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Abstract Background Chronic kidney disease (CKD) is a significant risk factor for pulmonary hypertension (PH), a complication that adversely affects patient prognosis. However, the mechanisms underlying this association remain poorly understood. A major obstacle to progress in this field is the lack of a reliable animal model replicating CKD-PH. Methods This study aimed to establish a stable rat model of CKD-PH. We employed a combined approach, inducing CKD through a 5/6 nephrectomy and concurrently exposing the rats to a high-salt diet. The model's hemodynamics were evaluated dynamically, alongside a comprehensive assessment of pathological changes in multiple organs. Lung tissues and serum samples were collected from the CKD-PH rats to analyze the expression of angiotensin-converting enzyme 2 (ACE2), evaluate the activity of key vascular components within the renin–angiotensin–aldosterone system (RAAS), and characterize alterations in the serum metabolic profile. Results At 14 weeks post-surgery, the CKD-PH rats displayed significant changes in hemodynamic parameters indicative of pulmonary arterial hypertension. Additionally, right ventricular hypertrophy was observed. Notably, no evidence of pulmonary vascular remodeling was found. Further analysis revealed RAAS dysregulation and downregulated ACE2 expression within the pulmonary vascular endothelium of CKD-PH rats. Moreover, the serum metabolic profile of these animals differed markedly from the sham surgery group. Conclusions Our findings suggest that the development of pulmonary arterial hypertension in CKD-PH rats is likely a consequence of a combined effect: RAAS dysregulation, decreased ACE2 expression in pulmonary vascular endothelial cells, and metabolic disturbances. Graphical Abstract

Abstract Delta‐like ligand 4 (DLL‐4) inhibitor drugs are an emerging cancer treatment. In clinical trials for solid organ malignancies, intravenous administration of monoclonal antibodies that inhibit DLL‐4 is associated with development of pulmonary hypertension, in the absence of left ventricular dysfunction. Analysis of 13 clinical trials showed that pulmonary hypertension is a complication of DLL‐4 inhibition.

Abstract Exposure to the spike protein or receptor-binding domain (S-RBD) of SARS-CoV-2 significantly influences endothelial cells and induces pulmonary vascular endotheliopathy. In this study, angiotensin-converting enzyme 2 humanized inbred (hACE2 Tg) mice and cultured pulmonary vascular endothelial cells were used to investigate how spike protein/S-RBD impacts pulmonary vascular endothelium. Results show that S-RBD leads to acute-to-prolonged induction of the intracellular free calcium concentration ([Ca2+]i) via acute activation of TRPV4, and prolonged upregulation of mechanosensitive channel Piezo1 and store-operated calcium channel (SOCC) key component Orai1 in cultured human pulmonary arterial endothelial cells (PAECs). In mechanism, S-RBD interacts with ACE2 to induce formation of clusters involving Orai1, Piezo1 and TRPC1, facilitate the channel activation of Piezo1 and SOCC, and lead to elevated apoptosis. These effects are blocked by Kobophenol A, which inhibits the binding between S-RBD and ACE2, or intracellular calcium chelator, BAPTA-AM. Blockade of Piezo1 and SOCC by GsMTx4 effectively protects the S-RBD-induced pulmonary microvascular endothelial damage in hACE2 Tg mice via normalizing the elevated [Ca2+]i. Comparing to prototypic strain, Omicron variants (BA.5.2 and XBB) of S-RBD induces significantly less severe cell apoptosis. Transcriptomic analysis indicates that prototypic S-RBD confers more severe acute impacts than Delta or Lambda S-RBD. In summary, this study provides compelling evidence that S-RBD could induce persistent pulmonary vascular endothelial damage by binding to ACE2 and triggering [Ca2+]i through upregulation of Piezo1 and Orai1. Targeted inhibition of ACE2-Piezo1/SOCC-[Ca2+]i axis proves a powerful strategy to treat S-RBD-induced pulmonary vascular diseases.

Idiopathic pulmonary arterial hypertension is a rare and fatal disease with a heterogeneous treatment response. Here the authors show that unsupervised machine learning of whole blood transcriptomes from 359 patients with idiopathic pulmonary arterial hypertension identifies 3 subgroups (endophenotypes) that improve risk stratification and provide new molecular insights.

