RESUMO
Platelet derived growth factor beta and its receptor, Pdgfrb, play essential roles in the development of vascular mural cells, including pericytes and vascular smooth muscle cells. To determine if this role was conserved in zebrafish, we analyzed pdgfb and pdgfrb mutant lines. Similar to mouse, pdgfb and pdgfrb mutant zebrafish lack brain pericytes and exhibit anatomically selective loss of vascular smooth muscle coverage. Despite these defects, pdgfrb mutant zebrafish did not otherwise exhibit circulatory defects at larval stages. However, beginning at juvenile stages, we observed severe cranial hemorrhage and vessel dilation associated with loss of pericytes and vascular smooth muscle cells in pdgfrb mutants. Similar to mouse, pdgfrb mutant zebrafish also displayed structural defects in the glomerulus, but normal development of hepatic stellate cells. We also noted defective mural cell investment on coronary vessels with concomitant defects in their development. Together, our studies support a conserved requirement for Pdgfrb signaling in mural cells. In addition, these zebrafish mutants provide an important model for definitive investigation of mural cells during early embryonic stages without confounding secondary effects from circulatory defects.
Assuntos
Músculo Liso Vascular/metabolismo , Pericitos/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Diferenciação Celular , Vasos Coronários/metabolismo , Desenvolvimento Embrionário , Músculo Liso Vascular/embriologia , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Proteínas Proto-Oncogênicas c-sis/fisiologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Smooth muscle cells (SMCs) represent a major structural and functional component of many organs during embryonic development and adulthood. These cells are a crucial component of vertebrate structure and physiology, and an updated overview of the developmental and functional process of smooth muscle during organogenesis is desirable. Here, we describe the developmental origin of SMCs within different tissues by comparing their specification and differentiation with other organs, including the cardiovascular, respiratory and intestinal systems. We then discuss the instructive roles of smooth muscle in the development of such organs through signaling and mechanical feedback mechanisms. By understanding SMC development, we hope to advance therapeutic approaches related to tissue regeneration and other smooth muscle-related diseases.
Assuntos
Desenvolvimento Embrionário , Músculo Liso/crescimento & desenvolvimento , Miócitos de Músculo Liso/fisiologia , Vertebrados/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Sistema Cardiovascular , Diferenciação Celular/fisiologia , Trato Gastrointestinal , Pulmão , Mesoderma , Músculo Liso/citologia , Músculo Liso/embriologia , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/crescimento & desenvolvimento , Miócitos de Músculo Liso/citologia , Organogênese/fisiologia , Sistema Respiratório , Vertebrados/embriologiaRESUMO
OBJECTIVE: Myh11 encodes a myosin heavy chain protein that is specifically expressed in smooth muscle cells (SMCs) and is important for maintaining vascular wall stability. The goal of this study is to generate a Myh11 dual reporter mouse line for definitive visualization of MYH11+ SMCs in vivo. Approach and Results: We generated a Myh11 knock-in mouse model by inserting LoxP-nlacZ-4XpolyA-LoxP-H2B-GFP-polyA-FRT-Neo-FRT reporter cassette into the Myh11 gene locus. The nuclear (n) lacZ-4XpolyA cassette is flanked by 2 LoxP sites followed by H2B-GFP (histone 2B fused green fluorescent protein). Upon Cre-mediated recombination, nlacZ-stop cassette is removed thereby permitting nucleus localized H2B-GFP expression. Expression of the nuclear localized lacZ or H2B-GFP is under control of the endogenous Myh11 promoter. Nuclear lacZ was expressed specifically in SMCs at embryonic and adult stages. Following germline Cre-mediated deletion of nuclear lacZ, H2B-GFP was specifically expressed in the nuclei of SMCs. Comparison of nuclear lacZ expression with Wnt1Cre and Mef2cCre mediated-H2B-GFP expression revealed heterogenous origins of SMCs from neural crest and second heart field in the great arteries and coronary vessels adjacent to aortic root. CONCLUSIONS: The Myh11 knock-in dual reporter mouse model offers an exceptional genetic tool to visualize and trace the origins of SMCs in mice.
