A7r5 Vascular Smooth Muscle Cells Research Articles

Identification and Characterization of Potential Anti-Hypertensive Therapeutic Candidates in Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin 3 (CUL3) Axis

Blood pressure (BP) is regulated by a variety of cellular mechanisms. We previously reported the regulation of endothelial nitric oxide (NO)-dependent vessel tone and BP through RhoBTB1-CUL3 mediated proteasomal turnover of Phosphodiesterase 5 (PDE5). We determined that the C-terminal half of RhoBTB1 consisting of two BTB-domains (B1 & B2) and the C-terminal (C) domain of RhoBTB1 can deliver PDE5 to proteasomal pathway. The cellular expression of RhoBTB1 and CUL3 is ubiquitous whereas expression of PDE5 is cell specific. Moreover, vessel tone is regulated through NO-independent mechanisms. Therefore, we hypothesized that RhoBTB1 may have other interacting partners, potentially functioning as regulators of vessel tone and BP in vascular smooth muscle (VSM) cells, through a similar mode of binding as PDE5. Therefore, we utilized ascorbate peroxidase (APEX2) proximity labelling system comprising APEX2 tagged B1B2C domain (B1B2C-APEX2) to identify unknown potential regulators of vasomotor tone in RhoBTB1-CUL3 axis in A7R5 VSM cells. The background of the proteomic screen was minimized by utilizing APEX2-B1B2 which lacks the final C-terminal region of the protein and cannot bind PDE5. We validated the interaction of several identified proteins with RhoBTB1 and the B1B2C domains using co-immunoprecipitation (Co-IP). Our Co-IP data corroborated the proteomic screen as several proteins demonstrated differential interaction with B1B2C and B1B2. Among them, we examined RbFox2, a regulator of mRNA splicing for further investigation. Further data established that RbFox2 is upregulated under the following conditions: 1) pharmacological inhibition of the proteasomal pathway, 2) in CRISPR edited CUL3KO HEK293 cells, 3) in aorta of VSM cell-specific CUL3 deleted mice, 4) by siRNA mediated knock-down of RhoBTB1 in SMCs, and 5) in Ang-II treated SMCs and aorta of Ang-II treated animals. We hypothesized that RbFox2 could modulate actin and actinin-dependent cytoskeletal dynamics, an important regulator of arterial stiffness. First, Co-IP data suggest an interaction between RbFox2 and SMC-specific alpha actinin. Next, siRNA-mediated knock down of RbFox2 altered the actin and actinin-dependent cytoskeletal dynamics in SMCs. Collectively, this establishes the potential regulation of RbFox2 through the RhoBTB1-CUL3 proteasomal pathway and that RbFox2 may play a mechanistic role in arterial stiffness in hypertension. Funding: NIH R35 HL144807 (to Dr. Curt D. Sigmund). 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.

Zinc Deficiency Promotes Calcification in Vascular Smooth Muscle Cells Independent of Alkaline Phosphatase Action and Partly Impacted by Pit1 Upregulation.

Inorganic phosphate (Pi) is a critical determinant of calcification, and its concentration is regulated by alkaline phosphatase (ALP) and Pit1. ALP is a key regulator of osteogenic calcification and acts by modulating local inorganic phosphate (Pi) concentrations through hydrolyzing pyrophosphate in the extracellular matrix (ECM). Pit1, a sodium-dependent phosphate transporter, regulates calcification via facilitating phosphate uptake within the cells. To investigate whether zinc differentially regulates osteoblastic and vascular calcifications, we examined ALP activity and Pit1 in osteoblastic and vascular smooth muscle cells (VSMCs). Our findings demonstrate that calcification in osteoblastic MC3T3-E1 cells is decreased via diminished ALP action under zinc deficiency. In contrast, zinc-deficiency-induced calcification in VSMCs is independent of ALP action, as demonstrated by very weak ALP activity and expression in calcified VSMCs. In zinc-deficient A7r5 VSMC, P accumulation increased with increasing Na phosphate concentration (3-7 mM) but not with β-GP treatment, which requires ALP activity to generate Pi. Ca deposition also increased with Na phosphate in a dose-dependent manner; in contrast, β-GP did not affect Ca deposition. In osteoblastic cells, Pit1 expression was not affected by zinc treatments. In contrast, Pit1 expression is highly upregulated in A7r5 VSMC under zinc deficiency. Using phosphonoformic acid, a competitive inhibitor of Pit1, we showed that calcification is inhibited in both A7r5 and MC3T3-E1 cells, indicating a requirement for Pit1 in both calcifications. Moreover, the downregulation of VSMC markers under zinc deficiency was restored by blocking Pit1. Taken together, our results imply that zinc-deficiency-induced calcification in VSMC is independent of ALP action in contrast to osteoblastic calcification. Moreover, Pit1 expression in VSMCs is a target for zinc deficiency and may mediate the inhibition of VSMC marker expression under zinc deficiency.

