The American Microcirculatory Society Landis Award lecture. Endothelial cells, inflammatory edema, and the microvascular barrier: comments by a "free radical".
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Awards and Prizes↗
Biomedical subjects
D Shepro
Publications and source records attributed to D Shepro.
Phalloidin enhances endothelial barrier function and reduces inflammatory permeability in vitro.
Phalloidin, a potent microfilament toxin, induces polymerization of actin in vitro and in vivo. In a permeability assay, bovine aortic endothelial cell cultured on microcarrier beads exclude significantly more serum albumin after 30 min treatment with 10(-6), 10(-8), and 10(-10) M phalloidin than controls. Furthermore, pretreatment of microcarriers with 10(-8) M phalloidin significantly reduces permeability increases by histamine, bradykinin, thromboxane A2 mimetic, and cytochalasin B, (all at 10(-6) M). Phalloidin also causes significant surface area and perimeter increases in cultured endothelial cells. The cells also display increased acting stress fibers and show a weblike cytoskeletal pattern of microfilaments. These data suggest that in vitro the endothelial junctional barrier may be enhanced in part by assembly of actin filaments.
Animals↗
Limb ischemia-induced increase in permeability is mediated by leukocytes and leukotrienes.
This study tests the role of white blood cells (WBC) and leukotrienes in mediating the increased microvascular permeability following ischemia and reperfusion. Anesthetized dogs (n = 23) underwent 2 hours of hind limb ischemia induced by tourniquet inflation to 300 mmHg. In untreated animals (n = 7), tourniquet release led after 5 minutes to a rise in plasma thromboxane (Tx) B2 levels from 360 to 1702 pg/ml (p less than 0.05); after 2 hours, lymph TxB2 concentration had risen from 412 to 1598 pg/ml (p less than 0.05). There were decreases in circulating WBC from 11,766 to 6550/mm3 and platelets from 230 to 155 x 10(3)/mm3. During reperfusion, popliteal lymph flow (QL) increased from 0.07 to 0.24 ml/hour (p less than 0.05), while the lymph/plasma (L/P) protein ratio was unchanged from 0.39, changes consistent with increased microvascular permeability. WBC depletion (n = 7) to 302/mm3 by hydroxyurea or nitrogen mustard attentuated (p less than 0.05) the reperfusion induced rise in plasma TxB2 from 91 to 248 pg/ml and prevented the increase in lymph TxB2 concentration. Within 5 minutes of tourniquet release WBC counts further decreased to 191/mm3 (p less than 0.05) and platelets declined from 175 to 93 x 10(3)/mm3 (p less than 0.05). QL increased from 0.07 to 0.12 ml/hour (p less than 0.05), lower than untreated animals (p less than 0.05), and the L/P protein ratio declined from 0.49 to 0.37 (p less than 0.05), dilutional changes consistent with increased filtration pressure but not permeability to protein. Pretreatment with the lipoxygenase inhibitor diethylcarbamazine (DEC) (n = 8) prevented the reperfusion-induced increase in plasma and lymph TxB2 levels (p less than 0.05) and the fall in WBC counts (p less than 0.05), while platelet counts declined from 381 to 210 x 10(3)/mm3 (p less than 0.05). QL rose from 0.09 to 0.23 ml/hour (p less than 0.05) during reperfusion, and the L/P protein ratio of 0.3 remained unchanged, a value lower than in untreated dogs (p less than 0.05). In two animals of each group, vascular recruitment was induced by tourniquet inflation to 50 mmHg. This led to a high QL of 0.25 ml/hour and a low L/P ratio of 0.18. In untreated animals during reperfusion, QL further increased to 1.3 ml/hour, and L/P ratio rose to 0.44, documenting increased vascular permeability. In contrast, reperfusion in leukopenic or diethylcarbamazine (DEC)-treated dogs with vascular recruitment, was not associated with increases in QL or the L/P protein ratio.(ABSTRACT TRUNCATED AT 400 WORDS)
6-Ketoprostaglandin F1 alpha↗
Lower torso ischemia-induced lung injury is leukocyte dependent.
