A familiar stranger:CD34 expression and putative functions in SVF cells of adipose tissue

Human adipose tissue obtained by liposuction is easily accessible and an abundant potential source of autologous cells for regenerative medicine applications. After digestion of the tissue and removal of differentiated adipocytes, the so-called stromal vascular fraction (SVF) of adipose, a mix of various cell types, is obtained. SVF contains mesenchymal fibroblastic cells, able to adhere to culture plastic and to generate large colonies in vitro , that closely resemble bone marrow-derived colony forming units-fibroblastic, and whose expanded progeny, adipose mesenchymal stem/stromal cells (ASC), show strong similarities with bone marrow mesenchymal stem cells. The sialomucin CD34, which is well known as a hematopoietic stem cell marker, is also expressed by ASC in native adipose tissue but its expression is gradually lost upon standard ASC expansion in vitro . Surprisingly little is known about the functional role of CD34 in the biology and tissue forming capacity of SVF cells and ASC. The present editorial provides a short introduction to the CD34 family of sialomucins and reviews the data from the literature concerning ex- pression and function of these proteins in SVF cells and their in vitro expanded progeny.     

Expression and function of leptin and its receptor in mouse mammary gland

Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-, lacta-tion-and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway.     

The effect of bafilomycin A1 and protease inhibitors on the degradation and recycling of a Class 5-mutant LDLR

The low-density lipoprotein receptor （LDLR） mediates cholesterol homeostasis through endocytosis of lipoprotein particles, particularly low-density lipoproteins （LDLs）. Normally, the lipoprotein particles are released in the endosomes and the receptors recycle to the cell surface. Familial hypercholesterolemia （FH） is an autosomal dominant disease caused by mutations in the gene encoding the LDLR. These mutations are divided into five functional classes where Class 5 mutations encode receptors that suffer from ligand-induced degradation and recycling deficiency. The aim of this study was to investigate whether it is possible to prevent the fast ligand-induced degradation of Class 5-mutant LDLR and to restore its ability to recycle to the cell surface. E387K is a naturally occurring Class 5 mutation found in FH patients, and in the present study, we used Chinese hamster ovary cells transfected with an E387K-mutant LDLR. Abrogation of endosomal acidification by adding bafdomycin A1 or addition of the irreversible serine protease inhibitors, 4-（2-aminoethyl）-benzenesulfonyl fluoride （AEBSF） and 3,4-dichloroisocoumarin （DCI）, prevented the degradation of the E387K-mutant LDLR. However, the undegraded receptor did not recycle to the cell surface in the presence of LDL. Unexpectedly, AEBSF caused aggregation of early endosome antigen-1positive endosomes and the intracellular trapped LDLR co-localized with these aggregated early endosomes.     

Polymorphism analyses of hepatitis B virus X gene in hepatocellular carcinoma patients from southern China

Chronic hepatitis B virus(HBV)infection is one of the major causes of hepatocellularcarcinoma(HCC),and the HBV X(HBx)gene plays a critical role in the molecular pathogenesis ofHBV-related HCC.We have investigated whether there are particular HBx gene mutations associated withHCC in patients from southern China.The HBx gene was examined in 51 paraffin-embedded tumor tissuesamples from patients with HCC and 25 serum samples from the HBV carrier by nested polymerase chainreaction(PCR),single-stranded conformational polymorphism and heteroduplex analysis.The HBx geneswith potentially important mutations from tumor tissue samples were cloned,sequenced and aligned withthe published HBx gene sequence.HBV genotypes in tumor tissue samples were analyzed by nested PCR.Analyses of HBx gene polymorphism showed that 31.3% of HBx gene fragments in tumor tissue sampleshad a special pattern.A common deletion at nt 382-400 of the HBx gene accompanied by 29 point mutationswas detected in four randomly selected tumor tissue samples with this pattern which caused a frame-shiftin the HBx open reading frame with a new stop codon at nt 1818,resulting in an HBx polypeptide chaintruncated at the C end in these cases.Among the four randomly selected samples,three were HBV genotypeB,and one was not detected by our present assay.In another tumor tissue sample,amplification of thefull-length HBx gene yielded a shorter fragment.Sequencing of this fragment revealed a 264 bp deletionbetween nt 1577 and 1840 of the HBV gene.These results suggest that HBx gene mutation occurs frequentlyin HCC samples,and the deletion at nt 382-400 of the HBx gene might play a role in carcinogenesis of HCCin southern China.     

