Abstract
Endothelial cell migration is a key process in angiogenesis. Progress of endothelial cell migration is orchestrated by coordinated generation of Ca2+ signals through a mechanism organized in caveolar microdomains. Connexins (Cx) play a central role in coordination of endothelial cell function, directly by cell-to-cell communication via gap junction and, indirectly, by the release of autocrine/paracrine signals through Cx-formed hemichannels. However, Cx hemichannels are also permeable to Ca2+ and Cx43 can be associated with caveolin-1, a structural protein of caveolae. We proposed that endothelial cell migration relies on Cx43 hemichannel opening. Here we show a novel mechanism of Ca2+ signaling in endothelial cell migration. The Ca2+ signaling that mediates endothelial cell migration and the subsequent tubular structure formation depended on Cx43 hemichannel opening and is associated with the translocation of Cx43 with caveolae to the rear part of the cells. These findings indicate that Cx43 hemichannels play a central role in endothelial cell migration and provide new therapeutic targets for the control of deregulated angiogenesis in pathological conditions such as cancer.
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Introduction
Tissue functional integrity and viability relies on the accurate delivery of oxygen and nutrients through the blood flow, according to the cellular activity along the time. Although regulation of blood flow distribution through dynamic control of vasomotor tone of resistance arteries in the microcirculation can timely match the changes in cellular metabolic demand, the vessel density and the microvascular network architecture must be coherent with the physiological function of the tissue. In this context, long-term regulation of tissue irrigation depends on the growth of new blood vessels from pre-existing ones through a process known as angiogenesis [1, 2], which is stimulated by tissue requirements of oxygen as observed in different physiological and pathological conditions such as wound healing, tissue regeneration, embryonic development, and tumor growth [3].
Angiogenesis is a complex process that involves a cascade of events initiated by coordinated endothelial cell migration [4] in response to an increase in intracellular Ca2+ concentration ([Ca2+]i). Ca2+ has been recognized as a key signaling mechanism in angiogenesis [5, 6] and although Ca2+ release from intracellular stores plays an important role in endothelial cell migration, the prevalence of the response depends on Ca2+ entry from the extracellular compartment. However, the mechanisms involved in this process are controversial and have not been clearly determined [7,8,9]. In addition, it has been proposed that cell migration also depends on the intercellular coordination of Ca2+ signals and, consistent with this, blockade of gap junctions results in a reduction in both migration and tubular structure formation by endothelial cells [10, 11].
Gap junctions are intercellular channels that directly connect the cytoplasm of adjacent cells, allowing the exchange of current, ions and small signaling molecules (< 1.4 nm of diameter), such as second messengers. These intercellular channels are made up by serial docking of two hemichannels, each one provided by each neighboring cell and, in turn, hemichannels are formed by the assembly of six connexin (Cx) proteins [12]. It should be noted, however, that, in addition to form gap junctions, individual hemichannels can also be functional [13, 14]. Thus, the opening of these channels contributes to the transmembrane communication of the intra- and extracellular compartments [15, 16], which may be relevant in the control of endothelial cell function, since Cx43 hemichannels have been reported to be activated by nitric oxide (NO) through direct S-nitrosylation of this Cx protein [Endothelial cell-mediated formation of tubular structures The analysis of endothelial cells tube formation was performed in 12 mm coverslips covered with 100 µl Matrigel® (Corning, NY, USA) according to the manufacturer’s protocol. Matrigel®-solution was added to coverslips located into a 96 wells plate and allowed to solidify and polymerize at 37ºC in a 5% CO2-95% air atmosphere. Then, endothelial cells were seeded on top of the Matrigel and tubular-like structure formation was evaluated for 6 and 12 h in control conditions or in the presence of 300 µM TAT-Gap19. Seven fields per coverslip were examined using a Nikon Eclipse E600 FN1 microscope and the results were expressed as the angiogenic index according to the following formula: (Total cells + connected cells)/ total cells x (1-non-connected cells), where Total cells is the number of total cell in the field, connected cells is the number of the cells that form a tubular structures and non-connected cells is the number of the cells outside of tubular structures. Results are expressed as mean ± SEM. All values represent data from at least three independent cultures. Comparison between groups was performed using unpaired or paired Student t-test, one-way ANOVA followed by Bonferroni post-hoc test or two-ways ANOVA as appropriate. P < 0.05 was considered significant.Statistical analysis
Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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This work was supported by Grant Anillo ANID/ACT210057 from the Agencia Nacional de Investigación y Desarrollo, Grant #1150530 from Fondo Nacional de Desarrollo Científico y Tecnológico – FONDECYT and by PhD and Thesis support scholarship #21170977 from Comisión Nacional de Investigación Científica y Tecnológica – CONICYT.
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Hilda Espinoza. Collected and analyzed the data, prepared figures and participated in the manuscript preparation. Xavier Figueroa. Directed and supervised the investigation, designed the experimental protocols, contributed to data analysis and manuscript writing and editing.
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All studies and experimental procedures were approved by the Institutional Bioethics Committee (protocol ID 170823033).
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Espinoza, H., Figueroa, X.F. Opening of Cx43-formed hemichannels mediates the Ca2+ signaling associated with endothelial cell migration. Biol Direct 18, 52 (2023). https://doi.org/10.1186/s13062-023-00408-3
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DOI: https://doi.org/10.1186/s13062-023-00408-3