Estrogen depletion modulates aortic prothrombotic signaling in normotensive and spontaneously hypertensive female rats

Abstract

Aim: In this study, we investigated how platelets and aorta contribute to the creation and maintenance of a prothrombotic state in an experimental model of postmenopausal hypertension in ovariectomized rats.

Methods: Bilateral ovariectomy was performed in both 14-week-old female spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. The animals were kept in phytoestrogen free diet. Vascular parameters, platelet, coagulation and aortic prothrombotic functions and mechanisms were assessed.

Results: Exacerbated platelet aggregation was observed in both SHR and WKY animals after ovariectomy. The mechanism was related to aortic COX2 downregulation and reduction in AMP, ADP, and ATP hydrolysis in serum and platelets. A procoagulant potential was observed in plasma from ovariectomized rats and this was confirmed by kallikrein and factor Xa generation in aortic rings. Aortic rings derived from ovariectomized SHR presented a greater thrombin generation capacity compared to equivalent rings from WKY animals. The mechanism involved tissue factor and PAR-1 upregulation as well as an increase in extrinsic coagulation and fibrinolysis markers in aorta and platelets. Aortic smooth muscle cells pre-treated with a plasma pool derived from estrogen-depleted animals developed a procoagulant profile with tissue factor upregulation. This procoagulant profile was dependent on inflammatory signalling, since NFκB inhibition attenuated the procoagulant activity and tissue factor expression.

Conclusions: A prothrombotic phenotype was observed in both WKY and SHR ovariectomized rats being associated with platelet hyperreactivity and tissue factor upregulation in aorta and platelets. The mechanism involves proinflammatory signalling that supports greater thrombin generation in aorta and vascular smooth muscle cells.

Keywords: Estrogen; Hypertension; Menopause; Platelet; Thrombosis; Tissue factor.

Figure 1.. Experimental model of menopausal hypertension.

(A) Twenty female spontaneously hypertensive (SHR) and 20 normotensive Wistar Kyoto (WKY) rats arrived at our institution 60 days after birth. This date was considered as the day zero of our protocol. Before day zero, the animals received a standard diet. During the entire protocol after day zero, they received a phytoestrogen free diet. At day 40 (at approximately 14 weeks of age), the animals were submitted to a bilateral ovariectomy (OVX) or SHAM surgery (referred here as gonadal-intact animals). The experimental groups (n = 10/group) were WKY-SHAM, WKY-OVX, SHR-SHAM and SHR-OVX. Five days before either surgery (at day 35) or euthanasia (at day 85), blood pressure, body weight and estrous cycle phase were evaluated. At day 90 (50 days after surgery), all animals were euthanised for sample collection. (B) Rat body weight variation between animals post-OVX surgery (at day 85) and pre-OVX (at day 35). (C) Ratio between uterus weight and body weight was determined as an index of OVX surgery efficiency.

Figure 2.. Platelet aggregation function in estrogen-depleted normo and hypertensive rats.

A bilateral ovariectomy (OVX) or SHAM surgical procedure was performed in 14-week-old female spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. After 50 days, animals were euthanized and blood was collected for ex-vivo platelet aggregation functional tests. (A) ADP (10 μM)-induced platelet aggregation in platelet rich plasma (PRP). (B) Representative profile of ADP-induced aggregation curves for normotensive WKY rats. (C) Representative profile of ADP-induced aggregation curves for SHR rats. (D) Collagen (3 μg/mL)-induced platelet aggregation in PRP. (E) Representative profile of collagen-induced aggregation curves for normotensive WKY rats. (F) Representative profile of collagen-induced aggregation curves for SHR rats. Data are presented as mean ± SE and (*) represents a statistically significant difference between indicated groups (one-way ANOVA followed by Bonferroni’s-post hoc test).

Figure 3.. Nitrate/nitrite, nitric oxide synthase and cyclooxygenase 2 levels in normo and hypertensive ovariectomized rats.