Pharmacologic interventions to halt/reverse the vascular remodeling and right ventricular dysfunction in pulmonary arterial hypertension (PAH) remains an unmet need. We previously demonstrated extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a DAMP (damage‐associated molecular pattern protein) contributing to PAH pathobiology via TLR4 ligation. We examined the role of endothelial cell (EC)‐specific eNAMPT in experimental PH and an eNAMPT‐neutralizing mAb as a therapeutic strategy to reverse established PH. Hemodynamic/echocardiographic measurements and tissue analyses were performed in Sprague Dawley rats exposed to 10% hypoxia/Sugen (three weeks) followed by return to normoxia and weekly intraperitoneal delivery of the eNAMPT mAb (1 mg/kg). WT C57BL/6J mice and conditional EC‐cNAMPTec−/− mice were exposed to 10% hypoxia (three weeks). Biochemical and RNA sequencing studies were performed on rat PH lung tissues and human PAH PBMCs. Hypoxia/Sugen‐exposed rats exhibited multiple indices of severe PH (right ventricular systolic pressure, Fulton index), including severe vascular remodeling, compared to control rats. PH severity indices and plasma levels of eNAMPT, IL‐6, and TNF‐α were all significantly attenuated by eNAMPT mAb neutralization. Compared to hypoxia‐exposed WT mice, cNAMPTec−/− KO mice exhibited significantly reduced PH severity and evidence of EC to mesenchymal transition (EndMT). Finally, biochemical and RNAseq analyses revealed eNAMPT mAb‐mediated rectification of dysregulated inflammatory signaling pathways (TLR/NF‐κB, MAP kinase, Akt/mTOR) and EndMT in rat PH lung tissues and human PAH PBMCs. These studies underscore EC‐derived eNAMPT as a key contributor to PAH pathobiology and support the eNAMPT/TLR4 inflammatory pathway as a highly druggable therapeutic target to reduce PH severity and reverse PAH.

Pulmonary arterial hypertension is a progressive and fatal disease and rodents with experimental pulmonary hypertension (PH) are often used to study pathogenic mechanisms, identify therapeutic targets, and develop novel drugs for treatment. Here we describe a hands‐on set of experimental approaches including ex vivo lung angiography and histology and in vivo right heart catheterization (RHC) to phenotypically characterize pulmonary hemodynamics and lung vascular structure in normal mice and mice with experimental PH. We utilized Microfil polymer as contrast in our ex vivo lung angiogram to quantitatively examine pulmonary vascular remodeling in mice with experimental PH, and lung histology to estimate pulmonary artery wall thickness. The peripheral lung vascular images were selected to determine the total length of lung vascular branches, the number of branches and the number of junctions in a given area (mm−2). We found that the three parameters determined by angiogram were not significantly different among the apical, middle, and basal regions of the mouse lung from normal mice, and were not influenced by gender (no significant difference between female and male mice). We conducted RHC in mice to measure right ventricular systolic pressure, a surrogate measure for pulmonary artery systolic pressure and right ventricle (RV) contractility (RV ± dP/dtmax) to estimate RV function. RHC, a short time (4–6 min) procedure, did not alter the lung angiography measurements. In summary, utilizing ex vivo angiogram to determine peripheral vascular structure and density in the mouse lung and utilizing in vivo RHC to measure pulmonary hemodynamics are reliable readouts to phenotype normal mice and mice with experimental PH. Lung angiogram and RHC are also reliable approaches to examine pharmacological effects of new drugs on pulmonary vascular remodeling and hemodynamics.

