Battling Imatinib Resistance in CML
Shah NP, Tran C, Lee FY, Chen P, Norris D, Sawyers CL. Overriding Imatinib resistance with a novel ABL kinase inhibitor. Science 2004; 305:399-401.
By Nancy Andrews, M.D., Ph.D., and Michael Williams, M.D.
Clinical trials have established imatinib mesylate (Gleevec) as standard front-line therapy for chronic myelogenous leukemia (CML) in chronic phase. Although the clinical and hematologic response is dramatic in most patients, resistance to therapy may be present at diagnosis or may develop during therapy, usually mediated by a variety of point mutations in the drug's BCR-ABL binding site. Shah and colleagues tested a novel, orally bioavailable second-generation BCR-ABL tyrosine kinase inhibitor, BMS-354825, for its ability to inhibit mutant isoforms resistant to imatinib. Fourteen of 15 BCR-ABL isoforms were inhibited in vitro at low nanomolar concentrations. Regression of established disease and improved survival were also demonstrated in a murine model for all but the single resistant T315I mutation. The drug did not inhibit in vitro growth of normal human marrow progenitors, but did inhibit growth of neoplastic marrow progenitors from imatinib-sensitive and -resistant CML patients.
Molecularly targeted therapies combining enhanced anti-tumor efficacy with decreased toxicity to normal tissues are being pursued for many cancer types. The seminal studies reported by Dr. Brian Druker and colleagues established the therapeutic targeting paradigm of imatinib for CML, demonstrating remarkable clinical responses in most patients to a single daily dose, oral regimen. Identifying the mechanisms of resistance mediated via mutations in the enzymatically active BCR-ABL binding site and understanding the resulting conformational changes in the protein permit a rational approach to the design and selection of novel therapeutic agents. The preclinical studies reported by Shah and colleagues in the UCLA group led by Dr. Charles Sawyers hold promise for a second generation of highly effective targeted therapies, now being tested in a phase I clinical trial of BMS-354825 for patients with imatinib-resistant CML. If efficacy and safety are established for the new agent, possibilities are opened for combination tyrosine kinase inhibitor therapy that may improve rates of molecular remission and decrease the emergence of resistant CML clones. Furthermore, the insights as to conformational and functional relationships between active and inactive BCR-ABL isoforms and inhibitory small molecules may facilitate the development of analogous therapies with application in breast, lung, and other cancers with mutated tyrosine kinases, such as SRC or the epidermal growth factor receptor (EGFR).
Could BMS-354825, or another kinase inhibitor that is less selective than imatinib, be used with imatinib for first-line, multidrug therapy of CML? Assuming that it proves safe and effective in human patients, that seems like a reasonable approach. However, it will not solve all problems. About 15-20 percent of imatinib-resistant CML patients have the stubborn T315I mutation, which appears to block drug binding of both imatinib and BMS-354825. And, less commonly, resistance arises through genomic amplification of BCR-ABL; it is not yet clear whether that type of resistance would yield to the new kinase inhibitor. Nonetheless, this appears to be a significant step forward in curing a highly malignant disease.
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Recycling Vitamin K: An Old Story Gets a New Look
Rost S, Fregin A, Ivaskevicius V, Conzelmann E, HF6rtnagel K, Pelz HJ, Lappegard K, Seifried E, Scharrer I, Tuddenham EGD, MFCller CR, Strom TM, Oldenburg J. Mutations in VKORC1 cause warfarin resistance and multiple coagulation deficiency type 2. Nature 2004 Feb; 427: 537 - 541.
Li T, Chang CY, Jin DY, Lin PJ, Khvorova A, Stafford DW. Identification of the gene for vitamin K epoxide reductase. Nature 2004 Feb; 427: 541 - 544.
By Lilli Petruzzelli, M.D., Ph.D.
