Publications by authors named "Chandra C Ghosh"

28 Publications

  • Page 1 of 1

Inhibiting Airway Smooth Muscle Contraction Using Pitavastatin: A Role for the Mevalonate Pathway in Regulating Cytoskeletal Proteins.

Front Pharmacol 2020 6;11:469. Epub 2020 May 6.

Department of Emergency Medicine, Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States.

Despite maximal use of currently available therapies, a significant number of asthma patients continue to experience severe, and sometimes life-threatening bronchoconstriction. To fill this therapeutic gap, we examined a potential role for the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitor, pitavastatin. Using human airway smooth muscle (ASM) cells and murine precision-cut lung slices, we discovered that pitavastatin significantly inhibited basal-, histamine-, and methacholine (MCh)-induced ASM contraction. This occurred reduction of myosin light chain 2 (MLC2) phosphorylation, and F-actin stress fiber density and distribution, in a mevalonate (MA)- and geranylgeranyl pyrophosphate (GGPP)-dependent manner. Pitavastatin also potentiated the ASM relaxing effect of a simulated deep breath, a beneficial effect that is notably absent with the β2-agonist, isoproterenol. Finally, pitavastatin attenuated ASM pro-inflammatory cytokine production in a GGPP-dependent manner. By targeting all three hallmark features of ASM dysfunction in asthma-contraction, failure to adequately relax in response to a deep breath, and inflammation-pitavastatin may represent a unique asthma therapeutic.
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http://dx.doi.org/10.3389/fphar.2020.00469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218099PMC
May 2020

-Secreted Alkaline Protease 1 Mediates Airways Hyperresponsiveness in Severe Asthma.

Immunohorizons 2019 08 6;3(8):368-377. Epub 2019 Aug 6.

Lung and Vascular Inflammation Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases/National Institutes of Health, Bethesda, MD 20892;

The hallmark features of allergic asthma are type 2 (eosinophilic) inflammation and airways hyperresponsiveness (AHR). Although these features often comanifest in mouse lungs in vivo, we demonstrate in this study that the serine protease Alp1 from the ubiquitous mold and allergen, , can induce AHR in mice unable to generate eosinophilic inflammation. Strikingly, Alp1 induced AHR in mice devoid of protease-activated receptor 2/F2 trypsin-like receptor 1 (PAR2/F2RL1), a receptor expressed in lung epithelium that is critical for allergic responses to protease-containing allergens. Instead, using precision-cut lung slices and human airway smooth muscle cells, we demonstrate that Alp1 directly increased contractile force. Taken together, these findings suggest that Alp1 induces bronchoconstriction through mechanisms that are largely independent of allergic inflammation and point to a new target for direct intervention of fungal-associated asthma.
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http://dx.doi.org/10.4049/immunohorizons.1900046DOI Listing
August 2019

Calcineurin inhibitors augment endothelial-to-mesenchymal transition by enhancing proliferation in association with cytokine-mediated activation.

Biochem Biophys Res Commun 2019 11 18;519(4):667-673. Epub 2019 Sep 18.

The Transplant Research Program and the Division of Nephrology, Boston Children's Hospital, Boston, MA, 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA, 02115, USA. Electronic address:

Calcineurin Inhibitors (CNIs) are routinely used for immunosuppression following solid organ transplantation. However, the prolonged use of these agents lead to organ fibrosis which limits their efficacy. CNIs induce TGFβ expression, which is reported to augment endothelial-to-mesenchymal transition (EndMT), but their role in this process is not known. In these studies, we find that the CNIs FK506 and cyclosporine (CsA) are potent to increase endothelial cell (EC) proliferation using established in vitro assays (P < 0.05). Furthermore, using phosphokinase arrays, we find that each CNI activates the MAPK and Akt/mTOR signaling pathways, and that pharmacological inhibition of each pathway targets CNI-induced proliferative responses (P < 0.001). EndMT was evaluated by FACS for N-cadherin and CD31 expression and by qPCR for the expression of α-smooth muscle actin, N-cadherin and Snail. We find that CNIs do not directly induce dedifferentiation, while TGFβ and hypoxia induce EndMT in small numbers of EC. In contrast, the treatment of EC with the inflammatory cytokine TNFα was potent to elicit an EndMT response, and its effects were most notably in EC following proliferation/doubling. Taken together, these observations suggest that CNIs elicit proliferative responses, which enhance EndMT in association with local inflammation. The clinical implications of these findings are that anti-proliferative therapeutics have high potential to target the initiation of this EndMT response.
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http://dx.doi.org/10.1016/j.bbrc.2019.09.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7119266PMC
November 2019

Treatment with 2,4-Dihydroxybenzoic Acid Prevents FSGS Progression and Renal Fibrosis in Podocyte-Specific Knockout Mice.

J Am Soc Nephrol 2019 Feb 8. Epub 2019 Feb 8.

Division of Nephrology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts;

Background: Although studies have identified >55 genes as causing steroid-resistant nephrotic syndrome (SRNS) and localized its pathogenesis to glomerular podocytes, the disease mechanisms of SRNS remain largely enigmatic. We recently reported that individuals with mutations in COQ6, a coenzyme Q (also called CoQ, CoQ, or ubiquinone) biosynthesis pathway enzyme, develop SRNS with sensorineural deafness, and demonstrated the beneficial effect of CoQ for maintenace of kidney function.

Methods: To study function in podocytes, we generated a podocyte-specific knockout mouse ( ) model and a transient siRNA-based knockdown in a human podocyte cell line. Mice were monitored for development of proteinuria and assessed for development of glomerular sclerosis. Using a podocyte migration assay, we compared motility in knockdown podocytes and control podocytes. We also randomly assigned 5-month-old mice and controls to receive no treatment or 2,4-dihydroxybenzoic acid (2,4-diHB), an analog of a CoQ precursor molecule that is classified as a food additive by health authorities in Europe and the United States.