Patients with diabetes with coronary microvascular disease (CMD) exhibit higher cardiac mortality than patients without CMD. However, the molecular mechanism by which diabetes promotes CMD is poorly understood. RNA-binding protein human antigen R (HuR) is a key regulator of mRNA stability and translation; therefore, we investigated the role of HuR in the development of CMD in mice with type 2 diabetes. Diabetic mice exhibited decreases in coronary flow velocity reserve (CFVR; a determinant of coronary microvascular function) and capillary density in the left ventricle. HuR levels in cardiac endothelial cells (CECs) were significantly lower in diabetic mice and patients with diabetes than the controls. Endothelial-specific HuR-KO mice also displayed significant reductions in CFVR and capillary density. By examining mRNA levels of 92 genes associated with endothelial function, we found that HuR, Cx40, and Nox4 levels were decreased in CECs from diabetic and HuR-KO mice compared with control mice. Cx40 expression and HuR binding to Cx40 mRNA were downregulated in CECs from diabetic mice. Cx40-KO mice exhibited decreased CFVR and capillary density, whereas endothelium-specific Cx40 overexpression increased capillary density and improved CFVR in diabetic mice. These data suggest that decreased HuR contributes to the development of CMD in diabetes through downregulation of gap junction protein Cx40 in CECs.

Excessive pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and migration are implicated in the development of pathogenic pulmonary vascular remodeling characterized by concentric arterial wall thickening and arteriole muscularization in patients with pulmonary arterial hypertension (PAH). Pulmonary artery smooth muscle cell contractile-to-proliferative phenotypical transition is a process that promotes pulmonary vascular remodeling. A rise in cytosolic Ca2+ concentration [(Ca2+)cyt] in PASMCs is a trigger for pulmonary vasoconstriction and a stimulus for pulmonary vascular remodeling. Here, we report that the calcium homeostasis modulator (CALHM), a Ca2+ (and ATP) channel that is allosterically regulated by voltage and extracellular Ca2+, is upregulated during the PASMC contractile-to-proliferative phenotypical transition. Protein expression of CALHM1/2 in primary cultured PASMCs in media containing serum and growth factors (proliferative PASMC) was significantly greater than in freshly isolated PA (contractile PASMC) from the same rat. Upregulated CALHM1/2 in proliferative PASMCs were associated with an increased ratio of pAKT/AKT and pmTOR/mTOR and an increased expression of the cell proliferation marker PCNA, whereas serum starvation and rapamycin significantly downregulated CALHM1/2. Furthermore, CALHM1/2 were upregulated in freshly isolated PA from rats with monocrotaline (MCT)-induced PH and in primary cultured PASMC from patients with PAH in comparison to normal controls. Intraperitoneal injection of CGP 37157 (0.6 mg/kg, q8H), a non-selective blocker of CALHM channels, partially reversed established experimental PH. These data suggest that CALHM upregulation is involved in PASMC contractile-to-proliferative phenotypical transition. Ca2+ influx through upregulated CALHM1/2 may play an important role in the transition of sustained vasoconstriction to excessive vascular remodeling in PAH or precapillary PH. Calcium homeostasis modulator could potentially be a target to develop novel therapies for PAH.

Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS), a refractory lung disease with an unacceptable high mortality rate. Paradoxically, mechanical ventilation also creates excessive mechanical stress that directly augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The specific mechanisms involved in VILI-induced pulmonary capillary leakage, a key pathologic feature of VILI are still far from resolved. The mechanoreceptor, transient receptor potential cation channel subfamily V member 4, TRPV4 plays a key role in the development of VILI through unresolved mechanism. Endothelial nitric oxide synthase (eNOS) uncoupling plays an important role in sepsis-mediated ARDS so in this study we investigated whether there is a role for eNOS uncoupling in the barrier disruption associated with TRPV4 activation during VILI. Our data indicate that the TRPV4 agonist, 4α-Phorbol 12,13-didecanoate (4αPDD) induces pulmonary arterial endothelial cell (EC) barrier disruption through the disruption of mitochondrial bioenergetics. Mechanistically, this occurs via the mitochondrial redistribution of uncoupled eNOS secondary to a PKC-dependent phosphorylation of eNOS at Threonine 495 (T495). A specific decoy peptide to prevent T495 phosphorylation reduced eNOS uncoupling and mitochondrial redistribution and preserved PAEC barrier function under 4αPDD challenge. Further, our eNOS decoy peptide was able to preserve lung vascular integrity in a mouse model of VILI. Thus, we have revealed a functional link between TRPV4 activation, PKC-dependent eNOS phosphorylation at T495, and EC barrier permeability. Reducing pT495-eNOS could be a new therapeutic approach for the prevention of VILI.

