Female sex protects against renal edema, but not lung edema, in mice with partial deletion of the endothelial barrier regulator Tie2 compared to male sex

Anoek L I van Leeuwen; Elise Beijer; Roselique Ibelings; Nicole A M Dekker; Marjolein R A van der Steen; Joris J T H Roelofs; Matijs van Meurs; Grietje Molema; Charissa E van den Brom

doi:10.1371/journal.pone.0293673

. 2023 Nov 16;18(11):e0293673. doi: 10.1371/journal.pone.0293673

Female sex protects against renal edema, but not lung edema, in mice with partial deletion of the endothelial barrier regulator Tie2 compared to male sex

Anoek L I van Leeuwen ^1,², Elise Beijer ^1,^3,⁴, Roselique Ibelings ^1,⁴, Nicole A M Dekker ¹, Marjolein R A van der Steen ¹, Joris J T H Roelofs ⁵, Matijs van Meurs ^6,⁷, Grietje Molema ⁷, Charissa E van den Brom ^1,^4,^8,^*

Editor: Keiko Hosohata⁹

PMCID: PMC10653528 PMID: 37972011

Abstract

Background

The endothelial angiopoietin/Tie2 system is an important regulator of endothelial permeability and targeting Tie2 reduces hemorrhagic shock-induced organ edema in males. However, sexual dimorphism of the endothelium has not been taken into account. This study investigated whether there are sex-related differences in the endothelial angiopoietin/Tie2 system and edema formation.

Methods

Adult male and female heterozygous Tie2 knockout mice (Tie2^+/−) and wild-type controls (Tie2^+/+) were included (n = 9 per group). Renal and pulmonary injury were determined by wet/dry weight ratio and H&E staining of tissue sections. Protein levels were studied in plasma by ELISA and pulmonary and renal mRNA expression levels by RT-qPCR.

Results

In Tie2^+/+ mice, females had higher circulating angiopoietin-2 (138%, p<0.05) compared to males. Gene expression of angiopoietin-1 (204%, p<0.01), angiopoietin-2 (542%, p<0.001) were higher in females compared to males in kidneys, but not in lungs. Gene expression of Tie2, Tie1 and VE-PTP were similar between males and females in both organs. Renal and pulmonary wet/dry weight ratio did not differ between Tie2^+/+ females and males. Tie2^+/+ females had lower circulating NGAL (41%, p<0.01) compared to males, whereas renal NGAL and KIM1 gene expression was unaffected.

Interestingly, male Tie2^+/- mice had 28% higher renal wet/dry weight ratio (p<0.05) compared to Tie2^+/+ males, which was not observed in females nor in lungs. Partial deletion of Tie2 did not affect circulating angiopoietin-1 or angiopoietin-2, but soluble Tie2 was 44% and 53% lower in males and females, respectively, compared to Tie2^+/+ mice of the same sex. Renal and pulmonary gene expression of angiopoietin-1, angiopoietin-2, estrogen receptors and other endothelial barrier regulators was comparable between Tie2^+/- and Tie2^+/+ mice in both sexes.

Conclusion

Female sex seems to protect against renal, but not pulmonary edema in heterozygous Tie2 knock-out mice. This could not be explained by sex dimorphism in the endothelial angiopoietin/Tie2 system.

Introduction

Sex-related differences in outcome of traumatic hemorrhagic shock (THS) patients gained more attention since 2010. Although THS remains associated with high mortality, several studies have demonstrated a protective effect of female gender in response to THS [1–5]. Female gender is associated with a 2% decreased risk of acute kidney injury [2–5], a 9% decreased risk of lung injury [5] and a 14–21% decreased mortality rate in patients following THS [2, 6–8]. To date, sex-related differences in outcome following THS are unexplained and understanding the differences between male and female THS patients could contribute to the development of novel treatment strategies.

THS causes a systemic inflammatory response that activates the endothelium [9]. This results in increased endothelial permeability, leakage of fluids into the interstitium and tissue edema [10]. We have previously shown that plasma from male THS patients induces endothelial hyperpermeability in vitro [11] and that THS in male rats induces vascular leakage in both lung and kidney [12]. One of the molecular systems involved in regulation of endothelial permeability is the endothelial angiopoietin/Tie2 system (Fig 1).

Fig 1 — In quiescence (A), angiopoietin-1 (ang-1) is released from pericytes and activates, and thereby phosphorylates tyrosine kinase receptor Tie2. Activation of Tie2 strengthens endothelial barrier function via Rac-1/Rho kinase/vascular endothelial (VE)-cadherin signaling. In contrast, in response to hemorrhagic shock (B) angiopoietin-2 (ang-2) is rapidly released from weibel palade bodies (WPB), leading to increased endothelial permeability via inhibition of Tie2 activation. Other transmembrane proteins that affect Tie2 phosphorylation include Tie1 and vascular endothelial-protein tyrosine phosphatase (VE-PTP), which both inhibit Tie2 phosphorylation upon a stress stimulus.

The activation of the endothelial receptor Tie2 is tightly controlled by the interplay between the paralogous endothelial receptor Tie1, the tyrosine phosphatase vascular endothelial protein tyrosine phosphatase (VE-PTP), and two secreted ligands, angiopoietin-1 and angiopoietin-2 [13]. The latter can act as agonist, partial agonist, or antagonist depending upon context [14]. In healthy conditions, angiopoietin-1 phosphorylates and thereby activates the endothelial Tie2 receptor, which assists in maintaining endothelial barrier function via inhibition of RhoA kinase and activation of small GTPase Rac-1 [15, 16]. During stress, circulating angiopoietin-2 and the expression of the VE-PTP receptor are increased. Both can inhibit phosphorylation and thereby prevent activation of Tie2. This consequently disrupts the endothelial barrier with vascular leakage and edema as a result [15, 17]. Additionally, Tie2 signaling and endothelial barrier function can be affected by other receptors, such as integrin α5β1, and vascular endothelial growth factor (VEGF). VEGF can increase permeability via inhibition of vascular endothelial (VE)-cadherin adherens junctions [18], and is a known promotor of shedding of the Tie2 receptor [19]. On the other hand integrin α5β1 promotes Tie2 phosphorylation when angiopoietin-1 levels are low [20].

We and others have previously shown that THS disturbs endothelial angiopoietin/Tie2 signaling, including reduced expression of the endothelial Tie2 receptor in the kidney and lungs [12, 21] and increased circulating levels of angiopoietin-2 [12]. Interestingly, we have been able to reduce vascular leakage by pharmacologically activating endothelial Tie2 in male THS rats [12], suggesting that targeting Tie2 is a promising strategy to reduce organ injury [22, 23]. Unfortunately, sexual dimorphism of the endothelium in THS has not been taken into account [24]. Here, we investigated the effect of sex on renal and pulmonary edema in heterozygous mice with genetically reduced endothelial Tie2 expression thereby mimicking the suppressive effect of THS on expression of the endothelial Tie2 receptor. In addition, we investigated whether expression of components of the endothelial angiopoietin/Tie2 system in kidneys and lungs differs between healthy female and male mice.

Materials and methods

Study approval

Analyses were performed on tissue samples of terminated animals from breeding surplus provided by the Amsterdam Animal Research Center of the VU University Amsterdam, the Netherlands. These experiments do not require an animal research permit from the Central Committee for Animal Experiments (Centrale Commissie Dierproeven) in the Netherlands. These experiments make an important contribution to the replacement and reduction principles in animal experimentation according to animal welfare regulations in full agreement with the Directive 2010/63/EU. All procedures were conducted following the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes and the ARRIVE 2.0 guidelines on animal research [25]. Euthanasia was performed under isoflurane anesthesia, and all efforts were made to minimize suffering.

Animals and genotyping

Heterozygous Tie2 knockout mice (exon 9 deletion, C57BL6/J background) were generated by Jongman et al. and bred as previously described [26]. In the current study, heterozygous Tie2 knockout mice (Tie2^+/-) with partially reduced Tie2 expression and wild-type littermate controls (Tie2^+/+) with normal Tie2 expression were included.

Mouse genomic DNA was extracted from ear punches using standard protocols. The genotype of Tie2^+/− mice was determined by PCR using the primers 5′-GGGCTGCTACAATAGCTTTGG-3′ and 5′-GTTATGTCCAGTGTCAATCAC-3′ resulting in a 644 bp PCR product when exon 9 is still present (Tie2^+/+) and in a 309 bp PCR product when exon 9 of Tie2 was excised by Cre-recombinase (Tie2^+/−). PCR products were run on a 1.5% (w/v) agarose gel in Tris-borate-EDTA-buffer with 0.005% (v/v) ethidium bromide, and visualized under UV light (S1 Fig).

