C-reactive protein (CRP) is a powerful independent risk factor for cardiovascular diseases. Elevated mechanical strain on vessels induces the local expression of proinflammatory cytokines. We hypothesized that mechanical strain on vessels may induce local CRP expression. Human saphenous vein and internal mammary artery (IMA) rings were stretched in vitro with a mechanical strength of 1, 3, or 5 g. Reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay results showed that mechanical stretching significantly induced CRP mRNA and protein expression in the saphenous vein and IMA rings in a strength-dependent manner reaching a maximum at a mechanical strength of 3 g, but CRP expression returned at strengths of >5 g. In vessels, mechanical strain-induced CRP expression was blocked by two stretch-activated ion channel (SAC) blockers: GdCl3 and streptomycin. Mechanical strain also increased activation of nuclear factor κB (NF-κB), which was detected with a nonradioactive NF-κB p50/p65 EZ-TFA transcription factor assay. Mechanical strain-induced NF-κB activation was blocked by SAC blockers and the NF-κB inhibitor (SN50, H-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-OH). SN50 also blocked mechanical strain-induced CRP expression in vessels. In conclusion, mechanical strain induces CRP expression in IMAs and saphenous veins by activating the SAC-induced NF-κB pathway.
C-reactive protein (CRP) is a nonspecific marker of inflammation and has been shown to have a close relationship with vascular diseases. CRP is a powerful independent risk factor for atherosclerosis and atherosclerosis-related diseases (Lusic et al., 2006; Verma et al., 2006). Elevated high-sensitivity CRP (hsCRP) has been measured in the blood of patients with essential hypertension (Li et al., 2005) or abdominal aortic aneurysms (Vainas et al., 2003; Tambyraja et al., 2007) with enhanced systemic or local arterial strain. Elevated serum hsCRP independently correlates with blood pressure (Sung et al., 2003), arterial stiffness (Kim et al., 2007), and aneurysmal size (Vainas et al., 2003). Although several investigations have demonstrated that aneurysmal tissues and diseased coronary artery venous bypass grafts (Jabs et al., 2003) produce CRP, little is known about its mechanism.
Blood vessels are dynamically subjected to mechanical strain in the forms of stretch and shear stress that result from blood pressure and blood flow. Mechanical strain on the vessel wall can increase from 15 to 30% in hypertensive individuals (Safar et al., 1981; Shaw and Xu, 2003). Biomechanical stress on the vein graft wall was shown to increase by 91% after graft installation (Karayannacos et al., 1980). Mechanical strain can therefore have very important pathological roles in blood vessels. For instance, excessive hemodynamic strain is involved in vascular remodeling and atherosclerosis genesis (Thubrikar and Robicsek, 1995; Shaw and Xu, 2003). Mechanical strain has been shown to be an important regulator of structure and function in mammalian cells, tissues, and organs (Shaw and Xu, 2003). Mechanical strain, such as hydrostatic pressure and stretch, was shown to induce DNA synthesis and interleukin-6 (IL-6) expression in vascular smooth muscle cells (VSMCs) (Hishikawa et al., 1994; Zampetaki et al., 2005).
Stretch-activated ion channels (SACs) are one of the major classes of molecules involved in mechanosensitive signal transduction. SACs activate several intracellular mechanosensitive signaling pathways, including the nuclear factor κB (NF-κB) pathway (Kumar et al., 2003; Amma et al., 2005). NF-κB regulates the expression of many genes involved in inflammatory and acute stress responses (Piva et al., 2006). NF-κB activation was reported in stretched VSMCs and human aneurysmal walls (Lindeman et al., 2008), but reports concerning stretch-induced CRP expression are lacking.
We examined the hypothesis that mechanical strain induces CRP expression via SAC activation of the NF-κB signaling pathway in human saphenous veins and internal mammary arteries (IMAs).
Materials and Methods
Sample Collection. Our study conformed to the ethical principles outlined in the Declaration of Helsinki. The study protocol was approved by the ethics committee of the First Affiliated Hospital to Sun Yat-sen University (Guangzhou, China). Written informed consent was obtained from all study participants.
Saphenous veins and IMAs were collected from 11 male patients with unstable angina pectoris (coronary stenosis ≥70% as measured by angiography) who had coronary bypass surgery between August and November 2008 at the First Affiliated Hospital. Patients with acute myocardial infarction and other diseases causing increased CRP (e.g., inflammatory disorders, malignancies, infections) were excluded from the study. Saphenous veins or IMAs with atherosclerotic plaques were also excluded.
Preparation of the Vascular Ring. Immediately after removal, tissues were placed in ice-cold Krebs-Henseleit solution containing 118 mM NaCl, 4.76 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 1.25 mM CaCl2, and 11.0 mM glucose. Vessels were dissected, and endothelial cells were removed by gently rotating the vascular section with the tip of a pair of forceps. Each vessel was cut into 3-mm ring segments, which were divided into different groups according to our study protocol. Ring samples were suspended vertically on stainless steel hooks in a tissue chamber containing Krebs-Henseleit solution at 37°C in an atmosphere of 95% O2 and 5% CO2. The mechanical strength generated by the vascular smooth muscle was measured by using a force transducer (JH-2 type; Aerospace Medical Institute, Beijing, China) and recorded with the BL-420 Experimental System of Biological Function (Chengdu TME Technology Company, Chengdu, China). The resting tension was set to 0.2 g. After 90 min of equilibration, rings were activated with 50 mM KCl to test their integrity. The removed endothelial cells were assessed at the beginning of each experiment with 0.5 μM acetylcholine [2-(acetyloxy)-N,N,N-trimethylethanaminium chloride] not producing relaxation of vascular rings precontracted with 1.5 μM prostaglandin F2α [O-2-deoxy-2-(methylamino)-α-l-glucopyranosyl-(1→2)-O-5-deoxy-3-C-formyl-α-l-lyxofuranosyl-(1→4)-N,N′-bis-(aminoiminomethyl)-streptamine sulfate]. Vascular rings were stretched with 0- (0-g group), 1- (1-g group), 3- (3-g group), or 5-g (5-g group) mechanical strengths for 20 min. There were five rings in each group. Lengths of the vascular rings were measured by using a micrometer before and after stretching.
Gadolinium III chloride hexahydrate (GdCl3), sulfate streptomycin [O-2-deoxy-2-(methylamino)-α-l-glucopyranosyl-(1→2)-O-5-deoxy-3-C-formyl-α-l-lyxofuranosyl-(1→4)-N,N′-bis(aminoiminomethyl)-streptamine sulfate], SN50 (a cell-permeable peptide inhibitor of NF-κB), and its mutant SN50M (a cell-permeable inactive control peptide) were applied to evaluate the effects of the mechanical strength-induced SAC/NF-κB pathway on vascular hsCRP expression. GdCl3, streptomycin, SN50, and SN50M were each dissolved in phosphate-buffered saline as stock solutions and diluted in Krebs-Henseleit solution to prepare working solutions to pretreat the vascular rings. Vascular rings in the control group were pretreated with the same volume and concentrations of vehicle solutions. After pretreatment for 1 h, the vascular rings were stretched with a mechanical strength of 3 g for 20 min. Rings were then frozen in liquid nitrogen and stored at -80°C until analysis.
CRP Measurement. Cytoplasmic and nuclear proteins in the vascular rings (five rings per group) were extracted using a protein extraction kit (Viagene Biotech Company, Ninbo, China) according to manufacturer's instructions and stored at -80°C until analysis. The amount of hsCRP was detected from the cytoplasmic proteins by using an immunoassay kit specific for human CRP (R&D Systems, Minneapolis, MN). This assay is highly sensitive and employs the quantitative sandwich enzyme-linked immunosorbent assay. According to the manufacturer's instructions, the minimal detectable concentration of CRP was 0.010 ng/ml. All experiments were carried out in triplicate.
Determination of NF-κB Activation. Two NF-κB subunits, p50 and p65, were detected in the nuclear protein extract from the vascular rings using the NF-κB p50/p65 EZ-TFA Transcription Factor Assay kit (Millipore Corporation, Billerica, NY). This assay combines the principles of the electrophoretic mobility shift assay with a 96-well based enzyme-linked immunosorbent assay. During the assay, the capture probe, a double-stranded biotinylated oligonucleotide containing the DNA binding consensus sequence for NF-κB (5′-GGGACTTTCC-3′), was mixed with the nuclear extract. The active form of NF-κB contained in the nuclear extract bound to its consensus sequence. The extract/probe/buffer mixture was then directly transferred to a streptavidin-coated plate. The active NF-κB protein was immobilized on the capture probe bound to the streptavidin plate well, and inactive unbound material was washed away. The bound NF-κB transcription factor subunits, p50 and p65, were detected with specific primary antibodies. A highly sensitive horseradish peroxidase (HRP)-conjugated secondary antibody was then used for detection. This provides sensitive colorimetric detection that can be read in a spectrophotometric plate reader at 450 nm. The wild-type consensus oligonucleotide (not biotinylated) was used as a specific competitor for NF-κB binding to monitor the specificity of the assay. A mutated consensus oligonucleotide that had no effect on NF-κB binding was the internal negative control.
Reverse Transcription-Polymerase Chain Reaction for CRP mRNA. Total RNA was isolated from the vascular rings with TRIzol reagent (Invitrogen, Carlsbad, CA). Reverse transcription-polymerase chain reaction (RT-PCR) was done by using the Prime-Script RT-PCR Kit (TaKaRa Biotechnology Company, Dalian, China). Total RNA (1 μg) was reverse-transcribed into cDNA by using oligo(dT) and PrimeScript RTase. Human CRP primers (forward, 5′-TCGTATGCCACCAAGAGACAAGACA-3′; reverse, 5′-AACACTTCGCCTTGCACTTCATACT-3′) (Vainas et al., 2003) were designed to amplify a 440-bp fragment from GenBank accession number M11725. Human β-actin primers (forward, 5′-GATTCCTATGTGGGCGACGAG-3′; reverse, 5′-CCATCTCTTGCTCGAAGTCC-3′) were designed to amplify a 532-bp fragment. PCR was performed under the following conditions: denaturation for 5 min at 94°C, 32 cycles at 94°C for 30 s, 57°C for 30 s, 72°C for 1 min, and elongation at 72°C for 5 min. Amplification fragments were separated on ethidium bromide staining 1.5% agarose gels and were confirmed by sequencing. The resulting bands were imaged with a charge-coupled device (CCD) camera (UVItec Limited, Cambridge, UK) and analyzed by using the ImageJ 1.37 system from Wayne Rasband (National Institutes of Health, Bethesda, MD). CRP mRNA expression was normalized to the amount of β-actin mRNA in each sample.
Drugs. Acetylcholine, prostaglandin F2α, GdCl3, and sulfate streptomycin were purchased from Sigma-Aldrich (St. Louis, MO). SN50 and SN50M (Calbiochem, San Diego, CA) were purchased from Merck (Darmstadt, Germany).
Statistical Analysis. Data are indicated as mean ± S.D. Differences between means were evaluated by using analysis of variance (ANOVA) followed by Scheffe's post hoc test for intergroup comparisons. P < 0.05 was considered significant. Statistical analysis was carried out with the Stata 6.0 computer package (Stata Corporation, College Station, TX).
All 11 patients were male with a mean age of 63 ± 7.4 years and a mean body mass index of 23.2 ± 3.1 kg/m2 (Table 1). All patients had a diagnosis of unstable angina due to coronary heart disease; 63.6% (7/11) had a history of hypertension, 36.4% (4/11) had a history of hyperlipidemia, and 36.4% (4/11) had a history of diabetes. According to their medical histories, 100% patients were given aspirin and 72.7% had nitroglycerin, but only 27.3% were given statins. Paired IMAs and saphenous veins were isolated from each patient.
Applying mechanical strengths of 1, 3, and 5 g to IMA and saphenous vein rings induces ring length extensions from the basal lengths in a strength-dependent manner, and saphenous vein rings have more ring length extension than IMA rings under the same mechanical strengths (Table 2). Figure 1 shows that application of mechanical strength resulted in significant up-regulation of CRP mRNA and protein expression in arterial and venous rings. In the IMA rings, application of 1, 3, and 5 g induced increases of 3.27 ± 0.90-, 9.67 ± 3.03-, and 2.80 ± 1.04-fold, respectively, in CRP mRNA expression from the basal level (0-g group) (P < 0.05 versus basal level, n = 11; Fig. 1, A1 and A2). In the venous rings, application of 1, 3, and 5 g induced increases of 3.45 ± 0.71-, 10.27 ± 2.80-, and 2.98 ± 0.92-fold, respectively, in CRP mRNA expression from the basal level (P < 0.05 versus basal level, n = 11; Fig. 1, B1 and B2). The level of hsCRP reached a peak on application of a mechanical strength of 3 g, resulting in increases of 7.28 ± 1.84- and 7.60 ± 2.05-fold from basal levels in the arterial rings and venous rings, respectively (P < 0.05 versus 0-, 1-, and 5-g groups).
The role of SAC activation during mechanical stretch-induced CRP expression in the vascular rings was evaluated with two SAC blockers: GdCl3 and streptomycin. Pretreatment with GdCl3 (25 μM) or streptomycin (200 μM) completely blocked the increases in CRP mRNA and protein expressions induced by 3 g in arterial and venous rings (P < 0.05 versus 3-g group; P > 0.05 versus 0-g group) (Fig. 2).
The effects of mechanical stretch on NF-κB activation were evaluated by detecting activations of NF-κB p65 and p50 in the nuclear extract from vascular rings stretched by mechanical force. NF-κB p65/p50 activations increased 2.34 ± 0.46/2.20 ± 0.48-, 3.81 ± 0.83/3.58 ± 0.54-, and 1.65 ± 0.46/1.67 ± 0.33-fold from the basal level in arterial rings and increased 2.57 ± 0.64/2.40 ± 0.75-, 4.14 ± 1.70/3.86 ± 1.11-, and 1.78 ± 0.44/1.75 ± 0.46-fold from the basal level in venous rings after being stretched by 1, 3, or 5 g, respectively, for 20 min (P < 0.05 versus basal level; Fig. 3). NF-κB p65 and p50 activations reached peak levels in both types of rings after being stretched by a 3-g mechanical force (P < 0.05 versus 1- and 5-g group; Fig. 3).
We then examined whether SAC was involved in mechanical stretch-induced NF-κB activation. In arterial and venous rings stretched by 3 g, activations of NF-κB p65 and p50 were completely blocked by treatment with streptomycin (200 μM) or GdCl3 (25 μM) (Fig. 4). These effects of streptomycin and GdCl3 were similar to those of the NF-κB inhibitor SN50 (18 μM) in inhibiting NF-κB activation (P < 0.05 versus 3-g group; P > 0.05, versus 0-g group). The inactive control peptide, SN50M, had no such effect (P > 0.05 versus 3-g group; P < 0.05 versus 0-g group). These results clearly demonstrated that mechanical strain increased NF-κB p65 and p50 activation by activating SAC.
To further clarify that the NF-κB signaling pathway was involved in the induction of CRP expression by mechanical stretch, we tested the effect of SN50 on CRP expression in the vascular rings. SN50 (18 μM) completely inhibited CRP mRNA and protein expression induced by 3 g in the IMA rings and saphenous vein rings (P < 0.05 versus 3-g group; P > 0.05 versus 0-g group), but SN50M (18 μM) did not affect CRP mRNA and protein expression (P > 0.05 versus 3-g group; P < 0.05 versus 0-g group) (Fig. 5).
The primary finding of our study was that mechanical strain increased CRP expression in the saphenous vein and IMAs in a strength-dependent manner, reaching a peak at a mechanical strength of 3 g. CRP expression partially recovered at strengths of >5 g. CRP expression induced by mechanical strain was blocked by the SAC blockers GdCl3 and streptomycin. Mechanical strain also increased NF-κB activation; this effect was blocked by SAC blockers and by an NF-κB inhibitor (SN50). SN50 also blocked CRP expression induced by mechanical strain in vessels.
This is the first time that mechanical strain has been shown to induce CRP expression in vessels, although the phenomenon has been suggested indirectly from clinical phenomena. Increased serum CRP was observed in patients with atherosclerosis (Lusic et al., 2006), hypertension (Li et al., 2005), aneurysms (Vainas et al., 2003), and vein grafts in coronary bypass surgery (Parolari et al., 2007) in which mechanical strain increased. More directly, patients with elevated pulse pressure, which indicates significantly increased mechanical strain on vessels, have been shown to have higher serum hsCRP levels (Abramson et al., 2002). The sources of increased CRP in patients with these diseases are controversial. Evidence has shown that CRP is present in atherosclerotic plaques (Wilson et al., 2007), aneurysmal tissues (Vainas et al., 2003), and diseased vein grafts; VSMCs can also express CRP (Jabs et al., 2003). Increased hsCRP was believed to be derived from the systemic circulation or release by local vessels (Sun et al., 2005; Gulkarov et al., 2006). The mechanism and relationship between increased mechanical strain and increased hsCRP were unclear. Our work clarifies this relationship by showing that vessel tissues, in the absence of endothelial cells and adventitial connective tissues, express CRP due to mechanical strain. VSMCs are the major constituent of blood vessels that sense mechanical strain, whereas endothelial cells sense shear stress (Kang et al., 2008). To avoid interference from endothelial cells and adventitial connective tissues, vessel rings were prepared without these cells and tissues in our experiments.
In the present study, stretch-induced CRP expression was mediated by opening SAC and inducing NF-κB activation. SACs are present in various cells, including VSMCs (Shaw and Xu, 2003), and can be blocked by gadolinium and streptomycin (Franco et al., 1991; Amma et al., 2005; Yeung et al., 2005). SAC opening can activate downstream signaling pathways, such as mitogen-activated protein kinases and NF-κB pathways, that are involved in the inflammatory reaction (Kumar et al., 2003; Amma et al., 2005; Zampetaki et al., 2005; Piva et al., 2006). Research has shown that mechanical strain opens SACs in lung fibroblast cells, causing phosphorylation of an inhibitor of κB(IκB) and leading to translocation of NF-κB to the nucleus (Amma et al., 2005). SAC activation seems to be rapid, occurring within several seconds (Bett and Sachs, 2000). It was shown that 30 min of high tidal volume ventilation, which increased the mechanical strain in the lung, was sufficient to cause significant up-regulation of 10 genes encoding transcriptional factors, stress proteins, and inflammatory mediators and significant suppression of 12 genes mainly encoding metabolic enzymes (Copland et al., 2003). Our study showed that sustained mechanical strain for 20 min sufficiently induced CRP expression in blood vessels, and we observed similar results after 1 h (data not shown). The vessels of patients with hypertension or aneurysms generally undergo persistently enhanced mechanical strain.
Mechanical strain-induced hsCRP expression in vessels may be involved in pathological processes in atherosclerosis. Although a few studies suggested that the proinflammatory effects of commercial CRP in vessels were caused by contamination with lipopolysaccharide, not by CRP itself (Pepys et al., 2005), numerous studies have shown that highly purified CRP (in the absence of lipopolysaccharides and azides) directly activate neutrophils and mononuclear cells, resulting in the production of oxygen free radicals, matrix metalloproteinase, and various cytokines, such as tumor necrosis factor-α and IL-1β (Prasad, 2004; Verma et al., 2006; Nabata et al., 2008). One study has shown that the minimal amount of lipopolysaccharide derived from commercial CRP does not induce an inflammatory response in vivo (Bisoendial et al., 2005). Locally produced CRP in vessel walls may therefore be an important mediator in the stretch-induced inflammation response.
Our demonstration of stretch-induced CRP expression provides new insights into the associations between mechanical strain, inflammatory diseases, and vascular diseases. Mechanical strain is a key modulator of the morphology and function of VSMCs and can lead to apoptosis, hypertrophy, and proliferation, which contribute to the development of atherosclerosis, hypertension, and restenosis (Shaw and Xu, 2003). Atherosclerotic lesions occur mainly in areas where vessels experience elevated stretch stress and low shear stress (Zou et al., 1998; Long et al., 2000; Xu, 2000). In their native environment, veins usually do not develop atherosclerosis, but if they are grafted from a low-pressure environment into high-pressure arterial circulation, veins are likely to develop arteriosclerosis (Zou et al., 1998), which is the main reason for late vein graft failure (Motwani and Topol, 1998; Shaw and Xu, 2003). Essential hypertension (Li et al., 2005), aneurysms (Shimizu et al., 2006; Lindeman et al., 2008), and diseased vein grafts (Zou et al., 1998 and 2000), which lead to increased mechanical strain in vessels, are related to inflammation and atherosclerosis. The latter is believed to be an inflammatory disease that involves leukocyte recruitment and production of proinflammatory mediators, such as adhesion molecules, IL-6, and CRP (Libby et al., 2002; Verma et al., 2006). Mechanical strain has been shown to increase production of IL-6 and adhesion molecules in mouse aortic smooth muscle cells, human vascular endothelial cells, and vein grafts (Nagel et al., 1994; Zou et al., 2000; Zampetaki et al., 2005).
In conclusion, mechanical strain triggers CRP expression in IMAs and saphenous veins. Its mechanism involves the alteration of SAC gating leading to the transformation of the mechanical stimulus into an electrical or biochemical signal, subsequently leading to translocation of NF-κB to the nucleus of VSMCs.
We thank Jianwen Chen and Cheng Li for technical assistance and for providing the apparatus to prepare the vascular rings.
This work was supported by the National Natural Science Foundation of China [Grant 30770897/C03030201].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CRP, C-reactive protein; hsCRP, high-sensitivity C-reactive protein; IL-6, interleukin-6; VSMCs, vascular smooth muscle cells; SAC, stretch-activated ion channel; NF-κB, nuclear factor-κB; IMAs, internal mammary arteries; RT-PCR, reverse transcription-polymerase chain reaction; SN50, H-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-OH; SN50M, H-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gln-Arg-Asn-Gly-Gln-Lys-Leu-Met-Pro-OH.
- Received January 14, 2009.
- Accepted April 24, 2009.
- The American Society for Pharmacology and Experimental Therapeutics