Increased Antielastase Activity in Idiopathic Pulmonary Arterial Hypertension and Chronic Thromboembolic Pulmonary Hypertension

To the Editor:

Pulmonary arterial hypertension (PAH) is characterized by abnormal remodeling and occlusion of precapillary arterioles in the lung with a subsequent increase in pulmonary vascular resistance. This can lead to right ventricular hypertrophy and ultimately right heart failure. Elastase is implicated in the pathobiology of PAH, with evidence including ultrastructural studies showing increased elastase activity in pulmonary arteries from children with congenital heart disease–associated PAH (1), increased elastase release from peripheral blood neutrophils isolated from patients with pulmonary hypertension compared with healthy control subjects (2), and elevated plasma concentrations of elastase in patients with idiopathic PAH (IPAH) (3).

The major elastase inhibitor in the circulation is alpha-1 antitrypsin (AAT). Produced by the liver, AAT is one of the most abundant serine protease inhibitors in the blood, circulating in micromolar concentrations, and accounts for over 90% of neutrophil elastase inhibition in plasma (4–6). AAT protects the lungs against proteolytic damage from elastase, and its deficiency can cause unopposed proteolytic parenchymal damage and emphysema (6).

Increased pulmonary artery elastolytic activity in rat models of PAH can be reversed with elastase inhibitors (7). This suggests that the elastase/AAT axis may be dysregulated in PAH. Although elastase has been measured in plasma from patients with IPAH (3), the levels of endogenous circulating elastase inhibitor AAT remain elusive. One study reported reduced levels of AAT in pooled sera from 20 patients with IPAH based on two-dimensional (2D) gel electrophoresis coupled with mass spectrometry (8). However, 2D gel electrophoresis separates proteins by isoelectric point and size. Plasma AAT is known to exhibit microheterogeneity (9) and is present in multiple isoforms with different isoelectric points due to differential glycosylation (9). Hence, when pooled sera are examined by 2D gel electrophoresis, AAT is represented by multiple spots and the intensity change in one spot is unlikely to reflect the changes in the total amount of AAT.

We measured AAT concentrations in plasma from 29 patients with IPAH, 29 healthy control subjects, and 21 patients with chronic thromboembolic pulmonary hypertension (CTEPH) as a disease comparator (for details regarding the materials and methods used, see the data supplement). The baseline characteristics for the groups are summarized in Table 1. Age and sex did not vary when all three groups were considered (P = 0.081 and P = 0.392). However, there were individual group differences, with patients with IPAH being younger (median ± interquartile range: 47 ± 24 yr) than patients with CTEPH and healthy control subjects, and predominantly female (76%). In contrast to what was reported previously (8), AAT concentrations in plasma assessed by ELISA did not vary significantly between patients with IPAH (mean ± SEM: 1.91 ± 0.04 g/L) and healthy control subjects (1.79 ± 0.05 g/L; P = 0.052; Figure 1A). There was also no difference between healthy control subjects and patients with CTEPH (1.81 ± 0.09 g/L; P = 0.775; Figure 1A). We found slightly higher elastase-inhibitory activities in plasma from patients with IPAH and patients with CTEPH (Figure 1B). The ELISA did not show a similar increase in AAT levels, possibly because elastase inhibitors other than AAT were present, or the heterogeneity of the AAT glycosylation resulted in some isoforms being more reactive with the antibodies used in the ELISA measurement.

Table 1.

Baseline Characteristics for the Idiopathic Pulmonary Arterial Hypertension, Chronic Thromboembolic Pulmonary Hypertension, and Healthy Control Groups

		Healthy Control	IPAH	CTEPH	P Value
Subjects		29	29	21
Sex, female		16 (55)	22 (76)	6 (33)	0.081
Age, yr		58 ± 24.2	47 ± 24	64 ± 23	0.392
WHO functional class, 1/2/3/4 (%)			4/29/63/4	0/29/71/0	1.000
6mwd, m			412 ± 174	322 ± 125	0.447
Pulmonary hemodynamics
mPAP, mm Hg			54 ± 18	43 ± 9	0.007
CI, L/min/m2			2 ± 0.7	1.8 ± 0.4	0.632
PVR, dyn⋅s/cm−5			875 ± 474	696 ± 334	0.081
Pulmonary function tests
FEV1, %			90 ± 22	93 ± 32	0.872
FVC, %			102 ± 23	106 ± 24	0.817
TlCO, %			67 ± 26	65 ± 13	0.674
Kco, %			81 ± 16	75 ± 15	0.817
Clinical blood tests
WCC, ×109/L			7.1 ± 3.5	8.1 ± 1.7	0.232
Lymphocyte, %			23 ± 15	22 ± 6	0.632
Neutrophil, %			66 ± 15	69 ± 6	0.651
CRP, mg/L			4 ± 7	5 ± 12	0.651
Smoker*			10 (34)	5 (24)	0.743
Emphysema on CT			1 (4)	0 (0)	1.000
Pulmonary vasodilating medication†			17 (59)	9 (43)	0.651

Definition of abbreviations: 6mwd = 6-min walk distance; CI = cardiac index; CRP = C-reactive protein; CT = computed tomography; CTEPH = chronic thromboembolic pulmonary hypertension; FEV₁ = forced expiratory volume in 1 second; FVC = forced vital capacity; IPAH = idiopathic pulmonary arterial hypertension; Kco = gas transfer coefficient; mPAP = mean pulmonary arterial pressure; PVR = pulmonary vascular resistance; Tl_CO = transfer factor for carbon monoxide; WCC = white cell count; WHO = World Health Organization.