Experimental set-up

Male and female mature adult mice from breeding surplus (bred in the animal facility of the VU University, Amsterdam, the Netherlands, generation N2/F3 and N2/F4) were included at the age of 3 to 6 months. Mature mice of at least 3 months old are considered sexual mature and represent humans between 20 and 30 years of age [27]. Mice were housed in a temperature-controlled room (12/12 h day/night cycle, 20–23 °C, 40–60% humidity) with food (Teklad global 18% protein, catalog nr 2918CS, Envigo, Indianapolis, USA) and autoclaved tap-water ad libitum. Mice were terminated via decapitation after sedation with 5% isoflurane in air at the animal facility. For both sexes, Tie2^+/+ and Tie2^+/- mice were included (n = 9 per group). Upon sacrifice, the right kidney and lung were used to determine edema formation by wet/dry weight ratio. The left kidney and lung were snap frozen in liquid nitrogen and stored at -80°C for further analyses. Whole blood was collected in heparin tubes, centrifuged twice at 4°C (10 min at 4.000×g and 15 min at 10.000×g) to obtain platelet free plasma and stored at -80°C. The investigators (AvL, NAMD, MRAvdS, JJTHR) performing the ex vivo analyses were blinded to group allocation.

Renal and pulmonary edema formation

Renal and pulmonary tissue was harvested immediately upon sacrifice. Wet tissue was weighed and subsequently dried at 70°C. After 24 hours, dry tissue was weighed and wet/dry weight ratio was calculated as estimate of tissue water content.

Plasma analyses

Levels of circulating angiopoietin-1 (LS-F2956, LSBio, Seattle, USA), angiopoietin-2 (MANG20, R&D systems, Minneapolis, MN, USA), soluble Tie2 (MTE200, R&D systems, Minneapolis, MN, USA), and neutrophil gelatinase-associated lipocalin (NGAL; KIT042, Bioporto, Hellerup, Denmark) were measured with Enzyme-linked immunosorbent assay (ELISA) in accordance to the manufacturer.

RNA analyses

Total RNA was extracted from 10–20 mg frozen whole kidney and lung tissue and isolated using the RNeasy mini kit (Qiagen, Venlo, the Netherlands) as previously described [12]. RNA concentration and purity were determined using NanoDrop 1000 (NanoDrop Technologies, Wilmington, DE, USA). A total of 1 μg RNA was transcribed into complementary DNA using an iScript cDNA synthesis kit (Bio-Rad, Veenendaal, the Netherlands) using oligo-dT priming. mRNA abundance was measured using a CFX384 Touch real-time PCR detection system (Bio-Rad, Veenendaal, the Netherlands). mRNA abundance of angiopoietin-1, angiopoietin-2, Tie2, Tie1, vascular endothelial protein tyrosine phosphatase (VE-PTP), estrogen receptor α, estrogen receptor β, integrin α5, integrin β1, Rac1, RhoA, vascular endothelial growth factor α (VEGFα), kidney injury molecule 1 (KIM1), neutrophil gelatinase-associated lipocalin (NGAL) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems, Foster City, CA) was determined using assay-on-demand primers/probe sets (TaqMan Gene Expression, Thermo Fisher Scientific, Bleiswijk, The Netherlands; Table 1). Gene expression was normalized to the expression of the housekeeping gene GAPDH, yielding the ΔCT value.

Table 1. Real time-qPCR primers.

Gene	Assay ID	Encoded protein
Gapdh	Mm99999915_g1	Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Angpt1	Mm00456503_m1	Angiopoietin-1
Angpt2	Mm00545822_m1	Angiopoietin-2
Tek	Mm00443243_m1	Endothelial-specific receptor tyrosine kinase (Tie2)
Tie1	Mm00441786_m1	Tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (Tie1)
Ptprb	Mm00459467_m1	Protein tyrosine phosphatase, receptor type, B (VE-PTP)
Esrra	Mm00433143_m1	Estrogen related receptor, alpha
Esrrb	Mm00442411_m1	Estrogen related receptor, beta
Itga5	Mm00439797_m1	Integrin alpha 5 (fibronectin receptor alpha)
Itgb1	Mm01253230_m1	Integrin beta 1 (fibronectin receptor beta)
Rac1	Mm01201653_mH	RAS-related C3 botulinum substrate 1
RhoA	Mm00834507_g1	Ras homolog gene family, member A
Vegfa	Mm00437306_m1	Vascular endothelial growth factor A
Havcr1	Mm00506686_m1	Hepatitis A virus cellular receptor 1 (Havcr1), Kidney injury marker 1 (KIM-1)
Lcn2	Mm01324470_m1	Lipocalin 2 (NGAL)

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Histology

Four-micrometer-thick paraffin sections of lung and kidney tissue were stained with hematoxylin and eosin. Pulmonary sections were scored in a semi-quantitative fashion on a scale of 0–3 for the following parameters: perivascular edema, alveolar edema and interstitial inflammation. Kidney tissue was assessed for interstitial edema and signs of tubular injury on a scale of 0–3. A score of 0 represented normal tissue, less than 25% presence of above described parameters; 1 represented mild, 25 to 50%; 2 represented moderate, 50 to 75%; 3 represented severe, more than 75% presence.

Statistical analysis

The normality of data distribution was determined using the Shapiro-Wilk test. As data appeared non-normally distributed, all data are expressed as median with interquartile range and analyzed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). Primary outcome was renal edema formation. At least an increase in wet/dry weight ratio from 3.6 ± 0.4 to 4.1 was expected based on pilot experiments. With a two-sided significance level (α) of 0.05 and β of 0.9, group sizes of 9 mice were calculated to reach statistical significant differences. For every experimental group, all 9 mice were included for analysis. Sex differences in male and female Tie2^+/+ and Tie2^+/- mice were evaluated by Kruskal-Wallis testing with Dunn’s post hoc analyses. P values less than 0.05 were considered as statistically significant. The experimental data that support the findings of this study are available in DANS EASY with the identifier https://doi.org/10.17026/dans-zea-fs7p [28].

Results

Sex differences in wild type mice with normal Tie2 expression

We first investigated whether sex-related differences exist in mice with normal Tie2 expression by comparing male and female Tie2^+/+ mice. In general, female mice had a significantly lower body weight (23.0 [21.5–24.2] vs. 27.9 [26.7–36.3] gram, p = 0.0031) compared to male mice.

To provide insight in basal expression levels of the endothelial angiopoietin/Tie2 system, we determined whether circulating and renal and pulmonary gene expression levels of key molecules involved in endothelial angiopoietin/Tie2 signaling were differently expressed between males and females. In wild-type mice, circulating levels of angiopoietin-1 (p = 0.06) and angiopoietin-2 were higher in females compared to males (Fig 2A and 2B), whereas angiopoietin-2/angiopoietin-1 ratio or soluble Tie2 levels were comparable between males and females (Fig 2C and 2D).

Fig 2 — Circulating levels of angiopoietin-1 (A), angiopoietin-2 (B), angiopoietin-2/1 ratio (C) and soluble Tie2 (D) in plasma of Tie2^+/+ male (white bars), Tie2^+/+ female (grey bars), Tie2^+/- male (striped white bars) and Tie2^+/- female (striped grey bars) mice. Each dot represents an individual mouse. Data are presented as median with interquartile range. * p<0.05, ** p<0.01, *** p<0.001.

Renal gene expression of angiopoietin-1 and angiopoietin-2 was higher in females compared to males (Fig 3A and 3B). Renal gene expression of Tie2 and Tie1 was also higher in females compared to males, however, both did not reach statistical significance (both p = 0.09, Fig 3C and 3D), whereas renal VE-PTP gene expression did not differ between female and male mice (Fig 3E). No differences were found between male and female mice with regard to pulmonary gene expression of angiopoietin-1, angiopoietin-2, Tie2, Tie1 or VE-PTP (Fig 4A–4E).

Fig 4 — Pulmonary gene expression of angiopoietin-1 (A), angiopoietin-2 (B), Tie2 (C), Tie1 (D) and vascular endothelial protein tyrosine phosphatase (VE-PTP; E) in Tie2^+/+ male (white bars), Tie2^+/+ female (grey bars), Tie2^+/- male (striped white bars) and Tie2^+/- female (striped grey bars) mice. Each dot represents an individual mouse. Data are presented as median with interquartile range.

