PDZ-Binding Kinase, a Novel Regulator of Vascular Remodeling in Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is high blood pressure in the lungs that originates from structural changes in small resistance arteries. A defining feature of PAH is the inappropriate remodeling of pulmonary arteries (PA) leading to right ventricle failure and death. Although treatment of PAH has improved, the long-term prognosis for patients remains poor, and more effective targets are needed. METHODS: Gene expression was analyzed by microarray, RNA sequencing, quantitative polymerase chain reaction, Western blotting, and immunostaining of lung and isolated PA in multiple mouse and rat models of pulmonary hypertension (PH) and human PAH. PH was assessed by digital ultrasound, hemodynamic measurements, and morphometry. RESULTS: Microarray analysis of the transcriptome of hypertensive rat PA identified a novel candidate, PBK (PDZ-binding kinase), that was upregulated in multiple models and species including humans. PBK is a serine/threonine kinase with important roles in cell proliferation that is minimally expressed in normal tissues but significantly increased in highly proliferative tissues. PBK was robustly upregulated in the medial layer of PA, where it overlaps with markers of smooth muscle cells. Gain-of-function approaches show that active forms of PBK increase PA smooth muscle cell proliferation, whereas silencing PBK, dominant negative PBK, and pharmacological inhibitors of PBK all reduce proliferation. Pharmacological inhibitors of PBK were effective in PH reversal strategies in both mouse and rat models, providing translational significance. In a complementary genetic approach, PBK was knocked out in rats using CRISPR/Cas9 editing, and loss of PBK prevented the development of PH. We found that PBK bound to PRC1 (protein regulator of cytokinesis 1) in PA smooth muscle cells and that multiple genes involved in cytokinesis were upregulated in experimental models of PH and human PAH. Active PBK increased PRC1 phosphorylation and supported cytokinesis in PA smooth muscle cells, whereas silencing or dominant negative PBK reduced cytokinesis and the number of cells in the G2/M phase of the cell cycle. CONCLUSIONS: PBK is a newly described target for PAH that is upregulated in proliferating PA smooth muscle cells, where it contributes to proliferation through changes in cytokinesis and cell cycle dynamics to promote medial thickening, fibrosis, increased PA resistance, elevated right ventricular systolic pressure, right ventricular remodeling, and PH.

Purine synthesis suppression reduces the development and progression of pulmonary hypertension in rodent models.

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.

Calpain Promotes LPS-induced Lung Endothelial Barrier Dysfunction via Cleavage of Talin.

Acute lung injury (ALI) is characterized by lung vascular endothelial cell (EC) barrier compromise resulting in increased endothelial permeability and pulmonary edema. The infection of gram-negative bacteria that produce toxins like LPS is one of the major causes of ALI. LPS activates Toll-like receptor 4, leading to cytoskeleton reorganization, resulting in lung endothelial barrier disruption and pulmonary edema in ALI. However, the signaling pathways that lead to the cytoskeleton reorganization and lung microvascular EC barrier disruption remain largely unexplored. Here we show that LPS induces calpain activation and talin cleavage into head and rod domains and that inhibition of calpain attenuates talin cleavage, RhoA activation, and pulmonary EC barrier disruption in LPS-treated human lung microvascular ECs in vitro and lung EC barrier disruption and pulmonary edema induced by LPS in ALI in vivo. Moreover, overexpression of calpain causes talin cleavage and RhoA activation, myosin light chain (MLC) phosphorylation, and increases in actin stress fiber formation. Furthermore, knockdown of talin attenuates LPS-induced RhoA activation and MLC phosphorylation and increased stress fiber formation and mitigates LPS-induced lung microvascular endothelial barrier disruption. Additionally, overexpression of talin head and rod domains increases RhoA activation, MLC phosphorylation, and stress fiber formation and enhances lung endothelial barrier disruption. Finally, overexpression of cleavage-resistant talin mutant reduces LPS-induced increases in MLC phosphorylation in human lung microvascular ECs and attenuates LPS-induced lung microvascular endothelial barrier disruption. These results provide the first evidence that calpain mediates LPS-induced lung microvascular endothelial barrier disruption in ALI via cleavage of talin.

