The role of vascular endothelial growth factor in pulmonary arterial hypertension. The angiogenesis paradox.

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AJRCMB Articles in Press. Published on 16-June-2014 as 10.1165/rcmb.2014-0045TR

The Role of Vascular Endothelial Growth Factor in Pulmonary Arterial Hypertension: The angiogenesis paradox

Norbert F. Voelkel M.D1 and Jose Gomez-Arroyo M.D., Ph.D2

Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia, USA

Corresponding Author: Norbert F. Voelkel, Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, 1220 East Broad Street, Richmond, VA 23298. Telephone: 804-6283334 and Fax: 804-6280325. E-mail: [email protected]

Running Title: The role of VEGF in PAH

1 Copyright © 2014 by the American Thoracic Society


Abstract

Pulmonary arterial hypertension (PAH) is characterized by dysfunctional angiogenesis leading to lung vessel obliteration. PAH is widely considered a pro-angiogenic disease, however, the role of angiogenic factors such as the vascular endothelial factor (VEGF) and its receptors in the pathobiology of PAH remains incompletely understood. This review attempts to untangle some of the complex multilayered actions of VEGF, in order to provide a VEGF-centered perspective of PAH. Furthermore, we provide a cogent explanation for the paradox of VEGF receptor blockade-induced pulmonary hypertension that characterizes the SU5416-hypoxia rat model of PAH and attempt to translate the knowledge gained from the experimental model to the human disease by postulating the potential role of endogenous (SU5416-like) VEGF inhibitors. The main objective of this review is to promote discussion and investigation of the opposing and complementary actions of VEGF in PAH. Understanding the balance between angiogenic and anti-angiogenic factors and their role in the pathogenesis of PAH will be necessary before antiangiogenic drugs can be considered for the treatment of PAH.

Keywords: Sugen; angiogenesis; VEGFA.


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Introduction In severe forms of pulmonary arterial hypertension (PAH), the pulmonary vascular resistance is elevated because of pulmonary vasoconstriction, lung vessel remodeling or both(1). In recent years, our understanding of the mechanisms of pulmonary vascular remodeling in PAH has increased substantially. Indeed, in addition to the time-proven mechanical concepts of pressure, flow and shear stress, other concepts such as cell growth and death and cell phenotype plasticity have been applied to elucidate what drives the process of pulmonary vascular remodeling(2) (some of these concepts have recently been reviewed(3)). Whereas a large number of genes and proteins potentially involved in the pathobiology of PAH have been discussed, two particular proteins have been the focus of attention in both human and experimental PAH: 1) The bone morphogenetic protein receptor 2 (BMPR2) and the vascular endothelial growth factor (VEGF). While a molecular disease concept has been built around the BMPR2 gene, which is mutated in a significant number of patients with hereditary and non-hereditary forms of PAH(4), we suggest that the powerful angiogenic growth- and maintenance factor VEGF has not received adequate consideration in the recent discussions of the pathobiology of PAH. VEGF is abundant in the lung and its roles and actions as a lung-structure maintenance factor are numerous and have been recently reviewed(5). In addition to its role in normal organ biology, VEGF is important for tumor angiogenesis(6) and intraocular neovascular disorders, among other conditions(7). VEGF plasma levels are elevated in patients with severe PAH(8-11), and VEGF as well as the VEGF receptor 2 (VEGFR-2) are robustly expressed in the complex vascular lesions in the lungs from patients with PAH(12, 13). However, whether or how VEGF plays a mechanistic role in the development of PAH remains unclear, particularly in view of the fact that, paradoxically, treatment of rodents with an anti-angiogenic VEGF receptor blocker when combined with a second hit causes angioobliterative PAH.



In this review we provide a VEGF-centered perspective of PAH and attempt to untangle the complex multilayered system of paracrine and autocrine actions of VEGF in the setting of severe angioproliferative PAH by asking two key questions: 1) How can antiangiogenic blockade induce PAH? And 2) is there a role for endogenous inhibitors of VEGF receptor signaling in the pathogenesis of severe human PAH?

