The impact of adiposity on adipose tissue-resident lymphocyte activation in humans.

BACKGROUND/OBJECTIVES: The presence of T lymphocytes in human adipose tissue has only recently been demonstrated and relatively little is known of their potential relevance in the development of obesity-related diseases. We aimed to further characterise these cells and in particular to investigate how they interact with modestly increased levels of adiposity typical of common overweight and obesity. SUBJECTS/METHODS: Subcutaneous adipose tissue and fasting blood samples were obtained from healthy males aged 35-55 years with waist circumferences in lean (<94 cm), overweight (94-102 cm) and obese (>102 cm) categories. Adipose tissue-resident CD4+ and CD8+ T lymphocytes together with macrophages were identified by gene expression and flow cytometry. T lymphocytes were further characterised by their expression of activation markers CD25 and CD69. Adipose tissue inflammation was investigated using gene expression analysis and tissue culture. RESULTS: Participants reflected a range of adiposity from lean to class I obesity. Expression of CD4 (T-helper cells) and CD68 (macrophage), as well as FOXP3 RNA transcripts, was elevated in subcutaneous adipose tissue with increased levels of adiposity (P<0.001, P<0.001 and P=0.018, respectively). Flow cytometry revealed significant correlations between waist circumference and levels of CD25 and CD69 expression per cell on activated adipose tissue-resident CD4+ and CD8+ T lymphocytes (P-values ranging from 0.053 to <0.001). No such relationships were found with blood T lymphocytes. This increased T lymphocyte activation was related to increased expression and secretion of various pro- and anti-inflammatory cytokines from subcutaneous whole adipose tissue explants. CONCLUSIONS: This is the first study to demonstrate that even modest levels of overweight/obesity elicit modifications in adipose tissue immune function. Our results underscore the importance of T lymphocytes during adipose tissue expansion, and the presence of potential compensatory mechanisms that may work to counteract adipose tissue inflammation, possibly through an increased number of T-regulatory cells.

Impairment of adipose tissue in Prader-Willi syndrome rescued by growth hormone treatment.

BACKGROUND: Prader-Willi syndrome (PWS) results from abnormalities in the genomic imprinting process leading to hypothalamic dysfunction with an alteration of growth hormone (GH) secretion. PWS is associated with early morbid obesity and short stature which can be efficiently improved with GH treatment. OBJECTIVES: Our aims were to highlight adipose tissue structural and functional impairments in children with PWS and to study the modifications of those parameters on GH treatment. SUBJECTS AND METHODS: Plasma samples and adipose tissue biopsies were obtained from 23 research centers in France coordinated by the reference center for PWS in Toulouse, France. Lean controls (n=33), non-syndromic obese (n=53), untreated (n=26) and GH-treated PWS (n=43) children were enrolled in the study. Adipose tissue biopsies were obtained during scheduled surgeries from 15 lean control, 7 untreated and 8 GH-treated PWS children. RESULTS: Children with PWS displayed higher insulin sensitivity as shown by reduced glycemia, insulinemia and HOMA-IR compared with non-syndromic obese children. In contrast, plasma inflammatory cytokines such as TNF-α, MCP-1 and IL-8 were increased in PWS. Analysis of biopsies compared with control children revealed decreased progenitor cell content in the stromal vascular fraction of adipose tissue and an impairment of lipolytic response to ß-adrenergic agonist in PWS adipocytes. Interestingly, both of these alterations in PWS seem to be ameliorated on GH treatment. CONCLUSION: Herein, we report adipose tissue dysfunctions in children with PWS which may be partially restored by GH treatment.

15-Lipoxygenase promotes resolution of inflammation in lymphedema by controlling Treg cell function through IFN-ß.

