Genetic lineage tracing analysis of c-kit+ stem/progenitor cells revealed a contribution to vascular injury-induced neointimal lesions.

AIMS: Accumulating evidence indicates the presence of vascular stem/progenitor cells that may play a role in endothelial repair and lesion formation in the injured artery, in which c-kit+ stem/progenitor cells have been reported to differentiate into endothelial and smooth muscle cells in vitro and in ischemic tissue. In this study, we investigated whether and how endogenous c-kit+ stem/progenitor cells contribute to vascular injury and neointima formation in vivo. METHODS AND RESULTS: We created Kit-CreERxRosa26-RFP mice and performed genetic lineage tracing analysis of c-kit+ stem/progenitor cells in injury-induced neointima formation in vivo. We provide direct evidence that endogenous c-kit+ stem/progenitor cells minimally differentiate into endothelial or smooth muscle cells facilitating vascular repair, but predominantly generate monocytes/macrophages and granulocytes contributing to vascular immuno-inflammatory response to endothelial injury. Although c-kit+ cells reside in both bone marrow and vessel wall, bone marrow transplantation data indicate that bone marrow-derived c-kit+ cells are the main source for enhancing neointima formation. Furthermore, treatment of ACK2, a c-kit receptor antagonizer, attenuates neointimal hyperplasia after injury at least in part by depleting c-kit+ cells and their generated progeny. CONCLUSIONS: c-kit+ stem/progenitor cells are not a main source for endothelial regeneration and smooth muscle accumulation of the large artery injury, but a plausible interventional approach to reduce vascular immuno-inflammatory response and subsequently to ameliorate vascular lesions.

Adipose-derived factor CTRP9 attenuates vascular smooth muscle cell proliferation and neointimal formation.

Obesity is closely associated with the progression of vascular disorders, including atherosclerosis and postangioplasty restenosis. C1q/TNF-related protein (CTRP) 9 is an adipocytokine that is down-regulated in obese mice. Here we investigated whether CTRP9 modulates neointimal hyperplasia and vascular smooth muscle cell (VSMC) proliferation in vivo and in vitro. Left femoral arteries of wild-type (WT) mice were injured by a steel wire. An adenoviral vector expressing CTRP9 (Ad-CTRP9) or ß-galactosidase as a control was intravenously injected into WT mice 3 d before vascular injury. Delivery of Ad-CTRP9 significantly attenuated the neointimal thickening and the number of bromodeoxyuridine-positive proliferating cells in the injured arteries compared with that of control. Treatment of VSMCs with CTRP9 protein attenuated the proliferative and chemotactic activities induced by growth factors including platelet-derived growth factor (PDGF)-BB, and suppressed PDGF-BB-stimulated phosphorylation of ERK. CTRP9 treatment dose-dependently increased cAMP levels in VSMCs. Blockade of cAMP-PKA pathway reversed the inhibitory effect of CTRP9 on DNA synthesis and ERK phosphorylation in response to PDGF-BB. The present data indicate that CTRP9 functions to attenuate neointimal formation following vascular injury through its ability to inhibit VSMC growth via cAMP-dependent mechanism, suggesting that the therapeutic approaches to enhance CTRP9 production could be valuable for prevention of vascular restenosis after angioplasty.

Lentiviral vector mediated expression of Bax and hepatocyte growth factor inhibits vein graft thickening in a rabbit vein graft model.

