Vascular Adventitial Fibroblasts Research Articles

Protection from hypertension phenotype by a mitochondrial division inhibitor in mice infused with angiotensin II

Mitochondrial dysfunction has been implicated in various types of cardiovascular diseases including hypertension. Mitochondrial fission fusion balance is critical for mitochondrial quality control, whereas enhanced fission has been reported in several models of cardiovascular diseases. However, limited information is available regarding the contribution of mitochondrial fission in hypertension. Here, we have tested our hypothesis that inhibition of mitochondrial fission attenuates development of hypertension and associated vascular remodeling. C57BL6 mice were infused with angiotensin II for 2 weeks with or without treatment of mitochondrial fission inhibitor mdivi1. Cultured vascular endothelial cells and adventitial fibroblasts from rat aortas were stimulated with angiotensin II in the presence or absence of mdivi1. In C57BL6 mice infused with angiotensin II for 2 weeks, co-treatment of mitochondrial fission inhibitor mdivi1 significantly inhibited angiotensin II-induced development of hypertension assessed by telemetry. Histological assessment of hearts and aortas demonstrated that mdivi1 inhibited vessel fibrosis and hypertrophy induced by angiotensin II. This was associated with attenuation of angiotensin II-induced decline in mitochondrial aspect ratio seen in both endothelium and media of aortas. Mdivi1 also mitigated angiotensin II-induced cardiac hypertrophy assessed by heart weight body weight ratio as well as by echocardiogram. Proteomic analysis on endothelial cell culture media with angiotensin II and/or mdivi1 treatment revealed that mdivi1 inhibited endothelial cell hypersecretory phenotype induced by angiotensin II. In addition, mdivi1 attenuated angiotensin II-induced protein induction of periostin, a myofibroblast marker in cultured vascular fibroblasts. In conclusion, these data suggest that mdivi1 prevented angiotensin II-induced hypertension and cardiovascular remodeling via multicellular mechanisms in the vasculature. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3136: Protection From Hypertension Phenotype By A Mitochondrial Division Inhibitor In Mice Infused With Angiotensin II

Mitochondrial dysfunction has been implicated in various types of cardiovascular diseases including hypertension. Mitochondrial fission fusion balance is critical for mitochondrial quality control, whereas enhanced fission has been reported in several models of cardiovascular diseases. However, limited information is available regarding the contribution of mitochondrial fission in hypertension. The goal of this study is to explore the role of mitochondrial fission in hypertension and associated vascular pathology. Accordingly, we have tested our hypothesis that inhibition of mitochondrial fission attenuates development of hypertension and associated vascular remodeling. C57BL6 mice were infused with 1000 ng/kg/min angiotensin II for 2 weeks with or without treatment of mitochondrial fission inhibitor mdivi1 (25 mg/kg/day). Cultured vascular endothelial cells and adventitial fibroblasts from rat aortas were stimulated with 100 nM angiotensin II in the presence or absence of mdivi1 (20 uM). In C57BL6 mice infused with angiotensin II for 2 weeks, co-treatment of mitochondrial fission inhibitor mdivi1 significantly inhibited angiotensin II-induced development of hypertension assessed by telemetry (mean 149 vs 119 mmHg, day 9 p<0.01). Histological assessment of hearts and aortas demonstrated that mdivi1 inhibited vessel fibrosis and hypertrophy induced by angiotensin II. This was associated with attenuation of angiotensin II-induced decline in mitochondrial aspect ratio seen in both endothelium and media of aortas (aortas 1.620 vs 2.168, p<0.001). Mdivi1 also mitigated angiotensin II-induced cardiac hypertrophy assessed by heart weight body weight ratio (7.823 vs 6.336 mg/g, p<0.001) as well as by echocardiogram. Proteomic analysis on endothelial cell culture media with angiotensin II and/or mdivi1 treatment revealed that mdivi1 inhibited endothelial cell hypersecretory phenotype induced by angiotensin II. In addition, mdivi1 attenuated angiotensin II-induced protein induction of periostin, a myofibroblast marker in cultured vascular fibroblasts. In conclusion, these data suggest that mdivi1 prevented angiotensin II-induced hypertension and cardiovascular remodeling via multicellular mechanisms in the vasculature.

