Glycated ɑ1-Antitrypsin Involvement in Impaired Wound Healing: In- Vivo and In-Vitro Models

doi:10.21203/rs.3.rs-4931520/v1

Download PDF

Research Article

Glycated ɑ1-Antitrypsin Involvement in Impaired Wound Healing: In- Vivo and In-Vitro Models

https://doi.org/10.21203/rs.3.rs-4931520/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Impaired wound healing causes considerable morbidity among patients with diabetes. Human ɑ1-antitrypsin (hAAT) directs inflammation in injured tissues toward resolution. Upon glycation, gly-hAAT loses anti-proteolytic activity, but whether it fails to modulate inflammation and to promote wound repair is unknown.

Objective: Explore the impact of clinical-grade hAAT on wound repair under hyperglycemic conditions, and the role of gly-hAAT in impaired wound healing pathophysiology.

Research Design and Methods: Mice were rendered hyperglycemic and excisional wounding was performed, treated with topical albumin or hAAT every three days from time of wounding. Wound area was followed and samples collected for histology and gene expression analysis. Gly-hAAT was generated from clinical-grade hAAT in laboratory settings. In-vitro, RAW 264.7 macrophage responses were assessed and re-epithelialization was tested using A549 and HaCaT cells in the presence of gly-hAAT, and in the presence of sera from individuals with poor glucose control, both supplemented with clinical-grade hAAT.

Results: Topical hAAT accelerated in-vivo and in-vitro wound closure. Vascular maturity appeared earlier in hAAT-rich conditions, and gene expression skewed towards anti-inflammatory IL-1β/IL-1Ra ratio. Gly-hAAT inhibited normoglycemic mouse wound closure and epithelial cell gap closure, both systems rescued by clinical-grade hAAT. Gly-hAAT evoked an inflammatory response in macrophages, and diabetic patient serum inhibited epithelial cell gap closure; both trends were reversed by clinical-grade hAAT.

Conclusions: Topical hAAT accelerates wound closure under hyperglycemic conditions, and gly-hAAT is inflammatory and fails to benefit wound repair. Considering its phenomenal safety profile, it is suggested that clinical-grade hAAT is primed for testing in clinical settings.

1. The study was conducted to address an unmet medical need: wounds that fail to heal due to poor blood glucose control, and because ɑ1-antitrypsin, a tissue protective agent that is neutralized by excessive glucose levels, is clinically-available and safe for usage.

2. The question posed was on the effectiveness of hAAT in hyperglycemic circumstances and the characteristics of gly-hAAT.

3. According to study outcomes, topical hAAT accelerates wound closure under hyperglycemic conditions; gly-hAAT is inflammatory and impedes wound repair.

4. The study suggests that clinical-grade hAAT holds promise for diabetic wound treatment; clinical studies are encouraged.

The skin's main function is to shield the body from environmental insults and prevent fluid loss. Therefore, upon injury, it is extremely important to restore skin integrity in a rapid and efficient manner. The physiological wound healing process is initiated by an inflammatory surge characterized by inflammatory cytokine production, including IL-1β, TNFα and IL-6, as well as the desirable migration of neutrophils and facilitation of inflammatory M1 macrophages for the elimination of pathogens and removal of cell debris (1). While the inflammatory wave activates fibroblasts, drives re-epithelialization, and promotes angiogenesis and revascularization, it must subside in order to allow these processes to unfold. Difficult-to-heal wounds occur when inflammatory triggers persist, be it infection, residual inflammatory agents, ischemia or mechanical disruption. Hyperglycemia represents an persistent inflammatory trigger and often causes difficult-to-heal wounds (2); as such, it poses a significant risk for infections and lower limb amputations. Wounds that develop in individuals with poor glycemic control depict inadequate revascularization, dysfunctional fibroblasts, endothelial and epithelial cells (2), a predominance of M1 macrophages that typically express inducible nitric oxide synthase (iNOS), and, at times, a bacterial biofilm (3).

The molecular and cellular profile of a desired inflammatory resolution process includes M2-like macrophages, which typically express arginase-1 (Arg-1) and CD206, and anti-inflammatory cytokines, such as IL-1 receptor antagonist (IL-1Ra) (4). The latter mitigates IL-1–elicited inflammatory processes by preventing inflammatory IL-1 family members from activating the IL-1 receptor (5). Thus, IL-1Ra/IL-1β ratio is of significance to wound repair outcomes (6).

Human ɑ1-antitrypsin (hAAT) is a 52 kDa circulating glycoprotein that belongs to the serine-protease inhibitor family (7). From steady-state levels, it elevates with infection, inflammation, hypoxia, and tissue injury lasting a week or more. It also rises in the blood during a healthy pregnancy. Its circulating levels depend on liver production, while mucosal tissues produce local hAAT. hAAT promotes multiple processes relevant to wound healing. hAAT promotes mature blood vessel development (8), decreases the amount of necrotic tissue (9), and accelerates epithelial gap closure in-vitro and in-vivo (10,11). Interestingly, corticosteroids limit the beneficial effects of hAAT, supporting its classification as an inflammation-driven pro-resolution agent (10,11).

