Intimal Hyperplasia Response Research Articles

Effect of sequential release of sirolimus and rosuvastatin using silk fibroin microneedle to prevent intimal hyperplasia

Intimal hyperplasia (IH) is a major cause of vascular restenosis after bypass surgery, which progresses as a series of processes from the acute to chronic stage in response to endothelial damage during bypass grafting. A strategic localized drug delivery system that reflects the pathophysiology of IH and minimizes systemic side effects is necessary. In this study, the sequential release of sirolimus, a mechanistic target of rapamycin (mTOR) inhibitor, and statin, an HMG-COA inhibitor, was realized as a silk fibroin-based microneedle device in vivo. The released sirolimus in the acute stage reduced neointima (NI) and vascular fibrosis through mTOR inhibition. Furthermore, rosuvastatin, which was continuously released from the acute to chronic stage, reduced vascular stiffness and apoptosis through the inactivation of Yes-associated protein (YAP). The sequential release of sirolimus and rosuvastatin confirmed the synergistic treatment effects on vascular inflammation, VSMC proliferation, and ECM degradation remodeling through the inhibition of transforming growth factor (TGF)-beta/NF-κB pathway. These results demonstrate the therapeutic effect on preventing restenosis with sufficient vascular elasticity and significantly reduced IH in response to endothelial damage. Therefore, this study suggests a promising strategy for treating coronary artery disease through localized drug delivery of customized drug combinations.

Deficiency of cysteinyl cathepsin K suppresses the development of experimental intimal hyperplasia in response to chronic stress.

Chronic psychological stress (CPS) is linked to cardiovascular disease initiation and progression. Given that cysteinyl cathepsin K (CatK) participates in vascular remodeling and atherosclerotic plaque growth in several animal models, we investigated the role of CatK in the development of experimental neointimal hyperplasia in response to chronic stress. At first, male wild-type (CatK) mice that underwent carotid ligation injury were subjected to chronic immobilization stress. On postoperative and stressed day 14, the results demonstrated that stress accelerated injury-induced neointima hyperplasia. On day 4, stressed mice showed following: increased levels of monocyte chemoattractant protein-1, gp91phox, toll-like receptor-2 (TLR2), TLR4, and CatK mRNAs or/and proteins, oxidative stress production, aorta-derived smooth muscle cell (SMC) migration, and macrophage infiltration as well as targeted intracellular proliferating-related molecules. Stressed mice showed increased matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA expressions and activities and elastin disruption in the injured carotid arteries. Second, CatK and CatK deficiency (CatK) mice received ligation injury and stress to explore the role of CatK. The stress-induced harmful changes were prevented by CatK. Finally, CatK mice that had undergone ligation surgery were randomly assigned to one of two groups and administered vehicle or CatK inhibitor for 14 days. Pharmacological CatK intervention produced a vascular benefit. These data indicate that CatK deletion protects against the development of experimental neointimal hyperplasia via the attenuation of inflammatory overaction, oxidative stress production, and VSMC proliferation, suggesting that CatK is a novel therapeutic target for the management of CPS-related restenosis after intravascular intervention therapies.

PCSK6 Is a Key Protease in the Control of Smooth Muscle Cell Function in Vascular Remodeling.