Obesity and weight loss could alter the properties of adipose stem cells?

The discovery that adipose tissue represents an interesting source of multipotent stem cells has led to many studies exploring the clinical potential of these cells in cell-based therapies. Recent advances in understanding the secretory capacity of adipose tissue and the role of adipokines in the development of obesity and associated disorders have added a new dimension to the study of adipose tissue biology in normal and diseased states. Subcutaneous adipose tissue forms the interface between the clinical application of regenerative medicine and the establishment of the pathological condition of obesity. These two facets of adipose tissue should be understood as potentially related phenomena. Because of the functional characteristics of adipose stem cells, these cells represent a fundamental tool for understanding how these two facets are interconnected and could be important for therapeutic applications. In fact, adipose tissue stem cells have multiple functions in obesity related to adipogenic, angiogenic and secretory capacities. In addition, we have also previously described a predominance of larger blood vessels and an adipogenic memory in the subcutaneous adipose tissue after massive weight loss subsequent to bariatric surgery(ex-obese patients). Understanding the reversibility of the behavior of adipose stem cells in obeses and in weight loss is relevant to both physiological studies and the potential use of these cells in regenerative medicine.     

New findings showing how DNA methylation influences diseases

In 1975, Holliday and Pugh as well as Riggs independently hypothesized that DNA methylation in eukaryotes could act as a hereditary regulation mechanism that influences gene expression and cell differentiation. Interest in the study of epigenetic processes has been inspired by their reversibility as well as their potentially preventable or treatable consequences. Recently, we have begun to understand that the features of DNA methylation are not the same for all cells.Major differences have been found between differentiated cells and stem cells.Methylation influences various pathologies, and it is very important to improve the understanding of the pathogenic mechanisms. Epigenetic modifications may take place throughout life and have been related to cancer, brain aging, memory disturbances, changes in synaptic plasticity, and neurodegenerative diseases,such as Parkinson's disease and Huntington's disease. DNA methylation also has a very important role in tumor biology. Many oncogenes are activated by mutations in carcinogenesis. However, many genes with tumor-suppressor functions are "silenced" by the methylation of CpG sites in some of their regions.Moreover, the role of epigenetic alterations has been demonstrated in neurological diseases. In neuronal precursors, many genes associated with development and differentiation are silenced by CpG methylation. In addition,recent studies show that DNA methylation can also influence diseases that do not appear to be related to the environment, such as IgA nephropathy, thus affecting,the expression of some genes involved in the T-cell receptor signaling. In conclusion, DNA methylation provides a whole series of fundamental information for the cell to regulate gene expression, including how and when the genes are read, and it does not depend on the DNA sequence.     

Leptin treatment markedly increased plasma adiponectin but barely decreased plasma resistin of ob/ob mice

Adiponectin (ApN) and leptin are two adipocytokines that control fuel homeostasis, body weight, and insulin sensitivity. Their interplay is still poorly studied. These hormones are either undetectable or decreased in obese, diabetic ob/ob mice. We examined the effects of leptin treatment on ApN gene expression, protein production, secretion, and circulating levels of ob/ob mice. We also briefly tackled the influence of this treatment on resistin, another adipocytokine involved in obesity-related insulin resistance. Leptin-treated (T) obese mice (continuous sc infusion for 6 days) were compared with untreated lean (L), untreated obese (O), and untreated pair-fed obese (PF) mice. Blood was collected throughout the study. At day 3 or day 6, fat pads were either directly analyzed (mRNA, ApN content) or cultured for up to 24 h (ApN secretion). The direct effect of leptin was also studied in 3T3-F442A adipocytes. Compared with L mice, ApN content of visceral or subcutaneous fat and ApN secretion by adipose explants were blunted in obese mice. Accordingly, plasma ApN levels of O mice were decreased by 50%. Leptin treatment of ob/ob mice increased ApN mRNAs, ApN content, and secretion from the visceral depot by 50-80%. Leptin also directly stimulated ApN mRNAs and secretion from 3T3-F442A adipocytes. After 6 days of treatment, plasma ApN of ob/ob mice increased 2.5-fold, a rise that did not occur in PF mice. Plasma resistin of T mice was barely decreased. Leptin treatment, but not mere calorie restriction, corrects plasma ApN in obese mice by restoring adipose tissue ApN concentrations and secretion, at least in part, via a direct stimulation of ApN gene expression. Such a treatment only minimally affects circulating resistin. ApN restoration could, in concert with leptin, contribute to the metabolic effects classically observed during leptin administration.     

