Collagen plays a major role in the structural organization of the heart and therefore direct visualization of collagen fibers is a crucial component of cardiac analysis. Although linearly polarized light has proven an effective tool for the examination of myocardial collagen in histologic sections, the use of circularly polarized light may offer advantages and additional possibilities. We examined the potential enhancement of collagen analysis using circularly polarized light in two ways. We first measured the brightness, and hence indirectly assessed the birefringence, of collagen fibers in scars examined at different times after myocardial infarction. Secondly, we measured collagen content in myocardial tissue and compared results obtained from brightfield analysis of trichrome stained sections with those obtained from circularly polarized light analysis of picrosirius red stained sections. We observed a progressive increase in the maximum brightness of collagen fibers in the scar with time, and a time-dependent shift in the relative distribution of collagen fiber brightness from lower to higher levels. We found consistently lower values of collagen content in trichrome stained versus picrosirius red stained tissue, and concluded that trichrome staining underestimated collagen content. The information provided by these studies could not be obtained by brightfield analysis and could be only partially obtained from linearly polarized light analysis. Thus, analysis using circularly polarized light has the ability to enhance histologic assessment of tissue and can provide additional insights into the composition and structure of myocardial collagen.

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...

Human atheromata obtained in vivo were used to test the hypothesis that transforming growth factor-(31 plays a role in the development of vascular restenosis.We analyzed 28 specimens from patients with primary atherosclerotic or restenotic lesions; 26 of these were obtained by directional atherectomy and 2 at the time of coronary bypass surgery. Seven control tissues included operatively excised segments of human internal mammary artery, myocardium, and unused portions of vein graft obtained intraoperatively. From these 35 specimens, 210 sections were examined using in situ hybridization. Measurement of silver grains/ nucleus disclosed that expression of transforming growth factor-ftl mRNA was highest in restenotic tissues (P < 0.001 vs. primary atherosclerotic tissues) and lowest in nonatherosclerotic (control) tissues. In cultures of human vascular smooth muscle cells grown from explants of internal mammary artery, expression of mRNA for transforming growth factor-ftl was significantly greater in subconfluent than in confluent smooth muscle cells (P = 0.05). Transforming growth factor type-ft III receptor was expressed in cell cultures and undetectable in the tissue specimens. Sections taken adjacent to those studied by in situ hybridization were examined by immunohistochemistry using antibodies against transforming growth factor-,6l and a-actin (as a marker for smooth muscle cells) and disclosed transforming growth factor-,Bl in smooth muscle cells present in these sections.These findings are consistent with the concept that transforming growth factor-,8l plays an important role in modulating repair of vascular injury, including restenosis, after balloon angioplasty. (J. Clin. Invest. 1992. 90:1582-1592 Key words:

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 ...