Unraveling the molecular identity of the target of warfarin has remained elusive despite its widespread clinical use as the mainstay of oral anticoagulation therapy over the last fifty years. These two papers not only chisel away at this issue but also present the melding of keen clinical observation amassed over many years with molecular approaches. Vitamin K recycles into its active, reduced form through the activity of vitamin K epoxide reductase (VKOR); warfarin targets this step and inhibits the regeneration of reduced vitamin K. In the manuscript by Li et al., the investigators utilized the clinical observation from rats, humans, and mice with warfarin resistance that the genetic regions in each species were homologous to that in humans with combined vitamin K-dependent protein deficiency that mapped to Chromosome 16. After eliminating proteins with known function, they refined their search to 13 genes because biochemical evidence suggested that the protein was transmembrane. Utilizing a cell line that expresses high amounts of VKOR activity, they disrupted expression of each of the 13 candidate genes with siRNA and were able to narrow their search to one gene encoding a protein with a molecular mass of 18,200. Expression of this gene conferred VKOR activity on an insect cell line that does not normally express it, and the activity was inhibited by warfarin in vitro. The work by Rost et al. converged on the same protein (VKORC1) as that identified by Li et al. by using a positional cloning approach in patients that were identified on a genetic basis to have reduced vitamin K-dependent clotting factors or warfarin resistance. Their characterization revealed that the protein is widely expressed in a number of tissues, including liver. Mutations in the gene encoding VKORC1 were confirmed in patients deficient in vitamin K-dependent clotting factors and with warfarin resistance. The investigators found the same point mutation in the unrelated families with combined deficiency of vitamin K-dependent clotting factors 2, and four distinct mutations in those with warfarin resistance. Of interest was that expression of the protein with mutations corresponding to those found in warfarin resistance in all but one case yielded reduced levels of VKOR activity that was sensitive to warfarin. Surprisingly, only the mutation observed in the rat that is resistant to warfarin demonstrated marked resistance to warfarin in vitro.
Both groups of investigators have elegantly honed in on the molecular identification of VKOR. The work by Li et al. supports a model where this protein alone is responsible for the VKOR activity. The expression studies and analysis of VKOR activity presented by Rost et al. suggest that understanding how warfarin resistance is orchestrated in vivo may be more complex than just a single protein. Proteins expressing warfarin resistant mutations exhibited less activity than wild type and all of the mutants exhibited some degree of sensitivity to warfarin. Thus, it may be that in vivo there are other factors that play a role in modulating this process. Nonetheless, this first step certainly sets the groundwork to identify alternative drugs, other enzymes or proteins involved in this complex event, and alternative means of assessing clinical efficacy.
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Salvation from Shortage of Platelets for Transfusion and Their Improved Safety May Be Near
Hoffmeister KM, Josefsson EC, Isaac NA, Clausen H, Hartwig JH, Stossel TP. Glycosylation Restores Survival of Chilled Blood Platelets. Science 2003; 301: 1531-1534.
By Josef Prchal, M.D.
Platelet transfusion practice has been hampered by our inability to extend their shelf-life by cold storage. In this paper, the authors showed that the enzymatic insertion of a galactose residue onto the platelet von Willebrand receptor (GPIb) precludes the recognition of the clustered GPIb by the Mac-1 receptor and that this manipulation leads to the normal survival and normal function of cold stored platelets in mice. Fortuitously, the enzyme that can accomplish this is present in platelets and plasma. The galactose containing substrate (UDP-Gal) used by the enzyme is also normally present in blood, but only in small concentrations.
The discovery of techniques to store and transfuse red blood cells has revolutionized the way we practice medicine today. However, red cell transfusion is not sufficient to save patients from hemorrhagic death. As recently as fifty years ago, the majority of patients with acute leukemia died of bleeding. Clearly, without platelet transfusions, modern potent myelosuppressive chemotherapy regimens and advances in cardiovascular and organ transplantation surgery would not be possible. Early attempts to store platelets led to the recognition that exposure to cold leads to a change in their shape and also to immediate removal of the platelets from the circulation after transfusion. This problem necessitates that platelets be stored at room temperature, reducing their shelf-life to a few days because of the rapid decline in their viability. Additionally, storage at 24°C markedly increases the risk of bacterial growth. The risk of an infected platelet infusion is estimated to be at least 50 times that of a red cell or plasma transfusion, and in some centers, each bag of concentrated or pheresed platelets is cultured for two days before their release, increasing the expense, and further decreasing the platelets' viability and function. The mechanism of the "cold" platelet injury has been recently clarified1: GPIb is clustered by cold and these GPIb clusters are recognized by liver Kupffer cells via so-called Mac-1 receptors and rapidly removed from the circulation.
