Caroline O'Neil

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.

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.

Lipid Incorporation Inhibits Src-Dependent Assembly of Fibronectin and Type I Collagen by Vascular Smooth Muscle Cells

A vital role of vascular smooth muscle cells (SMCs) is to stabilize the artery wall by elaborating fibrils of type I collagen. This is especially important in atherosclerotic lesions. However, SMCs in these lesions can be laden with lipids and the impact of this modification on collagen fibril formation is unknown. To address this, we converted human vascular SMCs to a foam cell state by incubating them with either LDL or VLDL. Biochemical markers of a SMC phenotype were preserved. However, microscopic tracking revealed a profound perturbation in the ability of the cells to assemble collagen fibrils, reducing assembly by up to 79%. This dysfunction was mirrored by an inability of smooth muscle foam cells to assemble fibronectin. Lipid-loaded SMCs did not display a generalized defect in the actin cytoskeleton and the formation of vinculin-containing focal adhesion complexes was preserved. However, lipid-loaded SMCs were unable to assemble fibrillar adhesion complexes and clustering of tensin and α5β1 integrin was disordered. Moreover, phosphorylation of tensin, required for fibrillar adhesion complex formation, was suppressed by up to 57%, with a concomitant decrease in activation of Src and FAK and restriction of activated Src to the cell edges. Forced activation of Src-FAK signaling in lipid-engorged SMCs rescued both fibrillar adhesion formation and fibrillogenesis. We conclude that lipid accumulation by SMCs disables the machinery for collagen and fibronectin assembly. This previously unknown relationship between atherogenic lipids and integrin-based signaling could underlie plaque vulnerability.

Collagenase-resistant collagen promotes mouse aging and vascular cell senescence

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.

The Oxysterol 24(S),25-Epoxycholesterol Attenuates Human Smooth Muscle–Derived Foam Cell Formation Via Reduced Low-Density Lipoprotein Uptake and Enhanced Cholesterol Efflux

Background Foam cell formation by intimal smooth muscle cells (SMCs) inhibits the elaboration of extracellular matrix, which is detrimental to plaque stabilization. In the present study, we examined the lipoproteins and receptors involved in human SMC foam cell formation and investigated the ability of 24(S),25-epoxycholesterol [24(S),25-EC], an oxysterol agonist of the liver X receptor, to attenuate SMC foam cell formation. Methods and Results Incubation of human internal thoracic SMCs with atherogenic lipoproteins demonstrated that low-density lipoprotein (LDL), but not oxidized or acetylated LDL, was the primary lipoprotein taken up, resulting in marked cholesteryl ester deposition (6-fold vs 1.8-fold; P<0.05; n=4). Exposure of SMCs to exogenous or endogenously synthesized 24(S),25-EC attenuated LDL uptake (−90% and −47% respectively; P<0.05; n=3) through decreased sterol regulatory element–binding protein-2 expression (−30% and −17%, respectively; P<0.001; n=3), decreased LDL receptor expression (−75% and −40%, respectively; P<0.05; n=3) and increased liver X receptor–mediated myosin regulatory light chain interacting protein expression (7- and 3-fold, respectively; P<0.05; n=4). Furthermore, exogenous 24(S),25-EC increased adenosine triphosphate–binding cassettes A1– and G1–mediated cholesterol efflux to apolipoprotein AI (1.9-fold; P<0.001; n=5) and high-density lipoprotein3 (1.3-fold; P<0.05; n=5). 24(S),25-EC, unlike a nonsteroidal liver X receptor agonist, T0901317, did not stimulate sterol regulatory element–binding protein-1c–mediated fatty acid synthesis or triglyceride accumulation. 24(S),25-EC preserved the assembly of fibronectin and type I collagen by SMCs. Conclusions The oxysterol 24(S),25-EC prevented foam cell formation in human SMCs by attenuation of LDL receptor–mediated LDL uptake and stimulation of cholesterol efflux, restoring the elaboration of extracellular matrix. In contrast to T0901317, 24(S),25-EC prevented the development of a triglyceride-rich foam cell phenotype. (J Am Heart Assoc. 2012;1:e000810 doi: 10.1161/JAHA.112.000810.)

