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The current standard for lung function evaluation in murine models is based on forced oscillation technology, which provides a measure of the total airway function but cannot provide information on regional heterogeneity in function. Limited detection of regional airflow may contribute to a discontinuity between airway inflammation and airflow obstruction in models of asthma. Here, we describe quantification of regional airway function using novel dynamic quantitative imaging and analysis to quantify and visualize lung motion and regional pulmonary airflow in four dimensions (4D). Furthermore, temporo-spatial specific ventilation (ml/ml) is used to determine ventilation heterogeneity indices for lobar and sublobar regions, which are directly compared to ex vivo biological analyses in the same sublobar regions. In contrast, oscillation-based technology in murine genetic models of asthma have failed to demonstrate lung function change despite altered inflammation, whereas 4D functional lung imaging demonstrated diminished regional lung function in genetic models relative to wild-type mice. Quantitative functional lung imaging assists in localizing the regional effects of airflow. Our approach reveals repeatable and consistent differences in regional airflow between lung lobes in all models of asthma, suggesting that asthma is characterized by regional airway dysfunctions that are often not detectable in composite measures of lung function. 4D functional lung imaging technology has the potential to transform discovery and development in murine models by mapping out regional areas heterogeneously affected by the disease, thus deciphering pathobiology with greater precision.

Quantitative computed tomography (qCT) is being increasingly incorporated in research studies and clinical trials aimed at understanding lung disease risk, progression, exacerbations, and intervention response. Menstrual cycle-based changes in lung function are recognized; however, the impact on qCT measures is currently unknown. We hypothesize that the menstrual cycle impacts qCT-derived measures of lung structure in healthy women and that the degree of measurement change may be mitigated in subjects on cyclic hormonal birth control.Thirty-one non-smoking, healthy women with regular menstrual cycles (16 of which were on cyclic hormonal birth control) underwent pulmonary function testing and qCT imaging at both menses and early luteal phase time points. Data were evaluated to identify lung measurements which changed significantly across the two key time points and to compare degree of change across metrics for the sub-cohort with versus without birth control.The segmental airway measurements were larger and mean lung density was higher at menses compared to the early luteal phase. The sub-cohort with cyclic hormonal birth control did not have less evidence of measurement difference over the menstrual cycle compared to the sub-cohort without hormonal birth control.This study provides evidence that qCT-derived measures from the lung are impacted by the female menstrual cycle. This indicates studies seeking to use qCT as a more sensitive measure of cross-sectional differences or longitudinal changes in these derived lung measurements should consider acquiring data at a consistent time in the menstrual cycle for pre-menopausal women and warrants further exploration.• Lung measurements from chest computed tomography are used in multicenter studies exploring lung disease progression and treatment response. • The menstrual cycle impacts lung structure measurements. • Cyclic variability should be considered when evaluating longitudinal change with CT in menstruating women.

Glucocorticoids are a highly effective first-line treatment option for many inflammatory diseases, including asthma. Some patients develop a steroid-resistant condition, yet, the cellular and molecular mechanisms underlying steroid resistance remain largely unknown. In this study, we used a murine model of steroid-resistant airway inflammation and report that combining systemic dexamethasone and intranasal IL-27 is able to reverse the inflammation. Foxp3+ regulatory T cells (Tregs) were required during dexamethasone/IL-27 treatment of steroid-resistant allergic inflammation, and importantly, direct stimulation of Tregs via glucocorticoid or IL-27 receptors was essential. Mechanistically, IL-27 stimulation in Tregs enhanced expression of the agonistic glucocorticoid receptor-α isoform. Overexpression of inhibitory glucocorticoid receptor-β isoform in Tregs alone was sufficient to elicit steroid resistance in a steroid-sensitive allergic inflammation model. Taken together, our results demonstrate for the first time, to our knowledge, that Tregs are instrumental during steroid resistance and that manipulating steroid responsiveness in Tregs may represent a novel strategy to treat steroid refractory asthma.

In vascular research, clinical samples and samples from animal models are often used together to foster translation of preclinical findings to humans. General concepts of endothelia and murine-specific endothelial phenotypes were discussed in part 1 of this two part series (1). Here, in part 2, we present a comprehensive overview of human-specific endothelial phenotypes. Pan-endothelial cell markers, organ specific endothelial antigens, and flow cytometric immunophenotyping of blood-borne endothelial cells are reviewed.

3β-hydroxysteroid dehydrogenase-1 (3βHSD1), catalyzing conversion of dehydroepiandrosterone (DHEA) to Δ 4-androstenedione, is an essential enzyme in the pathway toward production of biologically active androgens such as dihydrotestosterone from the adrenally produced precursor DHEA sulfate, the most predominant steroid hormone in circulation. We previously identified, in the gene (HSD3B1) that encodes 3βHSD1, a germline gain-of-function missense-encoding variant that has now been validated in several studies as predicting more rapid progression in prostate cancer patients treated with gonadal testosterone deprivation. Production of androgens from adrenal precursors is important not just in the context of prostate cancer but in other physiologic and pathophysiologic processes, which could include asthma. Androgens are associated with better lung function in both asthma and healthy cohorts, and increasing circulating androgen levels in males help explain the switchover in asthma being more common in boys than girls but then more common in women than men. A main treatment for asthma, as well as other inflammatory processes, is administration of glucocorticoids, yet unresponsiveness to glucocorticoids in a subset of patients remains a major problem. Systemic glucocorticoid administration suppresses adrenally produced DHEA and DHEA-S, suggesting a depleted pool for biologically active androgen production as a mechanism for glucocorticoid resistance. Our surprising preliminary data support a link between glucocorticoid responsiveness and the more active HSD3B1 allele: patients homozygous for the adrenal-permissive HSD3B1(1245C) allele exhibit better response to oral glucocorticoids than those homozygous for the adrenal-restrictive HSD3B1(1245A), with heterozygous patients falling in the middle. This suggests a model in which patients with the more active (adrenal-permissive) form of 3βHSD1 produce sufficient androgens despite the depleted pool of precursor hormones whereas patients with the less active (adrenal-restrictive) form cannot. To further elucidate the link between 3βHSD1 activity and immune response, we assayed HSD3B1 expression in different types of white blood cells. Leukocyte subsets from asthma patients and healthy controls were purified using fluorescence-activated cell sorting, and HSD3B1 expression was analyzed using qPCR. White blood cells of several types expressed HSD3B1 at levels comparable to or greater than both prostate cancer and placental choriocarcinoma cell lines with very robust 3βHSD1 activity. Further determining the cell type specific expression and activity of this key enzyme is an important step in unraveling the link between the HSD3B1 polymorphism and asthma along with potentially many other immune processes.

