Cultured bovine aortic endothelial cells show increased histamine metabolism when exposed to oscillatory shear stress.

Oscillatory shear stress applied to the lining of blood vessels causes endothelial cell injury, one of the essential postulated prerequisites to the development of atherosclerosis. The purpose of this investigation was to study effects of shear stress on bovine aortic endothelial cells (BAEC), in vitro, for varying lengths of time (6 h, 12 h, 24 h) on BAEC histamine content (HC) and histidine decarboxylase activity (HD). Low intensity stress (1.6 dynes/cm2) as well as intermediate and high intensity shear stresses (3.5 dynes/cm2 and 7.6 dynes/cm2) resulted in an accelerated HD (281%) and elevated HC (144%). These data indicate that oscillatory shear stress produces increases in histamine metabolism.

Relationship between inhibition of aortic histamine formation, aortic albumin permeability and atherosclerosis.

Effects of partial inhibition of aortic histamine formation on aortic albumin uptake and lipid deposition were examined in male, New Zealand white rabbits maintained on Purina Rabbit Chow containing 0.5% cholesterol for a 2-week period. Aortic histamine synthesis was inhibited by partial inhibition of aortic histidine decarboxylase (HD) through administration of alpha-hydrazinohistidine (alpha-HH, MK785, Regis Chemical Co., 25 mg/kg, i.p. at 12-h intervals). Additional rabbits were maintained on either the cholesterol diet or on Purina Rabbit Chow without cholesterol. Results indicate that administration of alpha-HH for the 2-week period produced a 31% reduction (P less than 0.05) in aortic HD activity in those rabbits maintained on the cholesterol diet, and that concurrently there was a 51% reduction in aorta albumin uptake (P less than 0.025) and a 63% reduction in the extent of oil red O staining. By regression analysis a significant correlation coefficient (r = 0.71, P less than 0.005) was obtained between the aortic albumin uptake and the aortic histamine forming capacity (HFC) in rabbits maintained on this cholesterol diet. These findings indicate that the aortic HD system may be an important enzymatic coupler involved in vascular permeability alterations occurring early in the atherogenic sequence.

Rabbit aortic histamine synthesis following short-term cholesterol feeding.

The histidine decarboxylase (HD) activity of thoracic and abdominal aortic segments obtained from male, Dutch-belted rabbits fed a diet containing 0.5 per cent cholesterol for periods of either 2 or 4 weeks was examined. Mean thoracic aortic HD activities, expressed as histamine-forming capacity (HFC), were 3911 plus or minus 492, 6254 plus or minus 656, and 6215 plus or minus 878 dpm/100 mg benzenesulfonylhistamine (BSH) for the control group and from rabbits fed cholesterol for 2 and 4 weeks, respectively. Both treatment means were significantly higher than the control (P smaller than 0.05). Similar examination of abdominal aortic HD activities yielded mean HFC's of 4029 plus or minus 399, 5694 plus or minus 521, and 4762 plus or minus 902 dpm/100 mg BSH for control animals and those of the 2- and 4-week treatment groups, respectively. The difference between mean HFC's of the control and 2-week treatment group was significant (P smaller than 0.05). All increases occurred in the absence of either aortic structural alterations or any lipid deposition. These results give credence to the concept that the atherogenic process represents, at least in part, a delayed-prolonged inflammatory response phenomenon of the arterial wall.

A simple fluorescent method for simultaneous determination of aortic permeability to horseradish peroxidase and bovine serum albumin.

