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Diabetic nephropathy is characterized by excessive deposition of extracellular matrix protein and disruption of the glomerular filtration barrier. Matrix metalloproteinases (MMPs) affect the breakdown and turnover of extracellular matrix protein, suggesting that altered expression of MMPs may contribute to diabetic nephropathy. Here we used an MMP-9 gene knockout mouse model, with in vitro experiments and clinical samples, to determine the possible role of MMP-9 in diabetic nephropathy. After 6 months of streptozotocin-induced diabetes, mice developed markedly increased albuminuria, glomerular and kidney hypertrophy, and thickening of the glomerular basement membrane. Gelatin zymographic analysis and western blotting showed that there was enhanced MMP-9 protein production and activity in the glomeruli. However, MMP-9 knockout in diabetic mice significantly attenuated these nephropathy changes. In cultured podocytes, various cytokines related to diabetic nephropathy including TGF-β1, TNF-α, and VEGF stimulated MMP-9 secretion. Overexpression of endogenous MMP-9 induced podocyte dedifferentiation. MMP-9 also interrupted podocyte cell integrity, promoted podocyte monolayer permeability to albumin, and extracellular matrix protein synthesis. In diabetic patients, the upregulation of urinary MMP-9 concentrations occurred earlier than the onset of microalbuminuria. Thus, MMP-9 seems to play a role in the development of diabetic nephropathy. © 2014 International Society of Nephrology.

One mechanism for organ damage in individuals with arterial hypertension may be due to oxygen free radical production. This study was designed to localize free radicals in a microvascular network of mature spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats. Because glucocorticoids play a role in pressure elevation of SHRs, we investigated their role in microvascular free radical formation. Oxygen radical production in mesentery was detected by tetranitroblue tetrazolium reduction to formazan aided by digital light-absorption measurements. Formazan deposits were observed in the endothelial cells and lumens of all microvessels and in lymphatic endothelia but were fewer in tissue parenchyma. The formazan distribution in younger (14-16 wk old) WKY rats and SHRs was heterogeneous with low values in capillaries and small arterioles/venules (<30 µm) but enhanced deposits in larger venules. Adrenalectomy served to reduce the formazan density in SHRs to the level of WKY rats, whereas dexamethasone supplementation of the adrenalectomized rats caused elevation in the larger venules of SHRs. In older (40 wk old) SHRs, formazan levels were elevated in all hierarchies of microvessels. After pressure reduction was employed with chronic hydralazine treatment, the formazan deposits were reduced in all locations of the microcirculation in both WKY rats and SHRs. Elevated formazan deposits were also found in lymphatic endothelium. These results suggest that oxygen free radical production is elevated in both high- and low-pressure regions of SHR microcirculation via a process that is controlled by glucocorticoids. Older SHRs have higher formazan levels than younger SHRs in all microvessels. Chronic hydralazine treatment, which serves to reduce arterial blood pressure, attenuates tetranitroblue tetrazolium reduction in WKY rats and SHRs even in venules of the microcirculation, which has no micropressure elevation. Free radical production may be a more global condition in SHRs and may not be limited to arteries and arterioles. [ABSTRACT FROM AUTHOR]

Recent evidence suggests that granulocytes play a key role in perfusion of the microcirculation. In this regard two phenomena are of specific importance: pseudopod formation and adhesion to the endothelium. Thus we studied the influence of AQA-39 on leukocyte adhesion to the endothelium in rat mesentery venules and on pseudopod formation in human granulocytes. Direct intravital estimates of granulocyte fracture stress were obtained by measurement of the ratio of leukocyte and midstream velocity. The marginating and circulating cell flux and the areal density of stationary granulocytes on the intraluminal side of the endothelium were measured simultaneously in the same venular segments. During administration of 10 mg kg-1 AQA-39 the fracture stress is significantly reduced resulting in decreased areal density of marginating cells and also washout of strongly adhering stationary leukocytes found frequently on the endothelium of such vessels. Furthermore it was observed on single granulocytes that AQA-39 leads to transient obliteration of pseudopod formation. These results suggest that the effect of AQA-39 on flow may be mediated in part by reduction in granulocyte adhesion to the endothelium and suppression of pseudopod formation. [ABSTRACT FROM PUBLISHER]