The work reviewed here has opened a possible avenue for storing blood platelets in the cold, which could decrease the infectious risk and markedly improve the availability of platelets for transfusion. Thus UDP-Gal could potentially be added to cold-stored platelets prior to their transfusion, protecting them from clearance by the liver. Although more work needs to be done2 (e.g., is UDP-Gal the best substrate, should it be added at the time of platelet collection, during, or after storage?), this discovery has the potential to radically change the availability and safety of platelet transfusions.
1. Andrews RK. Berndt MC:
Platelet physiology: in cold
blood. Curr Biol 2003;13:282-
284.
2. Couzin J. Sugary Cloak Protects
Platelets From the Cold.
Science 2003; 301:1457.
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Antineoplastic Therapy for Atherosclerosis?
Lassila M, Allen TJ, Cao Z, et al. Imatinib attenuates diabetes-associated atherosclerosis. Arterioscler Throm Vasc Biol 2004; 24: 953-942.
By Andrew Schafer, M.D.
Imatinib (Gleevec) is a selective inhibitor of specific protein tyrosine kinases, including the ABL kinase, the transmembrane receptor KIT, and the platelet-derived growth factor (PDGF) receptor. The drug has been demonstrated to have striking clinical efficacy in malignancies that utilize these tyrosine kinase pathways: (a) in chronic myeloid leukemia involving the chimeric BCR-ABL fusion oncoprotein; (b) in gastrointestinal stromal tumors involving KIT; and (c) in clonal eosinophilic disorders involving the PDGF receptor. But PDGF signaling also plays an important role in the pathogenesis of atherosclerosis. In this interesting paper, Lassila et al. demonstrate that imatinib treatment prevents the development of atherosclerosis. The authors used transgenic hyperlipidemic mice (apolipoprotein E knockout mice) that were also rendered diabetic by streptozotocin treatment. These mice developed advanced, spontaneous atherosclerotic plaques throughout the aorta. Atherosclerotic lesion formation was associated with increased aortic PDGF expression and PDGF receptor phosphorylation. When these mice were also treated with imatinib for 20 weeks, atherosclerosis was significantly attenuated throughout the entire aorta, and PDGF expression and PDGF receptor phosphorylation were blocked. The authors concluded that imatinib appears to be a novel therapeutic option to retard the development of atherosclerosis.
In addition to reducing the risk factors associated with atherosclerotic cardiovascular disease, will we enter a new era of targeted molecular therapy that has direct effects on blood vessels? Will there be a paradigm shift in the treatment of cardiovascular disease that follows the lead set in recent years for the treatment of malignancies? Indeed, there are striking similarities between the pathophysiology of cancer and that of atherosclerosis. They have such common molecular pathways of disease development and progression that atherosclerosis has been referred to as a "cancer of the blood vessels."1 Studies have suggested that atherosclerosis and cancer similarly develop from a clonal proliferation of altered cells at sites of tissue injury, inflammation, and genomic instability. They have in common many etiologic agents, predisposing genetic defects, and molecular pathways. This paper describing the protective role of imatinib in atherosclerosis demonstrates the central role of PDGF-dependent tyrosine kinase activity in atherosclerosis, as it also has in certain malignancies, further advancing the pathophysiological parallels between the two leading causes of death in the United States.
Intriguing as these developments are, it is important to note that there are also critical differences between the clinical expression of cancer and atherosclerosis that argue against a completely unified disease hypothesis.1 These include the apparent lack of impact on cancer incidence for some of the well-known risk factors for atherosclerotic cardiovascular disease and the apparently contradictory roles of angiogenesis and some specific signaling pathways (e.g. TGF- DF ) in disease progression. For the study under review, it will have to be determined if imatinib has clinical efficacy in human atherosclerotic cardiovascular disease; if it exerts antiatherogenic actions in coronary arteries as it does in the aorta; and, if clinical trials do demonstrate efficacy, how to deal with the problem of imatinib resistance in the cardiovascular setting. Nevertheless, clarifying and exploiting the molecular similarities between atherosclerosis and cancer may lead to the emergence of targeted molecular therapy as a new therapeutic option in cardiovascular disease.
1. Ross JS, Stagliano NE, Donovan MJ, et al. Atherosclerosis and cancer: common molecular pathways of disease development and progression. Ann NY Acad Sci 2001; 947:271-292.
