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IFNγ induces an endothelial pro-immunogenic phenotype through the JAK/STAT1 pathway, which can shape the activation of alloreactive leukocytes in transplant rejection. In immune cells, the DNA binding protein BCL6 controls transcription of inflammatory genes. This study tested if BCL6 modulates IFNγ-induced gene expression in endothelial cells (EC). In vitro, BCL6 was IFNγ-inducible in primary human endothelium, along with CXCR3 chemokines and HLA. BCL6 was also increased in human cardiac transplants during acute rejection, as were HLA II and CXCL9. Knockdown of BCL6 augmented, while overexpression and BTB domain inhibitors (BCL6-BTBi) suppressed, HLA II and CXCR3 chemokine expression but not HLA I. Further, BCL6 had a greater effect on HLA-DR and DP, but was less involved in regulating HLA-DQ expression. The effect correlated with BCL6 binding motifs in or near affected genes. The BCL6 DNA recognition sequence was highly similar to that of STAT1, and BTBi reduced STAT1's transcriptional activity in vitro. Our results show for the first time that BCL6 selectively controls IFNγ-induced endothelial gene expression, advancing our understanding of the endogenous mechanisms regulating donor immunogenicity.
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We previously reported that adding a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to a Western diet (WD) ameliorated systemic inflammation. To determine the mechanism(s) responsible for these observations, Ldlr-/- mice were fed chow, a WD, or WD plus Tg6F. We found that a WD altered the taxonomic composition of bacteria in jejunum mucus. For example, Akkermansia muciniphila virtually disappeared, while overall bacteria numbers and lipopolysaccharide (LPS) levels increased. In addition, gut permeability increased, as did the content of reactive oxygen species and oxidized phospholipids in jejunum mucus in WD-fed mice. Moreover, gene expression in the jejunum decreased for multiple peptides and proteins that are secreted into the mucous layer of the jejunum that act to limit bacteria numbers and their interaction with enterocytes including regenerating islet-derived proteins, defensins, mucin 2, surfactant A, and apoA-I. Following WD, gene expression also decreased for Il36γ, Il23, and Il22, cytokines critical for antimicrobial activity. WD decreased expression of both Atoh1 and Gfi1, genes required for the formation of goblet and Paneth cells, and immunohistochemistry revealed decreased numbers of goblet and Paneth cells. Adding Tg6F ameliorated these WD-mediated changes. Adding oxidized phospholipids ex vivo to the jejunum from mice fed a chow diet reproduced the changes in gene expression in vivo that occurred when the mice were fed WD and were prevented with addition of 6F peptide. We conclude that Tg6F ameliorates the WD-mediated increase in oxidized phospholipids that cause changes in jejunum mucus, which induce dysbiosis and systemic inflammation.
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DisbiosisRESUMEN
Since constant perfusion of blood throughout the brain is critical for neuronal health, the regulation of cerebral blood flow is complex and highly controlled. This regulation is controlled, in part, by the cerebral endothelium. In this review, multiple modes of endothelium-derived blood flow regulation is discussed, including chemical control of vascular tone, heterotypic and homotypic cell-cell interactions, second messenger signaling, and cellular response to physical forces and inflammatory mediators. Because cerebral small vessel disease is often associated with endothelial dysfunction and a compromised blood-brain barrier, understanding the endothelial factors that regulate vessel function to maintain cerebral blood flow and prevent vascular permeability may provide insights into disease prevention and treatment.
