- Email: [email protected]
598 HEMODYNAMIC AND RENAL EFFECTS OF AN ORAL PRODRUG OF NOREPINEPHRINE IN PORTAL HYPERTENSIVE RATS
POSTERS Results: In vivo study: NAC reduced plasmatic ammonia level (from 1.5±0.2 mg/dl to 1±0.3 mg/dl, p < 0.05) and decreased oxidative stress markers significantly The NAC improved TMT-B and SDT tests. In “in vivo study”: NH4 increased significantly MDA production that was reverted by NAC or exogenous GSH addition, NH4 induced a decreased brain Glutamate transport in astrocyte cells with Glutamate accumulation in extra-cellular compartment and a diminution of intracellular production of Glutamine The presence of antioxidant substance was able to revert the Glutamate accumulation in extracellular compartment and to promote the uptake of Glutamate in astrocyte and its conversion into Glutamine. Conclusions: NAC therapy improves the grade of hepatic encephalophaty and reduces the number of major episodes of HE. In “in vitro system NAC is able to improve NH4, metabolism decreasing the oxidative stress markers. Thus, this might explain how NAC improves HE in vivo. 596 NEUROPEPTIDE Y (NPY) CORRECTS PRONOUNCED VASCULAR SYMPATHOADRENERGIC DESENSIBILISATION OF MESENTERIC ARTERIES IN EXPERIMENTAL PORTAL HYPERTENSION P. Dietrich, L. Moleda, J. Schoelmerich, R.H. Straub, R. Wiest. Dept. Internal Medicine I University Hospital, Regensburg, Germany E-mail: [email protected] Splanchnic arterial vasodilation ist the pathophyisological hallmark of hemodynamic disturbances in portal hypertension. A compensatory increase in sympathetic nerve activity is well-known. However, whether (a) this leads to vascular desensibilisation and (b) the sympathoadrenergic neurotransmitter NPY plays any role is unclear so far. Therefore, we used portal-vein ligated (PVL) and CCl4-induced cirrhotic rats (LC) and sham-operated rats (Sham) for in-vitro perfusion of the mesenteric arterial bed. Desensibilisation was induced by repetitive, periarterial electrical sympathetic nerve stimulation (50 V, 1 ms, 30 s) and this protocol was repeated after incubation with NPY (50nM) (second perfusion cycle). Results: Maximal stimulated perfusion pressure (vascular contractility) and latest remaining minimal perfusion pressure at the end of stimulation series was significantly lower in PVL and LC rats as compared to Sham rats (table 1). The decrease in perfusion pressure due to repetitive nerve stimulation (percent from peak value: representing degree of desensibilisation) was significantly enhanced in PVL and LC rats as compared to sham rats (table 1). After incubation with NPY no significant difference between study groups was observed for these parameters. Portal hypertensive rats but not sham rats did present with a marked NPY-induced improvement in vascular contractility and responsiveness to nerve stimulation. Table 1 Perfusion pressure Group/Parameter Sham PVL Cirrhosis
Max. (mmHg) 140.6±12.9 99.4±16.1** 81.8±10.2**
Decrease in Perf. pressure Min. (mmHg)
Max. absolute (mmHg)
Max. in Percent (%)
102.8±30.3 44.5±8.8** 45.2±12.3**
37.8±23.3 54.9±25.2 36.5±13.8
28.9±17.3 55.3±26.2* 44.7±15.6*
*: p < 0.05 and **: p < 0.01 vs. Sham.
Conclusions: In experimental portal hypertension mesenteric arteries not only present with vascular hypocontractility but also pronounced desensibilsation to repetitive sympathoadrenergic stimulation. NPY restores vascular contractility as well as -sensibility for sympathetic nerve stimulation making it a potential promising therapeutic vasoactive agent in portal hypertension.