Differences in regional aortic net uptake of bovine serum albumin (BSA) and horseradish peroxidase (HP) have been examined by means of conjugation of these molecules to the fluorescent protein tracers fluorescein isothiocyanate (FITC) and lissamine rhodamine B (RB200). Using male Wistar rats, uptake of FITC-BSA under steady state conditions in the ascending aorta and aortic arch (14 +/- 2 micrograms/mg aorta) is significantly higher (p less than 0.05) than that of either the upper, middle, or lower third of the thoracic aorta (10 +/- 1 mu, 9 +/- 1 mu, and 8 +/- 1 micrograms/mg, respectively). No regional variation in net uptake of RB200-HP was observed in these same aortic regions, with respective mean values (+/- SE) being 69 +/- 2, 69 +/- 2, 68 +/- 4, and 68 +/- 4 micrograms/mg. Examination of fluorescence photomicrographs indicate that FITC-BSA is localized along the collagen-elastin bands, while the RB200-HP is found between these bands. Differences between FITC-BSA and RB200-HP uptake and deposition reflect such things as differences in uptake rates as influenced by initial concentrations, permeability coefficients, as well as possible differences in molecular charge and affinity. The results indicate that the fluorometric procedures described in this investigation are simple, sensitive, and quantitative, and suitable for simultaneous measurement of aortic uptake of molecules having different molecular sizes as well as for the intraaortic localization of these substances.

Feulgen-deoxyribonucleic acid analysis of rabbit aortic smooth muscle cells using scanning- integrating microdensitometry.

A procedure entailing the use of the Feulgen reaction is described for precise quantification of nuclear DNA levels in smooth muscle cells (SMC) of paraffin-processed microtome sections of the rabbit aorta. It was established that maximal, stable, and reproducible Feulgen-DNA (F-DNA) staining of SMC nuclei is achieved using 3.5 N HCl hydrolysis of 30-50 min prior to staining of aortic sections in Schiff reagent for 60 min at 22 degrees C. Scanning-integrating microdensitometry of Feulgen-stained SMC revealed that the tunica media is comprised of a relatively homogeneous population of cells with between 0.3 and 1% of the SMC nuclei yielding 3C or 4C (tetraploid) F-DNA levels, depending on location within the aortic wall. The nuclear chromatin in inner medial SMC was found to be in a more dispersed state than that of outer SMC (using nuclear area and nuclear susceptibility to acid hydrolysis as indices of chromatin dispersion). A linear correspondence was evidenced between nuclear area and nuclear F-DNA stainability throughout the tunica media. The observation that the lumenal portion of the tunica media contains a greater abundance of SMC with large, vesicular nuclei is interpreted as reflecting a greater metabolic reactivity of this compartment relative to that of SMC bordering the tunica adventitia.

Retinal histamine synthesis is increased in experimental diabetes.

We examined retinal de novo histamine synthesis mediated by retinal histidine decarboxylase in normal and streptozotocin-diabetic male, Sprague Dawley rats that were diabetic for 21 days. We also examined effects of insulin and alpha-hydrazinohistidine (alpha HH) treatments on retinal histamine synthesis in this diabetic model. alpha HH is a specific inhibitor of histidine decarboxylase. Results indicate that the retina contains an active histidine decarboxylase enzyme system, and that in streptozotocin diabetes retinal histamine synthesis is increased 197%. Both insulin and alpha HH independently reverse and normalize retinal histamine synthesis. These data thus indicate that the retinal inducible histamine pool is increased in experimental diabetes, and that insulin is an important modulator of retinal histamine metabolism. This newly described retinal metabolic alteration may be one factor responsible for increased retinal vascular permeability in diabetic retinopathy.

Histamine H1 receptors mediate increased blood-retinal barrier permeability in experimental diabetes.

To test the hypothesis that histamine receptors mediate increased blood-retinal barrier permeability in experimental diabetes, 51 rats were made diabetic by streptozocin injection (65 mg/kg; jugular vein) and were held for four weeks. The seven animal groups were as follows: untreated controls; untreated diabetic rats; diabetic rats receiving diphenhydramine hydrochloride (Benadryl); diabetic rats receiving cimetidine hydrochloride (Tagamet); diabetic rats receiving diphenhydramine and cimetidine; diabetic rats receiving purified pork insulin (Iletin II); and diabetic rats receiving insulin and diphenhydramine. All treatments were given during the last week. Blood-retinal barrier permeability was assessed through measurement of the vitreous content of fluorescein isothiocyanate conjugated to bovine serum albumin (FITCBSA) after 20 minutes of FITCBSA circulation. Vitreous FITCBSA content of the diabetic group was 64% greater than control content. Diabetic rats treated with either diphenhydramine or diphenhydramine and insulin had respective decreases of 43% and 40% in vitreous FITCBSA content. The vitreous content of the diabetic group receiving insulin was lowered 37% below untreated diabetic values, while the vitreous FITCBSA content of the diabetic group receiving both insulin and diphenhydramine was reduced 63%. These data indicate that retinal histamine H1-receptor activation may be partially responsible for initial blood-retinal barrier leakage of macromolecules into the vitreous and that this abnormal leakage can be prevented both by diphenhydramine and by insulin. Histamine H1 receptors may play an important role in mediating increased blood-retinal barrier permeability in experimental diabetes.

