Transcriptomics and proteomics revealed sex differences in human pulmonary microvascular endothelial cells.

Marked sexual dimorphism is displayed in the onset and progression of pulmonary hypertension (PH). Females more commonly develop pulmonary arterial hypertension, yet females with pulmonary arterial hypertension and other types of PH have better survival than males. Pulmonary microvascular endothelial cells play a crucial role in pulmonary vascular remodeling and increased pulmonary vascular resistance in PH. Given this background, we hypothesized that there are sex differences in the pulmonary microvascular endothelium basally and in response to hypoxia that are independent of the sex hormone environment. Human pulmonary microvascular endothelial cells (HPMECs) from healthy male and female donors, cultured under physiological shear stress, were analyzed using RNA sequencing and label-free quantitative proteomics. Gene set enrichment analysis identified a number of sex-different pathways in both normoxia and hypoxia, including pathways that regulate cell proliferation. In vitro, the rate of proliferation in female HPMECs was lower than in male HPMECs, a finding that supports the omics results. Interestingly, thrombospondin-1, an inhibitor of proliferation, was more highly expressed in female cells than in male cells. These results demonstrate, for the first time, important differences between female and male HPMECs that persist in the absence of sex hormone differences and identify novel pathways for further investigation that may contribute to sexual dimorphism in pulmonary hypertensive diseases.NEW & NOTEWORTHY There is marked sexual dimorphism in the development and progression of pulmonary hypertension. We show differences in RNA and protein expression between female and male human pulmonary microvascular endothelial cells grown under conditions of physiological shear stress, which identify sex-different cellular pathways both in normoxia and hypoxia. Importantly, these differences were detected in the absence of sex hormone differences. The pathways identified may provide novel targets for the development of sex-specific therapies.

High-Soluble-Fiber Diet Attenuates Hypoxia-Induced Vascular Remodeling and the Development of Hypoxic Pulmonary Hypertension.

BACKGROUND: Hypoxic pulmonary hypertension is a difficult disease to manage that is characterized by sustained elevation of pulmonary vascular resistance and pulmonary artery pressure due to vasoconstriction, perivascular inflammation, and vascular remodeling. Consumption of soluble-fiber is associated with lower systemic blood pressure, but little is known about its ability to affect the pulmonary circulation. METHODS: Mice were fed either a low- or high-soluble-fiber diet (0% or 16.9% inulin) and then exposed to hypoxia (FiO2, 0.10) for 21 days to induce pulmonary hypertension. The impact of diet on right ventricular systolic pressure and pulmonary vascular resistance was determined in vivo or in ex vivo isolated lungs, respectively, and correlated with alterations in the composition of the gut microbiome, plasma metabolome, pulmonary inflammatory cell phenotype, and lung proteome. RESULTS: High-soluble-fiber diet increased the abundance of short-chain fatty acid-producing bacteria, with parallel increases in plasma propionate levels, and reduced the abundance of disease-related bacterial genera such as Staphylococcus, Clostridioides, and Streptococcus in hypoxic mice with parallel decreases in plasma levels of p-cresol sulfate. High-soluble-fiber diet decreased hypoxia-induced elevations of right ventricular systolic pressure and pulmonary vascular resistance. These changes were associated with reduced proportions of interstitial macrophages, dendritic cells, and nonclassical monocytes. Whole-lung proteomics revealed proteins and molecular pathways that may explain the effect of soluble-fiber supplementation. CONCLUSIONS: This study demonstrates for the first time that a high-soluble-fiber diet attenuates hypoxia-induced pulmonary vascular remodeling and the development of pulmonary hypertension in a mouse model of hypoxic pulmonary hypertension and highlights diet-derived metabolites that may have an immuno-modulatory role in the lung.

CAPILLARY LEAK AND EDEMA AFTER RESUSCITATION: THE POTENTIAL CONTRIBUTION OF REDUCED ENDOTHELIAL SHEAR STRESS CAUSED BY HEMODILUTION.

