J. Geoffrey Pickering

Extension of Human Cell Lifespan by Nicotinamide Phosphoribosyltransferase

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 β-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.

Pre–B-Cell Colony–Enhancing Factor Regulates NAD+-Dependent Protein Deacetylase Activity and Promotes Vascular Smooth Muscle Cell Maturation

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+) content was increased in PBEF-overexpressing SMCs and decreased in PBEF-knockdown SMCs. Furthermore, NAD+-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+-dependent reactions in SMCs, reactions that promote, among other potential processes, the acquisition of a mature SMC phenotype.

Evidence from a novel human cell clone that adult vascular smooth muscle cells can convert reversibly between noncontractile and contractile phenotypes.

Smooth muscle cells (SMCs) perform diverse functions that can be categorized as contractile and synthetic. A traditional model holds that these distinct functions are performed by the same cell, by virtue of its capacity for bidirectional modulation of phenotype. However, this model has been challenged, in part because there is no physiological evidence that an adult synthetic SMC can acquire the ability to contract. We sought evidence for this by cloning adult SMCs from human internal thoracic artery. One clone, HITB5, expressed smooth muscle alpha-actin, smooth myosin heavy chains, heavy caldesmon, and calponin and showed robust calcium transients in response to histamine and angiotensin II, which confirmed intact transmembrane signaling cascades. On serum withdrawal, these cells adopted an elongated and spindle-shaped morphology, random migration slowed, extracellular matrix protein production fell, and cell proliferation and [(3)H]thymidine incorporation fell to near 0. Cell viability was not compromised, however; in fact, apoptosis rate fell significantly. In this state, agonist-induced elevation of cytoplasmic calcium was even more pronounced and was accompanied by SMC contraction. Readdition of 10% serum completely returned HITB5 cells to a noncontractile, proliferative phenotype. Contractile protein expression increased after serum withdrawal, although modestly, which suggested that the switch to contractile function involved reorganization or sensitization of existing contractile structures. To our knowledge, the physiological properties of HITB5 SMCs provide the first direct demonstration that cultured human adult SMCs can convert between a synthetic, noncontracting state and a contracting state. HITB5 cells should be valuable for characterizing the basis of this critical transition.

Vascular Smooth Muscle Cells Orchestrate the Assembly of Type I Collagen via α2β1 Integrin, RhoA, and Fibronectin Polymerization

Assembly of collagen into fibrils is widely studied as a spontaneous and entropy-driven process. To determine whether vascular smooth muscle cells (SMCs) impact the formation of collagen fibrils, we microscopically tracked the conversion of soluble to insoluble collagen in human SMC cultures, using fluorescent type I collagen at concentrations less than that which supported self-assembly. Collagen microaggregates were found to form on the cell surface, initially as punctate collections and then as an increasingly intricate network of fibrils. These fibrils displayed 67-nm periodicity and were found in membrane-delimited cellular invaginations. Fibril assembly was inhibited by an anti-α2β1 integrin antibody and accelerated by an α2β1 integrin antibody that stimulates a high-affinity binding state. Newly assembled collagen fibrils were also found to co-localize with newly assembled fibronectin fibrils. Moreover, inhibition of fibronectin assembly with an anti-α5β1 integrin antibody completely inhibited collagen assembly. Collagen fibril formation was also linked to the cytoskeleton. Fibrils formed on the stretched tails of SMCs, ran parallel to actin microfilament bundles, and formed poorly on SMCs transduced with retrovirus containing cDNA for dominant-negative RhoA and robustly on SMCs expressing constitutively active RhoA. Lysophosphatidic acid, which activates RhoA and stimulates fibronectin assembly, stimulated collagen fibril formation, establishing for the first time that collagen polymerization can be regulated by soluble agonists of cell function. Thus, collagen fibril formation is under close cellular control and is dynamically integrated with fibronectin assembly, opening new possibilities for modifying collagen deposition.

