Radiation-induced glomerular thrombus formation and nephropathy are not prevented by the ADP receptor antagonist clopidogrel.

PURPOSE: To assess the effects of kidney irradiation on glomerular adenosine diphosphatase (ADPase) activity and intraglomerular microthrombus formation, and their correlation to the development of renal functional impairment. METHODS AND MATERIALS: C3H/HenAf-nu(+) mice were given single-dose or fractionated kidney irradiations. Glomerular ADPase activity was measured using a cerium-based histochemical method. Microthrombus formation within the glomeruli was assessed by a semiquantitative immunohistochemical analysis of fibrinogen/fibrin deposits. Renal function was assessed by the [(51)Cr]EDTA retention assay. RESULTS: The ADPase activity was significantly reduced, to approximately 50% of pretreatment value, 4--40 weeks after 10--16 Gy single-dose irradiation and at 44 weeks after 20 x 2 Gy. No dose--effect relationship was found. An approximately fourfold increase in glomerular fibrinogen/fibrin staining was observed at 1 year after irradiation. This increase was not influenced by treating the mice with daily, oral clopidogrel, a platelet ADP receptor antagonist, which reduced platelet aggregation by more than 75%. Radiation-induced impairment of glomerular filtration was also not affected by the clopidogrel treatment. CONCLUSION: These data indicate that irradiation significantly reduced glomerular ADPase activity, which correlated with an increased glomerular fibrinogen/fibrin deposition. We were not able to reduce these prothrombotic changes, nor to protect against radiation nephropathy, by pharmacological intervention with an ADP-receptor antagonist.

Radiation nephropathy--the link between functional damage and vascular mediated inflammatory and thrombotic changes.

The extent of radiation-induced nephropathy, which develops progressively over periods of months to years after treatment, is strongly influenced by both total dose and dose per fraction. In this study we examined the relationship between the early expression of various thrombotic and inflammatory markers of endothelial cell (EC) damage in irradiated mouse kidneys and the subsequent development of nephropathy. Decreased levels of glomerular ADPase and increased levels of glomerular Vwf were seen from 4 or 20 weeks after irradiation, respectively. These pro-thrombotic changes were associated with increased fibrin/fibrinogen deposits, indicative of microthrombus formation, at later times. These events were, however, not sensitive to changes in total dose or dose per fraction, therefore they cannot be quantitatively linked to the development of radiation nephropathy. Increased leucocyte invasion of the renal cortex was also seen after irradiation; this was quantitatively dependent on both total dose and dose per fraction. Linear quadratic analysis of the leucocyte dose-response curves yielded an alpha/beta ratio of 7.7 Gy, which is significantly greater than the alpha/beta ratio or 2.7 Gy determined for nephropathy, indicating less fractionation sensitivity for the inflammatory response. We conclude that inflammatory changes contribute to, but do not entirely explain, radiation nephropathy. The role of thrombotic changes is less clear.

Influence of acetylsalicylic acid on development of radiation-induced nephropathy.

PURPOSE: Previous studies have demonstrated that long-term treatment with acetylsalicylic acid (ASA) can significantly reduce the renal functional impairment that develops after high doses of irradiation. The effect is hypothesized to be mediated by selective inhibition of thromboxane A2 synthesis and inhibition of platelet aggregation. The present study was undertaken to investigate this phenomenon further using more clinically relevant fractionated and re-irradiation schedules. METHODS AND MATERIALS: Groups of mice were given bilateral renal irradiation with a series of four or 20 daily fractions of X-rays, or 10 daily fractions with a single dose of re-irradiation (0-10 Gy) after 27 weeks. Half the mice received ASA in drinking water (2.4 g x l(-1)) from 1 week before the start of irradiation and continuously throughout the follow-up period. Renal function was assessed by clearance of [51Cr]EDTA, about every 4 weeks for up to 80 weeks after the start of treatment. Histological damage in representative groups of mice was also assessed. RESULTS: Oral administration of ASA caused inhibition of thromboxane A2 synthesis (to < 36% of controls) and a strong inhibition of platelet aggregation in whole mouse blood (ex vivo). Prolonged treatment with ASA also resulted in a small, non-significant reduction of radiation-induced renal functional damage. No reduction in histological damage was seen in the ASA treated mice. CONCLUSION: Long-term oral administration of ASA gave only a modest, non-significant reduction of renal radiation injury after clinically relevant fractionated irradiation schedules.

