Decoding Tie2 Signaling with AI-Based Designed Proteins to Mitigate Vascular Diseases

Abstract

Background Blood vessels are crucial for tissue regeneration and proper organ functions. Functional endothelial cells display heterogeneity in stability and permeability that facilitate organ function and tissue homeostasis. Tie2 signaling controls vascular remodeling, stability, and permeability. Understanding the angiopoietin-Tie2 pathway will allow the regeneration of stable, functional blood vessels to treat diseases related to vascular dysfunctions in the oral cavity, such as oral cancers1,2, diabetic oral complications3, pulpitis4, periodontitis, and tooth loss5. Thus, investigating the Tie2 pathway that regulates angiogenesis is paramount in regenerative dentistry and oral medicine. Angiopoietin-1 (Ang1) and -2 (Ang2) modulate Tie2 signaling via nearly identical receptor-binding domains (F-domain) but elicit opposing vascular outcomes. Ang1 stabilizes blood vessels and reduces permeability while Ang2 does the opposite despite sharing high structural similarity. To date, the mode of Ang-Tie2 activation and downstream molecular events leading to vascular stability is not fully understood. Objectives This study aims to dissect the molecular basis of the Tie2 pathway and the subsequent molecular events leading to vascular stability using artificial intelligence-based algorithms to computationally designed proteins as scaffolds for probing the mechanism of the Tie2 pathway. Methods A combination of RoseTTAFold and AlphaFold was employed to computationally design protein scaffolds for conjugating the Ang1 F-domain at various valencies and geometries to produce F-domain scaffolds. These F-domain constructs were used to probe (I) the molecular basis of Tie2 downstream signaling output and (II) the subsequent molecular events leading to tight junction formation. Tie2 signaling was analyzed using quantitative western blot, wound healing assay, tube formation assay, and a tight junction recovery assay in human umbilical vein endothelial cells (HUVEC) and human brain microvascular endothelial cells (HBMECs). Quantitative super-resolution microscopy was used to identify the molecular components that underly the Tie2 interaction upon receptor clustering in HUVECs and HBMECs. Controlled cortical impact to generate traumatic brain injury (CCI-TBI) in murine models was used to evaluate the in vivo effect of vascular repair upon Tie2 activation. Results Upon screening the designed F-domain scaffolds, two broad phenotypic classes were identified and distinguished by the number of presented F-domains. Scaffolds presenting 3 to 4 F-domains have Ang2-like activity, up-regulating pFAK and pERK, but not pAKT, and failing to induce cell migration and vascular stability. In contrast, scaffolds presenting 6, 8, 12, 30, or 60 F-domains have Ang1-like activity, up-regulating pAKT and inducing FOXO1 nuclear exclusion, cell migration, and vascular stability. When examined in vivo, superagonist icosahedral nanoparticles displaying 60 F-domains significantly improved revascularization in hemorrhagic brains after a controlled cortical impact injury. To further investigate how Tie2 stabilizes blood vessels at the molecular level, F-domains conjugated to two-dimensional protein sheets6 were employed to identify the molecular interactions that Tie2 makes. We found that clustering Tie2 receptors produces two classes of Tie2 clusters that recruit integrin α5β1, adherens junctions (VE-Cadherin), and tight junctions (ZO1, claudin-5, and occludin). The knockdown of α5 integrin using siRNA demonstrated that tight junction recruitment to Tie2 clusters does not require integrin, while VE-Cadherin is integrin-dependent. Upon detailed examination, super clustering Tie2 forms two functional classes of Tie2 complexes. The Tie2-integrin complex upregulates the pAKT/FOXO1 signaling axis to promote cell survival via transcriptional change and recruits pCAS and VE-cadherin to promote cell migration. The other Tie2 clusters localize at cell-cell junctions and orchestrate the assembly of ZO1, claudin-5, and occludin to form tight junctions. Tie2 activation using agonistic F-domain scaffolds accelerates tight junction (CLDN5 and ZO1) re-assembly after chemical disruptions in 2D endothelial cell culture, reinforcing the notion that Tie2 activation initiates tight junction formation. Conclusions For the first time, this study illustrates that F-domain valency determines Tie2 signaling and vascular outcome. Tie2 activation generates two functional classes of Tie2 clusters that regulate endothelial cell gene expression and junctional cell-cell adhesion to modulate vascular stability and permeability. In essence, this study utilized protein design as a novel tool for investigating biochemical processes involving receptor tyrosine kinases (RTKs). The Tie2 superagonists and antagonists characterized here hold tremendous therapeutic value for tissue regeneration, wound healing, and cancer therapy.