Bovine Coronary Artery Research Articles

Prevalence of anatomic variants in coronary arteries of Japanese Black cattle.

Coronary artery anomalies that can cause sudden cardiac death have been described in some mammals. However, few studies have investigated coronary anomalies and coronary artery branching patterns in cattle. Therefore, understanding of bovine coronary arteries is incomplete. The aim of this study was to clarify anatomic variants in coronary arteries of Japanese Black cattle. The gross anatomy of the coronary arteries was examined in the hearts of 100 cattle. A total of 14 of 100 hearts exhibited confirmed coronary anomalies in the left main trunk. These 14 hearts were classified into 5 types potentially associated with cardiac ischemia. Regarding the coronary branches, the subsinuosal interventricular branch originated from the left circumflex branch (LCX) in 98 hearts and from the right coronary artery in 2 hearts. The origin of the sinoatrial nodal branch was classified into 3 types. In 99 hearts, the atrioventricular nodal branch originated from the LCX at the heart crux. This study characterized the prevalence of coronary anomalies, branching patterns of coronary arteries, and coronary artery anatomic variants in Japanese Black cattle.

Arginase inhibition by (−)-Epicatechin reverses endothelial cell aging

Endothelial dysfunction (EnD) occurs with aging and endothelial nitric oxide (NO) production by NO synthase (NOS) can be impaired. Low NO levels have been linked to increased arginase (Ar) activity as Ar competes with NOS for L-arginine. The inhibition of Ar activity can reverse EnD and (−)-epicatechin (Epi) inhibits myocardial Ar activity. In this study, through in silico modeling we demonstrate that Epi interacts with Ar similarly to its inhibitor Norvaline (Norv). Using in vitro and in vivo models of aging, we examined Epi and Norv-inhibition of Ar activity and its endothelium-protective effects. Bovine coronary artery endothelial cells (BCAEC) were treated with Norv (10 μM), Epi (1 μM) or the combination (Epi + Norv) for 48 h. Ar activity increased in aged BCAEC, with decreased NO generation. Treatment decreased Ar activity to levels seen in young cells. Epi and Epi + Norv decreased nitrosylated Ar levels by ~25% in aged cells with lower oxidative stress (~25%) (dihydroethidium) levels. In aged cells, Epi and Epi + Norv restored the eNOS monomer/dimer ratio, protein expression levels and NO production to those of young cells. Furthermore, using 18 month old rats 15 days of treatment with either Epi (1 mg/kg), Norv (10 mg/kg) or combo, decreased hypertension and improved aorta vasorelaxation to acetylcholine, blood NO levels and tetra/dihydribiopterin ratios in cultured rat aortic endothelial cells. In conclusion, results provide evidence that inhibiting Ar with Epi reverses aged-related loss of eNOS function and improves vascular function through the modulation of Ar and eNOS protein levels and activity.

Study of biological properties of field isolates of cattle minor infections agents on homological cell cultures

Biological properties of field isolates of bovine immunodeficiency virus and bovine foamy virus on homological cell cultures (fetal bovine lung and bovine coronary artery endothelial cells) were investigated. Pathogens of bovine slow infections, namely bovine immunodeficiency virus and bovine foamy virus, are able to integrate into cell cultures of homologous to cattle type, which is confirmed by the results of PCR. There has been determined the presence of genetic material of pathogens of bovine immunodeficiency (BIV) and spumavirus infection (BFV) in the cultivation of lymphocytes of field isolates in the culture of bovine coronary artery endothelial cells (BCAEC) at the level of 5th passage, and in the cell culture of fetal bovine lung (FBL) — at the level of 10th passage. In the process of integration of pathogens of immunodeficiency and spumavirus infection of cattle in continuous cell cultures FBL and BCAEC, morphological changes in the state of the monolayer by the principle of syncytiation and vacuolation are observed

Potential role of cartilage oligomeric matrix protein in the modulation of pulmonary arterial smooth muscle superoxide by hypoxia.

