**6. Prevention of hypertensive pulmonary vascular remodeling through NOS/NO pathway**

with ATRA treatment, ATRA increases NO production, suggesting that ATRA increases activity of expressed eNOS indirectly through the decrease in ADMA due to increased DDHA2 [102]. ATRA also upregulates NO production in vascular endothelial cells through the PI3 kinase/Akt pathway [103]. ATRA induces eNOS phosphorylation at ser1177 and Akt phosphor‐ ylation at ser473 without changes in protein expression such as occur during DDAH2 upregu‐ lation. In terms of inducible NOS(iNOS), interleukin(IL)-1β increases iNOS mRNA levels and ATRA reduces this increase in vascular smooth muscle cell culture [104]. Because iNOS inhibition by the iNOS inhibitor N6-(1-iminoethyl-L-lysine, dihydrochloride(L-NIL) prevent‐ ed the development of PH [105], the inhibitory effect of ATRA on iNOS expression might reduce the development of PH. Peroxisome proliferator-activated receptors (PPARγs) are a nuclear hormone receptor superfamily of ligand-activated transcription factors of retinoid hormone receptors other than steroid and thyroid hormones. PPARγ or retinoid X receptor (RXR) agonists inhibit smooth muscle proliferation. The PPARγ agonist rosiglitazone attenu‐ ates the development of chronic hypoxia-induced vascular structural remodeling [106], although it has little effect on the vasoconstriction component of PH. Since PPARγ mediates effects through the RXR, retinoids might also ameliorate PH vascular changes. PPARγ ligands increase the release of NO from endothelial cells through a transcriptional mechanism probably through the increase in DDAH mRNA expression without changes in eNOS expression [107]. These results suggest that ATRA might prevent the development of experi‐ mental PH in rats. ATRA ameliorated the development of MCT-induced PH [108], but not chronic hypoxia-induced PH [109]. These differences in the effect of ATRA on the development of PH may be due to a more pronounced inflammatory response in MCT-induced PH and a more subtle inflammatory reaction in chronic hypoxia-induced PH; endothelial damage precedes the rise in PAP in MCT model whereas the rise in PAP precedes endothelial changes

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Mutation of the bone morphogenetic protein receptor type II (BMPIIR) gene is one of the causes of familial PAH. The link between BMPIIR and eNOS partly explains the mechanism for the development of PH caused by BMPIIR mutations. Stimulation of BMPIIR induces eNOS phosphorylation, primarily through the cyclic-AMP dependent protein kinase and partially through serine-threonine kinase Akt [112]. Stimulation of BMPIIR also causes dissociation of eNOS from caveolin-1 and increases the eNOS-HSP90 interaction, which facilitates electron transfer through eNOS[112]. Thus, impaired BMPIIR or loss of BMPIIR stimulation might

Vascular endothelial growth factor (VEGF) stimulates NO production initially by increasing intracellular Ca++ levels and subsequent Ca++-calmodulin dependent activation of eNOS, and later by increasing intracellular eNOS message and protein levels [113]. VEGF stimulates vasodilation, microvascular hyperpermeability, and angiogenesis. Plexiform lesions show striking expression of VEGF associated with endothelial proliferation. NOS inhibition prevents

disturb the pulmonary vascular homeostasis, thereby causing PH.

in the chronic hypoxic model [110,111].

