CCL21-CCR7 signaling promotes microglia/macrophage recruitment and chemotherapy resistance in glioblastoma.

Glioblastoma (GBM) is the most common and fatal primary tumor of the central nervous system (CNS) and current treatments have limited success. Chemokine signaling regulates both malignant cells and stromal cells of the tumor microenvironment (TME), constituting a potential therapeutic target against brain cancers. Here, we investigated the C-C chemokine receptor type 7 (CCR7) and the chemokine (C-C-motif) ligand 21 (CCL21) for their expression and function in human GBM and then assessed their therapeutic potential in preclinical mouse GBM models. In GBM patients, CCR7 expression positively associated with a poor survival. CCL21-CCR7 signaling was shown to regulate tumor cell migration and proliferation while also controlling tumor associated microglia/macrophage recruitment and VEGF-A production, thereby controlling vascular dysmorphia. Inhibition of CCL21-CCR7 signaling led to an increased sensitivity to temozolomide-induced tumor cell death. Collectively, our data indicate that drug targeting of CCL21-CCR7 signaling in tumor and TME cells is a therapeutic option against GBM.

FIBER-ML, an Open-Source Supervised Machine Learning Tool for Quantification of Fibrosis in Tissue Sections.

Pathologic fibrosis is a major hallmark of tissue insult in many chronic diseases. Although the amount of fibrosis is recognized as a direct indicator of the extent of disease, there is no consentaneous method for its quantification in tissue sections. This study tested FIBER-ML, a semi-automated, open-source freeware that uses a machine-learning approach to quantify fibrosis automatically after a short user-controlled learning phase. Fibrosis was quantified in sirius red-stained tissue sections from two fibrogenic animal models: acute stress-induced cardiomyopathy in rats (Takotsubo syndrome-like) and HIV-induced nephropathy in mice (chronic kidney disease). The quantitative results of FIBER-ML software version 1.0 were compared with those of ImageJ in Takotsubo syndrome, and with those of inForm in chronic kidney disease. Intra- and inter-operator and inter-software correlation and agreement were assessed. All correlations were excellent (>0.95) in both data sets. The values of discriminatory power between the pathologic and healthy groups were <10-3 for data on Takotsubo syndrome and <10-4 for data on chronic kidney disease. Intra-operator agreement, assessed by intra-class coefficient correlation, was good (>0.8), while inter-operator and inter-software agreement ranged from moderate to good (>0.7). FIBER-ML performed in a fast and user-friendly manner, with reproducible and consistent quantification of fibrosis in tissue sections. It offers an open-source alternative to currently used software, including quality control and file management.

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke.

RATIONALE: Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. OBJECTIVE: To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. METHODS AND RESULTS: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. CONCLUSIONS: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

The BMS-LM ontology for biomedical data reporting throughout the lifecycle of a research study: From data model to ontology.

Biomedical research data reuse and sharing is essential for fostering research progress. To this aim, data producers need to master data management and reporting through standard and rich metadata, as encouraged by open data initiatives such as the FAIR (Findable, Accessible, Interoperable, Reusable) guidelines. This helps data re-users to understand and reuse the shared data with confidence. Therefore, dedicated frameworks are required. The provenance reporting throughout a biomedical study lifecycle has been proposed as a way to increase confidence in data while reusing it. The Biomedical Study - Lifecycle Management (BMS-LM) data model has implemented provenance and lifecycle traceability for several multimodal-imaging techniques but this is not enough for data understanding while reusing it. Actually, in the large scope of biomedical research, a multitude of metadata sources, also called Knowledge Organization Systems (KOSs), are available for data annotation. In addition, data producers uses local terminologies or KOSs, containing vernacular terms for data reporting. The result is a set of heterogeneous KOSs (local and published) with different formats and levels of granularity. To manage the inherent heterogeneity, semantic interoperability is encouraged by the Research Data Management (RDM) community. Ontologies, and more specifically top ontologies such as BFO and DOLCE, make explicit the metadata semantics and enhance semantic interoperability. Based on the BMS-LM data model and the BFO top ontology, the BioMedical Study - Lifecycle Management (BMS-LM) core ontology is proposed together with an associated framework for semantic interoperability between heterogeneous KOSs. It is made of four ontological levels: top/core/domain/local and aims to build bridges between local and published KOSs. In this paper, the conversion of the BMS-LM data model to a core ontology is detailed. The implementation of its semantic interoperability in a specific domain context is explained and illustrated with examples from small animal preclinical research.

Assessment of BOLD response in the fetal lung.

OBJECTIVE: Assessment of lung development and maturity is of utmost importance in prenatal counseling. Blood oxygen level-dependent (BOLD) effect MRI was developed for functional evaluations of organs. To date, no data are available in fetal lungs and nothing is known about the existence of a BOLD effect in the lungs. The aim of our study was to evaluate if a BOLD response could be detected in fetal lungs. MATERIALS AND METHODS: From January 2014 to December 2016, 38 healthy pregnant women were prospectively enrolled. After a routine scan on a 1.5-T MRI device (normoxic period), maternal hyperoxia was induced for 5 min before the BOLD sequence (hyperoxic period). R2* was evaluated by fitting average intensity of the signal, both for normoxic (norm) and hyperoxic (hyper) periods. RESULTS: A significant BOLD response was observed after maternal hyperoxia in the lungs with a mean R2* decrease of 12.1 ± 2.5% (p < 0.001), in line with the placenta response with a mean R2* decrease of 19.2 ± 5.9% (p < 0.0001), confirming appropriate oxygen uptake. Conversely, no significant BOLD effect was observed for the brain nor the liver with a mean ∆R2* of 3.6 ± 3.1% (p = 0.64) and 2.8 ± 3.7% (p = 0.23). CONCLUSION: This study shows for the first time in human that a BOLD response can be observed in the normal fetal lung despite its prenatal "non-functional status." If confirmed in congenital lung and chest malformations, this property could be used in addition to the lung volume for a better prediction of postnatal respiratory status. KEY POINTS: • Blood oxygen level-dependent (BOLD) effect MRI was developed for functional evaluations of organs and could have interesting implications for the fetal organs. • Assessment of lung development is of utmost importance in prenatal counseling, but to date no data are available in fetal lungs. • BOLD response can be observed in the normal fetal lung opening the way to studies on fetus with pathological lungs.

Succinate detection using in vivo 1H-MR spectroscopy identifies germline and somatic SDHx mutations in paragangliomas.

PURPOSE: Germline mutations in genes encoding succinate dehydrogenase (SDH) are frequent in patients with pheochromocytoma and paraganglioma (PPGL). They lead to SDH inactivation, mediating a massive accumulation of succinate, which constitutes a highly specific biomarker of SDHx-mutated tumors when measured in vitro. In a recent pilot study, we showed that magnetic resonance spectroscopy (1H-MRS) optimized for succinate detection (SUCCES) could detect succinate in vivo in both allografted mouse models and PPGL patients. The objective of this study was to prospectively assess the diagnostic performances of 1H-MRS SUCCES sequence for the identification of SDH deficiency in PPGL patients. METHODS: Forty-nine patients presenting with 50 PPGLs were prospectively enrolled in our referral center for 1H-MRS SUCCES. Two observers blinded to the clinical characteristics and genetic status analyzed the presence of a succinate peak and confronted the results to a composite gold standard combining PPGL genetic testing and/or in vitro protein analyses in the tumor. RESULTS: A succinate peak was observed in 20 tumors, all of which had proven SDH deficiency using the gold standard (17 patients with germline SDHx mutations, 2 with a somatic SDHD mutation, and 1 with negative SDHB IHC and SDH loss of function). A false negative result was observed in 3 tumors. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 1H-MRS SUCCES were respectively 87%, 100%, 100%, 90%, and 94%. CONCLUSIONS: Detection of succinate using 1H-MRS is a highly specific and sensitive hallmark of SDH-deficiency in PPGLs.

Pyruvate cellular uptake and enzymatic conversion probed by dissolution DNP-NMR: the impact of overexpressed membrane transporters.

Pyruvate membrane crossing and its lactate dehydrogenase-mediated conversion to lactate in cells featuring different levels of expression of membrane monocarboxylate transporters (MCT4) were probed by dissolution dynamic nuclear polarization-enhanced NMR. Hyperpolarized 13 C-1-labeled pyruvate was transferred to suspensions of rodent tumor cell carcinoma, cell line 39. The pyruvate-to-lactate conversion rate monitored by dissolution dynamic nuclear polarization-NMR in carcinoma cells featuring native MCT4 expression level was lower than the rate observed for cells in which the human MCT4 gene was overexpressed. The enzymatic activity of lactate dehydrogenase was also assessed in buffer solutions, following the real-time pyruvate-to-lactate conversion speeds at different enzyme concentrations. Copyright © 2016 John Wiley & Sons, Ltd.

Monitoring therapeutic efficacy of sunitinib using [(18)F]FDG and [(18)F]FMISO PET in an immunocompetent model of luminal B (HER2-positive)-type mammary carcinoma.

BACKGROUND: Clinical studies implying the sunitinib multi-kinase inhibitor have led to disappointing results for breast cancer care but mostly focused on HER2-negative subtypes. Preclinical researches involving this drug mostly concern Triple Negative Breast Cancer (TNBC) murine models. Here, we explored the therapeutic efficacy of sunitinib on a PyMT-derived transplanted model classified as luminal B (HER2-positive) and monitored the response to treatment using both in vivo and ex vivo approaches. METHODS: Tumour-induced animals were treated for 9 (n = 7) or 14 (n = 8) days with sunitinib at 40 mg/kg or with vehicle only. Response to therapy was assessed in vivo by monitoring glucose tumour metabolism and hypoxia using 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) and [(18)F]fluoromisonidazole ([(18)F]FMISO) Positron Emission Tomography (PET). After primary tumour excision, ex vivo digital microscopy was performed on treated and control samples to estimate vascular density (CD31), apoptosis (Tunel), proliferation (Ki-67), Tumour-Associated Macrophage (TAM) infiltration (F4/80), metabolism (GLUT1) and cellular response to hypoxia (HIF1 alpha). The drug impact on the metastasis rate was evaluated by monitoring the PyMT gene expression in the lungs of the treated and control groups. RESULTS: Concomitant with sunitinib-induced tumour size regression, [(18)F]FDG PET imaging showed a stable glycolysis-related metabolism inside tumours undergoing treatment compared to an increased metabolism in untreated tumours, resulting at treatment end in 1.5 less [(18)F]FDG uptake in treated (n = 4) vs control (n = 3) tumours (p < 0.05). With this small sample, [(18)F]FMISO PET showed a non-significant decrease of hypoxia in treated vs control tumours. The drug triggered a 4.9 fold vascular volume regression (p < 0.05), as well as a 17.7 fold induction of tumour cell apoptosis (p < 0.001). The hypoxia induced factor 1 alpha (HIF1 alpha) expression was twice lower in the treated group than in the control group (p < 0.05). Moreover, the occurrence of lung metastases was not reduced by the drug. CONCLUSIONS: [(18)F]FDG and [(18)F]FMISO PET were relevant approaches to study the response to sunitinib in this luminal B (HER2-positive) model. The sunitinib-induced vascular network shrinkage did not significantly increase tumour hypoxia, suggesting that tumour regression was mainly due to the pro-apoptotic properties of the drug. Sunitinib did not inhibit the metastatic process in this PyMT transplanted model.

Targeted therapy for capillary-venous malformations.

