Extending the productive lifespan of human cells could have major implications for diseases of aging, such as atherosclerosis. We identified a relationship between aging of human vascular smooth muscle cells (SMCs) and nicotinamide phosphoribosyltransferase (Nampt/PBEF/Visfatin), the rate-limiting enzyme for NAD ؉ salvage from nicotinamide. Replicative senescence of SMCs was preceded by a marked decline in the expression and activity of Nampt. Furthermore, reducing Nampt activity with the antagonist FK866 induced premature senescence in SMCs, assessed by serial quantification of the proportion of cells with senescence-associated ␤-galactosidase activity. In contrast, introducing the Nampt gene into aging human SMCs delayed senescence and substantially lengthened cell lifespan, together with enhanced resistance to oxidative stress. Nampt-mediated SMC lifespan extension was associated with increased activity of the NAD ؉ -dependent longevity enzyme SIRT1 and was abrogated in Nampt-overexpressing cells transduced with a dominant-negative form of SIRT1 (H363Y). Nampt overexpression also reduced the fraction of p53 that was acetylated on lysine 382, a target of SIRT1, suppressed an age-related increase in p53 expression, and increased the rate of p53 degradation. Moreover, add-back of p53 with recombinant adenovirus blocked the anti-aging effects of Nampt. These data indicate that Nampt is a longevity protein that can add stress-resistant life to human SMCs by optimizing SIRT1-mediated p53 degradation.Age is the greatest risk factor for myocardial infarctions and strokes (1). This risk is partly attributable to an age-related decline in the ability of vascular cells to resist stress and effectively remodel the arterial wall. Vascular smooth muscle cells (SMCs) 3 are especially important in this regard; the efficiency with which SMCs stabilize a developing atherosclerotic lesion determines whether the lesion will rupture, a potentially fatal event. Strategies to prevent the premature senescence of SMCs could be a promising approach for reducing vascular disease if molecular targets can be identified.Nicotinamide phosphoribosyltransferase (Nampt, also known as Pre-B-cell colony-enhancing factor and Visfatin (2)) is the rate-limiting enzyme for NAD ϩ biosynthesis from nicotinamide. The intracellular levels of NAD ϩ and nicotinamide have recently been identified as important for certain cell survival reactions, including those linked to the sirtuin family of protein deacetylases (3, 4). Sirtuins, such as Sir2 and its mammalian homolog SIRT1, consume NAD ϩ and generate nicotinamide as they hydrolytically remove a targeted acetyl group (3). Nicotinamide is a known inhibitor of NAD ϩ -dependent deacetylation reactions. Therefore, pathways that both replenish NAD ϩ and clear nicotinamide could be vital to SIRT1 activity.Recently, we discovered that Nampt was substantially upregulated when a uniquely long-lived human vascular SMC line was subjected to the stress of complete serum withdrawal (5). Here, we report that Nampt i...

Abstract-Conversion of vascular smooth muscle cells (SMCs) from a proliferative state to a nonproliferative, contractile state confers vasomotor function to developing and remodeling blood vessels. Using a maturation-competent human SMC line, we determined that this shift in phenotype was accompanied by upregulation of pre-B-cell colony-enhancing factor (PBEF), a protein proposed to be a cytokine. Knockdown of endogenous PBEF increased SMC apoptosis and reduced the capacity of synthetic SMCs to mature to a contractile state. In keeping with these findings, human SMCs transduced with the PBEF gene had enhanced survival, an elongated bipolar morphology, and increased levels of h-caldesmon, smoothelin-A, smoothelin-B, and metavinculin. Notwithstanding some prior reports, PBEF did not have attributes of a cytokine but instead imparted the cell with increased nicotinamide phosphoribosyltransferase activity. Intracellular nicotinamide adenine dinucleotide (NAD ϩ ) content was increased in PBEF-overexpressing SMCs and decreased in PBEF-knockdown SMCs. Furthermore, NAD ϩ -dependent protein deacetylase activity was found to be essential for SMC maturation and was increased by PBEF. Xenotransplantation of human SMCs into immunodeficient mice revealed an increased capacity for PBEF-overexpressing SMCs to mature and intimately invest nascent endothelial channels. This microvessel chimerism and maturation process was perturbed when SMC PBEF expression was lowered. These findings identify PBEF as a regulator of NAD ϩ -dependent reactions in SMCs, reactions that promote, among other potential processes, the acquisition of a mature SMC phenotype. Key Words: vascular smooth muscle Ⅲ pre-B-cell colony-enhancing factor Ⅲ maturation Ⅲ nicotinamide phosphoribosyltransferase Ⅲ deacetylation C onversion of smooth muscle cells (SMCs) from a proliferative, noncontractile state to a nonproliferative, contractile state is essential for conferring vasomotor function to developing arteries. 1,2 This shift toward a more mature SMC phenotype is also important for terminating SMC-mediated remodeling of diseased arteries. 2 Recently, we cloned adult vascular SMC lines that, in contrast to other human SMC preparations, could reversibly convert between a spread, proliferative, and synthetic state when cultured in the presence of serum to a highly elongated, nonproliferative state when serum was withdrawn. 3,4 In the latter state, the cells displayed decreased apoptosis, increased contractile protein expression, and the ability to contract in response to vasoactive agonists. This system, therefore, provided us with an opportunity to seek out factors that enabled a proliferative adult SMC to efficiently shift to a more quiescent state specialized to contract. Accordingly, we undertook differential display polymerase chain reaction (PCR) and high-density microarray analyses to identify genes that were differentially expressed as these human SMCs executed this key shift in phenotype.These surveys consistently identified pre-B cell colonyenhancing ...

