RESUMEN
Coronary artery disease (CAD) is a pandemic disease where up to half of the risk is explained by genetic factors. Advanced insights into the genetic basis of CAD require deeper understanding of the contributions of different cell types, molecular pathways, and genes to disease heritability. Here, we investigate the biological diversity of atherosclerosis-associated cell states and interrogate their contribution to the genetic risk of CAD by using single-cell and bulk RNA sequencing (RNA-seq) of mouse and human lesions. We identified 12 disease-associated cell states that we characterized further by gene set functional profiling, ligand-receptor prediction, and transcription factor inference. Importantly, Vcam1+ smooth muscle cell state genes contributed most to SNP-based heritability of CAD. In line with this, genetic variants near smooth muscle cell state genes and regulatory elements explained the largest fraction of CAD-risk variance between individuals. Using this information for variant prioritization, we derived a hybrid polygenic risk score (PRS) that demonstrated improved performance over a classical PRS. Our results provide insights into the biological mechanisms associated with CAD risk, which could make a promising contribution to precision medicine and tailored therapeutic interventions in the future.
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Aterosclerosis , Enfermedad de la Arteria Coronaria , Humanos , Aterosclerosis/genética , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/patología , Factores de Riesgo , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo/métodos , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
BACKGROUND: CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. METHODS: To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. RESULTS: We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. CONCLUSIONS: Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.
Asunto(s)
Proteína Similar al Receptor de Calcitonina , Enfermedad de la Arteria Coronaria , Células Endoteliales , Elementos de Facilitación Genéticos , Polimorfismo de Nucleótido Simple , Estrés Mecánico , Humanos , Células Endoteliales/metabolismo , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Proteína Similar al Receptor de Calcitonina/genética , Proteína Similar al Receptor de Calcitonina/metabolismo , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Mecanotransducción Celular , Células Cultivadas , Regulación de la Expresión Génica , Unión Proteica , Predisposición Genética a la Enfermedad , Sitios de UniónRESUMEN
Diabetes mellitus is one of the leading causes of chronic kidney disease and its progression to end-stage kidney disease (ESKD). Diabetic kidney disease (DKD) is characterized by glomerular hypertrophy, hyperfiltration, inflammation, and the onset of albuminuria, together with a progressive reduction in glomerular filtration rate. This progression is further accompanied by tubulointerstitial inflammation and fibrosis. Factors such as genetic predisposition, epigenetic modifications, metabolic derangements, hemodynamic alterations, inflammation, and inappropriate renin-angiotensin-aldosterone system (RAAS) activity contribute to the onset and progression of DKD. In this context, decades of work have focused on glycemic and blood pressure reduction strategies, especially targeting the RAAS to slow disease progression. Although much of the work has focused on targeting angiotensin II, emerging data support that the mineralocorticoid receptor (MR) is integral in the development and progression of DKD. Molecular mechanisms linked to the underlying pathophysiological changes derived from MR activation include vascular endothelial and epithelial cell responses to oxidative stress and inflammation. These responses lead to alterations in the microcirculatory environment, the abnormal release of extracellular vesicles, gut dysbiosis, epithelial-mesenchymal transition, and kidney fibrosis. Herein, we present recent experimental and clinical evidence on the MR in DKD onset and progress along with new MR-based strategies for the treatment and prevention of DKD.
