General theory of inflammation: patient self-administration of hydrocortisone safely achieves superior control of hydrocortisone-responding disorders by matching dosage with symptom intensity.

Objective: To determine if patient self-administration of hydrocortisone will safely achieve superior symptom control for all hydrocortisone-responding disorders as it does for Addison's disease and rheumatoid arthritis. Methods: Two thousand four hundred and twenty-eight participants with hydrocortisone-responding disorders were brought to a minimum symptom state using daily administered hydrocortisone tablets in a 24-week, open study. Thereafter, participants used 5-day, low-dose hydrocortisone regimens to quench subsequent disorder exacerbations (flares) to maintain the minimum symptom state. Stressors such as emotional traumas, infections, allergies, and injuries were minimized to reduce disorder intensity, hydrocortisone consumption, and participant discomfort. Results: Two thousand fifteen participants, 601 with fibromyalgia, 579 with osteoarthritis, 246 with rheumatoid arthritis, 226 with undifferentiated arthritis, 75 with back pain, 51 with Parkinson's disease, 44 with polymyalgia rheumatica, 25 with neuropathy, 25 with chronic fatigue syndrome, 25 with dementia, 21 with migraine headache, 19 with multiple sclerosis, and 78 with other disorders completed the 24-week study to achieve a composite average symptom improvement of 76% with equal response rates. The participants averaged ingesting 12 mg of hydrocortisone per day. No significant adverse reactions were observed. Conclusions: Patient self-administration of hydrocortisone safely achieves superior symptom control for 38 hydrocortisone-responding disorders at equal rates and symptom improvements to confirm and amplify an earlier double-blind study finding on rheumatoid arthritis. These results are consistent with the body having an inflammation control system and chronic inflammation being a disorder unto itself with differing symptoms sets dependent on its location. Clinical Trials Government Identifier: NCT03558971.

Mechanics of endothelial cell architecture and vascular permeability.

Blood vessel walls form a selective barrier to the transport of materials between blood and tissue, and the endothelium contributes significantly to this barrier function. The role of the endothelium is particularly important in thin-walled vessels, such as venules, because during tissue inflammation the endothelial junctions widen in localized areas and gaps form, thus compromising the barrier function. The mechanisms of endothelial gap formation are still under question. In this review we describe what is known about the structure of endothelial cell-cell junctions and how this structure can change during inflammation. We then consider two possible mechanisms by which endothelial gaps are formed: active endothelial cell contraction or breakdown of the junctional complex, followed by passive recoil. Using measured values of the mechanical properties of endothelial cells, and the forces to which they are subjected, we calculate that gap formation by breakdown of cellular adhesion, followed by passive recoil, is a feasible mechanism. Finally, since endothelial cell surfaces, including junctions, are coated with a glycocalyx, we consider the question of whether changes in the glycocalyx can markedly increase endothelial permeability. We conclude that gap formation can occur by active contraction or by breakdown of adhesion, depending on the inflammatory mediator, and that the responses of the glycocalyx may also play an important role in the regulation of microvascular permeability.

Introduction: a brief history of capillaries and some examples of their apparently strange behaviour.

1. Over the past decade, evidence has accumulated challenging the notion that the smallest blood vessels, the capillaries, are static, uniform, semipermeable pipes with no function except as a passive barrier marking the boundary between the vascular compartments from the tissue compartment. 2. Instead, capillaries are dynamic structures that participate in the active regulation of water, waste and nutrient exchange, the formation and destruction of exchange vessels and the sites for initiation of signals to regulate the flow of blood into the exchange vascular network. 3. The following papers will provide insight into the expanded appreciation of the varied nature of capillaries and how their functions are co-ordinated to achieve 'whole organ' exchange.

Nitric oxide protects venules against histamine-induced leaks.

OBJECTIVE: The goal of the present study was to investigate the prophylactic role of nitric oxide (NO) in the mesenteric microvasculature in preventing microvascular leakage subsequent to histamine application, and to evaluate the response of mast cells during these conditions. METHODS: Regions of the rat mesenteric microcirculation were flushed free of blood and pretreated with either the nitric oxide donor sodium nitroprusside (SSP 10(-6) M) or Hepes-buffered saline containing 0.5% bovine serum albumin (HBS-BSA) for 15 minutes, then exposed to histamine (10(-3) M) for another three minutes. In another set of experiments, the microvasculature was treated with either histamine (10(-3) M) for three minutes or SNP (10(-6) M) for 15 minutes. A control group was treated with HBS-BSA for 15 minutes. RESULTS: The protective role of NO was evaluated by its ability to reduce or prevent histamine-induced venular leaks. Mesenteric microvessels pretreated with SNP before histamine suffusion showed a significant decrease in both area and number of venular leaks following the perfusion of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA). Although SNP pretreatment did not reduce the percentage of mast cells that degranulated in the presence of histamine, it did somewhat reduce the severity of the degranulation. CONCLUSION: This study demonstrated that nitric oxide availability protects mesenteric venules against histamine-induced leaks, but does not prevent degranulation of mast cells. Therefore, nitric oxide probably acts directly on venular endothelial cells to prevent leak formation.