Shenlian extract improves atherosclerosis by relieving adventitial inflammation

Ethnopharmacological relevanceShenlian (SL) extract, a Chinese medicinal compound mainly pointing at inflammation response of atherosclerosis, is composed of Salvia miltiorrhizae Bunge and Andrographis paniculata (Burm.f.) Nees. Salvia miltiorrhizae Bunge has been reported to activate blood to remove stasis, while another herb, Andrographis paniculata (Burm.f.) Nees, has been revealed to clear endogenous heat toxins. The anti-atherosclerotic effects of these two herbs have been reported closely relating to inflammation. However, from the point of view of adventitial inflammation, the in-depth study of SL extract in anti-atherosclerotic effects by relieving adventitial inflammation is still unknown. Aim of the studyTo explore the effects of adventitial inflammation in atherosclerosis progression and if SL extract could reverse the process. Materials and methodsA novel atherosclerosis model based on adventitial inflammation was established. High-fat diet-fed ApoE-/- mice were implanted a cotton thread soaked with LPS on the right common carotid artery (RCCA). Meanwhile, three time points were set (week 2, 4, and 12) to accurately evaluate the effect of SL extract on the whole process of atherosclerosis with adventitial inflammation. The pathological changes of phenotype transformation of VAFs, vascular cell proliferation and collagen synthesis were observed dynamically by immunohistochemistry (IHC), BrdU method and sirius red staining. Then primary VAFs were stimulated by LPS to mirror the process of adventitial inflammation in vitro. The VAFS phenotype conversion and its function alterations including proliferation, migration, inflammatory secretion was assessed. Finally, we established a co-culture model of activated VAFs and vascular smooth muscle cells (VSMCs) to observe the impacts of activated VAFs on phenotype transformation and migration of VSMCs. ResultsSL extract improved atherosclerosis progression by reducing lipid content, adventitial inflammation and plaque formation. HE results showed sham-operated group (Sham) appeared light infiltrated inflammation only in adventitia at week 2, and the degree of inflammation infiltrated in model was more severe than that in Sham at week 2, 4, and 12. At week 12, the sham and model group showed evidently thickened media and intima. The phenotypic transformation, proliferation and migration of vascular adventitial fibroblasts (VAFs) as well as inflammatory secretion enhanced remarkably in vivo and vitro, but SL extract reversed these changes. Moreover, SL extract downregulated JAK2-STAT3-MMP2 signal pathway. The VSMCs transformed from contractile phenotype into synthetic phenotype and the migration of VSMCs increased after co-culture with activated VAFs. In contrast, SL extract could suppress theses effects. ConclusionsTaken together, atherosclerotic inflammation could be a “outside-in” signaling. Adventitial inflammation not only accelerated intimal plaque formation in atherosclerosis, but also worsened the degree of vascular lesion. And SL extract improved atherosclerosis by relieving adventitial inflammation, and the underlying mechanisms could be associated with curbing phenotypic transformation, proliferation and migration of VAFs and VSMCs.

Effects of norepinephrine‑induced activation of rat vascular adventitial fibroblasts on proliferation and migration of BMSCs involved in vascular remodeling.

Vascular remodeling caused by vascular injury such as hypertension and atherosclerosis is a complex process involving a variety of cells and factors, and the mechanism is unclear. A vascular injury model was simulated by adding norepinephrine (NE) to culture medium of vascular adventitial fibroblasts (AFs). NE induced activation and proliferation of AFs. To investigate the association between the AFs activation and bone marrow mesenchymal stem cells (BMSCs) differentiation in vascular remodeling. BMSCs were cultured with supernatant of the AFs culture medium. BMSC differentiation and migration were observed by immunostaining and Transwell assay, respectively, while cell proliferation was measured using the Cell Counting Kit-8. Expression levels of smooth muscle actin (α-SMA), TGF-β1 and SMAD3 were measured using western blot assay. The results indicated that compared with those in the control group, in which BMSCs were cultured in normal medium, expression levels of α-SMA, TGF-β1 and SMAD3 in BMSCs cultured in medium supplemented with supernatant of AFs, increased significantly (all P<0.05). Activated AFs induced the differentiation of BMSCs into vascular smooth muscle-like cells and promoted proliferation and migration. AFs activated by NE may induce BMSCs to participate in vascular remodeling. These findings may help design and develop new approaches and therapeutic strategies for vascular injury to prevent pathological remodeling.

Emerging role of exosomes in vascular diseases.

Exosomes are biological small spherical lipid bilayer vesicles secreted by most cells in the body. Their contents include nucleic acids, proteins, and lipids. Exosomes can transfer material molecules between cells and consequently have a variety of biological functions, participating in disease development while exhibiting potential value as biomarkers and therapeutics. Growing evidence suggests that exosomes are vital mediators of vascular remodeling. Endothelial cells (ECs), vascular smooth muscle cells (VSMCs), inflammatory cells, and adventitial fibroblasts (AFs) can communicate through exosomes; such communication is associated with inflammatory responses, cell migration and proliferation, and cell metabolism, leading to changes in vascular function and structure. Essential hypertension (EH), atherosclerosis (AS), and pulmonary arterial hypertension (PAH) are the most common vascular diseases and are associated with significant vascular remodeling. This paper reviews the latest research progress on the involvement of exosomes in vascular remodeling through intercellular information exchange and provides new ideas for understanding related diseases.

