RESUMO
Atherosclerosis is the underlying pathology in a major part of cardiovascular disease, the leading cause of mortality in developed countries. The infiltration of monocytes into the vessel walls of large arteries is a key denominator of atherogenesis, making monocytes accountable for the development of atherosclerosis. With the development of high-throughput transcriptome profiling platforms and cytometric methods for circulating cells, it is now feasible to study in-depth the predicted functional change of circulating monocytes reflected by changes of gene expression in certain pathways and correlate the changes to disease outcome. Neuroimmune guidance cues comprise a group of circulating- and cell membrane-associated signaling proteins that are progressively involved in monocyte functions. Here, we employed the CIRCULATING CELLS study cohort to classify cardiovascular disease patients and healthy individuals in relation to their expression of neuroimmune guidance cues in circulating monocytes. To cope with the complexity of human datasets featured by noisy data, nonlinearity and multidimensionality, we assessed various machine-learning methods. Of these, the linear discriminant analysis, Naïve Bayesian model and stochastic gradient boost model yielded perfect or near-perfect sensibility and specificity and revealed that expression levels of the neuroimmune guidance cues SEMA6B, SEMA6D and EPHA2 in circulating monocytes were of predictive values for cardiovascular disease outcome.
Assuntos
Biomarcadores/sangue , Doenças Cardiovasculares/diagnóstico , Efrinas/sangue , Aprendizado de Máquina , Monócitos/metabolismo , Netrina-1/sangue , Semaforinas/sangue , Adulto , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/genética , Estudos de Casos e Controles , Estudos de Coortes , Efrinas/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Netrina-1/genética , Semaforinas/genética , TranscriptomaRESUMO
BACKGROUND: Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is an inherited genetic disorder characterized by vascular malformations and hemorrhage. HHT2 results from ACVRL1 haploinsufficiency, the remaining wild-type allele being unable to contribute sufficient protein to sustain endothelial cell function. Blood vessels function normally but are prone to respond to angiogenic stimuli, leading to the development of telangiectasic lesions that can bleed. How ACVRL1 haploinsufficiency leads to pathological angiogenesis is unknown. METHODS: We took advantage of Acvrl1+/- mutant mice that exhibit HHT2 vascular lesions and focused on the neonatal retina and the airway system after Mycoplasma pulmonis infection, as physiological and pathological models of angiogenesis, respectively. We elucidated underlying disease mechanisms in vitro by generating Acvrl1+/- mouse embryonic stem cell lines that underwent sprouting angiogenesis and performed genetic complementation experiments. Finally, HHT2 plasma samples and skin biopsies were analyzed to determine whether the mechanisms evident in mice are conserved in humans. RESULTS: Acvrl1+/- retinas at postnatal day 7 showed excessive angiogenesis and numerous endothelial "tip cells" at the vascular front that displayed migratory defects. Vascular endothelial growth factor receptor 1 (VEGFR1; Flt-1) levels were reduced in Acvrl1+/- mice and HHT2 patients, suggesting similar mechanisms in humans. In sprouting angiogenesis, VEGFR1 is expressed in stalk cells to inhibit VEGFR2 (Flk-1, KDR) signaling and thus limit tip cell formation. Soluble VEGFR1 (sVEGFR1) is also secreted, creating a VEGF gradient that promotes orientated sprout migration. Acvrl1+/- embryonic stem cell lines recapitulated the vascular anomalies in Acvrl1+/- (HHT2) mice. Genetic insertion of either the membrane or soluble form of VEGFR1 into the ROSA26 locus of Acvrl1+/- embryonic stem cell lines prevented the vascular anomalies, suggesting that high VEGFR2 activity in Acvrl1+/- endothelial cells induces HHT2 vascular anomalies. To confirm our hypothesis, Acvrl1+/- mice were infected by Mycoplasma pulmonis to induce sustained airway inflammation. Infected Acvrl1+/- tracheas showed excessive angiogenesis with the formation of multiple telangiectases, vascular defects that were prevented by VEGFR2 blocking antibodies. CONCLUSIONS: Our findings demonstrate a key role of VEGFR1 in HHT2 pathogenesis and provide mechanisms explaining why HHT2 blood vessels respond abnormally to angiogenic signals. This supports the case for using anti-VEGF therapy in HHT2.
