Topographic distribution of inflammation factors in a healing aneurysm.

BACKGROUND: Healing of intracranial aneurysms following endovascular treatment relies on the organization of early thrombus into mature scar tissue and neointima formation. Activation and deactivation of the inflammation cascade plays an important role in this process. In addition to timely evolution, its topographic distribution is hypothesized to be crucial for successful aneurysm healing. METHODS: Decellularized saccular sidewall aneurysms were created in Lewis rats and coiled. At follow-up (after 3 days (n = 16); 7 days (n = 19); 21 days (n = 8)), aneurysms were harvested and assessed for healing status. In situ hybridization was performed for soluble inflammatory markers (IL6, MMP2, MMP9, TNF-α, FGF23, VEGF), and immunohistochemical analysis to visualize inflammatory cells (CD45, CD3, CD20, CD31, CD163, HLA-DR). These markers were specifically documented for five regions of interest: aneurysm neck, dome, neointima, thrombus, and adjacent vessel wall. RESULTS: Coiled aneurysms showed enhanced patterns of thrombus organization and neointima formation, whereas those without treatment demonstrated heterogeneous patterns of thrombosis, thrombus recanalization, and aneurysm growth (p = 0.02). In coiled aneurysms, inflammation markers tended to accumulate inside the thrombus and in the neointima (p < 0.001). Endothelial cells accumulated directly in the neointima (p < 0.0001), and their presence was associated with complete aneurysm healing. CONCLUSION: The presence of proinflammatory cells plays a crucial role in aneurysm remodeling after coiling. Whereas thrombus organization is hallmarked by a pronounced intra-thrombotic inflammatory reaction, neointima maturation is characterized by direct invasion of endothelial cells. Knowledge concerning topographic distribution of regenerative inflammatory processes may pave the way for future treatment modalities which enhance aneurysm healing after endovascular therapy.

Zoledronate alters natural progression of tissue-engineered vascular grafts.

Macrophages are a critical driver of neovessel formation in tissue-engineered vascular grafts (TEVGs), but also contribute to graft stenosis, a leading clinical trial complication. Macrophage depletion via liposomal delivery of clodronate, a first-generation bisphosphonate, mitigates stenosis, but simultaneously leads to a complete lack of tissue development in TEVGs. This result and the associated difficulty of utilizing liposomal delivery means that clodronate may not be an ideal means of preventing graft stenosis. Newer generation bisphosphonates, such as zoledronate, may have differential effects on graft development with more facile drug delivery. We sought to examine the effect of zoledronate on TEVG neotissue formation and its potential application for mitigating TEVG stenosis. Thus, mice implanted with TEVGs received zoledronate or no treatment and were monitored by serial ultrasound for graft dilation and stenosis. After two weeks, TEVGs were explanted for histological examination. The overall graft area and remaining graft material (polyglycolic-acid) were higher in the zoledronate treatment group. These effects were associated with a corresponding decrease in macrophage infiltration. In addition, zoledronate affected the deposition of collagen in TEVGs, specifically, total and mature collagen. These differences may be, in part, explained by a depletion of leukocytes within the bone marrow that subsequently led to a decrease in the number of tissue-infiltrating macrophages. TEVGs from zoledronate-treated mice demonstrated a significantly greater degree of smooth muscle cell presence. There was no statistical difference in graft patency between treatment and control groups. While zoledronate led to a decrease in the number of macrophages in the TEVGs, the severity of stenosis appears to have increased significantly. Zoledronate treatment demonstrates that the process of smooth muscle cell-mediated neointimal hyperplasia may occur separately from a macrophage-mediated mechanism.

Patterns of Neointima Formation After Coil or Stent Treatment in a Rat Saccular Sidewall Aneurysm Model.

