Involvement of the excretory/secretory and surface-associated antigens of Dirofilaria immitis adult worms in the angiogenic response in an in-vitro endothelial cell model

doi:10.21203/rs.3.rs-2608205/v1

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Involvement of the excretory/secretory and surface-associated antigens of Dirofilaria immitis adult worms in the angiogenic response in an in-vitro endothelial cell model

https://doi.org/10.21203/rs.3.rs-2608205/v1

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published 31 May, 2023

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Background: Angiogenesis is a process by which new vessels are formed from pre-existing ones when the physiological conditions of the vascular endothelium are altered. Heartworm disease (Dirofilaria immitis) causes changes in the vascular endothelium of the pulmonary arteries due to obstruction, friction and hypoxia. The aim of this study was to analyze whether the excretory/secretory antigen of adult worms interacts and modulates the angiogenic mechanism, viable cell number and cell migration, as well as the formation of pseudo-capillaries.

Methods: Cultures of human vascular endothelial cells (HUVECs) stimulated with excretory/secretory antigens (DiES), surface-associated antigens (Cut) from D. immitis adult worms, VEGF, as well as DiES+VEGF and Cut+VEGF were used. The production of VEGF-A and other proangiogenic [soluble VEGFR-2 (sVEGFR-2), membrane Endoglin (mEndoglin)] and antiangiogenic [VEGFR-1/soluble Flt (sFlt), soluble Endoglin (sEndoglin)] molecules was assessed using commercial ELISA kits. Cell viability was analyzed by live cell count and cytotoxicity assays by a commercial kit. In addition, viable cell number by MTT-based assay, cell migration by wound-healing assay carrying out scratched wounds, and the capacity of pseudo-capillary formation to analyze cell connections and cell groups in Matrigel cell cultures, were evaluated. In all cases, non‑stimulated cultures were used as controls.

Results: DiES+VEGF and Cut+VEGF significantly increased the production of VEGF and VEGFR2, and only Cut+VEGF significantly increased the production of VEGFR1/sFlt compared to other groups and non-stimulated cultures. Moreover, only DiES+VEGF produced a significant increase in viable cell number and cell migration, as well as in the organization and number of cell connections.

Conclusions: Excretory/secretory and surface-associated antigens of adult D. immitis activated the angiogenic mechanism by mainly stimulating the synthesis of proangiogenic factors, and only excretory/secretory antigens increased viable cell number, activated cell migration and the formation of pseudo-capillaries. These processes could lead to vascular endothelial remodeling of the infected host and favor the long-term survival of the parasite.

Angiogenesis

excretory/secretory antigens

surface-associated antigens

adult D. immitis worms

cell proliferation

cell migration

pseudo-capillary formation

Angiogenesis is the formation of new blood vessels from pre-existing vessels, a key process in the resolution of physiological situations such as wound healing and obstructive inflammatory processes, among others. Angiogenesis begins with tissue hypoxia/ischemia, which leads to increased growth, proliferation and differentiation of vascular endothelium. These alterations produce physiological effects such as cardiac ischemia, hypertension, or respiratory distress [1–3]. These physiological alterations upregulate the expression of different genes involved in various steps of angiogenesis mediated by the balance and interaction between numerous pro- and anti-angiogenic factors [4]. Vascular Endothelial Growth Factor (VEGF), at which point migration, endothelial cell proliferation and tube formation begin [5–6], is the most potent angiogenic factor and is the first produced in "endothelial tip cells", which enables them to begin proliferation and migration and orient themselves according to the VEGF gradient produced, mediated by the interaction of VEGF-Receptor 2 (VEGFR-2) and soluble VEGFR-2 (sVEGFR-2), at which point migration, endothelial cell proliferation and tube formation begin [6]. This process ends with the inhibition of endothelial proliferation and migration and formation of new capillaries mediated by platelet-derived growth factor β (PDGF-B) and its receptor (PDGFR-B) [7]. However, both VEGFR-1 and its soluble form (sFlt1) exert negative regulation of signalling through VEGFR-2, as they act by sequestering the ligand and preventing it from binding to the receptor [8].