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Nefropatías Diabéticas , Receptores de Mineralocorticoides , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/fisiopatología , Humanos , Receptores de Mineralocorticoides/metabolismo , Animales , Sistema Renina-Angiotensina , Riñón/metabolismo , Riñón/patología , Antagonistas de Receptores de Mineralocorticoides/uso terapéutico , Antagonistas de Receptores de Mineralocorticoides/farmacología , Transducción de Señal , Progresión de la EnfermedadRESUMEN
INTRODUCTION: Ascending aortic dilatation is a well-known risk factor for aortic rupture. Indications for aortic replacement in its dilatation concomitant to other open-heart surgery exist; however, cut-off values based solely on aortic diameter may fail to identify patients with weakened aortic tissue. We introduce near-infrared spectroscopy (NIRS) as a diagnostic tool to nondestructively evaluate the structural and compositional properties of the human ascending aorta during open-heart surgeries. During open-heart surgery, NIRS could provide information regarding tissue viability in situ and thus contribute to the decision of optimal surgical repair. MATERIALS AND METHODS: Samples were collected from patients with ascending aortic aneurysm (n = 23) undergoing elective aortic reconstruction surgery and from healthy subjects (n = 4). The samples were subjected to spectroscopic measurements, biomechanical testing, and histological analysis. The relationship between the near-infrared spectra and biomechanical and histological properties was investigated by adapting partial least squares regression. RESULTS: Moderate prediction performance was achieved with biomechanical properties (r = 0.681, normalized root-mean-square error of cross-validation = 17.9%) and histological properties (r = 0.602, normalized root-mean-square error of cross-validation = 22.2%). Especially the performance with parameters describing the aorta's ultimate strength, for example, failure strain (r = 0.658), and elasticity (phase difference, r = 0.875) were promising and could, therefore, provide quantitative information on the rupture sensitivity of the aorta. For the estimation of histological properties, the results with α-smooth muscle actin (r = 0.581), elastin density (r = 0.973), mucoid extracellular matrix accumulation(r = 0.708), and media thickness (r = 0.866) were promising. CONCLUSIONS: NIRS could be a potential technique for in situ evaluation of biomechanical and histological properties of human aorta and therefore useful in patient-specific treatment planning.
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Aneurisma de la Aorta , Enfermedades de la Aorta , Humanos , Espectroscopía Infrarroja Corta , Aorta/fisiología , Aneurisma de la Aorta/cirugía , Elasticidad , Fenómenos Biomecánicos/fisiologíaRESUMEN
Smooth muscle cells and endothelial cells have a remarkable level of plasticity in vascular pathologies. In thoracic and abdominal aortic aneurysms, smooth muscle cells have been suggested to undergo phenotypic switching and to contribute to degradation of the aortic wall structure in response to, for example, inflammatory mediators, dysregulation of growth factor signaling or oxidative stress. Recently, endothelial-to-mesenchymal transition, and a clonal expansion of degradative smooth muscle cells and immune cells, as well as mesenchymal stem-like cells have been suggested to contribute to the progression of aortic aneurysms. What are the factors driving the aortic cell phenotype changes and how vascular flow, known to affect aortic wall structure and to be altered in aortic aneurysms, could affect aortic cell remodeling? In this review, we summarize the current literature on aortic cell heterogeneity and phenotypic switching in relation to changes in vascular flow and aortic wall structure in aortic aneurysms in clinical samples with special focus on smooth muscle and endothelial cells. The differences between thoracic and abdominal aortic aneurysms are discussed.
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Aneurisma de la Aorta Abdominal , Aneurisma de la Aorta Torácica , Aneurisma de la Aorta , Aneurisma de la Aorta/metabolismo , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Torácica/patología , Células Endoteliales/metabolismo , Humanos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismoRESUMEN
Super-enhancers are clusters of enhancers associated with cell lineage. They can be powerful gene-regulators and may be useful in cell-type specific viral-vector development. Here, we have screened for endothelial super-enhancers and identified an enhancer from within a cluster that conferred 5-70-fold increase in transgene expression. Importantly, CRISPR/Cas9 deletion of enhancers demonstrated regulation of ADAMTS18, corresponding to evidence of chromatin contacts between these genomic regions. Cell division-related pathways were primarily affected by the enhancer deletions, which correlated with significant reduction in cell proliferation. Furthermore, we observed changes in angiogenesis-related genes consistent with the endothelial specificity of this SE. Indeed, deletion of the enhancers affected tube formation, resulting in reduced or shortened sprouts. The super-enhancer angiogenic role is at least partly due to its regulation of ADAMTS18, as siRNA knockdown of ADAMTS18 resulted in significantly shortened endothelial sprouts. Hence, functional characterization of a novel endothelial super-enhancer has revealed substantial downstream effects from single enhancer deletions and led to the discovery of the cis-target gene ADAMTS18 and its role in endothelial function.