**6.4. VEGF increases eNOS expression**

**6.3. BMPIIR activates eNOS**

#### **6.1. NO precursor L-arginine ameliorates PH**

Arginase, an enzyme in the urea cycle, converts arginine to ornithine and urea. NOx concen‐ trations in exhaled gas and serum are decreased in PH patients compared with normal persons [95], suggesting decreased NO availability in PH. The deficiency of the NO precursor Larginine, the substrate depletion of NOS, might partly explain the decrease in NO availability. Lower levels of arginine in the cell might be due to the increased activity of arginase. In PH patients, lower levels of arginine correlate with higher pulmonary artery pressures. Serum arginase activity is higher and the serum arginine-ornithine ratio is lower in PH patients than in healthy controls, indirectly suggesting increased intracellular arginase activity [33]. Animal studies showed that prolonged administration of L-arginine ameliorated the development of monocrotaine-induced PH [24,96] and chronic hypoxia-induced PH [96]. In patients with PH L-arginine treatment reduces PAP [97]. In addition to functioning as the substrate for NO formation, L-arginine prevents eNOS uncoupling, serves as a direct radical scavenger, and competes with the endogenous eNOS inhibitor ADMA, which decreases superoxide and increases NO formation [41].

#### **6.2. ATRA increases NO production (Figure 5)**

The level of asymmetrical dimethylarginine (ADMA) is increased in patients with PAH and MCT-induced PH in rats [98]. Since ADMA is an endogenous competitive inhibitor of NOS and suppresses NOS activity, increases in ADMA inhibit NO production. In atherosclerotic arteries from patients with high serum ADMA, endothelium-dependent relaxation by acetylcholine was impaired and O2 •- production was increased [99]. Dysregulation of ADMA might cause PH through the decrease in NO in the lung as well. Dimethylarginine dimethya‐ minohydrolase (DDAH) is a metabolizing enzyme of ADMA. Thus the increase in DDAH activity reduces ADMA and induces subsequent increases in NOS activity. DDAH has two isoforms: DDAH 1 and DDAH 2. DDHA 1 and DDAH 2 are expressed predominantly in tissues containing neuronal NOS (nNOS) and eNOS, respectively [100]. Phosphodiesterase (PDE) 3/4 inhibitors reduce ADMA and raise NO/cGMP levels [2]; PDE3/4 inhibitors activate the cAMP/ protein kinase A (PKA) pathway and induce subsequent activation of the promoter region of DDAH2. Western blot analysis of lung from PH rats 28 days after the injection of MCT showed decreases in eNOS, pNOS, AKT, and DDAH2 and increases in lung and serum ADMA levels [101]. In this PH model, 1) decreased Akt reduces eNOS phosphorylation and thereby decreases eNOS activity 2) decreased DDAH2 reduces ADMA breakdown and thereby the increase in ADMA inhibits eNOS activity. This study showed that rosuvastatin ameliorates MCT-induced PH through the normalization of Akt, eNOS and DDAH2 expression and ADMA levels [101].

Endothelial cells express retinoid receptors and all-trans-retinoic acid (ATRA) increased DDHA2 mRNA levels in endothelial cells. Although eNOS mRNA expression is not increased with ATRA treatment, ATRA increases NO production, suggesting that ATRA increases activity of expressed eNOS indirectly through the decrease in ADMA due to increased DDHA2 [102]. ATRA also upregulates NO production in vascular endothelial cells through the PI3 kinase/Akt pathway [103]. ATRA induces eNOS phosphorylation at ser1177 and Akt phosphor‐ ylation at ser473 without changes in protein expression such as occur during DDAH2 upregu‐ lation. In terms of inducible NOS(iNOS), interleukin(IL)-1β increases iNOS mRNA levels and ATRA reduces this increase in vascular smooth muscle cell culture [104]. Because iNOS inhibition by the iNOS inhibitor N6-(1-iminoethyl-L-lysine, dihydrochloride(L-NIL) prevent‐ ed the development of PH [105], the inhibitory effect of ATRA on iNOS expression might reduce the development of PH. Peroxisome proliferator-activated receptors (PPARγs) are a nuclear hormone receptor superfamily of ligand-activated transcription factors of retinoid hormone receptors other than steroid and thyroid hormones. PPARγ or retinoid X receptor (RXR) agonists inhibit smooth muscle proliferation. The PPARγ agonist rosiglitazone attenu‐ ates the development of chronic hypoxia-induced vascular structural remodeling [106], although it has little effect on the vasoconstriction component of PH. Since PPARγ mediates effects through the RXR, retinoids might also ameliorate PH vascular changes. PPARγ ligands increase the release of NO from endothelial cells through a transcriptional mechanism probably through the increase in DDAH mRNA expression without changes in eNOS expression [107]. These results suggest that ATRA might prevent the development of experi‐ mental PH in rats. ATRA ameliorated the development of MCT-induced PH [108], but not chronic hypoxia-induced PH [109]. These differences in the effect of ATRA on the development of PH may be due to a more pronounced inflammatory response in MCT-induced PH and a more subtle inflammatory reaction in chronic hypoxia-induced PH; endothelial damage precedes the rise in PAP in MCT model whereas the rise in PAP precedes endothelial changes in the chronic hypoxic model [110,111].

