Portosystemic shunting prevents hepatocellular carcinoma in non-alcoholic fatty liver disease mouse models.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is one of the leading cause of hepatocellular carcinoma (HCC). This association is supported by the translocation of bacteria products into the portal system, which acts on the liver through the gut-liver axis. We hypothesize that portosystemic shunting can disrupt this relationship, and prevent NAFLD-associated HCC. METHODS: HCC carcinogenesis was tested in C57BL/6 mice fed a high-fat high-sucrose diet (HFD) and injected with diethylnitrosamine (DEN) at two weeks of age, and in double transgenic LAP-tTA and TRE-MYC (LAP-Myc) mice fed a methionine-choline-deficient diet. Portosystemic shunts were established by transposing the spleen to the sub-cutaneous tissue at eight weeks of age. RESULTS: Spleen transposition led to a consistent deviation of part of the portal flow and a significant decrease in portal pressure. It was associated with a decrease in the number of HCC in both models. This effect was supported by the presence of less severe liver steatosis after 40 weeks, and lower expression levels of liver fatty acid synthase. Also, shunted mice exhibited lower liver oxygen levels, a key factor in preventing HCC as confirmed by the development of less HCCs in mice with hepatic artery ligation. CONCLUSIONS: The present data show that portosystemic shunting prevents NAFLD-associated HCC, utilizing two independent mouse models. This effect is supported by the development of less steatosis, and a restored liver oxygen level. Portal pressure modulation and shunting deserve further exploration as potential prevention/treatment options for NAFLD and HCC.

Lymphatic Connexins and Pannexins in Health and Disease.

This review highlights current knowledge on the expression and function of connexins and pannexins, transmembrane channel proteins that play an important role in intercellular communication, in both the developing and mature lymphatic vasculature. A particular focus is given to the involvement of these proteins in functions of the healthy lymphatic system. We describe their influence on the maintenance of extracellular fluid homeostasis, immune cell trafficking to draining lymph nodes and dietary nutrient absorption by intestinal villi. Moreover, new insights into connexin mutations in primary and secondary lymphedema as well as on the implication of lymphatic connexins and pannexins in acquired cardiovascular diseases are discussed, allowing for a better understanding of the role of these proteins in pathologies linked to dysfunctions in the lymphatic system.

In vivo miR-138-5p inhibition alleviates monocrotaline-induced pulmonary hypertension and normalizes pulmonary KCNK3 and SLC45A3 expression.

BACKGROUND: The pathogenesis of pulmonary arterial hypertension (PAH) involves many signalling pathways. MicroRNAs are potential candidates involved in simultaneously coordinating multiple genes under such multifactorial conditions. METHODS AND RESULTS: MiR-138-5p is overexpressed in pulmonary arterial smooth muscle cells (PASMCs) from PAH patients and in lungs from rats with monocrotaline-induced pulmonary hypertension (MCT-PH). MiR-138-5p is predicted to regulate the expression of the potassium channel KCNK3, whose loss is associated with the development and progression of PAH. We hypothesized that, in vivo, miR-138-5p inhibition would restore KCNK3 lung expression and subsequently alleviate PAH. Nebulization-based delivery of anti-miR-138-5p to rats with established MCT-PH significantly reduced the right ventricular systolic pressure and significantly improved the pulmonary arterial acceleration time (PAAT). These haemodynamic improvements were related to decrease pulmonary vascular remodelling, lung inflammation and pulmonary vascular cell proliferation in situ. In vivo inhibition of miR-138-5p restored KCNK3 mRNA expression and SLC45A3 protein expression in the lungs. CONCLUSIONS: We confirmed that in vivo inhibition of miR-138-5p reduces the development of PH in experimental MCT-PH. The possible curative mechanisms involve at least the normalization of lung KCNK3 as well as SLC45A3 expression.

Comparison of Human and Experimental Pulmonary Veno-Occlusive Disease.

