Intramuscular gene transfer of CGRP inhibits neointimal hyperplasia after balloon injury in the rat abdominal aorta

CGRP is a well-known neuropeptide that has various protective effects on cardiovascular system. Our previous studies have shown that CGRP inhibits vascular smooth muscle cell (VSMC) proliferation in vitro. The present study aimed to explore the role of the CGRP in neointimal formation after balloon injury in the rat aortic wall and the underlying mechanism. Gene transfer of CGRP was performed with the use of intramuscular electroporation in a balloon-injured rat aorta model. Apoptosis in VSMCs was determined by electrophoresis assessment of DNA fragmentation and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay. Overexpression of the CGRP gene significantly inhibited the neointimal formation after balloon injury compared with the mock transfer, as assessed by the intima-to-media ratio 14 days after balloon injury (29.2 +/- 3.7% vs. 52.7 +/- 5.4%; n = 9-12, P < 0.05). In addition, CGRP gene expression increased the number of apoptotic cells in the neointima in vivo 14 days after balloon injury. Similarly, the addition of bioactive CGRP and the nitric oxide donor induced similar apoptosis in cultured VSMCs. The antagonist of the CGRP(1) receptor and inhibitors of cAMP-PKA and nitric oxide blocked CGRP-mediated apoptosis. Furthermore, CGRP gene transfer increased inducible nitric oxide synthase and p53 but decreased PCNA and Bcl-2 protein levels in balloon-injured rat aorta. Our data demonstrated that CGRP potently inhibited neointimal thickening in the rat aorta, at least in part through its distinct effects on apoptosis and proliferation of VSMCs both in vivo and in vitro. Therefore, delivery of the CGRP gene may have therapeutic implications in limiting vascular restenosis.     

Electrical stimulation inhibits neointimal hyperplasia after abdominal aorta balloon injury through the PTEN/p27Kip1 pathway

Electric fields (EFs) exert biological effects on promoting wound healing by facilitating cell division, cell proliferation, and cell directional migration toward the wound. In this study, we examined the inhibitory effect of direct-current (DC) EFs on the formation of neointimal hyperplasia and the possible mechanism in an abdominal aorta balloon injury rabbit model. Sixty rabbits were divided into normal, control, and experimental groups. After establishment of the abdominal aorta balloon injury model, electrodes were implanted into the bilateral psoas major muscle in control and experimental groups. Only the experimental group received electric stimulation (EFs applied at 3 or 4 V/cm for 30 min/day) for 1, 2, and 4 weeks, respectively. Neointimal hyperplasia of the abdominal aorta and proliferation of vascular smooth muscle cells (VSMCs) were measured. Expressions of collagen, p27(Kip1), and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) were detected. Results showed that the ratio of the tunica intima area to the tunica media area, the expression of type-I collagen in the neointimal, and the proliferating cell nuclear antigen index in experimental groups were significantly less than those in control groups 2 weeks post-operation (P< 0.01). Expressions of p27(Kip1) and PTEN were increased in experimental groups compared with control groups (P< 0.01). In conclusion, our results suggested that the application of DC EFs could inhibit neointimal hyperplasia and reduce collagen expression after abdominal aorta balloon injury. This was probably induced by upregulation of PTEN/p27(Kip1) expression, thereby inhibiting VSMC proliferation.     

Sex differences in intracranial arterial bifurcations

Background: Subarachnoid hemorrhage (SAH) is a serious condition, occurring more frequently in females than in males. SAH is mainly caused by rupture of an intracranial aneurysm, which is formed by localized dilation of the intracranial arterial vessel wall, usually at the apex of the arterial bifurcation. The female preponderance is usually explained by systemic factors (hormonal influences and intrinsic wall weakness); however, the uneven sex distribution of intracranial aneurysms suggests a possible physiologic factor—a local sex difference in the intracranial arteries.Objective: The aim of this study was to explore sex variation in the bifurcation anatomy of the middle cerebral artery (MCA) and internal carotid artery (ICA), and the subsequent hemodynamic impact.Methods: Vessel radii and bifurcation angles were measured in patients with MCA and ICA bifurcations. Data from a previously published study of 55 patients undergoing diagnostic cerebral digital subtraction angiography at Dalcross Private Hospital in Sydney, Australia, between 2002 and 2003, were available for analysis. The measurements were used to create idealized, averaged bifurcations of the MCA and ICA for females and males. Computational fluid dynamics simulations were performed to calculate hemodynamic forces in the models.Results: The vessel radii and bifurcation angles of 47 MCA and 52 ICA bifurcations in 49 patients (32 females, 17 males; mean age, 53 years; age range, 14–86 years) were measured. Statistically significant sex differences were found in vessel diameter (males larger than females; P < 0.05), but not in bifurcation angle. Computational fluid dynamics simulations revealed higher wall shear stress in the female MCA (19%) and ICA (50%) bifurcations compared with the male bifurcations.Conclusions: This study of MCA and ICA bifurcations in female and male patients suggests that sex differences in vessel size and blood flow velocity result in higher hemodynamic forces acting on the vessel wall in females. This new hypothesis may partly explain why intracranial aneurysms and SAH are more likely to occur in females than in males.     

