1. Attenuation of leukocyte recruitment via CXCR1/2 inhibition stops the progression of PAH in mice with genetic ablation of endothelial BMPR-II
by Victoria J. Burton, Alan M. Holmes, Loredana I. Ciuclan, Alexander Robinson, Jan S. Roger, Gabor Jarai, Andrew C. Pearce, and David C. Budd
Blood
Volume 118(17):
October 27, 2011
©2011 by American Society of Hematology

2. Assessment of vascular leakage and leukocyte infiltration in endothelial restricted Bmpr-II–deficient mice with PAH. To assess vascular leakage (A), L1Cre(−)Bmpr2f/f mice or L1Cre(+)Bmpr2f/f mice were treated with SCH or vehicle orally twice daily for...
Assessment of vascular leakage and leukocyte infiltration in endothelial restricted Bmpr-II–deficient mice with PAH. To assess vascular leakage (A), L1Cre(−)Bmpr2f/f mice or L1Cre(+)Bmpr2f/f mice were treated with SCH or vehicle orally twice daily for 21 days. Evans blue dye was then administered intravenously via the tail vein for 1 hour, after which the lungs were flushed with PBS and the heart/lung block excised. Evans blue was extracted from the right lung, quantified, and expressed as micrograms per milliliter Evans blue per right lung. To assess leukocyte infiltration (B), the concentration of MPO in the right lung of L1Cre(−)Bmpr2f/f and L1Cre(+)Bmpr2f/f mice treated with vehicle or SCH was determined by ELISA. Leukocyte infiltration was further assessed by observing leukocyte infiltration (arrows) (Ci) into the small pulmonary vessels by histologic sections stained with H&E and scoring the number and location of infiltrated leukocytes (Cii). Identification of specific leukocyte subsets was assessed in lung sections stained with specific antibodies targeting monocyte/macrophages, T cells, B cells, and neutrophils (Ciii). Data are the mean ± SEM for minimum 8 animals per group. (A) ANOVA showed a significant effect of treatment with SCH and genotype (P < .001). ***P < .001, compared with L1Cre(+)Bmpr2f/f animals treated with vehicle by Dunnett test. (B) ANOVA showed a significant effect of treatment with SCH and genotype (P < .001). ***P < .001, compared with L1Cre(+)Bmpr2f/f animals treated with vehicle by Dunnett test. Slides were mounted using VECTORSHIELD (Vector Laboratories) and viewed using a 20× objective lens. Images were captured using a DMLB microscope and digital camera. Digital image acquisition was performed using IM50 Version 4.0 imaging sofware (Leica Microsystems) and digital image slide viewing using Image Scope software (Aperio Technologies).
Victoria J. Burton et al. Blood 2011;118:
©2011 by American Society of Hematology

3. Assessment of plasma cytokine levels in L1Cre(−)Bmpr2f/f and L1Cre(−)Bmpr2f/f mice treated with SCH or vehicle.
Assessment of plasma cytokine levels in L1Cre(−)Bmpr2f/f and L1Cre(−)Bmpr2f/f mice treated with SCH or vehicle. Plasma from L1Cre(−)Bmpr2f/f mice or L1Cre(+)Bmpr2f/f mice treated with SCH or vehicle was assessed for levels of multiple proinflammatory cytokines using a commercial proteome profile array (A-B). The levels of each cytokine could be observed as a pair of duplicate spots (A) and semiquantified by subtracting the background pixel density from the total pixel density of each duplicate spot and represented as the average of both pixel densities for each pair of spots (B). (B) Hatched bars represent L1Cre(+)Bmpr2f/f mice treated with vehicle; and open bars, L1Cre(+)Bmpr2f/f mice treated with SCH KC levels in pooled plasma from each group of mice were then quantified by commercial ELISA (C). Data are the mean ± SEM for a minimum of 8 animals per group. ***P < .001, compared with L1Cre(−)Bmpr2f/f mice by Dunnett test. **P < .01, compared with vehicle-treated L1Cre(+)Bmpr2f/f mice by Dunnett test.
Victoria J. Burton et al. Blood 2011;118:
©2011 by American Society of Hematology

