F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.128621.1 Research Article Articles Sepsis decreases lung SVEP1 expression in a murine model

[version 1; peer review: 1 approved with reservations, 2 not approved]

 Kurita Takeo Conceptualization Formal Analysis Investigation Methodology Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-7555-1160 1 Oami Takehiko Conceptualization Formal Analysis Investigation Methodology Writing – Original Draft Preparation Writing – Review & Editing 1 Fujimura Lisa Formal Analysis Investigation Methodology Writing – Original Draft Preparation 2 Sakamoto Akemi Formal Analysis Investigation Methodology Writing – Original Draft Preparation 2 3 Sato-Nishiuchi Ryoko Formal Analysis Methodology Writing – Original Draft Preparation 4 Sekiguchi Kiyotoshi Formal Analysis Methodology Writing – Original Draft Preparation 4 Hatano Masahiko Formal Analysis Investigation Methodology Writing – Original Draft Preparation 2 3 Nakada Taka-aki Conceptualization Formal Analysis Investigation Methodology Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-4480-556X a 1 Department of Emergency and Critical Care Medicine, Chiba University Graduate School of Medicine, Inohana, Chuo, Chiba, 260-8677, Japan Biomedical Research Center, Chiba University, Inohana, Chuo, Chiba, 260-8670, Japan Department of Biomedical Science, Chiba University Graduate School of Medicine, Inohana, Chuo, Chiba, 260-8670, Japan Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka, 565-0871, Japan taka.nakada@nifty.com

No competing interests were disclosed.

 19 1 2023 2023 12 77 10 1 2023 Copyright: © 2023 Kurita T et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Genome-wide association studies have identified sushi, von Willebrand factor type A, EGF, and pentraxin domain-containing 1 ( SVEP1) polymorphism as a genetic risk factor for sepsis, as well as acute coronary syndrome. However, research on the role of SVEP1 in systemic inflammation, such as surgical invasion and sepsis, remains insufficient. Therefore, we investigated SVEP1 gene expression and protein levels after surgical invasion and sepsis in mice.

Methods: We compared the gene expression and protein levels of SVEP1 between the control (no surgery), sham operation model, and sepsis model with cecal ligation and puncture in mice. Samples were collected at 2, 6, and 24 h after surgery.

Results: The lungs had high gene expression and protein production of SVEP1 at baseline. Sham operation and sepsis decreased SVEP1 gene expression in the lungs immediately after stimulation. Furthermore, sepsis significantly downregulated the gene expression compared with sham operation. Flow cytometric analysis showed that mice with sepsis had a significantly decreased percentage of CD31 high / SVEP1 high and lymphatic vessel endothelial receptor 1 (LYVE-1) high / SVEP1 high cells and an increased percentage of CD45.2 high / SVEP1 high cells.

Conclusions: Sepsis decreased SVEP1 gene expression in the lungs. Mice with sepsis had a decreased percentage of SVEP1 high vascular and lymphatic endothelial cells and an increased percentage of SVEP1 high hematopoietic cells.

 SVEP1 Polydom sepsis vascular endothelial cell lymphatic endothelial cell Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science. JP18K08910 This work was supported by Grants-in-Aid for Scientific Research (C) (JP18K08910) from the Japan Society for the Promotion of Science assigned to Takeo Kurita. Introduction

Sepsis remains the leading cause of death despite the development of acute care and the widespread use of guidelines. 1 – 3 Among the various factors related to the severity of sepsis, 4 – 6 a recent genome-wide association study has revealed that sushi, von Willebrand factor type A, EGF, and pentraxin domain-containing 1 ( SVEP1) gene polymorphism is associated with altered mortality and organ dysfunction in septic shock. 7 , 8 The negative impact of genetic polymorphisms of SVEP1 has also been reported in coronary artery disease with promoted inflammation and atherosclerosis. 8 – 10 These findings suggest that SVEP1 plays a critical role in the progression of systemic inflammation. Therefore, investigating SVEP1 following surgical invasion and sepsis may contribute to further elucidation of the pathophysiology and development of novel treatments.

