RESUMO
Ab-mediated rejection of organ transplants remains a stubborn, frequent problem affecting patient quality of life, graft function, and grant survival, and for which few efficacious therapies currently exist. Although the field has gained considerable knowledge over the last two decades on how anti-HLA Abs cause acute tissue injury and promote inflammation, there has been a gap in linking these effects with the chronic inflammation, vascular remodeling, and persistent alloimmunity that leads to deterioration of graft function over the long term. This review will discuss new data emerging over the last 5 y that provide clues into how ongoing Ab-endothelial cell interactions may shape vascular fate and propagate alloimmunity in organ transplants.
Assuntos
Células Endoteliais , Qualidade de Vida , Humanos , Rejeição de Enxerto , Anticorpos , Inflamação , Antígenos HLARESUMO
NF-κB-mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through adhesion molecules Icam1 and Vcam1. The endothelium is primed for cytokine activation of NF-κB by exposure to low and disturbed blood flow (LDF)but the molecular underpinnings are not fully understood. In an experimental in vivo model of LDF, platelets were required for the increased expression of several RNA-binding splice factors, including polypyrimidine tract binding protein (Ptbp1). This was coordinated with changes in RNA splicing in the NF-κB pathway in primed cells, leading us to examine splice factors as mediators of priming. Using Icam1 and Vcam1 induction by tumor necrosis factor (TNF)-α stimulation as a readout, we performed a CRISPR Cas9 knockout screen and identified a requirement for Ptbp1 in priming. Deletion of Ptbp1 had no effect on cell growth or response to apoptotic stimuli, but reversed LDF splicing patterns and inhibited NF-κB nuclear translocation and transcriptional activation of downstream targets, including Icam1 and Vcam1. In human coronary arteries, elevated PTBP1 correlates with expression of TNF pathway genes and plaque. In vivo, endothelial-specific deletion of Ptbp1 reduced Icam1 expression and myeloid cell infiltration at regions of LDF in atherosclerotic mice, limiting atherosclerosis. This may be mediated, in part, by allowing inclusion of a conserved alternative exon in Ripk1 leading to a reduction in Ripk1 protein. Our data show that Ptbp1, which is induced in a subset of the endothelium by platelet recruitment at regions of LDF, is required for priming of the endothelium for subsequent NF-κB activation, myeloid cell recruitment and atherosclerosis.
Assuntos
Aterosclerose , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Processamento Alternativo , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Endotélio/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Inflamação/genética , Inflamação/metabolismo , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismoRESUMO
BACKGROUND: Whereas several interventions can effectively lower lipid levels in people at risk for atherosclerotic cardiovascular disease (ASCVD), cardiovascular event risks remain, suggesting an unmet medical need to identify factors contributing to cardiovascular event risk. Monocytes and macrophages play central roles in atherosclerosis, but studies have yet to provide a detailed view of macrophage populations involved in increased ASCVD risk. METHODS: A novel macrophage foaming analytics tool, AtheroSpectrum, was developed using 2 quantitative indices depicting lipid metabolism and the inflammatory status of macrophages. A machine learning algorithm was developed to analyze gene expression patterns in the peripheral monocyte transcriptome of MESA participants (Multi-Ethnic Study of Atherosclerosis; set 1; n=911). A list of 30 genes was generated and integrated with traditional risk factors to create an ASCVD risk prediction model (30-gene cardiovascular disease risk score [CR-30]), which was subsequently validated in the remaining MESA participants (set 2; n=228); performance of CR-30 was also tested in 2 independent human atherosclerotic tissue transcriptome data sets (GTEx [Genotype-Tissue Expression] and GSE43292). RESULTS: Using single-cell transcriptomic profiles (GSE97310, GSE116240, GSE97941, and FR-FCM-Z23S), AtheroSpectrum detected 2 distinct programs in plaque macrophages-homeostatic foaming and inflammatory pathogenic foaming-the latter of which was positively associated with severity of atherosclerosis in multiple studies. A pool of 2209 pathogenic foaming genes was extracted and screened to select a subset of 30 genes correlated with cardiovascular event in MESA set 1. A cardiovascular disease risk score model (CR-30) was then developed by incorporating this gene set with traditional variables sensitive to cardiovascular event in MESA set 1 after cross-validation generalizability analysis. The performance of CR-30 was then tested in MESA set 2 (P=2.60×10-4; area under the receiver operating characteristic curve, 0.742) and 2 independent data sets (GTEx: P=7.32×10-17; area under the receiver operating characteristic curve, 0.664; GSE43292: P=7.04×10-2; area under the receiver operating characteristic curve, 0.633). Model sensitivity tests confirmed the contribution of the 30-gene panel to the prediction model (likelihood ratio test; df=31, P=0.03). CONCLUSIONS: Our novel computational program (AtheroSpectrum) identified a specific gene expression profile associated with inflammatory macrophage foam cells. A subset of 30 genes expressed in circulating monocytes jointly contributed to prediction of symptomatic atherosclerotic vascular disease. Incorporating a pathogenic foaming gene set with known risk factors can significantly strengthen the power to predict ASCVD risk. Our programs may facilitate both mechanistic investigations and development of therapeutic and prognostic strategies for ASCVD risk.