Long-term effects of total-body irradiation on the kidney of Rhesus monkeys.

PURPOSE: To investigate the long-term effects of total-body irradiation (TBI) on kidneys in non-human primates. METHODS AND MATERIALS: The kidneys of Rhesus monkeys were histologically examined at 6-8 years after TBI with low single doses of 4.5-8.5Gy or two fractions of 5.4Gy. The kidneys of age-matched non-irradiated monkeys served as controls. Irradiation was performed on adult monkeys aged about 3 years; 6-8 years later animals were sacrificed and the kidneys removed and processed for histology. A semi-quantitative scoring system was used to evaluate overall histological damage. Glomerular changes were also morphometrically analysed according to previously published criteria. In selected dose groups (pro)thrombotic and inflammatory changes were investigated by immunostaining cryosections with antibodies against von Willebrand factor (vWF), leukocytes and macrophages. RESULTS: Histological changes were generally mild and only seen in kidneys irradiated with doses higher than 7 Gy. Glomerular changes were characterized by increased mesangial matrix and capillary dilatation. Tubulo-interstitial changes included hypercellularity, fibrosis and mild tubular atrophy. The mean glomerular area expressing vWF protein in the irradiated kidneys was not different from that in the age-matched controls. Numbers of infiltrating leukocytes were not significantly different between irradiated kidneys and controls. However, slightly increased numbers of macrophages were present in the renal cortex after irradiation. CONCLUSIONS: Renal damage after TBI of Rhesus monkeys with single doses of 4.5-8.5 Gy or two fractions of 5.4 Gy was mild, even after follow-up times of 6-8 years.

Increased expression of glomerular von Willebrand factor after irradiation of the mouse kidney.

Ionizing irradiation has been shown to induce an increased release of von Willebrand factor (vWF) in human endothelial cells in vitro. The present study was undertaken to investigate whether an increase in expression of vWF also occurs in glomerular endothelial cells in vivo after irradiation of the kidney. Increased expression of vWF may initiate prothrombotic changes, and the resultant vascular damage could cause renal failure. The amount of adherent leukocytes in the renal cortex after irradiation was also quantified, since this may contribute to the histological changes that occur after irradiation. Changes in expression of glomerular vWF and in the amount of leukocytes were related to the development of impairment of renal function, as assessed with the [51Cr]EDTA retention assay. Mice were given bilateral irradiation (single dose of 16 Gy) or were sham-irradiated and were sacrificed at intervals of 1 day to 40 weeks after irradiation. Immunohistochemical analysis of kidney cryosections was performed using a polyclonal vWF antibody or monoclonal CD45 antibody (leukocyte common antigen). The amount of glomerular vWF staining and CD45 staining in the renal cortex (percentage surface coverage) was quantified using a computerized image analyzer. The mean glomerular vWF staining in the nonirradiated kidneys was 34.4 +/- 6.2% (mean +/- SEM, 10 weeks after sham treatment). After irradiation, the expression of glomerular vWF increased gradually from 10 weeks to 53.4 +/- 3.6% at 40 weeks. The total number of leukocytes in the renal cortex of nonirradiated mice at 10 weeks after sham treatment was low, with a mean area of 1.0 +/- 0.09%, whereas in the irradiated kidneys the relative tissue area covered by leukocytes increased to 7.6 +/- 2.1% at 40 weeks. These alterations preceded impairment of renal function. The extent to which these changes are causally related to impairment of function will be the subject of future study using specific antithrombotic and anti-inflammatory agents.