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Circulación Cerebrovascular/fisiología , Endotelio Vascular/fisiología , Animales , Barrera Hematoencefálica/fisiopatología , Enfermedades de los Pequeños Vasos Cerebrales/fisiopatología , HumanosRESUMEN
PURPOSE OF REVIEW: The formation of a hierarchical vascular network is a complex process that requires precise temporal and spatial integration of several signaling pathways. Amongst those, Notch has emerged as a key regulator of multiple steps that expand from endothelial sprouting to arterial specification and remains relevant in the adult. This review aims to summarize major concepts and rising hypotheses on the role of Notch signaling in the endothelium. RECENT FINDINGS: A wealth of new information has helped to clarify how Notch signaling cooperates with other pathways to orchestrate vascular morphogenesis, branching, and function. Endothelial vascular endothelial growth factor, C-X-C chemokine receptor type 4, and nicotinamide adenine dinucleotide phosphate oxidase 2 have been highlighted as key regulators of the pathway. Furthermore, blood flow forces during vascular development induce Notch1 signaling to suppress endothelial cell proliferation, enhance barrier function, and promote arterial specification. Importantly, Notch1 has been recently recognized as an endothelial mechanosensor that is highly responsive to the level of shear stress to enable differential Notch activation in distinct regions of the vessel wall and suppress inflammation. SUMMARY: Although it is well accepted that the Notch signaling pathway is essential for vascular morphogenesis, its contributions to the homeostasis of adult endothelium were uncovered only recently. Furthermore, its exquisite regulation by flow and impressive interface with multiple signaling pathways indicates that Notch is at the center of a highly interactive web that integrates both physical and chemical signals to ensure vascular stability.
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Células Endoteliales/metabolismo , Mecanotransducción Celular/fisiología , Neovascularización Fisiológica/fisiología , Receptor Notch1/metabolismo , Animales , Velocidad del Flujo Sanguíneo/fisiología , Proliferación Celular/fisiología , Células Endoteliales/citología , Humanos , NADPH Oxidasa 2/metabolismo , Receptores CXCR4/metabolismo , Resistencia al Corte , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: Perivascular cells, including pericytes, macrophages, smooth muscle cells, and other specialized cell types, like podocytes, participate in various aspects of vascular function. However, aside from the well-established roles of smooth muscle cells and pericytes, the contributions of other vascular-associated cells are poorly understood. Our goal was to ascertain the function of perivascular macrophages in adult tissues under nonpathological conditions. APPROACH AND RESULTS: We combined confocal microscopy, in vivo cell depletion, and in vitro assays to investigate the contribution of perivascular macrophages to vascular function. We found that resident perivascular macrophages are associated with capillaries at a frequency similar to that of pericytes. Macrophage depletion using either clodronate liposomes or antibodies unexpectedly resulted in hyperpermeability. This effect could be rescued when M2-like macrophages, but not M1-like macrophages or dendritic cells, were reconstituted in vivo, suggesting subtype-specific roles for macrophages in the regulation of vascular permeability. Furthermore, we found that permeability-promoting agents elicit motility and eventual dissociation of macrophages from the vasculature. Finally, in vitro assays showed that M2-like macrophages attenuate the phosphorylation of VE-cadherin upon exposure to permeability-promoting agents. CONCLUSIONS: This study points to a direct contribution of macrophages to vessel barrier integrity and provides evidence that heterotypic cell interactions with the endothelium, in addition to those of pericytes, control vascular permeability.
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Capilares/metabolismo , Permeabilidad Capilar , Comunicación Celular , Células Endoteliales/metabolismo , Macrófagos Peritoneales/metabolismo , Mesenterio/irrigación sanguínea , Peritoneo/irrigación sanguínea , Piel/irrigación sanguínea , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Movimiento Celular , Células Cultivadas , Técnicas de Cocultivo , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Ovalbúmina/metabolismo , Fenotipo , Fosforilación , Rodaminas/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
The luminal surface of the endothelium is exposed to dynamic blood flow patterns that are known to affect endothelial cell phenotype. While many studies have documented the phenotypic changes by gene or protein expression, less is known about the role of blood flow pattern on the endothelial cell (EC) lipidome. In this study, shotgun lipidomics was conducted on human aortic ECs (HAECs) exposed to unidirectional laminar flow (UF), disturbed flow (DF), or static conditions for 48 hrs. A total of 520 individual lipid species from 17 lipid subclasses were detected. Total lipid abundance was significantly increased for HAECs exposed to DF compared to UF conditions. Despite the increase in the total lipid abundance, HAECs maintained equivalent composition of each lipid subclass (% of total lipid) under both DF and UF. However, by lipid composition (% of total subclass), 28 lipid species were significantly altered between DF and UF. Complimentary RNA sequencing of HAECs exposed to UF or DF revealed changes in transcripts involved in lipid metabolism. Shotgun lipidomics was also performed on HAECs exposed to pro-inflammatory agonists lipopolysaccharide (LPS) or Pam3CSK4 (Pam3) for 48 hrs. Exposure to LPS or Pam3 reshaped the EC lipidome in both unique and overlapping ways. In conclusion, exposure to flow alters the EC lipidome and ECs undergo stimulus-specific lipid reprogramming in response to pro-inflammatory agonist exposure. Ultimately, this work provides a resource to profile the transcriptional and lipidomic changes that occur in response to applied flow that can be accessed by the vascular biology community to further dissect and extend our understanding of endothelial lipid biology.