Next, we have investigated whether basal differences exist between male and female mice in commonly used markers of organ injury, such as renal and pulmonary edema formation and renal ischemia (NGAL and KIM-1). Renal and pulmonary wet/dry weight ratio, as measure of organ edema, did not differ between female and male mice (Fig 5A and 5B). Basal circulating levels of NGAL were lower in females compared to males (Fig 5E). Renal gene expression of NGAL was higher and renal gene expression of KIM1 was lower in females compared to males, however, both did not reach statistical significance (p = 0.06 and p = 0.09, respectively; Fig 5F and 5G). Histopathological analysis revealed no renal or pulmonary damage for both sexes (Fig 5C and 5D).

Fig 5 — Renal wet/dry weight ratio (A), pulmonary wet/dry weight ratio (B), H&E stainings of one typical example of each group in kidney (C) and lung (D), circulating plasma levels of neutrophil gelatinase-associated lipocalin (NGAL; E) and renal gene expression of NGAL (F) and kidney injury molecule 1 (KIM1; G) in Tie2^+/+ male (white bars), Tie2^+/+ female (grey bars), Tie2^+/- male (striped white bars) and Tie2^+/- female (striped grey bars) mice. Each dot represents an individual mouse. Data are presented as median with interquartile range. * p<0.05, ** p<0.01, *** p<0.001.

As a first step in finding an explanation for the sex-differences in the endothelial angiopoietin/Tie2 system, we determined renal and pulmonary estrogen gene expression levels. Renal and pulmonary gene expression of estrogen receptor α was comparable between males and females (Figs 6A and 7A). Gene expression of estrogen receptor β was higher in female than in male kidneys (Fig 5B), whereas no differences were found between sexes in lungs (Fig 7B).

Fig 6 — Renal gene expression of estrogen receptor α (A), estrogen receptor β (B), integrin α5 (C), integrin β1 (D), vascular endothelial growth factor α (VEGFα; E), RhoA (F) and Rac1 (G) in Tie2^+/+ male (white bars), Tie2^+/+ female (grey bars), Tie2^+/- male (striped white bars) and Tie2^+/- female (striped grey bars) mice. Each dot represents an individual mouse. Data are presented as median with interquartile range. * p<0.05, *** p<0.001.

Fig 7 — Pulmonary gene expression of estrogen receptor α (A), estrogen receptor β (B), integrin α5 (C), integrin β1 (D), vascular endothelial growth factor α (VEGFα; E), RhoA (F) and Rac1 (G) in Tie2^+/+ male (white bars), Tie2^+/+ female (grey bars), Tie2^+/- male (striped white bars) and Tie2^+/- female (striped grey bars) mice. Each dot represents an individual mouse. Data are presented as median with interquartile range. ** p<0.01.

Additionally, as we found differences in basal expression levels in the angiopoietin/Tie2 system, we were interested whether other (downstream) endothelial barrier regulators differed between sexes due to the complex interaction with the endothelial angiopoietin/Tie2 system. Renal gene expression of vascular endothelial growth factor (VEGF) α was higher in females compared to males (Fig 6E). Renal gene expression of integrin α5, integrin β1, RhoA and Rac1 did not differ between females and males (Fig 6C, 6D, 6F and 6G). In pulmonary tissue, no differences in gene expression were found between males and females for integrin α5, integrin β1, VEGFα, RhoA and Rac1 (Fig 7C–7G).

Sex differences in heterozygous Tie2^+/- mice with partial deletion of Tie2

Following the confirmation of sex-related differences in basal expression of molecules involved in endothelial angiopoietin/Tie2 signaling, we subsequently investigated the effect of sex in heterozygous mice with lower basal endothelial Tie2 expression (Tie2^+/-), thereby mimicking the suppressive effect of THS on the endothelial Tie2 receptor. First, we confirmed the heterozygous Tie2 knockout by determining renal and pulmonary gene expression levels of Tie2. Renal Tie2 gene expression was reduced by 48% in heterozygous male and 60% in heterozygous female mice (Fig 3C). Pulmonary Tie2 gene expression was reduced by 51% in heterozygous male and 41% in heterozygous female mice (Fig 4C).

Next, we identified whether Tie2 heterozygosity resulted in differences in body weight between males and females. In Tie2^+/- mice, body weight was similar to Tie2^+/+ controls for both males (31.1 [30.2–33.5] vs. 27.9 [26.7–36.3] gram, p>0.99) and females (24.6 [21.9–28.3] vs. 23.0 [21.5–24.2] gram, p = 0.79). Similar to Tie2^+/+ controls, Tie2^+/- females had a lower body weight compared to Tie2^+/- males (24.6 [21.9–28.3] vs. 31.1 [30.2–33.5] gram, p = 0.05).

We also determined expression of molecules involved in endothelial angiopoietin/Tie2 signaling in heterozygous knockout of Tie2 and whether differences exist between males and females. Circulating levels of angiopoietin-1 did not differ between Tie2^+/+ and Tie2^+/- mice in both males and females (Fig 2A). Likewise, circulating plasma levels of angiopoietin-2 were not affected by heterozygosity, nevertheless they were higher in Tie2^+/- females compared to Tie2^+/- males (Fig 2B). This resulted in similar angiopoietin-2/angiopoietin-1 ratios in both sexes, which were not affected by partial deletion of Tie2 (Fig 2C). Soluble Tie2 levels were decreased in both female and male Tie2^+/- mice compared to Tie2^+/+ female and male mice (Fig 2D).

Renal gene expression of angiopoietin-1, angiopoietin-2, Tie1 and VE-PTP did not differ between Tie2^+/+ and Tie2^+/- in both female and male mice (Fig 3A, 3B, 3D and 3E), whereas renal angiopoietin-1 and angiopoietin-2 gene expression was higher in Tie2^+/- females compared to Tie2^+/- males (Fig 3A and 3B). No differences in pulmonary gene expression were found for angiopoietin-1, angiopoietin-2, Tie1 or VE-PTP between Tie2^+/+ and Tie2^+/- in both female and male mice (Fig 4A, 4B, 4D and 4E).

As THS is associated with reduced Tie2 expression and edema formation in kidneys and lungs, we subsequently investigated whether partial deletion of Tie2 resulted in renal or pulmonary edema and whether sex-related differences exist in renal or pulmonary edema and expression of renal ischemia markers in mice with lowered Tie2 expression. Tie2^+/- males had higher renal wet/dry weight ratios compared to Tie2^+/+ males (Fig 5A). Interestingly, this higher renal wet/dry weight ratio was not observed in female Tie2^+/- mice compared to Tie2^+/+ females (Fig 5A). In lungs, no differences in wet/dry weight ratio were found between all groups (Fig 5B). Histopathological analysis revealed no renal or pulmonary damage for all groups (Fig 5C and 5D). Circulating levels of NGAL did not differ between Tie2^+/- and Tie2^+/+ mice in both males and females (Fig 5E). However, female Tie2^+/- mice had significantly lower circulating levels of NGAL compared to Tie2^+/- males (Fig 5E). Renal gene expression of NGAL and KIM1 did not differ between Tie2^+/- and Tie2^+/+ mice of both sexes (Fig 5F and 5G), whereas female Tie2^+/- mice had significantly lower gene expression levels of KIM1 compared to Tie2^+/- male mice (Fig 5G).

As a first step in finding an explanation for the suggested protective effect of female sex in renal, but not pulmonary edema formation in heterozygous Tie2 knockout mice, we determined whether expression levels of estrogen receptors and receptors that closely interact with the endothelial angiopoietin/Tie2 system differ between male and female Tie2^+/- mice. Renal gene expression levels of estrogen receptor β did not differ between Tie2^+/- and Tie2^+/+ mice, but were higher in female Tie2^+/- mice compared to Tie2^+/- male mice (Fig 6B), whereas expression of estrogen receptor α was comparable (Fig 6A). No differences were found in pulmonary gene expression of estrogen receptor α or β (Fig 7A and 7B). Renal gene expression of integrin α5, integrin β1, VEGFα, RhoA and Rac1 was not affected by sex and did not differ between Tie2^+/- and Tie2^+/+ mice (Fig 6C–6G). In lungs, RhoA gene expression was higher in female Tie2^+/- mice compared to female Tie2^+/+ mice (Fig 7F), whereas gene expression of integrin α5, integrin β1, VEGFα, and Rac1 was not affected by sex and did not differ between Tie2^+/- and Tie2^+/+ mice (Fig 7C–7E and 7G).