Changes in reactive oxygen species and extracellular matrix seem to participate in pulmonary hypertension development. Because we recently reported evidence for chronic hypoxia decreasing expression of cartilage oligomeric matrix protein (COMP) and evidence for this controlling loss of pulmonary arterial smooth muscle bone morphogenetic protein receptor-2 (BMPR2) and contractile phenotype proteins, we examined if changes in superoxide metabolism could be an important factor in a bovine pulmonary artery (BPA), organoid cultured under hypoxia for 48 h model. Hypoxia (3% O2) caused a depletion of COMP in BPA, but not in bovine coronary arteries. Knockdown of COMP by small-interfering RNA (siRNA) increased BPA levels of mitochondrial and extra-mitochondrial superoxide detected by MitoSOX and dihydroethidium (DHE) HPLC products. COMP siRNA-treated BPA showed reduced levels of SOD2 and SOD3 and increased levels of NADPH oxidases NOX2 and NOX4. Hypoxia increased BPA levels of MitoSOX-detected superoxide and caused changes in NOX2 and SOD2 expression similar to COMP siRNA, and exogenous COMP (0.5 μM) prevented the effects of hypoxia. In the presence of COMP, BMPR2 siRNA-treated BPA showed increases in superoxide detected by MitoSOX and depletion of SOD2. Superoxide scavengers (0.5 μM TEMPO or mitoTEMPO) maintained the expression of contractile phenotype proteins calponin and SM22α decreased by 48 h hypoxia (1% O2). Adenoviral delivery of BMPR2 to rat pulmonary artery smooth muscle cells prevented the depletion of calponin and SM22α by COMP siRNA. Thus, COMP regulation of BMPR2 appears to have an important role in controlling hypoxia-elicited changes in BPA superoxide and its potential regulation of contractile phenotype proteins.

Novel Anti-inflammatory and Vasodilatory ω-3 Endocannabinoid Epoxide Regioisomers.

Accumulating evidence suggests that diets rich in ω-3 polyunsaturated fatty acids (PUFAs) offer protection against vascular inflammation, neuroinflammation, hypertension, and thrombosis. Recently, biochemical studies have demonstrated that these benefits are partially mediated by their conversion to ω-3 endocannabinoid epoxide metabolites. These lipid metabolites originate from the epoxidation of ω-3 endocannabinoids, docosahexanoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) by cytochrome P450 (CYP) epoxygenases to form epoxydocosapentaenoic acid-ethanolamides (EDP-EAs) and epoxyeicosatetraenoic acid-ethanolamides (EEQ-EAs), respectively. The EDP-EAs and EEQ-EAs are endogenously produced in rat brain and peripheral organs. Additionally, EDP-EAs and EEQ-EAs dose-dependently decrease pro-inflammatory IL-6 cytokine and increased anti-inflammatory IL-10 cytokine. Furthermore, the EEQ-EAs and EDP-EAs attenuate angiogenesis and cell migration in cancer cells, induce vasodilation in bovine coronary arteries, and reciprocally regulate platelet aggregation in washed human platelets. Taken together, the ω-3 endocannabinoid epoxides represent a new class of dual acting molecules that display unique pharmacological properties.

Inhibition of ferrochelatase impairs vascular eNOS/NO and sGC/cGMP signaling.

Ferrochelatase (FECH) is an enzyme necessary for heme synthesis, which is essential for maintaining normal functions of endothelial nitric oxide synthase (eNOS) and soluble guanylyl cyclase (sGC). We tested the hypothesis that inhibition of vascular FECH to attenuate heme synthesis downregulates eNOS and sGC expression, resulting in impaired NO/cGMP-dependent relaxation. To this end, isolated bovine coronary arteries (BCAs) were in vitro incubated without (as controls) or with N-methyl protoporphyrin (NMPP; 10−5–10-7M; a selective FECH antagonist) for 24 and 72 hours respectively. Tissue FECH activity, heme, nitrite/NO and superoxide levels were sequentially measured. Protein expression of FECH, eNOS and sGC was detected by western blot analysis. Vascular responses to various vasoactive agents were evaluated via isometric tension studies. Treatment of BCAs with NMPP initiated a time- and dose-dependent attenuation of FECH activity without changes in its protein expression, followed by significant reduction in the heme level. Moreover, ACh-induced relaxation and ACh-stimulated release of NO were significant reduced, associated with suppression of eNOS protein expression in NMPP-treated groups. Decreased relaxation to NO donor spermine-NONOate reached the statistical significance in BCAs incubated with NMPP for 72 hours, concomitantly with downregulation of sGCβ1 expression that was independent of heat shock protein 90 (HSP90), nor did it significantly affect BCA relaxation caused by BAY 58–2667 that activates sGC in the heme-deficiency. Neither vascular responses to non-NO/sGC-mediators nor production of superoxide was affected by NMPP-treatment. In conclusion, deletion of vascular heme production via inhibiting FECH elicits downregulation of eNOS and sGC expression, leading to an impaired NO-mediated relaxation in an oxidative stress-independent manner.

