Rat Endothelium Research Articles

Adenine incorporation in human and rat endothelium

Adenine (ADE) reutilisation is an important pathway of adenylate pool regeneration. Data on the rate of this process in different types of cells, its regulation and the importance of species differences is limited. In this study we evaluated adenine incorporation rate and the effect of metabolic factors on this process in human and rat endothelium and compared it to adenine phosphoribosyltransferase (APRT) activity. Microvascular endothelial cells from human (HE) and rat (RE) hearts and a transformed human microvascular endothelial cell line (HMEC-1) were investigated. The rate of adenine incorporation into the adenine nucleotide pool under control conditions was 3.1±0.3, 82.8±11.1 and 115.1±11.2 pmol/min per mg protein for HE, RE and HMEC-1, respectively. In the presence of 2.5 mM ribose or elevated inorganic phosphate concentration in the medium (4.8 mM), few changes were observed in all types of cells. In the presence of both ribose and high inorganic phosphate, the rate of adenine incorporation for RE and HMEC-1 was not significantly different from control, while in HE the rate of adenine incorporation into adenine nucleotides was increased by 75%. Activities of APRT in RE and HMEC-1 were 237.7±23.2 and 262.0±30.6 pmol/min per mg protein respectively while the activity in HE was markedly lower 48.7±3.0 pmol/min per mg protein. In conclusion, nucleotide synthesis from adenine seems to be a slow process in human cardiac microvascular endothelium but it is fast and efficient in rat heart microvascular endothelial cells. Low APRT activity in normal human endothelial cells seems to be the most likely mechanism for this. However, adenine incorporation rate and APRT activity could be greatly enhanced in human endothelium, as demonstrated in transformed cells.

Evidence of a Nonendothelial Source of Nitric Oxide in the Isolated Perfused Hindlimb Vasculature of the Pregnant Rat

Objective: This study was undertaken to further elucidate the roles of the vascular endothelium and nitric oxide (NO) in the regulation of vascular tone and constrictor responsiveness in pregnancy.Methods: The perfusion pressure-flow relationship was measured in isolated, perfused, norepinephrine-constricted (1) endothelium-intact, (2) endothelium-denuded, and (3) A-nitro-L-arginine methyl ester (L-NAME)-treated hindlimbs from nonpregnant and term-pregnant rates.Results: Baseline perfusion pressure at a flow rate of 2 mL/min was similar (-20 min Hg) in all hindlimbs. Norepinephrine (0.5 μM) increased perfusion pressure in both nonpregnant (+21.6 ± 2.4 mm Hg) and pregnant (+13.6 ± 0.9 mm Hg) endothelium-intact rat hindlimbs. In nonpregnant rat hindlimbs, endothelium removal and L-NAME increased norepinephrine vasoconstriction similarly (+44.3 ± 4.0 mm Hg and +46.4 ± 8.6 mm Hg, respectively). In pregnant-rat hindlimbs, L-NAME increased norepinephrine vasoconstriction by 43.5 ± 10.8 mm Hg, similar to that in nonpregnant-rat hindlimbs, but endothelium removal only increased norepinephrine vasoconstriction by 28.0 ± 2.2 mm Hg. Perfusion pressure increased linearly as the flow rate was increased from 2 to 4 mL/min, and the slope of the regression line of the endothelium-intact pregnant-rat hindlimbs (7.0 ± 0.6) was slightly, but not significantly, lower than that of the nonpregnant-rat hindlimbs (9.6 ± 0.9). Endothelium removal increased the slopes of the regression lines, but that of pregnant-rat hindlimbs (12.8 ± 1.6) was significantly lower (p ± 0.05) than that of the nonpregnant-rat hindlimbs (23.8 ± 1.8). L-NAME caused a similar increase in the pressure-flow slopes of nonpregnant-rat (36.5 ± 3.4) and pregnant-rat (32.1 ± 5.3) hindlimbs.Conclusions: These results suggest that nonendothelial nitric oxide production may be increased in the hindlimb resistance vasculature of the pregnant rat, which may play a role in the normal pregnancy blunting of constrictor responsiveness and reduction of vascular resistance.