Assuntos
Linhagem da Célula , Rastreamento de Células , Proteínas de Fluorescência Verde/metabolismo , Óperon Lac , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Fatores Etários , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Genes Reporter , Idade Gestacional , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/embriologia , Cadeias Pesadas de Miosina/genéticaRESUMO
The thin endothelial wall of a newly formed vessel is under enormous stress at the onset of blood flow, rapidly acquiring support from mural cells (pericytes and vascular smooth muscle cells; vSMCs) during development. Mural cells then develop vasoactivity (contraction and relaxation) but we have little information as to when this first develops or the extent to which pericytes and vSMCs contribute. For the first time, we determine the dynamic developmental acquisition of vasoactivity in vivo in the cerebral vasculature of zebrafish. We show that pericyte-covered vessels constrict in response to α1-adrenergic receptor agonists and dilate in response to nitric oxide donors at 4â days postfertilization (dpf) but have heterogeneous responses later, at 6 dpf. In contrast, vSMC-covered vessels constrict at 6 dpf, and dilate at both stages. Using genetic ablation, we demonstrate that vascular constriction and dilation is an active response. Our data suggest that both pericyte- and vSMC-covered vessels regulate their diameter in early development, and that their relative contributions change over developmental time.
Assuntos
Músculo Liso Vascular/embriologia , Miócitos de Músculo Liso/fisiologia , Pericitos/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Animais , Animais Geneticamente Modificados , Encéfalo/irrigação sanguínea , Encéfalo/diagnóstico por imagem , Encéfalo/embriologia , Células Endoteliais/fisiologia , Endotélio Vascular/embriologia , Inativação Gênica , Metronidazol/farmacologia , Contração Muscular/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Vasodilatação/efeitos dos fármacosRESUMO
OBJECTIVE: While GFAP (glial fibrillary acidic protein) is commonly used as a classical marker for astrocytes in the central nervous system, GFAP-expressing progenitor cells give rise to other cell types during development. The goal of this study was to investigate whether GFAP-expressing progenitor cells contribute to the development of vascular cells in major arteries. Approach and Results: To label GFAP-expressing progenitor cells and their progeny, we crossed GFAP promoter-driven Cre recombinase mice (GFAP-Cre) with transgenic mice expressing the Cre-dependent mTmG dual fluorescent reporter gene. Using this genetic fate-mapping approach, here we demonstrate that GFAP-positive progenitor cells contribute to the development of vascular smooth muscle cells in both neural crest- and non-neural crest-derived vascular beds. In addition, GFAP-positive progenitor cells contribute to a subset of endothelial cells in some vasculature. Furthermore, fate-mapping analyses at multiple time points of mouse development demonstrate a time-dependent increase in the contribution of GFAP-positive progenitors to vascular smooth muscle cells, which mostly occurs in the postnatal period. CONCLUSIONS: Our study demonstrates that vascular smooth muscle cells and endothelial cells within the same vascular segment are developmentally heterogeneous, where varying proportions of vascular smooth muscle cells and endothelial cells are contributed by GFAP-positive progenitor cells.
