Rat Carotid Artery Research Articles

Osthole Alleviates Neointimal Hyperplasia in Balloon-Induced Arterial Wall Injury by Suppressing Vascular Smooth Muscle Cell Proliferation and Downregulating Cyclin D1/CDK4 and Cyclin E1/CDK2 Expression.

Percutaneous coronary intervention (PCI) is the most widely used therapy for treating ischemic heart disease. However, intimal hyperplasia and restenosis usually occur within months after angioplasty. Modern pharmacological researchers have proven that osthole, the major active coumarin of Cnidium monnieri (L.) Cusson, exerts potent antiproliferative effects in lung cancer cells, the human laryngeal cancer cell line RK33 and TE671 medulloblastoma cells, and its mechanism of action is related to cell cycle arrest. The goal of the present study was to observe the effect of osthole on vascular smooth muscle cell (VSMC) proliferation using platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMCs isolated from rats and vascular balloon injury as models to further elucidate the molecular mechanisms underlying this activity. We detected the relative number of VSMCs by the MTT assay and EdU staining and examined cell cycle progression by flow cytometry. To more deeply probe the mechanisms, the protein expression levels of PCNA, the cyclin D1/CDK4 complex and the cyclin E1/CDK2 complex in balloon-treated rat carotid arteries and the mRNA and protein expression levels of the cyclin D1/CDK4 and cyclin E1/CDK2 complexes in VSMCs were detected by real-time RT-PCR and western blotting. The data showed that osthole significantly inhibited the proliferation of VSMCs induced by PDGF-BB. Furthermore, osthole caused apparent VSMC cycle arrest early in G0/G1 phase and decreased the expression of cyclin D1/CDK4 and cyclin E1/CDK2. Our results demonstrate that osthole can significantly inhibit PDGF-BB-induced VSMC proliferation and that its regulatory effects on cell cycle progression and proliferation may be related to the downregulation of cyclin D1/CDK4 and cyclin E1/CDK2 expression as well as the prevention of cell cycle progression from G0/G1 phase to S phase. The abovementioned mechanism may be responsible for the alleviation of neointimal hyperplasia in balloon-induced arterial wall injury by osthole.

Diabetes Attenuates the Contribution of Endogenous Nitric Oxide but Not Nitroxyl to Endothelium Dependent Relaxation of Rat Carotid Arteries.

Introduction:Endothelial dysfunction is a major risk factor for several of the vascular complications of diabetes, including ischemic stroke. Nitroxyl (HNO), the one electron reduced and protonated form of nitric oxide (NO•), is resistant to scavenging by superoxide, but the role of HNO in diabetes mellitus associated endothelial dysfunction in the carotid artery remains unknown. Aim: To assess how diabetes affects the role of endogenous NO• and HNO in endothelium-dependent relaxation in rat isolated carotid arteries. Methods: Male Sprague Dawley rats were fed a high-fat-diet (HFD) for 2 weeks prior to administration of low dose streptozotocin (STZ; 35 mg/kg i. p./day) for 2 days. The HFD was continued for a further 12 weeks. Sham rats were fed standard chow and administered with citrate vehicle. After 14 weeks total, rats were anesthetized and carotid arteries collected to assess responses to the endothelium-dependent vasodilator, acetylcholine (ACh) by myography. The combination of calcium-activated potassium channel blockers, TRAM-34 (1 μmol/L) and apamin (1 μmol/L) was used to assess the contribution of endothelium-dependent hyperpolarization to relaxation. The corresponding contribution of NOS-derived nitrogen oxide species to relaxation was assessed using the combination of the NO• synthase inhibitor, L-NAME (200 μmol/L) and the soluble guanylate cyclase inhibitor ODQ (10 μmol/L). Lastly, L-cysteine (3 mmol/L), a selective HNO scavenger, and hydroxocobalamin (HXC; 100 μmol/L), a NO• scavenger, were used to distinguish between NO• and HNO-mediated relaxation. Results: At study end, diabetic rats exhibited significantly retarded body weight gain and elevated blood glucose levels compared to sham rats. The sensitivity and the maximal relaxation response to ACh was significantly impaired in carotid arteries from diabetic rats, indicating endothelial dysfunction. The vasorelaxation evoked by ACh was abolished by L-NAME plus ODQ, but not affected by the apamin plus TRAM-34 combination, indicating that NOS-derived nitrogen oxide species are the predominant endothelium-derived vasodilators in sham and diabetic rat carotid arteries. The maximum relaxation to ACh was significantly decreased by L-cysteine in both sham and diabetic rats, whereas HXC attenuated ACh-induced relaxation only in sham rats, suggesting that diabetes impaired the contribution of NO•, whereas HNO-mediated vasorelaxation remained intact. Conclusion: Both NO• and HNO contribute to endothelium-dependent relaxation in carotid arteries. In diabetes, NO•-mediated relaxation is impaired, whereas HNO-mediated relaxation was preserved. The potential for preserved HNO activity under pathological conditions that are associated with oxidative stress indicates that HNO donors may represent a viable therapeutic approach to the treatment of vascular dysfunction.

