Chandra C. Ghosh

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

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.

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

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.

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

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.

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

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 VTs 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.

Bortezomib Induces Nuclear Translocation of IκBα Resulting in Gene-Specific Suppression of NF-κB–Dependent Transcription and Induction of Apoptosis in CTCL

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. Mol Cancer Res; 9(2); 183–94. ©2011 AACR.

Angiopoietin-1 Requires IQ Domain GTPase-Activating Protein 1 to Activate Rac1 and Promote Endothelial Barrier Defense

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.

Gene control of tyrosine kinase TIE2 and vascular manifestations of infections

Significance Major infections, such as influenza and bacterial sepsis, kill millions of individuals yearly, most commonly from complications affecting the vasculature, such as acute respiratory distress syndrome. Poor outcomes from rare infections, such as Ebola virus disease, have also been linked to the vasculature. The basis for prominent vascular involvement in infectious syndromes remains poorly understood. The present work shows that humans exhibit common, yet highly consequential, genetic variation in the ability to sustain expression of a key homeostatic vascular receptor called tunica interna endothelial cell kinase 2. The results suggest that host determinants of the molecular vascular response to infection may have a heretofore underappreciated impact on clinical outcomes. They also suggest new means to identify at-risk individuals and personalize future therapies. 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.

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

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.

Parmodulins inhibit thrombus formation without inducing endothelial injury caused by vorapaxar

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.

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

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.