Assuntos
Diferenciação Celular , Linhagem da Célula , Células Progenitoras Endoteliais/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Crista Neural/metabolismo , Animais , Feminino , Genes Reporter , Proteína Glial Fibrilar Ácida/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/embriologia , Crista Neural/embriologia , Fenótipo , Proteína Vermelha FluorescenteRESUMO
The etiology of pulmonary vascular abnormalities in CDH is incompletely understood. Studies have demonstrated improvement in pulmonary vasculature with prenatal therapy in animal models. We hypothesize that prenatal sildenafil may attenuate defective pulmonary vascular development via modulation of vSMC phenotype from undifferentiated, proliferative phenotype to differentiated, contractile phenotype. We utilized the nitrofen model of CDH to examine the effect of IA sildenafil on pulmonary vSMC phenotype during lung development. Timed-pregnant CD-1 mice were gavage fed 25 mg nitrofen or olive oil (control) at E8.5 of gestation. Single IA injections of Sildenafil (Revatio; 10 µL of 4 mg/4 ml solution) or dextrose control were performed at E12.5. Mice were sacrificed on various gestational days for embryonic lung harvest. Markers of vSMC development of undifferentiated and differentiated phenotypes were analyzed by immunostaining and western blot. Across all time points in gestation, nitrofen-treated embryonic lungs demonstrated increased vSMC expression of NOTCH3, Hes-5, PDGFR-ß, desmin and α-SMA and decreased expression of calponin and SMMHC, compared to oil controls. IA dextrose treatment had no effect on expression levels. However, IA Sildenafil treatment resulted in down-regulation of NOTCH3, Hes-5, PDGFR-ß, desmin and α-SMA and upregulation of calponin and SMMHC, comparable to oil controls. In the nitrofen model, vSMC express markers consistent with more undifferentiated proliferative phenotype, resulting in hypermuscularization of intrapulmonary arterioles in CDH. A single dose of IA Sildenafil treatment early in gestation, results in sustained normalization of vSMC phenotype. Pharmacologic modulation of the vSMC phenotype at key gestational points may have therapeutic potential.
Assuntos
Hérnias Diafragmáticas Congênitas/tratamento farmacológico , Músculo Liso Vascular/efeitos dos fármacos , Citrato de Sildenafila/uso terapêutico , Vasodilatadores/uso terapêutico , Âmnio , Animais , Feminino , Hérnias Diafragmáticas Congênitas/induzido quimicamente , Hérnias Diafragmáticas Congênitas/etiologia , Injeções , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Pulmão/embriologia , Camundongos , Músculo Liso Vascular/embriologia , Fenótipo , Éteres Fenílicos , Gravidez , Citrato de Sildenafila/administração & dosagem , Vasodilatadores/administração & dosagemRESUMO
The cardiovascular system develops during the early stages of embryogenesis, and differentiation of smooth muscle cells (SMCs) is essential for that process. SMC differentiation is critically regulated by transforming growth factor (TGF)-ß/SMAD family member 3 (SMAD3) signaling, but other regulators may also play a role. For example, long noncoding RNAs (lncRNAs) regulate various cellular activities and events, such as proliferation, differentiation, and apoptosis. However, whether long noncoding RNAs also regulate SMC differentiation remains largely unknown. Here, using the murine cell line C3H10T1/2, we found that brain cytoplasmic RNA 1 (BC1) is an important regulator of SMC differentiation. BC1 overexpression suppressed, whereas BC1 knockdown promoted, TGF-ß-induced SMC differentiation, as indicated by altered cell morphology and expression of multiple SMC markers, including smooth muscle α-actin (αSMA), calponin, and smooth muscle 22α (SM22α). BC1 appeared to block SMAD3 activity and inhibit SMC marker gene transcription. Mechanistically, BC1 bound to SMAD3 via RNA SMAD-binding elements (rSBEs) and thus impeded TGF-ß-induced SMAD3 translocation to the nucleus. This prevented SMAD3 from binding to SBEs in SMC marker gene promoters, an essential event in SMC marker transcription. In vivo, BC1 overexpression in mouse embryos impaired vascular SMC differentiation, leading to structural defects in the artery wall, such as random breaks in the elastic lamina, abnormal collagen deposition on SM fibers, and disorganized extracellular matrix proteins in the media of the neonatal aorta. Our results suggest that BC1 is a suppressor of SMC differentiation during vascular development.
Assuntos
Aorta/embriologia , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Músculo Liso Vascular/embriologia , Miócitos de Músculo Liso/metabolismo , RNA Longo não Codificante/biossíntese , Animais , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Aorta/citologia , Linhagem Celular , Humanos , Camundongos , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , RNA Longo não Codificante/genéticaRESUMO
Arterial vasculature distributes blood from early embryonic development and provides a nutrient highway to maintain tissue viability. Atherosclerosis, peripheral artery diseases, stroke and aortic aneurysm represent the most frequent causes of death and are all directly related to abnormalities in the function of arteries. Vascular intervention techniques have been established for the treatment of all of these pathologies, yet arterial surgery can itself lead to biological changes in which uncontrolled arterial wall cell proliferation leads to restricted blood flow. In this review we describe the intricate cellular composition of arteries, demonstrating how a variety of distinct cell types in the vascular walls regulate the function of arteries. We provide an overview of the developmental origin of arteries and perivascular cells and focus on cellular dynamics in arterial repair. We summarize the current knowledge of the molecular signaling pathways that regulate vascular smooth muscle differentiation in the embryo and in arterial injury response. Our review aims to highlight the similarities as well as differences between cellular and molecular mechanisms that control arterial development and repair.
