Background
Exposure to silica may occur in a variety of working and living environments since crystalline silica is one of the most abundant minerals on earth. For example, occupational expose to silica occurs during mining, stone cutting, tunneling and quarrying [
1]. Environmental exposure to silica may occur during sand storms, during inhalation of very fine particles of windblown soil, and following volcanic eruptions. Chronic inhalation of crystalline silica promotes the development of several diseases such as silicosis, chronic obstructive pulmonary diseases (COPD), and lung cancer [
2,
3]. Silicosis is a fibrotic pneumoconiosis characterized by nonneoplastic granulomatous and fibrotic changes in the lung. Silica-exposed patients remain asymptomatic for decades when eventually diagnosed by the presence of fine nodular opacities in the lung by chest X-ray or CT-scan [
4]. Depending of dose and time of exposure, silica may produce acute or various forms of chronic silicosis [
5].
In general, two major stages can be defined during silicosis progression. First, an inflammatory stage characterized by the release of inflammatory mediators such as IL-1β, IL-6, TNF-α that can continue to be released into the second fibrotic stage. The second state is a fibrotic stage characterized by excess deposition of extracellular matrix proteins such as collagen and fibronectin [
6,
7]. Although the exact mechanisms responsible for these changes remain unclear, it is well established that inhaled silica particles are engulfed by macrophages, which leads to cell activation and death followed by the release of intracellular silica that is then taken up by other macrophages. This recurring cycle of cell death and macrophage activation produces the influx of inflammatory cells and the production of cytokines and reactive oxygen and nitrogen species [
8]. These inflammatory mediators are able to enter the pulmonary and systemic circulations where they can produce vascular injury. Moreover, ultra-fine silica particles may cross the pulmonary epithelium into the vascular bed and directly affect the integrity of the vascular endothelium [
9,
10]. Interestingly, cardiovascular diseases are among the leading causes of death in patients with silicosis [
11].
The recurring injury to the pulmonary vasculature may lead to the development of pulmonary hypertension. Pulmonary hypertension results from a proliferative vasculopathy of the small pulmonary arteries and arterioles of the lung best characterized by vasoconstriction, cellular hyperplasia, fibrosis, and thrombosis. These constricted or blocked arteries lead to increased pressure in the vessels and in the right ventricle of the heart. If left untreated, the right ventricular chamber hypertrophies leading to premature right heart failure. In the United States, about 200,000 hospitalizations occur annually due to pulmonary hypertension as primary or secondary diagnosis. About 15,000 deaths per year are ascribed to pulmonary hypertension, although this is likely a low estimate [
12]. The contribution of silicosis to these statistics is likely small, but it has been shown that pulmonary hypertension in patients with silicosis is linked with poorer prognosis [
13]. Similarly, COPD and diffuse parenchymal lung diseases, including idiopathic lung fibrosis and sarcoidosis, are associated with a high incidence of pulmonary hypertension [
14,
15]. Patients with combined interstitial lung disease and pulmonary hypertension have significantly lower survival rate and quality of life.
Because of its clinical relevance, elucidating the mechanisms by which silicosis may lead to pulmonary hypertension is considered important. Animal models of silica exposure exist and, as observed in humans, silica induces granulomatous changes in the lungs of these animals, resulting from the loose aggregation of activated foamy histiocytes and lymphocytes or well organized nodular structures consisting of epitheloid macrophages and multinucleated giant cells [
16,
17]. However, there are currently no well-described in vivo models of silica exposure that demonstrate increased risk of pulmonary vascular remodeling and pulmonary hypertension. The objective of this study was to determine whether silica exposure leads to increased right ventricular systolic pressure (RVSP) and vascular remodeling in pulmonary arteries. We now provide evidence that RVSP and vascular abnormalities are markedly increased in silica-exposed mice compared to control mice.
Methods
Experimental animals and animal care
The research protocol was approved by the Institutional Animal Care and Use Committee of the University of Louisville, and the care and handling of the animals were in accordance with National Institutes of Health guidelines. C57BL6 mice were obtained from Jackson Laboratory (Bar Harbor, ME).
Animal model
Adult male C57BL6 mice (10 weeks of age) were separated into 5 experimental groups with 5 animals per group. Animals were anesthetized and placed in the supine position. Using sterile technique, the trachea was exposed via midline neck incision followed by instillation of silica or saline. Crystalline silica was sterilized at 200 °C for 2 h to inactivate endotoxin contamination. Silica suspension in sterile 0.9% NaCl was prepared by vigorous vortexing immediately prior to intratracheal administration. Using a 27-gauge needle attached to a microliter syringe, 0.2 g/kg, 0.3 g/kg and 0.4 g/kg of crystalline silica suspension or the equivalent volume of saline was instilled into the trachea. The fifth group of mice was instilled with 3.5 U/kg of bleomycin (APP Pharmaceuticals, Schaumburg, IL). The incision was then closed using surgical clips, and animals were allowed to recover. Twenty-eight days after intratracheal instillation of silica, RSVP parameters were measured and lung and heart tissues were harvested for morphological, biochemical, and histochemical analyses. Bleomycin-injected mice (3.5 U/kg) were used as positive control and were analyzed 21 days later. Tissue was immediately processed or quick-frozen in liquid nitrogen.
