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American journal of Pathology, Vol. 143, No. 3, September 1993 Copynight C) American Society for Investigative Pathology

Type 1 Plasminogen Activator Inhibitor Gene Expression Following Partial Hepatectomy

Jacob Schneiderman,*t Michael Sawdey,* Heather Craig,* Terri Thinnes,* Gerald Bordin,t and David J. Loskutoff* From the Committee for the Study of Vascular Biology* and Division of Vascular and 7Toracic Surge,yt The Scripps Research Institute, La Jolla, California; and the Department of Clinical Pathology,* Scripps Clinic, La Jolla, California

A murine model ofpartial hepatectomy (PH) was employed to investigate type 1 plasminogen activator inhibitor (PAI-1) gene expression in regenerating liver. Mice were anesthetized, and a portion of the left lobe of the liver was ligated and resected distal to the ligature, and at various times thereafter, total liver RNA was prepared and analyzed by Northern blotting. PH caused a transient increase in PAI-I messenger (m)RNA that was apparent within I to 2 hours after surgery, was maximal at 8 hours (ninefold increase over sham-operated controls), and then slowly declined. Analysis of discrete liver segments demonstrated much greater induction of PAI-I mRNA in the region adjacent to PH than in more distal regions. Further analysis ofthe adjacent tissue by in situ hybridization revealed that PAI-I mRNA was induced primarily in hepatocytes in the transition zone created by the occluding hemostatic ligature between viable and necrotic tissue. Expression of PAI-I mRNA could also be detected in this transition zone in capsular mesothelial ceUls, subcapsular hepatocytes, and venous endothelial ceUs bordering the area. A much weaker signal was evident in hepatocytes dispersed throughout the remaining intact lobes ofPH mice, and no signal was detected in the livers of sham-operated mice. These observations suggest that PAI-i may be of importance in local tissue remodeling events accompanying liver regeneration. (Am J Pathol

1993, 143:753-762)

Partial hepatectomy (PH) induces the liver to enter a regenerative stage. Regrowth of the ablated tissue involves both parenchymal and nonparenchymal

cells and is effected by humoral factors that act in a temporally and spatially defined manner to elicit hepatic regeneration (for review, see ref. 1). In the prereplicative phase of liver regeneration, PH results in the induction of cellular immediate early genes, such as the transcription factors c-fos2'3 and c-jun. For example, a 13-fold increase in hepatic c-jun messenger (m)RNA was detected 2 hours after PH in mice, whereas the mRNA of a related gene, jun B, was shown to be increased by approximately 50% during the same interval.4 The products of the c-fos and c-jun genes are known to form heterodimers (AP-1) capable of binding to specific DNA sequence elements in the promoter regions of target genes and stimulating their rate of transcription.5'6 Because the gene encoding the extracellular matrix-degrading enzyme transin contains an AP-1 site7 and the level of transin mRNA has been shown to rise following PH, AP-1 was postulated to mediate induction of the transin gene in regenerating liver.4 Type 1 plasminogen activator inhibitor (PAI-1) is the primary physiological inhibitor of both tissue type and urokinase type plasminogen activators (for review, see ref. 8). In vivo, PAI-1 negatively regulates thrombolysis by tissue-type plasminogen activator.9'10 More recently, it has been shown to be deposited within the extracellular matrix of cultured cells11'12 where it may function to inhibit extracellular proteolysis mediated by urokinase type plasminogen activator.13'14 In vitro, PAI-1 is synthesized by a variety of cells, including human hepatocytes15 and hepatoma cells. Its synthesis in hepatoma cells is positively regulated by glucocorticoids,16 and by transforming growth factor-,B (TGF-f3),17 epidermal growth factor,18 and interleukin-1 (11-1),19 agents implicated in the Supported by NIH grant HL47819 to DJL. Accepted for publication May 4, 1993. Address reprint requests to Dr. David J. Loskutoff, Committee for the Study of Vascular Biology (CVB-3), The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037. This is publication number 7824-CVB from The Scripps Research Institute. Present address of J. Schneiderman is Department of General and Vascular Surgery, Sheba Medical Center, Tel Hashomer 52621, Israel.

