Sparking angiogenesis by carbon monoxide-rich gold nanoparticles obtained by pulsed laser driven CO2 reduction reaction

Abstract

Endothelial tissue regeneration is a major challenge in the context of vascular disorders and tissue repair. One of the most recent and promising therapies for endothelial tissue disorders is the administration of carbon monoxide (CO) by direct injection or release by CO-releasing molecules (CORMs). Despite the great potential of CORMs, light instability and cytotoxicity associated with the heavy metal core are still major drawbacks that inhibit clinical application. Recently, we have shown the possibility to synthesize carbon monoxide rich gold nanoparticles (CO-rich AuNPs) by the pulsed laser driven CO₂ reduction reaction in water. In this work, we investigate the potential of this unique metal-organic complex as a therapeutic approach to promote endothelial tissue regeneration, by performing a comparative analysis between the CO releasing potential of CO-rich AuNPs and a well-known CO-releasing molecule, specifically CORM-2. Through a combination of in vitro and in vivo experiments, we elucidated the mechanisms by which the laser synthesized ligand-free or CO-rich AuNPs inhibit or enhance, respectively, the endothelial cell migration and angiogenesis. Notably, the biocompatible CO-rich AuNPs not only boost these cellular processes but also play a pivotal role in triggering histone acetylation, shedding light on an additional regulatory pathway.

Keywords: Capillary morphogenesis; Carbon monoxide; Endothelial colony forming cells; Gold nanoparticles; Histones acetylation; matrigel sponges; angiogenesis; Pulsed laser ablation in water.

Fig. 1

Transmission electron microscopy images of the different synthesized nanomaterials: (A) AuNPs_air; (B) AuNPs_CO2; (C) AuNPs_arg. The insets show the experimental statistical size (radius) log-normal distribution. (D) SERS spectra of the AuNPs synthesized by PLAL at different gas-water interfaces, and of the citrate-stabilized AuNP (ch, red line). Only the spectral region of interest between 1800 cm⁻¹ and 2700 cm⁻¹ is shown for convenience. In yellow is indicated the region corresponding to the gold-carbonyl vibration. The baseline of each spectra was artificially shifted for a better visualization

Fig. 2

DLS investigation on the stability of the colloidal dispersion of AuNPs_air and AuNPs_arg in cell culture media. (A) Hydrodynamic diameter distributions of AuNPs_air in PBS solution (black solid line) and AuNPs_air dispersed in EGM-2 culture medium supplemented with 10% FBS measured immediately (blue solid line) and after 2 h (blue dotted line). (B) Hydrodynamic diameter distributions of AuNPs_arg (gray solid line) and AuNPs_arg in the presence of EGM-2 culture medium supplemented with 10% FBS measured immediately (red solid line) and after 2 h (red dotted line). In all tests, AuNPs_air and AuNPs_arg contained 1 mg/mL of PF127. The distributions represent the average of two measurements

Fig. 3

Quantification of CoHB released by K562 treated with CORM-2 or AuNPs_air

Fig. 4

(A) Optical microscopy images of ECFCs treated with the vehicle or with AuNPs_air; (B) ECFC viability performed by trypan blue assay. (C) Transmission Electronic Microscopy Images of AuNPs_air; loaded ECFCS; (D) Capillary morphogenesis of ECFCs treated with the vehicle or AuNPs_sir;. The capillary network was quantified by Angiogenesis Analyzer Image J tool. Histograms represent the mean number of number of nodes, junctions, master junctions, meshes, segments, master segment, total master and segment length, total mesh area and mesh size respectively. Representative microphotographs (x4 and x20) of capillary-like structures at 24 h are shown. Data are representative of measures obtained from at least nine fields. (E) Histograms show the permeability activity of ECFCs expressed as % of FITC-BSA cleared across the filter with respect to Vehicle-treated ECFC. (F) NO concentration in CM of vehicle- or AuNPs_air-treated ECFCs. (G) Western blotting of total and GTP-loaded forms of small Rho-GTPase RhoA in control conditions and after ECFC treatment with AuNPs_air. RhoA-GTP, GTP-loaded form of small Rho GTP-ase; RhoA, total un-loaded form of small Rho GTP-ase, used as a reference loading control. Histograms report band densitometry. Results are the mean of 3 different experiments performed in duplicate. (H) Real-time qPCR of angiogenic molecules such as VEGF, uPAR and IL8 in ECFCs treated with the vehicle or AuNPs_air- Error bars: mean ± SD; *p < 0.05 indicates significant difference from each vehicle

Fig. 5

(A) Capillary morphogenesis of ECFCs treated with the vehicle, VEGF-A or AuNPs_air. The capillary network was quantified by Angiogenesis Analyzer Image J tool. Histograms represent the mean number of number of nodes, junctions, master junctions, meshes, segments, master segments. Representative microphotographs (x4) of capillary-like structures at 24 h are shown. (B) ELISA of IL-8, released in CM of ECFCs treated with the vehicle, VEGF-A or AuNPs_air respectively. (C) Western blot analysis of important angiogenic cues (KDR, HIF1α, bFGF) in ECFCs treated with the vehicle, VEGF-A or AuNPs_air. GAPDH is reported as loading control. Histograms report band densitometry. Results are the mean of 3 different experiments performed in duplicate. Error bars: mean ± SD; *p < 0.05 indicates significant difference from each vehicle

