Robert E. Monticone

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.

Proinflammatory Profile Within the Grossly Normal Aged Human Aortic Wall

Studies in animal models demonstrate that angiotensin II and its downstream signaling molecules, that is, matrix metalloproteinases and monocyte chemoattractant protein-1, increase within the diffusely thickened intima of central arteries with aging. Whether such age-related changes occur within the human arterial wall is unknown. We harvested “grossly normal thoracic aortas” from 5 young (20±3 years) and 5 old white males (65±6 years) at necropsy, after death from traumatic causes. The intimae of older samples were markedly and diffusely thickened compared with younger intimae and contained increased levels of angiotensin-converting enzyme, angiotensin II, angiotensin II receptor type 1, matrix metalloproteinases 2/9, monocyte chemoattractant protein-1, and collagen I and III proteins. In situ activities of metalloproteinases 2/9 were also significantly enhanced within old, normal aortas. The thickened intima of older aortas also contained a 5-fold increase in the embryonic form of smooth muscle myosin heavy chain–labeled cells than that of younger aortas, and these fetal-type cells were colocalized with angiotensin II protein staining. The ability of isolated smooth muscle cells to invade an artificial basement membrane in response to a monocyte chemoattractant protein-1 gradient increased with age. Furthermore, angiotensin II increased the invasive capacity of young smooth muscle cells, and this effect was reduced by a metalloproteinase inhibitor or an angiotensin II receptor blocker. Thus, in the absence of lipid infiltration, the aged human aortic wall exhibits a proinflammatory profile that renders it a fertile substrate for the development of arterial disease, for example, atherosclerosis and hypertension.

Matrix Metalloproteinase 2 Activation of Transforming Growth Factor-β1 (TGF-β1) and TGF-β1–Type II Receptor Signaling Within the Aged Arterial Wall

Objective—To study matrix metalloproteinase 2 (MMP-2) effects on transforming growth factor-&bgr;1 (TGF-&bgr;1) activation status and downstream signaling during arterial aging. Methods and Results—Western blotting and immunostaining showed that latent and activated TGF-&bgr;1 are markedly increased within the aorta of aged Fisher 344 cross-bred Brown Norway (30 months of age) rats compared with adult (8 months of age) rats. Aortic TGF-&bgr;1–type II receptor (T&bgr;RII), its downstream molecules p-similar to mad-mother against decapentaplegic (SMAD)2/3 and SMAD4, fibronectin, and collagen also increased with age. Moreover, TGF-&bgr;1 staining is colocalized with that of activated MMP-2 within the aged arterial wall and vascular smooth muscle cell (VSMC) in vitro, and this physical association was confirmed by coimmunoprecipitation. Incubation of young aortic rings ex vivo or VSMCs in vitro with activated MMP-2 enhanced active TGF-&bgr;1, collagen, and fibronectin expression to the level of untreated old counterparts, and this effect was abolished via inhibitors of MMP-2. Interestingly, in old untreated rings or VSMCs, the increased TGF-&bgr;1, fibronectin, and collagen were also substantially reduced by inhibition of MMP-2. Conclusions—Active TGF-&bgr;1, its receptor, and receptor-mediated signaling increase within the aortic wall with aging. TGF-&bgr;1 activation is dependent, in part at least, by a concomitant age-associated increase in MMP-2 activity. Thus, MMP-2–activated TGF-&bgr;1, and subsequently T&bgr;RII signaling, is a novel molecular mechanism for arterial aging.

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.

Rat Aortic MCP-1 and Its Receptor CCR2 Increase With Age and Alter Vascular Smooth Muscle Cell Function

Objective—With age, rat arterial walls thicken and vascular smooth muscle cells (VSMCs) exhibit enhanced migration and proliferation. Monocyte chemotactic protein-1 (MCP-1) affects these VSMC properties in vitro. Because arterial angiotensin II, which induces MCP-1 expression, increases with age, we hypothesized that aortic MCP-1 and its receptor CCR2 are also upregulated and affect VSMC properties. Methods and Results—Both MCP-1 and CCR2 mRNAs and proteins increased in old (30-month) versus young (8-month) F344×BN rat aortas in vivo. Cellular MCP-1 and CCR2 staining colocalized with that of &agr;-smooth muscle actin in the thickened aortas of old rats and were expressed by early-passage VSMCs isolated from old aortas, which, relative to young VSMCs, exhibited increased invasion, and the age difference was abolished by vCCI, an inhibitor of CCR2 signaling. MCP-1 treatment of young VSMCs induced migration and increased their ability to invade a synthetic basement membrane. The MCP-1–dependent VSMC invasiveness was blocked by vCCI. After MCP-1 treatment, migration and invasion capacities of VSMCs from young aortas no longer differed from those of VSMCs isolated from older rats. Conclusions—Arterial wall and VSMC MCP-1/CCR2 increase with aging. MCP-1 enhances VSMC migration and invasion, and thus, MCP-1/CCR2 signaling may play a role in age-associated arterial remodeling.

Arterial aging: a journey into subclinical arterial disease.

Purpose of reviewAge-associated arterial alterations in cells, matrix, and biomolecules are the foundation for the initiation and progression of cardiovascular diseases in older persons. This review focuses on the latest advances on the intertwining of aging and disease within the arterial wall at the cell and molecular levels. Recent findingsEndothelial dysfunction, vascular smooth muscle cell (VSMC) proliferation/invasion/secretion, matrix fragmentation, collagenization and glycation are characteristics of an age-associated arterial phenotype that creates a microenvironment enriched with reactive oxygen species (ROS) for the pathogenesis of arterial disease. This niche creates an age-associated arterial secretory phenotype (AAASP), which is orchestrated by the concerted effects of numerous age-modified angiotensin II signaling molecules. Most of these biomolecular, cell, and matrix modifications that constitute the AAASP can be elicited by experimental hypertension or atherosclerosis at a younger age. The arterial AAASP also shares features of a senescence-associated secretory phenotype (SASP) identified in other mesenchymocytes, that is, fibroblasts. SummaryA subclinical AAASP evolves during aging. Targeting this subclinical AAASP may reduce the incidence and progression of the quintessential age-associated arterial diseases, that is, hypertension and atherosclerosis.

