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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1189-1198

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1189.)
© 2000 American Heart Association, Inc.

Vascular Biology

Protein-Tyrosine Phosphatases in the Vessel Wall

Differential Expression After Acute Arterial Injury

Matthew B. Wright; Ronald A. Seifert; Daniel F. Bowen-Pope

From the Department of Pathology, University of Washington, Seattle. Dr Wright is now at the Department of Preclinical Cardiovascular Research, F. Hoffmann-La Roche, Basel, Switzerland.


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Abstract—Many protein-tyrosine phosphatases (PTPases) have now been identified, but little is known about PTPase expression and regulation in vascular tissue and in vascular disease. Polymerase chain reaction (PCR) amplification and cDNA fingerprinting of PTPase catalytic domains, combined with random sequencing of PCR product libraries, identified 18 (8 receptor-like and 10 cytosolic) PTPases in the rat carotid artery and revealed differential expression of 5 of these PTPases during neointima formation after balloon catheter injury. In situ hybridization was used to localize mRNA expression in vessel cross sections for the 5 differentially expressed PTPases. This revealed that for 3 PTPases (SHP1, CD45, and PTPß), differential transcript abundance was due to appearance/loss of the cell types by which they were expressed (leukocytes for SHP1 and CD45, endothelial cells for PTPß). However, mRNA expression of 2 PTPases (PTPL1 and PTP1B) was specifically upregulated by proliferating and migrating smooth muscle cells (SMCs) in characteristic temporal and regional patterns in response to vessel damage. Quantitative PCR analysis showed that PTP1B and PTPL1 were induced {approx}30-fold and {approx}60-fold, respectively, by 2 weeks after injury in the damaged vessels compared with the uninjured vessels. PTP1B was rapidly upregulated in the media after vessel injury and remained highly expressed in the developing neointima. By contrast, PTPL1 expression did not increase dramatically until the SMCs had migrated into the intima. The differential expression of PTP1B and PTPL1 by SMCs after injury suggests roles for these PTPases in the regulation of vessel wall remodeling.


Key Words: protein-tyrosine phosphatase • smooth muscle cells • injury • neointima • carotid arteries


*    Introduction
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 *Introduction
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Acute arterial injury stimulates smooth muscle cell (SMC) proliferation in the media, followed by the migration of SMCs into the intima. These events result in vessel wall remodeling and the formation of a neointima. Multiple secreted growth factors mediate the SMC proliferation and migration that occurs after balloon catheter injury of the normal rat carotid artery. Basic fibroblast growth factor appears to be the major mitogen driving early medial SMC proliferation, whereas platelet-derived growth factor has a lesser effect on SMC replication but plays a major role in promoting the migration of SMCs into the intima.1 2 3

Protein-tyrosine phosphatases (PTPases) reverse the activity of growth factor receptor tyrosine kinases and likely play important roles in the vessel wall; however, little is known of the identity of PTPases expressed by vascular tissue, and less is known about the potential roles of specific PTPases in active remodeling. Approximately 70 to 80 PTPases have been cloned and described.4 The family is broadly divided between receptor-like and cytosolic enzymes and is further divided by the similarities of accessory and regulatory motifs.5 The receptor PTPases generally contain an extracellular region with adhesion-like motifs, a single membrane-spanning segment, and 1 or 2 intracellular catalytic domains.6 The possibility that receptor PTPases play a role in cell-cell or cell-matrix adhesion and signaling has received support from the finding that the receptor PTPases rPTPµ, rPTP{kappa}, and rPTP{lambda} mediate homophilic adhesion between cells.7 8 9 In addition, the ectodomain of rPTP{zeta}/ß has been shown to interact with the matrix protein tenascin10 and the neuronal cell recognition molecule contactin.11 However, the mechanism(s) by which ligand binding to the ectodomain of receptor-like PTPases transduces cellular signals by modulating intracellular PTPase activity and/or substrate access remains unclear. The cytosolic PTPases are equally diverse. They contain a single PTPase catalytic segment and accessory modules, including cell junction-associated PDZ, src-homology 2, and band 4.1-like domains, which function to direct the appropriate subcellular compartmentalization and to mediate interactions with substrates and effector molecules.4

We chose the balloon catheter–injured rat carotid artery to study regulation of the expression of vascular PTPases because the kinetics of SMC proliferation and migration leading to neointima formation and lumen narrowing have been well defined in this model.12 We sequenced sufficient random clones from polymerase chain reaction (PCR) product libraries derived from injured vessels to identify the PTPases expressed in the normal and injured vessel wall. We used cDNA fingerprinting of degenerate PCR products amplified directly from vessel cDNA to reveal the pattern of PTPase expression during the period after injury.13 Using these methods, we identified 18 PTPases in the rat carotid artery and found that 5 of these are differentially expressed in the whole tissue after injury. In situ hybridization studies were performed for these 5 PTPases to identify the expressing cells. We found that the expression of PTP1B and PTPL1 was upregulated in proliferating and migrating SMCs during neointima formation. Taqman PCR analysis was used to quantify the extent of upregulation and showed that PTP1B and PTPL1 were induced {approx}30-fold and {approx}60-fold, respectively, by 2 weeks after injury in damaged vessels compared with uninjured control vessels. Changes in the transcript level of 3 other PTPases (PTPß, SHP1, and CD45) marked the appearance or loss of the cells by which they were specifically expressed (endothelial cells for PTPß or leukocytes for SHP1 and CD45). These results suggest that PTP1B and PTPL1 play roles in regulating SMC behavior after vascular injury and that many PTPases play constitutive or cell-type–specific functions in the vasculature.


