Human splanchnic amino-acid metabolism.

Plasma levels of several amino acids are correlated with metabolic dysregulation in obesity and type 2 diabetes. To increase our understanding of human amino-acid metabolism, we aimed to determine splanchnic interorgan amino-acid handling. Twenty patients planned to undergo a pylorus preserving pancreatico-duodenectomy were included in this study. Blood was sampled from the portal vein, hepatic vein, superior mesenteric vein, inferior mesenteric vein, splenic vein, renal vein, and the radial artery during surgery. The difference between arterial and venous concentrations of 21 amino acids was determined using liquid chromatography as a measure of amino-acid metabolism across a given organ. Whereas glutamine was significantly taken up by the small intestine (121.0 ± 23.8 µmol/L; P < 0.0001), citrulline was released (-36.1 ± 4.6 µmol/L; P < 0.0001). This, however, was not seen for the colon. Interestingly, the liver showed a small, but a significant uptake of citrulline from the circulation (4.8 ± 1.6 µmol/L; P = 0.0138) next to many other amino acids. The kidneys showed a marked release of serine and alanine into the circulation (-58.0 ± 4.4 µmol/L and -61.8 ± 5.2 µmol/L, P < 0.0001), and a smaller, but statistically significant release of tyrosine (-12.0 ± 1.3 µmol/L, P < 0.0001). The spleen only released taurine (-9.6 ± 3.3 µmol/L; P = 0.0078). Simultaneous blood sampling in different veins provides unique qualitative and quantitative information on integrative amino-acid physiology, and reveals that the well-known intestinal glutamine-citrulline pathway appears to be functional in the small intestine but not in the colon.

IL-17A up-regulates expression of endothelial tissue factor in liver cirrhosis via the ROS/p38 signal pathway.

Interleukin-17A (IL-17A), an inflammatory cytokine, is elevated in liver cirrhosis. Inflammation and coagulation dysfunction are closely related. Tissue factor (TF) is a bridge between endothelial activation, blood coagulation and inflammation. The aims of the present study were to evaluate endothelial TF expression in liver cirrhosis and identify the possible underlying role of IL-17A in TF expression. In the present study, we found that TF expression was increased on endothelium of splenic vein from cirrhotic patients and significantly correlated with intima/media ratios of splenic vein and coagulation parameters. Serum levels of IL-17A were significantly higher in cirrhotic patients as compared with normal controls. Cirrhotic serum and IL-17A stimulated TF expression in HUVECs, which was reduced by blockade of IL-17A, p38, and reactive oxygen species (ROS). Taken together, our data show that enhanced expression of endothelial TF, which plays an important role in coagulopathy and splenic vein remodeling in liver cirrhosis, is induced by IL-17A in a ROS dependent manner.

Dysregulated apoptosis of the venous wall in chronic venous disease and portal hypertension.

Introduction The etiology of varicose veins remains elusive. We hypothesized that abnormal cell cycle events in the vein wall may contribute to changes in the structural integrity, thus predisposing to the development of varicosities. The present study was designed to determine whether or not the same molecular apoptotic pathway exists between great saphenous and splenic veins. Methods Thirty-six samples of diseased splenic veins and varicose great saphenous veins were collected. Twenty-five samples of control splenic and great saphenous veins were also collected. The apoptotic cell proteins expression was immunohistochemically stained with antibodies (anti-Bax and anti-Bcl-xl). Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay and immunofluorescence staining. The morphology of apoptotic cells was observed with an electron microscope. Results The apoptotic ratio in walls (intima and media) of diseased splenic vein and varicose great saphenous vein groups were significantly lower than the corresponding regions in the splenic vein and great saphenous vein groups ( p < 0.01), respectively. A significant difference was not noted in the ratio change of apoptotic cells between the diseased splenic vein and varicose great saphenous vein groups ( p > 0.05). The high positive expression of Bcl-xl proteins was detected in the diseased splenic vein and varicose great saphenous vein groups, respectively. While the high positive expression of Bax proteins was also observed in the splenic vein and great saphenous vein groups, respectively. Electron microscopic observations confirmed that endothelial and smooth muscle cells in diseased splenic vein, varicose great saphenous vein, splenic vein, and great saphenous vein walls exhibited apoptotic morphologic features, such as fuzzy mitochondrial cristae, medullary changes, and margination of the nuclear chromatin. Conclusions Our results showed the same dysregulation of apoptosis via the intrinsic pathway in diseased splenic veins and varicose great saphenous veins. This observational study suggests that apoptotic down-regulation in the veins wall is a cause of diseased splenic veins and varicose great saphenous veins, but does not exclude the possibility that other mechanisms are involved.

