Rat Carotid Artery Research Articles

Establishment of a Modified and Standardized Ferric Chloride-Induced Rat Carotid Artery Thrombosis Model.

Background: Ferric chloride is widely utilized in inducing thrombosis in small vessels of experimental animals. However, the lack of its application in large blood vessels of experimental animals and inconsistent concentration has limited its application. Therefore, we systematically tested the most suitable concentration and reliable induction time in the experiment of using ferric chloride to induce rat carotid artery thrombosis. Methods: In this study, we selected the common carotid artery of 59 Sprague–Dawley rats as the target vessel. The exploration process was divided into three stages. First, to determine the optimum induction concentration, we compared the effects of 30–60% ferric chloride on thrombus formation within 24 h. Second, to confirm the handling time, we tested different induction times from 3 min to 10 min. Lastly, we used the thrombolytic drug rt-PA to detect whether the formed thrombus can be lysed. Doppler blood flow imaging and H–E staining were employed to estimate the blood flow and thrombus. The ATP levels in the brain were measured using a bioluminescence ATP assay kit. Results: We found that the application of 50% ferric chloride for 10 min was enough to successfully induce thrombosis in the rat carotid artery and without spontaneous thrombolysis after 24 h. It is better than other concentrations and will lead to the decline of the ATP content in the ischemic hemisphere. Conclusions: Our results indicate that the rat carotid artery thrombosis model induced by 50% ferric chloride for 10 min is stable and reliable.

Icariside II Attenuates Vascular Remodeling Via Wnt7b/CCND1 Axis.

Angioplasty often fails due to the abnormal proliferation of vascular smooth muscle cells (VSMCs). Success rates of angioplasty may increase following the administration of an agent that effectively ameliorates aberrant vascular remodeling. Icariside II (ICS-II) is a natural flavonol glycoside extract from the Chinese herbal medicine Epimedii that possesses several medicinal qualities that are beneficial in humans. Nevertheless, the role of ICS-II in addressing aberrant vascular remodeling have yet to be clarified. The current investigation studies the molecular effects of ICS-Ⅱ on balloon-inflicted neointimal hyperplasia in rats in vivo and on platelet-derived growth factor-induced vascular proliferation in primary rat aortic smooth muscle cells (VSMCs) in vitro. ICS-II was found to be as effective as rapamycin, the positive control used in this study. ICS-II inhibited neointimal formation in injured rat carotid arteries and notably reduced the expression of Wnt7b. ICS-Ⅱ significantly counteracted platelet-derived growth factor-induced VSMCs proliferation. Cell cycle analysis showed that ICS-II triggered cell cycle arrest during the G1/S transition. Western blot analysis further indicated that this cell cycle arrest was likely through Wnt7b suppression that led to CCND1 inhibition. In conclusion, our findings demonstrate that ICS-II possesses significant antiproliferative qualities that counteracts aberrant vascular neointimal hyperplasia. This phenomenon most likely occurs due to the suppression of the Wnt7b/CCND1 axis.

Selective neurodegeneration of the hippocampus caused by chronic cerebral hypoperfusion: F-18 FDG PET study in rats.

Chronic cerebral hypoperfusion (CCH) is known to induce Alzheimer's disease (AD) pathology, but its mechanism remains unclear. The purpose of this study was to identify the cerebral regions that are affected by CCH, and to evaluate the development of AD pathology in a rat model of CCH. A rat model of CCH was established by bilaterally ligating the common carotid arteries in adult male rats (CCH group). The identical operations were performed on sham rats without arteries ligation (control group). Regional cerebral glucose metabolism was evaluated at 1 and 3 months after bilateral CCA ligation using positron emission tomography with F-18 fluorodeoxyglucose. The expression levels of amyloid β40 (Aβ40), amyloid β42 (Aβ42), and hyperphosphorylated tau were evaluated using western blots at 3 months after the ligation. Cognitive function was evaluated using the Y-maze test at 3 months after the ligation. At 1 month after the ligation, cerebral glucose metabolism in the entorhinal, frontal association, motor, and somatosensory cortices were significantly decreased in the CCH group compared with those in the control group. At 3 months after the ligation, cerebral glucose metabolism was normalized in all regions except for the anterodorsal hippocampus, which was significantly decreased compared with that of the control group. The expression of Aβ42 and the Aβ42/40 ratio were significantly higher in the CCH group than those in the control group. The phosphorylated-tau levels of the hippocampus in the CCH group were significantly lower than those in the control group. Cognitive function was more impaired in the CCH group than that in the control group. Our findings suggest that CCH causes selective neurodegeneration of the anterodorsal hippocampus, which may be a trigger point for the development of AD pathology.

Regulation of bFGF-induced effects on rat aortic smooth muscle cells by β3-adrenergic receptors

BackgroundBasic fibroblast growth factor (bFGF)-mediated vascular smooth muscle cell (VSMC) proliferation and migration play an important role in vascular injury-induced neointima formation and subsequent vascular restenosis, a major event that hinders the long-term success of angioplasty. The function of β3-adrenergic receptors (β3-ARs) in vascular injury-induced neointima formation has not yet been defined. ObjectivesOur current study explored the possible role of β3-ARs in vascular injury-induced neointima formation by testing its effects on bFGF-induced VSMC migration and proliferation. Methodsβ3-AR expression in rat carotid arteries was examined at 14 days following a balloon catheter-induced injury. The effects of β3-AR activation on bFGF-induced rat aortic smooth muscle cell proliferation, migration, and signaling transduction (including extracellular-signal-regulated kinase/mitogen activated protein kinase, ERK/MAPK and Protein kinase B, AKT) were tested. ResultsWe found that vascular injury induced upregulation of β3-ARs in neointima. Pretreatment of VSMCs with a selective β3-AR agonist, CL316,243 significantly potentiated bFGF-induced cell migration and proliferation, and ERK and AKT phosphorylation. Our results also revealed that suppressing phosphorylation of ERK and AKT blocked bFGF-induced cell migration and that inhibiting AKT phosphorylation reduced bFGF-mediated cell proliferation. ConclusionOur results suggest that activation of β3-ARs potentiates bFGF-mediated effects on VSMCs by enhancing bFGF-mediated ERK and AKT phosphorylation and that β3-ARs may play a role in vascular injury-induced neointima formation.

MicroRNA-17-5p, a novel endothelial cell modulator, controls vascular re-endothelialization and neointimal lesion formation.

The functions of miR-17-5p in tumorigenesis have been explored. However, their functionalities in arterial endothelial cells (ECs) have not been investigated. Besides, the issue of vascular remodelling is barely addressed. The study aimed to determine the effect of overexpression or inhibition of miR-17-5p on arterial endothelial cells' (ECs) function and vascular remodelling in vitro and the rat carotid arteries model. Quantitative RT-PCR analysis was performed to examine the expression of miR-17-5p. Then, gain-of-function and loss-of-function approaches were employed to investigate the functional roles of miR-17-5p in cultured human coronary artery endothelial cells (HCAECs); further, TargetScan software analysis and luciferase reporter activity assay were performed to investigate the potential mechanism. Lastly, the results of the cell segment were verified in a rat carotid artery balloon injury model by Western blot analysis, measurement of the vascular cGMP level and plasma 8-iso-prostaglandin F2 (8-iso-PGF2) testing. Moreover, morphometric analysis was implemented to detect the re-endothelialization and neointimal formation in rat carotid artery after balloon injury. This study firstly found that miR-17-5p expression was upregulated in the injured vascular walls and highly expressive in ECs; overexpression of miR-17-5p inhibited HCAECs' proliferation and migration, whereas miR-17-5p knockdown strengthened its proliferative and migratory roles, influenced inflammatory response, through regulating VEGRA and VEGFR2. It was found that miR-17-5p bind to VEGFA and VEGFR2 at the 3'UTR. Next, downregulation of miR-17-5p promotes re-endothelialization, and attenuates neointimal formation as measured by the I/M ratio (0.63±0.05 vs 1.45±0.06, antagomiR-17-5p vs. Lenti-NC, p < 0.05). In addition, the functional recovery of the endothelium was also accelerated by miR-17-5p knockdown. Our study suggests that miR-17-5p is a feasible strategy for the selective modulation of endothelialization and vascular remodelling through regulating VEGFA and VEGFR2.

Portable fluid circuit device containing printed silicone microvessels as a training aid for arterial microanastomosis

Anastomosis of the microvessels requires high-level skills and extensive basic training. This study was performed to introduce and evaluate an inexpensive laboratory device as a training aid. Micro-tubes of 0.8 mm inner diameter and 0.5/0.8 mm wall thickness mimicking human vein/artery were printed from a silicon-containing hydrogel using three-dimensional printing technology. The hydrogel components are optimized to render the printed tubes biomechanical features resembling the blood vessels of a living organism. These artificial vessels were connected to a pump for fluid flow, simulating the blood circulation. Forty medical interns were assigned to two equal groups. The 20 interns in group A practiced anastomosis using the training aid for a total of 10 hours over 5 days. The 20 interns in group B practiced anastomosis using the traditional gum pieces and silicone tubes. Then, all interns performed anastomosis on rat carotid arteries, and their performance was scored by a team of five experienced maxillofacial surgeons. The average success score and time required for anastomosis were compared between the two groups. The mean success score of group A was significantly higher than that of group B (0.83 ± 0.12 vs 0.64 ± 0.10, P < 0.001). The mean anastomosis time of group A was significantly shorter than that of group B (10.2 ± 1.1 vs 17.2 ± 1.4 minutes, P < 0.001). This training device for vessel microanastomosis is an inexpensive, practical, and effective tool for use in laboratories and also reduces the use of animals.

Single-Cell RNA Sequencing of the Rat Carotid Arteries Uncovers Potential Cellular Targets of Neointimal Hyperplasia.

Aims: In-stent restenosis (ISR) remains an Achilles heel of drug-eluting stents despite technical advances in devices and procedural techniques. Neointimal hyperplasia (NIH) is the most important pathophysiological process of ISR. The present study mapped normal arteries and stenotic arteries to uncover potential cellular targets of neointimal hyperplasia.Methods and Results: By comparing the left (control) and right (balloon injury) carotid arteries of rats, we mapped 11 clusters in normal arteries and 11 mutual clusters in both the control and experimental groups. Different clusters were categorized into 6 cell types, including vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), macrophages, unknown cells and others. An abnormal cell type expressing both VSMC and fibroblast markers at the same time was termed a transitional cell via pseudotime analysis. Due to the high proportion of VSMCs, we divided them into 6 clusters and analyzed their relationship with VSMC phenotype switching. Moreover, N-myristoyltransferase 1 (NMT1) was verified as a credible VSMC synthetic phenotype marker. Finally, we proposed several novel target genes by disease susceptibility gene analysis, such as Cyp7a1 and Cdk4, which should be validated in future studies.Conclusion: Maps of the heterogeneous cellular landscape in the carotid artery were defined by single-cell RNA sequencing and revealed several cell types with their internal relations in the ISR model. This study highlights the crucial role of VSMC phenotype switching in the progression of neointimal hyperplasia and provides clues regarding the underlying mechanism of NIH.

Lipid-related protein NECTIN2 is an important marker in the progression of carotid atherosclerosis: An intersection of clinical and basic studies.

ABSTRACTBackground:The nectin cell adhesion molecule 2 (NECTIN2) protein is a cell adhesion molecule involved in lipid metabolism. We aimed to explore the potential role of NECTIN2 in carotid atherosclerosis (CA).Materials and Methods:Patients who underwent carotid endarterectomy (CEA) at the First Affiliated Hospital of Zhengzhou University were enrolled in this study. APOE-/- rats fed western or normal diet were used to model early pathological changes in CA. The relationship between patients’ lipid indices and plaque severity was assessed using ordinal regression analysis. Mendelian randomisation (MR) analysis was used to determine the causal links between low-density lipoprotein cholesterol (LDL-C) and atherosclerosis. After matching analysis of the single-cell transcriptome and microarray data of carotid plaques, NECTIN2 was identified as a key factor affecting CA. The importance of NECTIN2 was further verified by immunofluorescence staining of CEA and APOE-/- rat specimens.Results:A total of 108 patients were included. The traditional lipid indices did not correlate significantly with the plaque severity (P > 0.05). NECTIN2 provided a strong causal link between LDL-C level and CA (MR effect size >0). Deep-sequencing data illustrated that NECTIN2 expression was cell specific. In early-stage CA, NECTIN2 expression was increased in endothelial cells; however, in advanced-stage CA, NECTIN2 was overexpressed in macrophages located in fibrous caps. APOE-/- rat carotid artery and human carotid plaques modelled the entire atherosclerotic process, showing an upregulation of NECTIN2 expression in CA.Conclusions:Lipid-related protein NECTIN2 is a potential marker in CA progression and can potentially be a new therapeutic target for clinical prevention.

Sophocarpine Alleviates Injury-Induced Intima Hyperplasia of Carotid Arteries by Suppressing Inflammation in a Rat Model.

Introduction: Balloon angioplasty is a commonly applied procedure for treating atherosclerotic vascular diseases. However, the maintenance of long-term lumen patency is relatively difficult due to the occurrence of restenosis. Previous research has shown that the occurrence of vascular wall inflammation is associated with higher rates of restenosis. Sophocarpine (SPC) can exert various therapeutic effects such as anti-oxidation, anti-inflammation, anti-tumor, antivirus and immune regulation. This study aimed to investigate whether SPC can alleviate intimal hyperplasia following balloon injury in a rat carotid artery model. Methods: Twenty Sprague–Dawley rats were randomly assigned to four groups: (i) control, (ii) balloon injury, (iii) balloon injury followed by saline injection, and (iv) balloon injury followed by SPC administration. Each group contained five rats. A high-pressure balloon of 3 mm × 20 mm was placed in the carotid artery. The balloon was inflated to a pressure of 8 atmospheres to carry out rat carotid artery balloon injury model. The areas of neointimal and media were determined by Verhoeff_Van Gieson staining, and the intima-to-media (I:M) ratios were subsequently evaluated. After that, the protein levels of IL-6, IL-1β, MCP-1, NF-κB, TNF-α, VCAM-1, ICAM-1 and eNOS were measured. Results: The ratio of I:M was remarkably higher in the balloon injury group than in the control group (p < 0.01). SPC could significantly decrease the ratio of I:M compared with the balloon injury group (p < 0.01). Besides, the protein levels of IL-6, IL-1β, MCP-1, NF-κB, TNF-α, ICAM-1 and VCAM-1 were increased in rat carotid arteries exposed to balloon injury (p < 0.01), and treatment with SPC could attenuate these effects (p < 0.05). Furthermore, balloon injury inhibited the protein expression of eNOS (p < 0.01), and SPC could elevate its level (p < 0.05). Conclusions: SPC could alleviate an intimal hyperplasia in balloon-injured carotid artery, and the mechanisms underlying this protective effect might be due to its inhibitory potency against inflammation signals. Our study also implies the potential applicability of SPC in treating restenosis after balloon angioplasty.

Essential role of Orai1 and SARAF in vascular remodeling

Orai1 and STIM1, molecular components of store-operated calcium entry (SOCE), have been associated with vascular smooth muscle cell (VSMC) proliferation in vascular remodeling. Nevertheless, the role of SARAF (SOCE-associated regulatory factor), a regulatory protein involved in STIM1 inhibition, in vascular remodeling has not been examined. The aim of this study is to examine the role of SARAF and Orai1 in VSMC proliferation and neointima formation after balloon injury of rat carotid arteries. Experiments were conducted in an animal model of rat carotid angioplasty to characterize neointima formation. VSMC isolated from rat coronary arteries was also used to examine cell proliferation. The formation of neointima after balloon injury of rat carotid arteries was confirmed by hematoxylin and eosin staining of tissue sections up to 3 wk after surgery. Injured arteries showed significantly higher expression of SARAF, STIM1, and Orai1 compared with control tissues, corroborating the presence of these regulatory proteins in the neointima layer. Proximity ligation and coimmunoprecipitation assays revealed that SARAF interacts with Orai1 in the neointima. Furthermore, selective silencing of SARAF and Orai1 by small interfering RNA (siRNA) inhibited IGF-1-induced VSMC proliferation. Our data suggest that SARAF interacts with Orai1 to modulate SOCE and VSMC proliferation after vascular injury.

CARMN Is an Evolutionarily Conserved Smooth Muscle Cell-Specific LncRNA That Maintains Contractile Phenotype by Binding Myocardin.

Vascular homeostasis is maintained by the differentiated phenotype of vascular smooth muscle cells (VSMCs). The landscape of protein coding genes comprising the transcriptome of differentiated VSMCs has been intensively investigated but many gaps remain including the emerging roles of noncoding genes. We reanalyzed large-scale, publicly available bulk and single-cell RNA sequencing datasets from multiple tissues and cell types to identify VSMC-enriched long noncoding RNAs. The in vivo expression pattern of a novel smooth muscle cell (SMC)-expressed long noncoding RNA, Carmn (cardiac mesoderm enhancer-associated noncoding RNA), was investigated using a novel Carmn green fluorescent protein knock-in reporter mouse model. Bioinformatics and quantitative real-time polymerase chain reaction analysis were used to assess CARMN expression changes during VSMC phenotypic modulation in human and murine vascular disease models. In vitro, functional assays were performed by knocking down CARMN with antisense oligonucleotides and overexpressing Carmn by adenovirus in human coronary artery SMCs. Carotid artery injury was performed in SMC-specific Carmn knockout mice to assess neointima formation and the therapeutic potential of reversing CARMN loss was tested in a rat carotid artery balloon injury model. The molecular mechanisms underlying CARMN function were investigated using RNA pull-down, RNA immunoprecipitation, and luciferase reporter assays. We identified CARMN, which was initially annotated as the host gene of the MIR143/145 cluster and recently reported to play a role in cardiac differentiation, as a highly abundant and conserved, SMC-specific long noncoding RNA. Analysis of the Carmn GFP knock-in mouse model confirmed that Carmn is transiently expressed in embryonic cardiomyocytes and thereafter becomes restricted to SMCs. We also found that Carmn is transcribed independently of Mir143/145. CARMN expression is dramatically decreased by vascular disease in humans and murine models and regulates the contractile phenotype of VSMCs in vitro. In vivo, SMC-specific deletion of Carmn significantly exacerbated, whereas overexpression of Carmn markedly attenuated, injury-induced neointima formation in mouse and rat, respectively. Mechanistically, we found that Carmn physically binds to the key transcriptional cofactor myocardin, facilitating its activity and thereby maintaining the contractile phenotype of VSMCs. CARMN is an evolutionarily conserved SMC-specific long noncoding RNA with a previously unappreciated role in maintaining the contractile phenotype of VSMCs and is the first noncoding RNA discovered to interact with myocardin.

CircSOD2: A Novel Regulator for Smooth Muscle Proliferation and Neointima Formation.

Vascular smooth muscle cell (SMC) proliferation contributes to neointima formation following vascular injury. Circular RNA-a novel type of noncoding RNA with closed-loop structure-exhibits cell- and tissue-specific expression patterns. However, the role of circular RNA in SMC proliferation and neointima formation is largely unknown. The objective of this study is to investigate the role and mechanism of circSOD2 in SMC proliferation and neointima formation. Approach and Results: Circular RNA profiling of human aortic SMCs revealed that PDGF (platelet-derived growth factor)-BB up- and downregulated numerous circular RNAs. Among them, circSOD2, derived from back-splicing event of SOD2 (superoxide dismutase 2), was significantly enriched. Knockdown of circSOD2 by short hairpin RNA blocked PDGF-BB-induced SMC proliferation. Inversely, circSOD2 ectopic expression promoted SMC proliferation. Mechanistically, circSOD2 acted as a sponge for miR-206, leading to upregulation of NOTCH3 (notch receptor 3) and NOTCH3 signaling, which regulates cyclin D1 and CDK (cyclin-dependent kinase) 4/6. In vivo studies showed that circSOD2 was induced in neointima SMCs in balloon-injured rat carotid arteries. Importantly, knockdown of circSOD2 attenuated injury-induced neointima formation along with decreased neointimal SMC proliferation. CircSOD2 is a novel regulator mediating SMC proliferation and neointima formation following vascular injury. Therefore, circSOD2 could be a potential therapeutic target for inhibiting the development of proliferative vascular diseases.

Nootropic effect of herbal extract against impairment of spatial memory in rats subjected to cronic cerebral ischemia

Chronic cerebrovascular disease is progressive pathology with gradual development of cognitive disorders. In the treatment of cognitive disturbances, drugs with comprehensive antioxidant, angioprotective, neuroprotective and neurotrophic action are usually used. The present study is aimed at evaluating the effect of the multicomponent herbal extract on cognition disturbance after unilateral occlusion of common carotid artery in rats.

Thrombolytic Effect of Nattokinase and Its Mechanism

Objective: Exploring the anti-thrombotic effect and mechanism of nattokinase in vitro and vivo. Methods: The thrombolytic effect of NK on old thrombus was studied by blood clot lysis method in vitro, the rat carotid artery thrombosis model induced by ferric chloride and the rat venous thrombosis model induced by inferior vena cava ligation method were used to study the thrombolytic effect of NK in vivo. TXB2, 6-k, PGE2, t-PA, \begin{document}$\cdot {\rm{O}}_2^- $\end{document} , ·OH, XOD and other indicators were detected to explore the antithrombotic mechanism of NK. Results: Nattokinase could significantly dissolve old thrombus (P \begin{document}$\cdot {\rm{O}}_2^- $\end{document} , ·OH and XOD. The values of TXB2, PEG2, \begin{document}$\cdot {\rm{O}}_2^- $\end{document} , ·OH and XOD were increased significantly, while the values of 6-K-PGF1 α and t-PA were decreased significantly. Conclusion : NK had strong thrombolytic activity in vivo and vitro, and had effect on fresh thrombus and old thrombus. NK could inhibit thrombosis by inhibiting platelet aggregation, reduce oxidative damage and reducing the level of coagulation factors. NK could also play a direct thrombolytic role by stimulating the production of t-PA, dissolving fibrin, and might play an indirect thrombolytic role by inhibiting the production of PAI-1.

Hemodynamic characteristics of the right ventricle following gradient pulmonary artery banding in rats

Right ventricle/ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary disorders. Reliable and reproducible animal models of RV failure are crucial for investigating disease mechanisms and the effects of potential therapeutic strategies. In the present study, in order to establish a rat model of acute RV failure, the hemodynamic characteristics of the RV and the carotid artery in rats with varying degrees of pulmonary artery banding (PAB) were analyzed. For this purpose, rats were divided into 6 groups as follows: The control (0%), PAB (1‑30%), PAB (31‑60%), PAB (61‑70%), PAB (71‑80%), or PAB (100%). RV pressure (RVP) was measured using right heart catheterization when the pulmonary artery was ligated. The results revealed that the RVP gradually increased with the increasing degrees of banding; however, when the occlusion level exceeded 70%, a high‑pressure state was only maintained for a few minutes or seconds, and the RVP then rapidly declined to below normal pressure levels, which was particularly evident in the PAB (100%) group. On the whole, the present study demonstrates that RVP responds differently to changes in the occlusion level, and that &gt;70% ligation is a successful model of acute right heart failure. These results may have important implications for therapeutic strategies to prevent acute right heart failure.

Energy metabolism and redox status of the glutathione system in experimental brain ischemia and its correction by metabolic neuroprotectors

The changes in the parameters of oxidative stress, energy metabolism, and redox potential of the glutathione system in the rat brain following cerebral ischemia were studied. To correct metabolic disorders, the pantothenic acid derivatives were used in combination with precursors of glutathione biosynthesis and selenium substances.Cerebral ischemia was modeled by ligating the both common carotid arteries in rats for 2 h. Drugs were administered i.p. in the following doses: panthenol – 400 mg/kg, N-acetylcysteine – 150, nanoselen – 1 mg/kg, three times: 1 h before ligation of the carotid arteries, at the time of ligation and 1 hour after ligation. We showed that the development of oxidative stress caused by ischemia is accompanied by the changes in the parameters of energy metabolism and the pentose phosphate pathway in the cerebral hemispheres. Simultaneously, there are a decrease in the GSH level, an increase in the GSSG content, a decrease in the GSH/GSSG ratio, and the activation of enzymes of redox transformations of glutathione.The redox potential of the glutathione system decreases and shifts towards oxidation, while the level of S-glutathionylated proteins increases. Thus, the value of the GSH/GSSG ratio and the protein glutathionylation intensity are the sensitive indicators of the redox potential in the brain tissue and can be used as markers of the extent of changes in the redox balance. The panthenol injection to animals leads to a decrease in the content of free radical oxidation products, violations of oxidative phosphorylation and restoration of thiol-disulfide balance in the brain. When panthenol is administered together with N-acetylcysteine and nanoselen, the corrective effect of panthenol is enhanced.

Engineering a “three-in-one” hirudin prodrug to reduce bleeding risk: A proof-of-concept study

An ideal anticoagulant should have at least three properties including targeted delivery to the thrombosis site, local activation or releasing to centralize the anti-thrombosis effects and thus reduce the bleeding risks, and long persistence in circulation to avoid repeated administration. In the present study, we sought to test a “three-in-one” strategy to design new protein anticoagulants. Based on these criteria, we constructed two hirudin prodrugs, R824-HV-ABD and ABD-HV-R824. The R824 peptide can bind phosphatidylserine on the surface of the procoagulant platelets and thus guide the prodrug to the thrombosis sites; albumin-binding domain (ABDs) can bind the prodrug to albumin, and thereby increase its persistence in circulation; the hirudin (HV) core in the prodrug is flanked by factor Xa recognition sites, thus factor Xa at the thrombosis site can cleave the fusion proteins and release the activated hirudin locally. Hirudin prodrugs were able to bind with procoagulant platelets and human serum albumin in vitro with high affinity, targeted concentrated and prevented the formation of occlusive thrombi in rat carotid artery injury model. Their effective time was significantly extended compared to native hirudin, and R824-HV-ABD showed a significantly improved half-life of about 24 h in rats. The bleeding time of prodrug-treated mice was much shorter than that of hirudin-treated mice. The results from the proof-of-concept studies, for the first time, demonstrate that “three-in-one” prodrug strategy may be a good solution for protein or peptide anticoagulants to reduce their bleeding risks.

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) accelerates vascular remodelling via p53 and JAK2-STAT3 regulation in vascular smooth muscle cells.

Abnormal vascular smooth muscle cell (VSMC) proliferation and migration lead to neointima formation, which eventually results in cardiovascular hyperplastic diseases. The molecular mechanisms underlying these cellular processes have not been fully understood. Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) has been identified as an anti-apoptotic molecule, but little is known about its target genes and related pathways in VSMC dysfunction or its clinical implication in neointima formation following vascular injury. Determination, using loss/gain-of-function approaches by gene delivery, of whether CIAPIN1 modulates VSMC proliferation, migration and neointima formation and the underlying mechanisms was carried out. Balloon injury or ligation and local delivery of lentivirus were performed on rat or mouse carotid arteries. Rat aortic smooth muscle cells, the primary cell, was used as the model to evaluate the effect of CIAPIN1 on proliferation and migration. CIAPIN1 was overexpressed in the neointimal region of rat arteries. CIAPIN1 deficiency markedly inhibited injury-induced or ligation-induced intimal hyperplasia and suppressed PDGF-BB-induced VSMC proliferation, migration and cell cycle progression, while overexpression promoted proliferation, migration and neointima formation. CIAPIN1 negatively regulated Tp53 transcription, which promoted cell cycle progression and migration via cyclin E1-CDK2/pRb/PCNA and the MMP2 pathway. CIAPIN1 also increased JAK2 expression, enhancing JAK2 and STAT3 phosphorylation by vascular injury, which forced phenotypic switching from contractile to synthetic state in injured arteries. These findings provide new insights into the mechanism by which CIAPIN1 regulates vascular remodelling and suggest a novel therapeutic target for treating vascular proliferative diseases.

Pathological Role of Phosphoglycerate Kinase 1 in Balloon Angioplasty-Induced Neointima Formation

Restenosis is a common vascular complication after balloon angioplasty. Catheter balloon inflation-induced transient ischemia (hypoxia) of local arterial tissues plays a pathological role in neointima formation. Phosphoglycerate kinase 1 (PGK1), an adenosine triphosphate (ATP)-generating glycolytic enzyme, has been reported to associate with cell survival and can be triggered under hypoxia. The purposes of this study were to investigate the possible role and regulation of PGK1 in vascular smooth muscle cells (VSMCs) and balloon-injured arteries under hypoxia. Neointimal hyperplasia was induced by a rat carotid artery injury model. The cellular functions and regulatory mechanisms of PGK1 in VSMCs were investigated using small interfering RNAs (siRNAs), chemical inhibitors, or anaerobic cultivation. Our data indicated that protein expression of PGK1 can be rapidly induced at a very early stage after balloon angioplasty, and the silencing PGK1-induced low cellular energy circumstance resulted in the suppressions of VSMC proliferation and migration. Moreover, the experimental results demonstrated that blockage of PDGF receptor-β (PDGFRB) or its downstream pathway, the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) axis, effectively reduced hypoxia-induced factor-1 (HIF-1α) and PGK1 expressions in VSMCs. In vivo study evidenced that PGK1 knockdown significantly reduced neointima hyperplasia. PGK1 was expressed at the early stage of neointimal formation, and suppressing PGK1 has a potential beneficial effect for preventing restenosis.

Investigation into the role of Stmn2 in vascular smooth muscle phenotype transformation during vascular injury via RNA sequencing and experimental validation.

This study examined the effects of Stmn2 on phenotype transformation of vascular smooth muscle in vascular injury via RNA sequencing and experimental validation. Total RNA was extracted for RNA sequencing after 1, 3 and 5 days of injury to screen the differentially expressed genes (DEGs). Western blot was used to detect the protein expression of Stmn2 and its associated targets. The morphological changes of carotid arteries in rats were examined by hematoxylin and eosin (H&E) staining. The expression of vascular smooth muscle cell (VSMC) phenotype markers smooth muscle alpha-actin (α-SMA), vimentin and OPN were detected by immunohistochemistry. DEGs were related to the extracellular matrix and other cell components outside the plasma membrane. They were associated with protein binding, cytoskeleton protein binding, signal receptor binding and other molecular functions, actin cytoskeleton regulation and other Kyoto Encyclopedia of Genes and Genomes pathways. Stmn2 was identified as the hub gene of actin cytoskeleton pathway and vascular disease, and its expression followed the trend of decreasing initially and increasing afterwards during the progress of vascular injury. Western blot assay showed that the expression of Stmn2 and Tubulin decreased immediately after vascular injury; Stmn2 overexpression significantly up-regulated the expression of osteopontin and α-SMA and vimentin in VSMCs. The results of morphology analysis and immunostaining also showed that Stmn2 overexpression promoted the intima thickening and enhanced the proliferating cell nuclear antigen expression in the injured vascular tissues. In conclusion, our results implied that Stmn2 may play a potential role in vascular injury, which may be associated with VSMC phenotype transformation. Further studies are warranted to determine detailed molecular mechanisms of Stmn2 in vascular injury.