Oxidation-Dependent Effects of Alpha-1 Antitrypsin on Wound Healing and Inflammation: Implications for Tissue Repair

doi:10.21203/rs.3.rs-4975765/v1

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Oxidation-Dependent Effects of Alpha-1 Antitrypsin on Wound Healing and Inflammation: Implications for Tissue Repair

https://doi.org/10.21203/rs.3.rs-4975765/v1

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Background. Wound healing requires a delicate balance between cellular and molecular factors, all affected by reactive oxygen species (ROS). While ROS decontaminate, they also might lead to impaired wound healing, as evident in radiation-exposed skin and in venous insufficiency. Human alpha-1 antitrypsin (hAAT) is a circulating antiprotease that is anti-inflammatory and tissue-protective. Accordingly, tissue repair is enhanced in hAAT-rich conditions. hAAT is oxidized in oxidative conditions, and its antiprotease function is lost; whether its anti-inflammatory and tissue-protective properties remain unknown.

Methods. Excisional skin wound closure rates were first examined on irradiated skin and then tested using an iron-loading venous insufficiency model. The former was tested on hAAT transgenic mice, the latter on wild-type mice using topical clinical-grade hAAT. In-vitro, hAAT was oxidized using H₂O₂ (0.5, 5 and 25 mM), then tested for elastase inhibition and added to an in-vitro A549 epithelial cell gap closure assay and a RAW 264.7 macrophage response assay. ROS levels, inflammatory responses and NRF2/ARE activation were determined.

Results. Wound closure was impaired in wild-type mice by both radiation and iron. In contrast, hAAT-transgenic mice exhibited accelerated wound closure in both normal and irradiated skin, and topical hAAT improved wound healing in the venous insufficiency model. hAAT^OX lacked elastase inhibition across the three oxidation levels, yet highly oxidized hAAT (hAAT^{OX 25mM}) impaired epithelial gap closure and weakly oxidized hAAT (hAAT^{OX 0.5mM}) enhanced gap closure. All forms of hAAT^OX elevated ROS in macrophages, as well as the expression of iNOS and catalase, IL-1β, TNFα and CXCL-1. Unexpectedly, the NRF2/ARE pathway was activated by hAAT^{OX 25mM} and suppressed by hAAT^{OX 0.5mM}, and hAAT^{OX 0.5 mM} induced IL-1 receptor antagonist expression.

Conclusions. Oxidation levels of hAAT modify its effects on inflammation and tissue repair. While protease inhibition is lost, anti-inflammatory and repair attributes are maintained under low oxidative conditions, suggesting a molecular profile that is physiologically attuned to local signals. Considering its safety record, the study proposes that hAAT therapy is poised for trials in the context of defective tissue repair under oxidative conditions.

Elastase inhibition

epithelial gap closure

inflammation

IL-1 receptor antagonist

macrophage activation

NRF2/ARE pathway

oxidative stress

oxidized AAT

reactive oxygen species (ROS)

wound healing

Skin wounds heal through complex interactions between several cellular and molecular entities, some of which can change the course and the outcome of repair. For example, reactive oxygen species (ROS), also referred to as free radicals, regulate inflammation, cell proliferation, angiogenesis and extracellular matrix turnover (1). Generated by activated neutrophils, ROS act as antimicrobial and inflammatory agents (2). Failure to generate ROS results in a primary immunodeficiency, while overabundance of ROS can severely damage DNA and subcellular structures in local cells (3). To protect these cell structures, some enzymatic antioxidants are produced upon exposure to ROS, including a catalase that breaks down hydrogen peroxide (H₂O₂) into water and oxygen (4). The correct balance between the defense and distraction of local tissue is key for normal wound healing.

Skin cells generate ROS in reaction to X-ray and UV radiation. This response appears to be persistent and can induce chronic ulcerations, impaired wound healing and fibrosis long after exposure (5). Similarly, increased ROS production and impaired wound healing occur during venous insufficiency, whereby ferrum accumulates in the tissue in the form of hemosiderin that interacts with oxygen and leads to excessive levels of free radicals (6, 7). Aside from decontamination, excessive levels of ROS have no apparent physiological advantage to a tissue and can cause significant clinical challenge. In vascularized organ transplantation, ischemia-reperfusion injury (IRI) can develop due to abruptly restored blood flow and a sudden flux of oxygen, causing oxidative stress, inflammation, infiltration of immunocytes, and widespread tissue damage (8). A surge in upstream proinflammatory cytokines occurs, including IL-1β and TNFα, further driving the spread of inflammation (9, 10), and chemoattractants promptly diffuse, including the neutrophil-active chemokine, CXCL-1 (11). In immunocytes, an oxidative environment activates the NF-κB signaling pathway, which triggers inducible nitric oxide synthase (iNOS) expression and the release of inflammatory levels of nitric oxide (12). The Nrf2-ARE pathway is activated by ROS, and acts as a modulator of oxidative stress (13). ROS subsequently stimulate tissue-degrading proteases that impair tissue healing (14, 15), a physiological attribute intended to allow activated neutrophils to enzymatically break down tissues without restraint, as they migrate towards their target. Ideally, this intentional oxidative environment is limited in time, so as to allow tissue recovery once local neutrophils are replaced by macrophages (16).