*    Methods
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Carotid Artery Balloon Catheter Injury Model
Two sets of animals were used for these experiments. The first group of 48 male Sprague-Dawley rats (300 to 350 g, Bantin and Kingman, Kent, Washington) provided vessels for RNA extraction for PTPase cloning/cDNA fingerprint display and for fixation/embedding for in situ hybridization analysis. Individual vessels were taken from a second group of 21 animals for RNA extraction for Taqman quantitative PCR analysis. The rats were anesthetized with an intraperitoneal injection of xylazine (2.2 mg/kg body wt AnaSed, Lloyd Laboratories) and ketamine (50 mg/kg body wt Ketaset, Aveco Co, Inc). Acute injury was performed with a 2F Fogarty embolectomy catheter (Baxter) in the left and right carotid arteries essentially as described by Clowes et al.12 For the first set, animals were euthanized in groups of 6 at 6 hours, 24 hours, 4 days, 14 days, and 3 months after injury. Three additional groups of 6 animals were as follows: (1) an uninjured group, (2) a group deendothelialized ex vivo, and (3) a 12-day postinjury group, from which separated neointima and media samples were obtained. The experimental protocol was approved by the University of Washington Committee for Animal Care and Use.

Vessel Harvesting and RNA Isolation
Rats were killed by an intravenous injection of sodium pentobarbital (75 mg/kg body wt Nembutal, Abbott Laboratories) and perfused with lactated Ringer’s solution. Venous drainage was accomplished through bilateral jugular venotomy. Left and right carotid arteries were excised, stripped of most of the adventitia, and frozen in liquid nitrogen. Ex vivo–deendothelialized carotid arteries were prepared by opening excised uninjured arteries longitudinally and scraping away the endothelium with a polytetrafluoroethylene (Teflon) card. To isolate the neointima from the underlying media, excised arteries were opened longitudinally, and the neointima was stripped from the media at the internal elastic lamina.

For the cDNA fingerprint display analysis, RNA was isolated from pooled carotid arteries from 2 rats (4 arteries). The 12-day medial and neointimal samples were prepared by pooling dissected tissue from 3 vessels. For Taqman analysis, RNA was extracted from single carotid arteries. Vessels were ground in a mortar and pestle under liquid nitrogen, and RNA was extracted with TRIZol reagent (GIBCO-BRL). RNA yield and quality were determined by UV absorbance (A260) and/or visualization by ethidium bromide staining of 1 µg of each sample electrophoresed in MOPS-formaldehyde gels.

PCR amplification and cDNA Fingerprinting of PTPase Expression
A differential display PCR approach (restriction enzyme cDNA fingerprint display method) was used essentially as previously described to detect differential expression of PTPases after balloon catheter injury of the rat carotid artery.13 14 PTPases are first collectively amplified by PCR by use of unlabeled sense and labeled antisense degenerate primers to regions of sequence conservation in the catalytic domains. The resulting PCR product is a mixture of the catalytic domains of many expressed PTPases. A cDNA fingerprint of the amplified PTPases is produced by restriction enzyme digestion of the PCR product, followed by gel electrophoresis and autoradiography. The restriction fragmentation patterns are thus a blueprint of expressed PTPases within the experimental cDNA. Comparison of the cDNA fingerprints from an experimental time course is then performed to identify PTPases that may show differential or restricted expression. For the carotid balloon injury series, we compared PTPase expression by the cDNA fingerprint method in the following samples: uninjured whole carotid arteries; uninjured carotid arteries with endothelial cells removed by scraping ex vivo; whole carotid arteries at 6 hours, 24 hours, 4 days, 14 days, and 3 months after balloon catheter injury; separated neointima and media dissected apart at 12 days after balloon catheter injury; and cultured rat SMCs.