hAAT inhibits the activity of inflammatory serine-proteases, such as neutrophil elastase, which would otherwise degrade tissue elastin, activate stimulatory protease-activated receptors (PARs) on immunocytes, and activate pro–IL-1β released from injured cells (7,12). Accordingly, the major clinical characteristic of genetic hAAT deficiency (AATD) is lung alveolar wall deterioration (13). Although less common, patients with AATD exhibit dermatological manifestations, such as panniculitis, vasculitis, psoriasis, urticaria and angioedema; they also suffer from impaired wound healing and longer hospitalization times (14). Despite reducing inflammation, hAAT is an immunomodulator that directs, rather than blocks, neutrophils, macrophages, dendritic cells, B lymphocytes, and indirectly T and NK cells, toward tissue-protective phenotypes (15).

hAAT also acts by binding local agents (16). For example, hAAT increases the production of inflammation-induced IL-1Ra in a manner independent of protease inhibition (12), and binds to the neutrophil chemokine, IL-8, and to several danger-associated molecular pattern molecules (DAMPs) (15,17). A cysteine residue on its surface covalently binds to local nitric oxide and turns hAAT anti-bacterial (18). In this regard, the integrity of its surface amino acids is cardinal for its function.

The non-enzymatic covalent attachment of glucose to lysine and arginine residues of a protein yields advanced glycation end-products (AGEs) (19). AGEs are inflammatory; they induce IL-1β, TNFα and IL-6 production, promote oxidative stress and impair wound healing. hAAT undergoes glycation under hyperglycemic conditions, losing its ability to inhibit serine proteases (20); however, whether glycated hAAT (gly-hAAT) fails to promote inflammatory resolution is unknown.

The present study postulates that upon glycation, hAAT falls short of tissue protection, both by loss of its pro-resolution features, and by gaining a pro-inflammatory phenotype.

Animals

Studies were approved by the Institutional Animal Care and Use Committee (BGU302-1-2024D) and conducted in line with the Guide for the Care and Use of Laboratory Animals, 8^th Edition. C57BL/6 mice (8-12-week–old females, Envigo+ Laboratories, Inc., Rehovot, Israel) were housed at a standard vivarium. Mice transgenic for human AAT (hAAT^+/+; C57BL/6 background) were generated at the University of British Columbia, Canada and bred in house (21). hAAT^+/+ mice express constitutive levels of circulating hAAT (<1 µg/ml) and are used as a positive control for hAAT treatment.

In-vivo wounds in hyperglycemic mice

Mice were weighed prior to interventions and periodically thereafter. Hyperglycemia was induced using streptozotocin (STZ, Sigma Aldrich, Rehovot, Israel) in 50 mM sodium citrate, pH 4.5. Mice received a single intraperitoneal injection of either 100 or 225 mg/kg STZ (indicated per experiment) at a volume of 250 µl per mouse and blood glucose levels were measured daily to monitor hyperglycemia (≥120 mg/dl fasting or ≥250 mg/d non-fasting blood glucose). One week into hyperglycemia, mice were anesthetized and surgical excision was made at the center of the shaved dorsum. Human serum albumin (70024-90-7, Sigma-Aldrich) or hAAT (Glassia^®, Kamada LTD., Ness Ziona, Israel) were introduced directly to wound borders (4 mg/kg in 100 µl), on day of excision and then every three days. Wounds were photographed serially, and images were analyzed using ImageJ (MedCalc Software, Ostend, Belgium).

Histological Analysis

Animals were sacrificed, wounds were excised and immediately immersed in 10% neutral-buffered formalin (Sigma-Aldrich). Tissues were subsequently cut into 4-6μm sections, mounted on slides and stained with Hematoxylin and Eosin (H&E; Jackson ImmunoResearch, West Grove, PA, USA).

Glycation of hAAT in acellular conditions

Non-enzymatic glycation of hAAT was conducted, adapting the methodology described by Duell et al. (22). Briefly, 0.5 ml hAAT (0.59 mg protein) in 2 ml of 50 mM Tris-HCl buffer (pH 8.0) was combined with 12 mg/ml sodium borohydride. D-glucose was then added to concentrations of 400, 500, 800 and 4000 mM, the preparations sealed under nitrogen and incubated at 37°C for 7 days. Glycosylated solutions then underwent extensive dialysis against the initiating buffers. The extent of glycation was ascertained by assays using picryl sulfonic acid and trinitrobenzene sulfonic acid (TNBS).

Western blot analysis

Sample protein concentrations were determined using BCA protein assay kit (Cat#202389, Santa Cruz Biotechnology). For each sample, 20 μg protein were loaded and resolved on 7.5-18% SDS-PAGE. The proteins were subsequently electro-transferred onto nitrocellulose membranes (Cat#1620147, Bio-Rad, CA, USA), and membranes were blocked with 5% BSA in Tris-Buffered Saline with 0.1% Tween 20 (TBS/T) for 45 minutes at room temperature. The membranes were probed with mouse anti-human AAT (1/1000, Cat#VMA00662, Bio-Rad) followed by goat anti-mouse HRP-conjugated antibody (1/10,000, STAR207P), then visualized using enhanced chemiluminescence (ECL, Cat#XLS063, Cyanagen, Bologna, Italy).

Elastase activity assay

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

In-vitro epithelial gap repair assay and macrophage stimulation assay

In-vitro scratch assay was performed using A549 cells (Cat# CCL-185, ATCC) grown to confluence in 24-well plates and then scratched uniformly using a 200 µl pipette tip, creating a cell-free area (10,11). Cultures were then washed twice with complete RPMI 1640 supplemented with 2.5% FCS (both from Biological Industries Inc., Beit Haemek, Israel) and serial Images acquired by photomicroscope (Zeiss). Cell-free areas were analyzed by ImageJ.