Human alpha-1 antitrypsin (hAAT) is a 394-amino-acid–long circulating protease inhibitor that inhibits elastase and other serine proteases by way of an amino acid specific bait; it also acts as an immune-modulating protein that reduces tissue injury and ROS-associated damage (17, 18). The levels of circulating hAAT rise appropriately during inflammatory conditions, as well as during the 3rd trimester of a healthy pregnancy. Patients with genetic hAAT deficiency present with tissue-degrading pulmonary manifestations, as well as impaired wound healing, deep tissue necrosis (panniculitis) and multiple forms of vasculitis (19, 20). Some aspects of hAAT appear to be unrelated to its ability to inhibit proteases (21, 22)hAAT upregulates the expression of the anti-inflammatory cytokine, IL-1 receptor antagonist (IL-1Ra) in LPS-stimulated immunocytes (17) and in the context of islet cell transplantation (23), irrespective of its ability to block elastase (21, 24–26). In addition to its anti-inflammatory effects, treatment with hAAT accelerates epithelial gap repair and promotes wound healing in various clinical, and preclinical in-vivo and in-vitro systems (24, 27). hAAT appears to adopt multiple presentations based on its immediate environment. For instance, in the presence of high concentrations of nitric oxide, hAAT is S-nitrosylated, pro-inflammatory and it gains a capacity to inhibit cysteine proteases, such as caspase-3 (28, 29). In oxidative environments, hAAT loses its capacity to inhibit proteases, as a methionine residue in position 358 within its protease-binding site is oxidized (30–32). Oxidized hAAT (hAAT^OX) has been implicated in various pathologies, including chronic obstructive pulmonary disease (COPD), emphysema and various inflammatory conditions (33–35). While hAAT^OX exhibits reduced anti-protease activity, it has been shown to retain some anti-inflammatory properties in certain circumstances (33). For example, hAAT^OX can inhibit TNFα-induced gene expression in human lung endothelial cells (36). Despite this, the extent to which hAAT^OX maintains its anti-inflammatory attributes across a gradient of oxidative environments and the role it plays in disease, remain a subject of ongoing research (35).

The current study hypothesizes that hAAT may have different functional profiles depending on its level of oxidation, as it is exposed to an oxidative gradient in an affected tissue. Since hAAT is safe for clinical use and presently explored for a wide range of clinical tissue-injury–related conditions, as reviewed elsewhere (37), it is speculated that treatment with native hAAT may partially override the possibly damaging effects of hAAT^OX. Here, two in-vivo models of impaired skin wound healing are examined, namely, post-radiation skin wound repair, and a wound inflicted in the context of venous insufficiency. The study investigates how cells react to ROS when exposed to hAAT and hAAT^OX, specifically under varying levels of oxidative stress.

Animals

Animal studies were approved by the Institutional Animal Care and Use Committee (IL-155-10-2022D) and conducted in line with the Guide for the Care and Use of Laboratory Animals, 8th Edition. C57BL/6 mice (8-12-week–old females, Envigo + Laboratories, Inc., Rehovot, Israel) were housed at a standard viva. Mice transgenic for human AAT (hAAT^+/+) on a C57BL/6 background were generated at the University of British Columbia, Canada (38). hAAT^+/+ mice express constitutive levels of hAAT in their blood (< 1 µg/ml) and as a result, they are frequently used as a positive control for hAAT treatment.

Radiation-induced impaired wound healing model

To establish a model of radiation-induced impaired wound healing, mice were anesthetized using a combination of intraperitoneal ketamine (100 mg/kg) and xylazine (10 mg/kg). The sedated animals were then securely fixed to a custom-designed lead-coated apparatus that allows for targeted irradiation of the dorsal skin while protecting non-exposed areas with a 5 mm lead wall. The skin of the mice was then exposed to a single dose of 5 Gray (Gy) X-ray radiation using a linear accelerator (RS-2000 Small Animal Irradiator, Rad Source Technologies Suwanee, Georgia, USA). Radiation parameters were set at energy 160 kVp, current 25 mA, filter 0.3 mm Cu, source-to-skin distance 30.5 cm, and dose rate 1.25 Gy/min. These settings were chosen based on data from preliminary studies to inflict tissue damage that may cause a delay in wound healing without inducing excessive skin toxicity (39, 40). Twenty-one days post-irradiation, a uniform surgical excision was performed on the radiation-exposed sites to create a wound. Briefly, mice were anesthetized (isoflurane inhalation 2.5% for induction 2% for maintenance) and dorsal hair was removed by electric razor and skin was disinfected with chlorhexidine gluconate (0.5% w/v) in a 70% v/v ethanol solution. A circular 8-mm surgical skin excision was performed on the treated site. The healing process was monitored through photographs at indicated time points, and subsequent digital image analysis was conducted using ImageJ software (MedCalc Software, Ostend, Belgium).

Ferrum loading impaired wound healing model

Six- to eight-week-old mice were anesthetized with inhalational isoflurane and disinfected at the injection site, then treated subcutaneously along the dorsolateral aspect of the middle back with either 100 µl saline or 50 mg/ml iron-dextran (Venofer INJ) at 100 µl per mouse (41). Injections were repeated twice a week for 3 weeks. Two days following the final injection, a uniform surgical excision was performed on the treated site as described in the previous paragraph.

