A quinazoline-derivative compound with PARP inhibitory effect suppresses hypertension-induced vascular alterations in spontaneously hypertensive rats.

AIMS: Oxidative stress and neurohumoral factors play important role in the development of hypertension-induced vascular remodeling, likely by disregulating kinase cascades and transcription factors. Oxidative stress activates poly(ADP-ribose)-polymerase (PARP-1), which promotes inflammation and cell death. We assumed that inhibition of PARP-1 reduces the hypertension-induced adverse vascular changes. This hypothesis was tested in spontaneously hypertensive rats (SHR). METHODS AND RESULTS: Ten-week-old male SHRs and wild-type rats received or not 5mg/kg/day L-2286 (a water-soluble PARP-inhibitor) for 32 weeks, then morphological and functional parameters were determined in their aortas. L-2286 did not affect the blood pressure in any of the animal groups measured with tail-cuff method. Arterial stiffness index increased in untreated SHRs compared to untreated Wistar rats, which was attenuated by L-2286 treatment. Electron and light microscopy of aortas showed prominent collagen deposition, elevation of oxidative stress markers and increased PARP activity in SHR, which were attenuated by PARP-inhibition. L-2286 treatment decreased also the hypertension-activated mitochondrial cell death pathway, characterized by the nuclear translocation of AIF. Hypertension activated all three branches of MAP-kinases. L-2286 attenuated these changes by inducing the expression of MAPK phosphatase-1 and by activating the cytoprotective PI-3-kinase/Akt pathway. Hypertension activated nuclear factor-kappaB, which was prevented by PARP-inhibition via activating its nuclear export. CONCLUSION: PARP-inhibition has significant vasoprotective effects against hypertension-induced vascular remodeling. Therefore, PARP-1 can be a novel therapeutic drug target for preventing hypertension-induced vascular remodeling in a group of patients, in whom lowering the blood pressure to optimal range is harmful or causes intolerable side effects.

Age determines the magnitudes of angiotensin II-induced contractions, mRNA, and protein expression of angiotensin type 1 receptors in rat carotid arteries.

In this study, we hypothesized that aging alters angiotensin II (Ang II)-induced vasomotor responses and expression of vascular mRNA and protein angiotensin type 1 receptor (AT1R). Thus, carotid arteries were isolated from the following age groups of rats: 8 days, 2-9 months, 12-20 months, and 20-30 months, and their vasomotor responses were measured in a myograph after repeated administrations of Ang II. Vascular relative AT1R mRNA level was determined by quantitative reverse-transcriptase polymerase chain reaction and the AT1R protein density was measured by Western blot. Contractions to the first administration of Ang II increased from 8 days to 6 months and then they decreased to 30 months. In general, second administration of Ang II elicited reduced contractions, but they also increased from 8 days until 2 months and then they decreased to 30 months. Similarly the AT1R mRNA level increased from 8 days to 12 months and then decreased to 30 months. Similarly the AT1R protein density increased from 8 days until 16 months and then they decreased to 30 months. The pattern of these changes correlated with functional vasomotor data. We conclude that aging (newborn to senescence) has substantial effects on Ang II-induced vasomotor responses and AT1R signaling suggesting the importance of genetic programs.

Asymmetric dimethylarginine reduces nitric oxide donor-mediated dilation of arterioles by activating the vascular renin-angiotensin system and reactive oxygen species.

INTRODUCTION: We tested the hypothesis that asymmetric dimethylarginine (ADMA) interferes with other mechanisms in addition to inhibition of nitric oxide synthase (NOS). Thus, in skeletal muscle arterioles, in the presence of ADMA, we investigated the dilator effect of an NO donor and increases in flow and aimed to elucidate the underlying mechanisms, including the role of oxidative stress, which is known to reduce the bioavailability of NO. METHODS AND RESULTS: In isolated rat gracilis skeletal muscle arterioles (∼160 µm at 80 mm Hg), ADMA (similarly to pyrogallol) reduced dilations to sodium nitroprusside (SNP), which was significantly prevented by the presence of superoxide dismutase (SOD) and catalase (CAT): SNP 10(-8)M; control: 43.2 ± 3%, ADMA: 4.9 ± 1%, ADMA + SOD/CAT: 30.2 ± 9% (p < 0.05). Also, ADMA reduced basal diameter and flow-induced dilations, which were not restored by L-arginine, but prevented by SOD/CAT and by inhibition of NAD(P)H oxidase (but not xanthine oxidase) and by an angiotensin-converting enzyme inhibitor or an angiotensin type 1 receptor blocker (ARB). ADMA increased the production of reactive oxygen species detected by lucigenin-enhanced chemiluminescence, which was significantly inhibited by SNP or ARB. CONCLUSION: We suggest that by activating the vascular renin-angiotensin-NAD(P)H oxidase pathway, ADMA elicits oxidative stress, which interferes with the bioavailability of NO and consequently reduces NO-mediated dilations.