PCSKs (Proprotein convertase subtilisins/kexins) are a protease family with unknown functions in vasculature. Previously, we demonstrated PCSK6 upregulation in human atherosclerotic plaques associated with smooth muscle cells (SMCs), inflammation, extracellular matrix remodeling, and mitogens. Here, we applied a systems biology approach to gain deeper insights into the PCSK6 role in normal and diseased vessel wall. Genetic analyses revealed association of intronic PCSK6 variant rs1531817 with maximum internal carotid intima-media thickness progression in high-cardiovascular risk subjects. This variant was linked with PCSK6 mRNA expression in healthy aortas and plaques but also with overall plaque SMA+ cell content and pericyte fraction. Increased PCSK6 expression was found in several independent human cohorts comparing atherosclerotic lesions versus healthy arteries, using transcriptomic and proteomic datasets. By immunohistochemistry, PCSK6 was localized to fibrous cap SMA+ cells and neovessels in plaques. In human, rat, and mouse intimal hyperplasia, PCSK6 was expressed by proliferating SMA+ cells and upregulated after 5 days in rat carotid balloon injury model, with positive correlation to PDGFB (platelet-derived growth factor subunit B) and MMP (matrix metalloprotease) 2/MMP14. Here, PCSK6 was shown to colocalize and cointeract with MMP2/MMP14 by in situ proximity ligation assay. Microarrays of carotid arteries from Pcsk6-/- versus control mice revealed suppression of contractile SMC markers, extracellular matrix remodeling enzymes, and cytokines/receptors. Pcsk6-/- mice showed reduced intimal hyperplasia response upon carotid ligation in vivo, accompanied by decreased MMP14 activation and impaired SMC outgrowth from aortic rings ex vivo. PCSK6 silencing in human SMCs in vitro leads to downregulation of contractile markers and increase in MMP2 expression. Conversely, PCSK6 overexpression increased PDGFBB (platelet-derived growth factor BB)-induced cell proliferation and particularly migration. PCSK6 is a novel protease that induces SMC migration in response to PDGFB, mechanistically via modulation of contractile markers and MMP14 activation. This study establishes PCSK6 as a key regulator of SMC function in vascular remodeling. Visual Overview: An online visual overview is available for this article.

Increased Intimal Hyperplasia After Vascular Injury in Male Androgen Receptor-Deficient Mice.

Intimal hyperplasia is a vascular pathological process involved in the pathogenesis of atherosclerosis. Data suggest that T, the most important sex steroid hormone in males, protects men from atherosclerotic cardiovascular disease. T mainly acts via the androgen receptor (AR), and in this study we evaluated formation of intimal hyperplasia in male AR knockout (ARKO) mice using a vascular injury model. Two weeks after ligation of the carotid artery, male ARKO mice showed increased intimal area and intimal thickness compared with controls. After endothelial denudation by an in vivo scraping injury, there was no difference in the reendothelialization in ARKO compared with control mice. Ex vivo, we observed increased outgrowth of vascular smooth muscle cells from ARKO compared with control aortic tissue explants; the number of outgrown cells was almost doubled in ARKO. In vitro, stimulation of human aortic vascular smooth muscle cells with a physiological T concentration inhibited both migration and proliferation of the cells. Analyzing the expression of central regulators of cell proliferation and migration, we found that mRNA and protein levels of p27 were lower in uninjured arteries from ARKO mice and that T replacement to castrated male mice increased p27 mRNA in an AR-dependent manner. In conclusion, AR deficiency in male mice increases intimal hyperplasia in response to vascular injury, potentially related to the effects of androgens/AR to inhibit proliferation and migration of smooth muscle cells.

Effect of Hormone Replacement Therapy in Matrix Metalloproteinase Expression and Intimal Hyperplasia Development After Vascular Injury