Endocannabinoid and Cannabinoid Metabolism by CYP Epoxygenases

The human body contains endocannabinoids that elicit similar psychoactive and anti-nociceptive effects to phytocannabinoids in cannabis. Herein we report on the endogenous production of a previously unknown class of ω-3 PUFA–derived endocannabinoid epoxides that originate from the crosstalk between endocannabinoid and cytochrome P450 (CYP) epoxygenase metabolic pathways. The ω-3 endocannabinoid epoxides are derived from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to form epoxyeicosatetraenoic acid-ethanolamide (EEQ-EA) and epoxydocosapentaenoic acid-ethanolamide (EDP-EA), respectively. Both EEQ-EAs and EDP-EAs are endogenously present in rat brain and peripheral organs as determined via targeted lipidomics methods. These metabolites were directly produced by direct epoxygenation of the ω- 3 endocannabinoids, docosahexanoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) by activated BV-2 microglial cells, and by human CYP2J2 epoxygenase. Neuroinflammation studies revealed that the terminal epoxides 17,18-EEQ-EA and 19,20-EDP-EA dose-dependently abated proinflammatory IL-6 cytokines while increasing anti-inflammatory IL-10 cytokines, in part through cannabinoid receptor-2 and PPAR gamma activation. Furthermore, the ω-3 endocannabinoid epoxides 17,18-EEQ-EA and 19,20-EDP-EA exerted antiangiogenic effects in human microvascular endothelial cells (HMVEC) and vasodilatory actions on bovine coronary arteries and reciprocally regulated platelet aggregation in washed human platelets. Taken together, the ω-3 endocannabinoid epoxides' physiological effects are mediated through both endocannabinoid and epoxyeicosanoid signaling pathways. Furthermore, we examined the anti-inflammatory and anti-apoptotic role of the six different regioisomers of EDP-EA (19,20, 16,17-, 13,14-, 10,11-, 7,8- & 3,4) that showed wide range of activity towards cannabinoid receptors 1 & 2. In a separate studies we show that cannabinoids inhibit metabolism of endocannabinoids by CYPs. Support or Funding Information American Heart Association Scientist Development grant 15SDG25760064 National Institute of Health (NIH) R01 grant 1R01GM115584-01A1 and NIH R03 grant 1R03DA042365-01A1 Endocannabinoid metabolism by P450s This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synthesis of novel (−)-epicatechin derivatives as potential endothelial GPER agonists: Evaluation of biological effects

To potentially identify proteins that interact (i.e. bind) and may contribute to mediate (−)-epicatechin (Epi) responses in endothelial cells we implemented the following strategy: 1) synthesis of novel Epi derivatives amenable to affinity column use, 2) in silico molecular docking studies of the novel derivatives on G protein-coupled estrogen receptor (GPER), 3) biological assessment of the derivatives on NO production, 4) implementation of an immobilized Epi derivative affinity column and, 5) affinity column based isolation of Epi interacting proteins from endothelial cell protein extracts. For these purposes, the Epi phenol and C3 hydroxyl groups were chemically modified with propargyl or mesyl groups. Docking studies of the novel Epi derivatives on GPER conformers at 14 ns and 70 ns demostrated favorable thermodynamic interactions reaching the binding site. Cultures of bovine coronary artery endothelial cells (BCAEC) treated with Epi derivatives stimulated NO production via Ser1179 phosphorylation of eNOS, effects that were attenuated by the use of the GPER blocker, G15. Epi derivative affinity columns yielded multiple proteins from BCAEC. Proteins were electrophoretically separated and inmmunoblotting analysis revealed GPER as an Epi derivative binding protein. Altogether, these results validate the proposed strategy to potentially isolate and identify novel Epi receptors that may account for its biological activity.