Nuclear receptor subfamily 1 group D member 1 suppresses the proliferation, migration of adventitial fibroblasts, and vascular intimal hyperplasia via mammalian target of rapamycin complex 1/β-catenin pathway

ABSTRACT Background In-stent restenosis hardly limits the therapeutic effect of the percutaneous vascular intervention. Although the restenosis is significantly ameliorated after the application of new drug-eluting stents, the incidence of restenosis remains at a high level. Objective Vascular adventitial fibroblasts (AFs) play an important role in intimal hyperplasia and subsequent restenosis. The current study was aimed to investigate the role of nuclear receptor subfamily 1, group D, member 1 (NR1D1) in the vascular intimal hyperplasia. Methods and Results We observed increased expression of NR1D1 after the transduction of adenovirus carrying Nr1d1 gene (Ad-Nr1d1) in AFs. Ad-Nr1d1 transduction significantly reduced the numbers of total AFs, Ki-67-positive AFs, and the migration rate of AFs. NR1D1 overexpression decreased the expression level of β-catenin and attenuated the phosphorylation of the effectors of mammalian target of rapamycin complex 1 (mTORC1), including mammalian target of rapamycin (mTOR) and 4E binding protein 1 (4EBP1). Restoration of β-catenin by SKL2001 abolished the inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs. Surprisingly, the restoration of mTORC1 activity by insulin could also reverse the decreased expression of β-catenin, attenuated proliferation, and migration in AFs induced by NR1D1 overexpression. In vivo, we found that SR9009 (an agonist of NR1D1) ameliorated the intimal hyperplasia at days 28 after injury of carotid artery. We further observed that SR9009 attenuated the increased Ki-67-positive AFs, an essential part of vascular restenosis at days 7 after injury to the carotid artery. Conclusion These data suggest that NR1D1 inhibits intimal hyperplasia by suppressing the proliferation and migration of AFs in a mTORC1/β-catenin-dependent manner.

Paracrine Signals in Calcified Conditioned Media Elicited Differential Responses in Primary Aortic Vascular Smooth Muscle Cells and in Adventitial Fibroblasts.

Our goal was to determine if paracrine signals from different aortic layers can impact other cell types in the diabetic microenvironment, specifically medial vascular smooth muscle cells (VSMCs) and adventitial fibroblasts (AFBs). The diabetic hyperglycemic aorta undergoes mineral dysregulation, causing cells to be more responsive to chemical messengers eliciting vascular calcification. Advanced glycation end-products (AGEs)/AGE receptors (RAGEs) signaling has been implicated in diabetes-mediated vascular calcification. To elucidate responses shared between cell types, pre-conditioned calcified media from diabetic and non-diabetic VSMCs and AFBs were collected to treat cultured murine diabetic, non-diabetic, diabetic RAGE knockout (RKO), and non-diabetic RKO VSMCs and AFBs. Calcium assays, western blots, and semi-quantitative cytokine/chemokine profile kits were used to determine signaling responses. VSMCs responded to non-diabetic more than diabetic AFB calcified pre-conditioned media. AFB calcification was not significantly altered when VSMC pre-conditioned media was used. No significant changes in VSMCs signaling markers due to treatments were reported; however, genotypic differences existed. Losses in AFB α-smooth muscle actin were observed with diabetic pre-conditioned VSMC media treatment. Superoxide dismutase-2 (SOD-2) increased with non-diabetic calcified + AGE pre-conditioned VSMC media, while same treatment decreased diabetic AFBs levels. Overall, non-diabetic and diabetic pre-conditioned media elicited different responses from VSMCs and AFBs.

Metformin Attenuates Hyperglycaemia-Stimulated Pro-Fibrotic Gene Expression in Adventitial Fibroblasts via Inhibition of Discoidin Domain Receptor 2.

Molecular mechanisms underlying the diverse therapeutic effects of anti-diabetic metformin, beyond its anti-hyperglycaemic effects, remain largely unclear. Metformin is reported to reduce the long-term complications of diabetes, including cardiovascular fibrosis and remodelling. Our recent investigations show that Discoidin Domain Receptor 2 (DDR2), a Collagen receptor tyrosine kinase, has an obligate regulatory role in Collagen type I gene expression in cardiac and vascular adventitial fibroblasts, and that it may be a molecular link between arterial fibrosis and metabolic syndrome in rhesus monkeys. Using gene knockdown and overexpression approaches, the present study examined whether DDR2 is a target of metformin and whether, by targeting DDR2, it inhibits Fibronectin and Collagen type I expression in rat aortic adventitial fibroblasts exposed to hyperglycaemic conditions. Metformin was found to attenuate hyperglycaemia-induced increase in DDR2 mRNA and protein expression by inhibiting TGF-β1/SMAD2/3 signalling that mediates the stimulatory effect of hyperglycaemia on DDR2 expression. Metformin also inhibited DDR2-dependent expression of Fibronectin and Collagen type I, indicating that it regulates these matrix proteins via DDR2 inhibition. The findings identify DDR2, a mediator of cardiovascular remodelling, as a molecular target of metformin, thereby uncovering the molecular basis of its protective role in vascular fibrosis and possibly cardiac fibrosis associated with diabetic cardiomyopathy.