Background Abnormal endothelial function in the lungs is implicated in the development of pulmonary hypertension; however, there is little information about the difference of endothelial function between small distal pulmonary artery (PA) and large proximal PA and their contribution to the development of pulmonary hypertension. Herein, we investigate endothelium‐dependent relaxation in different orders of PAs and examine the molecular mechanisms by which chronic hypoxia attenuates endothelium‐dependent pulmonary vasodilation, leading to pulmonary hypertension. Methods and Results Endothelium‐dependent relaxation in large proximal PAs (second order) was primarily caused by releasing NO from the endothelium, whereas endothelium‐dependent hyperpolarization (EDH)–mediated vasodilation was prominent in small distal PAs (fourth–fifth order). Chronic hypoxia abolished EDH‐mediated relaxation in small distal PAs without affecting smooth muscle–dependent relaxation. RNA‐sequencing data revealed that, among genes related to EDH, the levels of Cx37, Cx40, Cx43, and IK were altered in mouse pulmonary endothelial cells isolated from chronically hypoxic mice in comparison to mouse pulmonary endothelial cells from normoxic control mice. The protein levels were significantly lower for connexin 40 (Cx40) and higher for connexin 37 in mouse pulmonary endothelial cells from hypoxic mice than normoxic mice. Cx40 knockout mice exhibited significant attenuation of EDH‐mediated relaxation and marked increase in right ventricular systolic pressure. Interestingly, chronic hypoxia led to a further increase in right ventricular systolic pressure in Cx40 knockout mice without altering EDH‐mediated relaxation. Furthermore, overexpression of Cx40 significantly decreased right ventricular systolic pressure in chronically hypoxic mice. Conclusions These data suggest that chronic hypoxia‐induced downregulation of endothelial Cx40 results in impaired EDH‐mediated relaxation in small distal PAs and contributes to the development of pulmonary hypertension.

Using RNAseq, we identified a 61 gene-based circulating transcriptomic profile most correlated with four indices of pulmonary arterial hypertension severity. In an independent dataset, 13/61 (21%) genes were differentially expressed in lung tissues of pulmonary arterial hypertension cases versus controls, highlighting potentially novel candidate genes involved in pulmonary arterial hypertension development.

Hypoxic Pulmonary Vasoconstriction (HPV) is an important physiological mechanism of the lungs that matches perfusion to ventilation thus maximizing O 2 saturation of the venous blood within the lungs. This study emphasizes on principal pathways in the initiation and modulation of hypoxic pulmonary vasoconstriction with a primary focus on the role of Ca 2+ signaling and Ca 2+ influx pathways in hypoxic pulmonary vasoconstriction. We used an ex vivo model, isolated perfused/ventilated mouse lung to evaluate hypoxic pulmonary vasoconstriction. Alveolar hypoxia (utilizing a mini ventilator) rapidly and reversibly increased pulmonary arterial pressure due to hypoxic pulmonary vasoconstriction in the isolated perfused/ventilated lung. By applying specific inhibitors for different membrane receptors and ion channels through intrapulmonary perfusion solution in isolated lung, we were able to define the targeted receptors and channels that regulate hypoxic pulmonary vasoconstriction. We show that extracellular Ca 2+ or Ca 2+ influx through various Ca 2+ -permeable channels in the plasma membrane is required for hypoxic pulmonary vasoconstriction. Removal of extracellular Ca 2+ abolished hypoxic pulmonary vasoconstriction, while blockade of L-type voltage-dependent Ca 2+ channels (with nifedipine), non-selective cation channels (with 30 µM SKF-96365), and TRPC6/TRPV1 channels (with 1 µM SAR-7334 and 30 µM capsazepine, respectively) significantly and reversibly inhibited hypoxic pulmonary vasoconstriction. Furthermore, blockers of Ca 2+ -sensing receptors (by 30 µM NPS2143, an allosteric Ca 2+ -sensing receptors inhibitor) and Notch (by 30 µM DAPT, a γ-secretase inhibitor) also attenuated hypoxic pulmonary vasoconstriction. These data indicate that Ca 2+ influx in pulmonary arterial smooth muscle cells through voltage-dependent, receptor-operated, and store-operated Ca 2+ entry pathways all contribute to initiation of hypoxic pulmonary vasoconstriction. The extracellular Ca 2+ -mediated activation of Ca 2+ -sensing receptors and the cell–cell interaction via Notch ligands and receptors contribute to the regulation of hypoxic pulmonary vasoconstriction.

