Endothelial Denudation Research Articles

Mechanical Fingerprint of Senescence in Endothelial Cells.

Endothelial senescence entails alterations of the healthy cell phenotype, which accumulate over time and contribute to cardiovascular disease. Mechanical aspects regulating cell adhesion, force generation, and the response to flow contribute to the senescence-associated drift; however, they remain largely unexplored. Here, we exploit force microscopy to resolve variations of the cell anchoring to the substrate and the tractions generated upon aging in the nanonewton (nN) range. Senescent endothelial cells display a multifold increase in the levels of basal adhesion and force generation supported by mature and strong focal adhesions. The enhanced mechanical interaction with the substrate yields static endothelial monolayers that polarize in response to flow but fail the process of coordinated cell shape remodeling and reorientation. The emerging picture indicates that senescence reinforces the local cell interaction with the substrate and may therefore prevent endothelial denudation; however, it compromises the ability to functionally adapt to the local hemodynamic conditions.

Intracellular glutamine level determines vascular smooth muscle cell-derived thrombogenicity

Background and aimsThe everolimus-eluting stent (EES), one of the effective stents for in-stent restenosis (ISR), has a lower incidence of stent thrombosis; however, the underlying mechanism remains unknown. This study aimed to identify the effects of everolimus on vascular metabolism and thrombogenicity and examine their mechanistic link. MethodsEESs and bare-metal stents were implanted in rabbit iliac arteries with smooth muscle cell (SMC)-rich neointima induced by endothelial denudation. Four weeks after stent implantation, the stented arteries were examined for histological analysis and metabolomics. Additionally, everolimus effects in coronary artery SMCs metabolism, tissue factor (TF) expression, and procoagulant activity were assessed in vitro. ResultsEES-implanted arteries showed decreased neointima formation, less SMCs infiltration, and reduced TF expression. Concomitantly, they were metabolically characterized by increased levels of metabolites in amino acids, such as glutamine. Similarly, everolimus increased intracellular glutamine levels, decreased TF expression, and reduced procoagulant activity in SMCs in vitro. On the contrary, exogenous glutamine administration also increased intracellular glutamine level, decreased TF expression, and reduced procoagulant activity despite enhanced mammalian target of rapamycin (mTOR) activity. ConclusionsIntracellular glutamine level is likely to determine vascular SMC-related thrombogenicity regardless of mTOR pathway activity. Therefore, increased intracellular glutamine level might contribute partially to the beneficial effect of EES use on stent thrombosis.

Indoxyl sulfate enhances endothelin-1-induced contraction via impairment of NO/cGMP signaling in rat aorta.

The microbiome-derived tryptophan metabolite, indoxyl sulfate, is considered a harmful vascular toxin. Here, we examined the effects of indoxyl sulfate on endothelin-1 (ET-1)-induced contraction in rat thoracic aortas. Indoxyl sulfate (10-3 M, 60 min) increased ET-1-induced contraction but did not affect isotonic high-K+-induced contraction. The ET-1-induced contraction was enhanced by endothelial denudation in both control and indoxyl sulfate-treated groups. BQ123 (10-6 M), an ETA receptor antagonist, reduced the ET-1-induced contraction in both control and indoxyl sulfate groups. BQ788 (10-6 M), an ETB receptor antagonist, increased the contraction in the control group but had no effect on the indoxyl sulfate group. Conversely, indoxyl sulfate inhibited relaxation induced by IRL1620, an ETB receptor agonist. L-NNA, an NO synthase (NOS) inhibitor, increased the ET-1-induced contractions in both the control and indoxyl sulfate groups, whereas L-NPA (10-6 M), a specific neuronal NOS inhibitor, did not affect the ET-1-induced contraction in both groups. However, ODQ, an inhibitor of soluble guanylyl cyclase, increased the ET-1-induced contraction in both groups. Organic anion transporter (OAT) inhibitor probenecid (10-3 M) and antioxidant N-acetyl-L-cysteine (NAC; 5 × 10-3 M) inhibited the effects of indoxyl sulfate. A cell-permeant superoxide scavenger reduced the ET-1-induced contraction in the indoxyl sulfate group. The aortic activity of SOD was reduced by indoxyl sulfate. The present study revealed that indoxyl sulfate augments ET-1-induced contraction in rat aortae. This enhancement may be due to the impairment of NO/cGMP signaling and may be attributed to impairment of the antioxidant systems via cellular uptake through OATs.