Pulmonary veno-occlusive disease (PVOD) occurs in humans either as a heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2) or as a sporadic form in older age (sPVOD). The chemotherapeutic agent mitomycin C (MMC) is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here, we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular, and molecular levels to unravel common altered pathomechanisms. MMC exposure in rats was associated primarily with arterial and microvessel remodeling, and secondarily by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there was convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of HO-1 (heme oxygenase 1) and CHOP (CCAAT-enhancer-binding protein [C/EBP] homologous protein), two downstream effectors of GCN2 signaling and endoplasmic reticulum stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 (EIF2AK4 mutations carriers and Eif2ak4-/- rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells using pharmacological and siRNA approaches demonstrated that GCN2 loss of function negatively regulates BMP (bone morphogenetic protein)-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced proliferation of pulmonary artery endothelial cells. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9, as potential therapeutic options for PVOD.

Selective inhibition of Panx1 channels decreases hemostasis and thrombosis in vivo.

BACKGROUND: Hemostasis is a tightly regulated physiological process to rapidly induce hemostatic plugs at sites of vascular injury. Inappropriate activation of this process may lead to thrombosis, i.e. pathological blood clot formation in uninjured vessels or on atherosclerotic lesions. ATP release through Pannexin1 (Panx1) membrane channels contributes to collagen-induced platelet aggregation in vitro. OBJECTIVE: To investigate the effects of genetic and pharmacological inhibition of Panx1 on hemostasis and thrombosis in vivo. RESULTS: Bleeding time after tail clipping was increased by 2.5-fold in Panx1-/- mice compared to wild-type controls, suggesting that Panx1 deficiency impairs primary hemostasis. Wire myography on mesenteric arteries revealed diminished vasoconstriction in response to phenylephrine or U446619 in Panx1-/- mice. Mice with platelet-specific deletion of Panx1 (Panx1PDel) displayed 2-fold longer tail bleeding times than Panx1fl/fl controls. Moreover, venous thromboembolism (VTE) after injection of collagen/epinephrine in the jugular vein was reduced in Panx1-/- and Panx1PDel mice. Panx1PDel mice also showed reduced FeCl3-induced thrombosis in mesenteric arteries. BrilliantBlue-FCF, a Panx1 channel inhibitor, decreased collagen-induced platelet aggregation in vitro, increased tail bleeding time and reduced VTE in wild-type mice. Furthermore, we developed a specific Panx1 blocking antibody targeting a Panx1 extracellular loop, which reduced ATP release from platelets in vitro. Treating wild-type mice with this antibody increased tail bleeding time and decreased VTE compared to control antibody. CONCLUSIONS: Panx1 channel deletion or inhibition diminishes clot formation during hemostasis and thrombosis in vivo. Blocking Panx1 channels may be an attractive strategy for modulating platelet aggregation in thrombotic disease.

Characterization of Kcnk3-Mutated Rat, a Novel Model of Pulmonary Hypertension.

