Novel Approaches to Anti-atherosclerotic Therapy: Cell-based Models and Herbal Preparations (Review of Our Own Data).

Atherosclerosis is a chronic arterial disease characterized by vascular inflammation, accumulation of lipids in the arterial wall, and formation and growth of atherosclerotic plaques followed by ischemia. In subclinical atherosclerosis, cholesterol retention in subendothelial cells leads to induction of local inflammation, generation of foam cells and lesion formation, followed by a chain of other pathogenic events. Atherosclerotic progression can frequently be fatal, since plaque rupture may lead to thrombosis and acute events, such as myocardial infarction, stroke and sudden death. Traditional anti-atherosclerotic therapy is mainly focused on improving the blood lipid profile and does not target various stages of plaque progression. Obviously, treating the disease at initial stages is better than beginning treatment at advanced stages and, in that regard, current atherosclerosis management can be improved. Cholesterol retention is an important component of atherogenesis that precedes plaque formation. Therapeutic targeting of cholesterol retention may be beneficial for preventing further atherogenic progression. For this purpose, we suggest using herbal preparations due to good tolerability and suitability for long-lasting treatment. We developed test systems based on cultured human intimal aortic cells for rapid screening of potential anti-atherogenic drugs. With the help of these test systems, we selected several natural substances with significant anti-atherogenic activity and further use these compounds to prepare herbal preparations for anti-atherosclerotic therapy. These preparations were clinically tested and showed good safety and a potent anti-atherogenic potential.

S100B is required for maintaining an intermediate state with double-positive Sca-1+ progenitor and vascular smooth muscle cells during neointimal formation.

INTRODUCTION: Accumulation of vascular smooth muscle cells (VSMCs) within the neointimal region is a hallmark of atherosclerosis and vessel injury. Evidence has shown that Sca-1-positive (Sca-1+) progenitor cells residing in the vascular adventitia play a crucial role in VSMC assemblages and intimal lesions. However, the underlying mechanisms, especially in the circumstances of vascular injury, remain unknown. METHODS AND RESULTS: The neointimal formation model in rats was established by carotid artery balloon injury using a 2F-Forgaty catheter. Most Sca-1+ cells first appeared at the adventitia of the vascular wall. S100B expressions were highest within the adventitia on the first day after vessel injury. Along with the sequentially increasing trend of S100B expression in the intima, media, and adventitia, respectively, the numbers of Sca-1+ cells were prominently increased at the media or neointima during the time course of neointimal formation. Furthermore, the Sca-1+ cells were markedly increased in the tunica media on the third day of vessel injury, SDF-1α expressions were obviously increased, and SDF-1α levels and Sca-1+ cells were almost synchronously increased within the neointima on the seventh day of vessel injury. These effects could effectually be reversed by knockdown of S100B by shRNA, RAGE inhibitor (SPF-ZM1), or CXCR4 blocker (AMD3100), indicating that migration of Sca-1+ cells from the adventitia into the neointima was associated with S100B/RAGE and SDF-1α/CXCR4. More importantly, the intermediate state of double-positive Sca-1+ and α-SMA cells was first found in the neointima of injured arteries, which could be substantially abrogated by using shRNA for S100B or blockade of CXCR4. S100B dose-dependently regulated SDF-1α expressions in VSMCs by activating PI3K/AKT and NF-κB, which were markedly abolished by PI3K/AKT inhibitor wortmannin and enhanced by p65 blocker PDTC. Furthermore, S100B was involved in human umbilical cord-derived Sca-1+ progenitor cells' differentiation into VSMCs, especially in maintaining the intermediate state of double-positive Sca-1+ and α-SMA. CONCLUSIONS: S100B triggered neointimal formation in rat injured arteries by maintaining the intermediate state of double-positive Sca-1+ progenitor and VSMCs, which were associated with direct activation of RAGE by S100B and indirect induction of SDF-1α by activating PI3K/AKT and NF-κB.

