PreeCB-Cell ColonyeCEnhancing Factor Regulates NAD+-Dependent Protein Deacetylase Activity and Promotes Vascular Smooth Muscle Cell Maturati
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循环研究杂志 2005年第7期
the Robarts Research Institute (Vascular Biology Group) (E.v.d.V., Z.N., C.O., J.G.P.) and London Health Sciences Centre (B.U., D.F., J.G.P.), Departments of Medicine (Cardiology), Biochemistry, Medical Biophysics, Physiology
Pharmacology, University of Western Ontario, London, Canada.
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 preeCB-cell colonyeCenhancing 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.
Key Words: vascular smooth muscle;preeCB-cell colonyeCenhancing factor;maturation;nicotinamide phosphoribosyltransferase;deacetylation
Introduction
Conversion 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 preeCB cell colonyeCenhancing factor (PBEF) as being upregulated as SMCs shifted toward maturity. PBEF is a 52- to 55-kDa protein that has been proposed to be a cytokine.5 Reported actions in this regard include synergizing with other cytokines to stimulate the maturation of preeCB cells,5 stimulating the expression of inflammatory cytokines in epithelial cells,6 prolonging neutrophil survival,7 and acting as an adipokine that lowers plasma glucose levels.8 However, the contention that PBEF is a secreted cytokine is controversial. PBEF does not have a signal sequence for secretion, and the presence of PBEF in culture media has been suggested to be a consequence of activation-induced cell death, rather than secretion by either a classical or alternative pathway.9,10 Moreover, PBEF has sequence similarity with bacterial NadV, a protein that confers bacteria with the ability to grow in nicotinamide adenine dinucleotide (NAD+)-deficient conditions.11 In keeping with this, Rongvaux et al have reported that mouse PBEF functions within the cell as a nicotinamide phosphoribosyltransferase.9 This enzyme catalyzes the rate-limiting step in the salvage pathway for NAD+ biosynthesis, whereby nicotinamide that is generated during NAD+-consuming reactions is used to regenerate NAD+.12,13
We report here that human PBEF is not a cytokine but an intracellular regulator of NAD+-consuming reactions, including NAD+-dependent protein deacetylation. We further report that PBEF can promote the efficient acquisition of a stable and mature SMC phenotype and can increase the capacity of SMCs to invest developing blood vessels.
Materials and Methods
An expanded Materials and Methods can be found in the online data supplement available at http://circres.ahajournals.org
Cell Culture
Experiments were performed using the maturation-competent human vascular SMC lines HITB5 and HITC6 generated from the human internal thoracic artery, as described previously.3,4
Overexpression of PBEF in Human SMCs
A retroviral geneeCdelivery system was used to generate human SMCs stably overexpressing PBEF, using methods described previously.14 A PBEF-IRES-EGFP bicistronic fragment was cloned into the retroviral expression vector pLNCX2 (Clontech). A second retroviral expression construct was generated by inserting PBEF cDNA into pQCXIP-IRES-PURO (Clontech). Stable transductants were selected and overexpression of PBEF was confirmed before each experiment by Western blot analysis.
Western Blot Analysis
Expression of PBEF and SMC differentiation markers was assessed by Western blot analysis with chemiluminescence detection, as described.14
Apoptosis
SMC apoptosis was assessed by fluorescence in situ end-labeling of DNA fragments, as described.3
Knockdown of PBEF by RNA Interference
PBEF knockdown was accomplished by infecting human SMCs with retrovirus-containing sequences encoding hairpin small interfering (siRNA) fragments. Three different targeting sequences were used, each consisting of 19 nucleotides starting at nucleotides 147, 384, and 1278 of the PBEF coding sequence (siRNA147, 5'-GGAAGGTGAAATATGAGGA-3'; siRNA384, 5'-ATGTTCTCTTCACGGTGGA-3'; siRNA1278, 5'-AGGG CCGATTATCTTTACA-3'). Each sequence was separated by a 9-nucleotide noncomplementary spacer from the reverse complement of the same 19-nucleotide sequence. Blast search confirmed that only the PBEF gene was targeted. Control inserts contained the gene-specific 19-nucleotide sequence and hairpin loop sequence but not the antisense component.
Real-Time Reverse TranscriptioneCPCR
Probe (5'-CAGTTGCTGATCCCA- 3') and flanking primers (5'-primer, 5'-TGCAGCTATGTTGTAACCAATGG-3'; 3'-primer, 5'-ACAAAAGGTCGAAAAAGGGCC-3') were used for TaqMan real-time reverse transcription (RT)-PCR for PBEF. GAPDH transcript abundance was used as an endogenous RNA control to which PBEF transcript abundance was normalized.
Nicotinamide Phosphoribosyltransferase Activity
Cell lysates were reacted with 5 eol/L [carbonyl-14C]nicotinamide in the presence of 0.5 mmol/L phosphoribosylpyrophosphate and labeled, acetone-precipitable nicotinamide mononucleotide (NMN) quantified by scintillation counting.9
NAD+ Analysis by High-Performance Liquid Chromatography
Cellular nucleotides were extracted using perchloric acid, neutralized with KOH, and deproteinized cell lysate residues were analyzed by high-performance liquid chromatography (HPLC). NAD+ retention time, determined from a NAD+ standard, was 10 minutes.
Histone Deacetylase Assay
Histone H4 peptide (Upstate) was labeled with [3H]acetyl coenzyme A (ICN) using PCAF histone acetylase. Cell lysates were incubated with [3H]acetyl histone and released [3H]acetate assessed by scintillation counting (SCID, Charles River Laboratories, Wilmington, Mass).
Human-Mouse Chimeric Angiogenesis In Vivo
HITC6 SMCs in M199 with 10% FBS and fibroblast growth factor (FGF)-2 were mixed with an equal volume of growth factor-reduced matrigel (BD Discovery Labware) yielding final concentrations of 5x105 cells/mL and 250 ng/mL FGF-2. The cell-matrigel suspension (500 e蘈) was subcutaneously injected into the abdomen of mice with severe combined immunodeficiency syndrome (SCID). After 8 days, implants were harvested, fixed for 8 hour in Tris-buffered zinc,15 and paraffin-embedded tissues were sectioned at 5 e. Sections were double-immunostained for CD31 (BD Biosciences) and enhanced green fluorescent protein (EGFP) (BD Clontech) using diaminobenzidine (Vector Laboratories Ltd) and red alkaline phosphatase substrates, respectively, and counterstained with Harris hematoxylin. Apoptosis of human SMCs in matrigel was assessed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay, after immunostaining for EGFP and incubating sections with proteinase K (100 e/mL).
