Aging Accelerates Endotoxin-Induced Thrombosis
2002; Elsevier BV; Volume: 161; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)64457-4
ISSN1525-2191
AutoresKoji Yamamoto, T Shimokawa, Hong Yi, Ken‐ichi Isobe, Tetsuhito Kojima, David J. Loskutoff, Hidehiko Saito,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoAlthough older subjects are susceptible to thrombosis under septic conditions, the underlying molecular mechanisms have not been fully elucidated. Since elevated plasminogen activator inhibitor-1 (PAI-1) primarily contributes to endotoxin-induced thrombosis, we first compared the induction of PAI-1 by lipopolysaccharide (LPS) between young and aged mice. The higher induction of PAI-1 antigen and mRNA with increased renal glomerular fibrin deposition was observed in LPS-treated aged mice compared to young mice. In situ hybridization analysis showed that the aging-associated induction of PAI-1 mRNA by LPS was pronounced in hepatocytes and in renal glomerular cells. The increased magnitude of the response of aged mice to lower doses of LPS was observed in terms of renal glomerular fibrin deposition and PAI-1 mRNA induction in the tissues. Furthermore, older PAI-1 deficient mice treated with LPS developed much less fibrin deposition in kidneys. Importantly, a larger induction of receptor molecules for LPS (eg, CD14 and Toll-like receptor 4) was demonstrated in LPS-treated aged mice as compared with young mice. The enhanced LPS signaling in aged mice was also demonstrated by the marked induction of nuclear factor-κB in the tissues after endotoxin treatment. As a consequence, increases in an inflammatory cytokine, tumor necrosis factor-α, were pronounced in plasma and tissues of LPS-treated aged mice. These results emphasize the key role played by PAI-1 in aging-associated deterioration in this thrombosis model, and suggest that the hyperresponse of PAI-1 gene to LPS results from the enhanced LPS signaling and the subsequent inflammatory response in aged mice. Although older subjects are susceptible to thrombosis under septic conditions, the underlying molecular mechanisms have not been fully elucidated. Since elevated plasminogen activator inhibitor-1 (PAI-1) primarily contributes to endotoxin-induced thrombosis, we first compared the induction of PAI-1 by lipopolysaccharide (LPS) between young and aged mice. The higher induction of PAI-1 antigen and mRNA with increased renal glomerular fibrin deposition was observed in LPS-treated aged mice compared to young mice. In situ hybridization analysis showed that the aging-associated induction of PAI-1 mRNA by LPS was pronounced in hepatocytes and in renal glomerular cells. The increased magnitude of the response of aged mice to lower doses of LPS was observed in terms of renal glomerular fibrin deposition and PAI-1 mRNA induction in the tissues. Furthermore, older PAI-1 deficient mice treated with LPS developed much less fibrin deposition in kidneys. Importantly, a larger induction of receptor molecules for LPS (eg, CD14 and Toll-like receptor 4) was demonstrated in LPS-treated aged mice as compared with young mice. The enhanced LPS signaling in aged mice was also demonstrated by the marked induction of nuclear factor-κB in the tissues after endotoxin treatment. As a consequence, increases in an inflammatory cytokine, tumor necrosis factor-α, were pronounced in plasma and tissues of LPS-treated aged mice. These results emphasize the key role played by PAI-1 in aging-associated deterioration in this thrombosis model, and suggest that the hyperresponse of PAI-1 gene to LPS results from the enhanced LPS signaling and the subsequent inflammatory response in aged mice. The increased incidence of thromboembolism in the elderly suggests that age-related changes occur in the vascular and hemostatic systems, including platelets, coagulation and fibrinolytic proteins, and endothelium. For example, the plasma levels of some major risk factors for thrombotic tendency (ie, fibrinogen and factor VII) have been shown to increase in subjects 60 years of age and older.1Balleisen L Bailey J Epping PH Schulte H van de Loo J Epidemiological study on factor VII, factor VIII and fibrinogen in an industrial population. I. Baseline data on the relation to age, gender, body-weight, smoking, alcohol, pill-using, and menopause.Thromb Haemost. 