Endothelin-1 acutely increases nitric oxide production via the calcineurin mediated dephosphorylation of Caveolin-1.

Endothelin (ET)-1 is an endothelial-derived peptide that exerts biphasic effects on nitric oxide (NO) levels in endothelial cells such that acute exposure stimulates-while sustained exposure attenuates-NO production. Although the mechanism involved in the decrease in NO generation has been identified but the signaling involved in the acute increase in NO is still unresolved. This was the focus of this study. Our data indicate that exposing pulmonary arterial endothelial cells (PAEC) to ET-1 led to an increase in NO for up to 30min after which levels declined. These effects were attenuated by ET receptor antagonists. The increase in NO correlated with significant increases in pp60Src activity and increases in eNOS phosphorylation at Tyr83 and Ser1177. The ET-1 mediated increase in phosphorylation and NO generation were attenuated by the over-expression of a pp60Src dominant negative mutant. The increase in pp60Src activity correlated with a reduction in the interaction of Caveolin-1 with pp60Src and the calcineurin-mediated dephosphorylation of caveolin-1 at three previously unidentified sites: Thr91, Thr93, and Thr95. The calcineurin inhibitor, Tacrolimus, attenuated the acute increase in pp60Src activity induced by ET-1 and a calcineurin siRNA attenuated the ET-1 mediated increase in eNOS phosphorylation at Tyr83 and Ser1177 as well as the increase in NO. By using a Caveolin-1 celluSpot peptide array, we identified a peptide targeting a sequence located between aa 41-56 as the pp60Src binding region. This peptide fused to the TAT sequence was found to decrease caveolin-pp60Src interaction, increased pp60Src activity, increased eNOS pSer1177 and NO levels in PAEC and induce vasodilation in isolated aortic rings in wildtype but not eNOS knockout mice. Together, our data identify a novel mechanism by which ET-1 acutely increases NO via a calcineurin-mediated dephosphorylation of caveolin-1 and the subsequent stimulation of pp60Src activity, leading to increases in phosphorylation of eNOS at Tyr83 and Ser1177.

PFKFB3-mediated endothelial glycolysis promotes pulmonary hypertension.

Increased glycolysis in the lung vasculature has been connected to the development of pulmonary hypertension (PH). We therefore investigated whether glycolytic regulator 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFKFB3)-mediated endothelial glycolysis plays a critical role in the development of PH. Heterozygous global deficiency of Pfkfb3 protected mice from developing hypoxia-induced PH, and administration of the PFKFB3 inhibitor 3PO almost completely prevented PH in rats treated with Sugen 5416/hypoxia, indicating a causative role of PFKFB3 in the development of PH. Immunostaining of lung sections and Western blot with isolated lung endothelial cells showed a dramatic increase in PFKFB3 expression and activity in pulmonary endothelial cells of rodents and humans with PH. We generated mice that were constitutively or inducibly deficient in endothelial Pfkfb3 and found that these mice were incapable of developing PH or showed slowed PH progression. Compared with control mice, endothelial Pfkfb3-knockout mice exhibited less severity of vascular smooth muscle cell proliferation, endothelial inflammation, and leukocyte recruitment in the lungs. In the absence of PFKFB3, lung endothelial cells from rodents and humans with PH produced lower levels of growth factors (such as PDGFB and FGF2) and proinflammatory factors (such as CXCL12 and IL1ß). This is mechanistically linked to decreased levels of HIF2A in lung ECs following PFKFB3 knockdown. Taken together, these results suggest that targeting PFKFB3 is a promising strategy for the treatment of PH.

Galectin-3 Promotes ROS, Inflammation, and Vascular Fibrosis in Pulmonary Arterial Hypertension.

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.

PFKFB3 in Smooth Muscle Promotes Vascular Remodeling in Pulmonary Arterial Hypertension.