Platelet-derived growth factor is one of the major growth factors found in human and mammalian serum and tissues. Abnormal activation of platelet-derived growth factor signaling pathway through platelet-derived growth factor receptors may contribute to the development and progression of pulmonary vascular remodeling and obliterative vascular lesions in patients with pulmonary arterial hypertension. In this study, we examined the expression of platelet-derived growth factor receptor isoforms in pulmonary arterial smooth muscle and pulmonary arterial endothelial cells and investigated whether platelet-derived growth factor secreted from pulmonary arterial smooth muscle cell or pulmonary arterial endothelial cell promotes pulmonary arterial smooth muscle cell proliferation. Our results showed that the protein expression of platelet-derived growth factor receptor α and platelet-derived growth factor receptor β in pulmonary arterial smooth muscle cell was upregulated in patients with idiopathic pulmonary arterial hypertension compared to normal subjects. Platelet-derived growth factor activated platelet-derived growth factor receptor α and platelet-derived growth factor receptor β in pulmonary arterial smooth muscle cell, as determined by phosphorylation of platelet-derived growth factor receptor α and platelet-derived growth factor receptor β. The platelet-derived growth factor-mediated activation of platelet-derived growth factor receptor α/platelet-derived growth factor receptor β was enhanced in idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell compared to normal cells. Expression level of platelet-derived growth factor-AA and platelet-derived growth factor-BB was greater in the conditioned media collected from idiopathic pulmonary arterial hypertension-pulmonary arterial endothelial cell than from normal pulmonary arterial endothelial cell. Furthermore, incubation of idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell with conditioned culture media from normal pulmonary arterial endothelial cell induced more platelet-derived growth factor receptor α activation than in normal pulmonary arterial smooth muscle cell. Accordingly, the conditioned media from idiopathic pulmonary arterial hypertension-pulmonary arterial endothelial cell resulted in more pulmonary arterial smooth muscle cell proliferation than the media from normal pulmonary arterial endothelial cell. These data indicate that (a) the expression and activity of platelet-derived growth factor receptor are increased in idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell compared to normal pulmonary arterial smooth muscle cell, and (b) pulmonary arterial endothelial cell from idiopathic pulmonary arterial hypertension patients secretes higher level of platelet-derived growth factor than pulmonary arterial endothelial cell from normal subjects. The enhanced secretion (and production) of platelet-derived growth factor from idiopathic pulmonary arterial hypertension-pulmonary arterial endothelial cell and upregulated platelet-derived growth factor receptor expression (and function) in idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell may contribute to enhancing platelet-derived growth factor/platelet-derived growth factor receptor-associated pulmonary vascular remodeling in pulmonary arterial hypertension.