RATIONALE: Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of function mutations in KCNK3 (potassium channel subfamily K member 3) gene, which encodes an outward rectifier K+ channel, have been identified in pulmonary arterial hypertension patients. OBJECTIVE: We have demonstrated that KCNK3 dysfunction is common to heritable and nonheritable pulmonary arterial hypertension and to experimental pulmonary hypertension (PH). Finally, KCNK3 is not functional in mouse pulmonary vasculature. METHODS AND RESULTS: Using CRISPR/Cas9 technology, we generated a 94 bp out of frame deletion in exon 1 of Kcnk3 gene and characterized these rats at the electrophysiological, echocardiographic, hemodynamic, morphological, cellular, and molecular levels to decipher the cellular mechanisms associated with loss of KCNK3. Using patch-clamp technique, we validated our transgenic strategy by demonstrating the absence of KCNK3 current in freshly isolated pulmonary arterial smooth muscle cells from Kcnk3-mutated rats. At 4 months of age, echocardiographic parameters revealed shortening of the pulmonary artery acceleration time associated with elevation of the right ventricular systolic pressure. Kcnk3-mutated rats developed more severe PH than wild-type rats after monocrotaline exposure or chronic hypoxia exposure. Kcnk3-mutation induced a lung distal neomuscularization and perivascular extracellular matrix activation. Lungs of Kcnk3-mutated rats were characterized by overactivation of ERK1/2 (extracellular signal-regulated kinase1-/2), AKT (protein kinase B), SRC, and overexpression of HIF1-α (hypoxia-inducible factor-1 α), survivin, and VWF (Von Willebrand factor). Linked with plasma membrane depolarization, reduced endothelial-NOS expression and desensitization of endothelial-derived hyperpolarizing factor, Kcnk3-mutated rats presented predisposition to vasoconstriction of pulmonary arteries and a severe loss of sildenafil-induced pulmonary arteries relaxation. Moreover, we showed strong alteration of right ventricular cardiomyocyte excitability. Finally, Kcnk3-mutated rats developed age-dependent PH associated with low serum-albumin concentration. CONCLUSIONS: We established the first Kcnk3-mutated rat model of PH. Our results confirm that KCNK3 loss of function is a key event in pulmonary arterial hypertension pathogenesis. This model presents new opportunities for understanding the initiating mechanisms of PH and testing biologically relevant therapeutic molecules in the context of PH.

KLF4-Induced Connexin40 Expression Contributes to Arterial Endothelial Quiescence.

Shear stress, a blood flow-induced frictional force, is essential in the control of endothelial cell (EC) homeostasis. High laminar shear stress (HLSS), as observed in straight parts of arteries, assures a quiescent non-activated endothelium through the induction of Krüppel-like transcription factors (KLFs). Connexin40 (Cx40)-mediated gap junctional communication is known to contribute to a healthy endothelium by propagating anti-inflammatory signals between ECs, however, the molecular basis of the transcriptional regulation of Cx40 as well as its downstream effectors remain poorly understood. Here, we show that flow-induced KLF4 regulated Cx40 expression in a mouse EC line. Chromatin immunoprecipitation in ECs revealed that KLF4 bound to three predicted KLF consensus binding sites in the Cx40 promoter. HLSS-dependent induction of Cx40 expression was confirmed in primary human ECs. The downstream effects of Cx40 modulation in ECs exposed to HLSS were elucidated by an unbiased transcriptomics approach. Cell cycle progression was identified as an important downstream target of Cx40 under HLSS. In agreement, an increase in the proportion of proliferating cell nuclear antigen (PCNA)-positive ECs and a decrease in the proportion of ECs in the G0/G1 phase were observed under HLSS after Cx40 silencing. Transfection of communication-incompetent HeLa cells with Cx40 demonstrated that the regulation of proliferation by Cx40 was not limited to ECs. Using a zebrafish model, we finally showed faster intersegmental vessel growth and branching into the dorsal longitudinal anastomotic vessel in embryos knock-out for the Cx40 orthologs Cx41.8 and Cx45.6. Most significant effects were observed in embryos with a mutant Cx41.8 encoding for a channel with reduced gap junctional function. Faster intersegmental vessel growth in Cx41.8 mutant embryos was associated with increased EC proliferation as assessed by PH3 immunostaining. Our data shows a novel evolutionary-conserved role of flow-driven KLF4-dependent Cx40 expression in endothelial quiescence that may be relevant for the control of atherosclerosis and diseases involving sprouting angiogenesis.

Bmpr2 Mutant Rats Develop Pulmonary and Cardiac Characteristics of Pulmonary Arterial Hypertension.

