Porcine Coronary Arteries Research Articles

Abstract 489: Ex vivo Porcine Coronary Artery Remodeling With Seeded Endothelial Cells

Coronary heart disease is the most common cardiovascular problem that causes 370,000 deaths in America annually. Replacing the diseased coronary artery with a conduit obtained from another part of the patients’ body is the gold standard for the current surgical interventions. The great saphenous veins are widely preferred as coronary bypass grafts since they are easy to harvest and can replace multiple arteries due to their abundance. However, the endothelial cells in saphenous vein lose their venous characteristics and cannot function properly, which causes frequent graft failure. Conversely, arterial grafts have higher patency rate due to better function of endothelium. Yet, arterial grafts are not abundantly available as saphenous veins. Tissue engineered blood vessels can potentially serve as an ample source for bypass conduits. However, small-diameter engineered vessels are also tending to fail without a healthy endothelium. Here we propose to investigate the potential of Sox17-overexpressing endothelial cells in the vessel lumens to prevent intimal hyperplasia. Sox17 expression leads to upregulation of arterial, anti-thrombotic and anti-inflammatory markers in endothelial cells. We used an ex-vivo porcine coronary artery model where we cultured the vessels in perfusion bioreactors to mimic arterial flow conditions. Our preliminary results show that the vessel wall injury, seeded endothelial cells and arterial shear acted synergistically on smooth muscle cell death in the media layer (Fig. 1). The present ex vivo vessel culture platform is a promising tool to study remodeling by isolating factors like endothelial cells, soluble and mechanical factors.

Six-month evaluation of novel bioabsorbable scaffolds composed of poly-L-lactic acid and amorphous calcium phosphate nanoparticles in porcine coronary arteries.

Objective Using coronary angiography and intravascular ultrasound methods to evaluate the performance of the novel fully bioabsorbable scaffold (NFBS) composed of poly-L-lactic acid/amorphous calcium phosphate (PLLA/ACP) at six-month follow-up by comparing with PLLA scaffolds Methods Twelve PLLA/ACP scaffolds and 12 PLLA scaffolds were implanted into the coronary arteries of 12 miniature pigs. Quantitative coronary angiography (QCA) was used to measure the reference vessel diameter (RVD), mean lumen diameter (MLD) and late lumen loss (LLL). According to IVUS images, we calculated the strut malapposition rate (SMR) at post implantation, strut overlap rate (SOR), reference vessel area (RVA), mean stent area (MSA), mean lumen area (MLA) and luminal patency rate (LPR) at six-month follow-up. The radial strength of the scaffold was evaluated using a catheter tensile testing machine. Results QCA results indicated that, at six month, MLD of PLLA/ACP scaffolds was greater than those of PLLA scaffolds (2.47 ± 0.22 mm vs. 2.08 ± 0.25 mm, P < 0.05); LLL of PLLA/ACP scaffolds was less than those of PLLA scaffolds (0.42 ± 0.20 mm vs. 0.75 ± 0.22 mm, P < 0.05). IVUS results showed the SMR and SOR were all significantly less with the PLLA/ACP scaffolds than the PLLA scaffolds (5.84% ± 3.56% vs. 17.72% ± 4.86%, P < 0.05) (6.17% ± 4.63% vs. 17.65% ± 4.29%, P < 0.05). MSA, MLA and LPR of the PLLA/ACP scaffolds were all greater than those of PLLA scaffolds (6.35 ± 0.45 mm2 vs. 5.35 ± 0.51 mm2, P < 0.05) (4.76 ± 0.46 mm2 vs. 3.77 ± 0.46 mm2, P < 0.05) (78.01% ± 12.29% vs. 61.69% ± 9.76%, P < 0.05). Radial strength of PLLA/ACP scaffold at six month was greater than that of PLLA scaffold (76.33 ± 3.14 N vs. 67.67 ± 3.63 N). Conclusion The NFBS had less stent recoil, better lumen patency rate and greater radial strength than PLLA scaffolds. The results suggest the NFBS scaffolds can maintain the structural strength and functional performance, which are effective for up to six months when implanted in porcine coronary arteries.