Lower torso ischemia leads on reperfusion to sequestration of polymorphonuclear leukocytes (PMN) in the lungs and increased permeability. This study tests the role of circulating leukocytes (WBC) in mediating this lung injury. Anesthetized sheep prepared with chronic lung lymph fistulae underwent 2 hours of bilateral hind limb tourniquet ischemia. In untreated controls (n = 7), 1 minute after reperfusion there were transient increases in mean pulmonary arterial pressure (MPAP) from 13 to 38 mmHg (p less than 0.05) and pulmonary microvascular pressure (Pmv) from 7 to 18 mmHg (p less than 0.05), changes temporally related to a rise in plasma thromboxane (Tx) B2 levels from 211 to 735 pg/ml (p less than 0.05). Lung lymph TxB2 levels rose from 400 to 1005 pg/ml at 30 minutes (p less than 0.05), and remained elevated longer than plasma levels. Lung lymph flow (QL) rose from 4.3 to 8.3 ml/30 minutes (p less than 0.05) after 30 minutes of reperfusion and remained elevated for 2 hours. The lymph/plasma (L/P) protein ratio was unchanged from 0.6, while the lymph protein clearance increased from 2.6 to 4.6 ml/30 minutes (p less than 0.05), suggesting increased microvascular permeability. WBC counts decreased within the first hour of reperfusion from 6853 to 3796/mm3 (p less than 0.05), and lung histology after 2 hours showed proteinaceous exudates and leukosequestration of 62 PMN/10 high-powered fields (HPF), higher than the 22 PMN/10 HPF (p less than 0.05) in sham animals (n = 3). Recruitment of the pulmonary vasculature by left atrial balloon inflation (n = 3) resulted in a rise in MPAP to 20 mmHg. After 3 hours of balloon inflation, QL stabilized at 9.8 ml/15 minutes, and a pressure-independent L/P protein ratio of 0.3 was achieved. During reperfusion, QL increased further to 11.2 ml/15 minutes, the L/P ratio rose to 0.56 and the calculated osmotic reflection coefficient decreased from 0.70 to 0.44, documenting an increase in lung microvascular permeability. In contrast to these untreated ischemic controls, sheep (n = 7) rendered leukopenic with hydroxyurea or nitrogen mustard and having a total WBC count of 760/mm3 and PMN count of 150/mm3 did not manifest reperfusion-induced increases in MPAP, Pmv, QL, lymph protein clearance, or lung lymph. TxB2 level (p less than 0.05). Plasma TxB2 levels rose slightly at 30 minutes from 199 to 288 pg/ml (p less than 0.05). Lung histology was normal. These data indicate that WBC mediate the ischemia-induced increase in pulmonary microvascular permeability.
Animals↗
Inhibition of thromboxane (Tx) synthesis by free radical scavengers.
Treatment with thromboxane (Tx) synthase inhibitors or free radical scavengers has been shown to afford protection from renal ischemia. Since free radicals are closely associated with thromboxane (Tx) synthesis, this study examines the thesis that free radical scavengers inhibit formation of Tx. Anesthetized rats (n = 42) underwent right nephrectomy. By random choice, before 45 min of left renal pedicle clamping, rats received: 0.5 ml dextrose placebo IV (n = 6); the hydroxyl radical scavenger dimethyl-thiourea (DMTU), 500 mg/kg IV (n = 10); or the superoxide scavenger superoxide dismutase (SOD), 24,000 Sigma Units (SU)/kg IV (n = 12). This dose of SOD was repeated before release of the clamp. Treatment with DMTU and SOD decreased plasma TxB2 levels following 5 min of reperfusion from 2,480 pg/ml in dextrose treated controls to 1,155 pg/ml (p less than 0.01) and 1,419 pg/ml (p less than 0.03), respectively. At 24 hr, DMTU and SOD therapy decreased creatinine from 3.0 mg/dl in controls to 1.6 mg/dl (p less than 0.01) and 2.1 mg/dl (p less than 0.05), respectively. At 24 hr, DMTU but not SOD decreased left renal weight from 113 to 94% (p less than 0.0003) of the weight of the previously removed right kidney, and histologically prevented acute tubular necrosis (p less than 0.05). In nephrectomized but nonischemic sham control rats (n = 7) plasma TxB2 and 6-keto-PGF1 alpha concentrations were 757 pg/ml and 82 pg/ml, creatinine level 0.9 mg/dl and kidney weight 94% of the previously removed right kidney.(ABSTRACT TRUNCATED AT 250 WORDS)
6-Ketoprostaglandin F1 alpha↗
Reperfusion of ischemic lower limbs increases pulmonary microvascular permeability.
