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In this study, we present a versatile, scaffold-free approach to create ring-shaped engineered vascular tissue segments using human mesenchymal stem cell-derived smooth muscle cells (hMSC-SMCs) and endothelial cells (ECs). We hypothesized that incorporation of ECs would increase hMSC-SMC differentiation without compromising tissue ring strength or fusion to form tissue tubes. Undifferentiated hMSCs and ECs were co-seeded into custom ring-shaped agarose wells using four different concentrations of ECs: 0, 10, 20, and 30%. Co-seeded EC and hMSC rings were cultured in SMC differentiation medium for a total of 22 days. Tissue rings were then harvested for histology, western blotting, wire myography, and uniaxial tensile testing to examine their structural and functional properties. Differentiated hMSC tissue rings comprised of 20 and 30% ECs exhibited significantly greater SMC contractile protein expression, endothelin-1 (ET-1)-meditated contraction, and force at failure compared to the 0% EC rings. On average, the 0, 10, 20, and 30% EC rings exhibited a contractile force of 0.745 ± 0.117, 0.830 ± 0.358, 1.31 ± 0.353, and 1.67 ± 0.351 mN (mean ± SD) in response to ET-1, respectively. Additionally, the mean maximum force at failure for the 0, 10, 20, and 30% EC rings was 88.5 ± 36.2, 121 ± 59.1, 147 ± 43.1, and 206 ± 20.8 mN (mean ± SD), respectively. Based on these results, 30% EC rings were fused together to form tissue engineered blood vessels (TEBVs) and compared to 0% EC TEBV controls. The addition of 30% ECs in TEBVs did not affect ring fusion but did result in significantly greater SMC protein expression (calponin and smoothelin). In summary, co-seeding hMSCs with ECs to form tissue rings resulted in greater contraction, strength, and hMSC-SMC differentiation compared to hMSCs alone and indicates a method to create functional 3D human vascular cell co-culture model.
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The transgenerational effects of exposing male mice to chronic social instability (CSI) stress are associated with decreased sperm levels of multiple members of the miR-34/449 family that persist after their mating through preimplantation embryo (PIE) development. Here we demonstrate the importance of these miRNA changes by showing that restoring miR-34c levels in PIEs derived from CSI stressed males prevents elevated anxiety and defective sociability normally found specifically in their adult female offspring. It also restores, at least partially, levels of sperm miR-34/449 normally reduced in their male offspring who transmit these sex-specific traits to their offspring. Strikingly, these experiments also revealed that inducing miR-34c levels in PIEs enhances the expression of its own gene and that of miR-449 in these cells. The same induction of embryo miR-34/449 gene expression likely occurs after sperm-derived miR-34c is introduced into oocytes upon fertilization. Thus, suppression of this miRNA amplification system when sperm miR-34c levels are reduced in CSI stressed mice can explain how a comparable fold-suppression of miR-34/449 levels can be found in PIEs derived from them, despite sperm containing ~50-fold lower levels of these miRNAs than those already present in PIEs. We previously found that men exposed to early life trauma also display reduced sperm levels of miR-34/449. And here we show that miR-34c can also increase the expression of its own gene, and that of miR-449 in human embryonic stem cells, suggesting that human PIEs derived from men with low sperm miR-34/449 levels may also contain this potentially harmful defect.