Mechanisms underlying vasorelaxation induced in the porcine coronary arteries by Thymus linearis, Benth

Ethnopharmacological relevanceThymus linearis, Benth indigenous to Pakistan has been traditionally used for the treatment of various diseases including hypertension. Aim of the studyPresent study aims to investigate vasorelaxant effect of Thymus linearis and its underlying vasorelaxation mechanisms in porcine coronary artery rings. Materials and methodsAqueous-methanolic extract of aerial parts of Thymus linearis was prepared by maceration process and then bio-guided fractionation was carried out using different solvents. The effects of extract and subsequent fractions were assessed on coronary artery rings with intact and denuded endothelium. The mechanisms of vasorelaxant effect were investigated using different pharmacological tools. The in-vitro inhibitory effects of the test fractions were also assessed on purified phophodiestrases using radioenzymatic assay. Phytochemical studies were carried out using GCMS. ResultsThe aqueous-methanolic extract elicited similar relaxations in coronary artery rings with and without endothelium in dose dependent fashion and removal of endothelium did not alter this response. Further, n-butanolic fraction of Thymus liniaris (TLB) was found to be the most potent among other derived fractions. TLB did not alter the relaxation produced by endothelium dependent vasodilators in rings with intact endothelium. However, TLB significantly potentiated the relaxation elicited by cyclic AMP and cyclic GMP elevating drugs but not those to soluble guanylyl cyclase activators (YC-1 and BAY 41–2272) and K+ channel openers (levcromakalim and 1-EBIO). Pretreatment with TLB inhibited in a concentration-dependent manner contractions to KCl, CaCl2 and U46619 in coronary artery rings without endothelium. Further, TLB was found to non-selectively inhibit the PDE activity in concentration manner. Conclusionn-Butanolic fraction of Thymus linearis possesses endothelium independent vasorelaxant effects in coronary artery by direct acting on the smooth muscles. These effects involve the elevation of the cyclic AMP and cyclic GMP possibly through the inhibition of various PDEs. GCMS analysis revel presence of thymole and carvacrol as major constituents. Furthermore, these investigations also support the folklore use of Thymus linearis in hypertension.

Drug Distribution and Basic Pharmacology of Paclitaxel/Resveratrol-Coated Balloon Catheters

PurposeTo experimentally investigate a new homogenously paclitaxel/resveratrol-coated balloon catheter in terms of transport of the coating to the treated tissue and local effects including histology and functional tests.MethodsAdherence of the coating to the balloon was explored by in vitro simulation of its passage to the lesion. Paclitaxel and resveratrol transfer to the vessel wall was investigated in porcine coronary and peripheral arteries. Matrix-assisted laser desorption/ionization (MALDI) was used for direct microscopic visualization of paclitaxel in arterial tissue. Inhibition of neointimal proliferation and tolerance of complete coating and resveratrol-only coating was investigated in pigs 4 weeks after treatment, and the effect of resveratrol on inflammation and healing after 3 and 7 days.ResultsDrug loss on the way to the lesion was < 10% of dose, while 65 ± 13% was detected at the site of balloon inflation. After treatment similar proportions of drug were detected in coronary and peripheral arteries, i.e., 7.4 ± 4.6% of dose or 125 ± 74 ng/mg tissue. MALDI showed circumferential deposition. Inhibition of neointimal proliferation by paclitaxel/resveratrol coating was significant (p = 0.001) whereas resveratrol-only coating did not inhibit neointimal proliferation. During the first week after treatment of peripheral arteries with resveratrol-only balloons, we observed nominally less inflammation and fibrin deposition along with a significant macrophage reduction and more pronounced re-endothelialization. No safety issues emerged including left ventricular ejection fraction for detection of potential distal embolization after high-dose treatment of coronary arteries.ConclusionsPaclitaxel/resveratrol-coated balloons were effective and safe in animal studies. Beyond acting as excipient resveratrol may contribute to vascular healing.

Design and testing of hydrophobic core/hydrophilic shell nano/micro particles for drug-eluting stent coating

In this study, we designed a novel drug-eluting coating for vascular implants consisting of a core coating of the anti-proliferative drug docetaxel (DTX) and a shell coating of the platelet glycoprotein IIb/IIIa receptor monoclonal antibody SZ-21. The core/shell structure was sprayed onto the surface of 316L stainless steel stents using a coaxial electrospray process with the aim of creating a coating that exhibited a differential release of the two drugs. The prepared stents displayed a uniform coating consisting of nano/micro particles. In vitro drug release experiments were performed, and we demonstrated that a biphasic mathematical model was capable of capturing the data, indicating that the release of the two drugs conformed to a diffusion-controlled release system. We demonstrated that our coating was capable of inhibiting the adhesion and activation of platelets, as well as the proliferation and migration of smooth muscle cells (SMCs), indicating its good biocompatibility and anti-proliferation qualities. In an in vivo porcine coronary artery model, the SZ-21/DTX drug-loaded hydrophobic core/hydrophilic shell particle coating stents were observed to promote re-endothelialization and inhibit neointimal hyperplasia. This core/shell particle-coated stent may serve as part of a new strategy for the differential release of different functional drugs to sequentially target thrombosis and in-stent restenosis during the vascular repair process and ensure rapid re-endothelialization in the field of cardiovascular disease.