Pulmonary vascular remodeling, including proliferation and migration of pulmonary artery endothelial cells (PAEC), is a pathologic hallmark of pulmonary arterial hypertension (PAH). Multiple studies have shown evidence of increased levels of DNA damage and lineage-specific genetic changes in PAH lung vascular cells, suggesting increased genomic instability. Highly proliferative endothelial colony-forming cell (ECFC) clones can be isolated from PAEC. Here we utilized ECFC to track chromosomal copy number of 20 PAH and eight control clones across serial passages using genome-wide microarrays. All PAH clones were genomically stable for at least 20-22 population doublings. At very late passages, ECFC developed a highly aneuploid karyotype, but this was generally associated with senescence and was common to both PAH and controls. We also utilized ECFC to isolate the chromosomally abnormal cells from a mixed population of PAH PAEC. Analysis of PAEC harboring two different changes affecting chromosomes 1 and X demonstrated that both abnormalities were present in the same clone, indicating they originated in a common ancestral cell. In a second case, with a partial duplication of chromosome 17, clones carrying the duplication were more frequent at later passages than chromosomally normal clones from the same PAEC culture, suggesting the rearrangement may confer a proliferative advantage. Overall, this small study suggests that endothelial cells from PAH lungs are stable in culture, but that when chromosome abnormalities do occur, they may confer a selective advantage that allows expansion of the abnormal cell population and could contribute to lung vascular remodeling in vivo.

Mechanical cell–matrix interactions can drive the innate immune responses to infection; however, the molecular underpinnings of these responses remain elusive. This study was undertaken to understand the molecular mechanism by which the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), alters the in vivo response to lung infection. For the first time, to our knowledge, we show that TRPV4 protects the lung from injury upon intratracheal Pseudomonas aeruginosa in mice. TRPV4 functions to enhance macrophage bacterial clearance and downregulate proinflammatory cytokine secretion. TRPV4 mediates these effects through a novel mechanism of molecular switching of LPS signaling from predominant activation of the MAPK, JNK, to that of p38. This is accomplished through the activation of the master regulator of inflammation, dual-specificity phosphatase 1. Further, TRPV4’s modulation of the LPS signal is mechanosensitive in that both upstream activation of p38 and its downstream biological consequences depend on pathophysiological range extracellular matrix stiffness. We further show the importance of TRPV4 on LPS-induced activation of macrophages from healthy human controls. These data are the first, to our knowledge, to demonstrate new roles for macrophage TRPV4 in regulating innate immunity in a mechanosensitive manner through the modulation of dual-specificity phosphatase 1 expression to mediate MAPK activation switching.

The incidence of chronic, non-healing skin wounds is accelerating, largely due to the epidemic of obesity-related Type 2 diabetes. Abnormal inflammation in wounds contributes to delayed healing. During wound repair, blood monocytes are recruited into the wound bed where they differentiate into macrophages that secrete cytokines and regulate subsequent repair events. Because the study of wound macrophages via immunohistochemistry is often unsatisfactory due to nonspecific antibody staining, the ability to isolate and analyze single cells is important for determining the phenotypes of the wound macrophages. In this article, we have expanded upon a protocol originally described by Wilson et al, 2002 [1], and optimized it for isolation of large numbers of viable macrophages from murine skin wounds that are suitable for flow cytometric cell sorting or analysis. Several parameters were found to be critical for improved macrophage yields, including: (1) The proper amount of starting material (skin tissue); (2) The optimal time for addition of Brefeldin A during enzymatic digestion; (3) Revamped guidelines for centrifugation to maximize cell pellet recovery. This optimized protocol could be further modified to perform cell sorting and flow-based immunophenotyping of any cell type involved in wound healing and inflammation., Graphical abstract Image, graphical abstract

Preparation of a single cell suspension from solid tissue is vital for a successful flow cytometry experiment. We report a detailed and reproducible method to produce a quality cell suspension from the zebrafish retina. Zebrafish retinas, especially their Muller glia cells, are of particular interest for their inherent regenerative capacity, making them a useful model for regenerative medicine and cell therapy research. Here, we detail a papain-based dissociation that is gentle enough to keep cells intact, but strong enough to disrupt cell-cell and cell-matrix interactions to yield a cell suspension that produces clean and reliable flow cytometric cell sorting results. This procedure consistently results in over 90% viability and three populations of cells based on GFP expression. The dissociation procedure described herein has been optimized for the collection of Muller glia from Tg(apoe:gfp) zebrafish retinas; however, the overall process may be applicable to other cell types in the fish retina, additional flow cytometric techniques, or preparing cell suspensions from similar tissues. © 2019 International Society for Advancement of Cytometry.

