Myeloid-specific IκB kinase ß deficiency decreases atherosclerosis in low-density lipoprotein receptor-deficient mice.

OBJECTIVE: Inflammatory responses are the driving force of atherosclerosis development. IκB kinase ß (IKKß), a central coordinator in inflammation through regulation of nuclear factor-κB, has been implicated in the pathogenesis of atherosclerosis. Macrophages play an essential role in the initiation and progression of atherosclerosis, yet the role of macrophage IKKß in atherosclerosis remains elusive and controversial. This study aims to investigate the impact of IKKß expression on macrophage functions and to assess the effect of myeloid-specific IKKß deletion on atherosclerosis development. METHODS AND RESULTS: To explore the issue of macrophage IKKß involvement of atherogenesis, we generated myeloid-specific IKKß-deficient low-density lipoprotein receptor-deficient mice (IKKß(ΔMye)LDLR(-/-)). Deficiency of IKKß in myeloid cells did not affect plasma lipid levels but significantly decreased diet-induced atherosclerotic lesion areas in the aortic root, brachiocephalic artery, and aortic arch of low-density lipoprotein receptor-deficient mice. Ablation of myeloid IKKß attenuated macrophage inflammatory responses and decreased atherosclerotic lesional inflammation. Furthermore, deficiency of IKKß decreased adhesion, migration, and lipid uptake in macrophages. CONCLUSIONS: The present study demonstrates a pivotal role for myeloid IKKß expression in atherosclerosis by modulating macrophage functions involved in atherogenesis. These results suggest that inhibiting nuclear factor-κB activation in macrophages may represent a feasible approach to combat atherosclerosis.

Transcriptional regulation of S phase kinase-associated protein 2 by NR4A orphan nuclear receptor NOR1 in vascular smooth muscle cells.

Members of the NR4A subgroup of the nuclear hormone receptor superfamily have emerged as key transcriptional regulators of proliferation and inflammation. NOR1 constitutes a ligand-independent transcription factor of this subgroup and induces cell proliferation; however, the transcriptional mechanisms underlying this mitogenic role remain to be defined. Here, we demonstrate that the F-box protein SKP2 (S phase kinase-associated protein 2), the substrate-specific receptor of the ubiquitin ligase responsible for the degradation of p27(KIP1) through the proteasome pathway, constitutes a direct transcriptional target for NOR1. Mitogen-induced Skp2 expression is silenced in vascular smooth muscle cells (VSMC) isolated from Nor1-deficient mice or transfected with Nor1 siRNA. Conversely, adenovirus-mediated overexpression of NOR1 induces Skp2 expression in VSMC and decreases protein abundance of its target p27. Transient transfection experiments establish that NOR1 transactivates the Skp2 promoter through a nerve growth factor-induced clone B response element (NBRE). Electrophoretic mobility shift and chromatin immunoprecipitation assays further revealed that NOR1 is recruited to this NBRE site in the Skp2 promoter in response to mitogenic stimulation. In vivo Skp2 expression is increased during the proliferative response underlying neointima formation, and this transcriptional induction depends on the expression of NOR1. Finally, we demonstrate that overexpression of Skp2 rescues the proliferative arrest of Nor1-deficient VSMC. Collectively, these results characterize Skp2 as a novel NOR1-regulated target gene and detail a previously unrecognized transcriptional cascade regulating mitogen-induced VSMC proliferation.

Deficiency of the NR4A orphan nuclear receptor NOR1 decreases monocyte adhesion and atherosclerosis.