Biocompatibility Evaluation of Electrospun Scaffolds of Poly (L-Lactide) with Pure and Grafted Hydroxyapatite

<p>The objective of this work was to evaluate the biocompatibility of scaffolds of poly(<em>L</em>-lactide) with pure and grafted hydroxyapatite, at various concentrations of reinforcement. The biocompatibility tests were carried out <em>in vivo </em>in Wistar rats by implanting the material into the subcutaneous and muscle tissues from 1 to 14 weeks and evaluating the surrounding tissue stained with hematoxylin-eosin. For <em>in vitro </em>assays, MTT and neutral red assay were used to evaluate any cytotoxicity in Mioblast Muscle C2C12 Cells (ATCC® CRL-1772™) and Bovine Coronary Artery Endothelial Cells (BCAEC); <em>Escherichia coli </em>and <em>Staphylococcus aureus </em>were used to evaluate bacterial adhesion. All variants of scaffolds provoked a mild inflammatory response, without showing necrosis. No evidence of cytotoxicity was presented in cell viability tests and good bacterial cell adhesion was visualized for all of the materials studied.</p>

Anti-inflammatory ω-3 endocannabinoid epoxides

Clinical studies suggest that diets rich in ω-3 polyunsaturated fatty acids (PUFAs) provide beneficial anti-inflammatory effects, in part through their conversion to bioactive metabolites. Here we report on the endogenous production of a previously unknown class of ω-3 PUFA-derived lipid metabolites that originate from the crosstalk between endocannabinoid and cytochrome P450 (CYP) epoxygenase metabolic pathways. The ω-3 endocannabinoid epoxides are derived from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to form epoxyeicosatetraenoic acid-ethanolamide (EEQ-EA) and epoxydocosapentaenoic acid-ethanolamide (EDP-EA), respectively. Both EEQ-EAs and EDP-EAs are endogenously present in rat brain and peripheral organs as determined via targeted lipidomics methods. These metabolites were directly produced by direct epoxygenation of the ω-3 endocannabinoids, docosahexanoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) by activated BV-2 microglial cells, and by human CYP2J2. Neuroinflammation studies revealed that the terminal epoxides 17,18-EEQ-EA and 19,20-EDP-EA dose-dependently abated proinflammatory IL-6 cytokines while increasing anti-inflammatory IL-10 cytokines, in part through cannabinoid receptor-2 activation. Furthermore the ω-3 endocannabinoid epoxides 17,18-EEQ-EA and 19,20-EDP-EA exerted antiangiogenic effects in human microvascular endothelial cells (HMVEC) and vasodilatory actions on bovine coronary arteries and reciprocally regulated platelet aggregation in washed human platelets. Taken together, the ω-3 endocannabinoid epoxides' physiological effects are mediated through both endocannabinoid and epoxyeicosanoid signaling pathways. In summary, the ω-3 endocannabinoid epoxides are found at concentrations comparable to those of other endocannabinoids and are expected to play critical roles during inflammation in vivo; thus their identification may aid in the development of therapeutics for neuroinflammatory and cerebrovascular diseases.

Modulation of Heme Biosynthesis by Ferrochelatase Inhibition Controls Soluble Guanylate Cyclase Expression and Superoxide Production in Bovine Coronary Arteries

Nitric oxide interacts with the Fe2+ heme prosthetic group in soluble guanylate cyclase (sGC) stimulating the conversion of guanosine 5′‐triphosphate to cyclic guanosine 3′,5′‐monophosphate (cGMP). Adequate cGMP production is associated with protective mechanisms against multiple cardiovascular disease processes. Thus, disturbances in this signaling pathway can lead to many pathological consequences. Since there has been minimal investigation on the consequences of altering heme biosynthesis on the cardiovascular system, we investigated if mitochondrial heme biosynthesis is an important factor in controlling the expression and function of sGC and systems influencing superoxide generation and actions. For the purpose of this study, we induced a heme deficiency by inhibiting ferrochelatase (FECH) with N‐methyl protoporphyrin IX (NMPP), potent FECH inhibitor. Bovine coronary arteries (BCA) were treated for 24 hours with 10 μM NMPP. Chemiluminescence (5μM lucigenin) was used to measure superoxide production; western blot analysis was used to measure expression of FECH, sGC and other heme‐containing proteins; and a QuantiChrom heme assay kit was used to measure heme levels. NMPP treatment promoted sGC depletion and attenuation of its activity based on it decreasing protein kinase G‐mediated phosphorylation of VASP and attenuation of relaxation to a nitric oxide donor. NMPP also decreased the expression of heme proteins including cytochrome oxidase subunit 4 and catalase. In addition, superoxide production was significantly elevated in NMPP treated coronary arteries when compared to that in control samples, and scavenging extra mitochondrial superoxide with Tempol and mitochondrial matrix superoxide with MitoTempol attenuated the increase in superoxide. NMPP stimulated increases in superoxide were associated with a decrease expression of mitochondrial FECH expression. Thus, inhibition of FECH increased extra mitochondrial and mitochondrial superoxide and depleted sGC. Our studies suggest that disruption of heme biosynthesis resulting in a loss of cGMP production may serve as a contributing mechanism to the progression of cardiovascular disease.Support or Funding InformationFunded by NIH grants R01HL115124 & R01HL129797