Astemizole reduces blood-retinal barrier leakage in experimental diabetes.

We examined the potential of astemizole, a histamine H1-receptor antagonist that does not cross the blood-brain barrier, to reverse blood-retinal barrier leakage to albumin in streptozotocin diabetic rats. Four groups of nondiabetic and four groups of diabetic rats received vehicle or astemizole at dosages of 5, 10, or 20 mg/kg body weight for days 22-28 of a 28-day holding period. There were no significant differences in nondiabetic plasma-vitreous albumin ratios between animals receiving vehicle or any of the three astemizole dosages. Only diabetic rats receiving vehicle showed a significant (p < 0.05) 100% increase in the plasma-vitreous albumin ratio over their nondiabetic counterparts. Diabetic rats receiving either 5, 10, or 20 mg/kg astemizole exhibited total normalization of vitreous albumin accumulation, despite persistence of diabetes. These data indicate that astemizole, an H1-receptor antagonist that does not cross the blood-retinal barrier, is effective in reversing blood-retinal barrier leakage of albumin in experimental diabetes.

Physiological transport properties of cultured retinal microvascular endothelial cell monolayers.

PURPOSE: To characterize baseline transport properties: hydraulic conductivity (Lp), albumin permeability (Pe), and transendothelial electrical resistance (TER) of bovine retinal microvascular endothelial cells (RMEC) in the development of an in vitro model of the blood-retinal barrier (BRB). METHODS: RMEC were grown on porous, polycarbonate filters for determination of the number of days required to achieve minimal transport rates. Lp, Pe, and TER were measured by utilizing a bubble tracking spectrophotometer, by quantifying the diffusional movement of fluorescein isothiocyanate-labeled albumin, and by utilizing a Millipore electrical resistance meter, respectively. RESULTS: Lp decreased significantly from 7.82 +/- 0.85 x 10(-7) (mean +/- SEM) cm/sec/cm H2O at post-plating Day 5 to 1.44 +/- 0.26 x 10(-7) cm/sec/cm H2O at Day 9. Pe of the monolayer also decreased progressively with days post-plating from 3.44 +/- 0.53 x 10(-6) cm/sec at Day 7 to a minimum of 1.95 +/- 0.29 x 10(-6) cm/sec at Day II. Peak TER fluctuated until Day 7, when it began to steadily increase from 17.14 ohm-cm2 to a peak value of 25.42 ohm-cm2 at Day 10, decreasing from then on to 22.24 ohm.cm2 on Day 12. Known disrupters of the BRB, NECA and VEGF, elicited significant increase in RMEC Lp showing the sensitivity of this model to pharmacological alterations. CONCLUSIONS: Our data indicate that RMEC grown on polycarbonate filters form a restrictive monolayer of cells, which exhibit dynamic alterations in response to pharmacological agents, thus demonstrating an in vitro model of the BRB. Future studies with the model may offer insights into the pathogenesis of retinal vascular diseases and allow convenient testing of pharmacological interventions.

Local aortic histamine metabolism and albumin accumulation. Differences between blue and white areas.

