Figure 1.
Depiction of internal elastic lamina fenestration and conversion for fenestrae quantification.
Autofluorescence of the internal elastic lamina was imaged via laser scanning confocal microscopy. Following 3-dimensional volume rendering, images were recorded and reconstructed using Volocity 6.0.0 to produce a black and white map which allowed for detection of fenestrae via Image J software analysis.
Figure 2.
Internal elastic lamina in mesenteric vs. skeletal muscle arteries.
Autofluorescence (green) of the internal elastic lamina (IEL) of mesenteric first, second, and third order arteries (top), and skeletal muscle popliteal, feed, first and second order arteries (bottom). Note that areas without green demonstrate lack of IEL, indicating fenestration and area of myoendothelial space. Bar = 10 µm.
Figure 3.
IEL fenestrae number, size, and total area.
While all vessel segments had fenestrae, skeletal muscle feed arteries had a significantly greater number than all other vessel segments within skeletal muscle (black) and mesentery (gray); (A). Individual hole area for skeletal muscle first and second order arterioles and first order mesenteric arterioles was significantly different from all other vessel segments (B). Compilation of total fenestrae number and individual fenestrae size produced total MEC area as a percent. First and second order skeletal muscle arterioles had significantly greater area than all other artery segments. * = P<0.05 vs all other artery segments; † = P<0.05 vs 1st order skeletal muscle arteriole; ‡ = P<0.05 vs 1st order mesenteric artery.
Figure 4.
Vessel diameter vs. total IEL fenestration.
A: Diameter (black bars) and total IEL Fenestration (gray bars) for skeletal muscle and mesenteric arteries for all vessels. B: Diameter (black bars) and total IEL Fenestration (gray bars) for size-matched 1st order skeletal muscle arterioles and 3rd order mesenteric arteries. * = P<0.05 vs skeletal muscle.
Figure 5.
KCa2.3 in skeletal muscle arteries.
A: Skeletal muscle popliteal (left) and 1st order (right) arteries immunolabeled for KCa2.3 (top 3 panels) or no primary antibody control (bottom panel). The top image represents a composite of DAPI-stained nuclei in the endothelium, autofluorescence of the internal elastic lamina (green), and KCa2.3 immunolabeling (red), and a compressed z-stack image endothelial cell surface (EC, top panel). The second image is a compressed z-stack image from the perspective of the smooth muscle face (SM), the third image is a cross-sectional slice, and the fourth is a compressed z-stack image of a no primary antibody control (NC). Red staining indicates endothelial expression of KCa2.3. B: Magnified image of the cross-sectional view of the IEL. Note the sections highlighted by dotted square boxes indicating larger MEC and area of KCa2.3 staining in 1st order arterioles. Bar = 10 µm. Representative of images from tissue isolated from at least 3 animals.
Figure 6.
KCa3.1 in skeletal muscle arteries.
A: skeletal muscle popliteal (left) and 1st order (right) arteries immunolabeled for KCa3.1 (top 3 panels) or no primary antibody control (bottom panel). The top image represents a composite of DAPI-stained nuclei in the endothelium, autofluorescence of the internal elastic lamina (green), and KCa3.1 immunolabeling immunolabeling (red), and a compressed z-stack image endothelial cell surface (EC, top panel). The second image is a compressed z-stack image from the perspective of the smooth muscle face (SM), the third image is a cross-sectional slice, and the fourth is a compressed z-stack image of a no primary antibody control (NC).Red staining indicates endothelial expression of KCa3.1. B: Magnified image of the cross-sectional view of the IEL. Note the sections highlighted by dotted square boxes indicating larger MEC and area of KCa3.1 staining in 1st order arterioles. Bar = 10 µm. Representative of images from tissue isolated from at least 3 animals.
Figure 7.
IP3R in skeletal muscle arteries.
A: skeletal muscle popliteal (left) and 1st order (right) arteries immunolabeled for IP3R (top 3 panels) or no primary antibody control (bottom panel). The top image represents a composite of DAPI-stained nuclei in the endothelium, autofluorescence of the internal elastic lamina (green), and IP3R immunolabeling immunolabeling (red), and a compressed z-stack image endothelial cell surface (EC, top panel). The second image is a compressed z-stack image from the perspective of the smooth muscle face (SM), the third image is a cross-sectional slice, and the fourth is a compressed z-stack image of a no primary antibody control (NC).Red staining indicates endothelial expression of IP3R. B: Magnified image of the cross-sectional view of the IEL. Note the sections highlighted by dotted square boxes indicating larger MEC and area of IP3R staining in 1st order arterioles. Bar = 10 µm. Representative of images from tissue isolated from at least 3 animals.
Figure 8.
TRPC3 in skeletal muscle arteries.
A: skeletal muscle popliteal (left) and 1st order (right) arteries immunolabeled for TRPC3 (top 3 panels) or no primary antibody control (bottom panel). The top image represents a composite of DAPI-stained nuclei in the endothelium, autofluorescence of the internal elastic lamina (green), and TRPC3 immunolabeling immunolabeling (red), and a compressed z-stack image endothelial cell surface (EC, top panel). The second image is a compressed z-stack image from the perspective of the smooth muscle face (SM), the third image is a cross-sectional slice, and the fourth is a compressed z-stack image of a no primary antibody control (NC).Red staining indicates endothelial expression of TRPC3. B: Magnified image of the cross-sectional view of the IEL. Note the sections highlighted by dotted square boxes indicating larger MEC and area of TRPC3 staining in 1st order arterioles. Bar = 10 µm. Representative of images from tissue isolated from at least 3 animals.
Figure 9.
“Working hypothesis” schematic for electrical communication within large and small skeletal muscle arteries.
Proposed vascular signaling among skeletal muscle popliteal (left) and 1st order (right) arteries. ER, endoplasmic reticulum; EC PM, endothelial cell plasma membrane; IEL, internal elastic lamina; SMC PM, smooth muscle cell plasma membrane; IP3R, inositol trisphosphate receptor; SK, small conductance calcium-activated potassium channel (KCa2.3); IK, intermediate conductance calcium-activated potassium channel (KCa3.1); TRPC3, canonical transient receptor potential channel 3; MEGJ, myoendothelial gap junction.