Abstract Background Obesity is a state of subclinical inflammation resulting in loss of function of insulin receptors and decreased insulin sensitivity. Inhibition of the inflammatory enzymes, matrix metalloproteinases (MMPs), for 6 months in rodent models restores insulin receptor function and insulin sensitivity. Methods This 12-week double-blind, randomized, placebo (PL)-controlled proof-of-concept study was performed to determine if the MMP inhibitor (MMPI), doxycycline, decreased global markers of inflammation and enhanced muscle insulin sensitivity in obese people with type 2 diabetes (DM2). The study included non-DM2 controls (n = 15), and DM2 subjects randomized to PL (n = 13) or doxycycline 100 mg twice daily (MMPI; n = 11). All participants were evaluated on Day 1; MMPI and PL groups were also evaluated after 84 days of treatment. Results There was a significant decrease in inflammatory markers C-reactive protein (P Conclusions This study demonstrated short term treatment of people with diabetes with an MMPI resulted in decreased inflammation and improved insulin sensitivity. Larger, longer studies are warranted to determine if doxycycline can improve glucose control in people with diabetes. Trial Registration Clinicaltrials.gov NCT01375491

Presents information pertaining to the mechanisms of cell injury in the rat mesentery and cremaster muscle. Reference to the events responsible for cell injury after a tissue stimulation; Description of these tissues; Proposal of several mechanisms of tissue injury; Production of significant levels of oxygen free radicals by endothelial cells.

Background: Inflammation and hypertension are primary mechanisms involving in obesity-associated adverse effects of a high-fat diet. The aim of this study was to evaluate the effects of rice bran extract (RBE) on arterial blood pressure, hepatic steatosis, inflammation, and oxidative stress in high-fat diet (HFD)-induced obese mice., Methods: Male ICR mice were divided into four groups, including a normal-diet control group, a high-fat diet (HFD) (60% kcal from fat) group, an HFD group treated with RBE (220 mg/kg/day), and an HFD group treated with 1100 mg/kg/day for eight weeks. Besides body weight and arterial blood pressure, we determined liver values of total cholesterol, triglyceride, as well as percent body fat, tumor necrosis factor- α (TNF- α ), malondialdehyde (MDA), nuclear factor kappa-B (NF- κ B), matrix metalloprotease-9 (MMP-9), cyclooxygenase-2 (COX-2), and mRNA endothelial nitric oxide synthase (eNOS)., Results: The HFD group had increased body weight, increased systolic and diastolic blood pressure, liver total cholesterol, triglyceride, NF- κ B, COX-2 and MMP-9 protein levels, and decreased mRNA eNOS in the aorta. Mice of the HFD group receiving RBE had reduced diastolic blood pressure, as well as significantly decreased liver and serum TNF- α and MDA levels in the liver, and reduced NF- κ B levels in both the liver and heart., Conclusions: These results demonstrate that RBE decreases diastolic blood pressure, the liver lipid droplet accumulation, liver and myocardial NF- κ B, myocardial COX-2 and MMP-9 protein levels, and oxidative stress. Moreover, RBE may improve endothelial function and may alleviate adverse health effects associated with obesity including obesity-associated hypertension., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Naphatsanan Duansak et al.)

Background: Uncontrolled proteolysis contributes to cell injury and organ dysfunction in animal models of circulatory shock. We investigated in humans the relationship between septic shock, proteolysis, and outcome., Methods: Intensive care patients with septic shock (n=29) or sepsis (n=6) and non-hospitalised subjects (n=9) were recruited as part of the prospective observational trial 'ShockOmics' (ClinicalTrials.gov Identifier NCT02141607). A mass spectrometry-based approach was used to analyse the plasma peptidomes and the origin of circulating peptides from proteolysis in the enrolled subjects., Results: Evidence of systemic proteolysis was indicated by a larger number of circulating peptides in septic shock patients, compared with septic patients and non-hospitalised healthy subjects. The peptide count and abundance in the septic shock patients were greater in patients who died (n=6) than in survivors (n=23), suggesting an association between magnitude of proteolysis and outcome. In silico analysis of the peptide sequences and of the sites of cleavage on the proteins of origin indicated a predominant role for serine proteases, such as chymotrypsin, and matrix metalloproteases in causing the observed proteolytic degradation., Conclusions: Systemic proteolysis is a novel fundamental pathological mechanism in septic shock. Plasma peptidomics is proposed as a new tool to monitor clinical trajectory in septic shock patients., Clinical Trial Registration: NCT02141607., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Growing interest in gut and digestive processes and their potential link to brain and peripheral based inflammation or biobehavioral phenotypes has led to an increasing number of basic and translational scientific reports focused on the role of gut microbiota within the context of neuropsychiatric disorders. However, the effect of dietary modification on specific gut metabolites, in association with immune, metabolic, and psychopathological functioning in schizophrenia spectrum disorders has not been well characterized. The short chain fatty acids (SCFA) acetate, butyrate, and propionate, major metabolites derived from fermentation of dietary fibers by gut microbes, interact with multiple immune and metabolic pathways. The specific pathways that SCFA are thought to target, are dysregulated in cardiovascular disease, type II diabetes, and systemic inflammation. Most notably, these disorders are consistently linked to an attenuated lifespan in schizophrenia. Although, unhealthy dietary intake patterns and increased prevalence of immune and metabolic dysfunction has been observed in people with schizophrenia; dietary interventions have not been well utilized to target immune or metabolic illness. Prior schizophrenia patient trials primarily focused on the effects of gluten free diets. Findings from these studies indicate that a diet avoiding gluten benefits a limited subset of patients, individuals with celiac disease or non-celiac gluten sensitivity. Therefore, alternative dietary and nutritional modifications such as high-fiber, Mediterranean style, diets that enrich the production of SCFA, while being associated with a minimal likelihood of adverse events, may improve immune and cardiovascular outcomes linked to premature mortality in schizophrenia. With a growing literature demonstrating that SCFA can cross the blood brain barrier and target key inflammatory and metabolic pathways, this article highlights enriching dietary intake for SCFA as a potential adjunctive therapy for people with schizophrenia.

Objective: Our aim is to determine the effect of paced eating, exposure to an educational programme that promotes healthy eating habits and allowing the satiety reflex to limit food intake in controlling weight gain in healthy adolescents., Methods: Fifty-four healthy individuals consisting of 18 adolescent girls and 36 boys aged 12 ± 2 years were given recommendations for reducing eating rate without changing diet or meal size according to the educational programme 'good manners for a healthy future'. Each participant was provided with a 30-s portable hourglass to pace time between bites. Individuals using and not using the hourglass were placed either into an 'adhering' or a 'non-adhering' group, respectively. Control data were obtained from a similar population., Results: Initially, the adhering group had higher weight compared with the non-adhering group (64.1 ± 13.2 vs. 56.2 ± 11.7 kg). Control group weight was no different from the study group at baseline (56.3 ± 10.3 kg). Weight in the adhering group decreased after the first semester of participation by 2.0 ± 5.7% and after a year by 3.4 ± 4.8%, while the non-adhering group gained weight by 5.8 ± 4.5% and 12.6 ± 8.3%. The control group increased weight after a year by 8.2 ± 6.5%. In total, 18 non-adhering and 14 adhering adolescents completed the study., Conclusions: This 1-year study shows a statistically significant association between rate of food intake and weight control in adherence to an educational programme directed at developing healthy eating habits. The proposed behavioural training may serve as an option for weight control in adolescents., (© 2015 The Authors. Pediatric Obesity published by John Wiley & Sons Ltd on behalf of World Obesity Federation.)

Objective: The mortality rate among patients with septic shock is high despite current therapy. We present a case of Fournier's gangrene and septic shock at 4 years post-heart transplantation that was reversed by "continuous enteral feeding" of the digestive enzyme inhibitor, gabexate mesilate. Recently, powerful pancreatic digestive proteases in the lumen of the intestine have been identified as initiators of the systemic inflammatory response. Intraluminal inhibitions of the proteases significantly attenuates intestinal damage, system inflammation, and multiorgan failure in experimental forms of shock but it has not been tested in man., Methods and Results: Gabexate mesilate, a synthetic digestive protease inhibitor, was continuously administered in two liters of crystalloid solution to a patient by enteral feeding during septic shock. The condition and markers for shock due to sepsis reversed in a few days., Conclusion: This case suggested that "enteral" digestive protease inhibition may decrease and even reverse the sequelae of shock and sepsis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