BACKGROUND AND PURPOSE: Endovascular aneurysm treatment relies on a biological process, including cell migration for thrombus organization and growth of a neointima. To better understand aneurysm healing, our study explores the origin of neointima-forming and thrombus-organizing cells in a rat saccular sidewall aneurysm model. METHODS: Saccular aneurysms were transplanted onto the abdominal aorta of male Lewis rats and endovascularly treated with coils (n=28) or stents (n=26). In 34 cases, GFP+ (green fluorescent protein)-expressing vital aneurysms were sutured on wild-type rats, and in 23 cases, decellularized wild-type aneurysms were sutured on GFP+ rats. Follow-up at 3, 7, 14, 21, and 28 days evaluated aneurysms by fluorescence angiography, macroscopic inspection, and microscopy for healing and inflammation status. Furthermore, the origin of cells was tracked with fluorescence histology. RESULTS: In animals with successful functional healing, histological studies showed a gradually advancing thrombus organization over time characterized by progressively growing neointima from the periphery of the aneurysm toward the center. Cell counts revealed similar distributions of GFP+ cells for coil or stent treatment in the aneurysm wall (54.4% versus 48.7%) and inside the thrombus (20.5% versus 20.2%) but significantly more GFP+ cells in the neointima of coiled (27.2 %) than stented aneurysms (10.4%; P=0.008). CONCLUSIONS: Neointima formation and thrombus organization are concurrent processes during aneurysm healing. Thrombus-organizing cells originate predominantly in the parent artery. Neointima formation relies more on cell migration from the aneurysm wall in coiled aneurysms but receives greater contributions from cells originating in the parent artery in stent-treated aneurysms. Cell migration, which allows for a continuous endothelial lining along the parent artery's lumen, may be a prerequisite for complete aneurysm healing after endovascular therapy. In terms of translation into clinical practice, these findings may explain the variability in achieving complete aneurysm healing after coil treatment and the improved healing rate in stent-assisted coiling.

Immunomodulation of IL-33 and IL-37 with Vitamin D in the Neointima of Coronary Artery: A Comparative Study between Balloon Angioplasty and Stent in Hyperlipidemic Microswine.

Inflammation is a major contributor to the development and progression of atherosclerosis. Interleukin (IL)-33 and IL-37, members of the IL-1 family, modulate inflammation, with IL-33 having a pro-inflammatory effect and IL-37 having anti-inflammatory properties. IL-37 is constitutively expressed at low levels but upregulated in inflammatory contexts. The aim of this study was to evaluate the effect of vitamin D on the expression of IL-33, IL-37, macrophages, and caspase-1 in the neointimal tissue of coronary artery in Yucatan microswine with vitamin D deficient, sufficient, and supplemented status. The intimal injury was induced by balloon angioplasty and stenting in the coronary artery, and tissues were harvested after 6 months. The expression of various proteins of interest was evaluated by immunostaining. Increased expression of IL-33 and IL-37 in the neointimal tissue of the vitamin D deficient, as compared to the sufficient and supplemented microswine, as revealed by histological evaluation and semi-quantitative analysis, suggested the immunomodulatory effect of vitamin D on the expression of IL-33 and IL-37. The minimal expression or absence of IL-33 and IL-37 expression in stented arteries is suggestive of an attenuated inflammatory response in stented arteries, compared to balloon angioplasty. The decreased IL-33 expression in the sufficient and supplemented microswine could be a potential mechanism for controlling the inflammatory process and neointima formation leading to attenuated luminal narrowing of the coronary artery. Overall, these results support supplementation of vitamin D to attenuate inflammation, neointima formation, and restenosis.

The Role of MicroRNA-21 in Venous Neointimal Hyperplasia: Implications for Targeting miR-21 for VNH Treatment.