Protease-activated receptor 2 (PAR-2), an airway epithelial pattern recognition receptor (PRR), participates in the genesis of house dust mite-induced (HDM-induced) asthma. Here, we hypothesized that lung endothelial cells and proangiogenic hematopoietic progenitor cells (PACs) that express high levels of PAR-2 contribute to the initiation of atopic asthma. HDM extract (HDME) protease allergens were found deep in the airway mucosa and breaching the endothelial barrier. Lung endothelial cells and PACs released the Th2-promoting cytokines IL-1α and GM-CSF in response to HDME, and the endothelium had PAC-derived VEGF-C-dependent blood vessel sprouting. Blockade of the angiogenic response by inhibition of VEGF-C signaling lessened the development of inflammation and airway remodeling in the HDM model. Reconstitution of the bone marrow in WT mice with PAR-2-deficient bone marrow also reduced airway inflammation and remodeling. Adoptive transfer of PACs that had been exposed to HDME induced angiogenesis and Th2 inflammation with remodeling similar to that induced by allergen challenge. Our findings identify that lung endothelium and PACs in the airway sense allergen and elicit an angiogenic response that is central to the innate nonimmune origins of Th2 inflammation.

Background Right-sided heart failure is the leading cause of death in pulmonary arterial hypertension (PAH). Similar to left heart failure, sympathetic overactivation and β-adrenoreceptor (βAR) abnormalities are found in PAH. Based on successful therapy of left heart failure with β-blockade, the safety and benefits of the nonselective β-blocker/vasodilator carvedilol were evaluated in PAH. Methods PAH Treatment with Carvedilol for Heart Failure (PAHTCH) is a single-center, double-blind, randomized, controlled trial. Following 1-week run-in, 30 participants were randomized to 1 of 3 arms for 24 weeks: placebo, low-fixed-dose, or dose-escalating carvedilol. Outcomes included clinical measures and mechanistic biomarkers. Results Decreases in heart rate and blood pressure with carvedilol were well tolerated; heart rate correlated with carvedilol dose. Carvedilol-treated groups had no decrease in exercise capacity measured by 6-minute walk, but had lower heart rates at peak and after exercise, and faster heart rate recovery. Dose-escalating carvedilol was associated with reduction in right ventricular (RV) glycolytic rate and increase in βAR levels. There was no evidence of RV functional deterioration; rather, cardiac output was maintained. Conclusions Carvedilol is likely safe in PAH over 6 months of therapy and has clinical and mechanistic benefits associated with improved outcomes. The data provide support for longer and larger studies to establish guidelines for use of β-blockers in PAH. Trial registration ClinicalTrials.gov NCT01586156FUNDING. This project was supported by NIH R01HL115008 and R01HL60917 and in part by the National Center for Advancing Translational Sciences, UL1TR000439.

Life-sustaining responses to low oxygen, or hypoxia, depend on signal transduction by HIFs, but the underlying mechanisms by which cells sense hypoxia are not completely understood. Based on prior studies suggesting a link between the β-adrenergic receptor (β-AR) and hypoxia responses, we hypothesized that the β-AR mediates hypoxia sensing and is necessary for HIF-1α accumulation. Beta blocker treatment of mice suppressed hypoxia induction of renal HIF-1α accumulation, erythropoietin production, and erythropoiesis in vivo. Likewise, beta blocker treatment of primary human endothelial cells in vitro decreased hypoxia-mediated HIF-1α accumulation and binding to target genes and the downstream hypoxia-inducible gene expression. In mechanistic studies, cAMP-activated PKA and/or GPCR kinases (GRK), which both participate in β-AR signal transduction, were investigated. Direct activation of cAMP/PKA pathways did not induce HIF-1α accumulation, and inhibition of PKA did not blunt HIF-1α induction by hypoxia. In contrast, pharmacological inhibition of GRK, or expression of a GRK phosphorylation-deficient β-AR mutant in cells, blocked hypoxia-mediated HIF-1α accumulation. Mass spectrometry-based quantitative analyses revealed a hypoxia-mediated β-AR phosphorylation barcode that was different from the classical agonist phosphorylation barcode. These findings indicate that the β-AR is fundamental to the molecular and physiological responses to hypoxia.

High levels of arginine metabolizing enzymes, including inducible nitric oxide synthase (iNOS) and arginase (ARG), are typical in asthmatic airway epithelium; however, little is known about the metabolic effects of enhanced arginine flux in asthma. Here, we demonstrated that increased metabolism sustains arginine availability in asthmatic airway epithelium with consequences for bioenergetics and inflammation. Expression of iNOS, ARG2, arginine synthetic enzymes, and mitochondrial respiratory complexes III and IV was elevated in asthmatic lung samples compared with healthy controls. ARG2 overexpression in a human bronchial epithelial cell line accelerated oxidative bioenergetic pathways and suppressed hypoxia-inducible factors (HIFs) and phosphorylation of the signal transducer for atopic Th2 inflammation STAT6 (pSTAT6), both of which are implicated in asthma etiology. Arg2-deficient mice had lower mitochondrial membrane potential and greater HIF-2α than WT animals. In an allergen-induced asthma model, mice lacking Arg2 had greater Th2 inflammation than WT mice, as indicated by higher levels of pSTAT6, IL-13, IL-17, eotaxin, and eosinophils and more mucus metaplasia. Bone marrow transplants from Arg2-deficient mice did not affect airway inflammation in recipient mice, supporting resident lung cells as the drivers of elevated Th2 inflammation. These data demonstrate that arginine flux preserves cellular respiration and suppresses pathological signaling events that promote inflammation in asthma.