BACKGROUND: Monoallelic mutations in the gene encoding bone morphogenetic protein receptor 2 ( Bmpr2) are the main genetic risk factor for heritable pulmonary arterial hypertension (PAH) with incomplete penetrance. Several Bmpr2 transgenic mice have been reported to develop mild spontaneous PAH. In this study, we examined whether rats with the Bmpr2 mutation were susceptible to developing more severe PAH. METHODS: The zinc finger nuclease method was used to establish rat lines with mutations in the Bmpr2 gene. These rats were then characterized at the hemodynamic, histological, electrophysiological, and molecular levels. RESULTS: Rats with a monoallelic deletion of 71 bp in exon 1 (Δ 71 rats) showed decreased BMPRII expression and phosphorylated SMAD1/5/9 levels. Δ 71 Rats develop age-dependent spontaneous PAH with a low penetrance (16%-27%), similar to that in humans. Δ 71 Rats were more susceptible to hypoxia-induced pulmonary hypertension than wild-type rats. Δ 71 Rats exhibited progressive pulmonary vascular remodeling associated with a proproliferative phenotype and showed lower pulmonary microvascular density than wild-type rats. Organ bath studies revealed severe alteration of pulmonary artery contraction and relaxation associated with potassium channel subfamily K member 3 (KCNK3) dysfunction. High levels of perivascular fibrillar collagen and pulmonary interleukin-6 overexpression discriminated rats that developed spontaneous PAH and rats that did not develop spontaneous PAH. Finally, detailed assessments of cardiomyocytes demonstrated alterations in morphology, calcium (Ca2+), and cell contractility specific to the right ventricle; these changes could explain the lower cardiac output of Δ 71 rats. Indeed, adult right ventricular cardiomyocytes from Δ 71 rats exhibited a smaller diameter, decreased sensitivity of sarcomeres to Ca2+, decreased [Ca2+] transient amplitude, reduced sarcoplasmic reticulum Ca2+ content, and short action potential duration compared with right ventricular cardiomyocytes from wild-type rats. CONCLUSIONS: We characterized the first Bmpr2 mutant rats and showed some of the critical cellular and molecular dysfunctions described in human PAH. We also identified the heart as an unexpected but potential target organ of Bmpr2 mutations. Thus, this new genetic rat model represents a promising tool to study the pathogenesis of PAH.

Endothelial connexins in vascular function.

Gap junctions are essential for intercellular crosstalk in blood and lymphatic vasculature. These clusters of intercellular channels ensure direct communication among endothelial cells and between endothelial and smooth muscle cells, and the synchronization of their behavior along the vascular tree. Gap junction channels are formed by connexins; six connexins form a connexon or hemichannel and the docking of two connexons result in a full gap junction channel allowing for the exchange of ions and small metabolites between neighboring cells. Recent evidence indicates that the intracellular domains of connexins may also function as an interaction platform (interactome) for other proteins, thereby regulating their function. Interestingly, fragments of Cx proteins generated by alternative internal translation were recently described, although their functions in the vascular wall remain to be uncovered. Variations in connexin expression are observed along different types of blood and lymphatic vessels; the most commonly found endothelial connexins are Cx37, Cx40, Cx43 and Cx47. Physiological studies on connexin-knockout mice demonstrated the essential roles of these channel-forming proteins in the coordination of vasomotor activity, endothelial permeability and inflammation, angiogenesis and in the maintenance of fluid balance in the body.

Loss of KCNK3 is a hallmark of RV hypertrophy/dysfunction associated with pulmonary hypertension.

Aims: Mutations in the KCNK3 gene, which encodes for an outward-rectifier K+ channel, have been identified in patients suffering from pulmonary arterial hypertension (PAH), and constitute the first described channelopathy in PAH. In human PAH and experimental pulmonary hypertension (PH), we demonstrated that KCNK3 expression and function are severely reduced in pulmonary vascular cells, promoting PH-like phenotype at the morphologic and haemodynamic levels. Since KCNK3 channel is also expressed in both the human and rodent heart, we aimed to elucidate the pathophysiological role of KCNK3 channel in right ventricular (RV) hypertrophy (RVH) related to PH. Methods and results: Using whole-cell Patch-clamp technique, we demonstrated that KCNK3 is predominantly expressed in adult rat RV cardiomyocytes compared to the left ventricle cardiomyocytes and participates in the repolarizing phase of the RV action potential. We revealed a reduction in KCNK3 function prior to development of RVH and the rise of pulmonary vascular resistance. KCNK3 function is severely reduced in RV cardiomyocytes during the development of RVH in several rat models of PH (exposure to monocrotaline, chronic hypoxia, and Sugen/hypoxia) and chronic RV pressure overload (pulmonary artery banding). In experimental PH, we revealed a reduction in KCNK3 function before any rise in pulmonary vascular resistance and the development of RVH. KCNK3 mRNA level is also reduced in human RV tissues from PAH patients compared to non-PAH patients. In line with these findings, chronic inhibition of KCNK3 in rats with the specific inhibitor (A293) induces RV hypertrophy which is associated with the re-expression of foetal genes, RV fibrosis, RV inflammation, and subsequent loss of RV performance as assessed by echocardiography. Conclusion: Our data indicate that loss of KCNK3 function and expression is a hallmark of the RV hypertrophy/dysfunction associated with PH.

