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. 2016 Apr;173(7):1179-95.
doi: 10.1111/bph.13415. Epub 2016 Mar 6.

Induction of endothelium-dependent constriction of mesenteric arteries in endotoxemic hypotensive shock

Tzu-Ling Tseng  1   2   3 , Mei-Fang Chen  1   3   4 , Chin-Hung Liu  1   2   3 , Cheng-Yoong Pang  1   2 , Yung-Hsiang Hsu  2   5 , Tony J F Lee  1   2   3   6
Affiliations

Induction of endothelium-dependent constriction of mesenteric arteries in endotoxemic hypotensive shock

Tzu-Ling Tseng et al. Br J Pharmacol. 2016 Apr.

Abstract

Background and purpose: Effective management of hypotension refractory to vasoconstrictors in severe sepsis is limited. A new strategy to ameliorate endotoxemic hypotension by inducing endothelium-dependent constriction of large arteries was assessed.

Experimental approach: Endotoxemia in rats was induced by injection of LPS (10 mg·kg(-1), i.v.). Haemodynamics were measured in vivo, reactivity of isolated mesenteric arteries by myography and expression of proteins and enzyme activities by immunohistochemistry, biochemistry and molecular biology.

Key results: Six hours after LPS, the hypotension was promptly reversed following injection (i.v. or i.p.) of oroxylin-A (OroA) . In isolated LPS-treated but not normal mesenteric arteries, OroA (1-10 μM) induced endothelium-dependent, sustained constriction, blocked by endothelin-1 (ET-1) receptor antagonists. OroA further enhanced LPS-induced expression of endothelin-converting enzyme, ET-1 mRNA and proteins and ET-1 release, OroA also enhanced phosphorylation of Rho-associated protein kinase (ROCK) and reversed LPS-induced suppression of RhoA activities in smooth muscle of arteries with endothelium. Activated- phosphorylation of smooth muscle ROCK was blocked by ET-1-receptor antagonists and ROCK inhibitors. Moreover, OroA post-treatment suppressed, via inhibiting NF-κB activation, inducible NOS expression and circulating NO.

Conclusions and implications: Reversal of endotoxemic hypotensive by OroA was due to release of endothelial ET-1, upregulated by LPS, from mesenteric arteries, inducing prompt and sustained vasoconstriction via activation of vascular smooth muscle RhoA/ROCK-pathway. In late endotoxemia, OroA-induced vasoconstriction was partly due to decreased circulating NO. Activation of endothelium-dependent constriction in large resistance arteries and suppression of systemic inflammation offer new strategies for acute management of endotoxemic hypotensive shock.