Structural alterations in the rat kidney after acute arsine exposure.

The mechanism of arsine (AsH3) toxicity is not completely understood. In this investigation, the toxicity of AsH3 and AsH3-produced hemolytic products was determined in primary culture of renal cortical epithelial cells and in the in situ isolated rat kidney. The objective of this study was to model kidney dysfunction caused by AsH3 exposure. The hypothesis was that unchanged AsH3 and AsH3-produced hemolysate that may contain arsenite (As(III)) as metabolite are both responsible for renal toxicity. Toxicity in isolated cells was determined by 2, 3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxa nilide inner salt (XTT) bioreduction, intracellular potassium (K+), and lactate dehydrogenase (LDH) leakage. Data from XTT bioreduction showed that most toxicity occurred at 1 hour and was independent of the arsenic species. At 4 hours, the observed toxicity depended on the arsenic species and was generated by As(III). In the isolated cells, the As(III)-spiked hemolysate produced similar toxicities with regard to intracellular K and LDH. The AsH3-hemolysate only affected LDH at 1 hour. Unchanged AsH3 was very toxic to the isolated rat kidney. In this system, after 10 minutes exposure to AsH3, the effects of toxicity were observed mainly in the glomerular and peritubular endothelial cells. Tubular epithelial cells also presented early signs of toxicity. The AsH3-hemolysate was not toxic after a 1 -minute exposure. These data suggested that early cytotoxicity caused by unchanged AsH3 results in kidney dysfunction, produced by AsH3, and later by the formation of a hemolysate that may contain As(III). These data may be important in understanding the renal toxic effects after AsH3 intoxication.

Nitric oxide: role in venular permeability recovery after histamine challenge.

Histamine is an inflammatory mediator produced by mast cells that reside close to blood vessels. It causes a transient increase in venular permeability and stimulates endothelial production of nitric oxide (NO). In this study, we investigated the role that NO plays in the permeability recovery and evaluated the response of mast cells. The mesenteric microvasculature of anesthetized rats was suffused with 10(-3) M histamine for 3 min and then perfused with the NO donor sodium nitroprusside (SNP; 10(-6) M), the NO inhibitor N(G)-monomethyl-L-arginine (L-NMMA; 10(-5) M), its enantiomer (D-NMMA; 10(-5) M), or HEPES-buffered saline containing 0.5% BSA for 15 min. This was replaced by FITC-albumin for 3 min, followed by fixative. The vasculature was visualized using epifluorescence microscopy and was stained for mast cells. Preparations treated with histamine only showed discrete FITC-albumin leaks. Subsequent inhibition of NO increased venular FITC-albumin leaks and prevented permeability recovery, whereas subsequent treatment with SNP decreased the histamine-induced venular leaks. Mast cells degranulated due to histamine and the other treatment combinations. In conclusion, inhibition of NO prevented permeability recovery and depleted mast cells of their histamine content.

Environmental stress causes mast cell degranulation, endothelial and epithelial changes, and edema in the rat intestinal mucosa.

OBJECTIVE: Mental stress has been shown to produce intestinal disease, but the effects of a mild environmental stress on intestinal physiology have not been elucidated. This study was performed to determine the effects of environmental stress on the ultrastructure of the intestinal mucosa, using the rat as an experimental model. METHODS: One group of rats (group A, n = 3) was examined immediately upon arrival at the animal care facility. Groups B (n = 6) and C (n = 6) were housed in rooms with high and low personnel activity, respectively, for up to 4 wk. Group D (n = 8) was housed in the high activity room for 3 to 4 wk followed by 1, 2, or 3 in the low activity room. RESULTS: Rats in group B had the greatest number of degranulated intestinal mucosal mast cells, and activated goblet cells. Intestinal villi were edematous and epithelial cells were detaching from the basement membrane at villus tips. Changes were observed in capillary endothelial ultrastructure. In group B there were greater numbers of vesicles and multilamellar fenestral diaphragms compared to group C. Rats in groups A and C had the lowest numbers of degranulated mast cells and activated goblet cells. Intestinal villi showed normal ultrastructure. Group D was in a recovery phase and the condition of the intestinal mucosa was improved relative to group B, but the number of degranulated mast cells was not significantly reduced. CONCLUSIONS: This study demonstrates that environmentally induced stress causes pathological changes in the rat intestinal mucosa that compromise the epithelial-endothelial exchange barrier. These results emphasize the importance of closely monitoring the environment of experimental animals and provide evidence to stimulate further research into the mechanisms linking mental stress to gastrointestinal dysfunction in humans.

