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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: Acta Biomater. 2019 Jul 26;97:374–384. doi: 10.1016/j.actbio.2019.07.042

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

Alban Longchamp 1, Kuljeet Kaur 2, Diane Macabrey 1, Celine Dubuis 1, Jean-Marc Corpataux 1, Sébastien Déglise 1, John B Matson 2,*, Florent Allagnat 1,*
PMCID: PMC6801028  NIHMSID: NIHMS1535800  PMID: 31352106

Abstract

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 (H2S) works as a vasculoprotective gasotransmitter in vivo. However, given its reactive and hazardous nature, H2S is difficult to administer systemically. Here, we developed a hydrogel capable of localized slow release of precise amounts of H2S and tested its benefits on IH. The H2S-releasing hydrogel was prepared from a short peptide attached to an S-aroylthiooxime H2S donor. Upon dissolution in aqueous buffer, the peptide self-assembled into nanofibers, which formed a gel in presence of calcium. This new hydrogel delivered H2S over the course of several hours, in contrast with fast-releasing NaHS. The H2S-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 H2S-releasing gel limited IH in human great saphenous vein segments obtained from vascular patients undergoing bypass surgery, with the H2S-releasing gel showing efficacy at a 5× lower dose than NaHS. These results suggest local perivascular H2S release as a new strategy to limit VSMC proliferation and IH while promoting EC proliferation, hence re-endothelialization.

Keywords: Intimal hyperplasia, Smooth muscle cells, Proliferation, Hydrogen sulfide, Hydrogel

Graphical Abstract

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1. Introduction

Despite remarkable technological advances, the rate of restenosis due to intimal hyperplasia (IH) one year following endovascular reconstruction or bypass surgery reaches 30% [1]. IH is driven by the dysfunction of endothelial cells (EC) lining the inner part of the vessels, which results in a reprogramming of the vascular smooth muscle cells (VSMCs) from a differentiated phenotype to a proliferating and migrating phenotype and the formation of an occlusive neo-intima layer at the site of injury [2]. The available systemic drug therapies used to prevent restenosis are generally poorly tolerated and show narrow therapeutic ranges [3, 4]. Localized treatments include drug-eluting angioplasty balloons and stents, which limit VSMC proliferation and reduce IH, but they also delay re-endothelization, limiting their efficacy and prolonging the need for anti-thrombotic medication [5].

Hydrogen sulfide (H2S) is an endogenous gasotransmitter with important cytoprotective and anti-inflammatory properties, which may protect against atherosclerosis, chronic heart failure and vascular restenosis [6, 7]. In humans, plasma H2S concentration declines with age [8], and the circulating levels of H2S are reduced in patients suffering from cardiovascular diseases [9, 10]. Administration of exogenous H2S may reduce the symptoms of cardiovascular diseases, but its gaseous nature and rapid in vivo half-life nature create a delivery challenge.

To tap the therapeutic potential of this gasotransmitter, numerous sulfur-containing small molecules called H2S donors have been developed that release H2S either by hydrolysis or in response to a specific trigger [11]. However, H2S delivery via small molecule donors is often limited by short release periods, low water solubility, lack of target specificity, and toxicity in some cases [11, 12]. To limit toxicity and extend the H2S release period, H2S donors have been incorporated into water-soluble polymers, micelles, hydrogels, nanofibers, and films [13–17]. However, there is still a growing need for H2S-releasing materials capable of delivering H2S directly at a site of interest for an extended period of time [18, 19]. Peptide-based hydrogels have been widely used for tissue engineering and regenerative medicine [20], but only a handful of reports detail their use in delivering gasotransmitters [21–23].

Here we developed and tested the safety and therapeutic potential of a self-assembling aromatic peptide amphiphile, employing an H2S-releasing S-aroylthiooxime (SATO) group as the aromatic component. We found that our H2S-releasing hydrogel, which is capable of localized H2S delivery, promotes EC function while inhibiting VSMC expansion and IH formation in human veins at a significantly lower dose than NaHS.

2. Materials and methods

2.1. Chemicals

Rink Amide MBHA resin and 9-fluorenylmethoxy carbonyl (Fmoc) protected L-amino acids were purchased from P3Biosystems and used as received. HBTU, N-methylpiperidine, DBU, and other reagents for peptide synthesis were purchased from commercial vendors and used as received unless otherwise noted. The solvents employed for peptide synthesis were reagent grade.

2.2. Peptide synthesis and purification

Peptides were synthesized either manually or using a Libertyl microwave-assisted peptide synthesizer (CEM) using solid-phase peptide synthesis (SPPS) via standard Fmoc protocols as described previously [23]. 4-Formyl benzoic acid was coupled to the N-terminus of the peptide on resin using HBTU and DIEA in DMF. After cleavage and isolation, peptides were dissolved in water containing 0.1% NH4OH and filtered through a 0.45 μm PTFE filter before purification. Purification by preparative-scale reverse phase high-performance liquid chromatography (RP-HPLC) was carried out on an Agilent Technologies 1260 Infinity HPLC system, eluting with a gradient of 2% ACN to 90% ACN in milliQ H2O over 33 min using an Agilent PLRP-S column (100 Å particle size, 25 × 150 mm) and monitoring at 220 nm. To both mobile phases was added 0.1% NH4OH to aid in solubility. Fractions were analyzed by mass spectrometry (Advion ExpressIon Compact Mass Spectrometer), and product-containing fractions were combined, rotovapped to remove ACN, and lyophilized (LabConco).