RNA was reverse-transcribed to first-strand cDNA with oligo(dT) and Superscript II reverse transcriptase (GIBCO-BRL). For PCR amplification, 3 sets of degenerate PCR primers were derived to conserved patches of amino acid sequence in the core PTPase catalytic domain. The forward primer (F1), corresponding to the (S/T)DYINA motif, was 5'-CTCTGGATCCACIGA(C/T)TA(C/T)AT(ACT)AA(CT)GC-3'. Two slightly different forward primers, designed to the less well conserved KC(V/A/H)(K/Q)YWP motif, were as follows: F2 5'-CTCTGGATCCAA(A/G)TG(T/C)GT (G/A/T/C)AA(A/G)TA(T/C)TGGC-3' and F3 5'-CTCTGGATCCAA(A/G)TG(T/C) GC(G/A/T)CA(A/G)TA(T/C)TGGC-3'. The single reverse primer (R1) corresponding to the HCSAG(I/V)GR motif was 5'-CTCTAAGCTTC(G/T)ICCIA(T/C)ICCIGCI(C/G)(T/A)(A/G)CA(G/A)TG-3'. PCR products were labeled by incorporation of the radioactively labeled primer R1 in the PCR reaction. Primer R1 was labeled by phosphorylation by T4 kinase with [{gamma}-32P]ATP. Radioactive counts (4x106 cpm) of the labeled reverse primer were added to each 40 µL PCR reaction. Each PCR used 2 µL of experimental first-strand cDNA (cDNA from 100 ng total RNA) with 50 pmol each of unlabeled forward and reverse primers. Three separate PCR reactions were performed with the F1/R1, F2/R1, and F3/R1 primer pairs for each cDNA. The optimized PCR amplification conditions were as follows: 5 cycles of 94°C for 30 seconds, 44°C for 2 minutes, and 72°C for 2 minutes and then 35 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 45 seconds, followed by an extension at 72°C for 4 minutes. The products of each PCR reaction were resolved on a DNA sequencing gel to identify the heterogeneous bands of {approx}500 bp or 300 bp derived from PTPase catalytic domain coding regions.

We normalized for the variation in product yield after first-round amplification by separating the PTPase products on an acrylamide gel and recovering the labeled PTPase fragments by the crush-and-soak method.15 Recovered material was normalized to 100 cpm/µL and then amplified in a second PCR under the same conditions for 25 cycles. Equivalent amounts (in counts per minute) of the PTPase products after second PCR were digested to completion with a panel of frequently cutting restriction enzymes (HinfI, RsaI, TaqI, and Sau3AI) in separate reactions. The restricted PCR products were separated side by side on a DNA sequencing gel. Complete digestion of the heterogeneous PTPase amplification products revealed smaller, labeled restriction fragments that uniquely identified specific PTPases within the mixture. The presence or absence and the intensity of each unique band were compared over the experimental time course to identify potentially differentially expressed PTPases.

DNA Cloning and Sequence Analysis
The F1/R1, F2/R1, and F3/R1 PTPase amplification products from the second PCR amplification reaction, with RNA from carotid arteries injured on day 4 used as a template, were subcloned into the PCR-Script plasmid (Stratagene) to generate 3 plasmid libraries. Plasmid DNA was purified from 70 to 80 randomly chosen subclones from each library, and a partial DNA sequence of the subcloned inserts was determined by single-pass dideoxy chain-termination sequencing.16 For each unique PTPase identified, 1 representative subclone was selected for complete double-stranded sequence determination. Sequence analysis was performed with the Geneworks program (Intelligenetics). Homology and database searches were performed with use of the BLAST program at the National Center for Biotechnology Information (NCBI) web page (http://www.ncbi.nlm.nih.gov/BLAST/).

Cell Culture
SMC lines from 12-day-old Wistar-Kyoto rats were kindly supplied by Dr C. Giachelli, University of Washington, Seattle. Cells were cultured in DMEM supplemented with 10% bovine calf serum.

Riboprobes
For the 5 PTPases that appeared to be differentially expressed by PCR restriction enzyme cDNA fingerprint display analysis (see Results), we obtained larger fragments by PCR amplification from carotid artery cDNA to produce constructs for generating in situ hybridization probes. All amplified fragments were subcloned into the pGEM7Z plasmid (Promega). A 1450-bp fragment of rat PTPß was obtained by PCR with a reverse primer specific for the rat PTPß catalytic domain and a forward primer specific for a conserved region of human and mouse PTPß (GenBank accession Nos. X54131 and X58289).17 A 2500-bp fragment of rat PTPL1 was obtained by PCR with a reverse primer for the rat catalytic domain and a forward primer for the full-length human and partial mouse sequences (GenBank accession Nos. X80289 and D28529). A 2300-bp internal EcoRI fragment of the resulting PCR product, corresponding to nucleotides 4482 to 6851, was recovered in pGEM7Z.18 A 1989-bp fragment of rat PTP1B, corresponding to positions 615 to 2604, was amplified with primers for the rat sequence (GenBank accession No. M33962).19 A 1650-bp fragment of rat CD45, corresponding to nucleotides 1702 to 3336, was obtained by amplification with primers for the partial sequence (GenBank accession No. M10072).20 A 1051-bp fragment of rat SHP1 was obtained by nested 3'-RACE PCR using Marathon cDNA Amplification Kit (Clontech) with primary and nested primers to the rat catalytic domain coding sequence. The receptor-like PTPase, rPTP-GMC1, is expressed specifically by glomerular mesangial cells and served as a negative control for the in situ experiments. The rPTP-GMC1 construct for producing in situ probes has been described previously.22

In Situ Hybridization
Antisense riboprobes were generated by in vitro transcription of linearized plasmid DNA with SP6 or T7 RNA polymerase in a 10-µL reaction with 200 µCi of [33P]UTP (2000 Ci/mmol, DuPont NEN). The probes were purified from unincorporated label with G-50 NICK columns (Pharmacia), hydrolyzed to an average length of 150 bp, precipitated with sodium acetate and ethanol, and resuspended in Tris-EDTA buffer.