Vascular, molecular and cellular mechanisms of development of cognitive impairments after occlusion of carotid arteries

The study was aimed at determining the role of vascular endothelial dysfunction in induction of molecular and cellular mechanisms of the development of cognitive impairments after occlusion of common carotid arteries. The experiments were carried out on Wistar rats. The animals were divided into 2 groups: group 1 - control, sham-operated animals; group 2 - rats with cerebral ischaemia-reperfusion. The model of cerebral ischaemia-reperfusion was reproduced by means of simultaneous occlusion of common carotid arteries (for 5 minutes). The degree of endothelial dysfunction was assessed by determining the amount of circulating endotheliocytes and concentration of nitrogen oxide products in blood plasma. In the brain, we measured the content of malonic dialdehyde, the activity of superoxide dismutase and catalase. Pathomorphological studies of cerebral structures and morphometric analysis of the number of damaged neurons in ischaemic exposure were carried out by means of light microscopy. Cognitive functions in rats were assessed by means of conditioned reaction of passive avoidance and the test of pattern identification. Common carotid arteries occlusion in rats induced an increase in the level of circulating endotheliocytes and a decrease in end products of nitrogen oxide - nitrites in blood plasma as compared with the control (p≤0.05); the content of malonic dialdehyde in the brain increased 2.6-fold, the activity of superoxide dismutase and catalase decreased 5.9-fold and 2.8-fold, respectively (p≤0.05). The findings of pathomorphological examination registered signs of vasoconstrictive reactions, endothelial oedema, an increase in the proportion of damaged neurons in the cortex of greater hemispheres and hippocampus (p≤0.05). It was determined that occlusion of common carotid arteries in rats resulted in memory disorders revealed in tests of conditioned passive avoidance response and pattern identification (p≤0.05). Occlusion of common carotid arteries in rats becomes a cause of endothelial dysfunction, neurometabolic alterations, damage of neurons in vulnerable regions of the cerebral cortex, hippocampus and cognitive impairments. Damaging factors of the neurovascular system are intensification of oxidative processes and a decrease in the level of antioxidant defence, presenting important targets of neuroprotection.

Microvascular Patch Graft Angioplasty of the Common Carotid Arteries in Rats.

Microvascular patch graft angioplasty is one of the most important revascularization techniques in cerebrovascular neurosurgery. It is necessary for surgeons to develop the microvascular patch graft angioplasty technique in the laboratory before performing it in a real human body. To provide a training model for microvascular patch graft angioplasty of the common carotid arteries (CCAs) in rats. Using male Sprague-Dawley rats (n=20), an oval-shaped arterial patch 3 mm in length and 1.2 mm in width was prepared from a segment of left CCA, and a linear longitudinal arteriotomy 3 mm in length was made along the anterior aspect of the right CCA, then the arterial patch graft was anastomosed to the right CCA with 10-0 sutures in an interrupted fashion. Patency was assessed immediately and 30 minutes after the procedure. All microvascular patch graft angioplasties of the rat common carotid arteries were successful, and all the patency rates immediately after the operation and thirty minutes after the restoration of blood flow were 100%. The training model for microvascular patch angioplasty with rat CCAs can serve as a training tool for mastering the procedure, and this technique could provide an alternative strategy for the surgical repair of microvascular aneurysms and microvascular vessel injuries.

