Changes in endothelial connexin 43 expression inversely correlate with microvessel permeability and VE-cadherin expression in endotoxin-challenged lungs

Abstract

Endothelial barrier restoration reverses microvessel hyperpermeability and facilitates recovery from lung injury. Because inhibiting connexin 43 (Cx43)-dependent interendothelial communication blunts hyperpermeability in single microvessels, we determined whether endothelial Cx43 levels correlate with changes in microvessel permeability during recovery from lung injury. Toward this, bacterial endotoxin was instilled intratracheally into rat lungs, and at different durations postinstillation the lungs were isolated and blood perfused. Microvessel Cx43 expression was quantified by in situ immunofluorescence and microvessel permeability via a fluorescence method. To supplement the immunofluorescence data, protein levels were determined by immunoblots of lung tissue from endotoxin-instilled rats. Immunofluorescence and immunoblot together revealed that both Cx43 expression and microvessel permeability increased above baseline within a few hours after endotoxin instillation but declined progressively over the next few days. On day 5 postendotoxin, microvessel Cx43 declined to negligible levels, resulting in complete absence of intermicrovessel communication determined by photolytic uncaging of Ca2+. However, by day 14, both Cx43 expression and microvessel permeability returned to baseline levels. In contrast to Cx43, expression of microvessel vascular endothelial (VE)-cadherin, a critical determinant of vascular barrier integrity, exhibited an inverse trend by initially declining below baseline and then returning to baseline at a longer duration. Knockdown of vascular Cx43 by tail vein injection of Cx43 shRNA increased VE-cadherin expression, suggesting that reduction in Cx43 levels may modulate VE-cadherin levels in lung microvessels. Together, the data suggest that endotoxin challenge initiates interrelated changes in microvessel Cx43, VE-cadherin, and microvessel permeability, with changes in Cx43 temporally leading the other responses.

Early disruption of the alveolar-capillary barrier in a ricin-induced ARDS mouse model: neutrophil-dependent and -independent impairment of junction proteins

“…Regarding connexin 43, the data in the literature are controversial. While some studies showed increased expression of connexin 43 following ALI/ARDS (21,26), others reported an absence of connexin 43 within a few days after exposure to endotoxin (17). Unlike VE-cadherin, claudin 18 and 5, and connexin 43, in our hands, occludin levels failed to show a sign of decline at early time points after exposure to ricin, their reduction being observed…”

Connexin 43 gap junctions contribute to brain endothelial barrier hyperpermeability in familial cerebral cavernous malformations type III by modulating tight junction structure

“…We sought to identify proteins that are aberrantly expressed in stage 0 lesions that promote BBB hyperpermeability and enable lesion maturation. Aberrant elevation of Cx43 protein has recently been linked to increased barrier permeability (10, 11). Immunofluorescence staining of Cx43 in ccm3 +/– p53 –/– lesions revealed dramatically increased Cx43 expression in all lesion stages, particularly at stage 0, which correlates with lesion progression (Pearson coefficient r = 0.7074; P < 0.01; Fig.…”

“…Elevated Cx43 has recently been suggested to be detrimental to barrier permeability in GJ‐ and HC‐dependent mechanisms. Multiple studies in lung endothelia have demonstrated the rescue of barrier properties after suppression of inflammation‐induced Cx43 expression (11, 26). In brain endothelia, elevated GJIC is suggested to be pathogenic to barrier function because of the effects of Ca 2+ propagation on cytoskeletal integrity or localized endothelial cell death (27, 28).…”

“…Cx family members display tissue‐specific expression, with Cx37, Cx40, and Cx43 expressed in brain endothelia (8, 9). Aberrant elevation of Cx43 protein has recently been shown to be detrimental to barrier integrity, but whether this effect is dependent on Cx43 GJs or other functions of Cx43, including hemichannels (HCs) that do not directly oppose another Cx43 channel, has not been thoroughly described (10, 11).…”

“…Multiple hemorrhages of variable size are visible in the ccm3 +/2 p53 2/2 mouse (black arrows). Compiled lesion volumes of 3 ccm3 +/2 p53 2/2 mice assessed at age5,8,9,11, and 13 wk. B) Representative image of ccm3 +/2 p53 2/2 mouse lesions with Gd-DTPA leakage only (top) and Gd-DTPA + hemorrhage (bottom) as assessed by T1-weighted (Gd-DTPA) or T2*-weighted (hemorrhage) imaging.…”