Characterization of pressure-mediated vascular tone in resistance arteries from bile duct-ligated rats

Abstract

In cirrhosis, changes in pressure-mediated vascular tone, a key determinant of systemic vascular resistance (SVR), are unknown. To address this gap in knowledge, we assessed ex vivo dynamics of pressurized mesenteric resistance arteries (diameter ~ 260 μm) from bile duct-ligated (BDL) and sham-operated (SHAM) rats and determined the underlying mechanisms. At isobaric intraluminal pressure (70 mmHg) as well as with step-wise increase in pressure (10-110 mmHg), arteries from SHAM-rats constricted more than BDL-rats, and had reduced luminal area. In both groups, incubation with LNAME (a NOS inhibitor) had no effect on pressure-mediated tone, and expression of NOS isoforms were similar. TEA, which enhances Ca2+ influx, augmented arterial tone only in SHAM-rats, with minimal effect in those from BDL-rats that was associated with reduced expression of Ca2+ channel TRPC6. In permeabilized arteries, high-dose Ca2+ and γGTP enhanced the vascular tone, which remained lower in BDL-rats that was associated with reduced ROCK2 and pMLC expression. Further, compared to SHAM-rats, in BDL-rats, arteries had reduced collagen expression which was associated with increased expression and activity of MMP-9. BDL-rats also had increased plasma reactive oxygen species (ROS). In vascular smooth muscle cells in vitro, peroxynitrite enhanced MMP-9 activity and reduced ROCK2 expression. These data provide evidence that in cirrhosis, pressure-mediated tone is reduced in resistance arteries, and suggest that circulating ROS play a role in reducing Ca2+ sensitivity and enhancing elasticity to induce arterial adaptations. These findings provide insights into mechanisms underlying attenuated SVR in cirrhosis.

Keywords: Pathology Section; cirrhosis; mesenteric arteries; myogenic tone; portal hypertension; vascular dysfunction.

Figure 1. Effect of isobaric conditions and increasing intraluminal pressure on diameter of small mesenteric resistance arteries from SHAM- and BDL-rats

A. Compared to SHAM-rats, fourth-order mesenteric arteries from BDL-rats pressurized at 70 mmHg and incubated with warm (37^ᵒC) PSS for 1 h developed reduced tone. B. Change in diameters of pressurized arterial segments are shown. Passive (maximal) diameter was determined by incubating the arteries in Ca²⁺-free PSS. C. To determine myogenic response, intraluminal pressure was increased in steps between 10-110 mmHg and spontaneous tone was allowed to develop until a stable diameter was achieved. The pressure-response was repeated in Ca²⁺-free PSS to determine the corresponding passive diameters. The myogenic tone is expressed as a percent of passive diameter (PD) and calculated as: (PD-achieved diameter)/PD×100. D. Arterial diameter at various pressure steps in PSS with and without Ca²⁺ are shown. **P < 0.01 when compared to SHAM-rats. † P < 0.05, ††† P < 0.001 when compared within treatment groups (n = 5-6/group).

Figure 2. Effect of NOS inhibition on myogenic response in small mesenteric resistance arteries from SHAM- and BDL-rats

Arteries were subjected to a series of intraluminal pressure steps between 10-110 mmHg before and after incubating with 300 μM LNAME (a NOS inhibitor for 60 min). The pressure-response was repeated in Ca²⁺-free PSS as described above. The arterial diameter at various pressure steps from A. SHAM-rats and B. BDL-rats are shown. C. Myogenic response was determined as described above. Incubation with LNAME improved MR minimally and non-significantly in both groups (n = 4-6/group). D. In mesenteric arteries from SHAM- and BDL-rats, the expression of mRNA for NOS isoforms was similar. E. The expression of NOS was also determined by immunoblotting; the immunoblots for eNOS and loading control are shown; The bar graph shows densitometry analysis. **P < 0.01, ***P < 0.001 when compared to SHAM-rats. † P < 0.05, †† P < 0.01 when compared within treatment groups; ns-not significant.

Figure 3. Effect of an agonist and TEA on vascular tone in pressurized mesenteric arteries from SHAM- and BDL-rats

A. In the arteries from BDL-rats, U46619 (a thromboxane analogue and a ROCK activator)-mediated tone is reduced when compared to SHAM-rats. At 70 mmHg, arteries developed spontaneous vasoconstriction that was further augmented by 10 μM U46619. Incubation with 300 μM LNAME for 60 min (a NOS inhibitor) enhanced U46619-mediated vascular tone in the SHAM-rats only and had no effect in those from the BDL-rats. Arterial diameters in the B. absence and C. presence of LNAME are shown (n = 5-6/group). D. TEA (5 mM, a Ca²⁺-activated K⁺ channel blocker) enhanced vascular tone in pressurized arteries from the SHAM-rats and had minimal effect on those from the BDL-rats; peak and plateau responses are shown (n = 4-6/group). Effect of TEA was determined in arteries incubated with LNAME. *P < 0.05, ***P < 0.001 when compared to SHAM-rats. †† P < 0.01, ††† P < 0.001 when compared within treatment groups.