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.

Role of Calcium/Calmodulin-Dependent Protein Kinase II in the Regulation of Vascular Smooth Muscle Cell Migration

BACKGROUND The migration of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of many vascular diseases. We have previously shown that VSMC migration in response to platelet-derived growth factor (PDGF) is suppressed when cultured cells are growth-arrested and induced to differentiate. The present study was undertaken to elucidate the mechanism of this suppression. METHODS AND RESULTS While both proliferating and growth-arrested VSMCs upregulated expression of the immediate early response genes, c-fos and JE (monocyte chemoattractant protein 1), growth-arrested VSMCs exhibited much smaller changes in intracellular calcium in response to PDGF and failed to activate the calcium/calmodulin-dependent protein kinase II (CaM kinase II). Blocking calcium-calmodulin interactions (50 mumol/L W7) or the activation of CaM kinase II (10 mumol/L KN62) in proliferating cells blocked their migration by more than 90%, whereas inhibition of protein kinase C activation had no significant effect on migration. Pretreatment of growth-arrested VSMCs with the calcium ionophore ionomycin resulted in an approximately 2.5-fold activation of CaM kinase II and increased migration of growth-arrested cells to 84 +/- 6% that of proliferating cells. These effects of ionomycin were blocked by inhibitors of CaM kinase II. Constitutively activated (ie, calcium/calmodulin-independent) CaM kinase II introduced by gene transfection into growth-arrested cells significantly increased migration toward PDGF from < 20% to > 70% that of proliferating cells. CONCLUSIONS These results demonstrate that activation of CaM kinase II is required for VSMC migration, that its activation in response to PDGF is suppressed in growth-arrested VSMCs, and that this suppression of CaM kinase II activation is responsible, in large part, for the failure of growth-arrested VSMCs to migrate toward PDGF.

Age-Associated Vascular Oxidative Stress, Nrf2 Dysfunction, and NF-κB Activation in the Nonhuman Primate Macaca mulatta

Aging promotes oxidative stress in vascular endothelial and smooth muscle cells, which contribute to the development of cardiovascular diseases. NF-E2-related factor 2 (Nrf2) is a transcription factor, which is activated by reactive oxygen species in the vasculature of young animals, leading to adaptive upregulation of numerous reactive oxygen species detoxifying and antioxidant genes. The present study was designed to elucidate age-associated changes in the homeostatic role of Nrf2-driven free radical detoxification mechanisms in the vasculature of nonhuman primates. We found that carotid arteries of aged rhesus macaques (Macaca mulatta, age: ≥20 years) exhibit significant oxidative stress (as indicated by the increased 8-iso-PGF2α and 4-HNE content and decreased glutathione and ascorbate levels) as compared with vessels of young macaques (age:~10 years) that is associated with activation of the redox-sensitive proinflammatory transcription factor, nuclear factor-kappaB. However, age-related oxidative stress does not activate Nrf2 and does not induce Nrf2 target genes (NQO1, GCLC, and HMOX1). In cultured vascular smooth muscle cells (VSMCs) derived from young M mulatta, treatment with H(2)O(2) and high glucose significantly increases transcriptional activity of Nrf2 and upregulates the expression of Nrf2 target genes. In contrast, in cultured vascular smooth muscle cells cells derived from aged macaques, H(2)O(2)- and high glucose-induced Nrf2 activity and Nrf2-driven gene expression are blunted. High glucose-induced H(2)O(2) production was significantly increased in aged vascular smooth muscle cells compared with that in vascular smooth muscle cells from young M mulatta. Taken together, aging is associated with Nrf2 dysfunction in M mulatta arteries, which likely exacerbates age-related cellular oxidative stress, promoting nuclear factor-kappaB activation and vascular inflammation in aging.

The inhibition of vascular smooth muscle cell migration by peptide and antibody antagonists of the alphavbeta3 integrin complex is reversed by activated calcium/calmodulin- dependent protein kinase II.

The migration of vascular smooth muscle cells (VSMCs) is thought to play a key role in the pathogenesis of many vascular diseases and is regulated by soluble growth factors/ chemoattractants as well as interactions with the extracellular matrix. We have studied the effects of antibodies to rat beta3 and human alphavbeta3 integrins on the migration of VSMCs. Both integrin antibodies as well as cyclic RGD peptides that bind to the vitronectin receptors alphavbeta3 and alphavbeta5 significantly inhibited PDGF-directed migration. This resulted in a reduction in the accumulation of inositol (1,4,5) trisphosphate and the activation of calcium/calmodulin-dependent protein kinase II (CamKII), an important regulatory event in VSMC migration identified previously. PDGF-directed VSMC migration in the presence of the anti-integrin antibodies and cyclic RGD peptides was restored when intracellular CamKII activity was elevated by either raising intracellular calcium levels with the ionophore, ionomycin, or infecting with a replication-defective recombinant adenovirus expressing a constitutively activated CamKII cDNA (AdCMV.CKIID3). Rescue of rat VSMCs was also observed in stably transfected cell lines expressing constitutively activated but not wild-type CamKII. These observations identify a key intermediate in the regulation of VSMC migration by outside-in signaling from the integrin alphavbeta3.