Mechanism of direct regulation of cerebral artery contractility by statins

Statins are amongst the most widely prescribed drugs in the world with a range of vascular effects that have been primarily attributed to the inhibition of cholesterol and mevalonate biosynthesis, and the inhibition of mevalonate-dependent Rho/ROCK signaling upon long-term treatment. However, no studies have investigated the direct effects of acute statin application on fresh isolated resistance cerebral arteries using therapeutic concentrations of statins. We examined acute vascular effects of therapeutically relevant concentrations (0.01-10nM) of rosuvastatin and simvastatin on Sprague Dawley rat cerebral arteries and underlying molecular mechanisms. Cannulated arterial segments were maintained at 37°C in a perfusion chamber and intraluminal pressure slowly increased to 60 mmHg. For calcium imaging, the isolated arteries were preincubated in Fura-2AM (10μM) and 0.02% Kolliphor solution at room temperature for two hours before mounting in the perfusion chamber. At 60mmHg, cerebral arteries developed ~36% myogenic tone, after which increasing concentrations of statins were applied. Our data showed that the application of 1nM rosuvastatin and simvastatin constricted cerebral arteries by ~26 μm and ~24μm, respectively, within 2-3 minutes of drug application. Such statin-induced vasoconstriction remained unaltered upon endothelium denudation (intact ~23μm vs denuded ~25μm), suggesting an endothelium-independent mechanism. Co-application of mevalonate did not alter the vasoconstriction either (control ~28μm vs mevalonate ~29 μm), indicating that the effect is HMG-CoA reductase-independent. However, removal of extracellular Ca2+ with EGTA or the application of nimodipine, a selective blocker of smooth muscle cell voltage-gated Ca2+ channel, CaV1.2, each abolished cerebral artery vasoconstriction by statins, indicating that the Ca2+ entry through CaV1.2 plays a critical role here. Since Ca2+ entry into smooth muscle cells induces Ca2+ release from intracellular Ca2+ stores such as sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER), we next examined the role of these Ca2+ release pathways. We found that co-application of ryanodine, a blocker of ryanodine receptor-mediated Ca2+ release, had no effects on statin-induced constriction. In contrast, statin-evoked vasoconstriction of cerebral arteries was significantly attenuated upon co-application of thapsigargin, a blocker of SR/ER membrane Ca2+-ATPase pump (SERCA), highlighting the involvement of thapsigargin-sensitive Ca2+ stores in regulating [Ca2+]i and vasoconstriction. Simultaneous measurement of arterial Ca2+ fluorescence and diameter further confirmed the involvement of CaV1.2 channel in mediating Ca2+ entry and subsequent Ca2+ release, leading to cerebral artery vasoconstriction. Altogether, our data suggests that smooth muscle cell CaV1.2 opening and Ca2+ influx is the primary mechanism underlying statin-induced constriction of cerebral arteries.

Histamine causes endothelium‐independent dilation of mouse bladder feeder arterioles

In addition to being highly vascularized, the urinary bladder vasculature has unique properties that help to maintain blood flow during voiding and filling. Unlike other vessels of similar size, however, bladder arterioles do not contract to increases in intraluminal pressure. Prolonged decreases in bladder blood flow are linked to many types of bladder dysfunction, including painful bladder syndrome and interstitial cystitis. Although the vasculature has an important role in bladder dysfunction, the mechanisms regulating vasculature diameter in the bladder are still widely unknown. We tested the hypothesis that histamine, an important pro-inflammatory compound released from mast cells in the bladder wall, also alters bladder arteriolar diameter. Using pressure myography with isolated bladder feed arterioles, concentration-response curves to histamine (1 nM – 10 µM) were performed in vessels constricted with the thromboxane mimetic U-46619 (10 nM). Histamine caused a concentration-dependent vasodilation that was inhibited by the histamine H2 receptor antagonist cimetidine (10 µM) but not the H1 receptor antagonist fexofenadine (10 µM). Histamine-dependent vasodilation was not inhibited by the NOS inhibitor L-NAME (200 µM), the inward rectifier K+ channel blocker BaCl2 (100 µM), or endothelial denudation, suggesting the dilation was endothelium-independent. Alternatively, increasing extracellular K+ (35 mM) or exposure to the KV channel blocker XE-991 (10 µM) attenuated dilations to histamine. These findings suggest the H2 receptors in bladder vascular smooth muscle, and not the endothelium, mediate histamine-induced dilation in bladder arterioles. Since H2 receptors are coupled to GaS, these findings also suggest that histamine dilation results from an increase in cAMP that may hyperpolarize smooth muscle cells via protein kinase A-mediated activation of smooth muscle KV channels.

