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Lysophosphatidic acid contributes to angiogenic homeostasis
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Review Article
Lysophosphatidic acid contributes to angiogenic homeostasis Andrius Kazlauskas Schepens Eye Research Institute/Massachusetts Eye and Ear Infirmary/Harvard Medical School, 20 Staniford St., Boston, MA 02114, USA
article information Article Chronology: Received 23 October 2014 Accepted 5 November 2014 Available online 26 November 2014 Keywords: LPA ATX Angiogenesis Blood vessel stability/regression
Contents Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 LPA plays multiple roles in governing angiogenic homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 ATX/LPA is required for developmental blood vessels formation in zebrafish and mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 ATX/LPA promotes both formation and regression of blood vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 LPA modulates extracellular matrix (ECM) components to regulate blood vessel formation . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Pericytes enhance metabolism of LPA and thereby prevent regression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Multiple modes of blood vessels regression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Regulating the level of LPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Responsiveness to LPA is variable . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Angiogenesis Angiogenesis is a deliberate sequence of events that starts with destabilization of an existing vascular bed, and ends when the newly generated vessels quiesce [1,2]. During angiogenesis, endothelial cell E-mail address: [email protected] http://dx.doi.org/10.1016/j.yexcr.2014.11.012 0014-4827/& 2014 Elsevier Inc. All rights reserved.
growth, migration, and tube formation are regulated by pro- and anti-angiogenic factors, matrix-degrading proteases, and cell–extracellular matrix interactions. This complex orchestra of processes is essential for normal physiologic development and is aberrant during disease. For instance, the importance of angiogenesis during the
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formation of an embryo is highlighted by the fact that embryonic lethality is observed in mice that lack even a single allele of genes encoding proteins that govern angiogenesis (such as vascular endothelial cell growth factor A (VEGF) or delta-like 4 (Dll4)) [3–7]. Angiogenesis also underlies a variety of endemic diseases such as solid tumors, which require formation of new blood vessels within the growing tumor [8]. These examples, which illustrate the importance of angiogenesis in both physiology and pathology, suggest that this process is tightly regulated. This is not a surprise given that a variety of cellular responses must be precisely coordinated for the angiogenic program to respond to changing needs of a tissue. Indeed, VEGF, arguably the best-studied angiomodulator, is highly regulated at the levels of synthesis, spatial distribution, receptor and co-receptor availability, and signaling downstream of the receptors [1,9,10]. While many agents that contribute to angiogenesis have been identified and studied in depth, others, such as lysophosphatidic acid (LPA), are relatively unexplored.
LPA plays multiple roles in governing angiogenic homeostasis ATX/LPA is required for developmental blood vessels formation in zebrafish and mice The following series of observations support the concept that ATX and LPA are important for vascular development and function. First, endothelial cells express LPA receptors and respond to LPA [11]. Second, ATX null mice die at embryonic day 9.5–10.5 with profound vascular defects [12,13]. ATX (autotaxin) is the enzyme responsible for generating the vast majority of LPA [12–16]. Third, knockdown of ATX in zebrafish results in abnormal vascular development [17]. While these studies reveal the indispensability of ATX/LPA for blood vessel formation in both zebrafish and mice, the precise contribution to angiogenesis requires additional studies such as those described below.
