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Aims Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of pulmonary hypertension (PH). Proliferative cells utilize purine bases from the de novo purine synthesis (DNPS) pathways for nucleotide synthesis; however, it is unclear whether DNPS plays a critical role in VSMC proliferation during development of PH. The last two steps of DNPS are catalysed by the enzyme 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC). This study investigated whether ATIC-driven DNPS affects the proliferation of pulmonary artery smooth muscle cells (PASMCs) and the development of PH. Methods and results Metabolites of DNPS in proliferative PASMCs were measured by liquid chromatography-tandem mass spectrometry. ATIC expression was assessed in platelet-derived growth factor-treated PASMCs and in the lungs of PH rodents and patients with pulmonary arterial hypertension. Mice with global and VSMC-specific knockout of Atic were utilized to investigate the role of ATIC in both hypoxia- and lung interleukin-6/hypoxia-induced murine PH. ATIC-mediated DNPS at the mRNA, protein, and enzymatic activity levels were increased in platelet-derived growth factor-treated PASMCs or PASMCs from PH rodents and patients with pulmonary arterial hypertension. In cultured PASMCs, ATIC knockdown decreased DNPS and nucleic acid DNA/RNA synthesis, and reduced cell proliferation. Global or VSMC-specific knockout of Atic attenuated vascular remodelling and inhibited the development and progression of both hypoxia- and lung IL-6/hypoxia-induced PH in mice. Conclusion Targeting ATIC-mediated DNPS compromises the availability of purine nucleotides for incorporation into DNA/RNA, reducing PASMC proliferation and pulmonary vascular remodelling and ameliorating the development and progression of PH.

Pulmonary arterial hypertension (PAH) is a complex and fatal disorder characterized by an unrelenting increase in pulmonary arterial pressure. Excessive proliferation of pulmonary artery smooth muscle cells (PASMC) leads to increased vascular resistance and eventually right ventricular failure and death. Our lab has hypothesized that circulating Galectin-3 (Gal-3), a readily secreted b-galactoside-binding lectin protein, previously investigated as a biomarker of PAH, has a key role in the pathology of PAH. Co-culture experiments as well as recombinant protein supplementation show that soluble GAL-3 is biologically active and induces SMC growth and proliferation(P This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic inflammation underlies many chronic diseases such as obesity and diabetes and can lead to vascular and organ dysfunction, disability and death. Inflammation has been widely studied due to its broad involvement in numerous disease states, but current models of inflammation suffer from limitations. Models such as LPS treatment are non-specific and require multiple doses over extended periods of time which can lead to septic shock. The goal of our study was to develop an alternative model offering cell and organ specificity using an AAV-based approach. We hypothesize that delivering Ikk2, a potent activator of NF-KB will lead to chronic inflammation in the targeted cells and tissues. This approach has the benefit of cell and tissue specificity using specialized promoters and modified AAV capsids. Additionally, it will allow for consistent and regulatable expression over time. In preliminary studies I compared two know activators of NFKB, IKBKE and IKK2 to investigate differences in expression of inflammatory markers. Western blot analysis revealed ICAM-1 and IRF-1 protein levels were increased with IKK2 expression. In comparison, the inhibitior of apoptosis, CIAP-2, was more highly increased under IKBKE expression. We conclude that IKK2 would be more suitable to induce inflammation in target cells. Future studies will employ qPCR, Western blot, cytokine analysis and vascular function studies to characterize the effects in vivo. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pulmonary arterial hypertension is a rare and debilitating condition with a five-year survival rate of only 34% without treatment. The origins of pulmonary arterial hypertension are complex and incompletely understood and elevated pulmonary artery pressures result from excessive vascular remodeling due to functional changes in endothelial, vascular smooth muscle cells (VSMCs), and adventitial cells. Recent papers have explored the importance of metabolic switching of VSMCs in pulmonary hypertension in the transition towards a proliferative, apoptotic resistant phenotype which involves a shift from fatty acid oxidation to increased glycolysis. We hypothesized that ATPase Inhibitory Factor 1 (ATPIF1) in VSMCs could function as a metabolic switch that promotes a shift from fatty acid oxidation to glycolysis to increase the severity of pulmonary arterial hypertension. This hypothesis will be investigated using Western blot, Seahorse extracellular flux assays, qRT-PCR, and imaging of GFP-labelled ATPIF1. In preliminary data, we have cloned ATPIF1 and transfected cells to show overexpression of ATPIF1 decreases the maximal respiratory rate while increasing extracellular acidification, a measure of glycolysis. Future goals are to investigate the links between hypoxia, hypoxia-inducible factors and ATPIF1, the role of increased glycolysis in pulmonary smooth muscle cells, and ultimately whether ATPIF1 impacts of the development of pulmonary arterial hypertension in mice. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Significance: Pulmonary arterial hypertension (PAH) is a progressive disease arising from the narrowing of pulmonary arteries (PAs) resulting in high pulmonary arterial blood pressure and ultimately right ventricle (RV) failure. A defining characteristic of PAH is the excessive and unrelenting inward remodeling of PAs that includes increased proliferation, inflammation, and fibrosis. Critical Issues: There is no cure for PAH nor interventions that effectively arrest or reverse PA remodeling, and intensive research over the past several decades has sought to identify novel molecular mechanisms of therapeutic value. Recent Advances: Galectin-3 (Gal-3) is a carbohydrate-binding lectin remarkable for its chimeric structure, composed of an N-terminal oligomerization domain and a C-terminal carbohydrate-recognition domain. Gal-3 has been identified as a regulator of numerous changes in cell behavior that contributes to aberrant PA remodeling, including cell proliferation, inflammation, and fibrosis, but its role in PAH has remained poorly understood until recently. In contrast, pathological roles for Gal-3 have been proposed in cancer and inflammatory and fibroproliferative disorders, such as pulmonary vascular and cardiac fibrosis. Herein, we summarize the recent literature on the role of Gal-3 in the development of PAH. We provide experimental evidence supporting the ability of Gal-3 to influence reactive oxygen species production, NADPH oxidase enzyme expression, and redox signaling, which have been shown to contribute to both vascular remodeling and increased pulmonary arterial pressure. Future Directions: While several preclinical studies suggest that Gal-3 promotes hypertensive pulmonary vascular remodeling, the clinical significance of Gal-3 in human PAH remains to be established. Antioxid. Redox Signal. 00, 000–000.