Paraffin-embedded formaldehyde-fixed tissue sections (6-µm sections) were deparaffinized and treated with 20 µg/mL proteinase K at 37°C for 7.5 minutes. Hybridization with 5x107 cpm of probe per milliliter of hybridization solution was performed overnight at 65°C in a humidified chamber. Slides were washed to remove unbound probe, treated with RNase A, dehydrated with ethanol, air-dried, and dipped in emulsion (Kodak NTB2). After 12 days to 6 weeks, slides were developed in Kodak D19 and counterstained with hematoxylin and eosin. In situ hybridization for each PTPase was performed on 6 sections per rat carotid artery, with 3 carotid arteries (3 animals) per time point for normal (uninjured) vessels and for damaged vessels 6 hours, 24 hours, 2 days, 4 days, 7 days, 14 days, and 20 weeks after balloon catheter injury.

Taqman Quantitative PCR Analysis
Taqman quantitative PCR (Perkin Elmer) exploits the 5' nuclease activity of Taq DNA polymerase to cleave a fluorescently labeled probe during the DNA polymerization steps of the PCR cycle.23 The probe is labeled with a quencher dye (TAMRA-6-carboxytetramethyl-rhodamine) and a reporter dye (FAM-6-carboxyfluorescein). The proximity of the quencher and donor in the intact probe suppresses fluorescence. During the PCR cycle, the probe hybridizes to the template DNA and is cleaved by liberation of the reporter by Taq polymerase. The increase in fluorescence of the reporter in relation to a passive reference dye reflects product formation. The threshold value for positivity (CT value) is the point at which signal intensity is detectable above background and occurs during the log phase of the PCR reaction, a point at which product formation is directly dependent on the amount of target cDNA molecules in the sample. Thus, comparison of CT values between samples for an mRNA of interest gives relative expression levels. Primers and Taqman probes were designed with the use of Primer Express Software (PE Applied Biosystems) to rat PTP1B and PTPL1. RNA was isolated from 5 or 6 individual injured carotid arteries at days 2, 4, 7, 10, 12, and 14 after injury and from uninjured control arteries and was reverse-transcribed to cDNA (Preamplification Kit, GIBCO-BRL). The cDNAs were diluted with water. We used Extaq DNA polymerase with the supplied nucleotides and buffer (Takara) for PCR at the recommended concentrations but added MgCl2 to a final concentration of 6 mmol/L and ROX as a passive reference to a final concentration of 5 µmol/L. PCR was carried out in an ABI Prism 7700 Sequence Detector with standard cycling parameters. CT values were calculated by use of the ABI Sequence Detector software.


*    Results
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Identification of PTPases in Rat Carotid Artery
To identify PTPases expressed in the rat carotid artery, we subcloned the undigested PCR products obtained with each of the PTPase degenerate primer pairs (F1+R1, F2+R1, and F3+R1) from vessels 4 days after injury to create 3 small plasmid libraries. DNA sequence analysis of 237 independent subclones identified 20 unique PTPase catalytic domains represented by the 3 libraries. The 20 unique sequences defined 18 PTPase cDNAs, because both the first and second catalytic domains of 2 known receptor PTPases were represented (TableDown). Each pair of degenerate PTPase primers identified different (but overlapping) subsets of PTPases. Thus, the use of multiple primer sets increased the comprehensiveness of the study. However, a PTPase differing significantly in sequence from the consensus may remain undetected. Homology searches of the PTPase sequences with public databases indicated that all are known enzymes that have been cloned in the rat or are the orthologues of mouse and/or human PTPases. The data revealed that a diverse repertoire of cytosolic and receptor-like protein-tyrosine phosphatases is expressed in blood vessels (Figure 1Down).


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  		Table 1. Number of Representations of Each Carotid PTPase Identified in Random PCR Libraries



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  		Figure 1. PTPases expressed in the rat carotid artery. Diagrammatic representations of the proteins predicted from the largest splice form of each of the PTPases identified in injured rat carotid artery and the most commonly used name for each PTPase are shown. Structural domains are indicated in the box. FN-III indicates fibronectin type III–like repeat; PTP, protein-tyrosine phosphatase domain; Ig, immunoglobulin-like domain; MAM, homology to meprin; CA, carbonic anhydrase-like domain; and SH2, src-homology 2 domain.

cDNA Fingerprint Display Analysis of PTPase Expression
We used the restriction enzyme cDNA fingerprint display method to make a preliminary evaluation of changes in the PTPase expression level in the carotid artery during the response to balloon catheter injury.13 14 PTPases were amplified by 2 rounds of degenerate PCR by using primers to the conserved regions in the PTPase catalytic domain. Restriction enzyme digestion and gel electrophoresis of the mixed products revealed unique fragment patterns representing the repertoire of PTPases expressed in each sample. The relative intensity of each fragment in the cDNA fingerprinting patterns was evaluated over the time course of the balloon injury to identify PTPases that are potentially regulated at the level of mRNA expression during vessel wall remodeling.