Other main molecule is endoglin, a transmembrane glycoprotein, essential for angiogenesis, cell proliferation and vascular development, which is predominantly expressed in vascular endothelial cells and the suppression of which produces the inhibition of angiogenesis [9]. Several endoglin isoforms have been described. One of them, the circulating form of endoglin, soluble endoglin (s-Endoglin), negatively regulates the production of several proangiogenic and promigratory proteins involved in angiogenesis [10–12]. However, the membrane-bound form (m-Endoglin) is involved in processes with proangiogenic effects [13].

Heartworm disease is a vector-borne zoonotic disease, caused by Dirofilaria immitis, mainly affecting domestic and wildcanines and felines, with the main reservoir being the domestic dog, and where humans can be infected [14]. Adult worms are found in the pulmonary artery and right ventricle of the heart where they can live for several years. Their presence causes chronic pathology due to anatomical and histological changes in the arterial wallscausing proliferative endarteritis, characterized by a loss of elasticity, decreased arterial lumen and increased permeability of the vascular wall. These changes can lead to hypoxia, pulmonary hypertension and congestive heart failure. When the adult worms die, an acute pathology occurs as a result of the massive release of antigens from the parasite and the endosymbiont bacteria of the genus Wolbachia, which may increase clot formation (pulmonary thromboembolism), exacerbate endothelial and lung damage, accelerate heart failure, or cause the death of the infected host [15–16]. Moreover, microfilariae, which can live up to 2 years [15], are found in the glomeruli and can cause renal dysfunction [17].

Dirofilaria immitis participates in several processes that contribute to its survival within the host, limiting short-term damage, although long-term vascular damage is inevitable [18]. Excretory/secretory antigens promote vasodilation [19] and in the short term regulate the host fibrinolysis system by preventing or promoting the lysis of fibrin clots [20, 21], as well as cuticle surface-associated antigens of adult worms [22]. In the long term, plasmin production increases in-vitro endothelial and muscle proliferation, as well as migration and destruction of extracytoplasmic matrix, all of which are related to vascular remodeling [23]. On the other hand, when adult worms die, naturally or induced by adulticidal treatment, Wolbachia somatic antigens promote a vasoconstrictive and chemotactic endothelial response that activates anti-angiogenic mechanisms and inhibits in-vitro formation of pseudo-capillaries; this seems to be related to the amount of Wolbachia antigens released into the bloodstream [24–26].

Taking into account that D. immitis is involved in the modulation of inflammatory mechanisms that allow the parasite to survive in the host for years, the aim of this study was to analyze the influence of excretory/secretory and surface-associated antigens of adult D. immitis in the production of some factors involved in the angiogenic process, in the viable cell number and migration, and in the formation of pseudo-capillaries, using an in-vitro model of vascular endothelial cells.

Reagents

Excretory/secretory antigens of adult D. immitis (DiES) were prepared as previously described [19]. In brief, 23 live worms (8 males and 15 females) obtained from a naturally infected dog were washed in sterile PBS pH 7.2 and incubated for 1 day in 50 ml of Eagle’s minimum essential medium (MMEE) (Sigma Chemical Company, St. Louis, MO, USA) supplemented with 0.04% gentamycin and 0.01% nistatin, at 37 ºC. Later, it was dialyzed against 0.01% PBS pH 7.2 and filtered through an Amicon YC05 (Amicon Corporation Scientific System Division, Danvers, MA, USA). The final concentration obtained was 0.4 µg/µl.

Cuticle antigens of adult D. immitis (Cut) were obtained following the methodology described by Wedrychowicz et al. [27]. In brief, 10 live worms (4 males and 6 females) obtained from a naturally infected dog were washed and then incubated in saline solution containing 0.25% cetyltrimethylammonium bromide (CTAB) with a cocktail of protease inhibitors [28] at 37 ºC for 4 hours. The worms were separated from detergent and extracted proteins were precipitated with sodium acetate 0.002 mM with nine volumes of 96% ethanol, at -20 ºC for 48 h followed. Finally, Cut was centrifuged at 10,000 xg during 10 min and the resulting pellets were resuspended in PBS pH 7.2.

Protein concentration of both antigens (DiES and Cut) were measured by detergent compatible (DC) protein assay commercial kit (Bio-Ras) and were stored at -80 ºC until use.

