Altered reactivity and nitric oxide signaling in the isolated thoracic duct from an ovine model of congenital heart disease with increased pulmonary blood flow

Abstract

We have previously shown decreased pulmonary lymph flow in our lamb model of chronically increased pulmonary blood flow, created by the in utero placement of an 8-mm aortopulmonary shunt. The purpose of this study was to test the hypothesis that abnormal lymphatic function in shunt lambs is due to impaired lymphatic endothelial nitric oxide (NO)-cGMP signaling resulting in increased lymphatic vascular constriction and/or impaired relaxation. Thoracic duct rings were isolated from 4-wk-old shunt (n = 7) and normal (n = 7) lambs to determine length-tension properties, vascular reactivity, and endothelial NO synthase protein. At baseline, shunt thoracic duct rings had 2.6-fold higher peak to peak tension and a 2-fold increase in the strength of contractions compared with normal rings (P < 0.05). In response to norepinephrine, shunt thoracic duct rings had a 2.4-fold increase in vascular tone compared with normal rings (P < 0.05) and impaired relaxation in response to the endothelium-dependent dilator acetylcholine (63% vs. 13%, P < 0.05). In vivo, inhaled NO (40 ppm) increased pulmonary lymph flow (normalized for resistance) ∼1.5-fold in both normal and shunt lambs (P < 0.05). Inhaled NO exposure increased bioavailable NO [nitrite/nitrate (NOx); ∼2.5-fold in normal lambs and ∼3.4-fold in shunt lambs] and cGMP (∼2.5-fold in both) in the pulmonary lymph effluent (P < 0.05). Chronic exposure to increased pulmonary blood flow is associated with pulmonary lymphatic endothelial injury that disrupts NO-cGMP signaling, leading to increased resting vasoconstriction, increased maximal strength of contraction, and impaired endothelium-dependent relaxation. Inhaled NO increases pulmonary lymph NOx and cGMP levels and pulmonary lymph flow in normal and shunt lambs. Therapies that augment NO-cGMP signaling within the lymphatic system may provide benefits, warranting further study.

Keywords: lymphatic endothelial function; nitric oxide; nitric oxide-cGMP signaling.

Fig. 1.

A: representative graph of a normal (A) and shunt (B) thoracic duct ring in response to increasing log doses of norepinephrine (NE). Active contractions of lymph vessels demonstrate two dynamic components: 1) a low-frequency dynamic trend in developed force (thick solid tracing) that reaches a steady state over time similar to arteries (34) plus 2) a superimposed spontaneous pumping-like force of contraction that is pulse like, reflective of a lymph's pumping ability (24). Dynamic contraction behavior was evaluated from the dose response to NE at selected doses (baseline or passive stretch to 0.2-mN force followed by 9 progressive molar concentrations of 1 × 10⁻⁹ to 1 × 10⁻⁵ M NE). Note the different y-axis scales. C: shunt thoracic duct rings have 2.4 times greater response to log fold increases in NE than normal rings (§P < 0.05). Values are means ± SE; n = 7 normal lambs and 7 shunt lambs. *P < 0.05, significant difference from baseline for shunt rings; †P < 0.05, significant difference from baseline for normal rings.

Fig. 2.

Peak to peak (PP) tension (A and B) and strength of spontaneous contractions (dT/dt) (C and D) in normal and shunt thoracic duct rings at baseline [80% maximum constriction (EC₈₀)] and in a dose-response relationship to ACh (A and C) or S-nitroso-N-acetylpenicillamine (SNAP; B and D). Analysis was based on the high-frequency component of developed tension. §P < 0.05, significant difference in peak to peak tension or dT/dt between normal and shunt rings. Shunt thoracic duct rings have 2.6-fold higher peak to peak tension and 2-fold greater dT/dt than normal rings (P < 0.05). Values are means ± SE; n = 6 normal lambs and 7 shunt lambs. *P < 0.05, significant difference from baseline for shunt rings. Note that at higher doses of SNAP but not ACh, the behavior of normal and shunt rings is equivalent.

Fig. 3.

Dynamic relaxation behavior was evaluated by preconstriction with NE to EC₈₀ and a dose-response relationship to ACh (5 doses from 10⁻⁷ to 10⁻⁴ M; A) and SNAP (4 doses from 10⁻⁸ to 10⁻⁵ M; B). Shunt thoracic duct rings have an attenuated response to ACh compared with normal rings (A; §P < 0.05) but have a similar response to SNAP, an NO donor (B; P > 0.05). Values are mean ± SE; n = 6 normal lambs and 7 shunt lambs. *P < 0.05, significant difference from baseline for shunt rings; †P < 0.05, significant difference from baseline for normal rings.

Fig. 4.

Staining of isolated and paraformaldehyde-fixed thoracic duct sections from normal (A, C, E, G, and I) and shunt (B, D, F, H, and J) lambs. A and B: hematoxylin and eosin staining. C–J: immunostaining with 4′,6-diamidino-2-phenylindole (DAPI; blue; C and D), secondary antibody (2°) alone (red; E and F), endothelial nitric oxide (NO) synthase (eNOS; red; G and H), and actin (pink; I and J). K: eNOS protein expression in pooled homogenates from normal (n = 7) or shunt (n = 7) isolated thoracic duct tissue normalized to β-actin. The dashed line denotes the discontinuity between normal and shunt lanes. eNOS expression was higher in the shunt thoracic duct, and there was increased cellularity and smooth muscle.

Fig. 5.

Lymph flow normalized for resistance (pulmonary vascular resistance) increased significantly from baseline (Bsln) in both normal (†) and shunt (*) lambs by 15 min (15') after the start of inhaled NO (iNO) at 40 ppm and continued for the duration of the study period [45 min (45')] (P < 0.05 for each). Values are mean percent changes ± SE from Bsln; n = 6 normal lambs and 6 shunt lambs. Note that the Bsln percent change (%Δ) represents the variation in lymph flow over the 45-min “steady-state” period and before any study interventions. Lymph flow increased in both normal and shunt lambs in response to iNO.

Fig. 6.

A and B: bioavailable NO [nitrite/nitrate (NO_x); A] and cGMP (B) increased in the pulmonary lymph effluent 15 and 45 min after the start of iNO at 40 ppm in both normal and shunt lambs. *P < 0.05, significant increase from Bsln for shunt lambs; †P < 0.05, significant increase from Bsln for normal lambs. Values are mean percent changes ± SD from Bsln in NO_x (A) or cGMP (B); n = 6 normal lambs and 6 shunt lambs. NO_x and cGMP increased in the pulmonary lymph in both normal and shunt lambs in response to iNO.