Assuntos
Aterosclerose/terapia , Doenças Cardiovasculares/terapia , Células Espumosas/citologia , Macrófagos/citologia , Idoso , Idoso de 80 Anos ou mais , Aterosclerose/etiologia , Aterosclerose/genética , Doenças Cardiovasculares/complicações , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/terapia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Placa Aterosclerótica/complicações , Placa Aterosclerótica/genética , Placa Aterosclerótica/terapia , Curva ROC , Risco , Calcificação Vascular/complicações , Calcificação Vascular/genética , Calcificação Vascular/terapiaRESUMO
Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.
Assuntos
Encéfalo/metabolismo , Núcleo Celular/metabolismo , Citometria de Fluxo/métodos , Células Endoteliais da Veia Umbilical Humana/metabolismo , RNA/metabolismo , Análise de Célula Única/métodos , Animais , Encéfalo/citologia , Fracionamento Celular/métodos , Células Cultivadas , Criopreservação/métodos , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , RNA/isolamento & purificação , Reprodutibilidade dos Testes , Análise de Sequência de RNA/métodos , Bancos de Tecidos , Regulador Transcricional ERG/metabolismoRESUMO
OBJECTIVE: Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered composition of the extracellular matrix at the site of lesions, and to contribute to inflammatory processes within the lesions. We have previously shown that alternative splicing of FN (fibronectin) protects against flow-induced hemorrhage. However, the impact of alternative splicing of FN on extracellular matrix composition remains unknown. Approach and Results: Here, we perform quantitative proteomic analysis of the matrisome of murine carotid arteries in mice deficient in the production of FN splice isoforms containing alternative exons EIIIA and EIIIB (FN-EIIIAB null) after exposure to low and disturbed flow in vivo. We also examine serum-derived and endothelial-cell contributions to the matrisome in a simplified in vitro system. We found flow-induced differences in the carotid artery matrisome that were impaired in FN-EIIIAB null mice. One of the most interesting differences was reduced recruitment of FBLN1 (fibulin-1), abundant in blood and not locally produced in the intima. This defect was validated in our in vitro assay, where FBLN1 recruitment from serum was impaired by the absence of these alternatively spliced segments. CONCLUSIONS: Our results reveal the extent of the dynamic alterations in the matrisome in the acute response to low and disturbed flow and show how changes in the splicing of FN, a common response in vascular inflammation and remodeling, can affect matrix composition.