Cell Culture And Stimulation Of Endothelial Cells

Human Umbilical Vein Endothelial Cells (HUVECs) were grown in Endothelial Basal Medium 2 (Lonza, Walkersville, MD, USA) supplemented with SingleQuots® (Lonza, Walkersville, MD, USA): 20% fetal bovine serum, 22.5 µg /ml, heparin (22.5 µg /ml), VEGF (0.5 ng/ml), ascorbic acid (1 µg /ml), hFGF-B (10 ng/ml), hydrocortisone (0.2 µg /ml), hEGF (5 ng/ml), Gentamicin (30 mg/ml), amphotericin B (15 µg/ml) and R³-IGF-3 (20 ng/ml). Plates were pre-coated with 0.1% pig gelatine (Sigma Chemical Co., San Luis, USA), 0.01% fibronectin (Sigma-Aldrich, Misuri, USA) and 0.01% collagen (Corning). Cells were cultured at 37°C in a humidified atmosphere in the presence of 5% CO₂/95% air. The medium was changed every 3 days. Expansion was carried out by trypsinizing the cells (Trypsin/EDTA, Lonza, USA) and replating them when the proliferating cells had reached sufficient density. Passaging was performed the ratio of 1:3. Cell counts were performed using a Countess® Automated Cell Counter (Invitrogen, California, USA) following the manufacturer’s instructions.

HUVECs were treated as previously described [24]. In brief, endothelial cells (10⁶ cells/plate) were plated on 60 mm culture plates and were grown for 4 days to obtain confluent cultures and treated with 5 different stimuli: 1 µg/ml of DiES, Cut or recombinant Vascular Endothelial Growth Factor protein (VEGF) (RRD SYSTEMS), 1 µg/ml of DiES plus 1 µg/ml of VEGF and 1 µg/ml of Cut plus 1 µg/ml of VEGF. Non-stimulated cells were used as controls in the same conditions. Finally, the supernatant of the cell cultures was collected and HUVECs were lysed in ice-cold lysis buffer [20 mM Tris-HCl (pH 7.5); 140 mM NaCl; 10 mM ethylenediaminetetraacetic acid; 10% glycerol; 1% Igepal CA-630; aprotinin, pepstatin, and leupeptin at 1 µg/ml each; 1 mM phenylmethylsulfonyl fluoride and 1 mM sodium orthovanadate].

Cell Viability And Cytotoxicity Assays

Cell viability was analyzed through cell counts using the equipment Countess® Automated Cell Counter (Invitrogen) following the manufacturer’s instructions. Cytotoxicity was assessed in the supernatant of the stimulated and control cell cultures by the Toxilight BioAssay Kit (Cambrex, Verviers, Belgium) following commercial instructions. This commercial kit quantitatively measures the release of adenylate kinase from damaged cells. The results were presented as the mean ± standard deviation (SD) of three experiments performed in duplicates.

Angiogenic Factors Assays

VEGF-A, soluble VEGFR-1 (VEGFR-1/sFlt), soluble VEGFR-2 (sVEGFR-2) and sEndoglin (sEng) concentration in the HUVECs culture medium were measured by ELISA using a Human VEGF Quantikine ELISA kit (R&D Systems, Minneapolis, USA), Human VEGFR-1/sFlt Quantikine ELISA kit (R&D Systems, Minneapolis, USA), Human VEGFR-2 Quantikine ELISA kit (R&D Systems, Minneapolis, USA) and Human Endoglin Quantikine ELISA kit (R&D Systems, Minneapolis, USA) respectively; mEndoglin (mEng) concentration in the listed endothelial cells was measured by Human Endoglin Quantikine ELISA kit (R&D Systems, Minneapolis, USA) following the manufacturers’ instructions for the first 24 hours. The results were presented as the mean ± SD of three experiments performed in duplicate.