The prevalent use of electronic cigarettes (e-cigarettes) has increased exponentially in recent years, especially in youth who are attracted to flavored e-cigarettes. Indeed, e-cigarette or vaping product use-associated lung injury (EVALI) cases started to emerge in the United States in August 2019, resulting in 2807 hospitalized cases and 68 deaths as of 18 February 2020. In the present study, we investigated, for the first time, whether flavored and nicotine containing e-cigarettes induce endothelial dysfunction to result in impaired angiogenesis and wound healing particularly under diabetic condition. Nicotine containing e-cigarettes with various contents of nicotine (0, 1.2%, 2.4%), and flavored e-cigarettes of classic tobacco, mint, menthol, and vanilla or fruit from BLU (nicotine 2.4%) or JUUL (nicotine 3%), were used to treat endothelial cells in vitro and streptozotocin-induced diabetic mice in vivo. Endothelial cell superoxide production, determined by dihydroethidium (DHE) fluorescent imaging and electron spin resonance (ESR), was markedly increased by exposure to e-cigarette extract (e-CSE) in a nicotine-content dependent manner, while nitric oxide (NO) bioavailability detected by DAF-FM fluorescent imaging was substantially decreased. All of the different flavored e-cigarettes examined also showed significant effects in increasing superoxide production while diminishing NO bioavailability. Endothelial cell apoptosis evaluated by caspase 3 activity was markedly increased by exposure to e-CSE prepared from flavored and nicotine containing e-cigarettes. Endothelial monolayer wound assays revealed that nicotine-containing and flavored e-cigarettes induced impaired angiogenic wound repair of endothelial cell monolayers. Furthermore, vascular endothelial growth factor (VEGF) stimulated wound healing in diabetic mice was impaired by exposure to e-CSEs prepared from nicotine-containing and flavored e-cigarettes. Taken together, our data demonstrate for the first time that flavored and nicotine-containing e-cigarettes induce endothelial dysfunction through excessive ROS production, resulting in decreased NO bioavailability, increased endothelial cell apoptosis, and impairment in angiogenesis and wound healing, especially under diabetic condition. These responses of endothelial dysfunction likely underlie harmful effects of e-cigarettes in endothelial-rich organs, such as heart and lungs. These data also indicate that rigorous regulation on e-cigarette use should be enforced in diabetic and/or surgical patients to avoid severe consequences from impaired angiogenesis/wound healing.

Summary: Concentric lung vascular wall thickening due to enhanced proliferation of pulmonary arterial smooth muscle cells is an important pathological cause for the elevated pulmonary vascular resistance reported in patients with pulmonary arterial hypertension. We identified a differential role of mammalian target of rapamycin (mTOR) complex 1 and complex 2, two functionally distinct mTOR complexes, in the development of pulmonary hypertension (PH). Inhibition of mTOR complex 1 attenuated the development of PH; however, inhibition of mTOR complex 2 caused spontaneous PH, potentially due to up-regulation of platelet-derived growth factor receptors in pulmonary arterial smooth muscle cells, and compromised the therapeutic effect of the mTOR inhibitors on PH. In addition, we describe a promising therapeutic strategy using combination treatment with the mTOR inhibitors and the platelet-derived growth factor receptor inhibitors on PH and right ventricular hypertrophy. The data from this study provide an important mechanism-based perspective for developing novel therapies for patients with pulmonary arterial hypertension and right heart failure. Key Words: mTOR, pulmonary hypertension, Raptor, Rictor, right ventricle

Right ventricular (RV) function strongly associates with mortality in patients with pulmonary arterial hypertension (PAH). Current methods to determine RV function require temporal measurements of pressure and volume. The aim of the study was to investigate the feasibility of using right heart catheterization (RHC) measurements to estimate systolic and diastolic RV function. RV pressure and volume points were fit to P = α(e βV -1) to assess diastolic stiffness coefficient (β) and end-diastolic elastance (Eed). Single-beat methods were used to assess RV contractility (Ees). The effects of a non-zero unstressed RV volume (V 0 ), RHC-derived stroke volume (SV RHC ), and normalization of the end-diastolic volume (EDV) on estimates of β, Eed, and Ees were tested using Bland–Altman analysis in an incident PAH cohort (n = 32) that had both a RHC and cardiac magnetic resonance (CMR) test. RHC-derived measures of RV function were used to detect the effect of prostacyclin therapy in an incident PAH cohort and the severity of PAH in prevalent PAH (n = 21). A non-zero V 0 had a minimal effect on β with a small bias and limits of agreement (LOA). Stroke volume (SV) significantly influenced estimates of β and Ees with a large LOA. Normalization of EDV had minimal effect on both β and Eed. RHC-derived β and Eed increased due to the severity of PAH and decreased due to three months of prostacyclin therapy. It is feasible to detect therapeutic changes in specific stiffness and elastic properties of the RV from signal-beat pressure-volume loops by using RHC-derived SV and normalizing RV EDV.

