Extracellular Vesicle ASC: A Novel Mediator for Lung-Brain Axis in Preterm Brain Injury.

Bronchopulmonary dysplasia (BPD) and neurodevelopmental impairment (NDI) are among the most common morbidities affecting preterm infants. Although BPD is a predictor of poor NDI, it is currently uncertain how BPD contributes to brain injury in preterm infants. Extracellular vesicles (EVs) are involved in inter-organ communication in diverse pathological processes. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly and activation of inflammatory response. We assessed expression profiles of alveolar macrophage (AM) markers, CD11b, CD11c, and CD206, and ASC in EVs isolated from the plasma of preterm infants at risk for BPD at 1 week of age. We found that infants on higher fraction inspired oxygen (FiO2) therapy (HO2, ≥30%) had increased levels of AM-derived EV-ASC compared with infants on lower FiO2 (LO2, <30%). To assess the function of these EVs, we performed adoptive transfer experiments by injecting them into the circulation of newborn mice. We discovered that mice that received EVs from infants on HO2 had increased lung inflammation, decreased alveolarization, and disrupted vascular development, the hallmarks of BPD. Importantly, these EVs crossed the blood-brain barrier and the EVs from infants on HO2 caused inflammation, reduced cell survival, and increased cell death with features of pyroptosis and necroptosis in the hippocampus. These results highlight a novel role for AM-derived EV-ASC in mediating the lung-to-brain crosstalk that is critical in the pathogenesis of BPD and brain injury and identify potential novel targets for preventing and treating BPD and brain injury in preterm infants.

IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice.

BACKGROUND: Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation. METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR. RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2. CONCLUSION: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

GSDMD gene knockout alleviates hyperoxia-induced hippocampal brain injury in neonatal mice.

BACKGROUND: Neonatal hyperoxia exposure is associated with brain injury and poor neurodevelopment outcomes in preterm infants. Our previous studies in neonatal rodent models have shown that hyperoxia stimulates the brain's inflammasome pathway, leading to the activation of gasdermin D (GSDMD), a key executor of pyroptotic inflammatory cell death. Moreover, we found pharmacological inhibition of caspase-1, which blocks GSDMD activation, attenuates hyperoxia-induced brain injury in neonatal mice. We hypothesized that GSDMD plays a pathogenic role in hyperoxia-induced neonatal brain injury and that GSDMD gene knockout (KO) will alleviate hyperoxia-induced brain injury. METHODS: Newborn GSDMD knockout mice and their wildtype (WT) littermates were randomized within 24 h after birth to be exposed to room air or hyperoxia (85% O2) from postnatal days 1 to 14. Hippocampal brain inflammatory injury was assessed in brain sections by immunohistology for allograft inflammatory factor 1 (AIF1) and CD68, markers of microglial activation. Cell proliferation was evaluated by Ki-67 staining, and cell death was determined by TUNEL assay. RNA sequencing of the hippocampus was performed to identify the transcriptional effects of hyperoxia and GSDMD-KO, and qRT-PCR was performed to confirm some of the significantly regulated genes. RESULTS: Hyperoxia-exposed WT mice had increased microglia consistent with activation, which was associated with decreased cell proliferation and increased cell death in the hippocampal area. Conversely, hyperoxia-exposed GSDMD-KO mice exhibited considerable resistance to hyperoxia as O2 exposure did not increase AIF1 + , CD68 + , or TUNEL + cell numbers or decrease cell proliferation. Hyperoxia exposure differentially regulated 258 genes in WT and only 16 in GSDMD-KO mice compared to room air-exposed WT and GSDMD-KO, respectively. Gene set enrichment analysis showed that in the WT brain, hyperoxia differentially regulated genes associated with neuronal and vascular development and differentiation, axonogenesis, glial cell differentiation, hypoxia-induced factor 1 pathway, and neuronal growth factor pathways. These changes were prevented by GSDMD-KO. CONCLUSIONS: GSDMD-KO alleviates hyperoxia-induced inflammatory injury, cell survival and death, and alterations of transcriptional gene expression of pathways involved in neuronal growth, development, and differentiation in the hippocampus of neonatal mice. This suggests that GSDMD plays a pathogenic role in preterm brain injury, and targeting GSDMD may be beneficial in preventing and treating brain injury and poor neurodevelopmental outcomes in preterm infants.

