Rat Carotid Artery Research Articles

Long-term biocompatibility of fluorescent diamonds-(NV)-Z~800 nm in rats: survival, morbidity, histopathology, particle distribution and excretion studies (part IV).

BackgroundThromboembolic events are a major cause of heart attacks and strokes. However, diagnosis of the location of high risk vascular clots is hampered by lack of proper technologies for their detection. We recently reported on bio-engineered fluorescent diamond-(NV)-Z~800nm (FNDP-(NV)) conjugated with bitistatin (Bit) and proven its ability to identify iatrogenic blood clots in the rat carotid artery in vivo by Near Infra-Red (NIR) monitored by In Vivo Imaging System (IVIS).PurposeThe objective of the present research was to assess the in vivo biocompatibility of FNDP-(NV)-Z~800nm infused intravenously to rats. Multiple biological variables were assessed along this 12 week study commissioned in anticipation of regulatory requirements for a long-term safety assessment.MethodsRats were infused under anesthesia with aforementioned dose of the FNDP-(NV), while equal number of animals served as control (vehicle treated). Over the 12 week observation period rats were tested for thriving, motor, sensory and cognitive functions. At the termination of study, blood samples were obtained under anesthesia for comprehensive hematology and biochemical assays. Furthermore, 6 whole organs (liver, spleen, brain, heart, lung and kidney) were collected and examined ex vivo for FNDP-NV) via NIR monitored by IVIS and histochemical inspection.ResultsAll animals survived, thrived (no change in body and organ growth). Neuro-behavioral functions remain intact. Hematology and biochemistry (including liver and kidney functions) were normal. Preferential FNDP-(NV) distribution identified the liver as the main long-term repository. Certified pathology reports indicated no outstanding of finding in all organs.ConclusionThe present study suggests outstanding biocompatibility of FNDP-(NV)-Z~800nm after long-term exposure in the rat.

Cognitive impairments and microvascular endothelial dysfunction in unilateral occlusion of the carotid artery

The study was aimed at determining cognitive impairments, vascular endothelial dysfunction and morphofunctional alterations of neurons in brain structures (neocortex and hippocampus) in unilateral occlusion of the common carotid artery in albino rats. The experiments were carried out on Wistar albino rats. The animals were divided into two groups: group 1 - the control group composed of sham-operated rats and group 2 - the study group consisting of rats with cerebral ischaemia. The model of cerebral ischaemia was reproduced by occlusion of the left common carotid artery. Cognitive functions in rats were assessed by means of the object recognition test and conditioned passive avoidance response (CPAR). The degree of vascular endothelial dysfunction was evaluated by the number of circulating endotheliocytes and concentration of end products of nitric oxide - nitrites in blood plasma. Pathomorphological studies of the brain and morphometric analysis of the number of damaged neurons after ischaemic exposure were carried out by means of light microscopy. It was determined that unilateral occlusion of the common carotid artery in rats resulted in memory impairment revealed by the object recognition test and CPAR (p≤0.05). Cerebral ischaemia induced an elevated level of circulating endotheliocytes and a decrease in end products of nitric oxide - nitrates as compared with the controls (p≤0.05). Morphological study demonstrated signs of vasoconstrictive reactions, microvascular endothelial oedema, as well as an increase in the proportion of damaged neurons localized in the fronto-bregmatic region of the cortex of the cerebral hemispheres and hippocampus on the ipsilateral side (p≤0.05). Unilateral occlusion of the common carotid artery in albino rats resulted in cognitive impairments, damage of neurons in the most vulnerable areas of the cortex of the cerebral hemispheres and hippocampus predominantly on the ipsilateral side. Cognitive impairments and ischaemic lesions of the brain structures are induced by endothelial dysfunction, enhanced desquamation of endotheliocytes and prevalence of vasoconstrictive reactions resulting from decreased production of the major vasorelaxing factor - nitric oxide.

Effects of Periodontal Intervention on Levels of Serum High-sensitivity C-reactive Protein and Interleukin 6, and on Carotid Artery in Rats with Chronic Periodontitis and Hyperlipidemia.

To investigate the effects of nonsurgical periodontal therapy on the serum interleukin 6 (IL-6) and high-sensitivity C-reactive protein (hsCRP) and on the carotid artery in rats with chronic periodontitis (CP) and with or without hyperlipidemia (HL). A total of 29 Sprague-Dawley rats were randomly divided into two groups: group A (CP, n = 14) and B (CP + HL, n = 15), and subjected to the corresponding treatment. Groups A and B were further divided into groups A1/B1 (without periodontal interventions) and A2/B2 (with periodontal interventions). The serum IL-6 and hsCRP levels were evaluated before periodontal intervention and at 1, 3, 5, and 7 weeks after periodontal intervention. The rats were euthanised 8 weeks after the periodontal intervention and the histopathologic changes in the carotid artery were observed. The serum hsCRP and serum IL-6 levels in groups A1 and B1 were elevated with time; they were significantly higher in group B1 than in group A1 (P < 0.001) at all time points. The hsCRP and IL-6 levels in groups A2 and B2 increased with time and reached the maximum level 1 week after the second intervention, and then gradually decreased. Atherosclerotic plaques, fibrous cap, and calcium salt deposits were apparent in the rats of group B1, whereas no obvious atherosclerotic changes were observed in the rats of groups A2 and B2. Periodontal interventions resulted in acute, short-term systemic inflammation. However, it was beneficial in long-term as it improved the carotid artery integrity.

Effect and Mechanism of Kir2.1 Channel Overexpression on Transdifferentiation of Endothelial Progenitor Cells

Objective The propose of the study is to investigate the specific effects of the mechanically sensitive channel Kir2.1 on the transdifferentiation of EPCs so as to understand the molecular mechanism of pathological vascular remodeling. Methods Endothelial progenitor cells (EPCs) were isolated from rat bone marrow and cultured in EGM2 medium in vitro. The recombinant lentiviral vectors carrying Kir2.1 (NM_017296.1) gene was designed and constructed in order to overexpress the gene. The smooth muscle cells (SMCs) molecules marker on EPCs, such as α-SMA, FSP1 and α-SM22, were detected by RT-PCR and cellular immunofluorescence. In addition, cell angiogenic capacity and migration in vitro were assessed by Matrigel and Transwell methods respectively. Moreover, neointimal thickening was evaluated in the surgery model of balloon injury of rat carotid artery in vivo. Result The results showed that the expression levels of α-SM22, FSP1 and α-SMA were up-regulated in the Kir2.1 overexpression group compared with the control. The number of migrating cells in the Kir2.1 overexpression group was significantly higher than that in the scramble group, while quantitative assessment further confirmed that the Kir2.1 overexpression strongly attenuated the ability of bone marrow-derived EPC to form tube-like structures in Matrigel assay. Compared with the control group, morphometric analysis showed ratio of intimal area/medial area (I/M) in rats was increased in rats transplanted with Lenti-Kir2.1 overexpression. Conclusion It is indicated that the overexpression of channel Kir2.1 induces EPCs transdifferentiated into mesenchymal transition SMCs (EndoMT). It may provide a potential target for the treatment or prevention of pathological vascular remodeling disease.

Dihydroartemisinin ameliorates balloon injury-induced neointimal formation in rats.

This study aims to evaluate the effects of dihydroartemisinin (DHA) on the balloon injury-induced neointimal formation in rats and to investigate the underlying mechanism. The balloon-induced carotid artery injury model was established in male Sprague-Dawley rats, immediately after which the DHA solution was injected into the tail vein of rats. In in vitro assays, primary rat vascular smooth muscle cells (VSMCs) were pretreated with DHA and then coincubated with LPS. DHA ameliorated the induced neointimal formation and fibrosis but enhanced apoptosis in rat carotid artery after balloon injury. Furthermore, DHA suppressed migration and enhanced apoptosis of the lipopolysaccharide (LPS)-treated primary VSMCs in vitro. Moreover, in both the balloon injury-induced rat sera and the LPS-treated VSMCs, DHA significantly inhibited proinflammatory cytokines, including interleukin-1β, tumor necrosis factor-ɑ, and matrix metalloproteinase-1. Importantly, DHA significantly decreased the balloon injury-increased expression of nuclear factor kappa B (NF-κB) subunit NF-κB p65 expression, and increased the balloon injury-reduced expression of inhibitor of NF-κB-alpha, indicating the inhibition of the IκB/NF-κB pathway. DHA significantly inhibited neointimal formation in balloon-induced rat carotid artery injury and the mechanism may be related to the inhibition of IκB/NF-κB signaling, which alleviates the inflammatory response.