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Proteínas ADAMTS/genética , Cromatina/genética , Elementos de Facilitación Genéticos/genética , Neovascularización Fisiológica/genética , Sistemas CRISPR-Cas/genética , División Celular/genética , Linaje de la Célula/genética , Células Endoteliales/metabolismo , Humanos , ARN Interferente Pequeño/genética , Transducción de Señal/genéticaRESUMEN
[Figure: see text].
Asunto(s)
Aorta/metabolismo , Enfermedades de la Aorta/genética , Aterosclerosis/genética , Perfilación de la Expresión Génica , MicroARNs/genética , Transcriptoma , Aorta/patología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Macrófagos/metabolismo , Macrófagos/patología , MicroARNs/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Placa Aterosclerótica , RNA-SeqRESUMEN
The BMP/TGFß-Smad, Notch and VEGF signaling guides formation of endothelial tip and stalk cells. However, the crosstalk of bone morphogenetic proteins (BMPs) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling has remained largely unknown. We demonstrate that BMP family members regulate VEGFR2 and Notch signaling, and act via TAZ-Hippo signaling pathway. BMPs were found to be regulated after VEGF gene transfer in C57/Bl6 mice and in a porcine myocardial ischemia model. BMPs 2/4/6 were identified as endothelium-specific targets of VEGF. BMP2 modulated VEGF-mediated endothelial sprouting via Delta like Canonical Notch Ligand 4 (DLL4). BMP6 modulated VEGF signaling by regulating VEGFR2 expression and acted via Hippo signaling effector TAZ, known to regulate cell survival/proliferation, and to be dysregulated in cancer. In a matrigel plug assay in nude mice BMP6 was further demonstrated to induce angiogenesis. BMP6 is the first member of BMP family found to directly regulate both Hippo signaling and neovessel formation. It may thus serve as a target in pro/anti-angiogenic therapies.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Morfogenética Ósea 6/metabolismo , Células Endoteliales/metabolismo , Neovascularización Fisiológica , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 2/metabolismo , Hipoxia de la Célula , Núcleo Celular/metabolismo , Vía de Señalización Hippo , Humanos , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Transporte de Proteínas , Porcinos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: Previous studies have demonstrated that the expression of several lysine (K)-specific demethylases (KDMs) is induced by hypoxia. Here, we sought to investigate the exact mechanisms underlying this regulation and its functional implications for endothelial cell function, such as angiogenesis. Approach and Results: We analyzed the expression changes of KDMs under hypoxia and modulation of HIF (hypoxia-inducible factor) expression using GRO-Seq and RNA-Seq in endothelial cells. We provide evidence that the majority of the KDMs are induced at the level of nascent transcription mediated by the action of HIF-1α and HIF-2α. Importantly, we show that transcriptional changes at the level of initiation represent the major mechanism of gene activation. To delineate the epigenetic effects of hypoxia and HIF activation in normoxia, we analyzed the genome-wide changes of H3K27me3 using chromosome immunoprecipitation-Seq. We discovered a redistribution of H3K27me3 at ≈2000 to 3000 transcriptionally active loci nearby genes implicated in angiogenesis. Among these, we demonstrate that vascular endothelial growth factor A (VEGFA) expression is partly induced by KDM4B- and KDM6B-mediated demethylation of nearby regions. Knockdown of KDM4B and KDM6B decreased cell proliferation, tube formation, and endothelial sprouting while affecting hundreds of genes associated with angiogenesis. These findings provide novel insights into the regulation of KDMs by hypoxia and the epigenetic regulation of VEGFA-mediated angiogenesis. CONCLUSIONS: Our study describes an additional level of epigenetic regulation where hypoxia induces redistribution of H3K27me3 around genes implicated in proliferation and angiogenesis. More specifically, we demonstrate that KDM4B and KDM6B play a key role in modulating the expression of the major angiogenic driver VEGFA.
