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Keywords:

  • progenitor cells;
  • CRP;
  • apoptosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In vitro, C-reactive protein (CRP) impairs endothelial progenitor cell (EPC) function; however, the influence of CRP on EPCs in vivo is unclear. We determined whether EPC function is impaired in adults with elevated plasma CRP concentrations, independent of other risk factors. EPCs were harvested from 75 adults (43 males, 32 females): 25 with low CRP (<1.0 mg/L); 25 with moderate CRP (1.0–3.0 mg/L); and 25 with high CRP (>3.0 mg/L). The capacity of EPCs to form colonies (colony assay), migrate (Boyden chamber), release angiogenic growth factor (ELISA) and resist apoptosis (active caspase-3) was determined. There were no significant differences between the CRP groups in EPC colony formation (CFU), migration (AU) or the ability to release vascular endothelial growth factor (VEGF; pg/mL): low (13 ± 3 CFU; 1255 ± 100 AU; 126 ± 24 pg/mL); moderate (11 ± 3 CFU; 1137 ± 85 AU; 97 ± 14 pg/mL); and high (13 ± 4 CFU; 1071 ± 80 AU; 119 ± 22 pg/mL) CRP. Staurosporine-stimulated activation of caspase-3 was also similar between the low (2.3 ± 0.2 ng/mL), moderate (2.1 ± 0.3 ng/mL), and high (2.2 ± 0.2 ng/mL) CRP groups. These results indicate that elevations in plasma CRP are not associated with impaired EPC function. EPC dysfunction may not play a role in CRP-related cardiovascular risk.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The acute phase reactant C-reactive protein (CRP) has emerged as an independent predictor of future atherothrombotic events.[1] CRP appears to be a pro-atherogenic factor that is mechanistically involved in the initiation and progression of atherosclerotic vascular diseases.[2-4] Although the exact mechanisms by which CRP promote atherothrombosis are not fully understood, CRP is thought to directly promote vascular remodeling, atherosclerotic plaque instability and endothelial cell dysfunction.[4] Circulating endothelial progenitor cells (EPCs) represent a key mediator of vascular repair and have the capacity to migrate to local sites of ischemia and endothelial damage, resist cellular apoptosis, secrete potent angiogenic growth factors, and participate in neovascularization.[5, 6]

In vitro, CRP has been shown to adversely alter EPC migration and angiogenic growth factor release.[7] Thus, EPC dysfunction has been suggested to be a contributing factor to the atherogenic effects of CRP. Currently, it is unknown if physiologic elevations in plasma CRP concentrations in healthy adult humans are associated with impaired EPC function. The experimental aim of this study was to determine whether EPC function is impaired in adults with elevated plasma CRP concentrations, independent of other risk factors.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Subjects

Seventy-five, healthy, sedentary, nonsmoking, nonmedicated adults (43 males/32 females) were studied: 25 with CRP < 1.0 mg/L (low CRP; 15M/10F), 25 with CRP 1.0–3.0 mg/L (moderate CRP; 15M/10F), and 25 with CRP > 3.0 mg/L (high CRP; 13M/12F). CRP classification tertiles were based on published guidelines regarding plasma CRP concentrations and associated cardiovascular risk.[8, 9] Before participation, all of the subjects had the research study and its potential risks and benefits explained fully before providing written informed consent according to the guidelines of the University of Colorado at Boulder.

Body composition and metabolic measurements

Body mass, body mass index, minimal waist circumference were measured according to standard techniques. Percent body fat was determined by dual energy x-ray absorptiometry (Lunar Radiation Corporation, Madison, WI, USA). Fasting plasma lipid, lipoprotein, glucose and insulin concentrations were determined by the clinical laboratory affiliated with the Clinical Translational Research Center at the University of Colorado, Boulder.

Plasma CRP concentration

Blood samples were collected in chilled EDTA tubes with minimal venostasis, after a 12-hour overnight fast as previously described.[10] Plasma concentrations of high sensitivity CRP were determined in duplicate by enzyme immunoassay (ALPCO Diagnostics, Salen, NH, USA). Intra- and interassay coefficients of variation were <7% and <8%, respectively.

Putative EPC isolation, characterization, and function

Putative EPCs were isolated as previously described by our laboratory.[11] Endothelial phenotype of these cells was confirmed by immunofluorescent staining for the uptake of DiI-ac-LDL and expression of von Willebrand factor, VE-cadherin, CD31, and VEGFR-2. EPC colony-forming capacity was determined as described previously by our laboratory.[12] Only colonies consisting of multiple thin, flat cells emanating from a central cluster of rounded cells were counted. EPC migration was determined using a modified Boyden chamber with calcein AM labeling.[12] Phytohemagglutinin (PHA; 10 μg/mL) was used to stimulate EPC release of vascular endothelial growth factor (VEGF).[10] VEGF concentration in EPC-conditioned growth medium was determined by enzyme immunoassay. Activation of intracellular caspase-3 was used as biomarker of apoptotic susceptibility. EPCs were incubated with the apoptotic stimulus staurosporine (1 μmol/L) for 3 h at 37°C. The cells were lysed and the concentration of active caspase-3 in the supernatant was determined by enzyme immunoassay.[11] VEGF release and intracellular caspase-3 concentrations were determined in a subgroup of 15 subjects in each of the three groups.

Statistical analysis

Differences in subject baseline characteristics and the primary outcome variables were determined by analysis of variance. Relations between variables of interest were determined by linear regression analysis. There were no significant gender interactions in any of the primary outcome variables; therefore, the data were pooled and presented together. Because of the skewed distribution of plasma CRP concentrations, the data were log transformed to satisfy basic assumptions for parametric testing. However, per joint American College of Cardiology and Centers for Disease Control recommendations,[8] the absolute values for CRP are presented to facilitate clinical interpretation. Data are reported as mean ± SEM. Statistical significance was set at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Selected subject characteristics are presented in the Table 1. There were no differences in any anthropometric, hemodynamic, or metabolic variables between the groups. EPC colony-forming units (CFU) and migration (AU) were not different between the low (13 ± 3 CFU; 1255 ± 100 AU), moderate (11 ± 3 CFU; 1137 ± 85 AU) and high (13 ± 4 CFU; 1071 ± 80 AU) CRP groups (Figure 1). There were no significant CRP-related differences in either VEGF release or caspase-3 activity between the groups. Ability of EPCs to release VEGF between the low (126 ± 24 pg/mL), moderate (97 ± 14 pg/mL) and high (119 ± 22 pg/mL) CRP groups was comparable across the groups (Figure 1). Active caspase-3 concentrations under basal or staurosporine-stimulated conditions were almost identical between the low (0.3 ± 0.1 and 2.3 ng/mL), moderate (0.3 ± 0.07 and 2.1 ± 0.3 ng/mL) and high (0.3 ± 0.1 and 2.2 ± 0.2 ng/mL) CRP groups (Figure 1).

Table 1. Selected subject characteristics
VariableLow CRP (<1.0mg/L) (n = 25)Moderate CRP (1.0–3.0 mg/L) (n = 25)High CRP (>3.0mg/L) (n = 25)
  1. Values are mean ± SEM.

  2. BMI = body mass index; BP = blood pressure; LDL = low-density lipoprotein;

  3. HDL = high-density lipoprotein.

  4. aP < 0.05 vs. low CRP. b†P < 0.05 vs. moderate CRP

Age (year)52 ± 251 ± 256 ± 2
Gender (M/F)15/1015/1013/12
CRP (mg/L)0.4 ± 0.12.0 ± 0.1a5.8 ± 0.4a,b
Body mass (kg)76.6 ± 3.679.0 ± 2.277.5 ± 2.4
BMI (kg/m2)25.4 ± 0.826.4 ± 0.526.7 ± 0.6
Body fat (%)28.8 ± 1.931.9 ± 1.933.7 ± 1.8
Waist circumference (cm)87.1 ± 2.889.9 ± 2.190.9 ± 1.7
Systolic BP (mmHg)119 ± 2119 ± 2120 ± 2
Diastolic BP (mmHg)76 ± 174 ± 277 ± 1
Total cholesterol (mg/dL)195.7 ± 5.3204.4 ± 6.0208.0 ± 4.9
LDL-cholesterol (mg/dL)124.4 ± 4.6127.4 ± 5.2134.6 ± 4.7
HDL-cholesterol (mg/dL)49.5 ± 2.552.4 ± 2.851.8 ± 2.8
Triglycerides (mg/dL)109.4 ± 8.8123.5 ± 13.2108.0 ± 8.0
Glucose (mg/dL)88.6 ± 2.093.6 ± 1.789.6 ± 1.5
Insulin mU/mL6.6 ± 0.77.3 ± 0.86.4 ± 0.6
image

Figure 1. EPC colony forming units (Panel A), migration (Panel B), release of VEGF (Panel C), and Intracellular concentrations of active caspase-3 in the absence and presence of staurosporine stimulation (Panel D) in the low, moderate and high CRP groups. Values are mean ± SEM.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The novel finding of this study is that elevations in CRP concentrations is not associated with EPC dysfunction in healthy adults. The ability of EPCs to form colonies, migrate, release angiogenic growth factors, and resist apoptosis is not influenced by CRP concentrations. To our knowledge, this is the first study to investigate the relation between circulating CRP levels and EPC function.

