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1.
Int J Mol Sci ; 22(9)2021 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-33946583

RESUMEN

Molecular imaging of pathologic lesions can improve efficient detection of cancer and cardiovascular diseases. A shared pathophysiological feature is angiogenesis, the formation of new blood vessels. Endoglin (CD105) is a coreceptor for ligands of the Transforming Growth Factor-ß (TGF-ß) family and is highly expressed on angiogenic endothelial cells. Therefore, endoglin-based imaging has been explored to visualize lesions of the aforementioned diseases. This systematic review highlights the progress in endoglin-based imaging of cancer, atherosclerosis, myocardial infarction, and aortic aneurysm, focusing on positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), near-infrared fluorescence (NIRF) imaging, and ultrasound imaging. PubMed was searched combining the following subjects and their respective synonyms or relevant subterms: "Endoglin", "Imaging/Image-guided surgery". In total, 59 papers were found eligible to be included: 58 reporting about preclinical animal or in vitro models and one ex vivo study in human organs. In addition to exact data extraction of imaging modality type, tumor or cardiovascular disease model, and tracer (class), outcomes were described via a narrative synthesis. Collectively, the data identify endoglin as a suitable target for intraoperative and diagnostic imaging of the neovasculature in tumors, whereas for cardiovascular diseases, the evidence remains scarce but promising.


Asunto(s)
Enfermedades Cardiovasculares/diagnóstico por imagen , Endoglina/análisis , Neoplasias/diagnóstico por imagen , Animales , Enfermedades Cardiovasculares/cirugía , Humanos , Imagen por Resonancia Magnética/métodos , Neoplasias/cirugía , Imagen Óptica/métodos , Tomografía de Emisión de Positrones/métodos , Cirugía Asistida por Computador/métodos , Tomografía Computarizada de Emisión de Fotón Único/métodos , Ultrasonografía/métodos
2.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-33670533

RESUMEN

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a severe vascular disorder caused by mutations in the TGFß/BMP co-receptor endoglin. Endoglin haploinsufficiency results in vascular malformations and impaired neoangiogenesis. Furthermore, HHT1 patients display an impaired immune response. To date it is not fully understood how endoglin haploinsufficient immune cells contribute to HHT1 pathology. Therefore, we investigated the immune response during tissue repair in Eng+/- mice, a model for HHT1. Eng+/- mice exhibited prolonged infiltration of macrophages after experimentally induced myocardial infarction. Moreover, there was an increased number of inflammatory M1-like macrophages (Ly6Chigh/CD206-) at the expense of reparative M2-like macrophages (Ly6Clow/CD206+). Interestingly, HHT1 patients also showed an increased number of inflammatory macrophages. In vitro analysis revealed that TGFß-induced differentiation of Eng+/- monocytes into M2-like macrophages was blunted. Inhibiting BMP signaling by treating monocytes with LDN-193189 normalized their differentiation. Finally, LDN treatment improved heart function after MI and enhanced vascularization in both wild type and Eng+/- mice. The beneficial effect of LDN was also observed in the hind limb ischemia model. While blood flow recovery was hampered in vehicle-treated animals, LDN treatment improved tissue perfusion recovery in Eng+/- mice. In conclusion, BMPR kinase inhibition restored HHT1 macrophage imbalance in vitro and improved tissue repair after ischemic injury in Eng+/- mice.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Modelos Animales de Enfermedad , Endoglina/metabolismo , Infarto del Miocardio/prevención & control , Pirazoles/farmacología , Pirimidinas/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Receptores de Proteínas Morfogenéticas Óseas/genética , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Células Cultivadas , Endoglina/genética , Femenino , Heterocigoto , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Telangiectasia Hemorrágica Hereditaria/genética , Telangiectasia Hemorrágica Hereditaria/inmunología , Telangiectasia Hemorrágica Hereditaria/metabolismo , Cicatrización de Heridas/genética
3.
Molecules ; 24(1)2019 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-30621310