1985; 54: 475-479PubMed Google Scholar, 2Aillaud MF Pignol F Alessi MC Harle JR Escande M Mongin M Juhan-Vague I Increase in plasma concentration of plasminogen activator inhibitor, fibrinogen, von Willebrand factor, factor VIII: C and in erythrocyte sedimentation rate with age.Thromb Haemost. 1986; 55: 330-332PubMed Google Scholar Recently, the molecular mechanism of the age-associated increase in factor IX was investigated, and the age-responsive regulatory elements were identified in this gene.3Kurachi S Deyashiki Y Takeshita J Kurachi K Genetic mechanisms of age regulation of human blood coagulation factor IX.Science. 1999; 285: 739-743Crossref PubMed Scopus (50) Google Scholar The fibrinolytic system is impaired by aging since a progressive prolongation of the euglobulin lysis time4Abbate R Prisco D Rostagno C Boddi M Gensini GF Age-related changes in the hemostatic system.Int J Clin Lab Res. 1993; 23: 1-3Crossref PubMed Scopus (73) Google Scholar and an increase in plasminogen activator inhibitor-1 (PAI-1), a principal regulator of fibrinolysis,5Loskutoff DJ Sawdey M Mimuro J Type 1 plasminogen activator inhibitor.in: Coller B Progress in Hemostasis and Thrombosis. WB Saunders Company, Philadelphia1988: 87-115Google Scholar have been observed with aging.2Aillaud MF Pignol F Alessi MC Harle JR Escande M Mongin M Juhan-Vague I Increase in plasma concentration of plasminogen activator inhibitor, fibrinogen, von Willebrand factor, factor VIII: C and in erythrocyte sedimentation rate with age.Thromb Haemost. 1986; 55: 330-332PubMed Google Scholar, 6Hashimoto Y Kobayashi A Yamazaki N Sugawara Y Takada Y Takada A Relationship between age and plasma t-PA, PA-inhibitor, and PA activity.Thromb Res. 1987; 46: 625-633Abstract Full Text PDF PubMed Scopus (89) Google Scholar Clinically, elevated levels of plasma PAI-1 are observed in a variety of thrombotic conditions,7Yamamoto K Saito H A pathological role of increased expression of plasminogen activator inhibitor-1 in human or animal disorders.Int J Hematol. 1998; 68: 371-385Crossref PubMed Google Scholar including myocardial infarction,8Hamsten A Wiman B de Faire U Blomback M Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction.N Engl J Med. 1985; 313: 1557-1563Crossref PubMed Scopus (1236) Google Scholar deep vein thrombosis,9Wilman B Hamsten A The fibrinolytic enzyme system and its role in the etiology of thrombo-embolic disease.Semin Thromb Haemost. 1990; 26: 207-216Crossref Scopus (156) Google Scholar and disseminated intravascular coagulation.10Pralong G Calandra T Glauser M-P Schellekens J Verhoef J Bachmann F Kruithof EKO Plasminogen activator inhibitor 1: a new prognostic marker in septic shock.Thromb Haemost. 1989; 61: 459-462PubMed Google Scholar The inappropriate expression of PAI-1 in the tissues may underlie the occurrence of local thrombotic events (eg, cerebral infarction and cardiovascular disease), which are frequently observed in the elderly. Sepsis caused by gram-negative bacteria is frequently associated with thrombotic complications and is characterized histologically by microvascular fibrin deposition in several organs,11Coalson JJ Pathology of sepsis, septic shock, and multiple organ failure.in: Perspective on Sepsis and Septic Shock. Anonymous Society of Critical Care Medicine, Fullerton1986: 27-54Google Scholar tissue necrosis, and vascular disruption. Endotoxin (lipopolysaccharide; LPS) profoundly alters the fibrinolytic system of both humans12Suffredini AF Harpel PC Parrillo JE Promotion and subsequent inhibition of plasminogen activation after administration of intravenous endotoxin to normal subjects.N Engl J Med. 1989; 320: 1165-1172Crossref PubMed Scopus (388) Google Scholar and experimental animals,13Quax PHA van den Hoogen CM Verheijen JH Padro T Zeheb R Gelehrter TD van Berkel TJC Kuiper J Emeis JJ Endotoxin induction of plasminogen activator and plasminogen activator inhibitor type 1 mRNA in rat tissues in vivo.J Biol Chem. 1990; 265: 15560-15563Abstract Full Text PDF PubMed Google Scholar frequently leading to a procoagulant state. Elderly individuals are susceptible to endotoxin-induced effects than the young,14Horan MA Hendriks HFJ Brouwer A Organisms under stress: infectious agents and host defences.in: Horan MA Brouwer A Gerontology: Approaches to Biomedical and Clinical Research. Edward Arnold, London1990: 105-134Google Scholar and aged rats demonstrate increased susceptibility to hemorrhage and intravascular hypercoagulation following endotoxin administration.15Carthew P Dorman BM Edwards RE Increased susceptibility of aged rats to haemorrhage and intravascular hypercoagulation following endotoxin administered in a generalized Shwartzman regime.J Comp Pathol. 