Reagents
Primers and probes for real-time PCR were obtained from Integrated DNA and ThermoFisher Scientific. All other chemicals and enzymes were from Sigma Chemical Co. (St. Louis, MO), or Invitrogen (Carlsbad, CA). Crystalline silica was a gift from US Silica (Min-U-Sil-5, US Silica, Frederick, MD).
Hemodynamic measurements
RVSP was determined with a 1 F pressure transducer catheter (Millar Instruments) and LabChart 8 software (AD Instruments). Briefly, the 1 F pressure transducer was inserted through the right external jugular vein of anesthetized mice (100 mg ketamine/5 mg xylazine/kg of body weight, i.p.). Mice were placed on thermal plates to keep body temperature constant at 37 °C. Then, a pressure catheter was threaded into the right ventricle and RVSP was recorded using PowerLab 4/35 (AD Instruments) and analyzed using LabChart 8 software.
Lung and heart histology
Heart and lungs were flushed with PBS and inflated and fixed with 10% formalin overnight, then embedded in paraffin, sectioned at a thickness of 5 μm, and stained with Mason’s trichrome to visualize lung morphology, fibrosis and vascular remodeling. Images were captured by a high-resolution digital camera connected to a light microscope using 4× and 40× magnification lenses. The evaluation and image analysis procedures were performed using ImageJ software.
Immunohistochemical staining of mouse lungs for smooth muscle, von Willebrand Factor (vWF), LY-6B and CD107b
Longitudinal sections (5 μm) of left lung lobe were hydrated and antigen retrieval was first performed by incubating with 0.1% pronase for 5 min at 37 °C and then heating the slides in 10 mM sodium citrate (pH 6.0) plus 0.05% Tween 20 at 98 °C for 10 min. Sections were stained with anti-smooth muscle actin-alpha antibodies clone 1A4 (Sigma) at concentration 23 ng/μl, anti-vWF antibodies H-300 (Sigma), anti-LY-6B and anti CD-107b antibodies (Bio-Rad) at concentration 10 ng/μl. After washing, the slides were incubated with secondary antibodies labeled with either AlexaFluor 488 or AlexaFluor 594. To determine the specificity of staining, lung sections were incubated with control, non-immune IgG. Slides were analyzed with fluorescent microscopy. Images were processed using ImageJ (National Institutes of Health, Bethesda, MA). Pulmonary arteries were defined as vessels that accompanied airways (veins are interlobular). To measure percent of area stained with specific marker of neutrophils and macrophages we used ImageJ software. For silica and bleomycin treated lung at least four different areas showing pulmonary arteries and silicotic granulomas or fibrotic lesions were selected. The stained areas were selected by thresholding and then calculated using particles analysis extension of ImageJ. The same parameters for thresholding and for calculation of particles were applied to all images.
Right ventricular hypertrophy
After hemodynamic measurements, the hearts were removed and right and left ventricles and septum were separated. The ratio of the right ventricular weight to the sum of left ventricular and septal weight (RV/[LV + S]) served as a measure for right ventricular hypertrophy.
Granuloma area calculation
In sections stained with Mason’s trichrome, the total area of lung section and granuloma area were determined using ImageJ software. Three to four images of each lung were taken at 40× magnification to cover entire lung lobe. Granuloma area percentage was calculated by dividing granuloma area by total lung area and multiplying by 100.
Pulmonary vessels morphometry
To assess muscularization of pulmonary vessels, all blood vessels ranging from 10–100 μm in diameter were counted in at least four fields at 40× magnification. The counted vessels were categorized as fully muscularized (95–100% of medial layer covered by anti-αSMA staining), partially muscularized (1–95% of medial layer is covered by anti-αSMA staining), or nonmuscularized vessels. The percentage of pulmonary vessels in each category was calculated by dividing the number of vessels in the category by the total number of counted vessels in the same field.
Morphometric analysis
The diameter and wall thickness of arteries were measured using ImageJ software, after the number of pixels were calibrated according to the scale bars for each magnification. The values of medial wall thickness were calculated as outer diameter minus inner diameter divided by 2. At least four vessels were counted for each mouse lung. The analysis and measurement of α-SMA staining was evaluated by an investigator blinded to treatment groups.