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Angiogenesis with a Twist
Streubel B, Chott A, Huber D, Exner M, Jager U, Wagner O, Schwarzinger I. Lymphoma-specific genetic aberrations in microvascular endothelial cells in B-cell lymphomas. N Engl J Med. 2004 Jul 15; 351(3):250-9.
By Peter Lee, M.D.
Angiogenesis - the formation of new blood vessels - is now widely recognized as an important event in cancer progression. The significance of this is illustrated by recent FDA approval of the first anti-cancer drug which specifically targets angiogenesis, Avastin, which treats metastatic carcinoma of the colon or rectum. Angiogenesis is not limited to solid tumors; evidence suggests that angiogenesis also plays a role in hematologic malignancies, including myeloma and lymphoma. This article adds an important twist to this story. The authors asked an intriguing question - whether genetic abnormalities may be found not only within tumor cells, but also in endothelial cells within tumors. Using a combined immunohistochemical and fluorescence in situ hybridization assay (Fluorescence immunophenotyping and interphase cytogenetics, or 'FICTION'), they examined endothelial cells within 27 B cell lymphomas for cytogenetic alterations known to be present in the lymphoma cells. Endothelial cells were identified and distinguished from lymphoma cells by staining for two to four endothelial cell markers: CD31, CD34, von Willebrand factor, and Ulex europaeus lectin. Intriguingly, they found that 15-85 percent of endothelial cells within these lymphoma lesions harbored the same chromosomal translocations as the lymphoma cells. These data strongly suggest that the endothelial cells within these lymphomas are in part tumor-related. The authors proposed four possible explanations for these findings. First, that lymphoma and endothelial cells may derive from a common progenitor. This fits well with the notion of “cancer stem cells,” which has been generating considerable momentum in recent years. Second, that lymphoma cells may give rise to endothelial cells through plasticity. Immature B cells with Pax-5 gene deletion have been shown to differentiate into all known hematopoietic lineages (except mature B cells) in vitro and in vivo. Third, that there may be lymphoma-endothelial cell fusions. The authors pointed out that if this were the case, endothelial cells with chromosomal abnormalities would be tetraploid, which was not observed. Finally, the authors proposed that uptake of apoptotic lymphoma cells by endothelial cells may lead to gene transfer. However, this would not explain the loss of chromosomal material in endothelial cells, which was observed in two cases.
While novel, these findings build upon several prior observations. Melanoma cells have been shown to mimic endothelial cells in forming fluid-conducting, vascular-like networks. Stromal cells in breast cancer have been shown to carry genetic alterations associated with carcinogenesis. Collectively, these intriguing clinicopathological observations hold implications and raise additional questions. From a scientific perspective, the mechanisms by which support cells such as endothelial or stromal cells are recruited in cancer now warrant a fresh look. The observation that a fraction (15-85 percent) but not all endothelial cells harbored genetic abnormalities also raises questions regarding the interplay between “cancerous” and “non-cancerous” endothelial cells in forming new vascular structures within tumors. From a therapeutic perspective, these data suggest that inhibition of “normal” angiogenesis may not be sufficient; strategies may be needed to specifically target “cancerous” endothelial cells or block their formation. Together, these findings illustrate that cancer is more complex and cunning that we ever imagined - what other cancer cells in disguise could be lurking?
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Gene Expression Profiling May Enlighten Prognostication and Treatment for AML
Bullinger L, Dohner K, Bair E, et al. Use of Gene-Expression profiling to identify prognostic subclasses in adult acute myeloid leukemia. N Engl J Med 2004;350:1605-1616.
Valk PJM, Verhaak RGW, Beijen MA, et al. Prognostically useful gene-expression profiles in acute myeloid leukemia. N Engl J Med 2004; 350:1617-1628.
By J. Douglas Rizzo, M.D.
