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Lung endothelium resides at the interface between the circulation and the underlying tissue, where it senses biochemical and mechanical properties of both the blood as it flows through the vascular circuit and the vessel wall. Endothelium performs the bidirectional signaling between the blood and tissue compartments that is necessary to maintain homeostasis while physically separating both, facilitating a tightly regulated exchange of water, solutes, cells, and signals. Disruption in endothelial function contributes to vascular disease, which can manifest in discrete vascular locations along the arterial-to-capillary-to-vein axis. While our understanding of mechanisms that contribute to endothelial cell injury and repair in acute and chronic vascular disease have advanced, pathophysiological mechanisms that underlie site-specific vascular disease remain incompletely understood. In an effort to improve the translatability of mechanistic studies of the endothelium, the American Thoracic Society convened a workshop to optimize rigor, reproducibility, and translation of discovery to advance our understanding of endothelial cell function in health and disease.
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Pulmonary arterial hypertension (PAH) is characterized by endothelial cell (EC) dysfunction. There are no data from living patients to inform whether differential gene expression of pulmonary artery ECs (PAECs) can discern disease subtypes, progression and pathogenesis. We aimed to further validate our previously described method to propagate ECs from right heart catheter (RHC) balloon tips and to perform additional PAEC phenotyping. We performed bulk RNA sequencing of PAECs from RHC balloons. Using unsupervised dimensionality reduction and clustering we compared transcriptional signatures from PAH to controls and other forms of pulmonary hypertension. Select PAEC samples underwent single cell and population growth characterization and anoikis quantification. Fifty-four specimens were analyzed from 49 subjects. The transcriptome appeared stable over limited passages. Six genes involved in sex steroid signaling, metabolism, and oncogenesis were significantly upregulated in PAH subjects as compared to controls. Genes regulating BMP and Wnt signaling, oxidative stress and cellular metabolism were differentially expressed in PAH subjects. Changes in gene expression tracked with clinical events in PAH subjects with serial samples over time. Functional assays demonstrated enhanced replication competency and anoikis resistance. Our findings recapitulate fundamental biological processes of PAH and provide new evidence of a cancer-like phenotype in ECs from the central vasculature of PAH patients. This "cell biopsy" method may provide insight into patient and lung EC heterogeneity to advance precision medicine approaches in PAH.
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Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Doenças Vasculares , Humanos , Hipertensão Pulmonar/patologia , Artéria Pulmonar/patologia , Células Endoteliais/metabolismo , Hipertensão Arterial Pulmonar/patologia , Hipertensão Pulmonar Primária Familiar/metabolismo , Doenças Vasculares/patologia , Via de Sinalização Wnt/genéticaRESUMO
Objective: Protein kinase C (PKC) influences myocardial contractility and susceptibility to long-term cardiac dysfunction after ischemia-reperfusion injury. In diabetes, PKC inhibition has a protective effect in terms of microvascular dysfunction. SK-channel dysfunction also influences endothelial dysfunction in cardioplegic hypoxia-reoxygenation (CP-H/R). Here, we examine whether acute inhibition of PKC beta protects against CP-H/R-induced coronary endothelial and SK channel dysfunction. Methods: Isolated mouse coronary arterioles, half pretreated with selective PKC inhibitor ruboxistaurin (RBX), were subjected to hyperkalemic, cardioplegic hypoxia (1 hour), and reoxygenation (1 hour) with Krebs buffer. Sham control vessels were continuously perfused with oxygenated Krebs buffer without CP-H/R. After 1 hour of reoxygenation, responses to the endothelium-dependent vasodilator adenosine-diphosphate (ADP) and the SK-channel activator NS309 were examined. Endothelial SK-specific potassium currents from mouse heart endothelial cells were examined using whole-cell path clamp configurations in response to NS309 and SK channel blockers apamin and TRAM34. Results: CP-H/R significantly decreased coronary relaxation responses to ADP (P = .006) and NS309 (P = .0001) compared with the sham control group. Treatment with selective PKC beta inhibitor RBX significantly increased recovery of coronary relaxation responses to ADP (P = .031) and NS309 (P = .004) after CP-H/R. Treatment with RBX significantly increased NS309-mediated potassium currents following CP-H/R (P = .0415). Apamin and TRAM34 sensitive currents were significantly greater in CP-H/R + RBX versus CP-H/R mouse heart endothelial cells (P = .0027). Conclusions: Acute inhibition of PKC beta significantly protected mouse coronary endothelial function after CP-H/R injury. This suggests that acute PKC beta inhibition may be a novel approach for preventing microvascular dysfunction during CP-H/R.