Lymphedema (LD) is characterized by the accumulation of interstitial fluid, lipids and inflammatory cell infiltrate in the limb. Here, we find that LD tissues from women who developed LD after breast cancer exhibit an inflamed gene expression profile. Lipidomic analysis reveals decrease in specialized pro-resolving mediators (SPM) generated by the 15-lipoxygenase (15-LO) in LD. In mice, the loss of SPM is associated with an increase in apoptotic regulatory T (Treg) cell number. In addition, the selective depletion of 15-LO in the lymphatic endothelium induces an aggravation of LD that can be rescued by Treg cell adoptive transfer or ALOX15-expressing lentivector injections. Mechanistically, exogenous injections of the pro-resolving cytokine IFN-ß restores both 15-LO expression and Treg cell number in a mouse model of LD. These results provide evidence that lymphatic 15-LO may represent a therapeutic target for LD by serving as a mediator of Treg cell populations to resolve inflammation.

Semaphorin 3C is a novel adipokine linked to extracellular matrix composition.

AIMS/HYPOTHESIS: Alterations in white adipose tissue (WAT) function, including changes in protein (adipokine) secretion and extracellular matrix (ECM) composition, promote an insulin-resistant state. We set out to identify novel adipokines regulated by body fat mass in human subcutaneous WAT with potential roles in adipose function. METHODS: Adipose transcriptome data and secretome profiles from conditions with increased/decreased WAT mass were combined. WAT donors were predominantly women. In vitro effects were assessed using recombinant protein. Results were confirmed by quantitative PCR/ELISA, metabolic assays and immunochemistry in human WAT and adipocytes. RESULTS: We identified a hitherto uncharacterised adipokine, semaphorin 3C (SEMA3C), the expression of which correlated significantly with body weight, insulin resistance (HOMA of insulin resistance [HOMAIR], and the rate constant for the insulin tolerance test [KITT]) and adipose tissue morphology (hypertrophy vs hyperplasia). SEMA3C was primarily found in mature adipocytes and had no direct effect on human adipocyte differentiation, lipolysis, glucose transport or the expression of ß-oxidation genes. This could in part be explained by the significant downregulation of its cognate receptors during adipogenesis. In contrast, in pre-adipocytes, SEMA3C increased the production/secretion of several ECM components (fibronectin, elastin and collagen I) and matricellular factors (connective tissue growth factor, IL6 and transforming growth factor-ß1). Furthermore, the expression of SEMA3C in human WAT correlated positively with the degree of fibrosis in WAT. CONCLUSIONS/INTERPRETATION: SEMA3C is a novel adipokine regulated by weight changes. The correlation with WAT hypertrophy and fibrosis in vivo, as well as its effects on ECM production in human pre-adipocytes in vitro, together suggest that SEMA3C constitutes an adipocyte-derived paracrine signal that influences ECM composition and may play a pathophysiological role in human WAT.

Arterio-venous differences in peripheral blood mononuclear cells across human adipose tissue and the effect of adrenaline infusion.

Recent evidence indicates that adipose tissue macrophages and lymphocytes have a major role in the pathophysiology of obesity. The arterio-venous (A-V) difference technique has been used very effectively to understand adipose tissue metabolism in humans in vivo, and we set out to investigate whether it is possible to apply and use this technique to determine A-V differences for peripheral blood mononuclear cells (PBMCs) across human adipose tissue. We used flow-cytometric analysis of arterial blood and venous blood draining upper- (abdominal) and lower-body (femoral) adipose tissue depots in middle-aged volunteers (age 45±8 years, BMI 25.9±4.1 kg m(-2)). We determined A-V differences for various PBMCs. In basal conditions, there was evidence of modest retention of some PBMCs in adipose tissue, whereas the infusion of low-dose (physiological) adrenaline led to a marked release of many PBMCs (with little evidence of depot-specific differences). In addition to the demonstration that this approach is technically feasible, these results also indicate that physiological stimuli that change adrenaline concentrations and/or adipose tissue blood flow (such as physical activity) provoke the release of PBMCs from femoral and abdominal adipose depots.

Macrophage gene expression is related to obesity and the metabolic syndrome in human subcutaneous fat as well as in visceral fat.

