Expression of Angiopoietin-1, Angiopoietin-2, and Tie Receptors after Middle Cerebral Artery Occlusion in the Rat

MCAO Model

All experiments were performed in male Fisher 344 rats (Iffa Credo, L’Abresle, France) weighing 220 to 300 g. Animals were housed under standard conditions with free access to rat chow and tap water before and after surgery.

Irreversible occlusion of the right middle cerebral artery was performed as described previously. 3,29,30 Briefly, animals were anesthetized with 2% isoflurane in a 70/30 (by volume) nitrous/oxygen mixture and, using an operating microscope, the right middle cerebral artery was exposed by subtemporal craniectomy. The artery and its lenticulostriate branches were then occluded by bipolar electrocoagulation. Sham-operated control animals were prepared in similar manner, except that the exposed middle cerebral artery was not occluded. Afterward, retracted soft tissues were replaced, wounds were sutured, anesthesia was discontinued, and the rats were put back into their cages. Body temperature was maintained at 37°C by means of a rectal probe connected to a heating pad (CMA 150; Carnegie Medicine, Stockholm, Sweden) during surgery and until animals regained consciousness. Thereafter, rectal temperature was checked frequently (every 10 to 15 minutes) during the following 2 hours and, if necessary, it was corrected to 37°C by placing a heating pad under the cage. After MCAO the animals survived for 3 hours, 6 hours, 12 hours, 24 hours, 3 days, and 7 days (n = 4 to 6 per time). Thereafter the animals were decapitated under deep anesthesia. Brains were removed within 5 minutes after decapitation, frozen in OCT embedding medium (Sakura Finetec, Torrance, CA) on dry ice, and stored at −80°C until further processing. Sham-operated animals (at 3 hours and 24 hours) served as controls in all experiments mentioned.

Magnetic Resonance Imaging

To verify successful MCAO and to visualize the lesioned territory all brains were characterized by nuclear magnetic resonance imaging before brain removal as previously described. 3 Briefly, in animals surviving 24 hours or longer, infarct volume was determined by means of T₂-weighted quantitative in vivo magnetic resonance imaging. 31 The rat was anesthetized with 1% to 1.5% isoflurane delivered via a face mask and positioned with its head in the resonator. Each animal was then subjected to one imaging cycle in which 13 contiguous T₂-weighted coronal slices of the brain (1.2 mm thick) were taken using a RARE sequence (optimized parameters: repetition time, 3,000 ms; effective echo time, 66 ms; spatial resolution in plane, 156 μm²). The total measuring time was 5 minutes.

At early times (up to 12 hours) after MCAO, the ischemic lesion was visualized in diffusion-weighted magnetic resonance images. Eleven 1.2-mm-thick contiguous coronal slices were acquired in 5 minutes (TR = 1,500 ms; TE = 37.2 ms, two averages) using a multislice Stejskal-Tanner-like spin echo sequence. 32 The diffusion gradient was applied along the left-right axis of the animal, choosing a nominal b value of 1,500 s/mm². The in-plane spatial resolution was 312 μm².