Effects of vasoactive substances on the neurovascular structures and microcirculation in the developing callus 10 and 15 days after bone injury.

The developing callus requires sufficient oxygen and substrate supply. Despite the importance of these processes, we have limited understanding of regulation of the callus microcirculation. We aimed to assess the role of vasoactive substances in the microcirculation of the callus in a gap osteotomy model in the rabbit detected by laser-Doppler flowmetry. The reactions were elicited with locally applied vasoactive substances: epinephrine (E), calcitonine-gene related protein (CGRP), substance P (SP), sodium nitroprusside (SNP) and Ebrantil (Ebr) on the 10th and 15th postoperative days. Changes of the circulatory parameters were compared to changes in the ipsilateral femoral bone marrow. Perfusion pressure, maximal change of the blood flow and 50% recovery time (50RT) of the flow reactions and peripheral micro vascular resistance (MVR) was calculated. Systemic blood pressure (BP) was measured directly with an arterial catheter. Reactive neurovascular structures, sensitive to neuropeptides and vasoactive substances, appear at a very early stage of callus formation. On the 10th postoperative day, 2/3 of the blood flow velocity of the intact tibia has already returned, and the values are higher on the 15th postoperative day than those of the intact tibia. The basal blood flow velocities (prior to administration of any substance) are significantly higher on the 15th postoperative day than on the 10th.

Calcitonin gene-related peptide, substance P, nitric oxide and epinephrine modulate bone marrow micro circulation of the rabbit tibia and femur.

Different bones have different blood supplies, which may influence bone healing. Therefore, elucidation of the mechanisms involved in the regulation of bone marrow blood flow in different bones is of high clinical importance. To assess the micro circulation of bone marrow of the femur and tibia simultaneously, flow velocities were continuously measured by a two-channel laser-Doppler flowmeter. The probes were introduced into the femoral and tibial diaphysis, respectively, in the anesthetized rabbit. Changes in micro circulation of the bone marrow were elicited by intra-arterial bolus injections of vasoactive substances: epinephrine (E), calcitonine-gene related peptide (CGRP), substance P (SP), sodium nitroprusside (SNP), E and Ebrantil. Systemic arterial blood pressure was recorded with an electro-manometer. Micro vascular resistance (MVR) and 50% recovery time (50RT) to baseline flow level were calculated from the measured data. Flow velocity in the femur was significantly higher. Epinephrine considerably reduced micro vascular blood flow, which could be significantly warded off by Ebrantil. CGRP and SP did not change MVR. Application of SNP resulted in reduction of flow velocity, but it also decreased MVR. No statistically significant differences were found between reactions of the micro circulation in the two marrows. These results suggest that there are no significant differences between the blood flow response patterns of these two bone marrow sites, thus the regulation patterns of the micro circulation of the two bones are also similar.

Effects of neuropeptides and vasoactive substances on microcirculation of the callus after tibial osteotomy in rabbits.

Previous studies have demonstrated a dynamic ingrowth of vessels into the developing callus. In this study, maturation and development of the regulation of microcirculation were followed in the callus of rabbits. In the first series, the effects of vasoactive substances on blood flow velocity, perfusion pressure, duration of effects and peripheral vascular resistance of the bone marrow in the femur and tibia were compared. In the second series, the same parameters were measured in the femur and in the developing callus 10 and 15 days following gap osteotomy of the tibia. There were no significant differences between the microcirculatory reactions of the intact femur and tibia. Basal blood flow could be verified in the callus on the 10th postoperative day. No vascular reactions could be elicited. Basal blood flow velocity was higher on the 15th day, when compared to the measurements on the 10th day. The substances elicited statistically significant differences in flow velocity, resistance and 50% recovery time in the callus on the 15th day. Blood flow reactions of the ipsilateral femoral and tibial bone marrow are identical, thus the femur can serve as a reference site for blood flow measurements in the callus. Regulation and maturation of callus microcirculation develop rapidly between the 10th and 15th days.

An easy-to-use practical method to measure coincidence in the flow cytometer--the case of platelet-granulocyte complex determination.

Cell complexes composed of two different cells labeled with different fluorophores can be detected as double positive events in the flow cytometer. Double positivity can originate not only from real complexes but from non-interacting coinciding cells as well. Coincidence has a high impact on the determination of the amount of platelet-granulocyte complexes since platelet concentration is in the orders of magnitude higher than that of the granulocytes. Mixtures of non-interacting fluorescent beads as well as EDTA anticoagulated or citrated blood samples were analyzed in the flow cytometer in the presence and absence of fluorescent beads at various dilutions. Experimental data were evaluated by mathematical means. The bead or platelet concentration dependence of double positivity was converted into linear functions using Poisson distribution. This linearised form contains information on the detection volume as well as on the presence/absence of dilution independent complexes. The presence of appropriate fluorescent beads in the blood sample makes possible to estimate the fraction of double positivity originating from coincidence if data collection is triggered by the granulocytes or by the fluorescent beads, alternatively. Mixing fluorescent beads into a blood sample is a simple experimental method to distinguish double positivity originating from real cell-cell complexes from the coincidence of cells in a flow cytometer, thus providing a tool for the determination of the real amount of cell-cell complexes.

Impact of coincidence on granulocyte-platelet complex determination by flow cytometry is evaluated by a novel computer simulation model of coincidence.

Cell complexes composed of two different cells labeled with different fluorophores can be detected as double positive events in the flow cytometer. Double positivity can originate not only from real complexes but from non-interacting coinciding cells as well. Coincidence has a high impact on the determination of the amount of platelet-granulocyte complexes since platelet concentration is in the orders of magnitude higher than that of the granulocytes. A computer model has been developed to simulate coincidence in the flow cytometer to reveal the contribution of coincidence to the overestimation of the total amount of platelet-granulocyte complexes. Mixtures of non-interacting fluorescent beads as well as EDTA-anticoagulated blood samples were analyzed in the flow cytometer. An excellent fit was found between computer simulated and measured data pairs. Bead mixture in the flow cytometer and simulation of that resulted in 37.3+/-1.3 and 35.7+/-0.6% double positivity, respectively. 30.2+/-4.3% double positivity was measured for EDTA-anticoagulated blood samples while simulation of that resulted in 28.3+/-0.6%. Double positivity attributed to platelet-granulocyte complexes in slightly diluted blood samples might originate in coincidence and not from true complexes.

Early systemic effects of hind limb ischemia-reperfusion on hemodynamics and acid-base balance in the rat.

We investigated the systemic hemodynamic effects and the early arteriovenous acid-base changes after 2-h tourniquet ischemia on left hind limb in rats during the first hour of reperfusion. The right femoral artery and vein were prepared and catheterized for direct blood pressure monitoring and blood sampling. In ischemia-reperfusion group, 5 min before releasing the tourniquet and during the first hour of the reperfusion (5', 10', 15', 30', 45', and 60'), arterial and venous blood samples were taken in parallel with a sham operated control group. In the ischemia-reperfusion group venous pH continuously decreased during reperfusion and was significantly lower compared to control and base in the 60th min, while arterial pH remained almost unchanged. PCO2 and pO2 showed moderate signs of a parallel respiratory compensation. Mean arterial pressure decreased almost by 20%, heart rate slightly increased during reperfusion. Our data indicates that besides the general effects anesthesia, limb ischemia-reperfusion results in hemodynamic and acid-base changes during the first hour of reperfusion.

Prolonged intestinal mucosal acidosis is associated with multiple organ failure in human acute pancreatitis: gastric tonometry revisited.

