Sage Journals: Discover world-class research

Abstract

Pulmonary arterial hypertension (PAH) is a major complication of sickle cell disease (SCD). Low levels of apolipoprotein A1 (Apo-A1) have been implicated in the development of PAH in SCD. We speculate that lower levels of Apo-A1 are related to dysregulation of the ubiquitin-proteasome pathway (UPP). Of 36 recruited patients with SCD, 14 were found to have PAH on the basis of right heart catheterization. Levels of Apo-A1 and Apo-B, polyubiquitin, total protease, and specific and normalized activity of chymotrypsin-like, trypsin-like, and caspase-like proteases in plasma were measured. Levels of Apo-A1 were found to be lower and polyubiquitin levels were found to be significantly higher in the PAH group (P < 0.05) in SCD. Apo-A levels were inversely correlated with polyubiquitin levels (r = −0.499, P = 0.009). These results indicate that lower levels of Apo-A1 in SCD patients with PAH are likely related to enhance degradation by UPP, potentially contributing to pulmonary vascular pathology. These findings may provide significant insight in identifying suitable therapeutic targets in these patients.

Keywords

pulmonary arterial hypertension (PAH)sickle cell disease (SCD)ubiquitin-proteasome pathway (UPP)apolipoprotein A (Apo-A)

INTRODUCTION

Pulmonary arterial hypertension (PAH) is a major complication of sickle cell disease (SCD) and a major risk factor for death in this population.^1,2 The prevalence of PAH in adults with SCD varies from 6% to 10%, on the basis of right heart catheterization (RHC).^3–5 Almost 75% of adult patients with SCD have histological evidence of PAH at the time of death.⁶

The specific mechanism by which PAH develops in SCD is not clear, but it is thought to be multifactorial. Serum apolipoprotein (Apo) levels are known to be lower in patients with SCD than in the general population.⁷ The most significant of these is Apo-A1, a major protein component of high-density lipoprotein particles (HDL-C) in plasma. A recent study indicated that SCD patients with low Apo-A1 levels have a higher prevalence of PAH.⁸ In addition, SCD patients with lower-than-median Apo-A1 levels showed deficient vasodilatory responses to the endothelial agonist acetylcholine. This suggests that SCD patients with low Apo-A1 levels have endothelial dysfunction, which is common to the systemic and pulmonary circulations and is a precursor to vascular diseases.^6,9

The exact cause of low levels of Apo-A1 in SCD patients is unknown. The ubiquitin-proteasome pathway (UPP) is known to play a central role in intracellular protein degradation.¹⁰ The pathway employs an enzymatic cascade by which multiple ubiquitin molecules are covalently attached to the protein substrate. The polyubiquitin modification marks the protein for destruction and directs it to the proteasome complex for degradation.¹¹ The 26S proteasome consists of a catalytic 20S proteasome core and two 19S (cap) regulatory complexes. The 20S proteasome is a 660–700-kDa multicatalytic proteinase complex consisting of seven α subunits and seven β subunits.^12,13 The caspase-like activity of the β1 subunit, the trypsin-like activity of the β2 subunit, and the chymotrypsin-like activity of the β5 subunit allow the proteasome to cleave different proteins into oligopeptides.

In the current study, we hypothesize that low levels of Apo-A1 in SCD patients are related to abnormalities of UPP and may play a role in the development of PAH in this population.

METHODS

Study population

Patients with known SCD were recruited from our hospital's SCD clinic. Patients with known causes of secondary pulmonary hypertension, chronic renal insufficiency, pregnancy, smoking or substance abuse, active sickle crisis, or acute chest syndrome at the time of their echo were excluded (Table 1). This study was approved by the Downstate–Kings County Institutional Review Board, and written informed consent was obtained from all individuals.

Table 1.

Demographics and biochemistry in patients with sickle cell disease and pulmonary arterial hypertension (PAH) versus no PAH