An investigation on the correlation between the mechanical property change and the alterations in composition and microstructure of a porcine vascular tissue underwent trypsin-based decellularization treatment.

The nonlinear pseudoelastic behavior of a native/decellularized vascular tissue is closely related to the detailed composition and microstructure of the extracellular matrix and is important in maintaining the patency of a small-caliber vascular graft. A commonly used enzyme-detergent based decellularization protocol is effective in cell component removal but it also changes the microstructure and composition of the decellularized tissues. Previous studies provide limited information to correlate the mechanical property change with the alterations in composition and microstructure in a decellularization process. In this study, the correlations were studied by implementing a previously established fiber-progressive-engagement model to describe the nonlinear pseudoelastic behavior of a vascular tissue and to evaluate the effects of trypsin concentration and exposure duration on porcine coronary artery decellularization RESULTS: Results showed that tissue length and width increased and thickness and wet weight decreased with the exposure of trypsin. The effects of trypsin exposure times on the four mechanical parameters, i.e. initial strain, turning strain, initial modulus and stiffness modulus, in the longitudinal and circumferential directions were similar, but stronger in the circumferential direction. Major components of the extracellular matrix were vulnerable to the trypsin-based decellularization process. The decreases in initial and turning strain and the increase in initial modulus in circumferential direction were correlated with the significant decrease of collagen and glycosaminoglycans in the media layer. Although trypsin-based decellularization achieved cell component removal and preservation of ultimate tensile stress, the microstructure and composition changed with alterations in the pseudoelastic behavior of the porcine coronary artery. Taken together, the current observations suggested less waviness, early engagement, or re-alignment of insoluble collagen fibers in the media layer, which resulted in turning from anisotropic into isotropic uniaxial mechanical property of porcine vascular tissue. Selecting the proper trypsin concentration (< 0.03-0.5%) and duration (< 12 h) of trypsin exposure in combination with other methods will achieve optimal porcine coronary artery decellularization.

Modified Magnesium Hydroxide Nanoparticles Inhibit the Inflammatory Response to Biodegradable Poly(lactide- co-glycolide) Implants.

Biodegradable polymers have been extensively used in biomedical applications, ranging from regenerative medicine to medical devices. However, the acidic byproducts resulting from degradation can generate vigorous inflammatory reactions, often leading to clinical failure. We present an approach to prevent acid-induced inflammatory responses associated with biodegradable polymers, here poly(lactide- co-glycolide), by using oligo(lactide)-grafted magnesium hydroxide (Mg(OH)2) nanoparticles, which neutralize the acidic environment. In particular, we demonstrated that incorporating the modified Mg(OH)2 nanoparticles within degradable coatings on drug-eluting arterial stents efficiently attenuates the inflammatory response and in-stent intimal thickening by more than 97 and 60%, respectively, in the porcine coronary artery, compared with that of drug-eluting stent control. We also observed that decreased inflammation allows better reconstruction of mouse renal glomeruli in a kidney tissue regeneration model. Such modified Mg(OH)2 nanoparticles may be useful to extend the applicability and improve clinical success of biodegradable devices used in various biomedical fields.

Arsenic Trioxide-Coated Stent Is an Endothelium-Friendly Drug Eluting Stent.

An ideal vascular stent would both inhibit in-stent restenosis (ISR) and promote rapid re-endothelialization. In the current study, the performance of arsenic trioxide (ATO)-drug eluting stent (AES) is compared with the bare metal stent, poly-lactic-co-glycolic acid-coating metal stent, and rapamycin-drug eluting stent (RES). In vivo AES is shown to prevent neointimal hyperplasia more efficiently than the others when implanted into the carotid arteries of rabbits. Moreover, AES promotes endothelial cells proliferation and re-endothelialization more quickly than RES. In vitro ATO exposure significantly increases the viability, proliferation, adhesion, and spreading of primary porcine coronary artery endothelial cells (PCAECs), which are critical for endothelialization. However, ATO exposure reduces the viability of porcine coronary artery smooth muscle cells (PCASMCs). The evaluation of mitochondrial morphology, membrane potential, and function demonstrates that ATO at 2 µmol L-1 causes enlargement of the mitochondrion, enhancement of mitochondrial membrane potential, and adenosine triphosphate (ATP) production in PCAECs but not in PCASMCs. Thus, both in vivo and in vitro studies demonstrate that AES is an effective strategy for rapid re-endothelialization and inhibition of ISR.

