Linda Cheng

Paclitaxel Stent Coating Inhibits Neointimal Hyperplasia at 4 Weeks in a Porcine Model of Coronary Restenosis

BackgroundDespite limiting elastic recoil and late vascular remodeling after angioplasty, coronary stents remain vulnerable to restenosis, caused primarily by neointimal hyperplasia. Paclitaxel, a microtubule-stabilizing drug, has been shown to inhibit vascular smooth muscle cell migration and proliferation contributing to neointimal hyperplasia. We tested whether paclitaxel-coated coronary stents are effective at preventing neointimal proliferation in a porcine model of restenosis. Methods and ResultsPalmaz-Schatz stents were dip-coated with paclitaxel (0, 0.2, 15, or 187 &mgr;g/stent) by immersion in ethanolic paclitaxel and evaporation of the solvent. Stents were deployed with mild oversizing in the left anterior descending coronary artery (LAD) of 41 minipigs. The treatment effect was assessed 4 weeks after stent implantation. The angiographic late loss index (mean luminal diameter) decreased with increasing paclitaxel dose (P <0.0028 by ANOVA), declining by 84.3% (from 0.352 to 0.055, P <0.05) at the highest level tested (187 &mgr;g/stent versus control). Accompanying this change, the neointimal area decreased (by 39.5%, high-dose versus control;P <0.05) with increasing dose (P <0.040 by ANOVA), whereas the luminal area increased (by 90.4%, high-dose versus control;P <0.05) with escalating dose (P <0.0004 by ANOVA). Inflammatory cells were seen infrequently, and there were no cases of aneurysm or thrombosis. ConclusionsPaclitaxel-coated coronary stents produced a significant dose-dependent inhibition of neointimal hyperplasia and luminal encroachment in the pig LAD 28 days after implantation; later effects require further study. These results demonstrate the potential therapeutic benefit of paclitaxel-coated coronary stents in the prevention and treatment of human coronary restenosis.

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.

Angiotensin II activates matrix metalloproteinase type II and mimics age-associated carotid arterial remodeling in young rats.

Increased angiotensin II (Ang II), matrix metalloproteinase type II (MMP2), and sympathetic activity accompany age-associated arterial remodeling. To analyze this relationship, we infused a low subpressor dose of Ang II into young (8 months old) rats. This increased carotid arterial MMP2 transcription, translation, and activation, as well as transforming growth factor-beta1 activity and collagen deposition. A higher Ang II concentration, which increased arterial pressure to that of old (30 months old) untreated rats, produced carotid media thickening and intima infiltration by vascular smooth muscle cells (VSMCs). Ex vivo, Ang II increased MMP2 activity in carotid rings from young rats to that of untreated old rats. Ang II also increased the ability of early passage VSMCs from young rats to invade a synthetic basement membrane, similar to that of untreated VSMCs from old rats. The MMP inhibitor GM6001 and the AT1 receptor antagonist Losartan inhibited these effects. The alpha-adrenoreceptor agonist phenylephrine increased arterial Ang II protein, causing MMP2 activation and intima and media thickening. Exposure of young VSMCs to phenylephrine in vitro increased Ang II protein and MMP2 activity to the levels of old VSMCs; Losartan abolished these effects. Thus, Ang II-induced effects on MMP2, transforming growth factor-beta1, collagen, and VSMCs are central to the arterial remodeling that accompanies advancing age.

Adenovirus-mediated gene transfer of the human tissue inhibitor of metalloproteinase-2 blocks vascular smooth muscle cell invasiveness in vitro and modulates neointimal development in vivo

