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

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.

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

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.

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

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.

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

Renal 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. * p<0.05, ** p<0.01, *** p<0.001.

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

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.

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

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.

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

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 other endothelial barrier regulators.

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.