A phase I study of CR002, a fully-human monoclonal antibody against platelet-derived growth factor-D.

OBJECTIVE: To investigate the safety, pharmacokinetics (PK), binding activity and immunogenicity of CR002, a human monoclonal antibody (mAb) directed against platelet-derived growth factor-D (PDGF-D), administered as a single intravenous (i.v.) infusion over a range of doses. SUBJECTS: 40 healthy male subjects received increasing doses of CR002 at 0.3, 1, 3, 10, 30 mg/kg or placebo. METHOD: This was a randomized, double-blind, placebo-controlled, dose-escalation Phase I study. The trial had a duration of 90 days, with dosing on Day 1 and follow-up visits on Days 2, 4, 7, 14, 21, 30, 45 and 90. Serum was collected for PK, binding activity and immunogenicity analysis at screening and up to Day 90. Safety was recorded throughout the study by performing laboratory tests, recording vital signs and electrocardiograms (ECGs), by monitoring the occurrence of adverse events (AEs). The use of concomitant medications was also recorded. RESULTS: All 40 subjects received CR002 or placebo, and completed the trial. No dose-limiting toxicities (DLTs) occurred, the maximum tolerated dose (MTD) was not reached and was estimated as > 30 mg/kg. There were no deaths during this study and no SAEs or other significant AEs reported. The most frequent drug-related treatment-emergent AE (TEAE) was headache in 4 of 30 subjects (13.3%) in the CR002 group vs. 0 of 10 subjects in the placebo group. CR002 exhibited linear PK parameters, had a long half-life (t1/2 in the range 15.5 â 48.1 days) and a volume of distribution at steady state in the range 4.7 â 6.5. Free PDGF-D in the serum bound to CR002 in a reversible manner, as shown in the lowest dose cohort. However, levels of total circulating PDGF-D remained constant throughout the study. There were no anti-CR002 antibodies detected in subjects dosed with CR002. CONCLUSIONS: CR002 was safe and well-tolerated at all doses tested as a single i.v. administration. The MTD was estimated to be above 30 mg/kg, the highest dose tested. CR002 had a long half-life, low clearance and a limited tissue distribution. Although total levels of PDGF-D at all dose levels remained relatively constant, there was no detectable circulating free PDGF-D after CR002 administration. At the lowest CR002 dose tested (0.3 mg/kg), PDGF-D was detectable again by Day 21 and the levels increased near to pre-infusion levels by Day 90. In this study, CR002 was not immunogenic during the 90-day study period.

Yield of human adipose-derived adult stem cells from liposuction aspirates.

BACKGROUND: Primary cultures of isolated human adipose-derived adult stem (ADAS) cells are multipotent and differentiate in vitro along the adipocyte, chondrocyte, neuronal, osteoblast, and skeletal muscle pathways. METHODS: We examined the ADAS cell yield per unit volume of liposuction tissue, and their surface protein phenotype by flow cytometry. Adipogenesis was assessed by Oil Red O staining and ELISA analysis of leptin secretion. RESULTS: The donor population was 87.5% female (n=18) with a mean age (+/-SD) of 44+/-10 years and body mass index (BMI) of 24.9+/-2.7. The mean cell yield was 404 000+/-206 000 cells per milliliter of lipoaspirate (n=18). Linear regression analysis of the cells derived from the female donors demonstrated a significant negative correlation between the number of cells obtained per milliliter of lipoaspirate with the BMI but not the age of the donor. The undifferentiated ADAS cells were homogeneously positive for the cell-surface markers CD10, CD13, CD29, CD44, CD49e, CD59, CD90, and HLA-ABC, and homogeneously negative for the cell surface markers CD11b, CD45, and HLA-DR. The absence of the panhematopoietic marker, CD45, indicates that the ADAS cells do not derive from circulating BM hematopoietic stem cells. Adipocyte differentiation led to a 5.1-fold increase in Oil Red O staining, and a 196-fold increase in leptin secretion levels. Culture of the cells in the presence of antibiotic and fungizone did not alter the undifferentiated ADAS cell immunophenotype based on flow cytometry, or their adipocyte differentiation based on leptin secretion. DISCUSSION: The ability to isolate a consistently homogeneous population of undifferentiated adult stem cells from adipose tissue of multiple donors supports their potential utility in future tissue-engineering applications.

Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells.