1991; 105: 323-330Crossref PubMed Scopus (7) Google Scholar These LPS-mediated changes result in an increased mortality of aged rats as compared to young rats.15Carthew P Dorman BM Edwards RE Increased susceptibility of aged rats to haemorrhage and intravascular hypercoagulation following endotoxin administered in a generalized Shwartzman regime.J Comp Pathol. 1991; 105: 323-330Crossref PubMed Scopus (7) Google Scholar In these studies, a larger increase in PAI-1 activity and a more significant decrease in the total PA activity were demonstrated in plasma of aged rats treated with endotoxin in comparison with young rats.16Emeis JJ Brouwer A Barelds J Horan MA Durham SK Kooistra T On the fibrinolytic system in aged rats, and its reactivity to endotoxin and cytokines.Thromb Haemost. 1992; 67: 697-701PubMed Google Scholar These observations suggest that aged animals may tend to develop thrombosis due to the high anti-fibrinolytic potential in endotoxemia and in inflammatory processes. We previously reported that fibrin deposition in the tissues of LPS-treated mice correlated with changes in the local expression level of key procoagulant and fibrinolytic genes, including PAI-1.17Yamamoto K Loskutoff DJ Fibrin deposition in tissues from endotoxin-treated mice correlates with decreases in the expression of urokinase-type but not tissue-type plasminogen activator.J Clin Invest. 1996; 97: 2440-2451Crossref PubMed Scopus (184) Google Scholar In the current study, we treated young (8 weeks old) and aged (24 months old) mice with LPS, and analyzed renal fibrin deposition in association with the expression of PAI-1 gene. Renal glomerular fibrin deposition and renal PAI-1 gene expression were markedly induced and sustained in LPS-treated aged mice compared with young mice. This increased response of the aged mice to LPS in the PAI-1 induction, together with the observation that little fibrin was detected in LPS-treated PAI-1 deficient mice, suggests that PAI-1 contributes to the observed thrombotic tendency in aged mice of endotoxemia. Finally, we investigated the expression of CD14 and Toll-like receptor 4 (TLR4), two major receptor molecules for LPS, and of transcription factor, nuclear factor-κB (NF-κB), in LPS-treated young and aged mice since the response of PAI-1 gene to LPS may be dependent on the LPS recognition and signaling via these molecules. The expression of these molecules were also markedly induced by LPS in aged mice as compared with young mice, suggesting that a larger induction of PAI-1 and subsequent increased fibrin deposition results from the enhanced LPS signaling in these mice. Male C57BL/6J mice, 8 weeks and 24 months of age, were obtained from SLC Japan and through the National Institute of Aging. Mice were injected intraperitoneally (i.p.) either with 5 μg of LPS (0.2 mg/kg) (Escherichia coli serotype O111:B4; Sigma Chemical Co., St. Louis, MO) in saline (Baxter, Deerfield, IL), or with saline alone. At 2, 4, 8, 16, 24 hours after LPS injection, the mice were sacrificed by overdose inhalation anesthesia with methoxyflurane (Pitman-Moore, Mundelein, MD). The blood was collected into 20 mmol/L EDTA (final concentration), centrifuged at 3000 × g for 5 minutes, and then the plasma was removed and stored at −70°C. Tissues were rapidly removed by standard dissection techniques, and either minced and immediately frozen in liquid nitrogen for preparation of total RNA and protein extracts, or fixed in chilled (4°C) 4% paraformaldehyde and embedded in paraffin for in situ hybridization. In the latter experiments, tissues were rapidly perfused with cold PBS (15 minutes) through the left ventricle into the opened vena cava at an approximate rate of 600 μl/min using a peristaltic pump to wash out fibrinogen. The tissues were then perfused with chilled 4% paraformaldehyde for 15 minutes, and then removed