Results: Abrogation of in mouse podocytes caused FSGS and proteinuria (>46-fold increases in albuminuria). studies revealed an impaired podocyte migration rate in knockdown human podocytes. Treating mice or cells with 2,4-diHB prevented renal dysfunction and reversed podocyte migration rate impairment. Survival of mice given 2,4diHB was comparable to that of control mice and significantly higher than that of untreated mice, half of which died by 10 months of age.

Conclusions: These findings reveal a potential novel treatment strategy for those cases of human nephrotic syndrome that are caused by a primary dysfunction in the CoQ biosynthesis pathway.
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http://dx.doi.org/10.1681/ASN.2018060625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405149PMC
February 2019

Multiplexed, high-throughput measurements of cell contraction and endothelial barrier function.

Lab Invest 2019 01 11;99(1):138-145. Epub 2018 Oct 11.

Center for Vascular Biology Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA.

Vascular leakage, protein exudation, and edema formation are events commonly triggered by inflammation and facilitated by gaps that form between adjacent endothelial cells (ECs) of the vasculature. In such paracellular gap formation, the role of EC contraction is widely implicated, and even therapeutically targeted. However, related measurement approaches remain slow, tedious, and complex to perform. Here, we have developed a multiplexed, high-throughput screen to simultaneously quantify paracellular gaps, EC contractile forces, and to visualize F-actin stress fibers, and VE-cadherin. As proof-of-principle, we examined barrier-protective mechanisms of the Rho-associated kinase inhibitor, Y-27632, and the canonical agonist of the Tie2 receptor, Angiopoietin-1 (Angpt-1). Y-27632 reduced EC contraction and actin stress fiber formation, whereas Angpt-1 did not. Yet both agents reduced thrombin-, LPS-, and TNFα-induced paracellular gap formation. This unexpected result suggests that Angpt-1 can achieve barrier defense without reducing EC contraction, a mechanism that has not been previously described. This insight was enabled by the multiplex nature of the force-based platform. The high-throughput format we describe should accelerate both mechanistic studies and the screening of pharmacological modulators of endothelial barrier function.
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http://dx.doi.org/10.1038/s41374-018-0136-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309267PMC
January 2019

Molecular Regulation of Acute Tie2 Suppression in Sepsis.

Crit Care Med 2018 09;46(9):e928-e936

Department of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany.

Objectives: Tie2 is a tyrosine kinase receptor expressed by endothelial cells that maintains vascular barrier function. We recently reported that diverse critical illnesses acutely decrease Tie2 expression and that experimental Tie2 reduction suffices to recapitulate cardinal features of the septic vasculature. Here we investigated molecular mechanisms driving Tie2 suppression in settings of critical illness.

Design: Laboratory and animal research, postmortem kidney biopsies from acute kidney injury patients and serum from septic shock patients.

Setting: Research laboratories and ICU of Hannover Medical School, Harvard Medical School, and University of Groningen.

Patients: Deceased septic acute kidney injury patients (n = 16) and controls (n = 12) and septic shock patients (n = 57) and controls (n = 22).

Interventions: Molecular biology assays (Western blot, quantitative polymerase chain reaction) + in vitro models of flow and transendothelial electrical resistance experiments in human umbilical vein endothelial cells; murine cecal ligation and puncture and lipopolysaccharide administration.

Measurements And Main Results: We observed rapid reduction of both Tie2 messenger RNA and protein in mice following cecal ligation and puncture. In cultured endothelial cells exposed to tumor necrosis factor-α, suppression of Tie2 protein was more severe than Tie2 messenger RNA, suggesting distinct regulatory mechanisms. Evidence of protein-level regulation was found in tumor necrosis factor-α-treated endothelial cells, septic mice, and septic humans, all three of which displayed elevation of the soluble N-terminal fragment of Tie2. The matrix metalloprotease 14 was both necessary and sufficient for N-terminal Tie2 shedding. Since clinical settings of Tie2 suppression are often characterized by shock, we next investigated the effects of laminar flow on Tie2 expression. Compared with absence of flow, laminar flow induced both Tie2 messenger RNA and the expression of GATA binding protein 3. Conversely, septic lungs exhibited reduced GATA binding protein 3, and knockdown of GATA binding protein 3 in flow-exposed endothelial cells reduced Tie2 messenger RNA. Postmortem tissue from septic patients showed a trend toward reduced GATA binding protein 3 expression that was associated with Tie2 messenger RNA levels (p < 0.005).

Conclusions: Tie2 suppression is a pivotal event in sepsis that may be regulated both by matrix metalloprotease 14-driven Tie2 protein cleavage and GATA binding protein 3-driven flow regulation of Tie2 transcript.
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http://dx.doi.org/10.1097/CCM.0000000000003269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095816PMC
September 2018

Flunarizine suppresses endothelial Angiopoietin-2 in a calcium - dependent fashion in sepsis.

Sci Rep 2017 03 9;7:44113. Epub 2017 Mar 9.

Division of Nephrology and Hypertension, Hannover Medical School, Germany.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to an infection leading to systemic inflammation and endothelial barrier breakdown. The vascular-destabilizing factor Angiopoietin-2 (Angpt-2) has been implicated in these processes in humans. Here we screened in an unbiased approach FDA-approved compounds with respect to Angpt-2 suppression in endothelial cells (ECs) in vitro. We identified Flunarizine - a well-known anti-migraine calcium channel (CC) blocker - being able to diminish intracellular Angpt-2 protein in a time- and dose-dependent fashion thereby indirectly reducing the released protein. Moreover, Flunarizine protected ECs from TNFα-induced increase in Angpt-2 transcription and vascular barrier breakdown. Mechanistically, we could exclude canonical Tie2 signalling being responsible but found that three structurally distinct T-type - but not L-type - CC blockers can suppress Angpt-2. Most importantly, experimental increase in intracellular calcium abolished Flunarizine's effect. Flunarizine was also able to block the injurious increase of Angpt-2 in murine endotoxemia in vivo. This resulted in reduced pulmonary adhesion molecule expression (intercellular adhesion molecule-1) and tissue infiltration of inflammatory cells (Gr-1). Our finding could have therapeutic implications as side effects of Flunarizine are low and specific sepsis therapeutics that target the dysregulated host response are highly desirable.
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http://dx.doi.org/10.1038/srep44113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343493PMC
March 2017

KLF2 and KLF4 control endothelial identity and vascular integrity.

