Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Androgen stimulates endothelial cell proliferation via an androgen receptor/VEGF/cyclin A-mediated mechanism

doi: 10.1152/ajpheart.01210.2010

Figure Lengend Snippet: Androgens, both dihydrotestosterone (DHT) and testosterone, but not 17β-estradiol, induced cell proliferation in primary human aortic endothelial cells (HAECs). A–C: time-course and dose-response analysis of DHT, 17β-estradiol, and testosterone effects on viable cell numbers in HAECs obtained from Lonza. D: DHT effects in HAECs obtained from Invitrogen. HAECs were seeded in 96-well plate and treated with various doses of DHT or 17β-estradiol (E) for 24 or 48 h (A and B) or testosterone (C) or DHT (D) for 48 h, and the viable cell number was determined at the end of experiments. The values are the means ± SE of 6–12 individual samples of 2–4 independent triplicate experiments. *P < 0.05 and **P < 0.01 compared with corresponding vehicle control (Student-Newman-Keuls test).

Article Snippet: A – C : time-course and dose-response analysis of DHT, 17β-estradiol, and testosterone effects on viable cell numbers in HAECs obtained from Lonza.

Techniques:

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Androgen stimulates endothelial cell proliferation via an androgen receptor/VEGF/cyclin A-mediated mechanism

doi: 10.1152/ajpheart.01210.2010

Figure Lengend Snippet: DHT upregulated androgen receptor (AR) (A and B), vascular endothelial growth factor (VEGF)-A (C and D), cyclin A (E), and cyclin D1 (F) gene expression in HAECs. Cells seeded in 6-well plates were treated with a vehicle control or DHT at 5 and 10 nmol/l for 6, 24, or 48 h as indicated. Total cellular RNAs or cellular proteins were extracted, and specific mRNA and protein levels were determined as described in materials and methods. The values of mRNAs are the means ± SE from 3–4 independent experiments, and the VEGF levels in the culture media (D) are the means ± SE of 6 individual samples from 2 independent triplicate experiments. B: representative Western blot analysis of AR levels in cells treated with or without DHT (10 nmol/l) or transfected with or without a small-interference RNA (siRNA) as indicated. β-Actin was used as an internal control. The numbers above the pictures are relative AR levels compared with vehicle control of lane 1 on the left. NS, nonspecific. *P < 0.05 and **P < 0.01 compared with corresponding vehicle control. The dotted line in D denotes the detection limit at 6 pg/ml of the assay.

Article Snippet: A – C : time-course and dose-response analysis of DHT, 17β-estradiol, and testosterone effects on viable cell numbers in HAECs obtained from Lonza.

Techniques: Expressing, Western Blot, Transfection

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Androgen stimulates endothelial cell proliferation via an androgen receptor/VEGF/cyclin A-mediated mechanism

doi: 10.1152/ajpheart.01210.2010

Figure Lengend Snippet: DHT-induced cell proliferation was mediated through AR. HAECs seeded in 96-well plates were treated with a vehicle control or DHT (10 nmol/l) in the presence or absence of various doses of casodex (Cdx), or a specific AR siRNA, or a nonspecific siRNA transfection as indicated for 48 h. At the end of treatment, either viable cell number or bromodeoxyuridine (BrdU) incorporation was determined. The data for viable cell numbers (A and C) are expressed as fold of vehicle control, and the values are the means ± SE of 9–12 individual samples from 3–4 independent triplicate experiments. The values for BrdU incorporation (B and D) are the means ± SE of 6 individual samples from 2 independent triplicate experiments. **P < 0.01 compared with corresponding vehicle control; and ##P < 0.01 compared with corresponding DHT treatment group without casodex or siRNA.

Article Snippet: A – C : time-course and dose-response analysis of DHT, 17β-estradiol, and testosterone effects on viable cell numbers in HAECs obtained from Lonza.

Techniques: Transfection, BrdU Incorporation Assay

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Androgen stimulates endothelial cell proliferation via an androgen receptor/VEGF/cyclin A-mediated mechanism

doi: 10.1152/ajpheart.01210.2010

Figure Lengend Snippet: DHT-induced gene expression was mediated through AR. HAECs were treated with a vehicle control or DHT (10 nmol/l) in the presence or absence of casodex (10 μmol/l, A–E), or a siRNA transfection at 200 nM (F) as indicated. The mRNA levels (A–D) quantitated by real-time qRT-PCR are expressed as fold of corresponding vehicle control group, and the values are the means ± SE of 3–4 independent experiments. The values of VEGF concentrations in culture media as determined by an electrochemiluminescence method in E and F are the means ± SE of 6 individual samples from 2 independent triplicate experiments. The dotted line in E and F denotes the detection limit of the assay. The DHT-induced increase in VEGF level in culture media was blocked by the addition of casodex (E), a specific AR and VEGF siRNA, but not a nonspecific siRNA (F). *P < 0.05 and **P < 0.01 compared with corresponding vehicle control; and #P < 0.05 compared with correspond 10 nmol/l DHT treatment without casodex.