One of the key features of cardiovascular complications, such as hypertension or diabetes, is that they often appear at the same time in the same individual together with other forms of co-morbidities. While clinically a recognized phenomenon, no molecular mechanism for such co-morbidities has received universal acceptance. We propose a new hypothesis that provides a molecular basis for co-morbidities in hypertension due to unchecked proteolytic activity and receptor destruction. Testing of the hypothesis in the spontaneously hypertensive rat reveals an unchecked matrix metalloproteinase and serine protease activity in plasma and on several cardiovascular and parenchymal cells. The elevated proteolytic activity causes extracellular cleavage of multiple receptor types, such that cleavage of one receptor type leads to loss of the function carried out by this receptor. Proteolytic cleavage of the extracellular domain of the β(2) adrenergic receptor in arteries and arterioles causes vasoconstriction and elevation of the central blood pressure while cleavage of the extracellular domain of the insulin receptor leads to insulin resistance and lack of transmembrane glucose transport. A diverse set of cell dysfunctions in the spontaneously hypertensive rat are accompanied by cleavage of the membrane receptors that are involved in these functions. Chronic inhibition of the unchecked protease activity in the spontaneously hypertensive rat serves to restore the extracellular receptor density and alleviates the corresponding cell dysfunctions. The mild unchecked proteolytic activity in the spontaneously hypertensive rat points towards a chronic autodigestion process as a contributor to the end organ injury encountered in this rat strain. The presence of various soluble receptors, which consist of extracellular fragments of membrane receptors, in the plasma of hypertensive and diabetic patients suggest that the autodigestion process may also be present in man.

Objectives: To characterize the acute response of the vein wall to venous hypertension and associated altered fluid shear stress and to test the effect of micronized purified flavonoid fraction (MPFF, Daflon 500), on this response., Material and Methods: A femoral arteriovenous fistula was created in Wistar rats (n=48). A cohort of 24 rats received oral treatment with MPFF (100 mg/kg/day body weight), 24 rats underwent the arteriovenous fistula procedure and received no treatment. At days 1, 7 and 21 the animals (n=8 at each time point) were killed. Experimental parameters measured included limb circumference, blood flow at the sapheno-femoral junction, leukocyte infiltration and gelatinase activity (matrix metalloproteinase, MMP)., Results: The acute rise in venous hypertension was accompanied by limb edema and venous reflux together with an eventual loss of valve leaflets in the saphenous vein. There was an increase in granulocyte and macrophage infiltration into the venous wall and the surrounding tissue, and a lesser increase in T- and B-lymphocyte infiltration. These changes were accompanied by a local increase in the proteolytic enzymes, MMP-2 and MMP-9. Administration of MPFF reduced the edema and lessened the venous reflux produced by the acute arteriovenous fistula. Decreased levels of granulocyte and macrophage infiltration into the valves were also observed compared with untreated animals., Conclusions: Venous hypertension caused by an arteriovenous fistula resulted in the development of venous reflux and an inflammatory reaction in venous valves culminating in their destruction. MPFF was able to delay the development of reflux and suppress damage to the valve structures in this rat model of venous hypertension.

Objectives: To identify possible mechanisms for destruction of valves in chronic venous hypertension and the results of treatment with an anti-inflammatory micronized purified flavonoid fraction., Material and Methods: The saphenous vein valves in a rat model of venous hypertension caused by a femoral arterial-venous fistula were studied. Studies included femoral venous pressure, valve morphology, femoral venous reflux and selected molecular inflammatory markers as examined by immunohistochemistry. The effects of treatment with the anti-inflammatory micronized purified flavonoid fraction (S 5628, Servier, 50 and 100 mg/kg/day) were investigated., Results: The femoral venous pressure was elevated close to arterial values for a period of 3 weeks. We then examined the morphology of the veins and selected molecular inflammatory markers were assessed. The results show that in this model venous reflux develops in response to venous hypertension. This can be inhibited by the administration of the anti-inflammatory micronized purified flavonoid fraction (S 5628, Servier, 50 and 100 mg/kg/day). The valve becomes incompetent by a combination of venous dilation and shortening of the valve leaflets. This is not inhibited by treatment with S 5628. The valve leaflets are infiltrated with granulocytes, monocytes and T-lymphocytes, and the endothelial cells express enhanced levels of P-selectin and ICAM-1. Cells in the valves are subject to extensive apoptosis although no enhancement of MMP 2,9 expression could be detected at the three-week time point examined in this study., Conclusions: These results indicate that in this model chronic elevation of venous pressure is associated with an inflammatory reaction in venous valves, a process that may lead to their dysfunction, reflux, and upstream elevation of venous pressure. These effects are mitigated by the anti-inflammatory micronized purified flavonoid fraction in a dose dependent manner.