Assuntos
Processamento Alternativo , Artérias Carótidas/metabolismo , Estenose das Carótidas/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Remodelação Vascular , Animais , Artérias Carótidas/patologia , Artérias Carótidas/fisiopatologia , Estenose das Carótidas/patologia , Estenose das Carótidas/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Matriz Extracelular/patologia , Fibronectinas/deficiência , Fibronectinas/genética , Mecanotransdução Celular , Camundongos Knockout , Isoformas de Proteínas , Fluxo Sanguíneo Regional , Estresse MecânicoRESUMO
Atherosclerosis is a chronic inflammatory pathology that precipitates substantial morbidity and mortality. Although initiated by physiological patterns of low and disturbed flow that differentially prime endothelial cells at sites of vessel branch points and curvature, the chronic, smoldering inflammation of atherosclerosis is accelerated by comorbidities involving inappropriate activation of the adaptive immune system, such as autoimmunity. The innate contributions to atherosclerosis, especially in the transition of monocyte to lipid-laden macrophage, are well established, but the mechanisms underpinning the infiltration, persistence, and effector dynamics of CD8 T cells in particular are not well understood. Adaptive immunity is centered on a classical cascade of antigen recognition and activation, costimulation, and effector cytokine secretion upon recall of antigen. However, chronic inflammation can generate alternative cues that supplant this behavior pattern and promote the retention and activation of peripherally activated T cells. Furthermore, the atherogenic foci that activated immune cell infiltrate are unique lipid-laden environments that offer a diverse array of stimuli, including those of survival, antigen hyporesponsiveness, and inflammatory cytokine expression. This review will focus on how known cardiovascular comorbidities may be influencing CD8 T-cell activation and how, once infiltrated within atherogenic foci, these T cells face a multitude of cues that skew the classical cascade of T-cell behavior, highlighting alternative modes of activation that may help contextualize associations of autoimmunity, viral infection, and immunotherapy with cardiovascular morbidity.
Assuntos
Artérias/imunologia , Aterosclerose/imunologia , Linfócitos T CD8-Positivos/imunologia , Inflamação/imunologia , Ativação Linfocitária , Placa Aterosclerótica , Imunidade Adaptativa , Animais , Artérias/metabolismo , Artérias/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Linfócitos T CD8-Positivos/metabolismo , Microambiente Celular , Humanos , Inflamação/metabolismo , Inflamação/patologia , Transdução de SinaisRESUMO
Effector CD8 T cells infiltrate atherosclerotic lesions and are correlated with cardiovascular events, but the mechanisms regulating their recruitment and retention are not well understood. CD137 (4-1BB) is a costimulatory receptor induced on immune cells and expressed at sites of human atherosclerotic plaque. Genetic variants associated with decreased CD137 expression correlate with carotid-intimal thickness and its deficiency in animal models attenuates atherosclerosis. These effects have been attributed in part to endothelial responses to low and disturbed flow (LDF), but CD137 also generates robust effector CD8 T cells as a costimulatory signal. Thus, we asked whether CD8 T cell-specific CD137 stimulation contributes to their infiltration, retention, and IFNγ production in early atherogenesis. We tested this through adoptive transfer of CD8 T cells into recipient C57BL/6J mice that were then antigen primed and CD137 costimulated. We analyzed atherogenic LDF vessels in normolipidemic and PCSK9-mediated hyperlipidemic models and utilized a digestion protocol that allowed for lesional T-cell characterization via flow cytometry and in vitro stimulation. We found that CD137 activation, specifically of effector CD8 T cells, triggers their intimal infiltration into LDF vessels and promotes a persistent innate-like proinflammatory program. Residence of CD137+ effector CD8 T cells further promoted infiltration of endogenous CD8 T cells with IFNγ-producing potential, whereas CD137-deficient CD8 T cells exhibited impaired vessel infiltration, minimal IFNγ production, and reduced infiltration of endogenous CD8 T cells. Our studies thus provide novel insight into how CD137 costimulation of effector T cells, independent of plaque-antigen recognition, instigates their retention and promotes innate-like responses from immune infiltrates within atherogenic foci. NEW & NOTEWORTHY Our studies identify CD137 costimulation as a stimulus for effector CD8 T-cell infiltration and persistence within atherogenic foci, regardless of atherosclerotic-antigen recognition. These costimulated effector cells, which are generated in pathological states such as viral infection and autoimmunity, have innate-like proinflammatory programs in circulation and within the atherosclerotic microenvironment, providing mechanistic context for clinical correlations of cardiovascular morbidity with increased CD8 T-cell infiltration and markers of activation in the absence of established antigen specificity.