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The luminal surface of the endothelium is exposed to dynamic blood flow patterns that are known to affect endothelial cell phenotype. While many studies have documented the phenotypic changes by gene or protein expression, less is known about the role of blood flow pattern on the endothelial cell (EC) lipidome. In this study, shotgun lipidomics was conducted on human aortic ECs (HAECs) exposed to unidirectional laminar flow (UF), disturbed flow (DF), or static conditions for 48 h. A total of 520 individual lipid species from 17 lipid subclasses were detected. Total lipid abundance was significantly increased for HAECs exposed to DF compared to UF conditions. Despite the increase in the total lipid abundance, HAECs maintained equivalent composition of each lipid subclass (% of total lipid) under DF and UF. However, by lipid composition (% of total subclass), 28 lipid species were significantly altered between DF and UF. Complimentary RNA sequencing of HAECs exposed to UF or DF revealed changes in transcripts involved in lipid metabolism. Shotgun lipidomics was also performed on HAECs exposed to pro-inflammatory agonists lipopolysaccharide (LPS) or Pam3CSK4 (Pam3) for 48 h. Exposure to LPS or Pam3 reshaped the EC lipidome in both unique and overlapping ways. In conclusion, exposure to flow alters the EC lipidome and ECs undergo stimulus-specific lipid reprogramming in response to pro-inflammatory agonist exposure. Ultimately, this work provides a resource to profile the transcriptional and lipidomic changes that occur in response to applied flow that can be accessed by the vascular biology community to further dissect and extend our understanding of endothelial lipid biology.
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Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, and this supports an antiinflammatory phenotype. High laminar shear stress also induces flow-aligned cell elongation and front-rear polarity, but whether these are required for the antiinflammatory phenotype is unclear. Here, we showed that caveolin-1-rich microdomains polarize to the downstream end of ECs that are exposed to continuous high laminar flow. These microdomains were characterized by high membrane rigidity, filamentous actin (F-actin), and raft-associated lipids. Transient receptor potential vanilloid (TRPV4) ion channels were ubiquitously expressed on the plasma membrane but mediated localized Ca2+ entry only at these microdomains where they physically interacted with clustered caveolin-1. These focal Ca2+ bursts activated endothelial nitric oxide synthase within the confines of these domains. Importantly, we found that signaling at these domains required both cell body elongation and sustained flow. Finally, TRPV4 signaling at these domains was necessary and sufficient to suppress inflammatory gene expression and exogenous activation of TRPV4 channels ameliorated the inflammatory response to stimuli both in vitro and in vivo. Our work revealed a polarized mechanosensitive signaling hub in arterial ECs that dampened inflammatory gene expression and promoted cell resilience.
