The circadian transcription factor ARNTL2 is regulated by weight-loss interventions in human white adipose tissue and inhibits adipogenesis.

Misalignment of physiological circadian rhythms promotes obesity which is characterized by white adipose tissue (WAT) expansion. Differentiation of Adipose stem/progenitor cells (ASCs) contributes to WAT increase but the importance of the cellular clock in this process is incompletely understood. In the present study, we reveal the role of the circadian transcription factor Aryl hydrocarbon receptor nuclear translocator-like 2 (ARNTL2) in human ASCs, isolated from subcutaneous (s)WAT samples of patients undergoing routine elective plastic abdominal surgery. We show that circadian synchronization by serum-shock or stimulation with adipogenic stimuli leads to a different expression pattern of ARNTL2 relative to its well-studied paralogue ARNTL1. We demonstrate that ARNTL2 mRNA is downregulated in ASCs upon weight-loss (WL) whereas ARNTL2 protein is rapidly induced in the course of adipogenic differentiation and highly abundant in adipocytes. ARNTL2 protein is maintained in ASCs cooperatively by mechanistic Target of Rapamycin (mTOR) and Mitogen-activated Protein Kinase (MAPK) signalling pathways while ARNTL2 functions as an inhibitor on both circuits, leading to a feedback mechanism. Consistently, ectopic overexpression of ARNTL2 repressed adipogenesis by facilitating the degradation of ARNTL1, inhibition of Kruppel-Like Factor 15 (KLF15) gene expression and down-regulation of the MAPK-CCAAT/enhancer-binding protein ß (C/EBPß) axis. Western blot analysis of sWAT samples from normal-weight, obese and WL donors revealed that ARNTL2 protein was solely elevated by WL compared to ARNTL1 which underscores unique functions of both transcription factors. In conclusion, our study reveals ARNTL2 to be a WL-regulated inhibitor of adipogenesis which might provide opportunities to develop strategies to ameliorate obesity.

Dipeptidyl peptidase-4 cell surface expression marks an abundant adipose stem/progenitor cell population with high stemness in human white adipose tissue.

The capacity of adipose stem/progenitor cells (ASCs) to undergo self-renewal and differentiation is crucial for adipose tissue homoeostasis, regeneration and expansion. However, the heterogeneous ASC populations of the adipose lineage constituting adipose tissue are not precisely known. In the present study, we demonstrate that cell surface expression of dipeptidyl peptidase-4 (DPP4)/cluster of differentiation 26 (CD26) subdivides the DLK1-/CD34+/CD45-/CD31- ASC pool of human white adipose tissues (WATs) into two large populations. Ex vivo, DPP4+ ASCs possess higher self-renewal and proliferation capacity and lesser adipocyte differentiation potential than DDP4- ASCs. The knock-down of DPP4 in ASC leads to significantly reduced proliferation and self-renewal capacity, while adipogenic differentiation is increased. Ectopic overexpression of DPP4 strongly inhibits adipogenesis. Moreover, in whole mount stainings of human subcutaneous (s)WAT, we detect DPP4 in CD34+ ASC located in the vascular stroma surrounding small blood vessels and in mature adipocytes. We conclude that DPP4 is a functional marker for an abundant ASC population in human WAT with high proliferation and self-renewal potential and low adipogenic differentiation capacity.

Adaptations of the 3T3-L1 adipocyte lipidome to defective ether lipid catabolism upon Agmo knockdown.