The lyophilized peptide FBA-IAVEE was dissolved in dry DMSO and reacted with S-benzoylthiohydroxylamine (SBTHA) in the presence of catalytic TFA to afford the final SATO-FBA-IAVEE peptide. Peptide FBA-IAVEEEE was similarly reacted with O-benzyl hydroxylamine hydrochloride (OBHA⋅HCl) in dry DMSO, but without using TFA, to afford the non-H2S-releasing control peptide OBHA-FBA-IAVEEEE. Peptides were dissolved in a mixture of phosphate buffer (100 mM at pH 7.4) and acetonitrile (5:2 v/v) and filtered through a 0.45 μm PTFE filter before purification. Purification was carried out using RP-HPLC, eluting with a gradient of 2% ACN to 90% ACN in milliQ H2O without any additives. The protocol for analysing and recovering the peptides was the same as described above. The final peptides were dissolved in milliQ water and distributed into aliquots (100 μg each). Aliquots were frozen, lyophilized, and stored at −20 °C.

2.3. Critical aggregation concentration (CAC) measurements

Nile red stock solution in acetone (1 mg/mL) was diluted in milliQ water to a concentration of 0.01 mg/mL and was used to make all peptide solutions. A peptide stock solution was prepared at 4 mg/mL in the Nile red stock solution and was further diluted to the concentration of 3 mg/mL, 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.1 mg/mL, 0.01 mg/mL, 0.001 mg/mL, and 0.0001 mg/mL. All peptide dilutions were vortexed/sonicated for a few seconds, then 300 μL of each was transferred to a 96-well plate, and the plate was allowed to sit in dark for 15–20 min. Florescence spectra were recorded using a Varian Cary Eclipse fluorescence spectrophotometer (FL1105M003) with an excitation wavelength of 550 nm. Fluorescence intensity measured at 628 nm was plotted against log[concentration], and the final CAC values were estimated to be the point of intersection between the linear fits of high and low concentration regimes.

2.4. Circular Dichroism (CD) Spectroscopy

CD spectra were measured at room temperature using a Jasco J-815 CD spectrometer (Jasco Inc.) with a preset N2 flow at 120 mL/min. The range of wavelengths employed was 250 to 190 nm (50 nm/min) with a response time of 8 s. Samples for both Pep-H2S and Pep-Ctrl were freshly prepared at 10 mM (20 μL) in 1X PBS (pH 7.4) and were analyzed using a dismountable quartz cuvette with path length of 0.2 mm with 3 iterations for each sample. Raw spectra were converted to mean residual ellipticity for comparison.

2.5. Hydrogelation

Both Pep-H2S and Pep-Ctrl formed hydrogels in PBS solutions (Gel-H2S and Gel-Ctrl) at physiological pH upon addition of 4 μL of CaCl2 solution (200 mM in water) to 90 μL of peptide solution (10 mM in 1X PBS). The final concentration of CaCl2 in the hydrogels was 8 mM.

2.6. Rheology

Rheological experiments were done on an AR-2000 (TA instruments) using a 25 mm parallel plate geometry. Buffered peptide solutions (240 μL, 1 wt.% peptide, ~10 mM) were prepared for each peptide in 1X PBS (pH 7.4) and quickly transferred to the rheometer’s bottom geometry. Gelation was initiated upon addition of 10 μL CaCl2 solution (200 mM in water), and the resulting solution was mixed thoroughly with a pipet tip to ensure homogeneity. After allowing the solution to gel for 10 min, the upper geometry was lowered to a pre-set gap of 500 μm, and a dynamic time sweep was performed at a frequency of 1 Hz and 0.5% strain to measure storage (G’) and loss (G”) moduli. Each time sweep was followed by a dynamic frequency sweep (0.010–100 Hz at 0.5% strain) and a strain sweep (1 Hz at 0.5–100% strain).

2.7. Morphology analysis via TEM

Peptide solutions (10 mM in 0.05 M phosphate buffer at pH 7.4) were prepared and allowed to age overnight, then diluted with water to 500 μM. Next, 10 μL of the peptide solution was deposited on a carbon-coated copper TEM grid (300 mesh, Electron Microscopy Sciences), allowed to sit for 5–6 min, and then gently blotted with filter paper. The grid was then washed by adding a drop of MilliQ water, allowing it to stand for 1 min, and then blotting with filter paper. Samples were stained with 10 μL of a 2% uranyl acetate aqueous solution for 5–6 min, blotted with filter paper, and allowed to dry in air before TEM observation. Images were taken on a Philips EM420 TEM with a slow scan CCD camera.