Human adipose tissue represents an abundant reservoir of stromal cells with potential utility for tissue engineering. The current study demonstrates the ability of human adipose tissue-derived stromal cells to display some of the hallmarks of osteoblast differentiation in vitro. Following treatment with ascorbate, beta-glycerophosphate, dexamethasone, and 1,25 dihydroxy vitamin D(3), adipose tissue-derived stromal cells mineralize their extracellular matrix based on detection of calcium phosphate deposits using Alizarin Red and von Kossa histochemical stains. Fourier transform infrared analysis demonstrates the apatitic nature of these crystals. Mineralization is accompanied by increased expression or activity of the osteoblast-associated proteins osteocalcin and alkaline phosphatase. These and other osteoblast-associated gene markers are detected based on polymerase chain reaction. In contrast, the adipocyte gene markers--leptin, lipoprotein lipase, and peroxisome proliferator activated receptor gamma2--are reduced under mineralization conditions, consistent with the reciprocal relationship postulated to exist between adipocytes and osteoblasts. The current work supports the presence of a multipotent stromal cell population within human extramedullary adipose tissue. These findings have potential implications for human bone tissue bioengineering.

Trans-10, cis-12, but not cis-9, trans-11, conjugated linoleic acid attenuates lipogenesis in primary cultures of stromal vascular cells from human adipose tissue.

We have previously shown that both a commercially available mixture of conjugated linoleic acid (CLA) isomers and the trans-10, cis-12 isomer of CLA reduced the triglyceride (TG) content and induced apoptosis in differentiating cultures of murine 3T3-L1 preadipocytes. However, the influence of CLA isomers on differentiating human (pre)adipocytes is unknown. Therefore, we conducted a series of studies using primary cultures of stromal vascular cells isolated from human adipose tissue to determine: 1) the influence of seeding density and thiazolidinedione (TZD) concentration on TG content; 2) the chronic dose response of cis-9, trans-11 CLA vs. trans-10, cis-12 CLA on TG content; 3) whether chronic linoleic acid supplementation could rescue the TG content of CLA-treated cultures; and 4) whether trans-10, cis-12-mediated reduction in cellular TG was due to decreased lipogenesis and/or increased lipolysis. In expt. 1, the TG content [micromol/(L x 10(6) cells)] increased as both seeding density and TZD concentration increased. For example, cultures seeded at 4 x 10(4) cells/cm(2) and supplemented with 10 micromol/L BRL 49653 had 10-fold more TG than similarly seeded cultures without BRL 49653. In expt. 2, TG content decreased as the level of trans-10, cis-12 CLA increased from 1 to 10 micromol/L, whereas the TG content increased with increasing concentrations of either linoleic acid or cis-9, trans-11 CLA. In expt. 3, linoleic acid supplementation restored the TG content of cultures treated with trans-10, cis-12 CLA compared with cultures treated with CLA alone, suggesting that attenuation of TG content by CLA is reversible. In expt. 4, glucose incorporation into total lipid decreased with increasing levels of trans-10, cis-12 CLA, whereas neither CLA isomer acutely affected lipolysis. These data suggest that the reported antiobesity actions of a supplement containing a crude mixture of CLA isomers given to humans may be due to inhibition of lipogenesis by the trans-10, cis-12 isomer.

Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: biochemical, cellular, and molecular analysis.

While adipocyte differentiation has been studied extensively in murine cultures, the lack of a readily available preadipocyte model has hindered equivalent studies in man. We describe methods for the isolation and culture of primary human stromal cells from surgical adipose tissue specimens. In vitro, the stromal cells rapidly differentiate in response to a combination of adipogenic agents. Among these, glucocorticoids and thiazolidinediones act together to induce the formation of lipid vacuoles within the cells. These morphologic changes accompany the increased expression of 2 characteristic adipocyte proteins, the cytoplasmic enzyme glycerol phosphate dehydrogenase (GPDH) and the secreted cytokine leptin. Likewise, stromal cell differentiation results in elevated mRNA levels for the fatty acid binding protein aP2 and the adipogenic regulatory transcription factors CCAAT/enhancer binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) in addition to leptin. The in vitro differentiated stromal cells exhibit a lipolytic response to beta-adrenergic agonists, comparable to that reported with primary human adipocytes. These studies demonstrate the validity of human adipose tissue-derived stromal cells as a reliable in vitro model for investigations of adipocyte metabolism in humans.

Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous.

