Smith, Rebecca (2014-05). Annexin A2 is Required for Endothelial Cell Junctional Response to S1P. Master's Thesis. Thesis uri icon

abstract

  • Endothelial cell (EC) junctions are critical for angiogenesis, the sprouting and growth of new blood vessels from existing vessels. Sphingosine 1-phosphate (S1P) is a proangiogenic factor that potently stimulates sprouting, fortifies EC junctions, and work by others has shown it stimulates VE-cadherin, ?-catenin, focal adhesion kinase (FAK) and paxillin junction localization among others. Annexin A2 (ANXA2), a calcium-regulated membrane-binding and adapter protein, has a known role in angiogenesis. Previously, we showed that ANXA2 is required for barrier integrity by binding to vascular endothelial (VE)-Cadherin and preventing its phosphorylation. Thus, we tested whether ANXA2 silencing in human endothelial cells alters localization of junctional and focal adhesion proteins with immunofluorescence staining. Removal of the ANXA2 protein in ECs resulted in the formation of wide reticular junctions in 2D, affecting localization of adherens junction proteins such as VE-cadherin, platelet endothelial cell adhesion molecule-1 (PECAM-1), filamin A, and ?- and ?-catenin. Additionally, when ANXA2 was silenced, neither FAK, paxillin, nor vinculin formed large focal adhesions near the cell-cell junctions, particularly, near the reticular cell-cell junctions. Reticular junctions were reported in the literature in non-transduced cells seeded on fibronectin coated glass coverslips. We showed that these reticular junctions were present in ECs seeded on collagen I-, collagen IV-, and Matrigel-coated glass coverslips in addition to fibronectin. We characterized these reticular junctions temporally, showing the number of reticular junctions increased over time, particularly after 8 hours in low serum culture medium. Levels of VE-cadherin and zonula occludens-1 (ZO-1) were also regulated over time in cells forming reticular junctions, upregulated at 4 hours and 12 hours, respectively. Most striking was the affect S1P had on reticular junction formation. The addition of S1P to ECs abrogated the formation of reticular junctions in 2D. Upon comparison of the proteins involved, the shANXA2 reticular junctions and the non-transduced EC reticular junctions appeared similar. In both groups only adherens junctions proteins participated in reticular localization while focal adhesion proteins and tight junction proteins did not localize in a reticular pattern. The junctions differed, however, in that the shANXA2 reticular junctions formed in the presence of S1P while the non-transduced reticular junctions did not, indicating ANXA2 is required for proper junctional response to S1P. Finally, we show the presence of reticular junctions in EC monolayers on 3D collagen matrices, reticular junctions contributing to EC sprout initiation, and reticular junctions present in mouse uterine tissue from pregnant mice 7.5 days after implantation. The discoveries detailed in this thesis illustrate the importance of ANXA2 in EC junctional response to S1P as well as the potential for future discoveries concerning the role of reticular junctions in sprouting angiogenesis.
  • Endothelial cell (EC) junctions are critical for angiogenesis, the sprouting and growth of new blood vessels from existing vessels. Sphingosine 1-phosphate (S1P) is a proangiogenic factor that potently stimulates sprouting, fortifies EC junctions, and work by others has shown it stimulates VE-cadherin, ?-catenin, focal adhesion kinase (FAK) and paxillin junction localization among others. Annexin A2 (ANXA2), a calcium-regulated membrane-binding and adapter protein, has a known role in angiogenesis. Previously, we showed that ANXA2 is required for barrier integrity by binding to vascular endothelial (VE)-Cadherin and preventing its phosphorylation. Thus, we tested whether ANXA2 silencing in human endothelial cells alters localization of junctional and focal adhesion proteins with immunofluorescence staining.

    Removal of the ANXA2 protein in ECs resulted in the formation of wide reticular junctions in 2D, affecting localization of adherens junction proteins such as VE-cadherin, platelet endothelial cell adhesion molecule-1 (PECAM-1), filamin A, and ?- and ?-catenin. Additionally, when ANXA2 was silenced, neither FAK, paxillin, nor vinculin formed large focal adhesions near the cell-cell junctions, particularly, near the reticular cell-cell junctions. Reticular junctions were reported in the literature in non-transduced cells seeded on fibronectin coated glass coverslips. We showed that these reticular junctions were present in ECs seeded on collagen I-, collagen IV-, and Matrigel-coated glass coverslips in addition to fibronectin. We characterized these reticular junctions temporally, showing the number of reticular junctions increased over time, particularly after 8 hours in low serum culture medium. Levels of VE-cadherin and zonula occludens-1 (ZO-1) were also regulated over time in cells forming reticular junctions, upregulated at 4 hours and 12 hours, respectively.

    Most striking was the affect S1P had on reticular junction formation. The addition of S1P to ECs abrogated the formation of reticular junctions in 2D. Upon comparison of the proteins involved, the shANXA2 reticular junctions and the non-transduced EC reticular junctions appeared similar. In both groups only adherens junctions proteins participated in reticular localization while focal adhesion proteins and tight junction proteins did not localize in a reticular pattern. The junctions differed, however, in that the shANXA2 reticular junctions formed in the presence of S1P while the non-transduced reticular junctions did not, indicating ANXA2 is required for proper junctional response to S1P. Finally, we show the presence of reticular junctions in EC monolayers on 3D collagen matrices, reticular junctions contributing to EC sprout initiation, and reticular junctions present in mouse uterine tissue from pregnant mice 7.5 days after implantation. The discoveries detailed in this thesis illustrate the importance of ANXA2 in EC junctional response to S1P as well as the potential for future discoveries concerning the role of reticular junctions in sprouting angiogenesis.

publication date

  • May 2014