Our data support the notion that HGF has a definite role in cardioprotection from ischemia/reperfusion injury as an endogenous cardiotrophic factor, and that this cardioprotection is exerted through the direct action of HGF on cardiomyocytes. Evidence for this proposal includes: (a) the neutralization of HGF in vivo resulted in exacerbated pathology and increased mortality due to cardiac failure; (b) HGF directly exhibited protective effects on cardiomyocytes in vitro, and the cardioprotective activity in the plasma from rats with induced ischemia/reperfusion injury was significantly suppressed by the neutralization of HGF; (c) both c-Met/HGF receptor expression in cardiomyocytes and plasma HGF levels rapidly increased in response to ischemia/reperfusion injury; and (d) administration of HGF to rats with ischemia/reperfusion injury resulted in a much lesser degree of myocardial apoptosis and infarct area, and better cardiac function, compared with control rats.
It is noteworthy that serum HGF levels in patients with myocardial infarction were over 50 times higher than those in healthy humans, but the biological significance of this remains to be addressed (26
). Our results suggest that the increased blood HGF may reflect defensive reactions and participate in cardioprotection against myocardial infarction, possibly also in humans. Although the source of plasma HGF that increased following ischemia/reperfusion injury remains to be defined, rapid induction of HGF mRNA expression was seen in distant intact organs such as the liver, kidney, lung, and spleen, as well as in the injured heart after ischemia/reperfusion injury in rats (ref. 36
, and our unpublished data), suggesting that endogenous HGF might be derived from these organs. A similar rapid induction of HGF expression in injured and distant intact organs was noted in the case of acute hepatic and renal injuries (37
), and the presence of a humoral factor that induces HGF expression was noted in plasma of rats with hepatic and renal injuries (39
). In addition to an increase in HGF levels in plasma, there was also rapid upregulation of c-Met receptor after ischemia/reperfusion injury. Since biological effects of HGF on cardiomyocytes, as revealed by serum CPK, Bcl-xL expression, and myocardial apoptosis, were seen as early as 3 hours after reperfusion, c-Met receptor expressed in normal cardiomyocytes at a relatively low level might be responsible for early signaling to protect the myocardium. Subsequent increase in c-Met mRNA may possibly relate to the autoinduction of gene expression triggered by HGF (38
Myocardial apoptosis occurs after ischemia/reperfusion injury (2
), and prevention of it has gained much attention as a new target for cardioprotection from ischemia/reperfusion injury (41
). We found that biochemical neutralization of HGF resulted in increased apoptosis, while HGF administration resulted in a much-decreased apoptosis in cardiomyocytes, and HGF was cytoprotective for cultured cardiomyocytes. Therefore, the cardioprotective effects of HGF may be mainly attributable to its antiapoptotic action on cardiomyocytes. With respect to mechanisms that relate to the antiapoptotic effects of HGF, we focused on Bcl-xL expression in cardiomyocytes, since Bcl-xL has a critical role in antiapoptosis, counteracting apoptotic signals in a variety of cells, including cardiomyocytes (43
). In addition, previous studies showed that prevention of massive hepatocyte apoptosis and fulminant hepatic failure by HGF were associated with a remarkable induction of Bcl-xL (23
). Therefore, the rapid induction of Bcl-xL expression in cardiomyocytes may be involved in the antiapoptosis achieved by HGF. On the other hand, although we focused here on the antiapoptotic action of HGF, necrotic cell death after myocardial infarction has to be considered. Recent studies revealed that intracellular signaling pathways leading to antiapoptotic and antinecrotic cell death are at least partially shared (44
). Overexpression of Bcl-2 in hepatocytes suppressed necrotic cell death (45
). Moreover, activation of ERK-1/2 participates in the reduction of necrotic cell death, as well as apoptotic cell death (46
). Therefore, rapid induction of Bcl-xL and activation of ERK by HGF in cardiomyocytes make way for the notion that HGF may give a signal to reduce necrosis in cardiomyocytes following myocardial infarction.
Other researchers reported that protective effects of cardiotrophic factors such as IGF-1 and leukemia inhibitory factor involve ERK activation (47
). Inhibition of ERK phosphorylation by a specific inhibitor resulted in increased apoptosis in cardiomyocytes (49
) and ischemia/reperfusion injury in isolated rat hearts (34
). In addition to the ERK-related pathway, stimulation of Akt downstream of phosphatidylinositol (PI) 3-kinase is involved in protection of cardiomyocytes by IGF-1 (50
). However, HGF did not induce Akt phosphorylation; thus the PI 3-kinase–Akt pathway does not seem to be involved in HGF-dependent antiapoptosis in cardiomyocytes (our unpublished data). Therefore, the intracellular signaling pathways leading to antiapoptosis in cardiomyocytes by HGF and to that by IGF-1 differ, at least in part, although HGF and IGF-1 exhibit similar cardioprotective actions, some of which are mediated by receptor tyrosine kinases. In this context, it is noteworthy that Bag-1, a partner of Bcl-2/Bcl-xL, is specifically associated with the c-Met receptor. Association of Bag-1 with Bcl-2/Bcl-xL enhances antiapoptotic action of Bcl-2/Bcl-xL, and expression of Bag-1 in cells promotes the cell survival activity of HGF (51
Although the cardioprotective and antiapoptotic effects of HGF on cardiomyocytes are likely to be largely attributable to direct actions on cardiomyocytes, previous studies revealed a potent angiogenic action of HGF in vivo (52
). Likewise, transfection of HGF gene into the rat myocardium resulted in a significant increase in the number of blood vessels (54
). Together with findings that administration or gene transfection of angiogenic growth factors into laboratory animals promoted coronary angiogenesis and resulted in a diminished pathology due to myocardial infarction (55
), biological actions of HGF for both myocardium and coronary vessels may further minimize the extensive myocardial injury due to prolonged ischemia.
In summary, endogenous and exogenous HGF protected the rat heart against ischemia/reperfusion injury. Preclinical and clinical trials of HGF for treatment of patients with myocardial infarction can be considered.