M199 (cat. no. 11310882), FBS (cat. no. A5256701) and DMSO (cat. no. 855190) were purchased from Gibco (Thermo Fisher Scientific, Inc.). Collagenase type I (cat. no. SCR103), nicotine (cat. no. N3876), choline (cat. no. C7527), neutral red (cat. no. N4638), sulforhodamine B (cat. no. S1402) and BSA (cat. no. A9418) were obtained from Sigma-Aldrich (Merck KGaA). Cultrex^® extracellular matrix (cat. no. 3533-010-02) was acquired from Trevigen, Inc. (BioTechne). CellTiter 96^® Aqueous One Solution Cell Proliferation Assay (cat. no. G3582) was obtained from Promega Corporation. ISO-1 was synthesized as described by Cortés et al (15).

All umbilical cords were obtained following delivery from full-term normal pregnancies from Gynecological-Obstetric Unit of the Carlos Van Buren Hospital (Valparaiso, Chile) with written informed consent of the mothers and the approval of the Ethics Committee of the Faculty of Pharmacy of Universidad de Valparaiso in Valparaiso, Chile (approval no. 19/2015) and the Ethics Committee of the Valparaiso-San Antonio Health Service in Valparaiso, Chile (approval no. 1435), which has responsibility for ethically approving studies on behalf of Carlos Van Buren Hospital.

ECs were isolated as described by Jaffe et al (19) HUVECs were isolated using collagenase I (0.5 mg/ml) digestion and cells were cultured in M199 supplemented with 2.5 mM L-glutamine, 14 mM HEPES, 200 UI/l penicillin, 400 UI/l streptomycin and 20% FBS at pH 7.42, at 37˚C and 5% CO₂ atmosphere. Experiments were performed in confluent cultures of ECs (~5 days of primary culture) or in cells at passage 1-3.

HUVECs were seeded in 96-well plates at a density of 3x10⁴ cells/well and incubated in M199 supplemented with 5% FBS (negative control) at 37˚C for 72 h. Cells were treated with solvent (S control, DMSO 0.3%) or ISO-1 in solvent medium at concentrations of 1x10^-3.5 M, 1x10^-4 M, 1x10^-5, 1x 10^-7 M and 1x10^-9 M and incubated for 6 or 24 h at 37˚C and 5% CO₂.

Lysosomal activity was evaluated using the neutral red uptake assay, as described by Repetto et al (20). Each treatment was removed, and the wells were washed twice with PBS. Subsequently, a control medium containing neutral red (40 µg/ml) was added, and the plate was incubated at 37˚C for 2 h. Neutral red was discarded, the plate was washed with PBS and neutral red destain solution [50% ethanol (96%), 49% deionized water, 1% glacial acetic acid] was added. Plates were agitated for 10 min using an orbital shaker and absorbance was determined at 540 nm using Thermo Fisher Scientific, Inc. Varioskan^® Flash microplate reader. Results were expressed as percentage of the negative control response.

Mitochondrial activity was assessed using the MTS assay. Mitochondrial activity, used as a marker of viability, was evaluated using CellTiter 96^® Aqueous One Solution Cell Proliferation Assay kit (Promega Corporation), following the manufacturer's instructions. Absorbance at 490 nm was determined in each well using Thermo Fisher Scientific, Inc. Varioskan Flash microplate reader. Results were expressed as percentage of the negative control response.

The proliferation of HUVECs was determined using the SRB assay described by Vichai and Kirtikara (21). HUVECs were treated as aforementioned, 10% trichloroacetic acid (Sigma-Aldrich; Merck KGaA) was added and the plate was incubated for 1 h at 4˚C. Each well was washed four times with deionized water and the plate was allowed to air-dry at room temperature. Then, 0.057% SRB solution (Sigma-Aldrich) was added to each well at room temperature for 30 min. After that, the plate was rinsed with 1% acetic acid and allowed to air-dry at room temperature. Finally, 10 mM Tris solution (pH 10.5) was added to each well and the plate was shaken on an orbital shaker for 10 min to solubilize the protein-bound dye. The absorbance at 510 nm of each well was measured using Thermo Fisher Scientific, Inc. Varioskan^® Flash microplate reader. Results obtained were expressed as percentage of control medium.

The migration of HUVECs was analyzed through the scratch wound assay (22). HUVECs were seeded in 96-well plates at confluence (3x10⁴ cells/well) and incubated in M199 supplemented with 20% FBS for 24 h at 37˚C. A confluent monolayer of HUVECs (100% confluence) in control conditions (M199 supplemented with 5% FBS) or treated with nicotine (1x10^-8 M), ISO-1 (1x10^-10 M, 1x10^-8 M, 1x10^-6 M, 1x10^-4 M) or choline (1x10^-5 M), alone or in combination with 100 nM α-Bungarotoxin (BTX, Sigma-Aldrich), was scratched using a P10 micropipette tip. The monolayer was washed with PBS to remove cell debris. Treatments were maintained throughout the experiment in M199 supplemented with 5% FBS, and the plate was incubated at 37˚C for 24 h. Images were captured at 0 and 24 h using Nikon Eclipse TE200 microscope (Nikon Corporation). Wound closure was analyzed using ImageJ version 1.52p (23) and HUVEC migration was expressed as the percentage of wound closure relative to the control.

