Serum inter-alpha-trypsin inhibitor and matrix hyaluronan promote angiogenesis in fibrotic lung injury

Abstract

Rationale: The etiology and pathogenesis of angiogenesis in idiopathic pulmonary fibrosis (IPF) is poorly understood. Inter-alpha-trypsin inhibitor (IaI) is a serum protein that can bind to hyaluronan (HA) and may contribute to the angiogenic response to tissue injury.

Objectives: To determine whether IaI promotes HA-mediated angiogenesis in tissue injury.

Methods: An examination was undertaken of angiogenesis in IaI-sufficient and -deficient mice in the bleomycin model of pulmonary fibrosis and in angiogenesis assays in vivo and in vitro. IaI and HA in patients with IPF were examined.

Measurements and main results: IaI significantly enhances the angiogenic response to short-fragment HA in vivo and in vitro. lal deficiency Ieads to decreased angiogenesis in the matrigel model, and decreases lung angiogenesis after bleomycin exposure in mice. IaI is found in fibroblastic foci in IPF, where it colocalizes with HA. The colocalization is particularly strong in vascular areas around fibroblastic foci. Serum levels of IaI and HA are significantly elevated in patients with IPF compared with control subjects. High serum IaI and HA levels are associated with decreased lung diffusing capacity, but not FVC.

Conclusions: Our findings indicate that serum IaI interacts with HA, and promotes angiogenesis in lung injury. IaI appears to contribute to the vascular response to lung injury and may lead to aberrant angiogenesis. Clinical trial registered with www.clinicaltrials.gov (NCT00016627).

Figures

Figure 1.

In vitro vascular tube formation by human microvascular endothelial cells type 1. (A) Short-fragment hyaluronan (sHA) induces vascular tube formation, an effect that is significantly enhanced by inter–α-trypsin inhibitor (IaI). Negative control was 0% fetal bovine serum (FBS). Positive control was 5% FBS. (B) Quantification of vascular tube formation (Dunnett's multiple comparison testing; n = 6 wells/group). Arrowheads indicate vascular nodes. Error bars represent SEM.

Figure 2.

In vivo matrigel angiogenesis assay. (A) Photographs of matrigel plugs explanted from inter–α-trypsin inhibitor (IaI)–deficient mice (top panels) or IaI-sufficient mice (bottom panels) 7 days after injection. There is a virtual absence of vascularization in plugs from IaI-deficient mice. (B) Quantification of plug hemoglobin content. No hemoglobin was detected in plugs from IaI-deficient mice. The difference from IaI-sufficient littermates is statistically significant. Error bars represent SEM. (C) Histologic examination of matrigel plugs. There were no visible cells or angiogenesis in plugs removed from IaI-deficient mice (top panel) compared with IaI-sufficient mice (bottom panel, arrows) (CD34 staining; original magnification = ×200; n = 7 mice/group).

Figure 3.

Expression of inter–α-trypsin inhibitor (IaI) heavy chains in liver and lung at baseline and after intratracheal bleomycin exposure in C57BL/6 mice. (A) inter–α-trypsin inhibitor heavy chain (ITIH1) and (B) ITIH2 are expressed in liver (arbitrarily set at 100% reference) more than 50-fold than in lung at baseline. (C) After bleomycin exposure, ITIH1 expression in the liver (open squares) is up-regulated twofold and ITIH2 expression (open triangles) is up-regulated more than sixfold. The highest up-regulation is seen between Days 7 and 14 after bleomycin (*P < 0.001). (D) Lung ITIH1 and ITIH2 expression decreases after bleomycin exposure. Error bars represent SEM. (E) IaI is detectable through immunohistochemistry (brown staining) in fibrotic lungs 3 weeks after bleomycin exposure (original magnification = ×40). (F) Isotype control for (E). (G) Staining of bleomycin-exposed lung for hyaluronan (left panel), and IaI (middle panel). There is hyaluronan–IaI colocalization evident in yellow (right panel) (original magnification = ×200). (H) Staining of unexposed lung for hyaluronan (left panel) and IaI (middle panel). Hyaluronan is mainly in the subepithelial space, and IaI mainly in bronchial epithelia, as well as in vessels (a result of serum IaI) with little colocalization (original magnifcation, ×200).

Figure 4.

Angiogenesis in bleomycin-exposed mice. No difference was observed in baseline vessels between (A) inter–α-trypsin inhibitor (IaI)-sufficient and (B) IaI-deficient mice. At 3 weeks after intratracheal bleomycin exposure, (C) IaI-sufficient mice show more vascularity in fibrotic areas than (D) IaI-deficient mice (factor VIII immunohistochemistry; original magnification = ×100). (E) The difference in vascularity is statistically significant when quantified. Two independent observers were involved in this measurement. One observer took photographs of all fibrotic areas in a longitudinal section of lung, and a second, blinded observer measured vessels in 10 high-power (×200 original magnification) fields (HPF) per mouse. The average per HPF for every mouse was used for statistical calculations (n = 10–14 per group). Error bars represent SEM.

Figure 5.

Inter–α-trypsin inhibitor (IaI) and hyaluronan localize in fibroblastic foci in usual interstitial pneumonitis (UIP). (A) Representative photomicrograph of a fibroblastic focus in a patient with UIP (hematoxylin and eosin; original magnification = ×400). (B) Same fibroblastic focus stained for IaI (original magnification = ×400). IaI is present in the fibroblastic focus, staining most strongly in the margins. (C and D). Alcian blue staining of a lung with UIP fibroblastic foci (arrows) with (C) or without (D) hyaluronidase pretreatment (original magnification = ×200). Hyaluronidase treatment significantly diminishes the blue–green staining within the fibroblastic focus.

Figure 6.

Inter–α-trypsin inhibitor (IaI) and hyaluronan colocalize in fibroblastic foci in usual interstitial pneumonitis (UIP). (A) Hematoxylin–eosin stain of a fibroblastic focus (original magnification = ×400). (B) Immunohistochemistry for IaI (red; original magnification = ×400). (C) Immunohistochemistry for hyaluronan (green; original magnification = ×400). (D) Merged image for IaI and hyaluronan colocalization (yellow; original magnification = ×400).

Figure 7.

Inter–α-trypsin inhibitor (IaI) and hyaluronan colocalize in the subendothelial space. (A) Fibroblastic focus stained for hematoxylin and eosin. The same fibroblastic focus stains positive for (B) hyaluronan and (C) IaI, particularly in the periphery (arrows), where vessels are marked with CD31 (D) (×200 original magnification). The subendothelial area and basal membrane of a small-sized blood vessel stain positive for (E) IaI and (F) hyaluronan, which colocalize in this location (G) (original magnification = ×630).

Figure 8.

Inter–α-trypsin inhibitor (IaI) serum concentrations. (A) Significantly higher serum IaI concentrations in patients with idiopathic pulmonary fibrosis (IPF; gray bars) compared with control subjects (white bars). (B) No correlation exists between serum IaI concentrations and FVC. (C) Correlation between serum IaI concentrations and diffusion capacity for carbon monoxide (DlCO). (D) No difference in FVC between patients with high (white bar) and low (gray bar) serum IaI concentrations. (E) DlCO is significantly higher in patients with low serum IaI concentrations (white bar) than in patients with high serum IaI concentrations (gray bar). Error bars represent SEM.