Effect of early transient adherent leukocytes on venular permeability and endothelial actin cytoskeleton.

In a time course study of the development and recovery of venular leaks, it was shown that, after as early as 3 min of histamine application, venular leak formation was identified [Baldwin, A. L., and G. Thurston. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1528-H1537, 1995]. This was accompanied by changes in the endothelial actin cytoskeleton and the presence of adherent leukocytes. The venular leaks remained elevated for at least 30 min, whereas the adherent leukocytes were decreased by 20 min. The present study was performed to determine the role that 3 min (early), transient histamine-associated adherent leukocytes play in the formation of venular leaks and changes in the endothelial actin cytoskeleton. In anesthetized rats, the microvasculature of a mesenteric window was perfused with buffered saline or fucoidin. FITC-BSA or FITC-BSA and 10(-4) M histamine was added to the perfusate for the last 3 min. The vasculature was perfusion fixed, stained for filamentous actin, and viewed microscopically. Fucoidin pretreatment significantly reduced the number of early, transient histamine-associated adherent leukocytes (P < 0.01). The number of adherent leukocytes in leaky venules was significantly greater than that seen in nonleaky venules (P < 0.01); however, the reduction in the number of histamine-associated adherent leukocytes with fucoidin pretreatment had no significant effect on the number (P > 0.05) or area (P > 0.05) of FITC-BSA leaks or on the endothelial actin cytoskeleton.

Modified hemoglobins produce venular interendothelial gaps and albumin leakage in the rat mesentery.

Cross-linked hemoglobin (alphaalpha-Hb) and polyethylene glycol (PEG)-conjugated Hb have both been considered as possible "blood substitutes." Previously, we showed that PEG-Hb extravasates rapidly in the intestinal mucosa and causes transient epithelial sloughing, resulting in temporary opening of the intestinal epithelial barrier. In the present study, the rat mesenteric preparation was used to quantify the effects of the two Hbs on microvascular leakage to albumin and to investigate possible changes in the integrity of the interendothelial cell junctions and the endothelial actin cytoskeleton. In anesthetized Sprague-Dawley rats, the microvasculature of a mesenteric window was perfused with HEPES-buffered saline (HBS) containing 0.5 mg/ml BSA and 2 mg/ml alphaalpha-Hb (n = 16) or PEG-Hb (n = 5) for 2 or 10 min. Controls (n = 4) just received HBS-BSA. In some experiments (n = 9 for alphaalpha-Hb; n = 5 for PEG-Hb), the perfusate was then replaced by FITC-albumin in HBS-BSA for the next 3 min. The vasculature was then perfusion fixed, stained for filamentous actin and for mast cells, and viewed microscopically. In the remaining experiments, the mesenteric microvasculature was stained with silver nitrate to determine the number of endothelial junctional gaps per length of venules. Both Hbs increased the number and area of leaks per micrometer of venular length compared with control, but alphaalpha-Hb increased to a greater extent than PEG-Hb. Formation of leaks was accompanied by changes in the endothelial actin cytoskeleton and by an increased number of endothelial gaps. Mast cell degranulation was significantly greater (P < 0.05) in Hb-treated preparations compared with controls, but there was no direct correlation between sites of degranulation and albumin leakage. These Hbs appear to induce venular leakage in the mesentery by mechanisms similar to those previously observed after treatment with histamine or nitric oxide synthase inhibitors.

Cadherin-5 redistribution at sites of TNF-alpha and IFN-gamma-induced permeability in mesenteric venules.