Postmenopausal women taking hormone replacement therapy (HRT) require secondary intervention after vascular reconstruction more frequently than women not taking HRT, often due to increased development of intimal hyperplasia (IH). Matrix metalloproteinases (MMPs) play a role in IH by degradation and remodeling of components of the vascular basement membrane. The MMP pathway is regulated by a balance between MMPs, membrane-type MMPs (MT-MMPs), and tissue inhibitor of MMPs (TIMPs). We have recently provided evidence for unbalanced regulation of the MT1-MMP/MMP-2 pathway in vascular smooth muscle cells (VSMCs) exposed to hormones in vitro. Herein we study the role of HRT in the development of IH in a postmenopausal rodent model of vascular injury and in the modulation of this MMP regulatory pathway in vivo. Female rats were aged to 12 months. Animals were ovariectomized (OVX) and 4 weeks later hormones or placebo was delivered via a 90-day slow-release pellet. After 6 weeks of HRT each rat underwent balloon angioplasty of the left common carotid artery. At 14 days postinjury tissue samples were collected and stained with trichrome elastin and for isoform-specific MMPs. After vascular injury, the intima:media (I:M) ratio was decreased in OVX rats receiving placebos as compared with non-OVX controls (P < 0.05). In OVX animals receiving HRT, estrogen with and without progesterone and progesterone alone slightly increased I:M ratio compared with placebo, although no significant difference was found in any HRT group. Injury-induced intimal expression of MMP-2 and -9 was decreased in OVX placebo animals compared with non-OVX controls (P < 0.05). MMP-2 and -9 levels were subsequently increased by each type of hormone therapy compared with placebo, with a significant increase in MMP-9 in response to estrogen with and without progesterone (P < 0.05). Conversely, TIMP-2 was decreased by estrogen compared with placebo (P < 0.05). There was no effect on intimal MT1-MMP in any group. In this study we detected a statistically significant decrease in IH as a result of OVX. Subsequent HRT exposure resulted in increased I:M ratios compared with OVX animals given placebo, although significance was not reached with the doses given. Long-term exogenous exposure may have a more deleterious effect compared with acute exposure and should be examined further. We also demonstrated a significant reduction in MMP-2 and -9 and TIMP-2 in response to OVX. Subsequent hormone exposure resulted in the upregulation of MMP-2 and -9 without a counterregulatory increase in TIMP, indicating that HRT modulates the MMP regulatory pathway in vivo. The data suggest that the lack of hormones after OVX protects against pathologic remodeling in our aged model of disease and that exposure to both natural and exogenous hormones could be a negative risk factor resulting in an exaggerated vascular response to injury. Future studies should focus on in vivo manipulation of unbalanced MMP regulation for prevention of IH in response to HRT and in general. Furthermore, the age-associated difference in response to the presence of natural hormones in young vs aged models should be investigated.

Abstract 121: Modulation of Apoptosis, Matrix Degradation and Matrix Synthesis Are the Primary Determinants for Intimal Hyperplasia in Response to Altered Shear

Introduction: The quantity of high throughput microarray data leads to unique challenges in understanding its biological significance. We have developed a novel statistical method of clustering dynamics to elucidate for the mapping of gene expression arcs. In the current study, we applied these techniques to the multifactorial process of vein graft intimal hyperplasia, where direct correlation of these patterns was integrated with detailed morphologic data to identify those pathways central to the disease process. Methods: NZW rabbits underwent bilateral carotid vein grafting and unilateral distal branch ligation to create high and low shear environments. Vein grafts were harvested at 2h, 1 d, 7 ds, 28 d, 90 d, and 180 d (n=4 per group), and mRNA expression analyzed using a rabbit Agilent microarray platform. Parallel experiments were used to determine wall morphology, cell proliferation, apoptosis, and matrix content at each time point. Results: Compared to high shear grafts, exposure to a low shear resulted in a 5-fold greater intimal thickness at 180 d. Although 3365 (out of 14,958) genes varied with either time or shear, no single gene was significantly different (at a false discovery rate &lt;0.001) as a function of shear only. To analyze the broad-based modulation of gene expression, a novel clustering technique was used to identify temporal patterns of genes expression. Eight clusters (A through H) were identified, with five of these clusters demonstrating significant variation between high and low shear grafts (Table; A &lt;0.0001, D &lt;0.0001, F &lt; 0.0001, G &lt;0.001, H &lt;0.0001). Ingenuity pathway analysis was used to map gene ontology groups within the clusters to the dominant biologic process observed on histologic analysis. Cell death (cluster D), matrix degradation (cluster G), and matrix synthesis (cluster H), and the associated gene network pathways, were identified being significantly different in the high and low shear grafts (Figure). Conclusions: Using high throughput genomics and detailed morphologic data, we identify cell death, matrix degradation, and matrix synthesis, and the specific pathways associated with these process, as the most critical components in the development of the intimal hyperplastic lesion.