Assuntos
Aorta Abdominal/metabolismo , Aterosclerose/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Artérias Carótidas/metabolismo , Imunidade Inata , Ativação Linfocitária , Placa Aterosclerótica , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Transferência Adotiva , Animais , Aorta Abdominal/imunologia , Aorta Abdominal/patologia , Aterosclerose/imunologia , Aterosclerose/patologia , Linfócitos T CD8-Positivos/imunologia , Artérias Carótidas/imunologia , Artérias Carótidas/patologia , Células Cultivadas , Dieta Hiperlipídica , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Hiperlipidemias/complicações , Interferon gama/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Transdução de Sinais , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/deficiência , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Objective- Alterations in extracellular matrix quantity and composition contribute to atherosclerosis, with remodeling of the subendothelial basement membrane to an FN (fibronectin)-rich matrix preceding lesion development. Endothelial cell interactions with FN prime inflammatory responses to a variety of atherogenic stimuli; however, the mechanisms regulating early atherogenic FN accumulation remain unknown. We previously demonstrated that oxLDL (oxidized low-density lipoprotein) promotes endothelial proinflammatory gene expression by activating the integrin α5ß1, a classic mediator of FN fibrillogenesis. Approach and Results- We now show that oxLDL drives robust endothelial FN deposition and inhibiting α5ß1 (blocking antibodies, α5 knockout cells) completely inhibits oxLDL-induced FN deposition. Consistent with this, inducible endothelial-specific α5 integrin deletion in ApoE knockout mice significantly reduces atherosclerotic plaque formation, associated with reduced early atherogenic inflammation. Unlike TGFß (transforming growth factor ß)-induced FN deposition, oxLDL does not induce FN expression (mRNA, protein) or the endothelial-to-mesenchymal transition phenotype. In addition, we show that cell-derived and plasma-derived FN differentially affect endothelial function, with only cell-derived FN capable of supporting oxLDL-induced VCAM-1 (vascular cell adhesion molecule 1) expression, despite plasma FN deposition by oxLDL. The inclusion of alternative exon EIIIA (EDA) of FN (EIIIA) and alternative exon EIIIB (EDB) of FN (EIIIB) domains in cell-derived FN mediates this effect, as EIIIA/EIIIB knockout endothelial cells show diminished oxLDL-induced inflammation. Furthermore, our data suggest that EIIIA/EIIIB-positive cellular FN is required for maximal α5ß1 recruitment to focal adhesions and FN fibrillogenesis. Conclusions- Taken together, our data demonstrate that endothelial α5 integrins drive oxLDL-induced FN deposition and early atherogenic inflammation. Additionally, we show that α5ß1-dependent endothelial FN deposition mediates oxLDL-dependent endothelial inflammation and FN fibrillogenesis.
Assuntos
Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Doenças das Artérias Carótidas/metabolismo , Células Endoteliais/metabolismo , Fibronectinas/metabolismo , Inflamação/metabolismo , Integrina alfa5beta1/metabolismo , Placa Aterosclerótica , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Doenças da Aorta/genética , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/patologia , Caderinas/genética , Caderinas/metabolismo , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Fibronectinas/deficiência , Fibronectinas/genética , Adesões Focais/metabolismo , Adesões Focais/patologia , Humanos , Inflamação/genética , Inflamação/patologia , Integrina alfa5beta1/deficiência , Integrina alfa5beta1/genética , Lipoproteínas LDL/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Transdução de SinaisRESUMO
Arteriovenous (AV) malformation (AVM) is a devastating condition characterized by focal lesions of enlarged, tangled vessels that shunt blood from arteries directly to veins. AVMs can form anywhere in the body and can cause debilitating ischemia and life-threatening hemorrhagic stroke. The mechanisms that underlie AVM formation remain poorly understood. Here, we examined the cellular and hemodynamic changes at the earliest stages of brain AVM formation by time-lapse two-photon imaging through cranial windows of mice expressing constitutively active Notch4 (Notch4*). AVMs arose from enlargement of preexisting microvessels with capillary diameter and blood flow and no smooth muscle cell coverage. AV shunting began promptly after Notch4* expression in endothelial cells (ECs), accompanied by increased individual EC areas, rather than increased EC number or proliferation. Alterations in Notch signaling in ECs of all vessels, but not arteries alone, affected AVM formation, suggesting that Notch functions in the microvasculature and/or veins to induce AVM. Increased Notch signaling interfered with the normal biological control of hemodynamics, permitting a positive feedback loop of increasing blood flow and vessel diameter and driving focal AVM growth from AV connections with higher blood velocity at the expense of adjacent AV connections with lower velocity. Endothelial expression of constitutively active Notch1 also led to brain AVMs in mice. Our data shed light on cellular and hemodynamic mechanisms underlying AVM pathogenesis elicited by increased Notch signaling in the endothelium.