Loss of Transforming Growth Factor Beta Signaling in Aortic Smooth Muscle Cells Causes Endothelial Dysfunction and Aortic Hypercontractility.

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Mechanical thrombectomy injury to the arterial wall: A comparison between catheter aspiration and stent retriever

PurposeTo compare the extent of arterial wall damage when SR and CA were used for treatment of AIS models to evaluate their efficacy and safety. MethodsA thrombin-induced thrombus was pre-injected into the right distal external carotid-maxillary artery (ECMA) in 12 dogs to create an acute thrombus occlusion model and were randomly divided into the SR group (n = 6; received SR treatment) and CA group (n = 6; received CA treatment). Device safety was also assessed by five passages through the normal left ECMA using each device. Device manipulation-related damage to arterial walls, final flow restoration, recanalization time and complications were recorded. ResultsSixteen retriever and 10 aspiration attempts were performed in the SR and CA groups. Reperfusion time was significantly reduced in the CA group (17.83 ± 1.96 vs. 28.33 ± 3.26 in the SR group; P = 0.02). Stent retriever thrombectomy resulted in an increased risk of endothelium denudation (1.17 ± 0.24 in SR group vs. 0.42 ± 0.15 in CA group; P = 0.01) and reduced frequency of vessel vasospasm (0.67 ± 0.14 in SR group vs. 0.25 ± 0.13 in CA group; P = 0.04). Injury score and thrombus deposition were similar between the two groups (P > 0.05). TICI 2b/3 flow restoration values of the right ECMA were 100 % in both groups. Device-related complications, including dissection (P = 0.21), side branch influence (P = 0.24), and distal thromboembolism (P = 1.00), did not differ between the two groups. ConclusionBoth devices had similar efficacy and caused minimal arterial wall damage in our dog models. SR was more likely to cause endothelium denudation, while CA had a greater risk of vasospasm.

The Combined Contribution of Vascular Endothelial Cell Migration and Adhesion to Stent Re-endothelialization.

Coronary stent placement inevitably causes mechanical damage to the endothelium, leading to endothelial denudation and in-stent restenosis (ISR). Re-endothelialization depends mainly on the migration of vascular endothelial cells (VECs) adjacent to the damaged intima, as well as the mobilization and adhesion of circulating VECs. To evaluate the combined contribution of VEC migration and adhesion to re-endothelialization under flow and the influence of stent, in vitro models were constructed to simulate various endothelial denudation scales (2 mm/5 mm/10 mm) and stent deployment depths (flat/groove/bulge). Our results showed that (1) in 2 mm flat/groove/bulge models, both VEC migration and adhesion combined completed the percentage of endothelial recovery about 27, 16, and 12%, and migration accounted for about 21, 15, and 7%, respectively. It was suggested that the flat and groove models were in favor of VEC migration. (2) With the augmentation of the injury scales (5 and 10 mm), the contribution of circulating VEC adhesion on endothelial repair increased. Taken together, endothelial restoration mainly depended on the migration of adjacent VECs when the injury scale was 2 mm. The adhered cells contributed to re-endothelialization in an injury scale-dependent way. This study is helpful to provide new enlightenment for surface modification of cardiovascular implants.