Approximately 12 to 14 PTPases were amplified with each PCR primer pair (TableUp). To assign each fragment in the cDNA fingerprint display as the product of a specific PTPase, we compared the actual fragment size determined by gel electrophoresis with the corresponding fragment size predicted by the 18 PTPases found by random cloning and sequencing. All major restriction fragments in the displays were unambiguously assigned to each of the 18 cloned PTPases. Most of the restriction fragments exhibited little variation in intensity during the response to balloon injury (Figure 2ADown), suggesting that transcript levels of the corresponding PTPases were relatively unchanged. These constitutively expressed PTPases thus served as internal standards with which to compare the expression profiles of the remaining transcripts. This analysis suggested that 5 of the 18 vascular PTPases were regulated at the mRNA level after balloon injury (Figure 2Down). These were the receptor PTPases PTPß and CD45 and the cytosolic PTPases SHP1, PTP1B, and PTPL1. The remaining 13 PTPases appeared not to be regulated in response to injury, as suggested by maintenance of relatively uniform expression over the time course. However, their constitutive expression in the whole vessel suggests that they play some role in the vasculature.



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  		Figure 2. Detection of differential expression of PTPases in the rat carotid balloon injury model by degenerate PCR, followed by restriction enzyme cDNA fingerprint display. PTPases were amplified from cDNA prepared from carotid RNA samples at the times after injury noted above the lanes in panel A. Equivalent amounts of each PCR product were subjected to restriction enzyme digestion, followed by PAGE and autoradiography. This revealed restriction fragments for specific PTPases representing their relative expression over the injury time course. A, Representative cDNA fingerprint display pattern of PTPases, in this case obtained by amplifying with the F1/R1 primer pair (see Methods) and displayed after digestion with RsaI. To increase the probability of revealing at least 1 diagnostic fragment for each amplified PTPase, we routinely applied a panel of 4 restriction enzymes in separate digests and displays to each series of PTPase products. The RsaI restriction fragment pattern in panel A also shows fragments from PTPases whose expression does not appear to be regulated, 1 of which is indicated by an arrowhead. In addition, PTPß expression is represented by a 164-bp RsaI restriction fragment. B, PTP1B expression represented by a 163-bp Sau3AI restriction fragment (F1/R1 primers used for PCR). C, PTPL1 expression represented by a 304-bp Sau3AI restriction fragment (F1/R1 primers used for PCR). D, SHP1 expression represented by an 82-bp Sau3AI restriction fragment (F1/R1 primers used for PCR). E, CD45 expression represented by a 153-bp HinfI restriction fragment (F2/R1 primers used for PCR).

Taqman PCR Analysis of Transcript Expression
Fingerprint display is an attractive method for initial identification of transcripts whose expression level changes in response to injury, but it is not ideal for quantifying the magnitude of the change or for evaluating large numbers of samples. To do this, we used Taqman real-time quantitative PCR to quantify the transcript levels for PTPL1 and PTP1B in 5 or 6 carotid arteries from each time point after injury, including additional time points at days 2, 10, and 12 to gain a more detailed view of the time course. The results of the Taqman analysis indicated that the maximum relative increase in expression for PTPL1 is 63-fold for injured vessels versus normal uninjured control vessels and that the increase for PTP1B is {approx}30- to 40-fold for injured versus control vessels (Figure 3Down). These results are discussed, along with in situ hybridization results, in the sections below.



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  		Figure 3. Quantification of PTPL1 and PTP1B transcript levels by Taqman analysis. RNA was prepared from carotid arteries from 5 or 6 rats per time point. Each sample was analyzed in triplicate PCR reactions for expression level of each of the test genes, PTP1B (•) and PTPL1 ({circ}), and for GAPDH as a normalizing housekeeping gene (GAPDH control reagents, PE Applied Biosystems). A minimum of 3 no-template controls was included in each run. The average CT value of triplicate reactions was determined for each cDNA gene pair. Assuming a 2-fold difference in mRNA expression for each unit difference in CT value, the relative level of PTP1B, PTPL1, and GAPDH expression was determined for each sample. The expression level of PTP1B and PTPL1 in each sample was normalized with respect to GAPDH expression in each sample, and the results are plotted as the average fold increase over the expression level in untreated controls. GAPDH levels changed little under any condition or time point. Mean±SEM values for 5 or 6 vessels from each time point are plotted.

In Situ Hybridization Analysis
To identify the vessel cell type(s) expressing the 5 putatively regulated PTPases, we used in situ hybridization analysis (Figures 4 to 6DownDownDown). To increase signal intensity, we produced riboprobes from longer cDNA clones instead of the short PCR-derived catalytic domain clones (see Methods). We used an antisense riboprobe to a nonexpressed PTPase (rPTP-GMC1) as a control.22 The rPTP-GMC1 probe produced a low background throughout (data not shown) comparable to the background seen off the tissue with other antisense probes. The lack of a detectable signal showed that homologous PTPase sequences do not cause background by cross hybridization with other expressed PTPases. Additionally, because we did not clone rPTP-GMC1 from carotid tissue by degenerate PCR or observe it in the display analysis, the lack of a signal by in situ analysis confirmed its absence from the balloon injury series and supported the specificity of the degenerate cloning and display results.