Article Snippet: A – C : time-course and dose-response analysis of DHT, 17β-estradiol, and testosterone effects on viable cell numbers in HAECs obtained from Lonza.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Electrochemiluminescence

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Androgen stimulates endothelial cell proliferation via an androgen receptor/VEGF/cyclin A-mediated mechanism

doi: 10.1152/ajpheart.01210.2010

Figure Lengend Snippet: DHT-induced cell proliferation was mediated through the VEGF-VEGFR pathway. HAECs seeded in 96-well plates were treated with a vehicle control or DHT (10 nmol/L) in the presence or absence of various doses of SU-5416 (SU, a selective inhibitor of VEGFR tyrosine kinase) or a specific VEGF-A siRNA (100–200 nM) or a nonspecific siRNA (NS siRNA, 100–200 nM) transfection for 48 h as indicated. At the end of treatment, the viable cell number (A and C) or BrdU incorporation (B and D) was determined as described in materials and methods. The data of viable cell number in A and C are presented as fold of vehicle control, and the values are the means ± SE of 9–12 individual samples from 3–4 independent triplicate experiments. The values of BrdU incorporation in B and D are the means ± SE of 6 individual samples from 2 independent triplicate experiments. **P < 0.01 compared with the corresponding vehicle control; #P < 0.05 and ##P < 0.01 compared with the corresponding 10 nmol/l DHT treatment without SU or siRNA treatment.

Article Snippet: A – C : time-course and dose-response analysis of DHT, 17β-estradiol, and testosterone effects on viable cell numbers in HAECs obtained from Lonza.

Techniques: Transfection, BrdU Incorporation Assay

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Androgen stimulates endothelial cell proliferation via an androgen receptor/VEGF/cyclin A-mediated mechanism

doi: 10.1152/ajpheart.01210.2010

Figure Lengend Snippet: DHT-induced gene expression was modulated by a specific VEGFR inhibitor (SU5416). HAECs seeded in 96-well plates were treated with a vehicle control or DHT (10 nmol/l) in the presence or absence of 10 or 100 nM of SU5416 for either 6 or 48 h as indicated. The mRNA levels (A–D) quantitated by real-time qRT-PCR are expressed as fold of corresponding vehicle control group, and the values are the means ± SE of 3–4 independent experiments. The values of VEGF concentrations in culture media as determined by an electrochemiluminescence method (E) are the means ± SE of 6 individual samples from 2 independent triplicate experiments. The dotted line in E denotes the detection limit of the assay. *P < 0.05 and **P < 0.01 compared with corresponding vehicle control; and #P < 0.05 and ##P < 0.01 compared with corresponding 10 nmol/l DHT treatment without SU.

Article Snippet: A – C : time-course and dose-response analysis of DHT, 17β-estradiol, and testosterone effects on viable cell numbers in HAECs obtained from Lonza.

Techniques: Expressing, Quantitative RT-PCR, Electrochemiluminescence

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Androgen stimulates endothelial cell proliferation via an androgen receptor/VEGF/cyclin A-mediated mechanism

doi: 10.1152/ajpheart.01210.2010

Figure Lengend Snippet: DHT-induced cell proliferation (A and B) and gene expression (Panels C–F) involve cyclin-cyclin-dependent kinase (CDK) activity. HAECs were treated with a vehicle control, or DHT (10 nmol/l) in the presence or absence of 1 or 10 μM of roscovitine (Ros), a specific CDK inhibitor, for either 6 or 48 h as indicated. At the end of treatment, the viable cell number (A), BrdU incorporation (B), and specific mRNA levels were determined as described in materials and methods. The data of viable cell number at 48 h in A are presented as fold of vehicle control, and the values are the means ± SE of 9–12 individual samples from 3–4 independent triplicate experiments. The values of BrdU incorporation at 48 h in B are the means ± SE of 6 individual samples from 2 independent triplicate experiments. The mRNA levels quantitated by real-time qRT-PCR are expressed as fold of corresponding vehicle control group, and the values are the means ± SE of 3–4 independent experiments. *P < 0.05 and **P < 0.01 compared with the corresponding vehicle control; and #P < 0.05 and ##P < 0.01 compared with the corresponding 10 nmol/l DHT treatment without roscovitine.

Article Snippet: A – C : time-course and dose-response analysis of DHT, 17β-estradiol, and testosterone effects on viable cell numbers in HAECs obtained from Lonza.

Techniques: Expressing, Activity Assay, BrdU Incorporation Assay, Quantitative RT-PCR