1. Hepatocyte Growth Factor in Cardiovascular System

368R. Morishita & T. Ogihara

a)HGF gene (70kb)

·transcript

b)HGF mRNA (6kb)

·translation

c)pro-HGF

·processing(activation)

d)HGF protein

α chain (69kD)

Arg

Val ss

Ser

β chain (34kD)

PyrGlu

Fig. 1. Structure of hepatocyte growth factor. HGF belongs to the kringle family. The pro-HGF polypeptide comprises an N-terminal secretory signal that is cleaved during the maturation process of the protein. Arrows point to the cleavage sites of proHGF. For gene therapy experiments/trials, the cDNA encoding full-length pro-HGF is inserted in an expression cassette; HGF gene transfer is performed with this HGF expression construct (HGF minigene).

may be elevated in response to high blood pressure as a counter-system against endothelial dysfunction, and may be viewed as an indicator of severity of hypertension.7

2. HGF Signaling in Endothelial Cells

HGF acts as a mitogen, dissociation factor, and motility factor for many epithelial cells in culture through its tyrosine kinase receptor, c-met.2,8 Various intracellular signaling pathways have been shown to be activated by tyrosine kinases linked to c-met. As shown in Fig. 2, the biological responses mediated by c-met are triggered by the tyrosine phosphorylation of a single multifunctional docking site located in the receptor’s carboxy terminal.9 This sequence, containing two phosphotyrosines, interacts with several cytoplasmic signal transducers either directly or indirectly through molecular adapters such as Grb2,

50 kD 140 kD

αβ

Dimer

-S-S-

Fig. 2. Scheme of c-met (HGF receptor) structure. HGF receptor (c-met) consists of an α (50 kDa) and a β (140 kDa) chain, which make a heterodimer of each other. The biological responses mediated by c-met are triggered by the tyrosine phosphorylation of a single multifunctional docking site located in the carboxy terminal tail of the α chain. This sequence, containing two phosphotyrosines, interacts with several cytoplasmic signal transducers either directly or indirectly through molecular adapters such as Grb2. After HGF stimulation, c-met binds and activates phosphatidylinositol-3-OH kinase (PI3K), which then activates Akt/PKB (protein kinase B), and recruits the Grb-SOS complex, stimulating the Ras-MAP kinase cascade.

Shc and Gab1.10,11 After HGF stimulation, c-met binds and activates phosphatidylinositol-3-OH kinase (PI3K) and recruits the Grb-SOS complex, stimulating the Ras-MAP kinase cascade.12,13 In addition, the induction of epithelial tubules by HGF is dependent on activation of the STAT pathway and, importantly, c-met/the HGF tyrosine receptor can bind and directly phosphorylate STAT3.14 HGF also stimulates cell proliferation through the ERK-STAT3 pathway and has an anti-apoptotic activity through the PI3K-Akt pathway in human aortic endothelial cells.15 Interestingly, HGF also increases expression of the anti-apoptotic gene bcl-2 and inhibits translocation of a trigger of apoptosis, bax protein, from cytosol to the mitochondrial membrane.16 It has also been reported that HGF can protect against cell death via inhibition of bad translocation, which is regulated by phosphorylation,

370 R. Morishita & T. Ogihara

Pro-apoptotic stimuli

Caspase 3 and 9 activation

Fig. 3. Potential mechanisms of anti-apoptotic action of HGF. Pro-apoptotic stimuli increase pro-apoptotic genes, such as bax, and also stimulate the translocation of bax and/or bad to the mitochondrial heavy membrane. This bad translocation was regulated by binding to 14-3-3 protein through phosphorylation of bad by PI3K-Akt/PKB pathway. Since HGF can activate PI3K-Akt/PKB pathway and significantly increase bcl-2 and/or bcl-x/L protein, it can block the translocation of bax and/or bad. These changes in bax and/or bad protein release cytochrome c from mitochondria, resulting in activation of the caspase cascade. Therefore, HGF can block the release of cytochrome c through both direct action on mitochondria and blockade of bax and/or bad translocation.

and bax translocation, regulated by the conformational change resulting in the exposure of its BH3 domain via PI3K (Fig. 3).17

3.Angiogenic Therapy for Ischemic Peripheral Arterial Diseases

Critical limb ischemia is estimated to develop in 500 to 1000 individuals per million people in the general population per year. In a large proportion of these patients, the anatomic extent and the distribution of arterial occlusive disease make these individuals unsuitable for operative or percutaneous revascularization. Thus the disease frequently