Mechanism of cytotoxicity induced by the cigarette smoke extract (CSE) of heated tobacco products in vascular smooth muscle cells: A comparative study of the cytotoxic effects of CSE and the ferroptosis inducer, erastin

Heated tobacco products (HTPs) are marketed worldwide as less harmful alternatives to combustible cigarettes; however, their cytotoxic mechanisms in vascular smooth muscle cells are poorly understood. Ferroptosis is defined as iron-dependent cell death caused by the accumulation of lipid peroxidation products. In this study, the cytotoxic effects of nicotine- and tar-free cigarette smoke extracts (CSE) derived from three types of HTPs and the ferroptosis inducer, erastin, on vascular smooth muscle A7r5 cells were compared. Cigarette smoke from all HTPs was generated according to the following puffing regime: 55 mL, puff volume; 30 s, puff interval; 2 s, puff duration; bell-shaped, puff profile; and no blocking of the ventilation holes. Erastin and CSE decreased mitochondrial metabolic activity and increased lactate dehydrogenase leakage. The cytotoxic effects of erastin were almost completely inhibited by the radical-trapping antioxidant, UAMC-3203; iron chelator, deferoxamine mesylate (DFO); 12/15-lipoxygenase (12/15-LOX) inhibitor, baicalein; and selective 15-LOX inhibitor, ML351. In contrast, CSE-induced cell damage was partially attenuated by UAMC-3203, baicalein, and ML351 but not by DFO. These results suggest that erastin induces ferroptosis via 15-LOX-mediated iron-dependent lipid peroxidation, whereas CSE causes iron-independent cell damage via 15-LOX-mediated lipid peroxidation-dependent and -independent mechanisms.

Ligustilide from Radix Angelica Sinensis prevents the migration of vascular smooth muscle A7r5 cells based on network pharmacology and experimental verification

ObjectiveLigustilide (Lig), a major component of Radix Angelica Sinensis, exhibits a protective effect on many cardiovascular diseases. Our previous study showed that Lig inhibited the proliferation of vascular smooth muscle cells. In this study, we aimed to investigate the effect of Lig on the migration of vascular smooth muscle A7r5 cells induced by Angiotensin II (Ang II) and explore the mechanism underlying based on network pharmacology. MethodsIn this study, scratch-wound healing assay and transwell migration assay were used to assess the migration activity of Lig on Ang II-induced vascular smooth muscle A7r5 cells, and network pharmacology method was used to predict the possible target molecules. Western blot analysis, Gelatin zymography assay, and small interfering RNA (siRNA) were used to determine the underlying mechanism of Lig on the migration in Ang II-induced A7r5 cells. ResultsWe found that Lig could prevented the migration induced by Ang II in A7r5 cells, which could be associated with c-Myc and MMPs from the results of network pharmacology. Our results revealed that Lig could significantly reduce the increase in the protein expression levels of c-Myc and MMP-2 in Ang II-induced A7r5 cells, but not affect the protein expression levels of MMP-9. Our data further showed that c-Myc siRNA, just as Lig, could obviously inhibit the cell migration accompanied by decreased MMP-2 expression. ConclusionThese findings suggested the anti-migratory effect of Lig could be associated with the c-Myc/MMP-2 pathway, and Lig administration had a promising potential for preventing cardiovascular diseases.