Vascular Smooth Muscle Cells of Recipient Origin Mediate Intimal Expansion after Aortic Allotransplantation in Mice

Intimal expansion by vascular smooth muscle cells (SMCs) is a characteristic feature of graft vascular disease. Whether graft intimal SMCs arise from donor or recipient tissue is not well established but has important pathogenetic implications. We examined for the presence of male cells in the expanded intima of sex-mismatched mouse aortic allografts (C57BL/6-to-BALB/c) at 30 or 60 days after transplant by in situ hybridization using a Y-chromosome probe. Study groups included male-to-female allografts, female-to-male allografts, and female-to-female allografts in recipients previously engrafted with male bone marrow. Although intimal expansion developed in all allografts, male-to-female allografts lacked Y-chromosome-positive intimal cells. In contrast, such cells were abundant in female-to-male allografts and most of these cells co-labeled for smooth muscle alpha-actin by immunostain. Female-to-female allografts in recipients with male bone marrow showed a limited number of intimal Y-chromosome-positive cells. However, none of these clearly co-labeled for smooth muscle alpha-actin and their numbers declined throughout time, consistent with graft-infiltrating inflammatory cells. We conclude that intimal expansion of mouse aortic allografts is mediated by SMCs that originated from the recipient. There was little evidence of their derivation from the bone marrow, suggesting instead the adjacent host aorta as the primary source of intimal SMCs.

Coordinated Effects of Fibroblast Growth Factor-2 on Expression of Fibrillar Collagens, Matrix Metalloproteinases, and Tissue Inhibitors of Matrix Metalloproteinases by Human Vascular Smooth Muscle Cells Evidence for Repressed Collagen Production and Activated Degradative Capacity

Fibroblast growth factor-2 (FGF-2) is an established mediator of smooth muscle cell (SMC) proliferation after vascular injury. However, the influence of FGF-2 on collagen fiber remodeling, which may be a prerequisite for vascular SMC accumulation, is not well understood. We determined that FGF-2 almost completely abrogated the formation of immunodetectable type I collagen fibers in the extracellular matrix of cultured human vascular SMCs. This was associated with reduced expression of pro alpha-chains for types I and III collagen, as assessed by Western blot analysis, and a corresponding reduction in collagen synthesis. Densitometry of Northern blots indicated a potent reduction of mRNA encoding pro alpha-chains for types I and III collagen and a minor reduction in mRNA for pro alpha-chains for type V collagen. Interstitial collagenase (MMP-1), which is required for degradation of collagen types I and III, was not expressed by SMCs under basal culture conditions, but expression was induced by FGF-2, with a potent, dose-dependent increase in MMP-1 protein in conditioned medium. Metalloproteinase inhibitors TIMP-1, TIMP-2, and TIMP-3 were expressed by unstimulated SMCs and were differentially regulated by FGF-2. TIMP-1 expression increased modestly, TIMP-2 expression was repressed, and TIMP-3 was relatively unaffected. The net effect on substrate degradation, as assessed by zymography of conditioned media, was induction of MMP-1 lytic activity by FGF-2, with no effect on the activity of MMP-2, MMP-3, or MMP-9. These data indicate that stimulation of human SMCs with FGF-2 establishes a phenotype in which collagen fiber production is repressed and the capacity for fiber degradation activated. This coordinated response may be critical for SMC accumulation during vascular remodeling as well as atherosclerotic plaque destabilization.

Nicotinamide phosphoribosyltransferase imparts human endothelial cells with extended replicative lifespan and enhanced angiogenic capacity in a high glucose environment

Endothelial dysfunction is a characteristic of aging‐related vascular disease and is worsened during diabetes. High glucose can impair endothelial cell (EC) function through cellular accumulation of reactive oxygen species, an insult that can also limit replicative lifespan. Nicotinamide phosphoribosyltransferase (Nampt), also known as PBEF and visfatin, is rate‐limiting for NAD+ salvage from nicotinamide and confers resistance to oxidative stress via SIRT1. We therefore sought to determine if Nampt expression could resist the detrimental effects of high glucose and confer a survival advantage to human vascular EC in this pathologic environment. Human aortic EC were infected with retrovirus encoding eGFP or eGFP‐Nampt, and FACS‐selected to yield populations with similar, modest transgene expression. Using a chronic glucose exposure model we tracked EC populations to senescence, assessed cellular metabolism, and determined in vitro angiogenic function. Overexpression of Nampt increased proliferation and extended replicative lifespan, and did so preferentially during glucose overload. Nampt expression delayed markers of senescence and limited reactive oxygen species accumulation in high glucose through a modest increase in aerobic glycolysis. Furthermore, tube networks formed by Nampt‐overexpressing EC were more extensive and glucose‐resistant, in accordance with SIRT1‐mediated repression of the anti‐angiogenic transcription factor, FoxO1. We conclude that Nampt enables proliferating human EC to resist the oxidative stress of aging and of high glucose, and to productively use excess glucose to support replicative longevity and angiogenic activity. Enhancing endothelial Nampt activity may thus be beneficial in scenarios requiring EC‐based vascular repair and regeneration during aging and hyperglycemia, such as atherosclerosis and diabetes‐related vascular disease.

