ABSTRACT
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.
Subject(s)
Adipocytes/metabolism , CD146 Antigen/biosynthesis , Stromal Cells/metabolism , Adipocytes/cytology , Antigens, CD34/biosynthesis , Antigens, CD34/genetics , CD146 Antigen/genetics , Calcium-Binding Proteins , Cell Differentiation/physiology , Cells, Cultured , Flow Cytometry , Humans , Intercellular Signaling Peptides and Proteins/biosynthesis , Intercellular Signaling Peptides and Proteins/genetics , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Stromal Cells/cytologyABSTRACT
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.
Subject(s)
Adipogenesis , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/metabolism , Weight Loss , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolism , Adult , Animals , Autophagy , Cell Differentiation , Cells, Cultured , Female , Gene Expression Regulation , Humans , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , MAP Kinase Signaling System , Mice , Middle Aged , Phosphorylation , Stem Cells/cytology , Stem Cells/metabolism , Subcutaneous Fat/cytology , Subcutaneous Fat/metabolism , Young AdultABSTRACT
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.
Subject(s)
Adipose Tissue, White/cytology , Antigens, CD34/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Stromal Cells/metabolism , Actins/metabolism , Aged , Aged, 80 and over , Animals , Calcium-Binding Proteins , Cell Differentiation , Cells, Cultured , Female , Flow Cytometry , Humans , Male , Mice , Microscopy, Fluorescence , Middle Aged , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Stromal Cells/cytologyABSTRACT
The antitumor activity of monoclonal antibodies in the treatment of chronic lymphocytic leukemia is mediated mainly by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Unfortunately, the efficacy of complement-dependent cytotoxicity is strongly restricted due to the expression and acquisition of regulators of complement activation by lymphocytic leukemia cells. Whereas the role of membrane regulators of complement activation, such as CD55 and CD59, has been investigated in detail in chronic lymphocytic leukemia, the involvement of soluble regulators of complement activation, such as complement factor H, has not yet been reported. Propidium iodide staining was performed to investigate the efficacy of ofatumumab and factor H-derived short-consensus-repeat 18-20 in the induction of complement-dependent cytotoxicity on primary chronic lymphocytic leukemia cells from 20 patients. Deposition of complement C3 fragments was monitored by western blot analysis. Expression of CD20, CD55 or CD59 was determined by FACS analysis. Replacement of factor H with short consensus repeat 18-20 significantly increased the susceptibility of primary chronic lymphocytic leukemia cells to ofatumumab-induced complement-dependent cytotoxicity. More importantly, addition of short-consensus-repeat 18-20 was able to overcome complement- resistance occurring during treatment with ofatumumab alone. Use of short consensus repeat 18-20 is likely to prolong the turnover time of active C3b fragments generated on the target cells following ofatumumab-induced complement activation, thereby improving specific killing of chronic lymphocytic leukemia cells by complement-dependent cytotoxicity. The relative contribution of factor H to the protection of chronic lymphocytic leukemia cells against complement-dependent cytotoxicity was comparable to that of CD55. Our data suggest that, by abrogating factor H function, short consensus repeat 18-20 may provide a novel approach that improves the complement-dependent efficacy of therapeutic monoclonal antibodies.
Subject(s)
Antibodies, Monoclonal/pharmacology , Complement Factor H/pharmacology , Consensus Sequence/drug effects , Cytotoxins/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized , Cells, Cultured , Complement Factor H/genetics , Consensus Sequence/genetics , Cytotoxins/therapeutic use , Dose-Response Relationship, Drug , Drug Synergism , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathologyABSTRACT
Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels.