Decorin mimic inhibits vascular smooth muscle proliferation and migration.
ABSTRACT: Over the past 10 years, the number of percutaneous coronary intervention procedures performed in the United States increased by 33%; however, restenosis, which inhibits complete functional recovery of the vessel wall, complicates this procedure. A wide range of anti-restenotic therapeutics have been developed, although many elicit non-specific effects that compromise vessel healing. Drawing inspiration from biologically-relevant molecules, our lab developed a mimic of the natural proteoglycan decorin, termed DS-SILY, which can mask exposed collagen and thereby effectively decrease platelet activation, thus contributing to suppression of vascular intimal hyperplasia. Here, we characterize the effects of DS-SILY on both proliferative and quiescent human SMCs to evaluate the potential impact of DS-SILY-SMC interaction on restenosis, and further characterize in vivo platelet interactions. DS-SILY decreased proliferative SMC proliferation and pro-inflammatory cytokine secretion in vitro in a concentration dependent manner as compared to untreated controls. The addition of DS-SILY to in vitro SMC cultures decreased SMC migration and protein synthesis by 95% and 37%, respectively. Furthermore, DS-SILY decreased platelet activation, as well as reduced neointimal hyperplasia by 60%, in vivo using Ossabaw swine. These results indicate that DS-SILY demonstrates multiple biological activities that may all synergistically contribute to an improved treatment paradigm for balloon angioplasty.
Project description:Following balloon injury, smooth muscle cells (SMCs) serve as targets for many of the pro-inflammatory and pro-fibrotic factors, including platelet-derived growth factor (PDGF) and interferon-? (IFN-?) released from activated inflammatory cells and platelets. Previously, our lab designed a mimic of the proteoglycan decorin, termed DS-SILY20, that suppressed vascular SMC proliferation, migration, and protein synthesis in vitro, and injured vessels treated with DS-SILY20 demonstrated reduced hyperplasia in vivo. Here we characterize the effects of DS-SILY20 on modulating PDGF and IFN-? stimulation in both proliferative and quiescent human SMCs to further evaluate the potential impact of DS-SILY20-SMC interaction on restenosis. Nanomolar dissociation constants were observed between DS-SILY20 and both PDGF and IFN-?. PDGF significantly increased migration, proliferation, and protein and cytokine expression, as well as increased ERK-1/2 and p38 MAPK phosphorylation in both quiescent and proliferative cultures. However, DS-SILY20 inhibited these increases, presumably through sequestration of the PDGF. Consistent with the complex responses seen with IFN-? in SMC physiology in the literature, the response of SMC cultures to IFN-? was variable and complex. However, where increased activity was seen with IFN-?, DS-SILY20 attenuated this activity. Overall, the results suggest that DS-SILY20 would be an ideal alternative to traditional therapeutics used and may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.
Project description:To date, there is no periadventitial drug delivery method available in the clinic to prevent restenotic failure of open vascular reconstructions. Resveratrol is a promising anti-restenotic natural drug but subject to low bioavailability when systemically administered. In order to reconcile these two prominent issues, we tested effects of periadventitial delivery of resveratrol on all three major pro-restenotic pathologies including intimal hyperplasia (IH), endothelium impairment, and vessel shrinkage. In a rat carotid injury model, periadventitial delivery of resveratrol either via Pluronic gel (2-week), or polymer sheath (3-month), effectively reduced IH without causing endothelium impairment and vessel shrinkage. In an in vitro model, primary smooth muscle cells (SMCs) were stimulated with elevated transforming growth factor (TGFβ) and its signaling protein Smad3, known contributors to IH. TGFβ/Smad3 up-regulated Kruppel-like factor (KLF5) protein, and SMC de-differentiation which was reversed by KLF5 siRNA. Furthermore, TGFβ/Smad3-stimulated KLF5 production and SMC de-differentiation were blocked by resveratrol via its inhibition of the Akt-mTOR pathway. Concordantly, resveratrol attenuated Akt phosphorylation in injured arteries. Taken together, periadventitial delivery of resveratrol produces durable inhibition of all three pro-restenotic pathologies - a rare feat among existing anti-restenotic methods. Our study suggests a potential anti-restenotic modality of resveratrol application suitable for open surgery.
Project description:Drug-eluting stents are the most commonly employed method to control post-angioplasty restenosis. Unfortunately, they exacerbate life-threatening stent thrombosis because of endothelium damage caused by both drug and stenting. To solve this major medical problem, an endothelium-protective and stent-free anti-restenotic method is highly desirable. Here we have generated a biomimetic intravenous delivery system using dendritic polymer-based nanoclusters, which were coated with platelet membranes for targeting to the injured arterial wall where restenosis occurs. These nanoclusters were loaded with an endothelium-protective epigenetic inhibitor (JQ1) or an endothelium-toxic status quo drug (rapamycin), and compared for their ability to mitigate restenosis without hindering the process of re-endothelialization. Fluorescence imaging of Cy5-tagged biomimetic nanoclusters indicated their robust homing to injured, but not uninjured arteries. Two weeks after angioplasty, compared to no-drug control, both rapamycin- and JQ1-loaded biomimetic nanoclusters substantially reduced (by >60%) neointimal hyperplasia, the primary cause of restenosis. However, whereas the rapamycin formulation impaired the endothelial re-coverage of the denuded inner arterial wall, the JQ1 formulation preserved endothelial recovery. In summary, we have created an endothelium-protective anti-restenotic system with biomimetic nanoclusters containing an epigenetic inhibitor. This system warrants further development for a non-thrombogenic and stent-free method for clinical applications.
