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BACKGROUND: Toll-like receptor 9 (TLR9) agonists induce inflammatory responses that promote the killing of infectious micro-organisms, cancer cells and develop adaptive immune responses. Their ability as immunomodulators to enhance the activity of checkpoint inhibitors (CPI) in treating liver tumors is limited in part by the distinctive biology of intrahepatic myeloid-derived suppressor cells (MDSC) and challenges with tumor-specific therapeutic delivery. We have shown that the regional delivery of type C TLR9 agonist via pressure-enabled drug delivery (PEDD) system improves delivery to the tumor, enhances depletion of MDSCs and overall, stimulates the immune system in combination with or without CPI. Currently, CPIs are delivered intravenously, although there is a growing interest in its subcutaneous (SQ) administration. We compared nelitolimod formerly known as SD-101 administered using PEDD in combination with systemic (Sys) or SQ CPI in murine liver metastases (LM). METHODS: The LM model was developed by injecting MC38-Luc cells via the spleen of 8-12 week old male C57/BL6 mice followed by splenectomy. After a week, fluorescently labeled nelitolimod (10 µg/mouse) was delivered via PEDD and co-administered anti-programmed cell death-1 (α-PD-1) either via Sys or SQ. Tumor burden was monitored by in vivo imaging system. Serum cytokine levels were analyzed by Luminex. Tissues were harvested on Day 3 (D3) or Day 10 (D10) post-PEDD to enrich CD45+ cells and were analyzed via NanoString targeted transcriptomics (D3) or flow cytometry (FC, D10) to interrogate immune cell populations (D10). For NanoString analysis, the innate immune panels were selected, and for FC, MDSCs (CD11b+Gr1+), B cells (B220+), dendritic cells (DC, CD11c+), T (CD3+) cells, and M1-like macrophages (F4/80+CD38+Egr2-) were quantified. RESULTS: Nelitolimod delivered via PEDD resulted in changes in innate and adaptive immune cells within LM, including depletion of liver MDSC and increased M1-like macrophages in the liver, which are supportive of antitumor immunity. While CPI monotherapy failed to control tumor progression, nelitolimod and CPI combination improved LM control, survival and antitumor immunity beyond the nelitolimod monotherapy effect, irrespective of CPI delivery route. CONCLUSION: The SQ route of CPI delivery was equivalent to Sys in combination with nelitolimod, suggesting SQ-CPI may be a rational choice in combination with PEDD of nelitolimod for liver tumor treatment.
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Inhibidores de Puntos de Control Inmunológico , Neoplasias Hepáticas , Células Supresoras de Origen Mieloide , Animales , Ratones , Células Supresoras de Origen Mieloide/efectos de los fármacos , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/secundario , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Humanos , Sistemas de Liberación de Medicamentos , Ratones Endogámicos C57BL , Línea Celular TumoralRESUMEN
Myeloid-derived suppressor cells (MDSCs) expand in response to malignancy and suppress responsiveness to immunotherapy, including checkpoint inhibitors (CPIs). Within the liver, MDSCs have unique immunosuppressive features. While TLR9 agonists have shown promising activities in enhancing CPI responsiveness in superficial tumors amenable to direct needle injection, clinical success for liver tumors with TLR9 agonists has been limited by delivery challenges. Here, we report that regional intravascular infusion of ODN2395 into mice with liver metastasis (LM) partially eliminated liver MDSCs and reprogrammed residual MDSC. TLR9 agonist regional infusion also induced an increase in the M1/M2 macrophage ratio. Enhanced TLR9 signaling was demonstrated by an increased activation of in NFκB (pP65) and production of IL6 compared with systemic infusion. Further, PBMC-derived human MDSCs express TLR9, and treatment with class C TLR9 agonists (ODN2395 and SD101) reduced the expansion of MDSC population. TLR9 stimulation induced MDSC apoptosis and increased the M1/M2 macrophage ratio. Regional TLR9 agonist infusion along with systemic anti-PD-1 therapy improved control of LM. With effective delivery, TLR9 agonists have the potential to favorably reprogram the liver TME through reduction of MDSCs and favorable macrophage polarization, which may improve responsiveness to systemic CPI therapy.