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Calcio , Células Endoteliales , Inflamación , Mecanotransducción Celular , Canales Catiónicos TRPV , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética , Animales , Inflamación/metabolismo , Inflamación/patología , Inflamación/genética , Células Endoteliales/metabolismo , Células Endoteliales/patología , Calcio/metabolismo , Ratones , Humanos , Microdominios de Membrana/metabolismo , Caveolina 1/metabolismo , Caveolina 1/genética , Señalización del Calcio , Estrés Mecánico , Aorta Torácica/metabolismo , Aorta Torácica/patologíaRESUMEN
The physical properties of cellular membranes, including fluidity and function, are influenced by protein and lipid interactions. In situ labeling chemistries, most notably proximity-labeling interactomics are well suited to characterize these dynamic and often fleeting interactions. Established methods require distinct chemistries for proteins and lipids, which limits the scope of such studies. Here we establish a singlet-oxygen-based photocatalytic proximity labeling platform (POCA) that reports intracellular interactomes for both proteins and lipids with tight spatiotemporal resolution using cell-penetrant photosensitizer reagents. Using both physiologically relevant lipoprotein-complexed probe delivery and genetic manipulation of cellular cholesterol handling machinery, cholesterol-directed POCA captured established and unprecedented cholesterol binding proteins, including protein complexes sensitive to intracellular cholesterol levels and proteins uniquely captured by lipoprotein uptake. Protein-directed POCA accurately mapped known intracellular membrane complexes, defined sterol-dependent changes to the non-vesicular cholesterol transport protein interactome, and captured state-dependent changes in the interactome of the cholesterol transport protein Aster-B. More broadly, we find that POCA is a versatile interactomics platform that is straightforward to implement, using the readily available HaloTag system, and fulfills unmet needs in intracellular singlet oxygen-based proximity labeling proteomics. Thus, we expect widespread utility for POCA across a range of interactome applications, spanning imaging to proteomics.
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The intrinsic pathways that control membrane organization in immune cells and the impact of such pathways on cellular function are not well defined. Here we report that the non-vesicular cholesterol transporter Aster-A links plasma membrane (PM) cholesterol availability in T cells to immune signaling and systemic metabolism. Aster-A is recruited to the PM during T-cell receptor (TCR) activation, where it facilitates the removal of newly generated "accessible" membrane cholesterol. Loss of Aster-A leads to excess PM cholesterol accumulation, resulting in enhanced TCR nano-clustering and signaling, and Th17 cytokine production. Finally, we show that the mucosal Th17 response is restrained by PM cholesterol remodeling. Ablation of Aster-A in T cells leads to enhanced IL-22 production, reduced intestinal fatty acid absorption, and resistance to diet-induced obesity. These findings delineate a multi-tiered regulatory scheme linking immune cell lipid flux to nutrient absorption and systemic physiology.
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Exercise promotes pulsatile shear stress in the arterial circulation and ameliorates cardiometabolic diseases. However, exercise-mediated metabolic transducers for vascular protection remain under-investigated. Untargeted metabolomic analysis demonstrated that wild-type mice undergoing voluntary wheel running exercise expressed increased endothelial stearoyl-CoA desaturase 1 (SCD1) that catalyzes anti-inflammatory lipid metabolites, namely, oleic (OA) and palmitoleic acids (PA), to mitigate NF-κB-mediated inflammatory responses. In silico analysis revealed that exercise augmented time-averaged wall shear stress but mitigated flow recirculation and oscillatory shear index in the lesser curvature of the mouse aortic arch. Following exercise, endothelial Scd1-deleted mice (Ldlr-/- Scd1EC-/-) on high-fat diet developed persistent VCAM1-positive endothelium in the lesser curvature and the descending aorta, whereas SCD1 overexpression via adenovirus transfection mitigated endoplasmic reticulum stress and inflammatory biomarkers. Single-cell transcriptomics of the aorta identified Scd1-positive and Vcam1-negative endothelial subclusters interacting with other candidate genes. Thus, exercise mitigates flow recirculation and activates endothelial SCD1 to catalyze OA and PA for vascular endothelial protection.