The ability of HUVECs to form capillary-like tubular structures was examined using the Matrigel-based tube formation assay (24). HUVEC tube formation assay was performed in 96-well plates coated with 50 µl Cultrex^® (Trevigen, Inc.; BioTechne), according to the manufacturer's protocol. Cultrex solution was added to the plates and allowed to solidify and polymerize at 37˚C for 30 min. HUVECs at density of 1x10⁴ cells/well were seeded on the top of the Cultrex matrix and tubular structure formation was monitored for 4 and 12 h in control conditions (M199 supplemented with 5% FBS) or in the presence of solvent medium (0.3% DMSO), positive control (nicotine,1x10^-8 M) or ISO-1 (1x10^-10, 1x10^-8, 1x10^-6 or 1x10^-4 M). A total of six fields/well were examined using Nikon Eclipse TE200 microscope (Nikon Corporation) and results were analyzed using the Angiogenesis Analyzer for ImageJ (25). Number of branches/field and total length of tubular structures were calculated relative to the control.

The data analysis was performed using SigmaPlot version 11.0 Build 11.0.0.77(26). All data are presented as the mean ± SEM of ≥3 independent experiments. Comparisons between groups were performed using unpaired or paired Student's t-test or one-way ANOVA followed by Holm-Sidak post hoc test as appropriate. P<0.05 was considered to indicate a statistically significant difference.

Before evaluating the biological effects of a synthetic molecule, it is necessary to assess its potential toxic effects. Viability of HUVECs exposed to increasing concentrations of ISO-1 (1x10^-9-10^-3.5 M) was assessed by measuring lysosomal activity through the neutral red uptake assay and mitochondrial activity using the MTS assay at 6 and 24 h. The highest concentration of ISO-1 tested (1x10^-3.5 M) corresponded to the solubility limit of ISO-1 in culture medium without exceeding non-toxic DMSO concentration, while the lowest concentration tested (1x10^-9 M) was selected due to the absence of cytotoxic effects at higher concentrations. The viability of ECs treated with ISO-1 was similar to that of the negative control (M199 supplemented with 5% FBS), indicating that ISO-1 did not affect cell viability (Fig. 1). As DMSO was used to dissolve ISO-1, a solvent control containing the maximum DMSO concentration (0.3% DMSO) was included; the solvent control did not show any differences compared with medium control conditions (Fig. 1A and B).

EC proliferation, which is key for the formation of new blood vessels, was evaluated using the SRB assay. ECs exposed to ISO-1 at concentrations ranging from 1x10^-9 to 1x10^-3.5 M. ISO-1 showed no significant differences in proliferation compared with negative or solvent control (Fig. 2). These results indicate that ISO-1 does not promote EC proliferation under these conditions.

To investigate the effects of ISO-1 on cell migration, ECs were treated with ISO-1 at concentrations ranging from 1x10^-10 to 10^-4 M. This range was selected based on previous findings from our group, which demonstrated that ISO-1 at concentrations of 10^-5 and 10^-7 M increases intracellular calcium levels [Ca²⁺]_i via activation of α7 nAChR) (15). Additionally, this concentration range falls within the non-cytotoxic window established in the present study (10^-9 to 10^-³^.5 M). A wound was introduced into the EC monolayer to assess migration. The results demonstrated a significant increase in EC migration when cells were treated with ISO-1 at 1x10^-6 M and 1x10^-4 M compared with the negative control. The effects of ISO-1 at these concentrations were similar to those observed with nicotine, a nAChR agonist, at 1x10^-8 M (Fig. 3A and B).

Since new blood vessel formation involves the formation of tubular structures, this was evaluated using the Cultrex tube formation assay at 4 and 12 h. Compared with the negative control, treatment with ISO-1 at concentrations ranging from 1x10^-10 to 1x10^-6 M led to an increase in the total length of the tubular structures (Fig. 4B). Additionally, ISO-1 at 1x10^-6 and 1x10^-4 M resulted in a significant increase in the number of branches within these structures (Fig. 4A and B). Furthermore, no significant differences were observed between ISO-1 at these concentrations and nicotine group.

To investigate the role of α7-nAChR signaling in the proangiogenic effects of ISO-1, HUVEC migration was assessed using the α7-nAChR selective antagonist, BTX. ISO-1-induced increase in EC migration was completely abolished by BTX. This effect was similar to the inhibition observed with choline, suggesting that activation of the α7-nAChR pathway was involved in the pro-angiogenic response to ISO-1 (Fig. 5A and B).