Discussion

Female sex is suggested to be a potential advantage in outcome following THS [1–5]. Endothelial hyperpermeability contributes to THS-induced acute kidney and lung injury. Endothelial permeability is, amongst others, regulated by the endothelial angiopoietin/Tie2 system. During THS, the endothelial angiopoietin/Tie2 system is disbalanced and pharmacological activation of Tie2 reduces THS-induced vascular leakage in males. However, sexual dimorphism of the endothelium has not been taken into account. In the current study, we investigated whether sex-related differences exist in basal expression of the endothelial angiopoietin/Tie2 system. Additionally, we investigated whether reduced Tie2 expression, as seen during THS, resulted in a sex-dependent change in expression levels of the endothelial angiopoietin/Tie2 system and whether it results in renal or pulmonary injury. To our knowledge, we are the first to show the existence of sexual dimorphism in the basal expression levels of key molecules involved in endothelial angiopoietin/Tie2 signaling in healthy mature mice as reflected by higher expression of angiopoietin-1 and angiopoietin-2 in plasma and kidney, but not lungs, in females compared to males. Genetically reduced Tie2 expression did not affect proteins and genes involved in the endothelial angiopoietin/Tie2 system. Interestingly, heterozygous male mice with partial deletion of Tie2 showed renal edema, while this was not observed in females. These differences could not be explained by sex-related differences in the endothelial angiopoietin/Tie2 system. Interestingly, pulmonary edema was comparable between all groups and no difference in angiopoietin/Tie2 expression was found in lungs, which suggests also organ-specific regulations of endothelial angiopoietin/Tie2 signaling. Future studies are warranted to elaborate on the relation between sex differences and angiopoietin/Tie2 signaling in the development of organ edema.

The endothelial angiopoietin/Tie2 system is involved in the regulation of endothelial permeability, and it is not known whether sex affects expression of components of this system. In the present study, we showed that sex differences in basal expression levels of the endothelial angiopoietin/Tie2 system exist. Circulating levels of angiopoietin-2 were higher and soluble levels of Tie2 were lower in females compared to males. This is in agreement with the findings of a community-based cohort study of almost 4000 participants in which females also showed higher circulating angiopoietin-2 and lower soluble Tie2 levels compared to males [29]. In our study, both circulating angiopoietin-1 and angiopoietin-2 levels were higher in females compared to males and therefore the angiopoietin-2/angiopoietin-1 ratio was comparable between males and females, suggesting that Tie2 is activated to a similar extend by the angiopoietins in males and females. However, lower soluble Tie2 levels in females suggest less shedding of the Tie2 receptor, while increased shedding has previously been linked to inflammation and complement activation in septic patients [30]. As far as we know, we are the first to show sexual dimorphism of the endothelial angiopoietin/Tie2 system in healthy conditions. Sexual dimorphism in the endothelial angiopoietin/Tie2 system and corresponding explanations are limitedly reported. One of the basal key differences between females and males are sex hormones, which most often emerge around the time of sexual maturation. Interestingly, a potential advantage of female gender in THS seems specifically during their reproductive phase [2, 5, 6], suggesting a role for estrogen. Due to limited plasma volumes, we were not able to measure circulating estrogen in the current study, but based on the age of mice we expect that estrogen levels were higher in female compared to male mice [27, 31]. Estrogens may regulate the endothelial angiopoietin/Tie2 system, however, contrasting results exist. As determined in the endometrium of ovariectomized animals, systemic administration of estrogen increases gene expression of angiopoietin-1, but not angiopoietin-2 or Tie2 [32], whereas longer exposure to estrogen reduces angiopoietin-1 and Tie2, but increased angiopoietin-2 expression [33]. In renal tissue of ovariectomized rats, Ye et al. showed that estrogen reduced angiopoietin-1 and increased angiopoioetin-2 gene expression, but did not affect Tie2 gene expression [34]. These apparent conflicting results might be explained by differences in exposure time to estrogen, organ systems and the fact that only ovariectomized animals were studied. In the current study we report higher renal estrogen receptor β expression in females, which is previously shown to increase angiopoietin-2 expression [35]. Interestingly, we found no differences between males and females in endothelial angiopoietin/Tie2 signaling in lungs, suggesting that sex mainly affects renal angiopoietin/Tie2 signaling. Taken together, our results suggest that sexual dimorphism of the endothelial angiopoietin/Tie2 system exists in kidneys of healthy mice. Further research on the role of estrogen in the regulation of the endothelial angiopoietin/Tie2 system is of interest.

THS induces systemic inflammation, leading to a permeable endothelium, tissue edema and organ injury [9, 10]. We and others have previously shown reduced endothelial Tie2 expression, endothelial hyperpermeability and organ injury in animals following HS [10, 12, 21] and sepsis [21, 30] and in postmortem renal tissue of septic patients [36]. To study the effect of reduced Tie2 expression without additional effects such as systemic inflammation, we have included heterozygous Tie2 knockout mice. As expected, we found renal edema in male mice with partial deletion of Tie2. This is in line with previous studies where suppression of Tie2 induced endothelial hyperpermeability in an in vitro setting [37]. Against our expectations, we found no pulmonary edema in male mice with partial deletion of Tie2. Although previous studies described the occurrence of pulmonary edema [37] and decreased pulmonary microcirculatory perfusion in vivo [38] in response to reduced Tie2 expression, this was all initiated by either sepsis [36] or pulmonary arterial hypertension [38], suggesting pulmonary edema only occurs in response to both reduced Tie2 expression and an additional inflammatory hit. Surprisingly, we have found that female mice with partial deletion of Tie2 did not have renal or pulmonary edema formation. This absence of renal edema in heterozygous Tie2 knockout mice cannot be explained by differences in the extent of Tie2 knockout as female mice tended to have an even bigger reduction in Tie2 gene expression. A possible explanation is that in healthy humans, mean arterial pressure, oncotic pressure and capillary pressure are lower in premenopausal females compared to males, which will offset a higher fluid movement out of the capillaries in males [39, 40]. Additionally, differences exist between male and female endothelial cells in the in vitro setting [41, 42]. Male endothelial cells express less vascular endothelial cadherin (VE-cadherin), a protein regulating cell-cell interactions, and form a less strong endothelial barrier layer compared to female endothelial cells [42]. In addition, female sex hormones have a protective role in THS-induced pulmonary leakage [43] and lung injury [44]. However, above described differences have not been studied in different organs. Taken together, female mice appear to have a protective mechanism against renal edema formation following partial deletion of endothelial barrier regulator Tie2. Sex-related endothelial dysfunction receives insufficient attention and further research is warranted to determine sexual dimorphism of the endothelium.

Female gender seems also protective in the development of renal failure [2, 5, 7] and the progression to end-stage renal failure [45]. Structural differences exist between males and females, as males for example have a larger kidney with larger glomeruli volume compared to females [46]. Moreover, sex hormones regulate several cellular processes that affect renal function, such as the release of cytokines and synthesis of proteins. For example, estradiol may protect against the progression of renal disease by inducing nitric oxide synthesis in glomerular endothelial cells [47]. In the current study, we have provided evidence for differences in basal expression of markers commonly used for renal ischemia as females presented lower circulating NGAL and expressed lower KIM1 compared to males. These results confirm the findings of another mouse study investigating the effect of sex on cisplatin-induced acute kidney injury, where females presented lower renal KIM-1 and circulating NGAL expression [48]. In summary, we have shown sex-related differences in the basal expression levels of commonly used renal ischemia markers, which should be further explored and confirmed in the clinical situation.

Limitations

The current study was designed to provide a first overview on sex-related differences in the expression levels of the endothelial angiopoietin/Tie2 system. Our findings significantly increase basic knowledge, however, it would be of interest to further investigate these parameters in response to THS, with corresponding effects on activation of the Tie2 receptor, to increase translatability to the clinical setting. Additionally, due to absence of specific antibodies to determine phosphorylation of Tie2 in mice and the limited amount of tissue available, the current study did not investigate the functional aspects of the endothelial angiopoietin/Tie2 system. The current study was carried out on breeding surplus, thereby contributing to the reduction and refinement of animal studies. However, a study with breeding surplus is limited to only using non-invasive techniques. As the current study showed interesting sex-related differences, future studies could investigate these differences more in depth by for example increasing or reducing estrogen levels and thereby study the relation between estrogen and Tie2 more closely.

Conclusions

The present study showed sexual dimorphism of the endothelial angiopoietin/Tie2 system, which is involved in the regulation of endothelial permeability. Additionally, the current study revealed a sex-related susceptibility to reduced basal Tie2 expression. Female sex protects against renal edema in mice with partial deletion of Tie2. Future studies should reveal whether comparable sex-related differences exist in humans, and whether these differences contribute to sex-related susceptibility to renal injury. As a relation between estrogen and the endothelial angiopoietin/Tie2 system is suggested, it would be of interest to further explore this relation. Upon confirmation of the findings of the current study in THS patients, these differences may be the basis of the development of sex-specific treatment strategies to improve outcome following traumatic hemorrhagic shock.

Supporting information

S1 Fig. Typical example of genotyping results.

