Pulmonary arterial hypertension (PAH) is a disease of the small pulmonary arteries characterized by a progressive increase in pulmonary vascular resistance leading to right ventricular failure and ultimately death. A diagnosis of idiopathic PAH (IPAH) is made when no known risk factor is documented (1). Impairment of endothelial function is considered to be an important factor in the pathogenesis of IPAH. Endothelial dysfunction affects the production of vasoconstrictors (i.e., increases production of endothelin I and thromboxane) and vasodilators (decreases nitric oxide [NO] and prostacyclin), leading to pulmonary vasoconstriction (2). In vitro studies have shown that the expression and release of vasodilators correlates inversely with increased vascular resistance in PAH, reflecting the clinical relevance of endothelial dysfunction in these patients (3). An in vivo method for direct invasive assessment of pulmonary endothelial function has been described previously using the vasodilator response to acetylcholine (Ach) (4- 6), a model that has been used frequently in adults with coronary artery disease to predict long-term cardiovascular events (7). Ach induces endothelium-dependent relaxation by receptor-mediated stimulation of endogenous NO; therefore, an impaired response reflects endothelial dysfunction (8). However, endothelium-dependent pulmonary artery relaxation by Ach has not been investigated systematically in children and adolescents with IPAH. We hypothesized that a relationship existed among pulmonary endothelial response to Ach, severity of the disease, and clinical outcome in these patients.

The study protocol was approved by the institutional committee on ethical practice. Forty-three children and adolescents (24 girls [56%], mean age: 10.4 ± 5.5 years) with IPAH undergoing cardiac catheterization to assess pulmonary hypertension, defined as mean pulmonary arterial pressure (mPAP) of more than 25 mm Hg, before PAH specific treatment was initiated, were included in the study after informed written consent was obtained. Most of these patients belonged to World Health Organization (WHO) functional class II (n = 23) or class III (n = 16). Only 3 patients were in WHO functional class I, and 2 patients in WHO functional class IV.

Diagnostic cardiac catheterization was performed under local anesthesia through a percutaneous femoral approach and was age dependent, with the patients under conscious sedation using low dosages of midazolam, benzodiazepine and ketamine, or propofol sedation. Arterial blood gas measurements were obtained at the beginning, during, and at the end of each study to exclude carbon dioxide retention and to document respiratory and metabolic stability throughout the study. Measurements were performed only when the partial pressure of carbon dioxide level was between 32 and 45 mm Hg and pH was between 7.35 and 7.45. Measurements of baseline hemodynamics included mixed venous and arterial saturations, blood gases, systemic and pulmonary artery pressures, left atrial (or pulmonary capillary wedge) pressure, and right atrial pressure in the standard manner with fluid-filled catheters.

The assessment of pulmonary vascular reactivity was carried out as follows. Measurements were obtained at baseline (as the usual fraction of inspired oxygen), then the effect of inhaled NO at 40 ppm for 10 minutes was recorded; after that, the additional effect of oxygen with fraction of inspired oxygen at approximately 0.8 for 10 min was assessed. The NO plus additional oxygen then was discontinued, and new baseline hemodynamics were measured after 10 min. Subsequently, aerosolized iloprost (0.3 μg/kg) was administered by a specific applicator. In awake children with a chance for active inhalation, iloprost was administered in a total dose of 5 μg. Measurements were repeated after 10 min with a combination of iloprost plus NO plus oxygen.

Before vasoreactivity testing was performed with the above-mentioned protocol, pulmonary endothelial function was assessed by vasodilator response to Ach. Dependent on the patient size, a 4-F or 5-F multipurpose catheter was inserted into the left or right lower lobe pulmonary artery. A 0.014-inch pulsed Doppler wire (Cardiometrics, Inc., Mountain View, California) was positioned through the multipurpose catheter into a straight segment of the medial or posterior branch of the lower lobe pulmonary artery with a diameter of 3 to 5 mm. The position of the Doppler wire in the center of this vessel was confirmed by a hand injection of contrast through a Thouy-Borst adapter (sighting angiography) (Figure 1) and a stable flow-velocity signal with minimal noise (4). Then, serial infusions were made via the multipurpose catheter into the segmental pulmonary artery using an infusion pump with Ach concentrations of 10⁻⁶ M and 10⁻⁵ M, respectively. Changes in pulmonary blood flow in response to Ach were assessed using intravascular Doppler blood flow velocity measurements. Pulmonary flow reserve (PFR) was calculated as the ratio of pulmonary blood flow velocity in response to Ach relative to baseline values.