Recipient c-Kit Lineage Cells Repopulate Smooth Muscle Cells of Transplant Arteriosclerosis in Mouse Models.

RATIONALE: Transplantation-accelerated arteriosclerosis is one of the major challenges for long-term survival of patients with solid organ transplantation. Although stem/progenitor cells have been implicated to participate in this process, the cells of origin and underlying mechanisms have not been fully defined. OBJECTIVE: The objective of our study was to investigate the role of c-Kit lineage cells in allograft-induced neointima formation and to explore the mechanisms underlying this process. METHODS AND RESULTS: Using an inducible lineage tracing Kit-CreER;Rosa26-tdTomato mouse model, we observed that c-Kit is expressed in multiple cell types in the blood vessels, rather than a specific stem/progenitor cell marker. We performed allograft transplantation between different donor and recipient mice, as well as bone marrow transplantation experiments, demonstrating that recipient c-Kit+ cells repopulate neointimal smooth muscle cells (SMCs) and leukocytes, and contribute to neointima formation in an allograft transplantation model. c-Kit-derived SMCs originate from nonbone marrow tissues, whereas bone marrow-derived c-Kit+ cells mainly generate CD45+ leukocytes. However, the exact identity of c-Kit lineage cells contributing to neointimal SMCs remains unclear. ACK2 (anti-c-Kit antibody), which specifically binds and blocks c-Kit function, ameliorates allograft-induced arteriosclerosis. Stem cell factor and TGF (transforming growth factor)-ß1 levels were significantly increased in blood and neointimal lesions after allograft transplantation, by which stem cell factor facilitated c-Kit+ cell migration through the stem cell factor/c-Kit axis and downstream activation of small GTPases, MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal-regulated kinase)/MLC (myosin light chain), and JNK (c-Jun N-terminal kinase)/c-Jun signaling pathways, whereas TGF-ß1 induces c-Kit+ cell differentiation into SMCs via HK (hexokinase)-1-dependent metabolic reprogramming and a possible downstream O-GlcNAcylation of myocardin and serum response factor. CONCLUSIONS: Our findings provide evidence that recipient c-Kit lineage cells contribute to vascular remodeling in an allograft transplantation model, in which the stem cell factor/c-Kit axis is responsible for cell migration and HK-1-dependent metabolic reprogramming for SMC differentiation.

Versican is differentially regulated in the adventitial and medial layers of human vein grafts.

Changes in extracellular matrix proteins may contribute significantly to the adaptation of vein grafts to the arterial circulation. We examined the production and distribution of versican and hyaluronan in intact human vein rings cultured ex vivo, veins perfused ex vivo, and cultured venous adventitial and smooth muscle cells. Immunohistochemistry revealed higher levels of versican in the intima/media compared to the adventitia, and no differences in hyaluronan. In the vasa vasorum, versican and hyaluronan associated with CD34+ progenitor cells. Culturing the vein rings for 14 days revealed increased versican immunostaining of 30-40% in all layers, with no changes in hyaluronan. Changes in versican accumulation appear to result from increased synthesis in the intima/media and decreased degradation in the adventitia as versican transcripts were increased in the intima/media, but unchanged in the adventitia, and versikine (the ADAMTS-mediated cleavage product of versican) was increased in the intima/media, but decreased in the adventitia. In perfused human veins, versican was specifically increased in the intima/media in the presence of venous pressure, but not with arterial pressure. Unexpectedly, cultured adventitial cells express and accumulate more versican and hyaluronan than smooth muscle cells. These data demonstrate a differential regulation of versican and hyaluronan in human venous adventitia vs. intima/media and suggest distinct functions for these extracellular matrix macromolecules in these venous wall compartments during the adaptive response of vein grafts to the arterial circulation.

Down-regulation of vascular GLP-1 receptor expression in human subjects with obesity.