JCI Insight 2017 02 23;2(4):e91700. Epub 2017 Feb 23.

Cardiovascular Research Institute, Department of Medicine, and.

Maintenance of vascular integrity in the adult animal is needed for survival, and it is critically dependent on the endothelial lining, which controls barrier function, blood fluidity, and flow dynamics. However, nodal regulators that coordinate endothelial identity and function in the adult animal remain poorly characterized. Here, we show that endothelial KLF2 and KLF4 control a large segment of the endothelial transcriptome, thereby affecting virtually all key endothelial functions. Inducible endothelial-specific deletion of and/or reveals that a single allele of either gene is sufficient for survival, but absence of both (EC-DKO) results in acute death from myocardial infarction, heart failure, and stroke. EC-DKO animals exhibit profound compromise in vascular integrity and profound dysregulation of the coagulation system. Collectively, these studies establish an absolute requirement for KLF2/4 for maintenance of endothelial and vascular integrity in the adult animal.
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http://dx.doi.org/10.1172/jci.insight.91700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5313061PMC
February 2017

A branched-chain amino acid metabolite drives vascular fatty acid transport and causes insulin resistance.

Nat Med 2016 Apr 7;22(4):421-6. Epub 2016 Mar 7.

Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Epidemiological and experimental data implicate branched-chain amino acids (BCAAs) in the development of insulin resistance, but the mechanisms that underlie this link remain unclear. Insulin resistance in skeletal muscle stems from the excess accumulation of lipid species, a process that requires blood-borne lipids to initially traverse the blood vessel wall. How this trans-endothelial transport occurs and how it is regulated are not well understood. Here we leveraged PPARGC1a (also known as PGC-1α; encoded by Ppargc1a), a transcriptional coactivator that regulates broad programs of fatty acid consumption, to identify 3-hydroxyisobutyrate (3-HIB), a catabolic intermediate of the BCAA valine, as a new paracrine regulator of trans-endothelial fatty acid transport. We found that 3-HIB is secreted from muscle cells, activates endothelial fatty acid transport, stimulates muscle fatty acid uptake in vivo and promotes lipid accumulation in muscle, leading to insulin resistance in mice. Conversely, inhibiting the synthesis of 3-HIB in muscle cells blocks the ability of PGC-1α to promote endothelial fatty acid uptake. 3-HIB levels are elevated in muscle from db/db mice with diabetes and from human subjects with diabetes, as compared to those without diabetes. These data unveil a mechanism in which the metabolite 3-HIB, by regulating the trans-endothelial flux of fatty acids, links the regulation of fatty acid flux to BCAA catabolism, providing a mechanistic explanation for how increased BCAA catabolic flux can cause diabetes.
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http://dx.doi.org/10.1038/nm.4057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4949205PMC
April 2016

Gene control of tyrosine kinase TIE2 and vascular manifestations of infections.

Proc Natl Acad Sci U S A 2016 Mar 16;113(9):2472-7. Epub 2016 Feb 16.

Center for Vascular Biology Research and Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215;

Ligands of the endothelial-enriched tunica interna endothelial cell kinase 2 (Tie2) are markedly imbalanced in severe infections associated with vascular leakage, yet regulation of the receptor itself has been understudied in this context. Here, we show that TIE2 gene expression may constitute a novel vascular barrier control mechanism in diverse infections. Tie2 expression declined rapidly in wide-ranging models of leak-associated infections, including anthrax, influenza, malaria, and sepsis. Forced Tie2 suppression sufficed to attenuate barrier function and sensitize endothelium to permeability mediators. Rapid reduction of pulmonary Tie2 in otherwise healthy animals attenuated downstream kinase signaling to the barrier effector vascular endothelial (VE)-cadherin and induced vascular leakage. Compared with wild-type littermates, mice possessing one allele of Tie2 suffered more severe vascular leakage and higher mortality in two different sepsis models. Common genetic variants that influence TIE2 expression were then sought in the HapMap3 cohort. Remarkably, each of the three strongest predicted cis-acting SNPs in HapMap3 was also associated with the risk of acute respiratory distress syndrome (ARDS) in an intensive care unit cohort of 1,614 subjects. The haplotype associated with the highest TIE2 expression conferred a 28% reduction in the risk of ARDS independent of other major clinical variables, including disease severity. In contrast, the most common haplotype was associated with both the lowest TIE2 expression and 31% higher ARDS risk. Together, the results implicate common genetic variation at the TIE2 locus as a determinant of vascular leak-related clinical outcomes from common infections, suggesting new tools to identify individuals at unusual risk for deleterious complications of infection.
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http://dx.doi.org/10.1073/pnas.1519467113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780619PMC
March 2016

Drug Repurposing Screen Identifies Foxo1-Dependent Angiopoietin-2 Regulation in Sepsis.

Crit Care Med 2015 Jul;43(7):e230-40

1Center for Vascular Biology Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA. 2Department of Nephrology and Hypertension, Medical School Hannover, Hannover, Germany. 3Department of Heart, Thoracic, Transplantation and Vascular Surgery, Medical School Hannover, Hannover, Germany. 4Silence Therapeutics AG, Berlin, Germany. 5Department of Reproductive and Vascular Biology, Aston University, Birmingham, United Kingdom. 6Academic Department of Anaesthesia, Pain, and Critical Care, Heart of England NHS Foundation Trust, Birmingham, United Kingdom. 7Division of Critical Care and Department of Emergency Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA.

Objective: The recent withdrawal of a targeted sepsis therapy has diminished pharmaceutical enthusiasm for developing novel drugs for the treatment of sepsis. Angiopoietin-2 is an endothelial-derived protein that potentiates vascular inflammation and leakage and may be involved in sepsis pathogenesis. We screened approved compounds for putative inhibitors of angiopoietin-2 production and investigated underlying molecular mechanisms.