The molecular mechanism of hemodialysis access arteriovenous fistula (AVF) failure due to venous neointimal hyperplasia (VNH) is not known. The role of microRNA-21 (miR-21) in VNH associated with AVF failure was investigated by performing in vivo and in vitro experiments. In situ hybridization results revealed that miR-21 expression increased and was associated with fibroblasts in failed AVFs from patients. In a murine AVF model, qRT-PCR gene expression results showed a significant increase in miR-21 and a decrease in miR-21 target genes in graft veins (GVs) compared to contralateral veins in mouse AVF. miR-21 knockdown in GVs was performed using a lentivirus-mediated small hairpin RNA (shRNA), and this improved AVF patency with a decrease in neointima compared to control GVs. Moreover, loss of miR-21 in GVs significantly decreased the Tgfß1, Col-Ia, and Col-Iva genes. Immunohistochemistry demonstrated a significant decrease in myofibroblasts and proliferation with an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in miR-21-knockdown vessels, along with a decrease in hypoxia-inducible factor-1 alpha (HIF-1α) and phospho-SMAD2 (pSMAD-2) and phospho-SMAD3 (pSMAD-3) and an increase in phosphatase and tensin homolog (PTEN) staining. Hypoxic fibroblast knockdown for miR-21 showed a significant decrease in Tgfß-1 expression and pSMAD-2 and -3 levels and a decrease in myofibroblasts. These results indicate that miR-21 upregulation causes VNH formation by fibroblast-to-myofibroblast differentiation.

Exosomes from mesenchymal stem cells expressing miR-125b inhibit neointimal hyperplasia via myosin IE.

Intercellular communication between mesenchymal stem cells (MSCs) and their target cells in the perivascular environment is modulated by exosomes derived from MSCs. However, the potential role of exosome-mediated microRNA transfer in neointimal hyperplasia remains to be investigated. To evaluate the effects of MSC-derived exosomes (MSC-Exo) on neointimal hyperplasia, their effects upon vascular smooth muscle cell (VSMC) growth in vitro and neointimal hyperplasia in vivo were assessed in a model of balloon-induced vascular injury. Our results showed that MSC-Exo were internalised by VSMCs and inhibited proliferation and migration in vitro. Further analysis revealed that miR-125b was enriched in MSC-Exo, and repressed the expression of myosin 1E (Myo1e) by targeting its 3' untranslated region. Additionally, MSC-Exo and exosomally transferred miR-125b repressed Myo1e expression and suppressed VSMC proliferation and migration and neointimal hyperplasia in vivo. In summary, our findings revealed that MSC-Exo can transfer miR-125b to VSMCs and inhibit VSMC proliferation and migration in vitro and neointimal hyperplasia in vivo by repressing Myo1e, indicating that miR-125b may be a therapeutic target in the treatment of vascular diseases.

Vascular endothelial growth factor-modified macrophages accelerate reendothelialization and attenuate neointima formation after arterial injury in atherosclerosis-prone mice.

Vascular endothelial growth factor (VEGF) is a promising molecule for cardiovascular diseases therapy. But lack of a targeted delivery system limits its translation into clinical application. This study aimed to develop stably overexpressing VEGF macrophages for targeted VEGF delivery to injured arteries and determine their potential for repairing of the damaged endothelium. Wire-induced carotid artery injury model was established in atherosclerosis-prone mice. It was observed that the VEGF-modified macrophages were recruited to the site of vascular injury and incorporated into new endothelium formation. VEGF-modified macrophages therapy accelerated reendothelialization and attenuated neointima formation. The VEGF protein level in tissues of injured arteries treated with VEGF-modified macrophages was increased. The upregulated C-C chemokine receptor type 5 (CCR5) and unaltered CCR2 protein levels were verified in VEGF-modified macrophages in vitro. Moreover, enhanced nitric oxide (NO) production in the culture medium of VEGF-modified macrophages was demonstrated. Our results indicated that VEGF-modified macrophages acted as vectors of VEGF targeting injured arteries, promoting the repairing directly by incorporating into new endothelium formation and indirectly by secreting sustainable VEGF and producing NO locally. This study represents a novel therapeutic application of targeted cell therapy with VEGF-modified macrophages for cardiovascular diseases.

MicroRNA-146a sponge therapy suppresses neointimal formation in rat vein grafts.

The long-term failure of vein grafts due to neointimal hyperplasia remains a difficult problem in cardiovascular surgery. Exploring novel approaches to prevent neointimal hyperplasia is important. MicroRNA-146a (miR-146a) plays an essential role in promoting vascular smooth muscle cell (VSMC) proliferation. Thus, the aim of the present study is to investigate whether adenovirus-mediated miR-146a sponge (Ad-miR-146a-SP) gene therapy could attenuate neointimal formation in rat vein grafts. (Ad-miR-146a-SP) was constructed to transfect cultured VSMCs and grafted veins. To improve the efficiency of transferring the miR-146a sponge gene into the grafted veins, 20% poloxamer F-127 gel incorporated with 0.25% trypsin was used to increase adenovirus contact time and penetration. miR-146a-SP transduction significantly reduced the expression of miR-146a both in cultured VSMCs and vein grafts. miR-146a sponge markedly attenuated VSMC proliferation and migration. Consistent with this, miR-146a sponge gene therapy significantly attenuated neointimal formation and also improved blood flow in the vein grafts. Mechanistically, we identified the Krüppel-like factor 4(KLF4) as a potential downstream target gene of miR-146a in vein grafts. Our data show that miR-146a sponge gene therapy could effectively reduce miR-146a activity and attenuate neointimal formation in vein grafts, suggesting its potential therapeutic application for prevention of vein graft failure. © 2018 IUBMB Life, 71(1):125-133, 2019.

Neointimal Hyperplasia.

Neointimal hyperplasia is a physiologic healing response to injury to the blood vessel wall, involving all the three arterial layers and it occurs in the presence of internal (endovascular) or external (surgical) injury. It is a highly complex process involving several tissues (perivascular, vessel wall, and blood) and numerous cell lineages with multiple molecular signaling networks. So, there is a number of possible targets for inhibition of this process. There are known risk factors for Intimal Hyperplasia, such as diabetes, female gender, presence of systemic inflammation, type of arteries treated, types of surgical and endovascular materials, presence of turbulent flow and genetic status. The present paper discusses the pathophysiology of neointimal hyperplasia and the strategies to prevention and treatment of it.

Local adventitial anti-angiogenic gene therapy reduces growth of vasa-vasorum and in-stent restenosis in WHHL rabbits.

BACKGROUND: Antiproliferative drugs in drug eluting stents (DES) are associated with complications due to impaired re-endothelialization. Additionally, adventitial neovascularization has been suggested to contribute to in-stent restenosis (ISR). Since Vascular Endothelial Growth Factors (VEGFs) are the key mediators of angiogenesis, we investigated feasibility and efficacy of local gene therapy for ISR utilizing soluble decoy VEGF receptors to reduce biological activity of adventitial VEGFs. METHOD: Sixty-nine adult WHHL rabbit aortas were subjected to endothelial denudation. Six weeks later catheter-mediated local intramural infusion of 1.5x10e10 pfu adenoviruses encoding soluble VEGF Receptor-1 (sVEGFR1), sVEGFR2, sVEGFR3 or control LacZ and bare metal stent implantation were performed in the same aortic segment. Marker protein expression was assessed at 6d in LacZ cohort. Immunohistochemistry, morphometrical analyses and angiography were performed at d14, d42 and d90. RESULTS: Transgene expression was localized to adventitia. All decoy receptors reduced the size of vasa-vasorum at 14d, AdsVEGFR2 animals also had reduced density of adventitial vasa-vasorum, whereas AdsVEGFR3 increased the density of vasa-vasorum. At d42, AdsVEGFR1 and AdsVEGFR2 reduced ISR (15.7 ±â€¯6.9% stenosis, P < 0.01 and 16.5 ±â€¯2.7%, P < 0.05, respectively) vs. controls (28.3 ±â€¯7.6%). Moreover, AdsVEGFR-3 treatment led to a non-significant trend in the reduction of adventitial lymphatics at all time points and these animals had significantly more advanced neointimal atherosclerosis at 14d and 42d vs. control animals. CONCLUSIONS: Targeting adventitial neovascularization using sVEGFR1 and sVEGFR2 is a novel strategy to reduce ISR. The therapeutic effects dissipate at late follow up following short expression profile of adenoviral vectors. However, inhibition of VEGFR3 signaling accelerates neoatherosclerosis.

Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation.

Inhibition of vascular smooth muscle cell (VSMC) proliferation by drug eluting stents has markedly reduced intimal hyperplasia and subsequent in-stent restenosis. However, the effects of antiproliferative drugs on endothelial cells (EC) contribute to delayed re-endothelialization and late stent thrombosis. Cell-targeted therapies to inhibit VSMC remodeling while maintaining EC health are necessary to allow vascular healing while preventing restenosis. We describe an RNA aptamer (Apt 14) that functions as a smart drug by preferentially targeting VSMCs as compared to ECs and other myocytes. Furthermore, Apt 14 inhibits phosphatidylinositol 3-kinase/protein kinase-B (PI3K/Akt) and VSMC migration in response to multiple agonists by a mechanism that involves inhibition of platelet-derived growth factor receptor (PDGFR)-ß phosphorylation. In a murine model of carotid injury, treatment of vessels with Apt 14 reduces neointimal formation to levels similar to those observed with paclitaxel. Importantly, we confirm that Apt 14 cross-reacts with rodent and human VSMCs, exhibits a half-life of ~300 hours in human serum, and does not elicit immune activation of human peripheral blood mononuclear cells. We describe a VSMC-targeted RNA aptamer that blocks cell migration and inhibits intimal formation. These findings provide the foundation for the translation of cell-targeted RNA therapeutics to vascular disease.

Diverse contribution of bone marrow-derived late-outgrowth endothelial progenitor cells to vascular repair under pulmonary arterial hypertension and arterial neointimal formation.

AIMS: It is still controversial whether bone marrow (BM)-derived endothelial progenitor cells (EPCs) can contribute to vascular repair and prevent the progression of vascular diseases. We aimed to characterize BM-derived EPC subpopulations and to evaluate their therapeutic efficacies to repair injured vascular endothelium of systemic and pulmonary arteries. METHODS AND RESULTS: BM mononuclear cells of Fisher-344 rats were cultured under endothelial cell-conditions. Early EPCs appeared on days 3-6. Late-outgrowth and very late-outgrowth EPCs (LOCs and VLOCs) were defined as cells forming cobblestone colonies on days 9-14 and 17-21, respectively. Among EPC subpopulations, LOCs showed the highest angiogenic capability with enhanced proliferation potential and secretion of proangiogenic proteins. To investigate the therapeutic effects of these EPCs, Fisher-344 rats underwent wire-mediated endovascular injury in femoral artery (FA) and were concurrently injected intraperitoneally with 60mg/kg monocrotaline (MCT). Injured rats were then treated with six injections of one of three EPCs (1×10(6) per time). After 4weeks, transplanted LOCs, but not early EPCs or VLOCs, significantly attenuated neointimal lesion formation in injured FAs. Some of CD31(+) LOCs directly replaced the injured FA endothelium (replacement ratio: 11.7±7.0%). In contrast, any EPC treatment could neither replace MCT-injured endothelium of pulmonary arterioles nor prevent the progression of pulmonary arterial hypertension (PAH). LOCs modified protectively the expression profile of angiogenic and inflammatory genes in injured FAs, but not in MCT-injured lungs. CONCLUSION: BM-derived LOCs can contribute to vascular repair of injured systemic artery; however, even they cannot rescue injured pulmonary vasculature under MCT-induced PAH.

Inactivation of nuclear factor-Y inhibits vascular smooth muscle cell proliferation and neointima formation.