Determination of prognosis for patients presenting with acute myeloid leukemia (AML) may soon take a leap forward thanks to recent discoveries in gene expression profiling. Bullinger and colleagues describe sophisticated techniques of gene expression profiling that were applied to specimens from patients with newly diagnosed AML to identify distinct gene expression signatures that appear to predict clinical outcome. Using c-DNA microarray analysis, the investigators were able to identify 133 genes (from over 26260 gene candidates) whose expression patterns were predictive of clinical outcome for 116 patients with AML: the "gene-expression outcome predictor." Significant survival differences could even be identified in patients with normal cytogenetics and intermediate AML prognostic features using these predictive gene expression profiles. Similarly, patients with t(8:21) and inv(16) could be separated into good and poor prognostic groups. In multivariate analyses using training and validation sample sets, the gene-expression outcome predictor provided statistically significant prognostic information independent of other AML risk factors, including antecedent hematologic disorder and cytogenetic risk stratification. Gene expression signatures could be characterized for some known cytogenetic subtypes and for patients with normal karyotypes. Some gene expression levels appeared to differentiate outcomes and may help define pathogenesis. Independent work using similar techniques to identify prognostic gene expression profiles in AML reported by Valk et al. reinforces the robustness of the findings.
If the findings emerging from gene expression profiling in AML are reproducibly correlated in hypothesis driven research, the implications for clinicians and basic scientists could be tremendous. First, they represent an opportunity to enhance our fundamental understanding of AML, including an enhanced understanding of progenitor cell abnormalities, and insight into biologic mechanisms arising from the molecular heterogeneity of some AML entities, including inv(16) and t(8;21) abnormalities. Secondly, as more is learned about specific gene profiles that reproducibly predict outcomes, clinicians will gain a better perspective for risk stratification for individuals with AML. Gene-expression profiles may allow better sub-classification of risk, particularly within the current intermediate risk category. This will lead to better strategies for assigning high intensity (and highly toxic) therapy like transplantation to patients with adverse prognosis, rather than consolidation with cytarabine followed by watchful waiting. Thirdly, as specific gene sequences that correlate with outcome are identified, insight may be gained into the roles these genes play in the pathogenesis of AML. Gene clusters that correlate with outcome could include those that confer chemotherapy resistance or detoxification, or those that control apoptosis or cellular differentiation, providing clues that promote tailoring of chemotherapeutic regimens or sequencing. Furthest from our current means, but perhaps most intriguing, meaningful individual gene sequences (rather than clusters of genes) may be identified that lead to development of therapeutics targeted at the responsible molecular defect in any given patient, rather than "standard" therapy applied across the board for AML classification groups.
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Hematopoietic Stem Cells: Tie-ed to Home, in Their Niche
Arai F, Hirao A, Ohmura M, Sato H, Matsuoka S, Takubo K, Ito K, Koh GY, Suda T. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004 Jul; 118(2):149-61.
By Robert Lowsky, M.D., FRCPC
Stem cells share the unique characteristic of balancing quiescence, self-renewal, and commitment to differentiation. For adult hematopoiesis, this characteristic is regulated by the interactions of hematopoietic stem cells (HSC) with their particular microenvironments, known as stem cell niches. Several studies have suggested that the niche for primitive HSCs is the inner surface of bone, alongside of osteoblasts (OB), but the precise molecular mechanism(s) defining the interrelationships remain poorly defined. In this paper, Arai et al. demonstrate that angiopoietin-1 (Ang-1) produced by osteoblasts (OB) activates the receptor tyrosine kinase Tie2 on HSCs and promotes tight adhesion of HSCs to the niche, resulting in quiescence and enhanced survival.
Using a murine model, the authors demonstrated that Tie2 expression segregates with a population of bone marrow cells exhibiting typical HSC surface markers that function as long-term repopulating cells when injected into lethally irradiated mice. They confirmed that HSCs localize to the bone surface of bone marrow, and Tie2 expression appears induced in these cells via their interaction with Ang-1+ OBs. Tie2+ activity prevents cell division, increases the proportion of quiescent HSC in vivo, and increases the resistance to myelosuppressive doses of 5-Fluorouracil (5-FU), a drug that induces apoptosis in actively cycling cells. Finally, the authors also demonstrated that the administration of Ang-1 in vivo protects HSC from myelosuppressive effects of radiation (thus is anti-apoptotic) or 5-FU.