Viable Cell Number Assays

Viable cell number assays were assessed as previously described [29], with some modifications. In brief, 1000 cells per well were seeded on a 96-well plate in HUVECs culture medium with 5 different stimuli for 10 days: 1 µg/ml of DiES, Cut or Vascular Endothelial Growth Factor (VEGF) (RRD SYSTEMS), 1 µg/ml of DiES plus 1 µg/ml of VEGF and 1 µg/ml of Cut plus 1 µg/ml of VEGF. Non-stimulated cells were used as controls in the same conditions. Viable cell number was estimated every 2 days up to a maximum of 10 days of culture was estimated by incubating cell cultures with 0.5 mg/ml 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) for 4 h. Then, 10 SDS in 0.01M HCl was added at a 1:1 (v/v) ratio and left overnight at 37°C. Finally, absorbance was measured at 570 nm. The results are presented as the mean ± SD of three experiments performed in triplicate.

Migration Assays

Wound-healing assays were assessed as previously described by Fernández-López et al., [23] with some modifications. In brief, in-vitro scratched wounds were created by scraping confluent cell monolayers in 60 mm sterile plates with a sterile disposable pipet tips. The remaining cells were washed with sterile PBS buffer and incubated with HUVECs culture medium with 5 different stimuli up to 6 hours. Non-stimulated cells were used as controls in the same conditions. Endothelial cell migration into the denuded area was monitored by photography of the plates every 30 min. The results were presented as the mean ± SD of three experiments performed in triplicate.

Endothelial Cell Tube Formation Assay

Endothelial cell tube formation was assessed as previously described by Jerkic et al [30], with modifications. In brief, a total of 8000 HUVECs per well were plated on Matrigel® precoated µ-Slide Angiogenesis® plates (Ibidi, Gräfelfing, Germany) in HUVECs culture medium with 5 different stimuli: DiSA, Cut, VEGF, DiES + VEGF and Cut + VEGF (1:10 dilution). After seeding on Matrigel®, cells spread and aligned with each other to develop hollow, tube-like structures. The cells and intercellular junctions were observed each 30 minutes for 3 hours of incubation and the morphological changes were photographed using a phase contrast inverted Leica Microscope (Leica, Wetzlar, Germany). Subsequently, the intercellular junctions were divided between the cell bodies to calculate the relationship between them (endothelial cell tube formation = cellular connections/cellular bodies). Non-stimulated cells were used as controls in the same conditions. Each experiment was performed in triplicate.

Statistical analysis

The GraphPad Prism v.7 was used for all data analyses. Analyses were performed by ANOVA and corrected for repeated measurements when appropriate. If ANOVA revealed overall significant differences, individual means were evaluated post-hoc using Tukey’s test. All results were expressed as the mean ± SD. In all experiments, a significant difference was defined as a P-value of < 0.01 for a confidence level of 99%.

Effect of DiES and Cut antigens on cell viability and cytotoxicity

No differences were found in cell viability (Fig. 1) and cytotoxicity (date not shown) of stimulated cultures with DiES, Cut, VEGF, DiES + VEGF or Cut + VEGF compared to non-stimulated cell cultures.

Effect Of Dies And Cut Antigens On Angiogenic Factors

VEGF

The stimulation of cell cultures with DiES + VEGF significantly increased the production of VEGF-A when compared to cell cultures stimulated with DiES (t = 175.5, df = 4, P < 0.0001) and VEGF (t = 66.87, df = 4, P < 0.0001) and non-stimulated cultures (t = 139.8, df = 4, P < 0.0001) for the first 24 hours. In addition, VEGF-stimulated cell cultures showed a significant increase in VEGF-A production compared to DiES (t = 22.31, df = 4, P < 0.0001) and non-stimulated cultures (t = 18.97, df = 4, P < 0.0001). The stimulation of cell cultures with Cut + VEGF significantly increased the production of VEGF-A when compared to cell cultures stimulated with Cut (t = 10.86, df = 4, P = 0.0004), VEGF (t = 4.68, df = 4, P = 0.0094) and non-stimulated cultures (t = 13.94, df = 4, P = 0.0002). In addition, VEGF-stimulated cell cultures showed a significant increase in VEGF-A production compared to Cut (t = 9.372, df = 4, P = 0.0007) (Fig. 2A). No significant differences between cell cultures stimulated with VEGF and culture medium with VEGF prior to cell stimulation were detected for the first 24 hours, being obtained similar values.

