The major findings of this study are: (1) Proteasome inhibitors as a class markedly stimulate endothelial TM expression resulting in enhanced capacity of endothelial cells to generate APC. (2) Proteasome inhibitors effectively prevent the downregulation of TM by inflammatory cytokines. (3) The upregulation of TM by proteasome inhibitors is independent of their NF-kB inhibitory properties but rather mediated by induction of Krüppel-like transcription factors. (4) Systemic administration of a proteasome inhibitor can enhance endothelial expression of KLF2, KLF4 and TM in vivo.
In response to injury or inflammation, the normal anticoagulant properties of the endothelium become impaired and are opposed by the nascent expression of tissue factor which initiates thrombin generation and thrombosis.21
The critical importance of an intact TM-protein C anticoagulant pathway to maintaining endothelial thromboresistance is highlighted by the observation that mice with deletions of TM, protein C or the endothelial protein C receptor (which facilitates APC generation by the thrombin/thrombomodulin complex) die in utero or in the perinatal period from thrombosis.22–24
The acquired loss of TM expression, with a consequent reduction in APC generating capacity, is thought to be a major contributor to the thrombotic manifestations of several inflammatory conditions, including bacterial sepsis, transplant rejection, and radiation enteropathy.25–27
Dysfunction of the TM-protein C anticoagulant pathway has been implicated in thrombosis associated with multiple myeloma and other malignancies. Levels of soluble TM, caused by the release of membrane-bound TM from injured endothelial cells, are elevated at the time of diagnosis and fall with treatment.4
TM gene expression is well-recognized to be negatively regulated by inflammatory cytokines, such as TNF-α and interleukin-1β.5
Serum levels of both cytokines are markedly elevated in patients diagnosed with multiple myeloma and, like soluble TM, fall during treatment.6,7
Approximately 10–20% of patients with multiple myeloma also develop resistance to the anticoagulant actions of APC which is associated with an increased incidence of VTE.28
In contrast to congenital APC resistance due to the presence of the factor V Leiden mutation, the mechanism of acquired APC resistance associated with cancer appears related to increased levels of factors V and VIII which overwhelms the anticoagulant effects of APC.29,30
It is therefore mechanistically plausible that agents such as proteasome inhibitors, with the potential to both increase endothelial TM expression and APC-generating capacity as well as inhibit tissue factor expression, could reduce the risk of VTE in patients with multiple myeloma. Our results provide a conceptual basis for more in-depth in vivo animal and human clinical studies investigating this possibility.
Proteasome inhibition was found to not only stimulate baseline TM expression but also blocks its downregulation by TNF-α. We previously reported that TM downregulation by cytokines and endotoxin is mediated by activation of NF-κB, which competes for limited cellular quantities of the transcriptional coactivator p300 necessary for TM gene expression.5
As the inhibition of NF-κB is thought to mediate the primary anti-tumor effects of bortezomib, it was reasonable to speculate that NF-κB signaling might be involved in modulation of TM expression by proteasome inhibitors. The fact that TM upregulation was not diminished by blocking relA expression () and required new protein synthesis () ruled out a role for NF-κB signaling in mediating TM upregulation by proteasome inhibitors. Rather, this effect was found to be mediated by induction of the Krüppel-like transcription factors KLF2 and KLF4. KLF2 is known to be capable of stimulating TM transcription by binding to a specific GC-rich site in the TM promoter.19
Over-expression of both KLF2 and KLF4 in endothelial cells recapitulates many of the phenotypic changes observed with proteasome inhibition, including a marked increases in baseline TM and eNOS expression and blunting of cytokine-induced upregulation of tissue factor, VCAM-1 and E-selectin.19,20
It remains possible that the NF-κB inhibitory properties of proteasome inhibitors might still play some role in blocking the downregulation of TM by inflammatory cytokines. However, this effect could also be explained by KLF upregulation, as over-expression of KLF2 also is known to block TM downregulation by inflammatory cytokines.19
While it has been previously reported that the ubiquitin-proteasome pathway controls the degradation of several of the KLF proteins31–33
, the ability of proteasome inhibitors to stimulate KLF gene expression is a novel finding of our study. The mechanism by which this occurs is currently unknown, but the subject of active investigation. The KLFs are a large family of zinc-finger transcription factors that play key roles in regulating a wide array of cellular processes including differentiation, proliferation, apoptosis and neoplastic transformation.34
It is possible that some of the anti-tumor effects of bortezomib may be related to its stimulation of KLF expression rather than solely due to its NF-κB inhibitory properties. Consistent with this concept are data indicating that KLF4 and KLF6 can inhibit cellular proliferation through activation of p21WAF1/CIP
and may act as a tumor suppressors in gastrointenstinal and prostatic tissue.35,36
An intriguing finding of our study was the observed differential effects of bortezomib on TM induction in various organs. TM induction was most pronounced in the liver and kidney with no significant induction observed in the heart or lung. TM upregulation was only observed in tissue where KLF2 and/or KLF4 were also upregulated. Susceptibility to thrombus formation in general, and TM expression in particular is known to vary between different vascular beds.37
It is therefore possible that there are tissue-specific differences in the molecular pathways by which proteasome inhibitors regulate endothelial thromboresistance. These results, however, may be better explained by the pharmacokinetics of bortezomib. Following intravenous injection, the drug is rapidly cleared from the blood with a large volume of distribution.38
Radiolabeling studies in rats revealed that the liver and kidney have the highest bortezomib uptake, with virtually no uptake in the brain.39
The maximum tolerated daily intraperitoneal dose (0.8 mg/kg) given to mice in our study was equivalent to approximately half the intravenous bolus dose (1.3 mg/m2
) administered to myeloma patients on a biweekly basis in clinical studies.40
It is possible that alternate dosing schemes utilizing chronic intermittent bolus injections, such as those used in clinical studies, would achieve more widespread and consistent organ effects.
In summary, proteasome inhibitors as a class stimulate the expression of TM and enhance endothelial cell thromboresistance. These effects are mediated via induction of the Krüppel-like transcription factors, KLF2 and KLF4 and may help explain the reduction in thromboembolic events observed in patients who receive proteasome inhibitors for the treatment of plasma cell malignancies.