Abstract
BackgroundAngiogenesis can occur under pathological conditions when stimuli such as inflammation, vascular obstruction or hypoxia exist. These stimuli are present in cardiopulmonary dirofilariosis (Dirofilaria immitis). The aim of this study was to analyze the capacity of D. immitis antigens to modify the expression of angiogenic factors and trigger the formation of pseudocapillaries (tube-like structures) in an in vitro model of endothelial cells.MethodsThe expression of VEGF-A, sFlt, mEndoglin and sEndoglin in cultures of canine microvascular endothelial cells stimulated with extract of adult worms of D. immitis obtained from an untreated dog (DiSA) and from a dog treated for 15 days with doxycycline (tDiSA), was determined by using commercial kits. The capacity of pseudocapillary formation was evaluated analyzing cell connections and cell groups in Matrigel cell cultures stimulated with DiSA and tDiSA. In both cases non-stimulated cultures were used as controls.ResultsFirst, we demonstrated that worms obtained from the dog treated with doxycycline showed a significantly lower amount of Wolbachia (less than 60%) than worms removed from the untreated dog. Only DiSA was able to significantly increase the expression of the proangiogenic factor VEGF-A in the endotelial cells cultures. None of the D. immitis extracts modified the expression of sFlt. tDiSA extract was able to modify the expression of the endoglins, significantly decreasing the expression of the pro-angiogenic mEndoglin and increasing the anti-angiogenic sEndoglin. The formation of pseudocapillaries was negatively influenced by tDiSA, which reduced the organization and number of cellular connections.ConclusionsThe ability of antigens from adult D. immitis worms to modify the expression of pro and anti-angiogenic factors in endotelial cell cultures was demonstrated, as well as the trend to form pseudocapillaries in vitro. The capacity of stimulation may be linked to the amount of Wolbachia present in the antigenic extracts.
Highlights
Angiogenesis can occur under pathological conditions when stimuli such as inflammation, vascular obstruction or hypoxia exist
The pathogenic processes of cardiopulmonary dirofilariosis trigger the appearance of key factors in the stimulation of angiogenesis such as inflammation and hypoxia. Considering all these facts, in this paper we examined the influence of different antigenic extracts of D. immitis adult worms on the expression of some pro- and anti-angiogenic factors and their ability to stimulate the formation of pseudocapillaries by using an in vitro model of microvascular endothelial cells
In a first step we demonstrated that the antigenic extract of worms from a dog treated with doxycycline had much less Wolbachia than the extract of worms removed from an untreated dog (DiSA)
Summary
Angiogenesis can occur under pathological conditions when stimuli such as inflammation, vascular obstruction or hypoxia exist. These stimuli are present in cardiopulmonary dirofilariosis (Dirofilaria immitis). Adult worms lodge in the pulmonary arteries of the infected dogs cause tortuosity, loss of elasticity, proliferative endarteritis and, in addition, pulmonary thromboembolisms when adult worms die. All these anatomical changes at the vascular level leads to luminal obstruction and infarction, decrease of the blood flow, hypoxia, chronic pulmonary edema and hypertension and congestive heart failure [8, 9]. Since doxycycline reduces Wolbachia populations, the administration of this tetracycline is recommended in addition of the adulticide protocol to decrease the inflammatory effects of bacteria released from dying worms [13, 14]
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