Design: Laboratory and animal research plus prospective placebo-controlled randomized controlled trial (NCT00529139) and retrospective analysis (NCT00676897).

Setting: Research laboratories of Hannover Medical School and Harvard Medical School.

Patients: Septic patients/C57Bl/6 mice and human endothelial cells.

Interventions: Food and Drug Administration-approved library screening.

Measurements And Main Results: In a cell-based screen of more than 650 Food and Drug Administration-approved compounds, we identified multiple members of the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor drug class (referred to as statins) that suppressed angiopoietin-2. Simvastatin inhibited 3-hydroxy-3-methyl-glutaryl-CoA reductase, which in turn activated PI3K-kinase. Downstream of this signaling, PI3K-dependent phosphorylation of the transcription factor Foxo1 at key amino acids inhibited its ability to shuttle to the nucleus and bind cis-elements in the angiopoietin-2 promoter. In septic mice, transient inhibition of angiopoietin-2 expression by liposomal siRNA in vivo improved absolute survival by 50%. Simvastatin had a similar effect, but the combination of angiopoietin-2 siRNA and simvastatin showed no additive benefit. To verify the link between statins and angiopoietin-2 in humans, we performed a pilot matched case-control study and a small randomized placebo-controlled trial demonstrating beneficial effects on angiopoietin-2.

Conclusions: 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors may operate through a novel Foxo1-angiopoietin-2 mechanism to suppress de novo production of angiopoietin-2 and thereby ameliorate manifestations of sepsis. Given angiopoietin-2's dual role as a biomarker and candidate disease mediator, early serum angiopoietin-2 measurement may serve as a stratification tool for future trials of drugs targeting vascular leakage.
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http://dx.doi.org/10.1097/CCM.0000000000000993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806531PMC
July 2015

Angiopoietin-1 requires oxidant signaling through p47phox to promote endothelial barrier defense.

PLoS One 2015 11;10(3):e0119577. Epub 2015 Mar 11.

Center for Vascular Biology Research and Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, United States of America.

Background: Reactive oxygen species (ROS) are largely considered to be pathogenic to normal endothelial function in disease states such as sepsis. We hypothesized that Angiopoietin-1 (Angpt-1), an endogenous agonist of the endothelial-specific receptor, Tie-2, promotes barrier defense by activating NADPH oxidase (NOX) signaling.

Methods And Findings: Using primary human microvascular endothelial cells (HMVECs), we found that Angpt-1 stimulation induces phosphorylation of p47phox and a brief oxidative burst that is lost when chemical inhibitors of NOX activity or siRNA against the NOX component p47phox were applied. As a result, there was attenuated ROS activity, disrupted junctional contacts, enhanced actin stress fiber accumulation, and induced gap formation between confluent HMVECs. All of these changes were associated with weakened barrier function. The ability of Angpt-1 to prevent identical changes induced by inflammatory permeability mediators, thrombin and lipopolysaccharides (LPS), was abrogated by p47phox knockdown. P47phox was required for Angpt-1 to activate Rac1 and inhibit mediator-induced activation of the small GTPase RhoA. Finally, Angpt-1 gene transfer prevented vascular leakage in wildtype mice exposed to systemically administered LPS, but not in p47phox knock out (p47-/-) littermates.

Conclusions: These results suggest an essential role for NOX signaling in Angpt-1-mediated endothelial barrier defense against mediators of systemic inflammation. More broadly, oxidants generated for signal transduction may have a barrier-promoting role in vascular endothelium.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119577PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4356555PMC
November 2015

Parmodulins inhibit thrombus formation without inducing endothelial injury caused by vorapaxar.

Blood 2015 Mar 13;125(12):1976-85. Epub 2015 Jan 13.

Division of Hemostasis and Thrombosis, Department of Medicine, and.

Protease-activated receptor-1 (PAR1) couples the coagulation cascade to platelet activation during myocardial infarction and to endothelial inflammation during sepsis. This receptor demonstrates marked signaling bias. Its activation by thrombin stimulates prothrombotic and proinflammatory signaling, whereas its activation by activated protein C (APC) stimulates cytoprotective and antiinflammatory signaling. A challenge in developing PAR1-targeted therapies is to inhibit detrimental signaling while sparing beneficial pathways. We now characterize a novel class of structurally unrelated small-molecule PAR1 antagonists, termed parmodulins, and compare the activity of these compounds to previously characterized compounds that act at the PAR1 ligand-binding site. We find that parmodulins target the cytoplasmic face of PAR1 without modifying the ligand-binding site, blocking signaling through Gαq but not Gα13 in vitro and thrombus formation in vivo. In endothelium, parmodulins inhibit prothrombotic and proinflammatory signaling without blocking APC-mediated pathways or inducing endothelial injury. In contrast, orthosteric PAR1 antagonists such as vorapaxar inhibit all signaling downstream of PAR1. Furthermore, exposure of endothelial cells to nanomolar concentrations of vorapaxar induces endothelial cell barrier dysfunction and apoptosis. These studies demonstrate how functionally selective antagonism can be achieved by targeting the cytoplasmic face of a G-protein-coupled receptor to selectively block pathologic signaling while preserving cytoprotective pathways.
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http://dx.doi.org/10.1182/blood-2014-09-599910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4366627PMC
March 2015

Inflammatory Markers of the Systemic Capillary Leak Syndrome (Clarkson Disease).

J Clin Cell Immunol 2014 ;5:1000213

Laboratory of Allergic Diseases, NIAID/NIH, Bethesda, MD, USA.

Objectives: The Systemic Capillary Leak Syndrome (SCLS) is a rare and potentially fatal disorder resembling systemic anaphylaxis that is characterized by transient episodes of hypotensive shock and peripheral edema. The pathogenesis of SCLS is unknown, and triggers for attacks are apparent only in a minority of patients. We introduce a clinical algorithm for the diagnosis of SCLS, and we investigated potential serum biomarkers of acute SCLS episodes.