A proliferation of mast cells around the small pulmonary blood vessels and the alveolar septae has been noted in models of pulmonary hypertension, and in plexiform lesions of pulmonary arterial hypertension (PAH) in patients. Here, we hypothesize that total mast cell numbers and activation are increased in PAH and that they contribute to vascular remodeling through cellular and soluble proangiogenic effectors. To test this, blood and urine were collected from patients with PAH (N=44), asthma (N=18) and healthy controls (N=29) to quantitate biomarkers of total body mast cell numbers and activation (total and mature tryptase, N-methyl histamine, leukotriene LTE(4) and prostaglandin PGD-M). Serum total tryptase was higher in PAH than that in controls suggesting greater numbers of mast cells, but indicators of mast cell activation (mature tryptase, LTE(4) and PGD-M) were similar among PAH, asthma, and controls. Immunohistochemistry of lung tissues identified mast cells as primarily perivascular and connective tissue chymase(+) type in PAH, rather than mucosal phenotype. Intervention with mast cell inhibitors cromolyn and fexofenadine was performed in 9 patients for 12 weeks to identify the influence of mast cell products on the pathologic proangiogenic environment. Treatment decreased total tryptase and LTE-4 levels over time of treatment. This occurred in parallel to a drop in vascular endothelial growth factor (VEGF) and circulating proangiogenic CD34+CD133+ progenitor cells, which suggests that mast cells may promote vascular remodeling and dysfunction. In support of this, levels of exhaled nitric oxide, a vasodilator that is generally low in PAH, increased at the end of the 12-week mast cell blockade and antihistamine. These results suggest that mast cells might contribute to the pulmonary vascular pathologic processes underlying PAH. More studies are needed to confirm their potential contribution to the disease.

Proliferative pulmonary vascular remodeling is the pathologic hallmark of pulmonary arterial hypertension (PAH) that ultimately leads to right heart failure and death. Highly proliferative endothelial cells known as endothelial colony-forming cells (ECFC) participate in vascular homeostasis in health as well as in pathological angiogenic remodeling in disease. ECFC are distinguished by the capacity to clonally proliferate from a single cell. The presence of ECFC in the human pulmonary arteries and their role in PAH pathogenesis is largely unknown. In this study, we established a simple technique for isolating and growing ECFC from cultured pulmonary artery endothelial cells (PAEC) to test the hypothesis that ECFC reside in human pulmonary arteries and that the proliferative vasculopathy of PAH is related to greater numbers and/or more proliferative ECFC in the pulmonary vascular wall. Flow cytometric forward and side scatter properties and aggregate correction were utilized to sort unmanipulated, single PAEC to enumerate ECFC in primary PAEC cultures derived from PAH and healthy lungs. After 2 weeks, wells were assessed for ECFC formation. ECFC derived from PAH PAEC were more proliferative than control. A greater proportion of PAH ECFC formed colonies following subculturing, demonstrating the presence of more ECFC with high proliferative potential among PAH PAEC. Human androgen receptor assay showed clonality of progeny, confirming that proliferative colonies were single cell-derived. ECFC expressed CD31, von Willebrand factor, endothelial nitric oxide synthase, caveolin-1 and CD34, consistent with an endothelial cell phenotype. We established a simple flow cytometry method that allows ECFC quantification using unmanipulated cells. We conclude that ECFC reside among PAEC and that PAH PAEC contain ECFC that are more proliferative than ECFC in control cultures, which likely contributes to the proliferative angiopathic process in PAH.

Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH.