	PAH (n = 14)	No PAH (n = 22)
Age, years	39.47 ± 14.62	37.97 ± 13.27
Sex, M:F	6:8	9:13
Height, cm	170.7 ± 7.98	177.6 ± 51.92
Weight, kg	68.19 ± 14.20	64.93 ± 13.26
Body mass index	21.34 ± 3.51	20.79 ± 4.17
Ethnicity	African American	African American
Genotype, no.
SS	14	22
SC	0	0
Sickle cell trait	0	0
Sickle β⁺ thalassemia	0	0
Total hemoglobin, g/dL	7.99 ± 1.47	8.83 ± 1.62
Fetal hemoglobin, g/dL	6.49 ± 5.26	5.91 ± 4.03
Reticulocyte count, %	24.78 ± 69.45	11.6 ± 5.53
Platelets, k/L	425.7 ± 203.8	410.5 ± 189.4
Mean platelet volume, fL	9.01 ± 0.91	9.23 ± 0.95
Blood urea, mg/dL	12.73 ± 7.23	5.94 ± 1.05
Serum creatinine, mg/dL	0.77 ± 0.29	0.65 ± 0.21
C-reactive protein, mg/L	35.12 ± 50.15	27.09 ± 36.22
Lactate dehydrogenase, IU/L	548.0 ± 262.1	436.8 ± 179.6
Iron, μg/dL	90.53 ± 38.88	85.71 ± 43.22
Ferritin, ng/mL	778.6 ± 1,398.7	1,151.1 ± 2,140.9
Total bilirubin, mg/dL	2.96 ± 2.07	2.95 ± 1.86
Direct bilirubin, mg/dL	1.04 ± 1.43	0.61 ± 0.29
Ejection fraction, %	57.24 ± 4.54	57.90 ± 4.23
Tricuspid regurgitation velocity, m/s	3.49 ± 0.35^*	2.10 ± 0.23
Right heart catheterization
mPAP, mmHg	56 ± 7
PVR, dyn s/cm⁵	875 ± 203
Cardiac output, L/min	3.5 ± 0.7
6-minute walk distance, m	283 ± 41^*	577 ± 68
Apo-A1, mg/dL	93.8 ± 35^*	146.8 ± 69
Apo-B, mg/dL	163.9 ± 41	154.2 ± 39
Polyubiquitin, ng/mL plasma	208 ± 39^*	176 ± 45
Proteasome concentration, ng/mL plasma	5,482 ± 2,295	4,941 ± 1,535
Specific activity
Caspase, pmol AMC/s/mL plasma	2.41 ± 1.17	2.49 ± 1.24
Trypsin, pmol AMC/s/mL plasma	1.96 ± 1.16	2.15 ± 1.36
Chymotrypsin, pmol AMC/s/mL plasma	2.14 ± 1.15	2.35 ± 1.10
Normalized activity
Trypsin-like/pg proteasome	0.40 ± 0.35	0.50 ± 0.34
Caspase-like/pg proteasome	0.51 ± 0.23	0.56 ± 0.34
Chymotrypsin-like/pg proteasome	0.53 ± 0.36	0.53 ± 0.36

Note: Data are mean ± standard deviation, unless otherwise indicated. mPAP: mean pulmonary arterial pressure; PVR: pulmonary vascular resistance; Apo: apolipoprotein; AMC: 7-amino-4-methylcoumarin.

P < 0.05 compared with no PAH.

Evaluation for PAH

Two-dimensional and Doppler echocardiographic studies (iE33 and 5500; Philips, Andover, MA) were performed according to standard American Society of Echocardiography protocol. We defined tricuspid regurgitation velocity (TRV) of ≥2.5 m/s as a marker of PAH in SCD. The presence of PAH was confirmed in patients with TRV of ≥2.5 m/s via RHC, defined as mean pulmonary arterial pressure of ≥25 mmHg. Cardiac output was obtained using triplicate measurements with the thermodilution method (Agilent, Böblingen, Germany). Fourteen patients were found to have PAH, and 22 patients were found not to have PAH.

Measurement of apolipoproteins and UPP

Serum samples from 14 patients with SCD-related PAH and 22 patients without PAH were analyzed for levels of UPP markers (proteasome, polyubiquitin, and proteasome enzymatic activities [pmol 7-amino-4-methylcoumarin/s/mL plasma] and normalized activity/pg proteasome), as described elsewhere.¹⁴ Methods were adjusted according to the current study. Serum Apo-A1 levels were measured by enzyme-linked immunosorbent assay in triplicate.

Statistical analysis

A statistical software package (SAS, ver. 9.2; SAS Institute, Cary, NC) was used for data management. Results were expressed as mean ± standard deviation. Statistical analyses were performed using the Wilcoxin t test and the Spearman correlation. Multivariate regression analysis was applied to determine the independent relationships of all clinical variables. A P value of <0.05 was considered statistically significant.

RESULTS AND DISCUSSION

This study showed that among patients with SCD, those with PAH had lower levels of Apo-A and higher levels of polyubiquitin than did those with normal pulmonary arterial pressure. Polyubiquitin levels were significantly higher in the PAH group. Caspase, trypsin, trypsin-like normalized proteasome activity per picogram, caspase-like, and, chymotrypsin-like normalized activity levels were similar between the two groups. There was an inverse relationship between Apo-A and polyubiquitin levels, which was confirmed on multivariate analysis after adjustment for pulmonary arterial pressure. These findings support and extend those of prior studies showing lower Apo-A1 levels related to higher pulmonary arterial pressure. To our knowledge, this is the first study investigating the relationship between UPP and Apo-A1 in the setting of SCD-related PAH.