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Previous studies have shown that atrial natriuretic peptide (ANP) attenuates agonist-induced pulmonary edema and that this effect may be mediated in part by the ANP clearance receptor, natriuretic peptide receptor-C (NPR-C). Obesity has been associated with lower plasma ANP levels due to increased expression of NPR-C, and with decreased severity of acute lung injury (ALI). Therefore, we hypothesized that increased expression of NPR-C may attenuate ALI severity in obese populations. To test this, we examined ALI in Npr3 wild-type (WT) and knockout (KO) mice fed normal chow (NC) or high-fat diets (HFD). After 12 weeks, ALI was induced with intra-tracheal administration of Pseudomonas aeruginosa strain 103 (PA103) or saline. ALI severity was determined by lung wet-to-dry ratio (W/D) along with measurement of cell count, protein levels from bronchoalveolar lavage fluid (BALF), and quantitative polymerase chain reaction was performed on whole lung to measure cytokine/chemokine and Npr3 mRNA expression. ANP levels were measured from plasma. PA103 caused ALI as determined by significant increases in W/D, BALF protein concentration, and whole lung cytokine/chemokine expression. PA103 increased Npr3 expression in the lungs of wild-type (WT) mice regardless of diet. There was a nonsignificant trend toward increased Npr3 expression in the lungs of WT mice fed HFD versus NC. No differences in ALI were seen between Npr3 knockout (KO) mice and WT-fed NC, but Npr3 KO mice fed HFD had a significantly greater W/D and BALF protein concentration than WT mice fed HFD. These findings support the hypothesis that Npr3 may help protect against ALI in obesity.
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Role of blood-based factors in development and progression of heart failure (HF) is poorly characterized. Blood contains factors released during pathophysiological states that may impact cellular function and provide mechanistic insights to HF management. We tested effects of blood from two distinct HF models on cardiac metabolism and identified possible cellular targets of the effects. Blood plasma was obtained from daunorubicin- and myocardial infarction-induced HF rabbits (Dauno-HF and MI-HF) and their controls (Dauno-Control and MI-Control). Effects of plasma on bioenergetics of myocardial tissue from healthy mice and cellular cardiac components were assessed using high-resolution respirometry and Seahorse flux analyzer. Since endothelial cell respiration was profoundly affected by HF plasma, effects of plasma on endothelial cell barrier function and death were further evaluated. Western-blotting and electron microscopy were performed to evaluate mitochondrial proteins and morphology. Brief exposure to HF plasma decreased cardiac tissue respiration. Endothelial cell respiration was most impacted by exposure to HF plasma. Endothelial cell monolayer integrity was decreased by incubation with Dauno-HF plasma. Apoptosis and necrosis were increased in cells incubated with Dauno-HF plasma for 24 h. Down-regulation of voltage-dependent anion-selective channel (VDAC)-1, translocase of outer membrane 20 (Tom20), and mitochondrial fission factor (MFF) in cells exposed to Dauno-HF plasma and mitochondrial signal transducer and activator of transcription 3 (Stat3) and MFF in cells exposed to MI-HF plasma were observed. Mitochondrial structure was disrupted in cells exposed to HF plasma. These findings indicate that endothelial cells and mitochondrial structure and function may be primary target where HF pathology manifests and accelerates. High-throughput blood-based screening of HF may provide innovative ways to advance disease diagnosis and management.