HDAC6 Regulates the MRTF-A/SRF Axis and Vascular Smooth Muscle Cell Plasticity

Cellular plasticity is fundamental in biology and disease. Vascular smooth muscle cell (SMC) dedifferentiation (loss of contractile proteins) initiates and perpetrates vascular pathologies such as restenosis. Contractile gene expression is governed by the master transcription factor, serum response factor (SRF). Unlike other histone deacetylases, histone deacetylase 6 (HDAC6) primarily resides in the cytosol. Whether HDAC6 regulates SRF nuclear activity was previously unknown in any cell type. This study found that selective inhibition of HDAC6 with tubastatin A preserved the contractile protein (alpha-smooth muscle actin) that was otherwise diminished by platelet-derived growth factor-BB. Tubastatin A also enhanced SRF transcriptional (luciferase) activity, and this effect was confirmed by HDAC6 knockdown. Interestingly, HDAC6 inhibition increased acetylation and total protein of myocardin-related transcription factor A (MRTF-A), a transcription co-activator known to translocate from the cytosol to the nucleus, thereby activating SRF. Consistently, HDAC6 co-immunoprecipitated with MRTF-A. Invivo studies showed that tubastatin A treatment of injured rat carotid arteries mitigated neointimal lesion, which is known to be formed largely by dedifferentiated SMCs. This report is the first to show HDAC6 regulation of the MRTF-A/SRF axis and SMC plasticity, thus opening a new perspective for interventions of vascular pathologies.

Noninvasive in vivo Assessment of the Re-endothelialization Process Using Ultrasound Biomicroscopy in the Rat Carotid Artery Balloon Injury Model.

Ultrasound biomicroscopy (UBM), or ultra high-frequency ultrasound, is a technique used to assess the anatomy of small research animals. In this study, UBM was used to assess differences in intimal hyperplasia thickness as a surrogate measurement of the re-endothelialization process after carotid artery balloon injury in rats. Ultrasound biomicroscopic data from 3 different experiments and rat strains (Sprague Dawley, Wistar, and diabetic Goto-Kakizaki) were analyzed. All animals were subjected to carotid artery balloon injury and examined with UBM (30-70 MHz) 2 and 4 weeks after injury. Re-endothelialization on UBM was defined as the length from the carotid bifurcation to the most distal visible edge of the intimal hyperplasia. En face staining with Evans blue dye was performed at euthanasia 4 weeks after injury, followed by tissue harvesting for histochemical and immunohistochemical evaluations. A significant correlation (Spearman r = 0.63; P < .0001) was identified when comparing all measurements of re-endothelialization obtained from UBM and en face staining. The findings revealed a similar pattern for all rat strains: Sprague Dawley (Spearman r = 0.70; P < .0001), Wistar (Spearman r = 0.36; P < .081), and Goto-Kakizaki (Spearman r = 0.70; P < .05). A Bland-Altman test showed agreement between en face staining and UBM. Immunohistochemical staining confirmed the presence of the endothelium in the areas detected as re-endothelialized by the UBM assessment. Ultrasound biomicroscopy can be used for repeated in vivo assessment of re-endothelialization after carotid artery balloon injury in rats.

MiR-140-3p is Involved in In-Stent Restenosis by Targeting C-Myb and BCL-2 in Peripheral Artery Disease.

Aim: In-Stent Restenosis (ISR) is the major reason for recurrent ischemia and amputation after endovascular treatment of Peripheral Artery Disease (PAD). Our previous study demonstrated that miR-140-3p is significantly down-regulated in PAD arteries. However, expression and function of miR-140-3p in ISR of human PAD are currently unclear.The aim of this study is to determine the miR-140-3p expression and its regulative role in ISR of PAD.Methods: The RNA level was determined by quantitative real-time polymerase chain Reaction (qRT-PCR) and in situ hybridization. Primary cultured ASMCs were isolated from human femoral arterial of the healthy donors or ISR patients. Cell proliferation was determined by Edu incorporation and CCK-8 assay. Apoptosis was determined by Annexin-V/PI Double-Staining assay and TUNEL assay. A rat carotid artery balloon angioplasty model was used to investigate the effect of miR-140-3p on restenosis.Results: MiR-140-3p was significantly down-regulated in PAD and ISR arteries than normal arteries. Primary cultured ISR ASMCs exhibited elevated proliferation and down-regulated miR-140-3p than normal ASMCs. Transfection of miR-140-3p mimic attenuated PDGF-BB-induced proliferation in cultured ASMCs and induced apoptosis. Luciferase reporter assay indicated that miR-140-3p transfection significantly down-regulated C-Myb and BCL-2 in ISR ASMCs by targeting to their 3′-UTRs. MiR-140-3p transfection induced anti-proliferation and apoptosis in ASMCs, which were ameliorated by over-expression of C-Myb or BCL-2. Moreover, the animal study showed that miR-140-3p can reduce restenosis following angioplasty via targeting C-Myb and BCL-2.Conclusions: The result suggests that miR-140-3p regulates ASMC function via targeting C-Myb and BCL-2 in the process of ISR in PAD. The novel findings may offer a hopeful therapeutic target for human PAD.

Arterial Thrombosis Is Accompanied by Elevated Mitogen-Activated Protein Kinase (MAPK) and Cyclooxygenase-2 (COX-2) Expression via Toll-Like Receptor 4 (TLR-4) Activation by S100A8/A9.

BackgroundThe aim of this study was to determine the involvement of S100A8/A9 in the development of arterial thrombosis.Material/MethodsA total of 303 patients were enrolled in this study, with 110 having acute coronary syndrome (ACS) and 110 having coronary heart disease (CHD), and 83 subjects served as healthy blood donors. The concentrations of Toll-like receptor 4 (TLR-4), cyclooxygenase-2 (COX-2), and S100A8/A9 protein were determined in the sera of the participants and in peripheral blood mononuclear cells (PBMCs) derived from a rat carotid artery thrombosis model and in human aortic endothelial cells (HAECs). The mitogen-activated protein kinase (MAPK) inhibitor SB203580 and the TLR-4 blocker CLI-095 were used to investigate the role of the TLR-4-MAPK-COX2 signaling axis in thrombosis.ResultsThe levels of COX-2, TLR-4, and S100A8/A9 in the sera of patients with ACS and CHD were significantly higher than in healthy controls (P<0.05). S100A8/A9 expression was significantly correlated with TLR-4 and COX-2 in the ACS group and with TLR-4 in the CHD group. In the rat carotid thrombosis model, the expressions of TLR-4, COX-2, and p-p38 MAPK significantly increased until 14 days after thrombosis induction, whereas S100A8/A9 expression increased until day 7, but then decreased. Administration of SB203580 to rats reduced COX-2 expression in PBMCs after thrombosis induction, and incubation of HAECs with CLI-095 reduced their p-p38 MAPK and COX-2 response to S100A8/A9 stimulation.ConclusionsS100A8/A9 is upregulated after blood vessel injury and is enhanced in combination with TLR-4 COX-2 induction via p38 MAPK activation.

Matrix stiffness modulates the differentiation of neural crest stem cells in vivo.

Stem cells are often transplanted with scaffolds for tissue regeneration; however, how the mechanical property of a scaffold modulates stem cell fate in vivo is not well understood. Here we investigated how matrix stiffness modulates stem cell differentiation in a model of vascular graft transplantation. Multipotent neural crest stem cells (NCSCs) were differentiated from induced pluripotent stem cells, embedded in the hydrogel on the outer surface of nanofibrous polymer grafts, and implanted into rat carotid arteries by anastomosis. After 3 months, NCSCs differentiated into smooth muscle cells (SMCs) near the outer surface of the polymer grafts; in contrast, NCSCs differentiated into glial cells in the most part of the hydrogel. Atomic force microscopy demonstrated a stiffer matrix near the polymer surface but much lower stiffness away from the polymer graft. Consistently, in vitro studies confirmed that stiff surface induced SMC genes whereas soft surface induced glial genes. These results suggest that the scaffold's mechanical properties play an important role in directing stem cell differentiation in vivo, which has important implications in biomaterials design for stem cell delivery and tissue engineering.

Requirement for sphingosine kinase 1 in mediating phase 1 of the hypotensive response to anandamide in the anaesthetised mouse

In the isolated rat carotid artery, the endocannabinoid anandamide induces endothelium-dependent relaxation via activation of the enzyme sphingosine kinase (SK). This generates sphingosine-1-phosphate (S1P) which can be released from the cell and activates S1P receptors on the endothelium. In anaesthetised mice, anandamide has a well-characterised triphasic effect on blood pressure but the contribution of SK and S1P receptors in mediating changes in blood pressure has never been studied. Therefore, we assessed this in the current study.The peak hypotensive response to 1 and 10 mg/kg anandamide was measured in control C57BL/6 mice and in mice pretreated with selective inhibitors of SK1 (BML-258, also known as SK1-I) or SK2 ((R)-FTY720 methylether (ROMe), a dual SK1/2 inhibitor (SKi) or an S1P1 receptor antagonist (W146). Vasodilator responses to S1P were also studied in isolated mouse aortic rings.The hypotensive response to anandamide was significantly attenuated by BML-258 but not by ROMe. Antagonising S1P1 receptors with W146 completely blocked the fall in systolic but not diastolic blood pressure in response to anandamide. S1P induced vasodilation in denuded aortic rings was blocked by W146 but caused no vasodilation in endothelium-intact rings.This study provides evidence that the SK1/S1P regulatory-axis is necessary for the rapid hypotension induced by anandamide. Generation of S1P in response to anandamide likely activates S1P1 to reduce total peripheral resistance and lower mean arterial pressure. These findings have important implications in our understanding of the hypotensive and cardiovascular actions of cannabinoids.

Linagliptin protects rat carotid artery from balloon injury and activates the NRF2 antioxidant pathway.

Percutaneous coronary intervention (PCI) is main treatment for acute coronary syndrome (ACS). However, restenosis caused by PCI-induced injury influences the outcome of patients. Linagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, has been reported to ameliorate intimal hyperplasia post vascular injury. The underlying mechanisms by which linagliptin protects against balloon injury are unclear and require to be explored. Herein, Wistar rats with carotid artery balloon injury were given 1, 2 or 3 mg/kg/day linagliprin for 6 weeks. We found that linagliptin attenuated vascular injury-mediated neointima formation in rats without affecting body weight and blood glucose levels. ELISA results indicated that linagliptin significantly reduced overproduction of cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 post balloon injury. By detecting the level of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), we found that linagliptin prevented balloon injury-induced oxidative stress. Additionally, linagliptin decreased the level of Kelch ECH-associating protein 1 (KEAP1) compared with injury group. Results of Western blots and electrophoretic mobility shift assay (EMSA) demonstrated that linagliptin augmented nuclear accumulation of nuclear factor-E2-related factor 2 (NRF2) and its binding ability to target genes in rats with balloon injury. Moreover, heme oxygenase-1 (HO-1) and NAD (P) H quinine oxidoreductase 1 (NQO1), two downstream targets of NRF2, were further up-regulated after linagliptin treatment compared with injury group. In conclusion, our data suggest that linagliptin protects carotid artery from balloon injury-induced neointima formation and activates the NRF2 antioxidant pathway.

Expression of endothelin type B receptors (EDNRB) on smooth muscle cells is controlled by MKL2, ternary complex factors, and actin dynamics.

