Tempol therapy attenuates medial smooth muscle cell apoptosis and neointima formation after balloon catheter injury in carotid artery of diabetic rats

Accumulating data support the hypothesis that reactive oxygen species (ROS) play a critical role in the vascular complications observed in diabetes. However, the mechanisms of ROS-mediated vascular complications in diabetes are not clear. We tested the hypothesis that ROS-mediated increase in proapoptotic factor Bax expression leads to medial smooth muscle cell (SMC) apoptosis that is associated with neointima formation. We used a fructose-rich diet for 4 wk to model Type 2 diabetes in rats. SOD mimetic membrane-permeable 4-hydroxy-2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempol, 1 mM) was administered in drinking water to scavenge superoxide starting 1 day before surgery and continued during the duration of the experiment. Vascular injury resulted in a significant increase in medial SMC apoptosis that was associated with neointima formation. The number of medial SMC positive for Bax immunostaining significantly increased in injured arteries compared with uninjured arteries. Superoxide scavenging by Tempol treatment inhibited both the Bax-positive index as well as the apoptotic index of medial SMC in response to vascular injury. Tempol treatment inhibited apoptotic loss of medial SMC, thus increasing their density in the injured arteries. These alterations in the media were associated with a marked decrease in neointima formation in injured arteries. We conclude that Bax expression may play an important role in vascular SMC apoptosis and, finally, that this regulatory mechanism is redox sensitive.     

Proliferation of neointimal smooth muscle cells after arterial injury. Dependence on interactions between fibroblast growth factor receptor-2 and fibroblast growth factor-9

The growth factor signaling mechanisms responsible for neointimal smooth muscle cell (SMC) proliferation and accumulation, a characteristic feature of many vascular pathologies that can lead to restenosis after angioplasty, remain to be identified. Here, we examined the contribution of fibroblast growth factor receptors (FGFRs) 2 and 3 as well as novel fibroblast growth factors (FGFs) to such proliferation. Balloon catheter injury to the rat carotid artery stimulated the expression of two distinctly spliced FGFR-2 isoforms, differing only by the presence or absence of the acidic box, and two distinctly spliced FGFR-3 isoforms containing the acidic box and differing only by the presence of either the IIIb or IIIc exon. Post-injury arterial administration of recombinant adenoviruses expressing dominant negative mutant forms of these FGFRs were used to assess the roles of the endogenous FGFR isoforms in neointimal SMC proliferation. Dominant negative FGFR-2 containing the acidic box inhibited such proliferation by 40%, whereas the dominant negative FGFR-3 forms had little effect. Expression of FGF-9, known to be capable of binding to all four neointimal FGFR-2/-3 isoforms, was abundant within the neointima. FGF-9 markedly stimulated both the proliferation of neointimal SMCs and the activation of extracellular signal-related kinases 1/2, effects which were abrogated by the administration of antisense FGF-9 oligonucleotides to injured arteries and the expression of the dominant negative FGFR-2 adenovirus in cultured neointimal SMCs. These studies demonstrate that, although multiple FGFRs are induced in neointimal SMCs following arterial injury, specific interactions between distinctly spliced FGFR-2 isoforms and FGF-9 contribute to the proliferation of these SMCs.     

Novel model of inflammatory neointima formation reveals a potential role of myeloperoxidase in neointimal hyperplasia

Atherosclerosis, which is characterized by neointima formation, is an inflammatory disease. However, there is no inflammatory product-elicited neointimal model to support the causal role of inflammation in atherogenesis. We reported previously that leukocyte-derived MPO induces vascular injury responses such as endothelial dysfunction. We now test the role of MPO in inflammatory neointima formation. We infused temporarily isolated rat common carotid arteries with MPO (200 nM) and incubated for 1 h. We found that although MPO itself did not induce any neointima formation 2 wk after treatment, in the presence of its substrate, hydrogen peroxide, MPO was able to elicit neointimal hyperplasia. We further confirmed that MPO-induced neointimal hyperplasia is mediated by its product, hypochlorous acid (HOCl). HOCl elicited apoptosis both in intima and media followed by vascular proliferative response and resulted in neointima formation with a heterogeneous cell population. Both histological and functional features of HOCl-treated vessels are similar to those in atherosclerotic lesions. To our knowledge, this is the first direct in vivo demonstration of neointimal formation induced by a product of the inflammatory cascade. The results suggest that MPO may be a mediator for pathological neointima growth. This novel neointimal model could be useful for studying inflammation and atherosclerosis.     

