AngII Stimulation Research Articles

Abstract 2020: Vascular Glucose Transporter 1 Is Dispensable For Abdominal Aortic Aneurysm Induced By Angiotensin II

Background: Pharmacological inhibition of glycolysis attenuated abdominal aortic aneurysm (AAA) development in mice. Glucose transporters (GLUTs) incorporate glucose as a first step of glycolysis. GLUT1 over-expression in vascular smooth muscle cells (VSMC) leads to enhancement of atherosclerosis. However, the role of VSMC GLUT1 in AAA remains unclear. Hypothesis: VSMC GLUT1 deletion attenuates AAA development and rupture in mice. Goals/Aims: Our goal was to establish contribution of VSMC GLUT1 in AAA. Methods: The glucose consumption of cultured VSMCs was evaluated in the presence or absence of AngII stimulation and GLUT1 inhibitor BAY-876. GLUT1 floxed mice were crossed with tamoxifen-inducible SMMHC-Cre mice. At 6 weeks of age, mice were injected with tamoxifen. As a control, tamoxifen-treated SMMHC-Cre male mice were utilized. At 8 weeks, angiotensin II (AngII, 1 μg/kg/min) was delivered for 4 weeks. In addition, mice received 1 mg/mL BAPN during the first 2 weeks of AngII infusion. After 4weeks, all animals were euthanized and maximal external diameter of the abdominal aorta was measured. Results: In cultured VSMC, the glucose consumption increased by AngII was attenuated by pretreatment with GLUT1 inhibitor (media glucose 257.9±4.5 vs 238.9±6.2 mg/dL, p<0.05). AngII plus BAPN significantly developed AAA in control mice (diameter 1.05±0.07 vs 2.37±0.31mm, p<0.05). However, silencing of VSMC GLUT1 did not affect AAA development by AngII plus BAPN (2.41±0.46mm, p=0.66). The survival rate due to aortic rupture in VSMC GLUT1 silenced mice and control mice treated with BAPN and AngII were 60.0% and 57.1%, respectively (p=0.820). AngII induced hypertension in control mice (102±3.8 vs 133±7.1mmHg, p<0.05) and VSMC GLUT1 silenced mice (149±11.4mmHg, p=0.99). Discussion Although GLUT1 may be a primary VSMC GLUT to enhance glucose utilization in response to AngII stimulation, VSMC GLUT1 silencing did not alter AngII-dependent AAA development or rupture in a mouse model. Since pharmacological inhibition of GLUT attenuated AAA and associated macrophage infiltration, it is possible that GLUT1 expressed in macrophages may contribute to development of AAA. Conclusion: VSMC GLUT1 silencing did not alter AngII-dependent AAA development in a mouse model.

HBI-8000 improves heart failure with preserved ejection fraction via the TGF-β1/MAPK signalling pathway.

Heart failure with preserved ejection fraction (HFpEF) accounts for approximately 50% of total heart failure patients and is characterized by peripheral circulation, cardiac remodelling and comorbidities (such as advanced age, obesity, hypertension and diabetes) with limited treatment options. Chidamide (HBI-8000) is a domestically produced benzamide-based histone deacetylase isoform-selective inhibitor used for the treatment of relapsed refractory peripheral T-cell lymphomas. Based on our invivo studies, we propose that HBI-8000 exerts its therapeutic effects by inhibiting myocardial fibrosis and myocardial hypertrophy in HFpEF patients. At the cellular level, we found that HBI-8000 inhibits AngII-induced proliferation and activation of CFs and downregulates the expression of fibrosis-related factors. In addition, we observed that the HFpEF group and AngII stimulation significantly increased the expression of TGF-β1 as well as phosphorylated p38MAPK, JNK and ERK, whereas the expression of the above factors was significantly reduced after HBI-8000 treatment. Activation of the TGF-β1/MAPK pathway promotes the development of fibrotic remodelling, and pretreatment with SB203580 (p38MAPK inhibitor) reverses this pathological change. In conclusion, our data suggest that HBI-8000 inhibits fibrosis by modulating the TGF-β1/MAPK pathway thereby improving HFpEF. Therefore, HBI-8000 may become a new hope for the treatment of HFpEF patients.

Reduced expression of transmembrane protein 43 during cardiac hypertrophy leads to worsening heart failure in mice.

Transmembrane protein 43 (TMEM43), a member of the transmembrane protein subfamily, was found to be associated with arrhythmogenic right ventricular cardiomyopathy. However, its role in cardiac hypertrophy has not been elucidated. Here, we used a pressure overload-induced cardiac hypertrophy model to explore the role of TMEM43 in heart failure. Mice were subjected to aortic banding (AB) to induce cardiac hypertrophy. The mice were also randomly selected to receive injection of adeno-associated virus 9 (AAV9)-shTMEM43 to knockdown TMEM43 in cardiomyocytes or control AAV9 (ScRNA). Four weeks after AB, the mice were subjected to echocardiography to evaluate cardiac function. Neonatal rat cardiomyocytes (NRCMs) were stimulated with angiotensin II (AngII, 1 μM) and transfected with an adenovirus to over-express TMEM43. We found that TMEM43 was downregulated in mouse hearts and cardiomyocytes poststimulation. Mice with TMEM43 knockdown showed worsening heart failure accompanied by deteriorating cardiac function and exacerbated cardiac hypertrophy and fibrosis at 4 weeks post-AB. NRCMs over-expressing TMEM43 exhibited an ameliorated hypertrophic response. Moreover, we found that TMEM43 deficiency increased nuclear factor kappa B (NF-κB) activation in mouse hearts post-AB, while TMEM43 over-expression reduced NF-κB activation in cardiomyocytes upon AngII stimulation. Thus, we conclude that reduced expression of TMEM43 during cardiac hypertrophy leads to worsening heart failure in mice.