RAW 264.7 cells (ATCC, Cat#SC-6003) were seeded in 48-well plates (1×10⁵ cells/well) in RPMI 1640 medium supplemented with 5% FCS. Cells were incubated for 4 hours with indicated concentrations of hAAT or gly-hAAT and then stimulated with 5 ng/ml LPS. Eight-hour TNFα levels were determined by DuoSet ELISA (Cat#DY410, R&D systems, Minneapolis, USA). All measurements were performed in triplicate.

Gene expression analysis

Wound samples were submerged in RNA Save (Biological Industries), homogenized in a polytron homogenizer and loaded onto RNase-free microcentrifuge tubes. RNA was extracted using Gynzol^® reagent (Invitrogen, Waltham, MA, USA) and isolated on columns (RNAqueous^®-Micro, Thermo Fisher Scientific, MA, USA), following manufacturer’s guidelines. Eluted RNA was quantified using NanoDrop (Wilmington, DL, USA), and 200 ng were reverse-transcribed using Prime Script RT Reagent kit (Quanta Biotech, USA). Quantitative PCR was performed at a 20-μl volume reaction using an RT-PCR system (StepOnePlus™ Real-Time PCR, ThermoFisher Scientific Corporation, Waltham, MA, USA), and SYBR Premix Ex Taq II (Quanta Biotech, USA). CFX96 manager software was used to determine threshold cycle values; β-actin was used as a reference gene. Primers were designed for murine transcripts as follows: IL-1β ‘5-CTTCCAGGATGAGGACATGAAGG-3′ (forward), ‘5-AGTGCAGTTGTCTAATGGGA-3′ (reverse); VEGF, 5′-TGGGACTGGATTCGCCATTT-3′ (forward), 5′-GTGGGTGGGTGTGTCTACAG-3′ (reverse); IL-1Ra, 5′-GACCCTGCAAGATGCAAGCC-3′ (forward), 5′-GAGCGGATGAAGGTAAAGCG-3′ (reverse); MCP-1 5′-AGGCATCACAGTCCGAGTCA-3′ (forward), ‘5-CCACAACCACCTCAAGCACT-3′ (reverse), ARG-1 5′-AACACGGCAGTGGCTTTAACC-3′ (forward), ‘5-GGTTTTCATGTGGCGCATTC′ (reverse), CD206 5′-GGCTGATTACGAGCAGTGGA-3′ (forward), ‘5-CATCACTCCAGGTGAACCCC-3′ (reverse), CD14 5′-CAGAGAACACCACCGCTGTA-3′ (forward), ‘5-ACACGCTCCATGGTCGGT A-3′ (reverse) and β-actin 5′-CATTGCTGACAGGATGCAGA-3′ (forward), ‘5-TGCTGGAAGGTGGACAGTGA-3′ (reverse).

Human serum collection and analysis

The study was approved by the Institutional Review Board of Soroka University Medical Center, Israel (SOR 1106-2018), ensuring adherence to ethical guidelines for biomedical research. After informed consent was obtained, 9 adult patients ages 60±7.2 yrs old, diagnosed with diabetes and eligible for or had already undergone limb amputation, were recruited for serum sampling by the diabetes clinic. Glycated hemoglobin (HbA1c) levels and fasting blood glucose measurements were obtained. Sera were added to the epithelial scratch assay at 1:10 volume and the assay was performed with 5% FCS as background condition, as a decline in closure rate was anticipated.

Statistical Analysis

Statistical significance of differences between groups was evaluated using ANOVA followed by post-hoc tests for multiple comparisons. A p-value <0.05 was considered statistically significant. Statistical processing was performed using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA).

Circulating transgenic hAAT accelerates wound closure in hyperglycemic mice

The impact of hAAT therapy on wound healing in the context of hyperglycemia was addressed using WT and hAAT^+/+. As shown (Fig. 1A), hyperglycemia was comparable between groups at time of implementing a 5-mm dorsal excisional wound (Fig. 1B). Normoglycemic WT mice (CT) began closing wounds after day 4 from wounding (wound area 81.50 ± 38.91 percent from day 0 compared to 38.00 ± 11.14% on day 6, mean ± SEM, p < 0.05); hyperglycemic WT mice did not close wounds for at least 8 days, afterwhich follow-up was discontinued for ethical concerns. Hyperglycemic hAAT^+/+ mice initiated wound closure between days 2 and 4, albeit without reaching statistical significance compared to hyperglycemic WT mice at those time points. Nonetheless, the two hyperglycemic groups diverged on days 6 and 8, where hAAT^+/+ mice exhibited a clear capacity for wound closure (WT and hAAT^+/+ wound area day 6, 106.30 ± 36.57% and 62.75 ± 12.50%, and day 8, 108.5 ± 23.63% and 48.25 ± 18.52%, mean ± SEM, p < 0.05).

Topical clinical-grade hAAT accelerates wound closure in hyperglycemic mice

After hAAT^+/+ mice provided a proof-of-concept for improved wound closure under hyperglycemic conditions, the possibility of local hAAT treatment was tested herein with WT mice. Human albumin was used at the same dose and timing to control for treatment. Low-dose STZ was applied and an 8-mm excisional wound performed so as to reduce morbidity and allow a longer follow-up. As shown (Fig. 1C), hyperglycemia was comparable between groups. According to imaging follow-up (Fig. 1D), hyperglycemic albumin-treated mice exhibited poor wound closure (normoglycemic and albumin-treated hyperglycemic mice day-3 wound area 30.35 ± 6.36% and 43.31 ± 19.35% from initial wound, day-6 22.80 ± 5.22% and 30.97 ± 9.86%, p = 0.015 and p = 0.022 per time point, respectively). In contrast, topical hAAT resulted in a significantly more robust healing response (day 6, 17.55 ± 7.98%, p < 0.001 from day-6 albumin group). A day-by-day comparison is provided in Fig. 1E.