RATIONALE: The orphan nuclear receptor NOR1 is a member of the evolutionary highly conserved and ligand-independent NR4A subfamily of the nuclear hormone receptor superfamily. Members of this subfamily have been characterized as early response genes regulating essential biological processes including inflammation and proliferation; however, the role of NOR1 in atherosclerosis remains unknown. OBJECTIVE: The goal of the present study was to determine the causal contribution of NOR1 to atherosclerosis development and to identify the mechanism by which this nuclear receptor participates in the disease process. METHODS AND RESULTS: In the present study, we demonstrate expression of NOR1 in endothelial cells of human atherosclerotic lesions. In response to inflammatory stimuli, NOR1 expression is rapidly induced in endothelial cells through a nuclear factor kappaB-dependent transactivation of the NOR1 promoter. Overexpression of NOR1 in human endothelial cells increased the expression of vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule-1, whereas NOR1 deficiency altered adhesion molecule expression in response to inflammatory stimuli. Transient transfection experiments and chromatin immunoprecipitation assays revealed that NOR1 induces VCAM-1 promoter activity by binding to a canonical response element for NR4A receptors in the VCAM-1 promoter. Further functional studies confirmed that NOR1 mediates monocyte adhesion by inducing VCAM-1 and intercellular adhesion molecule-1 expression in endothelial cells. Finally, we demonstrate that NOR1 deficiency reduces hypercholesterolemia-induced atherosclerosis formation in apoE(-/-) mice by decreasing the macrophage content of the lesion. CONCLUSIONS: In concert, these studies identify a novel pathway underlying monocyte adhesion and establish that NOR1 serves a previously unrecognized atherogenic role in mice by positively regulating monocyte recruitment to the vascular wall.

Epigenetic regulation of vascular smooth muscle cell proliferation and neointima formation by histone deacetylase inhibition.

OBJECTIVE: Proliferation of smooth muscle cells (SMC) in response to vascular injury is central to neointimal vascular remodeling. There is accumulating evidence that histone acetylation constitutes a major epigenetic modification for the transcriptional control of proliferative gene expression; however, the physiological role of histone acetylation for proliferative vascular disease remains elusive. METHODS AND RESULTS: In the present study, we investigated the role of histone deacetylase (HDAC) inhibition in SMC proliferation and neointimal remodeling. We demonstrate that mitogens induce transcription of HDAC 1, 2, and 3 in SMC. Short interfering RNA-mediated knockdown of either HDAC 1, 2, or 3 and pharmacological inhibition of HDAC prevented mitogen-induced SMC proliferation. The mechanisms underlying this reduction of SMC proliferation by HDAC inhibition involve a growth arrest in the G(1) phase of the cell cycle that is due to an inhibition of retinoblastoma protein phosphorylation. HDAC inhibition resulted in a transcriptional and posttranscriptional regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip). Furthermore, HDAC inhibition repressed mitogen-induced cyclin D1 mRNA expression and cyclin D1 promoter activity. As a result of this differential cell cycle-regulatory gene expression by HDAC inhibition, the retinoblastoma protein retains a transcriptional repression of its downstream target genes required for S phase entry. Finally, we provide evidence that these observations are applicable in vivo by demonstrating that HDAC inhibition decreased neointima formation and expression of cyclin D1 in a murine model of vascular injury. CONCLUSIONS: These findings identify HDAC as a critical component of a transcriptional cascade regulating SMC proliferation and suggest that HDAC might play a pivotal role in the development of proliferative vascular diseases, including atherosclerosis and in-stent restenosis.

Telomerase activation in atherosclerosis and induction of telomerase reverse transcriptase expression by inflammatory stimuli in macrophages.

OBJECTIVE: Telomerase serves as a critical regulator of tissue renewal. Although telomerase activity is inducible in response to various environmental cues, it remains unknown whether telomerase is activated during the inflammatory remodeling underlying atherosclerosis formation. To address this question, we investigated in the present study the regulation of telomerase in macrophages and during atherosclerosis development in low-density lipoprotein receptor-deficient mice. METHODS AND RESULTS: We demonstrate that inflammatory stimuli activate telomerase in macrophages by inducing the expression of the catalytic subunit telomerase reverse transcriptase (TERT). Reporter and chromatin immunoprecipitation assays identified a previously unrecognized nuclear factor-κB (NF-κB) response element in the TERT promoter, to which NF-κB is recruited during inflammation. Inhibition of NF-κB signaling completely abolished the induction of TERT expression, characterizing TERT as a bona fide NF-κB target gene. Furthermore, functional experiments revealed that TERT deficiency results in a senescent cell phenotype. Finally, we demonstrate high levels of TERT expression in macrophages of human atherosclerotic lesions and establish that telomerase is activated during atherosclerosis development in low-density lipoprotein receptor-deficient mice. CONCLUSIONS: These results characterize TERT as a previously unrecognized NF-κB target gene in macrophages and demonstrate that telomerase is activated during atherosclerosis. This induction of TERT expression prevents macrophage senescence and may have important implications for the development of atherosclerosis.