Biological Evaluation of the Nitric Oxide-Trapping Agent, N-Methyl-D-glucamine Dithiocarbamate-Fe2+, as a Probe of Nitric Oxide Activity Released From Control and Diabetic Rat Endothelium

We utilized the nitric oxide (NO) scavenger N-methyl-D-glucamine dithiocarbamate-Fe2+ (MGD-Fe) to characterize the role of NO in basal and acetylcholine (ACh)-stimulated relaxation arising from the endothelium of control vs diabetic rat aortic rings. In phenylephrine-contracted rings, MGD-Fe produced an additional increment in tension that was indomethacin-insensitive (i.e., excluding a role of prostanoids in this action). This MGD-Fe-sensitive component was more pronounced in control rings than diabetic rings and of the same magnitude achieved in rings without MGD-Fe treatment after removal of endothelium or treatment with the NO synthase inhibitor L-nitroarginine (L-NA). This suggests complete scavenging of basal NO by MGD-Fe and supports reduced basal NO in diabetic rings. ACh fully relaxed both control and diabetic rings. This relaxation was abolished by removal of the endothelium and was inhibited by L-NA (by 100% and 90% in control and diabetic rings, respectively). In contrast, MGD-Fe only partially inhibited ACh-induced relaxation in control (65 ±5% inhibition) and diabetic (41 ±11% inhibition) rings. The MGD-Fe-resistant component was not further modified by indomethacin. Addition of L-arginine (L-ARG) (but not D-arginine (D-ARG) enhanced the ACh-induced relaxation of MGD-Fe-treated diabetic (but not control) rings. These data provide evidence about endothelium-dependent relaxation in control and diabetic rings which cannot be discerned by use of L-NA alone. This study suggests that ACh produces a NO synthase-dependent vasodilation, a portion of which is due to free NO radical (·NO) or due to NO in a form or location that is unavailable for scavenging by MGD-Fe.

Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction.

Nitric oxide release from the endothelium plays an important role in regulation of vascular tone, inhibition of both platelet and leukocyte aggregation and adhesion, and inhibition of cell proliferation. These properties suggest that the level of NO production by the endothelium may play a pivotal role in the regulation of vascular disease. Analysis using mass spectrometry has revealed that NO is produced by NOS from the terminal guanidino nitrogen of the precursor amino acid L-arginine. Thus, utilization of L-arginine and conversion to NO may establish a regulatory site in the development of endothelial dysfunction. Endothelial dysfunction is characterized by defective endothelium-dependent relaxation, and some reviews regarding endothelial dysfunction in diabetes have been published. These reviews have focused on factors that might contribute to defective relaxation, some of which will not be addressed in detail in this review. The purpose of the present review is to summarize evidence that specifically supports either decreased NO production by diabetic vascular endothelium and/or impaired NO-mediated endothelium-dependent relaxation. Second, this review provides reasonable alternatives to explain some of the controversies in this research area. Third, since there is growing evidence that arginine appears to have some benefits for diabetes-associated abnormalities, this review summarizes the current state of knowledge of effects of acute and chronic administration of L-arginine on diabetesinduced endothelial dysfunction and discusses potential NOdependent and -independent mechanisms whereby therapeutic intervention with L-arginine might benefit the diabetic endothelium.

Interleukin-1α Regulates Thy-1 Expression on Rat Vascular Endothelial Cells

We recently reported that Thy-1, a surface molecule induced on the rat endothelium, regulates vascular permeability at sites of inflammation. Although the rat inferior vena cava (IVC) did not express Thy-1in vivo, cultured endothelial cells from the IVC did express Thy-1, thereby suggesting that the expression was acquired during cultivation of the cellsin vitro,possibly by autoactivation by cytokine-like substances. Interleukin (IL)-1α but not tumor necrosis factor (TNF)-α or interferon (IFN)-γ was detected in culture supernatants of rat endothelial cells (REC) by ELISA. The production of IL-1α by REC was augmented by exogenously added IL-1α, thereby implying the presence of autocrine regulation by IL-1α. The unaltered expression of Thy-1 by exogenously added IL-1α suggests that Thy-1 expression on REC had already been maximally induced by autologous cytokines; the expression of Thy-1 on REC was lowered by inhibiting protein kinase C and by depleting IL-1α activity from culture supernatants. Although cytokine-like regulators, other than IL-1α, TNF-α, or IFN-γ, produced by REC may also modulate the expression of Thy-1, it is at least in part mediated by IL-1αin vitro.Moreover, Thy-1 expression was induced on rat vascular endothelium at the subcutis where recombinant IL-1α was injected. The evidence indicates that IL-1α functions as one regulator responsible for the induction of Thy-1 on REC,in vitroas well asin vivo.