Vein graft failure caused by vein graft thickening of the arterialized vein after bypass surgery is a main problem in clinical vascular surgery. Gene therapy is increasingly being recognized as a relevant treatment option for vein graft failure. In this study, we aimed to develop a novel recombinant lentivirus for the delivery of hepatocyte growth factor (HGF) and Bax in a rabbit vein graft model of bypass grafting. A bypass model was made in rabbits using the right jugular vein interposed end-to-end to the ipsilateral carotid artery. A lentivirus vector harboring HGF and Bax cDNAs (Lenti-HGF-Bax) was constructed and transduced into the venous grafts. Vein grafts were stained with hematoxilyn and eosin, and Masson. HGF and Bax expression in vein grafts was detected by immunohistochemical and Western blot analysis. Our results showed that vein graft thickening was reduced by 47.2 ± 7.4% in lenti-HGF-Bax treated rabbits, compared to controls. Meanwhile, the ratio of intima/media area was reduced in lentil-HGF-Bax treated rabbits, compared to controls. The number of HGF and Bax positive cells was increased in vein grafts from rabbits treated by lenti-HGF-Bax, compared to those from controls. Furthermore, protein levels of HGF and Bax were both significantly increased in grafts derived from rabbits treated by lenti-HGF-Bax, compared to those from control. In conclusion, Lenti-HGF-Bax inhibits vein graft thickening in vein grafts and is a promising agent for preventing vein graft failure.

Apigenin attenuates neointima formation via suppression of vascular smooth muscle cell phenotypic transformation.

Abnormal proliferation, migration, and phenotypic modulation of vascular smooth muscle cells (VSMCs) are critical factors in neointima formation during restenosis. The purpose of this study is to determine the efficacy and possible cell signaling mechanisms of apigenin in VSMC activation induced by platelet-derived growth factor (PDGF)-BB and injury-induced neointima formation. Our data revealed a dose-dependent apigenin inhibition of PDGF-BB-induced proliferation of VSMCs by arresting cells in G0/G1-phase of the cell cycle as determined using 5-bromo-2'-deoxyuridine incorporation and flow cytometry. This was associated with the inhibition of cyclin-dependent kinase (CDK) 4,6 expression and an increase in p27Kip1 levels in PDGF-stimulated VSMCs. Moreover, apigenin was also found to regulate PDGF-induced migration and expression of smooth-muscle-specific contractile markers. Mechanistically, the PDGF-BB-induced phosphorylation of PDGF-receptor ß (PDGF-Rß), Akt/glycogen synthase kinase(GSK)3ß, extracellular signal-regulated kinase1/2 (ERK1/2), and signal transducers and activators of transcription 3 (STAT3) is negatively modulated by apigenin. For the in vivo studies using a mouse carotid arterial injury model, the administration of apigenin resulted in a significant inhibition of the neointima/media ratio and proliferating cell nuclear antigen (PCNA)-positive cells. These results demonstrate that apigenin can suppress PDGF-induced VSMC activation and neointima hyperplasia after vascular injury; these beneficial effects are probably the result of the blockade of PDGF-Rß phosphorylation and its downstream signal transduction, including the Akt/GSK-3ß, ERK1/2, and STAT3 pathways. The results suggest that apigenin may be a potential therapeutic candidate for the prevention of restenosis.

Smooth muscle Notch1 mediates neointimal formation after vascular injury.

BACKGROUND: Notch1 regulates binary cell fate determination and is critical for angiogenesis and cardiovascular development. However, the pathophysiological role of Notch1 in the postnatal period is not known. We hypothesize that Notch1 signaling in vascular smooth muscle cells (SMCs) may contribute to neointimal formation after vascular injury. METHODS AND RESULTS: We performed carotid artery ligation in wild-type, control (SMC-specific Cre recombinase transgenic [smCre-Tg]), general Notch1 heterozygous deficient (N1+/-), SMC-specific Notch1 heterozygous deficient (smN1+/-), and general Notch3 homozygous deficient (N3-/-) mice. Compared with wild-type or control mice, N1+/- and smN1+/- mice showed a 70% decrease in neointimal formation after carotid artery ligation. However, neointimal formation was similar between wild-type and N3-/- mice. Indeed, SMCs derived from explanted aortas of either N1(+/-)- or smN1+/- mice showed decreased chemotaxis and proliferation and increased apoptosis compared with control or N3-/- mice. This correlated with decreased staining of proliferating cell nuclear antigen-positive cells and increased staining of cleaved caspase-3 in the intima of N1(+/-)- or smN1+/- mice. In SMCs derived from CHF1/Hey2-/- mice, activation of Notch signaling did not lead to increased SMC proliferation or migration. CONCLUSIONS: These findings indicate that Notch1, rather than Notch3, mediates SMC proliferation and neointimal formation after vascular injury through CHF1/Hey2 and suggest that therapies that target Notch1/CHF1/Hey2 in SMCs may be beneficial in preventing vascular proliferative diseases.