RESUMEN

Sustained pacemaker function is a challenge in biological pacemaker engineering. Human cardiomyocyte progenitor cells (CMPCs) have exhibited extended survival in the heart after transplantation. We studied whether lentivirally transduced CMPCs that express the pacemaker current If (encoded by HCN4) can be used as functional gene delivery vehicle in biological pacing. Human CMPCs were isolated from fetal hearts using magnetic beads coated with Sca-1 antibody, cultured in nondifferentiating conditions, and transduced with a green fluorescent protein (GFP)- or HCN4-GFP-expressing lentivirus. A patch-clamp analysis showed a large hyperpolarization-activated, time-dependent inward current (-20 pA/pF at -140 mV, n = 14) with properties typical of If in HCN4-GFP-expressing CMPCs. Gap-junctional coupling between CMPCs and neonatal rat ventricular myocytes (NRVMs) was demonstrated by efficient dye transfer and changes in spontaneous beating activity. In organ explant cultures, the number of preparations showing spontaneous beating activity increased from 6.3% in CMPC/GFP-injected preparations to 68.2% in CMPC/HCN4-GFP-injected preparations (P < 0.05). Furthermore, in CMPC/HCN4-GFP-injected preparations, isoproterenol induced a significant reduction in cycle lengths from 648 ± 169 to 392 ± 71 ms (P < 0.05). In sum, CMPCs expressing HCN4-GFP functionally couple to NRVMs and induce physiologically controlled pacemaker activity and may therefore provide an attractive delivery platform for sustained pacemaker function.


Asunto(s)
Técnicas de Transferencia de Gen , Ventrículos Cardíacos/trasplante , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Proteínas Musculares/genética , Miocitos Cardíacos/trasplante , Canales de Potasio/genética , Células Madre/citología , Animales , Terapia Genética/métodos , Proteínas Fluorescentes Verdes/química , Ventrículos Cardíacos/patología , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/uso terapéutico , Proteínas Musculares/uso terapéutico , Técnicas de Placa-Clamp , Canales de Potasio/uso terapéutico , Ratas , Trasplante de Células Madre
4.
Haematologica ; 103(6): 1073-1082, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29545340

RESUMEN

Tissue Factor is a cell-surface glycoprotein expressed in various cells of the vasculature and is the principal regulator of the blood coagulation cascade and hemostasis. Notably, aberrant expression of Tissue Factor is associated with cardiovascular pathologies such as atherosclerosis and thrombosis. Here, we sought to identify factors that regulate Tissue Factor gene expression and activity. Tissue Factor gene expression is regulated by various transcription factors, including activating protein-1 and nuclear factor-κ B. The peptidyl-prolyl isomerase Pin1 is known to modulate the activity of these two transcription factors, and we now show that Pin1 augments Tissue Factor gene expression in both vascular smooth muscle cells and activated endothelial cells via activating protein-1 and nuclear factor-κ B signaling. Furthermore, the cytoplasmic domain of Tissue Factor contains a well-conserved phospho-Ser258-Pro259 amino-acid motif recognized by Pin1. Using co-immunoprecipitation and solution nuclear magnetic resonance spectroscopy, we show that the WW-domain of Pin1 directly binds the cytoplasmic domain of Tissue Factor. This interaction occurs via the phospho-Ser258-Pro259 sequence in the Tissue Factor cytoplasmic domain and results in increased protein half-life and pro-coagulant activity. Taken together, our results establish Pin1 as an upstream regulator of Tissue Factor-mediated coagulation, thereby opening up new avenues for research into the use of specific Pin1 inhibitors for the treatment of diseases characterized by pathological coagulation, such as thrombosis and atherosclerosis.


Asunto(s)
Coagulantes/metabolismo , Expresión Génica , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Tromboplastina/genética , Tromboplastina/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Complejos Multiproteicos/metabolismo , FN-kappa B/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Tromboplastina/química , Factor de Transcripción AP-1/metabolismo
6.
J Mol Cell Cardiol ; 87: 79-91, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26278995