Intrinsic directionality of migrating vascular smooth muscle cells is regulated by NAD+ biosynthesis

Summary Cell migration is central to tissue repair and regeneration but must proceed with precise directionality to be productive. Directional migration requires external cues but also depends on the extent to which cells can inherently maintain their direction of crawling. We report that the NAD+ biosynthetic enzyme, nicotinamide phosphoribosyltransferase (Nampt/PBEF/visfatin), mediates directionally persistent migration of vascular smooth muscle cells (SMCs). Time-lapse microscopy of human SMCs subjected to Nampt inhibition revealed chaotic motility whereas SMCs transduced with the Nampt gene displayed highly linear migration paths. Ordered motility conferred by Nampt was associated with downsizing of the lamellipodium, reduced lamellipodium wandering around the cell perimeter, and increased lamellipodial protrusion rates. These protrusive and polarity-stabilizing effects also enabled spreading SMCs to undergo bipolar elongation to an extent not typically observed in vitro. Nampt was found to localize to lamellipodia and fluorescence recovery of Nampt–eGFP after photobleaching revealed microtubule-dependent transport of Nampt to the leading edge. In addition, Nampt was found to associate with, and activate, Cdc42, and Nampt-driven directional persistence and lamellipodium anchoring required Cdc42. We conclude that high-fidelity SMC motility is coordinated by a Nampt–Cdc42 axis that yields protrusive but small and anchored lamellipodia. This novel, NAD+-synthesis-dependent control over motility may be crucial for efficient repair and regeneration of the vasculature, and possibly other tissues.

Type I Collagen Cleavage Is Essential for Effective Fibrotic Repair after Myocardial Infarction

Efficient deposition of type I collagen is fundamental to healing after myocardial infarction. Whether there is also a role for cleavage of type I collagen in infarct healing is unknown. To test this, we undertook coronary artery occlusion in mice with a targeted mutation (Col1a1(r/r)) that yields collagenase-resistant type I collagen. Eleven days after infarction, Col1a1(r/r) mice had a lower mean arterial pressure and peak left ventricular systolic pressure, reduced ventricular systolic function, and worse diastolic function, compared with wild-type littermates. Infarcted Col1a1(r/r) mice also had greater 30-day mortality, larger left ventricular lumens, and thinner infarct walls. Interestingly, the collagen fibril content within infarcts of mutant mice was not increased. However, circular polarization microscopy revealed impaired collagen fibril organization and mechanical testing indicated a predisposition to scar microdisruption. Three-dimensional lattices of collagenase-resistant fibrils underwent cell-mediated contraction, but the fibrils did not organize into birefringent collagen bundles. In addition, time-lapse microscopy revealed that, although cells migrated smoothly on wild-type collagen fibrils, crawling and repositioning on collagenase-resistant collagen was impaired. We conclude that type I collagen cleavage is required for efficient healing of myocardial infarcts and is critical for both dynamic positioning of collagen-producing cells and hierarchical assembly of collagen fibrils. This seemingly paradoxical requirement for collagen cleavage in fibrotic repair should be considered when designing potential strategies to inhibit matrix degradation in cardiac disease.

Fibroblast Growth Factor 9 Imparts Hierarchy and Vasoreactivity to the Microcirculation of Renal Tumors and Suppresses Metastases

Background: Tumor microvessels are non-hierarchical and regulate blood flow poorly. Results: Delivery of fibroblast growth factor 9 (FGF9) reconfigured the chaotic renal tumor vasculature into a fortified, hierarchical, and vasoreactive network by activating PDGFRβ+-stromal cells. Tumor hypoxia and metastases declined. Conclusion: FGF9 can generate a vasoreactive tumor microcirculation. Significance: This advanced state of microvascular differentiation could favorably impact tumor behavior. Tumor vessel normalization has been proposed as a therapeutic paradigm. However, normal microvessels are hierarchical and vasoreactive with single file transit of red blood cells through capillaries. Such a network has not been identified in malignant tumors. We tested whether the chaotic tumor microcirculation could be reconfigured by the mesenchyme-selective growth factor, FGF9. Delivery of FGF9 to renal tumors in mice yielded microvessels that were covered by pericytes, smooth muscle cells, and a collagen-fortified basement membrane. This was associated with reduced pulmonary metastases. Intravital microvascular imaging revealed a haphazard web of channels in control tumors but a network of arterioles, bona fide capillaries, and venules in FGF9-expressing tumors. Moreover, whereas vasoreactivity was absent in control tumors, arterioles in FGF9-expressing tumors could constrict and dilate in response to adrenergic and nitric oxide releasing agents, respectively. These changes were accompanied by reduced hypoxia in the tumor core and reduced expression of the angiogenic factor VEGF-A. FGF9 was found to selectively amplify a population of PDGFRβ-positive stromal cells in the tumor and blocking PDGFRβ prevented microvascular differentiation by FGF9 and also worsened metastases. We conclude that harnessing local mesenchymal stromal cells with FGF9 can differentiate the tumor microvasculature to an extent not observed previously.