Hiroshi Mitsuoka

Protease inhibition in the intestinal lumen: attenuation of systemic inflammation and early indicators of multiple organ failure in shock.

Our recent evidence suggests that pancreatic digestive enzymes in the lumen of the intestine may play a major role in the production of cardiovascular stimulatory factors during splachnic artery occlusion and reperfusion. These stimulators are detected in plasma, but their origin and mechanism of production has remained uncertain. We examine here in the rat the role of pancreatic enzymes with and without administration of a serine protease inhibitor (FOY) into the lumen of the small intestine during splanchnic artery occlusion (90 min) and reperfusion (120 min). In the presence of pancreatic enzyme inhibition in the lumen of the intestine, there is significantly enhanced survival rate, lower levels of inflammatory mediator production, the femoral artery blood pressure is maintained close to control levels, and there are significantly lower levels of cell activators in plasma. These results support the hypothesis that pancreatic enzymes may escape across the brush border barrier during intestinal ischemia and thereby initiate the production of a powerful set of cytotoxic mediators. Blockade of pancreatic enzymes in the lumen of the intestine may be a tool to interfere with inflammation and early indicators of multiorgan failure.

Association of a G994→T missense mutation in the plasma platelet-activating factor acetylhydrolase gene with risk of abdominal aortic aneurysm in Japanese

ObjectiveTo investigate a possible association with plasma platelet activating factor acetylhydrolase (PAF-AH) gene mutation with the risk of abdominal aortic aneurysm (AAA). Summary Background DataPlasma platelet activating factor acetylhydrolase is known to catalyze platelet activating factor (PAF), thereby inactivating its inflammatory function. Deficiency of this enzyme is caused by a missense mutation (G994 →T) in exon 9 of the plasma PAF-AH gene. MethodsWe did a case-control study including 131 patients (median age 73.4 [range 50–84] years) and 106 controls matched for age and sex. Genomic DNA was analyzed for the mutant allele by a specific polymerase-chain reaction. Plasma PAF-AH activity was measured in both groups. ResultsThe frequency of the mutant allele (T allele) in the plasma PAF-AH gene in AAA patients was significantly higher than in control subjects. The association of the missense mutation with AAA was statistically significant and independent of other risk factors. Among AAA patients with normal genomic type, plasma PAF-AH activity was strongly correlated to the plasma concentration of low density lipoprotein cholesterol (LDL-C), while the correlation was not observed among AAA patients with heterozygotes genotype. Patients having AAA with both T allele and hyperlipidemia were more likely to have other atherosclerotic diseases such as ischemic heart disease, stroke and peripheral arterial occlusive diseases than patients with the normal genomic type and normal lipid level. ConclusionsThe genetic mutation of plasma PAF-AH gene appear to be an independent risk factor for AAA. Our findings need to be confirmed in a larger, prospective study including patients from different populations.

Mechanisms for blockade of in vivo activator production in the ischemic intestine and multi-organ failure

Our recent results indicate that pancreatic enzymes in the ischemic intestine are involved in the production of in vivo activators, which stimulate cells in the cardiovascular system and initiate multiple organ failure. Since the intestine is a rich source of xanthine oxidase (XO), we investigated whether XO may be involved in the production of circulating activators in shock. The small intestine was perfused with saline (SAL group), a broad acting pancreatic enzyme inhibitor, ANGD (ANGD group), allopurinol (ALLOP group), or a combination of ANGD and allopurinol (ANGD + ALLOP group). 100 min of splanchnic arterial occlusion was followed by 120 min of reperfusion. Leukocytes from asymptomatic volunteers were incubated with plasma from experimental animals, and the fractions of pseudopod positive cells were counted as an indicator for the activator. ANGD served to preserve the arterial blood pressure (MAP) close to its control values (96.6 +/- 6.2 % in ANGD versus 60.9 +/- 6.2 % in SAL, 120 min after reperfusion, P < 0.05). In line with our previous experiments, ANGD decreased the formation of activator (30.5 +/- 4.8% in SAL versus 7.3 +/- 1.6 % in ANGD, 120 min after reperfusion, P< 0.05). Although allopurinol inhibited the XO in the small intestine, no protection from early indicators of multi-organ injury was detected. The recovery of MAP and reduced levels of plasma activator achieved in the ANGD + ANGD group was similar to those in the ALLOP group. These results indicate that XO may not serve as a significant source for plasma derived activators in an acute phase of shock after severe intestinal ischemia.