2.8. H2S release measurements using an electrode probe

H2S release from the peptides was measured amperometrically using an electrode probe (ISO-H2S-100-CXX, World Precision Instruments). A solution of either Pep-H2S or Pep-Ctrl (20 μL of 0.1 mM solution in 1X PBS) was placed in an inner well inside a specially designed glass vial equipped with a stir bar. An additional 76 μL of 10 mM PBS buffer at pH 7.4 was then added to the well, followed by 4 μL L-Cys solution (5 mM in water). The final concentrations in the inner well were 20 μM in peptide and 0.2 mM (10 eq.) in L-Cys. The well was immediately covered with the gas-permeable membrane (Breathe easier, Diversified Bioteck), and PBS buffer at pH 7.4 (4.95 mL) mixed with 50 μL of diethylenetriaminepentaacetic acid (DTPA) solution (10 mM in water) was added into the vial, covering the inner well. The H2S-selective microelectrode was then immersed in the PBS solution, and the output signal was recorded. Similarly, H2S release from Pep-H2S at 200 μM was measured by mixing 20 μL of peptide solution (1 mM) with 80 μL of PBS in the well followed by addition of 1 μL (10 eq.) L-Cys solution (200 mM in water). H2S release from Gel-H2S was measured by placing 96 μL of the peptide solution (10 mM in 1X PBS) in the inner well followed by 4 μL of CaCl2 to form a hydrogel. 10 μL L-Cys solution (200 mM in water) was added, the well was covered with the membrane, and output signal was measured as above. Calibration was carried out as previously reported. [24]

2.9. Cell culture

Human veins were obtained from donors who underwent lower limb bypass surgery [25]. For static vein cultures, 5 mm segments were kept for 7 days in RPMI-1640 Glutamax supplemented with 10 % FBS and 1% antibiotic solution (10,000 U/mL penicillin G, 10,000 U/mL streptomycin sulphate) at 37 °C, 5% CO2 and 5% O2. The cell culture medium was changed every 48 h. The hydrogels were prepared by dissolving the peptides at 10 mM in PBS, then adding 8 mM final concentration of CaCl2 to achieve gelation. The hydrogels (10 or 20 μL/mL) were then added to the vein segments in the media. No remaining gel was found after 48 h of culture. Freshly prepared gel was added when changing the media every 48 h.

The segments of vein were then fixed in 4% formalin and paraffin-embedded for histological analysis. Five distinct vein segments coming from five different patients were used in this study. Human smooth muscle cells were prepared from these human saphenous vein segments as previously described [25]. Briefly, 1–2 mm vein explants were plated on the dry surface of a 6-well culture plate coated with 1% Gelatin type B (Sigma-Aldrich) and maintained in RPMI, 10% FBS 1% antibiotic solution medium in a 37 °C, 5% CO2, 5% O2 environment. Because ECs could not be isolated from the vein segments, HUVECs purchased from Lonza were maintained in EGM™−2 (Endothelial Cell Growth Medium-2 BulletKit™) at 37 °C, 5% CO2 and 5% O2. Passages 1 to 8 were used for the experiments.

2.10. Histomorphometry and Immunohistochemistry

Segments of vein (5 mm) embedded in paraffin were cut into 5 μm sections. 2-mm out of 5-mm were cut in 4 series of 10 slides with 5 sections per slides with a 250 μm interval between the series. One slide per series was stained using Van Gieson-elastin (VGEL) staining. 3 images per section were taken at 100× magnification to cover the whole vein area. 8 measurements of the intima and media thicknesses were made from the images, evenly distributed along the length of the vein wall [26]. Thus, for each vein, the intima and media thicknesses values are a mean of 4 series × 3 images × 8 measures = 96 independent measures. Morphometric measurements were done by two independent researchers, one of them blind to the experimental groups, using the Leica Qwin® software (Leica, Switzerland).

PCNA (proliferating cell nuclear antigen) immunohistochemistry was performed on paraffin sections. After rehydration and antigen retrieval (TRIS-EDTA buffer, pH 9, 15 min in a microwave at 600 watts), human vein sections were incubated overnight with the proliferating cell nuclear antigen antibody (PCNA; M087901, Dako, Baar, Switzerland), washed and revealed using the EnVision +/HRP, DAB+ system according to manufacturer’s instructions (Dako, Baar, Switzerland), and counterstained with hematoxylin [27]. One slide per series was assessed and 3 images per section were taken at 100× magnification. The PCNA and hemotoxlin positive nuclei were manually counted by two independent observers unaware of the conditions.

2.11. Immunofluorescent staining

Cell immunostaining was performed on cells grown on glass coverslips (106 cells per well in 24 well plates) and fixed for 5 min in methanol at −20 °C. Cells were then permeabilized in PBS supplemented with 2 wt. % BSA and 0.3 vol. % Triton X-100 for 30 min, blocked in PBS supplemented with 2 wt. % BSA and 0.1 vol. % Tween 20 for another 30 min, and incubated overnight with the primary antibodies diluted in the same buffer. BrdU immunostaining was performed using mouse anti-BrdU (BD Bioscience 55627, 1:200) and fluorescent-labelled anti-mouse secondary antibodies (AlexaFluor 568; 1/500, Thermo Fisher Scientific). BrdU positive nuclei were automatically detected using the ImageJ software and normalized to the total number of DAPI-positive nuclei. Double calponin/SM22α immunofluorescent staining was performed using mouse anti human calponin (DAKO; M3556; 1/200) and rabbit anti-human SM22α, (Abcam; 4106; 1/400). Cells were then washed 3 times for 5 min in PBS supplemented with 0.1 vol. % Tween 20, and incubated for 1 h at room temperature with a mix of fluorescent-labelled secondary antibodies (anti-rabbit AlexaFluor 488 and anti-mouse AlexaFluor 568; 1/500).

2.12. Live-cell hydrogen sulfide measurement

Free sulfide was measured in cells using the SF7-AM fluorescent probe [28] (Sigma-Aldrich cat: 748110). The probe was dissolved in anhydrous DMF at 5 mM. 105 cells per well were plated in a 96 well plate. After 24 h, SF7-AM (5 μM) was added to VSMCs or HUVECs, and fluorescence intensity (λex = 495 nm; λem = 520 nm) was measured continuously in a Synergy Mx fluorescent plate reader at 37 °C before and after addition of various donors as indicated. Linear regressions of the SF7-AM fluorescent signal were calculated during the linear part of the curves generated to measure the H2S release rate.

