(Circulation. 1996;94:2728-2734.)
© 1996 American Heart Association, Inc.
Articles |
Expression of Kunitz Protease Inhibitor–Containing Forms of Amyloid ß-Protein Precursor Within Vascular Thrombi
the Division of Pulmonary and Critical Care Medicine (I.M.L., K.M.M.), University of California at San Diego, and The Department of Vascular Biology (I.M.L., R.R.S.), the Scripps Research Institute, La Jolla, Calif.
Correspondence to Raymond R. Schleef, PhD, Department of Vascular Biology (VB-1), The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037.
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Abstract |
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Background The presence of patent neovessels within vascular occlusions in chronic thromboembolic pulmonary hypertension suggests that local mechanisms exist to regulate the coagulation system. This study investigated the expression of a potent inhibitor of Factor IXa and Factor XIa (ie, protease nexin-2/amyloid ß-protein precursor, AßPP) in the organized vascular occlusions harvested from patients with this disease.
Methods and Results Immunohistochemical analysis revealed intense immunoreactivity for AßPP in the single layer of cells that line the neovessels. A positive signal was also detected by in situ hybridization analysis with the use of a 35S-UTP–labeled antisense riboprobe that recognizes the various alternatively spliced mRNA forms of this molecule. To identify the forms of AßPP produced within the thrombi, total RNA was extracted from the thrombi, reverse transcribed, and subjected to amplification with the use of the polymerase chain reaction (PCR) and primers that flank the region encoding the alternatively spliced 56–amino acid Kunitz-type protease inhibitor (KPI) domain. The major PCR products consisted of 255 bp and 312 bp and corresponded to transcripts encoding this domain (ie, AßPP751 and AßPP770). In situ hybridization analysis with the use of a 35S-UTP–labeled antisense riboprobe complementary to the region encoding the KPI domain confirmed the presence of these mRNA species in nucleated cells lining the neovessels.
Conclusions The expression of KPI-containing isoforms of AßPP in thrombus endothelial cells may represent one mechanism utilized in this disease to shift the local hemostatic balance and preserve regional vessel patency.
Key Words: thrombosis • amyloid • endothelium • polymerase chain reaction • pulmonary heart disease
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Introduction |
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Human pulmonary thromboemboli are resolved over a period of several days in the majority of cases; however,
1% of the embolic obstructions undergo a poorly understood process of organization and recanalization.1 2 3 The persistent obstruction of the central pulmonary arteries leads to increased ventricular workload and is clinically recognized as chronic thromboembolic pulmonary hypertension.1 2 3 4 5 6 This disease carries a high mortality ultimately caused by right ventricular failure.1 2 3 4 5 6 Restoration of pulmonary blood flow by pulmonary thromboendarterectomy is able to dramatically improve the long-term function and survival of patients with this disorder.1 6 Our group has observed histologically that the core of the thromboemboli is composed of numerous EC-lined neovessels that are surrounded by a smooth muscle cell/collagen–containing extracellular matrix and fresh red blood cell/platelet/fibrin-rich thrombi occupying the central pulmonary arteries.7 This study also indicated that ECs present within the chronic organized regions of thromboemboli express high levels of PAI-1. Because PAI-1 has been observed to be critical for the deposition of a rich collagen-containing matrix in a bleomycin-induced model for pulmonary fibrosis,8 the localized release of this inhibitor in chronic thromboembolic pulmonary hypertension may play a similar role in the production of the highly organized extracellular matrix by its ability to reduce the generation of a key proteolytic enzyme (ie, plasmin) through inactivation of both urokinase-type plasminogen activator and tissue-type plasminogen activator. The presence of patent neovessels within the organized tissue in close proximity to both ongoing thrombosis and cells expressing high levels of a potent inhibitor of the fibrinolytic cascade (ie, PAI-1) suggests that local mechanisms exist to regulate the coagulation system. Presently, no information exists regarding the localized expression of coagulation inhibitors that may play a role in the maintenance of neovessel patency in the chronic vascular occlusions that are characteristic of this disease.