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Neoplasias Hepáticas , Células Supresoras de Origen Mieloide , Receptor Toll-Like 9 , Animales , Humanos , Ratones , Línea Celular Tumoral , Leucocitos Mononucleares , Neoplasias Hepáticas/tratamiento farmacológico , Receptor Toll-Like 9/agonistas , Microambiente TumoralRESUMEN
The statin family of therapeutics is widely used clinically as cholesterol lowering agents, and their effects to target intracellular mevalonate production is a key mechanism of action. In this study, we performed full transcriptomic RNA sequencing and qPCR to evaluate the effects of mevalonate on the immunoregulatory phenotype of endothelial cells (EC). We find that mevalonate-dependent gene regulation includes a reduction in the expression of multiple pro-inflammatory genes including TNFSF4 (OX40-L) and TNFSF18 (GITR-L) and a co-incident induction of immunoregulatory genes including LGALS3 (Galectin-3) and LGALS9 (Galectin-9). In functional assays, pretreatment of EC with simvastatin to inhibit mevalonate metabolism resulted in a dose-dependent reduction in the costimulation of CD45RO+ CD4+ T cell proliferation as well as IL-2, IFNγ and IL-6 production versus vehicle-treated EC. In contrast, pre-treatment of EC with L-mevalonate in combination with simvastatin reversed phenotypic and functional responses. Collectively, these results indicate that relative mevalonate metabolism by EC is critical to sustain EC-dependent mechanisms of immunity. Our findings have broad relevance for the repurposing of statins as therapeutics to augment immunoregulation and/or to inhibit local tissue pro-inflammatory cytokine production following transplantation.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Células Endoteliales , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Ácido Mevalónico/metabolismo , Ácido Mevalónico/farmacología , Fenotipo , Simvastatina/farmacología , Linfocitos T/metabolismoRESUMEN
Myeloid-derived suppressor cells (MDSCs) promote immunosuppressive activities in the tumor microenvironment (TME), resulting in increased tumor burden and diminishing the anti-tumor response of immunotherapies. While primary and metastatic tumors are typically the focal points of therapeutic development, the immune cells of the TME are differentially programmed by the tissue of the metastatic site. In particular, MDSCs are programmed uniquely within different organs in the context of tumor progression. Given that MDSC plasticity is shaped by the surrounding environment, the proteomes of MDSCs from different metastatic sites are hypothesized to be unique. A bottom-up proteomics approach using sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used to quantify the proteome of CD11b+ cells derived from murine liver metastases (LM) and lung metastases (LuM). A comparative proteomics workflow was employed to compare MDSC proteins from LuM (LuM-MDSC) and LM (LM-MDSC) while also elucidating common signaling pathways, protein function, and possible drug-protein interactions. SWATH-MS identified 2516 proteins from 200 µg of sample. Of the 2516 proteins, 2367 have matching transcriptomic data. Upregulated proteins from lung and liver-derived murine CD11b+ cells with matching mRNA transcriptomic data were categorized based on target knowledge and level of drug development. Comparative proteomic analysis demonstrates that liver and lung tumor-derived MDSCs have distinct proteomes that may be subject to pharmacologic manipulation.
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Metastatic liver tumors have presented challenges with the use of checkpoint inhibitors (CPIs), with only limited success. We hypothesize that regional delivery (RD) of CPIs can improve activity in the liver and minimize systemic exposure, thereby reducing immune-related adverse events (irAE). Using a murine model of colorectal cancer liver metastases (LM), we confirmed high levels of PD-L1 expression on the tumor cells and liver myeloid-derived suppressor cells (L-MDSC). In vivo, we detected improved LM response at 3 mg/kg on PTD7 via portal vein (PV) regional delivery as compared to 3 mg/kg via tail vein (TV) systemic delivery (p = 0.04). The minimal effective dose at PTD7 was 5 mg/kg (p = 0.01) via TV and 0.3 mg/kg (p = 0.02) via PV. We detected 6.7-fold lower circulating CPI antibody levels in the serum using the 0.3 mg/kg PV treatment compared to the 5 mg/kg TV cohort (p < 0.001) without increased liver toxicity. Additionally, 3 mg/kg PV treatment resulted in increased tumor cell apoptotic signaling compared to 5 mg/kg TV (p < 0.05). Therefore, RD of an anti-PD-1 CPI therapy for CRCLM may improve the therapeutic index by reducing the total dose required and limiting the systemic exposure. These advantages could expand CPI indications for liver tumors.
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Viral-associated respiratory infectious diseases are one of the most prominent subsets of respiratory failures, known as viral respiratory infections (VRI). VRIs are proceeded by an infection caused by viruses infecting the respiratory system. For the past 100 years, viral associated respiratory epidemics have been the most common cause of infectious disease worldwide. Due to several drawbacks of the current anti-viral treatments, such as drug resistance generation and non-targeting of viral proteins, the development of novel nanotherapeutic or nano-vaccine strategies can be considered essential. Due to their specific physical and biological properties, nanoparticles hold promising opportunities for both anti-viral treatments and vaccines against viral infections. Besides the specific physiological properties of the respiratory system, there is a significant demand for utilizing nano-designs in the production of vaccines or antiviral agents for airway-localized administration. SARS-CoV-2, as an immediate example of respiratory viruses, is an enveloped, positive-sense, single-stranded RNA virus belonging to the coronaviridae family. COVID-19 can lead to acute respiratory distress syndrome, similarly to other members of the coronaviridae. Hence, reviewing the current and past emerging nanotechnology-based medications on similar respiratory viral diseases can identify pathways towards generating novel SARS-CoV-2 nanotherapeutics and/or nano-vaccines.
