低氧对兔主动脉和肺动脉损伤差异的探讨

将兔置于模拟海拔５ｋｍ（ＰＯ２＝１０．８ｋＰａ）低氧舱内２４ｈ,观察和比较低氧对主动脉和肺动脉形态学损伤的差异,结果显示５ｋｍ低氧２４ｈ,兔主动脉和肺动脉出现内膜下水肿,空泡增加,胞内线粒体和内质网不同程度损伤。但低氧对肺动脉的损伤明显重于主动脉,甚至已殃及平滑肌细胞。扫描电镜观察也显示模拟海拔５ｋｍ低氧２４ｈ,肺动脉内皮细胞出现损伤脱落,甚至出现断裂带,而在主动脉仅见内皮细胞轻微损伤。另又将离体     

The ultrastructural basis of the permeability of arterial endothelium to horseradish peroxidase

The thoracic aorta and basilar artery, in which the incidence of atherosclerosis is known to be different, were examined to elucidate the correlation between the structure of the intercellular cleft junction between adjacent endothelial cells and its permeability to HRP. Tannic acid or HRP in the vessel lumen passed through the intercellular clefts of the thoracic aorta into the subendothelial space, whereas in the basilar artery they were unable to penetrate beyond the tight junction of the intercellular clefts. Freeze-fracture replicas revealed that the tight junctions of the thoracic aorta consisted of one to two junctional strands in most areas of the cleaved planes, with discontinuities in some places, whereas those of the basilar artery consisted of a continuous belt-like meshwork of six anastomosing junctional strands on average. These observations confirm that the structure of endothelial junctions in arteries has a close correlation with the permeability of the intercellular clefts to HRP.     

Adrenomedullin is upregulated in the heart and aorta during the early and late stages of sepsis

Although circulating levels of adrenomedullin (ADM), a newly reported vasodilatory peptide with 52 amino acid residues in the human and 50 amino acid residues in the rat, are elevated during the early and late stages of sepsis, ADM levels in cardiovascular tissues and its precise localization remain to be determined. To study this, rats were subjected to sepsis by cecal ligation and puncture (CLP), followed by administration of 3 ml/100 g b.wt. normal saline to these and sham-operated animals. The heart and thoracic aorta were harvested at 5 h (i.e. the early stage of sepsis) and 20 h (late sepsis) after CLP. Tissue levels of ADM were determined by radioimmunoassay. The localization of ADM in the left ventricle and thoracic aorta was examined by using immunohistochemistry and electron microscopy techniques. The results indicated that ADM levels in the heart and thoracic aorta increased significantly at 5 h after CLP and remained elevated at 20 h after the onset of sepsis. Immunohistochemistry findings showed that ADM immunoreaction products were localized in the cytoplasm of the cardiac myocytes and aortic endothelial cells. Using electron microscopy, ADM immunoreaction products were found in the cytoplasmic matrixes. The immunostainings were also associated with the outer membranes of mitochondria and vesicles of the myocytes as well as vascular endothelial cells. It appears that the cardiovascular tissues, among other organ systems, contribute to the increased levels of plasma ADM under those conditions. Since ADM is localized in different cell populations in the heart and the large blood vessel (i.e. myocytes versus vascular endothelial cells), this peptide may play a differential role in regulating cardiac and vascular functions during sepsis as an autocrine and/or paracrine mediator.     

Demonstration of fibronectin in normal and injured aorta by an indirect immunoperoxidase technique

The presence and localization of fibronectin in normal and mechanically injured aorta in rabbits was studied using an indirect immunoperoxidase technique on tissue specimens fixed in formaldehyde, embedded in paraffin and pretreated with pepsin. The effect on staining quality of treatment with testicular hyaluronidase prior to immunoperoxidase staining was also examined. In the intima from normal aorta fibronectin was present in the subendothelial basal layer, along the internal and external elastic laminae, around and between the smooth muscle cells of the media and along the collagen and elastic fibres in the adventitia. Sixteen days after a single mechanical dilatation of the descending thoracic aorta all animals developed gross atherosclerotic-like changes. Microscopic examination revealed prominent neo-intimal hyperplasia with subendothelial, cushion-like thickenings but no medial or adventitial alterations. Fibronectin, in increased amounts, was found between and around the endothelial cells and in the subendothelial thickenings between the proliferating smooth muscle cells in relation to the fine, thin elastic and argyrophilic fibres. In the media and adventitia the amount and distribution of fibronectin was indistinguishable from uninjured control aortas. Treatment with testicular hyaluronidase before immunoperoxidase staining resulted in a higher staining resolution in normal and injured aorta. The conspicuous observation in the present study is that fibronectin exclusively accumulates in areas of tissue repair. The origins and functions of fibronectin during tissue injury and repair are discussed.     