ROS levels in UVB-exposed macrophages RAW 264.7 cells

Intracellular ROS levels were measured using cell-permeated 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Sigma, Israel), which, upon cleavage of the acetate groups by intracellular esterases and oxidation, is converted to fluorescent dichlorodihydrofluorescein. RAW 264.7 cells (0.5×10⁵ cells/well in 96-well plates in quadruplicates) were seeded and grown to 90% confluence. Cells were then incubated with 50 µl of H2DCFDA for 30 min at 37°C. H2DCFDA was replaced with PBS in the absence or presence of hAAT at indicated concentrations, and fluorescence intensity was determined using a fluorescent optical reader 485^em/531^ex.

Oxidation of clinical-grade hAAT

hAAT (Glassia®, Kamada LTD., Ness Ziona, Israel) was oxidized using a protocol adapted from Taggart et al. (32). Briefly, 20 mg of hAAT were incubated for 2 hours at room temperature in a 1 ml reaction mixture containing 50 mM potassium phosphate, 100 mM potassium chloride,1 mM magnesium chloride at pH 5.0, and with varying concentrations of hydrogen peroxide (H₂O₂; 0.5, 5, and 25 mM) (all from Sigma, Israel). The concentration of H₂O₂ was measured at 240 nm (ε = 39.4 ± 0.2 M^− 1 cm^− 1). At the end of the reaction, the sample was dialyzed overnight at 4°C against 5 mM HEPES and 10 mM sodium chloride at pH 7.4, using dialysis cassettes (Pierce), for a total of six buffer changes to remove residual H₂O₂. The dialyzed samples were then centrifuged to remove precipitates.

Elastase activity assay

Neutrophil elastase activity was determined in acellular conditions using a designated kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. Briefly, hAAT was incubated at indicated concentrations with elastase (0.39 µM), and the kinetics of the color product were determined.

In-vitro human epithelial A549 cells gap repair assay

Epithelial gap repair assay was performed using human A549 cells (ATCC #CCL-185). Cells were grown to confluence in 24-well plates (50,000 per well in quadruplicates), and uniform scratches were inflicted using a sterile 200-µl pipette tip, thus creating a cell-free area, as described elsewhere (24, 27). Cultures were washed twice with complete RPMI 1640 supplemented with 2.5% FCS (both from Biological Industries Inc., Beit Haemek, Israel). Treatments were introduced directly onto cells in 2.5% FCS. Cells were treated with PBS, or 0.5 mg/ml of either hAAT or hAAT^OX produced by exposure to 0.5, 5 and 25 mM H₂O₂. Images were acquired using Zeiss Inverted microscope Axio Observer 7 and Moticam 5 Camera. The gap area between cells was measured using ImageJ.

Macrophage activation assay

RAW 264.7 cells (Cat#SC-6003, ATCC) were seeded in 48-well plates (0.5×10⁵ cells/well) in RPMI 1640 medium supplemented with 5% FCS (Biological Industries). Cells were incubated with 0.5 mg/ml hAAT and hAAT^OX. Cells were harvested for gene expression analysis at indicated time points.

Gene expression analysis

RNA extraction from the mouse skin wounds was performed at indicated time points using Gynzol® reagent (Invitrogen, Waltham, MA, USA) at 1 ml/well following manufacturer's instructions. Tissue was transferred to a polytron homogenizer (gentelMACS Dissocoator, MACS Miltenyi Biotec), and the homogenized sample was loaded into a 1.5 ml RNase-free microcentrifuge tube. RNA isolation followed the manufacturer’s guidelines. RNA was then quantified using a NanoDrop device (Wilmington, DL, USA), and 500 ng of RNA was reverse transcribed into cDNA using a Prime Script RT Reagent kit (Takara, Dalian, China). Quantitative PCR was performed using an RT-PCR system (StepOnePlus™ Real-Time PCR System, ThermoFisher Scientific Corporation, Waltham, MA, USA), and SYBR Premix Ex Taq II (Takara) at a 10 µl volume reaction. CFX96 manager software was used to determine threshold cycle values. Primers were designed for murine transcripts, as follows: IL-1β: ‘5-CTT CCA GGA TGA GGA CAT GAA GG-3′ (forward), ‘5-AGT GCA GTT GTC TAA TGG GA-3′ (reverse); IL-1Ra: 5′-GAC CCT GCA AGA TGC AAG CC-3′ (forward), 5′-GAG CGG ATG AAG GTA AAG CG-3′ (reverse), TNF𝛂: 5′-GAA AAG CAA GCA GCC AAC CA-3′ (forward), 5′-CGC GGA TCA TGC TTT CTG TG-3′ (reverse), CXCL-1: 5′-GGT GTC CCC AAG TAA CGG AG-3′ (forward), 5′-TTG TCA GAA GCC AGC GTT CA-3′ (reverse), iNOS: 5′--3′ (forward), 5′--3′ (reverse), Catalase: 5′-GAA GGA CCG TGT TTG GTT GC-3′ (forward), 5′-CCG CTG GCG CTT TTA TTG TT-3′ (reverse), β Actin: 5′-CAT TGC TGA CAG GAT GCA GA-3′ (forward), ‘5-TGC TGG AAG GTG GAC AGT GA-3′ (reverse). Relative quantification of transcript levels was performed using the delta-delta Ct method. The variation of Ct in the reference gene across samples of a given assay was < 1 cycle. The efficiency of all primer pairs was 95%-110% using a 5-point standard curve.