Lower torso ischemia and reperfusion has been shown to stimulate the generation of thromboxane (Tx)A2, leading to transient pulmonary hypertension and later to polymorphonuclear leukocyte accumulations in the lungs. This study investigated whether hind limb ischemia leads to increased pulmonary microvascular permeability. Anesthetized sheep (n = 6) previously prepared with a lung lymph fistula underwent 2 hr of tourniquet ischemia of both lower limbs. One minute following tourniquet release mean pulmonary arterial pressure (MPAP) rose from 14 +/- 1 to 36 +/- 4 mm Hg (p less than 0.05) and returned to baseline within 30 min. The pulmonary arterial wedge pressure of 4 +/- 1 mm Hg was unchanged. Plasma TxB2 levels rose from 211 +/- 21 to 304 +/- 52 pg/ml (p less than 0.05) 10 min after tourniquet release and were back to baseline at 30 min. Lymph flow (QL) rose from 4.3 +/- 0.6 ml/30 min to 8.3 +/- 1.8 ml/30 min (p less than 0.05); the lymph/plasma (L/P) protein ratio rose slightly but not significantly. In three sheep, inflation of a left atrial balloon increased left atrial pressure from 3 to 16 mm Hg. MPAP rose from 14 to 24 mm Hg. There was an increase in QL from 3.6 to 17 ml/30 min; the L/P protein ratio declined from 0.63 to 0.41. These results indicate that reperfusion following 2 hr of bilateral hind limb ischemia results in increased pulmonary microvascular permeability.
Animals↗
Selective lung leukosequestration after complement activation.
This study tests whether activated complement leads to a selective entrapment of polymorphonuclear leukocytes (PMN's) in the lungs. Awake sheep were infused for 5 min with zymosan-activated plasma (ZAP, 2.5 mg/ml) at a rate of 5 ml/min into the superior vena cava (IV, n = 4) or intra-arterially into the aortic arch or femoral artery (IA, n = 8). At the end of IV infusion, leukocyte counts fell from 8,862 to 1,631/mm3 (P less than 0.01). PMN counts across the lungs decreased by 74%. There were increases in plasma thromboxane (Tx) B2 from 114 to 2,733 pg/ml (P less than 0.01), mean pulmonary arterial pressure from 12 to 42 mmHg (P less than 0.01), and physiological shunt from 13 to 25% (P less than 0.05). Within 1 h lymph TxB2 levels had risen from 301 to 4,916 pg/ml (P less than 0.01), lung lymph flow (QL) rose from 3.7 to 11.1 ml/30 min (P less than 0.05), lymph-to-plasma protein ratio (L/P) remained unchanged at 0.63, and lymph protein clearance increased from 2.3 to 7.5 ml/30 min (P less than 0.05). Leukosequestration, quantitated by capillary PMN counting and by assaying the granulocyte marker myeloperoxidase, occurred relative to sham animals (P less than 0.05) in the lung and spleen but not in other organs. Intra-arterial ZAP infusion led to changes that were similar in magnitude and timing to the IV group.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Endothelial-secreted arachidonic acid metabolites modulate polymorphonuclear leukocyte chemotaxis and diapedesis in vitro.
The influence of endothelial cells (ECs) on polymorphonuclear leukocyte (PMN) motility was examined using in vitro assays of PMN diapedesis and chemotaxis. ECs are seen to release arachidonic acid (20:4) metabolites that directly increase or decrease PMN movement, with their general effect being enhanced motility. This effect can be increased or decreased by treating ECs with stimulators or inhibitors of 20:4 metabolism, respectively. The metabolites include thromboxane B2, which increases PMN random motility, chemotaxis, and diapedesis in a dose-responsive manner and which acts as a chemoattractant; 6-keto-PGF1 alpha, which increases chemotaxis and diapedesis at high doses but decreases these responses at low doses; and a lipoxygenase-derived metabolite, suggested to be 5-hydroxyeicosatetraenoic acid, which increases chemotaxis and diapedesis. Thromboxane A2 and prostacyclin mimetics also affect chemotaxis in qualitatively similar manners as TxB2 and 6-keto-PGF1 alpha, respectively, but display greater potency. EC release of these metabolites is also seen to be substratum modulated, with an increased production by cells cultured on extracellular matrices. These results suggest that ECs are capable of modulating PMN motility and suggest a role for ECs in the control of PMN diapedesis.
Animals↗
Effects of nitroprusside and ketanserin upon pulmonary edema after acid injury.
