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.

Effects of high hemodynamics upon the morphology of the walls of the great saphenous vein and splenic vein.

AIM: Studies have shown that the incidence and development of pathological changes in the walls of the great saphenous vein and splenic vein are closely related to high venous pressure. Such changes are referred to as "vascular adaptive remodeling responses under high venous pressure". The proposition of the concept of vascular remodeling contributes to our knowledge of pathological changes in the venous wall (dilation of the venous lumen and thickening of the venous wall). In the present study, we compared the histomorphology and cytomorphology of the walls of varicose great saphenous veins (GSVs) and diseased splenic veins (SVs) to investigate the remodeling of the venous wall under high hemodynamic pressure. METHODS: We collected 34 samples of varicose great saphenous veins and diseased splenic veins. Thirty-four samples of normal great saphenous veins and splenic veins were also collected (control group). Samples were made into slices and observed under light microscopy and electron microscopy. The thickness of the tunica intima and tunica media as well as the inner diameter of the venous lumen were measured. RESULTS: Under light microscopy, the walls of varicose veins stained with H&E were unevenly thickened, and those of diseased splenic veins were evenly thickened; mucoid degeneration of the tunica intima of varicose veins was not obvious by Masson staining (2/20 cases). The boundary between the tunica intima and tunica media was clearly defined. Uneven hyperplasia of muscular connective tissues was observed. For the diseased splenic-vein group, mucoid degeneration of the tunica intima was obvious (8/14 cases), with an unclearly defined boundary between the tunica intima and tunica extima. Uneven hyperplasia of muscular connective tissues was also observed. Differences in the thickness and inner diameter of the tunica intima and tunica media between the great saphenous vein and the splenic vein were significantly different. Under electron microscopy, mitochondrial degeneration in endothelial cells was observed in both groups. Increased numbers of rough endoplasmic reticula in the cytoplasm of smooth muscle cells, ribosomes and mitochondria and decreased numbers of myofilaments were also observed. CONCLUSION: High hemodynamics affected the remodeling of varicose great saphenous veins and diseased splenic veins. The histomorphology of visceral veins showed more significant pathological changes than that of peripheral veins. Similar cytomorphological changes were observed in both groups.

Synergistic effect of lidocaine with pingyangmycin for treatment of venous malformation using a mouse spleen model.

AIMS: To explore whether lidocaine has the synergistic effect with pingyangmycin (PYM) in the venous malformations (VMs) treatment. METHODS: The mouse spleen was chosen as a VM model and injected with different concentration of lidocaine or PYM or jointly treated with lidocaine and PYM. After 2, 5, 8 or 14 days, the mouse spleen tissues were acquired for hematoxylin-eosin (HE) staining, transmission electron microscopy (TEM) analysis, TUNEL assay and quantitative RT-PCR analysis to examine the toxicological effects of lidocaine and PYM on splenic vascular endothelial cells. RESULTS: 0.4% of lidocaine mildly promoted the apoptosis of endothelial cells, while 2 mg/ml PYM significantly elevated the apoptotic ratios. However, the combination of 0.2% lidocaine and 0.5 mg/ml PYM notably elevated the apoptotic ratios of splenic cells and severely destroyed the configuration of spleen, compared to those of treatment with 0.5 mg/ml PYM alone. CONCLUSION: Lidocaine exerts synergistic effects with PYM in promoting the apoptosis of mouse splenic endothelial cells, indicating that lidocaine possibly promotes the therapeutic effects of PYM in VMs treatment via synergistically enhancing the apoptosis of endothelial cells of malformed venous lesions.

[Splenoportal blood flow with nonalcoholic fatty liver disease].

UNLABELLED: The aim of this study was to characterise by duplex Doppler sonography the splenoportal venous and arterial blood flow in patients with nonalcoholic fatty liver disease (NAFLD) depending on the grade of biopsy proven steatosis. MATERIALS AND METHODS: 37 patients with NAFLD were examined: 22 female and 15 male average age 46,8 +/- 10,2 (29-62), with IBM 33,5 +/- 4,8 (28-42). The grade of steatosis was estimated by morphological investigation according to Brunt classification (1999). Colour Doppler sonography was performed by the same researcher using Doppler system Vivid-pro-7, USA with a 3,5 MHz convex probe. RESULTS: Alterations were detected in the following parameters: increase of portal and splenic vein diameters, slow-down of blood flow velocity in the portal and splenic veins, increase of the congestion index, decrease of systolic and diastolic blood flow velocity in the common hepatic and splenic artery, increase of pulsatility and resistance indexes of these arteries, increase of portal hypertensive index and decrease of liver vascular index according to the progression of steatosis with maximal negative dynamics of all characteristics in patients with III grade of steatosis. The closest correlative connections were revealed between the grade of steatosis and congestion index (r = 0.81), portal hypertensive index (r = 0.79), and negative connection--with liver vascular index (r = -0.69). CONCLUSION: Using Doppler sonography we have detected a deterioration of venous and arterial splenoportal hemodynamics in patients with nonalcoholic fatty liver disease in connection with the progression of steatosis, therefore this method may be used as a noninvasive way to estimation the grade of steatosis, establish the diagnosis of portal hypertension and detect the formation of liver cirrhosis.