Self-assembled miRNA-switch nanoparticles target denuded regions and prevent restenosis

Cardiovascular disease is the leading cause of death and disability worldwide. Effective delivery of cell-selective therapies that target atherosclerotic plaques and neointimal growth while sparing the endothelium remains the Achilles heel of percutaneous interventions. The current study utilizes synthetic microRNA switch therapy that self-assembles to form a compacted, nuclease-resistant nanoparticle <200nM in size when mixed with cationic amphipathic cell-penetrating peptide (p5RHH). These nanoparticles possess intrinsic endosomolytic activity that requires endosomal acidification. When administered in a femoral artery wire injury mouse model invivo, the mRNA-p5RHH nanoparticles deliver their payload specifically to the regions of endothelial denudation and not to the lungs, liver, kidney, or spleen. Moreover, repeated administration of nanoparticles containing a microRNA switch, consisting of synthetically modified mRNA encoding for the cyclin-dependent kinase inhibitor p27Kip1 that contains one complementary target sequence of the endothelial cell-specific miR-126 at its 5' UTR, drastically reduced neointima formation after wire injury and allowed for vessel reendothelialization. This cell-selective nanotherapy is a valuable tool that has the potential to advance the fight against neointimal hyperplasia and atherosclerosis.

A Chemomechanobiological Model of the Long-Term Healing Response of Arterial Tissue to a Clamping Injury.

Vascular clamping often causes injury to arterial tissue, leading to a cascade of cellular and extracellular events. A reliable in silico prediction of these processes following vascular injury could help us to increase our understanding thereof, and eventually optimize surgical techniques or drug delivery to minimize the amount of long-term damage. However, the complexity and interdependency of these events make translation into constitutive laws and their numerical implementation particularly challenging. We introduce a finite element simulation of arterial clamping taking into account acute endothelial denudation, damage to extracellular matrix, and smooth muscle cell loss. The model captures how this causes tissue inflammation and deviation from mechanical homeostasis, both triggering vascular remodeling. A number of cellular processes are modeled, aiming at restoring this homeostasis, i.e., smooth muscle cell phenotype switching, proliferation, migration, and the production of extracellular matrix. We calibrated these damage and remodeling laws by comparing our numerical results to in vivo experimental data of clamping and healing experiments. In these same experiments, the functional integrity of the tissue was assessed through myograph tests, which were also reproduced in the present study through a novel model for vasodilator and -constrictor dependent smooth muscle contraction. The simulation results show a good agreement with the in vivo experiments. The computational model was then also used to simulate healing beyond the duration of the experiments in order to exploit the benefits of computational model predictions. These results showed a significant sensitivity to model parameters related to smooth muscle cell phenotypes, highlighting the pressing need to further elucidate the biological processes of smooth muscle cell phenotypic switching in the future.

Bazedoxifene increases the proliferation of human arterial endothelial cells but does not affect the expression of cyclins A, B, and D1 and of p27Kip1

Objective Endothelial dysfunction and denudation are considered a first step in atherosclerosis. Endothelial proliferation is key for cellular repair. The effect of bazedoxifene on the vascular endothelium has not been explored. We investigated the effect of bazedoxifene on endothelial cell proliferation. Methods Primary cultures from human umbilical artery endothelial cells were used in dose-response experiments (0.1, 1.0, and 10.0 EC50 dose) with bazedoxifene, estradiol, raloxifene and a combination of bazedoxifene and estradiol. Proliferation was assessed with the XTT colorimetric cell-proliferation assay. The possible participation of cyclins A, B, D1 and p27Kip1 was analyzed by the measurement of their expression at both the protein and the gene levels. Results A significant increase of similar size for cell proliferation was obtained with bazedoxifene, estradiol and raloxifene, but no significant change was observed for the association of bazedoxifene and estradiol. The impact was detected at the first 0.1 EC50 dose and was not dose-dependent. Estradiol achieved a significant increase in the protein expression of cyclin A and p27Kip1, but no change was detected for the other compounds at either the gene or protein level. Conclusion Bazedoxifene demonstrated a proliferative effect of similar size to estradiol in cultured human umbilical artery endothelial cells. The molecular mechanisms need further investigation.

Impairment of AMPK-α2 augments detrusor contractions in bladder ischemia.