Serum leptin concentrations during short-term administration of growth hormone and triiodothyronine in healthy adults: a randomised, double-blind placebo-controlled study.

The regulation of adipose tissue mass and energy expenditure is currently subject to intensive research, which primarily relates to the discovery of leptin. Leptin is a peptide, which is the product of the obese (ob) gene expressed in adipose tissue of several species icluding humans. Leptin is supposed to serve both as an index of fat mass and as a sensor of energy balance. Administration of recombinant murine leptin in ob/ob-mice, which do not produce leptin, decreases food intake and increases thermogenesis both of which result in a reduction in body weight and adipose tissue mass. The calorigenic effect of leptin presumably acts through an increase in sympathetic outflow which in turn activates the beta3 adrenergic receptor in brown adipose tissue. The regulation and action of endogenous leptin in humans are less well understood, and clinical grade recombinant human leptin is so far not available. Serum leptin correlates logarithmically with total body fat in both normal weight and obese subjects, which suggest insensitivity to leptin in obese patients. Furthermore, more rapid excursions in serum leptin have been reported following short-term changes in caloric intake and administration of insulin. Growth hormone (GH) exerts pronounced effects on lipid metabolism and resting energy expenditure. The lipolytic actions of GH appear to involve both increased sensitivity to the beta-adrenergic pathway, and a suppression of adipose tissue lipoprotein lipase activity. The calorigenic effects of GH have been shown not only to be secondary to changes in lean body mass. Growth hormone administration furthermore increases the peripheral conversion of thyroxine to triiodothyronine, which may contribute to the overall actions of GH on fuel and energy metabolism. So far, little is known about the effects of GH and iodothyronines on serum leptin levels in humans. We therefore measured serum leptin levels and energy expenditure before and after the administration of GH and triiodothyronine, alone and in combinaion, in a randomized double-blind placebo-controlled study in healthy young male adults. The dose of triiodothyronine was selected to obtain serum levels comparable to those seen after GH administration.     

Modulation of circulating leptin levels by its soluble receptor

Leptin is an adipocyte-derived hormone with potent weight reducing effects. Genetically obese rodents with mutations of leptin or the leptin receptor are defective in leptin signaling and develop morbid obesity and diabetes. Interestingly, the levels of both leptin mRNA and protein are increased by up to 20-fold in these animals, suggesting the existence of a feedback mechanism controlling the amount of leptin in circulation. In this report, we attempted to determine whether the up-regulation of circulating leptin in Zucker Diabetic Fatty rats, which are nonresponsive to leptin due to a receptor point mutation, is entirely due to increased expression of leptin. We demonstrate that the high level of circulating leptin in these rats is attributable to at least two factors: increased leptin expression by the adipose tissue and delayed clearance of leptin from circulation due to binding to its soluble receptor. The latter conclusion was supported by three lines of evidence: 1) The soluble leptin receptor is up-regulated by about 20-fold in Zucker Diabetic Fatty rats; 2) Adenovirus-mediated overexpression of the soluble leptin receptor results in a similar -fold increase of circulating leptin; 3) In ob/ob mice, which have no endogenous leptin, exogenously administered leptin reaches a higher level when the soluble leptin receptor is overexpressed. The weight-reducing effect of leptin is enhanced in C57Bl/6 ob/ob mice with overexpression of the soluble leptin receptor. Soluble leptin receptor may be a significant factor determining the amount of total leptin in circulation.     

