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5.
Cells ; 13(13)2024 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-38994980

RESUMO

The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PPi) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5'-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A2aR and A2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy.


Assuntos
5'-Nucleotidase , Adenosina , Proliferação de Células , Músculo Liso Vascular , Miócitos de Músculo Liso , Diester Fosfórico Hidrolases , Pirofosfatases , Transdução de Sinais , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/genética , Pirofosfatases/metabolismo , Pirofosfatases/genética , 5'-Nucleotidase/metabolismo , 5'-Nucleotidase/genética , Animais , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Adenosina/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Camundongos , Humanos , Monofosfato de Adenosina/metabolismo , Camundongos Endogâmicos C57BL , AMP Cíclico/metabolismo , Masculino , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia , Calcificação Vascular/genética
6.
PLoS One ; 19(7): e0303472, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38990864

RESUMO

Plasmid transfection in cells is widely employed to express exogenous proteins, offering valuable mechanistic insight into their function(s). However, plasmid transfection efficiency in primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs) is restricted with lipid-based transfection reagents such as Lipofectamine. The STING pathway, activated by foreign DNA in the cytosol, prevents foreign gene expression and induces DNA degradation. To address this, we explored the potential of STING inhibitors on the impact of plasmid expression in primary ECs and SMCs. Primary human aortic endothelial cells (HAECs) were transfected with a bicistronic plasmid expressing cytochrome b5 reductase 4 (CYB5R4) and enhanced green fluorescent protein (EGFP) using Lipofectamine 3000. Two STING inhibitors, MRT67307 and BX795, were added during transfection and overnight post-transfection. As a result, MRT67307 significantly enhanced CYB5R4 and EGFP expression, even 24 hours after its removal. In comparison, MRT67307 pretreatment did not affect transfection, suggesting the inhibitor's effect was readily reversible. The phosphorylation of endothelial nitric oxide synthase (eNOS) at Serine 1177 (S1177) by vascular endothelial growth factor is essential for endothelial proliferation, migration, and survival. Using the same protocol, we transfected wild-type and phosphorylation-incapable mutant (S1177A) eNOS in HAECs. Both forms of eNOS localized on the plasma membrane, but only the wild-type eNOS was phosphorylated by vascular endothelial growth factor treatment, indicating normal functionality of overexpressed proteins. MRT67307 and BX795 also improved plasmid expression in human and rat aortic SMCs. In conclusion, this study presents a modification enabling efficient plasmid transfection in primary vascular ECs and SMCs, offering a favorable approach to studying protein function(s) in these cell types, with potential implications for other primary cell types that are challenging to transfect.


Assuntos
Células Endoteliais , Proteínas de Membrana , Plasmídeos , Transfecção , Humanos , Plasmídeos/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/citologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Animais , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Células Cultivadas , Fosforilação , Ratos , Expressão Gênica , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo
7.
J Pineal Res ; 76(5): e12988, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38982751

RESUMO

Vulnerable atherosclerotic plaque rupture, the leading cause of fatal atherothrombotic events, is associated with an increased risk of mortality worldwide. Peroxisome proliferator-activated receptor delta (PPARδ) has been shown to modulate vascular smooth muscle cell (SMC) phenotypic switching, and, hence, atherosclerotic plaque stability. Melatonin reportedly plays a beneficial role in cardiovascular diseases; however, the mechanisms underlying improvements in atherosclerotic plaque vulnerability remain unknown. In this study, we assessed the role of melatonin in regulating SMC phenotypic switching and its consequential contribution to the amelioration of atherosclerotic plaque vulnerability and explored the mechanisms underlying this process. We analyzed features of atherosclerotic plaque vulnerability and markers of SMC phenotypic transition in high-cholesterol diet (HCD)-fed apolipoprotein E knockout (ApoE-/-) mice and human aortic SMCs (HASMCs). Melatonin reduced atherosclerotic plaque size and necrotic core area while enhancing collagen content, fibrous cap thickness, and smooth muscle alpha-actin positive cell coverage on the plaque cap, which are all known phenotypic characteristics of vulnerable plaques. In atherosclerotic lesions, melatonin significantly decreased the synthetic SMC phenotype and KLF4 expression and increased the expression of PPARδ, but not PPARα and PPARγ, in HCD-fed ApoE-/- mice. These results were subsequently confirmed in the melatonin-treated HASMCs. Further analysis using PPARδ silencing and immunoprecipitation assays revealed that PPARδ plays a role in the melatonin-induced SMC phenotype switching from synthetic to contractile. Collectively, we provided the first evidence that melatonin mediates its protective effect against plaque destabilization by enhancing PPARδ-mediated SMC phenotypic switching, thereby indicating the potential of melatonin in treating atherosclerosis.