Pro-caspase-8 is predominantly localized in mitochondria and released into cytoplasm upon apoptotic stimulation

The recruitment and cleavage of pro-caspase-8 to produce the active form of caspase-8 is a critical biochemical event in death receptor-mediated apoptosis. However, the source of pro-caspase-8 available for activation by apoptotic triggers is unknown. In human fibroblasts and mouse clonal striatal cells, confocal microscopy revealed that pro-caspase-8 immunofluorescence was colocalized with cytochrome c in mitochondria and was also distributed diffusely in some nuclei. Biochemical analysis of subcellular fractions indicated that pro-caspase-8 was enriched in mitochondria and in nuclei. Pro-caspase-8 was found in the intermembrane space, inner membrane, and matrix of mitochondria after limited digestion of mitochondrial fractions, and this distribution was confirmed by immunogold electron microscopy. Pro-caspase-8 and cytochrome c were released from isolated mitochondria that were treated with an inhibitor of the ADP/ATP carrier atractyloside, which opens the mitochondria permeability transition pore. Release was blocked by the mitochondria permeability transition pore inhibitor cyclosporin A (CsA). After clonal striatal cells were exposed for 6 h to an apoptotic inducer tumor necrosis factor-alpha (TNF-alpha), mitochondria immunoreactive for cytochrome c and pro-caspase-8 became clustered at perinuclear sites. Pro-caspase-8 and cytochrome c levels decreased in mitochondrial fractions and increased, along with pro-caspase-8 cleavage products, in the cytoplasm of the TNF-alpha-treated striatal cells. CsA blocked the TNF-alpha-induced release of pro-caspase 8 but not cytochrome c. Internucleosomal DNA fragmentation started at 6 h and peaked 12 h after TNF-alpha treatment. These results suggest that pro-caspase-8 is predominantly localized in mitochondria and is released upon apoptotic stimulation through a CsA-sensitive mechanism.     

Reversible changes in stress fiber expression and cell shape in regenerating rat and rabbit aortic endothelium

The influence of intimal de-endothelialization on stress fiber expression in regenerating rat and rabbit aortic endothelium was studied using immunofluorescence microscopy. Rat thoracic and abdominal aortae were balloon de-endothelialized, and endothelial cell shape and stress fiber expression was studied in both uninjured and de-endothelialized animals. In control animals, the majority of thoracic endothelial cells did not contain stress fibers while the majority of abdominal endothelial cells did. One week after injury, all the endothelial cells distal to the regenerating edge contained very prominent stress fibers. In areas directly adjacent to the still de-endothelialized surface, the endothelial cells had an intense, diffuse cytoplasmic staining without stress fibers. Regenerating endothelium also had a substantially higher length-to-width ratio, but smaller cell areas. Six weeks after injury, the endothelium had completely regenerated, and stress fibers were lost from the majority of the thoracic endothelial cells. Changes in abdominal aorta stress fiber expression were not as marked. In the rabbit, all the control thoracic endothelial cells had stress fibers; however, cells at the leading edge of a narrow region of de-endothelialization had few stress fibers. The results suggest that stress fibers do not play a primary role in cellular migration in situ. The transient increase in stress fiber expression in the rat may result from a temporary demand for greater adhesive capabilities until the subendothelial extracellular matrix is remodeled.     

Hypertensive structural changes in blood vessels: do endothelial cells hold the key?

