Cell Line A7r5 Research Articles

Fluoride impairs vascular smooth muscle A7R5 cell lines via disrupting amino acids metabolism

Given the insidious and high-fatality nature of cardiovascular diseases (CVDs), the emergence of fluoride as a newly identified risk factor demands serious consideration alongside traditional risk factors. While vascular smooth muscle cells (VSMCs) play a pivotal role in the progression of CVDs, the toxicological impact of fluoride on VSMCs remains largely uncharted. In this study, we constructed fluorosis model in SD rats and A7R5 aortic smooth muscle cell lines to confirm fluoride impaired VSMCs. Fluoride aggravated the pathological damage of rat aorta in vivo. Then A7R5 were exposed to fluoride with concentration ranging from 0 to 1200 μmol/L over a 24-h period, revealing a dose-dependent inhibition of cell proliferation and migration. The further metabolomic analysis showed alterations in metabolite profiles induced by fluoride exposure, notably decreasing organic acids and lipid molecules level. Additionally, gene network analysis underscored the frequency of fluoride's interference with amino acids metabolism, potentially impacting the tricarboxylic acid (TCA) cycle. Our results also highlighted the ATP-binding cassette (ABC) transporters pathway as a central element in VSMC impairment. Moreover, we observed a dose-dependent increase in osteopontin (OPN) and α-smooth muscle actin (α-SMA) mRNA level and a dose-dependent decrease in ABC subfamily C member 1 (ABCC1) and bestrophin 1 (BEST1) mRNA level. These findings advance our understanding of fluoride as a CVD risk factor and its influence on VSMCs and metabolic pathways, warranting further investigation into this emerging risk factor.

Role of miRNAs regulation of Orai1 and store operated Ca2+ entry in vascular restenosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación Background The main cause of restenosis is attributed to the excessive proliferation and migration of vascular smooth muscle cells (VSMCs). Orai1 is one of the main molecular components of Store-Operated Calcium Entry (SOCE), playing a key role in VSMCs proliferation during neointima formation and vascular remodeling. Recent studies have demonstrated the implication of microRNAs (miRNAs) in VSMCs proliferation and migration, however, the role of these miRNAs as regulators of SOCE during vascular remodeling has not been described. Purpose This study aims to determine whether miRNAs regulate the intracellular Ca2+ handling, focusing on SOCE, during neointima formation. Methods Experiments were conducted in an animal rat model following the "Principles of laboratory animal care" and according to the Spanish law. The neointima development was assessed after balloon injury of carotid arteries and confirmed by haematoxylin and eosin staining, and αSMA staining of tissue sections up to 3 weeks after surgery. miRNAs array and RT-qPCR was used to examine the expression of miRNAs. Dual-Luciferase reporter assay was performed in A7R5 VSMCs line. Results Injured arteries showed significant increase in the expression of Orai1, as compared to non-injured arteries, indicating their possible participation in the neointima layer formation. Microarray of miRNAs analysis of the injured carotids compared to control carotids showed significant increase in the expression of miRNAs related to signalling pathways involving cell proliferation and migration, cell differentiation, or VSMCs contraction. Of these, we confirmed the significant increase of miR-18a-5p, miR-20a-5p, miR-20b-5p and miR-17-5p, which belong to widely studied miR-17-92 cluster involved in different cell proliferation. Moreover, we found that mimics of those miRNAs were able to regulate differentially the activity of the Orai1 promotor in A7R5 cell line, indicating that Orai1 can be targeted by miRNAs during in VSMCs. Conclusions In conclusion, our data confirms that Orai1 is upregulated during neointima formation associated with miRNAs dysregulation. Moreover, we found that Orai1-dependent SOCE can be finely regulated by miRNAs during restenosis.

Molecular Mechanism of Calycosin Inhibited Vascular Calcification.

Vascular calcification (VC) is a pathological condition frequently observed in cardiovascular diseases. Primary factors contributing to VC are osteogenic differentiation of vascular smooth muscle and hydroxyapatite deposition. Targeted autophagy (a lysosome-mediated mechanism for degradation/recycling of unnecessary cellular components) is a useful approach for inhibiting VC and promoting vascular cell health. Calycosin has been shown to alleviate atherosclerosis by enhancing macrophage autophagy, but its therapeutic effect on VC has not been demonstrated. Using an in vitro model (rat thoracic aortic smooth muscle cell line A7r5), we demonstrated effective inhibition of VC using calycosin (the primary flavonoid component of astragalus), based on the enhancement of autophagic flux. Calycosin treatment activated AMPK/mTOR signaling to induce initiation of autophagy and restored mTORC1-dependent autophagosome-lysosome fusion in late-stage autophagy by promoting soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation, thereby preventing stoppage of autophagy in calcified cells. Calycosin substantially reduced degrees of both osteogenic differentiation and calcium deposition in our VC cell model by enhancing autophagy. The present findings clarify the mechanism whereby calycosin mitigates autophagy stoppage in calcified smooth muscle cells and provide a basis for effective VC treatment via autophagy enhancement.

