FASEB J 17: 1373C1375, 2003. Bleomycin did not impact pulmonary 5-HT 2A receptor (R) expression, but did increase pulmonary gene expression of the 5-HT 2BR and serotonin transporter. Treatment with ketanserin attenuated bleomycin-induced PH (increased RVSP and RVH) and pulmonary vascular remodeling (decreased vessel density and increased muscularization of small vessels). In addition, we found that treatment with ketanserin activated pulmonary MAPK and Akt signaling in mice exposed to bleomycin. We conclude that 5-HT signaling is usually increased in a murine model of neonatal PH and pharmacological inhibition of the 5-HT 2AR protects against the development of PH in neonatal lung injury. We speculate this occurs through restoration of MAPK signaling and increased Akt signaling. = 14C23. PBS, PBS uncovered; KET, ketanserin uncovered; BLEO, bleomycin uncovered; B/K, bleomycin + ketanserin uncovered. Pulmonary Tph1, Htr2B, and Slc6A4 expression increased in bleomycin-induced PH and BPD. We examined the major enzyme systems responsible for 5-HT U-93631 synthesis and degradation in the lung. Tph1, the rate-limiting enzyme in the synthesis of 5-HT from tryptophan, increased in the lungs of mice exposed to bleomycin (Fig. 2and = 5. = 5. = 5C8. = 10. = 8C10. relative to 18S, = 9. relative to 18S, **= 9. Analysis by unpaired = 5C8. = 9C10. Ketanserin attenuates pulmonary vascular remodeling following exposure to bleomycin. We performed immunohistochemistry (IHC) for the endothelial cell marker vWF and counted the number of small vessels ( 30 m) per high-powered field as an indication of vascular development. Representative images are shown for vWF immunostaining of lung sections from mice exposed to ip PBS (Fig. 4= 5C7. Analysis by one-way ANOVA with Bonferronis posttest. = 5C8. Analysis by one-way ANOVA with Bonferronis posttest. = 5C12. and and gene expression in mice cotreated with bleomycin vs. bleomycin alone. lung expression relative to -actin, *= 5. = 5. = 5C9. = 9C10. = 8C10. = 9. = 8C10. Analysis by one-way ANOVA with Bonferronis posttest. Ketanserin restores pulmonary MAPK signaling and increases pulmonary Akt signaling in neonatal mice cotreated with bleomycin. Both MAPK and Akt signaling have been implicated in 5-HT-mediated PAEC, PASMC, and fibroblast (PA Fib) growth (6, 41C43, 61, 72, 74). To evaluate whether alterations in pulmonary MAPK and Akt signaling could contribute to impaired vascular growth and PH in bleomycin-treated mice, we measured extracellular signal-related kinase (ERK) and Akt activation. Neonatal mice exposed to bleomycin experienced decreased phosphorylation of ERK44/42, indicating loss of MAPK pathway activation (Fig. 8= 5C11. = 5C7. Analysis by one-way ANOVA with Bonferronis posttest. DISCUSSION In this study, we hypothesized that 5-HT signaling is usually increased in experimental PH associated with BPD and that 5-HT 2A-R antagonism would ameliorate experimental PH in neonatal mice. We tested this hypothesis in wild-type neonatal mice treated with bleomycin. We (17) experienced previously reported that bleomycin treatment resulted in RVH, pulmonary vascular remodeling, and simplified alveolar development, mirroring the findings seen in many infants with BPD. We now also statement RVSP measurements in 3-wk-old mice. We demonstrate that expression of Tph1, the rate-limiting enzyme in the synthesis of 5-HT, is usually increased in the lungs of mice with experimental neonatal PH and that cotreatment with the 5-HT 2AR antagonist ketanserin, along with bleomycin, prevents bleomycin-induced PH and pulmonary vascular remodeling. These data provide further support for the role of 5-HT in regulating pulmonary vascular firmness and is the first evidence that modulation of 5-HT signaling promotes vascular development in a neonatal model of pulmonary vascular disease. One novel obtaining of our study is the increased 5-HT signaling in experimental PH associated with BPD, including increased pulmonary protein expression of Tph1 and gene expression of 5-HT 2B-R (Htr2b) and SERT (Slc6A4). In this study we found no difference in whole lung expression of MAO-A, the enzyme primarily responsible for metabolism of 5-HT, or the 5-HT 2A-R or 1B (Htr1b) R. Increased pulmonary artery Tph1, 2B-R and SERT expression are well explained in adults with PH (21, 23, 37). Furthermore, pharmacological or genetic strategies that inhibit Tph1 synthesis or 2B-R or SERT activation protect mature rodents from your development of experimental PH (22, 37, 49, 52). As the function of the SERT and receptors are known to vary with maturation, injury model, and between species, further work is needed to understand the contribution of 5-HT synthesis vs. receptor activation in disease pathogenesis. It is, for example, necessary to consider that, since.doi:10.1159/000111071. 