Volume 20, Issue 8, Pages 1516-1528 (August 2012) Gene Therapy by Targeted Adenovirus-mediated Knockdown of Pulmonary Endothelial Tph1 Attenuates Hypoxia-induced Pulmonary Hypertension  Ian Morecroft, Katie White, Paola Caruso, Margaret Nilsen, Lynn Loughlin, Raul Alba, Paul N Reynolds, Sergei M Danilov, Andrew H Baker, Margaret R MacLean  Molecular Therapy  Volume 20, Issue 8, Pages 1516-1528 (August 2012) DOI: 10.1038/mt.2012.70 Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Effect of hypoxia on expression of tryptophan hydroxylase-1 (Tph1) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in human pulmonary artery endothelial cells (hPAECs). (a) Representative western blot and quantitative analysis of Tph1 and (b) representative western blot and quantitative analysis of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) expression in hPAECs exposed to normoxic (N) and hypoxic [(H); 5%O2)] conditions in the presence or absence of the Tph inhibitor p-chlorophenylalanine (p-CPA) (10 µmol/l). Tph1 and p-ERK1/2 protein were detected using polyclonal anti-Tph1 and p-ERK1/2 antibodies, respectively, and antibody binding was visualized by a chemiluminescence detection kit. Immunoblot images were scanned and quantified using the TotalLab image analysis software. Tph2 could not be detected in any PAEC samples either under normoxia or hypoxia (data not shown). ***Value significantly greater than corresponding value under normoxic conditions (P < 0.001). Data shown as mean ± SEM of five experiments. Lane numbers on the blot images a and b represent hPAECs treated with the following: (1 and 2) normoxic, (3 and 4) normoxic + p-CPA, (5 and 6) hypoxic, (7 and 8) hypoxic + p-CPA. tERK1/2, total ERK1/2. Molecular Therapy 2012 20, 1516-1528DOI: (10.1038/mt.2012.70) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Effect of hypoxia on human pulmonary artery endothelial cell (hPAEC) conditioned media-induced proliferation in bovine pulmonary artery smooth muscle cells (bPASMCs). (a) Quantification of [3H]-thymidine incorporation and (b) cell counts in response to conditioned media (CM) from hPAECs exposed to hypoxia in bPASMCs. Conditioned media was produced in either the absence (CM) or presence (CM(p-CPA)) of the Tph inhibitor p-Chlorophenylalanine (p-CPA) (10 µmol/l). The bPASMCs were stimulated with conditioned media in the absence or presence of the 5-HT1B receptor antagonist (+SB224289, 200 nmol/l), the serotonin transporter (SERT) inhibitor (+citalopram, 10 µmol/l) or the 5-HT2A receptor antagonist (+ketanserin, 1 µmol/l). (c) Cell counts in response to serotonin (1 µmol/l) in bPASMCs and the effect of ketanserin (1 µmol/l), SB224289 (200 nmol/l), citalopram (10 µmol/l), and p-CPA (10 µmol/l). The bPASMCs were treated with the conditioned medium from the hPAECs or serotonin for 48 hours. (d) Quantitative analysis and (e) representative western blot of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) expression in bPASMCs in response to conditioned media from hPAECs exposed to normoxic and hypoxic (5%O2) conditions. The bPASMCs were treated with the conditioned medium from the hPAECs for 15 minutes. The increased phosphorylation of ERK1/2 in response to conditioned media was attenuated by p-CPA (10 µmol/l), SB224289 (200 nmol/l), citalopram (10 µmol/l), and ketanserin (1 µmol/l). p-ERK1/2 were detected using polyclonal anti-p-ERK1/2 antibodies and antibody binding was visualized by a chemiluminescence detection kit. Immunoblot images for p-ERK1/2 were scanned and quantified using the TotalLab image analysis software. Antagonists and inhibitors were added for 45 minutes before any treatment. Values are mean ± SEM of three to four experiments. Lane numbers on the image e represent bPASMCs treated with the following: (1) Untreated bPASMCs, (2) normoxic CM, (3) normoxic CM+citalopram, (4) normoxic CM+SB224289, (5) normoxic CM+ketanserin, (6) normoxic CM+p-CPA, (7) Un-treated bPASMCs, (8) hypoxic CM, (9) hypoxic CM+citalopram, (10) hypoxic CM+SB224289, (11) hypoxic CM+ketanserin and (12) hypoxic CM+p-CPA *Value significantly greater than corresponding value under basal conditions (0.2% FBS) (*P < 0.05, **P < 0.01, ***P < 0.001) §Value significantly greater than corresponding value in normoxic conditions (§P < 0.05). †Value significantly less than corresponding value in the absence of selective inhibitors and antagonists (†P < 0.05, ††P < 0.01). Data shown as mean ± SEM. FBS, fetal bovine serum. Molecular Therapy 2012 20, 1516-1528DOI: (10.1038/mt.2012.70) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Effect of hypoxia on human pulmonary artery endothelial cell (hPAEC) conditioned media-induced phosphorylation of platelet derived growth factor receptor β (PDGFRβ) in bovine pulmonary artery smooth muscle cells (bPASMCs). (a) Quantitative analysis of platelet-derived growth factor receptor β (PDGFRβ) expression in bPASMCs in response to conditioned media (CM) from hPAECs exposed to normoxic and hypoxic (5%O2) conditions and (b) representative western blot. PDGFRβ proteins were detected using polyclonal anti-PDGFRβ antibodies respectively and antibody binding was visualized by a chemiluminescence detection kit. Immunoblot images for PDGFRβ were scanned and quantified using the TotalLab image analysis software. Antagonists and inhibitors were added for 45 minutes before any treatment. Values are mean ± SEM of three to four experiments. Lane numbers on the blot image represent bPASMCs treated with the following: (1 and 10) Untreated bPASMCs, (2) normoxic CM, (3) normoxic CM+p-CPA (4) normoxic CM+citalopram, (5) normoxic CM+p-CPA+ citalopram, (6) hypoxic CM, (7) hypoxic CM+p-CPA (8) hypoxic CM+citalopram, (9) hypoxic CM+citalopram+p-CPA. Data shown as mean ± SEM. CM, conditioned media; p-CPA, p-chlorophenylalanine. Molecular Therapy 2012 20, 1516-1528DOI: (10.1038/mt.2012.70) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 In vivo validation for monoclonal antibody (mAb) 9B9 lung retargeting and for short hairpin RNA (shRNA) sequences against tryptophan hydroxylase-1 (Tph1). (a) In vivo assessment of mAb9B9 (+mAB9B) retargeting in rat lung and (b) in vivo assessment of mAb9B9 (+mAB9B) retargeting in rat liver compared to nontargeted vector (AdCMVLuc). AdCMVLuc alone or complexed to mA9B9 was injected into the tail vein of rats (n = 3 per group). Three days later rats were sacrificed, organs were harvested, and luciferase activity per milligram of protein was determined. (c) Quantitative analysis showing the effect of adenovirus vectors expressing shRNA sequences against Tph1 on the expression of Tph1 mRNA in the pulmonary arteries of normoxic rats 72 hours post-shRNA injection using reverse trancription (RT)-PCR. The Ad-shRNATph1D, Ad-shRNA Tph1B, scrambled sequence Ad-shRNAScr or saline vehicle were injected via the femoral vein under anesthesia. Total RNA was isolated from snap-frozen pulmonary arteries and quantitative real-time PCR (qRT-PCR) was performed with Tph1-specific primers; n = 3 per group. *Value significantly less than corresponding value in controls (**P < 0.01, ***P < 0.001). Data shown as mean ± SEM. Ad-shRNA Tph1B, adenoviral–mAb9B9 complex-containing shRNA sequence to Tph1; Ad-shRNA Tph1D, adenoviral–mAb9B9 complex-containing shRNA sequence to Tph1; Ad-shRNA Scr, adenoviral–mAb9B9 complex-containing scrambled sequence. Molecular Therapy 2012 20, 1516-1528DOI: (10.1038/mt.2012.70) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Effects of tryptophan hydroxylase-1 (Tph1) short hairpin RNA (shRNA), scrambled