Assuntos
Artérias/fisiologia , Músculo Liso Vascular/fisiologia , Neovascularização Fisiológica/fisiologia , Cicatrização/fisiologia , Animais , Artérias/citologia , Artérias/embriologia , Artérias/lesões , Biomarcadores , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/embriologia , Miócitos de Músculo Liso/fisiologia , Transdução de Sinais , Fatores de Transcrição/fisiologia , Vasa Vasorum/fisiologia , Vasa Vasorum/ultraestruturaRESUMO
RATIONALE: Vascular smooth muscle turnover has important implications for blood vessel repair and for the development of cardiovascular diseases, yet lack of specific transgenic animal models has prevented it's in vivo analysis. OBJECTIVE: The objective of this study was to characterize the dynamics and mechanisms of vascular smooth muscle turnover from the earliest stages of embryonic development to arterial repair in the adult. METHODS AND RESULTS: We show that CD146 is transiently expressed in vascular smooth muscle development. By using CRISPR-Cas9 genome editing and in vitro smooth muscle differentiation assay, we demonstrate that CD146 regulates the balance between proliferation and differentiation. We developed a triple-transgenic mouse model to map the fate of NG2+CD146+ immature smooth muscle cells. A series of pulse-chase experiments revealed that the origin of aortic vascular smooth muscle cells can be traced back to progenitor cells that reside in the wall of the dorsal aorta of the embryo at E10.5. A distinct population of CD146+ smooth muscle progenitor cells emerges during embryonic development and is maintained postnatally at arterial branch sites. To characterize the contribution of different cell types to arterial repair, we used 2 injury models. In limited wire-induced injury response, existing smooth muscle cells are the primary contributors to neointima formation. In contrast, microanastomosis leads to early smooth muscle death and subsequent colonization of the vascular wall by proliferative adventitial cells that contribute to the repair. CONCLUSIONS: Extensive proliferation of immature smooth muscle cells in the primitive embryonic dorsal aorta establishes the long-lived lineages of smooth muscle cells that make up the wall of the adult aorta. A discrete population of smooth muscle cells forms in the embryo and is postnatally sustained at arterial branch sites. In response to arterial injuries, existing smooth muscle cells give rise to neointima, but on extensive damage, they are replaced by adventitial cells.
Assuntos
Desenvolvimento Embrionário/fisiologia , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Animais , Antígeno CD146/fisiologia , Linhagem Celular , Proliferação de Células/fisiologia , Feminino , Camundongos , Camundongos Transgênicos , GravidezRESUMO
OBJECTIVE: Smooth muscle cells (SMCs) of the proximal thoracic aorta are embryonically derived from the second heart field (SHF) and cardiac neural crest (CNC). However, distributions of these embryonic origins are not fully defined. The regional distribution of SMCs of different origins is speculated to cause region-specific aortopathies. Therefore, the aim of this study was to determine the distribution of SMCs of SHF and CNC origins in the proximal thoracic aorta. APPROACH AND RESULTS: Mice with repressed LacZ in the ROSA26 locus were bred to those expressing Cre controlled by either the Wnt1 or Mef2c (myocyte-specific enhancer factor 2c) promoter to trace CNC- and SHF-derived SMCs, respectively. Thoracic aortas were harvested, and activity of ß-galactosidase was determined. Aortas from Wnt1-Cre mice had ß-galactosidase-positive areas throughout the region from the proximal ascending aorta to just distal of the subclavian arterial branch. Unexpectedly, ß-galactosidase-positive areas in Mef2c-Cre mice extended from the aortic root throughout the ascending aorta. This distribution occurred independent of sex and aging. Cross and sagittal aortic sections demonstrated that CNC-derived cells populated the inner medial aspect of the anterior region of the ascending aorta and transmurally in the media of the posterior region. Interestingly, outer medial cells throughout anterior and posterior ascending aortas were derived from the SHF. ß-Galactosidase-positive medial cells of both origins colocalized with an SMC marker, α-actin. CONCLUSIONS: Both CNC- and SHF-derived SMCs populate the media throughout the ascending aorta. The outer medial cells of the ascending aorta form a sleeve populated by SHF-derived SMCs.
