The endothelium plays a pivotal role in the maintenance of vascular
homeostasis and its dysregulation promotes vascular complications. This
thesis proposes that heme oxygenase-1 (HO-1), an anti-inflammatory enzyme
with antioxidant properties, is endothelial protective factor that prevents
endothelial injury induced by cisplatin or activated neutrophils. Specifically,
this thesis aimed to test (i) that overexpression of HO-1 prevents cisplatin-induced
endothelial injury and suppresses caspase activity; (ii) whether
neutrophil-endothelial cell activation resulted in the release of soluble Flt-1
(sFlt-1) and soluble endoglin (sEng), the two anti-angiogenic factors known
to induce the clinical signs of preeclampsia; (iii) whether HO-1 prevented
activated neutrophils from stimulating the release of these factors from the
endothelium; (iv) the relative contribution and the co-dependency of
neutrophil activation and anti-angiogenic growth factors in preeclampsia
where systemic endothelial dysfunction is known to occur. This thesis shows
that cisplatin inhibited human umbilical vein endothelial cells (HUVEC)
metabolism as measured by MTT assay and resulted in the release of
placenta growth factor (PlGF). Immunoblotting confirmed that cisplatin
increased cleaved caspase-3 expression in HUVEC. These effects of cisplatin
were attenuated in HUVEC infected with adenovirus encoding HO-1 and the
effects were exacerbated when HO-1 was silenced by siRNA. Furthermore,
cisplatin stimulated PlGF release was suppressed by the overexpression of
HO-1. In addition, HO-1 overexpression inhibited angiogenesis as
determined by vascular endothelial growth factor-induced capillary tube
formation on Matrigel coated plates. Thus these studies indicate that agents
which upregulate HO-1 could increase the effectiveness and tolerability to
cisplatin in cancer treatment. Although neutrophils are early contributors to
endothelial cell activation, no studies have determined their contribution to
the release of sFlt-1 and sEng. We therefore investigated the effect of
activated neutrophils on the release of sFlt-1 and sEng in
endothelial/neutrophil co-cultures and in the circulation of women with
normal pregnancy and preeclampsia. LPS-mediated neutrophil activation
stimulated the release of sEng but not sFlt-1 from endothelial cells in culture.
In the absence of neutrophils, overexpression of HO-1 in HUVEC downregulated
the release of sEng. In contrast, HO-1 overexpression failed to
inhibit the release of sEng in the presence of activated neutrophils. The
release of sEng by activated neutrophils-endothelial cell cocultures appears
to be mediated by metalloproteinases (MMP) as the broad-spectrum MMP
inhibitor (GM6001) attenuated sEng release. Clinical studies demonstrated
that sEng, pro-inflammatory interleukin-6 (IL-6) and the soluble markers of
neutrophil activation (α-defensins and calprotectin) were all elevated in
women with preeclampsia. We identified a direct correlation between
neutrophil activation and IL-6 release. However, no correlation could be
established between these factors and sEng release in preeclampsia. Hence,
these results provide compelling clinical evidence to show that the increase
in neutrophil activation and IL-6 release during preeclampsia are unlikely to
significantly contribute to the clinical signs of preeclampsia as they fail to
correlate directly with the anti-angiogenic factors, which form the final
common pathway to the clinical signs of preeclampsia and systemic
endothelial dysfunction