Methods: We analyzed serum cytokines in a cohort of 35 patients with an established diagnosis of SCLS and characterized the effects of SCLS sera on endothelial cell function. We investigated the cellular source(s) of CXCL10, a chemokine that was significantly elevated in both basal and acute SCLS sera, by flow cytometry.

Results: Several cytokines were elevated in acute SCLS sera compared to baseline or sera from healthy controls, including CXCL10, CCL2, IL-1β, IL-6, IL-8, IL-12 and TNFα. The majority of acute sera failed to activate endothelial cells as assessed by surface adhesion marker expression. Monocytes appear to be the major source of serum CXCL10, and the percentage of CXLC10+ monocytes in response to IFNγ stimulation was increased in SCLS subjects compared to controls.

Conclusions: The presence of proinflammatory cytokines in acute SCLS sera suggests that inflammation or infection may have a role in triggering episodes. The enhanced capacity of monocytes from SCLS patients to produce CXCL10 suggests a new therapeutic avenue for SCLS.
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http://dx.doi.org/10.4172/2155-9899.1000213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4232957PMC
January 2014

Lung-targeted RNA interference against angiopoietin-2 ameliorates multiple organ dysfunction and death in sepsis.

Crit Care Med 2014 Oct;42(10):e654-62

1Department of Nephrology and Hypertension, Medical School Hannover, Hannover, Germany. 2Silence Therapeutics GmbH, Berlin, Germany. 3Center for Vascular Biology Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA.

Objective: Angiopoietin-2, a protein secreted by stimulated endothelium and an antagonist of the endothelium-stabilizing receptor Tie2, contributes to the pathophysiology of septic multiple organ dysfunction. We tested the therapeutic potential of a pulmonary-endothelium-specific RNA interference-based angiopoietin-2 targeting strategy in sepsis.

Design: Laboratory and animal research.

Settings: Research laboratories of the Medical School Hannover, Department of Nephrology and Hypertension, Hannover and Silence Therapeutics GmbH, Berlin.

Subjects: C57Bl/6 mice.

Interventions: Lung-endothelium-specific angiopoietin-2 small interfering RNA was administered both before and after sepsis induction (cecal ligation and puncture or lipopolysaccharides) intravenously.

Measurements And Main Results: Angiopoietin-2 small interfering RNA was highly specific and reduced angiopoietin-2 expression in the septic murine lungs up to 73.8% (p = 0.01) and enhanced the phosphorylation of Tie2 both in control and septic animals. Angiopoietin-2 small interfering RNA reduced pulmonary interleukin-6 transcription, intercellular adhesion molecule expression, neutrophil infiltration, and vascular leakage. Manifestations of sepsis were also attenuated in distant organs, including the kidney, where renal function was improved without affecting local angiopoietin-2 production. Finally, angiopoietin-2 small interfering RNA ameliorated the severity of illness and improved survival in cecal ligation and puncture, both as a pretreatment and as a rescue intervention.

Conclusion: The Tie2 antagonist angiopoietin-2 represents a promising target against sepsis-associated multiple organ dysfunction. A novel RNA interference therapeutic approach targeting gene expression in the pulmonary endothelium could be a clinically relevant pharmacological strategy to reduce injurious angiopoietin-2 synthesis.
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http://dx.doi.org/10.1097/CCM.0000000000000524DOI Listing
October 2014

Detecting Tie2, an endothelial growth factor receptor, by using immunohistochemistry in mouse lungs.

Methods Mol Biol 2014 ;1172:201-8

Roger Williams Medical Center, Cancer Immunotherapy (GMP), Providence, RI, USA.

Immunohistochemical (IHC) staining is an invaluable, sensitive, and effective method to detect the presence and localization of proteins in the cellular compartment in tissues. The basic concept of IHC is detecting the antigen in tissues by means of specific antibody binding, which is then demonstrated with a colored histochemical reaction that can be observed under a light microscope. The most challenging aspect of IHC techniques is optimizing the precise experimental conditions that are required to get a specific and a strong signal. The critical steps of IHC are specimen acquisition, fixation, permeabilization, detection system, and selection of the antigen specific antibody and its optimization. Here, we elaborate the technique using the endothelial growth factor binding receptor Tie2 in mouse lungs.
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http://dx.doi.org/10.1007/978-1-4939-0928-5_18DOI Listing
January 2015

Mechanistic classification of the systemic capillary leak syndrome: Clarkson disease.

Am J Respir Crit Care Med 2014 May;189(9):1145-7

1 National Institute of Allergy and Infectious Diseases/National Institutes of Health Bethesda, Maryland.

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http://dx.doi.org/10.1164/rccm.201310-1746LEDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4098109PMC
May 2014

Angiopoietin-2 may contribute to multiple organ dysfunction and death in sepsis*.

Crit Care Med 2012 Nov;40(11):3034-41

Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA.

Objective: : In sepsis, quiescent blood vessels become leaky and inflamed by mechanisms that are incompletely understood. We hypothesized that angiopoietin-2, a partial antagonist of the endothelium-stabilizing receptor Tie-2 secreted by endothelium, contributes to adverse outcomes in this disease.

Design: : Laboratory and animal research.

Settings: : Research laboratories and Emergency Department of Beth Israel Deaconess Medical Center, Boston, MA.

Subjects: : Angiopoietin-2 heterozygous mice, emergency department patients.

Measurements And Main Results: : Mice with one functional angiopoietin-2 allele developed milder kidney and lung injury, less tissue inflammation, and less vascular leakage compared to wild-type counterparts. Heterozygotes experienced >40% absolute survival advantage following two different models of sepsis (p = .004 and .018). In human subjects presenting to our emergency department with suspected infection (n = 270 combined), circulating angiopoietin-2 was markedly elevated within the first hour of clinical care. First-hour angiopoietin-2 concentrations were proportional to current disease severity (p < .0001), rose further over time in eventual nonsurvivors (p < .0001), and predicted the future occurrence of shock (p < .0001) or death (p < .0001) in the original cohort and an independent validation group. Finally, septic human serum disrupted the barrier function of microvascular endothelial cells, an effect fully neutralized by an angiopoietin-2 monoclonal antibody.