Pulmonary endothelial cell DNA methylation signature in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a severe and incurable pulmonary vascular disease. One of the primary origins of PAH is pulmonary endothelial dysfunction leading to vasoconstriction, aberrant angiogenesis and smooth muscle cell proliferation, endothelial-to-mesenchymal transition, thrombosis and inflammation. Our objective was to study the epigenetic variations in pulmonary endothelial cells (PEC) through a specific pattern of DNA methylation. DNA was extracted from cultured PEC from idiopathic PAH (n = 11), heritable PAH (n = 10) and controls (n = 18). DNA methylation was assessed using the Illumina HumanMethylation450 Assay. After normalization, samples and probes were clustered according to their methylation profile. Differential clusters were functionally analyzed using bioinformatics tools. Unsupervised hierarchical clustering allowed the identification of two clusters of probes that discriminates controls and PAH patients. Among 147 differential methylated promoters, 46 promoters coding for proteins or miRNAs were related to lipid metabolism. Top 10 up and down-regulated genes were involved in lipid transport including ABCA1, ABCB4, ADIPOQ, miR-26A, BCL2L11. NextBio meta-analysis suggested a contribution of ABCA1 in PAH. We confirmed ABCA1 mRNA and protein downregulation specifically in PAH PEC by qPCR and immunohistochemistry and made the proof-of-concept in an experimental model of the disease that its targeting may offer novel therapeutic options. In conclusion, DNA methylation analysis identifies a set of genes mainly involved in lipid transport pathway which could be relevant to PAH pathophysiology.

BMPRII influences the response of pulmonary microvascular endothelial cells to inflammatory mediators.

Mutations in the bone morphogenetic protein receptor (BMPR2) gene have been observed in 70 % of patients with heritable pulmonary arterial hypertension (HPAH) and in 11-40 % with idiopathic PAH (IPAH). However, carriers of a BMPR2 mutation have only 20 % risk of developing PAH. Since inflammatory mediators are increased and predict survival in PAH, they could act as a second hit inducing the development of pulmonary hypertension in BMPR2 mutation carriers. Our specific aim was to determine whether inflammatory mediators could contribute to pulmonary vascular cell dysfunction in PAH patients with and without a BMPR2 mutation. Pulmonary microvascular endothelial cells (PMEC) and arterial smooth muscle cells (PASMC) were isolated from lung parenchyma of transplanted PAH patients, carriers of a BMPR2 mutation or not, and from lobectomy patients or lung donors. The effects of CRP and TNFα on mitogenic activity, adhesiveness capacity, and expression of adhesion molecules were investigated in PMECs and PASMCs. PMECs from BMPR2 mutation carriers induced an increase in PASMC mitogenic activity; moreover, endothelin-1 secretion by PMECs from carriers was higher than by PMECs from non-carriers. Recruitment of monocytes by PMECs isolated from carriers was higher compared to PMECs from non-carriers and from controls, with an elevated ICAM-1 expression. CRP increased adhesion of monocytes to PMECs in carriers and non-carriers, and TNFα only in carriers. PMEC from BMPR2 mutation carriers have enhanced adhesiveness for monocytes in response to inflammatory mediators, suggesting that BMPR2 mutation could generate susceptibility to an inflammatory insult in PAH.