A bilateral ovariectomy (OVX) or SHAM surgical procedure was performed in 14-week-old female spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. After 50 days, animals were euthanized for plasma and aorta collection. (A) Plasma and (B) Aorta nitrite/nitrate (NO⁻_x) levels as determined by the Griess method. (C) Aorta protein expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) as determined by western-blot (upper panel showing the representative blots from three independent experiments and lower panel showing its respective quantitative analysis normalized by the β-actin expression). Data are presented as mean ± SE and (*) represents statistically significant difference between indicated groups (one-way ANOVA followed by Bonferroni’s-post hoc test).

Figure 4.. Hydrolysis of the main nucleotides involved in platelet aggregation in estrogen-depleted normo and hypertensive rats.

A bilateral ovariectomy (OVX) or SHAM surgical procedure was performed in 14-week-old female spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. After 50 days, animals were euthanized and serum and platelets were collected to determine the enzymatic activity of ectonucleotidases involved in nucleotide metabolism that are relevant for platelet aggregation function. The activities of E-NTPDases (ectonucleoside triphosphate diphosphohydrolase), ecto-5’-nucleotidase and E-NPP (nucleotide pyrophosphatase/phosphodiesterase) were estimated in serum and platelets by the hydrolysis rate of AMP (A and E), ADP (B and F), ATP (C and G) and 5’-TMP (D and H). Data are presented as mean± SE and (*) represents statistically significant difference between indicated groups (one-way ANOVA followed by Bonferroni’s-post hoc test).

Figure 5.. Blood coagulation parameters in estrogen-depleted normo and hypertensive rats.

Citrated blood was collected through intracardiac puncture from ovariectomized (OVX) and SHAM-operated spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. The following coagulation parameters were determined in plasma: (A) activated partial thromboplastin time (aPTT); (B) kinetics of WKY rats aPTT; (C) kinetics of SHR rats aPTT; (D) prothrombin time (PT); (E) thrombin time (TT) and (F) fibrinogen levels. Data are presented as mean ± SE and (*) represents statistically significant difference between indicated groups (one-way ANOVA followed by Bonferroni’s-post hoc test).

Figure 6.. Procoagulant phenotype of aortic rings derived from normo and hypertensive ovariectomized rats.

The descending thoracic aorta was collected from ovariectomized (OVX) and SHAM-operated spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. Then, two mm ring segments were prepared to measure its potential increasing rate of kallikrein, factor Xa (FXa) and thrombin generation. (A) Kallikrein generation was determined by incubating the aortic rings with diluted prothrombin-deficient human plasma and adding the specific synthetic substrate S2302 to measured formed kallikrein. (B) FXa generation was determined by incubating the aortic rings with purified human FX, FVIIa and calcium ions and adding the specific synthetic substrate S2222 to measured formed FXa activity. (C) Thrombin generation was determined by incubating the aortic rings in the presence of purified human prothrombin, FVa, FVIIa, FX and calcium ions and adding the specific synthetic substrate S2238 to measured formed thrombin. Data are presented as mean ± SE and (*) represents statistically significant difference between indicated groups (one-way ANOVA followed by Bonferroni’s-post hoc test).

Figure 7.. Tissue factor (TF) protein expression in aorta and platelets from normo and hypertensive ovariectomized rats.

Abdominal aorta and platelets were collected from ovariectomized (OVX) and SHAM-operated spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. Aorta (A) and platelet (B) extracts were prepared, and TF protein expression were analysed by western blot. The upper panel shows representative images from three independent analyses, while the lower panel presents the quantitative data normalized by the β-actin expression. Data are presented as mean ± SE and (*) represents statistically significant difference between indicated groups (one-way ANOVA followed by Bonferroni’s-post hoc test).

Figure 8.. Prothrombotic and fibrinolytic molecular markers in aorta from estrogen-depleted normo and hypertensive rats.

