Expression of Connexin43 Stimulates Endothelial Angiogenesis Independently of Gap Junctional Communication In Vitro

Abstract

Connexins (Cx) form gap junctions (GJ) and allow for intercellular communication. However, these proteins also modulate gene expression, growth, and cell migration. The downregulation of Cx43 impairs endothelial cell migration and angiogenetic potential. Conversely, endothelial Cx43 expression is upregulated in an in vivo angiogenesis model relying on hemodynamic forces. We studied the effects of Cx43 expression on tube formation and proliferation in HUVECs and examined its dependency on GJ communication. Expectedly, intercellular communication assessed by dye transfer was linked to Cx43 expression levels in HUVECs and was sensitive to a GJ blockade by the Cx43 mimetic peptide Gap27. The proliferation of HUVECs was not affected by Cx43 overexpression using Cx43 cDNA transfection, siRNA-mediated knockdown of Cx43, or the inhibition of GJ compared to the controls (transfection of an empty vector, scrambled siRNA, and the solvent). In contrast, endothelial tube and sprout formation in HUVECs was minimized after Cx43 knockdown and significantly enhanced after Cx43 overexpression. This was not affected by a GJ blockade (Gap27). We conclude that Cx43 expression positively modulates the angiogenic potential of endothelial cells independent of GJ communication. Since proliferation remained unaffected, we suggest that Cx43 protein may modulate endothelial cell migration, thereby supporting angiogenesis. The modulation of Cx43 expression may represent an exploitable principle for angiogenesis induction in clinical therapy.

Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

“…Moreover, we observed that the knock-down of Cx43 in the endothelium in mouse aortas impaired the aortic vessel sprouting ex vivo. This is in line with the recently published study by Koepple et al, where Cx43 siRNA treatment impaired the ability of endothelial cells to form capillary like structures in vitro [ 5 ]. Of note, Koepple et al found the endothelial cell proliferation to be unaffected by Cx43, and therefore hypothesized that the angiogenesis promoting effect of Cx43 is derived from an influence on migration.…”

“…Cx43 has been shown to influence cellular responses, independently of its gap junctional channel function [ 5 , 11 , 12 , 13 , 14 , 39 ]. We previously showed that the Cx43 channel independent function is important for cellular migration [ 13 , 16 ].…”

“…The activation of endothelial cells to proliferate and migrate is dependent on environmental signals like oxygen supply [ 1 , 2 ], the release of growth factors [ 3 ], and several signalling pathways like the Notch system expressed in vascular cells [ 4 ]. The connexin 43 (Cx43) has repeatedly been shown to be involved in angiogenesis [ 5 , 6 , 7 , 8 , 9 ].…”

“…They enable direct intercellular communication by allowing the exchange of molecules smaller than 1 kDa, thereby coordinating vascular function and communication [ 10 ]. In recent years, it has been repeatedly demonstrated that Cx additionally possess gap junction-independent functions [ 5 , 11 , 12 , 13 , 14 ]. The observed effect on angiogenesis via Cx43 seems to be part of its gap-junction independent function.…”

“…For instance, ischemia dependent angiogenesis in the heart was shown to be unaffected by a gap-junction opener, but increased by Cx43 overexpression [ 8 ]. In addition, the formation of capillary like structures in vitro were shown to be affected by Cx43, but were independent of its gap-junctional communication [ 5 ]. Earlier studies have shown that the gap-junctional independent function of Cx43 is important for cell motility and migration [ 13 , 15 , 16 ].…”