[Show abstract][Hide abstract] ABSTRACT: The extracellular matrix (ECM) supports vascular integrity during embryonic development. Proteolytic degradation of ECM components is required for angiogenesis, but excessive ECM proteolysis causes blood vessel fragility and hemorrhage. Little is understood about how ECM proteolysis is transcriptionally regulated during embryonic vascular development. We now show that the NuRD ATP-dependent chromatin-remodeling complex promotes vascular integrity by preventing excessive ECM proteolysis in vivo. Mice lacking endothelial CHD4-a catalytic subunit of NuRD complexes-died at midgestation from vascular rupture. ECM components surrounding rupture-prone vessels in Chd4 mutants were significantly downregulated prior to embryonic lethality. Using qPCR arrays, we found two critical mediators of ECM stability misregulated in mutant endothelial cells: the urokinase-type plasminogen activator receptor (uPAR or Plaur) was upregulated, and thrombospondin-1 (Thbs1) was downregulated. Chromatin immunoprecipitation assays showed that CHD4-containing NuRD complexes directly bound the promoters of these genes in endothelial cells. uPAR and THBS1 respectively promote and inhibit activation of the potent ECM protease plasmin, and we detected increased plasmin activity around rupture-prone vessels in Chd4 mutants. We rescued ECM components and vascular rupture in Chd4 mutants by genetically reducing urokinase (uPA or Plau), which cooperates with uPAR to activate plasmin. Our findings provide a novel mechanism by which a chromatin-remodeling enzyme regulates ECM stability to maintain vascular integrity during embryonic development.
[Show abstract][Hide abstract] ABSTRACT: Neutrophils emigrate from venules to sites of infection or injury in response to chemotactic gradients. How these gradients form is not well understood. Some IL-6-family cytokines stimulate endothelial cells to express adhesion molecules and chemokines that recruit leukocytes. Receptors for these cytokines share the signaling subunit gp130. We studied knockout mice lacking gp130 in endothelial cells. Unexpectedly, gp130-deficient endothelial cells constitutively expressed more CXCL1 in vivo and in vitro and even more upon stimulation with TNF-α. Mobilization of this increased CXCL1 from intracellular stores to the venular surface triggered β2 integrin-dependent arrest of neutrophils rolling on selectins but impaired intraluminal crawling and transendothelial migration. Superfusing CXCL1 over venules promoted neutrophil migration only after intravenously injecting mAb to CXCL1 to diminish its intravascular function or heparinase to release CXCL1 from endothelial proteoglycans. Remarkably, mice lacking gp130 in endothelial cells had impaired histamine-induced venular permeability, which was restored by injecting anti-P-selectin mAb to prevent neutrophil rolling and arrest. Thus, excessive CXCL1 expression in gp130-deficient endothelial cells augments neutrophil adhesion yet hinders migration, most likely by disrupting chemotactic gradients. Our data define a role for endothelial cell gp130 in regulating integrin-dependent adhesion and de-adhesion of neutrophils during inflammation.
[Show abstract][Hide abstract] ABSTRACT: Rationale: Sepsis-induced inflammation of the lung leads to acute respiratory distress syndrome (ARDS), which may trigger persistent fibrosis. The pathology of ARDS is complex and poorly understood, and the therapeutic approaches are limited. Objectives: We used a baboon model of E. coli sepsis that mimics the complexity of human disease to study the pathophysiology of ARDS. Methods: We performed extensive biochemical, histological and functional analyses to characterize the disease progression and the long-term effects of sepsis on the lung structure and function. Measurements and Main Results: Similarly to humans, sepsis-induced ARDS in baboons displays an early inflammatory exudative phase, with extensive necrosis. This is followed by a regenerative phase dominated by proliferation of type 2 epithelial cells, expression of epithelial to mesenchymal transition markers, myofibroblast migration and proliferation, and collagen synthesis. Baboons that survived sepsis showed persistent inflammation and collagen deposition 6-27 months after the acute episodes. Long-term survivors had almost double the amount of collagen in the lung as compared to age-matched controls. Immunostaining for procollagens showed persistent active collagen synthesis within the fibroblastic foci and interalveolar septa. Fibroblasts expressed markers of TGFβ and PDGF signaling, suggesting their potential role as mediators of myofibroblast migration and proliferation, and collagen deposition. In parallel, upregulation of the inhibitors of extracellular proteases supports a deregulated matrix remodeling that may contribute to fibrosis. Conclusions: The primate model of sepsis-induced ARDS mimics the disease progression in humans, including chronic inflammation and long-lasting fibrosis. This model helps understanding the pathophysiology of fibrosis and testing new therapies.