Little is known about the physiological role of alkylglycerol monooxygenase (AGMO), the only enzyme capable of cleaving the 1-O-alkyl ether bond of ether lipids. Expression and enzymatic activity of this enzyme can be detected in a variety of tissues including adipose tissue. This labile lipolytic membrane-bound protein uses tetrahydrobiopterin as a cofactor, and mice with reduced tetrahydrobiopterin levels have alterations in body fat distribution and blood lipid concentrations. In addition, manipulation of AGMO in macrophages led to significant changes in the cellular lipidome, and alkylglycerolipids, the preferred substrates of AGMO, were shown to accumulate in mature adipocytes. Here, we investigated the roles of AGMO in lipid metabolism by studying 3T3-L1 adipogenesis. AGMO activity was induced over 11 days using an adipocyte differentiation protocol. We show that RNA interference-mediated knockdown of AGMO did not interfere with adipocyte differentiation or affect lipid droplet formation. Furthermore, lipidomics revealed that plasmalogen phospholipids were preferentially accumulated upon Agmo knockdown, and a significant shift toward longer and more polyunsaturated acyl side chains of diacylglycerols and triacylglycerols could be detected by mass spectrometry. Our results indicate that alkylglycerol catabolism has an influence not only on ether-linked species but also on the degree of unsaturation in the massive amounts of triacylglycerols formed during in vitro 3T3-L1 adipocyte differentiation.

An organoid model derived from human adipose stem/progenitor cells to study adipose tissue physiology.

We established a functional adipose organoid model system for human adipose stem/progenitor cells (ASCs) isolated from white adipose tissue (WAT). ASCs were forced to self-aggregate by a hanging-drop technique. Afterwards, spheroids were transferred into agar-coated cell culture dishes to avoid plastic-adherence and dis-aggregation. Adipocyte differentiation was induced by an adipogenic hormone cocktail. Morphometric analysis revealed a significant increase in organoid size in the course of adipogenesis until d 18. Whole mount staining of organoids using specific lipophilic dyes showed large multi- and unilocular fat deposits in differentiated cells indicating highly efficient differentiation of ASCs into mature adipocytes. Moreover, we found a strong induction of the expression of key adipogenesis and adipocyte markers (CCAAT/enhancer-binding protein (C/EBP) ß, peroxisome proliferator-activated receptor (PPAR) γ, fatty acid-binding protein 4 (FABP4), adiponectin) during adipose organoid formation. Secreted adiponectin was detected in the cell culture supernatant, underscoring the physiological relevance of mature adipocytes in the organoid model. Moreover, colony formation assays of collagenase-digested organoids revealed the maintenance of a significant fraction of ASCs within newly formed organoids. In conclusion, we provide a reliable and highly efficient WAT organoid model, which enables accurate analysis of cellular and molecular markers of adipogenic differentiation and adipocyte physiology.

Quiescence, Stemness and Adipogenic Differentiation Capacity in Human DLK1-/CD34+/CD24+ Adipose Stem/Progenitor Cells.

We explore the status of quiescence, stemness and adipogenic differentiation capacity in adipose stem/progenitor cells (ASCs) ex vivo, immediately after isolation from human subcutaneous white adipose tissue, by sorting the stromal vascular fraction into cell-surface DLK1+/CD34-, DLK1+/CD34dim and DLK1-/CD34+ cells. We demonstrate that DLK1-/CD34+ cells, the only population exhibiting proliferative and adipogenic capacity, express ex vivo the bonafide quiescence markers p21Cip1, p27Kip1 and p57Kip2 but neither proliferation markers nor the senescence marker p16Ink4a. The pluripotency markers NANOG, SOX2 and OCT4 are barely detectable in ex vivo ASCs while the somatic stemness factors, c-MYC and KLF4 and the early adipogenic factor C/EBPß are highly expressed. Further sorting of ASCs into DLK1-/CD34+/CD24- and DLK1-/CD34+/CD24+ fractions shows that KLF4 and c-MYC are higher expressed in DLK1-/CD34+/CD24+ cells correlating with higher colony formation capacity and considerably lower adipogenic activity. Proliferation capacity is similar in both populations. Next, we show that ASCs routinely isolated by plastic-adherence are DLK1-/CD34+/CD24+. Intriguingly, CD24 knock-down in these cells reduces proliferation and adipogenesis. In conclusion, DLK1-/CD34+ ASCs in human sWAT exist in a quiescent state, express high levels of somatic stemness factors and the early adipogenic transcription factor C/EBPß but senescence and pluripotency markers are barely detectable. Moreover, our data indicate that CD24 is necessary for adequate ASC proliferation and adipogenesis and that stemness is higher and adipogenic capacity lower in DLK1-/CD34+/CD24+ relative to DLK1-/CD34+/CD24- subpopulations.