Increased protein O-GlcNAc modification inhibits inflammatory and neointimal responses to acute endoluminal arterial injury

Inflammation plays a major role in vascular disease. We have shown that leukocyte infiltration and inflammatory mediator expression contribute to vascular remodeling after endoluminal injury. This study tested whether increasing protein O-linked-N-acetylglucosamine (O-GlcNAc) levels with glucosamine (GlcN) and O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) inhibits acute inflammatory and neointimal responses to endoluminal arterial injury. Ovariectomized rats were treated with a single injection of GlcN (0.3 mg/g ip), PUGNAc (7 nmol/g ip) or vehicle (V) 2 h before balloon injury of the right carotid artery. O-GlcNAc-modified protein levels decreased markedly in injured arteries of V-treated rats at 30 min, 2 h, and 24 h after injury but returned to control (contralateral uninjured) levels after 14 days. Both GlcN and PUGNAc increased O-GlcNAc-modified protein levels in injured arteries compared with V controls at 30 min postinjury; the GlcN-mediated increase persisted at 24 h but was not evident at 14 days. Proinflammatory mediator expression increased markedly after injury and was reduced significantly (30-50%) by GlcN and PUGNAc. GlcN and PUGNAc also inhibited infiltration of neutrophils and monocytes in injured arteries. Chronic (14 days) treatment with GlcN reduced neointima formation in injured arteries by 50% compared with V controls. Acute GlcN and PUGNAc treatment increases O-GlcNAc-modified protein levels and inhibits acute inflammatory responses in balloon-injured rat carotid arteries; 14 day GlcN treatment inhibits neointima formation in these vessels. Augmenting O-GlcNAc modification of proteins in the vasculature may represent a novel anti-inflammatory and vasoprotective mechanism.     

Roles of the Kinase TAK1 in CD40-Mediated Effects on Vascular Oxidative Stress and Neointima Formation after Vascular Injury

Although TAK1 has been implicated in inflammation and oxidative stress, its roles in vascular smooth muscle cells (VSMCs) and in response to vascular injury have not been investigated. The present study aimed to investigate the role of TAK1 in modulating oxidative stress in VSMCs and its involvement in neointima formation after vascular injury. Double immunostaining reveals that vascular injury induces a robust phosphorylation of TAK1 (Thr187) in the medial VSMCs of injured arteries in wildtype mice, but this effect is blocked in CD40-deficient mice. Upregulation of TAK1 in VSMCs is functionally important, as it is critically involved in pro-oxidative and pro-inflammatory effects on VSMCs and eventual neointima formation. In vivo, pharmacological inhibition of TAK1 with 5Z-7-oxozeaenol blocked the injury-induced phosphorylation of both TAK1 (Thr187) and NF-kB/p65 (Ser536), associated with marked inhibition of superoxide production, 3-nitrotyrosine, and MCP-1 in the injured arteries. Cell culture experiments demonstrated that either siRNA knockdown or 5Z-7-oxozeaenol inhibition of TAK1 significantly attenuated NADPH oxidase activation and superoxide production induced by CD40L/CD40 stimulation. Co-immunoprecipitation experiments indicate that blockade of TAK1 disrupted the CD40L-induced complex formation of p22phox with p47phox, p67phox, or Nox4. Blockade of TAK1 also inhibited CD40L-induced NF-kB activation by modulating IKKα/β and NF-kB p65 phosphorylation and this was related to reduced expression of proinflammatory genes (IL-6, MCP-1 and ICAM-1) in VSMCs. Lastly, treatment with 5Z-7-oxozeaenol attenuated neointimal formation in wire-injured femoral arteries. Our findings demonstrate previously uncharacterized roles of TAK1 in vascular oxidative stress and the contribution to neointima formation after vascular injury.     