BACKGROUND Endovascular injury induced by balloon withdrawal leads to the increased activation of matrix metalloproteinases (MMPs) in the vascular wall, allowing smooth muscle cells (SMCs) to digest the surrounding extracellular matrix (ECM) and migrate from the media into the intima. The objective of this study was to examine the effects of a replication-deficient adenovirus carrying the cDNA for human tissue inhibitor of metalloproteinase-2 (AdCMV.hTIMP-2) on SMC function in vitro and neointimal development in the injured rat carotid artery. METHODS AND RESULTS Infection of cultured rat aortic SMCs at a multiplicity of infection of 100 with AdCMV.hTIMP-2 resulted in high-level expression of hTIMP-2 mRNA and protein secretion into the medium. Conditioned media (CM) from AdCMV. hTIMP-2-infected but not control virus (AdCMV.null or AdCMV. betagal)-infected SMCs inhibited MMP-2 activity on gelatin zymograms as well as the chemoattractant-directed migration of SMCs across reconstituted basement membrane proteins in the Boyden chamber assay. In contrast, AdCMV.hTIMP-2 CM had no effect on chemoattractant-directed migration of SMCs occurring in the absence of an ECM barrier or on the proliferation of cultured neointimal SMCs. Delivery of AdCMV.hTIMP-2 (2.5x10(9) pfu) to the carotid artery wall at the time of balloon withdrawal injury inhibited SMC migration into the intima by 36% (P<0.05) at 4 days and neointimal area by 53% (P<0.01) at 8 days and by 12% (P=NS) at 21 days after injury. AdCMV.hTIMP-2 had no effect on medial area. CONCLUSIONS Adenovirus-mediated hTIMP-2 gene transfer inhibits SMC invasiveness in vitro and in vivo and delays neointimal development after carotid injury.

Increased expression of membrane-type matrix metalloproteinase and preferential localization of matrix metalloproteinase-2 to the neointima of balloon-injured rat carotid arteries

BACKGROUND Remodeling of the injured vascular wall is dependent on the action of several extracellular proteases. Previous studies have shown that expression of matrix metalloproteinases (MMP-2 and MMP-9) is upregulated after vascular injury and that MMP-2 is required for the migration of cultured vascular smooth muscle cells across complex extracellular matrix barriers. The present study examined changes in the expression of membrane-type metalloproteinase (MT-MMP-1), a putative regulator of MMP-2, in the tissue localization of MMP-2, and in the expression of activated and latent forms of MMP-2 and the tissue inhibitor of metalloproteinases, TIMP-2, in rat carotid arteries subjected to balloon catheter injury. METHODS AND RESULTS MT-MMP-1 mRNA levels increased sixfold after 3 days of injury, coinciding with an increase in MMP-2 activation assessed by gelatin zymography. Western blotting and gelatin zymography showed an increase in MMP-2 protein levels beginning 5 to 7 days after injury; immunocytochemistry and Western blotting showed that the increase occurred preferentially in the developing neointima. CONCLUSIONS These results show that increased expression of MT-MMP-1 and activation of MMP-2 occurs early after injury to the rat carotid artery and that at later times MMP-2 is preferentially localized to the developing neointima.

Vascular smooth muscle cell cultures.

Publisher Summary Vascular smooth muscle cells (VSMCs) are the predominant cell type in blood vessels of the arterial tree. They are responsible for maintaining vascular tone in response to various hormonal and hemodynamic stimuli and they are the major cell type involved in the process of vascular repair, growth, and response to injury. The development of cell culture systems has greatly aided in this pursuit by providing a controlled environment to study the unique characteristics of these cells in isolation from other influences. When the blood vessel is mechanically injured and its endothelial lining removed, there is significant remodeling of the vessel. Medial VSMCs migrate across the elastic laminae and basement membrane and accumulate in the intima. There they proliferate and secrete increased amounts of interstitial matrix proteins. This new intimal structure is appropriately referred to as the neointima and the cells within it are neointimal VSMCs. This chapter discusses methods for culturing both medial and neointimal VSMCs as well as for culturing VSMCs obtained from atheromatous lesions.

A functional ryanodine-sensitive intracellular Ca2+ store is present in vascular endothelial cells.

The presence of the ryanodine receptor was recently demonstrated in vascular and endocardial endothelium, but its function has not been established. We investigated whether functional ryanodine-sensitive Ca2+ stores are present in cultured endothelial cells from rat aorta (RAECs), human aorta (HAECs), human umbilical vein (HUVECs), and bovine pulmonary artery (BPAECs) and what role these may play in intracellular Ca2+ regulation. Under resting conditions, HAECs, BPAECs, and HUVECs demonstrated a slow increase in intracellular Ca2+ (indexed by indo 1 fluorescence) on exposure to 5 mumol/L ryanodine, whereas RAECs did not. However, after an initial bradykinin exposure in RAECs, ryanodine markedly blunted the rapid increase in Ca2+ on a second exposure to bradykinin. In HUVECs, ryanodine in buffer with 1.5 mmol/L Ca2+ did not inhibit the agonist-sensitive Ca2+ increase, whereas it blunted the rapid increase in Ca2+ on histamine exposure in buffer with 5 mmol/L Ca2+, suggesting that increasing [Ca2+] enhances the binding of ryanodine to its receptor. Thus, functional ryanodine-sensitive Ca2+ stores are present in vascular endothelial cells. These appear to be involved in regulation of Ca2+ storage and release from agonist-sensitive intracellular compartments.