Fig. 6

(A) Capillary morphogenesis of ECFCs treated with the vehicle, AuNPs_air or AuNPs_ch. The capillary network was quantified by Angiogenesis Analyzer Image J tool. Histograms represent the mean number of number of nodes, junctions, master junctions, meshes, segments, master segment respectively. Representative microphotographs (x4 and x10) of capillary-like structures at 24 h are shown. Data are representative of measures obtained from at least nine felds. (B) Histograms refer to quantification of Matrigel invasion assay obtained by counting the total number of migrated cells/filter. (C) Western blot analysis in ECFCs treated with the vehicle, AuNPs_air or AuNPs_ch. Histograms report band densitometry. Results are the mean of 3 different experiments performed in duplicate. Error bars: mean ± SD; Asterisks, significant difference (*, P < 0.05; ***p < 0.0001) of AuNPs_air samples from vehicle. Number signs, significant difference of AuNPs_ch (#, P < 0.05) from vehicle

Fig. 7

(A) Optical images of ECFCs treated with AuNPs_air in presence or absence of kinase inhibitors: L-name, Rapamycin and Wortmannin. (B) Capillary morphogenesis of ECFCs treated with the vehicle or AuNPs_air in presence of the kinase inhibitors. Histograms represent the mean number of segments, nodes, junctions and meshes respectively. Representative microphotographs (x4 and x10) of capillary-like structures at 24 h are shown. Data are representative of measures obtained from at least nine fields. (C) Western blot analysis of phosphorylated and total levels of eNOS, mTOR and AKT in ECFCs treated with AuNPs_air in presence or absence of kinase inhibitors. Histograms report band densitometry. GAPDH was also examined to ensure equal loading of samples in each lane. Results are the mean of 3 different experiments performed in duplicate. Error bars: mean ± SD; Asterisks, significant difference (*, P < 0.05) from vehicle

Fig. 8

A) Capillary morphogenesis of ECFCs treated with the vehicle, AuNPs_air or OMNCs. The capillary network was quantified by Angiogenesis Analyzer Image J tool. Histograms represent the mean number of nodes, junctions, master junctions, meshes, segments, master segments. Representative microphotographs (x4) of capillary-like structures at 6 h are shown. (B-D) Western blot analysis of acetylation levels in ECFCs treated with SAHA (B), or AuNPs_air or CORM-2 (C-D) respectively. Histograms report band densitometry. (E-F) Nitric oxide detection was assessed with DAF-FM DA probe: fluorescent microscopy images showing NO production after treatment with AuNPs_air or CORM-2 for 6 h. Quantification of NO production was performed with flow cytometry. (G) Calcium flux was evaluated with Fluo 4 AM. Cells were treated with AuNPs_air or CORM-2 together with the probe for 1 h. Results are the mean of 3 different experiments performed in duplicate. Error bars: mean ± SD; Asterisks, significant difference (*, P < 0.0; **p < 0.001) from vehicle

Fig. 9

(A) Optical microscopy images of ECFCs treated with the vehicle or with AuNPs_arg, AuNPs_air; or AuNPs_CO2. (B) ECFC viability performed by trypan blue assay. (C)ICP-AES analysis of AuNPs loaded ECFCs. (D) Capillary morphogenesis of ECFCs treated with the vehicle or with AuNPs_arg, AuNPs_air or AuNPs_CO2. The capillary network was quantified by Angiogenesis Analyzer Image J tool. Histograms represent the mean number of nodes, junctions, meshes and segments. Representative microphotographs (x4 and x20) of capillary-like structures at 24 h are shown. Data are representative of measures obtained from at least nine fields. (E) Optical images of migrated cells. Histograms refer to quantification of Matrigel invasion assay obtained by counting the total number of migrated cells/filter. (F)Representative photographs of individual Matrigel sponges recovered at autopsy for the corresponding condition (Fig. 9 F upper panel) and histological analysis (Fig. 9 F lower panel) with Hematoxylin/eosin staining (x20 and x40). Error bars: mean ± SD; *p < 0.05 indicates significant difference from vehicle.Lower panel: representative images of immunohistochemical staining of CD31 expression on tumor sponges tissue sections. (G) The graph bar represents the densitometric analysis of the CD31 staining positive pixels. (H) Histograms illustrates the distribution of network patterns derived from angiogenesis parameters. The results are expressed as mean expression ± SEM. * p ≤ 0.05, *** p ≤ 0.001

Fig. 10

Schematic representation of the pro-angiogenic signaling pathways and molecular features induced by AuNPs_air in ECFCs. The illustration, created with BioRender, summarizes the key intracellular mechanisms triggered by AuNPs_air treatment, including Ca²⁺ influx, activation of the PI3K/mTOR/Akt pathway, NO production, and subsequent effects on histone acetylation and endothelial activation leading to angiogenesis