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control of liver regeneration.20 In the case of TGF-, induction of the PAI-1 gene has been shown to occur at the transcriptional level21 and may involve AP-1like elements present in the PAI-1 promoter.22 Collectively, these observations suggest that PAI-1 may be of importance in liver response to injury. In the studies described below, we employ a mouse model system to demonstrate that PAI-1 mRNA is induced in the liver following PH, primarily in specific regions adjacent to areas of necrosis in the remnant of the resected lobe. These data imply a linkage between PAI-1 gene expression and the remodeling of the extracellular matrix during liver regeneration.

Materials and Methods

Surgical Procedures Twelve-week-old male BalbC/ByJ x C57BI6/J mice (CB6; Scripps Clinic Rodent Breeding Colony) were employed for all experiments. The animals were housed and treated in compliance with the guidelines set forth in "Principles of Laboratory Animal Care" and "Guide for the Care and Use of Laboratory Animals" (NIH publication no. 80-23, revised 1985). Surgical procedures were performed under general anesthesia, which was introduced and maintained with Metofane (methoxyflurane; PitmanMoore, Mundeleine, IL). Mice undergoing partial hepatectomy underwent a midline laparotomy, through which the left lobe of the liver was delivered. A hemostatic ligature was applied at the base of the lobe, and the lobe distal to it divided with a scalpel. A narrow rim of devascularized tissue beyond the hemostatic ligature (referred to as the necrotic tissue in the remnant of the left lobe) was left unresected. The abdomen was closed with a running suture, and the mice were allowed to recover from anesthesia. Control animals underwent a sham operation including midline laparotomy, delivery, and gentle rubbing of the left lobe of the liver without resection. At various times after surgery (0, 1, 2, 4, 8, 16, 24, and 120 hours), individual mice were euthanized, and various tissues were harvested. Harvested segments of the liver (see Figure 2) were designated as follows: A) the left lobe of the liver (sham-operated animals) or its remains (subtotal left hepatectomy); B) a wedge from the right lobe of the liver; and C) the rest of the remaining liver, including the right, median, and caudate lobes. Small portions of each segment were collected and processed for in situ hybridization. The remainder of the segment was stored for Northern blot analysis

as detailed below. In control experiments, heart, kidney, and spleen were also harvested for Northern analysis.

Northern Blot Analysis For preparation of total RNA, freshly isolated tissues were snap-frozen and stored in liquid nitrogen. Total RNA was extracted by the acid guanidium thiocyanate-phenol-chloroform method,23 and its concentration determined by measurement of sample absorbance at 260 nm. For Northern blotting, total RNA samples (30 pg) were denatured, electrophoresed in formaldehyde-containing agarose gels, transferred to nylon membranes (Biotrans; ICN, Irvine, CA), and fixed by ultraviolet irradiation as previously described.24 Equal RNA loading and transfer were confirmed by visualization of the ethidium bromide-stained RNA in the nylon membrane under ultraviolet light. The blots were prehybridized for 30 minutes at 65 C in a solution consisting of 50

mmol/L piperazine-N,N'-bis[2-ethanesulfonic acid] (PIPES) pH 6.8, 100 mmol/L NaCI, 20 mmol/L Na2PO4, 30 mmol/L NaHPO4, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 5% (wt/vol) sodium dodecyl sulfate, and then hybridized for 16 hours at 65 C in the same solution containing approximately 106 cpm/ml radiolabeled probe. The blots were washed at 65 C with prewarmed 100 mmol/L NaCI, 10 mmol/L Na citrate, 5% (wt/vol) sodium dodecyl sulfate, and then autoradiographed at -80 C employing Kodak XAR-5 film with intensifying screens.