Targeting cellular senescence based on interorganelle communication, multilevel proteostasis, and metabolic control.

Cellular senescence, a stable cell division arrest caused by severe damage and stress, is a hallmark of aging in vertebrates including humans. With progressing age, senescent cells accumulate in a variety of mammalian tissues, where they contribute to tissue aging, identifying cellular senescence as a major target to delay or prevent aging. There is an increasing demand for the discovery of new classes of small molecules that would either avoid or postpone cellular senescence by selectively eliminating senescent cells from the body (i.e., 'senolytics') or inactivating/switching damage-inducing properties of senescent cells (i.e., 'senostatics/senomorphics'), such as the senescence-associated secretory phenotype. Whereas compounds with senolytic or senostatic activity have already been described, their efficacy and specificity has not been fully established for clinical use yet. Here, we review mechanisms of senescence that are related to mitochondria and their interorganelle communication, and the involvement of proteostasis networks and metabolic control in the senescent phenotype. These cellular functions are associated with cellular senescence in in vitro and in vivo models but have not been fully exploited for the search of new compounds to counteract senescence yet. Therefore, we explore possibilities to target these mechanisms as new opportunities to selectively eliminate and/or disable senescent cells with the aim of tissue rejuvenation. We assume that this research will provide new compounds from the chemical space which act as mimetics of caloric restriction, modulators of calcium signaling and mitochondrial physiology, or as proteostasis optimizers, bearing the potential to counteract cellular senescence, thereby allowing healthy aging.

CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells.

The CRISPR/Cas9 system is a powerful tool to generate a specific loss-of-function phenotype by gene knockout (KO). However, this approach is challenging in primary human cells. In this technical report, we present a reliable protocol to achieve a functional KO in the genome of human adipose stem/progenitor cells (ASCs). Using Sprouty1 (SPRY1) as a model target gene for a CRISPR/Cas9 mediated KO, we particularize the procedure including the selection of the CRISPR/Cas9 target sequences and the employment of appropriate lentiviral vectors to obtain a functional gene KO. The efficiency of CRISPR/Cas9 to mutate the SPRY1 gene is determined by a PCR-based mutation detection assay and sequence analysis. Effects on mRNA and protein levels are studied by RT-qPCR and Western blotting. In addition, we demonstrate that CRISPR/Cas9 mediated SPRY1 KO and gene silencing by shRNA are similarly effective to deplete the Sprouty1 protein and to inhibit adipogenic differentiation. In summary, we show a reliable approach to achieve a gene KO in human ASCs, which could also apply to other primary cell types. Abbreviations: ASC: Adipogenic Stem/Progenitor Cell; Cas: CRISPR-associated system; CRISPR: Clustered Regularly Interspaced Palindromic Repeat; gDNA: Genomic DNA; GOI: Gene of interest; gRNA: Guide RNA; NHEJ: Non-homologous end joining; Indel: Insertion/Deletion; PAM: Protospacer adjacent motif; sWAT: Subcutaneous white adipose tissue; TIDE: Tracking of indels by decomposition.

Sprouty1 Prevents Cellular Senescence Maintaining Proliferation and Differentiation Capacity of Human Adipose Stem/Progenitor Cells.