Telomerase deficiency in bone marrow-derived cells attenuates angiotensin II-induced abdominal aortic aneurysm formation.

OBJECTIVE: Abdominal aortic aneurysms (AAA) are an age-related vascular disease and an important cause of morbidity and mortality. In this study, we sought to determine whether the catalytic component of telomerase, telomerase reverse transcriptase (TERT), modulates angiotensin (Ang) II-induced AAA formation. METHODS AND RESULTS: Low-density lipoprotein receptor-deficient (LDLr-/-) mice were lethally irradiated and reconstituted with bone marrow-derived cells from TERT-deficient (TERT-/-) mice or littermate wild-type mice. Mice were placed on a diet enriched in cholesterol, and AAA formation was quantified after 4 weeks of Ang II infusion. Repopulation of LDLr-/- mice with TERT-/- bone marrow-derived cells attenuated Ang II-induced AAA formation. TERT-deficient recipient mice revealed modest telomere attrition in circulating leukocytes at the study end point without any overt effect of the donor genotype on white blood cell counts. In mice repopulated with TERT-/- bone marrow, aortic matrix metalloproteinase-2 (MMP-2) activity was reduced, and TERT-/- macrophages exhibited decreased expression and activity of MMP-2 in response to stimulation with Ang II. Finally, we demonstrated in transient transfection studies that TERT overexpression activates the MMP-2 promoter in macrophages. CONCLUSIONS: TERT deficiency in bone marrow-derived macrophages attenuates Ang II-induced AAA formation in LDLr-/- mice and decreases MMP-2 expression. These results point to a previously unrecognized role of TERT in the pathogenesis of AAA.

Group X secretory phospholipase A2 negatively regulates ABCA1 and ABCG1 expression and cholesterol efflux in macrophages.

OBJECTIVE: GX sPLA(2) potently hydrolyzes plasma membranes to generate lysophospholipids and free fatty acids; it has been implicated in inflammatory diseases, including atherosclerosis. To identify a novel role for group X (GX) secretory phospholipase A(2) (sPLA(2)) in modulating ATP binding casette transporter A1 (ABCA1) and ATP binding casette transporter G1 (ABCG1) expression and, therefore, macrophage cholesterol efflux. METHODS AND RESULTS: The overexpression or exogenous addition of GX sPLA(2) significantly reduced ABCA1 and ABCG1 expression in J774 macrophage-like cells, whereas GX sPLA(2) deficiency in mouse peritoneal macrophages was associated with enhanced expression. Altered ABC transporter expression led to reduced cholesterol efflux in GX sPLA(2)-overexpressing J774 cells and increased efflux in GX sPLA(2)-deficient mouse peritoneal macrophages. Gene regulation was dependent on GX sPLA(2) catalytic activity, mimicked by arachidonic acid and abrogated when liver X receptor (LXR)α/ß expression was suppressed, and partially reversed by the LXR agonist T0901317. Reporter assays indicated that GX sPLA(2) suppresses the ability of LXR to transactivate its promoters through a mechanism involving the C-terminal portion of LXR spanning the ligand-binding domain. CONCLUSIONS: GX sPLA(2) modulates gene expression in macrophages by generating lipolytic products that suppress LXR activation. GX sPLA(2) may play a previously unrecognized role in atherosclerotic lipid accumulation by negatively regulating the genes critical for cellular cholesterol efflux.

Deficiency of the NR4A neuron-derived orphan receptor-1 attenuates neointima formation after vascular injury.

BACKGROUND: The neuron-derived orphan receptor-1 (NOR1) belongs to the evolutionary highly conserved and most ancient NR4A subfamily of the nuclear hormone receptor superfamily. Members of this subfamily function as early-response genes regulating key cellular processes, including proliferation, differentiation, and survival. Although NOR1 has previously been demonstrated to be required for smooth muscle cell proliferation in vitro, the role of this nuclear receptor for the proliferative response underlying neointima formation and target genes trans-activated by NOR1 remain to be defined. METHODS AND RESULTS: Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in NOR1(-/-) mice compared with wild-type mice. In vitro, NOR1-deficient smooth muscle cells exhibit decreased proliferation as a result of a G(1)-->S phase arrest of the cell cycle and increased apoptosis in response to serum deprivation. NOR1 deficiency alters phosphorylation of the retinoblastoma protein by preventing mitogen-induced cyclin D1 and D2 expression. Conversely, overexpression of NOR1 induces cyclin D1 expression and the transcriptional activity of the cyclin D1 promoter in transient reporter assays. Gel shift and chromatin immunoprecipitation assays identified a putative response element for NR4A receptors in the cyclin D1 promoter, to which NOR1 is recruited in response to mitogenic stimulation. Finally, we provide evidence that these observations are applicable in vivo by demonstrating decreased cyclin D1 expression during neointima formation in NOR1-deficient mice. CONCLUSIONS: These experiments characterize cyclin D1 as an NOR1-regulated target gene in smooth muscle cells and demonstrate that NOR1 deficiency decreases neointima formation in response to vascular injury.