Relationships between histamine metabolism, histamine content, and albumin accumulation were examined in Evans blue dye stained areas (blue) and unstained (white) areas of normal canine aortas. Results indicated that, while no differences existed in histamine methyltransferase-mediated catabolism, both histidine decarboxylase-mediated histamine synthesis and the histamine content of blue regions were significantly greater (p less than 0.005) than in contiguous white areas. Blue areas also showed significantly higher fluorescein-labeled albumin accumulation than white areas. By multiple regression analysis, a significant relationship (r = 0.81) was obtained between local aortic albumin accumulation and combined influences of local histidine decarboxylase activity and histamine content. The best predictor in this case was the local histidine decarboxylase activity. These data indicate that blue areas, believed to represent areas of spontaneous hemodynamic-induced vascular injury, have a larger nascent histamine pool than do contiguous white areas and that the distribution of histamine and histamine synthesis in the aorta is highly variable depending on the region examined. The data also suggest that local aortic histamine synthesis in blue areas may play a significantly role in mediation of the increased albumin accumulation observed in these regions.

Inhibition of aortic histamine synthesis by alpha-hydrazinohistidine inhibits increased aortic albumin accumulation in experimental diabetes in the rat.

We examined the interrelationship between inhibition of aortic histamine synthesis through inhibition of aortic histidine decarboxylase and intra-aortic albumin accumulation in rats made diabetic by a jugular vein injection of 60 mg/kg of streptozotocin under ether anesthesia. Animals were held for 4 weeks following overt manifestation of diabetes. At the end of 3 weeks, at least six animals in each of the diabetic and non-diabetic groups received intra-peritoneal injections of alpha-hydrazinohistidine (25 mg/kg at 12 h) for the last 7 days. Aortic albumin accumulation was measured by quantification of aortic uptake of fluorescein isothiocyanate conjugated to rat serum albumin injected in the jugular vein 1 h before sacrifice. The aortic albumin mass transfer and flux rates of the diabetic group were more than 300% higher than that of the control group; alpha-hydrazinohistidine treated diabetic rats had aortic albumin mass transfer rates equivalent to control values. The aortic albumin content was nearly tenfold higher in untreated diabetic rats, but again treatment with alpha-hydrazinohistidine returned this to control values. These data offer strong support to the premise that accelerated aortic histamine synthesis, which occurs in experimental diabetes, is an important mediator of increased aortic macromolecule uptake, and as such, may be one component of the multitude of factors responsible for increased susceptibility of atherosclerosis among individuals having diabetes mellitus.

Density-dependent contraction of the endothelial nascent histamine pool by exogenous heparin.

The aortic endothelial cell nascent histamine pool has been implicated in the control of vessel wall permeability under conditions of stress and injury. We report the contraction of this histamine pool in low density bovine aortic endothelial cell (BAEC) cultures by exogenous heparin. Untreated BAEC exhibit a decline in histamine content in 3-day cultures with increasing plating density between 1000 and 16,000 cells/cm2. Heparin abolished this density-related difference by effecting a 67% contraction of the histamine pool in the lowest density cultures. This effect was reversible and specific to heparin. At a confluent density, endothelial cells secrete heparin-like glycosaminoglycans which affect smooth muscle and endothelial metabolism. We propose that the metabolic effects of exogenous heparin, and perhaps endogenous heparins, extend to specific modulations of the BAEC nascent histamine pool.

Aortic histamine synthesis and aortic albumin accumulation in diabetes: activity-uptake relationships.

The relationship between inhibition of aortic histamine synthesis induced by varied dosages of alpha-hydrazinohistidine (alpha HH) and aortic uptake of fluorescein-labeled bovine serum albumin (FITCBSA) was examined in 40 male Wistar rats made diabetic by streptozotocin. Compared to nondiabetic animals, aortic uptake of FITCBSA in untreated diabetic animals was increased 43%. alpha HH administration produced essentially a complete inhibition of this accelerated histamine synthesis in diabetic animals at all dosages examined and likewise prevented an increase in aortic FITCBSA uptake among these animals. In diabetic animals, aortic histamine synthesis and aortic FITCBSA uptake showed a significant, strong positive correlation (r = 0.822, P less than 0.001) when examined in relation to the degree of inhibition of accelerated aortic histamine synthesis achieved among the individual animals. These data support the hypothesis that elevations in de novo aortic histamine formation, and thus elevations in the inducible histamine pool, are responsible, at least in part, for increased aortic uptake of macromolecules in diabetes. Since increases in various permeability characteristics occur early in atherogenesis, these data also indicate that expansion of the nascent histamine pool occurring in diabetes may be an important factor in the predisposition of diabetics to atherosclerotic vascular disease.

