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Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1811–1823 & 2008 ISCBFM All rights reserved 0271-678X/08 $30.00 www.jcbfm.com

Induction of cerebral arteriogenesis leads to early-phase expression of protease inhibitors in growing collaterals of the brain Philipp Hillmeister1,8, Kerstin E Lehmann1,8, Anja Bondke1,2, Henning Witt3, Andre´ Duelsner1, Clemens Gruber4, Hans-Jo¨rg Busch5, Joachim Jankowski6, Patricia Ruiz-Noppinger3, Konstantin-Alexander Hossmann7 and Ivo R Buschmann1,5 1

Research Group for Experimental and Clinical Arteriogenesis, Department of Internal Medicine/Cardiology, (CC13), CCR—Center for Cardiovascular Research, Charite´—Universita¨tsmedizin Berlin, Berlin, Germany; 2 Charite´ Universita¨tsmedizin Berlin, Center for Preclinical Studies (CC2), Institute of Physiology, Berlin, Germany; 3CCR—Center for Cardiovascular Research, Max-Planck-Institute for Molecular Genetics, Berlin, Germany; 4Department of Anatomy, Institute of Integrative Neuroanatomy, Charite´—Universita¨tsmedizin Berlin, Berlin, Germany; 5Department of Internal Medicine (Cardiology), University of Freiburg, Freiburg i. Br., Germany; 6Medical Clinic IV, Charite´-Universita¨tsmedizin Berlin, Berlin, Germany; 7Max-Planck Institute for Neurological Research, Cologne, Germany

Cerebral arteriogenesis constitutes a promising therapeutic concept for cerebrovascular ischaemia; however, transcriptional profiles important for therapeutic target identification have not yet been investigated. This study aims at a comprehensive characterization of transcriptional and morphologic activation during early-phase collateral vessel growth in a rat model of adaptive cerebral arteriogenesis. Arteriogenesis was induced using a three-vessel occlusion (3-VO) rat model of nonischaemic cerebral hypoperfusion. Collateral tissue from growing posterior cerebral artery (PCA) and posterior communicating artery (Pcom) was selectively isolated avoiding contamination with adjacent tissue. We detected differential gene expression 24 h after 3-VO with 164 genes significantly deregulated. Expression patterns contained gene transcripts predominantly involved in proliferation, inflammation, and migration. By using scanning electron microscopy, morphologic activation of the PCA endothelium was detected. Furthermore, the PCA showed induced proliferation (PCNA staining) and CD68 + macrophage staining 24 h after 3-VO, resulting in a significant increase in diameter within 7 days after 3-VO, confirming the arteriogenic phenotype. Analysis of molecular annotations and networks associated with differentially expressed genes revealed that early-phase cerebral arteriogenesis is characterised by the expression of protease inhibitors. These results were confirmed by quantitative real-time reverse transcription-PCR, and in situ hybridisation localised the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and kininogen to collateral arteries, showing that TIMP-1 and kininogen might be molecular markers for early-phase cerebral arteriogenesis. Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1811–1823; doi:10.1038/jcbfm.2008.69; published online 2 July 2008 Keywords: arteriogenesis; cerebrovascular; epithelium; proliferation; gene expression; stroke

Introduction Correspondence: P Hillmeister, Research Group for Experimental and Clinical Arteriogenesis, Department of Internal Medicine/ Cardiology, (CC13), CCR—Center for Cardiovascular Research, Charite´—Universita¨tsmedizin Berlin, Hessische Strasse 3-4, 10115 Berlin, Germany. E-mail: [email protected] 8 These authors contributed equally to this work. This study was supported by the Deutsche Forschungsgemeinschaft, Germany (Grant DFG BU1141/4-2) and the Volkswagen Foundation, Germany. Received 10 April 2008; revised 2 June 2008; accepted 6 June 2008; published online 2 July 2008

Arteriogenesis refers to a positive outward remodelling of preexisting collateral arteries into larger calibre vessels, which bypasses sites of occlusion (Buschmann and Schaper, 2000). Hence, arteriogenesis constitutes an effective biologic rescue mechanism against the detrimental effects of arterial stenosis. Once a steep pressure gradient develops within the occluded arterial tree, recruitment of collateral pathways allows perfusion of the endangered peripheral tissue with nutrient blood flow (Liebeskind, 2004).

