Edwin Chang

Microarray analysis of replicative senescence

BACKGROUND Limited replicative capacity is a defining characteristic of most normal human cells and culminates in senescence, an arrested state in which cells remain viable but display an altered pattern of gene and protein expression. To survey widely the alterations in gene expression, we have developed a DNA microarray analysis system that contains genes previously reported to be involved in aging, as well as those involved in many of the major biochemical signaling pathways. RESULTS Senescence-associated gene expression was assessed in three cell types: dermal fibroblasts, retinal pigment epithelial cells, and vascular endothelial cells. Fibroblasts demonstrated a strong inflammatory-type response, but shared limited overlap in senescent gene expression patterns with the other two cell types. The characteristics of the senescence response were highly cell-type specific. A comparison of early- and late-passage cells stimulated with serum showed specific deficits in the early and mid G1 response of senescent cells. Several genes that are constitutively overexpressed in senescent fibroblasts are regulated during the cell cycle in early-passage cells, suggesting that senescent cells are locked in an activated state that mimics the early remodeling phase of wound repair. CONCLUSIONS Replicative senescence triggers mRNA expression patterns that vary widely and cell lineage strongly influences these patterns. In fibroblasts, the senescent state mimics inflammatory wound repair processes and, as such, senescent cells may contribute to chronic wound pathologies.

Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype

Expression of the human telomerase catalytic component, hTERT, in normal human somatic cells can reconstitute telomerase activity and extend their replicative lifespan. We report here that at twice the normal number of population doublings, telomerase-expressing human skin fibroblasts (BJ-hTERT) and retinal pigment epithelial cells (RPE-hTERT) retain normal growth control in response to serum deprivation, high cell density, G1 or G2 phase blockers and spindle inhibitors. In addition, we observed no cell growth in soft agar and detected no tumour formation in vivo. Thus, we find that telomerase expression in normal cells does not appear to induce changes associated with a malignant phenotype.

Human endothelial cell life extension by telomerase expression.

Normal human endothelial cells, like other somatic cells in culture, divide a limited number of times before entering a nondividing state called replicative senescence. Expression of the catalytic component of human telomerase, human telomerase reverse transcriptase (hTERT), extends the life span of human fibroblasts and retinal pigment epithelial cells beyond senescence without causing neoplastic transformation (Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu, C. P., Morin, G. B., Harley, C. B., Shay, J. W., Lichtsteiner, S., and Wright, W. E. (1998) Science 279, 349–352; Jiang, X., Jimenez, G., Chang, E., Frolkis, M., Kusler, B., Sage, M., Beeche, M., Bodnar, A., Wahl, G., Tlsty, T., and Chiu, C.-P. (1999) Nat. Genet. 21, 111–114). Here, we show that both human large vessel and microvascular endothelial cells also bypass replicative senescence after introduction of hTERT. For the first time, we report that hTERT expression in these life-extended vascular cells does not affect their differentiated and functional phenotype and that these cells maintain their angiogenic potential in vitro. Furthermore, hTERT(+) microvascular endothelial cells have normal karyotype, and hTERT(+) endothelial cell strains do not exhibit a transformed phenotype. Relative to parental cells at senescence, hTERT-expressing endothelial cells exhibit resistance to induction of apoptosis by a variety of different conditions. Such characteristics are highly desirable for designing vascular transplantation and gene therapy delivery systemsin vivo.

IFATS Collection: Adipose Stromal Cells Adopt a Proangiogenic Phenotype Under the Influence of Hypoxia