ROS levels in RAW 264.7 cells exposed to H₂O₂ and oxidized hAAT

RAW 264.7 cells (96-well plates at 0.5×10⁵ cells/well in 6-plicates) were pre-incubated with medium alone or hAAT (0.5 mg/ml) for 24 hours prior to initiation of the experimental conditions. The medium in all wells was then replaced for 30 minutes with either H₂O₂ or oxidized hAAT at indicated concentrations. Control cells were incubated with medium alone. Intracellular ROS levels were determined using H2DCFDA staining over 3 hours using a fluorescent optical reader.

Transient transfection and Nrf2/ARE reporter gene assay

RAW 264.7 cells were seeded in 24-well plates at 1×10⁵ cells/well. Twenty-four hours later, cells were transfected using a jetPEI reagent (Polyplus Transfection, Illkrich, France). Cells were then rinsed once with serum-free medium, followed by the addition of 0.45 ml of medium and 50 µl of a mixture containing DNA and jetPEI reagent at a charge ratio of 1:5. The total amount of DNA was 0.25 µg, containing 0.2 g 4×ARE reporter construct (42) (kindly provided by Dr. M. Hannink; University of Missouri-Columbia, Columbia, MO, USA) and 0.05 µg Renilla luciferase (P-RL-null; Promega, Madison, WI, USA) expression vectors, which served as an internal transfection standard. Cells were then incubated for 6 hours at 37°C, after which the medium was replaced with DMEM-FBS plus the test compounds, and cells were incubated for an additional 16 hours. Cell extracts were prepared for luciferase reporter assay (Dual Luciferase Reporter Assay System, Promega) according to manufacturer’s instructions, and luminescence was determined using a Turner Biosystems luminometer (Sunnyvale, CA, USA).

Statistical analysis

All quantitative data are presented as mean ± standard error of means (SEM). The statistical significance of the differences between groups was evaluated using ANOVA followed by post-hoc tests for multiple comparisons. Statistical processing was performed using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). A p-value < 0.05 was considered statistically significant.

In-vivo wound closure in WT and hAAT^+/+ mice 3 weeks after skin irradiation

In examining the impact of hAAT-rich conditions on wound closure rates in the context of impaired wound healing, excisional wound closure was examined in normal skin and in skin pre-exposed to radiation 21 days earlier (Fig. 1). Under these conditions, the affected skin is assumed to generate local ROS. As shown in Fig. 1A, without exposure to radiation, hAAT^+/+ mice exhibited accelerated wound healing compared to WT mice. Radiated tissue healing, however, was significantly slower than naive tissue healing (Fig. 1B). In comparing pre-irradiated sites across genotypes (Fig. 1C), wound closure rates were significantly enhanced in hAAT^+/+ mice compared to WT mice; by day 12, all hAAT^+/+ mice had completely closed their wounds, whereas WT mice wounds remained only 50% closed.

Effect of clinical-grade hAAT on ROS levels in UVB-exposed macrophages

Since macrophages are pivotal players in wound repair and are also producers of ROS, the effect of clinical-grade hAAT on UVB-induced ROS production in the macrophage cell line, RAW 264.7, was examined (Fig. 2). As shown, exposure of cell cultures to UVB caused an increase in ROS levels and treatment with NAC reduced ROS levels. However, there was no change in UVB-induced ROS levels in the presence of hAAT throughout a wide range of concentrations.

Oxidized clinical-grade hAAT (hAAT^OX) exhibits impaired elastase inhibition in association with degree of oxidation

To directly investigate the effect of local oxidation on hAAT, clinical-grade hAAT was treated with 0.5, 5 or 25 mM hydrogen peroxide (H₂O₂), and then isolated from solutes to generate oxidized hAAT (hAAT^OX). The antiproteolytic capacity of hAAT^OX was tested in an elastase activity assay at three molar ratios between it and the enzyme (Fig. 3). As shown, H₂O₂ alone had a mild effect on elastase activity. As expected, non-oxidized hAAT inhibited elastase in direct association with their molar ratio. However, hAAT^OX failed to inhibit elastase in a manner that correlated with the degree of exposure to H₂O₂. For example, the product of exposing hAAT to 25 mM H₂O₂, termed hAAT^{OX 25mM}, allowed for near complete elastase activity to occur, while hAAT^{OX 5mM} and hAAT^{OX 0.5mM} only partially inhibited elastase, in both cases to a lesser extent compared to their corresponding concentrations of (non-oxidized) hAAT.

hAAT^OX impairs in-vitro epithelial gap closure in association with degree of oxidation

The effects of hAAT^OX on wound healing were assessed using an in-vitro A549 human lung epithelial cell line scratch assay (Fig. 4). Cells were treated with 0.5 mg/ml hAAT, or 0.5 mg/ml of three degrees of hAAT exposure to H₂O₂ (0.5, 5 and 25 mM), intended to signify a gradient of local oxidative stress, such that is assumed to occur in disrupted tissues. As shown in Fig. 4, higher level oxidation resulted in slower gap closure. Of note, both hAAT^{OX 0.5mM} and hAAT^{OX 5mM} allowed for progression of gap closure between 6 hours and 12 hours from insult. In contrast, hAAT^{OX 25mM} significantly stunted gap closure, as observed both at 6 and 12 hours from injury; in that degree of hAAT oxidation, epithelial cell cultures completely failed to close the gap.

