Rationale: Extremely preterm infants develop bronchopulmonary dysplasia (BPD), a chronic lung injury that lacks effective treatment. TSP-1 (thrombospondin-1) is an antiangiogenic protein that activates TGF-β1 (transforming growth factor-β1), a cytokine strongly linked to both experimental and human BPD.
Objectives: 1) To examine effects of inhibiting TSP-1–mediated TGF-β1 activation (LSKL [leucine–serine–lysine–leucine]) in neonatal rats with bleomycin-induced lung injury; 2) to examine effects of a TSP-1 mimic (ABT-510) on lung morphology; and 3) to determine whether TSP-1 and related signaling peptides are increased in lungs of human preterm infants at risk for BPD.
Methods: From Postnatal Days 1 to 14, rat pups received daily intraperitoneal bleomycin (1 mg/kg) or vehicle and were treated with daily subcutaneous LSKL (20 mg/kg) or vehicle alone. Separate animals were treated with vehicle or ABT-510 (30 mg/kg/d). Paraffin-embedded lung tissues from 47 autopsies (controls; death <28 d, n = 30 and BPD at risk; death ⩾28 d, n = 17) performed on infants born <29 completed weeks’ gestation were semiquantified for injury markers (collagen, macrophages, and 3-nitrotyrosine), TSP-1, and TGF-β1.
Measurements and Main Results: Bleomycin or ABT-510 increased lung TGF-β1 activity and macrophage influx, caused pulmonary hypertension, and led to alveolar and microvascular hypoplasia. Treatment with LSKL partially prevented abnormal lung morphology secondary to bleomycin. Lungs from human infants at risk for BPD had increased contents of TSP-1 and TGF-β1 when compared with controls. TGF-β1 content correlated with markers of lung injury.
Conclusions: TSP-1 inhibits alveologenesis in neonatal rats, in part via the upregulated activity of TGF-β1. Observations in human lungs suggest a similar pathogenic role for TSP-1 in infants at risk for BPD.
A clearer understanding of pathogenesis is necessary to inform new therapies for bronchopulmonary dysplasia (BPD), a chronic lung injury characterized by impaired pulmonary angiogenesis and alveolar hypoplasia that primarily affects infants born extremely preterm. TSP-1 (thrombospondin-1) is an antiangiogenic protein that is also known to activate TGF-β1 (transforming growth factor-β1), a cytokine strongly linked to both experimental and human BPD. To date, experimental evidence supporting a direct role for TSP-1 in experimental BPD has been lacking.
In neonatal rats with BPD-like lung injury secondary to systemic bleomycin, we report that inhibition of TSP-1–mediated upregulation of TGF-β1 activity prevented vascular hypoplasia and chronic pulmonary hypertension and partially improved alveolar hypoplasia. Furthermore, in normal newborn rats, treatment with a TSP-1 mimetic increased lung TGF-β activity and caused microvascular and alveolar hypoplasia. In autopsy-derived lung tissues from extremely premature humans, contents of both TSP-1 and TGF-β1 were increased in cases at high risk for BPD, relative to controls, and TGF-β1 content positively correlated with markers of lung injury. These findings implicate a role for the TSP-1–TGF-β1 axis in both experimental and human BPD. Limiting the adverse effects of pathologically upregulated TGF-β1 activity via suppression of TSP-1 may represent a promising therapeutic strategy for BPD.
Extreme prematurity (birth <29 completed weeks’ gestational age [GA]) accounts for the majority of deaths and chronic disease burden in North American children (1), incurring enormous personal and health system costs (2). A major source of this burden is bronchopulmonary dysplasia (BPD) (3), a chronic lung injury characterized by arrested postnatal development of the pulmonary microvasculature and distal airspaces, leading to respiratory insufficiency due to reduced lung surface area and O2 diffusion capacity (4, 5). Pathological features include arterial and microvascular hypoplasia, enlarged and fewer distal airspaces, septal wall thickening, inflammation, and varying degrees of fibrosis (4, 6). Associated chronic pulmonary hypertension (cPH) is common and heralds a greatly increased risk of morbidity and mortality (7, 8). Adult survivors of BPD are also now recognized as being at high risk for early-onset chronic obstructive pulmonary disease (9–11).
Given its impact and the current lack of specific therapies, BPD has been identified by the U.S. Food and Drug Administration as an urgent priority for the development of new treatments (12). Previous work from our group and others has identified inhibition of ROCK (Rho-kinase) as a promising strategy for the prevention and rescue of experimental neonatal lung injuries (13–17). In neonatal rats with bleomycin-mediated lung injury, a ROCK inhibitor improved abnormal lung morphology and completely prevented cPH (15). Upregulation of TSP-1 (thrombospondin-1) in the bleomycin-exposed lung was also prevented by ROCK inhibition (15). TSP-1 is a potent antiangiogenic protein (18, 19) that acts in part via inhibition of VEGF (vascular endothelial growth factor)–nitric oxide (NO) signaling, a major pathway contributing to angiogenesis, lung growth, and repair (20, 21). TSP-1 is also known to activate latent TGF-β1 (transforming growth factor-β1), a known causal factor in experimental BPD that is also strongly linked to BPD in humans (22–26). TSP-1 is produced by platelets and by multiple lung cell types in response to diverse experimental injuries (15, 18, 27–29). Increased TSP-1 mRNA expression, localized predominantly to platelets, was also reported in lungs of mechanically ventilated preterm infants (30). Thus, as a putative regulator of two pathways that have stood the test of time as pathogenic factors, we hypothesized that TSP-1 is a critical mediator of BPD.
