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Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR
Published on: November 23, 2012
Cell-Specific Gene Deletion Reveals the Antithrombotic Function of COX1 and Explains the Vascular COX1/Prostacyclin
Jane A Mitchell1, Fisnik Shala1, Youssef Elghazouli1
1From the National Heart and Lung Institute, Imperial College London, United Kingdom (J.A.M., F.S., Y.E., N.S.K.).
Insights
Endothelial cyclooxygenase-1 (COX1) produces antithrombotic prostacyclin, a role previously masked by platelet COX1. This study reveals endothelial COX1 is crucial for preventing thrombosis in vivo.
Area of Science:
- Vascular Biology
- Thrombosis Research
- Enzyme Function
Background:
- Endothelial cells (ECs) and platelets produce opposing factors: ECs produce antithrombotic prostacyclin, while platelets produce prothrombotic thromboxane A2.
- Both cell types express cyclooxygenase-1 (COX1), complicating the isolation of COX1's specific role in prostacyclin production and antithrombotic effects.
- The distinct role of COX2 in antithrombotic protection is well-established due to its absence in platelets, leading to an incomplete understanding of COX1's contribution.
Purpose of the Study:
- To elucidate the in vivo antithrombotic role of COX1-derived prostacyclin.
- To compare the antithrombotic significance of COX1 in ECs with the known role of COX2.
Main Methods:
- Development of vascular-specific COX1 knockout mice and comparison with endothelial-specific COX2 knockout mice.
- Assessment of prostacyclin production in isolated aortic cells and aortic rings.
- In vivo thrombosis assays following carotid artery injury using FeCl3, with genetic manipulation of COX1 and COX2 in specific cell types.
Main Results:
- COX1 in ECs, not smooth muscle cells, was the primary source of aortic prostacyclin and inhibited platelet aggregation.
- Endothelial COX1 deletion accelerated thrombosis in vivo, similar to prostacyclin receptor blockade, but this was abrogated by concurrent platelet COX1 deletion.
- Endothelial COX2 deletion also induced a prothrombotic phenotype, independent of local prostacyclin production.
Conclusions:
- Endothelial COX1 provides essential antithrombotic protection in healthy animals, a function previously obscured by platelet COX1 activity.
- A novel two-component model for thrombotic tone regulation is proposed, involving both COX1 and COX2 through distinct mechanisms.
Rationale:
Endothelial cells (ECs) and platelets, which respectively produce antithrombotic prostacyclin and prothrombotic thromboxane A2, both express COX1 (cyclooxygenase1). Consequently, there has been no way to delineate any antithrombotic role for COX1-derived prostacyclin from the prothrombotic effects of platelet COX1. By contrast, an antithrombotic role for COX2, which is absent in platelets, is straightforward to demonstrate. This has resulted in an incomplete understanding of the relative importance of COX1 versus COX2 in prostacyclin production and antithrombotic protection in vivo.
Objective:
We sought to identify the role, if any, of COX1-derived prostacyclin in antithrombotic protection in vivo and compare this to the established protective role of COX2.
Methods And Results:
We developed vascular-specific COX1 knockout mice and studied them alongside endothelial-specific COX2 knockout mice. COX1 immunoreactivity and prostacyclin production were primarily associated with the endothelial layer of aortae; freshly isolated aortic ECs released >10-fold more prostacyclin than smooth muscle cells. Moreover, aortic prostacyclin production, the ability of aortic rings to inhibit platelet aggregation and plasma prostacyclin levels were reduced when COX1 was knocked out in ECs but not in smooth muscle cells. When thrombosis was measured in vivo after FeCl3 carotid artery injury, endothelial COX1 deletion accelerated thrombosis to a similar extent as prostacyclin receptor blockade. However, this effect was lost when COX1 was deleted from both ECs and platelets. Deletion of COX2 from ECs also resulted in a prothrombotic phenotype that was independent of local vascular prostacyclin production.
Conclusions:
These data demonstrate for the first time that, in healthy animals, endothelial COX1 provides an essential antithrombotic tone, which is masked when COX1 activity is lost in both ECs and platelets. These results help us define a new 2-component paradigm wherein thrombotic tone is regulated by both COX1 and COX2 through complementary but mechanistically distinct pathways.
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