Capsaicin inhibits A7r5 cell senescence via the mitochondrial carrier protein Slc25a12

Aging of vascular smooth muscle cells (VSMCs) is the principal factor responsible for the loss of vascular function, and continuous exposure to high glucose is one of the key factors contributing to the aging of VSMCs. This study established a high glucose-induced senescence model of the A7r5 cell line and used transcriptome sequencing to screen the regulatory target genes of high glucose-induced cellular senescence. The study revealed that the expression of the Slc25a12 gene, which belongs to the solute carrier family 25 member 12, was notably reduced following damage caused by high glucose levels. This inhibition was shown to cause mitochondrial malfunction and cellular senescence. The encoded product of the Slc25a12 gene is a mitochondrial carrier protein that binds to calcium and aids in transporting aspartate for glutamate exchange within the inner mitochondrial membrane. Mitochondrial dysfunction compromises the cell's capacity to resist oxidation and repair damage, and is an inherent element in hastening cellular aging. Moreover, our findings validated that the transient receptor potential vanilloid 1 (TRPV1) agonist capsaicin hindered the decrease in Slc25a12 expression, prevented mitochondrial dysfunction, and blocked cellular senescence. Could the regulation of Slc25a12 expression by capsaicin restore cellular mitochondrial function and restrict senescence? In vitro tests have verified that interference with A7r5 Slc25a12 noticeably diminishes capsaicin's effectiveness in repairing mitochondrial function and inhibiting senescence. The findings indicate that capsaicin delays mitochondrial dysfunction and therefore hinders cellular senescence by regulating the mitochondrial membrane protein Slc25a12 in the A7r5 cell line.

#3986 THE ROLE AND MECHANISM OF EZH2 IN VASCULAR CALCIFICATION ASSOCIATED WITH CHRONIC KIDNEY DISEASE

Abstract Background and Aims Vascular calcification (VC) is a prevalent complication in chronic kidney disease and contributes to increased cardiovascular morbidity and mortality. EZH2 (Enhancer of zeste 2 homolog-2) is reported as a key epigenetic regulator involved in various kidney diseases such as acute kidney injury, renal fibrosis, diabetic nephropathy, and lupus nephritis. In this study, we aimed to investigated the role of EZH2 in chronic kidney disease associated vascular calcification. Method 1. Patients and Radial Artery Analysis Radial arteries with or without calcification from patients with end-stage renal disease that underwent arterial venous fistular surgery were collected for Von Kossa staining and immunohistochemical (IHC) staining. 2. Animal model of Chronic renal failure-Vascular Calcification (CRF-VC) (in vivo) An adenine and phosphate (1.2%) diet-induced CRF mouse model was designed following an 8-week program. The thoracoabdominal arteries of mice and rats were dissected to assay Ca deposition and histological analysis. Plasma levels of Alkaline phosphate (ALP), and phosphate (Pi) were measured. The transcription level of EZH2 and Runt-related transcription factor 2 (Runx2) were measured by real-time PCR and IHC staining. 3. Aortic ring calcification (ex vivo) Thoracic aortas were dissected from SD rat. To induce calcification, Pi (NaH2PO4/Na2HPO4 [pH = 7.4]) was added to HG-DMEM at a final concentration of 2.6 mmol/l. After the indicated incubation periods (3, 5, 7 days), samples were taken for Calcium deposition assay, Western blotting and histological analysis. 4. Cell culture and treatment (in vitro) Calcification of rat smooth muscle embryonic thoracic aorta cell line A7r5 (VSMC) was induced by incubation in calcifying media. 3-deazaneplanocin A (3-DZNeP), a carbocyclic analog of adenosine was added right after Pi to block EZH2 activation. After the indicated incubation periods, samples were collected for Calcium deposition assay and Western blotting analysis. Results 1. In vivo, EZH2 protein level was increased in calcified radial arteries from patients with end-stage renal disease that underwent arterial venous fistular operation. In CRF-VC mice model, the aortic calcium deposition markedly aggravated in the group induced by adenine diet. Severe vascular calcification was also induced as shown by increased intensity of von Kossa. Moreover, IHC and q-PCR results showed that the protein and transcriptional levels of EZH2 expression were upregulated in calcified aortas of CRF mice, in paralleled with the increased expression Runx2 in protein and transcriptional levels. 2. Ex vivo, histological analysis revealed significant medial vascular calcification in the aortic ring culture with this calcifying medium for 7 days. Similarly, WB results showed that the expression of EZH2 and its downstream, methylation of Histone H3 at lysine 27 (H3K27me3), were significantly enhanced after high Pi medium stimulation while the osteogenic markers Runx2, Osteopontin (OPN) and Msh homeobox 2 (Msx2) protein levels were upregulated and the expression of VSMC differentiation markers α-Smooth muscle actin (α-SMA), Calponin (CNN) and Smooth muscle protein 22 (SM22) were downregulated. 3. In vitro, the osteogenic markers Runx2, OPN and Msx2 were significantly upregulated in high Pi treated VSMCs compared to control VSMCs. Moreover, the epigenetic markers EZH2 and H3K27me3a were markedly repressed in calcifying VSMCs by time. 4. 3-DZNep treatment reduced the calcium deposition in a concentration dependent manner. Western blotting showed that 3-DZNep treatment blocked the increased expression of osteogenic markers Runx2, Bone morphogenetic protein 2 (BMP2) as well as epigenetic markers EZH2 and H3K27me3a by high Pi treatment, indicating that inhibition of EZH2 attenuates VSMCs Calcification. Conclusion Our study revealed that EZH2 is highly expressed in calcified vascular tissues of CRF patients and CRF mice, rat aorta culture and VSMCs calcification models, and EZH2 inhibitor 3-DZNep attenuated calcification of VSMCs. EZH2 could be a promising target for treatment of vascular calcification in CRF patients.