American Journal of Respiratory Cell and Molecular Biology 09/2013; · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Circulating lymphocytes continuously enter lymph nodes for immune surveillance through specialized blood vessels named high endothelial venules, a process that increases markedly during immune responses. How high endothelial venules (HEVs) permit lymphocyte transmigration while maintaining vascular integrity is unknown. Here we report a role for the transmembrane O-glycoprotein podoplanin (PDPN, also known as gp38 and T1α) in maintaining HEV barrier function. Mice with postnatal deletion of Pdpn lost HEV integrity and exhibited spontaneous bleeding in mucosal lymph nodes, and bleeding in the draining peripheral lymph nodes after immunization. Blocking lymphocyte homing rescued bleeding, indicating that PDPN is required to protect the barrier function of HEVs during lymphocyte trafficking. Further analyses demonstrated that PDPN expressed on fibroblastic reticular cells, which surround HEVs, functions as an activating ligand for platelet C-type lectin-like receptor 2 (CLEC-2, also known as CLEC1B). Mice lacking fibroblastic reticular cell PDPN or platelet CLEC-2 exhibited significantly reduced levels of VE-cadherin (also known as CDH5), which is essential for overall vascular integrity, on HEVs. Infusion of wild-type platelets restored HEV integrity in Clec-2-deficient mice. Activation of CLEC-2 induced release of sphingosine-1-phosphate from platelets, which promoted expression of VE-cadherin on HEVs ex vivo. Furthermore, draining peripheral lymph nodes of immunized mice lacking sphingosine-1-phosphate had impaired HEV integrity similar to Pdpn- and Clec-2-deficient mice. These data demonstrate that local sphingosine-1-phosphate release after PDPN-CLEC-2-mediated platelet activation is critical for HEV integrity during immune responses.
[Show abstract][Hide abstract] ABSTRACT: Free radicals are known to play a major role in sepsis. Combined immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI) were used to detect in vivo and in situ levels of free radicals in murine septic encephalopathy following cecal ligation and puncture (CLP). DMPO (5,5-dimethyl pyrroline N-oxide) was injected over 6h following CLP, prior to administration of an anti-DMPO probe (anti-DMPO antibody bound to albumin-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI targeting contrast agent). In vitro assessment of the anti-DMPO probe in oxidatively-stressed mouse astrocytes significantly decreased T1 relaxation (p<0.0001), compared to controls. MRI detected the presence of anti-DMPO adducts via a substantial decrease in %T1 change within the hippocampus, striatum, occipital and medial cortex brain regions (p<0.01 for all) in septic animals compared to shams, which was sustained for over 60min (p<0.05 for all). Fluorescently-labeled streptavidin was used to target the anti-DMPO probe biotin, which was elevated in septic brain, liver and lungs, compared to sham. Ex vivo DMPO adducts (qualitative), and oxidative products, including 4-hydroxynonenal and 3-nitrotyrosine (quantitative, p<0.05 for both), were elevated in septic brains compared to shams. This is the first study that has reported on the detection of in vivo and in situ levels of free radicals in murine septic encephalopathy.
Free Radical Biology and Medicine 08/2013; · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p<0.001) in MR signal intensity or a significant decrease (p<0.001) in T1 relaxation, measured as %T1 change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p<0.0001) in T1 relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin-Gd-DTPA-biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p<0.001) and 3-nitrotyrosine (3-NT) (p<0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.