Rationale: Glycolytic shift is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). It remains unknown how glycolysis is increased and how increased glycolysis contributes to pulmonary vascular remodeling in PAH.Objectives: To determine whether increased glycolysis is caused by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and how PFKFB3-driven glycolysis induces vascular remodeling in PAH.Methods: PFKFB3 levels were measured in pulmonary arteries of patients and animals with PAH. Lactate levels were assessed in lungs of animals with PAH and in pulmonary artery smooth muscle cells (PASMCs). Genetic and pharmacologic approaches were used to investigate the role of PFKFB3 in PAH.Measurements and Main Results: Lactate production was elevated in lungs of PAH rodents and in platelet-derived growth factor-treated PASMCs. PFKFB3 protein was higher in pulmonary arteries of patients and rodents with PAH, in PASMCs of patients with PAH, and in platelet-derived growth factor-treated PASMCs. PFKFB3 inhibition by genetic disruption and chemical inhibitor attenuated phosphorylation/activation of extracellular signal-regulated kinase (ERK1/2) and calpain-2, and vascular remodeling in PAH rodent models, and reduced platelet-derived growth factor-induced phosphorylation/activation of ERK1/2 and calpain-2, collagen synthesis and proliferation of PASMCs. ERK1/2 inhibition attenuated phosphorylation/activation of calpain-2, and vascular remodeling in Sugen/hypoxia PAH rats, and reduced lactate-induced phosphorylation/activation of calpain-2, collagen synthesis, and proliferation of PASMCs. Calpain-2 inhibition reduced lactate-induced collagen synthesis and proliferation of PASMCs.Conclusions: Upregulated PFKFB3 mediates collagen synthesis and proliferation of PASMCs, contributing to vascular remodeling in PAH. The mechanism is through the elevation of glycolysis and lactate that results in the activation of calpain by ERK1/2-dependent phosphorylation of calpain-2.

Galectin-3 is expressed in vascular smooth muscle cells and promotes pulmonary hypertension through changes in proliferation, apoptosis, and fibrosis.

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.

Adventitial Fibroblast Nox4 Expression and ROS Signaling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease arising from remodeling and narrowing of pulmonary arteries (PA) resulting in high pulmonary arterial blood pressure and ultimately right ventricular failure. Elevated production of reactive oxygen species (ROS) by NADPH oxidase 4 (Nox4), a constitutively active enzyme, has been associated with oxygen sensing, vasomotor control, cellular proliferation, differentiation, migration, apoptosis, senescence, fibrosis, and angiogenesis. Further, elevated expression of Nox4 has been reported in a number of cardiovascular diseases, including atherosclerosis, hypertension, cardiac failure, ischemic stroke, and PAH. However, the cellular location of Nox4 and its contribution to aberrant vascular remodeling in PAH remains poorly understood. The goal of this review is to summarize the recent literature on the enzymatic regulation of Nox4 in the production of ROS in PAH. In the vascular wall, Nox4 is present in fibroblasts, a primary cell of the adventitia, and matches the adventitial location of ROS production in PAH. Further, in adventitial fibroblasts, Nox4 overexpression stimulates migration and proliferation as well as matrix gene expression. Collectively, reports indicate that Nox4 contributes to altered fibroblast behavior, ROS production leading to hypertensive vascular remodeling and the development of PAH. Finally, we address the functional significance of Nox4 in fibroblasts, and also suggest an "outside in" (adventitial) process of vascular remodeling that is mediated by Nox4, which although has physiological roles in the intimal layer (i.e., endothelium), may also have pathologic importance in the adventitial layer of the vascular wall through signaling in fibroblasts.

NADPH oxidase 4 is expressed in pulmonary artery adventitia and contributes to hypertensive vascular remodeling.