ATX/LPA promotes both formation and regression of blood vessels Compelling evidence that ATX/LPA promotes angiogenesis was obtained when mice were injected with Matrigel that did or did not contain ATX. Substantially more blood vessels invaded the Matrigel that was loaded with ATX [18]. The observation that VEGF stimulates endothelial cell to produce ATX [19] provides additional, albeit indirect support for the concept that ATX/LPA drives formation of new blood vessels. In light of the early appreciation that ATX/LPA stimulates angiogenesis, it was a surprise to learn that ATX/LPA also induces regression of nascent blood vessels. For instance, LPA and/or ATX trigger/triggers regression of tubes that are organized from primary endothelial cell tubes in vitro [19]. A variety of experimental approaches indicated that this was the case, including the observation that endothelial cells expressing a VEGFR mutant that could not stimulate production of ATX organized into stable tubes, whereas tubes assembled with cells expressing wild type VEGFR regressed [19,20]. Similarly, LPA triggers regression of neovessels that sprout from tissue explants [21]. Finally, regression of blood vessels that surround the lens in the eye of a mouse is accelerated
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by overexpression of ATX, which increased the circulating level of LPA by approximately two-fold [19]. So how is it possible that ATX/LPA drives opposing facets of the angiogenesis program? A plausible explanation emerges when one considers that LPA instructs endothelial cells to migrate, which stimulates the early steps of the angiogenic program, but antagonizes the later phase. Initiation of the angiogenic process requires destabilization of the quiescent vasculature [8]. This is likely to involve changes in the composition of the basement membrane and dissociation of endothelial cells from pericytes, which stabilize vessels. Such steps are followed by migration and proliferation of endothelial cells, and their organization into tubes that are continuous with the existing vasculature. Finally, the nascent vessels recruit pericytes and become quiescent, i.e., show reduced responsiveness to angiogenic agents. The key point here is that migration of cells is a crucial and early step in the angiogenic program. By stimulating migration of cells in this context, LPA fosters angiogenesis. Late in the angiogenic program, i.e. once endothelial cells have organized into a new vessel, LPAdriven migration can disorganize the vessel and hence cause its disappearance/regression. Thus the status of a vascular bed determines whether LPA stimulates or antagonizes angiogenesis. Angiopoietin 2 (Ang2) is a second example of an agent that can either promote or antagonize angiogenesis, depending on the context [22–24]. Ang2 induces regression of hyaloid vessels of the developing mouse eye, provided that the action of VEGF is blocked. In contrast, Ang2 enhances formation of new vessels in the same setting if administered along with VEGF. Similarly, Ang2 triggers either vessel formation, or regression in other vascular beds, and the deciding parameters is the maturity of the vessels and the presence of other angiomodulators (such as VEGF). Some authors have suggested that Ang2 enhances the responsiveness of endothelial cells to other pro-angiogenic agents [25]. Ang2 functions as an antagonist of Ang1, which activates the Tie2 receptor tyrosine kinase [25]. By suppressing the output of Tie2, Ang2 makes endothelial cells vulnerable and hence more receptive to other agents present in the microenvironment. These observations led to the realization that growth factors/ cytokines do not necessarily act independently of each other, but rather influence each other's impact. Such is the case in the context of proliferative vitreoretinopathy, a blinding condition that is driven by a complex interaction between three classes of growth factors that are present in vitreous; VEGF, platelet-derived growth factors (PDGFs) and growth factors outside of the PDGF family (non-PDGFs) [26]. VEGF competitively inhibits PDGF-dependent activation of PDGFRs [27], and PDGFs block non-PDGF-dependent activation of PDGFRα [28]. Consequently, when all three are present, the action of non-PDGFs predominates because VEGF negates the antagonistic action of PDGFs on non-PDGFs.
LPA modulates extracellular matrix (ECM) components to regulate blood vessel formation Temporal and spatial regulation of ECM remodeling events allows for local changes in net matrix deposition or degradation, which in turn contributes to control of endothelial cell growth, migration, and differentiation during the different stages of angiogenesis. ECM remodeling can have either pro- or anti-angiogenic effects. For instance, proteases release factors that enhance endothelial migration and growth thereby promoting
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angiogenesis, while other extracellular components, such as thrombospondins and endostatin, can exert anti-angiogenic effects by inhibiting endothelial cell proliferation, migration and tube formation. In smooth muscle cells, LPA can induce the expression of the matricellular protein Cyr61, which promotes plasma membrane integrin signaling towards focal adhesion kinase (FAK) activation, cell rounding, and migration [29]. LPA also mediates endothelial cell adhesion strength to the ECM and migration through localization of ECM proteins such as Hic-5, and increased matrix metalloproteinase2 (MMP2) expression [30,31]
Pericytes enhance metabolism of LPA and thereby prevent regression As mentioned above, pericytes stabilize blood vessels, and one of the mechanisms by which they accomplish this feat is by accelerating metabolism of LPA [32]. For instance, endothelial cells tubes that are associated with pericytes persist in the face of a concentration of LPA that causes tubes without pericytes to regress. The underlying mechanism seems to involve LPPs (lipid phosphate phosphatases), which are phosphatases that convert LPA to monoacylglycerol [32]. In light of the fact that a cell's responsiveness to LPA can be reduced under certain conditions (see below), we considered if pericytes, in addition to reducing the concentration of LPA, also attenuated the ability of endothelial cells to respond to LPA. This did not turn out to be true. An analog of LPA that is resistant to LPPs induced regression equally well in EC and EC–pericyte tubes [32]. Thus pericytes stabilize blood vessels by reducing the level of LPA and thereby preventing LPAdriven disorganization/regression. It is important to note that this is not the only mechanism by which pericytes stabilize newlyformed blood vessels; growth factors, proteins that mediate cell–cell interaction and proteases are also involved [33,34]. For instance, during the final steps of angiogenesis, newly formed blood vessels are stabilized by the recruitment of pericytes, which induces expression of Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) in endothelial cells and TIMP-3 in pericytes thereby switching off the proteolytic phenotype in endothelial cells [35].
