Reversal of elastase-induced abdominal aortic aneurysm following the delivery of nanoparticle-based pentagalloyl glucose (PGG) is associated with reduced inflammatory and immune markers

Abstract

Objective:

An Abdominal aortic aneurysm (AAA), a deadly disease in elderly population, is featured by expansion of aortic diameter, degradation and weakening of vasculature. Its common and significant characteristics are disarray and inflammation in vasculature. We tested the hypothesis that the reversal of abdominal aortic aneurysm by pentagalloyl glucose-loaded nanoparticles (PGG-NPs) therapy that targets degraded elastin suppresses inflammatory and immune markers to ameliorate the pathophysiology of the disease in advance stage aneurysm in a porcine pancreatic elastase (PPE)-induced mouse model of AAA.

Methods and results:

After induction of aneurysm in pathogen-free C57BL/6 male mice by applying PPE peri-adventitially to the abdominal aorta, once a week for two doses of intravenous injections of pentagalloyl glucose-loaded nanoparticles (PGG-NPs) conjugated with elastin targeted antibody were used to reverse the aneurysms. We showed that PGG-NPs therapy could suppress infiltration of macrophages, CD8 and CD4 subsets of T cells, matrix metalloproteinases (MMPs), inflammatory cytokines interferon (IFN-γ) and interleukin (IL)-6 at the local and systemic level. Moreover, such PGG-NPs therapy increases the induction of anti-inflammatory cytokines IL-13, IL-27 and IL-10 at the local and systemic level. The therapy also led to remodeling of elastic lamina at the aneurysm site.

Conclusion:

Nanoparticles-loaded pentagalloyl glucose therapy can be an effective treatment option against advanced stage aneurysms to reverse the disease by ameliorating inflammation and restoring arterial homeostasis.

1. Introduction

An abdominal aortic aneurysm (AAA) is a multifactorial disease and vascular inflammation plays a significant role in its progression. It is the 13th leading cause of death in the elderly population, and only surgical interventions are available when AAA growth makes them vulnerable to rupture. The absence of candidate drugs for the complete healing of aneurysms has prompted scientists to develop an efficient therapeutical approach. Since vascular inflammation is one of AAA’s primary pathophysiological conditions, interventions in the activated inflam matory pathways and proteolytic cascades in already developed aneurysms are considered reasonable strategies for the management of aneurysms (Cifani et al., 2015). Expanded vessel diameter, mural neovascularization, chronic transmural inflammation, and regional wall weakening are the common characteristic of AAAs due to the breakdown of extracellular matrix proteins such as elastin and collagen (Abdul-Hussien et al., 2007). Concomitantly, infiltration of T cells, B cells, monocytes, macrophages, mast cells, and Natural Killer cells (NK cells) occurs (Abdul-Hussien et al., 2007) (Shimizu et al., 2006), with macrophages and granulocyte macrophage colony-stimulating factor (GM-CSF) being required for the development and rupture of AAA (Son et al., 2015). Intraluminal thrombus formation plays a prime role in transluminal inflammation in AAA.

Localized hypoxia occurs in the thicker layer of intraluminal thrombus in AAA, leading to increased mural neovascularization and inflammation. Subsequently, macrophages and other immune cells infiltrate transmural lesions from all sides of the vessel wall (Vorp et al., 2001). As a result, inflammatory mediators, including cytokines, chemokines, reactive oxygen species (ROS), growth factors, and immunoglobulins are released (Brophy et al., 1991), (Shimizu et al., 2006). These elements activate macrophages with the release of MMPs related to the acute progression of pathology of AAA. MMPs such as MMP-2, -9, and -12, degrade elastin and collagen, the major components of ECM in the vasculature (Longo et al., 2002). Among the MMPs, MMP-12 is a unique marker for aortic aneurysm disease (Longo et al., 2005).

Immunosuppressive drugs Cyclosporin A and Azathioprine effectively inhibit MMPs and IFN-γ through CD8 and CD4 modulation in AAA patients (Yamaguchi et al., 2000), (Marinkovic et al., 2013). We previously demonstrated that targeting damaged elastin with an elastin antibody-conjugated nanoparticle loaded with PGG (PGG-NPs) in an elastase-induced mouse model delivered sustained release of PGG at the AAA site (Nosoudi et al., 2016) (Dhital and Vyavahare, 2020). This reversed aneurysms by suppressing local MMPs and inflammation and restored vascular integrity by regenerating elastic fibers (Dhital and Vyavahare, 2020). We have also shown that PGG hinders aneurysms by binding proline-rich hydrophobic regions of arterial elastin and collagen and thus protecting MMP-mediated extracellular matrix (ECM) degradation in the rat (Isenburg et al., 2007). Others have shown that intraluminal PGG infusion resulted in elastin fiber restoration resulting in the prevention of AAA development in the elastase-induced rat model of AAA (Schack et al., 2020). Tea polyphenol, epigallocatechin gallate (EGCG) regenerates elastin, inhibits MMPs and inflammatory mediators in calcium chloride plus elastase-infused rat AAA model (Setozaki et al., 2017). Cytokine-stimulated expression of proatherogenic C-reactive protein in macrophages suppresses LPS-induced pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways in bone marrow-derived macrophages (Joo et al., 2012), and a free-radical scavenger, Edaravone alleviates oxidative stress to repair the aneurysm by suppressing oxidative DNA damage in rat (Morimoto et al., 2012).