Effects of chemical sympathectomy on angiotensin II-induced neointimal growth in the balloon-injured rat carotid artery.

To investigate the role of the sympathetic nervous system in angiotensin II (AngII)-stimulated medial and neointimal smooth muscle cell (SMC) replication, we sympathectomized rats with 6-hydroxydopamine (6-OHDA) in which the left carotid artery was injured by a balloon catheter. Balloon injury is associated with a loss of specific [3H]-prazosin binding. AngII (250 ng/kg/min), infused 2 weeks after balloon injury of the rat left carotid artery, increased systolic blood pressure by approximately 70 mm Hg. There was no effect of 6-OHDA on this pressor response. AngII increased the cumulative 5-bromo-2'-deoxyuridine (BrdU) labeling fraction (LF) in the uninjured right carotid media and the injured left carotid neointima as compared to controls (5.7+/-1.6% vs. 0.4+/-0.1%, p<0.05; 10.6+/-0.9% vs. 5.0+/-0.8%, p<0.05, respectively). 6-OHDA decreased the AngII-induced increase in LF in the media of the uninjured right carotid artery (AngII/6-OHDA 0.9+/-0.2% vs. AngII 5.7+/-1.6%, p < 0.05). 6-OHDA did not decrease the AngII-induced increase in LF in both the injured left carotid media and neointima at 4 weeks after balloon injury. The effects of chemical sympathectomy were comparable with those obtained 12 weeks after balloon injury. Thus, the data show that the sympathetic nervous system mediates the AngII-induced increase in SMC DNA synthesis, but only in the uninjured carotid media. This indicates a differential regulation of AngII-induced SMC replication in injured and uninjured vessels.

Neointimal smooth muscle cell phenotype is important in its susceptibility to cytomegalovirus (CMV) infection: a study in rat.

OBJECTIVE: Recently, we have found that rat CMV (RCMV) infected smooth muscle cells (SMCs) in rat carotid arteries when administered 14 days after balloon injury. In the present study we investigated (1) the long term effects of CMV infection on neointimal cross-sectional area, and (2) whether the phenotype of the intimal SMCs influences their susceptibility to active CMV infection. METHODS: In the first part of the study, rats received RCMV intravenously, two weeks after balloon catheterisation of the left carotid artery and were sacrificed twenty weeks after catheterisation. Continuous BrdU infusion was performed by subcutaneously implanted osmotic pumps during the last two weeks of life. In the second part RCMV was administered eight weeks after catheterisation and rats were sacrificed two weeks later. Immunohistochemistry was used to detect viral antigens, to determine BrdU incorporation as well as the contents of alpha-actin, desmin and vimentin in the carotid arteries. Intima and media cross-sectional areas were determined using computerized morphometry. RESULTS AND CONCLUSIONS: RCMV infection did not induce any differences in intima or media cross-sectional areas of the injured carotid artery, nor in the extent of SMC proliferation as shown by BrdU incorporation, 20 weeks after balloon catheterisation. Eight weeks after balloon catheterisation, RCMV no longer infected neointimal SMCs. This non-responsiveness to RCMV was associated with "re-differentiation" of the eight weeks old neointima, compared with two weeks after catheterization, as shown by the contents of alpha-actin, desmin and vimentin. Our data suggest that intimal SMC phenotype determines its susceptibility to active RCMV infection in vivo. Since de-differentiation of neointimal SMCs is associated with enhanced proliferation of these cells it is stated that de-differentiation or proliferation is prerequisite for infection.

Chronic alpha 1-adrenoreceptor stimulation increases DNA synthesis in rat arterial wall. Modulation of responsiveness after vascular injury.

Despite indirect evidence from studies using adrenergic antagonists or sympathectomy, catecholamines have never been shown directly to stimulate vascular smooth muscle cell (SMC) DNA replication in vivo. We studied whether a chronic infusion of catecholamine stimulates SMC replication in vivo in both uninjured arteries and arteries with a neointima formed after vascular injury. Animals were killed after 2 weeks of continuous infusion of bromodeoxyuridine (to label replicating DNA) and either phenylephrine, norepinephrine, or vehicle solution, starting early (third week) or late (ninth week) after balloon injury to the left common carotid artery. In catecholamine-infused animals, the uninjured carotid artery or thoracic aorta showed a marked increase in cross-sectional area (> 25%) and frequency of cells undergoing DNA synthesis among medial SMCs (4- to 10-fold) and endothelial cells (13-fold). With catecholamine infusion at 9 to 10 weeks after injury, the media or neointima of the injured carotid artery showed a smaller increase in SMC DNA replication (< or = 4-fold) than did the normal arterial media. In contrast, catecholamine infusion at 3 to 4 weeks did not cause significant SMC growth in the injured vessel. Catecholamine infusion caused labile elevations of systolic blood pressure. Taken together with our previous observation that alpha 1-blockers suppress arterial SMC replication without preventing severe hypertension in the rat, the present data strongly suggest that alpha 1-adrenoreceptors stimulate SMC DNA synthesis in vivo in arteries with or without intimal thickening, although not during the first weeks after balloon injury. The stimulation of DNA synthesis in vascular cells via the alpha 1-adrenoreceptor pathway may contribute to the vascular remodeling that occurs in hypertension and atherosclerosis.

Angiotensin II-induced progression of neointimal thickening in the balloon-injured rat carotid artery is AT1 receptor mediated.

To investigate the relative importance of AT1 and AT2 receptors in angiotensin II (Ang II)-induced restimulation of neointimal smooth muscle cell (SMC) DNA synthesis and increased neointimal cross-sectional area (CSA), male Wistar rats were subcutaneously infused for 2 weeks with Ang II and losartan, an AT1 receptor antagonist, or Ang II and PD123319, an AT2 receptor antagonist, during the third and fourth week after balloon injury of the left common carotid artery. Concomitantly, all rats received 5-bromo-2'-deoxyuridine to label DNA-synthesizing SMCs. Neointimal CSAs and SMC DNA synthesis were compared with control groups that received Ang II, 0.9% NaCl, losartan, or PD123319. Systolic blood pressure (SBP) was measured at different times during the infusion. Ang II induced an increase in SBP that was significantly different from the SBP in the NaCl group. Infusion of Ang II together with losartan reduced the Ang II-induced increase in SBP to levels comparable with those obtained in the NaCl group. Infusion of Ang II+PD123319 caused an increase in SBP that was comparable with the increase in SBP of the Ang II group and significantly different from the SBP of the NaCl group. Infusion of losartan or PD123319 alone did not affect SBP. Ang II significantly enhanced neointimal CSA (47%, P < .05) compared with the control group infused with NaCl. Losartan significantly reduced Ang II-induced neointimal thickening (neointimal CSA, -37%, P < .05). Infusion of PD123319 together with Ang II did not affect Ang II-induced neointimal thickening. Losartan or PD123319 alone did not reduce neointimal thickening, since the neointimal CSAs in these groups did not differ from the neointimal CSA of the NaCl group. Comparable effects were found for SMC DNA synthesis in the neointima. Ang II infusion increased neointimal SMC DNA synthesis. Addition of losartan reduced the fraction of DNA-synthesizing neointimal SMCs from 23.7 +/- 2.1% in the Ang II group to 12.8 +/- 1.8% in the Ang II+losartan group, whereas the labeling fraction in the neointima remained 26.6 +/- 3.1% in the Ang II+PD123319 group. The labeling fractions in the neointimas of the groups that received losartan or PD123319 alone did not differ from the labeling fraction in the NaCl group. These data indicate that AT1 but not AT2 receptors mediate the progression of neointimal thickening induced by delayed application of Ang II in the injured left carotid artery in the rat. Furthermore, these data suggest that AT1 and AT2 receptors are not involved in the regulation of normal growth of a neointima in the third and fourth week after balloon injury.