The endothelin type B receptor (ETB or EDNRB) is highly plastic and is upregulated in smooth muscle cells (SMCs) by arterial injury and following organ culture in vitro. We hypothesized that this transcriptional plasticity may arise, in part, because EDNRB is controlled by a balance of transcriptional inputs from myocardin-related transcription factors (MRTFs) and ternary complex factors (TCFs). We found significant positive correlations between the TCFs ELK3 and FLI1 versus EDNRB in human arteries. The MRTF MKL2 also correlated with EDNRB. Overexpression of ELK3, FLI1, and MKL2 in human coronary artery SMCs promoted expression of EDNRB, and the effect of MKL2 was antagonized by myocardin (MYOCD), which also correlated negatively with EDNRB at the tissue level. Silencing of MKL2 reduced basal EDNRB expression, but depolymerization of actin using latrunculin B (LatB) or overexpression of constitutively active cofilin, as well as treatment with the Rho-associated kinase (ROCK) inhibitor Y27632, increased EDNRB in a MEK/ERK-dependent fashion. Transcript-specific primers indicated that the second EDNRB transcript (EDNRB_2) was targeted, but this promoter was largely unresponsive to LatB and was inhibited rather than stimulated by MKL2 and FLI1, suggesting distant control elements or an indirect effect. LatB also reduced expression of endothelin-1, but supplementation experiments argued that this was not the cause of EDNRB induction. EDNRB finally changed in parallel with ELK3 and FLI1 in rat and human carotid artery lesions. These studies implicate the actin cytoskeleton and ELK3, FLI1, and MKL2 in the transcriptional control of EDNRB and increase our understanding of the plasticity of this receptor.

TRPV4 activation in rat carotid artery in DOCA hypertension involves eNOS and endothelium-derived contractile factor (EDCF)

ABSTRACTAim: Role of TRPV4 channel in regulation of endothelial function in the carotid artery in deoxycorticosterone acetate (DOCA) model of hypertension in rat was studied.Methods: 8–10 weeks old albino Wistar rats divided into three groups namely Control, UNX and hypertensive animals. Vascular smooth muscle response was studied in isolated carotid artery of rat with acetylcholine, sodium nitroprusside, GSK1016790A (GSK) in presence and absence of L-NAME and indomethacin.Results: At the end of the 6th week, the mean systolic blood pressure was increased in DOCA-treated hypertensive rats (166 ± 8 mm Hg) compared to Control and UNX (125 ± 5 mm Hg). ACh (10–9 to 10–5 M) produced almost 100% relaxation in Control (Emax = 97.48 ± 1.06 %) and UNX animals (Emax = 93.16 ± 2.33 %) which was attenuated in DOCA-treated hypertensive animals (Emax = 70.85 ± 1.65 %). No significant changes seen in SNP (10–12 to 10–5 M) induced relaxation. GSK1016790A (10–12 to 10–7 M)-mediated relaxation was significantly attenuated in DOCA-treated hypertensive animals (Emax = 25.58 ± 13.60%) compared to the control (Emax = 80.59 ± 6.86%) and UNX (Emax = 87.32 ± 2.01%) animals. L-NAME (10–4 M) potently blocked GSK-induced relaxation, and a contractile response to GSK was observed in presence of L-NAME in all the three groups of animals which was sensitive to indomethacin (10–5 M).Conclusion: TRPV4 may regulate the vascular tone of rat carotid artery through an attenuated NO pathway and stimulation of the release of contractile prostanoids in the DOCA hypertensive rats.

CircDiaph3 regulates rat vascular smooth muscle cell differentiation, proliferation, and migration.

Intimal hyperplasia is a reaction to vascular injury, which is the primary reason for vascular restenosis caused by the diagnostic or therapeutic procedure for cardiovascular diseases. Circular RNAs (circRNAs) are known to be associated with several cardiovascular conditions, but the expression of circRNAs in the neointima has not been reported in detail. In this study, we established the balloon-injured rat carotid artery model and detected the expression of circRNAs in the carotid arteries with a microarray. We found that the circRNA expression profile of the healthy carotid arteries and the injured arteries were significantly different. We investigated the role of rno-circ_005717 ( circDiaph3) in the differentiation of rat vascular smooth muscle cells (VSMCs). We found that knockdown of circDiaph3 up-regulated the level of diaphanous-related formin-3 and promoted the differentiation of VSMCs to contractile type. In addition, circDiaph3 up-regulated the transcription of Igf1r and supported the proliferation and migration of VSMCs. circDiaph3 could be a molecular target to combat intimal hyperplasia.-Xu, J.-Y., Chang, N.-B., Rong, Z.-H., Li, T., Xiao, L., Yao, Q.-P., Jiang, R., Jiang, J. circDiaph3 regulates rat vascular smooth muscle cell differentiation, proliferation, and migration.