To further elucidate how PGG nanoparticle therapy reverses AAA disease, we studied its role in the inflammatory cascade. Herein, we demonstrate that the nanoparticle-based PGG-NPs therapy suppresses the percentage of local and systemic macrophages, CD8⁺ and CD4⁺ subsets of T-cells, and the inflammatory cytokines IFN-γ and IL-6, as well as MMPs. We also show robust induction of local and systemic IL-10. It regulates T-cell and macrophage function and promotes CD8⁺ T cell activation, thereby restoring vascular cell homeostasis (Adam et al., 2018) (Fig. 1). Our results provide a further understanding of the mechanisms of action associated with polyphenol therapy for AAA.

Fig. 1. Schematic drawing showing AAA’s pathology before and after PGG treatment.

The illustration of the representative AAA on PPE application results in the formation of the bulge, increased infiltration of immune cells, macrophages, and increased MMPs and inflammatory cytokines. After PGG treatment, the inflammatory pathology of AAA decreases with increased anti-inflammatory and remodeling cytokines leading to repair of the aneurysmal tissue.

2. Materials and methods

2.1. Preparation of pentagalloyl glucose (PGG) loaded bovine serum (BSA) nanoparticles (PGG-NPs)

PGG-NPs and Blank-NPs (BLNK) were prepared by our previous methods (Dhital and Vyavahare, 2020) (Wang et al., 2021) that describe the preparation of PGG loaded bovine serum albumin (BSA) nanoparticles and methods to conjugate with the elastin-specific antibody on the surface that recognizes degraded elastin at AAA site. Details are provided in Supplementary data.

2.2. Elastase-induced AAA in mice

AAA was induced in pathogen-free C57BL/6 background male mice obtained from The Jackson Laboratory (Bar Harbor, ME) as our previous method (Dhital and Vyavahare, 2020). Briefly, to induce AAA, the mouse was anesthetized with isoflurane, and porcine pancreatic elastase (PPE; Sigma-Aldrich Co., St. Louis, MO, 7.6 mg/mL) or phosphate-buffered saline (PBS) was applied peri-adventitially for 12 min. The aorta was rinsed with sterile PBS, and the fascial layers were closed with sutures placing abdominal specimens in the original order. After the surgery, mice were kept for two weeks to allow aneurysm development. Progression of the aneurysm was monitored via high-frequency ultrasound imaging (Vevo2100, VisualSonics, Toronto, Canada). Two weeks after the surgery, when aneurysms were fully developed, mice received two tail-vein injections of freshly prepared antibody conjugated PGG-NPs or Blank-NPs (BLNK) (10 mg/kg body weight) one week apart. Control animals did not receive any therapy (n-10 per group) and sham groups got no PPE treatment and no nanoparticle therapy. All mice were sacrificed at four weeks. The studies were carried out ethically with the protocol number AUP2018–020 which was approved from the Clemson University Institutional Animal Care and Use Committee (IACUC). Clemson University animal facilities are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC). Mice received ethical and humane care in compliance with NIH Public Law 99–158 (revised 2015). All efforts were made to minimize the number of animals used and prevent their suffering.

2.3. Immunohistochemical analysis of aorta sections

Paraffin-embedded and formalin-fixed 5-μm aortic sections were subjected to heat-induced antigen retrieval with citrate buffer, pH6 (Thermo Scientific, MA). The slides were incubated overnight at 4 °C with mouse-specific primary antibodies for anti - CD163 cat # PA5–78961 Invitrogen, MMP-12 Cat# 50–173-3976 ProteinTech, and IFN-γ Cat# NBP2–66900 Novus Biologics. The sections were incubated with relevant secondary antibodies (Goat anti- Mouse) Invitrogen. Immunohistochemistry (IHC) staining was completed with IHC kit (Enzo Life Sciences, NY). Slides were visualized by 3-Amino-9-ethylcarbazole (AEC) (Vector Laboratories, Burlingame, CA) chromogens followed by an appropriate counterstain.

2.4. Flow cytometry

Mice were anesthetized and blood was collected by cardiac puncture, followed by whole-body PBS perfusion to clear tissue blood. Single-cell suspensions were obtained from the aorta, spleen, and thymuses by mincing and meshing on 70 μm filters. Briefly, for aortic specimens, 2–3 mm pieces were incubated for 1 hr in 1mL digestion media prepared in DMEM medium as a mixture of PPE, Collagenase D, and DNAse I and washed with PBS to stop the enzymatic reaction before mincing as described previously. Immunofluorescence staining was performed as previously described (Dhital et al., 2017) using mouse-specific antibodies. Briefly, cells were incubated with Fc block solution (purified anti-mouse CD16/CD32, clone 2.4G2, cat # 553142 BD Biosciences) for 15 min at room temperature to prevent nonspecific binding. Cells were washed then labeled to determine percentages of macrophages/myeloid-derived dendritic cells (CD68⁺), T-helper cells (CD4⁺), cytotoxic T-cells (CD8⁺), or intracellular expression of Transforming Growth Factor-beta (TGF-β1). The following antibodies were used as directed by the manufacturers (APC-rat anti-mouse CD68, clone FA-11, cat# 137007 eBioscience; PE-rat anti-mouse CD4, clone RM4.5, cat # 14–0042-82 Biolegend; PECy™7-rat anti-mouse CD8, clone 53–6.7, cat # 552877 BD Biosciences; FITC-rat anti-mouse TGF- β1, clone TW7–16B4, cat # 141404 Biolegend; PE-rat anti-mouse TGF- β1, clone TW7–20B9, cat # 141305 Biolegend). For intracellular expression of TGF-β1, cells were permeabilized with fix/permeabilization buffer (eBiosciences) for 15 min before incubation with the antibodies. Cytometry was performed on an LSR II CytoFlex (Becton Dickinson). Data analysis and gating were performed (Nichols et al., 2020) using FlowJo (TreeStar, Ashland, OR) software. Gating was performed using fluorescence minus one (FMO) control for each fluorochrome. As for the immune cells (CD8 and CD4), macrophages (CD68), and TGF- β1, the lymphocytes were selected using FSC and SSC gating. The singlets were selected, and then droplets were eliminated based on SSC-A/H and FSC-A/H characteristics. Then CD8⁺, CD4⁺, CD68⁺, and TGF-β1⁺ cells were selected by expression of CD8, CD4, CD68, and TGF- β1.

The significance was determined as * p < 0.05 as compared to the control with no nanoparticle therapy. BLNK groups received PPE treatment and two times intravenous Blank-NPs therapy like PGG-NPs therapy. Data are representative of triplicate experiments.

2.5. Enzyme-linked immunosorbent assay (ELISA)

Blood was collected via a heart puncture and allowed to clot for 30 min. The serum was collected by centrifuging at 14,000 rpm for 10 min at 4 °C. The tissue lysates were prepared using ProcartaPlex tissue lysis buffer {EPX-99999-000 (Invitrogen, Carlsbad, CA) according to the protocol. Cytokine amounts were quantified from the mouse serum and tissue lysates using customized standard curves for anti-mouse IFN-γ, IL-6, IL-10, IL-13, and IL-27 of ProcartaPlex Immunoassay Kit EPX17026087901(Invitrogen, Carlsbad, CA) measured by using MAGPiX luminescence technology according to the protocol. The kit consists of capture antibody, detection antibody with the known magnetic bead region area and Net Mean Fluorescence Intensity (NMFI) for standards. The significance was determined as * p < 0.05 as compared to the control with no nanoparticle therapy. Data are representative of triplicate results least three independent experiments.

2.6. Histological analysis of spleen

Six micrometer frozen optimal cutting temperature (OCT) compound sections were mounted on positively charged glass slides and placed in 100% pre-cooled acetone (Fisher Science Education, Nazareth, PA) for 10 minutes to fix the sections. Subsequently, the slides were rinsed with tap water for 5 minutes to remove the OCT compound and stained with 15% FeCl₃ solution in DI water for 5 minutes to detect PGG content under microscope (n = 3 in each group).

2.7. Statistical analysis

Statistical analysis was conducted using GraphPad Prism 8 (GraphPad Software, San Diego, CA). Comparisons were performed by one-way or two-way Analysis of Variance (ANOVA) as appropriate. A posthoc test for multiple comparisons was performed. The significance was determined as p < 0.05.

3. Results

We characterized PGG-NPs conjugated with elastin antibody that recognizes degraded elastin while sparing healthy elastic lamina. Size distribution and surface charge before and after the conjugation process were analyzed by a particle size analyzer. Antibody conjugated, PGG-NPs had an average size of 240.6 ± 18.2 nm and a Zeta potential of −15.3 ± 6.3 mV. The size of NPs was confirmed by Scanning Electron Microscopy (SEM). The detail morphology, size distribution and zeta potential of nanoparticles are shown in Supplementary Fig. 1. We previously reported PGG loading and controlled release from albumin nanoparticles over a period of 4 weeks (Sinha et al., 2014b). We have shown that such nanoparticles reverse already developed aneurysms in mice (Dhital and Vyavahare, 2020) (Supplementary Fig. 2).

3.1. PGG-NP therapy suppresses monocytes and macrophages

To see the influence of PGG-NP therapy on macrophages at the local and systemic level, we evaluated the presence of monocyte and macrophage markers CD163 and CD68. We found heavy staining of CD163 in the lamina of aneurysmal aortic sections treated elastase alone. It was significantly reduced in aortic sections in PGG-NPs group (Fig. 2A). Subsequently, we assessed the percentage of (macrophages/myeloid-derived dendritic cells) CD68⁺ in the aorta, spleen, and thymuses by immunofluorescence staining. We observed that the percentage of CD68⁺ cells was robustly suppressed in the aorta and spleen in PGG-NPs treated groups compared to that from the aneurysmal control. However, PGG-NPs treatment did not alter the percentage of CD68⁺ cells in the thymuses (Fig. 2B). We previously reported that intravenous injection PGG-NPs in elastase-induced AAA mice suppressed the infiltration of CD68⁺ and Mac-2⁺ cells in the aortic lamina in comparison to that of the control (Dhital and Vyavahare, 2020). The results presented here indicate that PGG suppresses macrophage recruitment in the aneurysmal aortic site and at the systemic level.