Doxazosin blocks the angiotensin II-induced smooth muscle cell DNA synthesis in the media, but not in the neointima of the rat carotid artery after balloon injury.

OBJECTIVE: Infusion of angiotensin II (AngII) during the third and fourth week after balloon injury of the left common carotid artery of the rat induces smooth muscle cell (SMC) DNA synthesis. In this study we wanted to investigate whether alpha 1-adrenoreceptors are involved in AngII-induced SMC DNA synthesis in the neointima. METHODS: Adult male Wistar Kyoto rats were subcutaneously infused for 2 weeks with AngII and the alpha 1-adrenoreceptor antagonist doxazosin during the 3rd and the 4th week after balloon injury of the left common carotid artery. Control groups received AngII, 0.9% NaCl, AngII + 50% dimethylsulfoxide (DMSO, the solvent of doxazosin), doxazosin or 50% dimethylsulfoxide. Each rat received 5-bromo-2'-deoxyuridine in a separate osmotic minipump to label DNA-synthesizing SMC. Systolic blood pressures were measured in all groups. RESULTS: Angiotensin II caused an increase in systolic blood pressure, whereas addition of doxazosin did not affect the increase in SBP caused by AngII. In the media of the non-injured carotid artery, AngII increased SMC DNA synthesis, as the BrdUrd labeling fraction increased from 0.2 +/- 0.1% (mean +/- s.e.m.) in the NaCl group towards 3.4 +/- 0.6% in the AngII group. Coinfusion with doxazosin reduced the AngII-induced increase in BrdUrd labeling fraction from 3.2 +/- 0.8% in the AngII + DMSO group towards 0.6 +/- 0.2% in the AngII+doxazosin group. A similar effect of doxazosin was found in the media of the injured left carotid artery, in which coinfusion with doxazosin also reduced the BrdUrd labeling fraction from 2.6 +/- 0.8% in the AngII+DMSO group towards 0.3 +/- 0.1% in the AngII+doxazosin group. In the neointima of the injured left carotid artery, AngII increased the BrdUrd labeling fraction from 11.7 +/- 1.6% in the NaCl group towards 28.0 +/- 3.4% in the AngII group. Coinfusion with doxazosin did not influence the AngII-induced SMC DNA synthesis, since the BrdUrd labeling fraction in the neointima of the AngII+doxazosin group was 22.5 +/- 2.9%, whereas the neointimal BrdUrd labeling fraction in the AngII+DMSO group was 22.9 +/- 2.3%. Little effect was found on the medial cross-sectional area. The neointimal cross-sectional area was increased as a result of infusion of AngII (0.12 +/- 0.01 mm2 vs. 0.18 +/- 0.01 mm2), and coinfusion of doxazosin did not reduce the AngII-induced increase in neointimal cross-sectional area (0.18 +/- 0.03 mm2). CONCLUSIONS: These data suggest that alpha 1-adrenoreceptors are not involved in AngII-induced neointimal SMC DNA synthesis and cross-sectional area, but only play a role in the media of the carotid artery.

Mesenteric small artery changes after vasoconstrictor infusion in young rats.