Assuntos
Fator 4 Semelhante a Kruppel , Melatonina , Miócitos de Músculo Liso , PPAR delta , Placa Aterosclerótica , Animais , Melatonina/farmacologia , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Camundongos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Fator 4 Semelhante a Kruppel/metabolismo , Humanos , PPAR delta/metabolismo , PPAR delta/genética , Camundongos Knockout , Masculino , Camundongos Knockout para ApoE , Fenótipo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Apolipoproteínas E/deficiência , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/efeitos dos fármacos , Camundongos Endogâmicos C57BL
8.
Ecotoxicol Environ Saf ; 281: 116681, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38964063

RESUMO

Fluoride exposure has been implicated as a potential risk factor for hypertension, but the underlying mechanisms remain unclear. This study investigated the role of the RhoA/ROCK signaling pathway in fluoride-induced hypertension. Male Wistar rats were divided into different groups and exposed to varying concentrations of sodium fluoride (NaF) or sodium chloride (NaCl) via drinking water. The rats' blood pressure was measured, and their aortic tissue was utilized for high-throughput sequencing analysis. Additionally, rat and A7r5 cell models were established using NaF and/or Fasudil. The study evaluated the effects of fluoride exposure on blood pressure, pathological changes in the aorta, as well as the protein/mRNA expression levels of phenotypic transformation indicators (a-SMA, calp, OPN) in vascular smooth muscle cells (VSMCs), along with the RhoA/ROCK signaling pathway (RhoA, ROCK1, ROCK2, MLC/p-MLC). The results demonstrated that fluoride exposure in rats led to increased blood pressure. High-throughput sequencing analysis revealed differential gene expression associated with vascular smooth muscle contraction, with the RhoA/ROCK signaling pathway emerging as a key regulator. Pathological changes in the rat aorta, such as elastic membrane rupture and collagen fiber deposition, were observed following NaF exposure. However, fasudil, a ROCK inhibitor, mitigated these pathological changes. Both in vitro and in vivo models confirmed the activation of the RhoA/ROCK signaling pathway and the phenotypic transformation of VSMCs from a contractile to a synthetic state upon fluoride exposure. Fasudil effectively inhibited the activities of ROCK1 and ROCK2 and attenuated the phenotypic transformation of VSMCs. In conclusion, fluoride has the potential to induce hypertension through the activation of the RhoA/ROCK signaling pathway and phenotypic changes in vascular smooth muscle cells. These results provide new insights into the mechanism of fluoride-induced hypertension.


Assuntos
Hipertensão , Músculo Liso Vascular , Ratos Wistar , Transdução de Sinais , Quinases Associadas a rho , Animais , Quinases Associadas a rho/metabolismo , Masculino , Hipertensão/induzido quimicamente , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Fluoreto de Sódio/toxicidade , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Fenótipo , Pressão Sanguínea/efeitos dos fármacos , Fluoretos/toxicidade , Proteínas rho de Ligação ao GTP
9.
Sci Rep ; 14(1): 16323, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-39009669

RESUMO

Vascular calcification, which is a major complication of diabetes mellitus, is an independent risk factor for cardiovascular disease. Osteogenic differentiation of vascular smooth muscle cells (VSMCs) is one of the key mechanisms underlying vascular calcification. Emerging evidence suggests that macrophage-derived extracellular vesicles (EVs) may be involved in calcification within atherosclerotic plaques in patients with diabetes mellitus. However, the role of macrophage-derived EVs in the progression of vascular calcification is largely unknown. In this study, we investigated whether macrophage-derived EVs contribute to the osteogenic differentiation of VSMCs under high glucose conditions. We isolated EVs that were secreted by murine peritoneal macrophages under normal glucose (EVs-NG) or high glucose (EVs-HG) conditions. miRNA array analysis in EVs from murine macrophages showed that miR-17-5p was significantly increased in EVs-HG compared with EVs-NG. Prediction analysis with miRbase identified transforming growth factor ß receptor type II (TGF-ß RII) as a potential target of miR-17-5p. EVs-HG as well as miR-17-5p overexpression with lipid nanoparticles inhibited the gene expression of Runx2, and TGF-ß RII. Furthermore, we demonstrated that VSMCs transfected with miR-17-5p mimic inhibited calcium deposition. Our findings reveal a novel role of macrophage-derived EVs in the negative regulation of osteogenic differentiation in VSMCs under high glucose conditions.