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Blastocisto , Epigênese Genética , MicroRNAs , Espermatozoides , Estresse Psicológico , MicroRNAs/genética , MicroRNAs/metabolismo , Masculino , Animais , Espermatozoides/metabolismo , Feminino , Camundongos , Blastocisto/metabolismo , Estresse Psicológico/metabolismo , Estresse Psicológico/genética , Humanos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: RAB27A is a member of the RAS oncogene superfamily of GTPases and regulates cell secretory function. It, is expressed within blood vessels and perivascular adipose tissue. We hypothesized that loss of RAB27A would alter cardiovascular function. METHODS: Body weight of Rab27aash mice was measured from 2 to 18 months of age, along with glucose resorption at 6 and 12 months of age and glucose sensitivity at 18 months of age. Body weight and cellular and molecular features of perivascular adipose tissue and aortic tissue were examined in a novel C57BL/6J Rab27a null strain. Analyses included morphometric quantification and proteomic analyses. Wire myography measured vasoreactivity, and echocardiography measured cardiac function. Comparisons across ages and genotypes were evaluated via 2-way ANOVA with multiple comparison testing. Significance for myography was determined via 4-parameter nonlinear regression testing. RESULTS: Genome-wide association data linked rare human RAB27A variants with body mass index and glucose handling. Changes in glucose tolerance were observed in Rab27aash male mice at 18 months of age. In WT (wild-type) and Rab27a null male mice, body weight, adipocyte lipid area, and aortic area increased with age. In female mice, only body weight increased with age, independent of RAB27A presence. Protein signatures from male Rab27a null mice suggested greater associations with cardiovascular and metabolic phenotypes compared with female tissues. Wire myography results showed Rab27a null males exhibited increased vasoconstriction and reduced vasodilation at 8 weeks of age. Rab27a null females exhibited increased vasoconstriction and vasodilation at 20 weeks of age. Consistent with these vascular changes, male Rab27a null mice experienced age-related cardiomyopathy, with severe differences observed by 21 weeks of age. CONCLUSIONS: Global RAB27A loss impacted perivascular adipose tissue and thoracic aorta proteomic signatures, altered vasocontractile responses, and decreased left ventricular ejection fraction in mice.
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Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas rab27 de Ligação ao GTP , Animais , Proteínas rab27 de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP/metabolismo , Masculino , Feminino , Camundongos , Fenótipo , Tecido Adiposo/metabolismo , Vasodilatação , Vasoconstrição , Fatores Etários , Proteômica , Fatores Sexuais , Aorta/metabolismo , Aorta/fisiopatologia , HumanosRESUMO
Type 1 diabetes (T1D) is characterized by lymphocyte infiltration into the pancreatic islets of Langerhans, leading to the destruction of insulin-producing beta cells and uncontrolled hyperglycemia. In the nonobese diabetic (NOD) murine model of T1D, the onset of this infiltration starts several weeks before glucose dysregulation and overt diabetes. Recruitment of immune cells to the islets is mediated by several chemotactic cytokines, including CXCL10, while other cytokines, including SDF-1α, can confer protective effects. Global gene expression studies of the pancreas from prediabetic NOD mice and single-cell sequence analysis of human islets from prediabetic, autoantibody-positive patients showed an increased expression of metallothionein (MT), a small molecular weight, cysteine-rich metal-binding stress response protein. We have shown that beta cells can release MT into the extracellular environment, which can subsequently enhance the chemotactic response of Th1 cells to CXCL10 and interfere with the chemotactic response of Th2 cells to SDF-1α. These effects can be blocked in vitro with a monoclonal anti-MT antibody, clone UC1MT. When administered to NOD mice before the onset of diabetes, UC1MT significantly reduces the development of T1D. Manipulation of extracellular MT may be an important approach to preserving beta cell function and preventing the development of T1D.
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Diabetes Mellitus Tipo 1 , Estado Pré-Diabético , Humanos , Camundongos , Animais , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/prevenção & controle , Camundongos Endogâmicos NOD , Metalotioneína/genética , Metalotioneína/metabolismo , Quimiocina CXCL12RESUMO
The Interleukin-17 (IL17) family is a group of cytokines implicated in the etiology of several inflammatory diseases. Interleukin-17 receptor D (IL17RD), also known as Sef (similar expression to fibroblast growth factor) belonging to the family of IL17 receptors, has been shown to modulate IL17A-associated inflammatory phenotypes. The objective of this study was to test the hypothesis that IL17RD promotes endothelial cell activation and consequent leukocyte adhesion. We utilized primary human aortic endothelial cells and demonstrated that RNAi targeting of IL17RD suppressed transcript levels by 83 % compared to non-targeted controls. Further, RNAi knockdown of IL17RD decreased the adhesion of THP-1 monocytic cells onto a monolayer of aortic endothelial cells in response to IL17A. Additionally, we determined that IL17A did not significantly enhance the activation of canonical MAPK and NFκB pathways in endothelial cells, and further did not significantly affect the expression of VCAM-1 and ICAM-1 in aortic endothelial cells, which is contrary to previous findings. We also determined the functional relevance of our findings in vivo by comparing the expression of endothelial VCAM-1 and ICAM-1 and leukocyte infiltration in the aorta in Western diet-fed Il17rd null versus wild-type mice. Our results showed that although Il17rd null mice do not have significant alteration in aortic expression of VCAM-1 and ICAM-1 in endothelial cells, they exhibit decreased accumulation of proinflammatory monocytes and neutrophils, suggesting that endothelial IL17RD induced in vivo myeloid cell accumulation is not dependent on upregulation of VCAM-1 and ICAM-1 expression. We further performed proteomics analysis to identify potential molecular mediators of the IL17A/IL17RD signaling axis. Collectively, our results underscore a critical role for Il17rd in the regulation of aortic myeloid cell infiltration in the context of Western diet feeding.