Assuntos
Linhagem da Célula , Coração/embriologia , Músculo Liso Vascular/fisiologia , Miocárdio , Miócitos de Músculo Liso/fisiologia , Crista Neural/fisiologia , Túnica Média/fisiologia , Fatores Etários , Animais , Aorta Torácica/embriologia , Aorta Torácica/metabolismo , Aorta Torácica/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Integrases/genética , Óperon Lac , Fatores de Transcrição MEF2/genética , Masculino , Camundongos Transgênicos , Morfogênese , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , Miócitos de Músculo Liso/metabolismo , Crista Neural/embriologia , Crista Neural/metabolismo , Fenótipo , Regiões Promotoras Genéticas , RNA não Traduzido/genética , Fatores Sexuais , Túnica Média/embriologia , Túnica Média/metabolismo , Proteína Wnt1/genética , beta-Galactosidase/metabolismoAssuntos
Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Actinas/metabolismo , Animais , Aneurisma Aórtico/fisiopatologia , Aterosclerose/fisiopatologia , Humanos , Inflamação/fisiopatologia , Músculo Liso Vascular/embriologia , Fenótipo , Polimerização , Fator de Crescimento Transformador beta/metabolismo , Remodelação Vascular/fisiologiaRESUMO
The fetal cardiovascular responses to acute hypoxia include a redistribution of the cardiac output toward the heart and the brain at the expense of other organs, such as the intestine. We hypothesized that hypoxia exerts a direct effect on the mesenteric artery (MA) that may contribute to this response. Using wire myography, we investigated the response to hypoxia (Po2 ~2.5 kPa for 20 min) of isolated MAs from 15- to 21-day chicken embryos (E15, E19, E21), and 1- to 45-day-old chickens (P1, P3, P14, P45). Agonist-induced pretone or an intact endothelium were not required to obtain a consistent and reproducible response to hypoxia, which showed a pattern of initial rapid phasic contraction followed by a sustained tonic contraction. Phasic contraction was reduced by elimination of extracellular Ca2+ or by presence of the neurotoxin tetrodotoxin, the α1-adrenoceptor antagonist prazosin, or inhibitors of L-type voltage-gated Ca2+ channels (nifedipine), mitochondrial electron transport chain (rotenone and antimycin A), and NADPH oxidase (VAS2870). The Rho-kinase inhibitor Y27632 impaired both phasic and tonic contraction and, when combined with elimination of extracellular Ca2+, hypoxia-induced contraction was virtually abolished. Hypoxic MA contraction was absent at E15 but present from E19 and increased toward the first days posthatching. It then decreased during the first weeks of life and P45 MAs were unable to sustain hypoxia-induced contraction over time. In conclusion, the results of the present study demonstrate that hypoxic vasoconstriction is an intrinsic feature of chicken MA vascular smooth muscle cells during late embryogenesis and the perinatal period.