In recent decades, the complexity of the endothelium and its major role in maintaining or altering blood vessel architecture are being revealed. In contrast, the vascular smooth muscle cell previously received the most attention. I suggest support of the hypothesis that the endothelium is the key to vascular disease. An altered endothelium in diabetes mellitus likewise is likely to be pivotal in vascular complications that develop. We have demonstrated that adherent monocytes, indicators of altered endothelium, occur in deoxycorticosterone acetate induced hypertension in male Wistar rats. The coronary artery and thoracic aorta were investigated using transmission electron microscopy. Details of hypertensive changes were revealed as well as early atherogenic pathology in the absence of dietary modifications. Scanning electron microscopy of thoracic aorta showed details of the luminal endothelial surface and adherent monocyte-macrophages in hypertensive animals. There were two cell types: numerous typical monocytes with upstream tails, and larger cells that may have been free grazing macrophages or macrophages that had returned to the circulation. Debris and amorphous material were particularly evident in vessels from hypertensive animals. Monocytes squeezed between intact endothelial plasma membranes (as seen in section), and were found as subendothelial foam cells and phagocytosing macrophages. The endothelial adherence of monocytes to the aortas from diabetic animals was significantly (p less than 0.05) elevated over that found in controls (but not different from control-hypertensive or diabetic-hypertensive animals) supporting the concept of altered endothelium in diabetes.     

Enhanced tumor cell adhesion to the subendothelial matrix resulting from 12(S)-HETE-induced endothelial cell retraction

A 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE), which is produced by platelets and tumor cells, was tested for its ability to induce retraction of endothelial cell monolayers. The induction of endothelial cell retraction is a critical step in tumor cell metastasis. Endothelial cells demonstrated reversible retraction in response to 12(S)-HETE, but did not respond to the stereoisomer 12(R)-HETE or to unrelated 5-lipoxygenase (i.e., 5[S]-HETE) or 15-lipoxygenase (i.e., 15[S]-HETE) metabolites. Endothelial cells did not demonstrate loss of viability in response to 12(S)-HETE. The induction of retraction was both dose and time dependent. Scanning electron microscopy confirmed that 12(S)-HETE induced endothelial cell retraction and revealed collapsed filopodia on their surface, the appearance of spaces between endothelial cells and the underlying subendothelial matrix, in addition to large gaps between adjacent endothelial cells. Tumor cell adhesion to endothelial cell monolayers was enhanced 1 h after pretreatment of monolayers with 12(S)-HETE but not after pretreatment with other lipoxygenase metabolites. Tumor cell adhesion to endothelial cell monolayers 36 h after pretreatment with 12(S)-HETE was not different from adhesion to untreated monolayers. Therefore we suggest that 12(S)-HETE generated during tumor cell-platelet-endothelial cell interactions may induce reversible endothelial cell retraction, allowing tumor cell access to the subendothelial matrix, which is a critical step in their eventual extravasation from the microvasculature during hematogenous metastasis.     

Susceptibility of endothelial cells derived from different blood vessels to common viruses

Summary We examined whether endothelial cells derived from different blood vessels vary in their susceptibility to viral infection. Five common viral pathogens of humans (herpes simplex 1, measles, mumps, echo 9, and coxsackie B4 viruses) were evaluated for growth in endothelial cells derived from bovine fetal pulmonary artery thoracic aorta, and vena cava. All five viruses replicated in each type of endothelial cell. There were apparent differences in the quantities of measles and mumps viruses produced in pulmonary artery endothelium compared with thoracic aorta and vena cava when endothelial cells were obtained from different animals. However when pulmonary artery endothelial cells were compared with vena cava cells from the same animal, growth of each virus was similar in the two cell types. Four of the viruses replicated in the various endothelial cells without producing appreciable changes in cell morphology. These results indicate that endothelial cells from different blood vessels are equally susceptible to the human viruses evaluated, and that viral replication can occur without major alteration in cell morphology. Endothelial cells could serve as permissive cells permitting viruses to leave the circulation and initiate infection in adjacent tissues, including subendothelial smooth muscle cells. This work was supported by Public Health Service grants HL28220, HL 29492, and HL 24914 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