Circulating extracellular vesicles activate the pyroptosis pathway in the brain following ventilation-induced lung injury.

BACKGROUND: Mechanical ventilation of preterm newborns causes lung injury and is associated with poor neurodevelopmental outcomes. However, the mechanistic links between ventilation-induced lung injury (VILI) and brain injury is not well defined. Since circulating extracellular vesicles (EVs) are known to link distant organs by transferring their cargos, we hypothesized that EVs mediate inflammatory brain injury associated with VILI. METHODS: Neonatal rats were mechanically ventilated with low (10 mL/kg) or high (25 mL/kg) tidal volume for 1 h on post-natal day 7 followed by recovery for 2 weeks. Exosomes were isolated from the plasma of these rats and adoptively transferred into normal newborn rats. We assessed the effect of mechanical ventilation or exosome transfer on brain inflammation and activation of the pyroptosis pathway by western blot and histology. RESULTS: Injurious mechanical ventilation induced similar markers of inflammation and pyroptosis, such as increased IL-1ß and activated caspase-1/gasdermin D (GSDMD) in both lung and brain, in addition to inducing microglial activation and cell death in the brain. Isolated EVs were enriched for the exosomal markers CD9 and CD81, suggesting enrichment for exosomes. EVs isolated from neonatal rats with VILI had increased caspase-1 but not GSDMD. Adoptive transfer of these EVs led to neuroinflammation with microglial activation and activation of caspase-1 and GSDMD in the brain similar to that observed in neonatal rats that were mechanically ventilated. CONCLUSIONS: These findings suggest that circulating EVs can contribute to the brain injury and poor neurodevelopmental outcomes in preterm infants with VILI through activation of GSDMD.

Caspase-1 Inhibition Attenuates Hyperoxia-induced Lung and Brain Injury in Neonatal Mice.

Hyperoxia plays a key role in the development of bronchopulmonary dysplasia (BPD), a chronic lung disease of preterm infants. Infants with BPD often have brain injury that leads to long-term neurodevelopmental impairment, but the underlying mechanisms that control BPD-induced neurodevelopmental impairment remain unclear. Our previous studies have shown that hyperoxia-induced BPD in rodents is associated with lung inflammasome activation. Here, we tested the hypothesis that hyperoxia-induced lung and brain injury is mediated by inflammasome activation, and that inhibition of caspase-1, a key component of the inflammasome, attenuates hyperoxia-induced lung and brain injury in neonatal mice. C57/BL6 mouse pups were randomized to receive daily intraperitoneal injections of Ac-YVAD-CMK, an irreversible caspase-1 inhibitor, or placebo during exposure to room air or hyperoxia (85% O2) for 10 days. We found that hyperoxia activated the NLRP1 inflammasome, increased production of mature IL-1ß, and upregulated expression of p30 gasdermin-D (GSDMD), the active form of GSDMD that is responsible for the programmed cell death mechanism of pyroptosis in both lung and brain tissue. Importantly, we show that inhibition of caspase-1 decreased IL-1ß activation and p30 GSDMD expression, and improved alveolar and vascular development in hyperoxia-exposed lungs. Moreover, caspase-1 inhibition also promoted cell proliferation in the subgranular zone and subventricular zone of hyperoxia-exposed brains, resulting in lessened atrophy of these zones. Thus, the inflammasome plays a critical role in hyperoxia-induced neonatal lung and brain injury, and targeting this pathway may be beneficial for the prevention of lung and brain injury in preterm infants.

Recombinant CCN1 prevents hyperoxia-induced lung injury in neonatal rats.

BackgroundCystein-rich protein 61 (Cyr61/CCN1) is a member of the CCN family of matricellular proteins that has an important role in tissue development and remodeling. However, the role of CCN1 in the pathogenesis of bronchopulmonary dysplasia (BPD) is unknown. Accordingly, we have investigated the effects of CCN1 on a hyperoxia-induced lung injury model in neonatal rats.MethodsIn experiment 1, newborn rats were randomized to room air (RA) or 85% oxygen (O2) for 7 or 14 days, and we assessed the expression of CCN1. In experiment 2, rat pups were exposed to RA or O2 and received placebo or recombinant CCN1 by daily intraperitoneal injection for 10 days. The effects of CCN1 on hyperoxia-induced lung inflammation, alveolar and vascular development, vascular remodeling, and right ventricular hypertrophy (RVH) were observed.ResultsIn experiment 1, hyperoxia downregulated CCN1 expression. In experiment 2, treatment with recombinant CCN1 significantly decreased macrophage and neutrophil infiltration, reduced inflammasome activation, increased alveolar and vascular development, and reduced vascular remodeling and RVH in the hyperoxic animals.ConclusionThese results demonstrate that hyperoxia-induced lung injury is associated with downregulated basal CCN1 expression, and treatment with CCN1 can largely reverse hyperoxic injury.