Osteopontin mediates obesity-induced adipose tissue macrophage infiltration and insulin resistance in mice.

Obesity is associated with a state of chronic, low-grade inflammation characterized by abnormal cytokine production and macrophage infiltration into adipose tissue, which may contribute to the development of insulin resistance. During immune responses, tissue infiltration by macrophages is dependent on the expression of osteopontin, an extracellular matrix protein and proinflammatory cytokine that promotes monocyte chemotaxis and cell motility. In the present study, we used a murine model of diet-induced obesity to examine the role of osteopontin in the accumulation of adipose tissue macrophages and the development of insulin resistance during obesity. Mice exposed to a high-fat diet exhibited increased plasma osteopontin levels, with elevated expression in macrophages recruited into adipose tissue. Obese mice lacking osteopontin displayed improved insulin sensitivity in the absence of an effect on diet-induced obesity, body composition, or energy expenditure. These mice further demonstrated decreased macrophage infiltration into adipose tissue, which may reflect both impaired macrophage motility and attenuated monocyte recruitment by stromal vascular cells. Finally, obese osteopontin-deficient mice exhibited decreased markers of inflammation, both in adipose tissue and systemically. Taken together, these results suggest that osteopontin may play a key role in linking obesity to the development of insulin resistance by promoting inflammation and the accumulation of macrophages in adipose tissue.

The PPARalpha/p16INK4a pathway inhibits vascular smooth muscle cell proliferation by repressing cell cycle-dependent telomerase activation.

Peroxisome proliferator-activated receptor (PPAR)alpha, the molecular target for fibrates used to treat dyslipidemia, exerts pleiotropic effects on vascular cells. In vascular smooth muscle cells (VSMCs), we have previously demonstrated that PPARalpha activation suppresses G(1)-->S cell cycle progression by targeting the cyclin-dependent kinase inhibitor p16(INK4a) (p16). In the present study, we demonstrate that this inhibition of VSMC proliferation by PPARalpha is mediated through a p16-dependent suppression of telomerase activity, which has been implicated in key cellular functions including proliferation. PPARalpha activation inhibited mitogen-induced telomerase activity by repressing the catalytic subunit telomerase reverse transcriptase (TERT) through negative cross-talk with an E2F-1-dependent trans-activation of the TERT promoter. This trans-repression involved the recruitment of the retinoblastoma (RB) family proteins p107 and p130 to the TERT promoter resulting in impaired E2F-1 binding, an effect that was dependent on p16. The inhibition of cell proliferation by PPARalpha activation was lost in VSMCs following TERT overexpression or knockdown, pointing to a key role of telomerase as a target for the antiproliferative effects of PPARalpha. Finally, we demonstrate that PPARalpha agonists suppress telomerase activation during the proliferative response following vascular injury, indicating that these findings are applicable in vivo. In concert, these results demonstrate that the antiproliferative effects of PPARalpha in VSMCs depend on the suppression of telomerase activity by targeting the p16/RB/E2F transcriptional cascade.

Interactions between gut microbiota and skeletal muscle.

The gut microbiota is now recognized as a major contributor to the host's nutrition, metabolism, immunity, and neurological functions. Imbalanced microbiota (ie, dysbiosis) is linked to undernutrition-induced stunting, inflammatory and metabolic diseases, and cancers. Skeletal muscle also takes part in the interorgan crosstalk regulating substrate metabolism, immunity, and health. Here, we review the reciprocal influence of gut microbiota and skeletal muscle in relation to juvenile growth, performance, aging, and chronic diseases. Several routes involving the vascular system and organs such as the liver and adipose tissue connect the gut microbiota and skeletal muscle, with effects on fitness and health. Therapeutic perspectives arise from the health benefits observed with changes in gut microbiota and muscle activity, further encouraging multimodal therapeutic strategies.