One of the most potent inhibitors of Factors IXa and XIa has recently been shown to be protease nexin-2/AßPP,9 10 11 12 a complex family of 100-135–kD membrane-bound and soluble glycoproteins. The membrane-bound forms of this family of glycoproteins are recognized as precursors of a 4.5-kD protein found in senile plaques and cerebral vessels of patients with Alzheimer's disease and patients with hereditary cerebral hemorrhage with amyloidosis Dutch-type (for reviews, see References 13 through 16). Proteolytic inhibitory activity is associated with those isoforms of AßPP that contain an alternatively spliced 158-nucleotide insert that encodes for a KPI domain (eg, AßPP751 and AßPP770) (reviews, References 13 through 16). AßPPs have been detected in neuronal cells,17 18 19 20 21 22 23 glial cells,19 24 mononuclear cells,25 26 27 megakaryocytes,28 29 heart muscle,30 brain vessels,21 31 32 33 and cultured ECs.12 23 24 34 35 36 37 Furthermore, several studies have shown that AßPP is an abundant platelet 
-granule protein that is secreted upon platelet activation.10 28 29 38 39 40 41 Taken together, these observations have raised the possibility that AßPP may function not only in the cerebrovascular system (review, Reference 42) but also in the peripheral vascular system as an anticoagulant (review, Reference 43). The current study was initiated to analyze the local expression of AßPP in the patent vascular clefts and neovascular structures within chronic pulmonary thromboemboli.
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Methods |
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Subjects
Organized vascular thrombi were obtained from patients undergoing pulmonary thromboendarterectomy at the University of California San Diego Medical Center between September 1989 and December 1991.7 The diagnosis of chronic major vessel thromboembolic pulmonary hypertension in each patient was confirmed by careful review of the medical history, measurement of pulmonary vascular resistance, pulmonary angiography, and angioscopy. Tissue samples were obtained from 16 consecutive patients who had provided informed consent. The distribution of age, sex, duration of disease, and preoperative and postoperative pulmonary vascular resistance was similar to those in larger series we have described previously.1 6 All patients experienced a dramatic decrease in pulmonary vascular resistance as documented 3 days after the surgical intervention, when all patients were off mechanical ventilation, off vasoactive drugs, and just before removal of monitoring catheters. The thrombi were fixed for 20 hours in paraformaldehyde (4% wt/vol in 0.1 mol/L sodium phosphate) at 4°C, embedded in paraffin blocks, and sectioned at 2-µm thickness with the use of a microtome. The sections were mounted onto polylysine-coated slides and stored at room temperature until analyzed. For comparative purposes, tissues were also obtained from heart or lung transplant organ donors at the time of explantation (n=7). Individuals from this latter group had been selected as organ donors on the basis of criteria that are currently used in our institution44 and include a noneventful previous medical history, normal findings on cardiac echocardiography, normal chemical analysis of blood and urine before the transplantation, and normal hemodynamics. More specifically, blood pressure of the seven donors was 123.4±22.8/71±12.1 mm Hg; heart rate, 84.7±15.5 bpm; central venous pressure, 6.9±3.1 mm Hg; systolic pulmonary pressure, 25.4±4.1 mm Hg; and mean pulmonary pressure, 14.1±2.8 mm Hg (all data obtained at the time of organ explantation, mean±SD). The experimental protocol for this study was approved by the University of California, San Diego, Human Subject Investigations Committee (Protocol No. 89-509).
