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Pharmacological Manipulation of HEG1-KRIT1 Protein Complex Modulates Endothelial Vasoprotective Functions through Expression of KFL4/2

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Endothelial cells (EC) line the entire circulatory system and control the passage of materials into and out of the bloodstream. EC dysfunction is damage to EC that impairs the function of the endothelium, and plays a central role in the development of vascular disease states such as atherosclerosis (plaque deposits/build-up), thrombosis (blood clots), and their pathological consequences, including heart attack or stroke. Kruppel-like factors 4 and 2 (KLF4 and KLF2) are transcription factors with critical roles in endothelial phenotypic and vascular homeostasis. Increased levels of endothelial KLF4/2 serve vasopotective functions. In this study we examined potential pharmacological inhibitors of HEG1-KRIT1 protein complex in order to upregulate KLF4/2 transcription factors in the EC. Analysis of GFP-KRIT1 FERM domain binding HEG1 coated beads through flow cytometry demonstrated that the KRIT with the mutated lysing residues displayed lower levels of KRIT1 binding when compared to the wild type. We evaluated whether the small molecule HKi002 would affect endothelial KLF2 and KLF4 gene expression. HUVEC and hCMEC/D3 human cell lines, were treated with the HKi002 inhibitor and KLF2 and KLF4 expression levels were determined. We observed that incubation of HUVEC cells with HKi002 upregulated both KLF2 and KLF4 expression in a dose-dependent manner. These results indicate that pharmacological inhibition of KRIT1-HEG1 protein interaction can be used to study the early and sustained effects of upregulation of KLF2 and KLF4. Future studies will focus on identifying other small molecule inhibitors that demonstrate high levels of KLF2/4 upregulation and in-vivo experimental trials on mice.

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Pharmacological Manipulation of HEG1-KRIT1 Protein Complex Modulates Endothelial Vasoprotective Functions through Expression of KFL4/2

Endothelial cells (EC) line the entire circulatory system and control the passage of materials into and out of the bloodstream. EC dysfunction is damage to EC that impairs the function of the endothelium, and plays a central role in the development of vascular disease states such as atherosclerosis (plaque deposits/build-up), thrombosis (blood clots), and their pathological consequences, including heart attack or stroke. Kruppel-like factors 4 and 2 (KLF4 and KLF2) are transcription factors with critical roles in endothelial phenotypic and vascular homeostasis. Increased levels of endothelial KLF4/2 serve vasopotective functions. In this study we examined potential pharmacological inhibitors of HEG1-KRIT1 protein complex in order to upregulate KLF4/2 transcription factors in the EC. Analysis of GFP-KRIT1 FERM domain binding HEG1 coated beads through flow cytometry demonstrated that the KRIT with the mutated lysing residues displayed lower levels of KRIT1 binding when compared to the wild type. We evaluated whether the small molecule HKi002 would affect endothelial KLF2 and KLF4 gene expression. HUVEC and hCMEC/D3 human cell lines, were treated with the HKi002 inhibitor and KLF2 and KLF4 expression levels were determined. We observed that incubation of HUVEC cells with HKi002 upregulated both KLF2 and KLF4 expression in a dose-dependent manner. These results indicate that pharmacological inhibition of KRIT1-HEG1 protein interaction can be used to study the early and sustained effects of upregulation of KLF2 and KLF4. Future studies will focus on identifying other small molecule inhibitors that demonstrate high levels of KLF2/4 upregulation and in-vivo experimental trials on mice.