surgically, fixed overnight in fresh 4% paraformaldehyde, and embedded in paraffin for fibrin immunohistochemistry. Kidneys and livers also were harvested at 2 and 4 hours after LPS injection and prepared for analysis by quantitative RT-PCR and fibrin immunohistochemistry using the same methods as above. To investigate the dose dependency of the response to LPS in young and aged mice, we injected i.p. 2-month-old and 24-month-old mice with increasing doses of LPS (ie, 0.05, 0.25, 1, and 5 μg), and analyzed them as above. In separate experiments, male or female PAI-1 deficient18Carmeliet P Stassen JM Schoonjans L Ream B van den Oord JJ de Mol M Mulligan RC Collen D Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis, and thrombolysis.J Clin Invest. 1993; 92: 2756-2760Crossref PubMed Scopus (350) Google Scholar and wild-type mice were obtained from Scripps Clinic Rodent Breeding Colony (these animals were originally provided by Drs. P. Carmeliet and D. Collen, University of Leuven, Belgium). They were injected i.p. with 5 μg of LPS as above, and 4 hours later, they were sacrificed for subsequent immunohistochemical analysis for fibrin as described below. Active PAI-1 antigen in plasma was determined by using the t-PA binding assay as previously described.19Schleef RR Sinha M Loskutoff DJ Immunoradiometric assay to measure the binding of a specific inhibitor to tissue-type plasminogen activator.J Lab Clin Med. 1985; 106: 408-415PubMed Google Scholar Active PAI-1 levels (ng/ml) were calculated from a standard curve constructed by using recombinant mouse PAI-1. Total tumor necrosis factor (TNF)-α antigen in plasma (pg/ml) was measured by ELISA-view kit (BioSource International, Inc., Camarillo, CA). We have developed a quantitative reverse transcription (RT)-PCR assay to determine the concentration of PAI-1 mRNA in murine tissues, as described previously.17Yamamoto K Loskutoff DJ Fibrin deposition in tissues from endotoxin-treated mice correlates with decreases in the expression of urokinase-type but not tissue-type plasminogen activator.J Clin Invest. 1996; 97: 2440-2451Crossref PubMed Scopus (184) Google Scholar Briefly, total tissue RNA was prepared using the Ultraspec RNA Isolation System (Biotech Laboratories, Inc., Houston, TX), and the integrity of the 18S/28S ribosomal RNA was monitored by electrophoresing 10 μg of total RNA through a 1.2% agarose/formaldehyde gel. The control RNA (cRNA), which contains a pair of primer sequences for PAI-1, was in vitro transcribed using the Riboprobe Gemini II (Promega, Madison, WI). Thereafter, 1 μg of total tissue RNA and a fixed amount of the cRNA, which function as the competitor for PAI-1 mRNA, were combined and reverse transcribed using a Gene Amp RNA PCR kit (Perkin-Elmer/Cetus, Norwalk, CT). The RT mixtures were then amplified using specific primers for PAI-1 in the presence of 32P-end-labeled sense primer (5 × 105 cpm). After PCR amplification of 30 to 32 cycles (95°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute), 20-μl aliquots of the PCR products were electrophoresed on a 1.5–2.5% agarose gel. The appropriate bands corresponding to the cRNA product (438 bp) and PAI-1 mRNA product (540 bp) were excised from the gel and the incorporated radioactivity in each was determined using a scintillation counter. The amounts of PAI-1 mRNA were determined by extrapolation using the cRNA standard curve. Variations in sample loading were assessed by measuring β-actin mRNA. A similar method using the cRNA competitor was used for the quantitation of CD14 mRNA levels in murine tissues. The sequences of primers for quantitation of mouse CD14 mRNA were as follows: sense, 5′-CCACCTTAGACCTGTCTGACAATCC-3′; antisense, 5′-CAGCCTGTTGTAACTGAGATCCAGC-3′.20Setoguchi M Nasu N Yoshida S Higuchi Y Akizuki S Yamamoto S Mouse and human CD14 (myeloid cell-specific leucine-rich glycoprotein) primary structure deduced from cDNA clones.Biochim Biophys Acta. 1989; 1008: 213-222Crossref PubMed Scopus (74) Google Scholar The band for the cRNA was 132 bp and that for CD14 mRNA was 331 bp. The expression of TLR4 mRNA in murine tissues was similarly analyzed by semiquantitative RT-PCR using primers as follows: sense, 5′-GGTCAAGGAACAGAAGCAGTTCTTG-3′; antisense, 5′-TCAAGGACAATGAAGATGACGCCAG-3′.21Qureshi ST Lariviere L Leveque G Clermont