Pulmonary microvascular response to LTB4: effects of perfusate composition

We examined the effects of leukotriene B4 (LTB4) on pulmonary hemodynamics and vascular permeability using isolated perfused guinea pig lungs and cultured monolayers of pulmonary arterial endothelial cells. In lungs perfused with Ringer solution, containing 0.5 g/100 ml albumin (R-alb), LTB4 (4 micrograms) transiently increased pulmonary arterial pressure (Ppa) and capillary pressure (Pcap). Pulmonary edema developed within 70 min after LTB4 injection despite a normal Pcap. The LTB4 metabolite, 20-COOH-LTB4 (4 micrograms), did not induce hemodynamic and lung weight changes. In lungs perfused with autologous blood hematocrit = 12 +/- 1%; protein concentration = 1.5 +/- 0.2 g/100 ml), the increases in Ppa and Pcap were greater, and both pressures remained elevated. The lung weight did not increase in blood-perfused lungs. In lungs perfused with R-alb (1.5 g/100 ml albumin) to match the blood perfusate protein concentration, LTB4 induced similar hemodynamic changes as R-alb (0.5 g/100 ml) perfusate, but the additional albumin prevented the pulmonary edema. LTB4 (10(-11)-10(-6) M) with or without the addition of neutrophils to the monolayer did not increase endothelial 125I-albumin permeability. Therefore LTB4 induces pulmonary edema when the perfusate contains a low albumin concentration, but increasing the albumin concentration or adding blood cells prevents the edema. The edema is not due to increased endothelial permeability to protein and is independent of hemodynamic alterations. Protection at higher protein-concentration may be the result of LTB4 binding to albumin.     

Insulin and nateglinide reduce monocyte adhesion to endothelial cells in Goto-Kakizaki rats exhibiting repetitive blood glucose fluctuation

Epidemiological studies demonstrated the importance of postprandial hyperglycemia on the progression of atherosclerosis. However, whether treatment of postprandial hyperglycemia by insulin or insulin secretagogues has a beneficial effect on atherosclerosis has not been elucidated. To elucidate the effects of reduction of postprandial rise of blood glucose by insulin and nateglinide on monocyte adhesion to endothelial cells, we used non-obese type 2 diabetic Goto-Kakizaki (GK) rats fed twice daily, as a model of repetitive postprandial hyperglycemia. We investigated the effects of insulin injection and nateglinide administration just before each meal for 12 weeks on monocyte adhesion to endothelial cells. By setting the doses of insulin and nateglinide, both treatment significantly reduced postprandial hyperglycemia without significant reduction of HbA1c. Nateglinide also reduced serum insulin level just after 1 h meal. Both nateglinide and insulin therapy reduced the number of monocytes adherent to the aortic endothelial layer. Nateglinide, but not insulin, reduced intimal thickness of the thoracic aorta. While increased serum insulin level might be regarded as a factor responsible for the progression of atherosclerosis, our data showed that treatment with pre-meal insulin or nateglinide, which reduces postprandial hyperglycemia, reduced monocyte adhesion to endothelial cells.     

Responses of baboons to prolonged hyperoxia: physiology and qualitative pathology.

Cardiopulmonary responses to prolonged hyperoxia and their relationships to the development of lung pathology have not been fully characterized in primates. In this study, circulatory hemodynamics and pulmonary function, vascular permeability, and leukocyte sequestration were measured in male baboons after 100% O2 exposure and related to ultrastructural changes of lung injury by electron microscopy. Three groups of animals were exposed to 100% O2 in an exposure cage for 40, 66, and 80 h, respectively. A fourth group of animals was exposed in a cage for 80 h and then anesthetized and ventilated with 100% O2 for additional time. These animals were exposed for a total duration of 110 h or until death from the injury. Physiological responses to hyperoxia were characterized by decreases in total lung capacity and inspiratory capacity at 80 and 110 h. A significant increase in pulmonary leukocyte accumulation was noted by 80 h. Extravascular lung water and permeability surface-area product increased at 80 and 110 h. Cardiac output and stroke volume also decreased, and systemic vascular resistance increased after 80 and 110 h of hyperoxia. Histopathological changes were present in the lungs of all but the 40-h exposure group. Animals exposed for 66 h showed endothelial injury and neutrophil accumulation. By 80 h, animals showed endothelial cell destruction, interstitial edema, and type I cell injury. At 110 h, animals showed substantial destruction of endothelial and type I epithelial cells, exposure of alveolar basement membrane, congestion of capillaries, and substantial interstitial edema. The data indicate that histological changes by electron microscopy precede physiological responses to hyperoxic pulmonary injury in baboons by as much as 14 h and that the physiological responses to early hyperoxic injury are relatively insensitive to the pathological injury.     