Data are presented as mean ± SD. Correlations were obtained by Pearson's correlation test. To compare PFR values of patients with different clinical status, we used the unpaired nonparametric Mann-Whitney U test. A receiver-operating characteristic (ROC) curve was used to identify the best threshold value (maximum sensitivity and specificity) for prediction of serious cardiovascular events, defined as lung transplantation or death. Freedom from serious cardiovascular events after catheterization was illustrated by Kaplan-Meier curves, and outcomes were compared using the log-rank test. Statistical analysis was performed using IBM (IBM Corp., Armonk, New York) SPSS Statistics software version 19.0 (SPSS, Inc., Chicago, Illinois). The null hypothesis was rejected when p < 0.05.

Among the 43 patients, the mean mPAP-to-mSAP ratio was 0.89 ± 0.28, reflecting the severity of PAH. Twenty-one (49%) of the 43 patients showed an acute response to global vasoreactivity testing defined as a 20% reduction in the mPAP-to-mSAP ratio. The mean reduction of the mPAP-to-mSAP ratio was 30 ± 18.2%. Mean cardiac index increased from 3.0 ± 0.9 l/min/m² to 3.8 ± 1.6 l/min/m².

Baseline mPAP was 67.1 ± 19.1 mm Hg, and minimum mPAP during vasoreactivity testing was 50.9 ± 23.9 mm Hg. The mean change in mPAP during vasoreactivity testing was 26.9 ± 19.3%. Twenty-five of the 43 patients showed a more than 20% reduction of the mPAP, 18 of them to a mPAP of <40 mm Hg. Basal mean indexed pulmonary vascular resistance was 23.5 ± 12.6 Wood units/m² body surface area, and minimum mean indexed pulmonary vascular resistance during vasoreactivity testing was 16.2 ± 11.2 Wood units/m² body surface area.

The mean baseline flow velocity in the segmental pulmonary artery of all patients was 18.1 ± 9.7 cm/s. During Ach infusion, the mean flow velocity increased to 30.6 ± 25.5 cm/s (p < 0.0001). The calculated mean PFR was 1.58 ± 0.67.

PFR correlated well with maximum percentage decrease in the ratio of mPAP to mSAP during vasoreactivity testing (r = 0.61, p < 0.0001) (Figure 2A). There was an inverse correlation between PFR and minimum mean PA pressure during vasoreactivity testing (r = −0.45, p = 0.003) (Figure 2B) and between PFR and minimum mean indexed pulmonary vascular resistance (r = −0.42, p = 0.006) (Figure 2C); however, no significant correlation between basal pulmonary vascular resistance and PFR was observed (r = −0.3, p > 0.05).

Mean follow-up after catheterization was 55.7 ± 41.9 months. Freedom from serious cardiovascular events (defined as lung transplantation or death) after catheterization was 83% after 2 years, 76% after 3 years, and 57% after 5 years (Figure 3).

To investigate the predictive value of PFR, we performed ROC curve analysis, which discriminated a PFR of 1.4 as the best cutoff value (area under the ROC curve: 0.753, 95% confidence interval: 0.602 to 0.905, sensitivity: 0.67, specificity: 0.875, p = 0.006). The PFR was less than 1.4 in 23 of 43 patients. The ROC curve analysis also was performed for reduction of mPAP-to-mSAP ratio and revealed a reduction of mPAP-to-mSAP ratio of 30% as the best cutoff value (area under the ROC curve: 0.753, 95% confidence interval: 0.603 to 0.904, sensitivity: 0.63, specificity: 0.81, p = 0.006) (Figure 4).

PFR correlated inversely with the patient age (r = −0.33, p = 0.01), demonstrating that younger patients are more likely to have a higher PFR. In addition, PFR correlated with WHO functional class. Patients with a better clinical status showed a higher PFR. A PFR of <1.4 was represented more frequently in WHO classes III and IV. PFR was able to differentiate between patients in WHO class II and class III (p = 0.04), who reflected most of our patients (Figure 5).