The role of Ras-Mitogen-activated protein kinase (MAPK) signaling in cellular aging is not precisely understood. Recently, we identified Sprouty1 (SPRY1) as a weight-loss target gene in human adipose stem/progenitor cells (ASCs) and showed that Sprouty1 is important for proper regulation of adipogenesis. In the present study, we show that loss-of-function of Sprouty1 by CRISPR/Cas9-mediated genome editing in human ASCs leads to hyper-activation of MAPK signaling and a senescence phenotype. Sprouty1 knockout ASCs undergo an irreversible cell cycle arrest, become enlarged and stain positive for senescence-associated ß-galactosidase. Sprouty1 down-regulation leads to DNA double strand breaks, a considerably increased number of senescence-associated heterochromatin foci and induction of p53 and p21Cip1. In addition, we detect an increase of hypo-phosphorylated Retinoblastoma (Rb) protein in SPRY1 knockout ASCs. p16Ink4A is not induced. Moreover, we show that Sprouty1 knockout leads to induction of a senescence-associated secretory phenotype as indicated by the activation of the transcription factors NFκB and C/EBPß and a significant increase in mRNA expression and secretion of interleukin-8 (IL-8) and CXCL1/GROα. Finally, we demonstrate that adipogenesis is abrogated in senescent SPRY1 knockout ASCs. In conclusion, this study reveals a novel mechanism showing the importance of Sprouty1 for the prevention of senescence and the maintenance of the proliferation and differentiation capacity of human ASCs.

Human bone marrow adipocytes display distinct immune regulatory properties.

BACKGROUND: The bone marrow (BM) is a major reservoir of resting memory T cells and long-lived plasma cells, capable of providing protection against recurrent infections. Whether the age-related accumulation of adipose tissue in the BM affects the functionality and maintenance of memory cells is not well understood. METHODS: For the first time, we compare human femur marrow adipose tissue (fMAT) and subcutaneous white adipose tissue of the thigh (tsWAT) obtained from the same donors. Therefore, we used microarrays for comparative global gene expression analysis, and employed assays to analyse parameters of adipocyte biology, inflammation and oxidative stress. FINDINGS: We show that fMAT adipocytes differ significantly from tsWAT adipocytes regarding specific gene expression profiles including inflammatory responses and adipogenesis/adipocyte phenotype. Concomitant with considerably lower levels of CD36, a membrane-associated protein important for long-chain fatty acid uptake that is used as maturation marker, fMAT adipocytes are smaller and contain less triglycerides. fMAT adipocytes secrete similar levels of adiponectin and leptin as tsWAT adipocytes, and express increased levels of pro-inflammatory molecules concomitant with an elevated generation of reactive oxygen species (ROS) and impaired function of plasma cells in the BM. INTERPRETATION: Our findings suggest that fMAT is a unique type of adipose tissue containing small adipocytes with lower CD36 protein and triglyceride levels than tsWAT but high adipokine secretion. Moreover, fMAT adipocytes secrete high levels of pro-inflammatory cytokines, contributing to inflammation and impairment of plasma cell function in the BM, suggesting that fMAT has more immune regulatory functions than tsWAT.

Sprouty1 is a weight-loss target gene in human adipose stem/progenitor cells that is mandatory for the initiation of adipogenesis.

The differentiation of adipose stem/progenitor cells (ASCs) into adipocytes contributes to adipose tissue expansion in obesity. This process is regulated by numerous signalling pathways including MAPK signalling. In the present study, we show that weight loss (WL) interventions induce upregulation of Sprouty1 (SPRY1), a negative regulator of MAPK signalling, in human ASCs and elucidate the role of the Sprouty1/MAPK interaction for adipogenic differentiation. We found that the Sprouty1 protein levels are low in proliferating ASCs, increasing in density arrested ASCs at the onset of adipogenic differentiation and decreasing in the course of adipogenesis. Knock-down (KD) of Sprouty1 by RNA interference led to elevated MAPK activity and reduced expression of the early adipogenic transcription factor CCAAT/enhancer-binding protein ß (C/EBP ß), concomitant with an abrogation of adipogenesis. Intriguingly, co-treatment of Sprouty1 KD ASCs with differentiation medium and the pharmacological MEK inhibitor U0126 blunted ERK phosphorylation; however, failed to rescue adipogenic differentiation. Thus, the effects of the Sprouty1 KD are not reversed by inhibiting MAPK signalling although the inhibition of MAPK signalling by U0126 did not prevent adipogenic differentiation in wild type ASCs. In conclusion, we show that Sprouty1 is induced after WL in ASCs of formerly obese people acting as a negative regulator of MAPK signalling, which is necessary to properly trigger adipogenesis at early stages by a C/EBP ß dependent mechanism.

