The Role of Vascular Endothelial Growth Factor in Lung Injury and Repair

in Lung Injury and Repair

J. Varet and A.B. Millar

Introduction

Acute lung injury (ALI), along with its most severe form acute respiratory distress syndrome (ARDS), is one of the most challenging conditions in critical care medi- cine. ARDS continues to have a mortality of more than 35 % despite improvements in ventilator strategies and management of sepsis [1]. Inflammation and increased vascular permeability are characteristics of ARDS. Vascular endothelial growth fac- tor-A (VEGF-A) is a multi-functional cytokine known to play a pivotal role in angio- genesis and vascular permeability leading to interest in its potential role in ARDS.

There is a body of work suggesting that VEGF plays a major role in lung develop- ment; however, it is expressed more highly in the healthy adult lung than any other organ suggesting a physiological role [2]. This apparent contradiction leads to con- troversy about the role of VEGF in ARDS.

Acute Lung Injury and Acute Respiratory Distress Syndrome

The characteristics of ARDS as first described by Ashbaugh et al. are well known [3].

A lack of stringency in the definition of this condition made it difficult to undertake comparative studies. In 1994, the American – European Consensus Conference Com- mittee proposed the currently used definition of ARDS, which is not limited to adults. They described ARDS as a ‘syndrome of inflammation and increased perme- ability’ and suggested the term ALI to describe the continuum of pathological responses to pulmonary parenchymal injury. They defined ARDS as a severe form of ALI and a syndrome of acute pulmonary inflammation and resultant increased cap- illary endothelial permeability [4].

ARDS is characterized by inflammation and pulmonary edema, resulting from

increased permeability of the alveolar-capillary membrane. The precise sequence of

events occurring during ARDS is still unclear, however, several phases can be distin-

guished in the time course of the ARDS process. The earliest morphological abnor-

malities are injury to the lung microvascular endothelial cells and diffuse aggrega-

tion of polymorphonuclear leukocytes [5]. Activation of the leukocytes into the pul-

monary interstitium causes lung injury resulting in plasma proteins leaking into the

interstitium and the alveolar spaces. This acute or exudative early phase lasts only

for a few hours. As early as 24 h afterwards, a proliferative phase starts, in which

fibroblasts infiltrate and remodel the site of inflammation. After the acute phase,

some patients will show a rapid resolution, however, other patients will develop a

fibrotic response that results in consolidation and fibrosis of the pulmonary paren-

chyma in the late phase of ARDS, around day 5 – 7. Importantly, several studies have emphasized the critical importance of the degree of alveolar epithelial injury and its potential for repair in the pathogenesis and recovery from lung injury [5].

VEGF Biology

In mammals, the superfamily of VEGF proteins consists of five members: Placental growth factor (PlGF), VEGF-A, VEGF-B, VEGF-C, and VEGF-D, that are structurally homologous. However, there are molecular and functional diversities among these subtypes [6]. This chapter is confined to the importance of VEGF-A, termed VEGF throughout the text. VEGF is a dimeric 34 – 46 kDa glycoprotein, produced in vari- ous cell types: Cancer cells, inflammatory cells, fibroblasts, smooth muscle cells and epithelial cells. VEGF stimulates endothelial cell proliferation and is also a well- known pro survival factor for endothelial cells, inducing the expression of anti-apo- ptotic factors such as Bcl2 [7]. Importantly, VEGF mediates the secretion and activa- tion of enzymes involved in matrix degradation. It also stimulates endothelial cell migration and their organization in capillary tubes in vitro and in vivo. VEGF criti- cally regulates vasculogenesis such that embryos lacking a single VEGF allele have a lethal phenotype due to abnormal vascular development, including that of the lung [8]. In addition, VEGF increases microvascular permeability, up to 20,000 times more potently than histamine. This VEGF-increased vascular permeability also accounts for its active role in inflammation. It also stimulates arteriole vasodilata- tion via enhanced production of nitric oxide (NO). Although initially described as a specific growth factor for endothelial cells, targets for VEGF bioactivity outside the vascular endothelium have been discovered [6].

VEGF Isoforms

Alternate splicing of the VEGF transcript leads to the generation of several isoforms of differing sizes, the subscript relating to the number of amino acids present (VEGF

, VEGF

, VEGF

, VEGF

, VEGF

, VEGF

and VEGF

). VEGF

is physiologically the most abundant splice variant. VEGF

, lacking exons 6 and 7, does not bind heparin sulfate and is freely diffusible. In contrast, the longer iso- forms (VEGF

and VEGF

) have heparin binding sites and are cell surface and extracellular matrix associated. VEGF

has intermediate properties [7]. Recently, a new family of VEGF isoforms has been identified, VEGF

b, with differing amino acids in the exon 8 position, and some inhibitory properties [9].

VEGF Receptors and Co-receptors

VEGF isoforms bind to the tyrosine kinase receptors, VEGF receptor 1 (VEGF-R1 or

flt1) and VEGF receptor 2 (VEGF-R2 or KDR or flk-1) [7]. Although VEGF affinity

for VEGF-R1 is ten-fold higher than that for VEGF-R2, VEGF-R1 is a weak kinase

compared to VEGF-R2 that exhibits a strong autophosphorylation in response to

VEGF binding. This has lead to the hypothesis that VEGF-R1 may act as a decoy

receptor, by preventing binding of VEGF to VEGF-R2. This hypothesis has been

reinforced by the fact that PlGF, which only binds VEGF-R1, potentiates the effect of

VEGF by displacement of VEGF from VEGF-R1 binding. Further evidence of differ- ing functions comes from knock out murine studies [6]. Several groups have reported crosstalk between VEGF-R1 and 2 [6]. Currently VEGF-R2 is regarded as the main signaling receptor for VEGF bioactivity, most evidence coming from endo- thelial cell studies.

The function and activity of the VEGF-R can be modulated by co-receptors. The binding of VEGF

to VEGF-R2 is enhanced by heparin and heparin amplifies sig- naling by VEGF

but not VEGF

[6]. In addition, VEGF can bind cell surface gly- coproteins called neuropilin (NRP): NRP-1 and NRP-2. In contrast to the VEGF-R, the neuropilins bind VEGF in an isoform specific manner [6]. They are expressed by endothelial cells in many adult tissues but lack the intracellular component contain- ing tyrosine kinase activity. NRP-1 is expressed mainly in arteries whereas NRP-2 is expressed on venous and lymphatic vessels. However, several studies have reported their presence on numerous other cell types [6]. NRP-1 binds VEGF

through its exon 7 and enhances the effect of VEGF

by increasing its binding to VEGF-R2 [10]. In addition, it has been shown recently that NP-1 is an essential mediator for VEGF

-mediated endothelial cell permeability through VEGF-R2 in the lung vascu- lature [11]. These results may account for the permeability properties and greater mitogenic potency of VEGF

compared with the VEGF

isoform, unable to bind NP-1. NRP-2 can bind VEGF

and VEGF

but not VEGF

. It has also been sug- gested that NRP-2 could interact with VEGF-R1.

Regulation of VEGF Bioactivity

Ubiquitous cell types produce VEGF, and hypoxia is currently regarded as the major factor inducing VEGF expression and is certainly the most widely studied. HIF-1 and HIF-2, the hypoxia inducible factors, are transcription factors regulating gene expression according to oxygen tension. HIF is composed of two subunits: HIF- q , a nuclear protein constitutively expressed and HIF- [ , whose activity is oxygen depen- dent. There are mainly two possible subunits for HIF- [ : 1 [ , which is ubiquitously expressed and 2 [ , notably expressed in endothelial cells and type II pneumocytes.

Under hypoxic conditions, HIF-1 [ stability and bioactivity are increased and after heterodimerization with HIF- [ it stimulates VEGF gene transcription by binding to the hypoxia-responsive element located in its promoter [12]. However, hypoxia can also stimulate VEGF expression by another mechanism, transcription independent.

This has been described in retinal epithelial cells, where hypoxia increased VEGF mRNA stability [13]. Several growth factors have been involved in stimulation of VEGF production, such as platelet-derived growth factor, transforming growth fac- tor (TGF) - [ and - q , insulin like growth factor and keratinocyte growth factor. In addition, VEGF can be induced by reactive oxygen species (ROS), glucose depriva- tion, inflammatory cytokines, such as tumor necrosis factor- [ (TNF- [ ), interleukin (IL)-6 and interferon (IFN) gamma and mechanical forces per se [14].

Another very important mechanism regulating VEGF activity is the splicing of VEGF RNA, leading to this diversity of isoforms [15]. However, the mechanisms by which splicing occurs and is regulated remain to be elucidated.

Post-translational control of VEGF activity has also been described. Proteolytic

processing also regulates the bioactivity of VEGF. It has been demonstrated that

native VEGF

requires maturation by urokinase to bind to VEGF-R2 and stimulate

endothelial cell proliferation. In contrast, plasmin digestion of VEGF

yields an

amino-terminal homodimer (VEGF1 – 110) containing binding sites for VEGF-R1 and VEGF-R2, and a carboxyl-terminal fragment having a binding site for NRP. This VEGF1 – 110 exhibits a reduced mitogenic activity [16].

Regulation of VEGF Receptor and Co-receptor Activity

Hypoxia induces VEGF-R1 expression, whereas VEGF-R2 does not have hypoxia responsive elements in its promoter [17]. However, hypoxia could indirectly up reg- ulate VEGF-R2 expression. Endothelial cell VEGF-R2 expression is increased by ischemia. TGF- q 1 has been shown to decrease endothelial VEGF-R2 expression [18].

Conflicting results about TNF- [ -regulated VEGF-R2 and NRP-1 expression in endo- thelial cells have been reported [19]. Like VEGF expression, VEGF-R expression could be modulated directly or indirectly by mechanical forces [14]. In macro- phages, lipopolysaccharide (LPS) upregulates the expression of VEGF-R1 mRNA and increases specific binding for VEGF [20]. However, the mechanisms regulating VEGF-R and NRP expression remain largely to explore. Alternate splicing or proteo- lytic processing of VEGF-R gives rise respectively to soluble variants of VEGF-R1 (sflt1) or VEGF-R2. sflt1 lacks the cytoplasmic and membrane part of VEGF-R1, but it still has the same binding capacity to VEGF as VEGF-R1 and acts as an inhibitor of VEGF activity [7].

It is readily apparent from this brief review that VEGF bioactivity is complex as befits such a potent molecule, particularly in an organ such as the lung.

VEGF in the Lung

The main function of the lung is gas exchange. This function critically depends on the ‘fine-tuning’ between ventilation and perfusion. The integrity and functionality of the alveolar capillary barrier is, therefore, crucial. The normal alveolar barrier is composed of three different structures: The capillary endothelium, the interstitial space (basement membrane and the extracellular matrix), and the alveolar epithe- lium. The alveolar epithelium is constituted of alveolar epithelial type I and type II cells (respectively pneumocytes type 1 and 2 or AT1 and AT11). The AT1 are flat cells and cover more than 90 % of the alveolar surface area. Their thin cytoplasm is optimized for respiratory gas exchange. The AT11 are cuboidal cells, located in the corners of the alveolar space, and they constitute about 60 % of alveolar epithelial cells while they cover only about 5 % of the alveolar surface in adult mammals.

These AT11 have several functions: They secrete surfactant, are the progenitor cells

of the alveolus, and possess the engineering required for active alveolar liquid clear-

ance [21]. However, the lung is the organ where the highest concentrations of VEGF

are found. Why is VEGF present in an organ where angiogenesis and vascular per-

meability are unusual? In healthy human subjects, VEGF protein is highly compart-

mentalized within the lung. The alveolar levels of VEGF are even 500 times higher

than in plasma. It has been hypothesized that this could function as a physiological

reservoir. VEGF would be slowly released across the alveolar epithelium to stimulate

the lung microvascular endothelial cells, maintaining the integrity of the capillary

structure. However, in case of alveolar injury, this spatial compartmentalization

would lead to a strong induction of VEGF-stimulated endothelial cell permeability

resulting in pulmonary edema [22].

The Epithelial Surface

In vitro studies have confirmed the abundant secretion of VEGF by human AT II [23].

In A549 cells (a tumor derived lung epithelial cell line), VEGF secretion is increased in response to LPS, neutrophil elastase, keratinocyte growth factor and TGF- q , and hyp- oxia [24, 25]. Moreover, hypoxia stimulates apical VEGF secretion by primary rat alve- olar AT II in vitro and VEGF in bronchoalveolar lavage (BAL) in vivo [23].

In another study, rats exposed to hyperoxia showed a significant decrease in VEGF expression [26]. Distal airway epithelial cells from human fetal lung express VEGF-R2 and NRP-1. Interestingly, both cytoplasmic and nuclear staining of VEGF-R2 were detected in many of the distal airway epithelial cells [27]. Immunohistochemical stud- ies in normal mice lungs have shown that AT I can weakly express VEGF-R1 but not VEGF-R2. In contrast, AT II display a strong expression of VEGF-R1 and a weak expression for VEGF-R2. Alveolar macrophages express VEGF-R1 and could occasion- ally express VEGF-R2 [28]. These results are in accordance with the work of Fehren- bach et al., done in rat lungs [29]. In addition, it has been shown by immunohisto- chemistry that, in the adult lungs, alveolar cells express NRP-1 [30].

The Endothelial Surface

Lung microvascular endothelial cells express VEGF-R1 and 2 and at least NRP-1. VEGF is well known for its ability to stimulate endothelial cell survival, proliferation and che- motaxis [6]. In addition, VEGF increases lung endothelial permeability, via a mechanism that strictly depends on the presence of NRP-1. Interestingly, NRP-1 enhances, but is not essential to, VEGF-induced cell proliferation and chemotaxis through VEGF-R2 [11].

Role of VEGF in the Alveolar Space Pneumotrophic Effect of VEGF

It has been shown that VEGF is mitogen for human retinal pigment epithelial cells and is a survival factor for podocytes, cells involved in the glomerular capillary bar- rier [31, 32]. Trophic paracrine activity of VEGF has also been described in the liver, where VEGF stimulates the release of hepatocyte growth factor (HGF) by endothelial cells, promoting the growth of hepatocytes [33]. Therefore, the potential of VEGF as a pneumotrophic factor has been considered.

The tumor derived epithelial cell line, A549, expresses both VEGF-R1 and VEGF-

R2. In an acid exposure model of injury in vitro leading to suppression of A549 pro-

liferation and VEGF secretion, exogenous VEGF

is capable of restoring cellular

proliferation. VEGF-R neutralizing antibodies of either VEGF-R1 and 2 suppressed

proliferation of acid exposed A549 without altering control cell proliferation. These

results suggest that healthy A549 cells are less dependent on VEGF for their prolifer-

ation than acid injured cells [34]. It has recently been shown that downregulation of

either VEGF or VEGF-R1 by small interfering RNA (siRNA) in A549 cells reduced

their proliferation and induced morphological changes [28]. These results suggest

that VEGF may be a survival factor for the A549. However, it is still not clear if these

effects occur via a direct autocrine pathway or indirectly via the stimulation of

secretion of other growth factors by these same cells. Similar mechanisms have been

observed in several other malignant cell types.

In non-cancerous models, it has been shown that exogenous VEGF acts as a growth factor on human fetal lung explants in vitro. This study suggests a possible role of VEGF as an epithelial cell growth factor [27]. In contrast, no such effect was detected in isolated fetal rat type 2 pneumocytes, one explanation being a paracrine rather than an autocrine role of VEGF [35].

In summary, there is conflicting and limited evidence regarding the pneumotro- phic role of VEGF in the human lung.

Regulation of Surfactant Production

This also remains a controversial topic. Compernolle et al., showed that HIF-2a -/- mice developed neonatal fatal respiratory distress syndrome due to insufficient sur- factant production by type 2 pneumocytes [36]. These mice had lower VEGF levels in alveolar cells than controls suggesting that HIF-2a primarily regulates VEGF expression in fetal type 2 pneumocytes and, therefore, VEGF might regulate surfac- tant production. Subsequently, VEGF directly increased the transcription of surfac- tant protein B and C, in cultured rat type 2 pneumocytes [36]. In contrast, Raoul et al., showed that VEGF could directly stimulate only surfactant B protein transcrip- tion, in fetal rat AE2 in vitro [35]. Brown et al., had reported enhanced surfactant protein A and C expression in fetal lung explants, but unchanged surfactant protein B expression [27]. Recently, a study on cultured ovine type II pneumocytes in vitro has reported no direct effect of VEGF on surfactant protein transcription [37]. Many of these contradictions may be related to differing species and experimental design.

Effect of VEGF on Alveolar Structure

Compernolle et al., have described HIF-2a -/- mice suffering from respiratory dis- tress syndrome and demonstrated abnormal alveolar epithelium, attributable to impaired cellular differentiation [36]. In a model of prematurity, the same group showed that intra-uterine delivery of VEGF prevented development of respiratory distress syndrome.

Furthermore, intra-amniotically injected VEGF-R2 neutralizing antibody, remain- ing in the alveolar space, led to the development of respiratory distress. The benefi- cial effect of VEGF was associated with more normal alveolar septa through differ- entiation of alveolar cells, crucial for gas exchange, and inhibited by the addition of VEGF-R2. Similarly, mice with a deficiency of VEGF

or VEGF

isoform or of the HIF-binding site in the VEGF promoter died from respiratory distress syndrome.

These data strongly suggest an essential role of VEGF (

or

) in lung maturation [36]. However, Zeng et al., have shown that over-expression of VEGF targeted to the developing pulmonary epithelium in transgenic mice resulted in disruption of the lung branching morphogenesis and a lack of type I cell differentiation [37, 38].

These apparent discrepancies suggest that a tight regulation of the VEGF system in the alveolus is crucial to lung maturation and, therefore, possibly to lung repair.

VEGF in Lung Injury and Repair (Figure 1) Lung Injury and the VEGF System

The main features of lung injury are inflammation and vascular leakage. Since VEGF

strongly stimulates microvascular endothelial cell permeability and is an inflamma-

Fig. 1. Diagrammatic representation of the human alveolar capillary unit in (a) normal health subjects, (b)

subjects with lung injury, and (c), during the recovery phase. From [48].

tory mediator (via its monocyte chemoatactic effects), its role during lung injury has been the focus of much investigation.

Adenoviral VEGF

delivery to murine neonatal lung led to pulmonary edema and increased pulmonary capillary permeability [39]. Similarly, VEGF

over- expressing mice, targeted to respiratory epithelial cells, demonstrated pulmonary hemorrhage, endothelial destruction, and alveolar remodelling in an emphysema- like phenotype [40]. However, a recent study has demonstrated that NRP-1 inhibi- tion reduced VEGF-induced permeability [11]. This result emphasizes the fact that the effects of VEGF are tightly modulated by the specific combination of its recep- tors.

Lung Repair after Injury

One of the main determinants of the outcome of lung injury is the degree of alveolar epithelial injury. After damage of the alveolar barrier, the proliferative phase of lung injury is characterized by hyperplastic AT II cells. The AT II cells migrate and prolif- erate trying to restore epithelial integrity. This is crucial since integrity of the alveo- lar epithelium is essential for alveolar fluid clearance, the AT II cells possessing the engineering required for active ion transport. Integrity of this epithelium is also important for surfactant metabolism and for immune functions [4].

Lung Repair and VEGF

VEGF has been widely studied in repair mechanisms of organs other than the lung in relation to its angiogenic effects. In the lung the necessity for close approxima- tion of endothelial and epithelial surfaces can be considered in an angiogenic con- text. It has been described that after hyperoxia injury, in the rat, there is a decreased VEGF expression in the lung associated with apoptotic endothelial cells and epithelial cells [26]. Inhibition of angiogenesis and specific blocking of VEGF signaling leads to abnormal lung structure in rats [41 – 43]. In contrast, hyperoxia- induced lung damage in newborn rats is rescued by intratracheal adenovirus-medi- ated VEGF [43]. In a similar model, the intramuscular injection of VEGF

tran- siently worsened the lung edema, but subsequently improved recovery of lung structure [44]. In adult rats treated with a VEGF-R blocker, increased apoptosis and emphysematous like changes were found in the lung [45]. Finally, in transgenic mice over-expressing IL-13 exposure to hyperoxia reduces lung injury. This protec- tive effect has been linked to increased VEGF production in the lung and adminis- tration of VEGF neutralizing antibody decreases this protective effect of IL-13 over- expression [46]. All these data suggest a complex crosstalk between the endothe- lium and the alveolar epithelium.

If we consider human data then it has been shown that in ARDS patients there

are increased numbers of apoptotic endothelial cells and a reduced endothelial area

compared to controls [47]. Moreover, in several models of lung injury decreased pul-

monary levels of VEGF are observed compared to control, with recovery of intrapul-

monary VEGF levels to pre-injury levels following recovery [48]. Several other stud-

ies of lung injury in humans also describe a reduction in free VEGF levels in epithe-

lial lining fluid in ARDS patients compared to controls, in addition to similar reduc-

tions in intrapulmonary VEGF levels in other forms of lung injury [49, 50]. All these

data suggest that impaired VEGF expression or bioactivity could lead to endothelial

cell loss and, therefore, might compromise alveolar repair. Since the major source of

VEGF in the lung is the alveolar epithelium, this adds to the concept that the degree of epithelial injury is determinant for the outcome of the disorder. However, a tight regulation of the VEGF system is certainly involved in this positive feedback between epithelial and endothelial cells.

Conclusion

VEGF is a multi-functional growth factor. Several isoforms and receptors of VEGF have been described with potential to regulate its bioactivity. The normal lung expresses high concentrations of VEGF and has got a strong compartmentalization of the growth factor in the alveolar space. VEGF is well recognized to be angiogenic and increase permeability of capillary endothelial cells leading to interest in its role in ALI/ARDS, characterized by a disruption of the alveolar capillary membrane and an inflammatory reaction. Furthermore, the degree of epithelial injury appears to be crucial to the outcome of the disorder. However, the precise role of VEGF in normal lung function and during lung injury and repair remain unclear. Increased under- standing of both the complex biology of the VEGF family and crosstalk between the cellular constituents within the lung are needed to establish the role of VEGF.

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