Splenic neurohormonal modulation of mesenteric vascular tone.

In portal hypertension, development of a hyperdynamic circulation is preceded by transient mesenteric vasoconstriction. Portal hypertension increases splenic venous outflow pressure. We hypothesized that this causes direct reflex activation of mesenteric vasoconstrictor nerves and splenorenal reflex-mediated activation of the renin-angiotensin system. In anaesthetized male rats, we measured mesenteric efferent nerve activity and mesenteric vascular conductance (MVC) after selectively elevating splenic venous pressure. Partial splenic vein occlusion raised splenic venous pressure (from 4.8 ± 0.4 to 24.1 ± 0.3 mmHg; n = 18) and induced a significant increase in mesenteric efferent nerve activity (from 23.2 ± 3.3 to 31.6 ± 3.5 spikes s(-1); n = 11); this response was abolished by prior splenic denervation (from 32.4 ± 2.4 to 31.2 ± 1.6 spikes s(-1); n = 7). Mesenteric vascular conductance, the ratio of superior mesenteric artery blood flow to mean arterial pressure, fell upon splenic vein occlusion (ΔMVC = -0.0120 ± 0.0014 ml min(-1)mmHg(-1); P < 0.05, n = 10). This was attenuated by splenic denervation (ΔMVC = -0.0044 ± 0.0018 ml min(-1)mmHg(-1); P < 0.05, n = 8), but unaffected by mesenteric denervation (ΔMVC = -0.0145 ± 0.0020 ml min(-1)mmHg(-1); n = 6) or bilateral renal denervation (ΔMVC = -0.0106 ± 0.0021 ml min(-1)mmHg(-1); n = 5). Localized blockade of mesenteric vascular angiotensin II type 1 (AT(1)) receptors significantly attenuated the response (ΔMVC = -0.0058 ± 0.0017 ml min(-1)mmHg(-1); P < 0.05, n = 5), whereas blockade of both AT(1) and α(1)-adrenergic receptors caused a significant increase in mesenteric conductance (ΔMVC = +0.0033 ± 0.0010 ml min(-1)mmHg(-1); P < 0.05, n = 6). Our evidence suggests that increased splenic venous outflow pressure reflexly activates adrenergic/angiotensinergic mesenteric nerves, vasodilator mesenteric nerves and the renin-angiotensin system. We propose that obstruction to splenic venous outflow, such as would normally accompany portal hypertension, induces reflex mesenteric vasoconstriction independently of the increase in portal venous pressure.

[Expression of local angiotensinogen mRNA and activation of local nuclear factor-kappaB in vasculopathy of portal hypertension].

OBJECTIVE: To investigate the expression of local angiotensinogen mRNA and activation of local nuclear factor-kappaB in vasculopathy of portal hypertension and to discuss their role in the pathogenesis of portal hypertensive vasculopathy. METHODS: RT-PCR was used to detect the expression of local angiotensinogen mRNA in 28 specimens of splenic artery and vein obtained during operation of elective separation and amputation on 28 portal hypertension (PH) patients, 20 males and 8 females, aged 51.7 +/- 10 (30 - 65), and in 12 specimens of normal vessel from 12 patients undergoing splenectomy due to traumatic rupture of spleen. Chemiluminescent electrophoretic mobility shift assay (EMSA) was used to detect the activation of NF-kappaB in splenic artery and vein. RESULTS: The levels of local angiotensinogen mRNA in the splenic artery and vein of PH group were 0.48 +/- 0.21 and 0.43 +/- 0.16 respectively, both significantly higher than those in the control group (0.23 +/- 0.12 and 0.18 +/- 0.10, both P < 0.05). There was no significant difference between the splenic artery and vein in the expression of local angiotensinogen mRNA in PH group. The activation levels of NF-kappaB in splenic artery and vein of the PH patients were 1.44 +/- 0.23 and 1.38 +/- 0.18 respectively, both significantly higher than those of the control group (0.19 +/- 0.20 and 0.25 +/- 0.16 respectively, both P < 0.05). However, there was no significant difference in the activation levels of NF-kappaB between the splenic artery and vein in the PH patients (P > 0.05). CONCLUSION: Local angiotensinogen and activation of NF-kappaB maybe one of the factors in the pathogenesis of portal hypertensive vasculopathy, which can cause and advance portal hypertensive vasculopathy.