Chicken Muscle-Derived ACE2 Upregulating Peptide VVHPKESF Inhibits Angiotensin II-Stimulated Inflammation in Vascular Smooth Muscle Cells via the ACE2/Ang (1–7)/MasR Axis

This study aimed to evaluate the modulatory effects of four chicken muscle-derived peptides [VRP, LKY, VRY, and VVHPKESF (V-F)] on angiotensin II (Ang II)-induced inflammation in rat vascular smooth muscle A7r5 cells. Only V-F could significantly attenuate Ang II-stimulated inflammation via the inhibition of NF-κB and p38 MAPK signaling, being dependent on the Mas receptor (MasR) not on the Ang II type 1 or type 2 receptor (AT1R or AT2R). V-F accelerated Ang II degradation by enhancing cellular ACE2 activity, which was due to ACE2 upregulation other than a direct ACE2 activation. These findings demonstrated that V-F ameliorated Ang II-induced inflammation in A7r5 cells via the ACE2/Ang (1-7)/MasR axis. Three peptide metabolites of V-F─VHPKESF, PKESF, and SF─were identified but were not considered major contributors to V-F's bioactivity. The regulation of peptide V-F on vascular inflammation supported its functional food or nutraceutical application in the prevention and treatment of hypertension and cardiovascular diseases.

Antioxidant peptides derived from hydrolysates of red tilapia (Oreochromis sp.) scale

Processing red tilapia (Oreochromis sp.) yields a large quantity of scale, a byproduct of the aquaculture industry. This study focuses on evaluating the potential of red tilapia scales as a source of antioxidant peptides. Scales were ground and enzymatically hydrolyzed by Alcalase® 2.4 L. The hydrolysate obtained was fractionated to purify and identify the antioxidant peptides by membrane ultrafiltration and chromatographic technology guided by the antioxidant activity in rat vascular smooth muscle A7r5 cells. After ultrafiltration, the fraction with molecular weight of 3–10 kDa showed the highest activity and their chemical nature was hydrophilic. The sequence of twenty antioxidant peptides, containing 6–16 amino acids, was identified by liquid chromatography–tandem mass spectrometry. The results indicate that the red tilapia (Oreochromis sp.) scale could be a new natural source of antioxidant peptides.

Transcriptome Analysis of the Anti-TGFβ Effect of Schisandra chinensis Fruit Extract and Schisandrin B in A7r5 Vascular Smooth Muscle Cells.

Schisandra chinensis fruit extract (SCE) has been used as a traditional medicine for treating vascular diseases. However, little is known about how SCE and schisandrin B (SchB) affect transcriptional output-a crucial factor for shaping the fibrotic responses of the transforming growth factor β (TGFβ) signaling pathways in in vascular smooth muscle cells (VSMC). In this study, to assess the pharmacological effect of SCE and SchB on TGFβ-induced transcriptional output, we performed DNA microarray experiments in A7r5 VSMCs. We found that TGFβ induced distinctive changes in the gene expression profile and that these changes were considerably reversed by SCE and SchB. Gene Set Enrichment Analysis (GSEA) with Hallmark signature suggested that SCE or SchB inhibits a range of fibrosis-associated biological processes, including inflammation, cell proliferation and migration. With our VSMC-specific transcriptional interactome network, master regulator analysis identified crucial transcription factors that regulate the expression of SCE- and SchB-effective genes (i.e., TGFβ-reactive genes whose expression are reversed by SCE and SchB). Our results provide novel perspective and insight into understanding the pharmacological action of SCE and SchB at the transcriptome level and will support further investigations to develop multitargeted strategies for the treatment of vascular fibrosis.

Spent Hen Muscle Protein-Derived RAS Regulating Peptides Show Antioxidant Activity in Vascular Cells.

Spent hens are egg-laying hens reaching the end of their egg-laying cycles, being a major byproduct of the egg industry. Recent studies have been focusing on finding new value-added uses for spent hens. We have previously identified four bioactive peptides from spent hen muscle proteins, including three angiotensin-converting enzyme (ACE) inhibitory (ACEi) peptides (VRP, LKY, and VRY), and one ACE2 upregulating (ACE2u) peptide (VVHPKESF (V-F)). In the current study, we further assessed their antioxidant and cytoprotective activities in two vascular cell lines—vascular smooth muscle A7r5 cells (VSMCs) and endothelial EA.hy926 cells (ECs)—upon stimulation by tumor necrosis factor alpha (TNFα) and angiotensin (Ang) II, respectively. The results from our study revealed that all four peptides attenuated oxidative stress in both cells. None of the investigated peptides altered the expression of TNFα receptors in ECs; however, VRY and V-F downregulated Ang II type 1 receptor (AT1R), while V-F upregulated the Mas receptor (MasR) in VSMCs. Further, we found that the antioxidant effects of VRP, LKY, and VRY were likely through acting as direct radical scavengers, while that of V-F was at least partially ascribed to increased endogenous antioxidant enzymes (GPx4 and SOD2) in both cells. Besides, as an ACE2u peptide, V-F exerted antioxidant effect in a MasR-dependent manner, indicating a possible involvement of the upregulated ACE2-MasR axis underlying its antioxidant action. The antioxidant activities of VRP, LKY, VRY, and V-F in vascular cells indicated their multifunctional properties, in addition to their ACEi or ACE2u activity, which supports their potential use as functional food ingredients against hypertension.

Replacement of heme by soluble guanylate cyclase (sGC) activators abolishes heme-nitric oxide/oxygen (H-NOX) domain structural plasticity.

The gasotransmitter nitric oxide (NO) is a critical endogenous regulator of homeostasis, in major part via the generation of cGMP (cyclic guanosine monophosphate) from GTP (guanosine triphosphate) by NO's main physiological receptor, the soluble guanylate cyclase (sGC). sGC is a heterodimer, composed of an α1 and a β1 subunit, of which the latter contains the heme-nitric oxide/oxygen (H-NOX) domain, responsible for NO recognition, binding and signal initiation. The NO/sGC/cGMP axis is dysfunctional in a variety of diseases, including hypertension and heart failure, especially since oxidative stress results in heme oxidation, sGC unresponsiveness to NO and subsequent degradation. As a central player in this axis, sGC is the focus of intense research efforts aiming to develop therapeutic molecules that enhance its activity. A class of drugs named sGC “activators” aim to replace the oxidized heme of the H-NOX domain, thus stabilizing the enzyme and restoring its activity. Although numerous studies outline the pharmacology and binding behavior of these compounds, the static 3D models available so far do not allow a satisfactory understanding of the structural basis of sGC's activation mechanism by these drugs. Herein, application NMR describes different conformational states during the replacement of the heme by a sGC activators. We show that the two sGC activators (BAY 58-2667 and BAY 60-2770) significantly decrease the conformational plasticity of the recombinant H-NOX protein domain of Nostoc sp. cyanobacterium, rendering it a lot more rigid compared to the heme-occupied H-NOX. NMR methodology also reveals, for the first time, a surprising bi-directional competition between reduced heme and these compounds, pointing to a highly dynamic regulation of the H-NOX domain. This competitive, bi-directional mode of interaction is also confirmed by monitoring cGMP generation in A7r5 vascular smooth muscle cells by these activators. We show that, surprisingly, heme's redox state impacts differently the bioactivity of these two structurally similar compounds. In all, by NMR-based and functional approaches we contribute unique experimental insight into the dynamic interaction of sGC activators with the H-NOX domain and its dependence on the heme redox status, with the ultimate goal to permit a better design of such therapeutically important molecules.

Vascular Kv7 channels control intracellular Ca2+ dynamics in smooth muscle

Voltage-gated Kv7 (or KCNQ) channels control activity of excitable cells, including vascular smooth muscle cells (VSMCs), by setting their resting membrane potential and controlling other excitability parameters. Excitation-contraction coupling in muscle cells is mediated by Ca2+ but until now, the exact role of Kv7 channels in cytosolic Ca2+ dynamics in VSMCs has not been fully elucidated. We utilised microfluorimetry to investigate the impact of Kv7 channel activity on intracellular Ca2+ levels and electrical activity of rat A7r5 VSMCs and primary human internal mammary artery (IMA) SMCs. Both, direct (XE991) and G protein coupled receptor mediated (vasopressin, AVP) Kv7 channel inhibition induced robust Ca2+ oscillations, which were significantly reduced in the presence of Kv7 channel activator, retigabine, L-type Ca2+ channel inhibitor, nifedipine, or T-type Ca2+ channel inhibitor, NNC 55-0396, in A7r5 cells. Membrane potential measured using FluoVolt exhibited a slow depolarisation followed by a burst of sharp spikes in response to XE991; spikes were temporally correlated with Ca2+ oscillations. Phospholipase C inhibitor (edelfosine) reduced AVP-induced, but not XE991-induced Ca2+ oscillations. AVP and XE991 induced a large increase of [Ca2+]i in human IMA, which was also attenuated with retigabine, nifedipine and NNC 55-0396. RT-PCR, immunohistochemistry and electrophysiology suggested that Kv7.5 was the predominant Kv7 subunit in both rat and human arterial SMCs; CACNA1C (Cav1.2; L-type) and CACNA1 G (Cav3.1; T-type) were the most abundant voltage-gated Ca2+ channel gene transcripts in both types of VSMCs. This study establishes Kv7 channels as key regulators of Ca2+ signalling in VSMCs with Kv7.5 playing a dominant role.

Polyphenolic Composition and Hypotensive Effects of Parastrephia quadrangularis (Meyen) Cabrera in Rat.

Parastrephia quadrangularis (Pq), commonly called “Tola”, is widely used in folk medicine in the Andes, including for altitude sickness. In this study, polyphenolic composition was determined, and hypotensive effects were measured; the ethnopharmacological use as hypotensive was related to the presence of phenolic compounds. For this purpose, male Sprague-Dawley rats (6 to 8 weeks of age, 160 to 190 g) were fed Pq extract (10 to 40 mg/kg) for 10 days through gavage. Blood pressures and heart rate were significantly (p < 0.01) reduced in normotensive rats receiving Pq extract (40 mg/kg body weight). Pq extract induced a negative inotropic effect, and endothelium-dependent vasodilation mediated by nitric oxide (NO). Furthermore, preincubation with Pq extract significantly decreased the cytosolic calcium on vascular smooth muscle cells A7r5 in response to L-phenylephrine (PE). Seven metabolites were isolated from the Pq extract, but three flavonoids (10−4 M) showed similar vasodilation to the extract in intact rat aorta as follows: 5,3′,4′-trihydroxy-7-methoxyflavanone (2); 3,5,4′-trihydroxy-7,8,3′-trimethoxyflavone (6); and 5,4′-dihydroxy-3,7,8,3′-tetramethoxyflavone (7). The Pq extract and compounds 2 and 7 significantly (p < 0.05) reduced the contraction to Bay K8644 (10 nM, an agonist of CaV1.2 channels). Administration of Pq decreased cardiac contractility and increased endothelium-dependent and -independent vasodilation.

High Effectiveness in Actions of Carfilzomib on Delayed-Rectifier K+ Current and on Spontaneous Action Potentials.

Carfilzomib (CFZ, Kyprolis®) is widely recognized as an irreversible inhibitor of proteasome activity; however, its actions on ion currents in electrically excitable cells are largely unresolved. The possible actions of CFZ on ionic currents and membrane potential in pituitary GH3, A7r5 vascular smooth muscle, and heart-derived H9c2 cells were extensively investigated in this study. The presence of CFZ suppressed the amplitude of delayed-rectifier K+ current (I K(DR)) in a time-, state-, and concentration-dependent manner in pituitary GH3 cells. Based on minimal reaction scheme, the value of dissociation constant for CFZ-induced open-channel block of I K(DR) in these cells was 0.33 µM, which is similar to the IC50 value (0.32 µM) used for its efficacy on inhibition of I K(DR) amplitude. Recovery from I K(DR) block by CFZ (0.3 µM and 1 µM) could be well fitted by single exponential with 447 and 645 ms, respectively. The M-type K+ current, another type of K+ current elicited by low-threshold potential, was slightly suppressed by CFZ (1 µM). Under current-clamp condition, addition of CFZ depolarized GH3 cells, broadened the duration of action potentials as well as raised the firing frequency. In A7r5 vascular smooth muscle cells or H9c2 cardiac cells, the CFZ-induced inhibition of I K(DR) remained efficacious. Therefore, our study led us to reflect that CFZ or other structurally similar compounds should somehow act on the activity of membrane KV channels through which they influence the functional activities in different types of electrically excitable cells such as endocrine, neuroendocrine cells, smooth muscle cells, or heart cells, if similar in vivo findings occur.

Vitamin D-regulated osteocytic sclerostin and BMP2 modulate uremic extraskeletal calcification.

We induced chronic kidney disease (CKD) with adenine in WT mice, mice with osteocyte-specific deletion of Cyp27b1, encoding the 25-hydroxyvitamin D 1(OH)ase [Oct-1(OH)ase-/-], and mice with global deletion of Cyp27b1 [global-1α(OH)ase-/-]; we then compared extraskeletal calcification. After adenine treatment, mice displayed increased blood urea nitrogen, decreased serum 1,25(OH)2D, and severe hyperparathyroidism. Skeletal expression of Cyp27b1 and of sclerostin and serum sclerostin all increased in WT mice but not in Oct-1α(OH)ase-/- mice or global-1α(OH)ase-/- mice. In contrast, skeletal expression of BMP2 and serum BMP2 rose in the Oct-1α(OH)ase-/- mice and in the global-1α(OH)ase-/- mice. Extraskeletal calcification occurred in muscle and blood vessels of mice with CKD and was highest in Oct-1α(OH)ase-/-mice. In vitro, recombinant sclerostin (100 ng/mL) significantly suppressed BMP2-induced osteoblastic transdifferentiation of vascular smooth muscle A7r5 cells and diminished BMP2-induced mineralization. Our study provides evidence that local osteocytic production of 1,25(OH)2D stimulates sclerostin and inhibits BMP2 production in murine CKD, thus mitigating osteoblastic transdifferentiation and mineralization of soft tissues. Increased osteocytic 1,25(OH)2D production, triggered by renal malfunction, may represent a "primary defensive response" to protect the organism from ectopic calcification by increasing sclerostin and suppressing BMP2 production.

A novel Ca2+ current blocker promotes angiogenesis and cardiac healing after experimental myocardial infarction in mice

We previously reported a novel danshensu derivative (R)-(3,5,6-Trimethylpyrazinyl) methyl-2-acetoxy-3-(3,4-diacetoxyphenyl) propanoate (ADTM) that exhibited promising cardiovascular protective activities, such as antioxidant and antiplatelet activities, as well as arterial relaxation. Particularly, ADTM treatment for 24 h exhibited anti-oxidative activity and effectively protected against acute myocardial infarction (MI) in a rat model. Here, we further investigated the pharmacological actions of 14 days of treatment with ADTM in alleviating and restoring the MI size by stimulating revascularization. The pro-angiogenesis activity of ADTM has been validated in multiple experimental models including MI mouse, zebrafish, human umbilical vein endothelial cells (HUVECs) and A7r5 vascular smooth muscle cells (VSMCs). In addition, the effect of ADTM on L-type Ca2+ current (ICaL) was determined. We demonstrated that ADTM (12–24 mg/kg) treatment for 14 days significantly decreased myocardial infarct size, increased the blood vessel density compared to vehicle in the myocardial peri-infarct area, and ADTM (24 mg/kg) enhanced the serum VEGF level in MI mice (P < 0.05). We also demonstrated that treatment with ADTM at 50–200 μM rescued chemical-induced blood vessel loss in zebrafish. Although ADTM did not directly promote the features of angiogenesis in HUVECs, ADTM significantly increased VEGF production in a dose-dependent manner in A7r5 cells (P < 0.05). A patch clamp experiment demonstrated that ADTM (200 μM) inhibited ICaL at all depolarizing voltages, with > 50% inhibition at + 10 mV. Taken together, our results indicated that ADTM served as a Ca2+ current blocker, promoted angiogenesis and reduced experimental myocardial infarct size in mice, probably through stimulation of VEGF production in VSMCs.

Docosahexaenoic acid suppresses angiotensin II-induced A7r5 vascular smooth muscle cell proliferation and migration under pulsatile pressure stress.

Elevated mechanical stress applied to vascular walls is well known to modulate vascular remodeling and plays a part in the pathogenesis of atherosclerosis. On the other hand, docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, has been shown to protect against several types of cardiovascular diseases including atherosclerosis and hypertension. The aim of this study was to clarify the effect of pulsatile pressure stress and DHA on angiotensin II-induced proliferation and migration in A7r5 vascular smooth muscle cells (VSMCs). Pulsatile pressure of between 80 and 160 mmHg was repeatedly applied to VSMCs at a frequency of 4 cycles per min using an apparatus that we developed. Cell proliferation and migration were evaluated using a live cell movie analyzer. Application of pulsatile pressure stress for 24 h significantly increased cell proliferation. Angiotensin II also significantly increased cell proliferation in the presence or absence of pressure stress. DHA significantly inhibited angiotensin II-induced cell proliferation regardless of the pressure load. Angiotensin II significantly induced cell migration regardless of the pulsatile pressure load. Pulsatile pressure stress alone slightly, but not significantly, induced cell migration. DHA inhibited angiotensin II-induced VSMC proliferation and migration under abnormal pressure conditions. Pressure stress tended to induce extracellular signal-regulated kinase (ERK) phosphorylation in the absence of angiotensin II, whereas it significantly induced ERK phosphorylation in the presence of angiotensin II. However, the pressure-induced ERK phosphorylation was not observed in the DHA-treated VSMCs. Our findings may contribute to the understanding of the beneficial effect of DHA on various cardiovascular disorders.

Purification and characterization of antioxidant peptides from cooked eggs using a dynamic in vitro gastrointestinal model in vascular smooth muscle A7r5 cells

Antioxidant peptides derived from food sources are considered as safer alternatives to commercially available antioxidant drugs. As one of the most abundant protein sources, hen’s egg proteins were extensively used to produce antioxidant peptides by enzymatic hydrolysis. Our previous work indicated that gastrointestinal digestion of cooked eggs significantly increased the antioxidant activity due to hydrolysis of egg proteins. To characterize the responsible antioxidant peptides, cooked eggs were digested in a simulated in vitro model of human gastro-intestinal digestion. Prepared digests were fractionated with FPLC (Fast Protein Liquid Chromatography) and RP-HPLC (Reverse-Phase High-Performance Liquid Chromatography) and the antioxidant activity was determined in A7r5 cells (vascular smooth muscle cell line). Further identification of peptides from peptide fractions with the highest antioxidant activity was carried out using LC-MS/MS. Four peptides derived from ovalbumin, DSTRTQ (48–53), DKLPG (61–65), DVYSF (96–100), and ESKPV (205–209), were identified; of which DKLPG did not show antioxidant activity in cells. Enzyme cleave analysis suggested that these four peptides were likely released from ovalbumin only by pepsin non-specific cleaves. It is postulated that egg consumption may exert protection against oxidative stress on human health due to release of antioxidant peptides during digestion.

Distinct regulatory domains on the N‐ and C‐termini of Kv7.4 and Kv7.5 contribute to both their voltage‐dependence of activation and their responses to PKA and PKC.

We previously determined that homomeric Kv7.4 potassium channels expressed in smooth muscle cells are insensitive to regulation by protein kinases A and C (PKA or PKC), whereas homomeric Kv7.5 channels are robustly enhanced by PKA and robustly inhibited by PKC activation. To determine the molecular basis for these different responses of Kv7.4 and Kv7.5 channels, using A7r5 vascular smooth muscle cells as an expression system, we examined the regulation of chimeric channels, constructed by inserting subdomains of Kv7.5 into Kv7.4, to determine whether particular parts of Kv7.5 can confer sensitivity onto Kv7.4. A putative PKA phosphorylation site, S53, is located on the N‐terminus of Kv7.5, but is missing from Kv7.4. We found that replacing the Kv7.4 N‐terminus with the Kv7.5 N‐terminus (Q5N‐Q4) rendered Kv7.4 significantly more responsive to a PKA‐activating stimulus than was wild‐type Kv7.4, but this Q5N‐Q4 chimera was still insensitive to AVP (a PKC‐activating stimulus). PKC is thought to target sites on the C‐terminus of Kv7.5, so we switched the C‐terminus of Kv7.4 with that from Kv7.5 (Q5C‐Q4). Preliminary results suggest that this Q5C‐Q4 chimera is sensitive to PKC‐dependent current suppression. The Q5N‐Q4 chimera had voltage‐dependence of activation similar to WT Kv7.4 (V0.5=−31 ± 3 mV for Q5N‐Q4 and −34 ± 2 mV for WT Kv7.4), whereas the Q5C‐Q4 chimera activated at voltages similar to WT Kv7.5 (V0.5=−44 ± 1 mV for Q5C‐Q4 and −45 ± 2 mV for WT Kv7.5). Our findings suggest that distinct regulatory domains on the N‐ and C‐termini of Kv7.5 and Kv7.4 contribute to both the voltage‐dependence of activation and the differential regulation of the channels by protein kinases.Support or Funding InformationThe project described was supported by Award Number I01BX007080 from the Biomedical Laboratory Research &amp; Development Service of the VA Office of Research and Development.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The chondroitin sulfate moiety mediates thrombomodulin-enhanced adhesion and migration of vascular smooth muscle cells

BackgroundThrombomodulin (TM), a transmembrane glycoprotein highly expressed in endothelial cells (ECs), is a potent anticoagulant maintaining circulation homeostasis. Under inflammatory states, TM expression is drastically reduced in ECs while vascular smooth muscle cells (VSMCs) show a robust expression of TM. The functional role of TM in VSMCs remains elusive.MethodsWe examined the role of TM in VSMCs activities in human aortic VSMCs stimulated with platelet-derived growth factor-BB (PDGF-BB). Using rat embryonic aorta-derived A7r5 VSMCs which do not express TM, the role of the chondroitin sulfate (CS) moiety of TM in VSMCs was delineated with cells expressing wild-type TM and the CS-devoid TM mutant.ResultsExpression of TM enhanced cell migration and adhesion/spreading onto type I collagen, but had no effect on cell proliferation. Knocking down TM with short hairpin RNA reduced PDGF-stimulated adhesion and migration of human aortic VSMCs. In A7r5 cells, TM-mediated cell adhesion was eradicated by pretreatment with chondroitinase ABC which degrades CS moiety. Furthermore, the TM mutant (TMS490, 492A) devoid of CS moiety failed to increase cell adhesion, spreading or migration. Wild-type TM, but not TMS490, 492A, increased focal adhesion kinase (FAK) activation during cell adhesion, and TM-enhanced cell migration was abolished by a function-blocking anti-integrin β1 antibody.ConclusionChondroitin sulfate modification is required for TM-mediated activation of β1-integrin and FAK, thereby enhancing adhesion and migration activity of VSMCs.

Emblic Leafflower (Phyllanthus emblica L.) Fruits Ameliorate Vascular Smooth Muscle Cell Dysfunction in Hyperglycemia: An Underlying Mechanism Involved in Ellagitannin Metabolite Urolithin A.

Ellagitannins in Phyllanthus emblica L. (emblic leafflower fruits) have been thought of as the beneficial constituents for ameliorating endocrinal and metabolic diseases including diabetes. However, the effect of emblic leafflower fruits on diabetic vascular complications involved in ellagitannin-derived urolithin metabolites is still rare. In this study, acetylcholine-induced endothelium-independent relaxation in aortas was facilitated upon emblic leafflower fruit consumption in the single dose streptozotocin-induced hyperglycemic rats. Emblic leafflower fruit consumption also suppressed the phosphorylation of Akt (Thr308) in the hyperglycemic aortas. More importantly, urolithin A (UroA) and its derived phase II metabolites were identified as the metabolites upon emblic leafflower fruit consumption by HPLC-ESI-Q-TOF-MS. Moreover, UroA reduced the protein expressions of phosphor-Akt (Thr308) and β-catenin in a high glucose-induced A7r5 vascular smooth muscle cell proliferation model. Furthermore, accumulation of β-catenin protein and activation of Wnt signaling in LiCl-triggered A7r5 cells were also ameliorated by UroA treatment. In conclusion, our data demonstrate that emblic leafflower fruit consumption facilitates the vascular function in hyperglycemic rats by regulating Akt/β-catenin signaling, and the effects are potentially mediated by the ellagitannin metabolite urolithin A.

Treatment of β-thujaplicin counteracts di(2-ethylhexyl)phthalate (DEHP)-exposed vascular smooth muscle activation, inflammation and atherosclerosis progression

The initiation of atherosclerosis involves up-regulation of molecules such as E-selectin, VCAM-1, and ICAM-1. The progression of atherosclerosis is linked to proliferation and migration of vascular smooth muscle cell via MMP-2 and MMP-9 activities. However, the etiology of atherosclerosis concerning plasticizers is unknown. We evaluated β-thujaplicin in preventing the development of atherosclerosis in a model induced by pro-inflammatory cytokines. Moreover, we established a new atherosclerosis model in vascular smooth muscle cells (VSMC) exposed to a common contact plasticizer, di(2-ethylhexyl)phthalate (DEHP). SEVC4-10 endothelial cells were treated with 50% RAW conditioned medium and A7r5 VSMC was treated with the plasticizer, with/without β-thujaplicin (4 or 12 μM). Production of E-selectin, ICAM-1, and VCAM-1 in SEVC4-10 cells as well as MMP-2/MMP-9 (both expression and activity) in VSMC were monitored. Results showed that the conditioned medium induced E-selectin and ICAM were significantly prevented by β-thujaplicin. However, inhibition on the production of VCAM by β-thujaplicin was only seen in a concentration of 12 μM. Both concentrations of β-thujaplicin also significantly prevented DEHP-induced MMP-2 and MMP-9 expression and activities. Evidence uncovers that β-thujaplicin has additional factors in amelioration of atherosclerosis and corroborates that β-thujaplicin is a strong candidate in preventing the initiation and progression of atherosclerosis.