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The AAPS 103rd Annual Meeting 2025
May 24-27, 2025 | Austin, Texas
D156
The Early Angiogenic Effect Of Arterial And Venous Micropuncture Using In Vivo And In Vitro Models
Poster Presenter
Part of Topic
Basic Science Research
The Early Angiogenic Effects of Arterial and Venous Micropuncture Using in vivo and in vitro Models
Mohammad Hossein Asgardoon1, MD,MPH, Summer Horchler1, DO, Mingjie Sun1, MD, Mary Landmesser1, BS, Shailaja Agarwal1, PhD, Neekita R. Jikaria1,2, MD, Maryam Abdelaal1, BS, Dino J. Ravnic1,2 ,DO,MPH
Irvin S. Zubar Laboratory, College of Medicine, The Pennsylvania State University 1; Department of Surgery, Penn State Health Milton S. Hershey Medical Center2
Introduction
•Attempts at engineered tissue replacements are limited by slow vascularization upon implantation.
•Recently, we described a microsurgical approach called micropuncture (MP) to induce rapid scaffold vascularization.
•In MP, the blood vessel wall is precisely disrupted to provide an immediate route for inflammatory and endothelial cell (EC) extravasation leading to subsequent capillary formation.
•Our initial in vivo studies1 demonstrated the utility of MP when applied concurrently to the femoral artery and vein (Figure 1). However, the specific contributions of each vessel remains unclear.
•We hypothesize that early MP-induced angiogenesis is dependent on concurrent arterial and venous targeting.
Methods
•In vivo, 60 μm MP was performed on the rat femoral artery (MPA), vein (MPV), or both (MPAV) before collagen scaffold implantation (Figure 2). Controls did not receive MP.
•Angiogenesis was assessed at Day 3 via in situ fluorescence angiography.
•EC and macrophage (Mϕ) infiltration was evaluated using CD-31, F4/80 and DAPI staining.
•CD-31 and F4/80 expression were quantified with ImageJ and analyzed with GraphPad Prism.
•In vitro, ECs were isolated from rat femoral artery (ECA) and vein (ECV) (Figure 3).
•ECA, ECV, or their coculture (ECAV, 1:1) were seeded (1×105 cells) on collagen scaffolds and punctured (20 MP/well). Non-MP controls were included.
•Conditioned media was collected at 24 h for cytokine analysis (ECA or ECV alone vs. non-MP controls).
•Tube formation was assessed at 72 h post-MP using ≥ 10 images per well.
•mRNA sequencing at 24 h was analyzed using R.
•Angiograms and tube formation were quantified using AI (MetaVi Labs, TX).
•P < 0.05 was considered statistically significant.
Results
•In vivo, MPAV profoundly increased scaffold vascularization (Figure 4A).
•MPV alone showed greater vascular density compared to MPA, confirmed with CD-31 expression (Figure 4B, C).
•Mϕ infiltration into the scaffold was also enhanced with MP and appeared to originate primarily from the vein (Figure 4D).
•However, the interaction between ECs and Mϕs during MP-induced angiogenesis remains unclear.
•In vitro, ECAV formed more tubes compared to the non-MP combined cohort (Figure 5A).
•Semi-quantitative ELISA analysis revealed substantial alterations in angiogenic protein secretion following MP (Figure 5B).
•Specifically, inflammatory cytokines such as IL-6, MCP-1, GRO, IFN-γ, and RANTES were higher in ECV compared to ECA (Figure 5B).
•mRNA sequencing demonstrated that cytokine genes such as IL-6 and CCl-2 were upregulated in MPV and MPAV, however the level of upregulation was greater in the MPV.
Conclusion
•MP appears to stimulate rapid capillary formation through differential protein expression in arterial and venous ECs.
•While arterial and venous MP alone can trigger microvascular formation, the angiogenic effects are most profound when they are targeted concurrently.
•Further refinement of microsurgical therapies to induce angiogenesis could play an important role in advancing tissue engineering.
•Future in vitro studies will aim to mechanistically determine the role of macrophages in MP-induced angiogenesis under both static and dynamic conditions.