(TIF)

Click here for additional data file.^{(60.7KB, tif)}

Data Availability

All data have been made available via: Sex differences and loss of Tie2 - EASY (knaw.nl) https://easy.dans.knaw.nl/ui/datasets/id/easy-dataset:315203/tab/2.

Funding Statement

This research was supported by the Dutch Heart Foundation (2016T064, to NAMD, https://www.hartstichting.nl/), Dutch Research Council (ZonMW, Veni Grant 2019, to CEvdB, https://www.zonmw.nl/nl), European Society of Intensive Care Medicine (Basic research Award 2021 to CEvdB, https://www.esicm.org/) and European Society of Anaesthesiology and Intensive Care (ESAIC Research project grant 2022 to CEvdB, https://www.esaic.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0293673.r001

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14 Jun 2023

PONE-D-23-11857Female sex protects against renal edema in mice with partial deletion of the endothelial barrier regulator Tie2PLOS ONE

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. The little should be modified to include male and female mice.

2. There is need for critical grammatical review.

3. Wet/dry weight ratio is not the ideal test for renal and pulmonary injury, but an indicator for renal and pulmonary edeme. Medications, injections, etc can also cause edema.

4. Abstract should be revised to give a comprehensive summary of background, method, result and conclusion.

5. Conclusion shows that female mice could protect against renal edema, though the mechanism could not be explained. The preresent finding is a clear confirmation of the established findings.

6. Angiopoeitin-1 is a growth factor produced by kidney and liver, whereas angiopoeitin-2is produced and stored in Weibel-Palade bodies in endothelial cells. It binds to integrin beta-2 by interacting with platelete-derived growth factor –BB in monocytes. Hence determination of integrin and PLDGF-BB is necessary. Angiopoetin-1 signals through Tie 2, whereas angiopoetin-2 is antagonistic, hence it is a regular disruptive agent. The study was on both kidney and lung of the rats, therefore the tittle does not reflect the content.

7. Introduction should be revised to include the existing discoveries, shortcomings and the way forward. The aim of the study should be redefined. The emphasis is on the kidney and the study on both lung and kidney. Liver should have been included.

8. The methods are technical but grossly inadequate. Source, age, weight and strain of the mice are not mentioned. Period of acclimatization; cyclical diurnal changes, feeds, period of experimentation, vital parameters and generation of the mice (1st filial or 2nd filial) are not mentioned. Euthanasia using isoflurane is controversial. It is not the best drug to use. It causes behavioral changes such as induction of attention deficit, increased anxiety etc. Subheadings under Methods should be rearranged as follows: Experimental set up, Animals and Genotyping, Renal and pulmonary edema formation, Plasma analysis, Renal analysis and study approval. The use of isoflurane and decapitation is a great cruelty to Animal kingdom especially the species of mice. It is quite disheartening. Inhaled isoflurane cause pulmonary edema by atenuating histologic lung injury. I am quite wary of the use of isoflurane/decapitation. Note that isoflurane (5%) can cause death in 60 sec (too long) for humane killing. Respiratory arrest takes longer time.

9. Statistical methods should be revised. All the data presented in barcharts should be revised. Some data generated are quantitative while others are qualitative. The conclusion derived from the study based on the result is quite unfounded. The presented results are not adequately comprehensive. Therefore, I am of opinion that some results should be changed to tabular form, since males and females were used for the study. Quantitative data could be analyzed using student test unpaired. Specific relevant changes in the gene of each mice should be computed and the differences in the genes could be ranked. The Results section requires critical grammatical review. Functional parameters of kidney should have been measured.

10. Discussion should be revised. Findings should be discussed with reference to the past findings.

11. Conclusion is grossly inadequate and biased. Hence kindly revised the conclusion.

Reviewer #2: Dear

1. Introduction. It is informative with compatible references

2. Methods: other than the sample size are small, it is clear and concise. Small sample size may bias the results.

3. Procedures of methods: clear and concise

4. Statistical analysis: I think with a small number of animals, it is difficult to get statistically significant.

5. The results were presented as figures, which appeared a little bit confused. The other option is to tabulate some of your findings , which be of high values to the readers as well as you impressed them.

6. Please, revised your conclusion to be more informative.

Reviewer #3: Title: Female sex protects against renal edema in mice with partial deletion of the endothelial barrier regulator Tie2

Authors: N/A

Review

Comments to Author: The role of biological sex as a risk factor for adverse outcomes following major trauma has been widely recognized. Females may confer a protective effect against organ failure, sepsis, and mortality in patients experiencing traumatic hemorrhage. The endothelial angiopoietin/Tie2 system is well recognized for controlling the permeability of endothelial cells. In this study, in heterozygous Tie2 knock-out mice, it appears that the female sex provides protection against renal edema while not exerting the same protective effect against pulmonary edema. This study suggests that there is a sexual dimorphism that provides protection against renal edema in female mice. The study indicated that Tie2 has exhibited potential in mitigating organ edema induced by hemorrhagic shock, specifically in males. However, it is imperative to acknowledge the potential existence of sexual dimorphism in the endothelium, an aspect that has not been investigated in this particular context before.

Major Comments:

• The authors of the paper have done an impressive job in carrying out their research and presenting their findings. They demonstrated a good level of skill and attention to detail in executing the study and analyzing the data. Additionally, they extensively reviewed the existing literature related to their research topic, providing a comprehensive understanding of the subject matter.

• However, it is important to note that the experimental design employed in the study could have been more carefully chosen. This means that there may be some limitations in how the study was designed. These flaws could potentially impact the reliability of the results obtained.

• The authors investigated "whether expression of components of the endothelial angiopoietin/Tie2 system in kidneys and lungs differs between healthy female and male mice. The effect of sex on renal and pulmonary injury in heterozygous mice with genetically reduced endothelial Tie2 expression, thereby mimicking the suppressive effect of THS on expression of the endothelial Tie2 receptor."

• Although the tie-2 deletion has been universal, the authors failed to explain the organ-specific effect of this particular phenotype.

• The discrepancy between gene and plasma expression has not been discussed.

• The abstract stated "estrogens and other endothelial barrier regulators," although only estrogen receptors have been analyzed. While estrogen has been briefly discussed with regards to its role in vasoprotective effects, the postulated selective role of TIE-2 has not been optimally elucidated in this paper.

• The sample size is relatively small for assessing the claimed hypotheses.

• Ln 159: Histology has not been presented anywhere in the present study, and the removal of figures has not been justified.

Minor Comments:

o Reference [5] are repeated many times.

o A pharmacological blocker could have a superior outcome, although this has not been utilized in the study and previously utilized by your group

o Ln 81, typo error

o The wet/dry weight ratio approach did not show any significant difference in the present study. I wonder if there is a better mechanistic approach to show the investigated phenotype (lung vs. renal edema).

o Ln 188: "Circulating levels of angiopoietin-1 (p=0.06) and angiopoietin-2 were higher in females compared to males (Fig 1A-B)" is not entirely supported by the present findings and referenced figures

o Ln 199 analyzed soluble tie2, I wondered if the cellular lysate of the compared organs might show a better conclusion.

o Ln 202 *, **, and *** symbols have not been included in the study.

o Not all selected genes are relevant to the proposed mechanism.

o Protein expression methods other than ELISA might also provide additional insight (e.g. western blotting).

o The selected cohort of genes is not fully justified. The molecules included in the analysis were estrogen receptor α, estrogen receptor β, integrin α5, integrin β1, Rac1, RhoA, vascular endothelial growth factor α (VEGFα) (why not other isoforms), kidney injury molecule 1 (KIM1), and neutrophil gelatinase-associated lipocalin (NGAL).

o Estrogen roles have been suggested in many reports but were not sufficiently discussed

o TIE-2 has been proposed as a protective factor, although in vitro studies have been lacking.

Reviewer #4: Good work. The paper is well written and discussed found that Female sex seems to protect against renal, but not pulmonary edema in heterozygous Tie2 knock-out mice. This could not be explained by sex dimorphism in the endothelial angiopoietin/Tie2 system.

Reviewer #5: Leeuwen and colleagues have investigated the effect of gender on edema formation in mice with partial deletion of Tie2.

Major concerns:

1- The authors have to justify why they did not use mice with full Tie2. In addition, please include the genotyping bands of three random mice in each group (in the supp material).

2- The authors relied on the w/d ratio and certain markers to conclude the edema formation. This needs further investigations such as, the albumin contents of the kidney tissues and BAL fluid as well as IHC analysis of the kidney and lung tissue to evaluate the the thickness of interalveolar septa or the tubules in the kidney tissues.

3- Tie2 receptor known to be extensively regulated on post-translational level, thus, the tissue levels of Tie2 need to be analysed (by WB or ELISA).