2.13. Transmigration assays

The chemotactic-induced cell transmigration across a matrix barrier was investigated using a Boyden chamber made of a polycarbonate membrane insert with 8 mm pores (Falcon; BD Biosciences) placed in 24-well culture plates. 105 VSMCs resuspended in 300 μL of RPMI 1640 medium were plated onto the transwell. Transwells with VSMCs were placed in a 24-well plate with 400 μL of EGM-2 culture medium after 48 h in contact with HUVECs. 105 HUVECs resuspended in 300 μL of EBM™−2 (Endothelial Cell Growth Basal Medium-2, no supplements from Lonza) were plated in a transwell and placed in a 24-well plate with 400 μL of fresh complete EGM-2 BulletKit™ culture medium (with full supplements). In experiments with gels (Gel-H2S and Gel-Ctrl), 10 μL/mL of gel (1 wt. %) was placed in the 24-well plate. After 8 h, calcein-AM (5 μg/mL, Thermo) was added to the well to stain the cells on the outer surface of the membrane of the transwell. After 30 min and two washes with PBS, fluorescence was measured using a fluorescent plate reader (λex = 485 nm; λem = 530 nm). Cells were also imaged using an inverted fluorescent microscope (Leica AG).

2.14. Statistical analyses

All experiments were quantitatively analysed using GraphPad Prism® 6, and results are shown as mean ± SEM. One-way ANOVA tests were performed followed by multiple comparisons using post-hoc t-tests with the appropriate correction for multiple comparisons.

Ethics statement

Written, informed consent was obtained from all vein donors for human vein and VSMC primary cultures. The study protocols for organ collection and use were reviewed and approved by the Centre Hospitalier Universitaire Vaudois (CHUV) and the Cantonal Human Research Ethics Committee (http://www.cer-vd.ch/, no IRB number, Protocol Number 170/02), and are in accordance with the principles outlined in the Declaration of Helsinki of 1975, as revised in 1983 for the use of human tissues.

3. RESULTS

3.1. Design and synthesis of an H2S-releasing peptide and a control peptide

Self-assembling aromatic peptide amphiphiles rely on a combination of aromatic stacking of N-terminal aromatic groups and hydrogen bonding in a short peptide chain consisting of 2–6 amino acids to drive self-assembly in aqueous solution [29–32]. We based our peptide design for this study on our recent report on the first H2S-releasing aromatic peptide amphiphile [22]. Using an N-terminal SATO group as the aromatic component and a pentapeptide sequence modified from our original design, the sequence used here for the H2S-releasing peptide (termed Pep-H2S) was SATO-FBA-IAVEE, where SATO represents the aromatic H2S donor, FBA represents 4-formylbenzoic acid, and IAVEE represents the peptide sequence using 1-letter codes. We chose the IAVEE sequence compared with our previously published IAVEEE sequence because we envisioned that removing one Glu residue would increase gel stiffness. We also prepared a control peptide (termed Pep-Ctrl) incapable of releasing H2S with the sequence OBHA-FBA-IAVEEEE, where OBHA represents O-benzyl hydroxylamine. Pep-Ctrl contains an oxime linkage in place of the acylthiooxime linkage in Pep-H2S, but the peptides are otherwise identical. In initial rheological studies, we found that two additional C-terminal Glu residues were needed in Pep-Ctrl compared with Pep-H2S to afford hydrogels with similar storage moduli. We speculate that stronger aromatic stacking and/or hydrogen bonding in the OBHA component vs. the SATO component leads to this requirement for a longer hydrophilic sequence, which tends to decrease storage modulus, in Pep-Ctrl.

Peptides FBA-IAVEE and FBA-IAVEEEE were synthesized using Fmoc-based solid-phase peptide synthesis. The FBA units were coupled to the peptides on-resin before cleavage and purification of these two peptide-aldehydes. The two peptide aldehydes were then further derivatized to form the final aromatic peptide amphiphile products (Figure 1). Pep-H2S was prepared by condensing the N-terminal aldehyde in FBA-IAVEE with S-benzoylthiohydroxylamine to form the final molecule. Pep-Ctrl was synthesized similarly, condensing FBA-IAVEEEE with hydroxylamine OBHA. Both peptides were purified by preparative HPLC after the condensation step, lyophilized, and aliquoted for further analysis.

Figure 1.

Figure 1.

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A) Synthesis of Pep-H2S (sequence SATO-FBA-IAVEE). B) Synthesis of Pep-Ctrl (sequence OBHA-FBA-IAVEEEE).

3.2. Peptides self-assembled into nanoribbons

Self-assembly is typically observed in amphiphilic molecules, where the hydrophobic component drives assembly as it excludes water. In dilute solution, amphiphiles are molecularly dissolved, but self-assembly occurs above a given concentration, termed the critical aggregation concentration (CAC). Here we used the Nile red assay to determine the CAC for both Pep-H2S and Pep-Ctrl. The hydrophobic Nile red dye, which is non-fluorescent in a hydrophilic environment, incorporates within the hydrophobic core of the self-assembled nanostructures, resulting in a fluorescence enhancement proportional to the peptide concentration. The CAC is then defined as the point of abrupt change in the fluorescence intensity and refers to a minimum concentration above which the molecule exists primarily in a self-assembled state. In our experiments on Pep-H2S, we measured a CAC of 0.9 mM (near 1 mg/mL) for both peptides.