Levels of Apo-A1 were found to be significantly lower in SCD patients with PAH than in those without PAH (P < 0.05). In addition, Apo-A1 showed a significant negative correlation with polyubiquitin levels in blood (r = −0.499, P = 0.009; Fig. 1), which were elevated in the PAH group (P < 0.05). No significant association was observed between Apo-A1 levels and other parameters, such as total proteasome concentration and specific and normalized activity of chymotrypsin-like, trypsin-like, and caspase-like protease levels. Polyubiquitin and Apo-A1 levels did not correlate with age, sex, body mass index, hemoglobin, white blood cell count, reticulocyte count, lactate dehydrogenase, iron/ferritin levels, blood urea nitrogen (BUN), and serum creatinine. Our data indicate that lower levels of Apo-A1 in SCD patients, especially those with PAH, are probably related to enhanced ubiquitination, thereby directing more Apo-A1 to proteolytic chambers.

Figure 1.

Negative correlation between apolipoprotein A (Apo-A) and polyubiquitin levels in patients with sickle cell disease (r=–0.499, P = 0.009). Multivariate regression analysis was applied to determine the independent relationships of all clinical variables. A P value of ≤0.05 was considered statistically significant.

Plasma cholesterol is transported within lipoproteins by two interrelated cascades that include the Apo-B-containing lipoproteins (chylomicrons and low-density lipoproteins) and the Apo-A1-containing lipoproteins (HDL). Plasma HDL-C level is considered to reflect the efficiency of the reverse cholesterol transport, a mechanism to remove excess cholesterol from the vascular macrophages. Apo-A1, the major structural apolipoprotein of HDL, is synthesized and secreted by the liver and intestine as a lipid-poor apolipoprotein, pre-β-HDL. Pre-β-HDL binds to the adenosine triphosphate–binding cassette (ABC) A1 transporter to initiate cholesterol efflux from cells.^16,17 After cholesterol efflux, pre-β-HDL is converted to α-HDL.

α-HDL is the ligand for both the ABCG1 transporter¹⁸ and the scavenger receptor class B type I (SR-BI).¹⁹ SR-BI not only has the capacity to transport the cholesterol in and out of the cells but is also known to activate a poorly understood signal transduction pathway that ultimately stimulates endothelial nitric oxide synthase (eNOS) to release nitric oxide (NO).²⁰ In hyperlipidemic subjects without SCD, niacin increases Apo-A1 and HDL-C levels, which causes improvement in blood flow physiology and enhanced eNOS expression.²¹ Thus, a low Apo-A1 level in SCD patients can result in a decrease in SR-BI activation and finally a decrease in NO release, causing endothelial dysfunction. These findings in humans are supported by animal experiments, where a peptide mimetic of Apo-A1, L4F, ameliorates endothelial dysfunction in a sickle cell mouse model.²² This defect in vascular function seems to be discrete from and adjuvant to the NO resistance due to NO scavenging.²³

UPP is the major intracellular protein turnover pathway in eucaryotic cells. It regulates central mediators of proliferation, inflammation, and apoptosis that are fundamental for survival.²⁴ Although it was previously understood that the proteasome was found only intracellularly,²⁵ it is now known that the proteasome can also exist in extracellular spaces, such as human blood plasma, bronchoalveolar fluid, and sperm.^26–28 Apo-A1 is usually cleared from plasma via the kidneys. However, our studies indicate a novel possibility, namely, that some Apo-A1 (and possibly HDL) might be catabolized in the plasma compartment by proteasomes.

Our study is limited, as we performed RHC in patients with TRV of ≥2.5 only to confirm PAH but not in patients with normal TRV. Therefore, patients with PAH but normal TRV on echo may have been misclassified. In summary, we show here for the first time that low levels of Apo-A1 in SCD patients with PAH are related to dysregulation of UPP. Low levels of Apo-A1 may contribute to endothelial dysfunction and pulmonary vascular pathology. Further studies are warranted to explore distinct patterns of UPP in SCD-related PAH. These findings not only might provide potential insight into pathophysiological mechanisms but also might be useful for identifying suitable therapeutic targets in these patients.

Footnotes

Source of Support: Empire Clinical Research Investigator Program grant.

Conflict of Interest: None declared.

References

Sutton

Castro

Cross

Spencer

Lewis

. Pulmonary hypertension in sickle cell disease. Am J Cardiol 1994;74:626–628.

Castro

Hoque

Brown

. Pulmonary hypertension in sickle cell disease: cardiac catheterization results and survival. Blood 2003;101:1257–1261.

Mehari

Gladwin

Tian

. Mortality in adults with sickle cell disease and pulmonary hypertension. JAMA 2012;307:1254–1256.

Parent

Bachir

Inamo

. A hemodynamic study of pulmonary hypertension in sickle cell disease. N Engl J Med 2011;365:44–53.

Fonseca

Souza

Salemi

Jardim

Gualandro

. Pulmonary hypertension diagnosed by right heart catheterisation in sickle cell disease. Eur Respir J 2011;39:112–118.