The objectives of this study were twofold: 1) to establish a direct role for TSP-1 in experimental chronic neonatal lung injury and 2) to examine for increased TSP-1 mRNA and protein content and contents of putatively related signaling proteins in lung tissues from extremely preterm human infants at-risk for BPD. Herein, we report that in bleomycin-exposed neonatal rats, treatment with a small peptide inhibitor of TSP-1–mediated TGF-β1 activation (leucine–serine–lysine–leucine [LSKL]) prevented cPH and arterial hypoplasia and improved capillary hypoplasia and abnormal distal airway morphology. Furthermore, neonatal rats receiving a TSP-1–mimetic heptapeptide (N-acetyl-sarcosyl-glycyl-l-valyl-d-alloisoleucyl-l-threonyl-l-norvalyl-l-isoleucyl-l-arginyl-l-proline ethylamide acetate; ABT-510) developed alveolar and capillary hypoplasia resembling BPD. TSP-1 and TGF-β1 were increased in lungs from preterm infants with established BPD or those at high risk of developing BPD when compared with gestational age-matched control cases with early/evolving lung injury. Together, our observations provide novel evidence for a direct pathogenic role for TSP-1 in arrested postnatal lung development in neonatal rats and add substantively to evidence implicating a role for TSP-1 in human BPD.
Materials, detailed methods, and supplemental data are provided in the online supplement.
This study was approved by the University of Ottawa Animal Care Committee and by the Research Ethics Board of the Children’s Hospital of Eastern Ontario (CHEO).
Commencing on the day after birth, Sprague-Dawley rat pups received daily intraperitoneal bleomycin sulfate (1 mg/kg) or saline vehicle (control) for 14 days, as previously reported (15). Concurrent with bleomycin or vehicle, animals received daily subcutaneous LSKL (20 mg/kg) or saline vehicle. Preliminary dose–response studies for LSKL are shown in Figure E1. A separate cohort of animals was treated with a TSP-1 mimic, ABT-510 (30 mg/kg daily, s.c.), or 1× phosphate-buffered saline vehicle from Postnatal Days 1 to 14. All measurements were conducted on Postnatal Day 14 lungs. Markers of cPH included Fulton index, percentage medial wall area, and total length of pulmonary resistance arteries. Artery length, alveolar and macrophage counts and distal airspace, and alveolar capillary surface densities were evaluated using design-based stereology (see Supplement Methods). Measurement of lung nitric oxide oxidation products (NOx) and 3-nitrotyrosine (ELISA) was performed as previously described (31).
Archived paraffin-embedded lung samples from matched lung lobes were obtained from autopsies conducted at CHEO between January 2000 and December 2017. Cases (<29 completed weeks’ GA at birth and <1,000 g birthweight without major congenital malformations) were identified by interrogation of the CHEO autopsy database followed by a detailed review of the autopsy record and patient chart. Forty-seven autopsies meeting the above criteria were identified and separated into two groups based on age at death: 1) 1–27 days (early/evolving lung disease [control] group; n = 30) and 2) ⩾28 days on positive pressure respiratory support and supplemental O2 (FiO2 ⩾0.3) at 28 days (if death occurred <36 completed weeks’ GA) or a clinical diagnosis of severe BPD at 36 weeks corrected GA using established criteria (32) (BPD group; n = 17). Two noncontiguous lung sections per patient per marker were stained to determine the degree of injury: collagen (Picrosirius red), immunoreactive CD68 (activated macrophages), or 3-nitrotyrosine (reactive oxygen/nitrogen species), or to quantify signaling markers: immunoreactive p-adducin (a marker of ROCK activity), ROCK I, ROCK II, TSP-1, or TGF-β1. Antibodies, dilutions, and conditions are detailed in Table E1. TSP-1 mRNA expression was evaluated by in situ hybridization on a subset of slides (10 per group in which mRNA integrity was confirmed using a positive control probe), according to the manufacturer’s instructions (ACD Bio-Techne). Collagen-area fraction was measured using a point-counting grid (see Supplement Methods). For other markers, staining intensity was assessed by a perinatal pathologist (DG) unaware of group allocation, using a semiquantitative scale (immunohistochemistry [IHC] score): 0 = no staining/minimal nonspecific staining (<10% of section); 1 = focal/patchy staining (10–50% of section); and 2 = diffuse/intense staining (>50% of section).
Statistical analyses were performed using Sigma Plot 14.5 (Systat). Numerical data are shown as means ± SEM. For animal studies, statistical significance was determined by two-way ANOVA and Tukey or Dunn’s post hoc test where significant (P < 0.05) differences were found or by a two-tailed t test where only two groups were compared. For studies on human tissue, statistical significance (P < 0.05) was determined by Wilcoxon rank-sum test. Spearman’s rank correlation was used to describe associations.
Body weights and lung stereological data are shown in Table 1. Neither exposure to bleomycin nor treatment with LSKL had any significant effects on body weight at 14 days (Table 1). Bleomycin-exposed animals treated with LSKL had significantly (P < 0.05) greater left lung volume than all other groups (Table 1). There were no significant (P > 0.05) differences between groups in lung shrinkage attributable to processing in paraffin (Table 1). Compared with controls, bleomycin-exposed animals had significantly increased lung collagen volume density (Vv collagen) (Table 1) and average distal airspace dimension (mean linear intercept) (Table 1). Compared with controls, bleomycin-exposed animals also had significantly decreased alveolar surface density (Sv) (Table 1), total alveolar surface area (Figure 1A), alveolar numerical density (Nv) (Table 1), and total alveolar number (Figure 1B). Total capillary surface area (Figure 1C) was also significantly (P < 0.05) decreased by exposure to bleomycin when compared with vehicle-exposed controls. Interstitial macrophage number was significantly (P < 0.05) increased by bleomycin (Figure 1D). Treatment of bleomycin-exposed animals with LSKL significantly (P < 0.05) increased alveolar Sv and Nv (Table 1) and decreased interstitial macrophage number (Figure 1D) when compared with bleomycin-exposed animals treated with vehicle. LSKL-mediated inhibition of macrophage influx was confirmed by Western blot for CD68 (Figure E2). Treatment of bleomycin-exposed animals with LSKL also partially increased total alveolar surface area (Figure 1A), alveolar number (Figure 1B), and total alveolar capillary surface area (Figure 1C), such that values were no longer significantly different (P > 0.05) from controls. These improvements were due to the combined effects of increased Sv and Nv (Table 1) and increased lung volume (Table 1). Treatment of bleomycin-exposed animals with LSKL had no significant effect (P > 0.05) on collagen volume density (Vv collagen) (Table 1) compared with vehicle-treated animals. Relative differences in lung morphology and interstitial macrophages between groups are illustrated by representative images of elastin-stained (Figure 1E) and immunofluorescent CD68 (Figure 1F).
|Parameter||Control Vehicle Treated (n = 8)||Control LSKL Treated (n = 8)||Bleomycin Exposed, Vehicle Treated (n = 10)||Bleomycin Exposed, LSKL Treated (n = 10)||P Value|
|Body weight, g||33.1 ± 0.48||33.2 ± 0.86||31.9 ± 0.53||33.1 ± 0.52||NS|
|V lung Cavalieri, cm3||0.197 ± 0.013||0.196 ± 0.001||0.215 ± 0.013||0.260 ± 0.020*||<0.05|
|Tissue shrinkage factor (V lung [Archimedes]/V lung [Cavalieri])||2.73 ± 0.08||2.96 ± 0.09||2.83 ± 0.12||2.70 ± 0.12||NS|
|Vv parenchyma/lung, %||87.4 ± 0.8||86.7 ± 1.0||90.0 ± 0.6||91.0 ± 0.7*||<0.05|
|Vv collagen/lung, %||2.41 ± 0.17||3.36 ± 0.23||5.08 ± 0.25*||5.04 ± 0.34*||<0.001|
|Vv airspaces/parenchyma, %||66.7 ± 1.8||71.6 ± 1.3||79.6 ± 1.3*||83.1 ± 0.9*||<0.001|
|Sv alveoli/parenchyma, μm–1||0.065 ± 0.003||0.070 ± 0.002||0.049 ± 0.001*||0.056 ± 0.001*†||<0.05|
|MLI, μm||41.0 ± 2.5||40.9 ± 1.5||65.0 ± 2.0*||59.4 ± 2.3*||<0.001|
|Nv alveoli/lung, ×106/cm3||8.5 ± 0.6||8.3 ± 0.9||2.2 ± 0.1*||2.9 ± 0.2*†||<0.01|
Consistent with previous reports (15, 33), exposure to bleomycin for 14 days causes cPH, as evidenced by increased Fulton index (Figure 2A) and medial wall area of pulmonary resistance arteries (Figure 2B), relative to vehicle-exposed controls. Bleomycin-exposed, vehicle-treated animals also had significantly decreased total small artery length (a marker of resistance artery complexity) when compared with controls (Figure 2C). Treatment of bleomycin-exposed animals with LSKL restored these parameters to values comparable to controls (Figures 2A–2C). Relative differences in the right ventricular free wall and pulmonary artery medial wall thickness between groups are illustrated by ultra-low-power images of hematoxylin and eosin–stained hearts oriented in the short axis (Figure 2D) and by high-power images of elastin-stained pulmonary resistance arteries (Figure 2E). Relative differences in small vessel density between groups are illustrated by low-power images using immunoreactive von Willebrand factor as a marker (Figure 2F).
As previously reported (15), exposure to bleomycin significantly (P < 0.05) increased lung TSP-1 relative to controls (Figure 3A). Treatment with LSKL led to a partial decrease in lung TSP-1 content in bleomycin-exposed animals, such that values were no longer significantly different (P > 0.05) from controls (Figure 3A). We have previously shown that TSP-1 immunoreactivity colocalized with microvascular endothelium in the bleomycin-exposed lung (15). As shown by representative co-IF staining, TSP-1 immunoreactivity was also present in distal airway epithelium (colocalization with CDH1 [E-cadherin]) (Figure 3B) of the bleomycin-exposed lung. Increased immunoreactive 3-nitrotyrosine (nitroxidative stress marker) mediated by bleomycin exposure was completely prevented by treatment with LSKL (Figure 3C). Active TGF-β1 (Figure 3D) and p-SMAD3 (phospho–mothers against decapentaplegic homolog 3) (52 kD marker of TGF-β receptor activity) (Figure 3E) contents were significantly increased in the bleomycin-exposed lung, relative to controls, which were both prevented by LSKL treatment (Figures 3D and 3E). Neither exposure to bleomycin nor treatment with LSKL had any significant (P > 0.05) effect on latent TGF-β1 content (data not shown). Immunofluorescence staining for TGF-β1 (Figure 3F) illustrates widespread immunoreactivity in both proximal and distal respiratory epithelium in vehicle-treated, bleomycin-exposed animals and patchy distal epithelial immunoreactivity in other groups. A known mechanism of TSP-1–mediated inhibition of angiogenesis is decreased VEGF–eNOS (endothelial NO synthase) function, leading to reduced nitric oxide content and bioavailability (15, 34). We observed no change in lung NOx after LSKL treatment in either vehicle- or bleomycin-exposed animals (P > 0.05; data not shown), consistent with regulation of the eNOS–VEGF pathway by TSP-1 being independent of its effects on TGF-β1 activation.