AIMS/HYPOTHESIS: Our goal was to identify a set of human adipose tissue macrophage (ATM)-specific markers and investigate whether their gene expression in subcutaneous adipose tissue (SAT) as well as in visceral adipose tissue (VAT) is related to obesity and to the occurrence of the metabolic syndrome. METHODS: ATM-specific markers were identified by DNA microarray analysis of adipose tissue cell types isolated from SAT of lean and obese individuals. We then analysed gene expression of these markers by reverse transcription quantitative PCR in paired samples of SAT and VAT from 53 women stratified into four groups (lean, overweight, obese and obese with the metabolic syndrome). Anthropometric measurements, euglycaemic-hyperinsulinaemic clamp, blood analysis and computed tomography scans were performed. RESULTS: A panel of 24 genes was selected as ATM-specific markers based on overexpression in ATM compared with other adipose tissue cell types. In SAT and VAT, gene expression of ATM markers was lowest in lean and highest in the metabolic syndrome group. mRNA levels in the two fat depots were negatively correlated with glucose disposal rate and positively associated with indices of adiposity and the metabolic syndrome. CONCLUSIONS/INTERPRETATION: In humans, expression of ATM-specific genes increases with the degree of adiposity and correlates with markers of insulin resistance and the metabolic syndrome to a similar degree in SAT and in VAT.

Native human adipose stromal cells: localization, morphology and phenotype.

OBJECTIVES: Beside having roles in energy homeostasis and endocrine modulation, adipose tissue (AT) is now considered a promising source of mesenchymal stromal cells (adipose-derived stromal cells or ASCs) for regenerative medicine. Despite numerous studies on cultured ASCs, native human ASCs are rarely investigated. Indeed, the phenotype of ASCs in their native state, their localization within AT and comparison with bone marrow-derived mesenchymal stromal cells (BM-MSCs) has been poorly investigated. DESIGN: To address these issues, the stroma vascular fraction (SVF) of human AT was extracted and native cell subtypes were isolated by immunoselection to study their clonogenic potential in culture. Immunohistology on samples of human AT in combination with reconstruction of confocal sections were performed in order to localize ASCs. RESULTS: Compared with BM-MNCs, all native ASCs were found in the CD34(+) cell fraction of the AT-SVF. Native ASCs expressed classical mesenchymal markers described for BM-MSCs. Interestingly, CD34 expression decreased during ASC cell culture and was negatively correlated with cell proliferation rate. Immunohistological analysis revealed that native ASCs exhibited specific morphological features with protrusions. They were found scattered in AT stroma and did not express in vivo pericytic markers such as NG2, CD140b or alpha-smooth muscle actin, which appeared during the culture process. Finally, ASCs spontaneous commitment to adipocytic lineage was enhanced in AT from obese humans. CONCLUSIONS: The use of complementary methodological approaches to study native human ASCs revealed their immunophenotype, their specific morphology, their location within AT and their stemness. Furthermore, our data strongly suggest that human ASCs participate in adipogenesis during AT development.

Expression and secretion of the novel adipokine tartrate-resistant acid phosphatase from adipose tissues of obese and lean women.

OBJECTIVE: Tartrate-resistant acid phosphatase (TRAP) expressed by adipose tissue macrophages (ATMs) induces mice obesity and human adipocyte differentiation in vitro. This study aimed to investigate whether TRAP was secreted differently from human obese versus lean adipose tissues and to identify the cellular source of adipose tissue TRAP. DESIGN: Subcutaneous adipose tissues obtained from healthy subjects. Enzyme-linked immunosorbent assays (ELISAs) for total (5a+5b) and cleaved TRAP (5b) were used. TRAP secretion was determined in adipose tissue biopsies, and mRNA expression was studied in cell types isolated from the same. SUBJECTS: Results of 24 lean and 24 obese women (in vitro) and 8 subjects (in vivo) were compared. The main outcome measurements were TRAP expression and secretion in vitro and in vivo. RESULTS: In-house total TRAP ELISA showed high sensitivity and a coefficient of variance of 11%. Adipose secretion of total TRAP was linear in vitro with time and was evident in vivo. Total TRAP secretion in vitro was similar in lean and obese women expressed per unit weight of the adipose tissue but correlated positively with the number/size of adipocytes (P ≤ 0.01) and with adipose secretion of tumor necrosis factor-α and interleukin-6 (P<0.01). TRAP 5b was not secreted from the adipose tissue. ATMs displayed highest cellular expression of TRAP mRNA in adipose tissue cells derived from lean or obese women. CONCLUSIONS: TRAP is a novel human adipokine produced by macrophages and secreted from the subcutaneous adipose tissue in vivo and in vitro. Secretion is linked to the size and number of adipocytes, as well as to concomitant secretion of inflammatory mediators, suggesting that TRAP is involved in fat accumulation and adipose inflammation.