Platelet-derived microvesicles induce calcium oscillations and promote VSMC migration via TRPV4.

Rationale: Abnormal migration of vascular smooth muscle cells (VSMCs) from the media to the interior is a critical process during the intimal restenosis caused by vascular injury. Here, we determined the role of platelet-derived microvesicles (PMVs) released by activated platelets in VSMC migration.Methods: A percutaneous transluminal angioplasty balloon dilatation catheter was used to establish vascular intimal injury. Collagen I was used to activate PMVs, mimicking collagen exposure during intimal injury. To determine the effects of PMVs on VSMC migration in vitro, scratch wound healing assays were performed. Fluorescence resonance energy transfer was used to detect variations of calcium dynamics in VSMCs.Results: Morphological results showed that neointimal hyperplasia was markedly increased after balloon injury of the carotid artery in rats, and the main component was VSMCs. PMVs significantly promoted single cell migration and wound closure in vitro. Fluorescence resonance energy transfer revealed that PMVs induced temporal and dynamic calcium oscillations in the cytoplasms of VSMCs. The influx of extracellular calcium, but not calcium from intracellular stores, was involved in the process described above. The channel antagonist GSK219 and specific siRNA revealed that a membrane calcium channel, transient receptor potential vanilloid 4 (TRPV4), participated in the calcium oscillations and VSMC migration induced by PMVs.Conclusions: TRPV4 participated in the calcium oscillations and VSMC migration induced by PMVs. PMVs and the related molecules might be novel therapeutic targets for vascular remodeling during vascular injury.

Vascular Endothelial Repair and the Influence of Circulating Antiplatelet Drugs in a Carotid Coil Model.

Background:Clinicians may choose to administer antiplatelet medications to patients with cerebral aneurysms following endovascular coiling to prevent thrombus formation and vascular occlusion, if they fear a thrombus will form on the platinum wire where it diverges into the vessel from the aneurysm sac. However, the mechanism by which vascular endothelial cells repair a vessel in the living body in the event of a coil deviation and the effects of antiplatelet drugs on these cells have not been fully elucidated. We aimed to investigate the association between endothelial progenitor cells (EPCs) and endothelium formation at the surface of the platinum coils deployed in the carotid artery of rats, and to determine the effects of different antiplatelet drugs on this process.Subjects and Methods:We established an experimental model using normal and diabetic rats at 12 months of age. The diabetic rats were assigned to 4 different diet groups, distinguished by whether they were fed plain rat feed, or the same feed supplemented by 1 of 3 antiplatelet drugs (cilostazol, aspirin, or clopidogrel: all 0.1%) for 2 weeks, and the carotid artery was perforated by an embolization coil (“carotid coil model”). We monitored the process by which vascular endothelial cells formed the new endothelium on the surface of the coil by sampling and evaluating the region at 1, 2, and 4 weeks after placement. This repair process was also compared among 3 groups treated with different antiplatelet drugs (i.e. aspirin, clopidogrel, and cilostazol). One-way analysis of variance tests were performed to evaluate the differences in vascular thickness between groups, and P < .05 was considered statistically significant.Results:The diabetic rats showed delayed neoendothelialization and marked intimal hyperplasia. Cilostazol and clopidogrel effectively counteracted this delayed endothelial repair process. Flk1 immunostaining revealed greater expression in the diabetic rats administered cilostazol, second only to normal rats, suggesting that this agent acted to recruit EPCs.Conclusion:Neoendothelialization is delayed when vascular endothelial cells fail to function normally, which consequently leads to the formation of hyperplastic tissue. Cilostazol may remedy this dysfunction by recruiting EPCs to the site of injury.

Ref-1 protects against FeCl3-induced thrombosis and tissue factor expression via the GSK3β-NF-κB pathway.