CONTRIBUTION OF ENDOTHELIN-1 TO MICROCIRCULATORY IMPAIRMENT IN TOTAL HEPATIC ISCHEMIA AND REPERFUSION INJURY

BACKGROUND Endothelin (ET)-1 may have a role in hepatic polymorphonuclear leukocyte infiltration as well as microcirculatory disturbance during hepatic ischemia-reperfusion (HIR) injury. This study was conducted to investigate the influence of ET-1 on the hepatic microcirculation after total HIR and to evaluate the effect of a nonselective ET receptor antagonist under these conditions. METHODS Male rats pretreated with either normal saline (NS group) or TAK-044, a nonselective ET receptor antagonist (TAK group), were subjected to 120 min of total hepatic ischemia with extracorporeal portosystemic shunting. RESULTS Plasma ET-1 levels increased significantly from 1 to 6 hr after reperfusion in the NS group when compared with the nonischemic control. In the early phase of reperfusion, the NS group showed significantly narrower sinusoids, lower hepatic tissue blood flow, a lower hepatic tissue oxy-hemoglobin concentration, and more hepatic neutrophil infiltration than the TAK group (P<0.05). Pretreatment with TAK-044 improved hepatic microcirculatory derangement, and resulted in significantly better 7-day survival (61.5%) with more bile production after reperfusion when compared with the NS group (P<0.01). CONCLUSIONS The present study demonstrated that ET-1 is involved in the development of HIR injury by causing deterioration of the hepatic microcirculation. A nonselective ET receptor antagonist successfully ameliorated HIR injury through improvement of hepatic oxygenation and of the microcirculation along with reduced hepatic neutrophil infiltration.

Superficial Thrombophlebitis of the Lower Limbs in Patients with Varicose Veins

AbstractPurpose. This study reviews 51 consecutive patients with superficial thrombophlebitis (STP) among 710 patients treated for varicose veins in our department. Methods. An assessment was made of various factors involved. Results. Of these 51 patients, 21 (41.1%) had systemic disorders, including 4 (7.8%) with malignant diseases. Six patients (11.8%) had deep vein thrombosis (DVT) and five (9.8%) had pulmonary embolism (PE). All of the patients with DVT and/or PE had a thrombus in either the greater saphenous vein or the lesser saphenous vein; however, none of the patients with STP and a thrombus in the distal saphenous branch had either DVT or PE. The levels of coagulofibrinolytic markers such as fibrin degradation product-D dimer, plasmin α2 plasmin inhibitor complex, and thrombin antithrombin III complex were elevated in patients with STP or DVT, compared with those with varicose veins only. The level of C-reactive protein (CRP) was also elevated in the patients with STP or DVT. These findings indicate that STP is not necessarily a localized disease, but may be a symptom of systemic disease. In addition to duplex scanning, the measurement of coagulofibrinolytic markers as well as CRP may be useful for detecting STP and/or DVT prior to the treatment of varicose veins.

Pancreatic proteases and inflammatory mediators in peritoneal fluid during splanchnic arterial occlusion and reperfusion.

Pancreatic enzymes in the ischemic intestine are involved in the production of in vivo inflammatory mediators. These mediators stimulate cells in the cardiovascular system during shock and initiate multiorgan failure. An important aspect that controls the extent of the inflammation is the dispersion of these mediators from the ischemic intestine. In the past, two pathways for dispersion of these inflammatory mediators have been identified, absorption into the intestinal venous circulation and uptake into the lymphatics. We hypothesize here that the inflammatory mediators produced by pancreatic digestive enzymes in the lumen of the intestine may also be released directly into the peritoneal space. To assess the presence of inflammatory mediators in the peritoneal cavity in response to splanchnic arterial occlusion (90 min) and reperfusion (SAO shock), we measured the ability of fluid collected from this cavity to activate naïve donor granulocytes. After SAO in control rats, peritoneal lavage fluid caused activation of naïve donor granulocytes when tested in vitro. In contrast, when the lumen of the small intestine was flushed with a broad-acting pancreatic enzyme inhibitor (6-amidino-2-naphtyl p-guanidinobenzoate dimethanesulfate), the fluid no longer caused leukocyte activation. Reduction of the levels of inflammatory mediators in the peritoneal fluid was associated with an attenuation in the fall of blood pressure after SAO shock. These results indicate that the inflammatory mediators, which are produced by pancreatic digestive enzymes, can be absorbed directly into the systemic circulation via a transperitoneal route and play a part in the development of multiorgan failure.

Leukocyte fluid shear response in the presence of glucocorticoid.

Leukocytes respond to physiological fluid shear stress (∼1.5 dyn/cm2) by cytoplasmic reorganization. The cytoplasm is also influenced, however, by glucocorticoids. In this study, we explore how glucocorticoids may affect the leukocyte fluid shear response. Normal leukocytes, exposed to fluid shear in vitro during active migration, retract pseudopods accompanied by modestly decreasing intracellular calcium ions. In contrast, dexamethasone (DX)‐treated leukocytes project pseudopods after shear exposure with a significant rise in intracellular calcium ions, an effect that can be blocked by voltage‐dependent calcium channel blockers. Although a cyclic adenine monophosphate analog blocks calcium influx and pseudopod projection by DX, inhibition of A‐kinase induces reversal of the shear response, as seen with DX treatment. DX also reverses the leukocyte shear response in vivo in the rat circulation. Leukocytes that adhere to the endothelium in postcapillary venules of control rats return into the circulation only after pseudopod retraction, and in DX‐treated rats, adherent leukocytes return into the circulation still with projecting pseudopods. The fraction of circulating leukocytes with pseudopods in DX‐treated rats is higher than in controls. Thus, the reversal of leukocyte shear response by glucocorticoids may contribute to an enhanced incidence of circulating leukocytes with pseudopods, a process that affects the kinetics of these cells in the microcirculation.