Dietary intervention-induced weight loss decreases macrophage content in adipose tissue of obese women.

OBJECTIVE: Accumulation of adipose tissue macrophages (ATMs) is observed in obesity and may participate in the development of insulin resistance and obesity-related complications. The aim of our study was to investigate the effect of long-term dietary intervention on ATM content in human adipose tissue. DESIGN: We performed a multi-phase longitudinal study. SUBJECTS AND MEASUREMENTS: A total of 27 obese pre-menopausal women (age 39 ± 2 years, body mass index 33.7 ± 0.5 kg m(-2)) underwent a 6-month dietary intervention consisting of two periods: 4 weeks of very low-calorie diet (VLCD) followed by weight stabilization composed of 2 months of low-calorie diet and 3 to 4 months of weight maintenance diet. At baseline and at the end of each dietary period, samples of subcutaneous adipose tissue (SAT) were obtained by needle biopsy and blood samples were drawn. ATMs were determined by flow cytometry using combinations of cell surface markers. Selected cytokine and chemokine plasma levels were measured using enzyme-linked immunosorbent assay. In addition, in a subgroup of 16 subjects, gene expression profiling of macrophage markers in SAT was performed using real-time PCR. RESULTS: Dietary intervention led to a significant decrease in body weight, plasma insulin and C-reactive protein levels. After VLCD, ATM content defined by CD45+/14+/206+ did not change, whereas it decreased at the end of the intervention. This decrease was associated with a downregulation of macrophage marker mRNA levels (CD14, CD163, CD68 and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1)) and plasma levels of monocyte-chemoattractant protein-1 (MCP-1) and CXCL5 (chemokine (C-X-C motif) ligand 5). During the whole dietary intervention, the proportion of two ATM subpopulations distinguished by the CD16 marker was not changed. CONCLUSION: A 6-month weight-reducing dietary intervention, but not VLCD, promotes a decrease in the number of the whole ATM population with no change in the relative distribution of ATM subsets.

Remodeling phenotype of human subcutaneous adipose tissue macrophages.

BACKGROUND: Adipose tissue macrophages (ATMs) have become a focus of attention recently because they have been shown to accumulate with an increase in fat mass and to be involved in the genesis of insulin resistance in obese mice. However, the phenotype and functions of human ATMs are still to be defined. METHODS AND RESULTS: The present study, performed on human subcutaneous AT, showed that ATMs from lean to overweight individuals are composed of distinct macrophage subsets based on the expression of several cell surface markers: CD45, CD14, CD31, CD44, HLA-DR, CD206, and CD16, as assessed by flow cytometry. ATMs isolated by an immunoselection protocol showed a mixed expression of proinflammatory (tumor necrosis factor-alpha, interleukin-6 [IL-6], IL-23, monocyte chemoattractant protein-1, IL-8, cyclooxygenase-2) and antiinflammatory (IL-10, transforming growth factor-beta, alternative macrophage activation-associated cc chemokine-1, cyclooxygenase-1) factors. Fat mass enlargement is associated with accumulation of the CD206+/CD16- macrophage subset that exhibits an M2 remodeling phenotype characterized by decreased expression of proinflammatory IL-8 and cyclooxygenase-2 and increased expression of lymphatic vessel endothelial hyaluronan receptor-1. ATMs specifically produced and released matrix metalloproteinase-9 compared with adipocytes and capillary endothelial cells, and secretion of matrix metalloproteinase-9 from human AT in vivo, assessed by arteriovenous difference measurement, was correlated with body mass index. Finally, ATMs exerted a marked proangiogenic effect on AT-derived endothelial and progenitor cells. CONCLUSIONS: The present results showed that the ATMs that accumulate with fat mass development exhibit a particular M2 remodeling phenotype. ATMs may be active players in the process of AT development through the extension of the capillary network and in the genesis of obesity-associated cardiovascular pathologies.