A cutaneous gene therapy approach to human leptin deficiencies: correction of the murine ob/ob phenotype using leptin-targeted keratinocyte grafts.

Leptin deficiency produces a phenotype of obesity, diabetes, and infertility in the ob/ob mouse. In humans, leptin deficiency occurs in some cases of congenital obesity and in lipodystrophic disorders characterized by reduced adipose tissue and insulin resistance. Cutaneous gene therapy is considered an attractive potential method to correct circulating protein deficiencies, since gene-transferred human keratinocytes can produce and secrete gene products with systemic action. However, no studies showing correction of a systemic defect have been reported. We report the successful correction of leptin deficiency using cutaneous gene therapy in the ob/ob mouse model. As a feasibility approach, skin explants from transgenic mice overexpressing leptin were grafted on immunodeficient ob/ob mice. One month later, recipient mice reached body weight values of lean animals. Other biochemical and clinical parameters were also normalized. In a second human gene therapy approach, a retroviral vector encoding both leptin and EGFP cDNAs was used to transduce HK and, epithelial grafts enriched in high leptin-producing HK were transplanted to immunosuppressed ob/ob mice. HK-derived leptin induced body weight reduction after a drop in blood glucose and food intake. Leptin replacement through genetically engineered HK grafts provides a valuable therapeutic alternative for permanent treatment of human leptin deficiency conditions.     

Expression of ob gene coding the production of the hormone leptin in hepatocytes of liver with steatosis

Leptin is a circulating pleiotropic hormone that play an important role in appetite control, fat metabolism, regulation of body weight, fetus growth, growth and aging of adults and hematopoiesis. It is expressed abundantly and specifically in the adipose tissue. A liver cell with developed steatosis represents a cell metabolism similar to metabolism of cells of adipose tissue. Analyses of serum leptin and free leptin receptor in the serum of patients with steatosis showed significant variations from reference limits of normal values. However in liver tissue with verified steatosis detection of mRNA gene for leptin was not proven. Such expression of ob gene for leptin was not found even in the liver tissue without steatosis. With respect to the absence of ob gene expression, the direct effect of ob gene expression on other parameters of leptin metabolism could not be evaluated. The RT-PCR method with verified specificity and satisfying sensitivity was developed. The results obtained from analysis of serum leptin and free leptin receptor in the serum are presented and evaluated. The used methods were verified and reference limits for Czech population were defined in dependence on age and other clinical parameters.     

Reduction of obesity, as induced by leptin, reverses endothelial dysfunction in obese (Lep(ob)) mice.

Obesity is a major health care problem and is associated with significant cardiovascular morbidity. Leptin, a neuroendocrine hormone released by adipose tissue, is important in modulating obesity by signaling satiety and increasing metabolism. Moreover, leptin receptors are expressed on vascular endothelial cells (ECs) and mediate angiogenesis. We hypothesized that leptin may also play an important role in vasoregulation. We investigated vasoregulatory mechanisms in the leptin-deficient obese (ob/ob) mouse model and determined the influence of leptin replacement on endothelial-dependent vasorelaxant responses. The direct effect of leptin on EC nitric oxide (NO) production was also tested by using 4, 5-diaminofluorescein-2 diacetate staining and measurement of nitrate and nitrite concentrations. Vasoconstrictor responses to phenylephrine, norepinephrine, and U-46619 were markedly enhanced in aortic rings from ob/ob mice and were modulated by NO synthase inhibition. Vasorelaxant responses to ACh were markedly attenuated in mesenteric microvessels from ob/ob mice. Leptin replacement resulted in significant weight loss and reversal of the impaired endothelial-dependent vasorelaxant responses observed in ob/ob mice. Preincubation of ECs with leptin enhanced the release of NO production. Thus leptin-deficient ob/ob mice demonstrate marked abnormalities in vasoregulation, including impaired endothelial-dependent vasodilation, which is reversed by leptin replacement. These findings may be partially explained by the direct effect of leptin on endothelial NO production. These vascular abnormalities are similar to those observed in obese, diabetic, leptin-resistant humans. The ob/ob mouse may, therefore, be an excellent new model for the study of the cardiovascular effects of obesity.     