In Situ Hybridization

Ten-μm-thick frozen sections were melted on silanized (3-aminopropyltriethoxysilane; Fluka, Buchs, Switzerland) glass slides, dried at 50°C, and fixed for 15 minutes in 4% paraformaldehyde/phosphate-buffered saline (PBS) followed by dehydration through ethanol (30%, 60%, 80%, 95%, and 100% ethanol, 5 minutes each). Slides were incubated in 0.2 mol/L HCl for 10 minutes at room temperature followed by digestion with Proteinase K (10 μg/ml) (Sigma Chemical Co., Deisenhofen, Germany) for 10 minutes at room temperature and acetylation with 0.1 mol/L triethanolamine (Sigma Chemical Co.) mixed with 0.25% acetic anhydride (Fluka) for 10 minutes at room temperature. Sections were then prehybridized in 4× standard saline citrate (SSC), 0.02% sodium dodecyl sulfate, 5× Denhardt’s solution, 50% ultrapure formamide (Life Technologies, Inc., Karlsruhe, Germany), 5% dextran sulfate (Sigma Chemical Co.), and 0.5 mg/ml yeast tRNA (Sigma Chemical Co.) for 5 hours at room temperature. Hybridization was performed with a digoxigenin-labeled (Boehringer Mannheim, Mannheim, Germany) cRNA generated by in vitro transcription using the following cDNA templates: a 560-bp NotI-EcoRI Ang-1 cDNA fragment, a 640-bp EcoRI-XhoI Ang-2 cDNA fragment, a 560-bp SacII-NotI tie-2 cDNA fragment encoding part of the murine tie-2 extracellular domain, and a 600-bp XbaI-XhoI tie-1 cDNA encoding part of the murine tie-1 extracellular domain. Labeled cRNA probes were used at a concentration of 0.5 ng RNA/μl. Hybridization with sense probe served as control. Tissue sections were incubated in a humidified chamber under glass coverslips at 70°C (hybridization oven, Biometra, Göttingen, Germany) for 16 hours to 18 hours. Posthybridization stringency washes included 0.2×SSC for 30 minutes at 70°C, 2×SSC for 2 minutes at room temperature, 0.2×SSC for 15 minutes at 70°C, and 2×SSC for 5 minutes at room temperature. Each wash was performed twice. Hybridized probes were detected by an anti-digoxigenin antibody conjugated to alkaline phosphatase (diluted 1:500, 1 hour at room temperature; Boehringer Mannheim) using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution as substrate (Boehringer Mannheim). Color reaction time ranged from 2 hours to 2 days, after which slides were rinsed in PBS, counterstained with hematoxylin or methyl green, finally rinsed in aqua dest, and mounted in elvanol.

Immunohistochemistry

Immunohistochemistry was performed as described previously. 3 The following antibodies were used: rabbit polyclonal anti-human VEGF-A20 (0.5 μg/ml; Santa Cruz, Heidelberg, Germany), mouse monoclonal anti-rat CD11b (20 μg/ml; Serotec, Oxford, UK), mouse monoclonal anti-rat ED1 (Serotec), polyclonal anti-Ibal antibody (14.6 μg/ml, gift of Dr. Yoshinori Imai, Tokyo, Japan), monoclonal anti-glial fibrillary acid protein (GFAP) (5 μg/ml; Boehringer), rabbit polyclonal anti-human von Willebrand Factor (29 μg/ml; DAKO, Glostrup, Denmark), mouse monoclonal anti-human tie-2 (10 μg/ml; gift of Dr Kevin Peters, Durham, NC), mouse monoclonal anti-human tie-1 (15 μg/ml; gift of Dr. Kari Alitalo, University of Helsinki, Helsinki, Finland), rabbit polyclonal anti-mouse Ki67 (Dianova, Hamburg, Germany) and mouse monoclonal anti-rat CD31 (6 μg/ml; Serotec). In all immunohistochemical experiments omission of the primary antibody served as control. According to the primary antibody the following secondary antibodies were used: biotinylated rat anti-mouse, rabbit anti-mouse, or goat anti-rabbit antibodies (diluted 1:300 in 20% goat serum/PBS/0.1% Triton; Dianova), respectively. Sections stained for tie-1 were additionally incubated with biotinylated goat anti-rabbit sandwich antibody for 1 hour. Thereafter, slides were washed and incubated with peroxidase-conjugated streptavidin (Vectastain Kit ABC; Vector Laboratories, Burlingame, CA) for 1 hour at room temperature and then rinsed in PBS/0.1%Triton. The immunoperoxidase reaction was visualized with 3,3′-diaminobenzidine-HCl buffer tablets (Sigma Chemical Co.) or 3-amino-9-ethylcarbazole (Vector Laboratories) and 0.006% H₂O₂. The slides were briefly counterstained with hematoxylin or methyl green, finally rinsed in aqua dest, and mounted in elvanol.

Double-Labeling Experiments

Semiquantitative Analysis (Table 1) ▶

For semiquantitative analysis two to four randomly chosen high magnifications fields (×200) in the peri-infarct and the corresponding area in the contralateral hemisphere at different times after MCAO were assessed. Areas analyzed are shown in Figure 1 ▶ .

Ang-2 mRNA, tie-2 mRNA, and protein-expressing blood vessels, as well as proliferating and apoptotic vessels were counted. In contrast, for tie-1 mRNA and protein analysis only vessels with increased staining levels compared to vessels in the contralateral hemisphere were taken for analysis. For Table 1 ▶ stained blood vessels were classified as follows: −, no staining; +, 1 to 3 blood vessels; ++, 4 to 9 blood vessels; +++, >9 blood vessels.

Ang-1 mRNA expression in astrocytes and neurons was assessed in a similar manner using the following classification: −, no staining, +, 1 to 3 cells; ++, 4 to 9 cells; +++, >9 cells.

Expression of Ang-1 mRNA and Ang-2 mRNA

Ang-1 mRNA was constitutively expressed in astrocytes and in cerebellar Purkinje cells, whereas other neurons showed little expression. Blood vessels did not express Ang-1. No alteration of Ang-1 mRNA expression was observed after occlusion of the middle cerebral artery (Table 1) ▶ .

Ang-2 mRNA signal was first detected in single cells in the infarct area 6 hours after MCAO and reached a maximum 12 to 24 hours after MCAO. Expression was confined to single cells localized in the infarct area, in the peri-infarct zone and, although in fewer cells, in the corresponding areas of the contralateral hemisphere (Figure 2 ▶ , expressing cells marked by arrows). Both the ipsilateral and contralateral hippocampal formation showed induction of Ang-2 mRNA as well. The majority of Ang-2 mRNA-expressing cells seemed to be ECs. No Ang-2 transcripts were found in sham-operated animals.

Three days after MCAO the Ang-2 mRNA expression pattern considerably changed. Ang-2 mRNA was expressed at high levels in small and larger vessels around the infarct area (marked by arrows and arrowhead, respectively, in Figure 2 ▶ ; compare low-expressing large vessel at 12 hours MCAO marked by arrowhead). However, in contrast to Ang-2 mRNA induction in single cells during the first 24 hours, transcripts were now detected in all ECs of an expressing vessel. Yet, Ang-2 mRNA in the contralateral hemisphere had totally disappeared. Seven days after MCAO Ang-2 mRNA expression had almost returned to baseline levels. Results are summarized in Table 1 ▶ and are illustrated in Figure 2 ▶ .

Characterization of Ang-2 mRNA-Expressing Cell Types

To characterize the cell types expressing Ang-2 mRNA, double-labeling experiments combining in situ hybridization and immunohistochemistry were performed. Anti-GFAP, anti-αIbaI, and anti-vWF antibodies were used for detection of astrocytes, macrophages/microglial cells, and ECs, respectively.

Most Ang-2 mRNA-expressing cells also expressed vWF and were thus identified as ECs (Figure 3a) ▶ . Ang-2 mRNA-expressing cells were mainly located at the tip of an EC cord, although after 3 days of MCAO larger vessels around the infarct area also expressed high levels of Ang-2 mRNA. In addition, GFAP immunohistochemistry detected Ang-2 mRNA up-regulation in astrocytes in the peri-infarct area 24 hours after MCAO (Figure 3b) ▶ . No co-localization of Ang-2 mRNA and IbaI protein was found at any time of investigation (not shown), suggesting that microglial cells/macrophages did not express Ang-2 mRNA.

Expression of Tie-1 mRNA and Immunolocalization of Tie-1 Protein

During the first 24 hours after MCAO tie-1 mRNA and protein were expressed at low levels (not shown). A marked up-regulation of both tie-1 mRNA and protein was observed 3 days after MCAO. Labeling was confined to small and larger vessels in the peri-infarct zone. Seven days after MCAO tie-1 mRNA and protein expression returned to baseline levels. Results are summarized in Table 1 ▶ and illustrated in Figure 4 ▶ .

Expression of Tie-2 mRNA and Immunolocalization of Tie-2 Protein

Tie-2 mRNA was first detected 3 days after MCAO in vessels immediately adjacent to the infarct area. At 7 days after MCAO tie-2 mRNA levels had declined and staining was no longer seen. In contrast, tie-2 protein was broadly expressed in the endothelium of the quiescent vasculature of the rat brain. Interestingly, no ischemia-induced up-regulation could be observed at any time. Results are summarized in Table 1 ▶ and are illustrated in Figure 5 ▶ .