Inhibition of ß-catenin signaling protects against CTGF-induced alveolar and vascular pathology in neonatal mouse lung.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is the most common and serious chronic lung disease of premature infants. Connective tissue growth factor (CTGF) plays an important role in tissue development and remodeling. We have previously shown that targeted overexpression of CTGF in alveolar type II epithelial cells results in BPD-like pathology and activates ß-catenin in neonatal mice. METHODS: Utilizing this transgenic mouse model and ICG001, a specific pharmacological inhibitor of ß-catenin, we tested the hypothesis that ß-catenin signaling mediates the effects of CTGF in the neonatal lung. Newborn CTGF mice and control littermates received ICG001 (10 mg/kg/dose) or placebo (dimethyl sulfoxide, equal volume) by daily i.p. injection from postnatal day 5 to 15. Alveolarization, vascular development, and pulmonary hypertension (PH) were analyzed. RESULTS: Administration of ICG001 significantly downregulated expression of cyclin D1, collagen 1a1, and fibronectin, which are the known target genes of ß-catenin signaling in CTGF lungs. Inhibition of ß-catenin signaling improved alveolar and vascular development and decreased pulmonary vascular remodeling. More importantly, the improved vascular development and vascular remodeling led to a decrease in PH. CONCLUSION: ß-Catenin signaling mediates the autocrine and paracrine effects of CTGF in the neonatal lung. Inhibition of CTGF-ß-catenin signaling may provide a novel therapy for BPD.

Connective tissue growth factor acts within both endothelial cells and beta cells to promote proliferation of developing beta cells.

Type 1 and type 2 diabetes result from an absolute or relative reduction in functional ß-cell mass. One approach to replacing lost ß-cell mass is transplantation of cadaveric islets; however, this approach is limited by lack of adequate donor tissue. Therefore, there is much interest in identifying factors that enhance ß-cell differentiation and proliferation in vivo or in vitro. Connective tissue growth factor (CTGF) is a secreted molecule expressed in endothelial cells, pancreatic ducts, and embryonic ß cells that we previously showed is required for ß-cell proliferation, differentiation, and islet morphogenesis during development. The current study investigated the tissue interactions by which CTGF promotes normal pancreatic islet development. We found that loss of CTGF from either endothelial cells or ß cells results in decreased embryonic ß-cell proliferation, making CTGF unique as an identified ß cell-derived factor that regulates embryonic ß-cell proliferation. Endothelial CTGF inactivation was associated with decreased islet vascularity, highlighting the proposed role of endothelial cells in ß-cell proliferation. Furthermore, CTGF overexpression in ß cells during embryogenesis using an inducible transgenic system increased islet mass at birth by promoting proliferation of immature ß cells, in the absence of changes in islet vascularity. Together, these findings demonstrate that CTGF acts in an autocrine manner during pancreas development and suggest that CTGF has the potential to enhance expansion of immature ß cells in directed differentiation or regeneration protocols.

GSDMD deficiency ameliorates hyperoxia-induced BPD and ROP in neonatal mice.

Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are among the most common morbidities affecting extremely premature infants who receive oxygen therapy. Many clinical studies indicate that BPD is associated with advanced ROP. However, the mechanistic link between hyperoxia, BPD, and ROP remains to be explored. Gasdermin D (GSDMD) is a key executor of inflammasome-induced pyroptosis and inflammation. Inhibition of GSDMD has been shown to attenuate hyperoxia-induced BPD and brain injury in neonatal mice. The objective of this study was to further define the mechanistic roles of GSDMD in the pathogenesis of hyperoxia-induced BPD and ROP in mouse models. Here we show that global GSDMD knockout (GSDMD-KO) protects against hyperoxia-induced BPD by reducing macrophage infiltration, improving alveolarization and vascular development, and decreasing cell death. In addition, GSDMD deficiency prevented hyperoxia-induced ROP by reducing vasoobliteration and neovascularization, improving thinning of multiple retinal tissue layers, and decreasing microglial activation. RNA sequencing analyses of lungs and retinas showed that similar genes, including those from inflammatory, cell death, tissue remodeling, and tissue and vascular developmental signaling pathways, were induced by hyperoxia and impacted by GSDMD-KO in both models. These data highlight the importance of GSDMD in the pathogenesis of BPD and ROP and suggest that targeting GSDMD may be beneficial in preventing and treating BPD and ROP in premature infants.