597 EFFECT OF NEONATAL CAPSAICIN TREATMENT ON SYMPATHETIC ATROPHY AND HEMODYNAMIC ALTERATIONS OF PORTAL HYPERTENSIVE RATS N. Ezkurdia, M. Coll, I. Raurell, S. Cuenca, J. Genesca, ` M. Martell. Liver Diseases Laboratory, Liver Unit-Department of Internal Medicine, Hospital Universitari Vall d’Hebron, Institut de Recerca (VHIR), Barcelona, Spain E-mail: [email protected] Background: During cirrhosis, pressure changes detected at portal level could be the afferent signal responsible for the sympathetic atrophy observed in the superior mesenteric artery (SMA) in portal hypertensive rats. The blockage of the afferent nerves in portal vein ligated (PVL) adult rats prevents sympathetic atrophy and hemodynamic alterations during portal hypertension. Objective: To clarify the role of afferent sensory innervation in the development of sympathetic atrophy and hyperdynamic circulation through permanent ablation of the afferent nerves. Methods: 1. Rat pups treated with capsaicin or vehicle at 48 hours of life and PVL surgery (n = 12, n = 11) or sham (n = 5, n = 9) during adulthood. Capsaicin causes selective degeneration of sensory afferent neurons releasing calcitonin gene related peptide (CGRP). 2. Hemodynamic measurements: mean arterial pressure (MAP) and portal pressure (PP), mesenteric flow (SMABF) and resistance (SMAR). 3. Immunohistochemistry of tyrosine hydroxylase (Th) in SMA. 4. Immunofluorescence of CGRP and neuronal nitric oxide synthase in SMA. 5. Western blot of Th in mesenteric resistance arteries. Results: In PVL rats, capsaicin produced a 70% response (n = 8). These animals were very similar to normal rats both in hemodynamic values (except PP) and sympathetic innervation parameters. In contrast, compared to vehicle treated PVL, they showed a higher MAP (122.4±5.2 vs. 106.5±1.8 mmHg, p = 0.006) and SMAR (24.1±1.2 vs. 14.4± 1.2 mmHg/mLmin100g, p < 0.001), and a lower SMABF (4.5±0.2 vs. 6.3±0.4 mL/min100g, p = 0.007) with a significant increase in total nervous area of SMA (p = 0.003), Th staining area (p < 0.001) and Th expression in mesenteric resistance arteries (63% increase, p < 0.001). All capsaicin treated animals showed loss of CGRP, regardless of their level of response, suggesting that prevention of hemodynamic changes does not depend on the absence of vasodilator CGRP, but most probably on the afferent sensory signal. Capsaicin treatment did not affect nitrergic nerves, which did not co-localize with CGRPergics. Conclusion: The contribution of the adrenergic system atrophy in the splanchnic vasodilation of portal hypertension is supported by the simultaneous prevention of the development of the sympathetic atrophy and hemodynamic alterations through blockage of afferent sensory nerves. The afferent signal might be the triggering factor for the alterations of the mesenteric vasculature. 598 HEMODYNAMIC AND RENAL EFFECTS OF AN ORAL PRODRUG OF NOREPINEPHRINE IN PORTAL HYPERTENSIVE RATS M. Coll, I. Raurell, N. Ezkurdia Garmendia, S. Cuenca, M. Martell, J. Genesca. ` Liver Diseases Laboratory, Liver Unit-Department of Internal Medicine, Hospital Universitari Vall d’Hebron, Institut de Recerca (VHIR), Barcelona, Spain E-mail: [email protected] Background: Oral administration of Droxidopa, a synthetic amino acid, increases blood pressure and improves orthostatic tolerance in patients with orthostatic hypotension, due to its conversion to norepinephrine (NE) catalyzed by dopa decarboxylase (DDC).