Assuntos
Capilares/patologia , Malformações Arteriovenosas Intracranianas/metabolismo , Malformações Arteriovenosas Intracranianas/fisiopatologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Notch/metabolismo , Animais , Bromodesoxiuridina , Capilares/metabolismo , Células Endoteliais/metabolismo , Citometria de Fluxo , Malformações Arteriovenosas Intracranianas/etiologia , Camundongos , Receptor Notch4 , Fluxo Sanguíneo Regional/fisiologia , Transdução de Sinais/fisiologia , Microtomografia por Raio-XRESUMO
OBJECTIVE: Abnormally low-flow conditions, sensed by the arterial endothelium, promote aneurysm rupture. Fibronectin (FN) is among the most abundant extracellular matrix proteins and is strongly upregulated in human aneurysms, suggesting a possible role in disease progression. Altered FN splicing can result in the inclusion of EIIIA and EIIIB exons, generally not expressed in adult tissues. We sought to explore the regulation of FN and its splicing and their possible roles in the vascular response to disturbed flow. APPROACH AND RESULTS: We induced low and reversing flow in mice by partial carotid ligation and assayed FN splicing in an endothelium-enriched intimal preparation. Inclusion of EIIIA and EIIIB was increased as early as 48 hours, with negligible increases in total FN expression. To test the function of EIIIA and EIIIB inclusion, we induced disturbed flow in EIIIAB(-/-) mice unable to include these exons and found that they developed focal lesions with hemorrhage and hypertrophy of the vessel wall. Acute deletion of floxed FN caused similar defects in response to disturbed flow, consistent with a requirement for the upregulation of the spliced isoforms, rather than a developmental defect. Recruited macrophages promote FN splicing because their depletion by clodronate liposomes blocked the increase in endothelial EIIIA and EIIIB inclusion in the carotid model. CONCLUSIONS: These results uncover a protective mechanism in the inflamed intima that develops under disturbed flow, by showing that splicing of FN mRNA in the endothelium, induced by macrophages, inhibits hemorrhage of the vessel wall.