Algorithms for quantifying EGFP-positive human SMCs, TUNEL-positive human SMCs, microvessels, and SMC investment of microvessels are described in the online data supplement. Human SMCs were deemed to be investing a microvessel if they assumed contiguous and close apposition with an endothelial cell(s) and oriented their long axis along a contour of the vessel wall.
Statistics
Values are expressed as mean±SEM. Comparisons were made by t test or analysis of variance with Bonferroni post hoc test. Significance of fold-changes was assessed by the Wilcoxon signed-rank test. Statistical significance was set at P<0.05.
Results
PBEF Is Upregulated During SMC Maturation
The generation of clonal populations of SMCs from the human internal thoracic artery, designated HITB5 and HITC6, that can convert from a proliferative to a contractile SMC3,4 enabled us to screen for endogenous factors involved in this phenotype conversion. These screens suggested that PBEF was upregulated as SMCs shifted to a mature, contractile state. To verify this finding, HITB5 SMCs were analyzed for PBEF mRNA and protein expression by Northern and Western blot analysis, respectively. As shown in Figure 1, 6 days after serum withdrawal, HITB5 SMCs converted from spread cells variably oriented on the dish to highly elongated cells that had crawled in a directed fashion into multilayered cell aggregates. Concurrently, the 3 major transcripts of PBEF (4.8, 2.9, and 2.2 kb) were markedly upregulated (Figure 1B). Intracellular PBEF protein abundance also increased in maturing HITB5 and HITC6 SMCs (3.9±0.5-fold after 6 days), as did the expression of the SMC contractile apparatus proteins h-caldesmon and smoothelin A (Figure 1C). PBEF was not detected in concentrated culture media at any stage of the maturation program (data not shown).
Overexpression of PBEF Promotes SMC Maturation
To determine whether PBEF was capable of promoting a more mature SMC phenotype, HITB5 SMCs were infected with retrovirus containing cDNA encoding PBEF and EGFP, or cDNA encoding EGFP alone, and stable transductants were selected. As shown in Figure 2, SMCs overexpressing PBEF developed a bipolar morphology and were longer and thinner than vector-infected SMCs. This relative elongation persisted as the SMCs further elongated in response to serum withdrawal (Figure 2B) and was also observed with HITC6 SMCs and primary SMCs expressing PBEF (data not shown). To determine whether the capacity for spatial organization of SMCs was impacted by PBEF, cells were plated at higher densities (12 000 cells/cm2) and subjected to serum withdrawal. As shown in Figure 2, by 3 days PBEF-overexpressing SMCs had aggregated and organized into discrete, multilayered ridges and nodules. In contrast, the extent of patterning in control SMCs was modest.
PBEF-overexpressing SMCs also displayed increased levels of h-caldesmon, smoothelin-A, and smoothelin-B compared with control SMCs assessed simultaneously (Figure 3). Withdrawal of serum from cultures of HITB5-PBEF SMCs led to further upregulation of smoothelin-A, and metavinculin expression was induced, whereas the latter remained undetected in control SMCs. Thus, augmented expression of PBEF shifted both the morphological and biochemical phenotype of SMCs closer to that of mature, contractile SMCs.
Overexpression of PBEF Reduces SMC Apoptosis
Differentiation of SMCs has been associated with a decline in SMC apoptosis.3,16 To determine whether SMC survival was affected by PBEF, apoptosis was assessed using TUNEL. We previously established that, in contrast to primary cells, apoptosis of HITB5 and HITC6 SMCs is significantly higher when cells are cultured in 10% FBS as opposed to 0% FBS.3 As shown in Figure 4, for SMCs in M199 supplemented with 10% FBS, the proportion of apoptotic PBEF-overexpressing SMCs was approximately half that of control SMCs (P<0.01). When SMCs were incubated in serum-free medium, apoptosis was substantially lower (<3%), with no difference between control and PBEF-overexpressing SMCs.
siRNA-Mediated PBEF Knockdown Impairs SMC Survival and Maturation
We next determined the role of endogenous PBEF on SMC survival and the capacity to mature. For this, HITC6 SMCs were infected with retrovirus containing cDNA encoding a hairpin-forming siRNA fragment. To ensure the siRNA responses reflected PBEF knockdown, 3 different targeting fragments were studied and both PBEF mRNA and protein were quantified. Two of the 3 siRNA constructs (siRNA147, siRNA1278) yielded a significant decrease in PBEF mRNA and protein (the latter to 0.44±0.03 and 0.45±0.04 that of cells expressing the corresponding nonsilencing RNA [nsRNA] fragment). PBEF-knockdown SMCs had a short, truncated morphology (Figure 5A), and they survived poorly, precluding serial passages. Furthermore, the PBEF-knockdown SMCs that remained adherent to the culture dish did not elongate following serum withdrawal (Figure 5B). Heavy-caldesmon and smoothelin-B expression were also significantly lower in PBEF-knockdown HITC6-siRNA SMCs than control HITC6-nsRNA SMCs (to 0.28 and 0.54 of control levels, respectively) (Figure 5C). In contrast, SMCs expressing the siRNA construct (siRNA384) that did not manifest a reduction in PBEF mRNA or protein maintained an elongated morphology (Figure 5A) and responded to serum withdrawal normally.
PBEF Increases Nicotinamide Phosphoribosyltransferase Activity and Intracellular NAD+ in Human SMCs
Sequence and phylogenetic analysis in silico (see online Results in the data supplement) revealed that the PBEF protein has been well conserved throughout evolution, which suggests a fundamental and invariant role. As this role has been shown in bacteria and rodent cells to be an enzymatic one, catalyzing the formation of nicotinamide mononucleotide (NMN) from nicotinamide,9,11 we determined whether nicotinamide phosphoribosyltransferase activity was increased in PBEF-overexpressing SMCs. As shown in Figure 6, PBEF-overexpressing HITC6 SMCs manifest a substantial increase in nicotinamide phosphoribosyltransferase activity compared with vector-infected SMCs.
To further assess the consequences of PBEF on NAD+ biosynthesis, the steady-state levels of NAD+ in human cells were quantified by HPLC. This revealed that NAD+ content in SMCs stably expressing the PBEF transgene was 1.45±0.02-fold greater than in control cells (P<0.01). Conversely, the intracellular NAD+ content of SMCs expressing siRNA1278 was reduced to 0.78±0.08 that of HITC6-nsRNA SMCs (P<0.05) (Figure 6).