Bone marrow-derived mesenchymal stem cells inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia after arterial injury in rats

We investigated whether mesenchymal stem cell (MSC)-based treatment could inhibit neointimal hyperplasia in a rat model of carotid arterial injury and explored potential mechanisms underlying the positive effects of MSC therapy on vascular remodeling/repair. Sprague-Dawley rats underwent balloon injury to their right carotid arteries. After 2 days, we administered cultured MSCs from bone marrow of GFP-transgenic rats (0.8 × 10⁶ cells, n = 10) or vehicle (controls, n = 10) to adventitial sites of the injured arteries. As an additional control, some rats received a higher dose of MSCs by systemic infusion (3 × 10⁶ cells, tail vein; n = 4). Local vascular MSC administration significantly prevented neointimal hyperplasia (intima/media ratio) and reduced the percentage of Ki67 + proliferating cells in arterial walls by 14 days after treatment, despite little evidence of long-term MSC engraftment. Notably, systemic MSC infusion did not alter neointimal formation. By immunohistochemistry, compared with neointimal cells of controls, cells in MSC-treated arteries expressed reduced levels of embryonic myosin heavy chain and RM-4, an inflammatory cell marker. In the presence of platelet-derived growth factor (PDGF-BB), conditioned medium from MSCs increased p27 protein levels and significantly attenuated VSMC proliferation in culture. Furthermore, MSC-conditioned medium suppressed the expression of inflammatory cytokines and RM-4 in PDGF-BB-treated VSMCs. Thus, perivascular administration of MSCs may improve restenosis after vascular injury through paracrine effects that modulate VSMC inflammatory phenotype.     

Urokinase stimulates whereas tissue plasminogen activator attenuates blood vessel stenosis]

Periadventitial application of the urokinase-plasminogen activator (uPA) in pluronic gel to an injured artery stimulated the neointima and neoadventitia formation as well as cell migration and proliferation in vivo. In contrast, the tissue-type plasminogen activator (tPA) reduced the number of neointimal smooth muscle cells and neointimal area and attenuated the lumen stenosis after a balloon catheter injury of the rat carotid artery. This ability to stimulate the neointima and neoadbentitia formation was found to be quite specific for the uPA. The findings suggest that this uPA property provides a specific functional target for attenuating growth of the damage.     

Effects of enalaprilat on neointimal growth of cultured rabbit aorta following balloon injury

Our objective was to determine if the ability of an angiotensin-converting enzyme (ACE) inhibitor to attenuate neointima formation in balloon-damaged vessel is expressed in an isolated organ culture model of neointimal growth. In vivo balloon angioplasty in combination with in vitro organ culture was used to produce a unique model of vascular neointima formation. Aortic segments were cultured in medium containing a broad concentration range of the ACE inhibitor enalaprilat (0-100 microM). Cell proliferative indices and neointima:media thickness ratios were determined from vessel segments after 1, 4, and 7 days in culture. We observed no significant effect on either parameter at any dose of enalaprilat. Linear regression analysis on the rate of increase in intima to media thickness ratios during the 7 days of culture also showed no effect of enalaprilat at any concentration. We conclude that enalaprilat has no effect on neointimal growth or cell proliferation in this vascular organ culture model, and it is suggested that ACE inhibitors may act by mechanisms other than local converting enzyme inhibition to attenuate neointimal growth in rabbits following vascular ballooning in vivo.     

Vanin-1 pantetheinase drives smooth muscle cell activation in post-arterial injury neointimal hyperplasia

The pantetheinase vanin-1 generates cysteamine, which inhibits reduced glutathione (GSH) synthesis. Vanin-1 promotes inflammation and tissue injury partly by inducing oxidative stress, and partly by peroxisome proliferator-activated receptor gamma (PPARγ) expression. Vascular smooth muscle cells (SMCs) contribute to neointimal hyperplasia in response to injury, by multiple mechanisms including modulation of oxidative stress and PPARγ. Therefore, we tested the hypothesis that vanin-1 drives SMC activation and neointimal hyperplasia. We studied reactive oxygen species (ROS) generation and functional responses to platelet-derived growth factor (PDGF) and the pro-oxidant diamide in cultured mouse aortic SMCs, and also assessed neointima formation after carotid artery ligation in vanin-1 deficiency. Vnn1(-/-) SMCs demonstrated decreased oxidative stress, proliferation, migration, and matrix metalloproteinase 9 (MMP-9) activity in response to PDGF and/or diamide, with the effects on proliferation linked, in these studies, to both increased GSH levels and PPARγ expression. Vnn1(-/-) mice displayed markedly decreased neointima formation in response to carotid artery ligation, including decreased intima:media ratio and cross-sectional area of the neointima. We conclude that vanin-1, via dual modulation of GSH and PPARγ, critically regulates the activation of cultured SMCs and development of neointimal hyperplasia in response to carotid artery ligation. Vanin-1 is a novel potential therapeutic target for neointimal hyperplasia following revascularization.     