It has been thought that incretin signaling prevents arteriosclerosis, and very recently anti-arteriosclerotic effects through GLP-1 receptor were finally demonstrated in clinical human study. The purpose of this study was to investigate how vascular GLP-1 receptor expression is influenced in human subjects. First, we evaluated GLP-1 receptor expression in human arteries in immunostaining. Next, we separated the artery into the intima and media, and evaluated gene expression levels of various factors. We divided the subjects into obesity and non-obesity group and compared their expression levels between them. Finally, we evaluated which factors determine vascular GLP-1 receptor expression. GLP-1 receptor expression in intima and media was lower in obesity group compared to non-obesity group which was correlated with the alteration of TCF7L2 expression. Multiple regression analyses showed that BMI was an independent determining factor for GLP-1 receptor expression in the intima and media. Furthermore, using small interfering RNA method and TCF7L2-EGFP adenovirus, we showed that TCF7L2 was involved in GLP-1 receptor expression in human vascular cells. Taken together, vascular GLP-1 receptor and TCF7L2 expression was significantly down-regulated in human subjects with obesity. In addition, it is likely that TCF7L2 functions as a modulator of vascular GLP-1 receptor expression.

Dipeptidyl peptidase IV (DPP-4) inhibition alleviates pulmonary arterial remodeling in experimental pulmonary hypertension.

Dipeptidyl peptidase IV (DPP-4) is well known for its role in glucose homeostasis, and DPP-4 inhibitor (DPP-4i) exhibits multiple actions in cardiovascular diseases. However, the effect of DPP-4i on pulmonary hypertension (PH) remains unclear. Therefore, this study aims to investigate the effect of DPP-4i on pulmonary arterial remodeling in rats with PH and the potential underlying mechanisms. Our results show that DPP-4 was expressed in epithelial cells, endothelial cells, smooth muscle cells, and inflammatory cells in lung. DPP-4i (Sitagliptin) attenuated right ventricular systolic pressure (RVSP), right ventricle remodeling, hypertrophy of pulmonary arterial medial layer, inflammatory cell infiltration, and endothelial-mesenchymal transition (EndMT) in monocrotaline (MCT)-induced PH rats. Similarly, DPP-4i also alleviated bleomycin- and chronic hypoxia-induced PH in rats. In cultured human pulmonary arterial smooth muscle cells (PASMCs), DPP-4i inhibited platelet derived growth factor (PDGF)-BB-induced proliferation and migration, which was abolished by phosphatase and tensin homolog deleted on chromosome ten (PTEN) knockout. These results demonstrate that DPP-4 inhibition alleviates pulmonary arterial remodeling in experimental PH by inhibiting proliferation and migration of PASMCs.

Biomechanical relevance of the microstructure in artery walls with a focus on passive and active components.

The microstructure of arteries, consisting, in particular, of collagen, elastin, and vascular smooth muscle cells, plays a very significant role in their biomechanical response during a cardiac cycle. In this article, we highlight the microstructure and the contributions of each of its components to the overall mechanical behavior. We also describe the changes of the microstructure that occur as a result of abdominal aortic aneurysms and disease, such as atherosclerosis. We also focus on how the passive and active constituents are incorporated into a mathematical model without going into detail of the mathematical formulation. We conclude by mentioning open problems toward a better characterization of the biomechanical aspects of arteries that will be beneficial for a better understanding of cardiovascular pathophysiology.

A single nucleotide polymorphism of cyclin-dependent kinase inhibitor 1B (p27Kip1) associated with human vein graft failure affects growth of human venous adventitial cells but not smooth muscle cells.