sequence, and vehicle control on indices of pulmonary hypertension in the chronic hypoxic rat pulmonary arterial hypertension (PAH) model. At 7 days after exposure to chronic hypobaric hypoxia or normoxia, a hemodynamic study was performed in anesthetized rats to measure systolic right ventricular pressure (sRVP). The Ad-shRNATph1D, scrambled sequence Ad-shRNAScr, or saline vehicle were injected via the femoral vein under anesthesia and full recovery from surgery allowed before exposure to hypoxia/normoxia. Graphs show the effects of Ad-shRNA Tph1D, and the scrambled sequence Ad-shRHA Scr on (a) sRVP (n = 6–9 rats per group), (b) pulmonary vascular remodeling (n = 4–5 per group), and (c) right ventricular hypertrophy (RVH) in normoxic (open square) and 7-day hypoxic (closed square) rats (n = 6–9 rats). (d) Representative photomicrographs showing the effects of Ad-shRNA Tph1D and Ad-shRNA Scr on hypoxia-induced remodeling in small pulmonary arteries from normoxic and hypoxic rats. Note the readily identifiable double elastic lamina (black arrow) and newly formed smooth muscle layer (white asterisk) in the hypoxic group. Bars = 50 µm. *Value significantly greater than corresponding value in normoxic vehicle control rats (*P < 0.05, **P < 0.01, ***P < 0.001). †Value significantly less than corresponding value in vehicle-treated rats (†P < 0.05, ††P < 0.01). ‡Value significantly less than corresponding value in Ad-shRNAScr-treated rats (‡P < 0.05, ‡‡P < 0.01). Data shown as mean ± SEM. Ad-shRNA Tph1D, adenoviral–mAb9B9 complex-containing shRNA sequence to Tph1; Ad-shRNA Scr, adenoviral–mAb9B9 complex-containing scrambled sequence. mAb, monoclonal antibody. Molecular Therapy 2012 20, 1516-1528DOI: (10.1038/mt.2012.70) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Evaluation of the efficiency of tryptophan hydroxylase-1 (Tph1) short hairpin RNA (shRNA) treatment in hypoxia-induced pulmonary arterial hypertension (PAH). (a) Representative photomicrographs and (b) quantitative analysis showing the effects of chronic hypoxia, Tph1 shRNA and scrambled sequence on expression of Tph1 in the pulmonary arteries of 7-day hypoxic (H) rats (closed square; n = 4 rats) compared to normoxic (N) controls (open square; n = 3 rats). (c) Western blot and quantitative analysis showing the effects of chronic hypoxia, Tph1 shRNA and scrambled sequence on expression of Tph1 in the lungs of 7-day hypoxic rats compared to normoxic controls. Tph1 protein was detected using a polyclonal anti-Tph1 antibody, and antibody binding was visualized by a chemiluminescence detection kit. Immunoblot images were scanned and quantified using the TotalLab image analysis software. Immunohistochemistry image analysis was performed on images of lung tissue sections using Metamorph software. *Value significantly greater than corresponding value in normoxic vehicle control rats (***P < 0.001). †Value significantly less than corresponding value in vehicle-treated rats (††P < 0.01). ‡Value significantly less than corresponding value in Ad-shRNAScr-treated rats (‡‡P < 0.01). Data shown as mean ± SEM. Arrows highlight examples of expression of Tph1 in rat pulmonary arteries. Lane numbers on the blot in image c represent: (1) normoxic vehicle-treated, (2) normoxic Ad-shRNA Scr-treated, (3) normoxic Ad-shRNA Tph1D-treated (4) hypoxic vehicle-treated, (5) hypoxic Ad-shRNA Scr-treated, and (6) hypoxic Ad-shRNA Tph1D-treated rats. N, normoxia; H, hypoxia. Ad-shRNA Tph1D, adenoviral–mAb9B9 complex-containing shRNA sequence to Tph1; Ad-shRNA Scr, adenoviral–mAb9B9 complex-containing scrambled sequence; Tph1, tryptophan hydroxylase-1. Bars are 25 µm. mAb, monoclonal antibody. Molecular Therapy 2012 20, 1516-1528DOI: (10.1038/mt.2012.70) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Effects