Published online before print October 5, 2006, doi: 10.1161/01.RES.0000248184.21644.20
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© 2006 American Heart Association, Inc.
Molecular Medicine |
Cellular Mechanism Through Which Parathyroid Hormone–Related Protein Induces Proliferation in Arterial Smooth Muscle Cells
Definition of an Arterial Smooth Muscle PTHrP/p27kip1 Pathway
From the Division of Endocrinology (N.F.-T., B.M.S., K.U., T.B., K.K.T., I.C.-C., A.B., A.F.S.), University of Pittsburgh School of Medicine, Pa; and Department of Pharmacology and Experimental Therapeutics (B.L.), University of Florida, Gainesville.
Correspondence to Nathalie Fiaschi-Taesch, PhD, Instructor, Division of Endocrinology and Metabolism, BST E-1140, Endocrinology, University of Pittsburgh School of Medicine, 3550 Terrace St, Pittsburgh, PA 15213. E-mail Taeschn{at}msx.dept-med.pitt.edu
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Abstract |
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Parathyroid hormone–related protein (PTHrP) is present in vascular smooth muscle (VSM), is markedly upregulated in response to arterial injury, is essential for normal VSM proliferation, and also markedly accentuates neointima formation following rat carotid angioplasty. PTHrP contains a nuclear localization signal (NLS) through which it enters the nucleus and leads to marked increases in retinoblastoma protein (pRb) phosphorylation and cell cycle progression. Our goal was to define key cell cycle molecules upstream of pRb that mediate cell cycle acceleration induced by PTHrP. The cyclin D/cdk-4,-6 system and its upstream regulators, the inhibitory kinases (INKs), are not appreciably influenced by PTHrP. In striking contrast, cyclin E/cdk-2 kinase activity is markedly increased by PTHrP, and this is a result of a specific, marked, PTHrP-induced proteasomal degradation of p27kip1. Adenoviral restoration of p27kip1 fully reverses PTHrP-induced cell cycle progression, indicating that PTHrP mediates its cell cycle acceleration in VSM via p27kip1. In confirmation, adenoviral delivery of PTHrP to murine primary vascular smooth muscle cells (VSMCs) significantly decreases p27kip1 expression and accelerates cell cycle progression. p27kip1 is well known to be a central cell cycle regulatory molecule involved in both normal and pathological VSM proliferation and is a target of widely used drug-eluting stents. The current observations define a novel "PTHrP/p27kip1 pathway" in the arterial wall and suggest that this pathway is important in normal arterial biology and a potential target for therapeutic manipulation of the arterial response to injury.
Key Words: cell cycle progression • p27Kip1/pRb • proliferation • PTHrP • vascular smooth muscle cell proliferation
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Introduction |
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Parathyroid hormone–related protein (PTHrP) was originally identified through the search for the humoral factor responsible for humoral hypercalcemia of malignancy.1–4 PTHrP is now known to be ubiquitously expressed by normal tissues.3,4 At the protein level, it is translated as a prohormone, giving rise, through posttranslational endoproteolytic protein processing, to a family of daughter peptides that are secreted via the regulated and constitutive secretory pathways, in a cell type–specific manner3–5 (Figure 1). These daughter peptides act in traditional endocrine, paracrine, and autocrine fashions to mediate the diverse physiological actions of PTHrP. In this model, PTHrP secretion results from trafficking of the precursor to the endoplasmic reticulum directed by a typical signal peptide.3–6
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In addition to this typical neuroendocrine trafficking and secretion, PTHrP is unusual in that it is also able to traffic to the nucleus of multiple cells7–14 (Figure 1). This "bidirectional trafficking" results from the presence in the PTHrP sequence of alternate translational initiation sites, 1 of which uses an AUG translational initiation codon directly upstream of the signal peptide, as well as a second functional CUG translational initiation codon, which is internal to, and therefore disrupts, the signal peptide.7–10 Thus, if the AUG translational start site is used, PTHrP enters the endoplasmic reticulum and the Golgi apparatus and is secreted. In contrast, if the downstream CUG is used, the signal peptide function is lost and the newly translated peptide remains in the cytoplasm. This event, in conjunction with the presence of a typical, and functionally well-defined, nuclear localization signal (NLS) within the PTHrP sequence (Figure 1), results in the trafficking of PTHrP to the nuclear compartment.7–14 The NLS function has been documented using multiple approaches, including its ability to direct non-native proteins into the nucleus of living cells and its ablation or deletion completely preventing nuclear access by PTHrP.7–14