5-HT synthesis, tryptophan hydroxylase-1 (Tph1), was significantly increased. Bleomycin did not impact pulmonary 5-HT 2A receptor (R) expression, but did increase pulmonary gene expression of the 5-HT 2BR and serotonin transporter. Treatment with ketanserin attenuated bleomycin-induced PH (increased RVSP and RVH) and pulmonary vascular remodeling (decreased vessel density and increased muscularization of small vessels). In addition, we found that treatment with ketanserin activated pulmonary MAPK and Akt signaling in mice exposed to bleomycin. We conclude that 5-HT signaling is usually increased in a murine model of neonatal PH and pharmacological inhibition of the 5-HT 2AR protects against the development of PH in neonatal lung injury. We speculate this occurs through restoration of MAPK signaling and increased Akt signaling. = 14C23. PBS, PBS exposed; KET, ketanserin exposed; BLEO, bleomycin exposed; B/K, bleomycin + ketanserin exposed. U-93631 Pulmonary Tph1, Htr2B, and Slc6A4 expression increased in bleomycin-induced PH and BPD. We examined the major enzyme systems responsible for 5-HT synthesis and degradation in the lung. Tph1, the rate-limiting enzyme in the synthesis of 5-HT from tryptophan, increased in the lungs of mice exposed to bleomycin (Fig. 2and = 5. = 5. = 5C8. = 10. = 8C10. relative to 18S, = 9. relative to 18S, **= 9. Analysis by unpaired = 5C8. = 9C10. Ketanserin attenuates pulmonary vascular remodeling following exposure to bleomycin. We performed immunohistochemistry (IHC) for the endothelial cell marker vWF and counted the number of small vessels ( 30 m) per high-powered field as an indicator of vascular development. Representative images are shown for vWF immunostaining of lung sections from mice exposed to ip PBS (Fig. 4= 5C7. Analysis by one-way ANOVA with Bonferronis posttest. = 5C8. Analysis by one-way ANOVA with Bonferronis posttest. = 5C12. and and gene expression in mice cotreated with bleomycin vs. bleomycin alone. lung expression relative to -actin, *= 5. = 5. = 5C9. = 9C10. = 8C10. = 9. = 8C10. Analysis by one-way ANOVA with Bonferronis posttest. Ketanserin restores pulmonary MAPK signaling and increases pulmonary Akt signaling in neonatal mice cotreated with bleomycin. Both MAPK and Akt signaling have been implicated in 5-HT-mediated PAEC, PASMC, and fibroblast (PA Fib) growth (6, 41C43, 61, 72, 74). To evaluate whether alterations in pulmonary MAPK and Akt signaling could contribute to impaired vascular growth and PH in bleomycin-treated mice, we measured extracellular signal-related kinase (ERK) and Akt activation. Neonatal mice exposed to bleomycin had decreased phosphorylation of ERK44/42, indicating loss of MAPK pathway activation (Fig. 8= 5C11. = 5C7. Analysis by one-way ANOVA with Bonferronis posttest. DISCUSSION In this study, we hypothesized that 5-HT signaling is U-93631 increased in experimental PH associated with BPD and that 5-HT 2A-R antagonism would ameliorate experimental PH in neonatal mice. We tested this hypothesis in wild-type neonatal mice treated with bleomycin. We (17) had previously reported that bleomycin treatment resulted in RVH, pulmonary vascular remodeling, and simplified alveolar development, mirroring the findings seen in many infants with BPD. We now also report RVSP measurements in 3-wk-old mice. We demonstrate that expression of Tph1, the rate-limiting enzyme in the synthesis of 5-HT, is increased in the lungs of mice with experimental neonatal PH and that cotreatment with the 5-HT 2AR antagonist ketanserin, along with bleomycin, prevents bleomycin-induced PH and pulmonary vascular remodeling. These data provide further support for the role of 5-HT in regulating pulmonary vascular tone and is the first evidence that modulation of 5-HT signaling promotes vascular development in a neonatal model of pulmonary vascular disease. One novel finding of our study is the increased 5-HT signaling in experimental PH associated with BPD, including increased pulmonary protein expression of Tph1 and gene expression of 5-HT 2B-R (Htr2b) and SERT (Slc6A4). In this study we found no difference in whole lung expression of MAO-A, the enzyme primarily responsible for metabolism of 5-HT, or the 5-HT 2A-R or 1B (Htr1b) R. Increased pulmonary artery Tph1, 2B-R and SERT expression are well described in adults with PH (21, 23, 37). Furthermore, pharmacological