Assuntos
Diferenciação Celular , Vesículas Extracelulares , Glucose , MicroRNAs , Músculo Liso Vascular , Miócitos de Músculo Liso , Osteogênese , Transdução de Sinais , Fator de Crescimento Transformador beta , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologia , Glucose/farmacologia , Glucose/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Fator de Crescimento Transformador beta/metabolismo , Camundongos , Miócitos de Músculo Liso/metabolismo , Vesículas Extracelulares/metabolismo , Calcificação Vascular/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/patologia , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Masculino , Camundongos Endogâmicos C57BL , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética
10.
J Cell Mol Med ; 28(13): e18454, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-39010253

RESUMO

Studies have demonstrated a close correlation between MicroRNA and the occurrence of aortic dissection (AD). However, the molecular mechanisms underlying this relationship have not been fully elucidated and further exploration is still required. In this study, we found that miR-485-3p was significantly upregulated in human aortic dissection tissues. Meanwhile, we constructed in vitro AD models in HAVSMCs, HAECs and HAFs and found that the expression of miR-485-3p was increased only in HAVSMCs. Overexpression or knockdown of miR-485-3p in HAVSMCs could regulate the expression of inflammatory cytokines IL1ß, IL6, TNF-α, and NLRP3, as well as the expression of apoptosis-related proteins BAX/BCL2 and Cleaved caspase3/Caspase3. In the in vivo AD model, we have observed that miR-485-3p regulates vascular inflammation and apoptosis, thereby participating in the modulation of AD development in mice. Based on target gene prediction, we have validated that SIRT1 is a downstream target gene of miR-485-3p. Furthermore, by administering SIRT1 agonists and inhibitors to mice, we observed that the activation of SIRT1 alleviates vascular inflammation and apoptosis, subsequently reducing the incidence of AD. Additionally, functional reversal experiments revealed that overexpression of SIRT1 in HAVSMCs could reverse the cell inflammation and apoptosis mediated by miR-485-3p. Therefore, our research suggests that miR-485-3p can aggravate inflammation and apoptosis in vascular smooth muscle cells by suppressing the expression of SIRT1, thereby promoting the progression of aortic dissection.


Assuntos
Dissecção Aórtica , Apoptose , MicroRNAs , Músculo Liso Vascular , Miócitos de Músculo Liso , Sirtuína 1 , Animais , Humanos , Masculino , Camundongos , Dissecção Aórtica/genética , Dissecção Aórtica/metabolismo , Dissecção Aórtica/patologia , Apoptose/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Sirtuína 1/metabolismo , Sirtuína 1/genética
11.
Nat Commun ; 15(1): 5985, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39013850

RESUMO

The mechanism by which aging induces aortic aneurysm and dissection (AAD) remains unclear. A total of 430 participants were recruited for the screening of differentially expressed plasma microRNAs (miRNAs). We found that miR-1204 is significantly increased in both the plasma and aorta of elder patients with AAD and is positively correlated with age. Cell senescence induces the expression of miR-1204 through p53 interaction with plasmacytoma variant translocation 1, and miR-1204 induces vascular smooth muscle cell (VSMC) senescence to form a positive feedback loop. Furthermore, miR-1204 aggravates angiotensin II-induced AAD formation, and inhibition of miR-1204 attenuates ß-aminopropionitrile monofumarate-induced AAD development in mice. Mechanistically, miR-1204 directly targets myosin light chain kinase (MYLK), leading to the acquisition of a senescence-associated secretory phenotype (SASP) by VSMCs and loss of their contractile phenotype. MYLK overexpression reverses miR-1204-induced VSMC senescence, SASP and contractile phenotypic changes, and the decrease of transforming growth factor-ß signaling pathway. Our findings suggest that aging aggravates AAD via the miR-1204-MYLK signaling axis.