ABSTRACT: Normal shear stress is essential for the normal structure and functions of the microcirculation. Hemorrhagic shock leads to reduced shear stress due to reduced tissue perfusion. Although essential for the urgent restoration of cardiac output and systemic blood pressure, large volume resuscitation with currently available solutions causes hemodilution, further reducing endothelial shear stress. In this narrative review, we consider how the use of currently available resuscitation solutions results in persistent reduction in endothelial shear stress, despite successfully increasing cardiac output and systemic blood pressure. We consider how this reduced shear stress causes (1) a failure to restore normal vasomotor function and normal tissue perfusion thus leading to persistent tissue hypoxia and (2) increased microvascular endothelial permeability resulting in edema formation and impaired organ function. We discuss the need for clinical research into resuscitation strategies and solutions that aim to quickly restore endothelial shear stress in the microcirculation to normal.

Gremlin 1 is required for macrophage M2 polarization.

Pro-proliferative, M2-like polarization of macrophages is a critical step in the development of fibrosis and remodeling in chronic lung diseases such as pulmonary fibrosis and pulmonary hypertension. Macrophages in healthy and diseased lungs express gremlin 1 (Grem1), a secreted glycoprotein that acts in both paracrine and autocrine manners to modulate cellular function. Increased Grem1 expression plays a central role in pulmonary fibrosis and remodeling, however, the role of Grem1 in M2-like polarization of macrophages has not previously been explored. The results reported here show that recombinant Grem1 potentiated M2-like polarization of mouse macrophages and bone marrow-derived macrophages (BMDMs) in response to the Th2 cytokines IL4 and IL13. Genetic depletion of Grem1 in BMDMs inhibited M2 polarization while exogenous gremlin 1 could partially rescue this effect. Taken together, these findings reveal that gremlin 1 is required for M2-like polarization of macrophages.NEW & NOTEWORTHY We show here that gremlin 1 potentiated M2 polarization of mouse bone marrow-derived macrophages (BMDMs) in response to the Th2 cytokines IL4 and IL13. Genetic depletion of Grem1 in BMDMs inhibited M2 polarization while exogenous gremlin 1 partially rescued this effect. Taken together, these findings reveal a previously unknown requirement for gremlin 1 in M2 polarization of macrophages and suggest a novel cellular mechanism promoting fibrosis and remodeling in lung diseases.

Shear Stress Markedly Alters the Proteomic Response to Hypoxia in Human Pulmonary Endothelial Cells.

Blood flow produces shear stress that homeostatically regulates the phenotype of pulmonary endothelial cells, exerting antiinflammatory and antithrombotic actions and maintaining normal barrier function. Hypoxia due to diseases, such as chronic obstructive pulmonary disease (COPD), causes vasoconstriction, increased vascular resistance, and pulmonary hypertension. Hypoxia-induced changes in endothelial function play a central role in the development of pulmonary hypertension. However, the interactive effects of hypoxia and shear stress on the pulmonary endothelial phenotype have not been studied. Human pulmonary microvascular endothelial cells were cultured in normoxia or hypoxia while subjected to physiological shear stress or in static conditions. Unbiased proteomics was used to identify hypoxia-induced changes in protein expression. Using publicly available single-cell RNA sequencing datasets, differences in gene expression between the alveolar endothelial cells from COPD and healthy lungs were identified. Sixty proteins were identified whose expression changed in response to hypoxia in conditions of physiological shear stress but not in static conditions. These included proteins that are crucial for endothelial homeostasis (e.g., JAM-A [junctional adhesion molecule A], ERG [ETS transcription factor ERG]) or implicated in pulmonary hypertension (e.g., thrombospondin-1). Fifty-five of these 60 have not been previously implicated in the development of hypoxic lung diseases. mRNA for 5 of the 60 (ERG, MCRIP1 [MAPK regulated corepressor interacting protein 1], EIF4A2 [eukaryotic translation initiation factor 4A2], HSP90AA1 [heat shock protein 90 alpha family class A member 1], and DNAJA1 [DnaJ Hsp40 (heat shock protein family) member A1]) showed similar changes in the alveolar endothelial cells of COPD compared with healthy lungs in females but not in males. These data show that the proteomic responses of the pulmonary microvascular endothelium to hypoxia are significantly altered by shear stress and suggest that these shear-hypoxia interactions are important in the development of hypoxic pulmonary vascular disease.