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Células Endoteliais , Insuficiência Cardíaca , Camundongos , Animais , Coelhos , Células Endoteliais/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miocárdio/metabolismo , Metabolismo EnergéticoRESUMO
It is unknown whether biological sex influences phenotypes of commercially available human pulmonary artery endothelial cells (HPAECs). Ten lots of commercial HPAECs were used (Lonza Biologics; PromoCell). Five (50%) were confirmed to be genotypically male (SRY+) and five (50%) were confirmed to be female (SRY-). Experiments were conducted between passages five and eight. HPAEC phenotype was confirmed with a panel of cell expression markers. Standard assays for proliferation, migration and tube formation were performed in triplicate with technical replicates, under three treatment conditions (EndoGRO; Sigma-Aldrich). Apoptosis was assessed by exposing cells treated with complete media or low serum media to hypoxic (1% oxygen) or normoxic (20% oxygen) conditions. Laboratory staff was blinded. The median (range) age of male and female donors from whom the HPAECs were derived was 58 (48-60) and 56 (33-67), respectively. Our results suggest decreased proliferation in genotypically female cells compared with male cells (p = 0.09). With increasing donor age, female cells were less proliferative and male cells were more proliferative (p = 0.001). Female cells were significantly more apoptotic than male cells by condition (p = 0.001). Female cells were significantly more migratory than male cells in complete media but less migratory than male cells under vascular endothelial growth factor enriched conditions (p = 0.001). There are subtle sex-based differences in the behavior of HPAECs that depend on donor sex and, less so, age. These differences may undermine rigor and reproducibility. Future studies should define whether biological sex is an important regulator of HPAEC function in health and disease.
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COVID-19 has impacted millions of patients across the world. Molecular testing occurring now identifies the presence of the virus at the sampling site: nasopharynx, nares, or oral cavity. RNA sequencing has the potential to establish both the presence of the virus and define the host's response in COVID-19. Single center, prospective study of patients with COVID-19 admitted to the intensive care unit where deep RNA sequencing (> 100 million reads) of peripheral blood with computational biology analysis was done. All patients had positive SARS-CoV-2 PCR. Clinical data was prospectively collected. We enrolled fifteen patients at a single hospital. Patients were critically ill with a mortality of 47% and 67% were on a ventilator. All the patients had the SARS-CoV-2 RNA identified in the blood in addition to RNA from other viruses, bacteria, and archaea. The expression of many immune modulating genes, including PD-L1 and PD-L2, were significantly different in patients who died from COVID-19. Some proteins were influenced by alternative transcription and splicing events, as seen in HLA-C, HLA-E, NRP1 and NRP2. Entropy calculated from alternative RNA splicing and transcription start/end predicted mortality in these patients. Current upper respiratory tract testing for COVID-19 only determines if the virus is present. Deep RNA sequencing with appropriate computational biology may provide important prognostic information and point to therapeutic foci to be precisely targeted in future studies.
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COVID-19 , Antígeno B7-H1/genética , Teste para COVID-19 , Antígenos HLA-C/genética , Humanos , Unidades de Terapia Intensiva , Estudos Prospectivos , RNA Viral/genética , SARS-CoV-2/genética , Análise de Sequência de RNARESUMO
Atrial natriuretic peptide (ANP) protects against acute lung injury (ALI), but the receptor that mediates this effect is not known. Transgenic mice with 0 (knockout), 1 (heterozygote), or 2 (wild-type) functional copies of Npr3, the gene that encodes for natriuretic peptide receptor-C (NPR-C), were treated with intravenous infusion of ANP or saline vehicle before oropharyngeal aspiration of Pseudomonas aeruginosa (PA103) or saline vehicle. Lung injury was assessed 4 h following aspiration by measurement of lung wet/dry (W/D) weight, whole lung leukocyte and cytokine levels, and protein, leukocyte, and cytokine concentration in bronchoalveolar lavage fluid (BALF). PA103 induced acute lung injury as evidenced by increases in lung W/D ratio and protein concentration in BALF. The severity of PA103-induced lung injury did not differ between NPR-C genotypes. Treatment with intravenous ANP infusion reduced PA103-induced increases in lung W/D and BALF protein concentration in all three NPRC genotypes. PA103 increased the percentage of leukocytes that were neutrophils and cytokine levels in whole lung and BALF in NPR-C wild-type and knockout mice. This effect was blunted by ANP in wild-type mice but not in the NPR-C knockout mice. NPR-C does not mediate the protective effect of ANP on endothelial cell permeability in settings of PA103-induced injury but may mediate the effect of ANP on inhibition of the recruitment of neutrophils to the lung and thereby attenuate the release of inflammatory cytokines.