MCP-1 deficiency is associated with reduced intimal hyperplasia after arterial injury

Monocyte chemoattractant protein (MCP)-1 is abundant in smooth muscle cells (SMC) and macrophages of atherosclerotic plaques and in the injured arterial wall. MCP-1 and its receptor, CCR2, are important mediators of macrophage accumulation and atherosclerotic plaque progression. We have recently reported that CCR2(-/-) mice have a approximately 60% decrease in intimal hyperplasia and medial DNA synthesis in response to femoral arterial injury. We have now examined the response to femoral arterial injury in MCP-1(-/-) mice. MCP-1 deficiency was associated with a approximately 30% reduction in intimal hyperplasia at 4 weeks and was not associated with diminished medial DNA synthesis. Despite inducing tissue factor in SMC culture, MCP-1 deficiency was not associated with a decrease in neointimal tissue factor after injury. These data suggest that MCP-1 and CCR2 deficiencies have distinct effects on arterial injury. The effects of MCP-1 on intimal hyperplasia may be mediated largely through SMC migration.     

Femoral artery neointimal hyperplasia is reduced after wire injury in Ref-1+/- mice

Redox factor-1 (Ref-1) is a multifunctional protein that regulates redox, DNA repair, and the response to cell stress. We previously demonstrated that Ref-1(+/-) mice exhibit a significantly reduced Ref-1 mRNA and protein levels within the vasculature, which are associated with increased oxidative stress. The goal of this study was to test the hypothesis that partial loss of Ref-1 altered the cellular response to vascular injury. Fourteen days after femoral artery wire injury, we found that vessel intima-to-media ratio was significantly reduced in Ref-1(+/-) mice compared with that in wild-type mice (P < 0.01). Bromodeoxyuridine labeling and transferase-mediated dUTP nick-end labeling staining at 14 days did not differ in the Ref-1(+/-) mice. In vitro studies found no significant changes in either serum-induced proliferation or baseline apoptosis in Ref-1(+/-) vascular smooth muscle cells. Exposure to Fas ligand; however, did result in increased susceptibility of Ref-1(+/-) vascular smooth muscle cells to apoptosis (P < 0.001). Ref-1(+/-) mice exhibited an increase in circulating baseline levels of IL-10, IL-1alpha, and VEGF compared with those in wild-type mice but a marked impairment in these pathways in response to injury. In sum, loss of a single allele of Ref-1 is sufficient to reduce intimal lesion formation and to alter circulating cytokine and growth factor expression.     

Systemic alpha 1A-adrenoceptor antagonist inhibits neointimal growth after balloon injury of rat carotid artery

Previous in vitro and in vivo studies have shown that norepinephrine, acting through alpha(1A)-adrenoceptors, stimulates hypertrophy, proliferation, and migration of vascular smooth muscle cells and adventitial fibroblasts and may contribute to neointimal growth, lumen loss, and inward remodeling caused by iatrogenic wall injury and vascular disease. Our present aim was to determine whether intravenous administration of the alpha(1A)-adrenoceptor antagonist KMD-3213, at dosages without systemic hemodynamic effects, inhibits wall growth after injury. Inhibition of alpha(1A)-adrenoceptors with 12.8 and 32 microg/kg KMD-3213 had no effect on arterial pressure or renal and hindquarter resistances in anesthetized rats. A second group then received carotid balloon injury and continuous intravenous KMD-3213 at 4 and 10 microg x kg(-1) x h(-1) for 2 wk. Mean, systolic, and diastolic arterial pressures and heart rate of conscious unrestrained rats were unaffected. KMD-3213 reduced neointima growth by approximately 30 and 46% at the two doses (P < 0.01). These data support the novel hypothesis that a direct alpha(1A)-adrenoceptor-dependent trophic action of catecholamines is augmented by injury and may contribute significantly to hypertrophic vascular disease.     