Project description:RATIONALE:Vascular smooth muscle cell (SMC) phenotypic modulation and vascular remodeling contribute to the development of several vascular disorders such as restenosis after angioplasty, transplant vasculopathy, and atherosclerosis. The mechanisms underlying these processes, however, remain largely unknown. OBJECTIVE:The objective of this study is to determine the role of dedicator of cytokinesis 2 (DOCK2) in SMC phenotypic modulation and vascular remodeling. METHODS AND RESULTS:Platelet-derived growth factor-BB induced DOCK2 expression while modulating SMC phenotype. DOCK2 deficiency diminishes platelet-derived growth factor-BB or serum-induced downregulation of SMC markers. Conversely, DOCK2 overexpression inhibits SMC marker expression in primary cultured SMC. Mechanistically, DOCK2 inhibits myocardin expression, blocks serum response factor nuclear location, attenuates myocardin binding to serum response factor, and thus attenuates myocardin-induced smooth muscle marker promoter activity. Moreover, DOCK2 and Kruppel-like factor 4 cooperatively inhibit myocardin-serum response factor interaction. In a rat carotid artery balloon-injury model, DOCK2 is induced in media layer SMC initially and neointima SMC subsequently after vascular injury. Knockdown of DOCK2 dramatically inhibits the neointima formation by 60%. Most importantly, knockout of DOCK2 in mice markedly blocks ligation-induced intimal hyperplasia while restoring SMC contractile protein expression. CONCLUSIONS:Our studies identified DOCK2 as a novel regulator for SMC phenotypic modulation and vascular lesion formation after vascular injury. Therefore, targeting DOCK2 may be a potential therapeutic strategy for the prevention of vascular remodeling in proliferative vascular diseases.
Project description:To characterize and compare primary and restenotic lesions of the superficial femoral artery and analyze the contribution of TGF-beta/Smad3 signaling to the pathophysiology of peripheral artery occlusive disease.Immunohistochemical studies were performed on specimens retrieved from the superficial femoral artery of patients undergoing either atherectomy for primary atherosclerotic or recurrent disease after stenting and/or prior angioplasty. Immunohistochemical analysis revealed a significantly higher smooth muscle cell (SMC) content (alpha-actin+) and expression of Smad3 in restenotic lesions while primary lesions contained significantly more leukocytes (CD45+) and macrophages (CD68+). Further studies demonstrated colocalization of Smad3 with alpha-actin and PCNA, suggesting a role for Smad3 in the proliferation observed in restenotic lesions. To confirm a role for Smad3 in SMC proliferation, we both upregulated Smad3 via adenoviral mediated gene transfer (AdSmad3) and inhibited Smad3 through transfection with siRNA in human aortic SMCs, then assessed cell proliferation with tritiated thymidine. Overexpression of Smad3 enhanced whereas inhibition of Smad3 decreased cell proliferation.Differences in cellular composition and cell proliferation in conjunction with the finding that Smad3 is expressed exclusively in restenotic disease suggest that TGF-beta, through Smad3 signaling, may play an essential role in SMC proliferation and the pathophysiology of restenosis in humans.
Project description:<h4>Background</h4>Glaucoma is characterized by the progressive dysfunction and loss of retinal ganglion cells. Recent work in animal models suggests that a critical neuroinflammatory event damages retinal ganglion cell axons in the optic nerve head during ocular hypertensive injury. We previously demonstrated that monocyte-like cells enter the optic nerve head in an ocular hypertensive mouse model of glaucoma (DBA/2?J), but their roles, if any, in mediating axon damage remain unclear.<h4>Methods</h4>To understand the function of these infiltrating monocyte-like cells, we used RNA-sequencing to profile their transcriptomes. Based on their pro-inflammatory molecular signatures, we hypothesized and confirmed that monocyte-platelet interactions occur in glaucomatous tissue. Furthermore, to test monocyte function we used two approaches to inhibit their entry into the optic nerve head: (1) treatment with DS-SILY, a peptidoglycan that acts as a barrier to platelet adhesion to the vessel wall and to monocytes, and (2) genetic targeting of Itgam (CD11b, an immune cell receptor that enables immune cell extravasation).<h4>Results</h4>Monocyte specific RNA-sequencing identified novel neuroinflammatory pathways early in glaucoma pathogenesis. Targeting these processes pharmacologically (DS-SILY) or genetically (Itgam / CD11b knockout) reduced monocyte entry and provided neuroprotection in DBA/2?J eyes.<h4>Conclusions</h4>These data demonstrate a key role of monocyte-like cell extravasation in glaucoma and demonstrate that modulating neuroinflammatory processes can significantly lessen optic nerve injury.