RESUMEN

Cardiomyocyte progenitor cells (CMPCs) are a candidate cell source for cardiac regenerative therapy. However, like other stem cells, after transplantation in the heart, cell retention and differentiation capacity of the CMPCs are low. Combining cells with biomaterials might overcome this problem. By serving as a (temporal) environment, the biomaterial can retain the cells and provide signals that enhance survival, proliferation and differentiation of the cells. To gain more insight into the effect that the encapsulation of CMPCs in a biomaterial has on their behavior, we cultured CMPCs in unidirectional constrained and stress-free collagen/Matrigel hydrogels. CMPCs cultured in 3D hydrogels stay viable and keep their cardiomyogenic profile independent of the application of strain. Moreover, the increased expression of Nkx2.5, myocardin and cTnT in 3D hydrogels compared to 2D cultures, suggests enhanced cardiomyogenic differentiation capacity of cells in 3D. Furthermore, increased expression of collagen I, collagen III, elastin and fibronectin and of the matrix remodeling enzymes MMP-1, MMP-2, MMP-9, and TIMP-1 and TIMP-2 in the 3D hydrogels is indicative of an enhanced matrix remodeling capacity of CMPCs in a 3D environment, independent of the application of strain. Interestingly, the additional application of static strain to the 3D hydrogels, as imposed by hydrogel constrainment, stabilized CMPC viability and proliferation, resulted in enhanced cardiac marker protein expression and appeared crucial for cellular organization and morphology. More specifically, CMPCs cultured in 3D collagen/Matrigel constrained hydrogels became readily mechanosensitive, had a rod-shaped morphology, and responded to the applied strain by orienting in the direction of the constraint. Overall, our data demonstrate the applicability of CMPCs in a 3D environment since encapsulation of CMPCs may stabilize survival and proliferation, can enhance the differentiation and remodeling capacity of the cells, and could induce cellular re-organization, which all may contribute to an improved efficiency of cardiac stem cell therapy.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Miocitos Cardíacos/trasplante , Medicina Regenerativa , Trasplante de Células Madre , Animales , Materiales Biocompatibles/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Colágeno/farmacología , Combinación de Medicamentos , Fibronectinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Laminina/farmacología , Metaloproteinasas de la Matriz/biosíntesis , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Proteoglicanos/farmacología , Células Madre/citología , Células Madre/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Inhibidor Tisular de Metaloproteinasa-2/biosíntesis
7.
Circ Res ; 108(9): 1093-101, 2011 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-21393577

RESUMEN

RATIONALE: Primary cilia are cellular protrusions that serve as mechanosensors for fluid flow. In endothelial cells (ECs), they function by transducing local blood flow information into functional responses, such as nitric oxide production and initiation of gene expression. Cilia are present on ECs in areas of low or disturbed flow and absent in areas of high flow. In the embryonic heart, high-flow regime applies to the endocardial cushion area, and the absence of cilia here coincides with the process of endothelial-to-mesenchymal transition (EndoMT). OBJECTIVE: In this study, we investigated the role of the primary cilium in defining the responses of ECs to fluid shear stress and in EndoMT. METHODS AND RESULTS: Nonciliated mouse embryonic ECs with a mutation in Tg737/Ift88 were used to compare the response to fluid shear stress to that of ciliated ECs. In vitro, nonciliated ECs undergo shear-induced EndoMT, which is accompanied by downregulation of Klf4. This Tgfß/Alk5-dependent transformation is prevented by blocking Tgfß signaling, overexpression of Klf4, or rescue of the primary cilium. In the hearts of Tg737(orpk/orpk) embryos, Tgfß/Alk5 signaling was activated in areas in which ECs would normally be ciliated but now lack cilia because of the mutation. In these areas, ECs show increased Smad2 phosphorylation and expression of α-smooth muscle actin. CONCLUSIONS: This study demonstrates the central role of primary cilia in rendering ECs prone to shear-induced activation of Tgfß/Alk5 signaling and EndoMT and thereby provides a functional link between primary cilia and flow-related endothelial performance.