Assuntos
Telangiectasia Hemorrágica Hereditária/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Activinas Tipo II , Adulto , Animais , Anticorpos/administração & dosagem , Anticorpos/imunologia , Malformações Arteriovenosas/etiologia , Modelos Animais de Doenças , Feminino , Heterozigoto , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Mycoplasma pulmonis/fisiologia , Neovascularização Fisiológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Vasos Retinianos/fisiologia , Transdução de Sinais , Pele/patologia , Telangiectasia Hemorrágica Hereditária/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
The bioengineering of a replacement kidney has been proposed as an approach to address the growing shortage of donor kidneys for the treatment of chronic kidney disease. One approach being investigated is the recellularization of kidney scaffolds. In this study, we present several key advances toward successful re-endothelialization of whole kidney matrix scaffolds from both rodents and humans. Based on the presence of preserved glycosoaminoglycans within the decelullarized kidney scaffold, we show improved localization of delivered endothelial cells after preloading of the vascular matrix with vascular endothelial growth factor and angiopoietin 1. Using a novel simultaneous arteriovenous delivery system, we report the complete re-endothelialization of the kidney vasculature, including the glomerular and peritubular capillaries, using human inducible pluripotent stem cell -derived endothelial cells. Using this source of endothelial cells, it was possible to generate sufficient endothelial cells to recellularize an entire human kidney scaffold, achieving efficient cell delivery, adherence, and endothelial cell proliferation and survival. Moreover, human re-endothelialized scaffold could, in contrast to the non-re-endothelialized human scaffold, be fully perfused with whole blood. These major advances move the field closer to a human bioengineered kidney.
Assuntos
Bioengenharia , Endotélio Vascular/citologia , Matriz Extracelular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Transplante de Rim/métodos , Rim/citologia , Alicerces Teciduais/química , Animais , Células Cultivadas , Endotélio Vascular/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Rim/metabolismo , Ratos , Ratos Endogâmicos LewRESUMO
Inhibition of monocyte chemotactic protein-1 (MCP-1) with the Spiegelmer emapticap pegol (NOX-E36) shows long-lasting albuminuria-reducing effects in diabetic nephropathy. MCP-1 regulates inflammatory cell recruitment and differentiation of macrophages. Because the endothelial glycocalyx is also reduced in diabetic nephropathy, we hypothesized that MCP-1 inhibition restores glomerular barrier function through influencing macrophage cathepsin L secretion, thus reducing activation of the glycocalyx-degrading enzyme heparanase. Four weeks of treatment of diabetic Apoe knockout mice with the mouse-specific NOX-E36 attenuated albuminuria without any change in systemic hemodynamics, despite persistent loss of podocyte function. MCP-1 inhibition, however, increased glomerular endothelial glycocalyx coverage, with preservation of heparan sulfate. Mechanistically, both glomerular cathepsin L and heparanase expression were reduced. MCP-1 inhibition resulted in reduced CCR2-expressing Ly6Chi monocytes in the peripheral blood, without affecting overall number of kidney macrophages at the tissue level. However, the CD206+/Mac3+ cell ratio, as an index of presence of anti-inflammatory macrophages, increased in diabetic mice after treatment. Functional analysis of isolated renal macrophages showed increased release of IL-10, whereas tumor necrosis factor and cathepsin L release was reduced, further confirming polarization of tissue macrophages toward an anti-inflammatory phenotype during mouse-specific NOX-E36 treatment. We show that MCP-1 inhibition restores glomerular endothelial glycocalyx and barrier function and reduces tissue inflammation in the presence of ongoing diabetic injury, suggesting a therapeutic potential for NOX-E36 in diabetic nephropathy.
Assuntos
Quimiocina CCL2/metabolismo , Nefropatias Diabéticas/metabolismo , Glicocálix/metabolismo , Macrófagos/metabolismo , Podócitos/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/patologia , Rim/patologia , Masculino , Camundongos Knockout , Monócitos/metabolismoRESUMO
AIMS: Atrial fibrillation (AF) produces a hypercoagulable state. Stimulation of protease-activated receptors by coagulation factors provokes pro-fibrotic, pro-hypertrophic, and pro-inflammatory responses in a variety of tissues. We studied the effects of thrombin on atrial fibroblasts and tested the hypothesis that hypercoagulability contributes to the development of a substrate for AF. METHODS AND RESULTS: In isolated rat atrial fibroblasts, thrombin enhanced the phosphorylation of the pro-fibrotic signalling molecules Akt and Erk and increased the expression of transforming growth factor ß1 (2.7-fold) and the pro-inflammatory factor monocyte chemoattractant protein-1 (6.1-fold). Thrombin also increased the incorporation of 3H-proline, suggesting enhanced collagen synthesis by fibroblasts (2.5-fold). All effects could be attenuated by the thrombin inhibitor dabigatran. In transgenic mice with a pro-coagulant phenotype (TMpro/pro), the inducibility of AF episodes lasting >1 s was higher (7 out of 12 vs. 1 out of 10 in wild type) and duration of AF episodes was longer compared with wild type mice (maximum episode duration 42.8 ± 68.4 vs. 0.23 ± 0.39 s). In six goats with persistent AF treated with nadroparin, targeting Factor Xa-mediated thrombin generation, the complexity of the AF substrate was less pronounced than in control animals (LA maximal activation time differences 23.3 ± 3.1 ms in control vs. 15.7 ± 2.1 ms in nadroparin, P < 0.05). In the treated animals, AF-induced α-smooth muscle actin expression was lower and endomysial fibrosis was less pronounced. CONCLUSION: The hypercoagulable state during AF causes pro-fibrotic and pro-inflammatory responses in adult atrial fibroblasts. Hypercoagulability promotes the development of a substrate for AF in transgenic mice and in goats with persistent AF. In AF goats, nadroparin attenuates atrial fibrosis and the complexity of the AF substrate. Inhibition of coagulation may not only prevent strokes but also inhibit the development of a substrate for AF.