With CAC values in hand, we next explored the molecular packing of the self-assembled aromatic peptide amphiphiles Pep-H2S and Pep-Ctrl. Circular dichroism (CD) spectra were measured for both Pep-H2S and Pep-Ctrl at 10 mM in 1X PBS buffer to evaluate secondary structure formation. Prominent minima at 217 nm and 220 nm for Pep-H2S and Pep-Ctrl, respectively, indicated the presence of β-sheets (Figure 2A). Typically, β-sheet signals in the CD spectra of short, self-assembling peptides indicates the formation of extended, 1-dimensional nanostructures, which are necessary for gelation [33].

Figure 2.

Figure 2.

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A) CD spectra for 10 mM Pep-H2S and Pep-Ctrl solutions in 1X PBS buffer. B and C) Conventional TEM images for Pep-H2S and Pep-Ctrl, respectively. Peptides were dissolved at 10 mM in phosphate buffer (50 mM, pH 7.4). Samples were diluted to 500 μM immediately before casting and stained with 2% uranyl acetate solution. D and E) Frequency sweep oscillatory rheology for hydrogels Gel-H2S and Gel-Ctrl, respectively, prepared at 1 wt% in 1X PBS at pH 7.4 and gelled with CaCl2. F) H2S release curves measured on an H2S-selective electrochemical probe comparing Gel-H2S, Pep-H2S (200 μM and 20 μM), and Pep-Ctrl.

To explore further the self-assembled structures, we used conventional TEM with negative staining to image the nanostructures formed by Pep-H2S and Pep-Ctrl (Figure 2B, C). Aromatic peptide amphiphiles can take on many types of morphologies, including spheres, cylinders, flat or twisted ribbons, sheets, and others [30, 34]. When long, one-dimensional objects are observed, they may entangle under certain conditions to form gels. In both cases we observed flat nanoribbons, many several microns in length, with widths around 6–7 nm, which was consistent with the CD spectra depicting β-sheets. The nanoribbons were uniform in width, likely due to the fact that both peptide nanostructures are assembled from pure, single molecules with no molecular weight dispersity.

3.2. Calcium salts triggered peptide gelation to form soft hydrogels

As noted above, aromatic peptide amphiphiles that form long, one-dimensional aggregates may gel under conditions that promote entanglement of the nanostructures. We found that addition of CaCl2 promoted the rapid gelation of both Pep-H2S and Pep-Ctrl. This is likely due to two factors: 1) Charge screening of the negatively charged Glu residues, which reside on the nanofiber surface and cause the fibers to repel each other under low salt conditions; and 2) Formation of salt bridges between the nanofibers due to the divalent nature of the calcium ion.

The gelation and viscoelastic properties of the hydrogels, termed Gel-H2S and Gel-Ctrl when in the gel state, were evaluated by rheological measurements. Peptide solutions were first prepared at 1 wt. % (~10 mM) in 1X PBS, and solutions were added directly to the rheometer. Next, a solution of CaCl2 in water was added to the peptide solutions to afford a final CaCl2 concentration of 8 mM. After a brief waiting period to allow for gelation throughout the sample, each hydrogel was measured. Frequency sweeps of both Gel-H2S and Gel-Ctrl revealed that the storage modulus (G’) was higher than the loss modulus (G”) throughout the range tested, indicating that both peptides formed robust, soft hydrogels (Figure 2D, E). Gel-H2S showed a storage modulus of 50 Pa at intermediate frequencies, while Gel-Ctrl showed a storage modulus of 100 Pa. Both hydrogels were soft and easily manipulated by a spatula or syringe. At frequencies above 10 Hz, both hydrogels showed increases in both G’ and G”. This behavior is characteristic of materials with non-covalent cross-linking such as peptide-based hydrogels [35].

3.3. Peptide gel exhibited slow and sustained H2S release

SATOs are thiol-triggered H2S donors [24], so we aimed to evaluate H2S release from the peptides in solution and gel form in the presence of thiols. To obtain H2S release curves using real-time monitoring, we used an H2S-selective microelectrode probe (Figure 2F). A solution of Pep-H2S at 20 μM, triggered with a 10-fold excess of L-Cys, showed a steady rise in H2S concentration up to 0.2 μM over 120 min, after which it remained steady. Because H2S is constantly oxidizing and volatilizing, we typically observe peaking concentrations at values much lower than the starting concentration of the H2S donor. We also measured release at a 10-fold higher concentration to evaluate how this increase would affect the release rate. Similar to the 20 μM case, the H2S release profile for Pep-H2S at 200 μM showed a gradual release with a peaking time at 130 min at a concentration of 0.5 μM. As expected, Pep-Ctrl did not show any H2S release. We also tested release from Gel-H2S, prepared by addition of CaCl2 to H2S-Pep, into a large volume of PBS. Release was slow and steady and continued rising slowly over 220 min up to a peak concentration of 1.3 μM before tapering off. As measuring H2S release from Pep-H2S and Gel-H2S required different conditions, the peaking times and concentrations cannot be directly compared; however, the peak shapes for the peptide under both solution and gel conditions show slow and steady release, which we expect may be ideal for localized delivery of H2S.

