Vascular Heme Oxygenase-1 Induction Suppresses Microvascular Thrombus Formation In Vivo

Abstract

Objective-By heme degradation, heme oxygenase-1 (HO-1) provides endogenous carbon monoxide and bilirubin, both of which play major roles in vascular biology. The current study aimed to examine whether induction of HO-1 and its byproducts modulate the process of microvascular thrombus formation in vivo. Methods and Results-In individual microvessels of mouse cremaster muscle preparations, ferric chloride-induced thrombus formation was analyzed using intravital fluorescence microscopy. When mice were pretreated with an intraperitoneal injection of hemin, a HO-1 inducer, immunohistochemistry and Western blot protein analysis of cremaster muscle tissue displayed a marked induction of HO-1. In these animals, superfusion with ferric chloride solution induced arteriolar and venular thrombus formation, which, however, was significantly delayed when compared with thrombus formation in animals without HO-1 induction. The delay in thrombus formation in hemin-treated mice was completely blunted by tin protoporphyrin-IX, a HO-1 inhibitor, but not by copper protoporphyrin-IX, which does not inhibit the enzyme. Coadministration of the vitamin E analogue Trolox in HO-1-blocked animals almost completely restored the delay in thrombus formation, implying that, besides CO, the antioxidant HO pathway metabolite bilirubin mainly contributes to the antithrombotic property of HO-1. This was further supported by the fact that bilirubin was found as effective as hemin in delay of ferric chloride-induced thrombus formation. Animals with HO-1 induction revealed reduced P-selectin protein expression in cremaster muscle tissue, which most probably presented the molecular basis for delayed thrombus growth. Conclusion-Local

Carbon Monoxide-Induced Early Thrombolysis Contributes to Heme Oxygenase-1-Mediated Inhibition of Neointimal Growth after Vascular Injury in Hypercholesterolemic Mice

“…This finding appears to be consistent with a recent study showing that systemic induction of HO-1 by prior treatment of animals with heme suppressed microvascular thrombus formation subsequent to FeCl 3 -induced vascular injury [20]. In that study, the anti-oxidant activity of bilirubin was shown to mediate the anti-thrombotic property of HO-1 through down regulating the expression of P-selectin.…”

Serum Total Bilirubin Level and Prevalent Lower-Extremity Peripheral Arterial Disease

“…33,34 Several investigators have found that the vascular-protective effects of HO can be reproduced by the administration of bilirubin or biliverdin, suggesting that the production of bilirubin is a major component of the vascular protection offered by HO. 8,25,28 Our result is similar to the inverse relationship reported between bilirubin level and CAD. 12,35 Two studies in this area are particularly noteworthy.…”

“…9 Bilirubin also inhibits monocyte transmigration, 25 prevents the formation of oxidized LDL, 26 inhibits endothelial inflammation and dysfunction, 8 inhibits vascular smooth muscle proliferation, 27 and prevents thrombus formation. 28 Human studies demonstrate that low bilirubin is associated with impaired endothelial function and increased carotid intima media thickness, 2 predictors of atherosclerosis, in healthy individuals free of cardiovascular risk factors. 29,30 These data provide a biological basis for the inverse association of bilirubin with PAD, a manifestation of systemic atherosclerosis.…”

C-peptide exerts antithrombotic effects that are repressed by insulin in normal and diabetic mice

“…For the study of vascular thrombus formation, we used the opened cremaster muscle preparation, as described originally by Baez [16] and applied as a model of microvascular thrombus formation in previous studies [17][18][19][20]. After the preparation of the cremaster muscle, the animals were allowed to recover from surgery for 15 min before induction of thrombus formation.…”