Assuntos
Fibrilação Atrial/etiologia , Receptores de Trombina/efeitos dos fármacos , Trombina/farmacologia , Trombofilia/fisiopatologia , Análise de Variância , Animais , Antitrombinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Dabigatrana/farmacologia , Feminino , Fibrinolíticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibrose/etiologia , Cabras , Átrios do Coração/patologia , Indazóis/farmacologia , Camundongos Transgênicos , Nadroparina/farmacologia , Peptídeo Hidrolases/efeitos dos fármacos , Pirróis/farmacocinética , Quinazolinas/farmacocinética , Ratos , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
Patients with von Willebrand disease (VWD) are often heterozygous for a missense mutation in the von Willebrand factor (VWF) gene. Investigating the pathogenic features of VWF mutations in cells directly derived from patients has been challenging. Here, we have used blood outgrowth endothelial cells (BOECs) isolated from human peripheral blood to analyze the storage and secretion of VWF. BOECs showed full endothelial characteristics and responded to Weibel-Palade body (WPB) secretagogues except desmopressin. We examined BOECs derived from a single subject heterozygous for a type 2N mutation (p.Arg854Gln) and from 4 patients with type 1 VWD who were, respectively, heterozygous for p.Ser1285Pro, p.Leu1307Pro, p.Tyr1584Cys, and p.Cys2693Tyr. Compared with normal BOECs, BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr showed morphologically abnormal WPB and retention of VWF in the endoplasmic reticulum, whereas BOECs heterozygous for p.Arg854Gln or p.Tyr1584Cys showed normal WPB. The agonist-induced exocytosis of WPB from BOECs and formation of VWF strings on BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr, but not for p.Arg854Gln or p.Tyr1584Cys, were reduced. In conclusion, VWD phenotype can be recapitulated in BOECs, and thus BOECs provide a feasible bona fide cell model to study the pathogenic effects of VWF mutations.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Corpos de Weibel-Palade/metabolismo , Doença de von Willebrand Tipo 1/metabolismo , Fator de von Willebrand/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Células Endoteliais/fisiologia , Exocitose/fisiologia , Feminino , Citometria de Fluxo , Genótipo , Heterozigoto , Humanos , Masculino , Mutação de Sentido Incorreto , Fenótipo , Doença de von Willebrand Tipo 1/genética , Doença de von Willebrand Tipo 1/patologia , Fator de von Willebrand/genéticaRESUMO
RATIONALE: RNA-binding proteins are critical post-transcriptional regulators of RNA and can influence pre-mRNA splicing, RNA localization, and stability. The RNA-binding protein Quaking (QKI) is essential for embryonic blood vessel development. However, the role of QKI in the adult vasculature, and in particular in vascular smooth muscle cells (VSMCs), is currently unknown. OBJECTIVE: We sought to determine the role of QKI in regulating adult VSMC function and plasticity. METHODS AND RESULTS: We identified that QKI is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Quaking viable mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering noncontractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome, where it interacts with the myocardin pre-mRNA and regulates the splicing of alternative exon 2a. This post-transcriptional event impacts the Myocd_v3/Myocd_v1 mRNA balance and can be modulated by mutating the quaking response element in exon 2a of myocardin. Furthermore, we identified that arterial damage triggers myocardin alternative splicing and is tightly coupled with changes in the expression levels of distinct QKI isoforms. CONCLUSIONS: We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.