As noted above, PTHrP is ubiquitously expressed and has been implicated in multiple physiological processes, including the regulation of transepithelial ion flux; multiple developmental processes; and cellular differentiation, proliferation, and survival.3,4 For example, in the growing skeleton, PTHrP has been shown to be critical for the proliferation and survival of growth plate chondrocytes. In particular, the ability of PTHrP to enter the chondrocyte nucleus appears to be required for epiphyseal chondrocyte survival.12
Importantly, whereas PTHrP is present at low levels in the arterial wall in the basal state, it is markedly, rapidly, and very reproducibly increased in response to angioplasty and other types of arterial injury.3,4,13–23 In the case of the vascular smooth muscle cell (VSMC), we have shown that this alternate ability to act both as a secretory protein as well as an "intracrine," nuclear targeted peptide, results in directionally opposite effects: targeting of PTHrP to the nucleus of arterial smooth muscle cells leads to accelerated VSMC proliferation, whereas targeting it to the secretory pathway leads to inhibition of arterial VSMC proliferation.8,13,14,21 We have demonstrated these effects not only in vitro in cell lines13,14,21 but also in vivo in the aorta of PTHrP-null mouse embryos,13 where PTHrP deficiency leads to a diminution of VSMC proliferation, and also in the adult rat carotid, where overexpression of PTHrP exacerbates neointima formation following balloon angioplasty.21 Moreover, we have recently demonstrated that overexpression of PTHrP in VSMC cell lines results in phosphorylation of the retinoblastoma protein pRb.21
Thus, it is very clear that PTHrP targeted to the nucleus drives cell cycle progression, and this appears to involve pRb and the G1/S transition. To date, however, the cellular mechanisms responsible for the checkpoint release by nuclear PTHrP remain undefined. In the current study, we report that a particular cyclin-inhibitory protein (CIP) or kinase inhibitory protein (KIP), p27kip1, is selectively, specifically, and markedly downregulated by PTHrP overexpression in VSMCs and that loss of p27kip1 protein entirely accounts for the cell cycle acceleration that results from PTHrP overexpression.
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Materials and Methods |
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Cell Culture and PTHrP-Expressing Cell Lines
The VSM cell line A-10 was purchased from American Type Culture Collection (Rockville, Md) and cultured as described previously13,14,21 and in the online data supplement, available at http://circres.ahajournals.org.
Cell Cycle Analysis
Exponentially growing cells were harvested, trypsinized, washed with PBS, and incubated in 70% ethanol at 4°C until used for flow cytometry. Cell cycle distribution was analyzed by flow cytometry, as described previously.21
Northern analysis and real-time PCR for p27kip1 mRNA were performed using standard methods, as detailed in the online data supplement.
Immunoblot analyses of cell cycle proteins were performed using standard methods as detailed in the online data supplement and as described previously.21 The primary antibodies used are shown in Table I in the online data supplement.
Coimmunoprecipitation, immunoblots, and cdk kinase assays were performed using standard methods and reagents, as detailed in the online data supplement.
p27kip1, GFP, Wild-Type PTHrP, and Signal Peptide–Deleted PTHrP Adenoviruses
Adenoviruses encoding green fluorescent protein (GFP) (Ad-GFP), p27kip1 (Ad-p27) containing a histidine epitope tag (His6), and PTHrP (wild-type [Ad-PTHrP] or signal peptide deleted [Ad-
SP]) containing an HA epitope tag (HA) were prepared as described previously.14,21,24 Multiplicity of infection was determined using an optical density at 260 nm (OD260) and plaque assay.
Statistics
Statistical analysis was performed using Student’s t test. All values are expressed as means±SEM. Significance was taken as P<0.05.