Calcium-channel blocker therapy (CCB) was started after the initial catheterization in all 21 responders to the vasoreactivity testing, in accordance with the current IPAH guidelines. After 1 year of CCB treatment, in 9 of the 21 responders, an add-on therapy was necessary or the treatment strategy had to be modified because of clinical worsening. The remaining 12 patients showed a favorable long-term response to CCB therapy. The mean PFR within the group of good responders to CCB therapy was not significantly higher compared with that of patients who needed therapy modification (p = 0.07) (Figure 6); however, only 3 of 12 patients in the CCB responder group had a PFR of <1.4, compared with 6 of 9 in the therapy failure group.

Within the subset with long-term response to CCB, all 9 patients with a PFR of more than 1.4 had a decreased PA pressure of <40 mm Hg. There was an inverse correlation between PFR and minimum mean PA pressure during vasoreactivity testing (r = −0.45, p = 0.003).

To evaluate the prognostic value, we compared the following parameters using Kaplan-Meier analysis: reduction in mPAP-to-mSAP ratio of more than 20%, reduction in mPAP-to-mSAP ratio of more than 30%, decrease in mPAP of <40 mm Hg, and PFR of more than 1.4 (Figure 7). All of these parameters have an impact on prognosis of patients with IPAH; however, PFR of more than 1.4 was most predictive for long-term cardiovascular events (log-rank [Mantel Cox] chi-square: 12.49, p < 0.0001).

In this study, we demonstrated that the pulmonary endothelial function test using the vasodilator response to Ach administered in a segmental pulmonary artery had considerable prognostic value in children and adolescents with IPAH. An impaired Ach response, defined as PFR of less than 1.4 (an increase of flow velocity of less than 40%), was a strong predictor for poor prognosis in this patient group.

The diagnostic work-up of patients with IPAH is extensive and challenging (9). Cardiac catheterization is still required for confirming the diagnosis and for guiding management of the disease (10- 12). Acute testing of vasoreactivity during catheterization is an important factor in the evaluation of PAH (13- 14). However, although the presence of an acute response has important clinical consequences, its definition remains controversial. In international guidelines, the criteria of Sitbon et al. (15) currently are recommended for adult PAH patients. These criteria include a decrease in mPAP of 10 mm Hg or more, reaching an mPAP value of 40 mm Hg or less with an increased or unchanged cardiac output. In children, the criteria of Barst et al. (16) generally are used, which include a decrease in mPAP or in mPAP-to-mSAP ratio of 20% or more, with an increased or unchanged cardiac output and unchanged or decreased pulmonary-to-systemic vascular resistance ratio. Recently, it has been shown that the impact of an acute response as a prognostic tool in PAH patients seems to be limited (17). One reason may be that because of the use of different response criteria, study data are difficult to compare. In our study, we compared the Barst et al. (16) criteria and the Sitbon et al. (15) criteria with vasodilator response to Ach. In addition, we used a ROC analysis to demonstrate that a stricter definition of the Barst et al. (16) criteria with a decrease in mPAP-to-mSAP ratio of 30% or more may be more reliable to predict outcome in IPAH patients. According to our data, vasodilator response to Ach is a strong predictor of prognosis in patients with IPAH. Because the usual vasoreagibility test reflects the more global response of the entire pulmonary vessel system, making it more sensitive to other potential influencing factors, the response to Ach represents the local segmental vessel endothelial response. Therefore, our results confirmed the clinical relevance of endothelial dysfunction in these patients (18).

Vasodilator response to Ach in segmental pulmonary arteries has been reported previously in children with congenital heart disease. In 1993, Celermajer et al. (4) demonstrated that the maximal increase of flow velocity in pulmonary arteries in response to Ach in children with normal pulmonary hemodynamics is usually approximately 90% of the baseline value. Patients with pulmonary vascular disease showed an impaired response with a reduced increase of flow velocity of <50%. However, in this previous proof-of-principle study, only 2 patients with IPAH were included. Our recent data confirmed, in a relatively large cohort of pediatric patients with IPAH, this impaired response to Ach. In addition, we were able to show that this parameter has a high impact on diagnosis and prognosis in these patients. Patients lacking a response to Ach seem to have a poor prognosis and a rapid progress of the disease, despite currently available therapeutic options. At this disease stage, pre-capillary vessels may be engaged by matrix and adventitial proliferation in which the endothelium may react only by a residual vasoconstriction, but without any chance for vasodilation (19).