ABT-510 treatment significantly (P < 0.05) increased TGF-β receptor activity (p-SMAD3, 52 kD) (Figure 4A) when compared with vehicle-treated controls. Treatment with ABT-510 did not affect lung TSP-1 content, evaluated by Western blot (P > 0.05 versus vehicle-treated lung; data not shown), when compared with vehicle-treated controls. Lungs from ABT-510–treated animals also had significantly (P < 0.05) increased interstitial macrophage counts (Figure 4B) and collagen content (Figure 4C) and decreased total alveolar (Figure 4D) and alveolar capillary (Figure 4E) surface areas when compared with vehicle-treated controls. Treatment with ABT-510 also caused cPH as evidenced by increased Fulton’s index (0.27 ± 0.018 versus 0.18 ± 0.008 in vehicle-treated animals; P < 0.01, by t test, n = 6 per group). Similar to animals exposed to bleomycin (Table 1), ABT-510 did not affect body weight or left lung volume (P > 0.05 versus vehicle-treated animals; data not shown). Changes in lung morphology and collagen secondary to ABT-510 are illustrated by low-power elastin-stained images (Figure 4F) and Picrosirius red–stained sections (Figure 4G).
GA, birth weight, age, and cause(s) of death are listed in Tables E2 (controls) and E3 (BPD at-risk cases). We collectively termed the “BPD” cases “BPD at-risk,” as 14 of 17 patients died prior to 36 weeks’ corrected GA. The three patients with severe BPD who survived to discharge were on home O2 therapy. There were no significant (P > 0.05) differences in median GA (26 and 25 completed weeks in control and BPD groups, respectively) or mean birthweight (720 ± 28 and 743 ± 30 g, in control and BPD groups, respectively) between groups. Among BPD cases, there was a greater proportion of males (15 males, 2 females) than among controls (17 males, 13 females); χ2 = 4.98 (P < 0.05).
Compared with controls, lung sections from BPD at-risk cases had significantly (P < 0.05) increased collagen area fraction (Figure 5A) and greater IHC scores for CD68+ macrophages (Figure 5B) and 3-nitrotyrosine content (Figure 5C).
IHC scores for p-adducin (Figure E3), TSP-1 (Figure 6A), and TGF-β1 (Figure 6C) were significantly (P < 0.05) higher in BPD cases when compared with controls. There were no differences in IHC scores for either ROCK I or II between control and BPD groups (P > 0.05; data not shown). As illustrated by representative images in Figure 6B, lung tissue from BPD at-risk cases showed diffuse expression of TSP-1 mRNA in multiple cell types. In contrast, TSP-1 mRNA in control lungs was most evident around blood vessels (Figure 6B, left panels).
Moderately positive correlations were observed between IHC scores for CD68+ macrophages and TGF-β1 (r = 0.393; P = 0.006, n = 47) or p-adducin (r = 0.363; P = 0.012, n = 47), but no correlation was found between CD68+ macrophages and TSP-1 (r = 0.228; P > 0.05, n = 47). A weakly positive correlation was observed between IHC scores for 3-nitrotyrosine and TGF-β1 (r = 0.312; P = 0.03, n = 47), but not between 3-nitrotyrosine and p-adducin or TSP-1 (r = 0.034 and 0.058, respectively; n = 47). A weakly positive correlation was also observed between IHC scores for TSP-1 and TGF-β1 (r = 0.325; P = 0.02, n = 47), but not between p-adducin and TSP-1 or TGF-β1 (r = 0.126 and 0.232, respectively; n = 47). No correlations were observed between collagen area fraction and IHC scores for any of the signaling markers (P > 0.05).
The present study is the first to directly implicate a role for TSP-1–mediated signaling in arrested postnatal lung development and neonatal cPH and to examine both the distribution and evolution of TSP-1 expression/content in the human preterm lung. Our main findings in bleomycin-exposed neonatal rats were that inhibition of TSP-1–mediated TGF-β1 activity with LSKL prevented cPH, arterial hypoplasia, and lung macrophage influx and partially improved distal airspace and capillary hypoplasia. Importantly, we did not observe any significant effects of treatment with LSKL on lung morphology in control animals, suggesting that normal lung development was not impaired. LSKL limits TSP-1–mediated TGF-β1 activity by preventing displacement of the latency-associated peptide, which makes TGF-β1 accessible to its receptors. This is normally dependent on the interaction between a specific TSP-1 sequence (lysine–arginine–phenylalanine–lysine; KRFK) and a conserved sequence (LSKL) near the amino terminus of the latency-associated peptide that is blocked by exogenous LSKL. In accord, effects of LSKL on bleomycin-exposed animals also included decreased active (but not latent) TGF-β1 content and decreased p-SMAD3, a marker of TGF-β receptor activity. Conversely, a TSP-1 mimic (ABT-510, a structurally modified peptide fragment derived from the second type-1 repeat of TSP-1) known to inhibit angiogenesis in vivo (35), upregulated TGF-β receptor activity, macrophage influx, and collagen deposition in the neonatal rat lung and caused changes in distal airspace and microvascular morphology similar to the effects of systemic bleomycin.