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Histona Demetilasas/metabolismo , Histonas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/enzimología , Neovascularización Fisiológica , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipoxia de la Célula , Células Cultivadas , Desmetilación , Histona Demetilasas/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Brain arteriovenous malformations (bAVM) may rupture causing disability or death. BAVM vessels are characterized by abnormally high flow that in general triggers expansive vessel remodeling mediated by cyclo-oxygenase-2 (COX2), the target of non-steroidal anti-inflammatory drugs. We investigated whether COX2 is expressed in bAVMs and whether it associates with inflammation and haemorrhage in these lesions. METHODS: Tissue was obtained from surgery of 139 bAVMs and 21 normal Circle of Willis samples. The samples were studied with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR). Clinical data was collected from patient records. RESULTS: COX2 expression was found in 78% (109/139) of the bAVMs and localized to the vessels' lumen or medial layer in 70% (95/135) of the bAVMs. Receptors for prostaglandin E2, a COX2-derived mediator of vascular remodeling, were found in the endothelial and smooth muscle cells and perivascular inflammatory cells of bAVMs. COX2 was expressed by infiltrating inflammatory cells and correlated with the extent of inflammation (r = .231, p = .007, Spearman rank correlation). COX2 expression did not associate with haemorrhage. CONCLUSION: COX2 is induced in bAVMs, and possibly participates in the regulation of vessel wall remodelling and ongoing inflammation. Role of COX2 signalling in the pathobiology and clinical course of bAVMs merits further studies.
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Encéfalo/metabolismo , Ciclooxigenasa 2 , Malformaciones Arteriovenosas Intracraneales , Remodelación Vascular , Encéfalo/patología , Ciclooxigenasa 2/genética , Humanos , Inflamación , Malformaciones Arteriovenosas Intracraneales/metabolismoRESUMEN
AIMS: Histamine and vascular endothelial growth factor A (VEGF) are central regulators in vascular pathologies. Their gene regulation leading to vascular remodeling has remained obscure. In this study, EC regulation mechanisms of histamine and VEGF were compared by RNA sequencing of primary endothelial cells (ECs), functional in vitro assays and in vivo permeability mice model. METHODS AND RESULTS: By RNA sequencing, similar transcriptional alterations of genes involved in activation of primary ECs, cell proliferation and adhesion were observed between histamine and VEGF. Seventy-six commonly regulated genes were found, representing ~53% of all VEGF-regulated transcripts and ~26% of all histamine-regulated transcripts. Both factors regulated tight junction formation and expression of pro-angiogenic transcription factors (TFs) affecting EC survival, migration and tube formation. Novel claudin-5 upstream regulatory genes were identified. VEGF was demonstrated to regulate expression of SNAI2, whereas pro-angiogenic TFs NR4A1, MYCN and RCAN1 were regulated by both histamine and VEGF. Claudin-5 was shown to be regulated VEGFR2/PI3K-Akt dependently by VEGF and PI3K-Akt independently by histamine. Interleukin-8 was shown to downregulate claudin-5 by histamine. Additionally, SNAI2, NR4A1 and MYCN were shown to mediate EC survival, migration and tube formation and to regulate expression of claudin-5. Further systemic delivery of VEGF and histamine was shown to induce a fast vascular hyperpermeability response in intact vasculature of C57/Bl6 mice followed by regulation of NR4A1 and MYCN. CONCLUSIONS: Our study identifies novel claudin-5 upstream regulatory genes of histamine and VEGF that induce cellular angiogenic processes. Our results increase knowledge of angiogenic EC phenotype and provide novel treatment targets for vascular pathologies.