Minor concerns:

1- please label in each figure the corresponding tissue (kidney, pulmonary...).

2- please show the IHC data (stated in the results as data not shown).

3- The authors can design a scheme to describe the proposed mechanism.

Reviewer #6: Thank you for nice work and research. I have couple of recommendations:

Line 72-82 Add a simplified and summarized figure about the angiopoietin/Tie2 signaling pathway

Line 305-307 rewrite the two sentences regarding the pulmonary and renal gene expression to avoid duplication

All Figs (1B, 1D, 2A-C, 4A, 4C, 4D, 5B, 5E, & 6F) need to be revised and fix typo of (1)

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Reviewer #1: No

Reviewer #2: Yes: Marwan S.M. Al-Nimer

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

Reviewer #6: No

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PLoS One. 2023 Nov 16;18(11):e0293673. doi: 10.1371/journal.pone.0293673.r002

Author response to Decision Letter 0

15 Sep 2023

PONE-D-23-11857

Response to the reviewers

Reviewer #1:

We would like to thank the reviewer for putting his/her time and care in providing valuable comments which have significantly improved the manuscript. We answered the comments below in a point-wise manner. Changes and additions in the manuscript based on the provided comments are shown via track changes.

1. The little should be modified to include male and female mice.

We assume the reviewer means ‘title’ with the word ‘little’. We have added ‘lung edema’ as suggested under point 6 and ‘compared to male sex’ and adjusted the title as follows: “Female sex protects against renal edema, but not lung edema, in mice with partial deletion of the endothelial barrier regulator Tie2 compared to male sex”.

2. There is need for critical grammatical review.

We have critically reviewed our manuscript and corrected the text if necessary.

3. Wet/dry weight ratio is not the ideal test for renal and pulmonary injury, but an indicator for renal and pulmonary edeme. Medications, injections, etc can also cause edema.

We agree with the reviewer that an increase in wet/dry weight ratio is an indication of edema formation. It depends on the setting whether edema can be an indicator of organ injury. For example in patients with sepsis, edema is a known complication and indicates organ injury. However, we agree with the reviewer that there are also settings where edema formation is not a marker for organ injury, indeed when edema occurs in response to for example medications. Therefore in the current study we combined measurements of organ edema with markers known to indicate organ injury, such as NGAL and KIM-1, but also IHC analysis. However, as we agree with the reviewer that edema can be a marker for organ injury, but is not per definition a marker for organ injury, we carefully revised our manuscript and changed terminology to ‘edema formation’ instead of ‘organ injury’ when only results of wet/dry weight ratios were discussed.

4. Abstract should be revised to give a comprehensive summary of background, method, result and conclusion.

We critically read our abstract and revised it where necessary.

5. Conclusion shows that female mice could protect against renal edema, though the mechanism could not be explained. The present finding is a clear confirmation of the established findings.

We thank the reviewer for this positive comment.

6. Angiopoeitin-1 is a growth factor produced by kidney and liver, whereas angiopoeitin-2 is produced and stored in Weibel-Palade bodies in endothelial cells. It binds to integrin beta-2 by interacting with platelete-derived growth factor –BB in monocytes. Hence determination of integrin and PLDGF-BB is necessary. Angiopoetin-1 signals through Tie 2, whereas angiopoetin-2 is antagonistic, hence it is a regular disruptive agent.

We have to kindly disagree with the reviewer. Angiopoietin-1 is primary expressed by pericytes and as far as current knowledge on this protein not expressed by only the kidney and liver. Additionally, although the by the reviewer described mechanism of angiopoietin/Tie2 signaling is correct in some cases, angiopoietin-2 may also function as agonist, depending on the context (PMID: 27038015; PMID: 19223473). Lastly, to our knowledge, primary endothelial Tie2 signaling goes via integrin α5/β1, and not integrin β2. Therefore, we included analysis of integrin a5 and integrin b1 in our manuscript (figure 6 and 7). The interaction with integrin beta-2 that is described by the reviewer is present in monocytes, whereas the current study focused on endothelial angiopoietin/Tie2 signaling, therefore the analysis of integrin a5 and integrin b1 is more informative in this context/

The study was on both kidney and lung of the rats, therefore the tittle does not reflect the content.

The reviewer is correct. The title was adjusted to include the results of the lungs.

7. Introduction should be revised to include the existing discoveries, shortcomings and the way forward.

We feel that our introduction gives a nice overview of the current literature and we emphasize that sex differences are not taken into account. Of course we might have missed an important subject and would therefore like to ask the reviewer to be more precise so we can adjust the introduction.

The aim of the study should be redefined.

We noticed that our primary and secondary outcome were reversed in the aim and have adjusted this as follows: “Here, we have investigated the effect of sex on renal and pulmonary edema in heterozygous mice with genetically reduced endothelial Tie2 expression, thereby mimicking the suppressive effect of THS on expression of the endothelial Tie2 receptor. In addition, we have investigated whether expression of components of the endothelial angiopoietin/Tie2 system in kidneys and lungs differs between healthy female and male mice.”

The emphasis is on the kidney and the study on both lung and kidney.

We thank the reviewer for his/her critical reading and have adjusted the introduction accordingly.

Liver should have been included.

In our opinion, all vital organs are important as traumatic hemorrhagic shock affects the whole body. We have however focused on the two main affected vital organs, the lung and kidney. We share the opinion that the liver is of interest and in our new study we have included the liver.

8. The methods are technical but grossly inadequate. Source, age, weight and strain of the mice are not mentioned.

We have added more information regarding the origin of the line to the methods section. In short, mice were bred in the animal facility of the VU University, Amsterdam, the Netherlands. The creation of the line is described in reference Jongman et al. (PMID: 30520765). Mice were included based on age, therefore weight of the mice is only mentioned in the results section as it was not an inclusion criterion.

Period of acclimatization; cyclical diurnal changes, feeds, period of experimentation, vital parameters and generation of the mice (1st filial or 2nd filial) are not mentioned.

More information regarding housing, food, water and generation of the mice was added to the methods section. As mice were breeding surplus coming from the animal facility of the VU University, acclimatization was not required as no transport was applied. Additionally, period of experimentation (‘mice were included at the age of 3 to 6 months’) was noted in the methods section. Vital parameters were not measured as mice were only sacrificed.

Euthanasia using isoflurane is controversial. It is not the best drug to use. It causes behavioral changes such as induction of attention deficit, increased anxiety etc. Subheadings under Methods should be rearranged as follows: Experimental set up, Animals and Genotyping, Renal and pulmonary edema formation, Plasma analysis, Renal analysis and study approval. The use of isoflurane and decapitation is a great cruelty to Animal kingdom especially the species of mice. It is quite disheartening. Inhaled isoflurane cause pulmonary edema by attenuating histologic lung injury. I am quite wary of the use of isoflurane/decapitation. Note that isoflurane (5%) can cause death in 60 sec (too long) for humane killing. Respiratory arrest takes longer time.

Termination by decapitation under brief isoflurane inhalation anesthesia is an accepted method in the Netherlands, elsewhere in the EU, and in full compliance with the directive 2010/63/EU. The statement 'Inhaled isoflurane cause pulmonary edema by attenuating histologic lung injury.’ is somewhat confusing. Providing the corresponding reference would have been informative. Generally, beneficial effect of brief isoflurane anesthesia has been reported (PMID: 26068207) and also in combination with the avoidance of stressors by acute brain injections in mice (PMID: 33607166). Even if isoflurane would promote renal or lung edema formation, the mice are killed in an unconscious state immediately (seconds) after inhalation. It is therefore highly unlikely that mice develop pulmonary edema in the few seconds of anesthesia. We have also discussed this issue with our local animal welfare body and there is absolutely no animal welfare concern regarding this procedure.

We thank the reviewer for this suggestion. We agree with the reviewer that a large amount of data is presented in figure format. To improve readability of the data we attempted to present a part of it, the PCR data to be specific, in tabular format. Unfortunately, during the execution we came to the conclusion that presenting this data in tabular format did not improve the readability due to the amount of decimals. In our opinion, the readability even decreased when presenting this data in tabular format. We considered presenting the data as normalized to WT males, however this would make it difficult to compare WT females vs Tie2+/- females or Tie2+/- males vs Tie2+/- females. To give an impression of the tables we created, we added tables below. If the reviewer has any other ideas on how to improve the presentation of our data, we are very open to receive any suggestion.

Quantitative data could be analyzed using student test unpaired.

We tested the normality of data distribution using the Shapiro-Wilk test. As data appeared non-normally distributed, it is our opinion that according to good statistical practices, the data should be evaluated with Kruskal-Wallis test.

Specific relevant changes in the gene of each mice should be computed and the differences in the genes could be ranked.