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POSTERS Aim: To evaluate the effects of the oral administration of droxidopa on the hemodynamic and renal alterations of portal hypertensive rats in portal vein ligated (PVL) and bile duct ligated (BDL) models. Methods: Droxidopa (25 mg/kg) (Chelsea Therapeutics Inc.) or vehicle were orally administrated to PVL (n = 7;n = 9), BDL (n = 6;n = 6) and sham (n = 5;n = 5) rats. A group of 4 PVL rats received carbidopa (DDC inhibitor, 30 mg/kg, i.p.) 30 minutes before drug administration. Hemodynamic parameters were continuously registered and urine collected during two hours after treatment. NE plasma levels were determined 40 min after treatment. Results: PVL rats treated with droxidopa significantly increased mean arterial pressure (MAP) and mesenteric resistance (SMAR), decreased mesenteric flow (SMABF) and did not modify portal pressure compared to vehicle. In BDL rats, droxidopa caused the same beneficial effects observed in PVL: Maximum increase in MAP was 113.1±5.8 vs. 74.3±2.2 mmHg (p < 0.001), in SMAR was 23.01±3.1 vs. 13.8±2.1 mmHg (p = 0.04) and maximum decrease in SMABF was 3.8±0.5 vs. 5.5±0.5 mmHg (p = 0.05). Droxidopa also caused a significant decrease in portal blood flow (4.2±0.4 vs. 7.3±0.9 ml/min100gr, p = 0.019) and an increase in intrahepatic resistance (4.4±0.4 vs. 2.3±0.3 mmHg/mL/min100gr, p = 0.003) in BDL rats. Droxidopa therapy produced a significant increase in 2-hour diuresis, in both models, PVL (1.4±0.3 vs. 0.3±0.1 mL, p < 0.001) and BDL (2.4±0.3 vs. 0.9±0.3 mL, p = 0.004), but did not cause significant changes in renal hemodynamics (renal flow or resistance). NE plasma levels increased 10 times after treatment. Carbidopa blunted the hemodynamic response to droxidopa and the increase of NE. Preliminary results of a chronic droxidopa administration study (15 mg/kg, bid, p.o, for 5 days) in BDL rats showed the same beneficial hemodynamic and diuretic effects determined in the acute study. Conclusion: Droxidopa markedly improves the splanchnic circulatory dysfunction and diuresis of portal hypertensive rats. These data suggest that droxidopa might be an effective therapeutic agent for the hemodynamic and renal alteration of cirrhosis.
Figure: Droxidopa effect on SMAR in BDL rats.
599 TRANSDUCTION OF ADENOVIRUS ENCODING DOMINANT-NEGATIVE SOLUBLE PDGFRb IMPROVES SYSTEMIC HEMODYNAMICS, PORTAL PRESSURE AND HEPATIC FIBROSIS IN RATS CHRONICALLY TREATED WITH CCL4 1 G. Fernandez-Varo ´ , V. Reichenbach1 , J. Ros1 , R. Weiskirchen2 , 3 G. Casals1 , P. Melgar-Lesmes1 , M. Morales-Ruiz1 , W. Jimenez ´ . 1 Biochemistry and Molecular Genetics Service, Hospital Cl´ınic i Provincial de Barcelona, IDIBAPS, CIBERehd, University of Barcelona, Barcelona, Spain; 2 Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital, Aachen, Germany; 3 Biochemistry and Molecular Genetics Service and Department of Physiology I, Hospital Cl´ınic i Provincial de Barcelona, IDIBAPS, CIBERehd, University of Barcelona, Barcelona, Spain E-mail: [email protected]
Background and Aims: During hepatic fibrogenesis, plateletderived growth factor b (PDGFb) is the most potent stimulus for proliferation and migration of stellate cells. PDGFb receptor (PDGFRb) expression is an important phenotypic change in myofibroblastic cells that mediates proliferation and chemotaxis. In hepatic tissue of patients with chronic liver disease, expression of PDGF and its receptor correlates with the degree of necroinflammation and fibrosis. First, we analyzed the relationship between the activation of PDGFRb, hemodynamic deterioration and increased liver fibrosis in rats subjected to chronic inhalation of CCl4 and second, we investigated the hemodynamic effects produced by an adenovirus encoding a dominant-negative soluble PDGFRb (sPDGFRb). Methods: The first protocol included 6 control and 21 rats induced to hepatic fibrosis for 13 (n = 10) or 18 weeks (n = 11). Thereafter, mean arterial pressure (MAP) and portal