Lobular architecture of human adipose tissue defines the niche and fate of progenitor cells.

Human adipose tissue (hAT) is constituted of structural units termed lobules, the organization of which remains to be defined. Here we report that lobules are composed of two extracellular matrix compartments, i.e., septa and stroma, delineating niches of CD45-/CD34+/CD31- progenitor subsets characterized by MSCA1 (ALPL) and CD271 (NGFR) expression. MSCA1+ adipogenic subset is enriched in stroma while septa contains mainly MSCA1-/CD271- and MSCA1-/CD271high progenitors. CD271 marks myofibroblast precursors and NGF ligand activation is a molecular relay of TGFß-induced myofibroblast conversion. In human subcutaneous (SC) and visceral (VS) AT, the progenitor subset repartition is different, modulated by obesity and in favor of adipocyte and myofibroblast fate, respectively. Lobules exhibit depot-specific architecture with marked fibrous septa containing mesothelial-like progenitor cells in VSAT. Thus, the human AT lobule organization in specific progenitor subset domains defines the fat depot intrinsic capacity to remodel and may contribute to obesity-associated cardiometabolic risks.

Endothelial dysfunction in chronic myocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: role of enhanced vascular superoxide production.

BACKGROUND: Endothelial dysfunction of the peripheral vasculature is a well-known phenomenon in congestive heart failure that contributes to the elevated peripheral resistance; however, the underlying mechanisms have not yet been clarified. METHODS AND RESULTS: Dilator responses, the expression of protein and mRNA of the endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and soluble guanylate cyclase (sGC), and superoxide anion (O(2)(-)) and peroxynitrite production were determined in aortic rings from Wistar rats 8 weeks after myocardial infarction and compared with those in sham-operated animals. In rats with heart failure, the concentration-response curve of the endothelium-dependent vasodilator acetylcholine (after preconstriction with phenylephrine) was significantly shifted to the right, and the maximum relaxation was attenuated. Determination of expression levels of the 2 key enzymes for NO-mediated dilations, eNOS and sGC, revealed a marked upregulation of both enzymes in aortas from rats with heart failure, whereas iNOS expression was not changed. Pretreatment with exogenous superoxide dismutase partially restored the acetylcholine-induced relaxation in aortas from rats with heart failure. Aortic basal and NADH-stimulated O(2)(-) production assessed by use of lucigenin-enhanced chemiluminescence was significantly elevated in rats with chronic myocardial infarction. Peroxynitrite-mediated nitration of protein tyrosine residues was not different between the 2 groups of rats. CONCLUSIONS: These results demonstrate that endothelial dysfunction in ischemic heart failure occurs despite an enhanced vascular eNOS and sGC expression and can be attributed to an increase in vascular O(2)(-) production by an NADH-dependent oxidase. By inactivation of NO, O(2)(-) production appears to be an essential mechanism for the endothelial dysfunction observed in heart failure.

Improvement of postnatal neovascularization by human adipose tissue-derived stem cells.

BACKGROUND: Several studies have suggested that stem cells are present in the stroma-vascular fraction (SVF) of adipose tissue (AT). METHODS AND RESULTS: To characterize the cell populations that compose the SVF of human AT originating from subcutaneous and visceral depots, fluorescence-activated cell sorter analysis was performed by use of fluorescent antibodies directed against the endothelial and stem cell markers CD31, CD34, CD133, and ABCG2. The freshly harvested SVF contained large numbers of CD34+ cells as well as cells expressing CD133 and ABCG2. Further analysis of the CD34+ cells revealed 2 CD34+ cell populations with differential expression of the endothelial cell marker CD31. Selection of the CD34+/CD31- cells by use of magnetic microbeads, followed by cell culture, demonstrated that this cell population could differentiate under appropriate conditions into endothelial cells. Moreover, in mouse ischemic hindlimb, intravenous injection of CD34(+)/CD31(-) cells was associated with an increase in the blood flow and the capillary density and an incorporation of the cells in the leg vasculature. CONCLUSIONS: Our data indicate the presence of a cell population within the SVF of human AT characterized as CD34+/CD31- exhibiting characteristics of endothelial progenitor cells. Therefore, human AT might represent a source of stem/progenitor cells useful for cell therapy to improve vasculogenesis in adults.