The response of the endothelial permeability barrier in microvascular networks of the rat mesentery to perfused immune inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) was examined. TNF-alpha (12.5 U/ml) treatment did not change albumin permeability, but in combination with IFN-gamma (20 U/ml), there was a marked increase in the number of sites of extravascular albumin in postcapillary venules. Endothelial integrity was characterized by cadherin-5 immunoreactivity, which was localized to the continuous intercellular junctions of endothelium in arterioles, capillaries, and venules. Perfusion with the combined cytokines showed that the increased albumin permeability was dose dependent and correlated with the focal disorganization of cadherin-5 at intercellular junctions of venular endothelium. No correlation was found between the increase in albumin permeability and the localization of intravascular leukocytes or extravascular mast cells. These results show that the combination of TNF-alpha and IFN-gamma induces an endothelial phenotype with focal loss of cadherin-5 intercellular adhesion, which, in part, facilitates passage of blood macromolecules and cells to the interstitium.

Financial and risk considerations for successful disease management programs.

Results for disease management [DM] programs have not been as positive as hoped because of clinical issues, lack of access to capital, and administrative issues. The financial experience of DM programs can be quite volatile. Financial projections that are protocol-based, rather than experience-based, may understate the revenue required and the range of possible costs for a DM program by understating the impact of complicating conditions and comorbidities. Actuarial tools (risk analysis and risk projection models) support better understanding of DM contracts. In particular, these models can provide the ability to quantify the impact of the factors that drive costs of a contract and the volatility of those costs. This analysis can assist DM companies in setting appropriate revenue and capital targets. Similar analysis by health plans can identify diseases that are good candidates for DM programs and can provide the basis for performance targets.

Effects of environmental stress on the architecture and permeability of the rat mesenteric microvasculature.

OBJECTIVE: This study was performed to determine the effects of environmental stress on the leakage to albumin and architecture of microvessels in the rat mesentery. METHODS: One group of rats (Group A, n = 6) were examined immediately upon arrival at the animal care facility. Groups B (n = 24) and C (n = 32) were housed in rooms with high and low personnel activity, respectively, for up to 7 weeks. Group D (n = 18) was housed in the high activity room for 2, 3, or 4 weeks followed by the low activity room. RESULTS: Rats in the low activity room for 3-4 weeks showed robust microvascular networks within 25% to 50% of the mesenteric windows (each window consisting of the tissue extending between two adjacent feeding arterioles in the mesentery), whereas rats in Group B only showed fragile vessels at the edges of the mesenteric windows within fat deposits. Groups A and C demonstrated little mesenteric fat and few fragile vessels, in contrast to group B. Group D showed increased mesenteric networks and decreased mesenteric fat as recovery progressed. The microvascular networks of 6 rats, randomly selected from Group C, showed few venular leaks following perfusion with fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA). Such leaks were abundant in the mesenteric microvasculature of 3 rats randomly selected from Group B. CONCLUSIONS: This study demonstrates that environmentally induced stress alters the architecture and leakage to albumin of the rat mesenteric microvasculature, and emphasizes the importance of closely monitoring the environment of experimental animals.

Ultrastructural effects of intravascularly injected polyethylene glycol-hemoglobin in intestinal mucosa.

Polyethylene glycol (PEG)-conjugated Hb (PEG-Hb) is being considered as a blood substitute. Previously, we showed that PEG-Hb extravasates rapidly from the intestinal mucosa and causes transient epithelial sloughing, resulting in temporary unimpeded passage of material between the intestinal lumen and the microcirculation. The present study quantifies the time course of factors related to this disturbance. Anesthetized Sprague-Dawley rats (350-450 g) were injected with a bolus of PEG-Hb (10 mg/ml) in saline. Control animals received saline, alone or with Dextran 70 (5 mg/ml). After 2, 8, 15, 60, or 90 min, the small intestine was perfusion fixed for microscopy (4 animals for each time point). Epithelial cell detachment and mucosal mast cell degranulation peaked at 2 and 8-15 min, respectively, but by 90 min were back to normal. Goblet cell secretion increased with time up to 8-15 min, after which it leveled off. Mean interstitial width was significantly greater 8 min after injection than for controls and continued to increase with time. In capillaries, endothelial fenestral diaphragms were replaced by thick, amorphous structures. Mesenteric mast cell degranulation was significantly greater 60-90 min after injection compared with controls. We propose that these results are consistent with intravascular injection of PEG-Hb invoking a transient inflammatory response in the intestine.

Inhibition of nitric oxide synthesis increases venular permeability and alters endothelial actin cytoskeleton.