An aqueous extract of the Anogeissus leiocarpus bark (AEAL) induces the endothelium-dependent relaxation of porcine coronary artery rings involving predominantly nitric oxide.

Anogeissus leiocarpus is a Sahel tree traditionally used by the residents of Burkina Faso for its antihypertensive properties. In this study, experiments were conducted to evaluate whether an aqueous extract of the Anogeissus leiocarpus (AEAL) trunk bark induces a vasorelaxant effect on porcine coronary artery rings and to investigate the underlying mechanism. AEAL-induced relaxations were assessed using porcine coronary artery rings suspended in organ chambers. The phosphorylation levels of Src, Akt and endothelial nitric oxide synthase (eNOS) were assessed in a primary endothelial cell culture by Western blot. The reactive oxygen species (ROS) formation was assessed using dihydroethidine. In porcine coronary artery rings, AEAL at 0.1-300 μg/mL induced endothelium-dependent relaxations, which were inhibited in the presence of inhibitors of nitric oxide (NO) and the endothelium-derived hyperpolarization pathways. Moreover, the AEAL-induced NO-mediated relaxations were significantly reduced by the inhibitors of Src and PI3-kinase as well as by the membrane-permeant ROS scavengers. In cultured porcine coronary artery endothelial cells, treatment with AEAL is associated with an intracellular generation of ROS. Moreover, the AEAL induced the phosphorylations of Akt (Ser473), eNOS (Ser1177) and a transient phosphorylation of Src (Ser17) in a time-dependent manner. These findings indicate that AEAL is a potent inducer of endothelium-dependent NO-mediated relaxations in porcine coronary arteries through the redox-sensitive Src/PI3-kinase/Akt pathway-dependent activation of eNOS.

In vivo and in vitro analyses of the effects of a novel high-nitrogen low-nickel coronary stent on reducing in-stent restenosis.

Currently, percutaneous coronary intervention is an important treatment for coronary heart disease. However, the in-stent restenosis rate is still approximately 10-30% after stenting. Nickel ions from the stent are considered to be associated with in-stent restenosis. Therefore, in the present study, we quantitatively evaluated in-stent restenosis after implanting the novel high-nitrogen low-nickel coronary stent (HNS) and studied the mechanism underlying the reduction in in-stent restenosis by using ELISA and Western blot. The in vivo results showed that the HNS could significantly reduce neointima formation and inflammation as compared to SUS316L stents (316L) at 180 days after implantation in porcine coronary arteries and that vascular endothelial growth factor-A expression in porcine coronary arteries after HNS implantation also decreased. The in vitro results showed that, in the case of the HNS, human umbilical vein endothelial cell (HUVEC) proliferation was lower and lesser IL-6 release was noted from HUVECs at one and three days after culture than in the 316L group. Furthermore, p-STAT3 expression in HUVECs on the HNS surface was downregulated after culture for seven days. Thus, we conclude that the HNS could be a promising alternative coronary stent for percutaneous coronary intervention.

P-119 - Ex-vivo vasorelaxation induced by polyphenol-rich extracts from plant, fruits and berries on porcine coronary artery rings

The aim of the present study was to investigate if the polyphenol content of various commercial plant, fruits and berries extracts was linked to vasorelaxation properties. A total of 24 commercial extracts (including black currant fruit, green tea, grape seed, leucocyanidins of grape seed, pine bark, apple, pomegranate, bilberry, red vine, citrus pericarp, bitter orange, cocoa, aronia, raspberry leaf, hibiscus, olive leaf, olive fruit and a mix of olive fruit and leaf) was assessed on porcine coronary artery rings. Among all extracts, the most important endothelium-dependent vasorelaxation were obtained with grape seed (EC50=0.87 µg/ml and 2.0 µg/ml), leucocyanidins of grape seed (EC50=1.6 µg/ml), pine bark (EC50=2.2 µg/ml and 3.1 µg/ml), apple (EC50=2.2 µg/ml), cocoa (EC50=3.6 µg/ml), black currant (EC50=6.6 µg/ ml), pomegranate (EC50=6.6 µg/ml) and aronia EC50=12.7 µg/ml). The efficiency of all extracts (EC50) were significantly correlated with their content in total polyphenols (r=0.70; p=0.0002; n=23), in flavanols (r=0.65; p=0.031; n=11) and in flavonols (r=0.57; p=0.016; n=17). Our results suggest that some polyphenol-rich extracts could be potentially interesting to maintain a good cardiovascular health.