Early response in cerebral arteriogenesis P Hillmeister et al 1812

We recently provided the first evidence that cerebral hypoperfusion induces a compensatory growth of collateral arteries in the brain (Busch et al, 2003). By occluding three out of four extracranial arteries to the brain, one carotid artery and both vertebral arteries (three-vessel occlusion (3-VO)), a significant redistribution of blood flow is induced via the posterior cerebral artery (PCA), which showed a significant positive outward remodelling, increasing its diameter by 38%. In addition, application of the proinflammatory cytokine GM-CSF (granulocyte–macrophage colony-stimulating factor) in the latter same hypoperfusion model led to a further therapeutic increase in collateral growth (Buschmann et al, 2003; Hossmann and Buschmann, 2005), and correlated with a significant reduction in experimentally induced stroke volume (Schneeloch et al, 2004). This was the first evidence for therapeutic arteriogenesis in the brain (Love, 2003). To find novel therapeutic pharmaceutical compounds to treat arterial occlusive disease, more and more efforts are being undertaken to unravel the molecular processes of adaptive arteriogenesis (Lee et al, 2004; Prior et al, 2004; van Royen et al, 2001). Therefore, the purpose of this study was to induce collateral growth in the rat brain by employing the 3-VO model and subsequently to investigate the early phase of gene expression of the selectively isolated proliferating collateral pathways. To our knowledge, this is the first study to analyse the molecular mechanisms of arteriogenesis in the brain. Here, we present a comprehensive analysis of the gene expression profile upon experimentally induced arterial high flow across the PCA (Coyle and Heistad, 1991; Hendrikse et al, 2001). Database for Annotation, Visualization, and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA; Redwood City, CA, USA) was used to investigate gene annotations, molecular networks, and canonical pathways activated as a consequence of collateral growth in the brain. We characterised gene expression patterns at early time points (24 h) of cerebral arteriogenesis and identified molecular markers (protease inhibitors t-kininogen (t-KNG) and tissue inhibitor of metalloproteinase-1 (TIMP-1)) specifically expressed at the sites of increased levels of arterial blood flow in the PCA. Furthermore, by using latex angiography, immunohistochemistry, and scanning electron microscopy (SEM), we were able to show novel data on the growth dynamics and morphology of collateral arteries in the brain (PCA).

Materials and methods Animal Model Experiments were performed in male Sprague–Dawley rats (300 to 350 g; Harlan-Winkelmann, Borchen, Germany) in accordance with the German Law for the Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1811–1823

Protection of Animals and the National Institute of Health Guidelines for Care and Use of Laboratory Animals. The nonischaemic brain hypoperfusion 3-VO was used to induce adaptive arteriogenesis in the brain; occlusion of the left carotid artery and both vertebral arteries was carried out as previously described in detail (Busch et al, 2003).

Surgical Methods Anaesthesia was induced by inhalation of 2% to 4% isoflurane and maintained with 2% isoflurane in oxygen. Analgesia was achieved by 0.1 mg/kg buprenorphine administered subcutaneously and intraoperatively, and 0.05 mg/kg BID administered subcutaneously for 2 days after surgery. Vascular occlusions were carried out by electrocoagulation of both vertebral arteries, using a paravertebral access (Pulsinelli et al, 1983), followed by left common carotid artery ligation. To ensure cerebral hypoperfusion, cerebral blood flow was measured by transcranial LDF (laser-Doppler flowmetry; PeriSoft, PeriMed, Ja¨rfa¨lla, Sweden). Continuous LDF of both hemispheres was performed throughout the experiment. The skull above the frontoparietal cortex was exposed and a laser probe placed directly onto the skull bone. The LDF device was kept in an identical position before and after 3-VO. Successful 3-VO surgery (results for LDF), without detectable neurologic damage (immunohistological evaluation), has been described in detail by Busch et al (2003) and Schneeloch et al (2004).

Visualisation of Cerebral Angioarchitecture Cerebrovascular anatomy was studied after maximal vasodilation by a modification of the postmortem latex perfusion method of Maeda et al (1998). The external PCA diameter was measured with a stereozoom microscope (Leica MZ6) equipped with a calibrated eyepiece micrometer. Data sets are presented as mean±s.e.m. Diameter changes were analysed for statistical significance by unpaired Student’s t-test. Statistical significance was assumed for P < 0.05.

Histochemical Analysis After 4% PFA fixation, samples were embedded in paraffin and 5 mm sections for immunohistochemistry and haematoxylin–eosin staining were prepared. After heat-induced epitope retrieval (citrate buffer, 10 mmol/L), proliferating cells were identified by a monoclonal mouse antibody against PCNA (1:100, SM1421P; Acris, Karlsruhe, Germany) using the ARK-Kit (K3954; Dako, Carpinteria, CA, USA) for secondary antibody staining. An ED-1 (CD68) antibody (1:100, BM 4000; Acris) was used to identify macrophages (1:200, PA43002; Little Chalfont, Buckinghamshire, UK). The cell nucleus was stained with Hoechst 33342 (Molecular Probes Inc., Eugene, OR, USA). Representative sections are shown for PCNA and CD68. The animals were enrolled into the study as follows:


control group (n = 6), 24 h post-3-VO (n = 6), 24 h postsham (n = 6), 3 days post-3-VO (n = 6), and 3 days postsham (n = 6). A total of 12 sections from each animal were analysed in a blinded approach by three independent investigators. Images were obtained using a Leica DM-R microscope.