DHEA‐induced Voltage‐dependent Inhibition of ICa,L Is Augmented by Cholesterol Depletion and Down‐regulated by GPCR Blockade in Aortic Smooth Muscle Cells

Dehydroepiandrosterone (DHEA) is a steroid hormone synthesized from cholesterol and abundantly secreted as DHEA sulfate from the adrenal gland. DHEA produces relaxation of systemic and pulmonary arterial smooth muscles (ASM) and epiandrosterone, a metabolite of DHEA, causes voltage‐dependent inhibition (VDI) of ICa,L in ventricular myocytes. Here, we characterized DHEA‐induced VDI of ICa,L recorded with Ba2+ as a charge carrier by whole cell clamp and studied underlying mechanism and modulation by signal transduction in ASM cells (ASMCs) from bovine coronary artery and A7r5 cells from rat aorta. DHEA decreased peak amplitude of ICa,L rapidly, reversibly and dose‐dependently and accelerated time course of current decay decreasing the ratio of current amplitude at 500 ms (r500) to peak amplitude and shifted steady‐state inactivation curve (f∞‐V) to the left. DHEA‐induced inhibition of ICa,L became prominent by depolarization of holding potential; e.g. at −30 mV, the inhibition was 29% at 10 μM and 89% at 100 μM. Blockade of GPCR by dialysis of GDP βS shifted both f∞‐V and I–V relationships to the depolarizing direction, and decreased 10 μM‐DHEA‐induced VDI obtained at HP of −40 mV by 40%. Depletion of membrane cholesterol by methyl‐b‐cyclodextrin increased r500 and shifted f∞‐V to the right. Conversely, loading PEG‐cholesterol, a non‐ionic amphiphile, to the membrane decreased r500 and shifted f∞‐V to the left. In the cholesterol‐depleted cells, DHEA produced more potent decrease of r500 and larger negative shift of f∞‐V than in control. Therefore, DHEA‐induced VDI of ICa,L in ASMCs is due, at least partly, to cholesterol‐like action of DHEA on CaV1.2 in the membrane lipid bilayer. GPCR‐mediated signaling regulates voltage‐dependence of basal ICa,L and indirectly augments DHEA‐induced VDI of ICa,L in ASMCs.

Improved in vitro angiogenic behavior on anodized titanium dioxide nanotubes

BackgroundNeovascularization over dental implants is an imperative requisite to achieve successful osseointegration onto implanted materials. The aim of this study was to investigate the effects on in vitro angiogenesis of anodized 70 nm diameter TiO2 nanotubes (NTs) on Ti6Al4V alloy synthesized and disinfected by means of a novel, facile, antibacterial and cost-effective method using super oxidized water (SOW). We also evaluated the role of the surface roughness and chemical composition of materials of materials on angiogenesis.MethodsThe Ti6Al4V alloy and a commercially pure Ti were anodized using a solution constituted by SOW and fluoride as electrolyte. An acid-etched Ti6Al4V was evaluated to compare the effect of micro-surface roughness. Mirror-polished materials were used as control. Morphology, roughness, chemistry and wettability were assessed by field emission scanning electron microscopy (FE-SEM), transmission electron microscopy, atomic force microscopy, energy dispersive X-ray spectroscopy (EDX) and using a professional digital camera. Bovine coronary artery endothelial cells (BCAECs) were seeded over the experimental surfaces for several incubation times. Cellular adhesion, proliferation and monolayer formation were evaluated by means of SEM. BCAEC viability, actin stress fibers and vinculin cellular organization, as well as the angiogenic receptors vascular endothelial growth factor 2 (VEGFR2) and endothelial nitric oxide synthase (eNOS) were measured using fluorescence microscopy.ResultsThe anodization process significantly increased the roughness, wettability and thickness of the oxidized coating. EDX analysis demonstrated an increased oxygen (O) and decreased carbon (C) content on the NTs of both materials. Endothelial behavior was solidly supported and improved by the NTs (without significant differences between Ti and alloy), showing that endothelial viability, adhesion, proliferation, actin arrangement with vinculin expression and monolayer development were evidently stimulated on the nanostructured surface, also leading to increased activation of VEGFR2 and eNOS on Ti6Al4V-NTs compared to the control Ti6Al4V alloy. Although the rougher alloy promoted BCAECs viability and proliferation, filopodia formation was poor.ConclusionThe in vitro results suggest that 70 nm diameter NTs manufactured by anodization and cleaned using SOW promotes in vitro endothelial activity, which may improve in vivo angiogenesis supporting a faster clinical osseointegration process.