Chemical constituents and cytotoxic activity of Ocimum gratissimum L.

• The leaves of Ocimum gratissimum L. was subjected to identification of its chemical constituents. • The in vitro cytotoxic activity of compounds against lung adenocarcinoma (A549), breast carcinoma (MCF-7), acute monocytic leukemia (THP-1), murine macrophages Raw 264.7 cancer cell lines and the normal fibroblast A7R5 cell line were studied. • THP-1 was the most sensitive to compounds 1-6 . • Compound 4 and paclitaxel have similar IC 50 value against THP-1 cells. Malignancies constitute a global health concern and chemotherapy remains the main mode of treatment. The present study was designed to evaluate the cytotoxicity of metabolites isolated from Ocimum gratissimum L. The phytochemical investigation of the leaves of O. gratissimum L. (Lamiaceae) led to the isolation and characterization of eight compounds. Structures of isolated compounds were elucidated using 1D and 2D NMR, as well as MS. The purified compounds, were identified as five triterpenes ( 1-5 ), one flavonoid ( 6 ) and two steroidal compounds ( 7-8 ). The cytotoxicity effects of purified compounds were assessed in vitro by the WST-1 assay on three cancer cell lines including lung adenocarcinoma (A549), breast carcinoma (MCF-7), acute monocytic leukemia (THP-1), murine macrophages (Raw 264.7) and the normal fibroblast (A7R5) cells. Compound 4 had an IC 50 value of 3.21±0.4 µM similar to that of the reference drug, paclitaxel against THP-1 cells. The THP-1 cell lines were the most sensitive to compounds 1-6 . All the compounds showed low cytotoxicity against the normal cell line A7R5 as compared to cancer cell lines. The constituents of Ocimum gratissimum L. and especially pomolic acid ( 4 ) is a potent cytotoxic compound that deserves more investigations toward developing novel antiproliferative drugs against acute monocytic leukemia (THP-1).