Assuntos
Hipóxia/fisiopatologia , Artérias Mesentéricas/embriologia , Artérias Mesentéricas/fisiopatologia , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/fisiopatologia , Vasoconstrição , Animais , Embrião de Galinha , Desenvolvimento Embrionário , Contração MuscularRESUMO
Recent identification of the neonatal 2nd coronary vascular population (2nd CVP) suggests that a subset of these vessels form de novo and mature in the inner myocardial wall of the postnatal heart. However, the origin of smooth muscle cells (SMCs) in the postnatal 2nd CVP remains undetermined. Using a tamoxifen-inducible Wt1-CreER driver and a Rosa26-RFP reporter line, we traced the lineage of epicardial cells to determine if they contribute to SMCs of the 2nd CVP. Late embryonic and postnatal induction of Wt1-CreER activity demonstrated that at these stages Wt1-labeled epicardium does not significantly migrate into the myocardium to form SMCs. However, following tamoxifen treatment at an early embryonic stage (E10.5), we detected Wt1 descendants (epicardium-derived cells, or EPDCs) in the outer myocardial wall at E17.5. When the 2nd CVP forms and remodels at postnatal stage, these early labeled EDPCs re-migrate deep into the inner myocardial wall and contribute to 2nd CVP-SMCs in the adult heart. Our findings reveal that SMCs in the postnatal 2nd CVP are pre-specified as EPDCs from the earliest wave of epicardial cell migration. Rather than the re-activation and migration of epicardial cells at later stages, these resident EPDCs mobilize and contribute to smooth muscle of the 2nd CVP during postnatal development.
Assuntos
Vasos Coronários/citologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/embriologia , Animais , Animais Recém-Nascidos , Movimento Celular , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/embriologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/citologia , Pericárdio/citologia , Pericárdio/embriologia , TamoxifenoRESUMO
The ductus arteriosus is an arterial vessel that shunts blood flow away from the lungs during fetal life, but normally occludes after birth to establish the adult circulation pattern. Failure of the ductus arteriosus to close after birth is termed patent ductus arteriosus, and is one of the most common congenital heart defects. Our previous work demonstrated that vascular smooth muscle cell expression of the Jag1 gene, which encodes a ligand for Notch family receptors, is essential for postnatal closure of the ductus arteriosus in mice. However, it was not known what cell population was responsible for receiving the Jag1-mediated signal. Here we show, using smooth muscle cell-specific deletion of the Rbpj gene, which encodes a transcription factor that mediates all canonical Notch signaling, that Notch signal reception in the vascular smooth muscle cell compartment is required for ductus arteriosus closure. These data indicate that homotypic vascular smooth muscle cell interactions are required for proper contractile smooth muscle cell differentiation and postnatal closure of the ductus arteriosus in mice.
Assuntos
Canal Arterial/embriologia , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Músculo Liso Vascular/citologia , Proteínas Serrate-JaggedRESUMO
The aortic arch and major branch arteries are formed from the three pairs of pharyngeal arch arteries (PAAs) during embryonic development. Their morphological defects are clinically observed as isolated diseases, as a part of complicated cardiovascular anomalies or as a manifestation of multi-organ syndromes such as 22q11.2 deletion syndrome. Although numerous genes have been implicated in PAA formation and remodeling, detailed mechanisms remain poorly understood. Here we report that the mice null for Hrt1/Hey1, a gene encoding a downstream transcription factor of Notch and ALK1 signaling pathways, show perinatal lethality on the C57BL/6N, C57BL/6N × C57BL/6J or C57BL/6N × 129X1/SvJ background. Hrt1/Hey1 null embryos display abnormal development of the fourth PAA (PAA4), which results in congenital vascular defects including right-sided aortic arch, interruption of the aortic arch and aberrant origin of the right subclavian artery. Impaired vessel formation occurs randomly in PAA4 of Hrt1/Hey1 null embryos, which likely causes the variability of congenital malformations. Endothelial cells in PAA4 of null embryos differentiate normally but are structurally disorganized at embryonic day 10.5 and 11.5. Vascular smooth muscle cells are nearly absent in the structurally-defective PAA4, despite the appropriate migration of cardiac neural crest cells into the fourth pharyngeal arches. Endothelial expression of Jag1 is down-regulated in the structurally-defective PAA4 of null embryos, which may be one of the mechanisms underlying the suppression of vascular smooth muscle cell differentiation. While the direct downstream phenomena of the Hrt1/Hey1 deficiency remain to be clarified, we suggest that Hrt1/Hey1-dependent transcriptional regulation has an important role in PAA formation during embryonic development.