Endoplasmic Reticulum Chemical Chaperone 3-Hydroxy-2-Naphthoic Acid Reduces Angiotensin II-Induced Vascular Remodeling and Hypertension In Vivo and Protein Synthesis In Vitro.

Background Investigations into alternative treatments for hypertension are necessary because current treatments cannot fully reduce the risk for the development of cardiovascular diseases. Chronic activation of unfolded protein response attributable to the endoplasmic reticulum stress has been proposed as a potential therapeutic target for hypertension and associated vascular remodeling. Triggered by the accumulation of misfolded proteins, chronic unfolded protein response leads to downstream signaling of cellular inflammation and dysfunction. Here, we have tested our hypothesis that a novel chemical chaperone, 3-hydroxy-2-naphthoic acid (3HNA) can attenuate angiotensin II (AngII)-induced vascular remodeling and hypertension. Methods and Results Mice were infused with AngII for 2 weeks to induce vascular remodeling and hypertension with or without 3HNA treatment. We found that injections of 3HNA prevented hypertension and increase in heart weight body weight ratio induced by AngII infusion. Histological assessment revealed that 3HNA treatment prevented vascular medial thickening as well as perivascular fibrosis in response to AngII infusion. In cultured vascular smooth muscle cells, 3HNA attenuated enhancement in protein synthesis induced by AngII. In vascular adventitial fibroblasts, 3HNA prevented induction of unfolded protein response markers. Conclusions We present evidence that a chemical chaperone 3HNA prevents vascular remodeling and hypertension in mice with AngII infusion, and 3HNA further prevents increase in protein synthesis in AngII-stimulated vascular smooth muscle cells. Using 3HNA may represent a novel therapy for hypertension with multiple benefits by preserving protein homeostasis under cardiovascular stress.

C1q/gC1qR‐Mediated Activation of Extrinsic Coagulation

Vascular diseases can be characterized by and are highly associated with inflammation and thrombosis. Understanding the relationship between these two processes would allow us to treat vascular diseases more effectively. Activation of complement component 1 (C1) is an important contributor to innate immunity and drives the lysis of pathogens. Upon C1 activation, significant concentrations of circulating C1q are observed. C1q has an affinity for gC1qR, a cell surface receptor molecule that is commonly found on cell types associated with cardiovascular disease development, including vascular smooth muscle cells and adventitial fibroblasts. When C1q interacts with gC1qR, many pathological pathways initiate including the inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. The intrinsic cascade plays a minor role in physiological and pathological thrombosis as compared with the extrinsic cascade; however, the effects of C1 and the role of C1q/gC1qR on extrinsic coagulation have not been studied. We hypothesized that C1q association with gC1qR would increase the amount of sub‐endothelial tissue factor expressed by both aortic adventitial fibroblasts (HAAFs) and coronary artery smooth muscle cells (HCASMCs). We investigated these cells as they are the primary TF bearing cells. Further, we hypothesized that the newly expressed TF would bind Factor VII (FVII), which is a crucial step in the initiation of the extrinsic coagulation cascade. The objective of this study was to elucidate the role of C1q/gC1qR‐mediated TF expression and its ability to bind FVII. Using a solid‐phase ELISA, TF expression was observed on both HAAFs and HCASMCs. Cells were conditioned for one hour with either platelet poor plasma (PPP), purified human C1q, LPS, or a combination of anti‐gC1qR antibodies (60.11 or 74.5.2 clones) and purified human C1q, PPP, or LPS. A capture ELISA approach was used to evaluate the binding of FVII to TF following exposure after the same conditions. Further, a capture ELISA was used to assess released TF. Finally, a live/dead assay to assess cell viability and cell density, as well as an MTT assay to assess cell metabolic activity were performed to monitor cell culture conditions. Our results indicate that HAAF and HCASMC expression of TF increased significantly by approximately 15% after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blocking antibodies prior to conditioning with C1q exhibited no change in TF expression when compared to unconditioned cells. Additionally, a 20‐30% increase in bound FVII was observed in both cell types when conditioned with C1q as compared to unconditioned cells. Our results show no significant differences in metabolic activity or cell viability under these conditions; however, cell density decreased by approximately 25% when conditioned with C1q. Overall, this data illustrates a role for C1q in activation of extrinsic coagulation. Furthermore, our data suggests that gC1qR acts a mediator for both inflammation and thrombosis. This work serves as a meaningful step in identifying a convergence of inflammatory and thrombotic pathways that can be therapeutically targeted to mitigate vascular diseases. Future work will determine if the expressed TF and the binding of FVII can support downstream extrinsic coagulation activity. Thanks to NIH for supporting this research.

