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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.

Allergic airway disease models use laboratory mice housed in highly controlled and hygienic environments, which provide a barrier between the mice and a predetermined list of specific pathogens excluded from the facility. In this study, we hypothesized that differences in facility barrier level and, consequently, the hygienic quality of the environment that mice inhabit impact the severity of pulmonary inflammation and lung function. Allergen-naive animals housed in the cleaner, high barrier (HB) specific pathogen-free facility had increased levels of inflammatory cytokines and higher infiltration of immune cells in the lung tissue but not in the bronchoalveolar lavage compared with mice housed in the less hygienic, low barrier specific pathogen-free facility. In both genders, house dust mite–induced airway disease was more severe in the HB than the low barrier facility. Within each barrier facility, female mice developed the most severe inflammation. However, allergen-naive male mice had worse lung function, regardless of the housing environment, and in the HB, the lung function in female mice was higher in the house dust mite model. Severe disease in the HB was associated with reduced lung microbiome diversity. The lung microbiome was altered across housing barriers, gender, and allergen-exposed groups. Thus, the housing barrier level impacts microbial-driven disease and gender phenotypes in allergic asthma. The housing of laboratory mice in more clean HB facilities aggravates lung immunity and causes a more severe allergic lung disease.

Intact cell-free mitochondria have been reported in microparticles (MPs) in murine and human bodily fluids under disease conditions. However, cellular origins of circulating extracellular mitochondria have not been characterized. We hypothesize that intact, cell-free mitochondria from heterogeneous cellular sources are present in the circulation under physiological conditions. To test this, circulating MPs were analyzed using flow cytometry and proteomics. Murine and human platelet-depleted plasma showed a cluster of MPs positive for the mitochondrial probe MitoTracker. Transgenic mice expressing mitochondrial-GFP showed GFP positivity in plasma MPs. Murine and human mitochondria-containing MPs were positive for the platelet marker CD41 and the endothelial cell marker CD144, while hematopoietic CD45 labeling was low. Both murine and human circulating cell-free mitochondria maintained a transmembrane potential. Circulating mitochondria were able to enter rho-zero cells, and were visualized using immunoelectron microscopic imaging. Proteomics analysis identified mitochondria specific and extracellular vesicle associated proteins in sorted circulating cell-free human mitochondria. Together the data provide multiple lines of evidence that intact and functional mitochondria originating from several cell types are present in the blood circulation.

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.

Preparing a single cell suspension is a critical step in any solid tissue flow cytometry experiment. Tissue dissection, enzymatic digestion, and mechanical dissociation are three significant steps leading to the degradation of the extracellular matrix and the isolation of single cells, allowing the generation of high-quality flow cytometry data. Cells and the extracellular matrix contain various proteins and other structures which must be considered when designing a tissue digestion protocol to preserve the viability of cells and the presence of relevant antigens while digesting matrix components and cleaving cell-cell junctions. Evaluation of the single cell suspension is essential before proceeding with the labeling of the cells as high viability and absence of cell debris and aggregates are critical for flow cytometry. The information presented should be used as a general guide of steps to consider when preparing a single cell suspension from solid tissues for flow cytometry experiments. PURPOSE: The purpose of this paper is to define the critical steps in the segregation of solid tissues by describing the composition of tissues and the common digestive enzymes used to digest extracellular matrix and cleave cell-cell junctions to obtain a single cell suspension.

Mechanisms that degrade inflammatory mRNAs are well known; however, stabilizing mechanisms are poorly understood. Here, we show that Act1, an interleukin-17 (IL-17)-receptor-complex adaptor, binds and stabilizes mRNAs encoding key inflammatory proteins. The Act1 SEFIR domain binds a stem-loop structure, the SEFIR-binding element (SBE), in the 3' untranslated region (UTR) of Cxcl1 mRNA, encoding an inflammatory chemokine. mRNA-bound Act1 directs formation of three compartmentally distinct RNA-protein complexes (RNPs) that regulate three disparate events in inflammatory-mRNA metabolism: preventing mRNA decay in the nucleus, inhibiting mRNA decapping in P bodies and promoting translation. SBE RNA aptamers decreased IL-17-mediated mRNA stabilization in vitro, IL-17-induced skin inflammation and airway inflammation in a mouse asthma model, thus providing a therapeutic strategy for autoimmune diseases. These results reveal a network in which Act1 assembles RNPs on the 3' UTRs of select mRNAs and consequently controls receptor-mediated mRNA stabilization and translation during inflammation.

Inducible nitric oxide synthase (iNOS) and arginase-2 (ARG2) share a common substrate, arginine. Higher expression of iNOS and exhaled NO are linked to airway inflammation in patients. iNOS deletion in animal models suggests that eosinophilic inflammation is regulated by arginine metabolism. Moreover, ARG2 is a regulator of Th2 response, as shown by the development of severe eosinophilic inflammation in ARG2(–/–) mice. However, potential synergistic roles of iNOS and ARG2 in asthma have not been explored. Here, we hypothesized that arginine metabolic fate via iNOS and ARG2 may govern airway inflammation. In an asthma cohort, ARG2 variant genotypes were associated with arginase activity. ARG2 variants with lower arginase activity, combined with levels of exhaled NO, identified a severe asthma phenotype. Airway inflammation was present in WT, ARG2(–/–), iNOS(–/–), and ARG2(–/–)/iNOS(–/–) mice but was greatest in ARG2(–/–). Eosinophilic and neutrophilic infiltration in the ARG2(–/–) mice was abrogated in ARG2(–/–)/iNOS(–/–) animals. Similarly, angiogenic airway remodeling was greatest in ARG2(–/–) mice. Cytokines driving inflammation and remodeling were highest in lungs of asthmatic ARG2(–/–) mice and lowest in the iNOS(–/–). ARG2 metabolism of arginine suppresses inflammation, while iNOS metabolism promotes airway inflammation, supporting a central role for arginine metabolic control of inflammation.

