Spatial distribution and mechanical function of elastin in resistance arteries: a role in bearing longitudinal stress

Abstract

Objective: Despite the role that extracellular matrix (ECM) plays in vascular signaling, little is known of the complex structural arrangement between specific ECM proteins and vascular smooth muscle cells. Our objective was to examine the hypothesis that adventitial elastin fibers are dominant in vessels subject to longitudinal stretch.

Methods and results: Cremaster muscle arterioles were isolated, allowed to develop spontaneous tone, and compared with small cerebral arteries. 3D confocal microscopy was used to visualize ECM within the vessel wall. Pressurized arterioles were fixed and stained with Alexa 633 hydrazide (as a nonselective ECM marker), anti-elastin, or anti-type 1 collagen antibody and a fluorescent nuclear stain. Exposure of cremaster muscle arterioles to elastase for 5 minutes caused an irreversible lengthening of the vessel segment that was not observed in cerebral arteries. Longitudinal elastin fibers were demonstrated on cremaster muscle arterioles using 3D imaging but were confirmed to be absent in cerebral vessels. The fibers were also distinct from type I collagen fibers and were degraded by elastase treatment.

Conclusions: These results indicate the importance of elastin in bearing longitudinal stress in the arteriolar wall and that these fibers constrain vascular smooth muscle cells. Differences between skeletal muscle and cerebral small arteries may reflect differences in the local mechanical environment, such as exposure to longitudinal stretch.

Figure 1

Effect of elastase treatment on the length of cannulated cremaster and cerebral small arteries. Panel a shows example images for a cremaster and cerebral vessel. The numbers 1–5 refer to specific time-points as shown in Panel b. Note that the cremaster vessel shows considerable lengthening while the cerebral vessel is relatively unaffected. Note also that in image 5 for the cremaster vessel the pipettes have been repositioned in the ‘x’ dimension to straighten the vessel. Images were collected in real-time using a stereomicroscope coupled to a CCD camera and digital video recorder. Avi files were subsequently imported into Image Pro software for continuous measurement of vessel length. Length of the cremaster vessel is shown on the left hand y-axis and the cerebral artery on the right hand axis. Panel c, group data showing that abluminal application of elastaste (0.05 U/ml, 5 min) led to a significant (p<0.05) increase in cremaster vessel segment length (n = 10).

Figure 2

Panel a, following elastase treatment vessels continued to show pressure-induced myogenic tone (left) although acute myogenic responsiveness was blunted (right). Panel b, following elastase treatment vessels (n = 6) showed dilation to ACh (10⁻⁶ M) and constriction to phenylephrine (10⁻⁶ M) (right) comparable to that under control conditions (left). Panel c, elastase treatment caused a leftward shift in the passive pressure – diameter relationship compared to baseline indicating an increase in vessel stiffness. Panel d - collagenase treatment caused dilation of myogenically active cremaster arterioles (n = 6). This effect was reversed by washing and was in contrast to the effect of elastase which caused irreversible lengthening of the cannulated vessel segments. Results are shown as mean ± SEM, * P < 0.05.

Figure 3

Panel a, elastase treatment (0.05 U/ml; 5 mins) does not cause lengthening of cerebral arteries (n = 8) despite causing lengthening of cremaster arterioles (n = 8). Panel b, elastase treatment causes an upward and leftward shift in the passive pressure – diameter relationship for cerebral vessels showing a decrease in distensibility (n = 6). Results are shown as mean ± SEM. * P < 0.05.

Figure 4

Example pseudocolored images of a cannulated and pressurized cremaster arteriole (A) and a similarly prepared small cerebral artery (B). The images represent adventitial (Panel a), mid-wall (Panel b) intimal (Panel c) and end view (Panel D) sections. Vessel segments were fixed while pressurized (see text for details) and stained with Alexa 633 (for ECM structures) and Yo-Pro-1 iodide (nuclei). Images are representative of n = 11 (cremaster) and 6 (cerebral). Movie files for the complete image stacks and 3D rotating representations are shown in the Supplementary Material.

Figure 4

Example pseudocolored images of a cannulated and pressurized cremaster arteriole (A) and a similarly prepared small cerebral artery (B). The images represent adventitial (Panel a), mid-wall (Panel b) intimal (Panel c) and end view (Panel D) sections. Vessel segments were fixed while pressurized (see text for details) and stained with Alexa 633 (for ECM structures) and Yo-Pro-1 iodide (nuclei). Images are representative of n = 11 (cremaster) and 6 (cerebral). Movie files for the complete image stacks and 3D rotating representations are shown in the Supplementary Material.

Figure 5

Panel a shows Alexa 633 hydrazide staining of adventitial fibers in a cannulated cremaster 1A segment. Panel b shows the same vessel segment stained with a specific elastin antibody and a secondary antibody conjugated with Alexa fluor 488. Insert shows secondary antibody only control (additional details of controls are provided in the Supplementary Material). Panel c shows the overlay image for panels a and b. Results are typical of n = 4. Yellow color indicates apparent colocalization suggesting that the Alexa 633 hydrazide stained fibers contain elastin. Panel d elastase (0.05 U/ml, 5 min) cuts the longitudinally arranged, elastin-containing fibers (typical of n = 3).

Figure 6

Example pseudocolored images showing adventitial (Panel a), mid-wall (Panel b) intimal (Panel c) and end view (Panel d) sections of a small cannulated mesenteric artery. Vessel segments were fixed while pressurized and stained with Alexa 633 (for ECM structures) and Yo-Pro-1 iodide (nuclei). Images are representative of n = 3 vessels. Movie files for the complete image stacks and 3D rotating representations are shown in the Supplementary Material.