Glucose consumption of vascular cell types in culture; toward optimization of experimental conditions

Any experimental outcomes are potentially influenced by extracellular glucose availability and its cellular metabolism. However, there is a lack of attention paid to the supply and utilization of glucose in many cultured experiments. Surveillance of vascular‐related journals for the past 5 years demonstrated that less than 20% of published articles declared the medium glucose concentration. The present studies were designed to seek ideal medium glucose concentration(s) in various cell types with particular attention paid to changes in glucose consumption. By using distinct glucose concentrations in Dulbecco's Modified Eagle's medium (DMEM), we identified ideal glucose media for stimulation experiments. We have compared glucose consumption in three vascular cell types, endothelial cells (EC), vascular smooth muscle cells (VSMC) and adventitial fibroblasts (AF) with or without angiotensin II stimulation. In all cell types after a 48‐hour incubation in relatively low glucose media (1 g/L), medium glucose concentration was reduced to almost 0 (EC 0.01±0.01 g/L, VSMC 0.13±0.05 g/L, AF 0.02±0.01 g/L). Whereas medium glucose concentration remained significantly higher (EC 2.77±0.05 g/L, VSMC 3.87±0.05 g/L, AF 3.32±0.01 g/L) when cells were incubated for 48 hours in high glucose (4.5 g/L) media. In middle glucose (2.75 g/L) media, medium glucose concentration remained in physiological ranges (EC 0.62±0.18 g/L, VSMC 1.98±0.07 g/L, AF 1.17±0.17 g/L). AngII treatment enhanced glucose consumption in AF and VSMC but not in EC. Enhanced extracellular acidification rate by AngⅡ was observed in AF as well as VSMC. In AF, AngII induction of target proteins at 48 hours varied depending on the glucose concentration used. In low glucose media induction of Grp78 or hexokinase II was highest, whereas induction of vascular cell adhesion molecule‐1 was lowest. However, no glucose preference was seen in AngII‐induced c‐Fos induction at 1 hour in AF. Utilization of specific inhibitors further suggest essential roles of AT1 receptor and glycolysis in AngII‐induced fibroblast activation. Overall, the present study demonstrates a high risk of hypo‐ or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Medium glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.

Interleukin 10 Attenuates Angiotensin II-Induced Aortic Remodelling by Inhibiting Oxidative Stress-Induced Activation of the Vascular p38 and NF-κB Pathways.

Interleukin 10 (IL-10) is a probable anti-inflammatory factor that can attenuate hypertrophic remodelling caused by overloaded pressure and improve cardiac function. In this study, IL-10 was decreased in both the plasma of hypertensive patients and the aortic vessels of angiotensin II (Ang II)-induced hypertensive mice. IL-10 was unable to alter blood pressure in the case of Ang II-induced hypertension. The aortic thickness, collagen deposition, and the levels of fibrosis-associated markers, including collagen type I α 1 (Col1α1), connective tissue growth factor (CTGF), transforming growth factor-β (TGF-β), and matrix metalloproteinase 2 (MMP2), were significantly reduced in the IL-10 treatment group compared with the vehicle group after Ang II treatment. Moreover, IL-10 treatment significantly inhibited the number of CD45+ positive cells and the mRNA expression levels of proinflammatory cytokines in the vascular tissue of Ang II-infused mice. Furthermore, dihydroethidium (DHE) and 4hydroxynonenal (4-HNE) staining showed that IL-10 decreased Ang II-induced vascular oxidative stress and lipid peroxidation. Furthermore, IL-10 suppressed Ang II-induced proliferation, fibrosis, and inflammation of mouse vascular adventitial fibroblasts (mVAFs). Mechanistically, IL-10 suppressed the phosphorylation of p38 mitogen-activated protein (MAP) kinase and nuclear factor-κB (NF-κB) in Ang II-induced vascular fibrosis. In summary, our data indicated that IL-10, as a potential therapeutic target treatment, could limit the progression of Ang II-induced aortic remodelling.

Urotensin II activates the ferroptosis pathway through circ0004372/miR-124/SERTAD4 to promote the activation of vascular adventitial fibroblasts.