Multiple modes of blood vessels regression The mechanism by which LPA induces regression is fundamentally different from how anti-VEGF accomplishes this feat. Nascent blood vessels are addicted to VEGF prior to the recruitment of pericytes [36]. Consequently, neutralizing VEGF kills the endothelial cells of such vessels, and this phenomenon is often called “regression”. Similarly, macrophages are recruited to blood vessels, and can induce apoptosis in a Wnt-dependent manner, which also results in regression [37–39]. In contrast, LPA promotes viability and proliferation instead of death of endothelial cells [11,40]. Thus regression, which correctly indicates the disappearance of blood vessels, can be driven by several distinct mechanisms.
Regulating the level of LPA The level of LPA in plasma appears to be a dynamic balance between synthesis and degradation. Intravenously injected LPA,
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which is radiolabeled so that it can be tracked, disappears with a half-life of 3 min [16]. The rapid clearance of LPA, taken together with its relatively high steady state concentration in plasma (0.1–1 μM [41]), suggests that LPA is actively being produced. Indeed, injecting an ATX inhibitor into the circulation reduces the plasma level of LPA by 48% within 2 min [16]. These studies reveal that LPA in plasma is being actively produced (by ATX) and degraded (probably by LPPs). Such insights beg the question of what regulates these enzymes and thereby governs the circulating level of LPA? While the answers to such questions are unknown at the present time, the issue seems to be physiologically relevant because ATX transgenic mice, which have an elevated circulating level of LPA, display a phenotype (attenuated clotting) [42]. Proteins that bind LPA and regulate its activity have been identified (e.g. gelsolin and albumin), and constitute an additional layer of regulation on the circulating pool of LPA. [43–46]. Finally, published studies strongly support the idea that the level of LPA is regulated locally. ATX binds to activated lymphocytes and platelets in an integrindependent manner, which is likely to elevate the concentration of LPA in close proximity to LPA receptors [42,47–50]. Similarly, local metabolism of LPA may reduce its concentration, as is the case with pericyte-mediated protection of endothelial cell tubes from LPA-driven regression [32].
