Spatial and temporal regulation of gap junction connexin43 in vascular endothelial cells exposed to controlled disturbed flows in vitro

Abstract

Hemodynamic regulation of the endothelial gap junction protein connexin43 (Cx43) was studied in a model of controlled disturbed flows in vitro. Cx43 mRNA, protein expression, and intercellular communication were mapped to spatial variations in fluid forces. Hemodynamic features of atherosclerotic lesion-prone regions of the vasculature (flow separation and recirculation) were created for periods of 5, 16, and 30 h, with laminar shear stresses ranging between 0 and 13.5 dynes/cm2. Within 5 h, endothelial Cx43 mRNA expression was increased in all cells when compared with no-flow controls, with highest levels (up to 6- to 8-fold) expressed in regions of flow recirculation corresponding to high shear stress gradients. At 16 h, Cx43 mRNA expression remained elevated in regions of flow disturbance, whereas in areas of fully developed, undisturbed laminar flow, Cx43 expression returned to control levels. In all flow regions, typical punctate Cx43 immunofluorescence at cell borders was disrupted by 5 h. After 30 h of flow, disruption of gap junctions persisted in cells subjected to flow separation and recirculation, whereas regions of undisturbed flow were substantially restored to normal. These expression differences were reflected in sustained inhibition of intercellular communication (dye transfer) throughout the zone of disturbed flow (84.2 and 68.4% inhibition at 5 and 30 h, respectively); in contrast, communication was fully reestablished by 30 h in cells exposed to undisturbed flow. Up-regulation of Cx43 transcripts, sustained disorganization of Cx43 protein, and impaired communication suggest that shear stress gradients in regions of disturbed flow regulate intercellular communication through the expression and function of Cx43.

Figure 1

(A) Parallel-plate flow chamber. (B) Glass coverslip with steel frame and surface step (flow disturbance).

Figure 2

Flow streamlines in the chamber showing regions of flow separation, recirculation, reattachment, and recovery. (Lower) The corresponding shear stress distribution on the coverslip surface. The average shear stress gradients (slope of shear stress curve) in regions SSGR I–IV are 188, 182, 22, and 0 dynes/cm², respectively.

Figure 3

Spatial distribution of Cx43 mRNA expression. (A) Endothelial monolayers exposed to disturbed flows for 5 h. (B) Endothelial monolayers exposed to disturbed flows for 16 h. Each data point represents the average number of grains per cell (normalized to the no-flow control) in three to seven high-power fields (0.21 × 0.15 mm) from three separate experiments. Bars = means ± SD. The horizontal axis, x (mm), is the distance measured from the downstream edge of the step. The vertical, dotted lines mark the boundaries of the four SSGRs. The shear stress gradient in SSGRI and II is 182–188 dynes/cm² cm. The shear stress gradient is 22 dynes/cm² cm in SSGRIII and negligible in SSGRIV.

Figure 4

Cx43 gap junction protein distribution. (A) No-flow control. (B) Five-hour SSGRI/II. (C) Five-hour SSGRIII/IV. (D) Thirty-hour SSGRI/II. (E) Thirty-hour SSGRIII/IV. (Bar = 10 μm.)

Figure 5

Endothelial cell morphology in disturbed and undisturbed flow regions. Phase-contrast images of endothelial cells after 30-h flow. (A) Disturbed flow region SSGRI/II. The step is visible on the left edge of the photograph. Average cell aspect ratio (AR) was 2.4; average angle of orientation with flow direction (θ) was 40°. (B) Cells from the downstream recovered laminar flow region SSGRIII/IV. AR was 3.4 and θ was 15°. Flow is from left to right.

Figure 6

Functional GJIC (dye transfer experiments). Immediately after flow exposure, cells were microinjected with a combination of dyes (see Materials and Methods). (A and B) Fluorescent images of representative dye spread (Lucifer yellow) within the recirculation region (SSGRI/II) and downstream recovered laminar flow region (SSGRIII/IV), respectively, after 5 h of flow. (C and D) Corresponding images after 30-h flow. (E) No-flow control. Each Inset marks the injected cell (nontransferable rhodamine dextran signal), and the extent of cell communication is shown in histogram form.