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.

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.

The β-adrenergic receptor (βAR) exists in an equilibrium of inactive and active conformational states, which shifts in response to different ligands and results in downstream signaling. In addition to cAMP, βAR signals to hypoxia-inducible factor 1 (HIF-1). We hypothesized that a βAR-active conformation (R**) that leads to HIF-1 is separable from the cAMP-activating conformation (R*) and that pulmonary arterial hypertension (PAH) patients with HIF-biased conformations would not respond to a cAMP agonist. We compared two cAMP agonists, isoproterenol and salbutamol, in vitro. Isoproterenol increased cAMP and HIF-1 activity, while salbutamol increased cAMP and reduced HIF-1. Hypoxia blunted agonist-stimulated cAMP, consistent with receptor equilibrium shifting toward HIF-activating conformations. Similarly, isoproterenol increased HIF-1 and erythropoiesis in mice, while salbutamol decreased erythropoiesis. βAR overexpression in cells increased glycolysis, which was blunted by HIF-1 inhibitors, suggesting increased βAR leads to increased hypoxia-metabolic effects. Because PAH is also characterized by HIF-related glycolytic shift, we dichotomized PAH patients in the Pulmonary Arterial Hypertension Treatment with Carvedilol for Heart Failure trial (NCT01586156) based on right ventricular (RV) glucose uptake to evaluate βAR ligands. Patients with high glucose uptake had more severe disease than those with low uptake. cAMP increased in response to isoproterenol in mononuclear cells from low-uptake patients but not in high-uptake patients’ cells. When patients were treated with carvedilol for 1 wk, the low-uptake group decreased RV systolic pressures and pulmonary vascular resistance, but high-uptake patients had no physiologic responses. The findings expand the paradigm of βAR activation and uncover a novel PAH subtype that might benefit from β-blockers.

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.

Airway fibrosis is a prominent feature of asthma, contributing to the detrimental consequences of the disease. Fibrosis in the airway is the result of collagen deposition in the reticular lamina layer of the subepithelial tissue. Myofibroblasts are the leading cell type involved with this collagen deposition. Established methods of collagen deposition quantification present various issues, most importantly their inability to quantify current collagen biosynthesis occurring in airway myofibroblasts. Here a novel method to quantify myofibroblast collagen expression in asthmatic lungs is described. Single cell suspensions of lungs harvested from C57BL/6 mice in a standard house dust mite model of asthma were employed to establish a flow cytometric method and compare collagen production in asthmatic and non-asthmatic lungs. Cells found to be CD45(−)αSMA(+), indicative of myofibroblasts, were gated, and median fluorescence intensity of the anti-collagen-I antibody labeling the cells was calculated. Lung myofibroblasts with no, medium, or high levels of collagen-I expression were distinguished. In asthmatic animals, collagen-I levels were increased in both medium and high expressers, and the number of myofibroblasts with high collagen-I content was elevated. Our findings determined that quantification of collagen-I deposition in myofibroblastic lung cells by flow cytometry is feasible in mouse models of asthma and indicative of increased collagen-I expression by asthmatic myofibroblasts.

Asthma airway remodeling is linked to Th2 inflammation. Angiogenesis is a consistent feature of airway remodeling, but its contribution to pathophysiology remains unclear. We hypothesized that nascent endothelial cells in newly forming vessels are sufficient to initiate Th2-inflammation. Vascular endothelial (VE)-cadherin is a constitutively expressed endothelial cell adhesion molecule that is exposed in its monomer form on endothelial tip cells prior to adherens junction formation. Abs targeted to VE-cadherin monomers inhibit angiogenesis by blocking this adherens junction formation. In this study, VE-cadherin monomer Ab reduced angiogenesis in the lungs of the allergen-induced murine asthma model. Strikingly, Th2 responses including, IgE production, eosinophil infiltration of the airway, subepithelial fibrosis, mucus metaplasia, and airway-hyperreactivity were also attenuated by VE-cadherin blockade, via mechanisms that blunted endothelial IL-25 and proangiogenic progenitor cell thymic stromal lymphopoietin production. The results identify angiogenic responses in the origins of atopic inflammation.

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.

The purpose of this study was to determine whether bone marrow-derived cells can differentiate into myofibroblasts, as defined by alpha smooth muscle actin (SMA) expression, that arise in the corneal stroma after irregular phototherapeutic keratectomy and whose presence within the cornea is associated with corneal stromal haze. C57BL/6J-GFP chimeric mice were generated through bone marrow transplantation from donor mice that expressed enhanced green fluorescent protein (GFP) in a high proportion of their bone marrow-derived cells. Twenty-four GFP chimeric mice underwent haze-generating corneal epithelial scrape followed by irregular phototherapeutic keratectomy (PTK) with an excimer laser in one eye. Mice were euthanized at 2 weeks or 4 weeks after PTK and the treated and control contralateral eyes were removed and cryo-preserved for sectioning for immunocytochemistry. Double immunocytochemistry for GFP and myofibroblast marker alpha smooth muscle actin (SMA) were performed and the number of SMA+GFP+, SMA+GFP−, SMA−GFP+ and SMA−GFP− cells, as well as the number of DAPI+ cell nuclei, per 400X field of stroma was determined in the central, mid-peripheral and peri-limbal cornea. In this mouse model, there were no SMA+ cells and only a few GFP+ cells detected in unwounded control corneas. No SMA+ cells were detected in the stroma at two weeks after irregular PTK, even though there were numerous GFP+ cells present. At 4 weeks after irregular PTK, all corneas developed mild to moderately severe corneal haze. In each of the three regions of the corneas examined, there were on average more than 9X more SMA+GFP+ than SMA+GFP− myofibroblasts. This difference was significant (p

Background Eosinophilic inflammation is closely related to angiogenesis in asthmatic airway remodeling. In ovalbumin (OVA)–sensitized mice bone marrow–derived, proangiogenic endothelial progenitor cells (EPCs) are rapidly recruited into the lungs after OVA aerosol challenge and promptly followed by mobilization and recruitment of eosinophils. Objective We hypothesized that bone marrow–derived EPCs initiate the recruitment of eosinophils through expression of the eosinophil chemoattractant eotaxin-1. Methods EPCs were isolated from an OVA murine model of allergic airway inflammation and from asthmatic patients. Endothelial and smooth muscle cells were isolated from mice. Eotaxin-1 expression was analyzed by means of immunofluorescence, real-time PCR, or ELISA. In vivo recruitment of eosinophils by EPCs was analyzed in mice. Results Circulating EPCs of asthmatic patients had higher levels of eotaxin-1 compared with those seen in control subjects. In the murine model OVA allergen exposure augmented eotaxin-1 mRNA and protein levels in EPCs. The EPCs from OVA-sensitized and OVA-challenged mice released high levels of eotaxin-1 on contact with lung endothelial cells from sensitized and challenged mice but not from control animals and not on contact with cardiac or hepatic endothelial cells from sensitized and challenged mice. Intranasal administration of the eotaxin-rich media overlying cultures of EPCs caused recruitment into the lungs, confirming functional chemoattractant activity. Conclusions Bone marrow–derived EPCs are early responders to environmental allergen exposures and initiate a parallel switch to a proangiogenic and proeosinophilic environment in the lungs of asthmatic patients.

Matrix metalloproteinases (MMPs), a specialized group of enzymes capable of proteolytically degrading extracellular matrix proteins, have been postulated to play an important role in angiogenesis. It has been suggested that MMPs can regulate neovascularization using mechanisms other than simple remodeling of the capillary basement membrane. To determine the interplay between vascular endothelial growth factor (VEGF) and MMPs, we investigated the induction of angiogenesis by recombinant active MMPs and VEGF in vivo. Using a rat corneal micropocket in vivo angiogenesis assay, we observed that the active form of MMP-9 could induce neovascularization in vivo when compared with the pro- form of the enzyme as a control. This angiogenic response could be inhibited by neutralizing VEGF antibody, which suggests that MMPs acts upstream of VEGF. Additional in vitro studies using extracellular matrix loaded with radiolabeled VEGF determined that active MMPs can enzymatically release sequestered VEGF. Interestingly, in vivo angiogenesis induced by VEGF could be inhibited by MMP inhibitors, indicating that MMPs also act downstream of VEGF. In addition, inflammation plays an important role in the induction of angiogenesis mediated by both VEGF and MMPs. Our results suggest that MMPs act both upstream and downstream of VEGF and imply that potential combination therapies of VEGF and MMP inhibitors may be a useful therapeutic approach in diseases of pathological neovascularization.

Purpose: The purpose of this study was to investigate expression and epigenetic regulation of CD9 in multiple myeloma (MM) cells during disease progression. Experimental Design: CD9 expression was retrospectively analyzed on bone marrow myeloma samples from 81 patients by immunophenotyping. CD9 expression by murine 5TMM cells was detected by flow cytometric staining and quantitative PCR. The methylation status of the CD9 promoter was determined by bisulfite PCR sequencing. Results: Primary plasma cells in the majority of MM patients with nonactive disease (n = 28) showed CD9 expression, whereas most cases with active disease (n = 53) were CD9 negative. CD9 expression in diagnostic bone marrow samples (n = 74) correlated with survival. Moreover, CD9 expression on murine 5T33 and 5T2MM cells was significantly down-regulated during disease development. Treatment of CD9-nonexpressing 5T33MMvt cells with the clinically relevant histone deacetylase inhibitor LBH589 resulted in a significant increase in CD9 expression. In contrast, cells treated with the demethylation agent 5-aza-2′deoxycytidine barely showed any increase. A combination study with both compounds resulted in a strong synergistic reactivation of CD9. CD9-expressing 5T33MMvv cells and 5T33MMvt cells stably transduced with a mCD9 lentiviral transferplasmid were shown to be more susceptible to natural killer cell–mediated cytolysis than CD9-negative 5T33MMvt cells. Conclusions: CD9 expression correlates with disease status and survival of MM patients. In the murine 5T33MM model, we show that histone modifications, and to a lesser extent CpG methylation, are key epigenetic events in CD9 down-regulation. Furthermore, as CD9 expression becomes down-regulated, 5T33MM cells become less susceptible to natural killer cell–mediated cytolysis.