Scanning Electron Microscopy For SEM, male adult (300 to 400 g) Sprague–Dawley rats (n = 16) were killed 24 h or 3 days after 3-VO or sham operation. In brief, animals were deeply anaesthetised with a mixture of ketamine, xylazine, and heparin (Geisler et al, 2007), and fixed by transaortical perfusion with formaldehyde, glutaraldehyde, and methylene blue (Andres et al, 1999). Posterior cerebral arteries were isolated and dehydrated in ethanol concentrations of 30% (2 h), 50% (2 h), and 75% (overnight). Samples were opened lengthwise, treated with OsO4 (4%, 2 h), critical-point dried (CPD 30; Bal-Tec), sputtered with ionised gold in a high-pressure argon atmosphere (CDC40), and images were obtained using SEM (Quanta 200, FEI, Kassel, Germany).

Area of Interest and Selective Tissue Isolation A total of 24 h and 3 days after 3-VO or sham surgery, the part of the PCA within the circle of Willis and the posterior communicating artery (Pcom) were isolated and considered as our area of interest to study cerebral arteriogenesis, as shown in Supplementary Figure 1. RNA was extracted from the area of interest, the combined PCA and Pcom, abbreviated as PCA/Pcom in this manuscript. RNA isolation for gene expression analysis was performed for five treatment groups: 24 h post-3-VO; 24 h post-sham, 3 days post-3-VO, 3 days post-sham, and untreated control group. For each treatment group, vessels (PCA/Pcom) from 24 animals were isolated and split into three independent pools of vessels. Each pool consisted of PCA/Pcom from eight animals. Three affymetrix cDNA microarrays were hybridised (one array for each pool) for each treatment group (24 h post-3-VO (n = 3), 24 h postsham (n = 3), 3 days post-3-VO (n = 3), 3 days post-sham (n = 3), and control (n = 3)).

RNA Isolation and Quantification Total RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany). The quantity and quality of extracted RNA were further assessed using the RNA 6000 Nano LabChips Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer’s instructions.

Microarray Hybridisation Gene expression was analysed using the commercially available Rat 230 A (Affymetrix) Gene Chip containing

15,866 probe sets. For each array, total RNA (see above) was processed and biotin-labelled cRNA was hybridised by the Institute for Functional Genomics (Charite´, Berlin, Germany). Hybridisation, washing, antibody amplification, staining, and scanning of arrays were performed according to the Affymetrix technical manual. The arrays were scanned using the GeneChip System (HewlettPackard, Santa Cruz, CA, USA) and raw data were processed using GCOS and normalised to a global intensity of 500.

Microarray Data Analysis All microarray data were analysed using significance analysis of microarrays (SAM (1.21)) with logarithmic transformation to identify differential gene expression after 3-VO (false discovery rate < 10%). The fold change in the gene expression levels of each gene was calculated as relative to data from sham-operated animals. Significantly deregulated genes were further analysed using IPA 3.0, which identifies physical, transcriptional, or enzymatic interaction networks. Ingenuity Pathways Analysis is a web-based software application containing most literature knowledge of biologic interactions between gene products (http://www.ingenuity.com/products/pathways_analysis.html). In this article, the gene expression profile is presented by the networks generated by IPA, displaying the significant deregulated genes in list form, together with the according affymetrix gene identification number and the fold-change expression value. Furthermore, expression profiles of those genes were visualised and presented using Gene Math 1.5. To understand the molecular and biologic relations of candidate genes, significantly deregulated genes were functionally annotated using web-based DAVID (Dennis et al, 2003).

Quantitative Real-Time Reverse Transcription-PCR Differential expression of candidate genes was confirmed by quantitative real-time reverse transcription-PCR (qRTPCR) using SYBR green (Applied Biosystems, Foster City, CA, USA). Quantification was performed using the GEDstandard curve method as described in Schefe et al (2006). Amplification was carried out in the ABI Prism 7000 thermocycler (Applied Biosystems). Quantitative RT-PCR was performed using custom primers as listed in the supplement section (Supplementary Table 1). Gene expression data were normalised against HPRT. Data are expressed as means±s.e.m. Differences were evaluated with the Mann–Whitney U-test; P-values < 0.05 were considered significant.

In Situ Hybridisation In situ hybridisation (ISH) was performed on 5 mm paraffin-embedded tissue sections after post-fixing them in 4% PFA in phosphate buffer (pH 7.4; 30 min), and thereafter rinsed with phosphate buffer and digested with Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1811–1823


proteinase K (10 mg/mL) in Tris-EDTA buffer (pH 8.0; 10 min). Sections were hybridised for 18 h at 651C using digoxigenin-labelled probes. Immunostaining for digoxigenin was performed with an anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche, Indianapolis, IN, USA) using NBT/X-phosphate as the substrate (blue staining). For TIMP-1, a probe of 336 bp, and for KNT, a probe of 496 bp, were generated. Primers are shown in Supplementary Table 1. The probes were cloned using the pGEM-T vector (Promega, Madison, WI, USA) and sequenced by the Institute for Genetics (Humboldt University, Berlin, Germany).

White Blood Cell Count: Serum Amyloid-a Measurements To quantify postoperative acute phase reaction, blood samples were taken from each animal group (n = 6) and analysed by the Institute for Veterinary Medicine Diagnostics (VMDI, Berlin, Germany) for differential white blood cell (WBC) count and serum amyloid-a (SAA) levels using flow cytometry and solid phase ELISA with biotinylated monoclonal anti-SAA antibody (Tridelta, Maynooth, Ireland), respectively. Analyses were performed in a blinded fashion and statistical significance analysed by Student’s t-test. Statistical significance was assumed for P < 0.05.