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Lesão Pulmonar Aguda , Fator Natriurético Atrial , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Fator Natriurético Atrial/farmacologia , Citocinas/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Infiltração de Neutrófilos , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismoRESUMO
Chitinase 3 like 1 (CHI3L1) is the prototypic chitinase-like protein mediating inflammation, cell proliferation, and tissue remodeling. Limited data suggest CHI3L1 is elevated in human pulmonary arterial hypertension (PAH) and is associated with disease severity. Despite its importance as a regulator of injury/repair responses, the relationship between CHI3L1 and pulmonary vascular remodeling is not well understood. We hypothesize that CHI3L1 and its signaling pathways contribute to the vascular remodeling responses that occur in pulmonary hypertension (PH). We examined the relationship of plasma CHI3L1 levels and severity of PH in patients with various forms of PH, including group 1 PAH and group 3 PH, and found that circulating levels of serum CHI3L1 were associated with worse hemodynamics and correlated directly with mean pulmonary artery pressure and pulmonary vascular resistance. We also used transgenic mice with constitutive knockout and inducible overexpression of CHI3L1 to examine its role in hypoxia-, monocrotaline-, and bleomycin-induced models of pulmonary vascular disease. In all 3 mouse models of pulmonary vascular disease, pulmonary hypertensive responses were mitigated in CHI3L1-null mice and accentuated in transgenic mice that overexpress CHI3L1. Finally, CHI3L1 alone was sufficient to induce pulmonary arterial smooth muscle cell proliferation, inhibit pulmonary vascular endothelial cell apoptosis, induce the loss of endothelial barrier function, and induce endothelial-mesenchymal transition. These findings demonstrate that CHI3L1 and its receptors play an integral role in pulmonary vascular disease pathobiology and may offer a target for the treatment of PAH and PH associated with fibrotic lung disease.
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Proteína 1 Semelhante à Quitinase-3 , Hipertensão Pulmonar , Animais , Bleomicina/efeitos adversos , Proteína 1 Semelhante à Quitinase-3/metabolismo , Humanos , Hipertensão Pulmonar/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Monocrotalina/efeitos adversos , Remodelação VascularRESUMO
One of the hallmarks of acute respiratory distress syndrome (ARDS) is an excessive increase in pulmonary vascular permeability. In settings of ARDS, the loss of barrier integrity is mediated by cell-cell contact disassembly and actin remodelling. Studies into molecular mechanisms responsible for improving microvascular barrier function are therefore vital in the development of therapeutic targets for reducing vascular permeability seen in ARDS. Bitter taste receptors (T2Rs) belong to the superfamily of G-protein-coupled receptors found in several extraoral systems, including lung epithelial and smooth muscle cells. In the present study, we show for the first time that several T2Rs are expressed in human pulmonary arterial endothelial cells (HPAECs). Our results focus on those which are highly expressed as: T2R10, T2R14 and T2R38. Agonists for T2R10 (denatonium) and T2R38 (phenylthiourea), but not T2R14 (noscapine), significantly attenuated lipopolysaccharide (LPS)-induced permeability and VE-cadherin internalisation in HPAECs. In T2R10- or T2R38-siRNA knockdown cells, these endothelial-protective effects were abolished, indicating a direct effect of agonists in regulating barrier integrity. Our further findings indicate that T2R10 and T2R38 exert their barrier-protective function through cAMP but via Rac1-dependent and independent pathways, respectively. However, using an in vivo model of ARDS, the T2R38 agonist, phenylthiourea, was not able to protect against pulmonary edema formation. Taken together, these studies identify bitter taste sensing in the pulmonary endothelium to regulate barrier integrity in vitro through cAMP-Rac1 signalling.