Sex Dimorphism in Pulmonary Hypertension: The Role of the Sex Chromosomes.

Pulmonary hypertension (PH) is a condition characterised by an abnormal elevation of pulmonary artery pressure caused by an increased pulmonary vascular resistance, frequently leading to right ventricular failure and reduced survival. Marked sexual dimorphism is observed in patients with pulmonary arterial hypertension, a form of pulmonary hypertension with a particularly severe clinical course. The incidence in females is 2-4 times greater than in males, although the disease is less severe in females. We review the contribution of the sex chromosomes to this sex dimorphism highlighting the impact of proteins, microRNAs and long non-coding RNAs encoded on the X and Y chromosomes. These genes are centrally involved in the cellular pathways that cause increased pulmonary vascular resistance including the production of reactive oxygen species, altered metabolism, apoptosis, inflammation, vasoconstriction and vascular remodelling. The interaction with genetic mutations on autosomal genes that cause heritable pulmonary arterial hypertension such as bone morphogenetic protein 2 (BMPR2) are examined. The mechanisms that can lead to differences in the expression of genes located on the X chromosomes between females and males are also reviewed. A better understanding of the mechanisms of sex dimorphism in this disease will contribute to the development of more effective therapies for both women and men.

Gremlin 1 depletion in vivo causes severe enteropathy and bone marrow failure.

The intestinal epithelium is perpetually renewed from a stem cell niche in the base of crypts to maintain a healthy bowel mucosa. Exit from this niche and maturation of epithelial cells requires tightly controlled gradients in BMP signalling, progressing from low BMP signalling at the crypt base to high signalling at the luminal surface. The BMP antagonist gremlin 1 (Grem1) is highly expressed by subepithelial myofibroblasts adjacent to the intestinal crypts but its role in regulating the stem cell niche and epithelial renewal in vivo has not been explored. To explore the effects of Grem1 loss in adulthood following normal growth and development, we bred mice (ROSA26CreER-Grem1 flx/flx ) in which Grem1 could be deleted by tamoxifen administration. While Grem1 remained intact, these mice were healthy, grew normally, and reproduced successfully. Following Grem1 depletion, the mice became unwell and were euthanised (at 7-13 days). Post-mortem examination revealed extensive mucosal abnormalities throughout the small and large intestines with failure of epithelial cell replication and maturation, villous atrophy, and features of malabsorption. Bone marrow hypoplasia was also observed with associated early haematopoietic failure. These results demonstrate an essential homeostatic role for gremlin 1 in maintaining normal bowel epithelial function in adulthood, suggesting that abnormalities in gremlin 1 expression can contribute to enteropathies. We also identified a previously unsuspected requirement for gremlin 1 in normal haematopoiesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The effects of genetic deletion of Macrophage migration inhibitory factor on the chronically hypoxic pulmonary circulation.

While it is well established that the haemodynamic cause of hypoxic pulmonary hypertension is increased pulmonary vascular resistance, the molecular pathogenesis of the increased resistance remains incompletely understood. Macrophage migration inhibitory factor is a pleiotropic cytokine with endogenous tautomerase enzymatic activity as well as both intracellular and extracellular signalling functions. In several diseases, macrophage migration inhibitory factor has pro-inflammatory roles that are dependent upon signalling through the cell surface receptors CD74, CXCR2 and CXCR4. Macrophage migration inhibitory factor expression is increased in animal models of hypoxic pulmonary hypertension and macrophage migration inhibitory factor tautomerase inhibitors, which block some of the functions of macrophage migration inhibitory factor, and have been shown to attenuate hypoxic pulmonary hypertension in mice and monocrotaline-induced pulmonary hypertension in rats. However, because of the multiple pathways through which it acts, the integrated actions of macrophage migration inhibitory factor during the development of hypoxic pulmonary hypertension were unclear. We report here that isolated lungs from adult macrophage migration inhibitory factor knockout (MIF-/- ) mice maintained in normoxic conditions showed greater acute hypoxic vasoconstriction than the lungs of wild type mice (MIF+/+ ). Following exposure to hypoxia for three weeks, isolated lungs from MIF-/- mice had significantly higher pulmonary vascular resistance than those from MIF+/+ mice. The major mechanism underlying the greater increase in pulmonary vascular resistance in the hypoxic MIF-/- mice was reduction of the pulmonary vascular bed due to an impairment of the normal hypoxia-induced expansion of the alveolar capillary network. Taken together, these results demonstrate that macrophage migration inhibitory factor plays a central role in the development of the pulmonary vascular responses to chronic alveolar hypoxia.