S Moore KJ Gros P Malo D Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4).J Exp Med. 1999; 189: 615-625Crossref PubMed Scopus (1363) Google Scholar The size of target band for mouse TLR4 mRNA was 540 bp. Each density of the RT-PCR band was measured by densitometry and the relative contents were calculated based on the density of control (0 hour) young liver as 1.0. All statistical analyses were performed with STATA ver.7 software (STATA Corp., College Station, TX). Comparison of all quantitative RT-PCR results between two age groups (8 weeks vs. 24 months) was performed with the two-sample t-test. Welch's method was applied when variance between two-group was unequal. One-way analysis of variance (analysis of variance) was applied to examine the difference among four LPS dosage groups (0.05, 0.25, 1.0, and 5.0 μg) within same age or sample groups. Considering the issue for multiple comparison, corrected P value, the obtained P value multiplied by the numbers of testing in the same analysis which may give a conservative estimation, was applied to define the statistical significance in t-test. Bonferroni's correction of P value for multiple comparison was applied for one-way analysis of variance. P < 0.05 was considered statistically significant. In situ hybridizations for PAI-1 mRNA were performed using riboprobes as described previously.22Keeton M Eguchi Y Sawdey M Ahn C Loskutoff DJ Cellular localization of type 1 plasminogen activator inhibitor mRNA and protein in murine renal tissue.Am J Pathol. 1993; 142: 59-70PubMed Google Scholar After hybridization, the slides were dehydrated by immersion in a graded alcohol series containing 0.3 mol/L NH4Ac, and dried. The slides were then coated with NTB2 emulsion (Kodak; 1:2 in water), and exposed in the dark at 4°C for 4 to 8 weeks. The slides were developed for 2 minutes in D19 developer (Kodak), fixed, washed in water, and counterstained with hematoxylin and eosin. No specific hybridization signal could be detected in parallel sections using 35S-labeled sense probes for nonspecific hybridization in each experiment (data not shown). Immunohistochemical staining was performed using the Histostain-SP Kit (Zymed Laboratories, South San Francisco, CA), as described previously.22Keeton M Eguchi Y Sawdey M Ahn C Loskutoff DJ Cellular localization of type 1 plasminogen activator inhibitor mRNA and protein in murine renal tissue.Am J Pathol. 1993; 142: 59-70PubMed Google Scholar Briefly, the tissue sections were deparaffinized, treated with 2% hydrogen peroxide, and incubated with 10% normal goat serum for 30 minutes. The slides were then incubated with 10 μg/ml of rabbit anti-mouse fibrinogen/fibrin antibody (a kind gift of Dr. E. Plow, Cleveland Clinic), containing 0.1% goat serum at 4°C overnight, followed by incubation for 1 hour at 25°C. In control experiments, tissues were incubated with preimmune (normal) rabbit IgG instead of the primary antibody. The slides were then washed and treated sequentially with biotinylated goat anti-rabbit IgG (Zymed), streptavidin-peroxidase conjugate (Zymed) and aminoethylcarbazole chromogen containing 0.03% hydrogen peroxide (Zymed). After rinsing in distilled water, the slides were counterstained with Gill's modified hematoxylin, rinsed well with tap water, and mounted in GVA-mount (Zymed). The specificity of the antibody for fibrin in the extensively perfused tissues was indicated by the absence of staining in all tissues from control mice (not shown; 17). Quantitative evaluation of fibrin was achieved by counting the number of fibrin-positive glomeruli in each kidney section (magnification, ×400) in a blinded fashion. The data are shown as an average of three mice in each group. Resident peritoneal exudative cells of LPS-treated young and aged mice were collected by peritoneal lavage with cold PBS(-). After washing with RPMI (ICN Biomedicals, Aurora, OH)-HEPES (10 mmol/L, pH 7.4), cells were plated and incubated for 2 hours at 37°C in 5% CO2. Non-adherent cells were removed by washing the plates with RPMI-HEPES twice and lysed in buffer consisting of 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% Triton X-100, 50 mmol/L iodoacetamide, 1 mmol/L PMSF, 10 μg/ml soybean trypsin inhibitor, 2 mmol/L MgCl2, and 2 mmol/L CaCl2. Thus, whole cell lysates of