Pulmonary Arteries Show Differences in the Effects of Angiotensin II Stimulation of Mitochondrial Superoxide on Regulation of Heme Biosynthesis and Soluble Guanylate Cyclase Expression

It is well established that Angiotensin II (AngII) increases mitochondrial superoxide levels through the AT1 receptor. Ferrochelatase (FECH), the mitochondrial heme‐generating enzyme, has an iron‐sulfur cluster required for its stability and activity, which could potentially be disrupted by superoxide. We have observed that 24 hr organoid culture of bovine coronary arteries (BCAs) with AngII caused a depletion of mitochondrial superoxide dismutase‐2 (SOD‐2), FECH and soluble guanylate cyclase (sGC), as well as an increase in deficiency in heme (detected by depletion of cytochrome oxidase subunit‐4 (COx4), but not catalase). The heme precursor δ‐aminolevulinic acid (ALA) prevents these effects of AngII through a mechanism that appears to involve promoting protoporphyrin IX (PpIX) accumulation and its stimulation of sGC/cGMP‐activation of protein kinase G (PKG). Thus, in this study we investigated the effects of AngII on 24 hr organoid cultured endothelium‐denuded bovine pulmonary arteries (BPAs). AngII increased mitochondrial (MitoSox/HPLC) and extra‐mitochondrial/cytosolic superoxide (DHE/HPLC) and protoporphyrin IX (PpIX) levels (detected by its surface fluorescence), and it depleted SOD‐2 (western blot) and Cox4 (western blot) protein expression. However AngII did not deplete protein expression of FECH, sGC and catalase (western blot). ALA prevented AngII‐elicited increases in mitochondrial superoxide, and depletion of SOD‐2 and COx4. Furthermore, co‐organoid culture of BPAs with AngII in presence of cGMP‐independent activation of PKG (DHEA (dehydroepiandrosterone)) and cGMP‐mediated activation of PKG (8‐bromo‐cGMP) reverses the increases in mitochondrial and extra‐mitochondrial superoxide induced by AngII in BPA. The increase in PpIX and depletion of heme, but not FECH, suggests that increased mitochondrial superoxide in AngII‐treated BPA is impairing the availability of iron for heme generation from PpIX. Since agents which bind the heme site of sGC appear to protect it from depletion when the sGC heme is oxidized, the observed AngII‐elicited increase in PpIX in BPA may prevent the depletion of sGC. In contrast, AngII did not increase PpIX in BCA, and the depletion of both FECH and heme appears to be a more dominant factor in AngII‐treated BCA. While increased PKG stimulation by PpIX generation from ALA may function to attenuate AngII‐induced increases in mitochondrial superoxide and depletion of heme in both BPA and BCA, and the increase in PpIX observed in BPA treated with AngII may function to prevent the depletion of sGC, and perhaps FECH.Support or Funding InformationSupported by NIH grant: R01HL115124

Potential role of mitochondrial superoxide decreasing ferrochelatase and heme in coronary artery soluble guanylate cyclase depletion by angiotensin II.