Unfortunately, we do not understand the suggestion of the reviewer. Perhaps he/she could explain us the proposed analysis?

The Results section requires critical grammatical review.

We critically read our results section and revised it where necessary.

Functional parameters of kidney should have been measured.

Although the amount of material was limited, as the study was executed on surplus mice coming from the breeding population, we tested several parameters of kidney function. Specifically, we analyzed circulating NGAL and gene expression levels of NGAL and Kidney Injury Marker-1 (KIM-1). Unfortunately, additional analyses such as the evaluation of creatinine or urine expression levels of NGAL appeared unfeasible due to the limited amount of blood or the absence of an urine sample. However NGAL and KIM-1 have been described by many others as indicators of renal injury.

10. Discussion should be revised. Findings should be discussed with reference to the past findings.

We critically reviewed our discussion and revised it were necessary.

11. Conclusion is grossly inadequate and biased. Hence kindly revised the conclusion.

We critically reviewed our conclusion and revised it to fit the presented results and discussion.

Reviewer #2:

1. Introduction. It is informative with compatible references

We thank the reviewer for this positive feedback.

2. Methods: other than the sample size are small, it is clear and concise. Small sample size may bias the results.

We agree with the reviewer that the samples size is small. We did however perform a sample size calculation based on preliminary results.

3. Procedures of methods: clear and concise

We thank the reviewer for this positive feedback.

4. Statistical analysis: I think with a small number of animals, it is difficult to get statistically significant.

Based on the samples size calculation we have included 9 mice per group. The sample size calculation is performed on the primary outcome, namely wet/dry weight ratios, and might therefore be not adequate for the secondary outcomes.

5. The results were presented as figures, which appeared a little bit confused. The other option is to tabulate some of your findings, which be of high values to the readers as well as you impressed them.

We thank the reviewer for this suggestion. We agree with the reviewer that a large amount of data is presented in figure format. To improve readability of the data we attempted to present a part of it, the PCR data, in tabular format. Unfortunately, during the execution we came to the conclusion that presenting this data in tabular format did not improve the readability due to the amount of decimals. In our opinion, the readability even decreased when presenting this data in tabular format. We considered presenting the data as normalized to WT males, however this would make it difficult to compare WT females vs Tie2+/- females or Tie2+/- males vs Tie2+/- females. To give an impression of the tables we created, we added the tables below.. If the reviewer has any other ideas on how to improve the presentation of our data, we are very open to receive any suggestion.

6. Please, revised your conclusion to be more informative.

We critically reviewed our conclusion and revised it to fit the presented results and discussion.

Reviewer #3:

Major Comments:

We would like to thank the reviewer for his/her kind words.

We agree with the reviewer that the chosen experimental design may not be the most optimal. We would like to emphasize that the study was carried out using breeding surplus, minimizing the possibilities in choice for experimental design. However, by doing so, animals coming from breeding that are otherwise sacrificed without inclusion in an experiment, are hereby effectively used as data is derived from these animals. This contributes to the reduction of animals and refinement of animal experiments (3R’s).

The reviewer addresses an interesting point. Based on the results of the current study, it is difficult to elaborate on the underlying mechanism leading to this organ-specific effect of the heterozygous Tie2 knockout. Although we do not fully understand the mechanism yet, our previous studies also showed an organ-specific effect of targeting Tie2 in a model for hemorrhagic shock (PMID: 28968277). In that specific study, we showed that targeting Tie2 with a Tie2 agonist could reduce pulmonary vascular leakage, but not renal vascular leakage after hemorrhagic shock, even when both organs had reduced Tie2 expression following hemorrhagic shock. Differences in the effectivity of a certain therapy, but also organ-specific differences in response to a heterozygous knock-out could be explained by several mechanisms, but still requires additional research to draw definitive conclusions.

• The discrepancy between gene and plasma expression has not been discussed.

We agree with the reviewer that there is a discrepancy between the expression levels measured in the circulation and on genetic level. Both measurements have advantages and disadvantages. Measuring protein level in plasma is a technique often used in the clinical setting, as plasma is then easily available but organ-specific tissue most often not. However, measurements perform in plasma only give insight in deviations on a circulatory level, not on organ-specific level. As we measured a broad spectrum of genes and proteins in circulation, lung and kidney, it is difficult to include the explanation of the discrepancy in the discussion as this differs per gene and per organ.

First of all, the text in the abstract was adjusted to “estrogen receptors and other endothelial barrier regulators”.

Second, to our knowledge, there are only a few studies discussing an (indirect) link between Tie2 and the possible protective effect of sex. As far as we know, the known literature is included in the discussion.

• The sample size is relatively small for assessing the claimed hypotheses.

We agree with the reviewer that the samples size is small. We did however perform a sample size calculation based on preliminary results.

• Ln 159: Histology has not been presented anywhere in the present study, and the removal of figures has not been justified.

Images of the immunohistochemical analyses were added to the results section (Figure 5C-D).

Minor Comments:

o Reference [5] are repeated many times.

We agree with the reviewer that reference [5] is used multiple times. To our knowledge, this is so far one of the few studies describing a link between sexual differences and organ failure in patients following traumatic hemorrhagic shock. If the reviewer has any other ideas, we are open to his/her suggestions.

o A pharmacological blocker could have a superior outcome, although this has not been utilized in the study and previously utilized by your group

We fully agree with the reviewer that the addition of an intervention, such as administration of an agonist of antagonist of the angiopoietin/Tie2 system would improve the impact of the study. As the study was carried out with breeding surplus, this was not a possibility in the current set-up. Additionally, the current study solely had as goal to provide insight in basal expression levels of the angiopoietin/Tie2 system and the existing differences between male and female mice. As we see the additional value of interfering in the angiopoietin/Tie2 system, this is part of one of our newly designed studies.

o Ln 81, typo error

We thank the reviewer for reading our manuscript in a thorough manner. However, we did not observe a typo error in line 81. Perhaps the reviewer could point out which typo error he/she detected.

We agree with the reviewer that the measurement of wet/dry weight ratio may not be the most ideal measurement to investigate edema. Ideally, one would like to measure fluid extravasation via determination of FITC-labeled dextrans or Evans Blue extravasations. These techniques have the advantage that only extravasation of a specific molecule size is determined. Unfortunately, with the current set-up we were limited to non-invasive techniques, as we performed the study with breeding surplus, not allowing us to inject any substances. Therefore, we were limited to the determination of wet/dry weight ratio.

In the first section of the results, we describe the differences between wild-type male and female mice. In these mice circulating angiopoietin-1 and angiopoietin-2 were significantly increased in female mice compared to male mice, as shown in figure 1A and 1B (non-striped bars). To emphasize that this part of the results only describes differences between wild-type makes and females, adjusted the sentence to: “In wild-type mice, circulating levels of angiopoietin-1 (p=0.06) and angiopoietin-2 were higher in females compared to males (Fig 2A-B), whereas angiopoietin-2/angiopoietin-1 ratio or soluble Tie2 levels were comparable between males and females (Fig 2C-D).”

o Ln 199 analyzed soluble tie2, I wondered if the cellular lysate of the compared organs might show a better conclusion.

We agree with the reviewer that analyzing cellular lysate would be of high value. Ideally, we would like to measure the phosphorylation status of Tie2 to investigate whether there is a difference in activated Tie2 rather than only measuring total Tie2 or soluble Tie2. Unfortunately, several limitations existed:

- To measure total Tie2 in cellular lysate: the amount of material to do so was not available. One lung/kidney was used for wet/dry weight ratio measurements, where the remaining parts were used for mRNA measurements and IHC stainings.

- To measure pTie2: Until now no specific antibodies are available to determine pTie2 in the kidney or lungs of mice. We recently tested several newly available antibodies, but none of them was specific enough to produce reliable, reproducible results.

o Ln 202 *, **, and *** symbols have not been included in the study.

We apologize for this inconsistency. The figures are adjusted to match the correct significance indication.

o Not all selected genes are relevant to the proposed mechanism.

In this study we tested a broad spectrum of genes, directly or indirectly connected to the purpose of the study:

- We analyzed KIM1 and NGAL mRNA levels to discuss the effect of heterozygous Tie2 knockout on renal injury.

- We analyzed angiopoietin-1, angiopoietin-2 and Tie2 to directly measure the effect of Tie2 knockout on the angiopoietin/Tie2 system.

- As downstream targets of the angiopoietin/Tie2 system we measured RhoA and Rac-1.

- Integrin α5 and β1 and VEGFα affect angiopoietin/Tie2 signaling and were therefore added as indirect targets.

- To elaborate on a link between sex and angiopoietin/Tie2 signaling, we investigated estrogen receptor α, estrogen receptor β.