Inositol trisphosphate dynamics underlie physiological calcium signals within the coronary artery endothelium

The arterial endothelium modulates smooth muscle tone in response to its local hemodynamic environment. This process is critical to normal vascular function, and is impaired by endothelial cell injury in coronary artery disease. Last year, we discovered a unique mode of calcium signaling of the porcine coronary artery endothelium, characterized by a pattern of continuous, cell‐wide calcium waves in the absence of agonist stimulation. Stimulation with substance P (10−13 to 10−6 ml/L) amplified this pattern to match dose‐dependent vessel relaxation responses. Here, we investigated the mechanism underlying the formation of this characteristic pattern of endothelial calcium waves with confocal microscopy, utilizing both pharmacological and computational approaches. We found that coronary endothelial cell calcium waves were mediated by inositol trisphosphate receptors (IP3R), which exhibited distinct intracellular isoform distributions. The distribution of the type 1 IP3R isoform corresponded to the area (33.9 ± 2.6 μm2) of propagating calcium waves, including perinuclear IP3R clustering at subcellular sites of constitutively elevated calcium. We utilized a computational model of calcium signaling that included this distinct IP3R distribution, and discovered that cell‐wide waves formed only if intracellular concentration gradients of inositol trisphosphate (IP3) were present along the longitudinal cell axis. Importantly, we found that production and diffusion of IP3 from a focal intracellular source was sufficient to initiate a calcium wave event with the same propagation velocity curve (mean: 6.08 ± 0.61 μm/s) of the experimentally‐observed wave. Furthermore, amplification of IP3 production in our model resulted in an increase of the frequency of elementary wave events which reflected the dose‐dependent expansion of calcium activity induced by substance P. These results indicate a fundamental role for IP3 dynamics as a physiological mediator of vascular responses, underlying both the initiation and expansion of unique endothelial calcium signal modalities.Support or Funding InformationThis work was supported by NIH grants: HL136869, HL085887, and S10RR027535.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Echocardiographic assessment of G‐protein coupled estrogen receptor (GPER) activation on coronary blood flow and cardiac function in mice

G protein coupled estrogen receptor (GPER) has been reported to have cardiovascular protective effects in animal models and human tissues. Our previous work also showed that activation of GPER induced porcine coronary artery relaxation. In this study, we used non‐invasive high resolution echocardiography to evaluate the in vivo effects of GPER on coronary blood flow (CBF) of left coronary artery and cardiac functions in C57BL/6 male mice. Animals were anesthetized with 3% isoflurane in 100% oxygen and anesthesia was maintained with 1% isoflurane. In this echocardiographic studies, pulse wave Doppler imaging and M mode Doppler imaging were performed to measure CBF, left anterior descending coronary artery diameter and cardiac function parameters. CBF was calculated by measuring coronary artery diameter and velocity time integral in pulse wave Doppler. Cardiac function parameters including heart rate (HR), stroke volume (SV), cardiac output (CO), ejection fraction (EF), end diastolic volume (EDV) and end systolic volume (ESV) were calculated by tracing the left ventricle long axis in M mode Doppler. Intraperitoneal injection of G‐1 (3 mg/kg) significantly increased CBF from 1.68 ± 0.1 mL/min to 2.37 ± 0.2 mL/min. G‐1 injection on the cardiac function parameters showed biphasic responses. Within the first two minutes of G‐1 injection, HR (from 423 ± 15 bpm to 563 ± 12 bpm) and EF (55% % to 77 %) were increased; while EDV (from 34 ± 3.5 μl to 18 ± 1.2 μl), ESV (from 15 ± 1.9 μl to 4 ± 1.1 μl) and SV (from 19 ± 1.8 μl to 14 ± 1.5 μl) were decreased. After two minutes of G‐1 injection, cardiac responses reversed. HR (from 563 ± 12 bpm to 399 ± 8 bpm) and EF (from 77 % to 56 %) were decreased; while EDV (from 18 ± 1.2 μl to 38 ± 2.2 μl), ESV (from 4 ± 1.1 μl to 10 ± 1.2 μl) and SV (from 14 ± 1.5 μl to 20 ± 2.6 μl) were increased. These findings demonstrated that activation of GPER showed biphasic responses that initial activation of GPER exerted a positive chronotropic effects, and then the chronotropic effect became negative, with a positive inotropic effects on both systolic and diastolic functions in mice. In conclusion, we revealed that GPER activation increases coronary blood flow and induces biphasic responses on cardiac functions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Upregulation of sodium-glucose cotransporter 2 (SGLT2) expression in cultured senescent endothelial cells and in arterial sites at risk in vivo in rats