Angiotensin II-induced miR-31-5p upregulation promotes vascular smooth muscle cell proliferation and migration

Angiotensin II (Ang II) plays a central role in vascular smooth muscle cell (VSMC) proliferation and migration, being key to regulate vascular function and promote vascular remodeling in cardiovascular diseases. We recently showed that miR-31-5p promoted oxidative stress in spontaneously hypertensive rats. In this study, we aim to investigate whether miR-31-5p and fibronectin type III domain-containing 5 (FNDC5) contribute to Ang II-induced VSMC proliferation and migration. Experiments were performed in primary VSMCs of wide-type (WT) and FNDC5−/− mice as well as the rat A7r5 cell line. We found that Ang II increased miR-31-5p level, reduced FNDC5 expression and stimulated VSMC proliferation and migration, which were aggravated by miR-31-5p mimic, and prevented by miR-31-5p inhibitor in VSMCs. The Ang II-induced VSMC proliferation were prevented by exogenous FNDC5 in both WT and FNDC5−/− mice, while the effects were more significant in FNDC5−/− mice. Furthermore, exogenous FNDC5 reversed the effects of miR-31-5p mimic on VSMC proliferation and migration in Ang II-treated VSMCs. Meanwhile, FNDC5 deficiency prevented the effects of miR-31-5p inhibitor on VSMC proliferation and migration in Ang II-treated VSMCs. In conclusion, our findings demonstrate that the miR-31-5p upregulation and the following FNDC5 downregulation contribute to Ang II-induced VSMC proliferation and migration.

Mulberry Leaf and Neochlorogenic Acid Alleviates Glucolipotoxicity-Induced Oxidative Stress and Inhibits Proliferation/Migration via Downregulating Ras and FAK Signaling Pathway in Vascular Smooth Muscle Cell.

Mulberry leaf (Morus alba L.) has been used as a health food and in traditional medicine to treat several metabolic diseases, including diabetes, hypertension, and hyperlipidemia. However, the mechanism by which mulberry leaf and its functional components mediate atherosclerosis remains unclear. This study aimed to determine the effect of mulberry leaf extract (MLE) and its major component, neochlorogenic acid (nCGA), on the proliferation and migration of rat aortic vascular smooth muscle cells (VSMCs, A7r5 cell line) under diabetic cultured conditions (oleic acid and high glucose, OH). Our findings showed that MLE and nCGA significantly inhibited cell proliferation and migration in A7r5 cells as determined by a scratch wound assay and a Transwell assay. Furthermore, we observed MLE and nCGA inhibited cell proliferation and migration, such as reducing the phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt), focal adhesion kinase (FAK), and small GTPase proteins using Western blot analysis. In conclusion, we confirmed the anti-atherosclerotic effects of MLE and nCGA in reducing vascular smooth muscle cell (VSMC) migration and proliferation under diabetic cultured conditions via inhibition of FAK/small GTPase proteins, PI3K/Akt, and Ras-related signaling.

Regulation of total LC3 levels by angiotensin II in vascular smooth muscle cells

Hypertension is associated with high circulating angiotensin II (Ang II). We have reported that autophagy regulates Ang II‐induced vascular smooth muscle cell (VSMC) hypertrophy, but the mechanism mediating this effect is still unknown. Therefore, we studied how Ang II regulates LC3 levels in VSMCs and whether Bag3, a co‐chaperone known to regulate LC3 total levels, may be involved in the effects elicited by Ang II. A7r5 cell line or rat aortic smooth muscle cell (RASMC) primary culture were stimulated with Ang II 100 nM for 24 h and LC3 I, LC3 II and Bag3 protein levels were determined by Western blot. MAP1LC3B mRNA levels were assessed by RT‐qPCR. Ang II increased MAP1LC3B mRNA levels and protein levels of LC3 I, LC3 II and total LC3 (LC3 I + LC3 II). Cycloheximide, but not actinomycin D, abolished LC3 II and total LC3 increase elicited by Ang II in RASMCs. In A7r5 cells, cycloheximide prevented the Ang II‐mediated increase of LC3 I and total LC3, but not LC3 II. Moreover, Ang II increased Bag3 levels, but this increase was not observed upon co‐administration with either losartan 1 μM (AT1R antagonist) or Y‐27632 10 μM (ROCK inhibitor). These results suggest that Ang II may regulate total LC3 content through transcriptional and translational mechanisms. Moreover, Bag3 is increased in response to Ang II by a AT1R/ROCK signalling pathway. These data provide preliminary evidence suggesting that Ang II may stimulate autophagy in VSMCs by increasing total LC3 content and LC3 processing.

MiR-31-5p Promotes Oxidative Stress and Vascular Smooth Muscle Cell Migration in Spontaneously Hypertensive Rats via Inhibiting FNDC5 Expression.