hAAT^OX stimulates macrophages in association with degree of oxidation and increases iNOS and catalase expression

Considering the substantial role of macrophages in wound repair, the responses of RAW 264.7 macrophage cells to hAAT^OX were examined (Fig. 5). Cells were treated with either hAAT, H₂O₂, hAAT^OX at 3 different degrees of oxidation, or a combination of hAAT and hAAT^OX. Cells were harvested for gene expression analysis after 3 and 12 hours. As shown in Fig. 5A, the presence of H₂O₂ alone caused a significant and early induction of IL-1β expression. The same effect was observed in IL-1Ra expression, although in the case of IL-1Ra expression, hAAT alone also caused a significant increase in expression, which was not observed in IL-1β. Upon exposing the cells to hAAT^OX at the same concentration as hAAT, significant induction of IL-1β was observed after 3 hours in the presence of hAAT^{OX 5mM} and hAAT^{OX 25mM}, and after 12 hours in the presence of all three hAAT^OX concentrations. In contrast to the pattern of IL-1β, an inverse response was observed in IL-1Ra expression levels, even though hAAT^{OX 0.5mM} mM was the only oxidation degree that induced IL-1Ra expression. In testing the effect of hAAT^{OX 5mM} with and without added hAAT, a time-dependent phenomenon was observed in which the 3-hr induction of IL-1β was blocked by adding hAAT to hAAT^{OX 5mM}, but the 12-hr levels remained similar between hAAT^{OX 5mM} alone and hAAT^{OX 5mM} with hAAT. Similarly, the 3-hr expression levels of IL-1Ra were elevated by adding hAAT to hAAT^{OX 5mM} but the 12-hr expression levels remained unaffected and resembled hAAT^{OX 5mM} alone.

The expression of TNFα and CXCL-1 closely mimicked those of IL-1β at 3 hours (Fig. 5B). However, at 12 hours, TNFα continued to rise in response to H₂O₂ alone, and the combination of hAAT^{OX 5mM} with hAAT was distinctly inflammatory at that time point. In addition, expression of CXCL-1 continued to rise in the presence of hAAT^{OX 5mM} at 12 hours post exposure.

The expression of iNOS and catalase was assessed at 12 hours of culture, as shown (Fig. 5C). H₂O₂ alone and also all forms of hAAT^OX, regardless of added native hAAT, caused a rise in iNOS and catalase expression, primarily at the higher degrees of oxidation (hAAT^{OX 5–25mM}). As expected, these two gene products exhibited strong association in every sample.

hAAT ^OX induces oxidative stress in macrophages and activates the Nrf2/ARE pathway in a biphasic manner, irrespective of added native hAAT

To explore the oxidative properties of hAAT^OX, relative ROS levels were determined in RAW 264.7 macrophages (Fig. 6A). As shown, after 14 minutes, ROS levels were elevated in cultures treated with H₂O₂ and with hAAT^OX; more soin the 5 mM groups than the 0.5 mM groups for both agents. Added native hAAT to both groups resulted in no change in ROS levels.

Activation of the intrinsic mechanism of defense against oxidative stress, namely the Nrf2/ARE signaling pathway, was assessed using a luciferase reporter (Fig. 6B). Interestingly, while H₂O₂ induced Nrf2/ARE signaling, hAAT^{OX 0.5mM} did not induce signaling while hAAT^{OX 5mM} did induce signaling. As in the case of ROS levels, no major change was observed by adding native hAAT to either H₂O₂ or to hAAT^OX.

In-vivo iron-induced oxidative stress: clinical-grade hAAT accelerates impaired wound healing

Since the goal of our research is to promote the healing of difficult-to-heal wounds, and our earlier research (22, 24) demonstrated that hAAT accelerates acute wound healing, we decided to explore the relevance of hAAT on wound healing under various clinical scenarios that cause chronic wounds. Radiation was first, and tissue exposure to ferrum, as in venous insufficiency, was tested after. In both of these scenarios, oxidative stress is thought to play a substantial role in wound pathogenesis.

Since hAAT did not modify oxidative stress in cultured cells across low and high degrees of oxidation, nor did it act as an antioxidant under those conditions, and considering that oxidized hAAT induced the Nrf2/ARE signaling pathway only at high degrees of oxidation, the impact of hAAT wound treatment was examined using a model of high oxidative conditions. Iron-induced oxidative stress caused in-vivo by venous insufficiency was represented by introducing local iron over a 21-day period. As shown in Fig. 7, wounds inflicted on iron-treated tissues exhibited poorer wound healing properties compared to regular saline. While control animals displayed 50% wound area closure before day 3 and had all completed wound closure by day 9, animals in which skin was pretreated with iron displayed 50% wound area closure on day 6, and did not completely close their wounds even by day 9. In contrast, wounds inflicted on iron-treated skin sites that were treated with topical hAAT displayed wound closure rates that compared to normal wound healing.

hAAT therapy has long been linked to accelerated wound healing, and so the question of whether it also accelerated wound healing in the presence of an underlying oxidative microenvironment drew interest. Although hAAT is not inherently a potent antioxidant (43), it consistently displays significant benefits in conditions of oxidative stress, as is evident in the impact of hAAT treatment on renal, hepatic, pulmonary and cardiac ischemia-reperfusion injuries, as well as in cerebral stroke (44–50). Feng et al. describe hAAT as preventing the development of preeclampsia in mice through suppression of oxidative stress, observing a dose-dependent impact between hAAT and the expression of superoxide dismutase (SOD), endothelial nitric oxide synthase (eNOS) and glutathione peroxidase (51). Here, in evaluating wound closure in mice, hAAT^+/+ mice exhibited accelerated wound healing compared to wild-type mice in wounds inflicted on previously irradiated skin. Despite hAAT not influencing ROS levels or oxidative stress in cultured macrophages, its topical application significantly enhanced wound healing in a model of wound repair in the context of venous insufficiency.