The current study was done to assess if heterogeneity existed in the degree of adipogenesis in stromal cells (preadipocytes) from multiple donors. In addition to conventional lipid-based methods, we have employed a novel signal amplification technology, known as branched DNA, to monitor expression of an adipocyte specific gene product aP2. The fatty acid binding protein aP2 increases during adipocyte differentiation and is induced by thiazolidinediones and other peroxisome proliferator activated receptor gamma ligands. The current work examined the adipogenic induction of aP2 mRNA levels in human adipose tissue stromal cells derived from 12 patients (mean age +/- SEM, 38.9 +/- 3.1) with mild to moderate obesity (mean body mass index +/- SEM, 27.8 +/- 2.4). Based on branched DNA technology, a rapid and sensitive measure of specific RNAs, the relative aP2 level in adipocytes increased by 679 +/- 93-fold (mean +/- SEM, n=12) compared to preadipocytes. Normalization of the aP2 mRNA levels to the housekeeping gene, glyceraldehyde phosphate dehydrogenase, did not significantly alter the fold induction in a subset of 4 patients (803.6 +/- 197.5 vs 1118.5 +/- 308.1). Independent adipocyte differentiation markers were compared between adipocytes and preadipocytes in parallel studies. Leptin secretion increased by up to three-orders of magnitude while measurements of neutral lipid accumulation by Oil Red O and Nile Red staining increased by 8.5-fold and 8.3-fold, respectively. These results indicate that preadipocytes isolated from multiple donors displayed varying degrees of differentiation in response to an optimal adipogenic stimulus in vitro. This work also demonstrates that branched DNA measurement of aP2 is a rapid and sensitive measure of adipogenesis in human stromal cells. The linear range of this assay extends up to three-orders of magnitude and correlates directly with independent measures of cellular differentiation.

Role of intracellular calcium in human adipocyte differentiation.

Intracellular calcium ([Ca(2+)](i)) modulates adipocyte lipid metabolism and inhibits the early stages of murine adipogenesis. Consequently, we evaluated effects of increasing [Ca(2+)](i) in early and late stages of human adipocyte differentiation. Increasing [Ca(2+)](i) with either thapsigargin or A23187 at 0-1 h of differentiation markedly suppressed differentiation, with a 40-70% decrease in triglyceride accumulation and glycerol-3 phosphate dehydrogenase (GPDH) activity (P < 0.005). However, a 1-h pulse of either agent at 47-48 h only modestly inhibited differentiation. Sustained, mild stimulation of Ca(2+) influx with either agouti protein or 10 mM KCl-induced depolarization during 0-48 h of differentiation inhibited triglyceride accumulation and GPDH activity by 20-70% (P < 0.05) and markedly suppressed peroxisome proliferator-activated receptor gamma (PPARgamma) expression. These effects were reversed by Ca(2+) channel antagonism. In contrast, Ca(2+) pulses late in differentiation (71-72 h or 48-72 h) markedly increased these markers of differentiation. Thus increasing [Ca(2+)](i) appears to exert a biphasic regulatory role in human adipocyte differentiation, inhibiting the early stages while promoting the late stage of differentiation and lipid filling.

Differential regulation of peroxisome proliferator activated receptor gamma1 (PPARgamma1) and PPARgamma2 messenger RNA expression in the early stages of adipogenesis.

Adipocyte differentiation is driven by the expression and activation of three transcription factor families: the differentially expressed CAAT/enhancer binding proteins (C/EBPs) alpha, beta, and delta; the helix-loop-helix adipocyte differentiation and determination factor-1; and peroxisome proliferator activated receptor gamma (PPARgamma), expressed as two isoforms, PPARgamma1 and the adipocyte-specific PPARgamma2. Overexpression of PPARgamma can induce adipocyte differentiation; therefore, we analyzed the expression of the two PPARgamma isoforms during early stages of differentiation to determine whether one was preferentially induced as an early determining event. Surprisingly, in the first 24 h, a 3-6-fold increase of PPARgamma2 mRNA was observed, whereas PPARgamma1 mRNA remained unchanged. PPARgamma1 was induced 1 day later. Overexpression of C/EBPbeta has also been shown to induce adipocyte differentiation. A C/EBP site was identified only in the human PPARgamma2 promoter. Its deletion blunted the response of PPARgamma2 promoter to cotransfected C/EBPbeta or methylisobutylxanthine treatment. We hypothesize that PPARgamma2 initiates adipocyte differentiation.

Dual DNA binding specificity of ADD1/SREBP1 controlled by a single amino acid in the basic helix-loop-helix domain.

Adipocyte determination- and differentiation-dependent factor 1 (ADD1), a member of the basic helix-loop-helix (bHLH) family of transcription factors, has been associated with both adipocyte differentiation and cholesterol homeostasis (in which case it has been termed SREBP1). Using PCR-amplified binding analysis, we demonstrate that ADD1/SREBP1 has dual DNA sequence specificity, binding to both an E-box motif (ATCACGTGA) and a non-E-box sequence previously shown to be important in cholesterol metabolism, sterol regulatory element 1 (SRE-1; ATCACCCCAC). The ADD1/SREBP1 consensus E-box site is similar to a regulatory sequence designated the carbohydrate response element, defined by its ability to regulate transcription in response to carbohydrate in genes involved in fatty acid and triglyceride metabolism in liver and fat. When expressed in fibroblasts, ADD1/SREBP1 activates transcription through both the carbohydrate response E-box element and SRE-1. Substitution of an atypical tyrosine in the basic region of ADD1/SREBP1 to an arginine found in most bHLH protein causes a restriction to only E-box binding. Conversely, substitution of a tyrosine for the equivalent arginine in another bHLH protein, upstream stimulatory factor, allows this factor to acquire a dual binding specificity similar to that of ADD1/SREBP1. Promoter activation by ADD1/SREBP1 through the carbohydrate response element E box is not sensitive to the tyrosine-to-arginine mutation, while activation through SRE-1 is completely suppressed. These data illustrate that ADD1/SREBP1 has dual DNA sequence specificity controlled by a single amino acid residue; this dual specificity may provide a novel mechanism to coordinate different pathways of lipid metabolism.