Histochemistry
Immunohistochemical staining was carried out with the use of a three-step avidin-biotin-peroxidase method as described in detail previously.7 In this procedure, sections of paraffin-embedded, paraformaldehyde-fixed tissues were incubated with either rabbit antibodies (eg, anti-vWF7 ) or mouse monoclonal antibodies (eg, anti-human AßPP, clone 22C11, Boehringer Mannheim) followed by the appropriate second antibody (ie, biotinylated goat anti-rabbit IgG or biotinylated goat anti-mouse IgG, Zymed Lab). Subsequent incubations included a streptavidin-peroxidase conjugate followed with the chromogen aminoethylcarbazole(/)hydrogen peroxide mixture (Zymed Lab) that results in a reddish-brown deposit indicative of positive immunoreactivity. To differentiate fresh thrombus from organized thrombus and to identify fibrin/fibrinogen and collagen, a trichrome stain was performed as described previously.7
In Situ Hybridization
Plasmid vector pGEM-9Zf containing the cDNA encoding AßPP695, AßPP751, or AßPP770 was generously provided by Dr Dimitri Goldgaber (State University of New York, Stony Brook, NY). Plasmid pGEM-9Zf-AßPP695 was digested with KpnI (nucleotide 57) to delete the region encoding the signal peptide and with TaqI (nucleotide 861), which cuts before the alternatively spliced KPI domain. The 804 bp KpnI/TaqI fragment of AßPP was gel isolated and ligated to pBluescript (Strategene) through the use of the vector KpnI site and a TaqI-compatible cohesive end generated by digestion with ClaI. The pBluescript-AßPP57-861 construct was digested with KpnI and HindIII, and the 804 bp region of AßPP was subcloned into KpnI/HindIII-digested pGem-3Z and pGem-4Z vectors (Promega Corp). Antisense and sense riboprobes were prepared from the construct pGem-3Z-APP57-804 and the construct pGem-4Z-APP57-804, respectively, by in vitro transcription with the use of SP6 RNA polymerase in the presence of 35S-UTP (specific activity, 1200 Ci/mmol, Amersham) as described previously.7 The antisense riboprobe was purified and used to detect AßPP mRNA in paraffin sections with the use of the in situ hybridization protocols described in detail previously.7 After hybridization of the 35S-labeled probes with the tissue sections, the slides were coated with Kodak NTB2 emulsion and exposed in the dark at 4°C for 10 weeks. Slides were developed for 2 minutes in Kodak D19 developer, fixed, washed, and counterstained with hematoxylin and eosin, which stains nuclei blue. Parallel sections were analyzed with the use of a sense probe as the control for nonspecific hybridization. Specimens were analyzed with the use of combined light/epiluminescence microscopy to permit a simultaneous visualization of the sample and exposed silver grains. The latter appear as black or green dots, depending on the illumination, and indicate the presence of AßPP mRNA.
For the preparation of probes specific for mRNA containing the sequences encoding the KPI domain, two primers were synthesized and used in the PCR to amplify the 167 bp region encoding the KPI region from the AßPP751 cDNA. A forward primer (ie, 5'-GAGTGAGAGCTCAGAGGTGTGCTCTGAACAAGC-3') was synthesized on an Applied Biosystems 391 DNA Synthesizer that contains an internal SacI site (underlined) followed by the sequence corresponding to nucleotides 990-1008 of the AßPP770 cDNA. A reverse primer (ie, 5'-GAGAGATCTAGATGGCGCTGCCACACACGGCCAT-3') was synthesized that contains an internal XbaI site (underlined) and corresponded to nucleotides 1,157-1,136 of the AßPP770 cDNA. The two primers (60 pmol/primer) were combined with 100 ng of plasmid pGem-9Zf-AßPP751, 2.5 units of Taq DNA polymerase (Perkin-Elmer Cetus), 1x GeneAmp dNTPs (Perkin-Elmer Cetus), and 1x GeneAmp PCR reaction buffer (Perkin-Elmer Cetus) in a final reaction volume of 100 µL. PCR was performed by denaturation at 94°C for 5 minutes in an automated thermocycler (Perkin-Elmer Cetus) followed by 4 cycles (94°C, 1 minute; 55°C, 1 minute; 72°C, 1 minute), 20 cycles (94°C, 1 minute; 55°C, 1 minute; 72°C, 3.5 minutes), and a final extension cycle at 72°C for 10 minutes. The PCR products were analyzed by agarose gel electrophoresis and the 167 bp PCR product was excised from the gel, eluted, digested with XbaI and SacI, and cloned into XbaI/SacI-digested pGem-3Z and pGem-4Z plasmids. Clones were isolated and confirmed by sequence analysis. These plasmids were utilized for the preparation of 35S-antisense and 35S-sense riboprobes and used for in situ hybridization analysis as described above.