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Antivirales/química , Portadores de Fármacos/química , Nanomedicina , Infecciones del Sistema Respiratorio/patología , Vacunas Virales/química , Virosis/patología , Antivirales/uso terapéutico , COVID-19/inmunología , COVID-19/patología , COVID-19/terapia , COVID-19/virología , Humanos , Sistema Inmunológico/metabolismo , Infecciones del Sistema Respiratorio/terapia , Infecciones del Sistema Respiratorio/virología , SARS-CoV-2/aislamiento & purificación , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Virosis/inmunología , Virosis/prevención & control , Virosis/terapiaRESUMEN
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 via 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 ß2-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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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, Aspergillus fumigatus, 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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Aspergillus fumigatus/inmunología , Asma/inmunología , Asma/microbiología , Proteínas Fúngicas/inmunología , Serina Endopeptidasas/inmunología , Alérgenos/inmunología , Animales , Aspergillus fumigatus/enzimología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Broncoconstricción/efectos de los fármacos , Broncoconstricción/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Humanos , Inflamación/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/inmunología , Receptor PAR-2/genética , Receptor PAR-2/inmunologíaRESUMEN
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 TGFß 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 (Pâ¯<â¯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 (Pâ¯<â¯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 TGFß 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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Inhibidores de la Calcineurina/farmacología , Proliferación Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Células CHO , Cadherinas/metabolismo , Células Cultivadas , Cricetinae , Cricetulus , Ciclosporina/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Tacrolimus/farmacologíaRESUMEN
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 CoQ10, 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 COQ6 function in podocytes, we generated a podocyte-specific Coq6 knockout mouse (Coq6podKO ) model and a transient siRNA-based COQ6 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 COQ6 knockdown podocytes and control podocytes. We also randomly assigned 5-month-old Coq6podKO 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 Coq6 in mouse podocytes caused FSGS and proteinuria (>46-fold increases in albuminuria). In vitro studies revealed an impaired podocyte migration rate in COQ6 knockdown human podocytes. Treating Coq6podKO mice or cells with 2,4-diHB prevented renal dysfunction and reversed podocyte migration rate impairment. Survival of Coq6podKO mice given 2,4diHB was comparable to that of control mice and significantly higher than that of untreated Coq6podKO 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 CoQ10 biosynthesis pathway.
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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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Citoesqueleto de Actina/fisiología , Células Endoteliales/fisiología , Ensayos Analíticos de Alto Rendimiento/métodos , Amidas , Angiopoyetina 1 , Antígenos CD/metabolismo , Cadherinas/metabolismo , Endotelio Vascular/fisiología , Humanos , Uniones Intercelulares/fisiología , Microscopía Fluorescente , Permeabilidad , Cultivo Primario de Células , PiridinasRESUMEN
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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Receptor TIE-2/fisiología , Sepsis/fisiopatología , Adulto , Anciano , Animales , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Estudios Prospectivos , Receptor TIE-2/biosíntesisRESUMEN
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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Angiopoyetina 2/biosíntesis , Calcio/metabolismo , Endotoxemia/tratamiento farmacológico , Flunarizina/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Endotoxemia/metabolismo , Endotoxemia/patología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , RatonesRESUMEN
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 Klf2 and/or Klf4 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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Coagulación Sanguínea/genética , Permeabilidad Capilar/genética , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , Animales , Trastornos de la Coagulación Sanguínea/genética , Insuficiencia Cardíaca/genética , Factor 4 Similar a Kruppel , Ratones , Ratones Noqueados , Infarto del Miocardio/genética , Accidente Cerebrovascular/genéticaRESUMEN
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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Aminoácidos de Cadena Ramificada/metabolismo , Hidroxibutiratos/metabolismo , Insulina/metabolismo , Obesidad/metabolismo , Animales , Ácidos Grasos/genética , Ácidos Grasos/metabolismo , Humanos , Insulina/genética , Resistencia a la Insulina/genética , Ratones , Ratones Endogámicos NOD , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Obesidad/genética , Obesidad/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
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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Permeabilidad Capilar , Infecciones/fisiopatología , Receptor TIE-2/genética , Animales , Endotelio Vascular/fisiopatología , RatonesRESUMEN
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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Angiopoyetina 2/antagonistas & inhibidores , Angiopoyetina 2/fisiología , Factores de Transcripción Forkhead/fisiología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Sepsis/tratamiento farmacológico , Simvastatina/uso terapéutico , Anciano , Animales , Estudios de Casos y Controles , Reposicionamiento de Medicamentos , Femenino , Proteína Forkhead Box O1 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Angiopoyetina 1/genética , Angiopoyetina 1/metabolismo , Endotelio Vascular/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Estrés Oxidativo , Animales , Células Cultivadas , Endotelio Vascular/citología , Humanos , Uniones Intercelulares , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/antagonistas & inhibidores , Ratones , NADPH Oxidasa 2 , NADPH Oxidasas/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacos , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Trombina/farmacologíaRESUMEN
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.
Asunto(s)
Endotelio Vascular/lesiones , Lactonas/efectos adversos , Piridinas/efectos adversos , Receptor PAR-1/antagonistas & inhibidores , Trombosis/tratamiento farmacológico , Trombosis/prevención & control , Animales , Apoptosis , Sitios de Unión , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , Endotelio Vascular/efectos de los fármacos , Exocitosis , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación , Ligandos , Inhibidores de Agregación Plaquetaria/química , Proteína C/química , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
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-1ß, 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.