Conclusions: : We conclude that angiopoietin-2 induction precedes and contributes to the adverse outcomes in sepsis, opening a new avenue for therapeutic investigation.
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http://dx.doi.org/10.1097/CCM.0b013e31825fdc31DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3705559PMC
November 2012

Impaired function of the Tie-2 receptor contributes to vascular leakage and lethality in anthrax.

Proc Natl Acad Sci U S A 2012 Jun 4;109(25):10024-9. Epub 2012 Jun 4.

Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.

The anthrax lethal toxin (LT) enters host cells and enzymatically cleaves MAPKKs or MEKs. How these molecular events lead to death from anthrax remains poorly understood, but published reports suggest a direct effect of LT on vascular permeability. We have found that LT challenge in mice disrupts signaling through Tie-2, a tonically activated receptor tyrosine kinase in the endothelium. Genetic manipulations favoring Tie-2 activation enhanced interendothelial junctional contacts, prevented vascular leakage, and promoted survival following a lethal dose of LT. Cleavage of MEK1/2 was necessary for LT to induce endothelial barrier dysfunction, and activated Tie-2 signaled through the uncleaved fraction of MEKs to prevent LT's effects on the endothelium. Finally, primates infected with toxin-secreting Bacillus anthracis bacilli developed a rapid and marked imbalance in the endogenous ligands that signal Tie-2, similar to that seen in LT-challenged mice. Our results show that B. anthracis LT blunts signaling through Tie-2, thereby weakening the vascular barrier and contributing to lethality of the disease. Measurement of circulating Tie-2 ligands and manipulation of Tie-2 activity may represent future prognostic and therapeutic avenues for humans exposed to B. anthracis.
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http://dx.doi.org/10.1073/pnas.1120755109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3382507PMC
June 2012

Vascular endothelial hyperpermeability induces the clinical symptoms of Clarkson disease (the systemic capillary leak syndrome).

Blood 2012 May 12;119(18):4321-32. Epub 2012 Mar 12.

Molecular Signal Transduction Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

The systemic capillary leak syndrome (SCLS) is a rare disorder characterized by transient episodes of hypotensive shock and anasarca thought to arise from reversible microvascular barrier dysfunction. Although the high prevalence of a monoclonal gammopathy of unknown significance in SCLS suggests a pathogenic contribution of endogenous immunoglobulins, the mechanisms of vascular hyperpermeability remain obscure. Herein, we report clinical and molecular findings on 23 patients, the largest SCLS case series to date. Application of episodic SCLS sera, but neither the purified immunoglobulin fraction nor sera obtained from patients during remission, to human microvascular endothelial cells caused vascular endothelial cadherin internalization, disruption of interendothelial junctions, actin stress fiber formation, and increased permeability in complementary functional assays without inducing endothelial apoptosis. Intravenous immunoglobulin, one promising therapy for SCLS, mitigated the permeability effects of episodic sera. Consistent with the presence of endogenous, nonimmunoglobulin, circulating permeability factor(s) constrained to SCLS episodes, we found that vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2), were elevated in episodic SCLS sera but not in remission sera. Ab-based inhibition of Ang2 counteracted permeability induced by episodic SCLS sera. Comparable experiments with anti-VEGF Ab (bevacizumab) yielded less interpretable results, probably because of endothelial toxicity of VEGF withdrawal. Our results support a model of SCLS pathogenesis in which nonimmunoglobulin humoral factors such as VEGF and Ang2 contribute to transient endothelial contraction, suggesting a molecular mechanism for this highly lethal disorder.
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http://dx.doi.org/10.1182/blood-2011-08-375816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3359743PMC
May 2012

Angiopoietin-1 requires IQ domain GTPase-activating protein 1 to activate Rac1 and promote endothelial barrier defense.

Arterioscler Thromb Vasc Biol 2011 Nov;31(11):2643-52

Center for Vascular Biology Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA.

Objective: IQ domain GTPase-activating protein 1 (IQGAP1) contributes to cytoskeletal network regulation in epithelial cells by its scaffolding properties and by binding the Rho GTPase Rac1 to maintain its activity. The functions of IQGAP1 in endothelial cells beyond angiogenesis remain unclear. We hypothesized that IQGAP1 participates in the regulation of endothelial barrier function.

Methods And Results: Silencing IQGAP1 in human microvascular endothelial cells resulted in a disruption of adherens junctions, formation of interendothelial gaps, and a reduction in barrier function. Furthermore, silencing of IQGAP1 abrogated the barrier enhancement effect of angiopoietin-1 (Angpt-1) and abolished the barrier-stabilizing effect of Angpt-1 on thrombin-stimulated cells. Coimmunoprecipitation detected binding of endogenous IQGAP1 with Rac1 at baseline that was stronger when Rac1 was activated and weaker when it was deactivated. Measurement of GTP-bound Rac1 revealed that Angpt-1 failed to activate Rac1 not only if IQGAP1 was silenced but also if cells were transfected with a mutant disabled in Rac1 binding (T1050AX2). Furthermore, a dominant-active Rac1 was sufficient to completely reverse the morphological and functional changes induced by reduction in IQGAP1.

Conclusion: These experiments are the first demonstration of IQGAP1 regulating barrier function in any cell type. Further, our data show that Angpt-1 requires IQGAP1 as an indispensable activator of Rac1.
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http://dx.doi.org/10.1161/ATVBAHA.111.233189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249617PMC
November 2011

Effects of a synthetic PEG-ylated Tie-2 agonist peptide on endotoxemic lung injury and mortality.

Am J Physiol Lung Cell Mol Physiol 2011 Jun 18;300(6):L851-62. Epub 2011 Mar 18.

Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

A synthetic 7-mer, HHHRHSF, was recently identified by screening a phage display library for binding to the Tie-2 receptor. A polyethylene-oxide clustered version of this peptide, termed vasculotide (VT), was reported to activate Tie-2 and promote angiogenesis in a mouse model of diabetic ulcer. We hypothesized that VT administration would defend endothelial barrier function against sepsis-associated mediators of permeability, prevent lung vascular leakage arising in endotoxemia, and improve mortality in endotoxemic mice. In confluent human microvascular endothelial cells, VT prevented endotoxin-induced (lipopolysaccharides, LPS O111:B4) gap formation, loss of monolayer resistance, and translocation of labeled albumin. In 8-wk-old male C57Bl6/J mice given a ∼70% lethal dose of endotoxin (15 mg/kg ip), VT prevented lung vascular leakage and reversed the attenuation of lung vascular endothelial cadherin induced by endotoxemia. These protective effects of VT were associated with activation of Tie-2 and its downstream mediator, Akt. Echocardiographic studies showed only a nonsignificant trend toward improved myocardial performance associated with VT. Finally, we evaluated survival in this mouse model. Pretreatment with VT improved survival by 41.4% (n = 15/group, P = 0.02) and post-LPS administration of VT improved survival by 33.3% (n = 15/group, P = 0.051). VT-mediated protection from LPS lethality was lost in Tie-2 heterozygous mice, in agreement with VT's proposed receptor specificity. We conclude that this synthetic Tie-2 agonist, completely unrelated to endogenous Tie-2 ligands, is sufficient to activate the receptor and its downstream pathways in vivo and that the Tie-2 receptor may be an important target for therapeutic evaluation in conditions of pathological vascular leakage.
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http://dx.doi.org/10.1152/ajplung.00459.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3119126PMC
June 2011

Bortezomib induces nuclear translocation of IκBα resulting in gene-specific suppression of NF-κB--dependent transcription and induction of apoptosis in CTCL.

Mol Cancer Res 2011 Feb 11;9(2):183-94. Epub 2011 Jan 11.

Department of Biological Sciences, St. John's University, 8000 Utopia Parkway, Queens, NY 11439, USA.

Cutaneous T-cell lymphoma (CTCL) is characterized by constitutive activation of nuclear factor κB (NF-κB), which plays a crucial role in the survival of CTCL cells and their resistance to apoptosis. NF-κB activity in CTCL is inhibited by the proteasome inhibitor bortezomib; however, the mechanisms remained unknown. In this study, we investigated mechanisms by which bortezomib suppresses NF-κB activity in CTCL Hut-78 cells. We demonstrate that bortezomib and MG132 suppress NF-κB activity in Hut-78 cells by a novel mechanism that consists of inducing nuclear translocation and accumulation of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), which then associates with NF-κB p65 and p50 in the nucleus and inhibits NF-κB DNA binding activity. Surprisingly, however, while expression of NF-κB-dependent antiapoptotic genes cIAP1 and cIAP2 is inhibited by bortezomib, expression of Bcl-2 is not suppressed. Chromatin immunoprecipitation indicated that cIAP1 and cIAP2 promoters are occupied by NF-κB p65/50 heterodimers, whereas Bcl-2 promoter is occupied predominantly by p50/50 homodimers. Collectively, our data reveal a novel mechanism of bortezomib function in CTCL and suggest that the inhibition of NF-κB-dependent gene expression by bortezomib is gene specific and depends on the subunit composition of NF-κB dimers recruited to NF-κB-responsive promoters.
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http://dx.doi.org/10.1158/1541-7786.MCR-10-0368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078042PMC
February 2011

Gene-specific repression of proinflammatory cytokines in stimulated human macrophages by nuclear IκBα.

J Immunol 2010 Sep 9;185(6):3685-93. Epub 2010 Aug 9.

Department of Biological Sciences, St. John's University, New York, NY 11439, USA.

We have previously shown that increased nuclear accumulation of IkappaBalpha inhibits NF-kappaB activity and induces apoptosis in human leukocytes. In this study, we wanted to explore the possibility that the nucleocytoplasmic distribution of IkappaBalpha can be used as a therapeutic target for the regulation of NF-kappaB-dependent cytokine synthesis. Treatment of LPS-stimulated human U937 macrophages with an inhibitor of chromosome region maintenance 1-dependent nuclear export, leptomycin B, resulted in the increased nuclear accumulation of IkappaBalpha and inhibition of NF-kappaB DNA binding activity, caused by the nuclear IkappaBalpha-p65 NF-kappaB interaction. Surprisingly, however, whereas mRNA expression and cellular release of TNF-alpha, the beta form of pro-IL-1 (IL-1beta), and IL-6 were inhibited by the leptomycin B-induced nuclear IkappaBalpha, IL-8 mRNA expression and cellular release were not significantly affected. Analysis of in vivo recruitment of p65 NF-kappaB to NF-kappaB-regulated promoters by chromatin immunoprecipitation in U937 cells and human PBMCs indicated that although the p65 recruitment to TNF-alpha, IL-1beta, and IL-6 promoters was inhibited by the nuclear IkappaBalpha, p65 recruitment to IL-8 promoter was not repressed. Chromatin immunoprecipitation analyses using IkappaBalpha and S536 phosphospecific p65 NF-kappaB Abs demonstrated that although the newly synthesized IkappaBalpha induced by postinduction repression is recruited to TNF-alpha, IL-1beta, and IL-6 promoters but not to the IL-8 promoter, S536-phosphorylated p65 is recruited to IL-8 promoter, but not to TNF-alpha, IL-1beta, or IL-6 promoters. Together, these data indicate that the inhibition of NF-kappaB-dependent transcription by nuclear IkappaBalpha in LPS-stimulated macrophages is gene specific and depends on the S536 phosphorylation status of the recruited p65 NF-kappaB.
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http://dx.doi.org/10.4049/jimmunol.0902230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078650PMC
September 2010

Analysis of nucleocytoplasmic shuttling of NF kappa B proteins in human leukocytes.

Methods Mol Biol 2008 ;457:279-92

Department of Biological Sciences, St. John's University, Queens, NY, USA.