#### **6.3. BMPIIR activates eNOS**

**6. Prevention of hypertensive pulmonary vascular remodeling through**

Arginase, an enzyme in the urea cycle, converts arginine to ornithine and urea. NOx concen‐ trations in exhaled gas and serum are decreased in PH patients compared with normal persons [95], suggesting decreased NO availability in PH. The deficiency of the NO precursor Larginine, the substrate depletion of NOS, might partly explain the decrease in NO availability. Lower levels of arginine in the cell might be due to the increased activity of arginase. In PH patients, lower levels of arginine correlate with higher pulmonary artery pressures. Serum arginase activity is higher and the serum arginine-ornithine ratio is lower in PH patients than in healthy controls, indirectly suggesting increased intracellular arginase activity [33]. Animal studies showed that prolonged administration of L-arginine ameliorated the development of monocrotaine-induced PH [24,96] and chronic hypoxia-induced PH [96]. In patients with PH L-arginine treatment reduces PAP [97]. In addition to functioning as the substrate for NO formation, L-arginine prevents eNOS uncoupling, serves as a direct radical scavenger, and competes with the endogenous eNOS inhibitor ADMA, which decreases superoxide and

The level of asymmetrical dimethylarginine (ADMA) is increased in patients with PAH and MCT-induced PH in rats [98]. Since ADMA is an endogenous competitive inhibitor of NOS and suppresses NOS activity, increases in ADMA inhibit NO production. In atherosclerotic arteries from patients with high serum ADMA, endothelium-dependent relaxation by

might cause PH through the decrease in NO in the lung as well. Dimethylarginine dimethya‐ minohydrolase (DDAH) is a metabolizing enzyme of ADMA. Thus the increase in DDAH activity reduces ADMA and induces subsequent increases in NOS activity. DDAH has two isoforms: DDAH 1 and DDAH 2. DDHA 1 and DDAH 2 are expressed predominantly in tissues containing neuronal NOS (nNOS) and eNOS, respectively [100]. Phosphodiesterase (PDE) 3/4 inhibitors reduce ADMA and raise NO/cGMP levels [2]; PDE3/4 inhibitors activate the cAMP/ protein kinase A (PKA) pathway and induce subsequent activation of the promoter region of DDAH2. Western blot analysis of lung from PH rats 28 days after the injection of MCT showed decreases in eNOS, pNOS, AKT, and DDAH2 and increases in lung and serum ADMA levels [101]. In this PH model, 1) decreased Akt reduces eNOS phosphorylation and thereby decreases eNOS activity 2) decreased DDAH2 reduces ADMA breakdown and thereby the increase in ADMA inhibits eNOS activity. This study showed that rosuvastatin ameliorates MCT-induced PH through the normalization of Akt, eNOS and DDAH2 expression and

Endothelial cells express retinoid receptors and all-trans-retinoic acid (ATRA) increased DDHA2 mRNA levels in endothelial cells. Although eNOS mRNA expression is not increased

•- production was increased [99]. Dysregulation of ADMA

**NOS/NO pathway**

86 Pulmonary Hypertension

increases NO formation [41].

acetylcholine was impaired and O2

ADMA levels [101].