peritoneal macrophages were collected, and then, TLR4 antigen in the lysates was analyzed by SDS-PAGE and immunoblotting. Briefly, each 2 μg of the lysates were electrophoresed under non-reduced conditions on a 10% SDS-PAGE and transferred to PDVF membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were soaked in TBS (20 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl) containing 5% nonfat milk and 0.1% Tween-20 for 2 hours at room temperature to block additional protein binding sites, and washed three times (15 minutes/wash) in TBS containing 0.1% Tween-20 (TBS-T). The membranes were then incubated with an MTS510 mAb,23Akashi S Shimazu R Ogata H Nagai Y Takeda K Kimoto M Miyake K Cell surface expression and lipopolysaccharide signaling via the Toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages.J Immunol. 2000; 164: 3471-3475PubMed Google Scholar a rat antiserum specific for mouse TLR4 associated with MD-2 (1.6 mg/ml, 1:1000 dilution in TBS-T; kindly provided by Drs. S. Akashi and K. Miyake, The University of Tokyo, The Institute of Medical Science, Division of Infectious Genetics, Tokyo, Japan), washed 4 times in TBS-T, and incubated for 2 hours with biotin-labeled goat anti-rat antibody (KPL, Gaithersburg, MD). After three washes in TBS-T, the membranes were incubated for 1 hour at 37°C with peroxidase-labeled streptavidin (0.2 μg/ml; KPL) and developed with the enhanced chemiluminescent (ECL) detection system (Amersham International, Buckinghamshire, UK) according to manufacturers' instructions. In separate experiments, the nuclear extracts were prepared from the livers of LPS-treated (2 hours, 4 hours) young and aged mice, as follows. The frozen liver tissues were homogenized in chilled hypotonic buffer (10 mmol/L HEPES pH 7.9, 10 mmol/L KCl, 0.5 mmol/L DTT, 0.5 mmol/L PMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin), and pelleted by centrifugation at 12000 rpm for 10 minutes at 4°C. The pellets were resuspended in high-salt buffer (20 mmol/L HEPES pH 7.9, 25% glycerol, 0.5 mmol/L DTT, 0.2 mmol/L EDTA, 0.42 mol/L NaCl, 0.5 mmol/L PMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin) and the nuclear proteins were extracted by incubation for 30 minutes on ice. The nuclear debris was pelleted by centrifugation at 12,000 rpm for 20 minutes at 4°C. Each 20 μg of the nuclear proteins was electrophoresed on a 10% SDS-PAGE, and then, the blot was prepared in the same way as above. The expression of NF-κB in the nuclear proteins was analyzed by using rabbit anti-NF-κB antibody (Cell Signaling Technology, Inc., Beverly, MA; 1:2000 dilution in TBS-T), which detects p50 and p65 of the mouse NF-κB. The blot was incubated with this specific antibody, followed by the incubation with biotin-labeled goat anti-rabbit antibody (KPL) and by the detection with ECL as described above. Figure 1 shows the time course changes in plasma PAI-1 antigen and PAI-1 mRNA in livers and kidneys of young and aged mice after i.p. administration of 5 μg of LPS. The aged mice had significantly higher active PAI-1 antigen in their plasma in the basal state. For example, 8-week-old mice had 3.66 ± 0.88 ng/ml of PAI-1 antigen in plasma, while 24-month-old mice contained 11.9 ± 3.72 ng/ml, respectively. On LPS treatment, the magnitude of the induction of plasma PAI-1 was much greater in aged mice than young mice (Figure 1, left). Active PAI-1 antigen in plasma of 24-month-old mice continued increasing for at least 8 hours after LPS treatment, and reached levels that were threefold higher than those in 8-week-old mice. A similar difference was observed at the mRNA level in livers, with a four- to fivefold greater induction in PAI-1 mRNA at 8 hours in the 24-month-old mice compared with 8-week-old mice (Figure 1, center). Again, the maximal induction of PAI-1 mRNA in the liver of 24-month-old mice occurred at later time point (ie, at 8 hours after LPS). In kidneys of both groups, the maximal induction of PAI-1 mRNA was observed at 2 hours after LPS, but again, the induction was dramatic in aged mice compared with young mice (Figure 1, right). The differences of PAI-1 expression levels in each time point according to each tissue remained significant even when corrected P values were applied. Although PAI-1 mRNA was induced severalfold in the heart and lung, the level of induction did not significantly differ in the young versus aged mice (data not shown). Meanwhile, endotoxin caused a two- to threefold increase in t-PA mRNA and a 50 to 60% decrease in u-PA mRNA in kidneys (not shown), in agreement with previous studies.17Yamamoto K Loskutoff DJ Fibrin deposition in tissues from endotoxin-treated mice correlates with decreases in the expression of urokinase-type but not tissue-type plasminogen activator.J Clin Invest. 