Adipocyte produces matrix metalloproteinases 2 and 9: involvement in adipose differentiation.

Adipocyte hypertrophy and hyperplasia together with angiogenesis contribute to the growth of the fat mass. Because changes in the extracellular matrix (ECM) components are often associated with such cellular remodeling, we studied the adipocyte expression of the matrix metalloproteinases (MMPs) 2 and 9, two key enzymes involved in the modulation of ECM. The present study provides the first evidence that human adipose tissue produces and secretes MMP-2 and -9 as shown by gelatin zymography analysis performed on media conditioned by human subcutaneous adipose tissue and human preadipocytes in primary cultures and by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on transcripts from mature human adipocytes. The further characterization performed on the murine 3T3F442A preadipocyte cell line demonstrates that MMP expression, assessed by RT-PCR and Western blot analysis, as well as activity, assessed by gelatin zymography analysis, increased during the adipocyte differentiation, whereas the expression of tissue inhibitor metalloproteinases 1 and 2 were abolished or not affected, respectively. Finally, preadipocyte treatment with MMP inhibitors such as batimastat and captopril, as well as neutralizing antibodies, markedly decreased adipocyte differentiation as demonstrated by the inhibition in the appearance of lipogenic (triglycerides) and lipolytic (glycerol release and hormone-sensitive lipase expression) markers. These data suggest that MMP-2 and -9 could be important key regulators of adipocyte differentiation. Thus, the adipocyte-derived MMPs might represent a new target for the inhibition of adipose tissue growth.

Vascular endothelial growth factor up-regulates nitric oxide synthase expression in endothelial cells.

OBJECTIVE: Vascular endothelial growth factor (VEGF), secreted by vascular cells and a variety of tumour cells, is a potent angiogenic factor. Since nitric oxide (NO) seems to play a key role in the VEGF-induced proliferation of endothelial cells, the aim of the present study was to determine whether VEGF stimulates endothelial NO synthase (eNOS) expression and hence results in a maintained increase in NO formation. METHODS: Experiments were performed using cultured human umbilical vein endothelial cells (HUVEC) and isolated rat aortic rings. eNOS expression was assessed by Western blotting and RT-PCR analysis. RESULTS: Exposure of either confluent HUVEC or rat aortic rings to VEGF165 significantly increased eNOS mRNA and protein levels. This stimulatory effect of VEGF165 on eNOS expression was associated with an elevation in the basal production of cGMP in HUVEC, and with a leftward shift of the concentration-relaxation curve to acetylcholine in aortic rings. The VEGF-induced increase in eNOS mRNA levels was abolished by tyrosine kinase inhibitors suggesting involvement of a tyrosine kinase-dependent pathway. Since eNOS mRNA levels remained elevated in VEGF-treated cells in the presence of actinomycin D. it is likely that the VEGF-induced up-regulation of eNOS expression may be a consequence of a post-transcriptional effect on eNOS mRNA stability. CONCLUSION: The results demonstrate that VEGF enhances the expression of eNOS in native and cultured endothelial cells, an effect which may be important in the process of VEGF-induced angiogenesis.

Vasodilator dysfunction in aged spontaneously hypertensive rats: changes in NO synthase III and soluble guanylyl cyclase expression, and in superoxide anion production.