Quantitative RT-PCR
Total RNA was prepared from the superior lobe of right lung using RNAqueous-Micro Kit (Applied Biosystems, Foster City, CA). The synthesis of single stranded DNA from RNA was performed using SuperScript First-Strand Synthesis System for RT-PCR and random hexamers (Invitrogen, Carlsbad, CA), according to the protocol provided by manufacturer. To quantitate the abundance of gene-specific mRNAs, quantitative PCR was undertaken using the StepOnePlus Real-Time PCR Detection System (Applied Biosystems) and an SYBR® Green Master Mix. The PCR cycles were 95 °C for 3 min, then 40 cycles of 95 °C for 15 s, 60 °C for 1 min. The mouse fibronectin primers were forward (5′- GAC TGT ACT TGT CTA GGC GAA G -3′) and reverse (5′-GTT TCC TCG GTT GTC CTT CT-3′), mouse PECAM-1 primers were forward (5′-AGA GAC GGT CTT GTC GCA GT-3′) and reverse (5′-TAC TGG GCT TCG AGA GCA TT-3′), mouse Endothelin 1 primers were forward (5′- TCT GCA CTC CAT TCT CAG C-3′) and reverse (5′- CGT GAT CTT CTC TCT GCT GTT C-3′), mouse Platelet Factor 4 (PF4) primers were forward (5′- ACC ATC TCC TCT GGG ATC CAT-3′) and reverse (5′-CCA TTC TTC AGG GTG GCT ATG AG-3′), mouse Nestin primers were forward (5′-GGA AAG CCA AGA GAA GCC T-3′) and reverse (5′-CAC CTC AAG ATG TCC CTT AGT C-3′). PCR assays were run in triplicate, and gene expression was normalized to β-Actin mRNA levels. Primers for β-Actin were forward (5′- ACA GCT TCT TTG CAG CTC CT-3′) and reverse (5′-CCA TCA CAC CCT GGT GCC TA-3′). Analysis of Col1a1, Timp1, Ctgf, Tnf and Mmp2 were performed using custom gene expression assays with FAM labeled probe obtained from Applied Biosystems. The mRNA levels for these genes were normalized for β-Actin mRNA levels that were detected with custom gene expression assay with VIC labeled probe.
Data analysis
Values were expressed as means ± SEM. Comparisons between multiple independent groups were made by using One-way ANOVA followed by post hoc analysis with the Holm-Sidak test. Data of two groups were compared with unpaired t-test. A p-value of <0.05 indicated statistically significant differences.
Discussion
Despite efforts to prevent occupational exposure to crystalline silica dust, silicosis continues to occur in many developing nations in Asia and South America, and still remains a significant hazard in advanced countries of North America and Europe [
18]. Although this disease can be prevented by introducing safer working conditions, equally important is the development of early diagnostic tools and safe and effective therapies for those diagnosed with this devastating disease. Animal models remain an important tool in studying the genesis and the progression pulmonary vascular diseases associated with chronic lung disorders. However, despite strong epidemiologic links between exposure to silica and development of pulmonary hypertension in humans [
19], there have been no mouse models described to our knowledge that confirm this association and can be used to elucidate the underlying mechanism(s) responsible. Several previous studies analyzed the effects of asbestos on pulmonary hemodynamic and vascular abnormalities in guinea pigs and rats [
20,
21]. The current work suggests that 4 weeks of exposure to crystalline silica is associated with increases in RVSP, vascular remodeling and dysregulation of genes involved in maintenance of vascular homeostasis.
To compare the effects of silica with other injurious agents, we used mice exposed to bleomycin as a model of severe pulmonary hypertension secondary to lung chronic diseases. While many similarities were observed between the two models, we identified several distinctive features that were only seen in silica-exposed mouse lungs. First, the increase in RVSP observed in the silica model was markedly less profound compared to bleomycin model. This difference can be attributed to dosing and exposure differences. How inhaled silica produces its effects on the pulmonary vasculature and hemodynamic is not well understood. It is possible that silica-particles trapped within the lung parenchyma are quickly surrounded by aggregates of thrombocytes and mononuclear leukocytes, raising the likelihood that regional vasoconstriction triggered in response to focally released thromboxane or serotonin could substantially amplify the overall increase in pulmonary vascular resistance [
22,
23]. On the other hand, it is possible that reduced cardiac output can significantly obscure pulmonary vascular resistance. Unfortunately, we do not have adequate state-of-the-art equipment to test these possibilities. During the last decade, the proinflammatory cytokines TNF-α and IL-1β have emerged as biomarkers and mediators of oxidative stress and endothelial dysfunction in several cardiovascular diseases [
24]. In the present study, we observed that pulmonary arteries from silica-exposed animals showed enhanced TNF-α gene expression despite there being no changes in IL1-β (data not shown). In addition, increased levels of TNF-α can be responsible for the recruitment of more inflammatory cells such as neutrophils and macrophages to the site of injury in silica-treated lungs. Our data indicate that elevated TNF-α gene expression correlates with infiltration of macrophages and neutrophils into silica-induced fibrotic lesions. On the other hand, levels of TNF-α and number of neutrophils did not increase significantly in bleomycin-induced fibrotic lesions. Thus, inhaled silica particles could directly induce endothelial dysfunction by stimulating TNF-α expression and/or by increasing inflammatory cell recruitment in response to elevated secretion of pro-inflammatory cytokines.
