We chose PAH for the first clinical application of the EPC capture chip for several reasons: (i) PAH is a prototype of proliferative cardiovascular diseases: Its pathobiology is characterized by endothelial cell death and progressive obliteration of the peripheral pulmonary arteries, and involves multiple signalling pathways which currently makes tailored PAH therapy extremely difficult. Novel PAH biomarkers that indicate disease severity, progression and prognosis of PAH and associated right ventricular (RV) dysfunction would be extremely helpful in guiding established and more experimental clinical therapies. (ii) The number of circulating EPCs represents a promising candidate biomarker for pulmonary vascular disease severity: Adult IPAH patients have reduced numbers of circulating EPCs when compared with healthy controls; a reduced number of EPCs (CD34+/KDR+) is associated with worse hemodynamics, and abnormally elevated concentrations of inflammatory markers, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-reactive protein (CRP) [16
], and asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor [16
]. Heightened plasma ADMA levels are observed in PAH patients, and negatively correlate with hemodynamic performance and survival rates [35
]. (iii) Pilot studies have demonstrated that autologous transplantation of EPCs is safe and leads to significant improvements in pulmonary hemodynamics, exercise capacity including 6-minute-walk-distance in children [36
] and adults with PAH [37
]. (iv.) EPC number indicates response to therapy: Treatment with the phosphodiesterase type 5 (PDE5)-inhibitor sildenafil, an established PAH medication, leads to a dose-dependent rise in EPC numbers in IPAH patients [16
]. In separate studies, peroxisome proliferator-activated receptor-γ (PPARγ) treatment inhibit the negative effects of CRP on human EPC survival, differentiation, and function [30
], and increase EPC number in culture [38
]. Recently, we demonstrated that PPARγ agonists reverse PAH, right ventricular hypertrophy and pulmonary vascular remodeling in rodents [39
] thereby revealing their potential as a new pharmacotherapy [40
]. We [43
] and others [44
] have since shown that metabolic dysregulation such as insulin resistance (IR) [43
] and dyslipidemia (low HDL-cholesterol [43
]) is more common in (female) PAH patients and associated with clinical worsening and poorer survival at six [43
] and twenty [44
] months follow up. Given our current results on the lower number of circulating EPCs in obese vs.
non-obese, and post- vs.
pre-menopausal PAH patients, and the previously described inverse relation between EPC number and hemodynamic status of PAH patients [16
], it will be important to explore the impact of metabolic regulators such as PPARs [42
], mitochochondrial regulatory proteins [33
], micro RNAs [45
], and sex hormones [34
] on pulmonary vascular disease and associated right ventricular dysfunction.
There are several limitations to our study. Although most of the published competitive techniques solely use cell surface markers [13
], it should be noted that the definition of a stem/progenitor cell optimally should be based on both surface markers and
functional assays. Specifically, EPC characteristics including cell surface proteins have been shown to differ depending on the culture techniques and stage of differentiation in which the cells are isolated (CFU-Hill, early-EPCs, or late-EPCs; see Supplementary Results and Discussion
for details). Among the existing EPC assays only the laborious colony forming unit (CFU) assays (processing time 5 days) give information on EPC function (see ). Hence, as an EPC characterization tool, the described device is somewhat limited because the captured cell population is defined solely based on surface markers. However, as a practical diagnostic device, characterization of EPC function
is secondary to quantifying a novel, reliable and validated cellular PAH biomarker such as EPC number that is inversely associated with the hemodynamic status of PAH patients and increased by the phospodiesterase inhibitor sildenafil [48
]. This study was not designed to comprehensively investigate the role of EPC and EPC function in PAH; rather this report merely presents a novel EPC enumeration modality as a potential “bedside test” and facile alternative to the conventional laborious techniques.
Currently conflicting data exist in the literature on the relative EPC number in PAH patients [11
]. A number of studies have described a reduction in circulating EPCs when compared with healthy controls [16
], consistent with our data presented herein. However, others have found an elevation [19
], or no difference [49
] in EPC numbers in PAH patients versus healthy controls. A possible explanation for this discord in the literature may be attributed to the various methods and cell surface protein markers used to identify, isolate and quantify the cells, as well as possible differences in patient selection. Secondly, it is possible that “early” (immature) EPCs (CD133+) may be released from the bone marrow as an early adaptive response to pulmonary vascular injury [48
]. We speculate that environmental factors such as inflammation [50
], sex hormone dysbalance [34
], insulin resistant state [31
], and/or dyslipidemia [31
] subsequently inhibit the differentiation of the circulating EPCs from “early” to the “late” (mature) EPC (CD31+) phenotype. Such a biphasic EPC response may account for the different findings in patients with IPAH published to date [16
]. Nevertheless, the beneficial role of EPCs in PAH is supported by recent reports on the successful autologous transplantation of CD34+/KDR+ cells that lead to hemodynamic and clinical improvement in children [36
] and adults [37
] with IPAH.
In summary, the new EPC capture chip captures a significant percentage of EPCs in a single step, requiring minimal blood volume (200 μL) which is important for pediatric clinical care and small animal research. To the best of our knowledge, this is the first report on the application of a polymeric cell-affinity microfluidic diagnostic platform in cardiovascular disease and the first on the validation and clinical application of such a microfluidic device for the analysis of human circulating EPCs. In addition to patients with idiopathic PAH, those with PAH associated with either connective tissue disease or drug use (appetite suppressants) have about half the number of circulating EPCs relative to healthy, age and gender matched controls. Beyond the technical aspects, we found that the clinically relevant number of circulating EPCs (CD34+/KDR+, CD34+/KDR+/CD31+/CD45−) is inversely related to BMI and postmenopausal status in PAH patients. The novel EPC capture chip has the potential to become a practical, diagnostic tool in the risk assessment and clinical monitoring of patients with PAH and other cardiopulmonary and neurodegenerative diseases [51
] as well as cancer [52
], requiring only small blood volumes and no laborious pre-processing steps.