Vasodilation of rat retinal microvessels induced by monobutyrin. Dysregulation in diabetes.

1-Butyryl-glycerol (monobutyrin) is a simple lipid product of adipocytes with angiogenic activity. Recent studies have shown that the biosynthesis of this compound is tightly linked to lipolysis, a process associated with changes in blood flow. We now present data indicating that monobutyrin is an effective vasodilator of rodent blood vessels using a fluorescent retinal angiogram assay. The vasodilatory activity of monobutyrin is potent (ED50 = 3.3 x 10(-7) M), dose dependent, and stereospecific. Because diabetes represents a catabolic, lipolytic state with numerous vascular complications, we examined the action and regulation of monobutyrin in insulin-deficient diabetic rats. Serum levels of monobutyrin in streptozotocin-induced diabetic rats were greatly elevated compared to normal animals. At the same time, the retinal vessels of the diabetic animals develop a resistance to the vasodilatory activity of monobutyrin. These results demonstrate a role for monobutyrin in the control of vascular tone and suggest a possible involvement in the pathology of diabetes.

Identification of base and backbone contacts used for DNA sequence recognition and high-affinity binding by LAC9, a transcription activator containing a C6 zinc finger.

The LAC9 protein of Kluyveromyces lactis is a transcriptional regulator of genes in the lactose-galactose regulon. To regulate transcription, LAC9 must bind to 17-bp upstream activator sequences (UASs) located in front of each target gene. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae, and the two proteins must bind DNA in a very similar manner. In this paper we show that high-affinity, sequence-specific binding by LAC9 dimers is mediated primarily by 3 bp at each end of the UAS: [Formula: see text]. In addition, at least one half of the UAS must have a GC or CG base pair at position 1 for high-affinity binding; LAC9 binds preferentially to the half containing the GC base pair. Bases at positions 2, 3, and 4 in each half of the UAS make little if any contribution to binding. The center base pair is not essential for high-affinity LAC9 binding when DNA-binding activity measured in vitro. However, the center base pair must play an essential role in vivo, since all natural UASs have 17, not 16, bp. Hydroxyl radical footprinting shows that a LAC9 dimer binds an unusually broad region on one face of the DNA helix. Because of the data, we suggest that LAC9 contacts positions 6, 7, and 8, both plus and minus, of the UAS, which are separated by more than one turn of the DNA helix, and twists part way around the DNA, thus protecting the broad region of the minor groove between the major-groove contacts.

The transcription factor LAC9 from Kluyveromyces lactis-like GAL4 from Saccharomyces cerevisiae forms a Zn(II)2Cys6 binuclear cluster.

The DNA binding domain of the transcription factor LAC9 contains 6 cysteine residues with spacing in the primary peptide sequence identical to that found in the DNA binding domain of the GAL4 transcription factor. In GAL4, the CysX2CysX6CysX6CysX2CysX6Cys motif has been shown to form a Zn(II)2Cys6 binuclear cluster (Pan, T. and Coleman, J. E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2077-2081), representing a new structure for a Zn(II)-containing transcription factor which differs from the "zinc finger" motif first described for TFIIIA. LAC9 has been shown to bind two Zn(II) ions (Halvorsen, Y. C., Nandabalan, K., and Dickson, R. D. (1990) J. Biol. Chem. 265, 13283-13289). The similarity of the amino acid sequence and the Cys spacing within the DNA binding domain suggest that LAC9 should also be capable of forming the Zn(II)2Cys6 cluster found in GAL4. A fragment of LAC9 consisting of 144 amino acid residues spanning the DNA binding domain has been prepared with 113Cd(II) substituted for the two native Zn(II) ions. 113Cd NMR of this fragment (denoted LAC9(85-228*] has been carried out in an attempt to test the hypothesis that LAC9, like GAL4, forms a binuclear cluster. The chemical shifts of the two bound 113Cd(II) ions, 705 and 692 ppm respectively, are consistent with ligation of each 113Cd(II) ion to 4 sulfur atoms. The best model for such ligation is that two of the cysteine S- form bridges between the two Cd(II) ions. Formation of a Zn(II)-Cd(II) hybrid form of LAC9(85-228*) has also been observed. We conclude that LAC9 contains a Zn(II)2Cys6 binuclear cluster as previously reported for GAL4.