We also observed increased expression of MMP-2 and TIMP-1 in lung tissue of silica-exposed mice. MMPs are involved in remodeling of the alveolar architecture near granulomatous lesions but, at the same time, can influence the composition and remodeling of vascular wall. In response to angiogenic stimulus, endothelial-derived MMPs mediate proteolytic degradation of endothelial cell-cell interactions, which promotes a proliferative and migratory phenotype in endothelial cells [
25]. In addition, increased MMP-2 and TIMP-1 expression were identified in pulmonary artery smooth muscle cells isolated from idiopathic PAH patients [
26]. Importantly, increased gelatinolytic activity was mainly observed in the medial layer, and correlated with increased MMP-2 expression in the pulmonary arteries of monocrotaline-treated animals [
27]. This gelatinolytic activity can be attributed to MMP-2 since expression of MMP-9 gene in the pulmonary hypertension model was not observed. The correlation between MMP-2 expression and progression of pulmonary hypertension in the described animal model indicated important roles of this proteinase in different vascular remodeling processes that involves smooth muscle cell proliferation, migration and intimal thickening. The other source of increased MMP-2 and TIMP-1 mRNA levels might be adventitial fibroblasts. Progressing granulomatosis can promote hypoxemia in the lung tissues of silica-treated mice. It has been shown that hypoxia significantly increased MMP-2, TIMP-1, TIMP-2 and α-smooth muscle actin gene expression in adventitial fibroblasts and promoted neointimal hyperplasia [
28].
The increase of collagen type I (Col1a1) mRNA levels coinciding with muscularization and thickening of pulmonary vascular wall indicates collagen accumulation in the vasculature. The phenotype switch of pulmonary smooth muscle cell to hypertrophic cells can be regulated by changes in homeostasis of extracellular matrix components like vascular collagen, elastin and fibronectin. Increased expression and deposition of collagen are known to be important determinants of medial thickening during the progression of PAH [
29]. Collagen accumulation increases pulmonary arterial stiffening, which translates into pulmonary hypertension progression and eventually, RV dysfunction [
30].
We observed significant downregulation in expression of endothelium specific genes such as Pecam 1, also known as CD31, platelet factor 4 (PF4), and nestin. Pecam-1 is expressed on the cell surface of endothelial cells as well as hematopoietic and immune cells including platelets, neutrophils and monocytes. TNF-α and IFN-gamma can reduce the expression of Pecam-1 and transmigration of leukocyte in endothelial cells [
31]. Mice deficient in Pecam-1 become hyperresponsive to stimulation with collagen and demonstrate enhanced aggregation and formation of larger thrombi in vitro under physiologic flow conditions [
32,
33]. Nestin downregulation in vascular smooth muscle cells represented an early event in vascular disease in experimental type I diabetes [
34]. On the other hand, vascular cells expressing nestin were implicated in the development of pulmonary hypertension [
35]. Platelet factor 4 (PF4) expression was downregulated in human lung tissue derived from patients diagnosed with pulmonary hypertension secondary to pulmonary fibrosis, but up-regulated in PAH patients [
36]. A major physiological role of PF4 is to neutralize heparin-like molecules on the endothelial surface of blood vessels, thereby promoting coagulation and inhibiting angiogenesis. One physiological role of PF4 in endothelial cells is to inhibit endothelial cell growth through multiple signaling mechanisms [
37]. Thus, down-regulation of PF4 gene expression in our silica-induced model of pulmonary hypertension might promote angiogenesis and vascular remodeling in affected lungs.
Conclusions
We demonstrated that exposure of mice to a single intratracheal instillation of crystalline silica causes pulmonary vascular remodeling and pulmonary hypertension in mice, although these changes were less severe than those observed in bleomycin-treated mice. To our knowledge, this is the first study to establish that silica exposure causes vascular abnormalities and mild elevated RVSP in mice. Additionally, we observed significant changes in the expression of genes responsible for inflammatory and fibrotic responses in pulmonary cells as well as genes involved in regulation of vascular function. Together, these observations begin to unveil a mechanistic link between silicosis and pulmonary hypertension. Furthermore, they suggest that the silica-induced murine model of pulmonary hypertension could become a valuable tool to explore the pathogenesis of this disease in humans.
Acknowledgments
We thank US Silica Inc. (Frederick, MD) for providing the sample of crystalline silica used in this study.