Probes For Northern blot hybridizations, a plasmid subclone of the murine PAI-1 complementary (c)DNA25 was obtained as a generous gift from Drs. L. Diamond and M. Cole (Princeton University, Princeton, NJ). The 0.5-kb PAI-1 cDNA insert, corresponding to the 5' terminal region of murine PAI-1 mRNA, was excised by digestion with the appropriate restriction enzymes and purified by preparative gel electrophoresis. A plasmid containing the cDNA for human glyceraldehyde 6-phosphate dehydrogenase (GAP) was obtained from American Type Culture Collection (Rockville, MD), and linearized before use. The probes were radiolabeled to specific activities >108 cpm/pg by the random primer technique,26 employing a[32P]dGTP (> 3,000 Ci/mmol; Amersham, Arlington Heights, IL). For in situ hybridization analysis, a fragment corresponding to bases 1 to 1,085 of the murine PAI-1 cDNA25 was subcloned into the

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vector pGEM 3Z (Promega Biotec, Madison, WI) and linearized with the appropriate restriction enzyme before use. Sense and anti-sense PAI-1 riboprobes were synthesized by in vitro transcription of the linearized template with T7 or SP6 RNA polymerase, respectively, in the presence of a[35S]UTP (> 1,000 Ci/mmol; Amersham, Arlington Heights,

IL).

Densitometric Analysis Northern blot autoradiographic signals were quantitated by densitometric scanning, utilizing an Ultroscan XL laser densitometer (LKB, Bromma, Sweden). Densitometric data were expressed as absorbance units/mm2 (AU/mm2) of peak area. All densitometric readings were within the linear range of measurement, as established by previous control experiments comparing AU/mm2 with cpm obtained by direct radioanalytic imaging of the same blot.27 Densitometry units were obtained by dividing AU/ mm2 obtained for PAI-1 mRNA by the corresponding AU/mm2 obtained for GAP mRNA.

Tissue Preparation Liver pieces (4 x 4 mm) were fixed in fresh 4% (wt/ vol) paraformaldehyde overnight at 4 C, incubated in 15% (wt/vol) sucrose for 4 hours at 4 C, and frozen in optimum cutting temperature compound (Tissue Tek, Miles) before freezing at -70 C.

In Situ Hybridization In situ hybridizations were carried out essentially as

described.28 Tissue sections (4 p) were fixed with (wt/vol) paraformaldehyde in phosphatebuffered saline for 10 minutes at 4 C and deproteinated with 1 pg/ml proteinase K in 500 mmol/L NaCI, 10 mmol/L Tris-HCI, pH 8.0, for 10 minutes at 23 C. Prehybridization was performed for 1 hour at 42 C in prehybridization buffer, consisting of 50% (wt/vol) formamide, 0.3 mol/L NaCI, 20 mmol/L Tris-HCI, pH 8.0, 5 mmol/L EDTA, 1 x Denhardt's solution, 10% (wt/vol) dextran sulphate, and 10 mmol/L dithiothreitol, followed by hybridization for 16 hours at 55 C in prehybridization buffer supplemented with 0.6 x 107 cpm/ml 35S-labeled riboprobe and 2.5 mg/ml yeast transfer RNA. The sections were washed with 2x standard saline citrate (SSC), 10 mmol/L 2-mercaptoethanol, 1 mmol/L EDTA (2 x 5 minutes, 23 C), incubated with RNAse A (20 pg/ml in 500 mmol/L NaCI/10 mmol/L Tris-HCI) for 30 minutes 4%

at 23 C, washed with 2x SSC, 10 mmol/L 2-mercaptoethanol, 1 mmol/L EDTA (2 x 5 minutes, 23 C), and then washed at high stringency in 0.1x SSC, 10 mmol/L 2-mercaptoethanol, 1 mmol/L EDTA for 2 hours at 60 C. The sections were washed again in 0.5x SSC (4 x 10 minutes, 23 C), dehydrated in a graded alcohol series containing 0.3 mol/L NH4OAc, dried, coated with NTB2 emulsion (Kodak, diluted 1:1 in water), and exposed at 4 C for 3 to 12 weeks. The slides were developed in D19 developer (Kodak) for 2 minutes, fixed, washed in water, and counterstained with hematoxylin and eosin. To control for nonspecific hybridization, the above procedures were performed employing a sense riboprobe (data not shown).

Results To investigate expression of the PAI-1 gene in regenerating liver, PH was performed on adult male CB6 mice. Mice underwent midline laparotomy, followed by ligation and resection of the portion of the left lobe distal to the ligature (approximately 25% of the liver mass). As a control for the effects of surgery, mice underwent otherwise identical sham operations in which the left lobe of the liver was exposed but not ligated or removed. At selected time points following surgery (ie, 0, 1, 2, 4, 8, 16, 24, and 120 hours), both the liver and selected extrahepatic tissues (eg, heart, kidney, and spleen) were harvested for analysis. Total RNA was prepared from the entire liver (sham mice) or its remains (PH mice), and PAI-1 gene expression was analyzed by Northern blotting employing a murine PAI-1 cDNA probe (Figure 1A). Little if any PAI-1 mRNA was detected in the livers of untreated (0 hour) mice. However, in the livers of both PH- and sham-operated mice, PAI-1 mRNA increased significantly within 1 hour of surgery, reaching a maximum at 8 hours and remaining elevated through 24 hours. Correspondingly greater increases in PAI-1 mRNA levels were observed in the livers of PH mice throughout this period. By 120 hours (ie, 5 days), the level of PAI-1 mRNA had returned to basal levels in both groups. To quantitate the response of PAI-1 mRNA to PH, replicate time course experiments were performed and analyzed by Northern blotting as above. To control for variations in total RNA content between samples, the blots were rehybridized with a GAP probe. The autoradiographic signals for PAI-1 and GAP mRNAs were quantitated by densitometric scanning, and the densitometric values for PAI-1

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A

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Figure 1. Northern blot analysis of PAI-1 mRVA in murine liver follou'ing partial hepatectomy. A: Male CB6 mice were sacrificed at designated S~~~~~~~~~~~~~~~~~~~~~~~~~~~~~61 time intervals following partial hepatectomy (PH mice) or sham surgerv (sham mice). Total RNA uas extracted from the entire liver (sham mice) or its remains (PH mice) and 30 jig analyzed by Northern blotting nith a -2P-labeled murine PAI- I cDNA probe. The lenigth of the band shown (3 0 kb) was estimatedfrom its nzigration relative to the 28s and 18s ribosomal RNA bands visualized by ethidiunm staining. Th7e anitoradiogram was exposed for 48 hours. B: Relative fold increase in PAI-I mRNA in PH mice conipared to shanm mice. Replicate time course experiments were performed, anid PAI-1 and GAP mRNA levels were determined by Northern blotting as above. The autoradiographic signals uwere quantitated by densitometric scanning and the densitometric valnies for PAI-1 mRNA were normalized to those of GAP. The relative Jbld itncrease in PAI-i mRNA following PH was then calculated from the meatn of the replicate values obtained for each time point. All densitometric readings were within the linear range of measurenient, as established by previons control ex-

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mRNA were normalized to those of GAP. The fold increase in PAI-1 mRNA in PH relative to sham mice was then calculated from the normalized data (Figure 1B). Some PH-specific induction of PAI-1 mRNA was evident within 1 to 2 hours. However, the majority of the increase was apparent at later times, rising to fivefold at 4 hours and ninefold at 8 hours. By 16 to 24 hours, the relative fold increase in PAI-1 mRNA had declined to approximately threefold or lower (data not shown). Experiments were performed to analyze this response within discrete regions of the liver (Figure 2). PH and sham mice were sacrificed at 8 hours following surgery, and specific segments of the liver

Mouse No.:

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depicted in Figure 2 were then removed. Segment A is the left lobe of the liver (sham mice), or its remains (PH mice); segment B is a wedge of tissue taken from the right lobe of the liver; and segment C is the sum of the right, median, and caudate lobes. Total RNA was prepared from each segment, and changes in PAI-1 and GAP mRNAs were assessed by Northern blotting as above. Quantitative analysis of the blot autoradiograms by densitometric scanning revealed that PAI-1 mRNA levels had increased an average of 17- and eightfold in segments A and B, respectively, in PH mice compared to the sham mice (ie, mice 1 and 2 vs. 5 and 6; Figure 2). The average level of PAI-1 mRNA in seg-