Vascular Remodeling, EC Apoptosis, and Proliferation

It is suggested that Ang-2 has a dual role in vascular remodeling. It may mediate vessel regression or vascular proliferation depending on the absence or presence of VEGF. Our interest was to determine whether the spatial and temporal expression pattern of Ang-2 and VEGF would suggest a similar mechanism in MCAO-induced angiogenesis. Apoptotic (regressing) ECs were detected by combining the TUNEL assay with lectin staining. In sham-operated control animals and in 3- and 6-hour MCAO animals no apoptotic ECs were detectable. Twelve hours after MCAO the first apoptotic ECs appeared in the ischemic area and in the peri-ischemic zone (Figure 6a) ▶ . Notably, single apoptotic cells were also located in the contralateral hemisphere (insert in 6a). All cells undergoing apoptosis in the contralateral hemisphere were invariably identified as ECs. By 24 hours the number of apoptotic cells including ECs in the center of the ischemic lesion had considerably increased, however no apoptotic ECs in the peri-infarct area or the contralateral hemisphere were detectable at that time (Figure 6b) ▶ . Three and 7 days after MCAO no apoptotic ECs could be identified in the peri-infarct zone or in the contralateral hemisphere.

To identify proliferating vessels, we performed double immunohistochemistry using anti-CD31 and anti-Ki67 an tibodies. Proliferating ECs were only detectable in the peri-infarct area. Co-expression started 24 hours after MCAO, reached a maximum at 3 days MCAO, and decreased thereafter (Figure 6, c and d) ▶ . At 24 hours proliferation was confined to small seized vessels, at 3 days also larger vessels showed proliferating ECs. Results are summarized in Table 1 ▶ .

Expression of Angiopoietins

Ang-1 is hypothesized to be required for the stabilization of peri-endothelial contacts with surrounding smooth muscle cells in mature vessels. 36 In our study, Ang-1 mRNA was constitutively expressed in a subset of glial and neuronal cells throughout the cortex and cerebellum as previously described. 34 Corresponding with reports showing that hypoxia fails to up-regulate Ang-1 mRNA 37-39 we observed no change in its expression after MCAO.

Ang-2 is thought to play a role at sites of vascular remodeling in the adult by blocking the constitutive expression of Ang-1 and allowing the vessel to revert to a more plastic and unstable state. 36 Our finding of an early Ang-2 mRNA up-regulation in the tips of EC cords is reminiscent of the Ang-2 induction at the invading front of vascular sprouts in the developing corpus luteum. 16

Ang-2 mRNA up-regulation may be because of various mechanisms. Hypoxia and VEGF have been reported to increase Ang-2 mRNA in ECs in vitro as early as 2 hours with VEGF acting in a time- and concentration-dependent manner. 39 After MCAO, VEGF mRNA is up-regulated as early as 3 hours with a peak at 12 hours after MCAO. VEGF protein is first detected by 6 hours reaching a maximum in protein expression at 24 hours. 3 Ang-2 induction by VEGF would be expected to occur later than 6 hours, because it would require diffusion to EC, binding to VEGF receptor, and cell signaling. Thus, it seems likely that during the first hours of ischemia induction of Ang-2 is mainly hypoxia-induced, whereas VEGF-induced up-regulation might be involved at later times. The early induction of Ang-2 by hypoxia would render ECs more accessible to angiogenic inducers such as VEGF by loosening the EC/pericyte cell contact. The observed Ang-2 expression in astrocytes, a component of the blood-brain barrier, may contribute to this process. This would lead to further Ang-2 up-regulation, successive vessel outgrowth, and vascular proliferation.

An unexpected observation was the increase of Ang-2 transcripts in ECs in both hippocampal formations. Interestingly, the hippocampal formation is known to be vulnerable to hypoxic and ischemic events. 40 Neither VEGF up-regulation nor reductions in cerebral blood flow 41 have been described in this area. It is tempting to speculate that the secretion of a yet unidentified factor, likely transmitted by neuronal signaling, contributes directly or indirectly to Ang-2 mRNA up-regulation in ECs. The presence of such a factor might also help to explain Ang-2 up-regulation as well as the reported VEGF induction and microglia activation in the contralateral hemisphere. 3,12

Potential Role of Ang-2 in Vascular Remodeling after MCAO

It is suggested that vessel destabilization by Ang-2 in the presence of high VEGF levels primes the vessel to mount a robust angiogenic response state. 36 In our model, double immunofluorescence with the EC marker CD31 and proliferation marker Ki67 was used to identify proliferating ECs. Vessel growth started at 24 hours after MCAO with a maximum induction at 3 days MCAO. Interestingly, Ang-2 expression pattern and vessel proliferation seemed to correlate, eg, both were seen in small vessels at 24 hours and in small and large blood vessels at 3 days after MCAO. These results suggest that Ang-2 up-regulation is an important component and prerequisite for the induction of vascular proliferation. Neither in the contralateral hemisphere nor in the hippocampal formation was vessel proliferation or an increase in vessel density detected. Possibly, VEGF expression in these areas is not sufficient to induce vascular proliferation.