AIM: To evaluate whether multiple determinations of intramucosal pH (pHi) in acute pancreatitis (AP) patients could provide additional information of the disease severity during early hospitalization. METHODS: Twenty-one patients suffering from acute pancreatitis were monitored by gastric tonometry in the first 72 h after hospital admission. RESULTS: In the survivor group (n = 15) the initially low pHi values returned to normal level (pHi > or = 7.32) within 48 h (median pHi: d 1: 7.21; d 2: 7.32; d 3: 7.33). In contrast, pHi values in the non-survivor group n = 6) were persistently either below or in the low normal range (median pHi 7.12; 7.12; 7.07 respectively), but pHi differences between the two groups reached significance only after 24 h (P<0.01). Mucosal acidosis detected at any time during the monitored period was associated with the emergence of single or multiple organ dysfunction (P<0.01). CONCLUSION: Prolonged gastric mucosal acidosis was associated with remote organ dysfunction and failure in Acute Pancreatitis, however, correlation with the fatal outcome became significant only 24 h after admission. Due to its non-invasive nature gastric tonometry may supplement the pro-inflammatory markers to achieve a multi-faceted monitoring of the disease.

Self-regulation of neutrophils during phagocytosis is modified after severe tissue injury.

Neutrophil (PMNL) function is influenced by factors released by other immune cells during the course of the immune response. We investigated the effect of neutrophil cell density and the effect of supernatant of the phagocytosis assay on the phagocytosis activity of PMNLs. The measurements were carried out with naive (control) PMNLs of healthy donors and with PMNLs obtained from patients with severe tissue injury. Phagocytosis index (FI) of PMNLs was determined at cell densities of 7.5 x 10(5)/ml and 15 x 10(5)/ml. E. coli phagocytosis of heparinized whole blood from healthy donors and patients with severe tissue injury was measured and evaluated at three different cell densities (normal, half, and double densities) by flow cytometry. Supernatants of phagocytosis assays of either control or trauma (ISS >18) patient PMNLs were added to the assay suspensions of control and trauma PMNLs. An increase in cell density of healthy donor PMNLs increased yeast phagocytic activity. In cases of tissue injury, PMNLs showed increased phagocytic activity at lower cell densities. E. coli phagocytosis was increased with the increase of cell density, and tissue injury PMNLs were more active at each cell concentration compared to naive cells. Polytrauma supernatants in most cases inhibited, while healthy supernatants mostly increased the yeast phagocytosis of healthy and trauma PMNLs. These results reinforce the idea that primed PMNLs in the presence of microbial agents produce factor(s) which inhibit some of the cell's antimicrobial functions contributing to immune-dysfunction, while unprimed PMNLs produce factor(s) which facilitate antimicrobial countermeasure. These results also demonstrate that reduced phagocytosis of tissue injury primed PMNLs is not due to cytoskeletal changes but to the humoral environment.

Small-volume fluid resuscitation with hypertonic saline prevents inflammation but not mortality in a rat model of hemorrhagic shock.

Hemorrhage remains a primary cause of death in civilian and military trauma. Permissive hypotensive resuscitation is a possible approach to reduce bleeding in patients until they can be stabilized in an appropriate hospital setting. Small-volume resuscitation with hypertonic saline (HS) is of particular interest because it allows one to modulate the inflammatory response to hemorrhage and trauma. Here, we tested the utility of permissive hypotensive resuscitation with hypertonic fluids in a rat model of hemorrhagic shock. Animals were subjected to massive hemorrhage [mean arterial pressure (MAP) = 30 - 35 mmHg for 2 h until decompensation] and partially resuscitated with a bolus dose of 4 mL/kg of 7.5% NaCl (HS), hypertonic hydroxyl ethyl starch (HHES; hydroxyl ethyl starch + 7.5% NaCl), or normal saline (NS) followed by additional infusion of Ringer solution to maintain MAP at 40 to 45 mmHg for 40 min (hypotensive state). Finally, animals were fully resuscitated with Ringer solution and the heparinized shed blood. Hypotensive resuscitation with NS caused a significant increase in plasma interleukin (IL)-1beta, IL-6, IL-2, interferon gamma (IFNgamma), IL-10, and granulocyte-macrophage colony stimulating factor (GM-CSF). This increase was blocked by treatment with HS. HHES treatment significantly reduced the increase of IL-1beta and IL-2 but not that of the other cytokines studied. Despite the strong effects of HS and HHES on cytokine production, both treatments had little effect on plasma lactate, base excess (BE), white blood cell (WBC) count, myeloperoxidase (MPO) content, and the wet/dry weight ratio of the lungs. Moreover, on day 7 after shock, the survival rate in rats treated with HS was markedly, but not significantly, lower than that of NS-treated animals (47% vs. 63%, respectively). In summary, hypotensive resuscitation with hypertonic fluids reduces the inflammatory response but not lung tissue damage or mortality after severe hemorrhagic shock.

Capsaicin delays regeneration of the neuromuscular junction of rat extensor digitorum longus muscle after ischemia.

Trauma or the tourniquet used in orthopedic surgery is often associated with ischemia-reperfusion (I/R) injury with a consequent decrease of muscle power. To explore whether components of the neuromuscular junction (NMJ) are involved in this muscle dysfunction, NMJs were ultrastructurally characterized in the extensor digitorum longus muscle of rats at reperfusion times of 1, 24, 72, and 168 h after a 120-min arterial occlusion. Disorganization of the presynaptic membrane and mitochondrial injury was noted at 1 h, followed by fragmentation and partial engulfment of nerve terminals by Schwann cells at 24 and 72 h. The magnitude of degenerative changes declined at 168 h, suggesting the commencement of regeneration. The postsynaptic membrane remained intact throughout the whole period. In our previous study, deafferentation with pretreatment of the sciatic nerve with capsaicin, which reduces neurogenic inflammation and has a selective effect on nociceptive fibers, improved functional recovery of the muscle after I/R. The present results document a significantly delayed structural regeneration of the motor nerve terminals after combined capsaicin and I/R treatment. Since capsaicin treatment alone had no discernible effect on the structure of NMJs, the findings point to a possibly indirect effect of capsaicin on the motor nerves, which may predispose them to increased susceptibility unmasked only by a subsequent injury. The mismatch between the enhanced functional improvement of the muscle and delayed regeneration of the nerve after capsaicin pretreatment questions the efficient use of such deafferentation to protect the integrity of neuromuscular junctions in I/R injury.

[Experimental "functional amputate model" in ischemia-reperfusion]. / Kísérletes "funkcionális amputációs modell" lehetosége az ischaemiareperfusio tükrében.

Limb amputation and ischemia-reperfusion (I/R) after trauma is a serious challenge, though there are few laboratory parameters that are available to predict the prognosis. It is even less so when possible adverse effects of preventive cooling may also influence the clinical outcome. We performed an experimental model earlier on mongrel dogs to investigate the local and systemic effects of cooled and non-cooled limb I/R. In this paper we describe the model and summarize the informative laboratory results. In the warm I/R group the femoral vessels were separately clamped for 3 hours, while steel-loop tourniquet for 7 hours was performed under the femoral vessels around the thigh. After ischemia releasing the clamps provided reperfusion for 4 hours then the steel-loop was removed. In the cooled I/R group similar procedure was performed but with cooling by ice bags. Cooled and non-cooled sham-operated groups also were used. Before operations, during the reperfusion and for 5 days blood samples were collected then haematology and chemistry parameters were determined. Blood rheology and certain coagulation factors were significantly different between the cool and non-cool ischemia-reperfusion groups, furthermore, the changes were also significant compared to sham-operated animals, suggesting that these parameters may be useful in the prognosis.

Antiapoptotic effect of (-)-deprenyl in rat kidney after ischemia-reperfusion.

BACKGROUND: Since apoptosis of renal tubular cells is the basis of the damage caused by ischaemia-reperfusion, the antiapoptotic effect on kidney tubular epithelial cells of the monoamino oxidase-B (MAO-B) inhibitor (-) deprenyl (selegiline), known neuroprotective agent, with antiapoptotic properties, was studied in a rat model. MATERIAL/METHODS: Warm renal ischaemia was caused by clamping the left renal artery of rats for 30 minutes. With the start of reperfusion 0.015 mg/kg, 0.15 mg/kg and 1.5 mg/kg of (-)-deprenyl was injected simultaneously into the carotid artery of the animals, respectively. Five rats served as control, in which renal artery clamping was performed, but the rats were only treated with the solvent (physiological saline). After 6 hours of reperfusion the rats were exsanguinated and the kidneys were histologically examined. RESULTS: Severe tubular damage characterised by apoptosis was found in the kidneys of the untreated rats. Apoptosis was verified on the basis of morphological features, methylgreen-pyronin staining and TUNEL reaction. (-)-Deprenyl diminished dose-dependently the apoptotic damage, 0.15 mg/kg being the most effective dose. The same dose of (-)-Deprenyl is used in the therapy of human Parkinson's disease. CONCLUSIONS: Our findings suggest, that (-)-deprenyl might have an impact on decreasing renal injury also in case of human cadaveric renal transplantation.