Development of a Cell-based Regenerative Strategy to Modulate Angiogenesis and Inflammation in Ischemic Muscle
Doctor of Philosophy
Musculoskeletal Health Research
Flynn, Lauren E.
The delivery of human adipose-derived stromal cells (hASCs) to ischemic tissues represents a promising strategy to promote vascular regeneration for patients with critical limb ischemia (CLI). This thesis focused on the evaluation of hydrogels to enhance the retention and pro-angiogenic capacity of hASCs following delivery in vivo. Additionally, priming strategies to augment the paracrine function of hASCs were developed and assessed.
Recognizing the importance of endogenous macrophages in the pro-regenerative function of hASCs, delivery using a previously-developed hydrogel system, composed of peptide-functionalized methacrylated glycol chitosan (MGC-RGD) and a copolymer of poly(ethylene glycol) and poly(trimethylene carbonate) (PEG(PTMC-A)2), was assessed in a femoral artery ligation-induced CLI (FAL-CLI) model in athymic nu/nu mice. In contrast to the response in more severely immunocompromised NOD/SCID mice, hASC retention was enhanced in nu/nu mice when delivered in saline in comparison to the composite hydrogels. However, enhanced cell retention was insufficient to augment vascular regeneration. Interestingly, a negative host response was observed when the hASCs were delivered in the hydrogels in the nu/nu mice, which was not observed in the other treatment groups or in the NOD/SCID mice.
Building from this work, the effects of hydrogel composition on hASC retention and pro-angiogenic function were assessed by comparing hydrogels comprised of MGC and methacrylated hyaluronic acid (MHA) to the previous composite system. Notably, greater levels of pro-angiogenic and immunomodulatory paracrine factors were detected in conditioned media from hASCs encapsulated in the MGC-based hydrogels. Following delivery to an FAL-CLI model in athymic nu/nu mice, hASCs were better retained within the MHA hydrogels, but this enhanced cell retention was not associated with augmented vascular regeneration.
Finally, co-culture with human peripheral blood-derived monocytes (hPBMs) and stimulation with the inflammatory cytokines IFNγ and TNFα was assessed as an in vitro priming strategy to augment hASC paracrine function. hASC secretion of pro-angiogenic and immunomodulatory factors was enhanced by inflammatory cytokine stimulation in combination with co-culture with hPBMs. Demonstrating the immunomodulatory and pro-angiogenic effects of the secretome, conditioned media generated by inflammatory cytokine-stimulated hASCs increased the phagocytic activity of human macrophages and may have modulated human endothelial cell survival under serum-free conditions.
Summary for Lay Audience
Peripheral arterial disease (PAD) is a devastating condition characterized by narrowing or blockages of blood vessels in the limbs, with the lower limbs being most commonly affected. Current treatment options for these patients are limited, resulting in high rates of amputation and mortality. As such, there is interest in the delivery of pro-regenerative cell populations to regenerate blood vessels in patients with PAD. In particular, human adipose-derived stromal cells (hASCs) are a promising pro-regenerative cell source that can be isolated from human fat tissue. Following delivery, hASCs can secrete pro-regenerative proteins that coordinate host immune cells to reduce inflammation and promote the growth of new blood vessels that may help to restore blood flow to the affected tissues.
However, limitations with the application of hASCs exist, including poor retention following delivery to the harsh environment within diseased tissues. To address this barrier, the use of injectable biomaterial scaffolds to deliver the cells is a promising strategy to augment their retention and therapeutic efficacy following injection. Within this complex system, careful refinement is necessary to successfully harness the hASCs to promote regeneration without eliciting a negative inflammatory response. In addition, assessment of the therapeutic system must be performed using appropriate animal models.
Building from previous work, this thesis assessed the delivery of hASCs within biomaterial scaffolds in a mouse model of PAD with more functional immune cells than the model that had been applied in previous testing. Moreover, several different biomaterials were compared to see how scaffold composition influenced the secretion of proteins by hASCs, as well as hASC retention and blood vessel regeneration in the mouse model of PAD. Finally, a new strategy was developed to augment the secretion of regenerative proteins by hASCs, with the goal of enhancing their ability to promote blood vessel regeneration and modulate inflammation. Combining the lessons learned from this project will contribute to improved strategies to augment the therapeutic potential of hASCs, which will be important for designing effective therapies that can be successfully translated to the clinic to improve the health and quality of life of patients with PAD.
Serack, Fiona E., "Development of a Cell-based Regenerative Strategy to Modulate Angiogenesis and Inflammation in Ischemic Muscle" (2023). Electronic Thesis and Dissertation Repository. 9116.
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