Oxidized clinical-grade hAAT (hAAT^OX) demonstrated diminished elastase inhibition and impaired in-vitro epithelial gap closure, in correlation to the degree of oxidation. The results of the present study suggest that the degree of oxidation of hAAT influences the functional properties of hAAT. Figure 8 graphically summarizes the differing effects associated with hAAT and hAAT^OX on tissue response to injury. On the one hand, they playa role in normal wound healing by promoting the resolution of inflammation, on the other hand, under pathologic circumstances such as radiation injury or hemosiderin tissue deposition, they may act as a pro-inflammatory and oxidative agent. The highly oxidized form of hAAT lacks elastase inhibition and contributes to a pro-inflammatory environment, while less oxidized forms of hAAT, though also lacking elastase inhibition, retain some anti-inflammatory properties and still support epithelial gap repair. How does the gradient effect occur at the molecular level? It is suggested that the methionine residue at position 358 in hAAT is sterically exposed and readily oxidized (52). Other methionine residues on the globular surface of the molecule are not as approachable and display unique selectivities to various oxidative agents, most probably due to their physical orientation and neighboring amino acid residues (43, 53). Once these methionine residues do become oxidized, they are likely to cause structural distortion that may result in changes to the properties of hAAT beyond the mere elimination of elastase inhibition, namely, its ability to bind to a plethora of molecular targets (54, 55).

The initial evaluation of hAAT in promoting wound healing in radiation-damaged skin pointed to a complex interaction with oxidative environments. Accordingly, the effect of hAAT on wound repair in radiation-damaged skin differed from its impact on iron-treated skin. In irradiated skin, hAAT initially provided no benefit and possibly exacerbated wound repair in the first seven days, but then led to significantly better wound closure compared to WT irradiated skin. In contrast, on iron-treated skin, topical hAAT treatment showed early benefits in wound healing as soon as three days post-injury. The disparity in response may be explained by the different sources of ROS in both models; radiation-induced ROS result from photochemical reactions causing damage to keratinocytes, fibroblasts and melanocytes, while iron-induced ROS are linked to dysfunction of vascular endothelial cells, local fibroblasts and smooth muscle cells. Future studies should investigate the relationship between ROS-related damage and hAAT treatment, particularly in fibroblasts and endothelial cells, given the intimate protective effects of hAAT on endothelial cell injury (56–60). It is plausible that in iron-treated models, hAAT improves vascular health that is the primary driver of the poor underlying conditions, whereas on irradiated skin, the beneficial effects of hAAT might require a gradual advancement of neovessels, as shown by Bellacen et al. to occur in the presence of hAAT over several days (61).

hAAT has consistently shown improved epithelial cell gap closure outcomes across various studies. Here, gap closure appeared unchanged between hAAT and hAAT^OX at low oxidative exposure (0.5 mM of H₂O₂), while at the intermediate degree of oxidation (5 mM of H₂O₂), hAAT^OX was far less beneficial per the same concentration of native hAAT. Notably, hAAT^OX generated in the presence of 25 mM H₂O₂ significantly stunted gap closure throughout the entire 12-hr follow-up. Cells appeared macroscopically stressed but viable in the presence of hAAT^{OX 25mM}, suggesting that the mechanism for the stunting of gap closure has to do with flawed repair pathways rather than epithelial cell death. Interestingly, focusing on progression in gap closure between 6 hours and 12 hours from monolayer disruption, both hAAT^{OX 0.5mM} and hAAT^{OX 5mM} allowed gap closure advancement, most notable in the hAAT^{OX 5mM} setting (closure advanced from 62.12 ± 5.05% gap area at 6 hrs to 23.39 ± 4.28% at 12 hrs, p = 0.0011); nonetheless, these values are still impaired compared to the impact of native hAAT, which had advanced the gap area from 21.36 ± 12.93% at 6 hrs to complete closure at 12 hrs.

In a macrophage stimulation assay, treatment of cells with H₂O₂ induced IL-1β, IL-1Ra, TNFα, CXCL-1, iNOS and catalase, at all three concentrations of H₂O₂ exposure (0.5, 5 and 25 mM). However, hAAT that was treated with 0.5 mM H₂O₂ did not cause this response, and, more importantly, induced IL-1Ra expression as early as 3 hours from treatment. The rise in IL-1Ra at a time when IL-1β expression is low renders the system anti-inflammatory. As expected, 12 hrs from treatment, genes that are downstream to IL-1β/TNFα exhibited more profound responses compared to their 3-hr state of expression. Cytokine concentrations in supernatants were not tested in the current experimental system and may be of interest in future studies. Interestingly, according to transcriptome data generated by Sivaraman et al., hAAT^OX primarily affects genes related to transcriptional regulation, while native hAAT affects genes involved in inflammatory pathways (62). NF-κB, the key transcription factor for regulating inflammatory responses and oxidative stress (63, 64), is typically inhibited by hAAT, as was recently revisited in a model of hAAT-transgenic Drosophila (65). Taken together, these findings suggest that oxidative modifications of hAAT can impair its anti-inflammatory properties (30, 66).