Stimulation of the human nasal passage with pungent vapor elicits motor responses in a zone around the eye. This investigation addressed whether quantification of such responses, particularly activity of the orbicularis oculi muscle, could yield a sensitive index of nasal pungency. We placed an array of small, high-contrast targets just beneath the lower eyelid and videotaped their movement to capture deformation of the skin atop the orbicularis oculi during 3 s stimulation with pungent concentrations of ethyl acetate. Eleven subjects participated. Analysis of the movements served to determine mechanical strain, which yielded a single index that we termed 'maximum strain'. This increased with concentration of the vapor and with time during and just after stimulation. Comparison with psychophysical data showed that the strain became evident at concentrations just detectable as pungent. Maximum strain measured on the skin shows promise as an objective index of pungency.

Chronic venous insufficiency (CVI) is inseparably linked to elevated venous pressure and is accompanied by vascular, dermal, and subcutaneous tissue damage and restructuring. Abundant evidence exists both in humans and in experimental models to suggest that the tissue damage may be initiated by generation of an inflammatory reaction. Inflammatory indicators include elevation of endothelial permeability; attachment of circulating leukocytes to the endothelium; infiltration of monocytes, lymphocytes, and mast cells into the connective tissue; and development of fibrotic tissue infiltrates and several molecular markers, such as growth factor or membrane adhesion molecule generation. Indicators of an inflammatory reaction are already detectable at early stages of CVI and may be involved in the development of primary venous valve dysfunction. One of the important questions is to identify trigger mechanisms for the inflammatory reaction in CVI. Current evidence suggests that, among several possible mechanisms (hypoxia, humoral stimulation), a shift in fluid shear stress from normal physiological levels and endothelial distension under the influence of elevated venous pressure may serve as trigger mechanisms for inflammation.

The mechanism for interstitial fluid uptake into the lymphatics remains speculative and unresolved. A system of intralymphatic valves exists that prevents reflow along the length of the lymphatic channels. However, these valves are not sufficient to provide unidirectional flow at the level of the initial lymphatics. We investigate here the hypothesis that initial lymphatics have a second, separate valve system that permits fluid to enter from the interstitium into the initial lymph channels but prevents escape back out into the tissue. The transport of fluorescent microspheres (0.31 microm) across endothelium of initial lymphatics in rat cremaster muscle was investigated with micropipette manipulation techniques. The results indicate that microspheres can readily pass from the interstitium across the endothelium into the lumen of the initial lymphatics. Once inside the lymphatic lumen, the microspheres cannot be forced out of the lumen even after elevation of the lymphatic pressure by outflow obstruction. Reaspiration of the microspheres inside the lymphatic lumen with a micropipette is blocked by the lymphatic endothelium. This blockade exists whether the aspiration is carried out at the microsphere entry site or anywhere along the initial lymphatics. Nevertheless, puncture of the initial lymphatic endothelium with the micropipette leads to rapid aspiration of intralymphatic microspheres. Investigation of lymphatic endothelial sections fixed during lymph pumping shows open interendothelial junctions not found in resting initial lymphatics. These results suggest that initial lymphatics have a (primary) valve system at the level of the endothelium. In conjunction with the classical (secondary) intralymphatic valves, the primary valves provide the mechanism that facilitates the unidirectional flow during periodic compression and expansion of initial lymphatics.