Project description:<h4>Aims</h4>Restenosis in drug-eluting stents (DESs) occurs infrequently, however, it remains a pervasive clinical problem. We interrogated our autopsy registry to determine the underlying mechanisms of DES restenosis, and further we investigated the neointimal characteristics of DESs and compared with bare metal stents (BMSs).<h4>Methods and results</h4>Coronary lesions from patients with DES implants (n = 82) were categorized into four groups based on cross-sectional area narrowing: patent (<50%), intermediate (50-74%), restenotic (≥ 75% with residual lumen), and total occlusion (organized thrombus within the stent). Restenosis and occlusion were significantly dependent on the total stented length: restenosis (26.7 mm) and occlusion (25.7 mm) compared with patent DESs (17.3 mm). Further, restenotic and occluded lesions were located more distally in the coronary arteries and had greater vessel injury and uneven strut distribution suggesting local drug gradient. Multivariate analysis revealed that normalized maximum inter-strut distance was associated with DES restenosis (OR: 17.4, P = 0.04) while medial tear length was a predictor of DES occlusion (OR: 5.1, P = 0.03). No differences were observed between different DESs (sirolimus-, paclitaxel-, and everolimus-eluting stents) for restenosis and occlusion. Further, neointimal compositions of restenotic DESs demonstrated greater proteoglycan deposition and less smooth muscle cellularity over time, when compared with BMS with greater cell density and collagen deposition.<h4>Conclusions</h4>Our study indicates the impacts of inadequate drug concentration due to wider inter-strut distance and vessel injury as primary mechanisms of DES restenosis and occlusion, respectively. Moreover, the differences in neointimal compositions between DESs and BMSs might serve as a potential target for the suppression of late neointima growth via inhibition of proteoglycans in DESs.
Project description:Intracoronary stent restenosis, characterized by excessive smooth muscle cell (SMC) proliferation and myointimal hyperplasia, remains a clinical challenge. Mitochondrial membrane potential has been linked to the proliferative rate of SMCs. This study aimed to screen a critical gene regulating mitochondrial potential and to confirm its effects on myointimal formation in preclinical animal models.We performed transcriptome screening for genes differentially expressed in ligated versus unligated mouse carotid arteries. We observed that uncoupling protein 2 gene (Ucp2) mRNA, encoding UCP2, was transiently upregulated during the first 3 days after ligation and then significantly downregulated from day 7 through day 21, during which time neointima formed remarkably. The UCP2 protein level also declined after day 7 of ligation. In ligated carotid arteries, Ucp2-/- mice, compared with wild-type littermates, exhibited accelerated myointimal formation, which was associated with increased superoxide production and can be attenuated by treatment with antioxidant 4-hydroxy-2,2,6,6-tetramethyl-piperidinoxyl (TEMPOL). Knockdown of UCP2 enhanced human aortic SMC migration and proliferation that can also be attenuated by TEMPOL, whereas UCP2 overexpression inhibited SMC migration and proliferation, along with decreased activity of nuclear factor-κB. Moreover, nuclear factor-κB inhibitor attenuated UCP2 knockdown-enhanced SMC proliferation. Adenovirus-mediated overexpression of UCP2 inhibited myointimal formation in balloon-injured carotid arteries of rats and rabbits and in-stent stenosis of porcine coronary arteries. Moreover, UCP2 overexpression also suppressed neointimal hyperplasia in cultured human saphenous vein ex vivo.UCP2 inhibits myointimal hyperplasia after vascular injury, probably through suppressing nuclear factor-κB-dependent SMC proliferation and migration, rendering UCP2 a potential therapeutic target against restenosis.
Project description:Despite the introduction of antiproliferative drug-eluting stents, coronary heart disease remains the leading cause of death in the United States. In-stent restenosis and bypass graft failure are characterized by excessive smooth muscle cell (SMC) proliferation and concomitant myointima formation with luminal obliteration. Here we show that during the development of myointimal hyperplasia in human arteries, SMCs show hyperpolarization of their mitochondrial membrane potential (??m) and acquire a temporary state with a high proliferative rate and resistance to apoptosis. Pyruvate dehydrogenase kinase isoform 2 (PDK2) was identified as a key regulatory protein, and its activation proved necessary for relevant myointima formation. Pharmacologic PDK2 blockade with dichloroacetate or lentiviral PDK2 knockdown prevented ??m hyperpolarization, facilitated apoptosis and reduced myointima formation in injured human mammary and coronary arteries, rat aortas, rabbit iliac arteries and swine (pig) coronary arteries. In contrast to several commonly used antiproliferative drugs, dichloroacetate did not prevent vessel re-endothelialization. Targeting myointimal ??m and alleviating apoptosis resistance is a novel strategy for the prevention of proliferative vascular diseases.