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.

"Intermediate zone" of mammalian spleens: light and electron microscopic study of three primitive mammalian species (platypus, shrew, and mole) with special reference to intrasplenic arteriovenous communication.

The intermediate zone (IZ) of nonperfused and perfused spleens in three species of primitive mammals (shrew, mole, platypus) was studied morphologically. The IZ is a tissue zone consisting of plexiform vessels, probably venous capillaries, and is located transitionally between the white and red pulp. The IZ is separated from the white pulp by the arterial net (AN), in which the white pulp arteries terminate. Development of the IZ differs between the three species examined being distinctive in the platypus and shrew. The IZ is thin in the mole spleen. A closed type of arteriovenous (A-V) anastomosis was demonstrated in or around the IZ in the two Insectivora species examined. In the shrew spleen, peripheral arterial branches running within the IZ anastomose with the AN around the follicle. The AN anastomoses eventually with venous plexiform vessels of the IZ around the nonfollicular area of the white pulp to form a closed system. In the mole spleen, A-V anastomoses were noted between white pulp arteries (follicular and AN) and veins of the red pulp, either by direct communication or through fenestrated IZ vessels compatible with the plexiform vessels of the shrew spleen. A-V anastomosis in the IZ is probable, but not confirmed, in the platypus spleen, as analysis was limited to a nonperfused specimen. Well-developed ellipsoids were noted around arterial terminals of the IZ in the shrew spleen. Ellipsoids were also noted around all arterial terminals of the mole spleen directed to the red pulp. Most ellipsoids of the mole spleen appeared located within the IZ. No ellipsoids were present around arterial terminals of the IZ in the platypus spleen. Closed circulation was noted in terminals of the pulp artery in spleens of all three species. All pulp arteries of the mole spleen are postellipsoid segments of white pulp (AN and follicle) arteries. No ellipsoids were found around terminals of the pulp artery (penicillar artery) in shrew and platypus spleens. The IZ is probably homologous to the perilymphatic sinusoid (vein) of the lungfish spleen and may be regarded as part of the red pulp. The IZ may be representative of primitive mammalian spleens that have closed circulation. The marginal zone (MZ) of common mammalian spleens is probably a modified IZ by differentiation (remodelling) of the intrasplenic vein. In this process, withdrawal of venous vessels from the IZ occurred, leaving a lymphoreticular zone with open circulation (MZ). The marginal sinus reported in some mammalian spleens is probably a modified AN formed during this process. Possible morphological alterations of the spleen in vertebrate phylogeny are discussed.

Fine architecture of the splenic terminal vascular bed as revealed by arterial and venous pressure-loading perfusion fixation.

The three-dimensional fine architecture of the red pulp of human and animal spleens, which as fixed by a modified version of the arterial and venous pressure-loading perfusion fixation (AVPL perfusion fixation) method, is demonstrated by scanning and transmission electron microscopy. In the human spleen, changes in splenomegalias associated with hereditary spherocytosis and chronic portal hypertension are also introduced in addition to the normal architecture of the red pulp of spleens removed from patients with stomach cancer. The AVPL perfusion fixation of these spleens clearly visualized complicated three-dimensional fine architecture of the red pulp and provided much important information on in situ morphology and dynamic change of the terminal vascular bed, including venous pressure-dependent size change of the stomata and three-dimensional shapes of the capillary terminal, with positive proof of their opening into the cordal reticular tissue. In studies of the spleen associated with portal hypertension, the AVPL perfusion fixation is considered a necessary technique for analysis of the structural deviation closely relating to a very high venous pressure.

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.

Morphological studies of the spleen in idiopathic portal hypertension (so-called Banti's syndrome without liver cirrhosis) using light microscopy, scanning electron microscopy and histometry.

Morphological changes in the spleens of patients with idiopathic portal hypertension (IPH) were studied and compared with the normal spleen. The study used (1) light microscopy with histometry, (2) scanning electron microscopy (SEM) of the splenic tissue with histometry and (3) SEM of the spleen vascular replica. Histometrical studies by light microscopy showed that the volume of red pulp of IPH was increased in a unit area and to a total of 12 times the normal in the whole spleen. The white pulp was scanty of lymphocytes and decreased in a unit area but it was increased in the whole spleen. SEM of the white pulp of IPH demonstrated many channels formed by reticulum cells and running parallel with each other along the central artery. This finding presumably corresponds to periarterial fibrosis in light microscopy. SEM histometry demonstrated that the venous sinuses of IPH were small but increased in number and occupied the same percentage area in a unit red pulp area as in the normal spleen. The Billroth cord of IPH was narrowed and occupied by thickened reticulum cells, which may cause increased pooling and destruction of blood cells in the enlarged spleen (hypersplenism). SEM of the tissue and vascular replica demonstrated open arterial termination in the Billroth cord in the spleen of IPH as well as in the normal spleen. Venous sinuses in the replica of IPH ran parallel with each other forming bundles with fewer intercommunications than normal.