2.3. Angiogenesis

2.3.1. Vascular endothelial growth factor-A

Vascular endothelial growth factor-A (later called VEGF), belonging to the platelet-derived growth factor (PDGF)/VEGF family of growth factors, is a key regulator of angiogenesis. VEGF is a heparin-binding glycoprotein of about 45 kDa molecular weight that stimulates proliferation, migration, and proteolytic activity of endothelial cells (Ferrara & Henzel 1989, Leung et al. 1989, Pepper et al. 1991, Unemori et al. 1992, Lamoreaux et al. 1998). VEGF is also necessary for the survival of endothelial cells due to its ability to inhibit apoptosis and capillary regression (Gerber et al. 1998). Through its capacity to induce nitric oxide, VEGF may mediate vasodilatation and increase blood flow that precede angiogenesis (Liu et al. 2002, reviewed by Ziche & Morbidelli 2000). VEGF is also a potent mediator of increased vascular permeability; hence its other name, vascular permeability factor (Senger et al. 1983).

VEGF is synthesized by numerous cell lines (reviewed by Grützkau et al. 1998) and secreted through conventional pathways (Leung et al. 1989). To date, six human VEGF mRNA species, encoding VEGF isoforms of 121, 145, 165, 183, 189 and 206 amino acids, are produced by alternative splicing of the VEGF mRNA. An important biological property that distinguishes the different VEGF isoforms is their heparin and heparan-sulphate-binding ability. VEGF₁₂₁ is the most soluble isoform and does not bind to heparin or extracellular matrix (ECM), whereas VEGF₁₈₉ and VEGF₂₀₆ are almost completely sequestered in the ECM. VEGF₁₆₅ is a heparin-binding protein, and 50–70% of VEGF₁₆₅ remains bound to cell surface and ECM. VEGF₁₂₁, VEGF₁₄₅ and VEGF₁₆₅ induce angiogenesis in vivo, but VEGF₁₄₅ is found mainly to be expressed in cells derived from reproductive organs, as is apparently also VEGF₂₀₆. VEGF proteins may become available to target cells as freely diffusible proteins (VEGF₁₂₁ or VEGF₁₆₅) or following protease activation and cleavage. VEGF isoforms in the ECM constitute a reservoir of growth factors that can be slowly released by exposure to heparin, heparan sulphate and heparinases, or more rapidly mobilized by specific proteolytic enzymes such as plasmin and urokinase-type plasminogen activator (uPA). The bioactive VEGF consisting of the first 110 NH₂ terminal amino acids is generated in the extracellular compartment (reviewed by Neufeld et al. 1999, Ferrara 2001, and Robinson & Stringer 2001). Alternative splicing of VEGF may have an important role in the regulation of VEGF activity in developing and injured lungs (Watkins et al. 1999).

In the lung of human fetus, VEGF has been expressed in bronchial epithelium and in smooth muscle cells and pericytes of vessels (Shifren et al. 1994, Acarregui et al. 1999). Similarly, in normal adult human lung, VEGF has been expressed in bronchial epithelial cells, type II pneumocytes, smooth muscle cells of arterioles and bronchioles, and in alveolar macrophages (Fehrenbach et al. 1999a). The major site of VEGF mRNA expression in the human lung is probably alveolar type II epithelial cells (Boussat et al. 2000). Also, the normal human epithelial lining fluid (ELF) contains high concentrations of VEGF (Kaner & Crystal 2001). These results suggest that in lung, VEGF is involved not only in angiogenesis, but also in the maintenance and regulation of existing vessels with a paracrine mechanism of action between endothelial and nearby cells. Hypoxia stimulates VEGF gene expression in human endothelial and vascular smooth muscle cells and in fibroblasts in vitro (Brogi et al. 1994, Namiki et al. 1995, Jackson et al. 1997). In animal models, both acute and chronic hypoxia increase lung tissue gene expression for VEGF and its receptors Flt-1 and Flk-1 (Tuder et al. 1995, Christou et al. 1998).

Recently, there have been observations suggesting that VEGF could also have actions not related to angiogenesis: VEGF may have nonendothelial target cells in testis regulating male fertility (Korpelainen et al. 1998) and act as a direct autocrine growth factor for tumor cells in non-small cell lung carcinomas (Decaussin et al. 1999), renal tubular cells (Kanellis et al. 2000) and regenerating myocytes (Rissanen et al. 2002).

2.3.2. Flt-1 and Flk-1

VEGF has at least two receptors, called Flt-1(fms-like tyrosine kinase)/VEGFR-1 and Flk-1(fetal liver kinase-1)/VEGFR-2/KDR(kinase domain region) (deVries et al. 1992, Terman et al. 1992). Flt-1 and Flk-1 are transmembrane tyrosine kinase receptors, which are both upregulated by hypoxia (Tuder et al. 1995, Christou et al. 1998). When activated, Flt-1 and Flk-1 promote angiogenesis, but with somewhat different functions and signal transduction cascades (Waltenberger et al. 1994). Flt-1 is more closely associated with cell differentiation, while Flk-1 is thought to have a more important role in VEGF-mediated endothelial cell proliferation (Shibuya et al. 1990). Originally, Flt-1 and Flk-1 were thought to be endothelial cell-specific. Subsequently, however, trophoblast cells, monocytes, renal mesangial and tubular cells, hematopoietic stem cells, megakaryocytes, retinal progenitor cells, pancreatic duct cells, non-small cell lung carcinoma cells, lung fibroblasts and muscle cells have also been shown to express one or both of these receptors (reviewed by Neufeld et al. 1999, Öberg et al. 1994, Decaussin et al.1999, Kanellis et al. 2000, Ishida et al. 2001, Rissanen et al. 2002). According to recent studies it seems possible that VEGF has autocrine actions both on endothelial and non-endothelial cells via Flt-1 or Flk-1 (Namiki et al. 1995, Decaussin et al. 1999, Kanellis et al. 2000, Rissanen et al. 2002).

2.3.3. Basic fibroblast growth factor

Basic fibroblast growth factor (later called bFGF; also called FGF-2) is a well-documented angiogenic growth factor and induces endothelial cell replication, migration and extracellular proteolysis (Gospodarowicz et al. 1983, Montesano et al. 1986, Tsuboi 1990). bFGF is produced by several normal and tumor cells, endothelial cells included, and has autocrine activities on angiogenesis (Moscatelli et al. 1986, Schweigerer et al. 1987, Sato & Rifkin 1988). bFGF may promote angiogenesis both by a direct effect on endothelial cells and indirectly by the upregulation of VEGF in endothelial cells (Stavri et al. 1995), and bFGF and VEGF have a synergistic effect in the induction of angiogenesis both in vitro (Pepper et al. 1992) and in vivo (Mattern et al. 1997). Also, induction of bFGF induced angiogenesis is partly dependent on the activation of VEGF (Tille et al. 2001).

bFGF belongs to the FGF superfamily, which contains at least twenty distinct FGFs. Four different bFGF polypeptides of 18–24.2 kDa can be formed from the one fgf-2 gene (reviewed by Powers et al. 2000). bFGF does not code for a signal sequence required for vectorial translocation into endoplasmic reticulum and can be released through yet unknown mechanism of exocytosis independent of the ER-Golgi pathway (Abraham et al. 1986, Mignatti et al. 1992). Mechanical damage such as wounding has also been proposed as one mechanism for release of biologically active bFGF from endothelial cells (McNeil et al. 1989). In the ECM and on the cell surface, bFGF is bound to heparan-like glycosaminoglycans (HLGAGs) of the ECM and is present in BMs in vivo (Folkman et al. 1988). The association to HLGAGs may afford bFGF protection from proteolysis, besides creating a localized and persistent reservoir of the growth factor. bFGF is released from ECM by enzymatic cleavage of proteolytic enzymes, or by binding to a carrier protein, which can then deliver bFGF to the tyrosine kinase transmembrane receptors (reviewed by Powers et al. 2000).

Like VEGF, bFGF also inhibits apoptosis of endothelial cells (Karsan et al. 1997). bFGF is also a well-characterized fibroblast growth factor inducing mitogenic and chemotactic activity and differentiation of some other cell types of mesodermal and neuroectodermal origin (reviewed by Mignatti & Rifkin 1991).

In normal adult human lung, bFGF has been expressed in the BMs of blood vessels, epithelial cells lining trachea and major bronchi, and variably in endothelial cells. In bleomycin-induced acute lung injury in the rat, there was strong bFGF positivity in vascular media and endothelial cells, mast cells, and in fibrotic areas (Liebler et al. 1997). On the other hand, in an animal model with oxygen stress induced fibrosing alveolitis, bFGF was upregulated after injury and synthesized by type II pneumocytes (Sannes et al 1996).

2.3.4. Angiogenesis in pulmonary fibrosis

There are only a few studies on angiogenesis in human pulmonary fibrosis, and the results must be evaluated in the light of former and current knowledge on pathogenesis of pulmonary fibrosis. When summarized, the results suggest not only loss of gas changing capillaries, but also focal increase in the amount of capillaries in fibrous areas. In 1953, Golden & Bronk observed a variable morphology of alveolar vasculature in diffuse pulmonary fibrosis (Golden & Bronk 1953). However, with current knowledge, some of the changes interpreted by them as alveolar wall hypertrophy probably represent collapse of alveolar walls. A decrease in the number of septal capillaries and in the effective gas exchanging surface of capillaries has been shown in fibrotic human lung (Gracey et al. 1968). Based on morphometric, angiographic, light microscopic and ultrastructural studies, Bignon et al. showed in 1974 that in diffuse pulmonary fibrosis, the number of capillaries was decreased in areas with severe fibrosis, but intensively increased in fibrous areas at some distance from air space surface. The results also suggested that these capillaries originate from bronchial circulation (Bignon et al. 1974). This was in line with the study of Turner-Warwick, who showed the presence of systemic pulmonary microvasculature anastomoses in pulmonary fibrosis (Turner-Warwick 1963). From studying the biopsy specimens of patients who underwent open lung biopsy for fibrosing alveolitis, Coalson (1982) found alteration of the capillary structure together with evidence for both endothelial cell death and regeneration.

The extent of capillarization in newly formed connective tissue has been unclear so far. The number of capillaries within newly formed connective tissue has been reported to be small despite the underlying disease (Kawanami et al. 1983, Basset et al. 1986). The observations of an early phase of the fibrous process with prominent proliferating capillaries (Anderson & Foraker 1960, Hasleton 1983) or a stereotyped response in lung injury with a granulation tissue response and budding of capillaries (Snider 1986) have been treated with caution after the emergence of the concept of the collapse of the alveolar walls. However, in a profound study Peyrol et al. showed that in BOOP there are intraluminal protrusions of altered capillaries in the areas of intra-alveolar fibrosis (Peyrol et al. 1990).

Despite the active research on growth factors in pulmonary fibrosis, little interest has been focused on angiogenic growth factors. In surgical wounds, functional VEGF is a key mediator in wound angiogenesis, fluid accumulation, and granulation tissue formation (Howdieshell et al. 2001). In human lung, bFGF inducing granulation tissue formation has been identified from bronchoalveolar lavage fluid obtained from patients suffering from an acute lung injury, and alveolar macrophages were identified to be the cellular source bFGF (Henke et al. 1991, Henke et al. 1993). Keane et al. showed in 1997 that human UIP fibroblasts induced an angiogenic response partly caused by a CXC chemokine, IL-8. They also observed that areas associated with IL-8 immunolocalization demonstrated significant vascular remodeling in UIP. In a rat model of bleomycin-induced pulmonary fibrosis, an increased number of VEGF-positive type II alveolar epithelial cells and myofibroblasts were identified in fibrotic lesions (Fehrenbach et al. 1999a).