SVEP1 is an extracellular matrix protein containing a signal peptide followed by multiple different protein domains: a pentraxin domain, a von Willebrand factor type A domain, ephrin2-like cysteine-rich repeats, a hyalin domain, domain similar to thyroglobulin type 2 repeats, 10 epidermal growth factor domains, and 34 complement control protein modules. 11 SVEP1, with a size greater than 300 kDa, is present in the cytoplasm and is degraded into N-terminal SVEP1 (90 kDa) after secretion into the extracellular space. 12 This cell adhesion molecule appears to play a key role in regulating intercellular adhesion and embryonic lymphatic development via the angiopoietin-2 and Tie1/Tie2 receptor systems. 12 – 15

Cell adhesion and vascular permeability have been recognized as important elements that alter the pathophysiology of sepsis. 16 , 17 An interaction between angiopoietin and Tie, which modulates endothelial permeability, has an impact on the progression of organ dysfunction and mortality in septic shock. 18 – 23 A previous study showed that inhibition of SVEP1 expression resulted in an increase in the levels of soluble intracellular adhesion molecules (sICAM) and soluble E-selectin in an in vitro model of endotoxemia, implicating that SVEP1 is responsible for regulating vascular permeability in sepsis. 24 Despite the potentially strong associations between SVEP1 and sepsis, the role of SVEP1 during systemic inflammation is yet to be determined.

Therefore, in this study, we tested the hypothesis that surgical invasion and sepsis alter SVEP1 gene expression and protein production in a murine model.

 Methods Mouse strain and conditions

C57BL/6 mice were purchased from Japan CLEA (Tokyo, Japan). All mice were housed in a controlled environment with a 12-h day and night cycle under specific pathogen-free conditions. Eight- to 12-week-old male and female mice were used for the experiments. The experimental procedures were approved by the Institutional Animal Care and Use Committee of Chiba University (approval number; 2-88; 3/13/2020). This study was carried out in accordance with the recommendations of the Chiba University Resolution on the Use of Animals in Research. The protocol was approved by the Institutional Animal Care and Use Committee of the Chiba University, School of Medicine. The mice were maintained under specific pathogen-free conditions at the Animal Center of the Chiba University Graduate School of Medicine. All efforts were made to ameliorate harm to animals. If necessary, we sacrificed mice under anesthesia to minimize any suffering of animals.

 Cecal ligation and puncture model, abdominal incision model, and tissue collection

The cecal ligation and puncture (CLP) procedure was used to create an intra-abdominal sepsis model. Briefly, a midline incision was made on the abdomen, and the cecum was exposed following anesthesia with 2% isoflurane. The cecum was ligated with a 15 mm length from the tip of the cecum and punctured once with a 20-gauge needle after squeezing feces into the cecum. The abdomen was closed in layers. Next, 1 mL of saline was injected subcutaneously for fluid resuscitation. The same procedures were used in the sham operation model, except for CLP. After the surgery, the mice were sacrificed by cervical dislocation at the time of sample collection under anesthesia. In the control (no surgery), sham operation model, and CLP model, samples were harvested at 2, 6, and 24 h after the surgery without perfusion.

 Number of mice used in each experiment

We compared the gene expression and protein levels of SVEP1 between the control (no surgery), sham operation model, and sepsis model with cecal ligation and puncture in mice. Samples were collected at 2, 6, and 24 h after surgery. We used eight mice in each model for reverse transcription-quantitative polymerase chain reaction and western-blotting, and four mice in each model for flow cytometry. We used a total of 92 mice in all experiments. We did not conduct priori sample size calculation. As we had no pre-defined exclusion criteria, no mice were excluded in each experiment.

 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated using a RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Single-stranded cDNA was transcribed from total RNA using a SuperScript III™ First-Strand Synthesis System for RT-PCR and random hexamers (Invitrogen, USA). RT-qPCR was performed with a Power SYBR ® Green PCR Master Mix (including Dual-Lock™ Taq DNA Polymerase) (Thermo Fisher Scientific, USA) and analyzed using an ABI PRISM ® 7000 Sequence Detection System (Applied Biosystems, USA). We used GAPDH as a housekeeping gene. Primers are shown in Table 1. The thermal cycling were run on 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds and 60°C for 1 minute, 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds.

Oligonucleotide primers for RT-qPCR.

RT-qPCR, reverse transcription-quantitative polymerase chain reaction; F, forward; R, reverse.