The therapeutic potential of angiogenic growth factors has not been realized. This may be because formation of endothelial sprouts is not followed by their muscularization into vasoreactive arteries. Using microarray expression analysis, we discovered that fibroblast growth factor 9 (FGF9) was highly upregulated as human vascular smooth muscle cells (SMCs) assemble into layered cords. FGF9 was not angiogenic when mixed with tissue implants or delivered to the ischemic mouse hind limb, but instead orchestrated wrapping of SMCs around neovessels. SMC wrapping in implants was driven by sonic hedgehog-mediated upregulation of PDGFRβ. Computed tomography microangiography and intravital microscopy revealed that microvessels formed in the presence of FGF9 had enhanced capacity to receive flow and were vasoreactive. Moreover, the vessels persisted beyond 1 year, remodeling into multilayered arteries paired with peripheral nerves. This mature physiological competency was attained by targeting mesenchymal cells rather than endothelial cells, a finding that could inform strategies for therapeutic angiogenesis and tissue engineering.

The cholesterol biosynthetic pathway produces numerous signaling molecules. Oxysterols through liver X receptor (LXR) activation regulate cholesterol efflux, whereas the non-sterol mevalonate metabolite, geranylgeranyl pyrophosphate (GGPP), was recently demonstrated to inhibit ABCA1 expression directly, through antagonism of LXR and indirectly through enhanced RhoA geranylgeranylation. We used HMG-CoA reductase inhibitors (statins) to test the hypothesis that reduced synthesis of mevalonate metabolites would enhance cholesterol efflux and attenuate foam cell formation. Preincubation of THP-1 macrophages with atorvastatin, dose dependently (1-10 M) stimulated cholesterol efflux to apolipoprotein AI (apoAI, 10 -60%, p < 0.05) and high density lipoprotein (HDL 3 ) (2-50%, p < 0.05), despite a significant decrease in cholesterol synthesis (2-90%). Atorvastatin also increased ABCA1 and ABCG1 mRNA abundance (30 and 35%, p < 0.05). Addition of mevalonate, GGPP or farnesyl pyrophosphate completely blocked the statin-induced increase in ABCA1 expression and apoAI-mediated cholesterol efflux. A role for RhoA was established, because two inhibitors of Rho protein activity, a geranylgeranyl transferase inhibitor and C3 exoenzyme, increased cholesterol efflux to apoAI (20 -35%, p < 0.05), and macrophage expression of dominant-negative RhoA enhanced cholesterol efflux to apoAI (20%, p < 0.05). In addition, atorvastatin increased the RhoA levels in the cytosol fraction and decreased the membrane localization of RhoA. Atorvastatin treatment activated peroxisome proliferator activated receptor ␥ and increased LXR-mediated gene expression suggesting that atorvastatin induces cholesterol efflux through a molecular cascade involving inhibition of RhoA signaling, leading to increased peroxisome proliferator activated receptor ␥ activity, enhanced LXR activation, increased ABCA1 expression, and cholesterol efflux. Finally, statin treatment inhibited cholesteryl ester accumulation in macrophages challenged with atherogenic hypertriglyceridemic very low density lipoproteins indicating that statins can regulate foam cell formation.

Despite robust neovascularization, the microcirculation formed by regenerative angiogenesis in skeletal muscle is profoundly flawed in both structure and function, with no evidence for normalizing over time. This network-level dysfunction must be recognized and overcome to advance regenerative approaches for ischemic disease.