Results Microarray and Functional Network Analysis of Early-Phase Cerebral Arteriogenesis

Induction of arteriogenesis in the brain by 3-VO led to an active growth of the PCA in the collateral system of the circle of Willis (Figure 4E) (Busch et al, 2003). To generate an expression profile for earlyphase collateral growth, we used an Affymetrix system with rat 230A arrays. Using SAM, at 24 h after 3-VO, we identified 91 genes as upregulated and 73 genes as downregulated (n = 164 out of 15,866 probe sets) in comparison with sham-operated controls. These changes are considered significant, as, for each gene, SAM incorporates random permutations of the expression values of all samples and the expression deviation between biologic replicas. Raw data are available at the NCBI Gene Expression Omnibus, where series record (GSE6189) provides access to all data. Next, we grouped genes identified as deregulated by SAM to genetic networks in the IPA software. Here, knowledge-based analysis of physical, transcriptional, or enzymatic interactions revealed six functionally related gene expression patterns in cerebral arteriogenesis 24 h after 3-VO. Deregulated genes involved in distinct networks are shown (Figure 1) together with fold-change values (24 h after 3-VO). Expression values of all arrays were visualised and show homogeneous distribution within the groups (Figure 1). Although 3 days after 3-VO, most genes were not significantly Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1811–1823

deregulated, graphical visualisation of the listed genes clearly shows a similar trend of genes 24 h after 3-VO and 3 days after 3-VO. The IPA analysis showed that deregulated genes in early-phase cerebral arteriogenesis could be assigned to three major functions, categories, or networks: cellular movement, inflammation, and cellular growth/proliferation (Figure 1). Functional expression networks generated by IPA are presented in Supplementary Figure 2. Networks show deregulated genes, where colours indicate the degree of increased (red), decreased (green), or unregulated (white) gene expression 24 h after 3-VO. All six functional networks were related to each other at a superordinate level to the development and function of the cardiovascular system (Supplementary Figure 2H).

Functional Annotation of Genes Deregulated in EarlyPhase Cerebral Arteriogenesis

Using a detection software for overexpressed GO categories and annotation for biologic function (DAVID database), the highest significance was assigned to proinflammatory genes (Table 1). Upon genes being significantly repressed, we found several genes related to muscle development and contraction (Table 1). To rule out systemic postoperative acute phase reaction as a relevant cause of changes in the gene expression profiles, WBC count was performed and SAA was determined. The WBC count was not altered in 3-VO animals and control animals after 24 h and 3 days after 3-VO (Supplementary Figure 3). Serum amyloid-a was below detection limit in all groups (data not shown). Annotation of deregulated genes for the molecular function 24 h after 3-VO (DAVID) revealed a significant cluster of which protease inhibitors, t-KNG, a-2 macroglobulin (A2M) and TIMP-1, had the lowest P-values (0.00071; Table 1). A total of 24 h after 3-VO, t-KNG was the most strongly upregulated gene in this expression profile (66.4-fold change) and was involved in network 3 (cellular growth and proliferation) and network 6 (inflammation) (Figure 1). TIMP-1 (6.1-fold change; Figure 1) was the third highest upregulated gene in the expression profile. TIMP-1 was related to network 1 (cellular movement and immune response). Furthermore, lipocalin 2 (LCN2), a nonprotease inhibitor and the second strongest deregulated gene in this profile (6.8-fold change; Figure 1), was shown to be functionally related to TIMP-1 in extracellular matrix degradation (Supplementary Figure 2A). For cluster 3, expression of protease inhibitor A2M is shown to be induced with a 3.2-fold change (Figure 1). Protease inhibitors clearly show the strongest deregulation and were involved in most functional networks generated by IPA. Furthermore, in silico analyses show that the protease inhibitors deregulated are multifunctional proteins involved


Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1811–1823

Figure 1 Gene expression profile of early-phase cerebral arteriogenesis. Significantly deregulated genes are visualised for their expression level of each microarray (upregulated genes in red, downregulated genes in green). Deregulated genes were clustered by using the IPA software. Here, six groups/networks were identified, being significantly functionally related and involved in cell proliferation, movement, and development. Affymetrix identifier and fold change of each gene is given for the 24 h post-3-VO time point.