A defining characteristic of pulmonary hypertension (PH) is the extensive remodeling of pulmonary arteries (PAs), which results in progressive increases in vascular resistance and stiffness and eventual failure of the right ventricle. There is no cure for PH and identification of novel molecular mechanisms that underlie increased proliferation, reduced apoptosis, and excessive extracellular matrix production in pulmonary artery smooth muscle cells (PASMCs) is a vital objective. Galectin-3 (Gal-3) is a chimeric lectin and potent driver of many aspects of fibrosis, but its role in regulating PASMC behavior in PH remains poorly understood. Herein, we evaluated the importance of increased Gal-3 expression and signaling on PA vascular remodeling and cardiopulmonary function in experimental models of PH. Gal-3 expression was quantified by qRT-PCR, immunoblotting, and immunofluorescence imaging, and its functional role was assessed by specific Gal-3 inhibitors and CRISPR/Cas9-mediated knockout of Gal-3 in the rat. In rat models of PH, we observed increased Gal-3 expression in PASMCs, which stimulated migration and resistance to apoptosis, whereas silencing or genetic deletion reduced cellular migration and PA fibrosis and increased apoptosis. Gal-3 inhibitors attenuated and reversed PA remodeling and fibrosis, as well as hemodynamic indices in monocrotaline (MCT)-treated rats in vivo. These results were supported by genetic deletion of Gal-3 in both MCT and Sugen Hypoxia rat models. In conclusion, our results suggest that elevated Gal-3 levels contribute to inappropriate PA remodeling in PH by enhancing multiple profibrotic mechanisms. Therapeutic strategies targeting Gal-3 may be of benefit in the treatment of PH.

Pulmonary Arterial Hypertension (PAH) is a progressive vascular disease arising from the narrowing of pulmonary arteries (PA) resulting in high pulmonary arterial blood pressure and ultimately right ventricular (RV) failure. A defining characteristic of PAH is the excessive remodeling of PA that includes increased proliferation, inflammation, and fibrosis. There is no cure for PAH nor interventions that effectively impede or reverse PA remodeling, and research over the past several decades has sought to identify novel molecular mechanisms of therapeutic benefit. Galectin-3 (Gal-3; Mac-2) is a carbohydrate-binding lectin that is remarkable for its chimeric structure, comprised of an N-terminal oligomerization domain and a C-terminal carbohydrate-recognition domain. Gal-3 is a regulator of changes in cell behavior that contribute to aberrant PA remodeling including cell proliferation, inflammation, and fibrosis, but its role in PAH is poorly understood. Herein, we summarize the recent literature on the role of Gal-3 in the development of PAH and provide experimental evidence supporting the ability of Gal-3 to influence reactive oxygen species (ROS) production, NOX enzyme expression, inflammation, and fibrosis, which contributes to PA remodeling. Finally, we address the clinical significance of Gal-3 as a target in the development of therapeutic agents as a treatment for PAH.

Pulmonary Arterial Hypertension (PAH) is a progressive vascular disease arising from the narrowing of pulmonary arteries (PA) resulting in high pulmonary arterial blood pressure and ultimately right ventricular (RV) failure. A defining characteristic of PAH is the excessive remodeling of PA that includes increased proliferation, inflammation, and fibrosis. There is no cure for PAH nor interventions that effectively impede or reverse PA remodeling, and research over the past several decades has sought to identify novel molecular mechanisms of therapeutic benefit. Galectin-3 (Gal-3; Mac-2) is a carbohydrate-binding lectin that is remarkable for its chimeric structure, comprised of an N-terminal oligomerization domain and a C-terminal carbohydrate-recognition domain. Gal-3 is a regulator of changes in cell behavior that contribute to aberrant PA remodeling including cell proliferation, inflammation, and fibrosis, but its role in PAH is poorly understood. Herein, we summarize the recent literature on the role of Gal-3 in the development of PAH and provide experimental evidence supporting the ability of Gal-3 to influence reactive oxygen species (ROS) production, NOX enzyme expression, inflammation, and fibrosis, which contributes to PA remodeling. Finally, we address the clinical significance of Gal-3 as a target in the development of therapeutic agents as a treatment for PAH.