Arterial thrombosis and its associated diseases are considered to constitute a major healthcare problem. Arterial thrombosis, defined as blood clot formation in an artery that interrupts blood circulation, is associated with many cardiovascular diseases. Oxidative stress is one of many important factors that aggravates the pathophysiological process of arterial thrombosis. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ref-1) has a multifunctional role in cells that includes the regulation of oxidative stress and anti-inflammatory function. The aim of this study was to investigate the therapeutic effect of adenovirus-mediated Ref-1 overexpression on arterial thrombosis induced by 60% FeCl3 solution in rats. Blood flow was measured to detect the time to occlusion, thrombus formation was detected by hematoxylin and eosin staining, reactive oxygen species (ROS) levels were detected by high-performance liquid chromatography, and the expression of tissue factor and other proteins was detected by Western blot. FeCl3 aggravated thrombus formation in carotid arteries and reduced the time to artery occlusion. Ref-1 significantly delayed arterial obstruction via the inhibition of thrombus formation, especially by downregulating tissue factor expression through the Akt-GSK3β-NF-κB signaling pathway. Ref-1 also reduced the expression of vascular inflammation markers ICAM-1 and VCAM-1, and reduced the level of ROS that contributed to thrombus formation. The results showed that adenovirus-mediated Ref-1 overexpression reduced thrombus formation in the rat carotid artery. In summary, Ref-1 overexpression had anti-thrombotic effects in a carotid artery thrombosis model and could be a target for the treatment of arterial thrombosis.

Evaluation of an In Vivo Rat Carotid Artery Interposition Study to Assess Functionalized Tissue Engineered Vascular Scaffolds

Evaluation of an In Vivo Rat Carotid Artery Interposition Study to Assess Functionalized Tissue Engineered Vascular Scaffolds

Cyclic Stretch Induces Vascular Smooth Muscle Cells to Secrete Connective Tissue Growth Factor and Promote Endothelial Progenitor Cell Differentiation and Angiogenesis

Endothelial progenitor cells (EPCs) play a vital role in endothelial repair following vascular injury by maintaining the integrity of endothelium. As EPCs home to endothelial injury sites, they may communicate with exposed vascular smooth muscle cells (VSMCs), which are subjected to cyclic stretch generated by blood flow. In this study, the synergistic effect of cyclic stretch and communication with neighboring VSMCs on EPC function during vascular repair was investigated. In vivo study revealed that EPCs adhered to the injury site and were contacted to VSMCs in the Sprague–Dawley (SD) rat carotid artery injury model. In vitro, EPCs were cocultured with VSMCs, which were exposed to cyclic stretch at a magnitude of 5% (which mimics physiological stretch) and a constant frequency of 1.25 Hz for 12 h. The results indicated that stretched VSMCs modulated EPC differentiation into mature endothelial cells (ECs) and promoted angiogenesis. Meanwhile, cyclic stretch upregulated the mRNA expression and secretion level of connective tissue growth factor (CTGF) in VSMCs. Recombinant CTGF (r-CTGF) treatment promoted endothelial differentiation of EPCs and angiogenesis, and increased their protein levels of FZD8 and β-catenin. CTGF knockdown in VSMCs inhibited cyclic stretch-induced EPC differentiation into ECs and attenuated EPC tube formation via modulation of the FZD8/β-catenin signaling pathway. FZD8 knockdown repressed endothelial differentiation of EPCs and their angiogenic activity. Wnt signaling inhibitor decreased the endothelial differentiation and angiogenetic ability of EPCs cocultured with stretched VSMCs. Consistently, an in vivo Matrigel plug assay demonstrated that r-CTGF-treated EPCs exhibited enhanced angiogenesis; similarly, stretched VSMCs also induced cocultured EPC differentiation toward ECs. In a rat vascular injury model, r-CTGF improved EPC reendothelialization capacity. The present results indicate that cyclic stretch induces VSMC-derived CTGF secretion, which, in turn, activates FZD8 and β-catenin to promote both differentiation of cocultured EPCs into the EC lineage and angiogenesis, suggesting that CTGF acts as a key intercellular mediator and a potential therapeutic target for vascular repair.

Hemodynamic Force as a Potential Regulator of Inflammation-Mediated Focal Growth of Saccular Aneurysms in a Rat Model.