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  		Figure 4. In situ hybridization demonstrates upregulation of PTP1B transcript expression after balloon catheter injury. A, PTP1B expression is not yet detectably upregulated at day 2 after injury. B, PTP1B expression is upregulated by day 4 in medial SMCs. C, PTP1B expression is further increased by day 7 (silver grain density is highest over neointimal SMCs and somewhat lower over the underlying media). D, By 14 days, expression has decreased in the neointima, and no medial expression is evident. The bright silver grains were visualized by dark-field illumination, which also revealed some underlying tissue structure via the slight refractivity of hematoxylin- and eosin-stained tissue. The red arrows show the position of the internal elastic lamina to delineate the media from the neointima. There were 3 animals per time point; exposure time was 12 days.



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  		Figure 5. In situ hybridization demonstrates increased PTPL1 expression in neointimal SMCs. A, No PTPL1 expression is evident 4 days after balloon catheter injury. B, By day 7, SMCs have appeared in the neointima, and only these cells express detectable PTPL1. C, By 14 days, proliferation is largely restricted to SMCs closest to the lumen, and this correlates with detectable expression of PTPL1. D, High-magnification (bright-field) autoradiograph from the 7-day postinjury section in panel B is shown. The red arrows show the position of the internal elastic lamina to delineate the media from the neointima. There were 3 animals per time point; exposure time was 6 weeks.



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  		Figure 6. In situ hybridization demonstrates expression of leukocyte-specific and endothelial cell–specific PTPases during neointima formation. A and B, Dark-field illumination revealed the presence of intensely hybridizing cells for SHP1 (A) and CD45 (B) antisense riboprobes early after injury in the adventitia and adherent to the vessel lumen (white arrows). By 14 days, SHP1- and CD45-positive cells are distributed throughout the maturing neointima. The insets are bright-field images of regions of 14-day neointimas at higher magnification to demonstrate association of the silver grains with specific cells (putative leukocytes) but not adjacent cells (putative SMCs). C, PTPß expression by endothelial cells is shown. A normal uninjured carotid artery shows intense PTPß expression by luminal endothelial cells (white arrowhead). The inset is a dark-field image at higher magnification. A second vessel with a mature neointima at 14 days after injury shows no luminal expression of PTPß because of the absence of luminal endothelium but shows strong PTPß expression by endothelial cells of small associated vessels in the adventitia (white arrowheads). The red arrows show the position of the internal elastic lamina to delineate the media from the neointima. There were 3 animals per time point for each PTPase; exposure time was 12 days.

For the 5 putatively regulated PTPases (each of which is discussed individually in the following sections), the results of the in situ analysis were concordant with the expression patterns suggested by the PCR fingerprint display data and the Taqman PCR analysis, with the qualification that PCR always detected the first increase at earlier time points than did in situ hybridization. This probably results from the lower sensitivity, and higher background, of the in situ analysis, which makes it virtually impossible to detect low-level expression and small changes in expression. In situ analysis revealed that 2 of the 5 PTPases (PTP1B and PTPL1) were upregulated in proliferating and/or migrating SMCs responding to injury. The remaining 3 PTPases (SHP1, CD45, and PTPß) were markers for the appearance or loss of specific cell types by which they were expressed in the whole vessel. For rPTP-GMC1, the negative control PTPase, which was not represented among the random clones or as a predicted restriction fragment in the fingerprint displays (data not shown), no expression above background was detected by in situ analysis over the experimental time course. Thus, the in situ results supported the results of the cDNA fingerprint display by demonstrating that each of the 5 putatively regulated PTPases were indeed markers of remodeling events that occur after injury, ie, endothelial denudation and regrowth, leukocyte infiltration, medial SMC proliferation, and neointima formation.

PTP1B Is Upregulated in Medial and Intimal SMCs After Injury
PTP1B is a cytosolic PTPase implicated in signal transduction through the insulin receptor and other growth factor receptor kinases. In situ hybridization analysis of balloon-injured carotid arteries revealed that injury initially upregulated PTP1B transcript expression by SMCs in the media (Figure 4Up). PTP1B was most highly expressed by SMCs that had migrated into the intima by day 7 (Figure 4CUp). During continued intimal growth (day 14, Figure 4DUp), PTP1B transcript levels remained high in SMCs closest to the lumen and decreased in a graded fashion deeper in the intima and the underlying media. By 20 weeks (data not shown), PTP1B expression was reduced throughout the intima. As calculated by Taqman quantitative PCR analysis (Figure 3Up), PTP1B was upregulated 13-fold by day 2 and 29-fold by day 4 and remained 30- to 40-fold more highly expressed until day 14 in damaged vessels compared with normal uninjured control vessels.

PTPL1 Is Upregulated in Intimal SMCs After Injury
PTPL1 is a cytosolic PTPase with structural similarities to junction-associated guanylate kinases and the tight junction-associated proteins ZO-1 and ZO-2.18 24 25 The cDNA fingerprint display did not detect PTPL1 in uninjured vessels but demonstrated detectable expression 4 days after injury (Figure 2CUp). PTPL1 also appeared to be more highly expressed in a 12-day dissected neointima compared with the underlying 12-day dissected media. Taqman PCR analysis confirmed that PTPL1 expression was upregulated 22-fold in damaged vessels compared with uninjured control vessels at day 4 and continued to increase until day 14, at which time it was upregulated 63-fold above the expression in uninjured vessels (Figure 3Up). We had observed that the in situ hybridization signal for PTPL1 was generally weaker than that for PTP1B. Taqman analysis required {approx}3-fold more cDNA template to produce CT values for PTPL1 that were comparable to the values obtained for PTP1B, which is also consistent with a generally lower expression of PTPL1 than of PTP1B.