Sporadic venous malformations are genetic conditions primarily caused by somatic gain-of-function mutation of PIK3CA or TEK, an endothelial transmembrane receptor signaling through PIK3CA. Venous malformations are associated with pain, bleedings, thrombosis, pulmonary embolism, esthetic deformities and, in severe cases, life-threatening situations. No authorized medical treatment exists for patients with venous malformations. Here, we created a genetic mouse model of PIK3CA-related capillary venous malformations that replicates patient phenotypes. We showed that these malformations only partially signal through AKT proteins. We compared the efficacy of different drugs, including rapamycin, a mTORC1 inhibitor, miransertib, an AKT inhibitor and alpelisib, a PI3Kα inhibitor at improving the lesions seen in the mouse model. We demonstrated the effectiveness of alpelisib in preventing vascular malformations' occurrence, improving the already established ones, and prolonging survival. Considering these findings, we were authorized to treat 25 patients with alpelisib, including 7 children displaying PIK3CA (n = 16) or TEK (n = 9)-related capillary venous malformations resistant to usual therapies including sirolimus, debulking surgical procedures or percutaneous sclerotherapies. We assessed the volume of vascular malformations using magnetic resonance imaging (MRI) for each patient. Alpelisib demonstrated improvement in all 25 patients. Vascular malformations previously considered intractable were reduced and clinical symptoms were attenuated. MRI showed a decrease of 33.4% and 27.8% in the median volume of PIK3CA and TEK malformations respectively, over 6 months on alpelisib. In conclusion, this study supports PI3Kα inhibition as a promising therapeutic strategy in patients with PIK3CA or TEK-related capillary venous malformations.

Imaging microglial/macrophage activation in spinal cords of experimental autoimmune encephalomyelitis rats by positron emission tomography using the mitochondrial 18 kDa translocator protein radioligand [¹8F]DPA-714.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS. Activated microglia/macrophages play a key role in the immunopathogenesis of MS and its corresponding animal models, experimental autoimmune encephalomyelitis (EAE). Microglia activation begins at early stages of the disease and is associated with elevated expression of the 18 kDa mitochondrial translocator protein (TSPO). Thus, positron emission tomography (PET) imaging of microglial activation using TSPO-specific radioligands could be valuable for monitoring disease-associated neuroinflammatory processes. EAE was induced in rats using a fragment of myelin basic protein, yielding acute clinical disease that reflects extensive spinal cord inflammation. Enhanced TSPO expression in spinal cords of EAE rats versus those of controls was confirmed by Western blot and immunohistochemistry. Biodistribution studies in control and EAE rats were performed using the TSPO radioligand [¹8F]DPA-714 [N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide]. At 1 h after injection, almost fivefold higher levels of [¹8F]DPA-714 were measured in spinal cords of EAE rats versus controls. The specific binding of [¹8F]DPA-714 to TSPO in spinal cords was confirmed in competition studies, using unlabeled (R,S)-PK11195 [(R,S)-N-methyl-N-(1-methylpropyl)-1-(2-chlorophenyl)isoquinoline-3-carboxamide)] or DPA-714 in excess. MicroPET studies affirm that this differential radioactivity uptake in spinal cords of EAE versus control rats could be detected and quantified. Using [¹8F]DPA-714, neuroinflammation in spinal cords of EAE-induced rats could be visualized by PET, offering a sensitive technique for monitoring neuroinflammatory lesions in the CNS and particularly in the spinal cord. In addition to current MRI protocols, this approach could provide molecular images of neuroinflammation for detection, monitoring, and research in MS.

Motion Rejection and Spectral Unmixing for Accurate Estimation of In Vivo Oxygen Saturation Using Multispectral Optoacoustic Tomography.