pressure (PP) were recorded, samples of liver tissue were collected and PDGFRb mRNA expression (real-time PCR) and collagen content (Sirius red) were assessed. The second protocol included 30 rats induced to hepatic fibrosis for 18 weeks. The animals were randomized into three groups (n = 10) that were iv given saline and adenovirus encoding sPDGFRb (5×1010 pfu) or b-gal (5×1010 pfu). Following 7 days, MAP, PP, serum sPDGFRb and hepatic collagen content were measured. Results: Animals treated for 18 weeks showed a significant increase in PDGFRb mRNA than those treated for 13 weeks (p < 0.01) and control rats (p < 0.01). In rats treated with CCl4 the percentage of fibrous tissue area ranged from moderate fibrosis (2.7%) to cirrhosis (19.1%). A significant correlation between the degree of fibrosis and hemodynamic changes (MAP: r2 = 0.6, p < 0.001; PP: r2 = 0.56, p < 0.001) or PDGFRb activation (r2 = 0.5, p < 0.05) were observed. Fibrotic rats transduced with adenovirus encoding for sPDGFRb showed increased MAP, decreased PP and reduced collagen content (124±2 mmHg; 6.7±0.3 mmHg; 7.54±0.27%) in comparison to fibrotic rats receiving b-gal (114±3 mmHg, p < 0.05; 8.7±0.6 mmHg, p < 0.05; 8.64±0.33%, p < 0.05) or saline (113±2 mmHg, p < 0.05; 8.6±0.4 mmHg, p < 0.05; 8.55±0.29%, p < 0.05). Conclusions: PDGFRb activation closely correlates with hemodynamic disorders and increased fibrosis in rats chronically treated with CCl4 . Transduction of adenovirus expressing sPDGFRb improves systemic hemodynamics and attenuates the degree of liver fibrosis in CCl4 treated rats.
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Max. (mmHg) 140.6±12.9 99.4±16.1** 81.8±10.2**
Decrease in Perf. pressure Min. (mmHg)
Max. absolute (mmHg)
Max. in Percent (%)
102.8±30.3 44.5±8.8** 45.2±12.3**
37.8±23.3 54.9±25.2 36.5±13.8
28.9±17.3 55.3±26.2* 44.7±15.6*
*: p < 0.05 and **: p < 0.01 vs. Sham.
Conclusions: In experimental portal hypertension mesenteric arteries not only present with vascular hypocontractility but also pronounced desensibilsation to repetitive sympathoadrenergic stimulation. NPY restores vascular contractility as well as -sensibility for sympathetic nerve stimulation making it a potential promising therapeutic vasoactive agent in portal hypertension.
597 EFFECT OF NEONATAL CAPSAICIN TREATMENT ON SYMPATHETIC ATROPHY AND HEMODYNAMIC ALTERATIONS OF PORTAL HYPERTENSIVE RATS N. Ezkurdia, M. Coll, I. Raurell, S. Cuenca, J. Genesca, ` M. Martell. Liver Diseases Laboratory, Liver Unit-Department of Internal Medicine, Hospital Universitari Vall d’Hebron, Institut de Recerca (VHIR), Barcelona, Spain E-mail: [email protected] Background: During cirrhosis, pressure changes detected at portal level could be the afferent signal responsible for the sympathetic atrophy observed in the superior mesenteric artery (SMA) in portal hypertensive rats. The blockage of the afferent nerves in portal vein ligated (PVL) adult rats prevents sympathetic atrophy and hemodynamic alterations during portal hypertension. Objective: To clarify the role of afferent sensory innervation in the development of sympathetic atrophy and hyperdynamic circulation through permanent ablation of the afferent nerves. Methods: 1. Rat pups treated with capsaicin or vehicle at 48 hours of life and PVL surgery (n = 12, n = 11) or sham (n = 5, n = 9) during adulthood. Capsaicin causes selective degeneration of sensory afferent neurons releasing calcitonin gene related peptide (CGRP). 2. Hemodynamic measurements: mean arterial pressure (MAP) and portal pressure (PP), mesenteric flow (SMABF) and resistance (SMAR). 