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OBJECTIVE: Cardioplegic ischemia-reperfusion and diabetes mellitus are correlated with coronary endothelial dysfunction and inactivation of small conductance calcium-activated potassium channels. Increased reactive oxidative species, such as mitochondrial reactive oxidative species, may contribute to oxidative injury. Thus, we hypothesized that inhibition of mitochondrial reactive oxidative species may protect coronary small conductance calcium-activated potassium channels and endothelial function against cardioplegic ischemia-reperfusion-induced injury. METHODS: Small coronary arteries and endothelial cells from the hearts of mice with and without diabetes mellitus were isolated and examined by using a cardioplegic hypoxia and reoxygenation model to determine whether the mitochondria-targeted antioxidant Mito-Tempo could protect against coronary endothelial and small conductance calcium-activated potassium channel dysfunction. The microvessels or mouse heart endothelial cells were treated with or without Mito-Tempo (0-10 µM) 5 minutes before and during cardioplegic hypoxia and reoxygenation. Microvascular function was assessed in vitro by vessel myography. K+ currents of mouse heart endothelial cells were measured by whole-cell patch clamp. The levels of intracellular cytosolic free calcium (Ca2+) concentration, mitochondrial reactive oxidative species, and small conductance calcium-activated potassium protein expression of mouse heart endothelial cells were measured by Rhod-2 fluorescence staining, MitoSox, and Western blotting, respectively. RESULTS: Cardioplegic hypoxia and reoxygenation significantly attenuated endothelial small conductance calcium-activated potassium channel activity, caused calcium overload, and increased mitochondrial reactive oxidative species of mouse heart endothelial cells in both the nondiabetic and diabetes mellitus groups. In addition, treating mouse heart endothelial cells with Mito-Tempo (10 µM) reduced cardioplegic hypoxia and reoxygenation-induced Ca2+ and mitochondrial reactive oxidative species overload in both the nondiabetic and diabetes mellitus groups, respectively (P < .05). Treatment with Mito-Tempo (10 µM) significantly enhanced coronary relaxation responses to adenosine 5'-diphosphate and NS309 (P < .05), and endothelial small conductance calcium-activated potassium channel currents in both the nondiabetic and diabetes mellitus groups (P < .05). CONCLUSIONS: Administration of Mito-Tempo improves endothelial function and small conductance calcium-activated potassium channel activity, which may contribute to its enhancement of endothelium-dependent vasorelaxation after cardioplegic hypoxia and reoxygenation.
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Diabetes Mellitus , Células Endoteliais , Adenosina/metabolismo , Animais , Antioxidantes/metabolismo , Cálcio/metabolismo , Diabetes Mellitus/metabolismo , Difosfatos/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Hipóxia , Camundongos , Mitocôndrias , Oxirredução , Potássio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismoRESUMO
This report describes the 10-year outcome of implementing practices that support and foster success of underrepresented students in science, technology, engineering, and math (STEM) graduate training at Brown University. The results show sustained improvements in compositional diversity, retention, and degree attainment of supported students relative to their peers. Among the outcomes is an increase in enrolled student diversity from 19 (35 of 179) to 26% (58 of 223) for historically underrepresented minority (URM) students and an increase in Ph.D. degree attainment from 4 (1 of 25) to 14% (6 of 44) for this group. These achievements follow the introduction and coordination of academic and co-curricular practices through the National Institutes of General Medical Sciences-funded Brown University Initiative to Maximize Student Development (IMSD) Program. At the center of these outcomes is the alignment of IMSD practices with recent diversity initiatives launched by the university. The outcomes described result from long-term commitments to building a culture that includes: (1) development of relationships that serve underrepresented students, (2) provision of a personalized education program of support and skills-based learning that supplements discipline-based research and coursework, and (3) investments in processes that build a culture that values and benefits from diversity. These practices may yield similar outcomes and success for students when applied elsewhere.
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The CardioPulmonary Vascular Biology Center for Biomedical Research Excellence (CPVB COBRE) was funded in 2013 by the National Institute for General Medical Sciences to establish a collaborative center for research excellence in vascular biology in Rhode Island. The CPVB COBRE has funded successful junior faculty investigators and pilot projects spanning the research spectrum from basic vascular development mechanisms using zebrafish to clinical research on pulmonary hypertension to the effects of mindfulness on hypertension in pregnancy. The Administrative Core has united the group with an active seminar program with visiting experts, a focus on career development, and the use of evaluation to support continuous improvement. The Cell Isolation and Organ Function Core has provided high-quality research services and expertise. Most importantly, hard-working and creative physicians and basic scientist investigators and mentors have worked together to expand the spectrum of vascular biology research in Rhode Island.
