Cyclic stretch-induced Crp3 sensitizes vascular smooth muscle cells to ...

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Cyclic stretch-induced Crp3 sensitizes vascular smooth muscle cells to apoptosis during vein arterialization remodeling Luciene Cristina Gastalho Campos*§, João Carlos Ribeiro-Silva*, Alessandra Santos Menegon, Valerio Garrone Barauna , Ayumi Aurea Miyakawa¹, Jose Eduardo Krieger¹ Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of São Paulo Medical School, São Paulo, Brazil

*authors contributed equally to this work §

LCGC present address: Department of Biological Science, Santa Cruz State University,

Ilhéus, BA, Brazil VGB present address: Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES, Brazil

AAM ([email protected]) & JEK ([email protected]) Laboratory of Genetics and Molecular Cardiology Heart Institute (InCor), University of Sao Paulo Medical School Av. Dr. Eneas C. Aguiar, 44 – 10 andar 05403-000 São Paulo - SP – Brazil Tel. +55 11 2661 5068 Fax. +55 11 2661 5022

Abbreviations Akt, Protein Kinase B; Crp3, cysteine-rich protein-3; Fak, Focal Adhesion Kinase; KO, knockout; SMC, Smooth Muscle Cell; TUNEL, Terminal deoxynucleotidyl transferase dUTP Nick End Labeling assay; WT, Wild Type

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¹Corresponding authors:

Abstract: Vein graft failure limits the long-term patency of the saphenous vein used as a conduit for coronary artery bypass graft. Early graft adaptation involves some degree of intima hyperplasia to sustain the hemodynamic stress, but the progress to occlusion in some veins remains unclear. We have demonstrated that stretch-induced up-regulation of cysteine and glycine-rich protein 3 (Crp3) in rat jugular vein and human saphenous vein in response to arterialization. Here, we developed a Crp3-KO rat to investigate the role of Crp3 in vascular remodeling. After 28 days jugular vein arterialization, the intima layer was 3-fold thicker in the Crp3-KO that showed comparable smooth muscle cells (SMC) proliferation but an absence of early apoptosis observed in the wild-type rat (WT). We then investigated the role of Crp3 in early integrin-mediated signaling apoptosis in isolated jugular SMC. Interestingly, under basal conditions, ceramide treatment failed to induce apoptosis in both WT and Crp3-KO SMC. Under stretch, Crp3 expression increased in WT SMC and ceramide induced apoptosis. Immunoblotting analysis indicated that ceramide stretch-induced apoptosis in SMC is accompanied by a decrease in the phosphorylation status of both Fak and Akt, leading to an increase in Bax expression and caspase-3 cleavage. In contrast, ceramide failed to decrease Fak and Akt phosphorylation in Crp3-KO SMC and, therefore, there was no downstream induction of Bax expression and effector caspase-3 cleavage. Taken together, we provide evidence that stretch-induced Crp3 modulates vein remodeling in response to arterialization by sensitizing SMC to apoptosis. Key words: vein arterialization, cysteine-rich protein 3, apoptosis, smooth muscle cells