Brief overview of VEGF signaling, VEGF splice variants, receptor partnerships and soluble receptors. Angiogenesis is a highly complex and regulated process. Interestingly, although factors such as Fibroblast Growth Factor (FGF), Transforming Growth Factor β (TGFβ), Hepatocyte Growth Factor (HGF) and angiopoietins have been implicated as positive regulators, angiogenesis is prominently regulated by a single growth factor: VEGF(14). VEGF activity is controlled by its own level of expression but also by multiple proteins that can sequester and inactivate the ligand. In addition, the ratio of the different ligands competing for binding to the VEGF receptors determines cell fate, angiogenesis or inhibition of angiogenesis(14). Indeed, the different VEGF receptors and co-receptors, their signaling versatility and their structural similarities with other receptor tyrosine kinases make VEGF biology highly complex. The objective of this review is not to describe the VEGF signaling pathways, as this has been the subject of extensive research and of previous reviews. Instead, we aim to discuss some lesser known aspects of VEGF receptor signaling and how these aspects could be of importance for the development of PAH. Since the description by Senger et al(15), thirty years ago, of a vascular permeability factor secreted by tumor cells (also shown to stimulate the growth of human umbilical vein endothelial cells(16)) several VEGF splice variants with either proangiogenic or antiangiogenic activity have been identified(14). Some of the structural differences between VEGF splice variants are depicted in Figure 1.


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VEGFA, usually the most abundant VEGF protein isoform, is recognized for its traditional activities of enhancing vascular permeability, angiogenesis and vascular cell survival(17). The human VEGFA gene is organized in eight exons and alternative splicing results in the generation of the four different subisoforms VEGF121, VEGF165, VEGF189 and VEGF206 having 121, 165, 189 and 206 amino acids respectively and being VEGF165 the predominant isoform(18). VEGFA binds two related receptor tyrosine kinases (RTKs), VEGF-R1 and VEGFR2. Both receptors have seven immunoglobulin-like domains as part of the extracellular domain, a single transmembrane region and a consensus tyrosine kinase domain(19). VEGF-R1 (also known as flt-1) was the first RTK identified as a VEGF receptor(20) however all of its functions have not been discovered. VEGF-R1 undergoes weak tyrosine autophosphorylation in response to VEGF ligand binding, however it does not seem to be the primary receptor transmitting or inducing a mitogenic/angiogenic signal, but rather, VEGF-R1 acts as a decoy receptor which negatively regulates VEGF activity by preventing activation of VEGF-R2(21). An alternatively spliced and soluble variant of flt-1 (sflt-1) can also sequester VEGF and decrease its activity(22). The role of flt-1 as a decoy receptor is underscored by the fact that Flt-1 knock-out mice (but not mice lacking the VEGF-R1 tyrosine kinase domain) develop excessive proliferation of angioblasts which ultimately leads to death in utero(23). VEGFR-2 (also known as KDR or flk-1) largely mediates the mitogenic, pro-angiogenic and permeability-enhancing functions of VEGFA(17). VEGFR-2 undergoes dimerization and liganddependent tyrosine phosphorylation and results in a mitogenic, prosurvival signal that is partially mediated by activation of the PI3-kinase-Akt pathway and inhibition of activation of Bad and caspases(24). Cell proliferation may alternatively be stimulated via the PLCγ, PKC, MEK, ERK1/2 pathway (Figure 2)(25). Additionally, COX-2 and cPLA2 may be VEGF-R2-dependently activated, illustrating the linkage between inflammation, hypoxia, hypoxia-inducible factor (HIF), VEGF, angiogenesis and prostacyclin production(17). Because of the seminal role of VEGF in cancer angiogenesis(26), a particular focus on signaling pathways distal to VEGFR2 as well as