Fibroblast feeder layer supports adipogenic differentiation of human adipose stromal/progenitor cells.

Adipose stromal/progenitor cells (ASCs) can differentiate into adipocytes in the course of adipogenesis. This process is governed by systemic factors and signals of the adipose stem cell niche. ASCs isolated from fat tissues and amplified in vitro provide an essential and reliable model system to study adipogenesis. However, current cell culture models routinely grow ASCs on plastic surfaces largely missing niche parameters. In the present communication, we employed human foreskin fibroblasts (HFFs) monolayers as feeder cells for ASCs, which were isolated from human subcutaneous white adipose tissue and amplified in vitro. We found that PPARγ2 and several adipocyte markers were significantly higher expressed in differentiated ASCs growing on feeder layers relative to plastic dishes. Moreover, a significant higher number of adipocytes was generated from ASCs cultured on feeder layer and these adipocytes contained larger fat droplets. Insulin strongly stimulated glucose uptake into adipocytes produced on feeder layer suggesting that these cells show characteristic metabolic features of fat cells.  Finally, we show that the HFF feeder layer allows adipogenic differentiation of low-density-seeded ASCs. In conclusion, we demonstrate that the HFF feeder layer increases adipocyte differentiation of ASCs and allows differentiation of low density seeded progenitor cells  into functional adipocytes.

Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells.

BACKGROUND: The proliferation and adipogenic differentiation of adipose stromal cells (ASCs) are complex processes comprising major phenotypical alterations driven by up- and downregulation of hundreds of genes. Quantitative RT-PCR can be employed to measure relative changes in the expression of a gene of interest. This approach requires constitutively expressed reference genes for normalization to counteract inter-sample variations due to differences in RNA quality and quantity. Thus, a careful validation of quantitative RT-PCR reference genes is needed to accurately measure fluctuations in the expression of genes. Here, we evaluated candidate reference genes applicable for quantitative RT-PCR analysis of gene expression during proliferation and adipogenesis of human ASCs with the immunophenotype DLK1+/CD34+/CD90+/CD105+/CD45-/CD31-. METHODS: We evaluated the applicability of 10 candidate reference genes (GAPDH, TBP, RPS18, EF1A, TFRC, GUSB, PSMD5, CCNA2, LMNA and MRPL19) using NormFinder, geNorm and BestKeeper software. RESULTS: The results indicate that EF1A and MRPL19 are the most reliable reference genes for quantitative RT-PCR analysis of proliferating ASCs. PSMD5 serves as the most reliable endogenous control in adipogenesis. CCNA2 and LMNA were among the least consistent genes. CONCLUSIONS: Applying these findings for future gene expression analyses will help elucidate ASC biology.

Carbonic anhydrase III protects osteocytes from oxidative stress.