Inhibition of nitric oxide (NO) synthesis using NG-nitro-L-arginine methyl ester (L-NAME) or NG-monomethyl-L-arginine (L-NMMA) increases venular permeability in the rat mesentery (I. Kurose, R. Wolf, M. B. Grisham, T. Y. Aw, R. D. Specian, and D. N. Granger. Circ. Res. 76: 30-39, 1995), but the cellular mechanisms of this response are not known. This study was performed to determine whether such venular leaks are associated with changes in the endothelial actin cytoskeleton. In anesthetized Sprague-Dawley rats, the microvasculature of a mesenteric window was perfused with buffered saline, with or without 10(-5) M L-NAME, L-NMMA, or the inactive enantiomer NG-nitro-D-arginine methyl ester for 3 or 30 min. FITC-albumin was added to the perfusate for the last 3 min. The vasculature was perfusion fixed, stained for filamentous actin and for mast cells, and viewed microscopically. In control preparations, venules showed few FITC-albumin leaks and the endothelial actin cytoskeleton consisted of a peripheral rim along the cell-cell junctions. Preparations treated with L-NAME or L-NMMA showed significantly more leakage, the actin rims in leaky venules were discontinuous, and short, randomly oriented fibers appeared within the cells. In nonleaky venules, the peripheral actin rims sometimes contained small, equally spaced discontinuities not seen in control preparations. Although a mast cell stabilizer was used, 27-70% of the mast cells were degranulated in the presence of L-NMMA. Thus inhibition of NO synthesis alters the endothelial cytoskeleton and increases albumin leakage from mesenteric venules, either directly or indirectly via the involvement of mast cells.

Acute blood stasis reduces interstitial uptake of albumin from intestinal microcirculatory networks.

Temporary blood flow stoppage occurs in a greater percentage of the capillaries when blood flow to organs is reduced. Previous studies on the small intestine have suggested that acute blood stasis (< or = 10 min) results in expression of negative charge, not present when blood flow is brisk, on the luminal surface of mucosal capillaries. Negative surface charge would tend to reduce transcapillary passage of albumin from blood to interstitium, since albumin is also negatively charged. Here we test the hypothesis that acute blood stasis reduces the interstitial uptake of albumin from mucosal capillary networks in rat small intestine in situ. Animals were subjected to two treatments, which included intestinal blood flow and acute stasis. After each treatment, fluorescent albumins were perfused into the intestinal circulation, and then interstitial fluorescence was recorded using fluorescence microscopy. Images were later quantified by computer analysis. After brisk blood flow, but not after acute blood stasis, fluorescence rapidly appeared in the interstitium and resulted in higher interstitial fluorescence intensity values. These results may have relevance to the mechanisms by which albumin flux in the small intestine is synchronized with digestion and fasting, which are associated with high and low intestinal blood flow, respectively.

Identification and determination of material properties for porohyperelastic analysis of large arteries.

A "porohyperelastic" (PHE) material model is described and the theoretical framework presented that allows identification of the necessary material properties functions for soft arterial tissues. A generalized Fung form is proposed for the PHE constitutive law in which the two fundamental Lagrangian material properties are the effective strain energy density function, W(e), and the hydraulic permeability, kij. The PHE model is based on isotropic forms using W(e) = Ue (phi) = 1/2C0(e phi - 1) and the radial component of permeability, kRR = kRR(phi), with phi = C1'(I1 - 3) + C2'(I2 - 3) + K'(J - 1)2. The methods for determination of these material properties are illustrated using experimental data from in situ rabbit aortas. Three experiments are described to determine parameters in Ue and kRR for the intima and media of the aortas, i.e., (1) undrained tests to determine C0, C1', and C2'; (2) drained tests to determine K'; and (3) steady-state pressurization tests of intact and de-endothelialized vessels to determine intimal and medial permeability (adventitia removed in these models). Data-reduction procedures are presented that allow determination of kRR for the intima and media and Ue for the media using experimental data. The effectiveness and accuracy of these procedures are studied using input "data" from finite element models generated with the ABAQUS program. The isotropic theory and data-reduction methods give good approximations for the PHE properties of in situ aortas. These methods can be extended to include arterial tissue remodeling and anisotropic behavior when appropriate experimental data are available.

Effect of atherosclerosis on transmural convection an arterial ultrastructure. Implications for local intravascular drug delivery.