3. Immunohistochemistry of tyrosine hydroxylase (Th) in SMA. 4. Immunofluorescence of CGRP and neuronal nitric oxide synthase in SMA. 5. Western blot of Th in mesenteric resistance arteries. Results: In PVL rats, capsaicin produced a 70% response (n = 8). These animals were very similar to normal rats both in hemodynamic values (except PP) and sympathetic innervation parameters. In contrast, compared to vehicle treated PVL, they showed a higher MAP (122.4±5.2 vs. 106.5±1.8 mmHg, p = 0.006) and SMAR (24.1±1.2 vs. 14.4± 1.2 mmHg/mLmin100g, p < 0.001), and a lower SMABF (4.5±0.2 vs. 6.3±0.4 mL/min100g, p = 0.007) with a significant increase in total nervous area of SMA (p = 0.003), Th staining area (p < 0.001) and Th expression in mesenteric resistance arteries (63% increase, p < 0.001). All capsaicin treated animals showed loss of CGRP, regardless of their level of response, suggesting that prevention of hemodynamic changes does not depend on the absence of vasodilator CGRP, but most probably on the afferent sensory signal. Capsaicin treatment did not affect nitrergic nerves, which did not co-localize with CGRPergics. Conclusion: The contribution of the adrenergic system atrophy in the splanchnic vasodilation of portal hypertension is supported by the simultaneous prevention of the development of the sympathetic atrophy and hemodynamic alterations through blockage of afferent sensory nerves. The afferent signal might be the triggering factor for the alterations of the mesenteric vasculature. 598 HEMODYNAMIC AND RENAL EFFECTS OF AN ORAL PRODRUG OF NOREPINEPHRINE IN PORTAL HYPERTENSIVE RATS M. Coll, I. Raurell, N. Ezkurdia Garmendia, S. Cuenca, M. Martell, J. Genesca. ` Liver Diseases Laboratory, Liver Unit-Department of Internal Medicine, Hospital Universitari Vall d’Hebron, Institut de Recerca (VHIR), Barcelona, Spain E-mail: [email protected] Background: Oral administration of Droxidopa, a synthetic amino acid, increases blood pressure and improves orthostatic tolerance in patients with orthostatic hypotension, due to its conversion to norepinephrine (NE) catalyzed by dopa decarboxylase (DDC).
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POSTERS Aim: To evaluate the effects of the oral administration of droxidopa on the hemodynamic and renal alterations of portal hypertensive rats in portal vein ligated (PVL) and bile duct ligated (BDL) models. Methods: Droxidopa (25 mg/kg) (Chelsea Therapeutics Inc.) or vehicle were orally administrated to PVL (n = 7;n = 9), BDL (n = 6;n = 6) and sham (n = 5;n = 5) rats. A group of 4 PVL rats received carbidopa (DDC inhibitor, 30 mg/kg, i.p.) 30 minutes before drug administration. Hemodynamic parameters were continuously registered and urine collected during two hours after treatment. NE plasma levels were determined 40 min after treatment. Results: PVL rats treated with droxidopa significantly increased mean arterial pressure (MAP) and mesenteric resistance (SMAR), decreased mesenteric flow (SMABF) and did not modify portal pressure compared to vehicle. In BDL rats, droxidopa caused the same beneficial effects observed in PVL: Maximum increase in MAP was 113.1±5.8 vs. 74.3±2.2 mmHg (p < 0.001), in SMAR was 23.01±3.1 vs. 13.8±2.1 mmHg (p = 0.04) and maximum decrease in SMABF was 3.8±0.5 vs. 5.5±0.5 mmHg (p = 0.05). Droxidopa also caused a significant decrease in portal blood flow (4.2±0.4 vs. 7.3±0.9 ml/min100gr, p = 0.019) and an increase in intrahepatic resistance (4.4±0.4 vs. 2.3±0.3 mmHg/mL/min100gr, p = 0.003) in BDL rats. Droxidopa therapy produced a significant increase in 2-hour diuresis, in both models, PVL (1.4±0.3 vs. 0.3±0.1 mL, p < 0.001) and BDL (2.4±0.3 vs. 0.9±0.3 mL, p = 0.004), but did not cause significant changes in renal hemodynamics (renal flow or resistance). NE plasma levels increased 10 times after treatment. Carbidopa blunted the hemodynamic response to droxidopa and the increase of NE. Preliminary results of a chronic droxidopa administration study (15 mg/kg, bid, p.o, for 5 days) in BDL rats showed the same beneficial hemodynamic and diuretic effects determined in the acute study. Conclusion: Droxidopa markedly improves the splanchnic circulatory dysfunction and diuresis of portal hypertensive rats. These data suggest that droxidopa might be an effective therapeutic agent for the hemodynamic and renal alteration of cirrhosis.