Increased hydrostatic pressure and microvascular permeability after acid aspiration as causes of pulmonary and tracheobronchial edema were studied. Eighteen dogs were anesthetized and 3.0 milliliters per kilogram of 0.1 normal hydrochloride instilled into the trachea. After one hour, the platelet count decreased 47,500 per cubic millimeter (p less than 0.05) from base line levels of 221,400 per cubic millimeter and platelet serotonin stores decreased from 2.09 to 1.65 micrograms per 10(9) platelets (p less than 0.05). Mean pulmonary arterial pressure increased progressively from a base line value of 11 to 21 millimeters of mercury four hours after acid instillation (p less than 0.05). Mean arterial pressure fluctuated from a high of 143 millimeters of mercury to the final value, at four hours, of 119 millimeters of mercury. Ketanserin, a serotonin receptor antagonist, infused one hour after acid injury did not prevent release of platelet 5 hydroxytryptamine but led to a 24 per cent decrease in mean pulmonary arterial pressure from 13 to 10 millimeters of mercury (p less than 0.05) and an 11 per cent decrease in mean arterial pressure from 135 to 120 millimeters of mercury (p less than 0.05). Nitroprusside produced a 30 per cent decrease in mean arterial pressure, but only a 20 per cent decline in mean pulmonary arterial pressure (p less than 0.05). Four hours after acid was instilled, untreated dogs produced 129 milliliters of edema fluid from the endotracheal tube. In proportion to the relative decline in mean pulmonary arterial pressure, nitroprusside therapy reduced the volume of edema to 72 milliliters (p less than 0.05) and ketanserin to 52 milliliters (p less than 0.05). These results suggest that 5 hydroxytryptamine contributes to the pulmonary hypertension associated with acid injury and that the volume of edema formed may be directly related to the increase in the hydrostatic pressure.
Animals↗
Serotonin induced actin polymerization and association with cytoskeletal elements in cultured bovine aortic endothelium.
Serotonin, (5-hydroxytryptamine, 5-HT) binds to cultured endothelial cell stress fibers as identified by fluorescence microscopy and in vitro induces actin polymerization as measured by DNase 1 inhibition and differential centrifugation; the structural change in actin in the presence of 5-HT resembles actin paracrystals as visualized by negative staining under electron microscopy. These observations indicate that 5-HT, which is taken up by endothelium by a non-mediated diffusion, may interact directly with actin to affect cytoskeletal changes.
Actins↗
Correlation among endothelial cell shape, F-actin arrangement, and prostacyclin synthesis.
Though many factors have been identified which modulate prostacyclin (PGI2) synthesis, there is little information on cellular mechanisms whereby endothelial cells (EC) regulate their basal eicosanoid metabolism. Using substrates of various adhesive capacities, bovine and porcine aortic EC shape and cytoskeletal F-actin arrangement could be modulated. Staining with rhodamine-phalloidin (R-P) permitted analysis of F-actin arrangement, while differences in cell shape were determined by measurement of cell perimeter surface area (CPSA). Spectrophotoflurometric measurements were used to quantitate the R-P binding capacity of the cultures. Cultures of reduced CPSA (225.2 +/- 13.5 mu2) generated the highest levels of basal PGl2 (6.14 +/- 0.51 pg/ug cell protein); had a diffuse arrangement of F-actin and an increased binding capacity for R-P (463.55 +/- 50.58 nmoles/ug cell protein). Cultures of enlarged CPSA (1399.3 +/- 148.3 mu2), with many actin cables and a significantly reduced (p less than 0.001) R-P binding capacity (74.941 +/- 11.79 nmoles/ug of cell protein) produced significantly smaller (p less than 0.001) basal quantities of PGl2 (1.33 +/- 0.14 pg/ug cell protein). Similarly, arachidonic acid stimulation of cultures of reduced CPSA resulted in an increased synthesis of PGl2 when compared to stimulated cultures of enlarged cells. These findings suggest a role for cell shape and the cytoskeleton in the mechanism controlling PGl2 production and indicate that alteration of the arrangement of F-actin may be of importance in regulation of EC eicosanoid metabolism.
Actins↗
Microvascular pericyte contractility in vitro: comparison with other cells of the vascular wall.