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Células Endoteliais , Molécula 1 de Adesão Intercelular , Humanos , Animais , Camundongos , Molécula 1 de Adesão Intercelular/metabolismo , Células Endoteliais/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Dieta Ocidental , Aorta/metabolismo , Células Mieloides/metabolismo , Monócitos/metabolismo , Adesão Celular , Receptores de Interleucina/metabolismoRESUMO
Perivascular adipose tissue (PVAT) regulates vascular function by secreting vasoactive substances. In mice, Notch signaling is activated in the PVAT during diet-induced obesity, and leads to the loss of the thermogenic phenotype and adipocyte whitening due to increased lipid accumulation. We used the Adiponectin-Cre (Adipoq-Cre) strain to activate a ligand-independent Notch1 intracellular domain transgene (N1ICD) to drive constitutive Notch signaling in the adipose tissues (N1ICD;Adipoq-Cre). We previously found that constitutive activation of Notch1 signaling in the PVAT phenocopied the effects of diet-induced obesity. To understand the downstream pathways activated by Notch signaling, we performed a proteomic analysis of the PVAT from control versus N1ICD;Adipoq-Cre mice. This comparison identified prominent changes in the protein signatures related to metabolism, adipocyte homeostasis, mitochondrial function, and ferroptosis. PVAT-derived stromal vascular fraction cells were derived from our mouse strains to study the cellular and molecular phenotypes during adipogenic induction. We found that cells with activated Notch signaling displayed decreased mitochondrial respiration despite similar levels of adipogenesis and mitochondrial number. We observed variable regulation of the proteins related to mitochondrial dynamics and ferroptosis, including PHB3, PINK1, pDRP1, and the phospholipid hydroperoxidase GPX4. Mitochondria regulate some forms of ferroptosis, which is a regulated process of cell death driven by lipid peroxidation. Accordingly, we found that Notch activation promoted lipid peroxidation and ferroptosis in PVAT-derived adipocytes. Because the PVAT phenotype is a regulator of vascular reactivity, we tested the effect of Notch activation in PVAT on vasoreactivity using wire myography. The aortae from the N1ICD;Adipoq-Cre mice had increased vasocontraction and decreased vasorelaxation in a PVAT-dependent and age-dependent manner. Our data provide support for the novel concept that increased Notch signaling in the adipose tissue leads to PVAT whitening, impaired mitochondrial function, increased ferroptosis, and loss of a protective vasodilatory signal. Our study advances our understanding of how Notch signaling in adipocytes affects mitochondrial dynamics, which impacts vascular physiology.
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Tecido Adiposo , Proteômica , Camundongos , Animais , Tecido Adiposo/metabolismo , Transdução de Sinais , Obesidade/metabolismo , Adiponectina/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismoRESUMO
Chronically stressing male mice can alter the behavior of their offspring across generations. This effect is thought to be mediated by stress-induced changes in the content of specific sperm miRNAs that modify embryo development after their delivery to oocytes at fertilization. A major problem with this hypothesis is that the levels of mouse sperm miRNAs are much lower than those present in preimplantation embryos. This makes it unclear how embryos could be significantly impacted without an amplification system to magnify changes in sperm miRNA content, like those present in lower organisms where transgenerational epigenetic inheritance is well established. Here, we describe such a system for Chronic Social Instability (CSI) stress that can explain how it reduces the levels of the miR-34b,c/449a,b family of miRNAs not only in sperm of exposed males but also in preimplantation embryos ( PIEs ) derived from their mating, as well as in sperm of male offspring. Sperm-derived miR-34c normally positively regulates expression of its own gene and that of miR-449 in PIEs. This feed forward, auto-amplification process is suppressed when CSI stress reduces sperm miR-34c levels. Its suppression is important for the transmission of traits to offspring because restoring miR-34c levels in PIEs from CSI stressed males, which also restores levels of miR-449 in them, suppresses elements of elevated anxiety and defective sociability normally found specifically in their female offspring, as well as reduced sperm miR-34 and miR-449 levels normally found in male offspring, who pass on these traits to their offspring. We previously published that the content of sperm miR-34/449 is also reduced in men raised in highly abusive and/or dysfunctional families. We show here that a similar miRNA auto-amplification system functions in human embryonic stem cells. This raises the possibility that PIEs in offspring of these men also display reduced levels of miR-34/449, enhancing the potential translational significance of these studies.
