Integrin-linked kinase regulates endothelial cell nitric oxide synthase expression in hepatic sinusoidal endothelial cells

Abstract

Background & aims: Portal hypertension results from endothelial dysfunction after liver injury caused in part by abnormal production of endothelial cell derived nitric oxide synthase (eNOS). Here, we have postulated that endothelial mechanosensing pathways involving integrin-linked kinase (ILK) may play a critical role in portal hypertension, eNOS expression and function. In this study, we investigated the role of ILK and the small GTP-binding protein, Rho, in sinusoidal endothelial cell (SEC) eNOS regulation and function.

Methods: Primary liver SECs were isolated using standard techniques. Liver injury was induced by performing bile duct ligation (BDL). To examine the expression of Rho and ILK in vivo during wound healing, SECs were infected with constitutively active Rho (V14), a dominant negative Rho (N19) and constructs encoding ILK and a short hairpin-inhibiting ILK.

Results: Integrin-linked kinase expression was increased in SECs after liver injury; endothelin-1, vascular endothelial growth factor, and transforming growth factor beta-1 stimulated ILK expression in SECs. ILK expression in turn led to eNOS upregulation and to enhance eNOS phosphorylation and NO production. ILK knockdown or ILK (kinase) inhibition reduced eNOS mRNA expression, promoter activity, eNOS expression and ultimately NO production. In contrast, ILK overexpression had the opposite effect. Inhibition of ILK activity also disrupted the actin cytoskeleton in isolated SECs. Rho overexpression suppressed phosphorylation of the serine-threonine kinase, Akt and inhibited eNOS phosphorylation. Finally, inhibition of Rho function with the RGS domain of the p115-Rho-specific GEF (p115-RGS) significantly increased eNOS phosphorylation.

Conclusions: Our data suggest a potential role for ILK, the cytoskeleton and ILK signalling partners including Rho in regulating intrahepatic SEC eNOS expression and function.

Keywords: cirrhosis; liver; portal hypertension; rho; rho kinase; signalling.

Figure 1. Increased expression of ILK after liver injury

In (A-B), liver injury was induced by bile duct ligation and CCl₄ as described in “Experimental procedures” and sinusoidal endothelial cells were isolated and allowed to adhere for 18 hours on collagen-coated culture plates. Cells were harvested and lysates (50 μg total protein) were subjected to immunoblotting with specific antibody directed against ILK or μ-actin. Below the representative, respective immunoblots, data were quantified, normalized to the signal for μ-actin, and presented graphically (n-5, *`p < 0.05 vs. normal animals). In (C-G), bile duct ligation was performed as in “Experimental Procedures.” During surgery, an adenovirus containing GFP (Ad-GFP), ILK (Ad-ILK), or shILK (Ad-shILK) were injected, also as in “Experimental Procedures” in to the inferior vena cava (concentration of 10¹⁰/plaque-forming units/kg in 500 μl of PBS). Ten days later, livers were harvested and subjected to picrosirius red staining as described under “Experimental Procedures.” Representative liver sections are shown from rats undergoing sham BDL + Ad-GFP (C), BDL + Ad-GFP (D), or those additionally exposed to Ad-ShILK (E) or Ad-ILK (F). The scale bar shown represents 50 microns. Histomorphometric analysis was performed on random picrosirius red-stained liver sections (G). In (H), sinusoidal endothelial cells were serum-starved for 18 hours and exposed to EGF (10μg/ml), ET-1 (10 nM) VEGF (30 ng/ml) TGF-β (10 ng/mL) for 24 hours and subjected to immunoblotting for ILK detection. A representative ILK immunoblot is shown in the upper panel, and below it, a stripped blot re-probed for μ-actin; below the immunoblots, data from independent experiments were quantified, normalized to the signal for β-actin and presented graphically (n =3; *p <0.05 vs. control).

Figure 2. Regulation of ILK expression

In (A), sinusoidal endothelial cells were isolated and then serum-starved and exposed to ET-1 at the indicated concentrations for 24 hours and subjected to immunoblotting to detect ILK (50 μg total protein from the cell lysate). A representative immunoblot is shown in the upper panel, and below it, a stripped blot re-probed to detect β-actin; below the immunoblots, data from independent experiments were quantified, normalized to the signal for β-actin and presented graphically (n =3; *p <0.05 vs. control (no endothelin-1)). In (B), sinusoidal endothelial cells were as in (A), and were subsequently exposed to 20 nM ET-1 for 24 hours in the absence or presence of BQ-788 (20 nM, an endothelin-B receptor antagonist). In the upper panel of (B), representative immunoblots as in (A) are shown (ILK, top panel, and β-actin, lower panel), and in the bottom panel, data were quantified, normalized to the signal for β-actin, and presented graphically (n =3; *p <0.05 vs. control). In (C), sinusoidal endothelial cells were transduced with the indicated recombinant adenoviruses (Ad-ILK-expressing ILK, Ad-shILK-expressing small interfering short hairpin ILK RNA, or adenovirus containing the identical adenovirus backbone without a cDNA insert Ad-GFP) as described in “Experimental Procedures”, each at a multiplicity of infection of 100. Cell lysates (50 μg total protein) were subjected to immunoblotting with anti-ILK antibody as described in “Experimental Procedures”. Representative immunoblots are shown in the top panel of the figure, and in the bottom panel of the figure, data from independent experiments were quantified, normalized, and presented graphically (n=3; *p <0.01 vs. Ad-GFP).