Abdominal aorta was collected from ovariectomized (OVX) and SHAM-operated spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. The tissue extracts were prepared and protein expression of prothrombotic markers – kallikrein (KLK1), factor X (FX), factor II (FII) and protease activated receptor – 1 (PAR-1) - (A and C) and fibrinolytic markers – plasminogen activator inhibitor – 1 (PAI-1) and urokinase plasminogen activator (uPA) - (B and D) were determined by western blot. The left panel shows representative images from three independent analyses, while the right panel presents the quantitative data normalized by the β-actin expression. Data are presented as mean ± SE and the symbols (* and #) represents statistically significant difference between indicated groups (one-way ANOVA followed by Bonferroni’s-post hoc test).

Figure 9.. Plasma-derived from normo and hypertensive ovariectomized rats induced a procoagulant phenotype in vascular smooth muscle cells in culture.

Plasma obtained from ovariectomized (OVX) and SHAM-operated spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats was used to treat a cell line (A7r5) of vascular smooth muscle cells derived from rat thoracic aorta. (A) A7r5 cells were treated with different concentrations of SHR or WKY-derived plasma for 1 h, then cells were washed, and coagulation time was determined through the aPTT assay by the addition of a rat plasma pool collected from healthy animals. (B) A7r5 cells were treated with diluted plasma (10 %) from SHR or WKY animals, at different time-points cells were washed and aPTT was determined as described above. (C) A7r5 cells treated with 10 % plasma from SHR or WKY animals were collected in each time-point, and tissue factor (TF) protein expression was determined by western blot. The panel shows representative images from three independent analyses. (D) Data from TF blots in each time-point were quantified and normalized by the β-actin expression. All data showed on graphs are mean ± SE analysed by one-way ANOVA followed by Bonferroni’s-post hoc test. Coagulation data are presented as a ratio between coagulation time from ovariectomized versus SHAM-operated animals. The dotted line on graphs A and B indicates the normal coagulation pattern of A7r5 cells incubated with rat plasma pool collected from healthy animals. The symbols (*) and (#) in panels A and B indicate respectively the statistical difference of WKY and SHR versus the normal coagulation time from cells treated with plasma pool of healthy animals. The same symbols (* and #) in panel D indicate respectively the statistical difference between KY SHAM versus KY OVX and SHR SHAM versus SHR OVX groups.

Figure 10.. The procoagulant phenotype induced by plasma derived from normo and hypertensive ovariectomized rats requires NF-κB pathway.

Vascular smooth muscle cells (A7r5) were pre-treated overnight with PBS or 0.1 μM PDTC to block the NF-κB pathway. Then the cells were incubated for 2 h with 10 % plasma obtained from ovariectomized (OVX) and SHAM-operated spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. After a washing step, coagulation time was determined through the aPTT assay by the addition of a rat plasma pool obtained from healthy animals (A). Similarly, in another set of experiments vascular cells were treated as above and collected for tissue factor (TF) protein expression analysis by western blot (B). The upper panel in B shows representative images from three independent experiments, while the lower panel presents the quantitative data normalized by the β-actin expression. All data are presented as mean ± SE and symbols (* and #) represents statistically significant difference between indicated groups (one-way ANOVA followed by Bonferroni’s-post hoc test).

Figure 11.. Overview of the prothrombotic mechanisms in an experimental model of menopausal hypertension.

Bilateral ovariectomy leads to an increase in blood pressure. This event is associated with platelet hyperreactivity and increased tissue factor expression in platelets, aorta and vascular cells. The aorta and vascular cells of hypertensive ovariectomized animals have a greater capacity to generate procoagulant enzymes such as thrombin and factor Xa, which are formed on the cell surface by binding to tissue factor. Thrombin and factor Xa are the main activators of PAR-type receptors. These receptors regulate, through NF-kB, the expression of the tissue factor itself, which is found in endothelial cells, smooth muscle cells and platelets. The entire process appears to be part of a positive feedback mechanism that maintain a prothrombotic baseline state. Abbreviations: TF - tissue factor; FVIIa - factor VIIa, FXa - factor Xa, THR - thrombin, PLT - platelets; EC - endothelial cell; VSMC - vascular smooth muscle cell; PAR - protease-activated receptor; NF-kB - nuclear factor kappa B.