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BACKGROUND AND AIMS: Sprouty1 (Spry1) regulates the differentiation of vascular smooth muscle cells (VSMC), and our aim was to determine its role in atherogenesis. A significant proportion of cells within atherosclerotic lesions are derived from migration and pathological adaptation of medial VSMC. METHODS: We used global Spry1 null mouse, and Myh11-CreERT2, ROSA26-STOPfl/fl-tdTomato-Spry1fl/fl mice to allow for lineage tracing and conditional Spry1 deletion in VSMC. Atherosclerosis was induced by injection of a mutant form of mPCSK9D377Y-AAV followed by Western diet. Human aortic VSMC (hVSMC) with shRNA targeting of Spry1 were also analyzed. RESULTS: Global loss of Spry1 increased inflammatory markers ICAM1 and Cox2 in VSMC. Conditional deletion of Spry1 in VSMC had no effect on early lesion development, despite increased Sca1high cells. After 26 weeks of Western diet, mice with VSMC deletion of Spry1 had increased plaque burden, with reduced collagen content and smooth muscle alpha actin (SMA) in the fibrous cap. Lineage tracing via tdTomato marking Cre-recombined cells indicated that VSMC with loss of Spry1 had decreased migration into the lesion, noted by decreased proportions of tdTomato+ and tdTomato+/SMA + cells. Loss-of-function of Spry1 in hVSMC increased mesenchymal and activation markers, including KLF4, PDGFRb, ICAM1, and Cox2. Loss of Spry1 enhanced the effects of PDGFBB and TNFa on hVSMC. CONCLUSIONS: Loss of Spry1 in VSMC aggravated plaque formation at later stages, and increased markers of instability. Our results indicate that Spry1 suppresses the mesenchymal and inflammatory phenotype of VSMC, and its expression in VSMC is protective against chronic atherosclerotic disease.
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Aterosclerose , Placa Aterosclerótica , Animais , Humanos , Camundongos , Aterosclerose/genética , Aterosclerose/prevenção & controle , Aterosclerose/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fenótipo , Placa Aterosclerótica/metabolismoRESUMO
Neural communication between the brain and adipose tissues regulates energy expenditure and metabolism through modulation of adipose tissue functions. We have recently demonstrated that under pathophysiological conditions (obesity, diabetes, and aging), total subcutaneous white adipose tissue (scWAT) innervation is decreased ('adipose neuropathy'). With advanced age in the C57BL/6J mouse, small fiber peripheral nerve endings in adipose tissue die back, resulting in reduced contact with adipose-resident blood vessels and other cells. This vascular neuropathy and parenchymal neuropathy together likely pose a physiological challenge for tissue function. In the current work, we used the genetically diverse HET3 mouse model to investigate the incidence of peripheral neuropathy and adipose tissue dysregulation across several ages in both male and female mice. We also investigated the anti-aging treatment rapamycin, an mTOR inhibitor, as a means to prevent or reduce adipose neuropathy. We found that HET3 mice displayed a reduced neuropathy phenotype compared to inbred C56BL/6 J mice, indicating genetic contributions to this aging phenotype. Compared to female HET3 mice, male HET3 mice had worse neuropathic phenotypes by 62 weeks of age. Female HET3 mice appeared to have increased protection from neuropathy until advanced age (126 weeks), after reproductive senescence. We found that rapamycin overall had little impact on neuropathy measures, and actually worsened adipose tissue inflammation and fibrosis. Despite its success as a longevity treatment in mice, higher doses and longer delivery paradigms for rapamycin may lead to a disconnect between life span and beneficial health outcomes.
