J. Clin. Invest. 118(1): 40-50 (2007)
In vivo proliferation assay. Pregnant females were injected intraperitoneally with 100 μg/g body weight BrdU (Sigma-Aldrich). After 1 hour, mice were euthanized and embryos recovered for immunohistochemistry as described in Supplemental Methods. BrdU-positive cells were detected with the BrdU staining kit (Zymed) followed by staining in Mayer hematoxylin. Percent proliferative cells was determined by blinded quantitation of BrdU-positive cells in the lymphatic or vascular endothelium divided by total hematoxylin-stained nuclei. Data were analyzed using the 2-tailed Student’s t test assuming unequal variance. A P value < 0.05 was considered significant.
J. Clin. Invest. 118(1): 40-50 (2007)
Cell culture. HUVECs were obtained from Lonza and maintained in endothelial cell medium provided by the supplier and used at passages 3–7. HMVEC-dLys (product no. CC-2810) were purchased from Lonza and maintained in endothelial cell medium provided by the supplier and used at passages 3–7. Culture media was changed every 2–3 days, and cells were passed when they achieved 90% confluency.
J. Clin. Invest. 118(1): 40-50 (2007)
Adrenomedullin signaling is necessary for murine lymphatic vascular developmentJ. Clin. Invest. Kimberly L. Fritz-Six, et al. 118:40 doi:10.1172/JCI33302 [Go to this article.] Figure 8AM signaling preferentially mediates enhanced ERK activation in HMVEC-dLys compared with HUVECs. (A) Cultured HMVEC-dLys and HUVECs are morphologically and genetically distinct cell lines based on histology and expression pattern of Prox1. Original magnification, ×400. (B) Stimulation of HUVECs and HMVEC-dLys with the potent growth factor VEFGA resulted in a dose-dependant increase in cell proliferation that was not significantly different between the 2 cell lines. Data represent averages of 4 independent experiments, each performed in duplicate. (C) Stimulation of HUVECs and HMVEC-dLys with AM peptide resulted in a dose-dependant increase in cell proliferation that was significantly greater in HMVEC-dLys compared with HUVECs. *P < 0.05. Data represent averages of 4 independent experiments, each performed in duplicate. (D) Stimulation of HUVECs and HMVEC-dLys with 10 nM AM peptide resulted in a significantly greater induction of ERK phosphorylation in HMVEC-dLys compared with HUVECs over a 30-minute time course. *P < 0.03 at 15- and 20-minute time points. Data represent averages of 3 independent experiments, each performed in duplicate. (E) Induction of ERK activation by AM stimulation in HMVEC-dLys was significantly reduced by the RAMP2-specific peptide inhibitor AM22-52 and completely blocked by the MAPK inhibitor PD98057 (PD). *P < 0.05 compared with untreated; #P < 0.05 compared with AM-treated. Data represent averages of 3 independent experiments, each performed in duplicate.
J. Clin. Invest. 118(1): 40-50 (2007)
Adrenomedullin signaling is necessary for murine lymphatic vascular developmentJ. Clin. Invest. Kimberly L. Fritz-Six, et al. 118:40 doi:10.1172/JCI33302 [Go to this article.] Figure 2Generation and characterization of conditional calcrl line. (A) Schematic diagram depicting strategy used for generation of a floxed calcrl allele by gene targeting. The top figure shows the endogenous wild-type calcrl allele. The targeting vector was designed so that loxP sites would flank the same exons that were deleted in the calcrl global knockout (36). The third line depicts the targeted calcrlFlox allele, and the fourth line depicts the calcrlLoxP allele after Cre-mediated excision. Primers used for isolation of correctly targeted ES cells and for routine genotyping are indicated by small arrows. (B) Correctly targeted ES cells were confirmed by Southern blot analysis using the probe depicted in A. (C) PCR genotyping for the calcrlFlox allele. (D) Quantitative RT-PCR was performed on RNA isolated from lungs and hearts of wild-type and homozygous calcrlFlox/Flox mice and revealed no significant differences in the expression of the calcrlFlox allele before Cre-mediated excision. (E) Schematic representation of breeding scheme used to generate mice with calcrl expression deleted specifically in endothelial cells by use of the Tie2Cre transgene. (F) Results of cross demonstrate that no viable calcrlLoxP/–Tie2Cre+ mice were found beyond E16.5. (G) Compared with calcrlLoxP/+Tie2Cre+ control littermates, the calcrlLoxP/–Tie2Cre+ mice displayed remarkable hydrops without hemorrhage, which phenocopied the global calcrl–knockout phenotype, yet often occurred substantially later, at E16.5. Original magnification, ×10.
J. Clin. Invest. 118(1): 51-63 (2007)
Breast cancer research offers a clinically important venue for exploring resistance to targeted therapy. Antagonists of estrogen receptor–dependent (ER-dependent) and human epidermal growth factor receptor 2 (HER2-dependent) signaling are mainstays of modern breast cancer treatment that enhance cure rates when applied against early-stage disease and contribute to disease remissions when applied against late-stage disease (1, 2).