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Results |
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A Survey of the G1/S Proteome in PTHrP-Overexpressing A-10 VSMCs
Previously, we have demonstrated that the ability of PTHrP to drive cell cycle progression requires 2 regions of PTHrP (the NLS in the 87 to 106 region and the carboxy-terminal [107 to 139] region) and that this activation of cell cycle progression is associated with nuclear appearance of PTHrP8,13,14,21 (Figure 1). We have also demonstrated that overexpression of PTHrP in A-10 cells leads to phosphorylation of the retinoblastoma protein, pRb, to its inactive, phosphorylated form, ppRb21 (Figure 2A). As can be seen in Figure 2B, and as reported previously,21 pRb is present in A-10 cells and overexpression of PTHrP leads to pRb phosphorylation. Figure 2B also demonstrates that the 2 other "pocket protein" family members, p107 and p130, are present in A-10 cells and are upregulated by PTHrP. As shown in the right panels of Figure 2B, the majority of the cyclin D, cdk-4/6 family, and the cyclin E/cyclin A/cdk-2 family members are unaffected by PTHrP overexpression. The sole exceptions to this are cyclin D2 and D3, both of which reproducibly decline.
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Given the apparent stability of G1/S regulatory molecules (Figure 2B), we elected to examine the kinase activity of cyclin D/cdk-4/6 complexes and the cyclin E/cdk-2 complex. As shown in Figure 2D, kinase activity of the cyclin D/cdk-4 complex was not significantly altered by PTHrP overexpression. In contrast, cdk-2 kinase activity was markedly (approximately 3-fold) higher in PTHrP-overexpressing A-10 cells, as compared with controls (Figure 2C). This suggests that PTHrP overexpression controls the G1/S transition via the cyclin E/cdk-2 complex.
p27kip1 Is Specifically Downregulated by PTHrP Overexpression and Leads to Cell Cycle Progression
As shown in Figure 2E, no change in the level of expression of the 4 inhibitory kinases, the "INK" family members, was observed. The cyclin inhibitory proteins, p21cip1 and p57kip2, were also unchanged (Figure 3A). In contrast, p27kip1 was reproducibly reduced by approximately 90% in PTHrP-overexpressing cells (Figure 3A and 3B). This was confirmed using immunohistochemistry: p27kip1 was markedly reduced in both the cytoplasmic and nuclear compartments of A-10 cells overexpressing PTHrP (Figure 3C).
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To determine whether the loss of p27Kip1 in PTHrP-overexpressing cells was related in a causal fashion to the cell cycle progression, we restored p27Kip1 to normal in PTHrP-overexpressing A-10 cells using a p27Kip1 adenovirus (Ad-p27Kip1), using a GFP-adenovirus (Ad-GFP) as a control. Figure 4A and 4B demonstrate that 100 multiplicities of infection (mois) of Ad-p27Kip1 was able to restore p27Kip1 to normal in VSMCs overexpressing PTHrP. Figure 4C is used to calibrate Figure 4D and shows that overexpression of PTHrP in A-10 VSMCs leads to a marked increase in S (28.1±3.4% in PTHrP versus 17.9±1.3% in A-10) and in G2M (16.6±2.2% in PTHrP versus 6.4±0.7% in A-10) phases of the cell cycle and a reduction in G0G1 (55.7±4.1% in PTHrP versus 75.8±1.7% in A-10). Restoring p27Kip1 to normal resulted in complete normalization of the cell cycle, as assessed by fluorescence-activated cell sorting (FACS) analysis (Figure 4C and 4D). As a control, 100 mois of Ad-GFP did not alter the cell cycle in VSMCs overexpressing PTHrP, suggesting that the adenovirus itself has no adverse effect on the cell cycle. These observations clearly demonstrate that it is the lack of p27KIP1 in PTHrP-overexpressing that causes or permits cell cycle acceleration.
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PTHrP Overexpression Leads to Destabilization and Proteasomal Degradation of p27kip1
Figure 5 demonstrates that there is either no (Figure 5A) or a small (Figure 5B) decline in steady-state mRNA levels for p27kip1 in PTHrP-overexpressing A-10 cells, as compared with control cells, suggesting that overexpression of PTHrP does not regulate p27kip1 in an important way at the mRNA level. Figure 6 shows that preventing new protein synthesis using the translation inhibitor cycloheximide results in a more rapid disappearance of p27kip1 in PTHrP-overexpressing cells than in control cells. Thus, PTHrP overexpression leads to p27kip1 instability.
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One pathway to p27kip1 degradation involves phosphorylation of p27kip1 on Thr187 by cdk-2. As shown in Figure 7A, bottom, coimmunoprecipitation confirmed the marked loss of p27kip1 in PTHrP-overexpressing cells. Yet, even in these p27kip1-depleted cells, phospho-Thr187 was easily observed (Figure 7A, top). When phospho-Thr187 is corrected for total p27kip1 (Figure 7B), one can readily appreciate that marked p27kip1 phosphorylation, presumably by cdk-2, occurs in these cells.