**6.2. ATRA increases NO production (Figure 5)**

**6.1. NO precursor L-arginine ameliorates PH**

Mutation of the bone morphogenetic protein receptor type II (BMPIIR) gene is one of the causes of familial PAH. The link between BMPIIR and eNOS partly explains the mechanism for the development of PH caused by BMPIIR mutations. Stimulation of BMPIIR induces eNOS phosphorylation, primarily through the cyclic-AMP dependent protein kinase and partially through serine-threonine kinase Akt [112]. Stimulation of BMPIIR also causes dissociation of eNOS from caveolin-1 and increases the eNOS-HSP90 interaction, which facilitates electron transfer through eNOS[112]. Thus, impaired BMPIIR or loss of BMPIIR stimulation might disturb the pulmonary vascular homeostasis, thereby causing PH.

#### **6.4. VEGF increases eNOS expression**

Vascular endothelial growth factor (VEGF) stimulates NO production initially by increasing intracellular Ca++ levels and subsequent Ca++-calmodulin dependent activation of eNOS, and later by increasing intracellular eNOS message and protein levels [113]. VEGF stimulates vasodilation, microvascular hyperpermeability, and angiogenesis. Plexiform lesions show striking expression of VEGF associated with endothelial proliferation. NOS inhibition prevents

**6.5. Elastase inhibition by NO**

by cGMP dependent protein kinase activation [120].

Earlier studies have shown that vascular elastase activity is increased in MCT-induced PH and chronic hypoxia-induced PH in rats [3,117], and that elastase inhibition prevents the develop‐ ment of pulmonary hypertension, right ventricular hypertrophy, muscularization of periph‐ eral pulmonary arteries and medial hypertrophy of muscular arteries [3,117,118]. NO might reduce the elastase activity through its scavenging effect of superoxide. Reactive oxygen species inactivates endogenous elastase inhibitor, α1-protease inhibitor, and might increase elastase activity [119]. Furthermore NO might reduce elastase expression by inhibiting its transcriptional factor, acute myeloid leukemia factor 1 (AML-1), through extracellular signalregulated kinase mitogen-activated protein kinase (ERK MAPK) inhibition which is mediated

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**Figure 6. Rho/Rho kinase pathway inhibits eNOS/NO/cGMP pathway** Rho kinase is activated by the guanosine triphosphate (GTP)-bound, active form of RhoA (GTP RhoA). Activated Rho kinase phosphorylates and subsequently inactivates myosin phosphatase, causing smooth muscle contraction, which is the RhoA/Rho kinase pathway. PKG phosphorylates Rho A at Ser188 and inhibits Rho A function, thereby inactivating the RhoA/Rho kinase pathway. Acti‐ vated RhoA/Rho kinase decreases eNOS mRNA and protein expression, inactivates Akt, and inhibits PKG activity, thereby supressing the eNOS/ NO/cGMP pathway. VEGF upregulates eNOS mRNA and protein expression. AML-1, acute myeloid leukemia factor 1(transcriptional factor); EVE, endogenous vascular elastase; PKG, cyclic GMP-depend‐ ent protein kinase(G kinase); PKB, protein kinase B(=Akt), AML-1, acute myeloid leukemia factor 1; ML, myosin light chain; pML, phosphorylated myosin light chain; MLCK, myosin light chain kinase; ERK MAPK, extracellular signal-regu‐

lated kinase mitogen activated protein kinase; VEGF, vascular endothelial growth factor.