According to histological analysis of day-3 wound samples (Fig. 1F), the hyperglycemic control group exhibited inflammatory infiltrates characterized by extensive nuclear debris and substantial NETosis. Inside the granulation site there were extravasated red blood cells without concomitant formation of mature blood vessels. In contrast, wounds treated with hAAT exhibited structured organization of the tissue, orderly regions of inflammatory infiltration and reduced nuclear debris and NETsis; mature blood vessels carrying red blood cells are clearly evident.

Day-3 wound samples were also collected for gene transcript analysis. As shown (Fig. 1G), compared to samples from normoglycemic mice, wounds from hyperglycemic mice treated with albumin exhibited significantly elevated transcript levels of VEGF and IL-1β. Mice treated with hAAT displayed VEGF transcript levels that were comparable to normoglycemic mice, yet IL-1β transcript levels were comparable to albumin-treated hyperglycemic wounds. Unlike VEGF and IL-1β expression, IL-1Ra expression was significantly higher in hAAT-treated wounds, and IL-1Ra/IL-1β ratio per animal was predominantly IL-1Ra. According to analysis of macrophage-related genes, wound samples from hyperglycemic mice exhibited greater CD14 transcript levels, representing macrophagic burden, and of MCP-1 transcripts, irrespective of wound treatment. Transcript levels of CD206 and Arg-1 normalized to CD14 transcripts provide an insight as to the presence of M2-like macrophages. As shown, hyperglycemic wounds lack in these markers, and treatment with hAAT did not alter this profile at this early time point.

Glycated hAAT (gly-hAAT) is inflammatory

A glycated form of hAAT was generated. As shown (Fig. 2A), upon prolonged exposure to glucose, hAAT increased in molecular weight in a glucose-concentration–dependent manner. Gly-hAAT generated with 800 mM glucose is used for further investigation; as shown (Fig. 2B), naïve hAAT neutralized elastase at a 1:1 molar ratio, while gly-hAAT required three-fold greater concentrations (72.86 ± 11.00%, 28.06 ± 2.15% and 4.74 ± 3.06% elastase activity, ×1, ×2 and ×3 gly-hAAT, respectively).

The inflammatory response of the RAW 264.7 macrophage cell line was tested in the presence of gly-hAAT (Fig. 2C). Glucose alone was introduced to cells to represent the effect of possible carryover from the glycation protocol. As shown (left panel), cells released baseline TNFα levels that were comparable between control, hAAT and glucose treatments, and cells exposed to gly-hAAT released significantly higher concentrations of TNFα (38.91 ± 7.30 pg/ml and 429.30 ± 247.6 pg/ml, control and gly-hAAT, respectively, p < 0.0001). Supplementing gly-hAAT with naïve hAAT significantly reduced TNFα release (147.70 ± 33.86 pg/ml, p = 0.018). Under LPS stimulation (right panel), TNFα levels increased both in the presence of control medium and hAAT. Glucose reduced TNFα release under LPS stimulation, albeit without reaching statistical significance. TNFα release was further elevated by gly-hAAT (3405.0 ± 1010.0 pg/ml and 856.0 ± 46.4 pg/ml, gly-hAAT/LPS and LPS, p = 0.009).

Gly-hAAT fails to promote epithelial gap closure in-vitro

Following assessment of the inflammatory activity of gly-hAAT on macrophages, its impact on epithelial gap closure was investigated (Fig. 3). According to the A549 epithelial cell scratch assay, under control conditions, gap area was reduced to 39.41 ± 8.50% within 24 hours, and in the presence of hAAT it was significantly smaller (29.32 ± 9.80%, p = 0.04). Glucose treatment (80 mg/dl) resulted in a residual gap area of 25.14 ± 4.96% at 24 hours (p = 0.015 compared to control). In contrast, gly-hAAT–treated cells depicted gap closure comparable to control (40.04 ± 10.52% compared to hAAT-treated cells at 24 hours, p = 0.07).

Gly-hAAT interferes with wound closure in normoglycemic mice

Extending on the results obtained with gly-hAAT in-vitro,excisional wounds were performed in normoglycemic mice. As shown (Fig. 4A), while albumin-treated wounds exhibited 21.35 ± 5.38% area from initial size on day 3, gly-hAAT–treated wounds left an area of 37.00 ± 4.58% from initial wounding (p < 0.001). A day-by-day depiction of the differences between groups is presented in Fig. 4B. The apparent negative impact of gly-hAAT on wound closure rates was challenged by supplementation with naïve hAAT; accordingly, wound closure was significantly accelerated, resulting in a profile comparable to albumin-treated wounds.