Multispectral optoacoustic tomography (MSOT) uniquely enables spatial mapping in high resolution of oxygen saturation (SO2), with potential applications in studying pathological complications and therapy efficacy. MSOT offers seamless integration with ultrasonography, by using a common ultrasound (US) detector array. However, MSOT relies on multiple successive acquisitions of optoacoustic (OA) images at different optical wavelengths and the low frame rate of OA imaging makes the MSOT acquisition sensitive to body/respiratory motion. Moreover, the estimation of SO2 is highly sensitive to noise, and artifacts related to the respiratory motion of the animal were identified as the primary source of noise in MSOT. In this work, we propose a two-step image processing method for SO2 estimation in deep tissues. First, to mitigate motion artifacts, we propose a method of selection of OA images acquired only during the respiratory pause of the animal, using ultrafast ultrasound (US) images acquired immediately after each OA acquisition (US image acquisition duration of 1.4 ms and a total delay of 7 ms). We show that gating is more effective using US images than OA images at different optical wavelengths. Second, we propose a novel method that can estimate directly the SO2 value of a pixel and at the same time evaluate the amount of noise present in that pixel. Hence, the method can efficiently eliminate the pixels dominated by noise from the final SO2 map. Our postprocessing method is shown to outperform conventional methods for SO2 estimation, and the method was validated by in vivo oxygen challenge experiments.

Machine Learning of Multi-Modal Tumor Imaging Reveals Trajectories of Response to Precision Treatment.

The standard assessment of response to cancer treatments is based on gross tumor characteristics, such as tumor size or glycolysis, which provide very indirect information about the effect of precision treatments on the pharmacological targets of tumors. Several advanced imaging modalities allow for the visualization of targeted tumor hallmarks. Descriptors extracted from these images can help establishing new classifications of precision treatment response. We propose a machine learning (ML) framework to analyze metabolic-anatomical-vascular imaging features from positron emission tomography, ultrafast Doppler, and computed tomography in a mouse model of paraganglioma undergoing anti-angiogenic treatment with sunitinib. Imaging features from the follow-up of sunitinib-treated (n = 8, imaged once-per-week/6-weeks) and sham-treated (n = 8, imaged once-per-week/3-weeks) mice groups were dimensionally reduced and analyzed with hierarchical clustering Analysis (HCA). The classes extracted from HCA were used with 10 ML classifiers to find a generalized tumor stage prediction model, which was validated with an independent dataset of sunitinib-treated mice. HCA provided three stages of treatment response that were validated using the best-performing ML classifier. The Gaussian naive Bayes classifier showed the best performance, with a training accuracy of 98.7 and an average area under curve of 100. Our results show that metabolic-anatomical-vascular markers allow defining treatment response trajectories that reflect the efficacy of an anti-angiogenic drug on the tumor target hallmark.

Correction: Full-field optical coherence tomography for the diagnosis of giant cell arteritis.

[This corrects the article DOI: 10.1371/journal.pone.0234165.].

Acute stress induces long-term metabolic, functional, and structural remodeling of the heart.

Takotsubo cardiomyopathy is a stress-induced cardiovascular disease with symptoms comparable to those of an acute coronary syndrome but without coronary obstruction. Takotsubo was initially considered spontaneously reversible, but epidemiological studies revealed significant long-term morbidity and mortality, the reason for which is unknown. Here, we show in a female rodent model that a single pharmacological challenge creates a stress-induced cardiomyopathy similar to Takotsubo. The acute response involves changes in blood and tissue biomarkers and in cardiac in vivo imaging acquired with ultrasound, magnetic resonance and positron emission tomography. Longitudinal follow up using in vivo imaging, histochemistry, protein and proteomics analyses evidences a continued metabolic reprogramming of the heart towards metabolic malfunction, eventually leading to irreversible damage in cardiac function and structure. The results combat the supposed reversibility of Takotsubo, point to dysregulation of glucose metabolic pathways as a main cause of long-term cardiac disease and support early therapeutic management of Takotsubo.

Hemifacial myohyperplasia is due to somatic muscular PIK3CA gain-of-function mutations and responds to pharmacological inhibition.