Yin Yang-1 inhibits vascular smooth muscle cell growth and intimal thickening by repressing p21WAF1/Cip1 transcription and p21WAF1/Cip1-Cdk4-cyclin D1 assembly.

Vascular injury initiates a cascade of phenotype-altering molecular events. Transcription factor function in this process, particularly that of negative regulators, is poorly understood. We demonstrate here that the forced expression of the injury-inducible GLI-Krüppel zinc finger protein Yin Yang-1 (YY1) inhibits neointima formation in human, rabbit and rat blood vessels. YY1 inhibits p21(WAF1/Cip1) transcription, prevents assembly of a p21(WAF1/Cip1)-cdk4-cyclin D1 complex, and blocks downstream pRb(Ser249/Thr252) phosphorylation and expression of PCNA and TK-1. Conversely, suppression of endogenous YY1 elevates levels of p21(WAF1/Cip1), PCNA, pRb(Ser249/Thr252) and TK-1, and increases intimal thickening. YY1 binds Sp1 and prevents its occupancy of a distinct element in the p21(WAF1/Cip1) promoter without YY1 itself binding the promoter. Additionally, YY1 induces ubiquitination and proteasome-dependent degradation of p53, decreasing p53 immunoreactivity in the artery wall. These findings define a new role for YY1 as both an inducer of p53 instability in smooth muscle cells, and an indirect repressor of p21(WAF1/Cip1) transcription, p21(WAF1/Cip1)-cdk4-cyclin D1 assembly and intimal thickening.

MicroRNA expression signature and antisense-mediated depletion reveal an essential role of MicroRNA in vascular neointimal lesion formation.

MicroRNAs (miRNAs) are a recently discovered class of endogenous, small, noncoding RNAs that regulate about 30% of the encoding genes of the human genome. However, the role of miRNAs in vascular disease is currently completely unknown. Using microarray analysis, we demonstrated for the first time that miRNAs are aberrantly expressed in the vascular walls after balloon injury. The aberrantly expressed miRNAs were further confirmed by Northern blot and quantitative real-time polymerase chain reaction. Modulating an aberrantly overexpressed miRNA, miR-21, via antisense-mediated depletion (knock-down) had a significant negative effect on neointimal lesion formation. In vitro, the expression level of miR-21 in dedifferentiated vascular smooth muscle cells was significantly higher than that in fresh isolated differentiated cells. Depletion of miR-21 resulted in decreased cell proliferation and increased cell apoptosis in a dose-dependent manner. MiR-21-mediated cellular effects were further confirmed in vivo in balloon-injured rat carotid arteries. Western blot analysis demonstrated that PTEN and Bcl-2 were involved in miR-21-mediated cellular effects. The results suggest that miRNAs are novel regulatory RNAs for neointimal lesion formation. MiRNAs may be a new therapeutic target for proliferative vascular diseases such as atherosclerosis, postangioplasty restenosis, transplantation arteriopathy, and stroke.

Comparison of neointimal coverage by optical coherence tomography of a sirolimus-eluting stent versus a bare-metal stent three months after implantation.