OBJECTIVE/METHODS: Genetic hypertension is associated with an apparent endothelial dysfunction and impaired endothelium-dependent vasodilatation in response to increased flow and receptor-dependent agonists. However, the link between impaired vasodilatation and nitric oxide (NO) synthase expression is still unclear. In the present study, dilator responses were determined in the aorta and coronary circulation of 16 month old spontaneously hypertensive (SHR) and Wistar Kyoto rats (WKY). Changes in vascular reactivity were compared with alterations in superoxide anion production as well as endothelial NO synthase (NOS III) and soluble guanylyl cyclase expression. RESULTS: In the isolated perfused heart both the bradykinin- and sodium nitroprusside-induced vasodilator responses were attenuated in SHR compared to WKY. Western blot analysis revealed a parallel reduction in NOS III expression in coronary microvascular endothelial cells from SHR. Superoxide anion production in aortae from SHR was markedly elevated over that of aortae from WKY, and was almost completely abolished by pretreatment with superoxide dismutase. Superoxide dismutase induced similar relaxations in phenylephrine-preconstricted aortic rings from both SHR and WKY, but failed to restore the attenuated acetylcholine- and sodium nitroprusside-induced relaxations in SHR. No difference in NOS III expression was detected in the aortae from either strain whereas soluble guanylyl cyclase expression was markedly decreased in SHR. CONCLUSIONS: These results demonstrate that NOS III expression in different tissues is differentially affected by hypertension. Moreover, although an elevated superoxide anion production is apparent in the aorta, a reduced soluble guanylyl cyclase expression appears to account for the observed vasodilator dysfunction in SHR.

alpha2-Adrenoceptor stimulation promotes actin polymerization and focal adhesion in 3T3F442A and BFC-1beta preadipocytes.

We previously demonstrated that in white fat cell precursors alpha2-adrenoceptor stimulation lead to the phosphorylation of p44 and p42 mitogen-activated protein kinases and an increase in cell number. Regulation of cell adhesion and cell cytoskeleton plays a crucial role in the control of cell growth by various growth factors. Here, we report that in mouse 3T3F442A preadipocytes expressing 2500 fmol/mg protein of the human alpha2C10-adrenoceptor (alpha2AF2 cells), alpha2-adrenergic stimulation rapidly restored the spreading of cells previously retracted by serum withdrawal. This effect was pertussis toxin sensitive and was blocked by pretreatment of the cells with dihydrocytochalasin B (a blocker of actin polymerization), genistein (a tyrosine kinase inhibitor), or agents that increase cell cAMP content. Spreading was accompanied by cell membrane ruffling, formation of lamelipodia and filipodia, appearance of focal adhesion plaques, and induction of actin stress fibers. alpha2-Adrenergic stimulation also lead to a rapid Gi- and actin-dependent tyrosine phosphorylation of the pp125 focal adhesion kinase (FAK) as well as of the p42 and p44 mitogen-activated protein kinases. alpha2-Adrenergic-dependent spreading and FAK and mitogen-activated protein kinase phosphorylation were also observed in 3T3F442A preadipocytes permanently expressing 20 fmol/mg protein of the human alpha2C10-adrenoceptor (alpha2AF3 cells) as well as in BFC-1beta preadipocytes, which constitutively express 25 fmol/mg protein of mouse alpha2A-adrenoceptors. In BFC-1beta preadipocytes, alpha2-adrenergic-dependent spreading and pp125FAK phosphorylation were counteracted by beta-adrenergic stimulation. Our results suggest that alpha2-adrenergic control of actin polymerization and focal adhesion assembly could play a crucial role in the regulation of preadipocyte growth by the sympathetic nervous system.

Androgenic regulation of adipocyte alpha 2-adrenoceptor expression in male and female Syrian hamsters: proposed transcriptional mechanism.

Adaptation of male hamsters to short daily (SD) photoperiod induced a reduction of the adipocyte alpha 2-adrenoceptor (alpha 2-AR) expression which was related to a sexual involution and could be reversed by testosterone administration. In the present paper, the possible mechanisms of such a physiological regulation are explored. The effect of testosterone on the adipocyte alpha 2-AR was rapid, dose-dependent, occurred at the physiological plasma concentrations of androgen, and was mimicked by dihydrotestosterone, but not by 17 beta-estradiol, progesterone, hydrocortisol, insulin, or T3. Adaptation of female hamsters to SD photoperiod also induced a sexual involution, but no modification of the adipocyte alpha 2-AR number was observed. Administration of testosterone induced a large up-regulation of the adipocyte alpha 2-AR. Testosterone was also able to up-regulate the adipocyte alpha 2-AR in male hamsters adapted to long day photoperiod whatever their age (6-, 12-, and 25-week old). Adaptation to SD photoperiod did not modify the adipocyte adenylyl cyclase activity (basal, forskolin-stimulated, GppNHp-inhibited). Conversely, UK14304-mediated inhibition of the adenylyl cyclase was suppressed in SD photoperiod and recovered after testosterone treatment. Administration of testosterone in young male hamsters adapted to long day photoperiod induced an increase in the amount of the alpha 2-AR messenger RNA, which coincided with the increase in the alpha 2-AR maximal number. The existence of a specific regulation of the adipocyte alpha 2-AR by the androgens, probably via a transcriptional mechanism, can be hypothesized. This regulation, which occurs in both male and female hamsters, appears to be physiologically relevant.