Purpose Neurofibromatosis type 1 (NF1) is the result of inherited mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin. Eye manifestations are common in NF1 with recent reports describing a vascular dysplasia in the retina and choroid. Common features of NF1 retinopathy include tortuous and dilated feeder vessels that terminate in capillary tufts, increased endothelial permeability, and neovascularization. Given the retinal vascular phenotype observed in persons with NF1, we hypothesize that preserving neurofibromin may be a novel strategy to control pathologic retinal neovascularization. Methods Nf1 expression in human endothelial cells (EC) was reduced using small hairpin (sh) RNA and EC proliferation, migration, and capacity to form vessel-like networks were assessed in response to VEGF and hypoxia. Wild-type (WT), Nf1 heterozygous (Nf1+/-), and Nf1flox/+;Tie2cre pups were subjected to hyperoxia/hypoxia using the oxygen-induced retinopathy model. Retinas were analyzed quantitatively for extent of retinal vessel dropout, neovascularization, and capillary branching. Results Neurofibromin expression was suppressed in response to VEGF, which corresponded with activation of Mek-Erk and PI3-K-Akt signaling. Neurofibromin-deficient EC exhibited enhanced proliferation and network formation in response to VEGF and hypoxia via an Akt-dependent mechanism. In response to hyperoxia/hypoxia, Nf1+/- retinas exhibited increased vessel dropout and neovascularization when compared with WT retinas. Neovascularization was similar between Nf1+/- and Nf1flox/+;Tie2cre retinas, but capillary drop out in Nf1flox/+;Tie2cre retinas was significantly reduced when compared with Nf1+/- retinas. Conclusions These data suggest that neurofibromin expression is essential for controlling endothelial cell proliferation and retinal neovascularization and therapies targeting neurofibromin-deficient EC may be beneficial.

Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and adenosine 5'-[γ-thio]-triphosphate (ATPγS) can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to Ado, ATPγS-induced PKA activation is accompanied by a modest, but significant decrease in cyclic adenosine monophosphate (cAMP) levels supporting the existence of an unconventional cAMP-independent pathway of PKA activation. Furthermore, ATPγS-induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP but is instead dependent upon PKA-anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase-targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS-induced increases in transendothelial electrical resistance. Ado-induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP-dependent manner. In summary, ATPγS-induced enhancement of the EC barrier is EPAC1-independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP-independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation.

Pulmonary arterial hypertension (PAH) is a progressive disease of the lung vasculature that involves the loss of endothelial function together with inappropriate smooth muscle cell growth, inflammation, and fibrosis. These changes underlie a progressive remodeling of blood vessels that alters flow and increases pulmonary blood pressure. Elevated pressures in the pulmonary artery imparts a chronic stress on the right ventricle which undergoes compensatory hypertrophy but eventually fails. How PAH develops remains incompletely understood and evidence for the altered production of reactive oxygen and nitrogen species (ROS, RNS respectively) in the pulmonary circulation has been well documented. There are many different types of ROS and RNS, multiple sources, and collective actions and interactions. This review summarizes past and current knowledge of the sources of ROS and RNS and how they may contribute to the loss of endothelial function and changes in smooth muscle proliferation in the pulmonary circulation.

The inhibitory phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) at Thr497 (eNOSpThr497) by protein kinase C or RhoA-activated kinase is a major regulatory determinant of eNOS activity. The signalling mechanisms involved in the dephosphorylation of eNOSpThr497 have not yet been clarified. This study identifies myosin phosphatase (MP) holoenzyme consisting of protein phosphatase-1 catalytic subunit (PP1c) and MP target subunit-1 (MYPT1) as an eNOSpThr497 phosphatase. In support of this finding are: (i) eNOS and MYPT1 interacts in various endothelial cells (ECs) and in in vitro binding assays (ii) MYPT1 targets and stimulates PP1c toward eNOSpThr497 substrate (iii) phosphorylation of MYPT1 at Thr696 (MYPT1pThr696) controls the activity of MP on eNOSpThr497. Phosphatase inhibition suppresses both NO production and transendothelial resistance (TER) of ECs. In contrast, epigallocatechin-3-gallate (EGCG) signals ECs via the 67 kDa laminin-receptor (67LR) resulting in protein kinase A dependent activation of protein phosphatase-2A (PP2A). PP2A dephosphorylates MYPT1pThr696 and thereby stimulates MP activity inducing dephosphorylation of eNOSpThr497 and the 20 kDa myosin II light chains. Thus an interplay of MP and PP2A is involved in the physiological regulation of EC functions implying that an EGCG dependent activation of these phosphatases leads to enhanced NO production and EC barrier improvement.