PurposeIschemia disrupts cellular energy homeostasis. Adenosine monophosphate-activated protein kinase alpha-2 (AMPK-α2) is a subunit of AMPK that senses cellular energy deprivation and signals metabolic stress. Our goal was to examine the expression levels and functional role of AMPK-α2 in bladder ischemia.Materials and MethodsIliac artery atherosclerosis and bladder ischemia were engendered in apolipoprotein E knockout rats by partial arterial endothelial denudation using a balloon catheter. After eight weeks, total and phosphorylated AMPK-α2 expression was analyzed by western blotting. Structural integrity of AMPK-α2 protein was assessed by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Functional role of AMPK-α2 was examined by treating animals with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D ribofuranoside (AICAR). Tissue contractility was measured in the organ bath and bladder nerve density was examined by immunostaining.ResultsTotal AMPK-α2 expression increased in bladder ischemia, while phosphorylated AMPK-α2 was significantly downregulated. LC-MS/MS suggested post-translational modification of AMPK-α2 functional domains including phosphorylation sites, suggesting accumulation of catalytically inactive AMPK-α2 in bladder ischemia. Treatment of rats with AICAR diminished the force of overactive detrusor contractions and increased bladder capacity but did not have a significant effect on the frequency of bladder contractions. AICAR diminished contractile reactivity of ischemic tissues in the organ bath and prevented loss of nerve fibers in bladder ischemia.ConclusionsIschemia induces post-translational modification of AMPK-α2 protein. Impairment of AMPK-α2 may contribute to overactive detrusor contractions and loss of nerve fibers in bladder ischemia. AMPK activators may have therapeutic potential against detrusor overactivity and neurodegeneration in bladder conditions involving ischemia.

Effects of trans- versus cis-resveratrol on adrenergic contractions of the rat tail artery and role of endothelium.

The health benefits of the natural polyphenol trans‐resveratrol may play an important role in preventing a variety of diseases. Resveratrol has been shown to reduce blood pressure and improve metabolic diseases such as type 2 diabetes mellitus and obesity. Our previous studies examined the role of K+ channels in the vasorelaxation responses to trans‐resveratrol in the rat tail artery. During these studies, we uncovered a novel transient contraction prior to the sustained relaxation effect of trans‐resveratrol. Thus, the purpose of this study was to determine the role of the endothelium in these vascular contraction and relaxation responses to trans‐resveratrol. We additionally sought to determine if the cis‐isomer of resveratrol exerts any of the same vascular effects as the trans‐isomer. The vascular responses to trans‐resveratrol were examined in rat tail arteries with intact or denuded endothelium over a 2‐hr period. Additionally, the vascular responses to trans‐ and cis‐resveratrol were compared in rat tail arteries with intact endothelium. Both the transient contractile response and the persistent relaxation response to trans‐resveratrol were similar in the arterial rings with intact or denuded endothelium. There was a significant correlation between the initial contraction‐enhancing action of trans‐resveratrol and the magnitude of the sustained relaxation for vessels with both intact and denuded endothelium. Moreover, we demonstrated that cis‐resveratrol produced a significantly greater relaxation response as compared to trans‐resveratrol without the initial contractile response. These data demonstrate the role of the vascular smooth muscle in the vascular responses to resveratrol and the potential clinical benefits of the cis‐isomer of resveratrol as compared to the trans‐isomer.

Vascular endothelial and smooth muscle cell galvanotactic response and differential migratory behavior

Chronic disease or injury of the vasculature impairs the functionality of vascular wall cells particularly in their ability to migrate and repair vascular surfaces. Under pathologic conditions, vascular endothelial cells (ECs) lose their non-thrombogenic properties and decrease their motility. Alternatively, vascular smooth muscle cells (SMCs) may increase motility and proliferation, leading to blood vessel luminal invasion. Current therapies to prevent subsequent blood vessel occlusion commonly mechanically injure vascular cells leading to endothelial denudation and smooth muscle cell luminal migration. Due to this dichotomous migratory behavior, a need exists for modulating vascular cell growth and migration in a more targeted manner. Here, we examine the efficacy of utilizing small direct current electric fields to influence vascular cell-specific migration (“galvanotaxis”). We designed, fabricated, and implemented an in vitro chamber for tracking vascular cell migration direction, distance, and displacement under galvanotactic influence of varying magnitude. Our results indicate that vascular ECs and SMCs have differing responses to galvanotaxis; ECs exhibit a positive correlation of anodal migration while SMCs exhibit minimal change in directional migration in relation to the electric field direction. SMCs exhibit less motility response (i.e. distance traveled in 4 h) compared to ECs, but SMCs show a significantly higher motility at low electric potentials (80 mV/cm). With further investigation and translation, galvanotaxis may be an effective solution for modulation of vascular cell-specific migration, leading to enhanced endothelialization, with coordinate reduced smooth muscle in-migration.