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Aorta , Actividad Motora , Animales , Ratones , Aorta/metabolismo , Dieta Alta en Grasa , Endotelio Vascular/metabolismo , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismoRESUMEN
A novel magnetically actuated scaffold was used to explore the effects of strain stimulus on the proliferation and spatial distribution of smooth muscle cells and improve cell viability in the scaffold interior by pumping nutrients throughout the structure. Magnetically actuable scaffolds were fabricated in a tube shape by winding electrospun sheets of a biodegradable polymer modified with magnetic Fe(2)O(3) nanoparticles. Prior to rolling, the sheets were seeded with smooth muscle cells and wound into tubes with diameter 5.2 mm and wall thickness 0.2 mm. The tubular scaffolds were actuated by a magnetic field to induce a cyclic crimping deformation, which applies strain stimulus to the cells and pumps nutrient fluid through the porous tube walls. Comparison with non-actuated controls shows that magnetic actuation increases the total cell count throughout the scaffold after 14 days of incubation. Furthermore, whereas cell density as a function of position through the tube wall thickness showed a minimum in the mid-interior in the controls after 14 days due to cell starvation, the actuated scaffolds displayed a maximum cell density. Comparison of cell distributions with the expected spatial variations in strain amplitude and nutrient flux implies that both strain stimulus and nutrient pumping are significant factors in cell proliferation.
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Proliferación Celular , Esguinces y Distensiones , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Técnicas de Cultivo de Célula/métodos , Supervivencia Celular , Magnetismo , Células Musculares/fisiología , Ratas , Estrés MecánicoRESUMEN
Transplant vasculopathy (TV) causes thickening of donor blood vessels in transplanted organs, and is a significant cause of graft loss and mortality in allograft recipients. It is known that patients with repeated acute rejection and/or donor specific antibodies are predisposed to TV. Nevertheless, the exact molecular mechanisms by which alloimmune injury culminates in this disease have not been fully delineated. As a result of this incomplete knowledge, there is currently a lack of effective therapies for this disease. The immediate intracellular signaling and the acute effects elicited by anti-donor HLA antibodies are well-described and continuing to be revealed in deeper detail. Further, advances in rejection diagnostics, including intragraft gene expression, provide clues to the inflammatory changes within allografts. However, mechanisms linking these events with long-term outcomes, particularly the maladaptive vascular remodeling seen in transplant vasculopathy, are still being delineated. New evidence demonstrates alterations in non-coding RNA profiles and the occurrence of endothelial to mesenchymal transition (EndMT) during acute antibody-mediated graft injury. EndMT is also readily apparent in numerous settings of non-transplant intimal hyperplasia, and lessons can be learned from advances in those fields. This review will provide an update on these recent developments and remaining questions in our understanding of HLA antibody-induced vascular damage, framed within a broader consideration of manifestations and implications across transplanted organ types.
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Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, which supports an anti-inflammatory phenotype that protects them from atherosclerosis. High laminar shear stress also supports flow-aligned cell elongation and front-rear polarity, but whether this is required for athero-protective signaling is unclear. Here, we show that Caveolin-1-rich microdomains become polarized at the downstream end of ECs exposed to continuous high laminar flow. These microdomains are characterized by higher membrane rigidity, filamentous actin (F-actin) and lipid accumulation. Transient receptor potential vanilloid-type 4 (Trpv4) ion channels, while ubiquitously expressed, mediate localized Ca 2+ entry at these microdomains where they physically interact with clustered Caveolin-1. The resultant focal bursts in Ca 2+ activate the anti-inflammatory factor endothelial nitric oxide synthase (eNOS) within the confines of these domains. Importantly, we find that signaling at these domains requires both cell body elongation and sustained flow. Finally, Trpv4 signaling at these domains is necessary and sufficient to suppress inflammatory gene expression. Our work reveals a novel polarized mechanosensitive signaling hub that induces an anti-inflammatory response in arterial ECs exposed to high laminar shear stress.