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Pesquisa Biomédica , Peixe-Zebra , Animais , Biologia , Humanos , Mentores , Pesquisadores , Rhode Island , Estados UnidosRESUMO
Diabetes is associated with coronary endothelial dysfunction. Persistent oxidative stress during diabetes contributes to coronary endothelial dysfunction. The mitochondria are main sources of reactive oxygen species (ROS) in diabetes, and mitochondria-targeted antioxidant mito-Tempo can prevent mitochondrial reactive oxygen species (mROS) generation in a variety of disorders. Inhibition/inactivation of small-conductance Ca2+-activated K+ (SK) channels contribute to diabetic downregulation of coronary endothelial function/relaxation. However, few investigated the role of mROS on endothelial dysfunction/vasodilation and endothelial SK channel downregulation in diabetes. The aim of present study was to investigate the chronic administration of mito-Tempo, on coronary vasodilation, and endothelial SK channel activity of mice with or without diabetes. Mito-Tempo (1 mg/kg/day) was applied to the mice with or without diabetes (n = 10/group) for 4 weeks. In vitro relaxation response of pre-contracted arteries was examined in the presence or absence of the vasodilatory agents. SK channel currents of the isolated mouse heart endothelial cells were measured using whole-cell patch clamp methods. At baseline, coronary endothelium-dependent relaxation responses to ADP and the selective SK channel activator NS309 and endothelial SK channel currents were decreased in diabetic mice compared with that in non-diabetic (ND) mice (p < 0.05). After a 4-week treatment with mito-Tempo, coronary endothelium-dependent relaxation response to ADP or NS309 and endothelial SK channel currents in the diabetic mice was significantly improved when compared with that in untreated diabetic mice (p < 0.05). Interestingly, coronary relaxation responses to ADP and NS309 and endothelial SK channel currents were not significantly changed in ND mice after mito-Tempo treatment, as compared to that of untreated control group. Chronic inhibition of endothelial mROS appears to improve coronary endothelial function/dilation and SK channel activity in diabetes, and mROS inhibitors may be a novel strategy to treat vascular complications in diabetes.
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PURPOSE: COVID-19 has impacted millions of patients across the world. Molecular testing occurring now identifies the presence of the virus at the sampling site: nasopharynx, nares, or oral cavity. RNA sequencing has the potential to establish both the presence of the virus and define the host's response in COVID-19. METHODS: Single center, prospective study of patients with COVID-19 admitted to the intensive care unit where deep RNA sequencing (>100 million reads) of peripheral blood with computational biology analysis was done. All patients had positive SARS-CoV-2 PCR. Clinical data was prospectively collected. RESULTS: We enrolled fifteen patients at a single hospital. Patients were critically ill with a mortality of 47% and 67% were on a ventilator. All the patients had the SARS-CoV-2 RNA identified in the blood in addition to RNA from other viruses, bacteria, and archaea. The expression of many immune modulating genes, including PD-L1 and PD-L2, were significantly different in patients who died from COVID-19. Some proteins were influenced by alternative transcription and splicing events, as seen in HLA-C, HLA-E, NRP1 and NRP2. Entropy calculated from alternative RNA splicing and transcription start/end predicted mortality in these patients. CONCLUSIONS: Current upper respiratory tract testing for COVID-19 only determines if the virus is present. Deep RNA sequencing with appropriate computational biology may provide important prognostic information and point to therapeutic foci to be precisely targeted in future studies. TAKE HOME MESSAGE: Deep RNA sequencing provides a novel diagnostic tool for critically ill patients. Among ICU patients with COVID-19, RNA sequencings can identify gene expression, pathogens (including SARS-CoV-2), and can predict mortality. TWEET: Deep RNA sequencing is a novel technology that can assist in the care of critically ill COVID-19 patients & can be applied to other disease.