Oxidation of the soluble guanylate cyclase (sGC) heme promotes loss of regulation by nitric oxide (NO) and depletion of sGC. We hypothesized that angiotensin II (ANG II) stimulation of mitochondrial superoxide by its type 1 receptor could function as a potential inhibitor of heme biosynthesis by ferrochelatase, and this could decrease vascular responsiveness to NO by depleting sGC. These processes were investigated in a 24-h organoid culture model of bovine coronary arteries (BCA) with 0.1 μM ANG II. Treatment of BCA with ANG II increased mitochondrial superoxide, depleted mitochondrial superoxide dismutase (SOD2), ferrochelatase, and cytochrome oxidase subunit 4, and sGC, associated with impairment of relaxation to NO. These processes were attenuated by organoid culture with 8-bromo-cGMP and/or δ-aminolevulinic acid (a stimulator of sGC by protoporphyrin IX generation and heme biosynthesis). Organoid culture with Mito-TEMPOL, a scavenger of mitochondrial matrix superoxide, also attenuated ANG II-elicited ferrochelatase depletion and loss of relaxation to NO, whereas organoid culture with Tempol, an extramitochondrial scavenger of superoxide, attenuated the loss of relaxation to NO by ANG II, but not ferrochelatase depletion, suggesting cytosolic superoxide could be an initiating factor in the loss of sGC regulation by NO. The depletion of cytochrome oxidase subunit 4 and sGC (but not catalase) suggests that sGC expression may be very sensitive to depletion of heme caused by ANG II disrupting ferrochelatase activity by increasing mitochondrial superoxide. In addition, cGMP-dependent activation of protein kinase G appears to attenuate these ANG II-stimulated processes through both preventing SOD2 depletion and increases in mitochondrial and extramitochondrial superoxide.

Effects of a natural extract of Aronia Melanocarpa berry on endothelial cell nitric oxide production.

These in vitro results may be translated into potential future clinical applications where Aronia extracts may be used for prevention and coadjuvant treatment of cardiovascular diseases via increases in endothelial NO synthesis and related improvements in vascular functions. Given the dose-response effect of Aronia extract in vitro and metabolism of polyphenols that occurs in humans, dose-response studies would be necessary to define the optimal daily amount to be consumed.

The effects of (−)-epicatechin on endothelial cells involve the G protein-coupled estrogen receptor (GPER)

We have provided evidence that the stimulatory effects of (−)-epicatechin ((−)-EPI) on endothelial cell nitric oxide (NO) production may involve the participation of a cell-surface receptor. Thus far, such entity(ies) has not been fully elucidated. The G protein-coupled estrogen receptor (GPER) is a cell-surface receptor that has been linked to protective effects on the cardiovascular system and activation of intracellular signaling pathways (including NO production) similar to those reported with (−)-EPI. In bovine coronary artery endothelial cells (BCAEC) by the use of confocal imaging, we evidence the presence of GPER at the cell-surface and on F-actin filaments. Using in silico studies we document the favorable binding mode between (−)-EPI and GPER. Such binding is comparable to that of the GPER agonist, G1. By the use of selective blockers, we demonstrate that the activation of ERK 1/2 and CaMKII by (−)-EPI is dependent on the GPER/c-SRC/EGFR axis mimicking those effects noted with G1. We also evidence by the use of siRNA the role that GPER has on mediating ERK1/2 activation by (−)-EPI. GPER appears to be coupled to a non Gαi/o or Gαs, protein subtype. To extrapolate our findings to an ex vivo model, we employed phenylephrine pre-contracted aortic rings evidencing that (−)-EPI can mediate vasodilation through GPER activation. In conclusion, we provide evidence that suggests the GPER as a potential mediator of (−)-EPI effects and highlights the important role that GPER may have on cardiovascular system protection.

Role of Angiotensin II‐associated Mitochondrial Superoxide in Inhibiting Ferrochelatase Activity and Disrupting Heme Biosynthesis Regulation of Coronary Artery Soluble Guanylate Cyclase Expression

Oxidation of the soluble guanylate cyclase (sGC) heme promotes loss of regulation by nitric oxide (NO) and the proteolytic degradation of sGC. Due to the mitochondrial heme generating enzyme ferrochelatase (FECH) having an iron‐sulfur cluster which could potentially be disrupted by superoxide, and Angiotensin II (AngII) promoting increased mitochondrial superoxide and a loss of NO‐stimulated sGC/cGMP‐mediated vasodilation, we hypothesized that AngII may influence the regulation of sGC through a superoxide‐mediated inhibition of the conversion of protoporphyrin IX (PpIX) into heme by FECH.In addition, since we previously reported that 24‐hour organoid culture of bovine arteries with δ‐aminolevulinic acid (ALA) caused an increase in sGC activity by generating PpIX, we investigated if ALA attenuates actions of AngII. Organoid culture of bovine coronary arteries (BCAs) with 0.1 µM AngII for 24‐hours caused increases in mitochondrial (MitoSox/HPLC) and extra‐mitochondrial/cytosolic superoxide (DHE/HPLC), but ALA only reversed the increase in mitochondrial superoxide. AngII also decreased FECH activity, and this was attenuated by ALA or the mitochondrial superoxide scavenger mitoTempol. Treatment of BCAs with AngII also decreased FECH and sGC expression, and ALA prevented these actions of AngII suggesting a role of increased mitochondrial superoxide in regulating the loss of FECH and sGC. These observations suggest that AngII stimulated mitochondrial superoxide generation plays an important role in inhibiting FECH activity and its influence on maintaining the expression of sGC.