Asunto(s)
Cilios/patología , Cilios/fisiología , Transición Epitelial-Mesenquimal/fisiología , Mecanotransducción Celular/fisiología , Animales , Diferenciación Celular/fisiología , Células Endoteliales/patología , Células Endoteliales/fisiología , Regulación de la Expresión Génica/fisiología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Mesodermo/patología , Mesodermo/fisiología , Ratones , Ratones Mutantes , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteína Smad2/metabolismo , Estrés Mecánico , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo
8.
iScience ; 26(4): 106423, 2023 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-37035009

RESUMEN

Environmental stiffness is a crucial determinant of cell function. There is a long-standing quest for reproducible and (human matrix) bio-mimicking biomaterials with controllable mechanical properties to unravel the relationship between stiffness and cell behavior. Here, we evaluate methacrylated human recombinant collagen peptide (RCPhC1-MA) hydrogels as a matrix to control 3D microenvironmental stiffness and monitor cardiac cell response. We show that RCPhC1-MA can form hydrogels with reproducible stiffness in the range of human developmental and adult myocardium. Cardiomyocytes (hPSC-CMs) and cardiac fibroblasts (cFBs) remain viable for up to 14 days inside RCPhC1-MA hydrogels while the effect of hydrogel stiffness on extracellular matrix production and hPSC-CM contractility can be monitored in real-time. Interestingly, whereas the beating behavior of the hPSC-CM monocultures is affected by environmental stiffness, this effect ceases when cFBs are present. Together, we demonstrate RCPhC1-MA to be a promising candidate to mimic and control the 3D biomechanical environment of cardiac cells.

9.
Dev Dyn ; 240(7): 1670-80, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21604321

RESUMEN

Endothelial cells (EC) translate biomechanical forces into functional and phenotypic responses that play important roles in cardiac development. Specifically, EC in areas of high shear stress, i.e., in the cardiac outflow tract and atrioventricular canal, are characterized by high expression of Krüppel-like factor 2 (Klf2) and by transforming growth factor-beta (Tgfß)-driven endothelial-to-mesenchymal transition. Extraembryonic venous obstruction (venous clip model) results in congenital heart malformations, and venous clip-induced alterations in shear stress-related gene expression are suggestive for an increase in cardiac shear stress. Here, we study the effects of shear stress on Klf2 expression and Tgfß-associated signaling in embryonic EC in vivo using the venous clip model and in vitro by subjecting cultured EC to fluid flow. Cellular responses were assessed by analysis of Klf2, Tgfß ligands, and their downstream signaling targets. Results show that, in embryonic EC, shear stress activates Tgfß/Alk5 signaling and that induction of Klf2 is an Alk5 dependent process.


Asunto(s)
Células Endoteliales/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Resistencia al Corte/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Pollos , Células Endoteliales/citología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Ratones , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Receptor Tipo I de Factor de Crecimiento Transformador beta , Venas Umbilicales/citología
10.
Front Cardiovasc Med ; 9: 758265, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36277772

RESUMEN

A role for cardiac sympathetic hyperinnervation in arrhythmogenesis after myocardial infarction (MI) has increasingly been recognized. In humans and mice, the heart receives cervical as well as thoracic sympathetic contributions. In mice, superior cervical ganglia (SCG) have been shown to contribute significantly to myocardial sympathetic innervation of the left ventricular anterior wall. Of interest, the SCG is situated adjacent to the carotid body (CB), a small organ involved in oxygen and metabolic sensing. We investigated the remodeling of murine SCG and CB over time after MI. Murine SCG were isolated from control mice, as well as 24 h, 3 days, 7 days and 6 weeks after MI. SCG and CBs were stained for the autonomic nervous system markers ß3-tubulin, tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), as well as for the neurotrophic factors brain derived neurotropic factor (BDNF), nerve growth factor (NGF) and their tyrosine receptor kinase (pan TRK). Results show that after MI a significant increase in neuron size occurs, especially in the region bordering the CB. Co-expression of TH and ChAT is observed in SCG neuronal cells, but not in the CB. After MI, a significant decrease in ChAT intensity occurs, which negatively correlated with the increased cell size. In addition, an increase of BDNF and NGF at protein and mRNA levels was observed in both the CB and SCG. This upregulation of neurotropic factors coincides with the upregulation of their receptor within the SCG. These findings were concomitant with an increase in GAP43 expression in the SCG, which is known to contribute to axonal outgrowth and elongation. In conclusion, neuronal remodeling toward an increased adrenergic phenotype occurs in the SCG, which is possibly mediated by the CB and might contribute to pathological hyperinnervation after MI.