Responsiveness to LPA is variable Our recent studies seeking to understand why diabetes induces unresponsiveness to vitreous-induced regression of retinal neovessels revealed that responsiveness to LPA is variable. One of the complications of diabetes is proliferative diabetic retinopathy (PDR), which is characterized by accumulation of neovessels within the vitreous cavity of the eye [51]. Vitreous is the substance that fills the globe of the eye and is in direct contact with the retina. Normally, vitreous has an intrinsic ability to eliminate neovessels because it contains regression activity, which is in large part due to LPA [21]. As patients develop PDR this regression activity wanes, but not because the level of LPA declines [52]. Our working hypothesis is that PDR vitreous contains agents that prevent endothelial cells within neovessels from responding to LPA. Consistent with this possibility is the finding that PDR vitreous prevents purified LPA from triggering regression [52]. While key mechanistic questions remain open, these observations indicate that responsiveness to LPA is variable. This concept is reinforced by the fact that vascular endothelial cells remain quiescent in the face of continuous exposure to LPA that is present at a high concentration in plasma. Studies focused on how diabetes influences endothelial cells (instead of the vitreal bioactivity, which was discussed above) led to mechanistic insights regarding what governs responsiveness to LPA. Retinal explants sprout neovessels when cultured in an ex vivo setting in the presence of VEGF. Such neovessels consist of primarily endothelial cells and are relatively stable until they are exposed to LPA, whereupon they regress [21]. When this experiment is repeated with retinal explants isolated from diabetic mice, the neovessels do not respond to LPA [21]. This is because diabetes rewires signaling pathways in a way that prevents LPA from triggering regression. As illustrated in Fig. 1, diabetes activates the reactive oxygen species (ROS)/Src family
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[7]
[8] [9]
Fig. 1 – Diabetes induces non-responsiveness to LPA by activating the RSE pathway. Using a combination of experimental approaches we learned that diabetic conditions elevated reactive oxygen species (ROS) in primary retinal endothelial cells [21], an observation that is consistent with the findings from many investigators [53]. Elevating ROS activates Src family tyrosine kinases (SFKs) that engage the Erk pathway [21], which intersects with LPA-stimulated signaling at the level of Rho/ROCK; ROCK is a ser/thr kinase that is essential for LPA-induced regression [54,55]. kinase (SFK)/Erk (RSE) pathway, which antagonizes Rho/ROCK, which is an essential step in LPA-mediated regression. It is tempting to speculate that agents that accumulate in PDR vitreous engage the RSE pathway and thereby induce non-responsiveness to LPA. Ongoing studies are designed to address this possibility. In summary, the observation that environmental factors influence the ability of cells to respond to LPA underscores the concept that the concentration of LPA is not the only parameter influencing the impact of LPA.
Acknowledgments I would like to thank Mohammed Anwar, Sarah Jacobo and Ashley Mackey for constructive input for improving this review article. Funding was provided by NIH Grant EY016385.
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Review Article
Lysophosphatidic acid contributes to angiogenic homeostasis Andrius Kazlauskas Schepens Eye Research Institute/Massachusetts Eye and Ear Infirmary/Harvard Medical School, 20 Staniford St., Boston, MA 02114, USA
article information Article Chronology: Received 23 October 2014 Accepted 5 November 2014 Available online 26 November 2014 Keywords: LPA ATX Angiogenesis Blood vessel stability/regression
Contents Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 LPA plays multiple roles in governing angiogenic homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 ATX/LPA is required for developmental blood vessels formation in zebrafish and mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 ATX/LPA promotes both formation and regression of blood vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 LPA modulates extracellular matrix (ECM) components to regulate blood vessel formation . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Pericytes enhance metabolism of LPA and thereby prevent regression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Multiple modes of blood vessels regression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Regulating the level of LPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Responsiveness to LPA is variable . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Angiogenesis Angiogenesis is a deliberate sequence of events that starts with destabilization of an existing vascular bed, and ends when the newly generated vessels quiesce [1,2]. During angiogenesis, endothelial cell E-mail address: [email protected] http://dx.doi.org/10.1016/j.yexcr.2014.11.012 0014-4827/& 2014 Elsevier Inc. All rights reserved.
growth, migration, and tube formation are regulated by pro- and anti-angiogenic factors, matrix-degrading proteases, and cell–extracellular matrix interactions. This complex orchestra of processes is essential for normal physiologic development and is aberrant during disease. For instance, the importance of angiogenesis during the
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formation of an embryo is highlighted by the fact that embryonic lethality is observed in mice that lack even a single allele of genes encoding proteins that govern angiogenesis (such as vascular endothelial cell growth factor A (VEGF) or delta-like 4 (Dll4)) [3–7]. Angiogenesis also underlies a variety of endemic diseases such as solid tumors, which require formation of new blood vessels within the growing tumor [8]. These examples, which illustrate the importance of angiogenesis in both physiology and pathology, suggest that this process is tightly regulated. This is not a surprise given that a variety of cellular responses must be precisely coordinated for the angiogenic program to respond to changing needs of a tissue. Indeed, VEGF, arguably the best-studied angiomodulator, is highly regulated at the levels of synthesis, spatial distribution, receptor and co-receptor availability, and signaling downstream of the receptors [1,9,10]. While many agents that contribute to angiogenesis have been identified and studied in depth, others, such as lysophosphatidic acid (LPA), are relatively unexplored.