Assuntos
Envelhecimento , Aneurisma Aórtico , Dissecção Aórtica , Senescência Celular , MicroRNAs , Músculo Liso Vascular , Quinase de Cadeia Leve de Miosina , Transdução de Sinais , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Camundongos , Quinase de Cadeia Leve de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/genética , Envelhecimento/genética , Envelhecimento/metabolismo , Masculino , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Dissecção Aórtica/metabolismo , Dissecção Aórtica/genética , Dissecção Aórtica/patologia , Aneurisma Aórtico/metabolismo , Aneurisma Aórtico/genética , Aneurisma Aórtico/patologia , Miócitos de Músculo Liso/metabolismo , Camundongos Endogâmicos C57BL , Feminino , Fator de Crescimento Transformador beta/metabolismo , Modelos Animais de Doenças , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Angiotensina II/metabolismo , Proteínas de Ligação ao Cálcio
12.
Clin Sci (Lond) ; 138(14): 883-900, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-38959295

RESUMO

Hypertension is a leading risk factor for disease burden worldwide. Vascular contraction and remodeling contribute to the development of hypertension. Glutathione S-transferase P1 (Gstp1) plays several critical roles in both normal and neoplastic cells. In this study, we investigated the effect of Gstp1 on hypertension as well as on vascular smooth muscle cell (VSMC) contraction and phenotypic switching. We identified the higher level of Gstp1 in arteries and VSMCs from hypertensive rats compared with normotensive rats for the first time. We then developed Adeno-associated virus 9 (AAV9) mediated Gstp1 down-regulation and overexpression in rats and measured rat blood pressure by using the tail-cuff and the carotid catheter method. We found that the blood pressure of spontaneously hypertensive rats (SHR) rose significantly with Gstp1 down-regulation and reduced apparently after Gstp1 overexpression. Similar results were obtained from the observations of 2-kidney-1-clip renovascular (2K1C) hypertensive rats. Gstp1 did not influence blood pressure of normotensive Wistar-Kyoto (WKY) rats and Sprague-Dawley (SD) rats. Further in vitro study indicated that Gstp1 knockdown in SHR-VSMCs promoted cell proliferation, migration, dedifferentiation and contraction, while Gstp1 overexpression showed opposite effects. Results from bioinformatic analysis showed that the Apelin/APLNR system was involved in the effect of Gstp1 on SHR-VSMCs. The rise in blood pressure of SHR induced by Gstp1 knockdown could be reversed by APLNR antagonist F13A. We further found that Gstp1 enhanced the association between APLNR and Nedd4 E3 ubiquitin ligases to induce APLNR ubiquitination degradation. Thus, in the present study, we discovered a novel anti-hypertensive role of Gstp1 in hypertensive rats and provided the experimental basis for designing an effective anti-hypertensive therapeutic strategy.


Assuntos
Pressão Sanguínea , Glutationa S-Transferase pi , Hipertensão , Músculo Liso Vascular , Ubiquitina-Proteína Ligases Nedd4 , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Sprague-Dawley , Ubiquitinação , Animais , Masculino , Ratos , Proliferação de Células , Glutationa S-Transferase pi/metabolismo , Glutationa S-Transferase pi/genética , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ubiquitina-Proteína Ligases Nedd4/genética
13.
FASEB J ; 38(13): e23707, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38995239

RESUMO

Abdominal aortic aneurysm (AAA) is a life-threatening disease characterized by extensive membrane destruction in the vascular wall that is closely associated with vascular smooth muscle cell (VSMC) phenotypic switching. A thorough understanding of the changes in regulatory factors during VSMC phenotypic switching is essential for managing AAA therapy. In this study, we revealed the impact of NRF2 on the modulation of VSMC phenotype and the development of AAA based on single-cell RNA sequencing analysis. By utilizing a murine model of VSMC-specific knockout of nuclear factor E2-related factor 2 (NRF2), we observed that the absence of NRF2 in VSMCs exacerbated AAA formation in an angiotensin II-induced AAA model. The downregulation of NRF2 promoted VSMC phenotypic switching, leading to an enhanced inflammatory response. Through genome-wide transcriptome analysis and loss- or gain-of-function experiments, we discovered that NRF2 upregulated the expression of VSMC contractile phenotype-specific genes by facilitating microRNA-145 (miR-145) expression. Our data identified NRF2 as a novel regulator involved in maintaining the VSMC contractile phenotype while also influencing AAA formation through an miR-145-dependent regulatory mechanism.