[The study on correlation of c-myc gene expression with vascular smooth muscle cell proliferation in patients with portal hypertension].

OBJECTIVE: To investigate c-myc proto-oncogene expression and the relationship of PCNA protein expression of extrahepatic vascular smooth muscle cell in patients with portal hypertension and normal vessels. METHODS: RT-PCR was used to demonstrate the expression of c-myc mRNA and immuno-chemistry strain was performed to detect the expression of PCNA protein in splenic veins of 28 patients with portal hypertension and 12 normal vessels. RESULTS: The straining of PCNA protein was (29.8 +/- 4.7)% in splenic veins with portal hypertension, Normal vessels did not detect PCNA protein expression (P < 0.01); RT-PCR showed that the expression of c-myc mRNA in PCNA-positive control and in negative control of splenic veins with portal hypertension were (7.61 +/- 1.04)% and (3.82 +/- 0.92)%, respectively. There ws different between two groups (P < 0.01) and significant different (P < 0.01) when compared with (1.01 +/- 0.21)% in normal vessels. CONCLUSIONS: The c-myc was immediate-early gene when it modulated proliferation of vascular smooth muscle cell. Hemodynamic disturbance of portal vein system activate the proto-oncogene of smooth muscle cells in splenic vein of patients with portal hypertension, promoting the proliferation, migrating and phenotypic change and resulting in vascular remodelling of splenic veins.

Pathological morphology alteration of the splanchnic vascular wall in portal hypertensive patients.

OBJECTIVE: To investigate the pathological morphology alteration of the splanchnic vascular wall in portal hypertensive patients. METHODS: Splenic arteries, veins and gastric coronary veins from portal hypertensive patients (n = 50) were removed during esophagogastric devascularization with splenectomy and were observed under optic and electron microscopes. The expression of iNOS in the splenic artery wall was analysed with immunohistochemistry. RESULTS: The internal elastic membrane and medial elastic fibers of the splenic artery wall were broken and degenerated. Atrophy, apoptosis and phenotypic changes were seen in smooth muscle cells of splenic arteries. Positive staining for iNOS was seen in the cytoplasm of smooth muscle cells and iNOS activity was elevated compared with the non-cirrhotic patients (P < 0.01). In the splenic and gastric coronary veins of cirrhotic patients, we found proliferative intima, extensive thrombi adhering to the venous wall, mimicked arteriosclerosis plaques accompanied with hypertrophy of smooth muscle cells, and thickened muscle fibers of veins with increase in extracellular matrix. CONCLUSION: Portal hypertension may be complicated by splanchnic arterial and venous vasculopathy. There may be an interactive relationship among portal hypertension, splanchnic hyperdynamic disturbances and splanchnic vasculopathy in the pathogenesis of portal hypertension.

Histochemical analysis of acidic glycoconjugates in the endothelium lining the splenic blood vessels in the rat.

An electron microscopic analysis with specific histochemical stainings for acidic glycoconjugates was carried out to examine the endothelium lining blood vessels of the rat spleen. Histochemical staining performed was the postembedding high or low iron diaminethiocarbohydrazide-silver protein-physical development (HID or LID-TCH-SP-PD) method, with or without prior digestion with acidic glycoconjugate-degrading enzymes, such as heparitinase, testicular hyaluronidase, chondroitinase B and neuraminidase. The results indicated that the acidic glycoconjugates in the basal lamina of the endothelial cells lining the four types of blood vessel (central arteries, arterial capillaries, splenic sinuses and pulp veins) were heparan sulfate, chondroitin sulfate A and/or C, chondroitin sulfate B and sialic acid residues. In the endothelial cells lining the central arteries, arterial capillaries and pulp veins, the surface coat of the luminal plasma membrane included heparan sulfate, chondroitin sulfate A and/or C, chondroitin sulfate B and sialic acid residues, whereas the corresponding ultrastructure of the splenic sinuses was devoid of detectable amounts of acidic glycoconjugates. This suggests that such characteristic histochemical features of the endothelium in the four types of the splenic blood vessel can be related to the possible physiological functions of the spleen.

Local modulation of neurotransmitter release in bovine splenic vein.

Bovine splenic vein has an abundant sympathetic innervation. Isolated strips were used to examine whether autoinhibition of norepinephrine release from the noradrenergic nerve terminals could be demonstrated under various experimental conditions and whether additional local regulatory modulators of transmitter release could also be implicated. In particular, the possibility of a histamine interaction with presynaptic inhibitory receptors was examined because ultrastructural evidence disclosed a close spatial relationship between mast cells and noradrenergic nerve terminals in the vessel wall. To investigate the presence of presynaptic alpha-receptors the competitive blocking agent phentolamine was included in the superfusion medium at concentrations ranging from 1 to 50 microM during electrical field stimulation at frequencies between 1 and 10 Hz. Transmitter outflow was measured as fractional tritium release. Low frequency stimulation (1 Hz) with 1 microM phentolamine resulted in the typical increase in norepinephrine release characteristics for presynaptic alpha-receptor inhibition. In contrast, high frequency (10 Hz) stimulation in the presence of 50 microM phentolamine caused an unexpected decrease in norepinephrine outflow. This unusual result can be explained by additional pharmacological actions of phentolamine unrelated to alpha-receptor blockade, e.g. histamine release from the mast cells which subsequently can act on presynaptic inhibitory histamine receptors. This effect, manifested at higher phentolamine concentrations, would overcome the alpha-receptor blockade. The presence of histamine receptors was supported by the results from electrical stimulation in the presence of exogenous histamine. Histamine decreased norepinephrine outflow while increasing basal tension and the contractile response of the vein strip. Unexpectedly, these effects appeared to be mediated by histamine receptors of the H1-type because they were reduced after pyrilamine but unaffected by agonists and antagonists to receptors of the H2-type. It is speculated that interactions between mast cells and noradrenergic nerve terminals may serve to maintain homeostasis in the bovine splenic vein.

Growth stimulation of human bone marrow cells in agar culture by vascular cells.

Human vascular cells are capable of stimulating granulopoiesis in agar culture of human bone marrow cells. This effect was obtained by including vein fragments in the culture or by using endothelial cells separated from the vein of human umbilical cords as feeder cells. Furthermore, the stimulatory capacity of conditioned medium obtained from cord veins was found to be highly active in comparison to that obtained from peripheral leukocytes. Endothelial cells within the bond marrow cavity are suggested as a local source of factors regulating granulopoiesis in humans in addition to the monocyte.

Metabolism of arterial plasma estrogens by the splanchnic organs of the dog in vivo.

In order to study the splanchnic metabolism of blood-borne estrogens, a constant infusion of estrone-6,7-(3)H was made in a series of dogs, and arteriovenous (A-V) differences at equilibrium were determined for estrone-6,7-(3)H and for its products estradiol-17beta, estrone sulfate, estrone glucosiduronate, and estradiol-17beta glucosiduronate across the splanchnic bed (artery-hepatic vein), the small intestine (artery-superior mesenteric vein), and the spleen (artery-splenic vein). Per cent extractions (100 - [V/A] 100) were calculated. The plasma metabolic clearance rate (MCR) for estrone was measured. Principal findings were as follows: mean MCR was 731 liters/day per m(2), SEM 50. By comparison with estimated hepatic plasma flow and using the observed splanchnic extraction of estrone, 45-71% of estrone metabolism was calculated to be extrasplanchnic. The significant mean per cent extractions were as follows (SEM in parentheses): splanchnic bedestrone 85.9 (1.92), estradiol-17beta 88.11 (3.36), estrone sulfate 27.9 (5.22), estrone glucosiduronate -48.5 (9.33), estradiol-17beta glucosiduronate -33.3 (80.3); small intestine-estrone 45.3 (2.60), estradiol-17beta 46.1 (12.9), estrone glucosiduronate - 30.8 (7.9); spleen-estrone 35 (3.8), estrone glucosiduronate 12 (3.7). These results lead to the following conclusions. Both estrone and estradiol-17beta are nearly completely extracted in one passage through the splanchnic bed. There is net uptake of estrone sulfate and net production of estrone glucosiduronate and of estradiol-17beta glucosiduronate by the splanchnic bed. There is net uptake of estrone and of estradiol-17beta by the intestine, associated with substantial net production of estrone glucosiduronate. There is net uptake of estrone by the spleen and a small but significant net uptake of estrone glucosiduronate.