Assuntos
Aorta Torácica/anormalidades , Proteínas de Ciclo Celular/genética , Animais , Aorta Torácica/embriologia , Apoptose , Região Branquial/irrigação sanguínea , Região Branquial/embriologia , Movimento Celular , Proliferação de Células , Regulação para Baixo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/fisiologia , Deleção de SequênciaRESUMO
OBJECTIVE: We and others have previously shown that RhoA-dependent stimulation of myocardin-related transcription factor nuclear localization promotes smooth muscle cell (SMC) marker gene expression. The goal of this study was to provide direct in vivo evidence that actin polymerization by the diaphanous-related formins contributes to the regulation of SMC differentiation and phenotype. APPROACH AND RESULTS: Conditional Cre-based genetic approaches were used to overexpress a well-characterized dominant-negative variant of mDia1 (DNmDia) in SMC. DNmDia expression in SM22-expressing cells resulted in embryonic and perinatal lethality in ≈20% of mice because of defects in myocardial development and SMC investment of peripheral vessels. Although most DNmDia(+)/SM22Cre(+) mice exhibited no overt phenotype, the re-expression of SMC differentiation marker gene expression that occurs after carotid artery ligation was delayed, and this effect was accompanied by a significant decrease in myocardin-related transcription factor-A nuclear localization. Interestingly, neointima growth was inhibited by expression of DNmDia in SMC and this was likely because of a defect in directional SMC migration and not to defects in SMC proliferation or survival. Finally, by using the tamoxifen-inducible SM MHC-CreER(T2) line, we showed that SMC-specific induction of DNmDia in adult mice decreased SMC marker gene expression. CONCLUSIONS: Our demonstration that diaphanous-related formin signaling plays a role in heart and vascular development and the maintenance of SMC phenotype provides important new evidence that Rho/actin/myocardin-related transcription factor signaling plays a critical role in cardiovascular function.
Assuntos
Proteínas de Transporte/metabolismo , Cardiopatias Congênitas/metabolismo , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais , Actinas/metabolismo , Animais , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Artérias Carótidas/cirurgia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Proteínas de Transporte/genética , Diferenciação Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Forminas , Coração/embriologia , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/patologia , Ligadura , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Desenvolvimento Muscular , Proteínas Musculares/metabolismo , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/patologia , Músculo Liso Vascular/cirurgia , Miocárdio/patologia , Miócitos de Músculo Liso/patologia , Neointima , Fenótipo , Polimerização , Fatores de Tempo , Transfecção , Lesões do Sistema VascularRESUMO
Hemodynamic forces regulate many aspects of blood vessel disease and development, including susceptibility to atherosclerosis and remodeling of primary blood vessels into a mature vascular network. Vessels of the lymphatic circulatory system are also subjected to fluid flow-associated forces, but the molecular and cellular mechanisms by which these forces regulate the formation and maintenance of lymphatic vessels remain largely uncharacterized. This issue of the JCI includes two articles that begin to address how fluid flow influences lymphatic vessel development and function. Sweet et al. demonstrate that lymph flow is essential for the remodeling of primary lymphatic vessels, for ensuring the proper distribution of smooth muscle cells (SMCs), and for the development and maturation of lymphatic valves. Kazenwadel et al. show that flow-induced lymphatic valve development is initiated by the upregulation of GATA2, which has been linked to lymphedema in patients with Emberger syndrome. Together, these observations and future studies inspired by these results have potential to lead to the development of strategies for the treatment of lymphatic disorders.