Name Sequence
mSVEP1_exon3to4_qPCR_F 5’-CAGCTGCAAATGTGGGACAC-3’
mSVEP1_exon3to4_qPCR_R 5’-ATGCAGGTGCTGATTCCTCC-3’
mGAPDH_qPCR_F 5’-TGTGTCCGTCGTGGATCTGA-3’
mGAPDH_qPCR_R 5’-TTGCTGTTGAAGTCGCAGGAG-3’
Collagen1_F 5’-AGGCTTCAGTGGTTTGGATG-3’
Collagen1_R 5’-CACCAACAGCACCATCGTTA-3’
Fibronectin_F 5’-CTTTGTGGTCTCATGGGTCTC-3’
Fibronectin_R 5’-AGCAGGTCAGGAATGTTCAC-3’
Western blotting

The rabbit anti-N-terminal region of mouse SVEP1 (polyclonal N antibody) developed at the Institute for Protein Research, Osaka University 12 was used to detect proteins. The proteins collected from the homogenized organs were run on polyvinylidene difluoride gels and transferred onto silver nitrate or polyvinylidene difluoride membranes. For immunoblotting, the membranes were probed with the poly N antibody and peroxidase-conjugated affinity pure donkey anti-rabbit IgG (Jackson Immuno Research, USA) and then reacted with ECL Prime Western Blotting Detection Reagent (GE Healthcare, USA). The signal intensity of SVEP1 in the organs was analyzed using ImageJ software (RRID:SCR_003070) (Bethesda, MD, USA). We showed representative densitometric images of western blotting that were cropped as protein bands corresponding to the size of SVEP1 and GAPDH. We did not splice the images.

 Flow cytometry

Whole lung tissue was collected without perfusion and treated with collagenase to isolate single cells. After labeling with cell-surface antibodies, including anti-mouse CD45.2, anti-mouse CD31 (BD Pharmingen, USA), and anti-mouse lymphatic vessel endothelial receptor 1 (LYVE-1) (LifeSpan Biosciences, USA), the cells were stained with poly N antibody as the primary antibody and goat anti-rabbit IgG-FITC (Santa Cruz Biotechnology, USA) as the secondary antibody. This was followed by permeabilization and fixation using the Foxp3/Transcription Factor Staining Buffer Set ® (eBioscience, USA). Samples were run on a FACSCalibur ® (BD Bioscience, USA) and analyzed using FlowJo ® (RRID:SCR_008520) (BD Bioscience). A freely available alternative for the analysis is R version 4.1.2 (R Foundation for Statistical Computing, Vienna, Austria, https://www.r-project.org/) using the flowCore package (RRID:SCR_002205).

 Statistical analysis

We compared the control with the sham operation model and CLP model using the Kruskal-Wallis test with Dunnett’s multiple comparison test. We compared the sham operation model with the CLP model at each point in time using the Student’s t-test or Mann-Whitney test according to normality. We used GraphPad Prism 8 (RRID:SCR_002798) (GraphPad software, USA) for statistical analysis. R version 4.1.2 (R Foundation for Statistical Computing) is a freely available alternative for statistical analysis. Results were considered statistically significant at p < 0.05.

 Results Comparison of SVEP1 gene and protein expression between organs at baseline

We first evaluated SVEP1 gene expression and protein production in vital organs, including the heart, lungs, liver, spleen, kidneys, and colon, in C57BL/6 mice at baseline. The lungs and spleen had 29.6- and 11.2-fold higher SVEP1 gene expression, respectively, compared with the average expression in the other four organs ( Figure 1A). 36

SVEP1 gene and protein expression in various organs at baseline.

(A) Relative SVEP1 gene expression levels in various organs (normalized by the average of four organs: heart, liver, kidneys, and colon) are shown. Data are shown as the mean ± SEM; n = 8 mice in each group. (B) Representative densitometric images of western blotting in various organs at baseline are shown. GAPDH was used for normalization. SVEP1, sushi, von Willebrand factor type A, EGF, and pentraxin domain-containing 1.

Subsequently, SVEP1 protein expression in the organs was evaluated using the poly N antibody. The target band for the lung tissue was approximately 90 kDa, which is the size of the N-terminal SVEP1 secreted into the extracellular space. No target bands were clearly detected for the heart, liver, spleen, kidneys, and colon ( Figure 1B). These results demonstrated that the gene expression and protein levels of SVEP1 were relatively high in the lungs at baseline.

 SVEP1 gene and protein expression in mice with sepsis

Since SVEP1 gene expression and protein levels were higher in the lungs at baseline than in other organs, we focused on the lung and examined the time course of SVEP1 gene and protein expression in the lungs after sham operation and CLP.

In the lungs, there was a significant decrease in SVEP1 gene expression 2 h after sham operation and 2/6/24 h after CLP surgery. SVEP1 gene expression returned to baseline levels at 6 and 24 h after sham operation. Furthermore, sepsis decreased SVEP1 gene expression until 24 h after CLP surgery. We found that SVEP1 gene expression was significantly reduced in the CLP model at all three time points compared with the sham operation model ( Figure 2). These results indicate transiently decreased gene expression of SVEP1 in surgical invasion and persistent suppression of gene expression after sepsis induction.

