Targeting the CD39/CD73 pathway: New insights into cardiac fibrosis and inflammation in female cardiac surgery patients

Abstract

Women undergoing cardiac surgery suffer from worse outcomes than their male counterparts. The reasons for this disparity are multifactorial, but the loss of the protective effects of estrogen likely plays a role. Estrogen acts on the CD39/CD73 purine pathway, and loss of estrogen effects may contribute to the increased inflammation seen in post-menopausal women. We aimed to compare CD39/CD73 expression and downstream fibrosis, and inflammation in men and women undergoing cardiac surgery and then used an ovariectomy/high fat diet mouse model to approximate women who present for cardiac surgery to test therapeutics. We found decreased CD39 and CD73 in women compared to men, which was associated with increased fibrosis. Apyrase supplementation (a CD39 mimetic) improved ejection fraction and decreased E/e'. Increased CD73 function (via dipyridamole) decreased fibrosis. This study demonstrates the importance of purinergic dysfunction in cardiovascular disease in women and presents two potential therapeutics to improve cardiac health via manipulation of purine pathways.

Keywords: Apyrase; CD39; CD73; Cardiac surgery; Diastolic function; Fibrosis.

Graphical abstract

Fig. 1

Gender differences in atrial fibrosis and myocardial Blush Grades among patients undergoing cardiac surgery correspond to the altered purinergic pathway and inflammation profile in females. A, PicoSIRIUS fibrosis staining on atrial tissue. Images taken at 20× on atrial tissue before CBP. B, myocardial Blush Grade assessed preoperatively. C, quantification of purinergic signaling markers pre- and post-CPB from human atrial tissue. (Western blotting for CD39, CD73, and ADORA2B; ADA assay for serum ADA levels). D, Immunofluorescence staining of P2X7 on atrial tissue. Scale bar = 50 μm. E and F, quantification of inflammatory markers on human samples. Data represented as median ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001. CBP = cardiopulmonary bypass; CD39 = cluster of differentiation 39 (ecto-nucleoside triphosphate diphosphohydrolase 1); CD73 = cluster of differentiation 73 (Ecto 5’nucleotidase); ADORA2B = adenosine receptor 2B; ADA = adenosine deaminase; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; IL-22 = interleukin 22; MDC = macrophage-derived chemokine; TNFα = tumor necrotizing factor α; ENA-78 = epithelial neutrophil-activating protein 78. In A, n = 40 for both female and male participants. In B, n = 17 for both female and male participants. In C n = 17 for each group in CD39, n = 7 for each group in CD73 and ADORA2B, n = 9 for each group in ADA. In D, n = 5 for Pre—F, Post-F, Pre-M, and Post-M in P2X7. In E, n = 19 for each group in IL-22, MDC, and TNFα. In F, n = 8 for each group in Leptin, n = 6 for each group in ENA-78. In all cases, n corresponds to one participant.

Fig. 2

Under hypoxia, the inhibition of estrogen, rather than estrogen treatment or saline control, demonstrated the highest extracellular ATP level. Human umbilical vein endothelial cells (HUVEC) were cultured with saline (control), estrogen, or tamoxifen for 30 min first (Baseline, each subgroup n = 8). Then, cobalt chloride was added to the Hypoxia subgroups to create hypoxia environment (Control Hypoxia, Estrogen Hypoxia, and Tamoxifen Hypoxia). Measurements were taken every 2 min until the extracellular ATP levels normalized. Data are represented as mean ± SD. In each group, n = 8. In all cases, n corresponds to one well of a 96-well cell culture plate.

Fig. 3

The mouse ovariectomy model demonstrated the same purinergic pathway changing pattern compared with human females both on normal or high-fat diets, which contributed to the elevating extracellular ATP levels, leading to higher inflammation markers, myocardial fibrosis, and ultimately systolic dysfunction. A, Body weight (g), EF (%), FS (%), and calculated LV mass (mg) among four groups of mice: female mice fed with control diet (Control Diet); female ovariectomized mice, control diet (OVX); female mice with high-fat diet (HFD); female ovariectomized mice with high-fat diet (HFD + OVX). B, Quantification of purinergic pathway markers and extracellular adenosine level among four groups. C, PicoSIRIUS fibrosis staining on mice myocardial tissue. Scale bar = 100 μm. D, Quantification of inflammatory markers. Data are represented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001. EF = ejection fraction; FS = fractional shortening; LV = left ventricle; CD39 = cluster of differentiation 39 (ecto-nucleoside triphosphate diphosphohydrolase 1); CD73 = cluster of differentiation 73 (Ecto 5’nucleotidase); ENT-1 = equilibrative nucleoside transporter 1; ADA = adenosine deaminase; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; IL-6 = interleukin 6; MIG = Monokine induced by gamma- interferon. In A, n = 10 for each group in Body Weight, EF, FS, and LV Mass. In B, n = 10 for each group in CD39, CD73, ENT-1, and ADA, n = 4 for Purine Adenosine Assay. In C, n = 3 for Control Diet, OVX, HFD, and HFD + OVX. In D, n = 10 for each group in IL-6, MIG, and Leptin. In A, B, and D, n corresponds to one mouse. In C, n corresponds to slices from different mouse in the same group.