OBJECTIVE: Atherosclerosis and restenosis are multifactorial diseases associated with abnormal vascular smooth muscle cell (VSMC) proliferation. Nuclear factor-Y (NF-Y) plays a major role in transcriptional activation of the CYCLIN B1 gene (CCNB1), a key positive regulator of cell proliferation and neointimal thickening. Here, we investigated the role of NF-Y in occlusive vascular disease. APPROACH AND RESULTS: We performed molecular and expression studies in cultured cells, animal models, and human tissues. We find upregulation of NF-Y and cyclin B1 expression in proliferative regions of murine atherosclerotic plaques and mechanically induced lesions, which correlates with higher binding of NF-Y to target sequences in the CCNB1 promoter. NF-YA expression in neointimal lesions is detected in VSMCs, macrophages, and endothelial cells. Platelet-derived growth factor-BB, a main inductor of VSMC growth and neointima development, induces the recruitment of NF-Y to the CCNB1 promoter and augments both CCNB1 mRNA expression and cell proliferation through extracellular signal-regulated kinase 1/2 and Akt activation in rat and human VSMCs. Moreover, adenovirus-mediated overexpression of a NF-YA-dominant negative mutant inhibits platelet-derived growth factor-BB-induced CCNB1 expression and VSMC proliferation in vitro and neointimal lesion formation in a mouse model of femoral artery injury. We also detect NF-Y expression and DNA-binding activity in human neointimal lesions. CONCLUSIONS: Our results identify NF-Y as a key downstream effector of the platelet-derived growth factor-BB-dependent mitogenic pathway that is activated in experimental and human vasculoproliferative diseases. They also identify NF-Y inhibition as a novel and attractive strategy for the local treatment of neointimal formation induced by vessel denudation.

Pseudotyping the adenovirus serotype 5 capsid with both the fibre and penton of serotype 35 enhances vascular smooth muscle cell transduction.

Ex vivo gene therapy during coronary artery bypass grafting (CABG) holds great potential to prevent excessive smooth muscle cell (SMC) proliferation, neointima formation and graft failure. The most successful preclinical strategies to date have utilised vectors based on the species C adenovirus, Ad5, which engages the Coxsackie and Adenovirus receptor (CAR) as its primary attachment receptor. Profiling receptors on human SMCs demonstrated the absence of CAR but substantial expression of the species B receptor CD46. We performed transduction experiments using Ad5 and the CD46-utilising adenovirus Ad35, and found Ad35 significantly more efficient at transducing SMCs. To evaluate whether transduction could be further augmented, we evaluated chimeric CD46-utilising Ad5/Ad35 vectors comprising the Ad5 capsid pseudotyped with the Ad35 fibre alone (Ad5/F35) or in combination with the Ad35 penton (Ad5/F35/P35). In human smooth muscle cells (hSMCs), Ad5/F35/P35 mediated significantly higher levels of transduction than either parental vector or Ad5/F35. Ex vivo transduction experiments using mouse aortas from CD46 transgenics demonstrated that Ad5/F35/P35 was significantly more efficient at transducing SMCs than the other vectors tested. Finally, ex vivo transduction and immunofluorescent colocalisation experiments using human tissue from CABG procedures confirmed the preclinical potential of Ad5/F35/P35 as an efficient vector for vascular transduction during CABG.

Sustained reduction of vein graft neointima formation by ex vivo TIMP-3 gene therapy.