Surgically induced deficiency of sex hormones modulates coronary vasodilation by estradiol in hypertension.

The effect of oestrogen in hormonal dysfunction is not clear, especially in the coronary vascular bed. This study aimed at estradiol action (E2) in the coronary vascular bed from sham-operated and gonadectomized female and male spontaneously hypertensive rats (SHRs). Male and female SHRs had their mean arterial pressure (MAP) and baseline coronary perfusion pressure (CPP) determined. The effects of E2 (10μM) were evaluated in isolated hearts by in bolus infusion before and after endothelium denudation (0.25μM sodium deoxycholate) or perfusion with 100μM NG-nitro-l-arginine methyl ester (L-NAME), 2.8μM indomethacin, 0.75μM clotrimazole, L-NAME after endothelium denudation, L-NAME plus indomethacin, or 4mM tetraethylammonium (TEA). MAP was higher in males than in females, with gonadectomy increasing in females and reducing in males. CPP was higher in female group, remaining unaltered after gonadectomy. E2-induced vasorelaxation was observed in all groups, with no differences having been found between sexes even after gonadectomy. Perfusion with TEA, L-NAME, L-NAME plus indomethacin, and L-NAME after endothelium removal attenuated the relaxing response in all groups. Clotrimazole inhibited vasorelaxation only in female groups, and indomethacin did so only in gonadectomized groups. Endothelium participation was confirmed in female groups and in the gonadectomized male group. Our results indicated that the vasodilator effect of E2 was mediated by an indirect mechanism- via endothelium- as well as by direct action- via vascular smooth muscle- in both groups. The characterization of these mechanisms in coronary arteries might shed light on the functional basis of hormonal dysfunction symptoms in hypertension.

Effect of heparin administration on heparin-induced thrombocytopenia (HIT) in atherosclerotic New Zealand Rabbit (Oryctolagus cuniculus)

Background: Platelets have been associated with the pathogenesis of atherosclerosis by excreting various chemokines (i.e. PF4), which systematically interact with multiple molecules. The formation of circulating heparin-platelet factor-4 (PF4) complexes have been observed in heparin-induced thrombocytopenia (HIT) cases, raising questions about the role of PF4-derived atherosclerosis in the predisposition of HIT.  Methods: An in vivo model of atherosclerosis was generated by feeding rabbits with a high-cholesterol diet for six weeks and a normal rabbit as a control group. HIT was induced by injecting heparin subcutaneously for ten days twice daily. Platelets count before and after heparinization was performed by hemocytometer, PF4 measurement was determined by ELISA assay. Histopathology examinations for artery thickness, foam cell, and endothelial denudation were conducted by Hematoxylin and Eosin (H&amp;E) staining. Results: There was no significant difference in the IgG PF4/heparin complex between the atherosclerotic and the normal groups (43.84±2.07 ng/mL vs. 41.87±3.44 ng/mL), platelet count was significantly reduced only in the atherosclerotic group (p=0.01; mean differences 7162.00±57311.02/mm3), endothelial denudation and foam cell formation were observed in the atherosclerotic group. Also, a significant difference observed in endothelial thickness between the atherosclerotic group compared to the normal group (470.32 ± 131.15 µm vs. 332.05±50.82 µm; p=0.032).Conclusion: Our results suggest that atherosclerosis was significantly associated with the development of heparin-induced thrombocytopenia. However, atherosclerosis is not a risk factor for the formation of PF4-heparin complex in HIT.

Mechanical testing of glutaraldehyde cross-linked mitral valves. Part one: In vitro mechanical behaviour.