The phylogenetic data, the increased activity of nicotinamide phosphoribosyltransferase, and the altered NAD+ levels strongly implicate PBEF as an intracellular regulator of NAD+ biosynthesis in SMCs. At the same time, they raise the question as to whether the upregulation of PBEF in maturing SMCs might, in part, be a response to suboptimal in vitro concentrations of NAD+ precursors, potentially exacerbated by removal of FBS. That this was not the case was demonstrated by strong upregulation of PBEF following serum withdrawal even when the medium was supplemented with nicotinic acid and nicotinamide, at levels up to 5000-fold greater than those found in M199 (Figure 6C). Thus, PBEF upregulation in maturing SMCs appears to be an inherent feature of the HIT SMC maturation program, rather than a response to substrate limitation, and suggests that NAD+ is consumed during SMC maturation.
NAD+-Dependent Deacetylase Activity Is Required for SMC Maturation and Is Increased in PBEF-Overexpressing SMCs
Recently, a number of vital NAD+-dependent reactions have been identified that depend on the ability of the cell to regenerate NAD+ from nicotinamide. These reactions include deacetylation of certain histones and other proteins, reactions critical to gene silencing and cell survival.17,18 To determine whether SMC maturation was dependent on NAD+-dependent deacetylase activity, we studied the maturation of HITC6 SMCs in the presence or absence of sirtinol, a noncompetitive inhibitor of NAD+-dependent protein deacetylases that has no known effect on NAD+-independent histone deacetylases (HDACs).19 As shown in Figure 7, 10 eol/L sirtinol had no discernable effect on the morphology or growth of HITC6 SMCs in the presence of 10% FBS. However, following serum withdrawal, SMCs incubated with sirtinol failed to elongate. This effect was reversible, and elongation was restored following sirtinol washout. Western blot analysis revealed that PBEF expression increased 1.9-fold, 4 days after serum withdrawal, associated with upregulation of h-caldesmon and smoothelin-B expression. However, in the presence of sirtinol, h-caldesmon, and smoothelin-B levels were substantially lower than in dimethyl sulfoxideeCtreated control SMCs (Figure 7C).
To determine whether PBEF influenced HDAC activity in SMCs, lysates from control and PBEF-overexpressing SMCs were incubated with [3H]-acetylated histone H4 peptide and HDAC activity quantified. As shown in Figure 7D, total HDAC activity was significantly greater in HITC6-PBEF SMCs than HITC6-Vector SMCs. Sirtinol significantly inhibited HDAC activity in lysates of PBEF-overexpressing SMCs, with a more modest inhibition of HDAC activity in control SMCs such that there was no longer a difference in HDAC activity between control and PBEF-overexpressing cells. We also examined the effect of 40 nmol/L trichostatin A, an inhibitor of class I and II HDACs, but not the NAD+-dependent (class III) HDACs. This substantially inhibited HDAC activity in both control and PBEF-overexpressing SMCs. However, the residual trichostatin AeCindependent HDAC activity remained significantly greater in PBEF-overexpressing SMCs than in control SMCs. Taken together, the findings establish the following: (1) that vascular SMC maturation requires NAD+-dependent protein deacetylase activity; (2) that PBEF increases HDAC activity in SMCs; and (3) that most, if not all, of the PBEF-induced HDAC activity in SMCs can be attributed to NAD+-dependent HDAC reactions.
Investment of Newly Formed Blood Vessels With SMCs Is Dependent on PBEF
To assess the effect of PBEF on SMC phenotype in vivo, we developed a human-mouse chimeric model of angiogenesis, whereby PBEF expression was manipulated exclusively in SMCs. Growth factoreCreduced matrigel, mixed with FGF-2 and either PBEF-overexpressing SMCs (HITC6-PBEF-EGFP) or vector-transduced SMCs (HITC6-EGFP), was injected subcutaneously into the abdominal regions of SCID mice. After 8 days, mice were euthanized and zinc-fixed sections were studied histologically. Microvessel density, assessed by quantifying CD31-positive channels, was not significantly different in implants containing PBEF-overexpressing SMCs (287±18 versus 268±24 vessels/mm2). However, the proportion of microvessels that were invested by human SMCs, determined by double-immunolabeling for mouse endothelial cells and human SMCs (anti-EGFP), was significantly higher in matrigel implants containing PBEF-overexpressing SMCs (17.8±2.5%) than in implants loaded with vector-infected SMCs (10.7±2.2%, P<0.05) (Figure 8). SMCs that invested microvessels were elongated with a more compact and elongated nucleus than SMCs in the interstitium. PBEF-overexpressing SMCs could be found aligned with the long axis of microvessels, extending toward and partially apposed to an endothelial cell, as if actively investing the vessel, or wrapped circumferentially around the microvessel (Figure 8C through 8E). The increased SMC investment of microvessels did not appear to be solely attributable to increased SMC survival afforded by PBEF because the total number of EGFP-expressing SMCs in the matrigel was not significantly increased (1079±67 versus 1177±75 cells/mm2). Consistent with this, the proportion of apoptotic human PBEF-overexpressing SMCs in the matrigel was not significantly lower than that for vector-infected SMCs (1.4±0.3 versus 2.1±0.4%, P=0.42), likely because the basal SMC apoptosis rate in the matrigel was very low. This is consistent with other studies showing little apoptosis of cells in matrigel.20 Thus, the enhanced wrapping of vascular channels by PBEF-overexpressing SMCs appears to reflect a heightened capacity of these cells to mature. As detailed in the online supplement, the proportion of microvessels invested by HITC6 SMCs was significantly reduced when PBEF levels were lowered with siRNA (3.1±0.6 versus 9.4±1.4%; Figure 8F).
Discussion
PBEF is a protein that has been suggested in several studies to be a cytokine.5,7,8,21 In this report, we have shown that PBEF, in vascular SMCs, is a dynamically expressed intracellular protein that regulates the production of NAD+. Decreasing the level of PBEF in SMCs impaired their survival and their ability to mature in culture. In contrast, overexpression of PBEF in SMCs yielded a highly elongated and bipolar cell with increased expression of h-caldesmon, smoothelin-A, smoothelin-B, and metavinculin. This shift toward a more mature phenotype was also seen in a tissue environment. Specifically, PBEF imparted SMCs with an augmented capacity to intimately associate with endothelial cells and invest newly formed vascular channels. These findings implicate NAD+-consuming reactions in the acquisition of a more specialized SMC phenotype and identify PBEF as a determinant of these reactions.