OBJECTIVE: Pulmonary hypertension (PH) is a progressive disease arising from remodeling and narrowing of pulmonary arteries (PAs) resulting in high pulmonary blood pressure and ultimately right ventricular failure. Elevated production of reactive oxygen species by NADPH oxidase 4 (Nox4) is associated with increased pressure in PH. However, the cellular location of Nox4 and its contribution to aberrant vascular remodeling in PH remains poorly understood. Therefore, we sought to identify the vascular cells expressing Nox4 in PAs and determine the functional relevance of Nox4 in PH. APPROACH AND RESULTS: Elevated expression of Nox4 was detected in hypertensive PAs from 3 rat PH models and human PH using qualititative real-time reverse transcription polymerase chain reaction, Western blot, and immunofluorescence. In the vascular wall, Nox4 was detected in both endothelium and adventitia, and perivascular staining was prominently increased in hypertensive lung sections, colocalizing with cells expressing fibroblast and monocyte markers and matching the adventitial location of reactive oxygen species production. Small-molecule inhibitors of Nox4 reduced adventitial reactive oxygen species generation and vascular remodeling as well as ameliorating right ventricular hypertrophy and noninvasive indices of PA stiffness in monocrotaline-treated rats as determined by morphometric analysis and high-resolution digital ultrasound. Nox4 inhibitors improved PH in both prevention and reversal protocols and reduced the expression of fibroblast markers in isolated PAs. In fibroblasts, Nox4 overexpression stimulated migration and proliferation and was necessary for matrix gene expression. CONCLUSION: These findings indicate that Nox4 is prominently expressed in the adventitia and contributes to altered fibroblast behavior, hypertensive vascular remodeling, and development of PH.

A Reappraisal of the Utility of L-012 to Measure Superoxide from Biologically Relevant Sources.

The detection of superoxide anion (O2●-) in biological tissues remains challenging. Barriers to convenient and reproducible measurements include expensive equipment, custom probes, and the need for high sensitivity and specificity. The luminol derivative, L-012, has been used to measure O2●- since 1993 with mixed results and concerns over specificity. The goal of this study was to better define the conditions for use and their specificity. We found that L-012 coupled with depolymerized orthovanadate, a relatively impermeable tyrosine phosphatase inhibitor, yielded a highly sensitive approach to detect extracellular O2●-. In O2●- producing HEK-NOX5 cells, orthovanadate increased L-012 luminescence 100-fold. The combination of L-012 and orthovanadate was highly sensitive, stable, scalable, completely reversed by superoxide dismutase, and selective for O2●- generating NOXes versus NOX4, which produces H2O2. Moreover, there was no signal from cells transfected with NOS3 (NO●) and NOS2(ONOO-). To exclude the effects of altered tyrosine phosphorylation, O2●- was detected using non-enzymatic synthesis with phenazine methosulfate and via novel coupling of L-012 with niobium oxalate, which was less active in inducing tyrosine phosphorylation. Overall, our data shows that L-012 coupled with orthovanadate or other periodic group 5 salts yields a reliable, sensitive, and specific approach to measuring extracellular O2●- in biological systems.

Expression and functional significance of NADPH oxidase 5 (Nox5) and its splice variants in human blood vessels.

The expression and functional significance of NADPH oxidase 5 (Nox5) and its five isoforms in vascular cells is poorly understood. The goal of this study was to determine whether Nox5-α, -ß, -δ, -γ, and -ε (short) are expressed in human blood vessels and evaluate their respective functions. Nox5 mRNA and protein were detected in human blood vessels, cultured human vascular smooth muscle (HVSMC) and endothelium, but not fibroblasts. The most abundant isoforms were α and ß, whereas δ and γ were not detected. Nox5-α and -ß produced reactive oxygen species (ROS), but -δ, -γ, and -ε were not catalytically active. Coexpression of the active Nox5 isoforms with inactive Nox5 variants suppressed ROS production, and coimmunoprecipitation revealed that Nox5-ß binds the inactive ε variant, which may account for reduced ROS production. In HVSMC, angiotensin II, endothelin-1 and TNF-α increased endogenous Nox5 mRNA levels, while adenovirus-mediated overexpression of Nox5 promoted p38 MAPK, JAK2, JNK, and ERK1/2 phosphorylation in endothelial cells (EC), but only increased ERK1/2 phosphorylation in HVSMC. At higher levels of Nox5, there was evidence of increased apoptosis in EC, but not in HVSMC, as detected by the presence of cleaved caspase-3 and cleaved poly(ADP-ribose)polymerase. Although catalytically inactive, Nox5-ε potently activated ERK in HVSMC, and increased expression of Nox5-ε promoted HVSMC proliferation. Nox5 is expressed in human blood vessels. The Nox5-α and -ß splice variants are the major isoforms that are expressed and the only variants capable of ROS production. Nox5-ε can inhibit Nox5 activity and activate ERK and HVSMC proliferation.