Abstract 8377: Cathepsin K Deficiency Suppresses the Development of Experimental Intimal Hyperplasia in Response to Injury

Background. Cathepsin K (Cat K), a potent elastinolytic and collagenolytic cysteine protease, is considered likely to participate in the development and destabilization of atherosclerotic plaques. In order to define the role of Cat K in arterial remodeling, we evaluated the influence of the targeted deletion of the Cat K gene (Cat K−/−) on vascular remodeling after flow cessation in murine carotid arteries. Methods and Results. Male Cat K+/+ and Cat K−/− mice receiving normal chow underwent ligation or ligation/polyethylene cuff-placement injuries to the left common carotid artery just proximal to its bifurcation, and were then processed for morphological and biochemical studies at specific time points. There were no changes in the expression of Cat K mRNA or protein in the basal or injured arteries. Histological analysis revealed that there was significantly less intimal hyperplasia in Cat K−/− mice than in control mice at 28 days after surgery. At Day 4 after surgery, the amounts of mRNA for MMP-9, MMP-14, toll-like receptor-4 (TLR-4), chemokine ligand 12 (CCL12) and the contents of macrophages were significantly lower in both the single- and double-injured arteries of Cat K−/− mice than in corresponding model of Cat K+/+ mice. At Day 7, gelatin zymography showed that MMP-9 activity increased in the injured arteries of both types in Cat K+/+ mice. An ex vivo migration assay showed that Cat K deficiency impaired smooth muscle cell migration from aortic explants. Although there were no differences in SMC adhesion or apoptosis in mice of either genotype, Cat K deficiency impaired chemoattractant-directed SMC and macrophage invasions. Synthetic cathepsin inhibitor E64d inhibited ligation-induced neointima formation in Cat K+/+ mice but a deficiency of endogenous inhibitor cystatin C deteriorated the neointima in apoliporotein E deficiency mice. ELISA demonstrated that Cat K−/− mice had lower levels of plasma stromal-derived factor-1 and interleukin-1β. Conclusions. This study demonstrates an essential role of Cat K in atherosclerotic lesion formation in response to injury, possibly via the reduction of inflammation and SMC formation, suggesting a novel therapeutic strategy for the control of atherosclerosis by regulating Cat K activity.

Mouse Vein Graft Hemodynamic Manipulations to Enhance Experimental Utility

Mouse models serve as a tool to study vein graft failure. However, in wild-type mice, there is limited intimal hyperplasia, hampering efforts to identify anti-intimal hyperplasia therapies. Furthermore, vein graft wall remodeling has not been well quantified in mice. We hypothesized that simple hemodynamic manipulations can reproducibly augment intimal hyperplasia and remodeling end points in mouse vein grafts, thereby enhancing their experimental utility. Mouse inferior vena cava-to-carotid interposition isografts were completed using an anastomotic cuff technique. Three flow restriction manipulations were executed by ligating outflow carotid branches, creating an outflow common carotid stenosis, and constructing a midgraft stenosis. Flowmetry and ultrasonography were used perioperatively and at day 28. All ligation strategies decreased the graft flow rate and wall shear stress. Morphometry showed that intimal thickness increased by 26% via carotid branch ligation and by 80% via common carotid stenosis. Despite similar mean flow rates and shear stresses among the three manipulations, the flow waveform amplitudes were lowest with common carotid stenosis. The disordered flow of the midgraft stenosis yielded poststenotic dilatation. The creation of an outflow common carotid stenosis generates clinically relevant (poor runoff) vein graft low wall shear stress and offers a technically flexible method for enhancing the intimal hyperplasia response. Midgraft stenosis exhibits poststenotic positive wall remodeling. These reproducible approaches offer novel strategies for increasing the utility of mouse vein graft models.