Intravital laser confocal microscopy of pulmonary edema resulting from intestinal ischemia-reperfusion injury in the rat.

OBJECTIVE To observe pulmonary edema resulting from intestinal ischemia-reperfusion injury. We used a newly developed laser confocal microscope to observe the subpleural capillary network and the superficial alveoli under intravital conditions, and created three-dimensional images of the pulmonary microcirculation to analyze the time course and spatial pattern of pulmonary exudative changes during intestinal ischemia-reperfusion injury in vivo. DESIGN Prospective, randomized, unblinded study. SETTING Laboratory of a university hospital. SUBJECTS Male Sprague-Dawley rats. INTERVENTIONS The rats were injected intravenously with bovine serum albumin labeled with fluorescein isothiocyanate and subjected to 60 mins of intestinal ischemia, followed by 180 mins of reperfusion. During mechanical ventilation, the upper lobe of the right lung was examined in the intravital state using a high-speed confocal fluorescence microscope. MEASUREMENTS AND MAIN RESULTS Interstitial edema and alveolar leakage were recognized as changes of interstitial fluorescence in the subpleural capillary network and as changes of alveolar fluorescence in the alveolar cross-sectional view. Although exudative changes in the interstitium and alveoli were observed during intestinal ischemia, there was a marked increase in both interstitial edema and alveolar leakage after intestinal reperfusion. CONCLUSION We observed pulmonary edema under intravital conditions and demonstrated the utility of a newly developed laser confocal microscope. This system not only enabled us to analyze the development of pulmonary edema three-dimensionally, but also allowed us to evaluate the pulmonary microcirculation.

Virtual angioscopy using 3-dimensional rotational digital subtraction angiography for endovascular assessment.

Purpose: To investigate the feasibility of 3-dimensional rotational digital subtraction angiography (3D DSA) and the creation of virtual angioscopic images from its data before and after endovascular treatment. Technique: Data sets from 3D DSA studies were used to create intraluminal images simulating angioscopy for 36 patients with arterial stenosis, aneurysm, or endoleak after stent-graft deployment. A biplanar DSA unit was used to acquire rotational angiography data, which was then processed with a surface-rendering technique to create maximum intensity projections, shaded surface displays, multiplanar reconstructions, and virtual angioscopy. 3D reconstructions were created in 2 minutes after angiography and provided realistic views adequate for vessel measurement, morphology assessment, and endoleak evaluation. Conclusions: 3D DSA and virtual angioscopy are novel techniques that have been successful in recreating images of blood vessels immediately after angiography. These techniques could be useful as additional imaging modalities to complement computed tomography or magnetic resonance imaging in the evaluation of vascular diseases after endovascular therapy.

Peritoneal lavage with oxygenated perfluorochemical preserves intestinal mucosal barrier function after ischemia-reperfusion and ameliorates lung injury.

ObjectiveTo evaluate the effect of peritoneal lavage with an oxygenated perfluorochemical (PFC) on intestinal ischemia-reperfusion injury (IIR), we assessed intestinal barrier function in terms of bacterial translocation and endotoxemia, morphologic changes, and changes of intestinal luminal pH in rats subjected to IIR. We also examined lung injury after IIR to test the effect of oxygenated PFC lavage on remote organ failure. DesignProspective, randomized, and controlled animal study. SettingLaboratory of a university hospital. SubjectMale Sprague-Dawley rats. InterventionsRats were subjected to ischemia by clipping the superior mesenteric artery. Reperfusion was achieved by release of the clip. Lavage of the abdominal cavity was performed by inflow and outflow of oxygenated PFC solution during ischemia. ResultsRats undergoing peritoneal lavage with oxygenated PFC (PFC group) showed significantly better survival after IIR. The frequency of bacterial translocation and the endotoxin concentration in superior mesenteric venous blood were significantly lower in the PFC group. Luminal acidosis also was alleviated in the PFC group. Furthermore, PFC lavage preserved the intestinal mucosal architecture and inhibited interstitial edema and infiltration of inflammatory cells in the lungs. ConclusionWe conclude that peritoneal lavage with oxygenated PFC protects the intestinal mucosa and maintains mucosal barrier function after IIR. Preservation of the intestinal mucosa ameliorates lung injury after IIR.