Protective role of Cav-1 in pneumolysin-induced endothelial barrier dysfunction.

Pneumolysin (PLY) is a bacterial pore forming toxin and primary virulence factor of Streptococcus pneumonia, a major cause of pneumonia. PLY binds cholesterol-rich domains of the endothelial cell (EC) plasma membrane resulting in pore assembly and increased intracellular (IC) Ca2+ levels that compromise endothelial barrier integrity. Caveolae are specialized plasmalemma microdomains of ECs enriched in cholesterol. We hypothesized that the abundance of cholesterol-rich domains in EC plasma membranes confers cellular susceptibility to PLY. Contrary to this hypothesis, we found increased PLY-induced IC Ca2+ following membrane cholesterol depletion. Caveolin-1 (Cav-1) is an essential structural protein of caveolae and its regulation by cholesterol levels suggested a possible role in EC barrier function. Indeed, Cav-1 and its scaffolding domain peptide protected the endothelial barrier from PLY-induced disruption. In loss of function experiments, Cav-1 was knocked-out using CRISPR-Cas9 or silenced in human lung microvascular ECs. Loss of Cav-1 significantly enhanced the ability of PLY to disrupt endothelial barrier integrity. Rescue experiments with re-expression of Cav-1 or its scaffolding domain peptide protected the EC barrier against PLY-induced barrier disruption. Dynamin-2 (DNM2) is known to regulate caveolar membrane endocytosis. Inhibition of endocytosis, with dynamin inhibitors or siDNM2 amplified PLY induced EC barrier dysfunction. These results suggest that Cav-1 protects the endothelial barrier against PLY by promoting endocytosis of damaged membrane, thus reducing calcium entry and PLY-dependent signaling.

Activation of G protein-coupled estrogen receptor induces endothelium-independent relaxation of coronary artery smooth muscle.

Estrogens can either relax or contract arteries via rapid, nongenomic mechanisms involving classic estrogen receptors (ER). In addition to ERα and ERß, estrogen may also stimulate G protein-coupled estrogen receptor 1 (GPER) in nonvascular tissue; however, a potential role for GPER in coronary arteries is unclear. The purpose of this study was to determine how GPER activity influenced coronary artery reactivity. In vitro isometric force recordings were performed on endothelium-denuded porcine arteries. These studies were augmented by RT-PCR and single-cell patch-clamp experiments. RT-PCR and immunoblot studies confirmed expression of GPER mRNA and protein, respectively, in smooth muscle from either porcine or human coronary arteries. G-1, a selective GPER agonist, produced a concentration-dependent relaxation of endothelium-denuded porcine coronary arteries in vitro. This response was attenuated by G15, a GPER-selective antagonist, or by inhibiting large-conductance calcium-activated potassium (BK(Ca)) channels with iberiotoxin, but not by inhibiting NO signaling. Last, single-channel patch-clamp studies demonstrated that G-1 stimulates BK(Ca) channel activity in intact smooth muscle cells from either porcine or human coronary arteries but had no effect on channels isolated in excised membrane patches. In summary, GPER activation relaxes coronary artery smooth muscle by increasing potassium efflux via BK(Ca) channels and requires an intact cellular signaling mechanism. This novel action of estrogen-like compounds may help clarify some of the controversy surrounding the vascular effects of estrogens.

Mice with a specific deficiency of Pfkfb3 in myeloid cells are protected from hypoxia-induced pulmonary hypertension.