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Claudina-5/metabolismo , Histamina/farmacología , Interleucina-8/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Permeabilidad Capilar/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Claudina-5/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Ontología de Genes , Factor de Crecimiento de Hepatocito/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Neovascularización Fisiológica/genética , Especificidad de Órganos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Factores de Transcripción/metabolismo , Transcriptoma , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Baculoviruses are insect-specific viruses commonly found in nature. They are not able to replicate in mammalian cells but can transduce them when equipped with an appropriate mammalian cell active expression cassette. Although the viruses have been studied in several types of mammalian cells from different origins, the receptor that baculovirus uses to enter or interact with mammalian cells has not yet been identified. Due to the wide tropism of the virus, the receptor has been suggested to be a generally found cell surface molecule. In this article, we investigated the interaction of baculovirus and mammalian cell surface heparan sulfate proteoglycans (HSPG) in more detail. Our data show that baculovirus requires HSPG sulfation, particularly N- and 6-O-sulfation, to bind to and transduce mammalian cells. According to our results, baculovirus binds specifically to syndecan-1 (SDC-1) but does not interact with SDC-2 to SDC-4 or with glypicans. Competition experiments performed with SDC-1 antibody or recombinant SDC-1 protein inhibited baculovirus binding, and SDC-1 overexpression enhanced baculovirus-mediated transduction. In conclusion, we show that SDC-1, a commonly found cell surface HSPG molecule, has a role in the binding and entry of baculovirus in vertebrate cells. The results presented here reveal important aspects of baculovirus entry and can serve as a basis for next-generation baculovirus vector development for gene delivery.
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Baculoviridae/fisiología , Receptores Virales/metabolismo , Sindecano-1/metabolismo , Acoplamiento Viral , Internalización del Virus , Línea Celular , Humanos , Sulfatos/metabolismo , Transducción GenéticaRESUMEN
Some cell types are more susceptible to viral gene transfer or virus infection than others, irrespective of the number of viral receptors or virus binding efficacy on their surfaces. In order to characterize the cell-line-specific features contributing to efficient virus entry, we studied two cell lines (Ea.hy926 and MG-63) that are nearly nonpermissive to insect-specific baculovirus (BV) and the human enterovirus echovirus 1 (EV1) and compared their characteristics with those of a highly permissive (HepG2) cell line. All the cell lines contained high levels of viral receptors on their surfaces, and virus binding was shown to be efficient. However, in nonpermissive cells, BV and its receptor, syndecan 1, were unable to internalize in the cells and formed large aggregates near the cell surface. Accordingly, EV1 had a low infection rate in nonpermissive cells but was still able to internalize the cells, suggesting that the postinternalization step of the virus was impaired. The nonpermissive and permissive cell lines showed differential expression of syntenin, filamentous actin, vimentin, and phosphorylated protein kinase C subtype α (pPKCα). The nonpermissive nature of the cells could be modulated by the choice of culture medium. RPMI medium could partially rescue infection/transduction and concomitantly showed lower syntenin expression, a modified vimentin network, and altered activities of PKC subtypes PKCα and PKCε. The observed changes in PKCα and PKCε activation caused alterations in the vimentin organization, leading to efficient BV transduction and EV1 infection. This study identifies PKCα, PKCε, and vimentin as key factors affecting efficient infection and transduction by EV1 and BV, respectively.
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Enterovirus Humano B/patogenicidad , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Vimentina/metabolismo , Animales , Baculoviridae/genética , Baculoviridae/patogenicidad , Baculoviridae/fisiología , Línea Celular , Medios de Cultivo , Enterovirus Humano B/fisiología , Células HEK293 , Células Hep G2 , Interacciones Huésped-Patógeno , Humanos , Integrina alfa2beta1/metabolismo , Ratones , Modelos Biológicos , Fosforilación , Receptores Virales/metabolismo , Sindecano-1/metabolismo , Transducción Genética , Virulencia , Internalización del VirusRESUMEN
BACKGROUND: The vascular endothelium serves as a semi-selective permeable barrier as a conduit for transport of fluid, solutes, and various cell populations between the vessel lumen and tissues. The endothelium thus has a dynamic role in the regulation of coagulation, immune system, lipid and electrolyte transport, as well as neurohumoral influences on vascular tone and end-organ injury to tissues such as the heart and kidney. SUMMARY: Within this framework, pharmacologic strategies for heart and kidney diseases including blood pressure, glycemic control, and lipid reduction provide significant risk reduction, yet certain populations are at risk for substantial residual risk for disease progression and treatment resistance and often have unwanted off-target effects leaving the need for adjunct, alternative targeted therapies. Recent advances in techniques in sequencing and spatial transcriptomics have paved the way for the development of new therapies for targeting heart and kidney disease that include various gene, cell, and nano-based therapies. Cell-specific endothelium-specific targeting of viral vectors will enable their use for the treatment of heart and kidney diseases with gene therapy that can avoid unwanted off-target effects, improve treatment resistance, and reduce residual risk for disease progression. KEY MESSAGES: The vascular endothelium is an important therapeutic target for chronic kidney and cardiovascular diseases. Developing endothelial-specific gene therapies can benefit patients who develop resistance to current treatments.