To the Editor: Glutathione (l-γ-glutamyl-l-cysteinylglycine) is found at exceptionally high levels in the human lung (1–3), where it serves as the primary redox couple in the reduced GSH and oxidized GSSG forms. We have identified that the airway epithelium of patients with asthma is shifted to greater reducing potential, recognized by a higher ratio of reduced to oxidized glutathione (4), which occurs in response to the episodic oxidative inflammation of asthma (1, 5–9). On this basis, we hypothesized that regional airway inflammation in asthma may be quantitatively evaluated by a nuclear imaging strategy based on uptake of a radiopharmaceutical (99mTc-exametazime [HMPAO]) selectively retained in tissues depending on GSH levels (10–13) (see Figure E1 in the online supplement). To test this concept, airway redox, inflammation, and 99mTc-HMPAO uptake determined by single-photon emission computed tomography (SPECT) were evaluated in individuals with asthma and in control subjects (Table E1). Some of the results of these studies have been previously reported in the form of abstracts (14–16). 99mTc-HMPAO (Ceretec; GE Healthcare, Arlington Heights, IL) readily diffuses into cells, where it is reduced to a hydrophilic form in the presence of GSH and retained in tissues (Figure E1) (10–13). 99mTc-HMPAO accumulation in the lung was confirmed to be glutathione dependent as indicated by markedly less uptake in mice depleted of glutathione with diethyl maleate (17) as compared with naive mice (Figure E1). Many studies report an increase in total glutathione in asthmatic lungs that occurs in an adaptive response to the episodic asthmatic exacerbations that are characterized by profound oxidative bursts of inflammatory cells in the airways (1, 5–9). Here, in anticipation of imaging, intracellular glutathione was determined in airway epithelial cells obtained at bronchoscopy from patients with asthma and control subjects to assess the magnitude of difference in redox potential between asthma and control lungs. Airway epithelium was obtained by gently brushing the airway, and the freshly obtained cells were immediately processed in the bronchoscopy suite for determination of reduced and oxidized glutathione and reducing potential in human airway epithelium of control subjects and patients with asthma in vivo. Similar to a prior report (4), GSH tended to be higher but GSSG lower in asthma (GSH, μM: patients with asthma, 17 ± 3 [n = 3]; control subjects, 8.9 ± 0.5 [n = 3]; P = 0.14; GSSG, μM: patients with asthma, 0.38 ± 0.04; control subjects, 0.48 ± 0.08; P = 0.2), so that the ratio of GSH to GSSG in the airway epithelium of patients with asthma was double that of control subjects (GSH/GSSG: patients with asthma, 43 ± 6; control subjects, 20 ± 4; P = 0.05) (Figure 1A). The reduction potential (Eh) of the GSH:GSSG couple calculated by Nernst equation (18, 19) revealed a significantly greater intracellular reducing state in the airway epithelium of patients with asthma as compared with control subjects (Eh, mV: patients with asthma, –323 ± 4; control subjects, –306 ± 3; P = 0.04) (Figure 1A). On this basis, we tested whether redox-based imaging might differentiate asthmatic from control lungs, and used SPECT and coregistered computed tomography (CT) imaging to quantitate 99mTc-HMPAO concentration and to determine anatomic sites of 99mTc-HMPAO retention (detailed imaging methods are presented in the online supplement). 99mTc-HMPAO SPECT/CT imaging revealed visibly greater HMPAO uptake in asthmatic lungs as compared with control lungs (Figures 1C and 1D; Movie E1). Normalized uptake values (NUVs) were significantly higher in patients with asthma than control subjects within central and middle transverse areas of lower lung regions (Figure 1E). Interestingly, there was significant variance in the 99mTc-HMPAO NUV of upper and lower lung regions of patients with asthma. Control lung uptake was similar among upper and lower lungs, and across the central, middle, and peripheral transverse regions of the lung. In contrast, patients with asthma had significantly greater 99mTc-HMPAO uptake in lower lung as compared with upper lung regions, and the greatest uptake within central lung regions as compared with middle and peripheral transverse regions (Figure 1E and Table 1). The finding of greater uptake in central lung regions is consistent with a greater distribution of conducting airways in central regions, and the notion of greater reducing potential in asthmatic airways. Overall, the 99mTc-HMPAO uptake findings indicate a greater than normal reducing potential in asthmatic airways as compared with normal airways, as well as significant heterogeneity of lung redox, with the most striking changes in the lower and central regions of the asthmatic lung. In this context, the lower and central regions tend to be the most prominent regions of air trapping (20, 21), which also supports the idea of more severe inflammation/airway disease in those regions. Figure 1. Redox imaging of asthmatic lungs. (A) Glutathione, GSH/GSSG, and glutathione (GSH:GSSG) redox potential (Eh) are greater in the airway epithelium of patients with asthma (n = 3) as compared with control subjects (n = 3). (B–D) Quantitation of ... Table 1: Regional Distribution of Inflammation and 99mTc-HMPAO Uptake in Asthmatic Lung The greater 99mTc-HMPAO uptake in lower regions of asthmatic lungs also suggested that inflammation might be greater in lower lung regions. To investigate this, bronchoalveolar lavage (BAL) was performed in the upper and lower lobes of patients with asthma to compare inflammatory cells and cytokines. BAL volume return, protein concentration, and total cell counts were similar among upper and lower lobes (Table 1). Eosinophils and cytokines that typify helper T-cell type 2 (Th2) inflammation, IL-4 and IL-13, were present in greater concentrations in lower lobes as compared with upper lobes (Table 1). In contrast, IFN-γ, a prototypic Th1-type cytokine, tended to be greater in upper lobes (Table 1). The IL-13 levels in BAL were inversely related to IFN-γ levels (R = –0.483, P = 0.02). This letter reveals that there is a greater reducing potential in the asthmatic airway as compared with control lungs but that there is also substantial heterogeneity of reducing potential across the lung in asthma. This supports the general concept that functional imaging based on redox potential may be used to identify intensity and regionality of airway inflammatory disease. It is important to note that the greater uptake of HMPAO may not necessarily be due to the airway epithelium in asthma. In fact, the endothelium is also inflamed in asthma and might also account for the uptake of the HMPAO (22–26). However, vascular endothelial growth factor was similar among upper and lower lung regions in asthma (Table 1). Further, to assess whether the variation of HMPAO uptake in asthma was related to variation in vascular perfusion, the blood flow in the pulmonary circulation was measured in the supine position, using 99mTc-macroaggregated albumin SPECT/CT (patients with asthma, n = 3; control subjects, n = 3). Pulmonary blood flows were not different in upper and lower regions of the lungs (all P > 0.5). Because all subjects underwent 99mTc-HMPAO SPECT/CT in the supine position (during injection, uptake, and scan), this validated that blood flow to upper and lower parts of the lungs was equally distributed and unlikely to be the cause of variance in upper and lower lobe HMPAO uptake in asthma. Likewise, there may be potential effects and/or dependence of 99mTc-HMPAO uptake on participant age or sex. Although numbers are small, there was no apparent association among 99mTc-HMPAO uptake by age or sex (all P > 0.3). Other studies have identified heterogeneity of remodeling and airflow obstruction in the lungs of patients with asthma as identified by anatomical and functional imaging modalities, for example, high-resolution CT imaging and hyperpolarized helium magnetic resonance imaging (21, 27). The participants in this study were patients with stable asthma, and further investigation is necessary to determine whether 99mTc-HMPAO uptake changes with asthma exacerbations and/or with greater asthma severity. Resolution limitation of SPECT imaging precludes the assignment of uptake to specific airways, limiting the generalized application of 99mTc-HMPAO SPECT/CT imaging in asthma. However, future development of redox-imaging compounds detectable by superior resolution modalities, for example, positron emission tomography, would be a valuable approach to visualize sites of remodeling by coregistered CT images and amounts of inflammation by redox imaging. Current studies rely on invasive bronchoscopic approaches to investigate regional asthma inflammation. The invasive nature and risk of these approaches can preclude enrollment of those patients with severe airflow limitation. A noninvasive functional nuclear imaging approach might offer an alternative quantitative solution to advance research in asthma and assess new biologic-based therapies.