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Figures

Figure 1
Figure 1
Oroxylin‐A (OroA) prevention and reversal of LPS‐induced cardiovascular dysfunction. LPS administration (10 mg·kg−1, i.v.) followed 1 h later by solvent1 (Solv1) (saline + Tween 80 at 9:1 ratio, i.v.) [ivLPS(1 h)/ivSolv1] drastically decreased mean arterial pressure (MAP), and, after a brief recovery, remained low over the course of 24 h (panel A). This LPS‐challenge also drastically decreased heart rate (HR) following an initial increase (panel B). OroA (15 mg·kg−1, i.v.) administered 1 h after LPS challenge [ivLPS(1 h)/ivOroA] completely prevented the fall in MAP (panel A) and HR (panel B) in the course of 24 h. The MAP and HR in normal control rats receiving normal saline (Sal, i.v.) followed 1 h later by Solv1 [ivSal(1 h)/ivSolv1] remained normal over the course of the experiment. OroA (15 mg·kg−1, i.v.) administered 6 h after LPS challenge [ivLPS(6 h)/ivOroA] almost immediately reversed the severely decreased MAP (panel C) and HR (panel D) to normal ranges. OroA did not affect the normal MAP or HR of the control group that received only saline challenge followed by OroA over the entire course [ivSal(6 h)/ivOroA] (panels C and D). Data are means ± SEM. n = number of experiments. *P < 0.05, significantly different from the normal control group. #P < 0.05, significantly different from the LPS alone group. Arrows in each panel indicate the injection time of LPS or OroA.
Figure 2
Figure 2
Oroxylin‐A (OroA)‐induced endothelium‐dependent, sustained constriction of LPS‐treated mesenteric arteries. Representative tracings showing the presence of intact endothelium (EC+) of arteries from normal control rats verified by ACh‐induced vasorelaxation (panels A, B and C). After wash (W), the artery pre‐contracted with phenylephrine (PE) was incubated with LPS (200 ng·mL−1) for 3 h. At the end of incubation, the mesenteric artery (panel A) constricted exclusively upon addition of OroA (dissolved in Solv2) in a concentration‐dependent manner. In the arteries with endothelium but without incubation with LPS, OroA did not induce vasoconstriction (panels B and C). In the (panel D) arteries denuded of the endothelium (EC−) as verified by lack of ACh‐induced vasorelaxation, OroA did not induce vasoconstriction following LPS incubation for 3 h. These endothelium‐dependent vasoconstrictions are summarized in panel E. The number in each column represents the number of rats examined. Data are means ± SEM. *P < 0.05, significantly different from the respective endothelium‐denuded artery. OroA‐induced vasoconstriction was estimated as percent of maximum contraction induced by KCl (75 mM). n = number of experiments.
Figure 3
Figure 3
Endothelin‐receptor antagonists block the Oroxylin‐A (OroA)‐induced vasoconstriction in LPS‐treated, endothelium‐intact, mesenteric arteries. The representative tracing showing that the presence of intact endothelium (EC+) of an arterial ring from a normal rat as verified by the presence of ACh‐induced vasorelaxation and in vitro incubation of this artery with LPS (200 ng·mL−1; 3 h) (panels A, B and C) were performed as described in Figure 2. OroA (1, 3 and 10 μM) in concentration‐dependent manner constricted the LPS‐incubated arteries (panels A, B and C). The vasoconstriction was reversed by BQ123 (1, 3 and 10 μM, panel A) and BQ788 (1, 3 and 10 μM, panel B) in a concentration‐dependent manner. These were summarized in panel C. Pre‐treatment with BQ788 (1–10 μM) (panels D and E) or BQ123 (1–10 μM) (panel F) also blocked OroA (3 and 10 μM)‐induced vasoconstriction. The number in each column represents the number of rats examined. Data are means ± SEM. *P < 0.05, significantly different as indicated. ns: not significant.
Figure 4
Figure 4