VEGFR-1/sFlt and sVEGFR-2

On the one hand, with regard to the VEGFR-1/sFlt content detected, no significant differences were observed between DiES + VEGF, DiES, VEGF stimulated and unstimulated cell cultures for the first 24 hours. However, a significant decrease was observed between Cut + VEGF stimulated cell cultures and Cut (t = 4.390, df = 4, P = 0.0118), VEGF (t = 14.88, df = 4, P = 0.0001) stimulated and unstimulated cell cultures (t = 14.75, df = 4, P = 0.0001) (Fig. 2B). On the other hand, with regard to the sVEGFR-2 content detected, DiES + VEGF stimulated cell cultures showed a significant increase compared with DiES (t = 5.826, df = 4, P = 0.0043), VEGF (t = 6.151, df = 4, P = 0.0035) and non-stimulated cultures respectively (t = 3.274, df = 4, P = 0.0307). In addition, Cut + VEGF stimulated cell cultures showed a significant increase compared with Cut (t = 7.543, df = 4, P = 0.0017), VEGF (t = 8.727, df = 4, P = 0.0009) and non-stimulated cultures respectively (t = 4.047, df = 4, P = 0.0155) (Fig. 2C).

mEndoglin and sEndoglin

Regarding the sEndoglin production detected, no significant differences were observed between stimulated and unstimulated cultures for the first 24 hours (Fig. 3). However, DiES + VEGF significantly increased the production of mEndoglin when compared to cell cultures stimulated with DiES (t = 3.434, df = 4, P = 0.0264), VEGF (t = 3.347, df = 4, P = 0.0286) and non-stimulated cultures (t = 4.561, df = 4, P = 0.0103). A similar result was obtained when mEndoglin production was compared between cell cultures stimulated with Cut + VEGF and Cut (t = 3.355, df = 4, P = 0.0284), VEGF (t = 2.866, df = 4, P = 0.0456) and non-stimulated cell cultures (t = 3.953, df = 4, P = 0.0168).

Dies But Not Cut Antigens Produces An Increase In Viable Cells

The effect of DiES or Cut and VEGF on the number of viable endothelial cells was analysed, estimating the viable cell number by MTT technique in a period of 10 days every 2 days (Fig. 4). All stimulated and unstimulated cell cultures showed typical curves of cell growth in all experimental groups with progressive growth between days 0 and 6 or 8 post-stimulation, experiencing a decrease in viable cells from there until day 10 post-stimulation. MTT technique showed a significant increase in the number of viable cells on day 6 post-stimulation in cultures stimulated with DiES + VEGF compared with cultures stimulated with DiES (t = 9.072, df = 4, P = 0.0008), VEGF (t = 3.778, df = 4, P = 0.0195) and unstimulated cultures (t = 4.338, df = 4, P = 0.0123), and on day 8 post-stimulation in cultures stimulated with DiES + VEGF compared with cultures stimulated with DiES (t = 15.27, df = 4, P = 0.0001), VEGF (t = 4.764, df = 4, P = 0.0089) and unstimulated cultures (t = 12.04, df = 4, P = 0.0003). However, there were no significant differences between cells stimulated with Cut + VEGF and the other stimulated groups (Cut and VEGF stimulated and unstimulated cultures).

DiES but not Cut antigens produces cell migration.

A Wound Healing assay was performed to assess cell migration (Fig. 5). The quantification was carried out by measuring the distance of cell migration in comparison with negative control (untreated cells) to 6 hours post-stimulation. A significant increase of cell migration distance after stimulation with DiES + VEGF respect to DiES (t = 18.83, df = 8, P < 0.0001), VEGF (t = 9.249, df = 8, P < 0.0001) and unstimulated cultures (t = 24.34, df = 8, P < 0.0001) was detected. However, cell migration distance in Cut, VEGF, Cut + VEGF stimulated and unstimulated cells was similar.

Effect Of Dies And Cut Antigens On Pseudo-capillary Formation

The capacity for pseudo-capillary formation was evaluated by analyzing the cell junctions (connections) and the cellular set (converging strengths of different cellular ramifications) that emerged in stimulated and unstimulated cell cultures (Fig. 6). The formation of pseudo-capillaries and the connections/set relationship in cultures stimulated with DiES + VEGF showed a significant increase for the first 3 hours compared to cell cultures stimulated with DiES (t = 7.974, df = 4, P = 0.0013), VEGF (t = 9.127, df = 4, P = 0.0008) and non-stimulated cultures (t = 10.67, df = 4, P = 0.0004). However, in the case of cultures stimulated with Cut + VEGF showed a moderate increase compared to cell cultures stimulated with Cut and VEGF and unstimulated cultures.