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To test whether p27 was degraded by the proteasome, we used the proteasomal inhibitor MG132. As shown in Figure 7C and 7D, this proteasome inhibitor led to a marked increase in the abundance of p27kip1 in PTHrP-expressing A-10 cells, indicating that PTHrP overexpression targets p27kip1 to proteasomal degradation.
Nuclear PTHrP Overexpression Stimulates Proliferation and p27 Downregulation in Murine Primary VSMCs
To determine whether the preceding observations might be an artifact of clonal selection in A-10 cells, or whether it was a more general phenomenon in primary VSMCs, we isolated primary VSMCs from mouse aortas. These VSMCs were characterized for smooth muscle actin expression (Figure 8
A): essentially all of the cells in cultures were VSMCs. As shown in Figure 8B, progressively increasing the moi of Ad-PTHrP progressively increased the expression of PTHrP-HA. At 1000 mois, almost all VSMCs were effectively transduced, and expressed PTHrP-HA (Figure 8B). We therefore determined the expression of p27 in primary VSMCs infected with 1000 mois of control adenovirus GFP (Ad-GFP) or with 1000 mois of Ad-PTHrP. Figure 8C shows that overexpressing PTHrP in murine primary VSMCs leads to a gradual loss of p27 (Figure 8C and 8D). In addition, overexpression of PTHrP in murine primary VSMCs resulted in a stimulation of proliferation as assessed by FACS analysis (Figure 8E). In contrast, 1000 mois of Ad-GFP did not alter cell cycle in primary murine VSMCs.
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Finally, we wanted to confirm, in mouse primary VSMC cultures, that the stimulation of VSMC proliferation and p27 loss was inducible by a PTHrP construct that targeted the nucleus but could not be secreted. To address this question, we overexpressed a mutant form of PTHrP deleted for the signal peptide (SP-PTHrP) (Figure 1). We have shown previously in A-10 cells that this mutant form of PTHrP is exclusively expressed in nuclei when stably overexpressed in A-10 VSMCs,14 cannot be secreted,13,14 and leads a dramatic stimulation of VSMC proliferation.14 We delivered SP-PTHrP adenovirally (Ad-SP) to murine primary VSMCs. As shown in Figure 8
F, increasing moi of the Ad-SP leads to an increased expression of nuclear PTHrP. In parallel, overexpressing this nuclear isoform of PTHrP decreased p27 expression (Figure 8F) and also dramatically stimulated primary VSMC proliferation (Figure 8G). These results collectively suggest that PTHrP overexpression stimulates proliferation via p27 loss not only in A-10 VSMCs but also in primary VSMCs in general. In addition, these events are associated with specific nuclear targeting, but not secretion, of PTHrP.
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Discussion |
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PTHrP has repeatedly been shown to be involved in the arterial response to injury in rats and humans and, specifically, in the development of the neointima following angioplasty.3,4,14–24 However, the precise molecular role played by PTHrP in the arterial wall in response to arterial injury or angioplasty has not been defined. Here we demonstrate that PTHrP is an upstream regulator of p27kip1 and that this regulation is accomplished by phosphorylation and proteasomal degradation of p27kip1. Because PTHrP is known to be an important regulator of arterial smooth muscle during developmental physiology,13 as well as under pathophysiological circumstances,18,21 and because p27kip1 is a central regulator of arterial smooth muscle proliferation in response to injury,25–27 these findings demonstrate that a "PTHrP/p27kip1 pathway" is activated in arterial smooth muscle in response to injury and suggests that this pathway may be central to the arterial VSMC proliferative response to balloon injury.
Ozeki et al have shown that PTHrP is markedly and rapidly upregulated in the media and neointima of the rat carotid artery following balloon angioplasty, both at the protein and mRNA level.18 Other investigators have demonstrated that PTHrP is upregulated in many arterial beds and vessel types, from very large to terminal arterioles, in response to mechanical stretch, endothelial injury, and vasoconstrictors such as angiotensin II and norepinephrine.3,4,15–22 Nakayama et al19 and Ishikawa et al20 have shown that PTHrP is upregulated in atherosclerotic human coronary arteries. Moreover, PTHrP/smooth muscle interactions are not limited to arterial smooth muscle, for mechanical distension of bladder, uterine, gastric, gastrointestinal, and tracheal smooth muscle also lead to quantitatively striking and temporally rapid upregulation of both PTHrP mRNA and protein.3,4 Despite the documentation of this phenomenon for almost 2 decades, the cellular mechanisms responsible for, or linking, mechanical smooth muscle stretch to upregulation of PTHrP expression remain speculative.