Oroxylin‐A (OroA) enhancement of LPS‐induced ET‐1 and endothelin‐converting enzyme (ECE) expression in isolated mesenteric arteries. ET‐1 gene and receptor expression in the control, LPS‐treated (200 ng·mL−1) and LPS plus OroA (3 μM)‐treated isolated mesenteric arteries with or without endothelium (EC+ or EC−, respectively) were examined 3 h after LPS challenge (panels A, B and C). The expression of both ET‐1 and ECE mRNA (panels A and B, respectively) and proteins for ET‐1 (panel D), ECE (panel E) and ET‐1 receptor (panel F) in arterial preparations (EC+) was increased 3 h after LPS‐challenge and was further enhanced 45 min following addition of OroA (panels A, B and C). Endothelial ET‐1‐immunofluorescence intensity (arrows) also was increased in LPS‐treated (200 ng·mL−1) for 8 h (panel G) and was further increased by addition of OroA (3 μM) (horizontal line 3 figure in panel G) in isolated mesenteric arteries with endothelium (EC+). In endothelium‐denuded arteries (EC−), no endothelial ET‐1‐immunofluorescence was detected after LPS and OroA treatments (panels G). Double staining with CD31 as a marker for endothelial cell (EC) were performed. These are summarized in panel H. Panel I indicates that ET‐1 concentrations in the Krebs solution containing segments of mesenteric arteries with endothelium were increased following incubation with LPS and was further increased by addition of OroA for 45 min. The nucleus in blue colour was stained with DAPI. SMC: smooth muscle cell. Scale bars represent 20 μm in Figure D. Data are means ± SEM. *P < 0.05, significantly different between the compared groups. Numbers in the columns represent number of experiments.
Figure 5
Figure 5
Oroxylin‐A (OroA)‐induced, endothelium‐dependent, constriction of LPS‐pre‐incubated arteries is mediated by endothelium‐dependent activation of the RhoA/ ROCK pathway invascular smooth muscle cells. The representative tracing in panel A showing the presence of intact endothelium (EC+) of a phenylephrine (PE)‐contracted mesenteric arterial ring from a normal rat as verified by the presence of ACh‐induced vasorelaxation. After wash (W) and contracted by PE again, the artery was incubated with LPS (200 ng·mL−1; 3 h) (tracings in panels A and B). At the end of the third hour (3 h), OroA (3 μM) repeatedly constricted LPS‐pretreated arteries (panels A and B). The vasoconstriction was blocked by Y27632 (0.05–1 μM) (panel A) and fasudil (1–10 μM) (panel B) concentration‐dependently. Results of maximum inhibition were summarized in panel C. Pre‐incubation with fasudil (1–10 μM) also blocked OroA‐induced constriction of arteries following LPS‐incubation for 3 h (panel D). These were summarized in panel E. In panel F, RhoA activities in arteries with (+) or without (−) endothelial cells (ECs) were decreased significantly following LPS incubation (200 ng·mL−1) for 3 h. Addition of OroA significantly reversed the diminished RhoA activities in arteries with endothelium (EC+) but not in those without endothelium (EC−), and the reversed activities were even greater than those of the control (panel F). The decreased RhoA activities in arteries with (+) or without (−) endothelium, on the other hand, were not reversed by wogonin (Wogo, 3 μM) or baicalein (Bai, 3 μM) (panel G). Representative immunofluorescence profiles in panel H showing that OroA‐enhanced phosphorylation of ROCK (indicative of ROCK activity) and contracted smooth muscle cells (SMCs, oval shape in horizontal row 3 pictures) in LPS‐treated arteries with endothelium, but did not do so in arteries without endothelium. The phosphorylated ROCK (p‐ROCK)‐immunoreactivities (red fluorescence, arrows) were shown by using antibodies against ROCKII‐Ser1366. p‐ROCK and β‐actin (a marker for smooth muscle cell/SMC) were double stained, showing both are almost coincident. The fluorescence intensity was measured by the grey values using Plot Z‐axis scale (see Methods). LPS incubation did not affect the basal p‐ROCK intensity in arteries with endothelium (panels H and I). Addition of OroA increased fluorescence intensity in LPS (200 ng·mL−1)‐treated endothelium‐intact (EC+) arteries (panels H and I) compared with the low fluorescence intensity in the control and LPS‐treated arteries. Wogo or Bai treatment did not reverse the LPS‐induced low fluorescence intensity (panel I). In endothelium‐denuded arteries (EC−), the decreased p‐ROCK‐immunoreactive cells and immunofluorescence intensity induced by LPS was not reversed by OroA, baicalein or wogonin (panel I). Fluorescence intensity of p‐ROCK‐immunoreactive SMCs was calculated from randomly selected four fields with a total of 10 cells, and the summary is shown in panel I. Scale = 20 μm. Data are means ± SEM. *P < 0.05, significantly different as indicated. In parallel, LPS treatment significantly decreased the ratio of contracted SMC (oval shape) versus relaxed SMCs (elongated shape) compared with that of the control [EC+, incubated in Sal/normal saline followed by solvent2 (Solv2)] in mesenteric arteries (panel J). The decreased ratio of the contracted SMC by LPS was significantly reversed by OroA (3 μM), but was not appreciably affected by Wogo (3 μM) or Bai (3 μM) (panel J). Data are means ± SEM. *P < 0.05, significantly different from the respective control. #P < 0.05, significantly different from the respective LPS group (panel J). The numbers in each column represents number of rats examined. ns: non‐significant.