Both vascular adventitial fibroblasts (VAFs) and urotensin II (UII) play important roles in vascular remodeling diseases, but the mechanism of UII in VAFs is still unclear. UII inhibited miR-124 expression through up-regulating circ0004372 expression, thereby promoting SERTAD4 expression. UII significantly promoted the generation of ROS, MDA and 4-HNE, reduced the activities of SOD, GST and GR, increased Fe2+ concentration and inhibited GPX4 expression through circ0004372/miR-124/SERTAD4. Both UII and ferroptosis inducer Erastin significantly promoted the expression of α-SMA, Collagen I and TGF-β1 in VAFs, but circ0004372 siRNA, miR-124 mimics, SERTAD4 siRNA or Ferrostatin-1 significantly inhibited the effect of UII and Erastin on cell activation. When co-transfected with circ0004372 siRNA and miR-124 inhibitors or miR-124 mimics and SERTAD4 overexpression vector, UII still significantly increased the expression of α-SMA, Collagen I and TGF-β1. After transfection with circ0004372 overexpression vector, miR-124 inhibitors or SERTAD4 overexpression vector and then treating with UII and Ferrostatin-1, the expression of α-SMA, Collagen I and TGF-β1 was still significant; when the circ0004372 overexpression vector and miR-124 mimics or miR-124 inhibitors and SERTAD4 siRNA were co-transfected and then UII and Ferrostatin-1 were added, the expression of α-SMA, Collagen I and TGF-β1 was not significantly increased. Therefore, these results indicate that UII promotes the activation of VAFs through the circ0004372/miR-124/SERTAD4/ferroptosis pathway.

Danlou Tablet Activates Autophagy of Vascular Adventitial Fibroblasts Through PI3K/Akt/mTOR to Protect Cells From Damage Caused by Atherosclerosis.

Danlou tablet (DLT), a commercial Chinese patent medicine, has been widely used to treat cardiovascular diseases for many years. Atherosclerosis (AS) is the leading cause of cardiovascular disease. Increasing evidence indicates that autophagy plays a vital role in the development of AS. Here we investigated whether DLT could activate autophagy to improve AS and further clarified its underlying mechanisms. In an ApoE−/− mice model, the results of Oil red O, Masson’s trichrome, and H&E staining techniques showed that DLT significantly inhibited lipid accumulation and fibrosis formation in atherosclerotic plaque tissue. DLT also inhibited serum triglyceride, cholesterol, and low-density lipoprotein levels and suppressed serum levels of inflammatory factors interleukin-6 and tumor necrosis factor-α in ApoE−/− mice. Moreover, DLT suppressed proliferation, migration, and invasion of human vascular adventitial fibroblasts (HVAFs) by inhibiting the PI3K/Akt/mTOR pathway. In addition, western blot analysis showed that Danlou tablet treatment decreased the expression of p62 and increased Beclin 1 and LC3 I -to-LC3 II ratios in HVAFs. The role of autophagy in treating atherosclerosis by DLT is confirmed by 3-methyladenine (autophagy inhibitor) and rapamycin (autophagy activator) in HVAFs. In summary, DLT activated PI3K/Akt/mTOR-mediated autophagy of vascular adventitial fibroblasts to protect cells from damage caused by atherosclerosis.

Targeted RNA Sequencing of VZV-Infected Brain Vascular Adventitial Fibroblasts Indicates That Amyloid May Be Involved in VZV Vasculopathy.

Background and ObjectivesCompared with stroke controls, patients with varicella zoster virus (VZV) vasculopathy have increased amyloid in CSF, along with increased amylin (islet amyloid polypeptide [IAPP]) and anti-VZV antibodies. Thus, we examined the gene expression profiles of VZV-infected primary human brain vascular adventitial fibroblasts (HBVAFs), one of the initial arterial cells infected in VZV vasculopathy, to determine whether they are a potential source of amyloid that can disrupt vasculature and potentiate inflammation.MethodsMock- and VZV-infected quiescent HBVAFs were harvested at 3 days postinfection. Targeted RNA sequencing of the whole-human transcriptome (BioSpyder Technologies, TempO-Seq) was conducted followed by gene set enrichment and pathway analysis. Selected pathways unique to VZV-infected cells were confirmed by enzyme-linked immunoassays, migration assays, and immunofluorescence analysis (IFA) that included antibodies against amylin and amyloid-beta, as well as amyloid staining by Thioflavin-T.ResultsCompared with mock, VZV-infected HBVAFs had significantly enriched gene expression pathways involved in vascular remodeling and vascular diseases; confirmatory studies showed secretion of matrix metalloproteinase-3 and -10, as well increased migration of infected cells and uninfected cells when exposed to conditioned media from VZV-infected cells. In addition, significantly enriched pathways involved in amyloid-associated diseases (diabetes mellitus, amyloidosis, and Alzheimer disease), tauopathy, and progressive neurologic disorder were identified; predicted upstream regulators included amyloid precursor protein, apolipoprotein E, microtubule-associated protein tau, presenilin 1, and IAPP. Confirmatory IFA showed that VZV-infected HBVAFs contained amyloidogenic peptides (amyloid-beta and amylin) and intracellular amyloid.DiscussionGene expression profiles and pathway enrichment analysis of VZV-infected HBVAFs, as well as phenotypic studies, reveal features of pathologic vascular remodeling (e.g., increased cell migration and changes in the extracellular matrix) that can contribute to cerebrovascular disease. Furthermore, the discovery of amyloid-associated transcriptional pathways and intracellular amyloid deposition in HBVAFs raise the possibility that VZV vasculopathy is an amyloid disease. Amyloid deposition may contribute to cell death and loss of vascular wall integrity, as well as potentiate chronic inflammation in VZV vasculopathy, with disease severity and recurrence determined by the host's ability to clear virus infection and amyloid deposition and by the coexistence of other amyloid-associated diseases (i.e., Alzheimer disease and diabetes mellitus).