Assuntos
Processamento Alternativo , Estenose das Carótidas/patologia , Endotélio Vascular/metabolismo , Fibronectinas/genética , Hemorreologia , Hemorragia/prevenção & controle , Túnica Íntima/lesões , Túnica Adventícia/metabolismo , Animais , Estenose das Carótidas/genética , Modelos Animais de Doenças , Progressão da Doença , Endotélio Vascular/patologia , Éxons , Matriz Extracelular/patologia , Fibronectinas/biossíntese , Fibronectinas/deficiência , Deleção de Genes , Hemorragia/etiologia , Hemorragia/fisiopatologia , Hipertrofia , Ligadura , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Túnica Média/metabolismoRESUMO
Atherosclerotic plaques are defined by the accumulation of lipids and immune cells beneath the endothelium of the arterial intima. CD8 T cells are among the most abundant immune cell types in plaque, and conditions linked to their activation correlate with increased levels of cardiovascular disease. As lethal effectors of the immune response, CD8 T cell activation is suppressed at multiple levels. These checkpoints are critical in dampening autoimmune responses, and limiting damage in cardiovascular disease. Endothelial cells are well known for their role in recruiting CD8 T and other hematopoietic cells to low and disturbed flow (LDF) arterial regions that develop plaque, but whether they locally influence CD8 effector functions is unclear. Here, we show that endothelial cells can actively suppress CD8 T cell responses in settings of chronic plaque inflammation, but that this behavior is governed by expression of the RNA-binding protein Embryonic Lethal, Abnormal Vision-Like 1 (Elavl1). In response to immune cell recruitment in plaque, the endothelium dynamically shifts splicing of pre-mRNA and their translation to enhance expression of immune-regulatory proteins including C1q and CD27. This program is immuno-suppressive, and limited by Elavl1. We show this by Cdh5(PAC)-CreERT2-mediated deletion of Elavl1 (ECKO), and analysis of changes in translation by Translating Ribosome Affinity Purification (TRAP). In ECKO mice, the translational shift in chronic inflammation is enhanced, leading to increased ribosomal association of C1q components and other critical regulators of immune response and resulting in a ~70% reduction in plaque CD8 T cells. CITE-seq analysis of the remaining plaque T cells shows that they exhibit lower levels of markers associated with T cell receptor (TCR) signaling, survival, and activation. To understand whether the immunosuppressive mechanism occurred through failed CD8 recruitment or local modulation of T cell responses, we used a novel in vitro co-culture system to show that ECKO endothelial cells suppress CD8 T cell expansion-even in the presence of wild-type myeloid antigen-presenting cells, antigen-specific CD8 T cells, and antigen. Despite the induction of C1q mRNA by T cell co-culture in both wild-type and ECKO endothelial cells, we find C1q protein abundantly expressed only in co-culture with ECKO cells. Together, our data define a novel immune-suppressive transition in the endothelium, reminiscent of the transition of T cells to T-regs, and demonstrate the regulation of this process by Elavl1.
RESUMO
Despite the importance of the endothelium in the regulation of the blood brain barrier (BBB) in aging and neurodegenerative disease, difficulties in extracting endothelial cell (EC) nuclei have limited analysis of these cells. In addition, nearly all Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Degeneration (FTD), and a large portion of Alzheimer's Disease (AD) exhibit neuronal TDP-43 aggregation, leading to loss of nuclear function, but whether TDP-43 is similarly altered in human BBB ECs is unknown. Here we utilize a novel technique for the enrichment of endothelial and microglial nuclei from human cortical brain tissues, combined with inCITE-seq, to analyze nuclear proteins and RNA transcripts in a large cohort of healthy and diseased donors. Our findings reveal a unique transcriptional signature in nearly half of the capillary endothelial cells across neurodegenerative states, characterized by reduced levels of nuclear ß-Catenin and canonical downstream genes, and an increase in TNF/NF-kB target genes. We demonstrate that this does not correlate with increased nuclear p65/NF-kB, but rather a specific loss of nuclear TDP-43 in these disease associated ECs. Comparative analysis in animal models with targeted disruption of TDP-43 shows that this is sufficient to drive these transcriptional alterations. This work reveals that TDP-43 is a critical governor of the transcriptional output from nuclear p65/NF-kB, which has paradoxical roles in barrier maintenance and also barrier compromising inflammatory responses, and suggests that disease specific loss in ECs contributes to BBB defects observed in the progression of AD, ALS and FTD.
