Derivation and characterisation of endothelial cells from patients with chronic thromboembolic pulmonary hypertension.

Pulmonary endarterectomy (PEA) resected material offers a unique opportunity to develop an in vitro endothelial cell model of chronic thromboembolic pulmonary hypertension (CTEPH). We aimed to comprehensively analyze the endothelial function, molecular signature, and mitochondrial profile of CTEPH-derived endothelial cells to better understand the pathophysiological mechanisms of endothelial dysfunction behind CTEPH, and to identify potential novel targets for the prevention and treatment of the disease. Isolated cells from specimens obtained at PEA (CTEPH-EC), were characterized based on morphology, phenotype, and functional analyses (in vitro and in vivo tubule formation, proliferation, apoptosis, and migration). Mitochondrial content, morphology, and dynamics, as well as high-resolution respirometry and oxidative stress, were also studied. CTEPH-EC displayed a hyperproliferative phenotype with an increase expression of adhesion molecules and a decreased apoptosis, eNOS activity, migration capacity and reduced angiogenic capacity in vitro and in vivo compared to healthy endothelial cells. CTEPH-EC presented altered mitochondrial dynamics, increased mitochondrial respiration and an unbalanced production of reactive oxygen species and antioxidants. Our study is the foremost comprehensive investigation of CTEPH-EC. Modulation of redox, mitochondrial homeostasis and adhesion molecule overexpression arise as novel targets and biomarkers in CTEPH.

The Inflammatory Profile of CTEPH-Derived Endothelial Cells Is a Possible Driver of Disease Progression.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a form of pulmonary hypertension characterized by the presence of fibrotic intraluminal thrombi and causing obliteration of the pulmonary arteries. Although both endothelial cell (EC) dysfunction and inflammation are linked to CTEPH pathogenesis, regulation of the basal inflammatory response of ECs in CTEPH is not fully understood. Therefore, in the present study, we investigated the role of the nuclear factor (NF)-κB pro-inflammatory signaling pathway in ECs in CTEPH under basal conditions. Basal mRNA levels of interleukin (IL)-8, IL-1ß, monocyte chemoattractant protein-1 (MCP-1), C-C motif chemokine ligand 5 (CCL5), and vascular cell adhesion molecule-1 (VCAM-1) were upregulated in CTEPH-ECs compared to the control cells. To assess the involvement of NF-κB signaling in basal inflammatory activation, CTEPH-ECs were incubated with the NF-κB inhibitor Bay 11-7085. The increase in pro-inflammatory cytokines was abolished when cells were incubated with the NF-κB inhibitor. To determine if NF-κB was indeed activated, we stained pulmonary endarterectomy (PEA) specimens from CTEPH patients and ECs isolated from PEA specimens for phospho-NF-κB-P65 and found that especially the vessels within the thrombus and CTEPH-ECs are positive for phospho-NF-κB-P65. In summary, we show that CTEPH-ECs have a pro-inflammatory status under basal conditions, and blocking NF-κB signaling reduces the production of inflammatory factors in CTEPH-ECs. Therefore, our results show that the increased basal pro-inflammatory status of CTEPH-ECs is, at least partially, regulated through activation of NF-κB signaling and potentially contributes to the pathophysiology and progression of CTEPH.

Protein network analyses of pulmonary endothelial cells in chronic thromboembolic pulmonary hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a vascular disease characterized by the presence of organized thromboembolic material in pulmonary arteries leading to increased vascular resistance, heart failure and death. Dysfunction of endothelial cells is involved in CTEPH. The present study describes for the first time the molecular processes underlying endothelial dysfunction in the development of the CTEPH. The advanced analytical approach and the protein network analyses of patient derived CTEPH endothelial cells allowed the quantitation of 3258 proteins. The 673 differentially regulated proteins were associated with functional and disease protein network modules. The protein network analyses resulted in the characterization of dysregulated pathways associated with endothelial dysfunction, such as mitochondrial dysfunction, oxidative phosphorylation, sirtuin signaling, inflammatory response, oxidative stress and fatty acid metabolism related pathways. In addition, the quantification of advanced oxidation protein products, total protein carbonyl content, and intracellular reactive oxygen species resulted increased attesting the dysregulation of oxidative stress response. In conclusion this is the first quantitative study to highlight the involvement of endothelial dysfunction in CTEPH using patient samples and by network medicine approach.

