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. 2015 May 15;308(10):L1002-13.
doi: 10.1152/ajplung.00383.2014. Epub 2015 Apr 3.

Compartment-specific expression of collagens and their processing enzymes in intrapulmonary arteries of IPAH patients

Julia Hoffmann  1 , Leigh M Marsh  1 , Mario Pieper  2 , Elvira Stacher  3 , Bahil Ghanim  4 , Gabor Kovacs  5 , Peter König  2 , Heinrike Wilkens  6 , Hans Michael Haitchi  7 , Gerald Hoefler  8 , Walter Klepetko  9 , Horst Olschewski  10 , Andrea Olschewski  11 , Grazyna Kwapiszewska  12
Affiliations

Compartment-specific expression of collagens and their processing enzymes in intrapulmonary arteries of IPAH patients

Julia Hoffmann et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

Alterations in extracellular matrix (ECM) have been implicated in the pathophysiology of pulmonary hypertension. Here, we have undertaken a compartment-specific study to elucidate the expression profile of collagens and their processing enzymes in donor and idiopathic pulmonary arterial hypertension (IPAH) pulmonary arteries. Predominant intimal, but also medial and perivascular, remodeling and reduced lumen diameter were detected in IPAH pulmonary arteries. Two-photon microscopy demonstrated accumulation of collagen fibers. Quantification of collagen in pulmonary arteries revealed collagen accumulation mainly in the intima of IPAH pulmonary arteries compared with donors. Laser capture-microdissected pulmonary artery profiles (intima+media and perivascular tissue) were analyzed by real-time PCR for ECM gene expression. In the intima+media of IPAH vessels, collagens (COL4A5, COL14A1, and COL18A1), matrix metalloproteinase (MMP) 19, and a disintegrin and metalloprotease (ADAM) 33 were higher expressed, whereas MMP10, ADAM17, TIMP1, and TIMP3 were less abundant. Localization of COLXVIII, its cleavage product endostatin, and MMP10, ADAM33, and TIMP1 was confirmed in pulmonary arteries by immunohistochemistry. ELISA for collagen XVIII/endostatin demonstrated significantly elevated plasma levels in IPAH patients compared with donors, whereas circulating MMP10, ADAM33, and TIMP1 levels were similar between the two groups. Endostatin levels were correlated with pulmonary arterial wedge pressure, and established prognostic markers of IPAH, right atrial pressure, cardiac index, 6-min walking distance, NH2-terminal pro-brain natriuretic peptide, and uric acid. Expression of unstudied collagens, MMPs, ADAMs, and TIMPs were found to be significantly altered in IPAH intima+media. Elevated levels of circulating collagen XVIII/endostatin are associated with markers of a poor prognosis.

Keywords: collagen XVIII; endostatin; laser microdissection; vascular fibrosis; vascular remodeling.

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Figures

Fig. 1.
Fig. 1.
Histological characterization of our idiopathic pulmonary arterial hypertension (IPAH) patient cohort. Representative images and zoom of vessels 20–200 μm (A) and 200–500 μm (B) in diameter from donors (left) and IPAH patients (right). Hematoxylin and eosin (H&E), α-smooth muscle actin (SMA, pink), double SMA (purple)/von Willebrand Factor (vWF, light brown) staining. Scale bar: 100 μm.
Fig. 2.
Fig. 2.
Vascular remodeling of pulmonary arteries of IPAH patients. A: mean percentage of perivascular tissue, media, intima and lumen for each patient presented for vessels 20–200 and 200–500 μm; n = 17–20 patients each, means with SE, *P < 0.05; each dot represents 16–88 vessel profiles. B: overlay of regression lines with 95% confidence bands showing the proportion of each respective compartment according to vessel diameter.
Fig. 3.
Fig. 3.
Collagen deposition in IPAH pulmonary arteries. A: representative images and zoom of donor (left) and IPAH pulmonary artery (right) of Masson's trichrome staining (collagen: blue). Scale bar: 100 μm. B: quantification of percentage of absolute collagen as percentage of vessel diameter in perivascular tissue, media, and intima; n = 20 donors and n = 17 IPAH. Means with SE; *P < 0.05. One dot represents 16–88 vessel profiles. C: visualization of perivascular and vascular collagen by autofluorescence 2-photon microscopy. Maximum intensity projections of 1 mm2 × 80 μm cuboits. Left: donor. Right: IPAH. Each side shows connective tissue in red, channel 495–560 nm at 860-nm excitation wavelength (left); images of collagen in green, channel 495–560 nm at 1,100-nm excitation (middle); and overlay (right). Scale bar: 50 μm.
Fig. 4.
Fig. 4.
Expression of collagens, matrix metalloproteinases (MMPs), a disintegrin and metalloproteases (ADAMs), and tissue inhibitors of MMPs (TIMPs) in intima+media and perivascular tissue of donor and IPAH pulmonary arteries. Expression of significantly differentially regulated collagens (A), MMPs (B), ADAMs (C), and TIMPs (D); n = 6–10 each, median, *P < 0.05.
Fig. 5.
Fig. 5.
Localization of collagens and collagen-processing enzymes in pulmonary vessels and plasma levels in donors and IPAH patients. A: immunohistochemical staining of COL18A1, COLXVIII/endostatin, MMP10, ADAM33, TIMP1. Right: donors. Left: IPAH. Black arrowheads point toward positive staining (light brown). Negative and positive controls (COL18A1: human kidney, COLXVIII/endostatin: human kidney, MMP10: human trachea, ADAM33: human placenta, TIMP1: human breast carcinoma) are provided. Scale bar: 100 μm. B: detection of endostatin, MMP10, ADAM33, and TIMP1 in donor and IPAH plasma by ELISA, n = 40 each, *P < 0.05.
Fig. 6.
Fig. 6.
Correlation of endostatin levels with clinical parameters of IPAH patients. Significant positive correlation of endostatin level and pulmonary arterial wedge pressure (PAWP; A), right atrial pressure (RAP; B), NH2-terminal pro-brain natriuretic peptide (NT-proBNP; E), and uric acid (F) and negative correlation with cardiac index (CI; C) and 6-min walking distance (6MWD; D). R, Spearman R; P, significance level.
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