Osteocytes are master orchestrators of bone remodeling; they control osteoblast and osteoclast activities both directly via cell-to-cell communication and indirectly via secreted factors, and they are the main postnatal source of sclerostin and RANKL (receptor activator of NF-kB ligand), two regulators of osteoblast and osteoclast function. Despite progress in understanding osteocyte biology and function, much remains to be elucidated. Recently developed osteocytic cell lines-together with new genome editing tools-has allowed a closer look at the biology and molecular makeup of these cells. By using single-cell cloning, we identified genes that are associated with high Sost/sclerostin expression and analyzed their regulation and function. Unbiased transcriptome analysis of high- vs. low-Sost/sclerostin-expressing cells identified known and novel genes. Dmp1 (dentin matrix protein 1), Dkk1 (Dickkopf WNT signaling pathway inhibitor 1), and Phex were among the most up-regulated known genes, whereas Srpx2, Cd200, and carbonic anhydrase III (CAIII) were identified as novel markers of differentiated osteocytes. Aspn, Enpp2, Robo2, Nov, and Serpina3g were among the transcripts that were most significantly suppressed in high-Sost cells. Considering that CAII was recently identified as being regulated by Sost/sclerostin and capable of controlling mineral homeostasis, we focused our attention on CAIII. Here, we report that CAIII is highly expressed in osteocytes, is regulated by parathyroid hormone both in vitro and in vivo, and protects osteocytes from oxidative stress.-Shi, C., Uda, Y., Dedic, C., Azab, E., Sun, N., Hussein, A. I., Petty, C. A., Fulzele, K., Mitterberger-Vogt, M. C., Zwerschke, W., Pereira, R., Wang, K., Divieti Pajevic, P. Carbonic anhydrase III protects osteocytes from oxidative stress.

CD146 (MCAM) in human cs-DLK1-/cs-CD34+ adipose stromal/progenitor cells.

To precisely characterize CD146 in adipose stromal/progenitor cells (ASCs) we sorted the stromal vascular faction (SVF) of human abdominal subcutaneous white adipose tissue (sWAT) according to cell surface (cs) expression of CD146, DLK1 and CD34. This test identified three main SVF cell populations: ~50% cs-DLK1-/cs-CD34+/cs-CD146- ASCs, ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146+ and ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146- cells. All cells contained intracellular CD146. Whole mount fluorescent IHC staining of small vessels detected CD146+ endothelial cells (CD31+/CD34+/CD146+) and pericytes (CD31-/CD34-/CD146+ ASCs). The cells in the outer adventitial layer showed the typical ASC morphology, were strongly CD34+ and contained low amounts of intracellular CD146 protein (CD31-/CD34+/CD146+). Additionally, we detected wavy CD34-/CD146+ and CD34dim/CD146+ cells. CD34dim/CD146+ cells were slightly more bulky than CD34-/CD146+ cells. Both CD34-/CD146+ and CD34dim/CD146+ cells were detached from the inner pericyte layer and protruded into the outer adventitial layer. Cultured early passage ASCs contained low levels of CD146 mRNA, which was expressed in two different splicing variants, at a relatively high amount of the CD146-long form and at a relatively low amount of the CD146-short form. ASCs contained low levels of CD146 protein, which consisted predominantly long form and a small amount of short form. The CD146 protein was highly stable, and the majority of the protein was localized in the Golgi apparatus. In conclusion, the present study contributes to a better understanding of the spatial localization of CD34+/CD146+ and CD34-/CD146+ cells in the adipose niche of sWAT and identifies CD146 as intracellular protein in cs-DLK1-/cs-CD34+/cs-CD146- ASCs.

Silencing of the small GTPase DIRAS3 induces cellular senescence in human white adipose stromal/progenitor cells.