This report is significant as it identifies cellular participants in the BM niche that affect HSC fate and also defines a key regulatory signal, namely the Tie2/Ang-1 pathway, that promotes HSC quiescence. The ramifications of understanding the interactions between bone and bone marrow cells are many. Practical methods can be developed for HSC harvesting and expansion ex vivo or in vivo. Do self-renewing "leukemia stem cells" localize to BM niches like non-malignant HSCs, and do they evade chemoradiation-induced apoptosis by acquiring senescence promoting pathways such as Tie2/Ang-1? As always, much work remains. More precise definitions of the BM niche are necessary as likely only a subset of OBs provide the Ang-1 stimulus. As well, other supporting stromal cells probably contribute key regulatory signals and these would need to be integrated with Tie2/Ang-1. Nonetheless, Arai et al. provide the groundwork to better understand HSC quiescence and how HSCs are fit to be tie-ed to their niches.
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Unexpected Role for an Endothelial Cell Protein in the Antiangiogenic Activity of Endostatin
Yu Y, Moulton KS, Khan MK, Vineberg S, Boye E, Davis VM, O'Donnell PE, Bischoff J, and DS Milstone. E-selectin is required fort he antiangiogenic activity of endostatin. Proc Nat Acad Sci (USA) 2004; 101:8005-8010.
By Roy Silverstein, M.D.
Endostatin, a peptide fragment derived by endogenous proteolysis from type XVIII collagen, was initially isolated from a mouse hemangioendothelioma cell line on the basis of its potent antiangiogenic and anti-tumor activities. Studies reported in this paper make elegant use of murine genetic models to probe the molecular mechanisms of endostatin action. The authors found that endostatin lost its ability to inhibit angiogenesis in mice genetically engineered to delete the gene for E-selectin, a vascular endothelial cell surface protein that functions in the initial stages of leukocyte transmigration into peripheral tissues. E-selectin is not expressed on most endothelial cells under resting conditions. In states of inflammation, however, mediators such as bacterial endotoxin, IL-1DF, and TNF induce transcription of the E-selectin gene and expression of the protein on the endothelial surface. There it acts as a lectin, recognizing specific carbohydrate antigens on leukocyte surfaces thereby tethering them to the vessel wall, slowing down their forward momentum and allowing them to attach to the endothelial surface.
The authors used two different mouse angiogenesis models, an in vivo corneal pocket assay and an ex vivo aorta ring assay, with two different potent stimulators of angiogenesis, bFGF and VEGF, to show that E-selectin null mice were totally resistant to endostatin. They also studied the angiogenic behavior of human vascular endothelial cells grown in tissue culture, focusing on cell migration, a key component of the angiogenic response. Endostatin did not inhibit migration of resting endothelial cells in response to VEGF, but when the cells were induced to express E-selectin, either by treatment with endotoxin or IL-1DF, or by gene transfer via an adenoviral vector, they became sensitive to inhibition by endostatin. The molecular mechanism of E-selectin's role in the endostatin response was not determined, although the authors did show that it was unrelated to leukocyte adhesion and that the intracellular portion of the molecule was required, suggesting that E-selectin may be transmitting a signal to endothelial cells.
Initial excitement about the potential value of antiangiogenic therapies for multiple human diseases, including cancer, macular degeneration, diabetic retinopathy, and even obesity, has been tempered by inconsistent and often disappointing results from clinical trials. Better understanding of the molecular mechanisms underlying antiangiogenesis is needed to overcome the discrepancy between hope and reality. This work, along with several other recent studies, points to several important biological and pharmacodynamic principles related to the class of anti-angiogenic agents that includes endostain, angiostatin, and thrombospondin. These proteins, unlike bevacizumab (Avastin), do not act as direct inhibitors of angiogenic growth factors or receptors, but rather function by interacting with specific endothelial proteins, initiating an active anti-angiogenic signaling cascade. In the case of thrombospondin, its receptor CD36 diverts an angiogenic response to an apoptotic response, thereby aborting angiogenesis. Whether E-selectin serves a similar role in the endostatin response remains to be seen, but these new studies point to potential therapies based on understanding the endothelial cell proteins involved in mediating antiangiogenic responses. For example, pharmacologic modulation of E-selectin expression in specific locations, such as tumor beds, could be used to augment endostatin activity. At the least, this study suggests that assays of E-selectin expression in tumors could be used to predict clinical responses to endostatin.
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Are Vitamins Really Good for You? The Yin and Yang of Folic Acid
Lange H, Suryapranata H, Giuseppe D, BF6rner C, Dille J, Kallmayer K, Pasalary MN, Scherer E, and Dambrink J-H E. Folate Therapy and In-Stent Restenosis after Coronary Stenting. N Engl J Med 2004; 350:2673-81.