Fig. 2. PGG treatment suppresses the CD163 and CD68 macrophages.

A. IHC results for CD 63; aortic sections were stained for CD163. PGG-NPs treatment sharply decreased the staining for CD163 macrophages in the lamina of aortic sections derived from PGG-NPs-treated mice groups in comparison to that derived from the untreated aneurysmal control (20X and 100X magnification). B. Dot plots and scatter plots (mean ± SEM) measured by flow cytometry showing the percentage of CD68 positive cells were significantly suppressed in aorta and spleen derived from PGG-NPs-treated mouse groups (PGG-NPs; n = 9), in comparison to that from the control (Control; n = 9), in contrast, the percentage of CD68 positive cells in thymuses was unaltered. Data represent triplicate results from three experiments. (*p < 0.05 compared to the control).

3.2. PGG-NP therapy down-regulates CD8 and CD4 positive T cells

CD68⁺ macrophages are known to play a key role in the pathophysiology of AAA (Boytard et al., 2013) (Wang et al., 2021) and they can affect T-cell function in pathophysiology of aneurysm (Miyata et al., 2017). Therefore, to assess the effect of PGG treatment, we measured the percentage of CD8⁺ and CD4⁺ cells in the aorta, spleen, and thymuses by immunofluorescence staining. The percentage of CD8⁺ and CD4⁺ cells were suppressed in the aorta and spleen in PGG-NPs treated-group. However, the percentage of CD8⁺ cells was not altered in the thymuses compared to the aneurysmal controls (Fig. 3A), while CD4⁺ cells were not altered in the spleen and thymuses compared to that from the control (Fig. 3B). Importantly, blank nanoparticles (BLNK) alone as a carrier did not reduce the CD8 and CD4 positive cells (Fig. 3). These results implicate that PGG-NPs treatment suppresses the number of macrophages and CD8⁺ and CD4⁺ T cells in AAA.

Fig. 3. PGG treatment robustly suppresses the percentage of CD8⁺ and CD4⁺ cells.

Mouse cells from aorta, spleen and thymuses derived from PGG-NPs-treated (PGG-NPs; n = 9), Blank-NPs-treated (BLNK-NPs; n = 9), and the untreated controls (Controls; n = 9), were stained for CD8 and CD4. Dot plots and scatter plots (mean ± SEM) of percentages of CD8⁺ and CD4⁺ cells measured by flow cytometry. A. The percentages of CD8⁺ cells were significantly suppressed in aorta and spleen in PGG group. B. The percentages of CD4⁺ cells were modestly decreased in aorta in comparison to control in PGG group. Data are representative of triplicate results from four experiments.

3.3. Suppression of metalloproteinases (MMPs) by PGG-NP therapy

Macrophage-produced MMPs are responsible for ECM degradation during the progression of AAA (Longo et al., 2002, 2005). MMP-12, also known as macrophage metalloelastase (MME), released by proinflammatory macrophages is found early in the AAA formation (Yamada et al., 2008). To test the hypothesis, whether PGG inhibits macrophage-specific MMP-12, we stained the aortic sections for MMP-12 expression by IHC. We discovered the heavy staining of MMP-12 present in aortic sections in the elastase treated group. It was significantly reduced in the lamina of aortic sections obtained from the PGG-NPs treated group (Fig. 4A). We have previously reported that PGG-NPs treatment against elastase-induced AAA mouse suppresses MMP-2 and MMP-9 in the aortic lamina (Dhital and Vyavahare, 2020). Together, our results advocate that PGG treatment suppresses macrophages and thus suppresses MMPs in elastase-induced AAA.

Fig. 4. Effect of PGG treatment on MMP-12 and IFN-γ.

Aortic sections were stained for MMP-12 and IFN-γ IHC. A. PGG treatment sharply decreased the staining for MMP-12 in aortic aneurysm (20X and 100X magnification). B. PGG treatment sharply decreased the staining for IFN-γ in aneurysmal aorta sections derived from PGG-NPs-treated groups in comparison to the aneurysmal controls (20X and 100X magnification).

3.4. PGG-NP treatment suppresses inflammatory cytokines IFN-γ and IL-6

Progression of AAA starts with increased macrophages in the adventitial layers of the AAA site, which secrete inflammatory cytokines such as IFN-γ and IL-6 (Zhou et al., 2013). These cytokines are produced in the inflammatory response during thrombus formation in AAA (Sagan et al., 2012). We assessed if PGG-NPs treatment suppresses IFN-γ and IL-6 in the local and systemic levels. Indeed, we observed suppressed staining for IFN-γ in the lamina of aortic sections in the PGG-NPs group compared to the aneurysmal controls (Fig. 4B). Moreover, we discovered that IFN-γ was robustly suppressed in the aorta and spleen in the PGG-NPs treated-group compared to that of the aneurysmal control, whereas the amount of IFN-γ was modestly suppressed in serum almost reaching to a sham group (Fig. 5A). IFN-γ was found mainly produced by CD8⁺ and CD4⁺ T cells. These T cells mediate the pathophysiology of AAAs during the early development of AAAs (Xiong et al., 2004), (Zhou et al., 2013).