Figure 3. ILK regulates P-eNOS and NO production

In (A), sinusoidal endothelial cells were infected with the indicated recombinant adenoviruses (Ad-ILK, Ad-shILK, or Ad-GFP), each at a multiplicity of infection of 100. Cells were washed and incubated in serum-free medium for 6 h, followed by exposure to ET-1 (10 nM) for 30 min. Cellular lysates (50 μg total protein) were subjected to immunoblotting with anti P-eNOS after which blots were stripped and reprobed with anti-total eNOS antibody and anti-β actin antibody. The data shown are representative of three different experiments, each performed with cells from a different isolation (n =3; *p < 0.05 vs. control; # p < 0.01 vs. Ad-ILK). Representative immunoblots are shown in the top panel of the figure, and in the bottom panel of the figure. In (B), conditioned medium from the same cells was collected, and nitrite levels were measured by chemiluminescence as in “Experimental Procedures” (n = 3; *p <0.005 versus control; # p < 0.01 versus Ad-ILK).

Figure 4. ILK inhibition disrupts the actin cytoskeleton

Sinusoidal endothelial cells were isolated from rat livers and exposed to 25μM QLT-0267 for 36h (bottom panel), DMSO was used as a control (top panel) since this is what QLT-0267 was mixed in prior to addition to medium. Cells were fixed and labeled with rhodamine-phalloidin (red) and DAPI (blue) as in Methods. Representative (of > 10 other examples) images are shown. The scale bar represents 5 microns.

Figure 5. ILK kinase inhibition negatively regulates P-eNOS and NO production

In (A), sinusoidal endothelial cells were serum-starved and exposed to QLT-0267 at the indicated concentration (μg/mL). Cells were harvested and lysates (50 μg total protein) were subjected to immunoblotting with specific antibody directed against P-eNOS. Below the representative, respective immunoblot, data were quantified, normalized to the signal for eNOS and β-actin, and presented graphically (n=5; *p < 0.05 vs. DMSO control). The immunoblots shown are representative of three different experiments, each performed with cells from a different isolation. In (B), NO production in the same sinusoidal endothelial cells was detected by chemiluminescence detection of nitrites in conditioned medium as described in “Experimental Procedures” (n =3; *, p <0.005 vs. DMSO control). In (C), sinusoidal endothelial cells were serum-starved and exposed to QLT-0267 as in (A), and luciferase activity was assayed as described in “Experimental Procedures” (n = 3; *P <0.05 vs. corresponding control). In (D), sinusoidal endothelial cells were infected with the indicated adenoviruses (Ad-ILK, Ad-shILK or Ad-GFP) as described under “Experimental Procedures,” each at a multiplicity of infection of 100 for 24 hours and luciferase activity was assayed as in C (n = 3; *p < 0.05 vs. corresponding control).

Figure 6. Rho differentially modulates Akt and eNOS phosphorylation

In (A & B), sinusoidal endothelial cells were infected with the indicated recombinant adenoviruses, respectively at a multiplicity of infection of 100 for 24 hours as in “Experimental Procedures”. Cell lysates (50 μg total protein) were subjected to immunoblotting with anti P-Akt antibody as described in “Experimental Procedures”. Below the representative, respective immunoblot, data were quantified, normalized to the signal for Akt, and presented graphically (n=5, *p < 0.05 vs. control). In (C), sinusoidal endothelial cells were infected with the indicated adenoviruses, each at a multiplicity of infection of 100 for 24 hours as in A &B, and mRNA levels were measured by RT-PCR as described as in “Experimental Procedures”, and the data presented graphically (n=3, * p < 0.05, vs. control). In (D), cells were as in (C), and cell lysates (50 μg total protein) were subjected to immunoblotting with the indicated antibodies (including a stripped blot reprobed for eNOS and β-actin); representative immunoblots are shown in the upper panel, and below it, data from independent experiments were quantified, normalized and presented graphically (n =3, *p < 0.05 vs. control).

Figure 7. Rho inhibition inhibits eNOS function

Sinusoidal endothelial cells were isolated and infected with recombinant RGS adenovirus at the indicated multiplicity of infection for 24 hours as in “Experimental Procedures”. Cell lysates (50 μg total protein) were subjected to immunoblotting with anti-phospho-eNOS antibody and a stripped blot re-probed for eNOS subsequently. Representative immunoblots are shown in the upper panel, and below it data from independent experiments were quantified, normalized and data presented graphically (n =3, *p <0.05 vs. control).

Figure 8. A working model for integrin linked kinase (ILK), Rho, and eNOS in sinusoidal endothelial cells

The figure highlights previously established ILK signaling patterns and emphasizes data from the current study. Specific mediators such as endothelin-1 (ET-1) and/or liver injury stimulate ILK expression. ET-1 and injury induced changes in ILK expression lead to upregulation of in Akt and eNOS signaling, and ultimately increased NO production. eNOS expression is also downregulated by Rho, leading to an effect that is opposite of ILK, which ultimately leads to reduced NO production.