By Samuel Silver, M.D., Ph.D.
Homocysteine is an independent risk factor for both coronary artery disease and deep vein thrombosis (for review, see Seshadri N and Robinson K. Med Clinics North America 2000;84:215-37). Serum homocysteine levels can be decreased by the use of vitamin B12 and folic acid via B12- and folate-dependent remethylation of homocysteine to methionine. In an attempt to prevent the occurrence of restenosis after percutaneous coronary angioplasty, Schnyder and colleagues in a previous study (N Engl J Med 2001;345:1593-600) gave patients a cocktail of vitamin B12, folic acid, and vitamin B6 (pyridoxine) and found that there was a reduction in both homocysteine levels and restenosis after balloon angioplasty compared to a control group. With the increasing use of coronary stenting, Lange et al. now report the results of a multi-center, randomized, double-blind, placebo-controlled trial testing the effect of a combination of folic acid and vitamins B6 and
B12 on the risk of angiographic restenosis after coronary-stent placement. The primary angiographic end points were minimal luminal diameter, late luminal loss, and restenosis rate (stenosis >50 percent of luminal diameter) as assessed angiographically at six months or, if clinically indicated, at an earlier time. 636 patients were randomized to receive the "folate" therapy vs. placebo. Despite a significant decline in homocysteine levels in the folate-treated group (mean 12.2 B5mol/l at baseline compared to 9.0 B5mol/l at 6 mo., p<0.001), folate treatment had an adverse risk on restenosis as defined by minimal luminal diameter (1.59B10.62 vs. 1.72B10.64 mm, p=0.008), extent of late luminal loss (0.90B1.55 v 0.76B1.58mm, p=0.004), and restenosis rate (34.5 percent v 26.5 percent, p=0.05) in the folate vs. placebo groups, respectively. A clinical end point of a second revascularization procedure was required in 15.8 percent in the folate group compared to 10.6 percent in the placebo group (p=0.05). The incidence of myocardial infarction and death were not different in the two groups.
Why did the earlier Schnyder study demonstrate a beneficial effect on restenosis (serving as a potential standard of care to use folic acid cocktails post angioplasty) while the present study showed an adverse effect? There are a number of differences in the two studies including differences in the "folate" cocktails. However, most importantly, in the former study, patients who benefited from folate therapy had undergone balloon angioplasty, while in the present study stenting was used. The vascular physiology of these procedures differs. Restenosis following balloon angioplasty is associated with predominantly thrombus formation in intimal cracks, whereas restenosis following coronary stenting is associated with predominantly proliferation of smooth muscle cells. The use of folate therapy to decrease homocysteine theoretically might prevent restenosis in balloon angioplasty by inhibiting thrombus formation. Homocysteine is known to be prothrombotic via a number of mechanisms including activation of factors V and inhibition of tissue plasminogen activator (t-PA) on the endothelial surface and via activation of tissue factor. On the other hand, folic acid therapy could be involved in intimal proliferation, exacerbating restenosis in coronary stenting. Obviously, simple vitamin therapy is all but simple. Even the basic preventive therapy of folic acid supplementation to decrease high homocysteine levels has not yet been shown to decrease vascular disease. The yin and yang of folate therapy for the prevention of restenosis and the prevention of vascular disease in general is open to many avenues of investigation: epidemiology, large clinical trials, animal models, and basic vascular biology research. We have much to learn about this field, and unfortunately, simple solutions do not appear to be immediately obvious... Are vitamins good for you? Time will tell.
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CD39 in Thrombosis and Transplantation; the Evidence Continues to Accumulate
Dwyer KM, Robson SC, Nandurkar HH, Campbell DJ, Gock H, Murray-Segal LJ, Fisicaro N, Mysore TB, Kaczmarek E, Cowan PJ, d'Apice AJ.Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation. J Clin Invest 2004; 113:1440-1446
By Kenneth Kaushansky, M.D.