Vascular remodeling in idiopathic pulmonary arterial hypertension (IPAH) involves hyperproliferative and apoptosis-resistant pulmonary artery endothelial cells. In this study, we evaluated the relative contribution of bone marrow-derived proangiogenic precursors and tissue-resident endothelial progenitors to vascular remodeling in IPAH. Levels of circulating CD34 + CD133 + bone marrow-derived proangiogenic precursors were higher in peripheral blood from IPAH patients than in healthy controls and correlated with pulmonary artery pressure, whereas levels of resident endothelial progenitors in IPAH pulmonary arteries were comparable to those of healthy controls. Colony-forming units of endothelial-like cells (CFU-ECs) derived from CD34 + CD133 + bone marrow precursors of IPAH patients secreted high levels of matrix metalloproteinase-2, had greater affinity for angiogenic tubes, and spontaneously formed disorganized cell clusters that increased in size in the presence of transforming growth factor-β or bone morphogenetic protein-2. Subcutaneous injection of NOD SCID mice with IPAH CFU-ECs within Matrigel plugs, but not with control CFU-ECs, produced cell clusters in the Matrigel and proliferative lesions in surrounding murine tissues. Thus, mobilization of high levels of proliferative bone marrow-derived proangiogenic precursors is a characteristic of IPAH and may participate in the pulmonary vascular remodeling process.

Angiogenesis is closely linked to and precedes eosinophilic infiltration in asthma. Eosinophils are recruited into the airway by chemoattractant eotaxins, which are expressed by endothelial cells, smooth muscles cells, epithelial cells, and hematopoietic cells. We hypothesized that bone marrow–derived proangiogenic progenitor cells that contain eotaxins contribute to the initiation of angiogenesis and inflammation in asthma. Whole-lung allergen challenge of atopic asthma patients revealed vascular activation occurs within hours of challenge and before airway inflammation. The eotaxin receptor CCR3 was expressed at high levels on submucosal endothelial cells in patients and a murine model of asthma. Ex vivo exposure of murine endothelial cells to eotaxins induced migration and angiogenesis. In mechanistic studies, wild-type mice transplanted with eotaxin-1/2–deficient bone marrow had markedly less angiogenesis and inflammation in an atopic asthma model, whereas adoptive transfer of proangiogenic progenitor cells from wild-type mice in an atopic asthma model into the eotaxin-1/2–deficient mice led to angiogenesis and airway inflammation. The findings indicate that Th2-promoting hematopoietic progenitor cells are rapidly recruited to the lung upon allergen exposure and release eotaxins that coordinately activate endothelial cells, angiogenesis, and airway inflammation.

Hematopoietic myeloid progenitors released into the circulation are able to promote vascular remodeling through endothelium activation and injury. Endothelial injury is central to the development of pulmonary arterial hypertension (PAH), a proliferative vasculopathy of the pulmonary circulation, but the origin of vascular injury is unknown. In the present study, mice transplanted with BM-derived CD133+ progenitor cells from patients with PAH, but not from healthy controls, exhibited morbidity and/or death due to features of PAH: in situ thrombi and endothelial injury, angioproliferative remodeling, and right ventricular hypertrophy and failure. Myeloid progenitors from patients with heritable and/or idiopathic PAH all produced disease in xenografted mice. Analyses of hematopoietic transcription factors and colony formation revealed underlying abnormalities of progenitors that skewed differentiation toward the myeloid-erythroid lineage. The results of the present study suggest a causal role for hematopoietic stem cell abnormalities in vascular injury, right ventricular hypertrophy, and morbidity associated with PAH.

Considering the high cost of dedicated small-animal positron emission tomography/computed tomography (PET/CT), an acceptable alternative in many situations might be clinical PET/CT. However, spatial resolution and image quality are of concern. The utility of clinical PET/CT for small-animal research and image quality improvements from super-resolution (spatial subsampling) were investigated. National Electrical Manufacturers Association (NEMA) NU 4 phantom and mouse data were acquired with a clinical PET/CT scanner, as both conventional static and stepped scans. Static scans were reconstructed with and without point spread function (PSF) modeling. Stepped images were postprocessed with iterative deconvolution to produce super-resolution images. Image quality was markedly improved using the super-resolution technique, avoiding certain artifacts produced by PSF modeling. The 2 mm rod of the NU 4 phantom was visualized with high contrast, and the major structures of the mouse were well resolved. Although not a perfect substitute for a state-of-the-art small-animal PET/CT scanner, a clinical PET/CT scanner with super-resolution produces acceptable small-animal image quality for many preclinical research studies.

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.

Gas transfer in the female lung varies over the menstrual cycle in parallel with the cyclic angiogenesis that occurs in the uterine endometrium. Given that vessels form and regress in the uterus under the control of hormones, angiogenic factors, and proangiogenic circulating bone marrow-derived progenitor cells, we tested the possibility that variation in pulmonary gas transfer over the menstrual cycle is related to a systemic cyclic proangiogenic state that influences lung vascularity. Women were evaluated over the menstrual cycle with weekly measures of lung diffusing capacity and its components, the pulmonary vascular capillary bed and membrane diffusing capacity, and their relation to circulating CD34+CD133+progenitor cells, hemoglobin, factors affecting hemoglobin binding affinity, and proangiogenic factors. Lung diffusing capacity varied over the menstrual cycle, reaching a nadir during the follicular phase following menses. The decline in lung diffusing capacity was accounted for by ∼25% decrease in pulmonary capillary blood volume. In parallel, circulating CD34+CD133+progenitor cells decreased by ∼24% and were directly related to angiogenic factors and to lung diffusing capacity and pulmonary capillary blood volume. The finding of a greater number of lung microvessels in ovariectomized female mice receiving estrogen compared with placebo verified that pulmonary vascularity is influenced by hormonal changes. These findings suggest that angiogenesis in the lungs may participate in the cyclic changes in gas transfer that occur over the menstrual cycle.

