Periprocedural Hydrogen Sulfide Therapy Improves Vascular Remodeling and Attenuates Vein Graft Disease

Abstract

Background Failure rates after revascularization surgery remain high, both in vein grafts (VG) and arterial interventions. One promising approach to improve outcomes is endogenous upregulation of the gaseous transmitter-molecule hydrogen sulfide, via short-term dietary restriction. However, strict patient compliance stands as a potential translational barrier in the vascular surgery patient population. Here we present a new therapeutic approach, via a locally applicable gel containing the hydrogen sulfide releasing prodrug (GYY), to both mitigate graft failure and improve arterial remodeling. Methods and Results All experiments were performed on C57BL/6 (male, 12 weeks old) mice. VG surgery was performed by grafting a donor-mouse cava vein into the right common carotid artery of a recipient via an end-to-end anastomosis. In separate experiments arterial intimal hyperplasia was assayed via a right common carotid artery focal stenosis model. All mice were harvested at postoperative day 28 and artery/graft was processed for histology. Efficacy of hydrogen sulfide was first tested via GYY supplementation of drinking water either 1 week before VG surgery (pre-GYY) or starting immediately postoperatively (post-GYY). Pre-GYY mice had a 36.5% decrease in intimal/media+adventitia area ratio compared with controls. GYY in a 40% Pluronic gel (or vehicle) locally applied to the graft/artery had decreased intimal/media area ratios (right common carotid artery) and improved vessel diameters. GYY-geltreated VG had larger diameters at both postoperative days 14 and 28, and a 56.7% reduction in intimal/media+adventitia area ratios. Intimal vascular smooth muscle cell migration was decreased 30.6% after GYY gel treatment, which was reproduced in vitro. Conclusions Local gel-based treatment with the hydrogen sulfide-donor GYY stands as a translatable therapy to improve VG durability and arterial remodeling after injury.

Keywords: hydrogen sulfide; vascular remodeling; vein graft disease.

Conflict of interest statement

None.

Figures

Figure 1. Systemic therapy with the H2S donor GYY limits vein graft disease.

A, Experimental design: 60% high‐fat diet (HFD), 1 week of preconditioning (pre‐GYY) or 4 weeks of postconditioning (post‐GYY) vs control. B, Schematic depiction of the vein graft procedure: end‐to‐end anastomosis of a donor mouse caval vein into a recipient mouse right common carotid artery. C, Representative images of control, pre‐GYY, and post‐GYY vein grafts harvested at POD 28 after Masson‐trichome staining, with yellow lining highlighting the intimal‐M+A border. Scale bars=1 mm or 500 µm as indicated. D through G, Morphometric analysis of vein grafts at POD 28 in control, pre‐GYY, and post‐GYY treated mice as indicated; n=7 to 8/group. Multiple comparisons are via Kruskal‐Wallis test with Dunn’s multiple comparisons test unless indicated otherwise. D, I/M+A area ratios. E, I/M+A thickness. F, Intimal area. G, Intimal thickness. F through G, One‐way analysis of variance with Dunnett’s multiple comparisons test. H, Vein grafts after VSMC+Ki‐67 IHC. Scale bars=100 µm as indicated. I, Percentage of intimal and M+A layers occupied by VSMCs; n=6–7/group; via 2‐way analysis of variance with Tukey’s multiple comparisons test. Data represented as Mean±SD. *P<0.05, **P<0.01. GYY indicates hydrogen sulfide prodrug; I/M+A, intimal/media+adventitia; IHC, immunohistochemistry; POD, postoperative day; and VSMC, vascular smooth muscle cell.

Figure 2. Development of a locally applicable Pluronic gel that ensures extended H2S release both ex‐vivo and in‐vivo.

A through C, All samples were incubated with 0.25 µmol/L SF7‐AM in a 96‐well plate in triplicates for 30 minutes at 37°C and then imaged. Resulting fluorescent intensity was corrected for background signal with a PBS+SF7‐AM control. A, NaHS (1 mmol/L), GYY (1 mmol/L, 250 µmol/L), and PBS dissolved in equal volume PBS, H2S release was measured at regular intervals. B, Repeated measurement of same wells in 96‐wells plate to assess release rate of respective H2S releasing compounds. C, H2S‐drugs dissolved in equal volume of 40% Pluronic gel and overlaid with equal volume PBS. At regular intervals H2S release in PBS supernatant was measured. GYY indicates hydrogen sulfide prodrug; H2S, hydrogen sulfide; NaHS, sodium hydrosulfide; and SF7‐AM, fluorescent probe binding free hydrogen sulfide.

Figure 3. Local application of the H2S donor GYY mitigates injury‐induced arterial intimal hyperplasia.