Endothelial senescence is thought to promote endothelial dysfunction and the subsequent development of cardiovascular diseases. The EMPA-REG trial has shown that sodium-glucose cotransporter 2 (SGLT2) inhibition is associated with a reduced risk of cardiovascular mortality in type 2 diabetic patients, but the protective mechanism remains unclear. SGLT2 mRNA has not been detected in control endothelial cells (ECs). This study examined the possibility that SGLT2 contributes to endothelial senescence and dysfunction and, if so, to characterize the underlying mechanism. Endothelial cells isolated from porcine coronary arteries were used at passage 1. Senescence was assessed using senescence-associated beta-galactosidase activity (SA-beta-gal activity), protein level by Western blot analysis, oxidative stress using dihydroethidium, and NO formation by electronic paramagnetic resonance. Exposure of ECs to HG (25 mM) for 96 h increased the level of SA-beta-gal activity and of senescence markers (p21 and p16) and oxidative stress, decreased eNOS expression and NO formation, and increased the expression of VCAM-1 and tissue factor (TF). Both HG and H 2 O 2 induced the appearance of SGLT2 mRNA, increased SGLT2 protein level and SGLT2-mediated glucose entry in ECs. An increased expression level of SGLT2 and VCAM-1, and a down-regulation of eNOS were observed at arterial sites at risk (aortic cross) compared with those at low risk (thoracic aorta) in young rats. These findings indicate that premature ECs ageing is characterized by the down-regulation of NO formation and the expression of pro-atherothrombotic factors, and is associated with the redox-sensitive upregulation of SGLT2 expression promoting excessive glucose entry. The fact that an increased SGLT2 expression level is observed in vivo at arterial sites at risk, suggests that inhibition of SGLT2 might be an attractive strategy to protect the cardiovascular system.

Leukocyte-derived microparticles exaggerate endothelial senescence and vascular dysfunction induced by high glucose

Microparticles (MPs) are plasma membrane vesicles and vascular effectors. High levels of pro-inflammatory cytokines and procoagulant endothelial-derived MPs circulate in diabetic patients. We have shown that (i) leukocyte-derived MPs shed in response to stress are pro-inflammatory, procoagulant and prosenescent endothelial effectors; (ii) high glucose induces premature endothelial senescence. To determine the possibility that leukocyte-derived MPs affect endothelial senescence and vascular function in response to high glucose. Leukocyte MPs were isolated from rat splenocytes with either 5 mg/ml LPS (MPLPS), 25 ng/ml PMA/1 mM A23187 ionophore (MPPMAi), or vehicle (MPCTL). Porcine coronary artery endothelial cells (ECs) at passage 1 were incubated for 48 h with 1–30 nM MPs in high or low glucose concentration (HG 25 mM, NG 5.5 mM). Senescence-associated β-galactosidase (SA-ß-GAL) activity was assessed by C12FDG, protein expression by Western blot analysis. Pig coronary artery rings were pre-incubated with HG or NG for 12 h prior to addition of 1–30 nM MPs for 12 h. Bradykinin (BK)-induced endothelium-dependent relaxations were assessed in organ chambers, and staining of target proteins by confocal microscopy. At 10 nM, MPLPS and MPPMAi enhanced SA-b-GAL activity both by about 2-fold in NG, and respectively 3 and 3.7-fold in HG. The expression of senescence markers p21, p16 doubled and that of eNOS decreased 2-fold. MPPMAi and MPLPS induced a concentration-dependent inhibition of BK-induced relaxation, inhibition by respectively 10 nM and 30 nM,being 65% in NG, amounting to about 85% in HG, whereas 30 nM MPCTL had no effect. MPPMAi and MPLPS reduced eNOS expression by 60% in NG and 80% in HG. Conversely, VCAM-1, COX-2 were up-regulated. Leukocyte-derived MPs enhance HG-induced alteration of the endothelial function by inducing premature senescence and might contribute to vascular dysfunction in diabetes patients.