3.4. SF7-AM signal in VSMC and HUVEC upon addition of Pep-Ctrl and Pep-H2S

Live VSMCs or HUVECs were incubated with the fluorescent H2S probe SF7-AM, and fluorescence was measured continuously before and after addition of 200 μM Pep-Ctrl, Pep-H2S, or 100 μM NaHS. Experiments were performed in VSMC or HUVEC culture media containing 200 μM L-Cys. No additional thiol was added to trigger H2S release. Time-lapse imaging of SF7-AM in the presence of VSMCs demonstrated that VSMCs produced little endogenous H2S, as evidenced by the slow and low buildup of the SF7-AM signal (Ctrl and Pep-Ctrl conditions, Figure 3A). The rate of endogenous H2S production was two-fold higher in HUVECs as compared to VSMCs (Figure 3B, C). As expected, addition of NaHS rapidly raised the SF7-AM signal to similar levels in both cell types, while Pep-H2S induced a 4-fold slower release in both cell types (Figure 3C). Of note, 200 μM Pep-H2S was not sufficient to surpass significant endogenous SF7-AM signal in HUVECs, but it significantly raised the signal in VSMCs above controls (Figure 3C). Lower Pep-H2S concentration (20 μM) was not sufficient to generate detectable amounts of H2S in either cell type (data not shown).

Figure 3.

Figure 3.

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A) SF7-AM fluorescent signal in live primary VSMCs exposed or not (control) to 100 μM NaHS, 200 μM Pep-Ctrl or 200 μM Pep-H2S for the indicated time. B) SF7-AM fluorescent signal in HUVECs exposed or not (control) to 100 μM NaHS, 200 μM Pep-Ctrl or 200 μM Pep-H2S for the indicated time. *P<0.05, ***P<0.001 vs. respective control; ###P<0.001 vs. Pep-H2S; as determined by repeated measures two-way ANOVA with post-hoc t-test with Tukey’s correction for multiple comparisons. C) Linear regression of SF7-AM fluorescence curves to estimate H2S-release rates. Data are mean ± SEM of 4 independent experiments. *P<0.05, ***P<0.001 vs. respective control; ###P<0.001 vs. Pep-H2S; °°°p<0.001 vs. respective condition in VSMCs, as determined by two-way ANOVA with post-hoc t-test with Tukey’s correction for multiple comparisons.

3.5. Ex vivo treatment with the H2S-releasing gel prevented development of IH in human saphenous vein segments

To explore the application of Gel-H2S in vascular diseases, we obtained human vein segments, which were placed in culture for 7 d with or without Gel-H2S or Gel-Ctrl (10–20 μL gel/mL media, equivalent to ~100 or ~200 μM sulfide, respectively). The RPMI culture medium contained 200 μM L-Cys, and no additional thiol was added to trigger H2S release. NaHS (100 μM) was used as a positive control of exogenous H2S supply.

Histomorphometric analysis of intima and media thickness after 7 d in culture revealed that treatment of human vein segments with Gel-H2S or NaHS fully blocked the increase in intima thickness or intima over media thickness (I/M) over 7 d in culture. (Figure 4A). Importantly, treatment with Gel-H2S, Gel-Ctrl, or NaHS did not affect the media thickness, suggesting no cytotoxic effect of exogenous H2S treatment. Next, we analyzed cell proliferation in the vein segments using PCNA immunostaining. We observed a two-fold increase in cell proliferation in the control conditions (D7 and Gel-Ctrl) compared to the baseline (D0), while the Gel-H2S treatment lowered the proliferation to D0 levels (Figure 4B).

Figure 4.

Figure 4.

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Human great saphenous vein segments were incubated or not (D0) in static culture for 7 d with or without (control), NaHS (100 μM), Gel-Ctrl (20 μL/mL) or Gel-H2S (10 or 20 μL/mL). A) Representative VGEL staining (scale bar represents 100 μm) and scatter plots with mean±SEM of media, intima thicknesses, and intima/media ratio (I/M) in 5 vein segments. B) Representative PCNA immunostaining (black nuclei) counterstained with hematoxylin (blue nuclei) and scatter plots with mean±SEM of PCNA immunostaining. Insets are 3× magnification of main image. Scale bar represents 60 μm. *P<0.05; **P<0.01 vs D7 as determined by one-way ANOVA with post-hoc t-test with Dunnet’s correction for multiple comparisons.

3.6. The H2S-releasing peptide/gel decreased VSMC proliferation and transmigration

To understand further the cell-based effects of Pep-H2S and Gel-H2S, we examined human EC and VSMC proliferation and migration in vitro. In line with our ex-vivo data on human vein segments, in vitro studies of VSMCs derived from human vein segments revealed that Pep-H2S inhibited cell proliferation in a dose-dependent manner, while Pep-Ctrl had no effect (Figure 5A). Importantly, 10 μM Pep-H2S had a similar effect to 100 μM NaHS. Media thickness data in human veins (Figure 4) suggested that the donors had no cytotoxic effect on VSMCs. To further test this observation, in vitro assessment of cell apoptosis using Hoechst-propidium iodide staining was conducted. The results confirmed that Pep-H2S, Pep-Ctrl, and NaHS had no toxic effect on VSMCs after 48 h exposure (Figure 5B). Thapsigargin (TG) was used as a positive control to induce apoptosis (Figure 5B). Finally, using a transmigration assay in Boyden chambers, we observed that, as compared to the pre-conditioned EC culture medium alone (control+), the addition of NaHS or Gel-H2S (10 μL/mL), but not Gel-Ctrl, inhibited VSMC transmigration. Interestingly, Gel-H2S was significantly more potent than NaHS, fully blocking VSMC transmigration (Figure 5C).