1996; 97: 2440-2451Crossref PubMed Scopus (184) Google Scholar However, the magnitude of these changes in t-PA and u-PA mRNA expression did not significantly differ between young and aged mice. Thus, the clearance of endotoxin-induced glomerular fibrin may be impaired in aged mice due to larger induction of systemic and regional PAI-1 expression. High resolution in situ hybridization analysis for PAI-1 mRNA demonstrated that the same cell populations in livers and kidneys responded to endotoxin between young and aged mice, even though the magnitude of induction is much greater in the older animals than in young mice. For example, PAI-1 mRNA was induced in hepatocytes and in sinusoidal endothelial cells in livers of young (Figure 2A) and aged (Figure 2B) mice, but the magnitude of induction was considerably higher in the older animals. Similar results were obtained in the kidneys where hybridization signals for PAI-1 mRNA were markedly increased in glomerular cells in aged mice (Figure 2D) compared with those in young mice (Figure 2C). Young (8 weeks) and aged (24 months) mice were injected i.p. with 5 μg of LPS, and then sacrificed at various times for subsequent analysis. In these experiments, 2 out of 11 of the older mice died within 8 hours after LPS injection. In contrast, none of young mice (0 out of 9) had died at this time, and in fact, they were all still alive 24 hours after endotoxin treatment. This significant difference (P < 0.05) in survival suggests that aged mice are more susceptible to endotoxin than young mice. Quantitative evaluation of renal glomerular fibrin deposition in the young and aged mice after LPS administration was achieved by performing fibrin immunohistochemistry and then counting positive glomeruli as described in Materials and Methods (Figure 3). Fibrin deposition at 2 and 4 hours after LPS was observed in 15 to 18% of the glomeruli in kidneys of young mice. In contrast, fibrin deposits were detected in approximately 30% of the glomeruli of aged mice at 2 hours, and by 4 hours, fibrin was detected in more than 50% of their glomeruli. More than 40% of the glomeruli from aged mice remained positive for fibrin at 8 hours after LPS compared to less than 5% in young mice. The inset shows an example of the fibrin staining. At 8 hours, fibrin was readily demonstrated in glomerular capillaries in the kidneys of aged mice (Figure 3, inset, right). However, almost all of glomerular fibrin present at 2 to 4 hours in young mice had disappeared at this time (Figure 3, inset, left). These results demonstrate that aged mice are more vulnerable to renal thrombosis than young mice in endotoxemia. Experiments were performed to investigate the dose-dependency of the response to LPS in terms of renal glomerular fibrin deposition and PAI-1 mRNA induction in livers and kidneys of both young and aged mice. Mice were injected i.p. with increasing doses of LPS, and 4 hours later, the kidneys and livers were removed and analyzed for PAI-1 mRNA by quantitative RT-PCR and for fibrin by immunohistochemistry. In all cases, the older mice showed larger responses to endotoxin than the younger animals in terms of glomerular fibrin deposition and PAI-1 mRNA induction (Figure 4). For example, fibrin deposition was induced at lower doses of LPS in the older mice and in more glomeruli (ie, 50 to 60% in aged mice treated with 1–5 μg of LPS versus less than 20% in young mice) (Figure 4, left). The magnitude of the induction of PAI-1 mRNA in livers (Figure 4, center) and kidneys (Figure 4, right) in each LPS dosage was greater in aged mice than young mice. The differences were statistically significant even when multiple comparisons were considered (not shown). Young (8 weeks old) and older (32 weeks old) wild-type and PAI-1 defic
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