This is the first study reporting on the design and development innovative inhaled formulations of the novel natural product antioxidant therapeutic, tetramethylpyrazine (TMP), also known as ligustrazine. TMP is obtained from Chinese herbs belonging to the class of Ligusticum. It is known to have antioxidant properties. It can act as a Nrf2/ARE activator and a Rho/ROCK inhibitor. The present study reports for the first time on the comprehensive characterization of raw TMP (non-spray dried) and spray dried TMP in a systematic manner using thermal analysis, electron microscopy, optical microscopy, and Raman spectroscopy. The in vitro aerosol dispersion performance of spray dried TMP was tested using three different FDA-approved unit-dose capsule-based human dry powder inhaler devices. In vitro human cellular studies were conducted on pulmonary cells from different regions of the human lung to examine the biocompatibility and non-cytotoxicity of TMP. Furthermore, the efficacy of inhaled TMP as both liquid and dry powder inhalation aerosols was tested in vivo using the monocrotaline (MCT)-induced PH rat model.

Pulmonary Arterial Hypertension (PAH) is a fatal disorder with limited treatment options and reduced life expectancy after diagnosis. Complex genetic backgrounds in PAH complicates identification of causative mutations that is essential for an understanding of the disease diagnostics and etiology especially for idiopathic PAH (iPAH). Hemolysis has been implicated as contributing to the pathobiology of PAH. Glucose-6-Phosphate Dehydrogenase (G6PD) expression and activity define erythrocyte's antioxidant capacity, and its decrease contributes to erythrocyte fragility. As G6PD deficiency was previously reported in a limited number of PAH cases, we tested whether iPAH patients exhibit underlying G6PD alterations in erythrocytes. A cohort of 22 PAH patients and 8 non-PAH patients were recruited for this study. DNA isolated from Peripheral Blood Mononuclear Cells (PBMC) was used for detection of mutations in the coding region of the G6PD gene. RNA isolated from PBMC was used for determination of G6PD mRNA expression level. G6PD activity was measured in Red Blood Cell (RBC) pellets. Three patients had missense mutations in G6PD (Val291Met, Asn126Asp, Asp194Glu), however, only one mutation (Val291Met) results in a severe G6PD deficiency. A single patient with mutation (Asn126Asp) showed a 21% decrease in G6PD activity, two subjects showed G6PD deficiency without mutations, and one patient had a decreased level of G6PD mRNA and reduced enzyme levels. This study demonstrates that a moderate decrease in G6PD activity is associated with PAH. Screening for G6PD activity and mutations in the G6PD gene may provide early detection of individuals predisposed to PAH.

Endothelial dysfunction is a major player in the development and progression of vascular pathology in pulmonary arterial hypertension (PAH), a disease associated with small vessel loss and obstructive vasculopathy that leads to increased pulmonary vascular resistance, subsequent right heart failure, and premature death. Over the past ten years, there has been tremendous progress in our understanding of pulmonary endothelial biology as it pertains to the genetic and molecular mechanisms that orchestrate the endothelial response to direct or indirect injury, and how their dysregulation can contribute to the pathogenesis of PAH. As one of the major topics included in the 2017 Grover Conference Series, discussion centered on recent developments in four areas of pulmonary endothelial biology: (1) angiogenesis; (2) endothelial-mesenchymal transition (EndMT); (3) epigenetics; and (4) biology of voltage-gated ion channels. The present review will summarize the content of these discussions and provide a perspective on the most promising aspects of endothelial dysfunction that may be amenable for therapeutic development.

Two pairs of enantiomers, (−) and (+)-securidanes A (1 and 2) and B (3 and 4) featuring unprecedented triarylmethane (TAM) skeletons, were isolated from Securidaca inappendiculata. Their structures were established by spectroscopic data, X-ray crystallography, and CD analysis. A plausible biosynthetic pathway for 1−4 based on the co-isolated precursors was proposed. Bioinspired total synthesis of 1−4 was completed in high yield, which in turn corroborated the biosynthetic hypothesis. Compounds 1−4 showed good inhibition against protein tyrosine phosphatase 1B (PTP1B). The molecular docking demonstrated that the strongest inhibitor 3 (IC50 = 7.52 μM) reaches deeper into the binding pocket and has an additional H-bond.