Fig. 5. Suppression of inflammatory cytokines IFN-γ and IL-6 by PGG treatment.

Representative dot plots (with mean ± SEM of triplicate wells from three experiments) showing amount of IFN-γ and IL-6 in aorta, and spleens lysates, and serum derived from the PGG-NPs-treated (PGG-NPs; n = 9), and the PPE-treated (Control n = 9), and sham (no elastase) (Sham; n = 9), quantified by ELISA (*p < 0.05 compared to the control). A. IFN-γ was significantly higher in control aneurysmal aorta and spleen while PGG treatment decreased its levels back to sham group. B. Dot plots (mean ± SEM) showing IL-6 in aorta, and spleen lysates, and serum, quantified by ELISA. Significantly increased amount of IL-6 was detected in aorta and spleen lysates and serum of aneurysmal control group, while PGG treatment led to decrease of IL-6 similar to sham (no elastase) group. C. Illustrative demonstration showing PGG suppresses activated macrophages and T cells down regulating MMPs, IL-6, and IFN-γ expression.

Next, we quantified the amount of dominant inflammatory cytokine IL-6 in the aorta, spleen, and serum by ELISA. We focused on IL-6 since it is produced by interactions between leukocytes and fibroblasts in the aortic adventitia, potentiating the activation and recruitment of local monocyte enhancing chemokine secretion, vascular inflammation, ECM remodeling, and aortic weakening (Tieu et al., 2009). In AAA, hypertension and oxidative stress in the vascular wall stimulate the induction of an increased amount of IL-6 by vascular smooth muscle cells (VSMCs), expanding the lumen diameter (Akerman et al., 2018). Free-radical scavengers decrease oxidative stress to ameliorate aneurysms in the vessel wall (Morimoto et al., 2012). In our study, IL-6 was significantly suppressed in aorta, spleen lysates and serum obtained from PGG-NPs-treated-group in comparison to that of the aneurysmal controls and returned to similar levels to that of the sham (no elastase) group (Fig. 5B). Since activated macrophages produce IL-6, these results imply that the PGG suppression of IL-6 is correlated with the suppression of macrophages (Dhital et al., 2011). Another prospect for decreased amount of IFN-γ and IL-6 production is correlated with the reduced production of CD8⁺ and CD4⁺ T cells in the AAA model (Sagan et al., 2012, 2019; Zhou et al., 2013) (Fig. 5C).

3.5. Effect of PGG-NP treatment on TGF-β1

TGF-β1 plays a pivotal role in the pathogenesis and aortic homeostasis in AAA, and activated macrophages and monocytes produce TGF-β1 in progressive aneurysms. To this point, we previously demonstrated that PGG moderately suppressed TGF-β1 in the lamina in AAA (Dhital and Vyavahare, 2020). Other reports have shown that TGF-β1 supplementation aggravates aneurysms (Lareyre et al., 2017). Thus, we measured the percentage of TGF-β1 expressing cells in the aorta, spleen, and thymuses by flow cytometry. Herein we found that the expression of TGF-β1 was moderately modulated in the aorta in PGG-NPs treated group while remaining unaltered in the spleen and thymuses compared to the aneurysmal control group (Fig. 6).

Fig. 6. Influence of PGG therapy on TGF-β1 expression.

Dot plots and scatter plots (mean ± SEM) measured by flow cytometry showing percentages of TGF-β1. Mouse cells from aorta, spleen and thymuses derived from PGG-NPs-treated (PGG-NPs; n = 9), and the controls (Control n = 9), were fixed and stained for TGF-β1. The percentage of TGF-β1 in aorta was modestly modulated in PGG-treated-groups in comparison to the aneurysmal controls.

3.6. PGG-NP treatment induces anti-inflammatory cytokines IL-10, IL-27, and IL-13 production to ameliorate inflammation

Since PGG suppresses macrophages, MMPs, and inflammatory cytokines, we hypothesized that PGG would exert its effect on the induction of anti-inflammatory cytokines IL-10, IL-13, and IL-27. Notably, IL-10 is often diminished in mouse and human aneurysm patients (Wang et al., 2018). In our assessments, PGG-NPs treatment induced a significantly higher amount of IL-10 in the aorta spleen and serum in comparison to the aneurysmal control groups (Fig. 7A). Furthermore, an increased amount of IL-27 was detected in the spleen and serum in PGG-NPs-treated-group in comparison to the control (Fig. 7B). Also, a significantly increased amount of IL-13 was detected in serum derived from PGG-NPs-treated-groups compared to the controls, whereas the amounts of IL-13 in aorta and spleen lysates were unaltered (Fig. 8A). Studies demonstrate that IL-27 regulates macrophages activation, stimulates the production of IL-10, and eventually controls the development of thrombosis (Hirase et al., 2013). Moreover, IL-13 is correlated with suppression of MMP-12 production and stimulation of the induction of IL-10 that regulates inflammation through Th2 driven signaling (Madala et al., 2010) (Kothari et al., 2014). Our study suggests that the anti-inflammatory effect of PGG leads to the expression of growth factors such as TGF-β1 and increases the production of IL-27 and IL-13. The increased IL-27 and IL-13 observed in our study likely stimulate the production of dominant anti-inflammatory cytokine IL-10 regulating inflammation in the elastase-induced AAA model (Fig. 8B).