Data are presented as n, n (%), or median ± interquartile range unless otherwise stated.

^*Current or ex-smoker.

^†Medication at the time of sampling.

Figure 1.

Characterization of plasma alpha-1 antitrypsin (AAT) from patients with idiopathic pulmonary arterial hypertension (IPAH) and healthy control subjects. (A) Measurement of plasma AAT concentrations in healthy control subjects (n = 29), patients with IPAH (n = 29), and patients with chronic thromboembolic pulmonary hypertension (CTEPH; n = 21). The data are presented as mean ± SEM, and an unpaired t test was used. (B) Measurement of elastase-inhibitory activity in plasma samples from healthy control subjects and patient groups as in A. Means ± SEM are shown, unpaired t test. (C) Mechanism of elastase inhibition by AAT, and structures of native and cleaved AAT. Cleaved AAT can be formed either before the formation of the final complex (arrow 1) or from the breaking down of the final complex (arrow 2). Note that cleaved AAT contains two polypeptide chains (green and blue). (The diagram is based on Gettins and Olson [10], and structures with the following PDB codes were used to generate the figure: 3ne4 for native AAT, 1ezx for cleaved AAT and the final complex, and 1oph for protease and the Michaelis complex). (D) AAT from patients with IPAH and healthy control subjects is primarily in the native form. Equal volumes of albumin/IgG-depleted plasma samples were preincubated with 1 μg of human neutrophil elastase at 37°C overnight before SDS-PAGE and immunoblotting, with recombinant native AAT used as a positive control. C1–C6 are six different healthy control subjects, and P1–P6 are six different patients with IPAH. (E and F) Plasma samples from both patients with IPAH and patients with CTEPH have more cleaved AAT. (E) Equal volumes of albumin/IgG-depleted plasma samples were fractionated on 12% SDS-PAGE and immunoblotted with anti-AAT antibody. Recombinant AAT, with or without preincubation with recombinant elastase, was used as a control. Two blots were run and processed in parallel. (F) Densitometry analysis of E, shown as the ratio of cleaved to native AAT, calculated by the sum of the two cleaved bands (C1 and C2) divided by the native band (N). This ratio reveals changes in the AAT cleavage but is not affected by the small difference in the amount of sample loaded. Conc = concentration; MW = molecular weight.

We next examined plasma AAT using SDS-PAGE and immunoblotting. AAT is a member of the serine protease inhibitor (SERPIN) family. Its native form has a long reactive center loop (RCL) acting as the bait; hence, SERPINs are suicidal protease inhibitors (10). Upon encountering a target protease, the RCL is recognized and bound by the protease, allowing the formation of a Michaelis complex (Figure 1C). A covalent bond is then formed between the RCL and the protease active-site residue, followed by cleavage of the RCL and insertion of the RCL into β-sheet A, which brings the covalently linked protease to the opposing end of the SERPIN molecule (10). During this process, cleaved AAT can be generated either by protease cleavage before the final complex formation (Figure 1C, arrow 1) or by breakdown of the final complex (Figure 1C, arrow 2) (10), both of which occur as a result of protease activity. The resulting cleaved AAT contains two peptide fragments (Figure 1C, cleaved AAT, green and blue), which can be separated by SDS-PAGE. We used an antihuman AAT antibody that can detect both native AAT and the larger fragment of the cleaved AAT, which appeared as a distinct lower band owing to its lower molecular weight. As shown in Figure 1D, AAT in plasma from both patients with IPAH and healthy control subjects is predominantly in the native form (upper band), which can be converted into the cleaved form (lower band) upon incubation with elastase, in a manner identical to that used for recombinant AAT. When equal volumes of plasma from healthy control subjects, patients with IPAH, and patients with CTEPH were run simultaneously on SDS-PAGE and with longer exposure, more cleaved AAT was detected in plasma from both patients with IPAH and patients with CTEPH (Figure 1E and F), suggesting more protease activity in the plasma from these patients. Interestingly, the two major cleaved bands C1 and C2 (Figure 1E) are smaller than the major elastase cleavage product C3, suggesting there are proteases in plasma other than elastase that could cleave AAT. It is well known that activated neutrophils release two other serine proteases, proteinase 3 and cathepsin G, both of which can be inhibited by AAT and produce cleaved AAT.

The significance of the findings in this report is twofold. First, we demonstrated that AAT levels were not significantly changed in patients with IPAH compared with healthy control subjects, refuting the only published report on this. Additionally, we found an increase in cleaved AAT and slightly increased antielastase activity in plasma from patients with IPAH as well as those with CTEPH, suggesting that such changes in the elastase/AAT axis are not specific to IPAH, which is in agreement with a previous observation of increased elastase levels in both IPAH and CTEPH groups (3). It is worth noting that the previously reported fold increase in elastase levels (3) is much greater than the fold increase in antielastase activity measured here. Therefore, our data still support the exploration of elastase inhibitors as a potential therapy for PAH.