Endothelial Dysfunction in Pulmonary Hypertension: Cause or Consequence?

Pulmonary arterial hypertension (PAH) is a rare, complex, and progressive disease that is characterized by the abnormal remodeling of the pulmonary arteries that leads to right ventricular failure and death. Although our understanding of the causes for abnormal vascular remodeling in PAH is limited, accumulating evidence indicates that endothelial cell (EC) dysfunction is one of the first triggers initiating this process. EC dysfunction leads to the activation of several cellular signalling pathways in the endothelium, resulting in the uncontrolled proliferation of ECs, pulmonary artery smooth muscle cells, and fibroblasts, and eventually leads to vascular remodelling and the occlusion of the pulmonary blood vessels. Other factors that are related to EC dysfunction in PAH are an increase in endothelial to mesenchymal transition, inflammation, apoptosis, and thrombus formation. In this review, we outline the latest advances on the role of EC dysfunction in PAH and other forms of pulmonary hypertension. We also elaborate on the molecular signals that orchestrate EC dysfunction in PAH. Understanding the role and mechanisms of EC dysfunction will unravel the therapeutic potential of targeting this process in PAH.

Therapeutic effects of soluble guanylate cyclase stimulation on pulmonary hemodynamics and emphysema development in guinea pigs chronically exposed to cigarette smoke.

We have analyzed the effect of the soluble guanylate cyclase (sGC) stimulator BAY 41-2272 in a therapeutic intervention in guinea pigs chronically exposed to cigarette smoke (CS). The effects of sGC stimulation on respiratory function, pulmonary hemodynamics, airspace size, vessel remodeling, and inflammatory cell recruitment to the lungs were evaluated in animals that had been exposed to CS for 3 mo. CS exposure was continued for an additional 3 mo in half of the animals and withdrawn in the other half. Animals that stopped CS exposure had slightly lower pulmonary artery pressure (PAP) and right ventricle (RV) hypertrophy than those who continued CS exposure, but they did not recover from the emphysema and the inflammatory cell infiltrate. Conversely, oral BAY 41-2272 administration stopped progression or even reversed the CS-induced emphysema in both current and former smokers, respectively. Furthermore, BAY 41-2272 produced a reduction in the RV hypertrophy, which correlated with a decrease in the PAP values. By contrast, the degree of vessel remodeling induced by CS remained unchanged in the treated animals. Functional network analysis suggested perforin/granzyme pathway downregulation as an action mechanism capable of stopping the progression of emphysema after sGC stimulation. The pathway analysis also showed normalization of the expression of cGMP-dependent serine/kinases. In conclusion, in guinea pigs chronically exposed to CS, sGC stimulation exerts beneficial effects on the lung parenchyma and the pulmonary vasculature, suggesting that sGC stimulators might be a potential alternative for chronic obstructive pulmonary disease treatment that deserves further evaluation.

Metabolic Alterations in Cardiopulmonary Vascular Dysfunction.

Cardiovascular diseases (CVD) are the leading cause of death worldwide. CVD comprise a range of diseases affecting the functionality of the heart and blood vessels, including acute myocardial infarction (AMI) and pulmonary hypertension (PH). Despite their different causative mechanisms, both AMI and PH involve narrowed or blocked blood vessels, hypoxia, and tissue infarction. The endothelium plays a pivotal role in the development of CVD. Disruption of the normal homeostasis of endothelia, alterations in the blood vessel structure, and abnormal functionality are essential factors in the onset and progression of both AMI and PH. An emerging theory proposes that pathological blood vessel responses and endothelial dysfunction develop as a result of an abnormal endothelial metabolism. It has been suggested that, in CVD, endothelial cell metabolism switches to higher glycolysis, rather than oxidative phosphorylation, as the main source of ATP, a process designated as the Warburg effect. The evidence of these alterations suggests that understanding endothelial metabolism and mitochondrial function may be central to unveiling fundamental mechanisms underlying cardiovascular pathogenesis and to identifying novel critical metabolic biomarkers and therapeutic targets. Here, we review the role of the endothelium in the regulation of vascular homeostasis and we detail key aspects of endothelial cell metabolism. We also describe recent findings concerning metabolic endothelial cell alterations in acute myocardial infarction and pulmonary hypertension, their relationship with disease pathogenesis and we discuss the future potential of pharmacological modulation of cellular metabolism in the treatment of cardiopulmonary vascular dysfunction. Although targeting endothelial cell metabolism is still in its infancy, it is a promising strategy to restore normal endothelial functions and thus forestall or revert the development of CVD in personalized multi-hit interventions at the metabolic level.