Our main observations in human lung tissues were that TSP-1 mRNA expression and protein content were increased in cases at risk for BPD when compared with GA-matched infants with early/evolving lung injury. Contents/activity of proteins potentially acting upstream (ROCK; p-adducin ) and downstream (TGF-β1) of TSP-1 were also found to be increased in BPD at-risk lungs when compared with controls. Unfortunately, relative quantities of active versus latent TGF-β1 could not be distinguished by IHC. Contents of TSP-1 and TGF-β1 positively correlated with each other and TGF-β1 content positively correlated with the severity of oxidative/nitrative stress and macrophage influx, but not with the degree of collagen deposition. Our collective observations are illustrated in Figure 7 as a working hypothesis. Evidence of increased ROCK activity in human lung tissues was unaccompanied by upregulation of either of its isoforms, ROCK I or II, similar to previously reported observations in neonatal rat models of BPD and/or cPH (13, 15–17, 37). However, unlike bleomycin-induced injury, in which ROCK activity upregulated TSP-1 (15), we observed no correlations between contents of p-adducin and TSP-1 or TGF-β1 in the preterm human lung.
TSP-1 is a large trimeric glycoprotein serine protease, which was the first endogenous protein discovered to have angiostatic properties (19). Of the five known isoforms, two inhibit angiogenesis: TSP-1 and TSP-2 (27), among which only TSP-1 is upregulated in the bleomycin-exposed neonatal rat lung (15). TSP-1 suppresses angiogenesis by inhibiting endothelial cell proliferation and migration, by inducing endothelial apoptosis (38), and by attenuating endothelial colony-forming cell function (39). TSP-1 also stimulates smooth muscle cell proliferation and migration (27), contributing to vascular remodeling in adult rodent models of cPH (40). Most effects of TSP-1 are mediated by interactions with CD36 (cluster of differentiation 36) and CD47 (integrin-associated peptide) receptors, leading to activation of latent TGF-β1 (41), disruption of VEGF-R2 activation (42), uncoupling of eNOS (43), and inhibited NO signaling (44) (Figure 7). TGF-β1 is known to be secreted by many lung cell types in a latent form that is complexed with two matrix-bound proteins: latent binding protein and latency-associated peptide. TSP-1 and other proteases, such as plasmin, cleave latent TGF-β1 to a mature homodimeric form that activates its receptors. TSP-1 may also indirectly upregulate TGF-β1 activity via proinflammatory effects, which enhance the release of active TGF-β1 in part by promoting the activation of plasmin (24). In chronic hypoxia-exposed mice, interstitial macrophage-derived TSP-1 was reported to contribute to cPH (28). In contrast, we observed significant TSP-1 colocalization with pneumocytes in the bleomycin-exposed neonatal rat lung but little or no colocalization with macrophages (data not shown).
LSKL was previously reported to limit fibrotic injuries affecting the kidney and nervous system in adult rats (45, 46). Unexpectedly, LSKL did not decrease bleomycin-induced lung collagen deposition, nor was there any correlation between the degree of collagen deposition and immunoreactivity for TSP-1 or TGF-β1 in the human lung. We have previously shown that an inhibitor of arginase activity completely prevented bleomycin-induced fibrosis (47). Upregulated activity of arginase suppresses NO function by diverting L-arginine toward the production of L-ornithine and proline, the precursor of collagen. Together, these observations suggest a mechanism for bleomycin-induced fibrosis in the neonatal lung that is linked to dysregulated arginine-NO metabolism rather than to upregulated TGF-β1 activity.
In the present study and previous work examining preventive effects of a ROCK inhibitor on bleomycin-induced injury (15), we observed a disparity between preventive effects on markers of cPH, which were complete, and abnormal distal airspace morphology, which was only partial. Our present observations are consistent with TSP-1 mediating pulmonary vascular remodeling and cPH via upregulated TGF-β1 activity. Our observation that lung NOx (NO function) was not increased by treatment with LSKL may help explain why collagen deposition was not prevented and perhaps why improvements in airway morphology were only partial. We speculate that globally limited TSP-1 function, via a TSP-1 neutralizing antibody or soluble CD47 receptor-ligand trap, which was reported to increase NO bioavailability (48), could be a more effective strategy to prevent lung fibrosis and inhibited alveolarization. Future studies examining the effects of LSKL in alternative BPD-like lung injury models will also clarify whether the disparity between vascular and airway effects is peculiar to the bleomycin model.
We acknowledge the limitations of the bleomycin model as a surrogate for human BPD, especially when compared with other models that more closely mimic the injuries (e.g., hyperoxia) contributing to the human condition (49). Our rationale for employing the bleomycin model in the present studies was to add to previously published observations that implicated TSP-1 (15) and to provide proof-of-concept of its central role prior to undertaking similar studies in other models. Our observation that a TSP-1 mimic led to biochemical and morphological changes in the neonatal rat lung that were similar to the effects of bleomycin support the relevance of this model in understanding the pathogenic role of TSP-1. We equalized our experiments for sex to avoid potential confounding effects based on this variable. With the caveat that our study was not sufficiently powered to determine sex differences, no major differences in response to bleomycin exposure, LSKL, or ABT-510 treatment based on sex were observed (data not shown). Finally, treatment with LSKL limited the inhibitory effects on TSP-1 activity only to those directly related to TGF-β1 activation. Unfortunately, receptor-ligand traps also have limitations, given that CD36 and CD47 bind ligands other than TSP-1. Alternative approaches to abrogating TSP function could in the future also include the use of morpholinos (50) or small molecule inhibitors of CD36 (51).