Effect of a dipeptidyl peptidase-IV inhibitor, des-fluoro-sitagliptin, on neointimal formation after balloon injury in rats

Background

Recently, it has been suggested that enhancement of incretin effect improves cardiac function. We investigated the effect of a DPP-IV inhibitor, des-fluoro-sitagliptin, in reducing occurrence of restenosis in carotid artery in response to balloon injury and the related mechanisms.

Methods and Findings

Otsuka Long-Evans Tokushima Fatty rats were grouped into four: control (normal saline) and sitagliptin 100, 250 and 500 mg/kg per day (n = 10 per group). Sitagliptin or normal saline were given orally from 1 week before to 2 weeks after carotid injury. After 3 weeks of treatment, sitagliptin treatment caused a significant and dose-dependent reduction in intima-media ratio (IMR) in obese diabetic rats. This effect was accompanied by improved glucose homeostasis, decreased circulating levels of high-sensitivity C-reactive protein (hsCRP) and increased adiponectin level. Moreover, decreased IMR was correlated significantly with reduced hsCRP, tumor necrosis factor-α and monocyte chemoattractant protein-1 levels and plasminogen activator inhibitor-1 activity. In vitro evidence with vascular smooth muscle cells (VSMCs) demonstrated that proliferation and migration were decreased significantly after sitagliptin treatment. In addition, sitagliptin increased caspase-3 activity and decreased monocyte adhesion and NFκB activation in VSMCs.

Conclusions

Sitagliptin has protective properties against restenosis after carotid injury and therapeutic implications for treating macrovascular complications of diabetes.     

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.     

Sex differences and estrogen modulation of the cellular immune response after injury

Cell-mediated immunity is extremely important for resolution of infection and for proper healing from injury. However, the cellular immune response is dysregulated following injuries such as burn and hemorrhage. Sex hormones are known to regulate immunity, and a well-documented dichotomy exists in the immune response to injury between the sexes. This disparity is caused by differences in immune cell activation, infiltration, and cytokine production during and after injury. Estrogen and testosterone can positively or negatively regulate the cellular immune response either by aiding in resolution or by compounding the morbidity and mortality. It is apparent that the hormonal dysregulation is dependent not only on the type of injury sustained but also the amount of circulating hormones. Therefore, it may be possible to design sex-specific therapies to improve immunological function and patient outcome.     

Regional differences in viral growth and central nervous system injury correlate with apoptosis

Infection of neonatal mice with reovirus T3 Dearing (T3D), the prototypic neurotropic reovirus, causes fatal encephalitis associated with neuronal injury and virus-induced apoptosis throughout the brain. T3D variant K (VarK) is an antigenic variant that has a nearly 1 million-fold reduction in neurovirulence following intracerebral (i.c.) inoculation compared to T3D and a restricted pattern of central nervous system injury with damage limited to the hippocampus, sparing other brain regions. We wished to determine whether the restricted pattern of VarK-induced injury was due to a reduced capacity to replicate in or injure cortical, as opposed to hippocampal, tissue. We found that following i.c. inoculation, VarK grew to similar titers as T3D in the hippocampus but had significantly lower titers in the cortex. Both viruses grew to identical titers and infected the same percentage of cells in mouse primary hippocampal cultures (MHC). In mouse primary cortical cultures (MCC) both the number of infected cells and the viral yield per infected cell were significantly lower for VarK than T3D. VarK-induced apoptosis was limited to the hippocampus in vivo, and in vitro both viruses induced apoptosis equally in MHC but VarK induced significantly less apoptosis than T3D in MCC. Growth of T3D in MCC was reduced to levels comparable to those of VarK following treatment of MCC with caspase inhibitors. Conversely, induction of apoptosis in VarK-infected MCC with fatty acid synthase-activating antibody significantly enhanced viral yield. These results suggest that the decreased neurovirulence of VarK may be due to its failure to efficiently induce apoptosis in cortical neurons.     

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.     

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.     

