Demonstration of ATP-dependent, transcellular transport of lipid across the lymphatic endothelium using an in vitro model of the lacteal.

PURPOSE: The lymphatic system plays crucial roles in tissue fluid balance, trafficking of immune cells, and the uptake of dietary lipid from the intestine. Given these roles there has been an interest in targeting lymphatics through oral lipid-based formulations or intradermal delivery of drug carrier systems. However the mechanisms regulating lipid uptake by lymphatics remain unknown. Thus we sought to modify a previously developed in vitro model to investigate the role of ATP in lipid uptake into the lymphatics. METHODS: Lymphatic endothelial cells were cultured on a transwell membrane and the effective permeability to free fatty acid and Caco-2 cell-secreted lipid was calculated in the presence or absence of the ATP inhibitor sodium azide. RESULTS: ATP inhibition reduced Caco-2 cell-secreted lipid transport, but not dextran transport. FFA transport was ATP-dependent primarily during early periods of ATP inhibition, while Caco-2 cell-secreted lipid transport was lowered at all time points studied. Furthermore, the transcellular component of transport was highly ATP-dependent, a mechanism not observed in fibroblasts, suggesting these mechanisms are unique to lymphatics. Total transport of Caco-2 cell-secreted lipid was dose-dependently reduced by ATP inhibition, and transcellular lipoprotein transport was completely attenuated. CONCLUSION: The transport of lipid across the lymphatic endothelium as demonstrated with this in vitro model occurs in part by an ATP-dependent, transcellular route independent of passive permeability. It remains to be determined the extent that this mechanism exists in vivo and future work should be directed in this area.

Diastolic dysfunction is associated with cardiac fibrosis in the senescence-accelerated mouse.

Diastolic heart failure is a major cause of mortality in the elderly population. It is often preceded by diastolic dysfunction, which is characterized by impaired active relaxation and increased stiffness. We tested the hypothesis that senescence-prone (SAMP8) mice would develop diastolic dysfunction compared with senescence-resistant controls (SAMR1). Pulsed-wave Doppler imaging of the ratio of blood flow velocity through the mitral valve during early (E) vs. late (A) diastole was reduced from 1.3 ± 0.03 in SAMR1 mice to 1.2 ± 0.03 in SAMP8 mice (P < 0.05). Tissue Doppler imaging of the early (E') and late (A') diastolic mitral annulus velocities found E' reduced from 25.7 ± 0.9 mm/s in SAMR1 to 21.1 ± 0.8 mm/s in SAMP8 mice and E'/A' similarly reduced from 1.1 ± 0.02 to 0.8 ± 0.03 in SAMR1 vs. SAMP8 mice, respectively (P < 0.05). Invasive hemodynamics revealed an increased slope of the end-diastolic pressure-volume relationship (0.5 ± 0.05 vs. 0.8 ± 0.14; P < 0.05), indicating increased left ventricular chamber stiffness. There were no differences in systolic function or mean arterial pressure; however, diastolic dysfunction was accompanied by increased fibrosis in the hearts of SAMP8 mice. In SAMR1 vs. SAMP8 mice, interstitial collagen area increased from 0.3 ± 0.04 to 0.8 ± 0.09% and perivascular collagen area increased from 1.0 ± 0.11 to 1.6 ± 0.14%. Transforming growth factor-ß and connective tissue growth factor gene expression were increased in the hearts of SAMP8 mice (P < 0.05 for all data). In summary, SAMP8 mice show increased fibrosis and diastolic dysfunction similar to those seen in humans with aging and may represent a suitable model for future mechanistic studies.

The role of NADPH oxidase in chronic intermittent hypoxia-induced pulmonary hypertension in mice.

Obstructive sleep apnea, characterized by intermittent periods of hypoxemia, is an independent risk factor for the development of pulmonary hypertension. However, the exact mechanisms of this disorder remain to be defined. Enhanced NADPH oxidase expression and superoxide (O2(-).) generation in the pulmonary vasculature play a critical role in hypoxia-induced pulmonary hypertension. Therefore, the current study explores the hypothesis that chronic intermittent hypoxia (CIH) causes pulmonary hypertension, in part, by increasing NADPH oxidase-derived reactive oxygen species (ROS) that contribute to pulmonary vascular remodeling and hypertension. To test this hypothesis, male C57Bl/6 mice and gp91phox knockout mice were exposed to CIH for 8 hours per day, 5 days per week for 8 weeks. CIH mice were placed in a chamber where the oxygen concentration was cycled between 21% and 10% O2 45 times per hour. Exposure to CIH for 8 weeks increased right ventricular systolic pressure (RVSP), right ventricle (RV):left ventricle (LV) + septum (S) weight ratio, an index of RV hypertrophy, and thickness of the right ventricular anterior wall as measured by echocardiography. CIH exposure also caused pulmonary vascular remodeling as demonstrated by increased muscularization of the distal pulmonary vasculature. CIH-induced pulmonary hypertension was associated with increased lung levels of the NADPH oxidase subunits, Nox4 and p22phox, as well as increased activity of platelet-derived growth factor receptor beta and its associated downstream effector, Akt kinase. These CIH-induced derangements were attenuated in similarly treated gp91phox knockout mice. These findings demonstrate that NADPH oxidase-derived ROS contribute to the development of pulmonary vascular remodeling and hypertension caused by CIH.