Oxidative stress and the migration of vascular smooth muscle cells (VSMCs) are important for vascular remodeling in a variety of vascular diseases. miR-31-5p promotes cell migration in colorectal cancer cells but inhibits cell migration in renal cell carcinoma. However, whether miR-31-5p is involved in oxidative stress and VSMC migration remains unknown. This study shows the crucial roles of miR-31-5p in oxidative stress and VSMC migration, as well as underlying mechanisms. Experiments were carried out in primary VSMCs from aortic media of Wistar–Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), as well as the A7r5 cell line. Oxidative stress was assessed by NADPH oxidase (NOX) expression, NOX activity, and reactive oxygen species (ROS) production. Cell migration was evaluated with a Boyden chamber assay and a wound healing assay. The miR-31-5p mimic and inhibitor promoted and attenuated oxidative stress and cell migration in the VSMCs of SHR, respectively. A dual-luciferase reporter assay indicated that miR-31-5p targeted the 3’UTR domain of FNDC5. The miR-31-5p level was raised and FNDC5 expression was reduced in the VSMCs of SHR compared with those of WKY. The miR-31-5p mimic reduced FNDC5 expression in the A7r5 cells and the VSMCs of both WKY and SHR, while the miR-31-5p inhibitor only increased FNDC5 expression in the VSMCs of SHR. Exogenous FNDC5 attenuated not only the oxidative stress and VSMC migration in SHR but also the roles of the miR-31-5p mimic in inducing oxidative stress and VSMC migration. These results indicate that miR-31-5p promotes oxidative stress and VSMC migration in SHR via inhibiting FNDC5 expression. The increased miR-31-5p and reduced FNDC5 in the VSMCs of SHR contribute to enhanced oxidative stress and cell migration.

Curcumin-mediated Photodynamic Therapy Inhibits the Phenotypic Transformation, Migration, and Foaming of Oxidized Low-density Lipoprotein-treated Vascular Smooth Muscle Cells by Promoting Autophagy

Vascular smooth muscle cells (VSMCs) are becoming a hot spot and target of atherosclerosis research. This study aimed to observe the specific effects of curcumin (CUR)-mediated photodynamic therapy (CUR-PDT) on oxidized low-density lipoprotein (ox-LDL)-treated VSMCs and confirm whether these effects are mediated by autophagy. In this study, the mouse aortic smooth muscle cell line and A7r5 cell lines were used for parallel experiments. VSMC viability was evaluated by Cell Counting Kit-8 assay. VSMCs were treated with ox-LDL to establish a model of atherosclerosis in vitro. The autophagy level and the expression of proteins related to phenotypic transformation were detected by western blotting. The migration ability of the cells was detected by using transwell assay. The presence of intracellular lipid droplets was detected by Oil Red O staining. The results showed that VSMCs transformed from the contraction phenotype to the synthetic phenotype when stimulated by ox-LDL, during which autophagy was inhibited. However, CUR-PDT treatment significantly promoted the level of autophagy and inhibited the process of phenotypic transformation induced by ox-LDL. In addition, ox-LDL significantly promoted VSMC migration and increased the number of lipid droplets, whereas CUR-PDT treatment significantly reduced the ox-LDL-induced increase in the migration ability of, and lipid droplet numbers in, VSMCs. When the VSMCs were pretreated with the autophagy inhibitor 3-methyladenine for 24 hours, the effects of CUR-PDT were reversed. Therefore, our study indicated that CUR-PDT can inhibit the phenotypic transformation, migration, and foaming of ox-LDL-treated VSMCs by inducing autophagy.

Acarbose attenuates migration/proliferation via targeting microRNA-143 in vascular smooth muscle cells under diabetic conditions.

Acarbose (an α-glucosidase inhibitor) has been demonstrated to reduce the progression of atherosclerosis without affecting serum levels of glucose in rabbits fed a high cholesterol diet. The main focus of recent atherosclerosis studies has been microRNA targets. However, the mechanism by which acarbose targets miRNA-mediated atherosclerosis remains unclear. This study aimed to evaluate the effect of acarbose on microRNA-related regulation of rat aortic vascular smooth cell line (A7r5 cell) migration and proliferation induced by diabetic conditions. We reported that acabose exhibit significantly inhibits proliferative and cell migration abilities in A7r5 cells. The expression of protein and levels of mRNA were measured by Western blot analysis and real-time PCR. Acarbose inhibited the phosphorylation of focal adhesion kinase (FAK) and phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt), Ras signals, small GTPase proteins expression to attenuate cell migration and proliferation. Furthermore, acarbose upregulated the expression of miR-143, and transfected miR-143 mimic and its inhibitor to explore its mechanism. In conclusion, acarbose reduces VSMC migration and proliferation via upregulating miR-143 to inhibit Ras-related signaling, and potentially prevention of atherosclerosis.