Collagen lattices containing bovine retinal pericytes (RPs), vascular smooth muscle cells (VSMCs), pulmonary microvessel endothelial cells (PMECs), or aortic endothelial cells (AECs) were prepared and contraction was quantitated by measuring the resulting change in lattice area. VSMCs were the most efficient at lattice contraction followed by RPs and then PMECs. AECs did not contract the lattices. To document further that these observations represent contraction, cells were grown on inert silicone rubber sheets. Substratum wrinkling was indicative of tension development and quantitated as percent of cells contracted. RPs were more contractile than PMECs, and AECs were incapable of developing tension. VSMCs were less contractile than RPs, unlike the comparative contractility observed with the lattice system. Alteration of actin-containing filaments by cytochalasin B significantly reduced RP contraction of silicone rubber and inhibited their contraction of collagen lattices in a dose-dependent manner. Rhodamine-phalloidin staining of contracting RPs revealed microfilament bundle orientations that suggested their association in the force applied for contraction. RP, VSMC and PMEC contraction of collagen lattices was directly proportional to the concentration of fetal calf serum. Also, RP contraction was greater in calf serum than calf plasma-derived serum, an indication that RPs respond to substances that appear continuously and episodically in blood. These in vitro findings support the theory that pericytes in vivo are contractile but that endothelial cells may also contribute to microvascular tonus.
Animals↗
Vasodilator prostaglandins (PG) prevent renal damage after ischemia.
Thromboxane (Tx) synthase but not cyclo-oxygenase inhibitors prevent acute tubular necrosis (ATN) after renal ischemia, a phenomenon believed to be due to stimulation of the endogenous production of vasodilating prostaglandins (PG). This study directly tests that vasodilating PG protect against the consequences of renal ischemia. Anesthetized, 500-g rats had right nephrectomy and 45 minutes of left renal pedicle clamping or sham clamping. The rats were treated with intravenous (I.V.) saline 1.9 mL/h starting 40 minutes after clamping or sham clamping. All rats except the sham group (N = 8) were pretreated 1 hour before ischemia with ibuprofen (12 mg/kg) to prevent prostanoid synthesis. Beginning 5 minutes before clamp release, the rats were treated intravenously for 2 hours with: saline vehicle (N = 9), PGE1 400 ng/kg/min (N = 6), nitroprusside 4 micrograms/kg/min (N = 8), or dopamine 3 micrograms/kg/min (N = 11). After 24 hours, sham rat creatinine level was 0.5 mg/dL and weight of the left kidney was 86.5% of the previously removed right kidney. Compared with sham rats, ischemia and saline treatment resulted in a rise in creatinine level to 2.7 mg/dL (p less than 0.05) and a rise in kidney weight to 101.9% (p less than 0.05); PGE1 led to a creatinine level of 1.1 mg/dL, a value lower than that of the rats treated with saline (p less than 0.05), and a kidney weight of 92.0%, a value similar to that of sham rats; nitroprusside and dopamine led to a rise in creatinine levels to 3.2 mg/dL (p less than 0.05) and 2.3 mg/dL (p less than 0.05), respectively, as well as a rise in kidney weight to 108.0% (p less than 0.05) and 105.4% (p less than 0.05), respectively. Histologic examination showed ATN in rats treated with saline, nitroprusside, and dopamine, but not in rats treated with PGE1. These results indicate that PGE1 protects the cyclo-oxygenase-treated kidney against ischemia-induced ATN.
Acute Kidney Injury↗
Pulmonary leukosequestration induced by hind limb ischemia.