Assuntos
Aneurisma da Aorta Abdominal , MicroRNAs , Músculo Liso Vascular , Miócitos de Músculo Liso , Fator 2 Relacionado a NF-E2 , Fenótipo , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Animais , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Masculino , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Camundongos Knockout , Análise de Célula Única , Camundongos Endogâmicos C57BL , Angiotensina II/farmacologia , Análise de Sequência de RNA , Modelos Animais de Doenças
14.
Ren Fail ; 46(2): 2367708, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38973391

RESUMO

BACKGROUND: Cellular senescence, macrophages infiltration, and vascular smooth muscle cells (VSMCs) osteogenic transdifferentiation participate in the pathophysiology of vascular calcification in chronic kidney disease (CKD). Senescent macrophages are involved in the regulation of inflammation in pathological diseases. In addition, senescent cells spread senescence to neighboring cells via Interferon-induced transmembrane protein3 (IFITM3). However, the role of senescent macrophages and IFITM3 in VSMCs calcification remains unexplored. AIMS: To explore the hypothesis that senescent macrophages contribute to the calcification and senescence of VSMCs via IFITM3. METHODS: Here, the macrophage senescence model was established using Lipopolysaccharides (LPS). The VSMCs were subjected to supernatants from macrophages (MCFS) or LPS-induced macrophages (LPS-MCFS) in the presence or absence of calcifying media (CM). Senescence-associated ß-galactosidase (SA-ß-gal), Alizarin red (AR), immunofluorescent staining, and western blot were used to identify cell senescence and calcification. RESULTS: The expression of IFITM3 was significantly increased in LPS-induced macrophages and the supernatants. The VSMCs transdifferentiated into osteogenic phenotype, expressing higher osteogenic differentiation markers (RUNX2) and lower VSMCs constructive makers (SM22α) when cultured with senescent macrophages supernatants. Also, senescence markers (p16 and p21) in VSMCs were significantly increased by senescent macrophages supernatants treated. However, IFITM3 knockdown inhibited this process. CONCLUSIONS: Our study showed that LPS-induced senescence of macrophages accelerated the calcification of VSMCs via IFITM3. These data provide a new perspective linking VC and aging, which may provide clues for diagnosing and treating accelerated vascular aging in patients with CKD.


Assuntos
Senescência Celular , Lipopolissacarídeos , Macrófagos , Proteínas de Membrana , Músculo Liso Vascular , Proteínas de Ligação a RNA , Calcificação Vascular , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Lipopolissacarídeos/farmacologia , Calcificação Vascular/patologia , Calcificação Vascular/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/metabolismo , Humanos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Células Cultivadas , Animais , Osteogênese , Transdiferenciação Celular
15.
Biol Pharm Bull ; 47(7): 1288-1295, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39010214

RESUMO

The active form of discoidin domain receptors (DDRs) is expressed in cell surface and regulated post-translationally by glucose. The DDR2 and DDR1 transfected in HEK293 cells were expressed mainly in their active forms with sizes of 130 and 120 kDa, respectively. DDRs were observed predominantly as 100 kDa proteins in glucose-depleted culture conditions. However, transfection of endothelial growth factor receptor (EGFR) in HEK293 cells resulted in the expression of only one form regardless of glucose concentration. Vascular smooth muscle cells, HT1080s, and MDA-MB-231 cancer cells expressed DDRs in their active forms in high glucose concentrations, which did not occur with EGFR. In diabetic rats, DDRs were expressed at high levels in arterial tissue but EGFR was not highly expressed. Taken together, these results suggest that DDRs expression depends on glucose concentration it may cooperate in the development of atherosclerosis and kidney fibroblasts, promoting nephropathy in diabetic rats.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Glucose , Animais , Humanos , Glucose/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/genética , Masculino , Diabetes Mellitus Experimental/metabolismo , Células HEK293 , Ratos , Artérias/metabolismo , Artérias/patologia , Receptores ErbB/metabolismo , Receptores ErbB/genética , Linhagem Celular Tumoral , Receptor com Domínio Discoidina 2/metabolismo , Receptor com Domínio Discoidina 2/genética , Músculo Liso Vascular/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Ratos Wistar
16.
Int J Mol Sci ; 25(13)2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-39000009

