Development of a Cell-based Regenerative Strategy to Modulate Angiogenesis and Inflammation in Ischemic Muscle
Abstract
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.