Hemifacial myohyperplasia (HFMH) is a rare cause of facial asymmetry exclusively involving facial muscles. The underlying cause and the mechanism of disease progression are unknown. Here, we identified a somatic gain-of-function mutation of PIK3CA in five pediatric patients with HFMH. To understand the physiopathology of muscle hypertrophy in this context, we created a mouse model carrying specifically a PIK3CA mutation in skeletal muscles. PIK3CA gain-of-function mutation led to striated muscle cell hypertrophy, mitochondria dysfunction, and hypoglycemia with low circulating insulin levels. Alpelisib treatment, an approved PIK3CA inhibitor, was able to prevent and reduce muscle hypertrophy in the mouse model with correction of endocrine anomalies. Based on these findings, we treated the five HFMH patients. All patients demonstrated clinical, esthetical, and radiological improvement with proof of target engagement. In conclusion, we show that HFMH is due to somatic alteration of PIK3CA and is accessible to pharmacological intervention.

In vivo evidence that the increase in matrix metalloproteinase activity occurs early after cerebral ischemia.

There is controversy over whether matrix metalloproteinases (MMPs) are activated during the early therapeutic window following ischemic stroke. Ex vivo, an increase was reported as early as 4 hours, whereas in vivo, no increase was found until 24 hours postischemia. We used fluorescence diffuse optical tomography to image MMP activity following experimental cerebral ischemia; increased MMP activity was observed in the ischemic area as early as 3 to 6 hours after ischemic onset and correlated with the volume of ischemic cerebral tissue. Therefore, MMP activation is an immediate early response to cerebral ischemia concurrent with the therapeutic window.

Serum-stable RNA aptamers to urokinase-type plasminogen activator blocking receptor binding.

The serine proteinase urokinase-type plasminogen activator (uPA) is widely recognized as a potential target for anticancer therapy. Its association with cell surfaces through the uPA receptor (uPAR) is central to its function and plays an important role in cancer invasion and metastasis. In the current study, we used systematic evolution of ligands by exponential enrichment (SELEX) to select serum-stable 2'-fluoro-pyrimidine-modified RNA aptamers specifically targeting human uPA and blocking the interaction to its receptor at low nanomolar concentrations. In agreement with the inhibitory function of the aptamers, binding was found to be dependent on the presence of the growth factor domain of uPA, which mediates uPAR binding. One of the most potent uPA aptamers, upanap-12, was analyzed in more detail and could be reduced significantly in size without severe loss of its inhibitory activity. Finally, we show that the uPA-scavenging effect of the aptamers can reduce uPAR-dependent endocytosis of the uPA-PAI-1 complex and cell-surface associated plasminogen activation in cell culture experiments. uPA-scavenging 2'-fluoro-pyrimidine-modified RNA aptamers represent a novel promising principle for interfering with the pathological functions of the uPA system.

In vivo cellular imaging pinpoints the role of reactive oxygen species in the early steps of adult hematopoietic reconstitution.

Few techniques are available to characterize in vivo the early cellular dynamics of long-term reconstitution of hematopoiesis after transplantation of hematopoietic stem cells (HSCs) after lethal irradiation. Using a fiber-optic imaging system, we track the early steps of in vivo recruitment and proliferation of Lin(-)Sca-1(+)c-Kit(+)CD34(-) (LSKCD34(-)) HSCs highly enriched in HSCs and transplanted into lethally irradiated mice. Recruitment of the transplanted LSKCD34(-) hematopoietic cells first occurs in the femoral head and is continuous during 24 hours. Quantification of the fluorescence emitted by the transplanted hematopoietic cells shows that proliferation of LSKCD34(-) hematopoietic cells in the femoral head was potent 3 days after transplantation. Using a development of this fiber-optic imaging system, we show that the transplanted LSKCD34(-) hematopoietic cells are associated with vascularized structures as early as 5 hours after transplantation. This early association is dependent on reactive oxygen species (ROS) partly through the regulation of vascular cell adhesion molecule-1 expression on endothelial cells and is followed by a ROS-dependent proliferation of LSKCD34(-) hematopoietic cells. This new in vivo imaging technique permits the observation of the early steps of hematopoietic reconstitution by HSCs in long bones and shows a new role of ROS in the recruitment of HSCs by bone marrow endothelial cells.