization and a counteracting, protective factor. Based on the phenotype of null mice, Core binding factor alpha-1 (Cbfa-1) plays a pivotal role in bone formation, whereas Matrix Gla Protein (MGP) is a potent inhibitor of vascular calcification. METHODS We investigated the expression of MGP and Cbfa-1 in cultured, human monocytic cells, endothelial cells and smooth muscle cells (SMC), as well as in normal and atherosclerotic vessel specimens. RESULTS In cultured cells MGP is expressed in endothelial cells and SMC, whereas Cbfa-1 mRNA is predominantly present in macrophages and to a lesser extent in SMC. In the normal vessel wall MGP expression is high at the luminal side and declines toward the center of the media, whereas Cbfa-1 is absent. Moderate, diffuse calcification of the aorta media was observed only in those regions where MGP is low or absent. In atherosclerotic lesions MGP is expressed in endothelial cells and SMC that form fibrous caps, but is never present in macrophages. Cbfa-1 is synthesized in regions without MGP, it is associated with calcified areas and Cbfa-1 may be considered a marker for osteoprogenitor-like cells in the vessel wall. CONCLUSIONS Our observations on MGP expression confirm and extend published data and are consistent with a protective function of MGP. Cbfa-1 expression is absent in normal medial SMC and co-localizes with neointimal macrophages and focal calcifications.

Bone Marrow–Specific Deficiency of Nuclear Receptor Nur77 Enhances Atherosclerosis

Rationale: Nuclear receptor Nur77, also known as NR4A1, TR3, or NGFI-B, is expressed in human atherosclerotic lesions in macrophages, endothelial cells, T cells and smooth muscle cells. Macrophages play a critical role in atherosclerosis and the function of Nur77 in lesion macrophages has not yet been investigated. Objective: This study aims to delineate the function of Nur77 in macrophages and to assess the effect of bone marrow–specific deficiency of Nur77 on atherosclerosis. Methods and Results: We investigated Nur77 in macrophage polarization using bone marrow-derived macrophages (BMM) from wild-type and Nur77-knockout (Nur77−/−) mice. Nur77−/− BMM exhibit changed expression of M2-specific markers and an inflammatory M1-phenotype with enhanced expression of interleukin-12, IFN&ggr;, and SDF-1&agr; and increased NO synthesis in (non)-stimulated Nur77−/− BMM cells. SDF-1&agr; expression in nonstimulated Nur77−/− BMM is repressed by Nur77 and the chemoattractive activity of Nur77−/− BMM is abolished by SDF-1&agr; inhibiting antibodies. Furthermore, Nur77−/− mice show enhanced thioglycollate-elicited migration of macrophages and B cells. The effect of bone marrow–specific deficiency of Nur77 on atherosclerosis was studied in low density lipoprotein receptor-deficient (Ldlr−/−) mice. Ldlr−/− mice with a Nur77−/−-deficient bone marrow transplant developed 2.1-fold larger atherosclerotic lesions than wild-type bone marrow–transplanted mice. These lesions contain more macrophages, T cells, smooth muscle cells and larger necrotic cores. SDF-1&agr; expression is higher in lesions of Nur77−/−-transplanted mice, which may explain the observed aggravation of lesion formation. Conclusions: In conclusion, in bone marrow–derived cells the nuclear receptor Nur77 has an anti-inflammatory function, represses SDF-1&agr; expression and inhibits atherosclerosis.

Flow-Dependent Remodeling of Small Arteries in Mice Deficient for Tissue-Type Transglutaminase: Possible Compensation by Macrophage-Derived Factor XIII

Chronic changes in blood flow induce an adaptation of vascular caliber. Thus, arteries show inward remodeling after a reduction in blood flow. We hypothesized that this remodeling depends on the crosslinking enzyme tissue-type transglutaminase (tTG). Flow-dependent remodeling was studied in wild-type (WT) and tTG-null mice using a surgically imposed change in blood flow in small mesenteric arteries. WT mice showed inward remodeling after 2 days of low blood flow, which was absent in arteries from tTG-null mice. Yet, after continued low blood flow for 7 days, inward remodeling was similar in arteries from WT and tTG-null mice. Studying the alternative pathways of remodeling, we identified a relatively high expression of the plasma transglutaminase factor XIII in arteries of WT and tTG-null mice. In addition, vessels from both WT and tTG-null mice showed the presence of transglutaminase-specific crosslinks. An accumulation of adventitial monocytes/macrophages was found in vessels exposed to low blood flow in tTG-null mice. Because monocytes/macrophages may represent a source of factor XIII, tTG-null mice were treated with liposome-encapsulated clodronate. Elimination of monocytes/macrophages with liposome-encapsulated clodronate reduced both the expression of factor XIII and inward remodeling in tTG-null mice. In conclusion, tTG plays an important role in the inward remodeling of small arteries associated with decreased blood flow. Adventitial monocytes/macrophages are a source of factor XIII in tTG-null mice and contribute to an alternative, delayed mechanism of inward remodeling when tTG is absent.

Activation of Nuclear Receptor Nur77 by 6-Mercaptopurine Protects Against Neointima Formation

Background— Restenosis is a common complication after percutaneous coronary interventions and is characterized by excessive proliferation of vascular smooth muscle cells (SMCs). We have shown that the nuclear receptor Nur77 protects against SMC-rich lesion formation, and it has been demonstrated that 6-mercaptopurine (6-MP) enhances Nur77 activity. We hypothesized that 6-MP inhibits neointima formation through activation of Nur77. Methods and Results— It is demonstrated that 6-MP increases Nur77 activity in cultured SMCs, which results in reduced [3H]thymidine incorporation, whereas Nur77 small interfering RNA knockdown partially restores DNA synthesis. Furthermore, we studied the effect of 6-MP in a murine model of cuff-induced neointima formation. Nur77 mRNA is upregulated in cuffed arteries, with optimal expression after 6 hours and elevated expression up to 7 days after vascular injury. Local perivascular delivery of 6-MP with a drug-eluting cuff significantly inhibits neointima formation in wild-type mice. Locally applied 6-MP does not affect inflammatory responses or apoptosis but inhibits expression of proliferating cell nuclear antigen and enhances protein levels of the cell-cycle inhibitor p27Kip1 in the vessel wall. An even stronger inhibition of neointima formation in response to local 6-MP delivery was observed in transgenic mice that overexpressed Nur77. In contrast, 6-MP does not alter lesion formation in transgenic mice that overexpress a dominant-negative variant of Nur77 in arterial SMCs, which provides evidence for the involvement of Nur77-like factors. Conclusions— Enhancement of the activity of Nur77 by 6-MP protects against excessive SMC proliferation and SMC-rich neointima formation. We propose that activation of the nuclear receptor Nur77 is a rational approach to treating in-stent restenosis.