Evolving evidence suggests a possible role for adipose stromal cells (ASCs) in adult neovascularization, although the specific cues that stimulate their angiogenic behavior are poorly understood. We evaluated the effect of hypoxia, a central mediator of new blood vessel development within ischemic tissue, on proneovascular ASC functions. Murine ASCs were exposed to normoxia (21% oxygen) or hypoxia (5%, 1% oxygen) for varying lengths of time. Vascular endothelial growth factor (VEGF) secretion by ASCs increased as an inverse function of oxygen tension, with progressively higher VEGF expression at 21%, 5%, and 1% oxygen, respectively. Greater VEGF levels were also associated with longer periods in culture. ASCs were able to migrate towards stromal cell‐derived factor (SDF)‐1, a chemokine expressed by ischemic tissue, with hypoxia augmenting ASC expression of the SDF‐1 receptor (CXCR4) and potentiating ASC migration. In vivo, ASCs demonstrated the capacity to proliferate in response to a hypoxic insult remote from their resident niche, and this was supported by in vitro studies showing increasing ASC proliferation with greater degrees of hypoxia. Hypoxia did not significantly alter the expression of endothelial surface markers by ASCs. However, these cells did assume an endothelial phenotype as evidenced by their ability to tubularize when seeded with differentiated endothelial cells on Matrigel. Taken together, these data suggest that ASCs upregulate their proneovascular activity in response to hypoxia, and may harbor the capacity to home to ischemic tissue and function cooperatively with existing vasculature to promote angiogenesis. STEM CELLS 2009;27:266–274

Pilot Pharmacokinetic and Dosimetric Studies of 18F-FPPRGD2: A PET Radiopharmaceutical Agent for Imaging αvβ3 Integrin Levels

PURPOSE To assess the safety, biodistribution, and dosimetric properties of the positron emission tomography (PET) radiopharmaceutical agent fluorine 18 ((18)F) FPPRGD2 (2-fluoropropionyl labeled PEGylated dimeric RGD peptide [PEG3-E{c(RGDyk)}2]), which is based on the dimeric arginine-glycine-aspartic acid (RGD) peptide sequence and targets α(v)β(3) integrin, in the first volunteers imaged with this tracer. MATERIALS AND METHODS The protocol was approved by the institutional review board, and written informed consent was obtained from all participants. Five healthy volunteers underwent whole-body combined PET-computed tomography 0.5, 1.0, 2.0, and 3.0 hours after tracer injection (mean dose, 9.5 mCi ± 3.4 [standard deviation] [351.5 MBq ± 125.8]; mean specific radioactivity, 1200 mCi/mmol ± 714 [44.4 GBq/mmol ± 26.4]). During this time, standard vital signs, electrocardiographic (ECG) readings, and blood sample values (for chemistry, hematologic, and liver function tests) were checked at regular intervals and 1 and 7 days after the injection. These data were used to evaluate tracer biodistribution and dosimetric properties, time-activity curves, and the stability of laboratory values. Significant changes in vital signs and laboratory values were evaluated by using a combination of population-averaged generalized estimating equation regression and exact paired Wilcoxon tests. RESULTS The administration of (18)F-FPPRGD2 was well tolerated, with no marked effects on vital signs, ECG readings, or laboratory values. The tracer showed the same pattern of biodistribution in all volunteers: primary clearance through the kidneys (0.360 rem/mCi ± 0.185 [0.098 mSv/MBq ± 0.050]) and bladder (0.862 rem/mCi ± 0.436 [0.233 mSv/MBq ± 0.118], voiding model) and uptake in the spleen (0.250 rem/mCi ± 0.168 [0.068 mSv/MBq ± 0.046]) and large intestine (0.529 rem/mCi ± 0.236 [0.143 mSv/MBq ± 0.064]). The mean effective dose of (18)F-FPPRGD2 was 0.1462 rem/mCi ± 0.0669 (0.0396 mSv/MBq ± 0.0181). With an injected dose of 10 mCi (370 MBq) and a 1-hour voiding interval, a patient would be exposed to an effective radiation dose of 1.5 rem (15 mSv). Above the diaphragm, there was minimal uptake in the brain ventricles, salivary glands, and thyroid gland. Time-activity curves showed rapid clearance from the vasculature, with a mean 26% ± 17 of the tracer remaining in the circulation at 30 minutes and most of the activity occurring in the plasma relative to cells (mean whole blood-plasma ratio, 0.799 ± 0.096). CONCLUSION (18)F-FPPRGD2 has desirable pharmacokinetic and biodistribution properties. The primary application is likely to be PET evaluation of oncologic patients-especially those with brain, breast, or lung cancer. Specific indications may include tumor staging, identifying patients who would benefit from antiangiogenesis therapy, and separating treatment responders from nonresponders early.

