C-terminal cleavage of human Foxp3 at a proprotein convertase motif abrogates its suppressive function.

Foxp3 plays a critical role in the development and function of regulatory T cells (Tregs). Differences in translational and post-translational processing of murine and human Foxp3 have been recently reported. Human Foxp3 exists as four isoforms generated by alternative splicing. Mouse Foxp3 only exists as one isoform, but can be proteolytically cleaved by N-terminal and/or C-terminal proprotein convertase subtilisin/kexins (PCSKs). Here, we show by transcriptome analysis that the proprotein convertases PCSK7, PCSK5 and Furin are present in human CD4(+) T cells with different expression patterns. Notably, after in vitro activation, only PCSK7 and Furin are expressed in Tregs and T effector cells (Teffs), with overexpression of PCSK7 in Tregs compared to Teffs. Human Foxp3 protein displays specific motifs that can be potentially cleaved by convertases. Consequently, we transduced human CD4(+) cells with Foxp3-expressing lentiviral vectors and assessed the generation of proteolytically cleaved Foxp3 forms by Western blot. Three different Foxp3 forms were detected, indicating that human Foxp3 can also be subjected to proteolytic cleavage at the N-terminal and C-terminal ends. These results prompted us to assess the suppressive activity associated with each forms. We observed that full length and N-cleaved Foxp3-transduced CD4(+) T cells similarly suppressed the in vitro proliferation of Teffs. However, the C-cleaved or N&C-cleaved Foxp3 forms afforded almost no suppressive function, indicating a crucial role of the human Foxp3 C-terminal region in Tregs suppressive activity, in marked contrast with the report of a superior suppressive activity for the C-cleaved murine Foxp3 compared to the full length.

Transfer of CD4(+) T cells aggravates atherosclerosis in immunodeficient apolipoprotein E knockout mice.

BACKGROUND: Atherosclerosis is associated with immune responses to oxidized lipoproteins and certain microorganisms, but the role of specific immunity has remained unclear. METHODS AND RESULTS: To study the role of immunity in atherosclerosis, we crossed atherosclerosis-prone apoE(-/-) mice with immunodeficient scid/scid mice. The offspring showed a 73% reduction in aortic fatty streak lesions when compared with immunocompetent apoE(-/-) mice. Transfer of CD4(+) T cells from apoE(-/-) to immunodeficient apoE(-/-)/scid/scid mice increased lesions by 164%. This was associated with the infiltration of transferred T cells into lesions, increased circulating interferon-gamma levels, and increased I-A expression in lesions. CONCLUSIONS: CD4(+) T cells carry disease-promoting immunity in atherosclerosis.

Expression of the interleukin-6 gene is constitutive and not regulated by estrogen in rat vascular smooth muscle cells in culture.

Vascular smooth muscle cells (SMC) are major constituents of the medial layer of blood vessels and are involved in the development of atherosclerotic plaque. SMC secrete copious IL-6 under basal conditions that can be increased by cytokines such as tumor necrosis factor-alpha and interleukin-1beta (IL-1beta). The goal of our studies was to define the role of estrogen in IL-6 production by SMC. In a first series of experiments, the expression of specific messenger RNAs as well as the production of IL-6 bioactivity by rat SMC in culture could be demonstrated in basal and IL-1-stimulated conditions, but was unaffected by estrogen treatment. Different constructs containing deleted or mutated fragments of the human IL-6 promoter driving luciferase or chloramphenicol acetyltransferase reporter gene were then transiently transfected in these cells. A significant basal activity that was increased 2- to 4-fold after IL-1beta stimulation was observed with the total IL-6 promoter. Deletion analysis indicated that the -158/+11 region containing activator protein-1 and cAMP response element sites was apparently the minimal region of IL-6 promoter to confer both constitutive and IL-1-inducible activities. Site-directed mutagenesis experiments suggest that basal activity is dependent upon the promoter sequence -158 to -112 containing the nuclear factor (NF)-IL6(-153) and Sp1 sites, whereas IL-1beta stimulation would depend on the residual -112 nucleotides containing NF-IL6(-75) and NF-kappaB sites. In contrast to the down-regulation of IL-6 expression by estrogen described in osteoblasts, ethinyl estradiol as well as 17beta-estradiol did not influence stimulated IL-6 activity in our experimental conditions whatever the construct tested, even when either estrogen receptor alpha or beta was overexpressed. Thus, the atheroprotective properties of estrogen are probably not mediated through the regulation of IL-6 production by SMC.