Lower torso ischemia leads to acute respiratory failure, an event associated with the accumulation of inflammatory cells in the lungs. This study tests whether ischemia-induced eicosanoid synthesis leads to polymorphonuclear leukocyte (PMN) accumulation in the lungs. Anesthetized rats (N = 51) were randomized into five groups: nonischemic sham rats (N = 10); the remaining four groups were rats made ischemic for 4 hours with bilateral thigh tourniquets treated just before tourniquet release with saline vehicle (N = 17): the thromboxane (Tx) synthase inhibitor OKY-046 (Ono Pharmaceutica, Osaka, Japan) 2 mg/kg intravenously every 2 hours (N = 8); the lipoxygenase inhibitor diethylcarbamazine (DEC) (Sigma, St. Louis, MO) 0.2 mg/kg/min intravenously (N = 8); the platelet-activating factor receptor antagonist SRI (Sandoz Inc., East Hanover, NJ) 63-072 3 mg/kg intravenously every 30 minutes (N = 8). Four hours after ischemia, plasma TxB2 levels in the ischemic placebo-treated group was 3570 +/- 695 pg/mL, compared with 495 +/- 73 pg/mL in sham rats (p less than 0.001). Lung microscopy showed foci of proteinaceous exudate in alveoli and 121 +/- 10 PMN/20 high power fields (HPF) compared with 59 +/- 9 PMN/20 HPF in the sham group (p less than 0.001). One day after ischemia PMN accumulations remained elevated at 119 PMN/20 HPF. Pretreatment with OKY-046 led to reduced TxB2 levels of 149 +/- 17 pg/mL, normal lung histology, and 83 +/- 13 PMN/20 HPF, a value similar to that of the sham group and lower than that of the placebo-treated group (p less than 0.05). Treatment with DEC yielded TxB2 levels of 1419 +/- 492 pg/mL, which was lower than that of the placebo group (p less than 0.05) but higher than that of the sham group (p less than 0.05). Microscopy showed normal lungs with 79 +/- 7 PMN/20 HPF lower than the placebo group (p less than 0.05). SRI 63-072 did not inhibit Tx synthesis or leukosequestration in the lungs. Platelet counts decreased in all groups relative to sham animals (p less than 0.05). The results indicate that Tx synthesis induced by ischemia moderates PMN accumulations in the lungs. Inhibition of lipoxygenase is believed to prevent PMN accumulations both by limiting leukotriene-induced Tx synthesis as well as by limiting production of chemoattractants.
Animals↗
Pulmonary hypertension and leukosequestration after lower torso ischemia.
Ischemia stimulates thromboxane (Tx) synthesis. This study tests the hypothesis that the cardiopulmonary dysfunction that may follow aortic declamping is related to Tx. Anesthetized dogs (N = 15) were subjected to 4 hours of infrarenal aortic cross-clamping. In untreated control animals (N = 7), plasma levels of TxB2 rose from 654 +/- 74 pg/mL to 1238 +/- 585 pg/mL at 5 min (p less than 0.05), and to 3174 +/- 912 pg/mL 3 hours after declamping (p less than 0.05). Mean pulmonary artery pressure (MPAP) rose 5 min after declamping from 13 +/- 2 mmHg to 21 +/- 2 mmHg (p less than 0.05). Cardiac Index (CI) declined during ischemia from 181 +/- 30 mL/kg.min to 128 +/- 16 mL/min.kg (p less than 0.05), and to 80 +/- 8 mL/min.kg after 4 hours of reperfusion (p less than 0.05). Platelet counts declined but platelets labeled with In 111 did not accumulate in the lungs, whereas quantitative counts of polymorphonuclear leukocytes (PMN) in the lungs 4 hours after declamping yielded 213 +/- 33 PMN/25 high power fields (HPF) in dependent areas of the lung and 153 +/- 26 PMN/25 HPF in nondependent areas. The wet/dry weight ratio of the lungs was not elevated, although foci of proteinaceous exudate and PMNs in alveoli were noted. Another group of dogs (N = 8) were pretreated by random choice with the Tx synthase inhibitor OKY-046 2 mg/kg IV every 2 hours, which led to: lower TxB2 levels at baseline 95 +/- 35 pg/mL (p less than 0.05), 5 min after ischemia 140 +/- 93 pg/mL and after 3 hours of reperfusion 122 +/- 36 (p less than 0.05); lower MPAP, 16 +/- 2 mmHg (p less than 0.05); higher CI throughout (p less than 0.05); normal histology and reduced pulmonary PMN sequestration both in dependent 127 +/- 15 PMN/25 HPF and nondependent areas of the lungs 95 +/- 11 PMN/25 HPF (p less than 0.05). In animals undergoing sham ischemia (N = 3), levels of TxB2 and cardiopulmonary function remained unchanged from baseline. There were 150 PMN/25 HPF in dependent and 85 PMN/25 HPF in nondependent lung areas. The results indicate that ischemia-generated Tx mediates a rise in MPAP, a fall in CI, and PMN entrapment in the lungs.
Animals↗
Vasoactive amines directly modify endothelial cells to affect polymorphonuclear leukocyte diapedesis in vitro.