Caveolin-1 Influences Vascular Protease Activity and Is a Potential Stabilizing Factor in Human Atherosclerotic Disease

Caveolin-1 (Cav-1) is a regulatory protein of the arterial wall, but its role in human atherosclerosis remains unknown. We have studied the relationships between Cav-1 abundance, atherosclerotic plaque characteristics and clinical manisfestations of atherosclerotic disease.We determined Cav-1 expression by western blotting in atherosclerotic plaques harvested from 378 subjects that underwent carotid endarterectomy. Cav-1 levels were significantly lower in carotid plaques than non-atherosclerotic vascular specimens. Low Cav-1 expression was associated with features of plaque instability such as large lipid core, thrombus formation, macrophage infiltration, high IL-6, IL-8 levels and elevated MMP-9 activity. Clinically, a down-regulation of Cav-1 was observed in plaques obtained from men, patients with a history of myocardial infarction and restenotic lesions. Cav-1 levels above the median were associated with absence of new vascular events within 30 days after surgery [0% vs. 4%] and a trend towards lower incidence of new cardiovascular events during longer follow-up. Consistent with these clinical data, Cav-1 null mice revealed elevated intimal hyperplasia response following arterial injury that was significantly attenuated after MMP inhibition. Recombinant peptides mimicking Cav-1 scaffolding domain (Cavtratin) reduced gelatinase activity in cultured porcine arteries and impaired MMP-9 activity and COX-2 in LPS-challenged macrophages. Administration of Cavtratin strongly impaired flow-induced expansive remodeling in mice.This is the first study that identifies Cav-1 as a novel potential stabilizing factor in human atherosclerosis. Our findings support the hypothesis that local down-regulation of Cav-1 in atherosclerotic lesions contributes to plaque formation and/or instability accelerating the occurrence of adverse clinical outcomes. Therefore, given the large number of patients studied, we believe that Cav-1 may be considered as a novel target in the prevention of human atherosclerotic disease and the loss of Cav-1 may be a novel biomarker of vulnerable plaque with prognostic value.

Viability of Engineered Vessels as Arterial Substitutes

The search for vessel substitutes to replace small-/medium-caliber vessels is an ongoing concern for vascular surgeons. Engineered vessels were designed for use as arterial equivalents and assessed in an in vivo model in dog. Three study groups were established: clinical expanded polytetrafluoroethylene (ePTFE; control, n = 24), ePTFE seeding with endothelial cells (EC graft, n = 12), and ePTFE with a fibroblast matrix seeded with EC (FM+EC graft, n = 12). Grafts were subjected to a custom-designed femoral ex vivo circuit and implanted in the carotid artery for 60 days. The viability of the prosthetic grafts was evaluated. The ex vivo circuit revealed that the presence of a fibroblast matrix induced over double the cell retention compared to EC grafts. A significant reduction in platelet adhesion in EC grafts was observed. After their in vivo implantation, the engineered vessels were more efficient at avoiding occlusion than the prosthetic grafts. The FM+EC grafts induced more endothelialization than those seeded with ECs alone. Intimal hyperplasia response was reduced in the EC substitutes. Significant differences in apoptotic cells emerged between the EC and control ePTFE grafts. In conclusion, engineered vessels showed improved initial patency over ePTFE grafts. The EC graft was best at combating restenosis, a good indicator of the long-term efficiency of the graft.

Caveolin-1: a critical regulator of pulmonary vascular architecture and nitric oxide bioavailability in pulmonary hypertension

caveolae are 50- to 100-nm ω-shaped invaginations of the cell surface plasma membrane enriched in glycosphingolipids and cholesterol that occur in a variety of cell types including epithelial and endothelial cells, fibroblasts, smooth muscle cells, and adipocytes ([1][1]). These structures

Angiographic and 3D intravascular ultrasound assessment of overlapping bare metal stent and three different formulations of drug-eluting stents in patients with diabetes mellitus