Astragaloside IV inhibits angiotensin II-induced aortic dissection formation by regulating miR-320a/matrix metallopeptidase 9 axis

This study explored the role of miR-320a and Matrix metallopeptidase 9 (MMP-9) in aortic dissection (AD) formation. Aortic tissue diameter (LD) and medial thickness (MT) were measured after the AD model was established. Aortic smooth muscle cells (VSMCs) were assigned into blank group, Astragaloside IV (AS-IV), which was treated with 100, 150 or 200 μg/mL and AngII group. Cells in the AngII group were added with MMP9 inhibitor (MMP9 inhibitor group) and mimics (MMP9 mimics group), followed by analysis of cell proliferation, apoptosis, and target gene for miR-320a. Aortic tissue diameter(LD) from the AD group was significantly higher and MT was lower than in the control group (p<0.05). VSMCs proliferative activity in the AS-IV group and AngII group was increased significantly (p <0.05), with lower activity in the AS-IV group than AngII group (p < 0.05). After transfection, the VSMCs apoptosis in the AngII group was decreased (p <0.05). In addition, cell migration in the AS-IV and AngII groups increased (p <0.05), with lower ability in the AS-IV group than in the AngII group (p <0.05). Alpha smooth muscle Actin (α-SMA) expression was elevated after AngII stimulation and decreased after AS-IV treatment (p <0.05). After AngII stimulation, miR-320a and MMP9 were upregulated (p <0.05) and downregulated by AS-IV (p <0.05). MMP9 is a target gene for miR-320a. miR-320a and MMP9 expressions in the AS-IV group were significantly decreased, with lowest levels in the AS-IV-200 μg/mL group. In addition, MMP9 overexpression reduced the AngII expression. The expression of miR-320a/MMP9 axis was low in the AD, and activity were increased after AngII stimulation. In conclusion, As-IV can relieve the occurrence of AD via regulation of miR-320a/MMP9 axis.

Anti-pyroptosis effect of Albiziae Cortex-Tribuli Fructus combination on hepatic stellate cell line LX2: based on network pharmacology

Based on network pharmacology, molecular docking, and in vitro experimental verification, this study aims to explore the effect of Albiziae Cortex-Tribuli Fructus combination on HSC-LX2 pyroptosis. Specifically, the targets of Albiziae Cortex, Tribuli Fructus, and hepatic fibrosis were retrieved from an online database and CNKI, and "drug-component-target" network and "drug-component-target-disease" network were constructed. Protein-protein interaction(PPI) network was established based on STRING. Metascape was employed for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and the mechanism of Albiziae Cortex-Tribuli Fructus combination against liver fibrosis was predicted. Molecular docking was used to verify some of the results of network pharmacology, and in vitro experiment was carried out to further verify the above conclusions. According to the results of network pharmacological analysis, 25 active components and 439 targets of Albiziae Cortex-Tribuli Fructus combination and 152 anti-liver fibrosis targets were screened out, including nucleotide-binding oligomerization domain and leucine-rich-repeat-and pyrin-domain-containing 3(NLRP3) and caspase-1. The key targets were involved in 194 KEGG pathways in which the NOD-like receptor signaling pathway topped. The binding common targets were related to pyroptosis. The results of in vitro experiment showed that the pair-containing serum reduced the proliferation rate of HSC-LX2 and the content of reactive oxygen species(ROS), interleukin-18(IL-18), and interleukin-1β(IL-1β)(P<0.05). Western blot and qRT-PCR suggested that the protein and gene expression of NLRP3, caspase-1, α-smooth muscle actin(α-SMA), and gasdermin D(GSDMD) in HSC-LX2 increased after AngⅡ stimulation, and the expression decreased after the intervention of pair-containing serum(P<0.05). In summary, the pair-containing serum can inhibit the classic pathway of pyroptosis, which may be the anti-liver fibrosis mechanism. This is consistent with the predicted results of network pharmacology.