Biochimica et Biophysica Acta 08/2013; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Free radicals associated with oxidative stress play a major role in amyotrophic lateral sclerosis (ALS). By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo trapped radical adducts were detected in the spinal cords of SOD1(G93A) transgenic (Tg) mice for ALS. For this study, the nitrone spin-trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) was administered (i.p.) over 5 days prior to administration (i.v.) of an anti-DMPO probe [anti-DMPO antibody covalently bound to an albumin (BSA)-Gd (gadolinium)-DTPA (diethylenetriamine pentaacetic acid)-biotin MRI contrast agent] to trap free radicals. MRI was used to detect the presence of the anti-DMPO radical adducts by a significant sustained increase in MR signal intensities (p<0.05) or anti-DMPO probe concentrations measured from T1 relaxations (p<0.01). The biotin moiety of the anti-DMPO probe was targeted with fluorescent-labeled streptavidin to locate the probe in excised tissues. Negative controls included either Tg ALS mice initially administered saline rather than DMPO followed by the anti-DMPO probe, or non-Tg mice initially administered DMPO and then the anti-DMPO probe. The anti-DMPO probe was found to bind to neurons via co-localization fluorescence microscopy. DMPO adducts were also confirmed in disease/non-disease tissues from animals administered DMPO. Apparent diffusion coefficients from diffusion-weighted images of spinal cords from Tg mice were significantly elevated (p<0.001) compared to wild-type controls. This is the first report regarding the detection of in vivo trapped radical adducts in an ALS model. This novel, non-invasive, in vivo, diagnostic method can be applied to investigate the involvement of free radical mechanisms in ALS rodent models.
Free Radical Biology and Medicine 05/2013; · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor receptor 2 (VEGFR2) is an important angiogenic marker over-expressed in gliomas. With the use of molecular magnetic resonance imaging (mMRI) differing levels of VEGFR2 can be characterized in vivo with in rodent gliomas varying in angiogenesis. VEGFR2 levels were assessed by intravenous administration of an anti-VEGFR2 probe (anti-VEGFR2-albumin-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin) into C6 or RG2 glioma-bearing rats, and visualized with mMRI. A non-specific IgG was coupled to the albumin-Gd-DTPA-biotin construct as a contrast agent molecular weight control. VEGFR2 levels are heterogeneous in different regions of C6 gliomas, whereas VEGFR2 was more homogenous or evenly distributed in RG2 gliomas. RG2 gliomas have less VEGFR2 within tumor periphery and peri-necrotic (p<0.05) regions, but more VEGFR2 within tumor interior regions (p<0.01), compared to C6 gliomas. mMRI results were confirmed with fluorescence staining and mean fluorescence intensity (MFI) quantification of the anti-VEGFR2 probe in excised glioma and brain tissues, as well as detection of VEGFR2 in C6 and RG2 gliomas and corresponding contalateral brain tissues. Ex vivo VEGFR2 levels were found to be significantly higher in C6 gliomas compared to RG2 tumors (p<0.001), which corresponded with in vivo detection using the VEGFR2 probe. Immunohistochemistry staining for HIF-1α (hypoxia inducible factor 1α), which is associated with angiogenesis, indicated higher levels in RG2 (p<0.01) compared to C6 gliomas. The data suggests that C6 gliomas have angiogenesis which is associated more with large blood vessels in tumor periphery and peri-necrotic regions, and less microvascular angiogenesis within the tumor interior, compared to RG2 gliomas.
American Journal of Nuclear Medicine and Molecular Imaging 01/2013; 3(4):300-11. · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND:: Glioblastoma multiforme (GBM), high-grade glioma, is characterized by being diffuse, invasive, and highly angiogenic, and has a very poor prognosis. Identification of new biomarkers could help in the further diagnosis of GBM. OBJECTIVE:: To identify ELTD1 ([epidermal growth factor (EGF), latrophilin and seven transmembrane domain-containing 1] on chromosome 1) as a putative glioma-associated marker via a bioinformatic method. METHODS:: We used advanced data mining and a novel bioinformatics method to predict ELTD1 as a potential novel biomarker that is associated with gliomas. Validation was done with immunohistochemistry (IHC), which was used to detect levels of ELTD1 in human high-grade gliomas, and rat F98 glioma tumors. In vivo levels of ELTD1 in rat F98 gliomas were assessed using molecular MRI (mMRI). RESULTS:: ELTD1 was found to be significantly higher (P=.03) in high-grade gliomas (50 patients) compared to low-grade gliomas (21 patients), and compared well to traditional IHC markers including VEGF, GLUT-1,CAIX, and HIF-1α. ELTD1 gene expression indicates an association with grade, survival across grade, and an increase in the mesenchymal subtype. Significantly high (P<0.001) in vivo levels of ELTD1 were additionally found in F98 tumors, compared to normal brain tissue. CONCLUSION:: This study strongly suggests that associative analysis was able to accurately identify ELTD1 as a putative glioma-associated biomarker. The detection of ELTD1 was also validated in both rodent and human gliomas, and may serve as an additional biomarker for gliomas in pre-clinical and clinical diagnosis of gliomas.