Abstract P171: Glucose Consumption Of Vascular Cell Types In Culture; Toward Optimization Of Experimental Conditions

Any experimental outcomes are potentially influenced by extracellular glucose availability and its cellular metabolism. However, there is a lack of attention paid to the supply and utilization of glucose in many cultured experiments. Surveillance of vascular related journals for the past 5 years demonstrated that less than 20% of published articles declared the medium glucose concentration. The present studies were designed to seek ideal medium glucose concentration(s) in various cell types with particular attention paid to changes in glucose consumption. By using distinct glucose concentrations in Dulbecco's Modified Eagle's medium (DMEM), we identified ideal glucose media for stimulation experiments. We have compared glucose consumption in three vascular cell types, endothelial cells (EC), vascular smooth muscle cells (VSMC) and adventitial fibroblasts (AF) with or without angiotensin II (AII) stimulation. In all cell types after a 48h incubation in relatively low glucose media (1 g/L), medium glucose concentration was reduced to almost 0 (EC 0.01±0.01 g/L, VSMC 0.13±0.05 g/L, AF 0.02±0.01 g/L). Whereas medium glucose concentration remained significantly higher (EC 2.77±0.05 g/L, VSMC 3.87±0.05 g/L, AF 3.32±0.01 g/L) when cells were incubated for 48h in high glucose (4.5 g/L) media. In middle glucose (2.75 g/L) media, medium glucose concentration remained in physiological ranges (EC 0.62±0.18 g/L, VSMC 1.98±0.07 g/L, AF 1.17±0.17 g/L). AII treatment enhanced glucose consumption in AF and low passage VSMC but not in EC. In AF, AII induction of target proteins varied depending on the glucose concentration used. In low glucose media induction of Grp78 or hexokinase II was highest, whereas induction of VCAM1 was lowest. Utilization of specific inhibitors further suggest essential roles of AT1 receptor and glycolysis in AII-induced fibroblast activation. Overall, the present study demonstrates a high risk of hypo- or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Medium glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.

RAGE Differentially Altered in vitro Responses in Vascular Smooth Muscle Cells and Adventitial Fibroblasts in Diabetes-Induced Vascular Calcification.

The Advanced Glycation End-Products (AGE)/Receptor for AGEs (RAGE) signaling pathway exacerbates diabetes-mediated vascular calcification (VC) in vascular smooth muscle cells (VSMCs). Other cell types are involved in VC, such as adventitial fibroblasts (AFBs). We hope to elucidate some of the mechanisms responsible for differential signaling in diabetes-mediated VC with this work. This work utilizes RAGE knockout animals and in vitro calcification to measure calcification and protein responses. Our calcification data revealed that VSMCs calcification was AGE/RAGE dependent, yet AFBs calcification was not an AGE-mediated RAGE response. Protein expression data showed VSMCs lost their phenotype marker, α-smooth muscle actin, and had a higher RAGE expression over non-diabetics. RAGE knockout (RKO) VSMCs did not show changes in phenotype markers. P38 MAPK, a downstream RAGE-associated signaling molecule, had significantly increased activation with calcification in both diabetic and diabetic RKO VSMCs. AFBs showed a loss in myofibroblast marker, α-SMA, due to calcification treatment. RAGE expression decreased in calcified diabetic AFBs, and P38 MAPK activation significantly increased in diabetic and diabetic RKO AFBs. These findings point to potentially an alternate receptor mediating the calcification response in the absence of RAGE. Overall, VSMCs and AFBs respond differently to calcification and the application of AGEs.

Vascular Adventitial Fibroblasts-Derived FGF10 Promotes Vascular Smooth Muscle Cells Proliferation and Migration in vitro and the Neointima Formation in vivo.