The aim of this study was to perform an initial assessment, in vitro, of the feasibility of using a glutaraldehyde cross-linked porcine mitral valve to retain acute functionality, focusing on assessing mitral regurgitation. Six porcine hearts were tested using an in vitro simulator. Testing was repeated following cross-linking of mitral valves; where cross-linking was achieved by placing them in a glutaraldehyde solution. The simulator enabled systolic pressure on the ventricular side of the valve to be mimicked. Following testing, mitral valve leaflets underwent Scanning Electron Microscopy of the ventricular surface of both the anterior and posterior leaflets (1 cm2 samples). The peak pressure withstood by cross-linked valves was significantly lower than for untreated valves (108 mmHg cf. 128 mmHg for untreated valves; p < 0.05). The peak pressure was typically reached 0.5 s later than for the untreated valve. While both cross-linked and untreated valves exhibited endothelium denudation, the unfixed valve had less endothelial loss. Glutaraldehyde cross-linking of porcine mitral valves may be of potential value in assessing improved bioprosthetic mitral valve replacements. However, a more immobile valve exhibiting endothelial denudation (i.e. sclerosis) was a possible concerns identified following in vitro acute assessment.

Abstract 15984: Rpl13a Small Nucleolar RNAs Promote Oxidative Stress and Atherosclerosis

Reactive oxygen species (ROS) contribute to atherogenesis. An unusual mechanism that increases cellular ROS levels and oxidative stress involves 4 ubiquitously expressed noncoding small nucleolar RNAs (snoRNAs) from introns of the ribosomal protein L13a ( Rpl13a ) locus: U32a , U33 , U34 , and U35a . We tested the hypothesis that these snoRNAs promote aortic smooth muscle cell (SMC) activation and vascular inflammation, by using “snoKO” mice with targeted deletion of the 4 snoRNAs (but not Rpl13a ). Compared with congenic WT SMCs, snoKO SMCs showed 40±20% lower ROS levels, assessed by DCF fluorescence ( p &lt;0.02). Congruently, ROS levels were 35±5% lower in snoKO than WT aorta and carotid frozen sections ( p &lt;0.01), assessed by CellROX Orange fluorescence. Proliferation and migration evoked by FBS and PDGF-BB, respectively, were each 30±10% less in snoKO than WT SMCs ( p &lt;0.01 for each). To assess SMC migration and proliferation in vivo, we performed carotid artery endothelial denudation. Before injury, snoKO and WT carotid arteries were morphologically equivalent. Four wk after injury, carotid neointimal hyperplasia was 57±9% less and luminal area was 40±20 % more in snoKO than in WT mice ( p &lt;0.01). WT and snoKO mice had equivalent heart rates and systolic blood pressures by tail-cuff plethysmography: 480±20 vs 420±80 beats/min; 133±5, 132±7 mm Hg, respectively (n=5/group). To test whether snoRNAs affect atherosclerosis, we orthotopically transplanted carotid arteries from WT and snoKO mice into congenic Apoe -/- mice. Six wk post-op, atherosclerotic neointima was 70±10% smaller in snoKO than in WT carotids ( p &lt;0.01). To assess SMC-to-foam-cell transdifferentiation, which is ROS-dependent, carotid cross-sections were stained for apoE to identify graft-derived cells and for cholesteryl ester with BODIPY. BODIPY + foam cells comprised 21±3% and 11±7% of neointimal area in WT and snoKO carotids, respectively ( p &lt;0.05). Confocal co-localization of apoE and BODIPY (optical slice thickness 1 μm) showed that graft-derived foam cells were 2.0±0.6-fold more prevalent in WT than in snoKO carotids ( p &lt;0.01). We conclude that Rpl13a snoRNAs promote SMC ROS levels, proliferation and migration in vitro and in vivo, and that these snoRNAs augment atherosclerosis.