Loss of atheroprotective effect of estradiol in immunodeficient mice.

Estradiol significantly decreases fatty streak formation in the aortic root of chow-fed apolipoprotein E-deficient mice. In contrast, immunodeficient mice with homozygous disruption at the recombinase activating gene 2 loci present fatty streak development that is insensitive to estradiol. Lymphocytes thus appear to be required for development of the atheroprotective effect of estradiol in this mouse model.

Involvement of interleukin-6 in atherosclerosis but not in the prevention of fatty streak formation by 17beta-estradiol in apolipoprotein E-deficient mice.

Interleukin-6 (IL-6) gene expressed in bone marrow-derived stromal cells and osteoblasts contributes to the state of mineralization and its control by estradiol may be involved in the development of post-menopausal osteoporosis. Since IL-6 is also expressed in the different cell populations of the arterial wall, the purpose of this study was to gain more insight into its involvement in the atherosclerotic process and the atheroprotective effect of estradiol by studying double deficient mice at the apolipoprotein E and IL-6 loci (IL-6(-/-)/E(-/-)). At 1 year of age, IL-6(-/-)/E(-/-) mice showed similar hypercholesterolemia to IL-6(+/+)/E(-/-) mice but presented significantly larger and more calcified lesions. In younger mice (sixteen weeks of age), no significant difference in fatty streaks could be detected in IL-6(+/+)/E(-/-), IL-6(+/-)/E(-/-) and IL-6(-/-)/E(-/-) mice on a normal chow diet. Estrogen supplementation at this age induced a decrease of fatty streak formation in all three genotypes. The combined data indicate that IL-6 expression is involved at the fibrous plaque stage of the atherosclerotic process but does not constitute a direct target for estradiol to prevent fatty streak formation.

Estrogens and atherosclerosis.

Numerous epidemiological as well as experimental studies have suggested that estradiol (E2) prevents atherosclerosis development. However two controlled prospective and randomized studies in women using hormone replacement therapy (HRT) did not confirm this beneficial effect. We then decided to use mouse models of atherosclerosis to define the possible mechanisms involved and the reasons for the discrepancy. We have shown that, although serum cholesterol decreases, this influence on lipid metabolism is negligible. Surprisingly, E2 induces an inflammatory-immune response towards a T helper cell (Th1) profile with increasing interferon-gamma production that could destabilize atheromatous plaques, and could account for the increase in the frequency of cardiovascular events in women undergoing HRT. At the level of the endothelium, E2 induces an increase in nitric oxide (NO) biodisponibility, but this phenomenon does not concern the development of fatty streaks. Nevertheless, the atheroprotective effect is apparently mediated at the level of the endothelium by a mechanism that has still to be characterized in molecular terms. These new acquisitions constitute a basis for new pharmacological developments allowing the prevention of deleterious effects and preserving the beneficial ones.

[Effect of estrogens on arterial physiology and mechanisms of their atheroprotective action in the animal]. / Effet des oestrogènes sur la physiologie artérielle et mécanismes de leur effet athéroprotecteur chez l'animal.

The two isoforms (alpha and beta) of the oestrogen receptor were identified in arterial wall cells. The heterogeneity of their expression is considered according to vascular regions and gender. Oestrogens can have a direct influence on vascular physiology through a "genomic" mechanism of action, although "extragenomic" mechanisms allowing very short-term hormonal action are also possible. Oestrogens potentiate endothelium-dependent relaxation by increasing the bioavailability of nitrogen monoxide (higher production and/or lesser degradation by the superoxide anion as a function of vascular beds). The atheromatous artery is the site of endothelial "dysfunction" (an anomaly of endothelium-dependent vasodilation), which can be prevented and even corrected by administration of oestradiol. In the monkey, this beneficial effect of oestrogens is not altered by addition of progesterone, but abolished by the addition of medroxyprogesterone. Finally, oestrogens prevent formation of the fatty streak. Studies of the mechanism(s) of this effect are now in progress.

