Subsequent Smooth Muscle Cell Research Articles

Elastin, arterial mechanics, and stenosis.

Elastin is a long-lived extracellular matrix protein that is organized into elastic fibers that provide elasticity to the arterial wall, allowing stretch and recoil with each cardiac cycle. By forming lamellar units with smooth muscle cells, elastic fibers transduce tissue-level mechanics to cell-level changes through mechanobiological signaling. Altered amounts or assembly of elastic fibers leads to changes in arterial structure and mechanical behavior that compromise cardiovascular function. In particular, genetic mutations in the elastin gene (ELN) that reduce elastin protein levels are associated with focal arterial stenosis, or narrowing of the arterial lumen, such as that seen in supravalvular aortic stenosis and Williams-Beuren syndrome. Global reduction of Eln levels in mice allows investigation of the tissue- and cell-level arterial mechanical changes and associated alterations in smooth muscle cell phenotype that may contribute to stenosis formation. A loxP-floxed Eln allele in mice highlights cell type- and developmental origin-specific mechanobiological effects of reduced elastin amounts. Eln production is required in distinct cell types for elastic layer formation in different parts of the mouse vasculature. Eln deletion in smooth muscle cells from different developmental origins in the ascending aorta leads to characteristic patterns of vascular stenosis and neointima. Dissecting the mechanobiological signaling associated with local Eln depletion and subsequent smooth muscle cell response may help develop new therapeutic interventions for elastin-related diseases.

Ta ion implanted nanoridge-platform for enhanced vascular responses

Bare metal stents are commonly used in interventional cardiology; they provide successful treatment because of their excellent mechanical properties, expandability ratios, and flexibility. However, their insufficient vascular affinity can induce the development of neointimal hyperplasia following arterial injury and subsequent smooth muscle cell overgrowth in the lumen of a stented vessel. Nanoengineering of the bare metal stent surface is a valuable strategy for eliciting favorable vascular responses. In this study, we introduce a target-ion-induced plasma sputtering (TIPS) technique to fabricate a platform with a favorable endothelial environment. This technique enables the simple single-step production of a Ta-implanted nanoridged surface on a stent with a complex 3D geometry that shows a clear tendency to become oriented parallel to the direction of blood flow. Moreover, the nanoridges developed show good structural integrity and mechanical stability, resulting in apparently stable morphologies under high strain rates. In vitro cellular responses to the Co–Cr, such as endothelialization, platelet activation, and blood coagulation, are considerably altered after TIPS treatment; endothelium formation is rapid and surface thrombogenicity is low. An in vivo rabbit iliac artery model is used to confirm that the nanoridged surface facilitates rapid re-endothelialization and limits the formation of neointima compared to the bare stent. These results indicate that the Ta ion implanted nanoridge platform fabricated using the TIPS technique has immense potential as a solution for in-stent restenosis and ensuring the long-term patency of bare metal stents.

Fas ligand and nitric oxide combination to control smooth muscle growth while sparing endothelium

Metallic stents cause vascular wall damage with subsequent smooth muscle cell (SMC) proliferation, neointimal hyperplasia, and treatment failure. To combat in-stent restenosis, drug-eluting stents (DES) delivering mTOR inhibitors such as sirolimus or everolimus have become standard for coronary stenting. However, the relatively non-specific action of mTOR inhibitors prevents efficient endothelium recovery and mandates dual antiplatelet therapy to prevent thrombosis. Unfortunately, long-term dual antiplatelet therapy leads to increased risk of bleeding/stroke and, paradoxically, myocardial infarction. Here, we took advantage of the fact that nitric oxide (NO) increases Fas receptors on the SMC surface. Fas forms a death-inducing complex upon binding to Fas ligand (FasL), while endothelial cells (ECs) are relatively resistant to this pathway. Selected doses of FasL and NO donor synergistically increased SMC apoptosis and inhibited SMC growth more potently than did everolimus or sirolimus, while having no significant effect on EC viability and proliferation. This differential effect was corroborated in ex vivo pig coronaries, where the neointimal formation was inhibited by the drug combination, but endothelial viability was retained. We also deployed FasL-NO donor-releasing ethylene-vinyl acetate copolymer (EVAc)-coated stents into pig coronary arteries, and cultured them in perfusion bioreactors for one week. FasL and NO donor, released from the stent coating, killed SMCs close to the stent struts, even in the presence of flow rates mimicking those of native arteries. Thus, the FasL-NO donor-combination has a potential to prevent intimal hyperplasia and in-stent restenosis, without harming endothelial restoration, and hence may be a superior drug delivery strategy for DES.