BACKGROUND: Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a cell-cycle inhibitor whose -838C>A single nucleotide polymorphism (rs36228499; hereafter called p27 SNP) has been associated with the clinical failure of peripheral vein grafts, but the functional effects of this SNP have not been demonstrated. METHODS: Human saphenous vein adventitial cells and intimal/medial smooth muscle cells (SMCs) were derived from explants obtained at the time of lower extremity bypass operations. We determined the following in adventitial cells and SMCs as a function of the p27 SNP genotype: (1) p27 promoter activity, (2) p27 messenger (m)RNA and protein levels, and (3) growth and collagen gel contraction. Deoxyribonuclease I footprinting was also performed in adventitial cells and SMCs. RESULTS: p27 promoter activity, deoxyribonuclease I footprinting, p27 mRNA levels, and p27 protein levels demonstrated that the p27 SNP is functional in adventitial cells and SMCs. Both cell types with the graft failure protective AA genotype had more p27 mRNA and protein. As predicted because of higher levels of p27 protein, adventitial cells with the AA genotype grew slower than those of the CC genotype. Unexpectedly, SMCs did not show this genotype-dependent growth response. CONCLUSIONS: These results support the functionality of the p27 SNP in venous SMCs and adventitial cells, but an effect of the SNP on cell proliferation is limited to only adventitial cells. These data point to a potential role for adventitial cells in human vein graft failure and also suggest that SMCs express factors that interfere with the activity of p27.

Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats.

Structural adaptation of arteries to weightlessness might lower the working ability or even threaten the physical health of astronauts, but the underlying mechanism is unclear. Acid sphingomyelinase (ASM) catalyzes ceramide (Cer) generation controlling arterial remodeling through multiple signaling pathways. In the present study, we aimed to investigate the contribution of ASM/Cer to the changes of common carotid artery intima-media thickness (CIMT) induced by simulated weightlessness. Hindlimb-unloaded tail-suspended (HU) rats were used to simulate the effect of weightlessness. Morphology of the carotid artery (CA) was examined by hematoxylin-eosin staining. Protein content of ASM or proliferating cell nuclear antigen (PCNA) was detected by Western blot. Cer level was measured by immunohistochemistry analysis. Apoptosis events were observed by transferase-mediated dUTP nick end labeling (TUNEL) staining. During 4 weeks of tail suspension, CIMT was increased gradually in HU but not in their synchronous control rats (P < 0.05). Correspondingly, the CA of HU rats had a lower apoptosis and higher proliferation of vascular smooth muscle cells (VSMCs). As compared to the control, both ASM protein expression and Cer content were reduced significantly in CA of HU rats (P < 0.05), incubation of which with permeable Cer reversed the changes in apoptosis and proliferation substantially. Furthermore, when the ASM protein content as well as Cer level in CA of control rats was diminished by using an ASM inhibitor, an increase of CIMT along with reduced apoptosis and enhanced proliferation of VSMCs was found. Our results suggest that by controlling the balance between apoptosis and proliferation, ASM/Cer plays an important role in the regulation of CIMT during simulated weightlessness.

[Analysis of specificity of Shenque (CV 8) based on vascular biology].

To analyze the structural specificity of Shenque (CV 8) in terms of vascular biology from the three aspects of structure, tissue and molecular anatomies. On structural anatomy Shenque (CV 8) possessed defined vascular structure and was the only acupoint directly affected vascular intima. The basis of the specific therapeutic effects was owing to its relation with vessel and abundant microcirculation. Endothelial cells and microvascular endothelial cells were the tissue basis of the starting of therapeutic specificity. Molecular anatomy involved in the functions of transient receptor potential vanilloid family (TRPV) pathway and neuronal peptide secretion of endothelial cells in the structural function of the point. The "restoring yang for collapse" effect of salt-partition moxibustion at Shenque (CV 8) was analyzed based on the local biological specificity of blood vessels. The mechanisms were concluded as effectively targeting the dysfunction of microvascular endothelial cells and acquiring the maximum quantity of moxibustion by repeated warm-heat stimulation. The vascular biological structural features of Shenque (CV 8) may contribute to direct therapeutic effects on endothelial cells by the point.