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Doenças do Sistema Nervoso Periférico , Sirolimo , Masculino , Feminino , Animais , Camundongos , Sirolimo/farmacologia , Longevidade/genética , Camundongos Endogâmicos C57BL , Tecido Adiposo/metabolismo , Obesidade/metabolismo , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/genéticaRESUMO
OBJECTIVE: Perivascular adipose tissue (PVAT) regulates vascular health. Dietary methionine restriction (MetR) impacts age-related adiposity, and this study addresses its effects in PVAT. METHODS: Male C57BL/6 mice at 8, 52, and 102 weeks of age were fed a standard (0.86%) or low-methionine (0.12%) diet for 52 weeks in 8-week-old and 52-week-old mice and for 15 weeks in 102-week-old mice. RESULTS: Mice with dietary MetR were resistant to weight gain and maintained a healthy blood profile. Aging increased lipid accumulation, and MetR reversed this phenotype. Notch signaling in inguinal white adipose tissue (iWAT) was decreased by MetR but increased in gonadal white adipose tissue. However, the Notch phenotype of brown adipose tissue (BAT) was not affected by MetR. Uncoupling protein 1 (UCP1) was increased in PVAT, iWAT, and BAT by MetR when initiated in young mice, but this effect was lost in middle-aged mice. CONCLUSIONS: Lipid in mouse PVAT peaked at 1 year of age, consistent with peak body mass. MetR reduced body weight, normalized metabolic parameters, and decreased lipid in PVAT in all age cohorts. Mice fed a MetR diet from early maturity to 1 year of age displayed an increased thermogenic adipocyte phenotype in iWAT, PVAT, and BAT, all tissues with thermogenic capacity.
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Tecido Adiposo Marrom , Metionina , Camundongos , Masculino , Animais , Metionina/metabolismo , Camundongos Endogâmicos C57BL , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Racemetionina/metabolismo , Racemetionina/farmacologia , LipídeosRESUMO
INTRODUCTION: We previously identified Notch2 in smooth muscle cells (SMC) in human atherosclerosis and found that signaling via Notch2 suppressed human SMC proliferation. Thus, we tested whether loss of Notch2 in SMC would alter atherosclerotic plaque progression using a mouse model. METHODS: Atherogenesis was examined at the brachiocephalic artery and aortic root in a vascular SMC null (inducible smooth muscle myosin heavy chain Cre) Notch2 strain on the ApoE-/- background. We measured plaque morphology and size, as well as lipid, inflammation, and smooth muscle actin content after Western diet. RESULTS: We generated an inducible SMC Notch2 null on the ApoE-/- background. We observed â¼90% recombination efficiency with no detectable Notch2 in the SMC. Loss of SMC Notch2 did not significantly change plaque size, lipid content, necrotic core, or medial area. However, loss of SMC Notch2 reduced the contractile SMC in brachiocephalic artery lesions and increased inflammatory content in aortic root lesions after 6 weeks of Western diet. These changes were not present with loss of SMC Notch2 after 14 weeks of Western diet. CONCLUSIONS: Our data show that loss of SMC Notch2 does not significantly reduce atherosclerotic lesion formation, although in early stages of plaque formation there are changes in SMC and inflammation.
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Aterosclerose , Miócitos de Músculo Liso , Placa Aterosclerótica , Receptor Notch2 , Animais , Camundongos , Actinas , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Receptor Notch2/genética , Receptor Notch2/metabolismo , Miosinas de Músculo LisoRESUMO
During blood vessel disease, vascular smooth muscle cell (VSMC) expansion and interaction with the matrix trigger changes in gene expression and phenotype. In this issue of the JCI, Dave et al. discover a signaling network that drives VSMC expansion and vascular obstruction caused by elastin insufficiency. Using a combination of gene-targeted mice, tissues and cells from patients with Williams-Beuren syndrome, and targeting of elastin in human VSMCs, the authors identified VSMC-derived NOTCH3 signaling as a critical mediator of aortic hypermuscularization and loss of vascular patency. NOTCH3-specific therapies or therapies that target downstream molecular pathways may provide opportunities to minimize VSMC growth and treat cardiovascular disease with minimal side effects.
