Hepatocyte growth factor plasma levels after myocardial infarction are not affected by recombinant tissue-type plasminogen-activator therapy

ARTICLE

INTRODUCTION

Hepatocyte growth factor is a multifunctional, pleiotropic cytokine composed of an alpha- (69 kDa) and beta-subunit (34 kDa) linked by a disulfide bond. HGF is produced mainly by mesenchymal cells such as fibroblasts and smooth muscle cells and acts predominantly on epithelial and endothelial cells. HGF is the most potent mitogen for mature parenchymal hepatocytes in primary culture and seems to be a hepatotrophic factor that acts as a trigger for liver regeneration after partial hepatectomy and liver injury [1, 2]. The regenerative effect of HGF is not liver-specific, but has also been described in the gastrointestinal tract [3, 4] and in the nephron [5, 6]. In addition, HGF possesses motogenic, morphogenic, tumor-suppressing activity and is thought to play an important role in embryogenesis and organogenesis [7]. HGF exerts its biological effect through the membrane-spanning high-affinity c-met receptor, which is a member of the tyrosine kinase receptor family [8, 9].

HGF, like plasminogen, is synthesized and secreted as an inactive, single-chain precursor (pro-HGF) with a molecular weight of 83 kDa. Pro-HGF is mostly found associated with the extracellular matrix or the cell surface while the mature form of HGF is mainly soluble [10]. As pro-HGF lacks mitogenic activity, proteolytical cleavage in the extracellular environment is a critical step in regulating the physiological functions of this cytokine [11]. Enzymes taking part in the digestion of pro-HGF are subject of several studies. Hepatocyte growth factor activator (HGFA), a plasma protein produced in the liver, is reported to activate HGF in vitro [12]. Blood coagulation factor XIIa also cleaves pro-HGF in vitro [13]. HGF features sequence and structural homology to plasminogen (about 38%) including an amino-terminal hairpin loop, four kringle domains and a serine protease-like region [14]. In contrast to plasminogen, HGF is devoid of proteolytic activity because histidine and serine, critical for the catalytic domain, are replaced in HGF by glutamine and tyrosine, respectively [15]. Because of the structural homology of plasminogen and HGF, the plasminogen activators uPA and tPA were supposed to be candidates for HGF activation. In vitro experiments carried out by Mars et al. [16] revealed that incubation of HGF with uPA and tPA causes time- and dose-dependent cleavage of single-chain HGF to mature, two-chain HGF. Also Naldini et al. [10] reported effective activation of pro-HGF by nanomolar concentrations of uPA indicating its high activity as a substrate and argue that urokinase, besides its well known ability to mediate matrix degradation, may activate matrix-bound HGF in vivo.

Tissue injury leads to conversion of pro-HGF and to increased levels of HGF in the serum in vivo [17, 18]. Therefore it is not surprising that elevated HGF levels could be detected in sera of patients with acute myocardial infarction [19] and in a rat model of myocardial ischemia and reperfusion [20]. As reported for the liver, HGF may play an important role in tissue repair also after acute myocardial infarction. In the present study, we compared HGF levels in rtPA-lysed and non-lysed patients in order to examine whether tPA is engaged in activating pro-HGF in vivo.

MATERIALS AND METHODS

We studied HGF serum levels in 14 patients with acute myocardial infarction who were hospitalized between November 1996 and February 1997. The group of patients without rtPA-treatment (n = 7) consisted of 2 women and 5 men, mean age was 62.6 ± 7.4 years (mean ± SE), range 37-78 years. At time of admission, serum creatine kinase levels were 892 ± 526 IE/l, CK-MB isoenzyme levels were 71 ± 25 IE/l and lactatdehydrogenase (LDH) levels were 489 ± 161 IE/l (mean ± SE). Due to a lack of ST elevation in the ECG of 6 patients and a status postcardiopulmonary reanimation for 30 min in 1 patient, systemic thrombolytic therapy was not undertaken in this group. These patients were subjected to conventional therapy with aspirin, heparin and beta-blockers. The group of patients with rtPA treatment (n = 7) consisted of 1 woman and 6 men with an age of 53.6 ± 6.9 years (mean ± SE), range 26-71 years. In this group, serum creatine kinase levels were 179 ± 106 IE/l, CK-MB isoenzyme 9 ± 3 IE/l and LDH 248 ± 29 IE/l (mean ± SE). Lysis was carried out with rtPA (Actilyse, Boehringer Ingelheim) using an initial bolus of 15 mg, followed by 50 mg over 30 min and finally 35 mg over a period of 60 min. No cardiac catheterization was carried out during the first 48 hours in either group. Blood was collected at time of admission and subsequently 12-16 hours, 20-30 hours and 50-60 hours after onset of chest pain. Blood was centrifuged at 2,000 rpm for 10 min at 20° C to separate serum. Serum was stored at ­ 80° C.

HGF detection: a commercially available "sandwich" enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Minneapolis, USA) was used to determine the amount of HGF in the serum. The sensitivity of the kit was 0.04 ng/ml. The ELISA is not able to distinguish between pro-HGF and mature HGF but due to centrifugation of the blood samples, extracellular matrix or cell surface-associated pro-HGF is thought to be reduced to a negligible minimum.

Statistics: data are presented as mean ± SEM. Statistical analysis was performed using the paired samples
t-test. P values less than 0.05 were considered to be significant.

RESULTS

The delay from onset of chest pain to admission to our hospital was 0-5 hours (2.3 ± 0.7) in the rtPA-lysed group. Serum HGF at time of admission was 16.8 ± 2.2 ng/ml (mean ± SE) in this group. In the non-lysed group the delay was 0-9 hours (7.4 ± 0.7) and the HGF levels were 20.7 ± 6.5 ng/ml. HGF levels 12-16 hours after MCI were 12.5 ± 4.2 ng/ml in the rtPA-lysed group and 11.9 ± 2.9 ng/ml in the non-lysed group. HGF levels further decreased to 7.4 ± 1.6 ng/ml in the lysed group and 12.6 ± 3.2 ng/ml in the non-lysed group 20-30 hours after MCI. After 50-60 hours HGF levels of 3.2 ± 1.3 ng/ml in the lysed versus 4.4 ± 0.9 ng/ml in the non-lysed group were observed (Table 1). At any blood collection time intervals no statistical significant difference could be detected between these two groups (p > 0,1) (Figure 1). There was no correlation between serum HGF and serum creatine kinase at 0-9 hours after the onset of chest pain.

DISCUSSION

HGF is synthesized as single-chain pro-HGF by mesenchymal cells and secreted into the extracellular environment, where the main part of pro-HGF is bound to the extracellular environment and to cell surfaces. Pro-HGF is able to bind to the HGF-receptor
c-met but does not possess biological activity. Activation by proteolytical conversion of pro-HGF into the active heterodimeric form is required for biological activity. In vitro serine proteases were found to take part in the conversion of pro-HGF since serine-protease inhibitors prevent activation of single-chain HGF [21, 22]. Two homologous proteases, blood coagulation factor XIIa and hepatocyte growth factor activator, possess high affinity for single-chain HGF. Low doses of factor XIIa (200 ng/ml) and HGF activator (10 ng/ml) were sufficient for complete conversion of 200 mug/ml single-chain HGF into active two-chain HGF in vitro [13]. HGF activator purified from human serum is produced by parenchymal liver cells and circulates in the plasma as inactive zymogen. In response to tissue injury, the HGF activator zymogen is converted to the active form by proteolytic processing [23]. In vitro, thrombin in the presence of negatively charged substances was able to cleave and therefore activate the HGF activator precursor at the bond between Arg407 and Ile408 [24]. Factor XIIa, an active form of factor XII, is involved in the initiation of blood coagulation and fibrinolysis [25].

In the present study, we can show that serum HGF levels measured in patients with acute myocardial infarction who underwent high dose thrombolytic therapy with rtPA revealed no statistical significant differences at any blood collection time interval, compared to those measured in patients receiving conventional therapy. These results suggest that supply of tPA in vivo causes no further rise in soluble two-chain HGF levels by additional activation of extracellular matrix or cell surface-associated single-chain HGF. Our data are confirmed by the results reported by Shimomura et al. [13] and Mizuno et al. [22] who did not detect any HGF-converting activity of urokinase and tPA in vitro under their experimental conditions, whereas factor XIIa and HGF activator showed strong HGF-activating potency. Even Mars et al. [16], who showed that uPA and tPA can generate two-chain HGF from pro-HGF in vitro, reported that tPA has a lesser activity than uPA and causes only partial conversion of pro-HGF. In 1996, published data showed that in rat hepatocyte cultures, uPA mRNA and protein is present and able to cleave single-chain HGF into two-chain HGF, while tPA mRNA could not be detected [26]. Therefore, we suppose that other serine proteases such as blood coagulation factor XIIa or HGF-activator are decisive for proteolytical cleavage of pro-HGF and thus for the biological effectiveness of this cytokine.

Acute myocardial infarction is associated with coronary thrombosis and tissue injury, both leading to activation of the blood coagulation cascade with the formation of thrombin. As mentioned above, thrombin is able to convert the HGF activator precursor, with consequent cleavage of pro-HGF into active heterodimeric HGF which is implicated in tissue repair by inducing cell growth, cell migration, angiogenesis and collateral vessel growth [27]. The early maximum of HGF levels after onset of MCI corresponds with published data [20, 28] and could support a role for thrombin in the conversion of the HGF activator precursor followed by upregulation of active HGF. In contrast, a slight but steady increase of serum HGF, peaking between 12 and 24 hours, is reported by Matsumori et al. [19]. The demonstration of elevated levels of HGF mRNA and c-met mRNA in regions of ischemic myocardium after reperfusion as seen in a rat model, supports the suggestion that HGF is implicated in regeneration during the healing process. Some of the patients taking part in the study received intravenous injection of heparin before admission to our hospital. An early increase in circulating HGF, as reported for heparin injections [29], seems to be possible but the number of patients was too small for a subgroup analysis. Further investigations should be aimed at the detection of HGF activator in sera of patients with acute myocardial infarction in order to obtain further information about the mechanisms of HGF activation in vivo. In addition, interest should be paid to the question of whether mature HGF is engaged in restoration of damaged tissues after acute myocardial infarction.

REFERENCES

1. Michalopoulus G, Cianciulli H D, Novotny A R, Kligerman A D, Strom S C, Jirtle R L. 1982. Liver regeneration studies with rat hepatocytes in primary culture. Cancer Res. 42: 4673.

2. Kinoshita T, Tashiro K, Nakamura T. 1989. Marked increase of HGF mRNA in non-parenchymal liver cells of rats treated with hepatotoxins. Biochem. Biophys. Res. Commun. 165: 1229.

3. Fukamachi H, Ichinose M, Tsukada S, Kakei N, Suzuki T, Miki K, Kurokawa K, Masui T. 1994. Hepatocyte growth factor region specifically stimulates gastro-intestinal epithelial growth in primary culture. Biochem. Biophys. Res. Commun. 205: 1445.

4. Takahashi M, Ota S, Terano A, Yoshiura K, Matsumura M, Niwa Y, Kawabe T, Nakamura T, Omata M. 1993. Hepatocyte growth factor induces mitogenic reaction to the rabbit gastric epithelial cells in primary culture. Biochem. Biophys. Res. Commun. 191: 528.

5. Kawaida K, Matsumoto K, Shimazu H, Nakamura T. 1994. Hepatocyte growth factor prevents acute renal failure and accelerates renal regeneration in mice. Proc. Natl. Acad. Sci. USA 91: 4357.

6. Nagaike M, Hirao S, Tajima H, Noji S, Taniguchi S, Matsumoto K, Nakamura T. 1991. Renotropic functions of hepatocyte growth factor in renal regeneration after unilateral nephrectomy. J. Biol. Chem. 266: 22781.

7. Montesano R, Matsumoto K, Nakamura T, Orci L. 1991. Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell 67: 901.

8. Bottaro D P, Rubin J S, Faletto D L, Chan A M, Kmiecik T E, Vande Woude G F, Aaronson S A. 1991. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 251: 802.

9. Naldini L, Vigna E, Narsimhan R P, Gaudino G, Zarnegar R, Michalopoulos G K, Comoglio P M. 1991. Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-met. Oncogene 6: 501.

10. Naldini L, Tamagnone L, Vigna E, Sachs M, Hartmann G, Birchmeier W, Daikuhara Y, Tsubouchi H, Blasi F, Comoglio P M. 1992. Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor. EMBO J. 11: 4825.

11. Gak E, Taylor W G, Chan A M, Rubin J S. 1992. Processing of hepatocyte growth factor to the heterodimeric form is required for biological activity. FEBS Lett 311: 17.

12. Miyazawa K, Shimomura T, Kitamura A, Kondo J, Morimoto Y, Kitamura N. 1993. Molecular cloning and sequence analysis of the cDNA for a human serine protease responsible for activation of hepatocyte growth factor. J. Biol. Chem. 268: 10024.

13. Shimomura T, Miyazawa K, Komiyama Y, Hiraoka H, Naka D, Morimoto Y, Kitamura N. 1995. Activation of hepatocyte growth factor by two homologous proteases, blood-coagulation factor XIIa and hepatocyte growth factor activator. Eur. J. Biochem. 229: 257.

14. Cioce V, Csaky K G, Chan A M L, Bottaro D P, Taylor W G, Jensen R, Aaronson S A, Rubin J S. 1996. Hepatocyte growth factor (HGF)/NK1 is a naturally occurring HGF/scatter factor variant with partial agonist/antagonist activity. J. Biol. Chem. 271: 13110.

15. Nakamura T, Nishizawa T, Hagiya M, Seki T, Shimonishi M, Sugimura A, Tashiro K, Shimizu S. 1989. Molecular cloning and expression of human hepatocyte growth factor. Nature 342: 440.

16. Mars W M, Zarnegar R, Michalopoulos G K. 1993. Activation of hepatocyte growth factor by the plasminogen activators uPA and tPA. Am. J. Pathol. 143: 949.

17. Sakon M, Monden M, Gotoh M, Kanai T, Umeshita K, Mori T, Tsubouchi H, Daikuhara Y. 1992. Hepatocyte growth factor concentrations after liver resection. Lancet 339: 818.

18. Tsubouchi H, Niitani Y, Hirono S, Nakayama H, Gohda E, Arakaki N, Sakiyama O, Takahashi K, Kimoto M, Kawakami S, et al. 1991. Levels of the human hepatocyte growth factor in serum of patients with various diseases determined by an enzyme-linked immunosorbent assay. Hepatology 13: 1.

19. Matsumori A, Furukawa Y, Hashimoto T, Ono K, Shioi T, Okada M, Iwasaki A, Nishio R, Sasayama S. 1996. Increased circulating hepatocyte growth factor in the early stage of acute myocardial infarction. Biochem. Biophys. Res. Commun. 221: 391.

20. Ono K, Matsumori A, Shioi T, Furukawa Y, Sasayama S. 1997. Enhanced expression of hepatocyte growth factor/c-Met by myocardial ischemia and reperfusion in a rat model. Circulation 95: 2552.

21. Naka D, Ishii T, Yoshiyama Y, Miyazawa K, Hara H, Hishida T, Kitamura N. 1992. Activation of hepatocyte growth factor by proteolytic conversion of a single chain form to a heterodimer. J. Biol. Chem. 267: 20114.

22. Mizuno K, Takehara T, Nakamura T. 1992. Proteolytic activation of a single-chain precursor of hepatocyte growth factor by extracellular serine-protease. Biochem. Biophys. Res. Commun 189: 1631.

23. Miyazawa K, Shimomura T, Kitamura N. 1996. Activation of hepatocyte growth factor in the injured tissues is mediated by hepatocyte growth factor activator. J. Biol. Chem. 271: 3615.

24. Shimomura T, Kondo J, Ochiai M, Naka D, Miyazawa K, Morimoto Y, Kitamura N. 1993. Activation of the zymogen of hepatocyte growth factor activator by thrombin. J. Biol. Chem. 268: 22927.

25. Tans G, Rosing J. 1987. Structural and functional characterization of factor XII. Semin. Thromb. Hemost. 13: 1.

26. Mars W M, Kim T H, Stolz D B, Liu M L, Michalopoulus G K. 1996. Presence of urokinase in serum-free primary rat hepatocyte cultures and its role in activating hepatocyte growth factor. Cancer Res. 56: 2837.

27. Grant D S, Kleinman H K, Goldberg I D, Bhargava M M, Nickoloff B J, Kinsella J L, Polverini P, Rosen E M. 1993. Scatter factor induces blood vessel formation in vivo. Proc. Natl. Acad. Sci. USA 90: 1937.

28. Sato Y, Yoshinouchi T, Sakamoto T, Fujieda H, Murao S, Sato H, Kobayashi H, Ohe T. 1997. Hepatocyte growth factor (HGF): a new biochemical marker for acute myocardial infarction. Heart Vessels 12: 241.

29. Matsumori A, Ono K, Okada M, Miyamoto T, Sato Y, Sasayama S. 1998. Immediate increase in circulating hepatocyte growth factor/scatter factor by heparin. J. Mol. Cell. Cardiol. 30: 2145.