Angiotensin II can trigger HSC-LX2 pyroptosis through both classical and non-classical pathways

BackgroundCurrent evidence suggests that liver fibrosis is reversible even at late stages. Pyroptosis is reportedly regulated by classical and non-classical pathways and is also involved in the activation of the human hepatic stellate cell line LX2. This study sought to identify regulatory pathways that pyroptosis of HSC during AngII-ROS-induced HSC activation and provides novel insights for anti-fibrosis therapy by targeting HSC. Materials and methodsAll experiments were conducted in HSC-LX2. The expressions of α-SMA, NLRP3, Caspases-1, -4, -5, ASC and GSDMD-N were detected in HSC-LX2 cells induced with AngII by Western blot and qRT-PCR. CCK8 was used to detect cell proliferation and activity. 2′-7′dichlorofluorescin diacetate (DCFH-DA) was used to measure ROS generation. An LDH assay kit was used to detect LDH released from damaged cells, and ELISA was used to quantify IL-18 and IL-1β levels. ResultsAfter AngII stimulation, HSC-LX2 cell viability, ROS, LDH, IL-18, and IL-1β were increased compared with Control group. At the same time, the protein and mRNA levels of α-SMA, NLRP3, Caspases-1, -4, -5, ASC and GSDMD-N were increased. In addition, after NAC and NSA treatment, LDH, IL-18 and IL-1β levels and the protein and mRNA expression of α-SMA, Caspases-4 and -5, and GSDMD-N were decreased. ConclusionHSC-LX2 pyroptosis induced by AngII-ROS is mediated by the classical pathway involving NLRP3/Caspase-1 and the non-classical pathway involving Caspases-4 and -5. Our results provide compelling evidence that AngII could activate Caspases-4 and -5 by producing ROS.

ADAM17‐enriched Exosomes Contribute to Neuronal Activation in Hypertension

ADAM17 is the prime mediator of Angiotensin Converting Enzyme 2 (ACE2) shedding, a process by which the ACE2 ectodomain is cleaved from the cell membrane and released in the surrounding milieu. ACE2 is a compensatory enzyme converting Angiotensin (Ang)-II to Ang-(1-7). Recent studies have reported the presence of activated ADAM17 on exosomes, suggesting the possibility of shedding propagation from one cell to another. Here, we hypothesized that in hypertensive conditions, exosomes are enriched in ADAM17 and contribute to neuronal activation, possibly leading to enhanced sympathetic activity. Human plasma and cerebrospinal fluid were collected from both African Americans (AA) and Caucasian Americans (CA), normotensive and hypertensive patients (n=3/group). Exosomes were extracted using total exosome isolation kit (Invitrogen) and quantified by estimation of proteins (BCA kit). The ADAM17 expression in exosomes was measured by capillary western (protein Simple) and the exosomes’ morphology was studied using electron microscopy (Cryo-TEM). To investigate the role of these exosomes, Neuro2A cells were treated with phorbol myristate, an ADAM17 activator, or Ang-II (100 nM) for 4 hours, followed by extraction of exosomes (Invitrogen) and estimation of their ADAM17 levels (capillary western). After 24 h, Neuro2A cells were transfected with GCaMP6s, a fluorescent calcium indicator, and exposed for 4 h with PBS (control), Ang-II (100 nM) or exosomes isolated from hypertensive patients before Ca++-mediated fluorescence was quantified (Cytation 5). Exosomes extracted from patients’ plasma showed a significant 3-fold increase in ADAM17 expression in hypertensive individuals when compared to normotensive counterparts (Normotensive AA: 3.1 ±0.6, CA: 4.5 ±0.3; Hypertensive AA: 7.1 ±0.2, CA: 7.0 ±0.5, au: arbitrary units; n=3: one-way ANOVA, P< 0.01 and P< 0.001 vs. normotensive). In normotensive patients ADAM17 content was higher in CA but was similar in AA and CA under hypertensive conditions. Exosomes extracted from Neuro2A cells showed a 3-fold increase in ADAM17 expression in both Ang-II- and PMA-treated groups when compared to control, thereby confirming the expression of ADAM17 in exosomes from stimulated neuronal cells. The Ca++-mediated fluorescence intensity in Neuro2A cells treated with exosomes from hypertensive patients or Ang-II was significantly increased when compared to control cells (control: 4.0 ±0.6, Ang-II: 20 ±0.2, exosomes: 15.1 ±0.2, au, n=3, one-way ANOVA, P<0.001 vs. control, n=3). Our data show that upon activation the mature form of ADAM17 is released in the form of exosomes in the brain or plasma from hypertensive patients, and in cells following Ang-II stimulation, possibly contributing to the enhanced ACE2 shedding in those individuals. In addition, neurons exposed to Ang-II and ADAM17-enriched exosomes (i.e. isolated from hypertensive patients) show increased Ca++-mediated fluorescence confirming an activated neuron phenotype that could contribute to elevated excitatory activity on neuronal cells and ultimately to an enhanced sympathetic activity.

TMEM16A inhibits angiotensin II-induced basilar artery smooth muscle cell migration in a WNK1-dependent manner

