Increased Soluble VCAM-1 and Normal P-Selectin in Cystic Fibrosis: a Cross-Sectional Study

VCAM-1 is a ligand of very late antigen-4 (CD49d/CD29; integrin α4β1), taking part in adhesion of leukocytes to the endothelium [29]. It was found to associate with coronary artery disease [30] and its concentrations were shown to be higher in patients with hypertension [31], obesity, and diabetes [32], and also in women with preeclampsia [33]. sVCAM-1 levels were also raised in diabetic women in early pregnancy having both retino- and nephropathy compared with those who did not have retinopathy [34]. Retinopathy associated with high sVCAM-1 concentrations in another study as well, suggesting a link not only to microangiopathy through endothelial damage, but also to neovascularization [35].

In renal-insufficient patients without diabetes and atherosclerosis, sVCAM-1 was correlated with carotid intima-media thickness [36]. In menopausal women, blood-borne microvesicle VCAM-1 was weakly associated with a positive change in carotid intima-media thickness over a period 4 years; microvesicle P-selectin was included in the principal component, predicting increases in reactive hyperemia [37]. A 6-month follow-up of 75 acute coronary syndrome patients demonstrated that sVCAM-1 levels predict the risk of future major cardiac events (OR 4.62; 95% CI 1.8–11.4) [38]. High serum sVCAM-1 associates with coronary artery disease as well as lower brain-derived neurotrophic factor values during oral glucose tolerance test [39]. VCAM-1 relates to the thickness of the carotid intima-media and its plaque in rheumatoid arthritis [40]. In women, serum sVCAM-1 is inversely correlated with visceral adipose tissue [41]. In persons with hyperglycemia, moderate exercise was shown to increase sVCAM-1 [42].

In a murine model of reduced VCAM-1 expression, the burden of aortic lesions was 48% lower compared with a control group [72]. In estrogen-deficient rats, sVCAM-1 decreased after augmenting the content of polyunsaturated fatty acids in the diet; this was also accompanied by reductions in both leukocyte adherence to the wall of the aorta and platelet adhesiveness [43]. VCAM-1 is overexpressed in atherotic plaque after acute hypoxia and its silencing with interfering RNA in vivo decreases granulocyte recruitment to the damaged tissue [44]. Interestingly, the association between a plasma-specific microRNA—miR-1185—and arterial stiffness was found to be partially mediated by VCAM-1 [45].

It should, however, be admitted that there is important evidence contrary to sVCAM-1 being an independent risk factor for cardiovascular events [46, 47]. Both soluble and membrane VCAM-1 are unspecific. VCAM-1 expression is raised in acute respiratory distress syndrome [48], in breast [49] and non-small cell lung cancer [50], and in rheumatoid arthritis [51], where it decreases following treatment with infliximab and methotrexate [52]. Plasma sVCAM-1 is known to respond to the exposure to air pollution with fine particulate matter (PM_2.5) [53]. The available research on the role of this molecule is mainly clinical. Overall, in patients with substantial endothelial damage sVCAM-1 may be considered a risk factor of cardiovascular disease and in other subjects an indicator of atherosclerosis progression [54].

We did not confirm the findings by De Rose et al., who found that sVCAM-1 levels did not differ in 29 patients with CF and 12 healthy volunteers [19]. In the light of the above-mentioned studies, it might be proposed that the higher sVCAM-1 concentration in CF—as found in this study—may reflect a state of chronic inflammation, which probably predisposes to cardiovascular disease.

sVCAM-1 concentrations found by various studies differ and may not be comparable. For instance, its serum levels in HS in our study were similar to that found by some other research groups [43, 55, 56]. They are, however, higher than reported in a number of other publications [30, 33, 57]. We suppose that this variability might be due to specificity of antibodies used in ELISA kits. In fact, while the main sVCAM-1 form has the molecular weight of about 100 kDa, smaller forms also exist. Curiously, Hahne et al. showed that in mice it was 42-kDa sVCAM-1 and not 100-kDa sVCAM-1 that responded to stimulation with lipopolysaccharide [58]. Garton et al. indicated that it is the 100-kDa sVCAM-1 that is predominant in the serum of mice and that it was cleaved from the cellular surface by ADAM (a disintegrin and metalloproteinase) metallopeptidase domain 17 (ADAM17) in response to stimulation by 12-O-tetradecanoylphorbol-13-acetate [59]. They also revealed that neutrophil elastase produces a 65-kDa sVCAM-1 cleavage product. Singh et al. demonstrated that ADAM17-mediated sVCAM-1 shedding could be cytokine-induced [60], but did not relate to the molecular weight of obtained sVCAM-1. While some antibody suppliers state that their antibodies yield only the 100 kDa band, we also found others that admitted that the antibodies detected unidentified 48–49-kDa proteins. Therefore it cannot be excluded that there is a systematic bias introduced by various ELISA kits, some of which might recognize only the epitopes that are present in large, but not in smaller forms of sVCAM-1. This renders difficult the direct comparisons of sVCAM-1 concentrations obtained in different studies.