Past studies have elucidated the crucial role of macrophage-mediated inflammation in the growth of intracranial aneurysms (IAs), but the contributions of hemodynamics are unclear. Considering the size of the arteries, we induced de novo aneurysms at the bifurcations created by end-to-side anastomoses with the bilateral common carotid arteries in rats. Sequential morphological data of induced aneurysms were acquired by magnetic resonance angiography. Computational fluid dynamics analyses and macrophage imaging by ferumoxytol were performed. Using this model, we found that de novo saccular aneurysms with a median size of 3.2 mm were induced in 20/45 (44%) of animals. These aneurysms mimicked human IAs both in morphology and pathology. We detected the focal growth of induced aneurysms between the 10th and 17th day after the anastomosis. The regional maps of hemodynamic parameters demonstrated the area exposed to low wall shear stress (WSS) and high oscillatory shear index (OSI) colocalized with the regions of growth. WSS values were significantly lower in the growing regions than in ones without growth. Macrophage imaging showed colocalization of macrophage infiltration with the growing regions. This experimental model demonstrates the potential contribution of low WSS and high OSI to the macrophage-mediated growth of saccular aneurysms.

Surfactant Protein A, a Novel Regulator for Smooth Muscle Phenotypic Modulation and Vascular Remodeling-Brief Report.

The objective of this study is to determine the role of SPA (surfactant protein A) in vascular smooth muscle cell (SMC) phenotypic modulation and vascular remodeling. Approach and Results: PDGF-BB (Platelet-derived growth factor-BB) and serum induced SPA expression while downregulating SMC marker gene expression in SMCs. SPA deficiency increased the contractile protein expression. Mechanistically, SPA deficiency enhanced the expression of myocardin and TGF (transforming growth factor)-β, the key regulators for contractile SMC phenotype. In vivo, SPA was induced in medial and neointimal SMCs following mechanical injury in both rat and mouse carotid arteries. SPA knockout in mice dramatically attenuated the wire injury-induced intimal hyperplasia while restoring SMC contractile protein expression in medial SMCs. These data indicate that SPA plays an important role in SMC phenotype modulation and vascular remodeling in vivo. SPA is a novel protein factor modulating SMC phenotype. Blocking the abnormal elevation of SPA may be a potential strategy to inhibit the development of proliferative vascular diseases.

Thick PCL Fibers Improving Host Remodeling of PGS-PCL Composite Grafts Implanted in Rat Common Carotid Arteries.

Vasculopathy and the consequential ischemia are major medical challenges. Grafting is an effective treatment to vascular occlusion. However, autologous grafting, despite scarcity, is the only choice for small diameter blood vessels. Synthetic grafts can fill the gap if they can work satisfactorily in arterial circulation. Electrospun polycaprolactone (PCL) sheathed porous poly(glycerol sebacate) (PGS) vascular grafts have good performances in arterial circulation in abdominal aortas and carotid arteries in rats. However, a major issue associated with the graft remodeling in vivo is limited neo-tissue formation inside PCL sheaths. Small pores of PCL sheaths inhibit cell infiltration and migration. To increase porosity of PCL sheaths of PGS-PCL composite grafts, diameters of electrospun PCL fibers are increased. The thick PCL fibers encourage cell migration and elicit a higher degree of CD206+ cells. In addition, some of the CD206+ cells co-express vascular cell markers in the thick-fiber grafts. The thick-fiber grafts also show improved mechanical properties and a higher elastin and collagen content. The data demonstrate the feasibility of improving graft vascular remodeling by increasing PCL fiber diameters and the critical role of CD206+ cells during graft vascular remodeling.