We have demonstrated that PTHrP is required for arterial smooth muscle proliferation, because smooth muscle cell proliferation is reduced in the aorta of PTHrP-null mouse embryos as compared with that of wild-type embryos.13 Conversely, forced overexpression of PTHrP in the arterial wall following angioplasty worsens neointimal hyperplasia.21 Thus, PTHrP is linked in the arterial wall to both physiological as well as pathological smooth muscle proliferation. This cell cycle acceleration requires that PTHrP have an intact nuclear localization sequence or signal (NLS), because NLS mutant forms of PTHrP fail to stimulate VSMC proliferation in vitro13,14,21 or to neointimal hyperplasia in vivo.13,21 In addition to the NLS, we have shown that proliferation in VSMCs requires the carboxy-terminal (107 to 139) region (Figure 1) and that key serines and threonines in this sequence are also necessary.14,21 These observations suggest that these residues must be phosphorylated or otherwise modified in order for PTHrP to drive proliferation. Thus, a picture emerges in which PTHrP, or at least the NLS together with the carboxy-terminal region of PTHrP, is translocated to the nucleus of the vascular smooth muscle cell. The nuclear presence of PTHrP triggers pRb phosphorylation, release of G1/S arrest, and thereby cell cycle progression. The goal of the current studies was to determine exactly how, at a molecular level, overexpression of PTHrP leads to cell cycle progression.
We surveyed at the protein level the major components of the G1/S transition in control and PTHrP-overexpressing A-10 cells and found a surprisingly stable array of cell cycle regulatory molecules. A few exceptions were observed: some molecules such as p107 and p130 increased, whereas others such as cyclins D2 and D3 declined. We have not explored the significance of these observations, choosing instead to focus on upstream targets more directly affected by PTHrP, because we believe the most interesting events in the nuclear PTHrP story are likely upstream of p27. We speculate, however, that the increases in p107 and p130, and the declines in cyclins D2 and D3, may reflect a response to the increased rate of proliferation imposed by PTHrP overexpression.
The most dramatic observation of this G1/S cell cycle proteome survey was a specific and dramatic decline in p27kip1. This loss of p27kip1 was shown to be causally related to the acceleration in proliferation, because restoration of p27kip1 to normal levels using a p27kip1 adenovirus led to an apparent normalization or reversal of the PTHrP-driven proliferation.
Although there was a mild to moderate decline in steady-state p27kip1 mRNA in response to PTHrP overexpression, possibly reflecting a decline in p27kip1 gene transcription, the major contributor to the decline in p27kip1 appeared to be accelerated proteasomal degradation of p27kip1. Thus, we show that p27kip1 is phosphorylated on Thr187, that its half-life is markedly reduced, and that its loss can be reversed by MG132, which inhibits the 28S proteasome. We speculate that p27kip1 may be induced to form complexes with proteasome-targeting machinery such as Jab-128 or the E1–3 ligase complex (including cullin 1, skp-1, skp-229–33), which has been shown to guide p27kip1 to proteasomal degradation in other systems. Preliminary data suggest that cullin-1 and skp2 are downstream targets of PTHrP.34
p27kip1 is known to be essential for cell cycle regulation and down-modulation in arterial smooth muscle cells.25–27 In addition, evidence would support the concept that upregulation of p27kip1, at least in part, mediates the favorable clinical response to rapamycin-coated stents in human arterial disease.33,35–37 Perhaps most elegantly, Nabel and colleagues have shown that angioplasty in p27kip1-null mice results in a dramatic and severe increase in neointimal proliferation that results in a rapid and complete obliteration of the arterial vascular lumen following arterial injury.25 Replacement of p27kip1 led to a reduction in the neointimal response and the rate of VSMC proliferation, indicating that p27kip1 is both necessary and sufficient to prevent severe neointimal hyperplasia in response to injury.25 The current study supports and extends these studies by suggesting that it is the increase in PTHrP in response to arterial injury, acting via a reciprocal decline in p27kip1, that accounts, perhaps wholly or at least in part, for the neointimal hyperplasia that occurs following arterial injury. Clearly, many factors such as angiotensin II, interleukin (IL)-6, platelet-derived growth factor (PDGF), and others also may participate the decline in p27kip1.38,39 Further studies will be required to clarify how PTHrP fits into this hierarchy.
