Hydrogen sulfide-releasing peptide hydrogel limits the development of intimal hyperplasia in human vein segments.

Currently available interventions for vascular occlusive diseases suffer from high failure rates due to re-occlusive vascular wall adaptations, a process called intimal hyperplasia (IH). Naturally occurring hydrogen sulfide (H <sub>2</sub> S) works as a vasculoprotective gasotransmitter in vivo. However, given its reactive and hazardous nature, H <sub>2</sub> S is difficult to administer systemically. Here, we developed a hydrogel capable of localized slow release of precise amounts of H <sub>2</sub> S and tested its benefits on IH. The H <sub>2</sub> S-releasing hydrogel was prepared from a short peptide attached to an S-aroylthiooxime H <sub>2</sub> S donor. Upon dissolution in aqueous buffer, the peptide self-assembled into nanofibers, which formed a gel in the presence of calcium. This new hydrogel delivered H <sub>2</sub> S over the course of several hours, in contrast with fast-releasing NaHS. The H <sub>2</sub> S-releasing peptide/gel inhibited proliferation and migration of primary human vascular smooth muscle cells (VSMCs), while promoting proliferation and migration of human umbilical endothelial cells (ECs). Both NaHS and the H <sub>2</sub> S-releasing gel limited IH in human great saphenous vein segments obtained from vascular patients undergoing bypass surgery, with the H <sub>2</sub> S-releasing gel showing efficacy at a 5x lower dose than NaHS. These results suggest local perivascular H <sub>2</sub> S release as a new strategy to limit VSMC proliferation and IH while promoting EC proliferation, hence re-endothelialization. STATEMENT OF SIGNIFICANCE: Arterial occlusive disease is the leading cause of death in Western countries, yet current therapies suffer from high failure rates due to intimal hyperplasia (IH), a thickening of the vascular wall leading to secondary vessel occlusion. Hydrogen sulfide (H <sub>2</sub> S) is a gasotransmitter with vasculoprotective properties. Here we designed and synthesized a peptide-based H <sub>2</sub> S-releasing hydrogel and found that local application of the gel reduced IH in human vein segments obtained from patients undergoing bypass surgery. This work provides the first evidence of H <sub>2</sub> S efficacy against IH in human tissue, and the results show that the gel is more effective than NaHS, a common instantaneous H <sub>2</sub> S donor

Electrospun Scaffolds Functionalized with a Hydrogen Sulfide Donor Stimulate Angiogenesis

Tissue-engineered constructs are currently limited by the lack of vascularization necessary for the survival and integration of implanted tissues. Hydrogen sulfide (H2S), an endogenous signaling gas (gasotransmitter), has been recently reported as a promising alternative to growth factors to mediate and promote angiogenesis in low concentrations. Yet, sustained delivery of H2S remains a challenge. Herein, we have developed angiogenic scaffolds by covalent attachment of an H2S donor to a polycaprolactone (PCL) electrospun scaffold. These scaffolds were engineered to include azide functional groups (on 1, 5, or 10% of the PCL end groups) and were modified using a straightforward click reaction with an alkyne-functionalized N-thiocarboxyanhydride (alkynyl-NTA). This created H2S-releasing scaffolds that rely on NTA ring-opening in water followed by conversion of released carbonyl sulfide into H2S. These functionalized scaffolds showed dose-dependent release of H2S based on the amount of NTA functionality within the scaffold. The NTA-functionalized fibrous scaffolds supported human umbilical vein endothelial cell (HUVEC) proliferation, formed more confluent endothelial monolayers, and facilitated the formation of tight cell-cell junctions to a greater extent than unfunctionalized scaffolds. Covalent conjugation of H2S donors to scaffolds not only promotes HUVEC proliferation in vitro, but also increases neovascularization in ovo, as observed in the chick chorioallantoic membrane assay. NTA-functionalized scaffolds provide localized control over vascularization through the sustained delivery of a powerful endogenous angiogenic agent, which should be further explored to promote angiogenesis in tissue engineering