Localization and effects of hepatocyte growth factor on smooth muscle cells during neointimal formation after balloon denudation

The migration and proliferation of smooth muscle cells (SMCs) may play a key role in tissue remodeling after arterial wall injury. We investigated the localization and effects of hepatocyte growth factor (HGF) in rabbit carotid arteries after balloon denudation. Immunoreactivity for HGF and the c-Met receptor was clearly observed in neointimal SMCs. The immunoreactivity was not restricted to proliferating cells but was seen even in non-dividing cells in the basal layer of the neointima 4 and 6 weeks after balloon denudation. The distribution of platelet-derived growth factor (PDGF)-positive cells paralleled that of proliferating SMCs. The SMCs in the basal layer of the neointima at 4 and 6 weeks were positive for matrix metalloproteinase (MMP)-2 and membrane type 1-MMP which can activate the proform of MMP-2. HGF significantly stimulated the migration but not proliferation of cultured SMCs. Our results suggest that HGF and PDGF act in coordination to promote the proliferation and migration of SMCs in the earlier phases of neointimal formation and that HGF as well as MMP-2 contribute to the later stages by facilitating the migration but not replication of SMCs. Accepted: 19 March 1999     

Cytomegalovirus aggravates intimal hyperplasia in rats by stimulating smooth muscle cell proliferation

Epidemiological and animal studies suggest a role for cytomegalovirus (CMV) in restenosis. Previously, we demonstrated that proliferating smooth muscle cells (SMCs) in the injured arterial wall are particularly susceptible to CMV-induced effects. Therefore, we hypothesised that, depending on the time point of infection after vascular injury, CMV infection may affect cell proliferation either in the media or in the neointima, thereby aggravating the process of restenosis. In the present study, we focused on the individual layers of the arterial wall by evaluating, besides the neointima-to-media ratio, the medial and neointimal area and cellularity in the rat femoral artery. Vascular injury was photochemically induced in rat femoral arteries. Immediately or 14 days thereafter, rats were infected with rat CMV (RCMV) or mock infected. The presence of RCMV in the vascular wall was determined at 3, 5, 14 and 35 days after infection by quantitative real-time PCR. When rats were infected immediately after injury, a significant increase was seen only in the medial but not in the neointimal cross-sectional area. On the other hand, when rats were infected 14 days after the initial injury, a significant increase was only seen in the neointimal area, thereby confirming our hypothesis that RCMV infection primary affects proliferating SMCs. As the mean number of SMCs per microm2 in both cell layers was unchanged despite an increase in cross-sectional area, this implies that RCMV stimulated SMC proliferation. Furthermore, these vascular effects were observed without the virus being abundantly present in the vascular wall, suggesting that inflammatory and immune-mediated responses to RCMV infection are more important in aggravating the response to vascular injury than the virus itself.     

Expression of heparan sulphate N-deacetylase/N-sulphotransferase by vascular smooth muscle cells

Heparan sulphate is an important mediator in determining vascular smooth muscle cell (SMC) phenotype. The sulphation pattern of the heparan sulphate chains is critical to their function. We have examined the initial step in the biosynthesis of the sulphated domains mediated by the enzyme heparan sulphate N-deacetylase/N-sulphotransferase (NDST). Rabbit aortic SMC in primary culture exhibited NDST enzyme activity and expressed NDST-1 in their Golgi apparatus, with maximal expression in SMC 2 days after dispersal in primary culture confirmed by Western blot analysis. Endothelial cells, macrophages and fibroblasts expressed NDST-1 but had generally less intense staining than SMC, although SMC expression decreased with culture. The uninjured rat aorta also showed widespread expression of NDST-1. After balloon de-endothelialisation, NDST-1 could not be detected in SMC of the neointima in the early stages of neointimal formation, but was re-expressed at later time points (after 12 weeks). In human coronary arteries, SMC of the media and the diffuse intimal thickening expressed NDST-1, while SMC in the atherosclerotic plaque were negative for NDST-1. We conclude that SMC may regulate their heparan sulphate sulphation at the level of expression of the enzyme heparan sulphate NDST in a manner related to their phenotypic state.     