Bovine aortic endothelial cells were cultured on the basement membrane surface of amnionic membrane and used as a substrate for polymorphonuclear leukocyte (PMN) diapedesis in vitro. Norepinephrine (NE), serotonin (5HT), or phalloidin treatment of the endothelial cells (ECs) reduces, whereas histamine or cytochalasin B increases, the number of PMNs migrating across the ECs and amnionic membrane. In contrast, amine treatment of PMNs or acellular amnionic membrane does not alter PMN diapedesis or chemotaxis. The NE and histamine effects are blocked by appropriate receptor antagonists, but the 5HT effect is not. All the agents' effects are also reversible. Qualitatively similar effects on EC permeability to Evan's blue-labeled albumin occur with all agents; however, PMN adhesion to ECs is not affected. Previously, we reported that NE and 5HT increase stress fiber numbers and decrease EC permeability to macromolecules in vitro, whereas histamine has the opposite effects, and that NE and 5HT eliminate the erythrocyte extravasation associated with thrombocytopenia in vivo. In this study, we propose that these vasoactive amines also alter PMN diapedesis in vitro through a direct effect on the EC, in part due to alterations in the EC cytoskeleton.
Animals↗
A high plasma prostaglandin to thromboxane ratio protects against renal ischemia.
Pretreatment with the thromboxane synthase inhibitor OKY-046 but not the cyclo-oxygenase inhibitor ibuprofen protects against ischemia-induced acute tubular necrosis. However, ibuprofen together with the vasodilating agent prostaglandin E1 is protective. This suggests that a high prostaglandin to thromboxane ratio is the major factor operative in preventing tubular necrosis, the subject of this study. Rats that had unilateral nephrectomy (n = 60) with the exception of rats that had sham operations (n = 8) underwent 45 minutes of left renal pedicle clamping. Thirty minutes before the operation, the rats received either a saline solution or a thromboxane synthase inhibitor that was given intravenously. The inhibitors OKY-046 (2 milligrams per kilogram, n = 10), UK38485 (1 milligram per kilogram, n = 9) and U63357A (10 milligrams per kilogram, n = 10) were given as a single bolus while the inhibitor CGS13080 (0.1 milligram per kilogram, n = 9, and 1.0 milligram per kilogram, n = 7) was given by constant infusion and continued for 60 minutes after reperfusion. With saline solution therapy, five minutes after reperfusion, thromboxane B2 increased from 154 to 2,537 picograms per milliliter (p less than 0.00001) and 6-keto-prostaglandin F1 alpha increased from 51 to 266 picograms per milliliter (p less than 0.004). At 24 hours, the creatinine level increased from 0.5 to 2.8 milligrams per deciliter (p less than 0.00001). Only OKY-046 yielded a creatinine level at 24 hours of 1.2 milligrams per deciliter, a value lower than that for those in the saline solution control group (p less than 0.002). Furthermore, OKY-046 led to the highest prostaglandin to thromboxane ratio (p less than 0.035). The five other ratios which occurred after drug therapy were inversely related to the decrease in the creatinine value (r = -0.93, p less than 0.02). Histologically, OKY-046 was the only thromboxane synthase inhibitor to prevent acute tubular necrosis (p less than 0.05). Results show that a high prostaglandin to thromboxane ratio protects against acute tubular necrosis.
6-Ketoprostaglandin F1 alpha↗
Serotonin, norepinephrine, and histamine mediation of endothelial cell barrier function in vitro.
The effects of serotonin (5-hydroxytryptamine, 5-HT), norepinephrine (NE), and histamine on endothelial cell barrier function were examined in vitro. Bovine aortic endothelial (BAE) cells grown to confluence on microcarriers formed a measurable barrier to the passage of a trypan blue dye-bovine serum albumin conjugate (TB-BSA) from the culture medium into the microcarrier matrix. Vascular smooth muscle (VSM) cells or Swiss 3T3 fibroblasts impeded TB-BSA diffusion only 42% and 56%, respectively, relative to BAE cells. These results suggest that barrier formation may be an endothelial cell-specific phenomenon. Treatment of BAE cells with histamine was associated with 2- to 3-fold increases in the rate of TB-BSA diffusion. In contrast, treatment with 5-HT or NE at concentrations ranging from normal to pathophysiological circulating plasma levels significantly impeded TB-BSA diffusion by up to 43% and 33%, respectively, relative to untreated controls. The barrier-modulating effects of the vasoactive amines were dose-dependent, cell-specific, and in some cases appear to be receptor-mediated. These results are consistent with previous reports that histamine increases vascular permeability in part by affecting diffusion between endothelial cells; they support the hypothesis that 5-HT and NE contribute to the maintenance of the endothelial barrier in vivo.
Animals↗