partnerships with the ligand or receptor have been investigated(27-30). For instance, binding of VEGF to neuropilin-1 (NRP1, a molecule implicated in neuronal guidance) enhances binding of VEGF165 to the VEGFR-2 and increases signal transduction(31). Of note, VEGF bound to the extracellular matrix promotes integrin-dependent cell spreading, migration and cell survival which do not require signaling via VEGF receptor tyrosine kinases. The integrin αvβ3 can also bind the VEGFR-2 (32, 33)and this integrin has been identified as the major galectin-3 binding protein (Figure 3). This interaction is of importance because VEGF-stimulated angiogenesis is reduced in galectin-3 KO cells(34). Another partnership exists between sphingosine1 (S1P) and VEGFR2. S1P can form complexes with VEGFR2 and phosphorylate VEGFR2(35). The interaction between VEGF and the Notch receptor is also important for VEGF activity regulation, in particular for the formation of new vessels(30). Upon activation, Notch decreases the expression levels of VEGF receptors 2 and 3 but increases levels of VEGFR-1, thus decreasing the overall angiogenic activity of VEGF(30). Until recently, VEGF signaling was thought to occur only at the plasma membrane, yet upon ligand binding, VEGF receptors are rapidly internalized in a clathrin- and dynamin dependent manner(36), and are targeted for proteasome degradation or recycling back to the plasma membrane. Recent data suggest that many signaling events associated with RTKs actually occur after endocytosis(37). Upon entry into the cell, VEGFR-2 continues to signal and the signaling varies depending on its location within a specific endosomal compartment. Many proteins are involved in the endosomal-dependent degradation of VEGFR-2, and, EphrinB2, one of the transmembrane ligands for the EphB receptor tyrosine kinases, is of critical importance. Indeed, virtually no uptake of VEGFR-2 occurs in the absence of Ephrin-B2, resulting in dysfunctional VEGF signaling(29). Lastly, although VEGFA is the predominant circulating ligand isoform, other related growth factors exist including VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF) have been described and characterized(14). VEGF isoform B, which binds to VEGFR1, serves


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multiple non-angiogenic functions; for example VEGFB prevents angiotensin II-induced cardiac diastolic dysfunction(38) and facilitates fatty acid transport in endothelial cells(39). VEGFC binds to the VEGF Receptor 3 (VEGFR3) and modulates lymphangiogenesis as well as tip cell sprouting during angiogenesis (Figure 3)(40). It is now readily appreciated that the large number of proteins that interact with VEGFR-2 and affect its signaling and endocytosis pose a significant challenge when investigating VEGF signaling in diseases such as PAH.

VEGF in Human and Experimental Pulmonary Arterial Hypertension The report that acute and chronic hypoxia cause an increase in the expression of VEGF and of the VEGF receptors VEGFR1 and VEGFR2 in the lung(41) set the stage for experimental pulmonary hypertension studies in rats and mice (Table 1). The majority of the studies confirmed that in the lung, VEGF and its receptors are increased in expression under chronic hypoxic conditions and also in lung tissue samples following monocrotaline (MCT)-induced lung injury. Table 1 provides a simple overview of the experimental PAH studies and their main results as they relate to VEGF isoforms. In human forms of angioobliterative PAH, the upregulated expression of VEGF and of VEGFR1 and VEGFR2 has been linked to pulmonary arteriolar endothelial cell growth(12), however, whether VEGF is causally involved in pulmonary vascular remodelling is not clear. Postmortem lung tissue samples from infants with PH associated with congenital diaphragmatic hernia have been assessed by immunohistochemistry, and increased expression of VEGF has been found in the “small, pressure-regulating pulmonary arteries, perhaps suggesting a role in vascular remodeling”(42). Lassus and coworkers subsequently measured VEGF levels in the tracheal aspirates of neonates with persistent pulmonary hypertension of the newborn (PPHN) and demonstrated a high expression of VEGF and VEGFR1(43). Similarly, the expression of the VEGF isoform C (VEGFC) and VEGFR3 is significantly increased in the lung tissue of fetuses and neonates with bronchopulmonary dysplasia, a condition frequently associated with PH(44).



Interestingly – and perhaps surprisingly – experimental overexpression of the VEGF isoform A (VEGFA) does not induce pulmonary hypertension and, conversely, blunts the development of hypoxia-induced PAH(45). Similar results were obtained by Farkas et al who showed that VEGF ameliorated pulmonary hypertension in a rat model of pulmonary fibrosis, while VEGF blockade worsened it(46). The role of other VEGF isoforms has so far only been partially addressed. Louzier et al have shown that adenovirus-mediated VEGFB overexpression attenuated chronic hypoxia-induced pulmonary PAH(47). It could be speculated that increased activation of VEGFR1 by VEGFB (which does not bind VEGFR2) negatively regulates the proangiogenic effects of VEGFR2 activation upon exposure to chronic hypoxia. Little is known about a potential role of VEGF-C in the development of PAH, however we have recently demonstrated that blocking VEGF-C’s main receptor (VEGFR3) ameliorates pulmonary hypertension in the SU5416/hypoxia rat model of severe PAH (A.Al Husseini et al data unpublished).

Development of severe PAH following VEGFR inhibition combined with chronic hypoxia: The paradox of VEGF receptor blockade-triggered pulmonary hypertension SU5416 (3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1, 3-dihydro-indol-2-one, commonly known as Sugen) is one of the early small molecule receptor tyrosine kinase inhibitors discovered through a screening process designed to identify compounds that inhibit VEGF-stimulated endothelial cell proliferation(48). The drug is highly lipophilic and stays active for a prolonged period of time when implanted subcutaneously in rats(49). SU5416 inhibits VEGFR1(50) and VEGFR2(48) and induces lung endothelial cell apoptosis, loss of small lung vessels and airspace enlargement(51). However, when combined with chronic hypoxia, SU5416 causes severe angioobliterative PAH and right heart failure(52). Indeed, that VEGF receptor blockade induces lung endothelial cell apoptosis and causes severe angioobliterative PAH when combined with chronic hypoxia continues to be surprising. Moreover, the fact that the model


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does not require any genetic manipulation underscores the importance of VEGF as a central and critically important component of lung vessel homeostasis(5). The different modalities of SU5416-based models of PAH have been recently discussed(53). That chronic hypoxia in combination with an angiogenesis-inhibiting drug causes PAH can be at first glance counterintuitive and paradoxical. However, the recent discovery of the long-term effects of receptor tyrosine kinase inhibitors, including the case-series of patients with dasatinibinduced PAH and the so-called ‘rebound’ effects (increased expression of growth factor receptor genes and proteins) after anti-VEGF therapies in cancer patients(54, 55), supplies the contextual underpinnings for the concept of VEGFR blockade-triggered angiogenesis. We propose that this framework may also be applied to expand the understanding of the pathobiology of the SU5416/Hypoxia rat model and human forms of severe angioproliferative PAH. While the initial discovery of the SU5416/Hypoxia rat model of severe PAH was a product of serendipity(52, 56), subsequent investigations of this non-genetic model have been systematic and have led to the formulation of new hypotheses about the pathogenesis of PAH(57). Indeed, the insights gained from SU5416/hypoxia model have raised the question whether in human forms of severe PAH one single or several endogenous ‘SU5416-like’ inhibitor(s) of VEGF signaling could be present and initiate the disease by inducing endothelial cell apoptosis in genetically susceptible individuals. As already mentioned, there are several candidates that could meet the criterion of endogenous VEGFR blockers (Table 3). The splice variant VEGF165b can bind the VEGFR-2 and block its signaling and the plasma cpncentration of this ligand protein has been found to be increased in patients with systemic sclerosis; it has been proposed that an imbalance between VEGF165a and VEGF165b is responsible for the characteristic endothelial cell apoptosis frequently observed in scleroderma patients(58). A potentially pathogenetic role for the soluble VEGFR-1 has also been proposed. Serum levels of the soluble receptor s-Flt are elevated in women with preeclampsia(59) and elevated levels of circulating s-Flt have recently been reported in patients with PAH(60, 61). Of interest,



pluripotent endothelial progenitor cells (EPC) express both isoforms of the VEGFR-1 (Flt), a membrane bound isoform (mFlt) and the soluble isoform (sFlt1), and it has been demonstrated that an endothelial-cell secreted cytokine TNFSF15 (also known as VEGI or TL1A) simultaneously promoted mFlt1 degradation and upregulated sFlt1 expression ,thus disrupting VEGF signaling(62). Whereas the mechanistic importance of EPC in PAH remain to be investigated, a hypothetical disruption of VEGF signaling in circulating cells as a potential mechanism in the pathogenesis of PAH should be investigated. As mentioned, ligands other than VEGF can bind VEGFR-2. Decorin is a secreted proteoglycan that belongs to a family of small leucin-rich proteoglycans, which regulate the activity of various growth factors and can act as a pan-receptor tyrosine kinase inhibitor(63). Buraschi and collaborators demonstrated that soluble decorin can bind the VEGFR-2 and induce Peg3dependent autophagy in microvascular endothelial cells and suppress angiogenesis(64). These findings may be of particular importance because increased VEGFA-independent, VEGFR-2Peg3-Beclin-1 mediated induction of autophagy leads to mitochondrial fragmentation, a phenomenon that has been associated with pathological lung vascular remodeling in pulmonary arterial hypertension(65). The Notch receptor modulates the expression of the three VEGF receptors and Notch signaling has been associated with the development of vascular remodeling in PAH. Human PAH is characterized by overexpression of the NOTCH3 gene in small pulmonary artery smooth muscle cells, and mice with homozygous deletion of Notch3 do not develop pulmonary hypertension in response to chronic hypoxia exposure(66). One can hypothesize that increased Notch signaling disrupts VEGF signaling by reducing the expression of VEGFR-2 and by reducing the levels of available VEGF ligand via the inducition of the expression of the ‘decoy’ receptor VEGF-R1. Although not mechanistically tested, VEGF-R2 endocytosis may also play a role in the pathogenesis of PAH. After ligand-dependent endocytosis, VEGF-R2 can still activate the Pi3K/Akt prosurvival pathway, even when endosome trafficking is impaired (67). In this


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particular scenario, disruption of the endosomal-dependent internalization of VEGFR-2 could lead to increased receptor activation within the cell, perhaps promoting proliferation of endothelial cells. Different potential roles of VEGF-R1 in addition to ligand sequestration should also be considered. Indeed, although the majority of the prosurvival and proangiogenic effects of VEGF are mediated by VEGF-R2, it has been demonstrated that under certain circumstances VEGFR1 may transmit a prosurvival signal in ECs via induction of the antiapoptotic protein survivin, which has been associated with the development of plexiform-like lesions in PAH(68, 69). If so, then VEGF receptor blockade by SU5416 could interrupt VEGFR-1 function by inhibiting its tyrosine kinase dependent signaling, but conceivably promote signaling via VEGFR-1, which remains functional even when its tyrosine kinase domain is blocked(70). Interestingly, although VEGF receptor blockade-induced endothelial cell apoptosis is necessary for the development of the disease in the SU5416-based models of angioobliterative PAH, a second component or second ‘hit’ is required ,and this second hit seems to drive the proliferation of apoptosis-resistant cells(71). If angioobliterative PAH can be understood as a process of vascular “wound healing gone awry”(57), one could postulate that these apoptosisresistant, quasi-malignant phenotypes result from an initial hit caused by pharmacological (i.e SU5416 or dasatinib) or endogenous (Table 3) VEGFR2 blockade, and proliferate under the influence of 1) “rebound” high levels of VEGF ligands secondary to VEGFR2 blockade or 2) physiological stressors associated with PAH such as shear stress, drugs, infections or inflammation that could increase circulating VEGF leves. In both scenarios, increased VEGF would signal through an un-inhibited VEGFR3(72), and/or alternatively signal via integrins(32). Whereas a VEGF ‘rebound’ component remains to be further characterized in patients with PAH, VEGF expression is elevated in SU5416 induced emphysema(51) and PAH(73). Cell proliferation could also be driven by intracellular mechanisms (where VEGF produced by the quasi-malignant cells is not secreted but affects signaling within the cells) or by other



angiogenic factors, for example Fibroblast Growth Factor (FGF), which is present in plexiformlike lesions(74). Cell proliferation could also be mediated by a VEGFR inhibition-triggered paradoxical upregulation of several receptor tyrosine kinases, as proposed by Ferrara and collaborators (see below)(24).

Receptor Tyrosine Kinase Remodeling VEGF receptor tyrosine kinase inhibitors are now in clinical use and it is known that patients treated with sunitinib or sorafenib have increased levels of circulating VEGF and placental growth factor (PLGF), indicating that inhibition of VEGFR can increase the levels of circulating VEGF ligands(75). There is also evidence that not only VEGF receptors but other RTK may play a role in the pathogenesis of PAH(76) such as the platelet-derived growth factor, c-kit and epidermal growth factor(77-79). Treatment with the tyrosine kinase inhibitor, imatinib was reported to be efficient in a single patient with PAH(80) and based on this case report and on experimental data, a proof-of-concept 24-week randomized, double-blind, placebo controlled pilot study treating patients with PAH has been conducted(81). The results were mixed mainly because imatinib treatment did not modify the primary end-point (6-minute walked distance) but did confer some hemodynamic improvements. Paradoxically, patients treated with the pantyrosine kinase inhibitor, dasatinib, have been reported to develop pulmonary hypertension, which was reversible upon discontinuation of dasatinib(55). Because imatinib and dasatinib target different kinases (Table 4), the discrepant treatment outcomes could be partially explained by blockade of specific kinases. Indeed, dasatinib can block Ephrins2-5 and thus potentially affect VEGF signaling by affecting VEGF endocytosis. Whether plasma levels of VEGF were elevated in the patients that developed PAH after treatment with the broadspectrum tyrosine kinase inhibitor dasatinib(55, 82), and thus contributed to the development of the disease, is unknown. Similar results have been reported when using bevacizumab, a humanized monoclonal antibody against VEGF used for the treatment of recurrent ovarian


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cancer. Of seventy-two participants enrolled in a clinical trial, 2 patients receiving bevacizumab developed pulmonary hypertension(83). Whether these patients developed pulmonary arterial hypertension was not reported. Mechanisms of resistance to anti-angiogenesis therapies and the consequences for vascular pathobiology are now being investigated in great detail(54, 75). Ferrara’s group recently investigated the VEGF-dependent phosphoproteome of endothelial cells incubated with VEGF and found that VEGF receptor signaling not only increased the phosphorylation of a large number of proteins, but also decreased the phosphorylation of many other peptides(24). The authors point out that the VEGF-regulated phosphoproteome is linked to the PI3 kinasemTORC2 (mammalian target of rapamycin complex 2) axis and the authors identified the forkhead box protein 01 (FoXO1) as a downstream regulator that mediated not only endothelial cell survival, but also the reprogramming of the VEGFR2 (Figure 4). Such a mechanism could facilitate the evolution of a drug-induced cell phenotype change. Upon binding of VEGF to the VEGFR2 FoXO1 is phosphorylated (and deactivated) resulting in a decreased expression of cleaved caspase3, and promoting endothelial cell growth induced by VEGF(24). Conversely, VEGFR2 blockade increased FoXO1 expression and induced apoptosis (Figure 4). The investigators also presented evidence for a FoXO1-dependent feedback loop which was responsible for the enhanced transcription of 16 different receptor-encoding genes, among them FGF-1R, TGF-βR2, ephrin-B2 and Kit. Taken together, it appears that a string of phosphorylation reactions initiated by VEGF ligand engagement with a VEGF receptor controls endothelial cell growth and death; likewise, VEGFR2 inhibition not only induces endothelial cell apoptosis, but also paradoxically triggers receptor tyrosine kinase reprogramming (Figure 4) that could participate in the emergence of apoptosis-resistant cells. These recently gained insights regarding the sequelae of chronic VEGFR blockade can now be applied to investigations designed to further unravel the paradox of VEGFR blockade-triggered angioproliferation in the SU5416/ hypoxia rat model of severe



PAH. These novel insights can also motivate investigators to search for evidence of endogenous inhibitors of VEGF signaling in patients with PAH. Specifically, it appears to be fruitful to utilize SU5416-dependent PAH models to examine the link between VEGFR blockade and inflammation. The mechanisms and roles of pulmonary vascular inflammation in pulmonary vascular remodeling remain largely unknown. Both animal and cell-based models should be used to evaluate whether fibroblast growth factor (FGF), TGF-β, ephrin and kit receptors promote angioobliteration, endothelial cell proliferation and cell-phenotype change. It is true that the role of endogenous SU5416-like inhibitors (Table 3) in the pathobiology of PAH is presently speculative, but, if supported by sufficient data, endogenous inhibitors of VEGFR signaling can be investigated in PAH. For instance, a proteome-wide analysis and quantitative data recently demonstrated that high blood levels of the antiangiogenic chemokine CXCL4(84) predicted the development of PAH in patients with systemic sclerosis(85).

Conclusion VEGF can be angiogenic or antiangiogenic depending on the ligand, the receptor, the signaling pathway and the duration of the ligand/receptor engagement(14). Perhaps, the most important lesson learned from SU5416-based models (particularly in combination with hypoxia) is that undisturbed VEGF signaling is necessary for the health of pulmonary vessels(5). Because VEGF, its splice variants together with its many complex signaling pathways are central to the homeostatic maintenance of pulmonary vessels, the VEGF receptors are, like BMPR2, of critical importance in severe PAH. An interaction between BMPR2 and VEGF should also be considered as it has been demonstrated that deletion of BMPR2 can suppress VEGF signaling(86). As in animal models, similarly the pathobiology of human angioproliferative PAH may be influenced by VEGF isoforms and receptor-dependent and VEGF-receptor-independent signaling.


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We sincerely hope that this perspective will inspire discussion and investigation of the hypothesis of opposing and complementary actions of VEGF in PAH. A deeper understanding of the role of angiogenesis and angiogenesis factors in the setting of PAH – with or without RV dysfunction – is necessary before anti-angiogenic drugs, in particular VEGF inhibitors, can be recommended for the treatment of PAH. Whether soluble endogenous SU5416-like VEGFR inhibitors and/or VEGF ligands signaling via VEGFR3 are involved in the pathogenesis of PAH or whether they contribute to the origin and nature of phenotypically altered apoptosis-resistant (perhaps stem cell-like) cells present in lung vascular lesions needs to be investigated.

Disclosures: An investigator-initiated preclinical research grant had been received from Actelion (NFV).

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Lung Cellular and Molecular Physiology 2012;302:L1023–36.

Tables Table 1: VEGF in experimental models of pulmonary hypertension Experimental Conditions

Animal

Readouts


Reference

Page 22 of 32


Page 23 of 32

Chronic hypoxia

Rat

Lung VEGF↑, VEGFR1↑,

Tuder et al (41)

VEGFR2↑ Chronic hypoxia

Rat

RV VEGF mRNA↑

Rat

RV

Partovian et al (87)

VEGF mRNA↓ Monocrotaline

Rat

Lung VEGF↑

Cho Y. et al (88)

Chronic hypoxia

Rat

Lung VEGF↑

Burke et al (89)

Chronic hypoxia

Mouse

↑ lung VEGFR2

IL-6 over

Steiner et al (90)

expression Chronic hypoxia

Rat

↓ VEGF lung

Yamamoto et al (91)


Mouse

↑ lung VEGFR1

Kwapiszewska et al (92)


Rat

↑ lung VEGF mRNA

Chronic hypoxia

Mouse

↑ lung VEGF

P53-/-

mRNA, increased

Christou et al (93)

Mizuno et al (94)

pulmonary hypertension Chronic hypoxia

Rat

↓ lung VEGFB

Sands et al (95)

protein expression Ductus arteriosus ligation

Fetal

↓ lung VEGF

lambs

expression


Grover et al (96)


Chronic hypoxia

Neonatal

↑ pulmonary artery

piglets

VEGF protein

Nadeau et al (97)

↓ VEGFR2 after 14 d of hypoxia Nitrofen-induced congenital

Rat

diaphragmatic hernia Nitrofen-induced congenital

Decreased lung VEGF expression

Rat

diaphragmatic hernia

Okazaki et al (98)

No change in lung VEGF, VEGFR1

Guilbert et al (99)

and VEGFR2 mRNA Nitrofen-induced congenital diaphragmatic hernia

Rat

Decreased lung VEGF


Sanz-Lopez et al (100)

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Table 2. VEGF expression in human PAH

VEGF Condition

Reference

Source expression/level

Pulmonary arterial

Lung tissue

Increased

(101)

Lung tissue

Increased

(42)

Tracheal aspirate

Increased

(43)

Lung tissue

Increased (VEGF-C)

(44)

hypertension Pulmonary hypertension and congenital diaphragmatic hernia Persistent pulmonary hypertension of the newborn Pulmonary hypertension and bronchopulmonary dysplasia



Table 3. Endogenous circulating inhibitors of VEGF signaling that could have a SU5416like effect

Protein/Factor VEGF 165b(58) sVEGFR1 (sflt-1) (22) Decorin(64) TNFSF15(62) CXCL4(85)


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Table 4. Tyrosine Kinase Receptor Inhibiting Profiles

Imatinib

Dasatinib

ABL

TXK

LIMK2

ARG

DDR1

MYT1

BCR-ABL

DDR2

PTK6/Brk

KIT

ACK

QIK

PDGFR

ACTR2B

QSK

DDR1

ACVR2

NQO2

BRAF

RET

EGFR/ERBB1

RIPK2

EPHA2-5

SLK

FAK

STK36/ULK

GAK

SYK

GCK

TAO3

HH498/TNNI3K

TESK2

ILK

LIMK1 *Table 4 was adapted from Montani et al. (55)


RAF-1

TYK2 ZAK


Figure Legends: Figure 1: Alternative splicing of VEGF-A pre-mRNA. The pre-mRNA of VEGF-A undergoes alternative splicing leading to pro-angiogenic isoforms notated with the number of amino acids and containing as last exon, the exon 8a stemming from the Proximal Splicing Site (PSS) located at the beginning of exon 8. The more recent subfamily of VEGF isoforms containing five members so far, are anti-angiogenic and have exon 8b as last exon, resulting from the splicing at the Distal Splicing Site (DSS) located after the exon 8a. (Reproduced with permission from Hilmi et al(102)). Figure 2: 1) In addition to the hypoxia-inducible transcription factors HIF-1α and HIF-2α other factors can modulate the expression of VEGF such as p53(103), PGC-1α(104), NfκB(105) among other. 2) Upon ligand binding signaling via the VEGF receptor 2 results in a sequence of kinase-dependent phosphorylation reactions, which result in blocking apoptosis and stimulating cell growth. 3) Activation of Notch receptor can prevent VEGFR2 expression. 4) VEGFR2 can also signal via Akt after endocytosis of the receptor. Endocytosed VEGFR2 can also undergo degradation or recycling. Figure 3. Binding of the different VEGF isoforms as well as the binding of soluble VEGFR1 (s-Flt) and placenta growth factor (PLGF) to the three VEGF receptors (R1-R3) initiates signaling. The integrin αvβ3 ligates the VEGFR2 and galectin 3. VEGF stimulated angiogenesis is reduced in cells where the galectin 3 gene has been knocked down. The VEGFR1 and R2 antagonist inhibits the cytoplasmic tyrosine kinase. Figure 4: The central role of the transcription factor Fox01 in the control of VEGFR2 controlled endothelial cell survival (1), VEGFR2 antagonist-induced endothelial cell death (2) and VEGFR2blockade-induced receptor tyrosine kinases (RTC) remodeling and development of VEGF receptor blockade-induced resistance (3). We postulate that continuous blockade leads to FoxO1-mediated upregulation of the VEGFR2. The VEGFR tyrosine kinase blocker Sugen 5416 (semaxinib) hypothetically leads to Fox01-dependent activation of apoptotic signals (for details see Zhuang et al(24).


Page 28 of 32


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Figure 1



Figure 2


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Page 31 of 32

Figure 3



Figure 4.


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Antifibrotic role of vascular endothelial growth factor in pulmonary fibrosis.

Vitronectin regulation of vascular endothelial growth factor-mediated angiogenesis.

Copper promotion of angiogenesis in isolated rat aortic ring: role of vascular endothelial growth factor.

Pivotal role of vascular endothelial growth factor pathway in tumor angiogenesis.

Expression and purification of functional human vascular endothelial growth factor-a121; the most important angiogenesis factor.

Plasma vascular endothelial growth factor A and placental growth factor: novel biomarkers of pulmonary hypertension in congenital diaphragmatic hernia.

New molecular targets of pulmonary vascular remodeling in pulmonary arterial hypertension: importance of endothelial communication.

The Critical Role of Pulmonary Arterial Compliance in Pulmonary Hypertension.

Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro.

Vascular Endothelial Growth Factor and Angiogenesis in the Regulation of Cutaneous Wound Repair.

The emerging role of the contractile and vascular reserves in pulmonary arterial hypertension.

Vascular endothelial growth factor receptor 3 signaling contributes to angioobliterative pulmonary hypertension.

Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells.

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Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo.

Prostaglandin E2 and vascular endothelial growth factor A mediate angiogenesis of human ovarian follicular endothelial cells.

Vascular endothelial growth factor-C: its unrevealed role in fibrogenesis.

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The imitation game in pulmonary arterial hypertension. Sex, bone morphogenetic protein receptor, and the estrogen paradox.

Vascular endothelial growth factor-dependent angiogenesis and dynamic vascular plasticity in the sensory circumventricular organs of adult mouse brain.

The role of combination therapy in managing pulmonary arterial hypertension.

Role of the serotonin transporter in pulmonary arterial hypertension.

Quercetin inhibits angiogenesis-mediated human retinoblastoma growth by targeting vascular endothelial growth factor receptor.