Vascular smooth muscle cell (VSMC) migration plays a critical role in the pathogenesis of many cardiovascular diseases. We recently showed that TMEM16A is involved in hypertension-induced cerebrovascular remodeling. However, it is unclear whether this effect is related to the regulation of VSMC migration. Here, we investigated whether and how TMEM16A contributes to migration in basilar artery smooth muscle cells (BASMCs). We observed that AngII increased the migration of cultured BASMCs, which was markedly inhibited by overexpression of TMEM16A. TMEM16A overexpression inhibited AngII-induced RhoA/ROCK2 activation, and myosin light chain phosphatase (MLCP) and myosin light chain (MLC20) phosphorylation. But AngII-induced myosin light chain kinase (MLCK) activation was not affected by TMEM16A. Furthermore, a suppressed activation of integrinβ3/FAK pathway, determined by reduced integrinβ3 expression, FAK phosphorylation and F-actin rearrangement, was observed in TMEM16A-overexpressing BASMCs upon AngII stimulation. Contrary to the results of TMEM16A overexpression, silencing of TMEM16A showed the opposite effects. These in vitro results were further demonstrated in vivo in basilar arteries from VSMC-specific TMEM16A transgenic mice during AngII-induced hypertension. Moreover, we observed that the inhibitory effect of TMEM16A on BASMC migration was mediated by decreasing the activation of WNK1, a Cl−-sensitive serine/threonine kinase. In conclusion, this study demonstrated that TMEM16A suppressed AngII-induced BASMC migration, thus contributing to the protection against cerebrovascular remodeling during AngII-infused hypertension. TMEM16A may exert this effect by suppressing the RhoA/ROCK2/MLCP/MLC20 and integrinβ3/FAK signaling pathways via inhibiting WNK1. Our results suggest that TMEM16A may serve as a novel therapeutic target for VSMC migration-related diseases, such as vascular remodeling.

Fus knockdown inhibits the profibrogenic effect of cardiac fibroblasts induced by angiotensin II through targeting Pax3 thereby regulating TGF-β1/Smad pathway

ABSTRACT The Angiotensin II/transforming growth factor-β1 (AngII/TGF-β1) signal axis is an important regulatory pathway for atrial fibrosis, which can contribute to atrial fibrillation (AF). Fused in sarcoma (FUS) was recently found to regulate cardiac diseases. This study aimed to investigate whether FUS could regulate AngII induced fibrosis and uncover the possible mechanisms. The expression of FUS in AF patients and AngII-induced cardiac fibroblasts was measured by RT-qPCR and western blot assays. Fus was silenced in cells using short hairpin RNA (shRNA), then cell proliferation, migration, collagen synthesis and TGF-β1/Smad signaling were detected by CCK-8, wound healing and western blot assays, respectively. The possible target for Fus was predicted by searching Starbase database and verified by RNA-binding protein immunoprecipitation (RIP) and RNA pull down. Cells were overexpressed with Pax3 in the presence of Fus silence and AngII stimulation, then the above cellular processes were further evaluated. Results showed that FUS was upregulated in AF patients and AngII-induced cardiac fibroblasts. Fus knockdown inhibited AngII-enhanced cell proliferation, migration, collagen synthesis and TGF-β1/Smad signaling activation. Furthermore, Fus functions as an RNA-binding protein to bind to Pax3 mRNA and positively regulate its expression. Further studies demonstrated that Pax3 overexpression canceled the above effects of Fus knockdown on cell proliferation, migration, collagen synthesis, and TGF-β1/Smad signaling activation in AngII-induced cells. In conclusion, Fus could target Pax3 to increase the pro-fibrotic effect of AngII in cardiac fibroblasts via activating TGF-β1/Smad signaling. Knockdown of Fus/Pax3 axis may provide a potential therapy for relieving AF.

Abstract MP22: GRP78 Protects Against Angiotensin II-induced Protein Aggregation, Profibrotic VSMC Phenotype, And Vascular Remodeling

Arterial stiffening is a critical risk factor contributing to the development of cardiovascular disease and end organ damage. Vascular smooth muscle cell (VSMC) hypercontractility, vessel fibrosis, and proinflammatory responses are involved. The renin angiotensin system plays critical roles in the development of VSMC stiffness phenotype, as well as CVD pathophysiology. Angiotensin II (Ang II) stimulation induces these stiffness-promoting mechanisms, however, the involvement of dysfunctional proteostasis governing Ang II contribution to vascular stiffness remains underdefined. While endoplasmic reticulum (ER) stress has been implicated in the development of arterial stiffness and hypertension, it is unknown whether the protein aggregation potentially due to maladapted unfolded protein response (UPR) in VSMCs is involved. We hypothesize AngII induced ER stress initiates protein misfolding and effects VSMC stiffness phenotype. We identify a significant increase in protein aggregate size (P&lt;0.001: from 1,000 to 4,500 pixel/aggregate) and number per cell (P&lt;0.01: from 20 to 100 aggregate #/cell) in rat VSMCs stimulated with AngII, which is attenuated with adenoviral overexpression of resident ER chaperone, GRP78 (P&lt;0.01: 2000 pixels/aggregate and P&lt;0.05: 25 aggregate #/cell). Furthermore, UPRosome associated IRE1α phosphorylation induced at 3 and 6 h following AngII stimulation is reduced in GRP78 overexpressing VSMCs (P&lt;0.05), which may contribute to reduced monocyte adhesion (P&lt;0.001), assessed utilizing THP-1 cells. In mice, pharmacological chaperone 3-HNA reduced AngII induced vascular hypertrophy (P=0.056), cardiac perivascular fibrosis (P=0.065), and pulse propagation velocity from 0.35 m/s to 0.23 m/s (P&lt;0.05). A trend in pre-amyloid oligomer accumulation was seen in the medial layer of thoracic aortas following 2 weeks of AngII infusion, which was reduced with 3-HNA treatment. Similar findings were investigated in a SM22α cre-GRP78 mouse model, where a trend in reduction of these parameters are also seen. Overall, GRP78 regulation over VSMC proteostasis influences vascular stiffness phenotype and therefore, these findings may elude new pharmacological intervention for arterial stiffness and aging associated CVD.

P6494Activation of focal adhesion kinase is involved in pathogenesis of aortic dissection in mice

Abstract Background Aortic dissection (AD) is a fatal disease where the media of the aorta suddenly fail. Currently, Molecular pathogenesis of AD is unknown. Recently, we discovered that the activity of MRTF-A, a mechanosensitive transcriptional regulator, promotes AD development. The activity of MRTF-A is regulated by mechanical stress to cells, which is transduced through focal adhesion and actin dynamics. However, it is currently unknown whether the mechanotransduction mechanism is involved in AD pathogenesis. Purpose We investigated the role of focal adhesion kinase (FAK), a signaling molecule that transduces mechanostress from focal adhesion to actin dynamics, in AD pathogenesis. Methods We created a mouse model of AD with a continuous infusion of beta-aminopropionitrile (150 mg/kg/day), a collagen crosslink inhibitor, and angiotensin II (1,000 ng/kg/min) (BAPN + AngII) by an osmotic pump. This model caused about 60% death in all mice due to AD rupture within 2 weeks. In this model, we examined the severity and mortality rate of aortic dissection after 2 weeks in mice administered with PND-1186, an orally available FAK inhibitor, and in those treated with vehicle (n=20 for each group). We performed immunochemical staining, immunofluorescence staining and Western blot for activated (phosphorylated) FAK (pFAK) to evaluate the activation status of FAK in the aortic tissue. We also performed transcriptome analysis of the aortic tissue in with and without PND-1186 with BAPN + AngII stimulation before AD development. Results Immunochemical staining revealed that FAK was inactive in normal mouse aorta, but was strongly activated in the aortic walls after AD development. Immunofluorescence staining showed that FAK was activated mainly in smooth muscle cells after AD development. Western blot analysis also revealed that FAK was activated in 3 days after BAPN + AngII infusion before AD development, followed by transient reduction at day 7, and re-activation after AD at day 14. Significantly, administration of PND-1186 resulted in a significant reduction in the severity of AD in the aortic arch (1.96±0.41 mm in vehicle group, 0.66±0.29 mm in PND group, P&lt;0.05). In addition, survival rate improved from 36.4% to 80.0% by administration of PND-1186 (P&lt;0.01). In immunofluorescence staining, the PND-1186 treated group showed weaker staining of pFAK. Transcriptome analysis showed that genes for hematopoiesis and immune system were suppressed in PND-1186 treated group. Conclusions These findings proved that FAK plays a central role in the pathogenesis of AD probably by transmitting pathological stress to the aortic wall to cause tissue destruction. We propose that FAK is a potential therapeutic target for limiting the fatal destruction of the aortic wall of AD.

Vps15 is critical to mediate autophagy in AngII treated HUVECs probably by PDK1/PKC signaling pathway

AimsVps15 is an important regulator on the activity of class III PI3K in autophagy induction. AngII plays a positive role of autophagy in the early protection of endothelial cells. In this study, the expression of Vps15 was knocked down using the specific shRNA to investigate the effects of Vps15 on cell autophagy, senescence and apoptosis in HUVECs stimulated by AngII. The associated cell signaling pathway was also explored. Materials and methodsMDC staining was applied to show autophagic bodies. Cell senescence was detected using β-galactosidase staining. Cell apoptosis was examined by flow cytometry using Annexin V-FITC/PI staining. And western blot was used to evaluate the ratio of LC3-II/I and the activation of associated cell signaling pathway. Key findingsCell autophagy induced by AngII was inhibited in HUVECs transfected with Vps15-shRNA, while cell senescence and apoptosis were enhanced. Rescue experiment revealed that cell autophagy was activated after Vps15 reexpression, while cell senescence and apoptosis were inhibited. Moreover, the phosphorylations of PDK1 and PKC substrates were increased after AngII treatment, which were decreased by Vps15 knockdown. Pretreatment of cells with the inhibitor for PDK1 or PKC attenuated cell autophagy after AngII stimulation, yet promoted cell senescence and apoptosis. The phosphorylations of both PDK1 and PKC were inhibited in cells pretreated with PDK1 inhibitor. Only the activation of PKC was inhibited when the inhibitor for pan-PKC was used. SignificanceThese results suggested that Vps15 was critical to the protective autophagy in HUVECs induced by AngII, and PDK1/PKC signaling pathway was probably involved.

ERK1/2 communicates GPCR and EGFR signaling pathways to promote CTGF-mediated hypertrophic cardiomyopathy upon Ang-II stimulation

BackgroundHypertrophic cardiomyopathy occurs along with pathological phenomena such as cardiac hypertrophy, myocardial fibrosis and cardiomyocyte activity. However, few of the specific molecular mechanisms underlying this pathological condition have been mentioned.MethodsAll target proteins and markers expression in the study was verified by PCR and western bloting. H9c2 cell morphology and behavior were analyzed using immunofluorescent and proliferation assays, respectively. And, the CTGF protein secreted in cell culture medium was detected by ELISA.ResultsWe found that high expression of CTGF and low expression of EGFR were regulated by ERK1/2 signaling pathway during the cardiac hypertrophy induced by Ang-II stimulation. CTGF interacted with EGFR, and the interaction is reduced with the stimulation of Ang-II. ERK1/2 serves as the center of signal control during the cardiac hypertrophy.ConclusionThe ERK1/2 cooperates with GPCR and EGFR signaling, and promotes the occurrence and development of cardiac hypertrophy by regulating the expression and binding states of CTGF and EGFR. The study revealed a regulation model based on ERK1/2, suggesting that ERK1/2 signaling pathway may be an important control link for mitigation of hypertrophic cardiomyopathy treatment.