Background & Aims In intestinal inflammation the gut microbiota induces an innate immune response by activating epithelial and immune cells that initiate or maintain inflammation. We investigated whether the microbiota also can activate local microvascular cells and induce angiogenesis. Methods Human intestinal microvascular endothelial cells (HIMEC) and human intestinal fibroblasts (HIF) were exposed to bacterial ligands specific for Toll-like receptor (TLR)2/6 and 4, and NOD1 and NOD2, and cell proliferation, migration, transmigration, tube formation, and production of pro-angiogenic factors were measured. The ability of the ligands to induce ex vivo vessel sprouting in an aortic ring assay and in vivo angiogenesis using a collagen gel assay also were assessed. Results Bacterial ligands induced proliferation, migration, transmigration, tube formation of HIMEC, vessel sprouting, and in vivo angiogenesis; they also stimulated production of angiogenic factors from HIMEC and HIF, and HIF-derived angiogenic factors promoted HIMEC proliferation. To various degrees, all ligands induced angiogenic responses, but these were ligand- and cell type–dependent. Responses were mediated through receptor interacting protein-2 (RIP2)– and tumor necrosis factor receptor–associated factor 6 (TRAF6)–dependent signaling, involved the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways and the up-regulation of vascular endothelial growth factor receptor 2 (VEGF-R2) and focal adhesion kinase (FAK). Knockdown of RIP2 and TRAF6 by RNA interference and neutralization of interleukin-8, basic fibroblast growth factor, and vascular endothelial growth factor inhibited TLR-/NOD-like receptor–induced HIMEC angiogenesis. Conclusions The gut microbiota can selectively activate mucosal endothelial and mesenchymal cells to promote specific angiogenic responses in a TLR- and NOD-like receptor–dependent fashion. This innate immunity-mediated response may expand the mucosal microvascular network, foster immune cell recruitment, and contribute to chronic intestinal inflammation.

Vascular remodeling in pulmonary arterial hypertension (PAH) involves proliferation and migration of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Previous studies have indicated that the endothelial cell proliferation is quasineoplastic, with evidence of monoclonality and instability of short DNA microsatellite sequences.To assess whether there is larger-scale genomic instability.We performed genome-wide microarray copy number analysis on pulmonary artery endothelial cells and smooth muscle cells isolated from the lungs of patients with PAH.Mosaic chromosomal abnormalities were detected in PAEC cultures from five of nine PAH lungs but not in normal (n = 8) or disease control subjects (n = 5). Fluorescent in situ hybridization analysis confirmed the presence of these abnormalities in vivo in two of three cases. One patient harbored a germline mutation of BMPR2, the primary genetic cause of PAH, and somatic loss of chromosome-13, which constitutes a second hit in the same pathway by deleting Smad-8. In two female subjects with mosaic loss of the X chromosome, methylation analysis showed that the active X was deleted. One subject also showed completely skewed X-inactivation in the nondeleted cells, suggesting the pulmonary artery endothelial cell population was clonal before the acquisition of the chromosome abnormality.Our data indicate a high frequency of genetically abnormal subclones within PAH lung vessels and provide the first definitive evidence of a second genetic hit in a patient with a germline BMPR2 mutation. We propose that these chromosome abnormalities may confer a growth advantage and thus contribute to the progression of PAH.

Proliferative diabetic retinopathy is a consequence of retinal ischemia due to capillary occlusion resulting from damage to the retinal microvascular endothelium. Recent evidence suggests that high levels of bone-marrow derived circulating endothelial progenitor cells (EPCs) contribute to the pathological neovascularization of ischemic tissues and are a critical risk factor for the development of these complications. In the absence of a consensus definition of a circulating EPC and its surface markers in humans we evaluated the functional properties of CD34+ CD45− endothelial colony forming cells (ECFCs) in patients with proliferative diabetic retinopathy (PDR). Higher levels of circulating CD34+ CD45− cells were observed in patients with PDR compared to controls. However, ECFCs from patients with PDR were impaired in their ability to migrate towards SDF-1 and human serum, incorporate into and form vascular tubes with human retinal endothelial cells. The results from these pilot studies suggest that ECFCs from patients with PDR are mobilized into the circulation but may be unable to migrate and repair damaged capillary endothelium. This suggests that ECFCs may be a potential therapeutic target in the prevention and treatment of diabetic vascular complications.

Plasticity of the lung vasculature is intrinsically more complex than other organs due to the presence of two blood supply systems under different arterial pressures, the pulmonary and bronchial arterial systems. The bronchial and pulmonary circulations may both contribute to vascular remodelling in lungs after injury or inflammation. Vascular remodelling in the airway is a long recognized component in asthma. Growing numbers of reports suggest that a pro-angiogenic milieu is not a consequence of, but rather dictates the chronic inflammation of asthma. The fairly recent discovery of EPCs (endothelial progenitor cells) has enabled us to study the bone-marrow-derived cells that regulate lung vascular plasticity in asthma. This mini review provides a concise synopsis of our present knowledge about vascular plasticity in adult lungs, summarizes our current view of angioplasticity in asthma and highlights yet unresolved areas of potential interest.