Vessel destabilization mediated by Ang-2 in the absence of VEGF is proposed to lead to frank vessel regression as seen in atretic follicles 26 or in tumor formation. 27,28 We supposed that Ang-2 mRNA-expressing EC cord tips might undergo regression at early times in the peri-infarct area or in the contralateral hemisphere, when VEGF protein expression has not yet reached its maximum. To investigate apoptotic EC death after MCAO we combined the TUNEL assay with a vessel-specific lectin labeling. The first apoptotic cells were seen in the peri-ischemic zone in 6-hour MCAO animals. Apoptotic ECs could first be detected 12 hours after MCAO, mainly located in the ischemic area and in the peri-ischemic zone. Notably, single apoptotic ECs were also located in the contralateral hemisphere. At 24 hours MCAO, the time showing maximal VEGF expression, no apoptotic vessels around the infarct zone or in the contralateral hemisphere were detectable any longer. In contrast, total apoptotic cell number in the infarct area was increased.

For technical reasons, we could not investigate induction of Ang-2 and apoptosis within the same sections. However, Ang-2 mRNA-expressing ECs and apoptotic ECs were localized in the same regions, namely the peri-infarct area and the contralateral hemisphere. In addition, ECs undergoing apoptosis and expressing Ang-2 were both situated predominantly in the tips of EC cords. Taken together, these findings are highly suggestive of an Ang-2-mediated induction of EC cell death at 12 hours after MCAO. Although VEGF protein is present in these brain regions starting at 6 hours, the very concentration at the EC might not be sufficient to rescue the cells from apoptosis. Ang-2-induced susceptibility of ECs for apoptosis would thus be a transient process, lasting less than 12 hours. Increasing VEGF concentrations at later times probably counteracts this signal. Our observations support the hypothesis that Ang-2 induction leads to vessel proliferation or regression depending on the presence or absence of VEGF. 42

Expression of the Tie-Receptor Family

The receptor tie-2 has been implicated in vascular maintenance as well as angiogenesis. 22,34 It has been shown that both hypoxia and VEGF only slightly modulate tie-2 expression in vitro. 38,39 In our MCAO model we observed differing regulation patterns of tie-2 mRNA and protein levels. Tie-2-protein was widely expressed in the quiescent adult vasculature of the rat brains consistent with previous reports, 22 whereas tie-2 mRNA transcripts were barely detectable. At 3 days after MCAO a marked induction of tie-2 mRNA was seen in vessels in the peri-infarct area, which, surprisingly, did not lead to a concomitant increase in tie-2 protein. A similar discrepancy between tie-2 mRNA and protein regulation was observed in postnatal brain vasculature of the rat (our own unpublished observations). However, as minor alterations in protein levels might not be detected by immunohistochemistry, small increases of tie-2 protein cannot be excluded. Tie-2 mRNA levels gradually decreased with brain maturation, yet tie-2 protein remained constitutively expressed at high levels.

Tie-1 is also known to be implicated in angiogenesis. 35 In most adult organs except lung and heart only little tie-1 message can be detected. 43 Hypoxia and VEGF have been shown to up-regulate tie-1 protein in vitro in a time-dependent manner as early as 1 hour on stimulus. 44 Further, tie-1-mRNA expression has recently been described to be increased in microvessels residing in the peri-infarct area in an embolic MCAO rat model. 45 In our model tie-1 mRNA and protein levels were up-regulated only at 3 days after MCAO, which interestingly coincides with high expressions levels of the Ang-2/tie-2 system. Tie-1 was subsequently down-regulated to baseline levels at 7 days.

The failure of angiogenesis and insufficient growth of collateral vessels is a major problem in vascular diseases, such as stroke. It is of considerable importance for therapeutic purposes to elucidate the exact mechanisms by which the growth factor signaling cascade can be recruited for an angiogenic response in adults. Our study might provide a new experimental basis for treating cerebro-ischemic diseases by rescuing ECs from undergoing apoptosis and stimulating vessel proliferation, which could probably reduce infarct size.