In our study, we assessed only the vasodilating effect of Ach. It is possible that other locally administered vasodilators (i.e., nitroprusside) also would be able to predict outcome. However, Celermajer et al. (4) were able to show that vasodilation was greater in response to Ach than to nitroprusside. This may be because for nitroprusside, the dose range is limited by its powerful systemic vasodilator effect. When we measured the PFR during inhalation of NO, no significant changes of flow velocities were observed.

One might expect an inverse relation between vessel diameter and change in velocity, and the same change in absolute flow could lead to a greater change in velocity, and therefore PFR, in a smaller vessel. With respect to this physiological phenomenon, we considered using in our protocol segment arteries with a diameter of only 3 to 5 mm. Within this range of diameter, no relation between vessel size, basal flow velocity (r = −0.12, p = 0.64), and change in velocity related to absolute flow (r = 0.38, p = 0.13) was observed. To minimize this potential limitation on the results, only relative changes, rather than absolute values, were used in our calculations of flow velocity, which makes each child its own control. Changes in relative, rather than absolute, flow velocity also have been used by investigators of coronary circulation (7).

Calculation of vascular resistance was based on assumed, rather than measured, oxygen consumption. The potential error thereby introduced can be minimized by using the Rp-to-Rs ratio (pulmonary-to-systemic vascular resistance ratio). However, more important for the interpretation of the vasoreactivity test in IPAH patients is the mPAP or the mPAP-to-mSAP ratio, which is the reason why these parameters are the most important criteria for vasoreactivity testing in the current PAH guidelines.

In addition to the prognostic value, vasodilator response to Ach has an impact on guiding PAH-specific therapy. According to our data, nonresponders to the global vasoreactivity testing method can have a positive vasodilator response to Ach, which implies preserved endothelial function and a more favorable outcome in this group of patients. These patients also may show a favorable long-term benefit to therapy with phosphodiesterase type 5 inhibitors by mobilizing their preserved endogenous NO system. This may explain why in some nonresponders, this drug may be more effective than in other patients with comparable negative global acute vasoreactivity test results. In addition, dual endothelin-receptor antagonists in patients with preserved vasodilator response to Ach should be used with care, because it has to be kept in mind that the potentially endogenous vasodilating effect of endothelial endothelin-B receptors also will be blocked (20).

Our data demonstrate that endothelium-dependent pulmonary artery relaxation obviously is age and disease dependent. Younger patients are more likely to have a positive vasodilator response to Ach than older children. An age-dependent progress of IPAH has been postulated previously, especially because the frequency of responders to vasoreactivity testing is reported to be significantly higher in children than in adults (15- 16,21). Therefore, it has been speculated that aging may be responsible for a progressive loss of vascular response to NO and Ach. Continuous shear stress may induce progress of the disease from the primary affected endothelium to a destruction of the complete vessel.

In patients with IPAH showing an acute response during vasoreactivity testing (responders), treatment with CCB is recommended, according to the current international guidelines (13). There is evidence from previous studies in adult IPAH patients that in some of the responders, the effect of CCB therapy does not persist (15). Published data concerning this issue in children and adolescents do not exist to date, and there was no effective tool to evaluate favorable long-term response to CCB therapy in patients with IPAH who responded to vasoreactivity testing. According to our results, PFR in response to Ach obviously was higher in patients with a favorable long-term response to CCB therapy, information that may help in guiding PAH-specific therapy. Interestingly, we found that within the subset of patients who demonstrated a long-term response to CCB, all 9 patients with a PFR of more than 1.4 had a decreased PA pressure of <40 mm Hg, a parameter that was suggested by Sitbon et al. (15) to predict favorable long-term response in adults. We also demonstrated an inverse correlation between PFR and minimum mean PA pressure during vasoreactivity testing, which suggests that decreasing mean PA pressure to <40 mm Hg also may be a reliable predictor of long-term response to CCB in children with IPAH and may even be a surrogate for preserved endothelial function.

We conclude that assessment of endothelium-dependent pulmonary artery relaxation as an adjunct to the usual testing protocol provides additional information and has diagnostic and prognostic implications in patients with IPAH, and also may guide the decision making for initiation of a specific therapy. Our study demonstrates an impaired vasodilator response to Ach in segmental pulmonary arteries in most children and adolescents with IPAH, reflecting pulmonary endothelial dysfunction. An impaired Ach response was a strong predictor for poor prognosis.