Cytosolic alkalinization of vascular endothelial cells produced by an abrupt reduction in fluid shear stress.

Reductions in fluid shear stress produce endothelium-dependent vasoconstriction and promote neointimal hyperplasia, but the intracellular signaling mechanisms involved in these processes are poorly understood. To examine whether decreases in fluid shear stress affect endothelial cytosolic pH, carboxy-seminaphthorhodafluor-1-loaded rat aortic endothelial cells were cultured in glass microcapillary tubes and examined during abrupt reductions in laminar flow. After a 30-minute exposure to a shear stress of 2.7 dyne/cm2 in bicarbonate buffer, the acute reduction of fluid shear stress from 2.7 to 0.3 dyne/cm2 transiently increased cytosolic pH from 7.20+/-0.02 to 7.47+/-0.07 (mean+/-SEM, P<.05 versus control). This was not affected by prior inhibition of the Na+-H+ exchanger with 10 micromol/L ethylisopropylamiloride but was abolished in bicarbonate-free buffer. Recovery from an ammonium chloride prepulse-induced acid load occurred more rapidly when fluid shear stress was abruptly reduced from 2.7 to 0.3 dyne/cm2 after maximal acidification (+0.04+/-0.02 pH unit at 2 minutes) than when shear stress was maintained at 2.7 dyne/cm2 continuously (0.00+/-0.00 pH unit at 2 minutes, P<.05). This accelerated cytosolic pH recovery was dependent on the presence of bicarbonate ion and was blocked by the addition of the exchange inhibitors DIDS (100 micromol/L) and ethylisopropylamiloride or by removal of buffer Na+, indicating that the acute reduction in fluid shear stress activates the extracellular Na+-dependent Cl(-)-HCO3- exchanger and the Na+-H+ exchanger and increases cytosolic pH in vascular endothelial cells.

Protein arginine methyltransferase 5 functions as an epigenetic activator of the androgen receptor to promote prostate cancer cell growth

Protein arginine methyltransferase 5 (PRMT5) is an emerging epigenetic enzyme that mainly represses transcription of target genes via symmetric dimethylation of arginine residues on histones H4R3, H3R8 and H2AR3. Accumulating evidence suggests that PRMT5 may function as an oncogene to drive cancer cell growth by epigenetic inactivation of several tumor suppressors. Here, we provide evidence that PRMT5 promotes prostate cancer cell growth by epigenetically activating transcription of the androgen receptor (AR) in prostate cancer cells. Knockdown of PRMT5 or inhibition of PRMT5 by a specific inhibitor reduces the expression of AR and suppresses the growth of multiple AR-positive, but not AR-negative, prostate cancer cells. Significantly, knockdown of PRMT5 in AR-positive LNCaP cells completely suppresses the growth of xenograft tumors in mice. Molecular analysis reveals that PRMT5 binds to the proximal promoter region of the AR gene and contributes mainly to the enriched symmetric dimethylation of H4R3 in the same region. Mechanistically, PRMT5 is recruited to the AR promoter by its interaction with Sp1, the major transcription factor responsible for AR transcription, and forms a complex with Brg1, an ATP-dependent chromatin remodeler, on the proximal promoter region of the AR gene. Furthermore, PRMT5 expression in prostate cancer tissues is significantly higher than that in benign prostatic hyperplasia tissues, and PRMT5 expression correlates positively with AR expression at both the protein and mRNA levels. Taken together, our results identify PRMT5 as a novel epigenetic activator of AR in prostate cancer. Given that inhibiting AR transcriptional activity or androgen synthesis remains the major mechanism of action for most existing anti-androgen agents, our findings also raise an interesting possibility that targeting PRMT5 may represent a novel approach for prostate cancer treatment by eliminating AR expression.

The Sustained Release of Galardin and Taxol from Gelatin Chondroitin Sulfate Coacervate Films

Coronary artery atherosclerosis is the leading cause of death in the United States. Restenosis following percutaneous transluminal balloon angioplasty (PTCA) remains the limiting factor in the use of this treatment for coronary artery disease. Restenosis occurs in 30% of patients within 6 months. The restenotic lesion is a fibroproliferative response with resulting smooth muscle cell migration, proliferation and extracellular matrix production. Despite a decade of research there is no effective strategy for preventing restenosis in man.