LPA plays multiple roles in governing angiogenic homeostasis ATX/LPA is required for developmental blood vessels formation in zebrafish and mice The following series of observations support the concept that ATX and LPA are important for vascular development and function. First, endothelial cells express LPA receptors and respond to LPA [11]. Second, ATX null mice die at embryonic day 9.5–10.5 with profound vascular defects [12,13]. ATX (autotaxin) is the enzyme responsible for generating the vast majority of LPA [12–16]. Third, knockdown of ATX in zebrafish results in abnormal vascular development [17]. While these studies reveal the indispensability of ATX/LPA for blood vessel formation in both zebrafish and mice, the precise contribution to angiogenesis requires additional studies such as those described below.
ATX/LPA promotes both formation and regression of blood vessels Compelling evidence that ATX/LPA promotes angiogenesis was obtained when mice were injected with Matrigel that did or did not contain ATX. Substantially more blood vessels invaded the Matrigel that was loaded with ATX [18]. The observation that VEGF stimulates endothelial cell to produce ATX [19] provides additional, albeit indirect support for the concept that ATX/LPA drives formation of new blood vessels. In light of the early appreciation that ATX/LPA stimulates angiogenesis, it was a surprise to learn that ATX/LPA also induces regression of nascent blood vessels. For instance, LPA and/or ATX trigger/triggers regression of tubes that are organized from primary endothelial cell tubes in vitro [19]. A variety of experimental approaches indicated that this was the case, including the observation that endothelial cells expressing a VEGFR mutant that could not stimulate production of ATX organized into stable tubes, whereas tubes assembled with cells expressing wild type VEGFR regressed [19,20]. Similarly, LPA triggers regression of neovessels that sprout from tissue explants [21]. Finally, regression of blood vessels that surround the lens in the eye of a mouse is accelerated
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by overexpression of ATX, which increased the circulating level of LPA by approximately two-fold [19]. So how is it possible that ATX/LPA drives opposing facets of the angiogenesis program? A plausible explanation emerges when one considers that LPA instructs endothelial cells to migrate, which stimulates the early steps of the angiogenic program, but antagonizes the later phase. Initiation of the angiogenic process requires destabilization of the quiescent vasculature [8]. This is likely to involve changes in the composition of the basement membrane and dissociation of endothelial cells from pericytes, which stabilize vessels. Such steps are followed by migration and proliferation of endothelial cells, and their organization into tubes that are continuous with the existing vasculature. Finally, the nascent vessels recruit pericytes and become quiescent, i.e., show reduced responsiveness to angiogenic agents. The key point here is that migration of cells is a crucial and early step in the angiogenic program. By stimulating migration of cells in this context, LPA fosters angiogenesis. Late in the angiogenic program, i.e. once endothelial cells have organized into a new vessel, LPAdriven migration can disorganize the vessel and hence cause its disappearance/regression. Thus the status of a vascular bed determines whether LPA stimulates or antagonizes angiogenesis. Angiopoietin 2 (Ang2) is a second example of an agent that can either promote or antagonize angiogenesis, depending on the context [22–24]. Ang2 induces regression of hyaloid vessels of the developing mouse eye, provided that the action of VEGF is blocked. In contrast, Ang2 enhances formation of new vessels in the same setting if administered along with VEGF. Similarly, Ang2 triggers either vessel formation, or regression in other vascular beds, and the deciding parameters is the maturity of the vessels and the presence of other angiomodulators (such as VEGF). Some authors have suggested that Ang2 enhances the responsiveness of endothelial cells to other pro-angiogenic agents [25]. Ang2 functions as an antagonist of Ang1, which activates the Tie2 receptor tyrosine kinase [25]. By suppressing the output of Tie2, Ang2 makes endothelial cells vulnerable and hence more receptive to other agents present in the microenvironment. These observations led to the realization that growth factors/ cytokines do not necessarily act independently of each other, but rather influence each other's impact. Such is the case in the context of proliferative vitreoretinopathy, a blinding condition that is driven by a complex interaction between three classes of growth factors that are present in vitreous; VEGF, platelet-derived growth factors (PDGFs) and growth factors outside of the PDGF family (non-PDGFs) [26]. VEGF competitively inhibits PDGF-dependent activation of PDGFRs [27], and PDGFs block non-PDGF-dependent activation of PDGFRα [28]. Consequently, when all three are present, the action of non-PDGFs predominates because VEGF negates the antagonistic action of PDGFs on non-PDGFs.