Key genes and proteins involved in CTCM-reducing microvascular endothelial cell permeability induced by SLT-IIv using gene chips and DIGE

An Affymetrix mouse genome array and differential in-gel electrophoresis (DIGE) techniques were used to investigate the pharmacological mechanisms of a mixture of herbs, designated CTCM, a compound of traditional Chinese medicine, for the treatment of increased permeability in mouse intestinal microvascular endothelial cells (MIMECs) induced by the Shiga-like toxin type II variant (SLT-IIv). MIMECs were challenged with 10 μg/ml SLT-IIv for 12 h and then treated with CTCM at a concentration of 200 μg/ml for 12 h. Total RNA and proteins from each treatment group were extracted from cultured MIMECs for analysis by the Affymetrix GeneChip® Mouse Genome 430 2.0 microarray and DIGE. The results obtained demonstrated that there were one genes downregulated and one genes upregulated, one protein downregulated and four proteins upregulated in the SLT-IIv group compared to the control group. In the CTCM group, four genes were upregulated, three genes were downregulated, a single protein was downregulated and a single protein was upregulated when compared to the control group. When the CTCM-treated group was compared to the SLT-IIv group, expression of one gene was found to be increased, and all other genes were decreased, with five proteins downregulated. Analysis of the data suggested that CTCM specifically and effectively reduced microvascular endothelial cell permeability to SLT-IIv in the treatment of pig edema disease. In the CTCM-treated group, hspa9 expression was increased in both gene chip and DIGE analysis, so it may be a key protein in reducing cell permeability and utilized in medical treatments.     

Tumor necrosis factor enhances the neutrophil-dependent increase in endothelial permeability

We examined the effect of tumor necrosis factor alpha (TNF alpha) on the increase in pulmonary microvascular endothelial monolayer permeability induced by activated neutrophils (PMN). Layering of PMN onto endothelial monolayers followed by activation of PMN with phorbol 12-myristate 13-acetate (PMA) increased 125I-albumin clearance rate across the monolayers. Pretreatment of endothelial monolayers for 6 hr with TNF alpha (200 U/ml) potentiated the PMN-dependent increase in endothelial permeability, whereas 1 hr or 6 hr pretreatment of endothelial monolayers with 200 U/ml and 100 U/ml, respectively, TNF alpha did not enhance the response. Adherence of PMN to the endothelial cells was increased at 1 and 6 hr after TNF alpha (200 U/ml) treatment, but the adherence response was markedly greater following 6 hr of TNF alpha. The TNF alpha treatment of endothelial cells did not enhance neutrophil activation responses to PMA. Pretreatment of PMN with IB4, a MAb to the CD18 integrin, the common beta subunit of the adhesion proteins LFA-1, Mac-1, and p150,95 of PMN, reduced the increases in PMN adherence and the endothelial monolayer permeability induced by the 6 hr TNF alpha treatment. In contrast, pretreatment of PMN with OKM-1, a MAb to the CD11b epitope (alpha-subunit), had no effect on the adherence and the potentiation of the increase in permeability. The potentiation of the PMN-dependent permeability increase and enhanced endothelial adhesivity at 6 hr after TNF alpha priming of endothelial cells was dependent on protein synthesis. The results indicate that protein synthesis-dependent expression of an endothelial ligand for CD18 and resultant endothelial hyperadhesiveness potentiates the PMN-mediated increase in endothelial permeability after TNF alpha activation of endothelial cells. The priming of endothelial cells by TNF alpha may be a critical step in the mediation of endothelial injury.     