P.059 Identifying the role of trace amine-associated receptor 9 in behaviour, brain neurochemistry and blood biochemistry

Accelerated smooth muscle cell (SMC) proliferation is the primary cause of intimal hyperplasia (IH) following vascular interventions. Forkhead Box M1 (FOXM1) is considered a proliferation-associated transcription factor. However, the presence and role of FOXM1 in IH following vascular injury have not been determined.We examined the expression of FOXM1 in balloon-injured rat carotid arteries and investigated the effect of FOXM1 inhibition in SMCs and on the development of IH.FOXM1 was detected by immunofluorescent staining in balloon-injured rat carotid arteries where we observed an upregulation at day 7, 14, and 28 compared to uninjured controls. Immunofluorescence staining revealed FOXM1 coincided with proliferating cell nuclear antigen (PCNA). FOXM1 was also detectable in human carotid plaque samples. Western blot showed an upregulation of FOXM1 protein in serum-stimulated SMCs. Inhibition of FOXM1 using siRNA or chemical inhibition led to the induction of apoptosis as measured by flow cytometry and western blot for cleaved caspase 3. Perturbations in survival signaling were measured by western blot following FOXM1 inhibition, which showed a decrease in phosphorylated AKT and β-catenin. The chemical inhibitor thiostrepton was delivered by intraperitoneal injection in rats that underwent balloon injury and led to reduced intimal thickening compared to DMSO controls.FOXM1 is an important molecular mediator of IH that contributes to the proliferation and survival of SMCs following vascular injury.

Quercetin modulates AMPK/SIRT1/NF-κB signaling to inhibit inflammatory/oxidative stress responses in diabetic high fat diet-induced atherosclerosis in the rat carotid artery.

Inflammation and oxidative stress serve interrelated roles in the development of atherosclerosis and other vascular diseases. Quercetin has been previously reported to exhibit numerous beneficial properties towards several metabolic conditions and cardiovascular disease. The present study aimed to evaluate the effects of quercetin on the 5'adenosine monophosphate-activated protein kinase (AMPK)/sirtuin 1 (SIRT1)/NF-κB signaling pathway and inflammatory/oxidative stress response in diabetic-induced atherosclerosis in the carotid artery of rats. Male Wistar rats were used to create a diabetes-induced atherosclerosis model by the administration of high fat diet (HFD) with streptozotocin, which lasted for 8 weeks. Control and diabetic rats received quercetin (30 mg/kg/day; orally) for the last 2 weeks of the diabetic period. Plasma lipid profile and vascular levels of oxidative stress markers, inflammatory cytokines, NF-κB signaling proteins and SIRT1 expression were evaluated using ELISA and western blotting. Quercetin treatment in HFD diabetic rats was reported to improve the lipid profile and reduce the number of atherosclerotic lesions, atherogenic index and malondialdehyde levels, whilst increasing the activity of enzymatic antioxidants in the carotid artery. Additionally, the inflammatory response was suppressed by quercetin administration, as indicated by the reduced NF-κB and IL-1β levels, and increased IL-10 levels. Furthermore, SIRT1 expression was revealed to be significantly increased in response to quercetin treatment compared with non-treated HFD rats. However, these effects of quercetin were abolished or reversed by the administration of compound-C (0.2 mg/kg), a specific AMPK blocker, in HFD rats. Therefore, quercetin may have promising potential in ameliorating atherosclerotic pathophysiology in the rat carotid artery by inhibiting oxidative stress and inflammatory responses mechanistically by modulating the AMPK/SIRT1/NF-κB signaling pathway.

The phosphorylation of CHK1 at Ser345 regulates the phenotypic switching of vascular smooth muscle cells both in vitro and in vivo

Background and aimsDNA damage and repair have been shown to be associated with carotid artery restenosis and atherosclerosis. The proliferation and migration of vascular smooth muscle cells (VSMCs) is the main cause of artery stenosis. This study aims to define the relationship between DNA damage and VSMCs proliferation. MethodsA rat carotid artery injury model was established, and human and rat VSMCs cultured in vitro. H2O2 was used to induce DNA damage in vitro. The selected CHK1 inhibitor, LY2603618, was used to inhibit CHK1 phosphorylation both in vivo and in vitro. γH2AX, αSMA and phosphorylated CHK1 were detected both in rat carotid artery and cultured VSMCs from different groups. Hyperplasia ratio of rat carotid artery intimal was measured. ResultsDNA double-strand breaks occur in the rat carotid artery after injury. DNA damage induces CHK1 phosphorylation and down-regulates αSMA expression in VSMCs both in vitro and in vivo. The inhibition of CHK1 phosphorylation rescues αSMA expression in VSMCs both in vitro and in vivo, and rat carotid intimal hyperplasia after injury was suppressed. ConclusionsOur data demonstrated that phosphorylation of CHK1 under DNA damage stress modulates VSMCs phenotypic switching. CHK1 inhibition may be a potential therapeutic strategy for intima hyperplasia treatment.