Stachydrine Ameliorates Cardiac Fibrosis Through Inhibition of Angiotensin II/Transformation Growth Factor β1 Fibrogenic Axis.

Cardiovascular diseases, the leading cause of death worldwide, are tightly associated with the pathological myocardial fibrosis. Stachydrine (Sta), a major active compound in Chinese motherwort Leonurus heterophyllus, was reported to effectively attenuate cardiac fibrosis, but the cellular and molecular mechanism remains unclear. In this study, the anti-fibrotic effect of Sta and mechanism underlying were explored in a mouse model of pressure overload and AngII stimulated cardiac fibroblasts (CFs). Mice were randomly divided into sham, transverse aorta constriction with saline (TAC+Sal), TAC with telmisartan (TAC+Tel), and TAC with Sta (TAC+Sta) groups. Cardiac morphological and functional changes were evaluated by echocardiography and histological methods, and the molecular alterations were detected by western blotting. Primary cultured neonatal mouse CFs were treated with or without angiotensin II (AngII, 10−7 M), transformation growth factor β1 (TGFβ1, 10 ng/mL), and different dosage of Sta (10−6–10−4 M) for up to 96 h, and cell proliferation, cytotoxicity, morphology and related signals were also detected. The in vivo results revealed that TAC prominently induced cardiac dysfunction, left ventricular dilation, myocardial hypertrophy, and elevated myocardial collagen deposition, accompanied with increased fibrotic markers including α-smooth muscle actin (α-SMA) and periostin. However, Sta treatment partially reversed cardiac morphological and functional deteriorations, and significantly blunted cardiac fibrosis as well as Tel. Increments of myocardial angiotensinogen (AGT), angiotensin converting enzyme (ACE), AngII type 1 receptor (AT1R), and TGFβ1 transcripts, together with increased protein levels of ACE and AngII, after TAC were dramatically down-regulated by Sta treatment. Coincidently, in vitro experiments demonstrated that AngII stimulation in CFs led to up-regulation of AT1R and TGFβ1, and therefore promoted CFs trans-differentiating into hyper-activated myocardial fibroblasts (MFs) as evidenced by increased cell proliferation, collagen and fibrotic makers. On the contrary, Sta potently down-regulated but not directly inhibited AT1R, suppressed TGFβ1 production, and the pro-fibrotic effect of AngII in CFs. Moreover, activation of TGFβ1/Smads signal in the fibrotic process were observed both TAC model and in AngII stimulated CFs, which were also notably blunted by Sta. However, Sta failed to abolish the activation of CFs triggered by TGFβ1. Taken together, it was demonstrated in this study that Sta suppressed ACE/AngII/AT1R-TGFβ1 profibrotic axis, especially on the de novo production of AngII via down-regulating AGT/ACE and AT1R, and therefore inactivated CFs and blunted MFs transition, which ultimately prevented cardiac fibrosis.

EET Analogs and the Dual‐Inhibition of sEH/COX‐2 for the Treatment of Focal Segmental Glomerular Sclerosis