The kinetics of PTPL1 upregulation suggested that PTPL1 transcript upregulation occurs largely in the developing intima. In situ hybridization analysis confirmed that PTPL1 is upregulated during this period and revealed that the highest level of expression is almost completely restricted to SMCs in the intima (Figure 5Up). SMCs in the media did not express PTPL1 transcripts detectable over background, even during the initial phase of medial SMC proliferation, during which PTP1B upregulation was observed. Taqman PCR analysis demonstrated that some upregulation of PTPL1 expression had occurred by day 2 (Figure 3Up), but the sensitivity and/or resolution of the in situ hybridization was not adequate to determine whether this was due to a rare SMC that had already entered the intima or to a very slight upregulation by the general population of medial SMCs.

Increased SHP1 and CD45 Expression After Injury Is Due to Leukocyte Accumulation
The SHP1 and CD45 PTPases are preferentially expressed by leukocytes. SHP1 is involved in the regulation of proliferation in myeloid and lymphoid cells and appears, under different circumstances, to positively and negatively regulate the activity of src-family kinases.26 27 28 29 CD45 is a receptor-like PTPase also expressed by hematopoietic cells that plays major roles in the antigen-stimulated proliferation of T lymphocytes. The cDNA fingerprint display patterns were similar for both PTPases (Figure 2DUp and 2EUp), revealing little expression in uninjured vessels but significantly increased expression after injury. In situ analysis showed that increased expression (Figure 6Up) is due to the appearance of scattered cells expressing high levels of SHP1 and CD45 transcripts after injury. These cells were first observed at day 4 to be adherent to the luminal surface of the vessel and associated with the adventitia. By 2 weeks after injury, SHP1- and CD45-expressing cells were observed embedded throughout the neointima. The appearance of SHP1- and CD45-expressing cells is consistent with the infiltration of T lymphocytes and macrophages during inflammation after balloon injury. In fact, immunohistochemical identification of infiltrating macrophages in intimal lesions is routinely performed by detecting CD45 protein.

PTPß Is Expressed Exclusively by Endothelial Cells
PTPß is a type III receptor PTPase with a single catalytic domain.17 The PCR cDNA fingerprint display revealed significant PTPß expression in control and injured carotid arteries throughout the time course. Interestingly, there was no detectable expression in either neointima that had been microdissected away from the underlying medial and adventitial layers or in cultured rat aortic SMCs (Figure 2AUp). The in situ hybridization analysis revealed that PTPß is expressed specifically by vascular endothelial cells (Figure 6CUp), including luminal endothelial cells of the uninjured carotid and endothelial cells of the small vessels in the adventitia. Thus, it is likely that PTPß transcripts, detected in vessels denuded of endothelium, are contributed by endothelial cells in small associated adventitial vessels (clearly distinguishable in Figure 6CUp) or perhaps by regenerating regions of luminal endothelium. Thus, PTPß expression is a specific marker for endothelial cells.


*    Discussion
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 *Discussion
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To identify PTPases that may participate in remodeling events after acute vascular injury, we applied the cDNA fingerprint display method to the well-characterized rat carotid balloon catheter injury model. A thorough sampling of random libraries derived by cloning the products of degenerate PCR identified 18 PTPases expressed in the carotid artery. cDNA fingerprint display analysis revealed that most of these PTPases did not detectably change in expression level after vascular injury during vessel remodeling. However, we did detect changes in transcript levels for 5 PTPases (CD45, SHP1, PTPß, PTP1B, and PTPL1). In the cases of PTP1B and PTPL1, the detected changes in expression after vessel injury were due to upregulated mRNA expression in SMCs as a function of cell location and/or activation state.

We detected substantial upregulation of PTP1B expression in medial and intimal SMCs after injury. PTP1B was the first PTPase to be cloned and characterized.30 31 Transfection of PTP1B in cultured cells has revealed roles in downregulating mitogenic signaling in response to activation of many different growth factor receptor tyrosine kinases.32 Mounting evidence has suggested a significant role for PTP1B in insulin receptor signaling.32 33 34 Elchebly et al35 have recently proven an in vivo role for PTP1B in insulin signaling by disrupting the PTP1B gene in mice. PTP1B-null mice develop normally but show increased sensitivity to insulin in target tissues and are resistant to becoming obese.