Increased numbers of submucosal vessels are a consistent pathologic component of asthmatic airway remodeling. However, the relationship between new vessel formation and asthmatic inflammatory response is unknown. We hypothesized that angiogenesis is a primary event during the initiation of airway inflammation and is linked to the recruitment of bone marrow-derived endothelial progenitor cells (EPC). To test this hypothesis, circulating EPC and EPC-derived endothelial cell colony formation of individuals with asthma or allergic rhinitis and health controls was evaluated. Circulating EPC were increased in asthma, highly proliferative, and exhibited enhanced incorporation into endothelial cell tubes as compared with controls. In an acute allergen challenge murine asthma model, EPC mobilization occurred within hours of challenge and mobilized EPC were selectively recruited into the challenged lungs of sensitized animals, but not into other organs. EPC recruitment was Th1 and Th2 dependent and was temporally associated with an increased microvessel density that was noted within 48 h of allergen challenge, indicating an early switch to an angiogenic lung environment. A chronic allergen challenge model provided evidence that EPC recruitment to the lung persisted and was associated with increasing microvessel density over time. Thus, a Th1- and Th2-dependent angiogenic switch with EPC mobilization, recruitment, and increased lung vessel formation occurs early but becomes a sustained and cumulative component of the allergen-induced asthmatic response.

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.

Multiple myeloma 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 to study these interactions. These are murine models that originate from spontaneously developed myeloma in elderly mice. They are propagated by in vivo transfer of the myeloma cells into young syngeneic mice. We report methods involving the maintenance of the 5T2MM model and the quantification of tumor burden (by determining serum paraprotein concentration and plasmacytosis), assessment of bone lesions, and quantification of angiogenesis. The combination of these different techniques in these models not only helps in unraveling basic biological processes but also in the testing of potentially new therapeutic targets.

Liver sinusoids harbor populations of 2 important types of immunocompetent cells, Kupffer cells (KCs) and natural killer (NK) cells, which are thought to play an important role in controlling hepatic metastasis in the first 24 hr upon arrival of the tumor cells in the liver. We studied the early interaction of KCs, NK and CC531s colon carcinoma cells in a syngeneic rat model by confocal laser scanning microscopy. Results showed a minority of KCs (19% periportal and 7% pericentral) involved in the interaction with 94% of tumor cells and effecting the phagocytosis of 92% of them. NK cell depletion decreased the phagocytosis of tumor cells by KCs by 33% over a period of 24 hr, leaving 35% of the cancer cells free, as compared to 6% in NK-positive rats. Surviving cancer cells were primarily located close to the Glisson capsule, suggesting that metastasis would initiate from this region.

Myeloma is a deadly B-cell neoplasm, characterized by the monoclonal proliferation of plasma cells, the development of osteolytic lesions, and the induction of angiogenesis. Myeloma cells are predominantly localized in the marrow where they receive the appropriate survival and proliferation signals. To reach or spread over the marrow, the myeloma cells need to migrate from the vascular to the extravascular compartment of the marrow. A process called "homing". In this review, the steps of the homing scheme, analyzed in the 5TMM model, will be described. These murine models originated from spontaneously developed myeloma in elderly mice and have since been propagated by intravenous injection of myeloma cells into young syngeneic mice. These models resemble the human condition closely. The different studies reported here demonstrate that adhesion of 5TMM cells to marrow endothelial cells is partially mediated by CD44v10 and to stromal cells by CD44v6. The 5TMM cells migrate to the marrow through the effects of MCP-1, laminin-1, and IGF-1. Once past the marrow endothelium, they invade the extravascular compartment of the marrow by secreting MMP-9 and uPA. When they have settled in the marrow, they become susceptible to the effects of IGF-1, which stimulates the cells to proliferate and produce VEGF. Furthermore, studies targeting the marrow with inhibitors will be highlighted. These studies show that the 5TMM models are useful for unraveling basic biological processes and for identifying new therapeutic targets.

The active role of angiogenesis during disease progression is well recognized in solid tumors. In hematologic malignancies such as multiple myeloma (MM), it is not known whether tumor neovascularization is an epiphenomenon or whether it is actively involved in disease progression. At clinical presentation, myeloma disease and the associated angiogenesis are both well established. Here the 5T2MM murine model was used to analyze angiogenesis during preclinical myeloma stages. Bone marrow (BM) of 5T2MM-inoculated mice was analyzed at weekly intervals until the end stage of the disease. Histologic analysis and assessment of microvessel density (MVD) by CD31 staining demonstrated a preangiogenic stage of small tumor aggregates followed by an angiogenic switch and subsequently an angiogenic stage of progressive tumor growth and large, confluent tumor nodules. Flow cytometric analysis that indicated an increase in percentage CD45- MM cells preceded the angiogenic switch. Real-time polymerase chain reaction (RT-PCR) of sorted CD45+ and CD45- MM cells indicated higher vascular endothelial growth factor 120 (VEGF120) and VEGF164 transcripts in CD45- MM cells. VEGF enzyme-linked immunosorbent assay (ELISA) revealed high secretion by CD45- MM cells but no protein secretion by CD45+ MM cells, indicating angiogenic heterogeneity among the MM cells. These data suggest that, like in solid tumors, angiogenic switch and angiogenic heterogeneity exist in MM. (Blood. 2004;103:3131-3137)

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.

