Thrombospondin-1 and CD47 regulate blood pressure and cardiac responses to vasoactive stress

Abstract

Nitric oxide (NO) locally regulates vascular resistance and blood pressure by modulating blood vessel tone. Thrombospondin-1 signaling via its receptor CD47 locally limits the ability of NO to relax vascular smooth muscle cells and increase regional blood flow in ischemic tissues. To determine whether thrombospondin-1 plays a broader role in central cardiovascular physiology, we examined vasoactive stress responses in mice lacking thrombospondin-1 or CD47. Mice lacking thrombospondin-1 exhibit activity-associated increases in heart rate, central diastolic and mean arterial blood pressure and a constant decrease in pulse pressure. CD47-deficient mice have normal central pulse pressure but elevated resting peripheral blood pressure. Both null mice show exaggerated decreases in peripheral blood pressure and increased cardiac output and ejection fraction in response to NO. Autonomic blockade also induces exaggerated hypotensive responses in awake thrombospondin-1 null and CD47 null mice. Both null mice exhibit a greater hypotensive response to isoflurane, and autonomic blockage under isoflurane anesthesia leads to premature death of thrombospondin-1 null mice. Conversely, the hypertensive response to epinephrine is attenuated in thrombospondin-1 null mice. Thus, the matricellular protein thrombospondin-1 and its receptor CD47 serve as acute physiological regulators of blood pressure and exert a vasopressor activity to maintain global hemodynamics under stress.

Fig. 1. Telemetry blood pressure and pulse recordings in conscious WT and TSP1 null mice

Following transmitter placement in age and sex matched mice, the mice were permitted to equilibrate for 7 days. Recordings of physiologic data were obtained the second post-operative week. Mean arterial pressure (MAP), diastolic blood pressure (DBP), and pulse pressure (PP), heart rate (HR), systolic blood pressure (SBP), and activity level data are presented ±SE for three consecutive wake (18:00 to 6:00 h) - sleep (6:00 to 1800 h) cycles over 72 h. Results are from 4 mice of each strain.

Fig. 2. Telemetry blood pressure and pulse recordings in conscious WT and CD47 null mice

Following transmitter placement in age and sex matched mice, mice were permitted to equilibrate for 7 days. Recordings of physiologic data were obtained the second post-operative week. Mean arterial pressure (MAP), diastolic blood pressure (DBP), and pulse pressure (PP), heart rate (HR), systolic blood pressure (SBP), and activity level data are presented ±SE for three consecutive wake (18:00 to 6:00 h) - sleep (6:00 to 1800 h) cycles over 72 h. Results are from 4 mice of each strain.

Fig. 3. TSP1 and CD47 limit blood pressure changes in response to NO

Age and sex matched awake WT, TSP1 and CD47 null mice underwent analysis of blood pressure (A) and pulse (B) via tail cuff. Blood pressure was assessed via a carotid arterial catheter in 5 WT and 4 CD47 -/- mice under anesthesia (C). Awake WT and TSP1 null were treated with a rapid releasing NO-donor (1 μl/g i.p. of 100 mM DEA/NO) and blood pressure measured by tail cuff (D). Awake WT, TSP1 and CD47 null mice underwent treatment with an intermediate releasing NO-donor (1 μl/g i.p of 100 mM PAPA/NO) and blood pressure (E) and pulse (F) measurements obtained via tail cuff. Results are of the mean ±SD of 8 mice each of WT and TSP1 null and 4 CD47-null. Awake WT and TSP1 null mice underwent telemetric analysis of physiologic data before and after treatment with PAPA/NO. Pulse pressure (PP ±SE, G) is presented from 4 mice of each strain. Experiments were repeated a minimum of 3 times. Asterisk (*) indicates pressure values following treatment that significantly differ from baseline pressure values (P < 0.05).

Fig. 4. TSP1 and CD47 modulate cardiac responses to vasoactive challenge

Age and sex matched WT (n = 4), TSP1 null (n = 4), and CD47 null (n = 4) underwent echocardiography. After baseline recordings, mice were challenged with NO (0.5 μl/g body weight of 100 mM DEA/NO i.p.) and data gathered at 2, 5 and 15 min. Heart rate (A) is presented as the mean values ±SD, whereas ejection fraction (B) and cardiac output (C) as presented as percent control. Asterisk (*) indicates curves are of statistically significant difference when compared with corresponding WT mice (A, B) or individual points of curves are statistically significant compared to WT (C) (P < 0.05). Age and sex matched WT, TSP1 and CD47 null mice underwent euthanasia via cervical dislocation. Hearts were excised, pulverized in liquid nitrogen and tissue cGMP (D) or cAMP (E) levels determined. Results are expressed as the mean ±SD for 6 of each strain (cGMP) and 4 of each strain (cAMP). Asterisk (*) indicates statistically significant difference between TSP1 and CD47 null when compared with corresponding WT samples (D, E) (P < 0.05).

Fig. 5. TSP1 protects against cardiovascular collapse following autonomic blockade and regulates response to epinephrine and general anesthesia

Telemetric transmitters were placed in age and sex matched WT, TSP1 and CD47 null mice (A). On post-operative day 7 mice were treated with hexamethonium chloride (25 μg/g in 100 μl of sterile PBS i.p.) and MAP±SE determined. Experiments were repeated a minimum of three times in 4 mice of each strain. (B) WT and TSP1 null mice underwent laser Doppler analysis of cutaneous perfusion at 2.5 min intervals ± hexamethonium (30 μg/g) i.p. Results are the mean ±SD for 4 mice of each strain. Asterisk indicates values are of statistically significant difference when compared with corresponding WT mice (P < 0.05). Age and sex matched mice underwent blood pressure (C) and pulse (D) analysis via tail cuff before and after treatment with epinephrine (0.05 μg/animal i.p.). Asterisk (*) indicates arterial pressure following treatment significantly differs compared with baseline (C) (P < 0.05). Age and sex matched mice at a core temperature of 35.5° C underwent blood pressure analysis before and after receiving 1.5% inhalation isoflurane (E). Asterisk (*) indicates significant difference in change in pressure between TSP1 compared to wild type following treatment (P < 0.05).