A novel PDGF receptor inhibitor-eluting stent attenuates in-stent neointima formation in a rabbit carotid model.

A novel drug-eluting stent (DES) is required to target vascular smooth muscle cells (SMCs) without harming endothelial cells (ECs). Platelet-derived growth factor (PDGF) is critical for the proliferation and migration of SMCs. Sunitinib [a PDGF receptor (PDGFR) tyrosine kinase inhibitor]­eluting stents may therefore inhibit neointimal formation. The aim of the present study was to examine the stent­based delivery of sunitinib in a rabbit carotid model; in addition, the effects of sunitinib were evaluated in vitro. Local administration of sunitinib markedly reduced neointimal formation without delaying re-endothelialization in the carotid artery model. In vitro, sunitinib inhibited SMC proliferation; however, no effects were observed on ECs. Sunitinib caused necrosis of SMCs. In addition, sunitinib attenuated PDGF-stimulated SMC migration in a scratch wound assay and inhibited α­SMA cytoskeleton polymerization. Furthermore, sunitinib inhibited PDGF-induced phosphorylation of extracellular signal-regulated kinase in vitro and in vivo. Therefore, this novel DES may be a potential strategy for the treatment of vascular disorders.

Pericardial patch venoplasty heals via attraction of venous progenitor cells.

Pericardial patches are commonly used during cardiovascular surgery to close blood vessels. In arteries, patches accumulate arterial progenitor cells; we hypothesized that venous patches would accumulate venous progenitor cells, in the absence of arterial pressure. We developed a novel rat inferior vena cava (IVC) venotomy model and repaired it with a pericardial patch. Cells infiltrated the patch to form a thick neointima by day 7; some cells were CD34(+)/VEGFR2(+) and CD31(+)/Eph-B4(+) consistent with development of venous identity in the healing patch. Compared to arterial patches, the venous patches had increased neointimal thickness at day 7 without any pseudoaneurysms. Addition of an arteriovenous fistula (AVF) to increase blood flow on the patch resulted in reduced patch neointimal thickness and proliferation, but neointimal thickness was not reversible with AVF ligation. These results show that rat patch venoplasty is a novel model of aggressive venous neointimal hyperplasia.

Intimal smooth muscle cells are a source but not a sensor of anti-inflammatory CYP450 derived oxylipins.

Vascular pathologies are associated with changes in the presence and expression of morphologically distinct vascular smooth muscle cells. In particular, in complex human vascular lesions and models of disease in pigs and rodents, an intimal smooth muscle cell (iSMC) which exhibits a stable epithelioid or rhomboid phenotype in culture is often found to be present in high numbers, and may represent the reemergence of a distinct developmental vascular smooth muscle cell phenotype. The CYP450-oxylipin - soluble epoxide hydrolase (sEH) pathway is currently of great interest in targeting for cardiovascular disease. sEH inhibitors limit the development of hypertension, diabetes, atherosclerosis and aneurysm formation in animal models. We have investigated the expression of CYP450-oxylipin-sEH pathway enzymes and their metabolites in paired intimal (iSMC) and medial (mSMC) cells isolated from rat aorta. iSMC basally released significantly larger amounts of epoxy-oxylipin CYP450 products from eicosapentaenoic acid > docosahexaenoic acid > arachidonic acid > linoleic acid, and expressed higher levels of CYP2C12, CYP2B1, but not CYP2J mRNA compared to mSMC. When stimulated with the pro-inflammatory TLR4 ligand LPS, epoxy-oxylipin production did not change greatly in iSMC. In contrast, LPS induced epoxy-oxylipin products in mSMC and induced CYP2J4. iSMC and mSMC express sEH which metabolizes primary epoxy-oxylipins to their dihydroxy-counterparts. The sEH inhibitors TPPU or AUDA inhibited LPS-induced NFκB activation and iNOS induction in mSMC, but had no effect on NFκB nuclear localization or inducible nitric oxide synthase in iSMC; effects which were recapitulated in part by addition of authentic epoxy-oxylipins. iSMCs are a rich source but not a sensor of anti-inflammatory epoxy-oxylipins. Complex lesions that contain high levels of iSMCs may be more resistant to the protective effects of sEH inhibitors.