Figure 5.

Figure 5.

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A) VSMCs were incubated (control) with or without 100 μM NaHS, 5 or 10 μM Pep-Ctrl or Pep-H2S for 24 h in the presence of BrdU. Scale bar represents 20 μm. Proliferation was calculated as the ratio of BrdU-positive nuclei over total DAPI-stained nuclei and expressed as % of proliferation in the control condition. B) VSMC apoptosis levels after 48 h exposure to 100 μM NaHS, Pep-Ctrl, Pep-H2S, or a 24 h exposure to 100 nM thapsigargin as a positive control. C) VSMC transmigration through an artificial membrane toward EBM-2 medium (control(−)) or pre-conditioned medium of HUVECs (EGM-2 medium) supplemented or not (control(+)) with 100 μM NaHS, 10 μL/mL of Gel-Ctrl or Gel-H2S for 16 h. Scale bar represents 50 μm. All data are scatter plots with mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs. control(+) as determined by one-way ANOVA with post-hoc t-test with Tukey’s correction for multiple comparisons.

Further Western blot analyses of VSMC phenotype using two VSMC-specific proteins, calponin and SM22α, did not reveal major changes, although calponin levels tended to decrease in VSMCs exposed to the highest dose of Pep-H2S (20 μM) (Figure 6A, B). Further immunocytochemistry analysis revealed that NaHS and Pep-H2S (20 μM) disrupted the typical cytoskeleton staining for calponin and SM22α (Figure 6C), which may account for the reduced proliferation and mobility of VSMCs exposed to H2S.

Figure 6.

Figure 6.

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VSMCs were incubated (control) with or without NaHS (100 μM), Pep-Ctrl (10 or 20 μM), or Pep-H2S (10 or 20 μM) for 24 h. A, B) Western blot analysis of calponin (A) and SM22α (B) over tubulin levels. C) VSMC immunofluorescent staining for calponin (red), SM22α (green), and nuclei (DAPI staining in blue). Images are overlays of the 3 channels, representative of 5 independent experiments. Scale bar represents 20 μm.

3.7. The H2S-releasing peptide/gel increased EC proliferation and transmigration

We then performed similar experiments on HUVECs. H2S promotes EC proliferation, migration, and angiogenesis [36]. As expected, NaHS and Pep-H2S increased HUVEC proliferation, Pep-H2S being effective at a 10-fold lower dose than NaHS (Figure 7A). We also tested HUVEC transmigration toward basic EC medium without growth factors (EBM™−2; control(−)) or full HUVEC culture medium (EGM™−2 Bullet kit™; control(+)). The EGM™−2 Bullet kit stimulated transmigration as expected. Consistently, and in contrast with VSMCs, NaHS and Gel-H2S (10 μL/mL) promoted HUVEC transmigration (Figure 7B).

Figure 7.

Figure 7.

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A) HUVECs were incubated (control) with or without NaHS (100 μM), Pep-Ctrl (10 μM) or Pep-H2S (5 or 10 μM) for 8 h in the presence of BrdU. Proliferation was calculated as the ratio of BrdU-positive nuclei over total DAPI-stained nuclei and expressed as % of proliferation in the control condition. Scale bar represents 20 μm. B) HUVEC transmigration through an artificial membrane toward EBM-2 medium (control(−)) or EGM-2 medium (control(+)) supplemented with 100 μM NaHS, 10 μL/mL Gel-Ctrl, or 10 μL/mL Gel-H2S for 6 h. Data are mean ± SEM of calcein-AM fluorescence signal measured on a fluorescence plate reader (λex = 495 nm; λem = 517 nm). Scale bar represents 50 μm. All data are scatter plots with mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs control(+) as determined by one-way ANOVA with post-hoc t-test with Dunnet’s correction for multiple comparisons.

4. DISCUSSION

Previous studies in animal models have reported protection from IH with exogenous NaHS administration in rats [37], rabbits [38] and mice [39, 40]. However, human evidence of H2S donor efficacy against IH was lacking. Here we report, for the first time in human tissue, that a peptide hydrogel with sustained release of low levels of H2S inhibits VSMC proliferation and IH in an ex-vivo model of human vein culture.

Controlling the amount of H2S released is critical as relatively high doses of exogenous H2S (over 500–1000 ppm) lead to respiratory distress and death, hindering the use of H2S medications in humans. H2S donors release H2S by either hydrolysis or in response to a specific trigger, such as a thiol or other nucleophiles [11]. Here we employed a thiol-triggered donor motif, which adds a level of control to the release and ensures more sustained release compared to passive hydrolysis. The peptide is highly soluble and self-assembles to form long nanoribbons stabilized by β-sheets, resulting in steady release in the range of hours, in stark contrast to the instantaneous release of H2S from NaHS. Importantly, Pep-H2S reduces IH at a 10-fold lower total sulfide dose than NaHS, which is likely due to the slower release kinetics, leading to better H2S bioavailability. In addition to reduced toxicity, a sustained release strategy is crucial for the prevention of IH in patients because the acute stage of IH development typically occurs during the first 30 days following the intervention [41].