Endothelium-derived hyperpolarizing factor and protein kinase G Iα activation: H2O2 versus S-nitrosothiols

Protein kinase G (PKG) Iα mediates the cyclic guanosine monophosphate-mediated vasodilatory effects induced by NO. Endothelium-derived hyperpolarizing factors (EDHFs), like H2O2 can activate PKGIα in a cyclic guanosine monophosphate-independent manner, but whether this is true for all EDHFs (e.g., S-nitrosothiols) is unknown. Here, we investigated the contribution of PKGIα to bradykinin-, H2O2-, L-S-nitrosocysteine-, and light-induced relaxation in porcine coronary arteries, making use of the fact that thioredoxin reductase inhibition with auranofin or 1-chloro-2,4-dinitrobenzene potentiates PKGIα. Thioredoxin reductase inhibition potentiated bradykinin and H2O2, but not L-S-nitrosocysteine or light. The relaxations by the latter 2 and bradykinin, but not those by H2O2, were prevented by the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Yet, after S-nitrosothiol depletion with ethacrynic acid, thioredoxin reductase inhibition also potentiated light-induced relaxation, and this was prevented by the Na+-K+ ATPase inhibitor ouabain. This indicates that photorelaxation depends on sGC activation by S-nitrosothiols, while only after S-nitrosothiol depletion oxidized PKGIα comes into play, and acts via Na+-K+ ATPase. In conclusion, both bradykinin- and light-induced relaxation of porcine coronary arteries depend, at least partially, on oxidized PKGIα, and this does not involve sGC. H2O2 also acts via oxidized PKGIα in an sGC-independent manner. Yet, S-nitrosothiol-induced relaxation is PKGIα-independent. Clearly, PKG activation does not contribute universally to all EDHF responses, and targeting PKGIα may only mimick EDHF under certain conditions. It is therefore unlikely that PKGIα activators will be universal vasodilators.

Defining drug and target protein distributions after stent-based drug release: Durable versus deployable coatings

BackgroundInnovations in drug eluting stent designs make it increasingly important to develop models for differentiating performance through spatial definition of drug, receptor binding and cell state. MethodsTwo designs of sirolimus analog eluting stents were implanted into porcine coronary arteries for 28, 60 or 90 days (n = 9/time point), durable coating (Xience) and deployable absorbable coating (MiStent). Explanted arteries were evaluated for drug content (n = 3/time point) by LC-MS/MS and for drug and target protein (mTOR) distributions by immunofluorescence (IF, n = 6/time point). A computational model was developed to predict drug release and arterial distribution maps. ResultsBoth stents released the majority of drug load by 28 days, with different tissue retention efficiencies (91.4 ± 4.9% MiStent versus 21.5 ± 1.9% Xience, P < 0.001). Computational modeling of MiStent coating deployment and microcrystal dissolution recapitulated in vivo drug release and net tissue content and predicted that >98.5% of deployed drug remains crystalline through 90 days. Immunofluorescence and computational modeling showed peristrut drug localization for both stents, with similar peaks, but high interstrut levels only at sites of coating deployment from the absorbable coating. Co-localization of mTOR-IF with drug-IF for both devices showed persistent drug effects, though with differential drug–receptor pharmacokinetics. ConclusionsImmunofluorescence and computational modeling provide insights into drug distribution and binding status that can help differentiate drug delivery technologies. Herein we found that tissue deployment of slow dissolving crystalline drug particles results in temporally and spatially more uniform drug delivery to interstrut zones that might otherwise be under-dosed without excess peristrut drug.

The potency of commercial blackcurrant juices to induce relaxation in porcine coronary artery rings is not correlated to their antioxidant capacity but to their anthocyanin content.