Pulmonary endothelial permeability and tissue fluid balance depend on the viscosity of the perfusion solution.

Fluid filtration in the pulmonary microcirculation depends on the hydrostatic and oncotic pressure gradients across the endothelium and the selective permeability of the endothelial barrier. Maintaining normal fluid balance depends both on specific properties of the endothelium and of the perfusing blood. Although some of the essential properties of blood needed to prevent excessive fluid leak have been identified and characterized, our understanding of these remains incomplete. The role of perfusate viscosity in maintaining normal fluid exchange has not previously been examined. We prepared a high-viscosity perfusion solution (HVS) with a relative viscosity of 2.5, i.e., within the range displayed by blood flowing in vessels of different diameters in vivo (1.5-4.0). Perfusion of isolated murine lungs with HVS significantly reduced the rate of edema formation compared with perfusion with a standard solution (SS), which had a lower viscosity similar to plasma (relative viscosity 1.5). HVS did not alter capillary filtration pressure. Increased endothelial shear stress produced by increasing flow rates of SS, to mimic the increased shear stress produced by HVS, did not reduce edema formation. HVS significantly reduced extravasation of Evans blue-labeled albumin compared with SS, indicating that it attenuated endothelial leak. These findings demonstrate for the first time that the viscosity of the solution perfusing the pulmonary microcirculation is an important physiological property contributing to the maintenance of normal fluid exchange. This has significant implications for our understanding of fluid homeostasis in the healthy lung, edema formation in disease, and reconditioning of donor organs for transplantation.

Amyotrophic lateral sclerosis patient iPSC-derived astrocytes impair autophagy via non-cell autonomous mechanisms.

Amyotrophic lateral sclerosis, a devastating neurodegenerative disease, is characterized by the progressive loss of motor neurons and the accumulation of misfolded protein aggregates. The latter suggests impaired proteostasis may be a key factor in disease pathogenesis, though the underlying mechanisms leading to the accumulation of aggregates is unclear. Further, recent studies have indicated that motor neuron cell death may be mediated by astrocytes. Herein we demonstrate that ALS patient iPSC-derived astrocytes modulate the autophagy pathway in a non-cell autonomous manner. We demonstrate cells treated with patient derived astrocyte conditioned medium demonstrate decreased expression of LC3-II, a key adapter protein required for the selective degradation of p62 and ubiquitinated proteins targeted for degradation. We observed an increased accumulation of p62 in cells treated with patient conditioned medium, with a concomitant increase in the expression of SOD1, a protein associated with the development of ALS. Activation of autophagic mechanisms with Rapamycin reduces the accumulation of p62 puncta in cells treated with patient conditioned medium. These data suggest that patient astrocytes may modulate motor neuron cell death by impairing autophagic mechanisms, and the autophagy pathway may be a useful target in the development of novel therapeutics.

Hypoxic pulmonary hypertension in chronic lung diseases: novel vasoconstrictor pathways.