GSDMD gene knockout alleviates hyperoxia-induced hippocampal brain injury in neonatal mice.

Background: Neonatal hyperoxia exposure is associated with brain injury and poor neurodevelopment outcomes in preterm infants. Our previous studies in neonatal rodent models have shown that hyperoxia stimulates the brain's inflammasome pathway, leading to the activation of gasdermin D (GSDMD), a key executor of pyroptotic inflammatory cell death. Moreover, we found inhibition of GSDMD activation attenuates hyperoxia-induced brain injury in neonatal mice. We hypothesized that GSDMD plays a pathogenic role in hyperoxia-induced neonatal brain injury and that GSDMD gene knockout (KO) will alleviate hyperoxia-induced brain injury. Methods: Newborn GSDMD knockout mice and their wildtype (WT) littermates were randomized within 24 h after birth to be exposed to room air or hyperoxia (85% O2) from postnatal day 1 to 14. Hippocampal brain inflammatory injury was assessed in brain sections by immunohistology for allograft inflammatory factor 1 (AIF1), a marker of microglial activation. Cell proliferation was evaluated by Ki-67 staining, and cell death was determined by TUNEL assay. RNA sequencing of the hippocampus was performed to identify the transcriptional effects of hyperoxia and GSDMD-KO, and qRT-PCR was performed to confirm some of the significantly regulated genes. Results: Hyperoxia-exposed WT mice had increased microglia consistent with activation, which was associated with decreased cell proliferation and increased cell death in the hippocampal area. Conversely, hyperoxia-exposed GSDMD-KO mice exhibited considerable resistance to hyperoxia as O2 exposure failed to increase either AIF1+ or TUNEL+ cell numbers, nor decrease cell proliferation. Hyperoxia exposure differentially regulated 258 genes in WT and only 16 in GSDMD-KO mice compared to room air- exposed WT and GSDMD-KO, respectively. Gene set enrichment analysis showed that in the WT brain, hyperoxia differentially regulated genes associated with neuronal and vascular development and differentiation, axonogenesis, glial cell differentiation, and core development pathways hypoxia-induced factor 1, and neuronal growth factor pathways. These changes were prevented by GSDMD-KO. Conclusion: GSDMD-KO alleviates hyperoxia-induced inflammatory injury, cell survival and death, and alterations of transcriptional gene expression of pathways involved in neuronal growth, development, and differentiation in the hippocampus of neonatal mice. This suggests that GSDMD plays a pathogenic role in preterm brain injury, and targeting GSDMD may be beneficial in preventing and treating brain injury and poor neurodevelopmental outcomes in preterm infants.

Hyperoxia-activated circulating extracellular vesicles induce lung and brain injury in neonatal rats.

Hyperoxia-induced lung injury plays a key role in the development of bronchopulmonary dysplasia (BPD), characterized by inflammatory injury and impaired lung development in preterm infants. Although BPD is a predictor of poor neurodevelopmental outcomes, currently it is uncertain how lung injury contributes to brain injury in preterm infants. Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures that regulate intercellular and inter-organ communications. Gasdermin D (GSDMD) has emerged as a key executor of inflammasome-mediated cell death and inflammation. In this study, we utilized a neonatal rat model of BPD to assess if hyperoxia stimulates lung release of circulating EVs and if these EVs induce lung and brain injury. We found that hyperoxia-exposed rats had elevated numbers of plasma-derived EVs compared to rats maintained in room air. These EVs also had increased cargos of surfactant protein C, a marker of type II alveolar epithelial cells (AEC), and the active (p30) form of GSDMD. When these EVs were adoptively transferred into normal newborn rats via intravenous injection, they were taken up both by lung and brain tissues. Moreover, EVs from hyperoxic animals induced not only the pathological hallmarks of BPD, but also brain inflammatory injury in recipient rats, as well as inducing cell death in cultured pulmonary vascular endothelial cells and neural stem cells (NSC). Similarly, hyperoxia-exposed cultured AEC-like cells released EVs that also contained increased GSDMD-p30 and these EVs induced pyroptotic cell death in NSC. Overall, these data indicate that hyperoxia-activated circulating EVs mediate a lung to brain crosstalk resulting in brain injury and suggest a mechanism that links lung injury and neurodevelopmental impairment in BPD infants.