We evaluated whether chronic alpha 1-adrenergic stimulation, angiotensin II (AII), or increased blood pressure (BP) alters resistance arterial structure and function. Structural parameters and wall tension were recorded in mesenteric small arteries (MrA) isolated from 6-week-old normotensive Wistar Kyoto rats that had been infused for 4 days with saline (WKY), 2 mg/kg/day phenylephrine (WKY + PHE), or 0.3 mg/kg/day AII (WKY + AII) and from saline-infused spontaneously hypertensive rats (SHR). During the experimental period, systolic BP (SBP) did not change in WKY but increased in WKY + PHE, WKY + AII, and SHR. Relative cardiac mass did not differ between SHR and WKY, but was increased in WKY + PHE and WKY + AII. Stiffness and optimal lumen diameter of MrA did not differ between WKY and SHR and were not altered in WKY + PHE or WKY + AII. Maximal contractile responses and sensitivities for vasconstrictors and calcium in vessels of WKY + AII and SHR did not differ from those in WKY. In vessels of WKY + PHE, maximal responses to vasoconstrictors and sensitivities for norepinephrine (NE) and PHE were reduced. Relaxing responses to isoproterenol (ISO) and Na-nitroprusside did not differ between SHR and WKY and were not altered in WKY + PHE and WKY + AII. Those to acetylcholine (ACh) were reduced in WKY + PHE. Media cross-sectional area and media thickness were significantly larger in WKY + AII and SHR as compared with WKY but were not altered in WKY + PHE. These data indicate that in young rats AII leads to small artery hypertrophy and that neither increased BP or increased vasconstriction appear to be involved therein. Chronic alpha 1-adrenergic stimulation, on the other hand, did not modify small artery structure but resulted in nonselective reduction of arterial smooth muscle contractile reactivity.

Alpha 1-adrenoreceptor blockade reduces the angiotensin II-induced vascular smooth muscle cell DNA synthesis in the rat thoracic aorta and carotid artery.

We explored effects of alpha 1-adrenoreceptor blockade with prazosin on the increased vascular smooth muscle cell (SMC) DNA synthesis induced by angiotensin II (Ang II) in rats. Ang II was infused with or without prazosin or its solvent. Observations were compared with those in rats receiving saline or solvent. In group A, Ang II was infused for 2 weeks by subcutaneously implanted osmotic minipumps at a rate of 35 ng/100 g per minute. Group B received Ang II together with the alpha 1-adrenoreceptor antagonist prazosin (0.35 micrograms/100 g per minute). Group C received Ang II and 50% dimethyl sulfoxide (DMSO), the solvent of prazosin; group D received 50% DMSO; and group E received 0.9% NaCl (Ang II vehicle). All animals were infused with 5-bromo-2'-deoxyuridine for 2 weeks via separate minipumps to measure DNA synthesis. Ang II significantly increased the fraction of DNA synthesizing SMCs in the media of the thoracic aorta from 0.4 +/- 0.1% (mean +/- SD) in group E (n = 6) to 10.8 +/- 7.0% in group A (n = 8). Addition of prazosin to Ang II reduced the labeling fraction of SMCs to 3.0 +/- 2.2% (group B, n = 9). The remaining SMC DNA synthesis in the prazosin-treated group was probably due to the effects of the solvent of prazosin, i.e., 50% DMSO, since infusion of 50% DMSO alone increased the labeling fraction to 4.1 +/- 2.0% (group D, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

A comparative study on the metabolic activation of 3,4-benzo[a]pyrene to mutagens by aorta smooth muscle cells of rat and rabbit.

The mutagenic activation of benzo[a]pyrene (BaP) after exposure to aorta smooth muscle cells of different origin was examined. Three test systems with different genetic endpoints--sister-chromatid exchange (SCE), gene mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus and unscheduled DNA synthesis (UDS)--were used. Treatment of rat and rabbit aorta smooth muscle cells with BaP (1-6 micrograms/ml) resulted in a significant increase of SCEs, HGPRT mutations and UDS. So smooth muscle cells are capable of converting BaP to metabolites with a DNA-damaging action. In order to investigate the relation between the formation of mutagenic BaP metabolites and the susceptibility to atherosclerosis we compared the mutagenic potential of BaP using aorta smooth muscle cells of different species (rat, rabbit) and locations (thoracic and abdominal aorta). Rabbits and abdominal aortas are more susceptible to atherosclerosis than rats and thoracic aortas. The SCE, HGPRT and UDS assays revealed that smooth muscle cells of different origin possessed the same metabolic potential towards BaP. There was no correlation between the mutagenic potency of BaP metabolites and the susceptibility to atherosclerosis. As smooth muscle cells have a low metabolic capacity towards BaP, probably other factors in addition to the metabolic capacity of smooth muscle cells are responsible for species and tissue differences in susceptibility to atherosclerosis.