Sera from patients with poorly-controlled blood glucose interferes with epithelial gap closure: attenuation by clinical-grade hAAT

Sera were collected from a cohort of patients with diabetes with poorly-controlled blood glucose (N = 10), and then directly introduced onto epithelial gap closure assay. Within this cohort, average blood glucose levels were 212.5 ± 98.14 mg% and HbA1c was 8.0 ± 0.63% (64.0 ± 12.2 mmol/mol; mean ± SD). In addition, clinical-grade hAAT and serum from healthy individuals (N = 3) were used for reference; all assays were performed in 5% FCS since a decline in gap closure capacity was anticipated (gap area 5% FCS per time point represented by a gray horizontal line, Fig. 4C). As shown, conditions that included healthy serum (purple) or hAAT treatment (blue) depicted an overall improved cell density compared to 5% FCS alone. In contrast, serum samples from patients with poorly-controlled blood glucose (dashed red line) appeared to reduce cell density, each individual, expectedly, at a unique amplitude (3 representative outcomes). The area under the curve (AUC) is calculated for the difference between cell densities at 5% FCS and cell densities under patient serum (red arrow), as well as patient serum in the absence or presence of hAAT supplementation (blue arrow). As a group (Fig. 4D), samples from 10 patients with poorly-controlled blood glucose caused a 20.81 ± 47.18 decline in cell density, and added hAAT increased cell density by 77.5 ± 22.64 (p = 0.039).

The effect of a hyperglycemic environment on the properties of hAAT in the context of wound healing was investigated. The premise of the study is that hAAT acts as a binding protein whose properties are compromised by chronic exposure to high glucose levels. The study further suggests that hAAT glycation takes part in the pathogenesis of difficult-to-heal wounds in patients with diabetes. By this, it is suggested that individuals with poorly controlled glucose levels, at least in the context of tissue injury, may benefit from either topical clinical-grade hAAT or standard hAAT infusion therapy.

That hAAT improves acute wound repair is consistent with literature. Accordingly, compared to control normoglycemic WT mice, the rate of excisional skin wound closure was enhanced in normoglycemic WT mice treated with topical hAAT, and in normoglycemic hAAT^+/+ mice. To control for topical hAAT, human serum albumin was used as it is similar in molecular weight and, like hAAT, is prone to glycation. It is unclear how hAAT-rich settings speed resolution processes, yet, in its presence, inflammatory signals attract resolution processes sooner (10). Here, on day 3, wound tissue samples exhibited the predominant expression of IL-1Ra over IL-1β, while, nonetheless, mature blood vessel formation was already observed. Since protein glycation takes days and happens mostly in liver cells, it is believed that newly introduced naïve hAAT remains generally unglycated in the immediate period following treatment (19). Additionally, hAAT^+/+ mice produce hAAT in the lung (21).

The observation that gly-hAAT fails to accelerate wound repair fits well with its requirement for intact surface interactions (16). Future studies should examine the affinities of binding partners to gly-hAAT. To explore whether, like other AGEs, glycated hAAT aggravates diabetic wounds, gly-hAAT was generated by exposing clinical-grade hAAT to high levels of glucose over time. Accordingly, its molecular weight increased, it lost its ability to inhibit elastase, and it stimulated macrophage cultures in the absence (and presence) of LPS; ultimately, gly-hAAT interfered with wound closure in-vivo. Interestingly, gly-hAAT was less of an interruption to cultured epithelial cells, suggesting that in-vivo outcomes most probably involve its effect on, at the least, macrophages. In both circumstances, however, mixed with naïve clinical-grade hAAT, the negative impact of gly-hAAT was markedly attenuated. In that regard, we asked whether it is possible to rescue the compromised capacity for wound repair contained in serum samples from individuals with poorly controlled blood glucose levels. In a small-scale exploratory clinical arm, clinical-grade hAAT was combined into serum samples from people with poorly controlled blood glucose and then used in an epithelial gap closure experiment. Indeed, patient sera interfered with re-epithelialization, and added hAAT displayed enhanced closure rates, suggesting a reduction in some of the re-epithelialization–limiting factors contained in chronic hyperglycemic serum. Knowingly, this is a qualitative observation and requires a large cohort to explore the phenomenon.

Irrespective of glucose homeostasis, a profound alteration in the functionality of hAAT is observed in difficult-to-heal wounds. A comparative proteome analysis of wound exudates suggests that hAAT is abundant in acute wounds, and scarce in chronic wounds. hAAT exhibits extensive degradation in chronic wounds (23) and decreased activity hAAT in venous ulcers (24). Accordingly, fluids from chronic wounds exhibit 10–40–fold greater elastase activity than acute wounds (23).

Considering the prospect of hAAT as a cell survival and tissue recovery agent, and that pancreatic islet β-cells are targets of autoimmunity in type 1 diabetes, hAAT was widely tested in both preclinical (25, 26) and clinical studies (27–29), depicting beneficial outcomes during the early stages of type 1 diabetes. Here, animals that received STZ were confirmed to have reached comparable hyperglycemic values across strains at the time of wounding and throughout follow-up. However, follow-up duration was limited by hyperglycemia-related morbidity.

WT mice are studied alongside the transgenic model because a topical approach is more clinically relevant and because hAAT^+/+ mice are exposed to constant, low-levels of ectopic lung-derived circulating hAAT. As such, they do not mimic an infused individual and may differ from their background WT strain in some ways. For example, in hAAT^+/+ mice, blood pressure is lower (30, 31). The possibility that mouse anti-hAAT antibodies are induced in WT mice exposed to topical hAAT is unlikely, as even in high-dose systemic administration, the first signs of anti-hAAT antibodies occur after 18 days (32).