Stimulation of angiogenesis by canine parathyroid tissue

Autotransplanted parathyroid tissue is capable of inducing neovascularization in vivo, restoring calcium homeostasis with regulatory control. The mechanisms of parathyroid-induced neovascularization remain to be determined. Using an unique three-dimensional in vitro model, we tested the ability of parathyroid tissue to stimulate angiogenesis. Healthy 1 mm3 fragments of normal canine parathyroid tissue were cocultured with freshly isolated microvessels that were embedded in a collagen I gel. After 7 days the gels were stained with Gs-1 lectin, a specific marker for rat endothelium. With image analysis the microvessel density (as the percentage of area) was determined. A significant increase in mean microvessel density (20.90% +/- 1.28 versus 16.51% +/- 1.66%) was seen with parathyroid coculture compared with control (n = 17, p < 0.05). There was no difference in microvessel density between controls and microvessels exposed to increasing concentrations of parathyroid hormone or calcium. The density of seeded microvessels influenced the effect of parathyroid stimulation of angiogenesis. The effect was apparent only at low seeding densities. We conclude that parathyroid tissue intrinsically stimulates angiogenesis in vitro by a secreted product, independent of calcium or parathyroid hormone.

Thy-1 induced on rat endothelium regulates vascular permeability at sites of inflammation.

We investigated the role of surface adhesion molecules on endothelial cells in regulating vascular permeability, in vitro and in vivo. Cultured rat endothelial cells (REC) express Thy-1, intercellular adhesion molecule-1 (ICAM-1), CD44 and RT1A. Permeability of albumin across the REC monolayer increased through the interaction of Thy-1 and anti-Thy-1 mAb, but not through ICAM-1 and anti-ICAM-1, CD44 and anti-CD44, and RT1A and anti-RT1A mAb. This anti-Thy-1-effect was completely inhibited when the calmodulin antagonist W-7 and the protein kinase inhibitor H-7 was combined, while the IL-6-mediated increase in REC permeability was blocked by either W-7 or H-7, independently. The anti-Thy-1-mediated permeability increase was additively augmented when IL-6 was admixed. These data suggest that intracellular signaling pathways of anti-Thy-1- and IL-6-mediated permeability regulation may be overlapping to some extent but are largely independent. As anti-Thy-1-treatment generated rearrangement of vimentin filaments within REC, alteration of the cytoskeleton distribution may possibly correlate with the regulation of permeability. Although Thy-1-expression on rat vascular endothelium in vivo was not evident, it was induced at sites of Freund's complete adjuvant-induced dermatitis. The administered anti-Thy-1 mAb exclusively located on vascular endothelial surface at the sites of inflammation. Vascular permeability in inflamed skin tissues was significantly augmented when anti-Thy-1 but not anti-ICAM-1, anti-CD44 or anti-RT1A mAb was administered i.v., without affecting populations of inflammatory cells. The collective evidence suggests that Thy-1 induced on rat endothelium is one important regulatory event in vascular permeability at sites of inflammation.

Decreased ascorbic acid entry into cornea of streptozotocin-diabetic rats and guinea-pigs