Functional alterations in endothelial NO, PGI₂ and EDHF pathways in aorta in ApoE/LDLR⁻/⁻ mice

Adequate endothelial production of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin (PGI?) is critical to the maintenance of vascular homeostasis. However, it is not clear whether alterations in each of these vasodilatory pathways contribute to the impaired endothelial function in murine atherosclerosis. In the present study, we analyze the alterations in NO-, EDHF- and PGI?-dependent endothelial function in the thoracic aorta in relation to the development of atherosclerotic plaques in apoE/LDLR?/? mice. We found that in the aorta of 2-month-old apoE/LDLR?/? mice there was no lipid deposition, subendothelial macrophage accumulation; and matrix metalloproteinase (MMP) activity was low, consistent with the absence of atherosclerotic plaques. Interestingly, at this stage the endothelium was already activated and hypertrophic as evidenced by electron microscopy, while acetylcholine-induced NO-dependent relaxation in the thoracic aorta was impaired, with concomitant upregulation of cyclooxygenase-2 (COX-2)/PGI? and EDHF (epoxyeicosatrienoic acids, EETs) pathways. In the aorta of 3-6-month-old apoE/LDLR?/? mice, lipid deposition, macrophage accumulation and MMP activity in the intima were gradually increased, while impairment of NO-dependent function and compensatory upregulation of COX-2/PGI? and EDHF pathways were more accentuated. These results suggest that impairment of NO-dependent relaxation precedes the development of atherosclerosis in the aorta and early upregulation of COX-2/PGI? and EDHF pathways may compensate for the loss of the biological activity of NO.     

Endothelium-dependent relaxation by cilostazol, a phosphodiesteras III inhibitor, on rat thoracic aorta

The relaxation effect of cilostazol, a phosphodiesterase III inhibitor, on the thoracic aorta was investigated. Cilostazol induced the relaxation of the thoracic aorta precontracted by phenylephrine in a concentration-dependent manner. The concentration-dependent relaxation was shifted to the right in the endothelium denuded aorta compared with that of intact endothelium, suggesting that this relaxation was partly dependent on endothelium. Cilostazol-induced relaxation of thoracic aorta tone was reversed by treatment with N(G)-nitro L-arginine (L-NNA), a competitive inhibitor of nitric oxide (NO) synthase. Cilostazol also significantly increased the NO level in the porcine thoracic aorta. In rats treated with cilostazol, the urinary excretion of nitrites, a stable metabolite of NO, and basal production of NO of the aortic ring were significantly greater than in those without treatment. These findings indicate that cilostazol-induced vasodilation of the rat thoracic aorta was dependent on the endothelium, which released NO from aortic endothelial cells.     

Pulmonary edema and pleural effusion in norepinephrine-stimulated rats – hemodynamic or inflammatory effect?

Stimulation with norepinephrine (NE) leads to pulmonary edema and pleural effusion in rats. These pulmonary fluid shifts may result from pulmonary congestion due to the hemodynamic effects of NE and/or inflammation with an increase in vascular permeability. The contribution of these two factors were investigated in the present study. Female Sprague–Dawley rats received continuous i.v. NE infusion (0.1 mg/kg/h) over time intervals between 90 min and 72 h. After heart catheterization, pleural fluid (PF) and lung tissue were obtained. In some of the animals, a bronchoalveolar lavage (BAL) was performed. Pulmonary edema and inflammation were shown histologically. We determined the expression of interleukin (IL)-6 as one of the most potent acute-phase protein mediators in serum, PF and BAL supernatant fluid (BALF) using ELISA as well as in the lung tissue using Western blotting. Total protein concentration in BALF and PF served as indicators of increased capillary permeability. Pulmonary edema and pleural effusion appeared coincidentally with an increase in total peripheral resistance (TPR) after 6 h of NE infusion. PF reached a maximum between 8 and 16 h (2.2 ± 0.3 ml, controls < 0.5 ml) and disappeared within 48 h. Activation of IL-6 in the fluids was observed after 8 h of NE stimulation. In the lung tissue it started after 12 h and reached 330% of the control value after 48 h. Pulmonary inflammation was documented histologically. It was accompanied by increased protein concentration in BALF after 24 h of NE treatment. Hemodynamic effects of NE are the main causative factors in the initial phase of the pulmonary fluid shifts. Additionally, NE leads to an activation of cytokines such as IL-6 and to inflammation and to an increase in capillary permeability. However, inflammation and increased capillary permeability occurred later than pulmonary edema and pleural effusion. Hence, we conclude that they are secondary factors which may contribute to maintain the fluid shifts over a longer period of time.     