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.

Lymphocyte function-associated antigen-1 (LFA-1) expression on multiple myeloma cells and its potential role in myeloma biology have been the subject of conflicting literature reports. In this study we used the 5T experimental mouse model to analyze the involvement of LFA-1 in myeloma cell bone marrow homing, survival, and growth.The 5T33MM vitro (5T33MMvt) myeloma line was used. LFA-1 and intercellular adhesion molecule-1 (ICAM-1) expression were analyzed by flow cytometry. A small molecule antagonist of LFA-1/ICAM interactions, BIRT 377, was used to block LFA-1 in vitro. Transendothelial migration was assessed by measuring migration through Transwells coated with bone marrow endothelial cells. Immediate in vivo homing was analyzed by tracing 51Cr-labeled cells. Invert microscopic cell counting was used to analyze homotypic cell adhesion. Cell cycle analysis was used to analyze apoptosis. S+G(2)/M phase analysis and 3H-thymidine incorporation were used to assess proliferation. Cells were separated into LFA-1(+) and LFA-1(-) fraction by magnetic activated cell sorting.5T33MMvt cells had a heterogeneous LFA-1 expression and all cells were positive for the LFA-1 ligand ICAM-1. LFA-1 inhibition with BIRT 377 did not affect transendothelial migration of the 5T33MMvt cells; however, it did result in cell cluster scattering, indicating LFA-1 involvement in homotypic cell-cell adhesion. No effect was observed on apoptosis, but the percentage of cells in S+G(2)/M phase was decreased by 39%. 3H-thymidine incorporation confirmed this effect on 5T33MMvt cell proliferation (38% reduction). When 5T33MMvt cells were injected into animals, all myeloma cells isolated at the end stage of the disease were LFA-1(+) in contrast to the situation before injection. LFA-1(+) and LFA-1(-) MM cells had similar in vivo bone marrow homing capacities. Mice injected with LFA-1(+) 5T33MMvt cells developed myeloma (5/5) within 12 weeks after injection. In contrast, LFA-1(-) recipients did not develop the disease (0/5), even 1 year after tumor inoculation.Our data suggest that LFA-1-mediated homotypic cell-cell adhesion is involved in myeloma cell proliferation and raises the possibility that this interaction may have a crucial role in in vivo myeloma cell growth. LFA-1 does not appear to play a role in the bone marrow homing of these 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.

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 of cancer 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.

At clinical presentation, multiple myeloma (MM) is already a well-established disease. The processes involved in earlier stages are, however, unknown. Here the 5T2MM murine model was used to analyze differentiation, proliferation, invasion, and apoptosis of MM cells during disease progression. Naive mice were injected with 5T2MM cells and from the onset of the experiment 3 mice were killed each week until the end stage. Myeloma cells were isolated from the bone marrow and selected by sequential gating of 5T2MM idiotype+ cells by flow cytometry. Microscopic analysis of these sorted 5T2MM idiotype+ cells confirmed their identity as true myeloma cells. Based on serum paraprotein concentration and bone marrow tumor load, 3 disease stages were distinguished: a quiescent stage, an intermediate stage, and an end stage, of slow, moderate, and accelerated tumor progression, respectively. In the quiescent stage, the majority of the myeloma cells were CD45+CD138−IL-6Rα+, corresponding to an immature, invasive, and apoptosis-resistant phenotype. In the end stage the majority of the myeloma cells had differentiated into CD45−CD138+IL-6Rα− cells, corresponding to a mature, less invasive, and apoptosis-sensitive phenotype. In the intermediate stage a gradual transition from the quiescent toward the end stage was observed. In line with these data, analysis of sorted 5T2MM cells demonstrated a significant decrease in invasive capacity and a significant increase in (dexamethasone-induced) apoptosis sensitivity and in proliferation during the disease progression. These data suggest that myeloma disease progression is a multistage and dynamic process of differentiation, proliferation, invasion, and apoptosis.

MM is a B-cell malignancy mainly characterized by monoclonal expansion of plasma cells in the BM, presence of paraprotein in serum and occurrence of osteolytic bone lesions. MMPs are a family of proteolytic enzymes that can contribute to cancer growth, invasion, angiogenesis, bone degradation and other processes important in the pathogenesis of MM. We investigated MMP-9 production in the 5T33MM murine model. Expression of MMP-9 protein in supernatant and cell extracts was analyzed by gelatin zymography. The in vitro, stroma-independent variant 5T33MMvt showed no protein expression of MMP-9 in contrast to in vivo growing MM cells, 5T33MMvv. However, when 5T33MMvt cells were injected into naive mice and isolated after tumor take (5T33MMvt-vv), they secreted a significant amount of MMP-9. These results were confirmed by specific staining of cytospins with an anti-MMP-9 antibody. The MMP-9 production by 5T33MMvt-vv cells disappeared when the cells were recultured in vitro. These data demonstrated that upregulation of MMP-9 occurs in vivo and that this process is dependent on the microenvironment. Cocultures of 5T33MMvt cells with STR10 BMECs induced MMP-9 in MM cells, as determined by both gelatin zymography and flow-cytometric analysis. In conclusion, our results demonstrate that MMP-9 production by MM cells is upregulated in vivo by the interaction of MM cells with BMECs.