Riociguat prevents hyperoxia-induced lung injury and pulmonary hypertension in neonatal rats without effects on long bone growth.

Bronchopulmonary dysplasia (BPD) remains the most common and serious chronic lung disease of premature infants. Severe BPD complicated with pulmonary hypertension (PH) increases the mortality of these infants. Riociguat is an allosteric soluble guanylate cyclase stimulator and is approved by the FDA for treating PH in adults. However, it has not been approved for use in neonates due to concern for adverse effects on long bone growth. To address this concern we investigated if administration of riociguat is beneficial in preventing hyperoxia-induced lung injury and PH without side effects on long bone growth in newborn rats. Newborn rats were randomized to normoxia (21% O2) or hyperoxia (85% O2) exposure groups within 24 hours of birth, and received riociguat or placebo by once daily intraperitoneal injections during continuous normoxia or hyperoxia exposure for 9 days. In the hyperoxia control group, radial alveolar count, mean linear intercept and vascular density were significantly decreased, the pathological hallmarks of BPD, and these were accompanied by an increased inflammatory response. There was also significantly elevated vascular muscularization of peripheral pulmonary vessels, right ventricular systolic pressure and right ventricular hypertrophy indicating PH. However, administration of riociguat significantly decreased lung inflammation, improved alveolar and vascular development, and decreased PH during hyperoxia by inducing cGMP production. Additionally, riociguat did not affect long bone growth or structure. These data indicate that riociguat is beneficial in preventing hyperoxia-induced lung injury and PH without affecting long bone growth and structure and hence, suggests riociguat may be a potential novel agent for preventing BPD and PH in neonates.

Individual domains of connective tissue growth factor regulate fibroblast proliferation and myofibroblast differentiation.

All members of the Ctgf, Cyr61, and Nov (CCN) family share a high degree of sequence homology and conservation of structural motifs and domains. Here, we present data about a structure function analysis of connective tissue growth factor (CTGF), a prototypic member of the CCN family, which has been shown to be a downstream mediator of transforming growth factor-beta activities on fibroblasts. Our findings demonstrate the two domains of CTGF function to mediate two distinct biological effects. The N-terminal domain of CTGF mediates myofibroblast differentiation and collagen synthesis. The C-terminal domain of CTGF mediates fibroblast proliferation. These data provide a molecular basis for the divergence of CTGF actions on connective tissue cell types and suggest a model for functional analysis of all of the CCN family gene products.

Combinatorial signaling pathways determine fibroblast proliferation and myofibroblast differentiation.

Fibroblast proliferation, differentiation into myofibroblasts, and increased collagen synthesis are key events during both normal wound repair and fibrotic lesion formation. Here we report that these biological responses to TGF-beta by fibroblasts are regulated via a CTGF-dependent pathway in concert with either EGF or IGF-2. Our studies indicate these responses to TGF-beta are mutually exclusive, and cells that are proliferating do not express alpha-SMA or elevated levels of collagen synthesis. Cells expressing alpha-SMA do not exhibit DNA synthesis but do coexpress higher levels of types I and III collagen mRNA. Thus, fibroblast proliferation and differentiation are controlled by combinatorial signaling pathways involving not only components of the TGF-beta/CTGF pathway, but also signaling events induced by EGF and IGF-2-activated receptors. Collectively, our studies indicate TGF-beta functions as a classic embryonic inducer, initiating a cascade that is controlled by other factors in the cellular environment. We propose a model for this process with regard to wound repair and fibrotic lesion formation that is likely applicable to other instances of CTGF action during embryogenesis.

Connective tissue growth factor expression and action in human corneal fibroblast cultures and rat corneas after photorefractive keratectomy.