Leptin is a potent stimulator of bone growth in ob/ob mice

Leptin, the product of the obese gene, is a circulating hormone secreted primarily from adipocytes. The lack of leptin in ob/ob mice, who are homozygous for the obese gene, results in hyperglycemia, hyperinsulinemia, hyperphagia, obesity, infertility, decreased brain size and decreased stature. To this end, we investigated the role of leptin as a hormonal regulator of bone growth. Leptin administration led to a significant increase in femoral length, total body bone area, bone mineral content and bone density in ob/ob mice as compared to vehicle treated controls. The increase in total body bone mass was a result of an increase in both trabecular and cortical bone mass. These results suggest that the decreased stature of the ob/ob mouse is due to a developmental defect that is readily reversible upon leptin administration. Our demonstration that the signalling or long form (Ob-Rb) of the leptin receptor is present in both primary adult osteoblasts and chondrocytes suggests that the growth promoting effects of leptin could be direct. In summary, these results indicate a novel role for leptin in skeletal bone growth and development.     

Potential role of leptin in endochondral ossification.

Leptin, a 16-kD circulating hormone secreted mainly by white adipose tissue, is a product of the obese (ob) gene. Leptin acts on human marrow stromal cells to enhance differentiation into osteoblasts and inhibit differentiation into adipocytes. Leptin also inhibits bone formation through a hypothalamic relay. To obtain a better understanding of the potential role of leptin in bone formation, the localization of leptin in endochondral ossification was examined immunohistochemically. High expression of leptin was identified in hypertrophic chondrocytes in the vicinity of capillary blood vessels invading hypertrophic cartilage and in a number of osteoblasts of the primary spongiosa beneath the growth plate. The hypertrophic chondrocytes far from the blood vessels were negative for leptin. Moreover, we detected the production and secretion of leptin by a mouse osteoblast cell line (MC3T3-E1) and a mouse chondrocyte cell line (MCC-5) by RT-PCR, immunocytochemistry, and Western blotting. Leptin enhanced the proliferation, migration, tube formation, and matrix metalloproteinase-2 (MMP-2) activity of human endothelial cells (HUVECs) in vitro. These findings suggest the possibility that leptin exerts its influence on endochondral ossification by regulating angiogenesis.     

Isoproterenol Decreases Leptin Expression in Adipose Tissue of Obese Humans

RICCI, MATTHEW R. AND SUSAN K. FRIED. Isoproterenol decreases leptin expression in adipose tissue of obese humans. Obes Res. Objective: We investigated the effects of the non-selective β-adrenergic agonist, isoproterenol (Iso), on leptin expression in human adipose tissue. Research Methods and Procedures: Subcutaneous (SQ) and omental adipose (OM) tissue taken during surgery from 12 morbidly obese subjects (10 women and 2 men) were cultured for up to 24 hours with insulin (7 nM) and/or dexamethasone (25 nM), a synthetic glucocorticoid, in the presence or absence of isoproterenol (10 μM). Adipose tissue was also acutely incubated for 3 hours in media alone with or without isoproterenol. Leptin secretion and leptin mRNA abundance were measured. Results: Iso acutely decreased leptin release by −30% (vs. no hormone controls) in fragments of OM and SQ adipose tissue. In 24-hour culture, addition of Iso (in the presence of insulin) resulted in lower leptin accumulation in the medium (−20–30%) and leptin mRNA levels (−40–50%) from both tissue depots. Culture with insulin and dexamethasone increased leptin expression vs. insulin alone. Addition of Iso with insulin and dexamethasone decreased media leptin (−40–60%) and leptin mRNA levels were lower (−65%) in Iso-treated adipose tissue from both depots after 24 hours. Iso effects were not detectable after 5 hours of culture. Discussion: We conclude that stimulation of β-adrenergic receptors may modulate leptin expression in human adipose tissue by two mechanisms: an acute effect on leptin release and a longer-term antagonism of stimulatory effects of insulin and dexamethasone on leptin mRNA expression. These mechanisms may contribute to the decline in serum leptin that occurs during fasting.