Assuntos
Proliferação de Células , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Lesões das Artérias Carótidas/metabolismo , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Movimento Celular , Reestenose Coronária/metabolismo , Reestenose Coronária/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Hiperplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Quaking , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Neointima , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Interferência de RNA , Proteínas de Ligação a RNA/genética , Transativadores/genética , Transativadores/metabolismo , TransfecçãoRESUMO
Ischemia/reperfusion injury (IRI) is a central phenomenon in kidney transplantation and AKI. Integrity of the renal peritubular capillary network is an important limiting factor in the recovery from IRI. MicroRNA-126 (miR-126) facilitates vascular regeneration by functioning as an angiomiR and by modulating mobilization of hematopoietic stem/progenitor cells. We hypothesized that overexpression of miR-126 in the hematopoietic compartment could protect the kidney against IRI via preservation of microvascular integrity. Here, we demonstrate that hematopoietic overexpression of miR-126 increases neovascularization of subcutaneously implanted Matrigel plugs in mice. After renal IRI, mice overexpressing miR-126 displayed a marked decrease in urea levels, weight loss, fibrotic markers, and injury markers (such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin). This protective effect was associated with a higher density of the peritubular capillary network in the corticomedullary junction and increased numbers of bone marrow-derived endothelial cells. Hematopoietic overexpression of miR-126 increased the number of circulating Lin(-)/Sca-1(+)/cKit(+) hematopoietic stem and progenitor cells. Additionally, miR-126 overexpression attenuated expression of the chemokine receptor CXCR4 on Lin(-)/Sca-1(+)/cKit(+) cells in the bone marrow and increased renal expression of its ligand stromal cell-derived factor 1, thus favoring mobilization of Lin(-)/Sca-1(+)/cKit(+) cells toward the kidney. Taken together, these results suggest overexpression of miR-126 in the hematopoietic compartment is associated with stromal cell-derived factor 1/CXCR4-dependent vasculogenic progenitor cell mobilization and promotes vascular integrity and supports recovery of the kidney after IRI.
Assuntos
Injúria Renal Aguda/prevenção & controle , Células-Tronco Hematopoéticas/fisiologia , Rim/irrigação sanguínea , MicroRNAs/fisiologia , Neovascularização Fisiológica/fisiologia , Traumatismo por Reperfusão/prevenção & controle , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Movimento Celular/fisiologia , Quimiocina CXCL12/metabolismo , Rim/metabolismo , Rim/patologia , Masculino , Camundongos Endogâmicos C57BL , Receptores CXCR4/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
OBJECTIVE: Therapeutic arteriogenesis, that is, expansive remodeling of preexisting collaterals, using single-action factor therapies has not been as successful as anticipated. Modulation of factors that act as a master switch for relevant gene programs may prove more effective. Transcriptional coactivator p300-CBP-associated factor (PCAF) has histone acetylating activity and promotes transcription of multiple inflammatory genes. Because arteriogenesis is an inflammation-driven process, we hypothesized that PCAF acts as multifactorial regulator of arteriogenesis. APPROACH AND RESULTS: After induction of hindlimb ischemia, blood flow recovery was impaired in both PCAF(-/-) mice and healthy wild-type mice treated with the pharmacological PCAF inhibitor Garcinol, demonstrating an important role for PCAF in arteriogenesis. PCAF deficiency reduced the in vitro inflammatory response in leukocytes and vascular cells involved in arteriogenesis. In vivo gene expression profiling revealed that PCAF deficiency results in differential expression of 3505 genes during arteriogenesis and, more specifically, in impaired induction of multiple proinflammatory genes. Additionally, recruitment from the bone marrow of inflammatory cells, in particular proinflammatory Ly6C(hi) monocytes, was severely impaired in PCAF(-/-) mice. CONCLUSIONS: These findings indicate that PCAF acts as master switch in the inflammatory processes required for effective arteriogenesis.
Assuntos
Arterite/fisiopatologia , Isquemia/fisiopatologia , Neovascularização Fisiológica/imunologia , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/imunologia , Acetilação , Animais , Arterite/imunologia , Arterite/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/imunologia , Membro Posterior/irrigação sanguínea , Histonas/metabolismo , Isquemia/imunologia , Isquemia/metabolismo , Camundongos , Camundongos Knockout , Monócitos/imunologia , Monócitos/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Terpenos/farmacologia , Transcriptoma , Fatores de Transcrição de p300-CBP/antagonistas & inibidoresRESUMO
AIMS: MicroRNA-126 (miR-126) facilitates angiogenesis and regulates endothelial cell function. Recent data suggest that miR-126 can serve as a biomarker for vascular disease. Although endothelial cells are enriched for miR-126, platelets also contain miR-126. In this paper, we investigated the contribution of platelets to the pool of miR-126 in plasma of patients with type 2 diabetes (DM2) and how this is affected by aspirin. METHODS AND RESULTS: In vitro platelet activation resulted in the transfer of miR-126 from the platelet to the plasma compartment, which was prevented by aspirin. In vivo platelet activation, monitored in patients with DM2 by measuring soluble P-selectin, correlated directly with circulating levels of miR-126. The administration of aspirin resulted both in platelet inhibition and concomitantly reduced circulating levels of platelet-derived microRNAs including miR-126. CONCLUSION: Platelets are a major source of circulating miR-126. Consequently, in patho-physiological conditions associated with platelet activation, such as diabetes type 2, the administration of aspirin may lead to reduced levels of circulating miR-126. Thus, the use of platelet inhibitors should be taken into account when using plasma levels of miR-126 as a biomarker.