Abstract-Smooth muscle cells (SMCs) are called on to proliferate during vascular restructuring but must return to a nonproliferative state if remodeling is to appropriately terminate. To identify mediators of the reacquisition of replicative quiescence, we undertook gene expression screening in a uniquely plastic human SMC line. As proliferating SMCs shifted to a contractile and nonproliferative state, expression of TIMP-3, Axl, and KIAA0098 decreased whereas expression of complement C1s, cathepsin B, cellular repressor of E1A-activated genes increased. Wilms' tumor 1-associating protein (WTAP), a nuclear constituent of unknown function, was also upregulated as SMCs became nonproliferative. Furthermore, WTAP in the intima of injured arteries was substantially upregulated in the late stages of repair. Introduction of WTAP complementary DNA into human SMCs inhibited their proliferation, with a corresponding decrease in DNA synthesis and an increase in apoptosis. Knocking down endogenous WTAP increased SMC proliferation, because of increased DNA synthesis and G 1 /S phase transition, together with reduced apoptosis. WTAP was found to associate with the Wilms' tumor-1 protein in human SMCs and WTAP overexpression inhibited the binding of WT1 to an oligonucleotide containing a consensus WT1 binding site, whereas WTAP knockdown accentuated this interaction. Expression of the WT1 target genes, amphiregulin and Bcl-2, was suppressed in WTAP-overexpressing SMCs and increased in WTAP-deficient SMCs. Moreover, exogenous amphiregulin rescued the antiproliferative effect of WTAP. These findings identify WTAP as a novel regulator of the cell cycle and cell survival and implicate a WTAP-WT1 axis as a novel pathway for controlling vascular SMC phenotype. (Circ Res. 2006;99:1338-1346.)Key Words: amphiregulin Ⅲ smooth muscle cells Ⅲ Wilms' tumor 1-associating protein Ⅲ vascular smooth muscle cell proliferation P henotype plasticity is a feature of adult vascular smooth muscle cells (SMCs). A widely studied example of this is the dedifferentiation of mature, nonproliferative SMCs into proliferative SMCs, a process central to vascular remodeling. 1,2 Although less well studied, an equally important manifestation of SMC plasticity is the reverse shift, whereby proliferative adult SMCs convert back to a nonproliferative state. This particular phenotype switch is essential for limiting SMC accumulation and for terminating vascular remodeling. As such, the regulatory factors that drive proliferative SMCs into a nonproliferative state, and hold them in that state, are critical for effective vascular remodeling and for limiting vascular disease.We have generated unique lines of nonimmortalized human SMCs that are capable of converting between proliferative and nonproliferative states. 2,3 In the presence of serum, these SMCs proliferate, migrate, and elaborate extracellular matrix similar to primary SMCs. On withdrawal of serum however they undergo a reproducible program of cellular maturation whereby they exit the cell cycle, migrate...

The aortic media depends on an intrinsic NAD fueling system to protect against DNA damage and premature SMC senescence, with relevance to human thoracic aortopathy.

Collagen fibrils become resistant to cleavage over time. We hypothesized that resistance to type I collagen proteolysis not only marks biological aging but also drives it. To test this, we followed mice with a targeted mutation (Col1a1r/r) that yields collagenase-resistant type I collagen. Compared with wild-type littermates, Col1a1r/r mice had a shortened lifespan and developed features of premature aging including kyphosis, weight loss, decreased bone mineral density, and hypertension. We also found that vascular smooth muscle cells (SMCs) in the aortic wall of Col1a1r/r mice were susceptible to stress-induced senescence, displaying senescence-associated ß-galactosidase (SA-ßGal) activity and upregulated p16INK4A in response to angiotensin II infusion. To elucidate the basis of this pro-aging effect, vascular SMCs from twelve patients undergoing coronary artery bypass surgery were cultured on collagen derived from Col1a1r/r or wild-type mice. This revealed that mutant collagen directly reduced replicative lifespan and increased stress-induced SA-ßGal activity, p16INK4A expression, and p21CIP1 expression. The pro-senescence effect of mutant collagen was blocked by vitronectin, a ligand for αvß3 integrin that is presented by denatured but not native collagen. Moreover, inhibition of αvß3 with echistatin or with αvß3-blocking antibody increased senescence of SMCs on wild-type collagen. These findings reveal a novel aging cascade whereby resistance to collagen cleavage accelerates cellular aging. This interplay between extracellular and cellular compartments could hasten mammalian aging and the progression of aging-related diseases.