Metformin partially reverses the inhibitory effect of co-culture with ER-/PR-/HER2+ breast cancer cells on biomarkers of monocyte antitumor activity.

BACKGROUND: Immune activities of monocytes (MOs) can be altered within the microenvironment of solid malignancies, including breast cancer. Metformin (1,1-dimethylbiguanide hydrochloride, MET), has been shown to decrease tumor cell proliferation, but its effects have yet to be explored with respect to MOs (monocytes) activity during their crosstalk with breast cancer cells. Here, we investigated the effects of MET on overall phenotypic functional activities, including cellular immunometabolism and protective redox signaling based-biomarkers, intracellular free calcium ions (ifCa2+), phagocytosis and co-operative cytokines (IFN-γ and IL-10) of autologous MOs before and during their interplay with primary ER-/PR-/HER2+ breast cancer cells. METHODS: Human primary breast cancer cells were either cultured alone or co-cultured with autologous MOs before treatment with MET. RESULTS: MET downregulated breast cancer cell proliferation and phagocytosis, while having no significant effect on the ratio of phosphorylated Akt (p-Akt) to total Akt. Additionally, we observed that, in the absence of MET treatment, the levels of lactate dehydrogenase (LDH)-based cytotoxicity, catalase, ifCa2+, IL-10 and arginase activity were significantly reduced in co-cultures compared to levels in MOs cultured alone whereas levels of inducible nitric oxide synthase (iNOS) activity were significantly increased. In contrast, MET treatment reduced the effects measured in co-culture on the levels of LDH-based cytotoxicity, arginase activity, catalase, ifCa2+, and IFN-γ. MET also induced upregulation of both iNOS and arginase in MO cells, although the increase did not reach significant difference for iNOS activity. Moreover, MET induced a robust increase of superoxide dismutase (SOD) activity in MOs, but not in MOs co-cultured with breast cancer cells. Furthermore, MET markedly upregulated the levels of IFN-γ production and downregulated those of IL-10 in isolated MOs, while inducing a slight opposing up-regulation of IL-10 production in co-cultures. CONCLUSIONS: Our results show that the biomarkers of phenotypic functional activities of MOs are modified after co-culturing with primary human breast cancer cells. Treatment of co-cultures with MET resulted in increased release of antitumor cytokine IFN-γ and ifCa2+, and increased cell necrosis during breast cancer cells-MOs crosstalk.

TReP-132 is a novel progesterone receptor coactivator required for the inhibition of breast cancer cell growth and enhancement of differentiation by progesterone.

The sex steroid progesterone is essential for the proliferation and differentiation of the mammary gland epithelium during pregnancy. In relation to this, in vitro studies using breast carcinoma T47D cells have demonstrated a biphasic progesterone response, consisting of an initial proliferative burst followed by a sustained growth arrest. However, the transcriptional factors acting with the progesterone receptor (PR) to mediate the progesterone effects on mammary cell growth and differentiation remain to be determined. Recently, it has been demonstrated that the transcriptional regulating protein of 132 kDa (TReP-132), initially identified as a regulator of steroidogenesis, is also a cell growth suppressor. Similar to progesterone-bound PR, TReP-132 acts by inducing the gene expression of the G1 cyclin-dependent kinase inhibitors p21WAF1/Cip1 (p21) and p27Kip1 (p27). The putative interaction between TReP-132 and progesterone pathways in mammary cells was therefore analyzed in the present study. Our results show that TReP-132 interacts in vitro and in T47D cells with progesterone-activated PR. TReP-132 synergizes with progesterone-bound PR to trans activate the p21 and p27 gene promoters at proximal Sp1-binding sites. Moreover, TReP-132 overexpression and knockdown, respectively, increased or prevented the induction of p21 and p27 gene expression by progesterone. As a consequence, TReP-132 knockdown also resulted in the loss of the inhibitory effects of progesterone on pRB phosphorylation, G1/S cell cycle progression, and cell proliferation. Furthermore, the knockdown of TReP-132 expression also prevented the induction of both early and terminal markers of breast cell differentiation which had been previously identified as progesterone target genes. As well, the progesterone-induced accumulation of lipid vacuoles was inhibited in the TReP-132-depleted cells. Finally, TReP-132 gene expression levels increased following progesterone treatment, indicating the existence of a positive auto-regulatory loop between PR and TReP-132. Taken together, these data identify TReP-132 as a coactivator of PR mediating the growth-inhibitory and differentiation effects of progesterone on breast cancer cells.