GW29-e1749 Delayed reendothelialization with rapamycin is rescued by the addition of nicorandil in balloon-injured rat carotid arteries

We build the hypothesis that nicorandil may promote reendothelialization impaired by rapamycin through inhibiting XO-generated ROS formation and promoting eNOS expression. In vivo carotid balloon injury model and in vitro CMECs were used to indentify this hypothesis. Sprague-Dawley (SD) rats (male

Inhibition of Neointima Hyperplasia, Inflammation, and Reactive Oxygen Species in Balloon-Injured Arteries by HVJ Envelope Vector-Mediated Delivery of Superoxide Dismutase Gene

Extracellular superoxide dismutase (EC-SOD) has been implicated in regulation of vascular function but its underlying molecular mechanism is largely unknown. These two-step experiments investigate whether hemagglutinating virus of Japan envelope (HVJ-E) vector-mediated EC-SOD gene delivery might protect against neointima formation, vascular inflammation, and reactive oxygen species (ROS) generation, and also explore cell growth signaling pathways. The first in-vitro experiment was performed to assess the transfection efficacy and safety of HVJ-E compared to lipofectamine®. Results revealed that HVJ-E has higher transfection efficiency and lower cytotoxicity than those of lipofectamine®. Another in-vivo study initially used balloon denudation to rat carotid artery, then delivered EC-SOD cDNA through the vector of HVJ-E. Arterial section with H&E staining from the animals 14 days after balloon injury showed a significant reduction of intima-to-media area ratio in EC-SOD transfected arteries when compared with control (empty vector-transfected arteries) (p < 0.05). Arterial tissue with EC-SOD gene delivery also exhibited lower levels of ROS, as assessed by fluorescent microphotography with dihydroethidium staining. Quantitative RT-PCR revealed that EC-SOD gene delivery significantly diminished mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (p < 0.05 in all comparisons). An immunoblotting assay from vascular smooth muscle cell (VSMC) cultures showed that the EC-SOD transfected group attenuated the activation of MEK1/2, ERK1/2, and Akt signaling significantly. In conclusion, EC-SOD overexpression by HVJ-E vector inhibits neointima hyperplasia, inflammation, and ROS level triggered by balloon injury. The modulation of cell growth-signaling pathways by EC-SOD in VSMCs might play an important role in these inhibitory effects.

Long Noncoding RNA XR007793 Regulates Proliferation and Migration of Vascular Smooth Muscle Cell via Suppressing miR-23b.

BackgroundLong noncoding RNAs (lncRNAs) were identified as potential regulatory factor in vascular disease. However, the role of XR007793 in the regulation of neointima formation after vascular injury remains largely unknown.Material/MethodsLncRNA expression levels were detected using real-time polymerase chain reaction (RT-PCR). In vivo and in vitro assay were performed in Sprague-Dawley rats and VSMCs. Cell Counting Kit-8 (CCK-8) assay, Transwell assay, and scratch wound healing assay were performed to detect cell proliferation and migration. Western blotting was used to detect protein expression.ResultsThe results of qRT-PCR indicated that XR007793 expression was significantly increased in the injured carotid artery of Sprague-Dawley rats and platelet-derived growth factor-BB induced rat aortic smooth muscle cells. Knockdown of XR007793 repressed the proliferation and migration of VSMC in vitro. The expression level of miR-23b was reduced in mouse carotid injured tissues and cell line. Bioinformatics analysis and luciferase reporter assay revealed that XR007793 directly bonds to miR-23b. Pearson correlation analysis showed that XR007793a and miR-23b were negatively correlated in carotid samples. Furthermore, bioinformatics analysis and luciferase assay indicated that miR-23b targeted the Forkhead box O 4 (FOXO4) 3′-UTR to inhibit FOXO4 expression. After transfecting miR-23b inhibitor, the expression both of XR007793 and FOXO4 was increased. The effects on expression were reversed after transfected with miR-23b mimics. Rescue experiments results indicated that miR-23b inhibitor reduced the expression of VSMC marker and promoted proliferation and migration of VSMC.ConclusionsThis study shows that XR007793 aggravates the loss of function of VSMCs by negatively regulating miR-23b. It does so by targeting FOXO4, which could serve as a novel therapeutic target in post-angioplasty restenosis.