Levetiracetam effect on behavioral and electrophysiological parameters in rat model of global brain ischemia

Post-stroke paroxysmal activity is a neurophysiological indicator of epileptogenesis and increase of seizure susceptibility, so treatments with neuroprotective activity and anti-paroxysmal activity can be more beneficial during post-ischemic period. The goal of this study was evaluation of levetiracetam (100 mg/kg, 7 days of administration) effect on behavior and brain bioelectric activity changes in the post-ischemic period. Global ischemia model was carried out with bilateral ligation of carotid arteries in rats. Neurological deficit and electrophysiological changes of brain structures (striatum, cortex, hypothalamus, hippocampus) were analyzed during 28 days. Paroxysmal activity was not observed on the 1st day after ischemia and had early (2nd day) and late (28th day) onsets. Spectral analysis showed that rats, that died by the 10th day, had delta wave increase and theta decrease on the 1st day and delta activity reduction on the 2-7th days. LEV did not affect survival rate, however, it contributed to neurological disorder regression towards lighter forms on the 1st day after ischemia. It suppressed paroxysmal activity with an early onset and affected delta and theta waves on the 1st day in all structures except hippocampus. On the 7th and 28th days LEV increased delta activity due to 1–3 Hz frequency. Thus, LEV eliminates early onset post-ischemic paroxysmal activity and contributes to normalization of delta waves activity on the 1st day after ischemia, that positively affects neurological status of animals in post-ischemic period. It allows one to make a conclusion about possible LEV application in the post-ischemic period.

PERK Inhibition Promotes Post-angioplasty Re-endothelialization via Modulating SMC Phenotype Changes

BackgroundDrug-eluting stents impair post-angioplasty re-endothelialization thus compromising restenosis prevention while heightening thrombotic risks. We recently found that inhibition of protein kinase RNA-like endoplasmic reticulum kinase (PERK) effectively mitigated both restenosis and thrombosis in rodent models. This motivated us to determine how PERK inhibition impacts re-endothelialization. MethodsRe-endothelialization was evaluated in endothelial-denuded rat carotid arteries after balloon angioplasty and periadventitial administration of PERK inhibitor in a hydrogel. To study whether PERK in smooth muscle cells (SMCs) regulates re-endothelialization by paracrinally influencing endothelial cells (ECs), denuded arteries exposing SMCs were lentiviral-infected to silence PERK; in vitro, the extracellular vesicles isolated from the medium of PDGF-activated, PERK-upregulating human primary SMCs were transferred to human primary ECs. ResultsTreatment with PERK inhibitor versus vehicle control accelerated re-endothelialization in denuded arteries. PERK-specific silencing in the denuded arterial wall (mainly SMCs) also enhanced re-endothelialization compared to scrambled shRNA control. In vitro, while medium transfer from PDGF-activated SMCs impaired EC viability and increased the mRNA levels of dysfunctional EC markers, either PERK inhibition or silencing in donor SMCs mitigated these EC changes. Furthermore, CXCL10, a paracrine cytokine detrimental to ECs, was increased by PDGF activation and decreased after PERK inhibition or silencing in SMCs. ConclusionsAttenuating PERK activity pharmacologically or genetically provides an approach to accelerating post-angioplasty re-endothelialization in rats. The mechanism may involve paracrine factors regulated by PERK in SMCs that impact neighboring ECs. This study rationalizes future development of PERK-targeted endothelium-friendly vascular interventions.