BACKGROUND AND PURPOSE: Macrophage infiltration into the lungs is a characteristic of pulmonary hypertension (PH). Glycolysis is the main metabolic pathway for macrophage activation. However, the effect of macrophage glycolysis on the development of PH remains unknown. We investigated the effect of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKBF3), a critical enzyme of macrophage glycolysis, on PH development. EXPERIMENTAL APPROACH: Lung tissues from PH patients were examined by immunostaining with macrophage markers. PH was induced in Wistar rats with SU5416/hypoxia and in mice with hypoxia. Lungs and macrophages were isolated for analysis by RT-PCR, western blot, flow cytometry, and immunostaining. KEY RESULTS: Expression of glycolytic molecules was increased in circulating peripheral blood mononuclear cells (PBMCs) and lung macrophages of PH patients. These results were also found in lung macrophages of SU5416/hypoxia (Su/Hx)-induced PH rats and hypoxia-induced PH mice. PH was ameliorated in myeloid-specific Pfkfb3-deficient mice (Pfkfb3ΔMϕ ) or mice treated with the PFKFB3 inhibitor 3PO, compared with their controls. Alveolar macrophages of PH Pfkfb3ΔMϕ mice produced lower levels of growth factors and pro-inflammatory cytokines than those of control mice. Circulating myeloid cells and lung myeloid cells were much fewer in PH Pfkfb3ΔMϕ mice than controls. Mechanistically, overexpression of Hif1a or Hif2a in bone marrow-derived macrophages (BMDMs) cultured with bone marrow of Pfkfb3ΔMϕ mice restored the decreased expression of pro-inflammatory cytokines and growth factors. CONCLUSIONS AND IMPLICATIONS: Myeloid Pfkfb3 deficiency protects mice from PH, thereby suggesting that myeloid PFKFB3 is one of the important targets in the therapeutic effect of PFKFB3 inhibition in PH treatment.

Mutation of protein kinase C phosphorylation site S1076 on alpha-subunits affects BK(Ca) channel activity in HEK-293 cells.

Large conductance, calcium- and voltage-activated potassium (BK(Ca)) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BK(Ca) channels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BK(Ca) channel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BK(Ca) channel alpha-subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BK(Ca) channels. Results showed that mutating S1076 altered the effect of PKC activation on BK(Ca) channels in HEK-293 cells. Specifically, the phospho-deficient mutation BK(Ca)-alpha(S1076A)/beta(1) attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BK(Ca) channels, whereas the phospho-mimetic mutation BK(Ca)-alpha(S1076E)/beta(1) increased the excitatory effect of PMA on BK(Ca) channels. In addition, the phospho-null mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BK(Ca) channels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BK(Ca) channel alpha-subunits influences BK(Ca) channel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.

Essential role of the 90-kilodalton heat shock protein in mediating nongenomic estrogen signaling in coronary artery smooth muscle.

Under normal physiological conditions, estrogen is a coronary vasodilator, and this response involves production of NO from endothelial cells. In addition, estrogen also stimulates NO production in coronary artery smooth muscle (CASM); however, the molecular basis for this nongenomic effect of estrogen is unclear. The purpose of this study was to investigate a potential role for the 90-kDa heat shock protein (Hsp90) in estrogen-stimulated neuronal nitric-oxide synthase (nNOS) activity in coronary artery smooth muscle. 17Beta-estradiol produced a concentration-dependent relaxation of endothelium-denuded porcine coronary arteries in vitro, and this response was attenuated by inhibiting Hsp90 function with 1 microM geldanamycin (GA) or 100 microg/ml radicicol (RAD). These inhibitors also prevented estrogen-stimulated NO production in human CASM cells and reversed the stimulatory effect of estrogen on calcium-activated potassium (BK(Ca)) channels. These functional studies indicated a role for Hsp90 in coupling estrogen receptor activation to NOS stimulation in CASM. Furthermore, coimmunoprecipitation studies demonstrated that estrogen stimulates bimolecular interaction of immunoprecipitated nNOS with Hsp90 and that either GA or RAD could inhibit this association. Blocking estrogen receptors with ICI182780 (fulvestrant) also prevented this association. These findings indicate an essential role for Hsp90 in nongenomic estrogen signaling in CASM and further suggest that Hsp90 might represent a prospective therapeutic target to enhance estrogen-stimulated cardiovascular protection.

Galectin-3: A Harbinger of Reactive Oxygen Species, Fibrosis, and Inflammation in Pulmonary Arterial Hypertension.

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.