Controlled nucleocytoplasmic localization regulates activity of NF kappa B as well as other transcription factors. Analysis of the nucleocytoplasmic protein shuttling has been greatly facilitated by the use of leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export. The authors have previously shown that LMB inhibits NF kappa B activity in human neutrophils by increasing the nuclear accumulation of NF kappa B inhibitor, I kappa B alpha. In this chapter, the authors describe a protocol that uses LMB to study the nucleocytoplasmic shuttling of I kappa B alpha in human macrophage-like U937 cells, thus inhibiting NF kappa B activity. This protocol should be readily adaptable to analyze the nucleocytoplasmic shuttling of other proteins in human leukocytes.
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http://dx.doi.org/10.1007/978-1-59745-261-8_21DOI Listing
January 2009

Saccharomyces cerevisiae phospholipase C regulates transcription of Msn2p-dependent stress-responsive genes.

Eukaryot Cell 2008 Jun 28;7(6):967-79. Epub 2008 Mar 28.

Department of Biological Sciences, St. John's University, 8000 Utopia Parkway, Queens, NY 11439, USA.

Phosphatidylinositol phosphates are involved in signal transduction, cytoskeletal organization, and membrane trafficking. Inositol polyphosphates, produced from phosphatidylinositol phosphates by the phospholipase C-dependent pathway, regulate chromatin remodeling. We used genome-wide expression analysis to further investigate the roles of Plc1p (phosphoinositide-specific phospholipase C in Saccharomyces cerevisiae) and inositol polyphosphates in transcriptional regulation. Plc1p contributes to the regulation of approximately 2% of yeast genes in cells grown in rich medium. Most of these genes are induced by nutrient limitation and other environmental stresses and are derepressed in plc1 Delta cells. Surprisingly, genes regulated by Plc1p do not correlate with gene sets regulated by Swi/Snf or RSC chromatin remodeling complexes but show correlation with genes controlled by Msn2p. Our results suggest that the increased expression of stress-responsive genes in plc1 Delta cells is mediated by decreased cyclic AMP synthesis and protein kinase A (PKA)-mediated phosphorylation of Msn2p and increased binding of Msn2p to stress-responsive promoters. Accordingly, plc1 Delta cells display other phenotypes characteristic of cells with decreased PKA activity. Our results are consistent with a model in which Plc1p acts together with the membrane receptor Gpr1p and associated G(alpha) protein Gpa2p in a pathway separate from Ras1p/Ras2p and converging on PKA.
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http://dx.doi.org/10.1128/EC.00438-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2446660PMC
June 2008

NFkappaB is persistently activated in continuously stimulated human neutrophils.

Mol Med 2007 Mar-Apr;13(3-4):134-42

Department of Biological Sciences, St. John's University, New York, NY 11439, and Center for Immunology and Inflammation, The Feinstein Institute for Medical Research, North Shore--Long Island Jewish Health System, New Hyde Park, NY, USA.

Increased activation of the transcription factor NFkappaB in the neutrophils has been associated with the pathogenesis of sepsis, acute lung injury (ALI), bronchopulmonary dysplasia (BPD), and other neutrophil-mediated inflammatory disorders. Despite recent progress in analyzing early NFkappaB activation in human neutrophils, activation of NFkappaB in persistently stimulated neutrophils has not been previously studied. Because it is the persistent NFkappaB activation that is thought to be involved in the host response to sepsis and the pathogenesis of ALI and BPD, we hypothesized that continuously stimulated human neutrophils may exhibit a late phase of NFkappaB activity. The goal of this study was to analyze the NFkappaB activation and expression of IkappaB and NFkappaB proteins during neutrophil stimulation with inflammatory signals for prolonged times. We demonstrate that neutrophil stimulation with lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNFalpha) induces, in addition to the early activation at 30-60 min, a previously unrecognized late phase of NFkappaB activation. In LPS-stimulated neutrophils, this NFkappaB activity typically had a biphasic character, whereas TNFalpha-stimulated neutrophils exhibited a continuous NFkappaB activity peaking around 9 h after stimulation. In contrast to the early NFkappaB activation that inversely correlates to the nuclear levels of IkappaBalpha, however, in continuously stimulated neutrophils, NFkappaB is persistently activated despite considerable levels of IkappaBalpha present in the nucleus. Our data suggest that NFkappaB is persistently activated in human neutrophils during neutrophil-mediated inflammatory disorders, and this persistent NFkappaB activity may represent one of the underlying mechanisms for the continuous production of proinflammatory mediators.
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http://dx.doi.org/10.2119/2006–00072.MiskolciDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1892764PMC
August 2007

TNFalpha release from peripheral blood leukocytes depends on a CRM1-mediated nuclear export.

Biochem Biophys Res Commun 2006 Dec 16;351(2):354-60. Epub 2006 Oct 16.

Department of Biology, St. John's University, NY 11439, USA.

Tumor necrosis factor-alpha (TNFalpha) is a potent pro-inflammatory cytokine that plays a major role in the pathogenesis of acute and chronic inflammatory disorders such as septic shock and arthritis, respectively. Leukocytes stimulated with inflammatory signals such as lipopolysaccharide (LPS) are the predominant producers of TNFalpha, and thus control of TNFalpha release from stimulated leukocytes represents a potential therapeutic target. Here, we report that leptomycin B (LMB), a specific inhibitor of CRM1-dependent nuclear protein export, inhibits TNFalpha release from LPS-stimulated human peripheral blood neutrophils and mononuclear cells. In addition, immunofluorescence confocal microscopy and immunoblotting analysis indicate that TNFalpha is localized in the nucleus of human neutrophils and mononuclear cells. This study demonstrates that the cellular release of TNFalpha from stimulated leukocytes is mediated by the CRM1-dependent nuclear export mechanism. Inhibition of CRM1-dependent cellular release of TNFalpha could thus provide a novel therapeutic approach for disorders involving excessive TNFalpha release.
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http://dx.doi.org/10.1016/j.bbrc.2006.10.045DOI Listing
December 2006