Re-vascularization was examined in the present study. Histology showed that hyperglycemic mice treated with topical hAAT developed mature blood vessels earlier than controls. This finding is consistent with other reports outside the context of hyperglycemia; Bellacen et al. demonstrated accelerated blood vessel maturation in pancreatic islet allografts (8), and Schuster et al. observed accelerated re-vascularization in a skin flap model (9). Potential targets for the mechanism of action include endothelial cells, which self-organize and form tubes in culture more rapidly under hAAT-rich conditions, and VEGF, which is expressed earlier in the presence of hAAT (8). Indeed, hAAT mitigates hypoxia-reperfusion injury and inhibits endothelial cell apoptosis (33), as well as facilitates mature blood vessel formation in normoglycemic settings (34); in the case of diabetic retinopathy, vessel maturation represents a desired outcome (35, 36). Interestingly, blockade of VEGF causes emphysema-like features in mice, regardless of intact AAT (37), and in a flap re-vascularization model, the effect of hAAT is only partially impeded by anti-VEGF (9), suggesting that hAAT may also promote supportive surroundings for vessel progression. AGEs increase VEGF expression and are likely present on day 3 wound examination, ten days after STZ was introduced. In this regard, further examination of hAAT in the flap model under hyperglycemic conditions would hold immense implications for surgical limitations in patients with hyperglycemia.

hAAT increases IL-1Ra levels in different conditions (10, 12, 38), but its ability to do so during hyperglycemia is unknown. Here, as all hyperglycemic mice displayed elevated IL-1β expression levels, IL-1Ra expression set apart hAAT-treated wounds from albumin-treated wounds. In line with examination of the IL-1–pathway, the expression of genes relating to macrophage polarity was explored. Upon introduction to steady-state RAW 264.7 cells, gly-hAAT elicited an inflammatory response, and in LPS-stimulated conditions gly-hAAT further aggravated the inflammatory response, agreeing with the general concept of inflammatory AGEs (19). The TNFα pathway was explored as one that depicts a consistent response to hAAT; specifically, while native hAAT interferes with TNFα pathway, its glycated form induced TNFα production even without added LPS. The possibility arises that gly-hAAT fails to bind to TNFα receptors (16, 39). Additionally, lack of protease inhibition by gly-hAAT may contribute to excess release of membrane-bound TNFα (32). Glucose alone did not elicit a pro-inflammatory response, drawing a clear distinction from gly-hAAT. The predominance of M1 macrophages in wound samples under hyperglycemic conditions and the failure of hAAT to promote M2-like macrophages in day-3 wounds, stands in contrast to effects of hAAT in normoglycemic settings (25). This observation may be attributed to the time point of sampling, when the shift towards M2-like cells is at its very beginning even in normoglycemic wounds (40). It is therefore possible that with hAAT treatment, the desired M1/M2 shift in hyperglycemic conditions occurs at a later time point. Further experiments are encouraged for assessing macrophage polarization at wound sites under hyperglycemic conditions and hAAT treatment, as well as using more prolonged experiments with less severe models of hyperglycemia.

In conclusion, the modulatory capacities of hAAT underscore its ability to navigate between inflammatory and pro-resolution states based on local molecular signals. The present study emphasizes that this modulation might be afflicted by excessive glucose, and that exogenous hAAT may restore a more physiological milieu for wound healing. Considering its phenomenal safety profile, it is suggested that clinical-grade hAAT is primed for testing in clinical settings of difficult-to-heal wounds in patients with poorly controlled blood glucose levels.

Ethical Approval and Consent to participate

Animal: Studies were approved by the Institutional Animal Care and Use Committee (BGU302-1-2024D) and conducted in line with the Guide for the Care and Use of Laboratory Animals, 8th Edition.

Human samples: The study was approved by the Institutional Review Board of Soroka University Medical Center, Israel (SOR 1106-2018), ensuring adherence to ethical guidelines for biomedical research. Informed consent was obtained from all subjects involved in the study.

Consent for publication

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

Availability of data and materials

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

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

This research was funded by the Israeli Ministry of Defense Grant 1884-2018 and The Diane Lynn Family Foundation both to E.C.L.

Authors' contributions

I.F., E.S. and E.C.L. conceptualized the study; I.F., A.N., Y.I., M.Z., Y.A., L.S., R.S., V.F., M.C. and D.H. contributed in methodology; I.F. prepared original draft; E.C.L. and E.S. wrote, reviewed and edited; I.F., S.C., 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.

Acknowledgements

The authors wish to thank Valeria Frishman for her excellent technical assistance and Hadar Eini and Dalit Galitsky for invaluable administrative support. We thank Rotem Tzur, MD, for performing serum collection.