Assuntos
Fator de Transcrição GATA2/metabolismo , Linfa/fisiologia , Vasos Linfáticos/embriologia , Linfedema/embriologia , Músculo Liso Vascular/embriologia , Mutação , Miócitos de Músculo Liso/metabolismo , Animais , HumanosRESUMO
Fluid shear forces have established roles in blood vascular development and function, but whether such forces similarly influence the low-flow lymphatic system is unknown. It has been difficult to test the contribution of fluid forces in vivo because mechanical or genetic perturbations that alter flow often have direct effects on vessel growth. Here, we investigated the functional role of flow in lymphatic vessel development using mice deficient for the platelet-specific receptor C-type lectin-like receptor 2 (CLEC2) as blood backfills the lymphatic network and blocks lymph flow in these animals. CLEC2-deficient animals exhibited normal growth of the primary mesenteric lymphatic plexus but failed to form valves in these vessels or remodel them into a structured, hierarchical network. Smooth muscle cell coverage (SMC coverage) of CLEC2-deficient lymphatic vessels was both premature and excessive, a phenotype identical to that observed with loss of the lymphatic endothelial transcription factor FOXC2. In vitro evaluation of lymphatic endothelial cells (LECs) revealed that low, reversing shear stress is sufficient to induce expression of genes required for lymphatic valve development and identified GATA2 as an upstream transcriptional regulator of FOXC2 and the lymphatic valve genetic program. These studies reveal that lymph flow initiates and regulates many of the key steps in collecting lymphatic vessel maturation and development.
Assuntos
Linfa/fisiologia , Vasos Linfáticos/embriologia , Músculo Liso Vascular/embriologia , Miócitos de Músculo Liso/metabolismo , Animais , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Resistência ao CisalhamentoRESUMO
AIM: Age and injury cause structural and functional changes in coronary artery smooth muscle cells (caSMCs) that influence the pathogenesis of coronary artery disease. Although paracrine signalling is widely believed to drive phenotypic changes in caSMCs, here we show that developmental origin within the fetal epicardium can have a profound effect as well. METHODS AND RESULTS: Fluorescent dye and transgene pulse-labelling techniques in mice revealed that the majority of caSMCs are derived from Wt1(+), Gata5-Cre(+) cells that migrate before E12.5, whereas a minority of cells are derived from a later-emigrating, Wt1(+), Gata5-Cre(-) population. We functionally evaluated the influence of early emigrating cells on coronary artery development and disease by Gata5-Cre excision of Rbpj, which prevents their contribution to coronary artery smooth muscle cells. Ablation of the Gata5-Cre(+) population resulted in coronary arteries consisting solely of Gata5-Cre(-) caSMCs. These coronary arteries appeared normal into early adulthood; however, by 5-8 months of age, they became progressively fibrotic, lost the adventitial outer elastin layer, were dysfunctional and leaky, and animals showed early mortality. CONCLUSION: Taken together, these data reveal heterogeneity in the fetal epicardium that is linked to coronary artery integrity, and that distortion of the coronaries epicardial origin predisposes to adult onset disease.
Assuntos
Doença da Artéria Coronariana/patologia , Miócitos de Músculo Liso/citologia , Pericárdio/patologia , Envelhecimento , Animais , Diferenciação Celular/fisiologia , Camundongos Transgênicos , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/crescimento & desenvolvimento , Pericárdio/embriologiaRESUMO
BACKGROUND: The ductus arteriosus (DA), a fetal arterial connection between the main pulmonary artery and the descending aorta, normally closes immediately after birth. The oxygen concentration in the blood rises after birth, and in the DA this increase in oxygen concentration causes functional closure, which is induced by smooth muscle contraction. Previous studies have demonstrated that hypoxia and/or oxygenation affect vascular remodeling of various vessels. Therefore, we hypothesized that the rise in oxygen concentration would affect the vascular structure of the DA due to production of proteins secreted from DA smooth muscle cells (SMC). METHODS AND RESULTS: Liquid chromatography-tandem mass spectrometry was used to comprehensively investigate the secreted proteins in the supernatant of rat DA SMC harvested under hypoxic conditions (1% oxygen) or under normoxic conditions (21% oxygen). We found that the rise in oxygen concentration reduced the secretion of elastin from DA SMC. On reverse transcription-polymerase chain reaction, the expression of elastin mRNA was not significantly changed in DA SMC from hypoxic to normoxic conditions. CONCLUSIONS: Given that elastin forms internal elastic lamina and elastic fibers in the vascular muscle layers, and that a rise in oxygen concentration reduced the secretion of elastin, this suggests that the rise in blood oxygen concentration after birth reduces the secretion of elastin, and therefore may play a role in DA structural remodeling after birth.