PURPOSE: Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues. In this study, the interactions between CTGF and transforming growth factor (TGF)-beta were assessed in human corneal fibroblasts, and the levels and location of CTGF protein and mRNA were measured during healing of excimer laser ablation wounds in rat corneas. METHODS: Human corneal fibroblasts were incubated with TGF-beta1, -beta2, and -beta3 isoforms, and CTGF mRNA and protein were measured. CTGF was immunolocalized in the cultured fibroblasts by using a specific antibody. Regulation of collagen synthesis by TGF-beta and CTGF was assessed in human corneal fibroblasts with a neutralizing antibody and an antisense oligonucleotide to CTGF. CTGF mRNA and protein were measured in rat corneas up to day 21 after excimer ablation of the cornea. CTGF protein was immunolocalized in rat corneas after photorefractive keratectomy (PRK), and the presence of CTGF mRNA and protein in ex vivo rat corneal scrapings was established. RESULTS: All three TGF-beta isoforms stimulated expression of CTGF in human corneal fibroblasts, and CTGF was immunolocalized in the cells. Both TGF-beta and CTGF increased collagen synthesis in corneal fibroblasts. Furthermore, CTGF antibody or antisense oligonucleotide blocked TGF-beta-stimulated collagen synthesis. CTGF protein and mRNA increased in rat corneas through day 21 after PRK. CTGF expression was also detected in ex vivo scrapings of rat corneas. CONCLUSIONS: These data demonstrate that CTGF is expressed by corneal cells after stimulation by TGF-beta, that CTGF expression increases significantly during corneal wound healing, and that CTGF mediates the effects of TGF-beta induction of collagen synthesis by corneal fibroblasts. These data support the hypothesis that CTGF promotes corneal scar formation and imply that regulating CTGF synthesis and action may be an important goal for reducing corneal scarring.

A connective tissue growth factor signaling receptor in corneal fibroblasts.

PURPOSE: To biochemically characterize the receptor for connective tissue growth factor (CTGF) of human corneal fibroblasts (HCF). METHODS: Radiolabeled recombinant human CTGF was used to determine the specificity and time course of binding to low-passage cultures of HCF. The affinity and number of receptors present were calculated by Scatchard and best-fit analyses. In vitro immunoprecipitation assays with radiolabeled CTGF and soluble mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF-2-R) alone, or with CTGF-related growth factors were conducted. Additionally, (125)I-CTGF-binding and CTGF-stimulated proliferation were measured in cultures of M6P/IGF-2-R knockout fibroblasts. RESULTS: Binding of (125)I-CTGF to fibroblast cultures was significantly displaced by CTGF, but not by related growth factors. Scatchard plot analysis indicated the presence of both a high-affinity, low-abundance binding site, and a low-affinity, high-abundance binding site; whereas, the best-fit analysis suggests a single high-affinity, low-abundance binding site. A 280 kDa complex containing cross-linked (125)I-CTGF was immunoprecipitated by antibodies to CTGF or M6P/IGF-2-R. M6P/IGF-2-R knockout cells have a reduced proliferative response to TGF-ß, and don't proliferate at all in response to CTGF. CONCLUSIONS: CTGF binds to the M6P/IGF-2-R with high affinity, and the M6P/IGF-2-R is required for CTGF-stimulated proliferation in fibroblasts. These observations suggest that the M6P/IGF-2-R may be a new antifibrotic target.

Inhalation of sulfur mustard causes long-term T cell-dependent inflammation: possible role of Th17 cells in chronic lung pathology.

Sulfur mustard (SM) is a highly toxic chemical warfare agent that remains a threat to human health. The immediate symptoms of pulmonary distress may develop into chronic lung injury characterized by progressive lung fibrosis, the major cause of morbidity among the surviving SM victims. Although SM has been intensely investigated, little is known about the mechanism(s) by which SM induces chronic lung pathology. Increasing evidence suggests that IL-17(+) cells are critical in fibrosis, including lung fibrotic diseases. In this study we exposed F344 rats and cynomolgus monkeys to SM via inhalation and determined the molecular and cellular milieu in their lungs at various times after SM exposure. In rats, SM induced a burst of pro-inflammatory cytokines/chemokines within 72 h, including IL-1ß, TNF-α, IL-2, IL-6, CCL2, CCL3, CCL11, and CXCL1 that was associated with neutrophilic infiltration into the lung. At 2 wks and beyond (chronic phase), lymphocytic infiltration and continued elevated expression of cytokines/chemokines were sustained. TGF-ß, which was undetectable in the acute phase, was strongly upregulated in the chronic phase; these conditions persisted until the animals were sacrificed. The chronic phase was also associated with myofibroblast proliferation, collagen deposition, and presence of IL-17(+) cells. At ≥30 days, SM inhalation promoted the accumulation of IL-17(+) cells in the inflamed areas of monkey lungs. Thus, SM inhalation causes acute and chronic inflammatory responses; the latter is characterized by the presence of TGF-ß, fibrosis, and IL-17(+) cells in the lung. IL-17(+) cells likely play an important role in the pathogenesis of SM-induced lung injury.