11.
Nat Biomed Eng ; 6(4): 389-402, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-34992271

RESUMEN

The lack of a scalable and robust source of well-differentiated human atrial myocytes constrains the development of in vitro models of atrial fibrillation (AF). Here we show that fully functional atrial myocytes can be generated and expanded one-quadrillion-fold via a conditional cell-immortalization method relying on lentiviral vectors and the doxycycline-controlled expression of a recombinant viral oncogene in human foetal atrial myocytes, and that the immortalized cells can be used to generate in vitro models of AF. The method generated 15 monoclonal cell lines with molecular, cellular and electrophysiological properties resembling those of primary atrial myocytes. Multicellular in vitro models of AF generated using the immortalized atrial myocytes displayed fibrillatory activity (with activation frequencies of 6-8 Hz, consistent with the clinical manifestation of AF), which could be terminated by the administration of clinically approved antiarrhythmic drugs. The conditional cell-immortalization method could be used to generate functional cell lines from other human parenchymal cells, for the development of in vitro models of human disease.


Asunto(s)
Fibrilación Atrial , Antiarrítmicos/metabolismo , Antiarrítmicos/uso terapéutico , Atrios Cardíacos , Humanos , Miocitos Cardíacos/metabolismo
12.
J Cardiovasc Dev Dis ; 8(5)2021 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-33925811

RESUMEN

Whilst knowledge regarding the pathophysiology of congenital heart disease (CHDs) has advanced greatly in recent years, the underlying developmental processes affecting the cardiac outflow tract (OFT) such as bicuspid aortic valve, tetralogy of Fallot and transposition of the great arteries remain poorly understood. Common among CHDs affecting the OFT, is a large variation in disease phenotypes. Even though the different cell lineages contributing to OFT development have been studied for many decades, it remains challenging to relate cell lineage dynamics to the morphologic variation observed in OFT pathologies. We postulate that the variation observed in cellular contribution in these congenital heart diseases might be related to underlying cell lineage dynamics of which little is known. We believe this gap in knowledge is mainly the result of technical limitations in experimental methods used for cell lineage analysis. The aim of this review is to provide an overview of historical fate mapping and cell tracing techniques used to study OFT development and introduce emerging technologies which provide new opportunities that will aid our understanding of the cellular dynamics underlying OFT pathology.

13.
Front Cardiovasc Med ; 8: 784980, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-35187106

RESUMEN

AIMS: Vein grafts are frequently used to bypass coronary artery occlusions. Unfortunately, vein graft disease (VGD) causes impaired patency rates. ALK1 mediates signaling by TGF-ß via TGFßR2 or BMP9/10 via BMPR2, which is an important pathway in fibrotic, inflammatory, and angiogenic processes in vascular diseases. The role of the TGF-ß pathway in VGD is previously reported, however, the contribution of ALK1 signaling is not known. Therefore, we investigated ALK1 signaling in VGD in a mouse model for vein graft disease using either genetic or pharmacological inhibition of the Alk1 signaling. METHODS AND RESULTS: Male ALK1 heterozygous (ALK1+/-), control C57BL/6, as well as hypercholesterolemic ApoE3*Leiden mice, underwent vein graft surgery. Histologic analyses of ALK1+/- vein grafts demonstrated increased outward remodeling and macrophage accumulation after 28 days. In hypercholesterolemic ApoE3*Leiden mice receiving weekly ALK1-Fc injections, ultrasound imaging showed 3-fold increased outward remodeling compared to controls treated with control-Fc, which was confirmed histologically. Moreover, ALK1-Fc treatment reduced collagen and smooth muscle cell accumulation, increased macrophages by 1.5-fold, and resulted in more plaque dissections. No difference was observed in intraplaque neovessel density. Flow cytometric analysis showed increased systemic levels of Ly6CHigh monocytes in ALK1-Fc treated mice, supported by in vitro increased MCP-1 and IL-6 production of LPS-stimulated and ALK1-Fc-treated murine monocytes and macrophages. CONCLUSION: Reduced ALK1 signaling in VGD promotes outward remodeling, increases macrophage influx, and promotes an unstable plaque phenotype. TRANSLATIONAL PERSPECTIVE: Vein graft disease (VGD) severely hampers patency rates of vein grafts, necessitating research of key disease-driving pathways like TGF-ß. The three-dimensional nature of VGD together with the multitude of disease driving factors ask for a comprehensive approach. Here, we combined in vivo ultrasound imaging, histological analyses, and conventional in vitro analyses, identifying the ambiguous role of reduced ALK1 signaling in vein graft disease. Reduced ALK1 signaling promotes outward remodeling, increases macrophage influx, and promotes an unstable plaque phenotype in murine vein grafts. Characterization of in vivo vascular remodeling over time is imperative to monitor VGD development and identify new therapies.