PPAR alpha inhibits vascular smooth muscle cell proliferation underlying intimal hyperplasia by inducing the tumor suppressor p16INK4a.

Vascular SMC proliferation is a crucial event in occlusive cardiovascular diseases. PPARalpha is a nuclear receptor controlling lipid metabolism and inflammation, but its role in the regulation of SMC growth remains to be established. Here, we show that PPARalpha controls SMC cell-cycle progression at the G1/S transition by targeting the cyclin-dependent kinase inhibitor and tumor suppressor p16(INK4a) (p16), resulting in an inhibition of retinoblastoma protein phosphorylation. PPARalpha activates p16 gene transcription by both binding to a canonical PPAR-response element and interacting with the transcription factor Sp1 at specific proximal Sp1-binding sites of the p16 promoter. In a carotid arterial-injury mouse model, p16 deficiency results in an enhanced SMC proliferation underlying intimal hyperplasia. Moreover, PPARalpha activation inhibits SMC growth in vivo, and this effect requires p16 expression. These results identify an unexpected role for p16 in SMC cell-cycle control and demonstrate that PPARalpha inhibits SMC proliferation through p16. Thus, the PPARalpha/p16 pathway may be a potential pharmacological target for the prevention of cardiovascular occlusive complications of atherosclerosis.

TReP-132 controls cell proliferation by regulating the expression of the cyclin-dependent kinase inhibitors p21WAF1/Cip1 and p27Kip1.

The transcriptional regulating protein of 132 kDa (TReP-132) has been identified in steroidogenic tissues, where it acts as a coactivator of steroidogenic factor 1 (SF-1). We show here that TReP-132 plays a role in the control of cell proliferation. In human HeLa cells, TReP-132 knockdown by using small interfering RNA resulted in increased G(1)-->S cell cycle progression. The growth-inhibitory effects of TReP-132 was further shown to be mediated by induction of G(1) cyclin-dependent kinase inhibitors p21(WAF1) (p21) and p27(KIP1) (p27) expression levels. As a consequence, G(1) cyclin/cyclin-dependent kinase activities and pRB phosphorylation were markedly reduced, and cell cycle progression was blocked in the G(1) phase. The stimulatory effect of TReP-132 on p21 and p27 gene transcription involved interaction of TReP-132 with the transcription factor Sp1 at proximal Sp1-binding sites in their promoters. Moreover, in different breast tumor cell lines, endogenous TReP-132 expression was positively related with a lower proliferation rate. In addition, TReP-132 knockdown resulted in enhanced cell proliferation and lowered p21 and p27 mRNA levels in the steroid-responsive and nonresponsive T-47D and MDA-MB-231 cell lines, respectively. Finally, a statistic profiling of human breast tumor samples highlighted that expression of TReP-132 is correlated with p21 and p27 levels and is associated with lower tumor incidence and aggressiveness. Together, these results identify TReP-132 as a basal cell cycle regulatory protein acting, at least in part, by interacting with Sp1 to activate the p21 and p27 gene promoters.

Effect of rosiglitazone treatment on plaque inflammation and collagen content in nondiabetic patients: data from a randomized placebo-controlled trial.

BACKGROUND: Therapeutic strategies to stabilize advanced arteriosclerotic lesions may prevent plaque rupture and reduce the incidence of acute coronary syndromes. Thiazolidinediones (TZDs), like rosiglitazone, are oral antidiabetic drugs with additional antiinflammatory and potential antiatherogenic properties. In a randomized, placebo-controlled, single-blind trial, we examined the effect of 4 weeks of rosiglitazone therapy on histomorphological characteristics of plaque stability in artery specimen of nondiabetic patients scheduled for elective carotid endarterectomy. METHODS AND RESULTS: A total of 24 nondiabetic patients with symptomatic carotid artery stenosis were randomly assigned to rosiglitazone (4 mg BID) or placebo in addition to standard therapy. In this population of nondiabetic patients, rosiglitazone treatment did not significantly change fasting blood glucose, fasting insulin, or lipid parameters. In contrast, rosiglitazone significantly reduced CD4-lymphocyte content as well as macrophage HLA-DR expression in the shoulder region, reflecting less inflammatory activation of these cells by lymphocyte interferon-gamma. Moreover, rosiglitazone significantly increased plaque collagen content (7.7+/-1.6% versus 3.7+/-0.7% of plaque area; P=0.036) compared with placebo, suggesting that TZD treatment may stabilize arteriosclerotic lesions. In addition, rosiglitazone reduced serum levels of 2 inflammatory arteriosclerosis markers: C-reactive protein and serum amyloid A. CONCLUSIONS: Four weeks of treatment with rosiglitazone significantly reduces vascular inflammation in nondiabetic patients, leading to a more stable type of arteriosclerotic lesion.

