Accumulating evidence points to a relationship between inflammation and blood pressure. Data suggest that WBC counts are associated with incident hypertension and correlated with blood pressure concentrations. We hypothesized that WBC count is a surrogate for leukocytic chemokine activity and that the CXCL5 gene, which encodes the neutrophil attractor ENA-78, may be an important determinant of blood pressure. We demonstrated a significant, independent relationship between CXCL5 polymorphisms and SBP and DBP in the overall population of CVD-free individuals. Variant carriers of the −156 G > C promoter SNP had 7-mmHg and 4-mmHg higher SBP and DBP, respectively, than those with the wild-type −156 G/G genotype. Because of the epidemiologically significant difference in CVD risk conferred by blood pressure differences of this magnitude, and since variant carriers represent approximately 30% of the population studied, CXCL5 polymorphisms should be considered as a potential novel biomarker of pre-hypertension, hypertension, and CVD risk requiring future study. However, it is important to emphasize that genetic associations are preliminary and will require confirmation in additional populations.
Of particular interest, WBC count (along with traditional variables such as age, sex, smoking status, and BMI) was significantly associated with SBP and DBP in univariate analysis among CVD-free individuals. This finding supports the report by Orakzai et al. that demonstrated a relationship between WBC counts and SBP among nearly 3,500 white individuals without CVD and with SBP < 140 mmHg on entry . It also supports data from other clinical cohorts showing an association between WBC count, major WBC components (e.g., neutrophils), and blood pressure [21, 22, 40, 41]. However, in our analysis WBC count was no longer a significant predictor of SBP when CXCL5 genotype was included in multivariable analysis, suggesting genotype may capture the contribution of inflammation to SBP more effectively than WBC count. WBC did, however, remain a significant predictor of DBP in multivariate analysis, along with age, sex, and CXCL5 -156 G > C genotype.
To determine whether there is any functional basis for an observed association between CXCL5 variant alleles and blood pressure, we performed allele expression imbalance experiments in a subset of participants. The exonic 398 G > A allele was chosen as the genetic marker given its location in the coding region of the mRNA. However, the 398 G/A heterozygous individuals (N = 18) were also heterozygous for the promoter polymorphism, which minimizes confounding of an association by differing genotypes at the upstream locus. It was noted that variant carriers displayed nearly threefold higher expression of variant CXCL5 mRNA transcripts from the 398A allele. This novel finding is consistent with our previous observation that variant carriers exhibited higher plasma and leukocyte-produced ENA-78 than wild-type homozygotes and that the promoter and exonic SNPs occur in transcription factor binding and splicing enhancer sites, respectively . Given that the −156 G > C and 398 G > A SNPs are in near perfect linkage disequilibrium, it is unclear which polymorphism is the causal variant and functionally contributes to the blood pressure phenotype. However, the −156 G > C promoter SNP was more significantly correlated with blood pressure in our study. Further functional studies of these SNPs are warranted.
In addition to genotype and traditional covariates, we included plasma CRP and ENA-78 protein concentrations in our analyses. While CRP and ENA-78 were significantly associated with SBP (and PP) in univariate analyses, they fell out of the models when CXCL5 genotype was included. This suggests that in our analyses, genotype is more significantly associated with the blood pressure phenotype than systemically circulating concentrations of the non-specific inflammatory mediator CRP and the CXCL5 protein product ENA-78. While this observation may appear somewhat contradictory, it can be postulated that CXCL5 gene polymorphisms may be better indicators of chemokine activity at the target organ (e.g., endothelium) level than a measurement in the circulation. Because of trans-acting influences on systemic biomarker expression, polymorphisms in CXCL5 may be more robustly associated with blood pressure. In fact, we have shown a similar finding in a different population for the endothelial nitric oxide synthase gene where NOS3 gene polymorphisms, but not measures of circulating NO activity, were associated with arterial stiffness in children with type 1 diabetes [42, 43]. Further support for this observation can be found in a case–control study of the role of ENA-78 in patients with ischemic stroke. Zaremba et al. demonstrated that serum ENA-78 protein concentrations were not different between stroke patients and controls; contrarily, it was demonstrated that ENA-78 concentrations were significantly higher (twofold) in the cerebrospinal fluid of stroke patients compared with controls . Taken in sum, it is possible that genotype more effectively captures the likelihood for local preponderance of chemokine activity than plasma protein level.
In general, there is biological plausibility for the role of CXCL5 in CVD. For example, the protein product of CXCL5, ENA-78, belongs to the same class of chemokines as IL-8, IP-10, and I-TAC, which have been previously implicated in atherosclerotic inflammation [23, 45]. ENA-78 has been shown to be chemotactic for neutrophils and stimulate neutrophilic degranulation causing release of myeloperoxidase and generating reactive oxygen species [24, 25]. In addition, ENA-78 is involved in platelet-dependent activation of monocytes, displays angiogenic properties, and has been implicated in diseases such as obesity, diabetes, subclinical atherosclerosis, acute coronary syndromes, ischemic stroke, abdominal aortic aneurysm, and thrombosis [27, 28, 32, 44, 46–51]. Hypertension is a risk factor for adverse events such as atherosclerosis, stroke, and abdominal aortic aneurysm, and ENA-78 is overexpressed in these situations. We have shown CXCL5 polymorphisms to be associated with ENA-78 concentrations, blood pressure, and prognosis following acute coronary syndromes [27, 33]. Thus, the role of CXCL5 in CVD should be further explored. As final hypothesis-generating evidence of a link between the CXCL5 pathway and blood pressure, statins have been hypothesized to have mild antihypertensive effects, and we have shown that atorvastatin reduces ENA-78 production from human endothelial cells in a dose-dependent fashion [52, 53]. Our findings, along with existing data, support the need for future investigation of CXCL5 as a hypertension- and CVD-susceptibility gene.