of tryptophan hydroxylase-1 (Tph1) short hairpin RNA (shRNA), scrambled sequence, and vehicle control on proliferating cell nuclear antigen (PCNA) expression in chronic hypoxic rat pulmonary arteries and lungs. (a) Representative photomicrograph and (b) quantitative analysis showing the effects of chronic hypoxia, Tph1 shRNA and scrambled sequence on expression of proliferating cell nuclear antigen (PCNA) in rat small pulmonary arteries where expression of PNCA is shown as black staining. (c) western blot and (d) quantitative analysis showing the effects of chronic hypoxia, Tph1 shRNA and scrambled sequence on expression of PCNA in lungs from (n = 4–5) rats. PCNA protein was detected using a polyclonal anti-PCNA antibody, and antibody binding was visualized by a chemiluminescence detection kit. Immunoblot images were scanned and quantified using the TotalLab image analysis software. Immunohistochemistry image analysis was performed on images of lung tissue sections using Metamorph software. *Value significantly greater than corresponding value in normoxic rats (**P < 0.01, ***P < 0.001). †Value significantly less than corresponding value in vehicle-treated control rats (†P < 0.05). Data shown as mean ± SEM of 4–8 vessels analyzed. Arrows highlight examples of expression of PCNA. Lane numbers on the image c represent: (1) normoxic vehicle-treated, (2) normoxic Ad-shRNA Scr-treated, (3) normoxic Ad-shRNA Tph1D-treated (4) hypoxic vehicle-treated, (5) hypoxic Ad-shRNA Scr-treated, and (6) hypoxic Ad-shRNA Tph1D-treated rats. N = normoxia; H = hypoxia Ad-shRNATph1D, adenoviral–mAb9B9 complex-containing shRNA sequence to Tph1; Ad-shRNAScr, adenoviral–mAb9B9 complex-containing scrambled sequence. Bars are 50 µm. mAb, monoclonal antibody. Molecular Therapy 2012 20, 1516-1528DOI: (10.1038/mt.2012.70) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Effects of tryptophan hydroxylase-1 (Tph1) short hairpin RNA (shRNA), scrambled sequence and vehicle control on phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) expression in chronic hypoxic rat lungs. (a) Representative photomicrograph and (b) quantitative analysis showing the effects of chronic hypoxia, Tph1 shRNA and scrambled sequence on expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) in rat small pulmonary arteries where expression of p-ERK1/2 is shown as black staining. (c) Western blot and (d) quantitative analysis showing the effects of chronic hypoxia, Tph1 shRNA and scrambled sequence on expression of p-ERK1/2 in lungs from (n = 4–5) rats. p-ERK1/2 protein was detected using a polyclonal anti-p-ERK1/2 antibody, and antibody binding was visualized by a chemiluminescence detection kit. Immunoblot images were scanned and quantified using the TotalLab image analysis software. Immunohistochemistry image analysis was performed on images of lung tissue sections using Metamorph software *Value significantly greater than corresponding value in normoxic rats (**P < 0.01, ***P < 0.001). †Value significantly less than corresponding value in vehicle-treated control rats (†P < 0.05). Data shown as mean ± SEM of 4–8 vessels analyzed. Arrows highlight examples of expression of p-ERK1/2. Lane numbers on the image c represent: (1) normoxic vehicle-treated, (2) normoxic Ad-shRNA Scr-treated, (3) normoxic Ad-shRNA Tph1D-treated (4) hypoxic vehicle-treated, (5) hypoxic Ad-shRNA Scr-treated, and (6) hypoxic Ad-shRNA Tph1D-treated rats. N = normoxia; H = hypoxia Ad-shRNATph1D, adenoviral–mAb9B9 complex-containing shRNA sequence to Tph1; Ad-shRNAScr, adenoviral–mAb9B9 complex-containing scrambled sequence. Bars are 50 µm. mAb, monoclonal antibody. Molecular Therapy 2012 20, 1516-1528DOI: (10.1038/mt.2012.70) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions