EC apoptosis is a prominent feature associated with acute and/or chronic inflammation such as it occurs during hyperoxia 44
, endotoxic shock 25
, arteriosclerosis 26
, ischemia reperfusion injury 4546
, and acute or chronic graft rejection 234748
. Presumably, EC apoptosis contributes to the development of these inflammatory reactions by sustaining inflammation and promoting vascular thrombosis 2545
. The mechanism by which apoptotic ECs promote vascular thrombosis is thought to involve the expression of procoagulant phospholipids by apoptotic bodies 49
and presumably the exposure of the procoagulant subendothelial matrix that is associated with EC apoptosis. In addition, apoptotic cells can activate the complement cascade directly through the binding of C1q to apoptotic bodies 50
, and promote platelet adhesion 49
that will sustain inflammation and thrombosis as well.
The observation that HO-1 prevents apoptosis induced by different proapoptotic stimuli (2324
; ) suggests that HO-1 suppresses one or several signaling pathways that are common to a broad spectrum of proapoptotic stimuli. The antiapoptotic effect of HO-1 has recently been associated with increased cellular iron efflux through the upregulation of an iron pump that remains to be fully characterized 34
. According to this study 34
, HO-1 inhibits apoptosis by limiting the availability of prooxidant-free iron to participate in the generation of reactive oxygen species through the Fenton reaction, a well-established component in the signaling cascades leading to apoptosis 51
. We hypothesized that HO-1 may have additional effects that could contribute to suppress EC apoptosis, such as by generating CO. Data from L.E. Otterbein, A.M.K. Choi, and colleagues have suggested that this may be the case in fibroblasts 52
. However, data from other laboratories have suggested that in 293 cells CO is not antiapoptotic 34
. Moreover, CO has been suggested to be proapoptotic in ECs 53
. This data suggests that HO-1 can suppress EC apoptosis and that the antiapoptotic effect of HO-1 is mediated through the generation of CO (, , and ).
The observation that the elimination of endogenous CO by Hb abrogates the cytoprotective effect of HO-1 ( and ) supports the notion that, in the absence of CO, other biological functions engendered by HO-1, i.e., upregulation of ferritin expression and subsequent iron chelation, are not sufficient per se to prevent EC apoptosis ( and ). Given the above, it is difficult to understand why iron chelation by DFO can protect ECs from apoptosis, even under conditions in which the action of CO is prevented (i.e., inhibition of HO-1 activity by SnPPIX or elimination of CO by Hb; ). At least two possible interpretations may explain these observations: (a) DFO may have a higher ability to “eliminate” free iron compared with ferritin, and/or (b) DFO may have additional effects that contribute to prevent EC apoptosis, independently of its ability to eliminate free iron. In any case, these observations suggest that DFO does not, at least fully, mimic the effect of HO-1–mediated ferritin expression in preventing EC apoptosis.
Our data also suggest that CO, generated by cells that express HO-1, acts as an intercellular signaling molecule to prevent apoptosis of cells that do not express HO-1 (). If a similar effect of CO would occur in vivo, these data would suggest that vascular ECs at sites of inflammation might protect neighboring cells, such as infiltrating leukocytes that immigrate into sites of inflammation or smooth muscle cells in blood vessels from undergoing apoptosis.
The antiapoptotic effect of HO-1 in ECs is not mediated by guanylylcyclase and/or by the generation of cGMP (). This is in contrast to data showing that inhibition of guanylcyclase suppresses the antiapoptotic effect of HO-1 and/or CO in fibroblasts 51
. Our interpretation is that the mechanism by which HO-1 and/or CO prevents apoptosis is cell type specific.
Based on the finding by Otterbein et al. that HO-1/CO activates p38 MAPK in M 33
, we tested whether HO-1 and/or CO would have similar effects in ECs. We found that this is the case (). However, contrary to M
, exogenous CO per se induced the activation of p38 MAPK in ECs, whereas HO-1 did not (). Possible explanations for the difference between HO-1 and CO in regulating p38 MAPK activation include the following. Whereas CO generated by HO-1 activates p38 MAPK, other end products of HO-1 activity may inhibit p38 activation. Alternatively, the level of CO, generated by HO-1, is significantly lower than exposure to exogenous CO as we used it. Whatever the explanation, these data show that CO can specifically modulate the activation of p38 MAPK.
We also found that the antiapoptotic effect of HO-1/CO acts via the activation of a transduction pathway involving the activation of p38 MAPK (). This is consistent with findings by others showing that activation of p38 MAPK is key in regulating apoptosis in a variety of cell types including the kidney epithelial cell line HeLa 54
, cardiac muscle cells 55
, and lymphoid Jurkat T cells 5456
. The mechanism by which the activation of p38 MAPK modulates the induction of apoptosis is not well understood.
In conclusion, our findings suggest that CO, generated through heme catabolism by HO-1, acts as an antiapoptotic molecule that can suppress EC apoptosis. We show that the mechanism of action of CO involves the activation of p38 MAPK. These findings further support the notion that HO-1 acts as a protective gene and thereby contributes to prevent a series of inflammatory reactions that are associated with EC apoptosis.