Inhibition of Akt-mTOR signaling protects from obesity and extends life span in animals. In the present study, we analyse the impact of the small GTPase, GTP-binding RAS-like 3 (DIRAS3), a recently identified weight-loss target gene, on cellular senescence in adipose stromal/progenitor cells (ASCs) derived from human subcutaneous white adipose tissue (sWAT). We demonstrate that DIRAS3 knock-down (KD) in ASCs induces activation of Akt-mTOR signaling and proliferation arrest. DIRAS3 KD ASCs lose the potential to form colonies and are negative for Ki-67. Moreover, silencing of DIRAS3 results in a premature senescence phenotype. This is characterized by senescence-associated ß-galactosidase positive enlarged ASCs containing increased p16INK4A level and activated retinoblastoma protein. DIRAS3 KD ASCs form senescence-associated heterochromatic foci as shown by increased level of γ-H2A.X positive foci. Furthermore, these cells express a senescence-associated secretory phenotype characterized by increased interleukin-8 secretion. Human DIRAS3 KD ASCs develop also a senescence phenotype in sWAT of SCID mice. Finally, we show that DIRAS3 KD in ASCs stimulates both adipogenic differentiation and premature senescence. In conclusion, our data suggest that silencing of DIRAS3 in ASCs and subsequently hyper-activation of Akt-mTOR drives adipogenesis and premature senescence. Moreover, differentiating ASCs and/or mature adipocytes may acquire features of cellular senescence.

Weight Loss Upregulates the Small GTPase DIRAS3 in Human White Adipose Progenitor Cells, Which Negatively Regulates Adipogenesis and Activates Autophagy via Akt-mTOR Inhibition.

Long-term weight-loss (WL) interventions reduce insulin serum levels, protect from obesity, and postpone age-associated diseases. The impact of long-term WL on adipose-derived stromal/progenitor cells (ASCs) is unknown. We identified DIRAS3 and IGF-1 as long-term WL target genes up-regulated in ASCs in subcutaneous white adipose tissue of formerly obese donors (WLDs). We show that DIRAS3 negatively regulates Akt, mTOR and ERK1/2 signaling in ASCs undergoing adipogenesis and acts as a negative regulator of this pathway and an activator of autophagy. Studying the IGF-1-DIRAS3 interaction in ASCs of WLDs, we demonstrate that IGF-1, although strongly up-regulated in these cells, hardly activates Akt, while ERK1/2 and S6K1 phosphorylation is activated by IGF-1. Overexpression of DIRAS3 in WLD ASCs completely inhibits Akt phosphorylation also in the presence of IGF-1. Phosphorylation of ERK1/2 and S6K1 is lesser reduced under these conditions. In conclusion, our key findings are that DIRAS3 down-regulates Akt-mTOR signaling in ASCs of WLDs. Moreover, DIRAS3 inhibits adipogenesis and activates autophagy in these cells.

Characterization of DLK1(PREF1)+/CD34+ cells in vascular stroma of human white adipose tissue.

Sorting of native (unpermeabilized) SVF-cells from human subcutaneous (s)WAT for cell surface staining (cs) of DLK1 and CD34 identified three main populations: ~10% stained cs-DLK1+/cs-CD34-, ~20% cs-DLK1+/cs-CD34+dim and ~45% cs-DLK1-/cs-CD34+. FACS analysis after permeabilization showed that all these cells stained positive for intracellular DLK1, while CD34 was undetectable in cs-DLK1+/cs-CD34- cells. Permeabilized cs-DLK1-/cs-CD34+ cells were positive for the pericyte marker α-SMA and the mesenchymal markers CD90 and CD105, albeit CD105 staining was dim (cs-DLK1-/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45-/CD31-). Only these cells showed proliferative and adipogenic capacity. Cs-DLK1+/cs-CD34- and cs-DLK1+/cs-CD34+dim cells were also α-SMA+ but expressed CD31, had a mixed hematopoietic and mesenchymal phenotype, and could neither proliferate nor differentiate into adipocytes. Histological analysis of sWAT detected DLK1+/CD34+ and DLK1+/CD90+ cells mainly in the outer ring of vessel-associated stroma and at capillaries. DLK1+/α-SMA+ cells were localized in the CD34- perivascular ring and in adventitial vascular stroma. All these DLK1+ cells possess a spindle-shaped morphology with extremely long processes. DLK1+/CD34+ cells were also detected in vessel endothelium. Additionally, we show that sWAT contains significantly more DLK1+ cells than visceral (v)WAT. We conclude that sWAT has more DKL1+ cells than vWAT and contains different DLK1/CD34 populations, and only cs-DLK1-/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45-/CD31- cells in the adventitial vascular stroma exhibit proliferative and adipogenic capacity.