**Figure 5. Possible pathway to enhance NO production by ATRA, DDAH, PDE3/4 inhibition, and BMPIIR.** ADMA supresses NOS activity. DDAH is the enzyme that metabolizes ADMA. The cAMP/PKA pathway activates the promoter region of DDAH2, thereby increasing DDAH2 expression. ATRA increases DDAH2 mRNA, stimulates RAR with a subse‐ quent increase in PI3K activity as well as PI3K protein and mRNA expression, and thereby enhances Akt and eNOS phosphorylation without increasing eNOS expression. Phosphorylated Akt(pAkt) phosphorylates eNOS making pe‐ NOS, the activated form of NOS. B2-AR stimulation activates SrcK via Gi/o protein. Activated SrcK phosphorylates PI3K and induces subsequent its downstream eNOS phosphorylation as well as phosphorylation of caveolin -1 to dissociate eNOS from caveola (Figure 3]. ADMA, asymmetric dimethylarginine; ATRA, all trans retinoic acid; B2-AR, beta 2-adre‐ nergic receptor; BMPIIR, bone morphogenetic ptotein II receptor; CREB, cAMP responsive element binding protein; cAMP, cyclic adenosine monophosphate; DDAH, dimethylarginine dimethylaminohydrolase; eNOS, endothelial nitric oxide synthase; peNOS, phosphorylated eNOS; ERK, extracellular signal-regulated kinase; pERK, phosphorylated ERK; Gi/o, GTP binding protein subunit Gi/o; PDE, phosphodiesteras; PI3K, phosphoinositide 3-kinase; PKA, cAMP depend‐ ent protein kinase; PKG, cyclic GMP-dependent protein kinase ; PPARγ, peroxisome proliferator-activated receptor; Srck, src kinase; RAR, retinoic acid receptor;

VEGF-induced proliferation in cultured microvascular endothelial cells, associated with the decrease in cGMP levels [114], suggesting that VEGF-induced proliferation is in part mediated by the NOS-NO-cGMP pathway. VEGF induces translocation of eNOS and caveolin-1 from caveola to the nucleus, where NO production activates transcriptional factors thereby inducing the early growth response gene, c-fos [115] and possibly inducing angiogenesis, and endothe‐ lial cell growth. VEGF receptor 2 (VEGF2R) blockade combined with chronic hypoxic exposure causes PH with plexiform like lesions, where decreased expression of VEGF2R, Src, Akt, phosphorylated Akt protein in lung have been demonstrated [116]. Studies have demonstrated that reduced Src and Akt attenuate eNOS phosphorylation [101].

#### **6.5. Elastase inhibition by NO**

VEGF-induced proliferation in cultured microvascular endothelial cells, associated with the decrease in cGMP levels [114], suggesting that VEGF-induced proliferation is in part mediated by the NOS-NO-cGMP pathway. VEGF induces translocation of eNOS and caveolin-1 from caveola to the nucleus, where NO production activates transcriptional factors thereby inducing the early growth response gene, c-fos [115] and possibly inducing angiogenesis, and endothe‐ lial cell growth. VEGF receptor 2 (VEGF2R) blockade combined with chronic hypoxic exposure causes PH with plexiform like lesions, where decreased expression of VEGF2R, Src, Akt, phosphorylated Akt protein in lung have been demonstrated [116]. Studies have demonstrated

**Figure 5. Possible pathway to enhance NO production by ATRA, DDAH, PDE3/4 inhibition, and BMPIIR.** ADMA supresses NOS activity. DDAH is the enzyme that metabolizes ADMA. The cAMP/PKA pathway activates the promoter region of DDAH2, thereby increasing DDAH2 expression. ATRA increases DDAH2 mRNA, stimulates RAR with a subse‐ quent increase in PI3K activity as well as PI3K protein and mRNA expression, and thereby enhances Akt and eNOS phosphorylation without increasing eNOS expression. Phosphorylated Akt(pAkt) phosphorylates eNOS making pe‐ NOS, the activated form of NOS. B2-AR stimulation activates SrcK via Gi/o protein. Activated SrcK phosphorylates PI3K and induces subsequent its downstream eNOS phosphorylation as well as phosphorylation of caveolin -1 to dissociate eNOS from caveola (Figure 3]. ADMA, asymmetric dimethylarginine; ATRA, all trans retinoic acid; B2-AR, beta 2-adre‐ nergic receptor; BMPIIR, bone morphogenetic ptotein II receptor; CREB, cAMP responsive element binding protein; cAMP, cyclic adenosine monophosphate; DDAH, dimethylarginine dimethylaminohydrolase; eNOS, endothelial nitric oxide synthase; peNOS, phosphorylated eNOS; ERK, extracellular signal-regulated kinase; pERK, phosphorylated ERK; Gi/o, GTP binding protein subunit Gi/o; PDE, phosphodiesteras; PI3K, phosphoinositide 3-kinase; PKA, cAMP depend‐ ent protein kinase; PKG, cyclic GMP-dependent protein kinase ; PPARγ, peroxisome proliferator-activated receptor;

that reduced Src and Akt attenuate eNOS phosphorylation [101].