Table 1 Gene functional annotation (A) Biological function annotation

No. of genes

Significants

Positively regulated 24 h after 3-VO Response to pest, pathogen, or parasite Response to other organism Response to wounding Immune response Defence response Nervous system development Taxis Negative regulation of cellular process Locomotory behaviour Cell–cell signalling Negative regulation of physiologic process Regulation of organismal physiologic process Cell proliferation Negatively regulated 24 h after 3-VO

Muscle development Muscle contraction Cell proliferation Regulation of cellular physiologic process Regulation of signal transduction Embryonic development (sensu Metazoa) Circulation Organ morphogenesis Positive regulation of cellular process (B) Molecular function annotation

10 10 9 10 10 9 4 9 4 7 7 4

3.90E 5.20E 9.20E 1.50E 2.90E 3.20E 6.40E 7.00E 2.00E 2.50E 4.70E 6.40E

05 05 05 03 03 03 03 03 02 02 02 02

6

7.30E 02

No. of genes

Significants

6 5 7 14 4 3 3 5 6 No. of genes

7.90E 1.10E 1.50E 3.50E 4.50E 5.80E 7.30E 7.50E 9.80E

05 03 02 02 02 02 02 02 02

Significants

Positive regulated 24 h after 3-VO Protease inhibitor activity Immunoglobulin binding Receptor binding

4 2 6

7.10E 04 5.10E 02 6.70E 02

Protease inhibitor activity a-2 macroglobulin RG Kininogen 1 Tissue inhibitor of metalloproteinase 1 Serine (or cysteine) peptidase inhibitor, I1 3-VO, three-vessel occlusion. Functional annotation of deregulated genes in early-phase cerebral arteriogenesis was performed using the DAVID bioinformatic database. Significance for overrepresented GO categories was assessed by Fisher’s t-test. Cluster of protease inhibitors showing lowest P-values.

in most processes for cardiovascular system development and, therefore, were analysed further.

Validation of Deregulated Genes Significantly Expressed in the PCA/Pcom 24 h after 3-VO

To validate the results of the microarray analysis, qRT-PCR was performed with the same RNA samples used for microarray analysis. Data obtained by qRT-PCR confirmed a significant increase in mRNA levels of the protease inhibitors t-KNG, TIMP-1, A2M and the nonprotease inhibitor LCN2 during early cerebral arteriogenesis in the ipsilateral PCA/Pcom 24 h after 3-VO, compared with the sham Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1811–1823

group (Figures 2A–4D). Furthermore, we analysed gene expression levels for t-KNG, TIMP-1, A2M, and LCN2 in the PCA/Pcom contralateral to the ligated carotid artery by qRT-PCR. Here, we also found a significant increase in the mRNA expression levels of all four genes, similar to the ipsilateral PCA/Pcom region. Importantly, expression levels of genes validated by qRT-PCR within each of the three replicates of each experimental condition were upregulated in a similar range, indicating a similar expression pattern within the same groups. On comparing the expression of sham-operated animals with intact controls, no significant expression differences were detectable (Figure 2). As TIMP-1 was identified as being upregulated during arteriogenesis in the dog heart (Cai et al, 2004), we therefore further analysed TIMP-1 mRNA expression by using ISH, which also allowed us to localise mRNA expression in the vascular tissue of the PCA region. Furthermore, kininogen mRNA was assessed by ISH, as kininogen was one of the strongest induced genes we identified. Results showed a high TIMP-1 and t-KNG mRNA staining intensity in the vascular wall of the PCA 24 h after 3-VO (Figures 3A and 3D) compared with sham control (Figures 3B and 3E). Increased expression levels of TIMP-1 and t-KNG mRNA were detected in endothelial cells, smooth muscle cells, and adjacent meningeal tissues (arachnoidea). Using sense probes for TIMP-1 and t-KNG, no staining was detectable (Figures 3C and 3F), indicating specificity of the probes.

Characterization of the Arteriogenic Phenotype in the 3-VO Model by Morphologic Analysis and Assessment of Monocyte Invasion and Vascular Proliferation

To confirm early-phase collateral vessel growth after 3-VO, major parameters of the arteriogenesis process were assessed, such as vascular cell proliferation and CD68-positive cell influx. Immunohistochemical evaluation of cell proliferation using a PCNA antibody showed an increase in vascular cell proliferation 24 h and 3 days after 3-VO (Figures 4A–D). Here, a protrusion of proliferating endothelial cells was detectable in the growing PCA 3 days after 3-VO (Figure 4C). Differences in the numbers of proliferating cells between the PCAs on the ipsilateral and contralateral sides of the ligated carotid artery could not be observed, showing that cell proliferation in the PCA occurs bilaterally, which is in line with the findings of gene expression data. Immunostaining for the macrophage marker CD68 (ED-1) showed a significantly increased number of invading macrophages 24 h and 3 days after 3-VO (Figures 4F and 4G) compared with controls (Figure 4H). Accumulation of CD68-positive cells was detected in the adventitia as well as in the perivascular tissue in close proximity to the growing PCA. Positive CD68 staining was also obtained in

Early response in cerebral arteriogenesis P Hillmeister et al 1817 A2M

6

t-KNT

120 * 100

4

fold change

fold change

* *

2

PCA/Pcom ipsilateral PCA/Pcom contralateral

80 * 60 40 20

0

0 24h 3VO

14

24h 3VO 25

TIMP-1

12

* *

8 6

15

Intact control 24h Sham LCN2

*

20

10

fold change

fold change

Intact control 24h Sham

*

10

4 5

2 0

0 24h 3VO


24h 3VO


Figure 2 Validation of differential gene expression by qRT-PCR. Data show similar expression levels as obtained in the array analysis. (A) a-2 macroglobulin (A2M), (B) t-kininogen (KNG), (C) tissue inhibitor of metalloproteinase 1 (TIMP-1), (D) lipocalin 2 (LCN2).

the endothelial cell layer, indicating adhesion of macrophages to endothelial cells (Figure 4I). The main feature of arteriogenesis is the increase in vessel diameter after active proliferation and remodelling, which we could confirm in our experimental setup. We observed an increase of the PCA diameter, which becomes significant 7 days after 3-VO compared with sham-operated controls (Figure 4E). Here, ipsilateral diameters after 3-VO increased from 162 mm (±40 mm) up to 205 mm (±45 mm). A total of 24 h after 3-VO, no significant changes in the PCA diameter were detectable as compared with sham control animals (data not shown). Furthermore, morphologic changes indicating activation of the collateral endothelial surface were investigated using SEM, as a major inducer of arteriogenesis is the biomechanical activation of the endothelial cell layer by altered blood flow conditions. By using SEM, we could visualise the morphology of single endothelial cells within the PCA of 3-VO and control animals (Figure 5). Protrusion of the endothelial cell nucleus and alignment of the endothelial cells were detected 3 days after 3-VO (Figures 5A and C), whereas endothelial surfaces of the vascular PCA wall from control animals were flat (Figures 5B and D). In summary, upon 3-VO, we showed in the PCA activation/proliferation of endothelial cells, macrophage invasion and vessel growth, which are major features of the arteriogenesis process. These findings confirm ongoing cerebral arteriogenesis within our experimental setup.

Discussion To our knowledge, this is the first comprehensive description of initial molecular mechanisms and morphologic features during early-phase collateral artery growth in the brain. Arterial collateral pathways in the brain provide alternative blood flow to support brain viability when a primary vessel in the cervicocephalic arterial tree is critically stenosed or occluded. By combining bilateral vertebral and unilateral carotid artery occlusion 3-VO, we previously established a nonischaemic model for cerebral arteriogenesis for the first time (Busch et al, 2003). In our study, novel findings after experimentally induced arterial high flow conditions (3-VO) with increased blood flow across the PCA are as follows: First, we analysed the gene expression profile of growing collateral vessels in the brain after 3-VO. Second, we characterised the gene expression profile by biologic and molecular function annotation and identified physical, transcriptional, or enzymatic interaction networks by knowledge-based analysis (IPA). Third, we observed characteristic features of the arteriogenesis process in the PCA, such as vascular proliferation and monocyte invasion. For the first time we showed direct evidence for endothelial cell activation on 3-VO by SEM in growing collaterals of the brain. Collateral circulation can significantly influence the occurrence and size of cerebral infarction (Liebeskind, 2004). Therefore, therapeutic stimulation of arteriogenesis might have important clinical Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1811–1823


Figure 3 Localisation of mRNA expression by ISH. (A) TIMP-1 antisense probe hybridisation on PCA 24 h after 3-VO. (B) TIMP-1 antisense probe hybridisation on PCA 24 h after sham operation. (C) TIMP-1 sense probe hybridisation on PCA 24 h after 3-VO. (D) KNG antisense probe hybridisation on PCA 24 h after 3-VO (E) KNG antisense probe hybridisation on PCA 24 h after sham op. (F) KNG sense probe hybridisation on PCA 24 h after 3-VO.

implications for the development of prophylactic and acute treatments of cerebrovascular disease (Love, 2003). In the brain, the circle of Willis is the most efficient collateral system, providing a lowresistance link between the four main supplying arteries (two carotid and two vertebral arteries) (Hossmann, 1993). 3-VO introduced an arteriogenic stimulus by the redistribution of blood flow via the PCA, resulting in a significantly enlarged PCA by 38% within 7 days (Figure 4E). Basically, increased flow through preexisting anastomoses leads to an increase in vessel wall shear stress, which biomechanically activates endothelial cells, resulting in a differential gene expression profile. Still, it is presently unclear what Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1811–1823

are the molecular mechanisms that initiate the enlargement of collateral vessels, and, in particular, little is known about the condition in brain circulation. Investigating gene expression patterns upon the introduction of an arteriogenic stimulus (enhanced shear stress) by 3-VO provides an important insight into the coherences of gene expression pattern involved in the onset of arteriogenesis in the rat brain. We show that 91 genes are upregulated and 73 genes are downregulated in growing collaterals 24 h after 3-VO, as compared with shamoperated groups. By using the DAVID bioinformatic database, biologic function annotation revealed that early-


Figure 4 Validation of proarteriogenic phenotype. (A) PCNA staining (red) shows that by 24 h after 3-VO, proliferating cells in the endothelial cell layer of the PCA are detectable. (B) A total of 24 h after sham surgery, no proliferating cells were detectable. (C) Three days after 3-VO, the number of PCNA-positive cells increased and characteristic protrusion of the cell nucleus was detectable. (D) Three days after sham surgery, no proliferating cells were detectable. (E) Diameter increase of the PCA after 3-VO versus sham control. (F) Macrophage staining for CD68. A total of 24 h after 3-VO, macrophages accumulate around the PCA and the perivascular space within the arachnoidea (CD68 in red, nucleus in blue). (G) Three days after 3-VO, macrophages still accumulate around the PCA and the perivascular space within the arachnoidea. (H) Control (24 h after sham op). (I) A total of 24 h after 3-VO, macrophages at the inner surface of vessel (CD68 in red, smooth-muscle actin in green, nucleus in blue).

phase cerebral arteriogenesis is characterised by a positive regulation of genes related to inflammatory response (Table 1). This finding correlates with the observed invasion of CD68-positive cells (mono-

cytes) in the perivascular space of the PCA after 3-VO (Figures 4F and 4G), and supports previous findings that proinflammatory cytokines such as GM-CSF stimulate arteriogenesis (Buschmann et al, Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1811–1823


Figure 5 Validation of endothelial cell activation by SEM. (A and C) Endothelial cell layer of 3-VO animals at day three showing characteristic protrusion of the cell nucleus and alignment in the direction of flow. (B and D) Morphology of the endothelial cell layer of sham control animals at day 3 shows that cells are flat and smooth.

2003). As expression profiles were obtained from materials of isolated vessels at spatial distance from the surgical wound (extracranial ligation), detection of gene expression caused by postoperative inflammatory response seemed unlikely, as WBC count (Supplementary Figure 3) and SAA analysis (comparable to CRP in humans) were unaffected. Yet, it has to be considered that any injury and surgery can cause a postoperative effect, which might be able to lead to an acute phase reaction. However, as earlyphase arteriogenesis by itself is characterised by the occurrence of features of inflammatory response and the expression of proinflammatory cytokines, we cannot completely rule out an overlap with an inflammatory reaction caused by surgery. However, local gene expression related to inflammatory response at 24 h after 3-VO is consistent with gene expression profiles in early-phase arteriogenesis of the periphery (Lee et al, 2004). Among negatively regulated genes, biologic function annotation identified components of muscle development and contraction (Table 1). This finding is in line with the results obtained by Schaper (2004), and is associated with SMC de-differentiation, the initial step for SMC proliferation and migration in processes of vascular growth. For an elaborate characterisation of genes deregulated in growing collaterals, IPA identified a superior Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1811–1823

expression network for early-phase cerebral arteriogenesis. This network consists of six functionally related gene expression patterns (24 h after 3-VO) (Figure 1). Genes within such patterns are reported to interact in cellular mobility, proliferation, growth and development, as well as inflammatory response and cardiovascular system development, which is in accordance with the concept of arteriogenesis. Further analysis of the identified expression networks for early-phase cerebral arteriogenesis by molecular function annotation showed that the Pvalues were lowest for the protease inhibitors A2M, TIMP-1, and t-KNG (Table 1). Most upregulated genes include kininogen (66.4-fold change), TIMP-1 (6.1-fold change), and LCN2 (6.3-fold change). The latter was functionally related to TIMP-1, as revealed by network analysis (Supplementary Figure 2A). Earlier, TIMP-1 has been reported to be transcriptionally upregulated in a model for coronary arteriogenesis in the dog heart (Cai et al, 2004). In our study, mRNA ISH analysis showed TIMP-1positive mRNA staining in the intima, media, and adventitia of the PCA during arteriogenesis 24 h after 3-VO (Figure 3A). Therefore, we suggest TIMP-1 as a marker for early-phase arteriogenesis in the brain. The TIMP-1 controls matrix-metalloprotease (MMP) activity (e.g., MMP-9 and MMP-2), thereby


mediating extracellular matrix degradation by proteolysis, an important step during adaptive arteriogenesis (Cai et al, 2000). However, in contrast to upregulation of TIMP-1, we could not detect regulation of MMPs on the gene expression level in the early phase of arteriogenesis. This might be because of the fact that many MMPs are regulated at the posttranslational level. As shown by network analysis (IPA), TIMP-1 is functionally related to LCN2 (Supplementary Figure 2A). LCN2 can be expressed in blood vessels upon inflammation, forming a protein complex with MMP-9 and TIMP-1 (Bu et al, 2006). Upon complex formation, degradation of MMP-9 by TIMP-1 is significantly inhibited, resulting in prolonged MMP-9 activity. Interestingly, the effect of LCN2 on MMP-9 activity does not deregulate MMP-9 gene expression (Deindl et al, 2001), pointing toward a regulation of MMP-9 at the posttranslational level. Besides MMP regulation, TIMPs were found to be involved in regulating cell proliferation, angiogenesis, and apoptosis (Mannello and Gazzanelli, 2001). Therefore, TIMP-1 might also mediate proliferation and protection from apoptosis in vascular cells during early-phase arteriogenesis. Interestingly, a second protease inhibitor, t-KNG, was the strongest upregulated gene. RNA ISH showed that t-KNG is highly expressed at 24 h after 3-VO selectively in regions of the intima, media, and adventitia of the growing PCA (Figure 3D). Kininogen is a cysteine protease inhibitor, preventing degradation of the extracellular vascular matrix by MMPs, thereby orchestrating the homoeostasis of the vessel wall. In kininogen-deficient rat, severe aneurysm formation can be observed, pointing toward a role for kininogen in vascular remodelling (Kaschina et al, 2004). Furthermore, it is well known that kininogen is cleaved by kallikrein, thereby releasing the vasoactive peptide bradykinin (Pelc et al, 1991). Cellular functions of bradykinin are mediated by activation of the kinin B1 and kinin B2 receptors leading to vasodilation, cell proliferation, and increased endothelial cell permeability (Pesquero and Bader, 2006). In general, activation of the kallikrein–kinin system has been shown to reduce cardiovascular ischaemia and protect against stroke in rat and mouse models (Chao et al, 2006). Recently, it has been reported that in a model for hindlimb ischaemia, neovascularisation is markedly reduced in B1 receptor knockout mice. (Emanueli et al, 2002). For kininogen, a dual role in angiogenesis is reported, showing both pro- and antiangiogenic properties (Colman et al, 2003). However, a role for kininogen in arteriogenesis has not been described so far. To confirm collateral growth after 3-VO, we morphologically examined the PCA, observing specific features of adaptive arteriogenesis (Schaper, 2004). Exactly 24 h after 3-VO (Figure 4A), endothelial cells of the PCA showed increased levels of

proliferation followed by endothelial cell protrusion at day 3 (Figure 4C) and resulting in an increased PCA diameter after 7 days (Figure 4E). Another main feature of arteriogenesis is monocyte adhesion and invasion (Arras et al, 1998), which we detected 24 h and 3 days after 3-VO (Figures 4F–I). Monocytes have been shown to exert an important paracrine function during adaptive arteriogenesis, accelerating the remodelling process of collateral arterial tissue (Hossmann and Buschmann, 2005). However, blocking transendothelial migration of monocytes via intracellular cell adhesion molecule inhibitory antibodies leads to a severe reduction in arteriogenesis (Arras et al, 1998). This is supported by the finding in osteopetrotic (op /op ) mice, which show a significant reduction in circulating monocytes, and only partially recover from arterial ligation, as compared with healthy controls in terms of peripheral tissue perfusion (Bergmann et al, 2006). These data were supported by the findings from Heil et al (2002), providing a direct positive correlation between collateral artery growth and the number of circulating monocytes in the blood stream. We previously showed that stimulation with GM-CSF enhances arteriogenesis and collateral blood flow in the rat brain upon 3-VO (Buschmann et al, 2003). Presumably, GM-CSF stimulated arteriogenesis because of an increase in monocyte recruitment and by prolonging the life span of macrophages (Hossmann and Buschmann, 2005). By employing SEM, we showed that flow changes in the PCA lead to a subsequent endothelial activation 3 days after 3-VO, characterised by nuclear protrusions (Figures 5A and 5C). Indeed, nuclear protrusions are observed specifically in proliferating endothelial cells on paraffin sections 3 days after 3-VO (Figure 4C), which indicate that cells are turning into the mitotic cell cycle (McCracken et al, 1979). Endothelial cell activation can occur by osmotic cell swelling via calcium influx or opening of volume-regulated chloride channels as reported in in vitro studies (Schwarz et al, 1992). Furthermore, 3 days after 3-VO, endothelial cells showed more pronounced cell borders compared with control animals. This might indicate the expression of junctional adhesion molecules in a process of inflammatory cell recruitment after 3-VO, which would facilitate monocyte invasion into growing vessels (Chavakis and Orlova, 2006). In conclusion, this study presents a functional gene expression analysis, as well as a comprehensive morphologic analysis of collateral pathways in the brain to get a deeper insight into the initial molecular processes during collateral growth. Upon 3-VO, we observe that induction of arteriogenesis in the brain can lead to the biomechanical activation of endothelial cells in the PCA, resulting in vascular cell proliferation as early as 24 h after 3-VO, with increasing cell proliferation and nuclear protrusion in endothelial cells 3 days after 3-VO. Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1811–1823


Proliferation is accompanied by macrophage invasion into the perivascular space of growing collaterals. After active cell proliferation, the PCA diameter increase becomes significant within 7 days after 3-VO. Morphologic features of early-phase cerebral arteriogenesis can be implicated to the molecular patterns. Early-phase cerebral arteriogenesis is characterised by the expression of genes involved in cell proliferation/migration and inflammation, notably by the expression of protease inhibitors. Here, we suggest that TIMP-1 and kininogen are potential biomarkers for cerebral arteriogenesis. Enhanced expression of protease inhibitors during early-phase arteriogenesis exemplifies their role in controlling extracellular matrix degradation and advancing vascular remodelling by the activation of cell proliferation. Their multifunctional role might indicate that TIMP-1 and kininogen have important functions during arteriogenesis. However, further studies are needed to examine the role of protease inhibitors during early-phase cerebral collateral growth.

Disclosure/conflict of interest The authors declare no potential conflict of interest.

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Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website (http:// www.nature.com/jcbfm)

Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1811–1823