Plasma CRP concentrations were first identified as a strong, independent predictor of future cardiovascular events in healthy men by Ridker et al.[13] with data from the Physicians Health Study in 1997. Since that initial report, the ability of CRP to predict myocardial infarction, ischemic stroke, and cardiovascular mortality has been confirmed in more than 20 diverse population cohorts.[1] There is great interest in CRP as a causal agent in the initiation and progression of atherosclerosis.[2, 4] However, whether CRP is a direct contributor to atherosclerosis or a marker of risk is unclear. The results of this study suggest that elevations in CRP do not adversely affect functional properties of EPCs that are associated with vascular disease. Indeed, colony-forming capacity, migratory ability and angiogenic growth factor release are important functional characteristics of EPCs that have been associated with endothelial function and cardiovascular risk in healthy and diseased populations.[14, 15] In this study, there were no differences in the number of EPC CFUs, migration or the release of the angiogenic growth factor VEGF between the low, moderate and high CRP groups. This is interesting considering that EPC colony forming capacity, migration and angiogenic growth factor release are often impaired in conditions associated with elevated CRP concentrations, such as aging[12] and obesity[16] and CRP has been shown to have negative effects of EPCs in vitro.[7, 17] Moreover, because EPCs must survive in toxic environments to promote vessel repair, it is important that these cells are capable of resisting apoptotic stimuli. We observed no differences between the CRP groups in EPC intracellular active caspase-3 concentrations under basal or stimulated conditions. In direct contrast to in vitro studies,[17] our findings suggest that elevations in CRP concentration do not promote increased EPC susceptibility to apoptosis.

The discrepancy between our findings and in vitro studies is not clear, but, may be attributed to the purity of CRP used in basic cell experiments. Several studies have questioned the integrity of commercial CRP preparations used in vitro experiments.[17-22] Many studies have used recombinant CRP from E.Coli, initially intended for use in assay calibration.[21] The presence of lipopolysaccharide (LPS) and azide preservatives in these commercial CRP preparations have been shown to induce pro-inflammatory and pro-apoptotic responses in endothelial cells; raising doubt whether these reported effects of CRP in vitro are true effects of the protein per se.[21, 23] Several studies have explored the effects of commercial CRP preparation on a variety of cellular phenotypes. With respect to EPCs, Liu et al.[20] demonstrated that dialyzed CRP without azide contamination had no effect on EPC function. However, both nondialyzed commercial CRP and sodium azide significantly impaired EPC function to a similar extent. Taylor et al.[19] reported that apoptosis in endothelial cells was increased only with CRP preparations containing LPS and sodium azide, not with highly purified CRP. Moreover, in separate studies Lafuente et al.[18] and Pepys et al.[23] abolished the inflammatory effects of commercial CRP on vascular cells when sodium azide was removed. In addition to concerns regarding the purity of CRP preparations used for in vitro studies, there is evidence that pentameric and momomeric conformations of commercial CRP have divergent effects on both EPCs[22] and endothelial cells.[24] These studies, taken together with our findings, suggest that treatment of EPCs with certain CRP preparations in vitro may not accurately reflect the interaction between circulating CRP concentrations and EPC function in vivo.

It is important to note, that the findings of this study should be considered within the context of our isolation, culture and characterization of putative EPCs. Indeed, a consensus definition (and methodology for isolation) of an EPC remains elusive. Some studies involving EPCs have utilized prolonged in vitro culture conditions (2–4 weeks), resulting in a proliferative endothelial phenotype that is morphologically similar to mature endothelial cells.[25] However, the potential clinical importance of these cells as a clinical biomarker or an in vivo vascular progenitor cell is unclear. In this study, we isolated putative EPCs with minimal time spent in culture to limit phenotypic drift from circulating cells in the vasculature. Importantly, these cells demonstrate several endothelial phenotypic characteristics including DiI-ac-LDL uptake, lectin binding and expression of von Willebrand factor, VE-cadherin, VEGFR-2, and CD31.[26, 27] Moreover, their role in angiogenesis and vascular repair has been documented,[26, 28] and their dysfunction has been linked to impaired endothelial function and cardiovascular risk.[29, 30]

In conclusion, elevations in circulating CRP concentrations in middle-aged and older adults are not associated with EPC colony formation, migratory capacity, growth factor release or susceptibility to apoptosis. It is plausible that the reported negative in vitro effects of CRP on EPCs may be because of contamination of commercially available CRP preparations with LPS and/or azide preservatives. Clinically, CRP-related increase in cardiovascular risk may not involve impairment in EPC function.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study was supported by National Institutes of Health awards HL077450, HL076434, MOI RR00051 and 1 UL1 RR025780 and American Heart Association award 0840167N.

References

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  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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