Protective roles of endogenous carbon monoxide in neointimal development elicited by arterial injury

We reported that carbon monoxide (CO) generated through heme oxygenase (HO) inhibits mitogen-induced proliferation of vascular smooth muscle cells (VSMCs). We report that balloon injury induces HO-1, the stress-inducible isozyme of HO, in VSMCs and inhibits neointimal formation through the action of endogenous CO. Northern blot analysis and immunohistochemistry revealed that HO-1 is markedly induced in the media as early as 1 day after injury, whereas only a little expression was detected in the intact carotid artery. The neointimal proliferative changes were augmented or inhibited by the HO inhibitors or inducer, respectively, and effects of these interventions were not altered by suppression of endogenous nitric oxide (NO), if any. To elucidate the mechanisms by which HO controls the proliferative changes, effects of alterations in the HO reaction were examined by determining angiotensin II-elicited VSMC proliferation in vitro: the HO inducer attenuated and its inhibitor restored the proliferative response to angiotensin II (1 nM and 100 nM). Hemoglobin, a reagent trapping both NO and CO, but not met-hemoglobin, which can capture NO but not CO, augmented the proliferative response. These data suggest that endogenous CO serves as a protective factor that limits the excessive VSMC proliferation associated with vascular diseases.     

HCMV-infection in a human arterial organ culture model: effects on cell proliferation and neointimal hyperplasia

Background

The impact of infections with the human cytomegalovirus (HCMV) for the development of atherosclerosis and restenosis is still unclear. Both a clear correlation and no correlation at all have been reported in clinical, mostly serological studies. In our study we employed a human non-injury ex vivo organ culture model to investigate the effect of an in vitro permissive HCMV-infection on cell proliferation and neointimal hyperplasia for a period of 56 days.

Results

During routine-nephrectomies parts of renal arteries from 71 patients were obtained and prepared as human organ cultures. Cell free HCMV infection was performed with the fibroblast adapted HCMV strain AD169, the endotheliotropic strain TB40E, and a clinical isolate (AN 365). After 3, 7, 14, 21, 28, 35, and 56 days in culture staining of HCMV-antigens was carried out and reactive cell proliferation and neointimal thickening were analysed. Successful HCMV-infection was accomplished with all three virus strains studied. During the first 21 days in organ culture no cell proliferation or neointimal hyperplasia was detected. At day 35 and day 56 moderate cell proliferation and neointimal hyperplasia was found both in HCMV-infected segments and mock infected controls. Neointimal hyperplasia in productively HCMV-infected segments was lower than in non infected at day 35 and day 56, but relatively higher after infection with the endotheliotropic TB40E in comparison with the two other strains.

Conclusion

The data do not support the hypothesis that HCMV-infection triggers restenosis via a stimulatory effect on cell proliferation and neointimal hyperplasia in comparison to non infected controls. Interestingly however, even after lytic infection, a virus strain specific difference was observed.     

Sex differences in children's free drawings: a study on girls with congenital adrenal hyperplasia

Sex differences are recognized in children's free drawings with respect to motifs, colors, figure compositions, and expression. Boys tend to draw mobile objects and mechanical objects with dark or cold colors and often use bird's-eye-view composition when they draw pictures, whereas girls like to draw human motifs (especially girls and women), flowers, and butterflies with light and warm colors and tend to arrange motifs in a row on the ground. Analyses were made on the drawings of girls with congenital adrenal hyperplasia (CAH) and unaffected boys and girls, using masculine and feminine indexes. Sex difference in masculine and feminine indexes was clear in the drawings by unaffected boys and girls. Their drawings do not or mostly do not contain characteristics typical of the opposite sex. Compared with those of unaffected girls, the pictures of CAH girls more strongly showed masculine characteristics. The feminine index for the pictures of CAH girls was significantly lower than that for unaffected girls, while the masculine index for CAH girls was significantly higher than that for unaffected girls. Furthermore, the masculine index for CAH girls was not significantly different from that of unaffected boys. These results suggest that androgen exposure during fetal life may contribute to shaping masculine characteristics in children's free drawings.     