Focal segmental glomerulosclerosis (FSGS) is the single most common form of kidney disease. This devastating disorder can lead to end stage renal disease (ESRD), a terminal diagnosis in the absence of a kidney transplant. The progression of FSGS is defined by sclerotic damage to the glomerulus, the filtering unit of blood in the kidney. Epoxyeicosatrienoic acids (EETs) are a family of arachidonic acid metabolites important to glomerular function. EETs are metabolized by enzymes such as soluble epoxide hydrolase (sEH) and cyclooxygenase (COX), and we have developed a novel dual‐ligand drug (PTUPB) that inhibits both sEH and COX‐2. We hypothesize that EET analogs and the dual‐inhibition of sEH and COX‐2 will mitigate glomerular damage caused by FSGS. The long‐term goal of this research is to better understand the role of 8,9‐EET and its metabolites on glomerular function so that we can develop new therapies to treat FSGS and other nephropathies.MethodsGlomerular Permeability Assay: Glomeruli were pre‐incubated in 5% albumin (50kDa dialyzed BSA), followed by a bath exchange to 1% albumin. Under normal conditions the glomeruli are non‐permeable to albumin, and as a result, when the 5% albumin bath is exchanged for 1% albumin, water is taken up into the intra‐glomerular space due to the rapid change in oncotic pressure. Glomerular permeability was tested using our non‐hydrolyzable 8,9‐EET analog (1μM), our non‐hydrolyzable 14,15‐EET analog (EET‐A, 1μM) or PTUPB (1μM) with isolated SD rat glomeruli in the presence or absence of angiotensin II (AngII, 10μM), a known glomerular permeabilizing agent. 3D‐Confocal imaging (72μm z‐depth, 27‐layer z‐stack) was utilized to measure the relative changes in glomerular volume.ResultsGlomerular permeability to albumin (Palb) is derived from the albumin reflection coefficient (σalb), where a value of 1 indicates high permeability to albumin, and 0 indicates low permeability (relative to control). A baseline response to a 5% to 1% albumin exchange resulted in very low albumin permeability (Palb = −0.21 ± 0.21, n=6). Conversely, the presence of AngII resulted high albumin permeability (Palb = 1.21 ± 0.12, n=15). Our 8,9‐EET analog, in the presence of AngII, mitigated the effects of AngII‐induced permeability (Palb = 0.13 ± 0.18, n=7), as did our dual sEH/COX2 inhibitor, PTUPB, under the same Ang‐II stimulation (Palb = 0.71% ± 0.35, n=7), but not the 14,15‐EET analog (Palb = 0.99% ± 0.21, n=4) . All values are mean ± SEM.ConclusionFSGS is complex multi‐model disease, and glomerular damage/dysfunction are at the core of this condition. The ability of our 8,9‐EET analog and PUTPB to decrease AngII‐associated glomerular permeability suggest and important role for EETs and their metabolism. Future work will investigate not only the effects of 8,9‐EET analogs and PTUPB on glomerular health, but also the consequences of EET metabolites and other EET‐analogs, on both glomerular and mesangial function using cell and animal models.Support or Funding InformationR01 DK103616‐01 (John D. Imig). Dr. Ralph and Marian Falk Medical Research Trust ‐ Phase II (John D. Imig)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 516: Endothelial Foxp1 Regulates Cardiac Remodeling via Tgfbeta and Et-1 Signal Pathway

Rationale: Cardiac fibrosis and hypertrophy is common features in left ventricular remodeling that leads to heart failure. Forkhead box protein P-1 (Foxp1), a large modular transcriptional repressor that binds to DNA via the highly conserved forkhead DNA-binding domain, is crucial in coordinating the balance of cardiomyocyte proliferation and differentiation through cell lineage-specific regulation during cardiac development. However, its role in adult pathological cardiac remodel remains to be clarified. Objective: We seek to determine the role and the underlying mechanisms of Foxp1 in cardiac fibrosis and hypertrophy. Methods and Results: Foxp1 was highly expressed in endocardium and cardiac microvascular endothelial cells, significantly reduced following AngII stimulation. Loss of endothelial Foxp1 increased AngII-mediated cardiac fibrosis, which was accompanied by myofibroblast proliferation and endothelial-mesenchymal transition (endMT) with elevated expression of fibrotic matrix genes. TGF-β signal pathway was identified as Foxp1 direct downstream target gene in endothelial cells to regulate the fibrosis. Increased AngII-mediated cardiac hypertrophy was observed when loss of endothelial Foxp1, with elevated cardiac hypertrophic genes and fetal myosin gene (myh7) expression but reduced adult mature myosin gene (myh6) expression via endothelial ET-1 signal. The pathological remodel in endothelial Foxp1 deficient mutant mice finally led to worsen cardiac diastolic dysfunction as shown by elevated E/e’ ratio in echocardiogram. Moreover, the similar pathological remodel and worsen cardiac diastolic and systolic dysfunction as shown by reduction of LV fractional shortening (FS) and ejection fraction (EF) were observed in pressure overload transverse aortic constriction (TAC) model when loss of endothelial Foxp1. Conclusions: This study uncovered a previously unrecognized anti-fibrotic and anti-hypertrophic role of Foxp1 in pathological cardiac remodel, which is achieved through the regulation of endothelial TGF-β/ET-1 signals, implying the promising therapeutic targets in pathological cardiac remodeling diseases and heart failure.

Vascular wall regulator of G-protein signalling-1 (RGS-1) is required for angiotensin II–mediated blood pressure control

G-Protein coupled receptors (GPCRs) activate intracellular signalling pathways by coupling to heterotrimeric G-proteins that control many physiological processes including blood pressure homeostasis. The Regulator of G-Protein Signalling-1 (RGS1) controls the magnitude and duration of downstream GPCR signalling by acting as a GTPase-activating protein for specific Gα-proteins. RGS1 has contrasting roles in haematopoietic and non-haematopoietic cells. Rgs1−/−ApoE−/− mice are protected from Angiotensin II (Ang II)-induced aortic aneurysm rupture. Conversely, Ang II treatment increases systolic blood pressure to a greater extent in Rgs1−/−ApoE−/− mice than ApoE−/− mice, independent of its role in myeloid cells. However the precise role of RGS1 in hypertension and vascular-derived cells remains unknown. We determined the effects of Rgs1 deletion on vascular function in ApoE−/− mice. Rgs1 deletion led to enhanced vasoconstriction in aortas and mesenteric arteries from ApoE−/− mice in response to phenylephrine (PE) and U46619 respectively. Rgs1 was shown to have a role in the vasculature, with endothelium-dependent vasodilation being impaired, and endothelium-independent dilatation to SNP being enhanced in Rgs1−/−ApoE−/− mesenteric arteries. To address the downstream signalling pathways in vascular smooth muscle cells (VSMCs) in response to Ang II-stimulation, we assessed pErk1/2, pJNK and pp38 MAPK activation in VSMCs transiently transfected with Rgs1. pErk1/2 signalling but not pJNK and pp38 signalling was impaired in the presence of Rgs1. Furthermore, we demonstrated that the enhanced contractile response to PE in Rgs1−/−ApoE−/− aortas was reduced by a MAPK/Erk (MEK) inhibitor and an L-type voltage gated calcium channel antagonist, suggesting that Erk1/2 signalling and calcium influx are major effectors of Rgs1-mediated vascular contractile responses, respectively. These findings indicate RGS1 is a novel regulator of blood pressure homeostasis and highlight RGS1-controlled signalling pathways in the vasculature that may be new drug development targets for hypertension.