The role of the rat submandibular gland in the excretion of bis (tributyltin) oxide: electron microscopy, X-ray microanalysis and atomic absorption analysis.

The role of the submandibular glands in the excretion of parenterally administered bis (tributyltin) oxide (TBTO) was studied. Fine structural alterations of the submandibular glands were observed with an electron microscope. Accumulation sites of TBTO were determined with an X-ray microanalyzer and tin concentrations in saliva and blood were measured by atomic absorption spectrophotometry.     

Vanadyl sulfate protects against streptozotocin-induced morphological and biochemical changes in rat aorta

The aim of this study was to investigate the protective effects of vanadyl sulfate on aorta tissue of normal and streptozotocin (STZ)-induced diabetic rats, morphologically and biochemically. The animals were made diabetic by an intraperitoneal injection of streptozotocin (65 mg/kg) and vanadyl sulfate (100 mg/kg) that was given every day for 60 days by gavage technique to rats. Under the light and transmission electron microscopes, hypertrophy of the vessel wall, focal disruption in the elastic lamellae, an increase in thickness of total aortic wall, tunica intima, subendothelial space and adventitial layer, and a disorganization in smooth muscular cells of the tunica media were observed in diabetic animals. The aorta lipid peroxidation (LPO) levels were significantly increased and the aorta glutathione (GSH) levels were significantly reduced in STZ diabetic rats. In diabetic rats administered vanadyl sulfate for 60 days, aorta LPO levels significantly decreased and the aorta GSH level significantly increased. In conclusion, in vivo treatment with vanadyl sulfate of diabetic rats prevented the morphological and biochemical changes observed in thoracic aorta of diabetic animals.     

Fibronectin, hyaluronan, and a hyaluronan binding protein contribute to increased ductus arteriosus smooth muscle cell migration.

"Intimal cushions" which develop in the late gestation lamb ductus arteriosus (DA) are characterized by smooth muscle cells migrating into a large subendothelial space. Our previous in vitro studies, comparing DA cells with those from the aorta (Ao), have shown, even in early gestation, a 10-fold increase in DA endothelial incorporation of hyaluronan into the subendothelial matrix, a 2-fold increase in smooth muscle fibronectin synthesis and, in response to endothelial conditioned medium, a 2-fold increase in chondroitin sulfate. To determine whether these extracellular matrix components may be playing a role in inducing DA smooth muscle migration, we seeded Da or Ao smooth muscle cells onto three-dimensional collagen (2.0 mg/ml) gels and assessed migration 2, 5, and 8 days later. After 8 days, significantly greater numbers of DA compared to Ao cells were found invading the gels (23.1 +/- 3.1% vs 16.2 +/- 2.3%, P less than 0.01). Addition of GRGDS peptides (0.5 mM) or antibodies against fibronectin significantly decreased migration in the DA cells, but had no effect on migration in the Ao. Addition of endothelial conditioned medium to induce smooth muscle chondroitin sulfate production had no effect on DA cell migration. Inclusion of hyaluronan in the gel (0.5-1.5 mg), however, further enhanced DA cell migration, being greatest (31.9 +/- 3.1%) at a concentration of 1 mg/ml. Hyaluronan was without effect on Ao smooth muscle cell migration. The ability of hyaluronan to promote migration in cultures of DA smooth muscle cells was blocked completely by the addition of antibodies (1:100 dilution, 1 micrograms/ml) to a cell surface hyaluronan binding protein (HABP). As well, addition of anti-HABP to cells on gels containing collagen only significantly reduced migration in the DA but not the Ao. Immunofluorescent staining revealed that in DA cells, HABP was more concentrated in lamellipodia and leading edges than in Ao cells. As well, DA smooth muscle cells synthesized greater amounts of HABP as determined by Western immunoblotting and immunoprecipitation using polyclonal antisera to HABP. Thus, our studies indicate that both increased fibronectin and HABP contribute to the enhanced migration of DA smooth muscle cells. These results, together with our previous studies showing a 10-fold increase in hyaluronan accumulation in the DA endothelial matrix, would suggest a mechanism for increased DA smooth muscle migration into the subendothelial matrix observed in vivo.