RESUMO

Yohimbine (YHB) has been reported to possess anti-inflammatory, anticancer, and cardiac function-enhancing properties. Additionally, it has been reported to inhibit the proliferation, migration, and neointimal formation of vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor (PDGF) stimulation by suppressing the phospholipase C-gamma 1 pathway. However, the transcriptional regulatory mechanism of YHB controlling the behavior of VSMCs is not fully understood. In this study, YHB downregulated the expression of cell cycle regulatory proteins, such as proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4), and cyclin E, by modulating the transcription factor FOXO3a in VSMCs induced by PDGF. Furthermore, YHB decreased p-38 and mTOR phosphorylation in a dose-dependent manner. Notably, YHB significantly reduced the phosphorylation at Y397 and Y925 sites of focal adhesion kinase (FAK), and this effect was greater at the Y925 site than Y397. In addition, the expression of paxillin, a FAK-associated protein known to bind to the Y925 site of FAK, was significantly reduced by YHB treatment in a dose-dependent manner. A pronounced reduction in the migration and proliferation of VSMCs was observed following co-treatment of YHB with mTOR or p38 inhibitors. In conclusion, this study shows that YHB inhibits the PDGF-induced proliferation and migration of VSMCs by regulating the transcription factor FOXO3a and the mTOR/p38/FAK signaling pathway. Therefore, YHB may be a potential therapeutic candidate for preventing and treating cardiovascular diseases such as atherosclerosis and vascular restenosis.


Assuntos
Movimento Celular , Proliferação de Células , Proteína Forkhead Box O3 , Músculo Liso Vascular , Miócitos de Músculo Liso , Fator de Crescimento Derivado de Plaquetas , Ioimbina , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Proteína Forkhead Box O3/metabolismo , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Animais , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ioimbina/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Células Cultivadas , Paxilina/metabolismo , Ratos Sprague-Dawley , Masculino
17.
Int J Mol Sci ; 25(13)2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-39000253

RESUMO

It has been reported that, in the spontaneously hypertensive rat (SHR) model of hypertension, different components of the G-protein/adenylate cyclase (AC)/Calcium-activated potassium channel of high conductance (BK) channel signaling pathway are altered differently. In the upstream part of the pathway (G-protein/AC), a comparatively low efficacy has been established, whereas downstream BK currents seem to be increased. Thus, the overall performance of this signaling pathway in SHR is elusive. For a better understanding, we focused on one aspect, the direct targeting of the BK channel by the G-protein/AC pathway and tested the hypothesis that the comparatively low AC pathway efficacy in SHR results in a reduced agonist-induced stimulation of BK currents. This hypothesis was investigated using freshly isolated smooth muscle cells from WKY and SHR rat tail artery and the patch-clamp technique. It was observed that: (1) single BK channels have similar current-voltage relationships, voltage-dependence and calcium sensitivity; (2) BK currents in cells with a strong buffering of the BK channel activator calcium have similar current-voltage relationships; (3) the iloprost-induced concentration-dependent increase of the BK current is larger in WKY compared to SHR; (4) the effects of activators of the PKA pathway, the catalytic subunit of PKA and the potent and selective cAMP-analogue Sp-5,6-DCl-cBIMPS on BK currents are similar. Thus, our data suggest that the lower iloprost-induced stimulation of the BK current in freshly isolated rat tail artery smooth muscle cells from SHR compared with WKY is due to the lower efficacy of upstream elements of the G-Protein/AC/BK channel pathway.


Assuntos
Cálcio , Hipertensão , Iloprosta , Canais de Potássio Ativados por Cálcio de Condutância Alta , Músculo Liso Vascular , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Vasodilatadores , Animais , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologia , Ratos , Cálcio/metabolismo , Iloprosta/farmacologia , Hipertensão/metabolismo , Hipertensão/tratamento farmacológico , Vasodilatadores/farmacologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Masculino , Artérias/efeitos dos fármacos , Artérias/metabolismo , Cauda/irrigação sanguínea , Transdução de Sinais/efeitos dos fármacos
18.
Int J Mol Sci ; 25(13)2024 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-39000533