or genetic strategies that inhibit U-93631 Tph1 synthesis or 2B-R or SERT activation protect mature rodents from the development of experimental PH (22, 37, 49, 52). As the function of the SERT and receptors are known to vary with maturation, injury model, and between species, further work is needed to understand the contribution of 5-HT synthesis vs. receptor activation in disease pathogenesis. It is, for example, necessary to consider that, since 5-HT is a potent vascular mitogen, strategies utilized in models of adult PH to completely block pulmonary 5-HT synthesis may have adverse consequences on the developing vasculature and airway (25, 30). We.Analysis by unpaired = 5C8. vascular remodeling (decreased vessel density and increased muscularization of small vessels). In addition, we found that treatment with ketanserin activated pulmonary MAPK and Akt signaling in mice exposed to bleomycin. We conclude that 5-HT signaling is increased in a murine model of neonatal PH and pharmacological inhibition of the 5-HT 2AR protects against the development of PH in neonatal lung injury. We speculate this occurs through restoration of MAPK signaling and increased Akt signaling. = 14C23. PBS, PBS exposed; KET, ketanserin exposed; BLEO, bleomycin exposed; B/K, bleomycin + ketanserin exposed. Pulmonary Tph1, Htr2B, and Slc6A4 expression increased in bleomycin-induced PH and BPD. We examined the major enzyme systems responsible for 5-HT synthesis and degradation in the lung. Tph1, the rate-limiting enzyme in the synthesis of 5-HT from tryptophan, increased in the lungs of mice exposed to bleomycin (Fig. 2and = 5. = 5. = 5C8. = 10. = 8C10. relative to 18S, = 9. relative to 18S, **= 9. Analysis by unpaired = 5C8. = 9C10. Ketanserin attenuates pulmonary vascular remodeling following exposure to bleomycin. We performed immunohistochemistry (IHC) for the endothelial cell marker vWF and counted the number of small vessels ( 30 m) per high-powered field as an indicator of vascular development. Representative images are shown for vWF immunostaining of lung sections from mice exposed to ip PBS (Fig. 4= 5C7. Analysis by one-way ANOVA with Bonferronis posttest. = 5C8. Analysis by one-way ANOVA with Bonferronis posttest. = 5C12. and and gene expression in mice cotreated with bleomycin vs. bleomycin alone. lung expression relative to -actin, *= 5. = 5. = 5C9. = 9C10. = 8C10. = 9. = 8C10. Analysis by one-way ANOVA with Bonferronis posttest. Ketanserin restores pulmonary MAPK signaling and increases pulmonary Akt signaling in neonatal mice cotreated with bleomycin. Both MAPK and Akt signaling have been implicated in 5-HT-mediated PAEC, PASMC, and fibroblast (PA Fib) growth (6, 41C43, 61, 72, 74). To U-93631 evaluate whether alterations in pulmonary MAPK and Akt signaling could contribute to impaired vascular growth and PH in bleomycin-treated mice, we measured extracellular signal-related kinase (ERK) and Akt activation. Neonatal mice exposed to bleomycin had decreased phosphorylation of ERK44/42, indicating loss of MAPK pathway activation (Fig. 8= 5C11. = 5C7. Analysis by one-way ANOVA with Bonferronis posttest. DISCUSSION In this study, we hypothesized that 5-HT signaling is increased in experimental PH associated with BPD and that 5-HT 2A-R antagonism would ameliorate experimental PH in neonatal mice. We tested this hypothesis in wild-type neonatal mice treated with bleomycin. We (17) had previously reported that bleomycin treatment resulted in RVH, pulmonary vascular remodeling, and simplified alveolar development, mirroring the findings seen in many infants with BPD. We now also report RVSP measurements in 3-wk-old mice. We demonstrate that expression of Tph1, the rate-limiting enzyme in the synthesis of 5-HT, is increased in the lungs of mice with experimental neonatal PH and that cotreatment with the 5-HT 2AR antagonist ketanserin, along with bleomycin, prevents bleomycin-induced PH and pulmonary vascular remodeling. These data provide further support for the role of 5-HT in regulating pulmonary vascular tone and is the first evidence that modulation of 5-HT signaling promotes vascular development in a neonatal model of pulmonary vascular disease. One novel finding of our study is the increased 5-HT signaling in experimental PH associated with BPD, including increased pulmonary protein expression of Tph1 and gene expression of 5-HT 2B-R (Htr2b) and SERT (Slc6A4). In this study we found no difference in whole lung expression of MAO-A, the enzyme primarily responsible for metabolism of 5-HT, or the 5-HT 2A-R or 1B (Htr1b) R. Increased