BACKGROUND: Coronary artery vein graft failure, resulting from thrombosis, intimal thickening, and atherosclerosis, is a significant clinical problem, with approximately 50% of vein grafts failing within 10 years. Intimal thickening is caused by migration of vascular smooth muscle cells from the media to the intima, where they proliferate. Interventions using gene transfer to inhibit vascular smooth muscle cells proliferation and migration are attractive because ex vivo access to the graft is possible. The involvement of matrix-degrading metalloproteinases in intimal thickening is well established, and we previously showed that adenoviral-delivered overexpression of an endogenous inhibitor, the tissue inhibitor of metalloproteinases-3 (TIMP-3), significantly retarded intimal thickening in short-term autologous porcine arteriovenous interposition grafts (28 days). However, it is essential to determine whether this approach will provide longer-term benefits. METHODS AND RESULTS: We assessed whether a recombinant adenovirus that overexpresses TIMP-3 (RAdTIMP-3) affects vein graft intimal thickening in the longer term (at 3 months). Porcine saphenous veins were subjected to luminal infection with 2.5×10(10) pfu/mL RAdTIMP-3 or RAd60 (control virus) or vehicle control, for 30 minutes before implantation into the carotid artery. Analysis of grafts harvested 3 months after delivery revealed that RAdTIMP-3-infected grafts had significantly reduced intimal areas compared with both controls (3.2 ± 0.4 mm(2) versus 5.6 ± 0.7 mm(2) and 5.9 ± 0.5 mm(2), RAdTIMP-3, RAd60, and vehicle, respectively). Medial areas were also significantly decreased by TIMP-3 (3.8 ± 0.3 mm(2) versus 6.7 ± 1.0 mm(2) and 5.2 ± 0.4 mm(2), RAdTIMP-3, RAd60, and vehicle, respectively). CONCLUSIONS: Overexpression of TIMP-3 provides a sustained retardation of vein graft intimal thickening and highlights the translational potential for ex vivo TIMP-3 gene therapy.

Dermatan sulfate and bone marrow mononuclear cells used as a new therapeutic strategy after arterial injury in mice.

BACKGROUND AIMS: Previously, we have demonstrated that administration of dermatan sulfate (DS) suppresses neointima formation in the mouse carotid artery by activating heparin co-factor II. A similar suppressive effect was observed by increasing the number of progenitor cells in circulation. In this study, we investigated the combination of DS and bone marrow mononuclear cells (MNC), which includes potential endothelial progenitors, in neointima formation after arterial injury. METHODS: Arterial injury was induced by mechanical dilation of the left common carotid artery. We analyzed the extension of endothelial lesion, thrombus formation, P-selectin expression and CD45(+) cell accumulation 1 and 3 days post-injury, and neointima formation 21 days post-injury. Animals were injected with MNC with or without DS during the first 48 h after injury. RESULTS: The extension of endothelial lesion was similar in all groups 1 day after surgery; however, in injured animals treated with MNC and DS the endothelium recovery seemed to be more efficient 21 days after lesion. Treatment with DS inhibited thrombosis, decreased CD45(+) cell accumulation and P-selectin expression at the site of injury, and reduced the neointimal area by 56%. Treatment with MNC reduced the neointimal area by 54%. The combination of DS and MNC reduced neointima formation by more than 91%. In addition, DS promoted a greater accumulation of MNC at the site of injury. CONCLUSIONS: DS inhibits the initial thrombotic and inflammatory processes after arterial injury and promotes migration of MNC to the site of the lesion, where they may assist in the recovery of the injured endothelium.

Lentivirus-mediated RNAi targeting CREB binding protein attenuates neointimal formation and promotes re-endothelialization in balloon injured rat carotid artery.