Figure 4. Expression of ion channels in small mesenteric resistance arteries from SHAM- and BDL-rats

A. Immunoblots for Ca_v1.2, TRPC3 and β-actin (loading control) from small mesenteric artery homogenates indicate no differences among both groups. The bar graphs show densitometry analyses for Ca_v1.2 and TRPC3. B. qPCR for Ca_v1.2. Representative IHC photographs and qPCR indicate that in the arteries from the BDL-rats, expressions of C., D. BK_Ca and E., F. TRPC6 are reduced when compared to SHAM-rats (n = 3-5/group). G. The immunoblots for both BK_Ca and TRPC6 and their loading controls are shown. The bar graphs show densitometry analyses for BK_Ca and TRPC6 (n = 3/group). *P < 0.05, **P < 0.01 when compared to SHAM-rats.

Figure 5. Ca²⁺ sensitivity of small mesenteric arteries from SHAM- and BDL-rats

In β-escin-permeabilized arteries pressurized at 70 mmHg, the effect of high-dose Ca²⁺ was assessed. A. In response to pCa4.5, vascular tone increased in all rats but remained lower in the BDL-rats compared to SHAM-rats. B. Arterial diameters at pCa9.0 and pCa4.5 are shown (n = 4-5/group). C. Immunoblots indicate reduced arterial ROCK2 and pMLC expression in the BDL-rats (n = 3/group). D.-F. Summary data for ROCK2, RhoA and pMLC. G. Compared to SHAM-rats, permeabilized arteries from the BDL-rats developed reduced tone when stimulated with 100 μM γGTP (in pCa7.5). H. Arterial diameters before and after incubation with γGTP are shown (n = 4-5/group). *P < 0.05, **P < 0.01, ***P < 0.001 when compared to SHAM-rats. † P < 0.05, ††† P < 0.001 when compared within treatment groups.

Figure 6. Effect of BDL on arterial elasticity

A. After development of MT in isobaric conditions (within 1 h), the luminal area decreased in both but significantly more in the arteries from SHAM-rats than BDL-rats; B. Respective changes in luminal area. C. Arterial wall thickness was more in the BDL-rats, but after development of MT, was equal to those in the SHAM-rats (n = 16-17/group). To determine the effect of pressure on passive distension, arteries were incubated in Ca²⁺-free PSS. D. Increasing intraluminal pressure led to enhanced arterial distension in the BDL-rats. E. Arterial wall thickness in Ca²⁺-free PSS across intraluminal pressure range is shown. F. In the arteries from BDL-rats, stress-strain relation curve shifted right- and up-ward (n = 16/group). Arterial expression of mRNA for G. collagen 1a1, H. collagen 3a1, I. MMP-9 and J. MMP-2 are shown (n = 5-6/group). K. The immunoblots for both MMP-9, collagen 1 and their loading controls are shown. L. The bar graphs show densitometry analyses for MMP-9 and collagen 1 (n = 3/group). *P < 0.05, **P < 0.01, ***P < 0.001 when compared to SHAM-rats. †† P < 0.01, ††† P < 0.001 when compared within treatment groups.

Figure 7. Effect of BDL and ROS on vascular MMP-9 activity

A. Representative gelatin zymography indicates that in arteries from BDL-rats, MMP-9 activity is increased. B. Summary data (n = 3/group). Circulating ROS levels were measured in the plasma from BDL- and SHAM-rats. In BDL-rats, the plasma C. MDA levels and D. DCDF fluorescence were markedly increased when compared to SHAM-rats (n = 4/group). These data indicated that in cirrhosis, systemic circulation is an ample source of oxidative stress. Representative zymography indicates that incubation of rat VSMCs with both E. peroxynitrite and F. SIN-1 induces MMP-9 activity. G. Immunoblots and their densitometry data (I) indicate that in rat VSMCs, peroxynitrite reduces ROCK2 expression (n = 3/group). Arabic numerals indicate densitometric values. *P < 0.05 when compared to vehicle-treated cells. ***P < 0.001 when compared to SHAM-rats.