A strength of our animal study is that we employed design-based stereological methods to evaluate lung structure and inflammation. A limitation to our specific approach was the use of paraffin-embedded tissues, which was necessitated by our inability when embarking on this work to embed and section tissues in plastic resin, which does not cause significant tissue shrinkage or deformation. While shrinkage and deformation related to processing for paraffin embedding do not affect the accuracy of numerical analyses, they will affect measurements of volume, surface area, and length. To determine whether global differences in tissue shrinkage could have affected intergroup analyses, we also quantified shrinkage factor (V Archimedes/V Cavalieri), which was found not to significantly differ between experimental groups. Thus, we are confident that differences in airway morphology between groups were not in any major way artifactual.
Our observations were restricted to autopsy tissues, which, unlike for animals, were not prepared in a standardized fashion, leading to differing levels of autolysis that affected mRNA integrity in many cases. It is more difficult to estimate any effect of autolysis on protein integrity and their effects on IHC results, though the potential exists. Quantification of phosphorylated proteins as a signaling marker is also challenging using IHC, likely more so with differing levels of autolysis. Indeed, in addition to p-adducin, which our data suggests was increased in the BPD at-risk lung, we attempted IHC of p-VEGF-R2 and p-SMAD 2/3 in human tissues but were unconvinced of the specificity of staining. Most patients classified as being at risk for BPD in our study died prior to 36 weeks of corrected GA when the presence and severity of BPD are typically determined. For this reason, we classified cases based on a requirement for positive pressure and supplemental O2 support at 28 days, a group that would likely have developed moderate-severe BPD if they survived to 36 weeks. In addition, most infants in the BPD at-risk group died of sepsis and other profoundly proinflammatory conditions such as necrotizing enterocolitis, which would be expected to contribute to injury and expression/content of the signaling markers examined. Finally, our control group, which was based on earlier age at death, also has obvious limitations; however, among infants who died beyond 28 days of life, there were very few not on positive pressure ventilatory support for an extended period prior to death.
Our results strongly implicate a role for the TSP-1–TGF-β1 axis in chronic neonatal lung injury. Directly targeting TGF-β1 for the prevention of BPD has long been considered an enticing strategy; however, TGF-β1 also plays critical roles in lung development and homeostasis (22), rendering untranslatable any approach which globally inhibits its function. Inhibition of TSP-1 signaling may therefore represent a more translatable means of limiting the adverse effects of pathologically upregulated TGF-β1 activity.
The authors thank Mr. Harry Coenraad (Department of Pathology, CHEO) for assistance with the acquisition and sectioning of human lung tissues; Dr. Ana Giassi and Ms. Sharlene Faulkes (Louise Pelletier Histology Core, University of Ottawa) for validation of antibodies and automated IHC staining; and Drs. Nicholas Barrowman and Anne Tsampalieros (Clinical Research Unit, CHEO) for helpful statistical advice. The authors also thank Dr. Christian Mühlfeld (University of Hannover, Germany) for helpful provision of stereological advice.
|1.||Johnston KM, Gooch K, Korol E, Vo P, Eyawo O, Bradt P, et al. The economic burden of prematurity in Canada. BMC Pediatr 2014;14:93.|
|2.||Liu L, Oza S, Hogan D, Perin J, Rudan I, Lawn JE, et al. Global, regional, and national causes of child mortality in 2000-13, with projections to inform post-2015 priorities: an updated systematic analysis. Lancet 2015;385:430–440.|
|3.||Shah PS, Sankaran K, Aziz K, Allen AC, Seshia M, Ohlsson A, et al.; Canadian Neonatal Network. Outcomes of preterm infants <29 weeks gestation over 10-year period in Canada: a cause for concern? J Perinatol 2012;32:132–138.|
|4.||Jobe AJ. The new BPD: an arrest of lung development. Pediatr Res 1999;46:641–643.|
|5.||Jakkula M, Le Cras TD, Gebb S, Hirth KP, Tuder RM, Voelkel NF, et al. Inhibition of angiogenesis decreases alveolarization in the developing rat lung. Am J Physiol Lung Cell Mol Physiol 2000;279:L600–L607.|
|6.||Husain AN, Siddiqui NH, Stocker JT. Pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia. Hum Pathol 1998;29:710–717.|
|7.||Parker TA, Abman SH. The pulmonary circulation in bronchopulmonary dysplasia. Semin Neonatol 2003;8:51–61.|
|8.||Farquhar M, Fitzgerald DA. Pulmonary hypertension in chronic neonatal lung disease. Paediatr Respir Rev 2010;11:149–153.|
|9.||Gough A, Linden M, Spence D, Patterson CC, Halliday HL, McGarvey LP. Impaired lung function and health status in adult survivors of bronchopulmonary dysplasia. Eur Respir J 2014;43:808–816.|
|10.||Saad NJ, Patel J, Burney P, Minelli C. Birth weight and lung function in adulthood: a systematic review and meta-analysis. Ann Am Thorac Soc 2017;14:994–1004.|
|11.||Baraldi E, Filippone M. Chronic lung disease after premature birth. N Engl J Med 2007;357:1946–1955.|
|12.||Offringa M, Davis JM, Turner MA, Ward R, Bax R, Maldonado S, et al. Applying regulatory science to develop safe and effective medicines for neonates: report of the US Food and Drug Administration first annual neonatal scientific workshop, October 28-29, 2014. Ther Innov Regul Sci 2015;49:623–631.|
|13.||Wong MJ, Kantores C, Ivanovska J, Jain A, Jankov RP. Simvastatin prevents and reverses chronic pulmonary hypertension in newborn rats via pleiotropic inhibition of RhoA signaling. Am J Physiol Lung Cell Mol Physiol 2016;311:L985–L999.|
|14.||Gosal K, Dunlop K, Dhaliwal R, Ivanovska J, Kantores C, Desjardins JF, et al. Rho kinase mediates right ventricular systolic dysfunction in rats with chronic neonatal pulmonary hypertension. Am J Respir Cell Mol Biol 2015;52:717–727.|
|15.||Lee AH, Dhaliwal R, Kantores C, Ivanovska J, Gosal K, McNamara PJ, et al. Rho-kinase inhibitor prevents bleomycin-induced injury in neonatal rats independent of effects on lung inflammation. Am J Respir Cell Mol Biol 2014;50:61–73.|
|16.||Xu EZ, Kantores C, Ivanovska J, Engelberts D, Kavanagh BP, McNamara PJ, et al. Rescue treatment with a Rho-kinase inhibitor normalizes right ventricular function and reverses remodeling in juvenile rats with chronic pulmonary hypertension. Am J Physiol Heart Circ Physiol 2010;299:H1854–H1864.|
|17.||Ziino AJ, Ivanovska J, Belcastro R, Kantores C, Xu EZ, Lau M, et al. Effects of rho-kinase inhibition on pulmonary hypertension, lung growth, and structure in neonatal rats chronically exposed to hypoxia. Pediatr Res 2010;67:177–182.|
|18.||Rogers NM, Sharifi-Sanjani M, Csányi G, Pagano PJ, Isenberg JS. Thrombospondin-1 and CD47 regulation of cardiac, pulmonary and vascular responses in health and disease. Matrix Biol 2014;37:92–101.|
|19.||Lawler PR, Lawler J. Molecular basis for the regulation of angiogenesis by thrombospondin-1 and -2. Cold Spring Harb Perspect Med 2012;2:a006627.|
|20.||Kunig AM, Balasubramaniam V, Markham NE, Morgan D, Montgomery G, Grover TR, et al. Recombinant human VEGF treatment enhances alveolarization after hyperoxic lung injury in neonatal rats. Am J Physiol Lung Cell Mol Physiol 2005;289:L529–L535.|
|21.||Le Cras TD, Markham NE, Tuder RM, Voelkel NF, Abman SH. Treatment of newborn rats with a VEGF receptor inhibitor causes pulmonary hypertension and abnormal lung structure. Am J Physiol Lung Cell Mol Physiol 2002;283:L555–L562.|
|22.||Aschner Y, Downey GP. Transforming growth factor-β: master regulator of the respiratory system in health and disease. Am J Respir Cell Mol Biol 2016;54:647–655.|
|23.||toti p, Buonocore G, Tanganelli P, Catella AM, Palmeri ML, Vatti R, et al. Bronchopulmonary dysplasia of the premature baby: an immunohistochemical study. Pediatr Pulmonol 1997;24:22–28.|
|24.||Belcastro R, Lopez L, Li J, Masood A, Tanswell AK. Chronic lung injury in the neonatal rat: up-regulation of TGFβ1 and nitration of IGF-R1 by peroxynitrite as likely contributors to impaired alveologenesis. Free Radic Biol Med 2015;80:1–11.|
|25.||Moore AM, Buch S, Han RN, Freeman BA, Post M, Tanswell AK. Altered expression of type I collagen, TGF-β 1, and related genes in rat lung exposed to 85% O2. Am J Physiol 1995;268:L78–L84.|
|26.||Nakanishi H, Sugiura T, Streisand JB, Lonning SM, Roberts JD Jr. TGF-β-neutralizing antibodies improve pulmonary alveologenesis and vasculogenesis in the injured newborn lung. Am J Physiol Lung Cell Mol Physiol 2007;293:L151–L161.|
|27.||Adams JC, Lawler J. The thrombospondins. Cold Spring Harb Perspect Biol 2011;3:a009712.|
|28.||Kumar R, Mickael C, Kassa B, Sanders L, Hernandez-Saavedra D, Koyanagi DE, et al. Interstitial macrophage-derived thrombospondin-1 contributes to hypoxia-induced pulmonary hypertension. Cardiovasc Res 2020;116:2021–2030.|
|29.||Warburton D, Kaartinen V. When the lung is stretched, could it be thrombospondin via TGFbeta1 peptide activation? J Physiol 2007;584:365.|
|30.||De Paepe ME, Greco D, Mao Q. Angiogenesis-related gene expression profiling in ventilated preterm human lungs. Exp Lung Res 2010;36:399–410.|
|31.||Jankov RP, Daniel KL, Iny S, Kantores C, Ivanovska J, Ben Fadel N, et al. Sodium nitrite augments lung S-nitrosylation and reverses chronic hypoxic pulmonary hypertension in juvenile rats. Am J Physiol Lung Cell Mol Physiol 2018;315:L742–L751.|
|32.||Walsh MC, Wilson-Costello D, Zadell A, Newman N, Fanaroff A. Safety, reliability, and validity of a physiologic definition of bronchopulmonary dysplasia. J Perinatol 2003;23:451–456.|