Local infusion of agents through perforated catheters may reduce neointimal formation following vascular angioplasty. Such treatment will succeed only if the drug is retained within the arterial intima long enough to promote repair. Drugs will be dispersed throughout the wall predominantly by transmural convection instead of diffusion if the Peclet number, Pe = J (1-delta f)/P, is greater than unity, where J is the transmural fluid flow per unit surface area and delta(f) and P are the reflection and permeability coefficients to the drug, respectively. Although the targets of local drug delivery will be atherosclerotic vessels, little is known about the transport properties of these vessels. Accordingly, we evaluated the effects of hypercholesterolemia and atherosclerosis on J per unit pressure (hydraulic conductance, Lp) and on ultrastructure in femoral arteries. Measurements were made at 30, 60, and 90 mm Hg in anesthetized New Zealand white rabbits fed a normal diet (n = 6) and after 3 weeks of lipid feeding (n = 19). Atherosclerosis was induced in six lipid-fed animals by air desiccation of a femoral artery. Hydraulic conductance was significantly greater in vessels from hypercholesterolemic than from normal animals and decreased with pressure only in hypercholesterolemic arteries. Atherosclerosis did not augment hydraulic conductance compared with hypercholesterolemia alone. Electron microscopic examination demonstrated damaged endothelium in hypercholesterolemic arteries and both altered endothelium and less tightly packed medial tissue, compared with controls, in atherosclerotic vessels, at least at lower pressures. Peclet numbers for macromolecules exceeded unity for all three groups of arteries and reached 0.3 to 0.4 for molecules as small as heparin. Thus, convection plays a dominant role in the distribution of macromolecular agents following local delivery and may result in their rapid transport to the adventitia in the femoral artery.

Changes in endothelial actin cytoskeleton in venules with time after histamine treatment.

In this study the time course of development and recovery of histamine-induced venular leaks was followed in conjunction with rearrangement of endothelial actin fibers. The microvasculature of a single mesenteric window of anesthetized Sprague-Dawley rats was perfused with buffered saline, with or without 10(-4) M histamine, for 3-30 min. Fluorescein isothiocyanate (FITC)-albumin was added for the last 3 min. The microvasculature was perfusion fixed, stained with rhodamine phalloidin (for filamentous actin), and viewed using confocal microscopy. The number and relative size of FITC-albumin leaks per venule length were measured. After 3 min of histamine application focal leaks appeared in some of the venules. Most focal leaks were accompanied by local breaks in the endothelial peripheral actin rim. Larger leaks were also present, accompanied by greater disruption of the venular endothelial peripheral actin rim, diffuse F-actin staining, and adherent platelets and leukocytes. Few central actin fibers were visible even in endothelial cells associated with large leaks. After 10-15 min of histamine exposure, larger leaks were more abundant but with fewer adherent cells. Central actin fibers in endothelial cells increased in number, peaking after 20 min of histamine, while the diffuse actin staining declined. Leak area per micrometer of venule peaked at 10-15 min, but the numbers of leaks per micrometer did not vary significantly from 3 to 30 min. These data suggest that the central fibers are not involved with the phase of increasing permeability, but they may play a role in the structural and functional recovery of endothelial cells perturbed by histamine.

Changes in endothelial actin cytoskeleton at leakage sites in the rat mesenteric microvasculature.

The purpose of the present study was to compare the endothelial actin cytoskeleton at sites of inflammation-induced macromolecular leakage with that of intact endothelium. The circulation of a selected mesenteric window of anesthetized rats was perfused for 3 min with histamine (100 microM) plus fluorescein isothiocyanate (FITC)-albumin, or FITC-albumin alone, and was fixed at physiological pressure. The vasculature was then perfusion stained with rhodamine phalloidin to label filamentous actin (F-actin) and examined with a confocal microscope. The leakage sites were divided into three categories based on the extent of FITC-albumin leakage: 1) focal leaks, 2) mid leaks, and 3) extended leaks. The leaks were identifiable with a particular endothelial cell(s), and the structure of the endothelial actin cytoskeleton was characterized at these sites. Focal leaks occurred along a small region of endothelial cell-cell contact and in most observed cases were located at a region of specific disruption of the endothelial peripheral actin rim (PAR). Mid leaks involved the disruption of one endothelial cell, and the affected cell was observed to have extended disruption of the PAR as well as increased diffuse F-actin staining throughout the cell (1.9-fold increase relative to adjacent cells). Extended leaks involved the disruption of two or more adjacent endothelial cells, and each cell exhibited an actin pattern similar to that seen at mid leaks. These results show that histamine-induced macromolecular leakage in situ is associated with significant changes in the endothelial actin cytoskeleton.