Initially considered a B lymphocyte activation marker involved in homotypic aggregation, CD39 is now known to regulate platelet aggregation and vascular reactivity in procoagulant and inflammatory conditions. In this paper, using a H-2Kb promoter to drive human CD39 expression, Dwyer and colleagues investigated the role of this endothelial cell class E ectonucleotidase in coagulation and transplantation rejection. They found that overexpression of CD39 significantly blunted the disseminated intravascular coagulation that occurs in normal mice intravenously infused with collagen. Moreover, in a murine model designed to simulate porcine to human xenotransplantation, they found that cardiac transplants were partially protected from lethal rejection if either endothelial cells or hematopoietic cells (likely platelets) expressed the molecule. Also, and importantly for its potential as an anti-thrombotic therapeutic, the transgenic mice displayed no spontaneous bleeding tendency.
This work supports the physiological role played by CD39 in controlling excessive and pathological thrombosis and strengthens the rationale for development of a soluble form of the ectonuclease for therapeutic benefit. Endothelial cells express multiple mediators that maintain an antithrombogenic state (see Figure). Prior to the 1990s the production of the autocoids nitric oxide and prostacyclin were felt to account for much of the capacity of an intact and quiescent vascular endothelial cell layer to inhibit platelet reactivity and maintain blood fluidity. However, the identification of CD39 on the endothelial cell surface, and its demonstration as an ectonucleotidase capable of metabolizing platelet-derived ADP to AMP,1 and thence through endothelial cell surface CD73 to the vasodilator adenosine, identified a third anti-thrombotic and vascular flow-promoting pathway. The physiological relevance of this pathway was established by genetic elimination of CD39, in which a hypercoagulable state was demonstrated by reduced post-ischemic perfusion and increased stroke volume in a murine model of ischemic stroke, or death in a bowel ischemia-reperfusion study. Moreover, fibrin deposition in cardiac allografts is enhanced in CD39 null mice, suggesting a role for platelet derived thrombosis in transplantation rejection.
It is well known that denudation of the endothelial layer of the vessel wall is a powerful thrombogenic stimulus. However, activation of intact endothelial cells can also induce thrombosis, and it is the immune mediated microvascular targeting and activation of tissue endothelium that contributes to allogenic and xenogenic organ rejection. Much work has been performed attempting to identify the antigenic stimulus for rejection in porcine organ xenotransplantation into man. One of the primary targets is the carbohydrate structures that differ between the two species that are prominently presented on the vasculature. The present work by Dwyer and colleagues, using a pseudodiscordant carbohydrate model of cardiac transplantation, suggests that in addition to preventing induced intravascular coagulation, CD39 confers a survival advantage to xenografts and confirms a role for coagulation in the rejection of transplanted organs. As oxidative damage and reduced activity of surface CD39 occurs during ischemia-reperfusion injury of donor organs, and adenoviral vector based expression of the cell surface ectonucleotidase is also beneficial in the transplantation setting,2 these data lend additional impetus to developing a strategy to transiently deliver CD39 as adjunctive therapy for graft rejection. Recent studies of soluble CD39 in ischemia induced thrombosis have also been presented and include favorable effects in a rat model of ischemic stroke.3 Each year, nearly 500,000 persons in the U.S. alone suffer from the complications of cerebrovascular disease, despite several available therapeutic interventions. Moreover, by virtue of their very transitory existence, the other two mechanisms by which endothelial cells maintain vascular patency and blood flow, prostacyclin and nitric oxide, are not amenable to chronic therapy. It is not yet certain whether CD39 will play a key role in the therapy of graft rejection or ischemic thrombosis of the future, but our understanding of the role of this molecule is increasing substantially.
1. Marcus AJ, Broekman MJ, Drosopoulos, Islam N, Alyonycheva TN, Safier LB, Hajjar KA, Posnett DN, Schoenborn MA, Schooley KA, Gayle RB, Maliszewski CR. The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39. J Clin Invest 1997; 99:1351-1360
2. Imai M, Takigami K, Guckelberger O, Kaczmarek E, Csizmadia E, Bach FH, Robson SC. Recombinant adenoviral mediated CD39 gene transfer prolongs cardiac xenograft survival. Transplantation 2000; 70:864-870
3. Belayev L, Khoutorova L, Deisher TA, Belayev A, Busto R, Zhang Y, Zhao W, Ginsberg MD. Neuroprotective effect of SolCD39, a novel platelet aggregation inhibitor, on transient middle cerebral artery occlusion in rats. Stroke. 2003 Mar;34(3):758-63. Epub 2003 Feb 06
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