Assuntos
Aspirina , Diabetes Mellitus Tipo 2/diagnóstico , Angiopatias Diabéticas/diagnóstico , MicroRNAs/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária , Análise de Variância , Ácido Araquidônico/farmacologia , Biomarcadores/metabolismo , Contraindicações , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Selectina-P/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Background and Aims: Endothelial cell (EC) dysfunction in response to circulating plasma factors is a known causal factor in many systemic diseases. However, no appropriate assay is available to investigate this causality ex vivo. In liver cirrhosis, systemic inflammation is identified as central mechanism in progression from compensated to decompensated cirrhosis (DC), but the role of ECs therein is unknown. We aimed to develop a novel ex vivo assay for assessing EC responses to patient-derived plasma (PDP) and assess the potential of this assay in a cohort of liver cirrhosis patients. Methods: Image-based morphological profiling was utilized to assess the impact of PDP on cultured ECs. Endothelial cell (EC) monolayers were exposed to 25% stabilized PDP (20 compensated cirrhoses, 20 DCs, and 20 healthy controls (HCs). Single-cell morphological profiles were extracted by automated image-analysis following staining of multiple cellular components and high-content imaging. Patient profiles were created by dimension reduction and cell-to-patient data aggregation, followed by multivariate-analysis to stratify patients and identify discriminating features. Results: Patient-derived plasma (PDP) exposure induced profound changes in EC morphology, displaying clear differences between controls and DC patients. Compensated cirrhosis patients showed overlap with healthy controls and DC patients. Supervised analysis showed Child-Pugh (CP) class could be predicted from EC morphology. Most importantly, CP-C patients displayed distinct EC phenotypes, in which mitochondrial changes were most discriminative. Conclusion: Morphological profiling presents a viable tool to assess the endothelium ex vivo. We demonstrated that the EC phenotype corresponds with disease severity in liver cirrhosis. Moreover, our results suggest the presence of mitochondrial dysfunction in ECs of CP-C patient.
RESUMO
Endothelial cells (ECs) are highly susceptible to hypoxia and easily affected upon ischemia-reperfusion (I/R) during renal transplantation. Pericytes and angiopoeitins play important role in modulating EC function. In the present study, we investigate the effect of renal I/R on the dynamics of angiopoietin expression and its association with pericytes and fibrosis development. Male Lewis rats were subjected to unilateral renal ischemia for 45 min followed by removal of the contralateral kidney. Rats were killed at different time points after reperfusion. Endothelial integrity (RECA-1), pericytes [platelet-derived growth factor receptor-ß (PDGFR-ß)], angiopoietin-2 (Ang-2)/angiopoietin-1 (Ang-1) expression, and interstitial collagen deposition (Sirius red and α-smooth muscle actin) were assessed using immunohistochemistry and RT-PCR. Our study shows an increase in protein expression of Ang-2 starting at 5 h and remaining elevated up to 72 h, with a consequently higher Ang-2/Ang-1 ratio after renal I/R (P < 0.05 at 48 h). This was accompanied by an increase in protein expression of the pericytic marker PDGFR-ß and a loss of ECs (both at 72 h after I/R, P < 0.05). Nine weeks after I/R, when renal function was restored, we observed normalization of the Ang-2/Ang-1 ratio and PDGFR-ß expression and increase in cortical ECs, which was accompanied by fibrosis. Renal I/R induces a dysbalance of Ang-2/Ang-1 accompanied by proliferation of pericytes, EC loss, and development of fibrosis. The Ang-2/Ang-1 balance was reversed to baseline at 9 wk after renal I/R, which coincided with restoration of cortical ECs and pericytes. Our findings suggest that angiopoietins and pericytes play an important role in renal microvascular remodeling and development of fibrosis.