Role for Runt-related Transcription Factor 2 in Proliferative and Calcified Vascular Lesions in Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). This is sustained in time by the down-regulation of microRNA (miR)-204. In systemic vascular diseases, reduced miR-204 expression promotes vascular biomineralization by augmenting the expression of the transcription factor Runt-related transcription factor 2 (RUNX2). Implication of RUNX2 in PAH-related vascular remodeling and presence of calcified lesions in PAH remain unexplored. OBJECTIVES: We hypothesized that RUNX2 is up-regulated in lungs of patients with PAH, contributing to vascular remodeling and calcium-related biomineralization. METHODS: We harvested human lung tissues in which we assessed calcification lesions and RUNX2 expression. We also isolated PASMCs from these tissues for in vitro analyses. Using a bidirectional approach, we investigated the role for RUNX2 in cell proliferation, apoptosis, and calcification capacity. Ectopic delivery of small interfering RNA against RUNX2 was used in an animal model of PAH to evaluate the therapeutic potential of RUNX2 inhibition in this disease. MEASUREMENTS AND MAIN RESULTS: Patients with PAH display features of calcified lesions within the distal pulmonary arteries (PAs). We show that RUNX2 is up-regulated in lungs, distal PAs, and primary cultured human PASMCs isolated from PAH and compared with patients without PAH. RUNX2 expression histologically correlates with vascular remodeling and calcification. Using in vitro gain- and loss-of-function approaches, we mechanistically demonstrate that miR-204 diminution promotes RUNX2 up-regulation and that sustained RUNX2 expression activates hypoxia-inducible factor-1α, leading to aberrant proliferation, resistance to apoptosis, and subsequent transdifferentiation of PAH-PASMCs into osteoblast-like cells. In the PAH Sugen/hypoxia rat model, molecular RUNX2 inhibition reduces PA remodeling and prevents calcification, thus improving pulmonary hemodynamic parameters and right ventricular function. CONCLUSIONS: RUNX2 plays a pivotal role in the pathogenesis of PAH, contributing to the development of proliferative and calcified PA lesions. Inhibition of RUNX2 may therefore represent an attractive therapeutic strategy for PAH.

Potassium Channel Subfamily K Member 3 (KCNK3) Contributes to the Development of Pulmonary Arterial Hypertension.

BACKGROUND: Mutations in the KCNK3 gene have been identified in some patients suffering from heritable pulmonary arterial hypertension (PAH). KCNK3 encodes an outward rectifier K(+) channel, and each identified mutation leads to a loss of function. However, the pathophysiological role of potassium channel subfamily K member 3 (KCNK3) in PAH is unclear. We hypothesized that loss of function of KCNK3 is a hallmark of idiopathic and heritable PAH and contributes to dysfunction of pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, leading to pulmonary artery remodeling: consequently, restoring KCNK3 function could alleviate experimental pulmonary hypertension (PH). METHODS AND RESULTS: We demonstrated that KCNK3 expression and function were reduced in human PAH and in monocrotaline-induced PH in rats. Using a patch-clamp technique in freshly isolated (not cultured) pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, we found that KCNK3 current decreased progressively during the development of monocrotaline-induced PH and correlated with plasma-membrane depolarization. We demonstrated that KCNK3 modulated pulmonary arterial tone. Long-term inhibition of KCNK3 in rats induced distal neomuscularization and early hemodynamic signs of PH, which were related to exaggerated proliferation of pulmonary artery endothelial cells, pulmonary artery smooth muscle cell, adventitial fibroblasts, and pulmonary and systemic inflammation. Lastly, in vivo pharmacological activation of KCNK3 significantly reversed monocrotaline-induced PH in rats. CONCLUSIONS: In PAH and experimental PH, KCNK3 expression and activity are strongly reduced in pulmonary artery smooth muscle cells and endothelial cells. KCNK3 inhibition promoted increased proliferation, vasoconstriction, and inflammation. In vivo pharmacological activation of KCNK3 alleviated monocrotaline-induced PH, thus demonstrating that loss of KCNK3 is a key event in PAH pathogenesis and thus could be therapeutically targeted.