Influence of locally altered in vivo shear stress on aortic histamine-forming capacity and aortic albumin uptake.

Effects of locally elevated shear stresses on thoracic histamine formation and transmural albumin uptake have been examined under in vivo conditions in anesthetized male, mongrel dogs. Results indicate that if endothelium remains intact, significant linear relationships exist between the shear intensity and aortic histamine-forming capacity and aortic content of fluorescein-labeled bovine serum albumin following 20 h of circulation time. These data suggest that in vivo the aortic (and endothelial) histidine decarboxylase system is sensitive to local alterations in applied shear stress, and that de novo aortic histamine synthesis in involved in mediating shear-induced increases in aortic transmural permeability.

Rat aortic histamine synthesis during short-term hypertension.

Thoracic aortic histamine synthesis has been examined in rats rendered hypertensive for periods ranging from 1/4 to 8 days through ligation of abdominal aorta between origins of the renal arteries, in a preliminary attempt to establish whether activity of the aortic nonmast-cell histidine decarboxylase system is increased in experimental hypertension. Results indicate that aortic histamine synthesis is increased 66, 75, 96 and 55% at postsurgical intervals of 1/4, 1/2, 1 and 8 days. These data suggest at least one biochemical explanation for both increased arterial wall permeability and the arterial inflammation consistently associated with both clinical and experimental hypertension by resolving these into those associated with the prolonged inflammatory response.

Shear stress and aortic histamine synthesis.

Aortic histamine-forming capacity (HFC) has been examined in relationship to the applied mean shear stress intensity created by pulsatile perfusion of rabbit aortas with platelet-free blood for a 1-h period. Mean shear stress intensities ranged from 22 to 109 dyn/cm2. Results indicate that a high correlation exists between the shear stress and the HFC which is described by the regression equation y = 0.28 x -6.1, where y = HFC and x = mean shear stress intensity. Results suggest that the rate of histamine formation is sensitive to the applied shear stress, and that the histidine decarboxylase system of the aorta may have the potential of serving as one coupling agent between applied hemodynamic stress and resultant alterations in aortic wall resistance to macromolecules.

Aortic endothelial and smooth muscle histamine metabolism in experimental diabetes.

We studied histamine metabolism, i.e., histidine decarboxylase (HD)-mediated synthesis and histaminase-mediated catabolism, in relation to intracellular histamine content in both aortic endothelial and subjacent smooth muscle cells of control and diabetic rats. Diabetes was induced by a single jugular vein injection of streptozotocin (55 mg/kg in acidified saline, pH 4.5), and animals were held for either 2 or 4 weeks following overt manifestation of diabetes. An additional 4-week diabetic group received insulin (Iletin NPH, 10 U per 24 hour) during the last week. With respect to control values, the histamine content of aortic endothelial cells increased 138%, HD activity increased 250%, and histaminase activity decreased 50% over the 4-week period. In subjacent smooth muscle cells, the histamine content increased in excess of 150%, HD activity increased more than 300%, and histaminase activity decreased in excess of 30%. Insulin treatment for the last week resulted in complete reversal of all these changes. These results support the concept that a large vessel response similar to the microcirculatory prolonged phase of inflammation occurs in experimental diabetes, a change similar to that occurring in experimental atherosclerosis. They also indicate that both synthetic and catabolic changes occur in histamine metabolism under these conditions, changes that alter arterial wall histamine pools, and suggest that insulin administration under conditions of experimental diabetes may modulate aortic histamine metabolism and the resultant intraaortic histamine pools.