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Rationale: Sex hormones play a role in pulmonary arterial hypertension (PAH), but the menstrual cycle has never been studied.Objectives: We conducted a prospective observational study of eight women with stable PAH and 20 healthy controls over one cycle.Methods: Participants completed four study visits 1 week apart starting on the first day of menstruation. Relationships between sex hormones, hormone metabolites, and extracellular vesicle microRNA (miRNA) expression and clinical markers were compared with generalized linear mixed modeling.Results: Women with PAH had higher but less variable estradiol (E2) levels (P < 0.001) that tracked with 6-minute walk distance (P < 0.001), N-terminal prohormone of brain natriuretic peptide (P = 0.03) levels, and tricuspid annular plane systolic excursion (P < 0.01); the direction of these associations depended on menstrual phase. Dehydroepiandrosterone sulfate (DHEA-S) levels were lower in women with PAH (all visits, P < 0.001). In PAH, each 100-µg/dl increase in DHEA-S was associated with a 127-m increase in 6-minute walk distance (P < 0.001) and was moderated by the cardioprotective E2 metabolite 2-methoxyestrone (P < 0.001). As DHEA-S increased, N-terminal prohormone of brain natriuretic peptide levels decreased (P = 0.001). Expression of extracellular vesicle miRNAs-21, -29c, and -376a was higher in PAH, moderated by E2 and DHEA-S levels, and tracked with hormone-associated changes in clinical measures.Conclusions: Women with PAH have fluctuations in cardiopulmonary function during menstruation driven by E2 and DHEA-S. These hormones in turn influence transcription of extracellular vesicle miRNAs implicated in the pathobiology of pulmonary vascular disease and cancer.
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Hipertensão Pulmonar , MicroRNAs , Hipertensão Arterial Pulmonar , Hipertensão Pulmonar Primária Familiar , Feminino , Humanos , Ciclo MenstrualRESUMO
The novel endosome protein, p18, and the early endosome GTPase, Rab4, play a significant role in protecting the pulmonary vasculature against permeability associated with acute respiratory distress syndrome. Recently, endothelial-derived extracellular vesicles have been identified to play a key role in the endothelial permeability associated with acute respiratory distress syndrome. Therefore, we investigated the effect of these microparticles, released from endothelial cells overexpressing p18 and Rab4, on pulmonary endothelial barrier function. Endothelial-derived extracellular vesicles isolated from lung microvascular endothelial cells which overexpressed cDNA for wild-type p18 protected a naïve monolayer against lipopolysaccharide-induced permeability. In contrast, endothelial-derived extracellular vesicles from cells overexpressing the non-endosomal binding p18 mutant (p18N39) exerted no protective effect on the endothelial monolayer. Cells overexpressing either dominant active or inactive Rab4 released endothelial-derived extracellular vesicles which had no effect on lipopolysaccharide-induced permeability. miRNA analysis and permeability studies of endothelial-derived extracellular vesicle isolated from wild-type p18-overexpressing cells demonstrates that let-7i-5p, miR-96-5p, and miR-137-3p are endothelial-derived extracellular vesicle cargo which exert protective effects on the pulmonary endothelium. Finally, we observed down-regulation of p18 protein expression in both the lung and endothelium in an in vivo and in vitro model of acute respiratory distress syndrome. These results demonstrate that endothelial-derived extracellular vesicle released from cells overexpressing p18, but not Rab4, contain miRNA cargo which likely promote a barrier-protective effect on the pulmonary endothelium in settings of acute respiratory distress syndrome. Findings indicate the importance of p18 in the pulmonary vasculature and demonstrate that targeting this protein may provide a novel therapeutic strategy to reduce endothelial permeability associated with acute respiratory distress syndrome.
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OBJECTIVE: To investigate coronary endothelial protection of a small-conductance calcium-activated potassium (SK) channel activator against a period of cardioplegic-hypoxia and reoxygenation (CP-H/R) injury in mice and patients with diabetes (DM) and those without diabetes (nondiabetic [ND]). METHODS: Mouse small coronary arteries/heart endothelial cells (MHECs) and human coronary arterial endothelial cells (HCAECs) were dissected from the harvested hearts of mice (n = 16/group) and from discarded right atrial tissue samples of patients with DM and without DM (n = 8/group). The SK current density of MHECs was measured. The in vitro small arteries/arterioles, MHECs, and HCAECs were subjected to 60 minutes of CP hypoxia, followed by 60 minutes of oxygenation. Vessels were treated with or without the selective SK activator NS309 for 5 minutes before and during CP hypoxia. RESULTS: DM and/or CP-H/R significantly inhibited the total SK currents of MHECs and HCAECs and significantly diminished the mouse coronary relaxation response to NS309. Administration of NS309 immediately before and during CP hypoxia significantly improved the recovery of coronary endothelial function, as demonstrated by increased relaxation responses to adenosine 5'-diphosphate and substance P compared with those seen in controls (P < .05). This protective effect was more pronounced in vessels from ND mice and patients compared with DM mice and patients (P < .05). Cell surface membrane SK3 expression was significantly reduced after hypoxia, whereas cytosolic SK3 expression was greater than that of the sham control group (P < .05). CONCLUSIONS: Application of NS309 immediately before and during CP hypoxia protects mouse and human coronary microvasculature against CP-H/R injury, but this effect is diminished in the diabetic coronary microvasculature. SK inhibition/inactivation and/or internalization/redistribution may contribute to CP-H/R-induced coronary endothelial and vascular relaxation dysfunction.