Abstract Elevated pulmonary arterial pressure in patients with pulmonary hypertension (PH) is mainly caused by increased pulmonary vascular resistance (PVR), due primarily to sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling. According to the current classification, PH has been classified into five categories based on etiology (Simonneau et al, 2013). Among them, group 1 or pulmonary arterial hypertension (PAH) is a rare but progressive and deadly disease affecting ~1–10 per 1 million people. Despite expanding treatment options to ameliorate patients' symptoms, PAH remains a devastating disease with a poor long‐term prognosis.

BackgroundPreviously we found that smooth muscle cell (SMC)‐specific knockout of miR‐17~92 attenuates hypoxia‐induced pulmonary hypertension. However, the mechanism underlying miR‐17~92‐mediated pulmonary artery SMC (PASMC) proliferation remains unclear. We sought to investigate whether miR‐17~92 regulates hypoxia‐inducible factor (HIF) activity and PASMC proliferation via prolyl hydroxylases (PHDs). Methods and ResultsWe show that hypoxic sm‐17~92−/− mice have decreased hematocrit, red blood cell counts, and hemoglobin contents. The sm‐17~92−/− mouse lungs express decreased mRNA levels of HIF targets and increased levels of PHD2. miR‐17~92 inhibitors suppress hypoxia‐induced levels of HIF1α, VEGF, Glut1, HK2, and PDK1 but not HIF2α in vitro in PASMC. Overexpression of miR‐17 in PASMC represses PHD2 expression, whereas miR‐17/20a inhibitors induce PHD2 expression. The 3′‐UTR of PHD2 contains a functional miR‐17/20a seed sequence. Silencing of PHD2 induces HIF1α and PCNA protein levels, whereas overexpression of PHD2 decreases HIF1α and cell proliferation. SMC‐specific knockout of PHD2 enhances hypoxia‐induced vascular remodeling and exacerbates established pulmonary hypertension in mice. PHD2 activator R59949 reverses vessel remodeling in existing hypertensive mice. PHDs are dysregulated in PASMC isolated from pulmonary arterial hypertension patients. ConclusionsOur results suggest that PHD2 is a direct target of miR‐17/20a and that miR‐17~92 contributes to PASMC proliferation and polycythemia by suppression of PHD2 and induction of HIF1α.

AbstractPulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary vascular resistance (PVR) leading to right heart failure and premature death. The increased PVR results in part from pulmonary vascular remodeling and sustained pulmonary vasoconstriction. Excessive pulmonary vascular remodeling stems from increased pulmonary arterial smooth muscle cell (PASMC) proliferation and decreased PASMC apoptosis. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) in PASMC is a major trigger for pulmonary vasoconstriction and a key stimulus for PASMC proliferation and migration, both contributing to the development of pulmonary vascular remodeling. PASMC from patients with idiopathic PAH (IPAH) have increased resting [Ca2+]cyt and enhanced Ca2+ influx. Enhanced Ca2+ entry into PASMC due to upregulation of membrane receptors and/or Ca2+ channels may contribute to PASMC contraction and proliferation and to pulmonary vasoconstriction and pulmonary vascular remodeling. We have shown that the extracellular Ca2+-sensing receptor (CaSR), which is a member of G protein-coupled receptor (GPCR) subfamily C, is upregulated and the extracellular Ca2+-induced increase in [Ca2+]cyt is enhanced in PASMC from patients with IPAH in comparison to PASMC from normal subjects. Pharmacologically blockade of CaSR significantly attenuate the development and progression of experimental pulmonary hypertension in animals. Additionally, we have demonstrated that dihydropyridine Ca2+ channel blockers (e.g., nifedipine), which are used to treat PAH patients but are only effective in 15-20% of patients, activate CaSR resulting in an increase in [Ca2+]cyt in IPAH-PASMC, but not normal PASMC. Our data indicate that CaSR functionally couples with transient receptor potential canonical (TRPC) channels to mediate extracellular Ca2+-induced Ca2+ influx and increase in [Ca2+]cyt in IPAH-PASMC. Upregulated CaSR is necessary for the enhanced extracellular Ca2+-induced increase in [Ca2+]cyt and the augmented proliferation of PASMC in patients with IPAH. This review will highlight the pathogenic role of CaSR in the development and progression of PAH.