BackgroundActivation of vascular adventitial fibroblasts (VAFs) upon vascular injury contributes greatly to the medial vascular smooth muscle cells (VSMCs) proliferation, migration and the subsequent neointima formation. A number of factors including fibroblast growth factors (FGFs) have been shown to control VSMC growth, proliferation and phenotypic switching, suggesting that they may function as paracrine signals for VAFs to modulate VSMCs functions. However, little is known about the signaling molecule(s) and its mechanism of action. This study is set to identify which and how FGF family members are involved in VAFs mediated vascular remodeling.MethodsWe used qPCR, Western blot and Immunohistochemistry to observe the spatiotemporal expression of FGF10 and FGFR2 in injured vascular tissue. The proliferation and migration assays of VSMCs were performed in a co-culture system. The activation of signaling pathway was detected by Western blot, immunohistochemistry and immunofluorescence. Hematoxylin-eosin and immunofluorescence were used to assess the effects of exogenous FGF10 and siFGF10 on the neointima formation.ResultsThe expression of FGF10 and FGFR2 were increased from day 3 through day 14 post injury. FGF10 was significantly upregulated in adventitia, and FGFR2 was detected in both media and neointima after injury. In vitro, FGF10 was most prominently expressed in VAFs and FGFR2 was significantly expressed in VSMCs. Both were regulated by PDGF. Co-culture of VAFs and VSMCs in vitro showed that VAF-derived FGF10 promoted the proliferation and migration of VSMCs. PDGF could synergistically enhance the process. VAF-derived FGF10 can significantly activate the FGFR2 in VSMCs and furthermore significantly activate the downstream MAPK/PI3K-AKT signaling pathways. Delivery of exogenous FGF10 potentiated the neointima formation, while siFGF10 attenuated the neointima formation.ConclusionVAFs-derived FGF10 promoted the proliferation and migration of VSMCs and neointima formation, and FGF10-FGFR2 signaling triggered the activation of MAPK/PI3K-AKT pathways in VSMCs and PDGF synergistically amplified FGF10 signaling.

Extracellular vesicle-mediated miR135a-5p transfer in hypertensive rat contributes to vascular smooth muscle cell proliferation via targeting FNDC5

BackgroundExtracellular vesicles (EVs) from vascular adventitial fibroblasts (AFs) contribute to the proliferation of vascular smooth muscle cells (VSMCs) and vascular remodeling in spontaneously hypertensive rat (SHR). This study shows the crucial roles of EVs-mediated miR135a-5p transfer in VSMC proliferation and the underlying mechanisms in hypertension.MethodsAFs and VSMCs were obtained from the aorta of Wistar-Kyoto rat (WKY) and SHR. EVs were isolated from the culture of AFs with ultracentrifugation method.ResultsMiR135a-5p level in SHR-EVs was significantly increased. MiR135a-5p inhibitor prevented the SHR-EVs-induced VSMC proliferation. Fibronectin type III domain containing 5 (FNDC5) was a target gene of miR135a-5p. FNDC5 level was lower in VSMCs of SHR. MiR135a-5p inhibitor not only increased FNDC5 expression, but reversed the SHR-EVs-induced FNDC5 downregulation in VSMCs of SHR. MiR135a-5p mimic inhibited FNDC5 expression, but failed to promote the SHR-EVs-induced FNDC5 downregulation in VSMCs of SHR. Exogenous FNDC5 prevented the SHR-EVs-induced VSMC proliferation of both WKY and SHR. Knockdown of miR135a-5p in fibroblasts completely prevented the upregulation of miR135a-5p in the EVs. The SHR-EVs from the miR135a-5p knockdown-treated fibroblasts lost their roles in inhibiting FNDC5 expression and promoting proliferation in VSMCs of both WKY and SHR.ConclusionsIncreased miR135a-5p in the SHR-EVs promoted VSMC proliferation of WKY and SHR via inhibiting FNDC5 expression. MiR135a-5p and FNDC5 are crucial targets for intervention of VSMC proliferation in hypertension.

Transient Receptor Potential Melastatin 7 Promotes Vascular Adventitial Fibroblasts Phenotypic Transformation and Inflammatory Reaction Induced by Mechanical Stretching Stress via p38 MAPK/JNK Pathway

Remodeling of the arteries is one of the pathological bases of hypertension. We have previously shown that transient receptor potential melastatin 7 (TRPM7) aggravates the vascular adventitial remodeling caused by pressure overload in the transverse aortic constriction (TAC) model. In this study, we sought to explore the functional expression and downstream signaling of TRPM7 in vascular adventitial fibroblasts (AFs) stimulated by mechanical stretching stress (MSS). The expression of TRPM7 was upregulated with a concomitant translocation to the cytoplasm in the AFs stimulated with 20% MSS. Meanwhile, the expression of α-smooth muscle actin (α-SMA), a marker of transformation from AFs to myofibroblasts (MFs) was also increased. Moreover, AF-conditioned medium caused a significant migration of macrophages after treatment with MSS and contained high levels of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α). Pharmacological and RNA interference approaches using the TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB) and specific anti-TRPM7 small interfering RNA (si­RNA-TRPM7) abrogated these changes significantly. Further exploration uncloaked that inhibition of TRPM7 reduced the phosphorylation of p38 MAP kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) in the AFs stimulated with MSS. Furthermore, inhibition of the phosphorylation of p38MAPK or JNK could also alleviate the MSS-induced expression of α-SMA and secretion of inflammatory factors. These observations indicate that activated TRPM7 participates in the phenotypic transformation and inflammatory action of AFs in response to MSS through the p38MAPK/JNK pathway and suggest that TRPM7 may be a potential therapeutic target for vascular remodeling caused by hemodynamic changes in hypertension.