Rodrigues M, Kosaric N, Bonham CA, Gurtner GC. Wound Healing: A Cellular Perspective. Physiol Rev. 2019;99:665–706.
Brem H, Tomic-Canic M. Cellular and molecular basis of wound healing in diabetes [Internet]. Vol. 117, Journal of Clinical Investigation. 2007. p. 1219–22. Available from: http://dx.doi.org/10.1172/JCI32169
Raziyeva K, Kim Y, Zharkinbekov Z, Kassymbek K, Jimi S, Saparov A. Immunology of Acute and Chronic Wound Healing. Biomolecules [Internet]. 2021 May 8;11(5). Available from: http://dx.doi.org/10.3390/biom11050700
Yazdi AS, Ghoreschi K. The Interleukin-1 Family. Adv Exp Med Biol. 2016;941:21–9.
Arend WP. The balance between IL-1 and IL-1Ra in disease. Vol. 13. 2002 p. 323–40.
Yan C, Gao N, Sun H, Yin J, Lee P, Zhou L, et al. Targeting Imbalance between IL-1β and IL-1 Receptor Antagonist Ameliorates Delayed Epithelium Wound Healing in Diabetic Mouse Corneas. Am J Pathol. 2016 Jun;186(6):1466–80.
Lewis E. a1-Antitrypsin Therapy for Non-Deficient Individuals: Integrating and Mitigating Cross-Pathology Inflammatory and Immune Responses To the Injured Cell. Internal Medicine Review. 2017;3(5):1–28.
Bellacen K, Kalay N, Ozeri E, Shahaf G, Lewis EC. Revascularization of pancreatic islet allografts is enhanced by α-1-antitrypsin under anti-inflammatory conditions. Cell Transplant. 2013;22(11):2119–33.
Schuster R, Bar-Nathan O, Tiosano A, Lewis EC, Silberstein E. Enhanced Survival and Accelerated Perfusion of Skin Flap to Recipient Site Following Administration of Human α1-Antitrypsin in Murine Models. Adv Wound Care. 2019 Jul 1;8(7):281–90.
Schuster R, Motola-Kalay N, Baranovski BM, Bar L, Tov N, Stein M, et al. Distinct anti-inflammatory properties of alpha1-antitrypsin and corticosteroids reveal unique underlying mechanisms of action. Cell Immunol. 2020;356(July):104177.
Gimmon A, Sherker L, Kojukarov L, Zaknoun M, Lior Y, Fadel T, et al. Accelerated Wound Border Closure Using a Microemulsion Containing Non-Inhibitory Recombinant α1-Antitrypsin. Int J Mol Sci. 2022 Jul 1;23(13):7364.
Joosten LAB, Crişan TO, Azam T, Cleophas MCP, Koenders MI, van de Veerdonk FL, et al. Alpha-1-anti-trypsin-Fc fusion protein ameliorates gouty arthritis by reducing release and extracellular processing of IL-1β and by the induction of endogenous IL-1Ra. Ann Rheum Dis. 2016 Jun;75(6):1219–27.
Strnad P, McElvaney NG, Lomas DA. Alpha ₁ -Antitrypsin Deficiency. Longo DL, editor. N Engl J Med. 2020 Apr 9;382(15):1443–55.
Greulich T, Nell C, Hohmann D, Grebe M, Janciauskiene S, Koczulla AR, et al. The prevalence of diagnosed α1-antitrypsin deficiency and its comorbidities: results from a large population-based database. Eur Respir J [Internet]. 2017 Jan;49(1). Available from: http://dx.doi.org/10.1183/13993003.00154-2016
Guttman O, Baranovski BM, Schuster R, Kaner Z, Freixo-Lima GS, Bahar N, et al. Acute-phase protein α1-anti-trypsin: diverting injurious innate and adaptive immune responses from non-authentic threats. Clin Exp Immunol. 2015 Feb;179(2):161–72.
O’Brien ME, Murray G, Gogoi D, Yusuf A, McCarthy C, Wormald MR, et al. A review of alpha-1 antitrypsin binding partners for immune regulation and potential therapeutic application. Int J Mol Sci. 2022 Feb 23;23(5):2441.
Ochayon DE, Mizrahi M, Shahaf G, Baranovski BM, Lewis EC. Human α1-Antitrypsin Binds to Heat-Shock Protein gp96 and Protects from Endogenous gp96-Mediated Injury In vivo. Front Immunol. 2013 Oct 28;4:320.
Kaner Z, Ochayon DE, Shahaf G, Baranovski BM, Bahar N, Mizrahi M, et al. Acute phase protein α1-antitrypsin reduces the bacterial burden in mice by selective modulation of innate cell responses. J Infect Dis. 2015 May 1;211(9):1489–98.
Twarda-Clapa A, Olczak A, Białkowska AM, Koziołkiewicz M. Advanced Glycation End-Products (AGEs): Formation, Chemistry, Classification, Receptors, and Diseases Related to AGEs. Cells [Internet]. 2022 Apr 12;11(8). Available from: http://dx.doi.org/10.3390/cells11081312
Austin GE, Mullins RH, Morin LG. Non-enzymic glycation of individual plasma proteins in normoglycemic and hyperglycemic patients. Clin Chem. 1987 Dec;33(12):2220–4.
Dhami R, Zay K, Gilks B, Porter S, Wright JL, Churg A. Pulmonary epithelial expression of human alpha1-antitrypsin in transgenic mice results in delivery of alpha1-antitrypsin protein to the interstitium. J Mol Med . 1999 Apr;77(4):377–85.
Duell PB, Oram JF, Bierman EL. Nonenzymatic glycosylation of HDL resulting in inhibition of high-affinity binding to cultured human fibroblasts. Diabetes. 1990 Oct;39(10):1257–63.
Rao CN, Ladin DA, Lin YY, Chilukuri K, Zheng Hou Z, Woodley DT. at-Antitrypsin Is Degraded and Non-Functional in Chronic Wounds But Intact and Functional in Acute Wounds: The Inhibitor Protects Fibronectin from Degradation by Chronic Wound Fluid Enzymes. 1995.