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-GAP Figure 2. Northern blot analysis of PAI-1 mRNA in murine liver following partial hepatectomy: segmental analvsis. PH or sham mice were sacrificed at 8 hours following surgery, and total RNA was prepared from the regions indicated in the schematic diagram of the liver shown at the left and analyzed by Northern blotting: A denotes the stump of the remaining left lobe after partial hepatectomy (shaded area), or the unresected left lobe in sham mice; B denotes a limited segment from the edge of the right hepatic lobe; C denotes the suim of the right, median, and caudate lobes (the latter tuo not illustrated in the schenie); A + B + C denotes the entire remaining liver tissuie afterpartial hepatectomy. The top auitoradiograni shou's the results uising the 32P-labeled murine PAI-1 cDNA probe. The bottom auitoradiogram shows the results obtained Jbllouwing rehybridization of the blot with a GAP probe. The size of the GAP mRNA species (:1.1 kb) was estimated from its migration relative to the 28s and 18s ribosomal RNA species visualized by ethidium broniide stainitng. The auitoradiograms uwere exposed for 48 hours. -

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nuclei and eosinophilic cytoplasm (Figure 4C). A few sinusoidal cells within the transition zone also exhibited a strong hybridization signal (Figure 4C), and positive venous endothelial cells could frequently be observed in large veins intersected by the transition zone (Figure 4, B and D). Expression of PAI-1 mRNA was also clearly evident within the viable subcapsular cells that border the region of necrosis in segment A (Figure 4E). This layer of cells probably retains its viability via the capsular microcirculation. The hybridization signal was present predominantly within the first and second layers of subcapsular hepatocytes closest to the hepatic mesothelium and to a lesser extent in surface mesothelial cells. Rare positive sinusoidal cells could also be observed within the viable subcapsular region. In the remaining viable portion of segment A, and throughout segment B, a less intense hybridization signal for PAI-1 mRNA was evident (Figure 2). This decrease in PAI-1 mRNA seemed to result from significantly fewer positive hepatocytes in these areas (Figure 4F). Occasionally, a mild subcapsular hybridization signal was also noted in segment B. A pattern of hybridization generally similar to that described above was detected at 16 hours after PH in both segments A and B. By 24 hours, the level of signal had declined markedly, with the exception of positive venous endothelial cells within the necrotic area in close proximity to the transition zone and capsular regions (Figure 4G). By 120 hours after PH, no positive signal could be discerned. No positive signal could be visualized at any time in liver sections of sham mice (Figure 4H), despite the presence of PAI-1 mRNA levels comparable to those of PH mice (eg, Figure 1; compare PAI-1 mRNA autoradiographic signals for 8-hour sham versus 24-hour PH mice). This finding may reflect a more diffuse induction of PAI-1 gene expression throughout the livers of sham mice (ie, more cells are induced but at levels undetectable by in situ

ments A + B + C (mice 3 and 4) after PH seemed to be intermediate between that observed for segments A and B alone, although some variability between animals existed. In the sham controls (mice 5 and 6), little difference in PAI-1 mRNA levels was evident between segments A and B, although variability between animals was again apparent. To examine the response of the PAI-1 gene in extrahepatic tissues to PH, total RNA was prepared from the heart, kidney, and spleen of PH- and sham-operated mice at various times after surgery and analyzed for PAI-1 mRNA as above. Although only data from the heart are presented (see Figure 3), similar results were obtained for both kidney and spleen (data not shown). Densitometric analysis of the data shown in Figure 3 revealed that the level of PAI-1 mRNA in the heart increased approximately twofold following surgery in both PH and sham mice. Thus, in contrast to the results obtained for the liver, no greater increase could be discerned in extrahepatic organs of PH mice compared to the shams. Histopathological examination of liver segment A at 8 hours after PH revealed an abrupt and welldelineated zone of transition between the viable and necrotic tissue (Figure 4A). Although the sinuses within this transition zone contained a few neutrophils, little if any infiltration by lymphocytes or macrophages was observed, and increased numbers of Kupffer cells were not evident. All of the hepatocytes within the ischemic tissue seemed to be necrotic, and the sinuses in this region contained Kupffer cells as well as erythrocytes in varying stages of degeneration. Analysis of segment A (obtained 8 hours after PH) by in situ hybridization revealed a strong positive hybridization signal for PAI-1 mRNA that extended along the length of the transition zone between viable and necrotic cells (Figure 4B). Examination of this region under higher power magnification confirmed that the majority of the positive cells were hepatocytes, identifiable by their large

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tectomY. Mice were sacrificed at designated time intervals flblowing PH or sham surgery and total RNA (30 pig) was extracted from the heart and analyzed by Northern blotting as described for Figure 1. The auitoradiograms depicting PAI-1 and GAP mRNAs were exposedjfr hours rest,ectivelv. 5 davs and }f-, sk

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hybridization). Sections analyzed with a sense PAI-1 riboprobe were also uniformly negative (data not shown).