[Effect of estrogens on arterial physiology and mechanisms of their atherosclerosis-protective effect in animals]. / Effect des oestrogènes sur la physiologie artérielle et mécanismes de son effet athéroprotecteur chez l'animal.

Two isoforms of oestrogen receptors (alpha and beta) have been identified in the cells of the arterial wall, and a heterogeneity of their expression according to the animal species, the vascular beds and the sex has been reported. Oestrogens can thus directly influence the vascular physiology through a 'genomic' mechanisms, but 'extra-genomic' mechanisms responsible for a short-term effect have also been suggested. Oestrogens potentiate endothelium-dependent relaxation through an increase in nitric oxide bioavailability (increase in its production and/or decrease in its degradation by superoxide anion according to the vascular beds). Endothelial 'dysfunction' (abnormality of the endothelium-dependent vasodilation) occurs in atheromatous arteries. Oestrogen replacement prevents and even corrects this endothelial dysfunction. In monkeys, this beneficial effect of oestrogens is not altered by coadministration of progesterone, but is abolished by coadministration of medroxyprogesterone. Finally, oestrogens prevent fatty streak deposit, and the mechanisms of this atheroprotective effect are being studied.

[Estrogens and the arterial wall]. / OEstrogènes et paroi artérielle.

Two isoforms of oestrogens receptor (alpha and B) have been identified in the cells of the arterial wall, and an heterogenity of their expression according to the animal species, to the vascular bed and to sex has been reported. Estrogens can thus directly influence the vascular physiology through a genomic mechanism, but extra-genomic mechanisms responsible for a short-term effect have also been suggested. Endothelium appears to be an important target for estradiol, because this hormone potentiates endothelium-dependant relaxation through an increase in NO bioavailability, and accelerates endothelial regrowth. In the model of apolipoprotein E-deficient mice, as the atrhroprotective effect deposit. The immune system appears to play a key role, as the athroprotective effect of estradiol is absent in mice deficient in T and B lymphocytes. Estrogens potentiate the endothelium-dependant relaxation through the increase in nitric oxide bioavailability. Endothelial dysfunction (abnormality of the endothelium-dependent vasodilation) occurs in atheromatous arteries. Estrogens prevent and even correct this endothelial dysfunction. In monkeys, this beneficial effect of estrogens is not altered by coadministration of progesterone, but is abolished.

[Reproducibility of CT scan evaluation of muscular fatty degeneration. Intra- and interobserver analysis of 56 shoulders presenting with a ruptured rotator cuff muscles]. / Reproductibilité de l'évaluation tomodensitométrique de la dégénérescence graisseuse des muscles. Analyse intra- et inter-observateurs sur 56 épaules présentant une rupture de la coiffe des rotateurs.

PURPOSE OF THE STUDY: This study was undertaken to determine the reproducibility of measurements of fatty degeneration of the rotator cuff using computed tomography (CT). MATERIAL AND METHODS: Fifty-six patients who had undergone surgery for rotator cuff tear were included in this retrospective study. The extent of fatty infiltration was evaluated on CT scans with soft tissue windows in all 56 shoulders using a five-stage scoring system described by Goutallier. Five independent observers made the assessments. The same operation was repeated one month later to test intraobserver agreement. Four parameters were recorded: fatty infiltration of three muscles (supraspinatus, infraspinatus, subscapularis), and overall fatty infiltration grading. Interobserver variability was determined for each parameter using the intercorrelation coefficient (a test of reproducibility of quantitative measurements). RESULTS: The most reproducible measurement was the overall fatty infiltration grade. For this parameter, interobserver agreement was good with an intercorrelation coefficient of 0.75. The interval of confidence was +/- 0.5. Intraobserver agreement depended on the observer's level of experience. It was good for overall fatty infiltration grade assess by three senior observers (r=0.78) and moderate for two junior observers. CONCLUSION: The overall fatty infiltration grade is a reproducible parameter that should be used to evaluate the degree of fatty infiltration as the safety margin of this value (graded 0 to 4) is about 0.5. Fatty infiltration of a torn cuff would not be the only criterion to improve indications for treatment of rotator cuff tears.