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Elastina , Doenças Vasculares , Animais , Artérias , Elastina/genética , Elastina/metabolismo , Humanos , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Notch3/genética , Receptor Notch3/metabolismo , Doenças Vasculares/genética , Doenças Vasculares/metabolismoRESUMO
Quantification of adipocyte size and number is routinely performed for white adipose tissues using existing image analysis software. However, thermogenic adipose tissue has multilocular adipocytes, making it difficult to distinguish adipocyte cell borders and to analyze lipid proportion using existing methods. We developed a simple, standardized method to quantify lipid content of mouse thermogenic adipose tissue. This method, using FIJI analysis of hematoxylin/eosin stained sections, was highly objective and highly reproducible, with â¼99% inter-rater reliability. The method was compared to direct lipid staining of adipose tissue, with comparable results. We used our method to analyze perivascular adipose tissue (PVAT) from C57BL/6 mice on a normal chow diet, compared to calorie restriction or a high fat diet, where lipid storage phenotypes are known. Results indicate that lipid content can be estimated within mouse PVAT in a quantitative and reproducible manner, and shows correlation with previously studied molecular and physiological measures.
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Tecido Adiposo/metabolismo , Processamento de Imagem Assistida por Computador , Metabolismo dos Lipídeos , Microscopia Confocal , Microscopia de Fluorescência , Termogênese , Animais , Aorta Torácica , Restrição Calórica , Dieta Hiperlipídica , Corantes Fluorescentes , Indóis , Camundongos Endogâmicos C57BL , Fenótipo , Reprodutibilidade dos Testes , Coloração e RotulagemRESUMO
Brown adipose tissue can expend large amounts of energy, and therefore increasing its size or activity is a promising therapeutic approach to combat metabolic disease. In humans, major deposits of brown fat cells are found intimately associated with large blood vessels, corresponding to perivascular adipose tissue (PVAT). However, the cellular origins of PVAT are poorly understood. Here, we determine the identity of perivascular adipocyte progenitors in mice and humans. In mice, thoracic PVAT develops from a fibroblastic lineage, consisting of progenitor cells (Pdgfra+, Ly6a+ and Pparg-) and preadipocytes (Pdgfra+, Ly6a+ and Pparg+), which share transcriptional similarity with analogous cell types in white adipose tissue. Interestingly, the aortic adventitia of adult animals contains a population of adipogenic smooth muscle cells (Myh11+, Pdgfra- and Pparg+) that contribute to perivascular adipocyte formation. Similarly, human PVAT contains presumptive fibroblastic and smooth muscle-like adipocyte progenitor cells, as revealed by single-nucleus RNA sequencing. Together, these studies define distinct populations of progenitor cells for thermogenic PVAT, providing a foundation for developing strategies to augment brown fat activity.
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Adipócitos Marrons/fisiologia , Tecido Adiposo Marrom/fisiologia , Linhagem da Célula/fisiologia , Termogênese/fisiologia , Adipócitos Brancos/fisiologia , Adipogenia/fisiologia , Tecido Adiposo Marrom/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Aorta/citologia , Aorta/fisiologia , Vasos Sanguíneos/fisiologia , Linhagem da Célula/genética , Fibroblastos/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Recém-Nascido , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/fisiologia , Células-Tronco/fisiologia , Termogênese/genéticaRESUMO
OBJECTIVE: Perivascular adipose tissue (PVAT) surrounding arteries supports healthy vascular function. During obesity, PVAT loses its vasoprotective effect. We study pathological conversion of PVAT, which involves molecular changes in protein profiles and functional changes in adipocytes. Approach and Results: C57BL6/J mice were fed a 60% high-fat diet for 12 weeks or a cardioprotective 30% calorie-restricted diet for 5 weeks. Proteomic analysis identified PVAT as a molecularly distinct adipose depot, and novel markers for thermogenic adipocytes, such as GRP75 (stress-70 protein, mitochondrial), were identified. High-fat diet increased the similarity of protein signatures in PVAT and brown adipose, suggesting activation of a conserved whitening pathway. The whitening phenotype was characterized by suppression of UCP1 (uncoupling protein 1) and increased lipid deposition, leptin, and inflammation, and specifically in PVAT, elevated Notch signaling. Conversely, PVAT from calorie-restricted mice had decreased Notch signaling and less lipid. Using the Adipoq-Cre strain, we constitutively activated Notch1 signaling in adipocytes, which phenocopied the changes in PVAT caused by a high-fat diet, even on a standard diet. Preadipocytes from mouse PVAT expressed Sca1, CD140a, Notch1, and Notch2, but not CD105, showing differences compared with preadipocytes from other depots. Inhibition of Notch signaling during differentiation of PVAT-derived preadipocytes reduced lipid deposition and adipocyte marker expression. CONCLUSIONS: PVAT shares features with other adipose depots, but has a unique protein signature that is regulated by dietary stress. Increased Notch signaling in PVAT is sufficient to initiate the pathological conversion of PVAT by promoting adipogenesis and lipid accumulation and may thus prime the microenvironment for vascular disease.