RESUMO
Loss of nuclear TDP-43 occurs in a wide range of neurodegenerative diseases, and specific mutations in the TARDBP gene that encodes the protein are linked to familial Frontal Temporal Lobar Dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS). Although the focus has been on neuronal cell dysfunction caused by TDP-43 variants, TARDBP mRNA transcripts are expressed at similar levels in brain endothelial cells (ECs). Since increased permeability across the blood brain barrier (BBB) precedes cognitive decline, we postulated that altered functions of TDP-43 in ECs contributes to BBB dysfunction in neurodegenerative disease. To test this hypothesis, we examined EC function and BBB properties in mice with either knock-in mutations found in ALS/FTLD patients (TARDBPG348C and GRNR493X) or EC-specific deletion of TDP-43 throughout the endothelium (Cdh5(PAC)CreERT2; Tardbpff) or restricted to brain endothelium (Slco1c1(BAC)CreERT2; Tardbpff). We found that TARDBPG348C mice exhibited increased permeability to 3kDa Texas Red dextran and NHS-biotin, relative to their littermate controls, which could be recapitulated in cultured brain ECs from these mice. Nuclear levels of TDP-43 were reduced in vitro and in vivo in ECs from TARDBPG348C mice. This coincided with a reduction in junctional proteins VE-cadherin, claudin-5 and ZO-1 in isolated ECs, supporting a cell autonomous effect on barrier function through a loss of nuclear TDP-43. We further examined two models of Tardbp deletion in ECs, and found that the loss of TDP-43 throughout the endothelium led to systemic endothelial activation and permeability. Deletion specifically within the brain endothelium acutely increased BBB permeability, and eventually led to hallmarks of FTD, including fibrin deposition, microglial and astrocyte activation, and behavioral defects. Together, these data show that TDP-43 dysfunction specifically within brain ECs would contribute to the BBB defects observed early in the progression of ALS/FTLD.
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Brain arteriovenous malformations (BAVMs) can cause devastating stroke in young people and contribute to half of all hemorrhagic stroke in children. Unfortunately, the pathogenesis of BAVMs is unknown. In this article we show that activation of Notch signaling in the endothelium during brain development causes BAVM in mice. We turned on constitutively active Notch4 (int3) expression in endothelial cells from birth by using the tetracycline-regulatable system. All mutants developed hallmarks of BAVMs, including cerebral arteriovenous shunting and vessel enlargement, by 3 weeks of age and died by 5 weeks of age. Twenty-five percent of the mutants showed signs of neurological dysfunction, including ataxia and seizure. Affected mice exhibited hemorrhage and neuronal cell death within the cerebral cortex and cerebellum. Strikingly, int3 repression resolved ataxia and reversed the disease progression, demonstrating that int3 is not only sufficient to induce, but also required to sustain the disease. We show that int3 expression results in widespread enlargement of the microvasculature, which coincided with a reduction in capillary density, linking vessel enlargement to Notch's known function of inhibiting vessel sprouting. Our data suggest that the Notch pathway is a molecular regulator of BAVM pathogenesis in mice, and offer hope that their regression might be possible by targeting the causal molecular lesion.
Assuntos
Endotélio Vascular/metabolismo , Malformações Arteriovenosas Intracranianas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Animais , Imuno-Histoquímica , Malformações Arteriovenosas Intracranianas/etiologia , Malformações Arteriovenosas Intracranianas/patologia , Camundongos , Receptor Notch4RESUMO
The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.
Assuntos
Processamento Alternativo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Éxons , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Domínios e Motivos de Interação entre Proteínas , Animais , Proteínas de Transporte , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/química , Citometria de Fluxo , Técnicas de Inativação de Genes , Genes Reporter , Camundongos , Ligação Proteica , RNA Mensageiro/genéticaRESUMO
Myxococcus xanthus bacteria are a model system for understanding pattern formation and collective cell behaviors. When starving, cells aggregate into fruiting bodies to form metabolically inert spores. During predation, cells self-organize into traveling cell-density waves termed ripples. Both phase-contrast and fluorescence microscopy are used to observe these patterns but each has its limitations. Phase-contrast images have higher contrast, but the resulting image intensities lose their correlation with cell density. The intensities of fluorescence microscopy images, on the other hand, are well-correlated with cell density, enabling better segmentation of aggregates and better visualization of streaming patterns in between aggregates; however, fluorescence microscopy requires the engineering of cells to express fluorescent proteins and can be phototoxic to cells. To combine the advantages of both imaging methodologies, we develop a generative adversarial network that converts phase-contrast into synthesized fluorescent images. By including an additional histogram-equalized output to the state-of-the-art pix2pixHD algorithm, our model generates accurate images of aggregates and streams, enabling the estimation of aggregate positions and sizes, but with small shifts of their boundaries. Further training on ripple patterns enables accurate estimation of the rippling wavelength. Our methods are thus applicable for many other phenotypic behaviors and pattern formation studies.