Angiogenesis is an endothelial process that is triggered after stimulation by obstructive processes, resulting in increased vascular endothelial growth, cell proliferation and cell differentiation [1–3]. The presence of adult D. immitis worms causes changes in the vascular endothelium leading to proliferative endarteritis, hypertension, oedema, and pulmonary thromboembolism, among other conditions [24, 31]. Previous studies have shown that both somatic antigens of D. immitis and recombinant Wolbachia Surface Major protein (rWSP) are able to promote inflammatory processes causing vasoconstriction and chemotaxis of immune cells in an in-vitro model of endothelial cells [24, 32]), as well as a strong inflammatory response when adult worms die and antigens (both parasite and Wolbachia) are released in infected dogs [17]. However, DiES is able to stimulate vasodilation [19], and to activate the fibrinolytic system, which promotes the lysis of fibrin clots [20], in the same way that Cut does [23]. Neither DiES nor Cut not contain Wolbachia antigens that have been identified by proteomic techniques such as Two-dimensional electrophoresis (2-DE) and Liquid chromatography and tandem mass spectrometry (LC-MS/MS) and [15, 20, 22, 28]. This data could be related to mechanisms that promote the survival of D. immitis in the vascular endothelium.

There are few studies that address the angiogenic process of heartworm disease. There are studies in which D. immitis adult worm somatic antigen (DiSA) promotes the production of proangiogenic molecules in endothelial cells according to the production of VEGF-A [25] as well as the stimulation of the anti-angiogenic mechanism by Wolbachia and inhibition of pseudo-capillary formation [26]. On the other hand, remodeling of lymphatic and blood vessels in other filariasis has been reported. Cultures of lymphatic endothelial cells in the presence of live worms or Wuchereria bancrofti antigens activated lymphatic vessel remodeling, causing cell proliferation and cell differentiation. These events have been linked to processes that facilitate vascular irrigation in damaged tissues [33]. However, some authors have shown that angiogenesis and/or lymphangiogenesis can be induced by various factors derived from adult worm-activated monocytes [34–36].

The aim of this study was to analyze the angiogenic capacity of DiES and Cut from live adult worms in an in-vitro model of endothelial cells. For this purpose, an in-vitro endothelial cell model was used to recreate the angiogenic process produced by live D. immitis adult worm obstruction of blood vessels in dogs with heartworm. For the angiogenic process to be initiated, the first molecule produced is the production of VEGF-A [37]. To simulate this, stimulations with DiES and Cut from live adult worms together with VEGF were performed on endothelial cells and stimulation with both antigens separately. Using this endothelial cell model, the results have shown that excretory/secretory antigens and surface molecules from adult live D. immitis worms are able to promote the stimulation of proangiogenic factors as well as cell viable number and cell migration and the formation of pseudo-capillaries, without altering cell viability.

The effects of both antigens on VEGF-A, the main angiogenic factor, and other pro-antiangiogenic (sVEGFR-2 and mEndoglin) and anti-angiogenic (VEGFR-1/sFlt1 and sEndoglin) molecules were analysed. On the one hand, DiES and Cut + VEGF were able to stimulate the production of VEGF-A in endothelial cells. A similar result has been observed in human endothelial cells stimulated with DiES after being subjected or not to hypoxia, and Wolbachia under normoxic conditions [15, 24–26]. Furthermore, the results showed an increase in the production of sVEGFR-2, a soluble VEGF receptor that promotes the proangiogenic pathway, when endothelial cells were stimulated with DiES + VEGF and Cut + VEGF, a date that occurs in similar situations where vascular damage occurs [38–40]. However, DiES + VEGF did not alter the production of VEGFR-1/sFlt1 except in the case of Cut + VEGF where its production was reduced, being an anti-angiogenic factor, as described in other studies [41, 42].