ANG II-induced neointimal growth is mediated via cPLA2- and PLD2-activated Akt in balloon-injured rat carotid artery

Angiotensin II (ANG II) promotes neointimal growth in the balloon-injured rat carotid artery. However, the mechanism by which ANG II stimulates neointimal growth during vascular injury is not known. In cultured vascular smooth muscle cells, ANG II activates Akt through cytosolic phospholipase A2 (cPLA2)-dependent phospholipase D2 (PLD2). This study was conducted to determine whether ANG II-induced neointimal thickening is mediated via cPLA2- and PLD2-activated Akt in balloon-injured rat carotid arteries. ANG II-stimulated neointimal growth was inhibited by exposure of the injured carotid arteries to an adenovirus containing a dominant negative Akt mutant (intima-to-media ratio from 3.01 +/- 0.31 to 1.44 +/- 0.14, P < 0.01) or a retrovirus containing cPLA2 small interfering RNA (siRNA; intima-to-media ratio from 3.01 +/- 0.31 to 1.16 +/- 0.36, P < 0.001) or PLD2 siRNA (intima-to-media ratio from 3.01 +/- 0.31 to 1.33 +/- 0.11, P < 0.001). The effect of cPLA2 and PLD2 siRNA to reduce the ANG II-induced increase in neointimal thickening was associated with reduced expression of cPLA2 and PLD2 as determined by immunohistochemical analysis in injured carotid arteries. Western blot analysis showed that Akt phosphorylation that was increased by ANG II was inhibited in injured carotid arteries 2 days after exposure to cPLA2 or PLD2 siRNA or in injured arteries isolated after exposure to these agents for 30 min and then placed in tissue culture media for 24 h in the presence of these agents. These data suggest that the ANG II-induced neointimal growth is mediated by the activation of Akt through a mechanism dependent on cPLA2 and PLD2 activation in balloon-injured rat carotid arteries.     

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

Background aimsPreviously, 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.MethodsArterial 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.ResultsThe 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.ConclusionsDS 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.     

Immunolocalization of matrix metalloproteinases in rabbit carotid arteries after balloon denudation

 Extracellular matrix-degrading enzymes may play a key role in vascular remodeling after arterial wall injury. We investigated the immunolocalization of matrix metalloproteinases (MMPs) in rabbit carotid arteries after balloon denudation. Positive immunostaining for MMP-1, -2, -3, and -9 appeared through the neointima 1 week after balloon denudation. The localization of immunopositive smooth muscle cells (SMCs) for MMP-1, -3, and -9, particularly for MMP-9, was almost similar to that of replicative SMCs and became confined to the luminal surface layer of the neointima at later time periods. However, MMP-2-positive SMCs appeared also in the basal layer of the neointima at 2 weeks, increased at 4 weeks, and then totally occupied the neointima at 6 weeks. The MMP-2-positive SMCs in the basal layer of the neointima at 4 and 6 weeks were negative for proliferation-associated antigens and were surrounded by extracellular matrix proteins. Our results suggest that all MMPs act in coordination to promote replication and migration of SMCs in the earlier phases of neointimal formation and that MMP-2 independently contributes to the later stages by facilitating the migration but not replication of SMCs from the media to the intima. Accepted: 25 June 1997     

The origin of neointimal cells in the rat carotid artery after balloon angioplasty

At present the issue of a possible role of circulating stem cells and precursors in pathological vascular wall remodeling after angioplasty remains unsolved. Therefore the origin of neointimal cells was examined in the rat carotid artery after balloon angioplasty using morphological and immunocytochemical approaches. It is shown that at the early stages (1-7 days) after vessel injury acute inflammatory response arises in the arterial wall recruiting neutrophils, monocytes, macrophages as well as large amounts of low-differentiated blood-derived cells. At the late stages (10-28 days), at the area of injured intima, a new hyperplastic intima (neointima) is formed, which consists of cells carrying specific smooth muscle markers--alpha-actin and smoothelin. The study on cell proliferative behaviour in the injured vessel wall by bromodeoxyuridine showed that in the process of neointima formation blood-born rather than resident cells are involved. Probably, early smooth muscle and endothelial precursor cells penetrate into injured area with blood stream, where they proliferative and differentiate into mature cells.