Figure: Droxidopa effect on SMAR in BDL rats.
599 TRANSDUCTION OF ADENOVIRUS ENCODING DOMINANT-NEGATIVE SOLUBLE PDGFRb IMPROVES SYSTEMIC HEMODYNAMICS, PORTAL PRESSURE AND HEPATIC FIBROSIS IN RATS CHRONICALLY TREATED WITH CCL4 1 G. Fernandez-Varo ´ , V. Reichenbach1 , J. Ros1 , R. Weiskirchen2 , 3 G. Casals1 , P. Melgar-Lesmes1 , M. Morales-Ruiz1 , W. Jimenez ´ . 1 Biochemistry and Molecular Genetics Service, Hospital Cl´ınic i Provincial de Barcelona, IDIBAPS, CIBERehd, University of Barcelona, Barcelona, Spain; 2 Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital, Aachen, Germany; 3 Biochemistry and Molecular Genetics Service and Department of Physiology I, Hospital Cl´ınic i Provincial de Barcelona, IDIBAPS, CIBERehd, University of Barcelona, Barcelona, Spain E-mail: [email protected]
Background and Aims: During hepatic fibrogenesis, plateletderived growth factor b (PDGFb) is the most potent stimulus for proliferation and migration of stellate cells. PDGFb receptor (PDGFRb) expression is an important phenotypic change in myofibroblastic cells that mediates proliferation and chemotaxis. In hepatic tissue of patients with chronic liver disease, expression of PDGF and its receptor correlates with the degree of necroinflammation and fibrosis. First, we analyzed the relationship between the activation of PDGFRb, hemodynamic deterioration and increased liver fibrosis in rats subjected to chronic inhalation of CCl4 and second, we investigated the hemodynamic effects produced by an adenovirus encoding a dominant-negative soluble PDGFRb (sPDGFRb). Methods: The first protocol included 6 control and 21 rats induced to hepatic fibrosis for 13 (n = 10) or 18 weeks (n = 11). Thereafter, mean arterial pressure (MAP) and portal pressure (PP) were recorded, samples of liver tissue were collected and PDGFRb mRNA expression (real-time PCR) and collagen content (Sirius red) were assessed. The second protocol included 30 rats induced to hepatic fibrosis for 18 weeks. The animals were randomized into three groups (n = 10) that were iv given saline and adenovirus encoding sPDGFRb (5×1010 pfu) or b-gal (5×1010 pfu). Following 7 days, MAP, PP, serum sPDGFRb and hepatic collagen content were measured. Results: Animals treated for 18 weeks showed a significant increase in PDGFRb mRNA than those treated for 13 weeks (p < 0.01) and control rats (p < 0.01). In rats treated with CCl4 the percentage of fibrous tissue area ranged from moderate fibrosis (2.7%) to cirrhosis (19.1%). A significant correlation between the degree of fibrosis and hemodynamic changes (MAP: r2 = 0.6, p < 0.001; PP: r2 = 0.56, p < 0.001) or PDGFRb activation (r2 = 0.5, p < 0.05) were observed. Fibrotic rats transduced with adenovirus encoding for sPDGFRb showed increased MAP, decreased PP and reduced collagen content (124±2 mmHg; 6.7±0.3 mmHg; 7.54±0.27%) in comparison to fibrotic rats receiving b-gal (114±3 mmHg, p < 0.05; 8.7±0.6 mmHg, p < 0.05; 8.64±0.33%, p < 0.05) or saline (113±2 mmHg, p < 0.05; 8.6±0.4 mmHg, p < 0.05; 8.55±0.29%, p < 0.05). Conclusions: PDGFRb activation closely correlates with hemodynamic disorders and increased fibrosis in rats chronically treated with CCl4 . Transduction of adenovirus expressing sPDGFRb improves systemic hemodynamics and attenuates the degree of liver fibrosis in CCl4 treated rats.
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