Srck, src kinase; RAR, retinoic acid receptor;

88 Pulmonary Hypertension

Earlier studies have shown that vascular elastase activity is increased in MCT-induced PH and chronic hypoxia-induced PH in rats [3,117], and that elastase inhibition prevents the develop‐ ment of pulmonary hypertension, right ventricular hypertrophy, muscularization of periph‐ eral pulmonary arteries and medial hypertrophy of muscular arteries [3,117,118]. NO might reduce the elastase activity through its scavenging effect of superoxide. Reactive oxygen species inactivates endogenous elastase inhibitor, α1-protease inhibitor, and might increase elastase activity [119]. Furthermore NO might reduce elastase expression by inhibiting its transcriptional factor, acute myeloid leukemia factor 1 (AML-1), through extracellular signalregulated kinase mitogen-activated protein kinase (ERK MAPK) inhibition which is mediated by cGMP dependent protein kinase activation [120].

**Figure 6. Rho/Rho kinase pathway inhibits eNOS/NO/cGMP pathway** Rho kinase is activated by the guanosine triphosphate (GTP)-bound, active form of RhoA (GTP RhoA). Activated Rho kinase phosphorylates and subsequently inactivates myosin phosphatase, causing smooth muscle contraction, which is the RhoA/Rho kinase pathway. PKG phosphorylates Rho A at Ser188 and inhibits Rho A function, thereby inactivating the RhoA/Rho kinase pathway. Acti‐ vated RhoA/Rho kinase decreases eNOS mRNA and protein expression, inactivates Akt, and inhibits PKG activity, thereby supressing the eNOS/ NO/cGMP pathway. VEGF upregulates eNOS mRNA and protein expression. AML-1, acute myeloid leukemia factor 1(transcriptional factor); EVE, endogenous vascular elastase; PKG, cyclic GMP-depend‐ ent protein kinase(G kinase); PKB, protein kinase B(=Akt), AML-1, acute myeloid leukemia factor 1; ML, myosin light chain; pML, phosphorylated myosin light chain; MLCK, myosin light chain kinase; ERK MAPK, extracellular signal-regu‐ lated kinase mitogen activated protein kinase; VEGF, vascular endothelial growth factor.

#### **6.6. Rho-kinase inhibitor upregulates NOS in PH (Figure 6)**

Myosin light chain (MLC) phosphorylation by myosin light chain kinase (MLCK) causes vascular smooth muscle contraction. In contrast, myosin light chain dephosphorylation by myosin light chain phosphatase causes relaxation. The phosphorylation status of MLC phosphatase determines the contractility of smooth muscle at the same Ca++ concentration, thereby regulating the Ca++ sensitivity for contraction; the stronger the phosphatase activity, the weaker the vascular tone at the same Ca++ concentration. RhoA/Rho-kinase activation augments the phosphorylation of MLC phosphatase, which results in inhibition of MLC phosphatase. Studies have shown that Rho-kinase in circulating neutrophils is increased in patients with PH and that Rho-kinase expression is upregulated in isolated lung tissue on transplantation [121]. Rho-kinase activity in pulmonary arteries is enhanced in experimental PH [122,123]. NO-cGMP-cGMP dependent protein kinase pathway suppresses Rho/Rho kinase activity [124]. On the other hand Rho/Rho-kinase activation downregulates eNOS expression and eNOS phosphorylation through the inhibition of the protein kinase B/Akt pathway [125].

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