Introduction We commonly use the saphenous vein as a conduit for aortocoronary bypass grafting (CABG) to improve symptoms and survival in patients with severe coronary artery disease, but the graft failure remains a challenge limiting its long-term patency (1–3). Vein grafts fail due to occlusive events, a direct or indirect consequence of neointimal hyperplasia, which develops as an exacerbated adaptive response to the arterial hemodynamic regimen of high pressure and flow (4,5). We have demonstrated that a member of the Cysteine-Rich Protein (CRP) family of LIM domain proteins – Crp3, is modulated during the vein graft arterialization in response to the increased stretch of smooth muscle cells in an arterialized human saphenous vein (6). The crp3 expression is present in arteries and virtually absent in veins. Interestingly, during vein arterialization process, Crp3 is upregulated in a stretch-dependent manner. Crp3 plays a crucial role in the architecture and mechanical function of cardiac myocytes. Mice lacking Crp3 gene develop cardiac hypertrophy, dilated cardiomyopathy and heart failure (7). Crp3 has been described to be one of the Z-disc components, which is thought to be a mechanical stress sensor (8). In the vascular context, Crp3 seems to have a regulatory role in vascular smooth muscle cells, but the mechanism whereby or its role in the vascular remodeling associated with arterialization remain unclear (6,9). It has been well established that vascular remodeling during disease or injury involves altered expression of extracellular matrix (ECM) proteins and cell surface integrins (10–12). Integrins are transmembrane mechanosensors functioning as the interface between SMC and the ECM (13,14). The interaction between integrins and ECM leads to integrin clustering, and it is an essential step to trigger intracellular signaling pathways. These pathways regulate cell behavior and fate through the association of many scaffolding and signaling proteins linked to the cytoskeleton (13,15,16). The LIM domain has emerged as the domain providing a bridge between the cytoskeleton and the nucleus. It is present in many proteins that have diverse cellular roles as regulators of gene expression, cytoarchitecture, cell adhesion, cell motility and signal transduction (17). Here, we demonstrated that the absence of Crp3 impairs the downstream signaling pathway triggered by ȕ1-integrin. In this context, Crp3 acts as a key modifier of the vein arterialization remodeling through its ability to sensitize stretched smooth muscle cells to apoptosis.

Materials & Methods

Generation of Crp3-KO rat Genetic deletion of Crp3 on a Wistar rat background was generated by using CompoZr® Zinc Finger Nuclease Technology (Sigma-Aldrich, St. Louis, MO). Pairs of custom nucleases designed to target the Crp3 gene were delivered to embryos by microinjection. The embryos were implanted into a foster mother, and the littermates were screened for mutation. The mutation generated was a 4-bp frameshift deletion (deleted CATGC and inserted A) overlapping the start codon in exon 1 (Supplementary Figure 1A) and the absence of Crp3 expression was confirmed by western blot (Supplementary Figure 1B and 1C). The knockout animals showed no statistical differences in body weight, heart rate, blood pressure and blood flow in ascendant aorta when compared to wild type (Supplementary Table 1). As described for Crp3-KO mice, the Crp3-KO rats showed cytoarchitectural disorganization in cardiomyocytes and dilated heart. The cardiac output and work (measured directly) remained unchanged between the two animals under basal conditions. In contrast, upon an afterload stress with phenylephrine, both animals decrease cardiac stroke volume and work and as expected the capacity to cope with the increased resistance is compromised in the Crp3 KO rat (Supplementary Figure 2). The colony was established by the Howard J. Jacob group at the Medical College of Wisconsin (Milwaukee, WI). Animals were transferred to University of Sao Paulo Medical School animal facility and housed at a constant temperature (25 °C) and humidity, and 12:12 light-dark cycle.

Rat model of vein arterialization The rat model of vein arterialization is based on the connection of rat jugular vein to the carotid artery as previously described (6,18). Male Wistar rats (3 months old, 250–350 g) were obtained from University of Sao Paulo Medical School animal facility. Before surgical procedures, 70 UI/Kg of heparin was administered by intraperitoneal injection followed by anesthesia with ketalar (50 mg/Kg) and rompum (10 mg/Kg). The right external jugular vein was connected to the common carotid artery by an end-to-end anastomosis with 10.0 nylon suture. After the surgery, blood flow was re-established, and the vein graft was harvested at one day (TUNEL assay), seven days (ki67) or 28

days (morphometric analysis) after surgery. This study protocol was approved by the local Ethics Committee (SDS 3299/09/050, CAPPesq 0824/09).

Histology, morphometric analysis, and immunohistochemistry Rats were euthanized by sodium pentobarbital overdose (200 mg/kg). Vein grafts were flushed with a heparinized saline solution, fixed by pressure perfusion with 4% formalin, and embedded in paraffin for immunohistochemical analysis. Transverse sections (3μm-thick) were analyzed starting 400μm from the suture. Verhoeff Van Gieson (VVG) staining was performed, examined by light microscopy and the area quantification was performed with the Leica Qwin software. To assess the apoptotic events, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was done to detect DNA fragmentation by using an in-situ cell death detection kit, AP (Roche Molecular Biochemicals), according to the manufacturer's instructions. The percentage of TUNEL-positive nuclei was quantified. For immunohistochemistry, the non-specific reactions were blocked with 5% BSA, and the sections were incubated for 18h at 4 °C with an anti-Ki-67 antibody (Abcam). The sections were then incubated with a solution from the LSAB HRP Universal kit (DAKO). Immunoreactions were detected with 3,3´-diaminobenzidine (DAB) and counterstained with hematoxylin. The cross-sections were examined by light microscopy, and the result expressed as a percentage of the positive cell. All analyses were performed by an observer blinded to experimental group and animal genotype.