Pulmonary hypertension is a well recognised complication of chronic hypoxic lung diseases, which are among the most common causes of death and disability worldwide. Development of pulmonary hypertension independently predicts reduced life expectancy. In chronic obstructive pulmonary disease, long-term oxygen therapy ameliorates pulmonary hypertension and greatly improves survival, although the correction of alveolar hypoxia and pulmonary hypertension is only partial. Advances in understanding of the regulation of vascular smooth muscle tone show that chronic vasoconstriction plays a more important part in the pathogenesis of hypoxic pulmonary hypertension than previously thought, and that structural vascular changes contribute less. Trials of existing vasodilators show that pulmonary hypertension can be ameliorated and systemic oxygen delivery improved in carefully selected patients, although systemic hypotensive effects limit the doses used. Vasoconstrictor pathways that are selective for the pulmonary circulation can be blocked to reduce hypoxic pulmonary hypertension without causing systemic hypotension, and thus provide potential targets for novel therapeutic strategies.

Altered Expression of Bone Morphogenetic Protein Accessory Proteins in Murine and Human Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is a chronic, progressive fibrotic disease with a poor prognosis. The balance between transforming growth factor ß1 and bone morphogenetic protein (BMP) signaling plays an important role in tissue homeostasis, and alterations can result in pulmonary fibrosis. We hypothesized that multiple BMP accessory proteins may be responsible for maintaining this balance in the lung. Using the bleomycin mouse model for fibrosis, we examined an array of BMP accessory proteins for changes in mRNA expression. We report significant increases in mRNA expression of gremlin 1, noggin, follistatin, and follistatin-like 1 (Fstl1), and significant decreases in mRNA expression of chordin, kielin/chordin-like protein, nephroblastoma overexpressed gene, and BMP and activin membrane-bound inhibitor (BAMBI). Protein expression studies demonstrated increased levels of noggin, BAMBI, and FSTL1 in the lungs of bleomycin-treated mice and in the lungs of idiopathic pulmonary fibrosis patients. Furthermore, we demonstrated that transforming growth factor ß stimulation resulted in increased expression of noggin, BAMBI, and FSTL1 in human small airway epithelial cells. These results provide the first evidence that multiple BMP accessory proteins are altered in fibrosis and may play a role in promoting fibrotic injury.

Elevated plasma CXCL12α is associated with a poorer prognosis in pulmonary arterial hypertension.

RATIONALE: Recent work in preclinical models suggests that signalling via the pro-angiogenic and pro-inflammatory cytokine, CXCL12 (SDF-1), plays an important pathogenic role in pulmonary hypertension (PH). The objective of this study was to establish whether circulating concentrations of CXCL12α were elevated in patients with PAH and related to mortality. METHODS: Plasma samples were collected from patients with idiopathic pulmonary arterial hypertension (IPAH) and PAH associated with connective tissue diseases (CTD-PAH) attending two pulmonary hypertension referral centres (n = 95) and from age and gender matched healthy controls (n = 44). Patients were subsequently monitored throughout a period of five years. RESULTS: CXCL12α concentrations were elevated in PAH groups compared to controls (P<0.05) and receiver-operating-characteristic analysis showed that plasma CXCL12α concentrations discriminated patients from healthy controls (AUC 0.80, 95% confidence interval 0.73-0.88). Kaplan Meier analysis indicated that elevated plasma CXCL12α concentration was associated with reduced survival (P<0.01). Multivariate Cox proportional hazards model showed that elevated CXCL12α independently predicted (P<0.05) earlier death in PAH with a hazard ratio (95% confidence interval) of 2.25 (1.01-5.00). In the largest subset by WHO functional class (Class 3, 65% of patients) elevated CXCL12α independently predicted (P<0.05) earlier death, hazard ratio 2.27 (1.05-4.89). CONCLUSIONS: Our data show that elevated concentrations of circulating CXCL12α in PAH predicted poorer survival. Furthermore, elevated circulating CXCL12α was an independent risk factor for death that could potentially be included in a prognostic model and guide therapy.

Pulmonary vascular dysfunction in ARDS.