High l-ascorbic acid (AA) levels in aqueous humor and intraocular tissues including lens and cornea are thought to protect against the harmful effects of the photochemical and ambient oxidation reactions involving oxygen and its radicals. Our pulse-chase studies follow a bolus of radiolabeled test molecules including [ 14C] l-ascorbic acid and [ 3H] l-glucose ( l-glu) introduced into the blood at time t = 0, and determine the time-dependent concentrations of these labeled molecules as they move into aqueous humor, corneal endothelium and stroma tissues. Calculated entry and exit rate constants provide a representative measure of the functional state of passive and carrier mediated transport mechanisms in situ in normal and diabetic animals. Diabetic rats were categorized in terms of length of time exposed to a uniform, monitored streptozotocin (stz) diabetes as: short term (10–20 days); mid-term (40–60 days); and long term (100 + days). In the rat, we observed little change in entry rate of l-glu (a passive marker) into aqueous humor [control K i = 0·0216 ± 0·0021 ( n = 14)/mid-term stz-diabetes K i = 0·0202 ± 0·0027 ( n = 10)] and a modest decrease in the entry rate of AA into aqueous humor [control K Ai = 0·0231 ± 0·0022 ( n = 14)/mid-term stz-diabetes K Ai = 0·0201 ± 0·0034 ( n = 10)]. At corneal endothelium, we noted a significant decrease in the active movement of AA [control K E = 0·614 ± 0·053 ( n = 14)/mid-term stz-diabetes K E = 0·220 ± 0·026 ( n = 9)] while the passive l-gly entry rate remained essentially unchanged. Thus, our data suggest that the stz-diabetic rat endothelium demonstrates impaired ability to bring AA into the cornea while passive movement, as monitored with l-glucose, remained unchanged. AA movement into stroma was apparently passive and decreased in the corneal stroma of diabetic rats. Experiments with diabetic guinea-pigs demonstrated a diminished ability to bring AA into aqueous humor and a further decrease in concentrating AA in corneal endothelium. Thus, in both species our data indicate that the ability to bring AA actively into corneal endothelium and stroma is compromised by diabetes while l-glucose passive movement into the cornea remained unchanged.

Immunohistological detection of Saruplase (recombinant single-chain urokinase-type plasminogen activator) in normal rat tissue

Saruplase--a recombinant single-chain urokinase-type plasminogen activator was identified immunohistochemically in normal rat tissue after intravenous administration by means of a polyclonal antibody. For this purpose, rat tissues were fixed in various ways (liquid nitrogen, ethanol, formaldehyd solution). Saruplase could be detected by the PAP method, streptavidinbiotin system and indirect immunofluorescence in the kidney (proximal tubule), liver (hepatocytes, Kupffer cells) and spleen (reticular cells). Saruplase was not localized in the rat endothelium. It is discussed that the rat-specific receptors for urokinase-type plasminogen activator on endothelial cells cannot bind Saruplase due to the extreme species specificity.

Protein kinase C-regulated production of prostacyclin by rat endothelium is increased in the presence of lipoxin A4

Prostacyclin is generated by cultured rat endothelial cells. Compound blocking activity of protein kinase C and cyclic nucleotide-dependent protein kinases (H7) and compound blocking interaction between Ca 2+ and calmodulin (W7) diminish generation of prostacyclin in rat endothelial cells. These compounds give a synergistic effect when they are introduced to the endothelial cell cultures simultaneously. Compound HA1004, an inhibitor ofcAMP- and cGMP-dependent protein kinases has no effect on prostacyclin generation. Lipoxin A4, a potent direct stimulator of protein kinase C, rapidly induces prostacyclin generation in rat endothelium in a dose- and time-dependent fashion. Lipoxin A4-induced generation of prostacyclin can be inhibited by H7 and W7 but not by HA1004. Lipoxin B4 has no significant effect on prostacyclin generation in rat endothelium. In conclusion, our results demonstrate that generation of prostacyclin by rat endothelial cells is regulated via a pathway involving protein kinase C and Ca 2+.

Class I antibody induction by rat endothelium

Supradiaphragmatic vein grafts were transplanted from ACI (RT1 a) to Fisher (RT1 1) rats to determine whether the endothelium and/or circulating passenger leukocytes were necessary for the induction of an antibody response. Vein grafts from immunosuppressed and irradiated donors induced lymphocytotoxic and hemagglutinating antibody in recipient rats. In addition, IgG antibody produced in Fisher recipients was found by indirect immunofluorescence to donor endothelium. When the endothelium was removed from donor veins either by collagenase or by scraping the intima, no antibody resulted after transplantation. The lymphocytotoxic and hemagglutinating antibodies and the antibodies identified by indirect immunofluorescence were removed by absorbing the Fisher serum in ACI red blood cells. The endothelium seems to be capable of inducing Class I antibodies when allogeneically transplanted as vein grafts.