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Síndrome Cardiorrenal , Endotelio Vascular , Humanos , Síndrome Cardiorrenal/fisiopatología , Síndrome Cardiorrenal/metabolismo , Endotelio Vascular/fisiopatología , Endotelio Vascular/metabolismo , Terapia Genética/métodos , AnimalesRESUMEN
BACKGROUND: Cerebral cavernous malformation (CCM) is a disease associated with an elevated risk of focal neurological deficits, seizures, and hemorrhagic stroke. The disease has an inflammatory profile and improved knowledge of CCM pathology mechanisms and exploration of candidate biomarkers will enable new non-invasive treatments. METHODS: We analyzed protein signatures in human CCM tissue samples by using a highly specific and sensitive multiplexing technique, proximity extension assay. FINDINGS: Data analysis revealed CCM specific proteins involved in endothelial dysfunction/inflammation/activation, leukocyte infiltration/chemotaxis, hemostasis, extracellular matrix dysfunction, astrocyte and microglial cell activation. Biomarker expression profiles matched bleeding status, especially with higher levels of inflammatory markers and activated astrocytes in ruptured than non-ruptured samples, some of these biomarkers are secreted into blood or urine. Furthermore, analysis was also done in a spatially resolving manner by separating the lesion area from the surrounding brain tissue. Our spatial studies revealed that although appearing histologically normal, the CCM border areas were pathological when compared to control brain tissues. Moreover, the functional relevance of CD93, ICAM-1 and MMP9, markers related to endothelial cell activation and extracellular matrix was validated by a murine pre-clinical CCM model. INTERPRETATION: Here we present a novel strategy for proteomics analysis on human CCMs, offering a possibility for high-throughput protein screening acquiring data on the local environment in the brain. Our data presented here describe CCM relevant brain proteins and specifically those which are secreted can serve the need of circulating CCM biomarkers to predict cavernoma's risk of bleeding.
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Biomarcadores , Hemangioma Cavernoso del Sistema Nervioso Central , Molécula 1 de Adhesión Intercelular , Proteómica , Humanos , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Proteómica/métodos , Biomarcadores/metabolismo , Biomarcadores/análisis , Animales , Ratones , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Femenino , Adulto , Persona de Mediana Edad , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de la Membrana , Proteínas Proto-Oncogénicas , Proteínas Reguladoras de la ApoptosisRESUMEN
AIMS: Vascular smooth muscle cells (SMCs) and their derivatives are key contributors to the development of atherosclerosis. However, studying changes in SMC gene expression in heterogeneous vascular tissues is challenging due to the technical limitations and high cost associated with current approaches. In this paper, we apply translating ribosome affinity purification sequencing to profile SMC-specific gene expression directly from tissue. METHODS AND RESULTS: To facilitate SMC-specific translatome analysis, we generated SMCTRAP mice, a transgenic mouse line expressing enhanced green fluorescent protein (EGFP)-tagged ribosomal protein L10a (EGFP-L10a) under the control of the SMC-specific αSMA promoter. These mice were further crossed with the atherosclerosis model Ldlr-/-, ApoB100/100 to generate SMCTRAP-AS mice and used to profile atherosclerosis-associated SMCs in thoracic aorta samples of 15-month-old SMCTRAP and SMCTRAP-AS mice. Our analysis of SMCTRAP-AS mice showed that EGFP-L10a expression was localized to SMCs in various tissues, including the aortic wall and plaque. The TRAP fraction demonstrated high enrichment of known SMC-specific genes, confirming the specificity of our approach. We identified several genes, including Cemip, Lum, Mfge8, Spp1, and Serpina3, which are known to be involved in atherosclerosis-induced gene expression. Moreover, we identified several novel genes not previously linked to SMCs in atherosclerosis, such as Anxa4, Cd276, inter-alpha-trypsin inhibitor-4 (Itih4), Myof, Pcdh11x, Rab31, Serpinb6b, Slc35e4, Slc8a3, and Spink5. Among them, we confirmed the SMC-specific expression of Itih4 in atherosclerotic lesions using immunofluorescence staining of mouse aortic roots and spatial transcriptomics of human carotid arteries. Furthermore, our more detailed analysis of Itih4 showed its link to coronary artery disease through the colocalization of genome-wide association studies, splice quantitative trait loci (QTL), and protein QTL signals. CONCLUSION: We generated a SMC-specific TRAP mouse line to study atherosclerosis and identified Itih4 as a novel SMC-expressed gene in atherosclerotic plaques, warranting further investigation of its putative function in extracellular matrix stability and genetic evidence of causality.