14.
Cells ; 10(8)2021 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-34440833

RESUMEN

RATIONALE: In recent decades, the great potential of human epicardium-derived cells (EPDCs) as an endogenous cell source for cardiac regeneration has been recognized. The limited availability and low proliferation capacity of primary human EPDCs and phenotypic differences between EPDCs obtained from different individuals hampers their reproducible use for experimental studies. AIM: To generate and characterize inducible proliferative adult human EPDCs for use in fundamental and applied research. METHODS AND RESULTS: Inducible proliferation of human EPDCs was achieved by doxycycline-controlled expression of simian virus 40 large T antigen (LT) with a repressor-based lentiviral Tet-On system. In the presence of doxycycline, these inducible EPDCs (iEPDCs) displayed high and long-term proliferation capacity. After doxycycline removal, LT expression ceased and the iEPDCs regained their cuboidal epithelial morphology. Similar to primary EPDCs, iEPDCs underwent an epithelial-to-mesenchymal transition (EMT) after stimulation with transforming growth factor ß3. This was confirmed by reverse transcription-quantitative polymerase chain reaction analysis of epithelial and mesenchymal marker gene expression and (immuno) cytochemical staining. Collagen gel-based cell invasion assays demonstrated that mesenchymal iEPDCs, like primary EPDCs, possess increased invasion and migration capacities as compared to their epithelial counterparts. Mesenchymal iEPDCs co-cultured with sympathetic ganglia stimulated neurite outgrowth similarly to primary EPDCs. CONCLUSION: Using an inducible LT expression system, inducible proliferative adult human EPDCs were generated displaying high proliferative capacity in the presence of doxycycline. These iEPDCs maintain essential epicardial characteristics with respect to morphology, EMT ability, and paracrine signaling following doxycycline removal. This renders iEPDCs a highly useful new in vitro model for studying human epicardial properties.


Asunto(s)
Pericardio/metabolismo , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/metabolismo , Movimiento Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Doxiciclina/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Ganglios Simpáticos/citología , Ganglios Simpáticos/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Modelos Biológicos , Neuritas/fisiología , Comunicación Paracrina/efectos de los fármacos , Pericardio/citología , Factor de Crecimiento Transformador beta3/farmacología
15.
Front Physiol ; 12: 557514, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33716758

RESUMEN

BACKGROUND: Right ventricular (RV) function and failure are key determinants of morbidity and mortality in various cardiovascular diseases. Myocardial fibrosis is regarded as a contributing factor to heart failure, but its importance in RV failure has been challenged. This study aims to assess whether myocardial fibrosis drives the transition from compensated to decompensated volume load-induced RV dysfunction. METHODS: Wistar rats were subjected to aorto-caval shunt (ACS, n = 23) or sham (control, n = 15) surgery, and sacrificed after 1 month, 3 months, or 6 months. Echocardiography, RV pressure-volume analysis, assessment of gene expression and cardiac histology were performed. RESULTS: At 6 months, 6/8 ACS-rats (75%) showed clinical signs of RV failure (pleural effusion, ascites and/or liver edema), whereas at 1 month and 3 months, no signs of RV failure had developed yet. Cardiac output has increased two- to threefold and biventricular dilatation occurred, while LV ejection fraction gradually decreased. At 1 month and 3 months, RV end-systolic elastance (Ees) remained unaltered, but at 6 months, RV Ees had decreased substantially. In the RV, no oxidative stress, inflammation, pro-fibrotic signaling (TGFß1 and pSMAD2/3), or fibrosis were present at any time point. CONCLUSIONS: In the ACS rat model, long-term volume load was initially well tolerated at 1 month and 3 months, but induced overt clinical signs of end-stage RV failure at 6 months. However, no myocardial fibrosis or increased pro-fibrotic signaling had developed. These findings indicate that myocardial fibrosis is not involved in the transition from compensated to decompensated RV dysfunction in this model.