[Long term results of the surgical treatment of bicondylar fractures of the distal humerus extremity in adults]. / Résultats à long terme du traitement chirurgical des fractures bicondyliennes de l'extrémité distale de l'humérus chez l'adulte.

PURPOSE OF THE STUDY: In intra-articular fractures of the distal humerus, full functional recovery is difficult to obtain. An osteosynthesis by plate is the treatment of choice, but location and type of plate always remain open for debate. We present a consecutive series of intra-articular fractures of the distal humerus treated by osteosynthesis. The aim of the study is to determine and to compare the results of various types of osteosynthesis. MATERIALS AND METHODS: We reviewed 55 patients at an average of 108 months after early internal fixation for intraarticular displaced fractures of the distal humerus type C according to the A.O. classification. Intraarticular osteotomy of olecranon was used in 37 subjects (67.27%). The osteosynthesis has been achieved with a precasted lateral plate for 31 patients, with two posterior plates in 18 subjects, with a screwing on triangulation in four patients and with pins in two patients. RESULTS: The osteosynthesis with two posterior plates obtained a good result between 78.57% and 92.86% of cases, whereas the osteosynthesis with a precasted lateral plate gave a good result between 73.68% and 76.32% of cases according to the score used for estimation. In the aggregate, the functional estimate has a good result in the most of the cases whatever the score of estimate. At follow up we observed an average range of motion of 103 degrees. This value is quite good because it corresponds to the sector of the useful functional mobility. DISCUSSION: The review of our cases and the literature prompt us to follow the way of the osteosynthesis in adjusting indications to the type of fracture and in using a well-codifed technology. As it is already the case, a minimal osteosynthesis by screwing or by pins must be left except for peculiar cases in osteopenic elderly patients and if they have a very poor health. Contrary to other studies, the osteosynthesis with two posterior plates has given us better results provided that it has been systematically associated with a triangulation screwing in order to increase the strength of fitting in the sagittal plan. The precasted lateral plate gives a stable fitting too, nevertheless it is well advised to associate it to an osteosynthesis of medium column especially when the fracture is type C3. We have statistically proved that the age is not contraindication for osteosynthesis. CONCLUSION: This surgery is difficult and entails complications. The dismantling of the synthesis always gives poor results particularly if it is succeeded by an immobilization in plaster. The poor reduction, origin of arthrosis, of loss of bony substance and of calcifications worsen the functional prognosis. Last but not least, a good result can be obtained in the most of the cases however the types of fractures.

Estrogens modulate bovine vascular endothelial cell permeability and HSP 25 expression concomitantly.

The atheroprotective properties of estrogens are supported by clinical data from postmenopausal women who use estrogen replacement therapy. However, the mechanisms mediating activity remain unknown, and it has been suggested that estrogens may help to modulate endothelial permeability to atherogenic lipoproteins. In these studies we used bovine vascular endothelial cells as an in vitro model to show that estrogens were able to regulate low-density lipoprotein transport and permeability of the endothelial monolayer. Macromolecular transport was observed to be a second-order polynomial function of estrogen concentration. Moreover, this regulation was correlated with expression of heat shock protein (HSP) 25, which is known to influence fluid phase pinocytosis and cytoskeleton remodeling, thus suggesting a role for HSP 25 in the estrogenic control of transcellular permeability of the endothelium monolayer.

17 beta-estradiol prevents fatty streak formation in apolipoprotein E-deficient mice.

The reality of the atheroprotective effect of estrogens is still a matter of debate, and its unknown mechanisms could involve favorable changes in blood lipids and lipoproteins and/or direct action at the level of the arterial wall. We used the recently developed animal model of atherosclerosis constituted by apolipoprotein E-deficient mice in an attempt to clarify these issues. Male and female animals, fed a low-fat chow diet, were treated with increasing doses of 17 beta-estradiol (E2) after castration and compared with testosterone treated and uncastrated (intact) animals. Total serum cholesterol, LDL-cholesterol, and HDL-cholesterol concentrations decreased under E2 treatment in each sex and were weakly correlated with lesion area. However, a highly significant correlation between lesion area and serum E2 levels also suggested a direct action of E2 on cells of the vascular wall. A dose-response curve analysis revealed that these activities were sex-dependent, with females being nearly twice as sensitive to E2 as males. It also revealed that the atheroprotective activity was recruited at higher E2 concentrations than those needed by other E2 target tissues such as uterus or functions such as apoA-1 and LDL production and/or clearance rates.