Abstract 3195: Quantification of Endothelial Cell Apoptosis in Pulmonary Arterial Hypertension Using Tc99m-Annexin V

Background - Pulmonary artery hypertension (PAH) is usually detected late in its course. Early detection may allow for the earlier initiation of disease-modifying therapies. Endothelial cell (EC) apoptosis is an early event that leads to unrestrained EC growth and subsequent smooth muscle cell proliferation. We tested the hypothesis that these early apoptotic changes may be detected non-invasively using Tc99m-labeled Annexin V (Tc99m-ANX) in the monocrotaline (MCT)-induced PAH rat model. Methods - To document disease course, 54 MCT treated Sprague Dawley rats (60mg/kg) were compared to 18 controls; 5– 6 treated rats and 3 controls were sacrificed weekly over 6 weeks for gross pathology and histopathologic sectioning. PA pressures were estimated by echocardiography. EC apoptosis was detected by caspase staining in arterioles smaller than 50 microns and quantified by random sampling. A second group of 16 rats were treated with MCT and underwent serial non-invasive SPECT imaging. They were injected with 55.5 MBq of Tc99m-ANX and imaged by HiSPECT at baseline (before MCT) and weekly for 6 weeks. Quantification of Tc99m-ANX uptake was assessed by biodistribution and by region of interest (ROI) analyses of SPECT images using MIP Tool. Results - See table below. Quantitative pulmonary tracer uptake peaked at day 14, correlated with peak positive caspase staining and preceded hemodynamic changes and RVH. Tc99m-ANX pulmonary uptake seen at later time points corresponded to caspase positive inflammatory cells in a dense intra-alveolar infiltrate. Conclusions - EC apoptosis can be detected in vivo with Tc99m-ANX during the early development of PAH in MCT treated rats before the onset of any detectable changes in pulmonary pressures or changes in RV architecture. Data Summary

VEGF and PDGF have distinct roles in development of experimental obliterative bronchiolitis—a therapeutic approach

Obliterative bronchiolitis (OB) is the leading cause of lung allograft loss but no efficient treatment for OB is exists. Inflammation and subsequent smooth muscle cell proliferation are central in OB development, leading to gradual occlusion of bronchioles. We examined the possibility to treat OB by selective inhibition of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinase activity in rat tracheal allografts with PTK787 and imatinib respectively.

Combined Vascular Endothelial Growth Factor and Platelet-Derived Growth Factor Inhibition in Rat Cardiac Allografts: Beneficial Effects on Inflammation and Smooth Muscle Cell Proliferation

Perivascular inflammation and subsequent smooth muscle cell (SMC) proliferation are central in the development of cardiac allograft arteriosclerosis. We examined the effect of combined inhibition of proinflammatory vascular endothelial growth factor (VEGF) and SMC mitogen platelet-derived growth factor (PDGF) in rat cardiac allografts. Heterotopic cardiac transplantations were performed between fully major histocompatibility mismatched rat strains receiving cyclosporine A immunosuppression. In situ hybridization and immunohistochemistry were performed to examine VEGF and PDGF ligand and receptor (R) expression. Protein tyrosine kinase inhibitors PTK787 and imatinib were used to inhibit VEGFR and PDGFR activity, respectively. Rat coronary artery SMC migration and proliferation assays were used to examine the effect of VEGF and PDGF and tyrosine kinase inhibitors in vitro. Both ligand and receptor expression of VEGF and PDGF were detected in chronically rejecting allografts. In vitro, PDGF-BB mediated rat coronary artery SMC migration and proliferation was completely inhibited with imatinib and partially with PTK787. In vivo, combined treatment with PTK787 and imatinib significantly reduced the formation of neointimal lesions in arteries of cardiac allografts at 8 weeks, producing a greater effect than either drug alone. PTK787, in contrast with imatinib, reduced the number of ED1 macrophages and PDGF-B immunoreactivity in the allografts at 4 weeks. Blocking VEGF and PDGF receptor signaling in cardiac allografts has distinctive effects on inflammation and SMC proliferation, suggesting that targeting both inflammation and pathologic vascular remodeling may be needed to inhibit cardiac allograft arteriosclerosis.

Efficient Inhibition of In-stent Restenosis by Controlled Stent-based Inhibition of Elastase: A Pilot Study

It is proposed that local elastase inhibition could suppress the extracellular matrix (ECM) degradation and subsequent smooth muscle cell migration and limit subsequent in-stent restenosis. This study evaluated the effect of stent-based controlled elastase inhibition on restenosis after stent implantation in a rabbit model. Biodegradable microspheres containing the potent elastase inhibitor alpha-1-antitrypsin (AAT) were prepared. Daily release of AAT from the microspheres was confirmed in vitro. The microspheres were loaded into stents with an abluminal polymer reservoir. Implantation of the stent with AAT microspheres and blank microspheres (control) was performed in the abdominal aortae of six rabbits in each group. After stent deployment, all stents were overdilated to 125% diameter. Stent-implanted arteries were harvested after 7 days (n = 3 each) or 28 days (n = 3 each). To assess the effect of local delivery of AAT, elastase activity and elastin content of the stent-implanted aortae were analyzed. As an endpoint, intima-to-media (I/M) ratio was determined in the 7-day and 28-day specimens. Significant inhibition of elastase was confirmed in treated vessels versus controls at 7 days after stent implantation (P < .05). This reduction in elastase activity was sufficient to afford early and late reduction of in-stent neointima. Plaque progression in the 28-day specimens decreased to 67% with elastase inhibition relative to controls (P < .05). Stent-based controlled release of elastase inhibitor may significantly reduce ECM degradation and might limit in-stent restenosis.

Role for matrix metalloproteinase-2 in oxidized low-density lipoprotein-induced activation of the sphingomyelin/ceramide pathway and smooth muscle cell proliferation.

Oxidized LDLs (oxLDLs) and matrix metalloproteinases (MMPs) are present in atherosclerotic lesions. OxLDLs activate various signaling pathways potentially involved in atherogenesis. OxLDLs induce smooth muscle cell (SMC) proliferation mediated by the activation of the sphingomyelin/ceramide pathway and tyrosine kinase receptors. MMPs are also able to induce SMC migration and proliferation in addition to extracellular matrix degradation. The present study was designed to investigate whether MMPs play a role in the mitogenic effect of oxLDLs. OxLDLs induce the release of activated MMP-2 in SMC culture medium. MMP-2 was identified by its 65-kDa gelatinase activity on zymography and by using specific blocking antibodies and MMP-2-/- cells. MMP inhibitors (batimastat and Ro28-2653) and the blocking antibodies anti-MMP-2 and anti-membrane type 1-MMP inhibited the oxLDL-induced sphingomyelin/ceramide pathway activation and subsequent activation of ERK1/2 and DNA synthesis but did not inhibit the oxLDL-induced epidermal growth factor receptor and platelet-derived growth factor receptor activation. Exogenously added activated MMP-2 or membrane type 1-MMP-1 triggered the activation of both sphingomyelin/ceramide and ERK1/2 pathways and DNA synthesis. Conversely, suppression of MMP-2 expression in MMP-2-/- cells or in SMCs treated by small-interference RNA also blocked both sphingomyelin/ceramide signaling and DNA synthesis. Together, these data demonstrate that MMP-2 plays a pivotal role in oxLDL-induced activation of the sphingomyelin/ceramide signaling pathway and subsequent SMC proliferation. These pathways may constitute a potential therapeutic target for modulating the oxLDL-induced proliferation of SMCs in atherosclerosis or restenosis.

Neointima formation on vascular elastic laminae and collagen matrices scaffolds implanted in the rat aortae

Synthetic polymers, including polytetrafluoroethylene and Dacron, and biomatrix proteins, including collagen and fibrin, have been used for the construction of vascular substitutes. However, these materials induce inflammatory reactions, contributing to thrombosis, smooth muscle cell (SMC) proliferation, and neointima formation, processes leading to the failure of vascular substitutes. Thus, a pressing issue in vascular reconstruction is to construct vascular substitutes with surface materials that are inflammation-ressistant. Here, we demonstrate that the vascular elastic laminae exhibit such a property. Aortic specimens from donor rats were treated with 0.1 m NaOH for various times, resulting in elastic lamina–collagen matrix scaffolds with and without the basal lamina. Matrix scaffolds were implanted into the host aorta with three different surface materials, including the elastic lamina, basal lamina, and adventitial collagen, and observed for leukocyte adhesion, endothelial migration, cell proliferation, and neointimal formation on these surfaces. It was found that the elastic lamina was associated with significantly lower leukocyte adhesion, BrdU incorporation, and neointima formation than the basal lamina and adventitial collagen, while the migration of endothelial cells was comparable on all three surfaces. The adventitial collagen matrix was associated with leukocyte infiltration from blood and subsequent SMC migration from the host aorta, whereas the elastic laminae were resistant to such processes. The morphology of the implanted elastic laminae appeared normal at all times. These observations suggest that the vascular elastic laminae exhibit inflammation-resistant properties and inhibit SMC mitogenic activities compared with collagen-containing matrices and may be considered a potential surface material for vascular reconstruction.

Do Pathogens Accelerate Atherosclerosis?

Infection with the pathogens human cytomegalovirus (HCMV) or Chlamydia pneumonia (CP) is linked to the development of vascular disease, including atherosclerosis. The role of pathogens in vasculopathies has been controversial. However, animal models have demonstrated a direct link between infection with CP and herpesviruses and the development of vascular disease. Clinical studies have shown a direct association of HCMV and CP with the acceleration of vascular disease. This article will review the evidence supporting the role for CP and HCMV in the development of vascular disease and will suggest a potential mechanism for HCMV acceleration of the disease process. Vascular diseases are the result of either mechanical or immune-related injury followed by inflammation and subsequent smooth muscle cell (SMC) proliferation and/or migration from the vessel media to the intima, which culminates in vessel narrowing. A number of in vitro and in vivo models have provided potential mechanisms involved in pathogen-mediated vascular disease. Recently, we have demonstrated that HCMV infection of arterial but not venous SMC results in significant cellular migration in vitro. Migration was dependent on expression of the HCMV-encoded chemokine receptors, US28, and the presence of the chemokines, RANTES or MCP-1. Migration involved chemotaxis and provided the first evidence that viruses may induce migration of SMC toward sites of chemokine production through the expression of a virally encoded chemokine receptor in infected SMC. Because SMC migration into the neointimal space is the hallmark of vascular disease, these observations provide a molecular link between HCMV and the development of vascular disease.

Role of polymorphonuclear leukocytes in collar-induced intimal thickening in the rabbit carotid artery.

In this study, the involvement of polymorphonuclear leukocytes (PMNs) in the development of intimal thickening was investigated. A fibromuscular intima was induced by placing a silicone collar around the rabbit carotid artery for 3 days or 2 weeks; the contralateral artery was sham operated. Rabbits received placebo treatments (groups 1 and 3), granulocyte-colony stimulating factor (group 2; G-CSF, 20 microg x kg(-1) x d(-1), delivered by subcutaneous osmotic pumps), or an anti-CD18 monoclonal antibody (group 4; 1.5 mg/kg i.v.). The G-CSF treatment raised the peripheral PMN count 5- to 12-fold but had no effect on intimal thickening on day 3, 12, or 14. A single injection of anti-CD18 prevented PMN extravasation 6 hours after collar implantation without influencing intimal hyperplasia on day 14. Repeated daily administration of anti-CD18 strongly bound to CD18 on peripheral PMNs and inhibited both PMN-dependent plasma extravasation in the skin and accumulation of CD14-immunoreactive leukocytes in the intima and media. However, anti-CD18 did not suppress early intimal thickening or accumulation of alpha-smooth muscle actin-immunoreactive cells by day 3. It thus appears that the PMN influx in the intima and media evoked by the perivascular collar is of little functional relevance to the subsequent smooth muscle cell migration and intimal thickening in this model.

Effects of hypertension on migration and proliferation of smooth muscle in culture.

Migratory and proliferative characteristics of explanted rat aortic smooth muscle cells were studied in response to hypertension induced by 4 weeks of deoxycorticosterone-salt administration. Under low serum conditions (0.1% fetal bovine serum), over 80% of aortic medial explants from hypertensive rats yielded smooth muscle cell colonies after 8 days of culture while fewer than 10% of the control explants were positive. Time lapse video analysis of subsequent growth in the presence of 10% serum revealed that interdivision times of smooth muscle cells from hypertensive animals were significantly shorter than those in controls (p less than 0.01). Significant differences in proliferative capacity of smooth muscle cells were evident, even after one subculture (p less than 0.01). Comparison of these results with data from mechanical injury suggests that 4 weeks of deoxycorticosterone-salt hypertension can potentiate subsequent smooth muscle cell migration and growth in vitro to an extent similar to that observed with the combined effects of total endothelial denudation and wall distention by a balloon catheter.

In vivo aortic muscle cell growth kinetics. Differences between thoracic and abdominal segments after intimal injury in the rabbit.

Focal proliferation of smooth muscle cell (SMC) is an integral part of atherosclerotic plaque formation: characterization of regional variation in SMC growth kinetics is therefore important to the understanding of atherogenesis. SMC growth kinetics of rabbit abdominal and thoracic segments were compared. Rabbit aortas were denuded of endothelium and the animals killed after 3H-thymidine and Evans blue injections at 0 to 48 days after denudation. Incorporation of 3H-thymidine into both aortic segments peaked at 48 hours; no detectable incorporation occurred in the first 24 hours. Abdominal segment DNA specific activity (SA, dpm/micrograms DNA) and total kinetic activity (TKA, dpm/0.1 mm internal elastic lamina) at 48 hours were significantly greater than values for the thoracic aorta. Abdominal SA and TKA curves decreased exponentially after the 48-hour peak and parallel thoracic levels after day 7. SA and TKA values for each segment reflected the subsequent SMC intimal growth rates as measured morphometrically. Therefore, both segments share similar growth kinetic characteristics; however, the abdominal response to initimal injury is greater than the thoracic and leads to greater myointimal proliferation. The difference in response to injury in the two segments suggests regional variation in SMC's which are phenotypically similar.