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Doença da Artéria Coronariana/etiologia , Vasos Coronários/patologia , Diabetes Mellitus Tipo 2/complicações , Endotélio Vascular/metabolismo , Indóis/farmacologia , Oximas/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Vasodilatação/efeitos dos fármacos , Idoso , Animais , Células Cultivadas , Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/metabolismo , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Feminino , Humanos , Masculino , Camundongos , Transdução de Sinais , Canais de Potássio Ativados por Cálcio de Condutância Baixa/efeitos dos fármacosRESUMO
Endothelial dysfunction is a hallmark of pulmonary arterial hypertension (PAH) but there are no established methods to study pulmonary artery endothelial cells (PAECs) from living patients. We sought to culture PAECs from pulmonary artery catheter (PAC) balloons used during right-heart catheterisation (RHC) to characterise successful culture attempts and to describe PAEC behaviour.PAECs were grown in primary culture to confluence and endothelial cell phenotype was confirmed. Standard assays for apoptosis, migration and tube formation were performed between passages three to eight. We collected 49 PAC tips from 45 subjects with successful PAEC culture from 19 balloons (39%).There were no differences in subject demographic details or RHC procedural details in successful versus unsuccessful attempts. However, for subjects who met haemodynamic criteria for PAH, there was a higher but nonsignificant (p=0.10) proportion amongst successful attempts (10 out of 19, 53%) versus unsuccessful attempts (nine out of 30, 30%). A successful culture was more likely in subjects with a lower cardiac index (p=0.03) and higher pulmonary vascular resistance (p=0.04). PAECs from a subject with idiopathic PAH were apoptosis resistant compared to commercial PAECs (p=0.04) and had reduced migration compared to PAECs from a subject with portopulmonary hypertension with high cardiac output (p=0.01). PAECs from a subject with HIV-associated PAH formed fewer (p=0.01) and shorter (p=0.02) vessel networks compared to commercial PAECs.Sustained culture and characterisation of PAECs from RHC balloons is feasible, especially in PAH with high haemodynamic burden. This technique may provide insight into endothelial dysfunction during PAH pathogenesis.
Assuntos
Artéria Pulmonar , Doenças Vasculares , Catéteres , Células Cultivadas , Células Endoteliais , Humanos , PulmãoRESUMO
INTRODUCTION: Because a primary focus of Centers of Biomedical Research Excellence (COBRE) is the development of junior-level investigators into competent and successful research scientists, evaluation of their skills, mentoring experiences, and usefulness of COBRE services is paramount to the transition of the Center to a self-sustaining, collaborative, multidisciplinary research environment. A formative evaluation, focused on the processes of a COBRE, was undertaken and is presented here. METHODS: Two instruments, one for completion by junior investigators and one for completion by mentors, were developed for the purpose of evaluating this COBRE. Areas of inquiry were relationships between junior investigators and mentors, research self-efficacy, mentee progress, and satisfaction with the COBRE. All eight of the COBRE's current junior investigators and six of their mentors completed the online questionnaires. RESULTS: Junior investigators were very positive about mentors and vice versa. Junior investigators were least positive about their progress as academicians and most positive about their abilities to develop collaborations with other scholars/professionals. Mentors felt as though junior investigators could benefit most by increasing the number of publications they had generated. CONCLUSIONS: Activities provided by the CardioPulmonary Vascular Biology (CPVB) COBRE were extremely positive. Junior investigators felt as though the scientific, academic, and professional development opportunities afforded by this COBRE were integral to their success as researchers; however they would like more assistance developing professional networks (i.e., serving on committees of professional societies). Leadership of the CPVB COBRE may consider expanding the role of their advisory committee to ensure these opportunities are provided.