RESUMO
Brain arteriovenous malformations (BAVMs) can cause lethal hemorrhagic stroke and have no effective treatment. The cellular and molecular basis for this disease is largely unknown. We have previously shown that expression of constitutively-active Notch4 receptor in the endothelium elicits and maintains the hallmarks of BAVM in mice, thus establishing a mouse model of the disease. Our work suggested that Notch pathway could be a critical molecular mediator of BAVM pathogenesis. Here, we investigated the hypothesis that upregulated Notch activation contributes to the pathogenesis of human BAVM. We examined the expression of the canonical Notch downstream target Hes1 in the endothelium of human BAVMs by immunofluorescence, and showed increased levels relative to either autopsy or surgical biopsy controls. We then analyzed receptor activity using an antibody to the activated form of the Notch1 receptor, and found increased levels of activity. These findings suggest that Notch activation may promote the development and even maintenance of BAVM. We also detected increases in Hes1 and activated Notch1 expression in our mouse model of BAVM induced by constitutively active Notch4, demonstrating molecular similarity between the mouse model and the human disease. Our work suggests that activation of Notch signaling is an important molecular candidate in BAVM pathogenesis and further validates that our animal model provides a platform to study the progression as well as the regression of the disease.
Assuntos
Endotélio Vascular/metabolismo , Malformações Arteriovenosas Intracranianas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor Notch1/biossíntese , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Modelos Animais de Doenças , Endotélio Vascular/patologia , Proteínas de Homeodomínio/metabolismo , Humanos , Malformações Arteriovenosas Intracranianas/patologia , Camundongos , Camundongos Mutantes , Microscopia de Fluorescência , Neovascularização Patológica , Receptor Notch4 , Fatores de Transcrição HES-1 , Regulação para CimaRESUMO
Low and disturbed blood flow drives the progression of arterial diseases including atherosclerosis and aneurysms. The endothelial response to flow and its interactions with recruited platelets and leukocytes determine disease progression. Here, we report widespread changes in alternative splicing of pre-mRNA in the flow-activated murine arterial endothelium in vivo. Alternative splicing was suppressed by depletion of platelets and macrophages recruited to the arterial endothelium under low and disturbed flow. Binding motifs for the Rbfox-family are enriched adjacent to many of the regulated exons. Endothelial deletion of Rbfox2, the only family member expressed in arterial endothelium, suppresses a subset of the changes in transcription and RNA splicing induced by low flow. Our data reveal an alternative splicing program activated by Rbfox2 in the endothelium on recruitment of platelets and macrophages and demonstrate its relevance in transcriptional responses during flow-driven vascular inflammation.
Assuntos
Adaptação Fisiológica , Processamento Alternativo , Endotélio Vascular/fisiologia , Regulação da Expressão Gênica , Fatores de Processamento de RNA/metabolismo , Fluxo Sanguíneo Regional , Animais , CamundongosRESUMO
Certain RGD-binding integrins are required for cell adhesion, migration, and proliferation and are overexpressed in most tumors, making them attractive therapeutic targets. However, multiple integrin antagonist drug candidates have failed to show efficacy in cancer clinical trials. In this work, we instead exploit these integrins as a target for antibody Fc effector functions in the context of cancer immunotherapy. By combining administration of an engineered mouse serum albumin/IL-2 fusion with an Fc fusion to an integrin-binding peptide (2.5F-Fc), significant survival improvements are achieved in three syngeneic mouse tumor models, including complete responses with protective immunity. Functional integrin antagonism does not contribute significantly to efficacy; rather, this therapy recruits both an innate and adaptive immune response, as deficiencies in either arm result in reduced tumor control. Administration of this integrin-targeted immunotherapy together with an anti-PD-1 antibody further improves responses and predominantly results in cures. Overall, this well-tolerated therapy achieves tumor specificity by redirecting inflammation to a functional target fundamental to tumorigenic processes but expressed at significantly lower levels in healthy tissues, and it shows promise for translation.