4. RVSP pressure, Fulton index, and cardiac output in L1Cre(−)BMPR2f/f and L1Cre(+)Bmpr2f/f mice treated with vehicle or SCH
RVSP pressure, Fulton index, and cardiac output in L1Cre(−)BMPR2f/f and L1Cre(+)Bmpr2f/f mice treated with vehicle or SCH L1Cre(−)Bmpr2f/f mice or L1Cre(+)Bmpr2f/f mice were treated with vehicle or the CXCR1/2 antagonist SCH for 21 days after which RVSP levels (A), Fulton indexes (B), and cardiac output (C) were assessed. Data are the mean ± SEM for minimum of 8 animals per group. *P < .05, **P < .01, ***P < .001 compared with L1Cre(+)Bmpr2f/f mice treated with vehicle by Dunnett test.
Victoria J. Burton et al. Blood 2011;118:
©2011 by American Society of Hematology

5. Inhibition of CXCR1/2 reduces muscularization and leukocyte infiltration into lungs of L1Cre(+)Bmpr2f/f mice.
Inhibition of CXCR1/2 reduces muscularization and leukocyte infiltration into lungs of L1Cre(+)Bmpr2f/f mice. The left lung from L1Cre(−)Bmpr2f/f (Ai) and L1Cre(+)Bmpr2f/f mice (Aii) treated with vehicle or SCH was inflated with 1% (volume/volume) formalin, processed for histologic assessment, and then stained with anti-VWF and anti–α-SMA. The extent of α-SMA staining as a measure of pulmonary artery muscularization was assessed in 100 small vessels (30-70 μm) per animal and assigned as nonmuscularized (no α-SMA staining), partially muscularized, or fully muscularized (thick unbroken wall of smooth muscle), and the percentage distribution of each calculated per group (B). Data are the mean ± SEM for a minimum of 8 animals per group. *P < .05, ***P < .001 compared with L1Cre(−)Bmpr2f/f mice by Dunnett test. Slides were mounted using VECTORSHIELD (Vector Laboratories) and viewed using a 20× objective lens. Images were captured using a DMLB microscope and digital camera. Digital image acquisition was performed using IM50 Version 4.0 imaging software (Leica Microsystems) and digital image slide viewing using ImageScope software (Aperio Technologies).
Victoria J. Burton et al. Blood 2011;118:
©2011 by American Society of Hematology

6. Effect of the CXCR1/2 antagonist SCH on TEER of HPAEC monolayers transduced with BMPR-IIsh, NTCsh, or an EV. Confirmation of efficient knockdown of BMPR-II using lentivirus transduction of HPAECs transduced with BMPR-II shRNA, NTC shRNA, or an EV was ...
Effect of the CXCR1/2 antagonist SCH on TEER of HPAEC monolayers transduced with BMPR-IIsh, NTCsh, or an EV. Confirmation of efficient knockdown of BMPR-II using lentivirus transduction of HPAECs transduced with BMPR-II shRNA, NTC shRNA, or an EV was confirmed at the RNA and protein level (A). HPAECs were then seeded into wells containing 40 gold electrodes and cultured for 24 hours. Capacitance (B,E) and TEER (C-D,F) were measured over a 2-hour period. Then vehicle (C,F) or the CXCR1/2 antagonist SCH (10nM) (D,F) was added to each well and TEER measured for a further 2 hours. (B-D) Representative traces of capacitance and TEER before the addition of vehicle (B-C) and after the addition of SCH (D). (E-F) Data are the mean ± SEM for a minimum 6 experiments. (A) One-way ANOVA showed a significant effect of transduction with shRNA targeting BMPR-II. ***P < .001, compared with EV and NTC shRNA-transduced cells by Tukey test. (B) One-way ANOVA showed a significant effect of transduction with shRNA targeting BMPR-II. *P < .05, **P < .01, and ***P < .001, compared with EV and NTC shRNA-transduced cells by Tukey test.
Victoria J. Burton et al. Blood 2011;118:
©2011 by American Society of Hematology

7. Effect of reduced BMPR-II expression on the expression of CXCR2 in vitro and in vivo.
Effect of reduced BMPR-II expression on the expression of CXCR2 in vitro and in vivo. RNA was harvested from the lungs of L1Cre(−)Bmpr2f/f and L1Cre(+)Bmpr2f/f mice (A) or HPAECs transduced with shRNA targeting Bmpr-II, NTC shRNA, or an EV (B). Expression of BMPR-II and CXCR2 was determined with quantitative PCR (TaqMan) using primers specific for BMPR-II and CXCR2 or Western blotting (C). For TaqMan, data are the mean ± SEM for a minimum 3 independent experiments.
Victoria J. Burton et al. Blood 2011;118:
©2011 by American Society of Hematology