[Show abstract][Hide abstract] ABSTRACT: The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal toxin-induced cleavage of multiple MEK's. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function, as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness.
Infection and immunity 10/2012; · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress plays a major role in diabetes. In vivo levels of membrane-bound radicals (MBRs) in a streptozotocin-induced diabetic mouse model were uniquely detected by combining molecular magnetic resonance imaging (mMRI) and immunotrapping techniques. An anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibody (Ab) covalently bound to an albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent (anti-DMPO probe), and mMRI, were used to detect in vivo levels of DMPO-MBR adducts in kidneys, livers, and lungs of diabetic mice, after DMPO administration. Magnetic resonance signal intensities, which increase in the presence of a Gd-based molecular probe, were significantly higher within the livers, kidneys, and lungs of diabetic animals administered the anti-DMPO probe compared with controls. Fluorescence images validated the location of the anti-DMPO probe in excised tissues via conjugation of streptavidin-Cy3, which targeted the probe biotin moiety, and immunohistochemistry was used to validate the presence of DMPO adducts in diabetic mouse livers. This is the first report of noninvasively imaging in vivo levels of MBRs within any disease model. This method can be specifically applied toward diabetes models for in vivo assessment of free radical levels, providing an avenue to more fully understand the role of free radicals in diabetes.
[Show abstract][Hide abstract] ABSTRACT: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult-onset neurovascular disorder caused by stereotyped mutations in the NOTCH3 receptor. Elucidation of its pathobiology is still incomplete and remains a challenge, in part because the available preclinical mouse models to date do not reproduce the full spectrum of CADASIL pathology and clinical disease.
Here, we report a novel knock-in mouse with Arg170Cys substitution in murine Notch3, corresponding to the prevalent Arg169Cys substitution in CADASIL. The Notch3(Arg170Cys) mice displayed late-onset, dominant CADASIL arteriopathy with typical granular osmiophilic material deposition and developed brain histopathology including thrombosis, microbleeds, gliosis, and microinfarction. Furthermore, Notch3(Arg170Cys) mice experienced neurological symptoms with motor defects such as staggering gait and limb paresis.
This model, for the first time, phenocopies the arteriopathy and the histopathologic as well as clinical features of CADASIL and may offer novel opportunities to investigate disease pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: Severe sepsis leads to massive activation of coagulation and complement cascades that could contribute to multiple organ failure and death. To investigate the role of the complement and its crosstalk with the hemostatic system in the pathophysiology and therapeutics of sepsis, we have used a potent inhibitor (compstatin) administered early or late after Escherichia coli challenge in a baboon model of sepsis-induced multiple organ failure. Compstatin infusion inhibited sepsis-induced blood and tissue biomarkers of complement activation, reduced leucopenia and thrombocytopenia, and lowered the accumulation of macrophages and platelets in organs. Compstatin decreased the coagulopathic response by down-regulating tissue factor and PAI-1, diminished global blood coagulation markers (fibrinogen, fibrin-degradation products, APTT), and preserved the endothelial anticoagulant properties. Compstatin treatment also improved cardiac function and the biochemical markers of kidney and liver damage. Histologic analysis of vital organs collected from animals euthanized after 24 hours showed decreased microvascular thrombosis, improved vascular barrier function, and less leukocyte infiltration and cell death, all consistent with attenuated organ injury. We conclude that complement-coagulation interplay contributes to the progression of severe sepsis and blocking the harmful effects of complement activation products, especially during the organ failure stage of severe sepsis is a potentially important therapeutic strategy.