Homing of multiple myeloma (MM) cells to the bone marrow (BM) requires transendothelial migration. In the present work we tested whether monocyte chemoattractant protein-1 (MCP-1) and CCR2, the high affinity receptor for MCP-1, are involved in this process. Murine 5T2 and 5T33MM cell lines were selected as source of MM cells and STR4, 10 and 12 of BM endothelial cells (BMEC). RT-PCR demonstrated transcripts for MCP-1 in BMEC and ELISA the presence of MCP-1 protein in culture medium. RNase protection assay showed mRNA expression for CCR2, and FACS analysis the presence of CCR2 protein on the MM cells. EC conditioned medium induced chemoattraction of MM cells, a phenomenon inhibited by anti-MCP-1 antibodies. In conclusion, MM cells express CCR2 and are attracted by MCP-1 secreted by BMEC. We suggest that local MCP-1 production by BMEC is one of the mechanisms involved in homing of myeloma cells to the BM.

Isotype-switch variants can easily be detected in a significant proportion of multiple myeloma (MM) patients. The biological significance of these isotype-switch variants remains obscure. Therefore, we studied the appearance of these isotype-switch variants in two murine MM models, 5T2MM and 5T33MM, both of IgG isotype. With a MM-specific PCR assay we could detect isotype-switch variants in the bone marrow of both the 5T2MM and the 5T33MM bearing mice, reflecting again the close resemblance of this mouse model to the human MM. These isotype-switch variants were not found in an in vitro stroma-independent variant of the 5T33MM line. However, when this 5T33MMvitro line was injected into young syngeneic mice, isotype-switch variants appeared thereafter in the isolated tumour cells. These isotype-switch variants could only originate from the MM-IgG expressing cell since IgG subclones from the 5T33MMvitro line again gave rise to isotype-switch variants. The appearance of IgA cells can be explained by down-stream switching of IgG to IgA, while the emergence of IgM cells have to occur via trans-switching to the sister chromatid as the Cmu region is deleted from the CIS-chromosome. This study demonstrates that isotype-switch variants originate from the major tumour clone suggesting no role for the MM-IgM expressing cell as a pre-switch precursor MM cell. The appearance of isotype-switch variants should be considered as a rare but normal event now becoming visible due to the high number of clonal cells present in MM.

The presence of myeloma cells in the blood circulation. implicates that these cells must have the potential to extravasate and home to the bone marrow environment. Using the 5T2 MM mouse model, we could demonstrate that the restricted localization of myeloma cells in the bone marrow is the result of selective migration of myeloma cells in the bone marrow combined with a selective growth of the tumour cells in the bone marrow microenvironment. Moreover, we showed that 5T2 MM cells bind in vitro selectively to bone marrow-derived endothelial cells (EC) and not to lung-derived EC. In order to identify which chemotactic molecules mediate the transendothelial migration of myeloma cells, we examined the motility-inducing effect of different extracellular matrix proteins on myeloma cell lines. We found that laminin-1 a major component of the basement membrane, triggers the motility of both human myeloma cells and 5T2 MM cells, through the 67 kD laminin receptor. Because of the broad distribution of laminin in extracellular matrices throughout the body, it is clear that this molecule on itself can not be the only factor that determines the specificity of myeloma cell homing. In the 5T2 MM model we identified IGF-1 as a more specific bone marrow derived chemoattractant for myeloma cells. In addition we demonstrated that the marrow microenvironment can upregulate the expression of the IGF-1 receptor on 5T mouse myeloma cells. In the end phase of the disease, increasing numbers of myeloma cells are detectable in the peripheral blood and extramedullary tumour growth can occur. We found that the stroma-independent variant of the human MM5 myeloma cell line showed an increased in vitro motility as compared to the stroma-dependent variant. By representational difference analysis we demonstrated that the stroma-dependent MM5 cells show a downregulation of the motility-related protein (MRP-I CD9) which might reflect the involvement of this molecule in the regulation of myeloma cell extravasation.

Our group recently reported that multiple myeloma (MM) cells preferentially adhere to bone marrow (BM) endothelial cells and selectively home to the BM, suggesting the involvement of specific adhesive interactions in this process. The highly regulated expression of CD44 variant isoforms (CD44v) on the MM cells makes them good candidate adhesion molecules involved in this homing. We addressed this in the 5T experimental mouse model of myeloma. Fluorescence-activated cell sorting analysis demonstrated expression of CD44v6, CD44v7, and CD44v10 on the in vivo growing 5T2MM and 5T33MM myeloma lines. Antibody blocking experiments revealed the involvement of CD44v10 in the adhesion of 5T2MM and 5T33MM cells to BM endothelial cells. Coinjection of anti-CD44v10 antibodies with the myeloma cells into syngeneic mice demonstrated a selective blocking of their BM homing which resulted in a decreased BM tumor load and serum paraprotein at the end stage of the disease. The highly restricted expression of CD44v10 on MM cells, the blocking of MM adhesion to BM endothelial cells and of homing to BM by anti-CD44v10, and the decreased BM tumor load suggest that myeloma cells home to the BM via interactions mediated by this specific region of the adhesion molecule CD44.