Abstract 480: Non-muscle Myosin II Regulates Aortic Stiffness via Tension-dependent Phosphorylation of Specific Focal Adhesion Proteins

Non-muscle myosin II plays a role in many fundamental cellular processes including cell adhesion, migration, and cytokinesis. However, its role in vascular function is not well understood. Here, we investigated the function of non-muscle myosin II in the biomechanical properties of mouse proximal aorta. We found that blebbistatin, a specific inhibitor of non-muscle myosin II decreases agonist-induced aortic stress and stiffness in a dose-dependent manner. We also specifically demonstrate, in freshly isolated contractile aortic smooth muscle cells, using deconvolution microscopy that the NM myosin IIA isoform co-localizes with contractile filaments in the core of the cell as well as in the non-muscle cell cortex. However, the NM myosin IIB isoform is only colocalized with contractile filaments, and is excluded from the cell cortex. Furthermore, both the siRNA knockdown of NMIIA and NMIIB isoforms in a differentiated smooth muscle cell line A7r5 and blebbistatin-mediated inhibition of NM myosin II suppresses agonist-activated increases in phosphorylation of FAK Y925 and paxillin Y118. Thus, in the present study, we show, for the first time, that NM myosin II regulates aortic stiffness and that this regulation is mediated at least in part through the tension-dependent phosphorylation of focal adhesion proteins FAK and paxillin.

Apatinib attenuates phenotypic switching of arterial smooth muscle cells in vascular remodelling by targeting the PDGF Receptor-β.

Apatinib (YN968D1) is a small‐molecule tyrosine kinase inhibitor（TKI）which can inhibit the activity of vascular endothelial growth factor receptor‐2 (VEGFR‐2). It has been reported that apatinib has anti‐tumour effect of inhibiting proliferation and inducing apoptosis of a variety of solid tumour cells, whereas its effect on vascular smooth muscle cells (VSMC) remains unclear. This study investigated the effect of apatinib on phenotypic switching of arterial smooth muscle cells in vascular remodelling. Compared to the vehicle groups, mice that were performed carotid artery ligation injury and treated with apatinib produced a reduction in abnormal neointimal area. For in vitro experiment, apatinib administration inhibited VSMC proliferation, migration and reversed VSMC dedifferentiation with the stimulation of platelet‐derived growth factor type BB (PDGF‐BB).In terms of mechanism, with the preincubation of apatinib, the activations of PDGF receptor‐β (PDGFR‐β) and phosphoinositide‐specific phospholipase C‐γ1 (PLC‐γ1) induced by PDGF‐BB were inhibited in VSMCs. With the preincubation of apatinib, the phosphorylation of PDGFR‐β, extracellular signal‐related kinases (ERK1/2) and Jun amino‐terminal kinases (JNK) induced by PDGF‐BB were also inhibited in rat vascular smooth muscle cell line A7r5. Herein, we found that apatinib attenuates phenotypic switching of arterial smooth muscle cells induced by PDGF‐BB in vitro and vascular remodelling in vivo. Therefore, apatinib is a potential candidate to treat vascular proliferative diseases.

8-Ethynylxanthines as promising antiproliferative agents, angiogenesis inhibitors, and calcium channel activity modulators

Synthetic procedures for the preparation of 8-ethynylxanthines by treating 8-bromocaffeine and 8-bromopentoxifylline with terminal acetylenes were elaborated. Certain ethynylxanthine derivatives exhibit high in vitro antiproliferative activity against a panel of cancer cell lines, matrix metalloproteinase and in vitro angiogenesis inhibitory activity. Ca2+ channel blocking and agonist activity of the synthesized ethynylxanthines was discussed based on data obtained on the H9C2, SH-SY5Y, and A7R5 cell lines.

Using 99mTc-(V)-DMSA to follow the vascular calcification process in vascular smooth muscle cells based on pit-1 expression.