The molecular pathways by which PBEF acts have been controversial. When initially identified, PBEF was proposed to be a cytokine.5 Support for the notion of PBEF as a cytokine has come from studies wherein recombinant PBEF, generated by bacteria, stimulated the expression of inflammatory genes6,21 and inhibited neutrophil apoptosis.7 However, whether these effects were mediated by a cytokine receptor is unclear. Recently, PBEF was reported to bind to the insulin receptor and was renamed visfatin, a putative adipokine.8 However, the physiological relevance of PBEF binding to the insulin receptor binding has been questioned.22 Moreover, the assignment of PBEF as a cytokine has been challenged because PBEF has no coding sequence homology to cytokines, no signal sequence for secretion, and does not appear to be secreted from cells by either a classical or alternative pathway.9,10 We were unable to detect PBEF protein in the concentrated conditioned media from PBEF-overexpressing cell lines despite substantial upregulation of intracellular protein.
The alternative proposal is that PBEF is a nicotinamide phosphoribosyltransferase, the rate-limiting, intracellular enzyme for generating NAD+ from nicotinamide.9 Sequence and phylogenetic data are entirely consistent with the identity of human PBEF as a nicotinamide phosphoribosyltransferase. This assignment of PBEF is further strengthened by our finding that overexpression of PBEF in human SMCs substantially increased nicotinamide phosphoribosyltransferase activity. Moreover, overexpression of PBEF increased the level of NAD+ in SMCs, whereas a decrease in PBEF expression was associated with reduced NAD+ levels. We propose that the sequence data, the close evolutionary relationship with prokaryotic orthologs, the fact that PBEF stimulates the conversion of nicotinamide to NMN and upregulates NAD+, and previous complementation and enzyme analyses of mouse PBEF9 bring the weight of evidence to the conclusion that human PBEF is a nicotinamide phosphoribosyltransferase and not a cytokine.
The current study also reveals that the mechanism by which PBEF impacts SMC behavior likely relates to intracellular reactions that consume NAD+. In contrast to redox reactions, which use, but do not consume NAD+, a number of enzymatic reactions within the nucleus and cytoplasm consume NAD+ as a cosubstrate. Degradation of NAD+ in these reactions liberates nicotinamide, from which NAD+ can be regenerated via a 2-step salvage pathway. Nicotinamide phosphoribosyltransferase/PBEF catalyzes the conversion of nicotinamide to nicotinamide mononucleotide. NMN is then converted to NAD+ by NMN adenylyltransferase-1 (Nmnat1).12,13 Both steps are necessary for sustaining NAD+-consuming reactions, including the deacetylation of histones and nonhistone proteins.23,24 Interestingly, Nmnat1 has recently been found to prevent axonal degeneration in explanted mouse neurons,24 a finding concordant with the observed anti-apoptotic actions of PBEF.
We found that SMC maturation was dependent on NAD+-dependent protein deacetylase activity. HITC6 SMCs failed to elongate and failed to upregulate SMC contractile proteins in the presence of the NAD+-dependent deacetylase inhibitor sirtinol. Conversely, overexpression of PBEF not only promoted SMC survival and the capacity to mature but stimulated NAD+-dependent HDAC activity. Interestingly, NAD+ salvage pathway genes in yeast and mammals have been found to activate Sir2 (or the mammalian ortholog SIRT1), an NAD+-dependent deacetylase involved in transcriptional silencing and lifespan extension in yeast23,25,26 and survival of mammalian cells.27 Further studies will be required to determine the extent to which specific sirtuins, or other NAD+-dependent enzymes, regulate SMC function. However, the current data suggest that the capacity with which NAD+ can be regenerated (or nicotinamide cleared) is a key determinant of SMC behavior, including the acquisition of a quiescent phenotype.
The more mature attributes of PBEF-overexpressing SMCs could be functionally linked to their increased survival. That is, the apoptosis-resistant state imparted by PBEF could allow cultured SMCs to reassume more "physiological" characteristics, including a slender morphology and increased expression of SMC-associated proteins. Although such a relationship is likely, the more mature characteristics of PBEF-overexpressing SMCs were also observed under circumstances where there was no difference in apoptosis between control and PBEF-overexpressing cells. In serum-free media, both control and PBEF-overexpressing SMC clones displayed equally low apoptosis prevalence, yet there were substantially higher levels of smoothelin-A, smoothelin-B, caldesmon, and metavinculin in the PBEF-overexpressing SMCs. Furthermore, the increased investment of microvessels in vivo by PBEF-overexpressing SMCs compared with control SMCs occurred under conditions where SMC apoptosis was low and not different between control and PBEF-overexpressing SMCs. Interestingly, PBEF expression has been observed to be upregulated during differentiation/maturation of other cell types, including dendritic cells and B-lymphocytes.5,28
The relationship between PBEF and specialized SMC performance was supported in an in vivo context. In order for newly formed vascular networks to survive, they must be ensheathed by SMCs for support and stabilization.1,29 Investment by SMCs also enables the vessels to respond to vasoactive stimuli and thereby appropriately distribute blood. We capitalized on this in vivo, integrative response as a functional readout for SMC maturation. Human SMCs with reduced PBEF expression displayed a reduced ability to invest nascent microvessels. In contrast, SMCs overexpressing PBEF responded to the angiogenic environment by intimately wrapping around new microvessels and assuming an elongated morphology with a compact nucleus. It remains possible that the enhanced microvessel investment was attributable, in part, to increased directed migration of PBEF-overexpressing SMCs. Indeed, maturation of HITB5/C6 SMCs in culture was closely linked with directed SMC migration and, in vivo, SMC migration to endothelial channels may similarly be integrated with SMC maturation. The investment of microvessels with maturing SMCs is important in ischemic tissues, where neovessels are prone to regression. Furthermore, tumor vasculature inadequately invested by SMCs can result in leaky vessels that are predisposed to tumor shedding.30 The current findings thus suggest that manipulating NAD+ biosynthesis pathways may be relevant to these problems.
In summary, PBEF is not a cytokine but an intracellular protein that regulates NAD+ biosynthesis, NAD+-dependent protein deacetylation, vascular SMC survival, and the capacity of SMCs to mature. Augmentation of NAD+ regeneration, via PBEF, could have therapeutic potential for vascular conditions that depend on stress-resistant SMCs that can efficiently differentiate.