Activators and stimulators of soluble guanylate cyclase counteract myofibroblast differentiation of prostatic and dermal stromal cells.

BACKGROUND: Fibrotic diseases encompass numerous systemic and organ-specific disorders characterized by the development and persistence of myofibroblasts. TGFß1 is considered the key inducer of fibrosis and drives myofibroblast differentiation in cells of diverse histological origin by a pro-oxidant shift in redox homeostasis associated with decreased nitric oxide (NO)/cGMP signaling. Thus, enhancement of NO/cGMP represents a potential therapeutic strategy to target myofibroblast activation and therefore fibrosis. METHODS: Myofibroblast differentiation was induced by TGFß1 in human primary prostatic (PrSCs) and normal dermal stromal cells (NDSCs) and monitored by α smooth muscle cell actin (SMA) and IGF binding protein 3 (IGFBP3) mRNA and protein levels. The potential of enhanced cGMP production by the sGC stimulator BAY 41-2272 or the sGC activator BAY 60-2770 to inhibit and revert myofibroblast differentiation in vitro was analyzed. Moreover, potential synergisms of BAY 41-2272 or BAY 60-2770 and inhibition of cGMP degradation by the PDE5 inhibitor vardenafil were investigated. RESULTS: BAY 41-2272 and BAY 60-2770 at doses of 30µM significantly inhibited induction of SMA and IGFBP3 levels in PrSCs and reduced myofibroblast marker levels in TGFß1-predifferentiated cells. At lower concentrations (3 and 10µM) only BAY 41-2272 but not BAY 60-2770 significantly inhibited and reverted myofibroblast differentiation. In NDSCs both substances significantly inhibited differentiation at all concentrations tested. Attenuation of SMA expression was more pronounced in NDSCs whereas reduction of IGFBP3 levels by BAY 41-2272 appeared more efficient in PrSCs. Moreover, administration of BAY 41-2272 or BAY 60-2770 enhanced the efficiency of the PDE5 inhibitor vardenafil to inhibit and revert myofibroblast differentiation in vitro. CONCLUSIONS: Increase of cGMP by sGC stimulation/activation significantly inhibited and reverted myofibroblast differentiation. This effect was even more pronounced when a combination treatment with a PDE5 inhibitor was applied. Thus, enhancement of NO/cGMP-signaling by sGC stimulation/activation is a promising strategy for the treatment of fibrotic diseases. Whereas, in NDSCs BAY 60-2770 and BAY 41-2272 exerted similar effects on myofibroblast differentiation, higher potency of BAY 41-2272 was observed in PrSCs, indicating phenotypical differences between fibroblasts form different organs that should be taken into account in the search for antifibrotic therapies.

Analysis of human papillomavirus E7 protein status in C-33A cervical cancer cells.

High-risk human papillomaviruses (HPV) are the main etiologic factor for the development of cervical cancer. Infections by these viruses have been detected in virtually all cervical cancers. C-33A is one of the rare cervical cancer derived cell lines considered as HPV-negative. Employing monoclonal antibodies raised against a conformational epitope of the HPV-16 E7 oncoprotein, we present evidence suggesting that E7-positive cells can be sporadically and transiently detected in C-33A cell cultures. Immunoblotting with affinity-purified rabbit polyclonal anti-HPV 16 E7 antisera and q-RT-PCR analysis suggest that these cells do probably not express HPV-16 E7. Moreover, we show that the HPV E7 protein level differs considerably between individual cells in cultures of several established cervical cancer cell lines. Our data suggest that expression of the E7 protein is variable in established cervical cancer cell lines including C-33A cells.