Progesterone inhibits human breast cancer cell growth through transcriptional upregulation of the cyclin-dependent kinase inhibitor p27Kip1 gene.

The effects of progesterone derivatives on breast cancer development are still controversial, probably accounting for their biphasic, opposed effects on mammary cell-cycle regulation. Here, we demonstrate in vitro that the growth-inhibitory effects of progesterone on breast cancer T-47D cells require the transcriptional upregulation of the cyclin-dependent kinase inhibitor p27(Kip1) (p27) gene. A statistical analysis of human tumor biopsies further indicates that p27 mRNA levels correlate to progesterone receptor (PR) levels. Moreover, p27 gene expression is inversely associated with tumor aggressiveness, and is a prognostic factor of favorable disease outcome. Thus, progesterone derivatives selectively activating the p27 gene promoter could be promising drugs against breast cancer progression.

[A role of PPARgamma in reproduction?]. / Un rôle pour PPARgamma dans la reproduction?

Synthetic molecules of the glitazone family are currently used in the treatment of type II diabetes. Glitazones also improve secondary pathologies that are frequently associated with insulin resistance such as the polycystic ovary syndrome (PCOS). Glitazones bind to the peroxysome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor which is highly expressed in adipose tissue. PPARgamma also binds natural ligands such as long-chain fatty acids. Recently, several groups have shown that PPARgamma is also highly expressed in ovarian granulosa cells, and that glitazones are able to modulate in vitro granulosa cell proliferation and steroidogenesis in several species. These recent data raise new questions concerning the underlying mechanism of the effect of glitazones on PCOS. One might hypothesize, as for other << glucophage >> molecules such as metformin, that it is the general improvement of glucose metabolism and insulin sensitivity by glitazones which indirectly, and via an unknown mechanism, ameliorates ovarian functionality. The data discussed here suggest now an alternative possibility, that glitazones act directly at the ovarian level. Moreover, PPARgamma also seems to play a key role in the maturation of the placenta. In particular, inactivation of PPARgamma in mice is lethal, since the foetus is unable to develop because of alterations of placental maturation. In women, the activation of PPARgamma in placenta leads to an increase of placental hormone secretion. Overall, these results raise some questions about the role of natural ligands of PPARgamma such as long chain fatty acids on female fertility and the interactions between energy metabolism and reproduction in general.

2',6'-Dihalostyrylanilines, pyridines, and pyrimidines for the inhibition of the catalytic subunit of methionine S-adenosyltransferase-2.

Inhibition of the catalytic subunit of the heterodimeric methionine S-adenosyl transferase-2 (MAT2A) with fluorinated N,N-dialkylaminostilbenes (FIDAS agents) offers a potential avenue for the treatment of liver and colorectal cancers where upregulation of this enzyme occurs. A study of structure-activity relationships led to the identification of the most active compounds as those with (1) either a 2,6-difluorostyryl or 2-chloro-6-fluorostyryl subunit, (2) either an N-methylamino or N,N-dimethylamino group attached in a para orientation relative to the 2,6-dihalostyryl subunit, and (3) either an N-methylaniline or a 2-(N,N-dimethylamino)pyridine ring. These modifications led to FIDAS agents that were active in the low nanomolar range, that formed water-soluble hydrochloride salts, and that possessed the desired property of not inhibiting the human hERG potassium ion channel at concentrations at which the FIDAS agents inhibit MAT2A. The active FIDAS agents may inhibit cancer cells through alterations of methylation reactions essential for cancer cell survival and growth.