17.
Dis Model Mech ; 13(9)2020 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-32801116

RESUMEN

Patients with a congenital bicuspid aortic valve (BAV), a valve with two instead of three aortic leaflets, have an increased risk of developing thoracic aneurysms and aortic dissection. The mechanisms underlying BAV-associated aortopathy are poorly understood. This study examined BAV-associated aortopathy in Nos3-/- mice, a model with congenital BAV formation. A combination of histological examination and in vivo ultrasound imaging was used to investigate aortic dilation and dissections in Nos3-/- mice. Moreover, cell lineage analysis and single-cell RNA sequencing were used to observe the molecular anomalies within vascular smooth muscle cells (VSMCs) of Nos3-/- mice. Spontaneous aortic dissections were found in ascending aortas located at the sinotubular junction in ∼13% of Nos3-/- mice. Moreover, Nos3-/- mice were prone to developing aortic dilations in the proximal and distal ascending aorta during early adulthood. Lower volumes of elastic fibres were found within vessel walls of the ascending aortas of Nos3-/- mice, as well as incomplete coverage of the aortic inner media by neural crest cell (NCC)-derived VSMCs. VSMCs of Nos3-/- mice showed downregulation of 15 genes, of which seven were associated with aortic aneurysms and dissections in the human population. Elastin mRNA was most markedly downregulated, followed by fibulin-5 expression, both primary components of elastic fibres. This study demonstrates that, in addition to congenital BAV formation, disrupted endothelial-mediated nitric oxide (NO) signalling in Nos3-/- mice also causes aortic dilation and dissection, as a consequence of inhibited elastic fibre formation in VSMCs within the ascending aorta.


Asunto(s)
Aorta/patología , Enfermedad de la Válvula Aórtica Bicúspide/metabolismo , Enfermedad de la Válvula Aórtica Bicúspide/patología , Óxido Nítrico/metabolismo , Transducción de Señal , Envejecimiento/patología , Disección Aórtica/genética , Disección Aórtica/patología , Animales , Aorta/embriología , Enfermedad de la Válvula Aórtica Bicúspide/genética , Dilatación Patológica , Regulación hacia Abajo/genética , Embrión de Mamíferos/patología , Regulación del Desarrollo de la Expresión Génica , Variación Genética , Ratones , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Cresta Neural/patología , Óxido Nítrico Sintasa de Tipo III/deficiencia , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fenotipo
18.
Front Physiol ; 11: 588679, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33488393

RESUMEN

Electronic pacemakers still face major shortcomings that are largely intrinsic to their hardware-based design. Radical improvements can potentially be generated by gene or cell therapy-based biological pacemakers. Our previous work identified adenoviral gene transfer of Hcn2 and SkM1, encoding a "funny current" and skeletal fast sodium current, respectively, as a potent combination to induce short-term biological pacing in dogs with atrioventricular block. To achieve long-term biological pacemaker activity, alternative delivery platforms need to be explored and optimized. The aim of the present study was therefore to investigate the functional delivery of Hcn2/SkM1 via human cardiomyocyte progenitor cells (CPCs). Nucleofection of Hcn2 and SkM1 in CPCs was optimized and gene transfer was determined for Hcn2 and SkM1 in vitro. The modified CPCs were analyzed using patch-clamp for validation and characterization of functional transgene expression. In addition, biophysical properties of Hcn2 and SkM1 were further investigated in lentivirally transduced CPCs by patch-clamp analysis. To compare both modification methods in vivo, CPCs were nucleofected or lentivirally transduced with GFP and injected in the left ventricle of male NOD-SCID mice. After 1 week, hearts were collected and analyzed for GFP expression and cell engraftment. Subsequent functional studies were carried out by computational modeling. Both nucleofection and lentiviral transduction of CPCs resulted in functional gene transfer of Hcn2 and SkM1 channels. However, lentiviral transduction was more efficient than nucleofection-mediated gene transfer and the virally transduced cells survived better in vivo. These data support future use of lentiviral transduction over nucleofection, concerning CPC-based cardiac gene delivery. Detailed patch-clamp studies revealed Hcn2 and Skm1 current kinetics within the range of previously reported values of other cell systems. Finally, computational modeling indicated that CPC-mediated delivery of Hcn2/SkM1 can generate stable pacemaker function in human ventricular myocytes. These modeling studies further illustrated that SkM1 plays an essential role in the final stage of diastolic depolarization, thereby enhancing biological pacemaker functioning delivered by Hcn2. Altogether these studies support further development of CPC-mediated delivery of Hcn2/SkM1 and functional testing in bradycardia models.