P-selectin enables leukocyte adhesion to the vascular wall [61]. Its major ligands include P-selectin glycoprotein ligand-1 and TIM-1—T cell immunoglobulin and mucin domain 1 [62, 63]. The role of sP-selectin in atherosclerosis development and its association with the risk of venous thromboembolism is supported by studies of SELP polymorphisms [64, 65] and research in animal models [66]. Additionally, SELP rs6128 major allele is associated with a higher sP-selectin concentration and diabetic retinopathy [67]. In dialysis patients, raised sP-selectin concentrations associate with atherosclerotic cardiovascular disease and mortality [68]. The Framingham Heart Study Offspring and Omni studies revealed correlates of sP-selectin levels: male sex, age, cigarette smoking, and other modifiable cardiovascular risk factors [69]. sP-selectin is also more abundant in the blood (serum and plasma) of patients with severe chronic venous insufficiency [70]. In community-acquired pneumonia plasma sP-selectin is increased [71] and predicts the occurrence of myocardial infarction [72]. Platelet P-selectin is associated with hypertension [73]; in patients with hypertension caused by primary aldosteronism serum, sP-selectin decreased after removal of aldosterone-secreting adrenal adenoma [74]. Plasma sP-selectin associates with waist-to-hip ratio and visceral adipose tissue in men [41]. In healthy persons, physical effort relates to lower sP-selectin [75]. However, it must be underscored that there are also large cohort studies, in which plasma sP-selectin was not linked to cardiological outcomes [7, 76].

P-selectin interaction with its ligand is considered a target for novel therapies aiming at the reduction of cardiovascular risk [77]. Inclacumab, which is a monoclonal antibody against P-selectin, protects against myocardial damage after reperfusion in non-ST-segment elevation myocardial infarction [78]. Platelet P-selectin expression is reduced by thienopyridine class inhibitors of the adenosine pyrophosphate receptor [79]. This is relevant because platelet-derived microparticles reduce the plasticity of FOXP3(+) regulatory T cells by interacting with their P-selectin(+) subset, creating a pro-inflammatory milieu [80].

Serum sP-selectin levels tend to be higher than in plasma since they include P-selectin released from platelets activated during clot formation. The correlation between serum and plasma sP-selectin concentrations was shown to be linear and moderate-to-strong. Moreover, the reproducibility of measurements in both serum and plasma is excellent (intraclass correlation coefficient 0.98 and 0.92, respectively). Serum and plasma sP-selectin does not correlate with platelet count or mean platelet volume [81].

It seems that the role of P-selectin is dependent on where it is localized: in the serum, the platelets, or the endothelium. A cross-sectional study by Cleanthis et al. indicated that soluble—but not platelet—P-selectin correlated with spontaneous platelet aggregation in patients with intermittent claudication or stroke [82]. On the other hand, platelet—and not endothelial—P-selectin is required for the development of acute lung injury after a chemical insult: it mediates a platelet-neutrophil interaction leading to thromboxane A2 production, neutrophil adhesion, and—consequently—greater tissue damage [83].

O’Sullivan et al. found that P-selectin expression on platelets obtained in CF patients was insignificantly greater before stimulation with adenosine diphosphate, significantly higher after this stimulation at all five concentrations employed, and not entirely abolished by prostaglandin E1, which was the case in normal platelets [84]. The incomplete inhibition of platelet aggregation in CF was known previously [85]. Because similar findings were reproduced in washed platelets, O’Sullivan et al. concluded that this was due to an intrinsic platelet property. Curiously, O’Sullivan et al. did not identify CFTR or its mRNA within normal platelets, which led them to propose that the observed CF platelet hyperactivity could be traced back to megakaryocyte or related to a different chloride channel.

Sturm et al. found a higher sP-selectin concentration in 54 CF patients—including children—compared with 55 age- and sex-matched healthy subjects [21]. In the subgroup of subjects aged 15–41 years the values compared as follows (n _CF = 28; n _HS = 29): 41 ng/mL [25‐56] versus 29 ng/mL [17‐60]. A similar difference was observed in children aged 3–14 years. Sturm et al. considered sP-selectin to be a platelet-derived inflammatory factor. Another study, by Romano et al., reported sP-selectin to be higher in 20 CF patients compared with 20 HS [20] and to correlate with worse FEV1%. In our study, sP-selectin levels in patients and HS did not differ and the medians were within the reference range established by Deneva-Koycheva et al. (102–210 ng/mL in the serum) [57].

Whereas Sturm et al. assessed the level of sP-selectin in the plasma, we measured serum P-selectin: initially present in the plasma and released from platelets on clot formation. This partly explains the higher values in our study and may hint at why Sturm et al. and Romano et al. found an effect while we did not. However, the difference between serum and plasma sP-selectin in a study by Valdes et al. was not threefold as in this case but twofold [81]. Considering that median CRP levels and FEV1% in the group assessed by Sturm et al. and in ours were very similar, and that they also included only clinically stable patients, we expect that our assessment methods may have differed as well.

An unsurprising finding is the increased hsCRP concentration. Although the CF patients were clinically stable, it seems plausible that their hsCRP levels reflect a chronic inflammatory process in the respiratory tract. This process may be expected to negatively affect cardiac health.

The main strengths of this research include a relatively large sample size and a comprehensive characterization of CF-related clinical factors. Its main limitations comprise: the cross-sectional design, which does not permit for establishment of causation, and the investigation of biomarkers, which may give insight into pathophysiology, but cannot replace clinical endpoints. It must also be considered that the varying sensitivity of the available tests (various ELISA kits and other antibody-based techniques) lowers the utility of direct comparisons of soluble cell adhesion molecules’ concentrations between studies.