Early manifestations of chronic venous insufficiency (CVI) are edema, hyperpigmentation, and lipodermatosclerosis. Late complications are cutaneous ulceration and delayed healing. The specific hallmarks of this inflammation include CD68-positive infiltration into the dermal tissue, monocytes, and lymphocytes and enhanced endothelial permeability. This may lead to "fibrin cuff" formation. In addition, membrane adhesion molecules are present and cytokine expression is seen. In one experimental model of mesenteric venous hypertension, the inflammatory process was detected in its earliest stages. This was evident in the form of neutrophilic leukocyte adhesion to venular endothelium as well as migration of cells across the endothelium and basement membrane into the interstitial space. Simultaneously, parenchymal cell death was detected. This suggests that the mechanism that triggers the inflammatory reaction is venous hypertension. This may cause venous distension and a shift in fluid shear stress. Our observations suggest that patients with venous insufficiency demonstrate circulatory humoral stimulators for leukocyte activation. Otherwise, there is evidence that the inflammatory reaction is limited to the region of the venous ulceration or at least to the skin areas with severe microangiopathy. It may be that activated leukocytes traverse perivascular cuffs and release active transforming growth factor-beta1 (TGF-beta1) which has been found to be elevated exclusively in areas of clinically active CVI. Surgical intervention markedly decreases the number of dysfunctional vein segments and allows pharmacologic agents to protect normal structures from continuing damage. Daflon 500 mg, the purified micronized flavonoid fraction containing 90% diosmin and 10% hesperidin, acts favorably in venous ulcer treatment by inhibiting the synthesis of prostaglandins and free radicals. It decreases bradykinin-induced microvascular leakage and may act favorably to inhibit leukocyte activation, trapping, and migration. Clinically, edema is reduced, ulcer healing is accelerated, and leukocyte trapping diminished. The action of micronized purified flavonoid fraction is beginning to be better understood, and as further knowledge is gained, better pharmacologic control of CVI is a tantalizing promise.

Objective: The objective was to quantify and to describe microvascular endothelial cell death in glucocorticoid-induced hypertension. Microvascular rarefaction, which has been shown in human and animal hypertension, may result from increased endothelial cell apoptosis., Methods: Wistar rats were administered the synthetic glucocorticoid dexamethasone (0.5 mg/kg b.w. per day, i.m.) for 5 days and were compared with a group of control rats treated with the vehicle. In vivo microvascular endothelial cell death was quantified by propidium iodide fluorescent labeling in the mesentery. Normal nuclear DNA (labeled with Hoechst 33342) and DNA with apoptotic characteristics in muscle endothelium (labeled with ethidium bromide) were visualized using confocal imaging. Microvessel length density was measured by using a fluorescein isothyocyanate-labeled lectin technique., Results: The dexamethasone-treated rats exhibit approximately a 10% increase in cell death along the mesenteric arteriolar and venular endothelium compared with controls. Confocal analysis of cremaster muscle in dexamethasone-treated rats demonstrated nuclear fragmentation and decreased nuclear volumes in dying endothelial cells, which are consistent with an apoptotic process. The capillary length density in cremaster muscle was decreased on average by 16% in the hypertensive rats., Conclusions: These results suggest capillary structural rarefaction by an increased rate of apoptotic endothelial cell death in glucocorticoid-induced hypertension.

Cell activation in the microcirculation leads to an inflammatory cascade and is accompanied by many cardiovascular complications. There is a need to identify the trigger mechanisms that lead to the production of in vivo activating factors. We review here mechanisms for cell activation in the microcirculation and specifically the production of humoral cell activators in physiological shock. The elevated levels of activating factors in plasma could be traced to the action of pancreatic enzymes in the ischemic intestine. New interventions against the production of the activators are proposed. The evidence suggests that pancreatic enzymes in the ischemic intestine may attack several tissue components and generate cellular activators that are associated with multiorgan dysfunction in physiological shock.