|33.||McNamara PJ, Murthy P, Kantores C, Teixeira L, Engelberts D, van Vliet T, et al. Acute vasodilator effects of Rho-kinase inhibitors in neonatal rats with pulmonary hypertension unresponsive to nitric oxide. Am J Physiol Lung Cell Mol Physiol 2008;294:L205–L213.|
|34.||Miller TW, Isenberg JS, Roberts DD. Thrombospondin-1 is an inhibitor of pharmacological activation of soluble guanylate cyclase. Br J Pharmacol 2010;159:1542–1547.|
|35.||Audet GN, Fulks D, Stricker JC, Olfert IM. Chronic delivery of a thrombospondin-1 mimetic decreases skeletal muscle capillarity in mice. PLoS One 2013;8:e55953.|
|36.||Fukata Y, Oshiro N, Kinoshita N, Kawano Y, Matsuoka Y, Bennett V, et al. Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility. J Cell Biol 1999;145:347–361.|
|37.||Peng G, Ivanovska J, Kantores C, Van Vliet T, Engelberts D, Kavanagh BP, et al. Sustained therapeutic hypercapnia attenuates pulmonary arterial Rho-kinase activity and ameliorates chronic hypoxic pulmonary hypertension in juvenile rats. Am J Physiol Heart Circ Physiol 2012;302:H2599–H2611.|
|38.||Iruela-Arispe ML, Bornstein P, Sage H. Thrombospondin exerts an antiangiogenic effect on cord formation by endothelial cells in vitro. Proc Natl Acad Sci USA 1991;88:5026–5030.|
|39.||Ligi I, Simoncini S, Tellier E, Vassallo PF, Sabatier F, Guillet B, et al. A switch toward angiostatic gene expression impairs the angiogenic properties of endothelial progenitor cells in low birth weight preterm infants. Blood 2011;118:1699–1709.|
|40.||Ochoa CD, Yu L, Al-Ansari E, Hales CA, Quinn DA. Thrombospondin-1 null mice are resistant to hypoxia-induced pulmonary hypertension. J Cardiothorac Surg 2010;5:32.|
|41.||Sweetwyne MT, Murphy-Ullrich JE. Thrombospondin1 in tissue repair and fibrosis: TGF-β-dependent and independent mechanisms. Matrix Biol 2012;31:178–186.|
|42.||Zhang X, Kazerounian S, Duquette M, Perruzzi C, Nagy JA, Dvorak HF, et al. Thrombospondin-1 modulates vascular endothelial growth factor activity at the receptor level. FASEB J 2009;23:3368–3376.|
|43.||Bauer PM, Bauer EM, Rogers NM, Yao M, Feijoo-Cuaresma M, Pilewski JM, et al. Activated CD47 promotes pulmonary arterial hypertension through targeting caveolin-1. Cardiovasc Res 2012;93:682–693.|
|44.||Isenberg JS, Ridnour LA, Perruccio EM, Espey MG, Wink DA, Roberts DD. Thrombospondin-1 inhibits endothelial cell responses to nitric oxide in a cGMP-dependent manner. Proc Natl Acad Sci USA 2005;102:13141–13146.|
|45.||Xie XS, Li FY, Liu HC, Deng Y, Li Z, Fan JM. LSKL, a peptide antagonist of thrombospondin-1, attenuates renal interstitial fibrosis in rats with unilateral ureteral obstruction. Arch Pharm Res 2010;33:275–284.|
|46.||Liao F, Li G, Yuan W, Chen Y, Zuo Y, Rashid K, et al. LSKL peptide alleviates subarachnoid fibrosis and hydrocephalus by inhibiting TSP1-mediated TGF-β1 signaling activity following subarachnoid hemorrhage in rats. Exp Ther Med 2016;12:2537–2543.|
|47.||Grasemann H, Dhaliwal R, Ivanovska J, Kantores C, McNamara PJ, Scott JA, et al. Arginase inhibition prevents bleomycin-induced pulmonary hypertension, vascular remodeling, and collagen deposition in neonatal rat lungs. Am J Physiol Lung Cell Mol Physiol 2015;308:L503–L510.|
|48.||Kaur S, Martin-Manso G, Pendrak ML, Garfield SH, Isenberg JS, Roberts DD. Thrombospondin-1 inhibits VEGF receptor-2 signaling by disrupting its association with CD47. J Biol Chem 2010;285:38923–38932.|
|49.||Mankouski A, Kantores C, Wong MJ, Ivanovska J, Jain A, Benner EJ, et al. Intermittent hypoxia during recovery from neonatal hyperoxic lung injury causes long-term impairment of alveolar development: a new rat model of BPD. Am J Physiol Lung Cell Mol Physiol 2017;312:L208–L216.|
|50.||Soto-Pantoja DR, Stein EV, Rogers NM, Sharifi-Sanjani M, Isenberg JS, Roberts DD. Therapeutic opportunities for targeting the ubiquitous cell surface receptor CD47. Expert Opin Ther Targets 2013;17:89–103.|
|51.||Geloen A, Helin L, Geeraert B, Malaud E, Holvoet P, Marguerie G. CD36 inhibitors reduce postprandial hypertriglyceridemia and protect against diabetic dyslipidemia and atherosclerosis. PLoS One 2012;7:e37633.|
Supported by operating funding from the Canadian Institutes of Health Research Institute of Human Development, Child and Youth Health (FRN162226, to R.P.J. and D.G.) and by infrastructure funding from the Canada Foundation for Innovation John R. Evans Leaders Fund (37315, to R.P.J.).
Author Contributions: B.A.R.: study design, data acquisition, and edited manuscript; Y.E., K.D., and C.G.: data acquisition and edited manuscript; B.Y.: data acquisition and analysis and edited manuscript; D.G.: study design, data acquisition, analysis and interpretation of data, and edited manuscript; R.P.J.: hypothesis generation, study design, analysis and interpretation of data, and wrote the manuscript.
The authors dedicate this paper to the memory of Dr. A. Keith Tanswell (Professor Emeritus, University of Toronto).
This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.
Originally Published in Press as DOI: 10.1164/rccm.202104-1021OC on January 12, 2022