α5β1 Integrin Expression and Luminal Edge Fibronectin Matrix Assembly by Smooth Muscle Cells after Arterial Injury

Fibronectin is secreted from the cell as a soluble protein that must then polymerize to regulate cell function. To elucidate the process of fibronectin matrix assembly in vascular disease, we immunostained sections of balloon-injured rat carotid artery for the fibronectin-binding α5β1 integrin. Whereas α5β1 integrin was not evident in the normal carotid artery, its expression was induced after a vascular injury. By 14 days, the α5β1 integrin was localized exclusively to the less differentiated smooth muscle cells (SMCs) at the luminal surface of the neointima. Platelet-derived growth factor-BB, dominant in neointimal formation, selectively increased the expression of the α5β1 integrin by human SMCs in culture. To track the assembly of fibronectin fibers, fluorescence-labeled soluble fibronectin protomers were added to cultured SMCs and to fresh segments of normal and balloon-injured rat carotid arteries. Fibronectin fiber formation in cultured SMCs could be detected within 10 minutes, and was blocked by an RGD peptide, an anti-β1 integrin antibody, and an anti-α5β1 integrin antibody, but not by an anti-β3 integrin antibody. En face confocal microscopy of arterial segments revealed that soluble fibronectin had polymerized on the α5β1 integrin-expressing SMCs of the luminal surface of the injured arterial neointima, but not on the α5β1 integrin-negative neointimal SMCs below this or on the endothelial cells of uninjured arteries. Furthermore, in situ fibronectin assembly by the neointimal SMCs was inhibited by an RGD peptide and by an anti-β1 integrin antibody. These studies indicate that a subpopulation of SMCs in the repairing artery wall orchestrates integrin-mediated fibronectin assembly.

Fibroblast Growth Factor-2 Potentiates Vascular Smooth Muscle Cell Migration to Platelet-Derived Growth Factor

Fibroblast growth factor-2 (FGF-2) has been implicated in vascular smooth muscle cell (SMC) migration, a key process in vascular disease. We demonstrate here that FGF-2 promotes SMC motility by altering beta1 integrin-mediated interactions with the extracellular matrix (ECM). FGF-2 significantly increased surface expression of alpha2beta1, alpha3beta1, and alpha5beta1 integrins on human SMCs, as assessed by flow cytometry. The greatest increase was for the collagen-binding alpha2beta1 integrin. Despite this, FGF-2 did not increase SMC adhesion to type I collagen but instead promoted SMC elongation and SMC motility. The latter was evaluated by using a microchemotaxis chamber and by digital time-lapse video microscopy. Although FGF-2 was not chemotactic for human SMCs, cells preincubated with FGF-2 displayed a 3.1-fold increase in migration to the undersurface of porous type I collagen-coated membranes and a 2.1-fold increase in migration speed on collagen. Furthermore, chemotaxis to platelet-derived growth factor-BB on collagen was significantly greater in SMCs exposed to FGF-2. FGF-2-induced elongation and migration on collagen were inhibited by a blocking anti-alpha2beta1 antibody; however, SMC adhesion to collagen was unaffected. SMC migration on fibronectin was also enhanced by FGF-2, although less prominently: migration through porous membranes increased 1.8-fold, and migration speed increased 1.3-fold. Also, FGF-2 completely disassembled the smooth muscle alpha-actin-containing stress fiber network contemporaneously with the change in integrin expression and cell shape. We conclude that (1) exogenous FGF-2 promotes SMC migration and potentiates chemotaxis to PDGF-BB; (2) the promigratory effect of FGF-2 is especially prominent on type I collagen and is mediated by upregulation of alpha2beta1 integrin; and (3) FGF-2 disassembles actin stress fibers, which may promote differential utilization of alpha2beta1 integrin for motility but not adhesion. This dynamic SMC-ECM interplay may be an important mechanism by which FGF-2 facilitates SMC motility in vivo.

Fibroblast growth factor 9 delivery during angiogenesis produces durable, vasoresponsive microvessels wrapped by smooth muscle cells

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.