Quantitation of the in situ hybridization signal was done by counting silver grains associated with 
200 nucleated cells at either x400 or x1000 magnification with the use of oil immersion. Data are expressed as the number of exposed silver grains per 100 nuclei. A paired Student's t test was used to evaluate the differences between the number of exposed silver grains associated with thrombus ECs in comparison to the level associated with thrombus smooth muscle cells, and an unpaired Student's t test was used to assess the differences between the number of exposed silver grains associated with ECs in the thrombus and the cells present in sections of pulmonary arteries harvested from organ donors.
PCR-Mediated Amplification of Thromboemboli mRNA
Identification of the KPI-containing forms of AßPP was performed with the use of primers that flanked two alternatively spliced inserts: a 168-nucleotide insert encoding the 56–amino acid KPI domain and a 57-nucleotide insert encoding a 19–amino acid domain homologous to the OX-2 antigen found on the surface of neurons and thymocytes (for reviews, see References 15 and 16). The selection of the primers was according to the studies of Golde et al45 and yield the following potential amplification products: APP695 mRNA yields a PCR product of 87 bp; APP714 mRNA yields a PCR product of 144 bp; APP751 mRNA yields a PCR product of 255 bp; and APP770 mRNA yields a PCR product of 312 bp. The sequence of the forward primer, upstream (5') APP+958, is 5-CACCACAGAGTCTGTGGAAG-3' and corresponds to bases 958-977 of the APP751 cDNA.46 The sequence of the reverse primer, AßPP-1213, is 5'-AGGTGTCTCGAGATACTTGT-3' and is complementary to bases 1213-1194 of the AßPP751 cDNA. Total cellular RNA was prepared from both freshly harvested thromboemboli (n=6) and thromboemboli stored in liquid nitrogen (n=10)7 by extraction into guanidine-isothiocyanate with the use of a protocol described previously.47 The RNA was reverse-transcribed with the use of a first-strand cDNA synthesis kit (Boehringer Mannheim). Two microliters of the reverse-transcriptase reaction mixture and 60 pmol of each primer were combined with the reagents of the GeneAmp PCR kit (Perkin Elmer) as described above. Alternatively, 100 ng of plasmid pGem-9Zf containing the cDNA encoding either AßPP695, AßPP751, or AßPP770 was subjected to PCR as markers for the reverse-transcribed reaction mixture. PCR was performed with the use of the cycle conditions that are described above.
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Detection of AßPP Antigen and mRNA in ECs Within Chronic Pulmonary Thromboemboli
Histochemical analysis with the use of a trichrome stain demonstrated that the thromboemboli harvested from patients with chronic thromboembolic pulmonary hypertension are composed of an organized collagen matrix (green-stained) with patent neovessels (Fig 1
, A and B). Immunohistochemical analysis with the use of a monoclonal antibody directed against the amino-terminal region of AßPP revealed that the lining of these neovessels stain positively for AßPP (Fig 1, C and E). As observed previously,7 analysis of parallel sections with antibodies to vWF indicated that the cells lining the neovessels stained positively for vWF (data not shown). Because ongoing thrombus formation is known to occur in the central pulmonary arteries,7 it is possible that AßPP antigen associated with the ECs may have been derived by the binding and/or uptake of soluble AßPP released from activated platelets. To document the presence of AßPP mRNA associated with the ECs, in situ hybridization analysis was performed with an 804 bp 35S-labeled riboprobe that was designed to recognize the various alternatively spliced forms of AßPP mRNA. Positive staining for AßPP mRNA was detected with the use of the 35S-labeled antisense probe (Fig 1G) in comparison to the background observed with the 35S-labeled sense probe (Fig 1H). To obtain quantitative data on the steady-state levels of AßPP mRNA, we determined the mean number of exposed silver grains per 100 nuclei (Table). AßPP mRNA levels in ECs revealed a significant elevation in comparison to either the levels expressed in the smooth muscle cells within the parenchymal tissue of the thromboemboli (P<.001) or the levels expressed in ECs within samples of pulmonary arteries harvested from organ donors (P<.005) (Table).