Despite the clear impairment to anti inflammatory properties, Schuster et al. (27) demonstrated that native hAAT selectively allows for nuclear localization of the p65 (RelA) subunit of NF-κB in favor of inducible IL-1Ra expression. This suggests that hAAT skews NF-κB activity rather than inhibits it altogether, as would occur in the case of, e.g., corticosteroid therapy (27, 67). Janciauskiene et al. (33) and Lechowicz et al. (35) reported that oxidative modifications of hAAT can both exacerbate and ameliorate inflammatory responses, depending on the experimental context. In addition to a possible biological bifurcation, there is variation in laboratory techniques for hAAT oxidation that may account for some of the different experimental outcomes. For instance, in a study by Janciauskiene et al. (68) hAAT was oxidized using N-chlorosuccinimide in a 25 molar excess, while in a study by Alam et al. (69) hAAT was oxidized by exposure to cigarette smoke extract. In some studies, both native and oxidized forms of hAAT were found equally effective (70) whereas in others, addition of native hAAT was required in order to attenuate the inflammatory response induced by hAAT^OX. hAAT^OX treatment led to increased expression of iNOS and catalase, as well as increased activation of the Nrf2/ARE pathway, and levels of ROS in macrophage cultures. This indicates that hAAT^OX induces oxidative stress in an oxidation-dependent manner. Native hAAT did not directly mitigate these oxidative effects, suggesting that its anti-inflammatory properties may be independent of its antioxidant implications. An intriguing exception is observed in the case of Nrf2/ARE pathway activation. At low oxidative conditions, the pathway is suppressed relative to the effect of the oxidative stress itself (Fig. 8 second column from right), yet it is significantly enhanced at intermediate and high oxidative conditions (two left columns). This phenomenon suggests that an environment with mild oxidative stress renders hAAT a putative singular agent distinct from its native form or its highly-oxidized form. In this state, it lacks elastase inhibition capacity thus allowing neutrophil migration through tissues, but allows for inflammatory induction of iNOS and catalase, all while still promoting epithelial gap closure. The close relationship between hAAT and neutrophils is perhaps best represented by the fact that neutrophils actively release hAAT upon degranulation, potentially serving to support adjacent tissue along their destructive path (71). At the other extreme, in conditions of high oxidative stress, such that most probably occur in the presence of an injured tissue laden with activated neutrophils and macrophages (left column), hAAT^OX exhibits proinflammatory activity. These findings indicate the possibility of using supplementary hAAT treatment in specific circumstances, like as difficult-to-heal wounds, where one's own hAAT may lose its tissue protective attributes and become a pro-inflammatory agent.

Clinical manifestations of hAAT treatment for irradiated skin may include addressing poor wound healing of a surgical resection on skin that underwent radiotherapy in advanced head and neck cancer (72), or potential minimization of severe oral complications after maxillary radiotherapy (73). Clinical manifestations of hAAT treatment for venous insufficiency may include prevention of ulcer development (74). In addition, hAAT oxidation is physiologically reversible, both enzymatically and in the presence of reducing agents (43). This has allowed for an improvement to hAAT activity, through the introduction of antioxidants(33). For example, in baboons, treatment of severe bronchopulmonary dysplasia with antioxidants resulted in the enhancement of the anti-elastase activity of AAT (75). In fact, an oxidation-resistant, recombinant hAAT was developed as early as in 1984 (76) and was recently revisited in a study that showed that it prevents neutrophil elastase-induced cell death (77). It has been proven safe for introducing oxidation-resistant hAAT to patients with genetic AAT deficiency via adenovirus technology (78). It would be interesting to explore the implementation of this technology in pathologies of excessive oxidation, particularly where impaired wound healing is involved and topical hAAT is considered.

Taken together, the present study demonstrates that the oxidation level of hAAT plays a key role in determining its effects on inflammation, oxidative stress and tissue repair. This adaptable and reversible molecular characteristic is readily influenced by local signals, and offers an additional explanation for the remarkable clinical safety record of hAAT augmentation therapy. This is observed in individuals who are not genetically deficient in hAAT and yet receive hAAT treatment for other indications (79–86). Lastly, the study highlights the ability of hAAT to function independent of elastase inhibition, an emerging field which presents opportunities to develop and implement novel recombinant forms of hAAT (24, 87, 88). Additional investigation is required to clarify the processes behind the varying impacts of oxidation on hAAT, with an emphasis on exploring changes in affinity between hAAT and its rich repertoire of binding partners (54, 55). Future research should also explore hAAT’s effect on other medical conditions, particularly in the context of oxidative stress and inadequate tissue repair.

ANOVA

Analysis of variance

COPD

Chronic obstructive pulmonary disease

CXCL-1

C-X-C motif chemokine ligand 1

eNOS

Endothelial nitric oxide synthase

FCS

Fetal calf serum

Gray (unit of radiation)

H2O2

Hydrogen peroxide

hAAT

Human alpha-1 antitrypsin

hAAT+/+

Transgenic mice homozygous for human alpha-1 antitrypsin

hAAT^OX

Oxidized alpha-1 antitrypsin

IL-1β

Interleukin-1 beta

IL-1Ra

Interleukin-1 receptor antagonist

iNOS

Inducible nitric oxide synthase

IRI

Ischemia-reperfusion injury

NAC

N-acetylcysteine

NRF2/ARE

Nuclear factor erythroid 2-related factor 2/Antioxidant response element

PCR

Polymerase chain reaction

ROS

Reactive oxygen species

SEM

Standard error of the mean

SOD

Superoxide dismutase

SVG

Scalable Vector Graphics

TNFα

Tumor necrosis factor alpha

Ultraviolet

Wild-type

Ethical Approval and Consent to participate

Animal: Studies were approved by the Institutional Animal Care and Use Committee (IL-155-10-2022D) and conducted in line with the Guide for the Care and Use of Laboratory Animals, 8th Edition.

Consent for publication

All authors have read and agreed to the published version of the manuscript.

Competing interests

The authors declare no conflict of interest. Additionally, the funders had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Funding

The study was funded by The Diane Lynn Family Foundation to E.C.L. and by Israel Science Foundation (ISF 2976/20) to S.E. and E.C.L.

Author Contribution

I.F.,L.S, E.S. and E.C.L. conceptualized the study; I.F., L.S, A.Y., N.P, A.D, N.O and D.H. contributed to methodology; S.C performed linguistic editing; I.F. prepared original draft; E.C.L., S.E and E.S. wrote, reviewed and edited; I.F., E.S. and E.C.L. contributed to study visualization; E.C.L. and E.S. supervised the study; E.C.L. acquired study funding and is the guarantor of this work, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors have read and agreed to the published version of the manuscript.

Acknowledgement

The authors wish to thank Valeria Frishman for her excellent technical assistance and Hadar Eini and Dalit Galitsky for invaluable administrative support.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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No competing interests reported.

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Oxidation-Dependent Effects of Alpha-1 Antitrypsin on Wound Healing and Inflammation: Implications for Tissue Repair

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Animals

Radiation-induced impaired wound healing model

Ferrum loading impaired wound healing model

ROS levels in UVB-exposed macrophages RAW 264.7 cells

Oxidation of clinical-grade hAAT

Elastase activity assay

In-vitro human epithelial A549 cells gap repair assay

Macrophage activation assay

Gene expression analysis

ROS levels in RAW 264.7 cells exposed to H₂O₂ and oxidized hAAT

Transient transfection and Nrf2/ARE reporter gene assay

Statistical analysis

Results

In-vivo wound closure in WT and hAAT^+/+ mice 3 weeks after skin irradiation

Effect of clinical-grade hAAT on ROS levels in UVB-exposed macrophages

Oxidized clinical-grade hAAT (hAAT^OX) exhibits impaired elastase inhibition in association with degree of oxidation

hAAT^OX impairs in-vitro epithelial gap closure in association with degree of oxidation

hAAT^OX stimulates macrophages in association with degree of oxidation and increases iNOS and catalase expression

In-vivo iron-induced oxidative stress: clinical-grade hAAT accelerates impaired wound healing

Discussion

Abbreviations

Declarations

Competing interests

Funding

Author Contribution

Acknowledgement

Availability of data and materials

References

Additional Declarations

Status:

Version 1

Oxidation-Dependent Effects of Alpha-1 Antitrypsin on Wound Healing and Inflammation: Implications for Tissue Repair

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Animals

Radiation-induced impaired wound healing model

Ferrum loading impaired wound healing model

ROS levels in UVB-exposed macrophages RAW 264.7 cells

Oxidation of clinical-grade hAAT

Elastase activity assay

In-vitro human epithelial A549 cells gap repair assay

Macrophage activation assay

Gene expression analysis

ROS levels in RAW 264.7 cells exposed to H2O2 and oxidized hAAT

Transient transfection and Nrf2/ARE reporter gene assay

Statistical analysis

Results

In-vivo wound closure in WT and hAAT+/+ mice 3 weeks after skin irradiation

Effect of clinical-grade hAAT on ROS levels in UVB-exposed macrophages

Oxidized clinical-grade hAAT (hAATOX) exhibits impaired elastase inhibition in association with degree of oxidation

hAATOX impairs in-vitro epithelial gap closure in association with degree of oxidation

hAATOX stimulates macrophages in association with degree of oxidation and increases iNOS and catalase expression

In-vivo iron-induced oxidative stress: clinical-grade hAAT accelerates impaired wound healing

Discussion

Abbreviations

Declarations

Competing interests

Funding

Author Contribution

Acknowledgement

Availability of data and materials

References

Additional Declarations

Status:

Version 1

ROS levels in RAW 264.7 cells exposed to H₂O₂ and oxidized hAAT

In-vivo wound closure in WT and hAAT^+/+ mice 3 weeks after skin irradiation

Oxidized clinical-grade hAAT (hAAT^OX) exhibits impaired elastase inhibition in association with degree of oxidation

hAAT^OX impairs in-vitro epithelial gap closure in association with degree of oxidation

hAAT^OX stimulates macrophages in association with degree of oxidation and increases iNOS and catalase expression