No detailed data regarding neointimal coverage of bare-metal stents (BMSs) at 3 months after implantation was reported to date. This investigation was designed to evaluate the neointimal coverage of BMSs compared with sirolimus-eluting stents (SESs) using optical coherence tomography. A prospective optical coherence tomographic follow-up examination was performed 3 months after stent implantation for patients who underwent BMS (n = 16) or SES implantation (n = 24). Neointimal hyperplasia (NIH) thickness on each stent strut and percentage of NIH area in each cross section were measured. Malapposition of stent struts to the vessel wall and the existence of in-stent thrombi were also evaluated. There were 5,076 struts of SESs and 2,875 struts of BMSs identified. NIH thickness and percentage of NIH area in the BMS group were higher than in the SES group (351 +/- 248 vs 31 +/- 39 mum; p <0.0001; 45.0 +/- 14% vs 10.0 +/- 4%; p <0.0001, respectively). The frequency of uncovered struts was higher in the SES group than the BMS group (15% vs 0.1%; p <0.0001). Malapposed struts were observed more frequently in the SES group than the BMS group (15% vs 1.1%; p <0.0001). In conclusion, there was no difference in incidence of in-stent thrombus between the 2 groups (14% vs 0%; p = 0.23). The present study showed almost all BMS struts to be well covered at a 3-month follow-up, suggesting that patients receiving BMS stents may not require dual-antiplatelet therapy >3 months after implantation.

Stem cell factor deficiency is vasculoprotective: unraveling a new therapeutic potential of imatinib mesylate.

Evidence suggests that bone marrow (BM) cells may give rise to a significant proportion of smooth muscle cells (SMCs) that contribute to intimal hyperplasia after vascular injury; however, the molecular pathways involved and the timeline of these events remain poorly characterized. We hypothesized that the stem cell factor (SCF)/c-Kit tyrosine kinase signaling pathway is critical to neointimal formation by BM-derived progenitors. Wire-induced femoral artery injury in mice reconstituted with wild-type BM cells expressing yellow fluorescent protein was performed, which revealed that 66+/-12% of the SMCs (alpha-smooth muscle actin-positive [alphaSMA(+)] cells) in the neointima were from BM. To characterize the role of the SCF/c-Kit pathway, we used c-Kit deficient W/W(v) and SCF-deficient Steel-Dickie mice. Strikingly, vascular injury in these mice resulted in almost a complete inhibition of neointimal formation, whereas wild-type BM reconstitution of c-Kit mutant mice led to neointimal formation in a similar fashion as wild-type animals, as did chronic administration of SCF in matrix metalloproteinase-9-deficient mice, a model of soluble SCF deficiency. Pharmacological antagonism of the SCF/c-Kit pathway with imatinib mesylate (Gleevec) or ACK2 (c-Kit antibody) also resulted in a marked reduction in intimal hyperplasia. Vascular injury resulted in the local upregulation of SCF expression. c-Kit(+) progenitor cells (PCs) homed to the injured vascular wall and differentiated into alphaSMA(+) cells. Vascular injury also caused an increase in circulating SCF levels which promoted CD34(+) PC mobilization, a response that was blunted in mutant and imatinib mesylate-treated mice. In vitro, SCF promoted adhesion of BM PCs to fibronectin. Additionally, anti-SCF antibodies inhibited adhesion of BM PCs to activated SMCs and diminished SMC differentiation. These data indicate that SCF/c-Kit signaling plays a pivotal role in the development of neointima by BM-derived PCs and that the inhibition of this pathway may serve as a novel therapeutic target to limit aberrant vascular remodeling.

2-Methoxyestradiol, an estradiol metabolite, inhibits neointima formation and smooth muscle cell growth via double blockade of the cell cycle.

2-Methoxyestradiol (2-ME), an endogenous metabolite of estradiol with no affinity for estrogen receptors, is a potent anticarcinogenic agent (in phase II clinical trials) and mediates the inhibitory effects of estradiol on smooth muscle cell (SMC) growth. Here we studied the intracellular mechanisms by which 2-ME inhibits SMC growth and whether 2-ME prevents injury-induced neointima formation. 2-ME concentrations that inhibit proliferation of cycling human aortic SMCs by >or=50% blocked cell-cycle progression in G(0)/G(1) and in G(2)/M phase, as determined by flow cytometry. Consistent with the cell-cycle effects, at a molecular level (Western blots), 2-ME inhibited cyclin D(1) and cyclin B(1) expression; cyclin-dependent kinase (cdk)-1 and cdk-2 activity; and retinoblastoma protein (pRb), extracellular signal-regulated kinase (ERK) 1/2, and Akt phosphorylation. 2-ME also upregulated the Cdk inhibitor p27 and interfered with tubulin polymerization. Moreover, 2-ME augmented COX-2 expression, suggesting that it may also inhibit SMC growth via prostaglandin formation. In rats, treatment with 2-ME abrogated injury-induced neointima formation; decreased proliferating SMCs; downregulated expression of proliferating-cell nuclear antigen (PCNA), c-myc, cyclin D(1), cyclin B(1), phosphorylated Akt, phosphorylated ERK1/2, p21, and pRb; inhibited cdk-1 and cdk-4 activity; and upregulated expression of cyclooxygenase (COX)-2 and p27. Caspase-3 cleavage assay and fluorescence-activated cell-sorting (FACS) analysis showed no evidence of apoptosis in 2-ME-treated SMCs, and TUNEL staining in carotid segments showed no evidence of 2-ME-induced apoptosis in vivo. The antimitotic effects of 2-ME on SMCs are mediated by the inhibition of key cell-cycle regulatory proteins and effects on tubulin polymerization and COX-2 upregulation. These effects of 2-ME most likely contribute to the antivasoocclusive actions of this endogenous compound.

Self-assembled Collagen-Fibrin Hydrogel Reinforces Tissue Engineered Adventitia Vessels Seeded with Human Fibroblasts.

Efforts for tissue engineering vascular grafts focuses on the tunica media and intima, although the tunica adventitia serves as the primary structural support for blood vessels. In surgery, during endarterectomies, surgeons can strip the vessel, leaving the adventitia as the main strength layer to close the vessel. Here, we adapted our recently developed technique of forming vascular tissue rings then stacking the rings into a tubular structure, to accommodate human fibroblasts to create adventitia vessels in 8 days. Collagen production and fibril cross-linking was augmented with TGF-ß and ascorbic acid, significantly increasing tensile strength to 57.8 ± 3.07 kPa (p = 0.008). Collagen type I gel was added to the base fibrin hydrogel to further increase strength. Groups were: Fibrin only; 0.7 mg/ml COL; 1.7 mg/ml COL; and 2.2 mg/ml COL. The 0.7 mg/ml collagen rings resulted in the highest tensile strength at 77.0 ± 18.1 kPa (p = 0.015). Culture periods of 1-2 weeks resulted in an increase in extracellular matrix deposition and significantly higher failure strength but not ultimate tensile strength. Histological analysis showed the 0.7 mg/ml COL group had significantly more, mature collagen. Thus, a hydrogel of 0.7 mg/ml collagen in fibrin was ideal for creating and strengthening engineered adventitia vessels.

Vasa Vasorum Angiogenesis: Key Player in the Initiation and Progression of Atherosclerosis and Potential Target for the Treatment of Cardiovascular Disease.

Plaque microvascularization and increased endothelial permeability are key players in the development of atherosclerosis, from the initial stages of plaque formation to the occurrence of acute cardiovascular events. First, endothelial dysfunction and increased permeability facilitate the entry of diverse inflammation-triggering molecules and particles such as low-density lipoproteins into the artery wall from the arterial lumen and vasa vasorum (VV). Recognition of entering particles by resident phagocytes in the vessel wall triggers a maladaptive inflammatory response that initiates the process of local plaque formation. The recruitment and accumulation of inflammatory cells and the subsequent release of several cytokines, especially from resident macrophages, stimulate the expansion of existing VV and the formation of new highly permeable microvessels. This, in turn, exacerbates the deposition of pro-inflammatory particles and results in the recruitment of even more inflammatory cells. The progressive accumulation of leukocytes in the intima, which trigger proliferation of smooth muscle cells in the media, results in vessel wall thickening and hypoxia, which further stimulates neoangiogenesis of VV. Ultimately, this highly inflammatory environment damages the fragile plaque microvasculature leading to intraplaque hemorrhage, plaque instability, and eventually, acute cardiovascular events. This review will focus on the pivotal roles of endothelial permeability, neoangiogenesis, and plaque microvascularization by VV during plaque initiation, progression, and rupture. Special emphasis will be given to the underlying molecular mechanisms and potential therapeutic strategies to selectively target these processes.

Differentiated vascular myocytes: are they involved in neointimal formation?

The role of differentiated vascular myocytes are neointimal formation in canine carotid artery was investigated. Using antibodies and cDNA probes, cells were characterized in situ and after isolation. In situ characterization indicated the majority of medial cells expressed both smooth muscle myosin and alpha actin but many cells were negative to these markers. All adventitial cells were negative for these proteins. The muscle protein-positive cells were designated differentiated, vascular myocytes (VSMC). The others were designated type 2 cells. Sequential enzyme digestion from lumenal surface yielded VSMC ( > 90%) while digestions from the adventitial surface yielded type 2 cells ( > 90%). VSMC were viable in culture but did not spread, proliferate, or alter expression of muscle proteins. Type 2 cells proliferated and increased their expression of muscle actin but did not express muscle myosin. Characterization of neointimal cells from injured carotid arteries indicated they were morphologically and immunologically identical to cultured type 2 cells. We concluded that: (a) canine carotid artery media consists of a heterogeneous cell population: (b) serum does not stimulate isolated VSMC to undergo phenotypic modulation or proliferate: and (c) type 2 cells may be responsible for neointimal formation because they proliferate and acquire a phenotype identical to in situ neointimal cells.

Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice.

To begin to dissect atherogenesis as a complex genetic disorder affected by genetic makeup and environment, we have (a) generated a reproducible mouse model of neointimal growth; (b) evaluated the effect of disruption of a single gene, endothelial nitric oxide synthase, believed to be central to intimal growth, and (c) examined the modifying effects of gender and pregnancy upon the vascular response. Cuff placement around the femoral artery causes reproducible intimal growth. We assessed the response to injury by quantitative morphometry, measuring the intimal to medial (I/M) volume ratio. In wild-type mice, cuff placement causes pronounced intimal proliferation without affecting the media, resulting in I/M ratios of 31% (SV129 males) and 27% (C57BL/6 males). eNOS mutant male mice have a much greater degree of intimal growth (I/M ratio of 70%). Female mice show less intimal response than do males, although eNOS mutant female mice still have more response than do wild-type females. Most dramatic, however, is the effect of pregnancy, which essentially abolishes the intimal response to injury, even overriding the effect of eNOS mutation. We conclude that eNOS deficiency is a genetic predisposition to intimal proliferation that is enhanced by male gender, and that may be overridden by pregnancy.

Regulation of cellular proliferation and intimal formation following balloon injury in atherosclerotic rabbit arteries.

Injury to atherosclerotic arteries induces the expression of growth regulatory genes that stimulate cellular proliferation and intimal formation. Intimal expansion has been reduced in vivo in nonatherosclerotic balloon-injured arteries by transfer of genes that inhibit cell proliferation. It is not known, however, whether vascular cell proliferation can be inhibited after injury in more extensively diseased atherosclerotic arteries. Accordingly, the purpose of this study was to investigate whether expression of recombinant genes in atherosclerotic arteries after balloon injury could inhibit intimal cell proliferation. To test this hypothesis, we examined the response to balloon injury in atherosclerotic rabbit arteries after gene transfer of herpesvirus thymidine kinase gene (tk) and administration of ganciclovir. Smooth muscle cells from hyperlipidemic rabbit arteries infected with adenoviral vectors encoding tk were sensitive to ganciclovir, and bystander killing was observed in vitro. In atherosclerotic arteries, a human placental alkaline phosphatase reporter gene was expressed in intimal and medial smooth muscle cells and macrophages, identifying these cells as targets for gene transfer. Expression of tk in balloon-injured hyperlipidemic rabbit arteries followed by ganciclovir treatment resulted in a 64% reduction in intimal cell proliferation 7 d after gene transfer (P = 0.004), and a 35-49% reduction in internal area 21 d after gene transfer, compared with five different control groups (P < 0.05). Replication of smooth muscle cells and macrophages was inhibited by tk expression and ganciclovir treatment. These findings indicate that transfer of a gene that inhibits cellular proliferation limits the intimal area in balloon-injured atherosclerotic arteries. Molecular approaches to the inhibition of cell proliferation in atherosclerotic arteries constitute a possible treatment for vascular proliferative diseases.

Induction of sustained expression of proto-oncogene c-fms by platelet-derived growth factor, epidermal growth factor, and basic fibroblast growth factor, and its suppression by interferon-gamma and macrophage colony-stimulating factor in human aortic medial smooth muscle cells.

Vascular medial smooth muscle cells migrate, proliferate and transform to foam cells in the process of atherosclerosis. We have reported that the intimal smooth muscle cells express proto-oncogene c-fms, a characteristic gene of monocyte-macrophages, which is not normally expressed in medial smooth muscle cells. In the present study, we demonstrated that combinations of platelet-derived growth factor (PDGF)-BB and either epidermal growth factor (EGF) or fibroblast growth factor (FGF) induced high expression of c-fms in normal human medial smooth muscle cells to the level of intimal smooth muscle cells or monocyte-derived macrophages, whereas c-fms expression by PDGF-BB alone was 1/10 and both EGF and FGF had no independent effect on c-fms expression. By contrast, interferon (IFN)-gamma and macrophage colony-stimulating factor (M-CSF) suppressed the induction of c-fms expression. These results indicate that multiple growth factors and cytokines may play a role in the phenotypic transformation of medial smooth muscle cells to intimal smooth muscle cells in atherosclerotic lesions by altering c-fms expression.

Taxol inhibits neointimal smooth muscle cell accumulation after angioplasty in the rat.

Despite significant improvements in the primary success rate of the medical and surgical treatments for atherosclerotic disease, including angioplasty, bypass grafting, and endarterectomy, secondary failure due to late restenosis continues to occur in 30-50% of individuals. Restenosis and the later stages in atherosclerotic lesions are due to a complex series of fibroproliferative responses to vascular injury involving potent growth-regulatory molecules (such as platelet-derived growth factor and basic fibroblast growth factor) and resulting in vascular smooth muscle cell (VSMC) proliferation, migration, and neointimal accumulation. We show here, based on experiments with both taxol and deuterium oxide, that microtubules are necessary for VSMCs to undergo the multiple transformations contributing to the development of the neointimal fibroproliferative lesion. Taxol was found to interfere both with platelet-derived growth factor-stimulated VSMC migration and with VSMC migration and with VSMC proliferation, at nanomolar levels in vitro. In vivo, taxol prevented medial VSMC proliferation and the neointimal VSMC accumulation in the rat carotid artery after balloon dilatation and endothelial denudation injury. This effect occurred at plasma levels approximately two orders of magnitude lower than that used clinically to treat human malignancy (peak levels achieved in this model were approximately 50-60 nM). Taxol may therefore be of therapeutic value in preventing human restenosis with minimal toxicity.

Stent implantation in coronary porcine arteries is associated with early activation of TNFalpha and TNFalpha receptor II expression.

Inflammation present in restenosis after angioplasty is associated with production of cytokines such as tumor necrosis factor (TNFalpha). However, limited data exist on the possible increase in TNFalpha and TNFalpha receptor expression induced during the chronic phase after stenting. To this end, swine underwent balloon denudation (PTCA) and stent implantation in coronary arteries. At day 1, 7 or 28 post-procedure, sections from injured and reference vessel segments were evaluated for extent of pathology and expression of TNFalpha and TNFalpha receptors (RI and RII). Restenosis assessed at days 7 and 28 showed, respectively, two- and six-fold more neointimal (NI) area in stented than in PTCA segments. Unlike reference segments, TNFalpha-positive cells were detected in both the media and the NI of injured segments, with a significant increase over the 28-day time frame. Stenting was associated with an eight-fold enhancement in TNFalpha expression over PTCA. TNFalpha expression and NI area tended to correlate in injured segments. Furthermore, the pattern of expression of TNFalpha-RII, but not TNFalpha-RI, resembled that of TNFalpha itself. These results implicate TNFalpha and TNFalpha-RII as important actors in both the acute and the chronic phases of inflammation following stent implantation.

Nanosuspensions of alendronate with gallium or gadolinium attenuate neointimal hyperplasia in rats.

Monocytes/macrophages play a pivotal role in the formation of neointinal hyperplasia following vascular injury. Transient depletion of circulating monocytes by particulate delivery systems containing bisphosphonates, such as alendronate, results in restenosis inhibition. We hypothesized that a self-suspendable nanoparticulate dosage form, with a minimum amount of expients, could be formulated by complexing the negatively charged alendronate with gallium or gadolinium. We further hypothesized that a synergistic biological effect could be obtained by nanosuspensions of alendronate with these counter ions. Nanosuspensions (150-250 nm) of alendronate-gallium and alendronate-gadolinium were successfully formulated with no additives except for the active agents and HCl for pH adjustment. Both nanosuspensions exhibited macrophage cell line growth inhibition in a dose-response relationship in comparison to the various agents in solution and in liposomes. A synergistic effect of the nanosuspensions was observed in the inhibition of raw264 macrophages, and in reducing IL-1beta and TNF-alpha secretion in cell culture. Single IV administration at the time of injury, of alendronate-gallium or alendronate-gadolinium nanosuspensions resulted in inhibition of neointimal hyperplasia and stenosis in the rat model of vascular injury. The results correlated with the significant reduction of circulating monocytes. The nanosuspensions possess the advantages of no additives for minimal provocation of side effects, and the potential of immunomodulating inflammatory disorders.

Plasminogen activator inhibitor-1 from bone marrow-derived cells suppresses neointimal formation after vascular injury in mice.

OBJECTIVE: To investigate the ability of bone marrow (BM)-derived cells to modulate neointimal growth after injury by expressing plasminogen activator inhibitor-1 (PAI-1). METHODS AND RESULTS: We performed BM transplantation (BMT) in lethally irradiated wild-type (WT) and PAI-1(-/-) mice. Three weeks after carotid injury with ferric chloride, analysis of Y-chromosome DNA expression in the vessel wall of female hosts revealed that 20.8+/-6.0% of the cells in the neointima and 37.6+/-5.7% of those in the media were of BM origin. Lack of PAI-1 in either the host or the donor cells did not affect recruitment of BM-derived cells into sites of vascular injury. The neointima consisted predominantly of smooth muscle cells, and a proportion of these cells expressed PAI-1. Overall, lack of PAI-1 was associated with enhanced neointimal formation. However, importantly, BMT(WT-->PAI-1(-/-)) mice exhibited reduced neointimal area (P=0.05) and luminal stenosis (P=0.04) compared with BMT(PAI-1(-/-)-->PAI-1(-/-)) mice. Although PAI-1-expressing cells were shown to be present in BMT(WT-->PAI-1(-/-)) lesions, these mice did not exhibit detectable levels of the inhibitor in the circulation, suggesting that local production of PAI-1 by cells in the neointima and media was sufficient to reduce luminal stenosis. CONCLUSIONS: PAI-1 from BM-derived cells appears capable of suppressing neointimal growth after vascular injury.