Discussion We have employed a murine model system to investigate the response of the PAI-1 gene to partial hepatectomy. Surgical resection of the majority of the left lobe of the liver (approximately 25% of the total liver mass) resulted in a dramatic increase in hepatic PAI-1 mRNA levels. However, a similar although lower response was evoked by the surgical procedure itself. Both PH- and sham-operated mice exhibited increases in PAI-1 mRNA levels within 1 hour of surgery, rising to maximal levels at 8 hours and declining thereafter (Figure 1A). This response is consistent with the known acute phase behavior of PAI-1 in man. For example, clinical studies have established that the concentration of PAI-1 in blood may rise sharply following major surgery,29 trauma,30 or experimental administration of lipopolysaccharide.3132 The biosynthetic origin of acute phase PAI-1 is presently unclear. Most acute phase proteins are believed to originate from parenchymal cells of the liver, and previous studies have demonstrated induction of PAI-1 mRNA in these cells.33 Other studies employing mice34 and rats35 have implicated vascular endothelial cells in the liver and other organs as the site of increased PAI-1 synthesis following lipopolysaccharide treatment. Thus whereas the cellular origin of acute phase PAI-1 remains to be established, the data presented above indicate the liver is likely to represent a significant source of PAI-1 in acute phase reactions following major surgery. Relative to sham-operated mice, PH mice exhibited a consistently greater increase in hepatic PAI-1 mRNA levels in the period from 1 to 24 hours after surgery, with maximal induction (ie, > ninefold increase relative to sham mice) evident at 8 hours (Figure 1 B). This induction seemed to be specific to the liver because no such difference in PAI-1 gene

expression was evident in extrahepatic tissues (Figure 3). Analysis of the response of discrete liver segments to PH (Figure 2) revealed that PAI-1 mRNA levels were induced 17-fold over the sham controls in the remnant of the resected left lobe (segment A). In situ hybridization of this region at 8 hours after PH (Figure 4) revealed sharply delineated rows of PAI-1 mRNA-positive cells, consisting predominately of hepatocytes bordering the region of ischemic necrosis. PAI-1 mRNA was also localized to venous endothelial cells, capsular mesothelial cells, and sinusoidal cells proximal to the necrotic tissue. The marked induction of PAI-1 mRNA in this region of the liver at 8 hours after PH (Figure 2) would thus seem to result from a highly localized increase in its expression immediately adjacent to the area of necrosis. The nature of the stimulus for this increase is unknown. Induction of PAI-1 mRNA in hepatic parenchymal cells has recently been demonstrated following treatment of rats with the synthetic glucocorticosteroid dexamethasone.33 However, in human subjects given adrenocorticotropic hormone, no cause-effect relationship could be demonstrated between plasma PAI-1 activity and cortisol levels, despite elevations in plasma cortisol levels comparable to those observed postoperatively.29 The PAI-1 gene also is known to be responsive to inflammatory cytokines such as interleukin124 and tumor necrosis factor-a,24 which may mediate in part the response of target genes in acute phase reactions and gram negative sepsis.36'37 Histopathological evidence of inflammation (eg, infiltration of lymphocytes or macrophages, or increased numbers of Kupffer cells) was not observed coincident with the localized increase of PAI-1 mRNA at 8 hours after PH. Thus, whereas systemic release of these cytokines may account for the more generalized increase in PAI-1 mRNA observed in hepatocytes within more remote regions of the liver, they would not seem likely to account for its induction in the transition zone or subcapsular areas at 8 hours after PH. However, it