Relative <italic toggle="yes">SVEP1</italic> gene expression in the lungs after abdominal incision and CLP surgery.

Relative SVEP1 gene expression in the lungs was compared between control (no surgery), 2 h after sham operation (Sham 2h), 6 h after sham operation (Sham 6h), 24 h after sham operation (Sham 24h), 2 h after CLP procedure (CLP 2h), 6 h after CLP procedure (CLP 6h), and 24 h after CLP procedure (CLP 24h). The data were normalized using the control. Error bars represent the mean ± SEM; n = 8 mice in each group. We compared the control to the Sham and CLP models using the Kruskal-Wallis test with Dunnett’s multiple comparison test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). We compared the sham operation model with the CLP model at each time point using the Student’s t-test or Mann-Whitney test according to the normality (†, p < 0.05; ††, p < 0.01; †††, p < 0.001; ††††, p < 0.0001). SVEP1, sushi, von Willebrand factor type A, EGF, and pentraxin domain-containing 1; CLP, cecal ligation and puncture.

We quantitatively evaluated the SVEP1 90 kDa protein in the lungs by western blotting ( Figure 3A). There was no change in the SVEP1 90 kDa protein level 2/24 h after sham operation and 2/6/24 h after CLP surgery. The SVEP1 90 kDa protein level was slightly elevated only 6 h after sham operation, and there was a significant difference between the Sham 6 h and CLP 6 h group ( Figure 3B).

Relative SVEP1 90 kDa protein expression in the lungs during sepsis.

(A) Representative densitometric images of western blotting in the lungs are shown. GAPDH was used for normalization. (B) Relative expression of 90 kDa SVEP1 protein was compared between control (no surgery), 2 h after sham operation (Sham 2h), 6 h after sham operation (Sham 6h), 24 h after sham operation (Sham 24h), 2 h after CLP procedure (CLP 2h), 6 h after CLP procedure (CLP 6h), and 24 h after CLP procedure (CLP 24h). The data were normalized using the control. Error bars represent the mean ± SEM; n = 8 mice in each group. We compared the control with the Sham and CLP models using the Kruskal-Wallis test with Dunnett’s multiple comparison test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). We compared the sham operation model with the CLP model at each time point using the Student’s t-test or Mann-Whitney test according to the normality (†, p < 0.05; ††, p < 0.01; †††, p < 0.001; ††††, p < 0.0001). SVEP1, sushi, von Willebrand factor type A, EGF, and pentraxin domain-containing 1; CLP, cecal ligation and puncture.

 Comparison with other extracellular matrices in the lungs during sepsis

Since SVEP1 is an extracellular matrix protein, 12 we examined the time course of gene expression of other extracellular matrix proteins, such as collagen1 and fibronectin, in the lungs after sham operation and CLP surgery. The gene expression of collagen1 was significantly decreased after sham operation and CLP surgery. At 6 and 24 h after the insult, gene expression was significantly lower in the CLP model than in the sham operation model ( Figure 4A). By contrast, the gene expression of fibronectin significantly increased after sham operation and CLP surgery. In the sham operation model, fibronectin gene expression returned to baseline levels after 6 h, whereas gene expression in the CLP model remained significantly higher than that in the sham operation model ( Figure 4B). The time course of collagen1, but not fibronectin, showed a similar trend with that of SVEP1 gene expression in sepsis.

Relative collagen1 and fibronectin gene expression in the lungs during sepsis.

Relative gene expression of (A) collagen1 and (B) fibronectin in the lungs was compared between control (no surgery), 2 h after sham operation (Sham 2h), 6 h after sham operation (Sham 6h), 24 h after sham operation (Sham 24h), 2 h after CLP procedure (CLP 2h), 6 h after CLP procedure (CLP 6h), and 24 h after CLP procedure (CLP 24h). The data were normalized using the control. Error bars represent the mean ± SEM; n = 8 mice in each group. We compared the control to the Sham and CLP models using the Kruskal-Wallis test with Dunnett’s multiple comparison test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). We compared the sham operation model with the CLP model at each time point using the Student’s t-test or Mann-Whitney test according to the normality (†, p < 0.05; ††, p < 0.01; †††, p < 0.001; ††††, p < 0.0001). CLP, cecal ligation and puncture.

 SVEP1 in lung cells in sepsis

Next, we performed flow cytometric analysis to identify the dynamics of SVEP1-expressing cells at the single-cell level in the lungs after surgery. At baseline, SVEP1 was expressed on CD31-positive vascular endothelial cells, LYVE-1-positive lymphatic endothelial cells, and CD45.2-positive hematopoietic cells ( Figure 5B, D and F; Control).

Flow cytometric analysis in lung cells.

The percentage of (A) CD31 high/SVEP1 high, (C) LYVE-1 high/SVEP1 high, and (E) CD45.2 high/SVEP1 high cells was compared between control (no surgery), 2 h after sham operation (Sham 2h), 6 h after sham operation (Sham 6h), 24 h after sham operation (Sham 24h), 2 h after CLP procedure (CLP 2h), 6 h after CLP procedure (CLP 6h), and 24 h after CLP procedure (CLP 24h). Representative flow plots of (B) CD31 high/SVEP1 high, (D) LYVE-1 high/SVEP1 high, and (F) CD45.2 high/SVEP1 high are shown. The data were normalized using the control. Error bars represent the mean ± SEM; n = 8 mice in each group. We compared the control with the Sham and CLP models using the Kruskal-Wallis test with Dunnett’s multiple comparison test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). We compared the sham operation model with the CLP model at each time point using the Student’s t-test or Mann-Whitney test according to the normality (†, p < 0.05; ††, p < 0.01; †††, p < 0.001; ††††, p < 0.0001). SVEP1, sushi, von Willebrand factor type A, EGF, and pentraxin domain-containing 1; CLP, cecal ligation and puncture; LYVE-1, lymphatic vessel endothelial receptor 1.

The percentage of CD31 high/SVEP1 high vascular endothelial cells was significantly lower at 24 h after sham operation, whereas CLP surgery decreased the percentage at 2 and 6 h after the operation compared with the baseline. We found that the percentage of CD31 high/SVEP1 high cells was significantly lower at 2 and 6 h after CLP surgery compared with the sham operation ( Figure 5A and B).

The percentage of LYVE-1 high/SVEP1 high lymphatic endothelial cells tended to decrease over 24 h after sham operation without significance. By contrast, the CLP model had a decreased percentage of LYVE-1 high/SVEP1 high compared with the control at 6 and 24 h and at all time points compared with the sham operation model ( Figure 5C and D).

Although the percentage of CD45.2 high/SVEP1 high lung hematopoietic cells demonstrated no significant differences in the sham operation or CLP model compared with the control, the CLP model had a significantly higher percentage than the sham operation model at 2 and 6 h, which was followed by a return to baseline levels 24 h after the surgery ( Figure 5E and F).

 Discussion

In this study, we demonstrated that SVEP1 is highly expressed in the lungs at baseline. SVEP1 expression was decreased over the course of sepsis, with a decreased percentage of SVEP1 high vascular endothelial cells and lymphatic endothelial cells and an increased percentage of SVEP1 high hematopoietic cells.

Since the 300 kDa full-length SVEP1 protein is secreted into the extracellular matrix and degraded into a 90 kDa protein, we examined the dynamics of 90 kDa SVEP1 protein expression in the lungs after CLP. Because there were no significant changes in the amount of the 90 kDa protein, the expression was potentially regulated at the transcriptional level rather than active degradation or consumption of the extracellularly distributed SVEP1. The present study revealed that SVEP1 gene expression in the lungs decreased not only after the CLP procedure, but also after sham operation. These findings suggest that the gene expression of SVEP1 responds not only to sepsis induction, but also to surgical invasion in the early phase after surgery. In addition, the decreased gene expression of collagen1, resembling the dynamics of SVEP1, was induced by surgical invasion and sepsis. The notable downregulation of SVEP1 by surgical stimulation without sepsis is intriguing. It has been reported that cytokines, such as TNF-α, are activated after surgical invasion and induce biological responses similar to sepsis. 25 – 28 Based on our results, we believe that SVEP1 gene expression may be associated with these cytokines. Although estrogen reportedly regulates the expression of SVEP1, 29 the exact mechanisms underlying the regulation of SVEP1 remain to be determined. Our findings that SVEP1 shows similar dynamics to collagen1 following the insult could be a critical clue to clarify the mechanisms underlying the regulation of SVEP1 expression.

Since SVEP1 is highly expressed in lung tissue consisting of heterogeneous cell types, we analyzed the expression of SVEP1 at the single-cell level by flow cytometry. In accordance with the finding that SVEP1 deficiency in coronary artery disease increases endothelial CXCL1 expression and promotes plaque formation, 30 the present study suggests that SVEP1 is secreted from endothelial cells. Since SVEP1 is supplied by vascular and lymphatic endothelial cells, the decreased percentage of cells with CD31 high or LYVE-1 high/SVEP1 high after CLP surgery indicates that sepsis reduces intracellular SVEP1 protein production in the vascular and lymphatic endothelial cells or promotes the extracellular release of the protein. In terms of the reduced percentage of cells with CD31 high and LYVE-1 high after sepsis induction, the endothelial-mesenchymal transition might contribute to decreasing the overall percentage of CD31-positive cells after sepsis induction. The decreased percentage of CD31-positive cells in septic shock patients because of endothelial-mesenchymal transition supports our speculation of the altered specific population. 31

In the time course of SVEP1 expression after surgery in lung cells of mice with sepsis, an increased percentage of CD45.2 high/SVEP1 high cells indicates that hematopoietic cells containing SVEP1 migrate to the lungs during sepsis. Although blood cells reportedly express less SVEP1, 15 our results demonstrated a different pathology of SVEP1 following sepsis. Further analysis is needed to identify the localization of SVEP1 and the cells responsible for producing SVEP1.

In sepsis, endothelial hyperpermeability because of disruption of the glycocalyx layer 16 , 19 and tight junctions 17 , 32 exacerbates the pathophysiology of sepsis. Angiopoietin-1 and angiopoietin-2 act antagonistically with each other and regulate the function of tight junctions. 18 – 21 , 33 – 35 An association between decreased expression of SVEP1 and increased expression of sICAM and E-selectin in a model of endotoxemia has been reported. 24 These findings suggest that SVEP1 may regulate vascular permeability in cooperation with angiopoietin and cell adhesion molecules. Although we have not demonstrated the exact role of SVEP1 in sepsis, the time course of SVEP1 expression in vascular endothelial cells and lymphatic endothelial cells indicates that SVEP1 potentially regulates vascular permeability.

This study has several limitations. First, since we did not perform perfusion fixation prior to tissue collection, blood cells in the tissue potentially affected the whole analysis. Second, we used both male and female mice in this study; therefore, sex-associated differences might have affected the results. Third, the detailed functions of SVEP1 were not investigated in this study. Future research should clarify the significant contribution of SVEP1 in sepsis pathophysiology.

 Conclusions

SVEP1 expression was highly upregulated in the lungs compared with other organs at baseline. Sepsis suppressed SVEP1 gene expression with a decreased percentage of SVEP1 high vascular endothelial cells and lymphatic endothelial cells and an increased percentage of SVEP1 high hematopoietic cells.

 Data availability Underlying data

BioStudies: Sepsis decreases lung SVEP1 expression in a murine model. Accession number S-BSST942, https://identifiers.org/biostudies:S-BSST942. 36

This project contains the following underlying data:

SVEP1 Author Checklist – Full.pdf (completed ARRIVE checklist)

SVEP1 Data.xlsx

SVEP1 Lung GAPDH western-blot all gel.tiff

SVEP1 Lung SVEP1-90kDa western-blot all gel.tiff

SVEP1 organs GAPDH western-blot all gel.tiff

SVEP1 organs SVEP1-90kDa western-blot all gel.tiff

Acknowledgments

The authors thank A. Goda and F. Iida for their technical support.

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Competing interests: No competing interests were disclosed.

 18 8 2023 Copyright: © 2023 Kessler T 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

I had the opportunity to review the paper by Kurita and colleagues. The authors investigate the role of the protein SVEP1 in a murine model of sepsis. It needs to be taken into account that not much is known about SVEP1. It was associated with sepsis before but also coronary artery disease. Here, there are contradictory hypotheses which are based on in vivo studies in mice. In my opinion, a key question regarding all SVEP1 studies is how its expression behaves under baseline as compared to disease conditions. As a matter of fact, most of the SVEP1 that is found in the circulation might be produced by fat tissue. However, the role of circulating SVEP1 also remains elusive.

The authors add an important point to the SVEP1 research when they show that in sepsis, SVEP1 expression is reduced in lung tissue. A reduction of SVEP1 expression might indeed lead to leakage and characteristic sepsis phenotypes and might be comparable to what happens in atherosclerosis.

Major:

The manuscript is very focused. It is of descriptive nature and mechanistic insight is lacking. I am in particular not persuaded by the analyses of the SVEP1 90 kDa fragment.

Vascular tissue should be included in the gene expression analysis.

The protein analysis in Fig. 3 is not as clear as the mRNA analysis. What is the half-life of SVEP1?

The rationale to also investigate collagen 1 and fibronectin remains unclear. Further ECM proteins should be included.

The FC data in Fig. 5 is limited by the poor quality of SVEP1 antibodies and that they are not able to differentiate between full-length SVEP1 and fragments.

 Minor:

Contradictory results in atherosclerosis should be discussed.

Grammar and orthography should be re-checked.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Molecular biology, cardiovascular sciences

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

 10.5256/f1000research.141231.r189451 Reviewer response for version 1 Morris Gavin E 1 Referee https://orcid.org/0000-0003-4054-6959 University of Leicester, Leicester, UK

Competing interests: No competing interests were disclosed.

 18 8 2023 Copyright: © 2023 Morris GE 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

Kurita and co-authors present the results of a study that investigates the expression of SVEP1 in various organs of mice at baseline and during a model of sepsis. The study concludes that SVEP1 expression changes in SVEP1-expressing cells in the lungs during sepsis. To model sepsis, the authors perform a cecal ligation and puncture (CLP) procedure and examined the effect of CLP upon SVEP1 expression using various gene and protein expression techniques. They find that SVEP1 expression is highest in the lung and spleen at baseline, with significant SVEP1 protein detected in the lung. During sepsis, SVEP1 gene expression decreased in the lungs at different time points. This decrease in SVEP1 expression may associate with changes in specific cell populations; however, further control experiments need to be performed before this can be stated. The study also reveals that SVEP1 protein expression remained relatively stable in the lungs during sepsis, suggesting that SVEP1 expression is regulated at the transcriptional level. Interestingly, surgical invasion without sepsis also led to the downregulation of SVEP1 gene expression, indicating its response to surgical stimuli.

This study is part of the group’s ongoing research on the polymorphism in SVEP1 as a genetic risk factor for sepsis, and begins to provide a mechanistic understanding to this genetic association. The findings indicate dynamic regulation of SVEP1 in response to sepsis, supports the validity to the model for further investigations into how SVEP1 specifically contributes to cell inflammatory responses, sepsis, lung function and pathology.

However, there are some concerns and suggestions for the authors to consider:

Figure 1: While representative blots are shown in B, there is no cumulative data presented as a bar chart (as in Figure 3) to quantitatively demonstrate SVEP1 expression across the organ types.

In all figure legends, there are P values stated that are not expressed in the data presented (e.g. **** P<0.0001)

The western blots throughout are cropped significantly, and there are no visible ladders. It would be beneficial to include the antibody catalogue numbers (for westerns and flow cytometry) for others who might wish to reproduce these data with these antibodies.

Given that SVEP1 is a ligand for integrin α4β1 and α9β1, and that integrins are targets of several pathogen-host interactions, it would be interesting to examine whether the expression of these integrins is also altered upon CLP.

Major concerns regarding the presented flow cytometric data:

The methods section does not specify whether the CD45.2, CD31 or LYVE-1 antibodies were conjugated-primary antibodies, or which secondary antibodies were used (catalogue numbers would help).

It is unclear why the samples were permeabilised since the proteins are expressed on the cell surface. The use of the Foxp3/transcription factor staining set also appears irrelevant here.

The data in Figure 5 requires reassurance that the data presented are not due to technical artifacts:

including a live/dead dye would remove dead cells prior to analysis. There is no indication that doublets or red blood cells were removed prior to analysis. Alternatively, staining the nuclei with DAPI (or similar) would serve as a universal control for the cell population, ensuring only nucleated cells are included in the analysis.

Technical controls such as unlabelled cells (to show any autofluorescence) and IgG control antibodies (to show non-specific antibody binding) should be included to ensure cleaner populations for analysis. These will give reassurance that proper channel compensation has been performed to avoid fluorescence emission bleeding into other channels.

The cytometry axes are in arbitrary units and are (at best) semi-quantitative. The term “high” expression does not seem to correlate to the fluorescence intensity range within each gate, which makes further analysis redundant. In addition, the use of the term high, medium or low is usually relative to another population of the same (or similar) cell type. It is unclear how the thresholds were determined to categorize e.g. CD45.2+ cells as “high” for SVEP1 expression. Technical controls for each antibody will help determine the threshold in both x and y axis. There also seems to be very few truly high SVEP1 expressing cells in any cell population. For example, the alterations in event percentages in panels D&F do not seem to be due to any high expressing SVEP1 events, but just more LYVE-1 negative (panel D) or CD45.2+ events (panel F).

The alterations in cell percentages may be meaningless without control over identifying, quantifying, or accounting for the denominator populations. For example, in panel D, have the CD45.2+ events and instrument “noise” been removed from the analysis so that they do not confound the interpretation? Additional controls should be performed and included (see above) before inferring alterations in specific cell types.

Determining the smooth muscle population and examining whether SVEP1 expression is altered in this cell type could provide additional insight, with SVEP1 highly expressed in this cell type within the vasculature.

The introduction and results sections are clearly written, but the discussion section needs improvement. The authors make statements or link the current results to the findings of other studies without performing the necessary experiments to validate these claims: i) While the authors “believe SVEP1 gene expression may be associated with these cytokines” due to TNFα activation after surgical invasion, no experiments showing any association between SVEP1 and pro-inflammatory cytokine levels are presented. ii) The significance of both SVEP1 and collagen I gene expression decreasing post CLP as a “critical clue to clarify the mechanisms underlying the regulation of SVEP1 expression” needs to be explained more clearly. iii) The claim that the present study “suggests SVEP1 is secreted from endothelial cells” should be supported by experimental data. iv) Although the current study shows an alteration in SVEP1 expression in vascular endothelial cells, there is no experimental data linking SVEP1 and vascular permeability, and this should be made clear.

 By addressing these concerns and suggestions, the authors can significantly strengthen their study and contribute valuable insight into SVEP1 expression within the lung in sepsis.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

No

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

I have experience in working with SVEP1 and flow cytometry.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.5256/f1000research.141231.r189449 Reviewer response for version 1 Elenbaas Jared S 1 Referee Patel Ved 1 Co-referee Washington University School of Medicine The Dominantly Inherited Alzheimer Network, St. Louis, Missouri, USA

Competing interests: No competing interests were disclosed.

 18 8 2023 Copyright: © 2023 Elenbaas JS and Patel V 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

This study by Kurita et al. is designed to determine the expression of SVEP1 in pulmonary tissue of mice under different conditions that may relate to the protein's disease associations. This is a clear question that could provide meaningful insight into the diseases that associate with SVEP1, including sepsis.

The manuscript is well-written, and the statistics are acceptable, except in the cases outlined below. Unfortunately, we have a number of significant concerns regarding the methodology. These cases of inadequate methodology may undermine the major conclusions of the manuscript.

Major concerns:

Several experiments rely on an antibody that has not been rigorously validated. Tissues and/or cells from animals lacking SVEP1 are critical for determining the specificity of the antibody in the respective tissue. The full gel images that the authors provide suggest the antibody is widely non-specific, and it is not clear that the band corresponding to 90kDa is truly staining SVEP1. Please see citation for appropriate antibody validation.

GAPDH is used as a reference gene for qPCR, as well as for immunoblot assays in the manuscript. Although GAPDH is frequently used as a reference gene, it is well-recognized that it is not appropriate in certain conditions, including sepsis. Please see citation. Indeed, figure 3 suggests GAPDH may increase in their CLP model. A reference gene and loading control that does not change in response to sham and CLP treatments are necessary to interpret the experiments.

The authors use flow cytometry to identify changes in SVEP1-expressing cells. The signal along the x-axis purportedly represents SVEP1 staining, yet there is no  separation between SVEP1+ and SVEP1- cells, suggesting the staining is non-specific. Neither endothelial cells, nor hematopoietic cells are thought to be major sources of SVEP1 expression, further raising concern about the specificity of the antibody (see point 1). Knockout or heterozygous mouse tissues should be used to validate this assay.

 Minor concerns:

Please clarify what the positive control material is in Figure 1.

Please clarify the significance of comparing SVEP1 expression to collagen1 and fibronectin. Why were these two proteins studied?

The statement beginning with "the negative impact of genetic polymorphisms of SVEP1..." has unclear meaning. Are the authors implying that these polymorphisms are loss of function?

Please clarify the significance/relevance of the claim, "Based on our results, we believe that SVEP1...".

The authors' claim that "SVEP1 gene expression returned to baseline levels at 6 and 24 h after sham operation", but do not use appropriate statistical tests to make this claim. Since they are claiming a decrease at 2 h, they should also test whether 6 and 24 h are significantly different than 2h to support their claim about returning to baseline.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

No

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

SVEP1, animal models, translational research.

We confirm that we have read this submission and believe that we have an appropriate level of expertise to state that we do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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