AMD3100 and SDF‑1 regulate cellular functions of endothelial progenitor cells and accelerate endothelial regeneration in a rat carotid artery injury model

The present study was conducted to assess the effects of AMD3100 and stromal cell-derived factor 1 (SDF-1) on cellular functions and endothelial regeneration of endothelial progenitor cells (EPCs). The cell proliferation and adhesion capacity of EPCs were evaluated in vitro following treatment with AMD3100 and SDF-1 using a Cell Counting Kit-8 assay. Furthermore, the expression levels of C-X-C motif chemokine receptor 4 (CXCR4) and C-X-C motif chemokine receptor 7 (CXCR7) were detected before and after treatment with AMD3100 and SDF-1 to elucidate their possible role in regulating the cellular function of EPCs. A rat carotid artery injury model was established to assess the influences of AMD3100 and SDF-1 on endothelial regeneration. AMD3100 reduced the proliferation and adhesion capacity of EPCs to fibronectin (FN), whereas it increased the adhesion capacity of EPCs to human umbilical vein endothelial cells (HUVECs). However, SDF-1 stimulated the proliferation and cell adhesion capacity of EPCs to HUVECs and FN. Additionally, the expression levels of CXCR7 but not CXCR4 were upregulated following AMD3100 treatment, whereas the expression levels of both CXCR4 and CXCR7 were upregulated after SDF-1 treatment. In vivo results demonstrated that AMD3100 increased the number of EPCs in the peripheral blood and facilitated endothelial repair at 7 days after treatment. However, local administration of SDF-1 alone did not enhance reendothelialization 7 and 14 days after treatment. Importantly, the combination of AMD3100 with SDF-1 exhibited superior therapeutic effects compared with AMD3100 treatment alone, accelerated reendothelialization 7 days after treatment, and attenuated neointimal hyperplasia at day 7 and 14 by recruiting more EPCs to the injury site. In conclusion, AMD3100 could positively regulate the adhesion capacity of EPCs to HUVECs via elevation of the expression levels of CXCR7 but not CXCR4, whereas SDF-1 could stimulate the proliferation and adhesion capacity of EPCs to FN and HUVECs by elevating the expression levels of CXCR4 and CXCR7. AMD3100 combined with SDF-1 outperformed AMD3100 alone, promoted early reendothelialization and inhibited neointimal hyperplasia, indicating that early reendothelialization attenuated neointimal hypoplasia following endothelial injury.

Slow degrading poly(glycerol sebacate) derivatives improve vascular graft remodeling in a rat carotid artery interposition model

Porous synthetic grafts made of poly (glycerol sebacate) (PGS) can transform into autologous vascular conduits in vivo upon degradation of PGS. A long-held doctrine in tissue engineering is the necessity to match degradation of the scaffolds to tissue regeneration. Here, we tested the impact of degradation of PGS and its derivative in an interposition model of rat common carotid artery (CCA). Previous work indicates a complete degradation of PGS within approximately 2 weeks, likely at the fast end of the spectrum. Thus, the derivation of PGS focuses on delay degradation by conjugating the free hydroxy groups in PGS with a long chain carboxylic acid: palmitic acid, one of the most common lipid components. We evaluated two of the resultant palmitate-PGS (PPGS) in this study: one containing 9% palmitate (9-PPGS) and the other16% palmitate (16-PPGS). 16-PPGS grafts had the highest patency. Ultrasound imaging showed that the lumens of 16-PPGS grafts were similar to CCA and smaller than 9-PPGS and PGS grafts 12 weeks post-operation. Immunohistological and histological examination showed an endothelialized lumens in all three types of grafts within 4 weeks. Inflammatory responses to 16-PPGS grafts were limited to the adventitial space in contrast to a more diffusive infiltration in 9-PPGS and PGS grafts in week 4. Examination of calponin+ and αSMA+ cells revealed that 16-PPGS grafts remodeled into a distinctive bi-layered wall, while the walls of 9-PPGS grafts and PGS grafts only had one thick layer of smooth muscle-like cells. Correspondingly, the expression of collagen III and elastin displayed an identical layered structure in the remodeled 16-PPGS grafts, in contrast to a more spread distribution in 9-PPGS and PGS grafts. All the three types of grafts exhibited the same collagen content and burst pressure after 12 weeks of host remodeling. However, the compliance and elastin content of 16-PPGS grafts in week 12 were closest to those of CCA. Overall, placing the degradation of PGS derived elastomer to a window of 4–12 weeks results in vascular conduits closer to arteries in a rat carotid artery interposition model over a 12-week observation period.

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.