Aldehyde dehydrogenase-independent bioactivation of nitroglycerin in porcine and bovine blood vessels

The vascular bioactivation of the antianginal drug nitroglycerin (GTN), yielding 1,2-glycerol dinitrate and nitric oxide or a related activator of soluble guanylate cyclase, is catalyzed by aldehyde dehydrogenase-2 (ALDH2) in rodent and human blood vessels. The essential role of ALDH2 has been confirmed in many studies and is considered as general principle of GTN-induced vasodilation in mammals. However, this view is challenged by an early report showing that diphenyleneiodonium, which we recently characterized as potent ALDH2 inhibitor, has no effect on GTN-induced relaxation of bovine coronary arteries (De La Lande et al., 1996). We investigated this issue and found that inhibition of ALDH2 attenuates GTN-induced coronary vasodilation in isolated perfused rat hearts but has no effect on relaxation to GTN of bovine and porcine coronary arteries. This observation is explained by low levels of ALDH2 protein expression in bovine coronary arteries and several types of porcine blood vessels. ALDH2 mRNA expression and the rates of GTN denitration were similarly low, excluding a significant contribution of ALDH2 to the bioactivation of GTN in these vessels. Attempts to identify the responsible pathway with enzyme inhibitors did not provide conclusive evidence for the involvement of ALDH3A1, cytochrome P450, or GSH-S-transferase. Thus, the present manuscript describes a hitherto unrecognized pathway of GTN bioactivation in bovine and porcine blood vessels. If present in the human vasculature, this pathway might contribute to the therapeutic effects of organic nitrates that are not metabolized by ALDH2.

Pegylated Cholesterol and Methyl-Beta-Cyclodextrin are Modulators of L-Type Calcium Channel Current and Decrease Membrane Capacitance in Vascular Smooth Muscle Cells

Loading of pegylated cholesterol (PEG-cholesterol) by pretreatment of several hours decreases current density and augments voltage-dependent inactivation of L-type Ca channel current (ICa,L) and depletion of cholesterol by similar long pretreatment with methyl-beta-cyclodextrin (MbCD) increases the ICa,L density in A7r5 aortic smooth muscle cells. On the other hand, dehydroepiandrosterone (DHEA), a cholesterol-derived steroid hormone, rapidly induces voltage-dependent inhibition of ICa,L. If endocytosis and exocytosis of CaV1.2-containing membrane vesicles are involved in these modulations, they should induce a change in membrane capacitance (Cm). Cm could be affected also by a change of thickness and dielectric constant of lipid bilayer. Here we studied acute effects of PEG-cholesterol, MbCD and DHEA on Cm of isolated bovine coronary artery smooth muscle cells by whole-cell patch clamp technique. Ramp steps of 5 or 10 ms were applied repetitively before, during and after application of the modulators. Control Cm was 18.3±4.5 pF (mean±SD.). PEG-cholesterol and MbCD induced gradual and small decrease of Cm and their washout decelerated the rate of the decrease. Cm after 10 min of wash-in normalized by the initial value was: control without modulator, 1.01±0.02 (mean±SD.); 1 mM PEG-cholesterol, 0.96±0.02 (p<0.01, compared with control); 10 mM MbCD, 0.90±0.05 (p<0.0001); 0.1 mM DHEA, 1.00±0.03. Since the change of Cm is small, it little affects qualitative conclusion of the modulation of ICa,L density obtained by the pretreatment experiments with PEG-cholesterol and MbCD. The small but significant decrease of Cm reflects structural changes of the lipid bilayer which may be involved in the modulation of ICa,L by PEG-cholesterol and MbCD.