Nanodiagnostics, nanopharmacology and nanotoxicology of platelet-vessel wall interactions.

In physiological conditions, the interactions between blood platelets and endothelial cells play a major role in vascular reactivity and hemostasis. By contrast, increased platelet activation contributes to the pathogenesis of vascular pathology such as atherosclerosis, thrombosis, diabetes mellitus, hypertension and carcinogenesis. Nanomedicine, including nanodiagnostics and nanotherapeutics is poised to be used in the management of vascular diseases. However, the inherent risk and potential toxicity resultant from the use of nanosized (<100 nm) materials need to be carefully considered. This review, basing on a systematic search of literature provides state-of-the-art and focuses on new discoveries, as well as the potential benefits and threats in the field of nanodiagnostics, nanopharmacology and nanotoxicology of platelet-vessel wall interactions.

Cryoplasty versus angioplasty in the treatment of arterial restenosis in an experimental model of atherosclerosis in rabbits.

Cryoplasty may reduce the incidence of post-angioplasty restenosis in peripheral atherosclerotic arteries. Our study is looking to investigate the mid-term effects (4 weeks) of an FDA-approved cryoplasty catheter (PolarCath(®), Boston Scientific) compared to a conventional angioplasty catheter using a hypercholesterolemic rabbit model of arterial restenosis based on diet plus vessel injury. Thirty-six normolipidemic, 3-month old male New Zealand White rabbits were used. Balloon angioplasty was performed on left external iliac arteries on day 1. Animals were fed with a hypercholesterolemic diet for 60 days. On day 120, three groups of animals were established: conventional PTA (percutaneous transluminal angioplasty) was applied on the PTA group; the CRY group was treated with the PolarCath(®) cryoplasty system and no treatment was given to a control (CTR) group. A broad variety of atheromatous lesions were observed 30 days after treatment, presenting significant differences between groups. Most of the complicated lesions were found in the CRY group, while advanced and early lesions were more often appreciated in the CTR and PTA groups, respectively. The histomorphometric evaluation of the arteries showed significant differences between the CRY group and the other two groups, with the highest percentage of IEM (internal elastic membrane) injury, vascular stenosis and ratio intima/media being registered on animals treated with cryoplasty. Intravascular cryotherapy induces complicated lesions in arterial walls 30 days after treatment in a hypercholesterolemic rabbit model based on diet plus vessel injury. Cryoplasty leads to the production of severe fibrosis and mineralisation and stenosis compared to a conventional angioplasty.

Isolation of blood-vessel-derived multipotent precursors from human skeletal muscle.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

TGF-ß/Smad3 inhibit vascular smooth muscle cell apoptosis through an autocrine signaling mechanism involving VEGF-A.