Vascular protective effect of aspirin and rivaroxaban upon endothelial denudation of the mouse carotid artery

While in recent trials the dual pathway inhibition with aspirin plus rivaroxaban has shown to be efficacious in patients with atherosclerotic cardiovascular disease, little is known about the effects of this combination treatment on thrombus formation and vascular remodelling upon vascular damage. The aim of this study was to examine the effects of aspirin and/or rivaroxaban on injury-induced murine arterial thrombus formation in vivo and in vitro, vessel-wall remodelling, and platelet-leukocyte aggregates. Temporary ligation of the carotid artery of C57BL/6 mice, fed a western type diet, led to endothelial denudation and sub-occlusive thrombus formation. At the site of ligation, the vessel wall stiffened and the intima-media thickened. Aspirin treatment antagonized vascular stiffening and rivaroxaban treatment led to a positive trend towards reduced stiffening. Local intima-media thickening was antagonized by both aspirin or rivaroxaban treatment. Platelet-leukocyte aggregates and the number of platelets per leukocyte were reduced in aspirin and/or rivaroxaban treatment groups. Furthermore, rivaroxaban restricted thrombus growth and height in vitro. In sum, this study shows vascular protective effects of aspirin and rivaroxaban, upon vascular injury of the mouse artery.

The GLP-1 receptor agonist Semaglutide decreases vascular inflammation in a rabbit model of advanced atherosclerosis

Abstract Introduction Atherosclerosis is one of the most common inflammatory disorders leading to cardiovascular disease. Incretin therapies such as Glucagon like peptide 1 (GLP-1) receptor agonists has demonstrated efficacy in reducing major adverse cardiovascular events among high risk populations, possibly due to a reduction in vascular inflammation. Positron emission tomography (PET) is a promising modality in the study of atherosclerosis since it has the ability to evaluate physiological processes in vivo. The somatostaton receptor 2 (SSTR2) targeting tracer [64Cu]Cu-DOTA-TATE (DOTATATE) has high specificity for activated macrophages, which are one on the key instigators of atherosclerosis. Two other radiotracers, more commonly used to study atherosclerosis are Na[18F]F (NaF) a radiotracer used for detection of vascular microcalcifications, and [18F]FDG (FDG) used for visualization of inflammatory metabolic activity. Purpose The purpose of this study was to evaluate the anti-atherosclerotic and anti-inflammatory effects of the GLP-1 receptor agonist Semaglutide, using molecular imaging with DOTATATE, NaF and FDG, in a rabbit model of advanced atherosclerosis. Methods A total of 23 female New Zealand White rabbits were fed a high cholesterol diet for 4 months and endothelial denudation of the aorta was performed twice (Fig 1A). The animals underwent baseline PET/CT scans using DOTATATE and FDG. They were then randomly allocated to an intervention group (n=12) or control group (n=11) receiving bi- weekly subcutaneous injections of either Semaglutide in a dose escalating regimen up to 44 μg/kg/week, or placebo (n=11). The intervention period was 16 weeks for both groups. At follow-up, the animals underwent PET/CT scans with DOTATATE, FDG and NaF. Regions of interest were drawn on all CT scans of the aorta from the right renal artery to the iliac bifurcation, and SUVmax was measured from the superimposed PET scans. Data are presented as means ± SEM. Results SUVmax for FDG and DOTATATE were similar in the 2 groups at baseline (DOTATATE: 7.59±0.48 vs 6.69±0.28, P=0.13 and FDG: 2.63±0.12 vs 2.86±0.19 P=0.29). At follow-up, the Semaglutide group had a significantly lower uptake of both DOTATATE and FDG, although the largest difference was observed for DOTATATE (DOTATATE: 5.83±0.24 vs 7.10±0.33, P=0.001 and FDG: 2.49±0.13 vs 2.99±0.15, P=0.034) (Fig 1BC). Microcalcifications visualized using NaF PET, showed no difference at follow-up between the Semaglutide and the control group (4.15±0.30 vs 3.92±0.34, P=0.62) (Fig 1D). Increase in body weight was significantly attenuated in the Semaglutide group compared to the control group at follow-up (0.25±3.29% vs 10.68±3.01%, P=0.0016). Conclusions Semaglutide decreases vascular uptake of the SSTR2 tracer, DOTATATE, and FDG but not NaF. This supports the hypothesis that Semaglutide reduces inflammation by means of decreased macrophage activity and metabolism in the arterial wall. Figure 1 Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Department of Clinical Physiology, Nuclear Medicine &amp; PET and Cluster for Molecular Imaging, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark