WISP1-β3 integrin pathway promotes early pulmonary fibrosis through EndMT induced by mechanical ventilation in an experimental sepsis model

doi:10.21203/rs.3.rs-3753532/v1

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WISP1-β3 integrin pathway promotes early pulmonary fibrosis through EndMT induced by mechanical ventilation in an experimental sepsis model

https://doi.org/10.21203/rs.3.rs-3753532/v1

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Mechanical ventilation (MV) is an indispensable life support for patients with sepsis-induced acute respiratory distress syndrome (ARDS), and is the only proven treatment for improving survival. Many ARDS patients survive sepsis but die of pulmonary fibrosis. In this study, we investigated whether the WISP1-β3 integrin pathway is involved in early pulmonary fibrosis through EndMT, manifesting as accelerating deterioration in moderate mechanical ventilation (MTV) in an experimental sepsis model. To investigate our hypothesis, C57BL/6 mice and β3 integrin knockout mice (β3^−/−) were used to perform the study. As expected, the EndMT markers, CD31 and CD34 for endothelial cells and vimentin and α-SMA for mesothelial cells, were markedly changed and collagen deposition increased significantly in cecal ligation and puncture (CLP) after 12h later MTV (10 ml/kg; 4h) groups. The protective effects of anti-WISP1 or β3^−/− were associated with decreased levels of collagen deposition and vimentin, α-SMA, whereas CD31 and CD34 were opposed in the complex model. Overall, this study demonstrated a new potential molecular mechanism by which the WISP1-β3 integrin pathway mediating EndMT facilitates positively regulates early pulmonary fibrosis induced by sepsis combined with MV.

WISP1

β3 integrin

sepsis

mechanical ventilation

lung fibrosis

Sepsis, the most common cause of acute lung injury or acute respiratory distress syndrome (ALI/ARDS), promotes or interferes with mechanisms involved in tissue repair [1]. Mechanical ventilation (MV) is an important part of supportive therapy in the management of ALI/ARDS [2], MV also promotes an iatrogenic complication referred to as ventilator-induced lung injury (VILI). Many patients with ALI/ARDS survive the underlying disease but die of pulmonary fibrosis. Recent reports suggest that sepsis may trigger the development of persistent fibrosis and that VILI may be a major contributor to lung fibrosis [3, 4].

We and others have identified that WNT1 inducible signaling pathway protein 1 (WISP1), a secreted extracellular matrix (ECM) protein that is ubiquitously expressed in multiple organs, plays an indispensable role in contributing to pro-inflammatory cascades; for example, WISP1 aggravates proinflammatory responses in sepsis supported by MV [5], hepatic ischemia-reperfusion injury (IRI) [6] and lung injury after liver IRI [7]. Klee et al. [8] reported that WISP1 could accelerate idiopathic pulmonary fibrosis (IPF), depending on IL-6 levels.

β3 integrin (CD61), a member of the integrin family, is a common receptor expressed on a wide variety of cell types [9]. Su et al. demonstrated that β3 integrin enhances vascular leak [10], a pathophysiological function central to sepsis and ALI in mice, by inhibiting endothelial cortical actin formation. In contrast, our previous study indicated that the WISP1-β3 pathway worsens exudation in VILI [11]. Furthermore, other studies have shown that β3 integrin is involved in lung fibrosis [12, 13].

Recent studies have confirmed that pulmonary vascular endothelial cells are involved in the progression of pulmonary fibrosis, which can differentiate into mesenchymal cells, which are called endothelium-mesenchymal transition (EndMT), and eventually become fibroblasts and myofibroblasts, thus accelerating the process of pulmonary fibrosis [14]. In addition, areas of fibrosis are adjacent to inflammation, especially alveolar exudation, in the early exudative phase of ARDS [15], the role of MV as an inciting factor for lung fibrosis is poorly understood, and studies have also shown that inflammatory responses and endothelial dysfunction significantly accelerate the progression of EndMT [16].

Therefore, we hypothesized that sepsis combined with MV activates the WISP1- β3 pathway in early pulmonary fibrosis through EndMT.

2.1 Animals. Wild-type mice (WT, C57BL/6, 8–12 weeks old) were purchased from Shanghai Laboratory Animal Co. Ltd. (SLAC, Shanghai, China) and β3 integrin mutant mice (β3^−/−) were generously presented by Professor Quan Li. All mice were bred in a specific pathogen-free environment and housed in an environment with a temperature of 22 ± 1°C, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h.

2.2 In-vivo experimental animal model. The mice were anesthetized via intraperitoneal (i.p.) administration of 100 mg/kg Ketamine hydrochloride. sepsis was induced by cecal ligation and puncture (CLP) as previously described, and moderate VT ventilation (MTV 10 ml/kg, 4h, zero positive end-expiratory pressure) was performed in anesthetized mice alone or after CLP (12h) [5]. For the WISP1-blockade study, mice were randomized to the WISP1 antibody (anti-WISP1, MAB1680, R&D Systems) through intraperitoneal injection (5 mg/kg, i.p., 4h) or not. Sham control mice were subjected to the same procedure but without CLP or MTV. At last, all the experimental mice were sacrificed by high carbon dioxide. All experimental protocols were approved by a Shanghai General Hospital affiliated with Shanghai Jiaotong University Institutional Animal Care. All animal study is compliance with arrive guidelines.

2.3 Cell lines and cell culture. Human pulmonary microvascular endothelial cells (HPMECs, CRL-3244TM) (5–8 passages) were used for this experiment as described previously [17] and were cultured in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin in a 37℃ incubator with 5% CO₂. HPMECs were cultured in a 6-well plate and adjusted to approximately 5×10⁵ cells/well before treatment.

2.4 In-vitro experimental cell model. HPMECs were cultured on BioFlex cultured plate. Some cell samples will obtain cellular mechanical traction (FX-5000; FlexcelI International Corporation, Hillsborough, NC) stimulation (Pre group or LPS + Pre group) for 4h after lipopolysaccharide (LPS, 100ng/mL) stimulated 24h or not. Mechanical drawing is set as: 30% tensile load, drawing frequency 30 times/min. The incubator continued routine culture for a certain period of time, and finally collected cells for related experiments

2.5 Western Blot Analysis. Proteins were extracted from the frozen lung tissues by grinding with protease inhibitors. The proteins were incubated on ice for 30 min. Western blotting for WISP1, β3 integrin, CD31, CD34, vimentin and α-SMA in lung tissues was performed according to a standard protocol [18]. Membranes were blocked with 5% skimmed milk, incubated with mouse primary antibodies against WISP1 (Abcam, USA), β3 integrin (Abcam, USA), CD31 and CD34 (CST, USA), vimentin and α-SMA (CST, USA), and β-actin (Abcam, USA) overnight, and then incubated with a secondary antibody (Licor Biosciences, USA). Membranes were detected using the Odyssey Two-Color Infrared Laser Imaging System (LI-COR Biosciences).

2.6 Immunohistochemistry (IHC). Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and then used to prepare 5.0µm sections. Subsequently, sections were deparaffinized, rehydrated, and incubated with primary antibodies overnight at 4°C. After incubation, the sections were washed three times with phosphate-buffered saline (PBS) and incubated with HRP (horseradish peroxidase)-conjugated secondary antibody (Servicebio, China). Finally, all sections were stained with DAB (3,3-diaminobenzidine, Servicebio, China) and visualized using a light microscope (Olympus, Japan). The primary antibodies used for IHC were anti-CD31, anti-CD34, anti-vimentin and anti-α-SMA (Servicebio, China) [18].

2.7 Measurement of Cytokines. Bronchoalveolar lavage fluid (BALF) levels of TGF-β1 and MMP9 protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA, R&D Systems, USA) [19].

2.8 Collagen deposition. Mice were euthanized and lung tissues harvested and fixed in 10% (v/v) neutral buffered formalin before preparation of paraffin sections. Paraffin-embedded sections were deparaffinized and stained with a Masson's trichrome stain kit (Sigma-Aldrich) to detect collagen. The Masson staining score was determined following the criteria for the estimation of lung fibrosis severity developed. All the samples were scan under a light microscope (Nikon Optiphot, Japan) and photographed with a Nikon Digital DS-5M camera [20].

2.9 Statistical Analysis. Data are expressed as mean ± standard error of the mean (SEM). Statistically significant differences were determined using two-way or one-way ANOVA, followed by Bonferroni’s multiple comparisons or Tukey’s post-test, respectively, using GraphPad Prism ver. 7.0 (GraphPad Software, San Diego, CA, USA). The significance level was set at ^***P < 0.001, ^**P < 0.01, ^*P < 0.05.

3.1 Sepsis with MV could further upregulate lung pro-inflammatory proteins and collagen deposition.

In our previous study, moderate mechanical ventilation (MTV) alone had no impact on lung injury or permeability, but when applied after CLP, it markedly enhanced both the lung injury score and alveolar-capillary permeability [5]. WISP1 and β3 were both tested in our published studies, and increased in CLP models, and the WISP1-β3 pathway augments the inflammation reaction [11]. As shown in Fig. 1A, the results demonstrated that WISP1 and β3 protein levels were both increased in the CLP model groups (P < 0.01), followed CLP with MV groups (P < 0.001) compared with WT mice. What is more, in terms of WISP1 and β3 protein levels were also both increased more onefold in CLP with MV groups than pure CLP model groups (P < 0.05). In order to explore WISP1 and β3 levels on endothelial cells in the complex model, the human pulmonary microvascular endothelial cells (HPMECs) were used to take further investigate. The result was showed that WISP1 and β3 levels were increased in LPS group (P < 0.01), acutely increased in LPS + Pre group (P < 0.001) compared to Control group. (Supplement Fig. 1)

In addition, to reflect collagen deposition, the lung tissue was marked with Masson-Goldner, and the results showed that collagen (marked by blue color) was increased in CLP groups compared with the Sham groups, while more vast area accumulation was observed in the CLP + MV groups (Fig. 1B).

3.2 EndMT existed and the pro-fibrotic factor increased significantly in complex models.

Under normal conditions, the lung capillary endothelial cell phenotype is precisely maintained; however, numerous studies have demonstrated that endothelial cells display remarkable phenotypic plasticity [21], including their ability to undergo endothelial-to-mesenchymal transition (EndMT) [22], a newly recognized type of cellular transdifferentiation. CD31 and CD34 are both unique marker for endothelial cells, on the other hand, vimentin and α-SMA are both special marker for mesenchymal cells. Western blotting and ELISA were performed to measure the levels of EndMT and profibrotic factors. As shown in Fig. 2A, the results revealed reduced CD31, CD34 and increased vimentin, α-SMA in the CLP groups (P < 0.01), and further changes occurred in the CLP + MV groups (P < 0.001) compared to Sham groups. In addition, there was also a significant difference between CLP groups and CLP + MV groups (P < 0.05).

Extensive studies have shown that members of the transforming growth factor-β (TGF-β) family of growth factors, most prominently the TGF-β1 isoform, are the main inducers of EndMT [14]. In addition, matrix metalloproteinase9 (MMP9) displays an important role in pulmonary fibrosis [23]. ELISA results showed that TGF-β1 (Fig. 2B) and MMP9 (Fig. 2C) levels in BALF were distinctively increased in CLP + MV group (P < 0.001), followed CLP groups (P < 0.01) compared to the Sham groups. Of course, there was also a significant difference between CLP groups and CLP + MV group (P < 0.05). Overall, these results implied that MV supplied later in sepsis could further affect EndMT to induce lung fibrosis.

3.3 anti-WISP1 attenuates EndMT phenomenon in lung tissue after CLP combined with MTV.

For the WISP1-blockade study, mice were treated with WISP1 antibody (anti-WISP1). The western blot findings of this study were as follows:1) CD31 and CD34 protein levels were markedly decreased in the CLP + MV groups compared to the Sham groups (P < 0.001), but anti-WISP1 treatment effectively improved this change compared to CLP + MV groups (P < 0.01) and to Sham groups (P < 0.05); 2) vimentin and α-SMA protein levels showed an opposite trend, namely, increased in the CLP + MV groups (P < 0.001) vs Sham groups, but decreased in anti-WISP1 treated group (P < 0.01) vs CLP + MV groups (Fig. 3A). However, although the anti-WISP1 treated groups could reduce or increase the relative protein levels, the expression level was still distinctive than the Sham groups (P < 0.05).

IHC was used to label CD31, CD34, vimentin and α-SMA in lung tissue from different groups, and it exhibited the similar results as the respective protein levels (Fig. 3B). The results also showed that WISP1 has a potential function in EndMT responses.

3.4 anti-WISP1 deregulated collagen deposition in lung tissue from mice.

Previous results indicated anti-WISP1 ameliorated lung EndMT following CLP + MV. We next sought in determine whether treatment with anti-WISP1 deregulated collagen deposition and cytokine profiles which contributed to the fibrosis in the lung tissue. The main inducers of EndMT, TGF-β1 and MMP9, were examined by ELISA. To meet our expectation, the TGF-β1 (Fig. 4A) and MMP9 (Fig. 4B) level were significantly decreased in CLP + MV with anti-WISP1 treated group compared to without (P < 0.01). Also, the antagonistic group could reduce the inducer’s level, the expression level was still higher than that of the control group (P < 0.05). Additionally, collagen deposition in lung tissue followed the TGF-β1 and MMP9 level changes in the BALF (Fig. 4C). Together, these data suggested anti-WISP1 antibody led to decreased pro-fibrosis cytokine production and deregulated collagen deposition.

3.5 β3^−/− suppresses WISP1 protein level in lung tissue.

In a previous publication, Co-IP showed that WISP1 and β3 integrin could hinge with each other in lung injury induced by poly (I:C) combined with MV [11]. As shown in Fig. 5, the Western blot apparently exhibited that WISP1 was upregulated after CLP + MV in WT mice (P < 0.001). In contrast, WISP1 expression level in β3^−/− mice after CLP + MV effectively decreased but still higher than the Sham group (P < 0.05). Besides, there are obvious differences between the WT mice and β3^−/− mice treated with CLP + MV (P < 0.01).

3.6 EndMT was Markedly disturbed in Lung Tissue from β3-Targeted Mice.

Previous studies have suggested that β3 integrin is involved in lung fibrosis. To comprehensively study the pathological role of β3 integrin in EndMT after CLP + MV. Therefore, lung tissues were harvested, and then we performed western blotting to examine CD31, CD34, vimentin and α-SMA levels. We found that CD31 and CD34 were markedly downregulated, whereas vimentin and α-SMA was upregulated in CLP + MV of WT mice (P < 0.001), however, this variation was reversed in β3^−/− mice (P < 0.05). Meanwhile, mice treated by CLP + MV between WT mice and β3^−/− mice showed significant difference (P < 0.01) (Fig. 6A). In addition, to further conform this changed, we used the IHC method to both mark CD31, CD34, vimentin and α-SMA in the lung samples, the results showed the same as the protein levels (Fig. 6B). Collectively, these dates demonstrated β3 integrin was dependent on WISP1 contributed to lung EndMT after CLP + MV.

3.7 β3^−/− attenuated early lung fibrosis procedure induced by MV supplied later sepsis.

Finally, we investigated whether β3 integrin could affect MV-induced early lung fibrosis after sepsis. ELISA results showed that the BALF of β3 knockout mice, treated by CLP + MV, showed a decrease in TGF-β1 (P < 0.01) (Fig. 7A) and MMP9 (P < 0.001) (Fig. 7B) levels vs WT mice. The collagen deposition assay showed that the β3 integrin mutant effectively retarded lung fibrosis (Fig. 7C). Collectively, these results demonstrated β3 knockout significantly inhibited anti-fibrosis cytokine production and lung fibrosis after CLP + MV.

Sepsis is one of the most significant challenges in critical care, which requires MV support to maintain patients’ lives, but MV can also cause VILI [24, 25]. Pulmonary fibrosis is becoming an important cause of death in surviving acute lung injury induced by sepsis and/or MV. The previous main research is still focus on the single factor, sepsis or mechanical ventilation, however, Villar et al. [26] portrayed that tryptase could improve lung fibrosis induced by sepsis accompanied with MV, but the mechanism was unknown in the early time.

WISP1 is a secreted matricellular protein associated with cell proliferation, differentiation [27]. We showed that WISP1 exacerbated alveolar-capillary permeability in sepsis supported by MV [5], and Konigshoff et al. reported that WISP1 was increased in bleomycin-induced pulmonary fibrosis [28]. β3 integrin is expressed in both circulating endothelial progenitor cells and in platelets, influencing endothelial cell (EC) regeneration and blocking neointimal formation in arteriovenous grafts in mice [10], Weisen Wang et al. illustrated that β3 participated the EndMT via the Notch pathway in human umbilical vein endothelial cells (HUVECs) [12].

In our previous works, WISP1-β3 integrin pathway has been seriously identified that it significantly accelerated lung inflammation and injury induced by sepsis or liver I/R or MV [11, 29]. Meanwhile, other studies have reported that sepsis may initiate the process of pulmonary fibrosis, especially inflammatory exudation in the lung [30], and subsequent VILI will further worsen later progression [31]. Overall, we first hypothesized that the WISP1-β3 integrin pathway could mediate pulmonary vascular EndMT in a murine model of pulmonary fibrosis after systemic sepsis combined with MV injury.

To our expectation, the major and novel findings of this investigation include the following: (a) we first discovered that WISP1 and β3 were markedly expressed, and that collagen deposition was much more obvious in lung tissue after sepsis combined with MV. (b) EndMT change, exhibited by decreased endothelial marker CD31 and CD34 level and increased mesenchymal marker vimentin and α-SMA level, was also high in lung tissue from complex mice model; (c) anti-WISP1 or/and β3^−/− attenuated the expression of pro-EndMT factor, TGF-β1 and MMP9, and reduced collagen deposition in lung tissue from the complex mice group. These results provide evidence that the WISP1-β3 integrin pathway could exaggerate the lung fibrosis process after MV treatment in sepsis. However, how the WISP1-β3 pathway mediates EndMT is still unknown, and our unpublished results suggest that integrin-linked kinase (ILK) may play an important role in the downstream pathway and that ILK is involved in EndMT differentiation of endothelial cells in the tumor microenvironment [32]. On the other hand, according to the given clues, the Notch pathway may be another right downstream pathway for the WISP1-β3 pathway, but all hypotheses need strict and scientific methods for further exploration.

Above all, our study demonstrated that the WISP1-β3 pathway can mediate EndMT to enhance the lung fibrosis procedure, and it is also considered an interesting point and needs further investigation.

The authors declare that they have no conflict of interest. All animal studies (including the euthanasia procedure) were performed in compliance with the regulations and guidelines of the Shanghai General Hospital affiliated with Shanghai Jiaotong University Institutional Animal Care.

Author Contribution

Xu, Hong and Liu do the main experimental results and help collate and analyze collate experimental results; Wu helps to collect experimental data and do some experimental results; The experimental funds and experimental ideas are provided by Yang; Yu designs the whole experimental framework and the final writing.

Acknowledgments

The authors appreciate financial support from the Key Disciplines Group Construction Project of Shanghai Pudong New Area Health Commission (PWZxq2022-06);Medical discipline Construction Project of Pudong Health Committee of Shanghai(PWYgf2021-02) and Jiangxi Provincial Health Commission science and technology plan (SKJP220218547). The authors also thank Professor Quan Li for his kind help with the β3 integrin knockout mice.

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No competing interests reported.

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You are reading this latest preprint version

WISP1-β3 integrin pathway promotes early pulmonary fibrosis through EndMT induced by mechanical ventilation in an experimental sepsis model

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

3. Results

3.1 Sepsis with MV could further upregulate lung pro-inflammatory proteins and collagen deposition.

3.2 EndMT existed and the pro-fibrotic factor increased significantly in complex models.

3.3 anti-WISP1 attenuates EndMT phenomenon in lung tissue after CLP combined with MTV.

3.4 anti-WISP1 deregulated collagen deposition in lung tissue from mice.

3.5 β3^−/− suppresses WISP1 protein level in lung tissue.

3.6 EndMT was Markedly disturbed in Lung Tissue from β3-Targeted Mice.

3.7 β3^−/− attenuated early lung fibrosis procedure induced by MV supplied later sepsis.

4. Discussion

Declarations

Author Contribution

Acknowledgments

References

Additional Declarations

Supplementary Files

Status:

Version 1

WISP1-β3 integrin pathway promotes early pulmonary fibrosis through EndMT induced by mechanical ventilation in an experimental sepsis model

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

3. Results

3.1 Sepsis with MV could further upregulate lung pro-inflammatory proteins and collagen deposition.

3.2 EndMT existed and the pro-fibrotic factor increased significantly in complex models.

3.3 anti-WISP1 attenuates EndMT phenomenon in lung tissue after CLP combined with MTV.

3.4 anti-WISP1 deregulated collagen deposition in lung tissue from mice.

3.5 β3−/− suppresses WISP1 protein level in lung tissue.

3.6 EndMT was Markedly disturbed in Lung Tissue from β3-Targeted Mice.

3.7 β3−/− attenuated early lung fibrosis procedure induced by MV supplied later sepsis.

4. Discussion

Declarations

Author Contribution

Acknowledgments

References

Additional Declarations

Supplementary Files

Status:

Version 1

3.5 β3^−/− suppresses WISP1 protein level in lung tissue.

3.7 β3^−/− attenuated early lung fibrosis procedure induced by MV supplied later sepsis.