Fig. 7. PGG treatment induces anti-inflammatory cytokines IL-10 and IL-27.

Representative dot plots (mean ± SEM) of triplicate wells from three experiments, showing IL-27 and IL-10 in aorta and spleen lysates, and serum, quantified by ELISA in PGG treated (PGG-NPs; n = 9), control (PPE alone) (Control n = 9) and sham (no elastase) groups (Sham; n = 9). *p < 0.05 compared to the control. A. Significantly increased amount of IL-10 in aorta, spleen and serum. B IL-27 was significantly increased in aorta and spleen lysates derived from PGG-NPs-treated mouse groups in comparison to that from the aneurysmal control group.

Fig. 8. PGG treatment induces IL-13 in spleen and serum.

A. Representative dot plots (with ±SEM) showing IL-13 in aorta, and spleen lysates, and serum were quantified by ELISA. Significantly increased amount of IL-13 was detected in serum derived from PGG-NPs treated mouse groups (PGG-NPs; n = 9) in comparison to the controls (Control n = 9) (Sham; n = 9). Whereas the amount of IL-13 in aorta was experimentally elevated in PGG-NPs-treated group in comparison to controls. B. Schematic illustration to show PGG regulates activated macrophages, neutrophils and T cells during AAA inflammation. IL-27 and IL-13 are produced by the influence of milieu of growth factors, interleukins, cytokines and other stimulants. IL-27 and IL-13 stimulates the induction of IL-10 regulating inflammation. Data represent triplicate results repeated thrice (*p < 0.05).

3.7. Detection of PGG in spleen

As we have seen systemic effects of PGG-NP therapy, we wanted to query if nanoparticles are going to the spleen and delivering PGG there as well. It is well-known for most targeted therapies that part of the dose is taken up by the reticuloendothelial system (RES) (Mangarova et al., 2020). To test this, we assessed the presence of PGG in spleen sections derived from the PGG-NPs treated and the controls. We observed brown staining for PGG in spleen sections one week later of two weekly injections of PGG-NPs injection (Fig. 9) while the brown staining was absent in the control spleen. Thus, part of the systemic effect that we see for the PGG-NPs group may be due to the release of PGG in the spleen.

Fig. 9. Representative histology of FeCl₃ staining for detection of PGG in spleen.

Clearly brown staining for PGG is detected in spleen sections derived from PGG-NPs-treated mouse group (a 100X and b 40X magnification), whereas the staining for PGG is not visible in spleen sections derived from the PPE-treated control group (c 100X and d 40X magnification). Spleens from PGG treated and control groups were harvested after one week of PGG-NPs (second dose of PGG-NPs) injection were stained with FeCl₃ to detect PGG.

4. Discussion

Inflammation in the aneurysmal aorta is primed by the adaptive and innate immune systems (Koch et al., 1990) (Yamaguchi et al., 2000), involving recruitment of macrophages, T cells and increase in inflammatory cytokines and MMPs. We have previously shown that nanoparticle-based PPG therapy reverses aneurysms in an elastase-induced aggressive AAA model. This study focused on understanding the effect of this therapy on inflammatory markers in AAA. We focused primarily on macrophages and lymphocytes since they are the key cell types involved in inflammation. We demonstrate that PGG treatment down-regulates macrophages, CD8⁺ and CD4⁺ subsets of T cells, and inflammatory cytokines IFN-γ and IL-6, as well as MMPs. We further demonstrate that PGG treatment increases anti-inflammatory cytokines IL-10, IL-27 and IL-13. Thus, PGG therapy modulates both local and systemic inflammatory responses.

4.1. Suppression of monocytes and macrophages by PGG

Macrophages are one of the key regulators of AAA pathophysiology, and their depletion suppresses aneurysms (Davis et al., 2021). Our studies demonstrate that PGG suppresses recruitment of CD163⁺, CD68⁺ and Mac-2⁺ cells at the aneurysmal site and at the systemic level (Dhital and Vyavahare, 2020). We assessed monocytes and macrophage recruitment because the proteins or microRNAs released by macrophages are found to be contributing factors for augmenting pathology and oxidative stress and increasing rupture in AAA (Boytard et al., 2013) (Ohno-Urabe et al., 2018). Thus, local PGG release by nanoparticles affected macrophage activities. We also observed a reduction of macrophages in the spleen but not in the thymuses. When we stained for PGG, the spleen did show PGG staining suggesting that part of the dose of nanoparticles went to the spleen, which could have caused the systemic effect as we have previously shown systemic PGG could be anti-inflammatory (Wang et al., 2021).