Assuntos
Angiopoietinas/metabolismo , Pericitos/patologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Angiopoietina-1/biossíntese , Angiopoietina-2/biossíntese , Animais , Células Endoteliais , Fibrose , Nefropatias/patologia , Masculino , Pericitos/metabolismo , Ratos , Ratos Endogâmicos Lew , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossínteseRESUMO
OBJECTIVE: The role of toll-like receptors (TLRs) in vascular remodeling is well established. However, the involvement of the endosomal TLRs is unknown. Here, we study the effect of combined blocking of TLR7 and TLR9 on postinterventional remodeling and accelerated atherosclerosis. METHODS AND RESULTS: In hypercholesterolemic apolipoprotein E*3-Leiden mice, femoral artery cuff placement led to strong increase of TLR7 and TLR9 presence demonstrated by immunohistochemistry. Blocking TLR7/9 with a dual antagonist in vivo reduced neointimal thickening and foam cell accumulation 14 days after surgery by 65.6% (P=0.0079). Intima/media ratio was reduced by 64.5% and luminal stenosis by 62.8%. The TLR7/9 antagonist reduced the arterial wall inflammation, with reduced macrophage infiltration, decreased cytoplasmic high-mobility group box 1 expression, and altered serum interleukin-10 levels. Stimulation of cultured macrophages with TLR7 and TLR9 ligands enhanced tumor necrosis factor-α expression, which is decreased by TLR7/9 antagonist coadministration. Additionally, the antagonist abolished the TLR7/9-enhanced low-density lipoprotein uptake. The antagonist also reduced oxidized low-density lipoprotein-induced foam cell formation, most likely not via decreased influx but via increased efflux, because CD36 expression was unchanged whereas interleukin-10 levels were higher (36.1 ± 22.3 pg/mL versus 128.9 ± 6.6 pg/mL; P=0.008). CONCLUSIONS: Blocking TLR7 and TLR9 reduced postinterventional vascular remodeling and foam cell accumulation indicating TLR7 and TLR9 as novel therapeutic targets.
Assuntos
Aterosclerose/etiologia , Movimento Celular , Vasos Coronários/patologia , Células Espumosas/fisiologia , Ativação de Macrófagos , Glicoproteínas de Membrana/fisiologia , Receptor 7 Toll-Like/fisiologia , Receptor Toll-Like 9/fisiologia , Angioplastia Coronária com Balão , Animais , Citocinas/biossíntese , Proteína HMGB1/análise , Lipoproteínas LDL/fisiologia , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Neointima/prevenção & controle , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor Toll-Like 9/antagonistas & inibidoresRESUMO
BACKGROUND AND AIMS: This study aims to identify sex-specific transcriptional differences and signaling pathways in circulating monocytes contributing to cardiovascular disease. METHODS AND RESULTS: We generated sex-biased gene expression signatures by comparing male versus female monocytes of coronary artery disease (CAD) patients (n = 450) from the Center for Translational Molecular Medicine-Circulating Cells Cohort. Gene set enrichment analysis demonstrated that monocytes from female CAD patients carry stronger chemotaxis and migratory signature than those from males. We then inferred cytokine signaling activities based on CytoSig database of 51 cytokine and growth factor regulation profiles. Monocytes from females feature a higher activation level of EGF, IFN1, VEGF, GM-CSF, and CD40L pathways, whereas IL-4, INS, and HMGB1 signaling was seen to be more activated in males. These sex differences were not observed in healthy subjects, as shown for an independent monocyte cohort of healthy subjects (GSE56034, n = 485). More pronounced GM-CSF signaling in monocytes of female CAD patients was confirmed by the significant enrichment of GM-CSF-activated monocyte signature in females. As we show these effects were not due to increased plasma levels of the corresponding ligands, sex-intrinsic differences in monocyte signaling regulation are suggested. Consistently, regulatory network analysis revealed jun-B as a shared transcription factor activated in all female-specific pathways except IFN1 but suppressed in male-activated IL-4. CONCLUSIONS: We observed overt CAD-specific sex differences in monocyte transcriptional profiles and cytokine- or growth factor-induced responses, which provide insights into underlying mechanisms of sex differences in CVD.
Assuntos
Doenças Cardiovasculares , Doença da Artéria Coronariana , Humanos , Masculino , Feminino , Monócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Caracteres Sexuais , Interleucina-4 , Citocinas/metabolismo , Transdução de SinaisRESUMO
Several missense mutations in the von Willebrand Factor (VWF) gene of von Willebrand disease (VWD) patients have been shown to cause impaired constitutive secretion and intracellular retention of VWF. However, the effects of those mutations on the intracellular storage in Weibel-Palade bodies (WPBs) of endothelial cells and regulated secretion of VWF remain unknown. We demonstrate, by expression of quantitative VWF mutants in HEK293 cells, that four missense mutations in the D3 and CK-domain of VWF diminished the storage in pseudo-WPBs, and led to retention of VWF within the endoplasmic reticulum (ER). Immunofluorescence and electron microscopy data showed that the pseudo-WPBs formed by missense mutant C1060Y are indistinguishable from those formed by normal VWF. C1149R, C2739Y, and C2754W formed relatively few pseudo-WPBs, which were often short and sometimes round rather than cigar-shaped. The regulated secretion of VWF was impaired slightly for C1060Y but severely for C1149R, C2739Y, and C2754W. Upon co-transfection with wild-type VWF, both intracellular storage and regulated secretion of all mutants were (partly) corrected. In conclusion, defects in the intracellular storage and regulated secretion of VWF following ER retention may be a common mechanism underlying VWD with a quantitative deficiency of VWF.