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Enfermedades de la Aorta , Aterosclerosis , Modelos Animales de Enfermedad , Músculo Liso Vascular , Miocitos del Músculo Liso , Placa Aterosclerótica , Proteínas Ribosómicas , Animales , Femenino , Humanos , Masculino , Ratones , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/metabolismo , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Fenotipo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , TranscriptomaRESUMEN
This study aimed to show the significance of capillary function in post-ischemic recovery from the perspective of physiological parameters, such as blood flow, hemoglobin oxygenation and tissue regeneration. Muscle-level microvascular alterations of blood flow and hemoglobin oxygenation, and post-ischemic myofiber and capillary responses were analyzed in aged, healthy C57Bl/6J mice (n = 48) and aged, hyperlipidemic LDLR-/-ApoB100/100 mice (n = 69) after the induction of acute hindlimb ischemia using contrast ultrasound, photoacoustic imaging and histological analyses, respectively. The capillary responses that led to successful post-ischemic muscle repair in C57Bl/6J mice included an early capillary dilation phase, preceding the return of arterial driving pressure, followed by an increase in capillary density that further supported satellite cell-induced muscle regeneration. Initial capillary enlargement was absent in the LDLR-/-ApoB100/100 mice with lifelong moderate hypercholesterolemia and led to an inability to recover arterial driving pressure, with a resulting increase in distal necrosis, chronic tissue damage and a delay in the overall recovery after ischemia. To conclude, this manuscript highlights, beyond arterial collateralization, the importance of the proper function of the capillary endothelium in post-ischemic recovery and displays how post-ischemic capillary dynamics associate beyond tissue blood flow to both hemoglobin oxygenation and tissue regeneration.
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Arterias , Isquemia , Animales , Ratones , Endotelio Vascular , Ratones Endogámicos C57BL , Músculos , Miembro PosteriorRESUMEN
Background: Sporadic venous malformation (VM) and angiomatosis of soft tissue (AST) are benign, congenital vascular anomalies affecting venous vasculature. Depending on the size and location of the lesion, symptoms vary from motility disturbances to pain and disfigurement. Due to the high recurrence of the lesions, more effective therapies are needed. Methods: As targeting stromal cells has been an emerging concept in anti-angiogenic therapies, here, by using VM/AST patient samples, RNA-sequencing, cell culture techniques, and a xenograft mouse model, we investigated the crosstalk of endothelial cells (EC) and fibroblasts and its effect on vascular lesion growth. Results: We report, for the first time, the expression and secretion of transforming growth factor A (TGFA) in ECs or intervascular stromal cells in AST and VM lesions. TGFA induced secretion of vascular endothelial growth factor (VEGF-A) in paracrine fashion, and regulated EC proliferation. Oncogenic PIK3CA variant in p.H1047R, a common somatic mutation found in these lesions, increased TGFA expression, enrichment of hallmark hypoxia, and in a mouse xenograft model, lesion size, and vascularization. Treatment with afatinib, a pan-ErbB tyrosine-kinase inhibitor, decreased vascularization and lesion size in a mouse xenograft model with ECs expressing oncogenic PIK3CA p.H1047R variant and fibroblasts. Conclusions: Based on the data, we suggest that targeting of both intervascular stromal cells and ECs is a potential treatment strategy for vascular lesions having a fibrous component. Funding: Academy of Finland, Ella and Georg Ehnrooth foundation, the ERC grants, Sigrid Jusélius Foundation, Finnish Foundation for Cardiovascular Research, Jane and Aatos Erkko Foundation, GeneCellNano Flagship program, and Department of Musculoskeletal and Plastic Surgery, Helsinki University Hospital.