Prevention of fatty streak formation of 17beta-estradiol is not mediated by the production of nitric oxide in apolipoprotein E-deficient mice.

BACKGROUND: Estrogens have atheroprotective properties, the mechanisms of which remain obscure. Estrogens have recently been reported to increase endothelial NO synthase expression in castrated animals and to prevent the degradation of NO by decreasing superoxide anion production in cultured endothelial cells. In both cases, increased NO bioavailability would promote vasodilation, inhibit proliferation of the adjacent vascular smooth muscle, reduce platelet aggregation, and inhibit monocyte adhesion to the endothelium and the inflammatory reaction induced by cytokines, all key contributors in the development of atherosclerosis. METHODS AND RESULTS: In the present work, the respective roles of 17beta-estradiol and NO in the development of the atherosclerotic process were investigated in castrated apolipoprotein E-deficient (apo E KO) mice, which spontaneously develop fatty streak lesions within 3 months. N(omega)-Nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, 50 mg x kg(-1) x d(-1), increased arterial blood pressure and decreased cerebellum cGMP content, demonstrating the blockade of NO production, but did not influence the atherogenic process in castrated apo E KO mice. CONCLUSIONS: 17Beta-estradiol decreased the size of the aortic lesions approximately threefold, and the magnitude of this vasculoprotective effect was not altered by L-NAME. Moreover, L-NAME increased circulating malonyldialdehyde (MDA)-modified LDL, which was not altered by 17beta-estradiol, leading to a complete dissociation between circulating MDA-modified LDL and parietal lesions.

Differential effects of interleukin-1 receptor antagonist and tumor necrosis factor binding protein on fatty-streak formation in apolipoprotein E-deficient mice.

BACKGROUND: The cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) are secreted by the different cell populations of the vascular wall and have been suggested to promote atherosclerosis. METHODS AND RESULTS: Their respective roles in fatty-streak formation in apolipoprotein E-deficient mice were investigated by use of IL-1 receptor antagonist and TNF binding protein. Estradiol-17beta was used as a positive control. Blocking TNF seemed to be active in female animals but not in males. IL-1 receptor antagonist was as effective as or more effective than estradiol in both sexes. CONCLUSIONS: IL-1 plays a crucial role in the initial step of the atherosclerotic process in this animal model, and blocking the activity of this cytokine should be considered as a therapeutic possibility.

The mitogenic effect of 17beta-estradiol on in vitro endothelial cell proliferation and on in vivo reendothelialization are both dependent on vascular endothelial growth factor.

In addition to their actions on reproductive function, estrogens have important effects on endothelial cells. The present study was designed to evaluate the mechanism(s) by which 17beta-estradiol (E2) promotes endothelial cell proliferation. The potential involvement of vascular endothelial growth factor (VEGF) was investigated by the coadministration of polyclonal anti-VEGF antibody. First, the effect of E2 on the proliferation of cultured foetal bovine aortic endothelial cells (FBAEC) was studied. E2 stimulated this proliferation with an EC50 between 10(-11) and 10(-10) M and this effect was inhibited by the anti-VEGF antibody. The effect of a physiological dose of E2 was then studied in the rat model of carotid injury. After deendothelializing balloon injury, reendothelialization of the denuded surface may influence the growth of the underlying smooth muscle cells. Male Sprague-Dawley rats were castrated and then received E2 from subcutaneously implanted pellets that released 3.2 microg/kg/day. Endothelial regrowth (Evans blue staining) and neointimal thickening were evaluated 2 weeks after the carotid injury. In comparison to the placebo group, E2 increased the extent of reendothelialization (p = 0.0002) and reduced neointimal thickening (p = 0.0007). Anti-VEGF antibody abolished the effect of E2 on reendothelialization as well as on neointimal thickening. Thoracic aorta VEGF content was increased in E2-treated rats compared to control rats. In conclusion, the present study demonstrates that E2 increases endothelial cell proliferation in vitro and reendothelialization in vivo by means of a mechanism dependent on endogenous VEGF. This effect could contribute to the antiatherogenic effect of a physiological dose of E2.