In this study we analysed blood samples taken from the dorsalis pedis vein and a brachial vein of 11 healthy test persons and 8 patients with venous leg ulcer under experimental venous hypertension in order to examine changes in the expression of leukocyte adhesion molecules (LFA-1 (CD11a), Mac-1 (CD11b), p150,95 (CD11c), CD18, VLA-4 (CD49d) and L-selectin (CD62L)) which are involved in the adhesion steps of leukocytes to endothelial cells for transmigration into tissues. Under orthostatic stress, lymphocytes in controls collected at the foot level showed a significant reduction in the expression of L-selectin (p=0.002), compared to those of patients. This finding suggests that venous stasis negatively influences the expression of L-selectin on leukocytes only in healthy volunteers. Patients with chronic venous insufficiency seem to suffer from a specific defect in the regulation of L-selectin shedding under orthostatic stress.

Activation of cells in the vascular compartment causes profound alteration of cell rheological properties with impairment of the microcirculation and initiation of inflammatory reactions. Many cardiovascular diseases have been shown to be associated with cell activation and inflammation. While this situation offers the opportunity for new interventions against the deleterious effects of cell activation, there is the need for a better understanding of the mechanisms that lead to cell activation in the first place. We review here several mechanisms for cell activation in the circulation. We show that in shock, a condition associated with severe forms of cell activation, humoral cell activation factors can be detected in plasma. Further analysis indicates that the source of these humoral activators may be due to the action of pancreatic digestive enzymes in the intestine. Ischemia may serve to open the intestinal brush border and permit entry of pancreatic enzymes into the wall of the intestine to initiate self digestion. In this process low molecular weight but potent cell activators are produced which may escape via the intestinal circulation and the lymphatics into the general circulation. Inhibition of pancreatic enzymes in the lumen of the intestine leads to complete attenuation of humoral activator production as well as many of the deleterious sequelae that accompany shock, such as inflammation and multi-organ failure. We outline a method to carry out biochemical isolation of the cell activators derived from pancreatic enzymes. This analysis shows that there are multiple species of cell activators above and beyond currently known species, many of which have molecular weights below 3000 Da. Identification of the mechanisms that lead to cell activation is an important part to understand the mechanisms that lead to alterations of rheological properties of blood cells in disease and dysfunction of the endothelium and parenchymal cells. Our current evidence suggests that pancreatic digestive enzymes and tissue enzymes may play a central role in humoral activator production.

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.

Oxygen free radicals, including hydrogen peroxide, may mediate oxidative stress in target organ tissues and contribute to cardiovascular complications in hypertension. To examine heritability of hydrogen peroxide production, we investigated this trait in a family-based cohort consisting of family members (n=236) ascertained through probands (n=57) with essential hypertension. Significant effects on hydrogen peroxide production were found for gender and ethnicity, with men having greater values than women (P<0.001) and white subjects having greater values than black subjects (P=0.025). Hydrogen peroxide production correlated directly with plasma renin activity (P=0.015), suggesting an important interaction between circulating oxygen radicals and the renin-angiotensin system and a potential mechanism for lower hydrogen peroxide values observed in blacks. Heritability estimates from familial correlations revealed that approximately 20% to 35% of the observed variance in hydrogen peroxide production could be attributed to genetic factors, suggesting a substantial heritable component to the overall determination of this trait. Hydrogen peroxide production negatively correlated with cardiac contractility (r=-0.214, P=0.001) and renal function (r=-0.194, P=0.003). In conclusion, these results indicate that hydrogen peroxide production is heritable and is related to target organ function in essential hypertension. Genetic loci influencing hydrogen peroxide production may represent logical candidates to investigate as susceptibility genes for cardiovascular target organ injury.

There is a need to monitor perfusion of the microcirculation in patients with minimal invasive approaches. The recent development of ultrasound contrast agents may open such an opportunity and permit to visualize online perfusion of cardiac muscle and other organs. An effective approach to achieve this objective is with gas filled microbubbles that provide strong ultrasound wave reflection and scatter. To interpret the ultrasound scattering profiles, it is necessary to understand the transport of microbubbles in the microcirculation, specifically their transport in blood stream, the attachment to blood cells and endothelium, as well as possible leakage across the microvascular wall. Each of these events provides an opportunity to monitor with ultrasound not only microvascular blood flow but also detect inflammatory sites in the circulation.

