**2. Pulmonary vascular physiology**

Monolayer formed by endothelial cells (EC) is a critical interface between the circulating blood and underlying SMC and provides a non-thrombogenic barrier. EC transform mechanical stimuli into biological responses and depending on the stimuli, EC secrete several transducing molecules that participate in a number of biological functions such as vascular tone, cell proliferation, apoptosis, inflammation and thrombosis. EC maintain vascular tone by activat‐ ing cGMP and cAMP pathways. Nitric oxide (NO) is synthesized from L-arginine through the catalytic activity of endothelial NO synthase (eNOS). NO activates soluble guanylate cyclase (sGC) that catalyzes guanylate triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), which via cGMP-dependent protein kinase (PKG) induces vascular relaxation, inhibits cell proliferation and modulates inflammation. Subsequently cGMP is metabolized and inactivated by Phosphodiesterase 5. Prostacyclin (PGI2), an arachidonic acid metabolite is produced by the enzymatic activity of cyclooxygenase and PGI2 synthase. The prostacyclin receptor found on EC and platelets belongs to the family of G-protein coupled receptors. PGI2 binds to the receptor and stimulates adenylyl cyclase which catalyzes the conversion of ATP to second messenger cAMP. In addition, the cAMP/PKA pathway activates NO produc‐ tion via phosphorylation of eNOS. Both cGMP and cAMP mechanisms induce vascular relaxation and inhibit platelet aggregation and DNA synthesis. Juxtaposition of EC and SMC facilitates crosstalk, and EC maintain SMC in a quiescent state. [Mathew 2011a]. SMC inhibits flow-mediated activation of the mammalian target of rapamycin (mTOR) in EC, and SMC also participate in altering the expression of the factors involved in coagulation and fibrinolysis [Balcells 2010].

Caveolae, a subset of specialized microdomains (omega shaped invaginations, 50-100 nm) are found on a variety of cells including EC, SMC, fibroblasts and epithelial cells. They serve as a platform and compartmentalize a number of signaling molecules that reside in or are recruited to caveolae. They are also involved in transcytosis, endocytosis and potocytosis. Caveolin-1 (22kD) is the major scaffolding protein that supports and maintains the structure of caveolae. It interacts and regulates a number of proteins including Src family of kinases, G-proteins (α subunits), G protein-coupled receptors, H-Ras, PKC, eNOS, integrins and growth factor receptors such as vascular endothelial growth factor-receptor (VEGF-R), and epidermal growth factor-receptor (EGF-R). Caveolin-1 stabilizes these signaling proteins, and negatively regulates the target proteins within caveolae, through caveolin-1-scaffolding domain (CSD, residue 82-101). For optimal activation, eNOS is targeted to caveolae. Although it is negatively regulated by caveolin-1, caveolin-1 is essential for NO-mediated angiogenesis. In addition, the downstream effector of NO, sGC has been shown to compartmentalize in caveolae to facilitate its activation. In caveolin-1 knockout mice, the loss of caveolin-1 is associated with the hyperactivation of eNOS, and increased cGMP production. The hyper-activation of eNOS subse‐ quently leading to PKG nitration-induced stress is considered responsible for PH in these mice; and re-expression of endothelial caveolin-1 restores vascular and cardiac abnormalities [*reviewed in* Mathew 2011b]. Caveolin-1 functions as an antiproliferative molecule; it negatively regulates proliferative pathways such as mitogen-activated protein kinase/extracellular signalregulated kinase (MAPK/ERK), tyrosine- phosphorylated signal transducer and activator of transcription (PY-STAT) 3, EGF and platelet-derived growth factor (PDGF). Caveolin-1 also regulates cell cycle and apoptosis. In addition, caveolin-1 interacts with major ion channels such as Ca2+ -dependent potassium channels, voltage-dependent K+ channels (Kv1.5), and a number of molecules responsible for Ca2+ handling such as inositol triphosphate receptor (IP3R), heterodimeric GTP binding protein, Ca2+ ATPase and several transient receptor potential channels in caveolae. Through these interactions, caveolin-1 modulates cell prolif‐ eration and cell cycle progression. In SMC, caveolin-1 regulates Ca2+ entry and enables vasoconstriction. The localization of Ca2+ regulating proteins in caveolae and the proximity to the sarcoplasmic reticulum suggests an important role for caveolae/caveolin-1 for Ca2+ homeostasis [*reviewed in* Mathew 2011b]. RhoA interacts directly with caveolin-1, and the translocation of RhoA to caveolae is essential for myogenic tone. The CSD peptide of caveolin-1 has been shown to inhibit the agonist-induced redistribution of RhoA and PKC-α. Caveolin-1 blockage results in impaired formation of capillary tubes, and the overexpression of caveolin-1 accelerates EC differentiation and tube formation [Santibanz 2008, Liu 2002]. Furthermore, caveolin-1 modulates inflammation. It has recently been shown that caveolin-1 inhibits HIV replication through NF-κB [Wang 2011].