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Exercise modulates vascular plasticity in multiple organ systems; however, the metabolomic transducers underlying exercise and vascular protection in the disturbed flow-prone vasculature remain under-investigated. We simulated exercise-augmented pulsatile shear stress (PSS) to mitigate flow recirculation in the lesser curvature of the aortic arch. When human aortic endothelial cells (HAECs) were subjected to PSS ( τ ave = 50 dyne·cm -2 , ∂τ/∂t = 71 dyne·cm -2 ·s -1 , 1 Hz), untargeted metabolomic analysis revealed that Stearoyl-CoA Desaturase (SCD1) in the endoplasmic reticulum (ER) catalyzed the fatty acid metabolite, oleic acid (OA), to mitigate inflammatory mediators. Following 24 hours of exercise, wild-type C57BL/6J mice developed elevated SCD1-catalyzed lipid metabolites in the plasma, including OA and palmitoleic acid (PA). Exercise over a 2-week period increased endothelial SCD1 in the ER. Exercise further modulated the time-averaged wall shear stress (TAWSS or τ ave) and oscillatory shear index (OSI ave ), upregulated Scd1 and attenuated VCAM1 expression in the disturbed flow-prone aortic arch in Ldlr -/- mice on high-fat diet but not in Ldlr -/- Scd1 EC-/- mice. Scd1 overexpression via recombinant adenovirus also mitigated ER stress. Single cell transcriptomic analysis of the mouse aorta revealed interconnection of Scd1 with mechanosensitive genes, namely Irs2 , Acox1 and Adipor2 that modulate lipid metabolism pathways. Taken together, exercise modulates PSS ( τ ave and OSI ave ) to activate SCD1 as a metabolomic transducer to ameliorate inflammation in the disturbed flow-prone vasculature.
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Intestinal cholesterol absorption is an important contributor to systemic cholesterol homeostasis. Niemann-Pick C1 Like 1 (NPC1L1), the target of the drug ezetimibe (EZ), assists in the initial step of dietary cholesterol uptake. However, how cholesterol moves downstream of NPC1L1 is unknown. Here we show that Aster-B and Aster-C are critical for non-vesicular cholesterol movement in enterocytes, bridging NPC1L1 at the plasma membrane (PM) and ACAT2 in the endoplasmic reticulum (ER). Loss of NPC1L1 diminishes accessible PM cholesterol in enterocytes and abolishes Aster recruitment to the intestinal brush border. Enterocytes lacking Asters accumulate cholesterol at the PM and display evidence of ER cholesterol depletion, including decreased cholesterol ester stores and activation of the SREBP-2 transcriptional pathway. Aster-deficient mice have impaired cholesterol absorption and are protected against diet-induced hypercholesterolemia. Finally, we show that the Aster pathway can be targeted with a small molecule inhibitor to manipulate dietary cholesterol uptake. These findings identify the Aster pathway as a physiologically important and pharmacologically tractable node in dietary lipid absorption. One-Sentence Summary: Identification of a targetable pathway for regulation of dietary cholesterol absorption.
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Intestinal absorption is an important contributor to systemic cholesterol homeostasis. Niemann-Pick C1 Like 1 (NPC1L1) assists in the initial step of dietary cholesterol uptake, but how cholesterol moves downstream of NPC1L1 is unknown. We show that Aster-B and Aster-C are critical for nonvesicular cholesterol movement in enterocytes. Loss of NPC1L1 diminishes accessible plasma membrane (PM) cholesterol and abolishes Aster recruitment to the intestinal brush border. Enterocytes lacking Asters accumulate PM cholesterol and show endoplasmic reticulum cholesterol depletion. Aster-deficient mice have impaired cholesterol absorption and are protected against diet-induced hypercholesterolemia. Finally, the Aster pathway can be targeted with a small-molecule inhibitor to manipulate cholesterol uptake. These findings identify the Aster pathway as a physiologically important and pharmacologically tractable node in dietary lipid absorption.
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Colesterol en la Dieta , Enterocitos , Absorción Intestinal , Proteínas de Transporte de Membrana , Animales , Ratones , Transporte Biológico , Colesterol en la Dieta/metabolismo , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/fisiología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones Endogámicos C57BL , Enterocitos/metabolismo , Receptores X del Hígado/metabolismo , Humanos , Yeyuno/metabolismo , Ratones NoqueadosRESUMEN
Mechano-responsive signaling pathways enable blood vessels within a connected network to structurally adapt to partition of blood flow between organ systems. Wall shear stress (WSS) modulates endothelial cell proliferation and arteriovenous specification. Here, we study vascular regeneration in a zebrafish model by using tail amputation to disrupt the embryonic circulatory loop (ECL) at 3 days post fertilization (dpf). We observed a local increase in blood flow and peak WSS in the Segmental Artery (SeA) immediately adjacent to the amputation site. By manipulating blood flow and WSS via changes in blood viscosity and myocardial contractility, we show that the angiogenic Notch-ephrinb2 cascade is hemodynamically activated in the SeA to guide arteriogenesis and network reconnection. Taken together, ECL amputation induces changes in microvascular topology to partition blood flow and increase WSS-mediated Notch-ephrinb2 pathway, promoting new vascular arterial loop formation and restoring microcirculation.