Local biodegradable sheaths, wraps, meshes, membranes, and cuffs have all been tested for perivascular applications. However, these solid solutions adapt poorly to the elasticity of vascular tissue. While solid forms are more stable over time, injectable semi-solid formulations, in particular hydrogels, present exciting alternatives as they are easily positioned and provide adequate coverage of the vessel [42]. Due to their ability to self-assemble into nanoribbons, both Pep-H2S and Pep-Ctrl spontaneously form soft hydrogels at 1 wt. %, as measured by rheology, in aqueous solution in the presence of CaCl2. Gel-H2S provides extended release compared to Pep-H2S, with potentially increased efficacy in reducing IH in-vivo [41]. Most current hydrogel formulations involve the combination of a gel and a drug. The H2S-releasing gel developed here has advantages over polymer-based gels because it is a fully biodegradable single small molecule with no polydispersity, and it requires no covalent crosslinking beyond addition of salt.

The protective effect of the H2S-releasing peptide against IH is probably largely imparted by a direct inhibition of VSMC proliferation. Indeed, in line with previous reports [39], we observed cell proliferation only in the media layer of vessels and show, in vitro, that Pep-H2S/Gel-H2S and NaHS inhibit human VSMC proliferation and transmigration, which are important features of pathogenic synthetic VSMCs involved in IH [2]. We further observed that the H2S-releasing peptide and NaHS disrupt the normal cytoskeleton architecture of VSMCs, as evidenced by a modified pattern of calponin and SM22α immunostaining. Given the prominent role of cytoskeleton dynamics and remodeling during mitosis and cell migration, this disrupted pattern likely contributes to reduced VSMC proliferation and migration. Whether or not the protection imparted by the donors is directly mediated by H2S remains unknown. S-Aroylthiooxime compounds release H2S in the presence of thiols with intermediate formation of thiocysteine (cysteine persulfide) [23]. Thus, the observed biological effects of Pep-H2S/Gel-H2S may be attributable, at least in part, to cysteine persulfide or other related reactive sulfide species. Further studies will be conducted to identify the mechanism underlying the effects of H2S/persulfide on the VSMC cytoskeleton.

In contrast to VSMCs, EC proliferation and transmigration is stimulated by Pep-H2S/Gel-H2S, consistent with previous reports showing that exogenous H2S stimulates HUVEC proliferation and migration in vitro [43, 44] and has pro-angiogenic properties in vivo [45, 46]. This feature is of particular interest in the context of IH. Indeed, EC dysfunction and death during vascular surgery plays a major role in the development of IH, and the pro-angiogenic effects of H2S might accelerate endothelium recovery following vascular trauma [2]. However, in our model of static vein culture, the absence of laminar flow leads to severe EC dysfunction [47], which makes it impossible to assess endothelium recovery. Further in vivo studies will be conducted to ascertain that H2S donors can accelerate endothelium recovery in large vessels.

Currently available local therapies, such as drug-eluting stents and balloons, are coated with non-specific cytotoxic (paclitaxel) and cytostatic (sirolimus) drugs. These devices may improve long-term vessel patency when compared with standard “bare” stents and balloons [48, 49]. However, their long-term effects on patient outcomes remain unclear, and recent evidence suggests a negative outcome for paclitaxel-coated devices and increased rate of complications [5]. Thus, local perivascular application of an H2S-releasing gel might provide a unique therapeutic opportunity, with benefits on both VSMCs and ECs and without systemic toxicity.

5. CONCLUSIONS

To summarize, we developed and evaluated the therapeutic potential of an H2S-releasing biodegradable hydrogel to limit the development of IH in human veins. The thiol-triggered, controlled H2S release from peptide hydrogels provides sustained H2S concentrations over a period of hours. Consequently, extended release of low levels of H2S from the peptide hydrogel inhibits VSMC proliferation and IH in human vein models more effectively than sulfide salts (NaHS). In contrast to its inhibitory effects on VSMCs, the H2S-releasing peptide hydrogel facilitates in vitro HUVEC proliferation and transmigration, which may further assist in preventing IH and help the recovery after vascular intervention. In future efforts, we will evaluate the therapeutic potential of the perivascular application of H2S-releasing gels at the site of vascular trauma following surgery in animal models of IH. Overall, we propose that application of such H2S-releasing self-assembling peptide hydrogels may constitute a viable solution to limit IH in human vein grafts.

Supplementary Material

1

Statement of Significance.

Arterial occlusive disease is the leading cause of death in Western countries, yet current therapies suffer from high failure rates due intimal hyperplasia (IH), a thickening of the vascular wall leading to secondary vessel occlusion. Hydrogen sulfide (H2S) is a gasotransmitter with vasculoprotective properties. Here we designed and synthesized a peptide-based H2S-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 H2S efficacy against IH in human tissue, and the results show that the gel is more effective than NaHS, a common instantaneous H2S donor.

6. ACKNOWLEDGEMENTS

We thank Martine Lambelet for her dedication and technical assistance. We thank the mouse pathology facility for their services in histomorphology (https://www.unil.ch/mpf). We thank Prof Tijana Grove and Dr Ann Norris for instrumental assistance and Yun Qian for helpful discussions. This work was supported by the following: The Swiss National Science Foundation (grant FN-310030_176158 to FA and SD), the United States National Science Foundation (DMR-1454754 to JBM), the United States National Institutes of Health (R01GM123508 to JBM), and the Union des Sociétés Suisses des Maladies Vasculaires (to SD). The funding sources had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

Footnotes

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