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis is essential to tumour progression and a precise evaluation of angiogenesis is important for tumour early diagnosis and treatment. The quantitative and dynamic in vivo assessment of tumour angiogenesis can be achieved by molecular magnetic resonance imaging (mMRI). Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are the main regulatory systems in angiogenesis and have been used as hot targets for radionuclide-based molecular imaging. However, little research has been accomplished in targeting VEGF/VEGFRs by mMRI. In our study, we aimed to assess the expression of VEGFR2 in C6 gliomas by using a specific molecular probe with mMRI. The differential uptake of the probe conjugated to anti-VEGFR2 monoclonal antibody, shown by varied increases in T(1) signal intensity during a 2 hr period, demonstrated the heterogeneous expression of VEGFR2 in different tumour regions. Microscopic fluorescence imaging, obtained for the biotin group in the probe with streptavidin-Cy3, along with staining for cellular VEGFR2 levels, laminin and CD45, confirmed the differential distribution of the probe which targeted VEGFR2 on endothelial cells. The angiogenesis process was also assessed using magnetic resonance angiography, which quantified tumour blood volume and provided a macroscopic view and a dynamic change of the correlation between tumour vasculature and VEGFR2 expression. Together these results suggest mMRI can be very useful in assessing and characterizing the expression of specific angiogenic markers in vivo and help evaluate angiogenesis associated with tumour progression.
Journal of Cellular and Molecular Medicine 05/2010; 15(4):837-49. · 3.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Increased iNOS expression is often found in brain tumors, such as gliomas. The goal of this study was to develop and assess a novel molecular MRI (mMRI) probe for in vivo detection of iNOS in rodent models for gliomas (intracerebral implantation of rat C6 or RG2 cells or ethyl nitrosourea-induced glioma). The probe we used incorporated a Gd-DTPA (gadolinium(III) complex of diethylenetriamine-N,N,N',N'',N''-pentaacetate) backbone with albumin and biotin moieties and covalent binding of an anti-iNOS antibody (Ab) to albumin (anti-iNOS probe). We used mMRI with the anti-iNOS probe to detect in vivo iNOS levels in gliomas. Nonimmune normal rat IgG coupled to albumin-Gd-DTPA-biotin was used as a control nonspecific contrast agent. By targeting the biotin component of the anti-iNOS probe with streptavidin Cy3, fluorescence imaging confirmed the specificity of the probe for iNOS in glioma tissue. iNOS levels in glioma tumors were also confirmed via Western blots and immunohistochemistry. The presence of plasma membrane-associated iNOS in glioma cells was established by transmission electron microscopy and gold-labeled anti-iNOS Ab. The more aggressive RG2 glioma was not found to have higher levels of iNOS compared to C6. Differences in glioma vascularization and blood-brain barrier permeability between the C6 and the RG2 gliomas are discussed. In vivo assessment of iNOS levels associated with tumor development is quite feasible in heterogeneous tissues with mMRI.
Free Radical Biology and Medicine 03/2010; 48(5):691-703. · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The levels of Met, a tyrosine kinase receptor for the hepatocyte growth factor or scatter factor, are elevated during tissue regeneration, and can be used to assess tissue regeneration associated with engineered tissue grafts. This study involved the development and assessment of a novel magnetic resonance imaging (MRI) molecular probe for the in vivo detection of Met in an experimental rodent (rat) model of disease (C6 glioma). The implication of using these probes in tissue engineering is discussed. The molecular targeting agent we used in our study incorporated a magnetite-based dextran-coated nanoparticle backbone covalently bound to an anti-Met antibody. We used molecular MRI with an anti-Met probe to detect in vivo Met levels as a molecular marker for gliomas. Tumor regions were compared to normal tissue, and found to significantly (p < 0.05) decrease MR signal intensity and T(2) relaxation in tumors. Nonimmune nonspecific normal rat IgG coupled to the dextran-coated nanoparticles was used as a control. Met levels in tumor tissues were confirmed in Western blots. Based on our results, in vivo evaluation of tissue regeneration using molecular MRI is possible in tissue engineering applications.
Tissue Engineering Part A 11/2009; 16(2):365-71. · 4.64 Impact Factor