Acute respiratory distress syndrome (ARDS) is characterised by diffuse alveolar damage and is frequently complicated by pulmonary hypertension (PH). Multiple factors may contribute to the development of PH in this setting. In this review, we report the results of a systematic search of the available peer-reviewed literature for papers that measured indices of pulmonary haemodynamics in patients with ARDS and reported on mortality in the period 1977 to 2010. There were marked differences between studies, with some reporting strong associations between elevated pulmonary arterial pressure or elevated pulmonary vascular resistance and mortality, whereas others found no such association. In order to discuss the potential reasons for these discrepancies, we review the physiological concepts underlying the measurement of pulmonary haemodynamics and highlight key differences between the concepts of resistance in the pulmonary and systemic circulations. We consider the factors that influence pulmonary arterial pressure, both in normal lungs and in the presence of ARDS, including the important effects of mechanical ventilation. Pulmonary arterial pressure, pulmonary vascular resistance and transpulmonary gradient (TPG) depend not alone on the intrinsic properties of the pulmonary vascular bed but are also strongly influenced by cardiac output, airway pressures and lung volumes. The great variability in management strategies within and between studies means that no unified analysis of these papers was possible. Uniquely, Bull et al. (Am J Respir Crit Care Med 182:1123-1128, 2010) have recently reported that elevated pulmonary vascular resistance (PVR) and TPG were independently associated with increased mortality in ARDS, in a large trial with protocol-defined management strategies and using lung-protective ventilation. We then considered the existing literature to determine whether the relationship between PVR/TPG and outcome might be causal. Although we could identify potential mechanisms for such a link, the existing evidence does not allow firm conclusions to be drawn. Nonetheless, abnormally elevated PVR/TPG may provide a useful index of disease severity and progression. Further studies are required to understand the role and importance of pulmonary vascular dysfunction in ARDS in the era of lung-protective ventilation.

The α and Δ isoforms of CREB1 are required to maintain normal pulmonary vascular resistance.

Chronic hypoxia causes pulmonary hypertension associated with structural alterations in pulmonary vessels and sustained vasoconstriction. The transcriptional mechanisms responsible for these distinctive changes are unclear. We have previously reported that CREB1 is activated in the lung in response to alveolar hypoxia but not in other organs. To directly investigate the role of α and Δ isoforms of CREB1 in the regulation of pulmonary vascular resistance we examined the responses of mice in which these isoforms of CREB1 had been inactivated by gene mutation, leaving only the ß isoform intact (CREB(αΔ) mice). Here we report that expression of CREB regulated genes was altered in the lungs of CREB(αΔ) mice. CREB(αΔ) mice had greater pulmonary vascular resistance than wild types, both basally in normoxia and following exposure to hypoxic conditions for three weeks. There was no difference in rho kinase mediated vasoconstriction between CREB(αΔ) and wild type mice. Stereological analysis of pulmonary vascular structure showed characteristic wall thickening and lumen reduction in hypoxic wild-type mice, with similar changes observed in CREB(αΔ). CREB(αΔ) mice had larger lungs with reduced epithelial surface density suggesting increased pulmonary compliance. These findings show that α and Δ isoforms of CREB1 regulate homeostatic gene expression in the lung and that normal activity of these isoforms is essential to maintain low pulmonary vascular resistance in both normoxic and hypoxic conditions and to maintain the normal alveolar structure. Interventions that enhance the actions of α and Δ isoforms of CREB1 warrant further investigation in hypoxic lung diseases.

Hypoxia-induced inflammation in the lung: a potential therapeutic target in acute lung injury?

Acute lung injury (ALI) is a severe form of hypoxic lung disease responsible for a large number of deaths worldwide. Despite recent advances in supportive care, no reduction in mortality has been evident since the introduction of a standard consensus definition almost two decades ago. New strategies are urgently required to help design effective therapies for this condition. A key pathological feature of ALI involves regional alveolar hypoxia. Because alveolar hypoxia in isolation, such as that encountered at high altitude, causes an inflammatory pulmonary phenotype in the absence of any other pathogenic stimuli, these regions may not be passive bystanders but may actually contribute to the pathogenesis and progression of lung injury. Unique transcriptional responses to hypoxia in the lung apparently allow it to express an inflammatory phenotype at levels of hypoxia that would not produce such a response in other organs. We will review recent advances in our understanding of these unique transcriptional responses to moderate levels of alveolar hypoxia, which may provide new insights into the pathogenesis of ALI.