Xenobiotic pregnane X receptor promotes neointimal formation in balloon-injured rat carotid arteries.

Pregnane X receptor (PXR) is a member of nuclear receptor superfamily and responsible for the detoxification of xenobiotics. Recent studies demonstrated that PXR was also expressed in the vasculature and protected the vessels from endogenous and exogenous insults, thus representing a novel gatekeeper in vascular defense. In this study, we examined the potential function of PXR in the neointimal formation following vascular injury. In the rat carotid artery after balloon injury, overexpression of a constitutively active PXR increased the intima-to-media ratio in the injured region. PXR increased cell proliferation and migration in cultured rat aortic smooth muscle cells (SMCs) by inducing the expressions of cyclins (cyclin A, D1, and E) and cyclin-dependent kinase 2. In addition, PXR increased the phosphorylation and activation of extracellular-signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Inactivation of ERK1/2 and p38 MAPK pathways using selective inhibitors (U0126 and SB203580) abrogated PXR-induced SMC proliferation and migration. Furthermore, cigarette smoke particles (CSP) activated PXR in SMCs. Knockdown of PXR by small interfering RNA suppressed the cell proliferation, migration, and activation of the MAPK pathways by CSP. These findings suggested a novel role for PXR in promoting SMC proliferation and migration, and neointimal hyperplasia. Therefore, PXR may be a potential therapeutic target for vascular disease related to xenobiotics such as cigarette smoking and other environmental pollutants.

Proresolving Lipid Mediators Resolvin D1 and Protectin D1 Isomer Attenuate Neointimal Hyperplasia in the Rat Carotid Artery Balloon Injury Model

BackgroundSpecialized proresolving mediators from ω-3 polyunsaturated fatty acid may control resolution of inflammation. We evaluated the influence of two specialized proresolving mediators, resolvin D1 (RvD1) and protectin D1 isomer (PD1 iso) on neointimal hyperplasia after balloon injury. Materials and methodsSprague Dawley male rats at 12-14 wk of age were injured as a model of balloon angioplasty. Then, 1 μg/rat of RvD1 or PD1 iso was administered intravenously via the tail vein immediately and 2 d after angioplasty. The proliferation of injured artery and the infiltration of leukocytes, monocytes, and macrophages at 3 d after injury were evaluated by immunostaining. The activity of the inflammatory transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in the injured artery at 3 d after injury was evaluated using an enzyme-linked immuno sorbent assay kit. The proliferation of the neointima was evaluated by calculating the ratio of the neointimal and medial areas using specimens at 14 d after injury. ResultsRvD1 and PD1 iso attenuated proliferation of medial cells (P < 0.05) and infiltration of leukocytes (P < 0.05) and monocytes/macrophages (P < 0.01). Although both RvD1 and PD1 iso mitigated NFκB activity (P < 0.01), RvD1 attenuated this activity more strongly (P < 0.01). RvD1 decreased neointimal hyperplasia by 37.3% (P < 0.01), whereas PD1 iso decreased neointimal hyperplasia by 31.8% (P < 0.05) (RvD1 versus PD1 iso: P = 0.51). ConclusionsRvD1 and PD1 iso reduced the activity of inflammatory transcription factor NFκB within the injured artery and attenuated inflammatory cell infiltration, leading to a reduction in early inflammation and subsequent neointimal hyperplasia.