Angiogenesis is a defining pathologic feature of airway remodeling and contributes to asthma severity. Women experience changes in asthma control over the menstrual cycle, a time when vessels routinely form and regress under the control of angiogenic factors. One vital function modulated over the menstrual cycle in healthy women is gas transfer, and this has been related to angiogenesis and cyclic expansion of the pulmonary vascular bed.We hypothesized that changes in gas transfer and the pulmonary vascular bed occur in women with asthma over the menstrual cycle and are associated with worsening airflow obstruction.Twenty-three women, 13 with asthma and 10 healthy control subjects, were evaluated over the menstrual cycle with weekly measures of spirometry, gas transfer, nitric oxide, hemoglobin, factors affecting hemoglobin binding affinity, and proangiogenic factors.Airflow and lung diffusing capacity varied over the menstrual cycle with peak levels during menses that subsequently declined to nadir in early luteal phase. In contrast to healthy women, changes in lung diffusing capacity (DL(CO)) were associated with changes in membrane diffusing capacity and DL(CO) was not related to proangiogenic factors. DL(CO) did not differ between the two groups, although methemoglobin and carboxyhemoglobin were higher in women with asthma than in healthy women.Women with asthma experience cyclic changes in airflow as well as gas transfer and membrane diffusing capacity supportive of a hormonal effect on lung function.

Insulin-like growth factor 1 (IGF-1) plays a pleiotropic role in multiple myeloma (MM), that is, in survival, proliferation, chemotaxis, and angiogenesis. Strategies targeting the IGF-1 receptor (IGF-1R) may therefore be important to develop efficient anti-MM agents. In this work we investigated the effect of an IGF-1R tyrosine kinase (IGF-1RTK) inhibitor (picropodophyllin or PPP) in the 5T33MM mouse model. In vitro data showed that PPP reduced IGF-1R autophosphorylation and downstream ERK activation, leading to inhibition of IGF-1–stimulated proliferation and vascular endothelial growth factor (VEGF) secretion of MM cells. In an in vivo study, PPP reduced the bone marrow tumor burden and serum paraprotein in 5T33MM mice by 77% and 90%, respectively, compared to vehicle-treated animals. Angiogenesis was assessed by quantifying the microvessel density on CD31-stained paraffin sections and this was reduced by 60% in the PPP-treated group. In a separate survival experiment, Kaplan-Meier analysis demonstrated a significant increase in survival in PPP-treated 5T33MM animals compared to the vehicle controls (28 versus 18 days). These data suggest that the IGF-1RTK inhibitor PPP possesses a marked antitumor activity and strongly points to the possibility of using IGF-1R inhibitors in the treatment of MM.

BACKGROUND AND OBJECTIVES: Multiple myeloma (MM) is a lethal plasma cell cancer characterized by the monoclonal growth of cells in the bone marrow. To reach the bone marrow, MM cells need to be attracted by chemokines. Recently, it has been shown that chemokines can also be involved in the growth of several cancer types. Stromal cell derived factor 1a (SDF1alpha) or CXCL12 is known to play an important role as a chemokine for hematopoietic progenitor cells and human MM cells. We studied the effects of SDF1a in the 5TMM murine model. DESIGN AND METHODS: The in vitro effects of SDF1alpha were analyzed by gelatin zymography, adhesion, migration, proliferation, and chemoinvasion assays and by blockade with the CXCR4 inhibitor, 4F-benzoyl-TN14003. In vivo, diseased mice were treated with either vehicle or 4F-benzoyl-TN14003. RESULTS: In vitro SDF1alpha was capable of attracting both 5T2MM and 5T33MM cells and inducing a 1.6-fold increase in MMP9 production by the 5TMM cells, which was correlated with an increased invasive capacity. In addition, SDF1alpha induced a 20% increase in DNA synthesis in the 5TMM cells. All these effects could be blocked by the CXCR4 inhibitor, 4Fbenzoyl- TN14003. An in vivo study in the 5T33MM model showed that blocking CXCR4 led to a 20% reduction in bone marrow tumor load. INTERPRETATION AND CONCLUSIONS: These data demonstrate that SDF1alpha/CXCR4 is involved in the homing and the expansion of MM cells. Blocking CXCR4 could be useful in synergy with other anti-neoplastic treatments targeting the bone marrow microenvironment.

The migration of multiple myeloma (MM) cells from the circulation into the bone marrow (BM) implicates that they must have the capacity to cross the BM endothelium including the subendothelial basement membrane. In this study, human CD138+ MM cells were immunomagnetically isolated from BM samples of MM patients and their invasion through Matrigel, that is, a reconstituted basement membrane, was determined. We demonstrated that primary MM cells have the capacity to transmigrate through basement membrane and that this invasiveness was considerably increased when assessed on Matrigel filters coated with BM endothelial cells (EC) (4LHBMEC line) (transendothelial invasion). The isolated MM cells were shown by zymography to secrete matrix metalloproteinase (MMP)-9 and anti-MMP-9 antibodies inhibited transendothelial invasion, indicating that MMP-9 is involved in this process. BM EC were found to increase the MMP-9 secretion in MM cells, indicating that EC enhance MM cell invasion through stimulation of MMP-9 secretion. BM EC were found to produce hepatocyte growth factor (HGF), and this cytokine also stimulated MMP-9 secretion in MM cells, while anti-HGF antibodies significantly inhibited EC-stimulated MM cell invasion. In summary, our findings provide evidence that MM cell-BM EC interactions enhance the invasion of human MM cells through stimulation of MMP-9 secretion.

Insulin-like growth factor-1 (IGF-1) has been described as an important factor in proliferation, cell survival and migration of multiple myeloma (MM) cells. Angiogenesis correlates with development and prognosis of the MM disease. Vascular endothelial growth factor (VEGF) is one of the prominent factors involved in this process. The different functions of IGF-1 were investigated in the 5TMM mouse model with emphasis on proliferation, migration and VEGF secretion, and the signalling pathways involved. Western Blot analysis revealed that ERK1/2 and Akt (PKB) were activated after IGF-1 stimulation. The activation of ERK1/2 was reduced by the PI3K inhibitor Wortmannin, implying that the PI3K pathway is involved in its activation. Insulin-like growth factor-1 induced an increase in DNA synthesis in MM cells, which was mediated by a PI3K/Akt-MEK/ERK pathway. Insulin-like growth factor-1 enhanced F-actin assembly and this process was only PI3K mediated. Stimulation by IGF-1 of VEGF production was reduced by PD98059, indicating that only the MEK–ERK pathway is involved in IGF-1-stimulated VEGF production. In conclusion, IGF-1 mediates its multiple effects on MM cells through different signal transduction pathways. In the future, we can study the potential in vivo effects of IGF-1 inhibition on tumour growth and angiogenesis in MM.

It is now well established that angiogenesis in multiple myeloma (MM) is associated with poor prognosis. The exact function of the newly formed vessels in MM is, however, a matter of debate. It is believed that, in contrast to solid tumor growth, the bone marrow (BM) is a sufficiently vascularized organ to support expansion of the MM clone with no need for additional blood vessels. From this point of view, it could be that MM-associated angiogenesis is rather an epiphenomenon and that the newly formed microvessels form a chaotic network that does not contribute to the blood flow. We investigated whether these newly formed microvessels in MM are connected to the blood circulation. The intravenously injected ferritin 30 min prior to sacrifice was detected in 100% of the BM vessels of control mice. In MM-bearing mice, the ferritin tracer was found in 31% of the MM-associated vessels, indicating a connection with the peripheral blood circulation in these vessels. We conclude that, comparable to the situation in solid tumors, at least part of the tumor-associated microvessels in MM is functionally connected to the blood circulation and, therefore, can participate in the transport of nutrients and in the dissemination of MM cells.

Multiple myeloma (MM) is a B cell neoplasm characterized by the monoclonal proliferation of plasma cells in the bone marrow, the development of osteolytic lesions and the induction of angiogenesis. These different processes require three-dimensional interactions, with both humoral and cellular contacts. The 5TMM models are suitable models to study these interactions. These murine models originate from spontaneously developed myeloma in elderly mice, which are propagated by in vivo transfer of the myeloma cells into young syngeneic mice. In this review we report on studies performed in the 5TMM models with special emphasis on the homing of the myeloma cells, the characterization of the migrating and proliferating clone and the identification of the isotype switch variants. The bone marrow microenvironment was further targeted with osteoprotegerin (OPG) to block the RANK/RANKL/OPG system and with potent bisphosphonates. Both treatments resulted in a significant protection against myeloma-associated bone disease, and they decreased myeloma disease, as evidenced by a lower tumor load and an increased survival of the mice. These different studies demonstrate the strength of these models, not only in unraveling basic biological processes but also in the testing of potentially new therapeutic targets.

Both CD45(+) and CD45(-) multiple myeloma (MM) cells are observed in the bone marrow (BM) of MM patients; however, their impact on the outcome of the disease is unknown. Most (92%) of the mice injected with murine 5T2MM cells develop myeloma in 10-12 weeks and show hind leg paralysis at the end phase of the disease. In the end stage 5T2MM cells are predominantly CD45(-), in analogy to the common human situation. Herein we report that 8% of the mice have a delayed tumor progression (14-24 weeks) with a complete different feature in the end stage of the disease. These animals had typically a bowed back and never got paralyzed. The MM cells in the BM of these mice were predominantly CD45(+). These data indicate that CD45 subsets are associated with the final outcome of myeloma disease.

The aim of the present study was to determine whether modifying the local bone environment with osteoprotegerin (OPG), the soluble decoy receptor for receptor activator of nuclear factor-kappaB (RANK) ligand, could affect tumor burden and survival in the 5T33MM murine model of multiple myeloma. Treatment of mice, injected with 5T33MM cells, with recombinant OPG (Fc-OPG) caused a significant decrease in serum paraprotein and tumor burden and a significant increase in time to morbidity. This was associated with a decrease in osteoclast number in vivo but had no effect on apoptosis and proliferation of 5T33MM cells in vitro. These data indicate that targeting the bone microenvironment by inhibiting the interaction between RANK ligand and RANK with Fc-OPG not only inhibits the development of myeloma bone disease but also decreases tumor growth and increases survival.

Multiple myeloma (MM) is an incurable plasma cell cancer, localized in the bone marrow (BM). The mechanisms used by these cells to (re-)enter this organ remain largely unknown. Recently, we reported that both CD45+ and CD45− myeloma cells home to the BM and induce myeloma disease. In this work, we investigated the underlying mechanisms involved in the homing of CD45+ and CD45− myeloma cells in the experimental 5T2MM and 5T33MM murine models. In vivo tracing of flow cytometric sorted and radioactively labeled CD45 subsets revealed a reduced homing of the CD45− 5TMM cells to the BM as compared to the CD45+ 5TMM cells. Migration assays demonstrated an impaired chemotaxis towards BM endothelial cell conditioned medium, BM stromal cell conditioned medium and towards the basement membrane component laminin-1 of the CD45− 5TMM cells compared to the CD45+ subset. Matrix metalloproteinase-9 (MMP-9) and urokinase type plasminogen activator (uPA) are key extracellular matrix proteases involved in the invasion ofcancer cells. Inhibitor and antibody blocking experiments demonstrated the involvement of both in the invasion of the 5TMM cells. CD45− 5TMM cells had a low secretion of MMP-9 and (for the non-aggressive line 5T2MM only) a low cell surface expression of uPA receptor, as revealed by gelatin zymography and flow cytometric analysis, respectively. Accordingly, the synthetic basement membrane invasive capacity of the CD45− 5TMM subpopulations was also impaired. Our results indicate that CD45+ and CD45− 5T myeloma cells have a differential BM homing attributable to differential migratory and invasive capacities.