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KPI-Containing Isoforms of AßPP mRNA Are Present Within Chronic Thromboemboli
Two different experimental approaches were undertaken to define the alternatively spliced isoforms of AßPP mRNA within the thromboemboli. One approach utilized PCR and primers that flank the alternatively spliced KPI domain to amplify reverse-transcribed total RNA either extracted from thromboemboli immediately after their harvest from the patient's vasculature (n=6) or extracted from a library of thromboemboli frozen in liquid nitrogen (n=10).7 PCR products were detected at 312 bp and 255 bp, which respectively correspond to transcripts encoding AßPP770 and AßPP751, with the use of reverse-transcribed total RNA extracted from nonfrozen thrombi (ie, Fig 2, lanes 6 and 7, represent samples processed immediately upon their harvest from two individuals). No PCR products were detected in samples that were not reverse transcribed (Fig 2, compare lane 8 with lane 7). Similar but fainter PCR products were obtained with the use of reverse-transcribed total RNA that was derived from samples frozen in liquid nitrogen and subsequently extracted (Fig 2, lane 5, shows the PCR products obtained from a representative sample). These data suggest that the majority of the AßPP mRNA species present within the thromboemboli contain the alternatively spliced KPI domain.
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A second series of experiments was performed to document the expression of KPI-containing isoforms of AßPP mRNA on a single cell level. For this purpose, the cDNA encoding the KPI region was amplified by PCR and cloned into pGEM-3Z and pGEM-4Z and these constructs used for preparation of 35S-labeled antisense and sense riboprobes. Fig 1I
shows a representative photomicrograph of a section of a thromboembolus analyzed by in situ hybridization with the use of the 35S-antisense probe for the KPI domain, whereas Fig 1J indicates the signal obtained after hybridization of a parallel section with the 35S-sense probe for this domain. A lower signal intensity was observed with the use of identical hybridization and exposure conditions with the KPI riboprobe, which was in vitro transcribed in the presence of 35S-UTP from a 167 bp template in comparison to the signal obtained with AßPP riboprobe that was labeled with 35S-UTP from an 804 bp template (Fig 1, compare I with G). Quantitation of the in situ hybridization signal obtained with this domain-specific riboprobe (Table) confirmed that the corresponding mRNA was elevated in the ECs lining vascular channels of these patients' thromboemboli.
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Discussion |
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Two potent inhibitors of the coagulation cascade that are produced by ECs are (1) tissue factor pathway inhibitor (for review, see Reference 48) and (2) protease nexin-2/AßPP.9 10 11 12 Because a series of initial experiments failed to detect endothelial tissue factor mRNA and antigen within chronic thromboemboli (I.M. Lang, personal observation, 1994), we subsequently directed our attention to the latter inhibitor that is capable of interacting with proteases activated during the initial phases of the coagulation cascade. More specifically, the KPI domain present in several forms of protease nexin-2/AßPP is a tight-binding cell surface inhibitor of two activated coagulation enzymes (ie, Factors XIa9 10 and IXa11 12 ), which play a key role in the intrinsic coagulation pathway leading to activation of prothrombin. The release of these forms of protease nexin-2/AßPP from both cultured human umbilical vein ECs12 24 34 35 36 37 and platelets10 28 29 38 39 40 41 has raised the possibility that this molecule plays a role in hemostasis. Our experiments document the expression of protease nexin-2/AßPP in ECs that line patent vascular lumina within the chronic organized thromboemboli. Because regions of chronic thromboemboli differ in their degree of organization and vascularization, we decided to quantitate expression of AßPP with the use of a technique that permits an examination at a single-cell level (ie, visual counting of exposed silver grains after in situ hybridization) rather than using a whole-tissue type of analysis (eg, Northern blotting). Our data indicate that AßPP is expressed at a higher level in thrombus ECs than in thrombus smooth muscle cells or in ECs of pulmonary arteries that were harvested from organ donors (Table