Acknowledgments
This work was supported by grants from the Heart and Stroke Foundation of Canada (T4458), the Heart and Stroke Foundation of Ontario (HSFO) (PRG 4854), the Canadian Institutes of Health Research (MT11715), and the Heart and Stroke Foundation of Ontario Career Investigator award (to J.G.P.).
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Pharmacology, University of Western Ontario, London, Canada.
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 preeCB-cell colonyeCenhancing 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.
Key Words: vascular smooth muscle;preeCB-cell colonyeCenhancing factor;maturation;nicotinamide phosphoribosyltransferase;deacetylation
Introduction
Conversion 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 preeCB cell colonyeCenhancing factor (PBEF) as being upregulated as SMCs shifted toward maturity. PBEF is a 52- to 55-kDa protein that has been proposed to be a cytokine.5 Reported actions in this regard include synergizing with other cytokines to stimulate the maturation of preeCB cells,5 stimulating the expression of inflammatory cytokines in epithelial cells,6 prolonging neutrophil survival,7 and acting as an adipokine that lowers plasma glucose levels.8 However, the contention that PBEF is a secreted cytokine is controversial. PBEF does not have a signal sequence for secretion, and the presence of PBEF in culture media has been suggested to be a consequence of activation-induced cell death, rather than secretion by either a classical or alternative pathway.9,10 Moreover, PBEF has sequence similarity with bacterial NadV, a protein that confers bacteria with the ability to grow in nicotinamide adenine dinucleotide (NAD+)-deficient conditions.11 In keeping with this, Rongvaux et al have reported that mouse PBEF functions within the cell as a nicotinamide phosphoribosyltransferase.9 This enzyme catalyzes the rate-limiting step in the salvage pathway for NAD+ biosynthesis, whereby nicotinamide that is generated during NAD+-consuming reactions is used to regenerate NAD+.12,13
We report here that human PBEF is not a cytokine but an intracellular regulator of NAD+-consuming reactions, including NAD+-dependent protein deacetylation. We further report that PBEF can promote the efficient acquisition of a stable and mature SMC phenotype and can increase the capacity of SMCs to invest developing blood vessels.
Materials and Methods
An expanded Materials and Methods can be found in the online data supplement available at http://circres.ahajournals.org
Cell Culture
Experiments were performed using the maturation-competent human vascular SMC lines HITB5 and HITC6 generated from the human internal thoracic artery, as described previously.3,4
Overexpression of PBEF in Human SMCs
A retroviral geneeCdelivery system was used to generate human SMCs stably overexpressing PBEF, using methods described previously.14 A PBEF-IRES-EGFP bicistronic fragment was cloned into the retroviral expression vector pLNCX2 (Clontech). A second retroviral expression construct was generated by inserting PBEF cDNA into pQCXIP-IRES-PURO (Clontech). Stable transductants were selected and overexpression of PBEF was confirmed before each experiment by Western blot analysis.
Western Blot Analysis
Expression of PBEF and SMC differentiation markers was assessed by Western blot analysis with chemiluminescence detection, as described.14
Apoptosis
SMC apoptosis was assessed by fluorescence in situ end-labeling of DNA fragments, as described.3
Knockdown of PBEF by RNA Interference
PBEF knockdown was accomplished by infecting human SMCs with retrovirus-containing sequences encoding hairpin small interfering (siRNA) fragments. Three different targeting sequences were used, each consisting of 19 nucleotides starting at nucleotides 147, 384, and 1278 of the PBEF coding sequence (siRNA147, 5'-GGAAGGTGAAATATGAGGA-3'; siRNA384, 5'-ATGTTCTCTTCACGGTGGA-3'; siRNA1278, 5'-AGGG CCGATTATCTTTACA-3'). Each sequence was separated by a 9-nucleotide noncomplementary spacer from the reverse complement of the same 19-nucleotide sequence. Blast search confirmed that only the PBEF gene was targeted. Control inserts contained the gene-specific 19-nucleotide sequence and hairpin loop sequence but not the antisense component.
Real-Time Reverse TranscriptioneCPCR
Probe (5'-CAGTTGCTGATCCCA- 3') and flanking primers (5'-primer, 5'-TGCAGCTATGTTGTAACCAATGG-3'; 3'-primer, 5'-ACAAAAGGTCGAAAAAGGGCC-3') were used for TaqMan real-time reverse transcription (RT)-PCR for PBEF. GAPDH transcript abundance was used as an endogenous RNA control to which PBEF transcript abundance was normalized.
Nicotinamide Phosphoribosyltransferase Activity
Cell lysates were reacted with 5 eol/L [carbonyl-14C]nicotinamide in the presence of 0.5 mmol/L phosphoribosylpyrophosphate and labeled, acetone-precipitable nicotinamide mononucleotide (NMN) quantified by scintillation counting.9
NAD+ Analysis by High-Performance Liquid Chromatography
Cellular nucleotides were extracted using perchloric acid, neutralized with KOH, and deproteinized cell lysate residues were analyzed by high-performance liquid chromatography (HPLC). NAD+ retention time, determined from a NAD+ standard, was 10 minutes.
Histone Deacetylase Assay
Histone H4 peptide (Upstate) was labeled with [3H]acetyl coenzyme A (ICN) using PCAF histone acetylase. Cell lysates were incubated with [3H]acetyl histone and released [3H]acetate assessed by scintillation counting (SCID, Charles River Laboratories, Wilmington, Mass).
Human-Mouse Chimeric Angiogenesis In Vivo
HITC6 SMCs in M199 with 10% FBS and fibroblast growth factor (FGF)-2 were mixed with an equal volume of growth factor-reduced matrigel (BD Discovery Labware) yielding final concentrations of 5x105 cells/mL and 250 ng/mL FGF-2. The cell-matrigel suspension (500 e蘈) was subcutaneously injected into the abdomen of mice with severe combined immunodeficiency syndrome (SCID). After 8 days, implants were harvested, fixed for 8 hour in Tris-buffered zinc,15 and paraffin-embedded tissues were sectioned at 5 e. Sections were double-immunostained for CD31 (BD Biosciences) and enhanced green fluorescent protein (EGFP) (BD Clontech) using diaminobenzidine (Vector Laboratories Ltd) and red alkaline phosphatase substrates, respectively, and counterstained with Harris hematoxylin. Apoptosis of human SMCs in matrigel was assessed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay, after immunostaining for EGFP and incubating sections with proteinase K (100 e/mL).