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Adipócitos Brancos/metabolismo , Adipogenia , Tecido Adiposo Branco/metabolismo , Lipogênese , Obesidade/metabolismo , Receptores Notch/metabolismo , Adipócitos Brancos/patologia , Tecido Adiposo Branco/patologia , Adiposidade , Animais , Ataxina-1/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Restrição Calórica , Dieta Hiperlipídica , Modelos Animais de Doenças , Endoglina/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/genética , Obesidade/patologia , Fenótipo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Proteômica , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Notch/genética , Transdução de SinaisRESUMO
Small RNA molecules in early embryos, delivered from sperm to zygotes upon fertilization, are required for normal mouse embryonic development. Even modest changes in the levels of sperm-derived miRNAs appear to influence early embryos and subsequent development. For example, stress-associated behaviors develop in mice after injection into normal zygotes sets of sperm miRNAs elevated in stressed male mice. Here, we implicate early embryonic miR-409-3p in establishing anxiety levels in adult female, but not male mice. First, we found that exposure of male mice to chronic social instability stress, which leads to elevated anxiety in their female offspring across at least three generations through the paternal lineage, elevates sperm miR-409-3p levels not only in exposed males, but also in sperm of their F1 and F2 male offspring. Second, we observed that while injection of a mimic of miR-409-3p into zygotes from mating control males was incapable of mimicking this effect in offspring derived from them, injection of a specific inhibitor of this miRNA led to the opposite, anxiolytic effect in female, but not male, and offspring. These findings imply that baseline miR-409-3p activity in early female embryos is necessary for the expression of normal anxiety levels when they develop into adult females. In addition, elevated embryo miR-409-3p activity, possibly as a consequence of stress-induced elevation of its expression in sperm, may participate in, but may not be sufficient for, the induction of enhanced anxiety.
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Ansiedade/metabolismo , Embrião de Mamíferos/metabolismo , Epigênese Genética/fisiologia , MicroRNAs/fisiologia , Espermatozoides/metabolismo , Estresse Psicológico/metabolismo , Zigoto/metabolismo , Animais , Ansiedade/etiologia , Comportamento Animal/fisiologia , Feminino , Masculino , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Zigoto/efeitos dos fármacosRESUMO
Temporomandibular joint osteoarthritis (TMJOA) is a prevalent source of temporomandibular joint disorder (TMD). Women are more commonly diagnosed with TMD and are more likely to seek care at tertiary orofacial pain clinics. Limited knowledge regarding mechanisms underlying temporomandibular joint (TMJ) pain impairs development of improved pain management strategies. In a rat model of unilateral TMJOA, monosodium iodoacetate (MIA) produces joint pathology in a concentration-dependent manner. Unilateral MIA produces alterations in meal patterns in males and females without altering overnight time spent eating or weight across 2 weeks. Monosodium iodoacetate (80 mg/mL)-treated males develop ongoing pain within 2 weeks after MIA injection. Females develop ongoing pain at a 5-fold lower MIA concentration (16.6 mg/m). Monosodium iodoacetate (80 mg/mL)-treated males show spread of tactile hypersensitivity across the face during the first week after injection and then to the fore paws and hind paws during the second week after injection, indicating development of central sensitization. At the lower dose, female rats demonstrate a similar spread of tactile hypersensitivity, whereas male rats do not develop ongoing pain or spread of tactile hypersensitivity outside the area of the ipsilateral temporomandibular joint. These observations indicate that females have a higher susceptibility to development of ongoing pain and central sensitization compared with male rats that is not due to differences in MIA-induced joint pathology. This model of TMJOA pain can be used to explore sex differences in pain processes implicated in development of neuropathic pain, ongoing pain, and central sensitization, allowing for development of individualized strategies for prevention and treatment of TMD joint pain.