Aging and chronic hypertension decrease expression of rat aortic soluble guanylyl cyclase.

We analyzed the influence of aging and genetic hypertension on the function and expression of soluble guanylyl cyclase (sGC) in the aortas of prehypertensive and old spontaneously hypertensive rats (SHR) as well as in age-matched normotensive Wistar-Kyoto rats (WKY). The expression of heterodimeric sGC (alpha(1) and beta(1)) was assessed at the mRNA and protein level, and its function was assessed by the relaxant responses of phenylephrine-contracted endothelium-denuded aortic rings to the nitric oxide (NO) donor sodium nitroprusside. The vasodilator potency of sodium nitroprusside was significantly reduced (P<0.05) with age (3- to 6-fold increase in the EC(50) in old WKY and SHR compared with their young counterparts) as well as with hypertension (3-fold increase in old SHR compared with age-matched WKY), whereas the vasodilator potency of sodium nitroprusside did not differ between young SHR and WKY. A similar influence of aging and hypertension on NO-stimulated GC activity was revealed at the GC expression level: Whereas the beta(1) protein content was similar in young rats of both strains, old WKY exhibited 60% lower and old SHR exhibited 80% lower beta(1) subunit protein compared with young rats (P<0.05). Moreover, the abundance of alpha(1) and beta(1) mRNA (assessed by reverse transcriptase-polymerase chain reaction) was similar in young rats but was 2.5-fold (alpha(1)) and 4.3-fold (beta(1)) lower in old SHR compared with old WKY. In conclusion, our findings show that both aging and hypertension decrease sGC expression and its NO-dependent activation in aortic tissue. Downregulation of sGC may therefore contribute to arterial dysfunction in senescence and chronic hypertension.

Endothelial dysfunction coincides with an enhanced nitric oxide synthase expression and superoxide anion production.

We investigated the effects of aortic banding-induced hypertension on the endothelium-dependent vasodilator responses in the aorta and coronary circulation of Sprague-Dawley rats. We studied the influence of hypertension on the endothelial nitric oxide synthase (NOS III) expression, assessed by Western blot and reverse transcription-polymerase chain reactions experiments, and on the superoxide anion (O2-) production. Two weeks after aortic banding, the endothelium-dependent relaxations were not altered. At this time, the expression of NOS III in the aorta and in confluent coronary microvascular endothelial cells (RCMECs) exhibited no marked changes, whereas O2- production was enhanced 1.9-fold in aortas from aortic-banded rats. Six weeks after aortic banding, the endothelium-dependent dilations were markedly impaired in the heart (50% decrease) and aorta (35% decrease). Analysis of NOS III protein and mRNA levels revealed marked increases in both aortas and confluent RCMECs (2.6- to 4-fold) from aortic-banded compared with sham-operated rats. There was no further increase in O2production in both the aorta and confluent RCMECs from aortic-banded rats. An enhanced nitrotyrosine protein level was also detected in the aorta from 6-week aortic-banded rats. These findings indicate that in hypertension induced by aortic banding, an enhanced O2- production alone is not sufficient to produce endothelial dysfunction. Endothelial vasodilator hyporesponsiveness was observed only when NOS III expression and O2- production were increased and was associated with the appearance of enhanced nitrotyrosine residues. This would suggest that the development of endothelial dysfunction is linked to an overproduction of not one, but two, endothelium-derived radicals that might lead to the formation of peroxynitrite.