Algorithms for quantifying EGFP-positive human SMCs, TUNEL-positive human SMCs, microvessels, and SMC investment of microvessels are described in the online data supplement. Human SMCs were deemed to be investing a microvessel if they assumed contiguous and close apposition with an endothelial cell(s) and oriented their long axis along a contour of the vessel wall.
Statistics
Values are expressed as mean±SEM. Comparisons were made by t test or analysis of variance with Bonferroni post hoc test. Significance of fold-changes was assessed by the Wilcoxon signed-rank test. Statistical significance was set at P<0.05.
Results
PBEF Is Upregulated During SMC Maturation
The generation of clonal populations of SMCs from the human internal thoracic artery, designated HITB5 and HITC6, that can convert from a proliferative to a contractile SMC3,4 enabled us to screen for endogenous factors involved in this phenotype conversion. These screens suggested that PBEF was upregulated as SMCs shifted to a mature, contractile state. To verify this finding, HITB5 SMCs were analyzed for PBEF mRNA and protein expression by Northern and Western blot analysis, respectively. As shown in Figure 1, 6 days after serum withdrawal, HITB5 SMCs converted from spread cells variably oriented on the dish to highly elongated cells that had crawled in a directed fashion into multilayered cell aggregates. Concurrently, the 3 major transcripts of PBEF (4.8, 2.9, and 2.2 kb) were markedly upregulated (Figure 1B). Intracellular PBEF protein abundance also increased in maturing HITB5 and HITC6 SMCs (3.9±0.5-fold after 6 days), as did the expression of the SMC contractile apparatus proteins h-caldesmon and smoothelin A (Figure 1C). PBEF was not detected in concentrated culture media at any stage of the maturation program (data not shown).
Overexpression of PBEF Promotes SMC Maturation
To determine whether PBEF was capable of promoting a more mature SMC phenotype, HITB5 SMCs were infected with retrovirus containing cDNA encoding PBEF and EGFP, or cDNA encoding EGFP alone, and stable transductants were selected. As shown in Figure 2, SMCs overexpressing PBEF developed a bipolar morphology and were longer and thinner than vector-infected SMCs. This relative elongation persisted as the SMCs further elongated in response to serum withdrawal (Figure 2B) and was also observed with HITC6 SMCs and primary SMCs expressing PBEF (data not shown). To determine whether the capacity for spatial organization of SMCs was impacted by PBEF, cells were plated at higher densities (12 000 cells/cm2) and subjected to serum withdrawal. As shown in Figure 2, by 3 days PBEF-overexpressing SMCs had aggregated and organized into discrete, multilayered ridges and nodules. In contrast, the extent of patterning in control SMCs was modest.
PBEF-overexpressing SMCs also displayed increased levels of h-caldesmon, smoothelin-A, and smoothelin-B compared with control SMCs assessed simultaneously (Figure 3). Withdrawal of serum from cultures of HITB5-PBEF SMCs led to further upregulation of smoothelin-A, and metavinculin expression was induced, whereas the latter remained undetected in control SMCs. Thus, augmented expression of PBEF shifted both the morphological and biochemical phenotype of SMCs closer to that of mature, contractile SMCs.
Overexpression of PBEF Reduces SMC Apoptosis
Differentiation of SMCs has been associated with a decline in SMC apoptosis.3,16 To determine whether SMC survival was affected by PBEF, apoptosis was assessed using TUNEL. We previously established that, in contrast to primary cells, apoptosis of HITB5 and HITC6 SMCs is significantly higher when cells are cultured in 10% FBS as opposed to 0% FBS.3 As shown in Figure 4, for SMCs in M199 supplemented with 10% FBS, the proportion of apoptotic PBEF-overexpressing SMCs was approximately half that of control SMCs (P<0.01). When SMCs were incubated in serum-free medium, apoptosis was substantially lower (<3%), with no difference between control and PBEF-overexpressing SMCs.
siRNA-Mediated PBEF Knockdown Impairs SMC Survival and Maturation
We next determined the role of endogenous PBEF on SMC survival and the capacity to mature. For this, HITC6 SMCs were infected with retrovirus containing cDNA encoding a hairpin-forming siRNA fragment. To ensure the siRNA responses reflected PBEF knockdown, 3 different targeting fragments were studied and both PBEF mRNA and protein were quantified. Two of the 3 siRNA constructs (siRNA147, siRNA1278) yielded a significant decrease in PBEF mRNA and protein (the latter to 0.44±0.03 and 0.45±0.04 that of cells expressing the corresponding nonsilencing RNA [nsRNA] fragment). PBEF-knockdown SMCs had a short, truncated morphology (Figure 5A), and they survived poorly, precluding serial passages. Furthermore, the PBEF-knockdown SMCs that remained adherent to the culture dish did not elongate following serum withdrawal (Figure 5B). Heavy-caldesmon and smoothelin-B expression were also significantly lower in PBEF-knockdown HITC6-siRNA SMCs than control HITC6-nsRNA SMCs (to 0.28 and 0.54 of control levels, respectively) (Figure 5C). In contrast, SMCs expressing the siRNA construct (siRNA384) that did not manifest a reduction in PBEF mRNA or protein maintained an elongated morphology (Figure 5A) and responded to serum withdrawal normally.
PBEF Increases Nicotinamide Phosphoribosyltransferase Activity and Intracellular NAD+ in Human SMCs
Sequence and phylogenetic analysis in silico (see online Results in the data supplement) revealed that the PBEF protein has been well conserved throughout evolution, which suggests a fundamental and invariant role. As this role has been shown in bacteria and rodent cells to be an enzymatic one, catalyzing the formation of nicotinamide mononucleotide (NMN) from nicotinamide,9,11 we determined whether nicotinamide phosphoribosyltransferase activity was increased in PBEF-overexpressing SMCs. As shown in Figure 6, PBEF-overexpressing HITC6 SMCs manifest a substantial increase in nicotinamide phosphoribosyltransferase activity compared with vector-infected SMCs.
To further assess the consequences of PBEF on NAD+ biosynthesis, the steady-state levels of NAD+ in human cells were quantified by HPLC. This revealed that NAD+ content in SMCs stably expressing the PBEF transgene was 1.45±0.02-fold greater than in control cells (P<0.01). Conversely, the intracellular NAD+ content of SMCs expressing siRNA1278 was reduced to 0.78±0.08 that of HITC6-nsRNA SMCs (P<0.05) (Figure 6).