BACKGROUND/AIMS: Lentiviral vectors provide a promising strategy for the treatment of cardiovascular diseases, owing to their ability to govern efficient and durable gene transfer. However, relatively few studies have been addressed on restenosis after balloon or stent associated arterial injury. We previously found that CREB binding protein (CBP), a powerful transcriptional coactivator, regulated cell proliferation and apoptosis in vascular endothelial and smooth muscle cells. Therefore, we investigated whether inhibition of CBP by lentivirus-mediated small interfering RNA can reduce neointimal formation after arterial injury. METHODS: The carotid arteries from Sprague-Dawley rats were injured by balloon catheter, followed by incubating with 100 microl lentivirus expressing CBP or negative control (NC)-specific short hairpin RNAs (shRNAs) or PBS solution for 30 minutes. The rats were euthanized for real-time PCR, Western blot, immunohistochemical staining, and morphometric analysis at 4 weeks after balloon injury and in vivo gene transfer. RESULTS: Lentiviral shRNA targeting CBP markedly reduced CBP expression. Moreover, CBP siRNA showed potent inhibition on balloon injury-induced Nuclear factor kappaB (NF-kappaB) acetylation. Compared with controls, the significant decrease of neointimal formation by CBP siRNA was accompanied by reduced cell proliferation in the neointima of injured arteries. However, no changes in medial area were observed among these different groups. Interestingly, endothelial cell marker CD31 immunostaining and morphometric analysis both showed that CBP knockdown significantly accelerated re-endothelialization. CONCLUSIONS: These findings suggest that CBP is involved in the control of neointimal formation and re-endothelialization via regulating NF-kappaB acetylation. Lentivirus-mediated CBP silencing may represent a novel therapeutic approach for the prevention of restenosis after vascular interventions.

Physical exercises decreases thrombus and neointima formation in atherosclerotic mice.

The practice of physical exercise is highly indicated to prevent cardiovascular diseases and is directly related to the improvement of endothelial function and the regulation of arterial blood pressure. The objective of this study was to analyze the effect of physical exercise in vascular remodeling after FeCl3 chemically induced arterial injury on atherosclerotic mice. To analyze the effect of exercises on thrombus formation, LDL receptor-deficient mice were fed for 6 weeks with a high-fat diet and performed or not physical exercises for 2 weeks before the arterial injury. To verify endothelium recovery the animals were exercised or not 2 weeks before the injury, and 3 weeks after it, when the vessels were analyzed. In this work, we observed that physical exercises done only before arterial injury reduced thrombosis time, protected the endothelial layer, promoted the recruitment of CD34 positive progenitor cells, increased the level of eNOS and gelatinases activities and decreased the number of inflammatory cells in the vessel, but do not avoid the growth of neointima. Otherwise exercises done before and continued after injury, increased gelatinase activities, reduced lipid deposition in the aortic arch and prevented neointima formation. Thus, we could conclude that physical exercises are done before and continued after endothelial injury stimulate endothelial recovery by promoting endothelial cell growth, matrix remodeling and decreasing inflammation in the vessel wall.

Copper-Based SURMOFs for Nitric Oxide Generation: Hemocompatibility, Vascular Cell Growth, and Tissue Response.

A coating that can generate nitric oxide (NO) for surface modification of cardiovascular stents with adaptable NO release is an efficient approach to prevent thrombosis and neointimal hyperplasia. Herein, we prepared a copper-based surface-attached metal-organic framework (Cu-SURMOFs) of copper(II) benzene-1,3,5-tricarboxylate (CuBTC) using a layer-by-layer assembly method (LBL) for NO generation on the surface of alkali-activated titanium. It was easy to control surface chemistry and NO release by changing the number of LBL deposition cycles. The obtained CuBTC coating was characterized by X-ray diffraction, scanning electron microscopy, Fourier transform infrared, and X-ray photoelectron spectroscopy analysis and was able to decompose endogenous S-nitrosoglutathoine (GSNO) to catalytically produce NO. The resulting NO flux increased with increased deposition cycles. The coating prepared with 10 cycles of deposition showed ideal NO release and promoted proliferation of endothelial cells, suppressed growth of smooth muscle cells and macrophages, and inhibited platelet adhesion and activation. Further evaluation of thrombogenicity in an arteriovenous shunt model showed that the CuBTC coating had great ability to prevent thrombosis, and in vivo implantation of CuBTC-coated titanium wire demonstrated a significant inhibition of intimal hyperplasia. The results showed that use of copper-based SURMOFs could be a promising strategy for the surface modification of cardiovascular stents.