Macrophage digesting dysfunction by Angiotensin II through lysosomal cathepsin B‐mediated Nlrp3 inflammasome activation

Angiotensin II (Ang II) is an octapeptide hormone that plays a significant role in mediating hypertension. Hypertension is considered a chronic inflammatory disease; however, the molecular basis underlying the sterile inflammatory response involved in the process of hypertension remains unclear. The present study aims to investigate the role of macrophage Nlrp3 inflammasomes in engulfing and digesting microbes, which are associated with macrophage function, an early onset of hypertension‐associated macrophage injury. Our findings demonstrate that AngII stimulation decreased the NO release and macrophage digestion in cultured RAW264.7 cell. Accompanied with these changes, AngII markedly increased the Nlrp3 inflammasome formation and activation. As AngII‐induced up‐regulation of Nlrp3 inflammasome in macrophage was attributed to lysosome dysfunction induced cathepsin B release. In addition, Losartan, a nonpeptide Ang II receptor antagonist, decreased AngII‐induced Nlrp3 inflammasome activation, lysosomal membrane permeability, lysosomal cathepsin B release, and macrophage digestion dysfunction in macrophage. Similarly, AngII induced macrophage digestion microbes and NO production, which were blocked by Nlrp3 gene silencing and ATI gene silencing. It is concluded that Angiotensin II enhanced lysosomal membrane permeabilization and consequent release of lysosomal cathepsin B, resulting in activation of the macrophage Nlrp3 inflammasome, which may contribute to NO mediated the development of digesting microbes dysfunction.Support or Funding InformationThis study was supported by the National Natural Science Foundation of China (81603587 and 81603668) and by the Guangdong Science and Technology Department (2017A020211016).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interleukin-6 deficiency attenuates angiotensin II-induced cardiac pathogenesis with increased myocyte hypertrophy

Interleukin-6 (IL-6) signaling is critical for cardiomyocyte hypertrophy, while the role of IL-6 in the pathogenesis of myocardium hypertrophy remains controversial. To determine the essential role of IL-6 signaling for the cardiac development during AngII-induced hypertension, and to elucidate the mechanisms, wild-type (WT) and IL-6 knockout (IL-6 KO) mice were infused subcutaneously with either vehicle or AngII (1.5 μg/h/mouse) for 1 week. Immunohistological and serum studies revealed that the extents of cardiac fibrosis, inflammation and apoptosis were reduced in IL-6 KO heart during AngII-stimulation, while cardiac hypertrophy was obviously induced. To investigate the underlying mechanisms, by using myocardial tissue and neonatal cardiomyocytes, we observed that IL-6/STAT3 signaling was activated under the stimulation of AngII both in vivo and in vitro. Further investigation suggested that STAT3 activation enhances the inhibitory effect of EndoG on MEF2A and hampers cardiomyocyte hypertrophy. Our study is the first to show the important role of IL-6 in regulating cardiac pathogenesis via inflammation and apoptosis during AngII-induced hypertension. We also provide a novel link between IL-6/STAT3 and EndoG/MEF2A pathway that affects cardiac hypertrophy during AngII stimulation.

HOTAIR functions as a competing endogenous RNA to regulate PTEN expression by inhibiting miR-19 in cardiac hypertrophy.

Sustained cardiac hypertrophy (CH) is related to a variety of physiological as well as pathological stimuli and eventually increases the risk of heart failure. HOTAIR has been identified as a competing endogenous RNA in multiple human biological processes. Whether lncRNA-HOTAIR is involved in the progress of CH and how it works still remain unknown. Herein, we found that HOTAIR was down-regulated, while miR-19 was up-regulated in both heart tissues from TAC-operated mice in vivo and cultural cardiomyocytes treated with Ang-II in vitro by real-time PCR. Meanwhile, HOTAIR expression was negatively correlated with miR-19 in TAC-operated mice. HOTAIR overexpression reduced cell surface area and the expression of hypertrophic markers ANP, BNP, and β-MHC in response to Ang-II stimulation as well as knockdown of miR-19. The further molecular mechanisms of HOTAIR action in CH demonstrated that HOTAIR may act as a competing endogenous RNA (ceRNA) for miR-19, thereby modulating the dis-inhibition of its endogenous target PTEN and playing an important role in inhibiting CH progress. These findings reveal a novel function of LncRNAs, which conduce to an extensive understanding of CH and provide novel research directions and therapeutic options for treating this disease.