). In light of the elevated expression of this molecule in the thromboemboli, we analyzed these tissues for the presence of amyloid deposits, which is one of the hallmark features of Alzheimer's disease that results from the deposition of amyloid ß-protein, a 39-42–amino acid fragment of protease nexin-2/AßPP, into extracellular neuritic plaques and within cerebral blood vessel walls (for reviews, see References 13 through 16). Similar deposits are also observed in brains of individuals with hereditary cerebral hemorrhage with amyloidosis Dutch type and in sporadic cerebral amyloid angiopathy (for reviews, see References 13 through 16). However, after performing stains with Congo red, a sulfonated dye that specifically binds to amyloid plaques, no birefringent areas, plaques, or perivascular amyloid were detected on serial sections (I.M. Lang, data not shown). Thus, if amyloid ß-protein is present in the thromboemboli, it is in a form that does not react with classic amyloid stains as observed for amyloid ß-protein–immunoreactive material in other nonneural tissues (eg, surrounding microvessels in skin and intestine).32 Thrombin, serum, or cytokines (eg, IL-1) that are formed in the course of ongoing thrombosis and inflammation24 34 35 are candidate molecules within the thromboemboli that can elevate the expression of protease nexin-2/AßPP. Thrombin has also been observed to induce secretion of protease nexin-2/AßPP from a glioblastoma cell line via activation of the thrombin receptor, which results in a concomitant decrease in the level of the amyloidogenic forms of AßPP.49 A similar pathway may exist within the thromboemboli and participate in the degradation of amyloidogenic forms of AßPP. Although AßPP immunoreactivity has been detected in a number of neural and nonneural tissues,30 analysis of molecular expression in ECs outside the cerebral vasculature has been confined to in vitro studies using cultured human umbilical vein ECs.12 24 34 35 36 37 These observations suggest that protease nexin-2/AßPP is expressed at a low level in ECs that can be elevated by a number of agonists24 34 35 or trauma/cellular stress.37 The concept that AßPP is expressed at low or undetectable levels in the systemic vasculature is supported by the survey of tissues by Arai et al30 indicating that if AßPP is present within the system, it is present at levels below the sensitivity of this group's immunohistochemical system. The absence of widespread expression of ß-galactosidase in a transgenic mouse model system prepared with the use of a construct containing the regulatory region of protease nexin-2/AßPP fused to the cDNA encoding this reporter gene50 further supports the concept that AßPP expression is at a low or undetectable level under nonstimulated conditions in the systemic vasculature. Our studies also extend the Northern blotting analysis of cultured human umbilical vein ECs24 and the PCR data obtained with the use of large cerebral blood vessels and cerebral microvessels,45 which suggest that the forms of AßPP expressed by ECs are primarily composed of the alternatively spliced variants containing the KPI domain.
Because coagulant reactions occur on and are modified by interactions with cell surfaces, the capacity of protease nexin-2/AßPP to inhibit Factor IXa on endothelial surfaces in vitro (Ki=6.7x10-8 mol/L in the presence of Factor VIIIa12 ) supports a role for this molecule in regulating hemostasis at the EC surface. The observation that surface factors (eg, platelet versus endothelial surfaces) and Factor VIIIa are able to modify the inhibition of Factor IXa by protease nexin-2/AßPP raises the possibility that regions distinct from the KPI domain may also contribute its interaction with Factor IXa.12 These data coupled with the fact that protease nexin-2/AßPP is not a plasma protein but is synthesized and displayed on the cell surface as a transmembrane protein13 14 15 16 has led Schmaier and coworkers12 to suggest that the importance of membrane-associated protease nexin-2/AßPP as an anticoagulant is presently not fully appreciated. Our detection of elevated expression of protease nexin-2/AßPP in ECs within chronic thromboemboli, a tissue available after pulmonary thromboendarterectomy, may provide a useful model system to investigate the role of this molecule in regulating the hemostatic system and the preservation of regional vessel patency.
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Acknowledgments |
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This research was supported by Austrian grant FWF P10559-MED (to Dr Lang) and National Institutes of Health grants HL-23584 (to Dr Moser) and HL-49563 (to Dr Schleef).
Received December 19, 1995; revision received June 17, 1996; accepted July 8, 1996.
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