Polyphenol-rich products such as fruit juices have been found to have strong antioxidant capacities and to induce potent endothelium-dependent relaxation. We evaluated whether the commercial blackcurrant juices induced endothelium-dependent relaxation of isolated coronary arteries can be related to their antioxidant capacity and/or phenolic content. Six different commercial blackcurrant juices were selected. Their main phenolic compounds were measured by ultra-performance liquid chromatography and antioxidant capacity was evaluated by spectrometric methods. Vascular reactivity studies with these juices were done using isolated porcine coronary arteries. The six different commercial blackcurrant juices induced relaxation ranging from 21% to 100% at the concentration of 0.5% volume per volume (v/v). The relaxation induced at 0.5% v/v was not correlated to their antioxidant capacity measured by either oxygen radical antioxidant capacity or DPPH (2,2-diphenyl-1-picrylhydrazyl) assays and also not to the ascorbic acid, total polyphenols, total flavanols, and total phenolic acid contents. In contrast, the amplitude of the relaxation was correlated to the total anthocyanins content and the individual anthocyanin concentration. Correlations between relaxation amplitude and total anthocyanin or individual anthocyanin contents are of interest for the development of functional blackcurrant beverages with the potential to promote vascular protection.

Towards Additive Manufacture of Functional, Spline-Based Morphometric Models of Healthy and Diseased Coronary Arteries: In Vitro Proof-of-Concept Using a Porcine Template.

The aim of this study is to assess the additive manufacture of morphometric models of healthy and diseased coronary arteries. Using a dissected porcine coronary artery, a model was developed with the use of computer aided engineering, with splines used to design arteries in health and disease. The model was altered to demonstrate four cases of stenosis displaying varying severity, based on published morphometric data available. Both an Objet Eden 250 printer and a Solidscape 3Z Pro printer were used in this analysis. A wax printed model was set into a flexible thermoplastic and was valuable for experimental testing with helical flow patterns observed in healthy models, dominating the distal LAD (left anterior descending) and left circumflex arteries. Recirculation zones were detected in all models, but were visibly larger in the stenosed cases. Resin models provide useful analytical tools for understanding the spatial relationships of blood vessels, and could be applied to preoperative planning techniques, but were not suitable for physical testing. In conclusion, it is feasible to develop blood vessel models enabling experimental work; further, through additive manufacture of bio-compatible materials, there is the possibility of manufacturing customized replacement arteries.

The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries.

Estrogen exerts protective effects against cardiovascular diseases in premenopausal women, but is associated with an increased risk of both coronary heart disease and stroke in older postmenopausal women. Studies have shown that activation of the G-protein-coupled estrogen receptor 1 (GPER) can cause either relaxation or contraction of arteries. It is highly likely that these dual actions of GPER may contribute to the seemingly paradoxical effects of estrogen in regulating coronary artery function. The objective of this study was to test the hypothesis that activation of GPER enhances agonist-stimulated porcine coronary artery contraction via epidermal growth factor receptor (EGFR) transactivation and its downstream extracellular signal-regulated kinases (ERK1/2) pathway. Isometric tension studies and western blot were performed to determine the effect of GPER activation on coronary artery contraction. Our findings demonstrated that G-1 caused concentration-dependent relaxation of ET-1-induced contraction, while pretreatment of arterial rings with G-1 significantly enhanced ET-1-induced contraction. GPER antagonist, G-36, significantly inhibited both the G-1-induced relaxation effect and G-1-enhanced ET-1 contraction. Gallein, a Gβγ inhibitor, significantly increased G-1-induced relaxation, yet inhibited G-1-enhanced ET-1-mediated contraction. Similarly, inhibition of EGFR with AG1478 or inhibition of Src with phosphatase 2 further increased G-1-induced relaxation responses in coronary arteries, but decreased G-1-enhanced ET-1-induced contraction. Western blot experiments in porcine coronary artery smooth muscle cells (PCASMC) showed that G-1 increased tyrosine phosphorylation of EGFR, which was inhibited by AG-1478. Furthermore, enzyme-linked immunosorbent assays showed that the level of heparin-binding EGF (HB-EGF) released by ET-1 treatment increased two-fold; whereas pre-incubation with G-1 further increased ET-1-induced HB-EGF release to four-fold over control conditions. Lastly, the role of ERK1/2 was determined by applying the MEK inhibitor, PD98059, in isometric tension studies and detecting phospho-ERK1/2 in immunoblotting. PD98059 potentiated G-1-induced relaxation response, but blocked G-1-enhanced ET-1-induced contraction. By western blot, G-1 treatment decreased phospho-ERK1/2, however, in the presence of the adenylyl cyclase inhibitor, SQ22536, G-1 significantly increased ERK1/2 phosphorylation in PCASMC. These data demonstrate that activation of GPER induces relaxation via cAMP as well as contraction via a mechanism involving transactivation of EGFR and the phosphorylation of ERK1/2 in porcine coronary arteries.