Assuntos
Corpos de Weibel-Palade/metabolismo , Doenças de von Willebrand/metabolismo , Fator de von Willebrand/metabolismo , Substituição de Aminoácidos , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Corpos de Weibel-Palade/genética , Doenças de von Willebrand/genética , Fator de von Willebrand/genéticaRESUMO
OBJECTIVE: Annexin A5 (AnxA5) has antithrombotic, antiapoptotic, and antiinflammatory properties; we investigated its effectiveness against vascular inflammation, remodeling, and dysfunction in accelerated atherosclerosis. METHODS AND RESULTS: AnxA5 (1 mg/kg per day or vehicle) was investigated in vascular injury models in hypercholesterolemic apolipoprotein E (ApoE)3*Leiden mice. AnxA5 treatment reduced adhesion and infiltration of leukocytes by 71% to 69% (P=0.015, P=0.031) and macrophages by 51% to 87% (P=0.014, P=0.018), as well as monocyte chemotactic protein-1 and tumor necrosis factor-α expression in a femoral artery inflammation model (perivascular cuff for 3 days), indicating reduced vascular inflammation. In a vein graft model, 28 days of AnxA5 treatment reduced vein graft thickening (48%; P=0.006) and leukocyte infiltration (46%; P=0.003). In these mice, reduced plasma concentrations of IFN-γ (-72%; P=0.040), granulocyte colony-stimulating factor (-41%; P=0.010), and macrophage inflammatory protein-1ß (MIP-1ß) (-66%; P=0.020) were measured, indicating reduced systemic inflammation. An in vitro endothelial cell model shows the importance of AnxA5's anticoagulant properties in reducing vascular inflammation. Endothelium-mediated dilatation in hypercholesterolemic ApoE((-/-)) mice was improved by 3 days of AnxA5 treatment, shown by improved systolic and diastolic blood pressure reductions in response to metacholine, which could be abolished by l-Nitro-Arginine-Methyl Ester (l-NAME), indicating nitric oxide involvement. CONCLUSIONS: AnxA5 reduced local vascular and systemic inflammation and vascular remodeling and improved vascular function, indicating that it has a therapeutic potential against atherosclerotic cardiovascular diseases.
Assuntos
Anexina A5/farmacologia , Anti-Inflamatórios/farmacologia , Aterosclerose/tratamento farmacológico , Endotélio Vascular/química , Oclusão de Enxerto Vascular/prevenção & controle , Inflamação/prevenção & controle , Vasodilatação/efeitos dos fármacos , Animais , Anexina A5/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Citocinas/sangue , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Oclusão de Enxerto Vascular/genética , Oclusão de Enxerto Vascular/metabolismo , Oclusão de Enxerto Vascular/patologia , Oclusão de Enxerto Vascular/fisiopatologia , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Hipercolesterolemia/fisiopatologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/fisiopatologia , Mediadores da Inflamação/sangue , Injeções Intraperitoneais , Camundongos , Camundongos Knockout , Camundongos Mutantes , Mutação , Óxido Nítrico/metabolismo , Fatores de Tempo , Grau de Desobstrução Vascular/efeitos dos fármacos , Veias/transplanteRESUMO
BACKGROUND: Atrial fibrillation (AF) can lead to the loss of microvascular integrity thereby enhancing AF progression. Mechanistically, the pro-coagulant state that drives the risk of stroke in patients with AF may also play a causal role in microvascular loss. Direct oral anticoagulants (DOACs), the preferred anticoagulants for AF, can target factors upstream (factor Xa [FXa]) or downstream (thrombin) in the coagulation cascade and mediate differential vascular effects through interaction with protease-activated receptors (PARs). OBJECTIVE: To investigate the potential effect of different DOACs on vascular integrity. METHODS: To model the impact of DOACs on vascular integrity, we utilized platelet-free plasma in thrombin generation assays and endothelial barrier assays under identical experimental conditions. These multifactorial systems provide all coagulation factors and their respective natural inhibitors in physiological ratios in combination with the pro-coagulant endothelial surface on which coagulation is initiated. Furthermore, the system provides pro- and anti-barrier factors and monitoring both assays simultaneously permits coupling of thrombin kinetics to endothelial barrier dynamics. RESULTS: We provide evidence that the anti-FXa DOAC rivaroxaban and the anti-thrombin DOAC dabigatran are efficient in blocking their target proteases. However, while rivaroxaban could preserve endothelial barrier function, dabigatran failed to protect endothelial integrity over time, which could be prevented in the presence of a custom-made peptide that blocks thrombin's exosite-I. CONCLUSIONS: Proteolytically inactive thrombin in complex with dabigatran evokes loss of barrier function that can be prevented by a protease-activated receptor-1 mimicking peptide blocking thrombin's exosite-I.