The phylogenetic data, the increased activity of nicotinamide phosphoribosyltransferase, and the altered NAD+ levels strongly implicate PBEF as an intracellular regulator of NAD+ biosynthesis in SMCs. At the same time, they raise the question as to whether the upregulation of PBEF in maturing SMCs might, in part, be a response to suboptimal in vitro concentrations of NAD+ precursors, potentially exacerbated by removal of FBS. That this was not the case was demonstrated by strong upregulation of PBEF following serum withdrawal even when the medium was supplemented with nicotinic acid and nicotinamide, at levels up to 5000-fold greater than those found in M199 (Figure 6C). Thus, PBEF upregulation in maturing SMCs appears to be an inherent feature of the HIT SMC maturation program, rather than a response to substrate limitation, and suggests that NAD+ is consumed during SMC maturation.
NAD+-Dependent Deacetylase Activity Is Required for SMC Maturation and Is Increased in PBEF-Overexpressing SMCs
Recently, a number of vital NAD+-dependent reactions have been identified that depend on the ability of the cell to regenerate NAD+ from nicotinamide. These reactions include deacetylation of certain histones and other proteins, reactions critical to gene silencing and cell survival.17,18 To determine whether SMC maturation was dependent on NAD+-dependent deacetylase activity, we studied the maturation of HITC6 SMCs in the presence or absence of sirtinol, a noncompetitive inhibitor of NAD+-dependent protein deacetylases that has no known effect on NAD+-independent histone deacetylases (HDACs).19 As shown in Figure 7, 10 eol/L sirtinol had no discernable effect on the morphology or growth of HITC6 SMCs in the presence of 10% FBS. However, following serum withdrawal, SMCs incubated with sirtinol failed to elongate. This effect was reversible, and elongation was restored following sirtinol washout. Western blot analysis revealed that PBEF expression increased 1.9-fold, 4 days after serum withdrawal, associated with upregulation of h-caldesmon and smoothelin-B expression. However, in the presence of sirtinol, h-caldesmon, and smoothelin-B levels were substantially lower than in dimethyl sulfoxideeCtreated control SMCs (Figure 7C).
To determine whether PBEF influenced HDAC activity in SMCs, lysates from control and PBEF-overexpressing SMCs were incubated with [3H]-acetylated histone H4 peptide and HDAC activity quantified. As shown in Figure 7D, total HDAC activity was significantly greater in HITC6-PBEF SMCs than HITC6-Vector SMCs. Sirtinol significantly inhibited HDAC activity in lysates of PBEF-overexpressing SMCs, with a more modest inhibition of HDAC activity in control SMCs such that there was no longer a difference in HDAC activity between control and PBEF-overexpressing cells. We also examined the effect of 40 nmol/L trichostatin A, an inhibitor of class I and II HDACs, but not the NAD+-dependent (class III) HDACs. This substantially inhibited HDAC activity in both control and PBEF-overexpressing SMCs. However, the residual trichostatin AeCindependent HDAC activity remained significantly greater in PBEF-overexpressing SMCs than in control SMCs. Taken together, the findings establish the following: (1) that vascular SMC maturation requires NAD+-dependent protein deacetylase activity; (2) that PBEF increases HDAC activity in SMCs; and (3) that most, if not all, of the PBEF-induced HDAC activity in SMCs can be attributed to NAD+-dependent HDAC reactions.
Investment of Newly Formed Blood Vessels With SMCs Is Dependent on PBEF
To assess the effect of PBEF on SMC phenotype in vivo, we developed a human-mouse chimeric model of angiogenesis, whereby PBEF expression was manipulated exclusively in SMCs. Growth factoreCreduced matrigel, mixed with FGF-2 and either PBEF-overexpressing SMCs (HITC6-PBEF-EGFP) or vector-transduced SMCs (HITC6-EGFP), was injected subcutaneously into the abdominal regions of SCID mice. After 8 days, mice were euthanized and zinc-fixed sections were studied histologically. Microvessel density, assessed by quantifying CD31-positive channels, was not significantly different in implants containing PBEF-overexpressing SMCs (287±18 versus 268±24 vessels/mm2). However, the proportion of microvessels that were invested by human SMCs, determined by double-immunolabeling for mouse endothelial cells and human SMCs (anti-EGFP), was significantly higher in matrigel implants containing PBEF-overexpressing SMCs (17.8±2.5%) than in implants loaded with vector-infected SMCs (10.7±2.2%, P<0.05) (Figure 8). SMCs that invested microvessels were elongated with a more compact and elongated nucleus than SMCs in the interstitium. PBEF-overexpressing SMCs could be found aligned with the long axis of microvessels, extending toward and partially apposed to an endothelial cell, as if actively investing the vessel, or wrapped circumferentially around the microvessel (Figure 8C through 8E). The increased SMC investment of microvessels did not appear to be solely attributable to increased SMC survival afforded by PBEF because the total number of EGFP-expressing SMCs in the matrigel was not significantly increased (1079±67 versus 1177±75 cells/mm2). Consistent with this, the proportion of apoptotic human PBEF-overexpressing SMCs in the matrigel was not significantly lower than that for vector-infected SMCs (1.4±0.3 versus 2.1±0.4%, P=0.42), likely because the basal SMC apoptosis rate in the matrigel was very low. This is consistent with other studies showing little apoptosis of cells in matrigel.20 Thus, the enhanced wrapping of vascular channels by PBEF-overexpressing SMCs appears to reflect a heightened capacity of these cells to mature. As detailed in the online supplement, the proportion of microvessels invested by HITC6 SMCs was significantly reduced when PBEF levels were lowered with siRNA (3.1±0.6 versus 9.4±1.4%; Figure 8F).
Discussion
PBEF is a protein that has been suggested in several studies to be a cytokine.5,7,8,21 In this report, we have shown that PBEF, in vascular SMCs, is a dynamically expressed intracellular protein that regulates the production of NAD+. Decreasing the level of PBEF in SMCs impaired their survival and their ability to mature in culture. In contrast, overexpression of PBEF in SMCs yielded a highly elongated and bipolar cell with increased expression of h-caldesmon, smoothelin-A, smoothelin-B, and metavinculin. This shift toward a more mature phenotype was also seen in a tissue environment. Specifically, PBEF imparted SMCs with an augmented capacity to intimately associate with endothelial cells and invest newly formed vascular channels. These findings implicate NAD+-consuming reactions in the acquisition of a more specialized SMC phenotype and identify PBEF as a determinant of these reactions.