4.2. Suppression of CD8⁺ and CD4⁺ T-cell subsets by PGG

Immune cell infiltration in the aneurysmal site is one of the pathological conditions of AAA disease (Lareyre et al., 2017). Among the lymphocytes, CD8⁺ and CD4⁺ are the dominant subtypes of T cells linked in the pathophysiology of AAA (Teo et al., 2018), (Yamaguchi et al., 2000). In human and mouse aneurysm studies, CD4⁺ T cell subset was predominantly found in aneurysm lesions (Teo et al., 2018), (Koch et al., 1990), (Yamaguchi et al., 2000). After administering the T cell function inhibitor and immunosuppressive agent Cyclosporin A, the pathogenesis of an intracranial aneurysm was ameliorated in a rat model (Miyata et al., 2017). Another immunosuppressive drug Azathioprine decreased aortic aneurysm formation through its suppressive effects on leukocyte and endothelial cells (Marinkovic et al., 2013). Kaempferol, a plant polyphenol, inhibits the development and expansion of type 1 effector CD8⁺ T cells in Graft-versus-host disease models (Okamoto et al., 2002). Indeed, PGG treatment suppressed both CD8⁺ and CD4⁺ percentages and reduced immune response at the AAA site. These CD8⁺ and CD4⁺ subsets of T cells are reported to play key roles in the production of IFN-γ, which stimulates MMPs release in AAA (Xiong et al., 2004).

4.3. Suppression of MMPs and inflammatory cytokines by PGG

MMPs are produced by activated VSMCs through the stimulation of macrophage-generated proteins such as netrin-1. MMPs degrade ECM in elastase-induced AAA (Hadi et al., 2018), and we have previously shown that PGG treatment suppresses MMP-9 and MMP-2 in the aorta (Dhital and Vyavahare, 2020). Here we show the suppression of macrophages and MMP-12, which is directly correlated with inflammation, and IL-6 production correlates with the severity of inflammation (Madala et al., 2010). IL-6 stimulates the release of MMPs by macrophages and SMCs, which are involved in elastin and collagen degradation in AAA (Kothari et al., 2014) (Longo et al., 2002, 2005) (Sugimoto et al., 2018).

We have shown earlier that PGG binds to the proline-rich hydrophobic regions in collagen and elastin, preventing their breakdown by MMPs (Isenburg et al., 2007). Elastin peptides released by elastin degradation are chemotactic to inflammatory cells (Senior et al., 1980), and we have also shown recently that PGG can directly suppress MMP activity in cell culture (Parasaram et al., 2018). Thus, local PGG release might have caused suppression of MMPs by directly inhibiting its activity, reducing inflammation, and by binding to the collagen and elastin, and preventing their degradation and release of chemotactic elastin peptides.

Next, we evaluated PGG’s effect on the key inflammatory cytokines IFN-γ and IL-6. PGG treatment robustly suppressed IFN-γ and IL-6 in both aorta and spleen, which correlates with the suppression of macrophages and CD8⁺ and CD4⁺ T cells. In the early stage of AAA progression, IFN-γ producing CD8⁺ and CD4⁺ cells are extensively present at the site (Zhou et al., 2013). IFN-γ, produced both by CD4⁺ and CD8⁺ cells, has been found to play a key role in aneurysm formation and stimulating MMP-9 and MMP-2 production both in vivo and in vitro (Xiong et al., 2004), (Longo et al., 2002), (Zhou et al., 2013). In support of our observation, another plant flavonoid Kaempferol was shown to suppress IFN-γ along with CD8⁺ T cells in a dose-dependent manner (Okamoto et al., 2002).

Others have shown that polyphenols inhibit macrophages through multiple key regulators of inflammation suppressing inflammatory cytokines such as IFN-γ and IL-6 (Hussain et al., 2016) (Yahfoufi et al., 2018). The IL-6, produced by activated macrophages (Dhital et al., 2011), is associated with the aortic size expansion and aortic dissection in mice and human AAA (Tieu et al., 2009) (Ohno-Urabe et al., 2018), and neutralizing IL-6 decreases aneurysm disease (Nishihara et al., 2017). In our study, robustly suppressed levels of IL-6 were found in the aorta and spleen, implying that one of the mechanisms of PGG induced suppression of inflammation might be through suppression of IL-6. This might be due to the reduction of macrophages cells and their mediator byproducts. Others have shown that grape seed polyphenol inhibits inflammatory IL-6 messenger Ribonucleic Acid (mRNA) expression in vitro in Tumor Necrosis Factor (TNF)-α stimulated mouse VSMCs (Wang et al., 2017).

4.4. PGG’s effect on TGF-β1 production

TGF-β1 is a multifunctional cytokine that is required for tissue morphogenesis and homeostasis. In AAA, latent TGF-β1 binds to its receptor and signals through Smad2/3 dependent or independent pathways to activate genes that maintain the ECM homeostasis and morphogenesis. Reports have shown that TGF-β1 supplementation alleviates already developed aneurysms, but the inactivation of TGF-β1 before the initiation of the aneurysm in elastase-induced AAA led to exacerbate the disease. In AAA, activated macrophages regulate the release of TGF-β1, and the amount and activation of TGF-β1 depend on pathological circumstances (Lareyre et al., 2017). We observed modest modulation of TGF-β1 in the aorta but unaltered in spleen and thymuses in PGG-NPs-treated mouse groups as compared to the control group. Thus, local TGF-β1 suppression may be partly due to local reduction in macrophages.