Assuntos
Fibrilação Atrial , Dabigatrana , Administração Oral , Anticoagulantes/uso terapêutico , Fibrilação Atrial/tratamento farmacológico , Dabigatrana/efeitos adversos , Fator Xa/uso terapêutico , Inibidores do Fator Xa/efeitos adversos , Humanos , Receptor PAR-1 , Rivaroxabana/efeitos adversos , Trombina/uso terapêuticoRESUMO
Background Arteriovenous fistula (AVF) maturation failure is a main limitation of vascular access. Maturation is determined by the intricate balance between outward remodeling and intimal hyperplasia, whereby endothelial cell dysfunction, platelet aggregation, and vascular smooth muscle cell (VSMC) proliferation play a crucial role. von Willebrand Factor (vWF) is an endothelial cell-derived protein involved in platelet aggregation and VSMC proliferation. We investigated AVF vascular remodeling in vWF-deficient mice and vWF expression in failed and matured human AVFs. Methods and Results Jugular-carotid AVFs were created in wild-type and vWF-/- mice. AVF flow was determined longitudinally using ultrasonography, whereupon AVFs were harvested 14 days after surgery. VSMCs were isolated from vena cavae to study the effect of vWF on VSMC proliferation. Patient-matched samples of the basilic vein were obtained before brachio-basilic AVF construction and during superficialization or salvage procedure 6 weeks after AVF creation. vWF deficiency reduced VSMC proliferation and macrophage infiltration in the intimal hyperplasia. vWF-/- mice showed reduced outward remodeling (1.5-fold, P=0.002) and intimal hyperplasia (10.2-fold, P<0.0001). AVF flow in wild-type mice was incremental over 2 weeks, whereas flow in vWF-/- mice did not increase, resulting in a two-fold lower flow at 14 days compared with wild-type mice (P=0.016). Outward remodeling in matured patient AVFs coincided with increased local vWF expression in the media of the venous outflow tract. Absence of vWF in the intimal layer correlated with an increase in the intima-media ratio. Conclusions vWF enhances AVF maturation because its positive effect on outward remodeling outweighs its stimulating effect on intimal hyperplasia.
Assuntos
Fístula Arteriovenosa , Derivação Arteriovenosa Cirúrgica , Miócitos de Músculo Liso , Fator de von Willebrand , Animais , Derivação Arteriovenosa Cirúrgica/métodos , Proliferação de Células , Humanos , Hiperplasia , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/citologia , Fator de von Willebrand/metabolismoRESUMO
This study provides a method to assess the impact of circulating plasma factors on microvascular integrity by using a recently developed microvessel-on-a-chip platform featuring the human endothelium that is partly surrounded by the extracellular matrix. The system is high-throughput, which allows parallel analysis of organ-level microvessel pathophysiology, including vascular leakage. Ethylenediaminetetraacetic acid plasma samples are mixed with inhibitors for recalcification of the plasma samples to avoid activation of the coagulation- or complement system. Moreover, the assay is validated by spiking vascular endothelial growth factor, histamine, or tumor necrosis factor alpha to recalcified plasma and confirms their modulation of microvessel barrier function at physiologically relevant concentrations. Finally, this study shows that perfusing the microvessels with recalcified plasma samples of coronavirus disease-2019 patients, with a confirmed proinflammatory profile, results in markedly increased leakage of the microvessels. The assay provides opportunities for diagnostic screening of inflammatory or endothelial disrupting plasma factors associated with endothelial dysfunction.
Assuntos
COVID-19 , Microfluídica , Endotélio Vascular , Humanos , Plasma , SARS-CoV-2 , Fator A de Crescimento do Endotélio VascularRESUMO
We previously found that low shear stress (LSS) induces atherosclerotic plaques in mice with increased lipid and matrix metalloproteinase content and decreased vascular smooth muscle and collagen content. Here, we evaluated the role of chemokines in this process, using an extravascular device inducing regions of LSS, high shear stress, and oscillatory shear stress (OSS) in the carotid artery. One week of shear stress alterations induced expression of IFN-gamma-inducible protein-10 (IP-10) exclusively in the LSS region, whereas monocyte chemoattractant protein-1 (MCP-1) and the mouse homolog of growth-regulated oncogene alpha (GRO-alpha) were equally upregulated in both LSS and OSS regions. After 3 weeks, GRO-alpha and IP-10 were specifically upregulated in LSS regions. After 9 weeks, lesions with thinner fibrous caps and larger necrotic cores were found in the LSS region compared with the OSS region. Equal levels of MCP-1 expression were observed in both regions, while expression of fractalkine was found in the LSS region only. Blockage of fractalkine inhibited plaque growth and resulted in striking differences in plaque composition in the LSS region. We conclude that LSS or OSS triggers expression of chemokines involved in atherogenesis. Fractalkine upregulation is critically important for the composition of LSS-induced atherosclerotic lesions.