The molecular pathways by which PBEF acts have been controversial. When initially identified, PBEF was proposed to be a cytokine.5 Support for the notion of PBEF as a cytokine has come from studies wherein recombinant PBEF, generated by bacteria, stimulated the expression of inflammatory genes6,21 and inhibited neutrophil apoptosis.7 However, whether these effects were mediated by a cytokine receptor is unclear. Recently, PBEF was reported to bind to the insulin receptor and was renamed visfatin, a putative adipokine.8 However, the physiological relevance of PBEF binding to the insulin receptor binding has been questioned.22 Moreover, the assignment of PBEF as a cytokine has been challenged because PBEF has no coding sequence homology to cytokines, no signal sequence for secretion, and does not appear to be secreted from cells by either a classical or alternative pathway.9,10 We were unable to detect PBEF protein in the concentrated conditioned media from PBEF-overexpressing cell lines despite substantial upregulation of intracellular protein.
The alternative proposal is that PBEF is a nicotinamide phosphoribosyltransferase, the rate-limiting, intracellular enzyme for generating NAD+ from nicotinamide.9 Sequence and phylogenetic data are entirely consistent with the identity of human PBEF as a nicotinamide phosphoribosyltransferase. This assignment of PBEF is further strengthened by our finding that overexpression of PBEF in human SMCs substantially increased nicotinamide phosphoribosyltransferase activity. Moreover, overexpression of PBEF increased the level of NAD+ in SMCs, whereas a decrease in PBEF expression was associated with reduced NAD+ levels. We propose that the sequence data, the close evolutionary relationship with prokaryotic orthologs, the fact that PBEF stimulates the conversion of nicotinamide to NMN and upregulates NAD+, and previous complementation and enzyme analyses of mouse PBEF9 bring the weight of evidence to the conclusion that human PBEF is a nicotinamide phosphoribosyltransferase and not a cytokine.
The current study also reveals that the mechanism by which PBEF impacts SMC behavior likely relates to intracellular reactions that consume NAD+. In contrast to redox reactions, which use, but do not consume NAD+, a number of enzymatic reactions within the nucleus and cytoplasm consume NAD+ as a cosubstrate. Degradation of NAD+ in these reactions liberates nicotinamide, from which NAD+ can be regenerated via a 2-step salvage pathway. Nicotinamide phosphoribosyltransferase/PBEF catalyzes the conversion of nicotinamide to nicotinamide mononucleotide. NMN is then converted to NAD+ by NMN adenylyltransferase-1 (Nmnat1).12,13 Both steps are necessary for sustaining NAD+-consuming reactions, including the deacetylation of histones and nonhistone proteins.23,24 Interestingly, Nmnat1 has recently been found to prevent axonal degeneration in explanted mouse neurons,24 a finding concordant with the observed anti-apoptotic actions of PBEF.
We found that SMC maturation was dependent on NAD+-dependent protein deacetylase activity. HITC6 SMCs failed to elongate and failed to upregulate SMC contractile proteins in the presence of the NAD+-dependent deacetylase inhibitor sirtinol. Conversely, overexpression of PBEF not only promoted SMC survival and the capacity to mature but stimulated NAD+-dependent HDAC activity. Interestingly, NAD+ salvage pathway genes in yeast and mammals have been found to activate Sir2 (or the mammalian ortholog SIRT1), an NAD+-dependent deacetylase involved in transcriptional silencing and lifespan extension in yeast23,25,26 and survival of mammalian cells.27 Further studies will be required to determine the extent to which specific sirtuins, or other NAD+-dependent enzymes, regulate SMC function. However, the current data suggest that the capacity with which NAD+ can be regenerated (or nicotinamide cleared) is a key determinant of SMC behavior, including the acquisition of a quiescent phenotype.
The more mature attributes of PBEF-overexpressing SMCs could be functionally linked to their increased survival. That is, the apoptosis-resistant state imparted by PBEF could allow cultured SMCs to reassume more "physiological" characteristics, including a slender morphology and increased expression of SMC-associated proteins. Although such a relationship is likely, the more mature characteristics of PBEF-overexpressing SMCs were also observed under circumstances where there was no difference in apoptosis between control and PBEF-overexpressing cells. In serum-free media, both control and PBEF-overexpressing SMC clones displayed equally low apoptosis prevalence, yet there were substantially higher levels of smoothelin-A, smoothelin-B, caldesmon, and metavinculin in the PBEF-overexpressing SMCs. Furthermore, the increased investment of microvessels in vivo by PBEF-overexpressing SMCs compared with control SMCs occurred under conditions where SMC apoptosis was low and not different between control and PBEF-overexpressing SMCs. Interestingly, PBEF expression has been observed to be upregulated during differentiation/maturation of other cell types, including dendritic cells and B-lymphocytes.5,28
The relationship between PBEF and specialized SMC performance was supported in an in vivo context. In order for newly formed vascular networks to survive, they must be ensheathed by SMCs for support and stabilization.1,29 Investment by SMCs also enables the vessels to respond to vasoactive stimuli and thereby appropriately distribute blood. We capitalized on this in vivo, integrative response as a functional readout for SMC maturation. Human SMCs with reduced PBEF expression displayed a reduced ability to invest nascent microvessels. In contrast, SMCs overexpressing PBEF responded to the angiogenic environment by intimately wrapping around new microvessels and assuming an elongated morphology with a compact nucleus. It remains possible that the enhanced microvessel investment was attributable, in part, to increased directed migration of PBEF-overexpressing SMCs. Indeed, maturation of HITB5/C6 SMCs in culture was closely linked with directed SMC migration and, in vivo, SMC migration to endothelial channels may similarly be integrated with SMC maturation. The investment of microvessels with maturing SMCs is important in ischemic tissues, where neovessels are prone to regression. Furthermore, tumor vasculature inadequately invested by SMCs can result in leaky vessels that are predisposed to tumor shedding.30 The current findings thus suggest that manipulating NAD+ biosynthesis pathways may be relevant to these problems.
In summary, PBEF is not a cytokine but an intracellular protein that regulates NAD+ biosynthesis, NAD+-dependent protein deacetylation, vascular SMC survival, and the capacity of SMCs to mature. Augmentation of NAD+ regeneration, via PBEF, could have therapeutic potential for vascular conditions that depend on stress-resistant SMCs that can efficiently differentiate.
Acknowledgments
This work was supported by grants from the Heart and Stroke Foundation of Canada (T4458), the Heart and Stroke Foundation of Ontario (HSFO) (PRG 4854), the Canadian Institutes of Health Research (MT11715), and the Heart and Stroke Foundation of Ontario Career Investigator award (to J.G.P.).
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