Recent studies have continued to support significant roles for PTP1B in regulating other growth factor receptors. Flint et al36 used mutant "substrate-trapping" forms of PTP1B to identify substrates by physical association and demonstrated that PTP1B associates with the epidermal growth factor receptor. For vascular SMCs in culture, epidermal growth factor (EGF) has been shown to be a potent mitogen.37 EGF receptor–targeted diphtheria toxin has been shown to selectively kill proliferating intimal SMCs, but not medial SMCs, in the rat carotid balloon injury model, suggesting an in vivo role of EGF signaling in intimal development.38 We have found that balloon injury results in a 30-fold increase in PTP1B expression in SMCs during neointima formation. Taken together, these data suggest that PTP1B may play an important role in regulating mitogenic signaling in activated vascular SMCs in vivo. This may occur by modulating signals through EGF or other growth factor receptors.

We found that PTPL1 mRNA expression increases 63-fold at 14 days after balloon injury of the rat carotid artery. PTPL1 was upregulated later than PTP1B and was detected in situ hybridization analysis only in intimal SMCs, whereas PTP1B was detected in medial and intimal SMCs. PTPL1 is a very large cytosolic PTPase that was cloned independently by several groups.18 39 40 41 42 Its function remains unclear, but it has been shown to be constitutively expressed by polarized epithelial cells in a variety of tissues, including lung, epidermis, and gut.40 PTPL1 has multiple protein-protein interaction domains in addition to its single PTPase domain. At the amino terminus is a band 4.1–like domain, suggesting cytoskeletal localization. After the band 4.1 domain are 5 tandem PDZ domains. PDZ domains mediate protein-protein interactions and are commonly found in proteins localizing to cellular junctions, such as junction-associated guanylate kinases and the tight-junction–associated proteins ZO-1 and ZO-2.24 25 Thus, the domain structure and epithelial cell–specific expression of PTPL1 suggest that it may be a component/regulator of membrane structures in epithelial cells. It is unclear how PTPL1 may function in SMCs during development of the neointima. However, the preferential expression of PTPL1 by neointimal SMCs versus medial SMCs is intriguing in light of suggestions that neointimal SMCs exhibit an epithelial morphology compared with underlying medial SMCs.43 44 Thus, transition of medial SMCs to an epithelium-like phenotype after migration to the intima may be due, in part, to the activity of PTPL1.

The cDNA fingerprint display patterns suggest that PTPß, SHP1, and CD45 are also differentially expressed in response to injury. However, the in situ hybridization analysis demonstrated that the changes detected in whole-tissue transcript levels reflected changes in abundance of the cell types in which each PTPase was expressed over the injury time course rather than changes in upregulation of expression per se. CD45 and SHP1 are PTPases expressed most highly in cells of hematopoietic origin. The elevated expression of SHP1 and CD45 that we detected in the carotid artery after injury correlated with the immigration of monocytes that normally accompanies the formation of the neointima.45 We did not colocalize transcript expression with other leukocyte markers, but it was clear from the in situ hybridization data that high-level expression was restricted to scattered cells and that the number of these cells was consistent with the report by Verheyen et al45 in 1988 that macrophages represent 6% to 12% of the cells in the neointima. In fact, CD45 is itself a commonly used immunohistochemical marker for identifying macrophages in lesions. We found that SHP1 expression, in contrast to CD45 expression, was not entirely restricted to inflammatory cells. The PTPase cDNA fingerprint display data showed some SHP1 expression in cultured SMCs, and the in situ hybridization analysis indicated that in addition to the intense SHP1 expression by the leukocyte population in the neointima, the remaining cells (SMCs) showed low but detectable SHP1 expression. Thus, the overall increase in SHP1 expression in the neointima can be attributed to high expression by immigrating monocytes and lower expression by SMCs.

The in situ hybridization studies revealed that PTPß, a large transmembrane PTPase,17 is specifically expressed by endothelial cells. PTPß has high catalytic activity against many artificial substrates in vitro.46 PTPß itself has been shown to be a substrate for serine phosphorylation by protein kinase C.47 However, little is known of potential in vivo functions of PTPß other than that provided by a report describing a 12-fold increase in total plasma membrane–associated PTPase activity in confluent versus sparse cultures of human umbilical vein endothelial cells, which was attributed to a parallel increase in PTPß mRNA expression.48 These observations and the endothelium-specific expression of PTPß mRNA revealed in the present study suggest that PTPß may be a specific regulator of endothelial cell adhesion or endothelial permeability.

In summary, many different receptor and nonreceptor PTPases are expressed in the carotid artery, some of which may play important roles in dynamic remodeling of the vessel wall in response to injury. Further analysis of these important signaling proteins will improve the understanding of cellular response in vascular pathologies.


*    Acknowledgments
 
This study was supported by a grant from F. Hoffmann-La Roche Ltd, Basel, Switzerland, for cardiovascular research and from the National Institutes of Health (DK-54857). We would like to thank Joyce Murphy for performing the in situ hybridization and Bill Hanson and Patty Moesner for help with DNA cloning and sequencing. Our appreciation is extended to Christopher Jackson and Eric Olson for performing the carotid balloon injury procedure and to Michael Reidy for additional material. Our gratitude is expressed to Luis Borges, Charles Murry, and Jürgen Fingerle for critical reading of the manuscript.


*    Footnotes
 
Reprint requests to Daniel F. Bowen-Pope, PhD, Department of Pathology, University of Washington, Box 357470, Seattle, WA 98195-7470.

Received July 2, 1999; accepted January 18, 2000.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
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 *References
 

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