disease, 8. Pediatric thromboembolic disease, pediatric hypobaric hypoxic exposure, 10. Pediatric pulmonary vascular disease associated with other systemic disorders [del Cerro 2011]. Irrespective of the underlying pathology, patients usually present with similar changes in the lungs including endothelial dysfunction, impaired vascular reactivity, activation of inflammatory processes, vascular remodeling, with subsequent neointima formation and

Monolayer formed by endothelial cells (EC) is a critical interface between the circulating blood and underlying SMC and provides a non-thrombogenic barrier. EC transform mechanical stimuli into biological responses and depending on the stimuli, EC secrete several transducing molecules that participate in a number of biological functions such as vascular tone, cell proliferation, apoptosis, inflammation and thrombosis. EC maintain vascular tone by activat‐ ing cGMP and cAMP pathways. Nitric oxide (NO) is synthesized from L-arginine through the catalytic activity of endothelial NO synthase (eNOS). NO activates soluble guanylate cyclase (sGC) that catalyzes guanylate triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), which via cGMP-dependent protein kinase (PKG) induces vascular relaxation, inhibits cell proliferation and modulates inflammation. Subsequently cGMP is metabolized and inactivated by Phosphodiesterase 5. Prostacyclin (PGI2), an arachidonic acid metabolite is produced by the enzymatic activity of cyclooxygenase and PGI2 synthase. The prostacyclin receptor found on EC and platelets belongs to the family of G-protein coupled receptors. PGI2 binds to the receptor and stimulates adenylyl cyclase which catalyzes the conversion of ATP to second messenger cAMP. In addition, the cAMP/PKA pathway activates NO produc‐ tion via phosphorylation of eNOS. Both cGMP and cAMP mechanisms induce vascular relaxation and inhibit platelet aggregation and DNA synthesis. Juxtaposition of EC and SMC facilitates crosstalk, and EC maintain SMC in a quiescent state. [Mathew 2011a]. SMC inhibits flow-mediated activation of the mammalian target of rapamycin (mTOR) in EC, and SMC also participate in altering the expression of the factors involved in coagulation and fibrinolysis

Caveolae, a subset of specialized microdomains (omega shaped invaginations, 50-100 nm) are found on a variety of cells including EC, SMC, fibroblasts and epithelial cells. They serve as a platform and compartmentalize a number of signaling molecules that reside in or are recruited to caveolae. They are also involved in transcytosis, endocytosis and potocytosis. Caveolin-1 (22kD) is the major scaffolding protein that supports and maintains the structure of caveolae. It interacts and regulates a number of proteins including Src family of kinases, G-proteins (α subunits), G protein-coupled receptors, H-Ras, PKC, eNOS, integrins and growth factor receptors such as vascular endothelial growth factor-receptor (VEGF-R), and epidermal growth factor-receptor (EGF-R). Caveolin-1 stabilizes these signaling proteins, and negatively regulates the target proteins within caveolae, through caveolin-1-scaffolding domain (CSD, residue 82-101). For optimal activation, eNOS is targeted to caveolae. Although it is negatively regulated by caveolin-1, caveolin-1 is essential for NO-mediated angiogenesis. In addition, the

eventually right heart failure.

50 Pulmonary Hypertension

[Balcells 2010].

**2. Pulmonary vascular physiology**

BMPRII is predominantly expressed in EC, and a part of BMPRII colocalizes with caveolin-1 in caveolae and also in golgi bodies. BMPRII signaling, essential for BMP-mediated regulation of vascular SMC growth and differentiation also protects EC from apoptosis [Yu 2008, Teichert-Kuliszewska 2006]. BMPRII directly modulates proteins involved in cytoskeletal organization, possibly through Mas1 (G-protein-coupled receptor) interaction with Rho GTPase. Recently discovered angiotensin converting enzyme (ACE) 2, an endogenous inhibitor of ACE, is endothelium-bound. ACE2 cleaves angiotensin (Ang) I to Ang 1-9 which is an inactive compound. ACE2 metabolizes Ang I to produce Ang 1-7 which is a physiological antagonist of Ang II. ACE2/Ang (1-7) pathway antagonizes Ang II acting through Mas1, increases NO production via the Akt-dependent pathway, releases PGI2 and it inhibits Ang II-induced reactive oxygen species (ROS) formation within the cell nucleus. Loss of ACE2 causes increased vascular permeability, pulmonary edema and worsening lung function. The over-expression of Ang-(1-7) has a protective effect on MCT-induced PH and bleomycin-induced lung fibrosis. Interestingly, inhibition of Rho kinase has been shown to activate the ACE2/Ang-(1-9) pathway resulting in increased eNOS expression and amelioration of hypertension [Johnson 2012, Burton 2011, Lovern 2008, Mathew 2011, Ocaranza 2011, Shenoy 2010]. Thus, under normal conditions EC maintain homeostasis by producing cell protective factors and inhibiting inflammation and cell proliferation.