A Central Role for Nicotinic Cholinergic Regulation of Growth Factor–Induced Endothelial Cell Migration

Objective—An endothelial nicotinic acetylcholine receptor (nAChR) participates in atherogenesis and tumorigenesis by promoting neovascularization. To date, the mechanisms of nAChR-mediated angiogenesis and their relationship to angiogenic factors, eg, VEGF and bFGF, are unknown. Methods and Results—Nicotine induced dose-dependent human microvascular endothelial cell (HMVEC) migration, a key angiogenesis event, to an extent which was equivalent in magnitude to bFGF (10 ng/mL) but less than for VEGF (10 ng/mL). Unexpectedly, nAChR antagonism not only abolished nicotine-induced HMVEC migration but also abolished migration induced by bFGF and attenuated migration induced by VEGF. Transcriptional profiling identified gene expression programs which were concordantly regulated by all 3 angiogens (nicotine, VEGF, and bFGF), a notable feature of which includes corepression of thioredoxin-interacting protein (TXNIP), endogenous inhibitor of the redox regulator thioredoxin. Furthermore, TXNIP repression by all 3 angiogens induced thioredoxin activity. Silencing thioredoxin by small interference RNA abrogated all angiogen-induced migration while silencing TXNIP strongly induced HMVEC migration. Interestingly, nAChR antagonism abrogates growth factor (VEGF and bFGF)–mediated induction of thioredoxin activity. Conclusions—Nicotine promotes angiogenesis via stimulation of nAChR-dependent endothelial cell migration. Furthermore, growth factor–induced HMVEC migration, a key angiogenesis event, requires nAChR activation—an effect mediated in part by nAChR-dependent regulation of thioredoxin activity.

Bone formation and degradation of a highly porous biphasic calcium phosphate ceramic in presence of BMP-7, VEGF and mesenchymal stem cells in an ectopic mouse model

INTRODUCTION Angiogenesis and mesenchymal stem cells (MSCs) promote osteogenesis. The aim of the present study was to evaluate whether bone morphogenetic protein (BMP-7) promoted osteoinduction could be enhanced by combining it with vascular endothelial growth factor (VEGF) or MSCs in highly porous biphasic calcium phosphate (BCP) ceramics. MATERIALS AND METHODS BCP ceramic blocks were implanted in an ectopic site in 24 mice (BMP-7 vs. BMP-7/VEGF; BMP-7 vs. BMP-7/MSCs and BMP-7 vs. Control; each group n=8). Specimens were analysed 12 weeks after surgery by environmental scanning electron microscopy (ESEM) and Giemsa staining. RESULTS In all implanted scaffolds, newly formed bone was observed, even in the control site. No statistical differences in the amount of new bone were found in the presence of BMP-7 compared to BMP-7/VEGF (p=1.0) or BMP-7/MSCs (p=0.786). ESEM revealed a degradation of the scaffolds. A higher degradation was observed in areas where no bone-implant contact was present compared to areas where the ceramic was integrated in newly formed bone. CONCLUSIONS Neither VEGF nor MSCs enhanced BMP-7 induced bone formation under the selected conditions. The present ceramic seemed to be osteoinductive and degradable, making this material suitable for bone tissue engineering.

Proof-of-Concept Study of Monitoring Cancer Drug Therapy with Cerenkov Luminescence Imaging