Assuntos
Modelos Animais de Doenças , Medição da Dor/métodos , Caracteres Sexuais , Síndrome da Disfunção da Articulação Temporomandibular/diagnóstico por imagem , Animais , Suscetibilidade a Doenças/diagnóstico por imagem , Suscetibilidade a Doenças/psicologia , Feminino , Masculino , Medição da Dor/psicologia , Ratos , Ratos Sprague-Dawley , Síndrome da Disfunção da Articulação Temporomandibular/psicologia , Microtomografia por Raio-X/métodosRESUMO
Overdevelopment of visceral adipose is positively correlated with the etiology of obesity-associated pathologies including cardiovascular disease and insulin resistance. However, identification of genetic, molecular, and physiological factors regulating adipose development and function in response to nutritional stress is incomplete. Fibroblast Growth Factor 1 (FGF1) is a cytokine expressed and released by both adipocytes and endothelial cells under hypoxia, thermal, and oxidative stress. Expression of Fibroblast Growth Factor 1 (FGF1) in adipose is required for normal depot development and remodeling. Loss of FGF1 leads to deleterious changes in adipose morphology, metabolism, and insulin resistance. Conversely, diabetic and obese mice injected with recombinant FGF1 display improvements in insulin sensitivity and a reduction in adiposity. We report in this novel, in vivo study that transgenic mice expressing an endothelial-specific FGF1 transgene (FGF1-Tek) are resistant to high-fat diet-induced abdominal adipose accretion and are more glucose-tolerant than wild-type control animals. Metabolic chamber analyses indicate that suppression of the development of visceral adiposity and insulin resistance was not associated with alterations in appetite or resting metabolic rate in the FGF1-Tek strain. Instead, FGF1-Tek mice display increased locomotor activity that likely promotes the utilization of dietary fatty acids before they can accumulate in adipose and liver. This study provides insight into the impact that genetic differences dictating the production of FGF1 has on the risk for developing obesity-related metabolic disease in response to nutritional stress.
Assuntos
Tecido Adiposo/metabolismo , Células Endoteliais/metabolismo , Fator 1 de Crescimento de Fibroblastos/genética , Locomoção/genética , Obesidade Abdominal/genética , Adipócitos/metabolismo , Adiposidade/efeitos dos fármacos , Adiposidade/genética , Animais , Glicemia/metabolismo , Dieta Hiperlipídica , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 1 de Crescimento de Fibroblastos/farmacologia , Insulina/sangue , Resistência à Insulina/genética , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Obesidade Abdominal/metabolismoRESUMO
Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson's Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.
Assuntos
Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/citologia , Aorta/citologia , Diferenciação Celular , Adipócitos/citologia , Adipogenia , Animais , Forma Celular , Células Cultivadas , Condrogênese , Humanos , Células-Tronco Mesenquimais/citologia , Osteogênese , Células Estromais/citologiaRESUMO
Atherosclerosis is the most common cause of heart disease and stroke. The use of animal models has advanced our understanding of the molecular signaling that contributes to atherosclerosis. Further understanding of this degenerative process in humans will require human tissue. Plaque removed during endarterectomy procedures to relieve arterial obstructions is usually discarded, but can be an important source of diseased cells. Resected tissue from carotid and femoral endarterectomy procedures were compared with carotid arteries from donors with no known cardiovascular disease. Vascular smooth muscle cells (SMC) contribute to plaque formation and may determine susceptibility to rupture. Notch signaling is implicated in the progression of atherosclerosis, and plays a receptor-specific regulatory role in SMC. We defined protein localization of Notch2 and Notch3 within medial and plaque SMC using immunostaining, and compared Notch2 and Notch3 levels in total plaques with whole normal arteries using immunoblot. We successfully derived SMC populations from multiple endarterectomy specimens for molecular analysis. To better define the protein signature of diseased SMC, we utilized sequential window acquisition of all theoretical spectra (SWATH) proteomic analysis to compare normal carotid artery SMC with endarterectomy-derived SMC. Similarities in protein profile and differentiation markers validated the SMC identity of our explants. We identified a subset of differentially expressed proteins that are candidates as functional markers of diseased SMC. To understand how Notch signaling may affect diseased SMC, we performed Jagged1 stimulation of primary cultures. In populations that displayed significant growth, Jagged1 signaling through Notch2 suppressed proliferation; cultures with low growth potential were non-responsive to Jagged1. In addition, Jagged1 did not promote contractile smooth muscle actin nor have a significant effect on the mature differentiated phenotype. Thus, SMC derived from atherosclerotic lesions show distinct proteomic profiles and have altered Notch signaling in response to Jagged1 as a differentiation stimulus, compared with normal SMC.