Fig. 4

Targeting the purinergic pathway alleviated cardiac dysfunction by reducing inflammation and extracellular ATP levels. A, Body weight (g), fat mass (g), lean mass (g), heart mass/body weight ratio (%) among three groups of female ovariectomized mice fed with high-fat diet: peritoneal injection with saline (Saline Control), apyrase or dipyridamole. B, Transthoracic echocardiography data compared between the three groups. C, PicoSIRIUS fibrosis staining on mice myocardial tissue. Scale bar = 100 μm. D, Quantification of purinergic pathway markers and extracellular adenosine level among three groups. Data are represented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001. EF = ejection fraction; FS = fractional shortening; E/e’ = ratio between early mitral inflow velocity and mitral lateral annular early diastolic velocity; CD39 = cluster of differentiation 39 (ecto-nucleoside triphosphate diphosphohydrolase 1); CD73 = cluster of differentiation 73 (Ecto 5’nucleotidase); GAPDH = glyceraldehyde 3-phosphate dehydrogenase; MIG = Monokine induced by gamma- interferon. In A and B, n = 5 for Saline Control, n = 9 for Apyrase and Dipyridamole in Body Weight, Fat Mass, Lean Mass, Heart/Body Weight, EF, FS, and E/e’. In C, n = 3 for Saline Control, Apyrase and Dipyridamole. In D, n = 4 for Saline Control, n = 9 for Apyrase in CD39 and CD73; n = 14 for Saline Control, n = 8 for Apyrase, n = 7 for Dipyridamole in MIG; n = 6 for Saline Control, n = 9 for Apyrase, n = 5 for Dipyridamole in Purine Adenosine Assay. In A, B, and D, n corresponds to one mouse. In C, n corresponds to slices from different mouse in the same group.

Fig. 5

Experimental Timeline for the Ovariectomy and High-Fat Diet Mouse Model. Female C57BL/6 mice (n = 40) were divided into two dietary groups at 6 weeks of age: high-fat diet (HFD, n = 20) and control diet (n = 20). At 12 weeks, half of each group underwent ovariectomy (OVX, n = 10 per diet), while the remaining mice underwent a sham procedure (control, n = 10 per diet). This created four groups: OVX + HFD, HFD, OVX, and Control. Mice were maintained on their respective diets for a total of 24 weeks, after which cardiac function and body composition were assessed using transthoracic echocardiography, Doppler ultrasound, and EchoMRI measurements. Blood, plasma, and cardiac tissue samples were collected for biochemical and histological analyses. This model was used to approximate postmenopausal women with metabolic dysfunction and assess the effects of estrogen loss and purinergic signaling dysregulation on cardiac function and fibrosis.

Fig. 6

Experimental Timeline for the Ovariectomy and High-Fat Diet Mouse Model with Purinergic Pathway Interventions. Female C57BL/6 mice (n = 39) were divided into high-fat diet (HFD, n = 29) and control diet (n = 10) groups at 8 weeks of age. At 12 weeks, all HFD-fed mice underwent ovariectomy (OVX), while control diet-fed mice remained intact. At 30 weeks, OVX-HFD mice were further divided into three treatment groups: Saline (n = 6), Dipyridamole (CD73 enhancer, n = 9), and Apyrase (CD39 mimetic, n = 9). Control diet-fed mice (n = 10) served as a baseline comparison. Cardiac function was assessed using transthoracic echocardiography and Doppler ultrasound, and body composition was measured via EchoMRI. Following the experimental period, blood, plasma, and cardiac tissues were collected for further analysis. This model was designed to evaluate the impact of CD39/CD73 modulation on cardiac fibrosis and dysfunction in an estrogen-deficient, high-fat diet environment.