Figure 4. In SitL hybridization analysis of PAI-1 mRNA in murine liver tissue followitng partial hepatectomy. Liver tissues were collected 8 hours after PH and analyzed by either May-Gruenuald Giemsa staining (A) or by in situ hybridization with a -35S-labeled anti-sense PAI 1 riboprobe (B to H), as described in Materials and Methods. A: May-Gruenwald Giemsa stain of PH liver segment A at 8 hours after PH, demonstrating the transition zone ( 7Z) between viable and nonviable tissue. Viable ( V) hepatocytes exhibit darker staining relative to necrotic (N) cells. Note Kupffer cells (arrows) and abundant erythrocytes within necrotic tissue (magnification: 200X). B: In situ hybridization signalfor PAI-1 mRNA in the transition zone of segment A at 8 hours after PH. Note intersection of transition zone with large hepatic vein (HV) at bottom left, and positive signal in luminal endothelium (magnification: 10OX). C: Higher power magnification of transition zone region shown in B. Arrows denote positive hepatocytes; arrowheads denote rare positive sinusoidal cells (magnification: 400X). D: In situ hybridization signal for PAI-1 mRNA in hepatic vein (HV) of segment A at 8 hours after PH. Arrows indicate positive endothelial cells (magnification: 400X). E: High-power view of segment A at 8 hours after PH, demonstrating PAI-1 mRNA hybridization signal in the subcapsular region (magnification: 400X). Note positive hybridization signtal in capsular mesothelial cells (arrowhead) and subcapsular hepatocytes (arrows) (magnification: 400X). F: Scattered PAI-1 mRNA positive hepatocytes (arrou') in segment B 8 hours after PH ( magnification: 400X). G: High-power vieu' of segment A at 24 hours after PH, demonstrating a strong signal within endothelial cells lining hepatic vein (HV) (magnificatiotn: 400X). H: Negative sham liver at 8 hours after suirgery ( magnification: 400X).

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presently cannot be excluded that interleukin-1 and tumor necrosis factor-a may in part stimulate endothelial PAI-1 synthesis in these regions at later times (Figure 4G), as significant infiltration of neutrophils could be visualized in the vicinity of the vessels expressing PAI-1 mRNA by 24 hours after PH. Expression of the PAI-1 gene in both cultured hepatoma38 and endothelial cells21 is strongly stimulated by TGF-f3, which is released from platelets in response to physiological stimuli.39 The release and diffusion of TGF-,B from platelets trapped within the region of ischemic necrosis could thus be hypothesized to induce PAI-1 mRNA in bordering hepatocytes and vascular cells, consistent with the observed pattern of PAI-1 gene expression (Figure 4). Alternatively, because TGF-,B mRNA has been reported to be induced in regenerating liver within 4 hours after PH and this induction correlates with increased accumulation of mRNAs for several extracellular matrix proteins,40 the response of PAI-i mRNA within the transition zone and subcapsular space may reflect de novo synthesis and release of TGF-4 from nonparenchymal cells in this region. Induction of PAI-1 mRNA in vascular cells may otherwise reflect induction of PAI-1 synthesis by basic fibroblast growth factor, which is released from vascular cells under hypoxic conditions41'42 or by

hypoxemia.443 Extensive remodeling of the extracellular matrix is known to occur during liver regeneration. Transin, the rat homologue of human stromelysin, is a secreted metalloproteinase with a broad specificity for extracellular matrix components. The mRNA encoding transin was recently shown to be expressed in hepatocytes within areas subsequently undergoing necrosis due to CC14 intoxication.44 Interestingly, transin also participates in the activation of interstitial collagenase in cooperation with plasmin.45'46 Peripheral to regions of necrosis, the induction of PAI-1 may serve to limit extracellular plasmin generation by cell-surface-bound urokinase. 13 The increased synthesis of PAI-1 in these regions may thus counteract plasmin-mediated extracellular matrix degradation within viable tissue regions bordering necrotic areas. The PAI-1 and transin genes both contain functional AP-1 sites. Moreover, induction of the protooncogene products c-fos and c-jun, which together activate gene transcription from AP-1 sites,5'6 occurs rapidly following PH.4 In situ hybridization studies have revealed that expression of c-fos and c-jun mRNAs following CC14 injection is detected first within pericentral hepatocytes, which later un-

dergo necrosis.47 However, the expression of these and other proto-oncogenes subsequently spreads to adjacent areas and ultimately involves the entire liver.447 Thus, whereas the induction of PAI-i mRNA adjacent to regions of necrosis may initially involve AP-1-responsive elements in the PAI-1 gene, other factors must also regulate its expression within these regions. The identity of these factors remains to be determined.

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