A, Schematic depiction of surgical procedure, with partial ligation of the RCCA and resulting remodeling proximal from the stenosis. B, Experimental outline. C, Prestenosis diameter of RCCA at POD 7 and POD 14 in vehicle and local GYY treated animals, via 2‐way analysis of variance, n=9 to 10/group. D, Masson‐Trichome staining of RCCA cross‐sections at POD 28 after focal stenosis, with yellow lining indicating the intima‐media border. Scale bars are 200 µm as indicated. E through F, Measurement of I/M area and thickness ratio respectively at regular intervals proximal of stenosis at POD 28. G through H, Morphometric analysis of prestenosis RCCA, via Mann–Whitney test, n=9 to 10/group. G, I/M area. H, I/M thickness ratio. I, Intimal area. J, Intimal thickness. Data represented as Mean±SD. *P<0.05, **P<0.01, ***P<0.001. GYY indicates hydrogen sulfide prodrug; HFD, high‐fat diet; H2S, hydrogen sulfide; I/M, intimal/media; POD, postoperative day; and RCCA, right common carotid artery.

Figure 4. Periprocedural local H2S therapy protects from vein graft disease by attenuating intimal VSMC migration.

A, Experimental outline with local GYY application during vein graft surgery. B, Ultrasound measurements of vein graft diameters at POD 14 and POD 28 by 2‐way analysis of variance with Sidak’s multiple comparisons test; n=10 to 12/group. C, Representative images of locally treated vein grafts at POD 28 after Masson‐trichome staining. Yellow lining indicating the intima‐media+adventitia border. Scale bars 1 mm/500 µm as indicated. D through G, Morphometric analysis of vein grafts at POD 28, n=16 to18/group, Student’s t test was performed unless indicated otherwise. D, I/M+A area ratios. E, I/M+A thickness ratios F, Intimal area. G, Intimal thickness. H, IHC for SMC‐α+Ki‐67. Scale bars are 100 µm as indicated. I, Percentage of area occupied by SMC‐α positive cells in intimal and M+A layers, via 2‐way analysis of variance with Sidak’s multiple comparisons test; n=8 to 12/group. J, Intimal colocalization of VSMC and Ki‐67. Data represented as Mean±SD. *P<0.05, **P<0.01, ****P<0.0001. GYY indicates hydrogen sulfide prodrug; HFD, high‐fat diet; H2S, hydrogen sulfide; I/M+A, intimal/media+adventitia; IHC, immunohistochemistry; POD, postoperative day; SMC‐α, smooth muscle cell‐α; and VSMC, vascular smooth muscle cell.

Figure 5. Periprocedural H2S therapy limits VSMC migration and proliferation in vitro.

A, Representative immunocytochemistry image (20× & 40× magnification) of primary VSMC SMC‐α staining. Scale bars 100 and 50 µm as indicated. B, VSMCs after 6 hours treatment with 250 µmol/L GYY, with/without SF7‐AM incubation (20× & 40× magnification). Scale bars 100 and 50 µm as indicated. C, VSMCs after Transwell migration assay (6 hours in presence or absence of PDGF‐BB and/or 250 µm GYY). D, Quantification of Transwell migration assay. Number of migrated VSMCs normalized to per mm2 after 6 hours PDGF‐BB and/or 250 µmol/L GYY incubation in triplicates via 1‐way analysis of variance with Sidak’s multiple comparisons test. E, Quantification of Transwell migration assay after 6 hours of PDGF‐BB incubation and/or 6 hours of 250 µmol/L GYY pretreatment. F, Live imaging of VSMC positive for PI during incubation with 0 to 1000 µmol/L GYY or DMSO. Number of PI+ VSMC in GYY versus control via 2‐way analysis of variance with Dunnett’s multiple comparison test. G through I, MTT assay of VSMC incubated with 0 to 1000 µmol/L GYY or VEGF (10 ng/mL), 6 replicates per condition. One‐way analysis of variance with Dunnett’s multiple comparison test. J, Live imaging of HUVECs positive for PI during incubation with 0 to 1000 µmol/L GYY or DMSO. Number of PI‐positive HUVEC in GYY versus control via 2‐way analysis of variance with Dunnett’s multiple comparison test. */#: significant decrease in PI+ HUVECs supplemented with 250 µmol/L and 1000 µmol/L GYY respectively compared with control at T=18 to 19 hour. K through M, MTT assay of HUVECs incubated with 0 to 1000 µmol/L GYY or VEGF (10 ng/mL), 6 replicates per condition. One‐way analysis of variance with Dunnett’s multiple comparison test. Data represented as Mean±SD. *P<0.05, **P<0.01. DMSO indicates dimethyl sulfoxide; GYY, hydrogen sulfide prodrug; H2S, hydrogen sulfide; HUVEC, human umbilical endothelial cells; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide; PDGF‐BB, platelet‐derived growth factor; PI, proprium iodide; SMC‐α, smooth muscle cell‐α; VEGF, vascular endothelial growth factor; and VSMC, vascular smooth muscle cell.

Figure 6. Periprocedural local hydrogen sulfide therapy limits vein graft disease.

GYY indicates hydrogen sulfide prodrug; H2S, hydrogen sulfide; VSMC, vascular smooth muscle cell.