Cigarette smoke challenges bone marrow mesenchymal stem cell capacities in guinea pig.

BACKGROUND: Cigarette smoke (CS) is associated with lower numbers of circulating stem cells and might severely affect their mobilization, trafficking and homing. Our study was designed to demonstrate in an animal model of CS exposure whether CS affects the homing and functional capabilities of bone marrow-derived mesenchymal stem cells (BM-MSCs). METHODS: Guinea pigs (GP), exposed or sham-exposed to CS, were administered via tracheal instillation or by vascular administration with 2.5 × 106 BM-MSCs obtained from CS-exposed or sham-exposed animal donors. Twenty-four hours after cell administration, animals were sacrificed and cells were visualised into lung structures by optical microscopy. BM-MSCs from 8 healthy GP and from 8 GP exposed to CS for 1 month were isolated from the femur, cultured in vitro and assessed for their proliferation, migration, senescence, differentiation potential and chemokine gene expression profile. RESULTS: CS-exposed animals showed greater BM-MSCs lung infiltration than sham-exposed animals regardless of route of administration. The majority of BM-MSCs localized in the alveolar septa. BM-MSCs obtained from CS-exposed animals showed lower ability to engraft and lower proliferation and migration. In vitro, BM-MSCs exposed to CS extract showed a significant reduction of proliferative, cellular differentiation and migratory potential and an increase in cellular senescence in a dose dependent manner. CONCLUSION: Short-term CS exposure induces BM-MSCs dysfunction. Such dysfunction was observed in vivo, affecting the cell homing and proliferation capabilities of BM-MSCs in lungs exposed to CS and in vitro altering the rate of proliferation, senescence, differentiation and migration capacity. Additionally, CS induced a reduction in CXCL9 gene expression in the BM from CS-exposed animals underpinning a potential mechanistic action of bone marrow dysfunction.

Influence of nutrient medium composition on uranium toxicity and choice of the most sensitive growth related endpoint in Lemna minor.

Uranium (U) toxicity is known to be highly dependent on U speciation and bioavailability. To assess the impact of uranium on plants, a growth inhibition test was set up in the freshwater macrophyte Lemna minor. First growth media with different compositions were tested in order to find a medium fit for testing U toxicity in L. minor. Following arguments were used for medium selection: the ability to sustain L. minor growth, a high solubility of U in the medium and a high percentage of the more toxic U-species namely UO2(2+). Based on these selection criteria a with a low phosphate concentration of 0.5 mg L(-1) and supplemented with 5 mM MES (2-(N-morpholino)ethanesulfonic acid) to ensure pH stability was chosen. This medium also showed highest U toxicity compared to the other tested media. Subsequently a full dose response curve for U was established by exposing L. minor plants to U concentrations ranging from 0.05 µM up to 150 µM for 7 days. Uranium was shown to adversely affect growth of L. minor in a dose dependent manner with EC10, EC30 and EC50 values ranging between 1.6 and 4.8 µM, 7.7-16.4 µM and 19.4-37.2 µM U, respectively, depending on the growth endpoint. Four different growth related endpoints were tested: frond area, frond number, fresh weight and dry weight. Although differences in relative growth rates and associated ECx-values calculated on different endpoints are small (maximal twofold difference), frond area is recommended to be used to measure U-induced growth effects as it is a sensitive growth endpoint and easy to measure in vivo allowing for measurements over time.