The direct targets of the angiopoietin/Tie2 system have also been visualized in figure 1.

o Protein expression methods other than ELISA might also provide additional insight (e.g. western blotting).

The reviewer is correct. However, due to a limitation in the amount of tissue and the absence of specific antibodies to determine e.g. p-Tie2, we were not able to include western blot analysis in the current study.

We feel like this question has been answered above.

o Estrogen roles have been suggested in many reports but were not sufficiently discussed

To our knowledge, there is limited data available regarding the link between Tie2 and estrogens. As far as we know, the literature available has been included in the discussion of our manuscript. Future research should elaborate more on the link between Tie2 and estrogen, for example by overexpressing estrogen via estrogen administration, or reducing estrogen levels by for example an ovariectomy.

o TIE-2 has been proposed as a protective factor, although in vitro studies have been lacking.

As Tie2 is a flow-responsive gene, and cell culture is mainly static, it is difficult to investigate the protective effects of Tie2 in an in vitro setting. However, there are several studies, performed by our group but also by other groups, that have investigated the protective effects of Tie2 in an in vivo setting. For example, administration of Tie2 agonist vasculotide improves microcirculatory perfusion and reduces microvascular leakage in animals following hemorrhagic shock or cardiopulmonary bypass (PMID: 28968277, PMID: 30336848). Also, in mice with acute kidney injury, enhancing Tie2 phosphorylation could improve renal perfusion and reduce renal microvascular leakage (PMID: 26911791).

Reviewer #4:

Good work. The paper is well written and discussed found that Female sex seems to protect against renal, but not pulmonary edema in heterozygous Tie2 knock-out mice. This could not be explained by sex dimorphism in the endothelial angiopoietin/Tie2 system.

We would like to thank the reviewer for putting his/her time in reading our manuscript critically. We also would like to thank the reviewer for the positive feedback.

Reviewer #5:

Leeuwen and colleagues have investigated the effect of gender on edema formation in mice with partial deletion of Tie2.

Major concerns:

1- The authors have to justify why they did not use mice with full Tie2.

We agree with the reviewer that it would be very interesting to investigate the effect of a full Tie2 knock-out. Unfortunately, these mice are lethal as the expression of Tie2 is essential in the development of, amongst others, the vascular system. Therefore, only a heterozygous knock-out is viable.

In addition, please include the genotyping bands of three random mice in each group (in the supp material).

We thank the reviewer for this suggestion. We included a supplementary figure to show a typical example of genotyping results in both males and females. (supplementary figure 1)

2- The authors relied on the w/d ratio and certain markers to conclude the edema formation. This needs further investigations such as, the albumin contents of the kidney tissues and BAL fluid as well as IHC analysis of the kidney and lung tissue to evaluate the thickness of interalveolar septa or the tubules in the kidney tissues.

Indeed additional analyses would be interesting to elaborate more on the level of organ injury. In the current study we were limited to the usage of non-invasive techniques, as the study was carried out on breeding surplus. Therefore, BAL fluid analysis was not feasible. We did, however, perform IHC analysis of kidney and lung tissue. Results of these analysis are visualized in figure 5C and 5D.

3- Tie2 receptor known to be extensively regulated on post-translational level, thus, the tissue levels of Tie2 need to be analysed (by WB or ELISA).

The reviewer addresses an important point. It would, indeed, be of high additional value to include data regarding the phosphorylation status of Tie2 in tissue. Unfortunately, due to the limited amount of tissue available and the absence of specific antibodies, this was not possible in the current study. We recently tested several newly available antibodies for p-Tie2, but none of them appeared specific enough to generate reliable, reproducible data.

Minor concerns:

1- please label in each figure the corresponding tissue (kidney, pulmonary...).

The figures were adjusted as suggested.

2- please show the IHC data (stated in the results as data not shown).

We agree with the reviewer that visualization of the IHC data improves the interpretation of the results for the reader. We therefore included a typical example of each group in Figure 5C and 5D..

3- The authors can design a scheme to describe the proposed mechanism.

With the current study we aimed to provide a first insight in basal differences between male and female mice and the difference in effect of heterozygous Tie2 knockout between males and females. Unfortunately, the literature that describes a possible link between sex and Tie2 signaling is very limited and also based on the results of the current study it is difficult to propose a possible underlying mechanism for the found differences. In the current stage of this research topic it is therefore difficult to propose a specific mechanism.

Reviewer #6:

Thank you for nice work and research.

We thank the reviewer for his/her kind words.

I have couple of recommendations:

- Line 72-82 Add a simplified and summarized figure about the angiopoietin/Tie2 signaling pathway

We thank the reviewer for this suggestion. We added a schematic overview of angiopoietin/Tie2 signaling to the introduction (Fig. 1).

- Line 305-307 rewrite the two sentences regarding the pulmonary and renal gene expression to avoid duplication

We thank the reviewer for reading the manuscript in such a detailed manner and changed the sentences to avoid duplication as follows: “Renal gene expression of integrin α5, integrin β1, VEGFα, RhoA and Rac1 was not affected by sex nor differed between Tie2+/- and Tie2+/+ mice (Fig 6C-G). In lungs, RhoA gene expression was higher in female Tie2+/- mice compared to female Tie2+/+ mice (Fig 7F), whereas gene expression of integrin α5, integrin β1, VEGFα, and Rac1 was not affected by sex nor differed between Tie2+/- and Tie2+/+ mice (Fig 7C-E,G).”

- All Figs (1B, 1D, 2A-C, 4A, 4C, 4D, 5B, 5E, & 6F) need to be revised and fix typo of (1)

We apologize for this inconsistency. The figures are adjusted to match the correct significance indication.

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PLoS One. doi: 10.1371/journal.pone.0293673.r003

Decision Letter 1

Keiko Hosohata

18 Oct 2023

Female sex protects against renal edema, but not lung edema, in mice with partial deletion of the endothelial barrier regulator Tie2 compared to male sex

PONE-D-23-11857R1

Dear Dr. van den Brom,

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Reviewer #7: All comments have been addressed

Reviewer #8: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #7: Yes

Reviewer #8: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

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4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #8: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #7: Yes

Reviewer #8: Yes

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6. Review Comments to the Author

Reviewer #7: (No Response)

Reviewer #8: This is interesting paper. The authors answered questions. Authors shown sex dimorphism in the endothelial angiopoietin/Tie2 system.

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PLoS One. doi: 10.1371/journal.pone.0293673.r004

Acceptance letter

Keiko Hosohata

7 Nov 2023

PONE-D-23-11857R1

Female sex protects against renal edema, but not lung edema, in mice with partial deletion of the endothelial barrier regulator Tie2 compared to male sex

Dear Dr. van den Brom:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Typical example of genotyping results.

(TIF)

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Data Availability Statement

All data have been made available via: Sex differences and loss of Tie2 - EASY (knaw.nl) https://easy.dans.knaw.nl/ui/datasets/id/easy-dataset:315203/tab/2.

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PERMALINK

Female sex protects against renal edema, but not lung edema, in mice with partial deletion of the endothelial barrier regulator Tie2 compared to male sex

Anoek L I van Leeuwen

Elise Beijer

Roselique Ibelings

Nicole A M Dekker

Marjolein R A van der Steen

Joris J T H Roelofs

Matijs van Meurs

Grietje Molema

Charissa E van den Brom

Roles

Abstract

Background

Methods

Results

Conclusion

Introduction

Fig 1. Schematic overview of the effect of angiopoietin/Tie2 signaling on endothelial barrier function.

Materials and methods

Study approval

Animals and genotyping

Experimental set-up

Renal and pulmonary edema formation

Plasma analyses

RNA analyses

Table 1. Real time-qPCR primers.

Histology

Statistical analysis

Results

Sex differences in wild type mice with normal Tie2 expression

Fig 2. Circulating markers of the endothelial angiopoietin/Tie2 system.

Fig 3. Renal gene expression of the endothelial angiopoietin/Tie2 system.

Fig 4. Pulmonary gene expression of the endothelial angiopoietin/Tie2 system.

Fig 5. Sex differences and effect of Tie2 heterozygosity in renal and pulmonary function.

Fig 6. Renal gene expression of other endothelial barrier regulators.

Fig 7. Pulmonary gene expression of other endothelial barrier regulators.

Sex differences in heterozygous Tie2+/- mice with partial deletion of Tie2

Discussion

Limitations

Conclusions

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Keiko Hosohata

Roles

Author response to Decision Letter 0

Decision Letter 1

Keiko Hosohata

Roles

Acceptance letter

Keiko Hosohata

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Sex differences in heterozygous Tie2^+/- mice with partial deletion of Tie2