Upregulation of proteinase-activated receptors and hypercontractile responses precede development of arterial lesions after balloon injury

Thrombin and other proteinases exert vascular effects by activating the proteinase-activated receptors (PARs). The expression of PARs has been shown to be upregulated after balloon injury and in human arteriosclerosis. However, the relationship between the receptor upregulation and the alteration of vasomotor function remains to be elucidated. We herein demonstrated that the contractile responses to the PAR-1 and PAR-2 agonist were markedly enhanced in the rabbit femoral arteries after balloon injury. Neointimal thickening was established 4 wk after the injury. No histological change was observed in the sham operation, where the saphenous artery was ligated without any balloon injury. The contractile response to K(+) depolarization was significantly attenuated 1 wk after the injury and then partly recovered after 4 wk. Thrombin, PAR-1-activating peptide, trypsin, and PAR-2-activating peptide induced no significant contraction in the control. All these stimulants induced enhanced responses 1 wk after balloon injury. Such enhanced responses were seen 4 wk after the injury, except for thrombin. There was no change in the Ca(2+) sensitivity of the contractile apparatus as evaluated in the permeabilized preparations. PAR-1-activating peptide (100 mumol/l), but no other stimulants, induced an enhanced contraction in the sham operation. The expression of PAR-1 and PAR-2 slightly increased after the sham operation, whereas it markedly and significantly increased after balloon injury. Our observations suggest that balloon injury induced the receptor upregulation, thereby enhancing the contractile response before the establishment of vascular lesions. The local inflammation associated with the sham operation may also contribute to the receptor upregulation.     

Isoeugenodilol inhibits smooth muscle cell proliferation and neointimal thickening after balloon injury via inactivation of ERK1/2 pathway

The purpose of this study was to determine the efficacy and the possible mechanism of action of the synthesized drug isoeugenodilol (a new third-generation β-adrenoceptor blocker) on the growth factor-induced proliferation of cultured rat vascular smooth muscle cells (VSMCs) and neointimal formation in a rat carotid arterial balloon injury model. Isoeugenodilol significantly inhibited 10% FBS, 20 ng/ml PDGF-BB, and 20 ng/ml vascular endothelial growth factor (VEGF)-induced proliferation. In accordance with these findings, isoeugenodilol revealed blocking of the FBS-inducible progression through the G₀/G₁ to the S phase of the cell cycle in synchronized cells. Neointimal formation, measured 14 days after injury, was reduced by the oral administration of isoeugenodilol (10 mg/kg/day). In an in vitro assay, isoeugenodilol inhibited the migration of VSMCs stimulated by PDGF-BB. These findings indicate that isoeugenodilol shows an inhibitory potency on neointimal formation due to inhibition of both migration and proliferation of VSMCs. In addition, isoeugenodilol in concentration-dependent manner decreased the levels of phosphorylated ERK1/2 in both VSMCs and balloon-injured carotid arteries. The levels of phosphorylated MEK1/2 and Pyk2 as well as intracellular Ca²⁺ and reactive oxygen species (ROS) were in concentration-dependent manner reduced by isoeugenodilol. Taken together, these results indicate that isoeugenodilol may suppress mitogen-stimulated proliferation and migration partially through inhibiting cellular ROS and calcium, and hence, through activation of the Pyk2-ERK1/2 signal pathway. This suggests that isoeugenodilol has potential for the prevention of atherosclerosis and restenosis.     

Sex differences in the myocardial inflammatory response to ischemia-reperfusion injury

The myocardium generates inflammatory mediators during ischemia-reperfusion (I/R), and these mediators contribute to cardiac functional depression and apoptosis. The great majority of these data have been derived from male animals and humans. Sex has a profound effect over many inflammatory responses; however, it is unknown whether sex affects the cardiac inflammatory response to acute myocardial I/R. We hypothesized the existence of inherent sex differences in myocardial function, expression of inflammatory cytokines, and activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway after I/R. Isolated rat hearts from age-matched adult males and females were perfused (Langendorff), and myocardial contractile function was continuously recorded. After I/R, myocardium was assessed for expression of TNF-alpha, IL-1beta, and IL-6 (RT-PCR, ELISA); IL-1alpha and IL-10 mRNA (RT-PCR); and activation of p38 MAPK (Western blot). All indexes of postischemic myocardial function [left ventricular developed pressure, left ventricular end-diastolic pressure, and maximal positive (+dP/dt) and negative (-dP/dt) values of the first derivative of pressure] were significantly improved in females compared with males. Compared with males, females had decreased myocardial TNF-alpha, IL-1beta, and IL-6 (mRNA, protein) and decreased activation of p38 MAPK pathway. These data demonstrate that hearts from age-matched adult females are relatively protected against I/R injury, possibly due to a diminished inflammatory response.