Multiple myeloma, a plasma cell malignancy, is predominantly localized in the bone marrow. These tumoral cells display a heterogeneous expression of CD45. It is, however, unclear which subpopulation is responsible for the homing and outgrowth of the myeloma cells. In this work, we investigated the in vivo homing, proliferation, and differentiation of both CD45+ and CD45- cells in two murine myeloma models.5T2MM and 5T33MM in vivo lines of murine multiple myeloma were used. CD45 and IGF-I receptor expression was analyzed by FACS. Proliferative capacity was assessed by in vivo bromodeoxyuridine incorporation. 5TMM cells were separated into CD45+ and CD45- fractions by MACS. Initial homing was investigated in vivo by tracing of radioactively labeled cells. Myeloma cells were detected by FACS and histology. Osteolytic lesions were analyzed by radiography. Both CD45+ and CD45- 5TMM cells were able to home to the bone marrow, although the migration of the latter subset was lower, which was related to a low IGF-I receptor expression. Recipients of both fractions developed myeloma as evidenced by the presence of serum paraprotein, osteolytic lesions, and bone marrow infiltration by myeloma cells. The tumor load in the recipients of CD45- cells was higher than the CD45+ cells, which could be explained by a lower proliferation rate of the latter population. While the separated cells before injection had a homogenous expression of CD45, cells isolated from the bone marrow of these terminally diseased mice had a heterogeneous expression pattern, indicating an in vivo differentiation pattern of CD45- to CD45+ cells and vice versa. We conclude that both CD45+ and CD45- 5TMM subpopulations contain clonogenic myeloma cells with bone marrow homing and proliferative capacity.

The 67 kD laminin receptor (67LR) binds laminin-1 (LN), major component of the basement membrane, with high affinity. In this study, we demonstrated that human multiple myeloma cell lines (HMCL) and murine 5T2MM cells express 67LR. CD38bright+ plasma cells in fresh multiple myeloma (MM) bone marrow (BM) samples showed weaker 67LR expression, but expression increased after direct exposure to a BM endothelial cell line (4LHBMEC). LN stimulated the in vitro migration of 3 HMCL (MM5.1, U266 and MMS.1), primary MM cells and the murine 5T2MM cells. 67LR has been shown to mediate the actions of LN through binding to CDPGYIGSR, a 9 amino acid sequence from the B1 chain of LN. MM cell migration was partially blocked by peptide 11, a synthetic nonapeptide derived from this amino sequence and also by a blocking antiserum against 67LR. Co-injection of peptide 11 with 5T2MM cells in a murine in vivo model of MM resulted in a decreased homing of 5T2MM cells to the BM compartment. In conclusion, LN acts as a chemoattractant for MM cells by interaction with 67LR. This interaction might be important during extravasation of circulating MM cells. © 2001 Cancer Research Campaign.

One of the main characteristics of multiple myeloma cells is their predominant localization in the bone marrow. It is, however, unclear whether this is due to a selective initial entry, or whether this entry is more random and other processes like survival and/or growth stimulation, only present in the medullar microenvironment, are unique. To investigate this, in vivo homing kinetics of murine 5T2MM cells shortly after injection were assessed in bone marrow, liver, spleen, lungs, heart, intestines, kidney and testis by tracing of radiolabelled cells, by immunostaining of isolated cells and by polymerase chain reaction analysis. We demonstrated the presence of 5T2MM cells in bone marrow, spleen and liver with all other organs being negative. Adhesion assays of 5T2MM cells to different types of endothelial cells demonstrated a selective adhesion of 5T2MM cells to bone marrow and liver and not to lung endothelial cells. We here demonstrate that the specific in vivo localization of the 5T2MM cells is a result of the combination of a selective entry/adhesion of the 5T2MM cells in the bone marrow, spleen and liver, and a selective survival and growth of these tumour cells in the bone marrow and spleen but not in the liver. © 2000 Cancer Research Campaign

Chemicals/CAS: Cancer Vaccines; Myeloma Proteins; pristane, 1921-70-6; Terpenes; Vaccines, DNA

BACKGROUND AND OBJECTIVES: Unlike most other tumors, multiple myeloma (MM) cells have to survive and to grow in a bone marrow (BM) microenvironment which is already hypoxic by nature. BM hypoxia is crucial for normal hematopoiesis. However, how BM hypoxia and MM affect each other is unknown. We addressed this topic in the 5T2MM mouse model. DESIGN AND METHODS: Levels of hypoxia in the BM of control and tumor-bearing mice were analyzed by flow cytometric analysis of pimonidazole hypoxyprobe binding and hypoxia-inducible factor 1alpha (HIF-1alpha) expression. Micro-vessel density was measured by CD31 staining of BM sections for immunohistochemistry. Apoptosis sensitivity of CD45 5T2MM subsets in hypoxic conditions were analyzed by detection of active caspase-3. RESULTS: Analysis of control and 5T2MM diseased mice injected with pimonidazole hypoxyprobe indicated that both normal BM and myeloma-infiltrated BM are hypoxic. However, the hypoxia in the myelomatous BM was significantly decreased. Analysis of HIF-1alpha expression, a surrogate marker of hypoxia, also demonstrated significantly lower levels of hypoxia in myeloma-infiltrated BM. HIF-1alpha expression was inversely correlated with the micro-vessel density. In vitro culture of 5T2MM cells under hypoxic conditions indicated induction of apoptosis in the CD45- MM-fraction, but not in the CD45+ 5T2MM-cells. INTERPRETATION AND CONCLUSIONS: These data suggest that native BM hypoxia is advantageous for the tumor-initiating CD45+ 5T2MM-cells. Together with the decreased hypoxia in myeloma-infiltrated BM it also indicates that myeloma-associated angiogenesis is functional and permissive for the expansion of CD45- 5T2MM-cells. All together the data raise the possibility of an important role of BM hypoxia in myeloma tumor progression.