Thus, this study permits the development of a model (Figure 8J) in which the nuclear arrival of PTHrP triggers the destabilization of p27kip1 through as yet incompletely defined mechanisms. This results in a release from inhibition of the cdk-2/cyclin E complex and phosphorylation/inactivation of pRb, with release of the G1/S checkpoint (Figure 4C). The key mechanistic questions in the model at this point relate to exactly how p27kip1 is destabilized, whether via ubiquitination and proteasomal destruction, mediated for example by the Skp-2 or Jab-1 pathways, and how the arrival of PTHrP in the nucleus activates this sequence of events.
This study raises a number of additional questions that will require further study. First, these studies, like those of Nabel and colleagues,25 focus on rodent cells and models. Thus, although we have demonstrated the effect of PTHrP in primary mouse VSMCs and the rat A-10 VSMC cell line, it remains to be defined whether the events we describe occur in human arterial smooth muscle cells. It is important to emphasize here, however, that PTHrP is upregulated in injured or diseased human coronary arterial smooth muscle.3,4,19,20 Second, although these studies unequivocally demonstrate biochemical linkage between PTHrP and p27kip1, they do not define the relative importance of PTHrP among the several growth factors and cytokines involved in the response to arterial injury, particularly with reference to p27kip1. It is possible, indeed likely, that PTHrP will prove to be among several growth factors and cytokines that govern p27kip1 expression in arterial smooth muscle. A third question relates to exactly how the presence of PTHrP within the nucleus might lead to destabilization of p27kip1. We and others have reviewed the potential mechanisms through which the entry of PTHrP into the nuclear compartment might elicit biological responses.7,8,15 There is some evidence from 2-hybrid experiments that PTHrP participates in protein/protein interactions,40 although exactly what the nuclear partners for PTHrP might be remains unknown. It is also formally possible that PTHrP might bind directly to DNA or RNA, serving to regulate RNA processing or gene expression.41 In this latter scenario, it is possible that nuclear PTHrP might directly upregulate the transcription of other genes, such as c-Myc, cullin, or Skp-2, that lead directly or indirectly to the degradation of p27kip1. Finally, these studies raise a "chicken and egg" question. Is it a cdk-2 activation that leads to p27kip1 phosphorylation and degradation, or is it an initial p27kip1 degradation that leads to cdk-2 activation? Again, clarifying these issues will require further study.
Finally, MacLean et al42 have shown that PTHrP can also influence proliferation in the epiphyseal chondrocyte via its action on p57kip2. In our studies in VSMCs, rather than influencing p57kip2, PTHrP affected p27kip1, suggesting that the intracellular cell cycle regulatory targets are likely cell type–specific.
In summary, we have demonstrated that upregulation of PTHrP in 2 arterial smooth muscle models leads to a rapid, dramatic, and selective reduction in ambient p27kip1 levels. We demonstrate that PTHrP overexpression causes this reduction in p27kip1 by targeting it for proteasomal degradation. We demonstrate that this upregulation of PTHrP, which has been repeatedly observed in models of arterial and other smooth muscle injury, via its reciprocal decline in p27kip1, results in pRb phosphorylation and enhanced arterial smooth muscle proliferation. Furthermore, we demonstrate that restoration of p27kip1 to normal levels reverses the proliferative effects of PTHrP overexpression on arterial smooth muscle. This reciprocal functional association between PTHrP and p27kip1 defines a novel PTHrP/p27kip1 pathway involved in the regulation of arterial smooth muscle response to injury.
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Acknowledgments |
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We thank Darinka Sipula for expert technical assistance; Beth Kaplan, MD, for help with the p27kip1 immunoblots; Adolfo Garcia-Ocaña, PhD, and Rupangi Vasavada, PhD, for invaluable discussion of this work; and Jeffrey Bender, MD, for important scientific input.
Sources of Funding
Supported in part by NIH/National Cancer Institute grant R01-CA93651 (to B.L.).
Disclosures
N.F.-T. and A.F.S. have a financial interest in Vasculostatin LLC. The other authors report no conflicts of interest.
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Footnotes |
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Original received May 9, 2006; revision received September 21, 2006; accepted September 21, 2006.
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References |
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