pulmonary artery Tph1, 2B-R and SERT expression are well described in adults with PH (21, 23, 37). Furthermore, pharmacological or genetic strategies that inhibit Tph1 synthesis or 2B-R or SERT activation protect mature rodents from the development of experimental PH (22, 37, 49, 52). As the function of the SERT and receptors are known to vary with maturation, injury model, and between species, further work is needed to understand the contribution of 5-HT synthesis vs. receptor activation in disease pathogenesis. It is, for example, necessary to consider that, since 5-HT is a potent vascular mitogen, strategies utilized in models of adult PH to completely block pulmonary 5-HT synthesis may have adverse consequences.Mol Ther 20: 1516C1528, 2012. to bleomycin. We conclude that 5-HT signaling is definitely improved inside a murine model of neonatal PH and pharmacological inhibition of the 5-HT 2AR shields against the development of PH in neonatal lung injury. We speculate this happens through repair of MAPK signaling and improved Akt signaling. = 14C23. PBS, PBS revealed; KET, ketanserin revealed; BLEO, bleomycin revealed; B/K, bleomycin + ketanserin revealed. Pulmonary Tph1, Htr2B, and Slc6A4 manifestation improved in bleomycin-induced PH and BPD. We examined the major enzyme systems responsible for 5-HT synthesis and degradation in the lung. Tph1, the rate-limiting enzyme in the synthesis of 5-HT from tryptophan, improved in the lungs of mice exposed to bleomycin (Fig. 2and = 5. = 5. = 5C8. = 10. = 8C10. relative to 18S, = 9. relative to 18S, **= 9. Analysis by unpaired = 5C8. = 9C10. Ketanserin attenuates pulmonary vascular redesigning following exposure to bleomycin. We performed immunohistochemistry (IHC) for the endothelial cell marker vWF and counted the number of small vessels ( 30 m) per high-powered field as an indication of vascular development. Representative images are demonstrated for vWF immunostaining of lung sections from mice exposed to ip PBS (Fig. 4= 5C7. Analysis by one-way ANOVA with Bonferronis posttest. = 5C8. Analysis by one-way ANOVA with Bonferronis posttest. = 5C12. and and gene manifestation in mice cotreated with bleomycin vs. bleomycin only. lung expression relative to -actin, *= 5. = 5. = 5C9. = 9C10. = 8C10. = 9. = 8C10. Analysis by one-way ANOVA with Bonferronis posttest. Ketanserin restores pulmonary MAPK signaling and raises pulmonary Akt signaling in neonatal mice cotreated with bleomycin. Both MAPK and Akt signaling have been implicated in 5-HT-mediated PAEC, PASMC, and fibroblast (PA Fib) growth Itgb1 (6, 41C43, 61, 72, 74). To evaluate whether alterations in pulmonary MAPK and Akt signaling could contribute to impaired vascular growth and PH in bleomycin-treated mice, we measured extracellular signal-related kinase (ERK) and Akt activation. Neonatal mice exposed to bleomycin experienced decreased phosphorylation of ERK44/42, indicating loss of MAPK pathway activation (Fig. 8= 5C11. = 5C7. Analysis by one-way ANOVA with Bonferronis posttest. Conversation In this study, we hypothesized that 5-HT signaling is definitely improved in experimental PH associated with BPD and that 5-HT 2A-R antagonism would ameliorate experimental PH in neonatal mice. We tested this hypothesis in wild-type neonatal mice treated with bleomycin. We (17) experienced previously reported that bleomycin treatment resulted in RVH, pulmonary vascular redesigning, and simplified alveolar development, mirroring the findings seen in many babies with BPD. We now also statement RVSP measurements in 3-wk-old mice. We demonstrate that manifestation of Tph1, the rate-limiting enzyme in the synthesis of 5-HT, is improved in the lungs of mice with experimental neonatal PH and that cotreatment with the 5-HT 2AR antagonist ketanserin, along with bleomycin, helps prevent bleomycin-induced PH and pulmonary vascular redesigning. These data provide further support for the part of 5-HT in regulating pulmonary vascular firmness and is the 1st evidence that modulation of 5-HT signaling promotes vascular development inside a neonatal model of pulmonary vascular disease. One novel getting of our study is the improved 5-HT signaling in experimental PH associated with BPD, including improved pulmonary protein manifestation of Tph1 and gene manifestation of 5-HT 2B-R (Htr2b) and SERT (Slc6A4). With this study we found no difference in whole lung manifestation of MAO-A, the enzyme primarily responsible for rate of metabolism of 5-HT, or the 5-HT 2A-R or 1B (Htr1b) R. Improved pulmonary artery Tph1, 2B-R and SERT manifestation are well explained.