Figure 6
Figure 6
OroA‐induced endothelium‐dependent activation of the RhoA/ ROCK pathway in vascular smooth muscle of LPS‐pretreated mesenteric arteries is sensitive to ET‐1 receptor blockade. Representative cross‐sections showing immunoreactivity to ET‐1 and phosphorylated Rho kinase (p‐ROCK) in the mesenteric arteries with endothelium (EC+) (panel A) of the control (incubated in Solv2 for 3 h, data not shown), and those following incubation for 3 h with LPS (200 ng·mL−1), LPS plus OroA (3 μM) and LPS plus OroA, BQ123 and BQ788 (10 μM). All control arteries exhibited basal immunoreactivity to ET‐1 and p‐ROCK (panel B) and ET‐1‐proteins and p‐ROCK‐proteins (panels C, D, E and F). ROCK activity in the smooth muscle cell (SMC) was estimated by immunoreactivity to p‐ROCK (red fluorescence, arrows) using antibodies against ROCKII‐Ser1366. Co‐staining of nuclei with DAPI (blue fluorescence, panel A) in arteries with endothelium (EC+) served as controls. Similar to the ET‐1 immunoreactivity shown in Figure 4, ET‐1 immunoreactivity (panels A and B), ET‐1‐proteins (panels C and D) and ET‐1 released in Krebs incubation medium (panel G) increased in the arteries (EC+) after LPS treatment, and the increase was further enhanced by addition of OroA (3 μM) in the LPS + OroA group. In arteries of this group (LPS + OroA), OroA also increased the immunoreactivity to p‐ROCK (middle bottom figure in panel A and panel B) and p‐ROCK‐proteins (panels C and F). The increased immunoreactivity to p‐ROCK and ‐proteins in these arteries were reversed to the basal levels (for fluorescence, see right bottom figure of panel A. and for proteins. see panels C and F) by combined treatment of BQ123 and BQ788 (10 μM) (the LPS + OroA + BQ123 + BQ788 group). The relative percent of ET‐1, RhoA and p‐ROCK proteins was normalized to actin, which served as an internal control and summarized as the relative density in percent (%) of ET‐1, RhoA and p‐ROCK expression. *P < 0.05, significantly different as indicated. Numbers in the columns represent number of experiments. L: lumen. Scale = 20 μm in ET‐1/CD31/DAPI panels and p‐ROCK/Actin/DAPI panels. The overall summary is depicted in panel H. Expression of ET‐1 vasoactive substances, which are in very low concentrations or absent in the endothelial cell (EC) of mesenteric arteries in normal rats, is increased in LPS‐treated, endotoxemic arteries. Upon application of OroA, ET‐1 is released to induce constriction of the vascular smooth muscle (VSMC) by acting on the ETA and ETB receptors, and RhoA. ET‐1 also activates the RhoA and ROCK pathway, leading to contraction of the VSMC. In addition, OroA inhibits expression of inducible NO synthase (iNOS) on the blood vessel wall with decreased NO synthesis from Arg (L‐arginine), providing additional mechanisms in directly diminishing vasodilation, and indirectly enhancing vasoconstriction via lessening NO inhibition of RhoA and ROCK activities.
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References

    1. Aird WC (2003). The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood 101: 3765–3777. - PubMed
    1. Alexander SPH, Fabbro D, Kelly E, Marrion N, Peters JA, Benson HE, et al. (2015a). The Concise Guide to PHARMACOLOGY 2015/16: Enzymes. Br J Pharmacol 172: 6024–6109. - PMC - PubMed
    1. Alexander SPH, Davenport AP, Kelly E, Marrion N, Peters JA, Benson HE, et al. (2015b). The Concise Guide to PHARMACOLOGY 2015/16: G Protein‐Coupled Receptors. Br J Pharmacol 172: 5744–5869. - PMC - PubMed
    1. Arqués O, Chicote I, Tenbaum S, Puig I, Palmer GH (2012). Standardized Relative Quantification of Immunofluorescence Tissue Staining. Protocol Exchange doi:10.1038/protex.2012.008. - DOI
    1. Azevedo LC, Park M, Schettino GP (2008). Novel potential therapies for septic shock. Shock 30: 60–66. - PubMed

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