LPA modulates extracellular matrix (ECM) components to regulate blood vessel formation Temporal and spatial regulation of ECM remodeling events allows for local changes in net matrix deposition or degradation, which in turn contributes to control of endothelial cell growth, migration, and differentiation during the different stages of angiogenesis. ECM remodeling can have either pro- or anti-angiogenic effects. For instance, proteases release factors that enhance endothelial migration and growth thereby promoting
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angiogenesis, while other extracellular components, such as thrombospondins and endostatin, can exert anti-angiogenic effects by inhibiting endothelial cell proliferation, migration and tube formation. In smooth muscle cells, LPA can induce the expression of the matricellular protein Cyr61, which promotes plasma membrane integrin signaling towards focal adhesion kinase (FAK) activation, cell rounding, and migration [29]. LPA also mediates endothelial cell adhesion strength to the ECM and migration through localization of ECM proteins such as Hic-5, and increased matrix metalloproteinase2 (MMP2) expression [30,31]
Pericytes enhance metabolism of LPA and thereby prevent regression As mentioned above, pericytes stabilize blood vessels, and one of the mechanisms by which they accomplish this feat is by accelerating metabolism of LPA [32]. For instance, endothelial cells tubes that are associated with pericytes persist in the face of a concentration of LPA that causes tubes without pericytes to regress. The underlying mechanism seems to involve LPPs (lipid phosphate phosphatases), which are phosphatases that convert LPA to monoacylglycerol [32]. In light of the fact that a cell's responsiveness to LPA can be reduced under certain conditions (see below), we considered if pericytes, in addition to reducing the concentration of LPA, also attenuated the ability of endothelial cells to respond to LPA. This did not turn out to be true. An analog of LPA that is resistant to LPPs induced regression equally well in EC and EC–pericyte tubes [32]. Thus pericytes stabilize blood vessels by reducing the level of LPA and thereby preventing LPAdriven disorganization/regression. It is important to note that this is not the only mechanism by which pericytes stabilize newlyformed blood vessels; growth factors, proteins that mediate cell–cell interaction and proteases are also involved [33,34]. For instance, during the final steps of angiogenesis, newly formed blood vessels are stabilized by the recruitment of pericytes, which induces expression of Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) in endothelial cells and TIMP-3 in pericytes thereby switching off the proteolytic phenotype in endothelial cells [35].
Multiple modes of blood vessels regression The mechanism by which LPA induces regression is fundamentally different from how anti-VEGF accomplishes this feat. Nascent blood vessels are addicted to VEGF prior to the recruitment of pericytes [36]. Consequently, neutralizing VEGF kills the endothelial cells of such vessels, and this phenomenon is often called “regression”. Similarly, macrophages are recruited to blood vessels, and can induce apoptosis in a Wnt-dependent manner, which also results in regression [37–39]. In contrast, LPA promotes viability and proliferation instead of death of endothelial cells [11,40]. Thus regression, which correctly indicates the disappearance of blood vessels, can be driven by several distinct mechanisms.