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BACKGROUND AND AIMS: Phagocytosis (efferocytosis) of apoptotic neutrophils by macrophages anchors the resolution of intestinal inflammation. Efferocytosis prevents secondary necrosis and inhibits further inflammation, and also reprograms macrophages to facilitate tissue repair and promote resolution function. Macrophage efferocytosis and efferocytosis-dependent reprogramming are implicated in the pathogenesis of inflammatory bowel disease. We previously reported that absence of macrophage cyclooxygenase 2 (COX2) exacerbates inflammatory bowel disease-like intestinal inflammation. To elucidate the underlying pathogenic mechanism, we investigated here whether COX2 mediates macrophage efferocytosis and efferocytosis-dependent reprogramming, including intestinal epithelial repair capacity. METHODS: Using apoptotic neutrophils and synthetic apoptotic targets, we determined the effects of macrophage specific Cox2 knockout and pharmacological COX2 inhibition on the efferocytosis capacity of mouse primary macrophages. COX2-mediated efferocytosis-dependent eicosanoid lipidomics was determined by liquid chromatography tandem mass spectrometry. Small intestinal epithelial organoids were employed to assay the effects of COX2 on efferocytosis-dependent intestinal epithelial repair. RESULTS: Loss of COX2 impaired efferocytosis in mouse primary macrophages, in part, by affecting the binding capacity of macrophages for apoptotic cells. This effect was comparable to that of high-dose lipopolysaccharide and was accompanied by both dysregulation of macrophage polarization and the inhibited expression of genes involved in apoptotic cell binding. COX2 modulated the production of efferocytosis-dependent lipid inflammatory mediators that include the eicosanoids prostaglandin I2, prostaglandin E2, lipoxin A4, and 15d-PGJ2; and further affected secondary efferocytosis. Finally, macrophage efferocytosis induced, in a macrophage COX2-dependent manner, a tissue restitution and repair phenotype in intestinal epithelial organoids. CONCLUSIONS: Macrophage COX2 potentiates efferocytosis capacity and efferocytosis-dependent reprogramming, facilitating macrophage intestinal epithelial repair capacity.
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Ciclooxigenasa 2/metabolismo , Enfermedades Inflamatorias del Intestino , Fagocitosis , Animales , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/farmacología , Inflamación/patología , Enfermedades Inflamatorias del Intestino/patología , Macrófagos/metabolismo , Ratones , Fagocitosis/genéticaRESUMEN
OBJECTIVE: In biomanufacturing there is a need for quantitative methods to map cell viability and density inside 3D bioreactors to assess health and proliferation over time. Recently, noninvasive MRI readouts of cell density have been achieved. However, the ratio of live to dead cells was not varied. Herein we present an approach for measuring the viability of cells embedded in a hydrogel independently from cell density to map cell number and health. METHODS: Independent quantification of cell viability and density was achieved by calibrating the 1H magnetization transfer- (MT) and diffusion-weighted NMR signals to samples of known cell density and viability using a multivariate approach. Maps of cell viability and density were generated by weighting NMR images by these parameters post-calibration. RESULTS: Using this method, the limits of detection (LODs) of total cell density and viable cell density were found to be 3.88 ×108 cells · mL -1· Hz -1/2 and 2.36 ×109 viable cells · mL -1· Hz -1/2 respectively. CONCLUSION: This mapping technique provides a noninvasive means of visualizing cell viability and number density within optically opaque bioreactors. SIGNIFICANCE: We anticipate that such nondestructive readouts will provide valuable feedback for monitoring and controlling cell populations in bioreactors.