SMC culture and stretch protocol Primary culture of vascular smooth muscle cells was established, in parallel, from wild-type and Crp3-KO rats by using the explant protocol. Briefly, the endothelial cells were removed by mechanical friction, and small fragments of vessels were plated on 6-well culture plates coated with gelatin. The fragments were let to adhere to the plate and cultured with Dulbecco's modified Eagle Medium supplemented with 20% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. The cells derived from the explant were isolated and expanded. For the stretch experiments, cells were harvested and seeded in Bioflex plates coated with Collagen I. After 24h the basal medium was replaced with the starving medium (0.5% FBS). The following day cells were submitted to the stretch protocol (10% stretch, 1 Hz, for 24 h) in a Flexcell 5000 cell stretching system (Flexcell International). Control non-stretched SMCs were also

cultured in Bioflex plates. For the apoptosis studies, the cells were exposed to ceramide (Sigma) at a final concentration of 100μM or DMSO and then submitted to stretch. Cells from at least three different animals were extracted and used up to passage 8.

Flow cytometry analysis To evaluate the rate of apoptotic SMC, the percentage of cells with hypodiploid DNA (sub-G1) was quantified 24h after stretch-associated ceramide treatment. Briefly, cells were washed with cold PBS and resuspended in binding buffer. The nuclei were stained with propidium iodide (Molecular Probes, Eugene, OR, USA) and fluorescein isothiocyanate–annexin V. DNA content was measured using a FACSCalibur flow cytometer and Cell Quest software (Becton Dickinson, Franklin Lakes, NJ, USA). The quantification was carried out in annexin V positive and propidium iodine negative cells. Twenty thousand cells were counted in all assays.

RNA extraction and real-time RT-PCR Total RNA was isolated from smooth muscle cells using Trizol Reagent (Life Technologies) and cDNA synthesis was performed with SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer's specifications. One hundred nanogram of cDNA was used for real time RT–PCR reaction (SYBR Green PCR Master Mix-PE Applied Biosystems) on the ABI Prism 7700 Sequence Detection System (Applied Biosystems). The oligonucleotide primers used were: Crp3, 5’-ATT GGG TTT GGA GGG CTT AC-3’ (foward) and 5’-TGT TGA CTA GCA GGG CTG TG-3’ (reverse), and the internal control GAPDH, 5’-ATG GTG AAG GTC GGT GTG -3’ (foward) and 5’-GAA CTT GCC GTG GGT AGA G-3’ (reverse) and cyclophilin A 5’-AAT GCT GGA CCA ACA CAA A-3’ (foward) and 5’-CCT TCT TTC ACC TTC CCA AA-3’(reverse). mRNA analyses of target genes were assayed in triplicate. The comparative threshold (CT) cycle method was used for data analyses.

Immunopreciptation and immunoblotting For the immunoprecipitation studies, stretched wild type jugular SMC were lised in ice cold RIPA buffer (50 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1% Triton X-100, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, protease and phosphatase inhibitors cocktails). Lysates were centrifuged at 10,000 × g for 10-15 min in the cold and clear supernatants were used as whole cell lysates. Pre-cleared

supernatants were incubated with 10ȝg of Fak antibody (Cell Signaling) at 4 °C overnight on a rotator. The next day, the whole cell lysates were incubated with 100ȝL of Protein G Dyna-beads (Invitrogen) at 4°C overnight to collect the immune complexes. The beads were washed three times with RIPA buffer and analyzed by immunoblotting using anti-Fak (1:1000 dilution; Cell Signaling) or anti-Crp3 antibody (1:1000 dilution; Santa Cruz Biotechnology). Twenty-four hours after ceramide treatment, jugular SMC were lysed with SDS buffer (60mM Tris-+&OS+JO\FHURO6'6 $IWHUௗPLQDWž&WKH lysates were clarified by centrifugation (10,000 g for 10ௗminutes DWௗƒC) and the supernatant was collected. Protein concentration was determined by the Pierce assay (Thermo Scientific). Equivalent amounts of protein were electrophoresed on SDSPAGE gels. Kaleidoscope prestained standards (Bio-Rad) were used to determine molecular weight. The gels were then electroblotted onto PVDF membranes. After blocking with 5% BSA, membranes were incubated with the primary antibodies overnight (anti-pFak397 (Invitrogen), anti-Bax, anti-cleaved caspase-3, anti-pAkt473 (Cell Signaling), anti-Gapdh (R&D Systems), anti-Crp3 (kindly provided by Silvia Arber and Pico Caroni from University of Basel, Switzerland). Finally, the relevant protein was visualized by staining with the appropriate secondary horseradish peroxidase-labeled antibody for 1h followed by enhanced chemiluminescence. To visualize Crp3 after immunoprecipitation, it was used mouse-anti-rabbit IgG (conformation specific) antibody (Cell Signaling). Densitometric analysis of the bands, relative to GAPDH, was performed using ImageJ software (National Health Institute, Bethesda, MD, USA).

Statistical analysis All data are expressed as mean ± SEM. Comparison among groups was performed using two-way analysis of variance (ANOVA) followed by Tukey´s post hoc test for comparison. Comparisons between two groups were analyzed via a student’s ttest. Values of P < 0.05 were considered statistically significant.

Results Crp3-KO rats showed increased vascular remodeling and blunted early apoptotic events in response to arterialization We have shown that Crp3 expression is modulated in arterialized human saphenous and rat jugular veins in response to arterialization (6). To determine the influence of Crp3 in vein arterialization remodeling, we have used an established model of arterialization (19) in wild type and Crp3-KO rats. After 28 days of arterialization, Crp3 knockout rats showed a 3-fold increase of the intima layer compared to wild-type animals and no difference in media and adventitia layer. Similarly, collagen and elastin contents in the extracellular matrix remained unchanged between Crp3-KO and wild type animals (Figure 1A). To understand the underlying mechanisms to the increased remodeling, we evaluated apoptosis and cell proliferation in wild type and Crp3-KO jugular veins in response to arterialization. We have demonstrated that apoptosis reaches the highest level at day 1 and cell proliferation at day 7 of rat jugular vein arterialization (19). As evidenced by TUNEL assay, the absence of Crp3 results in the absence of apoptotic events (Figure 1B), while no change in proliferation rate was observed between Crp3-KO and wild-type animals (Figure 1C). These data suggest that the increased vein arterialization remodeling in Crp3-KO rat occurs at least in part by inhibition of the apoptotic events taking place at the early stages of vein arterialization.

Stretch-induced Crp3 expression in jugular vein induces apoptosis response to ceramide In contrast to aorta SMC, jugular vein SMC do not express Crp3 at the basal condition, but upon vein arterialization, Crp3 expression increases (6) and this response is due to stretch stimulus (Figure 2 and Supplementary Figure 3). The stretch-induction of Crp3 expression failed to occur in SMC derived from Crp3-KO (Supplementary Figure 1D). To test the hypothesis that the apoptotic response was associated with the increase in Crp3 expression, we submitted wild type and Crp3-KO jugular smooth muscle cells to stretch in the presence of ceramide and vehicle. Ceramide acts as a second messenger in activating apoptosis and it was used as an inductor of apoptosis to evaluate the signaling pathway in SMCs derived from Crp3-KO and wild type cells.

Ceramide was not able to induce apoptosis in both, static wild type and Crp3-KO jugular SMC (Figure 3A). However, under stretch-induced Crp3 expression, we observed that wild type jugular SMC undergo apoptosis in response to ceramide, but not Crp3-KO jugular SMC (Figure 3B, 3C and Supplementary Figure 4). These findings suggest that stretch induced-Crp3 modulates apoptosis in response to the arterialization.

The pro-apoptotic downstream signaling activation by ceramide is blocked in the absence of Crp3 Cell death is an early response to stretch mediated by integrins (20). To better define the influence of Crp3 in the apoptotic process, we evaluated a signaling pathway activated by ceramide in the presence of stretch (Figure 4A to F). At the protein level, we found no difference in beta-1-integrin levels, but ceramide decreased Fak and Akt phosphorylation, leading to a subsequent increase in Bax expression and caspase-3 cleavage in stretched smooth muscle cells from a wild-type rat jugular vein. This phenomenon was not observed in cells from Crp3 knockout animals showing that the absence of Crp3 impairs the apoptotic downstream signaling elicited by ceramide in stretched smooth muscle cells. To determine the Crp3 role in this signaling pathway, we performed an immunoprecipitation assay to Fak and Crp3. As demonstrated in figure 4G, stretchinduced Crp3 is pulled down with Fak in wild type jugular SMC. Altogether, these data suggest that stretch-induced Crp3 expression is essential to the apoptotic response of veins at the early stages of the arterialization process. Crp3 interacts with Fak, and its absence impairs the regulation of the integrin/Fak/Akt signaling pathway (Figure 5), leading to an increased remodeling response.

Discussion We have previously demonstrated that Crp3 expression is induced during vein graft arterialization in vascular SMC in response to increased stretch (6). Here, we developed a Crp3-KO rat and the results show a key role of Crp3 as a modifier of vascular remodeling by sensitizing VSMC to apoptosis through a decrease in the integrin-mediated signaling pathway.

In physiological conditions, tissue cell apoptosis is strictly controlled, and there is a balance between apoptosis and proliferation. In vein arterialization remodeling, this balance is often disturbed, leading to the overgrowth of cells and vein graft occlusion. The vein graft adaptation to the new arterial environment is characterized by structural vessel wall remodeling and intimal thickening (21). Moderate intimal hyperplasia formation is necessary for proper arterialization and long-term graft patency. However, about 50% of the grafts fail after 5-10 years by uncontrolled remodeling, and the mechanism remains unknown (22). Our data show that upon arterialization of rat jugular vein for 28 days, the Crp3-KO displayed a threefold increase of the intima layer compared to wild-type animals. Consistent with our data, Hall et al. demonstrated that the intima/media ratio of wired-injured carotid arteries was significantly greater in Crp3-KO mice compared to wild-type (9). Interestingly, it was also described that the two Crp3 paralogs – CRP1 and CRP2 – influence vascular remodeling. Following wire injury of femoral arteries in mouse, the absence of CRP1 attenuates neointima formation (23) while CRP2-KO mice display increased vascular remodeling in response to vascular injury (24). Cells are pre-programmed to undergo apoptosis when they become irreparably damaged or no longer receive essential survival signals (25). Increasing in VSMCs apoptosis can decrease neointimal hyperplasia (26,27), and the fine tuning of this process is essential to control the remodeling. One of the well-established survival signals of vascular smooth muscle is the focal adhesion structure. Integrins are cell surface adhesions receptors that act as mechanoreceptors, detecting mechanical stimuli originating from the extracellular matrix and converting them to chemical signals that regulate cell viability pathways (28–30). The focal adhesion kinase (Fak) is one of the main integrin-associated signaling molecules, and it has been demonstrated that a ȕ1integrin/Fak/Akt pathway regulates fibroblast survival to mechanical stimuli (31). We observed that ceramide decreased phosphorylation of Fak (Y397) and Akt (S473) in stretched SMCs from the jugular vein. This event is associated with increased expression of Bax, the Bcl-2 antagonist protein, and subsequent caspase-3 activation. Because the impairment of the Fak/Akt pathway occurs with no changes of beta-1 integrin, we performed immunopreciptation studies with FAK and Crp3 in stretched jugular SMCs. Our data provide evidence that Crp3 interacts with Fak and this is necessary to ceramide-induced apoptosis in stretched jugular SMCs indicating that Crp3 is part of the focal adhesion structure. The evidence that Crp3 interacts with zyxin,

another key protein of focal adhesions (32,33), supports this interpretation. It has been proposed that the Crp3 controls the actin cytoskeleton structure by the formation of a ternary complex between zyxin, Crp3, and Į-actinin. Additionally, it was recently shown that Crp3 binds, stabilizes and crosslinks the actin cytoskeleton into bundles (34). These findings suggest that interaction between Crp3 and zyxin at the focal adhesion, connected to the actin cytoskeleton is responsible for force generation in response to mechanical force (35). Our results indicate a role for Crp3 beyond its interaction with the actin cytoskeleton. We demonstrated that Crp3 interacts with Fak, the main integrin downstream associated-signaling molecule, which then triggers intracellular pathways to control cell activity. Altogether, the knockout rat in vivo model indicted that lack of Crp3 gene sensitizes vein remodeling in response to arterialization. We then used Crp3 knockout cell model system to show that Crp3 interacts with Fak to sensitize stretched vein SMC to apoptosis. This response is due to a decrease in integrin-mediated downstream signaling, followed by a decrease in Fak (Y397) and Akt (S473) phosphorylation, with the subsequent increase in Bax expression and activation of effector caspase-3. Notably, these findings underscore the potential role of Crp3 as a modulator of vascular remodeling during the vein graft arterialization and other vascular remodeling processes.

Clinical Perspectives (i)

Unlike arteries, smooth muscle cells from veins do not express Crp3, which can be induced during vein arterialization remodeling. So, we developed a knockout rat to assess the influence of Crp3 in this process.

(ii)

In this study, we provide evidence that Crp3 in association with Fak activates the intracellular response to mechanical stretch and sensitizes SMC to apoptosis. In contrast, the absence of Crp3 leads to an increased remodeling of vein arterialization.

(iii)

Thus, it is tempting to speculate that Crp3 gene variants may influence vascular remodeling outcomes and that this pathway may be explored to prevent neointimal growth leading to pathological remodeling or to predict vascular therapeutic outcomes.

Acknowledgments The authors want to thank Christian Merkel for the generating Crp3-KO rats, Monica Nunes Bizerra for performing vein arterialization surgery, Leandro dos Santos for collecting vessels samples, Julliana Campos de Carvalho for processing vessel samples for morphological and immunohistochemistry analysis, Viviane Caceres for cardiac hemodynamic measurements, Leonardo Jensen for blood flow measurement, Diego Campos for blood pressure measurement and Luciano Figueredo Borges for the transmission electron microscopy analysis. We are grateful to Silvia Arber and Pico Caroni (Friedrich Miescher Institute, Basel, Switzerland) for providing the anti-Crp3 antibody.

Declarations of interest None declared

Funding This work was supported by Sao Paulo Research Foundation – FAPESP (2013/173680). LCGC and ASM were recipient of a fellowship from FAPESP. JCR-S is recipient of fellowship from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico CNPq (130142/2016-6)

Author Contribution Conception and design: LCGC, AAM, JEK Analysis and interpretation: LCGC, JCR-S, VGB, AAM, JEK Data collection: LCGC, JCR-S, ASM, VGB Writing the article: JCR-S, AAM Critical revision of the article: LCGC, AAM, JEK Statistical Analysis: LCGC, JCR-S, AAM Obtained funding: AAM, JEK Overall responsibility: AAM, JEK

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Figure Legends Figure 1. Crp3-KO rats exhibit increased vascular remodeling in response to arterialization and lack early apoptotic events. (A) Representative VVG stain performed on sections from wild type and Crp3-KO jugular veins harvested 28 days after arterialization. An increased remodeling response is observed in Crp3-KO jugular vein compared with wild type, as confirmed by quantitative morphometric analysis of intimal area in wild type and Crp3-KO jugular veins. We found no differences in media and adventitia layer, total amount of collagen and elastin (n=8) between Crp3-KO and wild type. (B) Representative images of TUNEL assay performed in arterialized wild type and Crp3-KO jugular veins one day after surgery. The wild type jugular vein displays a great number of TUNEL-positive cells, an event that is not observed in Crp3KO arterialized jugular vein (n=10). (C) No change in proliferation rate is observed in response to the knockout of Crp3, as evidenced by ki67 staining of wild type and Crp3KO jugular veins seven days after arterialization (n=10). The magnification of the image is 100X in (A) and 400X in (B) and (C). * indicates p