| COMMENTARIES |
The Importance of Gas6/Axl Anti-Apoptotic Signaling in Regulating Vascular Calcification
Andrew Sage and Ann Canfield, Wellcome Trust Centre for Cell Matrix Research, University of Manchester, Manchester, U.K., Email: ann.canfield@manchester.ac.uk
Introduction
Vascular calcification refers to the aberrant development of cartilage and bone-like tissue and associated mineralization of the extracellular matrix at distinct locations within the vascular system: atherosclerotic plaques, the medial layer of large and medium-sized arteries, and cardiac valves.
Calcification is a common and significant component of atherosclerotic plaques, with estimates of its occurrence ranging from 60-90% [1,2]. While vascular calcification has been recognized and described by pathologists for more than a century (reviewed in [3]), until recently it was regarded as a consequence of the aging and deterioration of the vascular system in the elderly [4]. Now, however, the prevalence and clinical importance of vascular calcification in younger people with major chronic systemic diseases including cardiovascular disease, diabetes, and chronic kidney disease is more widely recognized [2]. Vascular calcification is not only highly prevalent and progressive in these diseases, but there is now substantial evidence that calcification directly contributes to the morbidity and mortality associated with these common and serious conditions [1,2,4]. This concept has led to a greatly increased interest in vascular calcification in terms of molecular research, clinical studies as well as the modification of current treatment regimes.
It is now established that vascular calcification is an active, cell-mediated process that resembles embryonic bone development [1,5-7]. At the molecular level, the causes and inducing factors involved in the initiation and early progression of vascular calcification are complex and still not fully understood. Altered metabolic homeostasis, such as hyperlipidemia or hyperphosphatemia, inflammation, and associated oxidative stress may be considered important underlying conditions that promote vascular calcification in addition to other cardiovascular complications. These conditions lead to both the active promotion of bone formation through a variety of pathways, as well as the loss or dysfunction of inhibitory mechanisms that normally act to prevent calcification outside the skeleton [4,5]. For example, increased TNF-α promotes vascular calcification [8] whereas reduced fetuin-A promotes calcification [9].
The Role of Apoptosis in Vascular Calcification
Apoptosis is an inbuilt program for cell death which is carried out by the caspase family of proteases. Current evidence suggests that this process is required for vascular calcification. For example, apoptosis is co-localized with both medial and atherosclerotic calcification in humans [10,11]. Furthermore, mineral deposition by vascular smooth muscle cells (VSMC) and pericytes is associated with increased apoptosis, and incubation of these cells with the caspase inhibitor zVADfmk inhibits mineral deposition [12-14 and unpublished data]. A recent paper also demonstrated that inducing chronic low-grade apoptosis in VSMCs of ApoE-/- mice induces atherosclerotic calcification in vivo [15]
It is possible that vascular osteoprogenitor cells undergo a matrix vesicle and/or apoptotic body-mediated process of mineralization similar to that described for hypertrophic chondrocytes [16,17]. Persuasive evidence indicates that chondrocyte apoptosis is an integral part of the differentiation pathway of these cells and is the terminal (and inevitable) phase of the transition to the hypertrophic stage, when mineralization first begins [16,17]. Both in vivo and in vitro, terminal differentiation of chondrocytes are associated with a decrease in the anti-apoptotic protein Bcl-2 and an increase in the pro-apoptotic proteins Bax and caspase-3 [16-18]. These changes are detectable before morphological evidence of apoptosis, suggesting that hypertrophic chondrocytes are primed for apoptosis but are held in a pre-apoptotic state until induced by, for example, factors introduced during vascular invasion [16-18]. In further support of this hypothesis, increased Pi selectively induces apoptosis of terminally-differentiated but not undifferentiated chondrocytes in vitro [17,18]. Therefore, determining whether a similar sensitization to apoptosis is responsible for vascular osteoprogenitor mineralization is an important goal for future studies.
Gas6 – Axl Survival Signaling Inhibits Vascular Calcification
The receptor tyrosine kinase Axl is activated by the vitamin K-dependent GLA protein, growth arrest specific-6 (Gas6). Axl has emerged as a critical regulator of vascular wall homeostasis, particularly in situations of acute and chronic injury [19] and of apoptosis [20]. Furthermore, in vitro studies from our lab and others have demonstrated that Axl and Gas6 may play an important role in vascular calcification. Axl was first identified as a regulator of mineralization in microvascular pericytes that spontaneously undergo osteogenic differentiation and form mineralized nodules [21,22]. Axl is specifically down-regulated in mineralized pericyte nodules, and inhibition of Axl activation with a decoy Axl receptor (Axl-ECD) accelerated mineralization, indicating the loss of Axl expression may be functionally significant [21].
More recent work from our lab and others has demonstrated that Axl down-regulation also occurs in mineralized human and bovine aortic VSMCs [12,23]. To determine the function of Axl in VSMCs, we modulated Axl expression using recombinant adenoviruses encoding either wild-type or kinase-dead mutant Axl (KD-Axl). Wild-type Axl over-expression significantly inhibited calcification; in contrast, over-expression of KD-Axl significantly accelerated calcification, implicating active signaling as the inhibitory entity. Consistent with other reports [20], we demonstrated that Axl increases PI3K-Akt signaling in these cells and furthermore that signaling via this pathway is critical for the effects of Axl on calcification [12 and unpublished data]. Additional experiments from our lab and recent work by Son et al. have also determined that Axl-PI3K signaling regulates calcification by preventing apoptosis, which would otherwise promote matrix vesicle/apoptotic body-mediated mineral formation in these cells [23] (see above). We also demonstrated that the increased calcification induced by KD-Axl was abrogated in the presence of zVAD.fmk, indicating that KD-Axl stimulates calcification by promoting apoptosis [12].
The loss of Axl expression may, therefore, be one mechanism by which mineralizing VSMCs become sensitized to apoptosis, allowing the completion of the mineralization process. Interestingly, Gas6-Axl survival signaling has also been identified as a downstream target of both phosphate and TNF-α [24], known inducers of calcification in vivo and in vitro [8,25]. Like many regulatory molecules, Axl is likely to have temporal and cell-specific roles in atherosclerosis [19], and as such, may not be a candidate for direct intervention to prevent calcification. However, our studies highlight its potentially important role in this process and lend further support to the hypothesis that apoptosis is involved in vascular calcification.
References