N-cadherin and integrin blockade inhibit arteriolar myogenic reactivity but not pressure-induced increases in intracellular Ca

Abstract

The vascular myogenic response is characterized by arterial constriction in response to an increase in intraluminal pressure and dilatation to a decrease in pressure. This mechanism is important for the regulation of blood flow, capillary pressure, and arterial pressure. The identity of the mechanosensory mechanism(s) for this response is incompletely understood but has been shown to include the integrins as cell-extracellular matrix receptors. The possibility that a cell-cell adhesion receptor is involved has not been studied. Thus, we tested the hypothesis that N-cadherin, a cell-cell adhesion molecule in vascular smooth muscle cells (VSMCs), was important for myogenic responsiveness. The purpose of this study was to investigate: (1) whether cadherin inhibition blocks myogenic responses to increases in intraluminal pressure and (2) the effect of the cadherin or integrin blockade on pressure-induced changes in [Ca(2+)]i. Cadherin blockade was tested in isolated rat cremaster arterioles on myogenic responses to acute pressure steps from 60 to 100 mmHg and changes in VSMC Ca(2+) were measured using fura-2. In the presence of a synthetic cadherin inhibitory peptide or a function-blocking antibody, myogenic responses were inhibited. In contrast, during N-cadherin blockade, pressure-induced changes in [Ca(2+)]i were not altered. Similarly, vessels treated with function-blocking β1- or β3-integrin antibodies maintained pressure-induced [Ca(2+)]i responses despite inhibition of myogenic constriction. Collectively, these data suggest that both cadherins and integrins play a fundamental role in mediating myogenic constriction but argue against their direct involvement in mediating pressure-induced [Ca(2+)]i increases.

Keywords: cadherins; cell adhesion; integrins; mechanosensors; mechanotransduction; microcirculation; myogenic response; vascular smooth muscle.

Figure 1

Fluorescent images of cultured VSMCs fluorescently immunolabeled with N-cadherin (green). N-cadherin was distributed along cell edges (arrows in left panel) and in areas of cell–cell overlap between adjacent cells (dashed circle in right panel).

Figure 2

Myogenic responsiveness exhibited in isolated cremaster arterioles. Slope of myogenic pressure–diameter relationship as determined by regression analyses used as an index of myogenic gain. (A) Percentage control diameter of isolated arterioles exposed to increments in intraluminal pressure (time control-no drug treatment). (B) Slope before and after step increments in pressure for the two separate control series (n = 5). Data are presented as mean ± SEM.

Figure 3

Anti-N-cadherin treatment inhibited pressure-induced myogenic vasoconstriction. The reduced slope of myogenic pressure–diameter relationship (i.e., index of myogenic gain) as determined by regression analyses demonstrates the ability of anti-N-Cadherin treatment to inhibit the myogenic response. (A) Percentage control diameter of isolated arterioles exposed to increments in intraluminal pressure before and after the anti-N-Cadherin antibody treatment. (B) Slope of the pressure–diameter regression line before the addition of the antibody and while in the presence of the inhibitory antibody (n = 6). (C) Percentage control diameter of isolated arterioles exposed to increments in intraluminal pressure before and after addition of the anti-VE-Cadherin antibody treatment as a control. (D) Before the addition of the antibody and while in the presence of the abluminal control antibody (n = 4). Data are presented as mean ± SEM. *Significantly different (p < 0.05) from control (first series of step increments in pressure in the same vessel).

Figure 4

Histidine–alanine–valine peptide inhibited pressure-induced myogenic vasoconstriction. The reduced slope of myogenic pressure–diameter relationship (i.e., index of myogenic gain) as determined by regression analyses demonstrates the ability of HAV peptide to inhibit the myogenic response. (A) Percentage control diameter of isolated arterioles exposed to increments in intraluminal pressure before and after addition of the HAV peptide treatment. (B) Slope of the pressure–diameter regression line before the addition of the antibody and while in the presence of the abluminal inhibitory peptide (n = 6). (C) Percentage control diameter of isolated arterioles exposed to increments in intraluminal pressure before and after the control anti-VE-Cadherin antibody treatment. (D) Slope of the pressure–diameter regression line before the addition of the peptide and while in the presence of the abluminal control peptide (n = 6). Data are presented as mean ± SEM. *Significantly different from the first series of step increments in pressure in the same vessel.

Figure 5

Myogenic response exhibited in following a step change in intravascular pressure from 60 to 100 mmHg in isolated cremaster arterioles. [Ca²⁺]i was measured in these arterioles using fura-2 to estimate VSMC Ca²⁺ changes in the vessel wall. (A) Percentage of [Ca²⁺]i change in arterioles exposed to increases in intraluminal pressure. (B) Percentage of change in diameter before and after repeated step increase in pressure as a time control (n = 6). Data are presented as mean ± SEM.

Figure 6

The N-cadherin binding inhibitory peptide, HAV, inhibited pressure-induced myogenic vasoconstriction but not [Ca²⁺]i changes in the vessel wall. (A) Percentage of [Ca²⁺]i change in arterioles exposed to an increase in intraluminal pressure before and after HAV peptide treatment. (B) Percentage of change in diameter before and after treatment with the HAV inhibitory peptide (n = 9). (C) Percentage of [Ca²⁺]i change in arterioles exposed to an increase in intraluminal pressure before and after HGV (control) peptide treatment. (D) Percentage of change in diameter before and after treatment with the HGV control peptide (n = 7). Data are presented as mean ± SEM. *Significantly different from before peptide treatment (first series of step increments in pressure in the same vessel).

Figure 7

Anti-N-cadherin decreased pressure-induced myogenic vasoconstriction without a significant effect on [Ca²⁺]i increase in the vessel wall. (A) Percentage of [Ca²⁺]i change in arterioles exposed to an increase in intraluminal pressure before and after anti-N-cadherin treatment. (B) Percentage of change in diameter before and after treatment with the inhibitory antibody (n = 4). Data are presented as mean ± SEM.

Figure 8

β1- and β3-integrin blockade diminished pressure-induced myogenic without affecting [Ca²⁺]i increases in response to pressure. (A) Percentage of [Ca²⁺]i change in arterioles exposed to an increase in intraluminal pressure before and after anti-β1-integrin. (B) Percentage of change in diameter before and after treatment with the inhibitory antibody (n = 6). (C) Percentage of [Ca²⁺]i change in arterioles exposed to an increase in intraluminal pressure before and after anti-β3-Integrin. (D) Percentage of change in diameter before and after treatment with the inhibitory antibody (n = 5). Data are presented as mean ± SEM.