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Células Endoteliales , Malformaciones Vasculares , Humanos , Ratones , Animales , Células Endoteliales/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Transducción de Señal , Inhibidores de Proteínas Quinasas/farmacología , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Malformaciones Vasculares/tratamiento farmacológico , Malformaciones Vasculares/genética , Malformaciones Vasculares/patologíaRESUMEN
Virus-induced alterations in cell morphology play important roles in the viral life cycle. To examine the intracellular events of coxsackievirus B3 (CVB3) infection, green monkey kidney (GMK) cells were either inoculated with the virus or transfected with the viral RNA. Various microscopic and flow cytometric approaches demonstrated the emergence of CVB3 capsid proteins at 8 h posttransfection, followed by morphological transformation of the cells. The morphological changes included formation of membranous protrusions containing viral capsids, together with microtubules and actin. Translocation of viral capsids into these protrusions was sensitive to cytochalasin D, suggesting the importance of actin in the process. Three-dimensional (3D) live-cell imaging demonstrated frequent contacts between cellular protrusions and adjacent cells. Markedly, in spite of an increase in the cellular viral protein content starting 8 h postinfection, no significant decrease in cell viability or increase in the amount of early apoptotic markers was observed by flow cytometry by 28 h postinfection. Comicroinjection of viral RNA and fluorescent dextran in the presence of neutralizing virus antibody suggested that these protrusions mediated the spread of infection from one cell to another prior to virus-induced cell lysis. Altogether, the CVB3-induced cellular protrusions could function as a hitherto-unknown nonlytic mechanism of cell-to-cell transmission exploited by enteroviruses.
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Proteínas de la Cápside/metabolismo , Enterovirus Humano B/patogenicidad , Infecciones por Enterovirus/transmisión , Infecciones por Enterovirus/virología , Riñón/ultraestructura , Riñón/virología , Proteínas Virales/metabolismo , Animales , Línea Celular , Enterovirus Humano B/fisiología , Enterovirus Humano B/efectos de la radiación , Humanos , Imagenología Tridimensional , Riñón/citología , Microscopía ElectrónicaRESUMEN
Objectives: In thoracic aortic aneurysm (TAA) of the ascending aorta (AA), AA is progressively dilating due to the weakening of the aortic wall. Predicting and preventing aortic dissections and ruptures in TAA continues to be challenging, and more accurate assessment of the AA dilatation, identification of high-risk patients, and timing of repair surgery are required. We investigated whether wall shear stress (WSS) predicts pathological and biomechanical changes in the aortic wall in TAA. Methods: The study included 12 patients with bicuspid (BAV) and 20 patients with the tricuspid aortic valve (TAV). 4D flow magnetic resonance imaging (MRI) was performed a day before aortic replacement surgery. Biomechanical and histological parameters, including assessing of wall strength, media degeneration, elastin, and cell content were analyzed from the resected AA samples. Results: WSSs were greater in the outer curves of the AA compared to the inner curves in all TAA patients. WSSs correlated with media degeneration of the aortic wall (ρ = -0.48, p < 0.01), elastin content (ρ = 0.47, p < 0.01), and aortic wall strength (ρ = -0.49, p = 0.029). Subsequently, the media of the outer curves was thinner, more rigid, and tolerated lower failure strains. Failure values were shown to correlate with smooth muscle cell (SMC) density (ρ = -0.45, p < 0.02), and indicated the more MYH10+ SMCs the lower the strength of the aortic wall structure. More macrophages were detected in patients with severe media degeneration and the areas with lower WSSs. Conclusion: The findings indicate that MRI-derived WSS predicts pathological and biomechanical changes in the aortic wall in patients with TAA and could be used for identification of high-risk patients.