ALK-5 mediates endogenous and TGF-beta1-induced expression of connective tissue growth factor in embryonic lung.

Transforming growth factor-beta1 (TGF-beta1) has been implicated as a major negative regulator of lung branching morphogenesis. Since connective tissue growth factor (CTGF) is a downstream mediator of TGF-beta1 effects on mesenchymal cells, we hypothesized that TGF-beta1 induces CTGF expression in mouse embryonic lung explants and that CTGF mediates TGF-beta1 inhibition of branching morphogenesis. We show that addition of TGF-beta1 to the serum-free medium of embryonic day (E)12.5 lung explant cultures inhibited branching morphogenesis and induced CTGF mRNA expression in time- and dose-dependent manners. In contrast to basal endogenous CTGF protein, which was exclusively localized in the distal airway epithelium, TGF-beta1-induced CTGF protein was localized in both the epithelium and the mesenchyme. Addition of exogenous CTGF to culture medium directly inhibited branching morphogenesis. To identify the signal transduction pathway through which TGF-beta1 induces CTGF, we used SB431542, a specific inhibitor for TGF-beta type I receptor (TbetaRI)/ALK-5 to block TGF-beta1-induced Smad2/3 phosphorylation. Consequently, SB431542 stimulated normal branching morphogenesis and blocked TGF-beta1 inhibition of branching. Furthermore, SB-431542 blocked both endogenous and TGF-beta1-induced expression of CTGF mRNA and protein. These results demonstrate for the first time that TGF-beta1 induces CTGF expression in mouse embryonic lung explants, that CTGF inhibits branching morphogenesis, and that both endogenous and TGF-beta1-induced CTGF expression are mediated by the TbetaRI/ALK-5-dependent Smad2 signaling pathway.

Expression of connective tissue growth factor in intra-abdominal adhesions.

PURPOSE: Connective tissue growth factor stimulates fibroblast proliferation and extracellular matrix deposition in many fibrotic disorders. The aim of our study was to determine the expression pattern of connective tissue growth factor in postoperative intra-abdominal adhesions. METHODS: Adhesions were created in 46 Sprague-Dawley rats by complete dissection and resuturing of a peritoneal patch 2 cm in diameter, lateral from the midline incision. Animals were killed at postoperative Days 3, 6, 9, 12, 15, 18, and 21 and the adhesions scored on a scale of 0 to 5. Tissue samples from adhesion areas and from uninvolved peritoneum were evaluated by Northern and Western blotting for temporal connective tissue growth factor mRNA and protein expression, respectively. Immunohistochemical analysis was performed for connective tissue growth factor localization. RESULTS: Adhesions formed in all animals after surgery and were confined to the peritoneal patches. Adhesion formation increased across time, with significant correlation between adhesion scores and postoperative days (r = 0.329, P = 0.026). Connective tissue growth factor mRNA concentrations were significantly elevated in adhesion tissue throughout the three-week period when compared with normal peritoneum (P = 0.012); peak levels occurred between Days 6 and 15. Western blots demonstrated connective tissue growth factor protein expression in adhesions from Days 6 to 21, in contrast to negligible bands in normal peritoneum. Fibroblasts within the adhesive tissue, but not in uninjured peritoneum, stained positive for connective tissue growth factor by immunohistochemistry. CONCLUSIONS: We have demonstrated a specific temporal and spatial expression pattern for connective tissue growth factor in intra-abdominal adhesions during a three-week postoperative time course. According to what is known about the functional role of connective tissue growth factor in fibrogenesis, our findings warrant further investigations addressing a causal relationship between this growth factor and fibrous adhesion formation.