19.
Sci Transl Med ; 12(554)2020 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-32727916

RESUMEN

Pulmonary arterial hypertension (PAH) in congenital cardiac shunts can be reversed by hemodynamic unloading (HU) through shunt closure. However, this reversibility potential is lost beyond a certain point in time. The reason why PAH becomes irreversible is unknown. In this study, we used MCT+shunt-induced PAH in rats to identify a dichotomous reversibility response to HU, similar to the human situation. We compared vascular profiles of reversible and irreversible PAH using RNA sequencing. Cumulatively, we report that loss of reversibility is associated with a switch from a proliferative to a senescent vascular phenotype and confirmed markers of senescence in human PAH-CHD tissue. In vitro, we showed that human pulmonary endothelial cells of patients with PAH are more vulnerable to senescence than controls in response to shear stress and confirmed that the senolytic ABT263 induces apoptosis in senescent, but not in normal, endothelial cells. To support the concept that vascular cell senescence is causal to the irreversible nature of end-stage PAH, we targeted senescence using ABT263 and induced reversal of the hemodynamic and structural changes associated with severe PAH refractory to HU. The factors that drive the transition from a reversible to irreversible pulmonary vascular phenotype could also explain the irreversible nature of other PAH etiologies and provide new leads for pharmacological reversal of end-stage PAH.


Asunto(s)
Cardiopatías Congénitas , Hipertensión Arterial Pulmonar , Animales , Senescencia Celular , Células Endoteliales , Hipertensión Pulmonar Primaria Familiar , Humanos , Ratas
20.
Diabetes ; 69(4): 603-613, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32005705

RESUMEN

Insulin-mediated microvascular recruitment (IMVR) regulates delivery of insulin and glucose to insulin-sensitive tissues. We have previously proposed that perivascular adipose tissue (PVAT) controls vascular function through outside-to-inside communication and through vessel-to-vessel, or "vasocrine," signaling. However, direct experimental evidence supporting a role of local PVAT in regulating IMVR and insulin sensitivity in vivo is lacking. Here, we studied muscles with and without PVAT in mice using combined contrast-enhanced ultrasonography and intravital microscopy to measure IMVR and gracilis artery diameter at baseline and during the hyperinsulinemic-euglycemic clamp. We show, using microsurgical removal of PVAT from the muscle microcirculation, that local PVAT depots regulate insulin-stimulated muscle perfusion and glucose uptake in vivo. We discovered direct microvascular connections between PVAT and the distal muscle microcirculation, or adipomuscular arterioles, the removal of which abolished IMVR. Local removal of intramuscular PVAT altered protein clusters in the connected muscle, including upregulation of a cluster featuring Hsp90ab1 and Hsp70 and downregulation of a cluster of mitochondrial protein components of complexes III, IV, and V. These data highlight the importance of PVAT in vascular and metabolic physiology and are likely relevant for obesity and diabetes.


Asunto(s)
Tejido Adiposo/metabolismo , Arteriolas/metabolismo , Glucosa/metabolismo , Insulina/farmacología , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Tejido Adiposo/efectos de los fármacos , Animales , Arteriolas/efectos de los fármacos , Técnica de Clampeo de la Glucosa , Resistencia a la Insulina/fisiología , Ratones , Microcirculación/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos
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