The aim of this study was to examine the impact of overlapping bare-metal stent (BMS) and three different formulations of drug-eluting stent (DES) on intimal hyperplasia (IH) response of patients with diabetes mellitus (DM). Forty-nine DM patients treated with overlapping BMS (19 lesions), sirolimus-eluting stent (SES 12 lesions), paclitaxel-eluting stent (PES 8 lesions) or tacrolimus-eluting stent (TES 10 lesions) were studied. Baseline and 9-month follow-up volumetric intravascular vascular ultrasound (IVUS) and quantitative coronary angiography (QCA) analysis were performed in the entire stented segment and in the overlapped (OL) and non-overlapped (non-OL) subsegments. Clinical outcomes were evaluated at 1-year follow-up. Post-procedure (PO-) QCA measurements were similar in all stent groups, and between OL and non-OL subsegments in each individual type of stents. Percent IH was lower in SES and PES vs. BMS (p < 0.05). Percent IH was significantly greater in OL subsegment compared with non-OL subsegment in BMS (p < 0.05), but not in all type of DES groups. SES showed significantly less %IH compared with PES and TES in OL and non-OL subsegments. Vessel area at the OL remained unchanged from PO to FU in all type of DES and BMS groups. There were no aneurysm formation and no stent thrombosis up to 1-year follow-up. Overlapping BMS is associated with enhanced IH response in diabetic patients, whereas overlapping DES, particularly SES and PES, appear effective to inhibit IH without detectable late vascular adverse effects.

Deletion of KIS accelerates intimal hyperplasia in response to vascular injury and enhances migratory activity of VSMC

Deletion of KIS accelerates intimal hyperplasia in response to vascular injury and enhances migratory activity of VSMC

Interleukin-10 attenuates the response to vascular injury 1,2

The inflammatory response to vascular injury is characterized by expression of cytokines, growth factors, and chemokines that conspire to promote vessel remodeling and intimal hyperplasia (IH). Interleukin-10 (IL-10) is a multifunctional cytokine that has several anti-inflammatory properties in vitro. Few studies have evaluated the effects of IL-10 in experimental atherosclerosis. The purpose of the present study was to determine the influence of IL-10 on vascular inflammation and IH following mechanical injury. Wire carotid injury was performed in wild-type (WT) mice with and without IL-10 treatment. Immunohistochemistry, PCR, and ELISA assays were used to examine vessel production of basic fibroblast growth factor (bFGF), monocyte chemotactic protein-1 (MCP-1), and nuclear factor kappa B (NFkappaB). Vessels were morphometrically analyzed for IH. Carotid injury induced early expression of MCP-1 and bFGF that was abrogated in mice treated with IL-10. Similarly, injury-induced expression of NFkappaB message and protein was attenuated in mice receiving exogenous IL-10. Compared to untreated mice, IL-10 markedly decreased levels of IH. Interestingly, carotid injury in IL-10-deficient mice resulted in an augmented IH response compared to injured WT mice. In an in vivo model of direct vascular injury, IL-10 decreased expression of the pro-inflammatory transcription factor, NFkappaB, and the mitogenic chemokine and growth factor, MCP-1 and bFGF, respectively. These observations were associated with IL-10-induced attenuation of IH. Furthermore, endogenous IL-10 appeared to suppress the injury response. In conclusion, exogenously delivered IL-10 may represent a clinically relevant anti-inflammatory strategy for post-injury intimal hyperplasia.

Inhibition of Intimal Hyperplasia in Transgenic Mice Conditionally Expressing the Chemokine-Binding Protein M3

Chemokines have been implicated in the pathogenesis of a wide variety of diseases. This report describes the generation of transgenic mice that conditionally express M3, a herpesvirus protein that binds and inhibits chemokines. In response to doxycycline, M3 expression was induced in a variety of tissues and M3 was detectable in the blood by Western blotting. No gross or histological abnormalities were seen in mice expressing M3. To determine whether M3 expression could modify a significant pathophysiological response, we examined its effect on the development of intimal hyperplasia in response to femoral arterial injury. Intimal hyperplasia is thought to play a critical role in the development of restenosis after percutaneous transluminal coronary angioplasty and in the progression of atherosclerosis. Induction of M3 expression resulted in a 67% reduction in intimal area and a 68% reduction in intimal/medial ratio after femoral artery injury. These data support a role for chemokines in regulating intimal hyperplasia and suggest that M3 may be effective in attenuating this process. This transgenic mouse model should be a valuable tool for investigating the role of chemokines in a variety of pathological states.

Fas ligand gene therapy for vascular intimal hyperplasia.

Fas, a member of the tumor necrosis factor receptor super-family, is expressed in all cell types examined, while physiologic expression of Fas ligand (FasL) is found predominantly in activated T-lymphocytes, vascular endothelial cells, and "immune-privileged" tissues. Activation of Fas following FasL binding activates caspases, which results in apoptosis. In the vasculature, there may be a delicate balance between cell proliferation and apoptosis in vascular smooth muscle cells. Shifts in this balance could account for the accumulation of vascular smooth muscle cells in response to arterial injury, a major feature of vascular intimal hyperplasia. Intimal hyperplasia occurs in more than a third of patients receiving percutaneous transluminal balloon angioplasty. Stenting with or without coating significantly reduces the incidence rate of angiographic restenosis and that of target vessel revascularization. However, "in-stent" intimal hyperplasia/restenosis remains a challenge for clinical cardiologists. Although both the cell types and mechanisms that contribute to intimal hyperplasia in response to vascular injury remain controversial, vascular smooth muscle cell migration and proliferation appear to play an important role in the process. In animal models, cytotoxic and cytostatic gene therapy strategies targeted at the vascular smooth muscle cells have shown therapeutic potential for the treatment of vascular intimal hyperplasia. However, Fas ligand-based gene therapy appears to offer several advantages. In this review article, we will discuss the mode of FasL/Fas signaling in vascular smooth muscle cells and its therapeutic implications. We will also compare the relative merits of FasL with other cytotoxic and cytostatic gene therapy approaches for the treatment of intimal hyperplasia.

Mice deficient in tissue factor demonstrate attenuated intimal hyperplasia in response to vascular injury and decreased smooth muscle cell migration.

Tissue factor (TF) is the primary initiator of the coagulation cascade and is thought to play a key role in the generation of arterial thrombosis. Recent studies have suggested that TF mediates inflammatory processes in the arterial wall and may be an important regulator of intimal hyperplasia. We have employed genetically engineered mice (mTF(-/-) /hTF(+)) with markedly diminished TF activity ( approximately 1% normal levels) o examine the role of TF in mediating the response to arterial injury. mTF(-/-)/hTF(+) displayed a marked reduction in intimal hyperplasia (46% decrease in intimal area, 60% decrease in intimal/medial ratio) in response to femoral artery injury when compared to wild type controls. The decreased intimal hyperplasia seen in low TF mice was noted in a model of vascular injury not associated with significant thrombosis, suggesting that it may be mediated by non-procoagulant properties of TF. Smooth muscle cells from mTF(-/-)/hTF(+) mice grew normally in response to serum, but exhibited a marked defect in cell migration in a modified Boyden chamber assay. In contrast, there was no difference in platelet derived growth factor- induced migration, suggesting that the effect of TF on smooth muscle cell migration is agonist dependent. These data suggest that TF may mediate intimal hyperplasia by regulating smooth muscle cell migration.

A novel vein graft model: adaptation to differential flow environments.

Accelerated intimal hyperplasia in response to altered flow environment is critical to the process of vein bypass graft failure. Lack of a reproducible animal model for dissecting the mechanisms of vein graft (VG) remodeling has limited progress toward solving this clinically significant problem. Combining a cuffed anastomotic technique with other surgical manipulations, we developed a well-defined, more robust method for studying hemodynamic factors in VG arterialization. VG with fistula placement, complete occlusion, or partial distal branch ligation (DBL) was performed in the carotid artery of 56 rabbits. Extensive hemodynamic and physiological analyses were performed to define the hemodynamic forces and histological adaptations of the wall at 1-28 days. Anastomotic time averaged 12 min, with 100% patency of bilateral grafts and unilateral grafts plus no adjunct or delayed fistula. Bilateral VG-DBL resulted in an immediate disparity in wall shear (0.8 +/- 0.1 vs. 12.4 +/- 1.1 dyn/cm2, ligated vs. contralateral graft). Grafts exposed to low shear stress responded primarily through enhanced intimal thickening (231 +/- 35 vs. 36 +/- 18 microm, low vs. high shear). High-shear-stress grafts adapted through enhanced outward remodeling, with a 24% increase in lumen diameter at 28 days (3.0 +/- 0.1 vs. 3.7 +/- 0.2 mm, low vs. high shear). We have taken advantage of the cuffed anastomotic technique and combined it with a bilateral VG-DBL model to dissect the impact of hemodynamic forces on VG arterialization. This novel model offers a robust, clinically relevant, statistically powerful small animal model for evaluation of high- and low-shear-regulated VG remodeling.

Local infusion of the nitric oxide donor Sin-1 after angioplasty. Effects on intimal hyperplasia in porcine coronary arteries.

To investigate the development of intimal hyperplasia in response to percutaneous transluminal coronary angioplasty (PTCA) followed by local delivery of the nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1). Overdilation PTCA was performed in coronary arteries in 20 healthy pigs. One of the dilated segments was additionally treated with local delivery of SIN-1 for 10 min. Segments distal to the treated part of the arteries served as controls. Arteries were radiographically depicted and analyzed after 1 and 8 weeks for actin, myosin and intermediate filaments (IF), nitric oxide synthetase (NOS) and histological evaluation. Segments treated with PTCA+SIN-1 showed a significantly (p=0.03) larger luminal diameter compared with PTCA only treated segments. The luminal loss after SIN-1 was not significant compared with the diameter prior to treatment. Endothelial NOS content was significantly lower in the PTCA+SIN-1 group compared with the PTCA group after 1 (p=0.03) and 8 weeks (p=0.013). IF/actin ratio after 1 week was significantly increased in PTCA-treated segments compared with untreated controls (p=0.004), and compared with PTCA+SIN-1-treated segments (p=0.004). PTCA-induced intimal hyperplasia was potently inhibited by local delivery of the NO donor SIN-1. Momentary events at the time of injury play a significant role in the development of intimal hyperplasia and long-lasting down-regulation of the endothelial NOS expression after SIN-1 exposure is suggested. The IF/actin ratio can be useful as an early marker of intimal hyperplasia.

Local Infusion of the Nitric oxide Donor Sin-1 After Angioplasty . Effects on intimal hyperplasia in porcine coronary arteries

To investigate the development of intimal hyperplasia in response to percutaneous transluminal coronary angioplasty (PTCA) followed by local delivery of the nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1). Overdilation PTCA was performed in coronary arteries in 20 healthy pigs. One of the dilated segments was additionally treated with local delivery of SIN-1 for 10 min. Segments distal to the treated part of the arteries served as controls. Arteries were radiographically depicted and analyzed after 1 and 8 weeks for actin, myosin and intermediate filaments (IF), nitric oxide synthetase (NOS) and histological evaluation. Segments treated with PTCA+SIN-1 showed a significantly (p=0.03) larger luminal diameter compared with PTCA only treated segments. The luminal loss after SIN-1 was not significant compared with the diameter prior to treatment. Endothelial NOS content was significantly lower in the PTCA+SIN-1 group compared with the PTCA group after 1 (p=0.03) and 8 weeks (p=0.013). IF/actin ratio after 1 week was significantly increased in PTCA-treated segments compared with untreated controls (p=0.004), and compared with PTCA+SIN-1-treated segments (p=0.004). PTCA-induced intimal hyperplasia was potently inhibited by local delivery of the NO donor SIN-1. Momentary events at the time of injury play a significant role in the development of intimal hyperplasia and long-lasting down-regulation of the endothelial NOS expression after SIN-1 exposure is suggested. The IF/actin ratio can be useful as an early marker of intimal hyperplasia.