Cerenkov luminescence imaging (CLI) has emerged as a less expensive, easier-to-use, and higher-throughput alternative to other nuclear imaging modalities such as PET. It is expected that CLI will find many applications in biomedical research such as cancer detection, probe development, drug screening, and therapy monitoring. In this study, we explored the possibility of using CLI to monitor drug efficacy by comparisons against PET. To assess the performance of both modalities in therapy monitoring, 2 murine tumor models (large cell lung cancer cell line H460 and prostate cancer cell line PC3) were given bevacizumab versus vehicle treatments. Two common radiotracers, 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) and 18F-FDG, were used to monitor bevacizumab treatment efficacy. Methods: One group of mice (n = 6) was implanted with H460 xenografts bilaterally in the shoulder region, divided into treatment and control groups (n = 3 each), injected with 18F-FLT, and imaged with PET immediately followed by CLI. The other group of mice (n = 6) was implanted with PC3 xenografts in the same locations, divided into treatment and control groups (n = 3 each), injected with 18F-FDG, and imaged by the same modalities. Bevacizumab treatment was performed by 2 injections of 20 mg/kg at days 0 and 2. Results: On 18F-FLT scans, both CLI and PET revealed significantly decreased signals from H460 xenografts in treated mice from pretreatment to day 3. Moderately increased to unchanged signals were observed in untreated mice. On 18F-FDG scans, both CLI and PET showed relatively unchanged signals from PC3 tumors in both treated and control groups. Quantifications of tumor signals of Cerenkov luminescence and PET images showed that the 2 modalities had excellent correlations (R2 > 0.88 across all study groups). Conclusion: CLI and PET exhibit excellent correlations across different tumor xenografts and radiotracers. This is the first study, to our knowledge, demonstrating the use of CLI for monitoring cancer treatment. The findings warrant further exploration and optimization of CLI as an alternative to PET in preclinical therapeutic monitoring and drug screening.

Integrin-targeted molecular imaging of experimental abdominal aortic aneurysms by (18)F-labeled Arg-Gly-Asp positron-emission tomography.

Background—Both inflammation and neoangiogenesis contribute to abdominal aortic aneurysm (AAA) disease. Arg-Gly-Asp–based molecular imaging has been shown to detect the integrin &agr;v&bgr;3. We studied a clinical dimeric 18F-labeled Arg-Gly-Asp positron-emission tomography (PET) agent (18F-FPPRGD2) for molecular imaging of experimental AAAs. Methods and Results—Murine AAAs were induced in Apo-E–deficient mice by angiotensin II infusion, with monitoring of aortic diameter on ultrasound. AAA (n=10) and saline-infused control mice (n=7) were injected intravenously with 18F-FPPRGD2, as well as an intravascular computed tomography contrast agent, then scanned using a small-animal PET/computed tomography scanner. Aortic uptake of 18F-FPPRGD2 was quantified by percentage-injected dose per gram and target-to-background ratio. Focal increased PET signal was found in AAA lesions, but not in normal control aortae, confirmed by quantitative analysis (median percentage-injected dose per gram [interquartile range], 2.05 [1.05–2.85] versus 0.63 [0.43–0.83], P=0.003; median target-to-background ratio [interquartile range], 2.72 [2.31–3.49] versus 1.44 [1.10–1.52], P=0.0008). Ex vivo autoradiography demonstrated high uptake of 18F-FPPRGD2 into the AAA wall, with immunohistochemistry showing substantial cluster of differentiation (CD)-11b+ macrophages and CD-31+ neovessels. Target-to-background ratio of AAAs on PET did not correlate with AAA diameter (r=−0.29, P=0.41) but did strongly correlate with both mural macrophage density (r=0.79, P=0.007) and neovessel counts (r=0.87, P=0.001) on immunohistochemistry. Conclusions—PET imaging of experimental AAAs using 18F-FPPRGD2 detects biologically active disease, correlating to the degree of vascular inflammation and neoangiogenesis. This may provide a clinically translatable molecular imaging approach to characterize AAA biology to predict risk beyond size alone.

Identification and cloning of a sequence homologue of dopamine β-hydroxylase

We have identified and cloned a cDNA encoding a new member of the monooxygenase family of enzymes. This novel enzyme, which we call MOX (monooxygenase X; unknown substrate) is a clear sequence homologue of the enzyme dopamine b-hydroxylase (DBH ). MOX maintains many of the structural features of DBH, as evidenced by the retention of most of the disulfide linkages and all of the peptidyl ligands to the active site copper atoms. Unlike DBH, MOX lacks a signal peptide sequence and therefore is unlikely to be a secreted molecule. The steady-state mRNA levels of MOX are highest in the kidney, lung, and adrenal gland, indicating that the tissue distribution of MOX is broader than that of DBH. Antisera raised to a fusion protein of MOX identifies a single band of the expected mobility by Western blot analysis. MOX mRNA levels are elevated in some fibroblast cell strains at replicative senescence, through this regulation is not apparent in all primary cell strains. The gene for MOX resides on the q arm of chromosome 6 and the corresponding mouse homolog has been identified.