4.5. PGG induced anti-inflammatory cytokines IL-13, IL-27, and IL-10 that play a critical role against inflammation

In an elastase-induced AAA model, we found the local release of PGG significantly elevated anti-inflammatory cytokines IL-10, IL-27 and IL-13 compared to the controls. IL-10 maintains immune homeostasis, regulates T cell proliferation, and cytokine secretion in AAA, and its amount is less in the AAA disease (Wang et al., 2018). Notably, systemic upregulation of IL-10 using gene therapy has ameliorated aneurysms (Adam et al., 2018). Mechanistically, just how PGG modulates IL-10 modulates IL-10 is multifaceted. For one, IL-10 down-regulates pro-inflammatory cytokine IL-6 production and MMP-9 release by activated macrophages (Kothari et al., 2014). Predominantly, IL-10 up-regulates anti-inflammatory cytokines and limits cytokine receptors and their activation. Furthermore, IL-10 regulates T-cell and macrophage function by polarizing towards an M2 phenotype along the spectrum of M1 vs. M2. It also promotes CD8⁺ T cell activation, as well as epithelial cell repair (Adam et al., 2018). Moreover, IL-10 inhibits IFN-γ secretion by suppression of activated macrophages and related elements (Shimizu et al., 2006).

IL- 27 was found to decrease the activation of macrophages and was found to stimulate the production of anti-inflammatory cytokine IL-10 in an atherosclerosis model (Hirase et al., 2013). IL-13, which is an anti-inflammatory Th2 type cytokine, further stimulates the production of IL-10. IL-10 and IL-13 coordinately suppress inflammation through Th2 driven anti-inflammation signaling (Wilson et al., 2007). Summarizing these properties of anti-inflammatory cytokines, we hypothesize increased IL-13 and IL-27 production could have led to induction of the production of IL-10 by PGG therapy. This then suppressed inflammation and thus inhibited AAA progression. Analogous to our study, a polyphenol derived from Quince was shown to induce IL-10 and suppress LPS-induced IL-6 in human macrophages (Essafi-Benkhadir et al., 2012).

4.6. Elastin repair by PGG treatment

PGG not only suppressed inflammatory conditions but also inhibited MMPs leading to first stabilizing elastin. IL-6 produced by fibroblasts stimulates the degradation of elastin (Sugimoto et al., 2018), and PGG’s suppression of IL-6 may contribute to the inhibition of degradation. It is known that aneurysm disease leads to increased local tropoelastin secretion by fibroblasts and vascular smooth muscle cells (Sinha et al., 2014a). However, these tropoelastin molecules do not assemble to create functional elastin fibers and are washed out. AAA patients show higher elastin degradation products and tropoelastin in their blood and urine (Mordi et al., 2019) (Osakabe et al., 1999). We have previously shown that PGG bound to elastic fibers at the AAA site also binds to tropoelastin molecules secreted by local VSM cells, thus allowing accumulation of tropoelastin at the site of AAA (Parasaram et al., 2018). Once accumulated, the lysyl oxidase (LOX) enzyme crosslinks these tropoelastin molecules to create fibers. We clearly see the evidence of restoration of wavy elastin fibers after PGG therapy (Supplementary Fig. 2C). Moreover, we have shown that VSMCs also return to their contractile phenotype when AAA is reversed (Sinha et al., 2014a). Thus, such nanoparticle-based therapy not only reverses inflammation, AAA dilation but allows a return of homeostasis of the aorta.

5. Conclusion

We show that systemic delivery of nanoparticles targeting degraded elastin delivers PGG to the AAA site and produces the stimulatory cytokines IL-27 and IL-13. These stimulatory cytokines robustly induced the local and systemic induction of dominant anti-inflammatory cytokine IL-10, ameliorating inflammation. Moreover, PGG delivery suppressed monocytes and macrophages and the major inflammatory cytokine IL-6 at the local and systemic levels. Furthermore, PGG-NPs treatment down-regulated IFN-γ, MMP-12, and subsets of CD8 and CD4 T cells percentages in local and systemic levels. Such systemic targeted therapy could be utilized to reverse already developed aneurysms in patients if it can be translated to a clinical situation.

Abbreviations:

AAA

Abdominal aortic aneurysm

BLNK

Blank nanoparticles

BSA

Bovine serum albumin

PGG-NPs

Elastin antibody-conjugated and PGG-loaded albumin NPs

ECM

Extra Cellular Matrix

EGCG

Epigallocatechin Gallate

ELISA

Enzyme-linked immunosorbent assay

FACS

Fluorescence-activated cell sorting

GM-CSF

granulocyte macrophage colony-stimulating factor

IFN-γ

Interferon gamma

IHC

Immunohistochemistry

IL

Interleukin

LOX

Lysyl oxidase

MAPK

Mitogen-activated protein kinase

MMP

Matrix metalloproteinase

mRNA

messenger Ribonucleic acid

NFκB

nuclear factor kappa-light-chain-enhancer of activated B cells

NK cells

Natural Killer cells

OCT

Optimal cutting temperature compound

PBS

Phosphate-buffered saline

PGG

Pentagalloyl glucose

PPE

Porcine Pancreatic Elastase

ROS

Reactive Oxygen Species

TGF-β

Transforming Growth Factor-beta

TNF

Tumor Necrosis Factor

VSMC

Vascular Smooth muscle Cells