Haque

Gokhale

Rampy

Adegboyega

Duarte

Saldana

. Pulmonary hypertension in sickle cell hemoglobinopathy: a clinicopathologic study of 20 cases. Hum Pathol 2002;33:1037–1043.

Sasaki

Waterman

Cottam

. Decreased apolipoprotein A-I and B content in plasma of individuals with sickle cell anemia. Clin Chem 1986;32:226–227.

Yuditskaya

Tumblin

Hoehn

. Proteomic identification of altered apolipoprotein patterns in pulmonary hypertension and vasculopathy of sickle cell disease. Blood 2009;113:1122–1128.

Budhiraja

Tuder

Hassoun

. Endothelial dysfunction in pulmonary hypertension. Circulation 2004;109:159–165.

10.

Sommer

Jarosch

Lenk

. Compartment-specific functions of the ubiquitin-proteasome pathway. Rev Physiol Biochem Pharmacol 2001;142:97–160.

11.

Døskeland

Flatmark

. Conjugation of phenylalanine hydroxylase with polyubiquitin chains catalysed by rat liver enzymes. Biochim Biophys Acta 2001;1547:379–386.

12.

Kopp

Hendil

Dahlmann

Kristensen

Sobek

Uerkvitz

. Subunit arrangement in the human 20S proteasome. Proc Natl Acad Sci USA 1997;94:2939–2944.

13.

White

Guerra

Wang

Lanford

. Presecretory degradation of apolipoprotein [a] is mediated by the proteasome pathway. J Lipid Res 1999;40:275–286.

14.

Zhang

. Ubiquitin-proteasome profiling for enhanced detection of hepatocellular carcinoma in patients with chronic liver disease. J Gastroenterol Hepatol 2011;26:751–758.

15.

Glomset

. Physiological role of lecithin-cholesterol acyltransferase. Am J Clin Nutr 1970;23:1129–1136.

16.

Castro

Fielding

. Early incorporation of cell-derived cholesterol into pre-β-migrating high-density lipoprotein. Biochemistry 1988;27:25–29.

17.

Liu

Bortnick

Nickel

. Effects of apolipoprotein A-I on ATP-binding cassette transporter A1–mediated efflux of macrophage phospholipid and cholesterol: formation of nascent high density lipoprotein particles. J Biol Chem 2003;278:42976–42984.

18.

Wang

Lan

Chen

Matsuura

Tall

. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc Natl Acad Sci USA 2004;101:9774–9779.

19.

Duong

Collins

Jin

Zanotti

Favari

Rothblat

. Relative contribution of ABCA1 and SR-BI to cholesterol efflux to serum from fibroblasts and macrophages. Arterioscler Thromb Vasc Biol 2006;26:541–547.

20.

Yuhanna

Zhu

Cox

. High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase. Nat Med 2001;7:853–857.

21.

Kuvin

Ramet

Patel

Pandian

Mendelsohn

Karas

. A novel mechanism for the beneficial vascular effects of high-density lipoprotein cholesterol: enhanced vasorelaxation and increased endothelial nitric oxide synthase expression. Am Heart J 2002;144:165–172.

22.

Jones

. L-4F, an apolipoprotein A-1 mimetic, dramatically improves vasodilation in hypercholesterolemia and sickle cell disease. Circulation 2003;107:2337–2341.

23.

Hsu

Champion

Campbell-Lee

. Hemolysis in sickle cell mice causes pulmonary hypertension due to global impairment in nitric oxide bioavailability. Blood 2007;109:3088–3098.

24.

Glickman

Ciechanover

. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 2002;82:373–428.

25.

Peters

Franke

Kleinschmidt

. Distinct 19 S and 20 S subcomplexes of the 26 S proteasome and their distribution in the nucleus and the cytoplasm. J Biol Chem 1994;269:7709–7718.

26.

Roth

Moser

Krenn

. Heightened levels of circulating 20S proteasome in critically ill patients. Eur J Clin Invest 2005;35:399–403.

27.

Sixt

Beiderlinden

Jennissen

Peters

. Extracellular proteasome in the human alveolar space: a new housekeeping enzyme? Am J Physiol Lung Cell Mol Physiol 2007;292:L1280–L1288.

28.

Morales

Pizarro

Kong

Jara

. Extracellular localization of proteasomes in human sperm. Mol Reprod Dev 2004;68:115–124.

Dysregulation of Ubiquitin-Proteasome Pathway and Apolipoprotein a Metabolism in Sickle Cell Disease–Related Pulmonary Arterial Hypertension

Abstract

Keywords

INTRODUCTION

METHODS

Study population

Evaluation for PAH

Measurement of apolipoproteins and UPP

Statistical analysis

RESULTS AND DISCUSSION

Footnotes

References