Eming SA, Koch M, Krieger A, Brachvogel B, Kreft S, Bruckner-Tuderman L, et al. Differential proteomic analysis distinguishes tissue repair biomarker signatures in wound exudates obtained from normal healing and chronic wounds. J Proteome Res. 2010;9(9):4758–66.
Gou W, Wang J, Song L, Kim DS, Cui W, Strange C, et al. Alpha-1 antitrypsin suppresses macrophage activation and promotes islet graft survival after intrahepatic islet transplantation. Am J Transplant. 2021 May;21(5):1713–24.
Kalis M, Kumar R, Janciauskiene S, Salehi A, Cilio CM. α 1-antitrypsin enhances insulin secretion and prevents cytokine-mediated apoptosis in pancreatic β-cells. Islets. 2010;2(3):185–9.
Ghoreishi A, Mahmoodi M, Khoshdel A. Comparison of alpha 1- antitrypsin activity and phenotype in type 1 diabetic patients to healthy individuals. Journal of Family Medicine and Primary Care. 2022;11(4):1377.
Rachmiel M, Strauss P, Dror N, Benzaquen H, Horesh O, Tov N, et al. Alpha-1 antitrypsin therapy is safe and well tolerated in children and adolescents with recent onset type 1 diabetes mellitus. Pediatr Diabetes. 2016 Aug 1;17(5):351–9.
Fleixo-Lima G, Ventura H, Medini M, Bar L, Strauss P, Lewis EC. Mechanistic evidence in support of alpha1-antitrypsin as a therapeutic approach for type 1 diabetes. J Diabetes Sci Technol. 2014 Jan 1;8(6):1193–203.
Liu LP, Gholam MF, Elshikha AS, Kawakibi T, Elmoujahid N, Moussa HH, et al. Transgenic Mice Overexpressing Human Alpha-1 Antitrypsin Exhibit Low Blood Pressure and Altered Epithelial Transport Mechanisms in the Inactive and Active Cycles. Front Physiol. 2021 Sep 22;12:710313.
Lugo CI, Liu LP, Bala N, Morales AG, Gholam MF, Abchee JC, et al. Human Alpha-1 Antitrypsin Attenuates ENaC and MARCKS and Lowers Blood Pressure in Hypertensive Diabetic db/db Mice. Biomolecules [Internet]. 2022 Dec 29;13(1). Available from: http://dx.doi.org/10.3390/biom13010066
Lewis EC, Shapiro L, Bowers OJ, Dinarello CA. Alpha1-antitrypsin monotherapy prolongs islet allograft survival in mice. Proc Natl Acad Sci U S A. 2005 Aug 23;102(34):12153–8.
Jeong KH, Lim JH, Lee KH, Kim MJ, Jung HY, Choi JY, et al. Protective Effect of Alpha 1-Antitrypsin on Renal Ischemia-Reperfusion Injury. Transplant Proc. 2019 Oct;51(8):2814–22.
Kilickesmez KO, Abaci O, Kocas C, Yildiz A, Kaya A, Okcun B, et al. Dilatation of the ascending aorta and serum alpha 1-antitrypsin level in patients with bicuspid aortic valve. Heart Vessels. 2012 Jul;27(4):391–7.
Ortiz G, Salica JP, Chuluyan EH, Gallo JE. Diabetic retinopathy: could the alpha-1 antitrypsin be a therapeutic option? Biol Res. 2014 Nov 18;47:58.
Mironidou-Tzouveleki M, Tsartsalis S, Tomos C. Vascular endothelial growth factor (VEGF) in the pathogenesis of diabetic nephropathy of type 1 diabetes mellitus. Curr Drug Targets. 2011 Jan;12(1):107–14.
Petrache I, Fijalkowska I, Zhen L, Medler TR, Brown E, Cruz P, et al. A novel antiapoptotic role for alpha1-antitrypsin in the prevention of pulmonary emphysema. Am J Respir Crit Care Med. 2006 Jun 1;173(11):1222–8.
Ozeri E, Rider P, Rigbi S, Shahaf G, Nita II, Sekler I, et al. Differential signaling patterns of stimulated bone marrow-derived dendritic cells under α1-antitrypsin-enriched conditions. Cell Immunol. 2021 Mar;361(104281):104281.
Bergin DA, Reeves EP, Hurley K, Wolfe R, Jameel R, Fitzgerald S, et al. The circulating proteinase inhibitor α-1 antitrypsin regulates neutrophil degranulation and autoimmunity. Sci Transl Med. 2014 Jan 1;6(217):217ra1.
Kuninaka Y, Ishida Y, Ishigami A, Nosaka M, Matsuki J, Yasuda H, et al. Macrophage polarity and wound age determination. Sci Rep. 2022 Nov 25;12(1):20327.

No competing interests reported.

Download PDF

Reviews received at journal
26 Aug, 2024
Reviewers agreed at journal
24 Aug, 2024
Reviewers invited by journal
19 Aug, 2024
Editor assigned by journal
19 Aug, 2024
Submission checks completed at journal
19 Aug, 2024
First submitted to journal
17 Aug, 2024

You are reading this latest preprint version

Glycated ɑ1-Antitrypsin Involvement in Impaired Wound Healing: In- Vivo and In-Vitro Models

Status:

Version 1

Abstract

Figures

Article Highlights

Introduction

Materials and Methods

Results

Circulating transgenic hAAT accelerates wound closure in hyperglycemic mice

Topical clinical-grade hAAT accelerates wound closure in hyperglycemic mice

Glycated hAAT (gly-hAAT) is inflammatory

Gly-hAAT fails to promote epithelial gap closure in-vitro

Gly-hAAT interferes with wound closure in normoglycemic mice

Discussion

Declarations

References

Additional Declarations

Status:

Version 1