The objective of this study was to assess the relationship of signaling molecules to monocyte/ macrophages as a precursor to venous valve and venous wall dysfunction in patients with varicose veins. One of the hallmarks of venous dysfunction is destruction of venous valves with subsequent reflux and elevation of distal venous pressure. We recently observed that monocytes/macrophages migrate into the venous walls and valves of patients with venous insufficiency. There, they may play a role in the pathogenesis of primary venous insufficiency. If so, an important element in their performance would be the interaction between the monocytes and the endothelium as a precursor of damage to venous valves and the venous wall. To explore this interaction, immunohistochemistry was carried out to detect adhesion molecules and cytokines in surgical specimens removed during surgical therapy. Twenty-four surgical specimens consisting of proximal saphenous vein and subterminal valve were obtained using minimally traumatic technique in 6 males and 18 females who ranged in age from 31 to 79 years. Reflux was confirmed preoperatively by duplex technique, and severity was classified by the CEAP classification of the American Venous Forum. Ten patient limbs were class 2, eight were class 3, four were class 4, and two were class 6. The venous specimens were labeled using monoclonal antibody against ICAM-1, E-selectin, IL-1alpha, and TNF-alpha. CD68 was used for detection of monocytes/macrophages. Our results indicate that not only luminal venous endothelium but also endothelium in the vasa vasora of refluxing saphenous veins is activated, as indicated by the up-regulation of ICAM-1. However, IL-1alpha and TNF-alpha were increased in only selected specimens and are mainly detected in the vein wall. The factors that serve as trigger mechanisms to activate cells in the pathogenesis of primary venous dysfunction remain to be explored.

During circulatory shock, activating factors for cells in the microcirculation can be detected in plasma. But the source of such activators has remained uncertain. We have demonstrated recently that homogenates derived from the pancreas but not other peritoneal organs activate naive leukocytes. Production of such activating factors can be blocked by a serine protease inhibitor. Thus, factors generated by pancreatic proteases may possibly produce cellular activation in vivo. Rats were subjected to 90 min of superior mesenteric and celiac artery occlusion followed by reperfusion (SAO shock). In addition, rats were subjected to SAO shock for 120 min, after a 60-min pretreatment prior to occlusion with either saline or the serine protease inhibitor Futhan (nafamostat mesilate, 3.3 mg/kg b.w.). A sham SAO protocol was carried out as a control. Cellular activation was tested by neutrophil pseudopod formation and NBT reduction. Plasma from SAO-shocked animals but not sham shock rats exhibited a significant increase (P < 0.001) in the activation of naive leukocytes. Futhan-treated animals subjected to SAO shock exhibited a significantly higher post-reperfusion blood pressure than non-treated animals (P < 0.005 for all time points greater than 120 minutes), as well as significantly greater survival (P < 0.001). Neutrophil pseudopod formation and plasma peroxide production, an additional index of cellular activation, were significantly lower in Futhan-treated SAO shock plasma (P < 0.05) than levels in non-treated SAO shock animals. These results demonstrate that activating factors for leukocyte are released in SAO shock and can be mitigated by pretreatment with the serine protease inhibitor Futhan. Proteolytically derived plasma factors released during SAO shock may contribute to leukocyte activation and ensuing organ dysfunction.

Objective: We investigated the propagation of viral-size particles by lymph and blood after subcutaneous injection., Methods: In the canine model, transport of [99mTc] sulfur colloid particles of different sizes was studied in different settings in venous blood and lymph for 45 minutes after inoculation., Results: The mean arrival time of particles in the blood was 2.10+/-0.46 minutes and 8.87+/-1.72 minutes in the lymph. Lymph flow in the canine leg was 28.79 +/-2.09) microl/min and was increased by leg massage. The particle concentration was 1000 times higher in the lymph fluid than in blood. Particle flux values were comparable in blood and lymph. The accumulation of particles in blood initially rose faster than in lymph. Accumulation in lymph rises slower but continues longer and reaches higher values. Ninety percent of the inoculum remains at the injection site for at least 45 minutes. Particle size matters more in blood distribution. Leg massage enhances particle transport by lymph., Conclusions: After subcutaneous injection, viral-size particles initially arrive in the blood and later in the lymph. Accumulation in lymph and blood increases for a prolonged time after inoculation. Results suggest possibilities for limiting the spread of infectious matter by early local antiviral treatment.