On the other hand, only stimulation of both DiES + VEGF and Cut + VEGF was able to cause a significant increase in the production of mEndoglin, and not sEndoglin, in the present endothelial cell model. mEndoglin is the membrane-bound form related to proangiogenic processes, while sEndogline in the soluble form related to anti-angiogenic mechanisms and proinflammatory processes [13]. In addition, endoglin suppression in HUVECs produced the inhibition of angiogenesis in-vitro [9]. Therefore, the present results confirm previous findings, showing that DiES and Cut together with VEGF are able to activate proangiogenic processes, which could facilitate the survival of adult D. immitis worms.

Finally, the effect of DiES and Cut on endothelial cell viability during the first 10 days was studied, as well as cell migration and pseudo-capillary formation, processes directly related to angiogenesis [4, 6, 8, 9]. Only DiES + VEGF produced an increase in viable cells and stimulated cell migration, as well as the formation of pseudo-capillaries in the present endothelial cell model. The increase of VEGF levels after stimulation is beneficial for angiogenesis, which promotes the increase in both endothelial proliferation and migration [43, 44], processes involved in tumor vascularization and activation of inflammatory mechanisms [45, 46]. Moreover, the formation of pseudo-capillaries which resemble small blood vessels in formation during angiogenesis [47, 48], was not altered when endothelial cells were stimulated with DiSA [25] and was even inhibited by Wolbachia [26], which could confirm that DiES would facilitate the survival of adult D. immitis worms, since, upon their death, this process is altered.

This is the first study to demonstrate the ability of excretory/secretory and surface-associated antigens of adult D. immitis worms to activate the production of some proangiogenic factors, and only excretory/secretory antigens of adult D. immitis worms produces an increase in viable cells and stimulate cell migration, as well as formation of pseudo-capillaries in cultured endothelial cells in-vitro. Considering that these processes occur in dogs with heartworm, in which adult worms produce obstructive inflammatory processes and ischemia, and that somatic antigens of D. immitis and Wolbachia promote the production of anti-angiogenic factors, the present results suggest that the excretory/secretory antigens of adult D. immitis worms could favor parasite survival in the vascular endothelium by stimulating proangiogenic processes.

VEGF: Recombinant human vascular endothelial growth factor; VEGF-A: Vascular endothelial growth factor A; VEGFR-1/sFlt: Soluble vascular endothelial growth factor receptor 1; sVEGFR-2: Soluble vascular endothelial growth factor receptor 2; mEndoglin: Membrane endoglin; sEndoglin; Soluble endoglin; DiES: Excretory/secretory of adult D. immitis worms; Cut: Surface-associated antigens of adult D. immitis worms; CTAB: cetyltrimethylammonium bromide; SD: standard deviation; DC: detergent compatible; t: Tukey value; df: the number of degrees of freedom

Acknowledgements

Not applicable

Funding

This study was supported by Agencia de Desarrollo Económico de Castilla y León, Spain (cofinanced with FEDER funds) and General Foundation of University of Salamanca

Availability of data and materials

The data sets supporting the conclusions of this article are included within the article.

Authors’ contributions

CDCM and CAT performed the experiments. RM, EC and JAMA managed the dogs to obtain the antigenic extracts. RM and MP participated in the design of the experiments in the discussion of the results and in the correction of the manuscript. RM and MP coordinated the entire study. RM also wrote the manuscript. IRE, EC and MP revised the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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Involvement of the excretory/secretory and surface-associated antigens of Dirofilaria immitis adult worms in the angiogenic response in an in-vitro endothelial cell model

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Journal Publication

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Abstract

Figures

Introduction

Methods

Reagents

Cell Culture And Stimulation Of Endothelial Cells

Cell Viability And Cytotoxicity Assays

Angiogenic Factors Assays

Viable Cell Number Assays

Migration Assays

Endothelial Cell Tube Formation Assay

Statistical analysis

Results

Effect of DiES and Cut antigens on cell viability and cytotoxicity

Effect Of Dies And Cut Antigens On Angiogenic Factors

VEGF

VEGFR-1/sFlt and sVEGFR-2

mEndoglin and sEndoglin

Dies But Not Cut Antigens Produces An Increase In Viable Cells

Effect Of Dies And Cut Antigens On Pseudo-capillary Formation

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Journal Publication

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