RESUMO

Vascular calcification (VC) is a cardiovascular disease characterized by calcium salt deposition in vascular smooth muscle cells (VSMCs). Standard in vitro models used in VC investigations are based on VSMC monocultures under static conditions. Although these platforms are easy to use, the absence of interactions between different cell types and dynamic conditions makes these models insufficient to study key aspects of vascular pathophysiology. The present study aimed to develop a dynamic endothelial cell-VSMC co-culture that better mimics the in vivo vascular microenvironment. A double-flow bioreactor supported cellular interactions and reproduced the blood flow dynamic. VSMC calcification was stimulated with a DMEM high glucose calcification medium supplemented with 1.9 mM NaH2PO4/Na2HPO4 (1:1) for 7 days. Calcification, cell viability, inflammatory mediators, and molecular markers (SIRT-1, TGFß1) related to VSMC differentiation were evaluated. Our dynamic model was able to reproduce VSMC calcification and inflammation and evidenced differences in the modulation of effectors involved in the VSMC calcified phenotype compared with standard monocultures, highlighting the importance of the microenvironment in controlling cell behavior. Hence, our platform represents an advanced system to investigate the pathophysiologic mechanisms underlying VC, providing information not available with the standard cell monoculture.


Assuntos
Diferenciação Celular , Técnicas de Cocultura , Músculo Liso Vascular , Miócitos de Músculo Liso , Calcificação Vascular , Humanos , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Células Cultivadas , Sobrevivência Celular , Fator de Crescimento Transformador beta1/metabolismo , Sirtuína 1/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Reatores Biológicos
19.
Elife ; 132024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38985140

RESUMO

Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries and play a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular disease and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating dedifferentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that arterial specification of CoW endothelial cells (ECs) occurs after their migration from cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors after they were recruited to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity and wall shear stress. Furthermore, pulsatile flow induces differentiation of human brain PDGFRB+ mural cells into VSMCs, and blood flow is required for VSMC differentiation on zebrafish CoW arteries. Consistently, flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight blood flow activation of endothelial klf2a as a mechanism regulating initial VSMC differentiation on vertebrate brain arteries.


Assuntos
Diferenciação Celular , Círculo Arterial do Cérebro , Hemodinâmica , Músculo Liso Vascular , Peixe-Zebra , Animais , Círculo Arterial do Cérebro/embriologia , Músculo Liso Vascular/fisiologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Humanos , Miócitos de Músculo Liso/fisiologia , Miócitos de Músculo Liso/metabolismo , Células Endoteliais/fisiologia , Células Endoteliais/metabolismo
20.
Front Immunol ; 15: 1412022, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38881898

RESUMO

Abdominal aortic aneurysm (AAA) is a degenerative disease characterized by local abnormal dilation of the aorta accompanied by vascular smooth muscle cell (VSMC) dysfunction and chronic inflammation. VSMC dedifferentiation, transdifferentiation, and increased expression of matrix metalloproteinases (MMPs) are essential causes of AAA formation. Previous studies from us and others have shown that Anemoside B4 (AB4), a saponin from Pulsatilla chinensis, has anti-inflammatory, anti-tumor, and regulatory effects on VSMC dedifferentiation. The current study aimed to investigate whether AB4 inhibits AAA development and its underlying mechanisms. By using an Ang II induced AAA model in vivo and cholesterol loading mediated VSMC to macrophage transdifferentiation model in vitro, our study demonstrated that AB4 could attenuate AAA pathogenesis, prevent VSMC dedifferentiation and transdifferentiation to macrophage-like cells, decrease vascular inflammation, and suppress MMP expression and activity. Furthermore, KLF4 overexpression attenuated the effects of AB4 on VSMC to macrophage-like cell transition and VSMC inflammation in vitro. In conclusion, AB4 protects against AAA formation in mice by inhibiting KLF4 mediated VSMC transdifferentiation and inflammation. Our study provides the first proof of concept of using AB4 for AAA management.


Assuntos
Aneurisma da Aorta Abdominal , Transdiferenciação Celular , Inflamação , Fator 4 Semelhante a Kruppel , Miócitos de Músculo Liso , Saponinas , Animais , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/prevenção & controle , Aneurisma da Aorta Abdominal/induzido quimicamente , Transdiferenciação Celular/efeitos dos fármacos , Fator 4 Semelhante a Kruppel/metabolismo , Camundongos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Inflamação/metabolismo , Saponinas/farmacologia , Modelos Animais de Doenças , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Angiotensina II/farmacologia , Humanos
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