Mitomycin-Induced Pulmonary Veno-Occlusive Disease: Evidence From Human Disease and Animal Models.

BACKGROUND: Pulmonary veno-occlusive disease (PVOD) is an uncommon form of pulmonary hypertension characterized by the obstruction of small pulmonary veins and a dismal prognosis. PVOD may be sporadic or heritable because of biallelic mutations of the EIF2AK4 gene coding for GCN2. Isolated case reports suggest that chemotherapy may be a risk factor for PVOD. METHODS AND RESULTS: We reported on the clinical, functional, and hemodynamic characteristics and outcomes of 7 cases of PVOD induced by mitomycin-C (MMC) therapy from the French Pulmonary Hypertension Registry. All patients displayed squamous anal cancer and were treated with MMC alone or MMC plus 5-fluoruracil. The estimated annual incidence of PVOD in the French population that have anal cancer is 3.9 of 1000 patients, which is much higher than the incidence of PVOD in the general population (0.5/million per year). In rats, intraperitoneal administration of MMC induced PVOD, as demonstrated by pulmonary hypertension at right-heart catheterization at days 21 to 35 and major remodeling of small pulmonary veins associated with foci of intense microvascular endothelial-cell proliferation of the capillary bed. In rats, MMC administration was associated with dose-dependent depletion of pulmonary GCN2 content and decreased smad1/5/8 signaling. Amifostine prevented the development of MMC-induced PVOD in rats. CONCLUSIONS: MMC therapy is a potent inducer of PVOD in humans and rats. Amifostine prevents MMC-induced PVOD in rats and should be tested as a preventive therapy for MMC-induced PVOD in humans. MMC-induced PVOD in rats represents a unique model to test novel therapies in this devastating orphan disease.

Endothelial-to-mesenchymal transition in pulmonary hypertension.

BACKGROUND: The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of α-smooth muscle actin-expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-smooth muscle actin-expressing cells. METHODS AND RESULTS: In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2(Δ140Ex1/+) rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable. CONCLUSIONS: EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications.

T-helper 17 cell polarization in pulmonary arterial hypertension.

BACKGROUND: Inflammation may contribute to the pathobiology of pulmonary arterial hypertension (PAH). Deciphering the PAH fingerprint on the inflammation orchestrated by dendritic cells (DCs) and T cells, key driver and effector cells, respectively, of the immune system, may allow the identification of immunopathologic approaches to PAH management. METHODS: Using flow cytometry, we performed immunophenotyping of monocyte-derived DCs (MoDCs) and circulating lymphocytes from patients with idiopathic PAH and control subjects. With the same technique, we performed cytokine profiling of both populations following stimulation, coculture, or both. We tested the immunomodulatory effects of a glucocorticoid (dexamethasone [Dex]) on this immunophenotype and cytokine profile. Using an epigenetic approach, we confirmed the immune polarization in blood DNA of patients with PAH. RESULTS: The profile of membrane costimulatory molecules of PAH MoDCs was similar to that of control subjects. However, PAH MoDCs retained higher levels of the T-cell activating molecules CD86 and CD40 after Dex pretreatment than did control MoDCs. This was associated with an increased expression of IL-12p40 and a reduced migration toward chemokine (C-C motif) ligand 21. Moreover, both with and without Dex, PAH MoDCs induced a higher activation and proliferation of CD4+ T cells, associated with a reduced expression of IL-4 (T helper 2 response) and a higher expression of IL-17 (T helper 17 response). Purified PAH CD4+ T cells expressed a higher level of IL-17 after activation than did those of control subjects. Lastly, there was significant hypomethylation of the IL-17 promoter in the PAH blood DNA as compared with the control blood. CONCLUSIONS: We have highlighted T helper 17 cell immune polarization in patients with PAH, as has been previously demonstrated in other chronic inflammatory and autoimmune conditions.