Regulating the level of LPA The level of LPA in plasma appears to be a dynamic balance between synthesis and degradation. Intravenously injected LPA,
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which is radiolabeled so that it can be tracked, disappears with a half-life of 3 min [16]. The rapid clearance of LPA, taken together with its relatively high steady state concentration in plasma (0.1–1 μM [41]), suggests that LPA is actively being produced. Indeed, injecting an ATX inhibitor into the circulation reduces the plasma level of LPA by 48% within 2 min [16]. These studies reveal that LPA in plasma is being actively produced (by ATX) and degraded (probably by LPPs). Such insights beg the question of what regulates these enzymes and thereby governs the circulating level of LPA? While the answers to such questions are unknown at the present time, the issue seems to be physiologically relevant because ATX transgenic mice, which have an elevated circulating level of LPA, display a phenotype (attenuated clotting) [42]. Proteins that bind LPA and regulate its activity have been identified (e.g. gelsolin and albumin), and constitute an additional layer of regulation on the circulating pool of LPA. [43–46]. Finally, published studies strongly support the idea that the level of LPA is regulated locally. ATX binds to activated lymphocytes and platelets in an integrindependent manner, which is likely to elevate the concentration of LPA in close proximity to LPA receptors [42,47–50]. Similarly, local metabolism of LPA may reduce its concentration, as is the case with pericyte-mediated protection of endothelial cell tubes from LPA-driven regression [32].
Responsiveness to LPA is variable Our recent studies seeking to understand why diabetes induces unresponsiveness to vitreous-induced regression of retinal neovessels revealed that responsiveness to LPA is variable. One of the complications of diabetes is proliferative diabetic retinopathy (PDR), which is characterized by accumulation of neovessels within the vitreous cavity of the eye [51]. Vitreous is the substance that fills the globe of the eye and is in direct contact with the retina. Normally, vitreous has an intrinsic ability to eliminate neovessels because it contains regression activity, which is in large part due to LPA [21]. As patients develop PDR this regression activity wanes, but not because the level of LPA declines [52]. Our working hypothesis is that PDR vitreous contains agents that prevent endothelial cells within neovessels from responding to LPA. Consistent with this possibility is the finding that PDR vitreous prevents purified LPA from triggering regression [52]. While key mechanistic questions remain open, these observations indicate that responsiveness to LPA is variable. This concept is reinforced by the fact that vascular endothelial cells remain quiescent in the face of continuous exposure to LPA that is present at a high concentration in plasma. Studies focused on how diabetes influences endothelial cells (instead of the vitreal bioactivity, which was discussed above) led to mechanistic insights regarding what governs responsiveness to LPA. Retinal explants sprout neovessels when cultured in an ex vivo setting in the presence of VEGF. Such neovessels consist of primarily endothelial cells and are relatively stable until they are exposed to LPA, whereupon they regress [21]. When this experiment is repeated with retinal explants isolated from diabetic mice, the neovessels do not respond to LPA [21]. This is because diabetes rewires signaling pathways in a way that prevents LPA from triggering regression. As illustrated in Fig. 1, diabetes activates the reactive oxygen species (ROS)/Src family
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[7]
[8] [9]
Fig. 1 – Diabetes induces non-responsiveness to LPA by activating the RSE pathway. Using a combination of experimental approaches we learned that diabetic conditions elevated reactive oxygen species (ROS) in primary retinal endothelial cells [21], an observation that is consistent with the findings from many investigators [53]. Elevating ROS activates Src family tyrosine kinases (SFKs) that engage the Erk pathway [21], which intersects with LPA-stimulated signaling at the level of Rho/ROCK; ROCK is a ser/thr kinase that is essential for LPA-induced regression [54,55]. kinase (SFK)/Erk (RSE) pathway, which antagonizes Rho/ROCK, which is an essential step in LPA-mediated regression. It is tempting to speculate that agents that accumulate in PDR vitreous engage the RSE pathway and thereby induce non-responsiveness to LPA. Ongoing studies are designed to address this possibility. In summary, the observation that environmental factors influence the ability of cells to respond to LPA underscores the concept that the concentration of LPA is not the only parameter influencing the impact of LPA.
Acknowledgments I would like to thank Mohammed Anwar, Sarah Jacobo and Ashley Mackey for constructive input for improving this review article. Funding was provided by NIH Grant EY016385.
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