Vascular calcification is an established feature of atherosclerosis process. The sodium/phosphate transporter PiT-1 acts as a biosensor in vascular calcification of VSMCs. [99mTc]-Pentavalent dimercaptosuccinic acid (99mTc-(V)-DMSA) was mediated by PiT-1 transporter in tumoral cells and we propose its evaluation in a vascular calcification in vitro model. The aim of this study was to determine if 99mTc-(V)-DMSA can follow the vascular calcification process in vascular smooth muscle cells (VSMCs) based on PiT-1 expression. From a rat aortic VSMC cell line (A7r5), we set up a model of calcification within 7 days using a calcifying medium containing a high inorganic phosphate concentration. Phosphocalcic deposits were monitored with Alizarin red and Von Kossa staining and with phase contrast microscopy. PiT-1 expression was evaluated with an immunofluorescence assay and osteopontin expression, with whole cell ELISA assay. 99mTc-(V)-DMSA uptake was measured in control and calcifying conditions and compared with optical microscopy evaluation. Under hyperphosphatemia conditions, the VSMC cells progressively overexpressed osteopontin protein, PiT-1 transporter, and synthetized mineralized matrix with phosphocalcic deposition. 99mTc-(V)-DMSA uptake was to 2.8±2.08%DA/mg-protein in control cells and 42±24%DA/mg-protein in calcified cells (P<0.001). PiT-1 inhibition with phosphonoformic acid completely reverse the calcium deposition as well as the 99mTc-(V)-DMSA uptake. These results demonstrated that 99mTc-(V)-DMSA in-vitro uptake is mediated by PiT-1 transporter and follow the VSMC calcification process. These preliminary in-vitro results showed 99mTc-(V)-DMSA uptake follow the phospho-calcic deposition mediated by PiT-1 transporter. This radiotracer may have some potential to detect changes of VSMC metabolism occurring in the atherosclerosis process.

A Self-Assembling Lipidic Peptide and Selective Partial V2 Receptor Agonist Inhibits Urine Production

Lipidised analgesic peptide prodrugs self-assemble into peptide nanofibers; with the nanofiber morphology protecting the peptide from plasma degradation and improving therapeutic efficacy. Extending this learning, we hypothesised that a self-assembling lipidized peptide arginine vasopressin (AVP) receptor agonist, that had not been designed as a prodrug, could prove pharmacologically active and control urine production. The only approved AVP receptor agonist, desmopressin is indicated for the treatment of central diabetes insipidus (DI), bedwetting, haemophilia A and von Willebrand disease. Desmopressin is well tolerated by most patients, however adverse effects, such as hyponatraemia and water intoxication necessitate a strict fluid intake, thus motivating the search for alternative DI treatments. Selective V2 receptor agonism is required for anti-DI activity and we hypothesised that our new lipidized peptide (METx) would lead to selective AVP receptor agonism. METx was synthesised and characterised and then tested for activity against the V2, V1a and OT uterine receptors and not tested against the V1b receptor as METx was not expected to cross the blood brain barrier. METx was also tested in vivo in a healthy rat model. METx forms nanofibers and is a partial V2 receptor agonist (determined by measuring MDCK cell line cAMP accumulation), producing 57% of AVP’s maximal activity (EC50 = 2.7 nM) and is not a V1a agonist up to a concentration of 1 μM (determined by measuring A7r5 cell line D-myo-inositol-1-phosphate accumulation). METx is a weak OT receptor antagonist, reducing the frequency of OT induced contractions (EC50 = 350 nM) and increasing the OT EC50 from 0.081 nM to 21 nM at a concentration of 600 nM. METx (41 nM) had no effect on spontaneous uterine contractions and METx (100 nM) had no effect on OT induced uterine contractions. Simulated binding studies show that binding avidity to the receptors follows the trend: V2 > OT > V1a. On intravenous injection, a nanoparticle formulation of METx reduced urine production in a healthy rat model in a dose responsive manner, with 40 mg kg−1 METx resulting in no urine production over 4 hours. The lipidized self-assembling peptide – METx - is a selective competitive V2 receptor agonist and an anti-diuretic.

Purification and identification of angiotensin II type I receptor downregulating peptide from egg white hydrolysate.

The vasoconstricting effect of angiotensin II (Ang II) is mediated by Ang II type I receptor (AT1R); blocking or blunting AT1R is a key strategy for the development of antihypertensive drugs. Our previous study showed that the blood pressure lowering the activity of egg white hydrolysate (EWH) in spontaneously hypertensive rats was due to the downregulation of aortic AT1R expression, which promoted us to further identify the AT1R downregulating peptides in the present study. The protein expression of AT1R in a rat aortic vascular smooth muscle cell line A7r5 cells was used for the activity test. The hydrolysate was fractionated stepwise by C18 Sep-Pack cartridge and reverse-phase chromatography, and the peptide sequences were characterized by LC-MS/MS. Peptides including ITKPNDVYS, VVGSAEAGVDAAS, AVHAAHAEINEAGRE, AGREVVGSAEAGVD, and VVGSAEAGVD, were identified. ITKPNDVYS showed the most potent peptide for lowering the level of AT1R in A7r5 cells. These results suggested that ITKPNDVYS is the responsible peptide for lowering the AT1R level in EWH. PRACTICAL APPLICATIONS: A new peptide ITKPNDVYS was identified as a downregulator of AT1R from egg white hydrolysate, which provides a new approach for the development of food protein-derived antihypertensive peptides as nutraceuticals or functional food ingredients.