We have previously shown that in the presence of elevated Smad3, transforming growth factor-ß (TGF-ß) transforms from an inhibitor to a stimulant of vascular smooth muscle cell (SMC) proliferation and intimal hyperplasia (IH). Here we identify a novel mechanism through which TGF-ß/Smad3 also exacerbates IH by inhibiting SMC apoptosis. We found that TGF-ß treatment led to inhibition of apoptosis in rat SMCs following viral expression of Smad3. Conditioned media from these cells when applied to naive SMCs recapitulated this effect, suggesting an autocrine pathway through a secreted factor. Gene array of TGF-ß/Smad3-treated cells revealed enhanced expression of vascular endothelial growth factor (VEGF), a known inhibitor of endothelial cell apoptosis. We then evaluated whether VEGF is the secreted mediator responsible for TGF-ß/Smad3 inhibition of SMC apoptosis. In TGF-ß/Smad3-treated cells, VEGF mRNA and protein as well as VEGF secretion were increased. Moreover, recombinant VEGF-A inhibited SMC apoptosis and a VEGF-A-neutralizing antibody reversed the inhibitory effect of conditioned media on SMC apoptosis. Stimulation of SMCs with TGF-ß led to the formation of a complex of Smad3 and hypoxia-inducible factor-1α (HIF-1α) that in turn activated the VEGF-A promoter and transcription. In rat carotid arteries following arterial injury, Smad3 and VEGF-A expression were upregulated. Moreover, Smad3 gene transfer further enhanced VEGF expression as well as inhibited SMC apoptosis. Finally, blocking either the VEGF receptor or Smad3 signaling in injured carotid arteries abrogated the inhibitory effect of Smad3 on vascular SMC apoptosis. Taken together, our study reveals that following angioplasty, elevation of both TGF-ß and Smad3 leads to SMC secretion of VEGF-A that functions as an autocrine inhibitor of SMC apoptosis. This novel pathway provides further insights into the role of TGF-ß in the development of IH.

Upregulation of let-7a inhibits vascular smooth muscle cell proliferation in vitro and in vein graft intimal hyperplasia in rats.

BACKGROUND: Proliferation of vascular smooth muscle cells (VSMCs) is a crucial event in the pathogenesis of intimal hyperplasia, which is the main cause of restenosis after vascular reconstruction. In this study, we assessed the impact of let-7a microRNA (miRNA) on the proliferation of VSMCs. METHODS: Using miRNA microarrays analysis for miRNA expression in the vein graft model. Lentiviral vector-mediated let-7a was transfected into the vein grafts. In situ hybridization was performed to detect let-7a. Cultured rat VSMCs were transfected with let-7a mimics for different periods of time. Cell proliferation, migration and cell cycle activity were monitored following transfection of the let-7a mimics. Immunohistochemical and Western blotting analysis the expression levels of c-myc and K-ras. RESULTS: We found that let-7a was the most downregulated miRNA in the vein graft model. In vivo proliferation of VSMCs was assessed in a rat model of venous graft intimal hyperplasia. Let-7a was found to localize mainly to the VSMCs. Let-7a miRNA expression was increased in VSMCs in the neointima of the let-7a treated group. Intimal hyperplasia was suppressed by upregulation of let-7a via lentiviral vector-mediated mimics. In cultured VSMCs, the expression of let-7a increased upon starving, and the upregulation of let-7a miRNA significantly decreased cell proliferation and migration. Immunohistochemical and Western blotting analysis demonstrated that treatment with let-7a mimics resulted in decreased expression levels of c-myc and K-ras. CONCLUSIONS: The results indicate that let-7a miRNA is a novel regulator of VSMC proliferation in intimal hyperplasia. These findings suggest that let-7a miRNA is a promising therapeutic target for the prevention of intimal hyperplasia.

Local thermal injury induces general endothelial cell contraction through p38 MAP kinase activation.

Endothelial cells (ECs) of thin-walled blood vessels form a barrier between blood and tissue. As a response to inflammation, the EC junctions widen and gaps form, resulting in compromised barrier functions. Although the mechanisms behind the establishment of these changes are still incompletely understood, one known reason is actomyosin-dependent actin rearrangement. Here, by using atomic force microscopy and a combination of confocal microscopy methods, we are the first to report that thermal injury induces general venular hyperpermeability and that serum from burned rats induces EC actin rearrangement, contraction, as well as tight-junction damage. Inhibition of the p38 mitogen-activated protein kinase (p38MAPK) largely ameliorates resulting vascular dysfunction by significantly reducing EC stress-fiber formation, contraction, volume changes and tight-junction damage, thereby greatly reducing the appearance of EC gaps. The findings may be of importance for the design of future pharmacotherapies aiming to ease the severe general vascular dysfunction that follows extensive burns.