A ruthenium nitrosyl cyclam complex with appended anthracenyl fluorophore

The complex trans-[Ru(NO)(H2O)(L)](ClO4)3 (L = (1-anthracen-9-ylmethyl)-1,4,8,11-tetraazacyclotetradecane) (RuNOL) was synthesized and characterized, and its photophysics and reactivity were investigated. The FTIR spectrum displays a νNO band at 1840 cm−1, indicating a nitrosonium character. 1H and 13C signals in 1D and 2D NMR spectra were assigned and are consistent with the trans configuration. The UV–Vis spectrum displays maximal absorption bands at 341 nm (log ε 3.74), 363 nm (log ε 3.91), 378 nm (log ε 3.97) and 397 nm (log ε 3.91). Coordination of the [RuNO] moiety to the L ligand strongly quenches the anthracenyl fluorescence, and thus RuNOL exhibits only very weak emission at 390, 418, 440 and 472 nm. Electrochemical studies indicate that cathodic peak potentials at +1.10 V(Ic), −0.1 V(IIc) and −0.4 V(IIIc) (vs Ag/AgCl) are related to An+/An, {RuNO}6/7 and {RuNO}7/8 reduction processes, respectively. The pKa of coordinated water was estimated as 2.8 ± 0.2 by DPV. The emission of the pendant fluorophore increases upon electrochemical or chemical reduction of RuNOL, and the resulting switch-ON fluorescence is possibly due to NO release from the target complex. Spectroscopic titrations with DNA resulted in hypochromism and red shifts and allowed estimation of binding constants of 1.9 × 103 (L) and 1.8 × 103 (RuNOL). Molecular docking showed that RuNOL complex has a great affinity with DNA. The RuNOL complex showed low cytotoxicity toward MCF-7 and NIH-3T3 and was ineffective in healthy HUVEC and A7r5 cell lines in the range of concentration of 1 × 10−7–1 × 10−4 mol L−1.

Hydrogen sulfide inhibited L-type calcium channels (CaV1.2) via up-regulation of the channel sulfhydration in vascular smooth muscle cells

Hydrogen sulfide (H2S) exerts different effects on the cardiovascular system by modulating ion channels. The present study was to ascertain whether H2S affects L-type calcium (Ca2+) channels in vascular smooth muscle cells (VSMCs) and the subsequent signaling pathways. Here, CaV1.2 L-type Ca2+ currents (ICa, L) were inhibited by sodium hydrosulfide (NaHS, an H2S donor) in A7r5 cell lines using the whole-cell patch-clamp technique. Then NaHS significantly reduced intracellular Ca2+ concentration ([Ca2+]i) in Bayk8644-stimulated CaV1.2-HEK293 cells by using flow cytometry. However, NaHS did not affect the ryanodine-induced elevation of [Ca2+]i by means of confocal microscopy, ruling out its influence on the intracellular Ca2+ release. In the following, the sulfhydration of L-type Ca2+ channels was determined by Ellman's Test. The results showed that NaHS decreased the number of free sulfhydryls, which was further strengthened by the oxidant sulfhydryl modifier diamide (DM) and significantly counteracted by the reductant sulfhydryl modifier dithiothreitol (DTT). DTT also partly reversed the NaHS-reduced [Ca2+]i in CaV1.2-HEK293 cells. Additionally, NaHS did not change CaV1.2 expression. Furthermore, NaHS increased phosphorylation of PKC and ERK in both a concentration- and a time-dependent manner in VSMCs. Isradipine, L-type Ca2+ channel specific blocker, further increased H2S-induced phosphorylation of PKC and ERK, showing an additive effect with H2S. Therefore, our results suggest that H2S reduced ICa, L & [Ca2+]i and hence influenced the downstream PKC/ERK pathway, which was likely through regulating the sulfhydration of L-type Ca2+ channels in VSMCs.